Downregulation of connexin43 by microRNA-130a in cardiomyocytes results in cardiac arrhythmias

Abstract

MicroRNAs (miRNAs) are now recognized as critical regulators of diverse physiological and pathological processes; however, studies of miRNAs and arrhythmogenesis remain sparse. Connexin43 (Cx43), a major cardiac gap junction protein, has elicited great interest in its role in arrhythmias. Additionally, Cx43 was a potential target for miR-130a as predicted by several computational algorithms. This study investigates the effect of miR-130a overexpression in the adult heart and its effect on cardiac rhythm. Using a cardiac-specific inducible system, transgenic mice demonstrated both atrial and ventricular arrhythmias. We performed ventricular-programmed electrical stimulation and found that the αMHC-miR130a mice developed sustained ventricular tachycardia beginning 6weeks after overexpression. Western blot analysis demonstrated a steady decline in Cx43 after 2weeks of overexpression with over a 90% reduction in Cx43 levels by 10weeks. Immunofluorescent staining confirmed a near complete loss of Cx43 throughout the heart. To validate Cx43 as a direct target of miR-130a, we performed in vitro target assays in 3T3 fibroblasts and HL-1 cardiomyocytes, both known to endogenously express miR-130a. Using a luciferase reporter fused to the 3'UTR of Cx43, we found a 52.9% reduction in luciferase activity in 3T3 cells (p<0.0001) and a 47.6% reduction in HL-1 cells (p=0.0056) compared to controls. Addition of an antisense miR-130a inhibitor resulted in a loss of inhibitory activity of the Cx43 3'UTR reporter. We have identified an unappreciated role for miR-130a as a direct regulator of Cx43. Overexpression of miR-130a may contribute importantly to gap junction remodeling and to the pathogenesis of atrial and ventricular arrhythmias.

Keywords: Atrial arrhythmia; Connexin43; MicroRNA; Ventricular arrhythmia.

Figure 1. Expression of miR-130a in cardiomyocytes and induction of miR-130a in adult heart

Localization of miR-130a using fluorescent in situ hybridization in adult cardiomyocytes. Panel (A) shows U6 stained nuclei as a positive control, panel (B) shows staining with a miR-scramble as a negative control, and panel (C) with miR-130a staining in cardiomyocytes. (D) Schematic of inducible transgenic system for miR-130a overexpression using the αMHC promoter to drive expression of the tTA protein. In panel (E), fluorescent in situ hybridization of αMHC-miR130a heart demonstrating increased miR-130a signal confirming overexpression of miR-130a. (F) Quantitative PCR of relative miR130a levels in transgenic heart 6–8 weeks after doxycycline is removed from water supply (n=5). (G) Schematic outline of doxycycline treatment and study time points. * indicates p<0.0001. Doxy: doxycycline; TetO: tetracycline responsive promoter; tTA: tetracycline-controlled transactivator.

Figure 2. Echocardiography in αMHC-miR130a mice

Panels (A) and (B), m-mode echocardiography of representative control vs. αMHC-miR130a mouse maintained on doxycycline at 10 weeks after weaning. Cardiac dimensions and function was preserved. In panels (C) and (D), representative M-mode echocardiography of control vs. αMHC-miR130a mouse at 10 weeks after doxycycline removal. Note the irregularity of ventricular contractions in the αMHC-miR130a mice. In panels (E) and (F), 2D–guided pulsed Doppler (see inset) of the mitral inflow in control and the αMHC-miR130a mice respectively. (G) and (H), serial assessment in LV diameter and % fractional shortening at 6, 8, 10, and 12 weeks. In each group, n = 5. * indicates p < 0.001.

Figure 3. Simultaneous surface and intracardiac electrocardiograms in control and αMHC-miR130a mice

In (A), simultaneous atrial (top panel), ventricular (middle panel) electrograms and surface ECG (bottom panel) of control mouse demonstrating normal sinus rhythm. Note the presence of regular P waves (P) representing atrial depolarization corresponding to high amplitude signal seen in the atrial recording. High amplitude ventricular signal corresponds to the QRS complex (QRS) on the surface ECG representing ventricular depolarization. In (B), simultaneous atrial, ventricular, and surface ECG in αMHC-miR130a mice. In contrast to the control mouse, the atrial electrogram (B, top panel) shows a rapid irregular atrial signal. The ventricular electrogram (B, middle panel) shows a high amplitude signal corresponding to QRS complexes on the surface ECG with a variable cycle length. Tracings obtained 10–12 weeks off doxycycline. Eight animals were studied in each group. A: atrial signal; V: ventricular signal.

