The role of autophagy in the heart.

Autophagy has evolved as a conserving process that uses bulk degradation and recycling of cytoplasmic components, such as long-lived proteins and organelles. In the heart, autophagy is important for the turnover of organelles at low basal levels under normal conditions and it is upregulated in response to stresses such as ischemia/reperfusion and in cardiovascular diseases such as heart failure. Cardiac remodeling involves increased rates of cardiomyocyte cell death and precedes heart failure. The simultaneously occurring multiple features of failing hearts include not only apoptosis and necrosis but also autophagy as well. However, it has been unclear as to whether autophagy is a sign of failed cardiomyocyte repair or is a suicide pathway for failing cardiomyocytes. The functional role of autophagy during ischemia/reperfusion in the heart is complex. It has also been unclear whether autophagy is protective or detrimental in response to ischemia/reperfusion in the heart. In this review, we will summarize the role of autophagy in the heart under both normal conditions and in response to stress.

Chelerythrine rapidly induces apoptosis through generation of reactive oxygen species in cardiac myocytes.

The role of protein kinase C (PKC) inhibition in cardiac myocyte apoptosis has not been well understood. We investigated the mechanism, by which chelerythrine, a commonly used PKC inhibitor, induces potent myocyte death. Chelerythrine (6-30 microm) rapidly induced pyknosis, shrinkage and subsequent cell death in cardiac myocytes. Chelerythrine-induced myocyte death was accompanied by nuclear fragmentation and activation of caspase-3 and -9, while it was prevented by XIAP, suggesting that the cell death is due to apoptosis. Higher concentrations of chelerythrine caused necrotic cell death where neither cell shrinkage nor caspase activation was observed. Intravenous injection of chelerythrine (5 mg/kg) also increased apoptosis in adult rat hearts in vivo. Downregulation of the phorbol 12-myristate 13-acetate (PMA)-sensitive PKC failed to affect chelerythrine-induced apoptosis, while anti-oxidants, including N-acetyl-L-cysteine (NAC) and glutathione, inhibited it, suggesting that generation of reactive oxygen species (ROS) rather than inhibition of PMA-sensitive PKC mediates chelerythrine-induced cardiac myocyte apoptosis. Chelerythrine caused cytochrome c release from mitochondria, which was significantly inhibited in the presence of NAC, suggesting that ROS mediates chelerythrine-induced cytochrome c release. Partial inhibition of cytochrome c release by Bcl-X(L) significantly reduced chelerythrine-induced apoptosis. These results suggest that chelerythrine rapidly induces cardiac myocyte apoptosis and that production of ROS, possibly H(2)O(2), and subsequent cytochrome c release from mitochondria play an important role in mediating chelerythrine-induced rapid cardiac myocyte apoptosis.

ANG II type 1 receptor downregulation does not require receptor endocytosis or G protein coupling.

ANG II type 1 (AT(1)) receptors respond to sustained exposure to ANG II by undergoing downregulation of absolute receptor numbers. It has been assumed previously that downregulation involves endocytosis. The present study hypothesized that AT(1) receptor downregulation occurs independently of receptor endocytosis or G protein coupling. Mutant AT(1) receptors with carboxy-terminal deletions internalized <5% of radioligand compared with 65% for wild-type AT(1) receptors. The truncated AT(1) receptors retained the ability to undergo downregulation. These data suggest the existence of an alternative pathway to AT(1) receptor degradation that does not require endocytosis, per se. Point mutations in either the second transmembrane region or second intracellular loop impaired G protein (G(q)) coupling. These receptors exhibited a biphasic pattern of downregulation. The earliest phase of downregulation (0-2 h) was independent of coupling to G(q), but no additional downregulation was observed after 2 h of ANG II exposure in the receptors with impaired coupling to G(q). These data suggest that coupling to G(q) is required for the later phase (2-24 h) of AT(1) receptor downregulation.

Cyclosporine reduces left ventricular mass with chronic aortic banding in mice, which could be due to apoptosis and fibrosis.

