Nazira Ozgen

p66Shc Links α1-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 &agr;1-adrenergtic receptor (&agr;1-AR) pathway that requires the cooperative effects of protein kinase (PK)Cϵ and PKC&dgr; and leads to AKT-FOXO3a phosphorylation in cardiomyocytes. &agr;1-ARs promote p66Shc-YY239/240 phosphorylation via a ROS-dependent mechanism that is localized to caveolae and requires epidermal growth factor receptor (EGFR) and PKCϵ activity. &agr;1-ARs also increase p66Shc-S36 phosphorylation via an EGFR transactivation pathway involving PKC&dgr;. p66Shc links &agr;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 &agr;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 &agr;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.

Accessory Subunit KChIP2 Modulates the Cardiac L-Type Calcium Current

Complex modulation of voltage-gated Ca2+ currents through the interplay among Ca2+ channels and various Ca2+-binding proteins is increasingly being recognized. The K+ channel interacting protein 2 (KChIP2), originally identified as an auxiliary subunit for KV4.2 and a component of the transient outward K+ channel (Ito), is a Ca2+-binding protein whose regulatory functions do not appear restricted to KV4.2. Consequently, we hypothesized that KChIP2 is a direct regulator of the cardiac L-type Ca2+ current (ICa,L). We found that ICa,L density from KChIP2−/− myocytes is reduced by 28% compared to ICa,L recorded from wild-type myocytes (P<0.05). This reduction in current density results from loss of a direct effect on the Ca2+ channel current, as shown in a transfected cell line devoid of confounding cardiac ion currents. ICa,L regulation by KChIP2 was independent of Ca2+ binding to KChIP2. Biochemical analysis suggested a direct interaction between KChIP2 and the CaV1.2 &agr;1C subunit N terminus. We found that KChIP2 binds to the N-terminal inhibitory module of &agr;1C and augments ICa,L current density without increasing CaV1.2 protein expression or trafficking to the plasma membrane. We propose a model in which KChIP2 impedes the N-terminal inhibitory module of CaV1.2, resulting in increased ICa,L. In the context of recent reports that KChIP2 modulates multiple KV and NaV currents, these results suggest that KChIP2 is a multimodal regulator of cardiac ionic currents.

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) Ser133 phosphorylation, but the physiologically relevant CREB-Ser133 kinase(s) in the heart remains uncertain. This study identifies a novel role for protein kinase D (PKD) as an in vivo cardiac CREB-Ser133 kinase. We show that thrombin activates a PKCδ-PKD pathway leading to CREB-Ser133 phosphorylation in cardiomyocytes and cardiac fibroblasts. α1-Adrenergic receptors also activate a PKCδ-PKD-CREB-Ser133 phosphorylation pathway in cardiomyocytes. Of note, while the epidermal growth factor (EGF) promotes CREB-Ser133 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-Ser133 phosphorylation in this cell type. Adenoviral-mediated overexpression of PKCδ (but not PKCϵ or PKCα) activates PKD; PKCδ and PKD1-S744E/S748E overexpression both promote CREB-Ser133 phosphorylation. Pasteuralla multocida toxin (PMT), a direct Gαq agonist that induces robust cardiomyocyte hypertrophy, also activates the PKD-CREB-Ser133 phosphorylation pathway, leading to the accumulation of active PKD and Ser133-phosphorylated CREB in the nucleus, activation of a CRE-responsive promoter, and increased Bcl-2 (CREB target gene) expression in cardiomyocyte cultures. Cardiac-specific Gαq overexpression also leads to an increase in PKD-Ser744/Ser748 and CREB-Ser133 phosphorylation as well as increased Bcl-2 protein expression in the hearts of transgenic mice. Collectively, these studies identify a novel Gαq-PKCδ-PKD-CREB-Ser133 phosphorylation pathway that is predicted to contribute to cardiac remodeling and could be targeted for therapeutic advantage in the setting of heart failure phenotypes.

HCN2/SkM1 Gene Transfer Into Canine Left Bundle Branch Induces Stable, Autonomically Responsive Biological Pacing at Physiological Heart Rates

OBJECTIVES This study sought to test the hypothesis that hyperpolarization-activated cyclic nucleotide-gated (HCN)-based biological pacing might be improved significantly by hyperpolarizing the action potential (AP) threshold via coexpression of the skeletal muscle sodium channel 1 (SkM1). BACKGROUND Gene-based biological pacemakers display effective in vivo pacemaker function. However, approaches used to date have failed to manifest optimal pacemaker properties, defined as basal beating rates of 60 to 90 beats/min, a brisk autonomic response achieving maximal rates of 130 to 160 beats/min, and low to absent electronic backup pacing. METHODS We implanted adenoviral SkM1, HCN2, or HCN2/SkM1 constructs into left bundle branches (LBB) or left ventricular (LV) epicardium of atrioventricular-blocked dogs. RESULTS During stable peak gene expression on days 5 to 7, HCN2/SkM1 LBB-injected dogs showed highly stable in vivo pacemaker activity superior to SkM1 or HCN2 alone and superior to LV-implanted dogs with regard to beating rates (resting approximately 80 beats/min; maximum approximately 130 beats/min), no dependence on electronic backup pacing, and enhanced modulation of pacemaker function during circadian rhythm or epinephrine infusion. In vitro isolated LV of dogs overexpressing SkM1 manifested a significantly more negative AP threshold. CONCLUSIONS LBB-injected HCN2/SkM1 potentially provides a more clinically suitable biological pacemaker strategy than other reported constructs. This superiority is attributable to the more negative AP threshold and injection into the LBB.

