Y. V. Ladilov

Role of the reverse mode of the Na+/Ca2+ exchanger in reoxygenation-induced cardiomyocyte injury.

OBJECTIVE We have recently shown that spontaneous Ca2+ oscillations elicit irreversible hypercontracture of cardiomyocytes during reoxygenation. The aim of this study was to investigate whether influx of exterior Ca2+ through the reverse mode of the Na+/Ca2+ exchanger (NCE) contributes to the development of these oscillations and, therefore, to reoxygenation-induced hypercontracture. METHODS Isolated cardiomyocytes and hearts from rats were used as models. Cardiomyocytes were exposed to 60 min simulated ischemia (pH(o) 6.4) and 10 min reoxygenation (pH(o) 7.4). During reoxygenation cardiomyocytes were superfused with medium containing 1 mmol/l Ca2+ (control), with nominally Ca2+-free medium or with medium containing 10 micromol/l KB-R 7943 (KB), a selective inhibitor of the reverse mode of the NCE. RESULTS In reoxygenated cardiomyocytes rapid Ca2+ oscillations occurred which were reduced under Ca2+-free conditions or in presence of KB. Hypercontracture was also significantly reduced under Ca2+-free conditions or in presence of KB. After 30 min of normoxic perfusion isolated rat hearts were subjected to 60 min global ischemia and reperfusion. KB (10 micromol/l) was present during the first 10 min of reperfusion. LVEDP, LVdevP and lactate dehydrogenase (LDH) release were measured. Presence of KB reduced post-ischemic LVEDP and improved left ventricular function (LVdevP). In KB treated hearts the reperfusion induced release of LDH was markedly reduced from 81.1 +/- 9.9 (control) to 49.3 +/- 8.8 U/60 min/g dry weight. CONCLUSION Our study shows that inhibition of the reverse mode of the NCE, during reperfusion only, protects cardiomyocytes and whole hearts against reperfusion injury.

The role of Na+/H+ exchange in ischemia-reperfusion

In ischemia the cytosol of cardiomyocytes acidifies; this is reversed upon reperfusion. One of the major pHi-regulating transport systems involved is the Na+/H+ exchanger. Inhibitors of the Na+/H+ exchanger have been found to more effectively protect ischemic-reperfused myocardium when administered before and during ischemia than during reperfusion alone. It has been hypothesized that the protection provided by pre-ischemic administration is due to a reduction in Na+ and secondary Ca2+ influx. Under reperfusion conditions Na+/H+ exchange inhibition also seems protective since it prolongs intracellular acidosis which can prevent hypercontracture. In detail, however, the mechanisms by which Na+/H+ exchange inhibition provides protection in ischemic-reperfused myocardium are still not fully identified.

Simulated Ischemia Increases the Susceptibility of Rat Cardiomyocytes to Hypercontracture

The hypothesis that rat cardiomyocytes become susceptible to hypercontracture after anoxia/reoxygenation was investigated. The cells were gradually overloaded with Ca2+ after different periods of simulated ischemia (substrate-free anoxia, medium at pH 6.4) followed by 20 minutes of reoxygenation. The cytosolic Ca2+ concentration (measured with fura 2) at which the cells developed maximal hypercontracture (Camax) was used as an index for their susceptibility to hypercontracture (SH). SH was increased in cardiomyocytes after prolonged periods of simulated ischemia; ie, these cells developed hypercontracture at significantly lower cytosolic Ca2+ levels than did normoxic cells (Camax, 0.80 +/- 0.05 mumol/L versus 1.27 +/- 0.05 mumol/L; P < .01). To find the possible cause of increased SH, the influence of Ca2+ overload, acidosis, and protein dephosphorylation were studied. Prevention of cytosolic Ca2+ overload in anoxic cardiomyocytes or imitation of ischemic acidosis in normoxic cells did not influence Camax. In contrast, use of 10 mumol/L cantharidin (inhibitor of protein phosphatases 1 and 2A) during anoxic superfusion prevented the reduction of Camax. Furthermore, treatment of normoxic cardiomyocytes with 20 mmol/L of the chemical phosphatase 2,3-butanedione monoxime reduced Camax. Therefore, prolonged simulated ischemia increases susceptibility of cardio-myocytes to hypercontracture. This seems to be due to protein dephosphorylation.

