- Email: [email protected]
Remote Control of A-Band Cardiac Thin Filaments by the I-Z-I Protein Network of Cardiac Sarcomeres
Scott RA, Vardulaki KA, Walker NM, et al.: 2001. The long-term benefits of a single scan for abdominal aortic aneurysm (AAA) at age 65. Eur J Vasc Endovasc Surg 21: 535–540. Shapiro SD, Campbell EJ, Kobayashi DK, Welgus HG: 1990. Immune modulation of metalloproteinase production in human macrophages: Selective pretranslational suppression of interstitial collagenase and stromelysin biosynthesis by interferon-gamma. J Clin Invest 86: 1204–1210. Shi GP, Sukhova GK, Grubb A, et al.: 1999. Cystatin C deficiency in human atherosclerosis and aortic aneurysms. J Clin Invest 104:1191–1197. Shimizu K, Shichiri M, Libby P, et al.: 2004. Th2-predominant inflammation and blockade of IFN-gamma signaling induce aneurysms in allografted aortas. J Clin Invest 114:300–308. Shimizu K, Sugiyama S, Aikawa M, et al.: 2001. Host bone-marrow cells are a source of donor intimal smooth-muscle-like cells in murine aortic transplant arteriopathy. Nat Med 7:738–741. Sukhova GK, Zhang Y, Pan JH, et al.: 2003. Deficiency of cathepsin S reduces atherosclerosis in LDL receptor-deficient mice. J Clin Invest 111:897–906. Sukhova GK, Wang B, Libby P, et al.: 2005. Cystatin C deficiency increases elastic lamina degradation and aortic dilatation in apolipoprotein E-null mice. Circ Res 96: 368–375. Tellides G, Tereb DA, Kirkiles-Smith NC, et al.: 2000. Interferon-gamma elicits arteriosclerosis in the absence of leukocytes. Nature 403:207–211. Thompson RW: 1996. Basic science of abdominal aortic aneurysms: Emerging therapeutic strategies for an unresolved clinical problem. Curr Opin Cardiol 11: 504–518. Thompson RW, Parks WC: 1996. Role of matrix metalloproteinases in abdominal aortic aneurysms. Ann NY Acad Sci 800: 157–174. Wang H, Keiser JA: 2000. Hepatocyte growth factor enhances MMP activity in human endothelial cells. Biochem Biophys Res Commun 272:900–905. Watanabe T, Shimokama T, Haraoka S, Kishikawa H: 1995. T lymphocytes in atherosclerotic lesions. Ann NY Acad Sci 748:40 –55. Xie B, Dong Z, Fidler IJ: 1994. Regulatory mechanisms for the expression of type IV collagenases/gelatinases in murine macrophages. J Immunol 152:3637–3644.
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Remote Control of A-Band Cardiac Thin Filaments by the I-Z-I Protein Network of Cardiac Sarcomeres R. John Solaro* A conventional view of the role of sarcomeric thin filaments in cardiac function is that they react with cross-bridges that translate them toward the center of the sarcomere in a reaction triggered by Ca2+ and powered by ATP. However, thin filaments also engage in a complex network of protein–protein interactions in the Z-disc. Thus, in the modern context, understanding of what thin filaments do in the heart must take into account not only A-band regions that react with cross-bridges, but also I-Z-I regions that rarely, if ever, react with cross-bridges and dwell near and within the Z-disc. To highlight these multiplex functions of the thin filament, I discuss the hypothesis that physical and chemical reactions at the interface of the thin filaments with Z-disc proteins control the docking and activity of kinases and phosphatases that control the levels of phosphorylation of thin filament regulatory proteins. Testing this hypothesis has taken on new significance with the identification of multisite phosphorylation of thin filament proteins as a critical element in the control of cardiac contraction and relaxation reserve and in maladaptive mechanisms in heart failure. Moreover, multiple mutations in Z-disc proteins that link to prevalent cardiomyopathies are likely to alter this remote control of A-band thin filament function. (Trends Cardiovasc Med 2005;15:148–152) D 2005, Elsevier Inc.
