The ankle-brachial index, gastrocnemius mitochondrial respirometry, and walking performance in people with and without peripheral artery disease.

Background: Mitochondrial abnormalities exist in lower-extremity peripheral artery disease (PAD), yet the association of the ankle-brachial index (ABI) with mitochondrial respiration in gastrocnemius muscle is unknown. The association of gastrocnemius mitochondrial respiration with 6-minute walk distance in PAD is unknown. Objective: To describe associations of the ABI with mitochondrial respiratory function in gastrocnemius muscle biopsies and associations of gastrocnemius mitochondrial respirometry with 6-minute walk distance in people with and without PAD. Methods: People with (ABI ⩽ 0.90) and without (ABI 1.00-1.40) PAD were enrolled. ABI and 6-minute walk distance were measured. Mitochondrial function of permeabilized myofibers from gastrocnemius biopsies was measured with high-resolution respirometry. Results: A total of 30 people with PAD (71.7 years, mean ABI: 0.64) and 68 without PAD (71.8 years, ABI: 1.17) participated. In non-PAD participants, higher ABI values were associated significantly with better mitochondrial respiration (Pearson correlation for maximal oxidative phosphorylation PCI+II: +0.29, p = 0.016). In PAD, the ABI correlated negatively and not significantly with mitochondrial respiration (Pearson correlation for PCI+II: -0.17, p = 0.38). In people without PAD, better mitochondrial respiration was associated with better 6-minute walk distance (Pearson correlation: +0.51, p < 0.001), but this association was not present in PAD (Pearson correlation: +0.10, p = 0.59). Conclusions: Major differences exist between people with and without PAD in the association of gastrocnemius mitochondrial respiration with ABI and 6-minute walk distance. Among people without PAD, ABI and walking performance were positively associated with mitochondrial respiratory function. These associations were not observed in PAD.

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.

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.

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.

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.

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.

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.

Multiple pathways to the same end: mechanisms of myonuclear apoptosis in sarcopenia of aging.

Sarcopenia, the age-related decline in muscle mass and function, represents a significant health issue due to the high prevalence of frailty and disability associated with this condition. Nevertheless, the cellular mechanisms responsible for the loss of muscle mass in old age are still largely unknown. An altered regulation of myocyte apoptosis has recently emerged as a possible contributor to the pathogenesis of sarcopenia. Studies in animal models have shown that the severity of skeletal muscle apoptosis increases over the course of aging and correlates with the degree of muscle mass and strength decline. Several apoptotic pathways are operative in aged muscles, with the mitochondria- and TNF-alpha-mediated pathways likely being the most relevant to sarcopenia. However, despite the growing number of studies on the subject, a definite mechanistic link between myocyte apoptosis and age-related muscle atrophy has not yet been established. Furthermore, the evidence on the role played by apoptosis in human sarcopenia is still sparse. Clearly, further research is required to better define the involvement of myocyte apoptosis in the pathogenesis of muscle loss at advanced age. This knowledge will likely help in the design of more effective therapeutic strategies to preserve muscle mass into old age, thus fostering independence of the elderly population and reducing the socioeconomic burden associated with sarcopenia.

Mitochondrial oxygen consumption in early postmortem permeabilized skeletal muscle fibers is influenced by cattle breed.

Functional properties and integrity of skeletal muscle mitochondria (mt) during the early postmortem period may influence energy metabolism and pH decline, thereby impacting meat quality development. Angus typically produce more tender beef than Brahman, a Bos indicus breed known for heat tolerance. Thus, our objectives were to compare mt respiratory function in muscle collected early postmortem (1 h) from Angus and Brahman steers (n = 26); and to evaluate the effect of normal and elevated temperature on mt function ex vivo. We measured mt oxygen consumption rate (OCR) in fresh-permeabilized muscle fibers from Longissimus lumborum (LL) at 2 temperatures (38.5 and 40.0 °C) and determined citrate synthase (CS) activity and expression of several mt proteins. The main effects of breed, temperature, and their interaction were tested for mt respiration, and breed effect was tested for CS activity and protein expression. Breed, but not temperature (P > 0.40), influenced mt OCR (per tissue weight), with Brahman exhibiting greater complex I+II-mediated oxidative phosphorylation capacity (P = 0.05). Complex I- and complex II-mediated OCR also tended to be greater in Brahman (P = 0.07 and P = 0.09, respectively). Activity of CS was higher in LL from Brahman compared to Angus (P = 0.05). Expression of specific mt proteins did not differ between breeds, except for higher expression of adenosine triphosphate (ATP) synthase subunit 5 alpha in Brahman muscle (P = 0.04). Coupling control ratio differed between breeds (P = 0.05), revealing greater coupling between oxygen consumption and phosphorylation in Brahman. Our data demonstrate that both Angus and Brahman mt retained functional capacity and integrity 1-h postmortem; greater oxidative phosphorylation capacity and coupling in Brahman mt could be related to heat tolerance and impact early postmortem metabolism.

Mitochondrial DNA damage in calf skeletal muscle and walking performance in people with peripheral artery disease.

