Low myoglobin concentration in skeletal muscle of elite cyclists is associated with low mRNA expression levels.

Myoglobin is essential for oxygen transport to the muscle fibers. However, measurements of myoglobin (Mb) protein concentrations within individual human muscle fibers are scarce. Recent observations have revealed surprisingly low Mb concentrations in elite cyclists, however it remains unclear whether this relates to Mb translation, transcription and/or myonuclear content. The aim was to compare Mb concentration, Mb messenger RNA (mRNA) expression levels and myonuclear content within muscle fibers of these elite cyclists with those of physically-active controls. Muscle biopsies were obtained from m. vastus lateralis in 29 cyclists and 20 physically-active subjects. Mb concentration was determined by peroxidase staining for both type I and type II fibers, Mb mRNA expression level was determined by quantitative PCR and myonuclear domain size (MDS) was obtained by immunofluorescence staining. Average Mb concentrations (mean ± SD: 0.38 ± 0.04 mM vs. 0.48 ± 0.19 mM; P = 0.014) and Mb mRNA expression levels (0.067 ± 0.019 vs. 0.088 ± 0.027; P = 0.002) were lower in cyclists compared to controls. In contrast, MDS and total RNA per mg muscle were not different between groups. Interestingly, in cyclists compared to controls, Mb concentration was only lower for type I fibers (P < 0.001), but not for type II fibers (P > 0.05). In conclusion, the lower Mb concentration in muscle fibers of elite cyclists is partly explained by lower Mb mRNA expression levels per myonucleus and not by a lower myonuclear content. It remains to be determined whether cyclists may benefit from strategies that upregulate Mb mRNA expression levels, particularly in type I fibers, to enhance their oxygen supply.

Work and oxygen consumption of isolated right ventricular papillary muscle in experimental pulmonary hypertension.

Right-sided myocardial mechanical efficiency (work output/metabolic energy input) in pulmonary hypertension can be severely reduced. We determined the contribution of intrinsic myocardial determinants of efficiency using papillary muscle preparations from monocrotaline-induced pulmonary hypertensive (MCT-PH) rats. The hypothesis tested was that efficiency is reduced by mitochondrial dysfunction in addition to increased activation heat reported previously. Right ventricular muscle preparations were subjected to 5 Hz sinusoidal length changes at 37°C. Work and suprabasal oxygen consumption ( V ̇ O 2 ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ ) were measured before and after cross-bridge inhibition by blebbistatin. Cytosolic cytochrome c concentration, myocyte cross-sectional area, proton permeability of the inner mitochondrial membrane and monoamine oxidase and glucose 6-phosphate dehydrogenase activities and phosphatidylglycerol/cardiolipin contents were determined. Mechanical efficiency ranged from 23% to 11% in control (n = 6) and from 22% to 1% in MCT-PH (n = 15) and correlated with work (r2  = 0.68, P < 0.0001) but not with V ̇ O 2 ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ (r2  = 0.004, P = 0.7919). V ̇ O 2 ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ for cross-bridge cycling was proportional to work (r2  = 0.56, P = 0.0005). Blebbistatin-resistant V ̇ O 2 ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ (r2  = 0.32, P = 0.0167) and proton permeability of the mitochondrial inner membrane (r2  = 0.36, P = 0.0110) correlated inversely with efficiency. Together, these variables explained the variance of efficiency (coefficient of multiple determination r2  = 0.79, P = 0.0001). Cytosolic cytochrome c correlated inversely with work (r2  = 0.28, P = 0.0391), but not with efficiency (r2  = 0.20, P = 0.0867). Glucose 6-phosphate dehydrogenase, monoamine oxidase and phosphatidylglycerol/cardiolipin increased in the right ventricular wall of MCT-PH but did not correlate with efficiency. Reduced myocardial efficiency in MCT-PH is a result of activation processes and mitochondrial dysfunction. The variance of work and the ratio of activation heat reported previously and blebbistatin-resistant V ̇ O 2 ${\dot{V}}_{{{\rm{O}}}_{\rm{2}}}$ are discussed. KEY POINTS: Mechanical efficiency of right ventricular myocardium is reduced in pulmonary hypertension. Increased energy use for activation processes has been demonstrated previously, but the contribution of mitochondrial dysfunction is unknown. Work and oxygen consumption are determined during work loops. Oxygen consumption for activation and cross-bridge cycling confirm the previous heat measurements. Cytosolic cytochrome c concentration, proton permeability of the mitochondrial inner membrane and phosphatidylglycerol/cardiolipin are increased in experimental pulmonary hypertension. Reduced work and mechanical efficiency are related to mitochondrial dysfunction. Upregulation of the pentose phosphate pathway and a potential gap in the energy balance suggest mitochondrial dysfunction in right ventricular overload is a resiult of the excessive production of reactive oxygen species.

