Mitochondrial Protein Content Research Articles

Energy Metabolism Behavior and Response to Microenvironmental Factors of the Experimental Cancer Cell Models Differ From That of Actual Human Tumors.

Analysis of the biochemical differences in the energy metabolism among bi-dimensional (2D) and tri-dimensional (3D) cultured cancer cell models and actual human tumors was undertaken. In 2D cancer cells, the oxidative phosphorylation (OxPhos) fluxes range is 2.5-19 nmol O2/min/mg cellular protein. Hypoxia drastically decreased OxPhos flux by 2-3 times in 2D models, similar to what occurs in mature multicellular tumor spheroids (MCTS), a representative 3D cancer cell model. However, mitochondrial protein contents and enzyme activities were significantly different between both models. Moreover, glycolytic fluxes were also significantly different between 2D and MCTS. The glycolytic flux range in 2D models is 1-34 nmol lactate/min/mg cellular protein, whereas in MCTS the range of glycolysis fluxes is 60-80 nmol lactate/min/mg cellular. In addition, sensitivity to anticancer canonical and metabolic drugs was greater in MCTS than in 2D. Actual solid human tumor samples show lower (1.6-4.5 times) OxPhos fluxes compared to normoxic 2D cancer cell cultures. These observations indicate that tridimensional organization provides a unique microenvironment affecting tumor physiology, which has not been so far faithfully reproduced by the 2D environment. Thus, the analysis of the resemblances and differences among cancer cell models undertaken in the present study raises caution on the interpretation of results derived from 2D cultured cancer cells when they are extended to clinical settings. It also raises awareness about detecting which biological and environmental factors are missing in 2D and 3D cancer cell models to be able to reproduce the actual human tumor behavior.

Loss of atrial natriuretic peptide signaling causes insulin resistance, mitochondrial dysfunction, and low endurance capacity.

Type 2 diabetes (T2D) and obesity are strongly associated with low natriuretic peptide (NP) plasma levels and a down-regulation of NP guanylyl cyclase receptor-A (GCA) in skeletal muscle and adipose tissue. However, no study has so far provided evidence for a causal link between atrial NP (ANP)/GCA deficiency and T2D pathogenesis. Here, we show that both systemic and skeletal muscle ANP/GCA deficiencies in mice promote metabolic disturbances and prediabetes. Skeletal muscle insulin resistance is further associated with altered mitochondrial function and impaired endurance running capacity. ANP/GCA-deficient mice exhibit increased proton leak and reduced content of mitochondrial oxidative phosphorylation proteins. We further show that GCA is related to several metabolic traits in T2D and positively correlates with markers of oxidative capacity in human skeletal muscle. Together, these results indicate that ANP/GCA signaling controls muscle mitochondrial integrity and oxidative capacity in vivo and plays a causal role in the development of prediabetes.

A partial loss-of-function variant (Ile191Val) of the TAS1R2 glucose receptor is associated with enhanced responses to exercise training in older adults with obesity: A translational study

BackgroundThe TAS1R2 receptor, known for its role in taste perception, has also emerged as a key regulator of muscle physiology. Previous studies have shown that genetic ablation of TAS1R2 in mice enhances muscle fitness mimicking responses to endurance exercise training. However, the translational relevance of these findings to humans remains uncertain. MethodsWe explored responses to endurance exercise training in mice and humans with genetic deficiency of TAS1R2. First, we assessed the effects of muscle-specific deletion of TAS1R2 in mice (mKO) or wild type controls (mWT) following 4 weeks of voluntary wheel running (VWR). Next, we investigated the effects of the TAS1R2-Ile191Val (rs35874116) partial loss-of-function variant on responses to a 6-month diet-induced weight loss with exercise training (WLEX), weight loss alone (WL), or education control (CON) interventions in older individuals with obesity. Participants were retrospectively genotyped for the TAS1R2-Ile191Val polymorphism and classified as conventional function (Ile/Ile) or partial loss-of-function (Val carriers: Ile/Val and Val/Val). Body composition, cardiorespiratory fitness, and skeletal muscle mitochondrial function were assessed before and after the intervention. ResultsIn response to VWR, mKO mice demonstrated enhanced running endurance and mitochondrial protein content. Similarly, TAS1R2 Val carriers exhibited distinctive improvements in body composition, including increased muscle mass, along with enhanced cardiorespiratory fitness and mitochondrial function in skeletal muscle following the WLEX intervention compared to Ile/Ile counterparts. Notably, every Val carrier demonstrated substantial responses to exercise training and weight loss, surpassing all Ile/Ile participants in overall performance metrics. ConclusionsOur findings suggest that TAS1R2 partial loss-of-function confers beneficial effects on muscle function and metabolism in humans in response to exercise training, akin to observations in TAS1R2 muscle-deficient mice. Targeting TAS1R2 may help enhancing exercise training adaptations in individuals with compromised exercise tolerance or metabolic disorders, presenting a potential avenue for personalized exercise interventions.

