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The E3 ubiquitin ligase Parkin plays an important role in regulating clearance of dysfunctional or unwanted mitochondria in tissues, including the heart. However, whether Parkin also functions to prevent cardiac aging by maintaining a healthy population of mitochondria is still unclear. Here, we have examined the role of Parkin in the context of mitochondrial DNA (mtDNA) damage and myocardial aging using a mouse model carrying a proofreading-defective mtDNA polymerase γ (POLG). We observed both decreased Parkin protein levels and development of cardiac hypertrophy in POLG hearts with age; however, cardiac hypertrophy in POLG mice was neither rescued, nor worsened by cardiac-specific overexpression or global deletion of Parkin, respectively. Unexpectedly, mitochondrial fitness did not substantially decline with age in POLG mice when compared with that in WT mice. We found that baseline mitophagy receptor–mediated mitochondrial turnover and biogenesis were enhanced in aged POLG hearts. We also observed the presence of megamitochondria in aged POLG hearts. Thus, these processes may limit the accumulation of dysfunctional mitochondria as well as the degree of cardiac functional impairment in the aging POLG heart. Overall, our results demonstrate that Parkin is dispensable for constitutive mitochondrial quality control in a mtDNA mutation model of cardiac aging.

A vast body of literature has established G protein–coupled receptor kinase 2 (GRK2; family: β-adrenergic receptor kinases [βARKs]) as a key player in the development and progression of heart failure. Inhibition of GRK2 improves cardiac function after injury in numerous animal models. In recent years, discovery of several noncanonical GRK2 targets has expanded our view of this kinase. This article describes the exciting finding that cardiac GRK2 activity can regulate whole-body metabolism. Transgenic mice with cardiac-specific expression of a peptide inhibitor of GRK2 (TgβARKct) display an enhanced obesogenic phenotype when fed a high-fat diet (HFD). In contrast, mice with cardiac-specific overexpression of GRK2 (TgGRK2) show resistance to HFD-induced obesity. White adipose tissue (WAT) mass was significantly enhanced in HFD-fed TgβARKct mice. Furthermore, regulators of adipose differentiation were differentially regulated in WAT from mice with gain or loss of GRK2 function. Using complex metabolomics, we found that cardiac GRK2 signaling altered myocardial branched-chain amino acid (BCAA) and endocannabinoid metabolism. In addition, it modulated circulating BCAA and endocannabinoid metabolite profiles on mice fed an HFD. We also found that one of the BCAA metabolites identified here enhances adipocyte differentiation in vitro. These results suggest that metabolic changes in the heart due to GRK2 signaling on mice fed an HFD control whole-body metabolism.

Rationale: G protein–coupled receptor kinase 2 (GRK2) is an important molecule upregulated after myocardial injury and during heart failure. Myocyte-specific GRK2 loss before and after myocardial ischemic injury improves cardiac function and remodeling. The cardiac fibroblast plays an important role in the repair and remodeling events after cardiac ischemia; the importance of GRK2 in these events has not been investigated. Objective: The aim of this study is to elucidate the in vivo implications of deleting GRK2 in the cardiac fibroblast after ischemia/reperfusion injury. Methods and Results: We demonstrate, using Tamoxifen inducible, fibroblast-specific GRK2 knockout mice, that GRK2 loss confers a protective advantage over control mice after myocardial ischemia/reperfusion injury. Fibroblast GRK2 knockout mice presented with decreased infarct size and preserved cardiac function 24 hours post ischemia/reperfusion as demonstrated by increased ejection fraction (59.1±1.8% versus 48.7±1.2% in controls; P Conclusions: These novel data showing the benefits of inhibiting GRK2 in the cardiac fibroblast adds to previously published data showing the advantage of GRK2 ablation and reinforces the therapeutic potential of GRK2 inhibition in the heart after myocardial ischemia.

GRK2, a G protein-coupled receptor kinase, plays a critical role in cardiac physiology. Adrenergic receptors are the primary target for GRK2 activity in the heart; phosphorylation by GRK2 leads to desensitization of these receptors. As such, levels of GRK2 activity in the heart directly correlate with cardiac contractile function. Furthermore, increased expression of GRK2 after cardiac insult exacerbates injury and speeds progression to heart failure. Despite the importance of this kinase in both the physiology and pathophysiology of the heart, relatively little is known about the role of GRK2 in skeletal muscle function and disease. In this study we generated a novel skeletal muscle-specific GRK2 knock-out (KO) mouse (MLC-Cre:GRK2fl/fl) to gain a better understanding of the role of GRK2 in skeletal muscle physiology. In isolated muscle mechanics testing, GRK2 ablation caused a significant decrease in the specific force of contraction of the fast-twitch extensor digitorum longus muscle yet had no effect on the slow-twitch soleus muscle. Despite these effects in isolated muscle, exercise capacity was not altered in MLC-Cre:GRK2fl/fl mice compared with wild-type controls. Skeletal muscle hypertrophy stimulated by clenbuterol, a β2-adrenergic receptor (β2AR) agonist, was significantly enhanced in MLC-Cre:GRK2fl/fl mice; mechanistically, this seems to be due to increased clenbuterol-stimulated pro-hypertrophic Akt signaling in the GRK2 KO skeletal muscle. In summary, our study provides the first insights into the role of GRK2 in skeletal muscle physiology and points to a role for GRK2 as a modulator of contractile properties in skeletal muscle as well as β2AR-induced hypertrophy.

