Unusually severe muscular dystrophy upon in-frame deletion of the dystrophin rod domain and lack of compensation by membrane-localized utrophin.

BACKGROUND: Utrophin, a dystrophin homolog, is consistently upregulated in muscles of patients with Duchenne muscular dystrophy (DMD) and is believed to partially compensate for the lack of dystrophin in dystrophic muscle. Even though several animal studies support the idea that utrophin can modulate DMD disease severity, human clinical data are scarce. METHODS: We describe a patient with the largest reported in-frame deletion in the DMD gene, including exons 10-60 and thus encompassing the entire rod domain. FINDINGS: The patient presented with an unusually early and severe progressive weakness, initially suggesting congenital muscular dystrophy. Immunostaining of his muscle biopsy showed that the mutant protein was able to localize at the sarcolemma and stabilize the dystrophin-associated complex. Strikingly, utrophin protein was absent from the sarcolemmal membrane despite the upregulation of utrophin mRNA. CONCLUSIONS: Our results suggest that the internally deleted and dysfunctional dystrophin lacking the entire rod domain may exert a dominant-negative effect by preventing upregulated utrophin protein from reaching the sarcolemmal membrane and thus blocking its partial rescue of muscle function. This unique case may set a lower size limit for similar constructs in potential gene therapy approaches. FUNDING: This work was supported by a grant from MDA USA (MDA3896) and by grant number R01AR051999 from NIAMS/NIH to C.G.B.

Is the fundamental pathology in Duchenne's muscular dystrophy caused by a failure of glycogenolysis-glycolysis in costameres?

Duchenne muscular dystrophy (DMD) is the most common form of progressive childhood muscular dystrophy associated with weakness of limbs, loss of ambulation, heart weakness and early death. The mutations causing either loss-of-expression or function of the full-length protein dystrophin (Dp427) from the DMD gene are responsible for the disease pathology. Dp427 forms a part of the large dystroglycan complex, called DAPC, in the sarcolemma, and its absence derails muscle contraction. Muscle biopsies from DMD patients show an overactivation of excitation-contraction-coupling (ECC) activable calcium incursion, sarcolemmal ROS production, NHE1 activation, IL6 secretion, etc. The signalling pathways, like Akt/PBK, STAT3, p38MAPK, and ERK1/2, are also hyperactive in DMD. These pathways are responsible for post-mitotic trophic growth and metabolic adaptation, in response to exercise in healthy muscles, but cause atrophy and cell death in dystrophic muscles. We hypothesize that the metabolic background of repressed glycolysis in DMD, as opposed to excess glycolysis seen in cancers or healthy contracting muscles, changes the outcome of these 'growth pathways'. The reduced glycolysis has been considered a secondary outcome of the cytoskeletal disruptions seen in DMD. Given the cytoskeleton-crosslinking ability of the glycolytic enzymes, we hypothesize that the failure of glycogenolytic and glycolytic enzymes to congregate is the primary pathology, which then affects the subsarcolemmal cytoskeletal organization in costameres and initiates the pathophysiology associated with DMD, giving rise to the tissue-specific differences in disease progression between muscle, heart and brain. The lacunae in the regulation of the key components of the hypothesized metabolome, and the limitations of this theory are deliberated. The considerations for developing future therapies based on known pathological processes are also discussed.

Deficient Sarcolemma Repair in ALS: A Novel Mechanism with Therapeutic Potential.

The plasma membrane (sarcolemma) of skeletal muscle myofibers is susceptible to injury caused by physical and chemical stresses during normal daily movement and/or under disease conditions. These acute plasma membrane disruptions are normally compensated by an intrinsic membrane resealing process involving interactions of multiple intracellular proteins including dysferlin, annexin, caveolin, and Mitsugumin 53 (MG53)/TRIM72. There is new evidence for compromised muscle sarcolemma repair mechanisms in Amyotrophic Lateral Sclerosis (ALS). Mitochondrial dysfunction in proximity to neuromuscular junctions (NMJs) increases oxidative stress, triggering MG53 aggregation and loss of its function. Compromised membrane repair further worsens sarcolemma fragility and amplifies oxidative stress in a vicious cycle. This article is to review existing literature supporting the concept that ALS is a disease of oxidative-stress induced disruption of muscle membrane repair that compromise the integrity of the NMJs and hence augmenting muscle membrane repair mechanisms could represent a viable therapeutic strategy for ALS.

