ER Protein Quality Control and the Unfolded Protein Response in the Heart.

Cardiac myocytes are the cells responsible for the robust ability of the heart to pump blood throughout the circulatory system. Cardiac myocytes grow in response to a variety of physiological and pathological conditions; this growth challenges endoplasmic reticulum-protein quality control (ER-PQC), a major feature of which includes the unfolded protein response (UPR). ER-PQC and the UPR in cardiac myocytes growing under physiological conditions, including normal development, exercise, and pregnancy, are sufficient to support hypertrophic growth of each cardiac myocyte. However, the ER-PQC and UPR are insufficient to respond to the challenge of cardiac myocyte growth under pathological conditions, including myocardial infarction and heart failure. In part, this insufficiency is due to a continual decline in the expression levels of important adaptive UPR components as a function of age and during myocardial pathology. This chapter will discuss the physiological and pathological conditions unique to the heart that involves ER-PQC, and whether the UPR is adaptive or maladaptive under these circumstances.

Kinetics of the translocation and phosphorylation of alphaB-crystallin in mouse heart mitochondria during ex vivo ischemia.

alphaB-crystallin (alphaBC) is a small heat shock protein expressed at high levels in the myocardium where it protects from ischemia-reperfusion damage. Ischemia-reperfusion activates p38 MAP kinase, leading to the phosphorylation of alphaBC on serine 59 (P-alphaBC-S59), enhancing its ability to protect myocardial cells from damage. In the heart, ischemia-reperfusion also causes the translocation of alphaBC from the cytosol to other cellular locations, one of which was recently shown to be mitochondria. However, it is not known whether alphaBC translocates to mitochondria during ischemia-reperfusion, nor is it known whether alphaBC phosphorylation takes place before or after translocation. In the present study, analyses of mitochondrial fractions isolated from mouse hearts subjected to various times of ex vivo ischemia-reperfusion showed that alphaBC translocation to mitochondria was maximal after 20 min of ischemia and then declined steadily during reperfusion. Phosphorylation of mitochondrial alphaBC was maximal after 30 min of ischemia, suggesting that at least in part it occurred after alphaBC association with mitochondria. Consistent with this was the finding that translocation of activated p38 to mitochondria was maximal after only 10 min of ischemia. The overexpression of alphaBC-AAE, which mimics alphaBC phosphorylated on serine 59, has been shown to stabilize mitochondrial membrane potential and to inhibit apoptosis. In the present study, infection of neonatal rat cardiac myocytes with adenovirus-encoded alphaBC-AAE decreased peroxide-induced mitochondrial cytochrome c release. These results suggest that during ischemia alphaBC translocates to mitochondria, where it is phosphorylated and contributes to modulating mitochondrial damage upon reperfusion.

Localization of phosphorylated alphaB-crystallin to heart mitochondria during ischemia-reperfusion.

The cytosolic small heat shock protein alphaB-crystallin (alphaBC) is a molecular chaperone expressed in large quantities in the heart, where it protects from stresses such as ischemia-reperfusion (I/R). Upon I/R, p38 MAP kinase activation leads to phosphorylation of alphaBC on Ser(59) (P-alphaBC-S59), which increases its protective ability. alphaBC confers protection, in part, by interacting with and affecting the functions of key components in stressed cells. We investigated the hypothesis that protection from I/R damage in the heart by P-alphaBC-S59 can be mediated by localization to mitochondria. We found that P-alphaBC-S59 localized to mitochondria isolated from untreated mouse hearts and that this localization increased more than threefold when the hearts were subjected to ex vivo I/R. Mitochondrial P-alphaBC-S59 decreased when hearts were treated with the p38 inhibitor SB-202190. Moreover, SB-202190-treated hearts exhibited more tissue damage and less functional recovery upon reperfusion than controls. I/R activates mitochondrial permeability transition (MPT) pore opening, which increases cell damage. We found that mitochondria incubated with a recombinant mutant form of alphaBC that mimics P-alphaBC-S59 exhibited decreased calcium-induced MPT pore opening. These results indicate that mitochondria may be among the key components in stressed cells with which P-alphaBC-S59 interacts and that this localization may protect the myocardium, in part, by modulating MPT pore opening and, thus, reducing I/R injury.