Cardiac Thin Filaments are Involved in Diverse Functions in Control of Force Generation and Shortening in the A-Band and in Signaling at the I-Z-I Region of Sarcomeres
Until recently, most of the action in cardiac thin filaments was thought to
R. John Solaro is at the Department of Physiology and Biophysics and Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA. * Address correspondence to: R. John Solaro, PhD, Department of Physiology and Biophysics (M/C 901), College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Avenue, Chicago, IL 60612-7342, USA. Tel.: (+1) 312-996-7620; fax: (+1) 312996-1414; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
occur in the A-band region of the sarcomere in which thin filament proteins react with myosin cross-bridges and promote the generation of myocyte force and shortening responsible for the ejection of blood from cardiac chambers (Kobayashi and Solaro 2005). Rather than being actively involved in control mechanisms, the I-Z-I band region of the thin filaments was pictured as providing mechanical stability to the sarcomere as an anchoring site in a scaffold making up the Z-disc protein network. However, recent and expanding evidence (Epstein and Davis 2003, Ervasti 2003, Granzier and Labeit 2004, Pyle and Solaro 2004) now pictures the I-Z-I region as a center for reception, transduction, and transmission of mechanical and biochemical signals. My focus here is on how events involving regions of thin filaments in the I-Z-I region of the sarcomere regions may affect A-band regions of the thin TCM Vol. 15, No. 4, 2005
filament, thereby regulating cardiac function by remote control. An understanding of cross-talk between A-band and I-Z-I band regions of the sarcomere appears especially significant with regard to the maladaptive mechanisms leading to heart failure. Mechanisms of decompensation are poorly understood and may involve altered mechanochemical transduction at the Z-disc with the elevated strain on the sarcomere. To highlight this concept, I first briefly summarize alterations (especially phosphorylation) in the thin filament proteins troponin I (cTnI) and troponin (cTnT) that are important determinants of cardiac function. I then consider emerging evidence that mechanical and biochemical signaling at the level of the Z-disc may modify thin filament function by controlling the level of phosphorylation of cTnI and cTnT.
Phosphorylation of cTnI and cTnT Significantly Affects the Dynamics in Both Short- and Long-Term Regulation of Cardiac Function
Tension generation and shortening of cardiac sarcomeres are determined by the mass of sarcomeres in the cardiac cell, the level of activation of the thin filament, the force generated per crossbridge, and the displacement of the thin filament in each cycle. Regulation of protein synthesis and breakdown ultimately determines the mass of sarcomeres and their protein isoform population. Release and removal of Ca2+ into and from the sarcomeric space and Ca binding to thin filament regulatory proteins are major determinants of the level of activation of the thin filament (Bers 2002). As shown in Figure 1, Ca2+ binding to the thin filament Ca2+ receptor, cardiac troponin C (cTnC), induces a complex series of protein– protein interactions enabling crossbridges to react with actin (Kobayashi and Solaro 2005). However, the idea that before thin filaments go into action in cardiac sarcomeres they wait passively for these changes in intracellular Ca2+ has undergone extensive transformation, with convincing evidence that modifications in the thin filament response to Ca2+ are functionally significant. The level of thin filament activation varies independently of intracellular Ca2+ with isoform switching of sarcomere proteins TCM Vol. 15, No. 4, 2005
Figure 1. Schematic illustration and electron micrograph of a portion of the A-band and Z-disc regions of the sarcomere. The A-band region of the thin filament depicts a functional unit of the sarcomere consisting of a helical array of actin monomers with associated regulatory proteins, a heterotrimeric cardiac troponin complex (Tn complex) and tropomyosin (Tm). The functional unit also shows the major proteins of the thick filament—the myosin heavy chain (MHC) and light chains (MLC1 and MLC2). The head of myosin reacts with the thin filament, splitting ATP and impelling the thin filament toward the center of the sarcomere (away from the Z-disc). Myosin connects through myosin-binding protein C (MyBP-C) to titin, a giant elastic protein responsible for passive tension in the sarcomere. Thin filament-regulatory proteins determine whether cross-bridges are strongly reacting and generating force in a reaction controlled by Ca2+ binding to cTnC (shown in red). The Ca2+–cTnC signal is translated to activation of the thin filament by complex protein–protein interactions among cTnI (orange), an inhibitory unit, cTnT (blue), a tropomyosin binding unit, and Tm. These interactions are modified by multisite phosphorylations of TnI and TnT as described in the text and reviewed in Solaro (2001). The Z-disc region illustrates components discussed in the text. These include a-actinin, which crosslinks adjacent thin filaments, CapZ, which caps the barbed ends of the thin filament, ZASP (also known as cypher; a docking site for protein kinase C), and Pak1, which may move away from the Z-disc upon activation and activate PP2A. The Z-disc network also connects to the rest of the sarcomere through titin, which terminates in the Z-disc at one end and in the M-line at the other. See Pyle and Solaro (2004) for a more complete picture of Z-disc proteins, and see text for discussion of the hypothesis that events at the Z-disc may regulate the actin–myosin interaction by affecting the activity of PKC and Pak1.
as occurs during development and hypertrophy (Parmacek and Solaro 2004). Moreover, length-dependent Ca2+ sensitivity remains widely accepted as a major determinant of the Frank–Starling relation as reflected in the slope of the end systolic pressure–volume relation (Konhilas et al. 2003). Changes in the chemical environment surrounding the sarcomeres, such as altered intracellular pH, modify myocardial function with little or no change in the Ca2+ transients (Orchard and Kentish 1990); pH-dependent alterations in sarcomeric response to Ca2+ are responsible for this effect.
