10.25376/hra.7819949.v1 Laurel A. Grisanti Laurel A. Grisanti Toby P. Thomas Toby P. Thomas Rhonda L. Carter Rhonda L. Carter Claudio de Lucia Claudio de Lucia Erhe Gao Erhe Gao Walter J. Koch Walter J. Koch Jeffrey L. Benovic Jeffrey L. Benovic Douglas G. Tilley Douglas G. Tilley pepducin-mediated cardioprotection via beta-arrestin-biased beta2-adrenergic receptor-specific signaling.pdf Health Research Alliance 2019 Pepducin β-arrestin β2-adrenergic receptor cardiac ischemia/reperfusion cardiomyocyte Cardiology (incl. Cardiovascular Diseases) 2019-03-08 13:41:23 Journal contribution https://hra.figshare.com/articles/journal_contribution/pepducin-mediated_cardioprotection_via_beta-arrestin-biased_beta2-adrenergic_receptor-specific_signaling_pdf/7819949 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.