Faculty Bibliography

Introduction: Most cases of familial arrhythmogenic right ventricular cardiomyopathy (ARVC) associate with mutations in desmosomal proteins, most commonly plakophilin-2 (PKP2). A crosstalk between PKP2 and connexin43 (Cx43) has been proposed as a pathogenic mechanism. We speculate that a) Cx43 and PKP2 are in close physical proximity, allowing for direct intermolecular interaction and b) the structure of the Cx43- PKP2 complex depends on expression of the scaffolding protein ankyrin-G (AnkG). To test these hypotheses, we implemented a novel method (direct stochastic reconstruction microscopy; dSTORM) that allows for spatial resolution of fluorescence microscopy images in the nanoscale. Methods: Neonatal rat ventricular myocytes were labeled with antibodies to Cx43 and PKP2 and imaged using a custom- made microscopy system. On-off cycles of light emission were recorded in 2000 frames, and the image reconstructed by custom-made software. Cells were treated with siRNAfor AnkG, or non-targeted constructs, and the characteristics of Cx43 and PKP2 clusters compared to control. Results: Optical resolution of dSTORM images was 20 nm. Cx43 was found in circular clusters of two predominant sizes: 13313+/-328 and 25035+226 nm^2. PKP2 clusters were of various shapes and widespread size distribution, but consistently found less than 40 nm away from a Cx43 plaque, with signals overlapping on the edges of the plaques. Loss of AnkG expression drastically altered Cx43 cluster morphology becoming less circular and of a larger dimension. Close proximity to PKP2 was maintained. Yet, the total number of PKP2 clusters was significantly decreased. Conclusion: We implemented a method that breaks the optical resolution barrier imposed by the diffraction properties of light (~300 nm), to reach a range previously reserved to electron microscopy (~20 nm). We demonstrate that PKP2 populates the edge of Cx43 plaques (the perinexus). Cx43 cluster architecture depends on AnkG expression and likely, Cx43-cytoskeletal interacti!

Rationale: Compartmentation of ion channels on the cardiomyocyte surface is important for electric propagation and electromechanical coupling. The specialized T-tubule and costameric structures facilitate spatial coupling of various ion channels and receptors. Existing methods such as immunofluorescence and patch clamp techniques are limited in their ability to localize functional ion channels. As such, a correlation between channel protein location and channel function remains incomplete. Objective: To validate a method that permits routine imaging of the topography of a live cardiomyocyte and study clustering of functional ion channels from a specific microdomain. Methods and Results: We used scanning ion conductance microscopy and conventional cell-attached patch clamp with a software modification that allows controlled increase of pipette tip diameter. The sharp nanopipette used for topography scan was modified into a larger patch pipette that could be positioned with nanoscale precision to a specific site of interest (crest, groove, or T-tubules of cardiomyocytes) and sealed to the membrane for cell-attached recording of ion channels. Using this method, we significantly increased the probability of detecting activity of L-type calcium channels in the T-tubules of ventricular cardiomyocytes. We also demonstrated that active sodium channels do not distribute homogenously on the sarcolemma instead, they segregate into clusters of various densities, most crowded in the crest region, that are surrounded by areas virtually free of functional sodium channels. Conclusions: Our new method substantially increases the throughput of recording location-specific functional ion channels on the cardiomyocyte sarcolemma, thereby allowing characterization of ion channels in relation to the microdomain where they reside.

BACKGROUND: Arrhythmogenic cardiomyopathy (AC) is closely associated with desmosomal mutations in a majority of patients. Arrhythmogenesis in patients with AC is likely related to remodeling of cardiac gap junctions and increased levels of fibrosis. Recently, using experimental models, we also identified sodium channel dysfunction secondary to desmosomal dysfunction. OBJECTIVE: To assess the immunoreactive signal levels of the sodium channel protein Na1.5, as well as connexin43 (Cx43) and plakoglobin (PKG), in myocardial specimens obtained from patients with AC. METHODS: Left and right ventricular free wall postmortem material was obtained from 5 patients with AC and 5 controls matched for age and sex. Right ventricular septal biopsies were taken from another 15 patients with AC. All patients fulfilled the 2010 revised Task Force Criteria for the diagnosis of AC. Immunohistochemical analyses were performed using antibodies against Cx43, PKG, Na1.5, plakophilin-2, and N-cadherin. RESULTS: N-cadherin and desmoplakin immunoreactive signals and distribution were normal in patients with AC compared to controls. Plakophilin-2 signals were unaffected unless a plakophilin-2 mutation predicting haploinsufficiency was present. Distribution was unchanged compared to that in controls. Immunoreactive signal levels of PKG, Cx43, and Na1.5 were disturbed in 74%, 70%, and 65% of the patients, respectively. CONCLUSIONS: A reduced immunoreactive signal of PKG, Cx43, and Na1.5 at the intercalated disks can be observed in a large majority of the patients. Decreased levels of Na1.5 might contribute to arrhythmia vulnerability and, in the future, potentially could serve as a new clinically relevant tool for risk assessment strategies.

