Josephine E. Joya

Identification of FHL1 as a regulator of skeletal muscle mass: implications for human myopathy.

Regulators of skeletal muscle mass are of interest, given the morbidity and mortality of muscle atrophy and myopathy. Four-and-a-half LIM protein 1 (FHL1) is mutated in several human myopathies, including reducing-body myopathy (RBM). The normal function of FHL1 in muscle and how it causes myopathy remains unknown. We find that FHL1 transgenic expression in mouse skeletal muscle promotes hypertrophy and an oxidative fiber-type switch, leading to increased whole-body strength and fatigue resistance. Additionally, FHL1 overexpression enhances myoblast fusion, resulting in hypertrophic myotubes in C2C12 cells, (a phenotype rescued by calcineurin inhibition). In FHL1-RBM C2C12 cells, there are no hypertrophic myotubes. FHL1 binds with the calcineurin-regulated transcription factor NFATc1 (nuclear factor of activated T cells, cytoplasmic, calcineurin-dependent 1), enhancing NFATc1 transcriptional activity. Mutant RBM-FHL1 forms aggregate bodies in C2C12 cells, sequestering NFATc1 and resulting in reduced NFAT nuclear translocation and transcriptional activity. NFATc1 also colocalizes with mutant FHL1 to reducing bodies in RBM-afflicted skeletal muscle. Therefore, via NFATc1 signaling regulation, FHL1 appears to modulate muscle mass and strength enhancement.

Divergent regulation of the sarcomere and the cytoskeleton.

The existence of a feedback mechanism regulating the precise amounts of muscle structural proteins, such as actin and the actin-associated protein tropomyosin (Tm), in the sarcomeres of striated muscles is well established. However, the regulation of nonmuscle or cytoskeletal actin and Tms in nonmuscle cell structures has not been elucidated. Unlike the thin filaments of striated muscles, the actin cytoskeleton in nonmuscle cells is intrinsically dynamic. Given the differing requirements for the structural integrity of the actin thin filaments of the sarcomere compared with the requirement for dynamicity of the actin cytoskeleton in nonmuscle cells, we postulated that different regulatory mechanisms govern the expression of sarcomeric versus cytoskeletal Tms, as key regulators of the properties of the actin cytoskeleton. Comprehensive analyses of tissues from transgenic and knock-out mouse lines that overexpress the cytoskeletal Tms, Tm3 and Tm5NM1, and a comparison with sarcomeric Tms provide evidence for this. Moreover, we show that overexpression of a cytoskeletal Tm drives the amount of filamentous actin.

Hypertrophy and dietary tyrosine ameliorate the phenotypes of a mouse model of severe nemaline myopathy

Nemaline myopathy, the most common congenital myopathy, is caused by mutations in genes encoding thin filament and thin filament-associated proteins in skeletal muscles. Severely affected patients fail to survive beyond the first year of life due to severe muscle weakness. There are no specific therapies to combat this muscle weakness. We have generated the first knock-in mouse model for severe nemaline myopathy by replacing a normal allele of the α-skeletal actin gene with a mutated form (H40Y), which causes severe nemaline myopathy in humans. The Acta1(H40Y) mouse has severe muscle weakness manifested as shortened lifespan, significant forearm and isolated muscle weakness and decreased mobility. Muscle pathologies present in the human patients (e.g. nemaline rods, fibre atrophy and increase in slow fibres) were detected in the Acta1(H40Y) mouse, indicating that it is an excellent model for severe nemaline myopathy. Mating of the Acta1(H40Y) mouse with hypertrophic four and a half LIM domains protein 1 and insulin-like growth factor-1 transgenic mice models increased forearm strength and mobility, and decreased nemaline pathologies. Dietary L-tyrosine supplements also alleviated the mobility deficit and decreased the chronic repair and nemaline rod pathologies. These results suggest that L-tyrosine may be an effective treatment for muscle weakness and immobility in nemaline myopathy.

Aged skeletal muscle retains the ability to fully regenerate functional architecture

While the general understanding of muscle regenerative capacity is that it declines with increasing age due to impairments in the number of muscle progenitor cells and interaction with their niche, studies vary in their model of choice, indices of myogenic repair, muscle of interest and duration of studies. We focused on the net outcome of regeneration, functional architecture, compared across three models of acute muscle injury to test the hypothesis that satellite cells maintain their capacity for effective myogenic regeneration with age. Muscle regeneration in extensor digitorum longus muscle (EDL) of young (3 mo-old), old (22 mo-old) and senescent female mice (28 mo-old) was evaluated for architectural features, fiber number and central nucleation, weight, collagen and fat deposition. The 3 injury paradigms were: a myotoxin (notexin) which leaves the blood vessels and nerves intact, freezing (FI) that damages local muscle, nerve and blood vessels and denervation-devascularization (DD) which dissociates the nerves and blood vessels from the whole muscle. Histological analyses revealed successful architectural regeneration following notexin injury with negligible fibrosis and fully restored function, regardless of age. In comparison, the regenerative response to injuries that damaged the neurovascular supply (FI and DD) was less effective, but similar across the ages. The focus on net regenerative outcome demonstrated that old and senescent muscle has a robust capacity to regenerate functional architecture.

