Ribonucleicacid interference or small molecule inhibition of Runx1 in the border zone prevents cardiac contractile dysfunction following myocardial infarction

Abstract

Aims: Myocardial infarction (MI) is a major cause of death worldwide. Effective treatments are required to improve recovery of cardiac function following MI, with the aim of improving patient outcomes and preventing progression to heart failure. The perfused but hypocontractile region bordering an infarct is functionally distinct from the remote surviving myocardium and is a determinant of adverse remodelling and cardiac contractility. Expression of the transcription factor RUNX1 is increased in the border zone 1-day after MI, suggesting potential for targeted therapeutic intervention.

Objective: This study sought to investigate whether an increase in RUNX1 in the border zone can be therapeutically targeted to preserve contractility following MI.

Methods and results: In this work we demonstrate that Runx1 drives reductions in cardiomyocyte contractility, calcium handling, mitochondrial density, and expression of genes important for oxidative phosphorylation. Both tamoxifen-inducible Runx1-deficient and essential co-factor common β subunit (Cbfβ)-deficient cardiomyocyte-specific mouse models demonstrated that antagonizing RUNX1 function preserves the expression of genes important for oxidative phosphorylation following MI. Antagonizing RUNX1 expression via short-hairpin RNA interference preserved contractile function following MI. Equivalent effects were obtained with a small molecule inhibitor (Ro5-3335) that reduces RUNX1 function by blocking its interaction with CBFβ.

Conclusions: Our results confirm the translational potential of RUNX1 as a novel therapeutic target in MI, with wider opportunities for use across a range of cardiac diseases where RUNX1 drives adverse cardiac remodelling.

Keywords: Runx1; Cardiomyocytes; Myocardial Infarction.

Figure 1

Runx1 alters BZ Ca²⁺. (A) Protocol. (B) Typical calcium (Ca²⁺) transients pre-MI in C57BL/6J mice. Mean (C) Ca²⁺ transient peak, (D) Ca²⁺ transient amplitude, (E) caffeine-induced Ca²⁺ transient amplitude, and (F) SERCA activity (RZ, n = 76,8 hearts) (BZ n = 43,8 hearts). (G) Typical Ca²⁺ transients 1-day post-MI in C57BL/6J mice. Mean (H) Ca²⁺ transient peak, (I) Ca²⁺ transient amplitude, (J) caffeine-induced Ca²⁺ transient amplitude, and (K) SERCA activity (RZ, n = 64,7 hearts), (BZ n = 30,7 hearts). (L) Typical Ca²⁺ transients 1-day post-MI in Runx1^Δ/Δ-mice. Mean (M) Ca²⁺ transient peak, (N) Ca²⁺ transient amplitude, (O) caffeine-induced Ca²⁺ transient amplitude, and (P) SERCA activity (RZ, n = 16,4 hearts) (BZ n = 9,4 hearts). Error bars = mean ± SEM. **P < 0.01, ***P < 0.001. SEM, standard error of the mean.

Figure 2

Runx1 regulates oxidative phosphorylation. (A) Comparison and Venn diagram of gene differences in Runx1^Δ/Δ and Runx1^fl/fl-mice (n = 6). (B) Enriched biological pathways ranked by logP using IPA from unique differences between BZ and RZ in Runx1^fl/fl (circles) compared with BZ and RZ differences in Runx1^Δ/Δ-mice (triangles). The heatmap (blue to orange) represents predicted inhibition and activation or no change of pathways based on the Z-score. (C) Generation of Cbfβ^Δ/Δ and Cbfβ^fl/fl-mice. (D) Comparison and Venn diagram of gene expression in Cbfβ^Δ/Δ and Cbfβ^fl/fl-mice (n = 6). (E) The enriched biological pathway from IPA generated from unique differences and (F) Cbfβ-mice ranked on Z-score.

Figure 3

Runx1 deficiency protects mitochondria post-MI. (A) Genes involved in oxidative phosphorylation from BZ and RZ 1-day post-MI in IPA of Runx1^fl/fl (n = 6, 7166 differentially expressed genes) and (B) Runx1^Δ/Δ-mice (n = 6, 1748 differentially expressed genes). (C) Electron microscopy images from BZ of Runx1^fl/fl and (D) Runx1^Δ/Δ-mice. (E) Quantification of mitochondrial density (percentage of cell area) and (F) number of damaged mitochondria from the BZ from Runx1^fl/fl and Runx1^Δ/Δ-mice (n = 92 cells; two hearts) 1-day post-MI. Error bars = mean ± SEM. *P < 0.05. SEM, standard error of the mean.

Figure 4

Targeting Runx1 protects cardiac function post-MI. (A) Schematic of Ad. (B) Echocardiographic data for FS of Ad-Runx1-shRNA vs. Ad-scramble-shRNA. (C) Typical picrosirius-red-stained hearts and infarct size as the percentage of the LV. (D) Schematic of AAV. (E) FS of AAV-Runx1-shRNA vs. AAV-scramble-shRNA. (F) Typical picrosirius-red stained hearts and infarct size as a percentage of the LV AAV-Runx1-shRNA (n = 8) vs. AAV-scramble-shRNA (n = 6). (G) Protocols for Ro5-3335. (H) FS for mice receiving protocol 1 and (I) protocol 2. (J) FS from Cbfβ^Δ/Δ-MI vs. Cbfβ^fl/fl-MI mice. Error bars = mean ± SEM. *P < 0.05. SEM, standard error of the mean.