Chronic GPR30 agonist therapy causes restoration of normal cardiac functional performance in a male mouse model of progressive heart failure: Insights into cellular mechanisms

Abstract

Aims

G protein-coupled estrogen receptor 30 (GPR30) activation by its agonist, G1, exhibits beneficial actions in female with heart failure (HF). Recent evidence indicates its cardiovascular benefits may also include male as well. However, whether and how GPR30 activation may limit HF progression and have a salutary role in males is unknown. We hypothesized that chronic G1 treatment improves LV and cardiomyocyte function, [Ca²⁺]_i regulation and β-adrenergic reserve, thus limiting HF progression in male.

Main methods

We compared left ventricle (LV) and myocyte function, [Ca²⁺]_i transient ([Ca²⁺]_iT) and β-AR modulation in control male mice (12/group) and isoproterenol-induced HF (150 mg/kg s.c. for 2 days). Two weeks after isoproterenol injection, HF mice received placebo, or G1 (150 μg/kg/day s.c. mini-pump) for 2 weeks.

Key findings

Isoproterenol-treated mice exhibited HF with preserved ejection fraction (HFpEF) at 2-weeks and progressed to HF with reduced EF (HFrEF) at 4-weeks, manifested by significantly increased LV time constant of relaxation (τ), decreased EF and mitral flow (dV/dt_max), which were accompanied by reduced myocyte contraction (dL/dt_max), relaxation (dR/dt_max) and [Ca²⁺]_iT. Acute isoproterenol-superfusion caused significantly smaller increases in dL/dt_max, dR/dt_max and [Ca²⁺]_iT. G1 treatment in HF increased basal and isoproterenol-stimulated increases in EF and LV contractility of E_ES. Importantly, G1 improved basal and isoproterenol-stimulated dL/dt_max, dR/dt_max and [Ca²⁺]_iT to control levels and restored normal cardiac β-AR subtypes modulation.

Significance

Chronic G1 treatment restores normal myocyte basal and β-AR-stimulated contraction, relaxation, and [Ca²⁺]_iT, thereby reversing LV dysfunction and playing a rescue role in a male mouse model of HF.

Introduction

Heart failure (HF) with preserved ejection fraction (HFpEF) is outpacing other forms of HF because of the expanding elderly population. Large cohort studies reveal that almost 50% of the HF populations have HFpEF with a comparable poor prognosis compared to HF with reduced EF (HFrEF). These worrisome epidemiological trends contrast with the uncertainties concerning the pathophysiological mechanisms underlying HFpEF, diagnostic guidelines and therapeutic strategies [1], [2], [3]. So far, no randomized controlled trial has shown improved survival of HFpEF. There are no targeted, effective treatments available.

The G protein-coupled receptor 30 (GPR30), functions alongside traditional estrogen receptors, ERα and ERβ. ERα and GPR30 are expressed at similar levels in cardiac tissue from male and female rodents and humans [4], [5], [6]. GPR30 plays critical roles in the development of left ventricular (LV) dysfunction and HF [4], [7], [8]. Activation of GPR30 exhibits beneficial actions in ischemia/reperfusion injury, hypertension and HF [4], [7], [9], [10], [11], [12]. While reports on GPR30 have highlighted mostly female sex-specific health issues, recent evidence indicates equivalent benefits in males [4], [13], [14]. It was reported that male, but not female, GPR30-deficient mice suffered from impaired cardiac function [8]. Ejection fraction (EF) and fractional shortening (FS) were both decreased in an age-dependent manner only in GPR30-knockout male mice [13]. Also, cardiomyocyte-specific GPR30-knockout leads to LV dysfunction in both sexes, but with more adverse changes in systolic function and LV structural remodeling among male knockout mice [15], which suggests a potential salutary role of GPR30 activation in male HF [8], [13]. However, the changes caused by GPR30 activation during HF progression in males have not been systematically evaluated. The functional effect of chronic GPR30 activation on single-myocyte mechanics, the dynamics of the cytosolic [Ca²⁺]_i and contractility reserve have not been previously assessed in an integrated fashion in HF. The direct cardiac effects and cellular mechanisms of GPR30 activation-caused cardiac protective effects remain unclear. Moreover, although HFpEF is the most common form of HF, limited studies have explored the roles of GPR30 in the development and progression of diastolic dysfunction. Whether and to what extent chronic GPR30 activation can limit the progression of HFpEF to HFrEF, or even reverse HFpEF, thereby changing the natural history of HF, remains to be critically examined.

The aim of this study was to explore the impact of chronic GPR30 activation on the progression of HFpEF. We simultaneously assessed the direct cardiac effects and underlying cellular mechanisms on the early initiation of G1, a highly selective GPR30 agonist [9], [16], [17], [18], [19], in male mice with isoproterenol-induced HF. Isoproterenol-induced HF, a time-and dose-dependent method, mimics many of the structural, functional, and hormonal changes observed in clinical HF [20], [21], [22], [23], [24], [25]. In the current study, isoproterenol-treated mice exhibited HFpEF at 2 weeks, which progressed to HFrEF at 4 weeks following the last isoproterenol injection. We assessed the hypothesis that early initiation of chronic G1 therapy could interrupt the progression of HFpEF to HFrEF and lead to regression of HFpEF in a male mouse model of isoproterenol-induced progressive HF by: (a) restoring normal LV function; and (b) improving intrinsic myocyte contraction, relaxation, [Ca²⁺]_i transient ([Ca²⁺]_iT) and β-adrenergic reserve accompanied with reversing the abnormal cardiac β-adrenergic receptor (β-AR) subtype system modulation.