Aim: Mitochondrial dysfunction is considered to drive the development of inherited cardiomyopathies. The mitochondrial interactosome is a supercomplex formed by ATP synthase, adenine nucleotide translocase (ANT) and voltage dependent anion channel (VDAC)-tubulin that regulate energy metabolism. Previously we proposed that VDAC modulates mitochondrial function through a structural-functional association between the L-type calcium channel, the cytoskeleton and mitochondria. However the molecular mechanism linking the cytoskeleton and the mitochondrial interactosome is unknown.
Method: Cardiomyocytes were isolated from wild-type mice and mitochondrial membrane potential was measured as changes in JC-1 fluorescence following activation of the L-type calcium channel by the dihydropyridine agonist BayK(-). We targeted VDAC with a VDAC peptide, inhibited the adenine nucleotide translocase with bongkrekic acid or atractyloside and inhibited the ATP synthase with oligomycin. Experiments were performed in the presence or absence of 5µM latrunculin-A or 1µM colchicine to depolymerise F-actin filaments or microtubules respectively.
Results: An increase in JC-1 fluorescence occurred upon application of 10µM BayK(-) (15.73 ± 1.57%, n=16) which was attenuated when preincubated with latrunculin-A or colchicine. Application of 10µM VDAC peptide, 10µM bongkrekic acid, 10µM atractyloside or 20µM oligomycin mimicked the increase in ψm induced by BayK(-). Preincubation of the myocytes with colchicine attenuated the increase in JC-1 upon addition of oligomycin and atractyloside but preincubation with latrunculin-A did not significantly alter the increase in JC-1 with oligomycin.
Conclusion: We propose that the L-type calcium channel associates with the mitochondrial interactosome via microtubules to regulate mitochondrial energetics on a beat-to-beat basis in the heart and that disturbances in these interactions can contribute to cardiomyopathies.