ETV1 activates a rapid conduction transcriptional program in rodent and human cardiomyocytes

Figure 1. RNA Sequencing of fluorescence-activated cell sorted (FACS) postnatal day 21 (P21) mouse Purkinje cells (PC) and ventricular myocytes (VM). (A) Expression of PC (n = 7) and VM (n = 10) transcription factors (TFs) at P21. Differential expression (PC vs VM) on the X-axis and Purkinje transcript levels (normalized counts) are shown on the Y-axis. Significantly enriched (Padj < 0.05) and top ten abundant PC-TFs labeled in green while all remaining significantly different TFs were labeled in red. TFs that were not significantly different labeled in black. (B) Top ten significantly enriched and abundant TFs in PCs. Etv1 was the most enriched and one of the most abundant TFs within PCs. (C,D) Etv1-enhanced green fluorescent protein (EGFP) bacterial artificial chromosome (BAC) transgenic mice were used to confirm regional expression of Etv1 in embryonic (C) and adult (D) hearts. (C) Immunohistochemistry on E13.5 Etv1-EGFP hearts showing overlapping expression of Connexin-40 (Cx40) and GFP in trabecular and atrial myocytes. Bottom two rows are high-magnification views of the Bundle of His (red dashed box) and Trabecular/Compact zone myocytes (white dashed box) outlined in the top row (D)…

Figure 2. Cardiomyocyte-specific knockout of ETV1 slows atrial and His-Purkinje system conduction. Etv1flox/flox mice crossed with Myh6-Cre transgenic mice enabled generation of cardiac-specific Etv1 mutant mice. (A) Heart and tail RT-PCR analysis of DNA from Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice demonstrate heart specific deletion of Etv1 exon 11. (B) Immunoblot assessment of Etv1 WT and Etv1 cKO tissue extracts. Protein from cerebellum and right atrial lysates were electrophoresed and immunoblotted to detect ETV1 (top) and Vinculin (bottom). (C) Immunofluorescence evaluation of ETV1 expression in 10-week-old Etv1 WT and Etv1 cKO cerebellar Purkinje cells, atrial myocytes, and His-Purkinje system (HPS). Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). (D) Representative surface ECG traces of 10-week-old Etv1 WT and Etv1 cKO mice. Conduction intervals from 10–12-week-old Etv1 cKO mice compared to Etv1 WT mice showed significantly prolonged P wave and QRS intervals. 20% of Etv1 cKO mice displayed an RsR’ pattern. LA, left atria; LV, left ventricle. Data represent mean ± SEM. Scale bars: 25 um.

Figure 3. Cardiomyocyte deletion of ETV1 resulted in decreased expression of fast conduction genes in atrial and His-Purkinje system (HPS) myocytes. (A) Quantitative RT-PCR of fast conduction gene RNA levels (normalized to Gapdh) comparing 10–12-week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) FACS-purified ventricular, atrial, and Purkinje myocytes. Relative Nkx2–5, Gja5, and Scn5a expression displayed versus control, Etv1 WT (n = 4). (B) Immunoblot assessment of Etv1 WT and Etv1 cKO atrial tissue lysates detecting NKX2–5, Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Etv1 WT (n = 5). (D) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO atria/ventricular sections. (E) Immunofluorescence evaluation of NKX2–5, Cx40, and NaV1.5 expression in 10-week-old Etv1 WT and Etv1 cKO HPS sections. Positive CNTN2 expression identified HPS cells. Nuclei were identified by DAPI (blue). LA, left atria; LV, left ventricle. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Figure 4. ETV1 is required for normal development of the His-Purkinje system (HPS). Etv1flox/flox mice backcrossed with Cntn2-EGFP were used to visualize the left and right HPS. (A) Representative images of the left ventricle (LV) HPS of 10-week-old Etv1 WT (Etv1flox/flox) Cntn2-EGFP and Etv1 cKO (Etv1flox/flox, Myh6-Cre) Cntn2-EGFP hearts. Lower row is a high-magnification image of the white dashed boxes in the top row. (B) Quantification of EGFP+ Purkinje cell density (base, mid and apex) in Etv1 WT and Etv1 cKO hearts. Percentage of EGFP+ Purkinje cell area was significantly reduced on the left ventricular septal wall (LSW) in Etv1 cKO hearts versus control (n = 3). (C) Representative images of the right ventricle (RV) HPS of 10-week-old Etv1 WT Cntn2-EGFP and Etv1 cKO Cntn2-EGFP hearts. Middle and bottom rows are high-magnification views of red and white dashed boxes outlined in the top row. Yellow arrows highlight regional loss of HPS cells. (D) Percentage of EGFP+ Purkinje cell area was significantly reduced on the RV septum wall (RSW) and RV free wall (RVFW) in Etv1 cKO Cntn2-EGFP hearts versus control (n = 3). Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale…

