Cytoskeletal modulation of electrical and mechanical activity in cardiac myocytes

ARTICLE IN PRESS

Progress in Biophysics & Molecular Biology 84 (2004) 29–59

Review

Cytoskeletal modulation of electrical and mechanical activity in cardiac myocytes S.C. Calaghana, J.-Y. Le Guennecb, E. Whitea,* b

a School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK Nutrition Croissance et Cancer, Emi 0211 Inserm, Facult!e de M!edecine, 2 Bd Tonnelle!, 37032 Tours, France

Abstract The cardiac myocyte has an intracellular scaffold, the cytoskeleton, which has been implicated in several cardiac pathologies including hypertrophy and failure. In this review we describe the role that the cytoskeleton plays in modulating both the electrical activity (through ion channels and exchangers) and mechanical (or contractile) activity of the adult heart. We focus on the 3 components of the cytoskeleton, actin microfilaments, microtubules, and desmin filaments. The limited visual data available suggest that the subsarcolemmal actin cytoskeleton is sparse in the adult myocyte. Selective disruption of cytoskeletal actin by pharmacological tools has yet to be verified in the adult cell, yet evidence exists for modulation of several ionic currents, including ICaL ; INa ; IKATP ; ISAC by actin microfilaments. Microtubules exist as a dense network throughout the adult cardiac cell, and their structure, architecture, kinetics and pharmacological manipulation are well described. Both polymerised and free tubulin are functionally significant. Microtubule proliferation reduces contraction by impeding sarcomeric motion; modulation of sarcoplasmic reticulum Ca2þ release may also be involved in this effect. The lack of effect of microtubule disruption on cardiac contractility in adult myocytes, and the concentration-dependent modulation of the rate of contraction by the disruptor nocodazole in neonatal myocytes, support the existence of functionally distinct microtubule populations. We address the controversy regarding the stimulation of the b-adrenergic signalling pathway by free tubulin. Work with mice lacking desmin has demonstrated the importance of intermediate filaments to normal cardiac function, but the precise role that desmin plays in the electrical and mechanical activity of cardiac muscle has yet to be determined. r 2003 Elsevier Ltd. All rights reserved. Keywords: Cardiac muscle; Actin; Tubulin; Desmin; Cytoskeleton

*Corresponding author. Tel.: +44-1132-334248; fax: +44-1132-334228. E-mail address: [email protected] (E. White). 0079-6107/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0079-6107(03)00057-9

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

30 Contents

1. General introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

30

2. Actin . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Structure of the cardiac actin cytoskeleton . . . 2.2. Pharmacology . . . . . . . . . . . . . . . . . . 2.3. Modulation of mechanical activity and [Ca2+]i 2.4. Sodium channels . . . . . . . . . . . . . . . . 2.5. Calcium channels . . . . . . . . . . . . . . . . 2.6. Potassium channels . . . . . . . . . . . . . . . 2.7. Stretch-activated channels . . . . . . . . . . . 2.8. Cell volume and chloride channels . . . . . . . 2.9. Membrane exchangers . . . . . . . . . . . . . 2.10. Action potential duration . . . . . . . . . . . . 2.11. Summary . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

. . . . . . . . . . . .

31 31 33 33 35 35 37 38 39 40 40 40

3. Microtubules . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Structure of the tubulin cytoskeleton . . . . . . . . . . . . . . 3.2. Pharmacology of microtubules . . . . . . . . . . . . . . . . . 3.3. Modulation of mechanical activity and [Ca2+]i . . . . . . . . 3.4. Ca2+ channels . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Modulation of cardiac b-adrenergic signalling by microtubules 3.6. Microtubules and volume regulation . . . . . . . . . . . . . . 3.7. Other channels and exchangers . . . . . . . . . . . . . . . . . 3.8. Action potential duration and membrane potential . . . . . . 3.9. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

. . . . . . . . . .

41 41 42 43 45 45 47 47 47 47

4. Desmin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

49

5. General summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

50

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

51

1. General introduction The cardiac myocyte contains an intracellular scaffold, the cytoskeleton, which provides structural support and compartmentalisation of intracellular components (Watson, 1991), as well as playing a role in protein synthesis, intracellular trafficking and organelle transport within the cell (Rogers and Gelfand, 2000). Regulatory proteins such as ion channels, are often embedded within and physically bound to the cytoskeleton so that alterations in the cytoskeleton can directly affect their function (e.g. Cherksey et al., 1980; Cantiello, 1997; Janmey, 1998; Johnson, 1999). Furthermore, cytoskeletal modulation of second messenger signalling pathways, including the b-adrenergic pathway, has been demonstrated (e.g. Rasenick et al., 1981). Various theories of cell architecture can be used to explain cellular signalling via the cytoskeleton, including percolation (Forgacs, 1995), the continuum model (Schmidt-Schonbein et al., 1995), and tensegrity (Ingber, 1997, 2000, 2003a, b). Percolation describes the ability of a random array of interconnecting structures to signal as long as the number of connections is

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

31

above a critical threshold (Forgacs, 1995). The continuum model comprises an elastic cortex, that bears the stress of applied external forces, and a viscous cytoplasm (Schmidt-Schonbein et al., 1995). Tensegrity is based principally upon a pre-stressed structure containing compressionresisting and tension-producing elements (Ingber, 1997, 2000, 2003a, b). These models are not mutually exclusive and can share certain components (see Forgacs, 1995; Heidemann et al., 2000). Research has implicated the cytoskeleton in many aspects of cardiac physiology and pathology (Ganote and Armstrong, 1993; Borg et al., 2000; and the reviews prefaced by Schaper and Kostin, 2000). The changing perception of the cardiac cytoskeleton from a purely structural one to one including regulation of cell function is described vividly by Katz (2000): ‘Cytoskeletal proteins, therefore, do more than maintain cell architecture, their participation in cell signalling is analogous to using the steel framework of a building as the phone system’. It is a measure of the interest in this field that this review will concentrate simply upon the evidence for a role of the cytoskeleton in the regulation of electrical and mechanical activity in the myocytes of the atria and ventricles. It should be noted that the cytoskeleton has also been implicated in the functional regulation of cardiac smooth muscle and fibroblasts (e.g. Ingber, 2002; Kamkin et al., 2001, 2003b). We will use the definition of the cytoskeleton put forward by Kostin et al. (1998, 2000) and focus upon the non-sarcomeric actin microfilaments, the microtubules, and the major component of intermediate filaments, desmin, along with related proteins such as actin-binding proteins and microtubule-associated proteins. By necessity we will also address the pharmacology of agents that are used in the study of the cytoskeleton.

2. Actin 2.1. Structure of the cardiac actin cytoskeleton Actin exists in 6 isoforms: 4 a-actins (skeletal, cardiac, vascular and enteric) are found in sarcomeric structures, while b and g actins are thought to be predominantly cytoplasmic (Stromer, 1998). In both neonatal and adult cultured myocytes distinct sarcomeric and non-sarcomeric actin can be seen (e.g. Sadoshima et al., 1992; Messerli and Perriard, 1995; Larsen et al., 1999, 2000; see Fig. 1A). Indeed it seems that these non-sarcomeric structures are necessary for cell spreading and the generation of new myofibrils (Rothen-Rutishauser et al., 1998). However immunocytochemical imaging of actin in acutely isolated adult myocytes reveals uniquely sarcomeric patterning (e.g. Messerli and Perriard, 1995; Kostin et al., 1998; Fig. 1B), even when using antibodies to b-actin, one of the cytosolic actin isomers (Fig. 1C). Similarly, in adult skeletal muscle Otey et al. (1988) were able to detect a-skeletal actin and g-actin, but both isoforms were distributed in a striated pattern, and were not associated with the sarcolemma or mitochondria. While immunocytochemistry has failed to distinguish sarcomeric and non-sarcomeric actin, electron microscopy of adult mouse ventricular (but not atrial) cardiac muscle has identified actin in the dense bars (but not the fine filaments) of leptomeres (Hosokawa et al., 1994) which are patterns of filamentous structure with a markedly shorter periodicity than the sarcomere (Viragh and Challice, 1969). Using fluorescently tagged phalloidin, Yang et al. (2002) observed sparse cortical cytoskeletal actin in the adult rat myocardium, which was increased by hypertrophy.

ARTICLE IN PRESS 32

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

Fig. 1. Labelling of actin in cultured neonatal cardiac myocytes (A) and adult cardiac myocytes (B, C) from the rat. Labelling of cultured neonatal myocytes with an antibody to a-actin reveals faint striated patterning (sarcomeric actin), and thick and thin bundles of microfilaments (cytoskeletal actin). Scale bar represents 20 mm. Taken from Sadoshima et al. (1992). (B) Freshly isolated adult myocytes labelled with fluorescent phalloidin show striated patterning consistent with the sarcomeric actin of the contractile elements. Taken from Kostin et al. (1998). (C) Freshly isolated adult myocytes labelled with antibodies to a-(sarcomeric) and b-(cytoskeletal) actin followed by FITC-conjugated secondary antibody and visualised using confocal microscopy. The images represent planes of approximately 2 mm thickness taken through the mid-plane of the cell at the level of the nucleus. Both antibodies give a longitudinally striated patterning consistent with binding to actin of the contractile elements. Scale bar represents 20 mm.

The cytoskeleton forms connections with the extracellular matrix via membrane-spanning integrins at sites close to the Z-line known as costameres. Of the components of the cytoskeleton, it is actin that has been assigned the primary role in this respect, although microtubules and intermediate filaments may also be involved (Wang et al., 1993; Maniotis et al., 1997). Many of the proteins with which integrins associate at the costamere are actin binding proteins e.g. vinculin, a-actinin, talin, paxillin (Borg et al., 2000; Samuel et al., 2000). These proteins maintain the organisation of cytoskeletal actin in the cell by cross-linking microfilaments, providing links with the sarcolemma, and regulating filament length (Raman and Atkinson, 1999). Other membrane spanning proteins such as b-dystroglycan form connections with the actin cytoskeleton via dystrophin (see Kaprielian and Severs, 2000). For example, Rybakova et al. (2000) showed a costameric patterning of cytoskeletal g-actin in adult skeletal muscle which was absent in the dystrophin-deficient mdx-mouse. However, it is clear that there are differences between costameric (cytoskeletal) actin in cultured cells and in the intact myocardium. In cells cultured on extracellular matrix substrates, actin is always present in the focal adhesion site at the costamere,

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

33

however it has not been detected as part of the extracellular matrix-integrin axis in the intact myocardium (see Borg et al., 2000). Thus the structure of the actin cytoskeleton in the adult myocyte has not been well characterised, and it is clear that there are marked differences between the density and distribution of actin microfilaments in cultured or neonatal cardiac myocytes compared with that in adult myocytes, or in the intact myocardium. Because of difficulties in visualising cytoskeletal actin, verification of the effects of pharmacological intervention or alterations seen in pathological conditions is problematic (see Kostin et al., 1998). 2.2. Pharmacology Cytochalasin-D (Cyto-D) is the most commonly used pharmacological tool to study the actin cytoskeleton. Cyto-D binds to the barbed end of the actin filament at which net polymerisation occurs thereby preventing addition of actin monomers, and it may also bind to a subunit in the interior of an actin filament and ‘sever’ the filament in two (Brenner and Korn, 1979; Cooper, 1987). Cyto-B has also been used to disrupt the actin cytoskeleton (e.g. Dick and Lab, 1998), but unlike Cyto-D, Cyto-B also inhibits sugar transport through the membrane (Grenier et al., 1975). In vitro 0.4 mM Cyto-D inhibited the polymerisation of actin within 5 min (Brenner and Korn, 1979), while in neonatal rat myocytes treatment with 0.4 mM Cyto-D for 1 h can disrupt nonsarcomeric actin structures but leave the sarcomeres intact (e.g. Sadoshima et al., 1992). Exposure to Cyto-D at 2 mM for 4 days also leaves the sarcomeres intact in cultured myocytes, if added several days after plating (Rothen-Rutishauser et al., 1998). It is generally considered that the relative stability of sarcomeric actin protects it from the effects of Cyto-D, perhaps due to the presence of capping proteins at the filament ends which maintain constant filament length (see Lodish et al., 2000). However, although sarcomeres appear visibly intact after treatment with Cyto-D, this does not mean that they are fully functional. Several studies have shown that the acute application of Cyto-D can depress contractile activity of cardiac muscle, by a depression of myofilament Ca2þ sensitivity rather than an effect upon the cytoskeleton (Howarth et al., 1998; Calaghan et al., 2000; see Section 2.3). Other agents which can be used to disrupt actin include latrunculin A, DNase I, gelsolin and cofilin. Latrunculin A is a membrane-permeable drug whose method of action differs to that of Cyto-D as it acts to sequester actin monomers (Aplin and Juliano, 1999). Deoxyribonuclease I (DNase I) forms complexes with G actin and prevents elongation of actin filaments (Berdiev et al., 1996). Alternatively, proteins such as gelsolin and cofilin, that normally control the length of actin in vivo by severing and/or disassembling filamentous actin (Kwiatkowsky et al., 1989; Moon and Drublin, 1995), can be introduced into cells via a patch pipette. Conversely, filamentous actin can be stabilised using the drug phalloidin (Wieland and Faulstich, 1978), or the level of actin in cells can be increased by addition of short lengths of actin (Cantiello, 1997). 2.3. Modulation of mechanical activity and [Ca2+]i Cytochalasin is known to reduce cardiac contractility (Maltsev and Undrovinas, 1998; Skobel and Kammermeier, 1997; Biermann et al., 1998; Howarth et al., 1998; Dick and Lab, 1998). Indeed it has been used specifically to remove motion artefacts associated with contraction in

ARTICLE IN PRESS 34

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

studies using voltage-sensitive dyes to monitor electrical activity (Wu et al., 1998; Lee et al., 2001). The interpretation of this effect has sometimes been based on a cytoskeletal site of action as it was generally assumed that Cyto-D does not affect the more stable sarcomeric actin. However it has been shown that the decrease in contractility seen following 2 min exposure of rat ventricular myocytes to 40 mM Cyto-D is associated with an increase in resting cell length and the amplitude of the intracellular Ca2þ (½Ca2þ i ) transient, and a shift in the phase-plane relationship between shortening and ½Ca2þ i during relaxation (Howarth et al., 1998). In skinned myocytes, a short exposure to Cyto-D was seen to shift the tension-pCa relationship to the right (Calaghan et al., 2000). These data strongly suggest that the negative inotropic effect of Cyto-D can actually be ascribed to a decrease in myofilament sensitivity to Ca2þ through interaction with sarcomeric actin rather than any cytoskeletal effect. Indeed fluorescently labelled Cyto-D gives a labelling pattern in both skinned and intact myocytes consistent with myofibrillar binding (Calaghan et al., 2000; Fig. 2). The rapid effects of Cyto-D on contractility in the adult myocyte are not consistent with its binding to the barbed end of actin filaments and inhibiting polymerisation (Calaghan et al., 2000). The pattern of binding of fluorescent Cyto-D suggests that, in these circumstances, it may be binding along the length of the filament. As well as the effect of Cyto-D on myofilament Ca2þ sensitivity, there are other ways in which the state of actin polymerisation may affect Ca2þ : For example, it has been suggested that actin

Fig. 2. Binding of fluorescent cytochalasin D within an intact adult rat ventricular myocyte. Myocytes were exposed to 1 mM cytochalasin D, BODIPY fluorescent conjugate for 15 min. The clear longitudinal stripes evident in the section taken through the mid-plane of the cell (A) are consistent with myofibrillar labelling and interaction of cytochalasin D with sarcomeric actin. There was no pattern of binding in the sub-sarcolemmal region (B) consistent with the suggested structure of the actin cytoskeleton in the adult cardiac cell. Scale bars represents 10 mm.

