Intercalated Discs Research Articles

The significance of intercalated discs in the pathogenesis of Friedreich cardiomyopathy

Friedreich ataxia (FRDA) is an autosomal recessive disorder with a complex clinical and neuropathological phenotype, but the most frequent cause of death is cardiomyopathy. The principal autopsy findings in FRDA hearts are concentric hypertrophy, enlargement of cardiomyocytes, myofiber necrosis, inflammatory infiltration, scarring, and random accumulation of iron. In addition, the myocardium shows generalized disorganization of intercalated discs (ICD), the Velcro-like end-to-end connections of heart fibers that provide mechanical cohesion and ionic coupling. The principal components of ICD are fascia adherens junctions (FAJ), desmosomes, and gap junctions. Frataxin deficiency in FRDA may cause improper assembly of ICD early in life, making hearts vulnerable to mechanical stress in childhood and adolescence. We studied the ICD in the myocardium of left ventricular wall (LVW), right ventricular wall, and ventricular septum in 18 genetically confirmed FRDA patients (age of death, 10 to 87years) and 12 normal controls (age of death, 13 to 69years). In cases with juvenile onset, electron microscopy and immunohistochemistry of N-cadherin and vinculin, two abundant FAJ proteins, showed enlargement of ICD, discontinuity, and hyperconvolution. Reaction product of the desmosomal protein desmoglein 2 was similar. The distribution of the gap junction protein connexin 43 at ICD was also irregular and displayed abnormal lateralization to the plasma membranes of cardiomyocytes. Confocal immunofluorescence microscopy of α-actinin, affinity fluorescence microscopy of actin with rhodamine-labeled phalloidin, and electron microscopy, revealed the principal integrity of sarcomeres of the myocardium in FRDA. In two late-onset long-surviving FRDA patients (ages 79 and 87), clinical cardiomyopathy was absent, and ICD were normal. The described observations in patients with a broad range of disease onset and duration allow us to conclude that faulty assembly of ICD interferes with proper end-to-end adhesion of cardiomyocytes of the growing heart and contributes to the pathogenesis of FRDA cardiomyopathy.

Lmod2 piggyBac mutant mice exhibit dilated cardiomyopathy.

BackgroundLeiomodin proteins, Lmod1, Lmod2 and Lmod3, are key regulators of the thin filament length in muscles. While Lmod1 is specifically expressed in smooth muscles, both Lmod2 and Lmod3 are expressed in striated muscles including both cardiac and skeletal muscles. We and others have previously shown that Lmod3 mainly function in skeletal muscles and the mutant mice display disorganized sarcomere. Lmod2 protein has been found to act as an actin filament nucleator in both cell-free assays and in cultured rat and chicken cardiomyocytes.ResultsTo better understand the function of Lmod2 in vivo, we have identified and characterized a piggyBac (PB) insertional mouse mutant. Our analysis revealed that the PB transposon inserts in the first exon of the Lmod2 gene and severely disrupts its expression. We found that Lmod2PB/PB mice exhibit typical dilated cardiomyopathy (DCM) with ventricular arrhythmias and postnatal lethality. Electron microscope reveals that the Lmod2PB/PB hearts carry disordered sarcomere, disarrayed thin filaments, and distorted intercalated discs (ICDs). Those ICDs display not only decreased convolutions, but also reduced electron-dense staining, indicating less ICDs component proteins in Lmod2PB/PB hearts. Consistent with the phenotype, the expression of the ICD component genes, β-catenin and Connexin43, are down-regulated.ConclusionsTaken together, our data reveal that Lmod2 is required in heart thin filaments for integrity of sarcomere and ICD and deficient mice exhibit DCM with ventricular arrhythmias and postnatal lethality. The Lmod2PB/PB mutant offers a valuable resource for interrogation of pathogenesis and development of therapeutics for DCM.

Sarcoplasmic reticulum is an intermediary of mitochondrial and myofibrillar growth at the intercalated disc.

