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AP upstroke velocity is reduced in DKO mice. (A) Representative APs recorded from the cardiomyocytes of mdx and DKO mice. (B) AP upstrokes from (A) displayed on expanded time-scale reveal a slowing of depolarization velocity above threshold. (C) Maximal AP upstroke velocity is reduced by 30% in cardiomyocytes from DKO (black bar) compared with mdx mice (white bar). Neither resting potential (D) nor AP amplitude (E) was different between DKO and mdx cardiomyocytes. (F ) Bar graph showing AP duration to 30, 50, and 90% repolarization in mdx and DKO cardiomyocytes. n ¼ 12 cells (mdx) and 10 cells (DKO), respectively. **P , 0.01; two-tailed Student's t-test.
Source publication
Duchenne muscular dystrophy (DMD) is a severe striated muscle disease due to the absence of dystrophin. Dystrophin deficiency results in dysfunctional sodium channels and conduction abnormalities in hearts of mdx mice. Disease progression in the mdx mouse only modestly reflects that of DMD patients, possibly due to utrophin up-regulation. Here, we...
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... v 1.5 plays a key role in the conduction of the cardiac electrical impulse and determines the upstroke velocity (dV m /dt) of the cardiac AP. Considering the strong reduction in the Na v 1.5 protein and current in DKO compared with mdx mice, we investigated whether these alterations were reflected in the APs recorded in freshly isolated ventricular myocytes from these two groups ( Figure 5A). When analysing the AP upstrokes above threshold ( Figure 5B), we found a significant reduction of about 30% in the maximal upstroke velocity in DKO cardiomyocytes compared with mdx (110.6 + 9.2 mV/ms in DKO and 151.8 + 9.4 mV/ms in mdx, Figure 5C), consistent with the reduction in functional Na v 1.5 protein. ...
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... the strong reduction in the Na v 1.5 protein and current in DKO compared with mdx mice, we investigated whether these alterations were reflected in the APs recorded in freshly isolated ventricular myocytes from these two groups ( Figure 5A). When analysing the AP upstrokes above threshold ( Figure 5B), we found a significant reduction of about 30% in the maximal upstroke velocity in DKO cardiomyocytes compared with mdx (110.6 + 9.2 mV/ms in DKO and 151.8 + 9.4 mV/ms in mdx, Figure 5C), consistent with the reduction in functional Na v 1.5 protein. As shown in Figure 5D and E, no difference was found in either resting potential (270.6 + 0.8 and 272.2 + 0.5 mV in DKO and mdx, respectively) or AP amplitude (118.1 + 2.5 and 120.9 + 2.1 mV in DKO and mdx, respectively). ...
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... the strong reduction in the Na v 1.5 protein and current in DKO compared with mdx mice, we investigated whether these alterations were reflected in the APs recorded in freshly isolated ventricular myocytes from these two groups ( Figure 5A). When analysing the AP upstrokes above threshold ( Figure 5B), we found a significant reduction of about 30% in the maximal upstroke velocity in DKO cardiomyocytes compared with mdx (110.6 + 9.2 mV/ms in DKO and 151.8 + 9.4 mV/ms in mdx, Figure 5C), consistent with the reduction in functional Na v 1.5 protein. As shown in Figure 5D and E, no difference was found in either resting potential (270.6 + 0.8 and 272.2 + 0.5 mV in DKO and mdx, respectively) or AP amplitude (118.1 + 2.5 and 120.9 + 2.1 mV in DKO and mdx, respectively). ...
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... analysing the AP upstrokes above threshold ( Figure 5B), we found a significant reduction of about 30% in the maximal upstroke velocity in DKO cardiomyocytes compared with mdx (110.6 + 9.2 mV/ms in DKO and 151.8 + 9.4 mV/ms in mdx, Figure 5C), consistent with the reduction in functional Na v 1.5 protein. As shown in Figure 5D and E, no difference was found in either resting potential (270.6 + 0.8 and 272.2 + 0.5 mV in DKO and mdx, respectively) or AP amplitude (118.1 + 2.5 and 120.9 + 2.1 mV in DKO and mdx, respectively). Finally, although AP durations of 30 and 50% repolarization (APD30 and APD50) were unchanged, APD90 was slightly prolonged in DKO mice, although this difference was not significant ( Figure 5F). ...