Sarco/endoplasmic reticulum calcium ATPase-2 expression is regulated by ATF6 during the endoplasmic reticulum stress response: intracellular signaling of calcium stress in a cardiac myocyte model system.

The recently described transcription factor, ATF6, mediates the expression of proteins that compensate for potentially stressful changes in the endoplasmic reticulum (ER), such as reduced ER calcium. In cardiac myocytes the maintenance of optimal calcium levels in the sarcoplasmic reticulum (SR), a specialized form of the ER, is required for proper contractility. The present study investigated the hypothesis that ATF6 serves as a regulator of the expression of sarco/endoplasmic reticulum calcium ATPase-2 (SERCA2), a protein that transports calcium into the SR from the cytoplasm. Depletion of SR calcium in cultured cardiac myocytes fostered the translocation of ATF6 from the ER to the nucleus, activated the promoter for rat SERCA2, and led to increased levels of SERCA2 protein. SERCA2 promoter induction by calcium depletion was partially blocked by dominant-negative ATF6, whereas constitutively activated ATF6 led to SERCA2 promoter activation. Mutation analyses identified a promoter-proximal ER stress-response element in the rat SERCA2 gene that was required for maximal induction by ATF6 and calcium depletion. Although this element was shown to be responsible for all of the effects of ATF6 on SERCA2 promoter activation, it was responsible for only a portion of the effects of calcium depletion. Thus, SERCA2 induction in response to calcium depletion appears to be a potentially physiologically important compensatory response to this stress that involves intracellular signaling pathways that are both dependent and independent of ATF6.

p38 MAPK regulates group IIa phospholipase A2 expression in interleukin-1beta -stimulated rat neonatal cardiomyocytes.

Group IIa phospholipase A(2) (GIIa PLA(2)) is released by some cells in response to interleukin-1beta. The purpose of this study was to determine whether interleukin-1beta would stimulate the synthesis and release of GIIa PLA(2) from cardiomyocytes, and to define the role of p38 MAPK and cytosolic PLA(2) in the regulation of this process. Whereas GIIa PLA(2) mRNA was not identified in untreated cells, exposure to interleukin-1beta resulted in the sustained expression of GIIa PLA(2) mRNA. Interleukin-1beta also stimulated a progressive increase in cellular and extracellular GIIa PLA(2) protein levels and increased extracellular PLA(2) activity 70-fold. In addition, interleukin-1beta stimulated the p38 MAPK-dependent activation of the downstream MAPK-activated protein kinase, MAPKAP-K2. Treatment with the p38 MAPK inhibitor, SB202190, decreased interleukin-1beta stimulated MAPKAP-K2 activity, GIIa PLA(2) mRNA expression, GIIa PLA(2) protein synthesis, and the release of extracellular PLA(2) activity. Infection with an adenovirus encoding a constitutively active form of MKK6, MKK6(Glu), which selectively phosphorylates p38 MAPK, induced cellular GIIa PLA(2) protein synthesis and the release of GIIa PLA(2) and increased extracellular PLA(2) activity 3-fold. In contrast, infection with an adenovirus encoding a phosphorylation-resistant MKK6, MKK6(A), did not result in GIIa PLA(2) protein synthesis or release by unstimulated cardiomyocytes. In addition, infection with an adenovirus encoding MKK6(A) abrogated GIIa PLA(2) protein synthesis and release by interleukin-1beta-stimulated cells. These results provide direct evidence that p38 MAPK activation was necessary for interleukin-1beta-induced synthesis and release of GIIa PLA(2) by cardiomyocytes.

The cytoprotective effects of the glycoprotein 130 receptor-coupled cytokine, cardiotrophin-1, require activation of NF-kappa B.

