Dystrophic Heart Research Articles

Nestin Expression in End-Stage Disease in Dystrophin-Deficient Heart: Implications for Regeneration From Endogenous Cardiac Stem Cells

Nestin(+) cardiac stem cells differentiate into striated cells following myocardial infarct. Transplantation of exogenous stem cells into myocardium of a murine model for Duchenne muscular dystrophy (DMD) increased proliferation of endogenous nestin(+) stem cells and resulted in the appearance of nestin(+) striated cells. This correlated with, and may be responsible for, prevention of dilated cardiomyopathy. We examined nestin(+) stem cells in the myocardium of dystrophin/utrophin-deficient (mdx/utrn(-/-)) mice, a model for DMD. We found that 92% of nestin(+) interstitial cells expressed Flk-1, a marker present on cardiac progenitor cells that differentiate into the cardiac lineage, and that a subset expressed Sca-1, present on adult cardiac cells that become cardiomyocytes. Nestin(+) interstitial cells maintained expression of Flk-1 but lost Sca-1 expression with age and were present in lower numbers in dystrophin-deficient heart than in wild-type heart. Unexpectedly, large clusters of nestin(+) striated cells ranging in size from 20 to 250 cells and extending up to 500 μm were present in mdx/utrn(-/-) heart near the end stage of disease. These cells were also present in dystrophin-deficient mdx/utrn(+/-) and mdx heart but not wild-type heart. Nestin(+) striated cells expressed cardiac troponin I, desmin, and Connexin 43 and correlated with proinflammatory CD68(+) macrophages. Elongated nestin(+) interstitial cells with striations were observed that did not express Flk-1 or the late cardiac marker cardiac troponin I but strongly expressed the early cardiac marker desmin. Nestin was also detected in endothelial and smooth muscle cells. These data indicate that new cardiomyocytes form in dystrophic heart, and nestin(+) interstitial cells may generate them in addition to other cells of the cardiac lineage.

Long-term miR-669a therapy alleviates chronic dilated cardiomyopathy in dystrophic mice.

BackgroundDilated cardiomyopathy (DCM) is a leading cause of chronic morbidity and mortality in muscular dystrophy (MD) patients. Current pharmacological treatments are not yet able to counteract chronic myocardial wastage, thus novel therapies are being intensely explored. MicroRNAs have been implicated as fine regulators of cardiomyopathic progression. Previously, miR‐669a downregulation has been linked to the severe DCM progression displayed by Sgcb‐null dystrophic mice. However, the impact of long‐term overexpression of miR‐669a on muscle structure and functionality of the dystrophic heart is yet unknown.Methods and ResultsHere, we demonstrate that intraventricular delivery of adeno‐associated viral (AAV) vectors induces long‐term (18 months) miR‐669a overexpression and improves survival of Sgcb‐null mice. Treated hearts display significant decrease in hypertrophic remodeling, fibrosis, and cardiomyocyte apoptosis. Moreover, miR‐669a treatment increases sarcomere organization, reduces ventricular atrial natriuretic peptide (ANP) levels, and ameliorates gene/miRNA profile of DCM markers. Furthermore, long‐term miR‐669a overexpression significantly reduces adverse remodeling and enhances systolic fractional shortening of the left ventricle in treated dystrophic mice, without significant detrimental consequences on skeletal muscle wastage.ConclusionsOur findings provide the first evidence of long‐term beneficial impact of AAV‐mediated miRNA therapy in a transgenic model of severe, chronic MD‐associated DCM.

Long-term wheel running compromises diaphragm function but improves cardiac and plantarflexor function in the mdx mouse

Dystrophin-deficient muscles suffer from free radical injury, mitochondrial dysfunction, apoptosis, and inflammation, among other pathologies that contribute to muscle fiber injury and loss, leading to wheelchair confinement and death in the patient. For some time, it has been appreciated that endurance training has the potential to counter many of these contributing factors. Correspondingly, numerous investigations have shown improvements in limb muscle function following endurance training in mdx mice. However, the effect of long-term volitional wheel running on diaphragm and cardiac function is largely unknown. Our purpose was to determine the extent to which long-term endurance exercise affected dystrophic limb, diaphragm, and cardiac function. Diaphragm specific tension was reduced by 60% (P < 0.05) in mice that performed 1 yr of volitional wheel running compared with sedentary mdx mice. Dorsiflexor mass (extensor digitorum longus and tibialis anterior) and function (extensor digitorum longus) were not altered by endurance training. In mice that performed 1 yr of volitional wheel running, plantarflexor mass (soleus and gastrocnemius) was increased and soleus tetanic force was increased 36%, while specific tension was similar in wheel-running and sedentary groups. Cardiac mass was increased 15%, left ventricle chamber size was increased 20% (diastole) and 18% (systole), and stroke volume was increased twofold in wheel-running compared with sedentary mdx mice. These data suggest that the dystrophic heart may undergo positive exercise-induced remodeling and that limb muscle function is largely unaffected. Most importantly, however, as the diaphragm most closely recapitulates the human disease, these data raise the possibility of exercise-mediated injury in dystrophic skeletal muscle.

