Myofibroblasts impair myocardial impulse propagation by heterocellular connexin43 gap-junctional coupling through micropores.

Aim: Composite population of myofibroblasts (MFs) within myocardial tissue is known to alter impulse propagation, leading to arrhythmias. However, it remains unclear whether and how MFs alter their propagation patterns when contacting cardiomyocytes (CMs) without complex structural insertions in the myocardium. We attempted to unveil the effects of the one-sided, heterocellular CM-MF connection on the impulse propagation of CM monolayers without the spatial insertion of MFs as an electrical or mechanical obstacle. Methods and results: We evaluated fluo8-based spatiotemporal patterns in impulse propagation of neonatal rat CM monolayers cultured on the microporous membrane having 8-µm diameter pores with co-culture of MFs or CMs on the reverse membrane side (CM-MF model or CM-CM model, respectively). During consecutive pacing at 1 or 2 Hz, the CM monolayers exhibited forward impulse propagation from the pacing site with a slower conduction velocity (θ) and a larger coefficient of directional θ variation in the CM-MF model than that in the CM-CM model in a frequency-dependent manner (2 Hz >1 Hz). The localized placement of an MF cluster on the reverse side resulted in an abrupt segmental depression of the impulse propagation of the upper CM layer, causing a spatiotemporally non-uniform pattern. Dye transfer of the calcein loaded in the upper CM layer to the lower MF layer was attenuated by the gap-junction inhibitor heptanol. Immunocytochemistry identified definitive connexin 43 (Cx43) between the CMs and MFs in the membrane pores. MF-selective Cx43 knockdown in the MF layer improved both the velocity and uniformity of propagation in the CM monolayer. Conclusion: Heterocellular Cx43 gap junction coupling of CMs with MFs alters the spatiotemporal patterns of myocardial impulse propagation, even in the absence of spatially interjacent and mechanosensitive modulations by MFs. Moreover, MFs can promote pro-arrhythmogenic impulse propagation when in face-to-face contact with the myocardium that arises in the healing infarct border zone.

Generation of myocyte agonal Ca2+ waves and contraction bands in perfused rat hearts following irreversible membrane permeabilisation.

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.

Heat-shock protein 90 modulates cardiac ventricular hypertrophy via activation of MAPK pathway.

The Raf/MAPK/ERK kinase (Mek)/extracellular signal-regulated kinases (Erk) pathway is activated in cardiac hypertrophy after a myocardial infarction. Although heat-shock protein 90 (Hsp90) may regulate the Raf/Mek/Erk signal pathway, the role of Hsp90 in pathophysiological cardiac hypertrophy remains unclear. In this study, we examined the role of Hsp90 in this pathway in cardiac hypertrophy under in vivo and in vitro experimental conditions. Cultured rat cardiomyocytes were treated with the Hsp90 inhibitor 17-(allylamino)-17-dimethoxy-geldanamycin (17-AAG) and proteasome inhibitor MG-132, and then incubated with endothelin-1 (ET) to induce hypertrophy of the cells. The ET-induced increase in the cell size was attenuated by 17-AAG pretreatment. Immunoblot analysis revealed that the c-Raf content of ET-treated cardiomyocytes was decreased in the presence of 17-AAG. An increase in phosphorylation levels of Erk1/2 and GATA4 in ET-treated cardiomyocytes was also attenuated by the 17-AAG pretreatment. Myocardial infarction was produced by ligation of the left ventricular coronary artery in rats, and then 17-AAG was intraperitoneally administered to the animals starting from the 2ndweek after coronary artery ligation (CAL). CAL-induced increases in the heart weight and cross-sectional area were attenuated by 17-AAG treatment. CAL rats showed signs of chronic heart failure with cardiac hypertrophy, whereas cardiac function in CAL rats treated with 17-AAG was not reduced. Treatment of CAL rats with 17-AAG caused a decrease in the c-Raf content and Erk1/2 and GATA4 phosphorylation levels. These findings suggest that Hsp90 is involved in the activation of the Raf/Mek/Erk pathway via stabilization of c-Raf in cardiomyocytes, resulting in the development of cardiac hypertrophy following myocardial infarction.