Transcriptomic and Proteomic of Gastrocnemius Muscle in Peripheral Artery Disease.

BACKGROUND: Few effective therapies exist to improve lower extremity muscle pathology and mobility loss due to peripheral artery disease (PAD), in part because mechanisms associated with functional impairment remain unclear. METHODS: To better understand mechanisms of muscle impairment in PAD, we performed in-depth transcriptomic and proteomic analyses on gastrocnemius muscle biopsies from 31 PAD participants (mean age, 69.9 years) and 29 age- and sex-matched non-PAD controls (mean age, 70.0 years) free of diabetes or limb-threatening ischemia. RESULTS: Transcriptomic and proteomic analyses suggested activation of hypoxia-compensatory mechanisms in PAD muscle, including inflammation, fibrosis, apoptosis, angiogenesis, unfolded protein response, and nerve and muscle repair. Stoichiometric proportions of mitochondrial respiratory proteins were aberrant in PAD compared to non-PAD, suggesting that respiratory proteins not in complete functional units are not removed by mitophagy, likely contributing to abnormal mitochondrial activity. Supporting this hypothesis, greater mitochondrial respiratory protein abundance was significantly associated with greater complex II and complex IV respiratory activity in non-PAD but not in PAD. Rate-limiting glycolytic enzymes, such as hexokinase and pyruvate kinase, were less abundant in muscle of people with PAD compared with non-PAD participants, suggesting diminished glucose metabolism. CONCLUSIONS: In PAD muscle, hypoxia induces accumulation of mitochondria respiratory proteins, reduced activity of rate-limiting glycolytic enzymes, and an enhanced integrated stress response that modulates protein translation. These mechanisms may serve as targets for disease modification.

Cigarette smoking and mitochondrial dysfunction in peripheral artery disease.

BACKGROUND: This study evaluated the association of smoking with mitochondrial function in gastrocnemius muscle of people with peripheral artery disease (PAD). METHODS: Participants were enrolled from Chicago, Illinois and consented to gastrocnemius biopsy. Mitochondrial oxidative capacity was measured in muscle with respirometry. Abundance of voltage-dependent anion channel (VDAC) (mitochondrial membrane abundance), peroxisome proliferator-activated receptor-γ coactivator (PGC-1α) (mitochondrial biogenesis), and electron transport chain complexes I-V were measured with Western blot. RESULTS: Fourteen of 31 people with PAD (age 72.1 years, ABI 0.64) smoked cigarettes currently. Overall, there were no significant differences in mitochondrial oxidative capacity between PAD participants who currently smoked and those not currently smoking (complex I+II-mediated oxidative phosphorylation: 86.6 vs 78.3 pmolO2/s/mg, respectively [p = 0.39]). Among participants with PAD, those who currently smoked had a higher abundance of PGC-1α (p < 0.01), VDAC (p = 0.022), complex I (p = 0.021), and complex III (p = 0.021) proteins compared to those not currently smoking. People with PAD who currently smoked had lower oxidative capacity per VDAC unit (complex I+II-mediated oxidative phosphorylation [137.4 vs 231.8 arbitrary units, p = 0.030]) compared to people with PAD not currently smoking. Among people without PAD, there were no significant differences in any mitochondrial measures between currently smoking (n = 5) and those not currently smoking (n = 63). CONCLUSIONS: Among people with PAD, cigarette smoking may stimulate mitochondrial biogenesis to compensate for reduced oxidative capacity per unit of mitochondrial membrane, resulting in no difference in overall mitochondrial oxidative capacity according to current smoking status among people with PAD. However, these results were cross-sectional and a longitudinal study is needed.

Sodium dichloroacetate stimulates cardiac mitochondrial metabolism and improves cardiac conduction in the ovine fetus during labor.

