ABSTRACT
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.
Subject(s)
Arteriovenous Fistula , Arteriovenous Shunt, Surgical , Kidney Failure, Chronic , Renal Insufficiency, Chronic , Humans , Male , Female , Animals , Mice , Catalase , Hydrogen Peroxide , Mice, Inbred C57BL , Renal Insufficiency, Chronic/therapy , Renal Dialysis , Muscle Strength , Kidney Failure, Chronic/therapyABSTRACT
BACKGROUND: Accumulating evidence has demonstrated that chronic tobacco smoking directly contributes to skeletal muscle dysfunction independent of its pathological impact to the cardiorespiratory systems. The mechanisms underlying tobacco smoke toxicity in skeletal muscle are not fully resolved. In this study, the role of the aryl hydrocarbon receptor (AHR), a transcription factor known to be activated with tobacco smoke, was investigated. METHODS: AHR related gene (mRNA) expression was quantified in skeletal muscle from adult controls and patients with chronic obstructive pulmonary disease (COPD), as well as mice with and without cigarette smoke exposure. Utilizing both skeletal muscle-specific AHR knockout mice exposed to chronic repeated (5 days per week for 16 weeks) cigarette smoke and skeletal muscle-specific expression of a constitutively active mutant AHR in healthy mice, a battery of assessments interrogating muscle size, contractile function, mitochondrial energetics, and RNA sequencing were employed. RESULTS: Skeletal muscle from COPD patients (N = 79, age = 67.0 ± 8.4 years) had higher levels of AHR (P = 0.0451) and CYP1B1 (P < 0.0001) compared to healthy adult controls (N = 16, age = 66.5 ± 6.5 years). Mice exposed to cigarette smoke displayed higher expression of Ahr (P = 0.008), Cyp1b1 (P < 0.0001), and Cyp1a1 (P < 0.0001) in skeletal muscle compared to air controls. Cigarette smoke exposure was found to impair skeletal muscle mitochondrial oxidative phosphorylation by ~50% in littermate controls (Treatment effect, P < 0.001), which was attenuated by deletion of the AHR in muscle in male (P = 0.001), but not female, mice (P = 0.37), indicating there are sex-dependent pathological effects of smoking-induced AHR activation in skeletal muscle. Viral mediated expression of a constitutively active mutant AHR in the muscle of healthy mice recapitulated the effects of cigarette smoking by decreasing muscle mitochondrial oxidative phosphorylation by ~40% (P = 0.003). CONCLUSIONS: These findings provide evidence linking chronic AHR activation secondary to cigarette smoke exposure to skeletal muscle bioenergetic deficits in male, but not female, mice. AHR activation is a likely contributor to the decline in muscle oxidative capacity observed in smokers and AHR antagonism may provide a therapeutic avenue aimed to improve muscle function in COPD.
Subject(s)
Pulmonary Disease, Chronic Obstructive , Tobacco Smoke Pollution , Aged , Animals , Humans , Male , Mice , Middle Aged , Mitochondria/metabolism , Muscle, Skeletal/pathology , Nicotiana , Pulmonary Disease, Chronic Obstructive/pathology , Receptors, Aryl Hydrocarbon/genetics , Receptors, Aryl Hydrocarbon/metabolism , Smoking/adverse effects , Tobacco Smoking , FemaleABSTRACT
BACKGROUND: Lower extremity peripheral artery disease (PAD) is a growing epidemic with limited effective treatment options. Here, we provide a single-nuclei atlas of PAD limb muscle to facilitate a better understanding of the composition of cells and transcriptional differences that comprise the diseased limb muscle. METHODS: We obtained gastrocnemius muscle specimens from 20 patients with PAD and 12 non-PAD controls. Nuclei were isolated and single-nuclei RNA-sequencing was performed. The composition of nuclei was characterized by iterative clustering via principal component analysis, differential expression analysis, and the use of known marker genes. Bioinformatics analysis was performed to determine differences in gene expression between PAD and non-PAD nuclei, as well as subsequent analysis of intercellular signaling networks. Additional histological analyses of muscle specimens accompany the single-nuclei RNA-sequencing atlas. RESULTS: Single-nuclei RNA-sequencing analysis indicated a fiber type shift with patients with PAD having fewer type I (slow/oxidative) and more type II (fast/glycolytic) myonuclei compared with non-PAD, which was confirmed using immunostaining of muscle specimens. Myonuclei from PAD displayed global upregulation of genes involved in stress response, autophagy, hypoxia, and atrophy. Subclustering of myonuclei also identified populations that were unique to PAD muscle characterized by metabolic dysregulation. PAD muscles also displayed unique transcriptional profiles and increased diversity of transcriptomes in muscle stem cells, regenerating myonuclei, and fibro-adipogenic progenitor cells. Analysis of intercellular communication networks revealed fibro-adipogenic progenitors as a major signaling hub in PAD muscle, as well as deficiencies in angiogenic and bone morphogenetic protein signaling which may contribute to poor limb function in PAD. CONCLUSIONS: This reference single-nuclei RNA-sequencing atlas provides a comprehensive analysis of the cell composition, transcriptional signature, and intercellular communication pathways that are altered in the PAD condition.
