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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 Ser(670) 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.

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.

Aging is a natural biological process in living organisms characterized by receding bioenergetics. Mitochondria are crucial for cellular bioenergetics and thus an important contributor to age-related energetics deterioration. In addition, mitochondria play a major role in calcium signaling, redox homeostasis, and thermogenesis making this organelle a major cellular component that dictates the fate of a cell. To maintain its quantity and quality, mitochondria undergo multiple processes such as fission, fusion, and mitophagy to eliminate or replace damaged mitochondria. While this bioenergetics machinery is properly protected, the functional decline associated with age and age-related metabolic diseases is mostly a result of failure in such protective mechanisms. In addition, metabolic by-products like reactive oxygen species also aid in this destructive pathway. Mitochondrial dysfunction has always been thought to be associated with diseases. Moreover, studies in recent years have pointed out that aging contributes to the decay of mitochondrial health by promoting imbalances in key mitochondrial-regulated pathways. Hence, it is crucial to understand the nexus of mitochondrial dysfunction in age-related diseases. This review focuses on various aspects of basic mitochondrial biology and its status in aging and age-related metabolic diseases.

G protein-coupled receptors (GPCRs) are important regulators of various cellular functions via activation of intracellular signaling events. Active GPCR signaling is shut down by GPCR kinases (GRKs) and subsequent β-arrestin-mediated mechanisms including phosphorylation, internalization, and either receptor degradation or resensitization. The seven-member GRK family varies in their structural composition, cellular localization, function, and mechanism of action (see sect. II). Here, we focus our attention on GRKs in particular canonical and novel roles of the GRKs found in the cardiovascular system (see sects. III and IV). Paramount to overall cardiac function is GPCR-mediated signaling provided by the adrenergic system. Overstimulation of the adrenergic system has been highly implicated in various etiologies of cardiovascular disease including hypertension and heart failure. GRKs acting downstream of heightened adrenergic signaling appear to be key players in cardiac homeostasis and disease progression, and herein we review the current data on GRKs related to cardiac disease and discuss their potential in the development of novel therapeutic strategies in cardiac diseases including heart failure.

During cardiac injury or stress, G protein-coupled receptor (GPCR) kinase 2 (GRK2) expression levels and activity are increased, leading to a desensitization of myocardial β-adrenergic receptors (βARs) and contributing to the loss of contractile reserve. Up-regulated GRK2 has been shown to be pathogenic in the post-injured heart and is involved in the promotion of heart failure (HF). Consequently, inhibition of GRK2 has been shown to rescue several animal models of HF, as demonstrated by cardiac expression of the βARKct, a peptide inhibitor of GRK2. There is new evidence that GRK2 has other, non-GPCR dependent pathological functions within cardiomyocytes. For example, during conditions that increase oxidative stress, GRK2 has been shown to localize to the mitochondria, where it is a pro-death kinase. βARKct has also been shown to inhibit the mitochondrial translocation of GRK2 due to inhibition of Hsp90 binding, a chaperone found to bind and localize GRK2 to mitochondria. Our objective is to identify the functions and targets of GRK2 in the mitochondria and its role in the development of HF. We hypothesize that GRK2 has mitochondrial targets that impair mitochondrial function in vivo, contributing to the pathogenesis of HF. Our results show that ADP-stimulated and maximal respiration are significantly decreased in mitochondria isolated from mouse hearts 2 weeks post-myocardial infarction (MI). Cardiac-specific expression of the βARKct rescued the reduction in mitochondrial respiration post-MI, in the presence of pyruvate or palmitoylcarnitine. To identify the mitochondrial targets of GRK2, we performed interactome and phospho-interactome analyses on GRK2 immunoprecipitates from neonatal rat cardiomyocytes subjected to oxidative stress. These results revealed that GRK2 interacts with several mitochondrial proteins, including those involved in metabolism, oxidative stress, calcium handling and cell death. Thus we propose that during ischemia, an increase in mitochondrial GRK2 results in its association with proteins that compromise respiration and contribute to myocardial damage and HF. This demonstrates that understanding both the canonical and non-canonical roles of GRK2 in HF is essential for more targeted intervention for therapy.