Temporal decline in mitochondrial function is widely considered to be a driver of cardiomyocyte aging, which in turn contributes to the prevalence of cardiovascular disease in the aging population. The accumulation of dysfunctional mitochondria appears to be a direct consequence of reduced autophagy and mitochondrial quality control in the aging heart. Parkin, an E3 ubiquitin ligase, plays a critical role in this process, marking dysfunctional mitochondria for degradation by autophagosomes; however, whether Parkin functions to prevent cardiac aging by maintaining a healthy population of mitochondria is still unclear. To examine the role of Parkin in the context of mtDNA damage and myocardial aging, we used a mouse model carrying a proofreading defective mitochondrial DNA polymerase gamma (POLG). We observed a significant decrease in Parkin protein levels in the hearts of aged (6 months) POLG mice, in spite of elevated Parkin mRNA. Seahorse analysis revealed a decrease in cardiac mitochondrial respiration in 6-month POLG mice. While cardiac structure and function were similar in both genotypes, POLG mice displayed modest but significant cardiac hypertrophy at this age. Next, we generated mice with concomitant Parkin deletion or cardiac specific Parkin overexpression with the mutant POLG. However, loss of Parkin did not exacerbate the accelerated cardiac aging phenotype observed in the POLG mice, and enhancing cardiac Parkin protein levels did not rescue the mitochondrial dysfunction or the cardiac hypertrophy observed in POLG mice up to 12 months of age. Surprisingly, we found that Parkin levels were reduced in POLG hearts, even with cardiac specific overexpression of Parkin. Translation of Parkin was unperturbed in 12-month POLGxParkin TG hearts, suggesting instead that Parkin protein stability is compromised in aged POLG hearts, a hypothesis we are currently testing. We also found both diminished protein ubiquitination and reduced degradation of Mfn1, a known Parkin substrate, in POLGxParkin TG hearts compared to Parkin TG hearts. These results provide new insights into mitophagy and aging, and suggest that Parkin plays a minor role in baseline mitochondrial maintenance and that overexpression of Parkin fails to prevent the cardiac aging process.

As average life expectancy continues to rise in the developed world, age-associated pathologies are increasing in prevalence. The hallmarks of cardiac ageing include cardiomyocyte loss, fibrosis and hypertrophy, all of which contribute to an increased incidence of cardiac disease. At the molecular level, cellular ageing is characterized by increased ROS production, mitochondrial dysfunction and the accumulation of damaged proteins and organelles. Cardiomyocytes and other senescent cell types rely upon autophagy, a lysosome-mediated degradation pathway, to remove potentially toxic protein aggregates and damaged organelles from the cellular milieu. However, increasing lines of evidence point to an age-associated decrease in cardiomyocyte autophagy, with predictably negative consequences for cardiac function and health. Conversely, stimulation of autophagy has been shown to improve cellular health and cardiac function and to increase lifespan in numerous model organisms. Clearly, autophagy represents a critical pathway for cellular vitality, as well as a promising therapeutic target for the treatment of age-related cardiac pathologies. In this review, we will discuss the mechanism of autophagy and its regulation in the cell, the role of autophagy in the ageing heart, and how the autophagy pathway might be targeted to improve cardiac health.

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.

Endorepellin, the C-terminal module of perlecan, has angiostatic activity. Here we provide definitive genetic and biochemical evidence that the functional endorepellin receptor is the alpha2beta1 integrin. Notably, the specific endorepellin binding to the receptor was cation-independent and was mediated by the alpha2 I domain. We show that the anti-angiogenic effects of endorepellin cannot occur in the absence of alpha2beta1. Microvascular endothelial cells from alpha2beta1(-/-) mice, but not those isolated from either wild-type or alpha1beta1(-/-) mice, did not respond to endorepellin. Moreover, syngeneic Lewis lung carcinoma xenografts in alpha2beta1(-/-) mice failed to respond to systemic delivery of endorepellin. In contrast, endorepellin inhibited tumor growth and angiogenesis in the wild-type mice expressing integrin alpha2beta1. We conclude that the angiostatic effects of endorepellin in vivo are mediated by a specific interaction of endorepellin with the alpha2beta1 integrin receptor.

