Spontaneous Ca²⁺ transients in rat pulmonary vein cardiomyocytes are increased in frequency and become more synchronous following electrical stimulation

The pulmonary veins have an external sleeve of cardiomyocytes that are a widely recognised source of ectopic electrical activity that can lead to atrial fibrillation. Although the mechanisms behind this activity are currently unknown, changes in intracellular calcium (Ca²⁺) signalling are purported to play a role. Therefore, the intracellular Ca²⁺ concentration was monitored in the pulmonary vein using fluo-4 and epifluorescence microscopy. Electrical field stimulation evoked a synchronous rise in Ca²⁺ in neighbouring cardiomyocytes; asynchronous spontaneous Ca²⁺ transients between electrical stimuli were also present. Immediately following termination of electrical field stimulation at 3 Hz or greater, the frequency of the spontaneous Ca²⁺ transients was increased from 0.45 ± 0.06 Hz under basal conditions to between 0.59 ± 0.05 and 0.65 ± 0.06 Hz (P<0.001). Increasing the extracellular Ca²⁺ concentration enhanced this effect, with the frequency of spontaneous Ca²⁺ transients increasing from 0.45 ± 0.05 Hz to between 0.75 ± 0.06 and 0.94 ± 0.09 Hz after electrical stimulation at 3 to 9 Hz (P<0.001), and this was accompanied by a significant increase in the velocity of Ca²⁺ transients that manifested as waves. Moreover, in the presence of high extracellular Ca²⁺, the spontaneous Ca²⁺ transients occurred more synchronously in the initial few seconds following electrical stimulation. The ryanodine receptors, which are the source of spontaneous Ca²⁺ transients in pulmonary vein cardiomyocytes, were found to be arranged in a striated pattern in the cell interior, as well as along the periphery of cell. Furthermore, labelling the sarcolemma with di-4-ANEPPS showed that over 90% of pulmonary vein cardiomyocytes possessed T-tubules. These findings demonstrate that the frequency of spontaneous Ca²⁺ transients in the rat pulmonary vein are increased following higher rates of electrical stimulation and increasing the extracellular Ca²⁺ concentration.