Mitochondrial‐targeted peptide rapidly improves mitochondrial energetics and skeletal muscle performance in aged mice

Mitochondrial dysfunction plays a key pathogenic role in aging skeletal muscle resulting in significant healthcare costs in the developed world. However, there is no pharmacologic treatment to rapidly reverse mitochondrial deficits in the elderly. Here, we demonstrate that a single treatment with the mitochondrial‐targeted peptide SS‐31 restores in vivo mitochondrial energetics to young levels in aged mice after only one hour. Young (5 month old) and old (27 month old) mice were injected intraperitoneally with either saline or 3 mg kg^?1 of SS‐31. Skeletal muscle mitochondrial energetics were measured in vivo one hour after injection using a unique combination of optical and ³¹P magnetic resonance spectroscopy. Age‐related declines in resting and maximal mitochondrial ATP production, coupling of oxidative phosphorylation (P/O), and cell energy state (PCr/ATP) were rapidly reversed after SS‐31 treatment, while SS‐31 had no observable effect on young muscle. These effects of SS‐31 on mitochondrial energetics in aged muscle were also associated with a more reduced glutathione redox status and lower mitochondrial H₂O₂ emission. Skeletal muscle of aged mice was more fatigue resistant in situ one hour after SS‐31 treatment, and eight days of SS‐31 treatment led to increased whole‐animal endurance capacity. These data demonstrate that SS‐31 represents a new strategy for reversing age‐related deficits in skeletal muscle with potential for translation into human use.     

Dietary supplementation with docosahexaenoic acid, but not eicosapentaenoic acid, dramatically alters cardiac mitochondrial phospholipid fatty acid composition and prevents permeability transition

Treatment with the ω-3 polyunsaturated fatty acids (PUFAs) docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) exerts cardioprotective effects, and suppresses Ca²⁺-induced opening of the mitochondrial permeability transition pore (MPTP). These effects are associated with increased DHA and EPA, and lower arachidonic acid (ARA) in cardiac phospholipids. While clinical studies suggest the triglyceride lowering effects of DHA and EPA are equivalent, little is known about the independent effects of DHA and EPA on mitochondria function. We compared the effects of dietary supplementation with the ω-3 PUFAs DHA and EPA on cardiac mitochondrial phospholipid fatty acid composition and Ca²⁺-induced MPTP opening. Rats were fed a standard lab diet with either normal low levels of ω-3 PUFA, or DHA or EPA at 2.5% of energy intake for 8 weeks, and cardiac mitochondria were isolated and analyzed for Ca²⁺-induced MPTP opening and phospholipid fatty acyl composition. DHA supplementation increased both DHA and EPA and decreased ARA in mitochondrial phospholipid, and significantly delayed MPTP opening as assessed by increased Ca²⁺ retention capacity and decreased Ca²⁺-induced mitochondria swelling. EPA supplementation increased EPA in mitochondrial phospholipids, but did not affect DHA, only modestly lowered ARA, and did not affect MPTP opening. In summary, dietary supplementation with DHA but not EPA, profoundly altered mitochondrial phospholipid fatty acid composition and delayed Ca²⁺-induced MPTP opening.     

Docosahexaenoic acid but not eicosapentaenoic acid withstands dietary cholesterol-induced decreases in platelet membrane fluidity

To determine the differenetial effects of docosahexaenoic (DHA) and eicosapentaenoic (EPA) acid on platelet membrane fluidity under hypercholesterolemic conditions. DHA and EPA were orally administered (300 mg/kg body weight^.day) to hypercholesterolemic rats for 12 weeks. Membrane fluidity, evaluated by fluorescence polarization of nonpolar 1,6-diphenyl-1,3,5-hexatriene (DPH), of the platelets of high cholesterol (HC; 1%)-fed rats decreased significantly compared with that of the platelets of normocholesterolemic rats. In HC-fed rats, dietary administration of DHA, unlike that of EPA, significantly increased platelet membrane fluidity. A high cholesterol diet significantly increased platelet aggregation, compared with the platelet aggregation of normocholesterolemic rats. DHA administration significantly decreased the aggregation, whereas EPA had no effect. Levels of EPA in the platelets of the EPA-fed HC rats and those of DHA in the platelets of the DHA-fed HC rats increased by 482 and 174%, respectively, compared with those in the platelets of the HC-fed rats. The unsaturation index and the ratio of saturated to (poly)unsaturated fatty acid of the platelet membrane increased only in the DHA-fed rats. The phospholipid content in platelet membranes remained unaltered in all groups, whereas the cholesterol content decreased significantly in DHA-fed rats, resulting in a significant decrease in the cholesterol/phospholipid molar ratio only in the platelet membranes of DHA-fed rats. These results suggest that DHA is a more potent membrane-fluidizer than EPA in withstanding cholesterol-induced decreases in platelet membrane fluidity and a stronger ameliorative modulator of platelet hyperaggregation.     

