Abstract 16052: Non-Canonical Mechanisms of BARKct-Mediated GRK2 Inhibition in Heart Failure Rescue

Introduction: Significant GRK2 upregulation during HF results in desensitization of βARs. βARKct is a peptide inhibitor of GRK2 comprised of the last carboxyl terminal region of GRK2, and prevents GRK2 binding and βAR desensitization. βARKct overexpression attenuates HF and improves outcomes in animal models. Following oxidative stress, MAPKs phosphorylate the Ser670 (S670) residue of GRK2, which induces GRK2 binding to Hsp90 and localization to mitochondria, initiating pro-death pathways. As S670 is also found in βARKct, it may prevent endogenous GRK2 accumulation in the mitochondria.

Hypothesis: βARKct-mediated cardioprotection in HF is due primarily to mitochondrial GRK2 blockade by βARKct.

Methods: We generated cardiac-specific mutant βARKct-S670A transgenic mice harboring a Ser-to-Ala mutation at the S670 residue to prevent Hsp90 binding and allow for endogenous GRK2 to continue to translocate to the mitochondria upon ischemic injury, while retaining βARKct in the cytosol to act on βAR signaling pathways. Additionally, we have generated AAV vectors to deliver WT βARKct, βARKct-S670A, and phosphomimetic βARKct-S670D peptides in mice. We performed in vivo hemodynamic analysis to assess cardiac function in βARKct and βARKct-S670A transgenic mice. Additionally, intracellular cAMP levels in AC16 cardiomyocytes transfected with βARKct or βARKct-S670A, and βARKct-S670D plasmids were quantified in response to increasing doses of isoproterenol.

Results: Hemodynamic analysis of the βARKct-S670A mice has demonstrated increased baseline contractility in βARKct-S670A mice compared to NLC mice, indicating comparable cardioprotective effects to βARKct mice lacking the point mutation, which are mediated by canonical pathways. In vitro cAMP quantification revealed AC16s transfected with βARKct, βARKct-S670A, and βARKct-S670D plasmids increased cAMP accumulation in response to increasing doses of isoproterenol compared to control.

Conclusions: Studies characterizing the mitochondrial pathways involved in HF rescue following ischemic injury are ongoing. These findings will identify new mechanistic information of GRK2 inhibition for HF, and elucidate a new method for attenuating mitochondrial dysfunction during HF.