Long‐term wheel running improves cardiac function but has negative consequences for diaphragmatic function in the mdx mouse

Dystrophin‐deficient muscles suffer from free radical injury, mitochondrial dysfunction, apoptosis, and inflammation, among other pathologies, which contribute to muscle fiber injury. Endurance exercise has the capacity to counter many of these factors. Indeed, previous work indicates that endurance training decreased disease severity in dystrophic limb muscle. Our purpose was to determine the extent to which long‐term volitional wheel running (VWR) would affect dystrophic diaphragm and cardiac function. This is especially important as the diaphragm most closely recapitulates the human disease. One year of VWR increased cardiac mass by 15% (p&lt;0.05), left ventricle chamber size by 20% (diastole) and 18% (systole) (p&lt;0.05), and stroke volume by 2‐fold (p&lt;0.05) compared to sedentary mdx mice. VWR increased soleus mass by 20% (p&lt;0.05) and tetanic force by 36% (p&lt;0.05) while specific tension was similar between VWR and sedentary mdx mice. Notably, diaphragm specific tension was reduced by 60% (p&lt;0.05) following one year of VWR compared to sedentary mdx mice. These data suggest that the dystrophic heart undergoes beneficial exercise‐induced remodeling. Importantly, however, the severe negative impact on diaphragm function indicates that serious caution should be taken when prescribing exercise to DMD patients. Supported by NIH grants F32AR055005–01 (JTS) and U54AR052646–03 (HLS).

Dystrophic mdx mice develop severe cardiac and respiratory dysfunction following genetic ablation of the anti-inflammatory cytokine IL-10

Duchenne muscular dystrophy (DMD) is a progressive muscle-wasting disease that causes respiratory and cardiac failure. Inflammation is a key pathological characteristic of dystrophic muscle lesion formation, but its role and regulation in the disease time course has not been sufficiently examined. In the present study, we used IL-10(-/-)/mdx mice lacking both dystrophin and the anti-inflammatory cytokine, interleukin-10 (IL-10), to investigate whether a predisposition to inflammation affects the severity of DMD with advancing age. The IL-10 deficiency caused a profound DMD phenotype in the dystrophic heart such as muscle degeneration and extensive myofiber loss, but the limb muscle and diaphragm morphology of IL-10(-/) (-)/mdx mice was similar to that of mdx mice. Extensive infiltrates of pro-inflammatory M1 macrophages in regeneration of cardiotoxin-injured muscle, altered M1/M2 macrophage phenotype and increased pro-inflammatory cytokines/chemokines production were observed in the diaphragm and heart of IL-10(-/-)/mdx mice. We characterized the IL-10(-/-)/mdx mice as a dystrophic model with chronic inflammation and severe cardiorespiratory dysfunction, as evidenced by decreased percent fractional shortening (%FS) and ejection fraction percent (EF%) on echocardiography, reduced lower tidal volume on whole-body plethysmography. This study suggests that a predisposition to inflammation is an important indicator of DMD disease progression. Therefore, the development of anti-inflammatory strategies may help in slowing down the cardiorespiratory dysfunction on DMD.