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

35

may act, in part, as an intracellular Ca2þ store (Lange and Brandt, 1996). Actin monomers near to the cell membrane can bind Ca2þ with high affinity; polymerisation renders bound Ca2þ less available, effectively creating a Ca2þ store. However, when actin filaments depolymerise, for example following treatment with Cyto-D, this pool of bound Ca2þ can be released. Several studies have shown directly that Cyto-D affects ½Ca2þ i in the cardiac cell (e.g. Howarth et al., 1998; Maltsev and Undrovinas, 1997). These changes are clearly important, as several ion channels and exchangers in the heart are sensitive to ½Ca2þ i (e.g. the inactivation of ICaL ; Lee et al., 1985). Therefore it is important to bear in mind that changes in ½Ca2þ i caused by modulation of sarcomeric (or cytoskeletal) actin, especially if this occurs close to sarcolemma, may influence ion channel or exchanger activity directly. 2.4. Sodium channels Actin has been implicated in the regulation of ion channels in non-cardiac tissue (e.g. Cantiello et al., 1991; Cantiello, 1997). In whole cell patch-clamp experiments on rat ventricular myocytes, 20 mM Cyto-D caused a 20% reduction in peak INa and a slower decay of the current within 2 min of exposure (Undrovinas et al., 1995). Single channel experiments in cell-attached mode showed that 1 h exposure to Cyto-D resulted in channel openings well into the depolarisation, which were not seen in untreated cells. Such an effect could explain the slowed decay of the whole cell current. In excised patches, acute application of Cyto-D to the intracellular surface of the patch caused similar effects and decreased ensemble currents without affecting single channel conductance. Further studies by Maltsev and Undrovinas (1997) suggest that the relationship between voltagedependent, steady-state activation and channel availability following inactivation is modulated by the actin cytoskeleton such that disruption (4 h with 20 mM Cyto-D) depresses channel excitability while stabilization with phalloidin leads to sustained INa : In epithelilal cells it has been shown that it is the length of the actin filament which is pivotal for the modulation of Na+ channel activity, as only short oligomers (3–4 monomers) induced Naþ channel activity (Cantiello et al., 1991); longer filaments had no effect. 2.5. Calcium channels In adult ventricular myocytes, Cyto-D and phalloidin have been used to test for a role of the actin cytoskeleton in modulating ICaL : In the presence of mM concentrations of added intracellular Ca2þ buffers (EGTA or BAPTA), neither Cyto-D nor phalloidin had any effect on ICaL in rat (Undrovinas and Maltsev, 1998; Yang et al., 2002) or guinea pig (Pascarel et al., 1999; Rueckschloss and Isenberg, 2001) ventricular myocytes. Concentrations of Cyto-D ranged from 10–50 mM and phalloidin from 20–100 mM with exposure times of 10 min (or 2 h, Undrovinas and Maltsev, 1998). Cyto-D (100 mM) also had no effect on ICaL in excised patches from rabbit ventricular myocytes in the presence of 10 mM EGTA (Dzhura et al., 2002). The only study to report an effect of Cyto-D on ICaL in the adult myocyte was performed with reduced Ca2þ buffering, suggesting that Ca2þ may be a pivotal mediator in the effect of Cyto-D on ICaL : Rueckschloss and Isenberg (2001) reported that in the presence of 5 mM EGTA, Cyto-D decreases the amplitude of ICaL : This effect was sensitive to acidosis and Ser/Thr protein phosphatases.

ARTICLE IN PRESS 36

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

Exposure to Cyto-D also reduced phoshorylated cofilin and the authors suggested that dephosphorylated cofilin depolymerises F-actin causing a reduction in ICaL : In terms of ICaL ; the difference between actin cytoskeleton in adult and neonatal myocyctes is highlighted by the study of Lader et al. (1999). These workers showed that in neonatal rat ventricular myocytes, even in the presence of 10 mM EGTA, Cyto-D (167 mM) applied through the patch pipette markedly reduced ICaL ; phalloidin (10 mM) had the opposite effect (Lader et al., 1999). An actin microfilament network was clearly visible in these myocytes. Furthermore, in neonatal myocytes from gelsolin knock-out mice, in which the actin cytoskeleton was stabilised, ICaL was larger than in wild type myocytes; this effect was reversed by addition of 600 nM gelsolin and Cyto-D (Lader et al., 1999). These data are consistent with the shifting of the inactivation rate of the L-type Ca2þ channel to a more rapidly inactivating state when the actin cytoskeleton is disrupted (Lader et al., 1999). Wang et al. (2000a, b) have shown that in adult cat atrial myocytes, alterations in adenyl cyclase and b-adrenergic receptor-dependent modulation of ICaL ; caused by laminin stimulation of integrin receptors, is prevented by exposure to 20 mM Cyto-D or 1 mM lantruculin A for 2 h (with 0.5 mM EGTA). The effect of actin disruptors on ICaL in these myocytes in the absence of laminin binding was not assessed. However, this report suggests that the actin cytoskeleton can modulate ion channel function (or signalling pathways) in the adult myocyte when the extracellular matrix is intact. The cytoskeleton has been linked to the modulation of GTP-binding proteins (G-proteins) (see Janmey, 1998 and Section 3.5). In agreement with the general findings of Wang et al. (2000a, b), Bloch et al. (2001) showed that in mouse embryonic heart cells normal regulation of ICaL was sensitive to Gi and to b1 -integrin receptors. The regulation of ICaL and the localisation and function of Gi ; were modulated by Cyto-D in a manner that was similar to changes seen in b1 integrin null mice. Thus these studies suggest that actin forms an important part of the signalling pathway arising from the extracellular matrix in both adult and neonatal cardiac myocytes. There is evidence for an indirect link between the L-type Ca2þ channel and the actin cytoskeleton via actin-binding proteins. It has been recently suggested that in human ventricle the L-type Ca2þ channel is linked to the actin cytoskeleton by the large (700 kDa) protein Ahnak which binds both actin and the b sub-unit of the channel (Hohaus et al., 2002). Co-localisation of the L-type Ca2þ channel with the actin binding proteins a-actinin at the Z line, and dystrophin at the Z and M lines of the sarcomere has been demonstrated (Sadeghi et al., 2002). Furthermore in dystrophin-deficient mdx-mice, ICaL inactivation was reduced and its voltage-dependence shifted to more positive potentials. This effect may explain the observed prolongation of the QT phase of the mdx-mouse electrocardiogram (Sadeghi et al., 2002). It has been established that phosphorylation of the L-type calcium channel by a Ca2þ calmodulin-dependent protein kinase II (CaMK II, Dzhura et al., 2000), as well as a calmodulin binding ‘‘IQ’’ domain on the L-type Ca2þ channel (Wu et al., 2001), increases ICaL amplitude. The actin cytoskeleton (and microtubules, see Section 3.4) have been implicated in targeting CaMK II and the ‘IQ’ domain to the L-type Ca2þ channel (Dzhura et al., 2002). Exposure of rabbit ventricular myocytes to 100 mM Cyto-D for >2 h abolished CaM-dependent facilitation of ICaL : By contrast, facilitation of ICaL in response to protein kinase A was not modulated by Cyto-D, suggesting that the functional association of CaM kinase but not protein kinase A is dependent on the actin cytoskeleton. This is supported by the observation of Rueckschloss and Isenberg (2001) that Cyto-D does not modify the response of ICaL to the b-agonist isoprenaline.

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

37

The T-type Ca2þ channel is less abundant than the L-type channel in mammalian cardiac muscle (Nilius et al., 1985). ICaT has been shown to be activated by hypotonic swelling, and this response is blocked by a 12 min exposure to 20 mM Cyto-D (Pascarel et al., 2001). 2.6. Potassium channels The sub-sarcolemmal actin cytoskeleton has been assigned a role in the modulation of the ATPsensitive Kþ (KATP) channel, which opens at low levels of intracellular ATP (Noma, 1983). In adult guinea pig ventricular myocytes disruption of actin attached to the inside of excised membrane patches with DNase I enhanced IKATP in the presence of ATP; this effect was partially reversible by addition of purified actin subunits (Terzic and Kurachi, 1996). Cyto-B had similar effects to DNase I, but microtubule disruption or stabilisation did not modulate ATP sensitivity of IKATP (Terzic and Kurachi, 1996). The inhibition of IKATP by sulfonylurea was also decreased by DNase (Brady et al., 1996). However, channel rundown was concurrently enhanced under these conditions (Furukawa et al., 1996), and could be prevented by the actin stabiliser phalloidin. Similar effects on ATP and sulfonylurea sensitivity of the channel were found in rats by Yokoshiki et al. (1997). More recently it has been shown that the diadenosine tetraphosphate (Ap4A) inhibition of IKATP is prevented by Cyto-B or DNase I (Jovanovic and Jovanovic, 2001). IKATP is activated as a protective mechanism in response to metabolic depletion, such as that seen during ischaemia. Although it has been suggested that the activation of only a fraction (1%) of KATP channels is sufficient to radically alter the excitability of the cardiac myocyte (Faivre and Findlay, 1990), the levels of ATP recorded during ischaemia appear higher than those required for the activation of IKATP : However it is known that the cytoskeleton is disrupted during ischaemia (Ganote and Armstrong, 1993; Hori et al., 1994; Hein et al., 1995). Disruption of the actin cytoskeleton will decrease sensitivity of KATP channels to ATP and to Ap4A (which may alert cells to metabolic injury, Kisselev et al., 1998), thus allowing them to open at higher intracellular concentrations of ATP. Ischaemic pre-conditioning refers to the protective effect a brief ischaemic episode has upon the myocardium in response to a subsequent severe ischaemic insult (Reimer et al., 1990). It has been suggested that activation of IKATP may be involved in this response (Grover and Garlid, 2000; Cohen et al., 2000). Baines et al. (1999) showed that in rabbit myocardium, ischaemic preconditioning and the protective action of IKATP activation were blocked by 20 mM Cyto-D, and on the basis of the relative sensitivity of sarcolemmal and mitochondrial IKATP to diazoxide, they concluded that pre-conditioning was dependent upon a process coupling the actin cytoskeleton and mitochondrial IKATP channels. Actin disruption has also been shown to enhance Kv1.5 channel activity (a component of the delayed rectifier IK ). Maruoka et al. (2000) and Cukovic et al. (2001) showed that human cardiac Kv1.5 channels expressed in HEK cells, bind to the actin binding protein a-actinin-2. In a response similar to that of IKATP (see above) treatment with 5 mM Cyto-D for 2 h enhanced currents through these channels. The effect of Cyto-D was countered by phalloidin. Part of the increase in current seen after 72 h treatment with Cyto-D was thought to come from an increased number of functioning channels. Co-localisation of Kv1.5 and a-actinin, seen at the cell periphery, was lost following 48 h of Cyto-D treatment. In Kv1.5 channels expressed in Xenopus oocytes,