Many cell types mount elaborate, compensatory responses to stress that enhance survival; however, the intracellular signals that govern these responses are poorly understood. Cardiotrophin-1 (CT-1), a stress-induced cytokine, belongs to the interleukin-6/glycoprotein 130 receptor-coupled cytokine family. CT-1 is released from the heart in response to hypoxic stress, and it protects cardiac myocytes from hypoxia-induced apoptosis, thus establishing a central role for this cytokine in the cardiac stress response. In the present study, CT-1 activated p38 and ERK MAPKs as well as Akt in cultured cardiac myocytes; these three pathways were activated in a parallel manner. CT-1 also induced the degradation of the NF-kappa B cytosolic anchor, I kappa B, as well as the translocation of the p65 subunit of NF-kappa B to the nucleus and increased expression of an NF-kappa B-dependent reporter gene. Inhibitors of the p38, ERK, or Akt pathways each partially reduced CT-1-mediated NF-kappa B activation, as well as the cytoprotective effects of CT-1 against hypoxic stress. Together, the inhibitors completely blocked CT-1-dependent NF-kappa B activation and cytoprotection. A cell-permeable peptide that selectively disrupted NF-kappa B activation also completely inhibited the cytoprotective effects of CT-1. These results indicate that CT-1 signals through p38, ERK, and Akt in a parallel manner to activate NF-kappa B and that NF-kappa B is required for CT-1 to mediate its full cytoprotective effects in cardiac myocytes.

Ras reduces L-type calcium channel current in cardiac myocytes. Corrective effects of L-channels and SERCA2 on [Ca(2+)](i) regulation and cell morphology.

Heart failure is associated with dysregulation of intracellular calcium ([Ca(2+)](i)), reduction in myofibrils, and increased activation of Ras, a regulator of signal-transduction pathways. To evaluate the potential effects of Ras on [Ca(2+)](i), we expressed constitutively active Ras (Ha-Ras(V12)) in cardiac myocytes and monitored [Ca(2+)](i) via fluorescence and electrophysiological techniques. Ha-Ras(V12) reduced the magnitude of the contractile calcium transients. Unexpectedly, however, calcium loading of the sarcoplasmic reticulum was increased, suggesting that Ha-Ras(V12) introduces a defect in excitation-calcium release coupling. Consistent with this idea, L-channel calcium currents were reduced by Ha-Ras(V12), which also downregulated the activity of the L-channel gene promoter. Coexpression of L-channels and SERCA2 largely corrected Ha-Ras(V12)-induced dysregulation of [Ca(2+)](i). Furthermore, whereas Ha-Ras(V12) downregulated myofibrils, this effect was blocked by coexpression of L-channels. These results suggest that Ras downregulates L-channel expression, which may play a pathophysiological role in cardiac disease.

Expression and characterization of Edg-1 receptors in rat cardiomyocytes: calcium deregulation in response to sphingosine 1-phosphate.

Recent evidence indicates that sphingolipids are produced by the heart during hypoxic stress and by blood platelets during thrombus formation. It is therefore possible that sphingolipids may influence heart cell function by interacting with G-protein-coupled receptors of the Edg family. In the present study, it was found that sphingosine 1-phosphate (Sph1P), the prototypical ligand for Edg receptors, produced calcium overload in rat cardiomyocytes. The cDNA for Edg-1 was cloned from rat cardiomyocytes and, when transfected in an antisense orientation, effectively blocked Edg-1 protein expression and reduced the Sph1P-mediated calcium deregulation. Taken together, these results demonstrate that cardiomyocytes express an extracellular lipid-sensitive receptorsystem that can respond to sphingolipid mediators. Because the major source of Sph1P is from blood platelets, we speculate that Edg-mediated Sph1P negative inotropic and cardiotoxic effects may play important roles in acute myocardial ischemia where Sph1P levels are probably elevated in response to thrombus.

The Rho effector, PKN, regulates ANF gene transcription in cardiomyocytes through a serum response element.