In cardiomyocytes columns of intermyofibrillar mitochondria run up to the intercalated disc (ID); half are collinear with those in the neighbouring cell, suggesting coordinated addition of sarcomeres and mitochondria both within and between cells during cardiomyocyte growth. Recent evidence for an association between sarcoplasmic reticulum (SR) and mitochondria indicates that the SR may be an intermediary in this coordinated behaviour. For this reason we have investigated the arrangement of SR and t tubules with respect to mitochondria and myofibrils, particularly at the ID. In the body of the cardiomyocyte the mitochondrial columns are frequently intersected by transverse tubules. In addition, we find that a majority of axial tubules are sandwiched between mitochondria and myofibril. No tubules are found at the ID. SR coats mitochondrial columns and fibrils throughout their length and reaches towards the peaks of the ID membrane where it attaches in the form of junctional (j)SR. These peripheral ID couplings are often situated between mitochondria and ID membrane, suggesting an SR connection between the two. In dilated cardiomyopathy (DCM) the mitochondria are somewhat disordered and clumped. In a mouse model for DCM, the muscle LIM protein KO, we find that there is a lack of mitochondria near the ID, suggesting the uncoupling of the myofibril/mitochondria organisation during growth. SR still coats the fibrils and reaches the ID folds in a jSR coupling. Unlike in control tissue, however, loops and long fingers of ID membrane penetrate into the proximal sarcomere suggesting a possible intermediary state in cardiomyocyte growth.

A Proteomics Approach to Identify New Putative Cardiac Intercalated Disk Proteins.

AimsSynchronous beating of the heart is dependent on the efficient functioning of the cardiac intercalated disk (ID). The ID is composed of a complex protein network enabling electrical continuity and chemical communication between individual cardiomyocytes. Recently, several different studies have shed light on increasingly prevalent cardiac diseases involving the ID. Insufficient knowledge of its composition makes it difficult to study these disease mechanisms in more detail and therefore here we aim expand the ID proteome. Here, using a combination of general membrane enrichment, in-depth quantitative proteomics and an intracellular location driven bioinformatics approach, we aim to discover new putative ID proteins in rat ventricular tissue.Methods and ResultsGeneral membrane isolation, enriched amongst others also with ID proteins as based on presence of the established markers connexin-43 and n-cadherin, was performed using centrifugation. By mass spectrometry, we quantitatively evaluated the level of 3455 proteins in the enriched membrane fraction (EMF) and its counterpart, the soluble cytoplasmic fraction. These data were stringently filtered to generate a final set of 97 enriched, putative ID proteins. These included Cx43 and n-cadherin, but also many interesting novel candidates. We selected 4 candidates (Flotillin-2 (FLOT2), Nexilin (NEXN), Popeye-domain-containg-protein 2 (POPDC2) and thioredoxin-related-transmembrane-protein 2 (TMX2)) and confirmed their co-localization with n-cadherin in the ID of human and rat heart cryo-sections, and isolated dog cardiomyocytes.ConclusionThe presented proteomics dataset of putative new ID proteins is a valuable resource for future research into this important molecular intersection of the heart.

Central role for GSK3β in the pathogenesis of arrhythmogenic cardiomyopathy

Arrhythmogenic cardiomyopathy (ACM) is characterized by redistribution of junctional proteins, arrhythmias, and progressive myocardial injury. We previously reported that SB216763 (SB2), annotated as a GSK3β inhibitor, reverses disease phenotypes in a zebrafish model of ACM. Here, we show that SB2 prevents myocyte injury and cardiac dysfunction in vivo in two murine models of ACM at baseline and in response to exercise. SB2-treated mice with desmosome mutations showed improvements in ventricular ectopy and myocardial fibrosis/inflammation as compared with vehicle-treated (Veh-treated) mice. GSK3β inhibition improved left ventricle function and survival in sedentary and exercised Dsg2mut/mut mice compared with Veh-treated Dsg2mut/mut mice and normalized intercalated disc (ID) protein distribution in both mutant mice. GSK3β showed diffuse cytoplasmic localization in control myocytes but ID redistribution in ACM mice. Identical GSK3β redistribution is present in ACM patient myocardium but not in normal hearts or other cardiomyopathies. SB2 reduced total GSK3β protein levels but not phosphorylated Ser 9-GSK3β in ACM mice. Constitutively active GSK3β worsens ACM in mutant mice, while GSK3β shRNA silencing in ACM cardiomyocytes prevents abnormal ID protein distribution. These results highlight a central role for GSKβ in the complex phenotype of ACM and provide further evidence that pharmacologic GSKβ inhibition improves cardiomyopathies due to desmosome mutations.

Extracellular sodium dependence of the conduction velocity-calcium relationship: evidence of ephaptic self-attenuation.

