Abstract 335
Poster Session II, Sunday, 5/2 (poster 257)
Triggered release of sarcoplasmic reticulum (SR) Ca2+ stores by L-type Ca2+ channel-mediated Ca2+ entry is a hallmark of a mature, SR-dependent mechanism of EC coupling in the ventricle of an adult (AD) heart. In contrast, there is evidence for a more SR-independent EC coupling phenotype in the newborn (NB) heart in which transsarcolemmal Ca2+ influx can directly activate contraction. While previous data suggest "reverse" Na-Ca exchange is a likely candidate for Ca2+ influx at birth, the relative contribution of other potential pathways during cardiac development is unclear. To address this issue, we used a pharmacological approach to systematically characterize the contribution of several different mechanisms, including various SR Ca2+ influx routes and cAMP-dependent voltage-activated SR Ca2+ release (VACR) to EC coupling during postnatal development. Ventricular myocytes isolated from NB (1-5 day) and AD (>150days) rabbit hearts were voltage clamped (perforated-patch) using a cell-specific action potential as the command waveform (1-2 Hz at steady state; 34°C). Simultaneous recordings of whole-cell membrane currents and cell shortening amplitude (% resting length) were measured prior to, during and following each intervention. Blocking either the L-type (10 µM nifedipine) or T-type (50 µM Ni2+) Ca2+ current reduced twitch amplitude in AD but not NB myocytes. Inhibition of P-type Ca2+ channels (50 µM ω-agatoxin) had no effect on twitch amplitude in either age group. Blocking all known transsarcolemmal Ca2+ entry pathways (5 mM Ni2+ or 0 Ca2+) abolished twitches in both age groups. However, when 5 mM Ni2+ was added in the presence of isoproterenol (1µM), twitches were transiently maintained in AD myocytes (45.6±2% of control after 30s) but were instantly abolished in NB myocytes. The SR-independent nature of the NB EC coupling phenotype was confirmed by the insensitivity of twitch amplitude to SR Ca depletion by pre-treatment with thapsigargin (10 µM). These data suggest that neither transsarcolemmal Ca2+ influx through Ca2+ channels nor VACR are sufficient to sustain contractions in NB myocytes. Instead, a Ca2+ influx pathway sensitive to high concentrations of Ni2+ (i.e. Na-Ca exchange) appears to be both sufficient and necessary for EC coupling in neonatal rabbit heart.
