SMYD1 modulates the proliferation of multipotent cardiac progenitor cells derived from human pluripotent stem cells during myocardial differentiation through GSK3β/β-catenin&ERK signaling

Abstract

Background: The histone-lysine N-methyltransferase SMYD1, which is specific to striated muscle, plays a crucial role in regulating early heart development. Its deficiency has been linked to the occurrence of congenital heart disease. Nevertheless, the precise mechanism by which SMYD1 deficiency contributes to congenital heart disease remains unclear.

Methods: We established a SMYD1 knockout pluripotent stem cell line and a doxycycline-inducible SMYD1 expression pluripotent stem cell line to investigate the functions of SMYD1 utilizing an in vitro-directed myocardial differentiation model.

Results: Cardiomyocytes lacking SMYD1 displayed drastically diminished differentiation efficiency, concomitant with heightened proliferation capacity of cardiac progenitor cells during the early cardiac differentiation stage. These cellular phenotypes were confirmed through experiments inducing the re-expression of SMYD1. Transcriptome sequencing and small molecule inhibitor intervention suggested that the GSK3β/β-catenin&ERK signaling pathway was involved in the proliferation of cardiac progenitor cells. Chromatin immunoprecipitation demonstrated that SMYD1 acted as a transcriptional activator of GSK3β through histone H3 lysine 4 trimethylation. Additionally, dual-luciferase analyses indicated that SMYD1 could interact with the promoter region of GSK3β, thereby augmenting its transcriptional activity. Moreover, administering insulin and Insulin-like growth factor 1 can enhance the efficacy of myocardial differentiation in SMYD1 knockout cells.

Conclusions: Our research indicated that the participation of SMYD1 in the GSK3β/β-catenin&ERK signaling cascade modulated the proliferation of cardiac progenitor cells during myocardial differentiation. This process was partly reliant on the transcription of GSK3β. Our research provided a novel insight into the genetic modification effect of SMYD1 during early myocardial differentiation. The findings were essential to the molecular mechanism and potential interventions for congenital heart disease.

Keywords: GSK3β; Histone modification; Human pluripotent stem cells; Myocardial differentiation; SMYD1.

Fig. 1

SMYD1 deficiency drastically reduces the myocardial differentiation efficiency of hPSCs A Diagram of the sgRNA positions targeting SMYD1. B TC bases insertion resulting in frameshift mutation. C Schematic representation of hPSCs myocardial differentiation stages D, E Western blot of SMYD1 protein at day 15, quantification of protein expression was normalized by GAPDH. F, G Immunostaining of TNNT2 in WT and SMYD1 KO cardiomyocytes at day 15. Scalebar, 75 μm. H, I Flow cytometry analysis for TNNT2 in WT and SMYD1 KO cardiomyocytes at day 15

Fig. 2

SMYD1 deficiency leads to excessive cell proliferation capacity during the early myocardial differentiation stage. A, B Western blot showing SMYD1, NKX2-5 and ISL1 in WT and SMYD1 KO cells at day 5, quantification of protein expression was normalized by WT. C, D Flow cytometry analysis for EDU⁺ in WT and SMYD1 KO cells at day 5. E, F Representative immunostaining for ISL1 and Ki67 expression in WT and SMYD1 KO cells at day 5. Scalebar, 50 μm

Fig. 3

Restoration of SMYD1 expression rescues myocardial differentiation phenotypes. A Pattern of doxycycline-inducible SMYD1 expression. B, C Western blot of SMYD1 at day 5 of differentiation, quantification of protein expression was normalized by GAPDH. D, E Immunostaining for TNNT2 in WT, SMYD1 KO and doxycycline-inducible SMYD1 at day 15. Scalebar, 50 μm. F, G Flow cytometry analysis for TNNT2 in WT, SMYD1 KO and doxycycline-inducible SMYD1 at day 15. H, I Flow cytometry analysis for EDU⁺ in WT, SMYD1 KO and doxycycline-inducible SMYD1 cells at day 5. J, K Proliferation marker Ki67 and cardiac progenitor cells marker ISL1 of cell immunofluorescent staining at day 5. Scalebar, 50 μm.

Fig. 4

SMYD1 participates in myocardial differentiation regulation by suppressing GSK3β/β-catenin&ERK signaling A The volcano plot shows up- and down-regulated genes in SMYD1 KO compared with WT at day 5. Red denotes upregulated and blue denotes down-regulated genes, p < 0.05. B The top 10 significantly enriched KEGG pathways. C, D Western blot of  Wnt/β-catenin and RAS-ERK signaling pathway-related proteins at day 5, quantification of protein expression was normalized by GAPDH. E Immunostaining for β-catenin and p-ERK in WT, SMYD1 KO cells and doxycycline-inducible SMYD1 cells at day 5. Scalebar, 100 μm. F–I Flow cytometry for comparisons of TNNT2⁺ after drug intervention at day 15. J–M Western blot of β-catenin and p-ERK/ERK proteins after drug intervention, quantification of protein expression was normalized by GAPDH

Fig. 5

SMYD1 acts as a transcriptional activator of GSK3β through Histone H3 Lysine 4 trimethylation A, B Western blot of GSK3β and p-GSK3β protein in WT, SMYD1 KO cells and doxycycline-inducible SMYD1 cells at day 5, quantification of protein expression was normalized by GAPDH. C Quantified analysis of GSK3B mRNA. D H3K4me3 enrichment within promoter regions of GSK3B is predicted in the heart by the WashU EpiGenome Database. E ChIP-qPCR revealed that H3K4me3 enrichment was found in the promoter regions of GSK3β in WT cells. F Schematic diagram of SMYD1 binding sites during the GSK3B -1 kb promoter region. G Luciferase reporter assays using SMYD1 plasmid and GSK3B-wt or mutant promoters

Fig. 6

Insulin/IGF-1 improves myocardial differentiation of SMYD1-deficient cells A, B Immunostaining for TNNT2 in WT, KO, KO + insulin, or IGF-1 cardiomyocytes at day 15. Scalebar, 100 μm. C, D Flow cytometry analysis for TNNT2 in WT, KO, KO + insulin, or IGF-1 cardiomyocytes at day 15. E, F Western blot of p-ERK and ERK protein at day 5, quantification of protein expression was normalized by WT