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. 2011 Oct 5;478(7367):114-8.
doi: 10.1038/nature10490.

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function

Chris McDermott-Roe  1 , Junmei Ye, Rizwan Ahmed, Xi-Ming Sun, Anna Serafín, James Ware, Leonardo Bottolo, Phil Muckett, Xavier Cañas, Jisheng Zhang, Glenn C Rowe, Rachel Buchan, Han Lu, Adam Braithwaite, Massimiliano Mancini, David Hauton, Ramon Martí, Elena García-Arumí, Norbert Hubner, Howard Jacob, Tadao Serikawa, Vaclav Zidek, Frantisek Papousek, Frantisek Kolar, Maria Cardona, Marisol Ruiz-Meana, David García-Dorado, Joan X Comella, Leanne E Felkin, Paul J R Barton, Zoltan Arany, Michal Pravenec, Enrico Petretto, Daniel Sanchis, Stuart A Cook
Affiliations

Endonuclease G is a novel determinant of cardiac hypertrophy and mitochondrial function

Chris McDermott-Roe et al. Nature. .

Abstract

Left ventricular mass (LVM) is a highly heritable trait and an independent risk factor for all-cause mortality. So far, genome-wide association studies have not identified the genetic factors that underlie LVM variation, and the regulatory mechanisms for blood-pressure-independent cardiac hypertrophy remain poorly understood. Unbiased systems genetics approaches in the rat now provide a powerful complementary tool to genome-wide association studies, and we applied integrative genomics to dissect a highly replicated, blood-pressure-independent LVM locus on rat chromosome 3p. Here we identified endonuclease G (Endog), which previously was implicated in apoptosis but not hypertrophy, as the gene at the locus, and we found a loss-of-function mutation in Endog that is associated with increased LVM and impaired cardiac function. Inhibition of Endog in cultured cardiomyocytes resulted in an increase in cell size and hypertrophic biomarkers in the absence of pro-hypertrophic stimulation. Genome-wide network analysis unexpectedly implicated ENDOG in fundamental mitochondrial processes that are unrelated to apoptosis. We showed direct regulation of ENDOG by ERR-α and PGC1α (which are master regulators of mitochondrial and cardiac function), interaction of ENDOG with the mitochondrial genome and ENDOG-mediated regulation of mitochondrial mass. At baseline, the Endog-deleted mouse heart had depleted mitochondria, mitochondrial dysfunction and elevated levels of reactive oxygen species, which were associated with enlarged and steatotic cardiomyocytes. Our study has further established the link between mitochondrial dysfunction, reactive oxygen species and heart disease and has uncovered a role for Endog in maladaptive cardiac hypertrophy.

