Calcium is a versatile intracellular messenger that encodes information in frequency, amplitude, and subcellular distribution. We examined how the ultrastructure of rat atrial myocytes constrains action potential-evoked calcium responses and their ability to promote cellular contraction.
Rats were humanely killed following CO2 anaesthesia. Myocytes were stimulated with 40V pulses (2 ms duration) using two field electrodes (0.5 cm from the cell) at 1 Hz, and calcium changes were monitored using laser scanning confocal microscopy (NORAN Oz, Bicester, UK) of Fluo-4-loaded cells.
Under control conditions, only 10 % (n = 120 cells) of atrial cardiac myocytes examined showed homogenous global calcium transients following electrical stimulation. In the majority of cells calcium transients originated in sub-sarcolemmal locations, and gave rise to a sharply-defined ring of elevated calcium. Despite functional ryanodine receptors being expressed at regular (~2 µm) intervals throughout the cells, the subsarcolemmal calcium signal did not spread in a regenerative manner to the cellular interior. Rather, there was only a diminishing inward diffusion of calcium. The averaged subsarcolemmal calcium response was 1656 ± 219 nM (mean ± S.E.M.) and took 38 ± 3 ms to reach peak (n = 5 cells), whilst the calcium rise in the central region of the atrial cells was typically 400 ± 63 nM and was maximal after 91 ± 7 ms (n = 5 cells). Immunostaining atrial myocytes revealed that L-type voltage-operated calcium channels were exclusively located around on the sarcolemma around the outside of the cells (n = 30 cells). Therefore, in atrial myocytes excitation-contraction coupling takes place in subsarcolemmal regions where L-type voltage-operated calcium channels and ryanodine receptors are co-localised. Treating atrial cells with cyclopiazonic acid (10 µM) or antimycin (20 µM) + oligomycin (20 µM), to inhibit sarcoplasmic reticulum calcium ATPases and mitochondrial calcium sequestration respectively, allowed the subsarcolemmal calcium signal to propagate regeneratively into the central regions of atrial cells (n = 30 cells). Calcium ATPases and mitochondria therefore form a functional firewall in atrial myocytes, which limits the calcium signals during normal excitation-contraction coupling to the subsarcolemmal sites. Positive inotropic agents such as endothelin-1 (100 nM) and the β-adrenergic agonist isoproterenol (100 nM) also induced globalisation of action potential-evoked calcium signals (n = 20 cells). The functional consequence of globalising calcium signals was a significant increase in the contractility of the cells. For example, with endothelin-1 the twitch amplitude increased by 585 ± 52 % (n = 8). We therefore suggest that atrial myocytes have two functionally distinct populations of ryanodine receptors. The subsarcolemmal population is recruited during each action potential, but produces only a spatially limited calcium signal that triggers modest contraction. The central non-junctional ryanodine receptors represent an inotropic reserve, but are located behind a calcium ATPase and mitochondrial firewall. Physiological inotropes can activate this second population of ryanodine receptors to enhance the contractility of the cells.