It is also widely accepted that posttranslational modifications of sarcomeric proteins, especially protein phosphorylation, influence cardiac function (Solaro 2001, Metzger and Westfall 2004). At the level of thin filaments, phosphorylation of multiple sites on cardiac troponin I (cTnI) and troponin T (cTnT) differentially affects maximum Ca2+ activation, kinetics of the crossbridge cycle, and sensitivity to Ca2+ (Solaro 2001), pH (Wolska et al. 2001), and sarcomere length (Konhilas et al. 2003). These phosphorylations are important in short-term control of cardiac power (Herron et al. 2001) and in
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tuning the contraction/relaxation cycle dynamics to the prevailing heart rate (Pi et al. 2002, Layland et al. 2004, Takimoto et al. 2004). Kinases that determine the level of phosphorylation of cTnT and cTnI include protein kinases A (PKA) and C (PKC) (Solaro 2001, Sumandea et al. 2004), PKD (Haworth et al. 2004), PKG (Pfitzer et al. 1982), p21-activated kinase (Pak1) (Buscemi et al. 2002), and Rhodependent kinase (Vahebi et al. 2005). Pak1 isoform has also been shown to activate protein phosphatase 2A (PP2A) (Ke et al. 2004), a major phosphatase in heart. These kinase/phosphatase cascades may also be of significance in long-term changes in cardiac function leading to heart failure (Solaro et al. 2002). For example, part of the hypertrophy/failure process involves downregulation of signaling through h-adrenergic receptors, which fits with reports of a depression of cTnI phosphorylation at PKA sites, which would decrease cardiac power (Wolff et al. 1996). The hypertrophy/ failure process also involves activation of the PKC pathway. Activation of the PKC pathway results in phosphorylation of transcription factors that promote protein synthesis, resulting in growth and remodeling of the cardiac myocyte (Ruwhof and Van der Laarse 2000). We have hypothesized that maladaptive phosphorylation of cTnI and cTnT also occurs with activation of the PKC pathway (Solaro et al. 2002). These phosphorylations depress cross-bridge cycling and power as well as maximum tension (Sumandea et al. 2004). We think this leads to a malignant vicious cycle in which heart cells attempt to grow in the face of depressed function.
Remote Control of Thin Filament Phosphorylation and Function in the A-Band by Signaling at the Z-Disc
Control of cardiac function by phosphorylation of cTnI and cTnT serves to highlight concepts in an exciting new direction in research on thin filament control of cardiac function. A central aspect of these concepts is the idea that full understanding of thin filamentrelated control of cardiac function must take into account regions of the sarcomere where thin filaments enter into a realm of interactions with proteins
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of the Z-disc network (Figure 1). The complexity of the Z-protein network and its connection to the cytoskeleton and nucleus have been reviewed elsewhere in detail (Epstein and Davis 2003, Ervasti 2003, Granzier and Labeit 2004, Pyle and Solaro 2004). As illustrated in Figure 1, I focus here on a subset, which includes actin, actin-capping protein (CapZ), aactinin 2, titin, ZASP/cypher (a docking site for protein kinase C), and Pak1. The barbed ends of the thin filaments terminate in the Z-disc, where they link through a-actinin 2 and CapZ. CapZ is a heterodimer consisting of a-1 and h-1 units that localize to cardiac Z-discs (Hart and Cooper 1999). a-Actinin 2 functions as an antiparallel homodimer; N-terminal actin binding domains crosslink parallel thin filaments within the Z-disc (Stromer and Goll 1972) creating a support scaffold for parallel thin filaments. Yet, a-actinin does more than merely act to maintain the structural integrity of the thin filament lattice (Young and Gautel 2000). The domain structure of a-actinin also includes near N-terminal calponin homology domains and a C-terminal calmodulin-like domain. The central region of a-actinin consists of four spectrin-like repeats. As summarized in Pyle and Solaro (2004), aactinin also interacts with Z-repeats of titin, MLP, ALP, myotilin, and calsarcins. The region of the thin filament within the Z-disc also interacts with a-actinin through CapZ. The exact nature of the interaction is not clear (Papa et al. 1999). Evidence that the integrity of the Z-disc protein interactions affects the localization of PKC and its control of thin filament function comes from our studies of sarcomeres from a transgenic (TG) mouse model in which CapZ expression is modestly reduced (Pyle et al. 2002). Compared with controls, cardiac skinned fiber bundles from these TG mice exhibited increased Ca2+ sensitivity and blunted depression of maximum tension that occurred with agonist activation of PKC signaling pathways. Activation of these pathways in wild-type preparations increased the association of PKC-q with the sarcomere, but decreased PKC-q in the transgenic preparations. Loss of CapZ also resulted in a loss of localization of PKC-h at the sarcomeres, but whether this involves a direct or indirect interaction of CapZ with PKC remains unknown.