Reactive Oxygen Species Decrease cAMP Response Element Binding Protein Expression in Cardiomyocytes via a Protein Kinase D1-Dependent Mechanism That Does Not Require Ser133 Phosphorylation

Reactive oxygen species (ROS) exert pleiotropic effects on a wide array of signaling proteins that regulate cellular growth and apoptosis. This study shows that long-term treatment with a low concentration of H2O2 leads to the activation of signaling pathways involving extracellular signal-regulated kinase, ribosomal protein S6 kinase, and protein kinase D (PKD) that increase cAMP binding response element protein (CREB) phosphorylation at Ser133 in cardiomyocytes. Although CREB-Ser133 phosphorylation typically mediates cAMP-dependent increases in CREB target gene expression, the H2O2-dependent increase in CREB-Ser133 phosphorylation is accompanied by a decrease in CREB protein abundance and no change in Cre-luciferase reporter activity. Mutagenesis studies indicate that H2O2 decreases CREB protein abundance via a mechanism that does not require CREB-Ser133 phosphorylation. Rather, the H2O2-dependent decrease in CREB protein is prevented by the proteasome inhibitor lactacystin, by inhibitors of mitogen-activated protein kinase kinase or protein kinase C activity, or by adenoviral-mediated delivery of a small interfering RNA that decreases PKD1 expression. A PKD1-dependent mechanism that links oxidative stress to decreased CREB protein abundance is predicted to contribute to the pathogenesis of heart failure by influencing cardiac growth and apoptosis responses.

Cardiac memory: a work in progress.

Cardiac memory is a form of electrophysiological remodeling generally considered benign, although it shares transduction pathways with factors that may be pathological, such as angiotensin II and reactive oxygen species. When induced by electrical pacing, memory provides a window into the mechanisms engaged during cardiac device therapy. Emphasis is placed on the complexity of signaling processes occurring downstream to the simple intervention of cardiac pacing and the relationship of resultant ion channel changes to their expression in action potentials and body surface recordings.

Protein Kinase D Isoforms Are Activated in an Agonist-specific Manner in Cardiomyocytes

Protein kinase D (PKD) exists as a family of structurally related enzymes that are activated through similar phosphorylation-dependent mechanisms involving protein kinase C (PKC). While individual PKD isoforms could in theory mediate distinct biological functions, previous studies identify a high level of functional redundancy for PKD1 and PKD2 in various cellular contexts. This study shows that PKD1 and PKD2 are activated in a stimulus-specific manner in neonatal cardiomyocytes. The α1-adrenergic receptor agonist norepinephrine selectively activates PKD1, thrombin and PDGF selectively activate PKD2, and endothelin-1 and PMA activate both PKD1 and PKD2. PKC activity is implicated in the α1-adrenergic receptor pathway that activates PKD1 and the thrombin- and PDGF-dependent pathways that activate PKD2. Endothelin-1 activates PKD via both rapid PKC-dependent and more sustained PKC-independent mechanisms. The functional consequences of PKD activation were assessed by tracking phosphorylation of CREB and cardiac troponin I (cTnI), two physiologically relevant PKD substrates in cardiomyocytes. We show that overexpression of an activated PKD1-S744E/S748E transgene increases CREB-Ser133 and cTnI-Ser23/Ser24 phosphorylation, but agonist-dependent pathways that activate native PKD1 or PKD2 selectively increase CREB-Ser133 phosphorylation; there is no associated increase in cTnI-Ser23/Ser24 phosphorylation. Gene silencing studies provide unanticipated evidence that PKD1 down-regulation leads to a compensatory increase in PKD2 activity and that down-regulation of PKD1 (alone or in combination with PKD2) leads to an increase in CREB-Ser133 phosphorylation. Collectively, these studies identify distinct roles for native PKD1 and PKD2 enzymes in stress-dependent pathways that influence cardiac remodeling and the progression of heart failure.