Previous studies in our laboratory have suggested that the increase in stillbirth in pregnancies complicated by chronic maternal stress or hypercortisolemia is associated with cardiac dysfunction in late stages of labor and delivery. Transcriptomics analysis of the overly represented differentially expressed genes in the fetal heart of hypercortisolemic ewes indicated involvement of mitochondrial function. Sodium dichloroacetate (DCA) has been used to improve mitochondrial function in several disease states. We hypothesized that administration of DCA to laboring ewes would improve both cardiac mitochondrial activity and cardiac function in their fetuses. Four groups of ewes and their fetuses were studied: control, cortisol-infused (1 g/kg/day from 115 to term; CORT), DCA-treated (over 24 h), and DCA + CORT-treated; oxytocin was delivered starting 48 h before the DCA treatment. DCA significantly decreased cardiac lactate, alanine, and glucose/glucose-6-phosphate and increased acetylcarnitine/isobutyryl-carnitine. DCA increased mitochondrial activity, increasing oxidative phosphorylation (PCI, PCI + II) per tissue weight or per unit of citrate synthase. DCA also decreased the duration of the QRS, attenuating the prolongation of the QRS observed in CORT fetuses. The effect to reduce QRS duration with DCA treatment correlated with increased glycerophosphocholine and serine and decreased phosphorylcholine after DCA treatment. There were negative correlations of acetylcarnitine/isobutyryl-carnitine to both heart rate (HR) and mean arterial pressure (MAP). These results suggest that improvements in mitochondrial respiration with DCA produced changes in the cardiac lipid metabolism that favor improved conduction in the heart. DCA may therefore be an effective treatment of fetal cardiac metabolic disturbances in labor that can contribute to impairments of fetal cardiac conduction.

Effect of heat stress during the early and late dry period on mammary gland development of Holstein dairy cattle.

Dry period heat stress impairs subsequent milk yield. Our objective was to evaluate the effect of heat stress or cooling during the early and late dry period on mammary gland gene expression and microstructure. Cows were dried off ∼45 d before expected parturition and randomly assigned to 1 of 2 treatments: heat stress (HT, n = 39) or cooling (CL, n = 39) during the first 21 d of the dry period. On d 22, cows were switched or remained on HT and CL and this yielded 4 treatments: heat stress during the entire dry period (HTHT, n = 18); cooling during the entire dry period (CLCL, n = 20); HT for the first 21 d dry, then CL until calving (HTCL, n = 21); or CL for the first 21 d dry, then HT until calving (CLHT, n = 19). Data were analyzed in 2 periods: first 21 d dry (early dry period) and from 22 d until calving (late dry period) and analyzed using PROC MIXED or GLM in SAS (SAS Institute Inc., Cary, NC). Mammary biopsies (5-8 cows/treatment) were collected at -3, 3, 7, 14, and 25 d relative to dry-off to evaluate mammary gland gene expression and histology [i.e., cellular apoptosis (terminal deoxynucleotidyl transferase dUTP nick end labeling) and proliferation (Ki67)]. Mammary alveoli number and connective tissue were visualized by hematoxylin and eosin and Mason's trichrome staining, respectively. During the early dry period, CL upregulated expression of CASP3, IGF1R, HSP90, HSF1, BECN1, ATG3, ATG5, and PRLR-LF relative to HT. However, in the late dry period, CLHT treatment upregulated expression of CASP3, CASP8, HSP70, HSP90, PRLR-LF, STAT5, CSN2, and ATG3 relative to CLCL. During the early dry period, cows exposed to HT had reduced mammary and stroma cell apoptosis and proliferation relative to CL. In addition to these findings, cows exposed to HT had lower connective tissue 3 d after dry-off relative to CL. However, in the late dry period, HTHT cows had higher connective tissue relative to CLCL. Also, in the early dry period, cows exposed to HT had greater alveoli number relative to CL, and HT decreased expression of genes related to autophagy and apoptosis in the early dry period, consistent with a delay in involution with HT. Thus, cows exposed to HT have extended involution with delayed apoptosis and autophagy signaling. Also, HT compromises mammary gland cell proliferation and leads to higher connective tissue later in the dry period. These results provide evidence that heat stress impairs overall mammary gland turnover during the dry period, which then affects secretory activity and productivity in the next lactation.

Old Mice Demonstrate Organ Dysfunction as well as Prolonged Inflammation, Immunosuppression, and Weight Loss in a Modified Surgical Sepsis Model.