There is, however, evidence of a direct interaction of PKC with the Z-disc protein ZASP/cypher. N-Terminal domains of ZASP interact with a-actinin 2 and its C-terminal domains interact with PKC. Thus, ZASP is one of many Z-disc proteins that form struts between different proteins and would be expected to sense strain at the Z-disc. As with many Z-disc proteins, the existence of multiple isoforms of ZASP introduces another complexity in the puzzle (Huang et al. 2003, Vatta et al. 2003). Most isoforms of ZASP possess PDZ domains in the N-terminal region and LIM domains in the C-terminal regions. The PDZ domains anchor ZASP to a-actinin, whereas the LIM domain binds PKC (Zhou et al. 1999). A clue to the importance of this subset of Z-disc proteins in long- and short-term regulation of cardiac function has come from studies demonstrating a relation between the integrity of the Z-disc as a causal mechanism in the maladaptive process leading to dilated cardiomyopathy (DCM). Dilated cardiomyopathy involves both a depression in systolic function and a ventricular dilatation. Ablation of ZASP in mutant (knockout) mice leads to DCM (Zhou et al. 2001). Vatta et al. (2003) first identified multiple mutations in ZASP isoforms linked to DCM, an observation indicating these changes may be a common cause of left ventricular depressed function, dilation, and isolated noncompaction of the left ventricular myocardium. One of these mutations in a LIM domain of ZASP (D626N) has been demonstrated by Arimura et al. (2004) to enhance the affinity of ZASP for PKC. They hypothesize that this may reduce the amount of PKC-q docked at a receptor for PKC (RACK-2) and lead to heart failure. This hypothesis is based on evidence that the integrity of the PKCq–RACK-2 interaction is critical for protection of the heart against stresses and a decrease in the PKC- q–RACK-2 interaction leads to depressed cardiac function (Johnson et al. 1996). In contrast to the case with kinases that alter the phosphorylation of sites on cTnI and cTnT, relatively little is known concerning possible remote control of phosphatases by signaling at the Z-disc. Ke et al. (2004) have investigated a role for Pak1, a serine/threonine kinase, which is activated by Rac1/Cdc42 TCM Vol. 15, No. 4, 2005
(Manser and Lim 1999), interacts with PP2A (Westphal et al. 1999), and appears important in signaling of stress responses in heart (Clerk and Sugden 1997). Ke et al. (2004) also reported that active Pak1 stimulates protein phosphatase activity in heart, dephosphorylates cTnI, and colocalizes with PP2A. Moreover, endogenous Pak1 localized at the Zdisc and moved away from this location, when activated. Interactions of Pak1 with near neighbors in the Z-disc remains unknown.
Future Directions
Altered strain on proteins at the Z-disc and their connections to the sarcomeric network appear important as a determinant of signals that affect the actin– myosin interaction. Strain on the network may also be influenced by direct interactions between titin and thin filament actin (Jin 2004) and/or tropomyosin (Raynaud et al. 2004) in the I-band region. In addition to signaling via phosphorylation, altered strain in the network has been demonstrated to regulate interfilament spacing in a mechanism that may underlie length-dependent activation (Fukuda et al. 2005) and to alter localization of transcription factors (Knoll et al. 2002). A major challenge is to understand the temporal elaboration of these signals following stresses on the myocardium with acquired and genetic abnormalities and to know what the signals do to the reactions regulating force and shortening of the sarcomeres.
Acknowledgments
The author is grateful to many fine colleagues for discussions related to the ideas presented here. Our laboratory is supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute.
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STAT3-Mediated Activation of Myocardial Capillary Growth Denise Hilfiker-Kleiner*, Anne Limbourg, and Helmut Drexler
Proper perfusion and vessel integrity are key requisites for myocardial homeostasis. In this regard, myocardial angiogenesis occurs in physiologic and pathologic conditions. Failure in this process and the resulting deficient oxygen supply induce loss and degeneration of cardiomyocytes, atrophy, and interstitial fibrosis and are viewed as a primary cause of myocardial dysfunction and heart failure. In this review, signal transducer and activator of transcription 3 (STAT3) is highlighted as a regulator of proangiogenic circuits promoting vessel formation in the adult heart under physiologic and pathophysiologic conditions. Specifically, STAT3 regulates proangiogenic vascular endothelial growth factor (VEGF) expression and activity in the postnatal heart and suppresses an antiangiogenic and profibrotic gene program by controlling autocrine and paracrine circuits. In addition, signaling through STAT3 represents a necessary survival pathway for cardiomyocytes and endothelial cells and seems to promote cytokinemediated cardiac angiogenesis. In contrast, STAT3 seems not to be required for differentiation processes of embryonic or adult endothelial progenitor cells. In summary, the properly timed expression and activation of STAT3 play a critical role on cardiac angiogenesis and involve the subtle control of paracrine and autocrine mechanisms regulating angiogenic circuits and survival pathways of cardiomyocytes and endothelial cells. (Trends Cardiovasc Med 2005;15:152–157) D 2005, Elsevier Inc. STAT3 was initially identified as APRF (acute-phase response factor), a DNAbinding protein that binds to interleukin 6 (IL-6)-responsive elements within promoters of hepatic acute phase proteins
