Thrombospondin-1 promotes fibro-adipogenic stromal expansion and contractile dysfunction of the diaphragm in obesity.

Pulmonary disorders impact 40% to 80% of individuals with obesity. Respiratory muscle dysfunction is linked to these conditions; however, its pathophysiology remains largely undefined. Mice subjected to diet-induced obesity (DIO) develop diaphragmatic weakness. Increased intra-diaphragmatic adiposity and extracellular matrix (ECM) content correlate with reductions in contractile force. Thrombospondin-1 (THBS1) is an obesity-associated matricellular protein linked with muscular damage in genetic myopathies. THBS1 induces proliferation of fibro-adipogenic progenitors (FAPs) - mesenchymal cells that differentiate into adipocytes and fibroblasts. We hypothesized that THBS1 drives FAP-mediated diaphragm remodeling and contractile dysfunction in DIO. We tested this by comparing the effects of dietary challenge on diaphragms of wild-type (WT) and Thbs1 knockout (Thbs1-/-) mice. Bulk and single-cell transcriptomics demonstrated DIO-induced stromal expansion in WT diaphragms. Diaphragm FAPs displayed upregulation of ECM and TGF ß-related expression signatures and augmentation of a Thy1-expressing sub-population previously linked to type 2 diabetes. Despite similar weight gain, Thbs1-/- mice were protected from these transcriptomic changes and from obesity-induced increases in diaphragm adiposity and ECM deposition. Unlike WT controls, Thbs1-/- diaphragms maintained normal contractile force and motion after DIO challenge. These findings establish THBS1 as a necessary mediator of diaphragm stromal remodeling and contractile dysfunction in overnutrition and a potential therapeutic target in obesity-associated respiratory dysfunction.

FICD deficiency protects mice from hypertrophy-induced heart failure via BiP-mediated activation of the UPR ER and ER-phagy.

Cardiomyocytes require the HSP70 chaperone BiP to maintain proteostasis in the endoplasmic reticulum (ER) following cardiac stress. The adenylyl transferase (AMPylase) FICD is increasingly recognized to regulate BiP activity through the post-translational addition of an adenosine monophosphate moiety to BiP surface residues. However, the physiological impact of FICD-mediated BiP regulation in the context of cardiovascular health is unknown. Here, we find that FICD deficiency prevents pressure overload-associated heart failure, hypertrophy, and fibrosis, and that FICD knockout mice maintain normal cardiac function after cardiac pressure overload. At a cellular level, we observe that FICD-mediated BiP AMPylation blunts the induction of the unfolded protein response (UPR ER ) and impairs BiP interaction with FAM134B, an ER-phagy receptor, thus limiting ER-phagy induction under stress. In contrast, FICD loss significantly increases BiP-dependent UPR ER induction and ER-phagy in stressed cardiomyocytes. We also uncover cell type-specific consequences of FICD activity in response to ER stress, positioning FICD as a critical proteostasis regulator in cardiac tissue. Our results highlight a novel regulatory paradigm controlling stress resilience in cardiomyocytes and offer a rationale to consider FICD as a therapeutic target to treat cardiac hypertrophy.

Thrombospondin-1 promotes fibro-adipogenic stromal expansion and contractile dysfunction of the diaphragm in obesity.

Pulmonary disorders impact 40-80% of individuals with obesity. Respiratory muscle dysfunction is linked to these conditions; however, its pathophysiology remains largely undefined. Mice subjected to diet-induced obesity (DIO) develop diaphragmatic weakness. Increased intra-diaphragmatic adiposity and extracellular matrix (ECM) content correlate with reductions in contractile force. Thrombospondin-1 (THBS1) is an obesity-associated matricellular protein linked with muscular damage in genetic myopathies. THBS1 induces proliferation of fibro-adipogenic progenitors (FAPs)-mesenchymal cells that differentiate into adipocytes and fibroblasts. We hypothesized that THBS1 drives FAP-mediated diaphragm remodeling and contractile dysfunction in DIO. We tested this by comparing effects of dietary challenge on diaphragms of wild-type (WT) and Thbs1 knockout ( Thbs1 -/- ) mice. Bulk and single-cell transcriptomics demonstrated DIO-induced stromal expansion in WT diaphragms. Diaphragm FAPs displayed upregulation of ECM and TGFß-related expression signatures, and augmentation of a Thy1 -expressing sub-population previously linked to type 2 diabetes. Despite similar weight gain, Thbs1 -/- mice were protected from these transcriptomic changes, and from obesity-induced increases in diaphragm adiposity and ECM deposition. Unlike WT controls, Thbs1 -/- diaphragms maintained normal contractile force and motion after DIO challenge. These findings establish THBS1 as a necessary mediator of diaphragm stromal remodeling and contractile dysfunction in overnutrition, and potential therapeutic target in obesity-associated respiratory dysfunction.

