Simvastatin Induced Porcine Coronary Artery Relaxation: Role of Ca2+ Influx and Mitochondrial Function

Almukhtar HM, Smith PA, Roberts RE. University Of Nottingham, Nottingham, UK

HMG-CoA reductase inhibitors, statins, are the drug of first choice for treatment of hypercholesterolemia. Statins also have beneficial effects beyond lipid lowering and these include direct vascular effects, Previous studies have demonstrated that simvastatin (SIM) induced coronary vasodilators in the isolated bovine coronary arteries (1). We have previously demonstrated that SIM produced a slow relaxation of the coronary artery, which was independent of K+ channel, NO, PGI2 or the endothelium (2). We have also proved that SIM (10 μM) depolarized the membrane potential of mitochondria in both isolated smooth muscle cells and intact blood vessels (3). However, the mechanism of SIM relaxation remains to be elucidated. The aim of the present study was to determine the effects of SIM on Ca2+ influx, the role of mitochondria also has been examined. SIM effects on Ca2+ influx through voltage-gated Ca2+ channels were assessed using isolated tissue bath, concentration–response curves for CaCl2 were constructed. Proximal coronary artery segments were isolated from porcine hearts obtained from a local abattoir and set up in Krebs- buffer in isolated tissue baths for isometric tension recording. To assess the effect of SIM on Ca2+ influx, concentration–response curves to CaCl2 were constructed in Ca2+-free, high potassium Krebs’ solution in the absence or presence of SIM (10 µM). The concentration of intracellular Ca2+ was monitored with Fluo-4 using standard imaging techniques, smooth muscle cells were isolated from proximal coronary artery segments by incubation with type II collagenase (2 mg/ml) for 30min, and then plated on glass coverslips. Cells were loaded with fluo-4 AM (5 μM) in Hanks buffer containing (in mM): 5.6 KCl, 138 NaCl, 4.2 NaHCO3, 1.2 NaH2PO4, 2.6 CaCl2, 1.2 MgCl2, 10 glucose and 10 HEPES [pH 7.4] for 1 hr at room temperature. After 5min basal recording, the coverslips were superfused with high K+ Hanks containing (in mM): 50 KCl, 92.6 NaCl, 4.2 NaHCO3, 1.2 NaH2PO4, 2.6 CaCl2, 1.2 MgCl2, 10 glucose, and 10 HEPES [pH 7.4], which elicited a sustained increase in fluorescence, subsequently SIM (10 μM), or DMSO (0.1% v/v) as a control, was added to the perfusion media. The role of mitochondrial complex I was investigated by pre-incubation with rotenone (10 μM). In a further set of experiments, tissues were pre-contracted with L-type Ca2+ channel blocker nifedipine (1 μM) prior to the addition of SIM (10 μM). Re-addition of Ca2+ in the presence of 60 mM KCl produced a concentration-dependent contraction in the porcine coronary artery; incubation with SIM (10 μM) for 1 hour produced a concentration-dependent inhibition of the contractile response (76.4±8.4) compared to control (132.3±8.8), P <0.001 (2- way ANOVA). The inhibition was nearly reversed by incubation with rotenone (113.3±10.8). Similarly rotenone (10 μM) incubation inhibited SIM relaxation (5.6±3.9) of the KCl pre-contracted coronary vessels versus control relaxation (50.8±5.7), P <0.001 (2- way ANOVA). On the other hand, nifedipine had no effect on SIM relaxation. In freshly-isolated smooth muscle cells, SIM inhibited KCl-induced increase in intracellular Ca2+ (45.6±1.5) compared to control non-treated cells (108±18.5) p<0.0001, unpaired t-test. In conclusion, this study has demonstrated that the relaxation to SIM is dependent upon the presence of extracellular Ca2+. SIM inhibits the contraction in response to increasing concentrations of extracellular Ca2+. Rotenone effect on SIM relaxation suggested that SIM relaxation is dependent, in part, upon mitochondria. The effects on the mitochondria may lead to reduction in Ca2+ influx and hence relaxation of the blood vessel, although future studies are required to determine if this is the case.

(1) Lorkowska B et al. (2005) Prost Leukot Essent Fatty Aids 72:133-8

(2) Almukhtar HM et al. (2012) 2nd Joint Meeting of the British and American Microcirculation

(3) Almukhtar HM et al. (2013) pA2 online