Denise Hilfiker-Kleiner, Anne Limbourg, and Helmut Drexler are at the Department of Cardiology and Angiology, Medical School Hannover, 30625 Hannover, Germany. * Address correspondence to: Denise Hilfiker-Kleiner, PhD, Abt. Kardiologie und Angiologie, Medizinische Hochschule Hannover, Carl-Neuberg Str. 1, 30625 Hannover, Germany. Tel.: (+49) 511-532-2531; fax: (+49) 511-532-3263; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
in response to IL-6 (Wegenka et al. 1993). Subsequently, it has been shown that STAT3 is activated by the entire family of IL-6–related cytokines, peptide growth factors, and hormones (Levy and Lee 2002, O’Shea et al. 2002, Zhu et al. 2001). Although initially considered to be a mere target gene of the IL-6–mediated inflammatory response, STAT3 is by now known to direct a wide variety of biologic processes, such as cell survival and apoptosis, inflammation, angiogenesis, and cardiac hypertrophy (Hirano et al. 2000, Levy and Lee 2002, Takeda and Akira 2001, Takeda et al. 1997). Embryonic lethality in mice with a systemic deletion of STAT3 suggests an essential role for STAT3 early in developTCM Vol. 15, No. 4, 2005
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Remote Control of A-Band Cardiac Thin Filaments by the I-Z-I Protein Network of Cardiac Sarcomeres R. John Solaro* A conventional view of the role of sarcomeric thin filaments in cardiac function is that they react with cross-bridges that translate them toward the center of the sarcomere in a reaction triggered by Ca2+ and powered by ATP. However, thin filaments also engage in a complex network of protein–protein interactions in the Z-disc. Thus, in the modern context, understanding of what thin filaments do in the heart must take into account not only A-band regions that react with cross-bridges, but also I-Z-I regions that rarely, if ever, react with cross-bridges and dwell near and within the Z-disc. To highlight these multiplex functions of the thin filament, I discuss the hypothesis that physical and chemical reactions at the interface of the thin filaments with Z-disc proteins control the docking and activity of kinases and phosphatases that control the levels of phosphorylation of thin filament regulatory proteins. Testing this hypothesis has taken on new significance with the identification of multisite phosphorylation of thin filament proteins as a critical element in the control of cardiac contraction and relaxation reserve and in maladaptive mechanisms in heart failure. Moreover, multiple mutations in Z-disc proteins that link to prevalent cardiomyopathies are likely to alter this remote control of A-band thin filament function. (Trends Cardiovasc Med 2005;15:148–152) D 2005, Elsevier Inc.
Cardiac Thin Filaments are Involved in Diverse Functions in Control of Force Generation and Shortening in the A-Band and in Signaling at the I-Z-I Region of Sarcomeres
Until recently, most of the action in cardiac thin filaments was thought to
R. John Solaro is at the Department of Physiology and Biophysics and Center for Cardiovascular Research, College of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA. * Address correspondence to: R. John Solaro, PhD, Department of Physiology and Biophysics (M/C 901), College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Avenue, Chicago, IL 60612-7342, USA. Tel.: (+1) 312-996-7620; fax: (+1) 312996-1414; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
occur in the A-band region of the sarcomere in which thin filament proteins react with myosin cross-bridges and promote the generation of myocyte force and shortening responsible for the ejection of blood from cardiac chambers (Kobayashi and Solaro 2005). Rather than being actively involved in control mechanisms, the I-Z-I band region of the thin filaments was pictured as providing mechanical stability to the sarcomere as an anchoring site in a scaffold making up the Z-disc protein network. However, recent and expanding evidence (Epstein and Davis 2003, Ervasti 2003, Granzier and Labeit 2004, Pyle and Solaro 2004) now pictures the I-Z-I region as a center for reception, transduction, and transmission of mechanical and biochemical signals. My focus here is on how events involving regions of thin filaments in the I-Z-I region of the sarcomere regions may affect A-band regions of the thin TCM Vol. 15, No. 4, 2005
filament, thereby regulating cardiac function by remote control. An understanding of cross-talk between A-band and I-Z-I band regions of the sarcomere appears especially significant with regard to the maladaptive mechanisms leading to heart failure. Mechanisms of decompensation are poorly understood and may involve altered mechanochemical transduction at the Z-disc with the elevated strain on the sarcomere. To highlight this concept, I first briefly summarize alterations (especially phosphorylation) in the thin filament proteins troponin I (cTnI) and troponin (cTnT) that are important determinants of cardiac function. I then consider emerging evidence that mechanical and biochemical signaling at the level of the Z-disc may modify thin filament function by controlling the level of phosphorylation of cTnI and cTnT.