OBJECTIVES: Our goal was to "reverse translate" the human response to surgical sepsis into the mouse by modifying a widely adopted murine intra-abdominal sepsis model to engender a phenotype that conforms to current sepsis definitions and follows the most recent expert recommendations for animal preclinical sepsis research. Furthermore, we aimed to create a model that allows the study of aging on the long-term host response to sepsis. DESIGN: Experimental study. SETTING: Research laboratory. SUBJECTS: Young (3-5 mo) and old (18-22 mo) C57BL/6j mice. INTERVENTIONS: Mice received no intervention or were subjected to polymicrobial sepsis with cecal ligation and puncture followed by fluid resuscitation, analgesia, and antibiotics. Subsets of mice received daily chronic stress after cecal ligation and puncture for 14 days. Additionally, modifications were made to ensure that "Minimum Quality Threshold in Pre-Clinical Sepsis Studies" recommendations were followed. MEASUREMENTS AND MAIN RESULTS: Old mice exhibited increased mortality following both cecal ligation and puncture and cecal ligation and puncture + daily chronic stress when compared with young mice. Old mice developed marked hepatic and/or renal dysfunction, supported by elevations in plasma aspartate aminotransferase, blood urea nitrogen, and creatinine, 8 and 24 hours following cecal ligation and puncture. Similar to human sepsis, old mice demonstrated low-grade systemic inflammation 14 days after cecal ligation and puncture + daily chronic stress and evidence of immunosuppression, as determined by increased serum concentrations of multiple pro- and anti-inflammatory cytokines and chemokines when compared with young septic mice. In addition, old mice demonstrated expansion of myeloid-derived suppressor cell populations and sustained weight loss following cecal ligation and puncture + daily chronic stress, again similar to the human condition. CONCLUSIONS: The results indicate that this murine cecal ligation and puncture + daily chronic stress model of surgical sepsis in old mice adhered to current Minimum Quality Threshold in Pre-Clinical Sepsis Studies guidelines and met Sepsis-3 criteria. In addition, it effectively created a state of persistent inflammation, immunosuppression, and weight loss, thought to be a key aspect of chronic sepsis pathobiology and increasingly more prevalent after human sepsis.

Effect of heat stress during early, late, and entire dry period on dairy cattle.

Cooling during the entire dry period abates the negative effects of heat stress postpartum, yet the temporal relationship of cooling (i.e., early or late dry period) to performance is unknown. We evaluated the effect of heat stress early, late, and for the entire dry period on subsequent performance. Cows were selected based on mature-equivalent milk yield and dried off 45 d before expected calving. Cows were blocked by parity, previous 305-d mature equivalent milk yield, and body weight (BW) and randomly assigned to cooling (shade, fans, and soakers; CL) or heat stress (shade; HT). Treatments included CL (n = 20) or HT (n = 18) during the entire dry period, HT during the first 3 wk dry and then CL until calving (HTCL, n = 21), or CL during the first 3 wk dry period and then HT until calving (CLHT, n = 19). Heat stress increased rectal temperature (RT; CL, 38.8; HT, 39.1 ± 0.04°C) and respiration rate (RR; CL, 52.9; HT, 70.5 ± 1.9 breaths/min) during the early dry period. In the late dry period, HT increased RT and RR relative to CL cows (RT = CL, 38.7; HT, 39.1; CLHT, 39.1; HTCL, 38.9 ± 0.05°C; RR = CL, 47; HT, 64; CLHT, 66; HTCL, 53 ± 2.1 breaths/min). During the early dry period, HT decreased dry matter intake (CL, 11.8; HT, 10.5 ± 0.35 kg/d) but dry matter intake did not differ among treatments during late dry period (HT, 10.7; HTCL, 11.1; CL, 11.2; CLHT, 10.1 ± 0.55 kg/d). Cows exposed to prepartum cooling during the entire dry period had increased dry matter intake compared with cows exposed to heat stress during the late dry period (CL vs. CLHT, 11.2 ± 0.55 and 10.1 ± 0.55 kg/d, respectively). Heat stress at any time reduced gestation length compared with cows under prepartum cooling during the entire dry period (CL, 277 vs. HT, 274; CLHT, 273; and HTCL, 274 ± 1.17 d). Dry period length decreased by approximately 4 d if cows were exposed to HT at any time. During the early dry period, HT decreased BW, whereas CL increased BW relative to that at dry-off (CL, 6.9; HT, -9.4 ± 3.7 kg). In the late dry period, we detected no differences in BW gain among treatments, but cows exposed to prepartum cooling for the entire dry period tended to have increased BW gain compared with HT and HTCL. Prepartum cooling during the early or late dry period alone partially rescued milk yield only in the first 3 wk of lactation (CL, 32.9; HT, 26.6; CLHT, 29.7; HTCL, 30.7 ± 1.37 kg/d). Cooling for the entire dry period increased milk yield up to 30 wk into lactation compared with all other treatments. Thus, HT at any time during the dry period compromises performance of cows after calving.

