Tipifarnib Reduces Extracellular Vesicles and Protects From Heart Failure.

BACKGROUND: Heart failure (HF) is one of the leading causes of mortality worldwide. Extracellular vesicles, including small extracellular vesicles or exosomes, and their molecular cargo are known to modulate cell-to-cell communication during multiple cardiac diseases. However, the role of systemic extracellular vesicle biogenesis inhibition in HF models is not well documented and remains unclear. METHODS: We investigated the role of circulating exosomes during cardiac dysfunction and remodeling in a mouse transverse aortic constriction (TAC) model of HF. Importantly, we investigate the efficacy of tipifarnib, a recently identified exosome biogenesis inhibitor that targets the critical proteins (Rab27a [Ras associated binding protein 27a], nSMase2 [neutral sphingomyelinase 2], and Alix [ALG-2-interacting protein X]) involved in exosome biogenesis for this mouse model of HF. In this study, 10-week-old male mice underwent TAC surgery were randomly assigned to groups with and without tipifarnib treatment (10 mg/kg 3 times/wk) and monitored for 8 weeks, and a comprehensive assessment was conducted through performed echocardiographic, histological, and biochemical studies. RESULTS: TAC significantly elevated circulating plasma exosomes and markedly increased cardiac left ventricular dysfunction, cardiac hypertrophy, and fibrosis. Furthermore, injection of plasma exosomes from TAC mice induced left ventricular dysfunction and cardiomyocyte hypertrophy in uninjured mice without TAC. On the contrary, treatment of tipifarnib in TAC mice reduced circulating exosomes to baseline and remarkably improved left ventricular functions, hypertrophy, and fibrosis. Tipifarnib treatment also drastically altered the miRNA profile of circulating post-TAC exosomes, including miR 331-5p, which was highly downregulated both in TAC circulating exosomes and in TAC cardiac tissue. Mechanistically, miR 331-5p is crucial for inhibiting the fibroblast-to-myofibroblast transition by targeting HOXC8, a critical regulator of fibrosis. Tipifarnib treatment in TAC mice upregulated the expression of miR 331-5p that acts as a potent repressor for one of the fibrotic mechanisms mediated by HOXC8. CONCLUSIONS: Our study underscores the pathological role of exosomes in HF and fibrosis in response to pressure overload. Tipifarnib-mediated inhibition of exosome biogenesis and cargo sorting may serve as a viable strategy to prevent progressive cardiac remodeling in HF.

GRK5 is a regulator of fibroblast activation and cardiac fibrosis.

Pathological remodeling of the heart is a hallmark of chronic heart failure (HF) and these structural changes further perpetuate the disease. Cardiac fibroblasts are the critical cell type that is responsible for maintaining the structural integrity of the heart. Stress conditions, such as a myocardial infarction (MI), can activate quiescent fibroblasts into synthetic and contractile myofibroblasts. G protein-coupled receptor kinase 5 (GRK5) is an important mediator of cardiovascular homeostasis through dampening of GPCR signaling, and is expressed in the heart and up-regulated in human HF. Of note, GRK5 has been demonstrated to translocate to the nucleus in cardiomyocytes in a calcium-calmodulin (Ca2+-CAM)-dependent manner, promoting hypertrophic gene transcription through activation of nuclear factor of activated T cells (NFAT). Interestingly, NFAT is also involved in fibroblast activation. GRK5 is highly expressed and active in cardiac fibroblasts; however, its pathophysiological role in these crucial cardiac cells is unknown. We demonstrate using adult cardiac fibroblasts that genetic deletion of GRK5 inhibits angiotensin II (AngII)-mediated fibroblast activation. Fibroblast-specific deletion of GRK5 in mice led to decreased fibrosis and cardiac hypertrophy after chronic AngII infusion or after ischemic injury compared to nontransgenic littermate controls (NLCs). Mechanistically, we show that nuclear translocation of GRK5 is involved in fibroblast activation. These data demonstrate that GRK5 is a regulator of fibroblast activation in vitro and cardiac fibrosis in vivo. This adds to previously published data which demonstrate the potential beneficial effects of GRK5 inhibition in the context of cardiac disease.

