The entrapment of the Ca2+ indicator arsenazo III in the matrix space of rat liver mitochondria by permeabilization and resealing. Na+-dependent and -independent effluxes of Ca2+ in arsenazo III-loaded mitochondria.

The permeabilization-resealing technique [Al-Nasser & Crompton, Biochem. J. (1986) 239, 19-29] has been applied to the entrapment of arsenazo III in the matrix compartment of rat liver mitochondria. The addition of 10 mM-arsenazo III to mitochondria permeabilized with Ca2+ partially restores the inner-membrane potential (delta psi) and leads to the recovery of 3.9 nmol of arsenazo III/mg of protein in the matrix when the mitochondria are washed three times. The recovery of entrapped arsenazo III is increased 2-fold by 4 mM-Mg2+, which also promotes repolarization. ATP with or without Mg2+ decreased arsenazo III recovery. Under all conditions, less arsenazo III than [14C]sucrose is entrapped, in particular in the presence of ATP. The amount of arsenazo III entrapped is proportional to the concentration of arsenazo III used as resealant, and is equally distributed between heavy and light mitochondria. Arsenazo III-loaded permeabilized and resealed (PR) mitochondria develop delta psi values of 141 +/- 3 mV. PR mitochondria retain arsenazo III and [14C]sucrose for more than 2 h at 0 degrees C. At 25 degrees C, and in the presence of Ruthenium Red, PR mitochondria lose arsenazo III and [14C]sucrose at equal rates, but Ca2+ efflux is more rapid; this indicates that Ca2+ is released by an Na+-independent carrier in addition to permeabilization. The Na+/Ca2+ carrier of PR mitochondria is partially (60%) inhibited by extramitochondrial free Ca2+ stabilized with Ca2+ buffers; maximal inhibition is attained with 2 microM free Ca2+. A similar inhibition occurs in normal mitochondria with 3.5 nmol of matrix Ca2+/mg of protein, but the inhibition is decreased by increased matrix Ca2+. The data suggest the presence of Ca2+ regulatory sites on the Na+/Ca2+ carrier that change the affinity for matrix free Ca2+.     

Cardiac alternans in embryonic mouse ventricles

T-wave alternans, an important arrhythmogenic factor, has recently been described in human fetuses. Here we sought to determine whether alternans can be induced in the embryonic mouse hearts, despite its underdeveloped sarcoplasmic reticulum (SR) and, if so, to analyze the response to pharmacological and autonomic interventions. Immunohistochemistry confirmed minimal sarcoplasmic-endoplasmic reticulum Ca-ATPase 2a expression in embryonic mouse hearts at embryonic day (E) 10.5 to E12.5, compared with neonatal or adult mouse hearts. We optically mapped voltage and/or intracellular Ca (Ca(i)) in 99 embryonic mouse hearts (dual mapping in 64 hearts) at these ages. Under control conditions, ventricular action potential duration (APD) and Ca(i) transient alternans occurred during rapid pacing at an average cycle length of 212 +/- 34 ms in 57% (n = 15/26) of E10.5-E12.5 hearts. Maximum APD restitution slope was steeper in hearts developing alternans than those that did not (2.2 +/- 0.6 vs. 0.8 +/- 0.4; P < 0.001). Disabling SR Ca(i) cycling with thapsigargin plus ryanodine did not significantly reduce alternans incidence (44%, n = 8/18, P = 0.5), whereas isoproterenol (n = 14) increased the incidence to 100% (P < 0.05), coincident with steepening APD restitution slope. Verapamil abolished Ca(i) transients (n = 9). Thapsigargin plus ryanodine had no major effects on Ca(i)-transient amplitude or its half time of recovery in E10.5 hearts, but significantly depressed Ca(i)-transient amplitude (by 47 +/- 8%) and prolonged its half time of recovery (by 18 +/- 3%) in E11.5 and older hearts. Embryonic mouse ventricles can develop cardiac alternans, which generally is well correlated with APD restitution slope and does not depend on fully functional SR Ca(i) cycling.     