Increased MAO-A Activity Promotes Progression of Pulmonary Arterial Hypertension.

Monoamine oxidases (MAOs), a class of enzymes bound to the outer mitochondrial membrane, are important sources of reactive oxygen species. Increased MAO-A activity in endothelial cells and cardiomyocytes contributes to vascular dysfunction and progression of left heart failure. We hypothesized that inhibition of MAO-A can be used to treat pulmonary arterial hypertension (PAH) and right ventricular (RV) failure. MAO-A levels in lung and RV samples from patients with PAH were compared with levels in samples from donors without PAH. Experimental PAH was induced in male Sprague-Dawley rats by using Sugen 5416 and hypoxia (SuHx), and RV failure was induced in male Wistar rats by using pulmonary trunk banding (PTB). Animals were randomized to receive either saline or the MAO-A inhibitor clorgyline at 10 mg/kg. Echocardiography and RV catheterization were performed, and heart and lung tissues were collected for further analysis. We found increased MAO-A expression in the pulmonary vasculature of patients with PAH and in experimental experimental PAH induced by SuHx. Cardiac MAO-A expression and activity was increased in SuHx- and PTB-induced RV failure. Clorgyline treatment reduced RV afterload and pulmonary vascular remodeling in SuHx rats through reduced pulmonary vascular proliferation and oxidative stress. Moreover, clorgyline improved RV stiffness and relaxation and reversed RV hypertrophy in SuHx rats. In PTB rats, clorgyline had no direct clorgyline had no direct effect on the right ventricle effect. Our study reveals the role of MAO-A in the progression of PAH. Collectively, these findings indicated that MAO-A may be involved in pulmonary vascular remodeling and consecutive RV failure.

Meijer and Vloedman's histochemical demonstration of mitochondrial coupling obeys Lambert-Beer's law in the myocardium.

Uncoupling of mitochondrial proton pumping and adenosine triphosphate (ATP) production lowers mitochondrial efficiency. Current methods to determine mitochondrial efficiency require substantial amounts of tissue and permeabilization or isolation procedures. A simple histochemical method has been described by Meijer and Vloedman (Histochemistry 69:217-232, 1980, https://doi.org/10.1007/BF00489769 ), but this was not quantitative. We found linear correlations between (1) absorbance and sections thickness and (2) absorbance and incubation time. Because the method obeys Lambert-Beer's law, we can estimate ATP/O2 ratios for healthy and overloaded right-sided rat myocardium. We related mitochondrial efficiency to the ratio between cardiolipin and its precursor phosphatidylglycerol. We found a non-linear relationship between mitochondrial efficiency and this ratio, indicating that lower mitochondrial efficiency as found in experimental pulmonary hypertension may be due to altered composition of the mitochondrial inner membrane. We conclude that the histochemical method of Meijer and Vloedman can be applied to quantify mitochondrial efficiency.

Critical determinants of combined sprint and endurance performance: an integrative analysis from muscle fiber to the human body.

