Simvastatin Inhibits the Function of TRPC1 and Aameliorate Endothelin 1 Induced Cardiac Hypertrophy
Authors: Selvaraj Senthil , Singh Brij B , Al Suwaidi Jassim , Yacoub Magdi 
Published: July 2019

Background & Purpose

Intracellular Ca2+ plays an important role in the cardiac physiology and development. Augmentation in intracellular Ca2+ activates the hypertrophic response in cardiomyocytes, although the source of the Ca2+ responsible for this is still elusive. We have previously shown that calcium influx through Transient receptor potential canonical 1 (TRPC1) channel is a key mediator of the cardiac hypertrophic response and lipid raft facilitates the channels assembly. Statins have recently been shown to exert pleiotropic protective effects in cardiac hypertrophy. Here, we studied whether simvastatin interfere with the membrane lipid raft formation and affects the TRPC1 channel function to protect the cardiomyocytes against hypertrophic stimuli.

Methods

H9C2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and maintained in high glucose DMEM supplemented with heat-inactivated 10% fetal bovine serum (FBS) (Gibco, MD), 100 units/ml penicillin, 100 μg/ml streptomycin in a water-saturated atmosphere of 5% CO2 at 37 °C. To induce hypertrophy, cells were exposed to endothelin 1 for 48 h. Cells treated with a combination of simvastain and endothelin-1 were exposed to simvastatin 12 h prior to the addition of endothelin-1 to the treatment medium and were then incubated in the presence of both drugs for a further 48 h. protein expression was analysed by western blotting. H9C2 cells were loaded with Fura-2 to measure the TRPC1 channel function and images were taken by using EasyratioPro (PTI Tech). For transient transfection, cells were transfected with TRPC1 siRNAs or STIM1 siRNA or scrambled control siRNA using Lipofectamine 3000. To measure NFAT luciferase activity, cells were transiently transfected with pNFAT-Luc (containing firefly luciferase gene from Photinus Pyralis; Clontech, Mountain View, CA) and 0.06 μg of pRL-TK (containing Renilla luciferase gene from Renilla reniformis, used as internal control; Promega, Madison, WI). To isolate lipid raft, cells were lysed in TNE buffer and lysates were mixed with 80% sucrose (w/v), and overlaid with 6 ml of 35% sucrose followed by 4 ml of 5% sucrose (in TNE buffer). Samples were centrifuged at 34,000 rpm for 18 h at 4 °C using SW 41 Ti Swinging Bucket Rotor. Ten 1.2-ml fractions were collected from the top of the tube and used as required.

Results

Endothelin 1 treatment induced hypertrophic response in cardiomyocytes which were confirmed by measuring cell size and hypertrophic markers, ANF and BNP (60% increase compare to control). Endothelin 1 treatment significantly induced calcium overload as revealed by an increase intracellular Ca2+ and subsequent NFAT nuclear activation compared with control. Moreover, hypertrophic stimuli increases the expression and function of TRPC1 and activate the recruitment of TRPC1 to membrane lipid raft domain from non-raft in hypertrophic cardiomyocytes (HC). Co-immunoprecipitation of STIM1, a Ca2+ sensor in the sarcoplasmic reticulum (SR), revealed that the functional interaction between STIM1 and TRPC1 was increased in HC and lipid raft domain facilitates the interaction. Silencing TRPC1 or STIM1 by respective siRNAs significantly prevented the calcium overload and NFAT activation in HC. Simvastatin treatment prevent the calcium overload and attenuates the hypertrophic response in H9C2 cells. Moreover, cholesterol depletion by simvastatin markedly reduced the calcium influx via TRPC1 by affecting the lipid raft domain and decrease the interaction of TRPC1 and STIM1 in hypertrophic cardiomyocytes.

Conclusion

Our results indicate that Ca2+ influx through TRPC1 provides a unique source of Ca2+ to activate pathologic cardiac hypertrophy and blockade of TRPC1 function by affecting the lipid raft domain formation in hypertrophic cardiomyocytes may be another important therapeutic mechanism of simvastatin in preventing cardiac hypertrophy.