Subject(s)
Muscle, Skeletal , Peripheral Arterial Disease , Humans , Muscle, Skeletal/metabolism , Peripheral Arterial Disease/metabolism , Lower Extremity , RNA/metabolismABSTRACT
For end-stage kidney disease (ESKD) patients, hemodialysis requires durable vascular access which is often surgically created using an arteriovenous fistula (AVF). However, some ESKD patients that undergo AVF placement develop access-related hand dysfunction (ARHD) through unknown mechanisms. In this study, we sought to determine if changes in the serum metabolome could distinguish ESKD patients that develop ARHD from those that have normal hand function following AVF creation. Forty-five ESKD patients that underwent first-time AVF creation were included in this study. Blood samples were obtained pre-operatively and 6-weeks post-operatively and metabolites were extracted and analyzed using nuclear magnetic resonance spectroscopy. Patients underwent thorough examination of hand function at both timepoints using the following assessments: grip strength manometry, dexterity, sensation, motor and sensory nerve conduction testing, hemodynamics, and the Disabilities of the Arm, Shoulder, and Hand (DASH) questionnaire. Nineteen of the forty-five patients displayed overt weakness using grip strength manometry (P < 0.0001). Unfortunately, the serum metabolome was indistinguishable between patients with and without weakness following AVF surgery. However, a significant correlation was found between the change in tryptophan levels and the change in grip strength suggesting a possible role of tryptophan-derived uremic metabolites in post-AVF hand-associated weakness. Compared to grip strength, changes in dexterity and sensation were smaller than those observed in grip strength, however, post-operative decreases in phenylalanine, glycine, and alanine were unique to patients that developed signs of motor or sensory disability following AVF creation.
Subject(s)
Arteriovenous Fistula , Arteriovenous Shunt, Surgical , Kidney Failure, Chronic , Humans , Lipidomics , Tryptophan , Upper Extremity , Kidney Failure, Chronic/therapy , Renal Dialysis/adverse effects , Arteriovenous Shunt, Surgical/adverse effects , Arteriovenous Shunt, Surgical/methods , Retrospective Studies , Treatment OutcomeABSTRACT
The objective of the present study was to determine if treatment with N-acetylcysteine (NAC) could reduce access-related limb dysfunction in mice. Male and female C57BL6J mice were fed an adenine-supplemented diet to induce chronic kidney disease (CKD) prior to the surgical creation of an arteriovenous fistula (AVF) in the iliac vascular bundle. AVF creation significantly increased peak aortic and infrarenal vena cava blood flow velocities, but NAC treatment had no significant impact, indicating that fistula maturation was not impacted by NAC treatment. Hindlimb muscle and paw perfusion recovery and muscle capillary density in the AVF limb were unaffected by NAC treatment. However, NAC treatment significantly increased the mass of the tibialis anterior (P = 0.0120) and soleus (P = 0.0452) muscles post-AVF. There was a significant main effect of NAC treatment on hindlimb grip strength at postoperative day 12 (POD 12) (P = 0.0003), driven by significantly higher grip strength in both male (P = 0.0273) and female (P = 0.0031) mice treated with NAC. There was also a significant main effect of NAC treatment on the walking speed at postoperative day 12 (P = 0.0447), and post hoc testing revealed an improvement in NAC-treated male mice (P = 0.0091). The area of postsynaptic acetylcholine receptors (P = 0.0263) and motor endplates (P = 0.0240) was also increased by NAC treatment. Interestingly, hindlimb skeletal muscle mitochondrial oxidative phosphorylation trended higher in NAC-treated female mice but was not statistically significant (P = 0.0973). Muscle glutathione levels and redox status were not significantly impacted by NAC treatment in either sex. In summary, NAC treatment attenuated some aspects of neuromotor pathology in mice with chronic kidney disease following AVF creation.NEW & NOTEWORTHY Hemodialysis via autogenous arteriovenous fistula (AVF) is the preferred first-line modality for renal replacement therapy in patients with end-stage kidney disease. However, patients undergoing AVF surgery frequently experience a spectrum of hand disability symptoms postsurgery including weakness and neuromotor dysfunction. Unfortunately, no treatment is currently available to prevent or mitigate these symptoms. Here, we provide evidence that daily N-acetylcysteine supplementation can attenuate some aspects of limb neuromotor function in a preclinical mouse model of AVF.