Exclusive skeletal muscle correction does not modulate dystrophic heart disease in the aged mdx model of Duchenne cardiomyopathy

Duchenne muscular dystrophy (DMD) is characterized by severe degeneration and necrosis of both skeletal and cardiac muscle. While many experimental therapies have shown great promise in treating skeletal muscle disease, an effective therapy for Duchenne cardiomyopathy remains a challenge in large animal models and human patients. The current views on cardiac consequences of skeletal muscle-centered therapy are controversial. Studies performed in young adult mdx mice (a mild DMD mouse model) have yielded opposing results. Since mdx mice do not develop dystrophic cardiomyopathy until ≥21 months of age, we reasoned that old mdx mice may represent a better model to assess the impact of skeletal muscle rescue on dystrophic heart disease. Here, we aged skeletal muscle-specific micro-dystrophin transgenic mdx mice to 23 months and examined the cardiac phenotype. As expected, transgenic mdx mice had minimal skeletal muscle disease and they also outperformed original mdx mice on treadmill running. On cardiac examination, the dystrophin-null heart of transgenic mdx mice displayed severe cardiomyopathy matching that of non-transgenic mdx mice. Specifically, both the strains showed similar heart fibrosis and cardiac function deterioration in systole and diastole. Cardiac output and ejection fraction were also equally compromised. Our results suggest that skeletal muscle rescue neither aggravates nor alleviates cardiomyopathy in aged mdx mice. These findings underscore the importance of treating both skeletal and cardiac muscles in DMD therapy.

Resveratrol Improves Cardiomyopathy in Dystrophin-deficient Mice through SIRT1 Protein-mediated Modulation of p300 Protein*[S

Cardiomyopathy is the main cause of death in Duchenne muscular dystrophy. Here, we show that oral administration of resveratrol, which leads to activation of an NAD(+)-dependent protein deacetylase SIRT1, suppresses cardiac hypertrophy and fibrosis and restores cardiac diastolic function in dystrophin-deficient mdx mice. The pro-hypertrophic co-activator p300 protein but not p300 mRNA was up-regulated in the mdx heart, and resveratrol administration down-regulated the p300 protein level. In cultured cardiomyocytes, cardiomyocyte hypertrophy induced by the α(1)-agonist phenylephrine was inhibited by the overexpression of SIRT1 as well as resveratrol, both of which down-regulated p300 protein levels but not p300 mRNA levels. In addition, activation of atrial natriuretic peptide promoter by p300 was inhibited by SIRT1. We found that SIRT1 induced p300 down-regulation via the ubiquitin-proteasome pathway by deacetylation of lysine residues for ubiquitination. These findings indicate the pathological significance of p300 up-regulation in the dystrophic heart and indicate that SIRT1 activation has therapeutic potential for dystrophic cardiomyopathy.

Progress in gene therapy of dystrophic heart disease

The heart is frequently afflicted in muscular dystrophy. In severe cases, cardiac lesion may directly result in death. Over the years, pharmacological and/or surgical interventions have been the mainstay to alleviate cardiac symptoms in muscular dystrophy patients. Although these traditional modalities remain useful, the emerging field of gene therapy has now provided an unprecedented opportunity to transform our thinking/approach in the treatment of dystrophic heart disease. In fact, the premise is already in place for genetic correction. Gene mutations have been identified and animal models are available for several types of muscular dystrophy. Most importantly, innovative strategies have been developed to effectively deliver therapeutic genes to the heart. Dystrophin-deficient Duchenne cardiomyopathy is associated with Duchenne muscular dystrophy (DMD), the most common lethal muscular dystrophy. Considering its high incidence, there has been a considerable interest and significant input in the development of Duchenne cardiomyopathy gene therapy. Using Duchenne cardiomyopathy as an example, here we illustrate the struggles and successes experienced in the burgeoning field of dystrophic heart disease gene therapy. In light of abundant and highly promising data with the adeno-associated virus (AAV) vector, we have specially emphasized on AAV-mediated gene therapy. Besides DMD, we have also discussed gene therapy for treating cardiac diseases in other muscular dystrophies such as limb-girdle muscular dystrophy.

Intracellular Sodium Overload Contributes to Deterioration of Function in Dystrophic Heart

