Hindlimb Muscles Research Articles

Endomorphin-2 and oxycodone paradoxically potentiate the exercise pressor reflex via acid-sensing ion channel 3 (ASIC3) in decerebrated rat

The exercise pressor reflex is evoked by the stimulation of group III and IV muscle afferents in response to muscle contractions. When the contracting muscles are freely perfused, acid-sensing ion channel 3 (ASIC3) on the endings of group IV afferents appear to play a minor role in evoking the exercise pressor reflex. We recently showed in isolated dorsal root ganglion (DRG) neurons innervating the gastrocnemius muscles that two μ opioid receptor agonists, namely endomorphin-2, and oxycodone, potentiated the sustained inward ASIC3 current evoked by acidic saline (pH 6.0). This in vitro finding prompted us to investigate whether endomorphin-2 and oxycodone, when infused into the arterial supply of freely perfused contracting hindlimb muscles, could potentiate the exercise pressor reflex. We found that infusing endomorphin-2 and naloxone (a μ opioid receptor antagonist) into the superficial epigastric artery of decerebrated unanesthetized rats potentiated the peak and integrated pressor responses to static contraction of the triceps surae muscles. The opioid-induced potentiation of the pressor responses to contraction were prevented by infusion of APETx2, an ASIC3 antagonist. Specifically, the peak pressor response to contraction averaged 19.3 ± 5.6 mmHg for control (n=10), 27.2 ± 8.1 mmHg after naloxone and endomorphin-2 infusion (n=10), and 20 ± 8 mmHg after APETx2 and endomorphin-2 infusion (n=10). Infusion of endomorphin-2 and naloxone did not potentiate the pressor responses to contraction in ASIC3 knockout rats (n=6), averaging 21.5 ± 7.7 mmHg for control (n=6), 20 ± 6.2 mmHg after naloxone and endomorphin-2 infusion (n=6), and 19.8 ± 6.9 mmHg after APETx2 and endomorphin-2 infusion (n=4). Similar findings were observed when oxycodone was substituted for endomorphin-2. Specifically, the peak pressor response to contraction averaged 24.3 ± 8.9 mmHg for control (n=10), 33.9 ± 10.6 mmHg after naloxone and oxycodone were infused (n=10), and 26.8 ± 8.1 mmHg after APETx2 and oxycodone were infused (n=10). Altogether, our data strongly suggest that ASIC3 stimulation by mu opioid agonists can potentiate the exercise pressor reflex, provided that mu opioid receptors on group III and IV afferents are not operative. (Supported by HL156594 and HL 156513). HL156594 and HL 156513. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Exercise Pressor Reflex Function Is Exacerbated in Decerebrate Adenine-Induced Chronic Kidney Disease Rats

Cardiovascular disease is the leading cause of death in chronic kidney disease (CKD). While the underlying mechanisms are not completely understood, elevated sympathetic nerve activity (SNA) and exercise intolerance are thought to play an important role. In normal physiological conditions, sympathetic modulation of the cardiovascular system during physical activity is largely governed by a peripheral neural reflex originating in contracting skeletal muscle, known as the exercise pressor reflex (EPR). Pioneering work by Park et al. demonstrated that CKD patients exhibited abnormal SNA and blood pressure (BP) elevations during both static and rhythmic handgrip exercise, suggesting an overactive EPR. However, due to experimental design constraints inherent to humans, the contribution of the EPR to the augmented SNA and BP responses to exercise could not be fully addressed. Therefore, the purpose of this study was to specifically test EPR function in CKD using a reduced animal model. Sprague-Dawley rats were randomly assigned to receive either a powdered chow diet containing adenine (0.25%w/w in feed) to induce CKD or a powdered chow diet alone (control group) for 12 weeks. Subsequently, in decerbrate animals, we measured mean arterial pressure (MAP) and renal SNA (RSNA) in response to isolated EPR activation during hindlimb muscle contraction (i.e., simulated exercise). We further assessed cardiovascular and sympathetic responses to passive muscle stretch to stimulate the mechanoreflex (i.e., activation of the mechanically-sesnsitive component of the EPR), and intra-arterial capsaicin administratration to activate the metaboflex (i.e., stimulation of the metabolically-sensitive component of the EPR). After completion of the diet, levels of plasma creatinine were significantly increased in adenine-fed rats compared to control rats (2.57±1.05 vs. 0.34±0.05 mg/dL, P<0.01), suggesting impaired renal function (i.e., adenine-induced CKD). We found that CKD animals exhibited augmentations in RSNA (Δ=153±75 vs. 61±50 %, P<0.05) and MAP (Δ=35±18 vs. 17±8 mmHg, P<0.05) in response to muscle contraction when compared to control animals. Importantly, the muscle tension developed during contraction was similar between the two groups (Δ=0.9±0.2 vs. 1.0±0.3 kg, P>0.1, CKD vs. control). During stimulation of the mechanoreflex, CKD rats also exhibited significantly greater RSNA (Δ=100±54 vs. 32±26 %, P<0.05) and MAP responses (Δ=25±14 vs. 10±7 mmHg, P<0.05) than control rats. The developed muscle tension during passive stretch was similar between the two groups (Δ=0.9±0.2 vs. 1.0±0.4 kg, P>0.1, CKD vs. control). There was a tendency for the responses to metaboreflex activation to be heightened in the CKD group compared with the control group, but the difference did not reach statistical significance (ΔRSNA=212±68 vs. 107±105 %, P=0.06, ΔMAP=67±12 vs. 48±21 mmHg, P=0.06, CKD vs. control, respectively). Our findings suggest that EPR function, particularly muscle mechanoreflex function, is exaggerated in CKD, leading to sympathetic activation and hypertensive responses during exercise. P30 DK079307 Subaward to H.K. and R01HL-133179 to W.V. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

What Is the Role of the Carotid Baroreflex in Exaggerating the Exercise Pressor Reflex in Male UCD-Type 2 Diabetes Mellitus Rats?

