Intercalated Discs Research Articles

Mechanical stability of αT-catenin and its activation by force for vinculin binding.

αT (Testes)-catenin, a critical factor regulating cell–cell adhesion in the heart, directly couples the cadherin-catenin complex to the actin cytoskeleton at the intercalated disk (ICD), a unique cell–cell junction that couples cardiomyocytes. Loss of αT-catenin in mice reduces plakophilin2 and connexin 43 recruitment to the ICD. Since αT-catenin is subjected to mechanical stretch during actomyosin contraction in cardiomyocytes, its activity could be regulated by mechanical force. To provide insight in how force regulates αT-catenin function, we investigated the mechanical stability of the putative, force-sensing middle (M) domain of αT-catenin and determined how force impacts vinculin binding to αT-catenin. We show that 1) physiological levels of force, <15 pN, are sufficient to unfold the three M domains; 2) the M1 domain that harbors the vinculin-binding site is unfolded at ∼6 pN; and 3) unfolding of the M1 domain is necessary for high-affinity vinculin binding. In addition, we quantified the binding kinetics and affinity of vinculin to the mechanically exposed binding site in M1 and observed that αT-catenin binds vinculin with low nanomolar affinity. These results provide important new insights into the mechanosensing properties of αT-catenin and how αT-catenin regulates cell–cell adhesion at the cardiomyocyte ICD.

Enhancement of Cardiac Store Operated Calcium Entry (SOCE) within Novel Intercalated Disk Microdomains in Arrhythmic Disease

Store-operated Ca2+ entry (SOCE), a major Ca2+ signaling mechanism in non-myocyte cells, has recently emerged as a component of Ca2+ signaling in cardiac myocytes. Though it has been reported to play a role in cardiac arrhythmias and to be upregulated in cardiac disease, little is known about the fundamental properties of cardiac SOCE, its structural underpinnings or effector targets. An even greater question is how SOCE interacts with canonical excitation-contraction coupling (ECC). We undertook a multiscale structural and functional investigation of SOCE in cardiac myocytes from healthy mice (wild type; WT) and from a genetic murine model of arrhythmic disease (catecholaminergic ventricular tachycardia; CPVT). Here we provide the first demonstration of local, transient Ca2+entry (LoCE) events, which comprise cardiac SOCE. Although infrequent in WT myocytes, LoCEs occurred with greater frequency and amplitude in CPVT myocytes. CPVT myocytes also evidenced characteristic arrhythmogenic spontaneous Ca2+ waves under cholinergic stress, which were effectively prevented by SOCE inhibition. In a surprising finding, we report that both LoCEs and their underlying protein machinery are concentrated at the intercalated disk (ID). Therefore, localization of cardiac SOCE in the ID compartment has important implications for SOCE-mediated signaling, arrhythmogenesis and intercellular mechanical and electrical coupling in health and disease.

Defining a Unifying Mechanism for Select Cardiomyopathy-Linked Variants of Desmoplakin

Arrhythmogenic cardiomyopathy (AC) affects 1 in 2000 Americans every year and segregates with sudden cardiac death (SCD). AC is linked to genetic variants in desmosomal genes, however the molecular mechanisms underlying AC remain unclear. We identified a novel variant (R451G) in the desmoplakin (DSP) gene; heart autopsy revealed pronounced fibrofatty scarring and a significant loss of DSP protein at the intercalated disc (ID). Engineered heart tissues (EHTs) from induced-pluripotent stem cells generated from a symptomatic R451G-positive family member showed a significant loss of DSP protein compared to control EHT. Notably, the R451G-variant sits in a pocket containing several clinical DSP variants and we show that endogenous DSP is cleaved via a calpain-dependent mechanism. From this, we hypothesize that mutant DSP is more susceptible to calpain cleavage, resulting in cellular haploinsufficiency. In previous work, we defined a unifying mechanism for AC-linked variants by assessing the biomolecular properties of DSP variants. While these variants did not significantly perturb the structure of DSP, there were significant changes to stabilizing intramolecular interactions of select variants, which correlated with augmented calpain degradation due to increased exposure of an auto-inhibited calpain-cleavage site. We have now shown that by changing the molecular environment using site-directed mutagenesis, we can ameliorate the calpain sensitivity of clinical variants. Taken together we have identified a unifying mechanism for select AC-linked variants of DSP grounded in increased susceptibility to calpain-cleavage leading to decreased functional DSP. DSP protein expression was also decreased in end-stage failing human hearts compared to control donor hearts. The physiological and biochemical impact of this loss is being further evaluated to define the role DSP haploinsufficiency plays in the fundamental disease mechanism of AC.

