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 Ca_V1.2) 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.

Background: Plakophilin-2 (PKP2) is classically defined as a desmosomal protein. Mutations in PKP2 associate with most cases of gene-positive arrhythmogenic right ventricular cardiomyopathy. A better understanding of PKP2 cardiac biology can help elucidate the mechanisms underlying arrhythmic and cardiomyopathic events consequent to PKP2 deficiency. Here, we sought to capture early molecular/cellular events that can act as nascent arrhythmic/cardiomyopathic substrates. Methods: We used multiple imaging, biochemical and high-resolution mass spectrometry methods to study functional/structural properties of cells/tissues derived from cardiomyocyte-specific, tamoxifen-activated, PKP2 knockout mice (PKP2cKO) 14 days post-tamoxifen injection, a time point preceding overt electrical or structural phenotypes. Myocytes from right or left ventricular free wall were studied separately. Results: Most properties of PKP2cKO left ventricular myocytes were not different from control; in contrast, PKP2cKO right ventricular (RV) myocytes showed increased amplitude and duration of Ca ²⁺ transients, increased Ca ²⁺ in the cytoplasm and sarcoplasmic reticulum, increased frequency of spontaneous Ca ²⁺ release events (sparks) even at comparable sarcoplasmic reticulum load, and dynamic Ca ²⁺ accumulation in mitochondria. We also observed early- and delayed-after transients in RV myocytes and heightened susceptibility to arrhythmias in Langendorff-perfused hearts. In addition, ryanodine receptor 2 in PKP2cKO-RV cells presented enhanced Ca ²⁺ sensitivity and preferential phosphorylation in a domain known to modulate Ca ²⁺ gating. RNAseq at 14 days post-tamoxifen showed no relevant difference in transcript abundance between RV and left ventricle, neither in control nor in PKP2cKO cells. Instead, we found an RV-predominant increase in membrane permeability that can permit Ca ²⁺ entry into the cell. Connexin 43 ablation mitigated the membrane permeability increase, accumulation of cytoplasmic Ca ²⁺ , increased frequency of sparks and early stages of RV dysfunction. Connexin 43 hemichannel block with GAP19 normalized [Ca ²⁺ ] _i homeostasis. Similarly, protein kinase C inhibition normalized spark frequency at comparable sarcoplasmic reticulum load levels. Conclusions: Loss of PKP2 creates an RV-predominant arrhythmogenic substrate (Ca ²⁺ dysregulation) that precedes the cardiomyopathy; this is, at least in part, mediated by a Connexin 43-dependent membrane conduit and repressed by protein kinase C inhibitors. Given that asymmetric Ca ²⁺ dysregulation precedes the cardiomyopathic stage, we speculate that abnormal Ca ²⁺ handling in RV myocytes can be a trigger for gross structural changes observed at a later stage.

Rationale:: In cardiomyocytes, Na _V 1.5 and Kir2.1 channels interact dynamically as part of membrane bound macromolecular complexes. Objective:: The objective of this study was to test whether Na _V 1.5 and Kir2.1 preassemble during early forward trafficking and travel together to common membrane microdomains. Methods and Results:: In patch-clamp experiments, coexpression of trafficking-deficient mutants Kir2.1 ^Δ314-315 or Kir2.1 ^R44A/R46A with wild-type (WT) Na _V 1.5 ^WT in heterologous cells reduced inward sodium current compared with Na _V 1.5 ^WT alone or coexpressed with Kir2.1 ^WT . In cell surface biotinylation experiments, expression of Kir2.1 ^Δ314-315 reduced Na _V 1.5 channel surface expression. Glycosylation analysis suggested that Na _V 1.5 ^WT and Kir2.1 ^WT channels associate early in their biosynthetic pathway, and fluorescence recovery after photobleaching experiments demonstrated that coexpression with Kir2.1 increased cytoplasmic mobility of Na _V 1.5 ^WT , and vice versa, whereas coexpression with Kir2.1 ^Δ314-315 reduced mobility of both channels. Viral gene transfer of Kir2.1 ^Δ314-315 in adult rat ventricular myocytes and human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current and inward sodium current, maximum diastolic potential and action potential depolarization rate, and increased action potential duration. On immunostaining, the AP1 (adaptor protein complex 1) colocalized with Na _V 1.5 ^WT and Kir2.1 ^WT within areas corresponding to t-tubules and intercalated discs. Like Kir2.1 ^WT , Na _V 1.5 ^WT coimmunoprecipitated with AP1. Site-directed mutagenesis revealed that Na _V 1.5 ^WT channels interact with AP1 through the Na _V 1.5 ^Y1810 residue, suggesting that, like for Kir2.1 ^WT , AP1 can mark Na _V 1.5 channels for incorporation into clathrin-coated vesicles at the trans -Golgi. Silencing the AP1 ϒ-adaptin subunit in human induced pluripotent stem cell–derived cardiomyocytes reduced inward rectifier potassium current, inward sodium current, and maximum diastolic potential and impaired rate-dependent action potential duration adaptation. Conclusions:: The Na _V 1.5-Kir2.1 macromolecular complex pre-assembles early in the forward trafficking pathway. Therefore, disruption of Kir2.1 trafficking in cardiomyocytes affects trafficking of Na _V 1.5, which may have important implications in the mechanisms of arrhythmias in inheritable cardiac diseases.