Gap junctions are essential to the function of multicellular animals, which require a high degree of coordination between cells. In vertebrates, gap junctions comprise connexins and currently 21 connexins are known in humans. The functions of gap junctions are highly diverse and include exchange of metabolites and electrical signals between cells, as well as functions, which are apparently unrelated to intercellular communication. Given the diversity of gap junction physiology, regulation of gap junction activity is complex. The structure of the various connexins is known to some extent; and structural rearrangements and intramolecular interactions are important for regulation of channel function. Intercellular coupling is further regulated by the number and activity of channels present in gap junctional plaques. The number of connexins in cell-cell channels is regulated by controlling transcription, translation, trafficking, and degradation; and all of these processes are under strict control. Once in the membrane, channel activity is determined by the conductive properties of the connexin involved, which can be regulated by voltage and chemical gating, as well as a large number of posttranslational modifications. The aim of the present article is to review our current knowledge on the structure, regulation, function, and pharmacology of gap junctions. This will be supported by examples of how different connexins and their regulation act in concert to achieve appropriate physiological control, and how disturbances of connexin function can lead to disease.

Ventricular ATP-sensitive potassium (K(ATP)) channels link intracellular energy metabolism to membrane excitability and contractility. Our recent proteomics experiments identified plakoglobin and plakophilin-2 (PKP2) as putative K(ATP) channel-associated proteins. We investigated whether the association of K(ATP) channel subunits with junctional proteins translates to heterogeneous subcellular distribution within a cardiac myocyte. Co-immunoprecipitation experiments confirmed physical interaction between K(ATP) channels and PKP2 and plakoglobin in rat heart. Immunolocalization experiments demonstrated that K(ATP) channel subunits (Kir6.2 and SUR2A) are expressed at a higher density at the intercalated disk in mouse and rat hearts, where they co-localized with PKP2 and plakoglobin. Super-resolution microscopy demonstrate that K(ATP) channels are clustered within nanometer distances from junctional proteins. The local K(ATP) channel density, recorded in excised inside-out patches, was larger at the cell end when compared with local currents recorded from the cell center. The K(ATP) channel unitary conductance, block by MgATP and activation by MgADP, did not differ between these two locations. Whole cell K(ATP) channel current density (activated by metabolic inhibition) was approximately 40% smaller in myocytes from mice haploinsufficient for PKP2. Experiments with excised patches demonstrated that the regional heterogeneity of K(ATP) channels was absent in the PKP2 deficient mice, but the K(ATP) channel unitary conductance and nucleotide sensitivities remained unaltered. Our data demonstrate heterogeneity of K(ATP) channel distribution within a cardiac myocyte. The higher K(ATP) channel density at the intercalated disk implies a possible role at the intercellular junctions during cardiac ischemia.

There is abundant evidence showing that connexins form gap junctions. Yet this does not exclude the possibility that connexins can exert other functions, separate from that of gap junction (or even a permeable hemichannel) formation. Here, we focus on these noncanonical functions of connexin43 (Cx43), particularly in the heart. We describe two specific examples: the importance of Cx43 on intercellular adhesion, and the role of Cx43 in the function of the sodium channel. We propose that these two functions of Cx43 have important repercussions on the propagation of electrical activity in the heart, irrespective of the presence of permeable gap junction channels. Overall, the gap junction-independent functions of Cx43 in cardiac electrophysiology emerge as an exciting area of future research.

AIMS: The shRNA-mediated loss of expression of the desmosomal protein plakophilin-2 leads to sodium current (I(Na)) dysfunction. Whether pkp2 gene haploinsufficiency leads to I(Na) deficit in vivo remains undefined. Mutations in pkp2 are detected in arrhythmogenic right ventricular cardiomyopathy (ARVC). Ventricular fibrillation and sudden death often occur in the 'concealed phase' of the disease, prior to overt structural damage. The mechanisms responsible for these arrhythmias remain poorly understood. We sought to characterize the morphology, histology, and ultrastructural features of PKP2-heterozygous-null (PKP2-Hz) murine hearts and explore the relation between PKP2 abundance, I(Na) function, and cardiac electrical synchrony. METHODS AND RESULTS: Hearts of PKP2-Hz mice were characterized by multiple methods. We observed ultrastructural but not histological or gross anatomical differences in PKP2-Hz hearts compared with wild-type (WT) littermates. Yet, in myocytes, decreased amplitude and a shift in gating and kinetics of I(Na) were observed. To further unmask I(Na) deficiency, we exposed myocytes, Langendorff-perfused hearts, and anaesthetized animals to a pharmacological challenge (flecainide). In PKP2-Hz hearts, the extent of flecainide-induced I(Na) block, impaired ventricular conduction, and altered electrocardiographic parameters were larger than controls. Flecainide provoked ventricular arrhythmias and death in PKP2-Hz animals, but not in the WT. CONCLUSIONS: PKP2 haploinsufficiency leads to I(Na) deficit in murine hearts. Our data support the notion of a cross-talk between desmosome and sodium channel complex. They also suggest that I(Na) dysfunction may contribute to generation and/or maintenance of arrhythmias in PKP2-deficient hearts. Whether pharmacological challenges could help unveil arrhythmia risk in patients with mutations or variants in PKP2 remains undefined.

