Frantisek Kolar

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function.

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

Reciprocal changes in the postnatal expression of the sarcolemmal Na+-Ca2+-exchanger and SERCA2 in rat heart

The aim of this study was to examine the relationship between sarcolemmal Na(+)-Ca2+ exchangers and sarcoplasmic reticulum (SR) Ca(2+) -ATPase (SERCA2) expression and the developmental differences in cardiac Ca2+ handling. Postnatal steady-state mRNA and protein levels were analysed in rat ventricular myocardium by Northern and immunoblot analysis, respectively. This was compared to Na+ gradient-induced and SR oxalate-supported Ca2 transport in isolated membranes. Na(+)-Ca2+ exchanger mRNA declined by 75% between day 1 and 30, whereas SR Ca2+ ATPase mRNA levels increased by 97% during this period. The Na(+)-Ca2+ exchanger mRNA/Ca(2+)-ATPase mRNA ratio was found to be inversely related to post-natal age. The changes in mRNA levels were associated with corresponding developmental differences in the Ca2+ transport activities of the respective membrane proteins. In crude membranes, the Na(+)-dependent Ca2+ transport activity (at 75 microM Ca2+) declined gradually (P < 0.01; mean +/- S.E.) from 17.7 +/- 2.4 nmoles Ca2+/g wet tissue/2s at day 1-3 (n = 5) to a value of 4.2 +/- 1.1 at day 40 (n =4). Conversely, SR Ca2+ uptake increased (P < 0.01) 2.6-fold during this period. The inversely related changes in the post-natal expression and function of the Na(+)-Ca2+ exchanger and SR Ca(2+)-ATPase suggest a coordinated control at the pretranslational level of the cellular Ca2+ transport processes mediated by the two membrane proteins.

Effects of melatonin on ischemia and reperfusion injury of the rat heart.

Effects of melatonin on various manifestations of ischemia/reperfusion injury of the isolated perfused rat heart were examined. Ischemia- and reperfusion-induced ventricular arrhythmias were studied under constant flow in hearts subjected to 10, 15 or 25 min of regional ischemia (induced by LAD coronary artery occlusion) and 10-min reperfusion. Melatonin was added to the perfusion medium 5 min before ischemia at concentrations of 10 μmol/l or 10 nmol/l and was present throughout the experiment. Recovery of the contractile function was evaluated under constant perfusion pressure after 20-min global ischemia followed by 40-min reperfusion. Hearts were treated with melatonin at a high concentration (10 μmol/l) either 5 min before ischemia only (M1) or 5 min before ischemia and during reperfusion (M2) or only during reperfusion (M3). At the high concentration, melatonin significantly reduced the incidence of reperfusion-induced ventricular fibrillation and decreased arrhythmia score (10% and 2.2 ± 0.3, respectively) as compared with the corresponding untreated group (62% and 4.1 ± 0.3, respectively); the low concentration had no effect. This substance did not affect the incidence and severity of ischemic arrhythmias. Melatonin (M2, M3) significantly improved the recovery of the contractile function as compared with the untreated group; this protection did not appear if melatonin was absent in the medium during reperfusion (M1). Our results show that melatonin, in accordance with its potent antioxidant properties, effectively protects the rat heart against injury associated with reperfusion. It appears unlikely that melatonin is cardioprotective at physiological concentrations.

Ischemic preconditioning in chronically hypoxic neonatal rat heart.

Rat hearts isolated on d 1, 4, 7, and 10 of postnatal life were perfused (in Langendorff mode) with Krebs-Henseleit solution at constant pressure, temperature, and stimulation rate. Recovery of the contractile function after global ischemia was measured by an isometric force transducer and analyzed using an online computer. Ischemic preconditioning (IP) was induced by three 3-min periods of global ischemia, each separated by a 5-min period of reperfusion. Prenatal hypoxia was induced by exposure of pregnant mothers to intermittent high altitude (IHA), simulated in a barochamber (8 h/d, 5000 m) from d 15 to 20 of pregnancy. Postnatal hypoxia was simulated by the identical procedure from postnatal d 1 to 6 and 9. Prenatal exposure to IHA failed to improve ischemic tolerance on d 1, but postnatal exposure to IHA improved recovery of the developed force after ischemia on d 7 (33 ± 3%versus 43 ± 4%) and 10 (39 ± 2%versus 54 ± 2%). Combination of IHA and IP induced higher protective effects in all age groups, including postnatal d 1 (48 ± 2%versus 56 ± 3%), whereas IHA and IP applied separately failed to improve ischemic tolerance. Neither the mitochondrial KATP channel blocker 5-hydroxydecanoate nor the nitric oxide synthase inhibitor Nω-nitro-l-arginine methyl ester abolished protection by IP in normoxic animals, but they decreased significantly protection by IHA hypoxia. The final recovery was even lower than the corresponding normoxic values. It seems likely that mitochondrial KATP channels and nitric oxide may be involved in the protective mechanisms of adaptation to chronic hypoxia but not to that of IP, at least in neonates.