Amino acids trigger MDC-dependent mitochondrial remodeling by altering mitochondrial function.

Cells utilize numerous pathways to maintain mitochondrial homeostasis, including a recently identified mechanism that adjusts the content of the outer mitochondrial membrane (OMM) through formation of OMM-derived multilamellar domains called mitochondrial-derived compartments, or MDCs. MDCs are triggered by perturbations in mitochondrial lipid and protein content, as well as increases in intracellular amino acids. Here, we sought to understand how amino acids trigger MDCs. We show that amino acid-activation of MDCs is dependent on the functional state of mitochondria. While amino acid excess triggers MDC formation when cells are grown on fermentable carbon sources, stimulating mitochondrial biogenesis blocks MDC formation. Moreover, amino acid elevation depletes TCA cycle metabolites in yeast, and preventing consumption of TCA cycle intermediates for amino acid catabolism suppresses MDC formation. Finally, we show that directly impairing the TCA cycle is sufficient to trigger MDC formation in the absence of amino acid stress. These results demonstrate that amino acids stimulate MDC formation by perturbing mitochondrial metabolism.

Short-term aerobic exercise prevents development of glucocorticoid myopathic features in aged skeletal muscle in a sex-dependent manner.

Older adults are vulnerable to glucocorticoid-induced muscle atrophy and weakness, with sex potentially influencing their susceptibility to those effects. Aerobic exercise can reduce glucocorticoid-induced muscle atrophy in young rodents. However, it is unknown whether aerobic exercise can prevent glucocorticoid myopathy in aged muscle. The objectives of this study were to define the extent to which sex influences the development of glucocorticoid myopathy in aged muscle, and to determine the extent to which aerobic exercise training protects against myopathy development. Twenty-four-month-old female (n = 30) and male (n = 33) mice were randomized to either sedentary or aerobic exercise groups. Within their respective groups, mice were randomized to either daily treatment with dexamethasone (DEX) or saline. Upon completing treatments, the contractile properties of the triceps surae complex were assessed in situ. DEX marginally lowered muscle mass and soluble protein content in both sexes, which was attenuated by aerobic exercise only in females. DEX increased sub-tetanic force and rate of force development only in females, which was not influenced by aerobic exercise. Muscle fatigue was higher in both sexes following DEX, but aerobic exercise prevented fatigue induction only in females. The sex-specific differences to muscle function in response to DEX treatment coincided with sex-specific changes to the content of proteins related to calcium handling, mitochondrial quality control, reactive oxygen species production, and glucocorticoid receptor in muscle. These findings define several important sexually dimorphic changes to aged skeletal muscle physiology in response to glucocorticoid treatment and define the capacity of short-term aerobic exercise to protect against those changes. KEY POINTS: There are sexually dimorphic effects of glucocorticoids on aged skeletal muscle physiology. Glucocorticoid-induced changes to aged muscle contractile properties coincide with sex-specific differences in the content of calcium handling proteins. Aerobic exercise prevents glucocorticoid-induced fatigue only in aged females and coincides with differences in the content of mitochondrial quality control proteins and glucocorticoid receptors.

The influence of sex, hemoglobin mass, and skeletal muscle characteristics on cycling critical power.

Critical power (CP) represents an important threshold for exercise performance and fatiguability. We sought to determine the extent to which sex, hemoglobin mass (Hbmass), and skeletal muscle characteristics influence CP. Before CP determination (i.e., 3-5 constant work rate trials to task failure), Hbmass and skeletal muscle oxidative capacity (τ) were measured and vastus lateralis (VL) muscle biopsy samples were collected from 12 females and 12 males matched for aerobic fitness relative to fat-free mass (FFM) [means (SD); V̇o2max: 59.2 (7.7) vs. 59.5 (7.1) mL·kg·FFM-1·min-1, respectively]. Males had a significantly greater CP than females in absolute units [225 (28) vs. 170 (43) W; P = 0.001] but not relative to body mass [3.0 (0.6) vs. 2.7 (0.6) W·kg·BM-1; P = 0.267] or FFM [3.6 (0.7) vs. 3.7 (0.8) W·kg·FFM-1; P = 0.622]. Males had significantly greater W' (P ≤ 0.030) and greater Hbmass (P ≤ 0.016) than females, regardless of the normalization approach; however, there were no differences in mitochondrial protein content (P = 0.375), τ (P = 0.603), or MHC I proportionality (P = 0.574) between males and females. Whether it was expressed in absolute or relative units, CP was positively correlated with Hbmass (0.444 ≤ r ≤ 0.695; P < 0.05), mitochondrial protein content (0.413 ≤ r ≤ 0.708; P < 0.05), and MHC I proportionality (0.506 ≤ r ≤ 0.585; P < 0.05), and negatively correlated with τ when expressed in relative units only (-0.588 ≤ r ≤ -0.527; P < 0.05). Overall, CP was independent of sex, but variability in CP was related to Hbmass and skeletal muscle characteristics. The extent to which manipulations in these physiological parameters influence CP warrants further investigation to better understand the factors underpinning CP.NEW & NOTEWORTHY In males and females matched for aerobic fitness [maximal oxygen uptake normalized to fat-free mass (FFM)], absolute critical power (CP) was greater in males, but relative CP (per kilogram body mass or FFM) was similar between sexes. CP correlated with hemoglobin mass, mitochondrial protein content, myosin heavy chain type I proportion, and skeletal muscle oxidative capacity. These findings demonstrate the importance of matching sexes for aerobic fitness, but further experiments are needed to determine causality.