Flotillins in the intercalated disc are potential modulators of cardiac excitability

BackgroundThe intercalated disc (ID) is important for cardiac remodeling and has become a subject of intensive research efforts. However, as yet the composition of the ID has still not been conclusively resolved and the role of many proteins identified in the ID, like Flotillin-2, is often unknown. The Flotillin proteins are known to be involved in the stabilization of cadherins and desmosomes in the epidermis and upon cancer development. However, their role in the heart has so far not been investigated. Therefore, in this study, we aimed at identifying the role of Flotillin-1 and Flotillin-2 in the cardiac ID. MethodsLocation of Flotillins in human and murine cardiac tissue was evaluated by fluorescent immunolabeling and co-immunoprecipitation. In addition, the effect of Flotillin knockout (KO) on proteins of the ID and in electrical excitation and conduction was investigated in cardiac samples of wildtype (WT), Flotillin-1 KO, Flotilin-2 KO and Flotilin-1/2 double KO mice. Consequences of Flotillin knockdown (KD) on cardiac function were studied (patch clamp and Multi Electrode Array (MEA)) in neonatal rat cardiomyocytes (NRCMs) transfected with siRNAs against Flotillin-1 and/or Flotillin-2. ResultsFirst, we confirmed presence in the ID and mutual binding of Flotillin-1 and Flotillin-2 in murine and human cardiac tissue. Flotillin KO mice did not show cardiac fibrosis, nor hypertrophy or changes in expression of the desmosomal ID proteins. However, protein expression of the cardiac sodium channel NaV1.5 was significantly decreased in Flotillin-1 and Flotillin-1/2 KO mice compared to WT mice. In addition, sodium current density showed a significant decrease upon Flotillin-1/2 KD in NRCMs as compared to scrambled siRNA-transfected NRCMs. MEA recordings of Flotillin-2 KD NRCM cultures showed a significantly decreased spike amplitude and a tendency of a reduced spike slope when compared to control and scrambled siRNA-transfected cultures. ConclusionsIn this study, we demonstrate the presence of Flotillin-1, in addition to Flotillin-2 in the cardiac ID. Our findings indicate a modulatory role of Flotillins on NaV1.5 expression at the ID, with potential consequences for cardiac excitation.

Abstract 17384: Cardiac-Specific Overexpression of Human Mutant Desmoplakin in Mice Disrupts Cardiac Voltage-Gated Sodium Channel Expression

Introduction: Arrhythmogenic cardiomyopathy (AC) is characterized by bi-ventricular dilation, fibro-fatty infiltration and life-threatening arrhythmias. Disruptions in cardiac voltage-gated sodium channel (Nav1.5) expression and function are known to cause arrhythmias. We have demonstrated that cardiac-specific overexpression of human mutant desmoplakin (DSP, Tg-R2834H) in mice leads to AC. However, whether mutant DSP expression in the heart affects the Nav1.5 distribution and function are unknown Hypothesis: Here, we tested whether Nav1.5 localization and expression are altered in the R2834H-Tg mouse hearts. Methods: Primary cardiomyocytes and frozen myocardial sections from non-transgenic (NTg), wild-type DSP (Tg-DSP) and Tg-R2834H mice were used for immunofluorescence studies to assess subcellular localization of DSP, desmin, Nav1.5, Cx43, plakoglobin and β-catenin. Western blot and qPCR were used for quantitative analysis. Results: Double staining of cardiomyocytes from NTg mice with DSP and Nav1.5 revealed that Nav1.5 was colocalized with DSP at the intercalated discs (IDs). In contrast, Tg-R2834H cardiomyocytes exhibited marked increase of mutant DSP expression at the IDs concomitant with a reduction in Nav1.5 immunoreactive signals. Tg-R2834H cardiomyocytes also revealed an aberration of DSP and desmin colocalizations at the IDs. There were not obvious differences in Cx43 expression between the genotypes, although the redistribution of Cx43 from the IDs to the sarcolemma was evident in Tg-R2834H cardiomyocytes. qPCR results correlated with reduced Nav1.5 mRNA expression in the Tg-R2834H mouse hearts. Conclusions: Defective DSP protein expression in the heart disrupts Nav1.5 localization and expression, implying an interaction between DSP and Nav1.5 to orchestrate normal mechanical and electrical coupling. Further electrophysiology studies to assess whole-cell Na + currents in these cardiomyocytes will provide insight into DSP and Nav1.5 interaction.

