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Drp1 regulates transcription of ribosomal protein genes in embryonic hearts - PubMed

  • ️Sat Jan 01 2022

. 2022 Feb 15;135(4):jcs258956.

doi: 10.1242/jcs.258956. Epub 2022 Feb 21.

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Drp1 regulates transcription of ribosomal protein genes in embryonic hearts

Qiancong Zhao et al. J Cell Sci. 2022.

Abstract

Mitochondrial dysfunction causes severe congenital cardiac abnormalities and prenatal/neonatal lethality. The lack of sufficient knowledge regarding how mitochondrial abnormalities affect cardiogenesis poses a major barrier for the development of clinical applications that target mitochondrial deficiency-induced inborn cardiomyopathies. Mitochondrial morphology, which is regulated by fission and fusion, plays a key role in determining mitochondrial activity. Dnm1l encodes a dynamin-related GTPase, Drp1, which is required for mitochondrial fission. To investigate the role of Drp1 in cardiogenesis during the embryonic metabolic shift period, we specifically inactivated Dnm1l in second heart field-derived structures. Mutant cardiomyocytes in the right ventricle (RV) displayed severe defects in mitochondrial morphology, ultrastructure and activity. These defects caused increased cell death, decreased cell survival, disorganized cardiomyocytes and embryonic lethality. By characterizing this model, we reveal an AMPK-SIRT7-GABPB axis that relays the reduced cellular energy level to decrease transcription of ribosomal protein genes in cardiomyocytes. We therefore provide the first genetic evidence in mouse that Drp1 is essential for RV development. Our research provides further mechanistic insight into how mitochondrial dysfunction causes pathological molecular and cellular alterations during cardiogenesis.

Keywords: Drp1; Heart development; RP gene transcription.

© 2022. Published by The Company of Biologists Ltd.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.

Deletion of Dnm1l by Mef2c-AHF-Cre leads to severe defects in the right ventricle. Mef2c-AHF-Cre/Dnm1lloxp/+ male mice were crossed with Dnm1lloxp/loxp female mice to obtain mutant (Mut; Mef2c-AHF-Cre/Dnm1lloxp/loxp) and control (Ctrl; Dnm1lloxp/+ or Dnm1lloxp/loxp) embryos at different stages. (A) Sagittal sections of E10.5 embryos were immunostained with antibodies against Drp1 (red) and MLC2A (green, a cardiomyocyte marker). The outflow tract (OFT), atrial ventricular canal (AVC) and common atrium (CA) regions are shown. Expression of Drp1 was efficiently inactivated in the OFT and RV of mutant hearts but not in other areas. (B) Percentage of live mutant embryos at different stages. Data were acquired from at least four litters at each developmental stage. **P<0.01, χ2 test. Embryonic lethality began to occur between E15.5 and E16.5. For each stage, at least 5 litters of embryos were examined. (C) Whole-mount control and mutant hearts at E14.5. The arrowhead indicates the reduced size of the mutant RV. (D) HE staining of heart sections at different stages. Reduced size of mutant RVs began to be observed from E12.5. At E15.5, mutant hearts also displayed noncompaction cardiomyopathy. (E) Quantification of the size of RVs and LVs in mutant and control hearts at E12.5 (left, n=4) and E15.5 (right, n=5). The size of RV and LV was measured using NIH ImageJ. The area of each structure was calculated as the average of five consecutive sections with the largest area. ***P<0.001, ****P<0.0001, two-way ANOVA followed by Tukey's multiple comparisons test; n.s., not significant. Data are shown as mean±s.e.m. Scale bars: 100 μm.

Fig. 2.
Fig. 2.

Deletion of Dnm1l leads to abnormal cell proliferation and survival. (A) Sagittal sections of E12.5 hearts from mutant (Mut; Mef2c-AHF-Cre/Dnm1lloxp/loxp) and control (Ctrl; Dnm1lloxp/+ or Dnm1lloxp/loxp) embryos were co-stained with an anti-phospho-histone 3 (pH3) antibody (green) and an anti-myosin heavy chain (MHC) antibody (red). Total nuclei were visualized with DAPI staining (blue). Examples of both LVs and RVs are shown. (B) Quantification results of pH3 staining (n=4). (C) Sagittal sections of E12.5 control and mutant hearts were co-stained with an anti-cleaved caspase 3 antibody (green) and an anti-MHC antibody (red). (D) Quantification results of caspase 3 staining (n=4). ***P<0.001, two-way ANOVA followed by Tukey's multiple comparisons test; n.s., not significant. Data are shown as mean±s.e.m. In A and C, insets show examples of positive pH3 signals (high magnifications of the boxed areas). Scale bars: 50 μm.

Fig. 3.
Fig. 3.

