Abstract 19049: CASK Regulates Sodium Channels Distribution and Function in Costameric Microdomain

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Catherine A Eichel ◽  
Lin Xianming ◽  
Florent Louault ◽  
Gilles Dilanian ◽  
Mario Delmar ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Sodium Current ◽  
Systolic Dysfunction ◽  
Super Resolution ◽  
Adult Rats ◽  
Lateral Membrane ◽  
T Tubules ◽  
Conventional Cell ◽  
And Function

Introduction: Proteins of the MAGUK family have emerged as key components in ion channels organization and regulation within specialized submembrane domains of cardiomyocytes. In this context, we investigated for the first time the expression, localization and function in the heart of the MAGUK protein CASK. Methods and Results: Surprisingly, no signal was detectable for CASK at intercalated discs, making CASK the first MAGUK to be excluded from these structures. On the contrary, CASK was located at the lateral membrane where it belonged specifically to the costameric dystrophin/glycoproteins complex (DGC). Since a lateral sub-population of Nav1.5 channels have been reported to interact with syntrophin, a member of DGC, we hypothesized that CASK could modulate Nav1.5 channels. Using high resolution 3-D deconvolution microscopy, we observed a co-localization of CASK and Nav1.5 in cardiomyocytes and co-IP experiments confirmed that the two proteins are in the same complex. Whole-cell patch-clamp recordings revealed a negative regulation by CASK of the sodium current INa carried by Nav1.5 channels in cardiomyocytes. Using super resolution scanning coupled to conventional cell-attached patch-clamp, we investigated the involvement of CASK on Nav1.5 function and localization in highly confined microdomains of the lateral membrane. INa was recorded in crests and T-tubules on freshly isolated cardiomyocytes obtained from adult rats injected with Adeno Associated Virus (eGFP-AAV-ShCASK or eGFP-AAV-ShScr). CASK silencing caused a drastic reduction of INa at the crest whereas it increased the current at the T-tubules suggesting that CASK retains Nav1.5 channels at the crest and prevents their clustering in T-tubules. In vivo CASK silencing in rat using an eGFP-AAV-ShCASK-injected was associated with a conduction slowing as indicated by the prolongation of the QRS duration, cardiac dilation and left ventricle systolic dysfunction. Conclusion: Taken together these results indicate that CASK is a major determinant of the organization of a subpopulation of Nav1.5 at the costamere of cardiomyocytes and that CASK-silencing induces electrical and morphological remodeling.

scholarly journals A distinct pool of Nav1.5 channels at the lateral membrane of murine ventricular cardiomyocytes

2019 ◽  
Author(s):  
Jean-Sébastien Rougier ◽  
Maria C. Essers ◽  
Ludovic Gillet ◽  
Sabrina Guichard ◽  
Stephan Sonntag ◽  
...  
Keyword(s):  
Patch Clamp ◽  
Sodium Channels ◽  
Sodium Current ◽  
Pdz Domain ◽  
Adaptor Protein ◽  
Cardiac Cells ◽  
Binding Motif ◽  
Wild Type ◽  
Lateral Membrane ◽  
T Tubules

AbstractBackgroundIn cardiac ventricular muscle cells, the presence of voltage-gated sodium channels Nav1.5 at the lateral membrane depends in part on the interaction between the dystrophin-syntrophin complex and the Nav1.5 C-terminal PDZ-domain-binding sequence Ser-Ile-Val (SIV motif). α1-Syntrophin, a PDZ-domain adaptor protein, mediates the interaction between Nav1.5 and dystrophin at the lateral membrane of cardiac cells. Using the cell-attached patch-clamp approach on cardiomyocytes expressing Nav1.5 in which the SIV motif is deleted (ΔSIV), sodium current (INa) recordings from the lateral membrane revealed an SIV-motif-independent INa. Since immunostainings have suggested that Nav1.5 is expressed in transverse (T-) tubules, this remaining INa might be conducted by channels in the T-tubules. Of note, a recent study using heterologous expression systems showed that α1-syntrophin also interacts with the Nav1.5 N-terminus, which may explain the SIV-motif independent INa at the lateral membrane of cardiomyocytes.AimTo address the role of α1-syntrophin in regulating the INa at the lateral membrane of cardiac cells.Methods and resultsPatch-clamp experiments in cell-attached configuration were performed on the lateral membranes of wild-type, α1-syntrophin knock-down, and ΔSIV ventricular mouse cardiomyocytes. Compared to wild-type, a reduction of the lateral INa was observed in myocytes from α1-syntrophin knockdown hearts. However, similar to ΔSIV myocytes, a remaining INa was still recorded. In addition, cell-attached INa recordings from lateral membrane did not differ significantly between non-detubulated and detubulated ΔSIV cardiomyocytes. Lastly, we obtained evidence suggesting that cell-attached patch-clamp experiments on the lateral membrane cannot record currents conducted by channels in T-tubules such as calcium channels.ConclusionAltogether, these results suggest the presence of a sub-pool of sodium channels at the lateral membrane of cardiomyocytes that is independent of α1-syntrophin and the PDZ-binding motif of Na 1.5, located in membrane domains outside of T-tubules. The question of a T-tubular pool of Nav1.5 channels however remains open.