Muscle fiber type specific alterations of mitochondrial respiratory function and morphology in aged female mice.

Mitochondrial dysfunction is considered to be a major cause of sarcopenia, defined as age-related muscle fiber atrophy and muscle weakness, as reduced mitochondrial respiration and morphological changes such as ragged red fibers (RRFs) are observed in aging muscles. However, the role of mitochondrial dysfunction in sarcopenia is not fully elucidated. Although previous studies have suggested that aging has a fiber type-specific effect on mitochondrial function, little is known about mitochondrial changes in individual fiber types. Here, we used C57BL/6NCr female mice to identify fiber type-specific pathological changes, examine the significance of pathological changes in sarcopenia, and identify possible mechanisms behind mitochondrial changes in slow-twitch soleus muscle (SOL) and fast-twitch extensor digitorum longus muscle (EDL). We observed reduced type I fiber-specific mitochondrial respiratory enzyme activity, impaired respiration, and subsarcolemmal mitochondrial accumulation in aged SOL, which was different from RRFs. These pathological alterations were not directly associated with fiber atrophy. Additionally, we found increased oxidative stress markers in aged SOL, suggesting that oxidative stress is involved in the pathological and functional changes in mitochondria. Meanwhile, obvious mitochondrial changes were not seen in aged EDL. Thus, age-related mitochondrial dysfunction is specific to the fiber type and may correlate with the muscle quality rather than the muscle mass.

The administration of antisense oligonucleotide golodirsen reduces pathological regeneration in patients with Duchenne muscular dystrophy.

During the last decade, multiple clinical trials for Duchenne muscular dystrophy (DMD) have focused on the induction of dystrophin expression using different strategies. Many of these trials have reported a clear increase in dystrophin protein following treatment. However, the low levels of the induced dystrophin protein have raised questions on its functionality. In our present study, using an unbiased, high-throughput digital image analysis platform, we assessed markers of regeneration and levels of dystrophin associated protein via immunofluorescent analysis of whole muscle sections in 25 DMD boys who received 48-weeks treatment with exon 53 skipping morpholino antisense oligonucleotide (PMO) golodirsen. We demonstrate that the de novo dystrophin induced by exon skipping with PMO golodirsen is capable of conferring a histological benefit in treated patients with an increase in dystrophin associated proteins at the dystrophin positive regions of the sarcolemma in post-treatment biopsies. Although 48 weeks treatment with golodirsen did not result in a significant change in the levels of fetal/developmental myosins for the entire cohort, there was a significant negative correlation between the amount of dystrophin and levels of regeneration observed in different biopsy samples. Our results provide, for the first time, evidence of functionality of induced dystrophin following successful therapeutic intervention in the human.

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.

Microutrophin expression in dystrophic mice displays myofiber type differences in therapeutic effects.