n-3 polyunsaturated fatty acid supplementation reverses stress-induced modifications on brain monoamine levels in mice

The aim of this study was to examine the effects of supplementation with n-3 polyunsaturated fatty acids (PUFAs) on stress responses in mice subjected to an unpredictable chronic mild stress (UCMS) procedure. Stress-induced modifications in coat and aggressiveness were evaluated, and phospholipid PUFA profiles and monoamine levels were analyzed in the frontal cortex, hippocampus, and striatum. The results showed that repeated exposure to mild stressors induced degradation in the physical state of the coat, lowered body weight gain, and increased aggressiveness, without any effect of n-3 PUFA supplementation. The UCMS induced a significant decrease in the levels of norepinephrine in the frontal cortex and striatum, and a nonsignificant decrease in the hippocampus. The tissue levels of serotonin (5-HT) were 40% to 65% decreased in the three brain regions studied. Interestingly, the n-3 PUFA supplementation reversed this stress-induced reduction in 5-HT levels. These findings showed that supplementation in n-3 long-chain PUFAs might reverse certain effects of UCMS in cerebral structures involved in stress-related behaviors.     

Recombinant production of omega‐3 fatty acids in Escherichia coli using a gene cluster isolated from Shewanella baltica MAC1

Aim: To isolate eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) genes from Shewanella baltica MAC1 and to examine recombinant production of EPA and DHA in E. coli to investigate cost‐effective, sustainable and convenient alternative sources for fish oils. Methods and Results: A fosmid library was prepared from the genomic DNA of S. baltica MAC1 and was screened for EPA and DHA genes by colony hybridization using a partial fragment of the S. baltica MAC1 pfaA and pfaD genes as probes. Analysis of total fatty acids isolated from transgenic E. coli positive for pfaA and pfaD genes by gas chromatography and gas chromatography‐mass spectrometry indicated recombinant production of both EPA and DHA. Analysis of the complete nucleotide sequence for the isolated gene cluster showed 16 putative open reading frames (ORFs). Among those, four ORFs showed homology with pfaA, pfaB, pfaC and pfaD genes of the EPA and/or DHA biosynthesis gene clusters; however, the protein domains of these genes were different from other EPA/DHA biosynthesis genes. Conclusions: The EPA and DHA gene cluster was cloned successfully. The transgenic E. coli strain carrying the omega‐3 gene cluster was able to produce both EPA and DHA. The isolated gene cluster contained all the genes required for the recombinant production of both EPA and DHA in E. coli. Significance and Impact of the Study: These findings have implications for any future use of the EPA and DHA gene cluster in other micro‐organisms, notably those being used for fermentation. Recombinant production of both EPA and DHA by E. coli or any other micro‐organism has great potential to add economic value to a variety of industrial and agricultural products.     

The impact of aging on mitochondrial function and biogenesis pathways in skeletal muscle of sedentary high- and low-functioning elderly individuals

Age-related loss of muscle mass and strength (sarcopenia) leads to a decline in physical function and frailty in the elderly. Among the many proposed underlying causes of sarcopenia, mitochondrial dysfunction is inherent in a variety of aged tissues. The intent of this study was to examine the effect of aging on key groups of regulatory proteins involved in mitochondrial biogenesis and how this relates to physical performance in two groups of sedentary elderly participants, classified as high- and low-functioning based on the Short Physical Performance Battery test. Muscle mass was decreased by 38% and 30% in low-functioning elderly (LFE) participants when compared to young and high-functioning elderly participants, respectively, and positively correlated to physical performance. Mitochondrial respiration in permeabilized muscle fibers was reduced (41%) in the LFE group when compared to the young, and this was associated with a 30% decline in cytochrome c oxidase activity. Levels of key metabolic regulators, SIRT3 and PGC-1α, were significantly reduced (50%) in both groups of elderly participants when compared to young. Similarly, the fusion protein OPA1 was lower in muscle from elderly subjects; however, no changes were detected in Mfn2, Drp1 or Fis1 among the groups. In contrast, protein import machinery components Tom22 and cHsp70 were increased in the LFE group when compared to the young. This study suggests that aging in skeletal muscle is associated with impaired mitochondrial function and altered biogenesis pathways and that this may contribute to muscle atrophy and the decline in muscle performance observed in the elderly population.     

