Tissue-resident macrophages provide a pro-tumorigenic niche to early NSCLC cells.

Macrophages have a key role in shaping the tumour microenvironment (TME), tumour immunity and response to immunotherapy, which makes them an important target for cancer treatment1,2. However, modulating macrophages has proved extremely difficult, as we still lack a complete understanding of the molecular and functional diversity of the tumour macrophage compartment. Macrophages arise from two distinct lineages. Tissue-resident macrophages self-renew locally, independent of adult haematopoiesis3-5, whereas short-lived monocyte-derived macrophages arise from adult haematopoietic stem cells, and accumulate mostly in inflamed lesions1. How these macrophage lineages contribute to the TME and cancer progression remains unclear. To explore the diversity of the macrophage compartment in human non-small cell lung carcinoma (NSCLC) lesions, here we performed single-cell RNA sequencing of tumour-associated leukocytes. We identified distinct populations of macrophages that were enriched in human and mouse lung tumours. Using lineage tracing, we discovered that these macrophage populations differ in origin and have a distinct temporal and spatial distribution in the TME. Tissue-resident macrophages accumulate close to tumour cells early during tumour formation to promote epithelial-mesenchymal transition and invasiveness in tumour cells, and they also induce a potent regulatory T cell response that protects tumour cells from adaptive immunity. Depletion of tissue-resident macrophages reduced the numbers and altered the phenotype of regulatory T cells, promoted the accumulation of CD8+ T cells and reduced tumour invasiveness and growth. During tumour growth, tissue-resident macrophages became redistributed at the periphery of the TME, which becomes dominated by monocyte-derived macrophages in both mouse and human NSCLC. This study identifies the contribution of tissue-resident macrophages to early lung cancer and establishes them as a target for the prevention and treatment of early lung cancer lesions.

Structural and Functional Characterization of a Nav1.5-Mitochondrial Couplon.

RATIONALE: The cardiac sodium channel NaV1.5 has a fundamental role in excitability and conduction. Previous studies have shown that sodium channels cluster together in specific cellular subdomains. Their association with intracellular organelles in defined regions of the myocytes, and the functional consequences of that association, remain to be defined. OBJECTIVE: To characterize a subcellular domain formed by sodium channel clusters in the crest region of the myocytes and the subjacent subsarcolemmal mitochondria. METHODS AND RESULTS: Through a combination of imaging approaches including super-resolution microscopy and electron microscopy we identified, in adult cardiac myocytes, a NaV1.5 subpopulation in close proximity to subjacent subsarcolemmal mitochondria; we further found that subjacent subsarcolemmal mitochondria preferentially host the mitochondrial NCLX (Na+/Ca2+ exchanger). This anatomic proximity led us to investigate functional changes in mitochondria resulting from sodium channel activity. Upon TTX (tetrodotoxin) exposure, mitochondria near NaV1.5 channels accumulated more Ca2+ and showed increased reactive oxygen species production when compared with interfibrillar mitochondria. Finally, crosstalk between NaV1.5 channels and mitochondria was analyzed at a transcriptional level. We found that SCN5A (encoding NaV1.5) and SLC8B1 (which encode NaV1.5 and NCLX, respectively) are negatively correlated both in a human transcriptome data set (Genotype-Tissue Expression) and in human-induced pluripotent stem cell-derived cardiac myocytes deficient in SCN5A. CONCLUSIONS: We describe an anatomic hub (a couplon) formed by sodium channel clusters and subjacent subsarcolemmal mitochondria. Preferential localization of NCLX to this domain allows for functional coupling where the extrusion of Ca2+ from the mitochondria is powered, at least in part, by the entry of sodium through NaV1.5 channels. These results provide a novel entry-point into a mechanistic understanding of the intersection between electrical and structural functions of the heart.

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

The cardiac connexome: Non-canonical functions of connexin43 and their role in cardiac arrhythmias.

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.

Bioinformatic analysis of a plakophilin-2-dependent transcription network: implications for the mechanisms of arrhythmogenic right ventricular cardiomyopathy in humans and in boxer dogs.

