Phosphorylation In Cardiomyocytes Research Articles

Detection of cardiac myosin binding protein-C (cMyBP-C) by a phospho-specific PKD antibody in contracting rat cardiomyocytes

Protein phosphorylation plays an important role in physiological processes, such as muscle contraction. Phospho-specific antibodies have become powerful tools to study these processes. Cardiac myosin binding protein-C (cMyBP-C) is one of the proteins that make up the contractile apparatus of cardiomyocytes. Phosphorylation of cMyBP-C is essential for normal cardiac function, since dephosphorylation of this protein leads to its degradation and has been associated with cardiomyopathy. One of the upstream kinases, which phosphorylate cMyBP-C, is protein kinase D (PKD). While studying the role of PKD in cMyBP-C phosphorylation, we tried to analyze phosphorylation of PKD with a phospho-specific PKD-Ser744/748 antibody. Contrary to the expected 115 kDa, a signal was found for a 150-kDa protein. By MALDI-TOF mass spectrometry, we identified this protein to be cMyBP-C. These data were confirmed by immunostaining using the p-PKD-Ser744/748 antibody, which displayed a striated pattern similar to the one observed for a regular cMyBP-C antibody. To our knowledge there are no antibodies commercially available for phosphorylated cMyBP-C. Thus, the p-PKD-Ser744/748 antibody can accelerate research into the role of cMyBP-C phosphorylation in cardiomyocytes.

Dilation of human atria: Increased diffusion restrictions for ADP, overexpression of hexokinase 2 and its coupling to oxidative phosphorylation in cardiomyocytes

Cardiac energy metabolism with emphasis on mitochondria was addressed in atrial tissue from patients with overload-induced atrial dilation. Structural remodeling of dilated (D) atria manifested as intracellular accumulation of fibrillar aggregates, lipofuscin, signs of myolysis and autophagy. Despite impaired complex I dependent respiration and increased diffusion restriction for ADP, no changes regarding adenylate and creatine kinase occurred. We observed 7-fold overexpression of HK2 gene in D atria with concomitant 2-fold greater activation of mitochondrial oxygen consumption by glucose, which might represent an adaption to increased energy requirements and impaired mitochondrial function by effectively joining glycolysis and oxidative phosphorylation.

Abstract 19434: Early Ischemic Preconditioning Requires Akt- and PKA-Mediated Activation of eNOS via Serine 1176 Phosphorylation

Aims: The role of endothelial nitric oxide synthase (eNOS)/NO signaling is well-documented in late preconditioning or postconditioning; however, the role of eNOS and its activation in early IPC remains controversial and poorly defined. This study investigates the role of eNOS in early IPC and what signaling pathways and molecular interactions regulate eNOS activation during early IPC. Methods and Results: Rat hearts were subjected to 30-min global ischemia and reperfusion (I/R) with or without IPC (3 cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor, L-NAME, PI 3 K inhibitor, LY294002/LY, or PKA inhibitor, H89, during IPC induction. IPC improved postischemic contractile function and reduced infarction compared to I/R with this abrogated by NOS inhibition. eNOS Ser1176 , Akt Ser473 , and PKA Thr197 phosphorylation was increased following IPC. I/R decreased eNOS Ser1176 phosphorylation, while IPC increased it. Mass spectroscopy confirmed eNOS Ser1176 phosphorylation and quantative western blots showed that ∼24 % modification of eNOS Ser1176 was present following induction of early IPC. Immunoprecipitation demonstrated eNOS, Akt and PKA complexation. Immunohistology showed that IPC induced Akt and PKA phosphorylation in cardiomyocytes and endothelial cells. With eNOS activation, IPC increased NO production as measured by EPR spin trapping and fluorescence microscopy. LY or H89 not only greatly decreased Akt Ser473 or PKA Thr197 phosphorylation, respectively; but also abolished IPC-induced preservation of eNOS and eNOS Ser1176 phosphorylation as well as subsequent cardioprotection. Conclusion: Thus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection with eNOS-derived NO serving a critical role.

