Lovastatin Induces Cell Death in Cardiomyocytes that Is Not Reversible by Coenzyme Q 10

The adverse effects of competitive inhibitors of 3-hydroxy-3-methylgutamyl coenzyme A (HMG-CoA) reductase (statins) on skeletal muscle, specifically rhabdomyolysis, has focused interest on the toxicity of these useful agents for the treatment of hypercholesterolaemia. The ability of statins to induce cell death is well recognized and involves the pathways that mediate apoptosis (Guijarro et al. 1998; Wong et al. 2001). Indeed apoptosis may account for the beneficial effects of stains in atherosclerosis (Guijarro et al. 1998) and may lead to their use as adjunctive therapy in certain malignancies (Wong et al. 2001). The potential of statins to produce cardiomyocyte cell death, however, has not received attention. HMG-CoA reductase converts 3-hydroxy-3-methylglutaryl CoA to mevalonic acid which is then further metabolized to eventually produce cholesterol as well as other products that include such ubiquinones as decaprenyl ubiquinone or CoQ10 (Goldstein & Brown 1990). Treatment of patients with the HMG-Co A reductase inhibitor, lovastatin (20 to 40 mg per day), reduces serum concentrations of CoQ10 in man (Folkers et al. 1990). Cardiac concentrations of CoQ10 fell 47% in hamsters (from 98.3 to 51.9 μg/g heart) after lovastatin 50 mg/kg/day for 23 weeks (Belichard et al. 1993), 8.3% in rats after 4 weeks of lovastatin 400 mg/kg/day (Willis et al. 1990); while in guinea pig lovastatin, 40 mg/kg/day produced a 31% and 37% fall in CQ10 concentrations in respectively cardiac muscle and cardiac mitochondria (Diebold et al. 1994). CoQ10 supplementation blunts lovastatin-induced reductions in cardiac CoQ10 concentration and perhaps lovastatin-induced effects on cardiac contractile function (Folkers et al. 1990; Willis et al. 1990). In addition to replenishing CoQ10 depletion, exogenous CoQ10 has other potential actions namely as a membrane antioxidant that inhibits components of the mitochondrial pathway leading to cell death and also as a pro-oxidant that participates in redox signaling (Yamamura et al. 2001). Data on the potential of CoQ10 to blunt cell death, in non-cardiac cells, is controversial. CoQ10 protected cultured neurones against spontaneous and excitotoxin-induced cell death (Favit et al. 1992) but failed to alter ischaemia-induced cell death in liver or brain in the intact rat (Cho et al. 1990; Li et al. 2000). The beneficial effects of CoQ10 depend on the cell type and the kind of injury (Kagan et al. 1999). Data that lovastatin reduces CoQ10, coupled with the importance of mitochondria in actively metabolizing cardiomyocytes and the role of mitochondria in the production of apoptosis suggest the hypothesis that lovastatin would induce cell death in cardiomyocytes and that CoQ10 would prevent this type of cardiac cell death. Chick embryonic ventricular cells were cultured from 7-day chick embryos from white Leghorn eggs (Rabkin & Kong 2000). Cardiomyocytes were placed in multiwell Primaria plates (Becton Dickinson, Lincoln Park, NJ, USA), 20,000 cells per well and maintained in culture for 72 hr. The proportion of myocytes at this time was at least 90% as verified by the proportion of cells showing spontaneous contraction or displaying muscle specific markers on immunohistologic examination (Rabkin & Kong 2000). Media were removed and replaced with warm media with lovastatin (10 to 300 μM) (A.G. Scientific Ltd.,San Diego, CA, USA) or its diluent; CoQ10 (10 or 100 μM) (Sigma Chemical Co., St. Louis, Mo., USA) or its diluent; or the combination of lovastatin and CoQ10 for 24 hr. Cell viability was assayed using the MTT assay, that in cardiomyocytes correlates highly with cell death measured by the trypan blue assay and apoptosis determined by nuclear DNA content (Rabkin & Kong 2000). The MTT dye was added to each well for the last 4 hr of treatment. The reaction was stopped with a solubilization reagent (Promega, Madison, WI, USA). The absorbance (optical density – OD) and the difference in OD at 570 nm from the reference at 655 nm was determined on a multiwell plate reader (BioRad model#3550, BioRad, Mississauga, Canada) (Rabkin & Kong 2000). Background OD of medium, in the absence of cells, was subtracted. All samples were done in duplicate and averaged for each experiment. Hypothesis testing used one way analysis of variance. The null hypothesis was rejected if the probability