Small molecule disruption of G beta gamma signaling inhibits the progression of heart failure

Abstract

Rationale: Excess signaling through cardiac Gbetagamma subunits is an important component of heart failure (HF) pathophysiology. They recruit elevated levels of cytosolic G protein-coupled receptor kinase (GRK)2 to agonist-stimulated beta-adrenergic receptors (beta-ARs) in HF, leading to chronic beta-AR desensitization and downregulation; these events are all hallmarks of HF. Previous data suggested that inhibiting Gbetagamma signaling and its interaction with GRK2 could be of therapeutic value in HF.

Objective: We sought to investigate small molecule Gbetagamma inhibition in HF.

Methods and results: We recently described novel small molecule Gbetagamma inhibitors that selectively block Gbetagamma-binding interactions, including M119 and its highly related analog, gallein. These compounds blocked interaction of Gbetagamma and GRK2 in vitro and in HL60 cells. Here, we show they reduced beta-AR-mediated membrane recruitment of GRK2 in isolated adult mouse cardiomyocytes. Furthermore, M119 enhanced both adenylyl cyclase activity and cardiomyocyte contractility in response to beta-AR agonist. To evaluate their cardiac-specific effects in vivo, we initially used an acute pharmacological HF model (30 mg/kg per day isoproterenol, 7 days). Concurrent daily injections prevented HF and partially normalized cardiac morphology and GRK2 expression in this acute HF model. To investigate possible efficacy in halting progression of preexisting HF, calsequestrin cardiac transgenic mice (CSQ) with extant HF received daily injections for 28 days. The compound alone halted HF progression and partially normalized heart size, morphology, and cardiac expression of HF marker genes (GRK2, atrial natriuretic factor, and beta-myosin heavy chain).

Conclusions: These data suggest a promising therapeutic role for small molecule inhibition of pathological Gbetagamma signaling in the treatment of HF.

Figure 1. Acute M119 treatment enhances cardiomyocyte β-AR signaling

A) Isolated adult cardiomyocytes treated with M119 (10 μM) show enhanced cAMP generation at baseline and in response to Iso stimulation. B, C) M119 (10 μM) interferes with β-AR induced GRK2 membrane recruitment. Representative western blot analysis of GRK2 protein level in membrane fractions of cardiomyocytes treated with vehicle alone, Iso, M119, or M119 and Iso. Positive control lane of proteins from HEK293 cells transfected with GRK2 (B). Densitometric analysis of GRK2 membrane recruitment from four independent experiments demonstrates that M119 leads to ~50% decrease in Iso-induced GRK2 membrane recruitment (C). *P<.05, **P<.01, ***P<.001

Figure 2. M119 enhances cardiomyocyte contractility in vitro

A) Representative tracings of untreated isolated adult cardiomyocytes and cells treated with M119, Iso, or M119 and Iso. B) Averaging of 4-7 independent experiments (1 experiment = average of ≥ 7 cardiomyocytes per condition) showing that M119 treatment significantly increased percent contractility over baseline and enhanced Iso-stimulated contractility. Pretreatment with the general β-AR antagonist, propranolol, abolishes the effects of M119 and Iso on cardiomyocyte contractility. C) M119 also increases the rate of shortening both in the presence and absence of Iso. *P<.05, **P<.01, ***P<.001

Figure 3. M119 improves cardiac function, and reduces cardiac hypertrophy and interstitial fibrosis, in an acute pharmacologic HF model

A) Representative M-mode tracings of vehicle-only, Iso-treated and M119+Iso treated animals. B) Average fractional shortening data of animals in each treatment group (n=6-8 per group) shows significant normalization of cardiac function in Iso-treated animals that also received M119. C) HW to BW ratios of animals following 7-day treatment. Iso treated animals show significant cardiac hypertrophy compared to vehicle controls. M119 treatment significantly reduces cardiac hypertrophy when administered to Iso-pumped animals. D) Masson’s Trichrome staining of tissue sections showed that Iso+M119 animals exhibit reduced interstitial fibrosis compared to Iso-only animals. **P<.01, ***P<.001.

Figure 4. Gallein prevents progression of HF in CSQ mice, trends toward improvement

Two groups of five male CSQ mice at 8 weeks of age were initiated on once daily injections of 30 mg/kg/day gallein for one month, and were followed by serial conscious echocardiography. A) Quantitation of fractional shortening data. B) representative M-mode echocardiographic images at 4 weeks. *P<0.05 vs. baseline

Figure 5. Gallein improves cardiac morphology and HF marker gene expression in CSQ mice

A). Morphometric analysis of heart weight to body weight (HW:BW) ratio in CSQ mice treated with vehicle or gallein. B. Real-time PCR analysis of ANF and β-MHC RNA expression in CSQ+V or CSQ+G mice. *P<0.05 vs. vehicle.

Figure 6. Molecular markers of HF are reduced by small molecule Gβγ inhibitor treatment

A) Representative immunoblotting of GRK2 in Iso pumped mice concurrently treated with M119 for seven days (above, representative image of n=6 per group) and densitometric quantitation analysis of GRK2 normalized to β-Actin (below). B) Immunoblotting of GRK2 in CSQ mice with established HF treated with daily gallein for four weeks (above) and densitometric quantitation analysis of GRK2 normalized to β-Actin (below, n=5 per group). GRK2 protein levels were significantly reduced by small molecule Gβγ inhibitor treatment as determined by densitometric analysis of western blots. *P<.05, **P<.01