Developmental and sex differences in cardiac tolerance to ischemia-reperfusion injury: the role of mitochondria 1.

Age and sex play an essential role in the cardiac tolerance to ischemia-reperfusion injury: cardiac resistance significantly decreases during postnatal maturation and the female heart is more tolerant than the male myocardium. It is widely accepted that mitochondrial dysfunction, and particularly mitochondrial permeability transition pore (MPTP) opening, plays a major role in determining the extent of cardiac ischemia-reperfusion injury. We have observed that the MPTP sensitivity to the calcium load differs in mitochondria isolated from neonatal and adult myocardium, as well as from adult male and female hearts. Neonatal and female mitochondria are more resistant both in the extent and in the rate of mitochondrial swelling induced by high calcium concentration. Our data further suggest that age- and sex-dependent specificity of the MPTP is not the result of different amounts of ATP synthase and cyclophilin D: neonatal and adult hearts, similarly as the male and female hearts, contain comparable amounts of MPTP and its regulatory protein cyclophilin D. We can speculate that the lower sensitivity of MPTP to the calcium-induced swelling may be related to the higher ischemic tolerance of both neonatal and female myocardium.

Cardiotoxicity of ß-mimetic catecholamines during ontogenetic development - possible risks of antenatal therapy.

Catecholamines are involved in the regulation of a wide variety of vital functions. The ß-adrenergic receptor (ß-AR) - adenylyl cyclase system has been identified early in embryogenesis before the heart has received adrenergic innervation. The structure of ß-receptors in the immature myocardium is similar to that in adults; there are, however, significant quantitative developmental changes in the inotropic and chronotropic responsiveness. Information on the toxic effect of the ß-AR agonists in the immature heart is surprisingly scarce, even though these agents are used in clinical practice both during pregnancy and in early postnatal development. Large doses of ß-AR agonists induce malformations of the cardiovascular system; the type of change depends upon the time at which the ß-AR agonist was administered during embryogenesis. During postnatal ontogeny, the cardiotoxicity of ß-AR agonists increased from birth to adulthood. It seems likely that despite interspecies differences, developmental changes in the cardiac sensitivity to ß-AR agonists may exist in all mammals, depending on the degree of maturation of the system involved in ß-adrenergic signaling. All the existing data draw attention to the possible harmful consequences of the clinical use of ß-AR agonists during early phases of cardiac development. Late effects of the early disturbances of the cardiac muscle cannot be excluded.

Sixty Years of Heart Research in the Institute of Physiology of the Czech Academy of Sciences.

In 2023, six decades have elapsed since the first experimental work on the heart muscle was published, in which a member of the Institute of Physiology of the Czech Academy of Sciences participated as an author; Professor Otakar Poupa was the founder and protagonist of this research domain. Sixty years - more than half of the century - is certainly significant enough anniversary that is worth looking back and reflecting on what was achieved during sometimes very complicated periods of life. It represents the history of an entire generation of experimental cardiologists; it is possible to learn from its successes and mistakes. The objective of this review is to succinctly illuminate the scientific trajectory of an experimental cardiological department over a 60-year span, from its inaugural publication to the present. The old truth - historia magistra vitae - is still valid. Keywords: Heart, Adaptation, Development, Hypoxia, Protection.

Epitranscriptomic Regulations in the Heart.

RNA modifications affect key stages of the RNA life cycle, including splicing, export, decay, and translation. Epitranscriptomic regulations therefore significantly influence cellular physiology and pathophysiology. Here, we selected some of the most abundant modifications and reviewed their roles in the heart and in cardiovascular diseases: N6-methyladenosine (m6A), N6,2'-O-dimethyladenosine (m6Am), N1-methyladenosine (m1A), pseudouridine (?), 5 methylcytidine (m5C), and inosine (I). Dysregulation of epitranscriptomic machinery affecting these modifications vastly changes the cardiac phenotype and is linked with many cardiovascular diseases such as myocardial infarction, cardiomyopathies, or heart failure. Thus, a deeper understanding of these epitranscriptomic changes and their regulatory mechanisms can enhance our knowledge of the molecular underpinnings of prevalent cardiac diseases, potentially paving the way for novel therapeutic strategies. Keywords: Epitranscriptomics, RNA modifications, Epigenetics, m6A, RNA, Heart.