AIMS: Previous studies in murine hearts and in cell systems have shown that modifications in the expression or sequence integrity of the desmosomal molecule plakophilin-2 (PKP2) can alter the downstream expression of transcripts necessary for the electrical and mechanical function of the heart. These findings have provided support to mechanistic hypotheses that seek to explain arrhythmogenic right ventricular cardiomyopathy (ARVC) in humans. However, the relation between PKP2 expression and the transcriptome of the human heart remains poorly explored. Furthermore, while a number of studies have documented the clinical similarity between familial ARVC in humans and inheritable ARVC in boxer dogs, there is a puzzling lack of convergence as to the possible genetic causes of disease in one species vs. the other. METHODS AND RESULTS: We implemented bioinformatics analysis tools to explore the relation between the PKP2-dependent murine and human transcriptomes. Our data suggest that genes involved in intracellular calcium regulation, and others involved in intercellular adhesion, form part of a co-ordinated gene network. We further identify PROX1 and PPARA (coding for the proteins Prox1 and PPAR-alpha, respectively) as transcription factors within the same network. CONCLUSION: On the basis our analysis, we hypothesize that the molecular cascades initiated by the seemingly unrelated genetic mutations in humans and in boxers actually converge downstream into a common pathway. This can explain the similarities in the clinical manifestation of ARVC in humans and in the boxer dogs.

Mitochondrial Cx43 hemichannels contribute to mitochondrial calcium entry and cell death in the heart.

Mitochondrial connexin 43 (Cx43) plays a key role in cardiac cytoprotection caused by repeated exposure to short periods of non-lethal ischemia/reperfusion, a condition known as ischemic preconditioning. Cx43 also forms calcium (Ca2+)-permeable hemichannels that may potentially lead to mitochondrial Ca2+ overload and cell death. Here, we studied the role of Cx43 in facilitating mitochondrial Ca2+ entry and investigated its downstream consequences. To that purpose, we used various connexin-targeting peptides interacting with extracellular (Gap26) and intracellular (Gap19, RRNYRRNY) Cx43 domains, and tested their effect on mitochondrial dye- and Ca2+-uptake, electrophysiological properties of plasmalemmal and mitochondrial Cx43 channels, and cell injury/cell death. Our results in isolated mice cardiac subsarcolemmal mitochondria indicate that Cx43 forms hemichannels that contribute to Ca2+ entry and may trigger permeability transition and cell injury/death. RRNYRRNY displayed the strongest effects in all assays and inhibited plasma membrane as well as mitochondrial Cx43 hemichannels. RRNYRRNY also strongly reduced the infarct size in ex vivo cardiac ischemia-reperfusion studies. These results indicate that Cx43 contributes to mitochondrial Ca2+ homeostasis and is involved in triggering cell injury/death pathways that can be inhibited by RRNYRRNY peptide.

Missense mutations in plakophilin-2 cause sodium current deficit and associate with a Brugada syndrome phenotype.

BACKGROUND: Brugada syndrome (BrS) primarily associates with the loss of sodium channel function. Previous studies showed features consistent with sodium current (INa) deficit in patients carrying desmosomal mutations, diagnosed with arrhythmogenic cardiomyopathy (or arrhythmogenic right ventricular cardiomyopathy). Experimental models showed correlation between the loss of expression of desmosomal protein plakophilin-2 (PKP2) and reduced INa. We hypothesized that PKP2 variants that reduce INa could yield a BrS phenotype, even without overt structural features characteristic of arrhythmogenic right ventricular cardiomyopathy. METHODS AND RESULTS: We searched for PKP2 variants in the genomic DNA of 200 patients with a BrS diagnosis, no signs of arrhythmogenic cardiomyopathy, and no mutations in BrS-related genes SCN5A, CACNa1c, GPD1L, and MOG1. We identified 5 cases of single amino acid substitutions. Mutations were tested in HL-1-derived cells endogenously expressing NaV1.5 but made deficient in PKP2 (PKP2-KD). Loss of PKP2 caused decreased INa and NaV1.5 at the site of cell contact. These deficits were restored by the transfection of wild-type PKP2, but not of BrS-related PKP2 mutants. Human induced pluripotent stem cell cardiomyocytes from a patient with a PKP2 deficit showed drastically reduced INa. The deficit was restored by transfection of wild type, but not BrS-related PKP2. Super-resolution microscopy in murine PKP2-deficient cardiomyocytes related INa deficiency to the reduced number of channels at the intercalated disc and increased separation of microtubules from the cell end. CONCLUSIONS: This is the first systematic retrospective analysis of a patient group to define the coexistence of sodium channelopathy and genetic PKP2 variations. PKP2 mutations may be a molecular substrate leading to the diagnosis of BrS.