Adaptive thermogenesis of the brown adipose tissue in Apodemus chevrieri during cold acclimation

Abstract To investigate the effect of low temperature on body mass, thermogenic activity, and Uncoupling protein-1 (UCP1) content of brown adipose tissue (BAT) in Chevrier’s field mouse (Apodemus chevrieri), 50 healthy adult mice with similar body mass were used in our experiment. They were divided into five groups of ten individuals as follows: a control group (0 d), where animals were maintained under 25 ± 1°C and a 12L:12D (light:dark, lights on 08:00) photoperiod; the other groups were maintained under 5 ± 1°C and a 12L:12D photoperiod for 7 d, 14 d, 21 d and 28 d, respectively. At the end of the experiment, the changes in body mass, resting metabolic rate (RMR), nonshivering thermogenesis (NST), BAT mass, mitochondrial protein (MP) content, cytochrome c oxidase (COX) activity, and UCP1 content were measured. The results showed that compared with the control group (0 d), the body mass of the cold acclimation groups decreased significantly. In contrast, RMR, NST, BAT mass, MP content, COX activity, and UCP1 content of the cold acclimation group increased significantly. After cold acclimation for 28 days, RMR increased by 89.4%, NST increased by 50.4%, BAT mass increased by 44.6%, and UCP1 content increased by 36.0%. The ratio of (NST − RMR)/RMR was 1.03 after seven days and then dropped to 0.59 on day 21, and remained at a steady level thereafter. Evidently, NST was significantly positively correlated with BAT mass and UCP1 content. The results indicated that under continuous cold exposure, A. chevrieri could take appropriate measures to reduce body mass, increase RMR, induce BAT tissue proliferation, and unregulated UCP1 expression, thereby enhancing BAT thermogenic activity to cope with a low-temperature environment. In the early stage of cold acclimation, NST was dominant in thermogenesis, but as time continued, it reduced. This may represent a unique energy adaptation strategy of small rodents originating from the north and spreading southward toward the Hengduan Mountain regions.

Defects of mitochondria-lysosomes communication induce secretion of mitochondria-derived vesicles and drive chemoresistance in ovarian cancer cells

BackgroundAmong the mechanisms of mitochondrial quality control (MQC), generation of mitochondria-derived vesicles (MDVs) is a process to avoid complete failure of mitochondria determining lysosomal degradation of mitochondrial damaged proteins. In this context, RAB7, a late endocytic small GTPase, controls delivery of MDVs to late endosomes for subsequent lysosomal degradation. We previously demonstrated that RAB7 has a pivotal role in response to cisplatin (CDDP) regulating resistance to the drug by extracellular vesicle (EVs) secretion.MethodsWestern blot and immunofluorescence analysis were used to analyze structure and function of endosomes and lysosomes in CDDP chemosensitive and chemoresistant ovarian cancer cell lines. EVs were purified from chemosensitive and chemoresistant cells by ultracentrifugation or immunoisolation to analyze their mitochondrial DNA and protein content. Treatment with cyanide m-chlorophenylhydrazone (CCCP) and RAB7 modulation were used, respectively, to understand the role of mitochondrial and late endosomal/lysosomal alterations on MDV secretion. Using conditioned media from chemoresistant cells the effect of MDVs on the viability after CDDP treatment was determined. Seahorse assays and immunofluorescence analysis were used to study the biochemical role of MDVs and the uptake and intracellular localization of MDVs, respectively.ResultsWe observed that CDDP-chemoresistant cells are characterized by increased MDV secretion, impairment of late endocytic traffic, RAB7 downregulation, an increase of RAB7 in EVs, compared to chemosensitive cells, and downregulation of the TFEB-mTOR pathway overseeing lysosomal and mitochondrial biogenesis and turnover. We established that MDVs can be secreted rather than delivered to lysosomes and are able to deliver CDDP outside the cells. We showed increased secretion of MDVs by chemoresistant cells ultimately caused by the extrusion of RAB7 in EVs, resulting in a dramatic drop in its intracellular content, as a novel mechanism to regulate RAB7 levels. We demonstrated that MDVs purified from chemoresistant cells induce chemoresistance in RAB7-modulated process, and, after uptake from recipient cells, MDVs localize to mitochondria and slow down mitochondrial activity.ConclusionsDysfunctional MQC in chemoresistant cells determines a block in lysosomal degradation of MDVs and their consequent secretion, suggesting that MQC is not able to eliminate damaged mitochondria whose components are secreted becoming effectors and potential markers of chemoresistance.

