Cardiac troponin I R193H mutant interacts with HDAC1 to repress phosphodiesterase 4D expression in cardiomyocytes

Abstract

Cardiac Troponin I (cTnI) is a subunit of the thin filament involved in regulation of heart contraction. Mutated cTnI accounts for most genetic mutations associated with restrictive cardiomyopathy (RCM). We previously found phosphodiesterase 4D (PDE4D) decreased in RCM mice with cTnIR193H mutation and the mutant cTnI might be involved in PDE4D reduction. This study aims to elucidate a novel role of cTnIR193H mutant as a gene regulator. Overexpression of cTnIR193H mutant in cardiomyocytes showed decrease in PDED4D protein expression, while the enrichment of histone deacetylase 1 (HDAC1) was increased along with decreases in acetylated lysine 4 (acH3K4) and 9 (acH3K9) levels in the PDE4D promoter. HDAC1 overexpression could also downregulate PDE4D via reducing acH3K4 and acH3K9 levels. Co-IP assays showed that cTnIR193H mutant owed increased binding ability to HDAC1 compared with wild type cTnI. EGCG as a HDAC1 inhibitor could diminish the strength of cTnIR193H-HDAC1 interactions and alleviate the reduction in PDE4D expression. Together, our data indicated that cTnIR193H mutant could repress PDE4D expression in cardiomyocytes through HDAC1 associated histone deacetylation modification. Unlike the typical function of cTnI in cytoplasm, our study suggested a novel role of cTnI mutants in nuclei in regulating gene expression.

Keywords: EGCG; HDAC1; Histone modifications; PDE4D reduction; cTnIR93H.

Figure 1

The effects of cTnI or cTnIR193H on PDE4D expression. mRNA or protein were extracted from primary neonatal cardiomyocytes overexpressing cTnI and cTnIR193H respectively (A) PDE4D mRNA levels in control group (NC), cTnI overexpression group (Flag-cTnI) and cTnIR193H overexpression group (Flag-cTnIR193H). (B) PDE4D protein level in NC and Flag-cTnI groups and quantification of western blotting results. (C) PDE4D protein level in NC and Flag-cTnIR193H groups and quantification of western blotting results. Statistical significance was determined by ANOVA followed by Least-Significant Difference (LSD) tests. *P < 0.05 relative to NC; n = 6 per group.

Figure 2

Nuclear translocation of cTnIR193H/cTnI and their transcriptional activities to PDE4D. (A) Western blotting for detecting the nuclear protein and total cTnI or cTnIR193H in primary neonatal cardiomyocytes overexpressing cTnI or cTnIR193H, and column graph for the ratios of nuclear to total protein reflecting the translocation ability. 30ug nuclei and 100ug whole cells extraction were used to make H3 similar (B) 293T cells expressing cTnI or cTnIR193H for dual-luciferase reporter gene assays to test cTnI or cTnIR193H transcriptional activities to PDE4D. *P < 0.05 relative to vector group; n = 6 per group.

Figure 3

cTnIR193H enhances the enrichment of HDAC1 and reduces acH3K4 and acH3K9 levels in PDE4D promoter. (A) ChiP assays for identifying the region of PDE4D promoter with highest enrichment of cTnI in the −1000bp upstream regions of PDE4D in primary neonatal cardiomyocytes. (B) The enrichment of cTnI, cTnIR193H and HDAC1 in the promoter region of −47∼-141bp in primary neonatal cardiomyocytes with overexpression of cTnI or cTnIR193H. (C) The enrichment of acH3K4 and acH3K9 in the promoter region of −47∼-141bp in primary neonatal cardiomyocytes with overexpression of cTnI or cTnIR193H. The results are expressed as mean ± SD. Statistical significance was determined by ANOVA followed by LSD tests. *P < 0.05 relative to Flag-cTnI group; n = 6 per group.

Figure 4

HDAC1 represses PDE4D expression via histone deacetylation. Primary neonatal cardiomyocytes overexpressing HDAC1 were used for these experiments. (A) PDE4D protein level in blank group, NC group, and HDAC1 overexpression group (OE-HDAC1). (B) ChiP assays for detecting the enrichment of HDAC1, acH3K4 and acH3K9 among the three groups. The results are expressed as mean ± SD. Statistical significance was determined by ANOVA followed by LSD tests. *P < 0.05 as relative to NC group; n = 6 per group.

Figure 5

cTnIR193H mutant shows increased binding to HDAC1 compared with cTnI. (A) Immunofluorescence shows cTnI and HDAC1 coexist in the nuclei of primary neonatal cardiomyocytes. (B) Co-IP assays for detecting the interaction between HDAC1 and cTnI or cTnIR193H in Primary neonatal cardiomyocytes overexpressing cTnI or cTnIR193H. The ratio of immunoprecipitated-proteins reflecting the interaction strength shows with column graph. (C) Western blotting for detecting the free HDAC1 in the supernatant of the cells overexpressing cTnI or cTnIR193H. The results are expressed as mean ± SD. Statistical significance was determined by ANOVA followed by LSD tests. *P < 0.05 as compared with each other; n = 6 per group.

Figure 6

EGCG alleviates cTnIR193H induced PDE4D low expression. Primary neonatal cardiomyocytes overexpressing cTnIR193H with or without EGCG treatment were used for these experiments. (A) PDE4D protein level in NC group, cTnIR193H group and cTnIR193H + EGCG group. (B) ChiP assays for detecting the enrichment of cTnIR193H and HDAC1 with or without EGCG treatment. (C) ChiP assays for detecting the enrichment of acH3K4 and acH3K9 with or without EGCG treatment. The results are expressed as mean ± SD. Statistical significance was determined by ANOVA followed by LSD tests. *P < 0.05; n = 6 per group.

Figure 7

EGCG inhibits cTnIR193H-HDAC1 interactions but not HDAC1 activity. Primary neonatal cardiomyocytes overexpressing cTnIR193H with or without EGCG treatment were used for these experiments. (A) CoIP assays for detecting the interaction between HDAC1 and cTnIR193H in the presence and absence of EGCG, and column graph for the ratio of immunoprecipitated-proteins reflecting the interaction strength. (B) Western blot assays for detecting the free HDAC1 in the supernatant. (C) HDAC1 activities. The results are expressed as mean ± SD. Statistical significance was determined by ANOVA followed by LSD tests. *P < 0.05; n = 6 per group.