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... shown in Figure 5D and E, no difference was found in either resting potential (270.6 + 0.8 and 272.2 + 0.5 mV in DKO and mdx, respectively) or AP amplitude (118.1 + 2.5 and 120.9 + 2.1 mV in DKO and mdx, respectively). Finally, although AP durations of 30 and 50% repolarization (APD30 and APD50) were unchanged, APD90 was slightly prolonged in DKO mice, although this difference was not significant ( Figure 5F). ...
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Background:
Duchenne muscular dystrophy is a fatal cardiac and skeletal muscle disease resulting from mutations in the dystrophin gene. We have previously demonstrated that a dystrophin-associated protein, sarcospan (SSPN), ameliorated Duchenne muscular dystrophy skeletal muscle degeneration by activating compensatory pathways that regulate muscle...
Golden retriever muscular dystrophy (GRMD) is a genetic myopathy corresponding to Duchenne muscular dystrophy (DMD) in humans. Muscle atrophy is known to be associated with degradation of the dystrophin-glycoprotein complex (DGC) via the ubiquitin-proteasome pathway. In the present study, we investigated the effect of bortezomib treatment on the mu...
The Duchenne and Becker muscular dystrophies (DMD, BMD) show significant comorbid diagnosis for autism, and the genomic sequences encoding the proteins responsible for these diseases, the dystrophin and associated proteins, have been proposed as new candidate risk loci for autism. Dystrophin is expressed not only in muscles but also in central inhi...
Citations
... Apoptosis is a proteolytic process related to meat tenderization under oxidative stress [49,54,55], which can result from increased mitochondrial membrane permeability [50]. Utrophin, encoded by UTRN, the hub gene of M1, is a therapeutic target for Duchenne muscular disease [56,57]. The upregulation of this protein reduces oxidative stress and mitochondrial pathology [57], and this protein was related to the homeostasis of the sodium channel in the heart [56], attenuating the Duchenne's pathology [56]. ...
... Utrophin, encoded by UTRN, the hub gene of M1, is a therapeutic target for Duchenne muscular disease [56,57]. The upregulation of this protein reduces oxidative stress and mitochondrial pathology [57], and this protein was related to the homeostasis of the sodium channel in the heart [56], attenuating the Duchenne's pathology [56]. Among the 15 genes within M1 that showed DASE for the same enriched trait (WBSF0), there are Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), previously identified as a DEG for Ca [22], and Prune Homolog 2 With BCH Domain (PRUNE2), differentially expressed in extremes of RFI [24]. ...
... Utrophin, encoded by UTRN, the hub gene of M1, is a therapeutic target for Duchenne muscular disease [56,57]. The upregulation of this protein reduces oxidative stress and mitochondrial pathology [57], and this protein was related to the homeostasis of the sodium channel in the heart [56], attenuating the Duchenne's pathology [56]. Among the 15 genes within M1 that showed DASE for the same enriched trait (WBSF0), there are Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), previously identified as a DEG for Ca [22], and Prune Homolog 2 With BCH Domain (PRUNE2), differentially expressed in extremes of RFI [24]. ...
Traditional transcriptomics approaches have been used to identify candidate genes affecting economically important livestock traits. Regulatory variants affecting these traits, however, remain under covered. Genomic regions showing allele-specific expression (ASE) are under the effect of cis-regulatory variants, being useful for improving the accuracy of genomic selection models. Taking advantage of the better of these two methods, we investigated single nucleotide polymorphisms (SNPs) in regions showing differential ASE (DASE SNPs) between contrasting groups for beef quality traits. For these analyses, we used RNA sequencing data, imputed genotypes and genomic estimated breeding values of muscle-related traits from 190 Nelore (Bos indicus) steers. We selected 40 contrasting unrelated samples for the analysis (N = 20 animals per contrasting group) and used a beta-binomial model to identify ASE SNPs in only one group (i.e., DASE SNPs). We found 1479 DASE SNPs (FDR ≤ 0.05) associated with 55 beef-quality traits. Most DASE genes were involved with tenderness and muscle homeostasis, presenting a co-expression module enriched for the protein ubiquitination process. The results overlapped with epigenetics and phenotype-associated data, suggesting that DASE SNPs are potentially linked to cis-regulatory variants affecting simultaneously the transcription and phenotype through chromatin state modulation.
... This finding suggests that either the SIV-dependent regulation of Na + channel expression is not essential within the ID region or Nav1.5 localization within the ID region was so crucial that compensatory mechanisms were employed to retain sodium channel expression and function at the ID region [86]. The latter appears to be a more plausible explanation because a similar mechanism has been shown to occur for Nav1.5 channel lateral membrane pool in mdx mice, in which utrophin a homolog of dystrophin that also interacts with Nav1.5 was found to be upregulated in these cardiac cells [87]. ...
SAP97 is a member of the MAGUK family of proteins, but unlike other MAGUK proteins that are selectively expressed in the CNS, SAP97 is also expressed in peripheral organs, like the heart and kidneys. SAP97 has several protein binding cassettes, and this review will describe their involvement in creating SAP97-anchored multiprotein networks. SAP97-anchored networks localized at the inner leaflet of the cell membrane play a major role in trafficking and targeting of membrane G protein-coupled receptors (GPCR), channels, and structural proteins. SAP97 plays a major role in compartmentalizing voltage gated sodium and potassium channels to specific cellular compartments of heart cells. SAP97 undergoes extensive alternative splicing. These splice variants give rise to different SAP97 isoforms that alter its cellular localization, networking, signaling and trafficking effects. Regarding GPCR, SAP97 binds to the ß1-adrenergic receptor and recruits AKAP5/PKA and PDE4D8 to create a multiprotein complex that regulates trafficking and signaling of cardiac ß1-AR. In the kidneys, SAP97 anchored networks played a role in trafficking of aquaporin-2 water channels. Cardiac specific ablation of SAP97 (SAP97-cKO) resulted in cardiac hypertrophy and failure in aging mice. Similarly, instituting transverse aortic constriction (TAC) in young SAP97 c-KO mice exacerbated TAC-induced cardiac remodeling and dysfunction. These findings highlight a critical role for SAP97 in the pathophysiology of a number of cardiac and renal diseases, suggesting that SAP97 is a relevant target for drug discovery.
... The absence of dystrophin in DMD has the potential to alter trafficking, localization, and function of DAPC-associated proteins in skeletal and cardiac muscle (Lohan et al., 2005). For example, the expression and function of ion channels are defective in ventricular cardiomyocytes of the mdx mouse model (Gavillet et al., 2006;Koenig et al., 2014;Rubi et al., 2017;Koenig et al., 2011;Albesa et al., 2011). The absence of dystrophin in young mdx mice affects the function of Na V 1.5, leading to cardiac conduction defects (Gavillet et al., 2006). ...
... Results from our laboratory and others strongly suggest that Na V 1.5 and Kir2.1 control cardiac excitability by mutually modulating each other's surface expression (Milstein et al., 2012;Albesa et al., 2011;Petitprez et al., 2011;Leonoudakis et al., 2004;Matamoros et al., 2016;Ponce-Balbuena et al., 2018). At the lateral membrane, Na V 1.5 and Kir2.1 channels form macromolecular complexes ('channelosomes') that include α1-syntrophin, which is a part of the DAPC (Gavillet et al., 2006). ...
... As such, the reduction in the Na V 1.5 and Kir2.1 protein levels could be related to ubiquitylation and proteasome degradation as suggested previously in studies in dystrophin-deficient mdx 5cv mice (Rougier et al., 2013). However, our results in DMD iPSC-CMs strongly suggest that disruption of the DAPC due to lack of dystrophin significantly impairs ion channel expression and function (Gavillet et al., 2006;Koenig et al., 2011;Albesa et al., 2011). Specifically, we demonstrate that the decrease in ion channel current densities is the result of Na V 1.5 and Kir2.1 trafficking and membrane highlights the increased outward component of I K1 at less negative potentials upon syntrophin expression. ...
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.
