Differences in MEF2 and NFAT transcriptional pathways according to human heart failure aetiology

Abstract

Background: Ca(2+) handling machinery modulates the activation of cardiac transcription pathways involved in heart failure (HF). The present study investigated the effect of HF aetiology on Ca(+2) handling proteins and NFAT1, MEF2C and GATA4 (transcription factors) in the same cardiac tissue.

Methodology and principal findings: A total of 83 hearts from ischemic (ICM, n = 43) and dilated (DCM, n = 31) patients undergoing heart transplantation and controls (CNT, n = 9) were analyzed by western blotting. Subcellular distribution was analyzed by fluorescence and electron microscopy. When we compared Ca(+2) handling proteins according to HF aetiology, ICM showed higher levels of calmodulin (24%, p<0.01), calcineurin (26%, p<0.01) and Ca(2+)/Calmodulin-dependent kinase II (CaMKIIδ(b) nuclear isoform 62%, p<0.001) than the CNT group. However, these proteins in DCM did not significantly increase. Furthermore, ICM showed a significant elevation in MEF2C (33%, p<0.01), and GATA4 (49%, p<0.05); also NFAT1 (66%, p<0.001) was increased, producing the resultant translocation of this transcriptional factor into the nuclei. These results were supported by fluorescence and electron microscopy analysis. Whereas, DCM only had a significant increase in GATA4 (52%, p<0.05). Correlations between NFAT1 and MEF2C in both groups (ICM r = 0.38 and DCM r = 0.59, p<0.05 and p<0.01, respectively) were found; only ICM showed a correlation between GATA4 and NFAT1 (r = 0.37, p<0.05).

Conclusions/significance: This study shows an increase of Ca(2+) handling machinery synthesis and their cardiac transcription pathways in HF, being more markedly increased in ICM. Furthermore, there is a significant association between MEF2, NFAT1 and GATA4. These proteins could be therapeutic targets to improve myocardial function.

Figure 1. Western blots showing increased CaM, CaN and NCX1 levels, and decreased SERCA2 levels in human heart failure.

CaM (A), CaN (B), NCX1 (C) and SERCA2 (D) in left ventricular myocardium from patients with ICM (n = 43) and DCM (n = 31) versus CNT group (n = 9). The data are expressed as means ± SEM of five independent experiments. Values are normalized to β-actin and finally to CNT group, which was also normalized to β-actin before. CaM, calmodulin; CaN, calcineurin; CNT, control; DCM, dilated cardiomyopathy; ICM, ischemic cardiomyopathy. *p<0.05 versus control.

Figure 2. Detailed protein values of CaMKIIδ in cytosolic and nuclear fractions in human myocardium.

Western blots for cytosolic (A) and nuclear (B) CaMKIIδ in controls, ischemic and dilated cardiomyopathies. The data are expressed as means ± SEM of five independent experiments. Values were normalized to β-actin and finally to control myocardium, which was also normalized to β-actin before. CaMKIIδ, Ca²⁺/calmodulin-dependent kinase II isoform delta; CNT, control, DCM, dilated cardiomyopathy; ICM, ischemic cardiomyopathy. **p<0.01 vs CNT. ***p<0.001 vs. CNT.

Figure 3. Influence of heart failure on the MEF2C and HDAC4 transcriptional factor levels.

We determined the values of MEF2 and HDAC4 by Western blots. In A, values of MEF2C were significantly increased in ICM samples (n = 43). In B, similar results were obtained in LV myocardium of ICM for HDAC4. Values are expressed as mean ± SEM of five independent experiments and normalized to β-actin and finally to CNT myocardium, which was also normalized to β-actin before. ICM, ischemic cardiomyopathy; DCM, dilated cardiomyopathy; CNT, control. *p<0.05 versus CNT.

Figure 4. Protein levels of NFAT1 transcriptional factor in cytosolic and nuclear fractions.

As shown, both distribution of NFAT1, cytoplasm (A) and nucleus (B) were increased in ICM (n = 43), but only cytosolic fraction was increased in DCM (n = 30) compared to CNT (n = 9). Values are expressed as mean ± SEM of five independent experiments and normalized to β-actin and finally to CNT myocardium, which was also normalized to β-actin before. ICM, ischemic cardiomyopathy; DCM, dilated cardiomyopathy; CNT, control. **p<0.01 vs CNT and ***p<0.001 vs. CNT.

Figure 5. Microscopic analysis of the effect of ICM aetiology on NFAT1 nuclear translocation in human cardiomyocytes.

Representative fluorescence micrographs for NFAT1 in CNT (A–C) and ICM (D–F) samples. All the micrographs correspond to four independent experiments. The bar represents 10 µm. In micrograph G, Bar graph comparing the fluorescence intensity in cytoplasm and into nucleus of NFAT1, in CNT and ICM groups. The values from the cytoplasm were set to 100. The data are expressed as mean ± SEM of five experiments. ICM, ischemic cardiomyopathy; DCM, dilated cardiomyopathy; CNT, control. ***p<0.001 versus cytoplasm.

Figure 6. Effect of heart failure aetiology on GATA4 synthesis.

As shown protein levels of GATA4 were significant increased in both aetiologies, ischemic (ICM) and dilated (DCM) cardiomyopathies compared to controls (CNT). Values are expressed as mean ± SEM of five independent experiments and normalized to β-actin and finally to CNT myocardium, which was also normalized to β-actin before. *p<0.05 versus CNT.

Figure 7. Nuclear activity as heterochromatin mass by electron microscopy in human cardiomyocytes.

Cross-sectional micrographs of a nucleus in control (A), ischemic (B) and dilated (C) samples, showing a more heterochromatin condensation (hc) in controls, overall perinuclear chromatin (asterisk). N indicates nucleus. Bar represents: 400 nm.

Figure 8. Relationship between cardiac transcriptional factors in heart failure human hearts.

(A) Correlation plots between NFAT1 with MEF2 and GATA4 in pathological samples (n = 74).