The central role of T-tubule and sarcoplasmic reticulum (SR) diadic junctions in excitation-contraction (EC) coupling in adult (AD) ventricular myocytes suggests that their absence in newborn (NB) cells may manifest as an altered EC coupling phenotype. We used confocal microscopy to compare fluo-3 [Ca2+]i transients in the subsarcolemmal space and cell center of field-stimulated NB and AD rabbit ventricular myocytes. Peak systolic [Ca2+]i occurred sooner and was higher in the subsarcolemmal space compared with the cell center in NB myocytes. In AD myocytes, [Ca2+]i rose and declined with similar profiles at the cell center and subsarcolemmal space. Disabling the SR (10 micromol/L thapsigargin) slowed the rate of rise and decline of Ca2+ in AD myocytes but did not alter Ca2+ transient kinetics in NB myocytes. In contrast to adults, localized SR Ca2+ release events ("Ca2+ sparks") occurred predominantly at the cell periphery of NB myocytes. Immunolabeling experiments demonstrated overlapping distributions of the Na(+)-Ca2+ exchanger and ryanodine receptors (RyR2) in AD myocytes. In contrast, RyR2s were spatially separated from the sarcolemma in NB myocytes. Confocal sarcolemmal imaging of di-8-ANEPPS-treated myocytes confirmed an extensive T-tubule network in AD cells, and that T-tubules are absent in NB myocytes. A mathematical model of subcellular Ca2+ dynamics predicts that Ca2+ flux via the Na(+)-Ca2+ exchanger during an action potential can account for the subsarcolemmal Ca2+ gradients in NB myocytes. Spatial separation of sarcolemmal Ca2+ entry from SR Ca2+ release channels may minimize the role of SR Ca2+ release during normal EC coupling in NB ventricular myocytes.
Abstract-Trimetazidine acts as an effective antianginal clinical agent by modulating cardiac energy metabolism. Recent published data support the hypothesis that trimetazidine selectively inhibits long-chain 3-ketoacyl CoA thiolase (LC 3-KAT), thereby reducing fatty acid oxidation resulting in clinical benefit. The aim of this study was to assess whether trimetazidine and ranolazine, which may also act as a metabolic modulator, are specific inhibitors of LC 3-KAT. We have demonstrated that trimetazidine and ranolazine do not inhibit crude and purified rat heart or recombinant human LC 3-KAT by methods that both assess the ability of LC 3-KAT to turnover specific substrate, and LC 3-KAT activity as a functional component of intact cellular -oxidation. Furthermore, we have demonstrated that trimetazidine does not inhibit any component of -oxidation in an isolated human cardiomyocyte cell line. Ranolazine, however, did demonstrate a partial inhibition of -oxidation in a dose-dependent manner (12% at 100 mol/L and 30% at 300 mol/L). Both trimetazidine (10 mol/L) and ranolazine (20 mol/L) improved the recovery of cardiac function after a period of no flow ischemia in the isolated working rat heart perfused with a buffer containing a relatively high concentration (1.2 mmol/L) of free fatty acid. In summary, both trimetazidine and ranolazine were able to improve ischemic cardiac function but inhibition of LC 3-KAT is not part of their mechanism of action. Key Words: cardiac Ⅲ metabolism Ⅲ ischemia Ⅲ trimetazidine M yocardial metabolic modulation is a new approach for the treatment and management of ischemic heart disease (IHD) and angina pectoris. In the healthy heart, fatty acids are the primary fuel source for the generation of ATP, 1 with the balance of energy provision coming from glucose and lactate oxidation.Myocardial ischemia radically alters the balance of cardiac fuel metabolism. The primary consequence of moderate ischemia (ie, a 30% to 60% reduction in coronary blood flow as experienced by stable angina patients) is a reduction in oxygen delivery to the tissue resulting in a decrease in ATP production. 1 During ischemia, fatty acid and pyruvate oxidation both decrease as glycolysis becomes the predominant route for ATP production. 2 However, as oxygen provision declines, the pyruvate produced from glycolysis cannot be fully oxidized by the tricarboxylic acid (TCA) cycle and is reduced to lactate that can further uncouple glycolysis from pyruvate oxidation. 2 Glycolytic regeneration of ATP leads to intracellular acidosis via the production of cytosolic protons 3 that in turn activate the sodium-hydrogen exchanger and sodium-calcium exchanger leading to intracellular calcium overload and contractile dysfunction. 2 Ischemic injury elevates plasma catecholamine concentrations leading to activation of hormone sensitive lipase (HSL) resulting in an increase in plasma free fatty acid (FFA) concentrations (typically 0.8 to 1.4 mmol/L). 4 On reperfusion, fatty acid oxidation becomes the predominant source of ATP ...
Two competitive inhibitors of TAFIa (activated thrombin-activable fibrinolysis inhibitor), 2-guanidinoethylmercaptosuccinic acid and potato tuber carboxypeptidase inhibitor, variably affect fibrinolysis of clotted human plasma. Depending on their concentration, the inhibitors shortened, prolonged, or had no effect on lysis in vitro. The inhibitor-induced effects were both tissuetypeplasminogenactivator(tPA)andTAFIaconcentrationdependent. Inhibitor-dependent prolongation was favored at lower tPA concentrations. The magnitude of the prolongation increased with TAFIa concentration, and the maximal prolongation observed at each TAFIa concentration increased saturably with respect to TAFIa. A theoretical maximal prolongation of 20-fold was derived from a plot of the maximum prolongation versus TAFIa. This represents, for the first time, a measurement of the maximal antifibrinolytic potential of TAFIa in vitro.Because TAFIa spontaneously decays, the stabilization of TAFIa was investigated as a mechanism explaining the inhibitor-dependent prolongation of lysis. Both inhibitors stabilized TAFIa in a concentration-dependent, non-saturable manner. Although their K I values differed by three orders of magnitude, TAFIa was identically stabilized when the fraction of inhibitor-bound TAFIa was the same. The data fit a model whereby only free TAFIa decays. Therefore, the variable effects of competitive inhibitors of TAFIa on fibrinolysis can be rationalized in terms of free TAFIa and lysis time relative to the half-life of TAFIa.
Previous studies suggesting a greater functional role of cardiac Na+/Ca2+ exchange at birth were performed using tightly buffered free cytosolic Ca2+ concentration ([Ca2+]i). Because Na+/Ca2+ exchange current (INaCa) is influenced by physiological fluctuations in [Ca2+]i, we used conditions of minimally buffered [Ca2+]i to simultaneously record INaCa and cell contractions in single ventricular myocytes isolated from 1 to 27-day-old and adult rabbits. With conventional Cl(-)-containing solutions. Ni(2+)-sensitive outward and inward charge movements were unbalanced, suggesting the presence of a contaminating current (presumably the Ca(2+)-activated Cl- current). Removing Cl- abolished this discrepancy in all age groups and allowed for the accurate quantitation of INaCa. Under Cl(-)-free conditions, outward and inward charge movements were high at birth (4 days: 0.42 +/- 0.03 and -0.38 +/- 0.04 pC/pF, respectively) and decreased postnatally (adult: 0.08 +/- 0.01 and -0.07 +/- 0.01 pC/pF, respectively). Newborn but not adult myocytes contracted during depolarizations in the presence of nifedipine, ryanodine, and thapsigargin. The magnitudes of outward charge movement (Ca2+ influx) and cell shortening exhibited similar voltage dependence, consistent with INaCa-mediated contractions. These results indicate that INaCa can directly support contraction in newborn rabbit ventricular myocytes.
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