Previous studies have shown that exercise evokes an excessive increase in blood pressure in individuals with type 2 diabetes mellitus (T2DM), which is mediated, in part, by an altered exercise pressor reflex. Importantly, in healthy individuals the arterial baroreflex modulates the pressor response to exercise; however, the effects of T2DM on this modulatory effect of the baroreflex remain unclear. Therefore, the purpose of this study was to characterize the arterial baroreflex function in T2DM rats and to determine whether the carotid baroreflex plays a role in exaggerating the exercise pressor reflex. We hypothesized that T2DM rats would present a baroreflex dysfunction that likely contributes to the exaggerated pressor response during muscle contraction. Experiments were performed on adult (8 ± 2 months old), male University of California Davis (UCD)-T2DM ( n=7; body weight: 640 ± 43 g) and healthy Sprague-Dawley rats ( n=3; body weight: 485 ± 29 g). Female rats were not included in this study because they become diabetic when they are much older (>10 months old). Cardiac baroreflex function was characterized in anesthetized T2DM and healthy rats using the modified Oxford technique, which involved sequential bolus infusions of sodium nitroprusside (10 μg/kg) and phenylephrine (10 μg/kg) to induce blood pressure decreases and increases, respectively. Overall cardiac baroreflex gain (i.e., slope of the relationship between systolic blood pressure and R-R interval) was calculated using logistic regression, while gains to blood pressure falling and rising were calculated separately using piecewise and linear regression. The role of the carotid baroreflex in exaggerating the exercise pressor reflex was tested by statically contracting the hindlimb muscles for 30 s after surgical sinoaortic denervation (SAD) in decerebrated T2DM ( n=1; body weight: 555 g) and healthy ( n=1; body weight: 519 g) rats. Carotid barointact T2DM ( n=8; body weight: 521 ± 38 g) and healthy rats ( n=8; body weight: 482 ± 56 g) were included as controls. The pressor responses were calculated as a change (Δ) in peak mean arterial pressure during 30 s contraction relative to baseline. Data are presented as mean ± SD. T2DM rats presented with lower overall baroreflex sensitivity (T2DM: 0.07 ± 0.02 ms/mmHg, healthy: 0.61 ± 0.81 ms/mmHg, p=0.044), as well as sensitivity to increases in blood pressure (T2DM: 0.14 ± 0.10 ms/mmHg , healthy: 0.28 ± 0.15 ms/mmHg, p=0.049) compared to healthy controls. In contrast, cardiac baroreflex gain during decreases in blood pressure were not different between groups (T2DM: 0.16 ± 0.07 ms/mmHg, healthy: 0.10 ± 0.06 ms/mmHg, p=0.109). Notably, SAD did not alter blood pressure response to muscle contraction in the T2DM rat (SAD: Δ32 mmHg vs barointact: Δ32 ± 12 mmHg), but evoked an exaggerated blood pressure response in healthy rat (SAD: Δ34 mmHg vs barointact: Δ12 ± 3 mmHg). Our preliminary findings suggest that despite an attenuated cardiac baroreflex function in T2DM rats, specifically during acute increases in blood pressure, carotid baroreflex dysfunction may not contribute significantly to the exaggerated exercise pressor reflex in diabetes. This project was supported by NIH R01 HL144723. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Lactate and hydrogen ions play a predominant role in evoking the exercise pressor reflex during ischaemic contractions but not during freely perfused contractions.

