James Ervasti

Duchenne muscular dystrophy (DMD) is a lethal degenerative muscle wasting disease caused by the loss of the structural protein dystrophin with secondary pathological manifestations including metabolic dysfunction, mood and behavioral disorders. In the mildly affected mdx mouse model of DMD, brief scruff stress causes inactivity, while more severe subordination stress results in lethality. Here, we investigated the kynurenine pathway of tryptophan degradation and the nicotinamide adenine dinucleotide (NAD+) metabolic pathway in mdx mice and their involvement as possible mediators of mdx stress-related pathology. We identified downregulation of the kynurenic acid shunt, a neuroprotective branch of the kynurenine pathway, in mdx skeletal muscle associated with attenuated peroxisome proliferator-activated receptor-gamma coactivator 1 alpha (PGC-1α) transcriptional regulatory activity. Restoring the …

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ataxin-1 (ATXN1) protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockin mouse (f-ATXN1146Q/2Q) with mouse Atxn1 coding exons replaced by human ATXN1 exons encoding 146 glutamines. f-ATXN1146Q/2Q mice manifested SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. Central nervous system (CNS) contributions to disease were revealed using f-ATXN1146Q/2Q; Nestin-Cre mice, that showed improved rotarod, open field, and Barnes maze performance by 6-12 weeks-of-age. In contrast, striatal contributions to motor deficits using f-ATXN1146Q/2Q; Rgs9-Cre mice revealed that mice lacking ATXN1146Q/2Q in striatal medium-spiny neurons showed a trending improvement in rotarod performance at 30 weeks-of-age. Surprisingly, a prominent role for muscle contributions to disease was revealed in f-ATXN1146Q/2Q; ACTA1-Cre mice based on their recovery from kyphosis and absence of muscle pathology. Collectively, data from the targeted conditional deletion of the expanded allele demonstrated CNS and peripheral contributions to disease and highlighted the need to consider muscle in addition to the brain for optimal SCA1 therapeutics.

The cytoplasmic actin proteins, β- and γ-actin, are 99% identical but thought to perform non-redundant functions. The nucleotide coding regions of cytoplasmic actin genes, Actb and Actg1, are 89% identical. Knockout (KO) of Actb by Cre-mediated deletion of first coding exons 2 and 3 in mice is embryonic lethal and fibroblasts derived from KO embryos (MEFs) fail to proliferate. In contrast, Actg1 KO MEFs display with a much milder defect in cell proliferation and Actg1 KO mice are viable, but present with increased perinatal lethality. Recent studies have identified important protein-independent functions for both Actb and Actg1 and demonstrate that deletions within the Actb nucleotide sequence, and not loss of the β-actin protein, cause the most severe phenotypes in KO mice and cells. Here, we use a multi-omics approach to better understand what drives the phenotypes of Actb KO MEFs. RNA-sequencing and …

Spinocerebellar ataxia type 1 (SCA1) is a fatal neurodegenerative disease caused by an expanded polyglutamine tract in the widely expressed ATXN1 protein. To elucidate anatomical regions and cell types that underlie mutant ATXN1-induced disease phenotypes, we developed a floxed conditional knockout mouse model (f-ATXN1146Q/2Q) having mouse Atxn1 coding exons replaced by human exons encoding 146 glutamines. F-ATXN1146Q/2Q mice manifest SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. CNS contributions to disease were revealed using ATXN1146Q/2Q; Nestin-Cre mice, that showed improved rotarod, open field and Barnes maze performances. Striatal contributions to motor deficits were examined using f-ATXN1146Q/2Q; Rgs9-Cre mice. Mice lacking striatal ATXN1146Q/2Q had improved rotarod performance late in disease. Muscle contributions to disease were revealed in f-ATXN1146Q/2Q; ACTA1-Cre mice which lacked muscle pathology and kyphosis seen in f-ATXN1146Q/2Q mice. Kyphosis was not improved in f-ATXN1146Q/2Q; Nestin-Cre mice. Thus, optimal SCA1 therapeutics will require targeting mutant ATXN1 toxic actions in multiple brain regions and muscle.