Most commonly, arrhythmogenic cardiomyopathy (also known as arrhythmogenic right ventricular cardiomyopathy, or ARVC) is caused by mutations in desmosomal proteins. The question arises as to the mechanisms by which mutations in mechanical junctions, affect the rhythm of the heart. We have proposed that a component of the arrhythmogenic substrate may include changes in the function of both, gap junctions and sodium channels. Here, we review the relevant literature on this subject.

BACKGROUND: Desmosomes and adherens junctions provide mechanical continuity between cardiac cells, whereas gap junctions allow for cell-cell electrical/metabolic coupling. These structures reside at the cardiac intercalated disc (ID). Also at the ID is the voltage-gated sodium channel (VGSC) complex. Functional interactions between desmosomes, gap junctions, and VGSC have been demonstrated. Separate studies show, under various conditions, reduced presence of gap junctions at the ID and redistribution of connexin43 (Cx43) to plaques oriented parallel to fiber direction (gap junction "lateralization"). OBJECTIVE: To determine the mechanisms of Cx43 lateralization, and the fate of desmosomal and sodium channel molecules in the setting of Cx43 remodeling. METHODS: Adult sheep were subjected to right ventricular pressure overload (pulmonary hypertension). Tissue was analyzed by quantitative confocal microscopy and by transmission electron microscopy. Ionic currents were measured using conventional patch clamp. RESULT: Quantitative confocal microscopy demonstrated lateralization of immunoreactive junctional molecules. Desmosomes and gap junctions in lateral membranes were demonstrable by electron microscopy. Cx43/desmosomal remodeling was accompanied by lateralization of 2 microtubule-associated proteins relevant for Cx43 trafficking: EB1 and kinesin protein Kif5b. In contrast, molecules of the VGSC failed to reorganize in plaques discernable by confocal microscopy. Patch-clamp studies demonstrated change in amplitude and kinetics of sodium current and a small reduction in electrical coupling between cells. CONCLUSIONS: Cx43 lateralization is part of a complex remodeling that includes mechanical and gap junctions but may exclude components of the VGSC. We speculate that lateralization results from redirectionality of microtubule-mediated forward trafficking. Remodeling of junctional complexes may preserve electrical synchrony under conditions that disrupt ID integrity.

Introduction: Progressive cardiac conduction disturbance (PCCD) is a hereditary disorder of the His-Purkinje system that often leads to complete heart block, pacemaker implantation, or sudden death. Although mutations in genes SCN5A, SCN1B and TRPM4 have been identified in some PCCD pedigrees, large scale studies of its clinical features, prognosis, and genetic basis are not available. We studied a population of 63 Japanese PCCD patients (47 probands and 16 family members; male 37, female 26) without underlying structural heart diseases. Despite the assumption that PCCD predominates in the elderly, the age of onset of the proband showed a wide-range distribution (59.6+/-23.7 years) with two peaks in the 2nd and 6th decade of life. Patients were followed for a variable period of time (0.3 to 33 years; 11.6+/-10.0 years). Progressive aggravation of the conduction disturbance and cardiac dysfunction with LVEF<35% was evident in 46% and 33% of patients, respectively. Pacemaker or ICD was implanted in 44 patients (70%). Six patients, three with device therapy and three without, died suddenly. Genetic screening revealed mutations in SCN5A (coding for Nav1.5; n=9) and in GJA5 (coding for connexin40; n=1). Heterologous expression of the GJA5 mutant in N2A cells resulted in marked reduction of junctional conductance and diffuse localization of Cx40-immunoreactive proteins in the vicinity of the plasma membrane without formation of gap junctions. The proband of the GJA5 mutation and his mother died at the age of 10 and 30, respectively, indicating an early-onset and malignant variant of PCCD. Moreover, mutations were identified in LMNA(n=9), the gene encoding lamin A/C, an inner nuclear membrane protein involved in a number of reported cases of dilated cardiomyopathy. The electrophysiological defects preceded the cardiac dysfunction in all 9 LMNA carriers and in 2 family members, presumably due to an age-dependent enhancement of apoptosis in cells of the conduction system. These data show that the PCCD is a heterogeneous disease that can be associated with defects in the amino acid sequence of integral membrane proteins involved in cell excitability and/or action potential propagation. A separate group may represent a prodromal stage of lamin A/C-related dilated cardiomyopathy. Methods: N/A Results:N/A Conclusions: N/A