Perm1 promotes cardiomyocyte mitochondrial biogenesis and protects against hypoxia/reoxygenation-induced damage in mice

Abstract

Normal contractile function of the heart depends on a constant and reliable production of ATP by cardiomyocytes. Dysregulation of cardiac energy metabolism can result in immature heart development and disrupt the ability of the adult myocardium to adapt to stress, potentially leading to heart failure. Further, restoration of abnormal mitochondrial function can have beneficial effects on cardiac dysfunction. Previously, we identified a novel protein termed Perm1 (PGC-1 and estrogen-related receptor (ERR)-induced regulator, muscle 1) that is enriched in skeletal and cardiac-muscle mitochondria and transcriptionally regulated by PGC-1 (peroxisome proliferator-activated receptor gamma coactivator 1) and ERR. The role of Perm1 in the heart is poorly understood and is studied here. We utilized cell culture, mouse models, and human tissue, to study its expression and transcriptional control, as well as its role in transcription of other factors. Critically, we tested Perm1's role in cardiomyocyte mitochondrial function and its ability to protect myocytes from stress-induced damage. Our studies show that Perm1 expression increases throughout mouse cardiogenesis, demonstrate that Perm1 interacts with PGC-1α and enhances activation of PGC-1 and ERR, increases mitochondrial DNA copy number, and augments oxidative capacity in cultured neonatal mouse cardiomyocytes. Moreover, we found that Perm1 reduced cellular damage produced as a result of hypoxia and reoxygenation-induced stress and mitigated cell death of cardiomyocytes. Taken together, our results show that Perm1 promotes mitochondrial biogenesis in mouse cardiomyocytes. Future studies can assess the potential of Perm1 to be used as a novel therapeutic to restore cardiac dysfunction induced by ischemic injury.

Keywords: Perm1; cardiomyocytes; mitochondrial biogenesis; oxidative metabolism.

Figure 1

Expression level of Perm1 protein, mitochondrial DNA copy number, and Oxphos proteins increase during cardiac development. Whole tissue protein and DNA were extracted from cardiac tissues at various embryonic stages or from neonatal pups and adult mice. A and B, Upper panel, western blot analyses with antibodies indicated. Long-exposure images are shown to highlight the differences of Perm1 between embryonic, postnatal, and adult hearts. Lower panel, quantification of Perm1 protein level. (Expression of Perm1 protein was normalized first to GAPDH in each sample and then relative amounts were expressed versus that in P0 heart samples, which was set = 1.) (n = 4). C, the relative mtDNA content was determined as the ratio of mitochondrial (CoxII) to genomic (Dio3) DNA copy numbers and expressed relative to the ratio seen in P0 hearts. Data are the mean ± SD, expressed relative to P0 (n = 4). ∗p < 0.05; ∗∗∗p < 0.001 versus P0. D, representative images of western blots of OxPhos proteins are shown. Quantification is shown in Figure S1. (For all studies, whole heart tubes were sampled at E9.5, with ventricular tissue used at later stages.)

Figure 2

Transcript level of Perm1, PGC-1s, and ERRs increased during cardiac development and maturation. Protein and RNA were extracted from cardiac tissues at a different time points of embryogenesis, from neonatal pups and adult mice. mRNA levels for: (A) Perm1, (B) PGC-1α, (C) PGC-1β, (D) ERRα, (E) ERRγ, and (F) ERRβ were determined by RT-qPCR, normalized by 36B4 levels, and expressed relative to levels of each gene in P0 heart, which was set = 1. Data are the mean ± SD, expressed relative to P0 (n = 4). ∗p < 0.05 versus P0. (Whole heart tubes were sampled at E9.5, with ventricular tissue used at later stages.)

Figure 3

Cardiac Perm1 is enriched in the ventricle and becomes decreased in failing human heart. A, the protein levels of Perm1 in the atrium, ventricle, and GAST muscles were determined by western blot analysis and quantified (right panel). Arrows indicate the two major protein isoforms encoded for by Perm1. (Expression of Perm1 protein was first normalized to GAPDH in each sample and then relative amounts were determined relative to atrial expression, which was set = 1.) Data are the mean ± SD, expressed relative to the atrium (n = 3). ∗p < 0.05 versus ventricle. B, adult mice heart tissue was subjected to subcellular fractionation, followed by western blotting. Antibodies against Perm1 (top panel), cytoplasmic GAPDH, mitochondrial Cox, sarcoplasmic reticulum SERCA2a, and nuclear lamins (shown as four western blots beneath Perm1) were used to assess the purity of the cytoplasmic, mitochondrial, SR (sarcoplasmic reticulum), and nuclear fractions. Representative images from three independent experiments are shown. C, total RNA was extracted from the ventricles of adult mice subjected to Sham or TAC surgery. mRNA levels for Perm1 were determined by RT-qPCR, normalized by 36B4 levels, and expressed relative to normalized expression in Sham-treated mice that were set = 1. (n = 6–8). D, whole tissue protein extracted from the ventricles of Sham or TAC mice was subjected to western blot with antibodies indicated (Perm1 and GAPDH). (n = 4). E, whole tissue protein extracted from the left ventricle of DCM (dilated cardiomyopathy) patients and normal subjects (Control) was evaluated with western blot using the antibodies indicated. (n = 6) Data are the mean ± SD, expressed relative to sham or control. ∗p < 0.05; ∗∗p < 0.01.