Gene therapy approaches for DMD using recombinant adeno-associated viral (rAAV) vectors to deliver miniaturized (or micro) dystrophin genes to striated muscles have shown significant progress. However, concerns remain about the potential for immune responses against dystrophin in some patients. Utrophin, a developmental paralogue of dystrophin, may provide a viable treatment option. Here we examine the functional capacity of an rAAV-mediated microutrophin (µUtrn) therapy in the mdx4cv mouse model of DMD. We found that rAAV-µUtrn led to improvement in dystrophic histopathology & mostly restored the architecture of the neuromuscular and myotendinous junctions. Physiological studies of tibialis anterior muscles indicated peak force maintenance, with partial improvement of specific force. A fundamental question for µUtrn therapeutics is not only can it replace critical functions of dystrophin, but whether full-length utrophin impacts the therapeutic efficacy of the smaller, highly expressed µUtrn. As such, we found that µUtrn significantly reduced the spacing of the costameric lattice relative to full-length utrophin. Further, immunostaining suggested the improvement in dystrophic pathophysiology was largely influenced by favored correction of fast 2b fibers. However, unlike µUtrn, µdystrophin (µDys) expression did not show this fiber type preference. Interestingly, µUtrn was better able to protect 2a and 2d fibers in mdx:utrn-/- mice than in mdx4cv mice where the endogenous full-length utrophin was most prevalent. Altogether, these data are consistent with the role of steric hindrance between full-length utrophin & µUtrn within the sarcolemma. Understanding the stoichiometry of this effect may be important for predicting clinical efficacy.

Autoantibodies targeting TRIM72 compromise membrane repair and contribute to inflammatory myopathy.

Idiopathic inflammatory myopathies (IIM) involve chronic inflammation of skeletal muscle and subsequent muscle degeneration due to an uncontrolled autoimmune response; however, the mechanisms leading to pathogenesis are not well understood. A compromised sarcolemmal repair process could promote an aberrant exposure of intramuscular antigens with the subsequent initiation of an inflammatory response that contributes to IIM. Using an adoptive transfer mouse model of IIM, we show that sarcolemmal repair is significantly compromised in distal skeletal muscle in the absence of inflammation. We identified autoantibodies against TRIM72 (also known as MG53), a muscle-enriched membrane repair protein, in IIM patient sera and in our mouse model of IIM by ELISA. We found that patient sera with elevated levels of TRIM72 autoantibodies suppress sarcolemmal resealing in healthy skeletal muscle, and depletion of TRIM72 antibodies from these same serum samples rescues sarcolemmal repair capacity. Autoantibodies targeting TRIM72 lead to skeletal muscle fibers with compromised membrane barrier function, providing a continuous source of autoantigens to promote autoimmunity and further amplifying humoral responses. These findings reveal a potential pathogenic mechanism that acts as a feedback loop contributing to the progression of IIM.

Nanoscale regulation of L-type calcium channels differentiates between ischemic and dilated cardiomyopathies.

BACKGROUND: Subcellular localization and function of L-type calcium channels (LTCCs) play an important role in regulating contraction of cardiomyocytes. Understanding how this is affected by the disruption of transverse tubules during heart failure could lead to new insights into the disease. METHODS: Cardiomyocytes were isolated from healthy donor hearts, as well as from patients with cardiomyopathies and with left ventricular assist devices. Scanning ion conductance and confocal microscopy was used to study membrane structures in the cells. Super-resolution scanning patch-clamp was used to examine LTCC function in different microdomains. Computational modeling predicted the impact of these changes to arrhythmogenesis at the whole-heart level. FINDINGS: We showed that loss of structural organization in failing myocytes leads to re-distribution of functional LTCCs from the T-tubules to the sarcolemma. In ischemic cardiomyopathy, the increased LTCC open probability in the T-tubules depends on the phosphorylation by protein kinase A, whereas in dilated cardiomyopathy, the increased LTCC opening probability in the sarcolemma results from enhanced phosphorylation by calcium-calmodulin kinase II. LVAD implantation corrected LTCCs pathophysiological activity, although it did not improve their distribution. Using computational modeling in a 3D anatomically-realistic human ventricular model, we showed how LTCC location and activity can trigger heart rhythm disorders of different severity. INTERPRETATION: Our findings demonstrate that LTCC redistribution and function differentiate between disease aetiologies. The subcellular changes observed in specific microdomains could be the consequence of the action of distinct protein kinases. FUNDING: This work was supported by NIH grant (ROI-HL 126802 to NT-JG) and British Heart Foundation (grant RG/17/13/33173 to JG, project grant PG/16/17/32069 to RAC). Funders had no role in study design, data collection, data analysis, interpretation, writing of the report.