Myofiber adaptational response to exercise in a mouse model of nemaline myopathy

In some muscle diseases, such as muscular dystrophy, exercise can increase muscle damage and alter myofiber adaptation. We determined whether this is also true for the congenital muscle disease nemaline myopathy using our mouse model of this disease. Nemaline mice expressing a mutant α‐tropomyosinslow protein [α‐Tmslow(Met9Arg)] in skeletal muscle underwent 4 weeks of treadmill exercise. Exercise increased slow/oxidative myofibers, but different fibers were involved in these transformations in nemaline mice. Despite similar expression of the mutant α‐Tmslow protein in muscles of the nemaline mouse, muscles responded in a unique manner that did not reflect fiber‐type composition. For example, the particular fibers involved in fast‐to‐slow transformation were specific for each muscle examined. In contrast to the muscular dystrophies, exercise did not result in muscle damage nor did it cause an increase in rod‐containing fibers; however, the fiber‐type distribution of rod‐containing fibers was altered in a muscle‐specific fashion. That exercise did not exacerbate the pathology (i.e., nemaline rod formation) supports its use in nemaline myopathy patients. This study shows that fibers of a similar type respond to increased activity differently in different muscles and suggests that fibers of similar type may be functionally distinct in different muscles. Muscle Nerve 30: 470–480, 2004

Reappearance of the minor α-sarcomeric actins in postnatal muscle

The postnatal expression profiles of α-sarcomeric actin transcripts and protein are quantified in mouse striated muscles from birth to postnatal day 56 by Northern and Western blot analyses. α-Cardiac actin (α-CA) transcripts transiently increase between 12 and 21 days after birth in the quadriceps muscle, reaching ∼90% that found in the adult mouse heart. Although α-CA is the α-sarcomeric actin isoform expressed in the immature fiber, the expression profiles of other contractile protein isoforms indicate that this postnatal period is not reflective of an immature phenotype. α-Skeletal actin (α-SA) transcripts accumulate to ∼32% of the total α-sarcomeric actin transcripts in the adult heart. Our study shows that 1) there is a simultaneous reappearance of α-CA and α-SA in postnatal skeletal and heart muscles, respectively, and 2) the contractile protein gene expression profile characteristic of adult skeletal muscle is not achieved until after 42 days postnatal in the mouse. We propose there is a previously uncharacterized period of postnatal striated muscle maturation marked by the reappearance of the minor α-sarcomeric actins.

Methylguanine DNA Methyltransferase‐Mediated Drug Resistance‐Based Selective Enrichment and Engraftment of Transplanted Stem Cells in Skeletal Muscle

Cell replacement therapy using stem cell transplantation holds much promise in the field of regenerative medicine. In the area of hematopoietic stem cell transplantation, O6‐methylguanine‐DNA methyltransferase MGMT (P140K) gene‐mediated drug resistance‐based in vivo enrichment strategy of donor stem cells has been shown to achieve up to 75%–100% donor cell engraftment in the hosts hematopoietic stem cell compartment following repeated rounds of selection. This strategy, however, has not been applied in any other organ system. We tested the feasibility of using this MGMT (P140K)‐mediated enrichment strategy for cell transplantation in skeletal muscles of mice. We demonstrate that muscle cells expressing an MGMT (P140K) drug resistance gene can be protected and selectively enriched in response to alkylating chemotherapy both in vitro and in vivo. Upon transplantation of MGMT (P140K)‐expressing male CD34+ve donor stem cells isolated from regenerating skeletal muscle into injured female muscle treated with alkylating chemotherapy, donor cells showed enhanced engraftment in the recipient muscle 7 days following transplantation as examined by quantitative‐polymerase chain reaction using Y‐chromosome specific primers. Fluorescent in situ hybridization analysis using a Y‐chromosome paint probe revealed donor‐derived de novo muscle fiber formation in the recipient muscle 14 days following transplantation, with approximately 12.5% of total nuclei within the regenerated recipient muscle being of donor origin. Following engraftment, the chemo‐protected donor CD34+ve cells induced substantial endogenous regeneration of the chemo‐ablated host muscle that is otherwise unable to self‐regenerate. We conclude that the MGMT (P140K)‐mediated enrichment strategy can be successfully implemented in muscle. Stem Cells 2009;27:1098–1108

Local anaesthetic refinement of pentobarbital euthanasia reduces abdominal writhing without affecting immunohistochemical endpoints in rats

Sodium pentobarbital is a commonly used agent for euthanizing laboratory rats, however its high pH can cause abdominal discomfort after intraperitoneal injection. Previous studies suggest that the addition of a local anaesthetic may alleviate this discomfort, but the practice has not been widely adopted. We examined the effect of combining lidocaine with pentobarbital on abdominal writhing, defecation, ultrasonic vocalizations, the rat grimace scale and immunohistochemical staining for c-Fos in the nucleus accumbens and basolateral amygdala of the brain. We also compared the amount of abdominal writhing following intraperitoneal administration of pentobarbital–lidocaine with that of pentobarbital–bupivacaine. Our results show that lidocaine reduces abdominal writhing and defecation without affecting immunohistochemistry for c-Fos or latency to loss of posture. However, scores on the rat grimace scale were low in both situations and almost no ultrasonic vocalizations were recorded. Additionally, we found that the amount of abdominal writhing was not significantly different when bupivacaine was used rather than lidocaine. Our results suggest that pentobarbital-induced euthanasia can be refined with the addition of lidocaine or other local anaesthetics.