Deletion of Dnm1l leads to abnormal cardiomyocyte orientation. (A) Sagittal sections of E12.5 hearts from mutant (Mut; Mef2c-AHF-Cre/Dnm1lloxp/loxp) and control (Ctrl; Dnm1lloxp/+ or Dnm1lloxp/loxp) embryos were co-stained with an anti-N-cadherin (N-CAD) antibody (red) and an anti-MLC2A antibody (green). Four pairs of control and mutant embryos were examined. (B) Expression of N-cadherin in control and mutant RVs at E12.5 was examined by western blotting. Tubulin was used as a loading control. (C) E12.5 heart sections were stained with WGA to label the cell membrane (green) and an anti-MHC antibody (red) to label cardiomyocytes. Nuclei were visualized with DAPI staining. We measured the orientation of cardiomyocytes in the compact zone of control and mutant LVs and RVs following published procedures (Miao et al., 2019). A total of 50 cardiomyocytes for each structure of each genotype were measured. The white lines show the longitudinal axis of a cell. (D) The number of cardiomyocytes with different orientations relative to the myocardial wall (parallel, non-classified and perpendicular). The distribution of cardiomyocytes of mutant RVs in the three categories was significantly different from that of the control RVs (P<0.001, Fisher's exact test, ‘stats’ package in R 4.0.3), whereas no significant difference was observed between control and mutant LVs. n=50. Scale bars: 50 μm.

Fig. 4.
Fig. 4.

Deletion of Dnm1l impairs mitochondrial morphology and ultrastructure. (A) Cardiomyocytes were isolated from RVs of mutant (Mut; Mef2c-AHF-Cre/Dnm1lloxp/loxp) and control (Ctrl; Dnm1lloxp/+ or Dnm1lloxp/loxp) embryos at E10.5 and were cultured for 48 h. They were then co-stained with antibodies against TOM20 (green) and Drp1 (red). Nuclei were stained with DAPI (blue). Samples were examined using a confocal microscope. Mutant cells (without Drp1 expression) displayed tubular mitochondria. (B) Sagittal sections of E10.5 hearts were immunostained with antibodies against TOM20 (green) and Drp1 (red), and samples were examined using a confocal microscope. In many mutant cells, mitochondria were clustered to one side of the nucleus. Arrowheads indicate examples of such cells. (C) Quantification of the results shown in B. ****P<0.0001, two-tailed, unpaired Student's t-test. Three embryos of each genotype were quantified, and for each embryo, 100 cells were examined. (D) Sections of control and mutant embryonic hearts (E13.5) were examined by TEM. The red boxed areas show examples of mitochondria in control and mutant RVs. Three pairs of control and mutant hearts were examined. (E) Comparison of average mitochondrial length between control and mutant samples (n=250 for both groups). Data are shown as mean±s.e.m. **P<0.001, two-tailed, unpaired Student's t-test. (F) Oxygen consumption of E14.5 control and mutant RVs were examined using Oroboros. BL, base line. **P<0.01, two-tailed, unpaired Student's t-test; Benjamini–Hochberg adjusted P<0.05. n=8. (G) RCR was calculated as Vmax/V0. *P<0.05, two-tailed, unpaired Student's t-test. n=8. (H) The ATP level in E13.5 LVs and RVs was measured. Data were normalized against sample weight. The level of control samples was set at 1.0. Data are shown as mean±s.e.m. *P<0.05, two-tailed, unpaired Student's t-test, n=6. ns, not significant. Scale bars: 50 μm (A,B); 1 μm (C).

Fig. 5.
Fig. 5.

Expression of a group of RP genes in embryonic cardiomyocytes is impaired by Dnm1l deletion. (A) scRNA-Seq was performed using cells isolated from RVs of control and mutant embryos at E13.5. Three pairs of control and mutant hearts were pooled. This chart shows UMAP projection of various cell types, including cardiomyocytes (CM1, CM2), epicardial cells (EpC1, EpC2), red blood cells (RBC), endocardial cells (EnC), macrophages (MP) and endothelial cells (EnT). (B) Dot plot showing average expression of representative marker genes of different cell types across all clusters. (C) The two CM clusters were combined and 122 genes were identified that were differentially expressed between control and mutant cardiomyocytes by at least 25% with a Benjamini–Hochberg adjusted P<0.05 (red dots). The RP genes are indicated. The x-axis shows fold alteration and the y-axis shows the adjusted P-value. The dashed lines show the threshold (Benjamini–Hochberg adjusted P<0.05, expression altered by at least 25%). (D) These 122 genes underwent GO term enrichment analysis using Metascape. The top 20 terms are shown.

Fig. 6.
Fig. 6.

