Role of calcitonin gene-related peptide in ischaemic preconditioning in diabetic rat hearts

1. It has been suggested that calcitonin gene-related peptide (CGRP) is involved in the protection provided by ischaemic preconditioning in rat hearts and that ischaemic preconditioning is absent in diabetic rat hearts. 2. In the present study, we tested the relationship between sensory nerve function and ischaemic preconditioning in diabetic rats. 3. In 4- and 8-week diabetic rats and age-matched non- diabetic controls, 30 min global ischaemia and 40 min reperfusion caused a significant decrease in cardiac function and a marked increase in creatine kinase (CK) release. Ischaemic preconditioning, by three cycles of 5 min ischaemia and 5 min reperfusion, improved the recovery of cardiac function and decreased CK release during reperfusion in 4-week diabetic rat hearts. However, the cardioprotection afforded by ischaemic preconditioning was lost in 8-week diabetic rat hearts. Pretreatment with CGRP for 5 min also significantly improved the recovery of cardiac function and decreased CK release in rats subjected to 4 or 8 weeks of diabetes. 4. The content of CGRP in the coronary effluent during ischaemic preconditioning was significantly increased in 4-week diabetic rat hearts (P < 0.05). However, only a slight increase in the release of CGRP was shown in 8-week diabetic rat hearts (P > 0.05). 5. In summary, the present results suggest that the protection afforded by ischaemic preconditioning is attenuated in diabetic rats and that the change may be related to the reduction in CGRP release in diabetic rat hearts.     

Role of protein kinase C in the reduction of infarct size by N-methyl-1-deoxynojirimycin, an alpha-1,6-glucosidase inhibitor.

Preischaemic treatment with N-methyl-1-deoxynojirimycin (MOR-14), an alpha-1,6-glucosidase inhibitor, attenuates glycogenolysis and lactate accumulation during ischaemia and markedly reduces infarct size in rabbit hearts. In the present study, we have investigated whether protein kinase C (PKC), a principal mediator of ischaemic preconditioning, is also involved in the cardioprotective effect of MOR-14. To assess the effect of PKC inhibition on infarct size in MOR-14-treated hearts, 38 rabbits were subjected to 30 min of ischaemia followed by 48 h of reperfusion. Infarct size, as a per cent of area at risk, was significantly smaller in rabbits administered 100 mg kg(-1) of MOR-14 10 min before ischaemia (17+/-2%, n=10), than in a control group (46+/-5%, n=10). This beneficial effect of MOR-14 was abolished when 5 mg kg(-1) of chelerythrine, a PKC inhibitor, was given 10 min prior to MOR-14 injection (39+/-4%, n=10), although chelerythrine alone did not alter infarct size (43+/-4%, n=8). Further, chelerythrine had no effect on MOR-14-induced attenuation of glycogen breakdown and lactate accumulation in hearts excised at 30 min of ischaemia. Immunoblot analysis of PKC in homogenates of Langendorff-perfused rabbit hearts revealed that MOR-14 significantly increased levels of PKC-epsilon in the particulate fraction at 20 and 30 min of ischaemia and in the cytosolic fraction at 30 min of ischaemia. Taken as a whole, our data suggest that PKC acts downstream of the inhibition of glycogenolysis by MOR-14 to reduce infarct size. Thus, activation of PKC is a more direct mediator of the cardioprotection afforded by MOR-14 than is inhibition of glycogenolysis.     

Gene transfer of heat-shock protein 20 protects against ischemia/reperfusion injury in rat hearts

AIM: To explore whether overexpression of HSP20 in the myocardium could protect against ischemia/reperfusion injury in rats. METHODS: Rat hearts were injected with vector, recombinant adenovirus encoding green fluorescent protein (Ad.GFP) or recombinant adenovirus encoding wild-type HSP20 (Ad.HSP20) in the left ventricle. Four days later, hearts were removed and expression of HSP20 was measured in the left ventricle. Subsets of animals in the vector-, Ad.GFP- , and Ad.HSP20-treated groups were subjected to 20-min ischemia and 120-min reperfusion. Myocardial injury was evaluated by infarct size and level of serum cardiac troponin T and creatine phosphokinase. Apoptosis of cardiomyocytes was determined by TUNEL staining. Cardiac function was evaluated by hemodynamic indexes. RESULTS: Infarct size and serum cardiac troponin T and creatine phosphokinase levels were significantly reduced in Ad.HSP20-treated hearts compared with vector- and Ad.GFP-treated hearts. The ratio of TUNEL-positive cardiomyocytes to total number of cardiomyocytes in the Ad.HSP20 group was significantly reduced as compared with the vector and Ad.GFP groups. Left ventricular end systolic pressure, and maximal rate of pressure increase (+dp/dt(max)) and decrease (-dp/dt(min)) values were increased significantly, while left ventricular end diastolic pressure was decreased significantly in Ad.HSP20-treated hearts compared with vector- and Ad.GFP-treated hearts. CONCLUSION: These data indicate that the cardioprotective effects of HSP20 may contribute to the reduction of myocardial necrosis and apoptosis in ischemia/reperfusion injury in rats.     

