Local Ca2+ Releases Enable Rapid Heart Rates in Developing Cardiomyocytes

Homogeneous intracellular Ca2+ release repeated with high frequency is the basis of the rhythmic contractions of cardiac myocytes. In adult ventricular myocytes, the t-tubular system enables transient homogeneous Ca2+ signals. Interestingly, the developing cardiomyocytes do not have t-tubuli and Ca2+ signal propagation in the cytosol is based on the relatively slow diffusion of Ca2+ ions. This is likely to result in spatiotemporal heterogeneity of Ca2+, which limits the maximal frequency of the Ca2+ signals. We observed that intracellular Ca2+ signals of 12.5 days old mouse embryonic ventricular myocytes are more homogeneous than expected if the Ca2+ signals would propagate by pure diffusion. To study the propagation more accurately, we injected a small amount of Ca2+ to a single point in the cytosol via patch-clamp pipette while performing the line-scan imaging of the intracellular Ca2+. With this method we found that inhibition of the sarcoplasmic reticulum (SR) Ca2+ release channels results in 3-fold slowing of Ca2+ signal propagation (control: 10.1 ± 2.7 ms/μm vs. ryanodine (50 μM): 33.6 ± 9.2 ms/μm, P < 0.05). This suggested that the propagation of Ca2+ signals is amplified with local SR Ca2+ releases. Immunolabeling of SR Ca2+ release and uptake proteins revealed a regular structure throughout the cytosol at ∼2 μm intervals. These extensions of SR were equally functional in all parts of the cytosol. To further study the role of these local Ca2+ release sites in developing cardiomyocytes, we implemented a model of them into the previously published mathematical model of an embryonic cardiomyocyte. The computer simulations showed that the local Ca2+ releases are prerequisite for synchronizing the global intracellular Ca2+ releases upon electrical excitation and maintaining the capability of developing cardiomyocytes to generate spontaneous pacemaking at a sufficiently high frequency.