Activation of pyruvate dehydrogenase by electrical stimulation, and low-Na+ perfusion of guinea-pig heart

(1) Electrical stimulation (2 Hz) of guinea-pig hearts, perfused with medium containing 11 mM D-glucose plus 0.1 mM octanoate as substrate, resulted in an increase in the percentage of pyruvate dehydrogenase in the active form (PDHa) from 16 to 68%. (2) Rapid isolation of mitochondria by a technique designed to minimize net loss or gain of Ca2+ revealed an increase in mitochondrial Ca2+ content of the stimulated hearts, as measured with 45Ca (2.74 +/- 0.27 versus 1.37 +/- 0.11 nmol/mg protein; stimulated versus rested). (3) Perfusion of rested hearts with a medium containing a reduced Na+ concentration (20 mM, with the remainder replaced with Li+) also gave increased values of PDHa content (30.9% versus 16% for the normal, physiological medium). This procedure is known to raise cytosol Ca2+ concentrations and would be expected to give mitochondrial Ca2+ loading. (4) These results are consistent with a role of mitochondrial Ca2+ in activating pyruvate dehydrogenase in the intact heart.     

Different tolerance to hypothermia and rewarming of isolated rat and guinea pig hearts.

We examined the effect of hypothermia and rewarming on myocardial function and calcium control in Langendorff-perfused hearts from rat and guinea pig. Both rat and guinea pig hearts demonstrated a rise in myocardial calcium ([Ca]total) in response to hypothermic perfusion (40 min, 10 degrees C), which was accompanied by an increase in left ventricular end diastolic pressure (LVEDP). The elevation in [Ca]total was severalfold higher in guinea pig than in rat hearts, reaching 12.9 +/- 0.8 and 3.1 +/- 0.6 micromol.g dry wt-1, respectively. The rise in LVEDP, however, was comparable in the two species: 62.5 +/- 2.5 (guinea pig) and 52.5 +/- 5.1 mm Hg (rat). Following rewarming, [Ca]total remained elevated in guinea pig, whereas a moderate decline in [Ca]total was observed in the rat (13.6 +/- 1.9 and 2.2 +/- 0.3 micromol.g dry wt-1, respectively). Posthypothermic values of LVEDP were also significantly higher in guinea pig compared to rat hearts (42.5 +/- 6.8 vs 20.5 +/- 5.1 mm Hg, P < 0.027). Furthermore, whereas rat hearts demonstrated a 78 +/- 7% recovery of left ventricular developed pressure, there was only a 15 +/- 7% recovery in guinea pig hearts. Measurements of tissue levels of high energy phosphates and glycogen utilization indicated a higher metabolic requirement in guinea pig than in rat hearts in order to oppose the hypothermia-induced calcium load. Thus, we conclude that isolated guinea pig hearts are more sensitive to a hypothermic insult than rat hearts.     

Apelin decreases the SR Ca2+ content but enhances the amplitude of [Ca2+]i transient and contractions during twitches in isolated rat cardiac myocytes

Apelin has been reported to have a positive inotropic action in the isolated rat heart. However, the effect of apelin on sarcoplasmic reticulum (SR) Ca2+ content and its influence on intracellular Ca2+ transient during excitation-contraction coupling remains poorly understood. In the present study, we determined the effect of apelin on Ca2+ transient and contractions in isolated rat cardiomyocytes. When compared with control, treatment with apelin caused a 55.7 +/- 13.9% increase in sarcomere fraction shortening and a 43.6 +/- 4.56% increase in amplitude of electrical-stimulated intracellular Ca2+ concentration (E[Ca2+]i) transients (n = 14, P < 0.05). But SR Ca2+ content measured by caffeine-induced [Ca2+]i (C[Ca2+]i) transient was decreased 8.41 +/- 0.92% in response to apelin (n = 14, P < 0.05). Na+/Ca2+ exchanger (NCX) function was increased since half-decay time of C[Ca2+]i was decreased 16.22 +/- 1.36% in response to apelin. Sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) activity was also increased by apelin. These responses can be partially or completely blocked by chelerythrine chloride, a PKC inhibitor. In addition, to confirm our data, we used indo-1 as another Ca2+ indicator and rapid cooling as another way to measure SR Ca2+ content, and we observed similar results. So we conclude that apelin has a positive inotropic effect on isolated myocytes, and increased amplitude of E[Ca2+]i is at least partially involved in the mechanism. NCX function and SERCA activity are increased by apelin, and the SR Ca2+ content is decreased by apelin during twitches. PKC played an important role in these signaling mechanisms.     