Effect of perinatal hypoxia on cardiac tolerance to acute ischaemia in adult male and female rats

1. The number of adult patients undergoing surgery for congenital cyanotic defects in childhood has increased significantly. Therefore, the aim of the present study was to examine the effect of perinatal hypoxia on the tolerance of the adult myocardium to acute ischaemia-reperfusion injury. 2. Pregnant Wistar rats were exposed to intermittent hypobaric hypoxia 7 days before delivery; pups were born under normoxic conditions and exposed to hypoxia again for 10 postnatal days. After the last hypoxic exposure, all animals were kept for an additional 3 months under normoxic conditions. All experiments were performed on 90-day-old rats. 3. Ventricular arrhythmias were assessed on isolated perfused hearts during 30 min occlusion of the left anterior descending coronary artery. Infarct size was measured on isolated hearts (40 min regional ischaemia and 120 min reperfusion) and on open-chest animals (20 min regional ischaemia and 3 h reperfusion). 4. Perinatal exposure to hypoxia significantly increased cardiac tolerance to ischaemic injury in adult females, as evidenced by the lower incidence and severity of ischaemic ventricular arrhythmias, compared with the normoxic group. The effect of perinatal hypoxia on ischaemic arrhythmias in males was quite the opposite. Myocardial infarct size measured in open-chest animals only was significantly smaller in normoxic females compared with normoxic males. Perinatal exposure to hypoxia had no effect on infarct size in either setting or sex. 5. The results of the present study support the hypothesis that perinatal hypoxia is a primary programming stimulus in the heart that may lead to sex-dependent changes in cardiac tolerance to acute ischaemia in later adult life. This would have important implications for patients who have experienced prolonged hypoxaemia in early life.     

Myocardial adenosine does not correlate with the protection mediated by ischaemic or pharmacological preconditioning in rat heart

1. We tested the hypothesis that ischaemic preconditioning of the rat heart activates cardiovascular adenosine formation to provide enhanced cardioprotection. 2. Rat isolated perfused hearts were either non-preconditioned, preconditioned with 5 min ischaemia or treated for 5 min with the alpha1-adrenoceptor agonist phenylephrine (50 micro mol/L) before being subjected to 30 min sustained ischaemia followed by 30 min reperfusion. Isolated cardiomyocytes were either non-preconditioned, subjected to 10 min simulated ischaemia or treated for 10 min with phenylephrine (50 micro mol/L) before being subjected to 30 min simulated ischaemia. Functional recovery of hearts and cell viability were used as indices of the effects of ischaemia. 3. Myocardial adenosine, as well as intracellular pH, was determined at the end of the preconditioning period and at 10, 20 and 30 min of sustained ischaemia. Intracellular pH was also determined during the reperfusion. 4. Ischaemic or pharmacological preconditioning with phenylephrine correlated with an improved functional recovery of perfused hearts during reperfusion and increased cell viability during ischaemia. 5. In perfused hearts, ischaemic preconditioning resulted in increased adenosine production in the myocardium during the following sustained ischaemia. However, in isolated cardiomyocytes, adenosine levels during sustained ischaemia were lower in ischaemically preconditioned cells compared with the respective non-preconditioned cardiomyocytes. 6. The increase in adenosine production was not observed in hearts preconditioned with phenylephrine instead of transient ischaemia. Similarly, pharmacological preconditioning resulted in decreased adenosine levels during sustained ischaemia in isolated cardiomyocytes. 7. Intracellular pH was preserved during ischaemia to the same extent in both ischaemically or pharmacologically preconditioned hearts and cardiomyocytes, indicating that less acidosis during ischaemia is related to protection. 8. Taken together, the results suggest that cardioprotection does not necessarily correlate with increased adenosine production. Thus, adenosine concentration is not crucial to the beneficial effects of preconditioning in rat heart.     