Heterogeneity of ATP-sensitive K+ channels in cardiac myocytes: enrichment at the intercalated disk.

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 ∼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.

Molecular determinants of the crosstalk between endosomal microautophagy and chaperone-mediated autophagy.

Chaperone-mediated autophagy (CMA) and endosomal microautophagy (eMI) are pathways for selective degradation of cytosolic proteins in lysosomes and late endosomes, respectively. These autophagic processes share as a first step the recognition of the same five-amino-acid motif in substrate proteins by the Hsc70 chaperone, raising the possibility of coordinated activity of both pathways. In this work, we show the existence of a compensatory relationship between CMA and eMI and identify a role for the chaperone protein Bag6 in triage and internalization of eMI substrates into late endosomes. Association and dynamics of Bag6 at the late endosome membrane change during starvation, a stressor that, contrary to other autophagic pathways, causes a decline in eMI activity. Collectively, these results show a coordinated function of eMI with CMA, identify the interchangeable subproteome degraded by these pathways, and start to elucidate the molecular mechanisms that facilitate the switch between them.

The noncanonical functions of Cx43 in the heart.

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.

A proliferative to invasive switch is mediated by srGAP1 downregulation through the activation of TGF-ß2 signaling.

Many breast cancer (BC) patients suffer from complications of metastatic disease. To form metastases, cancer cells must become migratory and coordinate both invasive and proliferative programs at distant organs. Here, we identify srGAP1 as a regulator of a proliferative-to-invasive switch in BC cells. High-resolution light-sheet microscopy demonstrates that BC cells can form actin-rich protrusions during extravasation. srGAP1low cells display a motile and invasive phenotype that facilitates their extravasation from blood vessels, as shown in zebrafish and mouse models, while attenuating tumor growth. Interestingly, a population of srGAP1low cells remain as solitary disseminated tumor cells in the lungs of mice bearing BC tumors. Overall, srGAP1low cells have increased Smad2 activation and TGF-ß2 secretion, resulting in increased invasion and p27 levels to sustain quiescence. These findings identify srGAP1 as a mediator of a proliferative to invasive phenotypic switch in BC cells in vivo through a TGF-ß2-mediated signaling axis.

Reduced endosomal microautophagy activity in aging associates with enhanced exocyst-mediated protein secretion.

Autophagy is essential for protein quality control and regulation of the functional proteome. Failure of autophagy pathways with age contributes to loss of proteostasis in aged organisms and accelerates the progression of age-related diseases. In this work, we show that activity of endosomal microautophagy (eMI), a selective type of autophagy occurring in late endosomes, declines with age and identify the sub-proteome affected by this loss of function. Proteomics of late endosomes from old mice revealed an aberrant glycation signature for Hsc70, the chaperone responsible for substrate targeting to eMI. Age-related Hsc70 glycation reduces its stability in late endosomes by favoring its organization into high molecular weight protein complexes and promoting its internalization/degradation inside late endosomes. Reduction of eMI with age associates with an increase in protein secretion, as late endosomes can release protein-loaded exosomes upon plasma membrane fusion. Our search for molecular mediators of the eMI/secretion switch identified the exocyst-RalA complex, known for its role in exocytosis, as a novel physiological eMI inhibitor that interacts with Hsc70 and acts directly at the late endosome membrane. This inhibitory function along with the higher exocyst-RalA complex levels detected in late endosomes from old mice could explain, at least in part, reduced eMI activity with age. Interaction of Hsc70 with components of the exocyst-RalA complex places this chaperone in the switch from eMI to secretion. Reduced intracellular degradation in favor of extracellular release of undegraded material with age may be relevant to the spreading of proteotoxicity associated with aging and progression of proteinopathies.