Subject(s)
Arteriovenous Fistula , Arteriovenous Shunt, Surgical , Kidney Failure, Chronic , Renal Insufficiency, Chronic , Male , Female , Animals , Mice , Acetylcysteine/pharmacology , Renal Dialysis , Renal Insufficiency, Chronic/therapy , Renal Insufficiency, Chronic/etiology , Kidney Failure, Chronic/therapy , Arteriovenous Shunt, Surgical/adverse effects , Retrospective StudiesABSTRACT
Lower-extremity peripheral arterial disease (PAD) has increased in prevalence, yet therapeutic development has remained stagnant. Skeletal muscle health and function has been strongly linked to quality of life and medical outcomes in patients with PAD. Using a rodent model of PAD, this study demonstrates that treatment of the ischemic limb with insulin-like growth factor (IGF)-1 significantly increases muscle size and strength without improving limb hemodynamics. Interestingly, the effect size of IGF1 therapy was larger in female mice than in male mice, highlighting the need to carefully examine sex-dependent effects in experimental PAD therapies.
ABSTRACT
BACKGROUND: Chronic kidney disease (CKD) accelerates the development of atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the mechanisms underlying this pathobiology are ill-defined. Recent work has indicated that tryptophan-derived uremic solutes, which are ligands for AHR (aryl hydrocarbon receptor), are associated with limb amputation in PAD. Herein, we examined the role of AHR activation in the myopathy of PAD and CKD. METHODS: AHR-related gene expression was evaluated in skeletal muscle obtained from mice and human PAD patients with and without CKD. AHRmKO (skeletal muscle-specific AHR knockout) mice with and without CKD were subjected to femoral artery ligation, and a battery of assessments were performed to evaluate vascular, muscle, and mitochondrial health. Single-nuclei RNA sequencing was performed to explore intercellular communication. Expression of the constitutively active AHR was used to isolate the role of AHR in mice without CKD. RESULTS: PAD patients and mice with CKD displayed significantly higher mRNA expression of classical AHR-dependent genes (Cyp1a1, Cyp1b1, and Aldh3a1) when compared with either muscle from the PAD condition with normal renal function (P<0.05 for all 3 genes) or nonischemic controls. AHRmKO significantly improved limb perfusion recovery and arteriogenesis, preserved vasculogenic paracrine signaling from myofibers, increased muscle mass and strength, as well as enhanced mitochondrial function in an experimental model of PAD/CKD. Moreover, viral-mediated skeletal muscle-specific expression of a constitutively active AHR in mice with normal kidney function exacerbated the ischemic myopathy evidenced by smaller muscle masses, reduced contractile function, histopathology, altered vasculogenic signaling, and lower mitochondrial respiratory function. CONCLUSIONS: These findings establish AHR activation in muscle as a pivotal regulator of the ischemic limb pathology in CKD. Further, the totality of the results provides support for testing of clinical interventions that diminish AHR signaling in these conditions.
Subject(s)
Muscular Diseases , Peripheral Arterial Disease , Renal Insufficiency, Chronic , Animals , Humans , Mice , Ischemia/metabolism , Mice, Knockout , Muscle, Skeletal/metabolism , Muscular Diseases/metabolism , Peripheral Arterial Disease/genetics , Peripheral Arterial Disease/metabolism , Receptors, Aryl Hydrocarbon/genetics , Renal Insufficiency, Chronic/genetics , Renal Insufficiency, Chronic/metabolismABSTRACT
Chronic kidney disease (CKD) accelerates the development of atherosclerosis, decreases muscle function, and increases the risk of amputation or death in patients with peripheral artery disease (PAD). However, the cellular and physiological mechanisms underlying this pathobiology are ill-defined. Recent work has indicated that tryptophan-derived uremic toxins, many of which are ligands for the aryl hydrocarbon receptor (AHR), are associated with adverse limb outcomes in PAD. We hypothesized that chronic AHR activation, driven by the accumulation of tryptophan-derived uremic metabolites, may mediate the myopathic condition in the presence of CKD and PAD. Both PAD patients with CKD and mice with CKD subjected to femoral artery ligation (FAL) displayed significantly higher mRNA expression of classical AHR-dependent genes ( Cyp1a1 , Cyp1b1 , and Aldh3a1 ) when compared to either muscle from the PAD condition with normal renal function ( P <0.05 for all three genes) or non-ischemic controls. Skeletal-muscle-specific AHR deletion in mice (AHR mKO ) significantly improved limb muscle perfusion recovery and arteriogenesis, preserved vasculogenic paracrine signaling from myofibers, increased muscle mass and contractile function, as well as enhanced mitochondrial oxidative phosphorylation and respiratory capacity in an experimental model of PAD/CKD. Moreover, viral-mediated skeletal muscle-specific expression of a constitutively active AHR in mice with normal kidney function exacerbated the ischemic myopathy evidenced by smaller muscle masses, reduced contractile function, histopathology, altered vasculogenic signaling, and lower mitochondrial respiratory function. These findings establish chronic AHR activation in muscle as a pivotal regulator of the ischemic limb pathology in PAD. Further, the totality of the results provide support for testing of clinical interventions that diminish AHR signaling in these conditions.
