Mdx Cardiomyocytes Research Articles

Abstract Or101: Phospho-Mimic Mutation in Beta Tubulin Suppresses Arrhythmias and Improves Cardiac Connexin 43 Localization in Duchenne Muscular Dystrophy Mice

Duchenne Muscular Dystrophy (DMD) is a severe X-linked neuromuscular disorder, causing progressive muscle degeneration and premature death due to heart and skeletal muscle dysfunction. It is caused by mutations in the dmd gene, leading to the absence of dystrophin, a crucial protein for muscle integrity. There is no cure for DMD. Its associated dystrophic cardiomyopathy is the primary cause of mortality by age 20. Connexin-43 (Cx43) is a gap junction protein responsible for electrical signaling in the heart. Cx43 localizes at the intercalated disc of the cardiomyocyte (CM), forming an intercellular channel with adjacent CMs; In DMD, Cx43 hemichannel remodels to the lateral side of the cardiomyocyte. Our previous research indicated that phospho-mimic Cx43 carrying serine to glutamic acid substitutions at the triplet regulatory site S325, S328, S330 in mdx hearts suppressed Cx43 lateralization and improved cardiac rhythm. The phospho-mimic Cx43 also reduced pathological hyperdensity and transverse directionality of microtubules in mdx cardiomyocytes. Colchicine (Colch) treatment, known to inhibit microtubule polymerization, also improved cardiac rhythm and Cx43 localization in mdx mice. To phenocopy the effect observed with Colch, we generated a mouse model with a beta tubulin mutation at serine 172, which was replaced by glutamic acid (S172E). We found reduced arrhythmias and improved localization of Cx43 in mdx hearts carrying the S172E phospho-mimic mutant tubulin by electrocardiography and immunofluorescent staining, respectively. Moreover, the proteomics data showed that the level of phospho-Cx43 was also upregulated in mdx hearts with S172E tubulin mutation. Further investigation will explore how this mutation impacts protein regulation and electrophysiology, and elucidate its mechanism of action in microtubule polymerization. These studies investigate a regulatory link between microtubule dynamics and localization of Cx43, potentially suggesting a novel therapeutic target for DMD-associated cardiomyopathy.

Decreasing microtubule detyrosination modulates Nav1.5 subcellular distribution and restores sodium current in mdx cardiomyocytes.

The microtubule (MT) network plays a major role in the transport of the cardiac sodium channel Nav1.5 to the membrane, where the latter associates with interacting proteins such as dystrophin. Alterations in MT dynamics are known to impact on ion channel trafficking. Duchenne muscular dystrophy (DMD), caused by dystrophin deficiency, is associated with an increase in MT detyrosination, decreased sodium current (INa), and arrhythmias. Parthenolide (PTL), a compound that decreases MT detyrosination, has shown beneficial effects on cardiac function in DMD. We here investigated its impact on INa and Nav1.5 subcellular distribution. Ventricular cardiomyocytes (CMs) from wild-type (WT) and mdx (DMD) mice were incubated with either 10 µM PTL, 20 µM EpoY, or dimethylsulfoxide (DMSO) for 3-5 h, followed by patch-clamp analysis to assess INa and action potential (AP) characteristics in addition to immunofluorescence and stochastic optical reconstruction microscopy (STORM) to investigate MT detyrosination and Nav1.5 cluster size and density, respectively. In accordance with previous studies, we observed increased MT detyrosination, decreased INa and reduced AP upstroke velocity (Vmax) in mdx CMs compared to WT. PTL decreased MT detyrosination and significantly increased INa magnitude (without affecting INa gating properties) and AP Vmax in mdx CMs, but had no effect in WT CMs. Moreover, STORM analysis showed that in mdx CMs, Nav1.5 clusters were decreased not only in the grooves of the lateral membrane (LM; where dystrophin is localized) but also at the LM crests. PTL restored Nav1.5 clusters at the LM crests (but not at the grooves), indicating a dystrophin-independent trafficking route to this subcellular domain. Interestingly, Nav1.5 cluster density was also reduced at the intercalated disc (ID) region of mdx CMs, which was restored to WT levels by PTL. Treatment of mdx CMs with EpoY, a specific MT detyrosination inhibitor, also increased INa density, while decreasing the amount of detyrosinated MTs, confirming a direct mechanistic link. Attenuating MT detyrosination in mdx CMs restored INa and enhanced Nav1.5 localization at the LM crest and ID. Hence, the reduced whole-cell INa density characteristic of mdx CMs is not only the consequence of the lack of dystrophin within the LM grooves but is also due to reduced Nav1.5 at the LM crest and ID secondary to increased baseline MT detyrosination. Overall, our findings identify MT detyrosination as a potential therapeutic target for modulating INa and subcellular Nav1.5 distribution in pathophysiological conditions.