Optimizing physical performance is a major goal in current physiology. However, basic understanding of combining high sprint and endurance performance is currently lacking. This study identifies critical determinants of combined sprint and endurance performance using multiple regression analyses of physiologic determinants at different biologic levels. Cyclists, including 6 international sprint, 8 team pursuit, and 14 road cyclists, completed a Wingate test and 15-km time trial to obtain sprint and endurance performance results, respectively. Performance was normalized to lean body mass2/3 to eliminate the influence of body size. Performance determinants were obtained from whole-body oxygen consumption, blood sampling, knee-extensor maximal force, muscle oxygenation, whole-muscle morphology, and muscle fiber histochemistry of musculus vastus lateralis. Normalized sprint performance was explained by percentage of fast-type fibers and muscle volume ( R2 = 0.65; P < 0.001) and normalized endurance performance by performance oxygen consumption ( V̇o2), mean corpuscular hemoglobin concentration, and muscle oxygenation ( R2 = 0.92; P < 0.001). Combined sprint and endurance performance was explained by gross efficiency, performance V̇o2, and likely by muscle volume and fascicle length ( P = 0.056; P = 0.059). High performance V̇o2 related to a high oxidative capacity, high capillarization × myoglobin, and small physiologic cross-sectional area ( R2 = 0.67; P < 0.001). Results suggest that fascicle length and capillarization are important targets for training to optimize sprint and endurance performance simultaneously.-Van der Zwaard, S., van der Laarse, W. J., Weide, G., Bloemers, F. W., Hofmijster, M. J., Levels, K., Noordhof, D. A., de Koning, J. J., de Ruiter, C. J., Jaspers, R. T. Critical determinants of combined sprint and endurance performance: an integrative analysis from muscle fiber to the human body.

Muscle morphology of the vastus lateralis is strongly related to ergometer performance, sprint capacity and endurance capacity in Olympic rowers.

Rowers need to combine high sprint and endurance capacities. Muscle morphology largely explains muscle power generating capacity, however, little is known on how muscle morphology relates to rowing performance measures. The aim was to determine how muscle morphology of the vastus lateralis relates to rowing ergometer performance, sprint and endurance capacity of Olympic rowers. Eighteen rowers (12♂, 6♀, who competed at 2016 Olympics) performed an incremental rowing test to obtain maximal oxygen consumption, reflecting endurance capacity. Sprint capacity was assessed by Wingate cycling peak power. M. vastus lateralis morphology (volume, physiological cross-sectional area, fascicle length and pennation angle) was derived from 3-dimensional ultrasound imaging. Thirteen rowers (7♂, 6♀) completed a 2000-m rowing ergometer time trial. Muscle volume largely explained variance in 2000-m rowing performance (R2 = 0.85), maximal oxygen consumption (R2 = 0.65), and Wingate peak power (R2 = 0.82). When normalized for differences in body size, maximal oxygen consumption and Wingate peak power were negatively related in males (r = -0.94). Fascicle length, not physiological cross-sectional area, attributed to normalized peak power. In conclusion, vastus lateralis volume largely explains variance in rowing ergometer performance, sprint and endurance capacity. For a high normalized sprint capacity, athletes may benefit from long fascicles rather than a large physiological cross-sectional area.

IGF-1 Attenuates Hypoxia-Induced Atrophy but Inhibits Myoglobin Expression in C2C12 Skeletal Muscle Myotubes.

Chronic hypoxia is associated with muscle wasting and decreased oxidative capacity. By contrast, training under hypoxia may enhance hypertrophy and increase oxidative capacity as well as oxygen transport to the mitochondria, by increasing myoglobin (Mb) expression. The latter may be a feasible strategy to prevent atrophy under hypoxia and enhance an eventual hypertrophic response to anabolic stimulation. Mb expression may be further enhanced by lipid supplementation. We investigated individual and combined effects of hypoxia, insulin-like growth factor (IGF)-1 and lipids, in mouse skeletal muscle C2C12 myotubes. Differentiated C2C12 myotubes were cultured for 24 h under 20%, 5% and 2% oxygen with or without IGF-1 and/or lipid treatment. In culture under 20% oxygen, IGF-1 induced 51% hypertrophy. Hypertrophy was only 32% under 5% and abrogated under 2% oxygen. This was not explained by changes in expression of genes involved in contractile protein synthesis or degradation, suggesting a reduced rate of translation rather than of transcription. Myoglobin mRNA expression increased by 75% under 5% O2 but decreased by 50% upon IGF-1 treatment under 20% O2, compared to control. Inhibition of mammalian target of rapamycin (mTOR) activation using rapamycin restored Mb mRNA expression to control levels. Lipid supplementation had no effect on Mb gene expression. Thus, IGF-1-induced anabolic signaling can be a strategy to improve muscle size under mild hypoxia, but lowers Mb gene expression.

Increased in vivo mitochondrial oxygenation with right ventricular failure induced by pulmonary arterial hypertension: mitochondrial inhibition as driver of cardiac failure?