Docosahexaenoic acid, but not eicosapentaenoic acid, reduces the early inflammatory response following compression spinal cord injury in the rat

Docosahexaenoic acid (DHA, 22 : 6) and eicosapentaenoic acid (EPA, 20 : 5) are omega-3 polyunsaturated fatty acids (n-3 PUFAs) with distinct anti-inflammatory properties. Both have neuroprotective effects acutely following spinal cord injury (SCI). We examined the effect of intravenous DHA and EPA on early inflammatory events after SCI. Saline, DHA or EPA (both 250 nmol/kg) were administered 30 min after T12 compression SCI, to female Sprague-Dawley rats. DHA significantly reduced the number of neutrophils to some areas of the injured epicentre at 4 h and 24 h. DHA also reduced C-reactive protein plasma levels, whereas EPA did not significantly reduce neutrophils or C-reactive protein. Laminectomy and SCI elicited a sustained inflammatory response in the liver, which was not reversed by the PUFAs. The chemokine KC/GRO/CINC and the cytokine IL-6 provide gradients for chemotaxis of neutrophils to the epicentre. At 4 h after injury, there was a significant increase in IL-6, KC/GRO/CINC, IL-1β and tumour necrosis factor-α in the epicentre, with a return to baseline at 24 h. Neither DHA nor EPA returned their levels to control values. These results indicate that the acute neuroprotective effects of n-3 PUFAs in rat compression SCI may be only partly attributed to reduction of some of the early inflammatory events occurring after injury.     

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

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

Apoptosis and necrosis mediate skeletal muscle fiber loss in age‐induced mitochondrial enzymatic abnormalities

Sarcopenia, the age‐induced loss of skeletal muscle mass and function, results from the contributions of both fiber atrophy and loss of myofibers. We have previously characterized sarcopenia in FBN rats, documenting age‐dependent declines in muscle mass and fiber number along with increased fiber atrophy and fibrosis in vastus lateralis and rectus femoris muscles. Concomitant with these sarcopenic changes is an increased abundance of mitochondrial DNA deletion mutations and electron transport chain (ETC) abnormalities. In this study, we used immunohistological and histochemical approaches to define cell death pathways involved in sarcopenia. Activation of muscle cell death pathways was age‐dependent with most apoptotic and necrotic muscle fibers exhibiting ETC abnormalities. Although activation of apoptosis was a prominent feature of electron transport abnormal muscle fibers, necrosis was predominant in atrophic and broken ETC‐abnormal fibers. These data suggest that mitochondrial dysfunction is a major contributor to the activation of cell death processes in aged muscle fibers. The link between ETC abnormalities, apoptosis, fiber atrophy, and necrosis supports the hypothesis that mitochondrial DNA deletion mutations are causal in myofiber loss. These studies suggest a progression of events beginning with the generation and accumulation of a mtDNA deletion mutation, the concomitant development of ETC abnormalities, a subsequent triggering of apoptotic and, ultimately, necrotic events resulting in muscle fiber atrophy, breakage, and fiber loss.     

Chronic intake of proanthocyanidins and docosahexaenoic acid improves skeletal muscle oxidative capacity in diet-obese rats

Obesity has become a worldwide epidemic. The cafeteria diet (CD) induces obesity and oxidative-stress-associated insulin resistance. Polyunsaturated fatty acids and polyphenols are dietary compounds that are intensively studied as products that can reduce the health complications related to obesity. We evaluate the effects of 21 days of supplementation with grape seed proanthocyanidins extract (GSPE), docosahexaenoic-rich oil (DHA-OR) or both compounds (GSPE+DHA-OR) on skeletal muscle metabolism in diet-obese rats. The supplementation with different treatments did not reduce body weight, although all groups used more fat as fuel, particularly when both products were coadministered; muscle β-oxidation was activated, the mitochondrial functionality and oxidative capacity were higher, and fatty acid uptake gene expressions were up-regulated. In addition to these outcomes shared by all treatments, GSPE reduced insulin resistance and improved muscle status. Both treatments increased 5’-AMP-activated protein kinase (AMPK) phosphorylation, which was consistent with higher plasma adiponectin levels. Moreover, AMPK activation by DHA-OR was also correlated with an up-regulation of peroxisome proliferator-activated receptor alpha (Pparα). GSPE+DHA-OR, in addition to activating AMPK and enhancing fatty acid oxidation, increased the muscle gene expression of uncoupling protein 2 (Ucp2). In conclusion, GSPE+DHA-OR induced modifications that improved muscle status and could counterbalance the deleterious effects of obesity, and such modifications are mediated, at least in part, through the AMPK signaling pathway.     

