Developmental physiology of EC-coupling in postnatal rat

Abstract

Excitation-contraction (EC)-coupling in mammalian adult ventricular myocytes links electrical activity to contraction. EC-coupling depends on many cellular structures that organize an array of proteins which underlie, control and regulate the links between the action potential and the [Ca2+] i transient. The work reported here investigates the process of postnatal development of EC-coupling, its structures and proteins in the rat, which occur during the first three weeks of life. The central hypothesis of this thesis is that EC-coupling structural organization enables EC-coupling function. Three aspects are investigated: (i) the development of transverse tubules (TTs), (ii) the development of the sarcoplasmic reticulum (SR) and (iii) the functional joining of TT elements with SR components. By using immunohistochemistry with laser scanning confocal microscopy and digital image processing, I examined the postnatal development of the TT system, monitoring both membrane and protein organizational changes, and the organization of the developing SR by exploring changes in ryanodine receptor (RyR2), calsequestrin and SR/ER Ca2+ -ATPase localization. Further utilization of high resolution imaging and digital analysis tools allowed for quantification of the formation of structural links between the TTs and SR by analyzing the spatial relationship between RyR2 and junctophilin-2, an SR protein which anchors to the TT. Through the simultaneous use of whole cell patch clamp to record L-type Ca 2+ current (ICa) and confocal microscopy to monitor cytosolic Ca2+ dynamics using fluorescent Ca2+ indicators, I studied the functional effects of structural development. This work demonstrates the independent structural development of both the TTs and the SR followed by the assembly of mature TT-SR couplings. Functional experiments demonstrate that the SR Ca2+ release system (junctional SR, jSR) matures with distinctive features but final EC-coupling function appears to require the linking together of the TT with the jSR. This provocative work lays the foundation for specific future molecular investigations into TT-genesis, EC-coupling protein trafficking and structural cross-talk between Z-line, M-line and SR proteins.