Inhibition of the cardiac electrogenic sodium bicarbonate cotransporter reduces ischemic injury.

OBJECTIVE: Although it is believed that sodium-driven acid-base transport plays a central role in the development of the reperfusion injury that follows cardiac ischemia, research to date has demonstrated only a role for Na(+)/H(+) exchange (NHE). However, Na(+)-driven HCO(-)(3) transport, which is quantitatively as important as NHE in cardiac cells, has not been examined. METHODS AND RESULTS: Here the results show that a neutralizing antibody raised against the human heart electrogenic Na(+)/HCO(3)(-) cotransporter (hhNBC) blocked the recovery of pH after acidic pulse both in HEK-293 cells expressing hhNBC and in rat cardiac myocytes demonstrating the presence of an electrogenic NBC in rat cardiac myocytes similar to hhNBC. Administration of anti-NBC antibody to ischemic-reperfused rat hearts markedly protects systolic and diastolic functions of the heart during reperfusion. Furthermore, using a quantitative real-time RT-PCR (TaqMan) and Western blot analysis we demonstrated that in human cardiomyopathic hearts, mRNA and protein levels of hhNBC increase, whereas mRNA levels of the electroneutral Na(+)/HCO(3)(-) cotransporter (NBCn1) remain unchanged. CONCLUSION: Our data provide evidence that inhibition of hhNBC, whose role in cardiac pathologies could be amplified by overexpression, represents a novel therapeutic approach for ischemic heart disease.

Pharmacology of SB-273779, a nonpeptide calcitonin gene-related peptide 1 receptor antagonist.

Calcitonin gene-related peptide (CGRP), a potent vasodilatory and cardiotonic peptide, has a potential role for CGRP in diverse physiologic and pathophysiologic situations such as congestive heart failure, diabetes, migraine, and neurogenic inflammation. Although a peptide CGRP receptor antagonist, CGRP(8-37,) is available, its utility presents significant limitations for these indications. Here, we describe the properties of SB-(+)-273779 [N-methyl-N-(2-methylphenyl)-3-nitro-4-(2-thiazolylsulfinyl)nitrobenzanilide], a selective nonpeptide antagonist of CGRP(1) receptor. SB-(+)-273779 inhibited (125)I-labeled CGRP binding to SK-N-MC (human neuroblastoma cells) and human cloned CGRP(1) receptor with K(i) values of 310 +/- 40 and 250 +/-15 nM, respectively. SB-(+)-273779 also inhibited CGRP (3 nM)-activated adenylyl cyclase in these systems with IC(50) values of 390 +/-10 nM (in SK-N-MC) and 210 +/-16 nM (recombinant human CGRP receptors). Prolonged treatment (>30 min) of SK-N-MC cells with SB-(+)-273779 followed by extensive washing resulted in reduction in maximum CGRP-mediated adenylyl cyclase activity, suggesting that this compound has irreversible binding characteristics. In addition, SB-(+)-273779 antagonized CGRP-mediated 1) stimulation of intracellular Ca(2+) in recombinant CGRP receptors in HEK-293 cells, 2) inhibition of insulin-stimulated [(14)C]deoxyglucose uptake in L6 cells, 3) vasodilation in rat pulmonary artery, and 4) decrease in blood pressure in anesthetized rats. SB-(+)-273779 tested at 3 microM had no significant affinity for calcitonin, endothelin, angiotensin II, and alpha-adrenergic receptors under standard ligand binding assays. SB-(+)-273779 also did not inhibit forskolin and pituitary adenylate cyclase-activating polypeptide. These results suggest that SB-(+)-273779 is a valuable tool for studying CGRP-mediated functional responses in complex biological systems.

Role of endothelium in ischaemia-induced myocardial dysfunction of isolated working hearts: cardioprotection by activation of adenosine A(2A) receptors.

