Two operational modes of atomic force microscopy reveal similar mechanical properties for homologous regions of dystrophin and utrophin.

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by the absence of the protein dystrophin. Dystrophin is hypothesized to work as a molecular shock absorber that limits myofiber membrane damage when undergoing reversible unfolding upon muscle stretching and contraction. Utrophin is a dystrophin homologue that is under investigation as a protein replacement therapy for DMD. However, it remains uncertain whether utrophin can mechanically substitute for dystrophin. Here, we compared the mechanical properties of homologous utrophin and dystrophin fragments encoding the N terminus through spectrin repeat 3 (UtrN-R3, DysN-R3) using two operational modes of atomic force microscopy (AFM), constant speed and constant force. Our comprehensive data, including the statistics of force magnitude at which the folded domains unfold in constant speed mode and the time of unfolding statistics in constant force mode, show consistent results. We recover parameters of the energy landscape of the domains and conducted Monte Carlo simulations which corroborate the conclusions drawn from experimental data. Our results confirm that UtrN-R3 expressed in bacteria exhibits significantly lower mechanical stiffness compared to insect UtrN-R3, while the mechanical stiffness of the homologous region of dystrophin (DysN-R3) is intermediate between bacterial and insect UtrN-R3, showing greater similarity to bacterial UtrN-R3.

Mapping SCA1 regional vulnerabilities reveals neural and skeletal muscle contributions to disease.

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, which 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.

Deletion of exons 2 and 3 from Actb and cell immortalization lead to widespread, ß-actin independent alterations in gene expression associated with cell cycle control.

The cytoplasmic actin proteins, ß- 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 ß-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 mass spectrometry reveal largescale changes to the transcriptome, proteome, and phosphoproteome in cells lacking Actb but not those only lacking ß-actin protein. Pathway analysis of genes and proteins differentially expressed upon Actb KO suggest widespread dysregulation of genes involved in the cell cycle that may explain the severe defect in proliferation.

Retention of stress susceptibility in the mdx mouse model of Duchenne muscular dystrophy after PGC-1α overexpression or ablation of IDO1 or CD38.

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 kynurenic acid shunt by skeletal muscle-specific PGC-1α overexpression in mdx mice did not prevent scruff -induced inactivity, nor did abrogating extrahepatic kynurenine pathway activity by genetic deletion of the pathway rate-limiting enzyme, indoleamine oxygenase 1. We further show that reduced NAD+ production in mdx skeletal muscle after subordination stress exposure corresponded with elevated levels of NAD+ catabolites produced by ectoenzyme cluster of differentiation 38 (CD38) that have been implicated in lethal mdx response to pharmacological ß-adrenergic receptor agonism. However, genetic CD38 ablation did not prevent mdx scruff-induced inactivity. Our data do not support a direct contribution by the kynurenine pathway or CD38 metabolic dysfunction to the exaggerated stress response of mdx mice.

Delineating regional vulnerability in the neurodegenerative disease SCA1 using a conditional mutant ATXN1 mouse.

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-ATXN1 146Q/2Q ) having mouse Atxn1 coding exons replaced by human exons encoding 146 glutamines. F-ATXN1 146Q/2Q mice manifest SCA1-like phenotypes including motor and cognitive deficits, wasting, and decreased survival. CNS contributions to disease were revealed using ATXN1 146Q/2Q ; Nestin-Cre mice, that showed improved rotarod, open field and Barnes maze performances. Striatal contributions to motor deficits were examined using f-ATXN1 146Q/2Q ; Rgs9-Cre mice. Mice lacking striatal ATXN1 146Q/2Q had improved rotarod performance late in disease. Muscle contributions to disease were revealed in f-ATXN1 146Q/2Q ; ACTA1-Cre mice which lacked muscle pathology and kyphosis seen in f-ATXN1 146Q/2Q mice. Kyphosis was not improved in f-ATXN1 146Q/2Q ;Nestin - Cre mice. Thus, optimal SCA1 therapeutics will require targeting mutant ATXN1 toxic actions in multiple brain regions and muscle.

Phosphorylation alters the mechanical stiffness of a model fragment of the dystrophin homologue utrophin.

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 detectably phosphorylated. Using atomic force microscopy, we show that phosphorylated UtrN-R3 exhibited significantly higher unfolding forces compared to unphosphorylated UtrN-R3 without altering its actin-binding activity. Consistent with the effect of phosphorylation on mechanical stiffness, mutating the phosphorylated serine residues on insect eukaryotic protein to alanine decreased its stiffness to levels not different from unphosphorylated bacterial protein. Taken together, our data suggest that the mechanical properties of utrophin may be tuned by phosphorylation, with the potential to improve its efficacy as a protein replacement therapy for dystrophinopathies.

Enhancing interaction of actin and actin-binding domain 1 of dystrophin with modulators: Toward improved gene therapy for Duchenne muscular dystrophy.