Our laboratory previously demonstrated that perfusate sodium and potassium concentrations can modulate cardiac conduction velocity (CV) consistent with theoretical predictions of ephaptic coupling (EpC). EpC depends on the ionic currents and intercellular separation in sodium channel rich intercalated disk microdomains like the perinexus. We suggested that perinexal width (WP) correlates with changes in extracellular calcium ([Ca(2+)]o). Here, we test the hypothesis that increasing [Ca(2+)]o reduces WP and increases CV. Mathematical models of EpC also predict that reducing WP can reduce sodium driving force and CV by self-attenuation. Therefore, we further hypothesized that reducing WP and extracellular sodium ([Na(+)]o) will reduce CV consistent with ephaptic self-attenuation. Transmission electron microscopy revealed that increasing [Ca(2+)]o (1 to 3.4 mM) significantly decreased WP Optically mapping wild-type (WT) (100% Cx43) mouse hearts demonstrated that increasing [Ca(2+)]o increases transverse CV during normonatremia (147.3 mM), but slows transverse CV during hyponatremia (120 mM). Additionally, CV in heterozygous (∼50% Cx43) hearts was more sensitive to changes in [Ca(2+)]o relative to WT during normonatremia. During hyponatremia, CV slowed in both WT and heterozygous hearts to the same extent. Importantly, neither [Ca(2+)]o nor [Na(+)]o altered Cx43 expression or phosphorylation determined by Western blotting, or gap junctional resistance determined by electrical impedance spectroscopy. Narrowing WP, by increasing [Ca(2+)]o, increases CV consistent with enhanced EpC between myocytes. Interestingly, during hyponatremia, reducing WP slowed CV, consistent with theoretical predictions of ephaptic self-attenuation. This study suggests that serum ion concentrations may be an important determinant of cardiac disease expression.

Small Obscurins at the Intercalated Disc Mediate Structure, Cell Size, and Adhesion

The intercalated disc (ID) of cardiac muscle embodies a highly ordered, multifunctional network, essential for the transmission of electrical stimuli and mechanical force resulting in the synchronous beating of the heart. Recently, a plethora of proteins have been identified as novel components of the ID. The challenge now lies in their characterization as it relates to the coupling of neighbouring cardiomyocytes and whole heart function. Here we focus on the functional characterization of two novel ID proteins, obscurin-40 and obscurin-80.Obscurins are a family of proteins expressed in striated muscle cells where they localize to distinct substructures, contributing to their assembly and integration with other sarcoplasmic elements. Our previous studies have shown that the obscurin transcript, arising from the single OBSCN gene is extensively spliced, resulting in several obscurin isoforms. Complex splicing at the 3’ end of OBSCN results in at least two novel products, obsc-40 and obsc-80, which preferentially concentrate at the ID. Consistent with their ID subcellular localization, biochemical assays demonstrated that obsc-40 and obsc-80 are in a complex with major ID proteins, including N-cadherin, connexin-43, vinculin, and ankyrin-G. In addition, the obscurin pleckstrin homology (PH) domain present in both obsc-40 and obsc-80 binds specifically and directly to phosphatidylinositol 3,4 and 4,5 bisphosphates, likely targeting both proteins to the ID membrane. Overexpression of the obscurin-PH domain in a cardiac derived cell line results in decreased activation of the PI3K/Akt and mTOR pathways. These molecular alterations lead to decreased cellular aggregation and overall cardiomyocyte size. Furthermore, both obsc-40 and obsc-80, but not giant obscurins, exhibit reduced expression in myocardial infarction and transaortic constriction cardiac-stress models. Taken together these important findings implicate these novel small obscurins in the maintenance of heart size and the prevention of hypertrophy.

Novel Storm-Based Quantitative Assessment of Relative Protein Localization Reveals New Role for Sodium Channel β1 Subunit in Cardiac Conduction

