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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.

The β1 and β2 adrenergic receptors (βARs) are the predominant G-protein-coupled receptors (GPCRs) subtypes expressed in the heart. It is now appreciated that ligands can induce multiple distinct “active” receptor conformations with unique downstream functional signaling profiles or efficacies. Our current understanding of GPCR signaling is that ligands can be biased toward activating either a G protein or a β-arrestin-signaling pathway, a concept known as biased ligand signaling. To identify novel biased signaling pathways mediated by β1- and β2ARs, we performed a proteomic interactome study of β1AR and β2AR using stable isotope labeling by amino acids in cell culture (SILAC). We identified several hundred proteins that distinctly bind to the β1AR or β2AR following stimulation with the unbiased full agonist, isoproterenol, or the β-arrestin-biased ligand carvedilol. We found that stimulation by the β-arrestin-biased agonist carvedilol of only β1ARs resulted in the recruitment of Gαi. No of the other ligand tested promoted Gαi recruitment, suggesting that carvedilol may be unique in its ability to activate Gαi-biased signaling. The Gαi inhibitor pertussis toxin blocked β-arrestin-dependent extracellular signal-regulated kinase (ERK) activation and epidermal growth factor receptor (EGFR) transactivation stimulated by carvedilol, suggesting the involvement of Gαi in carvedilol-induced β-arrestin biased signaling of β1ARs. Using β1AR/β2AR chimeric receptors we show that the C-terminal tail of the β1AR is required for carvedilol stimulated Gαi recruitment, but is unable to rescue the lack of Gαi recruitment by the β2AR. Since our current conceptual framework for biased signaling is based on the “bar code hypothesis”, ongoing phosphoproteomic experiments are testing whether recruitment of Gαi by carvedilol induces a distinct phosphorylation pattern on the c-tail of the β1AR. Our study shows that activation of β-arrestin-biased signaling requires G proteins for signaling and provides a new mechanistic understanding of how biased β-blockers can activate signaling.

Cardiac Troponin I-interacting kinase (TNNI3K) is a cardiac specific kinase whose biological function remains largely unknown. We have recently shown that TNNI3K expression greatly accelerates cardiac dysfunction in mouse models of cardiomyopathy, indicating an important role in modulating disease progression. To further investigate TNNI3K kinase activity in vivo, we have generated transgenic mice expressing both wild-type and kinase-dead versions of the human TNNI3K protein. Importantly, we show that the increased TNNI3K kinase activity induces mouse cardiac hypertrophy, and the kinase activity is required to accelerate disease progression in a left-ventricular pressure overload model of mouse cardiomyopathy. We demonstrate the clinical relevance of these observations by identifying two potential missense mutations near the kinase activation loop of TNNI3K in idiopathic dilated cardiomyopathy (DCM) human patients. Using an in vitro kinase assay and proteomics analysis, we show that TNNI3K is a dual-function kinase with Tyr and Ser/Thr kinase activity. Using antisera to TNNI3K, we show that TNNI3K protein is located at the sarcomere Z disc. These combined data suggest that TNNI3K mediates cell signaling to modulate cardiac response to stress. The essential role of the kinase activity makes TNNI3K a strong potential pharmaceutical target of kinase inhibitors for heart disease.