Intracellular signaling pathways in cardiac myocytes induced by mechanical stress

Intracellular Signaling Pathways Cardiac Myocytes Induced by Mechanical Stress Tssei Komuro

in

and Yoshio Yazaki

Mechanical stress is a major cause for cardiac hypertrophy. Although the mechanisms by which mechanical load induces cardiomyocyte hypertrophy have long been a subject of great interest for cardiologists, the lack of a good in vitro system has hampered the understanding of the biochemical mechanisms. For these past several years, however, an in vitro neonatal cardiocyte culture system has made it possible to examine the biochemical basis for the signal transduction of mechanical stress. Passive stretch of cardiac myocytes cultured on silicone membranes activates phosphorylation cascades and induces the expression of specific genes as well as the increase in protein synthesis. Although an important question regarding how mechanical stimulus is converted into biochemical signals remains unanswered, cultured cardiac myocytes may be a good model to examine the signal transduction pathways of mechanical stress. (Trends Cardiovasc Med 1994;4:117-121) Mechanical load is a major factor for cardiac hypertrophy at the pressure or volume overload [Cooper et al. (1985), and references therein]. Recently there has been increasing evidence to suggest that mechanical stress itself evokes a variety of intracellular biochemical signals without any participation of humoral or neural factors, and induces the expression of specific genes as well as increasing rates of protein synthesis in cardiac myocytes [for a review, see Komuro and Yazaki (1993)]. In the present short review, we focus on intracellular signaling pathways activated by mechanical stress in cardiac myocytes cultured in vitro.

l

Mechanical Stress Is a Primary Stimulus for Cardiac Hypertrophy Induced by Hemodynamic Overload

Issei Komuro

and Yoshio Yazaki are at the Molecular Cardiology Division, Third Department of Medicine, University of Tokyo School of Medicine, Tokyo 113, Japan.

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It has been controversial whether a primary stimulus for hypertrophy is a mechanical stress itself or an accompanying increase in neural or humoral factors. Many studies have shown that the adrenoceptor activation accompanies cardiac hypertrophy induced by hemodynamic overload (Zak 1984). Evidence presented below, however, suggests that the direct mechanical stress might be a primary factor for cardiac hypertrophy in response to hemodynamic overload. First, cardiac hypertrophy can be induced by hemodynamic overload even after adrenoceptor blockade or sympathectomy (Cooper et al. 1985). Second, increased cardiac load stimulates protein synthesis in isolated hearts (Kira et al. 1984). Third, it has been shown that stretching the cultured cardiomyocytes with no involvement of neural or humoral factors stimulates protein synthesis and specific gene expression (Mann et al. 1989, Komuro et al. 1990 and 199 1b). Since most of extracellular stimuli are humoral factors such as growth factors

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and hormones, it may appear unconventional to suggest that mechanical stress itself can be a stimulus for cells. The process of receiving and responding to mechanical stimuli is critical, however, for growth and function of many living cells, such as endothelial cells of vessels, vascular and visceral smooth muscle cells, proximal tubule cells of the kidney, fetal lung cells, and the cells of many sensory organs (Fung 1988). Plants and bacteria also respond to mechanical stimuli. These observations suggest that mechanical stress is a highly conserved and important stimulus among many cells and species. Mechanical Stress to Cardiomyocytes the Cell

l

Can Be Applied by Stretching

How is mechanical stress perceived by a cell as a stimulus? With the use of a Langendorf preparation, it has been shown that stretch of the ventricular wall as a consequence of increased aortic pressure is the mechanical parameter most closely related to the increase in protein synthesis (Kira et al. 1984). This observation has been confirmed by the experiments using cardiocytes cultured in vitro with serum-free media. When cardiocytes cultured on deformable silicone membrane were stretched, protein synthesis increased (Mann et al. 1989). Stretching the cardiocytes also induced the expression of specific genes, such as immediate early genes (IEGs) and fetaltype genes [for a review, see Komuro and Yazaki (1993)]. These gene expression patterns are similar to those observed in the heart in vivo in response to hemodynamic overload. These observations suggest that mechanical stress (hemodynamic overload) might affect cardiocytes as the result of stretch.

