Klotho regulates the myogenic response of muscle to mechanical loading and exercise

AUTHORS

Eisuke Ochi, Alice Barrington, Michelle Wehling-Henricks, Marcus Avila, Makoto Kuro-o, James G. Tidball

ABSTRACT

Muscle growth is influenced by changes in the mechanical environment that affect the expression of genes that regulate myogenesis. We tested whether the hormone Klotho could influence the response of muscle to mechanical loading. Applying mechanical loads to myoblasts in vitro increased RNA encoding transcription factors that are expressed in activated myoblasts (Myod) and in myogenic cells that have initiated terminal differentiation (Myog). However, application of Klotho to myoblasts prevented the loading-induced activation of Myog without affecting loading-induced activation of Myod. This indicates that elevated Klotho inhibits mechanically-induced differentiation of myogenic cells. Elevated Klotho also reduced the transcription of genes encoding proteins involved in the canonical Wnt pathway or their target genes (Wnt9a, Wnt10a, Ccnd1). Because the canonical Wnt pathway promotes differentiation of myogenic cells, these findings indicate that Klotho inhibits the differentiation of myogenic cells experiencing mechanical loading. We then tested whether these effects of Klotho occurred in muscles of mice experiencing high-intensity interval training (HIIT) by comparing wild-type mice and klotho transgenic mice. The expression of a klotho transgene combined with HIIT synergized to tremendously elevate numbers of Pax7+ satellite cells and activated MyoD+ cells. However, transgene expression prevented the increase in myogenin+ cells caused by HIIT in wild-type mice. Furthermore, transgene expression diminished the HIIT-induced activation of the canonical Wnt pathway in Pax7+ satellite cells. Collectively, these findings show that Klotho inhibits loading- or exercise-induced activation of muscle differentiation and indicate a new mechanism through which the responses of muscle to the mechanical environment are regulated.

Nystagmus Associated With the Absence of MYOD Expression Across the Lifespan in Extraocular and Limb Muscles

AUTHORS

Laura L. Johnson; Sadie Hebert; Rachel B. Kueppers; Linda K. McLoon

ABSTRACT

Purpose: The extraocular muscles (EOMs) undergo significant levels of continuous myonuclear turnover and myofiber remodeling throughout life, in contrast to limb skeletal muscles. Activation of the myogenic pathway in muscle precursor cells is controlled by myogenic transcription factors, such as MYOD. Limb muscles from MyoD−/− mice develop normally but have a regeneration defect, and these mice develop nystagmus. We examined MyoD−/− mice to determine if they have an aging phenotype.

Methods: Eye movements of aging MyoD−/− mice and littermate controls (wild type) were examined using optokinetic nystagmus (OKN). We assessed limb muscle function, changes to myofiber number, mean cross-sectional area, and abundance of the PAX7 and PITX2 populations of myogenic precursor cells.

Results: Aging did not significantly affect limb muscle function despite decreased mean cross-sectional areas at 18+ months. Aging wild type mice had normal OKN responses; all aging MyoD−/− mice had nystagmus. With OKN stimulus present, the MyoD−/− mice at all ages had shorter slow phase durations compared to wild type age matched controls. In the dark, the MyoD−/− mice had a shorter slow phase duration with age. This correlated with significantly decreased fiber numbers and cross-sectional areas. The EOM in MyoD−/− mice had increased numbers of PAX7-positive satellite cells and significantly decreased PITX2-positive myonuclei.

Conclusions: The absence of MYOD expression in aging mice causes a decrease in on-going myofiber remodeling, EOM fiber size, and number, and is associated with the development of spontaneous nystagmus. These results suggest that muscle-specific mutations can result in nystagmus, with increasing aging-related changes in the MyoD−/− EOM.

Low-Magnitude Mechanical Signals Combined with Zoledronic Acid Reduce Musculoskeletal Weakness and Adiposity in Estrogen-Deprived Mice

AUTHORS

Gabriel M. Pagnotti, Trupti Trivedi, Laura E. Wright, Sutha K. John, Sreemala Murthy, Ryan R. Pattyn, Monte S. Willis, Yun She, Sukanya Suresh, William R. Thompson, Clinton T. Rubin, Khalid S. Mohammad, Theresa A. Guise

ABSTRACT

Combination treatment of Low-Intensity Vibration (LIV) with zoledronic acid (ZA) was hypothesized to preserve bone mass and muscle strength while reducing adipose tissue accrual associated with complete estrogen (E2)-deprivation in young and skeletally mature mice. Complete E2-deprivation (surgical-ovariectomy (OVX) and daily injection of aromatase inhibitor (AI) letrozole) were performed on 8-week-old C57BL/6 female mice for 4 weeks following commencement of LIV administration or control (no LIV), for 28 weeks. Additionally, 16-week-old C57BL/6 female E2-deprived mice were administered ±LIV twice daily and supplemented with ±ZA (2.5 ng/kg/week). By week 28, lean tissue mass quantified by dual-energy X-ray absorptiometry was increased in younger OVX/AI+LIV(y) mice, with increased myofiber cross-sectional area of quadratus femorii. Grip strength was greater in OVX/AI+LIV(y) mice than OVX/AI(y) mice. Fat mass remained lower in OVX/AI+LIV(y) mice throughout the experiment compared with OVX/AI(y) mice. OVX/AI+LIV(y) mice exhibited increased glucose tolerance and reduced leptin and free fatty acids than OVX/AI(y) mice. Trabecular bone volume fraction and connectivity density increased in the vertebrae of OVX/AI+LIV(y) mice compared to OVX/AI(y) mice; however, this effect was attenuated in the older cohort of E2-deprived mice, specifically in OVX/AI+ZA mice, requiring combined LIV with ZA to increase trabecular bone volume and strength. Similar improvements in cortical bone thickness and cross-sectional area of the femoral mid-diaphysis were observed in OVX/AI+LIV+ZA mice, resulting in greater fracture resistance. Our findings demonstrate that the combination of mechanical signals in the form of LIV and anti-resorptive therapy via ZA improve vertebral trabecular bone and femoral cortical bone, increase lean mass, and reduce adiposity in mice undergoing complete E2-deprivation.

One Sentence Summary: Low-magnitude mechanical signals with zoledronic acid suppressed bone and muscle loss and adiposity in mice undergoing complete estrogen deprivation.

Translational Relevance Postmenopausal patients with estrogen receptor-positive breast cancer treated with aromatase inhibitors to reduce tumor progression experience deleterious effects to bone and muscle subsequently develop muscle weakness, bone fragility, and adipose tissue accrual. Bisphosphonates (i.e., zoledronic acid) prescribed to inhibit osteoclast-mediated bone resorption are effective in preventing bone loss but may not address the non-skeletal effects of muscle weakness and fat accumulation that contribute to patient morbidity. Mechanical signals, typically delivered to the musculoskeletal system during exercise/physical activity, are integral for maintaining bone and muscle health; however, patients undergoing treatments for breast cancer often experience decreased physical activity which further accelerates musculoskeletal degeneration. Low-magnitude mechanical signals, in the form of low-intensity vibrations, generate dynamic loading forces similar to those derived from skeletal muscle contractility. As an adjuvant to existing treatment strategies, low-intensity vibrations may preserve or rescue diminished bone and muscle degraded by breast cancer treatment.

Inhibition of MicroRNA-122-5p Relieves Myocardial Ischemia-Reperfusion Injury via SOCS1

AUTHORS

Jun Zhang , Li Fu , Jing Zhang , Bo Zhou , Yanrong Tang , Zhenzhen Zhang , Tongqing Gu

ABSTRACT

Objective 

Evidence has shown that microRNA (miR)-122–5p is a diagnostic biomarker of acute myocardial infarction. Here, we aimed to uncover the functions of miR-122–5p in the pathological process of myocardial ischemia-reperfusion injury (MI/RI).

Methods 

An MI/RI model was established by left anterior descending coronary artery ligation in mice. The levels of miR-122–5p, suppressor of cytokine signaling-1 (SOCS1), phosphorylation of Janus kinase 2 (p-JAK2), and signal transducers and activators of transcription (p-STAT3) in the myocardial tissues of mice were measured. Downregulated miR-122–5p or upregulated SOCS1 recombinant adenovirus vectors were injected into mice before MI/RI modeling. The cardiac function, inflammatory response, myocardial infarction area, pathological damage, and cardiomyocyte apoptosis in the myocardial tissues of mice were evaluated. Cardiomyocytes were subjected to hypoxia/reoxygenation (H/R) injury and cardiomyocyte biological function was tested upon transfection of miR-122–5p inhibitor. The target relation between miR-122–5p and SOCS1 was evaluated.

Results 

miR-122–5p expression and p-JAK2 and p-STAT3 expression were high, and SOCS1 expression was low in the myocardial tissues of MI/RI mice. Decreasing miR-122–5p or increasing SOCS1 expression inactivated the JAK2/STAT3 pathway to alleviate MI/RI by improving cardiac function and reducing inflammatory reaction, myocardial infarction area, pathological damage, and cardiomyocyte apoptosis in mice. Silencing of SOCS1 reversed depleted miR-122–5p-induced cardioprotection for MI/RI mice. In vitro experiments revealed that the downregulation of miR-122–5p induced proliferative, migratory, and invasive capabilities of H/R cardiomyocytes while inhibiting apoptosis. Mechanically, SOCS1 was a target gene of miR-122–5p.

Conclusion 

Our study summarizes that inhibition of miR-122–5p induces SOCS1 expression, thereby relieving MI/RI in mice.

Development of Nystagmus With the Absence of MYOD Expression in the Extraocular Muscles

AUTHORS

Laura L. Johnson; Rachel B. Kueppers; Erin Y. Shen; Jolene C. Rudell; Linda K. McLoon

ABSTRACT

Purpose: Myoblast determination protein 1 (MYOD) is a critical myogenic regulatory factor in muscle development, differentiation, myofiber repair, and regeneration. As the extraocular muscles significantly remodel their myofibers throughout life compared with limb skeletal muscles, we hypothesized that the absence of MYOD would result in their abnormal structure and function. To assess structural and functional changes in the extraocular muscles in MyoD−/− mice, fiber size and number and optokinetic nystagmus reflex (OKN) responses were examined.

Methods: OKN was measured in MyoD−/− mice and littermate wild-type controls at 3, 6, and 12 months. The extraocular muscles were examined histologically for changes in mean myofiber cross-sectional area, total myofiber number, and nuclei immunostained for PAX7 and PITX2, markers of myogenic precursor cells.

Results: The MyoD−/− mice developed nystagmus, with both jerk and pendular waveforms, in the absence and in the presence of moving visual stimulation. At 12 months, there were significant losses in mean myofiber cross-sectional area and in total number of orbital layer fibers in all rectus muscles, as well as in global layer fibers in the superior and inferior rectus muscles. Haploinsufficient mice showed abnormal OKN responses. PITX2-positive cell entry into myofibers of the MyoD−/− mice was significantly reduced.

Conclusions: This study is the first demonstration of the development of nystagmus in the constitutive absence of expression of the muscle-specific transcription factor MYOD. We hypothesize that myofiber loss over time may alter anterograde and/or retrograde communication between the motor nerves and extraocular muscles that are critical for maintaining normalcy of extraocular muscle function.

Adipogenic Differentiation Alters Properties of Vascularized Tissue-Engineered Skeletal Muscle

AUTHORS

Francisca M. Acosta, Kennedy K. Howland, Katerina Stojkova, Elizabeth Hernandez, Eric M. Brey, and Christopher R. Rathbone

ABSTRACT

Advances in the engineering of comprehensive skeletal muscle models in vitro will improve drug screening platforms and can lead to better therapeutic approaches for the treatment of skeletal muscle injuries. To this end, a vascularized tissue-engineered skeletal muscle (TE-SkM) model that includes adipocytes was developed to better emulate the intramuscular adipose tissue that is observed in skeletal muscles of patients with diseases such as diabetes. Muscle precursor cells cultured with and without microvessels derived from adipose tissue (microvascular fragments) were used to generate TE-SkM constructs, with and without a microvasculature, respectively. TE-SkM constructs were treated with adipogenic induction media to induce varying levels of adipogenesis. With a delayed addition of induction media to allow for angiogenesis, a robust microvasculature in conjunction with an increased content of adipocytes was achieved. The augmentation of vascularized TE-SkM constructs with adipocytes caused a reduction in maturation (compaction), mechanical integrity (Young's modulus), and myotube and vessel alignment. An increase in basal glucose uptake was observed in both levels of adipogenic induction, and a diminished insulin-stimulated glucose uptake was associated with the higher level of adipogenic differentiation and the greater number of adipocytes.