Pepducin ICL1-9-Mediated ß2-Adrenergic Receptor-Dependent Cardiomyocyte Contractility Occurs in a Gi Protein/ROCK/PKD-Sensitive Manner.

PURPOSE: ß-Adrenergic receptors (ßAR) are essential targets for the treatment of heart failure (HF); however, chronic use of ßAR agonists as positive inotropes to increase contractility in a Gs protein-dependent manner is associated with increased mortality. Alternatively, we previously reported that allosteric modulation of ß2AR with the pepducin intracellular loop (ICL)1-9 increased cardiomyocyte contractility in a ß-arrestin (ßarr)-dependent manner, and subsequently showed that ICL1-9 activates the Ras homolog family member A (RhoA). Here, we aimed to elucidate both the proximal and downstream signaling mediators involved in the promotion of cardiomyocyte contractility in response to ICL1-9. METHODS: We measured adult mouse cardiomyocyte contractility in response to ICL1-9 or isoproterenol (ISO, as a positive control) alone or in the presence of inhibitors of various potential components of ßarr- or RhoA-dependent signaling. We also assessed the contractile effects of ICL1-9 on cardiomyocytes lacking G protein-coupled receptor (GPCR) kinase 2 (GRK2) or 5 (GRK5). RESULTS: Consistent with RhoA activation by ICL1-9, both Rho-associated protein kinase (ROCK) and protein kinase D (PKD) inhibition were able to attenuate ICL1-9-mediated contractility, as was inhibition of myosin light chain kinase (MLCK). While neither GRK2 nor GRK5 deletion impacted ICL1-9-mediated contractility, pertussis toxin attenuated the response, suggesting that ICL1-9 promotes downstream RhoA-dependent signaling in a Gi protein-dependent manner. CONCLUSION: Altogether, our study highlights a novel signaling modality that may offer a new approach to the promotion, or preservation, of cardiac contractility during HF via the allosteric regulation of ß2AR to promote Gi protein/ßarr-dependent activation of RhoA/ROCK/PKD signaling.

SPG7 Is an Essential and Conserved Component of the Mitochondrial Permeability Transition Pore.

Mitochondrial permeability transition is a phenomenon in which the mitochondrial permeability transition pore (PTP) abruptly opens, resulting in mitochondrial membrane potential (ΔΨm) dissipation, loss of ATP production, and cell death. Several genetic candidates have been proposed to form the PTP complex, however, the core component is unknown. We identified a necessary and conserved role for spastic paraplegia 7 (SPG7) in Ca(2+)- and ROS-induced PTP opening using RNAi-based screening. Loss of SPG7 resulted in higher mitochondrial Ca(2+) retention, similar to cyclophilin D (CypD, PPIF) knockdown with sustained ΔΨm during both Ca(2+) and ROS stress. Biochemical analyses revealed that the PTP is a heterooligomeric complex composed of VDAC, SPG7, and CypD. Silencing or disruption of SPG7-CypD binding prevented Ca(2+)- and ROS-induced ΔΨm depolarization and cell death. This study identifies an ubiquitously expressed IMM integral protein, SPG7, as a core component of the PTP at the OMM and IMM contact site.

Enhanced NCLX-dependent mitochondrial Ca2+ efflux attenuates pathological remodeling in heart failure.