ARTICLE IN PRESS 38

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

currents were reduced following a depression of basal protein kinase A activity and this effect was sensitive to Cyto-B and Cyto-D and required a-actinin-2 (Mason et al., 2002). However, by contrast to findings from Maruoka et al. (2000) and Cukovic et al. (2001), this study found no effect of cytochalasins or a-actinin-2 antisense on basal Kv1.5 currents. The expression system may account for the difference between these two studies. In the heart, inward rectification of IK1 is largely due to open channel block of outward current by intracellular polyamines and divalent cations such as Ca2þ and Mg2þ : In control conditions, the IK1 channel shows four subconductance states. In patches excised from guinea pig ventricular myocytes in the absence of Ca2þ or Mg2þ ; these channels lose their rectifying behaviour by opening preferentially at subconductance states of progressively increasing conductance. Rectification can be restored by adding Ca2þ (Mazzanti et al., 1996). Cyto-D (10 mM) accelerates loss of rectification in divalent cation-free solution, and abolishes Ca2þ -induced outward subconductance states and rectification (Mazzanti et al., 1996). It appears that an intact subsarcolemmal cytoskeleton plays a vital role in the control of sub-conductance states and rectification in these channels. For some Kþ channels, the modulatory role of the actin cytoskeleton is only revealed under pathological conditions. In normal adult rat myocytes, neither Cyto-D (50 mM; 10 min) nor phalloidin (20 mM; 10 min) affect the transient outward current (Ito1 ) (Yang et al., 2002). However in myocytes from hypertrophied rat hearts, in which Ito1 is depressed, Cyto-D causes a further reduction of Ito1 ; whereas phalloidin normalises Ito1 to values similar to that found in nonhypertrophied cells (Yang et al., 2002). Depression of Ito1 plays a role in the prolongation of the action potential duration which is commonly seen in hypertrophy, and may predispose to cardiac arrhythmia (see Section 2.10). The cytoskeleton is not only implicated in the regulation of channel function; it is also important for channel translocation. In rats with Type 1 diabetes, Shimoni et al. (1999) and Shimoni and Rattner (2001) have shown that a Ca2þ -activated transient outward Kþ current (It ) and a slowly inactivating Kþ current (ISS ) are depressed. Both these currents could be restored by insulin, whereas in control animals only ISS was enhanced by insulin. The effect of insulin was sensitive to 1 mM Cyto-D, (and the microtubule disruptor colchicine, see Section 3.2). The cytoskeleton-dependent action of insulin upon ISS was more rapid in control cells. A 6–10 h exposure to 1 mM Cyto-D alone had no effect upon the currents (whole cell, 10 mM EGTA/1 mM CaCl2 ) and the action of insulin was protected from Cyto-D by pre-exposure to 2.5 mM phalloidin. The authors suggest that insulin triggers the synthesis of new channels and that the role of the cytoskeleton is to convey these to the membrane. 2.7. Stretch-activated channels Stretch-activated channels (SACs) were first discovered in cultured embryonic skeletal muscle (Guharay and Sachs, 1984), but have since been reported in many cell types including cardiac myocytes (Hamill and Martinac, 2001). The activation of these channels may occur as a result of changes in tension within the lipid bilayer bringing about altered bilayer curvature and/or hydrophobic mismatch between the channel and bilayer (Perozo et al., 2002). Generation of tension within the cytoskeleton may influence the bilayer or the channel directly. In the original study by Guharay and Sachs (1984), Cyto-A, -B or -E (10 mM for 12 h) increased the sensitivity to

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

39

mechanical stimulation. This observation has also been reported in response to Cyto-B (10 mM, 6 h) in neonatal rat atrial cells (Kim, 1993). In adult rat atrial myocytes exposure to 20–40 mM Cyto-D for 20–30 min increased the amplitude of a non-selective cationic SAC but did not alter a background SAC (Zhang et al., 2000). In mammalian ventricular myocytes, Cyto-D (and 5 mM intracellular colchicine) have been reported to reduce the amplitude of SACs (Isenberg et al., 2003; Kamkin et al., 2003a). One explanation for the increased sensitivity to stretch of SACs following treatment with cytochalasins is that breaking the links between the cytoskeleton and sites of stress increases the radius of membrane response, thereby increasing mechanosensitivity to a given stimulus (Janmey, 1998). SACs have been implicated in the generation of stretch-activated arrhythmias and consistent with this role and the action of cytochalasins on SACs, Dick and Lab (1998) reported that stretch of the left ventricle of isolated rabbit hearts provoked ventricular fibrillation in the presence of 10 mM Cyto-B. It should be noted that if Ca2þ was elevated due to its release from myofilaments (see Section 2.3), this might contribute to any arrhythmic stimulus. 2.8. Cell volume and chloride channels In cultured chick ventricular myocytes exposure to hypo-osmotic solution results in cell swelling followed quickly by a regulatory volume decrease (RVD) in intact (i.e. not ruptured patchclamped) cells. During these responses, the actin cytoskeleton is able to maintain its integrity (Zhang et al., 1997; Larsen et al., 2000). In these cultured cells, Cyto-B was shown to disrupt cytoskeletal actin and displace it from the sarcolemma. Following actin disruption, there was a greater increase in cell volume upon swelling and the RVD was decreased, while phalloidin reduced the change in volume upon hypo-osmotic challenge (Zhang et al., 1997). The RVD is associated with activation of a Cl current (ICl ) (Zhang et al., 1993) that is suppressed by exposure to either Cyto-B or phalloidin (Hall et al., 1997; Zhang et al., 1997). Removal of cytochalasin from treated cells leads to a rapid increase in ICl and an associated re-distribution of actin (Zhang et al., 1997). The authors conclude that an intact actin cytoskeleton at the sarcolemma is required for the activation of ICl and RVD, yet the precise interaction of these components remains to be elucidated. In neonatal rat ventricular myocytes cell swelling has been shown to cause a translocation of actin from the membrane to the cytosolic fraction, while ICln, a protein possibly associated with the activation of ICl ; moves in the opposite direction (Musch et al., 1998). However in adult rat myocytes treatment with Cyto-D (10 mM for at least 1 h) did not influence the swelling in response to exposure to hypo-osmotic solution (Lovett et al., 2003; Fig. 4B). Another Cl channel, whose dysfunction is associated with the onset of cystic fibrosis, the cystic fibrosis transmembrane conductance regulator (cCFTR) is regulated by the actin cytoskeleton (Cantiello, 1996). Neonatal rat ventricular myocytes show a large increase in cCFTR following 5–15 min treatment with 20 mM Cyto-D; this response was independent of the established protein kinase A activation of the channel. Similar to the effect reported for Naþ channels (see Section 2.4), the length of actin filaments appear to be pivotal for modulation of this channel; increased availability of short actin filaments activates the channel whereas complete degradation of the actin cytoskeleton causes inhibition (Prat et al., 1993, 1995).

ARTICLE IN PRESS 40

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

2.9. Membrane exchangers The electrogenic sodium:calcium exchanger is an important regulator of ½Ca2þ i ; capable of extruding Ca2þ from the cell (forward mode) or driving Ca2þ influx into the cell (reverse mode) (Bers, 2002). When the cardiac Na:Ca exchanger protein was expressed in CHO cells, Cyto-D (1 mM, 60 min) and depletion of ATP were shown to reduce ½Naþ o inhibition of Ca2þ uptake (Condrescu et al., 1995). Under these conditions, both Cyto-D and ATP depletion disrupted the actin cytoskeleton of CHO cells (Condrescu et al., 1995). The effects of actin may be mediated through ankyrin, a protein that has been shown to bind to the exchanger (Li et al., 1993). However, the role of actin in modulating INaCa in the adult cardiac myocyte is less convincing. In adult cardiac myocytes, cytochalasins did not alter INaCa (Hilgemann and Ball, 1996), and Haworth and Biggs (1997) found no effect of Cyto-D (20 mM for up to 1 h) upon Ca2þ uptake via the exchanger. 2.10. Action potential duration Bierman et al. (1998), Wu et al. (1998), and Lee et al. (2001) have reported that Cyto-D at concentrations up to 80 mM does not influence the action potential duration (APD) of canine or pig ventricular muscle. In the normal adult rat myocyte, Cyto-D also has no effect on APD (Yang et al., 2002). Wu et al. (1998) further reported that Cyto-D did not modify the transmural conduction velocity or the wavefront of action potential propagation in perfused wedges of canine left ventricle, and Lee et al. (2001) saw no effect of Cyto-D on the restitution curves of the action potential and indices of ventricular fibrillation (induced by rapid stimulation). However in contrast to these findings, there are two reports of Cyto-D (5–10 mM) lengthening action potential duration in rabbit ventricular muscle (Banville and Gray, 2002; Kettleman et al., 2002). Kettleman et al. (2002) have also reported an increase in conduction delay by 1 mM Cyto-D. Although Cyto-D does not affect APD in the normal rat myocyte, the long APD of hypertrophied rat ventricular myocytes is further prolonged by Cyto-D, and shortened by phalloidin (Yang et al., 2002). In Sections 2.4–2.9 we have discussed evidence for the regulation of individual ion channels and exchangers by agents that modulate the actin cytoskeleton. The alteration of a given membrane current would be expected to influence the shape of the action potential, which represents the sum of all membrane currents flowing at a given time. The majority of studies that have reported no effect of actin disruption upon the action potential suggest that the overall role of the actin cytoskeleton in modulating ionic currents is negligible. For Cyto-D, it also suggests that its influence on Ca2þ activated currents and contraction/force dependent changes are not important. It should be noted that neither the reported lack of effect or lengthening of the APD is consistent with the reduction of ICaL and INa seen in some studies (see Sections 2.4 and 2.5). However, when considering the lack of reported effects of agents that influence the actin cytoskeleton upon the action potential, with reports of effects upon ion channels, it should be noted that certain ion channels may not be activated under ‘normal’ recording conditions e.g. KATP, SACs, Cl : 2.11. Summary The evidence for an important role of the actin cytoskeleton in modulating electrical activity of the heart is very convincing in neonatal and cultured myocytes in which an actin microfilament

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

41

network can be clearly visualised. In the adult myocyte and in intact myocardium there are problems associated with imaging of the actin cytoskeleton, and with the interpretation of the effects of Cyto-D which also interacts with sarcomeric actin. Despite the lack of clear evidence for modulation of the action potential by cytoskeletal actin, there are now many reports of effects upon cardiac ionic currents where the logical conclusion is modulation of channel activity by the non-sarcomeric actin cytoskeleton. The exact mechanisms by which actin microfilaments may exert their effects on channel activity are ill-defined. At present an intriguing, role for the actin cytoskeleton in the regulation of electrical (and thereby mechanical) activity of the heart is beginning to emerge.

3. Microtubules 3.1. Structure of the tubulin cytoskeleton Microtubules are a major component of the cardiac myocyte cytoskeleton and have been assigned many functional roles, for example, in protein synthesis, intracellular trafficking, and intracellular signalling (Rogers and Gelfand, 2000). Microtubules are hollow protein cylinders of a- and b-tubulin heterodimers about 25 nm in diameter (Goldstein and Entman, 1979; Rappaport and Samuel, 1988) aligned predominantly along the longitudinal axis of the adult myocyte (e.g. Kostin et al., 2000) and adjacent to the nucleus in the perinuclear space (Cartwright and Goldstein, 1985) (see Fig. 2). Roughly 25% of total tubulin is in the polymerised form (Palmer et al., 1998; Yamamoto et al., 1998). In most cells, microtubules exist as a large dynamic population and a small subset of drug- and cold-stable microtubules (Schulze and Kirschner, 1987). With the exception of this small stable population, microtubules are in a state of dynamic instability with addition of subunits to the A, or plus end and loss at the D or minus end (Wilson and Jordan, 1994). Addition of subunits is powered by GTP hydrolysis, each a- and b-tubulin monomer binds GTP, but it is the GTP on the b-tubulin which is hydrolysed. Microtubules are found in association with GTP-binding proteins such as Gi and Gs (Rasenick et al., 1981, 1990), microtubule associated proteins (MAPs) that promote microtubule stability (predominantly MAP4 in cardiac muscle, Olmsted, 1986; Sato et al., 1997), Tau protein (Goedert et al., 1994), kinases (Pitcher et al., 1998), and molecular motors such as kinesin (Olmsted, 1986; Liao and Gundersen, 1998). Microtubules also associate with other components of the cytoskeleton such as actin (Cunningham et al., 1997) and intermediate filaments (Gurland and Gundersen, 1995). Microtubule associated protein has been shown to reorganise both actin microfilaments and microtubules (Cunningham et al., 1997). The microtubular cytoskeleton has been linked with various pathological conditions. Disruption of the microtubular network has been reported in ischaemia (Hori et al., 1994; Hein et al., 1995) and irreversible cell damage may be associated with collapse of the microtubule cytoskeleton (Iwai et al., 1990). Proliferation of the microtubules with taxol has been reported to protect against hypoxia/re-oxygenation injury (Skobel and Kammermeier, 1997), whereas the microtubule disruptor colchicine has been shown to abolish ischaemic pre-conditioning (Sharma and Singh, 2000a, b). Proliferation of microtubules has been implicated in the depression of contractility seen in cardiac hypertrophy (e.g. Tsutsui et al., 1994, see Section 3.3). In diabetic

ARTICLE IN PRESS 42

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

cardiomyopathy, an increase in the population of drug-stable microtubules is seen (Howarth et al., 2002). Likewise, in a model of heart failure in the rat, an increase in the stability of the microtubular network has been reported (Belmadani et al., 2002). 3.2. Pharmacology of microtubules The most popular agents used to manipulate the microtubule cytoskeleton are colchicine, which causes disruption of the microtubules, and taxol, which causes proliferation and stabilisation. Immunocytochemistry and Western blotting studies have shown that exposure of adult cardiac myocytes to colchicine (1–10 mM for 1–4 h) leads to a decrease in intact microtubules and an increase in free tubulin (e.g. Tsutsui et al., 1994; Calaghan et al., 2001a; Kerfant et al., 2001, see Fig. 4A), whereas taxol (10 mM for 2–4 h) has the opposite effect (e.g. Tsutsui et al., 1994; Howarth et al., 1999). As the effects of agents on microtubules in the adult myocyte can be clearly visualised and quantified, the role of the microtubule cytoskeleton is somewhat easier to define than that of the actin cytoskeleton (see Section 2.2). Colchicine has the advantage of having an inactive stereo-isomer lumicolchicine which does not have an action on microtubules in cardiac myocytes (Nath et al., 1978; Limas and Limas, 1983; Tsutsui et al., 1994). Other interventions that are used to disrupt microtubules in myocytes include storage below 4 C (Tsutsui et al., 1994), nocodazole (Wang et al., 1999; Rothen-Rutishauser et al., 1998; Webster and Patrick, 2000), and vinblastine (Limas and Limas, 1983; Lampidis et al., 1992). These agents affect microtubules by different mechanisms. Colchicine binds to free tubulin heterodimers that become incorporated into microtubules inhibiting addition of further subunits. GTPase activity of tubulin is increased by colchicine (David-Pfeuty et al., 1979). Vinca alkaloids such as vinblastine bind directly to the growing end of microtubules and induce the formation of tubulin paracrystals (non-microtubule polymers). By contrast to the effects of colchicine, vinblastine inhibits GTPase activity of tubulin (David-Pfeuty et al., 1979). Nocodazole inhibits the addition of tubulin subunits to microtubules, targeting dynamic (and not stable) microtubules at low doses (1 mM for 2 h) but depolymerising all microtubules at higher concentrations (33 mM for 2 h; Webster and Patrick, 2000). Colchicine and nocodazole interact at the same site of tubulin but this interaction is reported to be reversible only with nocodazole (Hoebecke et al., 1976), although Kerfant et al. (2001) have reported a reversible depolymerisation of microtubules in adult cardiac myocytes with colchicine. In terms of agents which increase the number of microtubules, taxol stabilises and proliferates intact microtubules by reducing the critical concentration of tubulin subunits required for microtubule assembly (Schiff et al., 1979; Arnal and Wade, 1995). Heavy water (deuterium oxide) is also known to proliferate microtubules and has been used to chronically manipulate microtubules in adult cardiac muscle (Takahashi et al., 1998a). However its rapid and reversible negative inotropic effect is probably due to its ability to decrease myofilament Ca2þ sensitivity and ICaL rather than to rapidly proliferate microtubules (Allen et al., 1984; Hongo et al., 2000). For the microtubule cytoskeleton, both polymerised and free tubulin heterodimers have been assigned vital roles in cell signalling, by contrast to the actin microfilaments which are only thought to be biologically active when in a state of polymerisation. This is an important consideration when selecting pharmacological agents to manipulate microtubules. For example, both colchicine and vinblastine decrease the number of polymerised microtubules, but only