Impact of microtubule detyrosination on subcellular distribution of Nav1.5 in mdx cardiomyocytes

Abstract Background Subcellular trafficking and distribution of the cardiac sodium channel Nav1.5 in cardiomyocytes is dependent on the microtubule network as well as Nav1.5 interacting proteins. Duchenne muscular dystrophy (DMD) is associated with loss of dystrophin, a Nav1.5-interacting protein localized at the lateral membrane of cardiomyocytes, leading to sodium current (INa) reduction in this microdomain. Cardiomyocytes of DMD (mdx) mice also display increased microtubule detyrosination, but its impact on INa and Nav1.5 distribution is unclear. Purpose To investigate the effect of reducing microtubule detyrosination by the compound parthenolide (PTL) on INa and subcellular Nav1.5 distribution in mdx cardiomyocytes (CMs). Methods and Results Isolated ventricular CMs from wild type (WT) and mdx (DMD) mice were incubated with either 10 µM PTL or DMSO for 3-5 hours. INa properties were assessed using the patch-clamp technique, confocal microscopy was used to investigate microtubule detyrosination, and stochastic optical reconstruction microscopy (STORM) was employed to assess Nav1.5 cluster density and size, as well as Nav1.5 cluster distribution (crest vs groove) at the cardiomyocyte lateral membrane. Compared to WT. mdx CMs displayed increased levels of detyrosinated tubulin and decreased whole-cell INa. Incubation with PTL decreased the fraction of detyrosinated tubulin and significantly increased whole-cell INa magnitude in mdx CMs, but had no effect in WT CMs. No effect of PTL on INa gating properties were observed, indicating that it increased INa by enhancing Nav1.5 membrane trafficking. Indeed, STORM analysis showed that PTL increased Nav1.5 cluster density at both the lateral membrane and intercalated disc region in mdx CMs while it had no effect on WT CMs. Furthermore, Nav1.5-α-actinin cluster distance analysis showed that PTL increased Nav1.5 clusters specifically at the crest of the lateral membrane in mdx CMs. Conclusions In mdx CMs, reduction of MT detyrosination rescues INa density and increases Nav1.5 cluster density both at the intercalated disc and lateral membrane. Hence, in addition to loss of the Nav1.5-interacting protein dystrophin, MT remodelling contributes to INa reduction in DMD. The PTL-induced increase in Nav1.5 at the crest of the lateral membrane of mdx CMs is likely due to a dystrophin-independent mechanism since dystrophin is normally absent from this microdomain. Overall, our findings identify MT detyrosination as a potential therapeutic target for modulating INa and subcellular Nav1.5 distribution during pathophysiological conditions.

Decreasing microtubule detyrosination by parthenolide restores sodium current in mdx cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: None. Background The cardiac sodium channel Nav1.5 is transported to the membrane by the microtubule network. Alterations in microtubule dynamics are known to impact on ion channel trafficking. Pathophysiological conditions such as heart failure and Duchenne muscular dystrophy (DMD) are associated with an increase in microtubule detyrosination as well as a decreased sodium current (INa) and pro-arrhythmia. Parthenolide, a compound that decreases the fraction of detyrosinated microtubules, has been shown to have beneficial effects on cardiac function in DMD mice, but its impact on INa has not been investigated. Methods and Results Cardiomyocytes (CMs) from wild type (WT) and mdx (DMD) mice were used to investigate the effect of parthenolide. Cells were incubated with either 10 µM parthenolide or DMSO for 3-5 hours. INa and action potential (AP) characteristics were assessed using the patch-clamp technique, while immunofluorescence and stochastic optical reconstruction microscopy (STORM), were used to investigate microtubule detyrosination and Nav1.5 cluster density, respectively. In accordance with previous studies, we observed increased levels of detyrosinated microtubules and decreased INa in mdx CMs compared to WT. Treatment with parthenolide decreased the fraction of detyrosinated microtubules and significantly increased INa magnitude in mdx CMs, but had no effect on INa in WT CMs. Accordingly, parthenolide significantly increased AP maximal upstroke velocity in mdx CMs, without affecting other AP properties. Parthenolide did not affect INa gating properties, indicating that it increased INa by enhancing Nav1.5 membrane trafficking. Indeed, STORM analysis showed that parthenolide increased Nav1.5 cluster density at both the lateral membrane (crest) and intercalated disc region in mdx CMs while it had no effect on WT CMs. Conclusions Parthenolide restores INa and Nav1.5 membrane expression in mdx CMs, and may be of potential benefit in other pathophysiological conditions associated with increased microtubule detyrosination and reduced INa. Further elucidation of the impact of microtubule dynamics on Nav1.5 may identify additional therapeutic targets for restoring conduction and preventing arrhythmias.