Early ischaemic preconditioning requires Akt- and PKA-mediated activation of eNOS via serine1176 phosphorylation

The role of endothelial nitric oxide synthase (eNOS)/NO signalling is well documented in late ischaemic preconditioning (IPC); however, the role of eNOS and its activation in early IPC remains controversial. This study investigates the role of eNOS in early IPC and the signalling pathways and molecular interactions that regulate eNOS activation during early IPC. Rat hearts were subjected to 30-min global ischaemia and reperfusion (I/R) with or without IPC (three cycles 5-min I and 5-min R) in the presence or absence of the NOS inhibitor l-NAME, phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (LY), and protein kinase A (PKA) inhibitor H89 during IPC induction or prior endothelial permeablization. IPC improved post-ischaemic contractile function and reduced infarction compared with I/R with this being abrogated by l-NAME or endothelial permeablization. eNOS(Ser1176), Akt(Ser473), and PKA(Thr197) phosphorylation was increased following IPC. I/R decreased eNOS(Ser1176) phosphorylation, whereas IPC increased it. Mass spectroscopy confirmed eNOS(Ser1176) phosphorylation and quantitative Western blots showed ∼24% modification of eNOS(Ser1176) following IPC. Immunoprecipitation demonstrated eNOS, Akt, and PKA complexation. Immunohistology showed IPC-induced Akt and PKA phosphorylation in cardiomyocytes and endothelium. With eNOS activation, IPC increased NO production as measured by electron paramagnetic resonance spin trapping and fluorescence microscopy. LY or H89 not only decreased Akt(Ser473) or PKA(Thr197) phosphorylation, respectively, but also abolished IPC-induced preservation of eNOS and eNOS(Ser1176) phosphorylation as well as cardioprotection. Thus, Akt- and PKA-mediated eNOS activation, with phosphorylation near the C-terminus, is critical for early IPC-induced cardioprotection, with eNOS-derived NO from the endothelium serving a critical role.

Inhibition of Na/K-ATPase promotes myocardial tumor necrosis factor-alpha protein expression and cardiac dysfunction via calcium/mTOR signaling in endotoxemia

Tumor necrosis factor-α (TNF-α) is a major pro-inflammatory cytokine that causes cardiac dysfunction during sepsis. Na/K-ATPase regulates intracellular Ca(2+), which activates mammalian target of rapamycin (mTOR), a regulator of protein synthesis. The aim of this study was to investigate the role of Na/K-ATPase/mTOR signaling in myocardial TNF-α expression during endotoxemia. Results showed that treatment with LPS decreased Na/K-ATPase activity in the myocardium in vivo and in cultured neonatal cardiomyocytes. Inhibition of Na/K-ATPase by ouabain enhanced LPS-induced myocardial TNF-α protein production, but had no effect on TNF-α mRNA expression. More importantly, ouabain further decreased in vivo cardiac function in endotoxemic mice, which was blocked by etanercept, a TNF-α antagonist. LPS-induced reduction in Na/K-ATPase activity was prevented by inhibition of PI3K, Rac1 and NADPH oxidase using LY294002, a dominant-negative Rac1 adenovirus (Ad-Rac1N17) and apocynin, respectively. To assess the role of Rac1 in Ca(2+) handling, Ca(2+) transients in adult cardiomyocytes from cardiomyocyte-specific Rac1 knockout (Rac1(CKO)) and wild-type (WT) mice were determined. LPS increased intracellular Ca(2+) in WT but not in Rac1(CKO) cardiomyocytes. Furthermore, LPS rapidly increased mTOR phosphorylation in cardiomyocytes, which was blocked by Rac1N17 and an inhibitor of calmodulin-dependent protein kinases (CaMKs) KN93, but enhanced by ouabain. Rapamycin, an inhibitor of mTOR suppressed TNF-α protein levels without any significant effect on its mRNA expression or global protein synthesis. In conclusion, myocardial Na/K-ATPase activity is inhibited during endotoxemia via PI3K/Rac1/NADPH oxidase activation. Inhibition of Na/K-ATPase activates Ca(2+)/CaMK/mTOR signaling, which promotes myocardial TNF-α protein production and cardiac dysfunction during endotoxemia.