[Endogenous opioid system as a mediator of acute and long-term adaptation to stress. Prospects for clinical use of opioid peptides].

It has been well established that opioid peptides (OPs) affect various hormonal systems. Opioids exhibit stress-limiting and gastro-protective effects in stressed animals, acting via mu- and delta-opioid receptors (OR). Peripheral mu-OR stimulation by endogenous and exogenous opioids increases cardiac tolerance to pathological consequences of stress. Enhancement ofprostacyclin synthesis, decrease of thromboxane production as well as suppression of lipid peroxidation can be directly responsible for cardioprotective effects of OPs in stressed animals. Adaptive responses are accompanied by increased OP levels in blood and tissues. Reduction of ventricular arrhythmias induced by repeated short-term immobilization stress is mediated via mu-OR stimulation by endogenous opioids, while delta-OR account for an antiarrhythmic effect of adaptation to chronic intermittent hypobaric hypoxia. The mechanism of infarct size-limiting effect of continuous normobaric hypoxia involves both mu- and delta-OR stimulation. Peptide OR agonists can be considered in future clinical practice for treatment of withdrawal syndrome, stress-related cardiac disease or myocardial injury caused by ischemia-reperfusion insult.

Repeated colonization of alpine habitats by Arabidopsis arenosa viewed through freezing resistance and ice management strategies.

Success or failure of plants to cope with freezing temperatures can critically influence plant distribution and adaptation to new habitats. Especially in alpine environments, frost is a likely major selective force driving adaptation. In Arabidopsis arenosa (L.) Lawalrée, alpine populations have evolved independently in different mountain ranges, enabling studying mechanisms of acclimation and adaptation to alpine environments. We tested for heritable, parallel differentiation in freezing resistance, cold acclimation potential and ice management strategies using eight alpine and eight foothill populations. Plants from three European mountain ranges (Niedere Tauern, Fagaraș and Tatra Mountains) were grown from seeds of tetraploid populations in four common gardens, together with diploid populations from the Tatra Mountains. Freezing resistance was assessed using controlled freezing treatments and measuring effective quantum yield of photosystem II, and ice management strategies by infrared video thermography and cryomicroscopy. The alpine ecotype had a higher cold acclimation potential than the foothill ecotype, whereby this differentiation was more pronounced in tetraploid than diploid populations. However, no ecotypic differentiation was found in one region (Fagaraș), where the ancient lineage had a different evolutionary history. Upon freezing, an ice lens within a lacuna between the palisade and spongy parenchyma tissues was formed by separation of leaf tissues, a mechanism not previously reported for herbaceous species. The dynamic adjustment of freezing resistance to temperature conditions may be particularly important in alpine environments characterized by large temperature fluctuations. Furthermore, the formation of an extracellular ice lens may be a useful strategy to avoid tissue damage during freezing.

Myocardial m6A regulators in postnatal development: effect of sex.

N6-methyladenosine (m6A) is an abundant mRNA modification affecting mRNA stability and protein expression. It is a highly dynamic process, and its outcomes during postnatal heart development are poorly understood. Here we studied m6A machinery in the left ventricular (LV) myocardium of Fisher344 male and female rats (postnatal days one to ninety; P1-P90) using Western Blot. A downward pattern of target protein levels (demethylases FTO and ALKBH5, methyltransferase METTL3, reader YTHDF2) was revealed in male and female rat LVs during postnatal development. On P1, the FTO protein level was significantly higher in male LVs compared to females.

Interstitial pressure and lung oedema in chronic hypoxia.