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of the protein dystrophin. Utrophin is a dystrophin homologue currently under investigation as a protein replacement therapy for Duchenne muscular dystrophy. Dystrophin is hypothesized to function as a molecular shock absorber that mechanically stabilizes the sarcolemma. While utrophin is homologous with dystrophin from a molecular and biochemical perspective, we have recently shown that full-length utrophin expressed in eukaryotic cells is stiffer than what has been reported for dystrophin fragments expressed in bacteria. In this study, we show that differences in expression system impact the mechanical stiffness of a model utrophin fragment encoding the N terminus through spectrin repeat 3 (UtrN-R3). We also demonstrate that UtrN-R3 expressed in eukaryotic cells was phosphorylated while bacterial UtrN-R3 was not …

Dystrophin is an essential muscle protein that contributes to cell membrane stability by mechanically linking the actin cytoskeleton to the extracellular matrix via an adhesion complex called the dystrophin–glycoprotein complex. The absence or impaired function of dystrophin causes muscular dystrophy. Focal adhesions (FAs) are also mechanosensitive adhesion complexes that connect the cytoskeleton to the extracellular matrix. However, the interplay between dystrophin and FA force transmission has not been investigated. Using a vinculin-based bioluminescent tension sensor, we measured FA tension in transgenic C2C12 myoblasts expressing wild-type (WT) dystrophin, a nonpathogenic single nucleotide polymorphism (SNP) (I232M), or two missense mutations associated with Duchenne (L54R), or Becker muscular dystrophy (L172H). Our data revealed cross talk between dystrophin and FAs, as the …

Duchenne muscular dystrophy is a lethal muscle disease, caused by mutations in the gene encoding dystrophin, an actin-binding cytoskeletal protein. Absence of functional dystrophin results in muscle weakness and degeneration, eventually leading to cardiac and respiratory failure. Strategies to replace the missing dystrophin via gene therapy have been intensively pursued. However, the dystrophin gene is too large for current gene therapy approaches. Currently available micro-dystrophin constructs lack the actin-binding domain 2 and show decreased actin-binding affinity in vitro compared to full-length dystrophin. Thus, increasing the actin-binding affinity of micro-dystrophin, using small molecules, could be a beneficial therapeutic approach. Here, we have developed and validated a novel high-throughput screening (HTS) assay to discover small molecules that increase the binding affinity of dystrophin's actin …

Cytoplasmic β- and γ-actin proteins are 99% identical but support unique organismal functions. The cytoplasmic actin nucleotide sequences Actb and Actg1, respectively, are more divergent but still 89% similar. Actb–/– mice are embryonic lethal and Actb–/– cells fail to proliferate, but editing the Actb gene to express γ-actin (Actbc–g) resulted in none of the overt phenotypes of the knockout revealing protein-independent functions for Actb. To determine if Actg1 has a protein-independent function, we crossed Actbc–g and Actg1–/– mice to generate the bG/0 line, where the only cytoplasmic actin expressed is γ-actin from Actbc–g. The bG/0 mice were viable but showed a survival defect despite expressing γ-actin protein at levels no different from bG/gG with normal survival. A unique myopathy phenotype was also observed in bG/0 mice. We conclude that impaired survival and myopathy in bG/0 mice are due to loss of …

Fibroblasts can be reprogrammed into induced cardiomyocyte-like cells (iCMs) by forced expression of cardiogenic transcription factors. However, it remains unknown how fibroblasts adopt a cardiomyocyte (CM) fate during their spontaneous ongoing transdifferentiation toward myofibroblasts (MFs). By tracing fibroblast lineages following cardiac reprogramming in vitro, we found that most mature iCMs are derived directly from fibroblasts without transition through the MF state. This direct conversion is attributable to mutually exclusive induction of cardiac sarcomeres and MF cytoskeletal structures in the cytoplasm of fibroblasts during reprogramming. For direct fate switch from fibroblasts to iCMs, significant remodeling of actin isoforms occurs in fibroblasts, including induction of α-cardiac actin and decrease of the actin isoforms predominant in MFs. Accordingly, genetic or pharmacological ablation of MF-enriched …

Psychosocial stressors can cause physical inactivity, cardiac damage, and hypotension‐induced death in the mdx mouse model of Duchenne muscular dystrophy (DMD). Because repeated exposure to mild stress can lead to habituation in wild‐type mice, we investigated the response of mdx mice to a mild, daily stress to determine whether habituation occurred. Male mdx mice were exposed to a 30‐sec scruff restraint daily for 12 weeks. Scruff restraint induced immediate physical inactivity that persisted for at least 60 minutes, and this inactivity response was just as robust after 12 weeks as it was after one day. Physical inactivity in the mdx mice was not associated with acute skeletal muscle contractile dysfunction. However, skeletal muscle of mdx mice that were repeatedly stressed had slow‐twitch and tetanic relaxation times and trended toward high passive stiffness, possibly due to a small but significant increase …

Discussion-No significant difference was found between the phosphorylated version of NT-R3 Utr from insect cells and the same unphosphorylated protein from bacteria.

