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.

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.

Cardiac resistance against acute ischemia/reperfusion (I/R) injury can be enhanced by adaptation to chronic intermittent hypoxia (CIH), but the changes at the molecular level associated with this adaptation are still not fully explored. Phospholipase A (PLA) plays an important role in phospholipid metabolism and may contribute to membrane destruction under conditions of energy deprivation during I/R. The aim of this study was to determine the effect of CIH (7000 m, 8 h/day, 5 weeks) on the expression of cytosolic PLAα (cPLAα) and its phosphorylated form (p-cPLAα), as well as other related signaling proteins in the left ventricular myocardium of adult male Wistar rats. Adaptation to CIH increased the total content of cPLAα by 14 % in myocardial homogenate, and enhanced the association of p-cPLAα with the nuclear membrane by 85 %. The total number of β-adrenoceptors (β-ARs) did not change but the β/β ratio markedly increased due to the elevation of β-ARs and drop in β-ARs. In parallel, the amount of adenylyl cyclase decreased by 49 % and Gα proteins increased by about 50 %. Besides that, cyclooxygenase 2 (COX-2) and prostaglandin E (PGE) increased by 36 and 84 %, respectively. In parallel, we detected increased phosphorylation of protein kinase Cα, ERK1/2 and p38 (by 12, 48 and 19 %, respectively). These data suggest that adaptive changes induced in the myocardium by CIH may include activation of cPLAα and COX-2 via β-AR/G-mediated stimulation of the ERK/p38 pathway.

Desmin mutations cause familial and sporadic cardiomyopathies. In addition to perturbing the contractile apparatus, both desmin deficiency and mutated desmin negatively impact mitochondria. Impaired myocardial metabolism secondary to mitochondrial defects could conceivably exacerbate cardiac contractile dysfunction. We performed metabolic myocardial phenotyping in left ventricular cardiac muscle tissue in desmin knock-out mice. Our analyses revealed decreased mitochondrial number, ultrastructural mitochondrial defects, and impaired mitochondria-related metabolic pathways including fatty acid transport, activation, and catabolism. Glucose transporter 1 and hexokinase-1 expression and hexokinase activity were increased. While mitochondrial creatine kinase expression was reduced, fetal creatine kinase expression was increased. Proteomic analysis revealed reduced expression of proteins involved in electron transport mainly of complexes I and II, oxidative phosphorylation, citrate cycle, beta-oxidation including auxiliary pathways, amino acid catabolism, and redox reactions and oxidative stress. Thus, desmin deficiency elicits a secondary cardiac mitochondriopathy with severely impaired oxidative phosphorylation and fatty and amino acid metabolism. Increased glucose utilization and fetal creatine kinase upregulation likely portray attempts to maintain myocardial energy supply. It may be prudent to avoid medications worsening mitochondrial function and other metabolic stressors. Therapeutic interventions for mitochondriopathies might also improve the metabolic condition in desmin deficient hearts.