A single nucleotide polymorphism in COQ9 affects mitochondrial and ovarian function and fertility in Holstein cows.

A single missense mutation at position 159 of coenzyme Q9 (COQ9) (G→A; rs109301586) has been associated with genetic variation in fertility in Holstein cattle, with the A allele associated with higher fertility. COQ9 is involved in the synthesis of coenzyme COQ10, a component of the electron transport system of the mitochondria. Here we tested whether reproductive phenotype is associated with the mutation and evaluated functional consequences for cellular oxygen metabolism, body weight changes, and ovarian function. The mutation in COQ9 modifies predicted tertiary protein structure and affected mitochondrial respiration of peripheral blood mononuclear cells. The A allele was associated with low resting oxygen consumption and high electron transport system capacity. Phenotypic measurements for fertility were evaluated for up to five lactations in a population of 2273 Holstein cows. There were additive effects of the mutation (P < 0.05) in favor of the A allele for pregnancy rate, interval from calving to conception, and services per conception. There was no association of genotype with milk production or body weight changes postpartum. The mutation in COQ9 affected ovarian function; the A allele was associated with increased mitochondrial DNA copy number in oocytes, and there were overdominance effects for COQ9 expression in oocytes, follicle number, and antimullerian hormone concentrations. Overall, results show how a gene involved in mitochondrial function is associated with overall fertility, possibly in part by affecting oocyte quality.

Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver.

BACKGROUND: Aberrant mitochondrial function, including excessive reactive oxygen species (ROS) production, has been implicated in the pathogenesis of human diseases. The use of mitochondrial inhibitors to ascertain the sites in the electron transport chain (ETC) resulting in altered ROS production can be an important tool. However, the response of mouse mitochondria to ETC inhibitors has not been thoroughly assessed. Here we set out to characterize the differences in phenotypic response to ETC inhibitors between the more energetically demanding brain mitochondria and less energetically demanding liver mitochondria in commonly utilized C57BL/6J mice. RESULTS: We show that in contrast to brain mitochondria, inhibiting distally within complex I or within complex III does not increase liver mitochondrial ROS production supported by complex I substrates, and liver mitochondrial ROS production supported by complex II substrates occurred primarily independent of membrane potential. Complex I, II, and III enzymatic activities and membrane potential were equivalent between liver and brain and responded to ETC. inhibitors similarly. Brain mitochondria exhibited an approximately two-fold increase in complex I and II supported respiration compared with liver mitochondria while exhibiting similar responses to inhibitors. Elevated NADH transport and heightened complex II-III coupled activity accounted for increased complex I and II supported respiration, respectively in brain mitochondria. CONCLUSIONS: We conclude that important mechanistic differences exist between mouse liver and brain mitochondria and that mouse mitochondria exhibit phenotypic differences compared with mitochondria from other species.

The interplay between autophagy and mitochondrial dysfunction in oxidative stress-induced cardiac aging and pathology.