We recently demonstrated that cardiac sodium channels (Nav1.5) localized within gap junction (GJ) -adjacent microdomains within the intercalated disc (ID) may enable ephaptic coupling between cardiac myocytes. Further we demonstrated conduction slowing and elevated arrhythmia risk secondary to disruption of close membrane apposition (<10nm) within these microdomains. Confocal micrographs of guinea pig ventricular myocardium revealed expression of β1 (SCN1b), a sodium channel auxiliary subunit and cell adhesion molecule, throughout the interplicate regions of the ID. Therefore, we hypothesized that β1-mediated adhesion may be key to close membrane apposition within Nav1.5-rich ID microdomains and thereby, to ephaptic coupling. In order to assess β1-localization within the ID, we analyzed STORM images of guinea pig ventricular sections immunolabeled for β1 along with Cx43 and N-cadherin (N-Cad) using custom algorithms. Overall, 16±2% of Cx43 clusters overlapped with a β1 cluster while 30±3% had a β1 cluster <200nm away, with a median distance of 55±6nm between Cx43 and β1. In contrast, only 7±3% of N-Cad overlapped with β1 while 16±2% had β1 <200nm away (p<0.05 vs. Cx43-β1), with a median distance of 154±98nm between N-Cad and β1. These data suggest preferential β1 localization adjacent Cx43 GJ relative (interplicate ID regions) to N-cadherin (plicate ID regions). Next we examined β1-mediated adhesion using electric cell-substrate impedance spectroscopy (ECIS): 1610 cells heterologously overexpressing β1 revealed 3-fold higher barrier function relative to native 1610 cells. Further, βadp1, a peptide mimetic of the β1 adhesion domain, inhibited barrier function in dose dependent fashion in β1-overexpressing 1610 cells but not in native 1610 cells. Taken together our results support a role for β1-mediated adhesion in modulating ephaptic coupling in the heart and highlight it as a potential target for novel anti-arrhythmic therapy.

Myozap Deficiency Promotes Adverse Cardiac Remodeling via Differential Regulation of Mitogen-activated Protein Kinase/Serum-response Factor and β-Catenin/GSK-3β Protein Signaling

The intercalated disc (ID) is a "hot spot" for heart disease, as several ID proteins have been found mutated in cardiomyopathy. Myozap is a recent addition to the list of ID proteins and has been implicated in serum-response factor signaling. To elucidate the cardiac consequences of targeted deletion of myozap in vivo, we generated myozap-null mutant (Mzp(-/-)) mice. Although Mzp(-/-) mice did not exhibit a baseline phenotype, increased biomechanical stress due to pressure overload led to accelerated cardiac hypertrophy, accompanied by "super"-induction of fetal genes, including natriuretic peptides A and B (Nppa/Nppb). Moreover, Mzp(-/-) mice manifested a severe reduction of contractile function, signs of heart failure, and increased mortality. Expression of other ID proteins like N-cadherin, desmoplakin, connexin-43, and ZO-1 was significantly perturbed upon pressure overload, underscored by disorganization of the IDs in Mzp(-/-) mice. Exploration of the molecular causes of enhanced cardiac hypertrophy revealed significant activation of β-catenin/GSK-3β signaling, whereas MAPK and MKL1/serum-response factor pathways were inhibited. In summary, myozap is required for proper adaptation to increased biomechanical stress. In broader terms, our data imply an essential function of the ID in cardiac remodeling beyond a mere structural role and emphasize the need for a better understanding of this molecular structure in the context of heart disease.

Connexin 26 expression in mammalian cardiomyocytes

Connexins (Cxs) are a family of membrane-spanning proteins named according to their molecular weight. They have been known to form membrane channels mediating cell-cell communication, which play an essential role in the propagation of electrical activity throughout the heart. So far, expression of seven isoforms, namely Cx30.2, Cx37, Cx40, Cx43, Cx45, Cx46 and Cx57, have been found in cardiac myocytes (1,2). Cx26 has been described in a number of tissues but not yet in the heart, and its mutations are frequently associated with deafness and skin diseases (3,4). To our knowledge, the expression of Cx26 also in human, pig, rat and mouse cardiomyocytes has been demonstrated for the first time in the present study. Interestingly, this Cx was found as scattered throughout cell cytoplasm but not at level of the intercalated disks where the other cardiac Cxs are mainly located. Furthermore, in cardiomyocytes of a pig model of left ventricular dysfunction (LVD), Cx26 expression was modulated and dipyridamole treatment, which was previously demonstrated to have a protective action on left ventricular function (5), was associated to an increased Cx26 expression. Dipyridamole induced the same effect in cardiac rat cell line H9c2. For our study, paraffin embedded sections of human auricle, pig ventricle, mouse whole heart and H9c2 cells were used. Several methods were employed to test the expression of Cx26. In particular, different immunohistochemical and molecular biology techniques were performed by using two types of primary anti-Cx26 antibodies to ascertain the specificity of cardiomyocyte immunopositivity for Cx26 avoiding analysis-dependent artifacts.