Bioenergetic and Metabolic Impairments in Induced Pluripotent Stem Cell-Derived Cardiomyocytes Generated from Duchenne Muscular Dystrophy Patients.

Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene and dilated cardiomyopathy (DCM) is a major cause of morbidity and mortality in DMD patients. We tested the hypothesis that DCM is caused by metabolic impairments by employing induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) generated from four DMD patients; an adult male, an adult female, a 7-year-old (7y) male and a 13-year-old (13y) male, all compared to two healthy volunteers. To test the hypothesis, we measured the bioenergetics, metabolomics, electrophysiology, mitochondrial morphology and mitochondrial activity of CMs, using respirometry, LC-MS, patch clamp, electron microscopy (EM) and confocal microscopy methods. We found that: (1) adult DMD CMs exhibited impaired energy metabolism and abnormal mitochondrial structure and function. (2) The 7y CMs demonstrated arrhythmia-free spontaneous firing along with "healthy-like" metabolic status, normal mitochondrial morphology and activity. In contrast, the 13y CMs were mildly arrhythmogenic and showed adult DMD-like bioenergetics deficiencies. (3) In DMD adult CMs, mitochondrial activities were attenuated by 45-48%, whereas the 7y CM activity was similar to that of healthy CMs. (4) In DMD CMs, but not in 7y CMs, there was a 75% decrease in the mitochondrial ATP production rate compared to healthy iPSC-CMs. In summary, DMD iPSC-CMs exhibit bioenergetic and metabolic impairments that are associated with rhythm disturbances corresponding to the patient's phenotype, thereby constituting novel targets for alleviating cardiomyopathy in DMD patients.

Mechanisms of skeletal muscle repair and regeneration in health and disease.

Skeletal muscle is a structurally and functionally remarkable tissue composed of multinucleated post-mitotic muscle fibres. These fibres are filled with an exquisite, near crystalline array of assembled contractile proteins, capable of coupling ATP utilization to mechanical muscle contraction. Fully differentiated muscle has an incredible ability to protect and repair itself from significant muscle injuries. In fact, through activation of a resident population of stem cells known as satellite cells, muscle fibres can be completely regenerated, and normal function can be restored in a matter of a few weeks after a major myocellular necrotic injury. The loss of key mechanisms to protect muscle from injuries or loss of the capacity to repair muscle after injury is thought to underlie several forms of muscular dystrophy and also the age-related decline of muscle function. In this Subject Collection, The FEBS Journal highlights articles that review or investigate key mechanisms of muscle repair and regeneration in response to injuries, and the contributions of these pathways to health and disease of skeletal muscle.

SNTA1 gene rescues ion channel function and is antiarrhythmic in cardiomyocytes derived from induced pluripotent stem cells from muscular dystrophy patients.