A tacit assumption in studies of left ventricular (LV) hypertrophy is that left ventricular/body weight (LV/BW) reflects the extent of myocyte hypertrophy. The goal of the current investigation was to determine if there was another explanation for the reduced LV/BW observed after inhibiting calcineurin with cyclosporine during the development of pressure overload LV hypertrophy as compared with animals that did not receive cyclosporine. Accordingly, we examined the prevalence of fibrosis and apoptosis and measured cell size in the hearts from mice at 1 and 3 weeks after transverse aortic banding with and without chronic cyclosporine. Although LV/BW, compared to aortic banded vehicle treated mice, was reduced by 30% in aortic banded cyclosporine treated mice, myocyte cross sectional area was similar in both banded groups (346+/-9 microm2 v 336+/-13 microm2). The volume percent interstitial fibrosis was greater in aortic banded cyclosporine treated animals (1.4+/-0.2%) compared with aortic banded vehicle treated animals (0.9+/-0.2%, P<0.05) or in sham animals (0.6+/-0.1%). Surprisingly, lesions including myocytes containing iron were observed and were most prominent in aortic banded cyclosporine treated animals. Apoptosis, quantitated with TUNEL staining as percent of myocytes, was increased in aortic banded cyclosporine treated animals at 7 days (1.6+/-0.4%) compared with aortic banded vehicle treated animals (0.4+/-0.1%, P<0.01) and was still increased at 21 days. Immunoblotting demonstrated a decrease in the phosphorylation of Akt and Bad, and also Bcl-2 levels were reduced in aortic banded cyclosporine treated animals at 7 days compared with aortic banded vehicle treated animals. These proteins protect against apoptosis, and support the concept that cyclosporine inhibited the calcineurin pathway, resulting in enhanced apoptosis. Thus, the decrease in LV/BW in the aortic banded cyclosporine treated animals actually may be due, at least in part, to cell loss and death, as reflected by the enhanced fibrosis and apoptosis and the focal iron deposits in myocytes.

Glycogen synthase kinase 3beta regulates GATA4 in cardiac myocytes.

Inactivation of glycogen synthase kinase 3beta (GSK3beta) is critical for transcription of atrial natriuretic factor (ANF) by beta-adrenergic receptors in cardiac myocytes. We examined the mechanism by which GSK3beta regulates ANF transcription. Stimulation of beta-adrenergic receptors induced nuclear accumulation of GATA4, whereas beta-adrenergic ANF transcription was suppressed by dominant negative GATA4, suggesting that GATA4 plays an important role in beta-adrenergic ANF transcription. Interestingly, GATA4-mediated transcription was markedly attenuated by GSK3beta. GSK3beta physically associates with GATA4 and phosphorylates GATA4 in vitro. Overexpression of GSK3beta suppressed both basal and beta-adrenergic increases in nuclear expression of GATA4, whereas inhibition of GSK3beta by LiCl caused nuclear accumulation of GATA4, suggesting that GSK3beta negatively regulates nuclear expression of GATA4. The nuclear exportin Crm1 reduced nuclear expression of GATA4, and the reduction was enhanced by GSK3beta but not by kinase-inactive GSK3beta. Leptomycin B, an inhibitor for Crm1, increased basal nuclear GATA4 and suppressed GSK3beta-induced decreases in nuclear GATA4. These results suggest that GSK3beta negatively regulates nuclear expression of GATA4 by stimulating Crm1-dependent nuclear export. Inhibition of GSK3beta by beta-adrenergic stimulation abrogates GSK3beta-induced nuclear export of GATA4, causing nuclear accumulation of GATA4, which may represent an important signaling mechanism mediating cardiac hypertrophy.

Beta-adrenergic cardiac hypertrophy is mediated primarily by the beta(1)-subtype in the rat heart.

