Claudia Schäfer

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.

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.

Hypertrophic responsiveness of cardiomyocytes to α- or β-adrenoceptor stimulation requires sodium-proton-exchanger-1 (NHE-1) activation but not cellular alkalization

The influence of the sodium‐proton‐exchanger‐1 (NHE‐1) inhibitor HOE694 on α‐ or β‐adrenoceptor mediated stimulation of protein synthesis was investigated in cultured ventricular cardiomyocytes from adult rat pre‐treated with fetal calf serum to induce hypertrophic responsiveness to β‐adrenoceptor stimulation. Stimulation of α‐adrenoceptors with phenylephrine (10 μM) in bicarbonate‐free medium caused cellular alkalization (ΔpHi: +0.17±0.02, n‐5, P<0.05). HOE694, an NHE‐1 inhibitor, completely abolished this effect. [14C]phenylalanine incorporation into cellular protein mass increased in the presence of phenylephrine by 23±8%, and this effect was also abolished in the presence of HOE694. HOE694 (1 μM) neither influenced basal protein synthesis nor interfered with α‐adrenoceptor mediated activation of ERK2. Phorbol myristate acetate, a direct stimulator of protein kinase C, mimicked the effect of α‐adrenoceptor stimulation in regard to protein synthesis, but did not lead to cellular alkalization. Protein synthesis increased in the presence of isoprenaline, a β‐adrenoceptor agonist also. Again, HOE694 attenuated the stimulation of protein synthesis although isoprenaline did not cause cellular alkalization. In conclusion, the growth response to different hypertrophic stimuli, namely α‐ or β‐adrenoceptor stimulation, is attenuated in the presence of the NHE‐1 inhibitor HOE694 and this inhibition is independent from cellular alkalization.

Stimulation of cGMP signalling protects coronary endothelium against reperfusion-induced intercellular gap formation

AIMS Ischaemia-reperfusion provokes barrier failure of the coronary microvasculature, impeding functional recovery of the heart during reperfusion. The aim of the present study was to investigate whether the stimulation of cGMP signalling by activation of soluble guanylyl cyclase (sGC) can reduce reperfusion-induced endothelial intercellular gap formation and to determine whether this is due to an influence on endothelial cytosolic Ca(2+) homeostasis during reperfusion. METHODS AND RESULTS Experiments were performed with cultured coronary endothelial monolayers and isolated saline-perfused rat hearts. HMR1766 (1 micromol/L) or DEAnonoate (0.5 micromol/L) were used to activate sGC. After exposure to simulated ischaemic conditions, reperfusion of endothelial cells led to a pronounced increase in cytosolic calcium levels and intercellular gaps. Stimulation of cGMP signalling during reperfusion increased Ca(2+) sequestration in the endoplasmic reticulum (ER) and attenuated the reperfusion-induced increase in cytosolic [Ca(2+)]. Phosphorylation of phospholamban was also increased, indicating a de-inhibition of the ER Ca(2+) pump (SERCA). Reperfusion-induced intercellular gap formation was reduced. Reduction of myosin light chain phosphorylation indicated inactivation of the endothelial contractile machinery. Effects on cytsolic Ca(2+) and gaps were abrogated by inhibition of cGMP-dependent protein kinase (PKG) with KT5823. In reperfused hearts, stimulation of cGMP signalling led to decreased oedema development. CONCLUSION sGC/PKG activation during reperfusion reduces reperfusion-induced endothelial intercellular gap formation by attenuation of cytosolic calcium overload and reduction of contractile activation in endothelial cells. This mechanism protects the heart against reperfusion-induced oedema.