Remodeling of the intercalated disc related to aging in the mouse heart

BackgroundAging is related to declined cardiac hemodynamic function. As pumping performance may be significantly related to slowed ventricular depolarization and non-synchronous contraction, we hypothesized that aging may cause dysfunction of intercalated disc (ID), which is the structure responsible for intercellular electrical communication between cardiomyocytes. MethodsMale C57BL/6J mice were used for the study at two ages: 4 and 24 months. Electrocardiographic recording was made to analyze the time of ventricular depolarization. Then mice were killed, and the hearts were harvested for examination in transmission electron microscopy (TEM) and immunofluorescence imaging. The expression of connexin 43 (Cx43), N-cadherin, and β-catenin in the myocardium of the left ventricle was evaluated using Western blotting. ResultsIn senescent mice, analysis of averaged QRS complex showed its significant prolongation. At the ultrastructural level, we found frequent disruptions of the ID (affecting 29±5% of them), mainly at the site of adherens junction, with relatively preserved desmosomal intercellular connections and diminished number of gap junctions. Western blotting revealed significantly decreased abundance of Cx43 protein in aged animals, which may cause slowed impulse propagation through the gap junctions and contribute to the observed electrocardiographic alterations. The level of RNA for Cx43 is similar between young and old animals, which suggests a post-transcriptional mechanism of Cx43 protein downregulation. ConclusionsOur study shows age-related disorganization of ID, which may be responsible for slowed conduction of the depolarization wave within the heart, and supports the hypothesis of cardiac dysfunction in senescence.

Abstract 14952: Disruption of a Newly Identified Complex Between soluble Guanylyl Cyclase and Connexin 43 is Potentially Linked to Cardiac Contractile Dysfunction

Background: The role of nitric oxide (NO) in regulation of excitation-contraction (EC) coupling remains controversial. NO activates soluble Guanylyl Cyclase (sGC), a heme-containing heterodimer (α/ß subunits), to increase cGMP production and regulate downstream targets such as Protein Kinase G (PKG) and phosphodiesterases (PDE). Intercalated discs (ID) are essential for electro-mechanical coupling between cardiomyocytes. Connexin 43 (Cx43) via formation of gap junctions at the ID mediate the electrical coupling. Cx43 targeting and assembly at the ID involved phosphorylation by many protein kinases, including cAMP-activated PKA. In various cardiomyopathies such as arrhythmia, Cx43 is relocated from ID to the lateral membrane of cardiomyocytes. Recently, we discovered that sGCβ is localized at the ID with Cx43 and PDE3A, using immunofluorescence (IF) and Triton/Urea heart fractionation. Importantly, we showed by co-immunoprecipitation (co-IP) that sGC physically interacts with Cx43 in the heart. Hypothesis: Cx43 assembly and function at the ID through PKA-dependent Cx43 phosphorylation depends on NO-sGC-cGMP microdomain inducing inhibition of PDE3A. Methods and Results: To assess the role and relevance of the sGC-Cx43 complex, we used Angiotensin II (AngII) to induce cardiac hypertrophy and arrhythmia in wild-type (WT) mice and sGCα-KO mice, which are depleted for sGC activity. Co-IP indicates that sGC-Cx43 complex is decreased in AngII-treated WT (n=5), independently of decreased Cx43 expression and IF showed Cx43 lateralization. NO-dependent cGMP in membrane fraction of AngII-hearts was decreased by 50%. Conversely, in sGCα-KO mice Cx43-sGC complex was reduced and even more so with AngII treatment. In addition to a significant increased cardiac hypertrophy in sGCα-KO mice (heart to body weight ratio KO: 5.16±0.19 vs WT: 4.45±0.04, n=4-8, P&lt;0.05), preliminary EKG (n=4) indicates that sGCα-KO mice suffer from cardiac arrhythmia with, intriguingly, alteration in P waves. Conclusion: Altogether, our data suggest that sGC via modulation of Cx43 location at ID is potentially involved in EC regulation. We currently explore the mechanism underlying these observations with in vivo, biochemical approaches and imaging.