1 This study aimed to determine the role of the vascular endothelium on recovery of contractile function following global low-flow ischaemia of guinea-pig isolated working hearts and the effects of adenosine analogues on this recovery. 2 Guinea-pig isolated spontaneously beating or paced working hearts were set up and coronary flow (CF), aortic output (AO) (as an index of cardiac function), heart rate (HR), left ventricular pressure (LVP) and dP/dt max recorded. The endothelium was either intact or removed by a blast of oxygen. 3 In spontaneously beating hearts, low-flow ischaemia for 30 min reduced CF and cardiac contractility (LVP, dP/dt max) but not AO. On reperfusion, CF, LVP and dP/dt max recovered, while AO fell precipitously followed by a gradual recovery, indicative of myocardial stunning. The effects of ischaemia did not differ between endothelium-intact and -denuded hearts, indicating no role of the endothelium in the changes observed. 4 The adenosine analogues, N6-cyclopentyladenosine (CPA, A1 selective), 5'-N-ethylcarboxamidoadenosine (NECA, two-fold A2 selective over A1) and 2-p-((carboxyethyl)-phenethylamino)-5'carboxamidoadenosine (CGS21680, A2A selective) were infused (3 x 10-7 M) from 10 min into the 30-min low-flow ischaemia of denuded hearts and during reperfusion. 5 CGS21680 increased CF and improved the postischaemic functional recovery, as measured by the AO. NECA and CPA were not cardioprotective. The A2A selective antagonist, ZM241385, attenuated the coronary vasodilatation by CGS21680 and abolished the improved recovery of AO on reperfusion. 6 Reperfusion of paced working hearts caused a dramatic fall in AO which failed to recover. Infusion of CGS21680 from 15 min into the ischaemic period produced vasodilatation but failed to restore AO, presumably because the ischaemic damage was irreversible. 7 Thus, the endothelium plays no role in myocardial dysfunction following low-flow global ischaemia and reperfusion of guinea-pig working hearts. The A2A adenosine receptor-selective agonist but not the non-selective A2 receptor agonist, NECA, attenuated ischaemia- and reperfusion-induced stunning. This was attributed to increased CF and was independent of the endothelium.

Impaired endothelium-dependent relaxation by adrenomedullin in monocrotaline-treated rat arteries.

The effects of adrenomedullin were evaluated in isolated vascular rings from rats treated with monocrotaline (60 mg/kg, s.c.) causing pulmonary hypertension and right ventricular hypertrophy within 3 to 4 weeks. Sham animals (NaCl-treated rats) were used for comparison. The relaxing effects of adrenomedullin (10(-8) M) and acetylcholine (10(-6) M) were determined in thoracic aorta and pulmonary artery rings precontracted with phenylephrine (10(-7) M). In sham animals, adrenomedullin caused significant vasorelaxation of aorta and pulmonary artery although of different amplitude (24 +/- 3% and 40 +/- 2%, respectively). A greater relaxation was observed in response to acetylcholine. Monocrotaline-treated rats exhibited a reduction in adrenomedullin relaxation in pulmonary artery (54 and 68% loss of effect, at 3 and 4 weeks, respectively, P < 0.01 vs. sham) and comparable reductions in acetylcholine responses. The decrease in adrenomedullin relaxing effect was less pronounced in aorta than in pulmonary artery, suggesting a distinct tissue sensitivity to monocrotaline. In contrast, the relaxing effect of acetylcholine on aorta was decreased at 4 weeks (36% reduction, P < 0.01 vs. sham). In this model, the adrenomedullin-induced relaxation of the pulmonary artery was impaired due to a severe endothelial dysfunction which may contribute partly to the evolving pathophysiological process.

Cloning and characterization of a human electrogenic Na+-HCO-3 cotransporter isoform (hhNBC).