Na+ effects on mitochondrial respiration and oxidative phosphorylation in diabetic hearts

Intracellular Na+ is approximately two times higher in diabetic cardiomyocytes than in control. We hypothesized that the increase in Na+i activates the mitochondrial membrane Na+/Ca2+ exchanger, which leads to loss of intramitochondrial Ca2+, with a subsequent alteration (generally depression) in bioenergetic function. To further evaluate this hypothesis, mitochondria were isolated from hearts of control and streptozotocin-induced (4 weeks) diabetic rats. Respiratory function and ATP synthesis were studied using routine polarography and 31P-NMR methods, respectively. While addition of Na+ (1-10 mM) decreased State 3 respiration and rate of oxidative phosphorylation in both diabetic and control mitochondria, the decreases were significantly greater for diabetic than for control. The Na+ effect was reversed by providing different levels of extramitochondrial Ca2+ (larger Ca2+ levels were needed to reverse the Na+ depressant effect in diabetes mellitus than in control) and by inhibiting the Na+/Ca2+ exchanger function with diltiazem (a specific blocker of Na+/Ca2+ exchange that prevents Ca2+ from leaving the mitochondrial matrix). On the other hand, the Na+ depressant effect was enhanced by Ruthenium Red (RR, a blocker of mitochondrial Ca2+ uptake, which decreases intramitochondrial Ca2+). The RR effect on Na+ depression of mitochondrial bioenergetic function was larger in diabetic than control. These findings suggest that intramitochondrial Ca2+ levels could be lower in diabetic than control and that the Na+ depressant effect has some relation to lowered intramitochondrial Ca2+. Conjoint experiments with 31P-NMR in isolated superfused mitochondria embedded in agarose beads showed that Na+ (3-30 mM) led to significantly decreased ATP levels in diabetic rats, but produced smaller changes in control. These data support our hypothesis that in diabetic cardiomyocytes, increased Na+ leads to abnormalities of oxidative processes and subsequent decrease in ATP levels, and that these changes are related to Na+ induced depletion of intramitochondrial Ca2+.     

Targeted inhibition of sarcoplasmic reticulum CaMKII activity results in alterations of Ca2+ homeostasis and cardiac contractility

Transgenic (TG) mice expressing a Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitory peptide targeted to the cardiac myocyte longitudinal sarcoplasmic reticulum (LSR) display reduced phospholamban phosphorylation at Thr17 and develop dilated myopathy when stressed by gestation and parturition (Ji Y, Li B, Reed TD, Lorenz JN, Kaetzel MA, and Dedman JR. J Biol Chem 278: 25063-25071, 2003). In the present study, these animals (TG) are evaluated for the effect of inhibition of sarcoplasmic reticulum (SR) CaMKII activity on the contractile characteristics and Ca2+ cycling of myocytes. Analysis of isolated work-performing hearts demonstrated moderate decreases in the maximal rates of contraction and relaxation (+/-dP/dt) in TG mice. The response of the TG hearts to increases in load is reduced. The TG hearts respond to isoproterenol (Iso) in a dose-dependent manner; the contractile properties were reduced in parallel to wild-type hearts. Assessment of isolated cardiomyocytes from TG mice revealed 40-47% decrease in the maximal rates of myocyte shortening and relengthening under both basal and Iso-stimulated conditions. Although twitch Ca2+ transient amplitudes were not significantly altered, the rate of twitch intracellular Ca2+ concentration decline was reduced by approximately 47% in TG myocytes, indicating decreased SR Ca2+ uptake function. Caffeine-induced Ca2+ transients indicated unaltered SR Ca2+ content and Na+/Ca2+ exchange function. Phosphorylation assays revealed an approximately 30% decrease in the phosphorylation of ryanodine receptor Ser2809. Iso stimulation increased the phosphorylation of both phospholamban Ser16 and the ryanodine receptor Ser2809 but not phospholamban Thr17 in TG mice. This study demonstrates that inhibition of SR CaMKII activity at the LSR results in alterations in cardiac contractility and Ca2+ handling in TG hearts.     