Abstract 15489: Altered Biomechanical Properties of PKP2-deficient Hl-1 Cardiac Cells

Arrhythmogenic cardiomyopathy (AC) is a genetic disease caused by mutations in genes encoding the intercalated disk (ID) proteins. ID is a multiprotein complex providing cell-cell junction and connecting the nucleus to the extracellular matrix, and playing a pivotal role for maintaining the structural integrity of the heart tissue. Mutations in PKP2 encoding plakophilin 2 (PKP2) are the most common causes of AC. The objective of this study was to investigate the biomechanical properties of the PKP2-knock down HL-1 mouse cardiac myocytes (PKP2-KD). PKP2 protein levels were reduced by 66 to 75% upon shRNA-targeting of the Pkp2 mRNA. Nuclear stiffness (Young’s modulus) and the force required to deform the nucleus were assessed by atomic force microscopy (AFM). Nuclear stiffness was decreased significantly (5 times) in PKP2-KD HL-1 myocytes as compared to wild-type (WT) myocytes, indicating an increased elasticity and/or susceptibility of the nucleus to be deformed (2 times). Interestingly, cell adhesion was also reduced (10 times) in the PKP2-KD myocytes, indicating a pivotal role of ID proteins in maintaining cell adhesion and mechanical integrity in cardiomyocytes. In the stress-relaxation test, while the decay time value was comparable between WT and PKP2-KD cells, relaxation force and cell deformation values were decreased 3 and 8 times, respectively, in PKP2-KD HL-1 myocytes, confirming the finding in the Young’s modulus. Moreover, the relaxation test revealed a reduced viscosity, highlighting a compromised cytoskeletal stability induced by the PKP2 deficiency. RNA-Sequencing and Ingenuity Canonical Pathway analysis identified integrin, tight junction, and the canonical Wnt signaling, among the most disturbed pathways in the PKP2-KD HL-1 myocytes. The findings indicate that the deficiency of a component of the ID protein markedly affects the whole-cellular biomechanics, which is expected to affect tissue stability, leading to activation of the mechano-transduction pathways.

Abstract 11138: Superresolution Studies of Sodium Channels Within Intercalated Disk Microdomains Suggest Novel Arrhythmia Mechanism

Pore-forming (Nav1.5) and auxiliary (β1; SCN1b) subunits of cardiac sodium channels are enriched at the cardiomyocyte intercalated disk (ID). Mathematical models suggest that this may facilitate conduction via ephaptic mechanisms. We recently demonstrated Nav1.5 enrichment (gSTED superresolution microscopy) and close membrane apposition (&lt;10 nm; electron microscopy) within the perinexus, a microdomain surrounding connexin43 (Cx43) gap junctions (GJ). These data identified the perinexus as a candidate structure for the cardiac ephapse. Further studies using gSTED and STORM superresolution microscopy revealed Nav1.5 and β1 enrichment within ID regions not containing dense clusters of Cx43 and N-Cadherin. Notably, both were identified within the perinexus: Overall, 22% of Nav1.5 &amp; β1 were located within perinexal regions while only 2 and 5% respectively overlapped with Cx43 clusters. Importantly, acute interstitial edema (AIE) increased intermembrane distance at perinexal, but not at non-perinexal sites in adult guinea pig myocardium. Functionally, this correlated with decreased transverse conduction velocity (CV-T; 15.2±0.3 vs. 19.6±0.1cm/s) and increased anisotropic ratio (AR; 3.0±0.2 vs. 2.8±0.1) relative to control, in perfused guinea pig ventricles. Nav1.5 blockade (0.5 μM flecainide) by itself decreased CV (18%) without changing AR. However, Nav1.5 inhibition during AIE preferentially decreased CV-T (13.0±0.6cm/s), increased AR (3.3±0.2) and increased spontaneous arrhythmias (7/9 vs. 4/11) compared to AIE alone. Notably, only a computer model including ephaptic coupling and the ID localization of Nav1.5 could recapitulate these results. Next we investigated the role of β1 in ephaptic coupling: Electrical cell-substrate impedance spectroscopy of 1610 cells heterologously overexpressing β1 revealed 3-fold higher paracellular resistance relative to native 1610 cells. These data along with the known cell adhesion function of β1 in neural tissue suggest that β1-mediated adhesion may facilitate close membrane apposition within the perinexus. Taken together, our results identify β1-mediated adhesion as a novel determinant of anisotropic conduction and potential antiarrhythmic target.

