Yasuchika Takeishi

Transgenic Overexpression of Constitutively Active Protein Kinase C ε Causes Concentric Cardiac Hypertrophy

Abstract—To test the hypothesis that activation of the protein kinase C (PKC) e isoform leads to cardiac hypertrophy without failure, we studied transgenic mice with cardiac-specific overexpression...

Responses of Cardiac Protein Kinase C Isoforms to Distinct Pathological Stimuli Are Differentially Regulated

Currently at least 11 protein kinase C (PKC) isoforms have been identified and may play different roles in cell signaling pathways leading to changes in cardiac contractility, the hypertrophic response, and tolerance to myocardial ischemia. The purpose of the present study was to test the hypothesis that responses of individual PKC isoforms to distinct pathological stimuli were differentially regulated in the adult guinea pig heart. Isolated hearts were perfused by the Langendorff method and were exposed to ischemia, hypoxia, H(2)O(2), or angiotensin II. Hypoxia and ischemia induced translocation of PKC isoforms alpha, beta(2), gamma, and zeta, and H(2)O(2) translocated PKC isoforms alpha, beta(2), and zeta. Angiotensin II produced translocation of alpha, beta(2), epsilon, gamma, and zeta isoforms. Inhibition of phospholipase C with tricyclodecan-9-yl-xanthogenate (D609) blocked hypoxia-induced (alpha, beta(2), and zeta) and angiotensin II-induced (alpha, beta(2), gamma, and zeta) translocation of PKC isoforms. Inhibition of tyrosine kinase with genistein blocked translocation of PKC isoforms by hypoxia (beta(2) and zeta) and by angiotensin II (beta(2)). By contrast, neither D609 nor genistein blocked H(2)O(2)-induced translocation of any PKC isoform. We conclude that hypoxia-induced activation of PKC isoforms is mediated through pathways involving phospholipase C and tyrosine kinase, but oxidative stress may activate PKC isoforms independently of Galphaq-phospholipase C coupling and tyrosine kinase signaling. Because oxidative stress may directly activate PKC, and PKC activation appears to be involved in human heart failure, selective inhibition of the PKC isoforms may provide a novel therapeutic strategy for the prevention and treatment of this pathological process.

Activation of mitogen-activated protein kinases and p90 ribosomal S6 kinase in failing human hearts with dilated cardiomyopathy.

OBJECTIVE A new member of the MAP kinase family, big MAP kinase-1 (BMK1), has been recently identified to promote cell growth and attenuate apoptosis. P90 ribosomal S6 kinase (p90RSK), one of the potentially important substrates of extracellular signal regulated kinase (ERK), regulates gene expression in part via phosphorylation of CREB and the Na(+)/H(+) exchanger. Recently, we have demonstrated that the activity of BMK1, Src (the upstream regulator of BMK1) and p90RSK was increased in hypertrophied myocardium induced by pressure-overload in the guinea pig. However, the abundance and activity of these kinases in human hearts are unknown. METHODS In addition to the three classical MAP kinases (ERK, p38 kinase, and c-Jun NH(2)-terminal kinase (JNK)), we examined the protein expression and activity of Src, BMK1, and p90RSK in explanted hearts from patients with dilated cardiomyopathy (n=9). Normal donor hearts, which were not suitable for transplant for technical reasons, were used as controls (n=5). RESULTS There were no significant differences in the levels of protein expression of these kinases between normal and failing hearts. ERK1/2 and p90RSK were activated in heart failure compared to control (P<0.01 and P<0.03, respectively), while the activity of p38 kinase was decreased (P<0.05) and the activity of JNK was unchanged in heart failure. By contrast, the activities of Src and BMK1 were significantly reduced in end-stage heart failure compared to normal donor hearts (P<0.05). CONCLUSION These data suggest that multiple MAP kinases, p90RSK, and Src are differentially regulated in human failing myocardium of patients with idiopathic dilated cardiomyopathy and may be involved in the pathogenesis of this complex disease.