Ventricular arrhythmias following coronary artery occlusion in rats: is the diabetic heart less or more sensitive to ischaemia?

Abstract Rhythm disorders are common complications in diabetic patients, due to their enhanced sensitivity to ischaemia. However, experimental studies are inconsistent, and both higher and lower vulnerability to injury has been reported. Our objectives were to compare susceptibility to ventricular arrhythmias in rats with prolonged duration of diabetes induced hy streptozotocin (45 mg/kg, i. v.), utilising two different models. Following 8 weeks, either anaestetised open-chest rats in vivo or isolated Langendorff-perfused hearts were subjected to 30 min regional zero-flow ischaemia induced by occlusion of LAD coronary artery. In addition, cardiac glycogenolysis and lactate production were measured. In open-chest rats, 90% of the controls exhibited ventricular tachycardia (VT) which represented 55.4% of total arrhythmias, whereby only 19.9% of arrhythmias occurred as VT in 44% of the diabetic rats (P < 0.05 vs controls). Duration of VT and ventricular fibrillation (VF) was reduced from 35.5 ± 11.1 and 224.8 ± 153.9 s in the controls to 4.8 ± 2.5 and 2.2 ± 0.2 s in the diabetics, respectively (P < 0.05). Accordingly, severity of arrhythmias (arrhythmia score, AS) was also lower in the diabetics (2.0 ± 0.38 vs 3.3 ± 0.3 in the controls; P < 0.05). In the isolated hearts, high incidence of VF was decreased in the diabetic hearts, and although VT occurred in almost all of the diabetic hearts, the duration of VT and VF was substantially shorter (61.5 ± 14.5 and 5.5 ± 0.5 s vs 221.5 ± 37 and 398.5 ± 55 s in the controls, respectively; P < 0.05). AS was reduced to 2.9 ± 0.12 from 4.1 ± 0.3 in the controls (P < 0.05). Postischaemic accumulation of lactate was lower in the diabetic than in the non-diabetic myocardium (20.4 ± 1.9 vs 29.5 ± 2.9 μmol/l/g w.wt.; P < 0.05). These results suggest that rat hearts with chronic diabetes, despite some differences in the arrhythmia profiles between the in vivo model and isolated heart preparation, are less sensitive to ischaemic injury and exhibit lower susceptibility to ventricular arrhythmias and reduced accumulation of glycolytic metabolites.

Effect of increased pressure loading on heart growth in neonatal rats.

During embryonic and fetal development, the ventricular myocardium increases its mass principally by adding new cells (hyperplasia), while postnatally, it does so mainly through increase of cell size (hypertrophy). Switching between these two mechanisms of adaptation to increasing functional demand occurs in the early neonatal period. We investigated the response of the neonatal rat left ventricle to pressure overload induced by constriction of the abdominal aorta at postnatal day 2. Sampling for morphological examination with measurements of ventricular wall thickness and myocyte width was performed at days 2, 3, 5, 10, and 21. 3H-thymidine pre-labeling with label dilution was used to assess proliferative history at day 21, and bromodeoxyuridine labeling was used to measure the rates of DNA synthesis at each time point. The left ventricular wall was significantly thicker than in controls in the AC group from day 3, while thickness of individual myocytes was not increased until day 10. Label dilution showed evidence of higher number of cellular divisions correlating with severity of the phenotype in the AC group at day 21. Terminal DNA synthesis index was increased significantly at day 3, but there was no significant difference from controls at days 5, 10, or 21. Apoptotic rates were not different from controls at any sampling interval. Together, these results suggest that adaptation of the neonatal myocardium to increased pressure load is rapid, and is based on transitory hyperplasia followed by hypertrophy of myocytes.