Effect of D,L-carnitine on the response of the isolated heart of the rat to ischaemia and reperfusion: relation to mitochondrial function.

1. The effect of 100 microM (20 micrograms ml-1) of D,L-carnitine was studied on the isolated heart of the rat subjected to 30 min of low flow ischaemia followed by reperfusion. 2. In untreated hearts (n = 30) ischaemia produced an almost total loss of contractility (P less than 0.05 compared with non-ischaemic time control) which was accompanied by an increase in resting tension of approximately 235% (P less than 0.05). Ventricular arrhythmias developed during ischaemia in 100% (P less than 0.05) of untreated hearts studied. Following reperfusion, untreated hearts recovered 16.3% of contractile function and demonstrated a 60% elevation in resting tension. The incidence of reperfusion-associated ventricular fibrillation was 60%. 3. Carnitine treatment produced no effect on either the contractile depression or the elevation in resting tension during ischaemia but did significantly decrease the incidence of arrythmias at the termination of ischaemia to 63.3% (n = 30, P less than 0.05). In the presence of carnitine, contractile recovery at the end of reperfusion was significantly increased to 30.2% (n = 10, P less than 0.05) and the elevation in resting tension was decreased to 30% (n = 10, P greater than 0.05). The incidence of ventricular arrhythmias during reperfusion was significantly reduced by carnitine. 4. Two populations of mitochondria, subsarcolemmal (SLM) and interfibrillar (IFM) isolated at the end of the ischaemic period exhibited an overall increase in oxidative phosphorylation rates as well as uncoupled oxygen consumption; both phenomena were more pronounced with IFM. Carnitine generally potentiated this response.(ABSTRACT TRUNCATED AT 250 WORDS)     

The influence of nifedipine and mioflazine on mitochondrial calcium overload in normoxic and ischaemic guinea-pig hearts

The influence of nifedipine (20 nM) and mioflazine (300 nM), i.e. concentrations inducing a 60-70% recovery of cardiac function during reperfusion of globally ischaemic guinea-pig working hearts, on the mitochondrial calcium content was investigated in normoxic, globally ischaemic and reperfused globally ischaemic guinea-pig working hearts. Mitochondrial calcium was determined electronmicroscopically with oxalate-pyroantimonate method. In normoxic hearts both nifedipine and mioflazine reduced the mitochondrial calcium content. Global ischaemia for 45 min and subsequent reperfusion for 25 min resulted in a pronounced mitochondrial calcium overload and damage to the cellular structure. In ischaemic and in reperfusion hearts the drugs maintained mitochondrial calcium at pre-ischaemic levels and decreased the damage to the cellular structure.     

Paradoxically enhanced heart tolerance to ischaemia in type 1 diabetes and role of increased osmolarity

There is considerable controversy regarding the tolerance of diabetic hearts to ischaemia and the underlying mechanisms responsible for the increased heart tolerance to ischamia remain uncertain. In the present study, we observed, in vitro, type 1 diabetic heart responses to ischaemia and reperfusion at different degrees of hyperglycaemia. In addition, the possible role of increased osmolarity in cardioprotection due to hyperglycaemia was evaluated. Hearts from 3 week streptozocin-induced diabetic rats were isolated and perfused in a Langendorff apparatus and subjected to 30 min ischaemia and 30 min reperfusion. Cardiac function and the electrocardiogram were recorded. Myocardial content of osmolarity associated heat shock protein (hsp) 90, heme oxygenase (HO)-1 and anti-oxidant enzymes were determined in diabetic or hyperosmotic solution-perfused hearts using western blot. The hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG; 2 x 10(-7) mol/L) or the nitric oxide synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (1 x 10(-5) mol/L) was added to the perfusate to observe the effects of hsp90 inhibition and hsp90-associated endothelial NOS on ischaemic responses of diabetic hearts. Compared with normal control rats, diabetic hearts with severe hyperglycaemia (blood glucose > 20 mmol/L) showed markedly improved postischaemic heart function with fewer reperfusion arrhythmias. Mild hyperglycaemia (< 12 mmol/L) exhibited no significant cardioprotection. Elevated expression of hsp90 accompanied the enhanced resistance to ischaemia in diabetic hearts, which was abrogated by 17-AAG. In the presence of the NOS inhibitor, heart function was preserved, whereas reperfusion arrhythmias were increased in diabetes. Diabetic hearts also had markedly elevated HO-1 and catalase, with no significant change in superoxide dismutase. Hyperosmotic perfusion with glucose or mannitol also increased myocardial hsp90 and catalase. The present findings reveal that heart resistance to ischaemia is increased in short-term type 1 diabetes with severe hyperglycaemia. Elevated osmolarity caused by significant hyperglycaemia may contribute to the enhanced myocardial activity against oxidative injury during ischaemia and reperfusion.     

