Faculty Bibliography

Intracellular protons and calcium ions are two major chemical factors that regulate connexin43 (Cx43) gap junction communication and the synergism or antagonism between pH and Ca²⁺ has been questioned for decades. To assess the ability of Ca²⁺ ions to modulate Cx43 junctional conductance (g_j) in the absence of pH-sensitivity, patch clamp experiments were performed on Neuroblastoma-2a (N2a) cells or neonatal mouse ventricular myocytes (NMVMs) expressing either full length Cx43 or the Cx43-M257 (Cx43K258stop) mutant protein, a carboxyl-terminal (CT) truncated version of Cx43 lacking pH-sensitivity. Addition of 1 mM ionomycin to normal calcium saline reduced Cx43 or Cx43-M257 g_j to zero within 15 min of perfusion. This response was prevented by Ca²⁺-free saline or addition of 100 nM calmodulin (CaM) inhibitory peptide to the internal pipette solution. Internal addition of a connexin50 cytoplasmic loop calmodulin-binding domain (CaMBD) mimetic peptide (200 nM) prevented the Ca²⁺/ionomycin-induced decrease in Cx43 g_j, while 100 mM Gap19 peptide had minimal effect. Investigation of the transjunctional voltage (V_j) gating properties of NMVM Cx43-M257 gap junctions confirmed the loss of the fast inactivation of Cx43-M257 g_j, but also noted the abolishment of the previously reported facilitated recovery of g_j from inactivating potentials. We conclude that the distal CT domain of Cx43 contributes to the V_j-dependent fast inactivation and facilitated recovery of Cx43 gap junctions, but the Ca²⁺/CaM-dependent gating mechanism remains intact in its absence. Sequence-specific connexin CaMBD mimetic peptides act by binding Ca²⁺/CaM non-specifically and the Cx43 mimetic Gap19 peptide has negligible effect on this chemical gating mechanism.

Rapid impulse propagation is a defining attribute of the pectinated atrial myocardium and His-Purkinje system (HPS) that safeguards against atrial and ventricular arrhythmias, conduction block, and myocardial dyssynchrony. The complex transcriptional circuitry that dictates rapid conduction remains incompletely understood. Here, we demonstrate that ETV1 (ER81)-dependent gene networks dictate the unique electrophysiological characteristics of atrial and His-Purkinje myocytes. Cardiomyocyte-specific deletion of ETV1 results in cardiac conduction abnormalities, decreased expression of rapid conduction genes (Nkx2-5, Gja5, and Scn5a), HPS hypoplasia, and ventricularization of the unique sodium channel properties that define Purkinje and atrial myocytes in the adult heart. Forced expression of ETV1 in postnatal ventricular myocytes (VMs) reveals that ETV1 promotes a HPS gene signature while diminishing ventricular and nodal gene networks. Remarkably, ETV1 induction in human induced pluripotent stem cell-derived cardiomyocytes increases rapid conduction gene expression and inward sodium currents, converting them towards a HPS phenotype. Our data identify a cardiomyocyte-autonomous, ETV1-dependent pathway that is responsible for specification of rapid conduction zones in the heart and demonstrate that ETV1 is sufficient to promote a HPS transcriptional and functional program upon VMs.

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.

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.

Introduction: Adjuvant sunitinib (SU; 50 mg daily, schedule 4/2) significantly improved disease-free survival (DFS) vs. placebo (PBO) in patients (pts) with locoregional renal cell carcinoma (RCC) at high risk of tumour recurrence after nephrectomy (hazard ratio [HR], 0.76; 95% confidence interval [CI], 0.59-0.98; P=0.03, median: 6.8 vs. 5.6 years). We provide data on the study population, treatment, pattern of recurrence, and the relationship between baseline factors and DFS (blinded independent central review). Methods: Disease recurrence was based on centrally confirmed imaging and/or histological findings. Subgroup analyses of DFS by baseline risk factors were conducted using a Cox Proportional hazards model. The baseline risk factors explored included modified UISS criteria, age, gender, Eastern Cooperative Oncology Group performance status (ECOG PS), weight, neutrophil-to-lymphocyte ratio (NLR), and Fuhrman grade. Results: 615 pts were enrolled in S-TRAC trial from 97 sites. >70% of patients received SU treatment for >=6 cycles (~8 months) and 56% completed the full 1-year treatment. A total of 97 patients (31.4%) in the SU arm and 122 (39.9%) in the PBO arm developed metastatic disease recurrence. Most common sites of distant recurrence (SU:PBO) were lung (13%:16%), lymph node (7%:9%), and liver (4%:5%). The benefit of adjuvant SU over PBO was observed across several subgroups of pts, including: higher risk than the overall study population (HR 0.74; 95% CI, 0.55-0.99; P=0.04); ECOG PS 0 (HR 0.69; 95% CI, 0.51-0.93; P=0.01); Fuhrman grade 3/4 (HR 0.73; 95% CI, 0.55-0.98; P=0.04); and NLR <=3 (HR 0.72; 95% CI, 0.54-0.95; P=0.02). Conclusions: The majority of subgroups demonstrated longer DFS with adjuvant SU compared with PBO. These results are consistent with the primary analysis showing benefit for adjuvant SU in pts at high risk for recurrent RCC post nephrectomy

