No abstract

Ca 2+ overload is a cardinal feature of cardiomyocyte injury, and its progression to irreversible state leads to cell death. However, unknowns are the precise spatiotemporal changes in the myocyte Ca 2+ dynamics and the relevant cell morphology of irreversibly injured hearts. On the hypothesis that myocytes exhibit high-frequency Ca 2+ waves and contraction band necrosis in saponin-permeabilized injured heart, we observed changes in the Ca 2+ dynamics and the relevant morphological changes in the subepicardial myocardium of the Fluo4-loaded rat hearts (n = 14) by rapid-scanning confocal microscopy (100 frames/s) under Langendorff perfusion with 0.3 mM Ca 2+ -Tyrode solution including 0.4 % saponin at 30°C. Also performed was confocal imaging of tetramethylrhodamine methyl ester (TMRM) fluorescence of the myocardium. Under quasi-quiescence of the heart after dissection of the SA node, individual myocytes barely exhibited spontaneous Ca 2+ waves, whereas after commencement of saponin perfusion high-frequency (118 ± 9.7 /min/cell, mean ± SEM) Ca 2+ waves (hereafter, “agonal waves”) emerged within 1 min, showing asynchronous, oscillatory contractions in the individual myocytes with a V prop of 124 ± 2.5 μm/s (n = 60). Subsequently, the waves gradually decreased in frequency with concomitant slowing of its decay time course, and eventually, disappeared in 6 min; myocytes exhibited high, static Fluo4-fluorescence intensity. Along with the progression of Ca 2+ overload by saponin, the TMRM fluorescence intensity was discretely lost in individual myocytes. The myocytes showing the agonal waves exhibited contraction bands, i.e., band-like aggregations of the actin fibers. Under mechanical arrest of the heart by 2,3-butanedione monoxime (20 mM), saponin still induced the agonal waves with a frequency of 253 ± 10.6 /cell/min and V prop of 118 ± 2.1 μm/s (n = 60); however, contraction bands were barely seen.In conclusion, irreversible myocyte injury by saponin provoked agonal Ca 2+ waves and oscillatory contractions indicating progressive Ca 2+ overload and the following mitochondrial damage, which may provide deeper insights into understanding the mechanism of contraction band necrosis.

Although irreversible cardiomyocyte injury provokes intracellular Ca2+ ([Ca2+]i) overload, the underlying dynamics of this response and its effects on cellular morphology remain unknown. We therefore visualised rapid-scanning confocal fluo4-[Ca2+]i dynamics and morphology of cardiomyocytes in Langendorff-perfused rat hearts following saponin-membrane permeabilisation. Our data demonstrate that 0.4% saponin-treated myocytes immediately exhibited high-frequency Ca2+ waves (131.3 waves/min/cell) with asynchronous, oscillatory contractions having a mean propagation velocity of 117.8 μm/s. These waves slowly decreased in frequency, developed a prolonged decay phase, and disappeared in 10 min resulting in high-static, fluo4-fluorescence intensity. The myocytes showing these waves displayed contraction bands, i.e., band-like actin-fibre aggregates with disruption of sarcomeric α-actinin. The contraction bands were not attenuated by the abolition of Ca2+ waves under pretreatment with ryanodine plus thapsigargin, but were partially attenuated by the calpain inhibitor MDL28170, while mechanical arrest of the myocytes by 2,3-butanedione monoxime completely attenuated contraction-band formation. The depletion of adenosine 5′-triphosphate by the mitochondrial electron uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone also attenuated Ca2+ waves and contraction bands. Overall, saponin-induced myocyte [Ca2+]i overload provokes agonal Ca2+ waves and contraction bands. Contraction bands are not the direct consequence of the waves but are caused by cross-bridge interactions of the myocytes under calpain-mediated proteolysis.

No abstract

Raf/Mek/Erk pathway plays a crucial role in the development of cardiac fibrosis. It is assumed that heat shock protein 90 (Hsp90) may regulate the Raf/Mek/Erk signal pathway. However, the role of Hsp90 in cardiac fibrosis under pathophysiological conditions remains unclear. In this study, effects of Hsp90 inhibitor on signal transducers in cultured cardiac fibroblasts were examined. Cardiac fibroblasts prepared from neonatal rats were treated with combination of Hsp90 inhibitor 17-(allylamino)-17-dimethoxy-geldanamycin (17-AAG) and proteasome inhibitor MG132. Proliferation of cardiac fibroblasts was attenuated by 17-AAG treatment for 48 h. 17-AAG treatment also reduced an expression of collagen I and III. c-Raf content of cardiac fibroblasts was decreased in the presence of 17-AAG. An increase in phosphorylation levels of Erk1/2 in cardiac fibroblasts attenuated by 17-AAG treatment. MG132 reversed the loss of c-Raf in cardiac fibroblasts treated with 17-AAG. These findings suggest that Hsp90 involves an activation of Raf/Mek/Erk pathway via c-Raf stability in cardiac fibroblasts, leading to the development of cardiac fibrosis.