Chronic Intermittent Hypoxia Induces 11β-Hydroxysteroid Dehydrogenase in Rat Heart

Corticosteroids are known to not only regulate electrolyte homeostasis but also play a role in the cardiovascular system, including myocardial remodeling. Because transgenic mice that overexpress 11beta-hydroxysteroid dehydrogenase (11HSD) type 2 in cardiomyocytes have been shown to spontaneously develop cardiac hypertrophy and fibrosis, we investigated whether changes in the cardiac metabolism of glucocorticoids accompany remodeling of the heart under physiological conditions. In the present study, glucocorticoid metabolism and 11HSD2 were explored in the hearts of rats exposed to chronic intermittent hypobaric hypoxia (CIH), which induces hypertrophy and fibrosis of the right and less of the left ventricle. We first demonstrated that adaptation to CIH led to a significant increase in 11HSD2 transcript levels and activity in the myocardium. In contrast, neither 11HSD1 activity and mRNA level nor the abundance of mineralocorticoid and glucocorticoid receptor mRNA were up-regulated. The adaptation to CIH also led to an increase of 11HSD2 mRNA in isolated cardiomyocytes, whereas 11HSD1, glucocorticoid receptor, and mineralocorticoid receptor mRNA levels were not changed in comparison with the cardiomyocytes of control normoxic rats. The changes in cardiac metabolism of glucocorticoids were accompanied by inflammatory responses. The expression levels of the proinflammatory markers cyclooxygenase-2 and osteopontin were significantly increased in both the myocardium and the cardiomyocytes isolated from rats exposed to CIH. These findings suggest that myocardial remodeling induced by CIH is associated with the up-regulation of cardiac 11HSD2. Consequently, local metabolism of glucocorticoids could indeed play a role in cardiac hypertrophy and fibrosis.

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.

Role of endogenous opioid peptides in the infarct size-limiting effect of adaptation to chronic continuous hypoxia.

AIMS The objective of this study was to examine the involvement of endogenous opioid peptides and opioid receptor (OR) subtypes in the cardioprotective effect of adaptation to chronic hypoxia in rats. MAIN METHODS Rats were exposed to continuous normobaric hypoxia (CNH; 12% oxygen) for 3 weeks. Myocardial ischemia was induced by 20-min coronary artery occlusion followed by 3-h reperfusion in anesthetized open-chest animals. Various OR antagonists were administered to rats prior to ischemia. The size of myocardial infarction and the incidence of ischemic ventricular arrhythmias were assessed. Myocardial and plasma concentrations of opioid peptides (met-enkephalin, β-endorphin, and endomorphins) were determined. KEY FINDINGS Adaptation to CNH significantly increased myocardial and plasma concentrations of opioids, potentiated their further elevation by ischemia/reperfusion, and reduced myocardial infarct size, but it did not affect the incidence of ischemic arrhythmias. The infarct size-limiting effect of CNH was abolished by OR antagonists naltrexone (non-selective), naloxone methiodide (non-selective peripherally acting), TIPP[ψ] (δ-OR), naltriben (δ2-OR), or CTAP (μ-OR), while BNTX (δ1-OR) and nor-binaltorphimine (κ-OR) had no effect. SIGNIFICANCE The results suggest that the infarct size-limiting effect afforded by adaptation to CNH is mediated by activation of peripheral δ2- and μ-ORs by elevated levels of endogenous opioid peptides.

Mitochondrial KATP opening confers protection against lethal myocardial injury and ischaemia-induced arrhythmias in the rat heart via PI3K/Akt-dependent and -independent mechanisms.

Opening of mitochondrial KATP channels (mitoKATP) has been reported to underlie protection against ischaemia-reperfusion injury induced by ischaemic preconditioning (I-PC); however, the molecular mechanisms of its antiarrhythmic effect have not been fully elucidated. We explored the involvement of phosphatidylinositol 3-kinase (PI3K)/Akt in the PC-like effect of mitoKATP opener diazoxide with particular regard to its role in protection against ischaemia-induced arrhythmias. Langendorff-perfused rat hearts were subjected to 30 min LAD occlusion with or without a prior 15 min of perfusion with diazoxide (50 micromol/L) given either alone (D-PC) or in combination with the PI3K/Akt inhibitor wortmannin (100 nmol/L). In an additional protocol, ischaemia was followed by 2 h reperfusion for infarct size (IS) determination (tetrazolium staining). The total number of premature ventricular complexes over the whole period of ischaemia, episodes of ventricular tachycardia and its duration were significantly lower in the D-PC group than in the non-preconditioned controls (158 +/- 20, 2 +/- 0.6 and 4.6 +/- 1.8 s vs. 551 +/- 61, 11 +/- 2 and 42 +/- 8 s, respectively; p < 0.05), concomitant with a 62% reduction in the size of infarction. Wortmannin modified neither arrhythmogenesis nor IS in the non-preconditioned hearts. Bracketing of diazoxide with wortmannin did not reverse the antiarrhythmic protection, whereas the IS-limiting effect was blunted. The results indicate that in contrast with the positive role of PI3K/Akt in protection against lethal myocardial injury, its activity is not involved in suppression of ischaemia-induced arrhythmias conferred by mitoKATP opening in the rat heart.