We investigated the role played by lactate and hydrogen in evoking the exercise pressor reflex (EPR) in decerebrated rats whose hindlimb muscles were either freely perfused or ischaemic. Production of lactate and hydrogen by the contracting hindlimb muscles was manipulated by knocking out the myophosphorylase gene (pygm). In knockout rats (pygm-/-; n=13) or wild-type rats (pygm+/+; n=13), the EPR was evoked by isometrically contracting the triceps surae muscles. Blood pressure, tension, blood flow, renal sympathetic nerve activity and blood lactate concentrations were measured. Intramuscular metabolites and pH changes induced by the contractions were quantified by 31P-magnetic resonance spectroscopy (n=5). In a subset of pygm-/- rats (n=5), contractions were evoked with prior infusion of lactate (pH 6.0) in an attempt to restore the effect of lactate and hydrogen ions. Contraction of freely perfused muscles increased blood lactate and decreased muscle pH in pygm+/+ rats only. Despite these differences, the reflex pressor and sympathetic responses to freely perfused contraction did not differ between groups (P=0.992). During ischaemia, contraction increased muscle lactate and hydrogen ion production in pygm+/+ rats (P<0.0134), whereas it had no effect in pygm-/- rats (P>0.783). Likewise, ischaemia exaggerated the reflex pressor, and sympathetic responses to contraction in pygm+/+ but not in pygm-/- rats. This exaggeration was restored when a solution of lactate (pH 6.0) was infused prior to the contraction in pygm-/- rats. We conclude that lactate and hydrogen accumulation in contracting myocytes play a key role in evoking the metabolic component of the EPR during ischaemic but not during freely perfused contractions. KEY POINTS: Conflicting results exist about the role played by lactate and hydrogen ions in evoking the exercise pressor reflex. Using CRISP-Cas9, we rendered the myophosphorylase gene non-functional to block the production of lactate and hydrogen ions. The exercise pressor reflex was evoked in decerebrated rats by statically contracting the triceps surae muscles with or without muscle ischaemia. Static contraction elevated the concentration of lactate and hydrogen ions in pygm+/+ but not in pygm-/- rats. Despite these differences, the exercise pressor reflex was not different between groups. Acute muscle ischaemia exaggerated the concentration of lactate and hydrogen ions in pygm+/+ but not in pygm-/- rats. Likewise, acute muscle ischaemia exaggerated the exercise pressor reflex in pygm+/+ but not in pygm-/- rats. We conclude that lactate and hydrogen play a key role in evoking the exercise pressor reflex during ischaemic but not during freely perfused contractions.

Hindlimb myology in two piscivorous raptorial birds: a quantitative comparison of the osprey and the white‐tailed sea eagle (Aves, Accipitriformes)

AbstractThe hindlimbs of Accipitriformes are vital for capturing prey. Research on hindlimb musculature has primarily focused on species preferring avian and mammalian prey, whereas piscivorous species have received limited attention. This study aims to investigate the quantitative characteristics of hindlimb muscles in two piscivorous Accipitriformes, Pandion haliaetus and Haliaeetus albicilla, to discern potential muscular features associated with their specific food preference. The mass and proportion of all hindlimb muscles in both species were assessed based on their primary function (flexion or extension). A Kruskal‐Wallis test was employed to analyze possible differences in muscle mass between species. The allometric relationships between the muscles and body mass were explored with the reduced major axis method. Additionally, a study on the architectural parameters of the primary gripping muscles in P. haliaetus was conducted, using published information from other raptorial birds for comparison. The isometric scaling relationship predominated in the majority of individual muscles helping maintain a proportional relationship relative to body mass. Both species exhibited a similar pattern in terms of quantitative muscle features, implying a preservation of muscle characteristics linked to their predatory capabilities. The largest proportion of hindlimb muscle mass was dedicated to digit flexion in accordance with the grasping abilities of birds of prey. The muscles tibialis cranialis, flexor digitorum longus and flexor hallucis longus exhibited the greatest mass, high physiological cross‐sectional area values, and long fibre, categorizing them as high‐power specialists. These muscles are crucial in the flexion movements that produce the gripping action that characterizes birds of prey. Although no statistically significant differences were detected, each species displayed slightly distinct muscular characteristics, particularly in the architectural properties of the flexor muscles controlling digits II, III, and IV. These variations seem to be associated with differences in their prey preferences.

Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice.

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles (EOMs) are comparatively spared. While hindlimb and diaphragm muscles of end-stage SOD1G93A (G93A) mice (a familial ALS mouse model) exhibit severe denervation and depletion of Pax7+satellite cells (SCs), we found that the pool of SCs and the integrity of neuromuscular junctions (NMJs) are maintained in EOMs. In cell sorting profiles, SCs derived from hindlimb and diaphragm muscles of G93A mice exhibit denervation-related activation, whereas SCs from EOMs of G93A mice display spontaneous (non-denervation-related) activation, similar to SCs from wild-type mice. Specifically, cultured EOM SCs contain more abundant transcripts of axon guidance molecules, including Cxcl12, along with more sustainable renewability than the diaphragm and hindlimb counterparts under differentiation pressure. In neuromuscular co-culture assays, AAV-delivery of Cxcl12 to G93A-hindlimb SC-derived myotubes enhances motor neuron axon extension and innervation, recapitulating the innervation capacity of EOM SC-derived myotubes. G93A mice fed with sodium butyrate (NaBu) supplementation exhibited less NMJ loss in hindlimb and diaphragm muscles. Additionally, SCs derived from G93A hindlimb and diaphragm muscles displayed elevated expression of Cxcl12 and improved renewability following NaBu treatment in vitro. Thus, the NaBu-induced transcriptomic changes resembling the patterns of EOM SCs may contribute to the beneficial effects observed in G93A mice. More broadly, the distinct transcriptomic profile of EOM SCs may offer novel therapeutic targets to slow progressive neuromuscular functional decay in ALS and provide possible 'response biomarkers' in pre-clinical and clinical studies.