Myocardial beta-adrenergic receptors (beta -ARs) consist of beta(1)- and beta(2)-subtypes, which mediate distinct signaling mechanisms. We examined which beta-AR subtype mediates cardiac hypertrophy. The beta(2)-subtype is predominant in neonatal rat cardiac myocytes (beta(1), 36%vbeta(2), 64%), while the beta(1)-subtype predominates in the adult rat heart (59%v 41%). Stimulation of cultured cardiac myocytes in vitro with isoproterenol (ISO), an agonist for beta(1)- and beta(2)-ARs, caused hypertrophy of myocytes along with increases in transcription of atrial natriuretic factor (ANF) and actin reorganization. All of these ISO-mediated myocyte responses in vitro were inhibited by a beta(1)-AR antagonist, betaxolol, but not by a beta(2)-AR antagonist, ICI 118551. Pertussis toxin failed to affect ISO-induced increases in total protein/DNA content and ANF transcription in vitro. ISO increased LV weight/body weight and ANF transcription in the adult rat in vivo, which were also inhibited by betaxolol but not by ICI 118551. These results suggest that beta -AR stimulated hypertrophy is mediated by the beta(1)-subtype and by a pertussis toxin-insensitive mechanism

Phosphorylation of elk-1 by MEK/ERK pathway is necessary for c-fos gene activation during cardiac myocyte hypertrophy.

Cardiac hypertrophy is associated with specific alterations in myocardial gene expression; however, the exact mechanisms responsible for altered gene expression are poorly defined. The goal of this study was to investigate whether signaling kinases that are activated during cardiac hypertrophy directly modulate transcription factor activity and regulate gene expression. In an effort to understand this process, we focused our studies on the transcriptional activation of c-fos gene through the serum response element (SRE)/ternary complex factor (TCF) element, during phenylephrine-induced myocyte hypertrophy. In this study, we show that phosphorylated Elk-1, a TCF, binds to c-fos SRE and its binding to SRE is increased upon phenylephrine stimulation. Phenylephrine treatment activates phosphorylation of Elk-1 in the nucleus within five minutes and Elk-1-dependent transcriptional activation is abolished by inhibitors selective for MEK/ERK kinases. These studies implicate that phosphorylation of Elk-1 by ERK kinase pathway is important for early gene activation during phenylephrine-induced myocyte hypertrophy.

The Akt-glycogen synthase kinase 3beta pathway regulates transcription of atrial natriuretic factor induced by beta-adrenergic receptor stimulation in cardiac myocytes.

We examined the mechanism of atrial natriuretic factor (ANF) transcription by isoproterenol (ISO), an agonist for the beta-adrenergic receptor (betaAR), in cardiac myocytes. ISO only modestly activated members of the mitogen-activated protein kinase family. ISO-induced ANF transcription was not affected by inhibition of mitogen-activated protein kinases, whereas it was significantly inhibited by KN93, an inhibitor of Ca(2+)/calmodulin-dependent kinase (CaM kinase II). Production of 3'-phosphorylated phosphatidylinositides (3 phosphoinositides) was also required for ISO-induced ANF transcription. ISO caused phosphorylation (Ser-473) and activation of Akt through CaM kinase II- and 3 phosphoinositides-dependent mechanisms. Constitutively active Akt increased myocyte surface area, total protein content, and ANF expression, whereas dominant negative Akt blocked ISO-stimulated ANF transcription. ISO caused Ser-9 phosphorylation and decreased activities of GSK3beta. Overexpression of GSK3beta inhibited ANF transcription, which was reversed by ISO. ISO failed to reverse the inhibitory effect of GSK3beta(S9A), an Akt-insensitive mutant. Kinase-inactive GSK3beta increased ANF transcription. Cyclosporin A partially inhibited ISO-stimulated ANF transcription, indicating that calcineurin only partially mediates ANF transcription. These results suggest that both CaM kinase II and 3 phosphoinositides mediate betaAR-induced Akt activation and ANF transcription in cardiac myocytes. Furthermore, betaAR-stimulated ANF transcription is predominantly mediated by activation of Akt and subsequent phosphorylation/inhibition of GSK3beta.

Specific role of the extracellular signal-regulated kinase pathway in angiotensin II-induced cardiac hypertrophy in vitro.

