Physical and Functional Association of Lactate Dehydrogenase (LDH) with Skeletal Muscle Mitochondria

The intracellular lactate shuttle hypothesis posits that lactate generated in the cytosol is oxidized by mitochondrial lactate dehydrogenase (LDH) of the same cell. To examine whether skeletal muscle mitochondria oxidize lactate, mitochondrial respiratory oxygen flux (JO₂) was measured during the sequential addition of various substrates and cofactors onto permeabilized rat gastrocnemius muscle fibers, as well as isolated mitochondrial subpopulations. Addition of lactate did not alter JO₂. However, subsequent addition of NAD⁺ significantly increased JO₂, and was abolished by the inhibitor of mitochondrial pyruvate transport, α-cyano-4-hydroxycinnamate. In experiments with isolated subsarcolemmal and intermyofibrillar mitochondrial subpopulations, only subsarcolemmal exhibited NAD⁺-dependent lactate oxidation. To further investigate the details of the physical association of LDH with mitochondria in muscle, immunofluorescence/confocal microscopy and immunoblotting approaches were used. LDH clearly colocalized with mitochondria in intact, as well as permeabilized fibers. LDH is likely localized inside the outer mitochondrial membrane, but not in the mitochondrial matrix. Collectively, these results suggest that extra-matrix LDH is strategically positioned within skeletal muscle fibers to functionally interact with mitochondria.     

Mitochondrial energetics is impaired in vivo in aged skeletal muscle

With aging, most skeletal muscles undergo a progressive loss of mass and strength, a process termed sarcopenia. Aging‐related defects in mitochondrial energetics have been proposed to be causally involved in sarcopenia. However, changes in muscle mitochondrial oxidative phosphorylation with aging remain a highly controversial issue, creating a pressing need for integrative approaches to determine whether mitochondrial bioenergetics are impaired in aged skeletal muscle. To address this issue, mitochondrial bioenergetics was first investigated in vivo in the gastrocnemius muscle of adult (6 months) and aged (21 months) male Wistar rats by combining a modular control analysis approach with ³¹P magnetic resonance spectroscopy measurements of energetic metabolites. Using this innovative approach, we revealed that the in vivo responsiveness (‘elasticity’) of mitochondrial oxidative phosphorylation to contraction‐induced increase in ATP demand is significantly reduced in aged skeletal muscle, a reduction especially pronounced under low contractile activities. In line with this in vivo aging‐related defect in mitochondrial energetics, we found that the mitochondrial affinity for ADP is significantly decreased in mitochondria isolated from aged skeletal muscle. Collectively, the results of this study demonstrate that mitochondrial bioenergetics are effectively altered in vivo in aged skeletal muscle and provide a novel cellular basis for this phenomenon.     

A kinetic assay of mitochondrial ADP-ATP exchange rate in permeabilized cells

We previously described a method to measure ADP-ATP exchange rates in isolated mitochondria by recording the changes in free extramitochondrial [Mg²⁺] reported by an Mg²⁺-sensitive fluorescent indicator, exploiting the differential affinity of ADP and ATP to Mg²⁺. In the current article, we describe a modification of this method suited for following ADP-ATP exchange rates in environments with competing reactions that interconvert adenine nucleotides such as in permeabilized cells that harbor phosphorylases and kinases, ion pumps exhibiting substantial ATPase activity, and myosin ATPase activity. Here we report that the addition of BeF₃⁻ and sodium orthovanadate (Na₃VO₄) to medium containing digitonin-permeabilized cells inhibits all ADP-ATP-using reactions except the adenine nucleotide translocase (ANT)-mediated mitochondrial ADP-ATP exchange. An advantage of this assay is that mitochondria that may have been also permeabilized by digitonin do not contribute to ATP consumption by the exposed F₁F_o-ATPase due to its sensitivity to BeF₃⁻ and Na₃VO₄. With this assay, ADP-ATP exchange rate mediated by the ANT in permeabilized cells is measured for the entire range of mitochondrial membrane potential titrated by stepwise additions of an uncoupler and expressed as a function of citrate synthase activity per total amount of protein.     