Transcriptome changes during the initiation and progression of Duchenne muscular dystrophy in Caenorhabditis elegans.

Duchenne muscular dystrophy (DMD) is a lethal, X-linked disease characterized by progressive muscle degeneration. The condition is driven by nonsense and missense mutations in the dystrophin gene, leading to instability of the sarcolemma and skeletal muscle necrosis and atrophy. Resulting changes in muscle-specific gene expression that take place in dystrophin's absence remain largely uncharacterized, as they are potentially obscured by the chronic inflammation elicited by muscle damage in humans. Caenorhabditis elegans possess a mild inflammatory response that is not active in the muscle, and lack a satellite cell equivalent. This allows for the characterization of the transcriptome rearrangements affecting disease progression independently of inflammation and regeneration. In effort to better understand these dynamics, we have isolated and sequenced body muscle-specific transcriptomes from C. elegans lacking functional dystrophin at distinct stages of disease progression. We have identified an upregulation of genes involved in mitochondrial function early in disease progression, and an upregulation of genes related to muscle repair in later stages. Our results suggest that in C. elegans, dystrophin may have a signaling role early in development, and its absence may activate compensatory mechanisms that counteract muscle degradation caused by loss of dystrophin. We have also developed a temperature-based screening method for synthetic paralysis that can be used to rapidly identify genetic partners of dystrophin. Our results allow for the comprehensive identification of transcriptome changes that potentially serve as independent drivers of disease progression and may in turn allow for the identification of new therapeutic targets for the treatment of DMD.

A high-throughput digital script for multiplexed immunofluorescent analysis and quantification of sarcolemmal and sarcomeric proteins in muscular dystrophies.

The primary molecular endpoint for many Duchenne muscular dystrophy (DMD) clinical trials is the induction, or increase in production, of dystrophin protein in striated muscle. For accurate endpoint analysis, it is essential to have reliable, robust and objective quantification methodologies capable of detecting subtle changes in dystrophin expression. In this work, we present further development and optimisation of an automated, digital, high-throughput script for quantitative analysis of multiplexed immunofluorescent (IF) whole slide images (WSI) of dystrophin, dystrophin associated proteins (DAPs) and regenerating myofibres (fetal/developmental myosin-positive) in transverse sections of DMD, Becker muscular dystrophy (BMD) and control skeletal muscle biopsies. The script enables extensive automated assessment of myofibre morphometrics, protein quantification by fluorescence intensity and sarcolemmal circumference coverage, colocalisation data for dystrophin and DAPs and regeneration at the single myofibre and whole section level. Analysis revealed significant variation in dystrophin intensity, percentage coverage and amounts of DAPs between differing DMD and BMD samples. Accurate identification of dystrophin via a novel background subtraction method allowed differential assessment of DAP fluorescence intensity within dystrophin positive compared to dystrophin negative sarcolemma regions. This enabled surrogate quantification of molecular functionality of dystrophin in the assembly of the DAP complex. Overall, the digital script is capable of multiparametric and unbiased analysis of markers of myofibre regeneration and dystrophin in relation to key DAPs and enabled better characterisation of the heterogeneity in dystrophin expression patterns seen in BMD and DMD alongside the surrogate assessment of molecular functionality of dystrophin. Both these aspects will be of significant relevance to ongoing and future DMD and other muscular dystrophies clinical trials to help benchmark therapeutic efficacy.

Myositis with sarcoplasmic inclusions in Nakajo-Nishimura syndrome: a genetic inflammatory myopathy.