Mechanosensitive Gene Regulation by Myocardin-Related Transcription Factors Is Required for Cardiomyocyte Integrity in Load-Induced Ventricular Hypertrophy.

Hypertrophic cardiomyocyte growth and dysfunction accompany various forms of heart disease. The mechanisms responsible for transcriptional changes that affect cardiac physiology and the transition to heart failure are not well understood. The intercalated disc (ID) is a specialized intercellular junction coupling cardiomyocyte force transmission and propagation of electrical activity. The ID is gaining attention as a mechanosensitive signaling hub and hotspot for causative mutations in cardiomyopathy. Transmission electron microscopy, confocal microscopy, and single-molecule localization microscopy were used to examine changes in ID structure and protein localization in the murine and human heart. We conducted detailed cardiac functional assessment and transcriptional profiling of mice lacking myocardin-related transcription factor (MRTF)-A and MRTF-B specifically in adult cardiomyocytes to evaluate the role of mechanosensitive regulation of gene expression in load-induced ventricular remodeling. We found that MRTFs localize to IDs in the healthy human heart and accumulate in the nucleus in heart failure. Although mice lacking MRTFs in adult cardiomyocytes display normal cardiac physiology at baseline, pressure overload leads to rapid heart failure characterized by sarcomere disarray, ID disintegration, chamber dilation and wall thinning, cardiac functional decline, and partially penetrant acute lethality. Transcriptional profiling reveals a program of actin cytoskeleton and cardiomyocyte adhesion genes driven by MRTFs during pressure overload. Indeed, conspicuous remodeling of gap junctions at IDs identified by single-molecule localization microscopy may partially stem from a reduction in Mapre1 expression, which we show is a direct mechanosensitive MRTF target. Our study describes a novel paradigm in which MRTFs control an acute mechanosensitive signaling circuit that coordinates cross-talk between the actin and microtubule cytoskeleton and maintains ID integrity and cardiomyocyte homeostasis in heart disease.

Intercalated discs: cellular adhesion and signaling in heart health and diseases.

Intercalated discs (ICDs) are highly orchestrated structures that connect neighboring cardiomyocytes in the heart. Three major complexes are distinguished in ICD: desmosome, adherens junction (AJ), and gap junction (GJ). Desmosomes are major cell adhesion junctions that anchor cell membrane to the intermediate filament network; AJs connect the actin cytoskeleton of adjacent cells; and gap junctions metabolically and electrically connect the cytoplasm of adjacent cardiomyocytes. All these complexes work as a single unit, the so-called area composita, interdependently rather than individually. Mutation or altered expression of ICD proteins results in various cardiac diseases, such as ARVC (arrhythmogenic right ventricular cardiomyopathy), dilated cardiomyopathy, and hypotrophy cardiomyopathy, eventually leading to heart failure. In this article, we first review the recent findings on the structural organization of ICD and their functions and then focus on the recent advances in molecular pathogenesis of the ICD-related heart diseases, which include two major areas: i) the ICD gene mutations in cardiac diseases, and ii) the involvement of ICD proteins in signal transduction pathways leading to myocardium remodeling and eventual heart failure. These major ICD-related signaling pathways include Wnt/β-catenin pathway, p38 MAPK cascade, Rho-dependent serum response factor (SRF) signaling, calcineurin/NFAT signaling, Hippo kinase cascade, etc., which are differentially regulated in pathological conditions.