of a Type I error was less than 5% (P<0.05). Lovastatin treatment, for 24 hr, produced a significant (P<0.01) concentration-dependent reduction in OD570 (fig. 1). Considering the ability of viable cells to reduce MTT and the direct relationship between cardiomyocyte cell number and MTT OD570 (Rabkin & Kong 2000), lovastatin treatment produced a significant (P<0.01) dose-dependent increase in cell death. Cardiomyocytes were more resistant to lovastatin-induced changes in MTT than leukaemia cells that showed reductions in MTT absorbance and cell survival with lovastatin as well as atorvastatin, fluvastatin and especially cerivastatin (Wong et al. 2001). The ability of lovastatin to induce cardiomyocyte cell death may explain the reduced survival of cardiomyopathic hamsters treated with lovastatin (Marz et al. 2000). There has been little previous data on the direct effects of statins on the heart. Lovastatin depresses calcium channel activity in cardiomyocytes and blocks excitation-contraction coupling (Renaud et al. 1986). However this is not a likely mechanism to explain lovastatin-induced cell death as calcium channel blockers inhibit cell death in these cardiomyocytes (Rabkin & Kong 2000). Cardiomyocyte viability after treatment with lovastatin. Cardiomyocytes, seeded in multiwell microtitre plates, cells were treated with lovastatin (μM) 10 (N=10), 50 (N=11), 100 (N=14), 300 (N=8), or 0 control (N=17) for 24 hr. Cell viability was assayed by the MTT assay at optical density (absorbance) 570 nm. Data are the mean±S.E.M. Hypothesis testing compared lovastatin and control cells (*P<0.05; **P<0.01). Cardiomyocytes were next treated with CoQ10 for 24 hr concomitantly with lovastatin. CoQ10, from 10 to 100 μM, did not produce any significant concentration-dependent changes in lovastatin-induced cell death. A wide range of lovastatin concentrations were used as the effects of CoQ10 might have been more evident at lower lovastatin concentrations where there was less cell death-potentially reversible, or at higher lovastatin concentrations were there was more cell death and an effect might be more apparent. The CoQ10 concentrations comprised those used by others to demonstrate CoQ10 dependent increases in cell survival (Favit et al. 1992). There was, however, no evidence that CoQ10 might blunt cardiomyocyte cell death induced by lovastatin at any concentration of lovastatin or CoQ10. The absence of an effect of CoQ10 is not likely due to the inability to prevent cardiomyocyte cell death as other factors can modify cell death in these myocytes (Rabkin & Kong 2000). Instead these data support the contention that CoQ10 does not prevent cell death and are consistent with the findings with CoQ10 in ischaemic liver (Cho et al. 1990) and brain (Li et al. 2000). Alternatively it suggests that cardiomyocytes in culture are different from neurones in culture that appear to benefit from CoQ10 (Favit et al. 1992). As the beneficial effect of CoQ10 may be dependent on the cell type and mode of injury (Kagan et al. 1999), the present data indicate that CoQ10 is not beneficial in protection of cardiomyocytes against lovastatin-induced cell death. One might have anticipated that CoQ10 would play more of a role in lovastatin-induced cell death, because CoQ10 is an end-product of the mevalonate pathway, which is interrupted by lovastatin. This lack of an effect of CoQ10 suggests that the mechanism of lovastatin-induced cell death is independent of CoQ10. This proposition can be supported, in part, by data in skeletal muscle where myopathy induced by simvastatin, could not be attributed to a dysfunction of mitochondrial respiration due to low ubiquinone levels (Nakahara et al. 1998). These data also suggest that lovastatin-induced cell death in cardiomyocytes is not mediated through oxidative stress of the type that can be modified by CoQ10. In summary, lovastatin produced a loss of cell viability in cardiomyocytes. The inability of cardiomyocytes to regenerate has important ramifications for heart failure and suggests the possibility that the fatalities associated with statin-induced rhabdomyolysis might also involve attendant statin-induced cardiotoxicity. This study showed that CoQ10 fails to alter lovastatin-induced cardiomyocyte cell death. Support by a grant in aid from the Heart and Stroke Foundation of British Columbia and the Yukon.

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