Activation ofβ-Adrenoceptors by Dobutamine May Induce a Higher Expression of Peroxisome Proliferator-Activated Receptorsδ(PPARδ) in Neonatal Rat Cardiomyocytes

Recent evidence showed the role of peroxisome proliferator-activated receptors (PPARs) in cardiac function. Cardiac contraction induced by various agents is critical in restoring the activity of peroxisome proliferator-activated receptors δ (PPARδ) in cardiac myopathy. Because dobutamine is an agent widely used to treat heart failure in emergency setting, this study is aimed to investigate the change of PPARδ in response to dobutamine. Neonatal rat cardiomyocytes were used to examine the effects of dobutamine on PPARδ expression levels and cardiac troponin I (cTnI) phosphorylation via Western blotting analysis. We show that treatment with dobutamine increased PPARδ expression and cTnI phosphorylation in a time- and dose-dependent manner in neonatal rat cardiomyocytes. These increases were blocked by the antagonist of β1-adrenoceptors. Also, the action of dobutamine was related to the increase of calcium ions and diminished by chelating intracellular calcium. Additionally, dobutamine-induced action was reduced by the inhibition of downstream messengers involved in this calcium-related pathway. Moreover, deletion of PPARδ using siRNA generated the reduction of cTnI phosphorylation in cardiomyocytes treated with dobutamine. Thus, we concluded that PPARδ is increased by dobutamine in cardiac cells.

β-adrenergic stimulation activate PKCε induces ERK phosphorylation in cardiomyocytes

ObjectiveTo evaluate PKCε translocation after β-adrenergic stimulation in isolated cardiomyocytes and the cross-talk with Epac and ERK phosphorylation.MethodsRat neonatal cardiomyocytes were cultured and treated with isoproterenol stimulation (Iso, 1 µmol/l...

Absence of thrombospondin-2 increases cardiomyocyte damage and matrix disruption in doxorubicin-induced cardiomyopathy

Clinical use of the antineoplastic agent doxorubicin (DOX) is limited by its cardiomyocyte toxicity. Attempts to decrease cardiomyocyte injury showed promising results in vitro, but failed to reduce the adverse effects of DOX in vivo, suggesting that other mechanisms contribute to its cardiotoxicity as well. Evidence that DOX also induces cardiac injury by compromising extracellular matrix integrity is lacking. The matricellular protein thrombospondin-2 (TSP-2) is known for its matrix-preserving function, and for modulating cellular function. Here, we investigated whether TSP-2 modulates the process of doxorubicin-induced cardiomyopathy (DOX-CMP). TSP-2-knockout (TSP-2-KO) and wild-type (WT) mice were treated with DOX (2 mg/kg/week) for 12 weeks to induce DOX-CMP. Mortality was significantly increased in TSP-2-KO compared to WT mice. Surviving DOX-treated TSP-2-KO mice had depressed cardiac function compared to WT animals, accompanied by increased cardiomyocyte apoptosis and matrix damage. Enhanced myocyte damage in the absence of TSP-2 was associated with impaired activation of the Akt signaling pathway in TSP-2-KO compared to WT. The absence of TSP-2, in vivo and in vitro, reduced Akt activation both under non-treated conditions and after DOX. Importantly, inhibition of Akt phosphorylation in cardiomyocytes significantly reduced TSP-2 expression, unveiling a unique feedback loop between Akt and TSP-2. Finally, enhanced matrix disruption in DOX-treated TSP-2-KO hearts went along with increased matrix metalloproteinase-2 levels. Taken together, this study is the first to provide evidence for the implication of the matrix element TSP-2 in protecting against DOX-induced cardiac injury and dysfunction.

