Jessica Ibetti

Prodeath Signaling of G Protein–Coupled Receptor Kinase 2 in Cardiac Myocytes After Ischemic Stress Occurs Via Extracellular Signal–Regulated Kinase-Dependent Heat Shock Protein 90–Mediated Mitochondrial Targeting

Rationale: GRK2 is abundantly expressed in the heart and its expression and activity is increased in injured or stressed myocardium. This up-regulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes and its inhibition by a peptide comprised of the last 194 amino acids of GRK2 (known as βARKct) is cardioprotective. Objective: The aim of this study was to elucidate the signaling mechanism that accounts for the pro-death signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the βARKct. Methods and Results: Using in vivo mouse models of ischemic injury and also cultured myocytes we found that GRK2 localizes to mitochondria providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced pro-death signaling. Localization of GRK2 to mitochondria was dependent upon phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal-regulated kinases (ERKs), resulting in enhanced GRK2 binding to heat shock protein 90 (Hsp90), which chaperoned GRK2 to mitochondria. Mechanistic studies invivo and invitro showed that ERK regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent pro-death signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca2+-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. Conclusions: We identify GRK2 as a pro-death kinase in the heart acting in a novel manner through mitochondrial localization via ERK regulation.Rationale: G protein–coupled receptor kinase 2 (GRK2) is abundantly expressed in the heart, and its expression and activity are increased in injured or stressed myocardium. This upregulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes, and its inhibition by a peptide comprising the last 194 amino acids of GRK2 (known as carboxyl-terminus of &bgr;-adrenergic receptor kinase [bARKct]) is cardioprotective. Objective: The aim of this study was to elucidate the signaling mechanism that accounts for the prodeath signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the carboxyl-terminus of &bgr;-adrenergic receptor kinase. Methods and Results: Using in vivo mouse models of ischemic injury and also cultured myocytes, we found that GRK2 localizes to mitochondria, providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced prodeath signaling. Localization of GRK2 to mitochondria was dependent on phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal–regulated kinases, resulting in enhanced GRK2 binding to heat shock protein 90, which chaperoned GRK2 to mitochondria. Mechanistic studies in vivo and in vitro showed that extracellular signal–regulated kinase regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent prodeath signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca2+-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. Conclusions: We identify GRK2 as a prodeath kinase in the heart, acting in a novel manner through mitochondrial localization via extracellular signal–regulated kinase regulation.

Pro-Death Signaling of GRK2 in Cardiac Myocytes after Ischemic Stress Occurs via ERK-Dependent, Hsp90-Mediated Mitochondrial Targeting

Rationale: GRK2 is abundantly expressed in the heart and its expression and activity is increased in injured or stressed myocardium. This up-regulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes and its inhibition by a peptide comprised of the last 194 amino acids of GRK2 (known as βARKct) is cardioprotective. Objective: The aim of this study was to elucidate the signaling mechanism that accounts for the pro-death signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the βARKct. Methods and Results: Using in vivo mouse models of ischemic injury and also cultured myocytes we found that GRK2 localizes to mitochondria providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced pro-death signaling. Localization of GRK2 to mitochondria was dependent upon phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal-regulated kinases (ERKs), resulting in enhanced GRK2 binding to heat shock protein 90 (Hsp90), which chaperoned GRK2 to mitochondria. Mechanistic studies invivo and invitro showed that ERK regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent pro-death signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca2+-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. Conclusions: We identify GRK2 as a pro-death kinase in the heart acting in a novel manner through mitochondrial localization via ERK regulation.Rationale: G protein–coupled receptor kinase 2 (GRK2) is abundantly expressed in the heart, and its expression and activity are increased in injured or stressed myocardium. This upregulation has been shown to be pathological. GRK2 can promote cell death in ischemic myocytes, and its inhibition by a peptide comprising the last 194 amino acids of GRK2 (known as carboxyl-terminus of &bgr;-adrenergic receptor kinase [bARKct]) is cardioprotective. Objective: The aim of this study was to elucidate the signaling mechanism that accounts for the prodeath signaling seen in the presence of elevated GRK2 and the cardioprotection afforded by the carboxyl-terminus of &bgr;-adrenergic receptor kinase. Methods and Results: Using in vivo mouse models of ischemic injury and also cultured myocytes, we found that GRK2 localizes to mitochondria, providing novel insight into GRK2-dependent pathophysiological signaling mechanisms. Mitochondrial localization of GRK2 in cardiomyocytes was enhanced after ischemic and oxidative stress, events that induced prodeath signaling. Localization of GRK2 to mitochondria was dependent on phosphorylation at residue Ser670 within its extreme carboxyl-terminus by extracellular signal–regulated kinases, resulting in enhanced GRK2 binding to heat shock protein 90, which chaperoned GRK2 to mitochondria. Mechanistic studies in vivo and in vitro showed that extracellular signal–regulated kinase regulation of the C-tail of GRK2 was an absolute requirement for stress-induced, mitochondrial-dependent prodeath signaling, and blocking this led to cardioprotection. Elevated mitochondrial GRK2 also caused increased Ca2+-induced opening of the mitochondrial permeability transition pore, a key step in cellular injury. Conclusions: We identify GRK2 as a prodeath kinase in the heart, acting in a novel manner through mitochondrial localization via extracellular signal–regulated kinase regulation.

