Selective Loss of Fine Tuning of Gq/11 Signaling by RGS2 Protein Exacerbates Cardiomyocyte Hypertrophy

Abstract

Alterations in cardiac G protein-mediated signaling, most prominently G q/11 signaling, are centrally involved in hypertrophy and heart failure development. Several RGS proteins that can act as negative regulators of G protein signaling are expressed in the heart, but their functional roles are still poorly understood. RGS expression changes have been described in hypertrophic and failing hearts. In this study, we report a marked decrease in RGS2 (but not other major cardiac RGS proteins (RGS3-RGS5)) that occurs prior to hypertrophy development in different models with enhanced G q/11 signaling (transgenic expression of activated G␣ q * and pressure overload due to aortic constriction). To assess functional consequences of selective down-regulation of endogenous RGS2, we identified targeting sequences for effective RGS2 RNA interference and used lipid-based transfection to achieve uptake of fluorescently labeled RGS2 small interfering RNA in >90% of neonatal and adult ventricular myocytes. Endogenous RGS2 expression was dose-dependently suppressed (up to 90%) with no major change in RGS3-RGS5. RGS2 knockdown increased phenylephrine-and endothelin-1-induced phospholipase C␤ stimulation in both cell types and exacerbated the hypertrophic effect (increase in cell size and radiolabeled protein) in neonatal myocytes, with no major change in G q/11 -mediated ERK1/2, p38, or JNK activation. Taken together, this study demonstrates that endogenous RGS2 exerts functionally important inhibitory restraint on G q/11 -mediated phospholipase C␤ activation and hypertrophy in ventricular myocytes. Our findings point toward a potential pathophysiological role of loss of fine tuning due to selective RGS2 down-regulation in G q/11 -mediated remodeling. Furthermore, this study shows the feasibility of effective RNA interference in cardiomyocytes using lipid-based small interfering RNA transfection.

Regulator of G protein signaling 2 mediates cardiac compensation to pressure overload and antihypertrophic effects of PDE5 inhibition in mice

“…RGS2-mediated protection against myocyte hypertrophy has been previously reported in neonatal myocytes using RGS2 RNAi (18). Here, we examined adult myocytes from Rgs2 +/+ and Rgs2 -/-mouse hearts and found enhanced protein synthesis in cells incubated with the G q agonist endothelin-1 (ET1).…”

“…Mice globally lacking RGS2 were found to develop modest systemic hypertension, although they exhibit no major cardiac phenotype (17), which suggested that RGS2 had a modest role in the heart. Yet more recent studies found that knockdown of the gene encoding RGS2 amplifies hypertrophic responses in neonatal myocytes exposed to G q stimuli (18).…”

Ecto-5′-Nucleotidase Deficiency Exacerbates Pressure-Overload–Induced Left Ventricular Hypertrophy and Dysfunction

“…Neonatal rat cardiomyocytes were isolated from 2-day-old SpragueDawley rats by enzymatic digestion 19 and separated from nonmuscle cells on a discontinuous Percoll gradient as described previously. 19 Detailed methodology is included in the online supplementary data.…”

Inverse agonism: the classic concept of GPCRs revisited [Review]

“…Recognition of the deleterious effects of overactivation of the renin-angiotensin system (RAS) and AT1-Gq/G13 signaling [28][29][30], in addition to desensitization of cardioprotective βAR-Gs signaling [31][32][33], has made RAS a major therapeutic target, whether systemically or regionally [34,35]. In many randomized, controlled trials, ACE inhibitor (ACEi) reduced morbidity and mortality more than comparators in patients with congestive heart failure and coronary heart disease [36][37][38][39][40].…”