BACKGROUND: The leading cause of mortality due to pulmonary arterial hypertension (PAH) is failure of the cardiac right ventricle. It has long been hypothesized that during the development of chronic cardiac failure the heart becomes energy deprived, possibly due to shortage of oxygen at the level of cardiomyocyte mitochondria. However, direct evaluation of oxygen tension levels within the in vivo right ventricle during PAH is currently lacking. Here we directly evaluated this hypothesis by using a recently reported technique of oxygen-dependent quenching of delayed fluorescence of mitochondrial protoprophyrin IX, to determine the distribution of mitochondrial oxygen tension (mitoPO2) within the right ventricle (RV) subjected to progressive PAH. METHODS: PAH was induced through a single injection of monocrotaline (MCT). Control (saline-injected), compensated RV hypertrophy (30 mg/kg MCT; MCT30), and RV failure (60 mg/kg MCT; MCT60) rats were compared 4 wk after treatment. The distribution of mitoPO2 within the RV was determined in mechanically-ventilated, anaesthetized animals, applying different inspired oxygen (FiO2) levels and two increment dosages of dobutamine. RESULTS: MCT60 resulted in RV failure (increased mortality, weight loss, increased lung weight), MCT30 resulted in compensated RV hypertrophy. At 30% or 40% FiO2, necessary to obtain physiological arterial PO2 in the diseased animals, RV failure rats had significantly less mitochondria (15% of total mitochondria) in the 0-20 mmHg mitoPO2 range than hypertrophied RV rats (48%) or control rats (54%). Only when oxygen supply was reduced to 21% FiO2, resulting in low arterial PO2 for the MCT60 animals, or when oxygen demand increased with high dose dobutamine, the number of failing RV mitochondria with low oxygen became similar to control RV. In addition, metabolic enzyme analysis revealed similar mitochondrial mass, increased glycolytic hexokinase activity following MCT, with increased lactate dehydrogenase activity only in compensated hypertrophied RV. CONCLUSIONS: Our novel observation of increased mitochondrial oxygenation suggests down-regulation of in vivo mitochondrial oxygen consumption, in the absence of hypoxia, with transition towards right ventricular failure induced by pulmonary arterial hypertension.

Iron deficiency in systemic sclerosis patients with and without pulmonary hypertension.

OBJECTIVES: SSc-associated pulmonary hypertension (SSc-PH) has a worse prognosis compared with SSc without PH (SSc-nonPH). Iron deficiency (ID) was previously associated with worse clinical outcome and survival in other types of PH, but ID effects in SSc-PH are unknown. Therefore we investigated the prevalence and clinical significance of ID in systemic sclerosis patients with and without PH. METHODS: Body iron status was determined in SSc-PH (n = 47) and SSc-nonPH patients (n = 122). ID was defined by circulating soluble transferrin receptor (sTfR) levels >28.1 nmol/l. Clinical and exercise parameters were compared between the groups. Four-year survival after iron measurements was determined. RESULTS: ID prevalence was 46.1% in SSc-PH compared with 16.4% in SSc-nonPH patients (P < 0.001). Overall hepcidin levels were high compared with reference values and related to sTfR, but not with IL-6 (P = 0.82). Six-minute walking distance and maximal achieved work at ergometry was lower in SSc-PH compared with SSc-nonPH patients (P < 0.001 and P < 0.01, respectively) and was even further reduced in case of ID (P(interaction) < 0.05). In addition, ID SSc-PH patients had a poorer survival compared with non-ID patients [hazard ratio (HR) 0.34, 95% CI 0.14, 0.82, P < 0.05) and a similar trend was observed in SSc-nonPH patients (HR 0.16, 95% CI 0.02, 1.11, P = 0.06). CONCLUSION: ID is more prevalent in SSc-PH than in SSc-nonPH patients and is associated with exercise impairment in both SSc-PH and SSc-nonPH. In addition, ID SSc-PH patients have a significantly worse survival compared with non-ID patients.

Intrinsic cardiac adrenergic (ICA) cell density and MAO-A activity in failing rat hearts.

