Danielle Feuvray

Controversies on the sensitivity of the diabetic heart to ischemic injury: the sensitivity of the diabetic heart to ischemic injury is decreased

Controversy exists as to whether the diabetic heart is more or less sensitive to ischemic injury. Although a considerable number of experimental studies have directly determined the effects of ischemia on the diabetic heart, there is still no general agreement as to whether metabolic changes within the myocardium contribute to the severity of ischemic injury. This paper reviews the evidence suggesting that the diabetic heart can actually be less sensitive to an episode of severe ischemia. Possible reasons for this decreased sensitivity to injury are discussed, which include a decreased accumulation of glycolytic products during ischemia (lactate and protons), as well as alterations in the regulation of intracellular pH in the diabetic heart. Based on existing studies, we suggest that although impaired glucose metabolism in the diabetic heart contributes to injury in hypoxic hearts or in hearts subjected to low-flow ischemia, diabetes-induced decreases in glycolysis can actually be beneficial to the diabetic heart during and following a severe ischemic episode. A decreased clearance of protons via the Na+/H+ exchanger may also contribute to the decreased sensitivity to ischemic injury in the diabetic heart.

Changes in intracellular sodium and pH during ischaemia-reperfusion are attenuated by trimetazidine. Comparison between low- and zero-flow ischaemia.

OBJECTIVE The aim of this study was to investigate whether trimetazidine (TMZ; 10(-6)M), which has been shown to inhibit fatty acid oxidation, reduces the ionic imbalance induced by ischaemia and reperfusion, especially through an attenuation in intracellular changes in H(+) and Na(+). METHODS Isovolumic rat hearts receiving 5.5 mM glucose and 1.2 mM palmitate as metabolic substrates were exposed to zero-flow ischaemia (TI) or low-flow ischaemia (LFI - coronary flow decreased by an average of 90%) (30 min at 37 degrees C) and then reperfused. 23Na nuclear magnetic resonance (NMR) spectroscopy was used to monitor intracellular Na(+) (Na(+)(i)) and 31P NMR spectroscopy was used to monitor intracellular pH (pH(i)). RESULTS During LFI the major effect of TMZ was a significant reduction in intracellular acidosis, whereas during TI the main effect of TMZ was a significant reduction in Na(+)(i) gain. In addition, the further gain in Na(+)(i) that occurred during the first minutes of reperfusion following TI, and to a far lesser extent following LFI, was suppressed in TMZ-treated hearts and also suppressed when hearts were perfused without fatty acid. In both LFI and TI, TMZ-induced attenuation of ionic imbalance was associated with a significantly improved recovery of ventricular function on reperfusion, as assessed by a lower increase in diastolic pressure and an increased recovery of developed pressure. CONCLUSION Our data provide evidence that specific myocardial metabolic modulation plays a significant role in reducing ionic imbalance during ischaemia and reperfusion.

Modulation by pHo and Intracellular Ca2+ of Na+-H+ Exchange in Diabetic Rat Isolated Ventricular Myocytes