Mechanism of Ca2+ overload in endothelial cells exposed to simulated ischemia

OBJECTIVE Several studies have shown that myocardial ischemia leads to functional failure of endothelial cells (EC) whereby disturbance of Ca(2+) homeostasis may play an important role. The mechanisms leading to Ca(2+) disbalance in ischemic EC are not fully understood. The aim of this study was to test effects of different components of simulated ischemia (glucose deprivation, anoxia, low extracellular pH (pH(o)) and lactate) on Ca(2+) homeostasis in EC. METHODS Cytosolic Ca(2+) (Ca(i)), cytosolic pH (pH(i)) and ATP content were measured in cultured rat coronary EC. RESULTS In normoxic cells 60 min glucose deprivation at pH(o) 7.4 had no effect on pH(i). It only slightly increased Ca(i) and decreased ATP content. Reduction of pH(o) to 6.5 under these conditions led to marked cytosolic acidosis and Ca(i) overload, but had no effect on ATP content. Anoxia at pH(o) 6.5 had no additional effect on Ca(i) overload, but significantly reduced cellular ATP. Addition of 20 mmol/l lactate to anoxia at pH(o) 6.5 accelerated Ca(i) overload due to faster cytosolic acidification. Acidosis-induced Ca(i) overload was prevented by inhibition of Ca(2+) release channels of endoplasmic reticulum (ER) with 3 micromol/l ryanodine or by pre-emptying the ER with thapsigargin. Re-normalisation of pH(o) for 30 min led to recovery of pH(i), but not of Ca(i). CONCLUSION The ischemic factors leading to cytosolic acidosis (low pH(o) and lactate) cause Ca(i) overload in endothelial cells, while anoxia and glucose deprivation play only a minor role. The ER is the main source for this Ca(i) rise. Ca(i) overload is not readily reversible.

Protection of Rat Cardiomyocytes Against Simulated Ischemia and Reoxygenation by Treatment With Protein Kinase C Activator

The aim of this study was to investigate whether treatment with the protein kinase C (PKC) agonist 1,2-dioctanoyl-sn-glycerol (1,2DOG) can protect isolated adult Wistar rat cardiomyocytes against simulated ischemia and reoxygenation. Cytosolic Ca2+ (assessed by fura 2 fluorescence), pHi (assessed by BCECF fluorescence), and cell length were measured during 80 minutes of simulated ischemia (anoxia, pHo 6.4) and 20 minutes of reoxygenation (pHo 7.4) and compared between control cells and cells treated with 20 micromol/L 1,2DOG before anoxia (10-minute treatment and 10-minute washout), before and during anoxia (two-step treatment), or only during anoxia. Treatment before anoxia attenuated rigor contracture but did not influence anoxic Ca2+ overload. In contrast, two-step treatment before and during anoxia accelerated rigor contracture but reduced the rate of anoxic Ca2+ accumulation. During reoxygenation, control cells developed irreversible hypercontracture (reduction of cell length to 43+/-2% of the initial cell length, n=62), which was accompanied by spontaneous oscillations of cytosolic Ca2+ (19.6+/-1.6 per minute). Two-step treatment with 1,2DOG before and during anoxia significantly reduced hypercontracture (reduction of cell length to 60+/-2%, P<.01 versus control, n=41) and suppressed spontaneous Ca2+ oscillations (2.8+/-0.9 per minute, P<.01 versus control). These effects could not be reproduced by treatment with 1,2DOG before anoxia or during anoxia or by a two-step treatment with the PKC-inactive 1,3-dioctanoyl-sn-glycerol and were fully abolished with 1 micromol/L bisindolylmaleimide (PKC inhibitor). We conclude that a two-step activation of PKC before and during anoxia is required for effective protection of cardiomyocytes against anoxic Ca2+ overload and reoxygenation-induced hypercontracture.

Pretreatment with PKC activator protects cardiomyocytes against reoxygenation-induced hypercontracture independently of Ca2+ overload

OBJECTIVE Although several studies have shown that activation of protein kinase C (PKC) plays an important role in protection through ischemic preconditioning, little is known about the effects of direct PKC activation on the course of ischemia-reperfusion injury. The aim of this study was to analyse the effects of a pretreatment with the PKC activator 1,2-dioctanoyl-sn-glycerol (1,2DOG). METHODS Isolated adult Wistar rat cardiomyocytes were exposed to 80 min of simulated ischemia (anoxia, pHo 6.4) and 20 min of reoxygenation (pHo 7.4). Cytosolic Ca2+ (fura-2), cytosolic pH (BCECF), Mg2+ (Mg-fura-2), lactate and cell length were measured and compared between control cells and cells treated with 20 mumol/l 1,2DOG before anoxia (10 min treatment and 10 min wash out). RESULTS 1,2DOG-pretreatment delayed the time to extreme ATP depletion, but had no effect on lactate production and cytosolic pH. The accumulation of cytosolic Ca2+ was markedly accelerated in pretreated cells that developed rigor shortening, but reoxygenation-induced hypercontracture was significantly reduced. 1,2DOG, therefore, completely abolished Ca(2+)-dependence of hypercontracture. The effects of pretreatment were fully abolished with 1 mumol/l bisindolylmalcimide (PKC inhibitor). We conclude that PKC preactivation leads to (1) reduction of energy demand, (2) acceleration of Ca2+ overload during anoxia and (3) prevention of reoxygenation-induced hypercontracture independent of anoxic changes in cytosolic Ca2+ and pH.