Acetic acid enhances endurance capacity of exercise-trained mice by increasing skeletal muscle oxidative properties

Acetic acid has been shown to promote glycogen replenishment in skeletal muscle during exercise training. In this study, we investigated the effects of acetic acid on endurance capacity and muscle oxidative metabolism in the exercise training using in vivo mice model. In exercised mice, acetic acid induced a significant increase in endurance capacity accompanying a reduction in visceral adipose depots. Serum levels of non-esterified fatty acid and urea nitrogen were significantly lower in acetic acid-fed mice in the exercised mice. Importantly, in the mice, acetic acid significantly increased the muscle expression of key enzymes involved in fatty acid oxidation and glycolytic-to-oxidative fiber-type transformation. Taken together, these findings suggest that acetic acid improves endurance exercise capacity by promoting muscle oxidative properties, in part through the AMPK-mediated fatty acid oxidation and provide an important basis for the application of acetic acid as a major component of novel ergogenic aids.     

Sod2 overexpression preserves myoblast mitochondrial mass and function, but not muscle mass with aging

Mice lacking superoxide dismutase-2 (SOD2 or MnSOD) die during embryonic or early neonatal development, with diffuse superoxide-induced mitochondrial damage. Although stem and progenitor cells are exquisitely sensitive to oxidant stress, they have not been well studied in MnSOD2-manipulated mouse models. Patterns of proliferation and differentiation of cultured myoblasts (muscle progenitor cells), PI3-Akt signaling during differentiation, and the maintenance of mitochondrial mass with aging using myoblasts from young (3–4 week old) and aged (27–29 months old) MnSOD2-overexpressing ( Sod2- Tg) and heterozygote ( Sod2 ^{+/ −}) mice were characterized by us. Overexpression of MnSOD2 in myoblasts had a protective effect on mitochondrial DNA abundance and some aspects of mitochondrial function with aging, and preservation of differentiation potential. Sod2 deficiency resulted in defective signaling in the PI3-Akt pathway, specifically impaired phosphorylation of Akt at Ser473 and Thr308 in young myoblasts, and decreased differentiation potential. Compared with young myoblasts, aged myoblast Akt was constitutively phosphorylated, unresponsive to mitogen signaling, and indifferent to MnSOD2 levels. These data suggest that specific sites in the PI3K-Akt pathway are more sensitive to increased superoxide levels than to the increased hydrogen peroxide levels generated in Sod2 -transgenic myoblasts. In wild-type myoblasts, aging was associated with significant loss of mitochondrial DNA relative to chromosomal DNA, but MnSOD2 overexpression was associated with maintained myoblast mitochondrial DNA with aging.     

The ketogenic diet preserves skeletal muscle with aging in mice

The causes of the decline in skeletal muscle mass and function with age, known as sarcopenia, are poorly understood. Nutrition (calorie restriction) interventions impact many cellular processes and increase lifespan and preserve muscle mass and function with age. As we previously observed an increase in life span and muscle function in aging mice on a ketogenic diet (KD), we aimed to investigate the effect of a KD on the maintenance of skeletal muscle mass with age and the potential molecular mechanisms of this action. Twelve‐month‐old mice were assigned to an isocaloric control or KD until 16 or 26 months of age, at which time skeletal muscle was collected for evaluating mass, morphology, and biochemical properties. Skeletal muscle mass was significantly greater at 26 months in the gastrocnemius of mice on the KD. This result in KD mice was associated with a shift in fiber type from type IIb to IIa fibers and a range of molecular parameters including increased markers of NMJ remodeling, mitochondrial biogenesis, oxidative metabolism, and antioxidant capacity, while decreasing endoplasmic reticulum (ER) stress, protein synthesis, and proteasome activity. Overall, this study shows the effectiveness of a long‐term KD in mitigating sarcopenia. The diet preferentially preserved oxidative muscle fibers and improved mitochondrial and antioxidant capacity. These adaptations may result in a healthier cellular environment, decreasing oxidative and ER stress resulting in less protein turnover. These shifts allow mice to better maintain muscle mass and function with age.     