Phosphorylation of cTnI and cTnT Significantly Affects the Dynamics in Both Short- and Long-Term Regulation of Cardiac Function
Tension generation and shortening of cardiac sarcomeres are determined by the mass of sarcomeres in the cardiac cell, the level of activation of the thin filament, the force generated per crossbridge, and the displacement of the thin filament in each cycle. Regulation of protein synthesis and breakdown ultimately determines the mass of sarcomeres and their protein isoform population. Release and removal of Ca2+ into and from the sarcomeric space and Ca binding to thin filament regulatory proteins are major determinants of the level of activation of the thin filament (Bers 2002). As shown in Figure 1, Ca2+ binding to the thin filament Ca2+ receptor, cardiac troponin C (cTnC), induces a complex series of protein– protein interactions enabling crossbridges to react with actin (Kobayashi and Solaro 2005). However, the idea that before thin filaments go into action in cardiac sarcomeres they wait passively for these changes in intracellular Ca2+ has undergone extensive transformation, with convincing evidence that modifications in the thin filament response to Ca2+ are functionally significant. The level of thin filament activation varies independently of intracellular Ca2+ with isoform switching of sarcomere proteins TCM Vol. 15, No. 4, 2005
Figure 1. Schematic illustration and electron micrograph of a portion of the A-band and Z-disc regions of the sarcomere. The A-band region of the thin filament depicts a functional unit of the sarcomere consisting of a helical array of actin monomers with associated regulatory proteins, a heterotrimeric cardiac troponin complex (Tn complex) and tropomyosin (Tm). The functional unit also shows the major proteins of the thick filament—the myosin heavy chain (MHC) and light chains (MLC1 and MLC2). The head of myosin reacts with the thin filament, splitting ATP and impelling the thin filament toward the center of the sarcomere (away from the Z-disc). Myosin connects through myosin-binding protein C (MyBP-C) to titin, a giant elastic protein responsible for passive tension in the sarcomere. Thin filament-regulatory proteins determine whether cross-bridges are strongly reacting and generating force in a reaction controlled by Ca2+ binding to cTnC (shown in red). The Ca2+–cTnC signal is translated to activation of the thin filament by complex protein–protein interactions among cTnI (orange), an inhibitory unit, cTnT (blue), a tropomyosin binding unit, and Tm. These interactions are modified by multisite phosphorylations of TnI and TnT as described in the text and reviewed in Solaro (2001). The Z-disc region illustrates components discussed in the text. These include a-actinin, which crosslinks adjacent thin filaments, CapZ, which caps the barbed ends of the thin filament, ZASP (also known as cypher; a docking site for protein kinase C), and Pak1, which may move away from the Z-disc upon activation and activate PP2A. The Z-disc network also connects to the rest of the sarcomere through titin, which terminates in the Z-disc at one end and in the M-line at the other. See Pyle and Solaro (2004) for a more complete picture of Z-disc proteins, and see text for discussion of the hypothesis that events at the Z-disc may regulate the actin–myosin interaction by affecting the activity of PKC and Pak1.
as occurs during development and hypertrophy (Parmacek and Solaro 2004). Moreover, length-dependent Ca2+ sensitivity remains widely accepted as a major determinant of the Frank–Starling relation as reflected in the slope of the end systolic pressure–volume relation (Konhilas et al. 2003). Changes in the chemical environment surrounding the sarcomeres, such as altered intracellular pH, modify myocardial function with little or no change in the Ca2+ transients (Orchard and Kentish 1990); pH-dependent alterations in sarcomeric response to Ca2+ are responsible for this effect.
It is also widely accepted that posttranslational modifications of sarcomeric proteins, especially protein phosphorylation, influence cardiac function (Solaro 2001, Metzger and Westfall 2004). At the level of thin filaments, phosphorylation of multiple sites on cardiac troponin I (cTnI) and troponin T (cTnT) differentially affects maximum Ca2+ activation, kinetics of the crossbridge cycle, and sensitivity to Ca2+ (Solaro 2001), pH (Wolska et al. 2001), and sarcomere length (Konhilas et al. 2003). These phosphorylations are important in short-term control of cardiac power (Herron et al. 2001) and in
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tuning the contraction/relaxation cycle dynamics to the prevailing heart rate (Pi et al. 2002, Layland et al. 2004, Takimoto et al. 2004). Kinases that determine the level of phosphorylation of cTnT and cTnI include protein kinases A (PKA) and C (PKC) (Solaro 2001, Sumandea et al. 2004), PKD (Haworth et al. 2004), PKG (Pfitzer et al. 1982), p21-activated kinase (Pak1) (Buscemi et al. 2002), and Rhodependent kinase (Vahebi et al. 2005). Pak1 isoform has also been shown to activate protein phosphatase 2A (PP2A) (Ke et al. 2004), a major phosphatase in heart. These kinase/phosphatase cascades may also be of significance in long-term changes in cardiac function leading to heart failure (Solaro et al. 2002). For example, part of the hypertrophy/failure process involves downregulation of signaling through h-adrenergic receptors, which fits with reports of a depression of cTnI phosphorylation at PKA sites, which would decrease cardiac power (Wolff et al. 1996). The hypertrophy/ failure process also involves activation of the PKC pathway. Activation of the PKC pathway results in phosphorylation of transcription factors that promote protein synthesis, resulting in growth and remodeling of the cardiac myocyte (Ruwhof and Van der Laarse 2000). We have hypothesized that maladaptive phosphorylation of cTnI and cTnT also occurs with activation of the PKC pathway (Solaro et al. 2002). These phosphorylations depress cross-bridge cycling and power as well as maximum tension (Sumandea et al. 2004). We think this leads to a malignant vicious cycle in which heart cells attempt to grow in the face of depressed function.
