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PKCzeta-mediated GalphaQ stimulation of the ERK5 pathway plays a key role in cardiac hypertrophy
ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S37–S54
MitoKATP, H2O2, and PKCε – completing the cycle Alexandre D. Costa, Keith D. Garlid. Dept. Biology, Portland State University, Portland OR, USA Activation of protein kinase C epsilon (PKCε), opening of mitochondrial KATP channels (mitoKATP), and increase in H2O2 generation by mitochondria have been shown to be key events in the signaling pathways that underlie cardioprotective mechanisms. However, the interactions among these and other components of the signaling cascades are not fully understood. We have shown that mitoKATP is activated by cGMPdependent protein kinase (PKG) via a mitochondrial PKCε that is closely associated with mitoKATP, which we designate PKCε1. MitoKATP opening causes an increase in H2O2 production by complex I of the respiratory chain. In this study, we investigate the relationship between PKCε1, mitoKATP, and the H2O2 generated by mitoKATP activity. We show that the outer membrane is essential for PKG-induced mitoKATP opening and that PKCε1 is located on the outer side of the inner membrane. PKCε activators such as H2O2, phorbol ester, or the peptide ψεRACK were able to induce mitoKATP opening in isolated heart mitochondria. H2O2- or NO-induced mitoKATP opening was inhibited by mitoKATP or PKCε inhibitors, but not by cyclosporin A. mitoKATP opening induced by PKG, PMA or diazoxide was not sensitive to MPG, showing that H2O2 does not act directly to open mitoKATP. We show that the mitoKATP-generated H2O2 activates PKCε1 and induces mitoKATP opening in vitro and in vivo. This mitoKATP-dependent mitoKATP opening constitutes a positive feedback loop that maintains the channel open after the stimulus is no longer present. These results suggest that mitoKATP might be a key player in the maintenance of the preconditioning memory effect. Keywords: Mitochondria; Signal transduction; Free radicals doi:10.1016/j.yjmcc.2007.03.126
PKCzeta-mediated GalphaQ stimulation of the ERK5 pathway plays a key role in cardiac hypertrophy Carlota García-Hoz, Michel Herranz, M. Teresa Díaz-Meco, Jorge Moscat, Catalina Ribas, Federico Mayor, Jr. Centro de Biología Molecular, Universidad Autónoma de Madrid, Spain Gq-coupled G protein-coupled receptors (GPCR) mediate the actions of a variety of messengers that are key regulators of cardiovascular function, such as norepinephrine angiotensin or endothelin. Enhanced Gq-mediated signaling has been implicated in cardiac hypertrophy and in the transition to heart failure. Interestingly, ERK5, a novel member of the MAPK family, has recently been shown to be involved in cardiac hypertrophy and to be stimulated by Gq-coupled GPCR in epithelial cells via unknown mechanisms. We have identified the atypical protein kinase C zeta (PKCζ) as the functional link between these two key players in cardio-
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vascular cell signaling. PKCζ associates to GalphaQ upon GPCR activation, and stimulation of ERK5 by Gq-coupled GPCR is abolished upon pharmacological inhibition of PKCζ or when using embryonic fibroblasts or cardiomyocytes obtained from PKCζ-deficient mice. Moreover, these mice do not develop cardiac hypertrophy upon chronic challenge with angiotensin II (as assessed by heart to body weight ratios, ANP levels and electrocardiographic analysis), thus indicating that this novel GalphaQ/PKCζ/ERK5 pathway is required for the induction of this important pathological process by Gq-coupled GPCR agonists. Keywords: Cardiac hypertrophy; G alpha q-GPCR; PKC doi:10.1016/j.yjmcc.2007.03.127
PKC depresses cardiac myocyte power output and limits the phosphorylation and mechanical effects by PKA Laurin M. Hanft, F. Steven Korte, Kerry S. McDonald. Department of Pharmacology and Physiology, University of Missouri, Columbia, MO, USA Protein kinase C (PKC) targets and phosphorylates myofibrillar proteins in cardiac myocytes, and PKC activity appears to be up-regulated in failing human hearts (Bowling et al., 1999). Since PKC phosphorylation of myofibrillar proteins affects myocyte contractility, we sought to directly test the effects of PKC phosphorylation of myofilament proteins on myocyte power output. We compared isometric force, loaded shortening velocity, and power output in skinned rat cardiac myocytes before and after treatment with the catalytic subunit of PKC. PKC increased phosphorylation levels of myosin binding protein C (MyBP-C) and cardiac troponin I (cTnI), and decreased force during submaximal Ca2+ activations (force before PKC = 6.1 ± 1.6 μN; force after PKC = 3.6 ± 1.0 μN). Moreover, PKC decreased absolute power output by 58% (power before PKC = 58 ± 21 pW; power after PKC = 24 ± 10 pW (n = 6)), which arose from both the fall in force and slower loaded shortening velocities. PKC even reduced power output in experiments performed at higher [Ca2+] to match force before and after PKC. Additionally, PKC blunted phosphorylation of cTnI and MyBP-C by protein kinase A (PKA), reduced PKA-induced spontaneous tension oscillations (SPOCs), and diminished PKA-induced augmentation of power output. Overall, these results indicate that PKC phosphorylation of myofibrillar proteins not only directly decreases power output of cardiac myocytes but also attenuates myocyte responsiveness to PKA, which may result in limited ventricular reserve and impaired cardiac function. Keywords: Cardiac myocytes; Myocyte function; Power output doi:10.1016/j.yjmcc.2007.03.128
