Myocardial lipin1 protects the heart against ischemic injury by preserving lipid homeostasis

Figures

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  Figure 1. Single-cell analysis identifies cardiac LPIN1. (A) Uniform Manifold Approximation and Projection (UMAP) plot of single-cell transcriptomes of cardiomyocytes and noncardiomyocytes from 3 patients with heart failure (HF) and 3 control (CTRL) patients. The heart tissue cells were divided into 6 clusters which are color-coded by cell type. Cardiac LPIN1 is mainly expressed in cardiomyocytes. (B) Feature plots of expression distribution for cardiomyocytes (clusters 0 and 2). The expression level of LPIN1 is color-coded. (C) Pathway analysis indicated significant changes in gene expression related to lipid and fatty acid metabolism pathways between HF and CTRL cardiomyocytes. (D) The expression level of cardiac LPIN1 was decreased in patients with HF compared with the CTRL.

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  Figure 2. Lpin1/lipin1 expression levels after MI surgery. (A) Lpin1 mRNA expression was measured by quantitative PCR and exhibited no significant difference in sham group, but it showed diminished levels 1 day and 3 days after MI surgery in the border and remote zones of WT mice compared with pre-MI levels. n = 5 each (Sham), n = 3 each (MI). Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001 by ordinary 1-way ANOVA. (B) Lipin1 protein expression, assessed through Western blot 1 week after MI, showed reduced abundance in the border and remote zones. n = 5 each. Data are presented as the mean ± SEM. *P < 0.05, ***P < 0.001 by ordinary 1-way ANOVA. (C) The expression level of Lpin1 was measured in both heart and skeletal muscle of Lpin1 transgenic mice. Lpin1 mRNA expression was only changed in hearts, but there were no significant difference in skeletal muscle. n = 3 each. Data are presented as the mean ± SEM. *P < 0.05 by unpaired 2-tailed t test. (D) Lipin1 protein expression, assessed through Western blot using heart or skeletal muscle of Lpin1 transgenic mice, displayed no significant difference in skeletal muscle but increased or decreased significantly in the cKO/cOE…

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  Figure 3. Cardiac function dynamics after MI surgery. (A) Four weeks after MI, cKO mice showed exacerbated remodeling and a significantly elevated HW/BW ratio compared with their littermates. n = 12 [Ctl(cKO), sham], n = 11 (cKO, sham), n = 18 [Ctl(cKO), MI], n = 21 (cKO, MI). Data are presented as the mean ± SEM. **P < 0.01 by ordinary 1-way ANOVA. cOE mice displayed milder remodeling relative to their littermate controls, maintaining a comparable heart weight/body weight (HW/BW) ratio. n = 9 [Ctl(cOE), sham], n = 6 (cOE, sham), n = 13 [Ctl(cOE), MI], n = 14 (cOE, MI). Data are presented as the mean ± SEM by ordinary 1-way ANOVA. Scale bar: 1mm. (B) B-mode echocardiography images at diastole and systole over 4 weeks after MI displayed endocardial surfaces, revealing left ventricular end-diastolic diameter (LVDd) and fractional area change (FAC) in cKO/cOE and their littermate controls. cKO mice showed a decreased FAC with increased LVDd after MI than their littermates. n = 12 [Ctl(cKO), sham], n = 11 (cKO, sham), n = 16–18 [Ctl(cKO), MI], n = 20-21 (cKO, MI). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 by 2-way ANOVA. cOE mice showed an…

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  Figure 4. Accumulation of lipid droplets in infarcted hearts. (A) One week after MI, hearts were Bodipy-stained to calculate lipid droplet numbers using ImageJ. LDs were stained by Bodipy in green, the nucleus was stained by DAPI in blue, and membranes were stained by WGA in red. While no disparities were noted in the sham group, cKO mice displayed fewer lipid droplets in the remote zone compared with control mice, whereas cOE mice exhibited more lipid droplets in the border and remote zones. n = 3 each. Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 by ordinary 1-way ANOVA. Scale bars: 10 μm. (B) The content in infarct hearts of TAG was observed, which showed that the storage of TAG was reduced after MI in cKO mice compared with their littermate controls. On the contrary, there is an increased tendency of TAG in cOE mice than the controls. n = 12 [Ctl(cKO)], n = 4 (cKO), n = 5 [Ctl(cOE)], n = 4 (cOE). Data are presented as the mean ± SEM. *P < 0.05 by unpaired 2-tailed t test. (C) Reduced FFA storage was observed in cKO mice. Conversely, cOE mice exhibited increased FFA expression after MI. n = 9 [Ctl(cKO)], n = 4 (cKO), n = 7 [Ctl(cOE)], n = 3 (cOE). Data are presented as…