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Mechanical Stress Evokes a Variety of Biochemical Signals

Phospholipase C-Protein k’inase C Myocyte stretching stimulates expression of IEGs such as the c-fos gene at least in part at transcriptional levels. Transfection experiments using the chloramphenicol acetyltransferase (CAT) gene linked to upstream sequences of the c-fbs gene indicate that the sequences containing a serum-response element (SRE)

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Mechanical Fetch

of

Stress

Catdiac

1 Regulation

Myocytes

1

c-fos, c- jun, c- myc, Egr- 1

>

IEG

ANF, PMHC, MLC-2 Skeletal a - actin Figure 1. Phosphorylation cascades activated by mechanical stress. Mechanical stress induces many events, including gene expressions and hypertrophy in the cardiac myocytes through protein kinase cascade of phosphotylation. AH, angiotensin II; ANF, atria1 natriuretic factor; BMHC, fi myosin heavy chain; &/CM, calcium-calmodulin kinase; DG, 1,2-diacylglycerol; IEG, immediate early genes; IP,, inositoi 1,4,5-trisphosphate; MAPK, MAP kinase; MAPKK, MAP kinase kinase; MLC-2, myosin light chain 2; PKC, protein kinase C; PLC, phospholipase C; S6K, S6 kinase; and SR, sarcoplasmic reticulum.

are required for efficient transcription by stretch (Komuro et al. 199Ib). Recently, using c-fos-CAT constructs that have point mutations in SREs, activation of SRE-binding protein, ~62*~~, by protein kinase C (PKC) was shown to be important for stretch-induced c-fis expression (Sadoshima and Izumo 1993a). The importance of PKC in c-fos induction by mechanical stress is supported by pharmacologic studies (Komuro et al. 1991a and b). The c-fos induction is suppressed by the inhibitors for PKC and by downregulation of PKC. Although protooncogenes are known to be induced by catecholamines, c-fos gene expression by stretch is not inhibited by a and l3 adrenergic antagonists, and pertussis toxin does not reduce stretch-induced c-fos expression. Myocyte stretching results in small but significant increases in the levels of inositol phosphates and 1,2-diacylglycerol (DG) of cultured cardiocytes, suggesting that PKC can be activated subsequent to mechanical stress (Figure 1) (Komuro et al. 199Ib, Sadoshima and Izumo 1993b). Activation of PKC by phorbol esters stimulates the expression of c-fos and skeletal aactin genes (Komuro et al. 1991b), and

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PKC has been demonstrated to activate transcriptions of B myosin heavy chain, myosin light chain 2, and atrial natriuretie factor (ANF) directly in cotransfection experiments involving expression of constitutively activated PKC (Kariya et al. 1991, Shubeita et al. 1992). Recently, the predominant cardiac PKC was reported to be PKC-E, whose activity is independent of Ca*+ (Bogoyevitch et al. 1993). Since PKC might be involved in a number of physiologic and pathologic responses in the heart (Table l), further studies regarding the involvement of specific PKC isoforms in cardiac function are required. MAP Kinase

and S6 Kinase

A great number of intracellular signals are transduced into a nucleus through a protein kinase cascade of phosphotylation (Cantley et al. 1991). Once a mechanical stimulus is converted into a biochemical signal involving phosphorylation events, how do these signals then regulate protein synthesis and gene expression in cardiac myocytes? Increased protein synthesis is generally associated with an increase in phosphorylation of S6 protein in the 40s ribosome. The 01994,