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-binding domain 1 (ABD1). We engineered a novel FRET biosensor, consisting of the mClover3, fluorescent protein (donor) attached to the C-terminus of dystrophin ABD1, and Alexa Fluor 568 (acceptor) attached to the C-terminal cysteine of actin. We used this biosensor in small-molecule screening, using a unique high-precision, HTS fluorescence lifetime assay, identifying several compounds from an FDA-approved library that significantly increase the binding between actin and ABD1. This HTS assay establishes feasibility for the discovery of small-molecule modulators of the actin-dystrophin interaction, with the ultimate goal of developing therapies for muscular dystrophy.

Fibroblast fate determination during cardiac reprogramming by remodeling of actin filaments.

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 actin isoforms significantly enhances cardiac reprogramming. Our results demonstrate that remodeling of actin isoforms is required for fibroblast to CM fate conversion by cardiac reprogramming.

Dystrophin missense mutations alter focal adhesion tension and mechanotransduction.

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 expression of WT or I232M dystrophin increased FA tension compared to dystrophin-less nontransgenic myoblasts. In contrast, the expression of L54R or L172H did not increase FA tension, indicating that these disease-causing mutations compromise the mechanical function of dystrophin as an FA allosteric regulator. Decreased FA tension caused by these mutations manifests as defective migration, as well as decreased Yes-associated protein 1 (YAP) activation, possibly by the disruption of the ability of FAs to transmit forces between the extracellular matrix and cytoskeleton. Our results indicate that dystrophin influences FA tension and suggest that dystrophin disease-causing missense mutations may disrupt a cellular tension-sensing pathway in dystrophic skeletal muscle.

Nucleotide- and Protein-Dependent Functions of Actg1.

Cytoplasmic ß- 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 Actg1 nucleotide-dependent function(s). On the other hand, the bG/0 genotype rescued functions impaired by Actg1-/-, including cell proliferation and auditory function, suggesting a role for γ-actin protein in both fibroblasts and hearing. Together, these results identify nucleotide-dependent functions for Actg1 while implicating γ-actin protein in more cell-/tissue-specific functions.

Contraction-Induced Loss of Plasmalemmal Electrophysiological Function Is Dependent on the Dystrophin Glycoprotein Complex.

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 mouse lines (p<0.001) but not within WT mice (R 2=0.211; p=0.155). By comparing mouse lines that had varying amounts and functionality of dystrophin and other DGC proteins, we observed that (1) when all DGCs are intact, plasmalemmal action potential generation and conduction is maintained, (2) deficiency of the DGC protein ß-sarcoglycan is as disruptive to plasmalemmal excitability as is dystrophin deficiency and, (3) some functionally intact DGCs are better than none. Our results highlight the significant role of the DGC plays in maintaining plasmalemmal excitability and that a collective synergism (via each DGC protein) is required for this complex to function properly during ECC contractions.

Some dystrophy phenotypes of dystrophin-deficient mdx mice are exacerbated by mild, repetitive daily stress.

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 in muscle fibrosis. Elevated urinary corticosterone secretion, adrenal hypertrophy, and a larger adrenal cortex indicating chronic activation of the hypothalamic-pituitary-adrenal (HPA) axis were measured in 12-week stressed mdx mice relative to those unstressed. However, pharmacological inhibition of the HPA axis did not affect scruff-induced physical inactivity and acute corticosterone injection did not recapitulate the scruff-induced phenotype, suggesting the HPA axis is not the driver of physical inactivity. Our results indicate that the response of mdx mice to an acute mild stress is non-habituating and that when that stressor is repeated daily for weeks, it is sufficient to exacerbate some phenotypes associated with dystrophinopathy in mdx mice.

Tetrahydrobiopterin synthesis and metabolism is impaired in dystrophin-deficient mdx mice and humans.

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 patients. Mdx mice had low urinary and skeletal muscle dihydrobiopterin + tetrahydrobiopterin compared to wild-type mice. Overexpression of dystrophins that localize neuronal nitric oxide synthase restored dihydrobiopterin + tetrahydrobiopterin in mdx mice to wild-type levels while utrophin overexpression did not. Mdx mice and Duchenne muscular dystrophy patients did not increase tetrahydrobiopterin during exercise and in mdx mice tetrahydrobiopterin deficiency was likely because of lower levels of sepiapterin reductase in skeletal muscle. Tetrahydrobiopterin supplementation improved skeletal muscle strength, resistance to fatiguing and injurious contractions in vivo, increased utrophin and capillary density of skeletal muscle and lowered cardiac muscle fibrosis and left ventricular wall thickness in mdx mice. CONCLUSION: These data demonstrate that impaired tetrahydrobiopterin synthesis is associated with dystrophin loss and treatment with tetrahydrobiopterin improves striated muscle histopathology and skeletal muscle function in mdx mice.