The efficiency (work/oxygen consumption) of isolated papillary muscles from failing hearts is reduced. We investigated whether this can be due to an increase of intrinsic cardiac adrenergic (ICA) cell density. The number of ICA cells in the septum and both ventricular walls was determined by tyrosine hydroxylase immunohistochemistry in rats with monocrotaline-induced pulmonary hypertension. We found that the number of ICA cells is about 200,000 per rat heart. ICA cell density was significantly lower in right ventricular myocardium of hypertrophied hearts (P < 0.01). MAO-A enzyme histochemistry and inhibition experiments with clorgyline in papillary muscles were performed to localize the enzyme and to determine its oxygen consumption. Upregulation of MAO-A was found in the right ventricular wall and papillary muscles of failing hearts (P = 0.018). A positive correlation between ICA cell density and MAO-A activity was absent. Clorgyline (2 µM) decreased the basal rate of oxygen consumption of right ventricular papillary muscles by 65 µM O(2)/s (P = 0.027). This rate can only be maintained for several seconds judging from the catecholamine content of the preparations reported previously. High ICA cell activity rather than density and/or recycling of oxidized catecholamines are discussed as alternative explanations for the low myocardial efficiency in experimental pulmonary hypertension.

Development of an in vitro model to measure bioactivity of botulinum neurotoxin A in rat bladder muscle strips.

BACKGROUND: Botulinum toxin A (BoNT-A) is a new treatment modality in various causes of bladder dysfunction; like neurogenic detrusor overactivity and overactive bladder. The best technique of administrating BoNT-A in patients is unknown. A validated in vitro model could be used to investigate newer intravesical administration techniques of BoNT-A. In this study, we describe the development and validation of in vitro model to measure inhibitory effects of BoNT-A on bladder strip contractions. METHODS: Rat bladder strips were mounted in organ baths filled with Krebs' solution. The strips were stimulated chemically (80 mM potassium chloride, 1 µM carbachol) and electrically (Electrical Field Stimulation (EFS) 100 shocks, 50 V, 20 Hz, every 3 minutes). The viability of the strips was measured by carbachol stimulation at the beginning and at the end of the experiments. The strips were incubated in various concentrations of BoNT-A (0.03, 0.2, 0.3 nM). Controls were incubated in Krebs' solution only. The inhibition of strip contraction induced by EFS was measured. These measurements were statistically analyzed with a log-logistic model representing diffusion. RESULTS: All strips remained viable during the experiments. Inhibition of strip contraction was observed after incubation with 0.3 nM BoNT-A. The measurements fitted to a log-logistic model describing diffusion of BoNT-A in the bladder strip. The parameters of the log-logistic model representing diffusion were significant for 0.3 nM BoNT-A. Incubation with 0.2 nM BoNT-A showed insignificant results for 2 out of 3 runs. Incubation with 0.03 nM BoNT-A did not result in significant inhibition of strip contractions. CONCLUSIONS: An in vitro model was developed and validated in which the inhibitory effect of low concentrations of BoNT-A on bladder strip contractions can be measured.

On-off asymmetries in oxygen consumption kinetics of single Xenopus laevis skeletal muscle fibres suggest higher-order control.

The mechanisms controlling skeletal muscle oxygen consumption (V(o)2) during exercise are not well understood. We determined whether first-order control could explain V(o)2kinetics at contractions onset (V(o)2(on)) and cessation (V(o)2off)) in single skeletal muscle fibres differing in oxdidative capacity, and across stimulation intensities up to V(o)2(max). Xenopus laevis fibres (n = 21) were suspended in a sealed chamber with a fast response P(o)2 electrode to measure V(o)2 every second before, during and after stimulated isometric contractions. A first-order model did not well characterize on-transient V(o)2 kinetics. Including a time delay (TD) in the model provided a significantly improved characterization than a first-order fit without TD (F-ratio; P < 0.05), and revealed separate 'activation' and 'exponential' phases in 15/21 fibres contracting at V(o)2(max) (mean ± SD TD: 14 ± 3s). On-transient kinetics (τV(o)2(on)) was weakly and linearly related to V(o)2(max) (R² = 0.271, P = 0.015). Off-transient kinetics, however, were first-order, and τV(o)2(off) was greater in low-oxidative (V(o)2max < 0.05 nmol mm⁻³s⁻¹ than high-oxidative fibres (V(o)2(max > 0.10 nmol mm ⁻³ s⁻¹; 170 ± 70 vs. 29 ± 6 s, P < 0.001). 1/ τV(o)2(off) was proportional to V(o)2(max) (R² = 0.727, P < 0.001), unlike in the on-transient. The calculated oxygen deficit was larger (P < 0.05) than the post-contraction volume of consumed oxygen at all intensities except V(o)2(max). These data show a clear dissociation between the kinetic control of V(o)2at the onset and cessation of contractions and across stimulation intensities. More complex models are therefore required to understand the activation of mitochondrial respiration in skeletal muscle at the start of exercise.