Distinct transcriptomic profile of satellite cells contributes to preservation of neuromuscular junctions in extraocular muscles of ALS mice

Amyotrophic lateral sclerosis (ALS) is a fatal neuromuscular disorder characterized by progressive weakness of almost all skeletal muscles, whereas extraocular muscles (EOMs) are comparatively spared. While hindlimb and diaphragm muscles of end-stage SOD1G93A (G93A) mice (a familial ALS mouse model) exhibit severe denervation and depletion of Pax7+satellite cells (SCs), we found that the pool of SCs and the integrity of neuromuscular junctions (NMJs) are maintained in EOMs. In cell sorting profiles, SCs derived from hindlimb and diaphragm muscles of G93A mice exhibit denervation-related activation, whereas SCs from EOMs of G93A mice display spontaneous (non-denervation-related) activation, similar to SCs from wild-type mice. Specifically, cultured EOM SCs contain more abundant transcripts of axon guidance molecules, including Cxcl12, along with more sustainable renewability than the diaphragm and hindlimb counterparts under differentiation pressure. In neuromuscular co-culture assays, AAV-delivery of Cxcl12 to G93A-hindlimb SC-derived myotubes enhances motor neuron axon extension and innervation, recapitulating the innervation capacity of EOM SC-derived myotubes. G93A mice fed with sodium butyrate (NaBu) supplementation exhibited less NMJ loss in hindlimb and diaphragm muscles. Additionally, SCs derived from G93A hindlimb and diaphragm muscles displayed elevated expression of Cxcl12 and improved renewability following NaBu treatment in vitro. Thus, the NaBu-induced transcriptomic changes resembling the patterns of EOM SCs may contribute to the beneficial effects observed in G93A mice. More broadly, the distinct transcriptomic profile of EOM SCs may offer novel therapeutic targets to slow progressive neuromuscular functional decay in ALS and provide possible ‘response biomarkers’ in pre-clinical and clinical studies.

Focal Traumatic Brain Injury Impairs the Integrity of the Basement Membrane of Hindlimb Muscle Fibers Revealed by Extracellular Matrix Immunoreactivity.

Traumatic brain injury (TBI) stands as a prominent global cause of disability, with motor deficits being a common consequence. Despite its widespread impact, the precise pathological mechanisms underlying motor deficits after TBI remain elusive. In this study, hindlimb postural asymmetry (HL-PA) development in rats subjected to focal TBI was investigated to explore the potential roles of collagen IV and laminin within the extracellular matrix (ECM) of selected hindlimb muscles in the emergence of motor deficits following TBI. A focal TBI was induced by ablating the left sensorimotor cortex in rats and motor deficits were assessed by measuring HL-PA. The expression of laminin and collagen IV in eight selected muscles on each side of the hindlimbs from both TBI- and sham-operated rats were studied using immunohistochemistry and semi-quantitatively analyzed. The results indicated that the TBI rats exhibited HL-PA, characterized by flexion of the contralateral (right) hindlimb. In the sham-operated rats, the immunoreactive components of laminin and collagen IV were evenly and smoothly distributed along the border of the muscle fibers in all the investigated muscles. In contrast, in the TBI rats, the pattern was broken into aggregated, granule-like, immunoreactive components. Such a labeling pattern was detected in all the investigated muscles both from the contra- and ipsilateral sides of the TBI rats. However, in TBI rats, most of the muscles from the contralateral hindlimb showed a significantly increased expression of these two proteins in comparison with those from the ipsilateral hindlimb. In comparison to sham-operated rats, there was a significant increase in laminin and collagen IV expression in various contralateral hindlimb muscles in the TBI rats. These findings suggest potential implications of laminin and collagen IV in the development of motor deficits following a focal TBI.

Effect of the mitochondrial transaminase (GOT2) on membrane potential-sensitive respiration in mitochondria of differentiated C2C12 muscle cells.

We previously showed that the transaminase inhibitor, aminooxyacetic acid, reduced respiration energized at complex II (succinate dehydrogenase, SDH) in mitochondria isolated from mouse hindlimb muscle. The effect required a reduction in membrane potential with resultant accumulation of oxaloacetate (OAA), a potent inhibitor of SDH. To specifically assess the effect of the mitochondrial transaminase, glutamic oxaloacetic transaminase (GOT2) on complex II respiration, and to determine the effect in intact cells as well as isolated mitochondria, we performed respiratory and metabolic studies in wildtype (WT) and CRISPR-generated GOT2 knockdown (KD) C2C12 myocytes. Intact cell respiration by GOT2KD cells versus WT was reduced by adding carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) to lower potential. In mitochondria of C2C12 KD cells, respiration at low potential generated by 1 µM FCCP and energized at complex II by 10 mM succinate + 0.5 mM glutamate (but not by complex I substrates) was reduced versus WT mitochondria. Although we could not detect OAA, metabolite data suggested that OAA inhibition of SDH may have contributed to the FCCP effect. C2C12 mitochondria differed from skeletal muscle mitochondria in that the effect of FCCP on complex II respiration was not evident with ADP addition. We also observed that C2C12 cells, unlike skeletal muscle, expressed glutamate dehydrogenase, which competes with GOT2 for glutamate metabolism. In summary, GOT2 KD reduced C2C12 respiration in intact cells at low potential. From differential substrate effects, this occurred largely at complex II. Moreover, C2C12 versus muscle mitochondria differ in complex II sensitivity to ADP and differ markedly in expression of glutamate dehydrogenase.NEW & NOTEWORTHY Impairment of the mitochondrial transaminase, GOT2, reduces complex II (succinate dehydrogenase, SDH)-energized respiration in C2C12 myocytes. This occurs only at low inner membrane potential and is consistent with inhibition of SDH. Incidentally, we observed that C2C12 mitochondria compared with muscle tissue mitochondria differ in sensitivity of complex II respiration to ADP and in the expression of glutamate dehydrogenase.

