Desmoglein-2 mutations are detected in 5-10% of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC). Endurance training accelerates the development of the ARVC phenotype, leading to earlier arrhythmic events. Homozygous Dsg2 mutant mice develop a severe ARVC-like phenotype. The phenotype of heterozygous mutant (Dsg2mt/wt) or haploinsufficient (Dsg20/wt) mice is still not well understood. To assess the effects of age and endurance swim training, we studied cardiac morphology and function in sedentary one-year-old Dsg2mt/wt and Dsg20/wt mice and in young Dsg2mt/wt mice exposed to endurance swim training. Cardiac structure was only occasionally affected in aged Dsg20/wt and Dsg2mt/wt mice manifesting as small fibrotic foci and displacement of Connexin 43. Endurance swim training increased the right ventricular (RV) diameter and decreased RV function in Dsg2mt/wt mice but not in wild types. Dsg2mt/wt hearts showed increased ventricular activation times and pacing-induced ventricular arrhythmia without obvious fibrosis or inflammation. Preload-reducing therapy during training prevented RV enlargement and alleviated the electrophysiological phenotype. Taken together, endurance swim training induced features of ARVC in young adult Dsg2mt/wt mice. Prolonged ventricular activation times in the hearts of trained Dsg2mt/wt mice are therefore a potential mechanism for increased arrhythmia risk. Preload-reducing therapy prevented training-induced ARVC phenotype pointing to beneficial treatment options in human patients.

The intercalated disk is a cardiac specific structure composed of three main protein complexes-adherens junctions, desmosomes, and gap junctions-that work in concert to provide mechanical stability and electrical synchronization to the heart. Each substructure is regulated through a variety of mechanisms including proteolysis. Calpain proteases, a class of cysteine proteases dependent on calcium for activation, have recently emerged as important regulators of individual intercalated disk components. In this review, we will examine how calcium homeostasis regulates normal calpain function. We will also explore how calpains modulate gap junctions, desmosomes, and adherens junctions activity by targeting specific proteins, and describe the molecular mechanisms of how calpain dysregulation leads to structural and signaling defects within the heart. We will then examine how changes in calpain activity affects cardiomyocytes, and how such changes underlie various heart diseases.

Arrhythmogenic cardiomyopathy (ACM) is a heritable heart muscle disease characterized by syncope, palpitations, ventricular arrhythmias and sudden cardiac death (SCD) especially in young individuals. It is estimated to affect 1:5,000 individuals in the general population, with >60% of patients bearing one or more mutations in genes coding for desmosomal proteins. Desmosomes are intercellular adhesion junctions, which in cardiac myocytes reside within the intercalated disks (IDs), the areas of mechanical and electrical cell-cell coupling. Histologically, ACM is characterized by fibrofatty replacement of cardiac myocytes predominantly in the right ventricular free wall though left ventricular and biventricular forms have also been described. The disease is characterized by age-related progression, vast phenotypic manifestation and incomplete penetrance, making proband diagnosis and risk stratification of family members particularly challenging. Key protein redistribution at the IDs may represent a specific diagnostic marker but its applicability is still limited by the need for a myocardial sample. Specific markers of ACM in surrogate tissues, such as the blood and the buccal epithelium, may represent a non-invasive, safe and inexpensive alternative for diagnosis and cascade screening. In this review, we shall cover the most relevant biomarkers so far reported and discuss their potential impact on the diagnosis, prognosis and management of ACM.

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CMYA1 (cardiomyopathy-associated protein 1, also termed Xin) localizes to the intercalated disks (ICDs) of the myocardium and functions to maintain ICD structural integrity and support signal transduction among cardiomyocytes. Our previous study showed that CMYA1 overexpression impairs the function of gap junction intercellular communication processes. Successful model generation was verified based on PCR, western blot analysis, immunohistochemistry, and immunofluorescence analysis. Myocardial CMYA1 expression was confirmed at both the mRNA and the protein levels in the CMYA1-OE transgenic mice. Masson\'s trichrome staining and electron microscopy revealed myocardial fibrosis and uneven bead width or the interruption of ICDs in the hearts of the CMYA1-OE transgenic mice. Furthermore, the Cx43 protein level was reduced in the CMYA1-OE mice, and co-immunoprecipitation assays of heart tissue protein extracts revealed a physical interaction between CMYA1 and Cx43. Electrocardiogram analysis enabled the detection of an obvious ventricular bigeminy for the CMYA1-OE mice. In summary, analysis of our mouse model indicates that elevated CMYA1 levels may induce myocardial fibrosis, impair ICDs, and downregulate the expression of Cx43. The observed ventricular bigeminy in the CMYA1-OE mice may be mediated by the reduced Cx43 protein level.