Loss of ß-Cytoplasmic Actin in the Intestinal Epithelium Increases Gut Barrier Permeability in vivo and Exaggerates the Severity of Experimental Colitis.

Intestinal epithelial barrier is critical for the maintenance of normal gut homeostasis and disruption of this barrier may trigger or exaggerate mucosal inflammation. The actin cytoskeleton is a key regulator of barrier structure and function, controlling the assembly and permeability of epithelial adherens and tight junctions. Epithelial cells express two actin isoforms: a ß-cytoplasmic actin and γ-cytoplasmic actin. Our previous in vitro studies demonstrated that these actin isoforms play distinctive roles in establishing the intestinal epithelial barrier, by controlling the organization of different junctional complexes. It remains unknown, whether ß-actin and γ-actin have unique or redundant functions in regulating the gut barrier in vivo. To address this question, we selectively knocked out ß-actin expression in mouse intestinal epithelium. Mice with intestinal epithelial knockout of ß-actin do not display gastrointestinal abnormalities or gross alterations of colonic mucosal architecture. This could be due to compensatory upregulation of γ-actin expression. Despite such compensation, ß-actin knockout mice demonstrate increased intestinal permeability. Furthermore, these animals show more severe clinical symptoms during dextran sodium sulfate induced colitis, compared to control littermates. Such exaggerated colitis is associated with the higher expression of inflammatory cytokines, increased macrophage infiltration in the gut, and accelerated mucosal cell death. Consistently, intestinal organoids generated from ß-actin knockout mice are more sensitive to tumor necrosis factor induced cell death, ex vivo. Overall, our data suggests that ß-actin functions as an essential regulator of gut barrier integrity in vivo, and plays a tissue protective role during mucosal injury and inflammation.

Rapid, redox-mediated mechanical susceptibility of the cortical microtubule lattice in skeletal muscle.

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 ß-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, ß- or γ-cytoplasmic actin phenocopied the transverse microtubule instability of miniaturized dystrophins. Our data demonstrate that multiple dystrophin domains, α-dystrobrevin and cytoplasmic actins are necessary for mechanically stable microtubules.

PFKFB3-mediated glycolysis rescues myopathic outcomes in the ischemic limb.

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 data on muscle from CLI patients revealed a unique deficit in the glucose metabolism Reactome. Muscles from these patients express lower PFKFB3 protein, and their muscle progenitor cells possess decreased glycolytic flux capacity in vitro. Here, we show supplementary glycolytic flux as sufficient to protect against ischemic myopathy in instances where reduced blood flow-related mitochondrial function is compromised preclinically. Additionally, our data reveal reduced glycolytic flux as a common characteristic of the failing CLI patient limb skeletal muscle.

Social stress is lethal in the mdx model of Duchenne muscular dystrophy.

BACKGROUND: Duchenne 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. METHODS: The 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. FINDINGS: Subordination 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, as well as marked up-regulation of the hypothalamus-pituitary-adrenocortical axis. A novel cardiovascular phenotype emerged in mdx mice, in that scruffing caused a transient drop in arterial pressure, while subordination stress caused severe and sustained hypotension with concurrent tachycardia. Transgenic expression of dystrophin or utrophin in skeletal muscle protected mdx mice from scruffing and social stress-induced responses including mortality. INTERPRETATION: We have identified a robust new stress phenotype in the otherwise mildly affected mdx mouse that suggests relatively benign handling may impact the outcome of behavioural experiments, but which should also expedite the knowledge-based therapy development for DMD. FUNDING: Greg Marzolf Jr. Foundation, Summer's Wish Fund, NIAMS, Muscular Dystrophy Association, University of Minnesota and John and Cheri Gunvalson Trust.

Stem Cell-Derived Cardiomyocytes and Beta-Adrenergic Receptor Blockade in Duchenne Muscular Dystrophy Cardiomyopathy.