Ryanodine and dihydropyridine receptor binding in ventricular cardiac muscle of fish with different temperature preferences

Ca(2+)-induced Ca2+ release (CICR) mechanism of cardiac excitation-contraction (e-c) coupling is dependent on the close apposition between the sarcolemmal dihydropyridine receptors (DHPR) and the sarcoplasmic reticulum (SR) ryanodine receptors (RyR). In particular, high RyR/DHPR ratio is considered to reflect strong dependence on SR Ca2+ stores for the intracellular Ca2+ transient. To indirectly evaluate the significance of CICR in fish hearts, densities of cardiac DHPRs and RyRs were compared in ventricular homogenates of three fish species (burbot, rainbow trout, and crucian carp) and adult rat by [3H] PN200-110 and [3H] ryanodine binding. The density of RyRs was significantly (P<0.05) higher in the adult rat (124+/-10 channels/microm3 myocyte volume) than in any of the fish species. Among the fish species, cold-acclimated (4 degrees C) trout had more RyRs than burbot, and crucian carp. The density of DHPRs was highest in the trout heart. RyR/DHPR ratio was significantly (P<0.05) higher in rat (4.1+/-0.5) than in the fish hearts (varying from 0.97+/-0.16 to 1.91+/-0.49) suggesting that "mammalian type" CICR is less important during e-c coupling in fish ventricular myocytes. In rainbow trout, acclimation to cold did not affect the RyR/DHPR ratio, while in crucian carp it was depressed in cold-acclimated animals (4 degrees C; 0.97+/-0.16) when compared to warm-acclimated fish (23 degrees C; 1.91+/-0.49). Although RyR/DHPR ratios were relatively low in fish hearts, there was a close correlation (r2=0.78) between the RyR/DHPR ratio and the magnitude of the Ry-sensitive component of contraction in ventricular muscle among the fish species examined in this study.     

Mitochondrial and cytosolic calcium in rat hearts under high-K(+) cardioplegia and pyruvate: mechano-energetic performance

High-K(+)-cardioplegia (CPG) and pyruvate (Pyr) are used as cardioprotective agents. Considering that mitochondria play a critical role in cardiac dysfunction, we investigated the effect of CPG on mitochondrial Ca(2+) uptake and sarcorreticular (SR) calcium handling. Cytosolic and mitochondrial Ca(2+), as well as mitochondrial membrane potential (ΔΨm) were assessed in rat cardiomyocytes by confocal microscopy. Mechano-calorimetrical correlation was studied in perfused hearts. CPG did not modify JC-1 (ΔΨm), but transiently increased, by up to 1.8 times, the Fura-2 (intracellular Ca concentration, [Ca(2+)]i) and Rhod-2 (mitochondrial free Ca concentration [Ca(2+)]m) fluorescence of resting cells, with exponential decays. The addition of 5?μmol·L(-1) thapsigargin (Tpg) increased the Rhod-2 fluorescence in a group of cells without any effect on the Fura-2 signal. In rat hearts perfused with CPG, 1?μmol·L(-1) Tpg decreased resting heat rate (ΔH(r):?-0.44?± 0.07?mW·g(-1)), while the addition of 5?μmol·L(-1) KB-R7943 increased resting pressure (ΔrLVP by?+5.26?± 1.10?mm Hg; 1?mm Hg?= 133.322 Pa). The addition of 10?mmol·L(-1) Pyr to CPG increased H(r) (+3.30?± 0.24?mW·g(-1)) and ΔrLVP (+2.2?± 0.4?mm Hg), which are effects potentiated by KB-R7943. The results suggest that under CPG, (i) there was an increase in [Ca(2+)]i and [Ca(2+)]m (without changing ΔΨm) that decayed by exothermic removal mechanisms; (ii) mitochondrial Ca(2+) uptake contributed to the removal of cytosolic Ca(2+), in a process that was potentiated by inhibition of sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA), and reduced by KB-R7943; (iii) under these conditions, SERCA represents the main energetic consumer; (iv) Pyr increased the energetic performance of hearts,mainly by inducing mitochondrial metabolism.     

