RESUMO
Calcium-calmodulin dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac electrophysiology, calcium (Ca) balance, contraction, transcription, arrhythmias and progression to heart failure. CaMKII is readily activated at mouths of dyadic cleft Ca channels, but because of its low Ca-calmodulin affinity and presumed immobility it is less clear how CaMKII gets activated near other known, extra-dyad targets. CaMKII is typically considered to be anchored in cardiomyocytes, but while untested, mobility of active CaMKII could provide a mechanism for broader target phosphorylation in cardiomyocytes. We therefore tested CaMKII mobility and how this is affected by kinase activation in adult rabbit cardiomyocytes. We measured translocation of both endogenous and fluorescence-tagged CaMKII using immunocytochemistry, fluorescence recovery after photobleach (FRAP) and photoactivation of fluorescence. In contrast to the prevailing view that CaMKII is anchored near its myocyte targets, we found CaMKII to be highly mobile in resting myocytes, which was slowed by Ca chelation and accelerated by pacing. At low [Ca], CaMKII was concentrated at Z-lines near the dyad but spread throughout the sarcomere upon pacing. Nuclear exchange of CaMKII was also enhanced upon pacing- and heart failure-induced chronic activation. This mobilization of active CaMKII and its intrinsic memory may allow CaMKII to be activated in high [Ca] regions and then move towards more distant myocyte target sites.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Miócitos Cardíacos/metabolismo , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Células Cultivadas , Imuno-Histoquímica , Fosforilação , Coelhos , Retículo Sarcoplasmático/metabolismo , Transdução de Sinais/fisiologiaRESUMO
RATIONALE: Both ß-adrenergic receptor (ß-AR) and Gq-coupled receptor (GqR) agonist-driven signaling play key roles in the events, leading up to and during cardiac dysfunction. How these stimuli interact at the level of protein kinase D (PKD), a nodal point in cardiac hypertrophic signaling, remains unclear. OBJECTIVE: To assess the spatiotemporal dynamics of PKD activation in response to ß-AR signaling alone and on coactivation with GqR-agonists. This will test our hypothesis that compartmentalized PKD signaling reconciles disparate findings of PKA facilitation and inhibition of PKD activation. METHODS AND RESULTS: We report on the spatial and temporal profiles of PKD activation using green fluorescent protein-tagged PKD (wildtype or mutant S427E) and targeted fluorescence resonance energy transfer-based biosensors (D-kinase activity reporters) in adult cardiomyocytes. We find that ß-AR/PKA signaling drives local nuclear activation of PKD, without preceding sarcolemmal translocation. We also discover pronounced interference of ß-AR/cAMP/PKA signaling on GqR-induced translocation and activation of PKD throughout the cardiomyocyte. We attribute these effects to direct, PKA-dependent phosphorylation of PKD-S427. We also show that phosphomimetic substitution of S427 likewise impedes GqR-induced PKD translocation and activation. In neonatal myocytes, S427E inhibits GqR-evoked cell growth and expression of hypertrophic markers. Finally, we show altered S427 phosphorylation in transverse aortic constriction-induced hypertrophy. CONCLUSIONS: ß-AR signaling triggers local nuclear signaling and inhibits GqR-mediated PKD activation by preventing its intracellular translocation. PKA-dependent phosphorylation of PKD-S427 fine-tunes the PKD responsiveness to GqR-agonists, serving as a key integration point for ß-adrenergic and Gq-coupled stimuli.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Miócitos Cardíacos/enzimologia , Proteína Quinase C/metabolismo , Receptores Adrenérgicos beta/metabolismo , Transdução de Sinais , Agonistas Adrenérgicos beta/farmacologia , Animais , Cardiomegalia/enzimologia , Cardiomegalia/patologia , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Modelos Animais de Doenças , Ativação Enzimática , Transferência Ressonante de Energia de Fluorescência , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Fosforilação , Proteína Quinase C/genética , Transporte Proteico , Coelhos , Ratos , Receptores Adrenérgicos beta/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , TransfecçãoRESUMO
Aim: Aging and heart failure (HF) are each characterized by increased mitochondrial damage, which may contribute to further cardiac dysfunction. Mitophagy in response to mitochondrial damage can improve cardiovascular health. HF is also characterized by increased formation and consumption of ketone bodies (KBs), which may activate mitophagy and provide an endogenous mechanism to limit the adverse effects of mitochondrial damage. However, the role of KBs in activation of mitophagy in aging and HF has not been evaluated. Methods: We assessed mitophagy by measuring mitochondrial Parkin accumulation and LC3-mediated autophagosome formation in cardiomyocytes from young (2.5 months), aged (2.5 years), and aged rabbits with HF (2.5 years) induced by aortic insufficiency and stenosis. Levels of reactive oxygen species (ROS) generation and redox balance were monitored using genetically encoded sensors ORP1-roGFP2 and GRX1-roGFP2, targeted to mitochondrial or cytosolic compartments, respectively. Results: Young rabbits exhibited limited mitochondrial Parkin accumulation with small (~1 µm2) puncta. Those small Parkin puncta increased four-fold in aged rabbit hearts, accompanied by elevated LC3-mediated autophagosome formation. HF hearts exhibited fewer small puncta, but many very large Parkin-rich regions (4-5 µm2) with completely depolarized mitochondria. Parkin protein expression was barely detectable in young animals and was much higher in aged and maximal in HF hearts. Expression of mitofusin 2 (MFN2) and dynamin-related protein 1 (DRP1) was reduced by almost 50% in HF, consistent with improper fusion-fission, contributing to mitochondrial Parkin build-up. The KB ß-hydroxybutyrate (ß-OHB) enhanced mitophagy in young and aging myocytes, but not in HF where ß-OHB further increased the number of cells with giant Parkin-rich regions. This ß-OHB effect on Parkin-rich areas was prevented by cell-permeable TAT-MP1Gly peptide (thought to promote MFN2-dependent fusion). Basal levels of mitochondrial ROS were highest in HF, while cytosolic ROS was highest in aged compared to HF myocytes, suggesting that cytosolic ROS promotes Parkin recruitment to the mitochondria. Conclusion: We conclude that elevated KB levels were beneficial for mitochondrial repair in the aging heart. However, an impaired MFN2-DRP1-mediated fusion-fission process in HF reduced this benefit, as well as Parkin degradation and mitophagic signaling cascade.