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The pig is an important animal model increasingly used for biomedical research, particularly in transplantation strategies involving xenotransplantation or the development of human organs in pig for exotransplantation. Pigs, however, are less characterized than other animal models. In this study, we produced wildtype (WT) pig embryos via somatic cell nuclear transfer (SCNT) technology and compared them to skeletal muscle null embryos (lacking MYF5/MYOD/MYF6) at embryonic day 41, 62, and 90, critical stages of porcine myogenesis. Magnetic resonance imaging (MRI) and histological analyses revealed progressive development of skeletal muscle in WT embryos but not in null embryos whereas development of viscera progressed equally in both groups. Molecular analyses highlighted dynamic changes in myogenic gene expression and myofiber formation, demonstrating an organized progression of myogenesis in WT embryos. Morphologically, the null embryos exhibited abnormalities, including marked edema and underdeveloped limbs. MRI revealed severe skeletal abnormalities, including the absence of ribs, sternum, and associated vertebral malformations. In addition, histological analysis confirmed the complete lack of myofiber formation. Immunohistochemical analysis revealed the absence of myogenic stem cells and muscle differentiation, and RNA sequencing demonstrated that the skeletal muscle development process was entirely disrupted in null embryos. Additionally, analysis of neuromuscular junctions (NMJs) in the null embryos revealed that functional NMJ formation was absent, consistent with the lack of skeletal muscle formation. Importantly, these defects culminated in embryonic lethality after day 62 in the null embryos. We determined that the myogenic regulatory gene cascade is crucial for porcine embryo development and viability. The deletion of skeletal muscle is essential for the creating a vacant niche to allow for complementation of null porcine embryos with human induced pluripotent stem cells. Characterization of this skeletal muscle null pig model provide an important platform for engineering humanized muscle in gene-edited pigs. Triple knockout of myogenic regulatory genes (MYF5/MYOD/MYF6) completely disrupts skeletal muscle formation, causing embryonic lethality in pigs and highlighting the critical role of myogenesis, providing a platform for humanized muscle engineering in pigs. [ABSTRACT FROM AUTHOR] more...

This study's objective was to investigate the extent to which two different levels of low‐intensity vibration training (0.6 g or 1.0 g) affected musculoskeletal structure and function after a volumetric muscle loss (VML) injury in male C57BL/6J mice. All mice received a unilateral VML injury to the posterior plantar flexors. Mice were randomized into a control group (no vibration; VML‐noTX), or one of two experimental groups. The two experimental groups received vibration training for 15‐min/day, 5‐days/week for 8 weeks at either 0.6 g (VML‐0.6 g) or 1.0 g (VML‐1.0 g) beginning 3‐days after induction of VML. Muscles were analyzed for contractile and metabolic adaptations. Tibial bone mechanical properties and geometric structure were assessed by a three‐point bending test and microcomputed tomography (µCT). Body mass‐normalized peak isometric‐torque was 18% less in VML‐0.6 g mice compared with VML‐noTx mice (p = 0.030). There were no statistically significant differences of vibration intervention on contractile power or muscle oxygen consumption (p ≥ 0.191). Bone ultimate load, but not stiffness, was ~16% greater in tibias of VML‐1.0 g mice compared with those from VML‐noTx mice (p = 0.048). Cortical bone volume was ~12% greater in tibias of both vibration groups compared with VML‐noTx mice (p = 0.003). Importantly, cross‐section moment of inertia, the primary determinant of bone ultimate load, was 44% larger in tibias of VML‐0.6 g mice compared with VML‐noTx mice (p = 0.006). These changes indicate that following VML, bones are more responsive to the selected vibration training parameters than muscle. Vibration training represents a possible adjuvant intervention to address bone deficits following VML. [ABSTRACT FROM AUTHOR] more...