ABSTRACT
Significance: An estimated 700 million people globally suffer from chronic kidney disease (CKD). In addition to increasing cardiovascular disease risk, CKD is a catabolic disease that results in a loss of muscle mass and function, which are strongly associated with mortality and a reduced quality of life. Despite the importance of muscle health and function, there are no treatments available to prevent or attenuate the myopathy associated with CKD. Recent Advances: Recent studies have begun to unravel the changes in mitochondrial and redox homeostasis within skeletal muscle during CKD. Impairments in mitochondrial metabolism, characterized by reduced oxidative phosphorylation, are found in both rodents and patients with CKD. Associated with aberrant mitochondrial function, clinical and preclinical findings have documented signs of oxidative stress, although the molecular source and species are ill-defined. Critical Issues: First, we review the pathobiology of CKD and its associated myopathy, and we review muscle cell bioenergetics and redox biology. Second, we discuss evidence from clinical and preclinical studies that have implicated the involvement of mitochondrial and redox alterations in CKD-associated myopathy and review the underlying mechanisms reported. Third, we discuss gaps in knowledge related to mitochondrial and redox alterations on muscle health and function in CKD. Future Directions: Despite what has been learned, effective treatments to improve muscle health in CKD remain elusive. Further studies are needed to uncover the complex mitochondrial and redox alterations, including post-transcriptional protein alterations, in patients with CKD and how these changes interact with known or unknown catabolic pathways contributing to poor muscle health and function. Antioxid. Redox Signal. 38, 318-337.
Subject(s)
Muscular Diseases , Renal Insufficiency, Chronic , Humans , Quality of Life , Mitochondria/metabolism , Muscular Diseases/etiology , Muscular Diseases/metabolism , Muscle, Skeletal/metabolism , Renal Insufficiency, Chronic/complications , Renal Insufficiency, Chronic/metabolism , Oxidation-ReductionABSTRACT
Objective: Hand disability after hemodialysis access surgery has been common yet has remained poorly understood. Arteriovenous fistula (AVF) hemodynamic perturbations have not reliably correlated with the observed measures of hand function. Chronic kidney disease (CKD) is known to precipitate myopathy; however, the interactive influences of renal insufficiency and ischemia on limb outcomes have remained unknown. We hypothesized that CKD would contribute to access-related hand dysfunction via altered mitochondrial bioenergetics. Using a novel murine AVF model, we sought to characterize the skeletal muscle outcomes in mice with and without renal insufficiency. Methods: Male, 8-week-old C57BL/6J mice were fed either an adenine-supplemented diet to induce renal insufficiency (CKD) or a casein-based control chow (CON). After 2 weeks of dietary intervention, the mice were randomly assigned to undergo iliac AVF surgery (n = 12/group) or a sham operation (n = 5/group). Measurements of aortoiliac hemodynamics, hindlimb perfusion, and hindlimb motor function were collected for 2 weeks. The mice were sacrificed on postoperative day 14 to assess skeletal muscle histopathologic features and mitochondrial function. To assess the late outcome trends, 20 additional mice had undergone CKD induction and sham (n = 5) or AVF (n = 15) surgery and followed up for 6 weeks postoperatively before sacrifice. Results: The adenine-fed mice had had a significantly reduced glomerular filtration rate and elevated blood urea nitrogen, confirming the presence of CKD. The sham mice had a 100% survival rate and AVF cohorts an 82.1% survival rate with an 84.4% AVF patency rate. The aorta and inferior vena cava velocity measurements and the vessel diameter had increased after AVF creation (P < .0001 vs sham). The AVF groups had had a 78.4% deficit in paw perfusion compared with the contralateral limb after surgery (P < .0001 vs sham). Mitochondrial function was influenced by the presence of CKD. The respiratory capacity of the CKD-sham mice (8443 ± 1509 pmol/s/mg at maximal energy demand) was impaired compared with that of the CON-sham mice (12,870 ± 1203 pmol/s/mg; P = .0001). However, this difference was muted after AVF creation (CKD-AVF, 4478 ± 3685 pmol/s/mg; CON-AVF, 5407 ± 3582 pmol/s/mg; P = .198). The AVF cohorts had had impairments in grip strength (vs sham; P < .0001) and gait (vs sham; P = .012). However, the presence of CKD did not significantly alter the measurements of gross muscle function. The paw perfusion deficits had persisted 6 weeks postoperatively for the AVF mice (P < .0001 vs sham); however, the myopathy had resolved (grip strength, P = .092 vs sham; mitochondrial respiration, P = .108 vs sham). Conclusions: CKD and AVF-induced distal limb ischemia both impaired skeletal muscle mitochondrial function. Renal insufficiency was associated with a baseline myopathy that was exacerbated by the acute ischemic injury resulting from AVF creation. However, ischemia was the primary driver of the observed phenotype of gross motor impairment. This model reliably reproduced the local and systemic influences that contribute to access-related hand dysfunction and provides a platform for further mechanistic and therapeutic investigation.
