Michael C. Hogan

Loss of Skeletal Muscle HIF-1α Results in Altered Exercise Endurance

The physiological flux of oxygen is extreme in exercising skeletal muscle. Hypoxia is thus a critical parameter in muscle function, influencing production of ATP, utilization of energy-producing substrates, and manufacture of exhaustion-inducing metabolites. Glycolysis is the central source of anaerobic energy in animals, and this metabolic pathway is regulated under low-oxygen conditions by the transcription factor hypoxia-inducible factor 1α (HIF-1α). To determine the role of HIF-1α in regulating skeletal muscle function, we tissue-specifically deleted the gene encoding the factor in skeletal muscle. Significant exercise-induced changes in expression of genes are decreased or absent in the skeletal-muscle HIF-1α knockout mice (HIF-1α KOs); changes in activities of glycolytic enzymes are seen as well. There is an increase in activity of rate-limiting enzymes of the mitochondria in the muscles of HIF-1α KOs, indicating that the citric acid cycle and increased fatty acid oxidation may be compensating for decreased flow through the glycolytic pathway. This is corroborated by a finding of no significant decreases in muscle ATP, but significantly decreased amounts of lactate in the serum of exercising HIF-1α KOs. This metabolic shift away from glycolysis and toward oxidation has the consequence of increasing exercise times in the HIF-1α KOs. However, repeated exercise trials give rise to extensive muscle damage in HIF-1α KOs, ultimately resulting in greatly reduced exercise times relative to wild-type animals. The muscle damage seen is similar to that detected in humans in diseases caused by deficiencies in skeletal muscle glycogenolysis and glycolysis. Thus, these results demonstrate an important role for the HIF-1 pathway in the metabolic control of muscle function.

Sirtuin 1 (SIRT1) Deacetylase Activity Is Not Required for Mitochondrial Biogenesis or Peroxisome Proliferator-activated Receptor-γ Coactivator-1α (PGC-1α) Deacetylation following Endurance Exercise

The protein deacetylase, sirtuin 1 (SIRT1), is a proposed master regulator of exercise-induced mitochondrial biogenesis in skeletal muscle, primarily via its ability to deacetylate and activate peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α). To investigate regulation of mitochondrial biogenesis by SIRT1 in vivo, we generated mice lacking SIRT1 deacetylase activity in skeletal muscle (mKO). We hypothesized that deacetylation of PGC-1α and mitochondrial biogenesis in sedentary mice and after endurance exercise would be impaired in mKO mice. Skeletal muscle contractile characteristics were determined in extensor digitorum longus muscle ex vivo. Mitochondrial biogenesis was assessed after 20 days of voluntary wheel running by measuring electron transport chain protein content, enzyme activity, and mitochondrial DNA expression. PGC-1α expression, nuclear localization, acetylation, and interacting protein association were determined following an acute bout of treadmill exercise (AEX) using co-immunoprecipitation and immunoblotting. Contrary to our hypothesis, skeletal muscle endurance, electron transport chain activity, and voluntary wheel running-induced mitochondrial biogenesis were not impaired in mKO versus wild-type (WT) mice. Moreover, PGC-1α expression, nuclear translocation, activity, and deacetylation after AEX were similar in mKO versus WT mice. Alternatively, we made the novel observation that deacetylation of PGC-1α after AEX occurs in parallel with reduced nuclear abundance of the acetyltransferase, general control of amino-acid synthesis 5 (GCN5), as well as reduced association between GCN5 and nuclear PGC-1α. These findings demonstrate that SIRT1 deacetylase activity is not required for exercise-induced deacetylation of PGC-1α or mitochondrial biogenesis in skeletal muscle and suggest that changes in GCN5 acetyltransferase activity may be an important regulator of PGC-1α activity after exercise.

Oxygen uptake on-kinetics in dog gastrocnemius in situ following activation of pyruvate dehydrogenase by dichloroacetate.