We evaluated how the increase in lung interstitial pressure correlates with the pulmonary vascular response to chronic hypoxia. In control and hypoxic (30 days; 10% O2) Wistar male rats, we measured: pulmonary interstitial pressure (P(ip)), cardiac and haemodynamic parameters by echocardiography, and performed lung morphometry on tissue specimens fixed in situ. In control animals, mean ± sd P(ip), air/tissue volume ratio and capillary vascularity index in the air-blood barrier were -12 ± 2.03 cmH2O, 3.9 and 0.43, respectively. After hypoxia exposure, the corresponding values of these indices in apparently normal lung regions were 2.6 ± 1.7 cmH2O, 3.6, and 0.5, respectively. In oedematous regions, the corresponding values were 12 ± 4 cmH2O, 0.4 and 0.3, respectively. Furthermore, in normal regions, the density of pre-capillary vessels (diameter ~50-200 µm) increased and their thickness/internal diameter ratio decreased, while opposite results were found in oedematous regions. Pulmonary artery pressure increased in chronic hypoxia relative to the control (39.8 ± 5.9 versus 26.2 ± 2.2 mmHg). Heterogeneity in local lung vascular response contributes to developing pulmonary hypertension in chronic hypoxia. In oedematous regions, the decrease in capillary vascularity correlated with the remarkable increase in interstitial pressure and morphometry of the pre-capillary vessels suggested an increase in vascular resistance; the opposite was true in apparently normal regions.

[Hypoxic preconditioning as novel approach to prophylaxis of ischemic and reperfusion damage of brain and heart].

Analysis of published data indicates that delayed hypoxic preconditioning essentially increases a cardiac and brain tolerance to ischemia-reperfusion. There are no experimental data in the literature on the neuroprotective effect of early hypoxic preconditioning in vivo. Clinical observations indicated that early hypoxic preconditioning exerts cardioprotective and neuroprotective effects. The single works testify that cardioprotective effect of delayed hypoxic preconditioning depend on the activation of inducible NO-synthase, KATP-channels and KCa-channels. Neuroprotective effect of hypoxic preconditioning is a consequence: (1) erythropoietin receptor stimulation and (2) an elevation of activity of PI3-Akt and ERK1/2 kinases. The supposed end effector of brain hypoxic preconditioning is MPT-pore.

Conplastic strains for identification of retrograde effects of mitochondrial dna variation on cardiometabolic traits in the spontaneously hypertensive rat.

Mitochondrial retrograde signaling is a pathway of communication from mitochondria to the nucleus. Recently, natural mitochondrial genome (mtDNA) polymorphisms (haplogroups) received increasing attention in the pathophysiology of human common diseases. However, retrograde effects of mtDNA variants on such traits are difficult to study in humans. The conplastic strains represent key animal models to elucidate regulatory roles of mtDNA haplogroups on defined nuclear genome background. To analyze the relationship between mtDNA variants and cardiometabolic traits, we derived a set of rat conplastic strains (SHR-mtBN, SHR-mtF344 and SHR-mtLEW), harboring all major mtDNA haplotypes present in common inbred strains on the nuclear background of the spontaneously hypertensive rat (SHR). The BN, F344 and LEW mtDNA differ from the SHR in multiple amino acid substitutions in protein coding genes and also in variants of tRNA and rRNA genes. Different mtDNA haplotypes were found to predispose to various sets of cardiometabolic phenotypes which provided evidence for significant retrograde effects of mtDNA in the SHR. In the future, these animals could be used to decipher individual biochemical components involved in the retrograde signaling.

Transient upregulation of protein kinase C in pressure-overloaded neonatal rat myocardium.

Protein kinase C (PKC) appears to play a significant role in the signal transduction of cardiac growth and development. The aim of this study was to determine changes in the total PKC activity and the expression of PKC isoforms alpha, delta and epsilon in the rat heart that was affected by pressure overload imposed at postnatal day (d) 2. Three groups of Wistar rats were employed for the experiment: rats submitted to the abdominal aortic constriction (AC), sham-operated controls (SO) and intact controls. Animals were sacrificed at d2, d3, d5 and d10. The total PKC activity was measured by the incorporation of (32)P into histone IIIS and the expression of PKC was analyzed by immunoblotting in the homogenate of the left ventricular myocardium and in the cytosolic, membrane-enriched (10(5) g) and nuclear-cytoskeletal-myofilament-enriched (10(3) g) fractions. We observed the significant transient increase in both the total PKC activity and the expression of all isoforms at d5 (the third day after the operation) in the cardiac homogenate of AC rats as compared with SO animals. Aortic constriction did not significantly affect the distribution of activity and isoform abundance among individual cellular fractions except for PKCdelta, which increased significantly at d10 in the cytosolic fraction at the expense of the membrane-enriched fraction. It is concluded that PKCalpha, PKCdelta and PKCepsilon undergo transient upregulation associated with the accelerated cardiac growth induced by pressure overload imposed in the very early postnatal period.