Cannabidiol protects C2C12 myotubes against cisplatin-induced atrophy by regulating oxidative stress.

Cancer and chemotherapy induce a severe loss of muscle mass (known as cachexia), which negatively impact cancer treatment and patient survival. The aim of the present study was to investigate whether cannabidiol (CBD) administration may potentially antagonize the effects of cisplatin in inducing muscle atrophy, using a model of myotubes in culture. Cisplatin treatment resulted in a reduction of myotube diameter (15.7 ± 0.3 vs. 22.2 ± 0.5 µm, P < 0.01) that was restored to control level with 5 µM CBD (20.1 ± 0.4 µM, P < 0.01). Protein homeostasis was severely altered with a ≈70% reduction in protein synthesis (P < 0.01) and a twofold increase in proteolysis (P < 0.05) in response to cisplatin. Both parameters were dose dependently restored by CBD cotreatment. Cisplatin treatment was associated with increased thiobarbituric acid reactive substances (TBARS) content (0.21 ± 0.03 to 0.48 ± 0.03 nmol/mg prot, P < 0.05), catalase activity (0.24 ± 0.01 vs. 0.13 ± 0.02 nmol/min/µg prot, P < 0.01), whereas CBD cotreatment normalized TBARS content to control values (0.22 ± 0.01 nmol/mg prot, P < 0.01) and reduced catalase activity (0.17 ± 0.01 nmol/min/µg prot, P < 0.05). These changes were associated with increased mRNA expression of GPX1, SOD1, SOD2, and CAT mRNA expression in response to cisplatin (P < 0.01), which was corrected by CBD cotreatment (P < 0.05). Finally, cisplatin treatment increased the mitochondrial protein content of NDUFB8, UQCRC2, COX4, and VDAC1 (involved in mitochondrial respiration and apoptosis), and CBD cotreatment restored their expression to control values. Altogether, our results demonstrated that CBD antagonize the cisplatin-induced C2C12 myotube atrophy and could be used as an adjuvant in the treatment of cancer cachexia to help maintain muscle mass and improve patient quality of life.NEW & NOTEWORTHY In an in vitro model, cisplatin treatment led to myotube atrophy associated with dysregulation of protein homeostasis and increased oxidative stress, resulting in increased apoptosis. Cotreatment with cannabidiol was able to prevent this phenotype by promoting protein homeostasis and reducing oxidative stress.

Abstract WP18: Hepatic Copper Dysregulation Induced Neuronal Cuproptosis in Ischemic Stroke: Insights Into Mechanisms and Therapeutic Efficacy of Remote Liver Hypothermia

Objective: Serum copper level was increased in ischemic stroke, which was associated with disorder in hepatic copper metabolism. This study aims to determine the link between the elevation in serum copper levels following ischemic stroke, the consequent disorder in hepatic copper metabolism, and copper-induced neuronal death (cuproptosis). We sought to elucidate the role of remote administration of hypothermia (RAH) in liver, attenuating these detrimental processes and mitigating associated brain injuries. Methods: We subjected 80 adult Sprague-Dawley rats to 2-h middle cerebral artery occlusion (MCAO) followed by 6, 24 or 48 h reperfusion. Brain damage was evaluated by infarct volume, neurological deficit, and apoptotic cell death. Cuproptosis was measured by cerebral copper content and expressions of copper uptake protein (SLC31A1) and mitochondrial function proteins (SDHB, LIAS, FDX1, DPYD). Liver injury was determined by AST, ALT, and LDH in serum. Hepatic and serum copper metabolism, and regulatory molecules (HIF-1α, ceruloplasmin) were measured. In order to suppress liver copper metabolism and thus brain cuproptosis, RAH in liver was conducted 30 minutes pre-reperfusion. Results: Significant infarction, neurological deficit, and heightened apoptotic cell death were observed after stroke. Cerebral copper content and SLC31A1 expression were increased, while expressions of SDHB, LIAS, FDX1, and DPYD were diminished post-ischemic stroke, indicating a mitochondrial-related neuronal cuproptosis. 24 h post-reperfusion, there was a surge in serum liver injury markers. HIF-1α and ceruloplasmin were increased in liver post-stroke, with a concomitant decrease in hepatic copper and increase in serum copper levels. RAH mitigated hepatic copper imbalance and lowered serum copper via the HIF-1α/ceruloplasmin pathway, thus significantly attenuated neuronal cuproptosis, minimizing brain injury. Conclusions: The increase of cerebral copper content in ischemic stroke was derived by liver, which, in turn, intensified mitochondrial-related neuronal cuproptosis, exacerbating brain injury. RAH effectively counteracted the hepatic copper imbalances and hampered neuronal cuproptosis, producing neuroprotective benefits.