Myocardial pathology induced by aldosterone is dependent on non-canonical activities of G protein-coupled receptor kinases

Hyper-aldosteronism is associated with myocardial dysfunction including induction of cardiac fibrosis and maladaptive hypertrophy. Mechanisms of these cardiotoxicities are not fully understood. Here we show that mineralocorticoid receptor (MR) activation by aldosterone leads to pathological myocardial signalling mediated by mitochondrial G protein-coupled receptor kinase 2 (GRK2) pro-death activity and GRK5 pro-hypertrophic action. Moreover, these MR-dependent GRK2 and GRK5 non-canonical activities appear to involve cross-talk with the angiotensin II type-1 receptor (AT1R). Most importantly, we show that ventricular dysfunction caused by chronic hyper-aldosteronism in vivo is completely prevented in cardiac Grk2 knockout mice (KO) and to a lesser extent in Grk5 KO mice. However, aldosterone-induced cardiac hypertrophy is totally prevented in Grk5 KO mice. We also show human data consistent with MR activation status in heart failure influencing GRK2 levels. Therefore, our study uncovers GRKs as targets for ameliorating pathological cardiac effects associated with high-aldosterone levels.

GRK2 compromises cardiomyocyte mitochondrial function by diminishing fatty acid-mediated oxygen consumption and increasing superoxide levels.

The G protein-coupled receptor kinase-2 (GRK2) is upregulated in the injured heart and contributes to heart failure pathogenesis. GRK2 was recently shown to associate with mitochondria but its functional impact in myocytes due to this localization is unclear. This study was undertaken to determine the effect of elevated GRK2 on mitochondrial respiration in cardiomyocytes. Sub-fractionation of purified cardiac mitochondria revealed that basally GRK2 is found in multiple compartments. Overexpression of GRK2 in mouse cardiomyocytes resulted in an increased amount of mitochondrial-based superoxide. Inhibition of GRK2 increased oxygen consumption rates and ATP production. Moreover, fatty acid oxidation was found to be significantly impaired when GRK2 was elevated and was dependent on the catalytic activity and mitochondrial localization of this kinase. Our study shows that independent of cardiac injury, GRK2 is localized in the mitochondria and its kinase activity negatively impacts the function of this organelle by increasing superoxide levels and altering substrate utilization for energy production.

Mitochondrial fusion dynamics is robust in the heart and depends on calcium oscillations and contractile activity

Significance Mitochondrial function is supported by dynamic quality control processes, such as mitochondrial fusion. Cardiac contractility depends on mitochondrial metabolism, yet in cardiomyocytes, mitochondria are confined among myofibrils, raising questions about the possibility of mitochondrial physical communication. Here we demonstrate that mitochondrial continuity is robust and fusion is frequent in freshly isolated rat ventricular myocytes, manifesting both as rapid content mixing events between adjacent organelles and slower, often long-distance events. We show that mitochondrial fusion decreases dramatically in culture because of the decay in contractile activity and, more specifically, the underlying calcium oscillations, which involve mitofusin 1 (Mfn1) abundance. In addition, we show that attenuation of cardiac contractility in vivo in alcoholic animals is also associated with depressed mitochondrial fusion. Mitochondrial fusion is thought to be important for supporting cardiac contractility, but is hardly detectable in cultured cardiomyocytes and is difficult to directly evaluate in the heart. We overcame this obstacle through in vivo adenoviral transduction with matrix-targeted photoactivatable GFP and confocal microscopy. Imaging in whole rat hearts indicated mitochondrial network formation and fusion activity in ventricular cardiomyocytes. Promptly after isolation, cardiomyocytes showed extensive mitochondrial connectivity and fusion, which decayed in culture (at 24–48 h). Fusion manifested both as rapid content mixing events between adjacent organelles and slower events between both neighboring and distant mitochondria. Loss of fusion in culture likely results from the decline in calcium oscillations/contractile activity and mitofusin 1 (Mfn1), because (i) verapamil suppressed both contraction and mitochondrial fusion, (ii) after spontaneous contraction or short-term field stimulation fusion activity increased in cardiomyocytes, and (iii) ryanodine receptor-2–mediated calcium oscillations increased fusion activity in HEK293 cells and complementing changes occurred in Mfn1. Weakened cardiac contractility in vivo in alcoholic animals is also associated with depressed mitochondrial fusion. Thus, attenuated mitochondrial fusion might contribute to the pathogenesis of cardiomyopathy.