Although MAP (mitogen-activated protein) kinases are implicated in cell proliferation and differentiation in many cell types, the role of MAP kinases in cardiac hypertrophy remains unclear. We examined the role of extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 MAP kinase in angiotensin II (Ang II)-induced hypertrophy compared with phenylephrine-induced hypertrophy in neonatal rat cardiac myocytes. Both Ang II and phenylephrine activated ERKs to a similar extent, whereas phenylephrine caused stronger and more sustained activation of JNK and p38 than Ang II. PD98059, a specific inhibitor of MAPK/ERK kinase (MEK),inhibited Ang II-induced, but not phenylephrine-induced, expression of atrial natriuretic factor (ANF) at both the mRNA and polypeptide levels. SB203580, a specific inhibitor of p38 and some JNK isoforms, did not show significant effects on ANF expression induced by Ang II or phenylephrine. Although PD98059 and dominant-negative MEK1 blocked Ang II-induced activation of the ANF promoter, SB203580 or dominant-negative MEK kinase 1 (MEKK1) showed no effect. Phenylephrine-induced ANF promoter activation was significantly inhibited by SB203580 and dominant-negative MEKK1, but not by PD98059 or dominant-negative MEK1. Dominant-negative Ras inhibited both ERK activation and ANF up-regulation by Ang II, whereas constitutively active forms of Ras and MEK were sufficient to activate the ANF promoter. Dominant-negative Ras also partly inhibited the phenylephrine-induced activation of ANF promoter. PD98059 did not affect other markers of Ang II-induced hypertrophy, such as skeletal alpha-actin and c-fos expression, increases in the rate of protein synthesis or rapid sarcomeric actin organization. These results suggest that Ang II uses ERK for ANF expression, whereas phenylephrine uses other pathways. The Ras/ERK pathway selectively mediates ANF expression in various phenotypes observed in Ang II-induced hypertrophy. The ERK pathway mediates an agonist-specific and phenotype-specific response in cardiac hypertrophy.

Myosin light chain kinase mediates sarcomere organization during cardiac hypertrophy in vitro.

During the development of hypertrophy, cardiac myocytes increase organization of the sarcomere, a highly ordered contractile unit in striated muscle cells. Several hypertrophic agonists, such as angiotensin II, phenylephrine, and endothelin-1, have been shown to promote the sarcomere organization. However, the signaling pathway, which links extracellular stimuli to sarcomere organization, has not been clearly demonstrated. Here, we demonstrate that myosin light chain kinase specifically mediates agonist-induced sarcomere organization during early hypertrophic response. Acute administration of a hypertrophic agonist, phenylephrine, or angiotensin II, causes phosphorylation of myosin light chain 2v both in cultured cardiac myocytes and in the adult heart in vivo. We also show that both sarcomere organization and myosin light chain 2v phosphorylation are dependent on the activation of Ca2+/calmodulin pathway, a known activator of myosin light chain kinase. These results define a new and specific role of myosin light chain kinase in cardiac myocytes, which may provide a rapid adaptive mechanism in response to hypertrophic stimuli.

High efficiency gene transfer by multiple transfection protocol.

A high efficiency transfection protocol employing a common polycationic lipid is described. Using LipofectAMINE, a widely used transfection reagent, we transfected 293T cells with a plasmid harboring the beta-galactosidase (beta-gal) gene. The transfection efficiency was determined by direct staining for X-gal. The conventional transfection protocol achieved an efficiency of <40% while our protocol, which employs the repetition of transfection a few times, achieved an efficiency of approximately 80%. Thus, a dramatic increase in transfection efficiency can be obtained by simply repeating transfection with the use of a common polycationic lipid. This method will be useful in many molecular biological experiments.

Mechanical stretch and angiotensin II differentially upregulate the renin-angiotensin system in cardiac myocytes In vitro.

