Dystrophy Patients Research Articles

Abstract 13405: Prevalence of Cardiac Disease in Duchenne Muscular Dystrophy Patients by Age: A Cardiac Magnetic Resonance Natural History Study

Introduction: Duchenne muscular dystrophy (DMD) patients universally develop cardiomyopathy and considered a dilated cardiomyopathy. Cardiac magnetic resonance imaging (CMR) revealed LGE prior to ventricular dysfunction. Various novel trials including gene therapy are ongoing with growing interest on how it impacts the heart. There is paucity of CMR data at a given age. A major barrier is lack of age specific data. Hypothesis: We therefore, sought to determine the prevalence left ventricular (LV) dilation by endiastolic volume (LVEDV), ejection fraction (LVEF) and late gadolinium enhancement (LGE) status at a given age. Methods: Retrospective review of CMR studies in DMD. LV dilation is defined as LVEDVi z-score > 2, abnormal LVEF (<55%) and LGE (yes or no). Patients were group by age in one year interval (given smaller numbers patients < 7 years were grouped together as were those ≥ 20 years). Results: 683 DMD patients were identified with average of 48.8±24 (range 19-94) patients per group divided into 14 age groups. Mean age was 12.3±4 (range 4-39 years). Table 1 summarizes the CMR data by age. LV dilation was uncommon even in older age patients (mean 1.9±2.6%, range 0-8.7%). LVEF is rarely abnormal before 10 years (<5%) but increased with age (69.9% in the ≥ 20 year group). In all groups mean % LVEF < 55% is 24.6±20.6% (range 2.5-69.9). LGE can be found as young as 6.5 years but uncommon before age 10 years (<10%), increased to nearly 20% by age 10 years and incrementally increased to >50% after 11 (4.3% < 7 year group to > 80% by 18 years). Conclusions: This is the first and largest CMR natural history study by individual age showing low prevalence of LV dilation in DMD population. LVEF abnormality and LGE is uncommon before 10 years but increased with age. Future longitudinal assessment is ongoing to determine rate of change at a given age, quantifying LGE and myocardial strain for earlier cardiac disease assessment and modeling for disease progression at different age.

Abstract 12003: TN-C Promotes Cardiac Fibrosis and Endothelial Dysfunction in Duchenne Muscular Dystrophy

Background: Cardiac fibrosis and heart failure are major contributors to mortality in Duchenne muscular dystrophy (DMD) patients. Recently, it has been found that Tenascin C (TN-C) plays a maladaptive role in adverse left ventricular (LV) remodelling and fibrosis. Aims: To 1) elucidate the vascular dysfunction and cardiac fibrosis in link to TN-C in a mouse model of DMD and 2) explore the effect of knocking out TN-C in dystrophic mice. Methods: Male wt, mdx and mdx TN-C KO mice were used. Echocardiography was performed, and fibrosis was assessed on cardiac tissue sections. To test the vascular reactivity wire myography was used and CD31+ endothelial cells (ECs) were isolated from mouse lung tissues and characterized for oxidative stress and inflammatory marker expression. To explore further the signalling pathways contributing to cardiac fibrosis, human cardiac fibroblasts (hCFs) were treated with TN-C or TGF-β, then fibrotic markers and epigenetic regulation of p65 were assessed. Results: Cardiac fibrosis was markedly enhanced in mdx mice compared to age-matched wt animals. This was accompanied by upregulation of TN-C in cardiac tissue and plasma. In addition, vascular endothelial function was impaired in mdx mice. In line with that, endothelial cells isolated from mdx mice also showed a marked upregulation of oxidative stress and inflammatory markers. Interestingly, mdx- TN-C KO mice showed preserved vascular function as well as reduced cardiac fibrosis compared to age-matched mdx mice. hCFs treated with TN-C or TGF-β showed increased collagen production and elevated α-SMA and matrix metalloproteinase 2, 3 and 9 (MMP-2, 3 and 9) expressions. TN-C siRNA reduced the expression of collagen and α-SMA, but not MMPs. In addition, both TN-C and TGF-β promote p65/ NF-κB promoter demethylation and subsequently stimulate pro-inflammatory and pro-fibrotic signalling. These effects were reversed by applying p38 MAPK inhibitor in hCFs. Conclusions: TN-C facilitates oxidative stress and inflammation in ECs and fibroblasts, contributing to severe endothelial dysfunction and cardiac fibrosis. In addition, activation of NF-κB p65 signalling pathway may play a role in TN-C induced fibrosis. Thus, TN-C may be a critical mediator and potential target for therapy in DMD.