Therapeutic inhibition of miR-375 attenuates post-myocardial infarction inflammatory response and left ventricular dysfunction via PDK-1-AKT signalling axis

Aims Increased miR-375 levels has been implicated in rodent models of myocardial infarction (MI) and with patients with heart failure. However, no prior study had established a therapeutic role of miR-375 in ischemic myocardium. Therefore, we assessed whether inhibition of MI-induced miR-375 by LNA anti-miR-375 can improve recovery after acute MI. Methods and results Ten weeks old mice were treated with either control or LNA anti miR-375 after induction of MI by LAD ligation. The inflammatory response, cardiomyocyte apoptosis, capillary density and left ventricular (LV) functional, and structural remodelling changes were evaluated. Anti-miR-375 therapy significantly decreased inflammatory response and reduced cardiomyocyte apoptosis in the ischemic myocardium and significantly improved LV function and neovascularization and reduced infarct size. Repression of miR-375 led to the activation of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) and increased AKT phosphorylation on Thr-308 in experimental hearts. In corroboration with our in vivo findings, our in vitro studies demonstrated that knockdown of miR-375 in macrophages modulated their phenotype, enhanced PDK-1 levels, and reduced pro-inflammatory cytokines expression following LPS challenge. Further, miR-375 levels were elevated in failing human heart tissue. Conclusion Taken together, our studies demonstrate that anti-miR-375 therapy reduced inflammatory response, decreased cardiomyocyte death, improved LV function, and enhanced angiogenesis by targeting multiple cell types mediated at least in part through PDK-1/AKT signalling mechanisms.

Differential Role of G Protein–Coupled Receptor Kinase 5 in Physiological Versus Pathological Cardiac Hypertrophy

RATIONALE G protein-coupled receptor kinases (GRKs) are dynamic regulators of cellular signaling. GRK5 is highly expressed within myocardium and is upregulated in heart failure. Although GRK5 is a critical regulator of cardiac G protein-coupled receptor signaling, recent data has uncovered noncanonical activity of GRK5 within nuclei that plays a key role in pathological hypertrophy. Targeted cardiac elevation of GRK5 in mice leads to exaggerated hypertrophy and early heart failure after transverse aortic constriction (TAC) because of GRK5 nuclear accumulation. OBJECTIVE In this study, we investigated the role of GRK5 in physiological, swimming-induced hypertrophy (SIH). METHODS AND RESULTS Cardiac-specific GRK5 transgenic mice and nontransgenic littermate control mice were subjected to a 21-day high-intensity swim protocol (or no swim sham controls). SIH and specific molecular and genetic indices of physiological hypertrophy were assessed, including nuclear localization of GRK5, and compared with TAC. Unlike after TAC, swim-trained transgenic GRK5 and nontransgenic littermate control mice exhibited similar increases in cardiac growth. Mechanistically, SIH did not lead to GRK5 nuclear accumulation, which was confirmed in vitro as insulin-like growth factor-1, a known mediator of physiological hypertrophy, was unable to induce GRK5 nuclear translocation in myocytes. We found specific patterns of altered gene expression between TAC and SIH with GRK5 overexpression. Further, SIH in post-TAC transgenic GRK5 mice was able to preserve cardiac function. CONCLUSIONS These data suggest that although nuclear-localized GRK5 is a pathological mediator after stress, this noncanonical nuclear activity of GRK5 is not induced during physiological hypertrophy.

Dynamic mass redistribution analysis of endogenous β-adrenergic receptor signaling in neonatal rat cardiac fibroblasts.