Pressure overload in vivo results in left ventricular hypertrophy and activation of the renin-angiotensin system in the heart. Mechanical stretch of neonatal rat cardiac myocytes in vitro causes secretion of angiotensin II (Ang II), which in turn plays a pivotal role in mechanical stretch-induced hypertrophy. Although in vivo data suggest that the stimulus of hemodynamic overload serves as an important modulator of cardiac renin-angiotensin system (RAS) activity, it is not clear whether observed upregulation of RAS genes is a direct effect of hemodynamic stress or is secondary to neurohumoral effects in response to hemodynamic overload. Moreover, it is unclear whether activation of the local RAS in response to hemodynamic overload predominantly occurs in cardiac myocytes or fibroblasts or both. In the present study, we examined the effect of mechanical stretch on expression of angiotensinogen, renin, angiotensin-converting enzyme (ACE), and Ang II receptor (AT(1A), AT(1B), and AT(2)) genes in neonatal rat cardiac myocytes and cardiac fibroblasts in vitro. The level of expression of angiotensinogen, renin, ACE, and AT(1A) genes was low in unstretched cardiac myocytes, but stretch upregulated expression of these genes at 8 to 24 hours. Stimulation of cardiac myocytes with Ang II also upregulated expression of angiotensinogen, renin, and ACE genes, whereas it downregulated AT(1A) and did not affect AT(1B) gene expression. Although losartan, a specific AT(1) antagonist, completely inhibited Ang II-induced upregulation of angiotensinogen, renin, and ACE genes, as well as stretch-induced upregulation of AT(1A) expression, it did not block upregulation of angiotensinogen, renin, and ACE genes by stretch. Western blot analyses showed increased expression of angiotensinogen and renin protein at 16 to 24 hours of stretch. The ACE-like activity was also significantly elevated at 24 hours after stretch. Radioligand binding assays revealed that stretch significantly upregulated the AT(1) density on cardiac myocytes. Interestingly, stretch of cardiac fibroblasts did not result in any discernible increases in the expression of RAS genes. Our results indicate that mechanical stretch in vitro upregulates both mRNA and protein expression of RAS components specifically in cardiac myocytes. Furthermore, components of the cardiac RAS are independently and differentially regulated by mechanical stretch and Ang II in neonatal rat cardiac myocytes.

Angiotensin II activates RhoA in cardiac myocytes: a critical role of RhoA in angiotensin II-induced premyofibril formation.

The organization of actin into striated fibers (myofibrils) is one of the major features of cardiac hypertrophy. However, its signal transduction mechanism is not well understood. Although Rho-family small G proteins have been implicated in actin organization in many cell types, it is not fully elucidated whether Rho mediates the organization of actin fibers by hypertrophic stimuli in cardiac myocytes. Therefore, we examined (1) whether Rho is activated by the hypertrophic stimulus, angiotensin II (Ang II), and (2) whether Rho mediates the Ang II-induced organization of actin fibers in cultured neonatal rat cardiac myocytes. Treatment of myocytes with Ang II caused a rapid formation of both striated (mature myofibrils) and nonstriated (premyofibrils) actin fibers within 30 minutes, as determined by phalloidin stainings of the polymerized actin and troponin T stainings. Immunoblot analyses and immunostainings have indicated that cardiac myocytes express RhoA, but RhoB is undetectable. In the control state, RhoA was observed predominantly in the cytosolic fraction, but it was translocated in part to the particulate fraction in response to Ang II, consistent with activation of RhoA by Ang II. Incubation of myocytes with exoenzyme C3 for 48 hours completely ADP-ribosylated Rho in vivo. The C3 treatment abolished formation of premyofibrils induced by Ang II, suggesting that Ang II causes premyofibril formation via a Rho-dependent mechanism. The Ang II-induced mature myofibril formation was only partly abolished by C3. Expression of constitutively active RhoA (V14RhoA) caused the formation of premyofibrils but not mature myofibrils. The C3 treatment inhibited Ang II-induced atrial natriuretic factor induction, whereas it had no effect on c-fos induction. These results indicate that RhoA is activated by Ang II and mediates the Ang II-induced formation of premyofibrils and induction of a subset of genes. Distinct signaling mechanisms seem to be responsible for striated mature myofibril formation by Ang II.

Molecular mechanisms for somatostatin inhibition of c-fos gene expression.