Comparative evaluation of cannabinoid receptors, apelin and S100A6 protein in the heart of women of different age groups

BackgroundRecent studies have shown a significant role of the endocannabinoid system, apelin and S100A6 protein in the regulation of cardiovascular system functioning. The aim of the study was to compare and evaluate the distribution of cannabinoid receptors (CB1 and CB2), apelin and S100A6 protein in the heart of healthy women in different age groups.MethodsThe study was conducted on the hearts of 10 women (organ donors) without a history of cardiovascular disease, who were divided into two age groups: women older than 50 years and women under 50 years of age. Paraffin heart sections were processed by immunohistochemistry for detection of cannabinoids receptors (CB1 and CB2), apelin and S100A6 protein.ResultsCB1 and CB2 immunoreactivity in the cytoplasm of cardiomyocytes in the heart of women over 50 was weaker than in younger individuals. There was also strong immunoreactivity of CB1 in intercalated discs (ICDs) of the heart, only in women over 50. The presence of this receptor in this location was not found in women under 50. Apelin- and S100A6-immunoreactivity in the cardiomyocytes was stronger in older women compared to women under 50.The CB1, apelin and S100A6 immunostaining in the endothelium of myocardial vessels was weaker in women over 50 than in younger women, while intensity of CB2- immunoreaction in coronary endothelium was similar in both groups of women.The results of the study indicate the important role of endocannabinoids, apelin, and S100A6 protein in cardiac muscle function.ConclusionThis report might contribute to a better understanding of the role of endocannabinoid system, apelin and S100 proteins in heart function as well as shed new light on processes involved in age-related cardiomyopathy.

Roles of Talin2 in Traction Force Generation, Tumor Metastasis and Cardiovascular Integrity.

There are two vertebrate talin genes, TLN1 and TLN2, which encode talin1 and talin2. Talin1 governs integrin activation, thus regulating focal adhesion (FA) assembly, cell migration and invasion, but the biological function of talin2 remains to be elucidated and not too long ago talin2 was presumed to function redundantly with talin1. Recent studies have shown distinct differences between talin2 and talin1. The promoter of TLN2 is different from that of TLN1 in their size and binding to different transcription factors. Talin2 has a higher affinity to β -integrins than talin1. Talin2 regulates traction force generation, focal adhesion dynamics and invadopodium formation, thus controlling tumor cell migration, invasion and metastasis. Also, talin2 is enriched in the myotendinous junction (MTJ) in striated muscle, costameres and intercalated disks (ICDs) of cardiac myofibrils, and atherosclerotic plaques of blood vessels, thus regulating cardiovascular integrity. In this review, we discuss the differences between talin1 and talin2, in genome, protein expression pattern, affinity with integrins, traction force generation, and provide a glance at the roles of talin2 in cancer cell invasion and cardiovascular function.

Erucic acid, a component of Lorenzo's oil and PPAR-δ ligand modifies C6 glioma growth and toxicity of doxorubicin. Experimental data and a comprehensive literature analysis

BackgroundPPAR-δ is a transcription factor which has crucial roles in stimulating oligodendroglial differentiation and myelination and its activation was also shown to differentiate malignant C6 glioma cells into oligodendrocytes. ObjectiveOne of the ligands of PPAR-δ is erucic acid (EA), an edible omega-9 fatty acid consumed more by Asian populations and exists highly in Chinese womens milk. There exist epidemiological evidence that pediatric brain tumor incidence is among the lowest in the Chinese population. EA is also an ingredient of Lorenzo's oil used against adrenoleukodystrophy, a pediatric demyelinating disease. EA was inappropriately assumed as a strong cardiotoxin based on Spanish oil syndrome, caused by toxic-aniline dye refined rapeseed oil. In this study, we studied whether EA is capable to block growth of C6 glioma cells and modify cardiotoxicity of doxorubicin. Materials and methodsWe studied effects of EA on the 3-dimensional appearance of the adherent cells, soft agar colony formation and S-phase in the 3-dimensional spheroids in C6 glioma cell cultures. We also investigated the effects of EA on hepatic and cardiac toxicity of doxorubicin. ResultsEA decreased in vitro growth of C6 glioma cells at therapeutically achievable concentrations. EA effects were more prominent in 3D-assays (soft agar colonies and spheroids) and induced cell fusions in monolayer cultures. EA decreased S-phase inhibitory potency of doxorubicin (DOX), yet augmented its efficacy to induce a senescent morphology (as assessed by scanning electron microscopy) in monolayer and to increase iNOS and eNOS expression in spheroids. In our study, EA reduced DOX-induced necrosis in mice heart and liver and induced healthier morphology of heart mitochondria (as assessed by transmission electron microscopy); yet intercalated disks (ID) were more disturbed with DOX + EA. ConclusionsBoth the antitumor and cardiac effects of EA may associate with the cell-to-cell contact mechanisms. Combining systemic EA with intrathecal DOX-chemotherapy via Ommaya reservoirs may reduce DOX concentrations in systemic circulation, hinder toxic interactions with EA and induce selective kill of glioma cells.