Cardiomyocyte depolarization triggers NOS-dependent NO transient after calcium release, reducing the subsequent calcium transient

Cardiac excitation–contraction coupling and metabolic and signaling activities are centrally modulated by nitric oxide (NO), which is produced by one of three NO synthases (NOSs). Despite the significant role of NO in cardiac Ca2+ homeostasis regulation under different pathophysiological conditions, such as Duchenne muscular dystrophy (DMD), no precise method describes the production, source or effect of NO through two NO signaling pathways: soluble guanylate cyclase-protein kinase G (NO-sGC-PKG) and S-nitrosylation (SNO). Using a novel strategy involving isolated murine cardiomyocytes loaded with a copper-based dye highly specific for NO, we observed a single transient NO production signal after each electrical stimulation event. The NO transient signal started 67.5 ms after the beginning of Rhod-2 Ca2+ transient signal and lasted for approximately 430 ms. Specific NOS isoform blockers or NO scavengers significantly inhibited the NO transient, suggesting that wild-type (WT) cardiomyocytes produce nNOS-dependent NO transients. Conversely, NO transient in mdx cardiomyocyte, a mouse model of DMD, was dependent on inducible NOS (iNOS) and endothelial (eNOS). In a consecutive stimulation protocol, the nNOS-dependent NO transient in WT cardiomyocytes significantly reduced the next Ca2+ transient via NO-sGC-PKG. In mdx cardiomyocytes, this inhibitory effect was iNOS- and eNOS-dependent and occurred through the SNO pathway. Basal NO production was nNOS- and iNOS-dependent in WT cardiomyocytes and eNOS- and iNOS-dependent in mdx cardiomyocytes. These results showed cardiomyocyte produces NO isoform-dependent transients upon membrane depolarization at the millisecond time scale activating a specific signaling pathway to negatively modulate the subsequent Ca2+ transient.

Metformin Reverses the Enhanced Myocardial SR/ER-Mitochondria Interaction and Impaired Complex I-Driven Respiration in Dystrophin-Deficient Mice.

Besides skeletal muscle dysfunction, Duchenne muscular dystrophy (DMD) exhibits a progressive cardiomyopathy characterized by an impaired calcium (Ca2+) homeostasis and a mitochondrial dysfunction. Here we aimed to determine whether sarco-endoplasmic reticulum (SR/ER)–mitochondria interactions and mitochondrial function were impaired in dystrophic heart at the early stage of the pathology. For this purpose, ventricular cardiomyocytes and mitochondria were isolated from 3-month-old dystrophin-deficient mice (mdx mice). The number of contacts points between the SR/ER Ca2+ release channels (IP3R1) and the porine of the outer membrane of the mitochondria, VDAC1, measured using in situ proximity ligation assay, was greater in mdx cardiomyocytes. Expression levels of IP3R1 as well as the mitochondrial Ca2+ uniporter (MCU) and its regulated subunit, MICU1, were also increased in mdx heart. MICU2 expression was however unchanged. Furthermore, the mitochondrial Ca2+ uptake kinetics and the mitochondrial Ca2+ content were significantly increased. Meanwhile, the Ca2+-dependent pyruvate dehydrogenase phosphorylation was reduced, and its activity significantly increased. In Ca2+-free conditions, pyruvate-driven complex I respiration was decreased whereas in the presence of Ca2+, complex I-mediated respiration was boosted. Further, impaired complex I-mediated respiration was independent of its intrinsic activity or expression, which remains unchanged but is accompanied by an increase in mitochondrial reactive oxygen species production. Finally, mdx mice were treated with the complex I modulator metformin for 1 month. Metformin normalized the SR/ER-mitochondria interaction, decreased MICU1 expression and mitochondrial Ca2+ content, and enhanced complex I-driven respiration. In summary, before any sign of dilated cardiomyopathy, the DMD heart displays an aberrant SR/ER-mitochondria coupling with an increase mitochondrial Ca2+ homeostasis and a complex I dysfunction. Such remodeling could be reversed by metformin providing a novel therapeutic perspective in DMD.