Mitochondrial function in permeabilized cardiomyocytes is largely preserved in the senescent rat myocardium

The aging heart is characterized by a progressive decline in contractile function and diastolic relaxation. Amongst the factors implicated in these changes is a progressive replacement fibrosis secondary to cardiomyoctye death, oxidative damage, and energetic deficit, each of which may be secondary to impaired mitochondrial function. Here, we performed an in-depth examination of mitochondrial function in saponin-permeabilized cardiomyocyte bundles, a preparation where all mitochondria are represented and their structure intact, from young adult (YA) and senescent (SEN) rats (n = 8 per group). When accounting for increased fibrosis (+19%, P<0.01) and proportional decrease in citrate synthase activity in the SEN myocardium (−23%, P<0.05), mitochondrial respiration and reactive oxygen species (H₂O₂) emission across a range of energized states was similar between age groups. Accordingly, the abundance of electron transport chain proteins was also unchanged. Likewise, except for CuZnSOD (−37%, P<0.05), the activity of antioxidant enzymes was unaltered with aging. Although time to mitochondrial permeability transition pore (mPTP) opening was decreased (−25%, P<0.05) in the SEN heart, suggesting sensitization to apoptotic stimuli, this was not associated with a difference in apoptotic index measured by ELISA. Collectively, our results suggest that the function of existing cardiac ventricular mitochondria is relatively preserved in SEN rat heart when measured in permeabilized cells.     

Scavenging mitochondrial hydrogen peroxide by peroxiredoxin 3 overexpression attenuates contractile dysfunction and muscle atrophy in a murine model of accelerated sarcopenia

Age‐related muscle atrophy and weakness, or sarcopenia, are significant contributors to compromised health and quality of life in the elderly. While the mechanisms driving this pathology are not fully defined, reactive oxygen species, neuromuscular junction (NMJ) disruption, and loss of innervation are important risk factors. The goal of this study is to determine the impact of mitochondrial hydrogen peroxide on neurogenic atrophy and contractile dysfunction. Mice with muscle‐specific overexpression of the mitochondrial H₂O₂ scavenger peroxiredoxin3 (mPRDX3) were crossed to Sod1KO mice, an established mouse model of sarcopenia, to determine whether reduced mitochondrial H₂O₂ can prevent or delay the redox‐dependent sarcopenia. Basal rates of H₂O₂ generation were elevated in isolated muscle mitochondria from Sod1KO, but normalized by mPRDX3 overexpression. The mPRDX3 overexpression prevented the declines in maximum mitochondrial oxygen consumption rate and calcium retention capacity in Sod1KO. Muscle atrophy in Sod1KO was mitigated by ~20% by mPRDX3 overexpression, which was associated with an increase in myofiber cross‐sectional area. With direct muscle stimulation, maximum isometric specific force was reduced by ~20% in Sod1KO mice, and mPRDX3 overexpression preserved specific force at wild‐type levels. The force deficit with nerve stimulation was exacerbated in Sod1KO compared to direct muscle stimulation, suggesting NMJ disruption in Sod1KO. Notably, this defect was not resolved by overexpression of mPRDX3. Our findings demonstrate that muscle‐specific PRDX3 overexpression reduces mitochondrial H₂O₂ generation, improves mitochondrial function, and mitigates loss of muscle quantity and quality, despite persisting NMJ impairment in a murine model of redox‐dependent sarcopenia.     

Proteomic DIGE analysis of the mitochondria‐enriched fraction from aged rat skeletal muscle