Mitochondrial Transplantation's Role in Rodent Skeletal Muscle Bioenergetics: Recharging the Engine of Aging.

Mitochondria are the 'powerhouses of cells' and progressive mitochondrial dysfunction is a hallmark of aging in skeletal muscle. Although different forms of exercise modality appear to be beneficial to attenuate aging-induced mitochondrial dysfunction, it presupposes that the individual has a requisite level of mobility. Moreover, non-exercise alternatives (i.e., nutraceuticals or pharmacological agents) to improve skeletal muscle bioenergetics require time to be effective in the target tissue and have another limitation in that they act systemically and not locally where needed. Mitochondrial transplantation represents a novel directed therapy designed to enhance energy production of tissues impacted by defective mitochondria. To date, no studies have used mitochondrial transplantation as an intervention to attenuate aging-induced skeletal muscle mitochondrial dysfunction. The purpose of this investigation, therefore, was to determine whether mitochondrial transplantation can enhance skeletal muscle bioenergetics in an aging rodent model. We hypothesized that mitochondrial transplantation would result in sustained skeletal muscle bioenergetics leading to improved functional capacity. Fifteen female mice (24 months old) were randomized into two groups (placebo or mitochondrial transplantation). Isolated mitochondria from a donor mouse of the same sex and age were transplanted into the hindlimb muscles of recipient mice (quadriceps femoris, tibialis anterior, and gastrocnemius complex). The results indicated significant increases (ranging between ~36% and ~65%) in basal cytochrome c oxidase and citrate synthase activity as well as ATP levels in mice receiving mitochondrial transplantation relative to the placebo. Moreover, there were significant increases (approx. two-fold) in protein expression of mitochondrial markers in both glycolytic and oxidative muscles. These enhancements in the muscle translated to significant improvements in exercise tolerance. This study provides initial evidence showing how mitochondrial transplantation can promote skeletal muscle bioenergetics in an aging rodent model.

Mitochondrial targeted catalase improves muscle strength following arteriovenous fistula creation in mice with chronic kidney disease

Hand dysfunction is a common observation after arteriovenous fistula (AVF) creation for hemodialysis access and has a variable clinical phenotype; however, the underlying mechanism responsible is unclear. Grip strength changes are a common metric used to assess AVF-associated hand disability but has previously been found to poorly correlate with the hemodynamic perturbations post-AVF placement implicating other tissue-level factors as drivers of hand outcomes. In this study, we sought to test if expression of a mitochondrial targeted catalase (mCAT) in skeletal muscle could reduce AVF-related limb dysfunction in mice with chronic kidney disease (CKD). Male and female C57BL/6J mice were fed an adenine-supplemented diet to induce CKD prior to placement of an AVF in the iliac vascular bundle. Adeno-associated virus was used to drive expression of either a green fluorescent protein (control) or mCAT using the muscle-specific human skeletal actin (HSA) gene promoter prior to AVF creation. As expected, the muscle-specific AAV-HSA-mCAT treatment did not impact blood urea nitrogen levels (P = 0.72), body weight (P = 0.84), or central hemodynamics including infrarenal aorta and inferior vena cava diameters (P > 0.18) or velocities (P > 0.38). Hindlimb perfusion recovery and muscle capillary densities were also unaffected by AAV-HSA-mCAT treatment. In contrast to muscle mass and myofiber size which were not different between groups, both absolute and specific muscle contractile forces measured via a nerve-mediated in-situ preparation were significantly greater in AAV-HSA-mCAT treated mice (P = 0.0012 and P = 0.0002). Morphological analysis of the post-synaptic neuromuscular junction uncovered greater acetylcholine receptor cluster areas (P = 0.0094) and lower fragmentation (P = 0.0010) in AAV-HSA-mCAT treated mice. Muscle mitochondrial oxidative phosphorylation was not different between groups, but AAV-HSA-mCAT treated mice had lower succinate-fueled mitochondrial hydrogen peroxide emission compared to AAV-HSA-GFP mice (P < 0.001). In summary, muscle-specific scavenging of mitochondrial hydrogen peroxide significantly improves neuromotor function in mice with CKD following AVF creation.

Changes in intra- and interlimb reflexes from hindlimb cutaneous afferents after staggered thoracic lateral hemisections during locomotion in cats.