[Na+]i is found to be elevated in several models of heart failure. This is believed to favor Ca2+ extrusion from mitochondria via the mNCX and therefore reduce mitochondrial Ca2+ accumulation. As several dehydrogenases of the mitochondrial Krebs cycle are stimulated by Ca2+, the defects in cellular sodium homeostasis may induce mitochondrial oxidative stress. Previously we have estimated that resting [Na+]i is significantly elevated in ventricular myocytes isolated from dystrophic (mdx) mice compared to those from wild-type (WT) mice (24.2 ± 3.1 mM, n=13 vs. 14.0 ± 1.7 mM, n=9). Moreover, the mitochondrial matrix was significantly oxidized at resting conditions (35±3.1 %, n=27 vs. 53±5.2 %, of NADH reduction n=10, in mdx and WT respectively). Here maximal oxidation of NADH by FCCP/oligomycin was taken as 0% reduced NADH and maximal reduction by rotenone and β-hydroxy-butyrate was defined as 100% reduced NADH. We suggested that beat-to-beat mechanical activity in dystrophic heart might lead to greater [Na+]i accumulation and oxidation of the mitochondrial matrix. Here we investigated whether elevated [Na+]i affects mitochondrial energetics during excitation-contraction coupling in dystrophic myocytes. We measured NADH autofluorescence in WT and mdx cells under control conditions and during 4Hz stimulation in the presence of isoproterenol. An abrupt increase in workload resulted in significant oxidation of NADH both in mdx and WT cardiomyocytes. However, the extent of oxidation was significantly greater in dystrophic cells (50±9 %, n=10 vs 15±6 %, n=11 decrease in NADH, respectively). When workload was discontinued, the mitochondrial matrix quickly returned back to its base line levels in WT but not in mdx cells. These results support the notion that the elevated [Na+]i contributes to the impaired energetics in dystrophic cardiomyocytes and therefore contributes to the development of dystrophic cardiomyopathy.

Connexin43 and the regulation of intercalated disc function

Gap junctions mediate the passage of ions and small molecules between cells. In the adult working cardiac ventricles, gap junctions are formed predominantly by the oligomerization of the 43-kDa protein Connexin43 (Cx43). It is generally accepted that gap junction–mediated intercellular communication is modulated by changes in the intracellular environment. The activity of various kinases, the concentration of protons or calcium, and the association of Cx43 with other intracellular components all converge to determine the filtering capabilities of intercellular channels. The most studied posttranslational modification of Cx43 is the phosphorylation of amino acids in its C-terminal domain (see, eg, References 1 Marquez-Rosado L. Solan J.L. Dunn C.A. Norris R.P. Lampe P.D. Connexin43 phosphorylation in brain, cardiac, endothelial and epithelial tissues. Biochim Biophys Acta. 2011; PubMed Google Scholar and 2 Remo B.F. Qu J. Volpicelli F.M. et al. Phosphatase-resistant gap junctions inhibit pathological remodeling and prevent arrhythmias. Circ Res. 2011; 108: 1459-1466 Crossref PubMed Scopus (85) Google Scholar ). Additional studies have shown that Cx43 is also a substrate for Nε-lysine acetylation. 3 Colussi C. Rosati J. Straino S. et al. Nε-lysine acetylation determines dissociation from GAP junctions and lateralization of connexin 43 in normal and dystrophic heart. Proc Natl Acad Sci U S A. 2011; 108: 2795-2800 Crossref PubMed Scopus (79) Google Scholar Changes in the ionic intracellular milieu can also, directly or indirectly, regulate the conductive state of Cx43. These modulatory mechanisms are likely to be activated in various pathophysiological states. 2 Remo B.F. Qu J. Volpicelli F.M. et al. Phosphatase-resistant gap junctions inhibit pathological remodeling and prevent arrhythmias. Circ Res. 2011; 108: 1459-1466 Crossref PubMed Scopus (85) Google Scholar Given the key role of gap junctions in action potential propagation, Cx43 regulation is a subject of intense research, and it is seen as an important pharmacological target for the treatment and/or prevention of cardiac arrhythmias. 3 Colussi C. Rosati J. Straino S. et al. Nε-lysine acetylation determines dissociation from GAP junctions and lateralization of connexin 43 in normal and dystrophic heart. Proc Natl Acad Sci U S A. 2011; 108: 2795-2800 Crossref PubMed Scopus (79) Google Scholar , 4 Qu J. Volpicelli F.M. Garcia L.I. et al. Gap junction remodeling and spironolactone-dependent reverse remodeling in the hypertrophied heart. Circ Res. 2009; 104: 365-371 Crossref PubMed Scopus (79) Google Scholar , 5 Verma V. Larsen B.D. Coombs W. et al. Novel pharmacophores of connexin43 based on the “RXP” series of Cx43-binding peptides. Circ Res. 2009; 105: 176-184 Crossref PubMed Scopus (30) Google Scholar , 6 O'Quinn M.P. Palatinus J.A. Harris B.S. Hewett K.W. Gourdie R.G. A peptide mimetic of the connexin43 carboxyl terminus reduces gap junction remodeling and induced arrhythmia following ventricular injury. Circ Res. 2011; 108: 704-715 Crossref PubMed Scopus (109) Google Scholar Reply to the Editor—Atrial Fibrillation: An Inflammatory and Autoimmune DisorderHeart RhythmVol. 9Issue 2PreviewMany factors contribute to the pathogenesis of atrial fibrillation, including electrical, structural, neurohumoral, and inflammatory mechanisms.1 Our review indicated that various autoantibodies that may play a role in the development and maintenance of atrial fibrillation have been identified.2 Indeed, mounting evidence demonstrates correlations between diseases with autoimmune mechanisms, including Graves' disease,3 celiac disease,4 and psoriasis,5 and an increased risk of atrial fibrillation. Full-Text PDF