The neuro-cardiac junction defines an extracellular microdomain required for neurotrophic signaling

Abstract Background Sympathetic neurons (SNs) innervate the myocardium with a defined topology that allows physiological modulation of cardiac activity. Neurotrophins released by cardiac cells control SN viability and myocardial distribution, which are impaired in heart diseases with reduced (e.g. heart failure) or heterogenous sympathetic stimulation (e.g. arrhythmias). We previously demonstrated that SNs interact directly with cardiomyocytes (CMs) at neuro-cardiac junctions (NCJ), and such structured contact sites allow neurons to efficiently activate β-adrenoceptors on the myocyte membrane. Aims We here asked whether NCJs are functional for retrograde (myocyte to neuron) neurotrophic signaling. Methods and results Electron microscopy and immunofluorescence on mouse heart slices and SN/CM co-cultures showed that the NGF receptor, TrkA, is preferentially found in correspondence of the NCJ. Consistently, neurons taking structured contact with CMs showed fast TrkA activation and its retrograde transport to the soma, which was monitored using live confocal imaging in cells expressing TrkA-RFP. In accord with NGF dependent effects, CM-contacted SN showed larger synaptic varicosities and did not require NGF supplementation in the culture medium. In support that NGF locally released at NCJs sustains SN viability, the neurotrophin concentration in the culture medium was 1.61 pg/mL, and did not suffice to maintain neuronal viability, which was also perturbed (66% decrease of neuronal density) by silencing NGF expression in CMs. These results support that the NCJ is essential for intercellular neurotrophin signaling. Consistently, by applying competitive inhibition of TrkA with increasing doses of K252a, we estimated NGF concentration at the contact site to be about 1000-fold higher than that released by CM in the culture medium. To seek for the structural determinants of the NCJ, we focused on dystrophin, based on the finding that the protein accumulates on the CM membrane portion contacted by SNs, as observed in mouse heart slices, and co-cultured CMs. In support of a role of CM-expressed dystrophin in neurotrophic signaling, hearts from dystrophin-KO (mdx) mice showed 74.36% decrease of innervation, with no significant changes of NGF expression. In line with the purported role of NCJs, in co-cultures between wild type SNs and mdx CMs, TrkA activation (TrkA movements toward SN soma (%): WTCM-WTSN=18±4; MDXCM-WTSN= 12±3; p<0,05) and neuronal survival were reduced. Conclusions Taken together, our results suggest that NGF-dependent signaling to SNs requires a direct and specialized interaction with myocytes, and that loss of dystrophin at the CM membrane impairs retrograde signaling to the neurons leading to cardiac sympathetic dys-innervation. Funding Acknowledgement Type of funding source: Public Institution(s). Main funding source(s): University of Padova

Internal structure and remodeling in dystrophin-deficient cardiomyocytes using second harmonic generation

Duchenne muscular dystrophy (DMD) is a debilitating disorder related to dystrophin encoding gene mutations, often associated with dilated cardiomyopathy. However, it is still unclear how dystrophin deficiency affects cardiac sarcomere remodeling and contractile dysfunction. We employed second harmonic generation (SHG) microscopy, a nonlinear optical imaging technique that allows studying contractile apparatus organization without histologic fixation and immunostaining. Images were acquired on alive DMD (mdx) and wild type cardiomyocytes at different ages and at various external calcium concentrations. An automated image processing was developed to identify individual myofibrils and extract data about their organization. We observed a structural aging-dependent remodeling in mdx cardiomyocytes affecting sarcomere sinuosity, orientation and length that could not be anticipated from standard optical imaging. These results revealed for the first time the interest of SHG to evaluate the intracellular and sarcomeric remodeling of DMD cardiac tissue in an age-dependent manner that could participate in progressive contractile dysfunction.

P.309Mechanistic insights into Duchenne muscular dystrophy-associated cardiomyopathy

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disorder that is associated with progressive muscle wasting and cardiomyopathy. Utilizing cardiac MRI, we have demonstrated that adult DMD patients have small hearts with very low LV mass as compared to patients with non-ischemic cardiomyopathy or healthy patients. Thus, we hypothesize that DMD-associated cardiomyopathy develops secondary to dysregulation of cardiac signaling pathways that modulate cardiac growth, namely cardiomyocyte proliferation. Utilizing the mdx mouse, a model of DMD, decreased heart/body weight ratios were noted from birth (P1: Hrt/BW 5.6 ± 0.16 vs. 4.8 ± 0.18 NTg vs mdx p<0.005 n=32). NTg and mdx hearts have similar cardiomyocyte cell size (WGA staining: 0.08 ± 0.001 vs 0.08 ± 0.002 NTg vs mdx p<0.05; n=3). FACS analysis indicated mdx hearts have fewer cardiomyocytes (35% reduction; 51.7e ± 4.9e vs. 33.8e ± 3.5e NTg vs mdx p<0.05 n=6). Transcriptome profiling demonstrated that molecular pathways governing cardiac proliferation were significantly reduced in mdx hearts (Ki67: 4.8 ± 0.3 vs 2.0 ± 0.1 NTg vs mdx p<0.005 n=3), with a corresponding increase in cardiac atrophy gene expression at later time points (Foxo3 1.4 ± 0.2 vs 2.8 ± 0.3 NTg vs mdx p<0.005 n=3). Likewise, immunohistochemical (IHC) analyses showed significantly reduced proliferation in mdx hearts (Ki67: 13.7 ± 1.2 vs. 6.0 ± 1.0 NTg vs mdx p<0.005, n=3; pH3: 118 ± 5.3 vs 59 ± 4.5 p<0.005 n=3). RNA-Seq data revealed a disruption of the Yap signaling pathway, an important co-transcriptional mediator of cardiomyocyte proliferation, in mdx P4 hearts. By quantitative RT-PCR, cell cycle targets of Yap were reduced in mdx hearts. Finally, western blot and IHC revealed less nuclear localization of Yap in mdx cardiomyocytes. Collectively, this data suggests a novel mode of cardiac remodeling that contributes to the development of DMD-associated cardiomyopathy.

Calcium current properties in dystrophin-deficient ventricular cardiomyocytes from aged mdx mice.

Duchenne muscular dystrophy (DMD), caused by mutations in the gene encoding for the cytoskeletal protein dystrophin, is linked with severe cardiac complications including cardiomyopathy development and cardiac arrhythmias. We and others recently reported that currents through L‐type calcium (Ca) channels were significantly increased, and channel inactivation was reduced in dystrophin‐deficient ventricular cardiomyocytes derived from the mdx mouse, the most commonly used animal model for human DMD. These gain‐of‐function Ca channel abnormalities may enhance the risk of Ca‐dependent arrhythmias and cellular Ca overload in the dystrophic heart. All studies, which have so far investigated L‐type Ca channel properties in dystrophic cardiomyocytes, have used hearts from either neonatal or young adult mdx mice as cell source. In consequence, the dimension of the Ca channel abnormalities present in the severely‐diseased aged dystrophic heart has remained unknown. Here, we have studied potential abnormalities in Ca currents and intracellular Ca transients in ventricular cardiomyocytes derived from aged dystrophic mdx mice. We found that both the L‐type and T‐type Ca current properties of mdx cardiomyocytes were similar to those of myocytes derived from aged wild‐type mice. Accordingly, Ca release from the sarcoplasmic reticulum was normal in cardiomyocytes from aged mdx mice. This suggests that, irrespective of the presence of a pronounced cardiomyopathy in aged mdx mice, Ca currents and Ca release in dystrophic cardiomyocytes are normal. Finally, our data imply that dystrophin‐ regulation of L‐type Ca channel function in the heart is lost during aging.

0346 : Characterization of the calcium deregulation in cardiomyocytes from mdx mice, the main rodent model of the Duchenne muscular dystrophy

The Duchenne muscular dystrophy (DMD) is caused by an absence or mutation of the protein dystrophin, which leads to smooth, skeletal and cardiac muscles degeneration. Intracellular calcium deregulation, notably abnormally high cytosolic calcium concentration, is thought to contribute to muscle necrosis and to cardiomyopathy development. Despite its importance in cell necrosis, the mechanisms involved in this calcium deregulation are not fully understood. In our study, we have investigated several parameters of the calcium homeostasis in cardiomyocytes from mdx mice, the main rodent model of the DMD. All the parameters have been measured by using confocal microscopy calcium imaging experiments in cardiomyocytes loaded with the calcium indicator Fluo-3AM. Our results show an increase of the occurrence of spontaneous calcium events such as a higher frequency of calcium sparks and waves in the mdx cardiomyocytes compared with wild type mice. We also found that the calcium sensitivity of the ryanodine receptors (RyRs) is increased in cardiomyocytes from mdx mice. Finally, the sarcoplasmic reticulum (SR) calcium content has been evaluated by caffeine-induced calcium release. We found reduced calcium content of the SR in the mdx mice, which could be explained, at least in part, by an excessive spontaneous calcium activity due to the enhanced RyRs sensitivity. Our data suggest that targeting this SR calcium “leak” with pharmacological tools could be beneficial for the DMD patients. The author hereby declares no conflict of interest

