Tbx5 variants disrupt Nav1.5 function differently in patients diagnosed with Brugada or Long QT Syndrome.

AIMS: The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. METHODS AND RESULTS: We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and ßIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and ßIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans-expressing mice. CONCLUSIONS: In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.

Digenic Heterozigosity in SCN5A and CACNA1C Explains the Variable Expressivity of the Long QT Phenotype in a Spanish Family.

INTRODUCTION AND OBJECTIVES: A known long QT syndrome-related mutation in Nav1.5 cardiac channels (p.R1644H) was found in 4 members of a Spanish family but only 1 of them showed prolongation of the QT interval. In the other 3 relatives, a novel missense mutation in Cav1.2 cardiac channels was found (p.S1961N). Here, we functionally analyzed p.S1961N Cav1.2 channels to elucidate whether this mutation regulates the expressivity of the long QT syndrome phenotype in this family. METHODS: L-type calcium current (ICaL) recordings were performed by using the whole-cell patch-clamp technique in Chinese hamster ovary cells transiently transfected with native and/or p.S1961N Cav1.2 channels. RESULTS: Expression of p.S1961N channels significantly decreased ICaL density. Using Ba as a charge carrier to suppress the Ca-dependent inactivation of Cav1.2 channels, we demonstrated that the mutation significantly accelerates the voltage-dependent inactivation of Cav1.2 channels decreasing the inactivation time constant. As a consequence, the total charge flowing through p.S1961N Cav1.2 channels significantly decreased. The effects of the p.S1961N Cav1.2 and p.R1644H Nav1.5 mutations alone or their combination on the action potential (AP) morphology were simulated using a validated model of the human ventricular AP. The p.S1961N Cav1.2 mutation shortens the AP duration and abrogates the prolongation induced by p.R1644H Nav1.5 channels. CONCLUSIONS: The p.S1961N mutation in Cav1.2 channels decreased the ICaL, an effect which might shorten ventricular AP. The presence of the loss-of-function Cav1.2 mutation could functionally compensate the prolonging effects produced by the Nav1.5 gain-of-function mutation.

Brugada syndrome trafficking-defective Nav1.5 channels can trap cardiac Kir2.1/2.2 channels.

Cardiac Nav1.5 and Kir2.1-2.3 channels generate Na (INa) and inward rectifier K (IK1) currents, respectively. The functional INa and IK1 interplay is reinforced by the positive and reciprocal modulation between Nav15 and Kir2.1/2.2 channels to strengthen the control of ventricular excitability. Loss-of-function mutations in the SCN5A gene, which encodes Nav1.5 channels, underlie several inherited arrhythmogenic syndromes, including Brugada syndrome (BrS). We investigated whether the presence of BrS-associated mutations alters IK1 density concomitantly with INa density. Results obtained using mouse models of SCN5A haploinsufficiency, and the overexpression of native and mutated Nav1.5 channels in expression systems - rat ventricular cardiomyocytes and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) - demonstrated that endoplasmic reticulum (ER) trafficking-defective Nav1.5 channels significantly decreased IK1, since they did not positively modulate Kir2.1/2.2 channels. Moreover, Golgi trafficking-defective Nav1.5 mutants produced a dominant negative effect on Kir2.1/2.2 and thus an additional IK1 reduction. Moreover, ER trafficking-defective Nav1.5 channels can be partially rescued by Kir2.1/2.2 channels through an unconventional secretory route that involves Golgi reassembly stacking proteins (GRASPs). Therefore, cardiac excitability would be greatly affected in subjects harboring Nav1.5 mutations with Golgi trafficking defects, since these mutants can concomitantly trap Kir2.1/2.2 channels, thus unexpectedly decreasing IK1 in addition to INa.

Kir2.1-Nav1.5 Channel Complexes Are Differently Regulated than Kir2.1 and Nav1.5 Channels Alone.

Cardiac Kir2.1 and Nav1.5 channels generate the inward rectifier K+ (IK1) and the Na+ (INa) currents, respectively. There is a mutual interplay between the ventricular INa and IK1 densities, because Nav1.5 and Kir2.1 channels exhibit positive reciprocal modulation. Here we compared some of the biological properties of Nav1.5 and Kir2.1 channels when they are expressed together or separately to get further insights regarding their putative interaction. First we demonstrated by proximity ligation assays (PLAs) that in the membrane of ventricular myocytes Nav1.5 and Kir2.1 proteins are in close proximity to each other (<40 nm apart). Furthermore, intracellular dialysis with anti-Nav1.5 and anti-Kir2.1 antibodies suggested that these channels form complexes. Patch-clamp experiments in heterologous transfection systems demonstrated that the inhibition of the Ca2+/calmodulin-dependent protein kinase II (CaMKII) decreased the INa and the IK1 generated by Nav1.5 and Kir2.1 channels when they were coexpressed, but not the IK1 generated by Kir2.1 channels alone, suggesting that complexes, but not Kir2.1 channels, are a substrate of CaMKII. Furthermore, inhibition of CaMKII precluded the interaction between Nav1.5 and Kir2.1 channels. Inhibition of 14-3-3 proteins did not modify the INa and IK1 densities generated by each channel separately, whereas it decreased the INa and IK1 generated when they were coexpressed. However, inhibition of 14-3-3 proteins did not abolish the Nav1.5-Kir2.1 interaction. Inhibition of dynamin-dependent endocytosis reduced the internalization of Kir2.1 but not of Nav1.5 or Kir2.1-Nav1.5 complexes. Inhibition of cytoskeleton-dependent vesicular trafficking via the dynein/dynactin motor increased the IK1, but reduced the INa, thus suggesting that the dynein/dynactin motor is preferentially involved in the backward and forward traffic of Kir2.1 and Nav1.5, respectively. Conversely, the dynein/dynactin motor participated in the forward movement of Kir2.1-Nav1.5 complexes. Ubiquitination by Nedd4-2 ubiquitin-protein ligase promoted the Nav1.5 degradation by the proteasome, but not that of Kir2.1 channels. Importantly, the Kir2.1-Nav1.5 complexes were degraded following this route as demonstrated by the overexpression of Nedd4-2 and the inhibition of the proteasome with MG132. These results suggested that Kir2.1 and Nav1.5 channels closely interact with each other leading to the formation of a pool of complexed channels whose biology is similar to that of the Nav1.5 channels.