Background: Patients with cardiomyopathy of Duchenne Muscular Dystrophy (DMD) are at risk of developing life-threatening arrhythmias, but the mechanisms are unknown. We aimed to determine the role of ion channels controlling cardiac excitability in the mechanisms of arrhythmias in DMD patients. Methods: To test whether dystrophin mutations lead to defective cardiac NaV1.5-Kir2.1 channelosomes and arrhythmias, we generated iPSC-CMs from two hemizygous DMD males, a heterozygous female, and two unrelated control males. We conducted studies including confocal microscopy, protein expression analysis, patch-clamping, non-viral piggy-bac gene expression, optical mapping and contractility assays. Results: Two patients had abnormal ECGs with frequent runs of ventricular tachycardia. iPSC-CMs from all DMD patients showed abnormal action potential profiles, slowed conduction velocities, and reduced sodium (INa) and inward rectifier potassium (IK1) currents. Membrane NaV1.5 and Kir2.1 protein levels were reduced in hemizygous DMD iPSC-CMs but not in heterozygous iPSC-CMs. Remarkably, transfecting just one component of the dystrophin protein complex (α1-syntrophin) in hemizygous iPSC-CMs from one patient restored channelosome function, INa and IK1 densities, and action potential profile in single cells. In addition, α1-syntrophin expression restored impulse conduction and contractility and prevented reentrant arrhythmias in hiPSC-CM monolayers. Conclusions: We provide the first demonstration that iPSC-CMs reprogrammed from skin fibroblasts of DMD patients with cardiomyopathy have a dysfunction of the NaV1.5-Kir2.1 channelosome, with consequent reduction of cardiac excitability and conduction. Altogether, iPSC-CMs from patients with DMD cardiomyopathy have a NaV1.5-Kir2.1 channelosome dysfunction, which can be rescued by the scaffolding protein α1-syntrophin to restore excitability and prevent arrhythmias. Funding: Supported by National Institutes of Health R01 HL122352 grant; 'la Caixa' Banking Foundation (HR18-00304); Fundación La Marató TV3: Ayudas a la investigación en enfermedades raras 2020 (LA MARATO-2020); Instituto de Salud Carlos III/FEDER/FSE; Horizon 2020 - Research and Innovation Framework Programme GA-965286 to JJ; the CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the Ministerio de Ciencia e Innovación (MCIN) and the Pro CNIC Foundation), and is a Severo Ochoa Center of Excellence (grant CEX2020-001041-S funded by MICIN/AEI/10.13039/501100011033). American Heart Association postdoctoral fellowship 19POST34380706s to JVEN. Israel Science Foundation to OB and MA [824/19]. Rappaport grant [01012020RI]; and Niedersachsen Foundation [ZN3452] to OB; US-Israel Binational Science Foundation (BSF) to OB and TH [2019039]; Dr. Bernard Lublin Donation to OB; and The Duchenne Parent Project Netherlands (DPPNL 2029771) to OB. National Institutes of Health R01 AR068428 to DM and US-Israel Binational Science Foundation Grant [2013032] to DM and OB.

Sarcolemma wounding activates dynamin-dependent endocytosis in striated muscle.

Plasma membrane repair is an evolutionarily conserved mechanism by which cells can seal breaches in the plasma membrane. Mutations in several proteins with putative roles in sarcolemma integrity, membrane repair, and membrane transport result in several forms of muscle disease; however, the mechanisms that are activated and responsible for sarcolemma resealing are not well understood. Using the standard assays for membrane repair, which track the uptake of FM 1-43 dye into adult skeletal muscle fibers following laser-induced sarcolemma disruption, we show that labeling of resting fibers by FM1-43 prior to membrane wounding and the induced FM1-43 dye uptake after sarcolemma wounding occurs via dynamin-dependent endocytosis. Dysferlin-deficient muscle fibers show elevated dye uptake following wounding, which is the basis for the assertion that membrane repair is defective in this model. Our data show that dynamin inhibition mitigates the differences in FM1-43 dye uptake between dysferlin-null and wild-type muscle fibers, suggesting that elevated wound-induced FM1-43 uptake in dysferlin-deficient muscle may actually be due to enhanced dynamin-dependent endocytosis following wounding, though dynamin inhibition had no effect on dysferlin trafficking after wounding. By monitoring calcium flux after membrane wounding, we show that reversal of calcium precedes the sustained, slower increase of dynamin-dependent FM1-43 uptake in WT fibers, and that dysferlin-deficient muscle fibers have persistently increased calcium after wounding, consistent with its proposed role in resealing. These data highlight a previously unappreciated role for dynamin-dependent endocytosis in wounded skeletal muscle fibers and identify overactive dynamin-dependent endocytosis following sarcolemma wounding as a potential mechanism or consequence of dysferlin deficiency.