Our group recently cloned the electrogenic Na+-HCO-3 cotransporter (NBC) from salamander kidney and later from mammalian kidney. Here we report cloning an NBC isoform (hhNBC) from a human heart cDNA library. hhNBC is identical to human renal NBC (hkNBC), except for the amino terminus, where the first 85 amino acids in hhNBC replace the first 41 amino acids of hkNBC. About 50% of the amino acid residues in this unique amino terminus are charged, compared with approximately 22% for the corresponding 41 residues in hkNBC. Northern blot analysis, with the use of the unique 5' fragment of hhNBC as a probe, shows strong expression in pancreas and expression in heart and brain, although at much lower levels. In Xenopus oocytes expressing hhNBC, adding 1.5% CO2/10 mM HCO-3 hyperpolarizes the membrane and causes a rapid fall in intracellular pH (pHi), followed by a pHi recovery. Subsequent removal of Na+ causes a depolarization and a reduced rate of pHi recovery. Removal of Cl- from the bath does not affect the pHi recovery. The stilbene derivative DIDS (200 microM) greatly reduces the hyperpolarization caused by adding CO2/HCO-3. In oocytes expressing hkNBC, the effects of adding CO2/HCO-3 and then removing Na+ were similar to those observed in oocytes expressing hhNBC. We conclude that hhNBC is an electrogenic Na+-HCO-3 cotransporter and that hkNBC is also electrogenic.

Consequences of the inhibition of the sarcoplasmic reticulum calcium ATPase on cardiac function and coronary flow in rabbit isolated perfused heart: role of calcium and nitric oxide.

The effects of thapsigargin (Tg) and cyclopiazonic acid (CPA), two selective blockers of the sarcoplasmic reticulum Ca2+-ATPase were studied in rabbit isolated perfused hearts. Tg and CPA were infused into the hearts for 60 min followed by 60 min of wash-out. Left-ventricular developed pressure (LVDP), left-ventricular end diastolic pressure (LVEDP) and the relaxation time constant,tau, were assessed with a fluid-filled LV intraventricular balloon. Both Tg and CPA induced a concentration-dependent reduction in LVDP and dose-dependently altered diastolic function parameters LVEDP and tau. After 60 min of perfusion, both Tg (0.01, 0.1 and 1.0 microM) and CPA (0.1, 1.0 and 10.0 microM) decreased LVDP from 98+/-1 mmHg in control to 83+/-4; 81+/-5 and 55+/-7 mmHg and to 91+/-3, 80+/-5 and 65+/-4 mmHg, respectively. LVEDP increased from 5+/-1 mmHg in controls to 6+/-0.2, 10+/-1 and 29+/-4 mmHg and to 7+/-0.2, 9+/-1 and 11+/- mmHg; while tau elevated from 28+/-1 ms to 32+/-1, 38+/-4 and 99+/-18 ms and to 34+/-1, 38+/-2 and 48+/-4 ms in Tg (0.01, 0.1 and 1.0 microM) and CPA (0.1, 1.0 and 10.0 microM), respectively. The effects of Tg were more pronounced than those of CPA and were modulated by extracellular Ca2+. With 1 mm Ca2+, both agents Tg (0.03 microM) and CPA (0.1 microM) produced a vasodilatation (81.7+/-2. 6 and 89.1+/-3.1% of pre-drug values, respectively). Pretreatment of the hearts with L-NMMA, a specific inhibitor of nitric oxide production, completely abolished the relaxing effect of Tg and CPA as well as the production of cGMP. These data show that the two SR-Ca2+ ATPase inhibitors, Tg and CPA, are negatively inotropic and lusitropic agents and that both Tg and CPA induce a vasodilatation mediated by a NO-dependent mechanism.

The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle.