Mitochondrial calcium (mCa2+) uptake couples changes in cardiomyocyte energetic demand to mitochondrial ATP production. However, excessive mCa2+ uptake triggers permeability transition and necrosis. Despite these established roles during acute stress, the involvement of mCa2+ signaling in cardiac adaptations to chronic stress remains poorly defined. Changes in NCLX expression are reported in heart failure (HF) patients and models of cardiac hypertrophy. Therefore, we hypothesized that altered mCa2+ homeostasis contributes to the hypertrophic remodeling of the myocardium that occurs upon a sustained increase in cardiac workload. The impact of mCa2+ flux on cardiac function and remodeling was examined by subjecting mice with cardiomyocyte-specific overexpression (OE) of the mitochondrial Na+/Ca2+ exchanger (NCLX), the primary mediator of mCa2+ efflux, to several well-established models of hypertrophic and non-ischemic HF. Cardiomyocyte NCLX-OE preserved contractile function, prevented hypertrophy and fibrosis, and attenuated maladaptive gene programs in mice subjected to chronic pressure overload. Hypertrophy was attenuated in NCLX-OE mice, prior to any decline in cardiac contractility. NCLX-OE similarly attenuated deleterious cardiac remodeling in mice subjected to chronic neurohormonal stimulation. However, cardiomyocyte NCLX-OE unexpectedly reduced overall survival in mice subjected to severe neurohormonal stress with angiotensin II + phenylephrine. Adenoviral NCLX expression limited mCa2+ accumulation, oxidative metabolism, and de novo protein synthesis during hypertrophic stimulation of cardiomyocytes in vitro. Our findings provide genetic evidence for the contribution of mCa2+ to early pathological remodeling in non-ischemic heart disease, but also highlight a deleterious consequence of increasing mCa2+ efflux when the heart is subjected to extreme, sustained neurohormonal stress.

Genomic Binding Patterns of Forkhead Box Protein O1 Reveal Its Unique Role in Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophic growth is mediated by robust changes in gene expression and changes that underlie the increase in cardiomyocyte size. The former is regulated by RNA polymerase II (pol II) de novo recruitment or loss; the latter involves incremental increases in the transcriptional elongation activity of pol II that is preassembled at the transcription start site. The differential regulation of these distinct processes by transcription factors remains unknown. Forkhead box protein O1 (FoxO1) is an insulin-sensitive transcription factor that is also regulated by hypertrophic stimuli in the heart. However, the scope of its gene regulation remains unexplored. METHODS: To address this, we performed FoxO1 chromatin immunoprecipitation-deep sequencing in mouse hearts after 7 days of isoproterenol injections (3 mg·kg-1·mg-1), transverse aortic constriction, or vehicle injection/sham surgery. RESULTS: Our data demonstrate increases in FoxO1 chromatin binding during cardiac hypertrophic growth, which positively correlate with extent of hypertrophy. To assess the role of FoxO1 on pol II dynamics and gene expression, the FoxO1 chromatin immunoprecipitation-deep sequencing results were aligned with those of pol II chromatin immunoprecipitation-deep sequencing across the chromosomal coordinates of sham- or transverse aortic constriction-operated mouse hearts. This uncovered that FoxO1 binds to the promoters of 60% of cardiac-expressed genes at baseline and 91% after transverse aortic constriction. FoxO1 binding is increased in genes regulated by pol II de novo recruitment, loss, or pause-release. In vitro, endothelin-1- and, in vivo, pressure overload-induced cardiomyocyte hypertrophic growth is prevented with FoxO1 knockdown or deletion, which was accompanied by reductions in inducible genes, including Comtd1 in vitro and Fstl1 and Uck2 in vivo. CONCLUSIONS: Together, our data suggest that FoxO1 may mediate cardiac hypertrophic growth via regulation of pol II de novo recruitment and pause-release; the latter represents the majority (59%) of FoxO1-bound, pol II-regulated genes after pressure overload. These findings demonstrate the breadth of transcriptional regulation by FoxO1 during cardiac hypertrophy, information that is essential for its therapeutic targeting.

Characterization of ßARKct engineered cellular extracellular vesicles and model specific cardioprotection.