ABSTRACT
End-stage kidney disease, the most advanced stage of chronic kidney disease (CKD), requires renal replacement therapy or kidney transplant to sustain life. To accomplish durable dialysis access, the creation of an arteriovenous fistula (AVF) has emerged as a preferred approach. Unfortunately, a significant proportion of patients that receive an AVF experience some form of hand dysfunction; however, the mechanisms underlying these side effects are not understood. In this study, we used nuclear magnetic resonance spectroscopy to investigate the muscle metabolome following iliac AVF placement in mice with CKD. To induce CKD, C57BL6J mice were fed an adenine-supplemented diet for 3 wk and then randomized to receive AVF or sham surgery. Two weeks following surgery, the quadriceps muscles were rapidly dissected and snap frozen for metabolite extraction and subsequent nuclear magnetic resonance analysis. Principal component analysis demonstrated clear separation between groups, confirming a unique metabolome in mice that received an AVF. AVF creation resulted in reduced levels of creatine, ATP, and AMP as well as increased levels of IMP and several tricarboxylic acid cycle metabolites suggesting profound energetic stress. Pearson correlation and multiple linear regression analyses identified several metabolites that were strongly linked to measures of limb function (grip strength, gait speed, and mitochondrial respiration). In summary, AVF creation generates a unique metabolome profile in the distal skeletal muscle indicative of an energetic crisis and myosteatosis.NEW & NOTEWORTHY Creation of an arteriovenous fistula (AVF) is the preferred approach for dialysis access, but some patients experience hand dysfunction after AVF creation. In this study, we provide a detailed metabolomic analysis of the limb muscle in a murine model of AVF. AVF creation resulted in metabolite changes associated with an energetic crisis and myosteatosis that associated with limb function.
Subject(s)
Arteriovenous Fistula , Arteriovenous Shunt, Surgical , Kidney Failure, Chronic , Renal Insufficiency, Chronic , Animals , Mice , Adenine , Adenosine Monophosphate , Adenosine Triphosphate , Arteriovenous Shunt, Surgical/adverse effects , Creatine , Muscles , Renal Dialysis/methods , Renal Insufficiency, Chronic/etiologyABSTRACT
Chronic kidney disease is a major public health problem, and the prevalence of end-stage renal disease (ESRD) requiring chronic renal replacement therapies such as hemodialysis continues to increase. Autogenous arteriovenous fistula (AVF) placement remains a primary vascular access option for ESRD patients. Unfortunately, approximately half of the hemodialysis patients experience dialysis access-related hand dysfunction (ARHD), ranging from subtle paresthesia to digital gangrene. Notably, the underlying biologic drivers responsible for ARHD are poorly understood, and no adequate animal model exists to elucidate the mechanisms and/or develop novel therapeutics for the prevention/treatment of ARHD. Herein, we describe a new mouse model in which an AVF is created between the left common iliac artery and vein, thereby facilitating the assessment of limb pathophysiology. The microsurgery includes vessel isolation, longitudinal venotomy, creation of arteriovenous anastomosis, and venous reconstruction. Sham surgeries include all the critical steps except for AVF creation. Iliac AVF placement results in clinically relevant alterations in central hemodynamics, peripheral ischemia, and impairments in hindlimb neuromotor performance. This novel preclinical AVF model provides a useful platform that recapitulates common neuromotor perturbations reported by hemodialysis patients, allowing researchers to investigate the mechanisms of ARHD pathophysiology and test potential therapeutics.
Subject(s)
Arteriovenous Shunt, Surgical , Kidney Failure, Chronic , Animals , Arteriovenous Shunt, Surgical/adverse effects , Arteriovenous Shunt, Surgical/methods , Disease Models, Animal , Humans , Kidney Failure, Chronic/therapy , Mice , Renal Dialysis/adverse effects , Renal Dialysis/methods , Retrospective Studies , Treatment Outcome , Upper Extremity , Vascular PatencyABSTRACT
BACKGROUND: Despite improved surgical approaches for chronic limb-threatening ischemia (CLTI), amputation rates remain high and contributing tissue-level factors remain unknown. The purpose of this study was twofold: (1) to identify differences between the healthy adult and CLTI limb muscle proteome, and (2) to identify differences in the limb muscle proteome of CLTI patients prior to surgical intervention or at the time of amputation. METHODS AND RESULTS: Gastrocnemius muscle was collected from non-ischemic controls (n = 19) and either pre-interventional surgery (n = 10) or at amputation outcome (n = 29) CLTI patients. All samples were subjected to isobaric tandem-mass-tag-assisted proteomics. The mitochondrion was the primary classification of downregulated proteins (> 70%) in CLTI limb muscles and paralleled robust functional mitochondrial impairment. Upregulated proteins (> 38%) were largely from the extracellular matrix. Across the two independent sites, 39 proteins were downregulated and 12 upregulated uniformly. Pre-interventional CLTI muscles revealed a robust upregulation of mitochondrial proteins but modest functional impairments in fatty acid oxidation as compared with controls. Comparison of pre-intervention and amputation CLTI limb muscles revealed mitochondrial proteome and functional deficits similar to that between amputation and non-ischemic controls. Interestingly, these observed changes occurred despite 62% of the amputation CLTI patients having undergone a prior surgical intervention. CONCLUSIONS: The CLTI proteome supports failing mitochondria as a phenotype that is unique to amputation outcomes. The signature of pre-intervention CLTI muscle reveals stable mitochondrial protein abundance that is insufficient to uniformly prevent functional impairments. Taken together, these findings support the need for future longitudinal investigations aimed to determine whether mitochondrial failure is causally involved in amputation outcomes from CLTI.