Acetylcholine inhibits hypoxia‐induced tumor necrosis factor‐α production via regulation of MAPKs phosphorylation in cardiomyocytes

Recent findings have reported that up-regulation of tumor necrosis factor-alpha (TNF-α) induced by myocardial hypoxia aggravates cardiomyocyte injury. Acetylcholine (ACh), the principle vagal neurotransmitter, protects cardiomyocytes against hypoxia by inhibiting apoptosis. However, it is still unclear whether ACh regulates TNF-α production in cardiomyocytes after hypoxia. The concentration of extracellular TNF-α was increased in a time-dependent manner during hypoxia. Furthermore, ACh treatment also inhibited hypoxia-induced TNF-α mRNA and protein expression, caspase-3 activation, cell death and the production of reactive oxygen species (ROS) in cardiomyocytes. ACh treatment prevented the hypoxia-induced increase in p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) phosphorylation, and increased extracellular signal-regulated kinase (ERK) phosphorylation. Co-treatment with atropine, a non-selective muscarinic acetylcholine receptor antagonist, or methoctramine, a selective type-2 muscarinic acetylcholine (M(2) ) receptor antagonist, abrogated the effects of ACh treatment in hypoxic cardiomyocytes. Co-treatment with hexamethonium, a non-selective nicotinic receptor antagonist, and methyllycaconitine, a selective alpha7-nicotinic acetylcholine receptor antagonist, had no effect on ACh-treated hypoxic cardiomyocytes. In conclusion, these results demonstrate that ACh activates the M(2) receptor, leading to regulation of MAPKs phosphorylation and, subsequently, down-regulation of TNF-α production. We have identified a novel pathway by which ACh mediates cardioprotection against hypoxic injury in cardiomyocytes.

E0674 Insulin induces phosphorylation of Ndrg2 through activation of Akt in cardiomyocytes during transient ischaemia/reperfusion

AimsThe protein kinase Akt mediates an important cell-survival signalling of insulin through inhibition of apoptosis post cardiac ischaemia/reperfusion (I/R) injury. As Ndrg2 (N-Myc downstream-regulated gene 2) protein is one of...

Regulation of VASP phosphorylation in cardiac myocytes: differential regulation by cyclic nucleotides and modulation of protein expression in diabetic and hypertrophic heart

Vasodilator-stimulated phosphoprotein (VASP) is a major substrate for cyclic nucleotide-dependent kinases that has been implicated in cardiac pathology, yet many aspects of VASP's molecular regulation in cardiomyocytes are incompletely understood. In these studies, we explored the role of VASP, both in signaling pathways in isolated murine myocytes, as well as in a model of cardiac hypertrophy in VASP(null) mice. We found that the beta-adrenergic agonist isoproterenol promotes the rapid and reversible phosphorylation of VASP at Ser157 and Ser239. Forskolin and the cAMP analog 8-(4-chlorophenylthio)-cAMP promote a similar pattern of VASP phosphorylation at both sites. The effects of isoproterenol are blocked by atenolol and by compound H-89, an inhibitor of the cAMP-dependent protein kinase. By contrast, phosphorylation of VASP only at Ser239 is seen following activation of particulate guanylate cyclase by atrial natriuretic peptide, or following activation of soluble guanylate cyclase by sodium nitroprusside, or following treatment of myocytes with cGMP analog. We found that basal and isoproterenol-induced VASP phosphorylation is entirely unchanged in cardiomyocytes isolated from either endothelial or neuronal nitric oxide synthase knockout mice. In cardiomyocytes isolated from diabetic mice, only basal VASP phosphorylation is increased, whereas, in cells isolated from mice subjected to ascending aortic constriction (AAC), we found a significant increase in basal VASP expression, along with an increase in VASP phosphorylation, compared with cardiac myocytes isolated from sham-operated mice. Moreover, there is further increase in VASP phosphorylation in cells isolated from hypertrophic hearts following isoproterenol treatment. Finally, we found that VASP(null) mice subjected to transverse aortic constriction develop cardiac hypertrophy with a pattern similar to VASP(+/+) mice. Our findings establish differential receptor-modulated regulation of VASP phosphorylation in cardiomyocytes by cyclic nucleotides. Furthermore, these studies demonstrate for the first time that VASP expression is upregulated in hypertrophied heart.