When the foot dorsum contacts an obstacle during locomotion, cutaneous afferents signal central circuits to coordinate muscle activity in the four limbs. Spinal cord injury disrupts these interactions, impairing balance and interlimb coordination. We evoked cutaneous reflexes by electrically stimulating left and right superficial peroneal nerves before and after two thoracic lateral hemisections placed on opposite sides of the cord at 9- to 13-week interval in seven adult cats (4males and 3 females). We recorded reflex responses in ten hindlimb and five forelimb muscles bilaterally. After the first (right T5-T6) and second (left T10-T11) hemisections, coordination of the fore- and hindlimbs was altered and/or became less consistent. After the second hemisection, cats required balance assistance to perform quadrupedal locomotion. Short-latency reflex responses in homonymous and crossed hindlimb muscles largely remained unaffected after staggered hemisections. However, mid- and long-latency homonymous and crossed responses in both hindlimbs occurred less frequently after staggered hemisections. In forelimb muscles, homolateral and diagonal mid- and long-latency response occurrence significantly decreased after the first and second hemisections. In all four limbs, however, when present, short-, mid- and long-latency responses maintained their phase-dependent modulation. We also observed reduced durations of short-latency inhibitory homonymous responses in left hindlimb extensors early after the first hemisection and delayed short-latency responses in the right ipsilesional hindlimb after the first hemisection. Therefore, changes in cutaneous reflex responses correlated with impaired balance/stability and interlimb coordination during locomotion after spinal cord injury. Restoring reflex transmission could be used as a biomarker to facilitate locomotor recovery. KEY POINTS: Cutaneous afferent inputs coordinate muscle activity in the four limbs during locomotion when the foot dorsum contacts an obstacle. Thoracic spinal cord injury disrupts communication between spinal locomotor centres located at cervical and lumbar levels, impairing balance and limb coordination. We investigated cutaneous reflexes during quadrupedal locomotion by electrically stimulating the superficial peroneal nerve bilaterally, before and after staggered lateral thoracic hemisections of the spinal cord in cats. We showed a loss/reduction of mid- and long-latency responses in all four limbs after staggered hemisections, which correlated with altered coordination of the fore- and hindlimbs and impaired balance. Targeting cutaneous reflex pathways projecting to the four limbs could help develop therapeutic approaches aimed at restoring transmission in ascending and descending spinal pathways.

Validation of positional candidates Rps6ka6 and Pou3f4 for a locus associated with skeletal muscle mass variability.

Genetic variability significantly contributes to individual differences in skeletal muscle mass; however, the specific genes involved in that process remain elusive. In this study, we examined the role of positional candidates, Rps6ka6 and Pou3f4, of a chromosome X locus, implicated in muscle mass variability in CFW laboratory mice. Histology of hindlimb muscles was studied in CFW male mice carrying the muscle "increasing" allele C (n = 15) or "decreasing" allele T (n = 15) at the peak marker of the locus, rs31308852, and in the Pou3f4y/- and their wild-type male littermates. To study the role of the Rps6ka6 gene, we deleted exon 7 (Rps6ka6-ΔE7) using clustered regularly interspaced palindromic repeats-Cas9 based method in H2Kb myogenic cells creating a severely truncated RSK4 protein. We then tested whether that mutation affected myoblast proliferation, migration, and/or differentiation. The extensor digitorum longus muscle was 7% larger (P < 0.0001) due to 10% more muscle fibers (P = 0.0176) in the carriers of the "increasing" compared with the "decreasing" CFW allele. The number of fibers was reduced by 15% (P = 0.0268) in the slow-twitch soleus but not in the fast-twitch extensor digitorum longus (P = 0.2947) of Pou3f4y/- mice. The proliferation and migration did not differ between the Rps6ka6-ΔE7 and wild-type H2Kb myoblasts. However, indices of differentiation (myosin expression, P < 0.0001; size of myosin-expressing cells, P < 0.0001; and fusion index, P = 0.0013) were significantly reduced in Rps6ka6-ΔE7 cells. This study suggests that the effect of the X chromosome locus on muscle fiber numbers in the fast-twitch extensor digitorum longus is mediated by the Rps6ka6 gene, whereas the Pou3f4 gene affects fiber number in slow-twitch soleus.

Early Movement Restriction Affects FNDC5/Irisin and BDNF Levels in Rat Muscle and Brain.

Interaction with the environment appears necessary for the maturation of sensorimotor and cognitive functions in early life. In rats, a model of sensorimotor restriction (SMR) from postnatal day 1 (P1) to P28 has shown that low and atypical sensorimotor activities induced the perturbation of motor behavior due to muscle weakness and the functional disorganization of the primary somatosensory and motor cortices. In the present study, our objective was to understand how SMR affects the muscle-brain dialogue. We focused on irisin, a myokine secreted by skeletal muscles in response to exercise. FNDC5/irisin expression was determined in hindlimb muscles and brain structures by Western blotting, and irisin expression in blood and cerebrospinal fluid was determined using an ELISA assay at P8, P15, P21 and P28. Since irisin is known to regulate its expression, Brain-Derived Neurotrophic Factor (BDNF) levels were also measured in the same brain structures. We demonstrated that SMR increases FNDC5/irisin levels specifically in the soleus muscle (from P21) and also affects this protein expression in several brain structures (as early as P15). The BDNF level was increased in the hippocampus at P8. To conclude, SMR affects FNDC5/irisin levels in a postural muscle and in several brain regions and has limited effects on BDNF expression in the brain.

Paradoxical potentiation of the exercise pressor reflex by endomorphin 2 in the presence of naloxone.