This study's objective was to investigate how contractile strength loss associated with a volumetric muscle loss (VML) injury affects the adjacent tibial bone structural and functional properties in male C57BL/6J mice. Mice were randomized into one of two experimental groups: VML‐injured mice that were injured at age 12 weeks and aged to 20 weeks (8 weeks postinjury, VML) and 20‐week‐old age‐matched uninjured mice (Uninjured‐20). Tibial bone strength, mid‐diaphysis cortical geometry, intrinsic material properties, and metaphyseal trabecular bone structure were assessed by three‐point bending and microcomputed tomography (µCT). The plantar flexor muscle group (gastrocnemius, soleus, plantaris) was analyzed for its functional capacities, that is, peak‐isometric torque and peak‐isokinetic power. VML‐injured limbs had 25% less peak‐isometric torque and 31% less peak‐isokinetic power compared to those of Uninjured‐20 mice (p < 0.001). Ultimate load, but not stiffness, was significantly less (10%) in tibias of VML‐injured limbs compared to those from Uninjured‐20 (p = 0.014). µCT analyses showed cortical bone thickness was 6% less in tibias of VML‐injured limbs compared to Uninjured‐20 (p = 0.001). Importantly, tibial bone cross‐section moment of inertia, the primary determinant of bone ultimate load, was 16% smaller in bones of VML‐injured limbs compared to bones from Uninjured‐20 (p = 0.046). Metaphyseal trabecular bone structure was also altered up to 23% in tibias of VML‐injured limbs (p < 0.010). These changes in tibial bone structure and function after a VML injury occur during a natural maturation phase between the age of 12 and 20 weeks, as evidenced by Uninjured‐20 mice having greater tibial bone size and strength compared to uninjured‐aged 12‐week mice. [ABSTRACT FROM AUTHOR] more...

New Findings: What is the central question of this study?First, how does physical rehabilitation influence recovery from traumatic muscle injury? Second, how does physical activity impact the rehabilitation response for skeletal muscle function and whole‐body metabolism?What is the main finding and its importance?The most salient findings were that rehabilitation impaired muscle function and range of motion, while restricting activity mitigated some negative effects but also impacted whole‐body metabolism. These data suggest that first, work must continue to explore treatment parameters, including modality, time, type, duration and intensity, to find the best rehabilitation approaches for volumetric muscle loss injuries; and second, restricting activity acutely might enhance rehabilitation response, but whole‐body co‐morbidities should continue to be considered. Volumetric muscle loss (VML) injury occurs when a substantial volume of muscle is lost by surgical removal or trauma, resulting in an irrecoverable deficit in muscle function. Recently, it was suggested that VML impacts whole‐body and muscle‐specific metabolism, which might contribute to the inability of the muscle to respond to treatments such as physical rehabilitation. The aim of this work was to understand the complex relationship between physical activity and the response to rehabilitation after VML in an animal model, evaluating the rehabilitation response by measurement of muscle function and whole‐body metabolism. Adult male mice (n = 24) underwent a multi‐muscle, full‐thickness VML injury to the gastrocnemius, soleus and plantaris muscles and were randomized into one of three groups: (1) untreated; (2) rehabilitation (i.e., combined electrical stimulation and range of motion, twice per week, beginning 72 h post‐injury, for ∼8 weeks); or (3) rehabilitation and restriction of physical activity. There was a lack of positive adaption associated with electrical stimulation and range of motion intervention alone; however, maximal isometric torque of the posterior muscle group was greater in mice receiving treatment with activity restriction (P = 0.008). Physical activity and whole‐body metabolism were measured ∼6 weeks post‐injury; metabolic rate decreased (P = 0.001) and respiratory exchange ratio increased (P = 0.022) with activity restriction. Therefore, restricting physical activity might enhance an intervention delivered to the injured muscle group but impair whole‐body metabolism. It is possible that restricting activity is important initially post‐injury to protect the muscle from excess demand. A gradual increase in activity throughout the course of treatment might optimize muscle function and whole‐body metabolism. [ABSTRACT FROM AUTHOR] more...

Volumetric muscle loss (VML) is a devastating orthopedic injury resulting in chronic persistent functional deficits, loss of joint range of motion, pathologic fibrotic deposition and lifelong disability. However, there is only limited mechanistic understanding of VML-induced fibrosis. Herein we examined the temporal changes in the fibrotic deposition at 3, 7, 14, 28, and 48 days post-VML injury. Adult male Lewis rats (n = 39) underwent a full thickness ~20% (~85 mg) VML injury to the tibialis anterior (TA) muscle unilaterally, the contralateral TA muscle served as the control group. All TA muscles were harvested for biochemical and histologic evaluation. The ratio of collagen I/III was decreased at 3, 7, and 14 days post-VML, but significantly increased at 48 days. Decorin content followed an opposite trend, significantly increasing by day 3 before dropping to below control levels by 48 days. Histological evaluation of the defect area indicates a shift from loosely packed collagen at early time points post-VML, to a densely packed fibrotic scar by 48 days. The shift from early wound healing efforts to a fibrotic scar with densely packed collagen within the skeletal muscle occurs around 21 days after VML injury through dogmatic synchronous reduction of collagen III and increase in collagen I. Thus, there appears to be an early window for therapeutic intervention to prevent pathologic fibrous tissue formation, potentially by targeting CCN2/CTGF or using decorin as a therapeutic. [ABSTRACT FROM AUTHOR] more...