Pharmacological inhibition of RUNX1 reduces infarct size after acute myocardial infarction in rats and underlying mechanism revealed by proteomics implicates repressed cathepsin levels

Abstract

Myocardial infarction (MI) results in prolonged ischemia and the subsequent cell death leads to heart failure which is linked to increased deaths or hospitalizations. New therapeutic targets are urgently needed to prevent cell death and reduce infarct size among patients with MI. Runt-related transcription factor-1 (RUNX1) is a master-regulator transcription factor intensively studied in the hematopoietic field. Recent evidence showed that RUNX1 has a critical role in cardiomyocytes post-MI. The increased RUNX1 expression in the border zone of the infarct heart contributes to decreased cardiac contractile function and can be therapeutically targeted to protect against adverse cardiac remodelling. This study sought to investigate whether pharmacological inhibition of RUNX1 function has an impact on infarct size following MI. In this work we demonstrate that inhibiting RUNX1 with a small molecule inhibitor (Ro5-3335) reduces infarct size in an in vivo rat model of acute MI. Proteomics study using data-independent acquisition method identified increased cathepsin levels in the border zone myocardium following MI, whereas heart samples treated by RUNX1 inhibitor present decreased cathepsin levels. Cathepsins are lysosomal proteases which have been shown to orchestrate multiple cell death pathways. Our data illustrate that inhibition of RUNX1 leads to reduced infarct size which is associated with the suppression of cathepsin expression. This study demonstrates that pharmacologically antagonizing RUNX1 reduces infarct size in a rat model of acute MI and unveils a link between RUNX1 and cathepsin-mediated cell death, suggesting that RUNX1 is a novel therapeutic target that could be exploited clinically to limit infarct size after an acute MI.

Keywords: Cardiac protection; Cathepsin; Myocardial infarction; Runx1; Therapeutic target.

Fig. 1

Infarct size in rat hearts treated with the RUNX1 inhibitor Ro5-3335 in vivo. Data were collected at 24 hours after infarction. (A) Schematic of the protocol used. (B) Representative TTC staining of heart slices. The red staining is viable tissue, and the pale color represents dead tissue (5 mm scale bar). (C) The mean infarct size for MI group (n= 8) and Ro group (n= 8). *P < 0.05. Study groups: MI, MI rat hearts treated by vehicle; Ro, MI rat hearts treated by Ro5-3335. Figure 1A was prepared using BioRender.com under a publication license

Fig. 2

Inhibition of RUNX1 changes protein expression profile. (A) Heart tissue samples taken from the BZ at 24 h post-MI was analyzed by proteomics. (B) The heatmap (blue to red) represents downregulated or upregulated proteins based on the Z-score. See enlarged heatmap in supplementary material. (C) The volcano plot shows the distribution of downregulated and upregulated proteins. The x-axis represents the expression of each protein, reported as log2 fold change, while the y-axis represents the −log10 (P-value). The red and blue dots highlight the proteins with significant differential expression (fold change>1.5 or <0.67, P<0.05). (D) Cluster analysis of identified proteins by Mfuzz illustrates 9 clusters with discrete expression changes. Study groups: MI, MI rat hearts treated by vehicle (n= 3 hearts); Ro, MI rat hearts treated by Ro5-3335 (n= 5 hearts); Sh, sham-operated rat hearts treated by vehicle (n= 3 hearts). Figure 2A was created using BioRender.com under a publication license

Fig. 3

Subcellular and GO enrichment analysis of proteomic data. (A) Prediction of subcellular locations of differentially expressed proteins between vehicle- and Ro5-3335-treated MI hearts. (B) GO enrichment analysis of differentially expressed proteins in Ro5-3335-treated hearts relative to control MI hearts. X axis shows rich factor which represents the ratio of the number of enriched proteins in a category to the total number of proteins in that category. Y axis shows category names. Area of each node represents the number of enriched proteins differentially expressed upon inhibiting RUNX1. P-values are represented by color scale

Fig. 4

KEGG analysis of proteomic data. (A) KEGG enrichment analysis of differentially expressed proteins. X axis shows rich factor which represents the ratio of the number of enriched proteins. Y axis shows category names. Area of each node represents the number of enriched proteins. P-values are represented by color scale. (B) Top KEGG pathways of differentially expressed proteins upon Ro5-3335-treatment relative to control ranked by enrichment P-value. Downregulation and upregulation are represented by blue and red, respectively

Fig. 5

The KEGG lysosome pathway map. Green boxes represent proteins downregulated in the BZ of Ro5-3335-treated hearts in comparison to control MI samples. White boxes indicate unchanged proteins

Fig. 6

Proteomic quantification of cathepsins and caspases. (A) Overall cathepsin levels reflected by the average of identified cathepsin members and protein levels for (B) individual cathepsin levels, (C) caspase 3, (D) caspase 7, (E) caspase 8 and (F) caspase 9. *P < 0.05. Study groups: MI, MI rat hearts treated by vehicle (n=3 hearts); Ro, MI rat hearts treated by Ro5-3335 (n= 5 hearts); Sh, sham-operated rat hearts treated by vehicle (n=3 hearts)

Fig. 7

Schematic depicting roles of RUNX1 and cathepsins in cardiomyocytes following myocardial infarction or ischemia/reperfusion injury. RUNX1 is a master-regulator transcription factor which controls gene expression. Cathepsins seem to be regulated by RUNX1 and act as executioners to mediate contractile dysfunction and various forms of cell death. NCX, sodium-calcium exchanger; RyR, ryanodine receptor calcium release channels; SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase. Figure 7 was created using BioRender.com under a publication license, based on information from this study and various sources (McCarroll et al. ; He et al. ; Yadati et al. ; Nagakannan et al. ; Xie et al. 2023)