The low-molecular-weight GTP-binding protein RhoA mediates hypertrophic growth and atrial natriuretic factor (ANF) gene expression in neonatal rat ventricular myocytes. Neither the effector nor the promoter elements through which Rho exerts its regulatory effects on ANF gene expression have been elucidated. When constitutively activated forms of Rho kinase and two protein kinase C-related kinases, PKN (PRK1) and PRK2, were compared, only PKN generated a robust stimulation of a luciferase reporter gene driven by a 638-bp fragment on the ANF promoter. This ANF promoter fragment contains a proximal serum response element (SRE) and an Sp-1-like element required for the transcriptional response to phenylephrine (PE). This response was inhibited by dominant negative Rho. The ability of dominant negative Rho to inhibit the response to PE and the ability of PKN to stimulate ANF reporter gene expression were both lost when the SRE was mutated. Mutation of the Sp-1-like element also attenuated the response to PKN. A minimal promoter driven by ANF SRE sequences was sufficient to confer Rho- and PKN-mediated gene expression. Interestingly, PKN preferentially stimulated the ANF versus the c-fos SRE reporter gene. Thus PKN and Rho are able to regulate transcriptional activation of the ANF SRE by a common element that could implicate PKN as a downstream effector of Rho in transcriptional responses associated with hypertrophy.

alpha B-crystallin gene induction and phosphorylation by MKK6-activated p38. A potential role for alpha B-crystallin as a target of the p38 branch of the cardiac stress response.

The MAPK kinase MKK6 selectively stimulates p38 MAPK and confers protection against stress-induced apoptosis in cardiac myocytes. However, the events lying downstream of p38 that mediate this protection are unknown. The small heat shock protein, alphaB-crystallin, which is expressed in only a few cell types, including cardiac myocytes, may participate in MKK6-mediated cytoprotection. In the present study, we showed that, in cultured cardiac myocytes, expression of MKK6(Glu), an active form of MKK6, led to p38-dependent increases in alphaB-crystallin mRNA, protein, and transcription. MKK6(Glu) also induced p38-dependent activation of the downstream MAPK-activated protein kinase, MAPKAP-K2, and the phosphorylation of alphaB-crystallin on serine-59. Initially, exposure of cells to the hyperosmotic stressor, sorbitol, stimulated MKK6, p38, and MAPKAP-K2 and increased phosphorylation of alphaB-crystallin on serine 59. However, after longer times of exposure to sorbitol, the cells began to undergo apoptosis. This sorbitol-induced apoptosis was increased when p38 was inhibited in a manner that would block alphaB-crystallin induction and phosphorylation. Thus, under these conditions, the activation of MKK6, p38, and MAPKAP-K2 by sorbitol can provide a degree of protection against stress-induced apoptosis. Supporting this view was the finding that sorbitol-induced apoptosis was nearly completely blocked in cells expressing MKK6(Glu). Therefore, the cytoprotective effects of MKK6 in cardiac myocytes are due, in part, to phosphorylation of alphaB-crystallin on serine 59 and to the induction of alphaB-crystallin gene expression.

p38 MAPK and NF-kappa B collaborate to induce interleukin-6 gene expression and release. Evidence for a cytoprotective autocrine signaling pathway in a cardiac myocyte model system.

In cardiac myocytes, the stimulation of p38 MAPK by the MAPKK, MKK6, activates the transcription factor, NF-kappaB, and protects cells from apoptosis. In the present study in primary neonatal rat cardiac myocytes, constitutively active MKK6, MKK6(Glu), bound to IkappaB kinase (IKK)-beta and stimulated its abilities to phosphorylate IkappaB and to activate NF-kappaB. MKK6(Glu) induced NF-kappaB-dependent interleukin (IL)-6 transcription and IL-6 release in a p38-dependent manner. IL-6 protected myocardial cells against apoptosis. Like IL-6, TNF-alpha, which activates both NF-kappaB and p38, also induced p38-dependent IL-6 expression and release and protected myocytes from apoptotis. While TNF-alpha was relatively ineffective, IL-6 activated myocardial cell STAT3 by about 8-fold, indicating a probable role for this transcription factor in IL-6-mediated protection from apoptosis. TNF-alpha-mediated IL-6 induction was inhibited by a kinase-inactive form of the MAPKKK, TGF-beta activated protein kinase (Tak1), which is known to activate p38 and NF-kappaB in other cell types. Thus, by stimulating both p38 and NF-kappaB, Tak1-activating cytokines, like TNF-alpha, can induce IL-6 expression and release. Moreover, the myocyte-derived IL-6 may then function in an autocrine and/or paracrine fashion to augment myocardial cell survival during stresses that activate p38.