Effects of Eicosapentaenoic Acid and Docosahexaenoic Acid Diet Supplement on Tolerance to the Cardiotoxicity of Epirubicin and to Ischaemia Reperfusion in the Isolated Rat Heart

Abstract: We compared the effects of 2 weeks dietary supplement of docosahexaenoic acid, eicosapentaenoic acid or olive oil on myocardial tolerance to the cardiotoxicity of the anthracycline epirubicin and to ischaemia reperfusion. Isolated rat hearts from the dietary groups were perfused at a constant flow rate of 12.5 ml/min. The hearts were subjected to a 20 min. period of epirubicin infusion by a side arm of the perfusion system at a rate of 0.2 mg/min. or a 20 min. period of global ischaemia. After 10 min. of epirubicin infusion a significantly (P<0.05) higher aortic pressure (an index of coronary resistance during constant flow perfusion) was observed in the olive oil group; 130±22% (mean±S.D.) compared to hearts in the docosahexaenoic acid; 108±9% (mean±S.D.), and eicosapentaenoic acid; 105±7% (mean±S.D.), group. Hearts from docosahexaenoic acid-fed rats showed a significantly increased left ventricular end-diastolic pressure (an index of contracture); of 66±30 mmHg (mean±S.D.) after 15 min. of global ischaemia compared to eicosapetaenoic acid fed rats; 37±18 mmHg (mean±S.D.), and significantly higher release of lactate dehydrogenase during the following 30 min. period of reperfusion compared to olive oil-fed rats. We conclude that eicosapentaenoic acid and docosahexaenoic acid could be useful during epirubicin infusion and that docosahexaenoic acid could be harmful during ischaemia reperfusion.     

Cardioprotective effects of a non-alcoholic extract of red wine during ischaemia and reperfusion in spontaneously hypertensive rats

1. We reported recently the cardioprotection conferred by a non-alcoholic extract of Cabernet-Sauvignon red wine (RWE) against alterations derived from ischaemia and reperfusion in normotensive rats. The aim of the present study was to assess the effects of RWE on ischaemia/reperfusion injury in hearts isolated from spontaneously hypertensive rats (SHR). 2. After stabilization, rat isovolumic perfused hearts were exposed to a 20 min global ischaemic period followed by 30 min reperfusion in the absence (ischaemic control (IC) hearts) or presence of RWE infused prior to ischaemia and early in reperfusion. In other hearts, N(G)-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide synthase, was administered prior to RWE infusion (L-NAME + RWE). 3. Left ventricular developed pressure (LVDP), dP/dt(max) and left ventricular end-diastolic pressure (LVEDP) were used to assess myocardial function. 4. At the end of reperfusion, LVDP and dP/dt(max) decreased to 47 +/- 9 and 46 +/- 9% of baseline values, respectively, in IC. Treatment with the RWE significantly improved systolic postischaemic recovery (LVDP = 85 +/- 8%; dP/dt(max) = 83 +/- 5%) and attenuated the increase in LVEDP (23 +/- 6 and 53 +/- 8 mmHg in RWE and IC, respectively; P < 0.05). 5. In the L-NAME + RWE group, L-NAME completely abolished the systolic and diastolic protection induced by RWE (LVDP = 44 +/- 13%; dP/dt(max) = 43 +/- 13%; LVEDP = 60 +/- 10 mmHg). 6. These data are the first demonstration that a non-alcoholic extract of Cabernet-Sauvignon red wine protects SHR hearts from systolic and diastolic alterations induced by ischaemia and reperfusion through a nitric oxide-dependent mechanism.     

