We aimed to study the impact of altered thyroid status on myocardial expression of electrical coupling protein connexin-43 (Cx43), the susceptibility of rats to ventricular fibrillation (VF) and the effects of antioxidant-rich red palm oil (RPO). Adult male and female euthyroid, hyperthyroid (treated with T/T), hypothyroid (treated with methimazole) Wistar rats supplemented or not with RPO for 6 weeks were used. Function of isolated perfused heart and VF threshold were determined. Left ventricular tissue was used for assessment of mRNA and protein levels of Cx43, its phosphorylated forms and topology. Protein kinase C signaling (PKC) and gene transcripts of some proteins related to cardiac arrhythmias were assessed. Hyperthyroid state resulted in decrease of total and phosphorylated forms of Cx43 and suppression of PKC-ε expression in males and females, decrease of Cx43 mRNA in females, decrease of VF threshold and increase of functional parameters in male rat hearts. In contrast, hypothyroid status resulted in the increase of total and phosphorylated forms of Cx43, enhancement PKC-ε expression in males and females, increase of Cx43 mRNA in females, increase of VF threshold and decrease of functional parameters in male rat hearts. Function of the heart was partially normalized by RPO intake, which also enhanced myocardial Cx43 and PKC-ε expression as well as increased VF threshold in hyperthyroid male rats. We conclude that there is an inverse relationship between myocardial expression of Cx43, including its functional phosphorylated forms, and susceptibility of male rat hearts to VF in condition of altered thyroid status. RPO intake partly ameliorated adverse changes caused by excess of thyroid hormones.

Background: Creatine kinase (CK) and hexokinase (HK) play a key role in myocardial energy homeostasis. We aimed to determine CK and HK expression and enzyme activity in the left (LV) and right (RV) ventricles of rats adapted for 3 weeks to normobaric hypoxia (10 % O₂) either continuously (CNH) or intermittently with 1-h or 16-h normoxic episode per day. Methods: The Real-Time RT-PCR, Western blot, and enzyme-coupled assays were used. In addition, the effect of CNH on the HK co-localization with mitochondria, which can inhibit apoptosis, was assessed using immunofluorescence techniques. Results: CK and HK activities increased in the LV during all hypoxic adaptations, which was consistent with elevated protein levels of mitochondrial mtCKs, cytosolic CKB, HK1, and HK2 isoforms. Enzyme activities also increased in the hypoxic RV, but only CKB protein was elevated. No effect of CNH on HK1 or HK2 co-localization with mitochondria was observed. Conclusion: Up-regulation of mtCKs and HK isoforms may stimulate the respiratory chain and help to maintain energy homeostasis of chronically hypoxic myocardium and prevent oxidative stress. In this way, CK and HK enzymes can possibly participate in the establishment of ischemia-resistant phenotype of chronically hypoxic hearts.

Cardioprotective and nonprotective regimens of chronic hypoxia diversely affect the myocardial antioxidant systems. Physiol Genomics 47: 612-620, 2015. First published October 13, 2015; doi:10.1152/physiolgenomics.00058.2015.-It has been documented that adaptation to hypoxia increases myocardial tolerance to ischemia-reperfusion (I/R) injury depending on the regimen of adaptation. Reactive oxygen species (ROS) formed during hypoxia play an important role in the induction of protective cardiac phenotype. On the other hand, the excess of ROS can contribute to tissue damage caused by I/R. Here we investigated the relationship between myocardial tolerance to I/R injury and transcription activity of major antioxidant genes, transcription factors, and oxidative stress in three different regimens of chronic hypoxia. Adult male Wistar rats were exposed to continuous normobaric hypoxia (FIO 2 0.1) either continuously (CNH) or intermittently for 8 h/day (INH8) or 23 h/day (INH23) for 3 wk period. A control group was kept in room air. Myocardial infarct size was assessed in anesthetized open-chest animals subjected to 20 min coronary artery occlusion and 3 h reperfusion. Levels of mRNA transcripts and the ratio of reduced and oxidized glutathione (GSH/GSSG) were analyzed by real-time RT-PCR and by liquid chromatography, respectively. Whereas CNH as well as INH8 decreased infarct size, 1 h daily reoxygenation (INH23) abolished the cardioprotective effect and decreased GSH/GSSG ratio. The majority of mRNAs of antioxidant genes related to mitochondrial antioxidant defense (manganese superoxide dismutase, glutathione reductase, thioredoxin/thioredoxin reductase, and peroxiredoxin 2) were upregulated in both cardioprotective regimens (CNH, INH8). In contrast, INH23 increased only PRX5, which was not sufficient to induce the cardioprotective phenotype. Our results suggest that the increased mitochondrial antioxidant defense plays an important role in cardioprotection afforded by chronic hypoxia. adaptation to hypoxia; cardioprotection; ischemia-reperfusion injury; oxidative stress; antioxidant defense ADAPTATION TO CHRONIC HYPOXIA may improve cardiac tolerance to acute ischemia-reperfusion (I/R) injury under certain conditions. Whereas chronic continuous hypoxia induces a protective cardiac phenotype, a brief daily interruption of hypoxic adaptation for 1 h eliminates the protective effect (31). Tissue damage caused by I/R insult is tightly related to the excess of reactive oxygen species (ROS), but the exact molecular mechanism underlying these adverse effects is still not fully elucidated. Intracellular ROS initiate a sequence of oxidation reactions that can seriously damage cell structures. ROS differ in their reactivity, as well as in mechanisms and sites of their production. These harmful species can be eliminated by specific scavengers. If production of ROS exceeds the capacity limit of antioxidant systems or if the systemic capacity is insufficient, the level of ROS in individual cell compartments increases and generates ...