AIMS: Nakajo-Nishimura syndrome (NNS) is an autosomal recessive disease caused by biallelic mutations in the PSMB8 gene that encodes the immunoproteasome subunit ß5i. There have been only a limited number of reports on the clinicopathological features of the disease in genetically confirmed cases. METHODS: We studied clinical and pathological features of three NNS patients who all carry the homozygous p.G201V mutations in PSMB8. Patients' muscle specimens were analysed with histology and immunohistochemistry. RESULTS: All patients had episodes of typical periodic fever and skin rash, and later developed progressive muscle weakness and atrophy, similar to previous reports. Oral corticosteroid was used for treatment but showed no obvious efficacy. On muscle pathology, lymphocytes were present in the endomysium surrounding non-necrotic fibres, as well as in the perimysium perivascular area. Nearly all fibres strongly expressed MHC-I in the sarcolemma. In the eldest patient, there were abnormal protein aggregates in the sarcoplasm, immunoreactive to p62, TDP-43 and ubiquitin antibodies. CONCLUSIONS: These results suggest that inflammation, inclusion pathology and aggregation of abnormal proteins underlie the progressive clinical course of the NNS pathomechanism.

A fundamental evaluation of the electrical properties and function of cardiac transverse tubules.

This work discusses active and passive electrical properties of transverse (T-)tubules in ventricular cardiomyocytes to understand the physiological roles of T-tubules. T-tubules are invaginations of the lateral membrane that provide a large surface for calcium-handling proteins to facilitate sarcomere shortening. Higher heart rates correlate with higher T-tubular densities in mammalian ventricular cardiomyocytes. We assess ion dynamics in T-tubules and the effects of sodium current in T-tubules on the extracellular potential, which leads to a partial reduction of the sodium current in deep segments of a T-tubule. We moreover reflect on the impact of T-tubules on macroscopic conduction velocity, integrating fundamental principles of action potential propagation and conduction. We also theoretically assess how the conduction velocity is affected by different T-tubular sodium current densities. Lastly, we critically assess literature on ion channel expression to determine whether action potentials can be initiated in T-tubules.

Uniform sarcolemmal dystrophin expression is required to prevent extracellular microRNA release and improve dystrophic pathology.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a fatal muscle-wasting disorder caused by genetic loss of dystrophin protein. Extracellular microRNAs (ex-miRNAs) are putative, minimally invasive biomarkers of DMD. Specific ex-miRNAs (e.g. miR-1, miR-133a, miR-206, and miR-483) are highly up-regulated in the serum of DMD patients and dystrophic animal models and are restored to wild-type levels following exon skipping-mediated dystrophin rescue in mdx mice. As such, ex-miRNAs are promising pharmacodynamic biomarkers of exon skipping efficacy. Here, we aimed to determine the degree to which ex-miRNA levels reflect the underlying level of dystrophin protein expression in dystrophic muscle. METHODS: Candidate ex-miRNA biomarker levels were investigated in mdx mice in which dystrophin was restored with peptide-PMO (PPMO) exon skipping conjugates and in mdx-XistΔhs mice that express variable amounts of dystrophin from birth as a consequence of skewed X-chromosome inactivation. miRNA profiling was performed in mdx-XistΔhs mice using the FirePlex methodology and key results validated by small RNA TaqMan RT-qPCR. The muscles from each animal model were further characterized by dystrophin western blot and immunofluorescence staining. RESULTS: The restoration of ex-myomiR abundance observed following PPMO treatment was not recapitulated in the high dystrophin-expressing mdx-XistΔhs group, despite these animals expressing similar amounts of total dystrophin protein (~37% of wild-type levels). Instead, ex-miRNAs were present at high levels in mdx-XistΔhs mice regardless of dystrophin expression. PPMO-treated muscles exhibited a uniform pattern of dystrophin localization and were devoid of regenerating fibres, whereas mdx-XistΔhs muscles showed non-homogeneous dystrophin staining and sporadic regenerating foci. CONCLUSIONS: Uniform dystrophin expression is required to prevent ex-miRNA release, stabilize myofiber turnover, and attenuate pathology in dystrophic muscle.

Elimination of fukutin reveals cellular and molecular pathomechanisms in muscular dystrophy-associated heart failure.