Aging is accompanied by a progressive increase in the incidence and prevalence of cardiovascular disease (CVD). Prolonged exposure to cardiovascular risk factors, together with intrinsic age-dependent declines in cardiac functionality, increases the vulnerability of the heart to both endogenous and exogenous stressors, ultimately enhancing the susceptibility to developing CVD in late life. Both increased levels of oxidative damage and the accumulation of dysfunctional mitochondria have been observed in a wide range of cardiac diseases, which may therefore represent a common ground upon which many aspects of CVD develop. In this review, we summarize the current knowledge on the mechanisms whereby oxidative stress arising from mitochondrial dysfunction is involved in the process of cardiac aging and in the pathogenesis of CVD highly prevalent in late life (e.g., heart failure and ischemic heart disease). Special emphasis is placed on recent evidence about the role played by alterations in cellular quality control systems, in particular autophagy/mitophagy and mitochondrial dynamics (fusion and fission), and their interconnections in the context of age-related CVD. Cardioprotective interventions acting through the modulation of mitochondrial autophagy (calorie restriction, calorie restriction mimetics, and the gasotransmitter hydrogen sulfide) are also presented. This article is part of a Special Issue entitled "Protein Quality Control, the Ubiquitin Proteasome System, and Autophagy".

Postmortem mitochondria function in longissimus lumborum of Angus and Brahman steers.

Mitochondria function and integrity may impact postmortem metabolism and meat quality development. Adaptations in heat tolerant Brahman may persist to limit cellular stress postmortem. Our objective was to evaluate glycolysis, pH decline, and mitochondria function in longissimus lumborum (LL) from Angus and Brahman steers (N = 28) early postmortem (1 to 6 h) and after rigor (24 h). We evaluated metabolites of anaerobic glycolysis, ATP, pH, and temperature, and determined mitochondria oxygen consumption rate (OCR) in permeabilized fibers. The main effects of breed (b) and time (t) and the interaction were tested. Brahman LL contained greater ATP during the first 6 h postmortem; Brahman also tended to exhibit a slower pH decline (b × t, P = 0.07) and more rapid temperature decline (b × t, P < 0.001), but metabolites of anaerobic glycolysis were not different. Mitochondria in Brahman and Angus LL were well-coupled and respired at 1 h postmortem. However, outer membrane integrity became increasingly compromised postmortem (t, P < 0.001). Brahman tended to exhibit greater electron transport system capacity (b, P < 0.1) and had greater capacity for oxidative phosphorylation (complex I and II substrates) at 6 h compared with Angus (P < 0.001). In totality, greater ATP, slower pH decline, and enhanced mitochondria capacity indicate that Brahman possess mitochondrial properties or cellular adaptations that help protect the cell during energy stress postmortem. Slower pH and more rapid temperature decline in LL from Brahman may also help preserve mitochondria function postmortem.

Use of Minimally Invasive Methods to Assess Fuel Utilization and Circadian Rhythms in Older Adults.

Aging is associated with multiple physiological changes that contribute synergistically and independently to physical disability and the risk of chronic disease. Although the etiology of age-related physical disability is complex and multifactorial, the decline in mitochondrial function appears to coincide with the progression of functional decline in many older adults. The reason why there is a decrease in mitochondrial function with aging remains elusive, but emerging science indicates that both fuel metabolism and circadian rhythms can influence mitochondrial function. Recent studies have established that circadian rhythms become disturbed with aging, and that disrupted circadian rhythms have pathological consequences that impact mitochondrial function and overlap with many age-associated chronic diseases. Current quantitative methods for direct assessment of mitochondrial function are invasive and typically require a muscle biopsy, which can pose difficulties with participant recruitment and study adherence, given the perceived levels of potential pain and risk. Thus, an innovative and relatively noninvasive protocol to assess changes in mitochondrial function at the cellular level and circadian patterns in older adults was adapted. Specifically, a real-time metabolic flux analyzer is used to assess the mitochondrial bioenergetic function of white blood cells under differential substrate availability. The expression of circadian clock genes in white blood cells to cross-correlate with the mitochondrial bioenergetics and circadian rhythm outcomes are also analyzed. It is believed that these innovative methodological approaches will aid future clinical trials by providing minimally invasive methods for studying mitochondrial substrate preference and circadian rhythms in older adults.

Nicotinamide riboside for peripheral artery disease: the NICE randomized clinical trial.