Abstract 375: Defining a Unifying Mechanism for Select Cardiomyopathy-Linked Variants of Desmoplakin

Arrhythmogenic cardiomyopathy (AC) is a disease that affects 1 in 2000 Americans every year and segregates with sudden cardiac death (SCD). AC is linked to genetic variants in desmosomal genes, however the molecular mechanisms underlying AC remain unclear. To probe the mechanisms of an AC-linked desmosomal mutation, we identified a candidate family with a history of SCD and an unknown variant (R451G) in the desmoplakin (DSP) gene. Statistically significant linkage between the R451G-variant and symptomatic family members (LOD score = 3.4) was established and heart autopsy tissue from a genotype-phenotype positive family member revealed pronounced fibrofatty scarring and a significant loss of DSP protein at the intercalated disc (ID). Notably, the R451G-variant sits in a pocket containing several clinical DSP variants and we show that endogenous DSP is cleaved via a calpain-dependent mechanism. Together this led us to hypothesize that mutant DSP is more susceptible to calpain cleavage, resulting in cellular haploinsufficiency. To define a unifying mechanism for AC-linked variants, we assessed the biomolecular properties of DSP variants. While these variants do not significantly perturb the global structure of DSP, there are significant changes to local stabilizing intramolecular interactions of select variants, which correlates with augmented calpain degradation due to increased exposure of an auto-inhibited calpain-cleavage site. Taken together we have identified a unifying mechanism for select AC-linked variants of DSP grounded in increased susceptibility to calpain-cleavage leading to decreased functional DSP. To test the hypothesis that DSP haploinsufficiency results in poor ID organization and reduced electrical conduction of the myocardium we created engineered heart tissues (EHTs) from induced pluripotent stem cells generated from a symptomatic R451G-positive family member. Surprisingly, we found that conduction velocities were unchanged however, mutant tissues had a significantly slower rate of contraction despite unchanged peak contractile force. This biomechanical deficiency in DSP haploinsuffient cells may indicate poor coupling between the sarcomere lattice and the ID as a fundamental disease mechanism in AC.

Abstract 340: Cardiomyocyte-Derived Mechanical Signals Function in a Paracrine Manner to Regulate Tissue Stiffness and Myocardial Proliferation

Cells sense and transduce mechanical signals through cell-cell adhesions and cell-extracellular matrix adhesions. After birth, contractile force generation and tissue stiffness increase as cardiac output increases to support the needs of the newborn organism. During this postnatal period, the N-cadherin adhesion complex assembles at the intercalated disc (ID) as integrin-fibronectin (FN) adhesions decrease, accompanying the switch from hyperplastic to hypertrophic growth. However, little is known regarding the reciprocity between integrin and N-cadherin adhesions in the regulation of cell cycle withdrawal that occurs in cardiomyocytes after birth. α-catenins function as mechanosensors and transduce the intercellular force from N-cadherin to the actin cytoskeleton. Cardiac-specific αE- and αT-catenins double knockout (DKO) mice exhibit sustained cardiomyocyte proliferation beyond the first week of life. To investigate the role of cell-matrix interactions in DKO hearts, the spatial distribution of integrin-FN complexes was examined at postnatal day (P) 4, P7, P14, and P60. DKO hearts exhibited aberrant N-cadherin localization accompanied by altered distribution of α5/β1 integrin, the primary FN receptor. Normally found at the lateral membrane, α5 and β1 accumulated at the ID while N-cadherin was reduced at the termini. FN matrix assembly, as monitored by immunofluorescent staining and its insolubility in the detergent deoxycholate, was increased in DKO hearts. Meanwhile, focal adhesion kinase (FAK) was activated in early postnatal DKO hearts. Complementary experiments performed with deformable substrata demonstrated that stiffness-mediated proliferation was dependent on FAK activity. Finally, treatment of DKO pups with the lysyl oxidase inhibitor, β-aminopropionitrile (BAPN), was sufficient to reduce cardiomyocyte proliferation to wild-type levels suggesting that matrix assembly and tissue stiffness feedback to promote cardiomyocyte proliferation in DKO hearts. This data demonstrates that α-catenins regulate the balance between cell-cell and cell-matrix adhesions, which, in turn, controls cardiomyocyte proliferation, thus providing a molecular explanation for loss of regenerative potential shortly after birth.