The aim of the present study was to determine whether the activation of the pyruvate dehydrogenase complex (PDC) by dichloroacetate (DCA) is associated with faster O2 uptake (V̇O2) on‐kinetics. V̇O2 on‐kinetics was determined in isolated canine gastrocnemius muscles in situ (n= 6) during the transition from rest to 4 min of electrically stimulated isometric tetanic contractions, corresponding to ∼60–70 % of peak V̇O2. Two conditions were compared: (1) control (saline infusion, C); and (2) DCA infusion (300 mg (kg body mass)−1, 45 min before contraction). Muscle blood flow (Q̇) was measured continuously in the popliteal vein; arterial and popliteal vein O2 contents were measured at rest and at 5–7 s intervals during the transition. Muscle V̇O2 was calculated as Q̇ multiplied by the arteriovenous O2 content difference. Muscle biopsies were taken before and at the end of contraction for determination of muscle metabolite concentrations. DCA activated PDC at rest, as shown by the 9‐fold higher acetylcarnitine concentration in DCA (vs. C; P < 0.0001). Phosphocreatine degradation and muscle lactate accumulation were not significantly different between C and DCA. DCA was associated with significantly less muscle fatigue. Resting and steady‐state V̇O2 values during contraction were not significantly different between C and DCA. The time to reach 63 % of the V̇O2 difference between the resting baseline and the steady‐state V̇O2 values during contraction was 22.3 ± 0.5 s in C and 24.5 ± 1.4 s in DCA (n.s.). In this experimental model, activation of PDC by DCA resulted in a stockpiling of acetyl groups at rest and less muscle fatigue, but it did not affect ‘anaerobic’ energy provision and V̇O2 on‐kinetics.

Increased [lactate] in working dog muscle reduces tension development independent of pH

The purpose of this work was to examine the effect of the lactate ion on the fatigue process in working muscle independent of muscle [H+]. L-(+)-lactate was infused, at a pH that did not change arterial pH, into the blood perfusing an isolated, in situ dog gastrocnemius (N = 5) working at a submaximal intensity (isometric contractions at 2 Hz) and compared with control (C) conditions without lactate infusion. Each muscle was stimulated to work for two 60-min periods (separated by 45 min rest), consisting of three 20-min time periods with either the high arterial lactate condition (high [La]) or C condition sequentially ordered within each 60-min work period. Blood flow and O2 delivery were held constant between the C and high [La] conditions. Arterial and venous blood measurements and muscle biopsies were taken (7 biopsies from each condition) during each condition. Lactate infusion significantly increased arterial [La] (C = 4.2 +/- 0.2 mM vs high [La] = 14.4 +/- 0.2; mean +/- SE) and muscle [La] (C = 8.1 +/- 0.8 mM w.w. vs high [La] = 12.0 +/- 1.4) while arterial and muscle pH were unchanged between conditions. Muscle tension development was significantly reduced (C = 94 +/- 2 N.100 g-1 vs high [La] = 80 +/- 3) during lactate infusion and muscle O2 uptake changed proportionally with tension. These findings support an effect of the lactate anion on tension development which is independent of pH.

Contraction duration affects metabolic energy cost and fatigue in skeletal muscle