Synergistic celecoxib and dimethyl-celecoxib combinations block cervix cancer growth through multiple mechanisms.

The synergistic inhibitory effect of celecoxib (CXB) and dimethyl-celecoxib (DMC) plus paclitaxel (PA) or cisplatin (CP) on human cervix HeLa and SiHa cells was assessed at multiple cellular levels in order to elucidate the biochemical mechanisms triggered by the synergistic drug combinations. The effect of CXB (5 μM)/CP (2 μM) or CXB (5 μM)/PA (15 μM) and DMC (15 μM)/CP (5 μM) or DMC (15 μM)/PA (20 μM) for 24 h was assayed on cancer cell proliferation, energy metabolism, mitophagy, ROS production, glycoprotein-P activity, DNA stability and apoptosis/necrosis. Drug combinations synergistically decreased HeLa and SiHa cell proliferation (>75%) and arrested cellular cycle by decreasing S and G2/M phases as well as the Ki67 content (HeLa) by 7.5-30 times. Cell viability was preserved (>90%) and no apparent effects on non-cancer cell growth were observed. Mitochondrial and glycolytic protein contents (44-95%) and ΔΨm (45-50%) in HeLa cells and oxidative phosphorylation and glycolysis fluxes (70-90%) in HeLa and SiHa cells were severely decreased, which in turn promoted a drastic fall in the ATP supply (85-88%). High levels of mitophagy proteins in HeLa cells and active mitochondrial digestion in HeLa and SiHa cells was observed. Mitochondrial fission and microtubule proteins were also affected. Intracellular ROS content (2-2.3-fold) and ROS production was stimulated (2.3-4 times), whereas content and activity of glycoprotein-P (45-85%) were diminished. DNA fragmentation was not observed and apoptosis/necrosis was not detected suggesting that cell death could be mainly associated to mitophagy induction. CXB or DMC combination with canonical chemotherapy may be a promising chemotherapy strategy against cervical cancer growth, because it can selectively block multiple cell processes including inhibition of energy pathways and in consequence ATP-dependent processes such as cell proliferation, glycoprotein-P activity, ROS production and mitophagy, with no apparent effects on non-cancer cells.

Contraction intensity affects NIRS-derived skeletal muscle oxidative capacity but not its relationships to mitochondrial protein content or aerobic fitness.

To further refine the near-infrared spectroscopy (NIRS)-derived measure of skeletal muscle oxidative capacity in humans, we sought to determine whether the exercise stimulus intensity affected the τ value and/or influenced the magnitude of correlations with in vitro measures of mitochondrial content and in vivo indices of exercise performance. Males (n = 12) and females (n = 12), matched for maximal aerobic fitness per fat-free mass, completed NIRS-derived skeletal muscle oxidative capacity tests for the vastus lateralis following repeated contractions at 40% (τ40) and 100% (τ100) of maximum voluntary contraction, underwent a skeletal muscle biopsy of the same muscle, and performed multiple intermittent isometric knee extension tests to task failure to establish critical torque (CT). The value of τ100 (34.4 ± 7.0 s) was greater than τ40 (24.2 ± 6.9 s, P < 0.001), but the values were correlated (r = 0.688; P < 0.001). The values of τ40 (r = -0.692, P < 0.001) and τ100 (r = -0.488, P = 0.016) correlated with myosin heavy chain I percentage and several markers of mitochondrial content, including COX II protein content in whole muscle (τ40: r = -0.547, P = 0.006; τ100: r = -0.466, P = 0.022), type I pooled fibers (τ40: r = -0.547, P = 0.006; τ100: r = -0.547, P = 0.006), and type II pooled fibers (τ40: r = -0.516, P = 0.009; τ100: r = -0.635, P = 0.001). The value of τ40 (r = -0.702, P < 0.001), but not τ100 (r = -0.378, P = 0.083) correlated with critical torque (CT); however, neither value correlated with W' (τ40: r = 0.071, P = 0.753; τ100: r = 0.054, P = 0.812). Overall, the NIRS method of assessing skeletal muscle oxidative capacity is sensitive to the intensity of skeletal muscle contraction but maintains relationships to whole body fitness, isolated limb critical intensity, and mitochondrial content regardless of intensity.NEW & NOTEWORTHY Skeletal muscle oxidative capacity measured using near-infrared spectroscopy (NIRS) was lower following high-intensity compared with low-intensity isometric knee extension contractions. At both intensities, skeletal muscle oxidative capacity was correlated with protein markers of mitochondrial content (in whole muscle and pooled type I and type II muscle fibers) and critical torque. These findings highlight the importance of standardizing contraction intensity while using the NIRS method with isometric contractions and further demonstrate its validity.