INTRODUCTION & OBJECTIVES: Adjuvant sunitinib (SU) treatment (50 mg daily; schedule 4/2) demonstrated longer disease-free survival (DFS) vs. placebo (PBO) in patients with locoregional renalcell carcinoma (RCC) at high risk of tumor recurrence post nephrectomy (hazard ratio [HR], 0.76; 95% confidence interval [CI], 0.59-0.98; P=0.03, median: 6.8 vs. 5.6 years, respectively). We provide new details on the population, treatment, and the relationship between baseline factors and DFS (blinded independent central review). MATERIAL & METHODS: Study treatment exposure was assessed by treatment group during cycles. Subgroup analyses of DFS by baseline risk factors were conducted using a Cox Proportional hazards model. The baseline risk factors explored in the subgroup analyses included UISS criteria, Fuhrman grade and ECOG performance status (PS). This study was approved by Ethical Committee. RESULTS: Overall, 615 patients were enrolled from 97 sites, including 73 in the European Union. >70% of patients received SU treatment for at least 6 cycles (9 months) and 56% completed the full 1-year treatment. In a subgroup analysis of patients at higher risk (n=388) than the overall study population (i.e., T3, N0 or NX, M0, Fuhrman's grade >=2, ECOG PS >=1, and T4 and/or N1-2), median (95% CI) DFS was 6.2 (4.9-not reached) years with sunitinib and 4.0 (2.6-6.0) years with placebo (HR, 0.74; 95% CI, 0.55-0.99; P=0.04). The benefit of adjuvant sunitinib over placebo was also observed in patients with ECOG PS 0 (n=448; HR 0.69, 95% CI 0.51-0.93, P=0.01) but not with ECOG PS >=1 (n=164; HR 0.99, 95% CI 0.63-1.56, P=0.96). Additional subgroups analyses, including detailed Fuhrman grades, will be presented. CONCLUSIONS: SU demonstrated a significant improvement in DFS compared with PBO in several subgroup analyses. These results are consistent with the primary analysis, showing benefit for adjuvant SU in patients at high risk of recurrent RCC post-nephrectomy

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.

