Abstract

Introduction: The induction of heart failure (HF) after aortic banding (TAC) in mice encompasses aberrations in gene regulation, leading to maladaptive cardiac hypertrophy and contractile dysfunction. Previously, our lab has shown that the nuclear targeting of GRK5, which is required for maladaptive hypertrophy, is controlled by its N-terminal (NT) calmodulin-binding domain. Hypothesis: We hypothesize that expression of a peptide encoding the NT of GRK5 will act as a GRK5-calmodulin inhibitor preventing the nuclear accumulation of GRK5 and inhibit its pathological activities. Methods: Neonatal rat ventricular cardiomyocytes (NRCM) overexpressing GRK5-NT or control virus were subjected to α-adrenergic stress with 50μM phenylephrine (PE) or vehicle for 30 minutes. Subcellular fractionation and western blotting were used to detect nuclear GRK5. Novel cardiac-specific transgenic mice were generated expressing HA-tagged GRK5-NT and these mice along with their control mice were characterized 8 weeks after TAC or sham surgery. Echocardiography, fibrosis, and lung and heart weight to tibia length metrics were used to evaluate hypertrophy and heart failure response post-TAC. Other cohorts were subjected to TAC for 1 week followed by Western blotting to assess acute signaling and nuclear accumulation of GRK5. Results: In vitro, expression of the GRK5-NT peptide reduced PE-induced nuclear accumulation of GRK5. In both NRCM and tissue, the GRK5-NT peptide was found to bind to calmodulin, which disrupts the endogenous calmodulin-GRK5 interaction. GRK5-NT expression abrogates nuclear GRK5 accumulation following hypertrophic stress in vitro and in vivo. Following 8 weeks of TAC, cardiac function, hypertrophy, and pulmonary congestion were significantly attenuated in GRK5-NT mice compared to NLC mice. Conclusion: We show that the GRK5-NT peptide binds to calmodulin and prevents nuclear accumulation of GRK5 following hypertrophic stress. Our in vivo data show that cardiac-restricted expression of GRK5-NT prevents HF by partially restoring cardiac dysfunction, hypertrophy, and pulmonary congestion following 8 weeks of TAC. Further in vitro work will reveal the calmodulin-dependence of GRK5-NT and the peptide’s effects on G-protein signaling.

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