MitoKATP, H2O2, and PKCε – completing the cycle Alexandre D. Costa, Keith D. Garlid. Dept. Biology, Portland State University, Portland OR, USA Activation of protein kinase C epsilon (PKCε), opening of mitochondrial KATP channels (mitoKATP), and increase in H2O2 generation by mitochondria have been shown to be key events in the signaling pathways that underlie cardioprotective mechanisms. However, the interactions among these and other components of the signaling cascades are not fully understood. We have shown that mitoKATP is activated by cGMPdependent protein kinase (PKG) via a mitochondrial PKCε that is closely associated with mitoKATP, which we designate PKCε1. MitoKATP opening causes an increase in H2O2 production by complex I of the respiratory chain. In this study, we investigate the relationship between PKCε1, mitoKATP, and the H2O2 generated by mitoKATP activity. We show that the outer membrane is essential for PKG-induced mitoKATP opening and that PKCε1 is located on the outer side of the inner membrane. PKCε activators such as H2O2, phorbol ester, or the peptide ψεRACK were able to induce mitoKATP opening in isolated heart mitochondria. H2O2- or NO-induced mitoKATP opening was inhibited by mitoKATP or PKCε inhibitors, but not by cyclosporin A. mitoKATP opening induced by PKG, PMA or diazoxide was not sensitive to MPG, showing that H2O2 does not act directly to open mitoKATP. We show that the mitoKATP-generated H2O2 activates PKCε1 and induces mitoKATP opening in vitro and in vivo. This mitoKATP-dependent mitoKATP opening constitutes a positive feedback loop that maintains the channel open after the stimulus is no longer present. These results suggest that mitoKATP might be a key player in the maintenance of the preconditioning memory effect. Keywords: Mitochondria; Signal transduction; Free radicals doi:10.1016/j.yjmcc.2007.03.126
PKCzeta-mediated GalphaQ stimulation of the ERK5 pathway plays a key role in cardiac hypertrophy Carlota García-Hoz, Michel Herranz, M. Teresa Díaz-Meco, Jorge Moscat, Catalina Ribas, Federico Mayor, Jr. Centro de Biología Molecular, Universidad Autónoma de Madrid, Spain Gq-coupled G protein-coupled receptors (GPCR) mediate the actions of a variety of messengers that are key regulators of cardiovascular function, such as norepinephrine angiotensin or endothelin. Enhanced Gq-mediated signaling has been implicated in cardiac hypertrophy and in the transition to heart failure. Interestingly, ERK5, a novel member of the MAPK family, has recently been shown to be involved in cardiac hypertrophy and to be stimulated by Gq-coupled GPCR in epithelial cells via unknown mechanisms. We have identified the atypical protein kinase C zeta (PKCζ) as the functional link between these two key players in cardio-
S45
vascular cell signaling. PKCζ associates to GalphaQ upon GPCR activation, and stimulation of ERK5 by Gq-coupled GPCR is abolished upon pharmacological inhibition of PKCζ or when using embryonic fibroblasts or cardiomyocytes obtained from PKCζ-deficient mice. Moreover, these mice do not develop cardiac hypertrophy upon chronic challenge with angiotensin II (as assessed by heart to body weight ratios, ANP levels and electrocardiographic analysis), thus indicating that this novel GalphaQ/PKCζ/ERK5 pathway is required for the induction of this important pathological process by Gq-coupled GPCR agonists. Keywords: Cardiac hypertrophy; G alpha q-GPCR; PKC doi:10.1016/j.yjmcc.2007.03.127
PKC depresses cardiac myocyte power output and limits the phosphorylation and mechanical effects by PKA Laurin M. Hanft, F. Steven Korte, Kerry S. McDonald. Department of Pharmacology and Physiology, University of Missouri, Columbia, MO, USA Protein kinase C (PKC) targets and phosphorylates myofibrillar proteins in cardiac myocytes, and PKC activity appears to be up-regulated in failing human hearts (Bowling et al., 1999). Since PKC phosphorylation of myofibrillar proteins affects myocyte contractility, we sought to directly test the effects of PKC phosphorylation of myofilament proteins on myocyte power output. We compared isometric force, loaded shortening velocity, and power output in skinned rat cardiac myocytes before and after treatment with the catalytic subunit of PKC. PKC increased phosphorylation levels of myosin binding protein C (MyBP-C) and cardiac troponin I (cTnI), and decreased force during submaximal Ca2+ activations (force before PKC = 6.1 ± 1.6 μN; force after PKC = 3.6 ± 1.0 μN). Moreover, PKC decreased absolute power output by 58% (power before PKC = 58 ± 21 pW; power after PKC = 24 ± 10 pW (n = 6)), which arose from both the fall in force and slower loaded shortening velocities. PKC even reduced power output in experiments performed at higher [Ca2+] to match force before and after PKC. Additionally, PKC blunted phosphorylation of cTnI and MyBP-C by protein kinase A (PKA), reduced PKA-induced spontaneous tension oscillations (SPOCs), and diminished PKA-induced augmentation of power output. Overall, these results indicate that PKC phosphorylation of myofibrillar proteins not only directly decreases power output of cardiac myocytes but also attenuates myocyte responsiveness to PKA, which may result in limited ventricular reserve and impaired cardiac function. Keywords: Cardiac myocytes; Myocyte function; Power output doi:10.1016/j.yjmcc.2007.03.128