... In the present study, the protein expression levels of Plakophilin-2 (PKP2) and desmoglein-2 (DSG2) were slightly down-regulated (18%, 19%, respectively), but the expression of dystrophin was significantly increased (65%) in heart tissue of Mog1 -/mice. All these proteins (PKP2, DSG2 and dystrophin) co-localizes within the Nav1.5 channel complex, and regulate the proper cell surface expression of Nav1.5 and its targeting to distinct cell membrane domains [21,[42][43][44]. ...
Our earlier studies identified MOG1 as a Nav1.5-binding protein that promotes Nav1.5 intracellular trafficking to plasma membranes. Genetic studies have identified MOG1 variants responsible for cardiac arrhythmias. However, the physiological functions of MOG1 in vivo remain incompletely characterized. In this study, we generated Mog1 knockout (Mog1−/−) mice. Mog1−/− mice did not develop spontaneous arrhythmias at the baseline, but exhibited a prolongation of QRS duration. Mog1−/− mice treated with isoproterenol (ISO), but not with flecainide, exhibited an increased risk of arrhythmias and even sudden death. Mog1−/− mice had normal cardiac morphology, however, LV systolic dysfunction was identified and associated with an increase in ventricular fibrosis. Whole-cell patch-clamping and Western blotting analysis clearly demonstrated the normal cardiac expression and function of Nav1.5 in Mog1−/− mice. Further RNA-seq and iTRAQ analysis identified critical pathways and genes, including extracellular matrix (Mmp2), gap junction (Gja1), and mitochondrial components that were dysregulated in Mog1−/− mice. RT-qPCR, Western blotting, and immunofluorescence assays revealed reduced cardiac expression of Gja1 in Mog1−/− mice. Dye transfer assays confirmed impairment of gap-junction function; Cx43 gap-junction enhancer ZP123 decreased arrhythmia inducibility in ISO-treated Mog1−/− mice. Transmission electron microscopy analysis revealed abnormal sarcomere ultrastructure and altered mitochondrial morphology in Mog1−/− mice. Mitochondrial dynamics was found to be disturbed, and associated with a trend toward increased mitochondrial fusion in Mog1−/− mice. Meanwhile, the level of ATP supply was increased in the hearts of Mog1−/− mice. These results indicate that MOG1 plays an important role in cardiac electrophysiology and cardiac contractile function.
... At the lateral membrane, Na V 1.5 interacts with the syntrophin-dystrophin complex, and both loss of dystrophin and disruption of the Na V 1.5-syntrophin interaction have been shown to decrease I Na in this subcellular domain and cause cardiac conduction slowing. [38][39][40] Similarly, disruption of the ID-localized interacting proteins PKP2 (plakophilin-2) and CAR (coxsackie-and adenovirus receptor) reduces I Na specifically at the ID. 1,41 Because I Na is larger at the ID than at the lateral membrane, 42 loss of Na V 1.5 at the ID results in a larger reduction of wholecell I Na and is consequently more detrimental to cardiac conduction. ...
Rationale: Loss-of-function of the cardiac sodium channel NaV1.5 causes conduction slowing and arrhythmias. NaV1.5 is differentially distributed within subcellular domains of cardiomyocytes, with sodium current (INa) being enriched at the intercalated discs (ID). Various pathophysiological conditions associated with lethal arrhythmias display ID-specific I Na reduction, but the mechanisms underlying microdomain-specific targeting of NaV1.5 remain largely unknown.
Objective: To investigate the role of the microtubule (MT) plus-end tracking proteins end binding protein 1 (EB1) and CLIP-associated protein 2 (CLASP2) in mediating NaV1.5 trafficking and subcellular distribution in cardiomyocytes.
Methods and Results: EB1 overexpression in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) resulted in enhanced whole-cell INa, increased action potential (AP) upstroke velocity (Vmax), and enhanced Na V 1.5 localization at the plasma membrane as detected by multi-color stochastic optical reconstruction microscopy (STORM). Fluorescence recovery after photobleaching (FRAP) experiments in HEK293A cells demonstrated that EB1 overexpression promoted NaV1.5 forward trafficking. Knockout of MAPRE1 in hiPSC-CMs led to reduced whole-cell INa , decreased V max and AP duration (APD) prolongation. Similarly, acute knockout of the MAPRE1 homolog in zebrafish (mapre1b) resulted in decreased ventricular conduction velocity and Vmax as well as increased APD. STORM imaging and macropatch INa measurements showed that subacute treatment (2-3 hours) with SB216763 (SB2), a GSK3β inhibitor known to modulate CLASP2-EB1 interaction, reduced GSK3β localization and increased NaV1.5 and I Na preferentially at the ID region of wild type murine ventricular cardiomyocytes. By contrast, SB2 did not affect whole cell INa or NaV1.5 localization in cardiomyocytes from Clasp2-deficient mice, uncovering the crucial role of CLASP2 in SB2-mediated modulation of NaV1.5 at the ID.
Conclusions: Our findings demonstrate the modulatory effect of the MT plus-end tracking protein EB1 on NaV1.5 trafficking and function, and identify the EB1-CLASP2 complex as a target for preferential modulation of INa within the ID region of cardiomyocytes.
... robustly decreased in DMD 21,22 . In various DMD mouse models it has been demonstrated that loss of dystrophin leads to a decrease in sodium current (I Na ), a known risk factor for arrhythmias and sudden cardiac death 20,23 . Crucially, a combination of I Na loss and myocardial fibrosis may result in a markedly disturbed cardiac conduction, greatly increasing the risk for potentially lethal cardiac arrhythmias in DMD patients. ...
... In accordance with previous studies 19,20,23 , we here show loss of I Na upon dystrophin absence, leading to conduction slowing and potentially to arrhythmias. In our current study, I Na loss was combined with a positive shift in the V 1/2 of Na V 1.5 inactivation, which was also found in a previous study using a different DMD mouse model 23 . This shift in inactivation theoretically results in increased Na V 1.5 availability, possibly compensating for the loss of I Na . ...
Duchenne muscular dystrophy (DMD) is a progressive neuromuscular disorder caused by loss of dystrophin. This lack also affects cardiac structure and function, and cardiovascular complications are a major cause of death in DMD. Newly developed therapies partially restore dystrophin expression. It is unclear whether this will be sufficient to prevent or ameliorate cardiac involvement in DMD. We here establish the cardiac electrophysiological and structural phenotype in young (2-3 months) and aged (6-13 months) dystrophin-deficient mdx mice expressing 100% human dystrophin (hDMD), 0% human dystrophin (hDMDdel52-null) or low levels (~ 5%) of human dystrophin (hDMDdel52-low). Compared to hDMD, young and aged hDMDdel52-null mice displayed conduction slowing and repolarisation abnormalities, while only aged hDMDdel52-null mice displayed increased myocardial fibrosis. Moreover, ventricular cardiomyocytes from young hDMDdel52-null animals displayed decreased sodium current and action potential (AP) upstroke velocity, and prolonged AP duration at 20% and 50% of repolarisation. Hence, cardiac electrical remodelling in hDMDdel52-null mice preceded development of structural alterations. In contrast to hDMDdel52-null, hDMDdel52-low mice showed similar electrophysiological and structural characteristics as hDMD, indicating prevention of the cardiac DMD phenotype by low levels of human dystrophin. Our findings are potentially relevant for the development of therapeutic strategies aimed at restoring dystrophin expression in DMD.
... Together with other groups, these authors concluded that the altered AP properties were due to a significant reduction in Na + currents (i.e. Na + current densities) in dystrophic cardiomyocytes when compared to the WT controls (Albesa et al., 2011;Gavillet et al., 2006;Koenig et al., 2011;Rougier et al., 2013). The reduced Na + currents were due to a down regulation of Nav 1.5 protein expression and loss of Na + channel function (Albesa et al., 2011;Colussi et al., 2010;Koenig et al., 2011). ...