scholarly journals Role of type 2A phosphatase regulatory subunit B56α in regulating cardiac responses to β-adrenergic stimulation in vivo

2018 ◽  
Vol 115 (3) ◽  
pp. 519-529 ◽  
Author(s):  
Sarah-Lena Puhl ◽  
Kate L Weeks ◽  
Alican Güran ◽  
Antonella Ranieri ◽  
Peter Boknik ◽  
...  
Keyword(s):  
Systolic Dysfunction ◽  
Heart Tissue ◽  
Left Ventricular ◽  
Regulatory Subunit ◽  
Adrenergic Stimulation ◽  
Hypertrophic Response ◽  
Cardiac Responses ◽  
Cardiac Phenotype ◽  
And Function

Abstract Aims B56α is a protein phosphatase 2A (PP2A) regulatory subunit that is highly expressed in the heart. We previously reported that cardiomyocyte B56α localizes to myofilaments under resting conditions and translocates to the cytosol in response to acute β-adrenergic receptor (β-AR) stimulation. Given the importance of reversible protein phosphorylation in modulating cardiac function during sympathetic stimulation, we hypothesized that loss of B56α in mice with targeted disruption of the gene encoding B56α (Ppp2r5a) would impact on cardiac responses to β-AR stimulation in vivo. Methods and results Cardiac phenotype of mice heterozygous (HET) or homozygous (HOM) for the disrupted Ppp2r5a allele and wild type (WT) littermates was characterized under basal conditions and following acute β-AR stimulation with dobutamine (DOB; 0.75 mg/kg i.p.) or sustained β-AR stimulation by 2-week infusion of isoproterenol (ISO; 30 mg/kg/day s.c.). Left ventricular (LV) wall thicknesses, chamber dimensions and function were assessed by echocardiography, and heart tissue collected for gravimetric, histological, and biochemical analyses. Western blot analysis revealed partial and complete loss of B56α protein in hearts from HET and HOM mice, respectively, and no changes in the expression of other PP2A regulatory, catalytic or scaffolding subunits. PP2A catalytic activity was reduced in hearts of both HET and HOM mice. There were no differences in the basal cardiac phenotype between genotypes. Acute DOB stimulation induced the expected inotropic response in WT and HET mice, which was attenuated in HOM mice. In contrast, DOB-induced increases in heart rate were unaffected by B56α deficiency. In WT mice, ISO infusion increased LV wall thicknesses, cardiomyocyte area and ventricular mass, without LV dilation, systolic dysfunction, collagen deposition or foetal gene expression. The hypertrophic response to ISO was blunted in mice deficient for B56α. Conclusion These findings identify B56α as a potential regulator of cardiac structure and function during β-AR stimulation.

scholarly journals Toward Biological Pacing by Cellular Delivery of Hcn2/SkM1

2021 ◽  
Vol 11 ◽  
Author(s):  
Anna M. D. Végh ◽  
Arie O. Verkerk ◽  
Lucía Cócera Ortega ◽  
Jianan Wang ◽  
Dirk Geerts ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Patch Clamp ◽  
Computational Modeling ◽  
Sodium Current ◽  
Pacemaker Activity ◽  
Cellular Delivery ◽  
Lentiviral Transduction ◽  
Biological Pacemaker