Background: Arrhythmogenic cardiomyopathy (also known as "ARVC") is an inherited disease characterized by fibrous or fibrofatty infiltration of the heart muscle, commonly of right ventricular (RV) predominance, ventricular arrhythmias, and high propensity for sudden death. Sudden cardiac arrest frequently associates with exercise and most often occurs in early adulthood during the subclinical ("concealed") phase of the disease. Understanding electrical remodeling in the early stage of the disease is paramount to understand sudden death mechanisms. Methods: We generated a cardiomyocyte-specif-ic, tamoxifen-activated, PKP2 knockout murine line (alphaMHC-Cre-ERT2/PKP2 fl/fl) which allowed us to control the onset of PKP2 loss of expression, limit it to adult cardiomyocytes, and establish a time line for progression of molecular and functional events. Results: The first consequence of PKP2 loss was RV mechanical dysfunction (14 days post-tamoxifen injection, 14 dpi), followed by fibrosis of RV predominance and RV dilation (21 dpi), then biventricular dilated cardiomyopa-thy and left ventricular (LV) failure (28 dpi and beyond). End-stage failure and death occurred between 30 and 49 dpi. Isoproterenol (ISO)-induced ventricular arrhythmias were first detected prior to LV dysfunction (17/17 mice), and ISO-induced fatal ventricular fibrillation was observed only at 16 dpi, i.e., during the concealed stage (3/9). Differential tran-scriptome analysis at 21 dpi revealed reduced transcript levels for a gene network involved in intracellular calcium ([Ca²⁺]i) cycling, most critically genes encoding Ca²⁺ channel proteins (RyR2 and CaV1.2) and structural molecules that scaffold the dyad (ankyrin-B and triadin). Nanoscale imaging (3D super-resolution microscopy, SICM, and FIB-SEM) showed preservation of T-tubular structure, reduced size and increased separation of CaV1.2 clusters, and displacement of functional CaV1.2 channels from the T-tubular domain. Calcium imaging showed disruption of [Ca²⁺]i homeostasis, potentially causative of ventricular arrhythmias. Flecainide i.p. prevented ISO-induced arrhythmias in all animals. Retrospective analysis of clinical cases showed instances of sudden cardiac arrest without structural disease and suspect diagnosis of catechol-aminergic polymorphic ventricular tachycardia (CPVT) later revealed to foster PKP2 nonsense mutations. Conclusions: Our data provide the first evidence that PKP2 deficiency in adult ventricular myocytes is sufficient to cause an arrhythmo-genic cardiomyopathy of RV predominance. Adrenergic-induced arrhythmias and sudden death occur before the onset of overt structural disease and can mimic a CPVT phenotype. Our data also document a transcript-based [Ca²⁺]i dysfunction as a new key mechanism of arrhythmias in PKP2-deficient hearts and suggest flecainide as potential effective antiarrhyth-mic treatment

With recent advances in stem cell technology, it is becoming efficient to differentiate human pluripotent stem cells (hPSCs) into cardiomyocytes, which can subsequently be used for myriad purposes, ranging from interrogating mechanisms of cardiovascular disease, developing novel cellular therapeutic approaches, as well as assessing the cardiac safety profile of compounds. However, the relative inability to acquire abundant pure and mature cardiomyocytes still hinders these applications. Recently, it was reported that glucose-depleted culture medium supplemented with lactate can facilitate purification of hPSC-derived cardiomyocytes. Here, we report that fatty acid as a lactate replacement has not only a similar purification effect but also improves the electrophysiological characteristics of hPSC-derived cardiomyocytes. Glucose-depleted culture medium supplemented with fatty acid and 3,3',5-Triiodo-l-thyronine (T3) was used during enrichment of hPSC-derived cardiomyocytes. Compared to untreated control cells, the treated cardiomyocytes exhibited enhanced action potential (AP) maximum upstroke velocity (as shown by a significant increase in dV/dtmax), action potential amplitude, as well as AP duration at 50% (APD50) and 90% (APD90) of repolarization. The treated cardiomyocytes displayed higher sensitivity to isoproterenol, more organized sarcomeric structures, and lower proliferative activity. Expression profiling showed that various ion channel and cardiac-specific genes were elevated as well. Our results suggest that the use of fatty acid and T3 can facilitate purification and maturation of hPSC-derived cardiomyocytes.

Rapid impulse propagation in the heart is a defining property of pectinated atrial myocardium (PAM) and the ventricular conduction system (VCS) and is essential for maintaining normal cardiac rhythm and optimal cardiac output. Conduction defects in these tissues produce a disproportionate burden of arrhythmic disease and are major predictors of mortality in heart failure patients. Despite the clinical importance, little is known about the gene regulatory network that dictates the fast conduction phenotype. Here, we have used signal transduction and transcriptional profiling screens to identify a genetic pathway that converges on the NRG1-responsive transcription factor ETV1 as a critical regulator of fast conduction physiology for PAM and VCS cardiomyocytes. Etv1 was highly expressed in murine PAM and VCS cardiomyocytes, where it regulates expression of Nkx2-5, Gja5, and Scn5a, key cardiac genes required for rapid conduction. Mice deficient in Etv1 exhibited marked cardiac conduction defects coupled with developmental abnormalities of the VCS. Loss of Etv1 resulted in a complete disruption of the normal sodium current heterogeneity that exists between atrial, VCS, and ventricular myocytes. Lastly, a phenome-wide association study identified a link between ETV1 and bundle branch block and heart block in humans. Together, these results identify ETV1 as a critical factor in determining fast conduction physiology in the heart.