Influence of long-term treatment of imidapril on mortality, cardiac function, and gene expression in congestive heart failure due to myocardial infarction

Although it is generally accepted that the efficacy of imidapril, an angiotensin-converting enzyme inhibitor, in congestive heart failure (CHF) is due to improvement of hemodynamic parameters, the significance of its effect on gene expression for sarcolemma (SL) and sarcoplasmic reticulum (SR) proteins has not been fully understood. In this study, we examined the effects of long-term treatment of imidapril on mortality, cardiac function, and gene expression for SL Na+/K+ ATPase and Na+ -Ca2+ exchanger as well as SR Ca2+ pump ATPase, Ca2+ release channel (ryanodine receptor), phospholamban, and calsequestrin in CHF due to myocardial infarction. Heart failure subsequent to myocardial infarction was induced by occluding the left coronary artery in rats, and treatment with imidapril (1 mg.kg(-1).day(-1)) was started orally at the end of 3 weeks after surgery and continued for 37 weeks. The animals were assessed hemodynamically and the heart and lung were examined morphologically. Some hearts were immediately frozen at -70 degrees C for the isolation of RNA as well as SL and SR membranes. The mortality of imidapril-treated animals due to heart failure was 31% whereas that of the untreated heart failure group was 64%. Imidapril treatment improved cardiac performance, attenuated cardiac remodeling, and reduced morphological changes in the heart and lung. The depressed SL Na+/K+ ATPase and increased SL Na+-Ca2+ exchange activities as well as reduced SR Ca2+ pump and SR Ca2+ release activities in the failing hearts were partially prevented by imidapril. Although changes in gene expression for SL Na+/K+ ATPase isoforms as well as Na+-Ca2+ exchanger and SR phospholamban were attenuated by treatments with imidapril, no alterations in mRNA levels for SR Ca2+ pump proteins and Ca2+ release channels were seen in the untreated or treated rats with heart failure. These results suggest that the beneficial effects of imidapril in CHF may be due to improvements in cardiac performance and changes in SL gene expression.     

Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms

Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca(2+) concentration ([Ca(2+)](i)) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca(2+) handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (I(Ca,L)) density (-0.964 +/- 0.172 vs. -0.745 +/- 0.128 pA/pF at +10 mV) and Na(+)/Ca(2+) exchanger (NCX) currents (2.1 +/- 0.8 vs. 2.3 +/- 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca(2+)](i) was depressed (at +10 mV, 0.72 +/- 0.07 and 0.56 +/- 0.04 microM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca(2+) spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca(2+) in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the alpha(1C)-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). alpha(1C) protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca(2+) uptake and release synergistically contribute to the depressed [Ca(2+)](i) and the altered AP profile phenotype.     

Evidence for an intracellular site of action in the heart for two hydrophobic cardiac steroids