Subject(s)
Chronic Limb-Threatening Ischemia/physiopathology , Proteome/pharmacology , Aged , Aged, 80 and over , Chronic Limb-Threatening Ischemia/complications , Chronic Limb-Threatening Ischemia/pathology , Cross-Sectional Studies , Extremities/blood supply , Extremities/innervation , Extremities/physiopathology , Female , Florida , Humans , Male , Muscle, Skeletal/drug effects , Muscle, Skeletal/physiopathology , North Carolina , Proteome/metabolism , Risk FactorsABSTRACT
OBJECTIVE: Hemodialysis access-related hand dysfunction is a common clinical feature of patients with chronic kidney disease (CKD) after arteriovenous fistula (AVF) placement. The heterogeneity in symptoms and the lack of a predictive association with changes in hemodynamic alterations precipitated by the AVF suggest that other factors are involved in the mechanisms responsible for causing hand and limb dysfunction postoperatively. To the best of our knowledge, no suitable animal models have provided a platform for performing preclinical experiments designed to elucidate the biologic drivers of access-related hand dysfunction. Therefore, our objective was to develop a novel murine AVF model that could be used to study dialysis access-related limb dysfunction. METHODS: Male 8-week-old C57BL/6J mice (n = 15/group) were exposed to either an adenine-supplemented diet to induce CKD or casein-based chow (control). Four weeks after the diet intervention, the mice were randomly assigned to receive an iliac AVF (n = 10/group) or sham surgery (n = 5/group) on the left hindlimb. The mice were sacrificed 2 weeks after surgery, and AVF specimens and hindlimb skeletal muscles were collected for further analysis. RESULTS: Before AVF or sham surgery, the glomerular filtration rates were significantly reduced and the blood urea nitrogen levels were significantly elevated in the CKD groups compared with the controls (P < .05). AVF surgery was associated with an â¼80% patency rate among the survivors (four control and three CKD mice died postoperatively). Patency was verified by changes in hemodynamics using Doppler ultrasound imaging and altered histologic morphology. Compared with sham surgery, AVF surgery reduced ipsilateral hindlimb perfusion to the tibialis anterior muscle (20%-40%) and paw (40%-50%), which remained stable until euthanasia. Analysis of gastrocnemius muscle mitochondrial respiratory function uncovered a significant decrease (40%-50%) in mitochondrial function in the AVF mice. No changes were found in the muscle mass, myofiber cross-sectional area, or centrally nucleated fiber proportion in the extensor digitorum longus and soleus muscles between the sham and AVF mice. CONCLUSIONS: The results from the present study have demonstrated that iliac AVF formation is a practical animal model that facilitates examination of hemodialysis access-related limb dysfunction. AVF surgery produced the expected hemodynamic changes, and evaluation of the limb muscle revealed a substantial mitochondrial impairment that was present without changes in muscle size.