Mechanical ventilation with high tidal volumes attenuates myocardial dysfunction by decreasing cardiac edema in a rat model of LPS-induced peritonitis.

BACKGROUND: Injurious mechanical ventilation (MV) may augment organ injury remote from the lungs. During sepsis, myocardial dysfunction is common and increased endothelial activation and permeability can cause myocardial edema, which may, among other factors, hamper myocardial function. We investigated the effects of MV with injuriously high tidal volumes on the myocardium in an animal model of sepsis. METHODS: Normal rats and intraperitoneal (i.p.) lipopolysaccharide (LPS)-treated rats were ventilated with low (6 ml/kg) and high (19 ml/kg) tidal volumes (Vt) under general anesthesia. Non-ventilated animals served as controls. Mean arterial pressure (MAP), central venous pressure (CVP), cardiac output (CO) and pulmonary plateau pressure (Pplat) were measured. Ex vivo myocardial function was measured in isolated Langendorff-perfused hearts. Cardiac expression of endothelial vascular cell adhesion molecule (VCAM)-1 and edema were measured to evaluate endothelial inflammation and leakage. RESULTS: MAP decreased after LPS-treatment and Vt-dependently, both independent of each other and with interaction. MV Vt-dependently increased CVP and Pplat and decreased CO. LPS-induced peritonitis decreased myocardial function ex vivo but MV attenuated systolic dysfunction Vt-dependently. Cardiac endothelial VCAM-1 expression was increased by LPS treatment independent of MV. Cardiac edema was lowered Vt-dependently by MV, particularly after LPS, and correlated inversely with systolic myocardial function parameters ex vivo. CONCLUSION: MV attenuated LPS-induced systolic myocardial dysfunction in a Vt-dependent manner. This was associated with a reduction in cardiac edema following a lower transmural coronary venous outflow pressure during LPS-induced coronary inflammation.

Early myocardial dysfunction is not caused by mitochondrial abnormalities in a rat model of peritonitis.

BACKGROUND: Patients with complicated intra-abdominal infections are prone to develop multiple organ failure, including myocardial dysfunction. We hypothesized that early dysfunction during sepsis is associated with inflammation, mitochondrial injury, impaired mitochondrial function, and activation of mitochondrial biogenesis. MATERIALS AND METHODS: Rats received lipopolysaccharide (LPS, n = 11) intraperitoneally. Healthy rats (n = 6) served as controls. Myocardial function was measured ex vivo in an isolated Langendorff-perfused heart set-up. Myocardial vascular cell adhesion molecule-1 (VCAM-1) expression was determined by immunofluorescence microscopy. Cytochrome c release and cytochrome c oxidase (COX IV) activity were measured by immunohistochemistry and enzyme histochemistry, respectively. Protein expression of tumor necrosis factor-α (TNF-α), B-cell lymphoma (Bcl)-2, peroxisome proliferator activated receptor γ cofactor 1α (PGC-1α), and mitochondrial transcription factor A (TFAM) were analyzed by Western blot technique. Mitochondria were studied by electron microscopy. RESULTS: Two hours after LPS injection, developed pressure had decreased and after 4 h myocardial contractility (+dP/dt) and relaxation (-dP/dt) also had decreased. TNF-α protein expression was increased after 2 h and returned to normal at 4 h, whereas after 4 h VCAM-1 expression was higher in LPS-treated animals. At 2 h a substrate-dependent increase in COXIV-activity was seen, but no mitochondrial damage occurred as cytochrome c release, COX IV activity and Bcl-2, PGC-1α or TFAM expression were not changed. Electron microscopy did not reveal differences in myocardial mitochondrial characteristics between LPS-treated and control rats. CONCLUSIONS: Early myocardial dysfunction in sepsis is associated with myocardial inflammation but not with mitochondrial injury, impaired mitochondrial function, or activated mitochondrial biogenesis.

Diaphragm muscle fiber weakness in pulmonary hypertension.