Parathyroid hormone–related peptide improves contractile responsiveness of adult rat cardiomyocytes with depressed cell function irrespectively of oxidative inhibition

AbstractParathyroid hormone–related peptide (PTHrP) was found to improve contractile function of stunned myocardium in pigs. The peptide is released from coronary endothelial cells during ischemia and significantly improves post–ischemic recovery. The present study was aimed to decide whether such an induction of contractile responsiveness of the heart requires co–activation of adjacent cells or is a genuine phenomenon of cardiomyocytes. A second aim of this study was to decide whether such an improvement is linked to depressed cell function in general or oxidative inhibition. Isolated adult ventricular cardiomyocytes from rats were constantly paced at 0.5 Hz for 10 min. Cells were exposed to a brief oxidative inhibition by addition of 0.5 mmol/l potassium cyanide (KCN) in the presence of glucose. Under these conditions, cells stopped beating after 280 s on average. 30 s before they stopped to beat, cells had already developed a reduction in cell shortening, maximal relaxation and contraction velocity. In the co–presence of PTHrP (1–34) (100 nmol/l) cells continued to beat regular and did not develop reduced cell shortening, irrespectively of oxidative inhibition. In a second attempt, cells were exposed to the NO donor SNAP (100 µmol/l) or 8–bromocGMP (1 mmol/l). As expected both agents reduced cell shortening significantly. This reduction in cell shortening was attenuated in co–presence of PTHrP, too. Finally, we investigated the effect of PTHrP on cell shortening at different extracellular concentrations of calcium. Although, PTHrP increased intracellular calcium at 2 and 5 mmol/l extracellular calcium, respectively, it improved cell shortening only at 5 mmol/l extracellular calcium. Thus, the beneficial effect of PTHrP on cell shortening was independent from intracellular calcium but dependent on the steepness of the calcium gradient between intra– and extracellular calcium. In conclusion, our study shows that PTHrP is able to improve cell shortening rapidly and directly irrespectively of the reason for the reduced cell function. Improved electromechanical coupling rather than intracellular calcium handling seems to be the most important mechanism.

Inhibition of contractile activation reduces reoxygenation-induced endothelial gap formation

OBJECTIVE Barrier function of coronary endothelium becomes disturbed by ischemia-reperfusion. We investigated the mechanism of reperfusion-induced endothelial gap formation in monolayers of cultured endothelial cells (CEC) of the rat, exposed to simulated ischemia (40 min anoxia, pH(o) 6.4) and reperfusion (30 min reoxygenation, pH(o) 7.4). METHODS Cytosolic Ca(2+) (fura-2) and intercellular gap formation (planimetrical analysis) were determined. Reoxygenation conditions were varied: (a) continuing perfusion at pH(o) 6.4, (b) with or without glucose (2.5 mM), (c) in presence of NaCN (2 mM), (d) with Ca(2+) (10 mM) or BAPTA/AM (25 microM), (e) in the presence of myosin light chain kinase inhibitors ML-7 (5 microM) or wortmannin (1 microM). RESULTS During anoxia, CEC developed cytosolic Ca(2+) overload which was not reversed during 30 min reoxygenation. Intercellular gap formation started during anoxia, but was increased during reoxygenation. Reoxygenation-related gap formation was largest in presence of glucose, lower when glucose was withdrawn or NaCN was added. Presence of ML-7 or wortmannin also reduced gap formation during reoxygenation. CONCLUSIONS Reoxygenation induces gap formation. This is dependent on (i) Ca(2+) overload during reoxygenation, (ii) energy production and (iii) activation of myosin light chain kinase. Together these results indicate that activation of the endothelial contractile machinery is the underlying cause.

Cell Biology of Acute Reperfusion Injury

In ischemic-reperfused myocardium, necrosis of cardiomyocytes may develop not only due to the ischemic conditions but also due to the specific circumstances of reperfusion. Reperfusion-induced hypercontracture is a major cause of lethal reperfusion-injury of myocardium. Hypercontracture of myofibrils is caused by reenergetisation of Ca2+-overloaded cardiomyocytes. Ca2+-overload is due to the preceding ischemic period. Upon reperfusion, Ca2+-overload leads to rapid oscillations of cytosolic Ca2+-concentration. Rapid normalisation of pH favors hypercontracture, prolonged acidosis protects against it. Inhibition of reperfusion-induced Ca2+-oscillations (inhibition of Ca2+-uptake or Ca2+-release by the sarcoplasmic reticulum; inhibition of the reverse mode of the sarcolemmal Na+/Ca2+-exchanger) or of pHi recovery (simultaneous inhibition of the Na+/H+-exchanger and the Na+/HC03 - symporter of sarcolemma) during the vulnerable phase of reperfusion can protect the ischemic-reperfused cardiomyocyte against reperfusion injury.