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. 2025 Apr 8;16(1):3345.
doi: 10.1038/s41467-025-58722-5.

GPAT4 sustains endoplasmic reticulum homeostasis in endocardial cells and safeguards heart development

Tianyang Zhao #  1 , Kuipei Jin #  2   3 , Xiaodong Wang  1 , Xiong Su  4 , Youjun Wang  5 , Mingming Gao  2 , Wen Luo  6 , Hongyuan Yang  7   8 , Zhongzhou Yang  9
Affiliations

GPAT4 sustains endoplasmic reticulum homeostasis in endocardial cells and safeguards heart development

Tianyang Zhao et al. Nat Commun. .

Abstract

The endocardium plays a pivotal role in governing myocardial development, and understanding the intrinsic regulatory insights will help apprehend pathological cardiomyopathy. Glycerol-3-phosphate acyltransferase 4 (GPAT4) is an endoplasmic reticulum (ER) membrane anchored protein. While the role of GPAT4 in glycerophospholipid biosynthesis is well established, its function in the ER is less explored. Here, we generate Gpat4 global and tissue-specific knockout mice and identify the essential role of GPAT4 in endocardial development. Deficiency of GPAT4 provokes endocardial ER stress response and enhances ER-mitochondrial (ER-mito) communications, leading to mitochondrial DNA (mtDNA) escape. As a result, the cGAS-STING pathway is triggered to stimulate type-I-interferon response, which affects heart development. Finally, abolishment of the cGAS-STING-type-I-interferon pathway rescues the heart defects of Gpat4 deletion mice. These findings uncover the pivotal role of GPAT4 in the maintenance of ER homeostasis during endocardial and heart development. Meanwhile, this study highlights the importance of the cGAS-STING pathway in cardiac organogenesis.

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Conflict of interest statement

Competing interests: The authors declare no competing interests.