Long-Term Systemic Administration of Unconjugated Morpholino Oligomers for Therapeutic Expression of Dystrophin by Exon Skipping in Skeletal Muscle: Implications for Cardiac Muscle Integrity

Duchenne muscular dystrophy (DMD) is a lethal X-linked inherited disease caused by mutations in the dystrophin gene and consequent lack of dystrophin in the skeletal, cardiac, and smooth musculature and in the nervous system. Patients die during their mid-twenties because of severe muscle loss and life-threatening respiratory and cardiac complications. The splicing modulation approach mediated by antisense oligonucleotides can restore the production of a partially functional quasi-dystrophin in skeletal muscles. We recently showed that a chronic, 12-month treatment with phosphorodiamidate morpholino oligomers efficiently restored dystrophin in widespread skeletal muscles and led to normal locomotor activity indistinguishable from that of dystrophin-expressing C57 mice. However, no detectable dystrophin expression was observed in the hearts of treated mice. In the present study, histological analyses show a more severe cardiac pathology compared with untreated animals in the face of enhanced locomotor behavior. This observation implies that the increase in locomotor activity of treated mdx mice may have a paradoxical detrimental effect on the dystrophic heart. In the context of skeletal muscle-centric therapies for DMD, our data suggest that particular vigilance should be instigated to monitor emergence of accelerated cardiac dysfunction.

Voltage-Gated Ion Channel Dysfunction Precedes Cardiomyopathy Development in the Dystrophic Heart

BackgroundDuchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is associated with severe cardiac complications including cardiomyopathy and cardiac arrhythmias. Recent research suggests that impaired voltage-gated ion channels in dystrophic cardiomyocytes accompany cardiac pathology. It is, however, unknown if the ion channel defects are primary effects of dystrophic gene mutations, or secondary effects of the developing cardiac pathology.Methodology/Principal FindingsTo address this question, we first investigated sodium channel impairments in cardiomyocytes derived from dystrophic neonatal mice prior to cardiomyopahty development, by using the whole cell patch clamp technique. Besides the most common model for DMD, the dystrophin-deficient mdx mouse, we also used mice additionally carrying an utrophin mutation. In neonatal cardiomyocytes, dystrophin-deficiency generated a 25% reduction in sodium current density. In addition, extra utrophin-deficiency significantly altered sodium channel gating parameters. Moreover, also calcium channel inactivation was considerably reduced in dystrophic neonatal cardiomyocytes, suggesting that ion channel abnormalities are universal primary effects of dystrophic gene mutations. To assess developmental changes, we also studied sodium channel impairments in cardiomyocytes derived from dystrophic adult mice, and compared them with the respective abnormalities in dystrophic neonatal cells. Here, we found a much stronger sodium current reduction in adult cardiomyocytes. The described sodium channel impairments slowed the upstroke of the action potential in adult cardiomyocytes, and only in dystrophic adult mice, the QRS interval of the electrocardiogram was prolonged.Conclusions/SignificanceIon channel impairments precede pathology development in the dystrophic heart, and may thus be considered potential cardiomyopathy triggers.