Decades of research have identified G Protein Coupled Receptor Kinase 2 (GRK2) as an important molecule that is upregulated in the cardiomyocyte after myocardial injury and during heart failure development. Research from our lab has convincingly demonstrated that myocyte-specific loss of GRK2 both before and after myocardial ischemic injury improves cardiac function and remodeling. Recent studies have reported that GRK2 is also upregulated in the cardiac fibroblast in the failing heart, suggesting a potential role for this molecule in the most abundant cell type in the heart. However, the in vivo implications of GRK2 expression in the fibroblast following cardiac stress remain a mystery. Tamoxifen inducible, fibroblast-specific GRK2 knockout mice (Col1α2CreER/GRK2flox) were treated with tamoxifen along with their control murine counterparts (GRK2flox alone) for 10 days to induce deletion of GRK2 in fibroblasts. Two weeks later mice were subjected to ischemia/reperfusion (I/R) injury via coronary artery occlusion for 30 minutes followed by periods of reperfusion. Fibroblast GRK2 knockout mice presented with preserved cardiac function 24 hours post-I/R compared to control mice as demonstrated by increased ejection fraction (58.1±1.8% vs. 48.7±1.2%, respectively, N=11-14, p=0.0005). GRK2 knockout mice also presented with decreased fibrosis in the infarcted area 72 hours following I/R injury as shown by Masson’s Trichrome staining. In line with decreased fibrosis, these mice also expressed decreased amounts of TGFβ1 and Collagen I. Additionally, α-smooth muscle actin expression is significantly diminished, indicating reduced fibroblast to myofibroblast transformation. These data suggest that GRK2 plays a key role up-stream in fibroblast activation and function in the ischemic heart and indicate that, like in the cardiomyocyte, inhibition of GRK2 in the cardiac fibroblast is a potential therapeutic target to limit cardiac dysfunction and remodeling after ischemic injury.

A vast body of literature has established GRK2 as a key player in the development and progression of heart failure. GRK2 levels increase in the heart post injury in an attempt to normalize sympathetic overdrive. While initially beneficial, this response becomes maladaptive. Inhibition of GRK2 improves cardiac function post injury in numerous animal models. Recently, discovery of several non-canonical GRK2 targets has expanded our view of this kinase. Amongst these, our lab has identified GRK2 as a negative regulator of cardiac insulin signaling and this is through direct interaction and phosphorylation of the insulin receptor substrate-1 (IRS1). While continuing to study cardiac metabolic aspects of GRK2 manipulation we have uncovered the novel and exciting finding that cardiac GRK2 activity can regulate whole body metabolism. We found that transgenic mice with cardiac-specific overexpression of GRK2 (Tg-GRK2) show resistance to high fat diet (HFD) induced obesity. In contrast, Tg-βARKct mice with cardiac-specific expression of a peptide inhibitor of GRK2, known as the βARKct display an enhanced obesogenic phenotype when fed a HFD. We placed transgenic mice (and their non-transgenic littermate controls) on a high fat diet (60% kcal% fat) or a control diet (10% kcal% fat) for up to 16 weeks. Mice were housed in grouped cages at room temperature (18-22°C). HFD feeding increased white adipose mass in Tg-BARKct mice vs. NLC, whereas adipose mass in Tg-GRK2 mice is lower than NLC animals. Moreover, HFD Tg-βARKct mice exhibit elevated serum leptin levels relative to NLC, indicating development of leptin resistance. The novelty of this finding could bear great clinical significance, not least because these cardiac specific transgenes elicit a global metabolic phenotype implicating the heart as an endocrine organ and GRK2 activity is essential for this new and unexpected finding.

Heart failure (HF) causes a tremendous burden on the worldwide healthcare system, affecting >23 million people. There are many cardiovascular disorders that contribute to the development of HF and multiple risk factors that accelerate its occurrence, but regardless of its underlying cause, HF is characterized by a marked decrease in myocardial contractility and loss of pump function. One biomarker molecule consistently shown to be upregulated in human HF and several animal models is G protein–coupled receptor kinase-2 (GRK2), a kinase originally discovered to be involved in G protein–coupled receptor desensitization, especially β-adrenergic receptors. Higher levels of GRK2 can impair β-adrenergic receptor–mediated inotropic reserve and its inhibition, or molecular reduction has shown to improve pump function in several animal models including a preclinical pig model of HF. Recently, nonclassical roles for GRK2 in cardiovascular disease have been described, including negative regulation of insulin signaling, a role in myocyte cell survival and apoptotic signaling, and it has been shown to be localized in/on mitochondria. These new roles of GRK2 suggest that GRK2 may be a nodal link in the myocyte, influencing both cardiac contractile function and cell metabolism and survival and contributing to HF independent of its canonical role in G protein–coupled receptor desensitization. In this review, classical and nonclassical roles for GRK2 will be discussed, focusing on recently discovered roles for GRK2 in cardiomyocyte metabolism and the effects that these roles may have on myocardial contractile function and HF development.