Skeletal muscle aging is associated with a loss in tissue mass and contractile strength, as well as fiber type shifting and bioenergetic adaptation processes. Since mitochondria represent the primary site for energy generation via oxidative phosphorylation, we investigated potential changes in the expression pattern of the mitochondrial proteome using the highly sensitive DIGE approach. The comparative analysis of the mitochondria‐enriched fraction from young adult versus aged muscle revealed an age‐related change in abundance for 39 protein species. MS technology identified the majority of altered proteins as constituents of muscle mitochondria. An age‐dependent increase was observed for NADH dehydrogenase, the mitochondrial inner membrane protein mitofilin, peroxiredoxin isoform PRX‐III, ATPase synthase, succinate dehydrogenase, mitochondrial fission protein Fis1, succinate‐coenzyme A ligase, acyl‐coenzyme A dehydrogenase, porin isoform VDAC2, ubiquinol‐cytochrome c reductase core I protein and prohibitin. Immunoblotting, enzyme testing and confocal microscopy were used to validate proteomic findings. The DIGE‐identified increase in key mitochondrial elements during aging agrees with the concept that sarcopenia is associated with a shift to a slower contractile phenotype and more pronounced aerobic‐oxidative metabolism. This suggests that mitochondrial markers are reliable candidates that should be included in the future establishment of a biomarker signature of skeletal muscle aging.     

Mitochondrial structure and function are disrupted by standard isolation methods

Mitochondria regulate critical components of cellular function via ATP production, reactive oxygen species production, Ca(2+) handling and apoptotic signaling. Two classical methods exist to study mitochondrial function of skeletal muscles: isolated mitochondria and permeabilized myofibers. Whereas mitochondrial isolation removes a portion of the mitochondria from their cellular environment, myofiber permeabilization preserves mitochondrial morphology and functional interactions with other intracellular components. Despite this, isolated mitochondria remain the most commonly used method to infer in vivo mitochondrial function. In this study, we directly compared measures of several key aspects of mitochondrial function in both isolated mitochondria and permeabilized myofibers of rat gastrocnemius muscle. Here we show that mitochondrial isolation i) induced fragmented organelle morphology; ii) dramatically sensitized the permeability transition pore sensitivity to a Ca(2+) challenge; iii) differentially altered mitochondrial respiration depending upon the respiratory conditions; and iv) dramatically increased H(2)O(2) production. These alterations are qualitatively similar to the changes in mitochondrial structure and function observed in vivo after cellular stress-induced mitochondrial fragmentation, but are generally of much greater magnitude. Furthermore, mitochondrial isolation markedly altered electron transport chain protein stoichiometry. Collectively, our results demonstrate that isolated mitochondria possess functional characteristics that differ fundamentally from those of intact mitochondria in permeabilized myofibers. Our work and that of others underscores the importance of studying mitochondrial function in tissue preparations where mitochondrial structure is preserved and all mitochondria are represented.     

Operation of the Permeability Transition Pore in Rat Heart Mitochondria in Aging

The functioning of the mitochondrial permeability transition pore (mPTP) is involved in the mechanism of programmed cell death and mitochondrial dysfunction observed with aging. In this work, the functional state of heart mitochondria isolated from young (mature and 2–3-month-old) and old (20–22-month-old) rats under conditions of mPTP opening was studied. In the mitochondria of old rats, the rates of Ca²⁺ and TPP⁺ absorption decreased by 40 and 42%, respectively, the threshold concentration of Ca²⁺ decreased by 20%, and the swelling rate of mitochondria from old animals was by 40% higher than that of mitochondria from young ones. In the heart mitochondria of old animals, the content and production of reactive oxygen species (ROS) varied, the superoxide anion content was increased, and the level of hydroperoxide (H₂O₂) increased at a threshold calcium concentration. Electron microscopy revealed a decrease in the number of cristae in mitochondria of the rat heart during aging. To study the potential role of proteins modulating the mPTP functioning, the content of 2',3'-cyclonucleotide-3'-phosphodiesterase (CNPase) and translocator protein (TSPO) in the heart mitochondria of rats of different ages was measured. A significant age-related decrease in the level of CNPase and an increase in the amount of TSPO were detected. The role of these proteins in mitochondrial dysfunction observed during aging is discussed.     