People with lower extremity peripheral artery disease (PAD) have increased oxidative stress, impaired mitochondrial activity, and poor walking performance. NAD+ reduces oxidative stress and is an essential cofactor for mitochondrial respiration. Oral nicotinamide riboside (NR) increases bioavailability of NAD+ in humans. Among 90 people with PAD, this randomized double-blind clinical trial assessed whether 6-months of NR, with and without resveratrol, improves 6-min walk distance, compared to placebo, at 6-month follow-up. At 6-month follow-up, compared to placebo, NR significantly improved 6-min walk (+7.0 vs. -10.6 meters, between group difference: +17.6 (90% CI: + 1.8,+∞). Among participants who took at least 75% of study pills, compared to placebo, NR improved 6-min walk by 31.0 meters and NR + resveratrol improved 6-min walk by 26.9 meters. In this work, NR meaningfully improved 6-min walk, and resveratrol did not add benefit to NR alone in PAD. A larger clinical trial to confirm these findings is needed.

Relationship between Mitochondrial Quality Control Markers, Lower Extremity Tissue Composition, and Physical Performance in Physically Inactive Older Adults.

Altered mitochondrial quality and function in muscle may be involved in age-related physical function decline. The role played by the autophagy-lysosome system, a major component of mitochondrial quality control (MQC), is incompletely understood. This study was undertaken to obtain initial indications on the relationship between autophagy, mitophagy, and lysosomal markers in muscle and measures of physical performance and lower extremity tissue composition in young and older adults. Twenty-three participants were enrolled, nine young (mean age: 24.3 ± 4.3 years) and 14 older adults (mean age: 77.9 ± 6.3 years). Lower extremity tissue composition was quantified volumetrically by magnetic resonance imaging and a tissue composition index was calculated as the ratio between muscle and intermuscular adipose tissue volume. Physical performance in older participants was assessed via the Short Physical Performance Battery (SPPB). Protein levels of the autophagy marker p62, the mitophagy mediator BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (BNIP3), the lysosomal markers transcription factor EB, vacuolar-type ATPase, and lysosomal-associated membrane protein 1 were measured by Western immunoblotting in vastus lateralis muscle biopsies. Older adults had smaller muscle volume and lower tissue composition index than young participants. The protein content of p62 and BNIP3 was higher in older adults. A negative correlation was detected between p62 and BNIP3 and the tissue composition index. p62 and BNIP3 were also related to the performance on the 5-time sit-to-stand test of the SPPB. Our results suggest that an altered expression of markers of the autophagy/mitophagy-lysosomal system is related to deterioration of lower extremity tissue composition and muscle dysfunction. Additional studies are needed to clarify the role of defective MQC in human muscle aging and identify novel biological targets for drug development.

Intraoperative Hemi-Diaphragm Electrical Stimulation Demonstrates Attenuated Mitochondrial Function without Change in Oxidative Stress in Cardiothoracic Surgery Patients.

Mechanical ventilation during cardiothoracic surgery is life-saving but can lead to ventilator-induced diaphragm dysfunction (VIDD) and prolong ventilator weaning and hospital length of stay. Intraoperative phrenic nerve stimulation may preserve diaphragm force production to offset VIDD; we also investigated changes in mitochondrial function after stimulation. During cardiothoracic surgeries (n = 21), supramaximal, unilateral phrenic nerve stimulation was performed every 30 min for 1 min. Diaphragm biopsies were collected after the last stimulation and analyzed for mitochondrial respiration in permeabilized fibers and protein expression and enzymatic activity of biomarkers of oxidative stress and mitophagy. Patients received, on average, 6.2 ± 1.9 stimulation bouts. Stimulated hemidiaphragms showed lower leak respiration, maximum electron transport system (ETS) capacities, oxidative phosphorylation (OXPHOS), and spare capacity compared with unstimulated sides. There were no significant differences between mitochondrial enzyme activities and oxidative stress and mitophagy protein expression levels. Intraoperative phrenic nerve electrical stimulation led to an acute decrease of mitochondrial respiration in the stimulated hemidiaphragm, without differences in biomarkers of mitophagy or oxidative stress. Future studies warrant investigating optimal stimulation doses and testing post-operative chronic stimulation effects on weaning from the ventilator and rehabilitation outcomes.

Mitochondrial Complex Abundance, Mitophagy Proteins, and Physical Performance in People With and Without Peripheral Artery Disease.

