Metabolic inhibition alters subcellular calcium release patterns in rat ventricular myocytes: implications for defective excitation-contraction coupling during cardiac ischemia and failure.

Metabolic inhibition (MI) contributes to contractile failure during cardiac ischemia and systolic heart failure, in part due to decreased excitation-contraction (E-C) coupling gain. To investigate the underlying mechanism, we studied subcellular Ca2+ release patterns in whole cell patch clamped rat ventricular myocytes using two-dimensional high-speed laser scanning confocal microscopy. In cells loaded with the Ca2+ buffer EGTA (5 mmol/L) and the fluorescent Ca2+-indicator fluo-3 (1 mmol/L), depolarization from -40 to 0 mV elicited a striped pattern of Ca2+ release. This pattern represents the simultaneous activation of multiple Ca2+ release sites along transverse-tubules. During inhibition of both oxidative and glycolytic metabolism using carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 50 nmol/L) and 2-deoxyglucose (2-DG, 10 mmol/L), there was a decrease in inward Ca2+ current (ICa), the spatially averaged Ca2+ transient, and E-C coupling gain, but no reduction in sarcoplasmic reticulum Ca2+ content. The striped pattern of subcellular Ca2+ release became fractured, or disappeared altogether, corresponding to a marked decrease in the area of the cell exhibiting organized Ca2+ release. There was no significant change in the intensity or kinetics of local Ca2+ release. The mechanism is not fully explained by dephosphorylation of L-type Ca2+ channels, because a similar degree of ICa"rundown" in control cells did NOT result in fracturing of the Ca2+ release pattern. We conclude that metabolic inhibition interferes with E-C coupling by (1) reducing trigger Ca2+, and (2) directly inhibiting sarcoplasmic reticulum Ca2+ release site open probability.

Action potential duration restitution and alternans in rabbit ventricular myocytes: the key role of intracellular calcium cycling.

Action potential duration (APD) restitution properties and repolarization alternans are thought to be important arrhythmogenic factors. We investigated the role of intracellular calcium (Ca2+i) cycling in regulating APD restitution slope and repolarization (APD) alternans in patch-clamped rabbit ventricular myocytes at 34 to 36 degrees C, using the perforated or ruptured patch clamp techniques with Fura-2-AM to record Ca2+i. When APD restitution was measured by either the standard extrastimulus (S1S2) method or the dynamic rapid pacing method, the maximum APD restitution slope exceeded 1 by both methods, but was more shallow with the dynamic method. These differences were associated with greater Ca2+i accumulation during dynamic pacing. The onset of APD alternans occurred at diastolic intervals at which the APD restitution slope was significantly <1 and was abolished by suppressing sarcoplasmic reticulum (SR) Ca2+i cycling with thapsigargin and ryanodine, or buffering the global Ca2+i transient with BAPTA-AM or BAPTA. Thapsigargin and ryanodine flattened APD restitution slope to <1 when measured by the dynamic method, but not by the S1S2 method. BAPTA-AM or BAPTA failed to flatten APD restitution slope to <1 by either method. In conclusion, APD alternans requires intact Ca2+i cycling and is not reliably predicted by APD restitution slope when Ca2+i cycling is suppressed. Ca2+i cycling may contribute to differences between APD restitution curves measured by S1S2 versus dynamic pacing protocols by inducing short-term memory effects related to pacing-dependent Ca2+i accumulation.

Functional adult myocardium in the absence of Na+-Ca2+ exchange: cardiac-specific knockout of NCX1.

