Vitalyi O. Rybin

Regulation of Myocardial Contractility and Cell Size by Distinct PI3K-PTEN Signaling Pathways

The PTEN/PI3K signaling pathway regulates a vast array of fundamental cellular responses. We show that cardiomyocyte-specific inactivation of tumor suppressor PTEN results in hypertrophy, and unexpectedly, a dramatic decrease in cardiac contractility. Analysis of double-mutant mice revealed that the cardiac hypertrophy and the contractility defects could be genetically uncoupled. PI3Kalpha mediates the alteration in cell size while PI3Kgamma acts as a negative regulator of cardiac contractility. Mechanistically, PI3Kgamma inhibits cAMP production and hypercontractility can be reverted by blocking cAMP function. These data show that PTEN has an important in vivo role in cardiomyocyte hypertrophy and GPCR signaling and identify a function for the PTEN-PI3Kgamma pathway in the modulation of heart muscle contractility.

Protein kinase C isoform expression and regulation in the developing rat heart.

To determine whether age-dependent differences in cardiac responses to autonomic agonists could result from developmental changes in protein kinase C (PKC) isoform expression, we probed extracts from the fetal, neonatal, and adult heart as well as cultured neonatal and isolated adult ventricular myocytes with specific antisera to calcium-dependent (alpha and beta) and calcium-independent (delta, epsilon and zeta) isoforms of the enzyme. Although PKC-beta immunoreactivity could not be detected in cultured neonatal or isolated adult ventricular myocytes, adult and neonatal myocytes expressed multiple other isoforms of PKC. Our studies revealed an age-dependent decline in the immunoreactivity for three PKC isoforms. PKC-alpha was detected in extracts from the fetal and 2-day-old neonatal heart as well as cultured neonatal rat ventricular myocytes. Only faint PKC-alpha immunoreactivity was detected in extracts from the adult heart, and PKC-alpha was not detected in extracts from isolated adult ventricular myocytes, suggesting that PKC-alpha resides in nonmyocyte elements in the adult heart. PKC-delta also was detected in greater abundance in fetal and neonatal than in adult myocardial extracts. The decline in PKC-alpha and PKC-delta expression occurred during the first 2 postnatal weeks. PKC-zeta was detected in greatest abundance in extracts from the fetal heart. PKC-zeta expression declined markedly by the second postnatal day, and only faint PKC-zeta immunoreactivity was detected in extracts from adult myocardium. Failure to detect PKC-zeta in extracts from isolated adult ventricular myocytes suggests that PKC-zeta resides primarily in nonmyocyte elements in the adult heart. PKC-epsilon was detected in all preparations, but it was detected in greatest abundance in extracts from neonatal hearts. In vitro sympathetic innervation of previously noninnervated neonatal ventricular myocytes or in vivo chemical sympathectomy of the neonatal heart did not modulate PKC isoform expression, suggesting that sympathetic innervation does not significantly regulate PKC isoform expression. PKC-alpha partitioned to the soluble fraction of unstimulated myocytes and was selectively translocated to the particulate fraction by Ca2+. In contrast, a major portion of the novel PKC isoforms partitioned to the particulate fraction of unstimulated myocytes. The subcellular distribution of novel PKC isoforms was not influenced by Ca2+. 12-O-Tetradecanoylphorbol 13-acetate (TPA, 300 nmol/L) induced translocation of soluble PKC-alpha, PKC-delta, and PKC-epsilon to the particulate fraction at 30 minutes and complete (PKC-alpha and PKC-delta) or 80% (PKC-epsilon) downregulation at 24 hours. PKC-zeta was not affected by TPA.(ABSTRACT TRUNCATED AT 400 WORDS)

Activated Protein Kinase C Isoforms Target to Cardiomyocyte Caveolae: Stimulation of Local Protein Phosphorylation

Protein kinase C (PKC) isoforms constitute an important component of the signal transduction pathway used by cardiomyocytes to respond to a variety of extracellular stimuli. Translocation to distinct intracellular sites represents an essential step in the activation of PKC isoforms, presumably as a prerequisite for stable access to substrate. Caveolae are specialized subdomains of the plasma membrane that are reported to concentrate key signaling proteins and may represent a locus for PKC action, given that PKC activators have been reported to dramatically alter caveolae morphology. Accordingly, this study examines whether PKC isoforms initiate signaling in cardiomyocyte caveolae. Phorbol ester-sensitive PKC isoforms were detected at very low levels in caveolae fractions prepared from unstimulated cardiomyocytes; phorbol 12-myristate 13-acetate (PMA) (but not 4alpha-PMA, which does not activate PKC) recruited calcium-sensitive PKCalpha and novel PKCdelta and PKCepsilon to this compartment. The subcellular localization of the phorbol ester-insensitive PKClambda isoform was not influenced by PMA. Endothelin also induced the selective translocation of PKCalpha and PKCepsilon (but not PKCdelta or PKClambda) to caveolae. Multiple components of the extracellular signal-regulated protein kinase (ERK) cascade, including A-Raf, c-Raf-1, mitogen-activated protein kinase kinase, and ERK, were detected in caveolae under resting conditions. Although levels of these proteins were not altered by PMA, translocation of phorbol ester-sensitive PKC isoforms to caveolae was associated with the activation of a local ERK cascade as well as the phosphorylation of a approximately 36-kDa substrate protein in this fraction. Finally, a minor fraction of a protein that has been designated as a receptor for activated protein kinase C resides in caveolae and (along with caveolin-3) could represent a mechanism to target PKC isoforms to cardiomyocyte caveolae. These studies identify cardiomyocyte caveolae as a meeting place for activated PKC isoforms and their downstream target substrates.

