Studies of excitation-contraction coupling in developing cultured embryonic amphibian skeletal muscle cells: A role of I(Ca) in excitation-contraction coupling

Abstract

Adult frog phasic skeletal muscle cells have slow inward calcium currents (ICa) with no obvious role in excitation-contraction coupling (ECC). We have found that embryonic skeletal myocytes are capable of contracting in the early stages of development, at the time when the T-system and the SR may not be fully developed. Contractions could then be triggered at this stage by Ca2+ flowing into the cells through ICa. The present thesis was undertaken to test the hypothesis that ICa may have a role in ECC during early stages of skeletal muscle cell development. This study was carried out in embryonic skeletal myocytes from Xenopus laevis cultured for 1-15 days. We recorded ICa with the patch-clamp technique and, Ba2+ as the carrier. Cell capacitance and current density indicated that ICa and total membrane area increase with time, reaching a plateau at around days 12-15. Cytosolic Ca2+ changes were observed from fluorescence images. While [Ca2+]i increased continuously during 700 ms depolarizing pulses in young cells, older cells showed an early [Ca2+]i rise followed by a steady lower [Ca2+]i. In young cells [Ca2+]i did not increase when the cells were field stimulated in the presence of 10-7 M [Ca2+]0, while 1-week-old cells showed an increase in [Ca2+]i. We studied the SR functional development by measuring contracture thresholds and [Ca2+]i in response to caffeine. Young cells responded to higher caffeine concentrations than older cells. Development of the T-tubular and SR membranes was investigated with fluorescence confocal microscopy. One-day-cultured myocytes are devoid of T-tubular membrane, except for short and sparse invaginations. Within 3 days, a T-tubular system forms and continues to grow until day 12 in culture. The SR is present in the periphery of the cell from day one. At this stage the SR is scanty but more abundant than the T-tubules and does not form a particular pattern. The longitudinal and transverse pattern of the SR forms within two weeks as a complex and highly dense structure. Our results indicate that (1) a different process of ECC is taking place during the early stages of embryonic skeletal muscle development and (2) the role of ICa is to provide an influx of calcium that may enable the cell to contract, probably by a mechanism similar to calcium-induced calcium release.