MICU3 regulates mitochondrial Ca2+-dependent antioxidant response in skeletal muscle aging

Age-related loss of skeletal muscle mass and function, termed sarcopenia, could impair the quality of life in the elderly. The mechanisms involved in skeletal muscle aging are intricate and largely unknown. However, more and more evidence demonstrated that mitochondrial dysfunction and apoptosis also play an important role in skeletal muscle aging. Recent studies have shown that mitochondrial calcium uniporter (MCU)-mediated mitochondrial calcium affects skeletal muscle mass and function by affecting mitochondrial function. During aging, we observed downregulated expression of mitochondrial calcium uptake family member3 (MICU3) in skeletal muscle, a regulator of MCU, which resulted in a significant reduction in mitochondrial calcium uptake. However, the role of MICU3 in skeletal muscle aging remains poorly understood. Therefore, we investigated the effect of MICU3 on the skeletal muscle of aged mice and senescent C2C12 cells induced by d-gal. Downregulation of MICU3 was associated with decreased myogenesis but increased oxidative stress and apoptosis. Reconstitution of MICU3 enhanced antioxidants, prevented the accumulation of mitochondrial ROS, decreased apoptosis, and increased myogenesis. These findings indicate that MICU3 might promote mitochondrial Ca²⁺ homeostasis and function, attenuate oxidative stress and apoptosis, and restore skeletal muscle mass and function. Therefore, MICU3 may be a potential therapeutic target in skeletal muscle aging.Subject terms: Ageing, Calcium and phosphate metabolic disorders     

Proteomic signatures of in vivo muscle oxidative capacity in healthy adults

Adequate support of energy for biological activities and during fluctuation of energetic demand is crucial for healthy aging; however, mechanisms for energy decline as well as compensatory mechanisms that counteract such decline remain unclear. We conducted a discovery proteomic study of skeletal muscle in 57 healthy adults (22 women and 35 men; aged 23–87 years) to identify proteins overrepresented and underrepresented with better muscle oxidative capacity, a robust measure of in vivo mitochondrial function, independent of age, sex, and physical activity. Muscle oxidative capacity was assessed by ³¹P magnetic resonance spectroscopy postexercise phosphocreatine (PCr) recovery time (τ_PCr) in the vastus lateralis muscle, with smaller τ_PCr values reflecting better oxidative capacity. Of the 4,300 proteins quantified by LC‐MS in muscle biopsies, 253 were significantly overrepresented with better muscle oxidative capacity. Enrichment analysis revealed three major protein clusters: (a) proteins involved in key energetic mitochondrial functions especially complex I of the electron transport chain, tricarboxylic acid (TCA) cycle, fatty acid oxidation, and mitochondrial ABC transporters; (b) spliceosome proteins that regulate mRNA alternative splicing machinery, and (c) proteins involved in translation within mitochondria. Our findings suggest that alternative splicing and mechanisms that modulate mitochondrial protein synthesis are central features of the molecular mechanisms aimed at maintaining mitochondrial function in the face of impairment. Whether these mechanisms are compensatory attempt to counteract the effect of aging on mitochondrial function should be further tested in longitudinal studies.     

Mitochondrial death effectors: Relevance to sarcopenia and disuse muscle atrophy

Accelerated apoptosis in skeletal muscle is increasingly recognized as a potential mechanism contributing to the development of sarcopenia of aging and disuse muscle atrophy. Given their central role in the regulation of apoptosis, mitochondria are regarded as key players in the pathogenesis of myocyte loss during aging and other atrophying conditions. Oxidative damage to mitochondrial constituents, impaired respiration and altered mitochondrial turnover have been proposed as potential triggering events for mitochondrial apoptotic signaling. In addition, iron accumulation within mitochondria may enhance the susceptibility to apoptosis during the development of sarcopenia and possibly acute muscle atrophy, likely through exacerbation of oxidative stress. Mitochondria can induce myocyte apoptosis via both caspase-dependent and independent pathways, although the apoptogenic mediators involved may be different depending on age, muscle type and specific atrophying conditions. Despite the considerable advances made, additional research is necessary to establish a definite causal link between apoptotic signaling and the development of sarcopenia and acute atrophy. Furthermore, a translational effort is required to determine the role played by apoptosis in the pathogenesis of sarcopenia and disuse-induced muscle loss in human subjects.     