Recent data supporting any benefit of stem cell therapy for ischemic heart disease have suggested paracrine-based mechanisms via extracellular vesicles (EVs) including exosomes. We have previously engineered cardiac-derived progenitor cells (CDCs) to express a peptide inhibitor, ßARKct, of G protein-coupled receptor kinase 2, leading to improvements in cell proliferation, survival, and metabolism. In this study, we tested whether ßARKct-CDC EVs would be efficacious when applied to stressed myocytes in vitro and in vivo. When isolated EVs from ßARKct-CDCs and control GFP-CDCs were added to cardiomyocytes in culture, they both protected against hypoxia-induced apoptosis. We tested whether these EVs could protect the mouse heart in vivo, following exposure either to myocardial infarction (MI) or acute catecholamine toxicity. Both types of EVs significantly protected against ischemic injury and improved cardiac function after MI compared with mice treated with EVs from mouse embryonic fibroblasts; however, ßARKct EVs treated mice did display some unique beneficial properties including significantly altered pro- and anti-inflammatory cytokines. Importantly, in a catecholamine toxicity model of heart failure (HF), myocardial injections of ßARKct-containing EVs were superior at preventing HF compared with control EVs, and this catecholamine toxicity protection was recapitulated in vitro. Therefore, introduction of the ßARKct into cellular EVs can have improved reparative properties in the heart especially against catecholamine damage, which is significant as sympathetic nervous system activity is increased in HF.NEW & NOTEWORTHY ßARKct, the peptide inhibitor of GRK2, improves survival and metabolic functions of cardiac-derived progenitor cells. As any benefit of stem cells in the ischemic and injured heart suggests paracrine mechanisms via secreted EVs, we investigated whether CDC-ßARKct engineered EVs would show any benefit over control CDC-EVs. Compared with control EVs, ßARKct-containing EVs displayed some unique beneficial properties that may be due to altered pro- and anti-inflammatory cytokines within the vesicles.

Transient Introduction of miR-294 in the Heart Promotes Cardiomyocyte Cell Cycle Reentry After Injury.

RATIONALE: Embryonic heart is characterized of rapidly dividing cardiomyocytes required to build a working myocardium. Cardiomyocytes retain some proliferative capacity in the neonates but lose it in adulthood. Consequently, a number of signaling hubs including microRNAs are altered during cardiac development that adversely impacts regenerative potential of cardiac tissue. Embryonic stem cell cycle miRs are a class of microRNAs exclusively expressed during developmental stages; however, their effect on cardiomyocyte proliferation and heart function in adult myocardium has not been studied previously. OBJECTIVE: To determine whether transient reintroduction of embryonic stem cell cycle miR-294 promotes cardiomyocyte cell cycle reentry enhancing cardiac repair after myocardial injury. METHODS AND RESULTS: miR-294 is expressed in the heart during development, prenatal stages, lost in the neonate, and adult heart confirmed by qRT-PCR and in situ hybridization. Neonatal ventricular myocytes treated with miR-294 showed elevated expression of Ki67, p-histone H3, and Aurora B confirmed by immunocytochemistry compared with control cells. miR-294 enhanced oxidative phosphorylation and glycolysis in Neonatal ventricular myocytes measured by seahorse assay. Mechanistically, miR-294 represses Wee1 leading to increased activity of the cyclin B1/CDK1 complex confirmed by qRT-PCR and immunoblot analysis. Next, a doxycycline-inducible AAV9-miR-294 vector was delivered to mice for activating miR-294 in myocytes for 14 days continuously after myocardial infarction. miR-294-treated mice significantly improved left ventricular functions together with decreased infarct size and apoptosis 8 weeks after MI. Myocyte cell cycle reentry increased in miR-294 hearts analyzed by Ki67, pH3, and AurB (Aurora B kinase) expression parallel to increased small myocyte number in the heart. Isolated adult myocytes from miR-294 hearts showed increased 5-ethynyl-2'-deoxyuridine+ cells and upregulation of cell cycle markers and miR-294 targets 8 weeks after MI. CONCLUSIONS: Ectopic transient expression of miR-294 recapitulates developmental signaling and phenotype in cardiomyocytes promoting cell cycle reentry that leads to augmented cardiac function in mice after myocardial infarction.