We reported previously that somatostatin inhibits the expression of the immediate early gene c-fos. Accordingly, we characterized the molecular mechanisms by which somatostatin inhibits c-fos gene expression. Because growth factors activate c-fos through a region of its promoter known as the serum response element [SRE; base pairs (bp) -357 to -276] we transfected rat pituitary adenoma cells (GH3) with plasmids containing the SRE or the SRE core fragment (bp -320 to -298) upstream of the luciferase reporter gene. Epidermal growth factor (EGF) stimulated SRE-luciferase activity, and this effect was inhibited by somatostatin and by the analog MK-678. Identical results were obtained with the SRE core plasmid, demonstrating that the sequence between bp -320 and -298 of the c-fos promoter is a somatostatin response element. Because the extracellular signal-regulated protein kinases (ERKs) induce the SRE via phosphorylation of transcription factors such as Elk-1, we examined the effect of somatostatin on ERK phosphorylation and activation. EGF stimulated tyrosine phosphorylation of ERK2, and MK-678 attenuated this effect. In experiments using in-gel kinase assays, MK-678 also inhibited EGF-stimulated ERK activity via a pertussis toxin sensitive pathway, and this effect resulted in inhibition of Elk-1 transcriptional activity. Our data suggest that one mechanism of somatostatin action involves inhibition of ERK activity, Elk-1 phosphorylation and transcriptional activation, and ultimately c-fos gene transcription.

Angiotensin II and serum differentially regulate expression of cyclins, activity of cyclin-dependent kinases, and phosphorylation of retinoblastoma gene product in neonatal cardiac myocytes.

The hypertrophic response in cardiac myocytes and the mitogenic response in other cell types share various early cellular responses. However, how the subsequent cell growth response, such as cell cycle machinery, is regulated in cardiac hypertrophy is not understood. Using cultured neonatal rat cardiac myocytes, we examined the effect of angiotensin II (Ang II), a hypertrophic stimulus, on mRNA and protein expression of cyclins and cyclin-dependent protein kinases (cdks), activity of cdks, and phosphorylation of retinoblastoma gene product (pRb). The effect of FCS, a stimulus that was previously reported to initiate both protein and DNA synthesis in cardiac myocytes, was also examined for comparison. Ang II activated cdk4 and caused phosphorylation of pRb, peaking at 12 hours, but subsequently downregulated cyclin D1, D3, and A expression and cdk2 activity. FCS increased the expression of G1-S cyclins, caused activation of cdk4, cdk2, and cdc2, and strongly phosphorylated pRb but failed to significantly stimulate DNA synthesis in neonatal cardiac myocytes. These results suggest that Ang II transiently activates but subsequently downregulates cell cycle regulators. Induction of G1 and G1-S cyclins and activation of cdks by FCS are not sufficient to drive cardiac myocytes into S phase. The functional role of pRb phosphorylation by Ang II and serum stimulation and, by inference, the subsequent liberation of E2F in terminally differentiated myocytes remain to be elucidated.

Tyrosine kinases mediation of c-fos expression by cell swelling in cardiac myocytes.

We investigated the signal transduction mechanism initiated by hypotonic cell swelling in cardiac myocytes using c-fos expression as a nuclear marker. Treatment of myocytes with hypotonic culture media rapidly induced c-fos expression, whereas hypertonic stress had no effect on it. Extensive pharmacological studies indicated that only tyrosine kinase inhibitors suppressed the hypotonic swelling-induced c-fos expression; down-regulation of protein kinase C or a Ca2+ chelator (EGTA) had no effect. Hypotonic stress immediately (within 5s) increased tyrosine kinase activity. Thus tyrosine kinase is one of the earliest signaling molecules activated by hypotonic stress and plays an essential role in hypotonic swelling-induced c-fos expression. Rapid activation of tyrosine kinase may be a common early signaling mechanism in response to mechanical stresses that increase cell membrane tension.

The cellular and molecular response of cardiac myocytes to mechanical stress.

External load plays a critical role in determining muscle mass and its phenotype in cardiac myocytes. Cardiac myocytes have the ability to sense mechanical stretch and convert it into intracellular growth signals, which lead to hypertrophy. Mechanical stretch of cardiac myocytes in vitro causes activation of multiple second messenger systems that are very similar to growth factor-induced cell signaling systems. Stretch of neonatal rat cardiac myocytes stimulates a rapid secretion of angiotensin II which, together with other growth factors, mediates stretch-induced hypertrophic responses in vitro. In this review, various cell signaling mechanisms initiated by mechanical stress on cardiac myocytes are summarized with emphasis on potential mechanosensing mechanisms and the relationship between mechanical loading and the cardiac renin-angiotensin system.