Label‐free systems for the agnostic assessment of cellular responses to receptor stimulation have been shown to provide a sensitive method to dissect receptor signaling. β‐adenergic receptors (βAR) are important regulators of normal and pathologic cardiac function and are expressed in cardiomyocytes as well as cardiac fibroblasts, where relatively fewer studies have explored their signaling responses. Using label‐free whole cell dynamic mass redistribution (DMR) assays we investigated the response patterns to stimulation of endogenous βAR in primary neonatal rat cardiac fibroblasts (NRCF). The EPIC‐BT by Corning was used to measure DMR responses in primary isolated NRCF treated with various βAR and EGFR ligands. Additional molecular assays for cAMP generation and receptor internalization responses were used to correlate the DMR findings with established βAR signaling pathways. Catecholamine stimulation of NRCF induced a concentration‐dependent negative DMR deflection that was competitively blocked by βAR blockade and non‐competitively blocked by irreversible uncoupling of Gs proteins. Subtype‐selective βAR ligand profiling revealed a dominant role for β2AR in mediating the DMR responses, consistent with the relative expression levels of β2AR and β1AR in NRCF. βAR‐mediated cAMP generation profiles revealed similar kinetics to DMR responses, each of which were enhanced via inhibition of cAMP degradation, as well as dynamin‐mediated receptor internalization. Finally, G protein‐independent βAR signaling through epidermal growth factor receptor (EGFR) was assessed, revealing a smaller but significant contribution of this pathway to the DMR response to βAR stimulation. Measurement of DMR responses in primary cardiac fibroblasts provides a sensitive readout for investigating endogenous βAR signaling via both G protein‐dependent and –independent pathways.

The increase in maternal expression of axin1 and axin2 contribute to the zebrafish mutant ichabod ventralized phenotype.

β‐catenin is a central effector of the Wnt pathway and one of the players in Ca+‐dependent cell‐cell adhesion. While many wnts are present and expressed in vertebrates, only one β‐catenin exists in the majority of the organisms. One intriguing exception is zebrafish that carries two genes for β‐catenin. The maternal recessive mutation ichabod presents very low levels of β‐catenin2 that in turn affects dorsal axis formation, suggesting that β‐catenin1 is incapable to compensate for β‐catenin2 loss and raising the question of whether these two β‐catenins may have differential roles during early axis specification. Here we identify a specific antibody that can discriminate selectively for β‐catenin1. By confocal co‐immunofluorescent analysis and low concentration gain‐of‐function experiments, we show that β‐catenin1 and 2 behave in similar modes in dorsal axis induction and cellular localization. Surprisingly, we also found that in the ich embryo the mRNAs of the components of β‐catenin regulatory pathway, including β‐catenin1, are more abundant than in the Wt embryo. Increased levels of β‐catenin1 are found at the membrane level but not in the nuclei till high stage. Finally, we present evidence that β‐catenin1 cannot revert the ich phenotype because it may be under the control of a GSK3β‐independent mechanism that required Axins RGS domain function. J. Cell. Biochem. 116: 418–430, 2015.

FOXD1-dependent MICU1 expression regulates mitochondrial activity and cell differentiation

Although many factors contribute to cellular differentiation, the role of mitochondria Ca2+ dynamics during development remains unexplored. Because mammalian embryonic epiblasts reside in a hypoxic environment, we intended to understand whether mCa2+ and its transport machineries are regulated during hypoxia. Tissues from multiple organs of developing mouse embryo evidenced a suppression of MICU1 expression with nominal changes on other MCU complex components. As surrogate models, we here utilized human embryonic stem cells (hESCs)/induced pluripotent stem cells (hiPSCs) and primary neonatal myocytes to delineate the mechanisms that control mCa2+ and bioenergetics during development. Analysis of MICU1 expression in hESCs/hiPSCs showed low abundance of MICU1 due to its direct repression by Foxd1. Experimentally, restoration of MICU1 established the periodic cCa2+ oscillations and promoted cellular differentiation and maturation. These findings establish a role of mCa2+ dynamics in regulation of cellular differentiation and reveal a molecular mechanism underlying this contribution through differential regulation of MICU1.Genetic ablation of Mitochondrial Ca2+ uptake protein 1 (MICU1) in mouse induces higher rates of perinatal lethality. Here the authors show that MICU1 expression is regulated by hypoxia in a FOXD1-dependent manner, establishing a cyclic switch between glycolytic and oxidative metabolism during development.