Potential role of intermittent functioning of baroreflexes in the etiology of hypertension in spontaneously hypertensive rats.

The spontaneously hypertensive rat (SHR) is a genetic model of primary hypertension with an etiology that includes sympathetic overdrive. To elucidate the neurogenic mechanisms underlying the pathophysiology of this model, we analyzed the dynamic baroreflex response to spontaneous fluctuations in arterial pressure in conscious SHRs, as well as in the Wistar-Kyoto (WKY), the Dahl salt-sensitive, the Dahl salt-resistant, and the Sprague-Dawley rat. Observations revealed the existence of long intermittent periods (lasting up to several minutes) of engagement and disengagement of baroreflex control of heart rate. Analysis of these intermittent periods revealed a predictive relationship between increased mean arterial pressure and progressive baroreflex disengagement that was present in the SHR and WKY strains but absent in others. This relationship yielded the hypothesis that a lower proportion of engagement versus disengagement of the baroreflex in SHR compared with WKY contributes to the hypertension (or increased blood pressure) in SHR compared with WKY. Results of experiments using sinoaortic baroreceptor denervation were consistent with the hypothesis that dysfunction of the baroreflex contributes to the etiology of hypertension in the SHR. Thus, this study provides experimental evidence for the roles of the baroreflex in long-term arterial pressure regulation and in the etiology of primary hypertension in this animal model.

Impaired Myocardial Energetics Causes Mechanical Dysfunction in Decompensated Failing Hearts.

Cardiac mechanical function is supported by ATP hydrolysis, which provides the chemical-free energy to drive the molecular processes underlying cardiac pumping. Physiological rates of myocardial ATP consumption require the heart to resynthesize its entire ATP pool several times per minute. In the failing heart, cardiomyocyte metabolic dysfunction leads to a reduction in the capacity for ATP synthesis and associated free energy to drive cellular processes. Yet it remains unclear if and how metabolic/energetic dysfunction that occurs during heart failure affects mechanical function of the heart. We hypothesize that changes in phosphate metabolite concentrations (ATP, ADP, inorganic phosphate) that are associated with decompensation and failure have direct roles in impeding contractile function of the myocardium in heart failure, contributing to the whole-body phenotype. To test this hypothesis, a transverse aortic constriction (TAC) rat model of pressure overload, hypertrophy, and decompensation was used to assess relationships between metrics of whole-organ pump function and myocardial energetic state. A multiscale computational model of cardiac mechanoenergetic coupling was used to identify and quantify the contribution of metabolic dysfunction to observed mechanical dysfunction. Results show an overall reduction in capacity for oxidative ATP synthesis fueled by either fatty acid or carbohydrate substrates as well as a reduction in total levels of adenine nucleotides and creatine in myocardium from TAC animals compared to sham-operated controls. Changes in phosphate metabolite levels in the TAC rats are correlated with impaired mechanical function, consistent with the overall hypothesis. Furthermore, computational analysis of myocardial metabolism and contractile dynamics predicts that increased levels of inorganic phosphate in TAC compared to control animals kinetically impair the myosin ATPase crossbridge cycle in decompensated hypertrophy/heart failure.

Enhanced dimethylarginine degradation improves coronary flow reserve and exercise tolerance in Duchenne muscular dystrophy carrier mice.