De novo protein synthesis is impaired by deletion of Dnm1l. (A) Total RNA was isolated from RVs of mutant (Mut; Mef2c-AHF-Cre/Dnm1lloxp/loxp) and control (Ctrl; Dnm1lloxp/+ or Dnm1lloxp/loxp) hearts at E13.5 followed by qRT-PCR analysis to examine expression of multiple RP genes. Data were normalized against Hprt. The level of control samples was set at 1.0. Consistent with the scRNA-Seq data in Fig. 5, expression of multiple RP genes was significantly reduced by Dnm1l deletion. *P<0.05; **P<0.01; ***P<0.001, two-tailed, unpaired Student's t-test. For all genes examined, Benjamini–Hochberg adjusted P<0.05. n=4. (B) Protein was isolated from the RVs of control and mutant samples at E13.5. Western blot analysis was then performed using the indicated antibodies. Tubulin was used as a loading control. Quantification data are shown in

Fig. S4

. (C) De novo protein synthesis was examined in cultured cardiomyocytes isolated from E13.5 hearts. Nuclei were visualized with DAPI staining (blue). The red signal represents newly synthesized proteins. Control cells treated with cycloheximide (cyclo; a protein synthesis blocker) were included as a negative control. Scale bar: 10 μm. (D) Quantification of protein synthesis. We measured the signal intensity of >100 cells for each culture. To obtain intensity level per cell, the total intensity was divided by the number of cells. Control values were set at 1.0. **P<0.01, one-way ANOVA followed by Tukey's multiple comparisons test. n=5.

Fig. 7.
Fig. 7.

Deletion of Dnm1l leads to reduced protein synthesis through the AMPK-SIRT7-GABPB axis. (A) Western analysis was performed on protein lysates isolated from RVs of mutant (Mut; Mef2c-AHF-Cre/Dnm1lloxp/loxp) and control (Ctrl; Dnm1lloxp/+ or Dnm1lloxp/loxp) embryos (E13.5) using the indicated antibodies. Tubulin was used as a loading control. Quantification data are shown in

Fig. S5A

. (B) GABPB1 was immunoprecipitated from E13.5 RVs using an anti-GABPB1 antibody followed by western blotting using various antibodies. Association of GABPA with GABPB1 was reduced in mutant samples. Immunoprecipitated samples were re-probed with an anti-phospho-serine antibody or an anti-acetyl-lysine antibody. The level of acetylated GABPB1 (Ac-GABPB1) was increased in mutant samples. (C) Western blotting was performed using E13.5 RV lysates. The level of SIRT7 was reduced in mutant samples. The total AMPK level remained the same, but active AMPK was increased by Dnm1l deletion. Quantification data are shown in

Fig. S5B

. (D) E13.5 hearts were isolated, cultured, and treated with DMSO (control) or Compound C (Comp.C) for 6 h. SIRT7 was immunoprecipitated from the lysates of RVs followed by western analysis using an anti-phospho-serine antibody. Compound C treatment reduced the level of phosopho-SIRT7 (P-SIRT7) to the control level. Western analysis of total lysates showed that Compound C treatment restored the total SIRT7 level in mutant samples to the normal level. Quantification data are shown in

Fig. S5C

. For A-D, three pairs of control and mutant embryonic hearts were examined. (E,F) Protein synthesis was measured in cultured cardiomyocytes treated with DMSO or Compound C. Quantification results (F) show that Compound C treatment (Mut-C) partially rescued the protein synthesis defect in mutant samples. Scale bar: 50 μm. *P<0.05; ***P<0.001, one-way ANOVA followed by Tukey's multiple comparisons test. n=6.

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References

    1. Baes, M., Gressens, P., Baumgart, E., Carmeliet, P., Casteels, M., Fransen, M., Evrard, P., Fahimi, D., Declercq, P. E., Collen, D.et al. (1997). A mouse model for Zellweger syndrome. Nat. Genet. 17, 49-57. 10.1038/ng0997-49 - DOI - PubMed
    1. Baker, M. J., Frazier, A. E., Gulbis, J. M. and Ryan, M. T. (2007). Mitochondrial protein-import machinery: correlating structure with function. Trends Cell Biol. 17, 456-464. 10.1016/j.tcb.2007.07.010 - DOI - PubMed
    1. Becht, E., McInnes, L., Healy, J., Dutertre, C.-A., Kwok, I. W. H., Ng, L. G., Ginhoux, F. and Newell, E. W. (2018). Dimensionality reduction for visualizing single-cell data using UMAP. Nat. Biotechnol. 37, 38-44. 10.1038/nbt.4314 - DOI - PubMed
    1. Berardo, A., Musumeci, O. and Toscano, A. (2011). Cardiological manifestations of mitochondrial respiratory chain disorders. Acta Myol. 30, 9-15. - PMC - PubMed
    1. Beutner, G., Eliseev, R. A. and Porter, G. A.Jr. (2014). Initiation of electron transport chain activity in the embryonic heart coincides with the activation of mitochondrial complex 1 and the formation of supercomplexes. PLoS ONE 9, e113330. 10.1371/journal.pone.0113330 - DOI - PMC - PubMed

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