Proceedings of The Physiological Society

University of Cambridge (2008) Proc Physiol Soc 11, PC16

Poster Communications

The role of reverse-mode NCX in the contractility of the rainbow trout heart.

H. Shiels1, A. Allen1, A. Smith1, J. Hall1, R. Birkedal1

1. Faculty of Life Sciences, University of Manchester, Manchester, United Kingdom.

The Ca2+ that initiates contraction in the rainbow trout heart arrives at the myofilaments primarily through L-type Ca2+ channels in the sarcolemmal membrane (Vornanen et al., 2000). However, at specific times during the action potential, Ca2+ can also be brought across the sarcolemma by the Na+-Ca2+ exchanger (NCX) operating in reverse-mode (revNCX)(Hove-Madsen et al., 2000). The relative importance of this route of Ca2+ influx was assessed in single isolated cardiac myocytes and isolated muscle preparations using the specific inhibitor of revNCX, KB-R7943. Rainbow trout (range 75 g to 600 g body weight) were killed humanely (UK Home Office Schedule 1 method). The heart was removed and prepared for either dissection of trabecular bundles or for enzymatic digestion. We evaluated the role of KB-R7943 on contractility by assessing isometric force production of isolated atrial and ventricular muscle preparations, and by assessing the percentage shortening of freshly isolated atrial and ventricular myocytes responding to field stimulation. The contribution of revNCX to the Ca2+-transient was assessed using whole-cell voltage-clamp and Fura-2 fluorescence measurements. In ventricular myocytes, KB-R7943 significantly reduce cell shortening (by 40% from control, n=7, Mann-Whitney Rank Sum, P<0.05) at a concentration of 0.3 μM. The decrease in shortening was greater (by 75% from control, n=6, Mann-Whitney Rank Sum, P<0.05) at a concentration of 5 μM. This reduction is probably due to a decrease in intracellular Ca2+ concentration as our preliminary data shows 5 μM KB-R7943 reduces the amplitude of the Ca2+-transient by approximately 50 %. The reduction in contractility in response to treatment with KB-R7943 was greater in atrial than ventricular cells such that 5 μM KB-7943 completely abolished contraction (n=3). The relative importance of revNCX decreased as contraction frequency was increased (from 0.2 to 0.8 Hz) in both atrial and ventricular cells. We attribute this to a frequency-induced shortening of action potential duration which reduces opportunity for Ca2+ to enter the cell on revNCX (see Birkedal & Shiels, 2007). In isolated muscle preparations, application of 5 μM KB-R7943 had no effect on peak isometric tension in either atrial (n=7, Mann-Whitney Rank Sum, P<0.05) or ventricular (n=6, Mann-Whitney Rank Sum, P<0.05) muscle over the entire range of contractile frequencies examined (0.2 - 2.0 Hz). This dose may be too low for trabecular muscle preparations. We conclude that revNCX is an important Ca2+ influx pathway in trout myocytes and is relative importance is affected by the frequency of contraction.

Where applicable, experiments conform with Society ethical requirements