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In mammals, hypoxic stress management is under the control of the Hypoxia Inducible Factors, whose activity depends on the stabilization of their labile α subunit. In particular, the skeletal muscle appears to be able to react to changes in substrates and O2 delivery by tuning its metabolism. The present study provides a comprehensive overview of skeletal muscle metabolic adaptation to hypoxia in mice and in human subjects exposed for 7/9 and 19 days to high altitude levels. The investigation was carried out combining proteomics, qRT-PCR mRNA transcripts analysis, and enzyme activities assessment in rodents, and protein detection by antigen antibody reactions in humans and rodents. Results indicate that the skeletal muscle react to a decreased O2 delivery by rewiring the TCA cycle. The first TCA rewiring occurs in mice in 2-day hypoxia and is mediated by cytosolic malate whereas in 10-day hypoxia the rewiring is mediated by Idh1 and Fasn, supported by glutamine and HIF-2α increments. The combination of these specific anaplerotic steps can support energy demand despite HIFs degradation. These results were confirmed in human subjects, demonstrating that the TCA double rewiring represents an essential factor for the maintenance of muscle homeostasis during adaptation to hypoxia.

It was previously found that H9c2 cardiomyocytes exposed to 24-h ischemia (1% O2 with glucose deprivation), were rescued by administration of 2.5 mM creatine + 5 mM D-ribose, while creatine or D-ribose alone were ineffective (1). These findings correlated with up-regulation of protein kinase B (Akt) phosphorylation. Creatine+D-ribose also blunted adenosine monophosphate-activated protein kinase (AMPK) and down-regulated apoptosis by reducing caspase-3 activation and poly (ADP-ribose) polymerase cleavage. In order to test the existence of an analogous mechanism in an in vivo context, five week-old mice were exposed to an atmosphere containing 10% O2 for 10 days, sacrificed and myocardial and pulmonary tissue harvested for structural and biochemical analyses. Mice were gavaged daily with vehicle, creatine, D-ribose or both. Results showed that hypoxia induced marked right ventricle hypertrophy and left ventricle apoptosis. Both phenotypes were slightly reduced by the administration of either creatine or D-ribose, whereas the simultaneous administration almost completely reverted the effects of hypoxia. Furthermore, creatine+D-ribose contributed to blunt the increases in the activity of AMPK, Akt and JNK, but not of ERK, caused by hypoxia. The increase in AMPK during hypoxia is an expected finding secondary to inadequate O2 supply with respect to needs. AMPK is probably upstream of Akt activation; in turn, Akt exerts pro-survival activities, however, by modulating the PI3-kinase pathway, Akt regulates cardiomyocyte size, thus inducing physiological cardiac hypertrophy (2). In addition, the development of right ventricle hypertrophy was found to be associated to left ventricle apoptosis (3), as reported here. During hypoxia, pulmonary hypertension, causing pressure overload, contributes to right ventricle hypertrophy. We show here that the mRNA expression of endothelin-1, a short-lived peptide responsible for the pulmonary and heart disease (4), is increased by hypoxia. The administration of creatine+D-ribose led to reduction of the hypoxia-induced pulmonary overexpression of endothelin-1 mRNA. Whereas creatine improves bioenergetics by recycling ADP into ATP via its shuttle property (5), ribose protects ischemic hearts by replenishing building blocks for ATP synthesis (6). By re-energization of not irreversibly-damaged myocardial cells. creatine+ribose appear to counteract myocardial injury by blunting the pathways originated from AMPK and Akt activation. This may constitute a useful therapeutic approach in several human diseases that involve systemic hypoxia, as for example chronic pulmonary obstructive diseases, heart failure as well as various forms of anemia. References: 1- Caretti A et al, Cell Physiol Biochem.(2010), 26:831-838. 2- Rigor DL et al, Am J Physiol Heart Circ Physiol. (2009);296:H566-72. 3- Kitahori K et al, Circ Heart Fail. (2009);2:599-607. 4- Karmouty-Quintana H et al, FASEB J. (2012); 26:2546-2557. 5- Kammermeier H. J Mol Cell Cardiol. (1987);19:115-118. 6- Smolenski RT et al,. J Mol Cell Cardiol. (1998);30:673-683.