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

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.

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.

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.

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

ß-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 ß-catenin exists in the majority of the organisms. One intriguing exception is zebrafish that carries two genes for ß-catenin. The maternal recessive mutation ichabod presents very low levels of ß-catenin2 that in turn affects dorsal axis formation, suggesting that ß-catenin1 is incapable to compensate for ß-catenin2 loss and raising the question of whether these two ß-catenins may have differential roles during early axis specification. Here we identify a specific antibody that can discriminate selectively for ß-catenin1. By confocal co-immunofluorescent analysis and low concentration gain-of-function experiments, we show that ß-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 ß-catenin regulatory pathway, including ß-catenin1, are more abundant than in the Wt embryo. Increased levels of ß-catenin1 are found at the membrane level but not in the nuclei till high stage. Finally, we present evidence that ß-catenin1 cannot revert the ich phenotype because it may be under the control of a GSK3ß-independent mechanism that required Axin's RGS domain function.

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: 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 ß-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 ß-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 Ca(2+)-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.

G protein-coupled receptor kinase 5 (GRK5) contributes to impaired cardiac function and immune cell recruitment in post-ischemic heart failure.

AIMS: Myocardial infarction (MI) is the most common cause of heart failure (HF) worldwide. G protein-coupled receptor kinase 5 (GRK5) is upregulated in failing human myocardium and promotes maladaptive cardiac hypertrophy in animal models. However, the role of GRK5 in ischemic heart disease is still unknown. In this study, we evaluated whether myocardial GRK5 plays a critical role post-MI in mice and included the examination of specific cardiac immune and inflammatory responses. METHODS AND RESULTS: Cardiomyocyte-specific GRK5 overexpressing transgenic mice (TgGRK5) and non-transgenic littermate control (NLC) mice as well as cardiomyocyte-specific GRK5 knockout mice (GRK5cKO) and wild type (WT) were subjected to MI and, functional as well as structural changes together with outcomes were studied. TgGRK5 post-MI mice showed decreased cardiac function, augmented left ventricular dimension and decreased survival rate compared to NLC post-MI mice. Cardiac hypertrophy and fibrosis as well as fetal gene expression were increased post-MI in TgGRK5 compared to NLC mice. In TgGRK5 mice, GRK5 elevation produced immuno-regulators that contributed to the elevated and long-lasting leukocyte recruitment into the injured heart and ultimately to chronic cardiac inflammation. We found an increased presence of pro-inflammatory neutrophils and macrophages as well as neutrophils, macrophages and T-lymphocytes at 4-days and 8-weeks respectively post-MI in TgGRK5 hearts. Conversely, GRK5cKO mice were protected from ischemic injury and showed reduced early immune cell recruitment (predominantly monocytes) to the heart, improved contractility and reduced mortality compared to WT post-MI mice. Interestingly, cardiomyocyte-specific GRK2 transgenic mice did not share the same phenotype of TgGRK5 mice and did not have increased cardiac leukocyte migration and cytokine or chemokine production post-MI. CONCLUSIONS: Our study shows that myocyte GRK5 has a crucial and GRK-selective role on the regulation of leucocyte infiltration into the heart, cardiac function and survival in a murine model of post-ischemic HF, supporting GRK5 inhibition as a therapeutic target for HF.

A peptide of the N terminus of GRK5 attenuates pressure-overload hypertrophy and heart failure.