Heart failure is the major cause of death for muscular dystrophy patients, however, the molecular pathomechanism remains unknown. Here, we 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 α-dystroglycan glycosylation and dystrophin-glycoprotein complex proteins in sarcolemma at all developmental stages, cardiac dysfunction was observed only in later adulthood, suggesting that membrane fragility is not the sole etiology of cardiac dysfunction. During young adulthood, Fktn-deficient mice were vulnerable to pathological hypertrophic stress with downregulation of Akt and the MEF2-histone deacetylase axis. 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. These data reveal fukutin is crucial for maintaining myocyte physiology to prevent heart failure, and thus, the results may lead to strategies for therapeutic intervention.

Dysregulation of Mitochondrial Ca2+ Uptake and Sarcolemma Repair Underlie Muscle Weakness and Wasting in Patients and Mice Lacking MICU1.

Muscle function is regulated by Ca2+, which mediates excitation-contraction coupling, energy metabolism, adaptation to exercise, and sarcolemmal repair. Several of these actions rely on Ca2+ delivery to the mitochondrial matrix via the mitochondrial Ca2+ uniporter, the pore of which is formed by mitochondrial calcium uniporter (MCU). MCU's gatekeeping and cooperative activation are controlled by MICU1. Loss-of-protein mutation in MICU1 causes a neuromuscular disease. To determine the mechanisms underlying the muscle impairments, we used MICU1 patient cells and skeletal muscle-specific MICU1 knockout mice. Both these models show a lower threshold for MCU-mediated Ca2+ uptake. Lack of MICU1 is associated with impaired mitochondrial Ca2+ uptake during excitation-contraction, aerobic metabolism impairment, muscle weakness, fatigue, and myofiber damage during physical activity. MICU1 deficit compromises mitochondrial Ca2+ uptake during sarcolemmal injury, which causes ineffective repair of the damaged myofibers. Thus, dysregulation of mitochondrial Ca2+ uptake hampers myofiber contractile function, likely through energy metabolism and membrane repair.

Dystrophin R16/17 protein therapy restores sarcolemmal nNOS in trans and improves muscle perfusion and function.

BACKGROUND: Delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma leads to functional muscle ischemia. This contributes to the pathogenesis in cachexia, aging and muscular dystrophy. Mutations in the gene encoding dystrophin result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). In many BMD patients and DMD patients that have been converted to BMD by gene therapy, sarcolemmal nNOS is missing due to the lack of dystrophin nNOS-binding domain. METHODS: Dystrophin spectrin-like repeats 16 and 17 (R16/17) is the sarcolemmal nNOS localization domain. Here we explored whether R16/17 protein therapy can restore nNOS to the sarcolemma and prevent functional ischemia in transgenic mice which expressed an R16/17-deleted human micro-dystrophin gene in the dystrophic muscle. The palmitoylated R16/17.GFP fusion protein was conjugated to various cell-penetrating peptides and produced in the baculovirus-insect cell system. The best fusion protein was delivered to the transgenic mice and functional muscle ischemia was quantified. RESULTS: Among five candidate cell-penetrating peptides, the mutant HIV trans-acting activator of transcription (TAT) protein transduction domain (mTAT) was the best in transferring the R16/17.GFP protein to the muscle. Systemic delivery of the mTAT.R16/17.GFP protein to micro-dystrophin transgenic mice successfully restored sarcolemmal nNOS without inducing T cell infiltration. More importantly, R16/17 protein therapy effectively prevented treadmill challenge-induced force loss and improved muscle perfusion during contraction. CONCLUSIONS: Our results suggest that R16/17 protein delivery is a highly promising therapy for muscle diseases involving sarcolemmal nNOS delocalizaton.

Sodium-Hydrogen Exchanger Isoform-1 Inhibition: A Promising Pharmacological Intervention for Resuscitation from Cardiac Arrest.

