Targeted Gene Therapy with RXFP1 Attenuates Myocardial Infarction and Preserves Left Ventricular Function in Mice

Background Several studies have shown that recombinant relaxin targets various mechanisms of cell injury and is protective in models of ischemia/reperfusion (I/R) by reducing oxidative stress, calcium overload, neutrophil activity, NLRP3 inflammasome, endothelial adhesive protein expression, mast cell degranulation, apoptosis, platelet adhesion, as well as improving coronary blood flow. However, there is no information on the role of relaxin receptor 1 (RXFP1) signaling in the heart in the context of myocardial infarction (MI). We sought to determine the effect of RXFP1 gain‐of‐function using novel cardiotropic AAV9 vectors as a therapeutic modality against MI. Methods RXFP1 cDNA (pCMV6 backbone) was purchased from Origene technologies and introduced into AAV9 cis plasmids following KpnI and AgeI digestion. Plasmids were transformed into NEB stable E. Coli and following propagation, they were transfected into 293T cells with the helper plasmid pDG‐9 to produce AAV9‐RXFP1. Viral particles were isolated post cell lysis and purified. 1.0 × 10 11 viral genomes in 100μL were intravenously administered to 8‐week‐old CD1 male mice through tail vein. Four weeks later, a subset of mice was sacrificed and cardiac tissue was extracted for qPCR, western blot, or adult cardiomyocyte isolation. Another subset of mice was subjected to LAD ligation for 30 minutes followed by 24 hours of reperfusion. Cardiac function was assessed via echocardiography prior to sacrifice and infarct size was subsequently determined using triphenyl tetrazolium (TTC) staining. Results Cardiac tissue from mice injected with AAV9‐RXFP1 showed increased mRNA and protein levels of RXFP1 (Fig. A&B). qPCR analysis also revealed overexpression of RXFP1 in adult cardiomyocytes isolated from mice that received AAV9‐RXFP1 (Fig. C). Phosphorylation of P44/42 MAP kinase downstream of RXFP1 activation was also evident in hearts overexpressing RXFP1 (Fig. D). Moreover, gene therapy with RXFP1 reduced infarct size and preserved cardiac function following MI as compared to control (Fig. E&F). Conclusion Our novel AAV9 construct successfully upregulates cardiac RXFP1, which in turn attenuates infarct size and preserves cardiac function. These findings provide strong rationale for investigating the therapeutic potential of gene therapy with RXFP1 in a more clinically relevant model of established ischemic HF. Support or Funding Information NHLBI (R01HL133167) and NIA (R21AG053654) to Dr. Salloum. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .