methods-for-situ-quantification-mitochondrial-morphology-muscle

Methods for <em>In situ</em> Quantification of Mitochondrial Morphology in Muscle and Terminal Schwann Cells of Mice

Mitochondrial network morphology is widely used as an indicator of cellular health; however, quantifying mitochondrial architecture within intact tissues ...

Texas A&M University, College Station

Acute injury of skeletal muscle disrupts myofibres, microvessels and motor innervation. Myofibre regeneration is well characterized, however its relationship with the regeneration of microvessels and motor nerves is undefined. Endothelial cell (EC) ephrin-B2 (Efnb2) is required for angiogenesis during embryonic development and promotes neurovascular regeneration in the adult. We hypothesized that, following acute injury to skeletal muscle, loss of EC Efnb2 would impair microvascular regeneration and the recovery of neuromuscular junction (NMJ) integrity. Mice (aged 3-6 months) were bred for EC-specific conditional knockout (CKO) of Efnb2 following tamoxifen injection with non-injected CKO mice as controls (CON). The gluteus maximus, tibialis anterior or extensor digitorum longus muscle was then injured with local injection of BaCl. Intravascular staining with wheat germ agglutinin revealed diminished capillary area in the gluteus maximus of CKO vs. CON at 5 days post-injury (dpi); both recovered to uninjured (0 dpi) level by 10 dpi. At 0 dpi, tibialis anterior isometric force of CKO was less than CON. At 10 dpi, isometric force was reduced by half in both groups. During intermittent contractions (75 Hz, 330 ms s, 120 s), isometric force fell during indirect (sciatic nerve) stimulation whereas force was maintained during direct (electrical field) stimulation of myofibres. Neuromuscular transmission failure correlated with perturbed presynaptic (terminal Schwann cells) and postsynaptic (nicotinic acetylcholine receptors) NMJ morphology in CKO. Resident satellite cell number on extensor digitorum longus myofibres did not differ between groups. Following acute injury of skeletal muscle, loss of Efnb2 in ECs delays capillary regeneration and attenuates recovery of NMJ structure and function. KEY POINTS: The relationship between microvascular regeneration and motor nerve regeneration following skeletal muscle injury is undefined. Expression of Efnb2 in endothelial cells (ECs) is essential to vascular development and promotes neurovascular regeneration in the adult. To test the hypothesis that EfnB2 in ECs is required for microvascular regeneration and myofibre reinnervation, we induced conditional knockout of Efnb2 in ECs of mice. Acute injury was then induced by BaCl injection into gluteus maximus, tibialis anterior or extensor digitorum longus (EDL) muscle. Capillary regeneration was reduced at 5 days post-injury (dpi) in gluteus maximus of conditional knockout vs. controls; at 10 dpi, neither differed from uninjured. Nerve stimulation revealed neuromuscular transmission failure in tibialis anterior with perturbed neuromuscular junction structure. Resident satellite cell number on EDL myofibres did not differ between groups. Conditional knockout of EC Efnb2 delays capillary regeneration and attenuates recovery of neuromuscular junction structure and function.

Inducible deletion of endothelial cell Efnb2 delays capillary regeneration and attenuates myofibre reinnervation following myotoxin injury in mice.

Metabolic conditions, such as gout, can result from elevated uric acid (UA) levels. Consuming high-purine meals increases UA levels. Therefore, people with hyperuricemia typically must avoid ingesting such foods. Polyphenols have been shown to reduce uric acid levels and tart cherries (TCs) are a rich source of phenolic and anthocyanin compounds. This proof-of-concept study evaluated whether ingesting TCs with a purine-rich meal affects the uricemic response.  A total of 25 adults (15 males and 10 females, 85.0 ± 17 kg, 40.6 ± 9 years, 29.1 ± 4.9 kg/m) with elevated fasting UA levels (5.8 ± 1.3 mg/dL) donated a fasting blood sample. In a randomized, double-blind, crossover, placebo-controlled, counterbalanced manner, participants ingested capsules containing 960 mg of a placebo (PLA) or concentrated TC powder containing 20.7 mg of proanthocyanins with a serving of hot soup (10 g of carbohydrate, 2 g protein, and 1 g fat) containing 3 g of purines (1 g of adenosine 5'-monophosphate, 1 g of disodium 5'-guanylate, and 1 g of disodium 5'-inosinate). Blood samples were obtained at 0, 60, 120, 180, and 240 min after ingestion to assess changes in uric acid levels and pharmacokinetic profiles. Cell blood counts, a comprehensive metabolic panel, cytokines, inflammatory markers, and subjective side effects ratings were analyzed on baseline (0 min) and post-treatment (240 min) samples. Participants continued consuming two capsules/day of the assigned treatment for one week and then repeated the experiment. Participants observed a 14-day washout and then repeated the experiment while ingesting the alternate treatment. Data were analyzed using general linear model (GLM) statistics with repeated measures, pairwise comparisons, and percentage change from baseline with 95% confidence intervals (CIs).  No statistically significant interaction effects or differences between treatments were seen in uric acid levels or PK profiles. Analysis of percent changes from baseline revealed that TC ingestion reduced the blood glucose levels following the ingestion of the high-purine meal (-4.2% [-7.7, -0.7],  = 0017). Additionally, there was some evidence that TC ingestion attenuated the increase from baseline in IL-1β and IL-10 and increased INF-γ. No significant differences were seen in the remaining health markers or subjective side effects ratings.  Acute and one-week TC supplementation did not affect the uricemic response to ingesting a high-purine meal in individuals with mildly elevated UA levels. However, there was some evidence that TC supplementation may blunt the glycemic response to ingesting a meal and influence some inflammatory cytokines. Registered clinical trial NCT04837274.