Neuronal nitric oxide synthase localizes to utrophin expressing intercalated discs and stabilizes their structural integrity

The neuronal nitric-oxide synthase (nNOS) splice variant nNOSµ is essential for skeletal muscle function. Its localization is dependent on dystrophin, which stabilizes the dystrophin glycoprotein complex (DGC) at the sarcolemma of skeletal muscle fibers. In Duchenne muscular dystrophy (DMD) dystrophin is absent and sarcolemmal nNOS is lost. This leads to functional ischemia due to a decrease in contraction-induced vasodilation. In cardiomyocytes, nNOSµ is believed to be the predominant NOS isoform. However, the association of nNOS with the DGC in the heart is unclear. Here, we report nNOS localization at the intercalated discs (IDs) of cardiomyocytes, where utrophin is highly expressed. In mdx, mdx:utr, nNOSµ knock-out (KO), and mdx:nNOSµ KO mice, we observed a gradual reduction of nNOS at IDs and disrupted ID morphology, compared to wild-type. In mdx:nNOSµ KO mice, but not in mdx or nNOSµ KO mice, we also observed an early development of cardiac fibrosis. These findings suggest that nNOS localization in the heart may not depend exclusively on the presence of dystrophin. Additionally, the β1 subunit of soluble guanylyl cyclase (sGC), responsible for the production of cGMP through nitric oxide (NO) signaling, was also detected at the IDs. Together, our results suggest a new role of nNOS at the IDs for the cGMP-dependent NO pathway and the maintenance of ID morphology.

Direct Exposure to Ethanol Disrupts Junctional Cell-Cell Contact and Hippo-YAP Signaling in HL-1 Murine Atrial Cardiomyocytes.

Direct exposure of cardiomyocytes to ethanol causes cardiac damage such as cardiac arrythmias and apoptotic cell death. Cardiomyocytes are connected to each other through intercalated disks (ID), which are composed of a gap junction (GJ), adherens junction, and desmosome. Changes in the content as well as the subcellular localization of connexin43 (Cx43), the main component of the cardiac GJ, are reportedly involved in cardiac arrythmias and subsequent damage. Recently, the hippo-YAP signaling pathway, which links cellular physical status to cell proliferation, differentiation, and apoptosis, has been implicated in cardiac homeostasis under physiological as well as pathological conditions. This study was conducted to explore the possible involvement of junctional intercellular communication, mechanotransduction through cytoskeletal organization, and the hippo-YAP pathway in cardiac damage caused by direct exposure to ethanol. HL-1 murine atrial cardiac cells were used since these cells retain cardiac phenotypes through ID formation and subsequent synchronous contraction. Cells were exposed to 0.5–2% ethanol; significant apoptotic cell death was observed after exposure to 2% ethanol for 48 hours. A decrease in Cx43 levels was already observed after 3 hours exposure to 2% ethanol, suggesting a rapid degradation of this protein. Upon exposure to ethanol, Cx43 translocated into lysosomes. Cellular cytoskeletal organization was also dysregulated by ethanol, as demonstrated by the disruption of myofibrils and intermediate filaments. Coinciding with the loss of cell-cell adherence, decreased phosphorylation of YAP, a hippo pathway effector, was also observed in ethanol-treated cells. Taken together, the results provide evidence that cells exposed directly to ethanol show 1) impaired cell-cell adherence/communication, 2) decreased cellular mechanotransduction by the cytoskeleton, and 3) a suppressed hippo-YAP pathway. Suppression of hippo-YAP pathway signaling should be effective in maintaining cellular homeostasis in cardiomyocytes exposed to ethanol.

Tumor necrosis factor-α confers cardioprotection through ectopic expression of keratins K8 and K18.

Tumor necrosis factor-α (TNF-α), one of the major stress-induced proinflammatory cytokines, is upregulated in the heart after tissue injury, and its sustained expression can contribute to the development of heart failure. Whether TNF-α also exerts cytoprotective effects in heart failure is not known. Here we provide evidence for a cardioprotective function of TNF-α in a genetic heart failure model, desmin-deficient mice. The cardioprotective effects of TNF-α are a consequence of nuclear factor-κB (NF-κB)-mediated ectopic expression in cardiomyocytes of keratin 8 (K8) and keratin 18 (K18), two epithelial-specific intermediate filament proteins. In cardiomyocytes, K8 and K18 (K8/K18) formed an alternative cytoskeletal network that localized mainly at intercalated discs (IDs) and conferred cardioprotection by maintaining normal ID structure and mitochondrial integrity and function. Ectopic induction of K8/K18 expression in cardiomyocytes also occurred in other genetic and experimental models of heart failure. Loss of the K8/K18 network resulted in a maladaptive cardiac phenotype following transverse aortic constriction. In human failing myocardium, where TNF-α expression is upregulated, K8/K18 were also ectopically expressed and localized primarily at IDs, which did not contain detectable amounts of desmin. Thus, TNF-α- and NF-κB-mediated formation of an alternative, stress-induced intermediate filament cytoskeleton has cardioprotective function in mice and potentially in humans.