Sex-Specific Effect of CARM1 in Skeletal Muscle Adaptations to Exercise.

The purpose of this study was to determine how the intersection of coactivator-associated arginine methyltransferase 1 (CARM1) and biological sex affects skeletal muscle adaptations to chronic physical activity. Twelve-week-old female (F) and male (M) wild-type (WT) and CARM1 skeletal muscle-specific knockout (mKO) mice were randomly assigned to sedentary (SED) or voluntary wheel running (VWR) experimental groups. For 8 wk, the animals in the VWR cohort had volitional access to running wheels. Subsequently, we performed whole-body functional tests, and 48 h later muscles were harvested for molecular analysis. Western blotting, enzyme activity assays, as well as confocal and transmission electron microscopy were used to examine skeletal muscle biology. Our data reveal a sex-dependent reduction in VWR volume caused by muscle-specific ablation of CARM1, as F CARM1 mKO mice performed less chronic, volitional exercise than their WT counterparts. Regardless of VWR output, exercise-induced adaptations in physiological function were similar between experimental groups. A broad panel of protein arginine methyltransferase (PRMT) biology measurements, including markers of arginine methyltransferase expression and activity, were unaffected by VWR, except for CARM1 and PRMT7 protein levels, which decreased and increased with VWR, respectively. Changes in myofiber morphology and mitochondrial protein content showed similar trends among animals. However, a closer examination of transmission electron microscopy images revealed contrasting responses to VWR in CARM1 mKO mice compared with WT littermates, particularly in mitochondrial size and fractional area. The present findings demonstrate that CARM1 mKO reduces daily running volume in F mice, as well as exercise-evoked skeletal muscle mitochondrial plasticity, which indicates that this enzyme plays an essential role in sex-dependent differences in exercise performance and mitochondrial health.

PPTC7 maintains mitochondrial protein content by suppressing receptor-mediated mitophagy

PPTC7 is a resident mitochondrial phosphatase essential for maintaining proper mitochondrial content and function. Newborn mice lacking Pptc7 exhibit aberrant mitochondrial protein phosphorylation, suffer from a range of metabolic defects, and fail to survive beyond one day after birth. Using an inducible knockout model, we reveal that loss of Pptc7 in adult mice causes marked reduction in mitochondrial mass and metabolic capacity with elevated hepatic triglyceride accumulation. Pptc7 knockout animals exhibit increased expression of the mitophagy receptors BNIP3 and NIX, and Pptc7-/- mouse embryonic fibroblasts (MEFs) display a major increase in mitophagy that is reversed upon deletion of these receptors. Our phosphoproteomics analyses reveal a common set of elevated phosphosites between perinatal tissues, adult liver, and MEFs, including multiple sites on BNIP3 and NIX, and our molecular studies demonstrate that PPTC7 can directly interact with and dephosphorylate these proteins. These data suggest that Pptc7 deletion causes mitochondrial dysfunction via dysregulation of several metabolic pathways and that PPTC7 may directly regulate mitophagy receptor function or stability. Overall, our work reveals a significant role for PPTC7 in the mitophagic response and furthers the growing notion that management of mitochondrial protein phosphorylation is essential for ensuring proper organelle content and function.

The Effect of SERCA Activation on Functional Characteristics and Signaling of Rat Soleus Muscle upon 7 Days of Unloading.

Skeletal muscle abnormalities and atrophy during unloading are accompanied by the accumulation of excess calcium in the sarcoplasm. We hypothesized that calcium accumulation may occur, among other mechanisms, due to the inhibition of sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) activity. Consequently, the use of the SERCA activator will reduce the level of calcium in the sarcoplasm and prevent the negative consequences of muscle unloading. Wistar rats were randomly assigned into one of three groups (eight rats per group): control rats with placebo (C), 7 days of unloading/hindlimb suspension with placebo (7HS), and 7 days of unloading treated with SERCA activator CDN1163 (7HSC). After seven days of unloading the soleus muscle, the 7HS group displayed increased fatigue in the ex vivo test, a significant increase in the level of calcium-dependent CaMK II phosphorylation and the level of tropomyosin oxidation, as well as a decrease in the content of mitochondrial DNA and protein, slow-type myosin mRNA, and the percentage of slow-type muscle fibers. All of these changes were prevented in the 7HSC group. Moreover, treatment with CDN1163 blocked a decrease in the phosphorylation of p70S6k, an increase in eEF2 phosphorylation, and an increase in MuRF-1 mRNA expression. Nevertheless, there were no differences in the degree of fast and slow muscle fiber atrophy between the 7HS and 7HSC groups. Conclusion: SERCA activation during 7 days of unloading prevented an increase in soleus fatigue, the decrease of slow-type myosin, mitochondrial markers, and markers of calcium homeostasis but had no effect on muscle atrophy.