Phosphoinositide 3-Kinase γ–Deficient Mice Are Protected From Isoproterenol-Induced Heart Failure

Background—We have recently shown that genetic inactivation of phosphoinositide 3-kinase &ggr; (PI3K&ggr;), the isoform linked to G-protein–coupled receptors, results in increased cardiac contractility with no effect on basal cell size. Signaling via the G-protein–coupled &bgr;-adrenergic receptors has been implicated in cardiac hypertrophy and heart failure, suggesting that PI3K&ggr; might play a role in the pathogenesis of heart disease. Methods and Results—To determine the role for PI3K&ggr; in hypertrophy induced by G-protein–coupled receptors and cardiomyopathy, we infused isoproterenol, a &bgr;-adrenergic receptor agonist, into PI3K&ggr;-deficient mice. Compared with controls, isoproterenol infusion in PI3K&ggr;-deficient mice resulted in an attenuated cardiac hypertrophic response and markedly reduced interstitial fibrosis. Intriguingly, chronic &bgr;-adrenergic receptor stimulation triggered impaired heart functions in wild-type mice, whereas PI3K&ggr;-deficient mice retained their increased heart function and did not develop heart failure. The lack of PI3K&ggr; attenuated the activation of Akt/protein kinase B and extracellular signal-regulated kinase 1/2 signaling pathways in cardiac myocytes in response to isoproterenol. &bgr;1- and &bgr;2-adrenergic receptor densities were decreased by similar amounts in PI3K&ggr;-deficient and control mice, suggesting that PI3K&ggr; isoform plays no role in the downregulation of &bgr;-adrenergic receptors after chronic &bgr;-adrenergic stimulation. Conclusions—Our data show that PI3K&ggr; is critical for the induction of hypertrophy, fibrosis, and cardiac dysfunction function in response to &bgr;-adrenergic receptor stimulation in vivo. Thus, PI3K&ggr; may represent a novel therapeutic target for the treatment of decreased cardiac function in heart failure.

Thyroid Hormone Represses Protein Kinase C Isoform Expression and Activity in Rat Cardiac Myocytes

We have previously demonstrated that at least four isoforms of protein kinase C (PKC; alpha, delta, epsilon, and zeta) are expressed in neonatal rat ventricular myocytes and that development is associated with a decline in their expression. The mechanism(s) regulating PKC isoform expression in ventricular myocytes is completely unknown. The developmental decline in PKC expression occurs, in large part, during the first 2 weeks of postnatal life, while thyroid hormone levels are known to be progressively increasing. Accordingly, this study examined the influence of thyroid hormone on PKC isoform expression to determine whether thyroid hormone can be implicated as a potential physiological regulator of PKC gene expression during normal cardiac development. Hypothyroidism was induced in adult rats by surgical thyroidectomy; thyroid status was manipulated in cultured neonatal ventricular myocytes by growth in serum-free medium with varying triiodothyronine (T3) levels. In each case, hypothyroidism was verified by a 10- to 50-fold increase in steady state mRNA for beta-myosin heavy chain. In hypothyroid adult ventricular myocardium, there was a selective 60% increase in the expression of PKC epsilon protein that corresponded to an increase in maximally stimulated PKC enzyme activity with PKC epsilon substrate peptide (epsilon pep) but not with histone as substrate. Northern blot analysis revealed a 70% increase in PKC epsilon mRNA, indicating that the regulatory effects of thyroid hormone are mediated, at least in part, at the message level. In neonatal ventricular myocytes, there was a T3-dependent reduction in immunoreactivity for both PKC alpha and PKC epsilon that was associated with significant reductions in both histone- and epsilon pep-kinase activities. The concentration of T3 that half-maximally repressed PKC alpha and PKC epsilon expression was approximately 0.5 nmol/L. Thyroid hormone had no effect on PKC delta and PKC zeta expression in neonatal or adult ventricular myocytes. PKC isoform expression in cardiac fibroblasts was not influenced by variations in the thyroid hormone concentration during culture. These results provide evidence that thyroid hormone specifically represses PKC alpha and PKC epsilon in the neonatal heart and PKC epsilon in the adult heart. Thyroid hormone-induced changes in PKC may play an important permissive role in the modulation of autonomic responsiveness in ventricular cardiomyocytes.