Responses to ischaemia and reperfusion in the mouse isolated perfused heart and the phenomenon of 'contractile cycling'

1. The aims of the present study were to examine the response of the murine heart to ischaemia and reperfusion and to determine whether these responses are influenced by the strain of mouse. 2. Isolated, paced (600 b.p.m.) murine (T/O mice) hearts were perfused aerobically (2.6 mL/min) with buffer for 40 min before being subjected to whole-heart (global) ischaemia (37 degrees C) for 20, 30, 35, 40 or 50 min prior to 90 min reperfusion. Contracture was measured during ischaemia and the reperfusate was collected and assayed for creatine kinase. 3. With increasing durations of ischaemia, there was a progressive decline in postischaemic recovery such that left ventricular developed pressure (LVDP) after 20, 30, 35, 40 or 50 min ischaemia was 75 +/- 4, 65 +/- 4, 38 +/- 6, 18 +/- 2 and 18 +/- 2% of pre-ischaemic controls, respectively. 4. There was a reciprocal increase in creatine kinase leakage, indicative of a time-dependent increase in tissue injury. 5. To compare the ischaemic vulnerability of different strains, hearts from Swiss and C57BL/6 mice were perfused for 20 min, followed by 40 min global ischaemia (37 degrees C) and 60 min reperfusion. Functional recovery of LVDP in Swiss mouse hearts was significantly higher than in C57BL/6 mouse hearts (39 +/- 7 vs 20 +/- 4%, respectively; P < 0.04, t-test; 10 d.f.). 6. During our investigations, we encountered and characterized the phenomenon of 'contractile cycling' (cyclical patterns of declining and increasing left ventricular systolic pressure of variable severity and duration). 7. We have shown that this confounding phenomenon is a manifestation of an underlying metabolic disturbance of unknown origin that can be attenuated by the addition of substrates, such as pyruvate or acetate, to the standard glucose-containing perfusion buffer.     

Pretreatment with Ischaemia Attenuates Acute Epirubicin-Induced Cardiotoxicity in Isolated Rat Hearts

Abstract: We investigated whether a brief ischaemic episode (ischaemic pretreatment) preconditioning might attenuate the acute cardiotoxicity of the anthracycline, epirubicin. Isolated rat hearts perfused at a constant flow rate of 10 ml/min. were preconditioned with 5 min. of global ischaemia and 10 min. of reperfusion (preconditoned hearts), or were perfused for 15 min. (control hearts). The hearts were then subjected to 20 min. of infusion with epirubicin (2 mg/ml) or vehicle by a side arm of the perfusion system at a rate of 0.1 ml/min. (0.2 mg epirubicin/min.). Attenuation of cardiotoxicity of a total dose of 4 mg of epirubicin was assessed by functional and metabolic parameters during infusion and during the following 30 min. recovery period. Cardiotoxic effects were reduced in preconditioned hearts compared to control hearts. Thus left ventricular developed pressure and heart rate product after 20 min. of epirubicin infusion was depressed to 27±7% (mean±S.D.) and 40±4% (mean±S.D.) of baseline values in the control group and the preconditioned group, respectively (P<0.05). Furthermore, we observed less contracture during epirubicin infusion and more effective reversal of contracture during the recovery period in the preconditioned hearts. Improvement in cardiac function was associated with a significantly lower (P<0.05) myocardial content of epirubicin in the preconditioned group at the end of the infusion period. We conclude that ischaemic preconditioning attenuates the acute cardiotoxicity of epirubicin, probably by reducing the myocardial accumulation of the anthracycline.     

Sevoflurane post-conditioning protects against myocardial reperfusion injury by activation of phosphatidylinositol-3-kinase signal transduction

The mechanisms underlying myocardial protection by sevoflurane post-conditioning are unclear. In the present study, we tested two hypotheses: (i) that sevoflurane post-conditioning produces cardioprotection via a phosphatidylinositol-3-kinase (PI3-K)-dependent pathway; and (ii) combining sevoflurane and ischaemic post-conditioning offers an additional benefit against reperfusion injury. Rat isolated perfused hearts were exposed to 25 min ischaemia followed by 90 min reperfusion. Sevoflurane post-conditioning was induced by administration of sevoflurane (3.0 vol%) for 15 min from the onset of reperfusion. In some groups, 15 micromol/L LY294002, a selective PI3-K inhibitor, was coadministrated with sevoflurane. Other groups of hearts were exposed to ischaemic post-conditioning or combined sevoflurane plus ischaemic post-conditioning in the presence and absence of LY294002. After 15 min reperfusion, phosphorylation of Akt and glycogen synthase kinase 3beta (GSK3beta) was determined by Western blot analysis. Infarct size was determined by 2,3,5-triphenyltetrazolium chloride staining and subsarcolemmal mitochondrial lesions were assessed by electron microscopy after 90 min reperfusion. Sevoflurane post-conditioning significantly decreased infarct size compared with control hearts (31 +/- 2 vs 42 +/- 3%, respectively; P < 0.05), diminished mitochondrial lesions and increased phosphorylation of Akt and GSK3beta, as did ischaemic post-conditioning. However, combined sevoflurane plus ischaemic post-conditioning did not further improve the cardioprotective effects compared with either intervention alone. Sevoflurane-mediated cardioprotection was abolished or inhibited by 15 micromol/L LY294002. In conclusion, sevoflurane acts during early reperfusion after ischaemia to salvage the myocardium by activating PI3-K. The combination of sevoflurane plus ischaemic post-conditioning does not offer any additional benefit over either intervention alone.     