p38 Mitogen-activated protein kinase mediates the transcriptional induction of the atrial natriuretic factor gene through a serum response element. A potential role for the transcription factor ATF6.

In various cell types certain stresses can stimulate p38 mitogen-activated protein kinase (p38 MAPK), leading to the transcriptional activation of genes that contribute to appropriate compensatory responses. In this report the mechanism of p38-activated transcription was studied in cardiac myocytes where this MAPK is a key regulator of the cell growth and the cardiac-specific gene induction that occurs in response to potentially stressful stimuli. In the cardiac atrial natriuretic factor (ANF) gene, a promoter-proximal serum response element (SRE), which binds serum response factor (SRF), was shown to be critical for ANF induction in primary cardiac myocytes transfected with the selective p38 MAPK activator, MKK6 (Glu). This ANF SRE does not possess sequences typically required for the binding of the Ets-related ternary complex factors (TCFs), such as Elk-1, indicating that p38-mediated induction through this element may take place independently of such TCFs. Although p38 did not phosphorylate SRF in vitro, it efficiently phosphorylated ATF6, a newly discovered SRF-binding protein that is believed to serve as a co-activator of SRF-inducible transcription at SREs. Expression of an ATF6 antisense RNA blocked p38-mediated ANF induction through the ANF SRE. Moreover, when fused to the Gal4 DNA-binding domain, an N-terminal 273-amino acid fragment of ATF6 was sufficient to support trans-activation of Gal4/luciferase expression in response to p38 but not the other stress kinase, N-terminal Jun kinase (JNK); p38-activating cardiac growth promoters also stimulated ATF6 trans-activation. These results indicate that through ATF6, p38 can augment SRE-mediated transcription independently of Ets-related TCFs, representing a novel mechanism of SRF-dependent transcription by MAP kinases.

The Raf-MEK-ERK cascade represents a common pathway for alteration of intracellular calcium by Ras and protein kinase C in cardiac myocytes.

Ras and protein kinase C (PKC), which regulate the Raf-MEK-ERK cascade, may participate in the development of cardiac hypertrophy, a condition characterized by diminished and prolonged contractile calcium transients. To directly examine the influence of this pathway on intracellular calcium ([Ca2+]i), cardiac myocytes were cotransfected with effectors of this pathway and with green fluorescent protein, which allowed the living transfected myocytes to be identified and examined for [Ca2+]i via indo-1. Transfection with constitutively active Ras (Ha-RasV12) increased cell size, decreased expression of the myofibrils and the calcium-regulatory enzyme SERCA2, and reduced the magnitude and prolonged the decay phase of the contractile [Ca2+]i transients. Similar effects on [Ca2+]i were obtained with Ha-RasV12S35, a Ras mutant that selectively couples to Raf, and with constitutively active Raf. In contrast, Ha-RasV12C40, a Ras mutant that activates the phosphatidylinositol 3-kinase pathway, had a lesser effect. The PKC-activating phorbol ester, phorbol 12-myristate 13-acetate, also prolonged the contractile [Ca2+]i transients. Cotransfection with dnMEK inhibited the effects of Ha-RasV12, Raf, and phorbol 12-myristate 13-acetate on [Ca2+]i. The effects of Ha-RasV12 and Raf on [Ca2+]i were also counteracted by SERCA2 overexpression. Both Ras and PKC may thus regulate cardiac [Ca2+]i via the Raf-MEK-ERK cascade, and this pathway may represent a critical determinant of cardiac physiological function.

Beta1 integrins participate in the hypertrophic response of rat ventricular myocytes.