The excitation-contraction coupling cycle in cardiac muscle is initiated by an influx of Ca2+ through voltage-dependent Ca2+ channels. Ca2+ influx induces a release of Ca2+ from the sarcoplasmic reticulum and myocyte contraction. To maintain Ca2+ homeostasis, Ca2+ entry is balanced by efflux mediated by the sarcolemmal Na+-Ca2+ exchanger. In the absence of Na+-Ca2+ exchange, it would be expected that cardiac myocytes would overload with Ca2+. Using Cre/loxP technology, we generated mice with a cardiac-specific knockout of the Na+-Ca2+ exchanger, NCX1. The exchanger is completely ablated in 80% to 90% of the cardiomyocytes as determined by immunoblot, immunofluorescence, and exchange function. Surprisingly, the NCX1 knockout mice live to adulthood with only modestly reduced cardiac function as assessed by echocardiography. At 7.5 weeks of age, measures of contractility are decreased by 20% to 30%. We detect no adaptation of the myocardium to the absence of the Na+-Ca2+ exchanger as measured by both immunoblots and microarray analysis. Ca2+ transients of isolated myocytes from knockout mice display normal magnitudes and relaxation kinetics and normal responses to isoproterenol. Under voltage clamp conditions, the current through L-type Ca2+ channels is reduced by 50%, although the number of channels is unchanged. An abbreviated action potential may further reduce Ca2+ influx. Rather than upregulate other Ca2+ efflux mechanisms, the myocardium appears to functionally adapt to the absence of the Na+-Ca2+ exchanger by limiting Ca2+ influx. The magnitude of Ca2+ transients appears to be maintained by an increased gain of sarcoplasmic reticular Ca2+ release. The myocardium of the NCX1 knockout mice undergoes a remarkable adaptation to maintain near normal cardiac function.

Cost-effectiveness of implementation methods for ELISA serology testing of Trypanosoma cruzi in California blood banks.

The first U.S. ELISA test for T. cruzi antibodies was licensed by the Food and Drug Administration (FDA) on December 13, 2006. Blood banks have begun screening in absence of FDA recommendations for best implementation methods. We surveyed 2,029 blood donors at five California sites with three risk-based Chagas risk-screening questions. Semi-Markov models compared the cost-effectiveness of three testing strategies. 30% of donors screened positively. Screening all dominated doing nothing, being less costly, and saving more lives. The choice to "screen and test" compared with "testing all" varied by Chagas prevalence, "screening and testing" being cost-effective for high (0.004) and low (0.00004) prevalences, and "testing all" cost-effective for moderate risk (0.0004). It is cost-effective to screen by ELISA rather than do nothing. The best strategy depends on site-specific risk. Census estimates of Hispanics do not predict donor risk well. We suggest using our screening questions to determine risk level and most cost-effective testing strategy.

Mice overexpressing the cardiac sodium-calcium exchanger: defects in excitation-contraction coupling.

Homozygous overexpression of the cardiac Na(+)-Ca(2+) exchanger causes cardiac hypertrophy and increases susceptibility to heart failure in response to stress. We studied the functional effects of homozygous overexpression of the exchanger at the cellular level in isolated mouse ventricular myocytes. Compared with patch-clamped myocytes from wild-type animals, non-failing myocytes from homozygous transgenic mice exhibited increased cell capacitance (from 208 +/- 16 pF to 260 +/- 15 pF, P < 0.05). Intracellular Ca(2+) oscillations were readily elicited in homozygous transgenic animals during depolarizations to +80 mV, consistent with rapid Ca(2+) overload caused by reverse Na(+)-Ca(2+) exchange. After normalization to cell capacitance, transgenic myocytes had significant increases in Na(+)-Ca(2+) exchange activity (318%) and peak L-type Ca(2+) current (8.2 +/- 0.7 pA pF(-1) at 0 mV test potential) compared to wild-type (5.8 +/- 0.9 pA pF(-1) at 0 mV, P < 0.02). The peak Ca(2+) current amplitude and its rate of inactivation could be modulated by rapid reversible block of the exchanger. Thus, we describe an unexpected direct influence of Na(+)-Ca(2+) exchange activity on the L-type Ca(2+) channel. Despite intact sarcoplasmic reticular Ca(2+) content and larger peak L-type Ca(2+) currents, homozygous transgenic animals exhibited smaller Ca(2+) transients (Delta[Ca(2+)](i)= 466 +/- 48 nm in transgenics versus 892 +/- 104 nm in wild-type, P < 0.0005) and substantially reduced gain of excitation-contraction coupling. These alterations in excitation-contraction coupling may underlie the tendency for these animals to develop heart failure following haemodynamic stress.