Atrogin-1/MAFbx Enhances Simulated Ischemia/Reperfusion-induced Apoptosis in Cardiomyocytes through Degradation of MAPK Phosphatase-1 and Sustained JNK Activation

Atrogin-1/MAFbx is a major atrophy-related E3 ubiquitin ligase that is expressed specifically in striated muscle. Although the contribution of atrogin-1 to cardiac and muscle hypertrophy/atrophy has been examined extensively, it remains unclear whether atrogin-1 plays an essential role in the simulated ischemia/reperfusion-induced apoptosis of primary cardiomyocytes. Here we showed that atrogin-1 markedly enhanced ischemia/reperfusion-induced apoptosis in cardiomyocytes via activation of JNK signaling. Overexpression of atrogin-1 increased phosphorylation of JNK and c-Jun and decreased phosphorylation of Foxo3a. In addition, atrogin-1 decreased Bcl-2, increased Bax, and enhanced the activation of caspases. Furthermore, JNK inhibitor SP600125 markedly blocked the effect of atrogin-1 on cell apoptosis and the expression of apoptotic-related proteins and caspases. Importantly, atrogin-1 induced sustained activation of JNK through a mechanism that involved degradation of MAPK phosphatase-1 (MKP-1) protein. Atrogin-1 interacted with and triggered MKP-1 for ubiquitin-mediated degradation. In contrast, proteasome inhibitors markedly blocked the degradation of MKP-1. Taken together, these results demonstrate that atrogin-1 promotes degradation of MKP-1 through the ubiquitin-proteasome pathway, thereby leading to persistent activation of JNK signaling and further cardiomyocyte apoptosis following ischemia/reperfusion injury.

P66Shc Links α 1 -Adrenergic Receptors to a Reactive Oxygen Species-Dependent AKT-FOXO3A Phosphorylation Pathway in Cardiomyocytes

p66Shc is an adapter protein that is induced by hypertrophic stimuli and has been implicated as a major regulator of reactive oxygen species (ROS) production and cardiovascular oxidative stress responses. This study implicates p66Shc in an alpha(1)-adrenergtic receptor (alpha(1)-AR) pathway that requires the cooperative effects of protein kinase (PK)Cepsilon and PKCdelta and leads to AKT-FOXO3a phosphorylation in cardiomyocytes. alpha(1)-ARs promote p66Shc-YY(239/240) phosphorylation via a ROS-dependent mechanism that is localized to caveolae and requires epidermal growth factor receptor (EGFR) and PKCepsilon activity. alpha(1)-ARs also increase p66Shc-S(36) phosphorylation via an EGFR transactivation pathway involving PKCdelta. p66Shc links alpha(1)-ARs to an AKT signaling pathway that selectively phosphorylates/inactivates FOXO transcription factors and downregulates the ROS-scavenging protein manganese superoxide dismutase (MnSOD); the alpha(1)-AR-p66Shc-dependent pathway involving AKT does not regulate GSK3. Additional studies show that RNA interference-mediated downregulation of endogenous p66Shc leads to the derepression of FOXO3a-regulated genes such as MnSOD, p27Kip1, and BIM-1. p66Shc downregulation also increases proliferating cell nuclear antigen expression and induces cardiomyocyte hypertrophy, suggesting that p66Shc exerts an antihypertrophic action in neonatal cardiomyocytes. The novel alpha(1)-AR- and ROS-dependent pathway involving p66Shc identified in this study is likely to contribute to cardiomyocyte remodeling and the evolution of heart failure.