Abnormal Sodium Handling and Mitochondrial Metabolism in Cardiac Dystrophy

Lack of dystrophin in striated muscle results in the activation of several stretch-induced transplasmalemmal ion influx pathways. Recently we demonstrated that mechanical challenges produce substantial inward currents in dystrophic (mdx) but not in WT cardiomyocytes. We also detected a significant increase in cytosolic [Na+] following stretch in mdx cells. We suggested that beat-to-beat mechanical activity of dystrophic heart might lead to accumulation of Na+ inside the cardiomyocytes (Na+ overload). Here we measured resting [Na+]i in mdx and WT cells using the ratiometric Na+ indicator SBFI. The averaged value of [Na+]i was indeed significantly greater in mdx than in WT myocytes (24.2 ± 3.1, n=13 vs. 14.0 ± 1.7 mM, n=9). Na+ overload can have a profound effect on cellular functions. E.g. it can change the reversal potential of NCXsl, reducing its ability to remove Ca2+. This is in agreement with our recent report that the reverse mode of NCXsl contributes to cytosolic Ca2+ overload following mechanical stress, despite little change in the resting potential. Elevated [Na+]i can also eventually affect mitochondrial metabolism as it could enhance Ca2+ extrusion from the mitochondria via NCXmt. Therefore we measured NADH autofluorescence. Maximal oxidation of NADH by FCCP/oligomycin was taken as 0% reduced NADH, whereas maximal reduction by rotenone and β-hydroxy-butyrate was defined as 100% reduced NADH. The averaged values show that mitochondrial matrix is significantly more oxidized in resting mdx myocytes compared with WT cells (35% ±3.1, n=27 vs. 53 ± 5.2 %, NADH reduction n=10). The oxidation of mitochondrial matrix can contribute to the cellular oxidative stress and favor the opening of mPTP and cell death -observations that we previously reported for dystrophic cardiomyocytes. Taken together, our findings suggest that elevated [Na+]i may contribute to the development of dystrophic cardiomyopathy.

N ε -lysine acetylation determines dissociation from GAP junctions and lateralization of connexin 43 in normal and dystrophic heart

Wanting to explore the epigenetic basis of Duchenne cardiomyopathy, we found that global histone acetylase activity was abnormally elevated and the acetylase P300/CBP-associated factor (PCAF) coimmunoprecipitated with connexin 43 (Cx43), which was N(ε)-lysine acetylated and lateralized in mdx heart. This observation was paralleled by Cx43 dissociation from N-cadherin and zonula occludens 1, whereas pp60-c-Src association was unaltered. In vivo treatment of mdx with the pan-histone acetylase inhibitor anacardic acid significantly reduced Cx43 N(ε)-lysine acetylation and restored its association to GAP junctions (GJs) at intercalated discs. Noteworthy, in normal as well as mdx mice, the class IIa histone deacetylases 4 and 5 constitutively colocalized with Cx43 either at GJs or in the lateralized compartments. The class I histone deacetylase 3 was also part of the complex. Treatment of normal controls with the histone deacetylase pan-inhibitor suberoylanilide hydroxamic acid (MC1568) or the class IIa-selective inhibitor 3-{4-[3-(3-fluorophenyl)-3-oxo-1-propen-1-yl]-1-methyl-1H-pyrrol-2-yl}-N-hydroxy-2-propenamide (MC1568) determined Cx43 hyperacetylation, dissociation from GJs, and distribution along the long axis of ventricular cardiomyocytes. Consistently, the histone acetylase activator pentadecylidenemalonate 1b (SPV106) hyperacetylated cardiac proteins, including Cx43, which assumed a lateralized position that partly reproduced the dystrophic phenotype. In the presence of suberoylanilide hydroxamic acid, cell to cell permeability was significantly diminished, which is in agreement with a Cx43 close conformation in the consequence of hyperacetylation. Additional experiments, performed with Cx43 acetylation mutants, revealed, for the acetylated form of the molecule, a significant reduction in plasma membrane localization and a tendency to nuclear accumulation. These results suggest that Cx43 N(ε)-lysine acetylation may have physiopathological consequences for cell to cell coupling and cardiac function.

Increased connective tissue growth factor associated with cardiac fibrosis in the mdx mouse model of dystrophic cardiomyopathy

Cardiomyopathy contributes to morbidity and mortality in Duchenne muscular dystrophy (DMD), a progressive muscle-wasting disorder. A major feature of the hearts of DMD patients and the mdx mouse model of the disease is cardiac fibrosis. Connective tissue growth factor (CTGF) is involved in the fibrotic process in many organs. This study utilized the mdx mouse model to assess the role of CTGF and other extracellular matrix components during the development of fibrosis in the dystrophic heart. Left ventricular function of mdx and control mice at 6, 29 and 43 weeks was measured by echocardiography. Young (6 weeks old) mdx hearts had normal function and histology. At 29 weeks of age, mdx mice developed cardiac fibrosis and increased collagen expression. The onset of fibrosis was associated with increased CTGF transcript and protein expression. Increased intensity of CTGF immunostaining was localized to fibrotic areas in mdx hearts. The upregulation of CTGF was also concurrent with increased expression of tissue inhibitor of matrix metalloproteinases (TIMP-1). These changes persisted in 43 week old mdx hearts and were combined with impaired cardiac function and increased gene expression of transforming growth factor (TGF)-β1 and matrix metalloproteinases (MMP-2, MMP-9). In summary, an association was observed between cardiac fibrosis and increased CTGF expression in the mdx mouse heart. CTGF may be a key mediator of early and persistent fibrosis in dystrophic cardiomyopathy.