At the heart of inter- and intracellular signaling: the intercalated disc

Proper cardiac function requires the synchronous mechanical and electrical coupling of individual cardiomyocytes. The intercalated disc (ID) mediates coupling of neighboring myocytes through intercellular signaling. Intercellular communication is highly regulated via intracellular signaling, and signaling pathways originating from the ID control cardiomyocyte remodeling and function. Herein, we present an overview of the inter- and intracellular signaling that occurs at and originates from the intercalated disc in normal physiology and pathophysiology. This review highlights the importance of the intercalated disc as an integrator of signaling events regulating homeostasis and stress responses in the heart and the center of several pathophysiological processes mediating the development of cardiomyopathies.

Three-dimensional reconstruction of the intercalated disc including the intercellular junctions by applying volume scanning electron microscopy.

The intercalated disc (ID) contains different kinds of intercellular junctions: gap junctions (GJs), desmosomes and areae compositae, essential for adhesion and communication between adjacent cardiomyocytes. The junctions can be identified based on their morphology when imaged using transmission electron microscopy (TEM), however, only with very limited information in the z-dimension. The application of volume EM techniques can give insight into the three-dimensional (3-D) organization of complex biological structures. In this study, we generated 3-D datasets using serial block-face scanning electron microscopy (SBF-SEM) and focused ion beam SEM (FIB-SEM), the latter resulting in datasets with 5nm isotropic voxels. We visualized cardiomyocytes in murine ventricular heart tissue and, for the first time, we could three-dimensionally reconstruct the ID including desmosomes and GJs with 5nm precision in a large volume. Results show in three dimensions a highly folded structure of the ID, with the presence of GJs and desmosomes in both plicae and interplicae regions. We observed close contact of GJs with mitochondria and a variable spatial distribution of the junctions. Based on measurements of the shape of the intercellular junctions in 3-D, it is seen that GJs and desmosomes vary in size, depending on the region within the ID. This demonstrates that volume EM is essential to visualize morphological changes and its potential to quantitatively determine structural changes between normal and pathological conditions, e.g., cardiomyopathies.

α-Catenin-dependent cytoskeletal tension controls Yap activity in the heart.

Shortly after birth, muscle cells of the mammalian heart lose their ability to divide. At the same time, the N-cadherin/catenin cell adhesion complex accumulates at the cell termini, creating a specialized type of cell-cell contact called the intercalated disc (ICD). To investigate the relationship between ICD maturation and proliferation, αE-catenin (Ctnna1) and αT-catenin (Ctnna3) genes were deleted to generate cardiac-specific α-catenin double knockout (DKO) mice. DKO mice exhibited aberrant N-cadherin expression, mislocalized actomyosin activity and increased cardiomyocyte proliferation that was dependent on Yap activity. To assess effects on tension, cardiomyocytes were cultured on deformable polyacrylamide hydrogels of varying stiffness. When grown on a stiff substrate, DKO cardiomyocytes exhibited increased cell spreading, nuclear Yap and proliferation. A low dose of either a myosin or RhoA inhibitor was sufficient to block Yap accumulation in the nucleus. Finally, activation of RhoA was sufficient to increase nuclear Yap in wild-type cardiomyocytes. These data demonstrate that α-catenins regulate ICD maturation and actomyosin contractility, which, in turn, control Yap subcellular localization, thus providing an explanation for the loss of proliferative capacity in the newborn mammalian heart.

Cardiomyocyte ZO-1 Regulates Intercalated Disc Organization and Whole Heart Physiology

