H. M. Piper

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.

Apoptosis induction by nitric oxide in adult cardiomyocytes via cGMP-signaling and its impairment after simulated ischemia

OBJECTIVE Nitric oxide (NO) has been shown to induce apoptosis in cardiomyocytes under normoxic conditions. The ability of NO to induce apoptosis after ischemia-reperfusion, a situation of increased NO release in vivo, has not been investigated. The present study was undertaken to characterize the pathway of induction of apoptosis by NO and the influence of ischemia on this pathway in cardiomyocytes. METHODS The study was performed on isolated adult cardiomyocytes of the rat. Ischemia was simulated by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected (1) by annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide and (2) by laddering of genomic DNA. RESULTS Incubation of cardiomyocytes with the NO-donor (+/-)-S-nitroso-N-acetylpenicillamine (SNAP, 100 microM) induced apoptosis in 14.1 +/- 1.9% of the cells and necrosis in 24.4 +/- 4.6%. The induction of apoptosis but not necrosis could be blocked by inhibition of soluble guanylyl cyclase or of protein kinase G. Apoptosis induction was mimicked by incubation of cardiomyocytes with 8-pCPT-cGMP (100 microM, 9.6 +/- 0.6% apoptotic cells) or YC-1 (75 microM, 14.6 +/- 2.8% apoptotic cells), a direct activator of soluble guanylyl cyclase. After 3 h of anoxia, cardiomyocytes were transiently protected against apoptosis induced by NO, but not by 8-pCPT-cGMP or YC-1 (8.9 +/- 0.7% or 13.4 +/- 2.4% apoptotic cells). A correlation of the apoptotic response to SNAP or YC-1 with an increased activity of soluble guanylyl cyclase, determined by measurements of intracellular cGMP contents, was found. CONCLUSIONS NO induces apoptosis in a cGMP dependent manner in isolated adult cardiomyocytes whereas induction of necrosis seems cGMP-independent. After simulated in vitro ischemia the activation of soluble guanylyl cyclase by NO is transiently inhibited resulting in a transient anti-apoptotic protection.

Peroxisome Proliferator‐Activated Receptor α Agonists Enhance Cardiomyogenesis of Mouse ES Cells by Utilization of a Reactive Oxygen Species‐Dependent Mechanism

Peroxisome proliferator‐activated receptors (PPARα, ‐β and ‐γ) are nuclear receptors involved in transcriptional regulation of lipid and energy metabolism. Since the energy demand increases when cardiac progenitor cells are developing rhythmic contractile activity, PPAR activation may play a critical role during cardiomyogenesis of embryonic stem (ES) cells. It is shown that ES cells express PPARα, ‐β, and ‐γ mRNA during differentiation of ES cells towards cardiac cells. Treatment with PPARα agonists (WY14643, GW7647, and ciprofibrate) significantly increased cardiomyogenesis and expression of the cardiac genes MLC2a, ANP, MHC‐β, MLC2v, and cardiac α‐actin. Furthermore, WY14643 increased PPARα gene expression and the expression of the cardiogenic transcription factors GATA‐4, Nkx2.5, DTEF‐1, and MEF 2C. In contrast, the PPARα antagonist MK886 decreased cardiomyogenesis, whereas the PPARβ agonist L‐165,041 as well as the PPARγ agonist GW1929 were without effects. Treatment with PPARα, but not PPARβ, and PPARγ agonists and MK886, resulted in generation of reactive oxygen species (ROS), which was inhibited in the presence of the NADPH oxidase inhibitors diphenylen iodonium (DPI) and apocynin and the free radical scavengers vitamin E and N‐(2‐mercapto‐propionyl)‐glycine (NMPG), whereas the mitochondrial complex I inhibitor rotenone was without effects. The effect of PPARα agonists on cardiomyogenesis of ES cells was abolished upon preincubation with free radical scavengers and NADPH oxidase inhibitors, indicating involvement of ROS in PPARα, mediated cardiac differentiation. In summary, our data indicate that stimulation of PPARα but not PPARβ and ‐γ enhances cardiomyogenesis in ES cells using a pathway that involves ROS and NADPH oxidase activity.

Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat

OBJECTIVES Apoptosis is one feature of myocardial damage after ischemia-reperfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is directly initiated by their sub-lethal injury that results from ischemia-reperfusion. METHODS Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling) and (3) annexinV-fluorescein isothiocyanate (annexinV-FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified by (1) staining with both annexinV-FITC and propidium iodide, (2) unspecific DNA degradation and (3) enzyme release. RESULTS Simulated ischemia caused a > 75% loss of high-energy phosphates within 2 h, which was reversible upon reoxygenation at pH 7.4. Even after 18 h of simulated ischemia, creatine phosphate contents recovered to 55.2 +/- 7.3% of control within 1 h. Apoptosis could be induced by UV irradiation (80 J/m2), H2O2 and the NO-donor N2-acetyl-S-nitroso-D,L-penicillinaminamide. In contrast to this, simulated ischemia and reoxygenation could not induce apoptosis in the cells, but with prolonged ischemia more cells became necrotic. After 18 hours of simulated ischemia and 4 h of reoxygenation 41.2 +/- 10.2% myocytes were necrotic (vs. 6.3 +/- 4.4% of control) and only 1.7 +/- 0.5% (vs. 8.7 +/- 4.6% of control) were apoptotic. The percentage of necrotic cells correlated with an increase in lactate dehydrogenase release from 9.9 +/- 0.6% (of total activity) of normoxic controls to 37.9 +/- 5.1% after 18 h of simulated ischemia and 12 h of reoxygenation. CONCLUSIONS Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis.

The calcium paradox revisited: an artefact of great heuristic value.

The 1967 paper by Zimmerman et al. [1] is a sequel to one published in the previous year [2] in which Ariaen Zimmerman and Willem Hulsmann had described an artefact produced on an isolated rat heart preparation. It is exceptional that an artefact receives so much attention and is not forgotten three decades later. The observation they made is that, when an isolated heart is perfused for 2 min with a Ca2+ free, otherwise normal Krebs-Henseleit buffer and then with buffer containing a physiological Ca2+ concentration, it rapidly deteriorates. Massive enzyme release occurs and the heart becomes pale due to myoglobin loss. The 1967 paper demonstrates that these changes are accompanied with dramatic alterations of myocardial ultrastructure, i.e. membrane disruption, myofibrillar hypercontracture and mitochondrial damage. The discoverers named this impressive artefact ‘calcium paradox’. Since the original description this phenomenon has fascinated hundreds of researchers, with the highest research activity in the 1980s. This brief review is an attempt to explain 33 years after the original description the main reasons for this long-lasting fascination. It is not intended to duplicate previous scientific reviews [3–6]. In the 1970s and 1980s the pathophysiological importance of calcium for the heart was in the centre of scientific awareness. The calcium paradox was soon regarded as a paradigm in this area of research as it became clear that repletion of the once Ca2+ depleted heart leads to massive Ca2+ influx into the myocardial cells, a phenomenon also observed in other situations of severe myocardial cell injury. In particular, it was a widely accepted hypothesis that the calcium paradox represents a paradigm for the pathomechanism of severe ischemia-reperfusion injury. In the words of Albrecht Fleckenstein [7]: “Certainly, with restoring the blood perfusion of the previous ischemic region, an unlimited Ca2+ …

SMAD‐proteins as a molecular switch from hypertrophy to apoptosis induction in adult ventricular cardiomyocytes

Heart failure development goes along with a transition from hypertrophic growth to apoptosis induction. In adult cardiomyocytes SMAD proteins are only activated under apoptotic, but not under hypertrophic conditions and are increased at the transition to heart failure. Therefore, SMADs could be candidates that turn the balance from hypertrophic growth to apoptosis resulting in heart failure development. To test this hypothesis we infected isolated rat ventricular cardiomyocytes with adenovirus encoding SMAD4 (AdSMAD4) and investigated the impact of SMAD4 overexpression on the development of apoptosis and hypertrophy under stimulation with phenylephrine (PE). Infection of cardiomyocytes with AdSMAD4 significantly enhanced SMAD‐binding activity while apoptosis after 24 and 36 h infection did not rise. But when SMAD4 overexpressing cardiomyocytes were incubated with PE (10 µM), the number of apoptotic cells increased (Ctrl: 94.97 ± 6.91%; PE: 102.48 ± 4.78% vs. AdSMAD4 + PE: 118.64 ± 3.28%). Furthermore expression of caspase 3 as well as bax/bcl2 ratio increased in SMAD4 overexpressing, PE‐stimulated cardiomyocytes. In addition, the effects of SMAD4 overexpression on PE‐induced hypertrophic growth were analyzed. Protein synthesis 36 h after AdSMAD4 infection was comparable to control cells, whereas the increase in protein synthesis stimulated by phyenylephrine was significantly reduced in SMAD4 overexpressing cells (134.28 ± 10.02% vs. 100.57 ± 8.86%). SMAD4 triggers the transition from hypertrophy to apoptosis in ventricular cardiomyocytes. Since SMADs are increased under several pathophysiological conditions in the heart, it can be assumed that it triggers apoptosis induction and therefore contributes to negative remodeling and heart failure progression. J. Cell. Physiol. 220: 515–523, 2009.