Dystrophin deficiency affects human astrocyte properties and response to damage.

In addition to progressive muscular degeneration due to dystrophin mutations, 1/3 of Duchenne muscular dystrophy (DMD) patients present cognitive deficits. However, there is currently an incomplete understanding about the function of the multiple dystrophin isoforms in human brains. Here, we tested the hypothesis that dystrophin deficiency affects glial function in DMD and could therefore contribute to neural impairment. We investigated human dystrophin isoform expression with development and differentiation and response to damage in human astrocytes from control and induced pluripotent stem cells from DMD patients. In control cells, short dystrophin isoforms were up-regulated with development and their expression levels changed differently upon neuronal and astrocytic differentiation, as well as in 2-dimensional versus 3-dimensional astrocyte cultures. All DMD-astrocytes tested displayed altered morphology, proliferative activity and AQP4 expression. Furthermore, they did not show any morphological change in response to inflammatory stimuli and their number was significantly lower as compared to stimulated healthy astrocytes. Finally, DMD-astrocytes appeared to be more sensitive than controls to oxidative damage as shown by their increased cell death. Behavioral and metabolic defects in DMD-astrocytes were consistent with gene pathway dysregulation shared by lines with different mutations as demonstrated by bulk RNA-seq analysis. Together, our DMD model provides evidence for altered astrocyte function in DMD suggesting that defective astrocyte responses may contribute to neural impairment and might provide additional potential therapeutic targets.

Reduced plasticity and microtubule densification in muscular dystrophy-related cardiomyopathy

The dystrophin-glycoprotein complex (DGC) links the intracellular cytoskeleton to the extracellular basement membrane, thereby providing structural support for the sarcolemma. Many patients with muscular dystrophies, particularly those with defects in cardiomyopathies with chamber dilation and myocardial dysfunction. Heart failure is the major cause of death for muscular dystrophy patients; however, the molecular pathomechanism remains unknown. Here, I show the detailed molecular pathogenesis of muscular dystrophy-associated cardiomyopathy in mice lacking the fukutin gene (Fktn), the causative gene for Fukuyama muscular dystrophy. Although cardiac Fktn elimination markedly reduced the glycosylation of α-dystroglycan and the expression of DGC proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that the physiological contribution of DGC proteins in the heart increases after 6 mo of age. In addition, Fktn-deficient mice maintain normal cardiac function at young age, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. Young Fktn-deficient mice did not show a compensative hypertrophic response to hemodynamic stress and quickly developed heart failure with chamber dilation and contractile dysfunction. In these mice, Ca2+-calcineurin signaling was already elevated under physiological conditions, and MEF2-HDAC axes essential for the hypertrophic response were unable to function under stress conditions. Acute Fktn elimination caused severe cardiac dysfunction and accelerated mortality with myocyte contractile dysfunction and disordered Golgi-microtubule networks, which were ameliorated with colchicine treatment. Microarray analysis in control and Fktn-deficient hearts suggest that elimination of Fktn impacts the expression profile of Golgi-related genes, and that the pathological mechanism of cardiac dysfunction induced by Fktn elimination partly overlaps with that of neurodegenerative disease. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and, thus, the results may lead to strategies for intervention.

Muscle inflammatory pattern in alpha- and gamma-sarcoglycanopathies.