BACKGROUND: Although cardiomyopathy has emerged as a leading cause of death in Duchenne muscular dystrophy (DMD), limited studies and therapies have emerged for dystrophic heart failure. OBJECTIVES: The purpose of this study was to model DMD cardiomyopathy using DMD patient-specific human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and to identify physiological changes and future drug therapies. METHODS: To explore and define therapies for DMD cardiomyopathy, the authors used DMD patient-specific hiPSC-derived cardiomyocytes to examine the physiological response to adrenergic agonists and ß-blocker treatment. The authors further examined these agents in vivo using wild-type and mdx mouse models. RESULTS: At baseline and following adrenergic stimulation, DMD hiPSC-derived cardiomyocytes had a significant increase in arrhythmic calcium traces compared to isogenic controls. Furthermore, these arrhythmias were significantly decreased with propranolol treatment. Using telemetry monitoring, the authors observed that mdx mice, which lack dystrophin, had an arrhythmic death when stimulated with isoproterenol; the lethal arrhythmias were rescued, in part, by propranolol pre-treatment. Using single-cell and bulk RNA sequencing (RNA-seq), the authors compared DMD and control hiPSC-derived cardiomyocytes, mdx mice, and control mice (in the presence or absence of propranolol and isoproterenol) and defined pathways that were perturbed under baseline conditions and pathways that were normalized after propranolol treatment in the mdx model. The authors also undertook transcriptome analysis of human DMD left ventricle samples and found that DMD hiPSC-derived cardiomyocytes have dysregulated pathways similar to the human DMD heart. The authors further determined that relatively few patients with DMD see a cardiovascular specialist or receive ß-blocker therapy. CONCLUSIONS: The results highlight mechanisms and therapeutic interventions from human to animal and back to human in the dystrophic heart. These results may serve as a prelude for an adequately powered clinical study that examines the impact of ß-blocker therapy in patients with dystrophinopathies.

Mechanical factors tune the sensitivity of mdx muscle to eccentric strength loss and its protection by antioxidant and calcium modulators.

BACKGROUND: Dystrophin deficiency sensitizes skeletal muscle of mice to eccentric contraction (ECC)-induced strength loss. ECC protocols distinguish dystrophin-deficient from healthy, wild type muscle, and test the efficacy of therapeutics for Duchenne muscular dystrophy (DMD). However, given the large lab-to-lab variability in ECC-induced strength loss of dystrophin-deficient mouse skeletal muscle (10-95%), mechanical factors of the contraction likely impact the degree of loss. Therefore, the purpose of this study was to evaluate the extent to which mechanical variables impact sensitivity of dystrophin-deficient mouse skeletal muscle to ECC. METHODS: We completed ex vivo and in vivo muscle preparations of the dystrophin-deficient mdx mouse and designed ECC protocols within physiological ranges of contractile parameters (length change, velocity, contraction duration, and stimulation frequencies). To determine whether these contractile parameters affected known factors associated with ECC-induced strength loss, we measured sarcolemmal damage after ECC as well as strength loss in the presence of the antioxidant N-acetylcysteine (NAC) and small molecule calcium modulators that increase SERCA activity (DS-11966966 and CDN1163) or lower calcium leak from the ryanodine receptor (Chloroxine and Myricetin). RESULTS: The magnitude of length change, work, and stimulation duration ex vivo and in vivo of an ECC were the most important determinants of strength loss in mdx muscle. Passive lengthening and submaximal stimulations did not induce strength loss. We further showed that sarcolemmal permeability was associated with muscle length change, but it only accounted for a minimal fraction (21%) of the total strength loss (70%). The magnitude of length change also significantly influenced the degree to which NAC and small molecule calcium modulators protected against ECC-induced strength loss. CONCLUSIONS: These results indicate that ECC-induced strength loss of mdx skeletal muscle is dependent on the mechanical properties of the contraction and that mdx muscle is insensitive to ECC at submaximal stimulation frequencies. Rigorous design of ECC protocols is critical for effective use of strength loss as a readout in evaluating potential therapeutics for muscular dystrophy.

Variable cytoplasmic actin expression impacts the sensitivity of different dystrophin-deficient mdx skeletal muscles to eccentric contraction.

Eccentric contractions (ECCs) induce force loss in several skeletal muscles of dystrophin-deficient mice (mdx), with the exception of the soleus (Sol). The eccentric force : isometric force (ECC : ISO), expression level of utrophin, fiber type distribution, and sarcoendoplasmic reticulum calcium ATPase expression are factors that differ between muscles and may contribute to the sensitivity of mdx skeletal muscle to ECC. Here, we confirm that the Sol of mdx mice loses only 13% force compared to 87% in the extensor digitorum longus (EDL) following 10 ECC of isolated muscles. The Sol has a greater proportion of fibers expressing Type I myosin heavy chain (MHC) and expresses 2.3-fold more utrophin compared to the EDL. To examine the effect of ECC : ISO, we show that the mdx Sol is insensitive to ECC at ECC : ISO up to 230 ± 15%. We show that the peroneus longus (PL) muscle presents with similar ECC : ISO compared to the EDL, intermediate force loss (68%) following 10 ECC, and intermediate fiber type distribution and utrophin expression relative to EDL and Sol. The combined absence of utrophin and dystrophin in mdx/utrophin-/- mice rendered the Sol only partially susceptible to ECC and exacerbated force loss in the EDL and PL. Most interestingly, the expression levels of cytoplasmic ß- and γ-actins correlate inversely with a given muscle's sensitivity to ECC; EDL < PL < Sol. Our data indicate that fiber type, utrophin, and cytoplasmic actin expression all contribute to the differential sensitivities of mdxEDL, PL, and Sol muscles to ECC.