Although cardiac steroids (CS) have long been used to treat cardiac insufficiency, the mechanism(s) of action of these agents remain open to question. While many results indicate that inhibition of Na+,K+-ATPase underlies both the therapeutic and toxic actions of CS, other studies suggest that actions on the SR membrane system may be important. We used two experimental approaches and measurements of left ventricular diastolic pressure (LVDP) in isolated guinea pig hearts to test whether CS had an intracellular site of action. In the first approach, we compared the inotropic effects of a hydrophilic CS, ouabain, and a hydrophobic CS, digitoxin, after the activity of the Na+ pump was reduced by perfusing hearts with solutions maintained at 5 degrees C. Under these conditions, exposure of hearts to 1 microM ouabain for 60 min did not increase LVDP above control levels. In contrast, an equi-effective concentration of digitoxin (0.3 microM) increased LVDP by 40 +/- 8.5% (p < 0.01) over pre-drug control levels. In the second experimental approach, we compared the inotropic effects of ouabain and digitoxin in the presence of rapid-cooling contractures (RCC), which result in the release of SR Ca2+. Hearts were perfused with Tyrode solution or Tyrode solution containing either digitoxin (0.3 microM) or ouabain (1 microM) for 180 sec, rapidly cooled and the RCC responses were analyzed. Compared to RCC elicited in Tyrode solution alone, or in Tyrode solution containing ouabain, RCC in the presence of digitoxin reached peak amplitudes more rapidly, but elicited reduced peak amplitude values. Based on these findings, we suggest that: 1) the ability of the hydrophobic CS, digitoxin, but not the hydrophilic CS, ouabain, to produce a positive inotropic effect at 5 degrees C, when the activity of the Na+ pump is markedly reduced, is consistent with a mechanism other than Na+ pump inhibition and involves an intracellular location; and 2) the diminished RCC observed in the presence of the hydrophobic CS, digitoxin, indicate that this alternative mechanism may involve effects on the SR Ca2+ release channel.     

Limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation prevents excess mitochondrial Ca2+ accumulation and attenuates myocardial injury.

BACKGROUND: intracellular Na+ accumulation during ischemia and reperfusion leads to cytosolic Ca2+ overload through reverse-mode operation of the sarcolemmal Na+ -Ca2+ exchanger. Cytosolic Ca2+ accumulation promotes mitochondrial Ca2+ (Ca2+ m) overload, leading to mitochondrial injury. We investigated whether limiting sarcolemmal Na+ entry during resuscitation from ventricular fibrillation (VF) attenuates Ca2+ m overload and lessens myocardial dysfunction in a rat model of VF and closed-chest resuscitation. METHODS: hearts were harvested from 10 groups of 6 rats each representing baseline, 15 min of untreated VF, 15 min of VF with chest compression given for the last 5 min (VF/CC), and 60 min postresuscitation (PR). VF/CC and PR included four groups each randomized to receive before starting chest compression the new NHE-1 inhibitor AVE4454B (1.0 mg/kg), the Na+ channel blocker lidocaine (5.0 mg/kg), their combination, or vehicle control. The left ventricle was processed for intracellular Na+ and Ca2+ m measurements. RESULTS: limiting sarcolemmal Na+ entry attenuated cytosolic Na+ increase during VF/CC and the PR phase and prevented Ca2+ m overload yielding levels that corresponded to 77% and 71% of control hearts at VF/CC and PR, without differences among specific Na+ -limiting interventions. Limiting sarcolemmal Na+ entry attenuated reductions in left ventricular compliance during VF and prompted higher mean aortic pressure (110 +/- 7 vs. 95 +/- 11 mmHg, P < 0.001) and higher cardiac work index (159 +/- 34 vs. 126 +/- 29 g x m x min(-1) x kg(-1), P < 0.05) with lesser increases in circulating cardiac troponin I at 60 min PR. CONCLUSIONS: Na+ -limiting interventions prevented excess Ca2+ m accumulation induced by ischemia and reperfusion and ameliorated myocardial injury and dysfunction.     

Enhanced gene expression of Na(+)/Ca(2+) exchanger attenuates ischemic and hypoxic contractile dysfunction