AMP-activated protein kinase (AMPK) is a multifunctional kinase that regulates microtubule (MT) dynamic instability through CLIP-170 phosphorylation; however, its physiological relevance in vivo remains to be elucidated. In this study, we identified an active form of AMPK localized at the intercalated disks in the heart, a specific cell-cell junction present between cardiomyocytes. A contractile inhibitor, MYK-461, prevented the localization of AMPK at the intercalated disks, and the effect was reversed by the removal of MYK-461, suggesting that the localization of AMPK is regulated by mechanical stress. Time-lapse imaging analysis revealed that the inhibition of CLIP-170 Ser-311 phosphorylation by AMPK leads to the accumulation of MTs at the intercalated disks. Interestingly, MYK-461 increased the individual cell area of cardiomyocytes in CLIP-170 phosphorylation-dependent manner. Moreover, heart-specific CLIP-170 S311A transgenic mice demonstrated elongation of cardiomyocytes along with accumulated MTs, leading to progressive decline in cardiac contraction. In conclusion, these findings suggest that AMPK regulates the cell shape and aspect ratio of cardiomyocytes by modulating the turnover of MTs through homeostatic phosphorylation of CLIP-170 at the intercalated disks.

We investigated targeting mechanisms of Na+ and KATP channels to the intercalated disk (ICD) of cardiomyocytes. Patch clamp and surface biotinylation data show reciprocal downregulation of each other\'s surface density. Mutagenesis of the Kir6.2 ankyrin binding site disrupts this functional coupling. Duplex patch clamping and Angle SICM recordings show that INa and IKATP functionally co-localize at the rat ICD, but not at the lateral membrane. Quantitative STORM imaging show that Na+ and KATP channels are localized close to each other and to AnkG, but not to AnkB, at the ICD. Peptides corresponding to Nav1.5 and Kir6.2 ankyrin binding sites dysregulate targeting of both Na+ and KATP channels to the ICD, but not to lateral membranes. Finally, a clinically relevant gene variant that disrupts KATP channel trafficking also regulates Na+ channel surface expression. The functional coupling between these two channels need to be considered when assessing clinical variants and therapeutics.

Background: Mutations in genes encoding intercalated disk/desmosome proteins, such as plakophilin 2 (PKP2), cause arrhythmogenic cardiomyopathy (ACM). Desmosomes are responsible for myocyte-myocyte attachment and maintaining mechanical integrity of the myocardium. Methods: We knocked down Pkp2 in HL-1 mouse atrial cardiomyocytes (HL-1Pkp2-shRNA) and characterized their biomechanical properties. Gene expression was analyzed by RNA-Sequencing, microarray, and qPCR. Immunofluorescence was used to detect changes in cytoskeleton and focal adhesion. Antagomirs were used to knock down expression of selected microRNA (miR) in the rescue experiments. Results: Knockdown of Pkp2 was associated with decreased cardiomyocyte stiffness and work of detachment, and increased plasticity index. Altered mechanical properties were associated with impaired actin cytoskeleton in HL-1Pkp2-shRNA cells. Analysis of differentially expressed genes identified focal adhesion and actin cytoskeleton amongst the most dysregulated pathways, and miR200 family (a, b, and 429) as the most upregulated miRs in HL-1Pkp2-shRNA cells. Knockdown of miR-200b but not miR-200a, miR-429, by sequence-specific shRNAs partially rescued integrin-α1 (Itga1) levels, actin organization, cell adhesion (on collagen), and stiffness. Conclusions: PKP2 deficiency alters cardiomyocytes adhesion through a mechanism that involves upregulation of miR-200b and suppression of Itga1 expression. These findings provide new insights into the molecular basis of altered mechanosensing in ACM.

The intercalated disk (ID), a highly organized adhesion structure connecting neighboring cardiomyocytes, fulfills mechanical and electrical signaling communication to ensure normal heart function. Lipoprotein receptor-related protein 6 (LRP6) is a co-receptor inducing canonical Wnt/β-catenin signaling. It was recently reported that LRP6 deficiency in cardiomyocytes predisposes to arrhythmia independent of Wnt signaling. However, whether LRP6 directly regulates the structure of IDs requires further investigation. The aim of the present study was to explore the role of LRP6 in IDs and the potential underlying mechanisms by inducible cardiac-specific LRP6 knockout mice. The results revealed that LRP6 was predominately expressed in the cell membrane, including the IDs of cardiomyocytes. Tamoxifen-inducible cardiac-specific LRP6 knockout mice displayed overt cardiac dysfunction and disruption of ID structure. Further analysis revealed that cardiac LRP6 deficiency induced the imbalance of ID component proteins, characterized by the sharply decreased expression of connexin 43 (Cx43) and the significantly increased expression of N-cadherin, desmoplakin and γ-catenin in tissue lysates or membrane fraction from the left ventricle. STRING database analysis indicated that β-catenin, but no other ID-associated proteins, interacted with LRP6. Our immunoprecipitation analysis demonstrated that LRP6 strongly interacted with Cx43, N-cadherin and γ-catenin, and weakly interacted with β-catenin, whereas there was no association with desmoplakin. In response to LRP6 deficiency, the recruitment of β- or γ-catenin to N-cadherin was increased, but they displayed little interaction with Cx43. In conclusion, LRP6 is required to maintain the integrity of ID structure and the balance of ID proteins, and the interaction between LRP6 and Cx43, N-cadherin and γ-catenin may be involved in this process.

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