Gradual cold acclimation induces cardioprotection without affecting ß-adrenergic receptor-mediated adenylyl cyclase signaling.

Novel strategies are needed that can stimulate endogenous signaling pathways to protect the heart from myocardial infarction. The present study tested the hypothesis that appropriate regimen of cold acclimation (CA) may provide a promising approach for improving myocardial resistance to ischemia/reperfusion (I/R) injury without negative side effects. We evaluated myocardial I/R injury, mitochondrial swelling, and ß-adrenergic receptor (ß-AR)-adenylyl cyclase-mediated signaling. Male Wistar rats were exposed to CA (8°C, 8 h/day for a week, followed by 4 wk at 8°C for 24 h/day), while the recovery group (CAR) was kept at 24°C for an additional 2 wk. The myocardial infarction induced by coronary occlusion for 20 min followed by 3-h reperfusion was reduced from 56% in controls to 30% and 23% after CA and CAR, respectively. In line, the rate of mitochondrial swelling at 200 µM Ca2+ was decreased in both groups. Acute administration of metoprolol decreased infarction in control group and did not affect the CA-elicited cardiprotection. Accordingly, neither ß1-AR-Gsα-adenylyl cyclase signaling, stimulated with specific ligands, nor p-PKA/PKA ratios were affected after CA or CAR. Importantly, Western blot and immunofluorescence analyses revealed ß2- and ß3-AR protein enrichment in membranes in both experimental groups. We conclude that gradual cold acclimation results in a persisting increase of myocardial resistance to I/R injury without hypertension and hypertrophy. The cardioprotective phenotype is associated with unaltered adenylyl cyclase signaling and increased mitochondrial resistance to Ca2+-overload. The potential role of upregulated ß2/ß3-AR pathways remains to be elucidated.NEW & NOTEWORTHY We present a new model of mild gradual cold acclimation increasing tolerance to myocardial ischemia/reperfusion injury without hypertension and hypertrophy. Cardioprotective phenotype is accompanied by unaltered adenylyl cyclase signaling and increased mitochondrial resistance to Ca2+-overload. The potential role of upregulated ß2/ß3-adrenoreceptor activation is considered. These findings may stimulate the development of novel preventive and therapeutic strategies against myocardial ischemia/reperfusion injury.

Ontogenetic development of cardiac tolerance to oxygen deprivation - possible mechanisms.

Our present focus on the hypoxic immature heart is driven by clinical urgency: cyanotic congenital cardiac malformations remain the single largest cause of mortality from congenital defects and ischemic heart disease is no more the disease of the fifth and older decades but its origin as well as risk factors are present already during early ontogeny. Moreover, the number of adult patients operated for cyanotic congenital heart disease during infancy steadily increases. This group approaches the age of the rising risk of serious cardiovascular diseases, particularly ischemic heart disease. Experimental results have clearly shown that the immature heart is significantly more tolerant to oxygen deficiency than the adult myocardium. However, the mechanisms of this difference have not yet been satisfactorily clarified; they are likely the result of developmental changes in cardiac energy metabolism, including mitochondrial function. The high resistance of the newborn heart cannot be further increased by ischemic preconditioning or adaptation to chronic hypoxia; these protective mechanisms appear only with decreasing tolerance during development. Resistance of the adult myocardium to acute oxygen deprivation may be significantly influenced by perinatal hypoxia. These results suggest that the developmental approach offers new possibilities in the studies of pathogenesis, prevention and therapy of critical cardiovascular diseases.

Reduced susceptibility to ischemia-induced arrhythmias in the preconditioned rat heart is independent of PI3-kinase/Akt.