Beta1- and beta2-adrenoceptors in heart muscle cells mediate the catecholamine-induced increase in the force and frequency of cardiac contraction. Recently, in addition, we demonstrated the functional expression of beta3-adrenoceptors in the human heart. Their stimulation, in marked contrast with that of beta1- and beta2-adrenoceptors, induces a decrease in contractility through presently unknown mechanisms. In the present study, we examined the role of a nitric oxide (NO) synthase pathway in mediating the beta3-adrenoceptor effect on the contractility of human endomyocardial biopsies. The negative inotropic effects of a beta3-adrenoceptor agonist, BRL 37344, and also of norepinephrine in the presence of alpha- and beta1-2-blockade were inhibited both by a nonspecific blocker of NO, methylene blue, and two NO synthase (NOS) inhibitors, L-N-monomethyl-arginine and L-nitroarginine-methyl ester. The effect of the NOS inhibitors was reversed by an excess of L-arginine, the natural substrate of NOS, but not by D-arginine. Moreover, the effects of the beta3-adrenoceptor agonist on contractility were associated with parallel increases in the production of NO and intracellular cGMP, which were also inhibited by NOS inhibitors. Immunohistochemical staining of human ventricular biopsies showed the expression of the endothelial constitutive (eNOS), but not the inducible (iNOS) isoform of NOS in both ventricular myocytes and endothelial cells. These results demonstrate that beta3-adrenoceptor stimulation decreases cardiac contractility through activation of an NOS pathway. Changes in the expression of this pathway may alter the balance between positive and negative inotropic effects of catecholamines on the heart potentially leading to myocardial dysfunction.

Comparative effects of carvedilol and metoprolol on cardiac ischemia-reperfusion injury.

The effects of carvedilol, a multiple-action neurohormonal antagonist, and metoprolol, a highly selective beta1 antagonist, were compared on postischemic contractile recovery and contracture. Isolated rabbit hearts were aerobically perfused for 45 min and subjected to zero-flow normothermic ischemia for 30 or 60 min followed by reperfusion for 30 min. Carvedilol and metoprolol were added to the perfusion solution 10 min before inducing ischemia and were maintained in the perfusate throughout reperfusion. Left ventricular developed pressure (LVDP) and left ventricular end-diastolic pressure (LVEDP) were assessed with an intraventricular balloon. Because the volume of the balloon was held constant, an increase in LVEDP reflected an increase in diastolic chamber stiffness or "contracture." After 30 min of ischemia, the carvedilol-treated hearts exhibited a significantly better cardiac function than did control or metoprolol-treated hearts. At the end of reperfusion, the control group LVDP recovered to 21.4+/-9.9% of the preischemic value. With 0.03, 0.1, and 0.3 microM metoprolol, LVDP recovered to 33.2+/-13.6%, 41.7+/-13.0%, and 48.8+/-13.3% of initial developed pressure, respectively. In the carvedilol group, a greater recovery of LVDP was obtained at 0.03, 0.1, and 0.3 microM: 64.0+/-2.5%, 60.4+/-6.3%, and 68.0+/-2.0% of preischemic values, respectively (p < 0.05 vs. controls). Within the first 5 min of reperfusion, LVEDP increased to 70.3+/-2.7 mm Hg in control hearts, indicating a pronounced contracture, whereas metoprolol reduced LVEDP when given at high concentration, 0.3 microM (41.9+/-10.7 mm Hg). Carvedilol, even at the lowest concentration, 0.03 microM, almost completely inhibited the postischemic contracture (16.5+/-4.0 mm Hg; p < 0.05 vs. control and metoprolol). The cardioprotection provided by carvedilol also is observed in hearts subjected to more severe ischemic periods. After 60 min of ischemia, control hearts failed to restore LVDP function; in the metoprolol group, ventricular function recovered to only 4.6+/-3.1%, whereas carvedilol-treated hearts exhibited 23.6+/-1.9% of preischemic values at the end of reperfusion. In addition, carvedilol induced a reduction in ischemic contracture: control, 36.7+/-3 mm Hg; metoprolol, 38.7+/-3.7 mm Hg; and carvedilol, 15.7+/-8.4 mm Hg at 50 min of ischemia. Similarly, carvedilol reduced contracture during the reperfusion compared with metoprolol and control groups (83.2+/-3.4 mm Hg, 106.9+/-3.3 mm Hg, and 107.6+/-4.1 mm Hg, respectively). These data clearly demonstrate that carvedilol was markedly more effective than metoprolol to protect systolic function after ischemia and to reduce postischemic contracture.