Axial stretch-dependent cation entry in dystrophic cardiomyopathy: Involvement of several TRPs channels

In Duchenne muscular dystrophy (DMD), deficiency of the cytoskeletal protein dystrophin leads to well-described defects in skeletal muscle but also to dilated cardiomyopathy (DCM). In cardiac cells, the subsarcolemmal localization of dystrophin is thought to protect the membrane from mechanical stress. The dystrophin deficiency leads to membrane instability and a high stress-induced Ca2+ influx due to dysregulation of sarcolemmal channels such as stretch-activated channels (SACs). In this work divalent cation entry has been explored in isolated ventricular Wild Type (WT) and mdx cardiomyocytes in two different conditions: at rest and during the application of an axial stretch. At rest, our results suggest that activation of TRPV2 channels participates to a constitutive basal cation entry in mdx cardiomyocytes.Using microcarbon fibres technique, an axial stretchwas applied to mimic effects of physiological conditions of ventricular filling and study on cation influx bythe Mn2+-quenching techniquedemonstrated a high stretch-dependentcationic influx in dystrophic cells, partially due to SACs. Involvement of TRPs channels in this excessive Ca2+ influx has been investigated using specific modulators and demonstratedboth sarcolemmal localization and an abnormal activity of TRPV2 channels.In conclusion, TRPV2 channels are demonstrated here to play a key role in cation influx and dysregulation in dystrophin deficient cardiomyocytes, enhanced in stretching conditions.

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.

Targeting Mechanically‐Activated NOS Signaling in Dystrophic Cardiomyopathy

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. Our objective was to identify the mechanisms of how the DGC regulates cardiac muscle nitric oxide synthase (NOS) activity. Isolated adult wild‐type (WT) or dystrophin‐deficient mdx mouse cardiomyocytes were plated on deformable membranes, and live‐cell DAF‐FM dye fluorescence and biochemical approaches were used to investigate how the DGC regulates mechanically‐activated NO signaling. 1Hz cyclic stretch of WT cardiomyocytes significantly increased NO production and neuronal NOS (nNOS) S1412 phosphorylation. In contrast, stretch‐induced NO production and nNOS phosphorylation were impaired in mdx cardiomyocytes, suggesting that dystrophin regulates the mechanical activation of NOS. We found that inhibition of AMP‐activated protein kinase (AMPK) inhibited stretch‐induced NO production in WT cardiomyocytes, indicating that AMPK plays a critical role in mechanoactivation of NOS. Importantly, direct pharmacological activation of AMPK increased nNOS S1412 phosphorylation and was sufficient to enhance NO production in mdx cardiomyocytes. We conclude that the DGC regulates the mechanical activation of cardiac NOS, and that pharmacological AMPK activation may be a therapeutic strategy to restore NOS activity in dystrophin‐deficient cardiomyocytes. Supported by the Muscular Dystrophy Association, the University of Michigan CVRE Training Program, and NIH T32 GM‐08322.

Morpholino Oligomer Peptide Therapy Improves Mitochondrial Function in mdx Cardiomyopathy

Duchenne Muscular Dystrophy (DMD) is a fatal muscle wasting disorder. It is commonly associated with cardiomyopathy that is characterised by disrupted cytoskeletal architecture and mitochondrial dysfunction. Activation of the L-type Ca2+ channel (LTCC) provides the Ca2+ influx required for contraction, but also causes an increase in mitochondrial membrane potential (Ψm) in a calcium-independent manner. This involves the movement of cytoskeletal proteins and regulation of mitochondrial VDAC. The increase in Ψm (JC-1 fluorescence) after activation of LTCC is absent in cardiomyocytes isolated from hearts of mdx mice, a murine model of DMD (1.5±1.0%, n=4 vs. 12.1±1.4%, n=10 in C57BL/10ScSnArc controls) but can be restored using antisense morpholino oligomers to induce exon skipping of dystrophin exon 23 (M23D). Here we investigate the effects of M23D peptides on changes in Ψm and mitochondrial oxygen consumption (flavoprotein autofluorescence) induced by LTCC activation. Male (M) and female (F) neonatal mdx mice were injected (i.p.) for 3 weeks and divided into two treatment groups: (i) 120mg/kg once per week; (ii) 30mg/kg four times per week. Activation of LTCC with the dihydropyridine agonist BayK(-) induced an increase in Ψm in cardiomyocytes from animals treated with a single weekly dose (M: 17±2%, n=5; F: 16±3%, n=12) and with multiple weekly doses (M: 16±2%, n=6; F: 13±2%, n=15). BayK(-) increased flavoprotein oxidation in mdx cardiomyocytes from animals treated with a single weekly dose (M:13±3%, n=13; F: 10±3%, n=9) and with multiple doses per week (M: 17±2%, n=16; F: 14±2, n=24). These data indicate that either single or multiple i.p. injections per week of morpholino oligo peptides are effective at restoring cardiac mitochondrial function in both male and female mdx mice.