It has been suggested that during a skeletal muscle contraction the metabolic energy cost at the onset may be greater than the energy cost related to holding steady-state force. The purpose of the present study was to investigate the effect of contraction duration on the metabolic energy cost and fatigue process in fully perfused contracting muscle in situ. Canine gastrocnemius muscle (n = 6) was isolated, and two contractile periods (3 min of isometric, tetanic contractions with 45-min rest between) were conducted by each muscle in a balanced order design. The two contractile periods had stimulation patterns that resulted in a 1:3 contraction-to-rest ratio, with the difference in the two contractile periods being in the duration of each contraction: short duration 0.25-s stimulation/0.75-s rest vs. long duration 1-s stimulation/3-s rest. These stimulation patterns resulted in the same total time of stimulation, number of stimulation pulses, and total time in contraction for each 3-min period. Muscle O2 uptake, the fall in developed force (fatigue), the O2 cost of developed force, and the estimated total energy cost (ATP utilization) of developed force were significantly greater (P < 0.05) with contractions of short duration. Lactate efflux from the working muscle and muscle lactate concentration were significantly greater with contractions of short duration, such that the calculated energy derived from glycolysis was three times greater in this condition. These results demonstrate that contraction duration can significantly affect both the aerobic and anaerobic metabolic energy cost and fatigue in contracting muscle. In addition, it is likely that the greater rate of fatigue with more rapid contractions was a result of elevated glycolytic production of lactic acid.It has been suggested that during a skeletal muscle contraction the metabolic energy cost at the onset may be greater than the energy cost related to holding steady-state force. The purpose of the present study was to investigate the effect of contraction duration on the metabolic energy cost and fatigue process in fully perfused contracting muscle in situ. Canine gastrocnemius muscle ( n = 6) was isolated, and two contractile periods (3 min of isometric, tetanic contractions with 45-min rest between) were conducted by each muscle in a balanced order design. The two contractile periods had stimulation patterns that resulted in a 1:3 contraction-to-rest ratio, with the difference in the two contractile periods being in the duration of each contraction: short duration 0.25-s stimulation/0.75-s rest vs. long duration 1-s stimulation/3-s rest. These stimulation patterns resulted in the same total time of stimulation, number of stimulation pulses, and total time in contraction for each 3-min period. Muscle O2 uptake, the fall in developed force (fatigue), the O2 cost of developed force, and the estimated total energy cost (ATP utilization) of developed force were significantly greater ( P < 0.05) with contractions of short duration. Lactate efflux from the working muscle and muscle lactate concentration were significantly greater with contractions of short duration, such that the calculated energy derived from glycolysis was three times greater in this condition. These results demonstrate that contraction duration can significantly affect both the aerobic and anaerobic metabolic energy cost and fatigue in contracting muscle. In addition, it is likely that the greater rate of fatigue with more rapid contractions was a result of elevated glycolytic production of lactic acid.

The role of oxygen in determining phosphocreatine onset kinetics in exercising humans

31P‐magnetic resonance spectroscopy was used to study phosphocreatine (PCr) onset kinetics in exercising human gastrocnemius muscle under varied fractions of inspired O2 (FIO2). Five male subjects performed three identical work bouts (5 min duration; order randomised) at a submaximal workload while breathing 0.1, 0.21 or 1.0 FIO2. Either a single or double exponential model was fitted to the PCr kinetics. The phase I τ (0.1, 38.6 ± 7.5; 0.21, 34.5 ± 7.9; 1.0, 38.6 ± 9.2 s) and amplitude, A1 (0.1, 0.34 ± 0.03; 0.21, 0.28 ± 0.05; 1.0, 0.28 ± 0.03,% fall in PCr) were invariant (both P > 0.05) across FIO2 trials. The initial rate of change in PCr hydrolysis at exercise onset, calculated as A1/τ1 (%PCr reduction s−1), was the same across FIO2 trials. A PCr slow component (phase II) was present at an FIO2 of 0.1 and 0.21; however, breathing 1.0 FIO2 ablated the slow component. The onset of the slow component resulted in a greater (P≤ 0.05) overall percentage fall in PCr (both phase I and II) as FIO2 decreased (0.43 ± 0.05, 0.34 ± 0.05, 0.28 ± 0.03) for 0.1, 0.21 and 1.0 FIO2, respectively. These data demonstrate that altering FIO2 does not affect the initial phase I PCr onset kinetics, which supports the notion that O2 driving pressure does not limit PCr kinetics at the onset of submaximal exercise. Thus, these data imply that the manner in which microvascular and intracellular PO2 regulates PCr hydrolysis in exercising muscle is not due to the initial kinetic fall in PCr at exercise onset.