Protective effect of the sodium/hydrogen exchange inhibitors during global low-flow ischemia.

We investigated the potential role of the Na+/H+ exchanger (NHE) during global low-flow ischemia. Isolated working rat hearts were subjected to a low-flow ischemic period of 30 or 60 min at 37 degrees C and then reperfused for 30 min. Under those conditions, the effects of two NHE inhibitors 3-methylsulphonyl-4-piperidinobenzoyl guanidine methanesulphonate (HOE-694, 1 microM) and 5-(N-ethyl-N-isopropyl) amiloride (EIPA, 1 microM), were compared. When added to the perfusion fluid 15 min before induction of ischemia, EIPA partially preserved aortic output (AO) during either a 30- or 60-min low-flow period. A lesser effect, which was not statistically significant, was observed with HOE-694. Therefore, after 30-min ischemia, AO was 18.7 +/- 2.7, 31.4 +/- 3.3% (p < 0.05 vs. control group) and 25.8 +/- 3.2% of the preischemic value in control and EIPA- and HOE-694-treated groups, respectively. Similarly, after 60-min low-flow ischemia, AO was 15.7 +/- 1.8, 32.7 +/- 4.2% (p < 0.05 vs. control group) and 23.3 +/- 5.6% in control and EIPA- and HOE-694-treated groups, respectively. When EIPA and HOE-694 were added to the perfusion solution during the 60-min ischemic period, i.e., at 15 min of low-flow ischemia, AO was maintained at 38.9 +/- 4.9 and 30.2 +/- 2.4% (vs. 15.7 +/- 1.8% in the controls) in HOE-694- and EIPA-treated groups, respectively. EIPA but not HOE-694 also significantly (p < 0.05) improved the AO recovery during reperfusion. When administered later during ischemia, EIPA but not HOE-694 caused some recovery of AO during the remainder of the ischemic period but did not aid recovery during reperfusion. Our data suggest that although inhibition of NHE may be of some benefit during low-flow ischemia, additional effects may be necessary to provide a more efficient cardioprotection. An additional action, e.g., inhibition of the Na+/HCO3- cotransporter, could explain the superior effect of EIPA with respect to HOE-694.

Mechanisms of intracellular pH regulation during postischemic reperfusion of diabetic rat hearts.

A marked decrease in the activity of the amiloride-sensitive Na+/H+ exchanger has been demonstrated in hearts from streptozotocin (STZ)-induced diabetic rats. The aim of this study was to investigate the contribution of other specific sarcolemmal transport mechanisms to intracellular pH (pHi) recovery upon reperfusion in STZ-induced diabetic rat hearts and their relation to recovery of ventricular function. Isovolumic rat hearts were submitted to a zero-flow ischemic period of 28 min at 37 degrees C and then reperfused for 28 min. The time course of pHi decline during ischemia and of recovery on reperfusion was followed by means of 31P-labeled NMR. The perfusion buffers used were either HEPES or CO2/HCO3-. An HCO3(-)-dependent (amiloride-insensitive) mechanism contributed to pHi recovery after ischemia in the diabetic rat hearts. Even when the Na+/H+ exchanger was blocked by amiloride in nominally HCO3(-)-free solution, a rapid rise in pHi occurred during the first 3 min of reperfusion. The early rise in pHi was reduced by external lactate and inhibited by alpha-cyano-4-hydroxycinnamate. This suggested that a coupled H(+)-lactate efflux may be a major mechanism for acid extrusion in the initial stage of reperfusion. The observation of a higher functional recovery on reperfusion in diabetic hearts is in accordance with previous studies using HCO3- buffer. However, this study shows that a good recovery of function occurred even more rapidly in diabetic hearts receiving HEPES-buffered solution than in those receiving HCO3(-)-buffered solution. This suggests that the HCO3(-)-dependent mechanism of regulation may be depressed in diabetic rat hearts.