Since the immune system plays a role in the pathogenesis of several muscular dystrophies, we aim to characterize several muscular inflammatory features in α- (LGMD R3) and γ-sarcoglycanopathies (LGMD R5). We explored the expression of major histocompatibility complex class I molecules (MHCI), and we analyzed the composition of the immune infiltrates in muscle biopsies from 10 patients with LGMD R3 and 8 patients with LGMD R5, comparing the results to Duchenne muscular dystrophy patients (DMD). A consistent involvement of the immune response was observed in sarcoglycanopathies, although it was less evident than in DMD. LGMD R3-R5 and DMD shared an abnormal expression of MHCI, and the composition of the muscular immune cell infiltrate was comparable. These findings might serve as a rationale to fine-tune a disease-specific immunomodulatory regimen, particularly relevant in view of the rapid development of gene therapy for sarcoglycanopathies.

Perturbation of PI3K/Akt signaling affected autophagy modulation in dystrophin-deficient myoblasts

BackgroundThe absence of dystrophin has gave a massive impact on myotube development in Muscular Dystrophy pathogenesis. One of the conserved signaling pathways involved in skeletal muscle differentiation is the PI3K/Akt/mTOR pathway that plays a vital role in autophagy regulation. To further understand and establish targeted therapy in dystrophin-deficient myoblasts, protein expression profiling has been determined which provides information on perturbed autophagy modulation and activation.MethodsIn this study, a dystrophin-deficient myoblast cell line established from the skeletal muscle of a dystrophic (mdx) mouse was used as a model. The dfd13 (dystrophin-deficient) and C2C12 (non-dystrophic) myoblasts were cultured in low mitogen conditions for 10 days to induce differentiation. The cells were subjected to total protein extraction prior to Western blotting assay technique. Protein sub-fractionation has been conducted to determine protein localization. The live-cell analysis of autophagy assay was done using a flow cytometer.ResultsIn our culture system, the dfd13 myoblasts did not achieve terminal differentiation. PTEN expression was profoundly increased in dfd13 myoblasts throughout the differentiation day subsequently indicates perturbation of PI3K/Akt/mTOR regulation. In addition, rictor-mTORC2 was also found inactivated in this event. This occurrence has caused FoxO3 misregulation leads to higher activation of autophagy-related genes in dfd13 myoblasts. Autophagosome formation was increased as LC3B-I/II showed accumulation upon differentiation. However, the ratio of LC3B lipidation and autophagic flux were shown decreased which exhibited dystrophic features.ConclusionPerturbation of the PTEN-PI3K/Akt pathway triggers excessive autophagosome formation and subsequently reduced autophagic flux within dystrophin-deficient myoblasts where these findings are of importance to understand Duchenne Muscular Dystrophy (DMD) patients. We believe that some manipulation within its regulatory signaling reported in this study could help restore muscle homeostasis and attenuate disease progression.1FSGSuke3ozHkrGhS-2dsfVideo

Global longitudinal strain detects subtle left ventricular systolic dysfunction in Duchenne muscular dystrophy patients and carriers

BackgroundWith the continuous improvement of the respiratory care of Duchenne muscular dystrophy patients, cardiac manifestations (heart failure and arrhythmias) become the leading causes of morbidity and mortality. Early identification of cardiac muscle affection is crucial to start anti-failure drugs that reverse remodeling and improve prognosis. This study aimed to detect subtle cardiac changes in Duchenne muscular dystrophy patients and carriers using electrocardiography and echocardiography.ResultsThis study included genetically diagnosed Duchenne muscular dystrophy patients (28 males) and carriers (25 females) and compared them to healthy gender-matched control groups. All study participants underwent clinical assessment, 12-lead electrocardiography, and global longitudinal strain augmented echocardiography. In the current study, Duchenne muscular dystrophy patients had higher heart rates, smaller left ventricular internal diameters, left atrial diameter, lower ejection fraction, and worse left ventricular global longitudinal strain in comparison with the control group. The global longitudinal strain inversely correlated with the age of Duchenne muscular dystrophy patients. The number of exon mutations did not affect electrocardiography and echocardiographic findings. Exon mutations 45–47 and 51–54 were significantly associated with an ejection fraction less than 60%. Duchenne muscular dystrophy carriers had smaller left ventricular wall diameters, left ventricular end-diastolic diameter, left atrial diameter, and worse left ventricular global longitudinal strain in comparison with the control group.ConclusionsLeft ventricular global longitudinal strain could detect subtle left ventricular systolic dysfunction in Duchenne muscular dystrophy patients and carriers before the decline of left ventricular ejection fraction.