Chronic hypoxia increases the myocardial resistance to acute ischemia-reperfusion injury by affecting the mitochondrial redox balance. Hexokinase (HK) bears a high potential to suppress the excessive formation of reactive oxygen species because of its increased association with mitochondria, thereby inhibiting the membrane permeability transition pore opening and preventing cell death. The purpose of this study was to determine the effect of severe intermittent hypobaric hypoxia (7,000 m, 8 h/day, 5 wk) on the function and colocalization of HK isoforms with mitochondria in the left (LV) and right ventricles of rat myocardium. The real-time RT-PCR, Western blot, enzyme coupled assay, and quantitative immunofluorescence techniques were used. Our results showed significantly elevated expression of HK isoforms (HK1 and HK2) in the hypoxic LV. In addition, intermittent hypoxia increased the total HK activity and the association of HK isoforms with mitochondria in both ventricles. These findings suggest that HK may contribute to the cardioprotective phenotype induced by adaptation to severe intermittent hypobaric hypoxia.

Background/Aims: Hexokinase (HK) is a key glycolytic enzyme which promotes the maintenance of glucose homeostasis in cardiomyocytes. HK1 isoform is predominantly bound to the outer mitochondrial membrane and highly supports oxidative phosphorylation by increasing the availability of ADP for complex V of the respiratory chain. HK2 isoform is under physiological conditions predominantly localized in the cytosol and upon stimulation of PI3K/ Akt pathway associates with mitochondria and thus can prevent apoptosis. The purpose of this study was to investigate expression and subcellular localization of both HK isoforms in left (LV) and right (RV) heart ventricles of adult male Wistar rats. Methods: Real-Time RT-PCR, Western blotting, and quantitative immunofluorescence microscopy were used. Results: Our results showed a significantly higher expression of both HK1 and HK2 at mRNA and protein levels in the RV compared to the LV. These findings were corroborated by immunofluorescence staining which revealed substantially higher fluorescence signals of both HKs in the RV than in the LV. The ratios of phospho-Ser473-Akt/non-phospho-Akt and phospho-Thr308-Akt/non-phospho-Akt were also markedly higher in the RV than in the LV. Conclusion: These results suggest that the RV has a higher activity of aerobic glycolytic metabolism and may be able to respond faster and more powerfully to stressful stimuli than the LV.

Adaptation to chronic hypoxia represents a potential cardioprotective intervention reducing the extent of acute ischemia/reperfusion (I/R) injury, which is a major cause of death worldwide. The main objective of this study was to investigate the anti-apoptotic Akt/hexokinase 2 (HK2) pathway in hypoxic hearts subjected to I/R insult. Hearts isolated from male Wistar rats exposed either to continuous normobaric hypoxia (CNH; 10% O) or to room air for 3 weeks were perfused according to Langendorff and subjected to 10 min of no-flow ischemia and 10 min of reperfusion. The hearts were collected either after ischemia or after reperfusion and used for protein analyses and quantitative fluorescence microscopy. The CNH resulted in increased levels of HK1 and HK2 proteins and the total HK activity after ischemia compared to corresponding normoxic group. Similarly, CNH hearts exhibited increased ischemic level of Akt protein phosphorylated on Ser. The CNH also strengthened the interaction of HK2 with mitochondria and prevented downregulation of mitochondrial creatine kinase after reperfusion. The Bax/Bcl-2 ratio was significantly lower after I/R in CNH hearts than in normoxic ones, suggesting a lower probability of apoptosis. In conclusion, the Akt/HK2 pathway is likely to play a role in the development of a cardioprotective phenotype of CNH by preventing the detachment of HK2 from mitochondria at reperfusion period and decreases the Bax/Bcl-2 ratio during I/R insult, thereby lowering the probability of apoptosis activation in the mitochondrial compartment.

Our study suggests that APOE may not be associated with spontaneous abortions in Caucasians.

Chronic hypoxia may produce a cardioprotective phenotype characterized by increased resistance to ischemia-reperfusion injury. Nevertheless, the molecular basis of cardioprotective effects of hypoxia is still not quite clear. The present study investigated the consequences of a 3-week adaptation to cardioprotective (CNH, continuous normobaric hypoxia) and nonprotective (INH, intermittent normobaric hypoxia; 23 h/day hypoxia followed by 1 h/day reoxygenation) regimen of hypoxia on β-adrenergic signaling in the rat myocardium. Both regimens of hypoxia lowered body weight and led to marked right ventricular (RV) hypertrophy, which was accompanied by 25% loss of β1-adrenergic receptors (β1-ARs) in the RV. No significant changes were found in β-ARs in left ventricular (LV) preparations from animals adapted to hypoxia. Although adenylyl cyclase (AC) activity stimulated through the G proteins was decreased in the RV and increased in the LV after exposure to hypoxia, there were no significant changes in the expression of the dominant myocardial AC 5/6 isoforms and the stimulatory G proteins. These data suggest that chronic normobaric hypoxia may strongly affect myocardial β-adrenergic signaling but adaptation to cardioprotective and nonprotective regimens of hypoxia does not cause notably diverse changes.