Figures

Fig. 1
Fig. 1. Defective heart development in Gpat4 global knockout mice.
A Gross analysis of WT and Gpat4 KO embryos and hearts at E14.5. The red arrows indicate subcutaneous edema. Scale bars, 500 µm. B Immuno-staining of heart sections at E14.5. Endomucin (EMCN) stains endocardium (in red) and DAPI stains the nuclei (in blue). Scale bars, 500 µm. C Quantification of heart width, n = 3. **P = 0.0066. D Higher magnification of the boxed areas in (B). Endomucin (EMCN) stains endocardium (in red) and DAPI stains the nuclei (in blue). Tra, trabecular layer; Myo, myocardial layer. E Quantification of the Tra and Myo thickness of the heart sections in (D). Note the substantially reduced thickness of the Myo but markedly increased thickness of the Tra in the heart of KO mice. LV, left ventricle; RV, right ventricle, n = 3. Scale bars, 100 µm. Left-Right, **P = 0.0024, **P = 0.0083, **P = 0.0040, ***P < 0.0001. F EdU staining. Scale bars, 100 µm. Edu (in green), Endomucin (EMCN) stains endocardium (in red) and DAPI stains the nuclei (in blue). G Quantification of EdU+ Cells in Tra and Myo, n = 3. Left-Right, ***P < 0.0001, **P = 0.0084. H, I Western blotting analysis and quantification (heart tissues from E14.5 WT and KO mice), n = 3. Left-Right, ***P < 0.0001, **P = 0.0013, **P = 0.0014. J–N Heart whole mount staining of the coronary vasculature and quantification of the vessels, n = 3. J displays the coronary veins. J1–J4 are higher magnification of the boxed areas in (J) Endomucin (EMCN) stains coronary veins (in green). K Quantification of vein diameter, n = 3, ***P = 0.0004. L Quantification of Vascular density, n = 3, ***P = 0.0009. M manifests the coronary arteries that was stained by JAG1 antibody (in green). Scale bars, 100 µm. N Quantification of JAG1+ diameter, n = 3, ***P = 0.0002. Data are mean ± s.e.m. Two-tailed unpaired Student’s t-test.
Fig. 2
Fig. 2. Essential role of GPAT4 in endocardial development.
A Gross analysis of Gpat4F/F and Tie2-Cre; Gpat4F/F embryos (E14.5) and hearts. The red arrows indicate subcutaneous edema. Scale bars, 500 µm. B Immuno-staining of heart sections. Endomucin (EMCN) stains endocardium (in red) and DAPI stains the nuclei (in blue). Scale bars, 500 µm. C Quantification of heart width, n = 3, **P = 0.0019. D Higher magnification of the boxed areas in (B). GPAT4 (in green), Endomucin (EMCN) stains endocardium (in red) and DAPI stains the nuclei (in blue). Tra, trabecular layer; Myo, myocardial layer. E Quantification of the Tra and Myo thickness of the heart sections in (D). Note the substantially reduced thickness of the Myo but markedly increased thickness of the Tra in the heart of KO mice, n = 3. Scale bars, 100 µm. Left-Right, ***P < 0.0001, ***P < 0.0001, ***P < 0.0001, ***P = 0.0002. F EdU staining. Scale bars, 100 µm. Edu (in green), Endomucin (EMCN) stains endocardium (in red) and DAPI stains the nuclei (in blue). G Quantification of EdU+ Cells in Tra and Myo, n = 3. Left-Right, ***P < 0.0001, **P = 0.0045. H, I Western blotting analysis and quantification, n = 3. Left-Right, **P = 0.0019, **P = 0.0022, ***P < 0.0001. J–N Heart whole mount staining of the coronary vasculature and quantification of the vessels, n = 3. J displays the coronary veins. (J1–J4) are higher magnification of the boxed areas in (J). Endomucin (EMCN) stains coronary veins (in gray). K Quantification of vein diameter, n = 3, ***P = 0.0003. L Quantification of Vascular density, n = 3, **P < 0.0015. M manifests the coronary arteries that was stained by JAG1 antibody (in red). N Quantification of JAG1+ diameter, n = 3, ***P < 0.0001. Scale bars, 100 µm. O Gross analysis of Gpat4F/F and Nfatc1-Cre; Gpat4F/F embryos (E14.5) and hearts. The red arrows indicate subcutaneous edema. Scale bars, 500 µm. P Quantification of heart width, n = 3, **P = 0.0055). Immuno-staining to display the trabeculae (Tra) and myocardium (Myo) layers of hearts from E14.5 embryos. Endomucin (EMCN) stains endocardium (in red) and DAPI stains the nuclei (in blue). Scale bars, 100 µm. R Quantification of the Tra and Myo thickness of the heart sections in (Q), n = 3. Left-Right, **P = 0.0013, ***P = 0.0003, **P = 0.0024, **P = 0.0074. Data are mean ± s.e.m. Two-tailed unpaired Student’s t-test.
Fig. 3
Fig. 3. GPAT4 regulates ER homeostasis and ER-Mito communications in the endocardium.