Altered sodium channel function in dystrophin/utrophin-deficient cardiomyocytes

Background Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is an inherited disease characterized by progressive muscle weakness and degeneration. Besides the relatively well-described skeletal muscle degenerative processes, DMD and some other muscular dystrophy types are also associated with cardiovascular complications including cardiomyopathy and cardiac arrhythmias. The current understanding of the patho-mechanisms underlying these cardiovascular complications is still very limited, but recent research points to a contribution of dysfunctional ion channels in dystrophic cardiomyocytes. Materials and methods By using the whole cell patch-clamp technique, the functional properties of voltage-gated sodium channels were studied in cardiomyocytes derived from normal and dystrophic mice. In addition, a computer model was used to simulate the effects of altered sodium channel properties on the cardiac action potential. Besides the most common mouse model for human DMD, the dystrophin-deficient mdx mouse, we also used mice additionally carrying a mutation in the utrophin gene. The mdx-utr double mutant mouse exhibits a more severe cardiac disease phenotype than the mdx mouse, and is thought to represent a more suitable animal model for human DMD. Results We found that dystrophic cardiomyocytes show a reduced sodium current density compared to wild-type cardiomyocytes. In addition, extra utrophin deficiency significantly shifted both the sodium channel activation and inactivation curve to more depolarised potentials, which was not observed in only dystrophin-deficient mdx cardiomyocytes. Computer modelling revealed that the described sodium channel impairments in dystrophic cardiomyocytes suffice to affect the action potential. Conclusions Sodium channel dysfunction may perturb electrical impulse propagation in the dystrophic heart, and thus contribute to cardiac complications associated with the muscular dystrophies.

Arginine Metabolism by Macrophages Promotes Cardiac and Muscle Fibrosis in mdx Muscular Dystrophy

BackgroundDuchenne muscular dystrophy (DMD) is the most common, lethal disease of childhood. One of 3500 new-born males suffers from this universally-lethal disease. Other than the use of corticosteroids, little is available to affect the relentless progress of the disease, leading many families to use dietary supplements in hopes of reducing the progression or severity of muscle wasting. Arginine is commonly used as a dietary supplement and its use has been reported to have beneficial effects following short-term administration to mdx mice, a genetic model of DMD. However, the long-term effects of arginine supplementation are unknown. This lack of knowledge about the long-term effects of increased arginine metabolism is important because elevated arginine metabolism can increase tissue fibrosis, and increased fibrosis of skeletal muscles and the heart is an important and potentially life-threatening feature of DMD.MethodologyWe use both genetic and nutritional manipulations to test whether changes in arginase metabolism promote fibrosis and increase pathology in mdx mice. Our findings show that fibrotic lesions in mdx muscle are enriched with arginase-2-expressing macrophages and that muscle macrophages stimulated with cytokines that activate the M2 phenotype show elevated arginase activity and expression. We generated a line of arginase-2-null mutant mdx mice and found that the mutation reduced fibrosis in muscles of 18-month-old mdx mice, and reduced kyphosis that is attributable to muscle fibrosis. We also observed that dietary supplementation with arginine for 17-months increased mdx muscle fibrosis. In contrast, arginine-2 mutation did not reduce cardiac fibrosis or affect cardiac function assessed by echocardiography, although 17-months of dietary supplementation with arginine increased cardiac fibrosis. Long-term arginine treatments did not decrease matrix metalloproteinase-2 or -9 or increase the expression of utrophin, which have been reported as beneficial effects of short-term treatments.Conclusions/SignificanceOur findings demonstrate that arginine metabolism by arginase promotes fibrosis of muscle in muscular dystrophy and contributes to kyphosis. Our findings also show that long-term, dietary supplementation with arginine exacerbates fibrosis of dystrophic heart and muscles. Thus, commonly-practiced dietary supplementation with arginine by DMD patients has potential risk for increasing pathology when performed for long periods, despite reports of benefits acquired with short-term supplementation.