Figure 5. ETV1 regulates the diversity of sodium channel biophysical properties between ventricular, atrial, and Purkinje myocytes. Whole-cell patch clamp data from dissociated cardiomyocytes (ventricular, right atrial, Purkinje myocytes) using 10–12 week-old Etv1 WT (Etv1flox/flox) and Etv1 cKO (Etv1flox/flox, Myh6-Cre) mice in a Cntn2-EGFP background (n = 4). (A) Comparison of sodium current–voltage (I–V) relationship. Maximum conductance was calculated to assess significant differences among experimental groups. (B) Voltage dependence of steady-state activation. Voltage at half activation (V0.5, activation) was calculated to assess significant differences among experimental groups. (C) Voltage dependence of steady-state inactivation. Voltage at half inactivation (V0.5, inactivation) was calculated to assess significant differences among experimental groups. (D) Time course of recovery from inactivation. Tau of recovery (τrecovery) was calculated to assess significant differences among experimental groups. Number of cells analyzed per cell type (ventricle, right atria, Purkinje) included in each graph legend. Patch clamp protocol diagrams are included for each endpoint. Data represent…

Figure 6. ETV1-transduced neonatal rat ventricular myocytes (NRVMs) upregulates a His-Purkinje system gene signature. (A) Volcano plot of relative transcript expression from NRVMs transduced with either Ad-Etv1-EGFP or Ad-EGFP. RNA-sequencing (RNA-seq) comparison revealed a total of 9,236 differentially expressed genes (normalized counts ≥ 5, padj < 0.05). All significantly different genes (padj < 0.05) are labeled blue (downregulated) or red (enriched) and all nonsignificantly different transcripts labeled in gray. Of these there were 4,696 upregulated and 4,540 downregulated genes in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs. (B) Functional clustering of upregulated genes in Ad-Etv1 transduced NRVMs highlighted significantly enriched ETV1-dependent cellular processes (top 20 non-redundant categories are shown). Pathways are color coded to represent genes clustered into functional classes for heat maps in C. (C) Comparative RNA-seq between 21-day-old (P21) wild-type mouse FACS-purified Purkinje cell (PC)/ventricular myocytes (VM) and Ad-Etv1-EGFP/Ad-EGFP transduced NRVMs. Heat map representation of 88 genes differentially expressed in Ad-Etv1-EGFP versus Ad-EGFP transduced NRVMs (n =…

Figure 7. ETV1 overexpression leads to marked enrichment of fast conduction genes in postnatal ventricular myocytes (VMs). (A) Quantitative RT-PCR (qPCR) validation of RNA sequencing (RNA-seq) gene expression data comparing known cardiac conduction associated genes from endogenous Purkinje cells (PCs) and VMs versus Ad-Etv1-EGFP or Ad-EGFP transduced neonatal rat ventricular myocytes (NRVMs). Log2 fold change displayed relative to VM levels (P21 PC/VM, n = 7,10 respectively) or Ad-EGFP levels (NRVM Ad-Etv1-EGFP/Ad-EGFP, n = 3) (Supplemental Table 10). (B) Western blots of Ad-EGFP and Ad-Etv1-EGFP transduced NRVM lysates detecting Cx40, NaV1.5, and Vinculin (loading control). (C) Protein level densitometric quantification (normalized to vinculin), displayed relative to Ad-EGFP (n = 5). (D,E) Immunofluorescence staining of (D) Cx40 and (E) NaV1.5 in Ad-Etv1-EGFP and Ad-EGFP transduced NRVMs. ETV1-transduced cells displayed ectopic expression of Cx40 and marked enrichment of NaV1.5 compared to Ad-GFP-transduced NRVMs. Nuclei were co-stained with DAPI. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test. Scale bars: 50 um.

Figure 8. Activation of ETV1 in human induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) leads to increased expression of rapid conduction genes and sodium current. (A) Schematic representation of hiPSC-CM generation and maturation (day 0–21), transduction of Ad-Etv1-EGFP or Ad-EGFP (day 24), and timepoint for experimentation (day 38–40). (B) Quantitative RT-PCR analysis of Etv1, NKX2–5, GJA5, SCN5A, and MYL2 in hiPSC-CM transduced with either Ad-Etv1-EGFP or Ad-EGFP (n = 4). (C) Whole-cell patch clamp was performed on Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Sodium current–voltage (I–V) relationship comparison. (D) hiPSC-CM NaV peak conductance (gNaV-peak). gNaV-peak following −120 mV to −35 mV depolarization step was measured for Ad-Etv1-EGFP (n = 12) or Ad-EGFP (n = 9) transduced hiPSC-CMs. Data represent mean ± SEM. *P < 0.05, 2-tailed Student’s t test.