From Fish To Mammals: New Insights In Cardiac Excitation-Contraction Coupling

Abstract

ABSTRACTRationale and objectivesMouse is currently the main model to study human diseases. However several aspects of murine biology limit its routine use in large-scale genetic. Recently, zebrafish (ZF) emerged as popular model for studying embryologically and genetically tractable diseases. Surprisingly, there is no information about the electrical activity of freshly isolated ventricular myocyte. The first part of the project addresses this point. Further in fish, cardiac Ca cycling is almost exclusively driven by sarcolemmal Ca, such that excitation-contraction coupling occurs with little to no sarcoplasmic reticulum (SR) Ca release. Interestingly, fish cardiac cells have a well-developed SR loaded with Ca. Thus, the second part of the project addresses the physiological function of the Ca stored in the SR of fish myocytes. Finally, β-adrenergic stimulation is the main modulator of cardiac myocytes contraction. Although, immunocytochemistry and biochemistry data have shown that key proteins of the β-adrenergic pathway are present at the transverse-tubules (TT) of myocytes (vs. surface sarcolemma (SS)), it is still unclear how β1-and β2-adrenergic receptors (AR) are located at the sub-cellular level (i.e. SS vs TT) and how they influence cell cardiac physiology. The last part of the project determined the functional localization of β1-and β2-AR at the SS and TT.Materials and MethodsExperiments were performed on ventricular myocytes freshly isolated from adults ZF, rainbow trout and rat hearts. Ca current (ICa) and action potential (AP) were recorded in ZF and trout using whole-cell configuration of the patch clamp technique. In trout, ICa was recorded with either EGTA or BAPTA. Field stimulated Ca transient (CaTr) were recorded in trout and rat cells using Ca fluorescent dye (fura-2 AM). In rat, CaTr were recorded with specific β-AR agonists in intact myocytes and after removal of TT. In trout caffeine-induced Ca transient were also evoked.Results and DiscussionFirst, we present an enzymatic method to isolate ZF ventricular myocytes that yield a large number of Ca tolerant cells. AP closely resembles those from large mammalian ventricular myocytes, notably human with the clear presence of a plateau phase. ICa density and voltage properties were similar to other cardiac species. Regarding trout myocytes, inactivation of ICa was used as an index of SR Ca release. In basal condition ICa inactivation is voltage-dependent (61%). SR Ca release does not occur despite the presence of Ca in the SR. In contrast, during β-adrenergic stimulation, ICa inactivation is mainly due to Ca from the SR (46%). Finally, in rat myocytes, β1-AR stimulation is ~2 times more effective at SS compare to TT (299% vs 125% increase). In contrast, β2-AR stimulation is mainly effective (~95 times) at SS (83% vs 1% increase).ConclusionTo conclude, we believe that ZF is a species ideally suited for investigation of ion channels related mutation screening of cardiac alteration important in human. From the trout study, we propose that Ca stored in the SR of fish cardiac cells acts as a safety mechanism, allowing greater contraction when needed by the environment. Finally, in rat ventricular myocytes, we believe that β1-and β2-AR are more efficient to regulate cell physiology at SS than at TT.

Date of Award	1 Aug 2011
Original language	English
Awarding Institution	The University of Manchester
Supervisor	Alison Gurney (Supervisor)