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

43

colchicine increases free tubulin. Therefore, a comparison of the response to these two agents may provide an assessment of the role of increased free tubulin (although colchicine and vinblastine have opposite effects on GTPase activity, David-Pfeuty et al., 1979). It is also worth noting that immunolabelling of cytoskeletal components often entails a cell permeabilisation step that may allow the escape of free component sub-units (Hatcher et al., 1999). 3.3. Modulation of mechanical activity and [Ca2+]i The microtubular cytoskeleton is a load-bearing structure (e.g. Ives et al., 1986) that makes only a small contribution to passive properties of the myocyte compared to the sarcomeric protein titin (Granzier and Irving, 1995). However, in a linear polymer, such as a microtubule, an extending force is predicted to lower the critical concentration for sub-unit (heterodimer) assembly and enhance polymer (microtubule) stability (Hill and Kirschner, 1982; Joshi et al., 1985). Indeed, in vivo, microtubules have been shown to proliferate in response to stress (pressure) overload (e.g. Tsutsui et al., 1993, 1994; see Cooper, 2000 for a review). It seems that proliferation occurs when hypertrophy no longer compensates for the increase in pressure, leading to an increase in wall stress (Cooper, 2000). Although this effect is not seen in all models of hypertrophy, it does appear relevant to certain situations of human pressure-overload hypertrophy and failure (Schaper et al., 1991; Heling et al., 2000; Kostin et al., 2000; Zile et al., 2001). As a consequence of microtubule proliferation, hypertrophied myocytes show increased internal apparent viscosity (Tagawa et al., 1997; Yamamoto et al., 1998; Cooper, 2000) and decreased contractility (Tsutsui et al., 1994; Zile et al., 1999; Cooper, 2000). The depression of contractility is mimicked in non-hypertrophied cells by microtubule proliferation with taxol (Tsutsui et al., 1994; Zile et al., 1999; Howarth et al., 1999), and reversed in hypertrophied cells by microtubule disruption with colchicine, while lumicolchicine is without effect. Dysfunction has been ascribed to microtubule interference with sarcomere motion (e.g. Tsutsui et al., 1993; Cooper, 2000). It is clear that the effects of hypertrophy and taxol on contraction can be ascribed to an increase in the number of microtubules, rather than an effect of free tubulin heterodimers, as the latter are increased in hypertrophy and decreased following treatment with taxol. Taxol has been shown to decrease the ½Ca2þ i transient in rat ventricular myocytes (Howarth et al., 1999). This reduction in the ½Ca2þ i transient was due, in part at least, to reduced Ca2þ release from the sarcoplasmic reticulum (Howarth et al., 1999). This reduction in ½Ca2þ i may play a role in the depression of contractility seen when microtubules are proliferated. However, the improvement in the contractile function of hypertrophied muscle caused by colchicine was not associated with an increase in the ½Ca2þ i transient (Zile et al., 1999) and colchicine did not increase the amplitude of the ½Ca2þ i transient in normal ventricular myocytes (Palmer et al., 1998; Calaghan et al., 2001a, but see Kerfant et al., 2001) or in papillary muscles (Zile et al., 1999). Consistent with a lack of effect upon the ½Ca2þ i transient, in the absence of prior microtubule proliferation, disruption of the microtubular cytoskeleton by colchicine does not significantly modulate cardiac contractility in adult cardiac muscle or myocytes (Tsutsui et al., 1993, 1994; Collins et al., 1996; Ishibashi et al., 1996; Bailey et al., 1997; Tagawa et al., 1998; Takahashi et al., 1998b; Yamamoto et al., 1998; Zile et al., 1999; Hongo et al., 2000; Calaghan et al., 2001a). However, in apparent contrast to these studies, it has also been reported that in rat ventricular myocytes colchicine decreased the amplitude of Ca2þ sparks and increased the amplitude of the

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

44

global ½Ca2þ i transient (Gomez et al., 2000; Kerfant et al., 2001; see Section 3.4). Although Gomez et al. (2000) and Kerfant et al. (2001) did not measure contractility, the increase in ½Ca2þ i transient which they observed with colchicine would be expected to increase contractility (unless there was a concomitant decrease in myofilament sensitivity, Calaghan et al., 2001b). No such increase in contractility with colchicine has been reported in the literature. Proliferated microtubules differ in appearance to microtubules present in the normal cardiac cell (Tsutsui et al., 1993; Howarth et al., 1999). Taxol-proliferated microtubules are resistant to the actions of cold, Ca2þ and colchicine (Kumar, 1981; Howarth et al., 1999) and proliferated microtubules in hypertrophy differ in terms of tubulin isoforms, post-translational modifications and the ratio of microtubule-associated protein to tubulin (Cooper, 2000; Belmadani et al., 2002). One line of reasoning that can reconcile the general finding that disruption of proliferated, but not normal, microtubules can modulate cardiac contractility is that there are functionally distinct populations of microtubules. Microtubules have also been implicated in the rate of beating of the neonatal heart. Early studies in this field have showed that colchicine, nocodazole and vinblastine (0.05–10 mM), but not lumicolchicine (10 mM), increased the spontaneous beating rate of cultured neonatal rat cardiac myocytes (Klein, 1983; Lampidis et al., 1992) (see Fig. 3). However, more recently, a concentration-dependent effect of nocodazole on beating rate was shown by Webster and Patrick (2000); after 2 h exposure, 1 mM nocodazole had no effect on beating rate, whereas 33 mM nocodazole significantly increased beating rate. These authors showed that under their experimental conditions, the lower concentration of nocodazole significantly reduced the number of polymerised microtubules, but that at the higher concentration all microtubules were

(B)

(A)

Fig. 3. Microtubular cytoskeleton in cultured neonatal and adult rat ventricular myocytes. Microtubules were labelled with an antibody to b-tubulin followed by FITC-conjugated secondary antibody and visualised using confocal microscopy in cultured neonatal myocytes (A) and freshly isolated rat ventricular myocytes (B). Neonatal and adult myocytes both show strong perinuclear staining, however, microtubules are primarily radial in the neonatal cells and longitudinally orientated in adult cells. Scale bars represents 20 mm. (A) from Sadoshima et al. (1992).

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

45

disassembled. These data suggest that it is the small population of stable microtubules that modulate the beating rate of neonatal cardiac cells, and provide further evidence for functionally distinct populations of microtubules. Others have assigned free tubulin heterodimers a role in modulating beating rate in neonatal cardiac cells. Motlagh et al. (2002) reported that colchicine (15 mM), but not vinblastine (0.5 mM), increased the rate of rise and decay of the ½Ca2þ i transient in spontaneously beating neonatal rat cardiac myocytes. Whilst both agents decreased polymerised microtubules in this study, only colchicine increased free tubulin heterodimers, and the authors suggested that free tubulin may be associated with the increase in rate via effects on INa (see Section 3.7). In spontaneously beating neonatal myocytes, consistent with previous findings (Lampidis et al., 1992), Webster and Patrick (2000) showed that 10 mM taxol was without effect upon rate, but that when cell tubulin content and microtubule density was increased by injection of tubulin into cells, beating rate fell. The lack of effect of taxol under these conditions was assigned to the limited magnitude of changes in the microtubular population compared with that seen following injection of tubulin (or hypertrophy) (Webster and Patrick, 2000), although it is interesting that of these three ‘interventions’, taxol is the only one that does not increase free tubulin. 3.4. Ca2+ channels A comprehensive role for the microtubule cytoskeleton in the regulation of the L-type Ca2þ channel has yet to emerge. Taxol and colchicine delay the inactivation of ICaL after a short period of exposure (10 min; Pascarel et al., 1999), and taxol produces similar effects after a longer period of exposure (2 h; Howarth et al., 1999). No effect of taxol on the amplitude of ICaL has been reported (Pascarel et al., 1999; Howarth et al., 1999; Gomez et al., 2000; Malan et al., 2003). Long (1–4 h) exposure to colchicine has been reported to increase ICaL in ruptured patch-clamped cells (Gomez et al., 2000; Calaghan et al., 2001a), but not in perforated patch-clamped cells (Calaghan et al., 2001a, see Section 3.5). Likewise, Malan et al. (2003) have reported an increase in ICaL with colchicine in ruptured patch clamped adult guinea-pig ventricular myocytes (although the concentration and exposure time for colchicine are not given). Long exposure to colchicine had no effect on ICaL in ruptured patch-clamped neonatal rat cardiac myocytes (Motlagh et al., 2002). In embryonic chick heart cells, taxol increased mean single Ca2þ channel open time and probability of opening, and decreased channel inactivation whereas colchicine tended to increase closed probability and speed channel inactivation (Galli and DeFelice, 1994). Regulation of L-type Ca2þ channel activity by CaMK II- and prepulse-dependent facilitation of ICaL are prevented by microtubule disruption with nocodazole (Dzhura et al., 2002). The swelling induced activation of ICaT (Pascarel et al., 2001) seen in adult guinea pig ventricular myocytes was prevented by a short exposure to colchicine, but not affected by taxol. 3.5. Modulation of cardiac b-adrenergic signalling by microtubules Microtubules have been linked to the b-adrenergic pathway in cardiac muscle (e.g. Palmer et al., 1998). Down-regulation of b-adrenergic receptors following exposure of cardiac cells to b-agonists, is prevented by colchicine and vinblastine, but not lumicolchicine (Limas and Limas,

ARTICLE IN PRESS 46

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

1983; Marsh et al., 1985). Electrically stimulated run-down of b-adrenergic receptor density has also been linked to the microtubule cytoskeleton and to mechanical stress (Yonemochi et al., 2000). Microtubules (or rather tubulin heterodimers) have also been implicated in post-receptor b-adrenergic signalling. Rasenick’s group have shown that in neuronal tissue, (which possesses 1000 times more tubulin than cardiac tissue, Rappaport and Samuel, 1988), free tubulin can transfer GTP to the a sub-unit of G proteins (e.g. Rasenick et al., 1981; Rasenick and Wang, 1988; Wang et al., 1990; Hatta et al., 1995; Yan et al., 2001). Transfer is initially to Gai and then to Gas ; thus, depending on experimental conditions either inhibiting or activating adenyl cyclase. It is the Gai 1 form of Gi that interacts with tubulin (Wang et al., 1990), a form that exists in the adult cardiac myocyte (Kumar et al., 1994). Malan et al. (2003) have also recently proposed an effect of proliferated microtubules on Gi signalling through the muscarinic receptor, possibly due to an effect on the interaction of the receptor and G protein. The role of microtubules in the regulation of the b-adrenergic signalling pathway in adult cardiac muscle is a subject of debate. At the level of the ventricular myocyte, microtubule disruption by colchicine has been reported to enhance basal ICaL measured using ruptured patchclamp, and to attenuate the response to b-adrenergic stimulation (Gomez et al., 2000; Calaghan et al., 2001a; Malan et al., 2003). However, as colchicine has no effect on basal or isoprenalinestimulated cell shortening or ½Ca2þ i transient amplitude in intact cells, or on basal or isoprenaline-stimulated ICaL in perforated-patch-clamped cells (Calaghan et al., 2001a), the reported modulation of ICaL could be ascribed to the use of the ruptured patch-clamp technique. It has been suggested that the combination of the ruptured patch configuration and microtubule disruption interact to activate adenyl cyclase and attenuate the response to b-adrenergic stimulation (Calaghan et al., 2001a). This might occur via increased mechanosensitivity of adenyl cyclase, or loss of tubulin/tubulin-associated components during cell dialysis in ruptured patchclamp. This would suggest that activation of adenyl cyclase would not be seen in cells with intact membranes. By contrast, Gomez et al. (2000) suggest that free tubulin activates Gs (and thereby adenyl cyclase), even in intact cells, and in support of this, Kerfant et al. (2001) have seen an increase in ½Ca2þ i transient with colchicine in intact rat myocytes, and Malan et al. (2003) have seen a 200% increase in cyclic AMP with colchicine in intact guinea-pig myocytes. However, these observations do not explain the lack of effect of microtubule disruption on the amplitude of contraction in a plethora of studies in cardiac muscle and myocytes (Tsutsui et al., 1993, 1994; Collins et al., 1996; Ishibashi et al., 1996; Bailey et al., 1997; Tagawa et al., 1998; Takahashi et al., 1998b; Yamamoto et al., 1998; Zile et al., 1999; Hongo et al., 2000; Calaghan et al., 2001a) or the lack of effect of colchicine on the amplitude of the ½Ca2þ i transient (Palmer et al., 1998; Zile et al., 1999; Calaghan et al., 2001a). Evidence to support the free tubulin-stimulation of adenyl cyclase is also lacking in studies performed with neonatal cardiac myocytes. In these cells, 15 mM colchicine had no effect on the amplitude of the ½Ca2þ i transient (Motlagh et al., 2002), and 1 mM colchicine had no effect upon adenyl cyclase activity (Klein, 1983). In addition, the hypertrophy model of Cooper shows elevated levels of free tubulin, but cyclic AMP, the product of adenyl cyclase stimulation does not increase in response to colchicine in either normal or hypertrophied muscle (Zile et al., 1999). Taxol decreases free tubulin heterodimers but has actions on contraction, ½Ca2þ i and ICaL inconsistent with a depression of adenyl cyclase (Tsutsui et al., 1994; Howarth et al., 1999; Gomez et al., 2000).