Electronic pacemakers still face major shortcomings that are largely intrinsic to their hardware-based design. Radical improvements can potentially be generated by gene or cell therapy-based biological pacemakers. Our previous work identified adenoviral gene transfer of Hcn2 and SkM1, encoding a “funny current” and skeletal fast sodium current, respectively, as a potent combination to induce short-term biological pacing in dogs with atrioventricular block. To achieve long-term biological pacemaker activity, alternative delivery platforms need to be explored and optimized. The aim of the present study was therefore to investigate the functional delivery of Hcn2/SkM1 via human cardiomyocyte progenitor cells (CPCs). Nucleofection of Hcn2 and SkM1 in CPCs was optimized and gene transfer was determined for Hcn2 and SkM1 in vitro. The modified CPCs were analyzed using patch-clamp for validation and characterization of functional transgene expression. In addition, biophysical properties of Hcn2 and SkM1 were further investigated in lentivirally transduced CPCs by patch-clamp analysis. To compare both modification methods in vivo, CPCs were nucleofected or lentivirally transduced with GFP and injected in the left ventricle of male NOD-SCID mice. After 1 week, hearts were collected and analyzed for GFP expression and cell engraftment. Subsequent functional studies were carried out by computational modeling. Both nucleofection and lentiviral transduction of CPCs resulted in functional gene transfer of Hcn2 and SkM1 channels. However, lentiviral transduction was more efficient than nucleofection-mediated gene transfer and the virally transduced cells survived better in vivo. These data support future use of lentiviral transduction over nucleofection, concerning CPC-based cardiac gene delivery. Detailed patch-clamp studies revealed Hcn2 and Skm1 current kinetics within the range of previously reported values of other cell systems. Finally, computational modeling indicated that CPC-mediated delivery of Hcn2/SkM1 can generate stable pacemaker function in human ventricular myocytes. These modeling studies further illustrated that SkM1 plays an essential role in the final stage of diastolic depolarization, thereby enhancing biological pacemaker functioning delivered by Hcn2. Altogether these studies support further development of CPC-mediated delivery of Hcn2/SkM1 and functional testing in bradycardia models.

scholarly journals Laminin-521 Protein Therapy for Glomerular Basement Membrane and Podocyte Abnormalities in a Model of Pierson Syndrome

2018 ◽  
Vol 29 (5) ◽  
pp. 1426-1436 ◽  
Author(s):  
Meei-Hua Lin ◽  
Joseph B. Miller ◽  
Yamato Kikkawa ◽  
Hani Y. Suleiman ◽  
Karl Tryggvason ◽  
...  
Keyword(s):  
Nephrotic Syndrome ◽  
Congenital Nephrotic Syndrome ◽  
Super Resolution ◽  
Diffuse Mesangial Sclerosis ◽  
Protein Therapy ◽  
Correct Orientation ◽  
Pierson Syndrome ◽  
Foot Process ◽  
And Function

Background Laminin α5β2γ1 (LM-521) is a major component of the GBM. Mutations in LAMB2 that prevent LM-521 synthesis and/or secretion cause Pierson syndrome, a rare congenital nephrotic syndrome with diffuse mesangial sclerosis and ocular and neurologic defects. Because the GBM is uniquely accessible to plasma, which permeates endothelial cell fenestrae, we hypothesized that intravenous delivery of LM-521 could replace the missing LM-521 in the GBM of Lamb2 mutant mice and restore glomerular permselectivity.Methods We injected human LM-521 (hLM-521), a macromolecule of approximately 800 kD, into the retro-orbital sinus of Lamb2−/− pups daily. Deposition of hLM-521 into the GBM was investigated by fluorescence microscopy. We assayed the effects of hLM-521 on glomerular permselectivity by urinalysis and the effects on podocytes by desmin immunostaining and ultrastructural analysis of podocyte architecture.Results Injected hLM-521 rapidly and stably accumulated in the GBM of all glomeruli. Super-resolution imaging showed that hLM-521 accumulated in the correct orientation in the GBM, primarily on the endothelial aspect. Treatment with hLM-521 greatly reduced the expression of the podocyte injury marker desmin and attenuated the foot process effacement observed in untreated pups. Moreover, treatment with hLM-521 delayed the onset of proteinuria but did not prevent nephrotic syndrome, perhaps due to its absence from the podocyte aspect of the GBM.Conclusions These studies show that GBM composition and function can be altered in vivovia vascular delivery of even very large proteins, which may advance therapeutic options for patients with abnormal GBM composition, whether genetic or acquired.