Enhanced gene expression of the Na(+)/Ca(2+) exchanger in failing hearts may be a compensatory mechanism to promote influx and efflux of Ca(2+), despite impairment of the sarcoplasmic reticulum (SR). To explore this, we monitored intracellular calcium (Ca(i)(2+)) and cardiac function in mouse hearts engineered to overexpress the Na(+)/Ca(2+) exchanger and subjected to ischemia and hypoxia, conditions known to impair SR Ca(i)(2+) transport and contractility. Although baseline Ca(i)(2+) and function were similar between transgenic and wild-type hearts, significant differences were observed during ischemia and hypoxia. During early ischemia, Ca(i)(2+) was preserved in transgenic hearts but significantly altered in wild-type hearts. Transgenic hearts maintained 40% of pressure-generating capacity during early ischemia, whereas wild-type hearts maintained only 25% (P < 0.01). During hypoxia, neither peak nor diastolic Ca(i)(2+) decreased in transgenic hearts. In contrast, both peak and diastolic Ca(i)(2+) decreased significantly in wild-type hearts. The decline of Ca(i)(2+) was abbreviated in hypoxic transgenic hearts but prolonged in wild-type hearts. Peak systolic pressure decreased by nearly 10% in hypoxic transgenic hearts and >25% in wild-type hearts (P < 0.001). These data demonstrate that enhanced gene expression of the Na(+)/Ca(2+) exchanger preserves Ca(i)(2+) homeostasis during ischemia and hypoxia, thereby preserving cardiac function in the acutely failing heart.     

Phosphorylation of phospholamban in ischemia-reperfusion injury: functional role of Thr17 residue

Phospholamban (PLB) is a sarcoplasmic reticulum (SR) protein that when phosphorylated at Ser16 by PKA and/or at Thr17 by CaMKII increases the affinity of the SR Ca2+ pump for Ca2+. PLB is therefore, a critical regulator of SR function, myocardial relaxation and myocardial contractility. The present study was undertaken to examine the status of PLB phosphorylation after ischemia and reperfusion and to provide evidence about the possible role of the phosphorylation of Thr17 PLB residue on the recovery of contractility and relaxation after a period of ischemia. Experiments were performed in Langendorff perfused hearts from Wistar rats. Hearts were submitted to a protocol of global normothermic ischemia and reperfusion. The results showed that (1) the phosphorylation of Ser16 and Thr17 residues of PLB increased at the end of the ischemia and the onset of reperfusion, respectively. The increase in Thr17 phosphorylation was associated with a recovery of relaxation to preischemic values. This recovery occurred in spite of the fact that contractility was depressed. (2) The reperfusion-induced increase in Thr17 phosphorylation was dependent on Ca2+ entry to the cardiac cell. This Ca2+ influx would mainly occur by the coupled activation of the Na+ / H+ exchanger and the Na+ / Ca2+ exchanger working in the reverse mode, since phosphorylation of Thr17 was decreased by inhibition of these exchangers and not affected by blockade of the L-type Ca2+ channels. (3) Specific inhibition of CaMKII by KN93 significantly decreased Thr17 phosphorylation. This decrease was associated with an impairment of myocardial relaxation. The present study suggests that the phosphorylation of Thr17 of PLB upon reflow, may favor the full recovery of relaxation after ischemia.     

白细胞介素-2对大鼠心肌Ca2+ATPase和Na+ /K+ATPase的影响

为了探讨IL－2对心肌细胞内钙影响的可能机制,用光学法检测心肌肌浆网Ca^2 ATPase的活性,以及细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性。结果：（1）用IL－2（10、40、200、800U/ml）灌流心脏后,其肌浆网Ca^2 ATPase的活性随IL－2浓度的升高而增强;（2）在ATP浓度为0.1-4mmol/L时,Ca^2 ATPase的活性随ATP浓度的升庙则增强,由IL－2（200U／ml）灌流后的心脏获得肌浆网（SR）,其Ca^2 ATPase的活性对ATP的反应强于对照组;（3）在[Ca^2 ]为1－40μmol/L时,心脏SR Ca^2 ATPase的活性随[Ca^2 ]增加而增强,而IL－2灌流心脏后分离的SR,其Ca^2 ATPase活性在[Ca^2 ]升高时没有明显改变;（4）用nor-BNI(10nmol/L）预处理5min后,IL－2（200U／ml）灌流后不再使SR Ca^2 ATPase的活性增强;（5）用PTX（5mg/L）预处理后,IL－2对SR Ca^2 ATPase的影响减弱;（6）用磷脂酶C（PLC）抑制剂U73122（5μmol/L）处理后,IL－2不再使SR Ca^2 ATPase活性增高;（7）用IL－2直接处理从正常大鼠分离的SR后,对SR Ca^2 ATPase活性无明显影响;（8）IL－2灌流后,对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase活性没有显著。上述结果表明,IL－2灌流心脏后使心肌肌浆网Ca^2 ATPase的活性增加,心肌细胞膜上的κ－阿片受体及其下游的G蛋白和PLC介导了IL－2的作用。尽管IL－2提高SR Ca^2 ATPase对ATP的反应性,但却抑制SR Ca^2 ATPase对钙离子的敏感性。IL－2对心肌细胞膜Ca^2 ATPase和Na^ /K^ ATPase的活性无明显影响。     