Remote Control of Thin Filament Phosphorylation and Function in the A-Band by Signaling at the Z-Disc
Control of cardiac function by phosphorylation of cTnI and cTnT serves to highlight concepts in an exciting new direction in research on thin filament control of cardiac function. A central aspect of these concepts is the idea that full understanding of thin filamentrelated control of cardiac function must take into account regions of the sarcomere where thin filaments enter into a realm of interactions with proteins
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of the Z-disc network (Figure 1). The complexity of the Z-protein network and its connection to the cytoskeleton and nucleus have been reviewed elsewhere in detail (Epstein and Davis 2003, Ervasti 2003, Granzier and Labeit 2004, Pyle and Solaro 2004). As illustrated in Figure 1, I focus here on a subset, which includes actin, actin-capping protein (CapZ), aactinin 2, titin, ZASP/cypher (a docking site for protein kinase C), and Pak1. The barbed ends of the thin filaments terminate in the Z-disc, where they link through a-actinin 2 and CapZ. CapZ is a heterodimer consisting of a-1 and h-1 units that localize to cardiac Z-discs (Hart and Cooper 1999). a-Actinin 2 functions as an antiparallel homodimer; N-terminal actin binding domains crosslink parallel thin filaments within the Z-disc (Stromer and Goll 1972) creating a support scaffold for parallel thin filaments. Yet, a-actinin does more than merely act to maintain the structural integrity of the thin filament lattice (Young and Gautel 2000). The domain structure of a-actinin also includes near N-terminal calponin homology domains and a C-terminal calmodulin-like domain. The central region of a-actinin consists of four spectrin-like repeats. As summarized in Pyle and Solaro (2004), aactinin also interacts with Z-repeats of titin, MLP, ALP, myotilin, and calsarcins. The region of the thin filament within the Z-disc also interacts with a-actinin through CapZ. The exact nature of the interaction is not clear (Papa et al. 1999). Evidence that the integrity of the Z-disc protein interactions affects the localization of PKC and its control of thin filament function comes from our studies of sarcomeres from a transgenic (TG) mouse model in which CapZ expression is modestly reduced (Pyle et al. 2002). Compared with controls, cardiac skinned fiber bundles from these TG mice exhibited increased Ca2+ sensitivity and blunted depression of maximum tension that occurred with agonist activation of PKC signaling pathways. Activation of these pathways in wild-type preparations increased the association of PKC-q with the sarcomere, but decreased PKC-q in the transgenic preparations. Loss of CapZ also resulted in a loss of localization of PKC-h at the sarcomeres, but whether this involves a direct or indirect interaction of CapZ with PKC remains unknown.
There is, however, evidence of a direct interaction of PKC with the Z-disc protein ZASP/cypher. N-Terminal domains of ZASP interact with a-actinin 2 and its C-terminal domains interact with PKC. Thus, ZASP is one of many Z-disc proteins that form struts between different proteins and would be expected to sense strain at the Z-disc. As with many Z-disc proteins, the existence of multiple isoforms of ZASP introduces another complexity in the puzzle (Huang et al. 2003, Vatta et al. 2003). Most isoforms of ZASP possess PDZ domains in the N-terminal region and LIM domains in the C-terminal regions. The PDZ domains anchor ZASP to a-actinin, whereas the LIM domain binds PKC (Zhou et al. 1999). A clue to the importance of this subset of Z-disc proteins in long- and short-term regulation of cardiac function has come from studies demonstrating a relation between the integrity of the Z-disc as a causal mechanism in the maladaptive process leading to dilated cardiomyopathy (DCM). Dilated cardiomyopathy involves both a depression in systolic function and a ventricular dilatation. Ablation of ZASP in mutant (knockout) mice leads to DCM (Zhou et al. 2001). Vatta et al. (2003) first identified multiple mutations in ZASP isoforms linked to DCM, an observation indicating these changes may be a common cause of left ventricular depressed function, dilation, and isolated noncompaction of the left ventricular myocardium. One of these mutations in a LIM domain of ZASP (D626N) has been demonstrated by Arimura et al. (2004) to enhance the affinity of ZASP for PKC. They hypothesize that this may reduce the amount of PKC-q docked at a receptor for PKC (RACK-2) and lead to heart failure. This hypothesis is based on evidence that the integrity of the PKCq–RACK-2 interaction is critical for protection of the heart against stresses and a decrease in the PKC- q–RACK-2 interaction leads to depressed cardiac function (Johnson et al. 1996). In contrast to the case with kinases that alter the phosphorylation of sites on cTnI and cTnT, relatively little is known concerning possible remote control of phosphatases by signaling at the Z-disc. Ke et al. (2004) have investigated a role for Pak1, a serine/threonine kinase, which is activated by Rac1/Cdc42 TCM Vol. 15, No. 4, 2005
(Manser and Lim 1999), interacts with PP2A (Westphal et al. 1999), and appears important in signaling of stress responses in heart (Clerk and Sugden 1997). Ke et al. (2004) also reported that active Pak1 stimulates protein phosphatase activity in heart, dephosphorylates cTnI, and colocalizes with PP2A. Moreover, endogenous Pak1 localized at the Zdisc and moved away from this location, when activated. Interactions of Pak1 with near neighbors in the Z-disc remains unknown.