We examined the involvement of phosphatidylinositol 3-kinase (PI3K) and its effector protein kinase B (Akt) in cardioprotective effects of ischemic preconditioning (PC) with particular regards to its role in the protection against ischemia-induced arrhythmias in isolated perfused rat heart. PI3K/Akt inhibitor wortmannin (100 nM) was administered 15 min prior to 30-min regional (left anterior descending coronary artery occlusion) ischemia for the study of ischemic arrhythmias in the hearts perfused at constant coronary flow or prior to 30-min global ischemia followed by 2-h reperfusion for the infarct size (IS) determination (tetrazolium staining) in the hearts perfused at constant pressure. PC procedure (one cycle of ischemia/reperfusion, 5 min each) significantly reduced the total number of ventricular premature complexes (PVC) and severity of arrhythmias (arrhythmia score; AS) over the whole period of left anterior descending coronary artery occlusion in comparison with non-PC controls (PVC 166+/-40; AS 1.6+/-0.2 vs. 550+/-60 and 3.2+/-0.2; respectively; P<0.05). In a setting of global ischemia/reperfusion, PC decreased IS (in % of the left ventricle, LV) by 73 %. Pretreatment with wortmannin modified neither arrhythmogenesis nor IS in the non-PC hearts. Bracketing of PC with wortmannin did not abolish antiarrhythmic protection (PVC 92+/-25; AS 1.7+/-0.2; P<0.05 vs. non-PC hearts). On the other hand, wortmannin increased IS/LV in the PC hearts to 24+/-1.2 % as compared with 9 +/- 0.6 % in the untreated ones (P<0.05). In conclusion, PI3K/Akt inhibition did not affect reduced arrhythmogenesis during ischemia in the PC hearts indicating that in contrast to its positive role in the irreversible myocardial injury, PI3K/Akt activity is not required for protection induced by PC against ischemic arrhythmias in the rat heart.

Myocardial phospholipid remodeling under different types of load imposed during early postnatal development.

Normal increase in hemodynamic load during early postnatal life is associated with heart growth and maturation of membrane structures that is accompanied by remodeling of membrane protein and lipid components. This review describes remodeling of phospholipids (PL) in rat myocardium during normal postnatal development and during accelerated cardiac growth induced by additional workload (aorta constriction, chronic hypoxia and hyperthyroidism) imposed on the heart early after birth. Normal physiological load after birth stimulates the development of membrane structures and synthesis of PL. While hyperthyroidism accelerates these processes, pressure overload has an inhibitory effect. These changes primarily influence the maturation of mitochondrial membranes as cardiolipin is one of the most affected PL species. The most sensitive part of PL structure in their remodeling process are PL acyl chains, particularly polyunsaturated fatty acids that are the key components determining the basic physicochemical properties of the membrane bilayer and thus the function of membrane-bound proteins and membrane-derived signaling lipid molecules. It is evident that PL remodeling may significantly influence both normal and pathological postnatal development of myocardium.

[Remote ischemic preconditioning].

It has been systematized modern data on the preconditioning at a distance and a tolerance of a heart, a brain and other organs to ischemia-reperfusion injury. It has been performed an analysis of the published works on the role of autonomic nervous system and humoral factors in the development of resistance to pathogenic action of ischemia and reperfusion. It has been shown that adenosine, bradykinin, opioid peptides, calcitonin gene-related peptide and also signaling cascade involved nitric oxide, free radicals, protein kinases, mitochondrial ATP-sensitive K+ -channel, mitochondrial permeability transition pore play an important role in the mechanism of cardioprotective action of remote preconditioning.

Participation of opioid receptors in the cytoprotective effect of chronic normobaric hypoxia.