Protein Kinase D1 Autophosphorylation via Distinct Mechanisms at Ser744/Ser748 and Ser916

Protein kinase D1 (PKD1) is a physiologically important signaling enzyme that is activated via protein kinase C-dependent trans-phosphorylation of the activation loop at Ser744 and Ser748 followed by PKD1 autophosphorylation at Ser916. Although PKD-Ser916 autophosphorylation is widely used to track cellular PKD activity, this study exposes conditions leading to increased PKD-Ser(P)916 immunoreactivity without an associated increase in PKD activity in cardiomyocytes that heterologously overexpress catalytically inactive PKD1 and in cardiomyocytes treated with Gö6976 (a PKD inhibitor that competes with ATP). In each case, PKD1 is detected as a Ser916-phosphorylated enzyme that lacks kinase activity. In vitro kinase assays reconcile these seemingly discrepant findings by demonstrating that PKD1-Ser916 autophosphorylation can proceed via either an intermolecular reaction or an intramolecular autophosphorylation that requires only very low ATP concentrations that do not support target substrate phosphorylation. Additional studies show that Ser744 and Ser748 are targets for a protein kinase C-independent autocatalytic phosphorylation and that the PKD1-S744A/S748A mutant is a Ser916-phosphorylated enzyme that is not active toward heterologous substrates. In contrast, PKD1-S916A is an active kinase that autophosphorylates at Ser744. However, the S916A substitution leads to a Ser748 phosphorylation defect and a prolonged cellular PKD1 signaling response. Collectively, these results implicate PKD1-Ser744 phosphorylation in the phorbol 12-myristate 13-acetate-dependent mechanism that increases PKD1 activity toward physiologically relevant substrates. We show that PKD1-Ser916 autophosphorylation does not necessarily correlate with PKD1 activity. Rather, autophosphorylation at Ser916 is required for subsequent autophosphorylation at Ser748. Finally, this study exposes a novel role for Ser916 and/or Ser748 autophosphorylation to terminate the cellular PKD1 signaling response.

Protein kinase Cepsilon (PKCepsilon) and Src control PKCdelta activation loop phosphorylation in cardiomyocytes.

Protein kinase Cδ (PKCδ) is unusual among AGC kinases in that it does not require activation loop (Thr505) phosphorylation for catalytic competence. Nevertheless, Thr505 phosphorylation has been implicated as a mechanism that influences PKCδ activity. This study examines the controls of PKCδ-Thr505 phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKCδ autophosphorylation in the “priming” maturational PKCδ-Thr505 phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKCδ-Thr505 phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the α1-adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKCϵ overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKCδ-Thr505 autophosphorylation and PKCδ-Thr505 phosphorylation in trans by PKCϵ. Our studies show that adenovirus-mediated PKCϵ overexpression leads to an increase in PKCδ-Thr505 phosphorylation. However, this cannot be attributed to an effect of PKCϵ to function as a direct PKCδ-Thr505 kinase, since the PKCϵ-dependent increase in PKCδ-Thr505 phosphorylation is accompanied by (and dependent upon) increased PKCδ phosphorylation at Tyr311 and Tyr332. Further studies implicate Src in this mechanism, showing that 1) PKCϵ overexpression increases PKCδ-Thr505 phosphorylation in cardiomyocytes and Src+ cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKCδ-Tyr311/Tyr332 phosphorylation), and 2) in vitro PKCδ-Thr505 autophosphorylation is augmented in assays performed with Src (which promotes PKCδ-Tyr311/Tyr332 phosphorylation). Collectively, these results identify a novel PKCδ-Thr505 autophosphorylation mechanism that is triggered by PKCϵ overexpression and involves Src-dependent PKCδ-Tyr311/Tyr332 phosphorylation.

Mechanisms for vagal modulation of ventricular repolarization and of coronary occlusion-induced lethal arrhythmias in cats.