ISCHEMIC POSTCONDITIONING PROTECTS MYOCARDIUM FROM ISCHEMIA/REPERFUSION INJURY THROUGH ATTENUATING ENDOPLASMIC RETICULUM STRESS

Ischemic postconditioning (I-postC) is a newly discovered endogenous protective phenomenon capable of protecting the myocardium from I/R injury. The cardioprotective mechanisms of I-postC involve protein synthesis and preventing an increase in cytosolic calcium. Endoplasmic reticulum (ER) is a principal site for secretory protein synthesis and calcium storage. Myocardial I/R causes ER stress and perturbations of ER function. The purpose of the present study was to determine whether I-postC's attenuation of I/R injury involves reductions in ER stress through mitogen-activated protein kinase (MAPK) pathway. In the present study, models of rat myocardial I/R and hypoxia/reoxygenation (H/R) of neonatal rat cardiomyocytes were used. Myocardial infarct size was measured by triphenyltetrazolium chloride staining, and flow cytometry was used to quantitate cardiomyocyte apoptosis. Calreticulin expression and activation of caspase 12, p38 MAPK, and c-Jun NH2-terminal kinase (JNK) in myocardium or cardiomyocytes were detected by Western blots. It is found that I-postC protects the I/R heart against myocardial infarction, and hypoxic postconditioning protects neonatal cardiomyocytes from H/R-induced apoptosis. Ischemic postconditioning suppressed I/R-induced ER stress, as shown by a decrease in calreticulin expression and caspase 12 activation. Hypoxic postconditioning up-regulates p38 MAPK phosphorylation and down-regulates JNK phosphorylation in cardiomyocytes subjected to H/R. These results indicate that I-postC protects myocardium from I/R injury by suppressing ER stress, and that p38 MAPK and JNK pathways are associated with the I-postC-induced suppression of ER stress.

Protein Phosphatase 1 beta is Most Abundant Isoform in the Longitudinal Sarcoplasmic Reticulum and Regulates Phospholamban Phosphorylation in Cardiomyocytes

Protein Phosphatase 1 beta is Most Abundant Isoform in the Longitudinal Sarcoplasmic Reticulum and Regulates Phospholamban Phosphorylation in Cardiomyocytes

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

Protein kinase Cdelta (PKCdelta) activation is generally attributed to lipid cofactor-dependent allosteric activation mechanisms at membranes. However, recent studies indicate that PKCdelta also is dynamically regulated through tyrosine phosphorylation in H(2)O(2)- and phorbol 12-myristate 13-acetate (PMA)-treated cardiomyocytes. H(2)O(2) activates Src and related Src-family kinases (SFKs), which function as dual PKCdelta-Tyr(311) and -Tyr(332) kinases in vitro and contribute to H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation in cardiomyocytes and in mouse embryo fibroblasts. H(2)O(2)-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation is defective in SYF cells (deficient in SFKs) and restored by Src re-expression. PMA also promotes PKCdelta-Tyr(311) phosphorylation, but this is not associated with SFK activation or PKCdelta-Tyr(332) phosphorylation. Rather, PMA increases PKCdelta-Tyr(311) phosphorylation by delivering PKCdelta to SFK-enriched caveolae. Cyclodextrin treatment disrupts caveolae and blocks PMA-dependent PKCdelta-Tyr(311) phosphorylation, without blocking H(2)O(2)-dependent PKCdelta-Tyr(311) phosphorylation. The enzyme that acts as a PKCdelta-Tyr(311) kinase without increasing PKCdelta phosphorylation at Tyr(332) in PMA-treated cardiomyocytes is uncertain. Although in vitro kinase assays implicate c-Abl as a selective PKCdelta-Tyr(311) kinase, PMA-dependent PKCdelta-Tyr(311) 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 PKCdelta to caveolae and increase PKCdelta-Tyr(311) 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 PKCdelta exclusively in non-caveolae membranes. Collectively, these results identify stimulus-specific PKCdelta localization and tyrosine phosphorylation mechanisms that could be targeted for therapeutic advantage.