Intercalated discs (IDs) in the heart provide both electrical and mechanical coupling between cells. Abnormalities in ID proteins are associated with arrhythmogenic cardiomyopathy, however the regulation of the ID and the role of ID proteins in heart physiology is still poorly understood. Tight junction proteins such as ZO-1 (Tjp1) are present and can bind to multiple ID proteins. However, its contributions to ID organization and heart function remain largely undefined. We aim to understand if and how the tight junction protein ZO-1 contributes to ID structure and heart physiology. We find ZO-1 expression was decreased in patients with paroxysmal atrial fibrillation (81±9% of control, P=0.03) by qPCR, suggesting ZO-1 expression change is associated with human disease. We then generated an inducible cardiomyocyte specific ZO-1 knockout mouse (ZO-1 KO) was by crossing Tjp1 floxed mice with myosin heavy chain 6-CreERT2 mice. 8-12 week old mice were injected with tamoxifen to induce Tjp1 gene deletion. Immunofluorescent staining showed decreased Coxsackie and adenovirus receptor (CAR) (56±2% decrease), and connexin 43 (67±3% decrease) distribution at IDs but no decrease of desmosome and adherens junction proteins was observed. Transmission and immunogold electron microscopy showed loss of gap junction structures. Surface ECG showed ZO-1 KO caused increased PR interval (28.0 vs. 45.3 ms) and intra-cardiac electrogram showed increased atrial-His interval (24.5±1.1 vs. 69.5±3.0 ms) and Wenckebach time (68.9±20.1 vs. 131.4±15.1 ms), without affecting His-ventricular interval. ZO-1 KO mice developed 3rd degree heart block 2 weeks after ZO-1 deletion. Loss of ZO-1 in adult heart results in altered ID protein composition, heart block, and heart failure. We propose a model in which ZO-1 stabilizes connexins and CAR to allow efficient cell-cell communication, and loss of ZO-1 contributes to the pathogenesis of heart disease.

Intercalated disc in failing hearts from patients with dilated cardiomyopathy: Its role in the depressed left ventricular function.

Alterations in myocardial structure and reduced cardiomyocyte adhesions have been previously described in dilated cardiomyopathy (DCM). We studied the transcriptome of cell adhesion molecules in these patients and their relationships with left ventricular (LV) function decay. We also visualized the intercalated disc (ID) structure and organization. The transcriptomic profile of 23 explanted LV samples was analyzed using RNA-sequencing (13 DCM, 10 control [CNT]), focusing on cell adhesion genes. Electron microscopy analysis to visualize ID structural differences and immunohistochemistry experiments of ID proteins was also performed. RT-qPCR and western blot experiments were carried out on ID components. We found 29 differentially expressed genes, most of all, constituents of the ID structure. We found that the expression of GJA3, DSP and CTNNA3 was directly associated with LV ejection fraction (r = 0.741, P = 0.004; r = 0.674, P = 0.011 and r = 0.565, P = 0.044, respectively), LV systolic (P = 0.003, P = 0.003, P = 0.028, respectively) and diastolic dimensions (P = 0.006, P = 0.001, P = 0.025, respectively). Electron microscopy micrographs showed a reduced ID convolution index and immunogold labeling of connexin 46 (GJA gene), desmoplakin (DSP gene) and catenin α-3 (CTNNA3 gene) proteins in DCM patients. Moreover, we observed that protein and mRNA levels analyzed by RT-qPCR of these ID components were diminished in DCM group. In conclusion, we report significant gene and protein expression changes and found that the ID components GJA3, DSP and CTNNA3 were highly related to LV function. Microscopic observations indicated that ID is structurally compromised in these patients. These findings give new data for understanding the ventricular depression that characterizes DCM, opening new therapeutic perspectives for these critically diseased patients.

Disturbed Desmoglein-2 in the intercalated disc of pediatric patients with dilated cardiomyopathy

Dilated cardiomyopathy (DCM) leads to disturbed contraction and force transduction, and is associated with substantial mortality in all age groups. Involvement of a disrupted composition of the intercalated disc (ID) has been reported. However, in children, little is established about such subcellular changes during disease, because of the pathological mix-up with the ongoing cardiac maturation. This leaves maladaptive remodeling often undetected. We aimed at illustrating subcellular alterations in children diagnosed with DCM compared to age-matched controls, focusing on ID proteins known to be crucially stable under healthy conditions and destabilized during cardiac injury in adults. Left ventricular or septal pediatric specimens were collected from 7 individuals diagnosed with DCM (age: 23 weeks in utero to 8 weeks postnatal) and age-matched controls that died of non-cardiovascular cause. We determined the amount of fibrosis and localization of ID proteins by immunohistochemistry. In pediatric DCM, most ID proteins follow similar spatiotemporal changes in localization as in controls. However, although no mutations were found, the signal of the desmosomal protein Desmoglein-2 was reduced in all pediatric DCM specimens, but not in controls or adult DCM patients. Endocardial and transmural fibrosis was increased in all pediatric DCM patients compared to age-matched controls. Composition of the ID in pediatric DCM patients is similar to controls, except for the localization of Desmoglein-2 and presence of severe fibrosis. This suggests that the architecture of desmosomes is already disturbed in the early stages of DCM. These findings contribute to the understanding of pediatric DCM.