Background Mitochondrial abnormalities exist in gastrocnemius muscle of people with peripheral artery disease (PAD). Whether abnormalities in mitochondrial biogenesis and autophagy are associated with greater ischemia or walking impairment in PAD is unknown. Methods and Results Protein markers of mitochondrial biogenesis and autophagy and the abundance of mitochondrial electron transport chain complexes were quantified in gastrocnemius muscle biopsies from people with and without PAD. Their 6-minute walk distance and 4-m gait speed were measured. Sixty-seven participants (mean age 65.0 years [±6.8], 16 [23.9%] women, 48 [71.6%] Black) were enrolled, including 15 with moderate to severe PAD (ankle brachial index [ABI] <0.60), 29 with mild PAD (ABI 0.60-0.90), and 23 without PAD (ABI 1.00-1.40). Abundance of all electron transport chain complexes was significantly higher in participants with lower ABI (eg, complex I: 0.66, 0.45, 0.48 arbitrary units [AU], respectively, P trend=0.043). Lower ABI values were associated with a higher LC3A/B II-to-LC3A/B I (microtubule-associated protein 1A/1B-light chain 3) ratio (2.54, 2.31, 2.15 AU, respectively, P trend=0.017) and reduced abundance of the autophagy receptor p62 (0.71, 0.69, 0.80 AU, respectively, P trend=0.033). The abundance of each electron transport chain complex was positively and significantly associated with 6-minute walk distance and 4-m gait speed at usual and fast pace only among participants without PAD (eg, complex I: r=0.541, P=0.008; r=0.477, P=0.021; r=0.628, P=0.001, respectively). Conclusions These results suggest that accumulation of electron transport chain complexes in gastrocnemius muscle of people with PAD may be because of impaired mitophagy in the setting of ischemia. Findings are descriptive, and further study in larger sample sizes is needed.

NMR Spectroscopy Identifies Chemicals in Cigarette Smoke Condensate That Impair Skeletal Muscle Mitochondrial Function.

Tobacco smoke-related diseases such as chronic obstructive pulmonary disease (COPD) are associated with high healthcare burden and mortality rates. Many COPD patients were reported to have muscle atrophy and weakness, with several studies suggesting intrinsic muscle mitochondrial impairment as a possible driver of this phenotype. Whereas much information has been learned about muscle pathology once a patient has COPD, little is known about how active tobacco smoking might impact skeletal muscle physiology or mitochondrial health. In this study, we examined the acute effects of cigarette smoke condensate (CSC) on muscle mitochondrial function and hypothesized that toxic chemicals present in CSC would impair mitochondrial respiratory function. Consistent with this hypothesis, we found that acute exposure of muscle mitochondria to CSC caused a dose-dependent decrease in skeletal muscle mitochondrial respiratory capacity. Next, we applied an analytical nuclear magnetic resonance (NMR)-based approach to identify 49 water-soluble and 12 lipid-soluble chemicals with high abundance in CSC. By using a chemical screening approach in the Seahorse XF96 analyzer, several CSC-chemicals, including nicotine, o-Cresol, phenylacetate, and decanoic acid, were found to impair ADP-stimulated respiration in murine muscle mitochondrial isolates significantly. Further to this, several chemicals, including nicotine, o-Cresol, quinoline, propylene glycol, myo-inositol, nitrosodimethylamine, niacinamide, decanoic acid, acrylonitrile, 2-naphthylamine, and arsenic acid, were found to significantly decrease the acceptor control ratio, an index of mitochondrial coupling efficiency.

Apparent Absence of BMAL1-Dependent Skeletal Muscle-Kidney Cross Talk in Mice.

BMAL1 is a core mammalian circadian clock transcription factor responsible for the regulation of the expression of thousands of genes. Previously, male skeletal-muscle-specific BMAL1-inducible-knockout (iMS-BMAL1 KO) mice have been described as a model that exhibits an aging-like phenotype with an altered gait, reduced mobility, muscle weakness, and impaired glucose uptake. Given this aging phenotype and that chronic kidney disease is a disease of aging, the goal of this study was to determine if iMS-BMAL1 KO mice exhibit a renal phenotype. Male iMS-BMAL1 KO and control mice were challenged with a low potassium diet for five days. Both genotypes responded appropriately by conserving urinary potassium. The iMS-BMAL1 KO mice excreted less potassium during the rest phase during the normal diet but there was no genotype difference during the active phase. Next, iMS-BMAL1 KO and control mice were used to compare markers of kidney injury and assess renal function before and after a phase advance protocol. Following phase advance, no differences were detected in renal mitochondrial function in iMS-BMAL1 KO mice compared to control mice. Additionally, the glomerular filtration rate and renal morphology were similar between groups in response to phase advance. Disruption of the clock in skeletal muscle tissue activates inflammatory pathways within the kidney of male mice, and there is evidence of this affecting other organs, such as the lungs. However, there were no signs of renal injury or altered function following clock disruption of skeletal muscle under the conditions tested.