Duchenne muscular dystrophy (DMD) is an X-linked disease caused by null mutations in dystrophin and characterized by muscle degeneration. Cardiomyopathy is common and often prevalent at similar frequency in female DMD carriers irrespective of whether they manifest skeletal muscle disease. Impaired muscle nitric oxide (NO) production in DMD disrupts muscle blood flow regulation and exaggerates postexercise fatigue. We show that circulating levels of endogenous methylated arginines including asymmetric dimethylarginine (ADMA), which act as NO synthase inhibitors, are elevated by acute necrotic muscle damage and in chronically necrotic dystrophin-deficient mice. We therefore hypothesized that excessive ADMA impairs muscle NO production and diminishes exercise tolerance in DMD. We used transgenic expression of dimethylarginine dimethylaminohydrolase 1 (DDAH), which degrades methylated arginines, to investigate their contribution to exercise-induced fatigue in DMD. Although infusion of exogenous ADMA was sufficient to impair exercise performance in wild-type mice, transgenic DDAH expression did not rescue exercise-induced fatigue in dystrophin-deficient male mdx mice. Surprisingly, DDAH transgene expression did attenuate exercise-induced fatigue in dystrophin-heterozygous female mdx carrier mice. Improved exercise tolerance was associated with reduced heart weight and improved cardiac ß-adrenergic responsiveness in DDAH-transgenic mdx carriers. We conclude that DDAH overexpression increases exercise tolerance in female DMD carriers, possibly by limiting cardiac pathology and preserving the heart's responses to changes in physiological demand. Methylated arginine metabolism may be a new target to improve exercise tolerance and cardiac function in DMD carriers or act as an adjuvant to promote NO signaling alongside therapies that partially restore dystrophin expression in patients with DMD.NEW & NOTEWORTHY Duchenne muscular dystrophy (DMD) carriers are at risk for cardiomyopathy. The nitric oxide synthase inhibitor asymmetric dimethylarginine (ADMA) is released from damaged muscle in DMD and impairs exercise performance. Transgenic expression of dimethylarginine dimethylaminohydrolase to degrade ADMA prevents cardiac hypertrophy, improves cardiac function, and improves exercise tolerance in DMD carrier mice. These findings highlight the relevance of ADMA to muscular dystrophy and have important implications for therapies targeting nitric oxide in patients with DMD and DMD carriers.

Continuous glucose monitoring reveals glycemic variability and hypoglycemia after vertical sleeve gastrectomy in rats.

OBJECTIVE: Post-bariatric surgery hypoglycemia (PBH) is defined as the presence of neuroglycopenic symptoms accompanied by postprandial hypoglycemia in bariatric surgery patients. Recent clinical studies using continuous glucose monitoring (CGM) technology revealed that PBH is more frequently observed in vertical sleeve gastrectomy (VSG) patients than previously recognized. PBH cannot be alleviated by current medication. Therefore, a model system to investigate the mechanism and treatment is required. METHODS: We used CGM in a rat model of VSG and monitored the occurrence of glycemic variability and hypoglycemia in various meal conditions for 4 weeks after surgery. Another cohort of VSG rats with CGM was used to investigate whether the blockade of glucagon-like peptide-1 receptor (GLP-1R) signaling alleviates these symptoms. A mouse VSG model was used to investigate whether the impaired glucose counterregulatory system causes postprandial hypoglycemia. RESULTS: Like in humans, rats have increased glycemic variability and hypoglycemia after VSG. Postprandial hypoglycemia was specifically detected after liquid versus solid meals. Further, the blockade of GLP-1R signaling raises the glucose nadir but does not affect glycemic variability. CONCLUSIONS: Rat bariatric surgery duplicates many features of human post-bariatric surgery hypoglycemia including postprandial hypoglycemia and glycemic variability, while blockade of GLP-1R signaling prevents hypoglycemia but not the variability.

A genetic mouse model of severe iron deficiency anemia reveals tissue-specific transcriptional stress responses and cardiac remodeling.