Cytochrome P450 2J3/epoxyeicosatrienoic acids mediate the cardioprotection induced by ischaemic post-conditioning, but not preconditioning, in the rat

1. Cytochrome P450 (CYP) epoxygenases and their arachidonic acid metabolites play a protective role against ischaemia-reperfusion injury. In the present study, we investigated whether endogenous CYP2J3/epoxyeicosatrienoic acid (EET) mediates the cardioprotective effects of ischaemic preconditioning (IPC) and ischaemic post-conditioning (IPost). 2. Male Wistar rats were subjected to two cycles of IPC, consisting of 5 min ischaemia and 5 min reperfusion, followed by 45 min occlusion and 2 h reperfusion; IPost consisted of three cycles of 30 s reperfusion and 30 s re-occlusion at the onset of reperfusion. The selective CYP epoxygenase inhibitor N-methylsulphonyl-6-(2-propargyloxyphenyl)hexanamide (MS-PPOH; 3 mg/kg) was administered 10 min before ischaemia or during ischaemia 10 min before reperfusion started. Cardiac function was measured continuously with a angiocatheter connected to a fluid-filled pressure transducer and myocardial infarct size was assessed by triphenyl tetrazolium chloride staining at the end of the experiment. 3. Subjecting rats to IPC and IPost similarly improved cardiac function and reduced myocardial infarct size. Interestingly, IPost, but not IPC, significantly increased CYP2J3 mRNA (1.75 ± 0.22 vs 1.0; P < 0.05) and protein (1.62 ± 0.22 vs 1.0; P < 0.05), as well as 11,12-EET synthesis compared to I/R (6.2 ± 0.2 vs 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01). Administration of MS-PPOH before ischaemia significantly decreased 11,12-EET synthesis in both IPC and IPost compared with I/R rats (2.1 ± 0.2, 3.2 ± 0.3 and 2.9 ± 0.2 ng/mg wet weight, respectively; P < 0.01), but decreased the cardioprotective effects, as evidenced by cardiac function and myocardial infarct size, of IPost only. 4. These data indicate that endogenous activation of CYP2J3/EET may be an essential trigger leading to the protective effects of IPost, but not IPC, in the rat heart.     

Dobutamine administration exacerbates postischaemic myocardial dysfunction in isolated rat hearts: an effect reversed by thyroxine pretreatment

The present study has investigated the effects of dobutamine on postischaemic dysfunction in the setting of global ischaemia and reperfusion in a model of isolated heart preparation. Isolated rat hearts were subjected to 20 min of zero-flow global ischaemia followed by 45 min of reperfusion. Dobutamine administration (10 μg/kg/min) during the reperfusion period resulted in deterioration of functional recovery, which was abolished by propranolol administration. Long-term thyroxine pretreatment (12.5 μg 100 g⁻¹ body weight, b.i.d., s.c., for 2 weeks) reversed the detrimental effect of dobutamine and increased postischaemic recovery of function. We conclude that the combination of thyroxine pretreatment and dobutamine administration could potentially be a new therapeutic strategy to improve postischaemic dysfunction particularly in clinical settings such as cardiopulmonary bypass and/or myocardial infarction.     

The effect of chemical sympathectomy on mitochondrial function in the ischaemic and reperfused myocardium.