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.

Preconditioning with diazoxide prevents reoxygenation-induced rigor-type hypercontracture.

Ischemic preconditioning has a powerful protective potential against a reperfusion-induced injury of the post-ischemic myocardium. Cardiomyocyte hypercontracture, i.e. excessive cell shortening, is an essential mechanism of the reperfusion-induced injury. Rigor contracture, i.e. Ca(2+)-independent contracture, has been shown to be an import component of the reperfusion-induced hypercontracture. Since rigor contracture is dependent on the rapidity of the metabolic recovery during reoxygenation, we hypothesized that preconditioning of the cardiomyocyte mitochondria may improve mitochondrial function to restore the energy balance during the initial phase of reoxygenation and may thus prevent rigor contracture. For this purpose adult rat cardiomyocytes were exposed to anoxia with subsequent reoxygenation. For preconditioning, cells were pre-treated with the mitochondrial ATP-sensitive K(+) channel opener diazoxide. Pre-treatment with 100 micromol/l diazoxide significantly reduced the reoxygenation-induced hypercontracture of cardiomyocytes due to an attenuation of the Ca(2+)-independent rigor-type contracture, which was accompanied by an acceleration of the phosphocreatine resynthesis during the initial phase of reoxygenation. Treatment with the mitochondrial ATP-sensitive K(+) channel antagonist 5-hydroxydecanoate (500 micromol/l) during preconditioning phase abolished these protective effects. Similarly, partial suppression of the mitochondrial function with 100 micromol/l NaCN during the reoxygenation phase abolished the diazoxide effects. Finally, in isolated rat hearts, preconditioning with diazoxide prior to global ischemia significantly improved left ventricular function and attenuated hypercontracture during reperfusion. This effect could be abolished by the treatment with 100 micromol/l NaCN during reperfusion. Taken together, pharmacological preconditioning of cardiomyocytes with diazoxide protects against the reoxygenation-induced rigor hypercontracture due to an improvement of the energy recovery at the onset of reoxygenation.

Protection of reoxygenated cardiomyocytes against sarcolemmal fragility: the role of glutathione.

Abstract This study addressed the question of whether the sarcolemmal fragility of cardiomyocytes after anoxia and subsequent reoxygenation can be altered by modulation of the cellular glutathione state. Isolated ventricular cardiomyocytes (from adult rats) were exposed to 120 min anoxia and subsequently to 30 min reoxygenation. Osmotic stress was generated by reduction of medium osmolarity from 270 to 80 mosmol/l and sarcolemmal fragility assessed by the leakage of lactate dehydrogenase (LDH). Under normoxic conditions 6.7±1.0 % of total LDH activity was found extracellularly. Hyposmolar reoxygenation, but not hypoosmolar anoxia, increased LDH release (17.9±2.7% of total, P<0.05). Increasing cellular glutathione content by pretreatment with N-acetylcysteine (1 mM) reduced LDH release following hyposmolar reoxygenation (12.3±1.9% vs. 18.2±2.9% of LDH in medium, P<0.05). Depletion of glutathione content by pretreatment with buthionine sulphoximine (BSO, 200 µM), increased LDH release following osmotic stress already in normoxia (10.5±1.8% of LDH in medium; P<0.05 vs. no BSO), and even further after reoxygenation (21.8±3.2%, P<0.05 vs. normoxia). We conclude that the increased sarcolemmal fragility in reoxygenated cardiomyocytes is due to reoxygenation in the presence of reduced antioxidant defence.

Direct effects of the angiotensin‐converting enzyme inhibitor ramiprilat on adult rat ventricular cardiomyocytes

Aim:  Angiotensin‐converting enzyme (ACE) inhibitors like ramiprilat bind to ACE expressed on the cell surface of endothelial cells and induce cell‐specific signalling including the activation of activator protein (AP)‐1. The present study addressed the question whether ramiprilat exerts a similar effect on adult ventricular cardiomyocytes, i.e. activates the AP‐1 or modifies contractile performance. It was further aimed to decide whether such effects depend on bradykinin receptors or whether they are directly mediated via ACE.