In vivo selective inhibition of TRPC6 by antagonist BI 749327 ameliorates fibrosis and dysfunction in cardiac and renal disease.

Transient receptor potential canonical type 6 (TRPC6) is a nonselective receptor-operated cation channel that regulates reactive fibrosis and growth signaling. Increased TRPC6 activity from enhanced gene expression or gain-of-function mutations contribute to cardiac and/or renal disease. Despite evidence supporting a pathophysiological role, no orally bioavailable selective TRPC6 inhibitor has yet been developed and tested in vivo in disease models. Here, we report an orally bioavailable TRPC6 antagonist (BI 749327; IC50 13 nM against mouse TRPC6, t1/2 8.5-13.5 hours) with 85- and 42-fold selectivity over the most closely related channels, TRPC3 and TRPC7. TRPC6 calcium conductance results in the stimulation of nuclear factor of activated T cells (NFAT) that triggers pathological cardiac and renal fibrosis and disease. BI 749327 suppresses NFAT activation in HEK293T cells expressing wild-type or gain-of-function TRPC6 mutants (P112Q, M132T, R175Q, R895C, and R895L) and blocks associated signaling and expression of prohypertrophic genes in isolated myocytes. In vivo, BI 749327 (30 mg/kg/day, yielding unbound trough plasma concentration ∼180 nM) improves left heart function, reduces volume/mass ratio, and blunts expression of profibrotic genes and interstitial fibrosis in mice subjected to sustained pressure overload. Additionally, BI 749327 dose dependently reduces renal fibrosis and associated gene expression in mice with unilateral ureteral obstruction. These results provide in vivo evidence of therapeutic efficacy for a selective pharmacological TRPC6 inhibitor with oral bioavailability and suitable pharmacokinetics to ameliorate cardiac and renal stress-induced disease with fibrosis.

Role of hypothalamic melanocortin 3/4-receptors in mediating chronic cardiovascular, renal, and metabolic actions of leptin.

The present study examined whether blockade of melanocortin receptors subtypes 3 and 4 (MC3/4-R) inhibits chronic cardiovascular and dietary responses to leptin infusion. A cannula was placed in the lateral ventricle of male Sprague-Dawley rats for chronic intracerebroventricular (ICV) infusion via osmotic minipump, and arterial and venous catheters were implanted for measurement of mean arterial pressure (MAP) and heart rate (HR) 24 h/d and IV infusions. After a 5-day control period, rats received (1) 0.9% saline vehicle ICV for 12 days plus leptin (1 microg/kg per minute IV, n=5) during the final 7 days; (2) MC3/4-R antagonist SHU-9119 (1 nmol/h ICV) for 12 days plus leptin (1 microg/kg per minute IV, n=6) during the final 7 days; and (3) SHU-9119 (1 nmol/h ICV, n=8) for 12 days. Leptin infusion in vehicle-treated rats caused a small increase in MAP (5+/-1 mm Hg) despite reduced food intake (23+/-1 to 10+/-1 g/d) and decreased body weight (-6%+/-1%). SHU-9119 infusion completely prevented the cardiovascular and dietary actions of leptin, leading to increased food intake (23+/-1 to 49+/-4 g/d) and body weight (+30%+/-2%), markedly decreased HR (-77+/-9 bpm), and caused a decrease in MAP (-6+/-1 mm Hg). Similar results were observed when SHU-9119 was infused alone in vehicle-treated rats. Leptin decreased plasma insulin to 30% of control values, an effect that was also abolished by SHU-9119 treatment, which caused a 5-fold increase in plasma insulin concentration. Thus, MC3/4-R antagonism completely blocked the chronic cardiovascular, satiety, and metabolic effects of leptin, suggesting that the hypothalamic melanocortin system plays an important role in mediating these actions of leptin.

Hypothalamic melanocortin receptors and chronic regulation of arterial pressure and renal function.

This study examined control of cardiovascular and renal function during chronic melanocortin-3/4 receptor (MC3/4-R) activation or inhibition. Arterial and venous catheters were implanted in Sprague-Dawley rats for measurements of mean arterial pressure (MAP) and heart rate (HR) 24 h/d and for intravenous infusions, and the lateral ventricle was cannulated for chronic intracerebroventricular (ICV) infusions. In experiment 1, after a 5-day control period, rats were administered the MC3/4-R agonist MTII (n=7, 10 ng/h ICV) or 0.9% saline (n=6, ICV) for 14 days, followed by a 5-day recovery period. In experiment 2, after a 5-day control period, rats were administered the MC3/4-R antagonist SHU-9119 (n=7, 1 nmol/h ICV) or 0.9% saline vehicle (n=7, ICV), or pair-fed during SHU-9119 infusion (n=5, 1 nmol/h ICV) for 12 days, followed by a 5-day recovery period. MC4-R activation transiently decreased food intake from 23+/-1 to 10+/-2 g/d. Despite the hypophagia, MC3/4-R activation increased MAP by 7+/-1 mm Hg. MC3/4-R inhibition for 12 days increased food intake from 21+/-1 to 35+/-4 g/d, decreased HR by 53+/-11 bpm, and caused no change in MAP despite the marked weight gain. In rats that were pair-fed to prevent increased food intake, MC3/4-R inhibition further decreased HR (-87+/-9 bpm), whereas MAP was unchanged. Thus, chronic hypothalamic MC3/4-R activation raises arterial pressure despite decreased food intake, whereas MC3/4-R inhibition causes marked weight gain without raising arterial pressure. These observations are consistent with the hypothesis that an intact hypothalamic MC3/4-R may be necessary for excess weight gain to raise arterial pressure.