... Na + current densities) in dystrophic cardiomyocytes when compared to the WT controls (Albesa et al., 2011;Gavillet et al., 2006;Koenig et al., 2011;Rougier et al., 2013). The reduced Na + currents were due to a down regulation of Nav 1.5 protein expression and loss of Na + channel function (Albesa et al., 2011;Colussi et al., 2010;Koenig et al., 2011). Therefore, if dystrophin deficiency impacted the Purkinje cell function in a similar way as in cardiomyocytes, it is conceivable that the sodium channel properties in dystrophic Purkinje cells may be altered and malfunctions, and consequently lead to the altered AP properties seen in the current study. ...
Duchenne muscular dystrophy (DMD) is a rapidly progressive X-linked recessive disease affecting about 1 in 3500 live male births. It is caused by mutations in the dystrophin gene, which result in the loss of dystrophin or expression of a non-functional truncated protein product. Full-length dystrophin is mainly expressed in muscles and the central nervous system. In addition to the degeneration of skeletal musculature, about one-third of patients with DMD display various degrees of intellectual impairment, commonly found with intelligence quotient (IQ) scores of one standard deviation below (IQ of 85) the normal population mean (IQ of 100). However, the mechanism underlying the cognitive deficits in DMD remains unclear and no effective treatment is available to reverse this condition in the affected individual. Recent studies showed that the life span of DMD patients today has increased from teens to their fourth decades. With longer survival, the quality of life becomes increasing important. Therefore, research on the cognitive aspect of DMD is as important as research on the muscular aspects because improvements in cognitive function will enhance the quality of life for the growing population of adult DMD patients. The aim of this thesis was to investigate the role of dystrophin in the central nervous system of the mdx mouse, a widely accepted murine model for DMD. This study employed the use of animal with different age groups, corresponding to young (3-4 months), adult (11-12 months), and aged (23-26 months). Adult and aged mdx mice are the focus in this study with findings from the older mouse especially valuable as, disease progression in aged mice closely resembling that of DMD. As numerous evidence has shown a high similarity between the specific cognitive dysfunctions seen in DMD (i.e. impaired verbal intelligence) and in patients with cerebellar lesions (i.e. language disorders), this study examined the function of cerebellar Purkinje cells in mdx mice using electrophysiological recording and calcium imaging. Overall, the data presented in this thesis provides new insights into the role of dystrophin in cerebellar Purkinje neurons. The findings suggest that dystrophin is important for normal inhibitory synaptic function, intrinsic electrophysiological properties, and calcium handling of the mature cerebellar Purkinje cells. The consequences of the absence of dystrophin including the altered GABAA receptor clustering and reduced peak amplitude of mIPSCs could be ameliorated when dystrophin was successfully rescued with Pip6f-PMO in an organotypic mdx cerebellar culture. If mdx mice and DMD patients share similar neuropathogenesis, the development of drugs targeting the altered functions in mdx Purkinje cells may serve as a potential therapy in alleviating the cognitive impairments seen in DMD.
... These specialized cells are reliant on dystrophin to support organization of ion channels and promote coordinated electrical activity 12 . In mouse models deficient in dystrophin, misregulation of critical ion channels in specialized conduction tissue cells disturbs electrical activity, contributing to the arrhythmogenesis in the dystrophin-deficient heart 13,14 . On electrocardiogram, both short and long PR intervals may be observed, and complete heart block has been observed 15 . ...
... We and others have previously reported that ventricular "working" cardiomyocytes from mdx mouse hearts exhibit an abnormally reduced Na ϩ current (I Na ), entailing a slowed action potential upstroke in these cells (1,10,20,21). According with this finding, Na v 1.5 Na ϩ channel protein expression in mdx compared with wild-type mouse hearts is diminished (1,10). ...
... We and others have previously reported that ventricular "working" cardiomyocytes from mdx mouse hearts exhibit an abnormally reduced Na ϩ current (I Na ), entailing a slowed action potential upstroke in these cells (1,10,20,21). According with this finding, Na v 1.5 Na ϩ channel protein expression in mdx compared with wild-type mouse hearts is diminished (1,10). Although reduced I Na in working cardiomyocytes may contribute to slowed ventricular conduction in the dystrophic heart, a functional impairment of this particular cell type cannot provide an explanation for prolonged His-ventricular intervals and bundle branch block in patients with DMD. ...