Effects of Acute and One-Week Supplementation with Montmorency Tart Cherry Powder on Food-Induced Uremic Response and Markers of Health: A Proof-of-Concept Study.

A time-release ion matrix (TRIM) restores damaged tissue following injury through local ion release to stimulate regenerative gene expression. Here we report the use of CoO-TRIM, an FDA designated Rare Pediatric Disease Drug, to restore muscle function and structure in the context of debilitating muscle disease. We demonstrate in an established animal model of Duchenne Muscular Dystrophy (DMD), the D2. mouse, that tibialis anterior (TA) muscles receiving a single injection of CoO-TRIM exhibit greater active force, myofiber size and regeneration through 70 days post treatment compared to  receiving vehicle alone. TRIM promoted upregulation of pro-angiogenic growth factor (vascular endothelial growth factor) and increased muscle microvasculature. These findings indicate that CoO-TRIM stimulates growth factors to promote the restoration of muscle structure and function of severely dystrophic mice  without toxicity. We conclude that CoO-TRIM is a first-in-class therapeutic compound to combat soft tissue disease by restoring tissue integrity. Moreover, this novel treatment strategy could benefit both early and late stage DMD patients.

Time Release Ion Matrix Regenerates Dystrophic Skeletal Muscle.

Vertebrate animal models of Becker muscular dystrophy (BMD) have been developed. Here, we characterized the gait kinematics and muscle function of a naturally occurring BMD pig model of dystrophin insufficiency. BMD pigs tended to have alterations in hip range of motion (ROM): hip (67%, 95% CI -0.64 to 14.12 degrees). While parameters were unaltered in extensor muscles, the dystrophin levels in flexor tibiotarsal joint muscles correlated with fatigue index as well as reduced isometric force (48%, 95% CI -1.86 to -0.61 N-m), and a 33% increase in fatigue index (95% CI -36.25 to 96.71 percent); the extensor muscles had no observable reductions in muscle force, with a 48% increase in fatigue index (95% CI -232.6 to 472.6 percent). Histological analysis of muscle biopsies supported a BMD phenotype in the flexor muscles of BMD pigs, with a 75% (95% CI -55.14 to -15.66 percent) decrease in large and a 43% (95% CI 17.74 to 57.38 percent) increase in small muscle fiber cross-sectional area. Dystrophin protein abundance was 28% less in flexor muscles from BMD pigs (95% CI -49.63 to 11.41 arbitrary units). Together, our model may serve as a clinically relevant model of BMD to assess safety and efficacy of therapeutics.

Characterization of Gait Kinematics and Muscle Function in Becker Muscular Dystrophy Pigs: a pilot study.

Skeletal muscle myopathy remains a significant cause of disability with limited treatment strategies. Advancements in tissue engineering have led to the development of borophosphate bioactive glasses (BPBGs) capable of enhancing skeletal muscle structure and function. Using a mouse model of severe myopathy (D2.), we investigated muscle force, regeneration, angiogenesis and inflammation at 14, 70 and 140 days post-treatment (dpt). Tibialis anterior (TA) muscles of D2. mice that received a single injection of cobalt oxide-doped BPBG (CoO-TRIM) particles exhibit greater active force, myofiber size, and regeneration through 70 dpt compared to control D2. mice injected with Saline. Vascular endothelial growth factor (VEGF) was elevated up to 70 dpt in D2. CoO-TRIM mice followed by increased muscle vascularity. As a marker of inflammation, interleukin (IL)-6 increased in D2. CoO-TRIM mice compared to D2. Saline controls at 14 dpt, with no differences at 70 or 140 dpt. No differences were observed in outcome measures between wild-type (WT) CoO-TRIM mice and WT Saline controls. We report that CoO-TRIM can stimulate VEGF production and promote restoration of muscle structure and function when inflammation is present. Local injection of an inorganic biomaterial alone can benefit myopathic skeletal muscle.

Article	Year
Inducible deletion of endothelial cell Efnb2 delays capillary regeneration and attenuates myofibre reinnervation following myotoxin injury in mice. The Journal of physiology\| PubMed ID: 39196901	2024
Effects of Acute and One-Week Supplementation with Montmorency Tart Cherry Powder on Food-Induced Uremic Response and Markers of Health: A Proof-of-Concept Study. Nutrients\| PubMed ID: 39408358	2024
Time Release Ion Matrix Regenerates Dystrophic Skeletal Muscle. Research square\| PubMed ID: 40166018	2025
Characterization of Gait Kinematics and Muscle Function in Becker Muscular Dystrophy Pigs: a pilot study. bioRxiv : the preprint server for biology\| PubMed ID: 41278853	2025
A Borophosphate Glass Doped with Cobalt Oxide Improves Skeletal Muscle Structure and Function in Myopathic Mice. Journal of functional biomaterials\| PubMed ID: 41893211	2026

Jacob A. Kendra

Biography

JoVE Journal Publications

Other Publications