Background: Aberrant calcium signaling may contribute to arrhythmias and adverse remodeling in hypertrophic cardiomyopathy (HCM). Mutations in sarcomere genes may distinctly alter calcium handling pathways. Methods: We analyzed gene expression, protein levels, and functional assays for calcium regulatory pathways in human HCM surgical samples with (n=25) and without (n=10) sarcomere mutations compared with control hearts (n=8). Results: Gene expression and protein levels for calsequestrin, L-type calcium channel, sodium-calcium exchanger, phospholamban, calcineurin, and calcium/calmodulin-dependent protein kinase type II (CaMKII) were similar in HCM samples compared with controls. CaMKII protein abundance was increased only in sarcomere-mutation HCM ( P <0.001). The CaMKII target pT17-phospholamban was 5.5-fold increased only in sarcomere-mutation HCM ( P =0.01), as was autophosphorylated CaMKII ( P <0.01), suggestive of constitutive activation. Calcineurin ( PPP3CB ) mRNA was not increased, nor was RCAN1 mRNA level, indicating a lack of calcineurin activation. Furthermore, myocyte enhancer factor 2 and nuclear factor of activated T cell transcription factor activity was not increased in HCM, suggesting that calcineurin pathway activation is not an upstream cause of increased CAMKII protein abundance or activation. SERCA2A mRNA transcript levels were reduced in HCM regardless of genotype, as was sarcoplasmic endoplasmic reticular calcium ATPase 2/phospholamban protein ratio (45% reduced; P =0.03). ⁴⁵ Ca sarcoplasmic endoplasmic reticular calcium ATPaseuptake assay showed reduced uptake velocity in HCM regardless of genotype ( P =0.01). The cardiac ryanodine receptor was not altered in transcript, protein, or phosphorylated (pS2808, pS2814) protein abundance, and [ ³ H]ryanodine binding was not different in HCM, consistent with no major modification of the ryanodine receptor. Conclusions: Human HCM demonstrates calcium mishandling through both genotype-specific and common pathways. Posttranslational activation of the CaMKII pathway is specific to sarcomere mutation–positive HCM, whereas sarcoplasmic endoplasmic reticular calcium ATPase 2 abundance and sarcoplasmic reticulum Ca uptake are depressed in both sarcomere mutation–positive and –negative HCM.