Abstract 77: The Role of Vinculin in Cardiomyocyte Adhesion and Mechanical Continuity

Cardiomyocytes are joined end-to-end by a complex adhesive structure known as the intercalated disc (ICD). The ICD is composed of mechanical and electrical junctional complexes including the adherens junction (AJ). The core of the AJ is the cadherin/catenin complex, which links the actin cytoskeletons of neighboring cells to provide mechanical continuity. Myofibrils – the contractile machinery of the heart – are coupled to the ICD, presumably through the AJ; however, this mechanical linkage is poorly defined. Tension applied to the AJ is sensed in part through alpha-catenin, which connects the cadherin/catenin complex to actin. Force alters the conformation of alpha-catenin to reveal binding sites for actin-binding ligands, including vinculin and afadin, which are both found at the ICD. We investigated the role of alpha-catenin and its ligands in linking the AJ to actin in cardiomyocytes. Using a Förester resonance energy transfer tension-sensing module inserted into N-cadherin, we found that N-cadherin is under tension at the ICD. We then measured protein mobility of EGFP-tagged N-cadherin and found that N-cadherin dynamics were reduced at sites of myofibril integration, suggesting that myofibrils stabilize N-cadherin. To examine the composition of AJs in cardiomyocytes, we used immunofluorescence and found that vinculin and afadin were preferentially recruited to myofibril integration sites at the ICD. To further examine the linkage between AJs and myofibrils, we built a series of EGFP-tagged N-cadherin:alpha-catenin fusion constructs to delineate the individual and combined roles of alpha-catenin, vinculin and afadin. A constitutively active alpha-catenin increased vinculin recruitment and decreased the mobility of the N-cadherin fusion, suggesting that vinculin recruitment stabilizes the AJ. In contrast, restricting the AJ-actin interface to afadin reduced stability, indicating afadin alone cannot replace vinculin in linking AJs to the myofibril network. Together, our data support a model in which alpha-catenin, vinculin and afadin cooperate to anchor myofibrils at the ICD to provide mechanical continuity between cells.

Abstract 114: Myocardin-Related Transcription Factors Are Required for Compensatory Signaling in Maladaptive Cardiac Remodeling

Intercellular communication is essential for coordinating the contraction of cardiomyocytes. This synchrony is established by coupling electrical activity and force transmission at specialized junctions called intercalated discs (ICDs). We have recently found that myocardin-related transcription factor A (MRTF-A) and –B (MRTFs) accumulate at ICDs of healthy mouse and human hearts, but are redistributed in cardiac pathology. MRTFs are mechanosensitive transcriptional co-activators of serum response factor (SRF) that regulate the actin cytoskeleton; however, the role of MRTFs in cardiomyocyte homeostasis remains unclear. We conditionally deleted Mrtfb in cardiomyocytes of mice harboring a homozygous null allele of Mrtfa (called MRTF cmdKO ). MRTF cmdKO mice exhibit no changes in cardiac function for up to a year; however, MRTF depletion results in conspicuous gap junction remodeling at ICDs and reduced cardiomyocyte communication. This phenotype stems from a significant reduction in genes important for trafficking gap junction proteins, including RP/EB family member 1 ( Mapre1 ), which we show is a direct transcriptional target of SRF/MRTFs. The absence of a functional phenotype amid gap junction displacement prompted us to model heart failure and mechanical stress at ICDs by subjecting MRTF cmdKO mice to transaortic constriction (TAC) surgery. Unexpectedly, MRTF cmdKO mice display partially penetrant sudden cardiac death within 2 weeks post-TAC surgery. MRTF cmdKO survivors undergo accelerated heart failure with a reduced adaptive gene response compared to controls. Future investigations are underway to determine the mechanisms governing accentuated heart failure in MRTF cmdKO mice. Taken together, the subcellular localization of MRTFs to ICDs creates a paradigm in which potent transcription factors are anchored to regions of electromechanical stress, which may represent a signaling circuit for ICD maintenance and remodeling in disease.