Assessing Cognitive Function in Neuromuscular Diseases: A Pilot Study in a Sample of Children and Adolescents.

Central nervous system (CNS) involvement has been variously studied in pediatric neuromuscular disorders (NMDs). The primary goal of this study was to assess cognitive functioning in NMDs, and secondary aims were to investigate possible associations of cognitive impairment with motor impairment, neurodevelopmental delay, and genotype. This was a cross-sectional study of 43 pediatric patients, affected by six NMDs. Myotonic dystrophy type 1 (DM1) and glycogen storage disease type 2 (GSD2) patients had a delay on the Bayley-III scales. On Wechsler scales, DMD and DM1 patients showed lower FSIQ scores, with an intellectual disability (ID) in 27% and 50%, respectively. FSIQ was normal in Becker muscular dystrophy (BMD), GSD2, and hereditary motor sensory neuropathy (HMSN) patients, while higher individual scores were found in the spinal muscular atrophy (SMA) group. In the DM1 cohort, lower FSIQ correlated with worse motor performance (ρ = 0.84, p < 0.05), and delayed speech acquisition was associated with ID (p = 0.048), with worse cognitive impairment in the congenital than in the infantile form (p = 0.04). This study provides further evidence of CNS in some NMDs and reinforces the need to include cognitive assessment in protocols of care of selected pediatric NMDs.

Targeted Next-Generation Sequencing Reveals Mutations in Non-coding Regions and Potential Regulatory Sequences of Calpain-3 Gene in Polish Limb-Girdle Muscular Dystrophy Patients.

Limb–girdle muscular dystrophy type R1 (LGMDR1) is caused by mutations in CAPN3 and is the most common type of recessive LGMD. Even with the use of whole-exome sequencing (WES), only one mutant allele of CAPN3 is found in a significant number of LGMDR patients. This points to a role of non-coding, intronic or regulatory, sequence variants in the disease pathogenesis. Targeted sequencing of the whole CAPN3 gene including not only intronic, 3′ and 5′ UTRs but also potential regulatory regions was performed in 27 patients suspected with LGMDR1. This group included 13 patients with only one mutated CAPN3 allele detected previously with exome sequencing. A second rare variant in the non-coding part of CAPN3 was found in 11 of 13 patients with previously identified single mutation. Intronic mutations were found in 10 cases, with c.1746-20C>G variant present in seven patients. In addition, a large deletion of exons 2–8 was found in one patient. In the patients with no causative mutation previously found, we detected rare CAPN3 variants in 5 out of 10 patients and in two of them in a compound heterozygous state. Rare variants within putative regulatory sequences distant from the CAPN3 gene were found in 15 patients, although in 11 of these cases, other variants are deemed causative. The results indicate that intronic mutations are common in Polish LGMDR patients, and testing for non-coding mutations in CAPN3 should be performed in apparently single heterozygous patients.

Electrocardiographic prediction of late gadolinium enhancement on cardiac magnetic resonance in Becker muscular dystrophy

Dystrophin deficiency results in cardiomyopathy and fibrosis with variable onset. Little is known about electrocardiographic abnormalities in Becker muscular dystrophy and their relationship to underlying cardiac pathology. We hypothesized QRS fragmentation is associated with myocardial fibrosis on cardiac magnetic resonance imaging in Becker muscular dystrophy patients. We retrospectively evaluated 44 patients, and extracted data from clinically obtained electrocardiogram and cardiac magnetic resonance. Ventricular function and presence or absence late gadolinium enhancement representing myocardial fibrosis were recorded from imaging. Nearly half (19/42, 45%) of patients interrogated had myocardial fibrosis on cardiac magnetic resonance. Total number of electrocardiogram leads with QRS fragmentation (median 1 vs 4, p < 0.001) and either right or left axis deviation from median was significantly increased (13.6 vs. 44.8°, p < 0.001) in patients with myocardial fibrosis. Decreased leftward voltage in V6 correlated to both increased fibrosis and decreased cardiac function (p < 0.01). The positive likelihood ratio for underlying myocardial fibrosis in patients with two of the three findings on electrocardiogram was 8.47 (p < 0.0001). QRS fragmentation and axis deviation on electrocardiography are strongly predictive of myocardial fibrosis in Becker muscular dystrophy and may inform the use of advanced imaging in evaluation of these patients.

