Premature MicroRNA-1 Expression Causes Hypoplasia of the Cardiac Ventricular Conduction System

Abstract

Mammalian cardiac Purkinje fibers (PFs) are specified from ventricular trabecular myocardium during mid-gestation and undergo limited proliferation before assuming their final form. MicroRNA-1 (miR-1), a negative regulator of proliferation, is normally expressed in the heart at low levels during the period of PF specification and outgrowth, but expression rises steeply after birth, when myocardial proliferation slows and postnatal cardiac maturation and growth commence. Here, we test whether premature up-regulation and overexpression of miR-1 during the period of PF morphogenesis influences PF development and function. Using a mouse model in which miR-1 is expressed under the control of the Myh6 promoter, we demonstrate that premature miR-1 expression leads to PF hypoplasia that persists into adulthood, and miR-1 TG mice exhibit delayed conduction through the ventricular myocardium beginning at neonatal stages. In addition, miR-1 transgenic embryos showed reduced proliferation within the trabecular myocardium and embryonic ventricular conduction system (VCS), a source of progenitor cells for the PF. This repression of proliferation may be mediated by direct translational inhibition by miR-1 of the cyclin dependent kinase Cdk6, a key regulator of embryonic myocardial proliferation. Our results suggest that altering the timing of miR-1 expression can regulate PF development, findings which have implications for our understanding of conduction system development and disease in humans.

Keywords: Purkinje fibers; cardiac conduction system; cardiac development; cardiac maturation; heart block; miR-1; microRNA.

FIGURE 1

Specific Overexpression of MicroRNA-1 in Myh6-MiR-1-TG Mice. (A) The Myh6 promoter was cloned upstream of pre-miR-1-2 for pronuclear injection. (B) Quantitative PCR demonstrated that 8 weeks old miR-1 TG mice had about threefold overexpression of miR-1 as compared to WT littermates. Expression of other cardiac-enriched microRNAs was unchanged. “^∗” denotes p < 0.05.

FIGURE 2

Hypoplasia of Ventricular Conduction System in miR-1 TG Embryos. (A) Quantitative PCR for mature miR-1 transcript in miR-1 TG hearts versus WT hearts at several stages of development shows that miR-1 expression is elevated in mid-gestation embryos and rises to adult levels in the perinatal period, anticipating the normal developmental upregulation of miR-1. The inset shows relative expression of miR-1 versus WT at E11.5. “^∗” denotes p < 0.05, “^∗∗” denotes p < 0.01, and “^∗∗∗” denotes p < 0.001. (B) Whole-mount in situ hybridization at E10.5 for Bmp10 in WT (top, left) and TG (bottom, left) and VCS development at E11.5 as assessed by whole-mount bluo-gal staining of a miR-1 TG heart (bottom, right) alongside a WT littermate (top, right) in the CCS-LacZ background. Note the similar distribution of reporter expression. (C) Light microscopic examination of embryonic hearts sectioned at E10.5 (left) and E12.5 (right) in WT (top) and miR-TG (bottom). Scale bars = 200 microns. (D) Whole-mount imaging of CCS-Lacz reporter activity at E13.5, after the onset of miR-1 upregulation in miR-1 TG mice, demonstrates a striking paucity of VCS tissue in the transgenic heart (bottom row) versus a WT littermate (top row). Panels 1–4 on the right show magnified images of developing RV (1,3) and LV (2,4) PF density in WT (1,2), and TG (3,4) hearts.

FIGURE 3

Abnormal Conduction System Structure and Function in MiR-1 TG Neonatal Mice. (A) Whole mount imaging of CCS-LacZ reporter activity in P5 hearts shows a reduction in PF density in the TG heart (bottom, left) versus the WT littermate (top, left). The magnified panels 1–4 (right) demonstrate atrioventricular bundle tissue (AVB; 1,3) and Purkinje Fiber (PF) density (PF; 2,4) in the WT (1,2) and miR-1 TG hearts (3,4). (B) Whole-mount imaging of reporter activity in WT (top) and TG (bottom) P5 hearts in the Irx3-LacZ background. Left panels show imaging of the left bundle branch and left-sided Purkinje fibers. Right panels show magnified images of right ventricular PFs. (C) Density of PFs in WT (n = 3) and miR-1 TG (n = 5) hearts were quantified from images of reporter activity in Irx3-LacZ hearts. Representative images for quantification are shown at top. The density of PFs was significantly reduced in miR-1 TG hearts, as shown in the bottom graph (“^∗∗” denotes p < 0.01). (D) Averaged lead aVF ECG tracings recorded from a neonatal WT mouse (top) and a miR-TG littermate show prolongation of the QRS duration in the miR-1 TG, reflecting slowed conduction through the VCS. (E) RR, PR, and QRS intervals quantified from WT and miR-1 TG mice (mice obtained from 3 distinct litters, n = 8 for WT and n = 5 for TG, “^∗” denotes p < 0.05).

FIGURE 4

Abnormal Conduction System Structure in Adult MiR-1 TG Mice. (A) Whole mount imaging after fixation and bluo-gal staining of the ventricular conduction system in WT (top) and miR-1 TG (bottom) hearts in the CCS-LacZ background. PF density is reduced in the miR-1 TG adult hearts. (B) Quantitative PCR for genes enriched in PFs demonstrates decreased expression of conduction system genes and either unchanged or only modest changes in core cardiac transcription factors. “^∗” denotes p < 0.05, “^∗∗” denotes p < 0.01, and “^∗∗∗” denotes p < 0.001; NS, non-significant. (C) Averaged ECG tracings derived from limb lead aVF from 8 weeks old WT (top) and miR-1 TG mice (bottom) demonstrated prolonged PR and QRS in the miR1-TG. (D) Comparison of electrocardiographic intervals in adult WT mice (n = 7) and miR-1 TG littermates (n = 8) demonstrate prolonged RR, PR, and QRS intervals in the miR-1 TG mice, reflecting widespread conduction system dysfunction.

FIGURE 5

MiR-1 Negatively Regulates VCS Proliferation and Cdk-6 Protein Expression. (A) Phosphohistone H3 staining in WT and miR-1 TG hearts at E12.5 shows a reduction in PH3+ cells in transgenic animals as compared to WT. The graph shows the percentage of trabecular nuclei that are PH3+ in each genotype. “^∗∗∗” denotes p < 0.001. (B) Co-staining of phosphohistone H3 and beta-galactosidase in Irx3-LacZ and Irx3-LacZ; MiR-1 TG E12.5 hearts demonstrates reduced mitotic index specifically in the developing VCS. The graph shows the percentage of beta-galactosidease nuclei that are also phosphohistone H3 positive “^∗∗∗” denotes p < 0.001. (C) mRNA level of Cdk6 is unchanged in postnatal hearts from miR-1 TG versus WT littermates, while protein level is significantly decreased, consistent with translational regulation. Knockdown of miR-1 in HL-1 cells results in upregulation of Cdk6 at the protein level. ^∗ denotes p < 0.05.

FIGURE 6

Working Model for Role of MiR-1 in VCS Proliferation. MiR-1 negatively regulates Cdk6, which binds to Cyclin D to inhibit the action of the pocket proteins, negative regulators of VCS cellularity. The net effect of Cdk6 reduction is increased activity of pocket proteins and thus reduced proliferation in the VCS.