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  Figure 5. Expression levels of fatty acid oxidation–related genes. Expression levels of Ppargc1a, Acaa2, Cpt1b, and Pnpla2 were evaluated both in cKO and cOE hearts. The expression of Ppargc1a and Acaa2 was diminished in cKO mice hearts, whereas the expression of Ppargc1a, Acaa2, Cpt1b, and Pnpla2 was elevated in cOE mice hearts. n = 8 [Ctl(cKO)], n = 8-9 (cKO), n = 5 each [Ctl(cOE)], n = 6–7 (cOE). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 by unpaired 2-tailed t test.

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  Figure 6. Inflammation after MI surgery. (A) One week after MI, hearts were CD45 stained, and ImageJ was employed to calculate positive cell numbers. The border zone of cKO mice exhibited more positive cells than the control mice, while cOE mice displayed fewer. n = 6 each [Ctl(cKO)], n = 5 each (cKO), n = 6 each [Ctl(cOE)], n = 7 each (cOE). Data are presented as the mean ± SEM. **P < 0.01, ***P < 0.001 by ordinary 1-way ANOVA. Scale bars: 200 μm. (B) One week after MI, proinflammatory and antiinflammatory factor expression levels were measured. In cKO hearts, the expression of proinflammatory factors including Il1a, Il6, and Tnf was increased, whereas the expression of antiinflammatory factor Il10 was decreased. In contrast, the expression of proinflammatory factors was decreased, whereas the expression of antiinflammatory factor was increased in cOE hearts. n = 4–8 [Ctl(cKO)], n = 5–6 (cKO), n = 6–9 [Ctl(cOE)], n = 5–7 (cOE). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 by unpaired 2-tailed t test.

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  Figure 7. Oxidative stress after MI surgery. (A) Examination of DHE fluorescence in different zones 1 week after MI or sham surgery showed no significant difference in sham group. In contrast, cKO hearts showed increased DHE fluorescence following MI, whereas cOE hearts showed a significant decrease compared with controls. n = 3 each [Ctl(cKO)], n = 3 each (cKO), n = 3 [Ctl(cOE), Sham], n = 3 (cOE, Sham), n = 4 each [Ctl(cOE), MI], n = 4 each (cOE, MI). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by ordinary 1-way ANOVA. Scale bars: 200 μm. (B) H2O2 levels were increased in cKO hearts, while decreased in cOE hearts. n = 4 [Ctl(cKO))] n = 4 (cKO); n = 7 [Ctl(cOE)], n = 3 (cOE). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 by unpaired 2-tailed t test.

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  Figure 8. NAC treatment in cKO mice after MI. (A) Echocardiographic analysis of cardiac function at 4 weeks after MI. NAC-treated cKO mice showed significantly improved systolic function, with decreased LVDd and increased FAC compared with untreated cKO mice. n = 3 (cKO), n = 4 (NAC-treated cKO). Data are presented as the mean ± SEM. *P < 0.05 by unpaired 2-tailed t test.Scale bar: 1mm. (B) EVG and H&E staining of heart sections at 4 weeks after MI. NAC treatment reduced fibrotic area in cKO hearts. Fibrotic area to LV free wall was measured. n = 3 (cKO), n = 4 (NAC-treated cKO). Data are presented as the mean ± SEM. *P < 0.05 by unpaired 2-tailed t test. (C) DHE staining showed reduced ROS levels in NAC-treated cKO mice at infarct, border, and remote zones. n = 3 (cKO), n = 3 (NAC-treated cKO). Data are presented as the mean ± SEM. *P < 0.05, **P < 0.01 by ordinary 1-way ANOVA. Scale bar: 200 μm.