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Historical aspects of muscle research in the Dubowitz Neuromuscular Centre: the Hammersmith days

Validity of Skeletal Muscle Ultrasound in Assessment of Suspected Limb Girdle Muscular Dystrophy Patients

Abstract Background Limb-girdle muscular dystrophies (LGMDs) are a clinically and genetically heterogeneous group of disorders characterized by progressive muscle weakness and degenerative muscle changes. Studies have shown that ultrasound can be useful both for diagnosis and follow-up of LGMDs patients. Objectives This study aims to measure the sensitivity and the specificity of muscle ultrasound in assessment of suspected limb girdle muscular dystrophy patients. Subjects and Methods This cross-sectional descriptive study was conducted on Fifty-five patients with suspected LGMD from neuromuscular unit, myology clinic, Ain Shams University hospitals and eight healthy subjects. Age was above 2 years. Both sexes were included in the study. They underwent real-time B-mode ultrasonography performed with using Logiq p9 General Electric ultrasound machine and General Electric 7-11.5 MHZ linear array ultrasound probe. All ultrasound images have been obtained and scored by a single examiner and muscle echo intensity was visually graded semiquantitative according to Heckmatt's scale. The examiner was blinded to the muscle biopsy results and clinical evaluations. Results Statistical analysis revealed that the diagnostic performance of muscle US (Heckmatt’s score) in LGMD is most sensitive when calculated in all examined upper limb and lower limb muscles, followed by lower limb muscles alone. US of upper limb was found to be the least sensitive. Conclusions Muscle ultrasound is a practical and reproducible and valid tool that can be used in assessment of suspected LGMD patients.

Clinical and genetic profile of duchenne and becker muscular dystrophy patients in the far west of Algeria

Duchenne's (DMD) and Becker's (BMD) muscular dystrophy are the principals causes of myopathy in male children. It is linked to a mutation in the dystrophygene and isinheritedin an X-linked mode. The aims of our study are to Describe the clinical and genetic phenotype of BMD and DMD patients in the far west of Algeria.

DMD – CLINICAL CARE: EP.110 Ambulatory performance of Duchenne muscular dystrophy patients on intermittent corticosteroid treatment – a single center study

DMD – CLINICAL CARE: EP.110 Ambulatory performance of Duchenne muscular dystrophy patients on intermittent corticosteroid treatment – a single center study

The variability in neurological deficits in Duchenne muscular dystrophy patients may be explained by differences in dystrophin glycoprotein complexes in the brain and muscle.

Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic neuromuscular disorder. The variability in neurologic deficits in DMD patients may be explained by the fact that (1) dystrophin containing complexes in the brain are more stable than dystrophin containing complexes in the muscle (2) neurons are not affected by the same stresses as muscle and (3) neurons have a greater capacity to buffer increases in intracellular calcium levels. In the muscle, the loss of dystrophin and subsequent loss of dystrophin-associated proteins (DAPs) affects the stability of the dystrophin-glycoprotein complex and calcium ion channels. It causes the sarcolemma of the muscle to tear and calcium ion leak. The subsequent calcium influx leads to calcium dependant proteolysis. In the brain, the structure of the dystrophin-containing complexes is completely different from the muscle. There are several dystrophin isoforms that combine with a completely different set of proteins compared to the muscle to form several different dystrophin-containing complexes. In addition, the loss of dystrophin does not affect the expression of DAPs. The heterogeneity of dystrophin-containing complexes and the continued expression of DAPs will result in more stable dystrophin-containing complexes in the DMD brain. Muscles are under more stress than neurons as they undergo contractions. This combined with txhe fact that the neurons have a better ability to buffer increases in calcium would suggest that neurons are less likely to be damaged despite the loss of dystrophin.