... We and others have previously reported that working cardiomyocytes from mdx mouse hearts exhibit abnormally reduced I Na (1,10,20,21). This finding has provided a potential explanation for the prolonged QRS complex observed in the electrocardiograms of dystrophic mdx when compared with wild-type mice (10,20). ...
Cardiac arrhythmias significantly contribute to mortality in Duchenne muscular dystrophy (DMD), a degenerative muscle disease triggered by mutations in the gene encoding for the intracellular protein dystrophin. A major source for the arrhythmias in DMD patients is impaired ventricular impulse conduction, which predisposes for ventricular asynchrony, decreased cardiac output, and the development of reentrant mechanisms. The reason for ventricular conduction impairments and the associated arrhythmias in the dystrophic heart has remained unidentified. In the present study, we explored the hypothesis that dystrophin-deficient cardiac Purkinje fibers have reduced Na currents (I Na ), which would represent a potential mechanism underlying slowed ventricular conduction in the dystrophic heart. Therefore, by using a Langendorff perfusion system, we isolated Purkinje fibers from the hearts of adult wild type control and dystrophin-deficient mdx mice. eGFP expression under control of the connexin 40 gene allowed us to discriminate Purkinje fibers from eGFP-negative ventricular working cardiomyocytes after cell isolation. Finally, we recorded I Na from wild type and dystrophic mdx Purkinje fibers for comparison by means of the whole cell patch clamp technique. We found substantially reduced I Na densities in mdx compared to wild type Purkinje fibers, suggesting that dystrophin deficiency diminishes I Na . Because Na channels in the Purkinje fiber membrane represent key determinants of ventricular conduction velocity, we propose that reduced I Na in Purkinje fibers at least partly explains impaired ventricular conduction and the associated arrhythmias in the dystrophic heart.
... [37][38][39] In contrast, mutant Na V 1.5 interacting proteins located at the lateral membranes of adult cardiomyocytes, such as dystrophin and syntrophin, do not show embryonic lethality. [40][41][42] Accordingly, the homozygous mutations Scn5a-D1275N and Scn5a-ΔSIV result in I Na loss mainly at the lateral membrane and these mice are viable. 43,44 However, whole cell I Na was moderately affected in these models, 77.2% and 49.2% respectively, which could also underlie the absence of embryonic lethality. ...
Aim
The voltage‐gated sodium channel NaV1.5, encoded by SCN5A, is essential for cardiac excitability and ensures proper electrical conduction. Early embryonic death has been observed in several murine models carrying homozygous Scn5a mutations. We investigated when sodium current (INa) becomes functionally relevant in the murine embryonic heart and how Scn5a/NaV1.5 dysfunction impacts on cardiac development.
Methods
Involvement of NaV1.5‐generated INa in murine cardiac electrical function was assessed by optical mapping in wild type embryos (Embryonic day (E)9.5 and E10.5) in the absence and presence of the sodium channel blocker tetrodotoxin (30 µM). INa was assessed by patch‐clamp analysis in cardiomyocytes isolated from wild type embryos (E9.5‐17.5). In addition, cardiac morphology and electrical function was assessed in Scn5a‐1798insD‐/‐ embryos (E9.5‐10.5) and their wild type littermates.
Results
In wild type embryos, tetrodotoxin did not affect cardiac activation at E9.5, but slowed activation at E10.5. Accordingly, patch‐clamp measurements revealed that INa was virtually absent at E9.5 but robustly present at E10.5. Scn5a‐1798insD‐/‐ embryos died in utero around E10.5, displaying severely affected cardiac activation and morphology. Strikingly, altered ventricular activation was observed in Scn5a‐1798insD‐/‐ E9.5 embryos before the onset of INa, in addition to reduced cardiac tissue volume compared to WT littermates.
Conclusion
We here demonstrate that NaV1.5 is involved in cardiac electrical function from E10.5 onwards. Scn5a‐1798insD‐/‐ embryos displayed cardiac structural abnormalities at E9.5, indicating that NaV1.5 dysfunction impacts on embryonic cardiac development in a non‐electrogenic manner. These findings are potentially relevant for understanding structural defects observed in relation to NaV1.5 dysfunction.
