Zfhx3 Transcription Factor Represses the Expression of SCN5A Gene and Decreases Sodium Current Density (INa)

The ZFHX3 and SCN5A genes encode the zinc finger homeobox 3 (Zfhx3) transcription factor (TF) and the human cardiac Na+ channel (Nav1.5), respectively. The effects of Zfhx3 on the expression of the Nav1.5 channel, and in cardiac excitability, are currently unknown. Additionally, we identified three Zfhx3 variants in probands diagnosed with familial atrial fibrillation (p.M1260T) and Brugada Syndrome (p.V949I and p.Q2564R). Here, we analyzed the effects of native (WT) and mutated Zfhx3 on Na+ current (INa) recorded in HL-1 cardiomyocytes. ZFHX3 mRNA can be detected in human atrial and ventricular samples. In HL-1 cardiomyocytes, transfection of Zfhx3 strongly reduced peak INa density, while the silencing of endogenous expression augmented it (from −65.9 ± 8.9 to −104.6 ± 10.8 pA/pF; n ≥ 8, p < 0.05). Zfhx3 significantly reduced the transcriptional activity of human SCN5A, PITX2, TBX5, and NKX25 minimal promoters. Consequently, the mRNA and/or protein expression levels of Nav1.5 and Tbx5 were diminished (n ≥ 6, p < 0.05). Zfhx3 also increased the expression of Nedd4-2 ubiquitin-protein ligase, enhancing Nav1.5 proteasomal degradation. p.V949I, p.M1260T, and p.Q2564R Zfhx3 produced similar effects on INa density and time- and voltage-dependent properties in WT. WT Zfhx3 inhibits INa as a result of a direct repressor effect on the SCN5A promoter, the modulation of Tbx5 increasing on the INa, and the increased expression of Nedd4-2. We propose that this TF participates in the control of cardiac excitability in human adult cardiac tissue.

Zfhx3 decreases expression of cardiac Nav1.5 channels and inhibits sodium current

Background Zfhx3 (zinc finger homeobox 3) is a transcription factor (TF) encoded by the ZFHX3 gene. GWAS and gene-based association studies showed that ZFHX3 is one of the major atrial fibrillation (AF) susceptibility-conferring genes. The sodium current (INa), carried by Nav1.5 channels encoded by SCN5A, is responsible for atrial and ventricular action potential depolarization and determines cardiac excitability. Zfhx3 interacts with other TFs, such as Tbx5 and Pitx2c that increase and decrease INa, respectively. However, the effects of Zfhx3 on cardiac INa are currently unknown. Purpose We aimed to determine the effects of Zfhx3 on the INa on HL-1 cardiomyocytes. Methods cDNAs encoding human Zfhx3 together or not with Pitx2c or Tbx5 were transfected in HL-1 cells. Endogenous Zfhx3 expression in HL-1 cells was silenced by means of siRNAs. INa was recorded at room temperature using the whole-cell patch-clamp and luciferase reporter assays, qPCR and Western-blot (WB) analyses were also conducted. Results Expression analysis of RNA-seq data from human ventricular (n=432) samples (GTEx) demonstrated that Zfhx3 mRNA can be detected in the adult working myocardium. Transfection of Zfhx3 strongly reduced peak INa density (from −75.0±6.6 to −30.9±2.9 pA/pF; n≥26, P&lt;0.001). In contrast, Zfhx3 silencing augmented INa density compared to cells transfected with scrambled siRNA (from −65.9±8.9 to −104.6±10.8 pA/pF; n≥8, P&lt;0.05). Neither Zfhx3 expression nor silencing modified time and voltage dependence of activation and inactivation or the reactivation kinetics. Zfhx3 significantly reduced transcriptional activity of human SCN5A, PITX2 and TBX5 minimal promoters and, consequently, the mRNA and protein expression levels of Nav1.5, Pitx2c, and Tbx5 were diminished (n≥6, P&lt;0.05). In cells transfected with Zfhx3 together with Pitx2c, but not with Tbx5, INa density was significantly smaller than in cells expressing WT Zfhx3 alone (n≥15, P&lt;0.05). Further WB experiments demonstrated that Zfhx3 increased the expression of Nedd4–2 ubiquitin-protein ligase, which ubiquitinates Nav1.5 channels and favors their proteasomal degradation. Conclusions Zfhx3 inhibits INa as a result of a direct repressor effect on the SCN5A promoter, the modulation of Tbx5-increasing and Pitx2-decreasing effects on the INa, and the enhancement of Nav1.5 channel degradation. We propose a novel and complex mechanism that regulates the expression of sodium channels and the density of the INa, which are critical for the control of cardiac excitability. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Ministerio de Economía y CompetitividadComunidad Autόnoma de Madrid

Proteomic and functional mapping of cardiac NaV1.5 channel phosphorylation sites

Phosphorylation of the voltage-gated Na+ (NaV) channel NaV1.5 regulates cardiac excitability, yet the phosphorylation sites regulating its function and the underlying mechanisms remain largely unknown. Using a systematic, quantitative phosphoproteomic approach, we analyzed NaV1.5 channel complexes purified from nonfailing and failing mouse left ventricles, and we identified 42 phosphorylation sites on NaV1.5. Most sites are clustered, and three of these clusters are highly phosphorylated. Analyses of phosphosilent and phosphomimetic NaV1.5 mutants revealed the roles of three phosphosites in regulating NaV1.5 channel expression and gating. The phosphorylated serines S664 and S667 regulate the voltage dependence of channel activation in a cumulative manner, whereas the nearby S671, the phosphorylation of which is increased in failing hearts, regulates cell surface NaV1.5 expression and peak Na+ current. No additional roles could be assigned to the other clusters of phosphosites. Taken together, our results demonstrate that ventricular NaV1.5 is highly phosphorylated and that the phosphorylation-dependent regulation of NaV1.5 channels is highly complex, site specific, and dynamic.

A Review on ECG Classification Methods

Arrhythmia is a main group of illnesses in cardiovascular disorder and it can occur on its own or with different cardiovascular diseases. The diagnosis of arrhythmia especially depends on the ECG (electrocardiogram). ECG is an important contemporary medical device that records the process of cardiac excitability, transmission, and recovery. The purpose of this study is to classify ECG signal using different methods.

KChIP2 is a core transcriptional regulator of cardiac excitability

Arrhythmogenesis from aberrant electrical remodeling is a primary cause of death among patients with heart disease. Amongst a multitude of remodeling events, reduced expression of the ion channel subunit KChIP2 is consistently observed in numerous cardiac pathologies. However, it remains unknown if KChIP2 loss is merely a symptom or involved in disease development. Using rat and human derived cardiomyocytes, we identify a previously unobserved transcriptional capacity for cardiac KChIP2 critical in maintaining electrical stability. Through interaction with genetic elements, KChIP2 transcriptionally repressed the miRNAs miR-34b and miR-34c, which subsequently targeted key depolarizing (INa) and repolarizing (Ito) currents altered in cardiac disease. Genetically maintaining KChIP2 expression or inhibiting miR-34 under pathologic conditions restored channel function and moreover, prevented the incidence of reentrant arrhythmias. This identifies the KChIP2/miR-34 axis as a central regulator in developing electrical dysfunction and reveals miR-34 as a therapeutic target for treating arrhythmogenesis in heart disease.