Intranuclear cardiac troponin I plays a functional role in regulating Atp2a2 expression in cardiomyocytes

Figures

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  Figure 1. Detecting of intranuclear cTnI in physiological and pathological conditions. (A) Immunoblotting of cTnI in nuclear (Nu) and cytoplasm (Cy) lysates of adult mice cardiomyocytes. (B) Immunoblotting of cTnI in nuclear (Nu) and cytoplasm (Cy) lysates of human fetal hearts (11- and 14-week's embryos). (C) cTnI was examined in neonatal mice ventricular myocytes through fluorescence microscope. Scale bars show 50 μm. (D–E) Intranuclear cTnI detection both in left and right ventricles of wild type adult mouse hearts. Western blot analysis were expressed as Mean ± SEM derived from n = 3 independent experiments analyzed by Mann–Whitney test. ∗P < 0.05 as compared with controls. (F–G) Intranuclear cTnI detection in left ventricles of mice with heart failure and wild type mice. The results are expressed as mean ± SEM from n = 3 separate experiments. ∗P < 0.05 as compared with controls analyzed by Mann–Whitney test.

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  Figure 2. Weighted gene co-expression network analysis (WGCNA) of cTnI and verification results in a knock-out mice model. (A) The flow charts of WGCNA of TNNI3. (B, C) Genes positive related to TNNI3 were analyzed by KEGG (Kyoto Encyclopedia of Genes and Genomes) pathways and GO (Gene ontology) in the Database for Annotation, Visualization and Integrated Discovery (DAVID). (D) RT-qPCR of candidate genes of PDE1A, PDE1B, PDE4D and ATP2a2 in TNNI3 knockout mice. The results are expressed as mean ± SEM from n = 4–6 separate experiments. ∗∗ means P < 0.01 as compared with cTnI+/+ group analyzed by Mann–Whitney test. (E) Correlation coefficient between ATP2a2 expression with TNNI3 in the dynamic process of heart failure was calculated. (Pearson's R = 0.919 95%CI [0.797, 0.969] P < 0.001, n = 19).

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  Figure 3. ATP2a2/SERCA2a expression levels after cTnI gain and loss in vivo.(A) mRNA levels of ATP2a2 in cTnI over-expression mice. The results are expressed as mean ± SE from n = 8–10 separate experiments. ∗∗∗ means P < 0.001 as compared with controls analyzed by Mann–Whitney test. (B, C) Immunoblotting of cTnI and SERCA2a in cTnI over-expression mice. Western blot analysis were expressed as Mean ± SEM derived from n = 3 independent experiments analyzed by Mann–Whitney test. ∗P < 0.05 as compared with controls. (D) mRNA levels of ATP2a2 in cTnI knockout mice(cTnI+/−). The results are expressed as mean ± SE from n = 11 separate experiments. ∗∗∗ means P < 0.001 as compared with controls analyzed by Mann–Whitney test. (E, F) Immunoblotting of cTnI and SERCA2a in cTnI+/− mice, and immunoblotting of cTnI in nuclear (Nu) lysates of cTnI ± mice. Western blot analysis were expressed as Mean ± SEM derived from n = 3 independent experiments analyzed by Mann–Whitney test. ∗P < 0.05, ∗∗P < 0.01 as compared with controls.

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  Figure 4. ATP2a2/SERCA2a expression levels after cTnI gain and loss in vitro.(A) mRNA levels of ATP2a2 in over-expression TNNI3 neonatal mice ventricular myocytes. The results are expressed as mean ± SE from n = 6 separate experiments. ∗∗ means P < 0.01 as compared with controls analyzed by Mann–Whitney test. (B, C) Immunoblotting of Flag-cTnI and SERCA2a in over-expression TNNI3 NMVMs. Western blot analysis were expressed as Mean ± SEM derived from n = 3 independent experiments analyzed by Mann–Whitney test. ∗P < 0.05 as compared with controls. (D) mRNA levels of ATP2a2 in NMVMs of TNNI3 knock-down by siRNA(siRNA-TNNI3). The results are expressed as mean ± SE from n = 7 separate experiments. ∗∗ means P < 0.01 as compared with controls analyzed by Mann–Whitney test. (E, F) Immunoblotting of cTnI and SERCA2a in siRNA-TNNI3 NMVMs. Western blot analysis were expressed as Mean ± SEM derived from n = 3 independent experiments analyzed by Mann–Whitney test. ∗P < 0.05 as compared with controls.

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  Figure 5. Calcium dyshomeostasis after cTnI loss in cardiomyocytes. (A) Beat frequencies of cardiomyocytes after transfecting siRNA-TNNI3. ∗∗∗ means P < 0.001 as compared with controls. (B) Total ATP contents in cardiomyocytes of siRNA-TNNI3 groups compared with controls. ∗∗ means P < 0.01 as compared with controls. (C) NMVMs loaded fluo4-AM were detected by laser confocal microscopy. Scale bars show 50 μm. (D, E) Intracellular calcium transient frequencies of siRNA-TNNI3 and control groups.

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  Figure 6. Binding affinity and binding regions of cTnI with promoter of ATP2a2.(A) Specific binding DNA sequences of cTnI were enriched in ATP2a2 promoter −239∼–889 region. (B, C) Binding sequence motif of cTnI was analyzed by software as "CCAT", which has been reported to be required for YY1 binding to the promoter region of YY1-related genes. (D) “CCAT” sequence was in the promoter region of ATP2a2. (E) The promoter region of ATP2a2 were divided into 6 segments. (F) Binding abilities of cTnI with each segment were verified by CHIP-qPCR. (G) Immunoblotting (IB) of cTnI in the lysates (input) or immunoprecipitates (IP; IgG or anti-YY1) of NMVMs. (H) In 293A cells, relative luciferase activity was shown after pcDNA3.1 plasmid/TNNI3 over-expression plasmid and ATP2a2 promoter luciferase reporter gene plasmid co-transfection. ∗∗∗ means P < 0.001 as compared with controls analyzed by Mann–Whitney test.