Neuron-specific chromosomal megadomain organization is adaptive to recent retrotransposon expansions.

Regulatory mechanisms associated with repeat-rich sequences and chromosomal conformations in mature neurons remain unexplored. Here, we map cell-type specific chromatin domain organization in adult mouse cerebral cortex and report strong enrichment of Endogenous Retrovirus 2 (ERV2) repeat sequences in the neuron-specific heterochromatic B2NeuN+ megabase-scaling subcompartment. Single molecule long-read sequencing and comparative Hi-C chromosomal contact mapping in wild-derived SPRET/EiJ (Mus spretus) and laboratory inbred C57BL/6J (Mus musculus) reveal neuronal reconfigurations tracking recent ERV2 expansions in the murine germline, with significantly higher B2NeuN+ contact frequencies at sites with ongoing insertions in Mus musculus. Neuronal ablation of the retrotransposon silencer Kmt1e/Setdb1 triggers B2NeuN+ disintegration and rewiring with open chromatin domains enriched for cellular stress response genes, along with severe neuroinflammation and proviral assembly with infiltration of dendrites . We conclude that neuronal megabase-scale chromosomal architectures include an evolutionarily adaptive heterochromatic organization which, upon perturbation, results in transcriptional dysregulation and unleashes ERV2 proviruses with strong neuronal tropism.

Single-Molecule Localization of the Cardiac Voltage-Gated Sodium Channel Reveals Different Modes of Reorganization at Cardiomyocyte Membrane Domains.

BACKGROUND: Mutations in the gene encoding the cardiac voltage-gated sodium channel Nav1.5 cause various cardiac arrhythmias. This variety may arise from different determinants of Nav1.5 expression between cardiomyocyte domains. At the lateral membrane and T-tubules, Nav1.5 localization and function remain insufficiently characterized. METHODS: We used novel single-molecule localization microscopy and computational modeling to define nanoscale features of Nav1.5 localization and distribution at the lateral membrane, the lateral membrane groove, and T-tubules in cardiomyocytes from wild-type (N=3), dystrophin-deficient (mdx; N=3) mice, and mice expressing C-terminally truncated Nav1.5 (ΔSIV; N=3). We moreover assessed T-tubules sodium current by recording whole-cell sodium currents in control (N=5) and detubulated (N=5) wild-type cardiomyocytes. RESULTS: We show that Nav1.5 organizes as distinct clusters in the groove and T-tubules which density, distribution, and organization partially depend on SIV and dystrophin. We found that overall reduction in Nav1.5 expression in mdx and ΔSIV cells results in a nonuniform redistribution with Nav1.5 being specifically reduced at the groove of ΔSIV and increased in T-tubules of mdx cardiomyocytes. A T-tubules sodium current could, however, not be demonstrated. CONCLUSIONS: Nav1.5 mutations may site-specifically affect Nav1.5 localization and distribution at the lateral membrane and T-tubules, depending on site-specific interacting proteins. Future research efforts should elucidate the functional consequences of this redistribution.

Adult human cardiac stem cell supplementation effectively increases contractile function and maturation in human engineered cardiac tissues.