Deleting the accessory subunit KChIP2 results in loss of Ito,f and increased IK,slow that maintains normal action potential configuration

BACKGROUND Four voltage-gated potassium currents, I(to,f) (K(V)4.2), I(to,s) (K(V)1.4), I(K,slow) (K(V)1.5+K(V)2.1), and I(SS) (TASK1), govern murine ventricular repolarization. Although the accessory subunit KChIP2 influences I(to,f) expression, in preliminary experiments we found that action potential duration (APD) is maintained in KChIP2 knockout mice. OBJECTIVE We tested the role of KChIP2 in regulating APD and studied the underlying ionic currents. METHODS We used microelectrode techniques, whole-cell patch clamp studies, and real-time polymerase chain reaction amplification to characterize ventricular repolarization and its determinants in wild-type and KChIP2(-/-) mice. RESULTS Despite comparable baseline action potentials, APD was more markedly prolonged by 4-aminopyridine (4-AP) in KChIP2(-/-) preparations. Peak K(+) current densities were similar in wild-type and KChIP2(-/-) cells (mean +/- SEM I(P): 28.3 +/- 2 (n = 27) vs. 29.2 +/- 2 pA/pF (n = 24), respectively; P > .05). Heteropodatoxin-2 (HpTx-2, 1 microM) had no effect on current amplitude in KChIP2(-/-) myocytes. The current fractions sensitive to 4-AP (50 microM and 1 mM) were larger in KChIP2(-/-) than wild-type (P < .05). Real-time polymerase chain reaction showed absence of KChIP2 and increased K(V)1.5 expression in KChIP2(-/-) ventricular myocardium. CONCLUSION KChIP2 deficiency eliminated HpTx-2-sensitive I(to,f), but had little impact on total APD, secondary to upregulation of 4-AP-sensitive I(K,slow) in association with increased K(V)1.5 expression. There is increased sensitivity to 4-AP-mediated APD prolongation in KChIP2(-/-). Thus, KChIP2 seems important for murine repolarization in circumstances of reduced repolarization reserve.

Expression of Skeletal Muscle Sodium Channel (Nav1.4) or Connexin32 Prevents Reperfusion Arrhythmias in Murine Heart

AIMS acute myocardial ischaemia induces a decrease in resting membrane potential [which leads to reduction of action potential (AP) V(max)] and intracellular acidification (which closes gap junctions). Both contribute to conduction slowing. We hypothesized that ventricular expression of the skeletal muscle Na(+) channel, Nav1.4 (which activates fully at low membrane potentials), or connexin32 (Cx32, which is less pH-sensitive than connexin43) would support conduction and be antiarrhythmic. We tested this hypothesis in a murine model of ischaemia and reperfusion arrhythmias. METHODS AND RESULTS empty adenovirus (Sham) or adenoviral constructs expressing either SkM1 (gene encoding Nav1.4) or Cx32 genes were injected into the left ventricular wall. Four days later, ventricular tachycardia (VT) occurred during reperfusion following a 5 min coronary occlusion. In Nav1.4- and Cx32-expressing mice, VT incidence and duration were lower than in Sham (P < 0.05). In vitro multisite microelectrode mapping was performed in the superfused right ventricular wall. To simulate ischaemic conditions, [K(+)] in solution was increased to 10 mmol/L and/or pH was decreased to 6.0. Western blots revealed Cx32 and Nav1.4 expression in both ventricles. Nav1.4 APs showed higher V(max) and conduction velocity (CV) than Shams at normal and elevated [K(+)]. Exposure of tissue to acid solution reduced intracellular pH to 6.4. There was no difference in CV between Sham and Cx32 groups in control solution. Acid solution slowed CV in Sham (P < 0.05) but not in Cx32. CONCLUSION Nav1.4 or Cx32 expression preserved normal conduction in murine hearts and decreased the incidence of reperfusion VT.

Determinants of CREB degradation and KChIP2 gene transcription in cardiac memory

BACKGROUND Left ventricular pacing (LVP) to induce cardiac memory (CM) in dogs results in a decreased transient outward K current (I(to)) and reduced mRNA and protein of the I(to) channel accessory subunit, KChIP2. The KChIP2 decrease is attributed to a decrease in its transcription factor, cyclic adenosine monophosphate response element binding protein (CREB). OBJECTIVE This study sought to determine the mechanisms responsible for the CREB decrease that is initiated by LVP. METHODS CM was quantified as T-wave vector displacement in 18 LVP dogs. In 5 dogs, angiotensin II receptor blocker, saralasin, was infused before and during pacing. In 3 dogs, proteasomal inhibitor, lactacystin, was injected into the left anterior descending artery before LVP. Epicardial biopsy samples were taken before and after LVP. Neonatal rat cardiomyocytes (NRCM) were incubated with H(2)O(2) (50 micromol/l) for 1 hour with or without lactacystin. RESULTS LVP significantly displaced the T-wave vector and was associated with increased lipid peroxidation and increased tissue angiotensin II levels. Saralasin prevented T-vector displacement and lipid peroxidation. CREB was significantly decreased after 2 hours of LVP and was comparably decreased in H(2)O(2)-treated NRCM. Lactacystin inhibited the CREB decrease in LVP dogs and H(2)O(2)-treated NRCM. LVP and H(2)O(2) both induced CREB ubiquitination, and the H(2)O(2)-induced CREB decrease was prevented by knocking down ubiquitin. CONCLUSION LVP initiates myocardial angiotensin II production and reactive oxygen species synthesis, leading to CREB ubiquitination and its proteasomal degradation. This sequence of events would explain the pacing-induced reduction in KChIP2, and contribute to altered repolarization and the T-wave changes of cardiac memory.