pepducin-mediated cardioprotection via beta-arrestin-biased beta2-adrenergic receptor-specific signaling.pdf

Reperfusion as a therapeutic intervention for acute myocardial infarction-induced cardiac injury itself induces<br>further cardiomyocyte death. β-arrestin (βarr)-biased β-adrenergic receptor (βAR) activation promotes<br>survival signaling responses in vitro; thus, we hypothesize that this pathway can mitigate cardiomyocyte death<br>at the time of reperfusion to better preserve function. However, a lack of efficacious βarr-biased orthosteric<br>small molecules has prevented investigation into whether this pathway relays protection against ischemic injury<br>in vivo. We recently demonstrated that the pepducin ICL1-9, a small lipidated peptide fragment designed from<br>the first intracellular loop of β2AR, allosterically engaged pro-survival signaling cascades in a βarr-dependent<br>manner in vitro. Thus, in this study we tested whether ICL1-9 relays cardioprotection against<br>ischemia/reperfusion (I/R)-induced injury in vivo.<br>Methods: Wild-type (WT) C57BL/6, β2AR knockout (KO), βarr1KO and βarr2KO mice received<br>intracardiac injections of either ICL1-9 or a scrambled control pepducin (Scr) at the time of ischemia (30 min)<br>followed by reperfusion for either 24 h, to assess infarct size and cardiomyocyte death, or 4 weeks, to monitor<br>the impact of ICL1-9 on long-term cardiac structure and function. Neonatal rat ventricular myocytes (NRVM)<br>were used to assess the impact of ICL1-9 versus Scr pepducin on cardiomyocyte survival and mitochondrial<br>superoxide formation in response to either serum deprivation or hypoxia/reoxygenation (H/R) in vitro and to<br>investigate the associated mechanism(s).<br>Results: Intramyocardial injection of ICL1-9 at the time of I/R reduced infarct size, cardiomyocyte death and<br>improved cardiac function in a β2AR- and βarr-dependent manner, which led to improved contractile function<br>early and less fibrotic remodeling over time. Mechanistically, ICL1-9 attenuated mitochondrial superoxide<br>production and promoted cardiomyocyte survival in a RhoA/ROCK-dependent manner. RhoA activation could<br>be detected in cardiomyocytes and whole heart up to 24 h post-treatment, demonstrating the stability of<br>ICL1-9 effects on βarr-dependent β2AR signaling.<br>Conclusion: Pepducin-based allosteric modulation of βarr-dependent β2AR signaling represents a novel<br>therapeutic approach to reduce reperfusion-induced cardiac injury and relay long-term cardiac remodeling<br>benefits.