Iron is a micronutrient fundamental for life. Iron homeostasis in mammals requires sustained postnatal intestinal iron absorption that maintains intracellular iron concentrations for central and systemic metabolism as well as for erythropoiesis and oxygen transport. More than 1 billion people worldwide suffer from iron deficiency anemia (IDA), a state of systemic iron insufficiency that limits the production of red blood cells and leads to tissue hypoxia and intracellular iron stress. Despite this tremendous public health concern, very few genetic models of IDA are available to study its progression. Here we developed and characterized a novel genetic mouse model of IDA. We found that tamoxifen-inducible deletion of the mammalian iron exporter ferroportin exclusively in intestinal epithelial cells leads to loss of intestinal iron absorption. Ferroportin ablation yielded a robust phenotype of progressive IDA that develops in as little as 3 months following disruption of intestinal iron absorption. We noted that, at end-stage IDA, tissue-specific transcriptional stress responses occur in which the heart shows little to no hypoxic and iron stress compared with other peripheral organs. However, morphometric and echocardiographic analysis revealed massive cardiac hypertrophy and chamber dilation, albeit with increased cardiac output at very low basal heart rates. We propose that our intestine-specific ferroportin knockout mouse model of end-stage IDA could be used in future studies to investigate IDA progression and cell-specific responses to hypoxic and iron stress.

Electrophysiological abnormalities in induced pluripotent stem cell-derived cardiomyocytes generated from Duchenne muscular dystrophy patients.

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease, caused by mutations in the dystrophin gene and resulting in death because of respiratory or cardiac failure. To investigate the cardiac cellular manifestation of DMD, we generated induced pluripotent stem cells (iPSCs) and iPSC-derived cardiomyocytes (iPSC-CMs) from two DMD patients: a male and female manifesting heterozygous carrier. Dystrophin mRNA and protein expression were analysed by qRT-PCR, RNAseq, Western blot and immunofluorescence staining. For comprehensive electrophysiological analysis, current and voltage clamp were used to record transmembrane action potentials and ion currents, respectively. Microelectrode array was used to record extracellular electrograms. X-inactive specific transcript (XIST) and dystrophin expression analyses revealed that female iPSCs underwent X chromosome reactivation (XCR) or erosion of X chromosome inactivation, which was maintained in female iPSC-CMs displaying mixed X chromosome expression of wild type (WT) and mutated alleles. Both DMD female and male iPSC-CMs presented low spontaneous firing rate, arrhythmias and prolonged action potential duration. DMD female iPSC-CMs displayed increased beat rate variability (BRV). DMD male iPSC-CMs manifested decreased If density, and DMD female and male iPSC-CMs showed increased ICa,L density. Our findings demonstrate cellular mechanisms underlying electrophysiological abnormalities and cardiac arrhythmias in DMD.

Fibro-Adipogenic Remodeling of the Diaphragm in Obesity-Associated Respiratory Dysfunction.

Respiratory dysfunction is a common complication of obesity, conferring cardiovascular morbidity and increased mortality and often necessitating mechanical ventilatory support. While impaired lung expansion in the setting of increased adipose mass and reduced central response to hypercapnia have been implicated as pathophysiological drivers, the impact of obesity on respiratory muscles-in particular, the diaphragm-has not been investigated in detail. Here, we demonstrate that chronic high-fat diet (HFD) feeding impairs diaphragm muscle function, as assessed in vivo by ultrasonography and ex vivo by measurement of contractile force. During an HFD time course, progressive adipose tissue expansion and collagen deposition within the diaphragm parallel contractile deficits. Moreover, intradiaphragmatic fibro-adipogenic progenitors (FAPs) proliferate with long-term HFD feeding while giving rise to adipocytes and type I collagen-depositing fibroblasts. Thrombospondin 1 (THBS1), a circulating adipokine, increases with obesity and induces FAP proliferation. These findings suggest a novel role for FAP-mediated fibro-adipogenic diaphragm remodeling in obesity-associated respiratory dysfunction.

Potential association of LMNA-associated generalized lipodystrophy with juvenile dermatomyositis.

