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.

Skeletal muscle microtissues are engineered to develop therapies for restoring muscle function in patients. However, optimal electrical field stimulation (EFS) parameters to evaluate the function of muscle microtissues remain unestablished. This study reports a protocol to optimize EFS parameters for eliciting contractile force of muscle microtissues cultured in micropost platforms. Muscle microtissues were produced across an opposing pair of microposts in polydimethylsiloxane and polymethyl methacrylate culture platforms using primary, immortalized, and induced pluripotent stem cell-derived myoblasts. In response to EFS between needle electrodes, contraction deflects microposts proportional to developed force. At 5 V, pulse durations used for native muscle (0.1-1 ms) failed to elicit contraction of microtissues; durations reported for engineered muscle (5-10 ms) failed to elicit peak force. Instead, pulse durations of 20-80 ms were required to elicit peak twitch force across microtissues derived from five myoblast lines. Similarly, although peak tetanic force occurs at 20-50 Hz for native human muscles, it varied across microtissues depending on the cell line type, ranging from 7 to 60 Hz. A new parameter, the dynamic oscillation of force, captured trends during rhythmic contractions, whereas quantifying the duration-at-peak force provides an extended kinetics parameter. Our findings indicate that muscle microtissues have cell line type-specific contractile properties, yet all contract and relax more slowly than native muscle, implicating underdeveloped excitation-contraction coupling. Failure to optimize EFS parameters can mask the functional potential of muscle microtissues by underestimating force production. Optimizing and reporting EFS parameters and metrics is necessary to leverage muscle microtissues for advancing skeletal muscle therapies. Electrical field stimulation (EFS) parameters remain to be standardized for engineered skeletal muscle. Herein, we report a protocol for defining EFS parameters that elicit the maximal contractile force of muscle microtissues cultivated in micropost devices and highlight the value of developing appropriate metrics. The dynamic oscillation of force and duration-at-peak force are introduced as novel metrics of contraction kinetics.

Optimizing electrical field stimulation parameters reveals the maximum contractile function of human skeletal muscle microtissues.

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.

Coenzyme Q10 (CoQ10), a vital mitochondrial antioxidant, may enhance cardiovascular recovery and delay fatigue in athletes. This double-blind, randomized, placebo-controlled trial investigated CoQ10's effects on cardiac stress markers and exercise performance in 16 professional soccer players.

Short-term CoQ10 supplementation reduces markers of cardiac stress in soccer players following heavy exercise: A randomized double-blind placebo-controlled trial.

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
Optimizing electrical field stimulation parameters reveals the maximum contractile function of human skeletal muscle microtissues. American journal of physiology. Cell physiology\| PubMed ID: 40019026	2025
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
Short-term CoQ10 supplementation reduces markers of cardiac stress in soccer players following heavy exercise: A randomized double-blind placebo-controlled trial. BMC sports science, medicine & rehabilitation\| PubMed ID: 41361495	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

Aaron B. Morton

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