BACKGROUND: Delivery of stem cells to the failing heart is a promising therapeutic strategy. However, the improvement in cardiac function in animal studies has not fully translated to humans. To help bridge the gap between species, we investigated the effects of adult human cardiac stem cells (hCSCs) on contractile function of human engineered cardiac tissues (hECTs) as a species-specific model of the human myocardium. METHODS: Human induced pluripotent stem cell-derived cardiomyoctes (hCMs) were mixed with Collagen/Matrigel to fabricate control hECTs, with an experimental group of hCSC-supplemented hECT fabricated using a 9:1 ratio of hCM to hCSC. Functional testing was performed starting on culture day 6, under spontaneous conditions and also during electrical pacing from 0.25 to 1.0 Hz, measurements repeated at days 8 and 10. hECTs were then frozen and processed for gene analysis using a Nanostring assay with a cardiac targeted custom panel. RESULTS: The hCSC-supplemented hECTs displayed a twofold higher developed force vs. hCM-only controls by day 6, with approximately threefold higher developed stress and maximum rates of contraction and relaxation during pacing at 0.75 Hz. The spontaneous beat rate characteristics were similar between groups, and hCSC supplementation did not adversely impact beat rate variability. The increased contractility persisted through days 8 and 10, albeit with some decrease in the magnitude of the difference of the force by day 10, but with developed stress still significantly higher in hCSC-supplemented hECT; these findings were confirmed with multiple hCSC and hCM cell lines. The force-frequency relationship, while negative for both, control (- 0.687 Hz- 1; p = 0.013 vs. zero) and hCSC-supplemented (- 0.233 Hz- 1;p = 0.067 vs. zero) hECTs, showed a significant rectification in the regression slope in hCSC-supplemented hECT (p = 0.011 vs. control). Targeted gene exploration (59 genes) identified a total of 14 differentially expressed genes, with increases in the ratios of MYH7/MHY6, MYL2/MYL7, and TNNI3/TNNI1 in hCSC-supplemented hECT versus controls. CONCLUSIONS: For the first time, hCSC supplementation was shown to significantly improve human cardiac tissue contractility in vitro, without evidence of proarrhythmic effects, and was associated with increased expression of markers of cardiac maturation. These findings provide new insights about adult cardiac stem cells as contributors to functional improvement of human myocardium.

Localized Myosin II Activity Regulates Assembly and Plasticity of the Axon Initial Segment.

The axon initial segment (AIS) is the site of action potential generation and a locus of activity-dependent homeostatic plasticity. A multimeric complex of sodium channels, linked via a cytoskeletal scaffold of ankyrin G and beta IV spectrin to submembranous actin rings, mediates these functions. The mechanisms that specify the AIS complex to the proximal axon and underlie its plasticity remain poorly understood. Here we show phosphorylated myosin light chain (pMLC), an activator of contractile myosin II, is highly enriched in the assembling and mature AIS, where it associates with actin rings. MLC phosphorylation and myosin II contractile activity are required for AIS assembly, and they regulate the distribution of AIS components along the axon. pMLC is rapidly lost during depolarization, destabilizing actin and thereby providing a mechanism for activity-dependent structural plasticity of the AIS. Together, these results identify pMLC/myosin II activity as a common link between AIS assembly and plasticity.

Sodium Channel Remodeling in Subcellular Microdomains of Murine Failing Cardiomyocytes.

BACKGROUND: Cardiac sodium channel (NaV1.5) dysfunction contributes to arrhythmogenesis during pathophysiological conditions. Nav1.5 localizes to distinct subcellular microdomains within the cardiomyocyte, where it associates with region-specific proteins, yielding complexes whose function is location specific. We herein investigated sodium channel remodeling within distinct cardiomyocyte microdomains during heart failure. METHODS AND RESULTS: Mice were subjected to 6 weeks of transverse aortic constriction (TAC; n=32) to induce heart failure. Sham-operated on mice were used as controls (n=20). TAC led to reduced left ventricular ejection fraction, QRS prolongation, increased heart mass, and upregulation of prohypertrophic genes. Whole-cell sodium current (INa) density was decreased by 30% in TAC versus sham-operated on cardiomyocytes. On macropatch analysis, INa in TAC cardiomyocytes was reduced by 50% at the lateral membrane (LM) and by 40% at the intercalated disc. Electron microscopy and scanning ion conductance microscopy revealed remodeling of the intercalated disc (replacement of [inter-]plicate regions by large foldings) and LM (less identifiable T tubules and reduced Z-groove ratios). Using scanning ion conductance microscopy, cell-attached recordings in LM subdomains revealed decreased INa and increased late openings specifically at the crest of TAC cardiomyocytes, but not in groove/T tubules. Failing cardiomyocytes displayed a denser, but more stable, microtubule network (demonstrated by increased α-tubulin and Glu-tubulin expression). Superresolution microscopy showed reduced average NaV1.5 cluster size at the LM of TAC cells, in line with reduced INa. CONCLUSIONS: Heart failure induces structural remodeling of the intercalated disc, LM, and microtubule network in cardiomyocytes. These adaptations are accompanied by alterations in NaV1.5 clustering and INa within distinct subcellular microdomains of failing cardiomyocytes.