Future Directions
Altered strain on proteins at the Z-disc and their connections to the sarcomeric network appear important as a determinant of signals that affect the actin– myosin interaction. Strain on the network may also be influenced by direct interactions between titin and thin filament actin (Jin 2004) and/or tropomyosin (Raynaud et al. 2004) in the I-band region. In addition to signaling via phosphorylation, altered strain in the network has been demonstrated to regulate interfilament spacing in a mechanism that may underlie length-dependent activation (Fukuda et al. 2005) and to alter localization of transcription factors (Knoll et al. 2002). A major challenge is to understand the temporal elaboration of these signals following stresses on the myocardium with acquired and genetic abnormalities and to know what the signals do to the reactions regulating force and shortening of the sarcomeres.
Acknowledgments
The author is grateful to many fine colleagues for discussions related to the ideas presented here. Our laboratory is supported by grants from the National Institutes of Health, National Heart, Lung, and Blood Institute.
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STAT3-Mediated Activation of Myocardial Capillary Growth Denise Hilfiker-Kleiner*, Anne Limbourg, and Helmut Drexler
Proper perfusion and vessel integrity are key requisites for myocardial homeostasis. In this regard, myocardial angiogenesis occurs in physiologic and pathologic conditions. Failure in this process and the resulting deficient oxygen supply induce loss and degeneration of cardiomyocytes, atrophy, and interstitial fibrosis and are viewed as a primary cause of myocardial dysfunction and heart failure. In this review, signal transducer and activator of transcription 3 (STAT3) is highlighted as a regulator of proangiogenic circuits promoting vessel formation in the adult heart under physiologic and pathophysiologic conditions. Specifically, STAT3 regulates proangiogenic vascular endothelial growth factor (VEGF) expression and activity in the postnatal heart and suppresses an antiangiogenic and profibrotic gene program by controlling autocrine and paracrine circuits. In addition, signaling through STAT3 represents a necessary survival pathway for cardiomyocytes and endothelial cells and seems to promote cytokinemediated cardiac angiogenesis. In contrast, STAT3 seems not to be required for differentiation processes of embryonic or adult endothelial progenitor cells. In summary, the properly timed expression and activation of STAT3 play a critical role on cardiac angiogenesis and involve the subtle control of paracrine and autocrine mechanisms regulating angiogenic circuits and survival pathways of cardiomyocytes and endothelial cells. (Trends Cardiovasc Med 2005;15:152–157) D 2005, Elsevier Inc. STAT3 was initially identified as APRF (acute-phase response factor), a DNAbinding protein that binds to interleukin 6 (IL-6)-responsive elements within promoters of hepatic acute phase proteins
Denise Hilfiker-Kleiner, Anne Limbourg, and Helmut Drexler are at the Department of Cardiology and Angiology, Medical School Hannover, 30625 Hannover, Germany. * Address correspondence to: Denise Hilfiker-Kleiner, PhD, Abt. Kardiologie und Angiologie, Medizinische Hochschule Hannover, Carl-Neuberg Str. 1, 30625 Hannover, Germany. Tel.: (+49) 511-532-2531; fax: (+49) 511-532-3263; e-mail: [email protected]. D 2005, Elsevier Inc. All rights reserved. 1050-1738/05/$-see front matter
in response to IL-6 (Wegenka et al. 1993). Subsequently, it has been shown that STAT3 is activated by the entire family of IL-6–related cytokines, peptide growth factors, and hormones (Levy and Lee 2002, O’Shea et al. 2002, Zhu et al. 2001). Although initially considered to be a mere target gene of the IL-6–mediated inflammatory response, STAT3 is by now known to direct a wide variety of biologic processes, such as cell survival and apoptosis, inflammation, angiogenesis, and cardiac hypertrophy (Hirano et al. 2000, Levy and Lee 2002, Takeda and Akira 2001, Takeda et al. 1997). Embryonic lethality in mice with a systemic deletion of STAT3 suggests an essential role for STAT3 early in developTCM Vol. 15, No. 4, 2005