Multiple signaling pathways have been implicated in the hypertrophic response of ventricular myocytes, yet the importance of cell-matrix interactions has not been extensively examined. Integrins are cell-surface molecules that link the extracellular matrix to the cellular cytoskeleton. They can function as cell signaling molecules and transducers of mechanical information in noncardiac cells. Given these properties and their abundance in cardiac cells, we evaluated the hypothesis that beta1 integrin function is involved in the alpha1-adrenergic mediated hypertrophic response of neonatal rat ventricular myocytes. The hypertrophic response of this model required interaction with extracellular matrix proteins. Specificity of these results was confirmed by demonstrating that ventricular myocytes plated onto an anti-beta1 integrin antibody supported the hypertrophic gene response. Adenovirus-mediated overexpression of beta1 integrin augmented the myocyte hypertrophic response when assessed by protein synthesis and atrial natriuretic factor production, a marker gene of hypertrophic induction. DNA synthesis was not altered by integrin overexpression. Transfection of cultured cardiac myocytes with either the ubiquitously expressed beta1A integrin or the cardiac/skeletal muscle-specific beta1 isoform (beta1D) activated reporter expression from both the atrial natriuretic factor and myosin light chain-2 ventricular promoters, genetic markers of ventricular cell hypertrophy. Finally, suppression of integrin signaling by overexpression of free beta1 integrin cytoplasmic domains inhibited the adrenergically mediated atrial natriuretic factor response. These findings show that integrin ligation and signaling are involved in the cardiac hypertrophic response pathway.

MKK6 activates myocardial cell NF-kappaB and inhibits apoptosis in a p38 mitogen-activated protein kinase-dependent manner.

In cardiac myocytes the stimulation of p38 mitogen-activated protein kinase activates a hypertrophic growth program and the induction of the cardiac-specific genes associated with this program. This study focused on determining whether these novel growth-promoting effects are accompanied by the p38-mediated inhibition of apoptosis, and if so, what signaling pathways might be responsible. Primary neonatal rat ventricular myocytes were driven into apoptosis by treatments known to induce apoptosis in other cell types, e.g. incubation with anisomycin or overexpression constitutively active MEKK-1 (MEKK-1COOH), a protein that strongly activates extracellular signal-regulated kinase and N-terminal c-Jun kinase, but not p38. Overexpression of constitutively active MKK6, MKK6 (Glu), which selectively activates p38 in cardiac myocytes, protected cells from either anisomycin- or MEKK-1COOH-induced apoptosis. This protection was blocked by SB 203580, a selective p38 inhibitor. MKK6 (Glu) also activated transcription mediated by NF-kappaB, a factor which protects other cell types from apoptosis. The activation of NF-kappaB and the protection from apoptosis mediated by MKK6 (Glu) were both blocked by SB 203580. Interestingly, overexpression of a mutant form of I-kappaBalpha, which inhibits nuclear translocation of NF-kappaB, completely blocked MKK6 (Glu)-activated NF-kappaB but had little effect on MKK6s anti-apoptotic effects. These findings suggest that, in part, the overexpression of MKK6 (Glu) may foster growth and survival of cardiac myocytes by protecting them from apoptosis in a p38-dependent manner. Additionally, while NF-kappaB is activated in myocardial cells by p38, this does not appear to be the major mechanism by which MKK6 (Glu) exerts its anti-apoptotic effects in this cell type, suggesting a novel pathway for p38-mediated protection from apoptosis.

LPS-induced TNF-alpha release from and apoptosis in rat cardiomyocytes: obligatory role for CD14 in mediating the LPS response.