A RNA-seq analysis of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, Heatmap of dysregulated genes. Red and blue colors represent upregulated and downregulated genes, n = 3. B qPCR analysis of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, n = 3. Left-Right, ***P = 0.0005, **P = 0.0010, ***P = 0.0002, ***P = 0.0007, ***P = 0.0002, ***P = 0.0006, ***P = 0.0007. C, D Western blotting analysis and quantification of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, n = 3. Left-Right, **P = 0.0014, ***P = 0.0004, **P = 0.0015, **P = 0.0013, *P = 0.0106. E, F Immunoprecipitation-mass spectrometry (GPAT4 antibody) analysis with KEGG/GO (HUVECs). G The association between GPAT4 protein and EIF2S1/eIF2α protein predicted by AF3. H Immuno-precipitation (GPAT4 and EIF2S1) and Western blotting analysis (HUVECs). I AF3 modeling of the association relationship among the three proteins of PERK, GPAT4 and EIF2S1/eIF2α. J AF3 modeling of association between PERK protein and EIF2S1/eIF2α protein. K Immuno-precipitation (PERK and EIF2S1) and Western blotting analysis in Ctrl and siGPAT4 groups (HUVECs). L Immuno-precipitation (PERK and EIF2S1) and Western blotting analysis in Ctrl and GPAT4 OE groups (HUVECs). M, N Ctrl and GPAT4 OE groups, Western blotting analysis and quantification (HUVECs), n = 3. Left-Right, **P = 0.0081, **P = 0.0012, *P = 0.0181, **P = 0.0016, **P = 0.0013. O, P TEM analysis of Gpat4F/F and Tie2-Cre; Gpat4F/F endocardial cells in E11.5 hearts and quantification of ER-Mito contacts, n = 3. Scale bars, 1 µm. Left-Right, *P = 0.0161, *P = 0.0158, *P = 0.0495, **P = 0.0078. Q, R Western blotting analysis and quantification of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, n = 3. Left-Right, *P = 0.0168, **P = 0.0042, ***P = 0.0004, **P = 0.0013, **P = 0.0036. Data are mean ± s.e.m. Two-tailed unpaired Student’s t-test.
Fig. 4
Fig. 4. Gpat4 deletion elicited cGAS-STING-TBK1-IRF3-typ I interferon response in the endocardium.
A RNA-seq analysis of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, Heatmap of dysregulated genes. Red and blue colors represent upregulated and downregulated genes, n = 3. B KEGG analysis of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts. C qPCR analysis of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, n = 3. Left-Right, ***P = 0.0003, ***P = 0.0004, ***P = 0.0002, ***P < 0.0001, ***P = 0.0003, ***P = 0.0009, ***P = 0.0007, ***P = 0.0002, ***P = 0.0009, **P = 0.0057. D, E Western blotting analysis and quantification of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts, n = 3. Left-Right, **P = 0.0050, **P = 0.0086, **P = 0.0022, *P = 0.0453. F cGAS staining of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts. Scale bars, 100 µm. cGAS (in green), Endomucin (EMCN) stains endocardium (in red) and DAPI stains the nuclei (in blue). G co-localization analysis of cGAS and endocardium. H STING staining of Gpat4F/F and Tie2-Cre; Gpat4F/F E14.5 hearts. Scale bars, 100 µm. STING (in green), Endomucin (EMCN) stains endocardium (in red) and DAPI stains the nuclei (in blue). I co-localization analysis of STING and endocardium. Data are mean ± s.e.m. Two-tailed unpaired Student’s t-test.
Fig. 5
Fig. 5. Loss of Gpat4 in endothelial cells caused impaired cell survival, ER stress activation, augmented ER-Mito communication and stimulation of cGAS-STING pathway.
A Cell survival was assessed in Ctrl and siGPAT4 groups by Cell Counting Kit-8 (CCK8), n = 6, ***P < 0.0001. B, C Western blotting analysis and quantification of Ctrl and siGPAT4 HUVECs, n = 3. Left-Right, **P = 0.0015, ***P = 0.0008, ***P = 0.0002, ***P = 0.0003, ***P = 0.0003. D qPCR analysis of Ctrl and siGPAT4 HUVECs, n = 3. Left-Right, **P = 0.0026, ***P = 0.0003, *P = 0.0349, **P = 0.0083, *P = 0.0104, **P = 0.0069, *P = 0.0420. E–H Western blotting analysis and quantification of Ctrl and siGPAT4 HUVECs, n = 3. F Left-Right, **P = 0.0044, ***P = 0.0003, ***P = 0.0007, **P = 0.0017, *P = 0.0181. H Left-Right, **P = 0.0070, ***P = 0.0003, **P = 0.0011, **P = 0.0026, ***P = 0.0007. I Proximity/overlapping analysis of mitochondria (Tom20 in red) and ER (Pdi in green). The left down boxed area is higher magnification of the right up boxed area. Note enhanced ER-Mito proximity in the siGPAT4 cells. Scale bars, 10 µm. J ER tracker (in red) and ER-Mito contact (in green) test using a fluorescence reporter to detect the contact. The left down boxed area is higher magnification of the right up boxed area. Note increased ER-Mito contacts in siGPAT4 cells, n = 3. ***P = 0.0003. Scale bars, 10 µm. K qPCR analysis of Ctrl and siGPAT4 HUVECs, n = 3. Left-Right, **P = 0.0043, *P = 0.0166, ***P = 0.0004, ***P = 0.0007, ***P = 0.0008, *P = 0.0151, **P = 0.0046, ***P = 0.0002, ***P = 0.0007, ***P = 0.0008. L, M Western blotting analysis and quantification of Ctrl and siGPAT4 HUVECs, n = 3. Left-Right, *P = 0.0106, **P = 0.0017, **P = 0.0098, **P = 0.0058. N, O Ca2+ probe signals. N Rhod-2 probe to display mitochondrial Ca2+ and