Aim Loss of dystrophin causes oxidative stress and affects nitric oxide synthase‐mediated vascular function in striated muscle. Because tetrahydrobiopterin is an antioxidant and co‐factor for nitric oxide synthase, we tested the hypothesis that tetrahydrobiopterin would be low in mdx mice and humans deficient for dystrophin. Methods Tetrahydrobiopterin and its metabolites were measured at rest and in response to exercise in Duchenne and Becker muscular dystrophy patients, age‐matched male controls as well as wild‐type, mdx and mdx mice transgenically overexpressing skeletal muscle‐specific dystrophins. Mdx mice were also supplemented with tetrahydrobiopterin and pathophysiology was assessed. Results Duchenne muscular dystrophy patients had lower urinary dihydrobiopterin + tetrahydrobiopterin/specific gravity1.020 compared to unaffected age‐matched males and Becker muscular dystrophy …

Weakness and atrophy are key features of Duchenne muscular dystrophy (DMD). Dystrophin is one of the many proteins within the dystrophin glycoprotein complex (DGC) that maintains plasmalemmal integrity and cellular homeostasis. The dystrophin-deficient mdx mouse is also predisposed to weakness, particularly when subjected to eccentric (ECC) contractions due to electrophysiological dysfunction of the plasmalemma. Here, we determined if maintenance of plasmalemmal excitability during and after a bout of ECC contractions is dependent on intact and functional DGCs rather than, solely, dystrophin expression. Wild-type (WT) and dystrophic mice (mdx, mL172H and Sgcb−/− mimicking Duchenne, Becker and Limb-girdle Type 2E muscular dystrophies, respectively) with varying levels of dystrophin and DGC functionality performed 50 maximal ECC contractions with simultaneous torque and electromyographic measurements (M-wave root-mean-square, M-wave RMS). ECC contractions caused all mouse lines to lose torque (p<0.001); however, deficits were greater in dystrophic mouse lines compared to WT mice (p<0.001). Loss of ECC torque did not correspond to a reduction in M-wave RMS in WT mice (p=0.080), while deficits in M-wave RMS exceeded 50% in all dystrophic mouse lines (p≤0.007). Moreover, reductions in ECC torque and M-wave RMS were greater in mdx mice compared to mL172H mice (p≤0.042). No differences were observed between mdx and Sgcb−/− mice (p≥0.337). Regression analysis revealed ≥98% of the variance in ECC torque loss could be explained by the variance in M-wave RMS in dystrophic …

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of dystrophin, a protein that directly links the cytoskeleton to the extracellular matrix through the dystrophin-glycoprotein complex. Cells generate, sense and respond to forces via mechanosensitive protein complexes, and altered force transmission via these complexes such as focal adhesions (FAs) are known to contribute to muscular dystrophy development and progression. However, the interplay between dystrophin loss and FAs force transmission remains unknown. Here we show that dystrophin deficient muscle cells transmit less tension via FAs and have decreased YAP activation. Using a vinculin bioluminescent tension sensor, we measured FAs tension in transgenic muscle cells expressing wild type (WT) dystrophin and two patient derived dystrophin missense mutants-L172H and L54R. We found that cells harboring …

Dystrophin is an essential muscle protein that contributes to cell membrane stability by linking the actin cytoskeleton to the extracellular matrix. The absence or impaired function of dystrophin causes muscular dystrophy. Focal adhesions are mechanosensitive adhesion complexes that also connect the cytoskeleton to the extracellular matrix. However, the interplay between dystrophin and focal adhesion force transmission has not been investigated. Using a bioluminescent tension sensor, we measured focal adhesion tension in transgenic C2C12 myoblasts expressing wild type (WT) dystrophin, a non-pathogenic SNP (I232M), or two missense mutations associated with Duchenne (L54R), or Becker muscular dystrophy (L172H). We found that myoblasts expressing WT or nonpathogenic I232M dystrophin showed increased focal adhesion tension compared to non-transgenic myoblasts, while myoblasts expressing L54R or L172H dystrophin presented with decreased focal adhesion tension. Moreover, myoblasts expressing L54R or L172H dystrophin showed decreased YAP activation and exhibited slower and less directional migration compared to cells expressing WT or I232M dystrophin. Our results suggest that disease-causing missense mutations in dystrophin may disrupt a cellular tension sensing pathway in dystrophic skeletal muscle.

The authors are grateful to María Paz Ramírez and W. Michael Southern for their helpful critiques. This work was supported by the National Institute of Arthritis and Musculoskeletal and Skin Diseases [RO1 AR042423 to JME and F31 AR073629 to DMN]. JME is both a consultant for, and receives project funding from Sarepta Therapeutics.

Duchenne muscular dystrophy is a lethal muscle wasting disease caused by the absence of dystrophin, a protein that links the actin cytoskeleton to the extracellular matrix. Cells generate, sense, and respond to forces via mechanosensitive protein complexes, and altered force transmission via these complexes such as focal adhesions (FAs) are known to contribute to the development and progression of muscular dystrophy. However, the interplay between loss of dystrophin and altered force transmission via FAs remains unknown. Here we show that FAs of dystrophin deficient muscle cells are under less mechanical load and have decreased activation of yes‐associated protein 1 (YAP). Using a vinculin bioluminescent tension sensor, we measured FAs tension in transgenic muscle cells expressing wild type (WT) dystrophin, two patient derived dystrophin missense mutants (L172H and L54R), and a non‐disease …

The highly ordered cortical microtubule lattice of skeletal muscle is disorganized in dystrophin-deficient mdx mice. Implicated mechanisms include loss of dystrophin binding, altered α-tubulin posttranslational modification, expression of a β-tubulin involved in regeneration, and reactive oxygen species (ROS). Here we show that the transverse microtubules in mdx muscle expressing miniaturized dystrophins are rapidly lost after eccentric contraction. Analysis of mdx lines expressing different dystrophin constructs demonstrate that spectrin-like repeats R4-15 and R20-23 were required for mechanically stable microtubules. Microtubule loss was prevented by the non-specific antioxidant N-acetylcysteine while inhibition of NADPH oxidase 2 had only a partial effect, suggesting that ROS from multiple sources mediate the rapid loss of transverse microtubules after eccentric contraction. Finally, ablation of α-dystrobrevin, β …

Compromised muscle mitochondrial metabolism is a hallmark of peripheral arterial disease, especially in patients with the most severe clinical manifestation—critical limb ischemia (CLI). We asked whether inflexibility in metabolism is critical for the development of myopathy in ischemic limb muscles. Using Polg mtDNA mutator (D257A) mice, we reveal remarkable protection from hind limb ischemia (HLI) due to a unique and beneficial adaptive enhancement of glycolytic metabolism and elevated ischemic muscle PFKFB3. Similar to the relationship between mitochondria from CLI and claudicating patient muscles, BALB/c muscle mitochondria are uniquely dysfunctional after HLI onset as compared with the C57BL/6 (BL6) parental strain. AAV-mediated overexpression of PFKFB3 in BALB/c limb muscles improved muscle contractile function and limb blood flow following HLI. Enrichment analysis of RNA sequencing …

BackgroundDuchenne muscular dystrophy (DMD) is caused by the loss of dystrophin. Severe and ultimately lethal, DMD progresses relatively slowly in that patients become wheelchair bound only around age twelve with a survival expectancy reaching the third decade of life.MethodsThe mildly-affected mdx mouse model of DMD, and transgenic DysΔMTB-mdx and Fiona-mdx mice expressing dystrophin or utrophin, respectively, were exposed to either mild (scruffing) or severe (subordination stress) stress paradigms and profiled for their behavioral and physiological responses. A subgroup of mdx mice exposed to subordination stress were pretreated with the beta-blocker metoprolol.FindingsSubordination stress caused lethality in ∼30% of mdx mice within 24 h and ∼70% lethality within 48 h, which was not rescued by metoprolol. Lethality was associated with heart damage, waddling gait and hypo-locomotion …