Differential Regulation of p90 Ribosomal S6 Kinase and Big Mitogen–Activated Protein Kinase 1 by Ischemia/Reperfusion and Oxidative Stress in Perfused Guinea Pig Hearts

Reactive oxygen species (ROS) activate members of the Src kinase and mitogen-activated protein kinase superfamily, including big mitogen-activated protein kinase 1 (BMK1) and extracellular signal-regulated kinases (ERK1/2). A potentially important downstream effector of ERK1/2 is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth through the activation of several transcription factors, as well as the Na(+)/H(+) exchanger. Previously, we showed that Src regulates BMK1 via a redox-sensitive signaling pathway. Because ROS are generated during ischemia and reperfusion after ischemia, we assessed the effects of these stimuli (H(2)O(2), ischemia, and reperfusion) in the activation of ERK1/2, p90RSK, Src, and BMK1 in perfused guinea pig hearts. H(2)O(2) (100 micromol/L) significantly activated all kinases. Ischemia alone stimulated p90RSK, Src, and BMK1 but not ERK1/2. These results suggest that p90RSK activation through ischemia occurs via a pathway other than ERK1/2. A role of Src in ischemia-mediated BMK1 activation was demonstrated through inhibition with the Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. Reperfusion after ischemia stimulated both p90RSK and ERK1/2. In contrast, although ROS increase during reperfusion after ischemia, the activities of both BMK1 and its upstream regulator, Src, were markedly attenuated by reperfusion after ischemia. The activation of C-terminal Src kinase during ischemia but not during reperfusion suggests that the attenuation of Src and BMK1 activity by reperfusion was not regulated by C-terminal Src kinase activity. The antioxidant N-2-mercaptopropionylglycine completely inhibited ERK1/2 and p90RSK activation by reperfusion but only partially inhibited ischemia-induced Src and BMK1 activation. The present study is the first to show the coregulation of Src and BMK1 by reperfusion after ischemia, which we propose to occur via a novel, ROS-independent pathway.

PKC translocation without changes in Gαq and PLC-β protein abundance in cardiac hypertrophy and failure

Activation of protein kinase C (PKC) has been implicated as playing a key role in the pathogenesis of cardiac hypertrophy. This study investigates the response of several signal transduction proteins responsible for PKC activation during the transition from compensated pressure-overload hypertrophy (POH) to congestive heart failure (CHF). Pressure overload was produced on male, adult, Hartley strain guinea pigs using a ligature around the descending thoracic aorta. Sham-operated controls, POH, and CHF groups were identified based on left ventricular hypertrophy, pulmonary congestion, and isolated heart Langendorff mechanics. Quantitative immunoblotting revealed phospholipase C (PLC)-βI and Gαq were unchanged during POH and CHF, as were RGS2, RGS3, and RGS4 (regulators of G protein signaling, which are activators of intrinsic GTPase activity). Translocation of PKC-α, -ε, and -γ from cytosolic to membranous fractions were significantly increased during POH and CHF. Cytosolic PKC activity was also elevated during POH. We conclude that differential PKC activation may be mediated by increases in Gαq and PLC-βI activity rather than upregulation of expression.

Signal transduction during cardiac hypertrophy: the role of Gαq, PLC βI, and PKC

Time for primary review 34 days. Currently the precise biochemical pathogenesis of cardiac hypertrophy remains unclear. A great deal of investigation has been directed toward the examination of various signal transduction pathways that are thought to be involved in stimulating this process. In particular, the signaling pathways that use heterotrimeric G protein-coupled receptors have been the focus for many investigations. This review will focus on the function of the Gαq family of proteins and its downstream effectors during cardiac hypertrophy. Hypertrophy is the final common pathway for a variety of insults to the normal cardiovascular system. Many mechanical and hormonal stimuli such as hypertension, valve disorders, and ischemic events, can produce a hypertrophic response. These pathologic stimuli cause an increase in the workload placed upon the heart resulting in hypertrophy and remodeling that has been observed at the myocyte [1] and the gross anatomical level. Current thought suggests that the hypertrophic response is a compensatory mechanism that allows normal cardiac function against a gradient of increasing workload. If the pathologic stimulus is sufficiently intense or prolonged, a period of failure characterized by impaired function, dilation of the left ventricle, and pulmonary congestion ensues. Recently, experiments using cardiac specific transgenesis to investigate G protein-mediated mechanisms of cardiac hypertrophy have yielded novel information that may give us cause to re-evaluate our traditional stratification of hypertrophic states [2]. It is important to evaluate these results along with other conventional and transgenic models so that we can develop a more comprehensive understanding of this complex disease. Heterotrimeric G protein-coupled receptors serve to convey an extracellular biochemical signal to intracellular effectors. These receptors are heptahelical structures with extracellular, transmembrane, and intracellular domains coupled to specific G proteins which are comprised of three (α, β, γ) subunits. When the G protein complex is in … * Corresponding author. Tel.: 1-216-844-3293; fax: 1-216-844-3145 raw19{at}po.cwru.edu