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

47

3.6. Microtubules and volume regulation Larsen et al. (2000) showed that in contrast to the effects on the actin cytoskeleton (see Section 2.8), osmotic swelling disrupts the microtubule cytoskeleton of cultured chick myocytes. Similar findings have been reported by Hall et al. (2001). Whether the swelling-induced disruption of the microtubular cytoskeleton in neonatal cardiac myocytes modifies the cell volume response to hypo-osmotic challenge is unclear. Hall et al. (2001) failed to observe a regulatory volume decrease following swelling in chick myocytes when microtubules were disrupted with colchicine or stabilised with taxol. By contrast, Larsen et al. (2000) observed no effect of treatment with either colchicine or taxol on the initial swelling response or subsequent regulatory volume decrease in these cells. In adult rat myocytes microtubule disruption with colchicine does not modify the contractile or swelling response to hypo-osmotic exposure (Bird et al., 2002, Fig. 4B). 3.7. Other channels and exchangers In adult cardiac myocytes, Maltsev and Undrovinas (1997) reported no effect of colchicine upon INa ; whilst taxol caused a significant acceleration of the time-dependent shift in voltagedependent activation, an effect that was blocked by the actin stabiliser phalloidin. In neonatal rat myocytes colchicine, but not vinblastine, has been shown to increase the amplitude of INa and it was suggested this was via the action of Gas following transfer of GTP to Gs by released tubulin dimers (Motlagh et al., 2002). The microtubule cytoskeleton has been shown to be important in the insulin-dependent synthesis of Kþ channels carrying It and ISS (Shimoni et al., 1999; Shimoni and Rattner, 2001; see Section 2.6). Regulation of Kv1.5 expressed in Xenopus oocytes was not sensitive to colchicine (Mason et al., 2002, see Section 2.6). In contrast to its sensitivity to Cyto-D, the Na:Ca exchanger expressed in CHO cells was not sensitive to either colchicine or nocodazole (10 mM; Condrescu et al., 1997; see Section 2.9). In terms of ionic currents which do not normally flow during the action potential, Isenberg et al. (2003) have reported that 5 mM colchicine applied intracellularly reduces the amplitude of SACs. 3.8. Action potential duration and membrane potential There is limited data on the effect of the microtubuIe cytoskeleton on action potential. In neonatal rat cardiac myocytes, colchicine has been shown to reduce the APD (Motlagh et al., 2002). Parker et al. (2001) have reported that treatment of whole rabbit hearts with taxol leads to an increase in stretch-induced arrhythmias, whereas colchicine has no effect (as reported previously by Dick and Lab, 1998). Given an absence of consistent evidence for an effect of microtubule disruption on ionic currents, we might predict that colchicine would have little effect on APD. 3.9. Summary The microtubule cytoskeleton is easily visualised in the adult cardiac myocyte, and the pharmacological tools used to manipulate it are well-characterised. Both polymerised and free

ARTICLE IN PRESS 48

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

Fluoresence intensity (arbitary units)

(A)

30

20

** 10

0 (B)

1.4 1.3 1.2

Relative cell volume

1.1 1.0 0.9 0

5

10

15

20

1.5 1.4 1.3 1.2 1.1 1.0 0.9

0

5

10

15

Time (min) Fig. 4. Microtubule disruption with colchicine and actin distruption with Cyto-D do not modify the volume change in response to hypo-osmotic challenge in the adult rat ventricular myocyte. Exposure of rat ventricular myocytes to 10 mM colchicine disrupts the microtubule cytoskeleton (A), but has no impact on the cell volume change seen during swelling (B). (A) b-tubulin was immunofluorescently labelled, and mean fluorescence intensity was measured in confocal sections of E2 mm thickness taken through myocytes at the level of the nucleus. Values are mean + S.E.M of 31 cells exposed to either vehicle (0.1% v/v methanol; open bar) or colchicine (filled bar). Po0.01 compared with vehicle-treated cells (Student’s t-test). Adapted from Calaghan et al. (2001a). (B) (Upper panel) Myocytes maintained in iso-osmotic solution (300 mosM) were exposed to an hypo-osmotic solution (165 mosM) at time zero and changes in relative volume measured over 18 min. Some cells were exposed to colchicine (10 mM; ) for 2 h previously and throughout the hypoosmotic challenge (n ¼ 15); others were exposed to vehicle (J) for 2 h previously and throughout the hypo-osmotic challenge (n ¼ 13). Adapted from Bird et al. (2002). (lower panel) Similar experiments performed in cells incubated (for at least 1 h) and swollen (for 10 min) in the presence of 10 mM cyto-D () or vehicle (J), (n ¼ 12 in each case). Adapted from Lovett et al. (2003). The amplitude and timecourse of the swelling response was not affected by either colchicine or cyto-D.

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

49

tubulin have been assigned roles in modulating intracellular signalling. No comprehensive role for the modulation of ion channels or exchangers by the microtubules and/or tubulin has emerged, but it is clear from measurements of contraction that microtubules are functionally relevant in the heart and that under certain circumstances, tubulin may modify the response to b-adrenergic stimulation. Changes in the density or characteristics of the microtubular cytoskeleton have been implicated in various pathological conditions.

4. Desmin Intermediate filaments are formed of a dimer composed of two a-helical chains orientated in parallel and intertwined in a coiled-coil rod, around 10 nm in diameter (Fuchs and Cleveland, 1998). Desmin forms the major component of cardiac intermediate filaments which form a physical link between the nucleus, contractile proteins (by surrounding the Z-discs), sarcolemma and extracellular matrix via costameres. They also have the potential to associate with other organelles including mitochondria and the sarcoplasmic reticulum (Capetanaki, 2002). Their cellular distribution makes them an ideal candidate for mechanical signalling into and out of the cell. They form links through the myocyte in both the longitudinal and transverse plane (Price and Sanger, 1983). Their presence at the Z-lines leads to a characteristic striated labelling pattern (Fig. 5). Certain human skeletal and cardiac myopathies are associated with changes in the intermediate filament network (Capetanaki, 2000). Pharmacological agents to selectively target desmin are lacking, however work with desmin null mice has yielded interesting information. Milner et al. (1999) showed that these mice had hypertrophied myocytes, the hypertrophy being concentric in nature, arising from an increase solely in transverse sectional area. The thickness of the free wall of the ventricles was increased at 6 months but there was a thinning of the wall, ventricular dilatation and reduced cardiac function at 12 months. Balogh et al. (2002) also reported a thicker ventricular wall at 5–6 months together with lower developed pressure in intact hearts. They also showed a reduced increase in force for a given level of filling (by balloon inflation) possibly indicating a depressed Frank-Starling mechanism. These authors further showed that skinned trabeculae from desmin null mice demonstrated reduced maximal force, but no change in myofilament Ca2þ sensitivity. At an ultra-structural level, cardiac muscle from desmin null mice shows sub-sarcolemmal clumping and proliferation of mitochondria (Milner et al., 2000), which is associated with a depression of mitochondrial respiration. The intermediate filaments have been implicated in the positioning of mitochondria, and desmin may allow anchorage of these organelles to areas of high energy demand, effectively regulating energy production (see Capetanaki, 2002). With respect to electrical activity Anumonwo et al. (1992) showed co-localisation of connexin 43, a major component of gap junctions, and desmin in rabbit sino-atrial node but a functional link is not supported by the lack of change in heart rate in desmin null mice compared to wild type (Milner et al., 1999; Balogh et al., 2002).

ARTICLE IN PRESS 50

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

Fig. 5. Distribution of the intermediate filament desmin in the adult rat cardiac myocyte. Labelling of myocytes with an antibody to desmin reveals a striated pattern consistent with concentration of desmin at the Z-line. There is also intense labelling at the intercalated discs. The image represents a shadow projection taken from Kostin et al. (1998).

5. General summary In this review, we have reported evidence for the modulation of both electrical and mechanical activity by the cytoskeleton in the adult cardiac cell. It is interesting to note that the flow of information also works in the opposite direction. Mechanical activity in turn has the ability to modify the cardiac cell cytoskeleton, for example by changing intermediate filament orientation (Bloom et al., 1996), or increasing the amount of cellular tubulin (Watson et al., 1996). There is limited evidence for a sparse actin cytoskeleton in the adult cardiac myocyte, although an abundant actin cytoskeleton is seen in cultured neonatal cardiac cells. Furthermore, the selective action of pharmacological tools such as Cyto-D on cytoskeletal actin has not been confirmed in the adult cell, although functional antagonism of Cyto-D by phalloidin and colocalisation of ion channels with actin binding proteins provides some evidence to support a role for cytoskeletal actin in these cells. Actin is only biologically active in a state of polymerisation, although the length of filaments has a marked impact on their function. The actin disruptor CytoD reduces contraction and increases the ½Ca2þ i transient, however we consider that this is due, in part at least, to an interaction with sarcomeric actin of the contractile machinery. There is convincing evidence for modulation of several ionic currents including ICaL (if Ca2þ is not heavily buffered), INa ; IKATP ; ISAC by cytoskeletal actin in the adult myocyte, although the nature of the interaction between actin and channels has yet to be described and no consistent effect of actin manipulation upon the action potential is reported. The actin cytoskeleton may also play a vital role in the mechanotransductive signalling pathway from the extracellular matrix of the cell exterior to the interior.

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

51

By contrast to the actin cytoskeleton, microtubules exist as a dense network throughout the adult cardiac cell. The pharmacological tools used to manipulate microtubules are well characterised and biological activity is not restricted to polymerised microtubules; free tubulin subunits are also active. Furthermore, polymerised microtubules exist in functionally distinct populations and the presence of associated proteins and post-translational modification may have important implications for their cellular role. There is convincing evidence in the adult cardiac cell for modulation of both force and rate of contraction by microtubules/tubulin, and under certain circumstances free tubulin has been shown to modify the response of the cardiac cell to b-adrenergic stimulation. There is little evidence to suggest that the impact of the microtubular cytoskeleton on cardiac contractility is mediated through an association with ionic channels, as is the case for the actin microfilaments, although a recent association between proliferated microtubules and muscarinic receptor-G1 signalling has been made. To date, the body of evidence suggests that in the adult cell, proliferation of microtubules interferes with sarcomeric motion and that this effect is important in the contractile dysfunction of some types of hypertrophy, also, it seems microtubule/tubulin can modulate G-protein dependent intracellular signalling pathways under ruptured patch-clamp conditions. The desmin intermediate filaments cannot be selectively manipulated by pharmacological tools, yet their importance in the cardiac cell is suggested by their wide cellular distribution and confirmed using desmin null mice which show cardiomyopathy. The way in which desmin controls cardiac activity has yet to be determined. Taken together, the 3 components of the cytoskeleton, actin microfilaments, microtubules and intermediate filaments, are clearly important for the normal electrical and mechanical activity of the heart and have been implicated in a wide range of cellular activities. Their importance is further suggested by the findings that many cardiac diseases are associated with alteration of cytoskeletal structure or function, although the relationship between pathological alterations in the cytoskeleton and changes in electrical activity are unknown. It may be in the pathological modulation of the cytoskeleton that effects upon ion channels are critical, for example altered sensitivity of SACs to mechanical stimuli might pre-dispose to cardiac arrhythmia. Certainly the contractile dysfunction of hypertrophied myocytes with a proliferated microtubule cytoskeleton or in myocytes lacking desmin has been clearly demonstrated.

Acknowledgements This work was partially funded by the British Heart Foundation and a Wellcome Trust Biomedical Collaborative Grant.

References Allen, D.G., Blinks, J.R., Godt, R.E., 1984. Influence of deuterium oxide on calcium transients and myofibrillar responses of frog skeletal muscle. J. Physiol. 354, 225–251. Anumonwo, J.M., Wang, H.Z., Trabka-Janik, E., Dunham, B., Veenstra, R.D., Delmar, M., Jalife, J., 1992. Gap junctional channels in adult mammalian sinus nodal cells. Immunolocalization and electrophysiology. Circ. Res. 71, 229–239.