Plakophilin-2 is required for transcription of genes that control calcium cycling and cardiac rhythm.

Plakophilin-2 (PKP2) is a component of the desmosome and known for its role in cell-cell adhesion. Mutations in human PKP2 associate with a life-threatening arrhythmogenic cardiomyopathy, often of right ventricular predominance. Here, we use a range of state-of-the-art methods and a cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mouse to demonstrate that in addition to its role in cell adhesion, PKP2 is necessary to maintain transcription of genes that control intracellular calcium cycling. Lack of PKP2 reduces expression of Ryr2 (coding for Ryanodine Receptor 2), Ank2 (coding for Ankyrin-B), Cacna1c (coding for CaV1.2) and Trdn (coding for triadin), and protein levels of calsequestrin-2 (Casq2). These factors combined lead to disruption of intracellular calcium homeostasis and isoproterenol-induced arrhythmias that are prevented by flecainide treatment. We propose a previously unrecognized arrhythmogenic mechanism related to PKP2 expression and suggest that mutations in PKP2 in humans may cause life-threatening arrhythmias even in the absence of structural disease.It is believed that mutations in desmosomal adhesion complex protein plakophilin 2 (PKP2) cause arrhythmia due to loss of cell-cell communication. Here the authors show that PKP2 controls the expression of proteins involved in calcium cycling in adult mouse hearts, and that lack of PKP2 can cause arrhythmia in a structurally normal heart.

Multilevel analyses of SCN5A mutations in arrhythmogenic right ventricular dysplasia/cardiomyopathy suggest non-canonical mechanisms for disease pathogenesis.

AIMS: Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy (ARVD/C) is often associated with desmosomal mutations. Recent studies suggest an interaction between the desmosome and sodium channel protein Nav1.5. We aimed to determine the prevalence and biophysical properties of mutations in SCN5A (the gene encoding Nav1.5) in ARVD/C. METHODS AND RESULTS: We performed whole-exome sequencing in six ARVD/C patients (33% male, 38.2 ± 12.1 years) without a desmosomal mutation. We found a rare missense variant (p.Arg1898His; R1898H) in SCN5A in one patient. We generated induced pluripotent stem cell-derived cardiomyocytes (hIPSC-CMs) from the patient's peripheral blood mononuclear cells. The variant was then corrected (R1898R) using Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 technology, allowing us to study the impact of the R1898H substitution in the same cellular background. Whole-cell patch clamping revealed a 36% reduction in peak sodium current (P = 0.002); super-resolution fluorescence microscopy showed reduced abundance of NaV1.5 (P = 0.005) and N-Cadherin (P = 0.026) clusters at the intercalated disc. Subsequently, we sequenced SCN5A in an additional 281 ARVD/C patients (60% male, 34.8 ± 13.7 years, 52% desmosomal mutation-carriers). Five (1.8%) subjects harboured a putatively pathogenic SCN5A variant (p.Tyr416Cys, p.Leu729del, p.Arg1623Ter, p.Ser1787Asn, and p.Val2016Met). SCN5A variants were associated with prolonged QRS duration (119 ± 15 vs. 94 ± 14 ms, P < 0.01) and all SCN5A variant carriers had major structural abnormalities on cardiac imaging. CONCLUSIONS: Almost 2% of ARVD/C patients harbour rare SCN5A variants. For one of these variants, we demonstrated reduced sodium current, Nav1.5 and N-Cadherin clusters at junctional sites. This suggests that Nav1.5 is in a functional complex with adhesion molecules, and reveals potential non-canonical mechanisms by which Nav1.5 dysfunction causes cardiomyopathy.