Mitochondria impairment correlates with increased sensitivity of aging RPE cells to oxidative stress

Impairment of mitochondria function and cellular antioxidant systems are linked to aging and neurodegenerative diseases. In the eye, the retinal pigment epithelium (RPE) is exposed to a highly oxidative environment that contributes to age-related visual dysfunction. Here, we examined changes in mitochondrial function in human RPE cells and sensitivity to oxidative stress with increased chronological age. Primary RPE cells from young (9–20)-, mid-age (48–60)-, and >60 (62–76)-year-old donors were grown to confluency and examined by electron microscopy and flow cytometry using several mitochondrial functional assessment tools. Susceptibility of RPE cells to H₂O₂ toxicity was determined by lactate dehydrogenase and cytochrome c release, as well as propidium iodide staining. Reactive oxygen species, cytoplasmic Ca²⁺ [Ca²⁺]_c, and mitochondrial Ca²⁺ [Ca²⁺]_m levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate, fluo-3/AM, and Rhod-2/AM, respectively, adenosine triphosphate (ATP) levels were measured by a luciferin/luciferase-based assay and mitochondrial membrane potential (ΔΨm) estimated using 5,5′,6,6′-tetrachloro 1,1′3,3′-tetraethylbenzimid azolocarbocyanine iodide. Expression of mitochondrial and antioxidant genes was determined by real-time polymerase chain reaction. RPE cells show greater sensitivity to oxidative stress, reduction in expression of mitochondrial heat shock protein 70, uncoupling protein 2, and superoxide dismutase 3, and greater expression of superoxide dismutase 2 levels with increased chronological age. Changes in mitochondrial number, size, shape, matrix density, cristae architecture, and membrane integrity were more prominent in samples obtained from >60 years old compared to mid-age and younger donors. These mitochondria abnormalities correlated with lower ATP levels, reduced ΔΨm, decreased [Ca²⁺]_c, and increased sequestration of [Ca²⁺]_m in cells with advanced aging. Our study provides evidence for mitochondrial decay, bioenergetic deficiency, weakened antioxidant defenses, and increased sensitivity of RPE cells to oxidative stress with advanced aging. Our findings suggest that with increased severity of mitochondrial decay and oxidative stress, RPE function may be altered in some individuals in a way that makes the retina more susceptible to age-related injury.     

Mitochondrial DNA Mutations Induce Mitochondrial Dysfunction,Apoptosis and Sarcopenia in Skeletal Muscle of Mitochondrial DNA Mutator Mice

Background

Aging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.

Methodology/Principal Findings

We investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψ_m). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.

Conclusions/Significance

These findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.     

How might you compare mitochondria from different tissues and different species?

Mitochondria were isolated from the liver, kidney and mixed hindlimb skeletal muscle of three vertebrate species; the laboratory rat Rattus norvegicus, the bearded dragon lizard Pogona vitticeps, and the cane toad Bufo marinus. These vertebrate species are approximately the same body mass and have similar body temperatures. The content of cytochromes B, C, C1, and A were measured in these isolated mitochondria by oxidised–reduced difference spectra. Adenine nucleotide translocase (ANT) was measured by titration of mitochondrial respiration with carboxyactractyloside and the protein and phospholipid content of isolated mitochondria were also measured. Fatty acid composition of mitochondrial phospholipids was measured. Mitochondrial respiration was measured at 37°C under states III and IV conditions as well as during oligomycin inhibition. Species differed in the ratios of different mitochondrial cytochromes. Muscle mitochondria differed from kidney and liver mitochondria by having a higher ANT content relative to cytochrome content. Respiration rates were compared relative to a number of denominators and found to be most variable when expressed relative to mitochondrial protein content and least variable when expressed relative to mitochondrial cytochrome A and ANT content. The turnover of cytochromes was calculated and found to vary between 1 and 94 electrons s⁻¹. The molecular activity of mitochondrial cytochromes was found to be significantly positively correlated with the relative polyunsaturation of mitochondrial membrane lipids.     

