Saponin-induced membrane permeabilisation generates Ca 2+ wavesįigure 1 shows the representative i dynamics of the subepicardial myocardium before and during saponin perfusion. Some of the results have been published as an abstract 13. We evaluated this theory using high-speed imaging of the subepicardial myocyte i dynamics in saponin-perfused rat hearts via rapid-scanning confocal microscopy together with confocal fluorescence imaging of myocyte morphology to address whether and how the Ca 2+ waves contribute to the formation of contraction bands. Here, we postulate that saponin-induced Ca 2+ waves contribute to the formation of contraction bands, a morphological pattern associated with reperfusion injuries following ischaemia 11, 12. The Ca 2+-paradox model, which has been widely studied as an alternative model of ischaemia‒reperfusion injury, shows detachment of cell‒cell junctions with hypercontracture of myocytes due to cadherin inhibition induced by Ca 2+ depletion 9. For example, the cryoinjured model exhibits diverse and complex myocyte i dynamics, including high-frequency Ca 2+ waves with or without diminution by Ca 2+ transients and high-static i, an indication of cell death 8 therefore, their morphological relevance is minimal. We chose saponin because this chemical simply permeabilises the sarcolemmal membrane 10, and the resultant entry of Ca 2+ through the membrane pores provides definitive, reproducible induction of irreversible i overload compared with other injury models. To address these issues, we sought to visualise the myocyte i dynamics induced by saponin in the Langendorff-perfused rat heart. However, the precise spatiotemporal i dynamics under the irreversible i overload and their effect on the cellular morphology leading to their eventual cell death remain unknown. We have also observed that irreversible membrane injury inflicted on cardiomyocytes by saponin initiates extremely high-frequency Ca 2+ waves, which are comparable to those induced by Ca 2+-paradox injury 9. Of these, extremely high-frequency Ca 2+ waves, called “agonal waves”, have been regarded as the representative i dynamics in irreversibly injured myocytes, e.g., myocytes subjected to cryoinjury 8 or Ca 2+-paradox injury 9. Our previous work demonstrated that myocytes in Langendorff-perfused rat hearts exhibit three different patterns of Ca 2+ waves, with each pattern depending on the degree of i overload 7. The myocytes suffering from i overload present with Ca 2+ waves, i.e., spontaneous propagating rises in the i concentration, which accompany localised contractions 3, 4, 5, 6. Intracellular Ca 2+ ( i) overload is a cardinal feature of cardiomyocyte injury, and its progression to an irreversible state leads to cell death 1, 2. Contraction bands are not the direct consequence of the waves but are caused by cross-bridge interactions of the myocytes under calpain-mediated proteolysis. Overall, saponin-induced myocyte i overload provokes agonal Ca 2+ waves and contraction bands. The depletion of adenosine 5′-triphosphate by the mitochondrial electron uncoupler carbonyl cyanide 4-trifluoromethoxy phenylhydrazone also attenuated Ca 2+ waves and contraction bands. The contraction bands were not attenuated by the abolition of Ca 2+ 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 myocytes showing these waves displayed contraction bands, i.e., band-like actin-fibre aggregates with disruption of sarcomeric α-actinin. These waves slowly decreased in frequency, developed a prolonged decay phase, and disappeared in 10 min resulting in high-static, fluo4-fluorescence intensity. Our data demonstrate that 0.4% saponin-treated myocytes immediately exhibited high-frequency Ca 2+ waves (131.3 waves/min/cell) with asynchronous, oscillatory contractions having a mean propagation velocity of 117.8 μm/s. We therefore visualised rapid-scanning confocal fluo4- i dynamics and morphology of cardiomyocytes in Langendorff-perfused rat hearts following saponin-membrane permeabilisation. Although irreversible cardiomyocyte injury provokes intracellular Ca 2+ ( i) overload, the underlying dynamics of this response and its effects on cellular morphology remain unknown.
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