Faculty Bibliography

Members of the desmosome protein family are integral components of the cardiac area composita, a mixed junctional complex responsible for electromechanical coupling between cardiomyocytes. In this study, we provide evidence that loss of the desmosomal armadillo protein Plakophilin-2 (PKP2) in cardiomyocytes elevates transforming growth factor beta1 (TGF-beta1) and p38 mitogen-activated protein kinase (MAPK) signaling, which together coordinate a transcriptional program that results in increased expression of profibrotic genes. Importantly, we demonstrate that expression of Desmoplakin (DP) is lost upon PKP2 knockdown and that restoration of DP expression rescues the activation of this TGF-beta1/p38 MAPK transcriptional cascade. Tissues from PKP2 heterozygous and DP conditional knockout mouse models also exhibit elevated TGF-beta1/p38 MAPK signaling and induction of fibrotic gene expression in vivo. These data therefore identify PKP2 and DP as central players in coordination of desmosome-dependent TGF-beta1/p38 MAPK signaling in cardiomyocytes, pathways known to play a role in different types of cardiac disease, such as arrhythmogenic or hypertrophic cardiomyopathy.

Connexin43 is the major component of gap junctions, an anatomical structure present in the cardiac intercalated disc that provides a low-resistance pathway for direct cell-to-cell passage of electrical charge. Recent studies have shown that in addition to its well-established function as an integral membrane protein that oligomerizes to form gap junctions, Cx43 plays other roles that are independent of channel (or perhaps even hemi-channel) formation. This article discusses non-canonical functions of Cx43. In particular, we focus on the role of Cx43 as a part of a protein interacting network, a connexome, where molecules classically defined as belonging to the mechanical junctions, the gap junctions and the sodium channel complex, multitask and work together to bring about excitability, electrical and mechanical coupling between cardiac cells. Overall, viewing Cx43 as a multi-functional protein, beyond gap junctions, opens a window to better understand the function of the intercalated disc and the pathological consequences that may result from changes in the abundance or localization of Cx43 in the intercalated disc subdomain.

Intercellular adhesion and electrical excitability are considered separate cellular properties. Studies of myelinated fibres, however, show that voltage-gated sodium channels (VGSCs) aggregate with cell adhesion molecules at discrete subcellular locations, such as the nodes of Ranvier. Demonstration of similar macromolecular organization in cardiac muscle is missing. Here we combine nanoscale-imaging (single-molecule localization microscopy; electron microscopy; and 'angle view' scanning patch clamp) with mathematical simulations to demonstrate distinct hubs at the cardiac intercalated disc, populated by clusters of the adhesion molecule N-cadherin and the VGSC NaV1.5. We show that the N-cadherin-NaV1.5 association is not random, that NaV1.5 molecules in these clusters are major contributors to cardiac sodium current, and that loss of NaV1.5 expression reduces intercellular adhesion strength. We speculate that adhesion/excitability nodes are key sites for crosstalk of the contractile and electrical molecular apparatus and may represent the structural substrate of cardiomyopathies in patients with mutations in molecules of the VGSC complex.

AIMS: 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). METHODS AND RESULTS: 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 increased total ID surface area by two orders of magnitude. In addition, PKP2-Hz samples showed increased average intercellular spacing, intercalated disc surface area and membrane tortuosity, as well as reduced number and length of mechanical junctions when compared to control. Finally, we observed membranous structures reminiscent of junctional sarcoplasmic reticulum at the ID, which were significantly more abundant in PKP2-Hz hearts. CONCLUSIONS: 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 behavior is discussed.

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.

KEY POINTS: Na(+) current (INa ) results from the integrated function of a molecular aggregate (the voltage-gated Na(+) channel complex) that includes the beta subunit family. Mutations or rare variants in Scn1b (encoding the beta1 and beta1B subunits) have been associated with various inherited arrhythmogenic syndromes, including Brugada syndrome and sudden unexpected death in patients with epilepsy. We used Scn1b null mice to understand better the relation between Scn1b expression, and cardiac electrical function. Loss of Scn1b caused, among other effects, increased amplitude of tetrodotoxin-sensitive INa , delayed after-depolarizations, triggered beats, delayed Ca(2+) transients, frequent spontaneous calcium release events and increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) homeostasis were prevented by 100 nm tetrodotoxin. We propose that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na(+) channel alpha subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis. ABSTRACT: Na(+) current (INa ) is determined not only by the properties of the pore-forming voltage-gated Na(+) channel (VGSC) alpha subunit, but also by the integrated function of a molecular aggregate (the VGSC complex) that includes the VGSC beta subunit family. Mutations or rare variants in Scn1b (encoding the beta1 and beta1B subunits) have been associated with various inherited arrhythmogenic syndromes, including cases of Brugada syndrome and sudden unexpected death in patients with epilepsy. Here, we have used Scn1b null mouse models to understand better the relation between Scn1b expression, and cardiac electrical function. Using a combination of macropatch and scanning ion conductance microscopy we show that loss of Scn1b in juvenile null animals resulted in increased tetrodotoxin-sensitive INa but only in the cell midsection, even before full T-tubule formation; the latter occurred concurrent with increased message abundance for the neuronal Scn3a mRNA, suggesting increased abundance of tetrodotoxin-sensitive NaV 1.3 protein and yet its exclusion from the region of the intercalated disc. Ventricular myocytes from cardiac-specific adult Scn1b null animals showed increased Scn3a message, prolonged action potential repolarization, presence of delayed after-depolarizations and triggered beats, delayed Ca(2+) transients and frequent spontaneous Ca(2+) release events and at the whole heart level, increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) homeostasis were prevented by 100 nm tetrodotoxin. Our results suggest that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na(+) channel alpha subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis in ventricular myocytes.