RATIONALE: Recently it was suggested that patients with pulmonary hypertension (PH) suffer from inspiratory muscle dysfunction. However, the nature of inspiratory muscle weakness in PH remains unclear. OBJECTIVES: To assess whether alterations in contractile performance and in morphology of the diaphragm underlie inspiratory muscle weakness in PH. METHODS: PH was induced in Wistar rats by a single injection of monocrotaline (60 mg/kg). Diaphragm (PH n = 8; controls n = 7) and extensor digitorum longus (PH n = 5; controls n = 7) muscles were excised for determination of in vitro contractile properties and cross-sectional area (CSA) of the muscle fibers. In addition, important determinants of protein synthesis and degradation were determined. Finally, muscle fiber CSA was determined in diaphragm and quadriceps of patients with PH, and the contractile performance of single fibers of the diaphragm. MEASUREMENTS AND MAIN RESULTS: In rats with PH, twitch and maximal tetanic force generation of diaphragm strips were significantly lower, and the force-frequency relation was shifted to the right (i.e., impaired relative force generation) compared with control subjects. Diaphragm fiber CSA was significantly smaller in rats with PH compared with controls, and was associated with increased expression of E3-ligases MAFbx and MuRF-1. No significant differences in contractility and morphology of extensor digitorum longus muscle fibers were found between rats with PH and controls. In line with the rat data, studies on patients with PH revealed significantly reduced CSA and impaired contractility of diaphragm muscle fibers compared with control subjects, with no changes in quadriceps muscle. CONCLUSIONS: PH induces selective diaphragm muscle fiber weakness and atrophy.

Tissue transglutaminase activity is involved in the differentiation of oligodendrocyte precursor cells into myelin-forming oligodendrocytes during CNS remyelination.

During normal brain development, axons are myelinated by mature oligodendrocytes (OLGs). Under pathological, demyelinating conditions within the central nervous system (CNS), axonal remyelination is only partially successful because oligodendrocyte precursor cells (OPCs) largely remain in an undifferentiated state resulting in a failure to generate myelinating OLGs. Tissue Transglutaminase (TG2) is a multifunctional enzyme, which amongst other functions, is involved in cell differentiation. Therefore, we hypothesized that TG2 contributes to differentiation of OPCs into OLGs and thereby stimulates remyelination. In vivo studies, using the cuprizone model for de- and remyelination in TG2(-/-) and wild-type mice, showed that during remyelination expression of proteolipid protein mRNA, as a marker for remyelination, in the corpus callosum lags behind in TG2(-/-) mice resulting in less myelin formation and, moreover, impaired recovery of motor behavior. Subsequent in vitro studies showed that rat OPCs express TG2 protein and activity which reduces when the cells have matured into OLGs. Furthermore, when TG2 activity is pharmacologically inhibited, the differentiation of OPCs into myelin-forming OLGs is dramatically reduced. We conclude that TG2 plays a prominent role in remyelination of the CNS, probably through stimulating OPC differentiation into myelin-forming OLGs. Therefore, manipulating TG2 activity may represent an interesting new target for remyelination in demyelinating diseases.

Reduced mechanical efficiency of rat papillary muscle related to degree of hypertrophy of cardiomyocytes.

Isolated rat papillary muscles of the right ventricle were used to discover the origin of reduced myocardial efficiency in chronic heart failure. Right ventricular hypertrophy was induced by monocrotaline injection, causing pulmonary hypertension. Control (n = 7) and hypertrophied (n = 11) papillary muscles were subjected to sinusoidal length changes at 37 degrees C and 5 Hz with a peak-to-peak amplitude of 15% of the length giving maximum force (L(max)) after being stretched to 92.5% of L(max). Isometric tension at L(max) was similar in control and hypertrophied muscles. Work was assessed from the area encompassed by force-length loops. Work per loop was 0.93 +/- 0.11 and 0.84 +/- 0.11 microJ/mm(3) (means +/- SE) for control and hypertrophied muscles, respectively (P = 0.591). Suprabasal O(2) uptake per work loop was 5.7 +/- 0.7 pmol/mm(3) in control muscles and 8.7 +/- 1.7 pmol/mm(3) in hypertrophied muscles (P = 0.133). Net mechanical efficiency was calculated from the ratio of work output and suprabasal O(2) uptake. The efficiency of hypertrophied muscles was 29.1 +/- 3.7% and was smaller than in control muscles (43.7 +/- 2.2%, P = 0.016). The right ventricular cardiomyocyte cross-sectional area increased from 272 +/- 17 microm(2) in control muscles to 396 +/- 31 microm(2) in hypertrophied muscles (P < 0.003). Mechanical efficiency correlated negatively with right ventricular wall thickness and cardiomyocyte cross-sectional area [Spearman rank correlation coefficients of -0.50 (P = 0.039) and -0.53 (P = 0.024), respectively]. We conclude that efficiency decreases with increasing cardiomyocyte hypertrophy. Thus, the reduced efficiency of diseased whole hearts can be at least partly explained by reduced efficiency at the cardiomyocyte level.