MITOCHONDRIA: Investigation of in vivo muscle mitochondrial function by 31P magnetic resonance spectroscopy

The most important function of mitochondria is the production of energy in the form of ATP. The socio-economic impact of human diseases that affect skeletal muscle mitochondrial function is growing, and improving their clinical management critically depends on the development of non-invasive assays to assess mitochondrial function and monitor the effects of interventions. ³¹P magnetic resonance spectroscopy provides two approaches that have been used to assess in vivo ATP synthesis in skeletal muscle: measuring P_i ⟶ ATP exchange flux using saturation transfer in resting muscle, and measuring phosphocreatine recovery kinetics after exercise. However, P_i ⟶ ATP exchange does not represent net mitochondrial ATP synthesis flux and has no simple relationship with mitochondrial function. Post-exercise phosphocreatine recovery kinetics, on the other hand, yield reliable measures of muscle mitochondrial capacity in vivo, whose ability to define the site of functional defects is enhanced by combination with other non-invasive techniques.     

Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport

Alloxan at millimolar concentrations slightly inhibited the velocity of Ca²⁺ uptake by isolated rat liver mitochondria irrespective of the free Ca²⁺ concentration between 1 and 10 µM and was an effective concentration-dependent stimulator of mitochondrial Ca²⁺ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca²⁺ uptake but only at free Ca²⁺ concentrations above 5 µM. However, ninhydrin was a strong stimulator of mitochondrial Ca²⁺ efflux even at micromolar concentrations, 10–50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10–20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca²⁺ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca²⁺ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca²⁺ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca²⁺ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca²⁺ uptake. The direct effects of ninhydrin on mitochondrial Ca²⁺ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca²⁺ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca²⁺ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan. Mol Cell Biochem 118: 141–151, 1992)     

Antioxidant effect of human placenta hydrolysate against oxidative stress on muscle atrophy

Sarcopenia, which refers to the muscle loss that accompanies aging, is a complex neuromuscular disorder with a clinically high prevalence and mortality. Despite many efforts to protect against muscle weakness and muscle atrophy, the incidence of sarcopenia and its related permanent disabilities continue to increase. In this study, we found that treatment with human placental hydrolysate (hPH) significantly increased the viability (approximately 15%) of H₂O₂-stimulated C2C12 cells. Additionally, while H₂O₂-stimulated cells showed irregular morphology, hPH treatment restored their morphology to that of cells cultured under normal conditions. We further showed that hPH treatment effectively inhibited H₂O₂-induced cell death. Reactive oxygen species (ROS) generation and Mstn expression induced by oxidative stress are closely associated with muscular dysfunction followed by atrophy. Exposure of C2C12 cells to H₂O₂ induced abundant production of intracellular ROS, mitochondrial superoxide, and mitochondrial dysfunction as well as myostatin expression via nuclear factor-κB (NF-κB) signaling; these effects were attenuated by hPH. Additionally, hPH decreased mitochondria fission–related gene expression (Drp1 and BNIP3) and increased mitochondria biogenesis via the Sirt1/AMPK/PGC-1α pathway and autophagy regulation. In vivo studies revealed that hPH-mediated prevention of atrophy was achieved predominantly through regulation of myostatin and PGC-1α expression and autophagy. Taken together, our findings indicate that hPH is potentially protective against muscle atrophy and oxidative cell death.     

The best approach: Homogenization or manual permeabilization of human skeletal muscle fibers for respirometry?

The number of studies on mitochondrial function is growing as a result of the recognition of the pivotal role of an intact mitochondrial function in numerous diseases. Measurements of oxygen consumption by the mitochondria in human skeletal muscle are used in many studies. There are several advantages of studying mitochondrial respiration in permeabilized fibers (Pfi), but the method requires a manual procedure of mechanical separation of the fiber bundles in the biopsy and chemical permeabilization of the cell membrane. This is time-consuming and subject to interpersonal variability. An alternative is to use a semiautomatic tool for preparation of a homogenate of the muscle biopsy. We investigated whether the PBI shredder is useful in preparing a muscle homogenate for measurements of mitochondrial respiratory capacity. The homogenate is compared with the Pfi preparation. Maximal respiratory capacity was significantly reduced in the homogenate compared with the Pfi from human skeletal muscle. A marked cytochrome c response was observed in the homogenate, which was not the case with the Pfi, indicating that the outer mitochondrial membrane was not intact. The mitochondria in the homogenate were more uncoupled compared with the Pfi. Manual permeabilization is an advantageous technique for preparing human skeletal muscle biopsies for respirometry.