We studied the role of the delta, micro, and kappa opioid receptor (OR) subtypes in the cardioprotective effect of chronic continuous normobaric hypoxia (CNH) in the model of acute anoxia-reoxygenation of isolated cardiomyocytes. Adaptation of rats to CNH was performed by their exposure to atmosphere containing 12 % of O(2) for 21 days. Anoxia-reoxygenation of cardiomyocytes isolated from normoxic control rats caused the death of 51 % of cells and lactate dehydrogenase (LDH) release. Adaptation of rats to CNH resulted in the anoxia/reoxygenation-induced cardiomyocyte death of only 38 %, and reduced the LDH release by 25 %. Pre-incubation of the cells with either the non-selective OR (opioid receptor) blocker naloxone (300 nM/l), the delta OR antagonist TIPP(psi) (30 nM/l), the selective delta(2) OR antagonist naltriben (1 nM/l) or the micro OR antagonist CTAP (100 nM/l) for 25 minutes before anoxia abolished the reduction of cell death and LDH release afforded by CNH. The antagonist of delta(1) OR BNTX (1 nM/l) or the kappa OR antagonist nor-binaltorphimine (3 nM/l) did not influence the cytoprotective effects of CNH. Taken together, the cytoprotective effect of CNH is associated with the activation of the delta(2) and micro OR localized on cardiomyocytes.

Acute but not chronic tempol treatment increases ischemic and reperfusion ventricular arrhythmias in open-chest rats.

The effect of the chronic and acute antioxidant tempol (superoxide dismutase mimetic) treatment on cardiac ischemic tolerance was investigated in adult male Wistar rats. The first experimental group was given tempol (1 mM) in drinking water for three weeks, the second group received tempol (100 mg/kg, i.v.) 10 min before test ischemia, and control rats received the same volume of solvent. Anesthetized open-chest animals (pentobarbitone 60 mg/kg, i.p.) were subjected to 20-min coronary artery occlusion and 3-h reperfusion for infarct size determination. Ventricular arrhythmias were monitored during ischemia and at the beginning (5 min) of reperfusion. Acute tempol administration shifted the time profile of ischemic arrhythmias to the later phase and significantly increased the number of ischemic and reperfusion premature ventricular complexes, respectively (504 +/- 127 and 84 +/- 21) as compared with the chronically treated group (218 +/- 36 and 47 +/- 7) or controls (197 +/- 26 and 31 +/- 7). Acute tempol-treated rats exhibited a tendency to decrease infarct size (P = 0.087). The mechanism of proarrhythmic tempol action during ischemia and reperfusion remains to be elucidated.

Effect of acute and chronic simvastatin treatment on post-ischemic contractile dysfunction in isolated rat heart.

Statins are powerful lipid-lowering drugs, widely used in patients with hyperlipidemia and coronary artery disease. It was found, however, that statins appear to have a pleiotropic effect beyond their lipid-lowering ability. They exert anti-inflammatory, antithrombotic and antioxidant effects, increase nitric oxide production and improve endothelial dysfunction. The aim of our study was to examine the effect of chronic and acute treatment with simvastatin on the contractile function of the isolated perfused rat heart after ischemia/reperfusion injury. Contractile function was measured on isolated rat hearts, perfused according to Langendorff under constant pressure. The hearts were subjected to 20 min of global ischemia, followed by 40 min of reperfusion. To investigate the acute effect, simvastatin at a concentration of 10 micromol/l was added to the perfusion solution during reperfusion. In chronic experiments the rats were fed simvastatin at a concentration of 10 mg/kg for two weeks before the measurement of the contractile function. Acute simvastatin administration significantly increased reparation of the peak of pressure development [(+dP/dt)(max)] (52.9+/-8.2 %) after global ischemia, as compared with the control group (28.8+/-5.2 %). Similar differences were also observed in the time course of the recovery of [(+dP/dt)(max)]. Chronic simvastatin was without any protective effect. Our results reveal that the acute administration of simvastatin during reperfusion, unlike the chronic treatment, significantly reduced contractile dysfunction induced by ischemia/reperfusion injury. This supports the idea of possible cardioprotective effect of statin administration in the first-line therapy of the acute coronary syndrome.

Cardiac adaptation to chronic high-altitude hypoxia: beneficial and adverse effects.

This review deals with the capability of the heart to adapt to chronic hypoxia in animals exposed to either natural or simulated high altitude. From the broad spectrum of related issues, we focused on the development and reversibility of both beneficial and adverse adaptive myocardial changes. Particular attention was paid to cardioprotective effects of adaptation to chronic high-altitude hypoxia and their molecular mechanisms. Moreover, interspecies and age differences in the cardiac sensitivity to hypoxia-induced effects in various experimental models were emphasized.