Age-dependent changes in diastolic Ca2+ and Na+ concentrations in dystrophic cardiomyopathy: Role of Ca2+ entry and IP3

Duchenne muscular dystrophy (DMD) is a lethal X-inherited disease caused by dystrophin deficiency. Besides the relatively well characterized skeletal muscle degenerative processes, DMD is also associated with a dilated cardiomyopathy that leads to progressive heart failure at the end of the second decade. The aim of the present study was to characterize the diastolic Ca2+ concentration ([Ca2+]d) and diastolic Na+ concentration ([Na+]d) abnormalities in cardiomyocytes isolated from 3-, 6-, 9-, and 12-month old mdx mice using ion-selective microelectrodes. In addition, the contributions of gadolinium (Gd3+)-sensitive Ca2+ entry and inositol triphosphate (IP3) signaling pathways in abnormal [Ca2+]d and [Na+]d were investigated. Our results showed an age-dependent increase in both [Ca2+]d and [Na+]d in dystrophic cardiomyocytes compared to those isolated from age-matched wt mice. Gd3+ treatment significantly reduced both [Ca2+]d and [Na+]d at all ages. In addition, blockade of the IP3-pathway with either U-73122 or xestospongin C significantly reduced ion concentrations in dystrophic cardiomyocytes. Co-treatment with U-73122 and Gd3+ normalized both [Ca2+]d and [Na+]d at all ages in dystrophic cardiomyocytes. These data showed that loss of dystrophin in mdx cardiomyocytes produced an age-dependent intracellular Ca2+ and Na+ overload mediated at least in part by enhanced Ca2+ entry through Gd3+ sensitive transient receptor potential channels (TRPC), and by IP3 receptors.

PGz Reverses Cardiac Dysfunction in Dystrophic Mice

Duchenne muscular dystrophy (DMD) is caused by the absence of dystrophin expression and is one of the most common genetic diseases of children worldwide. Duchenne cardiomyopathy (D-CM) is observed in all patients suffering from DMD as the disease progresses. Despite knowing the cause of DMD and D-CM, there are no treatments which reverse its inevitable progression.Periodic Acceleration (pGz) is a novel method that applies repetitive, sinusoidal, head-to-foot motion to a horizontally positioned body to induce increased pulsatile shear stress in fluid channels throughout the entire body. pGz increases the expression of eNOS, and nNOS and other endothelial derived proteins. Experiments were conducted in cardiomyocytes isolated from Wt and mdx mice (3, 6, 9 and 12-mo), treated or not with pGz (one hour for 16 consecutive days). Resting Ca2+ ([Ca2+]r) and Na+ ([Na+]r) were determined using Ca2+ and Na+ selective microelectrodes. pGz did not modify [Ca2+]r or [Na+]r in cardiomyocytes from Wt mice at any age (3, 6, 9 and 12-mo). In contrast, pGz restored [Ca2+]r and [Na+]r to Wt levels in cardiomyocytes from 3, 6 and 9-mo mdx mice and significantly reduced [Ca2+]r and [Na+]r in those from 12-mo old mdx mice. To assess the [NO]i Wt and mdx cardiomyocytes isolated from mice of 3, 6, 9 and 12-mo of age were loaded with 10µM DAF-FM DA (Molecular Probes, OR, USA). pGz did not significantly modify DAF fluorescence in Wt cardiomyocytes at any age. In contrast, pGz reduced DAF-Fluorescence in treated mdx cardiomyocytes at 3,6, 9 and 12 mo of age compared to untreated mdx cardiomyocytes of the same age. Our results suggest that pGz can be a drug free therapeutic approach to treat the cardiomyopathy observed in patients suffering from D-CM.