We have previously shown that diabetes is associated with a decrease in Na(+)-H+ exchange activity in rat cardiac papillary muscle. The present work has been carried out in order to elucidate the factors responsible for such an alteration. Thus, we have studied the effects of pH0 and intracellular Ca2+ on Na(+)-H+ exchange in ventricular myocytes isolated from streptozotocin-induced diabetic rat hearts. pH1 was recorded using carboxy-seminaphthorhodafluor (SNARF-1). The NH4+ (10 mmol/L) prepulse method was used to induce an acid load in order to activate Na(+)-H+ exchange in HEPES-buffered Tyrodes solution. Whereas diabetes did not change intracellular buffering power, it significantly decreased acid efflux through Na(+)-H+ exchange (acid efflux, 4.32 +/- 0.4 [n = 32, normal cells] versus 2.5 +/- 0.2 [n = 43, diabetic cells] meq/L per minute at pHi 6.9; P < .02). Upon changes of pH0 (at a range of 8.0 to 6.8), acid efflux similarly varied in normal and diabetic cells, thus pointing to an unchanged pH0 sensitivity of Na(+)-H+ exchange. Buffering of intracellular Ca2+ by pretreatment of the cells with BAPTA-AM (25 mumol/L Ca2(+)-chelator) resulted in a decrease by approximately 58% of acid efflux in the diabetic group. This decrease was even more marked in normal cells (by approximately 74%). Interestingly, the pH1 dependence of the acid efflux carried by Na(+)-H+ exchange then became identical in both groups of cells, thus pointing to a role for intracellular Ca2+ in the diabetes-related alterations of the exchange. Inhibition of calmodulin (by 1.5 mumol/L calmidazolium) and of Ca2+/calmodulin-dependent protein kinase II (by 2 mumol/L 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazin e [KN-62]) significantly slowed down pH1 recovery in both normal and diabetic cells. However, the effect of KN-62 was significantly lower in diabetic cells (efflux decreased by approximately 17%) compared with normal cells (decrease by 45%). In conclusion, these data, in light of recent observations showing a decreased [Ca2+]i associated with diabetes in isolated ventricular myocytes, suggest that changes in intracellular Ca2+ may play an important role in altering Na(+)-H+ exchange activity in diabetic ventricular myocytes. They also point to diabetes-related alterations in the Ca2+/calmodulin protein kinase II-dependent phosphorylation of Na(+)-H+ exchange.

Effects of trimetazidine on pHi regulation in the rat isolated ventricular myocyte

1 We have examined the effects of trimetazidine (TMZ) on intracellular pH (pHi) regulation in rat isolated ventricular myocytes. pHi was recorded ratiometrically by use of the pH‐sensitive fluoroprobe, carboxy‐SNARF‐1 (carboxy‐seminaphtorhodafluor). 2 Following an intracellular acid load (induced by 10 mM NH4Cl removal), pHi recovery in HEPES‐ buffered Tyrode solution was significantly slowed down upon application of 0.3 mM TMZ only when myocytes were pretreated for 5 h 30 min (slowing by ∼ 50%; P < 0.01). This effect of TMZ on pHi recovery was shown to be not only time‐ but also dose‐dependent with a large, quickly reversible, effect obtained with 1 mM TMZ applied for 2–3 h (slowing by ∼ 64%; P 0.001). This slowing of pHi recovery was also associated with a decrease of the NH4+ removal‐induced acidification. 3 Relationship between intracellular intrinsic buffering power (βi) and pHi was assessed in absence or presence of TMZ (0.3 mM or 1 mM). As expected, βi increased roughly linearly with a decrease in pHi in all cases. However, both concentrations of TMZ significantly increased βi (by ∼ 55 and 65% at pHi 7.1, respectively). 4 When Na+/H+ exchange was inhibited by dimethyl amiloride (DMA; 40 μm), trimetazidine (1 mM) did not change the H+ flux estimated at pHi 7.1 (0.31 ± 0.03 mequiv 1−1 min−1, n = 5, control, versus 0.30 ± 0.025 mequiv 1−1 min−1, n = 5, TMZ), ruling out any effect of TMZ on background acid loading. 5 Acid efflux carried by Na+/H+ exchange was significantly decreased only when myocytes were pretreated with 1 mM TMZ, for 2–3 h (JeH = 2.86 ± 0.38 mequiv 1−1 min−1, n = 26, control, versus 1.66 ± 0.26 mequiv 1−1 min−1, n = 10, TMZ, estimated at pHi 7.1; P < 0.05). 6 In conclusion, the present work demonstrates that, following an intracellular acid load in HEPES‐ buffered medium, trimetazidine slows down pHi recovery in rat isolated ventricular myocytes, primarily through an increase of βi. An effect on Na+/H+ exchange is also detected but only after long‐term incubation of the myocytes with TMZ.