Tbx20 controls the expression of the KCNH2 gene and of hERG channels.

Long QT syndrome (LQTS) exhibits great phenotype variability among family members carrying the same mutation, which can be partially attributed to genetic factors. We functionally analyzed the KCNH2 (encoding for Kv11.1 or hERG channels) and TBX20 (encoding for the transcription factor Tbx20) variants found by next-generation sequencing in two siblings with LQTS in a Spanish family of African ancestry. Affected relatives harbor a heterozygous mutation in KCNH2 that encodes for p.T152HfsX180 Kv11.1 (hERG). This peptide, by itself, failed to generate any current when transfected into Chinese hamster ovary (CHO) cells but, surprisingly, exerted "chaperone-like" effects over native hERG channels in both CHO cells and mouse atrial-derived HL-1 cells. Therefore, heterozygous transfection of native (WT) and p.T152HfsX180 hERG channels generated a current that was indistinguishable from that generated by WT channels alone. Some affected relatives also harbor the p.R311C mutation in Tbx20. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), Tbx20 enhanced human KCNH2 gene expression and hERG currents (IhERG) and shortened action-potential duration (APD). However, Tbx20 did not modify the expression or activity of any other channel involved in ventricular repolarization. Conversely, p.R311C Tbx20 did not increase KCNH2 expression in hiPSC-CMs, which led to decreased IhERG and increased APD. Our results suggest that Tbx20 controls the expression of hERG channels responsible for the rapid component of the delayed rectifier current. On the contrary, p.R311C Tbx20 specifically disables the Tbx20 protranscriptional activity over KCNH2 Therefore, TBX20 can be considered a KCNH2-modifying gene.

Population cardiovascular health and urban environments: the Heart Healthy Hoods exploratory study in Madrid, Spain.

BACKGROUND: Our aim is to conduct an exploratory study to provide an in-depth characterization of a neighborhood's social and physical environment in relation to cardiovascular health. A mixed-methods approach was used to better understand the food, alcohol, tobacco and physical activity domains of the urban environment. METHODS: We conducted this study in an area of 16,000 residents in Madrid (Spain). We obtained cardiovascular health and risk factors data from all residents aged 45 and above using Electronic Health Records from the Madrid Primary Health Care System. We used several quantitative audit tools to assess: the type and location of food outlets and healthy food availability; tobacco and alcohol points of sale; walkability of all streets and use of parks and public spaces. We also conducted 11 qualitative interviews with key informants to help understanding the relationships between urban environment and cardiovascular behaviors. We integrated quantitative and qualitative data following a mixed-methods merging approach. RESULTS: Electronic Health Records of the entire population of the area showed similar prevalence of risk factors compared to the rest of Madrid/Spain (prevalence of diabetes: 12 %, hypertension: 34 %, dyslipidemia: 32 %, smoking: 10 %, obesity: 20 %). The food environment was very dense, with many small stores (n = 44) and a large food market with 112 stalls. Residents highlighted the importance of these small stores for buying healthy foods. Alcohol and tobacco environments were also very dense (n = 91 and 64, respectively), dominated by bars and restaurants (n = 53) that also acted as food services. Neighbors emphasized the importance of drinking as a socialization mechanism. Public open spaces were mostly used by seniors that remarked the importance of accessibility to these spaces and the availability of destinations to walk to. CONCLUSION: This experience allowed testing and refining measurement tools, drawn from epidemiology, geography, sociology and anthropology, to better understand the urban environment in relation to cardiovascular health.

Assessing Walking and Cycling Environments in the Streets of Madrid: Comparing On-Field and Virtual Audits.

Audit tools are useful for exploring the urban environment and its association with physical activity. Virtual auditing options are becoming increasingly available potentially reducing the resources needed to conduct these assessments. Only a few studies have explored the use of virtual audit tools. Our objective is to test if the Madrid Systematic Pedestrian and Cycling Environment Scan (M-SPACES) discriminates between areas with different urban forms and to validate virtual street auditing using M-SPACES. Three areas (N = 500 street segments) were selected for variation in population density. M-SPACES was used to audit street segments physically and virtually (Google Street View) by two researchers in 2013-2014. For both physical and virtual audits, all analyzed features score significantly different by area (p < 0.05). Most of the features showed substantial (ICC = 0.6-0.8) or almost perfect (ICC ≥ 0.8) agreement between virtual and physical audits, especially neighborhood permeability walking infrastructure, traffic safety, streetscape aesthetics, and destinations. Intra-rater agreement was generally acceptable (ICC > 0.6). Inter-rater agreement was generally poor (ICC < 0.4). Virtual auditing provides a valid and feasible way of measuring residential urban environments. Comprehensive auditor training may be needed to guarantee good inter-rater agreement.