The bacterial endotoxin lipopolysaccharide (LPS) contributes to the cardiovascular collapse and death observed in patients with sepsis. Because LPS has such profound effects on cardiac performance, we speculate that direct effects of LPS could be demonstrated on cardiomyocytes in culture, and that these direct effects are mediated by the LPS receptor, CD14. Accordingly, in this study, we provide evidence for CD14-dependent cardiotoxic effects of LPS including the LPS-stimulated secretion of tumor necrosis factor alpha (TNF-alpha) from cardiomyocytes. TNF-alpha is an inflammatory cytokine which is known for its negative inotropic effects on cardiac performance, but has not until recently been shown to be produced by cardiac cells. In this study, LPS was found to stimulate strongly in a dose-dependent manner the secretion of TNF-alpha from cultured adult rat cardiomyocytes. Further, LPS-induced TNF-alpha secretion was blocked by an inhibitor of TNF-alpha processing, metallomatrix protease inhibitor (TAPI). Molecular and immunological evidence demonstrated the presence of LPS receptors (CD14) on cardiomyocytes. Attenuated TNF-alpha secretion following PI-PLC treatment confirmed the functional importance of CD14 for LPS-mediated myocardial effects. Importantly, LPS also triggered apoptosis in cultured cardiomyocytes as quantified by single-cell gel electrophoresis of nuclei exhibiting DNA fragmentation patterns characteristic of apoptosis (i.e. cardiac comets). Apoptotic cell death was blocked by pre-incubation with the soluble TNF-alpha receptor fragment (TNFRII:Fc), suggesting that LPS-induced apoptosis was TNF-alpha-dependent and probably involved an autocrine function for the TNF-alpha whose secretion was under LPS control. The results of this study suggest that the cardiodepressant effects of LPS are dependent on CD14 signaling and may not only be due to acute negative inotropic effects of TNF-alpha but also may be complicated by TNF-alpha-induced apoptotic cell death which effectively reduces the number of working myocardial cells.

A role for the p38 mitogen-activated protein kinase pathway in myocardial cell growth, sarcomeric organization, and cardiac-specific gene expression.

Three hallmark features of the cardiac hypertrophic growth program are increases in cell size, sarcomeric organization, and the induction of certain cardiac-specific genes. All three features of hypertrophy are induced in cultured myocardial cells by alpha1- adrenergic receptor agonists, such as phenylephrine (PE) and other growth factors that activate mitogen- activated protein kinases (MAPKs). In this study the MAPK family members extracellular signal-regulated kinase (ERK), c-jun NH2-terminal kinase (JNK), and p38 were activated by transfecting cultured cardiac myocytes with constructs encoding the appropriate kinases possessing gain-of-function mutations. Transfected cells were then analyzed for changes in cell size, sarcomeric organization, and induction of the genes for the A- and B-type natriuretic peptides (NPs), as well as the alpha-skeletal actin (alpha-SkA) gene. While activation of JNK and/or ERK with MEKK1COOH or Raf-1 BXB, respectively, augmented cell size and effected relatively modest increases in NP and alpha-SkA promoter activities, neither upstream kinase conferred sarcomeric organization. However, transfection with MKK6 (Glu), which specifically activated p38, augmented cell size, induced NP and alpha-Ska promoter activities by up to 130-fold, and elicited sarcomeric organization in a manner similar to PE. Moreover, all three growth features induced by MKK6 (Glu) or PE were blocked with the p38-specific inhibitor, SB 203580. These results demonstrate novel and potentially central roles for MKK6 and p38 in the regulation of myocardial cell hypertrophy.

Collaborative roles for c-Jun N-terminal kinase, c-Jun, serum response factor, and Sp1 in calcium-regulated myocardial gene expression.