Abstract 371: Down Regulation of ECM Genes Laminin and Collagen IV Lead to Preserved Cardiac Function With Age and Increased Lifespan in Drosophila

Increased deposition of extracellular matrix (ECM) is observed in all advanced age heart failure patients, but current model systems are complex and slow to age. To investigate the effect of extracellular remodeling on mechanical function in genetically tractable, rapidly aging, and simple model organisms, we employed Drosophila melanogaster, which has a simple trilayered heart tube. We found that two common wildtype strains of Drosophila, i.e. yellow-white (yw) and white-1118 (w1118), exhibit different cytoskeletal and ECM remodeling with age. Using a recently developed nanoindentation method to measure cardiomyocyte stiffness and high speed optical imaging to assess contractility of intact Drosophila hearts, we found that yw flies had stiffer intercalated discs (ICD) and exhibited diastolic dysfunction with age. On the other hand, w1118 flies had a shorter lifespan compared to yw, did not exhibit ICD stiffening, had a less severe diastolic dysfunction, and showed an increase in ECM layer thickness between ventral muscle (VM) and cardiomyocyte (CM) layers of the heart tube. To modulate ECM and assess its effect in the aged w1118 flies, we knocked-down ECM genes LamininA and Viking (homologous to Collagen IV). Both ECM KD genotypes exhibited diastolic dilation with increased fractional shortening at adult (1wk) and aged (5wk) time points. The LamininA KD resulted in decreased cardiomyocyte stiffness correlating with increased relaxation velocities in adult flies and preservation of shortening and relaxation velocities in aged flies over controls. However, both the LamininA and Collagen IV KD flies experienced a basal increase in the decoupling of their cardiomyocytes as determined by heart period variance and % fibrillar heart-beats. These conductance issues were not enough to counteract the increased cardiac output and performance with age, and the Collagen IV KD outlived controls by 1.5 weeks median survival and the LamininA KD by 3 weeks. This suggests that the cell-ECM contacts in the basement membrane are intimately tied not only to the coupling of the cardiomyocytes of the Drosophila heart tube but also to cytoskeletal remodeling, but perhaps different ECM proteins have different mechanisms for interacting with the cardiomyocyte cytoskeleton.

Ultrastructure of the intercellular space in adult murine ventricle revealed by quantitative tomographic electron microscopy.

Progress in tissue preservation (high-pressure freezing), data acquisition (tomographic electron microscopy, TEM), and analysis (image segmentation and quantification) have greatly improved the level of information extracted from ultrastructural images. Here, we combined these methods and developed analytical tools to provide an in-depth morphometric description of the intercalated disc (ID) in adult murine ventricle. As a point of comparison, we characterized the ultrastructure of the ID in mice heterozygous-null for the desmosomal gene plakophilin-2 (PKP2; mice dubbed PKP2-Hz). Tomographic EM images of thin sections of adult mouse ventricular tissue were processed by image segmentation analysis. Novel morphometric routines allowed us to generate the first quantitative description of the ID intercellular space based on three-dimensional data. We show that complex invaginations of the cell membrane significantly increased the total ID surface area. In addition, PKP2-Hz samples showed increased average intercellular spacing, ID surface area, and membrane tortuosity, as well as reduced number and length of mechanical junctions compared with control. Finally, we observed membranous structures reminiscent of junctional sarcoplasmic reticulum at the ID, which were significantly more abundant in PKP2-Hz hearts. We have developed a systematic method to characterize the ultrastructure of the intercellular space in the adult murine ventricle and have provided a quantitative description of the structure of the intercellular membranes and of the intercellular space. We further show that PKP2 deficiency associates with ultrastructural defects. The possible importance of the intercellular space in cardiac behaviour is discussed.

Gap junctions - guards of excitability.

Cardiomyocytes are connected by mechanical and electrical junctions located at the intercalated discs (IDs). Although these structures have long been known, it is becoming increasingly clear that their components interact. This review describes the involvement of the ID in electrical disturbances of the heart and focuses on the role of the gap junctional protein connexin 43 (Cx43). Current evidence shows that Cx43 plays a crucial role in organizing microtubules at the intercalated disc and thereby regulating the trafficking of the cardiac sodium channel NaV1.5 to the membrane.