Altered Gene Expression Profiles in Neural Stem Cells Derived from Duchenne Muscular Dystrophy Patients with Intellectual Disability.

Intellectual disability (ID) is a neurodevelopmental disorder defined by below-average intelligence (intelligence quotient of <70) accompanied by adaptive behavior deficits. Defects in the functions of neural stem cells during brain development are closely linked to the pathogenesis of ID. To understand the molecular etiology of ID, we examined neural stem cells from individuals with Duchenne muscular dystrophy (DMD), a genetic disorder in which approximately one-third of the patients exhibit ID. In this study, we generated induced pluripotent stem cells from peripheral blood mononuclear cells from a normal individual and DMD patients with and without ID to identify ID-specific functional and molecular abnormalities. We found defects in neural ectoderm formation in the group of DMD patients with ID. Our transcriptome analysis of patient-derived neural stem cells revealed altered expression of genes related to the hippo signaling pathway and neuroactive ligand-receptor interaction, implicating these in the pathogenesis of ID in patients with DMD.

Functional significance of gain-of-function H19 lncRNA in skeletal muscle differentiation and anti-obesity effects

BackgroundExercise training is well established as the most effective way to enhance muscle performance and muscle building. The composition of skeletal muscle fiber type affects systemic energy expenditures, and perturbations in metabolic homeostasis contribute to the onset of obesity and other metabolic dysfunctions. Long noncoding RNAs (lncRNAs) have been demonstrated to play critical roles in diverse cellular processes and diseases, including human cancers; however, the functional importance of lncRNAs in muscle performance, energy balance, and obesity remains elusive. We previously reported that the lncRNA H19 regulates the poly-ubiquitination and protein stability of dystrophin (DMD) in muscular dystrophy.MethodsHere, we identified mouse/human H19-interacting proteins using mouse/human skeletal muscle tissues and liquid chromatography–mass spectrometry (LC-MS). Human induced pluripotent stem-derived skeletal muscle cells (iPSC-SkMC) from a healthy donor and Becker Muscular Dystrophy (BMD) patients were utilized to study DMD post-translational modifications and associated proteins. We identified a gain-of-function (GOF) mutant of H19 and characterized the effects on myoblast differentiation and fusion to myotubes using iPSCs. We then conjugated H19 RNA gain-of-function oligonucleotides (Rgof) with the skeletal muscle enrichment peptide agrin (referred to as AGR-H19-Rgof) and evaluated AGR-H19-Rgof’s effects on skeletal muscle performance using wild-type (WT) C57BL/6 J mice and its anti-obesity effects using high-fat diet (HFD)- and leptin deficiency-induced obese mouse models.ResultsWe demonstrated that both human and mouse H19 associated with DMD and that the H19 GOF exhibited enhanced interaction with DMD compared to WT H19. DMD was found to associate with serine/threonine-protein kinase MRCK alpha (MRCKα) and α-synuclein (SNCA) in iPSC-SkMC derived from BMD patients. Inhibition of MRCKα and SNCA-mediated phosphorylation of DMD antagonized the interaction between H19 and DMD. These signaling events led to improved skeletal muscle cell differentiation and myotube fusion. The administration of AGR-H19-Rgof improved the muscle mass, muscle performance, and base metabolic rate of WT mice. Furthermore, mice treated with AGR-H19-Rgof exhibited resistance to HFD- or leptin deficiency-induced obesity.ConclusionsOur study suggested the functional importance of the H19 GOF mutant in enhancing muscle performance and anti-obesity effects.

Isolation of human fibroadipogenic progenitors and satellite cells from frozen muscle biopsies.

