Functional Expression and Inactivation of L-type Ca2+ Currents During Murine Heart Development -Implications for Cardiac Ca2+ Homeostasis

Abstract

Background/Aims: The expression and regulation of Ca²⁺ signaling in embryonic cardiomyocytes has been shown to be different from those in adult heart cells, particularly the L-type Ca²⁺channel current (I_CaL) increases during development. However, little is known about the underlying reasons for this increase of I_CaL density and developmental changes in the process of I_CaL inactivation, a critical regulator of intracellular Ca²⁺ homeostasis. In the present work, we therefore studied functional differences of I_CaL between embryonic and fetal cardiomyocytes and its interaction with intracellular Ca²⁺ homeostasis and Ca²⁺-induced Ca²⁺ release (CICR). Moreover, we examined the process of voltage- (VDI) and Ca²⁺-dependent inactivation (CDI) of I_CaL during murine embryonic heart development. Methods: The electrophysiological characteristics of I_CaL inactivation were analyzed in embryonic ventricular cardiomyocytes of early (E9.5-11.5) and late developmental, fetal (LDS, E16.5-18.5) stages and of adult mice using the whole-cell patch-clamp technique. Results: Fast, Ca²⁺-dependent inactivation kinetics (τ_f) were significantly accelerated in LDS-derived cardiomyocytes (2.53±1.43 ms, n=9) as compared to EDS (5.09±2.19 ms, n=8, p>=0.009), whereas slow, voltage-dependent inactivation time constants (&tau _s) were unchanged. In cardiomyocytes derived from LDS we observed an increase in the maximal gating charge (Q_max), suggesting an increase in the number of L-type Ca²⁺ channels at the sarcolemma, whereas the channel open probability (P_o) was unchanged. Accordingly a significantly higher I_CaL density was found in LDS (-14±2.26 pA/pF, n=14) versus> EDS-derived cardiomyocytes (-10.03±1.43 pA/pF, n=13, p< 0.05). Since ryanodine (10 µM) failed to alter τ_f at both developmental stages, a major contribution of CICR and bulk Ca²⁺ to the acceleration of the fast inactivation kinetics during heart development seems to be unlikely. Conclusion: Our data suggest that the increase of local subsarcolemmal Ca²⁺, evoked by the higher expression of L-type Ca²⁺ channels at the sarcolemma, rather than bulk Ca²⁺ accelerates I_CaL inactivation during embryonic heart development.