Endothelin receptor blockade combined with phosphodiesterase-5 inhibition increases right ventricular mitochondrial capacity in pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is often treated with endothelin (ET) receptor blockade or phosphodiesterase-5 (PDE5) inhibition. Little is known about the specific effects on right ventricular (RV) function and metabolism. We determined the effects of single and combination treatment with Bosentan [an ET type A (ET(A))/type B (ET(B)) receptor blocker] and Sildenafil (a PDE5 inhibitor) on RV function and oxidative metabolism in monocrotaline (MCT)-induced PAH. Fourteen days after MCT injection, male Wistar rats were orally treated for 10 days with Bosentan, Sildenafil, or both. RV catheterization and echocardiography showed that MCT clearly induced PAH. This was evidenced by increased RV systolic pressure, reduced cardiac output, increased pulmonary vascular resistance (PVR), and reduced RV fractional shortening. Quantitative histochemistry showed marked RV hypertrophy and fibrosis. Monotreatment with Bosentan or Sildenafil had no effect on RV systolic pressure or cardiac function, but RV fibrosis was reduced and RV capillarization increased. Combination treatment did not reduce RV systolic pressure, but significantly lowered PVR, and normalized cardiac output, RV fractional shortening, and fibrosis. Only combination treatment increased the mitochondrial capacity of the RV, as reflected by increased succinate dehydrogenase and cytochrome c oxidase activities, associated with an activation of PKG, as indicated by increased VASP phosphorylation. Moreover, significant interactions were found between Bosentan and Sildenafil on PVR, cardiac output, RV contractility, PKG activity, and mitochondrial capacity. These data indicate that the combination of Bosentan and Sildenafil may beneficially contribute to RV adaptation in PAH, not only by reducing PVR but also by acting on the mitochondria in the heart.

Metabolic Cost of Activation and Mechanical Efficiency of Mouse Soleus Muscle Fiber Bundles During Repetitive Concentric and Eccentric Contractions.

Currently available data on the energetics of isolated muscle preparations are based on bouts of less than 10 muscle contractions, whereas metabolic energy consumption is mostly relevant during steady state tasks such as locomotion. In this study we quantified the energetics of small fiber bundles of mouse soleus muscle during prolonged (2 min) series of contractions. Bundles (N = 9) were subjected to sinusoidal length changes, while measuring force and oxygen consumption. Stimulation (five pulses at 100 Hz) occurred either during shortening or during lengthening. Movement frequency (2-3 Hz) and amplitude (0.25-0.50 mm; corresponding to ± 4-8% muscle fiber strain) were close to that reported for mouse soleus muscle during locomotion. The experiments were performed at 32°C. The contributions of cross-bridge cycling and muscle activation to total metabolic energy expenditure were separated using blebbistatin. The mechanical work per contraction cycle decreased sharply during the first 10 cycles, emphasizing the importance of prolonged series of contractions. The mean ± SD fraction of metabolic energy required for activation was 0.37 ± 0.07 and 0.56 ± 0.17 for concentric and eccentric contractions, respectively (both 0.25 mm, 2 Hz). The mechanical efficiency during concentric contractions increased with contraction velocity from 0.12 ± 0.03 (0.25 mm 2 Hz) to 0.15 ± 0.03 (0.25 mm, 3 Hz) and 0.16 ± 0.02 (0.50 mm, 2 Hz) and was -0.22 ± 0.08 during eccentric contractions (0.25 mm, 2 Hz). The percentage of type I fibers correlated positively with mechanical efficiency during concentric contractions, but did not correlate with the fraction of metabolic energy required for activation.