In Vivo NGF Deprivation Reduces SNS Expression and TTX-R Sodium Currents in IB4-Negative DRG Neurons

1999 ◽  
Vol 81 (2) ◽  
pp. 803-810 ◽  
Author(s):  
Jenny Fjell ◽  
Theodore R. Cummins ◽  
Kaj Fried ◽  
Joel A. Black ◽  
Stephen G. Waxman
Keyword(s):  
Sodium Channel ◽  
Current Density ◽  
Sodium Current ◽  
High Antibody ◽  
Sodium Currents ◽  
Drg Neurons ◽  
Adult Rats ◽  
Antibody Titers

In vivo NGF deprivation reduces SNS expression and TTX-R sodium currents in IB4-negative DRG neurons. Recent evidence suggests that changes in sodium channel expression and localization may be involved in some pathological pain syndromes. SNS, a tetrodotoxin-resistant (TTX-R) sodium channel, is preferentially expressed in small dorsal root ganglion (DRG) neurons, many of which are nociceptive. TTX-R sodium currents and SNS mRNA expression have been shown to be modulated by nerve growth factor (NGF) in vitro and in vivo. To determine whether SNS expression and TTX-R currents in DRG neurons are affected by reduced levels of systemic NGF, we immunized adult rats with NGF, which causes thermal hypoalgesia in rats with high antibody titers to NGF. DRG neurons cultured from rats with high antibody titers to NGF, which do not bind the isolectin IB4 (IB4−) but do express TrkA, were studied with whole cell patch-clamp and in situ hybridization. Mean TTX-R sodium current density was decreased from 504 ± 77 pA/pF to 307 ± 61 pA/pF in control versus NGF-deprived neurons, respectively. In comparison, the mean TTX-sensitive sodium current density was not significantly different between control and NGF-deprived neurons. Quantification of SNS mRNA hybridization signal showed a significant decrease in the signal in NGF-deprived neurons compared with the control neurons. The data suggest that NGF has a major role in the maintenance of steady-state levels of TTX-R sodium currents and SNS mRNA in IB4− DRG neurons in adult rats in vivo.

scholarly journals Mitochondrial division, fusion and degradation

2019 ◽  
Vol 167 (3) ◽  
pp. 233-241 ◽  
Author(s):  
Daisuke Murata ◽  
Kenta Arai ◽  
Miho Iijima ◽  
Hiromi Sesaki
Keyword(s):  
Super Resolution ◽  
Healthy Population ◽  
Lipid Biochemistry ◽  
Mitochondrial Division ◽  
Cellular Processes ◽  
Size Number ◽  
Wide Range ◽  
Autophagic Degradation ◽  
And Function

Abstract The mitochondrion is an essential organelle for a wide range of cellular processes, including energy production, metabolism, signal transduction and cell death. To execute these functions, mitochondria regulate their size, number, morphology and distribution in cells via mitochondrial division and fusion. In addition, mitochondrial division and fusion control the autophagic degradation of dysfunctional mitochondria to maintain a healthy population. Defects in these dynamic membrane processes are linked to many human diseases that include metabolic syndrome, myopathy and neurodegenerative disorders. In the last several years, our fundamental understanding of mitochondrial fusion, division and degradation has been significantly advanced by high resolution structural analyses, protein-lipid biochemistry, super resolution microscopy and in vivo analyses using animal models. Here, we summarize and discuss this exciting recent progress in the mechanism and function of mitochondrial division and fusion.

scholarly journals Nav1.5 single-molecule localization reveals different reorganization modes at cardiomyocyte domains

2019 ◽  
Author(s):  
Sarah H. Vermij ◽  
Jean-Sébastien Rougier ◽  
Esperanza Agulló-Pascual ◽  
Eli Rothenberg ◽  
Mario Delmar ◽  
...  
Keyword(s):  
Single Molecule ◽  
Interacting Proteins ◽  
Wild Type ◽  
Gene Encoding ◽  
Localization Microscopy ◽  
Lateral Membrane ◽  
T Tubules ◽  
And Function ◽  
Nanoscale Features ◽  
Single Molecule Localization Microscopy