Our goal was to better understand the mechanisms underlying muscarinic receptor actions on the ventricle in vivo. Therefore, we studied the effects of vagal stimulation on ventricular repolarization and of vagal tone on lethal arrhythmias induced by 30 minutes of left anterior descending coronary artery ligation in anesthetized cats. Experimental groups included normal control cats subjected only to coronary ligation and cats pretreated with atropine, pertussis toxin (PTX), or propranolol. All cats received bilateral cervical vagal stimulation (Vstim) at 1, 3, and 5 Hz for 1 minute at 10-minute intervals. Before coronary ligation, Vstim slowed sinus rate, prolonged the PR interval, and lowered blood pressure. Most important from the point of view of electrophysiological function was a vagally induced acceleration of ventricular repolarization in paced and unpaced hearts, which could be explained by the effects of acetylcholine (ie, shortening the subepicardial muscle action potentials). The effect on repolarization was blocked by atropine or PTX but not by propranolol. The extent of sinus slowing and acceleration of repolarization was directly related to the level of functional PTX-sensitive G protein (P < .05). Coronary occlusion was performed during atrial pacing such that the heart rate in all groups was equal. The incidence of ventricular fibrillation (VF) was 10% in the control group and 50% and 54% in atropine and PTX groups, respectively (P < .05). During atrial pacing before coronary occlusion, a vagal index was calculated as percent QTc shortening during Vstim. When the vagal index was 13% to 26%, the incidence of VF during occlusion was zero. When the vagal index was 0% to 12%, VF was 52% (P < .01). Conclusions are as follows: (1) Vstim accelerates ventricular repolarization in cats via a pathway that incorporates a PTX-sensitive G protein and involves an altered gradient between epicardium and endocardium. (2) Removal of vagal tone during ischemia favors VF, as predicted by a vagal index.

α1-Adrenergic Receptors Activate AKT via a Pyk2/PDK-1 Pathway That Is Tonically Inhibited by Novel Protein Kinase C Isoforms in Cardiomyocytes

AKT is a potent antiapoptotic kinase, but its role in the cardioprotective actions of α1-adrenergic receptors (ARs) remains uncertain, because α1-ARs typically induce little-to-no AKT activation in most cardiomyocyte models. This study identifies a prominent α1-AR–dependent AKT activation pathway that is under tonic inhibitory control by novel protein kinase Cs (nPKCs) in neonatal rat cardiomyocyte cultures. We also implicate Pyk2, Pyk2 complex formation with PDK-1 and paxillin, and increased PDK-1–Y373/376 phosphorylation as the mechanism that links α1-AR activation to increased AKT phosphorylation. nPKCs (which are prominent α1-AR effectors) interfere with this α1-AR–dependent AKT activation by blocking Pyk2/PDK-1/paxillin complex formation and PDK-1–Y373/376 phosphorylation. Additional studies used an adenoviral-mediated overexpression strategy to show that Pyk2 exerts dual controls on antiapoptotic PDK-1/AKT and proapoptotic c-Jun N-terminal kinase (JNK) pathways. Although the high nPKC activity of most cardiomyocyte models favors Pyk2 signaling to JNK (and cardiac apoptosis), the cardioprotective actions of Pyk2 through the PDK-1/AKT pathway are exposed when PKC or JNK activation is prevented. Collectively, these studies identify JNK and AKT as functionally distinct downstream components of the α1-AR/Pyk2 signaling pathway. We also implicate nPKCs as molecular switches that control the balance of signaling via proapoptotic JNK and antiapoptotic PDK-1/AKT pathways, exposing a novel mechanism for nPKC-dependent regulation of cardiac hypertrophy and failure.

Phorbol 12-Myristate 13-Acetate-dependent Protein Kinase Cδ-Tyr311 Phosphorylation in Cardiomyocyte Caveolae

Protein kinase Cδ (PKCδ) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCδ also is dynamically regulated through tyrosine phosphorylation in H2O2- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H2O2 activates Src and related Src-family kinases (SFKs), which function as dual PKCδ-Tyr311 and -Tyr332 kinases in vitro and contribute to H2O2-dependent PKCδ-Tyr311/Tyr332 phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H2O2-dependent PKCδ-Tyr311/Tyr332 phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCδ-Tyr311 phosphorylation, but this is not associated with SFK activation or PKCδ-Tyr332 phosphorylation. Rather, PMA increases PKCδ-Tyr311 phosphorylation by delivering PKCδ to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCδ-Tyr311 phosphorylation, without blocking H2O2-dependent PKCδ-Tyr311 phosphorylation. The enzyme that acts as a PKCδ-Tyr311 kinase without increasing PKCδ phosphorylation at Tyr332 in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCδ-Tyr311 kinase, PMA-dependent PKCδ-Tyr311 phosphorylation persists in cardiomyocytes treated with the c-Abl inhibitor ST1571 and c-Abl is not detected in caveolae; these results effectively exclude a c-Abl-dependent process. Finally, we show that 1,2-dioleoyl-sn-glycerol mimics the effect of PMA to drive PKCδ to caveolae and increase PKCδ-Tyr311 phosphorylation, whereas G protein-coupled receptor agonists such as norepinephrine and endothelin-1 do not. These results suggest that norepinephrine and endothelin-1 increase 1,2-dioleoyl-sn-glycerol accumulation and activate PKCδ exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCδ localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.