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increase in S6 phosphorylation is thought to be caused by increased S6 kinase activity. S6 kinase is phosphorylated and activated by MAP kinase (Sturgill and Wu 1991). MAP kinase is activated by MAP kinase kinase, and Raf-1 kinase activates MAP kinase kinase (Blenis 199 1). Myocyte stretching was shown to increase phosphorylation of MAP kinase and MAP kinase activity toward myelin basic protein (MBP) (Yamazaki et al. 1993, Sadoshima and Izumo 1993a). As mentioned above, stretching myocytes stimulates PKC activity. Activation of PKC is known to increase the activity of MAP kinase in various cell types (Blenis 199 1). When PKC is depleted by preincubating myocytes with phorbol ester or its activity is blocked by the PKCinhibitor staurosporine, stretch-induced MBP kinase activity is decreased by -6O%-70%, suggesting that the stretchinduced increase in MAP kinase activity occurs through both PKC-dependent and -independent pathways (Figure 1). The stretch-induced MAP kinase activation is only partially dependent on transsarcolemmal Ca2+ influx through voltagedependent and -independent Ca*+ channels (Yamazaki et al. 1993). Myocyte stretching was shown to increase S6 peptide kinase activity two- to threefold (Sadoshima and Izumo 1993a, Yamazaki et al. 1993). Recently we also observed the activation of MAP kinase kinase and Raf-1 kinase by passive stretch (unpublished). Ras Protein Extensive evidence exists suggesting that the protooncogene Ras protein mediates a variety of signals from membrane receptors to cytoplasmic kinases [for reviews, see Satoh et al. (1992) and references therein]. Ras protein has been shown to be critical for three signaltransducing protein kinases, MAP kinase, Raf-1, and S6 kinase (Wood et al. 1992), resulting in interest in the role of Ras in cardiac hypertrophy. Recently, the importance of Ras protein in cardiac hypertrophy has been shown in cultured cardiomyocytes (Thorburn et al. 1993). Direct microinjection of activated Ras protein into primary neonatal rat cardiac myocytes resulted in a hypertrophic response as well as induction of c-fos and ANF gene expression. A dominant interfering Ras mutant inhibits the activation

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Table 1. Protein kinases activated by mechanical presumptive functions in the heart Kinases Protein

stress and their

Functions kinase

C

Induction Induction Activation Secretion Modulation Contraction

of IEG expression of cardiac-specific of MAP kinase of ANF of ion channels and relaxation

gene expression

Raf-1 kinase

Activation

of MAP kinase

MAP kinase kinase

Activation

of MAP kinase

MAP kinase

Induction Activation

and activation of S6 kinase

S6 kinase

Activation of IEG products Phosphorylation of ribosomal

ANF, atria1 natriuretic

factor;

and IEG, immediate

The Mechanisms by Which Mechanical Stress Is Converted into a Biochemical Stimulus

Stretch-Sensitive Channels Many cells respond to a variety of environmental stimuli by ion channels in the plasma membrane. Mechanosensitive ion channels have been observed with singlechannel recordings in >30 cell types of prokaryotes, plants, fungi, and all animals so far examined [for a review, see Morris (1990)]. The activation of stretchsensitive channels has been proposed as the transduction mechanism between load and protein synthesis in cardiac hypertrophy (Bustamante et al. 1991). The stretch-sensitive channels allow the TCM

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of IEG

S6 protein

early gene

of ANF promoter by phenylephrine. These results suggest that Ras might play critical roles in the hypertrophic response to a-adrenergic agonists, and that Ras might mediate the signals from a G-proteincoupled receptor as well as the signals from a tyrosine-phosphorylated receptor. Since imposition of mechanical stress and injection of activated Ras show very similar phenotypes (hypertrophy, IEG, and fetal gene expression), it is likely that activation of Ras protein exists in the signal transduction pathways of mechanical stress. Recently, mechanical stretching has been shown to increase the relative proportion of the activated form of the Ras protein in neonatal cardiac myocytes (Sadoshima and Izumo 1993a).