A biochemical and ultrastructural study of the species variation in myocardial cell damage

Abstract Species differences (rat, mouse, guinea pig and rabbit) to anoxic damage, glucose protection and reoxygenation damage were investigated using isolated perfused hearts arrested by K + during the anoxic period. Myocardial damage, as assessed by enzyme release and ultrastructural changes, was related to changes in myocardial glycogen, adenosine triphosphate, creatine phosphate and lactate. The results suggest that the speed of onset, magnitude of early tissue damage, and the extent to which reoxygenation can exacerbate damage are related to the characteristics of myocardial membranes, and that while rat and mouse hearts are resistant to the early onset of damage, reoxygenation can greatly exacerbate damage in these species. In contrast, guinea pig and rabbit hearts exhibit an early onset of damage and are therefore less susceptible to reoxygenation exacerbation. Major species differences also exist in the extent to which glucose can protect the anoxic myocardium and thereby reduce tissue damage and enzyme release. In the rat and the guinea pig, glucose consistently protected myocardial glycogen, adenosine triphosphate and creatine phosphate levels, reduced enzyme release and maintained myocardial ultrastructure. In contrast in the mouse, glucose was less able to protect myocardial energy supplies and extensive ultrastructural damage and enzyme release occurred. Variable protection was observed in the rabbit, in some instances ultrastructural damage and enzyme release were greatly reduced and in this sub-group, glucose maintained myocardial energy reserves. In other rabbits, glucose failed to protect energy supplies and extensive ultrastructural damage and enzyme release was observed. The results suggest that glucose protects the anoxic, arrested myocardium primarily through its ability to act as a substrate for glycolytic ATP production and that any variation in its efficacy either within or between species is due to variations in their maximum capacity for glycolytic flux.

Ultrastructural modifications induced by reoxygenation in the anoxic isolated rat heart perfused without exogenous substrate

Abstract Isolated potassium-arrested rat hearts were submitted to anoxic perfusions (30 or 100 min) at 37°C. After the period of anoxia, oxygen was re-introduced for 5 or 20 min. The ultrastructure of ventricular myocardium was studied: (1) in control experiments after 20 min stabilization period (standard perfusate, normal potassium concentration, with glucose); (2) after 35 or 105 min anoxia following the stabilization period; (3) after 5 min reoxygenation following 30 or 100 min anoxia; and (4) after 20 min reoxygenation following 100 min anoxia. During both anoxia and reoxygenation the perfusion fluid contained a high potassium concentration (17 m m ) and no glucose of other substrate. After 35 min of anoxic perfusion, or 30 min of anoxic perfusion and reoxygenation for 5 min, very slight ultrastructural modifications were observed. On the other hand, marked ultrastructural modifications in myofibrils and sarcoplasmic reticulum were encountered after 105 min anoxia. But most striking morphologic changes concerning the mitochondria were observed after 5 min of reoxygenation following 100 min of anoxia. However, after 20 min reoxygenation, these mitochondrial alterations were less marked. Our results indicate that in potassium-arrested rat hearts perfused without substrate, ultrastructural alterations appeared which were dependent on the duration of the anoxic period and were greatly enhanced by reoxygenation. It is concluded that enzyme release occurring in such experimental conditions may be related to these ultrastructural alterations.

Ischaemia-induced damage in the working rat heart preparation: The effect of perfusate substrate composition upon subendocardial ultrastructure of the ischaemic left ventricular wall

Ultrastructural modifications occuring in the subendocardium of the severely ischaemic part of the left ventricular wall following left coronary artery ligation, were studied in isolated working rat hearts perfused with different substrates. Substrates used were: glucose, glucose plus albumin, palmitate bound to albumin, and palmitate-albumin together with glucose and/or insulin. Non-ischaemic control hearts perfused with glucose as substrate showed a typical morphology. In contrast, control hearts perfused with either glucose-albumin or palminate-albumin exhibited dilatations of most of the unspecialized regions of the intercalated discs. In ischaemic hearts marked ultrastructural modifications were observed; these included extensive intracellular oedema, altered myofibrils, tubular system and nucleus as well as glycogen depletion and mitochondrial swelling. These changes occurred with all substrates. Moreover, typical mitochondrial changes occured which appeared to be dependent upon the nature of the substrate. In palmitate-perfused hearts, amorphous electron dense opacities appeared within 47% of the mitochondria. Such opacities were never observed in glucose or glucose-albumin perfused hearts and their number was considerably reduced when glucose and/or insulin, but mainly glucose, was added to palmitate-albumin solutions. These findings illustrate that in isolated working rat heart ischaemia-induced ultrastructural damage can be partly determined by the nature of the substrate.