β-adrenergic receptors (βARs) are critical regulators of cardiac contractility whose function are drastically impaired during heart failure (HF). Activated βARs are regulated via phosphorylation by G-protein coupled receptor kinase-2 (GRK2) and subsequent interaction with β-arrestin. Numerous studies have shown that GRK2 is elevated in human HF and animal models demonstrate that it contributes to HF pathogenesis after ischemic injury (IR). Further, our lab has uncovered non-canonical actions of GRK2 that are involved in HF development. One such novel action of GRK2 is within mitochondria where we have shown increased GRK2 localization after oxidative stress. Our lab has shown that in the heart, phosphorylation of GRK2 via Map kinase at residue Ser670 promotes binding to Hsp90 and further enhances translocation of GRK2 to the mitochondria after oxidative stress such as ischemia. To determine the ultimate role of this pathway in the post-ischemic heart we have generated novel GRK2-S670A mice where all endogenous GRK2 cannot be phosphorylated at this residue. Baseline characterization of these mice along with their controls, show a minimal phenotype, however we have found significant differences post-IR. Compared to control mice (NLC), GRK2-S670A have significantly less infarction size 24 hrs after IR and echocardiography and hemodynamics revealed significantly improved cardiac function in GRK2-S670A mice post-IR. GRK2-S670A mice showed improved ejection fraction (52.83%+/- 2.826 S670A vs 42.38%+/- 1.603 NLC) and smaller infarcted areas (9.942%+/- 1.763 S670A vs 20.78%+/- 3.579 NLC) when compared to NLC mice. Further, we found that mitochondrial GRK2 protein levels were lower in the GRK2-S670A mice at the area at risk (0.1428+/-0.02485 S670A vs 0.2327+/- 0.023 NLC). Overall, our data suggest that phosphorylation at Ser670 of GRK2 might act as a “biological switch” in cardiomyocytes, where un-phosphorylated GRK2 is readily available in the cytoplasm to desensitize receptors such as βARs, but upon phosphorylation, GRK2 translocates to the mitochondria leading to detrimental effects including cell death. This mechanism suggests a novel way to develop pharmacological interventions to aid in the treatment of HF.

Rationale: Plakophilin-2 (PKP2) is an essential component of the cardiac desmosome. Recent data show that it interacts with other molecules of the intercalated disc. Separate studies show preferential localization of the voltage-gated sodium channel (Na V 1.5) to this region. Objective: To establish the association of PKP2 with sodium channels and its role on action potential propagation. Methods and Results: Biochemical, patch clamp, and optical mapping experiments demonstrate that PKP2 associates with Na V 1.5, and that knockdown of PKP2 expression alters the properties of the sodium current, and the velocity of action potential propagation in cultured cardiomyocytes. Conclusions: These results emphasize the importance of intermolecular interactions between proteins relevant to mechanical junctions, and those involved in electric synchrony. Possible relevance to the pathogenesis of arrhythmogenic right ventricular cardiomyopathy is discussed.

β-adrenergic receptors (βARs) are powerful regulators of cardiovascular function and are impaired in heart failure (HF). Signal transduction of βARs is canonically shut down by phosphorylation via G protein-coupled receptor kinase 2 (GRK2) and the subsequent binding of β-arrestins. This process of receptor desensitization is enhanced in HF via the up-regulation of GRK2 and contributes to disease progression. We have recently reported non-canonical actions of GRK2, which contribute to the development of HF independent of βAR desensitization. We have previously shown that GRK2 can act as a pro-death kinase in cardiomyocytes bytranslocating to mitochondria and activating mitochondria permeability transition. This study was designed to gain more understanding of the mitochondrial function of GRK2. We isolated adult cardiomyocytes from cardiac-specific transgenic mice overexpressing GRK2 at levels found in human HF (TgGRK2), and examined superoxide production using the redox sensitive reporter MitoSox Red. Confocal imaging revealed a 4.6 fold increase in superoxide levels in cardiomyocytes overexpressing Grk2 as compared to non-transgenic (NLC) cardiomyocytes (corrected total cell fluorescence 11.59±1.06, TgGRK2 (n= 3 hearts, 88 cells) vs 2.54±0.02 NLC (n=3 hearts, 52 cells), (p

Presented here is the complete genome sequence of Thiomicrospira crunogena XCL-2, representative of ubiquitous chemolithoautotrophic sulfur-oxidizing bacteria isolated from deep-sea hydrothermal vents. This gammaproteobacterium has a single chromosome (2,427,734 base pairs), and its genome illustrates many of the adaptations that have enabled it to thrive at vents globally. It has 14 methyl-accepting chemotaxis protein genes, including four that may assist in positioning it in the redoxcline. A relative abundance of coding sequences (CDSs) encoding regulatory proteins likely control the expression of genes encoding carboxysomes, multiple dissolved inorganic nitrogen and phosphate transporters, as well as a phosphonate operon, which provide this species with a variety of options for acquiring these substrates from the environment. Thiom. crunogena XCL-2 is unusual among obligate sulfur-oxidizing bacteria in relying on the Sox system for the oxidation of reduced sulfur compounds. The genome has characteristics consistent with an obligately chemolithoautotrophic lifestyle, including few transporters predicted to have organic allocrits, and Calvin-Benson-Bassham cycle CDSs scattered throughout the genome., Secrets of the deep are revealed from the genome sequence ofThiomicrospira crunogena XCL-2, a chemolithoautotrophic sulfur-oxidizing gammaproteobacterium isolated from deep-sea hydrothermal vents.