Renal hypertension prevents run training modification of cardiomyocyte diastolic Ca2+ regulation in male rats.

The combined effects of endurance run training and renal hypertension on cytosolic Ca2+ concentration ([Ca2+]c) dynamics and Na+-dependent Ca2+ regulation in rat left ventricular cardiomyocytes were examined. Male Fischer 344 rats underwent stenosis of the left renal artery [hypertensive (Ht), n = 18] or a sham operation [normotensive (Nt), n = 20]. One-half of the rats from each group were treadmill trained for >16 wk. Cardiomyocyte fura 2 fluorescence ratio transients were recorded for 7 min during electrical pacing at 0.5 Hz, 2 mM extracellular Ca2+ concentration, and 29 degrees C. The rate of [Ca2+]c decline was not changed by run training in the Nt group but was reduced in the Ht group. At 7 min, cardiomyocytes were exposed to 10 mM caffeine in the absence of Na+ and Ca2+, which triggered sarcoplasmic reticular Ca2+ release and suppressed Ca2+ efflux via Na+/Ca2+ exchanger. External Na+ was then added, and Na+-dependent Ca2+ efflux rate was recorded. Treadmill training significantly enhanced Na+-dependent Ca2+ efflux rate under these conditions in the Nt group but not in the Ht group. These data provide evidence that renal hypertension prevents the normal run training-induced modifications in diastolic [Ca2+]c regulation mechanisms, including Na+/Ca2+ exchanger.     

Pyruvate potentiates inotropic effects of isoproterenol and Ca(2+) in rabbit cardiac muscle preparations

Catecholamines and elevated extracellular Ca(2+) concentration ([Ca(2+)](o)) augment contractile force by increased Ca(2+) influx and subsequent increased sarcoplasmic reticulum (SR) Ca(2+) release. We tested the hypothesis that pyruvate potentiates Ca(2+) release and inotropic response to isoproterenol and elevated [Ca(2+)](o), since this might be of potential importance in a clinical setting to circumvent deleterious effects on energy demand during application of catecholamines. Therefore, we investigated isometrically contracting myocardial preparations from rabbit hearts at 37 degrees C, pH 7.4, and a stimulation frequency of 1 Hz. At a [Ca(2+)](o) of 1.25 mM, pyruvate (10 mM) alone increased developed force (F(dev)) from 1.89 +/- 0.42 to 3.62 +/- 0.62 (SE) mN/mm(2) (n = 8, P < 0.05) and isoproterenol (10(-6) M) alone increased F(dev) from 2.06 +/- 0. 55 to 25.11 +/- 2.1 mN/mm(2) (P < 0.05), whereas the combination of isoproterenol and pyruvate increased F(dev) overproportionally from 1.89 +/- 0.42 to 33.31 +/- 3.18 mN/mm(2) (P < 0.05). In a separate series of experiments, we assessed SR Ca(2+) content by means of rapid cooling contractures and observed that, despite no further increase in F(dev) by increasing [Ca(2+)](o) from 8 to 16 mM, 10 mM pyruvate could still increase F(dev) from 26.4 +/- 6.8 to 29.7 +/- 7. 1 mN/mm(2) (P < 0.05, n = 9) as well as the Ca(2+) load of the SR. The results show that the positive inotropic effects of pyruvate potentiate the inotropic effects of isoproterenol or Ca(2+), because in the presence of pyruvate, Ca(2+) and isoproterenol induced larger increases in inotropy than can be calculated by mere addition of the individual effects.