The histone deacetylase inhibitor suberoylanilide hydroxamic acid reduces cardiac arrhythmias in dystrophic mice

The effect of histone deacetylase inhibitors on dystrophic heart function is not established. To investigate this aspect, dystrophic mdx mice and wild-type (WT) animals were treated 90 days either with suberoylanilide hydroxamic acid (SAHA, 5 mg/kg/day) or with an equivalent amount of vehicle. The following parameters were evaluated: (i) number of ventricular arrhythmias in resting and stress conditions (restraint test) or after aconitine administration; (ii) cardiac excitability, conduction velocity, and refractoriness; (iii) expression and distribution of connexins (Cxs) and Na(v)1.5 sodium channel. Ventricular arrhythmias were negligible in all resting animals. During restraint, however, an increase in the number of arrhythmias was detected in vehicle-treated mdx mice (mdx-V) when compared with SAHA-treated mdx (mdx-SAHA) mice or normal control (WT-V). Interestingly, aconitine, a sodium channel pharmacologic opener, induced ventricular arrhythmias in 83% of WT-V mice, 11% of mdx-V, and in 57% of mdx-SAHA. Epicardial multiple lead recording revealed a prolongation of the QRS complex in mdx-V mice in comparison to WT-V and WT-SAHA mice, paralleled by a significant reduction in impulse propagation velocity. These alterations were efficiently counteracted by SAHA. Molecular analyses revealed that in mdx mice, SAHA determined Cx remodelling of Cx40, Cx37 and Cx32, whereas expression levels of Cx43 and Cx45 were unaltered. Remarkably, Cx43 lateralization observed in mdx control animals was reversed by SAHA treatment which also re-induced Na(v)1.5 expression. SAHA attenuates arrhythmias in mdx mice by a mechanism in which Cx remodelling and sodium channel re-expression could play an important role.

Proteomic Profiling of the Dystrophin-Deficient MDX Heart Reveals Drastically Altered Levels of Key Metabolic and Contractile Proteins

Although Duchenne muscular dystrophy is primarily classified as a neuromuscular disease, cardiac complications play an important role in the course of this X-linked inherited disorder. The pathobiochemical steps causing a progressive decline in the dystrophic heart are not well understood. We therefore carried out a fluorescence difference in-gel electrophoretic analysis of 9-month-old dystrophin-deficient versus age-matched normal heart, using the established MDX mouse model of muscular dystrophy-related cardiomyopathy. Out of 2,509 detectable protein spots, 79 2D-spots showed a drastic differential expression pattern, with the concentration of 3 proteins being increased, including nucleoside diphosphate kinase and lamin-A/C, and of 26 protein species being decreased, including ATP synthase, fatty acid binding-protein, isocitrate dehydrogenase, NADH dehydrogenase, porin, peroxiredoxin, adenylate kinase, tropomyosin, actin, and myosin light chains. Hence, the lack of cardiac dystrophin appears to trigger a generally perturbed protein expression pattern in the MDX heart, affecting especially energy metabolism and contractile proteins.

Emergent Dilated Cardiomyopathy Caused by Targeted Repair of Dystrophic Skeletal Muscle

Duchenne muscular dystrophy (DMD) is a fatal disease characterized by deterioration of striated muscle, affecting skeletal and cardiac muscles. Recently, several therapeutic approaches have shown promise for repairing dystrophic skeletal muscles. However, these methods often leave the dystrophic heart untreated. Here we show that, in comparison to fully dystrophin-deficient animals, targeted transgenic repair of skeletal muscle, but not cardiac muscle, in otherwise dystrophin-deficient (mdx) mice paradoxically elicited a fivefold increase in cardiac injury and dilated cardiomyopathy in these animals in vivo. Skeletal muscle repair was shown to increase the voluntary activity of the mdx mice as quantified by voluntary running on the exercise wheel. Because the dystrophin-deficient heart is highly sensitive to increased stress, we hypothesize that increased activity (enabled by the repaired skeletal muscle) provided the stimulus for heightened cardiac injury and heart remodeling. In support of this hypothesis, the primary cellular compliance defect in dystrophin-deficient cardiac myocytes was found to be unchanged by skeletal muscle repair in the mdx mice. These findings provide new information on the evolution of cardiac disease in dystrophin-deficient animals and underscore the importance of implementing global striated muscle therapies for muscular dystrophy.