The role of the cytoskeleton in regulating mitochondrial function in cardiomyopathic states

H.M. Viola,¹ T. Tsoutsman,² C. Semsarian,^2,4 C.E. Seidman³ and L.C. Hool,¹ ¹School of Anatomy, Physiology and Human Biology, The University of Western Australia, Crawley, WA 6009, Australia, ²Agnes Ginges Centre for Molecular Cardiology, Centenary Institute, The University of Sydney, NSW 2006, Australia, ³Harvard Medical School Department of Genetics, 77 Avenue Louis Pasteur, Boston, MA 02115, USA. and ⁴Department of Cardiology, Royal Prince Alfred Hospital, Sydney, NSW 2066, Australia.

Cardiomyopathy is associated with alterations in calcium homeostasis and disrupted cytoskeletal architecture. We have previously demonstrated that the L-type calcium channel (LTCC) can regulate mitochondrial function through alterations in intracellular calcium and via the transmission of movement of cytoskeletal proteins associated with channel activation and inactivation. To determine whether the cytoskeleton is integral to this process, we investigated the role of the LTCC in regulating mitochondrial function in 30-50 wk old mice over expressing the human disease causing mutations Gly203Ser (cTnI-G203S) or Arg403Gln (αMHC^403/+), and exhibit characteristic features of Familial Hypertrophic Cardiomyopathy. LTCC current density recorded in cTnI-G203S and αMHC^403/+ myocytes was similar to wt myocytes. However the inactivation rate of the current was significantly faster in cTnI-G203S and αMHC^403/+ myocytes (cTnI-G203S: 22.9 ± 1.2 ms, n = 11 and MHC403: 20.2 ± 1.4 ms, n = 6 vs wt: 30.6 ± 1.4 ms, n = 7; P < 0 .05). We examined the effect of activation of LTCC on mitochondrial membrane potential assessed as changes in JC-1 fluorescence. Application of BayK(-) caused a 1 8.8 ± 2.0% increase in mitochondrial membrane potential in wt myocytes (n = 10). In contrast, BayK(-) caused a significantly greater increase in mitochondrial membrane potential in cTnI-G203S and αMHC^403/+ myocytes (cTnI-G203S: 29.2 ± 1.9 ms, n = 15; MHC403: 27.8 ± 3.2 ms, n = 9; P < 0.05). Consistent with this finding, application of BayK(-) caused a significantly greater increase in flavoprotein oxidation (an indirect measure of mitochondrial oxygen consumption) in cTnI-G203S and αMHC^403/+ myocytes (cTnI-G203S: 24.4 ± 6.5%, n = 8; MHC403: 24.6 ± 3.8%, n = 7) compared with wt myocytes (10.0 ± 1.6% increase, n = 9; P < 0.05) . These data indicate that L-type calcium channel current kinetics are altered in hearts of cTnI-G203S and αMHC^403/+ mice. The mice appear to display a hypermetabolic mitochondrial state in response to LTCC activation. These findings are in direct contrast to responses recorded in myocytes from mdx hearts, the murine model of Duchenne Muscular Dystrophy that demonstrate poor metabolic activity and dilated cardiomyopathy. We conclude that disruption of the cytoskeletal architecture contributes to altered regulation of mitochondria by the L-type calcium channel in the cardiomyopathic heart.