P.11.5 PMO-mediated dystrophin exon 23 skipping restores mitochondrial function in the mdx mouse heart

Approximately 20% of boys with Duchenne Muscular Dystrophy will die of dilated cardiomyopathy. The cardiomyopathy is characterised by disrupted structure and function of cardiac muscle cells and reduced energy production. However, the mechanisms responsible for the altered energy metabolism have been poorly understood. We have previously sought to identify the mechanisms for metabolic inhibition in mdx mouse cardiomyopathy. Calcium influx through the L-type Ca2+ channel (also known as the dihydropyridine receptor) in cardiac myocytes is essential for contraction. Calcium is also important for the regulation of mitochondrial function and production of ATP that is required to meet the energy demands of the heart. We have shown that the L-type Ca2+ channel can regulate mitochondrial function and metabolic activity in cardiac myocytes. In mdx heart, the communication between the L-type Ca2+ channel and the mitochondria is altered as a result of disruption of the cytoskeletal architecture. This contributes to metabolic inhibition in the mdx heart. We demonstrate that treatment of mdx mice with a phosphorodiamidate morpholino oligomer, designed to induce skipping of dystrophin exon 23, “restored” the increase in mitochondrial membrane potential in mdx cardiomyocytes after activation of the L-type Ca2+ channel with the dihydropyridine receptor agonist BayK (−). These results confirm that metabolic inhibition occurs as a result of the absence of dystrophin, and oligomer therapy may be able to normalise metabolic activity and restore contractility in mdx mouse heart.

Age‐Dependent Myocardial Sheet Dysfunction from 3 to 16 months in Duchenne Muscular Dystrophy Mice (mdx) Defined by Diffusion Tensor Magnetic Resonance Imaging (DTI)

INTROCardiomyocytes are organized in laminar structures (aka, sheets) that contribute to wall thickening/thinning. Diffusion tensor MRI (DTI) measures the sheet reorientation function in intact hearts. We have reported that hearts of 16‐mo old mdx mice exhibit abnormal Ca transients &amp; Ca‐dependent sheet dysfunction at the base but not apex. Because Ca mishandling in isolated mdx cardiomyocytes is age dependent, we hypothesized that sheet dysfunction in intact mdx hearts may also be age dependent.METHODSExcised hearts from 3 &amp; 16 mo old mdx and wildtype were prepared for Langendorff perfusion, and sequentially arrested in diastole then systole for DTI (Varian 11.7 T). The diastolic arrest was achieved with either normal [Ca] (1.2 mM, noted as NC) or low [Ca] (0.078 mM, noted as LC) cardioplegic solution to test the Ca dependency. The systolic arrest was achieved with barium.RESULTSSystolic |β| (defined below) was similar between WT and mdx, whereas diastolic |β| in mdx‐NC was lower for age matched WT‐NC at the base but not apex, indicating a regional failure to fully relax. Low [Ca] (mdx‐LC) perfusion restores the diastolic |β| to normal values at both ages. The Δ|β| (i.e., |β| of each group ‐ |β| of age matched WT‐NC) is 50% larger in 16 mo than for 3 mo old mice, indicating that Ca dependent sheet dysfunction is age dependent and regionally manifest despite the ubiquitous dystrophin deficiency.

Ultrastructural and Functional Alterations of EC Coupling Elements in mdx Cardiomyocytes: An Analysis from Membrane Surface to Depth

A dilated cardiomyopathy (DCM) is associated with Duchenne muscular dystrophy (DMD). The loss of dystrophin leads to membrane instability and calcium dysregulation in skeletal muscle but effects of such a loss are not elucidated at cardiomyocytes level. We sought to examine whether membrane and transverse tubules damages occur in ventricular myocytes from mdx mouse model of DMD and how they impact the function of single excitation-contraction coupling elements. Scanning ion conductance microscopy (SICM) was used to characterize the integrity loss of living mdx cardiomyocytes surface. 2D Fourier transform analysis of labeled internal networks (transverse tubules, alpha-actinin, dihydropyridine receptors, ryanodine receptors) was performed to evaluate internal alterations. During calcium measurements, "smart microperfusions" of depolarizing solutions were applied through SICM nanopipette, stimulating single tubules elements. These approaches revealed structural membrane surface (39% decrease for Z-groove ratio) and transverse tubules disorganization (21% transverse tubules ratio decrease) in mdx as compared to control. These disruptions were associated with functional alterations (sixfold increase of calcium signal duration and twofold increase of sparks frequency). In DCM associated with DMD, myocytes display evident membrane alterations at the surface level but also in the cell depth with a disruption of transverse tubules network as observed in other cases of heart failure. These ultrastructural changes are associated with changes in the function of some coupling elements. Thus, these profound disruptions may play a role in calcium dysregulation through excitation-contraction coupling elements perturbation and suggest a transverse tubules stabilizing role for dystrophin.