Key points: Spontaneous sarcoplasmic reticulum (SR) Ca²⁺ release events increased in fructose‐rich diet mouse (FRD) myocytes vs. control diet (CD) mice, in the absence of significant changes in SR Ca²⁺ load. In HEK293 cells, hyperglycaemia significantly enhanced [³H]ryanodine binding and Ca²⁺/calmodulin‐dependent protein kinase II (CaMKII) phosphorylation of RyR2‐S2814 residue vs. normoglycaemia. These increases were prevented by CaMKII inhibition. FRD significantly augmented cardiac apoptosis in WT vs. CD‐WT mice, which was prevented by co‐treatment with the reactive oxygen species scavenger Tempol. Oxidative stress was also increased in FRD‐SR‐autocamide inhibitory peptide (AIP) mice, expressing the SR‐targeted CaMKII inhibitor AIP, without any significant enhancement of apoptosis vs. CD‐SR‐AIP mice. FRD produced mitochondrial swelling and membrane depolarization in FRD‐WT mice but not in FRD‐S2814A mice, in which the CaMKII site on ryanodine receptor 2 was ablated. FRD decreased mitochondrial area, mean Feret diameter and the mean distance between SR and the outer mitochondrial membrane vs. CD hearts. This remodelling was prevented in AC3I mice, with cardiac‐targeted CaMKII inhibition. The impact of cardiac apoptosis in pre‐diabetic stages of diabetic cardiomyopathy is unknown. We show that myocytes from fructose‐rich diet (FRD) animals exhibit arrhythmias produced by exacerbated Ca²⁺/calmodulin‐protein kinase (CaMKII) activity, ryanodine receptor 2 (RyR2) phosphorylation and sarcoplasmic reticulum (SR) Ca²⁺ leak. We tested the hypothesis that this mechanism also underlies cardiac apoptosis in pre‐diabetes. We generated a pre‐diabetic model in FRD mice. FRD mice showed an increase in oxidative stress, hypertrophy and systolic dysfunction. FRD myocytes exhibited enhanced SR Ca²⁺ spontaneous events in the absence of SR Ca²⁺ load alterations vs. control‐diet (CD) myocytes. In HEK293 cells, hyperglycaemia significantly enhanced [³H]ryanodine binding and CaMKII phosphorylation of RyR2‐S2814 residue vs. normoglycaemia. CaMKII inhibition prevented hyperglycaemia‐induced alterations. FRD also evoked cardiac apoptosis in WT mice vs. CD‐WT mice. Co‐treatment with the reactive oxygen species scavenger Tempol prevented FRD‐induced apoptosis in WT mice. In contrast, FRD enhanced oxidative stress but not apoptosis in FRD‐SR‐AIP mice, in which a CaMKII inhibitor is targeted to the SR. FRD produced mitochondrial membrane depolarization in WT mice but not in S2814A mice, in which the CaMKII phosphorylation site on RyR2 was ablated. Furthermore, FRD decreased mitochondrial area, mean Feret diameter and mean SR–mitochondrial distance vs. CD‐WT hearts. This remodelling was prevented in AC3I mice, with cardiac‐targeted CaMKII inhibition. CaMKII phosphorylation of RyR2, SR Ca²⁺ leak and mitochondrial membrane depolarization are critically involved in the apoptotic pathway of the pre‐diabetic heart. The FRD‐induced decrease in SR–mitochondrial distance is likely to additionally favour Ca²⁺ transit between the two organelles.

The pathogenesis ofBrucellais related to the ability to multiply intracellularly, an event controlled by the two-component system BvrR/BvrS (TCS BvrRS) and the type IV secretion machinery VirB (T4SS VirB). We have hypothesized that the TCS BvrRS transcriptionally regulates the T4SS VirB. To test this hypothesis, we have compared the levels of VirB proteins in the wild-type strainBrucella abortus2308 and mutant strains devoid of the sensor and regulator genes (bvrSandbvrRmutants, respectively). While thebvrRandbvrSmutants showed low levels of the VirB1, VirB5, VirB8, and VirB9 proteins, the same proteins were overexpressed in thebvrRmutant complemented with a plasmid carrying a functionalbvrRgene. Quantitation ofvirB5mRNA confirmed these data and indicated that the influence of the TCS BvrRS on the T4SS VirB occurs at the transcriptional level. The expression of the transcriptional activator VjbR also depended on the TCS BvrRS. In addition, we demonstrate a direct interaction between the promoter region of the VirB operon and the response regulator BvrR. Altogether these data demonstrate that the TCS BvrRS controls the expression of the T4SS VirB through direct and indirect mechanisms.