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 β-adrenergic receptor (β-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 β1-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 β2- and β3-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 β2/β3-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 β2/β3-adrenoreceptor activation is considered. These findings may stimulate the development of novel preventive and therapeutic strategies against myocardial ischemia/reperfusion injury.

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: 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.

Remodeling of the cellular distribution of gap junctions formed mainly by connexin-43 (Cx43) can be related to the increased incidence of cardiac arrhythmias. It has been shown that adaptation to chronic intermittent hypobaric hypoxia (IHH) attenuates the incidence and severity of ischemic and reperfusion ventricular arrhythmias and increases the proportion of anti-arrhythmic n-3 polyunsaturated fatty acids (n−3 PUFA) in heart phospholipids. Wistar rats were exposed to simulated IHH (7,000 m, 8-h/day, 35 exposures) and compared with normoxic controls (N). Cx43 expression, phosphorylation, localization and n−3 PUFA proportion were analyzed in left ventricular myocardium. Compared to N, IHH led to higher expression of total Cx43, its variant phosphorylated at Ser368 [p-Cx43(Ser368)], which maintains “end to end” communication, as well as p-Cx43(Ser364/365), which facilitates conductivity. By contrast, expression of non-phosphorylated Cx43 and p-Cx43(Ser278/289), attenuating intercellular communication, was lower in IHH than in N. IHH also resulted in increased expression of protein kinase A and protein kinase G while casein kinase 1 did not change compared to N. In IHH group, which exhibited reduced incidence of ischemic ventricular arrhythmias, Cx43 and p-Cx43(Ser368) were more abundant at “end to end” gap junctions than in N group and this difference was preserved after acute regional ischemia (10 min). We further confirmed higher n-3 PUFA proportion in heart phospholipids after adaptation to IHH, which was even further increased by ischemia. Our results suggest that adaptation to IHH alters expression, phosphorylation and distribution of Cx43 as well as cardioprotective n-3PUFA proportion suggesting that the anti-arrhythmic phenotype elicited by IHH can be at least partly related to the stabilization of the “end to end” conductivity between cardiomyocytes during brief ischemia.

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.