The regulation of intracellular pH in the diabetic myocardium

Time for primary review 25 days. . In myocardial cells, as in any given cell, steady-state intracellular pH (pHi) is strictly maintained within a narrow range at relatively alkaline values. Resting pHi (7.1–7.2) is determined by the algebraic sum of acid-loading and acid-extruding processes. Whenever acid-loading exceeds acid extrusion, pHi falls. The degree to which pHi changes is inversely related to the intracellular buffering power (βi). The role of intracellular buffering power, which is the first line of defence of a cardiac cell against an intracellular acid–base disturbance, is to moderate pHi changes produced by acute acid (or alkali) load [1]. However, buffering mechanisms cannot prevent a change in pHi, but only reduce its amplitude. The regulation of pHi then largely depends upon the activity of plasma membrane carrier-mediated transport of acid/base equivalents [2, 3]. Intracellular pH is important for the activity of a number of enzymes with optimal pH within the physiological pH range, as well as for the conductivity of ion channels [4], calcium homeostasis, and the efficiency of contractile elements [5, 6]. Despite a vast literature demonstrating myocardial metabolic changes associated with diabetes (for reviews, see Refs. [7, 8]), until recent years relatively few studies have addressed the effects of diabetes on intracellular pH. Yet, disturbances in intracellular pH or in the processes regulating intracellular pH may be expected to occur in diabetic hearts as a result of either altered cellular metabolism and/or cellular and subcellular membrane changes. This review provides a summary of the data obtained with both multicellular and single cell preparations, as well as with isolated hearts from chemically-induced diabetic rats. The first study that allowed direct measurement of intracellular pH in cardiac cells of streptozotocin (STZ)-induced diabetic rat hearts was … * Tel.: +33 (1) 69 15 78 98; fax: +33 (1) 69 15 68 41.

Ionic and metabolic imbalance as potential factors of ischemia reperfusion injury

This study examined the influence of metabolic substrates on the effects of trimetazidine on functional and metabolic aspects of the ischemic reperfused heart. Isovolumic rat hearts were submitted to a 30-minute period of global mild ischemia (coronary flow decreased by an average of 70%) and then reperfused at constant preischemic coronary flow rate. Either glucose (11 mM) or glucose and palmitic acid (0.1 mM) were used as metabolic substrates. Trimetazidine (6 x 10(-7)M) markedly reduced the increase in diastolic pressure that occurred on reperfusion after the ischemic episode, whatever the exogenous substrate used. However, in those hearts that received fatty acid, the postischemic increase in diastolic pressure was abolished. Ischemia-induced increase in acyl carnitine levels-determined as indicators of fatty acid utilization by myocardial cells-was significantly decreased by trimetazidine in those hearts receiving fatty acid. Also, similar effects to those of trimetazidine on the postischemic increase in diastolic pressure and on tissue levels of acyl carnitine were obtained in the presence of dichloroacetate. Moreover, the presence of trimetazidine was associated with a reduction in the intracellular pH decrease during ischemia in those hearts receiving fatty acid. Combined with previous studies, these results suggest that an improved metabolic balance by trimetazidine may well consequently decrease the ionic imbalance after a transient period of ischemia.

Effect of short dimethylsulfoxide perfusions on ultrastructure of the isolated rat heart

Abstract Isolated rat hearts were perfused with dimethylsulfoxide (0.7 and 2.1 m ) for 10 min at 37°C. The ventricular ultrastructure was studied (i) after 10 min of dimethylsulfoxide perfusion; and (ii) 2 min after replacement with normal solution following dimethylsulfoxide perfusion. Ultrastructural alterations were found after the dimethylsulfoxide perfusion was over, when returning the heart to the normal solution; changes were dependent on the dimethylsulfoxide concentration. The T-system and mitochondria were chiefly involved. A hyperosmotic effect of dimethylsulfoxide could be responsible