Influence of the SERCA Activity on Rat’s Soleus Contractile Properties during Functional Unloading

Dysfunction of skeletal muscles and their atrophy during unloading are accompanied by excess calcium accumulation in the myoplasm of muscle fibers. We hypothesized that calcium accumulation may occur, among other reasons, due to inhibition of SERCA activity under muscle unloading. In this case, the use of a SERCA activator will reduce the calcium level in the myoplasm and prevent the consequences of unloading. Male Wistar rats were divided into 3 groups: vivarium control with placebo administration (C, n = 8), 7-day suspension group with placebo administration (7HS, n = 8) and 7-day suspension group with intraperitoneal administration of SERCA CDN1163 activator (50 mg/kg (7HS + CDN), n = 8). One m. soleus of each rat was frozen in liquid nitrogen, the second was tested for functional properties. In the 7HS group, increased soleus fatigue was found in the ex vivo test, a significant increase in mRNA and the number of fast muscle fibers, an increase in the level of calcium-dependent CaMK II phosphorylation and the level of tropomyosin oxidation, as well as a decrease in the content of mitochondrial DNA and protein. All these changes were prevented in the SERCA CDN1163 activator group. Conclusion: 7-day SERCA activator administration does not delay of soleus atrophy, but prevents the development of its fatigue, probably by preventing a decrease in the number of type I fibers and markers of mitochondrial biogenesis.

Dietary nitrate preserves mitochondrial bioenergetics and mitochondrial protein synthesis rates during short-term immobilization in mice.

Skeletal muscle disuse reduces muscle protein synthesis rates and induces atrophy, events associated with decreased mitochondrial respiration and increased reactive oxygen species. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation attenuates disuse-induced impairments in mitochondrial function and muscle protein synthesis rates. Female C57Bl/6N mice were subjected to single-limb casting (3 or 7days) and consumed drinking water with or without 1mM sodium nitrate. Compared with the contralateral control limb, 3days of immobilization lowered myofibrillar fractional synthesis rates (FSR, P< 0.0001), resulting in muscle atrophy. Although FSR and mitophagy-related proteins were higher in subsarcolemmal (SS) compared with intermyofibrillar (IMF) mitochondria, immobilization for 3days decreased FSR in both SS (P=0.009) and IMF (P=0.031) mitochondria. Additionally, 3days of immobilization reduced maximal mitochondrial respiration, decreased mitochondrial protein content, and increased maximal mitochondrial reactive oxygen species emission, without altering mitophagy-related proteins in muscle homogenate or isolated mitochondria (SS and IMF). Although nitrate consumption did not attenuate the decline in muscle mass or myofibrillar FSR, intriguingly, nitrate completely prevented immobilization-induced reductions in SS and IMF mitochondrial FSR. In addition, nitrate prevented alterations in mitochondrial content and bioenergetics after both 3 and 7days of immobilization. However, in contrast to 3days of immobilization, nitrate did not prevent the decline in SS and IMF mitochondrial FSR after 7days of immobilization. Therefore, although nitrate supplementation was not sufficient to prevent muscle atrophy, nitrate may represent a promising therapeutic strategy to maintain mitochondrial bioenergetics and transiently preserve mitochondrial protein synthesis rates during short-term muscle disuse. KEY POINTS: Alterations in mitochondrial bioenergetics (decreased respiration and increased reactive oxygen species) are thought to contribute to muscle atrophy and reduced protein synthesis rates during muscle disuse. Given that dietary nitrate can improve mitochondrial bioenergetics, we examined whether nitrate supplementation could attenuate immobilization-induced skeletal muscle impairments in female mice. Dietary nitrate prevented short-term (3day) immobilization-induced declines in mitochondrial protein synthesis rates, reductions in markers of mitochondrial content, and alterations in mitochondrial bioenergetics. Despite these benefits and the preservation of mitochondrial content and bioenergetics during more prolonged (7 day) immobilization, nitrate consumption did not preserve skeletal muscle mass or myofibrillar protein synthesis rates. Overall, although dietary nitrate did not prevent atrophy, nitrate supplementation represents a promising nutritional approach to preserve mitochondrial function during muscle disuse.

Invasive and noninvasive markers of human skeletal muscle mitochondrial function.