Electrical stimulation of contractions (pacing) of primary neonatal rat ventricular myocytes increases intracellular calcium and activates a hypertrophic growth program that includes expression of the cardiac-specific gene, atrial natriuretic factor (ANF). To investigate the mechanism whereby pacing increases ANF, pacing was tested for its ability to regulate mitogen-activated protein kinase family members, ANF promoter activity, and the trans-activation domain of the transcription factor, Sp1. Pacing and the calcium channel agonist BAYK 8644 activated c-Jun N-terminal kinase (JNK) but not extracellular signal-regulated kinase. Pacing stimulated ANF-promoter activity approximately 10-fold. Furthermore, transfection with an expression vector for c-Jun, a substrate for JNK, also activated the ANF promoter, and the combination of pacing and c-Jun was synergystic, consistent with roles for JNK and c-Jun in calcium-activated ANF expression. Proximal serum response factor and Sp1 binding sites were required for the effects of pacing or c-Jun on the ANF promoter. Pacing and c-Jun activated a GAL4-Sp1 fusion protein by 3- and 12-fold, respectively, whereas the two stimuli together activated GAL4-Sp1 synergistically, similar to their effect on the ANF promoter. Transfection with an expression vector for c-Fos inhibited the effects of c-Jun, suggesting that c-Jun acts independently of AP-1. These results demonstrate an interaction between c-Jun and Sp1 and are consistent with a novel mechanism of calcium-mediated transcriptional activation involving the collaborative actions of JNK, c-Jun, serum response factor, and Sp1.

Differential effects of protein kinase C, Ras, and Raf-1 kinase on the induction of the cardiac B-type natriuretic peptide gene through a critical promoter-proximal M-CAT element.

The cardiac genes for the A- and B-type natriuretic peptides (ANP and BNP) are coordinately induced by growth promoters, such as alpha1-adrenergic receptor agonists (e.g. phenylephrine (PE)). Although inducible elements in the ANP gene have been identified, responsible elements in the BNP gene are unknown. In this study, reporter constructs transfected into neonatal rat ventricular myocytes showed that in the context of 2.5 kilobase pairs of native BNP 5'-flanking sequences, a 2-base pair mutation in a promoter-proximal M-CAT site (CATTCT) disrupted basal and PE-inducible transcription by more than 98%. Expression of constitutively active forms of Ras, Raf-1 kinase, and protein kinase C, all of which are activated by PE in cardiac myocytes, strongly stimulated BNP reporter expression. Isolated M-CAT elements conferred PE, protein kinase C, and Ras inducibility to a minimal BNP promoter, however, they did not confer Raf-1 inducibility. These results show that M-CAT elements can serve as targets for Ras-dependent, Raf-1-independent pathways, implying the involvement of c-Jun N-terminal kinase and/or p38 mitogen-activated protein kinases, but not extracellular signal-regulated protein kinase/mitogen-activated protein kinase. Moreover, the essential M-CAT element distinguishes the BNP gene from the ANP gene, which utilizes serum response elements and an Sp1-like sequence.

Sphingosylphosphocholine modulates the ryanodine receptor/calcium-release channel of cardiac sarcoplasmic reticulum membranes.

Sphingosylphosphocholine (SPC) modulates Ca2+ release from isolated cardiac sarcoplasmic reticulum membranes; 50 microM SPC induces the release of 70 80% of the accumulated calcium. SPC release calcium from cardiac sarcoplasmic reticulum through the ryanodine receptor, since the release is inhibited by the ryanodine receptor channel antagonists ryanodine. Ruthenium Red and sphingosine. In intact cardiac myocytes, even in the absence of extracellular calcium. SPC causes a rise in diastolic Ca2+, which is greatly reduced when the sarcoplasmic reticulum is depleted of Ca2+ by prior thapsigargin treatment. SPC action on the ryanodine receptor is Ca(2+)-dependent. SPC shifts to the left the Ca(2+)-dependence of [3H]ryanodine binding, but only at high pCa values, suggesting that SPC might increase the sensitivity to calcium of the Ca(2+)-induced Ca(2+)-release mechanism. At high calcium concentrations (pCa 4.0 or lower), where [3H]ryanodine binding is maximally stimulated, no effect of SPC is observed. We conclude that SPC releases calcium from cardiac sarcoplasmic reticulum membranes by activating the ryanodine receptor and possibly another intracellular Ca(2+)-release channel, the sphingolipid Ca(2+)-release-mediating protein of endoplasmic reticulum (SCaMPER) [Mao, Kim, Almenoff, Rudner, Kearney and Kindman (1996) Proc.Natl.Acad.Sci. U.S.A 93, 1993-1996], which we have identified for the first time in cardiac tissue.