Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Loss of NAT10 Reduces the Translation of Kmt5a mRNA Through ac4C Modification in Cardiomyocytes and Induces Heart Failure

doi: 10.1161/JAHA.124.035714

Figure Lengend Snippet: A , An integrative heat map of TE‐altered genes in 2‐week‐old NAT10‐cKO and control hearts. B , A Venn diagram showing overlapped genes between TE‐altered genes in 2‐week‐old NAT10‐cKO hearts and the reactome term “Regulation of TP53 activity” (MM15226). The enrichment FDR value and gene names were presented. C , Ac4C modification sites on mRNAs of Kmt5a , Chek1 , Aurkb , and Ccna2 were predicted with PACES ( www.rnanut.net/paces ). D through F , Detection of Kmt5a protein expression by western blotting and mRNA expression by RT‐qPCR in NAT10‐cKO and control hearts (n=4 for each group), NAT10‐iKO and control hearts (n=4 for each group), and NAT10‐Ctrl, NAT10‐gRNA1 and NAT10‐gRNA2 H9c2 cardiomyocytes (n=4 for each group). The TE was calculated. α‐tubulin serves as loading control. G , Detection of Kmt5a protein expression by western blotting and mRNA expression by RT‐PCR in NRVCs infected with Ad‐Kmt5a in the treatment of DMSO (control) or remodelin (n=4 for each group). The TE was calculated. α‐tubulin serves as loading control. H , Ac4C modification level on Kmt5a mRNA in NAT10‐cKO and control hearts was detected by acRIP‐qPCR (n=4 for each group). I , Determination of the half‐life of Kmt5a mRNA in NAT10‐Ctrl, NAT10‐gRNA1, and NAT10‐gRNA2 H9c2 cardiomyocytes at 0, 3, and 6 hours after actinomycin D treatment by RT‐qPCR (n=3 for each group). The statistical significance of difference between groups in data with repeated measurements was tested by mixed model repeated measures. J , Dot blot of ac4C modification levels in total RNA from NRVCs infected with Ad‐GFP, Ad‐NAT10‐WT, and Ad‐NAT10‐G641E. Methylene blue staining serves as loading control. K , Detection and quantification of NAT10 and Kmt5a protein expression by western blotting in NRVCs infected with Ad‐GFP, Ad‐NAT10‐WT, and Ad‐NAT10‐G641E (n=4 for each group). α‐tubulin serves as loading control. Statistical significance indicated in this figure was determined using a 2‐tailed unpaired t test to assess differences between 2 groups unless indicated otherwise. *** P <0.001; ** P <0.01; * P <0.05. ac4C indicates N4‐acetylcytidine; acRIP, acetylated RNA immunoprecipitation; Ctrl, Control; FDR_B&H, false discovery rate_Benjamini & Hochberg; GFP, green fluorescent protein; MB, methylene blue; NAT10, N‐acetyltransferase 10; NAT10‐cKO, cardiac‐specific NAT10 knockout mice; NAT10‐iKO inducible cardiac‐specific NAT10 knockout mice; NRVC, neonatal rat cardiomyocytes; RT‐qPCR, real‐time quantitative polymerase chain reaction; TE, translational efficiency; and WT, wild type.

Article Snippet: Polyvinylidene difluoride membranes were blocked with 5% BSA for 1 hour and then incubated overnight at 4 °C with primary antibodies: anti‐NAT10 (Proteintech, 13 365‐1‐AP), anti‐ Kmt5a (Proteintech, 14 063‐1‐AP), anti‐ Bax (Proteintech, 50599‐2), anti‐ Bcl‐2 (Proteintech, 12789‐1‐AP), anti‐ p53 (CST, #2524), anti‐α‐tubulin (Proteintech, 11224‐1‐AP), and anti‐GAPDH (Proteintech, 60004‐1‐IG).

Techniques: Control, Activity Assay, Modification, Expressing, Western Blot, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Infection, Dot Blot, Staining, RNA Immunoprecipitation, Knock-Out, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Loss of NAT10 Reduces the Translation of Kmt5a mRNA Through ac4C Modification in Cardiomyocytes and Induces Heart Failure