Figure 4. Propensity for ventricular arrhythmias in αMHC-miR130a mice

In panel (A), representative P-QRS complexes in control and αMHC-miR130a mice at 6 and 10 weeks after doxycycline removal. In panel (B), representative programmed electrical ventricular stimulation studies in control mice (top panel) and αMHC-miR130a transgenic mice (bottom panel). In the control mouse, pacing stimulus could not induce VT. In contrast, sustained VT is seen in the αMHC-miR130a mouse following pacing stimulus. In (C), summary of programmed electrical stimulation studies with the number of inducible VT episodes in control (C) and miR-130a transgenic (Tg) animal cohorts at 4, 6, and 10 weeks. Sustained ventricular arrhythmias could not be induced in any of the controls. Beginning at 6 weeks after doxycycline removal, αMHC-miR130a mice could be induced into monomorphic VT. In panel (D), simultaneous intracardiac and surface ECG in αMHC-miR130a mice during VT episode. Atrial electrogram (top panel) demonstrates a slower atrial signal with a regular cycle length. Ventricular electrogram (middle panel) demonstrates a rapid high amplitude signal corresponding with the rapid wide QRS complexs on surface ECG. Arrows indicate potential atrial signal and corresponding surface ECG location within the QRS complex consistent with atrioventricular dissociation. These findings are supportive of VT. Shown in panel (E), an example of spontaneous VT initiated after premature ventricular complexes in αMHC-miR130a mice during ambulatory ECG monitoring 10 weeks after removal of doxycycline. Five animals in each group was studied for both stimulation studies and ambulatory ECG monitoring. * indicates p=0.026. NSVT: nonsustained ventricular tachycardia; A: atrial signal; VT: ventricular tachycardia; Tg: transgenic; C: control littermate.

Figure 5. Reduced connexin43 in αMHC-miR130a mice

In panel (A), computational alignment of a potential conserved target site in the Cx43 3’UTR and miR-130a. In (B), representative timecourse of Cx43 protein loss after doxycycline removal in αMHC-miR130a hearts compared to controls (n=3, each group). After 2 weeks, we found a steady reduction of Cx43 with near complete loss by 10 weeks after doxycycline removal. In (C), quantitative PCR demonstrated a 52% reduction in Cx43 mRNA at 12 weeks after doxycycline removal (n=5, each group). Western analysis on ventricular lysates (panel D) at the same time point shows >90% reduction in Cx43 protein in αMHC-miR130a hearts compared to controls (n=10 in each group). In panel (E), western blot for both Cx43 and phosphorylated-Cx43 at 12 weeks after doxycycline removal demonstrates significant reduction in both forms of Cx43. * indicates p < 0.001.

Figure 6. Loss of connexin43 in the atria and ventricles of αMHC-miR130a mice

In panel (A), representative immunofluorescent staining of Cx43 (green) in adult control atria and ventricles compared to αMHC-miR130a atria and ventricles (n=16 hearts studied). Cx43 is reduced throughout the myocardium of αMHC-miR130a mice. (B) Western blot of ventricular lysate at 10 weeks off doxycycline demonstrating >90% reduction in Cx43 levels as quantified in (C) (n=6 per group). In panel (D), representative immunofluorescent staining of atrial tissue for Cx40 (red) and Cx43 (green) at 10 weeks off doxycycline. Note the loss of Cx43 in αMHC-miR130a atria with preserved Cx40 expression. In panel (E), atrial lysates were used for western blotting confirming preserved Cx40 levels in αMHC-miR130a atria compared to controls and quantified in (F) (n=4 per group). Consistent with the ventricular lysates, Cx43 levels were significantly reduced in the atrial lysates. Nuclei are counterstained with DAPI (blue). * indicates p < 0.001.

Figure 7. MicroRNA-130a mediates translational repression via the Cx43 3’UTR

In panel (A), schematic of luciferase reporter constructs used for in vitro transfection assays. Firefly luciferase (F. luciferase) coding region is fused to the control 3’UTR vs. Cx43 3’UTR. Binding of miR-130a is predicted to inhibit translation of F. luciferase. Addition of a specific miR-130a inhibitor is predicted to allow translation of F. luciferase. As shown in (B), administration of a miR-130a inhibitor (300 picomoles) reduced miR-130a levels in 3T3 fibroblasts approximately 83%. Reporter constructs were transfected into NIH 3T3 fibroblasts (known to endogenously express miR-130a). In (C), luciferase assay with luciferase reporter constructs and miR-130a inhibitor vs. scramble. Inhibition of miR-130a had no effect on the translational efficiency of the control reporter. In contrast, translational repression of the reporter construct was relieved in a dose-dependent fashion. Co-transfection with a scramble inhibitor was performed as a control. Luciferase experiments were also carried out in HL-1 cardiomyocytes (also known to endogenously express miR-130a). Using quantitative PCR in (D), HL-1 cells contained 52.4% of the miR-130a compared to 3T3 cells. Administration of the miR-130a inhibitor resulted in a 73.2% reduction in miR-130a (E). Similar to 3T3 cells, luciferase assay demonstrated reduced luciferase activity in the presence of the Cx43 3’UTR reporter construct. Translational repression of the reporter construct was relieved with administration of a miR-130a inhibitor. Transfection with a scramble inhibitor was performed as a control. Experiments performed in triplicate × three independent experiments. * indicates p<0.01. 3’UTR: 3’ untranslated region; LNA: locked nucleic acid.