Aberrant changes in gene expression underlie the pathogenesis and progression of pressure-overload heart failure, leading to maladaptive cardiac hypertrophy, ventricular remodeling, and contractile dysfunction. Signaling through the G protein Gq triggers maladaptation and heart failure, in part through the activation of G protein-coupled receptor kinase 5 (GRK5). Hypertrophic stimuli induce the accumulation of GRK5 in the nuclei of cardiomyocytes, where it regulates pathological gene expression through multiple transcription factors including NFAT. The nuclear targeting of GRK5 is mediated by an amino-terminal (NT) domain that binds to calmodulin (CaM). Here, we sought to prevent GRK5-mediated pathology in pressure-overload maladaptation and heart failure by expressing in cardiomyocytes a peptide encoding the GRK5 NT (GRK5nt) that encompasses the CaM binding domain. In cultured cardiomyocytes, GRK5nt expression abrogated Gq-coupled receptor-mediated hypertrophy, including attenuation of pathological gene expression and the transcriptional activity of NFAT and NF-κB. We confirmed that GRK5nt bound to and blocked Ca2+-CaM from associating with endogenous GRK5, thereby preventing GRK5 nuclear accumulation after pressure overload. We generated mice that expressed GRKnt in a cardiac-specific fashion (TgGRK5nt mice), which exhibited reduced cardiac hypertrophy, ventricular dysfunction, pulmonary congestion, and cardiac fibrosis after chronic transverse aortic constriction. Together, our data support a role for GRK5nt as an inhibitor of pathological GRK5 signaling that prevents heart failure.

Author Correction: Circular RNA CircFndc3b modulates cardiac repair after myocardial infarction via FUS/VEGF-A axis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Circular RNA CircFndc3b modulates cardiac repair after myocardial infarction via FUS/VEGF-A axis.

Circular RNAs are generated from many protein-coding genes, but their role in cardiovascular health and disease states remains unknown. Here we report identification of circRNA transcripts that are differentially expressed in post myocardial infarction (MI) mouse hearts including circFndc3b which is significantly down-regulated in the post-MI hearts. Notably, the human circFndc3b ortholog is also significantly down-regulated in cardiac tissues of ischemic cardiomyopathy patients. Overexpression of circFndc3b in cardiac endothelial cells increases vascular endothelial growth factor-A expression and enhances their angiogenic activity and reduces cardiomyocytes and endothelial cell apoptosis. Adeno-associated virus 9 -mediated cardiac overexpression of circFndc3b in post-MI hearts reduces cardiomyocyte apoptosis, enhances neovascularization and improves left ventricular functions. Mechanistically, circFndc3b interacts with the RNA binding protein Fused in Sarcoma to regulate VEGF expression and signaling. These findings highlight a physiological role for circRNAs in cardiac repair and indicate that modulation of circFndc3b expression may represent a potential strategy to promote cardiac function and remodeling after MI.

Restricting mitochondrial GRK2 post-ischemia confers cardioprotection by reducing myocyte death and maintaining glucose oxidation.

Increased abundance of GRK2 [G protein-coupled receptor (GPCR) kinase 2] is associated with poor cardiac function in heart failure patients. In animal models, GRK2 contributes to the pathogenesis of heart failure after ischemia-reperfusion (IR) injury. In addition to its role in down-regulating activated GPCRs, GRK2 also localizes to mitochondria both basally and post-IR injury, where it regulates cellular metabolism. We previously showed that phosphorylation of GRK2 at Ser670 is essential for the translocation of GRK2 to the mitochondria of cardiomyocytes post-IR injury in vitro and that this localization promotes cell death. Here, we showed that mice with a S670A knock-in mutation in endogenous GRK2 showed reduced cardiomyocyte death and better cardiac function post-IR injury. Cultured GRK2-S670A knock-in cardiomyocytes subjected to IR in vitro showed enhanced glucose-mediated mitochondrial respiratory function that was partially due to maintenance of pyruvate dehydrogenase activity and improved glucose oxidation. Thus, we propose that mitochondrial GRK2 plays a detrimental role in cardiac glucose oxidation post-injury.

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.

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.