When contracting muscles are freely perfused, the acid-sensing ion channel 3 (ASIC3) on group IV afferents plays a minor role in evoking the exercise pressor reflex. We recently showed in isolated dorsal root ganglion neurons innervating the gastrocnemius muscles that two mu opioid receptor agonists, namely endomorphin 2 and oxycodone, potentiated the sustained inward ASIC3 current evoked by acidic solutions. This in vitro finding prompted us to determine whether endomorphin 2 and oxycodone, when infused into the arterial supply of freely perfused contracting hindlimb muscles, potentiated the exercise pressor reflex. We found that infusion of endomorphin 2 and naloxone in decerebrated rats potentiated the pressor responses to contraction of the triceps surae muscles. The endomorphin 2-induced potentiation of the pressor responses to contraction was prevented by infusion of APETx2, an ASIC3 antagonist. Specifically, the peak pressor response to contraction averaged 19.3 ± 5.6 mmHg for control (n = 10), 27.2 ± 8.1 mmHg after naloxone and endomorphin 2 infusion (n = 10), and 20 ± 8 mmHg after APETx2 and endomorphin 2 infusion (n = 10). Infusion of endomorphin 2 and naloxone did not potentiate the pressor responses to contraction in ASIC3 knockout rats (n = 6). Partly similar findings were observed when oxycodone was substituted for endomorphin 2. Oxycodone infusion significantly increased the exercise pressor reflex over its control level, but subsequent APETx2 infusion failed to restore the increase to its control level (n = 9). The peak pressor response averaged 23.1 ± 8.6 mmHg for control (n = 9), 33.2 ± 11 mmHg after naloxone and oxycodone were infused (n = 9), and 27 ± 8.6 mmHg after APETx2 and oxycodone were infused (n = 9). Our data suggest that after opioid receptor blockade, ASIC3 stimulation by the endogenous mu opioid, endomorphin 2, potentiated the exercise pressor reflex.NEW & NOTEWORTHY This paper provides the first in vivo evidence that endomorphin 2, an endogenous opioid peptide, can paradoxically increase the magnitude of the exercise pressor reflex by an ASIC3-dependent mechanism even when the contracting muscles are freely perfused.

PINK1 regulated mitophagy is evident in skeletal muscles.

PINK1, mutated in familial forms of Parkinson's disease, initiates mitophagy following mitochondrial depolarization. However, it is difficult to monitor this pathway physiologically in mice as loss of PINK1 does not alter basal mitophagy levels in most tissues. To further characterize this pathway in vivo, we used mito-QC mice in which loss of PINK1 was combined with the mitochondrial-associated POLGD257A mutation. We focused on skeletal muscle as gene expression data indicates that this tissue has the highest PINK1 levels. We found that loss of PINK1 in oxidative hindlimb muscle significantly reduced mitophagy. Of interest, the presence of the POLGD257A mutation, while having a minor effect in most tissues, restored levels of muscle mitophagy caused by the loss of PINK1. Although our observations highlight that multiple mitophagy pathways operate within a single tissue, we identify skeletal muscle as a tissue of choice for the study of PINK1-dependant mitophagy under basal conditions.

Celecoxib attenuates hindlimb unloading-induced muscle atrophy via suppressing inflammation, oxidative stress and ER stress by inhibiting STAT3.

Improving inflammation may serve as useful therapeutic interventions for the hindlimb unloading-induced disuse muscle atrophy. Celecoxib is a selective non-steroidal anti-inflammatory drug. We aimed to determine the role and mechanism of celecoxib in hindlimb unloading-induced disuse muscle atrophy. Celecoxib significantly attenuated the decrease in soleus muscle mass, hindlimb muscle function and the shift from slow- to fast-twitch muscle fibers caused by hindlimb unloading in rats. Importantly, celecoxib inhibited the increased expression of inflammatory factors, macrophage infiltration in damaged soleus muscle. Mechanistically, Celecoxib could significantly reduce oxidative stress and endoplasmic reticulum stress in soleus muscle of unloaded rats. Furthermore, celecoxib inhibited muscle proteolysis by reducing the levels of MAFbx, MuRF1, and autophagy related proteins maybe by inhibiting the activation of pro-inflammatory STAT3 pathway in vivo and in vitro. This study is the first to demonstrate that celecoxib can attenuate disuse muscle atrophy caused by hindlimb unloading via suppressing inflammation, oxidative stress and endoplasmic reticulum stress probably, improving target muscle function and reversing the shift of muscle fiber types by inhibiting STAT3 pathways-mediated inflammatory cascade. This study not only enriches the potential molecular regulatory mechanisms, but also provides new potential therapeutic targets for disuse muscle atrophy.

Mapping lumbar efferent and afferent spinal circuitries via paddle array in a porcine model