Dysregulated Genes, MicroRNAs, Biological Pathways, and Gastrocnemius Muscle Fiber Types Associated With Progression of Peripheral Artery Disease: A Preliminary Analysis.

Background Peripheral artery disease (PAD) is associated with gastrocnemius muscle abnormalities. However, the biological pathways associated with gastrocnemius muscle dysfunction and their associations with progression of PAD are largely unknown. This study characterized differential gene and microRNA (miRNA) expression in gastrocnemius biopsies from people without PAD compared with those with PAD. Participants with PAD included those with and without PAD progression. Methods and Results mRNA and miRNA sequencing were performed to identify differentially expressed genes, differentially expressed miRNAs, mRNA-miRNA interactions, and associated biological pathways for 3 sets of comparisons: (1) PAD progression (n=7) versus non-PAD (n=7); (2) PAD no progression (n=6) versus non-PAD; and (3) PAD progression versus PAD no progression. Immunohistochemistry was performed to determine gastrocnemius muscle fiber types and muscle fiber size. Differentially expressed genes and differentially expressed miRNAs were more abundant in the comparison of PAD progression versus non-PAD compared with PAD with versus without progression. Among the top significant cellular pathways in subjects with PAD progression were muscle contraction or development, transforming growth factor-beta, growth/differentiation factor, and activin signaling, inflammation, cellular senescence, and notch signaling. Subjects with PAD progression had increased frequency of smaller Type 2a gastrocnemius muscle fibers in exploratory analyses. Conclusions Humans with PAD progression exhibited greater differences in the number of gene and miRNA expression, biological pathways, and Type 2a muscle fiber size compared with those without PAD. Fewer differences were observed between people with PAD without progression and control patients without PAD. Further study is needed to confirm whether the identified transcripts may serve as potential biomarkers for diagnosis and progression of PAD.

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.

Comparative toxicities of BPA, BPS, BPF, and TMBPF in the nematode Caenorhabditis elegans and mammalian fibroblast cells.

Bisphenol A (BPA) is a chemical compound commonly used in the production of plastics for daily lives and industry. As BPA is well known for its adverse health effects, several alternative materials have been developed. This study comprehensively analyzed the toxicity of BPA and its three substitutes including bisphenol S (BPS), bisphenol F (BPF), and tetramethyl bisphenol F (TMBPF) on aging, healthspan, and mitochondria using an in vivo Caenorhabditis elegans (C. elegans) model animal and cultured mammalian fibroblast cells. C. elegans treated with 1 mM BPA exhibited abnormalities in the four tested parameters related to development and growth, including delayed development, decreased body growth, reduced reproduction, and abnormal tissue morphology. Exposure to the same concentration of each alternative including TMBPF, which has been proposed as a relatively safe BPA alternative, detrimentally affected at least three of these events. Moreover, all bisphenols (except BPS) remarkably shortened the organismal lifespan and increased age-related changes in neurons. Exposure to BPA and BPF resulted in mitochondrial abnormalities, such as reduced oxygen consumption and mitochondrial membrane potential. In contrast, the ATP levels were noticeably higher after treatment with all bisphenols. In mammalian fibroblast cells, exposure to increasing concentrations of all bisphenols (ranging from 50 µM to 500 µM) caused a severe decrease in cell viability in a dose-dependent manner. BPA increased ATP levels and decreased ROS but did not affect mitochondrial permeability transition pores (mPTP). Notably, TMBPF was the only bisphenol that caused a significant increase in mitochondrial ROS and mPTP opening. These results suggest that the potentially harmful physiological effects of BPA alternatives should be considered.