Résumé :
|
Delta-sarcoglycan deficiency induces muscular dystrophy and dilated cardiomyopathy (DCM) through pathological mechanisms that remain elusive. Lack of dystrophin, an other sarcolemmal protein, induces skeletal muscle fibers Ca2+ overload that may be responsible for fiber death by triggering over-activation of calcium-dependent proteases (ì- and/or m-calpain). In dystrophin and delta-sarcoglycan striated muscle cells, mean cytosolic [Ca2+], although elevated, is lower than the threshold needed to activate calpains. It has been proposed that in the sub-membrane area [Ca2+] could be higher that in the rest of the cytosol. We challenged herein this hypothesis in cardiomyocytes from delta-sarcoglycan deficient CHF147 hamsters by the use of two different techniques. Cardiomyocytes from healthy Syrian (Wt) and CHF147 hamsters were isolated with collagenase and then loaded with calcium fluorescent probes. Two types of acquisitions were made. Standard cytometry was used for measuring mean cytosolic Ca2+ with Fura-2 and sub-membrane Ca2+ with FFP-18. Additionally we used fluorescence image deconvolution to map cytosolic [Ca2+] within individual cardiomyocytes. When Fura-2 was used, the cytosolic [Ca2+] values obtained were similar, but each time higher in CHF147 (Cytometry/Deconvolution in nM: 144 ± 8 / 149 ± 15 (P> 0.05) for Wt and 215 ± 18/214 ± 36 (P> 0.05) for CHF147). Similar results were obtained in cytometry when [Ca2+] values were calculated in FFP-18 loaded cells. Nevertheless, FFP-18 exhibited a nonspecific location being homogeneously distributed in the cytosol. Finally, image deconvolution of Fura-2 loaded cells allowed the discrimination in CH147 cardiomyocytes of a sub-membrane area with a [Ca2+] 2-3 fold higher than that measured in the rest of the cytosol. Our results show that [Ca2+] is higher in the sub-sarcolemmal space of CHF147 cardiomyocytes. On the other hand, we show that the deconvolution method is suitable for the study of cytosolic calcium areas including sub-sarcolemmal space, unlike the use of FFP-18. The existence of [Ca2 +] higher beneath the plasma membrane is consistent with activation of calpain by Ca2+ in CHF147 cardiomyocytes.
|