ARTICLE IN PRESS 52

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

Aplin, A.E., Juliano, R.L., 1999. Integrin and cytoskeletal regulation of growth factor signaling to the MAP kinase pathway. J. Cell. Sci. 112, 695–706. Arnal, I., Wade, R.H., 1995. How does taxol stabilize microtubules? Curr. Biol. 5, 900–908. Bailey, B.A., Dipla, K., Li, S., Houser, S.R., 1997. Cellular basis of contractile derangements of hypertrophied feline ventricular myocytes. J. Mol. Cell. Cardiol. 29, 1823–1835. Baines, C.P., Liu, G.S., Birincioglu, M., Critz, S.D., Cohen, M.V., Downey, J.M., 1999. Ischemic preconditioning depends on interaction between mitochondrial KATP channels and actin cytoskeleton. Am. J. Physiol. 276, H1361–H1368. Balogh, J., Merisckay, M., Li, Z., Paulin, D., Arner, A., 2002. Hearts from mice lacking desmin have a myopathy with impaired active force generation and unaltered wall compliance. Cardiovasc. Res. 53, 439–450. Banville, I., Gray, R.A., 2002. Effect of action potential duration and conduction velocity restitution and their spatial dispersion on alternans and the stability of arrhythmias. J. Cardiovasc. Electrophysiol. 13, 1141–1149. Belmadani, S., Pous, . C., Ventura-Clapier, R., Fischmeister, R., Mery, P.F., 2002. Post-translational modifications of cardiac tubulin during chronic heart failure in the rat. Mol. Cell. Biochem. 237, 39–46. Berdiev, B.K., Prat, A.G., Cantiello, H.F., Ausiello, D.A., Fuller, C.M., Jovov, B., Benos, D.J., Ismailov, II., 1996. Regulation of epithelial sodium channels by short actin filaments. J. Biol. Chem. 271, 17704–17710. Bers, D.M., 2002. Cardiac excitation-contraction coupling. Nature 415, 198–205. Biermann, M., Rubart, M., Moreno, A., Wu, J., Josiah-Durant, A., Zipes, D., 1998. Differential effects of cytochalasin D and 2,3 butanedione monoxime on isometric twitch force and transmembrane action potential in isolated ventricular muscle: implications for optical measurements of cardiac repolarization. J. Cardiovasc. Electrophysiol. 9, 348–357. Bird, S.D., Calaghan, S.C., White, E., 2002. Microtubule disruption with colchicine does not modify the cell volume or contractile response to hyposmotic challenge in the adult rat ventricular myocyte. J. Physiol. 544p, 50p. Bloch, W., Fan, Y., Han, J., Xue, S., Schoneberg, T., Ji, G., Lu, Z.J., Walther, M., Fassler, R., Hescheler, J., Addicks, K., Fleischmann, B.K., 2001. Disruption of cytoskeletal integrity impairs Gi-mediated signaling due to displacement of Gi proteins. J. Cell. Biol. 154, 753–761. Bloom, S., Lockard, V.G., Bloom, M., 1996. Intermediate filament-mediated stretch-induced changes in chromatin: a hypothesis for growth initiation in cardiac myocytes. J. Mol. Cell. Cardiol. 28, 2123–2127. Borg, T.K., Goldsmith, E.C., Price, R., Carver, W., Terracio, L., Samarel, A.M., 2000. Specialization at the Z line of cardiac myocytes. Cardiovasc. Res. 46, 277–285. Brady, P.A., Alekseev, A.E., Aleksandrova, L.A., Gomez, L.A., Terzic, A., 1996. A disrupter of actin microfilaments impairs sulfonylurea-inhibitory gating of cardiac KATP channels. Am. J. Physiol. 271, H2710–H2716. Brenner, S.L., Korn, E.D., 1979. Substoichiometric concentrations of cytochalasin D inhibit actin polymerisation. J. Biol. Chem. 254, 9982–9985. Calaghan, S.C., White, E., Bedut, S., Le Guennec, J.Y., 2000. Cytochalasin D reduces Ca2+ sensitivity and maximum tension via interactions with myofilaments in rat skinned cardiac myocytes. J. Physiol. 529, 405–411. Calaghan, S.C., LeGuennec, J.Y., White, E., 2001a. Modulation of Ca2+ signalling by microtubules disruption in rat ventricular myocytes and its dependence on the ruptured patch-clamp configuration. Circ. Res. 88, e32–e37. Calaghan, S.C., White, E., LeGuennec, J.Y., 2001b. A unifying mechanism for the role of microtubules in the regulation of [Ca2+]i and contraction in cardiac myocytes. Circ. Res. 89, e31. Cantiello, H.F., 1996. Role of the actin cytoskeleton in the regulation of the cystic fibrosis transmembrane conductance regulator. Exp. Physiol. 81, 505–514. Cantiello, H.F., 1997. Role of actin filament organization in cell volume and ion channel regulation. J. Exp. Zool. 279, 425–435. Cantiello, H.F., Stow, J.L., Prat, A.G., Ausiello, D.A., 1991. Actin filaments regulate epithelial Na+ channel activity. Am. J. Physiol. 261, C882–C888. Capetanaki, Y., 2000. Desmin cytoskeleton in healthy and failing heart. Heart Failure Rev. 5, 203–220. Capetanaki, Y., 2002. Desmin cytoskeleton: a potential regulator of muscle mitochondrial behaviour and function. Trends Cardiovasc. Med. 12, 339–348. Cartwright, J., Goldstein, M.A., 1985. Microtubules in the heart muscle of the postnatal and neonatal rat. Mol. Cell. Cardiol. 17, 1–7.

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

53

Cherksey, B.D., Zadunaisky, J.A., Murphy, R.B., 1980. Cytoskeletal constrainst of the b-adrenergic receptor in frog erythrocyte membranes. Proc. Natl. Acad. Sci. USA 77, 6401–6405. Cohen, M.V., Baines, C.P., Downey, J.M., 2000. Ischemic preconditioning: from adenosine receptor of KATP channel. Ann. Rev. Physiol. 62, 79–109. Collins, J.F., Pawloski-Dahm, C., Davis, M.G., Ball, N., Dorn II, G.W., Walsh, R.A., 1996. The role of the cytoskeleton in left ventricular pressure overload hypertrophy and failure. J. Mol. Cell. Cardiol. 28, 1435–1443. Condrescu, M., Gardner, J.P., Chernaya, G., Aceto, J.F., Kroupis, C., Reeves, J.P., 1995. ATP-dependent regulation of sodium-calcium exchange in Chinese hamster ovary cells transfected with the bovine cardiac sodium-calcium exchanger. J. Biol. Chem. 270, 9137–9146. Condrescu, M., Chernaya, G., Kalaria, V., Reeves, J.P., 1997. Barium influx mediated by the cardiac sodium-calcium exchanger in transfected Chinese hamster ovary cells. J. Gen. Physiol. 109, 41–51. Cooper, J.A., 1987. Effects of cytochalasin and phalloidin on actin. J. Cell. Biol. 105, 1473–1478. Cooper, G., 2000. Cardiocyte cytoskeleton in hypertrophied myocardium. Heart Failure Rev. 5, 187–201. Cukovic, D., Lu, G.W., Wible, B., Steele, D.F., Fedida, D., 2001. A discrete amino terminal domain of Kv1.5 and Kv1.4 potassium channels interacts with the spectrin repeats of alpha-actinin-2. FEBS Lett. 498, 87–92. Cunningham, C.C., Leclerc, N., Flanagan, L.A., Lu, M., Janmey, P.A., Kosik, K.S., 1997. Microtubule-associated protein 2c reorganizes both microtubules and microfilaments into distinct cytological structures in an actin-binding protein-280-deficient melanoma cell line. J. Cell. Biol. 136, 845–857. David-Pfeuty, T., Simon, C., Pantaloni, D., 1979. Effect of antimitotic drugs on tubulin GTPase activity and selfassembly. J. Biol. Chem. 254, 11696–11702. Dick, D.J., Lab, M.J., 1998. Mechanical modulation of stretch-induced premature ventricular beats: induction of mechanoelectric adaptation period. Cardiovasc. Res. 38, 181–191. Dzhura, I., Wu, Y., Colbran, R.J., Balser, J.R., Anderson, M.E., 2000. Calmodulin kinase determines calciumdependent facilitation of L-type calcium channels. Nature Cell Biol. 2, 173–177. Dzhura, I., Wu, Y., Colbran, R.J., Corbin, J.D., Balser, J.R., Anderson, M.E., 2002. Cytoskeletal disrupting agents prevent calmodulin kinase, IQ domain and voltage-dependent facilitation of L-type Ca2+ channels. J. Physiol. 545, 399–406. Faivre, J.F., Findlay, I., 1990. Action potential duration and activation of ATP-sensitive potassium current in isolated guinea-pig ventricular myocytes. Biochim. Biophys. Acta. 1029, 167–172. Forgacs, G., 1995. On the possible role of cytoskeletal filamentous networks in intracellular signaling: an approach based on percolation. J. Cell. Sci. 108, 2131–2143. Fuchs, E., Cleveland, D.W., 1998. A structural scaffolding of intermediate filaments in health and disease. Science 279, 514–519. Furukawa, T., Yamane, T., Terai, T., Katayama, Y., Hiraoka, M., 1996. Functional linkage of the cardiac ATPsensitive K+ channel to the actin cytoskeleton. Pflugers Arch. 431, 504–512. Galli, A., DeFelice, L.J., 1994. Inactivation of L-type Ca channels in embryonic chick ventricle cells: dependence on the cytoskeletal agents colchicine and taxol. Biophys. J. 67, 2296–2304. Ganote, C., Armstrong, S., 1993. Ischaemia and the myocyte cytoskeleton: review and speculation. Cardiovasc. Res. 27, 1387–1403. Goedert, M., Jakes, R., Spillantini, M.G., Crowther, R.A., 1994. Tau proteins and Alzheimer’s disease in microtubules. In: Hyams, J.S., Lloyd, C.W. (Eds.), Modern Cell Biology, Vol. 13. Wiley-Liss, pp. 183–200. Goldstein, M.A., Entman, M.L., 1979. Microtubules in mammalian heart muscle. J. Cell. Biol. 80, 183–195. Gomez, A.M., Kerfant, B.G., Vassort, G., 2000. Microtubule disruption modulates Ca2+ signalling in rat cardiac myocytes. Circ. Res. 86, 30–36. Granzier, H.L., Irving, T.C., 1995. Passive tension in cardiac muscle: contribution of collagen, titin, microtubules and intermediate filaments. Biophys. J. 68, 1027–1044. Grenier, G., Van Sande, J., Willems, C., Neve, P., Dumont, J.E., 1975. Effects of microtubule inhibitors and cytochalasin B on thyroid metabolism in vitro. Biochimie 57, 337–341. Grover, G.J., Garlid, K.D., 2000. ATP-sensitive potassium channels: a review of their cardioprotective pharmacology. J. Mol. Cell. Cardiol. 32, 677–695.

ARTICLE IN PRESS 54

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

Guharay, F., Sachs, F., 1984. Stretch-activated single ion-channel currents in tissue-cultured embryonic chick skeletal muscle. J. Physiol. 352, 685–701. Gurland, G., Gundersen, G.G., 1995. Stable, detyrosinated microtubules function to localize vimentin intermediate filaments in fibroblasts. J. Cell. Biol. 131, 1275–1290. Hall, S.K., Zhang, J., Lieberman, M., 1997. An early transient current is associated with hyposmotic swelling and volume regulation in embryonic chick cardiac myocytes. Exp. Physiol. 82, 43–54. Hall, S.K., Rofe, C.J., Lewis, O., Evans, J.R., 2001. Dynamic rearrangement of microtubules is an important component of the volume regulatory response in embryonic chick cardiac myocytes. J. Physiol. 536p. Hamill, O.P., Martinac, B., 2001. Molecular basis of mechanotransduction in living cells. Physiol. Rev. 81, 685–740. Hatcher, C.J., Godt, R.E., Nosek, T.M., 1999. Excessive microtubules are not responsible for depressed force per crossbridge in cardiac neural-crest-ablated embryonic chick hearts. Pflugers Arch. 438, 307–313. Hatta, S., Ozawa, H., Saito, T., Ohshika, H., 1995. Participation of tubulin in the stimulatory regulation of adenyl cyclase in rat cerebral cortex membranes. J. Neurochem. 64, 1343–1350. Haworth, R.A., Biggs, A.V., 1997. Effect of ATP depletion on kinetics of Na/Ca exchange-mediated Ca influx in Na-loaded heart cells. J. Mol. Cell. Cardiol. 29, 503–514. Heidemann, S.R., Lamoureaux, P., Buxbaum, R.E., 2000. Opposing views on tensegrity as a structural framework for understanding cell mechanics. J. Appl. Physiol. 89, 1663–1678. Hein, S., Scheffold, T., Schaper, J., 1995. Ischaemia induces early changes to cytoskeletal and contractile proteins in diseased human myocardium. J. Thoracic Cardiovasc. Surg. 110, 89–98. Heling, A., Zimmermann, R., Kostin, S., Maeno, Y., Hein, S., Devaux, B., Bauer, E., Klovekorn, W.P., Schlepper, M., Schaper, W., Schaper, J., 2000. Increased expression of cytoskeletal, linkage, and extracellular proteins in failing human myocardium. Circ. Res. 86, 846–853. Hilgemann, D.W., Ball, R., 1996. Regulation of cardiac Na+, Ca2+ exchange and KATP potassium channels by PIP2. Science 273, 956–959. Hill, T.L., Kirschner, M.W., 1982. Bioenergetics and kinetics of microtubule and actin filament assembly-disassembly. Int. Rev. Cytol. 78, 1–125. Hoebecke, J., Van Nijen, G., De Brabander, M., 1976. Interaction of nocodazole (R 17934), a new antitumoral drug, with rat brain tubulin. Biochem. Biophys. Res. Comm. 69, 319–324. Hohaus, A., Person, V., Behlke, J., Schaper, J., Morano, I., Haase, H., 2002. The carboxyl-terminal region of ahnak provides a link between cardiac L-type Ca2+ channels and the actin-based cytoskeleton. FASEB J. 16, 1205–1216. Hongo, K., Brette, F., Haroon, M.M., White, E., 2000. Mechanisms associated with the negative inotropic effect of deuterium oxide in single rat ventricular myocytes. Exp. Physiol. 85, 133–142. Hori, M., Sato, H., Kitakaze, M., Iwai, K., Takeda, H., Inoue, M., Kamada, T., 1994. Beta-adrenergic stimulation disassembles microtubules in neonatal rat cultured cardiomyocytes through intracellular Ca2+ overload. Circ. Res. 75, 324–334. Hosokawa, T., Okada, T., Kobayashi, T., Hashimoto, K., Seguchi, H., 1994. Ultrastructural and immunocytochemical study of leptomeres in the mouse cardiac muscle fibre. Histol. Histopathol. 9, 85–94. Howarth, F.C., Boyett, M.R., White, E., 1998. Rapid effects of cytochalasin-D on contraction and intracellular calcium in single rat ventricular myocytes. Pflugers Arch. 436, 804–806. Howarth, F.C., Calaghan, S.C., Boyett, M.R., White, E., 1999. Effect of the microtubule polymerising agent on contraction, Ca2+ transient and L-type Ca2+ current in rat ventricular myocytes. J. Physiol. 516, 409–419. Howarth, F.C., Quereshi, M.A., White, E., Calaghan, S.C., 2002. Effect of streptozotocin-induced diabetes on the cardiac microtubular cytoskeleton. Pflug. Arch. 444, 432–437. Ingber, D.E., 1997. Tensegrity: the architectural basis of cellular mechanotransduction. Ann. Rev. Physiol. 59, 575–599. Ingber, D.E., 2000. Opposing views on tensegrity as a structural framework for understanding cell mechanics. J. Appl. Physiol. 89, 1663–1678. Ingber, D.M., 2002. Mechanical signalling and cellular response to extracellular matrix in angiogenesis and cardiovascular physiology. Circ. Res. 91, 877–887. Ingber, D.E., 2003a. Tensegrity I. Cell structure and hierarchical systems biology. J. Cell. Sci. 116, 1157–1173. Ingber, D.E., 2003b. Tensegrity II. How structural networks influence cellular information processing networks. J. Cell. Sci. 116, 1397–1408.

