Objective:

Human Relaxin-H2 (RLN) has been appreciated as a potential therapeutic for acute heart failure due to its cell protective properties and potential positive inotropy. However, RLN therapy failed to meet the endpoint criteria in the RELAX-AHF2 trial. A potential cause for the trial results could be the cardiac expression pattern of the cognate receptor of RLN, relaxin family peptide receptor 1 (RXFP1) which is restricted to atrial cells. Thus, the aim of the present study was to enlighten potential therapeutic effects of ventricular RXPF1 overexpression on cardiac function and remodeling in vivo and in vitro.

 

Methods and Results:

For in vitro studies in neonatal rat ventricular cardiomyocytes (NRVCM) and subsequent in vivo studies in mice an adeno-associated viral vector harboring RXFP1 (AAV.RXFP1) was generated. An AAV carrying a luciferase transgene served as control (AAV.Luc). Functionality of AAV.RXFP1 in transduced NRVCM could be confirmed by measurement of cAMP, an established downstream messenger of RXFP1. 

To evaluate in vivo efficacy of AAV.RXFP1, HF was induced by transverse aortic constriction (TAC) in wildtype mice. Following initial hypertrophic response animals were randomized to AAV.RXFP1 or AAV.Luc transduction via tail vein injection at day 7 after TAC. At day 28, when full transgene expression could be expected mice were implanted with mini osmotic pumps delivering recombinant RLN or saline for the duration of 4 weeks until day 56 post TAC. Follow-up by echocardiography revealed an attenuated HF progression in AAV.RXFP1 treated mice compared to AAV.Luc mice. Interestingly, additional exogenous RLN supplementation significantly enhanced this effect, restoring cardiac function. Moreover, RXFP1/RLN mitigated activation of the fetal gene program. While cardiac hypertrophy was similar in AAV.RXFP1 and AAV.Luc control mice, ventricular dilation was only observed in AAV.Luc treated animals upon TAC. In accordance with these findings, gene expression pattern of typical remodeling genes like collagens or periostin was restricted to AAV.Luc control animals. 

To enlighten the underlying mechanism, analysis on isolated NRVCM was performed. RLN stimulation of RXFP1 expressing NRVCMs resulted not only in an immediate dose dependent rise in cAMP levels, but also in a significant increase in phospholamban (PLN) phosphorylation. Although RXFP1 shares critical signaling mediators with classical beta-adrenergic receptors, we found that temporal activation, amplitude, and specificity of downstream targets differs considerably among these pathways. Classical beta-adrenergic inotropes like isoprenaline (ISO) induce an immediate surge in PLN phosphorylation at both, the protein dependent kinase (PKA) and Calcium/Calmodulin dependent Kinase II (CamKII) phospho-sites. In contrast, RXFP1 stimulation led to a less pronounced but specific PKA activation, resulting in predominant phosphorylation of PLN(S16) potentially explaining its protective properties. 

 

Conclusion: 

In vivo gene therapy mediated ventricular RXFP1 overexpression in combination with extrinsic RLN stimulation could rescue pressure overload induced heart failure. We assume the unique PKA-specific activation profile of RXFP1/RLN to beneficially influence cardiac inotropy and remodeling. Thus, harnessing RXFP1 dependent signaling represents a novel and adjustable approach for heart failure therapy.