Stress-Induced Down-Regulation of CPEB4 Disrupts Sodium Channel Regulation and Myocardial Excitability

Figures

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  Figure 1. CPEB4 Expression Is Reduced In Pathological And Stress-Associated Cardiac Conditions (A and B) Expression levels of CPEB isoforms in human heart confirmed by GTEx portal (https://gtexportal.org/). (A) Expression levels of CPEB isoforms in human heart bulk tissues. This panel displays a heatmap of transcripts per million (TPM) of different CPEB isoforms across different samples of cardiac tissue. Each row represents a different isoform, and each column represents a separate tissue sample. Color intensity indicates relative expression levels, ranging from low (pale yellow) to high (navy blue). (B) Single-cell expression analysis of CPEB isoforms in human heart tissues. A series of dot plots illustrates the distribution of CPEB isoform expression levels within individual cells of the heart. Each dot represents a single cell, with the color gradient from light to dark red indicating an increasing level of CPEB isoform expression. (C) Expression of CPEB4 in nonfailing (NF) and heart failure (HF) human hearts from the GSE141910 data set. Scatter plots show log2-transformed TPM values [log2(TPM+1)] for individual samples (NF, n = 161; HF, n = 195). Horizontal bars and error bars…

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  Figure 2. Targeted Knockdown of Cpeb4 in the Cardiac Tissue of Mice (A) Schematic representation of the genetic manipulation in mice. The ɑMHC (Myh6) promoter drives Cre recombinase expression selectively in cardiomyocytes (CMs). Upon tamoxifen (Tmx) induction, this system facilitates cardiac-specific Cpeb4 knockdown. Mice treated with Tmx are designated as the knockdown group (Tmx(+)), while vehicle-treated mice serve as the control group (Tmx(−)). (B) Western blot analysis comparing the protein levels of CPEB4 between Tmx(−) and Tmx(+) mice hearts, with tamoxifen treatment used to induce the knockdown in cKD mice. Band density corresponding to the protein levels was quantified and normalized to GAPDH across all samples. Data are presented as mean + SD (Tmx(−), n = 3; Tmx(+), n = 4). (C) Echocardiographic assessment of cardiac function. This panel presents the results from echocardiographic evaluation of cardiac function in a cohort of 8 Tmx(−) and 9 Tmx(+) mice. Key parameters assessed include left ventricular end-systolic (LVESV) and end-diastolic (LVEDV) volumes, and ejection fraction (EF). Data are presented as mean ± SD (Tmx(−), n = 8; Tmx(+), n = 9). ∗P < 0.05 when compared between…

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  Figure 3. Electrophysiological Changes Arising From Cardiac CPEB4 Down-Regulation (A) Electrocardiogram (ECG) parameters (RR interval, PR interval, QRS interval, QTc, R amplitude) analyzed in Tmx(+) mice, illustrating altered cardiac electrical activity. Data are presented as the mean + SD (Tmx(−), n = 8; Tmx(+), n = 7). (B) Expression levels of CPEB4 assessed by PCR in Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with control shRNA (shCon) and CPEB4-targeting shRNA (shCPEB4), demonstrating the knockdown efficiency. Data are presented as the mean + SD (shCon, n = 3; shCPEB4, n = 3). (C) Typical action potentials (APs) recorded from hiPSC-CMs treated with shCon and shCPEB4, indicating changes in electrical signaling. The dashed line represents 0 mV. (D) The average maximal rate of rise of the action potential at phase 0 (VMax), which reflects sodium channel availability and is influenced by the maximum sodium conductance (GNa, max) (left), and the average duration of the action potential at 30%, 60%, and 90% repolarization (APD30, APD60, APD90) (right) in hiPSC-CMs following shCon and shCPEB4 treatment, showing the impact of CPEB4 down-regulation on cardiac action…

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  Figure 4. Direct Binding Affinity of CPEB4 to SCN5A mRNA (A) CPE site variants (left). Schematic showing the sequence of CPE sites and poly(A) signal location in SCN5A 3'-UTR (right). (B) Binding of CPEB4 to SCN5A mRNA. SCN5A PCR products were detected in CPEB4 RNA immunoprecipitation (RIP) samples via endpoint (B, left) and real-time PCR (B, center and right). Three different primer sets were used for SCN5A detection. Data are presented as mean + SD. CPEB = cytoplasmic polyadenylation element binding protein; CPSF = cleavage and polyadenylation specificity factor; RT = reverse transcription.

