Single-Nuclei RNA-Sequencing of the Gastrocnemius Muscle in Peripheral Artery Disease.

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.

Activation of the Aryl Hydrocarbon Receptor in Muscle Exacerbates Ischemic Pathology in Chronic Kidney Disease.

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.

Use of Creatine and Creatinine to Minimize Doxorubicin-Induced Cytotoxicity in Cardiac and Skeletal Muscle Myoblasts.

Doxorubicin (DOX), an effective anticancer agent, can damage cardiac and skeletal muscle tissue via excessive generation of reactive oxygen species (ROS). Supplemental creatine (Cr) has been shown to have a therapeutic role in disease states characterized by increased oxidative stress. To investigate the effects of Cr and creatinine (CrN) on DOX-induced cytotoxicity. Cultured L6 and H9C2 myoblasts were exposed to 25 µM DOX, 10 mM Cr, 10 mM CrN, 25 µM DOX + 10 mM Cr, 25 µM DOX + 10 mM CrN, or control media for 12 h. Viability was assessed using Confocal and Widefield imaging. Immunoblotting was used to determine protein expression. Viability was lowest in the DOX-treated group regardless of cell type; however, when DOX was combined with Cr or CrN, viability was improved. Levels of oxidative stress, as measured by 4-hydroxynonenal (4HNE), were significantly (p < 0.05) higher in the DOX treated cells vs. controls; however, Cr + DOX and CrN + DOX significantly lowered 4HNE levels compared to DOX-treated cells. Creatine kinase (CK), a key marker of cellular damage, was significantly higher in DOX-treated H9c2 cells vs. controls. However, Cr or CrN in combination with DOX, resulted in no significant differences in CK vs. controls. Supplementation with Cr or CrN may preserve cell viability during DOX treatment.

Chronic activation of the aryl hydrocarbon receptor in muscle exacerbates ischemic pathology in chronic kidney disease.

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.

Profiling mouse cochlear cell maturation using 10× Genomics single-cell transcriptomics.

Juvenile and mature mouse cochleae contain various low-abundant, vulnerable sensory epithelial cells embedded in the calcified temporal bone, making it challenging to profile the dynamic transcriptome changes of these cells during maturation at the single-cell level. Here we performed the 10x Genomics single-cell RNA sequencing (scRNA-seq) of mouse cochleae at postnatal days 14 (P14) and 28. We attained the transcriptomes of multiple cell types, including hair cells, supporting cells, spiral ganglia, stria fibrocytes, and immune cells. Our hair cell scRNA-seq datasets are consistent with published transcripts from bulk RNA-seq. We also mapped known deafness genes to corresponding cochlear cell types. Importantly, pseudotime trajectory analysis revealed that inner hair cell maturation peaks at P14 while outer hair cells continue development until P28. We further identified and confirmed a long non-coding RNA gene Miat to be expressed during maturation in cochlear hair cells and spiral ganglia neurons, and Pcp4 to be expressed during maturation in cochlear hair cells. Our transcriptomes of juvenile and mature mouse cochlear cells provide the sequel to those previously published at late embryonic and early postnatal ages and will be valuable resources to investigate cochlear maturation at the single-cell resolution.

The Effect of a Multi-Ingredient Pre-Workout Supplement on Time to Fatigue in NCAA Division I Cross-Country Athletes.

This investigation aimed to determine the effect of a multi-ingredient pre-workout supplement (MIPS) on heart rate (HR), perceived exertion (RPE), lactate concentration, and time to fatigue (TTF) during a running task to volitional exhaustion. Eleven NCAA Division I cross-country runners (20 ± 2 year; height: 171 ± 14 cm; weight: 63.5 ± 9.1 kg) participated in this randomized, double-blind, placebo-controlled cross-over study. Bayesian statistical methods were utilized, and parameter estimates were interpreted as statistically significant if the 95% highest-density intervals (HDIs) did not include zero. TTF was increased in the MIPS condition with a posterior Meandiff = 154 ± 4.2 s (95% HDI: -167, 465) and a 0.84 posterior probability that the supplement would increase TTF relative to PL. Blood lactate concentration immediately post-exercise was also higher in the MIPS condition compared to PL with an estimated posterior Meandiff = 3.99 ± 2.1 mmol (95% HDI: -0.16, 7.68). There were no differences in HR or RPE between trials. These findings suggest that a MIPS ingested prior to sustained running at lactate threshold has an 84% chance of increasing TTF in highly trained runners and may allow athletes to handle a higher level of circulating lactate before reaching exhaustion.