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Figures

Figure 1
Figure 1. Positional cloning of Endog as the gene underlying the rat chromosome 3p cardiac mass quantitative trait locus (QTL)
a, Mapping of heart weight (HW) and left ventricular mass (LVM) corrected for body weight (BW) to chromosome 3p in the Brown Norway (BN) x Spontaneously Hypertensive (SHR) F2 population. The telomeric limits of the congenic strains (SHR.BN-(3L) and SHR.BN-(3S)) and the previously mapped cardiac mass (CM) QTLs, are shown; x-axis, physical position in Mbp. b, HW indexed to BW in the SHR (n=4) and the SHR.BN-(3L) congenic strains (n=5). c, Relative cardiomyocyte cross-sectional area in SHR and SHR.BN-(3L) congenic strains. d, In vivo telemetric systolic- and diastolic-blood pressure (SBP and DBP) measurements in the SHR (red circles) and SHR.BN-(3L) (black circles) (n=8 per genotype). e, Haplotype analysis of the refined QTL region. SNPs are depicted with reference to WKY/NCrl alleles (grey, identical; white, dissimilar) with numbers (1-5) denoting the polymorphic regions between strains with either high or low HW. QPCR of Endog mRNA expression (f) and immunoblot of Endog protein expression (g) in strains with low or high CM at the chromosome 3p locus. h, Nuclease activity in BN and SHR heart extracts over a range of cardiac protein extract amounts (grey wedge) (Supplementary Methods). i, Reversal of nuclease activity in cardiac lysates by a drosophila-derived inhibitor of Endog (range 1500nM-1.5nM, grey wedge). j, Association of the Endog indel with loss of Endog protein expression and diminished nuclease activity in the recombinant inbred (RI) strains. Upper, middle and lower panels display the DNA indel, protein expression and nuclease activity, respectively. k, Linkage mapping of nuclease activity in RI strains using a quantitative fluorescence-based assay (Supplementary Methods). All data are represented as mean+s.e.m. *, P<0.05, **, P<0.01, ***, P<0.001.
Figure 2
Figure 2. Endog regulates cardiac hypertrophy
a, Immunoblot of Endog expression in mouse and rat tissues (Endog: ~30 kDa). b, Immunoblot of Endog expression in myocyte and non-myocyte populations isolated from neonatal rat heart. c, Cardiomyocyte size (n≥100 cells, n=3 independent experiments) treated with shRNA against Endog (shEndog) or control shRNA (shControl) in the presence or absence of the hypertrophic stimulant phenylephrine (PE, 100μM, 24 h). d, Expression of the hypertrophic biomarker Anf in shEndog and shControl treated cells. e, Cardiomyocyte size (Supplementary Fig. 5) in Endog-/- and wildtype (WT) mice at baseline and following angiotensin II-induced cardiac hypertrophy. f, LVM/tibial length in Endog-/- and WT mice at baseline and following AngII stimulation. Data are represented as mean+s.e.m. *, P<0.05, **, P<0.01, ***, P<0.001.
Figure 3
Figure 3. ENDOG is co-expressed with a mitochondrial-specific gene network and regulated by Pgc1α and ERRα
a, Genes (8,490 from 210 datasets) are clustered and plotted based on the dissimilarity metric between their expression profiles (Supplementary Methods). From top to bottom: low-hanging branches in the dendrogram represent groups of genes (modules) that have a high similarity metric. Modules are shown beneath the dendrogram and are colour coded. The arrow indicates the module (also boxed) containing ENDOG. In the heat-map of the correlations between expression profiles, high and low similarities are coloured yellow and red, respectively. b, Weighted gene co-expression network analysis (WGCNA) for the module containing ENDOG, providing functional annotation by cellular localization by Gene Ontology classification (Supplementary Tables 2 and 3). Nodes represent genes and edges represent significant co-expression between genes. The node size is proportional to the relative degree of interconnectivity of each gene within the module. c, QPCR analysis of Endog expression in cultured cardiomyocytes following infection with adenovirus (ad) expressing GFP (ad.GFP) or Pgc1α (ad.Pgc1α). d, Immunoblot of Endog expression in ad.Pgc1α-infected cardiomyocytes (top panel), skeletal muscle of wild-type (WT) mice and transgenic mice expressing Pgc1α under the control of muscle creatine kinase (MCK-Pgc1α) (middle panel), and in hearts of WT and cardiac-specific Pgc1α deleted mice (Pgc1αΔC/ΔC) (bottom panel). e, Endog promoter activity in HEK293 cells infected with ad.Pgc1α and and/or ad.Errα. f, ERRα chromatin immunoprecipitation (ChIP) and PCR of two regions of the ENDOG promoter. Red arrows denote primers and ERRE specifies the location of a consensus ERR response element (1304 bases upstream). The experiment was repeated three times with similar results and PCR products quantified by QPCR.
Figure 4
Figure 4. Endog regulates mitochondrial function and cardiac lipid metabolism
a, Transmission electron micrographs and oil red O stained micrographs (high resolution, Supplementary Fig. 7) of left ventricular sections from WT and Endog-/- mice. b, Quantification of the number of mitochondrial-associated droplets in WT and Endog-/- mice. c, Quantification of cardiac triglyceride (TAG), phospholipid and cholesterol content in WT and Endog-/- mice (n=5). d, Ratio of mitochondrial DNA (mtDNA) to genomic DNA (gDNA) in hearts of WT and Endog-/- mice. e, Quantification of mitochondrial protein content in WT and Endog-/- mice (n=5). f, State 3 and state 4 oxygen consumption in the presence of complex I or complex II substrates in isolated cardiac mitochondria from WT (n=6) and Endog-/- (n=5) mice. g, Relative fluorescence-based measurement of ROS production by mitochondria isolated from WT (n=6) and Endog-/- (n=5) mice. h-k, Representative flow cytometry analysis of mitochondrial mass in HEK293 and H9C2 cells over-expressing ENDOG or Endog, respectively (n=4). h, Stable expression of ENDOG in HEK293 cells (HEK293-ENDOG). i, Flow cytometry analysis of HEK293 and HEK293-ENDOG cells stained with mitotracker. j, Adenovirus (ad)-mediated expression of GFP and Endog in myocytes and flow cytometry analysis of ad.Endog infected cells (Q2) and uninfected control cells (Q1). k, Number of events plotted against mitochondrial mass in ad.Endog infected (Q2) and control (Q1) H9C2 cells. l, QPCR of mtDNA-protein complexes following ChIP of mitochondrial chromatin using anti-ENDOG antibody or IgG. All data are represented as mean+s.e.m. *, P<0.05, **, P<0.01, ***, P<0.001.
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