Ischemic preconditioning on the cellular level

ConclusionThe studies using adult cardiomyocytes indicate that preconditioning is indeed a genuine phenomenon of the myocardial cell. Our own results show that a pharmacological precontioning protocol can provide a pronounced protective effect-comparable to the extent of protection in whole myocardium. The isolation procedure does, therefore, nor render cardio-myocytes unable to exhibit a clear preconditioning effect.

Protection of isolated cardiomyocytes against reoxygenation-induced hypercontracture by SIN-1C

Abstract Previous studies have shown that SIN-1C (N-morpholinoiminoacetonitrile) can protect ischemic-reperfused myocardium. The aim of the present study was to analyse on the cellular level the mechanism by which SIN-1C may exert this effect. To simulate ischemia-reperfusion, isolated adult rat cardiomyocytes were incubated at pH 6.4 under anoxia and reoxygenated at pH 7.4 in presence or absence of SIN-1C. Reoxygenation was started when intracellular Ca2+ (measured with fura-2) had increased to ≥10–5 mol/L and pHi (BCECF) decreased to 6.6. Development of hypercontracture was determined microscopically. In the control group reoxygenation provoked oscillations of cytosolic Ca2+ (60.9±9.6 min–1 at 5 min of reoxygenation) accompanied by development of hypercontracture (to 77.2±3.8% of end-ischemic cell length). When SIN-1C was added upon reoxygenation, Ca2+ oscillations were markedly reduced (27.0±4.5 min–1, p<0.001) and hypercontracture virtually abolished (90.6±2.0% of end-ischemic cell length, p<0.001). SIN-1C did not influence the recovery of pHi during reoxygenation. The results indicate that SIN-1C protects cardiomyocytes against reoxygenation-induced hypercontracture by its ability to suppress oscillations of intracellular Ca2+ during the early phase of reoxygenation.

Acute reperfusion injury of myocardium

Summary In ischemic-reperfused myocardium severe cellular injury may develop rapidly upon re-oxygenation (‘oxygen paradox’). Several causes of this acute form of reperfusion injury have now been identified. During oxygen depletion cardiomyocytes accumulate Ca2+ in the cytosol. When re-energized upon re-oxygenation high cytosolic Ca2+ causes uncontrolled contractile activation, leading to hypercontracture of the myofibrils. Protection against hypercontracture can be achieved by various means, for example, by prolongation of ischemic intracellular acidosis or by attenuation of the oscillatory elevations of cytosolic Ca2+ in the early phase of re-oxygenation. Structural damage provoked by hypercontracture is favored by increased cytoskeletal fragility of the cardiomyocytes. In reperfused myocardium cardiomyocytes are also challenged by osmotic stress. Oxidative injury reduces sarcolemmal stability upon re-oxygenation and thereby reduces the tolerance of cardiomyocytes to osmotic stress. All these causes contributing to acute reperfusion injury are rooted within the myocardial cells. They are independent of exogenous, blood-borne factors that can additionally modify the reperfusion situation.

Energy and Cation Control in the Reoxygenated Myocardial Cell

The development of cell injury in ischemic tissue starts with a deficit in the cellular balance of energy. The energetic deficit leads to a slowdown or cessation of important metabolic functions, among these the cellular control of Na+ and Ca 2+ ions. When the cellular reserves of energy are depleted, cation pumps regulating the normal intracellular ionic milieu fail due to a lack of energy. A lone-lasting overload of the cytosolic space and intracellular organelles with excess Car 2+ can be deleterious for the cell, as a number of structure degrading processes may become activated. In muscle cells, the activation of the myofibrillar contractile apparatus by high levels of Ca 2+ may additionally cause mechanical cell damage. The loss of cellular Ca 2+ homeostasis is a sign of advanced, but not necessarily irreversibly cell injury. For a better understanding of the pathogenesis of progressive myocardial injury the energy and cation control in the oxygen deprived and reoxygenated cardiomyocyte must be analyzed. This article provides a brief review.