l

kinase

passage of the major monovalent physiologic cations, Na+ and K+, and the divalent cation, Ca2+. With the use of a Ca2+-binding fluorescent dye (fluo3) and the patch-clamp technique, mechanically induced Ca2+ influx through stretch channels was shown to lead to waves of calcium-induced calcium release (Sigurdson et al. 1992). When an Na+ ionophore, monensin or veratridine, was added to cultured cardiomyocytes, c-fos expression was observed, possibly because of increased Ca2+ uptake by the Na+-Ca*+ exchange mechanism. However, the expression of fetaltype genes was not induced by Na+ increase [Komuro et al. (1991a) and Komuro unpublished]. Although we cannot rule out the existence of the inhibitorinsensitive stretch channels in cardiomyocytes, gadolinium as well as streptomycin do not inhibit IEG expression and protein synthesis by stretching (Komuro et al. 1991a; Sadoshima et al. 1992). Amiloride (a blocker of the Na+-H+ exchanger and stretch channels) and tetrodotoxin (an Na+ channel blocker) do not affect induction of c-fos expression by stretching (Komuro et al. 1991a). Preincubation with Ca*+ channel blockers or short exposure to EDTA also does not change the induction of c-fos mRNA after stretching. The induction of c-fos expression is observed even in Na+-free (Na+ replaced by choline) or Ca2+-free media. Although ion influx through stretch-sensitive channels may play certain roles such as stretch-induced arrhythmias (Hansen et al. 1991), the many 01994,

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biochemical events that are evoked by stretching cardiac myocytes cannot be explained by an opening of the stretchsensitive ion channels.

Extracellular Matrix and Cytoskeleton Much data have been accumulated that indicate that the mechanical stress is transduced into the cell from the sites at which cells attach to extracellular matrix (ECM) [for a review, see Juliano and Haskill (1993)]. Transmembrane ECM receptors, such as the integrin family, are good candidates for mechanoreceptors. A large extracellular domain of integrin receptor complex binds various ECM proteins, whereas a short cytoplasmic domain has been shown to interact with the cytoskeleton in the cell [for a review, see Hynes (1992)]. Since cytoskeleton proteins can potentially regulate plasma membrane proteins such as enzymes, ion channels, and antiporters, mechanical stress could modulate these membrane-associated proteins and stimulate second-messenger systems through the cytoskeleton. Integrins can transmit signals not only by organizing the cytoskeleton but also by altering biochemical properties such as the extent of tyrosine phosphorylation of a complex of proteins including ~~125’~~ (Juliano and Haskill 1993). Negative data about the role of integrincytoskeleton in cardiocyte mechanotransduction were reported (Sadoshima et al. 1992); however, further precise studies are necessary to allow conclusions about the roles of integrincytoskeleton in stretch-induced cardiac hypertrophy.

Autocrine and Paracrine Mechanisms Second-messenger cascades activated by mechanical stress on cardiac myocytes are very similar to those evoked by the addition of growth factors and cytokines. Cardiac myocytes and nonmuscle cells such as fibroblasts, endothelial cells, and smooth muscle cells may secrete some hypertrophy-promoting factors after a stretch stimulus. Mechanical stress on myocardial cells has been reported to increase the synthesis of some growthpromoting facto5 (Hammond et al. 1979). Many growth factors, including acidic and basic FGF, TGFl3, and insulinlike growth factors I and II, have been reported to exist in the heart, and some of them can induce cardiac hypertrophy