Figure 4

Perm1 enhances mitochondrial biogenesis in neonatal mouse cardiomyocytes. Primary cultured neonatal mouse cardiomyocytes (NCM) were infected with adenoviruses expressing LacZ or Perm1. Forty-eight hours later, the cells were harvested. A and B, whole cell protein lysates were subjected to western blot using antibodies as indicated. OxPhos protein levels were quantified with expression level of LacZ (control) infected cells = 1. C, the relative mtDNA content was determined in Perm1-infected cells, relative to that detected in control (LacZ)-infected NCM. (LacZ expression was set =1.) D and E, mRNA levels for the indicated mitochondrial genome-encoded OxPhos genes mt-CoxII and mt-CoxIII and other OxPhos component genes, NDUFS3, SDHb, UQCRC2, COXIV, and ATP5b were determined by RT-qPCR, normalized to 36B4 levels, and expressed relative to levels of each gene in control (LacZ) NCM, set = 1. B–E, data are the mean ± SD, expressed relative to LacZ. ∗p < 0.05 versus LacZ. Data are the mean of six experimental replicates from two representative experiments. F, oxygen consumption rates of NCM were measured in the absence and presence of 1 μM oligomycin (Oligo), 800 nM FCCP, and 1 μM rotenone/antimycin (RAA). Rates are normalized by 20 × 10³ cells. Data are the mean ± SD. ∗p < 0.05 versus LacZ. Data are the mean of nine experimental replicates from two representative experiments. Data are the mean ± SD, expressed relative to LacZ. ∗p < 0.05 versus LacZ.

Figure 5

Perm1 selectively regulates genes essential for oxidative metabolism. Primary cultured NCM were infected with adenoviruses expressing LacZ or Perm1. Forty-eight hours after infection later, the cells were harvested, followed by RNA extraction. A–D, mRNA levels for indicated genes were determined by qRT-PCR, normalized by 36B4 levels, and expressed relative to levels of each gene in LacZ infected cells = 1. Data are the mean ± SD. ∗p < 0.05 versus LacZ. Data are the mean of six experimental replicates from two representative experiments. E and F, whole cell protein lysates were subjected to western blot using antibodies as indicated (E). Sirt3 and Ckmt2 protein levels were quantified relative to expression level of LacZ (control) infected cells = 1 (F). Data are the mean of six experimental replicates from two representative experiments. ∗p < 0.05 versus LacZ.

Figure 6

Perm1 binds PGC-1α and increases ERR-dependent transcriptional activity. A, U2OS cells were transfected with the pERRE-LUC reporter (40 ng) and pcDNA3 (control) or ERR expression plasmids (10 ng), together with pcDNA3 control (−), PGC-1α (5 ng) (+), in the presence of pcDNA3 control (white bars), Perm1 (10 ng) (gray bars), or Perm1 (40 ng) (black bars), plasmids. ∗p < 0.05. Data are the mean ± SD, of eight experimental replicates from two representative experiments. B, interaction of Perm1 and PGC-1α in vitro. HEK293 cells were transfected with pcDNA3-PGC-1α in combination with control or FLAG-Perm1 plasmid (A Flag-tag with a Kozak sequence was inserted into the N-terminus of full-length Perm1). Protein complexes were immunoprecipitated with an anti-FLAG antibody and then immunoblotted with an anti-PGC-1α antibody or anti-Perm1 antibody. C, interaction of Perm1 and PGC-1α in vivo. Whole ventricular tissue protein lysates from adult C57Bl6 mice were subjected to immunoprecipitation with anti-PERM1 antibody and then then immunoblotted with an anti-PGC-1α antibody or anti-Perm1 antibody. Representative blots are from two independent experiments. IP, immunoprecipitation.

Figure 7

Perm1 enhances recruitment of PGC-1α to target gene promoter. A, C2C12 myotubes were infected with LacZ (white bars), PGC-1α (Flag-tagged) (black bars), Perm1 (striped bars), or both PGC-1α (Flag-tagged) and Perm1 (gray bars). ChIP assays were performed using anti-FLAG antibody, anti-PERM1 antibodies, or IgG (control). B, ChIPs were performed with adult mouse ventricular tissues using anti-PERM1, anti-ERRα, or anti-PGC-1α antibodies. The abundance of the Sirt3 ERRE, the Ckmt2 ERRE, and a negative control genomic region in the ChIPs were quantified by qPCR, normalized to the input signal, and expressed relative to the levels of each region in the control IgG sample. In A and B, data are the mean ± SD. A, ∗p < 0.05 versus LacZ. #p < 0.05 versus FLAG-PGC-1α. The data are the mean of eight experimental replicates from two representative experiments. B, ∗p < 0.05 versus IgG. The data are the mean of six different heart samples.

Figure 8

Perm1 protects cardiomyocytes from cellular damage induced by hypoxia-reoxygenation. Primary cultured NCM were infected adenoviruses (Ad) expressing LacZ or Perm1. Forty-eight hours later, the cells were subjected to hypoxia and reoxygenation (H/R). A, LDH released into the media was analyzed and Perm1-infected cell levels were detected, were quantified relative to that detected in Ad-LacZ infected cells. Data are the mean ± SD. ∗p < 0.05 versus Normoxia. #p < 0.05 versus LacZ. Data are the mean of eight experimental replicates from two representative experiments. B, cardiomyocyte apoptosis was determined by staining with TUNEL and DAPI, and representative images of TUENL-positive (green) and DAPI (blue) are shown. Scale bar: 50 μm. C, quantitative analysis of TUNEL-positive nuclei is shown. Data are the mean ± SD. ∗p < 0.05 versus Normoxia. #p < 0.05 and ##p < 0.01 versus LacZ. Data are the mean of eight experimental replicates from two representative experiments.