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

55

Ives, C.L., Eskin, S.G., McIntire, L.V., 1986. Mechanical effects on endothelial cell morphology: in vitro assessment. In Vitro Cell. Dev. Biol. 22, 500–507. Isenberg, G., Kazanski, V., Kondratev, D., Gallitelli, M.F., Kiseleva, I., Kamkin, A., 2003. Differential effects of stretch and compression on membrane currents and [Na+]c in ventricular myocytes. Prog. Biophys. Mol. Biol. 82, 43–56. Ishibashi, Y., Tsutsui, H., Yamamoto, S., Takahashi, M., Imanakayoshida, K., Yoshida, T., Urabe, Y., Sugimachi, M., Takeshita, A., 1996. Role of microtubules in myocyte contractile dysfunction during cardiac hypertrophy in the rat. Am. J. Physiol. 40, H1978–H1987. Iwai, K., Hori, M., Kitabatake, A., Kurihara, H., Uchida, K., Inoue, M., Kamada, T., 1990. Disruption of microtubules as an early sign of irreversible ischemic injury. Immunohistochemical study of in situ canine hearts. Circ. Res. 67, 694–706. Janmey, P.A., 1998. The cytoskeleton and cell signaling: component localization and mechanical coupling. Physiol. Rev. 78, 763–781. Johnson, B.D., 1999. The company they keep: ion channels and their intracellular regulatory partners. Adv. Second Messenger Phosphoprotein Res. 33, 203–228. Joshi, H.C., Chu, D., Buxbaum, R.E., Heidemann, S.R., 1985. Tension and compression in the cytoskeleton of PC 12 neurites. J. Cell. Biol. 101, 697–705. Jovanovic, S., Jovanovic, A., 2001. Diadenosine tetraphosphate-gating of cardiac K(ATP) channels requires intact actin cytoskeleton. Naunyn Schmiedebergs Arch. Pharmacol. 364, 276–280. Kamkin, A., Kiseleva, I., Wagner, K.D., Scholtz, H., Theres, H., Kazanski, V., Lozinsky, I., Gunther, J., Isenberg, G., 2001. Mechanically induced potentials in rat atrial fibroblasts depend on actin nd tubulin polymerisation. Pflugers. Arch. 442, 487–497. Kamkin, A., Kiseleva, I., Isenberg, G., 2003a. Ion selectivity of stretch-activated cation currents in mouse ventricular myocytes. Pflugers. Arch. 446, 220–231. Kamkin, A., Kiseleva, I., Isenberg, I., Wagner, K.-D., Gunther, . J., Theres, H., Scholz, H., 2003b. Cardiac fibroblasts and the mechano-electric feedback mechanism in healthy and diseased hearts. Prog. Biophys. Mol. Biol. 82, 111–120. Kaprielian, R.R., Severs, N.J., 2000. Dystrophin and the cardiomyocyte membrane cytoskeleton in the healthy and failing heart. Heart Failure Rev. 5, 221–238. Katz, A.M., 2000. Cytoskeletal abnormalities in the failing heart: out on a LIM? Circ. 101, 2672–2673. Kerfant, B.G., Vassort, G., Gomez, A.M., 2001. Microtubule disruption by colchicine reversibly enhances calcium signalling in intact rat cardiac myocytes. Circ. Res. 88, e59–e65. Kettleman, S., Walker, N.L., Burton, F.L., Cobbe, S.M., Smith, G.L., 2002. Mechanical and electrophysiological effects of BDM and cytochalasin-D in the rabbit heart. J. Physiol. 544p, 46p. Kim, D., 1993. Novel cation-selective mechanosensitive ion channel in the atrial cell membrane. Circ. Res. 72, 225–231. Kisselev, L.L., Justesen, J., Wolfson, A.D., Frolova, L.Y., 1998. Diadenosine oligophosphates (Ap(n)A), a novel class of signalling molecules? FEBS Lett. 427, 157–163. Klein, I., 1983. Colchicine stimulates the rate of contraction of heart cells in culture. Cardiovasc. Res. 17, 459–465. Kostin, S., Heling, A., Hein, S., Scholz, D., Klovekorn, W.-P., Schaper, J., 1998. The protein composition of the normal and diseased cardiac myocyte. Heart Failure Rev. 2, 245–260. Kostin, S., Hein, S., Arnon, E., Scholz, D., Schaper, J., 2000. The cytoskeleton and related proteins in the human failing heart. Heart Failure Rev. 5, 271–280. Kumar, N., 1981. Taxol-induced polymerisation of purified tubulin. J. Biol. Chem. 256, 10435–10441. Kumar, R., Joyner, R.W., Hartzell, C., 1994. Postnatal changes in the G-proteins, cyclic nucleotides and adenylyl cyclase activity in rabbit heart cells. J. Mol. Cell. Cardiol. 26, 1537–1550. Kwiatkowski, D.J., Janmey, P.A., Yin, H.L., 1989. Identification of critical functional and regulatory domains in gelsolin. J. Cell. Biol. 108, 717–1726. Lader, A.S., Kwiatkowski, D.J., Cantiello, H.F., 1999. Role of gelsolin in the actin filament regulation of cardiac L-type calcium channels. Am. J. Physiol. 277, C1277–C1283. Lampidis, T.J., Kolonias, D., Savaraj, N., Rubin, R.W., 1992. Cardiostimulatory and antiarrythmic activity of tubulinbinding agents. Proc. Natl. Acad. Sci. USA 89, 1256–1260.

ARTICLE IN PRESS 56

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

Lange, K., Brandt, U., 1996. Calcium storage and release properties of F-actin; evidence for involvement of F-actin in cellular calcium signalling. FEBS lett. 395, 137–142. Larsen, T.H., Dalen, H., Sommer, J.R., Boyle, R., Lieberman, M., 1999. Membrane skeleton in cultured chick cardiac myocytes revealed by high resolution immunocytochemistry. Histochem. Cell. Biol. 112, 307–316. Larsen, T.H., Dalen, H., Boyle, R., Souza, M.M., Lieberman, M., 2000. Cytoskeletal involvement during hypo-osmotic swelling and volume regulation in cultured chick cardiac myocytes. Histochem. Cell. Biol. 113, 479–488. Lee, K.S., Marban, E., Tsien, R.W., 1985. Inactivation of calcium channels in mammalian heart cells: joint dependence on membrane potential and intracellular calcium. J. Physiol. 364, 395–411. Lee, M.H., Lin, S.F., Ohara, T., Omichi, C., Okuyama, Y., Chudin, E., Garfinkel, A., Weiss, J.N., Karagueuzian, H.S., Chen, P.S., 2001. Effects of diacetyl monoxime and cytochalasin D on ventricular fibrillation in swine right ventricles. Am. J. Physiol. 280, H2689–H2696. Li, Z.P., Burke, E.P., Frank, J.S., Bennett, V., Philipson, K.D., 1993. The cardiac Na+Ca2+ exchanger binds to the cytoskeletal protein ankyrin. J. Biol. Chem. 268, 11489–11491. Liao, G., Gundersen, G.G., 1998. Kinesin is a candidate for cross-bridging microtubules and intermediate filaments. Selective binding of detyrosinated tubulin and vimentin. J. Biol. Chem. 273, 9797–9803. Limas, C.J., Limas, C., 1983. Involvement of microtubules in the isoproterenol-induced ‘down’-regulation of myocardial beta-adrenergic receptors. Biochim. Biophys. Acta. 735, 181–184. Lodish, H., 2000. Molecular Cell Biology, 4th Edition. W.H. Freeman, New York. Lovett, J., Calaghan, S.C., White, E., 2003. Effect of actin disruption and de-t-tubulation on the volume response of adult rat ventricular myocytes to hypo-osmotic challenge. J. Physiol., 551.P, PCI. Malan, D., PIa Gallo, M., Bedendi, I., Biasin, C., Levi, R.C., Alloatti, G., 2003. Microtubules mobility affects the modulation of L-type ICa by muscarinic and b-adrenergic agonists in guinea-pig cardiac myocytes. J. Mol. Cell. Cardiol. 35, 195–206. Maltsev, V.A., Undrovinas, A.I., 1997. Cytoskeleton modulates coupling between availability and activation of cardiac sodium channel. Am. J. Physiol. 273, H1832–H1840. Maniotis, A.J., Chen, C.S., Ingber, D.E., 1997. Demonstration of mechanical connections between integrins, cytoskeletal filaments, and nucleoplasm that stabilize nucleur structure. Proc. Natl. Acad. Sci. USA 94, 849–854. Marsh, J.D., Lachance, D., Kim, D., 1985. Mechanisms of beta-adrenergic receptor regulation in cultured chick heart cells. Role of cytoskeleton function and protein synthesis. Circ. Res. 57, 171–181. Maruoka, N.D., Steele, D.F., Au, B.P., Dan, P., Zhang, X., Moore, E.D., Fedida, D., 2000. Alpha-actinin-2 couples to cardiac Kv1.5 channels regulating current density and channel localization in HEK cells. FEBS Lett. 473, 188–194. Mason, H.S., Latten, M.J., Godoy, L.D., Horowitz, B., Kenyon, J.L., 2002. Modulation of Kv1.5 currents by protein kinase A, tyrosine kinase, and protein tyrosine phosphatase requires an intact cytoskeleton. Mol. Pharmacol. 61, 285–293. Mazzanti, M., Assandri, R., Ferroni, A., DiFrancesco, D., 1996. Cytoskeletal control of rectification and expression of four substates in cardiac inward rectifier K+ channels. FASEB J. 10, 357–361. Messerli, J.M., Perriard, J.C., 1995. Three-dimensional analysis and visualization of myofibrillogenesis in adult cardiomyocytes by confocal microscopy. Microsc. Res. Tech. 30, 521–530. Milner, D.J., Taffet, G.E., Wang, X., Pham, T., Tamura, T., Hartley, C., Gerdes, A.M., Capetanaki, Y., 1999. The absence of desmin leads to cardiomyocyte hypertrophy and cardiac dilation with compromised systolic function. J. Mol. Cell. Cardiol. 31, 2063–2076. Milner, D.J., Mavroidis, M., Weisleder, N., Capetanaki, Y., 2000. Desmin cytoskeleton linked to muscle mitochondrial distribution and respiratory function. J. Cell. Biol. 150, 1283–1298. Moon, A., Drubin, D.G., 1995. The ADF/cofilin proteins: stimulus-responsive modulators of actin dynamics. Mol. Biol. Cell. 6, 1423–1431. Motlagh, D., Alden, K.J., Russell, B., Garcia, J., 2002. Sodium current modulation by a tubulin/GTP coupled process in rat neonatal cardiac myocytes. J. Physiol. 540, 93–103. Musch, M.W., Davis-Amaral, E.M., Vandenburgh, H.H., Goldstein, L., 1998. Hypotonicity stimulates translocation of ICln in neonatal rat cardiac myocytes. Pflugers Arch. 436, 415–422. Nath, K., Shay, J.W., Bollon, A.P., 1978. Relationship between dibutyryl cyclic AMP and microtubule organization in contracting heart muscle cells. Proc. Natl. Acad. Sci. USA 75, 319–323.