Role of Na(+)-H+ exchange in mediating effects of endothelin-1 on normal and ischemic/reperfused hearts.

Endothelin (ET) has been shown to be elevated under conditions of cardiac pathology and to produce diverse cardiac effects, including coronary constriction and a positive inotropic influence. We characterized the concentration- and time-dependent effects of the most potent of the ET isoforms, ET-1 (0.4, 2, and 4 nmol/L), on myocardial contractility and coronary resistance and assessed its effects on the ischemic and reperfused heart. Because ET-1 has been shown to activate the Na(+)-H+ exchanger in cardiac myocytes, we determined the contribution of the antiport by examining the effects of ET-1 in the presence of the Na(+)-H+ exchange inhibitor methylisobutyl amiloride (MIA). At all three concentrations, ET-1 produced an initial positive inotropic effect that was reversed with continued perfusion, the degree of the reversal being dependent on ET-1 concentration. With 0.4 nmol/L, contractility returned to pre-ET-1 values, whereas after 75 minutes of perfusion with 4 nmol/L ET-1, contractility was depressed by 75%. At all concentrations, ET-1 produced a coronary-constricting effect, whereas an elevation in resting tension was observed only with 4 nmol/L ET-1. MIA significantly prevented the positive inotropic effect of ET-1 but had no effect on loss in function or elevation in resting tension produced by 4 nmol/L ET-1. Furthermore, MIA partially, but not significantly, attenuated the constricting effects of all ET-1 concentrations. In the ischemic heart, 0.4 nmol/L ET-1 appeared to delay the loss in contractility produced by cessation of flow, although the effect was not significant. Higher concentrations of ET-1 were without effect on ischemia-induced contractile depression, although their presence produced a marked elevation in resting tension during ischemia that was attenuated by MIA. Recovery in contractility was reduced by all concentrations of ET-1, although the effects of the lowest concentration were associated primarily with defective relaxation. The depressant effects of ET-1 either in normal or ischemic/reperfused hearts were irreversible. The inhibitory effects of ET-1 on contractile recovery were associated with diminished tissue glycogen and elevated lactate levels. High-energy phosphates after reperfusion were depressed in hearts treated with 4 nmol/L ET-1. The attenuation in contractile recovery and alterations in metabolite content were prevented by MIA. These results provide evidence that ET-1 produces complex effects on heart function that are likely mediated via different mechanisms and demonstrate its ability to aggravate ischemic and reperfusion injury through a mechanism possibly involving Na(+)-H+ exchange activation.

Adenosine-sensitive alpha 1-adrenoceptor effects on reperfused ischaemic hearts: comparison with phorbol ester.