Climate warming is predicted to reduce omega‐3, long‐chain,polyunsaturated fatty acid production in phytoplankton

Phytoplankton are the main source of energy and omega‐3 (n‐3) long‐chain essential fatty acids (EFA) in aquatic ecosystems. Their growth and biochemical composition are affected by surrounding environmental conditions, including temperature, which continues to increase as a result of climate warming. Increasing water temperatures may negatively impact the production of EFA by phytoplankton through the process of homeoviscous adaptation. To investigate this, we conducted an exploratory data synthesis with 952 fatty acid (FA) profiles from six major groups of marine and freshwater phytoplankton. Temperature was strongly correlated with a decrease in the proportion of n‐3 long‐chain polyunsaturated FA (LC‐PUFA) and an increase in omega‐6 FA and saturated FA. Based on linear regression models, we predict that global n‐3 LC‐PUFA production will be reduced by 8.2% for eicosapentaenoic acid (EPA) and 27.8% for docosahexaenoic acid (DHA) with an increase in water temperature of 2.5 °C. Using a previously published estimate of the global production of EPA by diatoms, which contribute to most of the world's supply of EPA, we predict a loss of 14.2 Mt of EPA annually as a result of ocean warming. The n‐3 LC‐PUFA are vitally important for an array of key physiological functions in aquatic and terrestrial organisms, and these FA are mainly produced by phytoplankton. Therefore, reduced production of these EFA, as a consequence of climate warming, is predicted to negatively affect species that depend on these compounds for optimum physiological function. Such profound changes in the biochemical composition of phytoplankton cell membranes can lead to cascading effects throughout the world's ecosystems.     

Glutathione peroxidase‐1 overexpression reduces oxidative stress,and improves pathology and proteome remodeling in the kidneys of old mice

This study investigated the direct roles of hydrogen peroxide (H₂O₂) in kidney aging using transgenic mice overexpressing glutathione peroxidase‐1 (GPX1 TG). We demonstrated that kidneys in old mice recapitulated kidneys in elderly humans and were characterized by glomerulosclerosis, tubular atrophy, interstitial fibrosis, and loss of cortical mass. Scavenging H₂O₂ by GPX1 TG significantly reduced mitochondrial and total cellular reactive oxygen species (ROS) and mitigated oxidative damage, thus improving these pathologies. The potential mechanisms by which ROS are increased in the aged kidney include a decreased abundance of an anti‐aging hormone, Klotho, in kidney tissue, and decreased expression of nuclear respiratory factor 2 (Nrf2), a master regulator of the stress response. Decreased Klotho or Nrf2 was not improved in the kidneys of old GPX1 TG mice, even though mitochondrial morphology was better preserved. Using laser capture microdissection followed by label‐free shotgun proteomics analysis, we show that the glomerular proteome in old mice was characterized by decreased abundance of cytoskeletal proteins (critical for maintaining normal glomerular function) and heat shock proteins, leading to increased accumulation of apolipoprotein E and inflammatory molecules. Targeted proteomic analysis of kidney tubules from old mice showed decreased abundance of fatty acid oxidation enzymes and antioxidant proteins, as well as increased abundance of glycolytic enzymes and molecular chaperones. GPX1 TG partially attenuated the remodeling of glomerular and tubule proteomes in aged kidneys. In summary, mitochondria from GPX1 TG mice are protected and kidney aging is ameliorated via its antioxidant activities, independent and downstream of Nrf2 or Klotho signaling.     

EPA,not DHA,prevents fibrosis in pressure overload-induced heart failure: potential role of free fatty acid receptor 4

Heart failure with preserved ejection fraction (HFpEF) is half of all HF, but standard HF therapies are ineffective. Diastolic dysfunction, often secondary to interstitial fibrosis, is common in HFpEF. Previously, we found that supra-physiologic levels of ω3-PUFAs produced by 12 weeks of ω3-dietary supplementation prevented fibrosis and contractile dysfunction following pressure overload [transverse aortic constriction (TAC)], a model that resembles aspects of remodeling in HFpEF. This raised several questions regarding ω3-concentration-dependent cardioprotection, the specific role of EPA and DHA, and the relationship between prevention of fibrosis and contractile dysfunction. To achieve more clinically relevant ω3-levels and test individual ω3-PUFAs, we shortened the ω3-diet regimen and used EPA- and DHA-specific diets to examine remodeling following TAC. The shorter diet regimen produced ω3-PUFA levels closer to Western clinics. Further, EPA, but not DHA, prevented fibrosis following TAC. However, neither ω3-PUFA prevented contractile dysfunction, perhaps due to reduced uptake of ω3-PUFA. Interestingly, EPA did not accumulate in cardiac fibroblasts. However, FFA receptor 4, a G protein-coupled receptor for ω3-PUFAs, was sufficient and required to block transforming growth factor β1-fibrotic signaling in cultured cardiac fibroblasts, suggesting a novel mechanism for EPA. In summary, EPA-mediated prevention of fibrosis could represent a novel therapy for HFpEF.     

NAD+ boosting reduces age-associated amyloidosis and restores mitochondrial homeostasis in muscle

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