P398Aggregate prone desmin mutations reduced mitochondrial calcium uptake in primary myotubes upon electrical stimulation

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Abstract

Purpose: Calcium (Ca2+) plays key role in muscle cell physiology, e.g. takes part in the contraction and acts as co-factor in Krebs cycle. Mitochondria are capable to uptake calcium due to close proximity to SR Ca2+ releasing microdomains. Mitochondrial calcium balance is crucial for cell destiny, increased calcium leads to apoptosis, when decreased drives autophagy. On the other hand mitochondria within the muscle cells tightly connected to desmin. In desmin knock-out mice mitochondria were non-functional. Desmin mutations are associated with developing of myopathies, however machinery remains to be elucidated. We proposed that mitochondrial failure in desmin-compromised cells might result in disturbed calcium balance. We aimed to assess impact of non-aggregate prone (D399Y) and aggregate prone (L345P, A357P, L370P) desmin mutations on mitochondrial calcium.

Materials and methods: Satellite cells were obtained from soleus muscle of young C57BL/6 males. Cells were transduced with lentiviral suspension, encoded desmin wild type or mutant and GFP as reporter gene. Cells were grown for one week, then differentiation was induced and continued two weeks more. Obtained myotubes were loaded with rhod-2 AM for 20 min, after that dye was washed out for 20 min. Electrical stimulation was applied for up to 30 s as follows: 1 Hz, rest, 10 Hz, rest, and 100 Hz, rest. Changes in rhod-2 fluorescence were detected using laser confocal microscopy. Intensity of fluorescent signal was measured in baseline (without stimulation (F0)) and during stimulation (F). Ratio F/F0 was calculated and represented peaks amplitudes of mitochondrial calcium transients.

Results: After electrical stimulation the peak amplitude of mitochondrial calcium uptake was similar in non-transduced and DesWT cells being 2.61 ± 0.19 and 3.12 ± 0.29 respectively. However, in DesL345P myotubes and DesA357P myotybes, the mitochondrial calcium uptake was markedly decreased by 50% and 44% respectively in comparison with DesWT cells. Interestingly, mitochondrial calcium uptake in the DesD399Y myotubes was similar to that seen in DesWT cells and was equaled 3.45 ± 0.53. DesL370P cells didn't decrease peak amplitude as much as DesA357P and DesL345P and showed transitional peak, F/F0 was 2.85 ± 0.31.

Conclusion: Therefore mitochondrial capacity to uptake calcium depended on conformational form of desmin protein and aggregate prone mutations markedly decreased calcium uptake in comparison with non-aggregate prone mutation. We assumed that desmin aggregates broke mitochondria localization within the calcium release microdomains.

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