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

57

Nilius, B., Hess, P., Lansman, J.B., Tsien, R.W., 1985. A novel type of cardiac calcium channel in ventricular cells. Nature 316, 443–446. Noma, A., 1983. ATP-regulated K+ channels in cardiac muscle. Nature 305, 147–148. Olmsted, J.B., 1986. Microtubule-associated proteins. Ann. Rev. Cell. Biol. 2, 421–457. Otey, C.A., Kalnoski, M.H., Bulinski, J.C., 1988. Immunolocalization of muscle and nonmuscle isoforms of actin in myogenic cells and adult skeletal muscle. Cell. Motil. Cytoskeleton 9, 337–348. Palmer, B.M., Valent, S., Holder, E.L., Weinberger, H.D., Bies, R.D., 1998. Microtubules modulate cardiomyocyte b-adrenergic response in cardiac hypertrophy. Am. J. Physiol. 275, H1707–H1716. Parker, K.K., Taylor, L.K., Atkinson, B., Hansen, D.E., Wikswo, J.P., 2001. The effects of tubulin-binding agents on stretch-induced ventricular arrhythmias. Eur. J. Pharmacol. 417, 131–140. Pascarel, C., Brette, F., Cazorla, O., Le Guennec, J.-Y., 1999. Effects on L-type calcium current of agents interfering with the cytoskeleton of isolated guinea-pig ventricular myocytes. Exp. Physiol. 84, 1043–1050. Pascarel, C., Brette, F., Le Guennec, J.-Y., 2001. Enhancement of the T-type calcium current by hyposmotic shock in isolated guinea-pig ventricular myocytes. J. Mol. Cell. Cardiol. 33, 1363–1369. Perozo, E., Cortes, D.M., Sompornpisut, P., Kloda, A., Martinac, B., 2002. Open channel structure of MscL and the gating mechanism of mechanosensitive channels. Nature 29;418(6901), 942–948. Pitcher, J.A., Hall, R.A., Daaka, Y., Zhang, J., Ferguson, S.S., Hester, S., Miller, S., Caron, M.G., Lefkowitz, R.J., Barak, L.S., 1998. The G protein-coupled receptor kinase 2 is a microtubule-associated protein kinase that phosphorylates tubulin. J. Biol. Chem. 273, 12316–12324. Prat, A.G., Bertorello, A.M., Ausiello, D.A., Cantiello, H.F., 1993. Activation of epithelial Na+ channels by protein kinase A requires actin filaments. Am. J. Physiol. 265, C224–C233. Prat, A.G., Xiao, Y.F., Ausiello, D.A., Cantiello, H.F., 1995. cAMP-independent regulation of CFTR by the actin cytoskeleton. Am. J. Physiol. 268, C1552–C1561. Price, M.G., Sanger, J.W., 1983. Intermediate filaments in striated muscle. A review of structural studies in embryonic and adult skeletal and cardiac muscle. Cell Muscle Motil. 3, 1–40. Raman, N., Atkinson, S.J., 1999. Rho controls actin cytoskeletal assembly in renal epithelial cells during ATP depletion and recovery. Am. J. Physiol. 276, C1312–C1324. Rappaport, L., Samuel, J.L., 1988. Microtubules in cardiac myocytes. Int. Rev. Cyto. 113, 101–143. Rasenick, M.M., Wang, N., 1988. Exchange of guanine nucleotides between tubulin and GTP-binding proteins that regulate adenylate cyclase: cytoskeletal modification of neuronal signal transduction. J. Neurochem. 51, 300–311. Rasenick, M.M., Stein, P.J., Bitensky, M.W., 1981. The regulatory subunit of adenylate cyclase interacts with cytoskeletal components. Nature 294, 560–562. Rasenick, M.M., Wang, N., Yan, K., 1990. Specific associations between tubulin and G proteins: participation of cytoskeletal elements in cellular signal transduction. Adv. Second Messenger Phosphoprotein Res. 24, 381–386. Reimer, K.A., Murry, C.E., Jennings, R.B., 1990. Cardiac adaptation to ischemia. Ischemic preconditioning increases myocardial tolerance to subsequent ischemic episodes. Circulation 82, 2266–2268. Rogers, S.L., Gelfand, V.I., 2000. Membrane trafficking, organelle transport, and the cytoskeleton. Curr Opin. Cell. Biol. 12, 57–62. Rothen-Rutishauser, B.M., Ehler, E., Perriard, E., Messerli, J.M., Perriard, J.-C., 1998. Different behaviour of the nonsarcomeric cytoskeleton in neonatal and adult rat cardiomyocytes. J. Mol. Cell. Cardiol. 30, 19–31. Rueckschloss, U., Isenberg, G., 2001. Cytochalasin D reduces Ca2+ currents via cofilin-activated depolymerization of F-actin in guinea-pig cardiomyocytes. J. Physiol. 537, 363–370. Rybakova, I.N., Patel, J.R., Ervasti, J.M., 2000. The dystrophin complex forms a mechanically strong link between the sarcolemma and costameric actin. J. Cell. Biol. 150, 1209–1214. Sadeghi, A., Doyle, A.D., Johnson, B.D., 2002. Regulation of the cardiac L-type Ca2+ channel by the actin-binding proteins alpha-actinin and dystrophin. Am. J. Physiol. 282, C1502–C1511. Sadoshima, J., Takahashi, T., Jahn, L., Izumo, S., 1992. Roles of mechano-sensitive ion channels, cytoskeleton, and contractile activity in stretch-induced immediate-early gene expression and hypertrophy of cardiac myocytes. Proc. Natl. Acad. Sci. USA 89, 9905–9909. Samuel, J.-L., Corda, S., Chassagne, C., Rappaport, L., 2000. The extracellular matrix and the cytoskeleton in heart hypertrophy and failure. Heart Failure Rev. 5, 239–250.

ARTICLE IN PRESS 58

S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

Sato, H., Nagai, T., Kuppuswamy, D., et al., 1997. Microtubule stabilization in pressure overload cardiac hypertrophy. J. Cell. Biol. 139, 963–973. Schaper, J., Kostin, S., 2000. Preface: the cytoskeleton and associated proteins in hypertrophy and heart failure. Heart Failure Rev. 5, 185. Schaper, J., Froede, R., Hein, S., Buck, A., Hashizume, H., Speiser, B., Freidl, A., Bleese, N., 1991. Impairment of the myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy. Circulation 83, 504–514. Schiff, P.B., Fant, J., Horwitz, S.B., 1979. Promotion of microtubule assembly in vitro by taxol. Nature 277, 665–667. Schmidt-Schonbein, G.W., Kosawada, T., Skalak, T., Chien, S., 1995. Membrane model of endothelial cell leukocytes. A proposal for the origin or cortical stress. J. Biomech. Eng. 117, 171–178. Schulze, E., Kirschner, M., 1987. Dynamic and stable populations of microtubules in cells. J. Cell. Biol. 104, 277–288. Sharma, A., Singh, M., 2000a. Possible mechanism of cardioprotective effect of angiotensin preconditioning in isolated rat heart. Eur. J. Pharmacol. 406, 85–92. Sharma, A., Singh, M., 2000b. Possible mechanism of cardioprotective effect of ischaemic preconditioning in isolated rat heart. Pharmacol. Res. 41, 635–640. Shimoni, Y., Rattner, J.B., 2001. Type 1 diabetes leads to cytoskeleton changes that are reflected in insulin action on rat cardiac K+ currents. Am. J. Physiol. 281, E575–E585. Shimoni, Y., Ewart, H.S., Severson, D., 1999. Insulin stimulation of rat ventricular K+ currents depends on the integrity of the cytoskeleton. J. Physiol. 514, 735–745. Skobel, E., Kammermeier, H., 1997. Relation between enzyme release and irreversible cell injury of the heart under the influence of the cytoskeletal modulating agents. Biochim. Biophys. Acta. 1362, 128–134. Stromer, M.H., 1998. The cytoskeleton in skeletal, cardiac and smooth muscle. Histol. Histopathol. 13, 283–291. Tagawa, H., Wang, N., Narishige, T., Ingber, D.E., Zile, M.R., Cooper, G., 1997. Cytoskeletal mechanics in pressureoverload cardiac hypertrophy. Circ. Res. 80, 281–289. Tagawa, H., Koide, M., Sato, H., Zile, M.R., Carabello, B.A., Cooper, G., 1998. Cytoskeletal role in the transition from compensated to decompensated hypertrophy during adult canine left ventricular pressure overloading. Circ. Res. 82, 751–761. Takahashi, M., Tsutsui, H., Tagawa, H., Igarahi-Saito, K., Imanaka-Yoshida, K., Takeshita, A., 1998a. Microtubules are involved in early hypotrophic responses of myocardium during pressure overload. Am. J. Physiol. 275, H341–H348. Takahashi, M., Tsutsui, H., Kinugawa, S., Igarashi-Saito, K., Yamamoto, S., Yamamoto, M., Tagawa, H., ImanakaYoshida, K., Egashira, K., Takeshita, A., 1998b. Role of microtubules in the contractile dysfunction of myocytes from tachycardia-induced dilated cardiomyopathy. J. Mol. Cell. Cardiol. 30, 1047–1057. Terzic, A., Kurachi, Y., 1996. Actin microfilament disrupters enhance K(ATP) channel opening in patches from guineapig cardiomyocytes. J. Physiol. 492, 395–404. Tsutsui, H., Ishihara, K., Cooper, G., 1993. Cytoskeletal role in the contractile dysfunction of hypertrophied myocardium. Science 260, 682–687. Tsutsui, H., Tagawa, H., Kent, R.L., McCollam, P.L., Ishihara, K., Nagatsu, M., Cooper, G., 1994. Role of microtubules in contractile dysfunction of hypertrophied cardiocytes. Circulation 90, 533–555. Undrovinas, A.I., Maltsev, V.A., 1998. Cytochalasin D alters kinetics of Ca2+ transient in rat ventricular cardiomyocytes: an effect of altered actin cytoskeleton? J. Mol. Cell. Cardiol. 30, 1665–1670. Undrovinas, A.I., Shander, G.S., Makielski, J.C., 1995. Cytoskeleton modulates gating of voltage-dependent sodium channel in heart. Am. J. Physiol. 269, H203–H214. Viragh, S., Challice, C.E., 1969. Variations in filamentous and fibrillar organization, and associated sarcolemmal structures, in cells of the normal mammalian heart. J. Ultrastruct. Res. 28, 321–334. Wang, N., Yan, K., Rasenick, M.M., 1990. Tubulin binds specifically to the signal-transducing proteins, Gs alpha and Gi alpha 1. J. Biol. Chem. 265, 1239–1242. Wang, N., Butler, J.P., Ingber, D., 1993. Mechanotransduction across the cell surface and through the cytoskeleton. Science 260, 1124–1127. Wang, X., Li, F., Campbell, S.E., Gerdes, A.M., 1999. Chronic pressure overload cardiac hypertrophy and failure in guinea pigs: II cytoskeletal remodelling. J. Mol. Cell. Cardiol. 13, 319–331.

ARTICLE IN PRESS S.C. Calaghan et al. / Progress in Biophysics & Molecular Biology 84 (2004) 29–59

59

Wang, Y.G., Samarel, A.M., Lipsius, S.L., 2000a. Laminin acts via 1 integrin signalling to alter cholinergic regulation of L-type Ca2+ current in cat atrial myocytes. J. Physiol. 526, 57–68. Wang, Y.G., Samarel, A.M., Lipsius, S.L., 2000b. Laminin binding to 1-integrins selectively alters 1 and 2-adrenoceptor signalling in cat atrial myocytes. J. Physiol. 527, 3–9. Watson, P.A., 1991. Function follows form: generation of intracellular signals by cell deformation. FASEB J. 5, 2013–2019. Watson, P.A., Hannan, R., Carl, L.L., Giger, K.E., 1996. Contractile activity and passive stretch regulate tubulin mRNA and protein content in cardiac myocytes. Am. J. Physiol. 271, C684–C689. Webster, D.R., Patrick, D.L., 2000. Beating rate of isolated neonatal cardiomyocytes is regulated by the stable microtubule subset. Am. J. Physiol. 278, H1653–H1661. Wieland, T., Faulstitch, H., 1978. Amatoxins, phallotoxins, phallolysin, and antaminide, the biologically active components of poisonous Amanita mushrooms. CRC Crit. Rev. Biochem. 5, 185–260. Wilson, L., Jordan, M.A., 1994. Pharmacological probes of microtubule function in microtubules. In: Hyams, J.S., Lloyd, C.W. (Eds.), Modern Cell Biology, 13th Edition. Wiley-Liss, pp. 59–83. Wu, J., Biermann, M., Rubart, M., Zipes, D.P., 1998. Cytochalasin D as excitation-contraction uncoupler for optically mapping action potentials in wedges of ventricular myocardium. J. Cardiovasc. Electrophysiol. 9, 1336–1347. Wu, Y., Dzhura, I., Colbran, R.J., Anderson, M.E., 2001. Calmodulin kinase and a calmodulin-binding ‘‘IQ’’ domain facilitate L-type Ca2+ current in rabbit ventricular myocytes by a common mechanism. J. Physiol. 535, 679–687. Yamamoto, S., Tsutsui, H., Takahashi, M., Ishibashi, Y., Tagawa, H., Imanaka-Yoshida, K., Saeki, Y., Takeshita, A., 1998. Role of microtubules in the viscoelastic properties of isolated cardiac muscle. J. Mol. Cell. Cardiol. 30, 1841–1853. Yan, K., Popova, J.S., Moss, A., Shah, B., Rasenick, M.M., 2001. Tubulin stimulates adenylyl cyclase activity in C6 glioma cells by bypassing the b-adrenergic receptor: a potential mechanism of G protein activation. J. Neurochem. 76, 182–190. Yang, X., Salas, P.J., Pham, T.V., Wasserlauf, B.J., Smets, M.J., Myerburg, R.J., Gelband, H., Hoffman, B.F., Bassett, A.L., 2002. Cytoskeletal actin microfilaments and the transient outward potassium current in hypertrophied rat ventriculocytes. J. Physiol. 541, 411–421. Yokoshiki, H., Katsube, Y., Sunugawa, M., Seki, T., Sperelakis, N., 1997. Disruption of actin cytoskeleton attenuates sulfonylurea inhibition of cardiac ATP-sensitive K+ channels. Pflugers Arch. 434, 203–205. Yonemochi, H., Yasunaga, S., Teshima, Y., Takahashi, N., Nakagawa, M., Ito, M., Saikawa, T., 2000. Rapid electrical stimulation of contraction reduces the density of beta-adrenergic receptors and responsiveness of cultured neonatal rat cardiomyocytes. Possible involvement of microtubule disassembly secondary to mechanical stress. Circ. 101, 2625–2630. Zhang, J., Rasmusson, R.L., Hall, S.K., Lieberman, M., 1993. A chloride current associated with swelling of cultured chick heart cells. J. Physiol. 472, 801–820. Zhang, J., Larsen, T.H., Lieberman, M., 1997. F-actin modulates swelling-activated chloride current in cultured chick cardiac myocytes. Am. J. Physiol. 273, C1215–C1224. Zhang, Y.H., Youm, J.B., Sung, H.K., Lee, S.H., Ryu, S.Y., Ho, W.K., Earm, Y.E., 2000. Stretch-activated and background non-selective cation channels in rat atrial myocytes. J. Physiol. 523, 607–619. Zile, M.R., Koide, M., Sato, H., Ishiguro, Y., Conrad, C.H., Buckley, M., Morgan, J.P., Cooper, G., 1999. Role of microtubules in the contractile dysfunction of hypertrophied myocardium. J. Am. Col. Cardiol. 33, 250–260. Zile, M.R., Green, G.R., Schuyler, G.T., Aurigemma, G.P., Miller, D.C., Cooper, G. 4th., 2001. Cardiomyocyte cytoskeleton in patients with left ventricular pressure overload hypertrophy. J. Am. Coll. Cardiol. 37, 1080–1084.