Abstract 20788: NCX Knockout Protects EC Coupling in Mice Exposed to Metabolic Inhibitors
Metabolic inhibition (MI), a major component of cardiac ischemia and failure, leads to defective excitation-contraction (EC) coupling and loss of synchronous couplon recruitment in ventricular myocytes (Chantawansri et al , Circ Res, 2008). To test the hypothesis that ablating sodium-calcium exchange (NCX) protects against couplon loss during MI, we recorded Ca transients and Ca sparks in ventricular myocytes isolated from WT and cardiac-specific NCX knockout (KO) mice. NCX KO mice live into adulthood, and have normal Ca transients despite smaller Ca current (ICa), in part because of elevated diadic cleft Ca (Pott et al , Biophys J, 2007). NCX KO hearts are also resistant to ischemia/reperfusion (Imahashi et al , Circ Res, 2005). We used a 3 min exposure to 50 nM FCCP and 10 mM 2-deoxyglucose to inhibit mitochondrial and glycolytic metabolism. Internal solutions in both control and MI groups contained substrates for ATP generation but no added ATP. In field stimulated myocytes loaded with 10 µM fluo-3 AM, MI reduced Ca transients 66.4 ± 3.3% in WT, but only 41.3 ± 5.8% in KO (p=0.017). Similarly, in patch clamped myocytes loaded with 1 mM fluo-3 salt, MI suppressed action potential evoked Ca spark activation by 64.2± 1.79% in WT (n=5), but only 43.9 ± 0.4% in KO (n=4, p=0.04). Since MI inhibited ICa at a test potential of 0 mV to a similar extent in both cell types (26.2± 8% for WT, n=7; 26.7± 2% for KO, n=6), preservation of ICa cannot explain the resistance of KO to MI. In myocytes permeabilized with the detergent saponin and bathed in a high K, low Ca ”internal” solution, MI had no effect on resting Ca spark frequency (CaSpF) in either WT or KO, suggesting that changes in the local microenvironment of the couplon are necessary to alter CaSpF during MI. To confirm this, we used the same permeabilized cell ”internal” solution as both the bath and pipette solution for intact whole cell patch clamped cells (current clamp mode, VRest = 0 mV, eliminating Ca entry through L-type Ca channels) exposed to MI. Under these conditions CaSpF again declined more in WT than KO. We conclude that knocking out NCX preserves Ca release during MI in myocytes with an intact sarcolemmal membrane, possibly by maintaining elevated diadic cleft Ca compared to WT.
- © 2010 by American Heart Association, Inc.