Rhod-2 (%) quantification, Rhod-2 in red and MitoTracker in green, n = 3. ***P = 0.0002. Scale bars, 10 µm. O Ca2+ probe to detect Ca2+ in the ER-Mito contacts and positive contacts (%) quantification, OMM-NEMOs for Ca2+ in green and mKate-ER for ER-Mito contacts in red, n = 3. ***P = 0.0003. Scale bars, 10 µm. P Representative curves of the time scan of MAMs Ca2+ after His treatment in Ctrl and siGPAT4 HUVECs, n = 6. Q, R Measurement of mitochondrial membrane potential. Q TMRE probe signal and TMRE MFI quantification of Ctrl and siGPAT4 HUVECs, n = 3. ***P < 0.0001. Scale bars, 10 µm. R JC-1 probe signal and JC-1 monomers MFI quantification of Ctrl and siGPAT4 HUVECs, n = 3. ***P = 0.0003. Scale bars, 10 µm. S MitoSox probe signal and MitoSOX MFI quantification of Ctrl and siGPAT4 HUVECs, n = 3. **P = 0.0010. Scale bars, 10 µm. T, U Mitochondria (Tom20 in red) and dsDNA staining (in green), and co-localization analysis of Ctrl and siGPAT4 HUVECs. Scale bars, 10 µm. V qPCR analysis of cytoplasmic mtDNA of Ctrl and siGPAT4 HUVECs, n = 3. *P = 0.0135. W ISG gene expression of Ctrl and siGPAT4 HUVECs, n = 3. Left-Right, ***P < 0.0001, *P = 0.0132, ***P = 0.0002, ***P = 0.0006, *** P < 0.0001, ***P = 0.0001. Data are mean±s.e.m. Two-tailed unpaired Student’s t-test. n represents three independent cell experiments.
Fig. 6
Fig. 6. Blockage of the cGAS-STING-type I interferon response pathway restored endocardial development in Gpat4-deficient mice.
A, B Gross analysis of embryos and hearts and quantification analysis of WT, Gpat4-/- and Gpat4-/-; Ifnar1-/- E14.5 hearts, n = 3. **P = 0.0046, **P = 0.0020. Scale bars, 500 µm. C, D Histological analysis of trabecular and myocardial layers, and quantification of WT, Gpat4-/- and Gpat4-/-; Ifnar1-/- E14.5 hearts, n = 3. ***P = 0.0006, ***P = 0.0005. Scale bars, 100 µm. E, F qPCR analysis of WT, Gpat4-/- and Gpat4-/-; Ifnar1-/- E14.5 hearts, n = 3. E Left-Right, **P = 0.0045, ***P < 0.0001, ***P = 0.0001, ns P = 0.1205, ***P < 0.0001, ns P = 0.5378, ***P = 0.0002, ns P = 0.6501, ***P = 0.0004, ns P = 0.6854, **P = 0.0011, ns P = 0.9643, **P = 0.0014, ns P = 0.0563, ***P = 0.0003, ns P = 0.6969. F Left-Right, ***P < 0.0001, **P = 0.0019, ***P = 0.0003, **P = 0.0025, **P = 0.0048, *P = 0.0175, **P = 0.0068, **P = 0.0048, **P = 0.0067, *P = 0.0325, ***P = 0.0007, **P = 0.0035, ***P = 0.0001, *P = 0.0113, **P = 0.0067, **P = 0.0063. G, H Western blotting analysis and quantification of WT, Gpat4-/- and Gpat4-/-; Sting-/- E14.5 hearts, n = 3. Left-Right, ***P < 0.0001, ns P = 0.1314, ***P < 0.0001, ***P < 0.0001, ***P = 0.0001, *P = 0.0178, *P = 0.0152, *P = 0.0269. I, J Gross analysis of embryos and hearts and quantification analysis of WT, Gpat4-/- and Gpat4-/-; Sting-/- E14.5 hearts, n = 3. *P = 0.0103, **P = 0.0352. Scale bars, 500 µm. K, L Histological analysis of trabecular and myocardial layers, and quantification of WT, Gpat4-/- and Gpat4-/-; Sting-/- E14.5 hearts, n = 3. *** P < 0.0001, *** P < 0.0001. Scale bars, 100 µm. M, N qPCR analysis of WT, Gpat4-/- and Gpat4-/-; Sting-/- E14.5 hearts, n = 3. M Left-Right, **P = 0.0038, ***P < 0.0001, ***P = 0.0003, ns P = 0.5043, ***P < 0.0001, ns P = 0.8883, ***P = 0.0002, ns P = 0.8013, ***P < 0.0001, ns P = 0.9270, ***P = 0.0006, ns P = 0.1899, ***P = 0.0003, ns P = 0.8434, ***P = 0.0001, ns P = 0.6958. N Left-Right, ***P = 0.0004, *P = 0.0207, ***P < 0.0001, ***P = 0.0004, **P = 0.0037, **P = 0.0084, **P = 0.0026, **P = 0.0050, **P = 0.0034, *P = 0.0208, **P = 0.0018, **P = 0.0095, ***P = 0.0001, *P = 0.0126, *P = 0.0139, *P = 0.0495. O, P Western blotting analysis and quantification of WT, Gpat4-/- and Gpat4-/-; Sting-/- E14.5 hearts, n = 3. Left-Right, ***P < 0.0001, ns P = 0.9582, ***P = 0.0009, ***P < 0.0001, ***P = 0.0002, ***P < 0.0001, ***P = 0.0009, *P = 0.0306. Data are mean ± s.e.m. Two-tailed unpaired Student’s t-test.
Fig. 7
Fig. 7. Working model.
(NORMAL) Under the normal condition, GPAT4 in the endocardial cells binding with EIF2S1/EIF2α to sustain ER homeostasis and fine-tunes ER-Mito communications to safeguard endocardial development. (GPAT4 LOSS) In the absence of GPAT4, ER homeostasis is disrupted and ER stress response is triggered by phosphorylating EIF2S1/EIF2α, leading to enhanced ER-Mito communications, which in turn, causes Mito Ca2+ overloading and mtDNA escape. As a result, cGAS-STING-type I interferon response is elicited, which induces endocardial apoptosis. In the end, this leads to defective trabecular compaction (poor myocardial development) and excessive trabeculation. (Created in BioRender. zhao, t. (2025) https://BioRender.com/m79n092).
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