Exploring cellular mechanisms underlying beneficial and detrimental responses to hypoxia represents the object of the present study. Signaling molecules controlling adaptation to hypoxia (HIF-1±), energy balance (AMPK), mitochondrial biogenesis (PGC-1±), autophagic/apoptotic processes regulation and proteomic dysregulation were assessed. Responses to acute hypoxia (AH) and chronic hypoxia (CH) in mouse heart proteome were detected by 2-D DIGE, mass spectrometry and antigen-antibody reactions. Both in AH and CH, the results indicated a deregulation of proteins related to sarcomere stabilization and muscle contraction. Neither in AH nor in CH the HIF-1± stabilization was observed. In AH, the metabolic adaptation to lack of oxygen was controlled by AMPK activation and sustained by an up-regulation of adenosylhomocysteinase and acetyl-CoA synthetase. AH was characterized by the mitophagic protein Bnip 3 increment. PGC-1±, a master regulator of mitochondrial biogenesis, was down-regulated. CH was characterized by the up-regulation of enzymes involved in antioxidant defense, in aldehyde bio-product detoxification and in misfolded protein degradation. In addition, a general down-regulation of enzymes controlling anaerobic metabolism was observed. After 10 days of hypoxia, cardioprotective molecules were substantially decreased whereas pro-apoptotic molecules increased accompained by down-regulation of specific target proteins.

Hypoxia-induced changes of rat skeletal muscle were investigated by two-dimensional difference in-gel electrophoresis (2D-DIGE) and mass spectrometry. The results indicated that proteins involved in the TCA cycle, ATP production, and electron transport are down-regulated, whereas glycolytic enzymes and deaminases involved in ATP and AMP production were up-regulated. Up-regulation of the hypoxia markers hypoxia inducible factor 1 (HIF-1alpha) and pyruvate dehydrogenase kinase 1 (PDK1) was also observed, suggesting that in vivo adaptation to hypoxia requires an active metabolic switch. The kinase protein, mammalian target of rapamycin (mTOR), which has been implicated in the regulation of protein synthesis in hypoxia, appears unchanged, suggesting that its activity, in this system, is not controlled by oxygen partial pressure. Keywords: skeletal muscle * hypoxia * 2D-DIGE * mass spectrometry.

The method described here allows the quantitative simultaneous determination of adenosine 5´-triphosphate, adenosine 5´-diphosphate, adenosine 5´-monophosphate, adenosine, guanosine 5´-triphosphate, guanosine 5´-diphosphate, guanosine, inosine 5´-monophosphate, inosine, uric acid, xanthine, hypoxanthine and b-nicotinamide adenine dinucleotide by ion-pair high performance liquid chromatography. The chromatographic analysis requires 26 min per sample and allows the separation of the mentioned metabolites in a time as short as 16 min. Primary cultures of rat cerebellar granule cells were incubated in serum-free medium containing 25 mM KCl for 1.5–48 h and their acid extracts were injected onto column. Uric acid, inosine 5´-monophosphate, inosine, b-nicotinamide adenine dinucleotide, adenosine, adenosine 5´-monophosphate, guanosine 5´-diphosphate, adenosine 5´-diphosphate, guanosine 5´-triphosphate and adenosine 5´-triphosphate were identified and quantified, while hypoxanthine, xanthine and guanosine were below the detection limit. This method makes use of a single-step sample pre-treatment procedure which allows a greater than 91% recovery of the compounds of interest and provides the assay of the metabolites of interest in little amounts of cell extracts. Therefore, this method is suitable to evaluate the energetic state in a variety of cell types, both under normal and dismetabolic conditions, such as after the induction of apoptosis or necrosis.

The objective of this study was to test the hypothesis that the beneficial effect of trimetazidine during reflow of ischemic hearts is mediated by energy sparing and ATP pool preservation during ischemia. Isolated rat hearts (controls and rats treated with 10(-6) M trimetazidine, n = 17 per group) underwent the following protocol: baseline perfusion at normal coronary flow (20 minutes), low-flow ischemia at 10% flow (60 minutes), and reflow (20 minutes). We measured contractile function, O2 uptake, lactate release, venous pH and PCO2, and the tissue content of high-energy phosphates and their metabolites. During baseline, trimetazidine induced higher venous pH and lower PCO2 without influencing performance and metabolism. During low-flow ischemia, trimetazidine reduced myocardial performance (P = 0.04) and ATP turnover (P = 0.02). During reflow, trimetazidine improved performance (91 +/- 6% versus. 55 +/- 6% of baseline), prevented the development of diastolic contracture and coronary resistance, and reduced myocardial depletion of adenine nucleotides and purines. ATP turnover during low-flow ischemia was inversely related to recovery of the rate-pressure product (P = 0.002), end-diastolic pressure (P = 0.007), and perfusion pressure (P = 0.05). We conclude that trimetazidine-induced protection of ischemic-reperfused hearts is also mediated by energy sparing during ischemia, which presumably preserves the ATP pool during reflow.