Alterations in Skeletal Muscle Indicators of Mitochondrial Structure and Biogenesis in Patients with Type 2 Diabetes and Heart Failure: Effects of Epicatechin Rich Cocoa

(‐)‐Epicatechin (Epi), a flavanol in cacao stimulates mitochondrial volume and cristae density and protein markers of skeletal muscle (SkM) mitochondrial biogenesis in mice. Type 2 diabetes mellitus (DM2) and heart failure (HF) are diseases associated with defects in SkM mitochondrial structure/function. A study was implemented to assess perturbations and to determine the effects of Epi‐rich cocoa in SkM mitochondrial structure and mediators of biogenesis. Five patients with DM2 and stage II/III HF consumed dark chocolate and a beverage containing approximately 100 mg of Epi per day for 3 months. We assessed changes in protein and/or activity levels of oxidative phosphorylation proteins, porin, mitofilin, nNOS, nitric oxide, cGMP, SIRT1, PGC1α, Tfam, and mitochondria volume and cristae abundance by electron microscopy from SkM. Apparent major losses in normal mitochondria structure were observed before treatment. Epi‐rich cocoa increased protein and/or activity of mediators of biogenesis and cristae abundance while not changing mitochondrial volume density. Epi‐rich cocoa treatment improves SkM mitochondrial structure and in an orchestrated manner, increases molecular markers of mitochondrial biogenesis resulting in enhanced cristae density. Future controlled studies are warranted using Epi‐rich cocoa (or pure Epi) to translate improved mitochondrial structure into enhanced cardiac and/or SkM muscle function. Clin Trans Sci 2012; Volume 5: 43–47

Effects of nitric oxide synthase inhibition by l-NAME on oxygen uptake kinetics in isolated canine muscle in situ

Nitric oxide (NO) has an inhibitory action on O2 uptake at the level of the mitochondrial respiratory chain. The aim of this study was to evaluate the effects of NO synthase (NOS) inhibition on muscle kinetics. Isolated canine gastrocnemius muscles in situ (n= 6) were studied during transitions from rest to 4‐min of electrically stimulated contractions corresponding to ∼60% of the muscle peak . Two conditions were compared: (i) Control (CTRL) and (ii) l‐NAME, in which the NOS inhibitor l‐NAME (20 mg kg−1) was administered. In both conditions the muscle was pump‐perfused with constantly elevated blood flow , at a level measured during a preliminary contraction trial with spontaneous self‐perfused. A vasodilatory drug was also infused. Arterial and venous O2 concentrations were determined at rest and at 5–7 s intervals during the transition. was calculated by Ficks principle. Muscle biopsies were obtained at rest and during contractions. Muscle force was measured continuously. Phosphocreatine hydrolysis and the calculated substrate level phosphorylation were slightly (but not significantly) lower in l‐NAME than in CTRL. Significantly (P < 0.05) less fatigue was found in l‐NAME versus CTRL. The time delay (TDf) and the time constant (τf) of the ‘fundamental’ component of kinetics were not significantly different between CTRL (TDf 7.2 ± 1.2 s; and τf 10.6 ± 1.3, ±s.e.m.) and l‐NAME (TDf 9.3 ± 0.6; and τf 10.4 ± 1.0). Contrary to our hypothesis, NOS inhibition did not accelerate muscle kinetics. The down‐regulation of mitochondrial respiration by NO does not limit the kinetics of adjustment of oxidative metabolism at exercise onset.

Kinetic control of oxygen consumption during contractions in self-perfused skeletal muscle

Non‐technical summary  The ability to sustain exercise is dependent on the ability to match muscular energy supply fuelled by oxygen to the energy demands of the activity (the ‘currency’ of biological energy is termed ATP). Experiments using muscle samples in a test tube suggest that the activation of muscle oxygen consumption is caused by accumulation of ADP (a breakdown product of ATP), which signals the need to increase ATP supply. The mechanism of this signalling process in vivo, however, is not well understood. We investigated the mechanism controlling oxidative ATP supply activation in canine muscle, by simultaneous measurements of oxygen consumption and ADP at the onset of muscle contractions. At the start of contractions muscle oxygen consumption increased more rapidly than predicted from the measured accumulation of ADP. These data suggest that an additional process (or processes) occurs to activate muscle oxidative ATP provision at the onset of exercise in vivo.