doi: 10.1161/JAHA.124.035714

Figure Lengend Snippet: A through C , Detection of Kmt5a protein expression by western blotting and mRNA expression by RT‐qPCR in failing and control human hearts, α‐tubulin serves as loading control. The TE was calculated (n=3 for control hearts, n=4 for failing hearts). D , Detection and quantification of Kmt5a protein expression in si‐Kmt5a and si‐NC treated NRVCs (n=3 for each group). α‐tubulin serves as loading control. E , Apoptotic cardiomyocytes were detected from NRVCs treated with si‐Kmt5a and si‐NC by TUNEL assay. cTnI marked cardiomyocytes, and DAPI marked nuclei. Scale bar=20 μm (n=3 for each group). F , Apoptotic cardiomyocytes were detected from NRVCs treated with si‐Kmt5a and si‐NC by flow cytometry (n=3 for each group). G , Detection and quantification of Bax and Bcl‐2 protein expressions in NRVCs treated with si‐Kmt5a and si‐NC. α‐tubulin serves as loading control. The ratio of Bax to Bcl‐2 was calculated (n=3 for each group). H , Determination of Bax , Bcl‐2 , and p21 transcript expressions in NRVCs treated with si‐Kmt5a and si‐NC by RT‐qPCR (n=3 for each group). Statistical significance indicated in this figure was determined using a q‐tailed unpaired t test to assess differences between 2 groups. *** P <0.001; ** P <0.01; * P <0.05. cTnI indicates cardiac troponin I; Ctrl, Control; HF, heart failure; NRVC, neonatal rat cardiomyocytes; RT‐qPCR, real‐time quantitative polymerase chain reaction; TE, translational efficiency; and WT, wild type.

Article Snippet: Polyvinylidene difluoride membranes were blocked with 5% BSA for 1 hour and then incubated overnight at 4 °C with primary antibodies: anti‐NAT10 (Proteintech, 13 365‐1‐AP), anti‐ Kmt5a (Proteintech, 14 063‐1‐AP), anti‐ Bax (Proteintech, 50599‐2), anti‐ Bcl‐2 (Proteintech, 12789‐1‐AP), anti‐ p53 (CST, #2524), anti‐α‐tubulin (Proteintech, 11224‐1‐AP), and anti‐GAPDH (Proteintech, 60004‐1‐IG).

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Control, TUNEL Assay, Flow Cytometry, Real-time Polymerase Chain Reaction

Journal: Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease

Article Title: Loss of NAT10 Reduces the Translation of Kmt5a mRNA Through ac4C Modification in Cardiomyocytes and Induces Heart Failure

doi: 10.1161/JAHA.124.035714

Figure Lengend Snippet: A , Apoptotic cardiomyocytes were detected from NRVCs infected with Ad‐GFP (control) or Ad‐Kmt5a in the treatment of DMSO (control) or remodelin by TUNEL assay. α‐actinin marked cardiomyocytes, and DAPI marked nuclei. Scale bar=20 μm (n=3 for each group). B , Apoptotic cardiomyocytes were detected from NRVCs infected with Ad‐GFP (control) or Ad‐Kmt5a in the treatment of DMSO (control) or remodelin by flow cytometry. (n=5 for each group). C , Bax and Bcl‐2 protein expressions in NRVCs infected with Ad‐GFP (control) or Ad‐Kmt5a in the treatment of DMSO (control) or remodelin were detected by western blotting and quantified (n=4 for each group). α‐tubulin serves as loading control. D , Bax , Bcl‐2 , and p21 transcript expressions in NRVCs infected with Ad‐GFP (control) or Ad‐Kmt5a in the treatment of DMSO (control) or remodelin was determined by RT‐qPCR (n=3 for each group). E , A working model postulating the function of NAT10 in heart failure. Statistical significance indicated in this figure was determined using a 2‐tailed unpaired t test to assess differences between 2 groups. *** P <0.001; ** P <0.01; * P <0.05. CM indicates cardiomyocyte; Ctrl, Control; GFP, green fluorescent protein; NAT10, N‐acetyltransferase 10; NRVC, neonatal rat cardiomyocytes; PI, propidium iodide; Re, remodelin; RT‐qPCR, real‐time quantitative polymerase chain reaction; and TE, translational efficiency.

Article Snippet: Polyvinylidene difluoride membranes were blocked with 5% BSA for 1 hour and then incubated overnight at 4 °C with primary antibodies: anti‐NAT10 (Proteintech, 13 365‐1‐AP), anti‐ Kmt5a (Proteintech, 14 063‐1‐AP), anti‐ Bax (Proteintech, 50599‐2), anti‐ Bcl‐2 (Proteintech, 12789‐1‐AP), anti‐ p53 (CST, #2524), anti‐α‐tubulin (Proteintech, 11224‐1‐AP), and anti‐GAPDH (Proteintech, 60004‐1‐IG).

Techniques: Infection, Control, TUNEL Assay, Flow Cytometry, Western Blot, Quantitative RT-PCR, Real-time Polymerase Chain Reaction