1. We have examined the effects of the alpha 1-adrenoceptor agonists, phenylephrine or methoxamine, on contractility in rat and rabbit isolated hearts as well as their effects on postischaemic ventricular recovery. We compared these effects to those of 12-phorbol 13-myristate acetate (PMA), a direct activator of protein kinase C (PKC). 2. The positive inotropic effect of alpha 1-receptor agonists was significantly attenuated in the presence of the Na/H exchange inhibitor, methylisobutyl amiloride (MIA, 1 microM), whereas the positive inotropic effect of PMA was unaffected. 3. Reperfusion of rat hearts subjected to either 30 or 60 min of zero-flow ischaemia, resulted in recovery of contractility to 91 +/- 2% and 57 +/- 7% of the preischaemic values, respectively which was unaffected by phenylephrine. In contrast, PMA at a concentration (10 pM) devoid of direct depressant effects, significantly decreased recovery following 60 min of ischaemia to 31 +/- 4% of pre-ischaemic value (P < 0.05 from control); an effect which was completely prevented by the PKC inhibitor, bisindolylmaleimide. A similar inhibitory effect of PMA and lack of effect of phenylephrine were seen in reperfused rabbit hearts. 4. As alpha 1-receptor activation has been shown previously to stimulate cardiac adenosine production, we assessed whether blockade of adenosine A1 receptors with the specific antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX, 0.5 microM) would unmask the actions of phenylephrine in hearts subjected to 30 min ischaemia and reperfusion. In the presence of DPCPX, phenylephrine reduced recovery to 44 +/- 9% compared to 82 +/- 10% recovery in the absence of phenylephrine (P < 0.05). Identical results were observed in rabbit hearts treated with DPCPX in which recovery was reduced from 57.1 +/- 11.2% to 17.8 +/- 6.8% by phenylephrine (P < 0.05). Another A1 receptor antagonist, (+/-)-N6-endonorbornan-2-yl-9-methyladenine (N-0861, 0.5 microM) produced virtually identical results to those observed with DPCPX. 5. MIA failed to modulate the inhibition of postischaemic recovery by phenylephrine. Bisindolylmaleimide, on the other hand, partially prevented the effects of phenylephrine on postischaemic contractile dysfunction. The inhibitory effect of either PMA or phenylephrine on postischaemic recovery of both rat and rabbit hearts was generally dissociated from alterations in energy metabolism, although in the case of rat hearts, inhibition by phenylephrine was associated with diminished high energy phosphate content. 6. Our results demonstrate that both alpha 1-receptor activation as well as direct activation of PKC with phorbol ester can attenuate post-ischaemic ventricular recovery. Moreover, our results strongly suggest that endogenous adenosine protects the heart against the deleterious effects of alpha 1-receptor activation during ischaemia and reperfusion.

Skinned cardiac fibres of diabetic rats: contractile activation and effects of 2,3-butanedione monoxime (BDM) and caffeine.

OBJECTIVE: The aim was to examine contractile properties of skinned cardiac fibres from rats with streptozotocin induced diabetes and to compare the effects of two agents, caffeine and 2,3-butanedione monoxime (BDM), on myocardial contractile characteristics of normal and diabetic cardiac fibres. METHODS: Small fibre bundles dissected from papillary muscles of the left ventricle were chemically skinned by exposure to Triton X-100. The tension-pCa (pCa = -log10 [Ca2+]) relationships were determined under isometric conditions. RESULTS: In skinned fibres from diabetic rats maximum Ca2+ activated force was unchanged in comparison with normal rats, but a significant, though small, increase in the Ca2+ sensitivity [pCa for one half maximal activation (pCa50)] of contraction was shown. Caffeine (5-20 mM) increased Ca2+ sensitivity in a dose dependent manner and to the same extent in the two groups of preparations. Up to 10 mM caffeine, maximum force was not affected. On the other hand, BDM (2 and 5 mM) decreased Ca2+ sensitivity in both normal and diabetic fibres, but the rightward shift of the tension-pCa relationship induced by BDM was more pronounced in diabetic than in normal fibres: pCa50 was 5.55(SEM 0.02), 5.51(0.01), and 5.46(0.01) in normal fibres, and 5.62(0.01), 5.51(0.02), and 5.45(0.02) in diabetic fibres for 0, 2, and 5 mM BDM, respectively. Maximum tension was similarly decreased by BDM in the two groups of fibres. CONCLUSIONS: (1) No change is induced by diabetes in the site of action of caffeine; (2) some drugs that affect myofilament Ca2+ sensitivity, such as BDM, may act differently in diabetic and control myocardium.

Intracellular pH and role of Na+/H+ exchange during ischaemia and reperfusion of normal and diabetic rat hearts.