Protein Kinase D Links Gq-coupled Receptors to cAMP Response Element-binding Protein (CREB)-Ser133 Phosphorylation in the Heart

Many growth regulatory stimuli promote cAMP response element-binding protein (CREB) Ser(133) phosphorylation, but the physiologically relevant CREB-Ser(133) kinase(s) in the heart remains uncertain. This study identifies a novel role for protein kinase D (PKD) as an in vivo cardiac CREB-Ser(133) kinase. We show that thrombin activates a PKCdelta-PKD pathway leading to CREB-Ser(133) phosphorylation in cardiomyocytes and cardiac fibroblasts. alpha(1)-Adrenergic receptors also activate a PKCdelta-PKD-CREB-Ser(133) phosphorylation pathway in cardiomyocytes. Of note, while the epidermal growth factor (EGF) promotes CREB-Ser(133) phosphorylation via an ERK-RSK pathway in cardiac fibroblasts, the thrombin-dependent EGFR transactivation pathway leading to ERK-RSK activation does not lead to CREB-Ser(133) phosphorylation in this cell type. Adenoviral-mediated overexpression of PKCdelta (but not PKCepsilon or PKCalpha) activates PKD; PKCdelta and PKD1-S744E/S748E overexpression both promote CREB-Ser(133) phosphorylation. Pasteuralla multocida toxin (PMT), a direct Galpha(q) agonist that induces robust cardiomyocyte hypertrophy, also activates the PKD-CREB-Ser(133) phosphorylation pathway, leading to the accumulation of active PKD and Ser(133)-phosphorylated CREB in the nucleus, activation of a CRE-responsive promoter, and increased Bcl-2 (CREB target gene) expression in cardiomyocyte cultures. Cardiac-specific Galpha(q) overexpression also leads to an increase in PKD-Ser(744)/Ser(748) and CREB-Ser(133) phosphorylation as well as increased Bcl-2 protein expression in the hearts of transgenic mice. Collectively, these studies identify a novel Galpha(q)-PKCdelta-PKD-CREB-Ser(133) phosphorylation pathway that is predicted to contribute to cardiac remodeling and could be targeted for therapeutic advantage in the setting of heart failure phenotypes.

Norepinephrine- and Epinephrine-induced Distinct β2-Adrenoceptor Signaling Is Dictated by GRK2 Phosphorylation in Cardiomyocytes

Agonist-dependent activation of G protein-coupled receptors induces diversified receptor cellular and signaling properties. Norepinephrine (NE) and epinephrine (Epi) are two endogenous ligands that activate adrenoceptor (AR) signals in a variety of physiological stress responses in animals. Here we use cardiomyocyte contraction rate response to analyze the endogenous beta(2)AR signaling induced by Epi or NE in cardiac tissue. The Epi-activated beta(2)AR induced a rapid contraction rate increase that peaked at 4 min after stimulation. In contrast, the NE-activated beta(2)AR induced a much slower contraction rate increase that peaked at 10 min after stimulation. Whereas both drugs activated beta(2)AR coupling to G(s) proteins, only Epi-activated receptors were capable of coupling to G(i) proteins. Subsequent studies showed that the Epi-activated beta(2)AR underwent a rapid phosphorylation by G protein-coupled receptor kinase 2 (GRK2) and subsequent dephosphorylation on serine residues 355 and 356, which was critical for sufficient receptor recycling and G(i) coupling. In contrast, the NE-activated beta(2)ARs underwent slow GRK2 phosphorylation, receptor internalization and recycling, and failed to couple to G(i). Moreover, inhibiting beta(2)AR phosphorylation by betaARK C terminus or dephosphorylation by okadaic acid prevented sufficient recycling and G(i) coupling. Together, our data revealed that distinct temporal phosphorylation of beta(2)AR on serine 355 and 356 by GRK2 plays a critical role for dictating receptor cellular events and signaling properties induced by Epi or NE in cardiomyocytes. This study not only helps us understand the endogenous agonist-dependent beta(2)AR signaling in animal heart but also offers an example of how G protein-coupled receptor signaling may be finely regulated by GRK in physiological settings.