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  Figure 5. Regulatory Influence of CPEB4 on SCN5A Expression (A and B) Impact of Cpeb4 down-regulation in Tmx(+) mice on Nav1.5 protein and SCN5A mRNA expression in mice. (A) The protein levels of Nav1.5 in heart tissues from Tmx(−) and Tmx(+) mice were investigated using Western blot analysis. Nav1.5 densities represent the sum of the upper and lower bands (total Nav1.5) and were normalized to GAPDH levels. Data are presented as the mean + SD (Tmx(−), n = 3; Tmx(+), n = 4). (B) The mRNA levels of SCN5A in heart tissues from Tmx(−) and Tmx(+) mice were investigated using PCR, normalized to GAPDH. Data are presented as the mean + SD (Tmx(−), n = 3; Tmx(+), n = 4). (C) Effects of CPEB4 down-regulation on SCN5A mRNA expression. RL14 cells were treated with control siRNA (siCon) or CPEB4-targeting siRNA (siCPEB4) for 24 hours. The mRNA levels of CPEB4 and SCN5A were assessed via PCR, normalized to GAPDH, and presented as fold change. Data are presented as the mean + SD (siCon, n = 3; siCPEB4, n = 3). (D) Consequences of CPEB4 down-regulation on Nav1.5 protein levels. RL14 cells were treated with siCon or siCPEB4 for 24 hours. Western blots for Nav1.5 and GAPDH were performed in 3 independent…

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  Figure 6. Control of SCN5A Expression and Function by CPEB4-Mediated CPE Sites (A) Diagram of luciferase reporter constructs with empty, wild type (WT), or deletion sequence variant at the CPE sites of SCN5A mRNA 3′-UTR (left). Effect of the CPE sequence variant of SCN5A mRNA 3′-UTR in RL14 cells (right). (B) Diagram of the Tet-On-operated CPEB4 overexpression construct (left). Control of CPEB4 expression via doxycycline (Dox) in a Tet-On CPEB4 overexpression system (right). (C) Diagram of coexpressed Tet-On-operated CPEB4 overexpression constructs and luciferase reporter constructs with WT or Del sequence variant in the CPE site (left). Effect of CPE sequence variant of SCN5A 3'-UTR by CPEB4 overexpressed in RL14 cells (right). qPCR was employed to assess expression levels, with GAPDH serving as an internal control. Results are presented as fold changes (FC) relative to control. Data are presented as mean + SD; n = 3 per condition for all panels. ∗∗P < 0.01; ∗∗∗P < 0.001 when compared between indicated groups by Student’s t-test or 1-way analysis of variance with Tukey multiple comparisons test. (D) Characterization of sodium currents in hiPSC-CMs. Comparison between WT control and…

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  Figure 7. Restorative Effects of CPEB4 Overexpression on SCN5A Expression and Function (A and B) Effect of CPEB4 on the hypoxia-mimetic chemical-induced SCN5A mRNA (A) and Nav1.5 protein reduction (B) in RL14 cells. Data represent all 3 replicates. All experiments were performed in triplicate. Data are presented as mean + SD (n = 3 per condition). ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 when compared between indicated groups by 1-way analysis of variance with Tukey multiple comparisons test. (C) AAV9 vector constructs expressing a Cpeb4 from a cTnT cardiac specific promoter. Cpeb4-AAV9 particles were injected into MI mice induced by LAD ligation via the jugular vein. The cardiac tissue was then removed, and RNA and protein were extracted. (D and E) Effect of cardiac-specific Cpeb4 expression on SCN5A mRNA (D) or Nav1.5 protein (E) expression. For Nav1.5, densities represent the sum of the upper and lower bands (total Nav1.5), and both Nav1.5 and CPEB4 densities were normalized to GAPDH. Data are presented as mean + SD (MI+Con, n = 3; MI+Cpeb4, n = 4). ∗P < 0.05; ∗∗P < 0.01 when compared between indicated groups by Student’s t-test. (F) Characterization of sodium currents in isolated CMs.…