1 Isolated rabbit hearts were perfused aerobically for 120 min, made ischaemic for 90 min, or made ischaemic for 90 min and then reperfused for 30 min. 2 Some rabbits were pretreated with 6-hydroxydopamine (6-OHDA), given as three separate intravenous doses of 30, 20 and 20 mg/kg, 20 to 48 h before they were killed; others (controls) received saline according to the same regime. 3 Mitochondria were harvested from left ventricular homogenates and their function assessed by measuring state 3O2 consumption (state 3 QO2), respiratory control index (RCI), phosphate: oxygen ratio (ADP:O), Ca2+ content, and ATP-producing activity. In other experiments peak left ventricular developed tension was recorded. 4 In hearts from saline-treated animals, mitochondrial state 3 QO2, RCI and ATP producing activities were reduced after global ischaemia, with or without reperfusion. There was a small gain in mitochondrial Ca2+ after ischaemia, and a large gain upon reperfusion. 5 6-OHDA pretreatment provided some protection against the effects of ischaemia and reperfusion on mitochondrial function and on peak developed tension. 6 It was concluded that chemical sympathectomy with 6-OHDA does not duplicate the effect of prolonged beta-adrenoceptor blockade in protecting mitochondrial function against the deleterious effects of ischaemia and reperfusion.     

Prolonged diuretic administration and myocardial tolerance to ischaemia

Hearts from rats, which received high doses of furosemide alone or the same doses of furosemide plus amiloride in a diet with low magnesium content for 4 weeks, were isolated and perfused in the Langendorff mode. After 15 min. of normoxic control perfusion no differences were found between the two groups of hearts with respect to cardiac physiology. After 20 min. of subtotal, global ischaemia and 15 min. of reperfusion the furosemide plus amiloride hearts showed a significantly higher recovery of function (judged by pressure rate product and coronary flow rate) than furosemide hearts. However, the myocardial content of adenosine triphosphate, creatine phosphate, and electrolytes at the end of the experiment exhibited no difference between the two groups. In separate experiments it was found that the addition of amiloride to the furosemide regimen significantly raised and almost normalized the values of plasma magnesium and potassium. Myocardial calcium was lower, whereas the magnesium and potassium content in the hearts was not different from the furosemide group. It is concluded that the administration of amiloride to rats provided high doses of furosemide and marginal magnesium supplies afforded some protection upon the ischaemic heart.     

Cardioprotective effects of N-hydroxyguanidine PR5 in myocardial ischaemia and reperfusion in rats

1. The potential for the N-hydroxyguanidine compound PR5 (N-(3, 4-dimethoxy-2-chlorobenzylideneamino)-N'-hydroxyguanidine) as a cardioprotective agent in heart ischaemia and reperfusion injury was investigated using rat models. 2. Administration of 1-10 mg kg-1 of PR5 5 min before 10 min of left coronary artery occlusion, followed by 20 min reperfusion, strongly inhibited reperfusion burst of arrhythmias and markedly improved the survival of the animals (e.g. ventricular fibrillation incidence 93 vs 43% (P<0.05); mortality 47 vs 0% (P<0.05), for controls and for 3 mg kg-1 of PR5, respectively). 3. Administration of 3 mg kg-1 of PR5 1 min before reperfusion to rats subjected to 10 min occlusion, 20 min reperfusion was most effective in reducing arrhythmias and decreasing mortality (43 vs 0%, P<0.05), but effects were also seen when PR5 was administered 0, 1 and 5 min after start of reperfusion. 4. Coronary occlusion/reperfusion (10 - 20 min) increased malondialdehyde (MDA) of rat hearts (0.88+/-0.13 for sham vs 1.45+/-0.12 nmol mg-1 protein for ischaemic; P<0.05). In rats where 3 mg kg-1 PR5 were administered 1 min before reperfusion the increase was attenuated (MDA being 1.04+/-0.12; P<0.05 vs ischaemic). 5. PR5 caused a substantial reduction of the infarction size in rats subjected to 180 min left coronary artery occlusion, followed by 120 min of reperfusion; the necrotic zone being 326+/-32 mg for controls vs 137+/-21 mg for animals treated with 3x3 mg kg-1 of PR5 (P<0.01). 6. PR5 reduced the elevation of the ST-segment of the ECGs, as well as caused pronounced attenuation of the rapid blood pressure drop seen at the start of reperfusion following coronary artery occlusion. 7 We conclude that the N-hydroxyguanidine PR5 provides remarkable protection against ischaemia and reperfusion induced myocardial necrosis and life-threatening arrhythmias. These effects of PR5 are discussed in relation to a recently discovered ability of N-hydroxyguanidines to function as electron acceptors at the xanthine oxidase enzyme.     