Regulation of Akt/PKB activity by P21-activated kinase in cardiomyocytes

Akt/PKB is a critical regulator of cardiac function and morphology, and its activity is governed by dual phosphorylation at active loop (Thr308) by phosphoinositide-dependent protein kinase-1 (PDK1) and at carboxyl-terminal hydrophobic motif (Ser473) by a putative PDK2. P21-activated kinase-1 (Pak1) is a serine/threonine protein kinase implicated in the regulation of cardiac hypertrophy and contractility and was shown previously to activate Akt through an undefined mechanism. Here we report Pak1 as a potential PDK2 that is essential for Akt activity in cardiomyocytes. Both Pak1 and Akt can be activated by multiple hypertrophic stimuli or growth factors in a phosphatidylinositol-3-kinase (PI3K)-dependent manner. Pak1 overexpression induces Akt phosphorylation at both Ser473 and Thr308 in cardiomyocytes. Conversely, silencing or inactivating Pak1 gene diminishes Akt phosphorylation in vitro and in vivo. Purified Pak1 can directly phosphorylate Akt only at Ser473, suggesting that Pak1 may be a relevant PDK2 responsible for AKT Ser473 phosphorylation in cardiomyocytes. In addition, Pak1 protects cardiomyocytes from cell death, which is blocked by Akt inhibition. Our results connect two important regulators of cellular physiological functions and provide a potential mechanism for Pak1 signaling in cardiomyocytes.

Protein Kinase Cɛ (PKCɛ) and Src Control PKCδ Activation Loop Phosphorylation in Cardiomyocytes

Protein kinase Cdelta (PKCdelta) is unusual among AGC kinases in that it does not require activation loop (Thr(505)) phosphorylation for catalytic competence. Nevertheless, Thr(505) phosphorylation has been implicated as a mechanism that influences PKCdelta activity. This study examines the controls of PKCdelta-Thr(505) phosphorylation in cardiomyocytes. We implicate phosphoinositide-dependent kinase-1 and PKCdelta autophosphorylation in the "priming" maturational PKCdelta-Thr(505) phosphorylation that accompanies de novo enzyme synthesis. In contrast, we show that PKCdelta-Thr(505) phosphorylation dynamically increases in cardiomyocytes treated with phorbol 12-myristate 13-acetate or the alpha(1)-adrenergic receptor agonist norepinephrine via a mechanism that requires novel PKC isoform activity and not phosphoinositide-dependent kinase-1. We used a PKCepsilon overexpression strategy as an initial approach to discriminate two possible novel PKC mechanisms, namely PKCdelta-Thr(505) autophosphorylation and PKCdelta-Thr(505) phosphorylation in trans by PKCepsilon. Our studies show that adenovirus-mediated PKCepsilon overexpression leads to an increase in PKCdelta-Thr(505) phosphorylation. However, this cannot be attributed to an effect of PKCepsilon to function as a direct PKCdelta-Thr(505) kinase, since the PKCepsilon-dependent increase in PKCdelta-Thr(505) phosphorylation is accompanied by (and dependent upon) increased PKCdelta phosphorylation at Tyr(311) and Tyr(332). Further studies implicate Src in this mechanism, showing that 1) PKCepsilon overexpression increases PKCdelta-Thr(505) phosphorylation in cardiomyocytes and Src(+) cells but not in SYF cells (that lack Src, Yes, and Fyn and exhibit a defect in PKCdelta-Tyr(311)/Tyr(332) phosphorylation), and 2) in vitro PKCdelta-Thr(505) autophosphorylation is augmented in assays performed with Src (which promotes PKCdelta-Tyr(311)/Tyr(332) phosphorylation). Collectively, these results identify a novel PKCdelta-Thr(505) autophosphorylation mechanism that is triggered by PKCepsilon overexpression and involves Src-dependent PKCdelta-Tyr(311)/Tyr(332) phosphorylation.