BackgroundPreclinical models are essential for identifying changes occurring after neurologic injury and assessing therapeutic interventions. Yucatan miniature pigs (minipigs) have brain and spinal cord dimensions like humans and are useful for laboratory-to-clinic studies. Yet, little work has been done to map spinal sensorimotor distributions and identify similarities and differences between the porcine and human spinal cords. New methodsTo characterize efferent and afferent signaling, we implanted a conventional 32-contact, four-column array into the dorsal epidural space over the lumbosacral spinal cord, spanning the L5–L6 vertebrae, in two Yucatan minipigs. Spinally evoked motor potentials were recorded bilaterally in four hindlimb muscles during stimulation delivered from different array locations. Then, cord dorsum potentials were recorded via the array by stimulating the left and right tibial nerves. ResultsUtilizing epidural spinal stimulation, we achieved selective left, right, proximal, and distal activation in the hindlimb muscles. We then determined the selectivity of each muscle as a function of stimulation location which relates to the distribution of the lumbar motor pools. Comparison with existing methodsMapping motoneuron distribution to hindlimb muscles and recording responses to peripheral nerve stimulation in the dorsal epidural space reveals insights into ascending and descending signal propagation in the lumbar spinal cord. Clinical-grade arrays have not been utilized in a porcine model. ConclusionsThese results indicate that efferent and afferent spinal sensorimotor networks are spatially distinct, provide information about the organization of motor pools in the lumbar enlargement, and demonstrate the feasibility of using clinical-grade devices in large animal research.

Myoglobin deficiency impairs maximal oxygen uptake and exercise performance: a lesson from Mb-/- mice.

Myoglobin (Mb) plays an important role at rest and during exercise as a reservoir of oxygen and has been suggested to regulate NO• bioavailability under hypoxic/acidic conditions. However, its ultimate role during exercise is still a subject of debate. We aimed to study the effect of Mb deficiency on maximal oxygen uptake ( ) and exercise performance in myoglobin knockout mice (Mb-/- ) when compared to control mice (Mb+/+ ). Furthermore, we also studied NO• bioavailability, assessed as nitrite (NO2 - ) and nitrate (NO3 - ) in the heart, locomotory muscle and in plasma, at rest and during exercise at exhaustion both in Mb-/- and in Mb+/+ mice. The mice performed maximal running incremental exercise on a treadmill with whole-body gas exchange measurements. The Mb-/- mice had lower body mass, heart and hind limb muscle mass (P<0.001). Mb-/- mice had significantly reduced maximal running performance (P<0.001). expressed in mlmin-1 in Mb-/ - mice was 37% lower than in Mb+/+ mice (P<0.001) and 13% lower when expressed in mlmin-1 kg body mass-1 (P=0.001). Additionally, Mb-/- mice had significantly lower plasma, heart and locomotory muscle NO2 - levels at rest. During exercise NO2 - increased significantly in the heart and locomotory muscles of Mb-/- and Mb+/+ mice, whereas no significant changes in NO2 - were found in plasma. Our study showed that, contrary to recent suggestions, Mb deficiency significantly impairs and maximal running performance in mice. KEY POINTS: Myoglobin knockout mice (Mb-/- ) possess lower maximal oxygen uptake ( ) and poorer maximal running performance than control mice (Mb+/+ ). Respiratory exchange ratio values at high running velocities in Mb-/- mice are higher than in control mice suggesting a shift in substrate utilization towards glucose metabolism in Mb-/- mice at the same running velocities. Lack of myoglobin lowers basal systemic and muscle NO• bioavailability, but does not affect exercise-induced NO2 - changes in plasma, heart and locomotory muscles. The present study demonstrates that myoglobin is of vital importance for and maximal running performance as well as explains why previous studies have failed to prove such a role of myoglobin when using the Mb-/- mouse model.

Effects of chronic alcohol intoxication on aerobic exercise-induced adaptations in female mice.

Chronic alcohol intoxication decreases muscle strength/function and causes mitochondrial dysfunction. Aerobic exercise training improves mitochondrial oxidative capacity and increases muscle mass and strength. Presently, the impact of chronic alcohol on aerobic exercise-induced adaptations was investigated. Female C57BL/6Hsd mice were randomly assigned to one of four groups: control sedentary (CON SED; n = 26), alcohol sedentary (ETOH SED; n = 27), control exercise (CON EX; n = 28), and alcohol exercise (ETOH EX; n = 25). Exercise mice had running wheel access for 2 h a day, 7 days a week. All mice were fed either control or an alcohol-containing liquid diet. Grip strength testing and EchoMRI were performed before and after the interventions. After 6 wk, hindlimb muscles were collected for molecular analyses. A subset of mice performed a treadmill run to fatigue (RTF), then abstained from alcohol for 2 wk and repeated the RTF. Alcohol decreased lean mass and forelimb grip strength compared with control-fed mice. Alcohol blunted the exercise-induced increase in muscle mass (plantaris and soleus), type IIa fiber percentage in the plantaris, and run time to fatigue. Mitochondrial markers (Citrate synthase activity and Complex I-IV, COXIV and Cytochrome C protein expression) were increased with exercise regardless of ETOH in the gastrocnemius but not tibialis anterior muscle. Two weeks of alcohol abstinence improved RTF time in ETOH EX but not in ETOH SED. These data suggest that alcohol impairs some exercise-induced adaptations in skeletal muscle, but not all were negatively affected, indicating that exercise may be a beneficial behavior even while consuming alcohol.NEW & NOTEWORTHY Alcohol consumption during an aerobic exercise training period prevented training-induced increases in run to fatigue time and grip strength. Cessation of alcohol allowed for recovery of endurance performance within 2 wk. The worsened exercise performance after alcohol was unrelated to impairments in markers of mitochondrial health. Therefore, some adaptations to exercise training are impaired with alcohol use (endurance performance, muscle growth, and strength), while others remain mostly unaffected (mitochondrial health).