Differential depression of myocardial function and metabolism by lactate and H

The effects of both high blood H+ concentration ([H+]) and high blood lactate concentration ([lactate]) under ischemia-reperfusion conditions are receiving attention, but little is known about their effects in nonischemic hearts. Isolated rat hearts were Langendorff perfused at constant flow with media at two pH values (7.4 and 7.0) and two [lactate] (0 and 20 mM) in various sequences ( n = 6/group). Coronary flow and arterial O2content were kept constant at levels that allowed hearts to function without O2 supply limitation. We measured contractility, O2 uptake, diastolic pressure, and at the end of the protocol, tissue [lactate] and pH. Perfusion with high [lactate] raised tissue [lactate] from 5.5 ± 0.1 to 17.5 ± 2.6 μmol/heart ( P < 0.0001), whereas decreasing the pH of the medium decreased tissue pH from 6.94 ± 0.02 to 6.81 ± 0.06 ( P = 0.002). Heart rate was not affected by high [lactate] but was reversibly depressed by high [H+] ( P = 0.004). Developed pressure declined by 20% in response to high [lactate], high [H+], and high [lactate] + high [H+] ( P = 0.002). After the high-[lactate] challenge was withdrawn, pressure continued to decline. In contrast, withdrawing the high [H+] challenge allowed partial recovery. The behavior of diastolic pressure mirrored that of developed pressure. Although unaffected by high [lactate], the O2 uptake was reversibly depressed by high [H+]. This suggests higher O2 cost per contraction in the presence of high [lactate]. We conclude that for similar acute contractility depression, high [lactate] induces irreversible damage, likely at some point in the pathway of O2 utilization. In contrast, the effect of high [H+] appears reversible. These differential behaviors may have implications for heart function during heavy exercise and ischemia-reperfusion events.The effects of both high blood H+ concentration ([H+]) and high blood lactate concentration ([lactate]) under ischemia-reperfusion conditions are receiving attention, but little is known about their effects in nonischemic hearts. Isolated rat hearts were Langendorff perfused at constant flow with media at two pH values (7.4 and 7.0) and two [lactate] (0 and 20 mM) in various sequences (n = 6/group). Coronary flow and arterial O2 content were kept constant at levels that allowed hearts to function without O2 supply limitation. We measured contractility, O2 uptake, diastolic pressure, and at the end of the protocol, tissue [lactate] and pH. Perfusion with high [lactate] raised tissue [lactate] from 5.5 +/- 0.1 to 17.5 +/- 2.6 micromol/heart (P < 0.0001), whereas decreasing the pH of the medium decreased tissue pH from 6.94 +/- 0.02 to 6.81 +/- 0.06 (P = 0.002). Heart rate was not affected by high [lactate] but was reversibly depressed by high [H+] (P = 0.004). Developed pressure declined by 20% in response to high [lactate], high [H+], and high [lactate] + high [H+] (P = 0.002). After the high-[lactate] challenge was withdrawn, pressure continued to decline. In contrast, withdrawing the high [H+] challenge allowed partial recovery. The behavior of diastolic pressure mirrored that of developed pressure. Although unaffected by high [lactate], the O2 uptake was reversibly depressed by high [H+]. This suggests higher O2 cost per contraction in the presence of high [lactate]. We conclude that for similar acute contractility depression, high [lactate] induces irreversible damage, likely at some point in the pathway of O2 utilization. In contrast, the effect of high [H+] appears reversible. These differential behaviors may have implications for heart function during heavy exercise and ischemia-reperfusion events.