BACKGROUND: Peripheral artery disease (PAD) is associated with mitochondrial dysfunction in calf skeletal muscle and a greater abundance of mitochondrial DNA (mtDNA) heteroplasmy. However, it is unknown whether calf skeletal muscle mtDNA of PAD participants harbors a greater abundance of mitochondrial DNA 4977-bp common deletion (mtDNA4977), strand breaks and oxidative damage (i.e., oxidized purines) compared to non-PAD participants and whether these mtDNA abnormalities are associated with poor walking performance in participants with PAD. METHODS: Calf muscle biopsies were obtained from 50 PAD participants (ankle-brachial index (ABI) < 0.95) and 25 non-PAD participants (ABI = 0.99-1.40) matched by age, sex, and race. The abundance of mtDNA copy number, mtDNA4977 deletion, strand breaks, and oxidized purines in selected mtDNA regions coding for electron transport chain (ETC) constituents and the non-coding D-Loop region was determined in calf muscle. All participants completed measurement of 6-min walk and usual and fast-paced 4-m walking velocity test. RESULTS: Participants with PAD (mean age = 65.4 years, SD = 6.9; 14 (28%) women, 38 (76%) black) and without PAD (mean age = 65.2 years, SD = 6.7; 7 (28%) women, 16 (64%) black) did not differ in the abundance of calf muscle mtDNA4977 deletion, mtDNA strand breaks, and oxidized purines. Though, a greater abundance of mtDNA strand breaks within ND4/5 genes was significantly associated with poorer 6-min walk distance, lower usual-paced 4-m walking velocity, and lower fast-paced 4-m walking velocity in non-PAD participants. Significant associations were also found in the density of strand break damage (i.e., damage per mtDNA copy) within ND1/2, ND4/5 and COII/ATPase 6/8 region with 6-min walk distance, usual-paced 4-m walking velocity and fast-paced 4-m walking velocity in non-PAD participants. Significant interactions were found between PAD presence vs. absence and density of strand break damage within ND1/2, ND4/5, COII/ATPase 6/8 regions for the associations with 6-min walk distance, usual-paced 4-m walking velocity, fast-paced 4-m walking velocity. Conversely, of the three walking performance measures only the usual-paced 4-m walking velocity showed a significant, although modest, negative association with the abundance of oxidized purines in the D-Loop (P = 0.031) and ND4/5 (P = 0.033) regions in the calf skeletal muscle of people with PAD. CONCLUSION: Overall, these data suggest that the abundance of calf muscle mtDNA strand breaks and mtDNA4977 common deletion are not associated with walking performance in people with PAD and may not be directly involved in the pathophysiology of PAD. Conversely, strand breaks in specific mtDNA regions may contribute to poor walking performance in people without PAD. Further study is needed to confirm whether usual-paced 4-m walking velocity is associated significantly with a greater abundance of oxidized purines in the D-loop, a "mutational hotspot" for oxidative damage, and why this association may differ from the association with 6-min walk distance and fast-paced 4-m walking velocity.

Altered Expression of Mitoferrin and Frataxin, Larger Labile Iron Pool and Greater Mitochondrial DNA Damage in the Skeletal Muscle of Older Adults.

Mitochondrial dysfunction and iron (Fe) dyshomeostasis are invoked among the mechanisms contributing to muscle aging, possibly via a detrimental mitochondrial-iron feed-forward loop. We quantified the labile Fe pool, Fe isotopes, and the expression of mitochondrial Fe handling proteins in muscle biopsies obtained from young and older adults. The expression of key proteins of mitochondrial quality control (MQC) and the abundance of the mitochondrial DNA common deletion (mtDNA4977) were also assessed. An inverse association was found between total Fe and the heavier Fe isotope (56Fe), indicating an increase in labile Fe abundance in cells with greater Fe content. The highest levels of labile Fe were detected in old participants with a Short Physical Performance Battery (SPPB) score ≤ 7 (low-functioning, LF). Protein levels of mitoferrin and frataxin were, respectively, higher and lower in the LF group relative to young participants and older adults with SPPB scores ≥ 11 (high-functioning, HF). The mtDNA4977 relative abundance was greater in old than in young participants, regardless of SPPB category. Higher protein levels of Pink1 were detected in LF participants compared with young and HF groups. Finally, the ratio between lipidated and non-lipidated microtubule-associated protein 1A/1B-light chain 3 (i.e., LC3B II/I), as well as p62 protein expression was lower in old participants regardless of SPPB scores. Our findings indicate that cellular and mitochondrial Fe homeostasis is perturbed in the aged muscle (especially in LF older adults), as reflected by altered levels of mitoferrin and frataxin, which, together with MQC derangements, might contribute to loss of mtDNA stability.

Skeletal muscle from aged American Quarter Horses shows impairments in mitochondrial biogenesis and expression of autophagy markers.

Aging is associated with decreased mitochondrial content and function in skeletal muscle, possibly due to compromised biogenesis and autophagic removal of dysfunctional mitochondria. The aim of this study was to compare markers of mitochondrial content and biogenesis and of autophagy between skeletal muscle from young and aged American Quarter Horses. Citrate synthase protein and mtDNA copy number were decreased in triceps brachii (TB) muscle (P<0.05) from aged horses, suggesting an age-related decline in mitochondrial content. Concomitantly, mRNA expression of PGC-1α and TFAM, regulators of mitochondrial biogenesis, was lower in aged compared to young TB (P<0.05). Expression of autophagy markers suggested an age-associated decline of autophagy. The autophagosomal cargo protein SQSTM/p62 accumulated with age in both muscles (P<0.05). Expression of Autophagy-related protein Atg5 (P<0.05) and the autophagosome-bound form of Microtubule-associated protein light chain 3 (LC3-II; P<0.05) were lower in aged compared to young TB. While LC3 transcript level was elevated in aged compared to young GM (P<0.001), protein expression of LC3-II was unaffected. Gene expression of Lysosomal Membrane-Associated Protein 2 (LAMP2) was not affected by age in either muscle. However, LAMP2 protein expression declined with age (P<0.05), suggesting a decline in autophagosome-lysosome fusion. Taken together, our data indicate that equine skeletal muscle mitochondrial content and biogenesis were impaired with age. Further, autophagosome formation and lysosomal degradation were negatively affected in aged TB and GM, respectively. Future research needs to explore whether interventions targeting these cellular processes can prolong health and performance of aging American Quarter Horses.