Effects of propionyl-carnitine on mitochondrial respiration and post-ischaemic cardiac function in the ischaemic underperfused diabetic rat heart

BACKGROUND AND OBJECTIVE: Carnitine and its derivatives, namely propionyl-carnitine (PC), have been shown to protect cardiac metabolism and function in diabetes mellitus and ischaemic heart disease. Since diabetes is associated with abnormalities in mitochondrial metabolism of fuels, we examined the effects of PC on mitochondrial respiration in ischaemic hearts from streptozotocin-diabetic rats. METHODS: Diabetes was induced in Sprague-Dawley rats by an intravenous injection of streptozotocin. Following the diagnosis of diabetes, oral PC treatment was initiated for a period of 6 weeks. After treatment, cardiac function was determined from working hearts perfused under aerobic conditions and in a separate group of hearts subjected to ischaemia and reperfusion. Mitochondrial respiration was determined under aerobic conditions and following low-flow ischaemia. RESULTS: Rates of state 3 mitochondria respiration with pyruvate were significantly lower in diabetic (n = 4) hearts compared with control (n = 6) hearts (80 +/- 5 vs 112 +/- 5 nanoatoms O2/mg protein/min, respectively), but those with palmitoylcarnitine were similar (101 +/- 11 vs 106 +/- 6 nanoatoms O2/mg protein/min). Diabetic rat heart (n = 8) function, expressed as rate pressure product, was also significantly decreased compared with control (n = 8) hearts (21.5 +/- 1.0 vs 29.5 +/- 0.9 beats x mm Hg x 10(-3)/min, respectively). In PC-treated diabetic (n = 6) hearts, state 3 respiration with pyruvate was increased, and a marked improvement in left ventricular function from 21.5 +/- 1.0 to 26.0 +/- 0.6 beats x mm Hg x 10(-3)/min was observed. During low-flow ischaemia, state 3 respiration with pyruvate remained lower in diabetic (n = 5) hearts compared with control (n = 5) hearts (64 +/- 3 vs 46 +/- 5 nanoatoms O2/mg protein/min, respectively). Following treatment with PC (n = 4), however, respiration with this substrate was significantly increased to 57 +/- 4 nanoatoms O2/mg protein/min. PC was also associated with a significant improvement in cardiac function in reperfused diabetic rat (n = 4) hearts (18.4 +/- 0.2 beats x mm Hg x 10(-3)/min). CONCLUSION: Our results showed that PC has a beneficial effect on cardiac function and increases ischaemic tolerance of the diabetic rat heart. This beneficial effect of PC can be explained, in part, as an improvement in mitochondrial metabolism of pyruvate during the actual ischaemic period.     

Mechanism of myocardial protective action of dilazep during ischaemia and reperfusion

The aim of this study was to investigate if dilazep is able to reduce with a direct protective action on the myocardium the deleterious effects caused by ischaemia and reperfusion. For this purpose we used an isolated rabbit heart preparation. The hearts were either perfused aerobically or made totally ischaemic for 60 min (by abolishing coronary flow) or made ischaemic for 60 min and then reperfused for 30 min. Ischaemic and reperfusion damage was measured in terms of alteration in mechanical function, lactate and CPK release, mitochondrial function and tissue content of Adenosine Triphosphate (ATP), Creatine Phosphate (CP) and calcium. Dilazep (10(-5) M) was administered in the perfusate either 20 minutes before ischaemia or only during post-ischaemic reperfusion. Ischaemia induced a decline of the endogenous stores of ATP and CP, followed by an alteration of calcium homeostasis with increase of diastolic pressure, mitochondria calcium overload and impairment of the oxidative phosphorylating capacities. On reperfusion, tissue and mitochondrial calcium increase the capacity of the mitochondria to use O2 for state III respiration was further impaired and the ATP-generating capacity reduced. Diastolic pressure increased and there was only a small recovery of active tension generation associated with massive CPK release. Administration of dilazep before ischaemia induced a negative inotropic effect which, in turn, resulted in a slowing of the rate of CP and ATP depletion during ischaemia. This protected the hearts against the ischemic, and reperfusion-induced decline in the ATP-generating and O2-utilizing capacities of the mitochondria. In addition, there was a less marked increase in tissue and mitochondrial Ca++, CPK and lactate release were reduced and the recovery of developed pressure on reperfusion was significantly increased. Administration of dilazep during reperfusion failed to modify the exacerbation of ischaemic damage caused by the readmission of coronary flow. These data suggest that dilazep benefits the ischaemic myocardium via an ATP sparing action.