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and specific gene expression in the cultured cardiomyocytes [for a review, see Parker and Schneider (1991)]. There have been only a few reports, however, regarding the expression of these growth factors during hypertrophy induced by hemodynamic overload. An increase in TGFl3 mRNA levels was reported in the rat heart overloaded by aortic banding (Komuro et al. 1991a, Villarreal and Dillmann 1992). Since this increase is recognized only 12 h after banding, it is unlikely that TGFl3 is a primary mediator for a variety of events induced by mechanical stress. The increased TGFfl may play some role at the later stage in increases in collagen synthesis or in the deposition of extracellular matrix (Komuro et al. 1991a, Villarreal and Dillmann 1992). A growing body of data suggests that the local renin-angiotensin system is important for cardiac hypertrophy [for a review, see Baker et al. (1992)]. All components of the renin-angiotensin system such as angiotensinogen, renin, and angiotensin-converting enzyme (ACE) have been identified at both the mRNA and protein levels in the heart. Angiotensin II (AII) stimulates protein synthesis in cultured cardiomyocytes. Increases in angiotensinogen and ACE mRNAs have been reported in hypertrophied left ventricle of rats. Subpressor doses of ACE inhibitors can prevent or cause regression of cardiac hypertrophy with no change in systemic systolic blood pressure. An increase in left ventricular mass that was produced by abdominal aortic constriction, without significant increase in plasma renin activity, was completely prevented with ACE inhibitor without any change in afterload (Baker et al. 1990). These results suggest that the local renin-angiotensin system may play a critical role in cardiac hypertrophy induced by pressure overload. AI1 increases c-fos gene expression as well as protein synthesis in neonatal rat cardiocytes. Upregulation of c-fox gene expression by AI1 is blocked by PKC inhibitor, and AI1 actually increases production of inositol phosphates and activates PKC (Katoh et al. 1989, Sadoshima and Izumo 1993b). These signals elicited by AH in cardiac myocytes are very similar to those evoked by mechanical stress as mentioned above. Therefore, we examined the possible involvement of AI1 in the stretch-induced increase in

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protein synthesis. Stretching cardiocytes induced a 1.5-fold increase in protein synthesis, as described previously (Komuro et al. 1990). The addition of an AI1 receptor antagonist partially blocked the stretch-induced increase in amino acid incorporation. The induction of c-fos gene expression and the activation of MAP kinase by stretching were also attenuated by the antagonist (Kojima et al. 1994). It was reported that mechanical stretch causes a release of AI1 from cultured cardiocytes (Sadoshima et al. 1993). These results suggest that AI1 is responsible in part for events evoked by mechanical stress on cardiac myocytes.

l

Summary

and Future

Directions

As shown in Figure 1, mechanical stress can evoke a variety of signals in cardiac myocytes, and the molecules that are involved in the signal transduction pathway of mechanical stress are similar to the molecules that play important roles in many other cells stimulated by growth factors. Recently, yeast genes encoding members of MAP kinase have been isolated by complementation of yeast mutations as an essential protein for restoring the osmotic gradient across the cell membrane in response to increased external osmorality (Brewster et al. 1993). This suggests that once cells receive external stimuli, intracellular signal transduction pathways are usually highly conserved among many cell types and many species. Although many biochemical events that occur in cardiac myocytes subsequent to mechanical stretch have been clarified, a main intriguing question remains unanswered, How is mechanical stress converted into biochemical signals? In other words, what is the mechanoreceptor or the transducer for mechanical stress in cardiac myocytes? It is conceivable that by stretching the plasma membrane, mechanical stress directly changes the conformations of the functional proteins such as enzymes and G proteins, or directly activates enzymes such as phospholipase by physically placing the enzymes close to their phospholipid substances in the plasma membrane. As just mentioned, the integrin-cytoskeleton complex seems to be an alternate candidate structure for a mechanoreceptor and a transducer. Inte-

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grin-cytoskeleton proteins not only play a “passive” role such as maintaining the cell structure, but also may play “dynamic” roles in regulation of cellular functions such as protein phosphorylation and activation of an antiporter. Integrin ct,$, was shown to activate the Na+-H+ antiporter by binding to fibronectin, suggesting that integrin can behave similarly to a growth factor receptor in activating signaling pathways (Schwartz et al. 1991). The cytoskeleton has been also shown to play an important role in secretion. Mechanical stress may stimulate secretion of some cytokines that may generate multiple intracellular signals as a secondary event. Further studies to identify specific signaling molecules, including mechanoreceptors and mechanotransducers, and characterization of their activities will be required for understanding of the physiologic functions of mechanical stimuli on cardiac myocytes and finally to clarify the mechanisms by which adaptive cardiac hypertrophy deteriorates into congestive heart failure. A lack of an immortalized cardiac cell line has hampered advances in biochemical research of the heart. Cultured primary cardiocytes, however, may be a good model for the investigation of the biochemical signals evoked by mechanical stress.

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