ABSTRACT
Preclinical animal models of chronic kidney disease (CKD) are critical to investigate the underlying mechanisms of disease and to evaluate the efficacy of novel therapeutics aimed to treat CKD-associated pathologies. The objective of the present study was to compare the adenine diet and 5/6 nephrectomy (Nx) CKD models in mice. Male and female 10-wk-old C57BL/6J mice (n = 5-9 mice/sex/group) were randomly allocated to CKD groups (0.2-0.15% adenine-supplemented diet or 5/6 Nx surgery) or the corresponding control groups (casein diet or sham surgery). Following the induction of CKD, the glomerular filtration rate was reduced to a similar level in both adenine and 5/6 Nx mice (adenine diet-fed male mice: 81.1 ± 41.9 µL/min vs. 5/6 Nx male mice: 160 ± 80.9 µL/min, P = 0.5875; adenine diet-fed female mice: 112.9 ± 32.4 µL/min vs. 5/6 Nx female mice: 107.0 ± 45.7 µL/min, P = 0.9995). Serum metabolomics analysis indicated that established uremic toxins were robustly elevated in both CKD models, although some differences were observed between CKD models (i.e., p-cresol sulfate). Dysregulated phosphate homeostasis was observed in the adenine model only, whereas Ca2+ homeostasis was disturbed in male mice with both CKD models. Compared with control mice, muscle mass and myofiber cross-sectional areas of the extensor digitorum longus and soleus muscles were â¼18-24% smaller in male CKD mice regardless of the model but were not different in female CKD mice (P > 0.05). Skeletal muscle mitochondrial respiratory function was significantly decreased (19-24%) in CKD mice in both models and sexes. These findings demonstrate that adenine diet and 5/6 Nx models of CKD have similar levels of renal dysfunction and skeletal myopathy. However, the adenine diet model demonstrated superior performance with regard to mortality (â¼20-50% mortality for 5/6 Nx vs. 0% mortality for the adenine diet, P < 0.05 for both sexes) compared with the 5/6 Nx surgical model.NEW & NOTEWORTHY Numerous preclinical models of chronic kidney disease have been used to evaluate skeletal muscle pathology; however, direct comparisons of popular models are not available. In this study, we compared adenine-induced nephropathy and 5/6 nephrectomy models. Both models produced equivalent levels of muscle atrophy and mitochondrial impairment, but the adenine model exhibited lower mortality rates, higher consistency in uremic toxin levels, and dysregulated phosphate homeostasis compared with the 5/6 nephrectomy model.
Subject(s)
Adenine/pharmacology , Glomerular Filtration Rate/genetics , Muscle, Skeletal/metabolism , Renal Insufficiency, Chronic/metabolism , Animals , Disease Models, Animal , Kidney/metabolism , Kidney/pathology , Male , Mice, Inbred C57BL , Muscle, Skeletal/pathology , Muscular Diseases/pathology , Muscular Diseases/physiopathology , Nephrectomy/methods , Renal Insufficiency, Chronic/drug therapy , Renal Insufficiency, Chronic/pathology , Uremia/physiopathologyABSTRACT
PURPOSE: To investigate the effects of a single session of either peristaltic pulse dynamic leg compressions (PPDC) or local heat therapy (HT) after prolonged intermittent shuttle running on skeletal muscle glycogen content, muscle function, and the expression of factors involved in skeletal muscle remodeling. METHODS: Twenty-six trained individuals were randomly allocated to either a PPDC (n = 13) or a HT (n = 13) group. After completing a 90-min session of intermittent shuttle running, participants consumed 0.3 g·kg-1 protein plus 1.0 g·kg-1 carbohydrate and received either PPDC or HT for 60 min in one randomly selected leg, while the opposite leg served as control. Muscle biopsies from both legs were obtained before and after exposure to the treatments. Muscle function and soreness were also evaluated before, immediately after, and 24 h after the exercise bout. RESULTS: The changes in glycogen content were similar (P > 0.05) between the thigh exposed to PPDC and the control thigh ~90 min (Control: 14.9 ± 34.3 vs PPDC: 29.6 ± 34 mmol·kg-1 wet wt) and ~210 min (Control: 45.8 ± 40.7 vs PPDC: 52 ± 25.3 mmol·kg-1 wet wt) after the treatment. There were also no differences in the change in glycogen content between thighs ~90 min (Control: 35.9 ± 26.1 vs HT: 38.7 ± 21.3 mmol·kg-1 wet wt) and ~210 min (Control: 61.4 ± 50.6 vs HT: 63.4 ± 17.5 mmol·kg-1 wet wt) after local HT. The changes in peak torque and fatigue resistance of the knee extensors, muscle soreness, and the mRNA expression and protein abundance of select factors were also similar (P > 0.05) in both thighs, irrespective of the treatment. CONCLUSIONS: A single 1-h session of either PPDC or local HT does not accelerate glycogen resynthesis and the recovery of muscle function after prolonged intermittent shuttle running.