Role of p90 Ribosomal S6 Kinase (p90RSK) in Reactive Oxygen Species and Protein Kinase C β (PKC-β)-mediated Cardiac Troponin I Phosphorylation

Protein kinase C (PKC)-induced phosphorylation of cardiac troponin I (cTnI) depresses the acto-myosin interaction and may be important during the progression of heart failure. Although both PKCβII and PKCϵ can phosphorylate cTnI, only PKCβ expression and activity are elevated in failing human myocardium during end-stage heart failure. Furthermore, although increased cTnI phosphorylation was observed in mice with cardiac-specific PKCβ II overexpression, no differences were observed in cTnI phosphorylation status between wild type and cardiac-specific PKCϵ overexpression mice. A potentially important downstream effector of PKCs is p90 ribosomal S6 kinase (p90RSK), which plays an important role in cell growth by activating several transcription factors as well as Na+/H+ exchanger. Since both Ser23 and Ser24 of cTnI are contained in putative consensus sequences of p90RSK phosphorylation sites, we hypothesized that p90RSK is downstream from PKCβ II and can be a cTnI (Ser23/24) kinase. p90RSK, but not ERK1/2 activation, was increased in PKCβII overexpression mice but not in PKCϵ overexpression mice. p90RSK could phosphorylate cTnI in vitro with high substrate affinity but not cardiac troponin T (cTnT). To confirm the role of p90RSK in cTnI phosphorylation in vivo, we generated adenovirus containing a dominant negative form of p90RSK (Ad-DN-p90RSK). We found that the inhibition of p90RSK prevented H2O2-mediated cTnI (Ser23/24) phosphorylation but not ERK1/2 and PKCα/βII activation. Next, we generated cardiac-specific p90RSK transgenic mice and observed that cTnI (Ser23/24) phosphorylation was significantly increased. LY333,531, a specific PKCβ inhibitor, inhibited both p90RSK and cTnI (Ser23/24) phosphorylation by H2 O2. Taken together, our data support a new redox-sensitive mechanism regulating cTnI phosphorylation in cardiomyocytes.

Protein Kinase C Activation in Cardiac Hypertrophy and Failure

It is clear from studies using neonatal rat ventricular myocytes that mechanical deformation activates the Gαq-phospholipase C signaling pathway and recapitulates the fetal gene program, followed by an increase in protein synthesis. In adult guinea pig heart, stretch stimulates phosphatidylinositol hydrolysis and translocation of protein kinase C (PKC). Cardiac-specific overexpression of the Gαq and PKCβ2 in transgenic mice demonstrate a gene-dose-dependent induction of cardiac hypertrophy and contractile depression. PKCβ2-mediated phosphorylation of cardiac troponin I may decrease myofilament responsiveness to calcium and thus cause cardiomyocyte dysfunction. Furthermore, in failed human hearts, the expression and activity of PKCα and PKCβ are elevated. These results suggest a critical role of phospholipase C-PKC signaling in cardiac hypertrophy and heart failure. Treatment of the PKCβ2 transgenic mouse with a highly selective inhibitor of the PKCβ largely prevents and/or reverses the phenotype. Thus, selective inhibition of the PKCβ isoform may provide a novel therapeutic strategy for the prevention and treatment of congestive heart failure.