Faculty Bibliography

Background: Mutations in plakophilin-2 (PKP2) are the most common cause of familial Arrhythmogenic Right Ventricular Cardiomyopathy, a disease characterized by ventricular arrhythmias, sudden death, and progressive fibrofatty cardiomyopathy. The relation between loss of PKP2 expression and structural cardiomyopathy remains under study, though paracrine activation of pro-fibrotic intracellular signaling cascades is a likely event. Previous studies have indicated that ATP release into the intracellular space, and activation of adenosine receptors, can regulate fibrosis in various tissues. However, the role of this mechanism in the heart, and in the specific case of a PKP2-initiated cardiomyopathy, remains unexplored. Objectives: To investigate the role of ATP/adenosine in the progression of a PKP2-associated cardiomyopathy. Methods: HL1 cells were used to study PKP2- and Connexin43 (Cx43)-dependent ATP release. A cardiac-specific, tamoxifen-activated PKP2 knock-out murine model (PKP2cKO) was used to define the effect of adenosine receptor blockade on the progression of a PKP2-dependent cardiomyopathy. Results: HL1 cells silenced for PKP2 showed increased ATP release compared to control. Knockout of Cx43 in the same cells blunted the effect. PKP2cKO transcriptomic data revealed overexpression of genes involved in adenosine-receptor cascades. Istradefylline (an adenosine 2A receptor blocker) tempered the progression of fibrosis and mechanical failure observed in PKP2cKO mice. In contrast, PSB115, a blocker of the 2B adenosine receptor, showed opposite effects. Conclusion: Paracrine adenosine 2A receptor activation contributes to the progression of fibrosis and impaired cardiac function in animals deficient in PKP2. Given the limitations of the animal model, translation to the case of patients with PKP2 deficiency needs to be done with caution.

BACKGROUND/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/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/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.

Connexins are proteins forming gap junction channels for intercellular communication. Connexin40 (Cx40) is highly expressed by endothelial cells (ECs) of healthy arteries but this expression is lost in ECs overlying atherosclerotic plaques. Low/oscillatory shear stress observed in bends and bifurcations of arteries is atherogenic partly through activation of the pro-inflammatory NFkappaB pathway in ECs. In this study, we investigated the relation between shear stress, Cx40 and NFkappaB. Shear stress-modifying casts were placed around carotid arteries of mice expressing eGFP under the Cx40 promoter (Cx40+/eGFP ). We found that Cx40 expression is decreased in carotid regions of oscillatory shear stress but conserved in high and low laminar shear stress regions. These results were confirmed in vitro. Using phage display, we retrieved a binding motif for the intracellular regulatory Cx40 C-terminus (Cx40CT), i.e. HS[I, L, V][K, R]. One of the retrieved peptides (HSLRPEWRMPGP) showed a 58.3% homology with amino acids 5-to-16 of IkappaBalpha, a member of the protein complex inhibiting NFkappaB activation. Binding of IkappaBalpha (peptide) and Cx40 was confirmed by crosslinking and en face proximity ligation assay on carotid arteries. TNFalpha-induced nuclear translocation of NFkappaB in ECs was enhanced after reducing Cx40 with siRNA. Transfection of HeLa cells with either full-length Cx40 or Cx40CT demonstrated that Cx40CT was sufficient for inhibition of TNFalpha-induced NFkappaB phosphorylation. Finally, Tie2CreTgCx40fl/flApoe-/- mice showed exaggerated shear stress-induced atherosclerosis and enhanced NFkappaB nuclear translocation. Our data show a novel functional IkappaBalpha-Cx40 interaction that may be relevant for the control of NFkappaB activation by shear stress in atherogenesis.

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.

Background and Rationale: Mutations in Plakophilin-2 (PKP2) are the most common cause of ARVC, an inherited disease characterized by fibro- or fibrofatty infiltration of RV predominance, ventricular arrhythmias and sudden death in the young. The relation between PKP2 expression and the heart transcriptome in vivo, is unknown. Furthermore, while splicing misregulation has been associated with other inherited diseases, PKP2-dependent exon usage differences remain unexplored. We generated a murine line of cardiac-restricted, tamoxifen activated PKP2 deficiency ("PKP2-cKO") and defined PKP2- dependent exon usage in adult non-failing hearts. Methods and Results: The first disease manifestation was an increase in RV area, detected by echocardiography 14 days after tamoxifen injection (14 days post-injection or "dpi"), followed by marked RV dilation and reparative fibrosis (21 dpi), then bi-ventricular dilated cardiomyopathy (28 dpi), heart failure and death (30-50 dpi). To capture the earliest molecular events, hearts 14 dpi were used for RNAseq and exon usage. Comparing RV vs LV revealed minor changes in transcript abundance, but significant differences in alternative splicing (AS) program. We found ~75% of differentially spliced exons flanked by sequences that bind RBFox2, an RNA-binding protein that acts as central AS regulator of the adult heart, and that is necessary to maintain cardiac structure. Western blot analysis at 14 dpi and thereafter showed reduced abundance of RBFox2. RNAseq at 21 dpi showed that in addition to RBFox2, transcripts were reduced for RBFox1, MBNL1, MBNL2 and RBM20 (also molecules that control the AS program). Exon usage analysis at 21 dpi identified massive AS misregulation, similar to that of a failing heart, even though ejection fraction at this stage was ~50%. Misregulated genes included several involved in electrical rhythm and intracellular calcium homeostasis. Conclusion: We generated a model of PKP2-dependent ARVC. Our studies point to a previously unrecognized association between a desmosomal molecule, a splicing regulator, and the control of electrical and mechanical function. AS misregulation may be a substrate for sudden unexpected arrhythmic death in the young

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.

Arrhythmogenic cardiomyopathy, or its most well-known subform arrhythmogenic right ventricular cardiomyopathy (ARVC), is a cardiac disease mainly characterised by a gradual replacement of the myocardial mass by fibrous and fatty tissue, leading to dilatation of the ventricular wall, arrhythmias and progression towards heart failure. ARVC is commonly regarded as a disease of the intercalated disk in which mutations in desmosomal proteins are an important causative factor. Interestingly, the Dutch founder mutation PLN R14Del has been identified to play an additional, and major, role in ARVC patients within the Netherlands. This is remarkable since the phospholamban (PLN) protein plays a leading role in regulation of the sarcoplasmic reticulum calcium load rather than in the establishment of intercellular integrity. In this review we outline the intracellular cardiac calcium dynamics and relate pathophysiological signalling, induced by disturbed calcium handling, with activation of calmodulin dependent kinase II (CaMKII) and calcineurin A (CnA). We postulate a thus far unrecognised role for Ca2+ sensitive signalling proteins in maladaptive remodelling of the macromolecular protein complex that forms the intercalated disk, during pro-arrhythmic remodelling of the heart.

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.