Cardiac Actions of a Small Molecule Inhibitor Targeting GATA4-NKX2-5 Interaction

Abstract

Transcription factors are fundamental regulators of gene transcription, and many diseases, such as heart diseases, are associated with deregulation of transcriptional networks. In the adult heart, zinc-finger transcription factor GATA4 is a critical regulator of cardiac repair and remodelling. Previous studies also suggest that NKX2-5 plays function role as a cofactor of GATA4. We have recently reported the identification of small molecules that either inhibit or enhance the GATA4-NKX2-5 transcriptional synergy. Here, we examined the cardiac actions of a potent inhibitor (3i-1000) of GATA4-NKX2-5 interaction in experimental models of myocardial ischemic injury and pressure overload. In mice after myocardial infarction, 3i-1000 significantly improved left ventricular ejection fraction and fractional shortening, and attenuated myocardial structural changes. The compound also improved cardiac function in an experimental model of angiotensin II -mediated hypertension in rats. Furthermore, the up-regulation of cardiac gene expression induced by myocardial infarction and ischemia reduced with treatment of 3i-1000 or when micro- and nanoparticles loaded with 3i-1000 were injected intramyocardially or intravenously, respectively. The compound inhibited stretch- and phenylephrine-induced hypertrophic response in neonatal rat cardiomyocytes. These results indicate significant potential for small molecules targeting GATA4-NKX2-5 interaction to promote myocardial repair after myocardial infarction and other cardiac injuries.

Figure 1

The effect of small molecule 3i-1000 on GATA–NKX2-5 interaction in cell-based reporter gene assay. (A) COS-1 cells were transfected with a reporter construct containing three high-activation binding sites for NKX2-5 together with protein expression vectors for GATA4 and NKX2-5. The cells were lysed, and the reporter gene activity was measured by a luminometer. The small molecule 3i-1000 inhibited GATA4–NKX2-5 transcriptional synergy in luciferase reporter assay at the concentration of 5 µM. The results are an average of three parallel samples ± SD. **p < 0.01 (independent samples Student’s t-test). (B) Dose-dependent inhibition of the GATA4–NKX2-5 transcriptional synergy with the small molecule 3i-1000. The molecular structure of the compound is also shown. The results are an average of two experiments with 4 or 8 replicates ± SEM.

Figure 2

The effect of small molecules acting on GATA4–NKX2-5 transcriptional synergy on the hypertrophic process in vitro in neonatal cardiac myocytes. (A,B) The effects of 3i-1000 and 3i-0777 on stretch-induced increase in ANP (A) and (B) BNP mRNA levels. Cultured neonatal rat cardiomyocytes were stretched cyclically up to 24 h. The compounds were added 1 h prior stretching to the cells. (C,D) Effects of the compounds on phenylephrine (PE) induced increase in ANP (C) and (D) BNP gene expression. Cells were treated for 24 h with PE and the compounds were added 1 h prior to PE. mRNA levels were measured by RT-PCR and normalised to housekeeping gene 18 S quantified from the same samples. The results are averages ± SD, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by a least significant difference post hoc test).

Figure 3

The effect of small molecule 3i-1000 on GATA4 protein levels and phosphorylation in vitro in neonatal rat cardiomyocytes. (A) Compound 3i-1000 at concentration of 50 µM had no influence on baseline levels of nuclear GATA4 or Ser-105 phosphorylation of GATA4 (pGATA4) protein. (B–E) Compound 3i-1000 (50 µM) inhibited the elevation of GATA4 and phospho-GATA4 protein levels produced with PE. The experiment was repeated three times, and the results presented here are an average of three parallel samples ± SD. The original whole blot images are presented in Supplementary Figure S1. *p < 0.05, **p < 0.01 (one-way ANOVA followed by a least significant difference post hoc test).

Figure 4

(A–D) The echocardiographic parameters and mRNA levels in the left ventricular tissue of mice that underwent acute myocardial infarction (AMI) or sham-operation (SHAM) and were treated either with vehicle (veh) or compound 3i-1000 (30 mg/kg/day i.p.) for 4 days. Echocardiographic measurements were performed at the end of the experiment one week after infarction. The number of animals was 15 in SHAM + veh, 4 in AMI + veh and 3 in AMI + 3i-1000 groups. (E–H) The echocardiographic parameters and the mRNA levels in the left ventricular tissue of rats that underwent acute myocardial infarction (AMI) or sham-operation (SHAM) and were treated either with vehicle or compound 3i-1000 (30 mg/kg/day i.p.) for one week. Echocardiographic measurements were performed at the end of the experiment at one week. The number of the animals was 6 in SHAM + veh, 7 in AMI + veh and 8 in AMI + 3i-1000 groups. mRNA levels were measured by RT-PCR and normalised to housekeeping gene 18 S quantified from the same samples. The results are averages ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by a least significant difference post hoc test).

Figure 5

(A–D) The echocardiographic parameters and mRNA levels in the left ventricular tissue of rats that were treated with angiotensin II (ANGII, 33.3 µg/kg/h s.c.) and vehicle or ANGII and the compound 3i-1000 (30 mg/kg/day i.p) for two weeks. Echocardiographic measurements were performed at 2 weeks. The number of animals in both groups was 6. mRNA levels were measured by RT-PCR and normalised to housekeeping gene 18 S quantified from the same samples. The results are averages ± SEM. *p < 0.05 (independent samples Student’s t-test).

Figure 6

(A,B) The left ventricular ejection fraction and fractional shortening analysed by echocardiography in rats treated either with vehicle or compound 3i-1000 (30 mg/kg/day i.p.) for one week before ischemia-reperfusion (I/R) or sham-operation (SHAM). Following 30 min of ischemia, the slipknot was released and the myocardium was re-perfused for 24 h. Echocardiographic measurements were performed at 24 h. The number of the animals was 4 in SHAM+veh, 15 in I/R+veh and 15 in I/R+3i-1000 groups. (C,D) The ANP and BNP mRNA levels in the left ventricular tissue. Number of animals was 4 in SHAM+veh, 13 in I/R+veh and 15 in I/R+3i-1000 groups. mRNA levels were measured by RT-PCR and normalised to housekeeping gene 18 S quantified from the same samples. The results are averages ± SEM. *p < 0.05 (one-way ANOVA followed by a least significant difference post hoc test).

Figure 7

(A,B) The echocardiographic parameters and (C–F) mRNA levels in the left ventricular tissue of rats that underwent acute myocardial infarction (AMI) or sham-operation (SHAM) and were treated either with vehicle or compound 3i-1000 loaded TOPSi 7 µm particles. Echocardiographic measurements were performed at one week. The number of animals was 7 in SHAM + veh, 7 in SHAM + 3i-1000, 6 in AMI + veh and 7 in AMI + 3i-1000 groups. mRNA levels were measured by RT-PCR and normalised to housekeeping gene 18 S quantified from the same samples. The results are averages ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way ANOVA followed by a least significant difference post hoc test).

Figure 8

(A–E) The mRNA levels in the left ventricular endocardial layer of rats in response to isoprenaline-induced myocardial ischemia after treatment with 3i-1000 loaded nanoparticles. Isoprenaline was injected s.c. 24 h before i.v. administration of the control particles (C) or 3i-1000 loaded particles. The samples were collected 4 h after particle injections. The number of animals was 4–5 in both groups. mRNA levels were measured by RT-PCR and normalised to housekeeping gene 18 S quantified from the same samples. The results are averages ± SEM. *p < 0.05, ***p < 0.001 (independent samples Student’s t-test).