STUDY OBJECTIVE: The aim was to investigate the role of intracellular pH (pHi) and ion exchange on the functional recovery of perfused hearts isolated from normal (N) rats either receiving or not receiving amiloride (an Na+/H+ exchange inhibitor), and from STZ induced diabetic (D) rats with decreased Na+/H+ exchange activity. DESIGN: Working heart preparations were submitted to a zero flow ischaemic period of 30 min at 37 degrees C and then reperfused for 30 min. The time course of pHi decline during ischaemia and of recovery on reperfusion was followed by means of 31P-NMR. MEASUREMENTS AND MAIN RESULTS: In N hearts without amiloride, ischaemia caused a progressive decrease in pHi. This was slightly, although not significantly, more abrupt in N hearts receiving amiloride. D hearts showed a slower fall in pHi, but the mean value reached after 30 min did not differ significantly from that of normal hearts. pHi recovery on reperfusion was markedly slower in the D hearts compared to N hearts. The mean value reached after 30 min did not differ significantly from that of N hearts. pHi recovery was also markedly slower in N hearts exposed to amiloride during both ischaemia and reperfusion. The higher functional recovery on reperfusion, as assessed by the recoveries of aortic flow and stroke volume, was observed for those hearts with slower pHi recovery. Improved recoveries of aortic flow and stroke volume as compared to normal non-treated hearts were 34% and 21% for the diabetic hearts, and 22% and 40% for the normal hearts receiving amiloride. CONCLUSIONS: The comparison of data from diabetic rat hearts with reduced activity of the Na+/H+ exchange process v normal hearts with pharmacological block of the exchanger provide support for a critical role of the Na+/H+ exchanger in the initial stage of reperfusion.

Influence of intracellular pH on mitochondrial calcium during ischaemia of the isolated rat heart.

Under physiological conditions cardiac mitochondria seem to play a minor role in maintaining intracellular Ca2+ homoeostasis. However, under conditions of cellular Ca2+ overload, mitochondria may accumulate large amounts of Ca2+. Using transmission and analytical electron microscopy, we investigated, in globally ischaemic rat heart preparations, the influence of intracellular pH on the development of Ca2+-containing intramitochondrial inclusions. We confirmed that under these experimental conditions Ca2+ was a major element of mitochondrial inclusions. The size of these inclusions increased with external Ca2+ concentration. An intracellular alkalinization, produced by addition of 20 mM NH4Cl to the perfusate prior to ischaemia, inhibited the formation of such inclusions. On the other hand, a pre-ischaemic intracellular acidification, produced by the addition and subsequent withdrawal of the 20 mM NH4Cl, increased the number of inclusions present at the end of an ischaemic episode. The presence of amiloride (10(-3) M), prior to and during ischaemia, increased the number of inclusions. These data suggest that cytoplasmic pH may be an important factor in mitochondrial Ca2+ accumulation in pathological conditions.

[Intermediate myocardial metabolism. Changes in ischemia, diabetes and hyperthyroidism]. / Métabolisme intermédiaire myocardique. Altérations dans l'ischémie, le diabète, l'hyperthyroïdie.

Long-chain free fatty acids (FFA) are oxidized, in preference to carbohydrates, by a myocardium with normal oxygen supply. Their utilization is increased in diabetes and most probably also in hyperthyroidism, since in both cases plasma FFA concentrations are augmented. Under conditions of ischaemia, the long-chain fatty acid esters of coenzyme A (CoA) and carnitine accumulate in cells. This accumulation depends on the degree of coronary blood flow reduction, being very high in moderate ischaemia and much reduced when the coronary flow is nul. The accumulation of acyl-CoA and acylcarnitine in ischaemic myocardium is amplified by diabetes. The presence in the cells of these amphophilic compounds (notably acylcarnitine) in high concentrations has been associated with changes in the structure and properties of mitochondrial and sarcolemmal membranes. Finally, the accumulation of glycolysis end-products (e.g. lactates and protons) may condition the degree of functional recovery from global and total ischaemia. In this respect, recently obtained in vitro data show that a decrease in cellular pH may be one of the determinant factors in reperfusion.

Abnormal cardiac rhythm in diabetic rats.

A significant occurrence of abnormal rhythm was observed in perfused working hearts of diabetic rats. The incidence of arrhythmias was 19/51 in diabetics as compared with 2/38 in normal controls. In considering possible pathogenetic mechanisms, conduction system defects appear to merit particular attention.