Out-of-hospital sudden cardiac arrest is a major public health problem with an overall survival of less than 5%. Upon cardiac arrest, cessation of coronary blood flow rapidly leads to intense myocardial ischemia and activation of the sarcolemmal Na+-H+ exchanger isoform-1 (NHE-1). NHE-1 activation drives Na+ into cardiomyocytes in exchange for H+ with its exchange rate intensified upon reperfusion during the resuscitation effort. Na+ accumulates in the cytosol driving Ca2+ entry through the Na+-Ca2+ exchanger, eventually causing cytosolic and mitochondrial Ca2+ overload and worsening myocardial injury by compromising mitochondrial bioenergetic function. We have reported clinically relevant myocardial effects elicited by NHE-1 inhibitors given during resuscitation in animal models of ventricular fibrillation (VF). These effects include: (a) preservation of left ventricular distensibility enabling hemodynamically more effective chest compressions, (b) return of cardiac activity with greater electrical stability reducing post-resuscitation episodes of VF, (c) less post-resuscitation myocardial dysfunction, and (d) attenuation of adverse myocardial effects of epinephrine; all contributing to improved survival in animal models. Mechanistically, NHE-1 inhibition reduces adverse effects stemming from Na+-driven cytosolic and mitochondrial Ca2+ overload. We believe the preclinical work herein discussed provides a persuasive rationale for examining the potential role of NHE-1 inhibitors for cardiac resuscitation in humans.

Stabilization of the cardiac sarcolemma by sarcospan rescues DMD-associated cardiomyopathy.

In the current preclinical study, we demonstrate the therapeutic potential of sarcospan (SSPN) overexpression to alleviate cardiomyopathy associated with Duchenne muscular dystrophy (DMD) utilizing dystrophin-deficient mdx mice with utrophin haploinsufficiency that more accurately represent the severe disease course of human DMD. SSPN interacts with dystrophin, the DMD disease gene product, and its autosomal paralog utrophin, which is upregulated in DMD as a partial compensatory mechanism. SSPN transgenic mice have enhanced abundance of fully glycosylated α-dystroglycan, which may further protect dystrophin-deficient cardiac membranes. Baseline echocardiography reveals SSPN improves systolic function and hypertrophic indices in mdx and mdx:utr-heterozygous mice. Assessment of SSPN transgenic mdx mice by hemodynamic pressure-volume methods highlights enhanced systolic performance compared to mdx controls. SSPN restores cardiac sarcolemma stability, the primary defect in DMD disease, reduces fibrotic response and improves contractile function. We demonstrate that SSPN ameliorates more advanced cardiac disease in the context of diminished sarcolemma expression of utrophin and ß1D integrin that mitigate disease severity and partially restores responsiveness to ß-adrenergic stimulation. Overall, our current and previous findings suggest SSPN overexpression in DMD mouse models positively impacts skeletal, pulmonary and cardiac performance by addressing the stability of proteins at the sarcolemma that protect the heart from injury, supporting SSPN and membrane stabilization as a therapeutic target for DMD.

Thrombospondin-3 augments injury-induced cardiomyopathy by intracellular integrin inhibition and sarcolemmal instability.

Thrombospondins (Thbs) are a family of five secreted matricellular glycoproteins in vertebrates that broadly affect cell-matrix interaction. While Thbs4 is known to protect striated muscle from disease by enhancing sarcolemmal stability through increased integrin and dystroglycan attachment complexes, here we show that Thbs3 antithetically promotes sarcolemmal destabilization by reducing integrin function, augmenting disease-induced decompensation. Deletion of Thbs3 in mice enhances integrin membrane expression and membrane stability, protecting the heart from disease stimuli. Transgene-mediated overexpression of α7ß1D integrin in the heart ameliorates the disease predisposing effects of Thbs3 by augmenting sarcolemmal stability. Mechanistically, we show that mutating Thbs3 to contain the conserved RGD integrin binding domain normally found in Thbs4 and Thbs5 now rescues the defective expression of integrins on the sarcolemma. Thus, Thbs proteins mediate the intracellular processing of integrin plasma membrane attachment complexes to regulate the dynamics of cellular remodeling and membrane stability.