BACKGROUND: Juvenile dermatomyositis (JDM) is an auto-immune muscle disease which presents with skin manifestations and muscle weakness. At least 10% of the patients with JDM present with acquired lipodystrophy. Laminopathies are caused by mutations in the lamin genes and cover a wide spectrum of diseases including muscular dystrophies and lipodystrophy. The p.T10I LMNA variant is associated with a phenotype of generalized lipodystrophy that has also been called atypical progeroid syndrome. CASE PRESENTATION: A previously healthy female presented with bilateral proximal lower extremity muscle weakness at age 4. She was diagnosed with JDM based on her clinical presentation, laboratory tests and magnetic resonance imaging (MRI). She had subcutaneous fat loss which started in her extremities and progressed to her whole body. At age 7, she had diabetes, hypertriglyceridemia, low leptin levels and low body fat on dual energy X-ray absorptiometry (DEXA) scan, and was diagnosed with acquired generalized lipodystrophy (AGL). Whole exome sequencing (WES) revealed a heterozygous c.29C > T; p.T10I missense pathogenic variant in LMNA, which encodes lamins A and C. Muscle biopsy confirmed JDM rather than muscular dystrophy, showing perifascicular atrophy and perivascular mononuclear cell infiltration. Immunofluroscence of skin fibroblasts confirmed nuclear atypia and fragmentation. CONCLUSIONS: This is a unique case with p.T10I LMNA variant displaying concurrent JDM and AGL. This co-occurrence raises the intriguing possibility that LMNA, and possibly p.T10I, may have a pathogenic role in not only the occurrence of generalized lipodystrophy, but also juvenile dermatomyositis. Careful phenotypic characterization of additional patients with laminopathies as well as individuals with JDM is warranted.

Generation of Duchenne muscular dystrophy patient-specific induced pluripotent stem cell line lacking exons 45-50 of the dystrophin gene (IITi001-A).

Duchenne muscular dystrophy (DMD) is an X-linked progressive muscle degenerative disease caused by mutations in the dystrophin gene. We generated induced pluripotent stem cells (iPSCs) from a 13-year-old male patient carrying a deletion mutation of exons 45-50; iPSCs were subsequently differentiated into cardiomyocytes. iPSCs exhibit expression of the pluripotent markers (SOX2, NANOG, OCT4), differentiation capacity into the three germ layers, normal karyotype, genetic identity to the skin biopsy dermal fibroblasts and the patient-specific dystrophin mutation.

Dilated cardiomyopathy mutations in δ-sarcoglycan exert a dominant-negative effect on cardiac myocyte mechanical stability.

Delta-sarcoglycan is a component of the sarcoglycan subcomplex within the dystrophin-glycoprotein complex located at the plasma membrane of muscle cells. While recessive mutations in δ-sarcoglycan cause limb girdle muscular dystrophy 2F, dominant mutations in δ-sarcoglycan have been linked to inherited dilated cardiomyopathy (DCM). The purpose of this study was to investigate functional cellular defects present in adult cardiac myocytes expressing mutant δ-sarcoglycans harboring the dominant inherited DCM mutations R71T or R97Q. This study demonstrates that DCM mutant δ-sarcoglycans can be stably expressed in adult rat cardiac myocytes and traffic similarly to wild-type δ-sarcoglycan to the plasma membrane, without perturbing assembly of the dystrophin-glycoprotein complex. However, expression of DCM mutant δ-sarcoglycan in adult rat cardiac myocytes is sufficient to alter cardiac myocyte plasma membrane stability in the presence of mechanical strain. Upon cyclical cell stretching, cardiac myocytes expressing mutant δ-sarcoglycan R97Q or R71T have increased cell-impermeant dye uptake and undergo contractures at greater frequencies than myocytes expressing normal δ-sarcoglycan. Additionally, the R71T mutation creates an ectopic N-linked glycosylation site that results in aberrant glycosylation of the extracellular domain of δ-sarcoglycan. Therefore, appropriate glycosylation of δ-sarcoglycan may also be necessary for proper δ-sarcoglycan function and overall dystrophin-glycoprotein complex function. These studies demonstrate that DCM mutations in δ-sarcoglycan can exert a dominant negative effect on dystrophin-glycoprotein complex function leading to myocardial mechanical instability that may underlie the pathogenesis of δ-sarcoglycan-associated DCM.

Differential protein expression and basal lamina remodeling in human heart failure.

