Abstract
In ventricular myocytes, excitation-contraction coupling (ECC), or the process by which an action potential leads to myocyte contraction, is reliant upon external influx of calcium (Ca+2) via sarcolemmal voltage-gated L-type Ca+2 channels to trigger Ca+2 release through nearby ryanodine receptors located in the sarcoplasmic reticulum (SR) via the mechanism of Ca+2-induced Ca+2 release. This causes a global increase in intracellular in Ca+2 concentration ([Ca+2]i) that activates contractile filaments. The level of expression of proteins and cellular organelles involved in cardiac ECC changes during development. These differences arise from adult myocytes being larger, having smaller surface-to-volume ratio and a developed transverse (T)-tubule system that tightly couples to the junctional SR. To investigate ECC developmental changes, we developed an adenovirus encoding a tagged red fluorescent protein with a retention signal for the sarcoplasmic reticulum (tRFP-SR). This approach allowed us for the first time to track in real-time SR structure and [Ca+2]i dynamics in neonatal and adult ventricular myocytes. Using confocal and total internal reflection fluorescence (TIRF) microscopy, we found that the SR within neonatal and adult ventricular cardiomyocytes forms a vast network that spans virtually the entire cell. Interestingly, we found that the SR is a highly dynamic network with relatively rapid changes in morphology in neonatal myocytes. Calcium sparks were observed from these SR structures, suggesting they harbor functional ryanodine receptors. In contrast to neonatal myocytes, the SR of adult ventricular myocytes is highly stable. Our findings suggest, that the fidelity of the [Ca+2]i transient during ECC in neonatal myocytes is not simply the result of ryanodine receptors in a static SR network. Instead, our data indicate it is the result of the activation of numerous couplings that form and dissolve rapidly between the sarcoplasmic reticulum and plasma membrane.
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