ABSTRACTMutations in the gene encoding the sodium channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Nav1.5 localization and function remain insufficiently characterized. We used novel single-molecule localization microscopy (SMLM) and modeling to define nanoscale features of Nav1.5 localization and distribution at the lateral membrane, groove, and T-tubules in wild-type, dystrophin-deficient (mdx) mice, and mice expressing C-terminally truncated Nav1.5 (ΔSIV). We show that Nav1.5 organizes as distinct clusters in the groove and T-tubules which density and distribution partially depend on SIV and dystrophin. We found that overall reduction in Nav1.5 expression in mdx and ΔSIV cells results in a non-uniform redistribution with Nav1.5 being specifically reduced at the groove of ΔSIV and increased in T-tubules of mdx cardiomyocytes. Nav1.5 mutations may therefore site-specifically affect Nav1.5 localization and distribution depending on site-specific interacting proteins.

scholarly journals Skill acquisition increases myelination and strengthens functional connectivity in the sensorimotor circuit of the adult rat

2019 ◽  
Author(s):  
Cassandra Sampaio-Baptista ◽  
Antoin de Weijer ◽  
Annette van der Toorn ◽  
Willem M. Otte ◽  
Anderson M. Winkler ◽  
...  
Keyword(s):  
Functional Connectivity ◽  
Motor Learning ◽  
Skill Acquisition ◽  
Structure And Function ◽  
Skill Learning ◽  
Structural Reorganization ◽  
Adult Rats ◽  
Whole Brain ◽  
And Function

ABSTRACTThe effects of skill acquisition on whole-brain structure and functional networks have been extensively investigated in humans but have yet to be explored in rodents. Forelimb reaching training in rodents results in well-established focal functional and structural reorganization within the motor cortex (M1) and cerebellum, indicating distributed alterations in both structure and function. However, it is unclear how local alterations in structure and function relate to distributed learning-related changes across motor networks. Here we trained adult rats in skilled reaching and used multimodal whole-brain in vivo MRI to assess both structural and functional plasticity over time.We detected increases in a myelin-related MRI metric in white matter, cortical areas, and to a lesser extent in the cerebellum, paralleled by strengthened functional connectivity between M1 and cerebellum, possibly reflecting a decrease in cerebellum inhibition over M1. Skill learning therefore leads to myelin increases in pathways that connect sensorimotor regions, and in functional connectivity increases between areas involved in motor learning, all of which correlate with performance. These findings closely mirror previous reports of network-level changes following motor learning in humans and underlines the correspondence between human and rodent brain circuits for motor learning, despite important differences in the anatomy of physiology of movement circuits between species.

High spatiotemporal resolution and low photo-toxicity fluorescence imaging in live cells and in vivo

2019 ◽  
Vol 47 (6) ◽  
pp. 1635-1650 ◽  
Author(s):  
Xiaohong Peng ◽  
Xiaoshuai Huang ◽  
Ke Du ◽  
Huisheng Liu ◽  
Liangyi Chen
Keyword(s):  
Fluorescence Microscopy ◽  
Spatial Resolution ◽  
Cell Biology ◽  
Critical Role ◽  
Super Resolution ◽  
Light Sheet ◽  
Live Cells ◽  
Spatiotemporal Resolution ◽  
And Function

Taking advantage of high contrast and molecular specificity, fluorescence microscopy has played a critical role in the visualization of subcellular structures and function, enabling unprecedented exploration from cell biology to neuroscience in living animals. To record and quantitatively analyse complex and dynamic biological processes in real time, fluorescence microscopes must be capable of rapid, targeted access deep within samples at high spatial resolutions, using techniques including super-resolution fluorescence microscopy, light sheet fluorescence microscopy, and multiple photon microscopy. In recent years, tremendous breakthroughs have improved the performance of these fluorescence microscopies in spatial resolution, imaging speed, and penetration. Here, we will review recent advancements of these microscopies in terms of the trade-off among spatial resolution, sampling speed and penetration depth and provide a view of their possible applications.

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