PURPOSE: A goal of this study was to identify and investigate previously unrecognized components of the remodeling process in the progression to heart failure by comparing protein expression in ischemic failing (F) and nonfailing (NF) human hearts. EXPERIMENTAL DESIGN: Protein expression differences were investigated using multidimensional protein identification and validated by Western analysis. This approach detected basal lamina (BL) remodeling, and further studies analyzed samples for evidence of structural BL remodeling. A rat model of pressure overload (PO) was studied to determine whether nonischemic stressors also produce BL remodeling and impact cellular adhesion. RESULTS: Differential protein expression of collagen IV, laminin α2, and nidogen-1 indicated BL remodeling develops in F versus NF hearts Periodic disruption of cardiac myocyte BL accompanied this process in F, but not NF heart. The rat PO myocardium also developed BL remodeling and compromised myocyte adhesion compared to sham controls. CONCLUSIONS AND CLINICAL RELEVANCE: Differential protein expression and evidence of structural and functional BL alterations develop during heart failure. The compromised adhesion associated with this remodeling indicates a high potential for dysfunctional cellular integrity and tethering in failing myocytes. Therapeutically targeting BL remodeling could slow or prevent the progression of heart disease.

Dystrophin-glycoprotein complex regulates muscle nitric oxide production through mechanoregulation of AMPK signaling.

Patients deficient in dystrophin, a protein that links the cytoskeleton to the extracellular matrix via the dystrophin-glycoprotein complex (DGC), exhibit muscular dystrophy, cardiomyopathy, and impaired muscle nitric oxide (NO) production. We used live-cell NO imaging and in vitro cyclic stretch of isolated adult mouse cardiomyocytes as a model system to investigate if and how the DGC directly regulates the mechanical activation of muscle NO signaling. Acute activation of NO synthesis by mechanical stretch was impaired in dystrophin-deficient mdx cardiomyocytes, accompanied by loss of stretch-induced neuronal NO synthase (nNOS) S1412 phosphorylation. Intriguingly, stretch induced the acute activation of AMP-activated protein kinase (AMPK) in normal cardiomyocytes but not in mdx cardiomyocytes, and specific inhibition of AMPK was sufficient to attenuate mechanoactivation of NO production. Therefore, we tested whether direct pharmacologic activation of AMPK could bypass defective mechanical signaling to restore nNOS activity in dystrophin-deficient cardiomyocytes. Indeed, activation of AMPK with 5-aminoimidazole-4-carboxamide riboside or salicylate increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC promotes the mechanical activation of cardiac nNOS by acting as a mechanosensor to regulate AMPK activity, and that pharmacologic AMPK activation may be a suitable therapeutic strategy for restoring nNOS activity in dystrophin-deficient hearts and muscle.

Voluntary Exercise Improves Estrous Cyclicity in Prenatally Androgenized Female Mice Despite Programming Decreased Voluntary Exercise: Implications for Polycystic Ovary Syndrome (PCOS).

Prenatal androgen (PNA) exposure in mice produces a phenotype resembling lean polycystic ovary syndrome. We studied effects of voluntary exercise on metabolic and reproductive parameters in PNA vs vehicle (VEH)-treated mice. Mice (8 wk of age) were housed individually and estrous cycles monitored. At 10 weeks of age, mice were divided into groups (PNA, PNA-run, VEH, VEH-run, n = 8-9/group); those in the running groups received wheels allowing voluntary running. Unexpectedly, PNA mice ran less distance than VEH mice; ovariectomy eliminated this difference. In ovary-intact mice, there was no difference in glucose tolerance, lower limb muscle fiber types, weight, or body composition among groups after 16 weeks of running, although some mitochondrial proteins were mildly up-regulated by exercise in PNA mice. Before running, estrous cycles in PNA mice were disrupted with most days in diestrus. There was no change in cycles during weeks 1-6 of running (10-15 wk of age). In contrast, from weeks 11 to 16 of running, cycles in PNA mice improved with more days in proestrus and estrus and fewer in diestrus. PNA programs reduced voluntary exercise, perhaps mediated in part by ovarian secretions. Exercise without weight loss improved estrous cycles, which if translated could be important for fertility in and counseling of lean women with polycystic ovary syndrome.