Skeletal muscle contains multiple cell types that work together to maintain tissue homeostasis. Among these, satellite cells (SC) and fibroadipogenic progenitors cells (FAPs) are the two main stem cell pools. Studies of these cells using animal models have shown the importance of interactions between these cells in repair of healthy muscle, and degeneration of dystrophic muscle. Due to the unavailability of fresh patient muscle biopsies, similar analysis of interactions between human FAPs and SCs is limited especially among the muscular dystrophy patients. To address this issue here we describe a method that allows the use of frozen human skeletal muscle biopsies to simultaneously isolate and grow SCs and FAPs from healthy or dystrophic patients. We show that while the purified SCs differentiate into mature myotubes, purified FAPs can differentiate into adipocytes or fibroblasts demonstrating their multipotency. We find that these FAPs can be immortalized and the immortalized FAPs (iFAPs) retain their multipotency. These approaches open the door for carrying out personalized analysis of patient FAPs and interactions with the SCs that lead to muscle loss.

Cardiac CIP protein regulates dystrophic cardiomyopathy

Heart failure is a leading cause of fatality in Duchenne muscular dystrophy (DMD) patients. Previously, we discovered that cardiac and skeletal-muscle-enriched CIP proteins play important roles in cardiac function. Here, we report that CIP, a striated muscle-specific protein, participates in the regulation of dystrophic cardiomyopathy. Using a mouse model of human DMD, we found that deletion of CIP leads to dilated cardiomyopathy and heart failure in young, non-syndromic mdx mice. Conversely, transgenic overexpression of CIP reduces pathological dystrophic cardiomyopathy in old, syndromic mdx mice. Genome-wide transcriptome analyses reveal that molecular pathways involving fibrogenesis and oxidative stress are affected in CIP-mediated dystrophic cardiomyopathy. Mechanistically, we found that CIP interacts with dystrophin and calcineurin (CnA) to suppress the CnA-Nuclear Factor of Activated Tcells (NFAT) pathway, which results in decreased expression of Nox4, a key component of the oxidative stress pathway. Overexpression of Nox4 accelerates the development of dystrophic cardiomyopathy in mdx mice. Our study indicates CIP is a modifier of dystrophic cardiomyopathy and a potential therapeutic target for this devastating disease.

Validation of Chemokine Biomarkers in Duchenne Muscular Dystrophy.

Duchenne muscular dystrophy (DMD) is a progressive muscle disease involving complex skeletal muscle pathogenesis. The pathogenesis is triggered by sarcolemma instability due to the lack of dystrophin protein expression, leading to Ca2+ influx, muscle fiber apoptosis, inflammation, muscle necrosis, and fibrosis. Our lab recently used two high-throughput multiplexing techniques (e.g., SomaScan® aptamer assay and tandem mass tag-(TMT) approach) and identified a series of serum protein biomarkers tied to different pathobiochemical pathways. In this study, we focused on validating the circulating levels of three proinflammatory chemokines (CCL2, CXCL10, and CCL18) that are believed to be involved in an early stage of muscle pathogenesis. We used highly specific and reproducible MSD ELISA assays and examined the association of these chemokines with DMD pathogenesis, age, disease severity, and response to glucocorticoid treatment. As expected, we confirmed that these three chemokines were significantly elevated in serum and muscle samples of DMD patients relative to age-matched healthy controls (p-value < 0.05, CCL18 was not significantly altered in muscle samples). These three chemokines were not significantly elevated in Becker muscular dystrophy (BMD) patients, a milder form of dystrophinopathy, when compared in a one-way ANOVA to a control group but remained significantly elevated in the age-matched DMD group (p < 0.05). CCL2 and CCL18 but not CXCL10 declined with age in DMD patients, whereas all three chemokines remained unchanged with age in BMD and controls. Only CCL2 showed significant association with time to climb four steps in the DMD group (r = 0.48, p = 0.038) and neared significant association with patients’ reported outcome in the BMD group (r = 0.39, p = 0.058). Furthermore, CCL2 was found to be elevated in a serum of the mdx mouse model of DMD, relative to wild-type mouse model. This study suggests that CCL2 might be a suitable candidate biomarker for follow-up studies to demonstrate its physiological significance and clinical utility in DMD.