Mitochondria are organelles that fuel cellular energy requirements by ATP formation via aerobic metabolism. Given the wide variety of methods to assess skeletal muscle mitochondrial capacity, we tested how well different invasive and noninvasive markers of skeletal muscle mitochondrial capacity reflect mitochondrial respiration in permeabilized muscle fibers. Nineteen young men (mean age: 24 ± 4 years) were recruited, and a muscle biopsy was collected to determine mitochondrial respiration from permeabilized muscle fibers and to quantify markers of mitochondrial capacity, content such as citrate synthase (CS) activity, mitochondrial DNA copy number, TOMM20, VDAC, and protein content for complex I-V of the oxidative phosphorylation (OXPHOS) system. Additionally, all participants underwent noninvasive assessments of mitochondrial capacity: PCr recovery postexercise (by 31 P-MRS), maximal aerobic capacity, and gross exercise efficiency by cycling exercise. From the invasive markers, Complex V protein content and CS activity showed the strongest concordance (Rc = 0.50 to 0.72) with ADP-stimulated coupled mitochondrial respiration, fueled by various substrates. Complex V protein content showed the strongest concordance (Rc = 0.72) with maximally uncoupled mitochondrial respiration. From the noninvasive markers, gross exercise efficiency, VO2max , and PCr recovery exhibited concordance values between 0.50 and 0.77 with ADP-stimulated coupled mitochondrial respiration. Gross exercise efficiency showed the strongest concordance with maximally uncoupled mitochondrial respiration (Rc = 0.67). From the invasive markers, Complex V protein content and CS activity are surrogates that best reflect skeletal muscle mitochondrial respiratory capacity. From the noninvasive markers, exercise efficiency and PCr recovery postexercise most closely reflect skeletal muscle mitochondrial respiratory capacity.

Mitochondrial respiration analysis in permeabilized porcine left ventricular and human right atrial specimens with ischemia-reperfusion.

Mitochondrial function is critical to myocardial ischemia-reperfusion injury and cardioprotection. The measurement of mitochondrial function in isolated mitochondria requires cardiac specimens of about 300 mg and is therefore only possible at the end of an animal experiment or during cardiosurgical interventions in humans. As an alternative, mitochondrial function can be measured in permeabilized myocardial tissue (PMT) specimens of about 2-5 mg, which are retrieved by sequential biopsies in animal experiments and during cardiac catheterization in humans. We attempted to validate measurements of mitochondrial respiration from PMT by comparison with those from isolated mitochondria of left ventricular myocardium from anesthetized pigs undergoing 60 min coronary occlusion and 180 min reperfusion. Mitochondrial respiration was normalized to the content of mitochondrial marker proteins [cytochrome-c oxidase 4 (COX4), citrate synthase, and manganese-dependent superoxide dismutase]. When normalized to COX4, mitochondrial respiration measurements in PMT and isolated mitochondria agreed well in Bland-Altman plots (bias score, -0.03 nmol/min/COX4; 95% confidence interval: 6.31 nmol/min/COX4 and -6.37 nmol/min/COX4) and correlated well (slope of 0.77 and Pearson's R of 0.87). Mitochondrial dysfunction by ischemia-reperfusion was equally reflected in PMT and isolated mitochondria (44 and 48% reduction of ADP-stimulated complex I respiration). Also in isolated human right atrial trabeculae, simulation of ischemia-reperfusion injury by exposure to 60 min hypoxia and 10 min reoxygenation reduced mitochondrial ADP-stimulated complex I respiration by 37% in PMT. In conclusion, mitochondrial function measurements in permeabilized cardiac tissue can substitute for that in isolated mitochondria to reflect mitochondrial dysfunction following ischemia-reperfusion.NEW & NOTEWORTHY Methods to quantify mitochondrial function in translationally relevant models and in human tissue are needed to improve the translation of cardioprotection to patients. Our present approach, using PMT instead of isolated mitochondria for the quantification of mitochondrial ischemia-reperfusion damage, provides a reference for further studies in translationally relevant large animal models and in human tissue, thus possibly improving the translation of cardioprotection to the benefit of patients with acute myocardial infarction.

Reducing mitochondrial ribosomal gene expression does not alter metabolic health or lifespan in mice

Maintaining mitochondrial function is critical to an improved healthspan and lifespan. Introducing mild stress by inhibiting mitochondrial translation invokes the mitochondrial unfolded protein response (UPRmt) and increases lifespan in several animal models. Notably, lower mitochondrial ribosomal protein (MRP) expression also correlates with increased lifespan in a reference population of mice. In this study, we tested whether partially reducing the gene expression of a critical MRP, Mrpl54, reduced mitochondrial DNA-encoded protein content, induced the UPRmt, and affected lifespan or metabolic health using germline heterozygous Mrpl54 mice. Despite reduced Mrpl54 expression in multiple organs and a reduction in mitochondrial-encoded protein expression in myoblasts, we identified few significant differences between male or female Mrpl54+/− and wild type mice in initial body composition, respiratory parameters, energy intake and expenditure, or ambulatory motion. We also observed no differences in glucose or insulin tolerance, treadmill endurance, cold tolerance, heart rate, or blood pressure. There were no differences in median life expectancy or maximum lifespan. Overall, we demonstrate that genetic manipulation of Mrpl54 expression reduces mitochondrial-encoded protein content but is not sufficient to improve healthspan in otherwise healthy and unstressed mice.