Subject(s)
Glycogen/biosynthesis , Hot Temperature/therapeutic use , Intermittent Pneumatic Compression Devices , Muscle, Skeletal/metabolism , Running/physiology , Adolescent , Adult , Female , Humans , Knee/physiology , Male , Muscle Fatigue , Muscle Proteins/metabolism , Muscle Strength , Myalgia/therapy , RNA, Messenger/metabolism , Torque , Young AdultABSTRACT
Chronic limb threatening ischemia (CLTI) is the most severe manifestation of peripheral atherosclerosis. Patients with CLTI have poor muscle quality and function and are at high risk for limb amputation and death. The objective of this study was to interrogate the metabolome of limb muscle from CLTI patients. To accomplish this, a prospective cohort of CLTI patients undergoing either a surgical intervention (CLTI Pre-surgery) or limb amputation (CLTI Amputation), as well as non-peripheral arterial disease (non-PAD) controls were enrolled. Gastrocnemius muscle biopsy specimens were obtained and processed for nuclear magnetic resonance (NMR)-based metabolomics analyses using solution state NMR on extracted aqueous and organic phases and 1H high-resolution magic angle spinning (HR-MAS) on intact muscle specimens. CLTI Amputation specimens displayed classical features of ischemic/hypoxic metabolism including accumulation of succinate, fumarate, lactate, alanine, and a significant decrease in the pyruvate/lactate ratio. CLTI Amputation muscle also featured aberrant amino acid metabolism marked by elevated branched chain amino acids. Finally, both Pre-surgery and Amputation CLTI muscles exhibited pronounced accumulation of lipids, suggesting the presence of myosteatosis, including cholesterol, triglycerides, and saturated fatty acids. Taken together, these metabolite differences add to a growing body of literature that have characterized profound metabolic disturbance's in the failing ischemic limb of CLTI patients.
ABSTRACT
Heat therapy (HT) has emerged as a potential adjunctive therapy to alleviate the symptoms of peripheral artery disease (PAD), but the mechanisms underlying the positive effects of this treatment modality remain undefined. Using a model of diet-induced obesity (DIO) and ischemia-induced muscle damage, we tested the hypothesis that HT would alter body composition, promote vascular growth and mitochondrial biogenesis, and improve skeletal muscle function. Male DIO C57Bl/6J mice underwent bilateral ligation of the femoral artery and were randomly allocated to receive HT or a control intervention for 30 min daily over 3 wk. When compared with a group of lean, sham-operated animals, ligated DIO mice exhibited increases in body and fat masses, exercise intolerance, and contractile dysfunction of the isolated soleus (SOL) and extensor digitorum longus (EDL) muscles. Repeated HT averted an increase in body mass induced by high-fat feeding due to reduced fat accrual. Fat mass was â¼25% and 29% lower in the HT group relative to controls after 2 and 3 wk of treatment, respectively. Muscle mass relative to body mass and maximal absolute force of the EDL, but not SOL, were higher in animals exposed to HT. There were no group differences in skeletal muscle capillarization, the expression of angiogenic factors, mitochondrial content, and the diameter of the gracilis arteries. These findings indicate that HT reduces diet-induced fat accumulation and rescues skeletal muscle contractile dysfunction. This practical treatment may prove useful for diabetic and obese PAD patients who are unable to undergo conventional exercise regimens.NEW & NOTEWORTHY The epidemic of obesity-related dyslipidemia and diabetes is a central cause of the increasing burden of peripheral artery disease (PAD), but few accessible therapies exist to mitigate the metabolic and functional abnormalities in these patients. We report that daily exposure to heat therapy (HT) in the form of lower-body immersion in water heated to 39 °C for 3 weeks attenuates fat accumulation and weight gain, and improves muscle strength in obese mice with femoral artery occlusion.
Subject(s)
Hot Temperature , Ischemia , Animals , Body Composition , Hindlimb , Ischemia/metabolism , Male , Mice , Muscle, Skeletal/metabolism , Obesity/metabolismABSTRACT
Peripheral artery disease (PAD) is caused by atherosclerosis in the lower extremities, which leads to a spectrum of life-altering symptomatology, including claudication, ischemic rest pain, and gangrene requiring limb amputation. Current treatments for PAD are focused primarily on re-establishing blood flow to the ischemic tissue, implying that blood flow is the decisive factor that determines whether or not the tissue survives. Unfortunately, failure rates of endovascular and revascularization procedures remain unacceptably high and numerous cell- and gene-based vascular therapies have failed to demonstrate efficacy in clinical trials. The low success of vascular-focused therapies implies that non-vascular tissues, such as skeletal muscle and oxidative stress, may substantially contribute to PAD pathobiology. Clues toward the importance of skeletal muscle in PAD pathobiology stem from clinical observations that muscle function is a strong predictor of mortality. Mitochondrial impairments in muscle have been documented in PAD patients, although its potential role in clinical pathology is incompletely understood. In this review, we discuss the underlying mechanisms causing mitochondrial dysfunction in ischemic skeletal muscle, including causal evidence in rodent studies, and highlight emerging mitochondrial-targeted therapies that have potential to improve PAD outcomes. Particularly, we will analyze literature data on reactive oxygen species production and potential counteracting endogenous and exogenous antioxidants.
ABSTRACT
The prolonged impairment in muscle strength, power, and fatigue resistance after eccentric exercise has been ascribed to a plethora of mechanisms, including delayed muscle refueling and microvascular and mitochondrial dysfunction. This review explores the hypothesis that local heat therapy hastens functional recovery after strenuous eccentric exercise by facilitating glycogen resynthesis, reversing vascular derangements, augmenting mitochondrial function, and stimulating muscle protein synthesis.