Recruitment of RNA Polymerase II to Metabolic Gene Promoters Is Inhibited in the Failing Heart Possibly Through PGC-1α (Peroxisome Proliferator-Activated Receptor-γ Coactivator-1α) Dysregulation
Proper dynamics of RNA polymerase II, such as promoter recruitment and elongation, are essential for transcription. PGC-1α (peroxisome proliferator-activated receptor [PPAR]-γ coactivator-1α), also termed PPARGC1a, is a transcriptional coactivator that stimulates energy metabolism, and PGC-1α target genes are downregulated in the failing heart. However, whether the dysregulation of polymerase II dynamics occurs in PGC-1α target genes in heart failure has not been defined.
Methods and Results:
Chromatin immunoprecipitation-sequencing revealed that reduced promoter occupancy was a major form of polymerase II dysregulation on PGC-1α target metabolic gene promoters in the pressure-overload–induced heart failure model. PGC-1α-cKO (cardiac-specific PGC-1α knockout) mice showed phenotypic similarity to the pressure-overload–induced heart failure model in wild-type mice, such as contractile dysfunction and downregulation of PGC-1α target genes, even under basal conditions. However, the protein levels of PGC-1α were neither changed in the pressure-overload model nor in human failing hearts. Chromatin immunoprecipitation assays revealed that the promoter occupancy of polymerase II and PGC-1α was consistently reduced both in the pressure-overload model and PGC-1α-cKO mice. In vitro DNA binding assays using an endogenous PGC-1α target gene promoter sequence confirmed that PGC-1α recruits polymerase II to the promoter.
These results suggest that PGC-1α promotes the recruitment of polymerase II to the PGC-1α target gene promoters. Downregulation of PGC-1α target genes in the failing heart is attributed, in part, to a reduction of the PGC-1α occupancy and the polymerase II recruitment to the promoters, which might be a novel mechanism of metabolic perturbations in the failing heart.
WHAT IS NEW?
Downregulation of genes involved in fatty acid oxidation and mitochondrial ATP production is a hallmark of heart failure, which is thought to promote the pathology because of insufficient energy production. PGC-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) is a transcriptional coactivator that induces metabolic genes. RNA polymerase II is an enzyme that transcribes genes, and therefore, proper polymerase II dynamics is essential for transcription.
Here, we show that (1) PGC-1α plays a role in the promoter recruitment of polymerase II and (2) PGC-1α dissociates from the promoters in the failing heart, thereby inhibiting polymerase II recruitment.
WHAT ARE THE CLINICAL IMPLICATIONS?
Previous studies aim to clarify the mechanism responsible for the metabolic disturbance largely focused on expressional changes of responsible transcriptional activators such as PGC-1α. However, downregulation of PGC-1α is not uniformly observed in the failing heart. In addition, forced expression of PGC-1α does not show any therapeutic effect.
Here, we show a novel mechanism responsible for the metabolic disturbance, which partly explains the downregulation of PGC-1α target genes independent of PGC-1α downregulation.
This study suggests that promoter recruitment of PGC-1α and polymerase II is a novel therapeutic target to normalize PGC-1α target gene expression in the failing heart.
The heart is an organ that demands high energy production and consumption to maintain blood flow throughout the body. To satisfy the high energy demand, cardiomyocytes harbor many mitochondria and utilize fatty acids as a primary source of energy. PGC-1α (peroxisome proliferator-activated receptor [PPAR]-γ coactivator-1α) is a transcriptional coactivator that broadly induces metabolic genes through coactivation of transcription factors such as nuclear receptors, including PPARs (peroxisome proliferator-activated receptors) and ERRs (estrogen-related receptors). Downregulation of PGC-1α target metabolic genes is a hallmark of heart failure, which may contribute to insufficient energy production and further progression of pathology.1 Pressure-overload (PO)–induced failing heart phenotypes and downregulation of metabolic genes are exacerbated in global PGC-1α knockout mice.2,3 Notably, despite the fact that significant metabolic actions of PGC-1α are well recognized not only in the heart but also other organs, such as adipose, liver, and skeletal muscle, the role of PGC-1α in the PO-induced heart failure model has not been investigated with cardiac-specific PGC-1α knockout mice. Therefore, whether PGC-1α in cardiomyocytes plays a role in cardiac function and metabolism has not been proven. To address this issue, the pathological characterization of cardiac-specific PGC-1α knockout under PO conditions is important.
Although PO-induced failing heart phenotypes are exacerbated in global PGC-1α knockout mice,2 this does not necessarily mean that PGC-1α is inactivated during heart failure development. It has been thought that downregulation of PGC-1α is a mechanism responsible for downregulation of the target genes in the failing heart. However, PGC-1α is downregulated neither in human heart failure patients nor in the PO-induced heart failure model in mice.3,4 Moreover, increased PGC-1α expression with the transgene in the failing heart does not rescue cardiac dysfunction and metabolic perturbations.5,6 Thus, it remains unclear whether downregulation of metabolic genes in the failing heart occurs through the dysregulation of PGC-1α.
RNA polymerase II is a multiple protein complex that transcribes a gene to synthesize mRNA. Polymerase II is recruited to promoter regions and moves to the gene body for transcription. Proper polymerase II dynamics, including promoter recruitment, promoter clearance, elongation, termination, and recycling, are required for transcription. Impairment of any of the processes could result in transcriptional suppression. However, it is largely unknown whether the downregulation of PGC-1α target genes in the failing heart is attributed to altered polymerase II dynamics.
PGC-1α binds to specific subunits of Mediator complexes and of general transcription factors, including TFIID and TFIIH.7–9 Mediators and general transcription factors are essential for proper polymerase II dynamics. Particularly, Mediators and TFIID play a crucial role in promoter recruitment of polymerase II, also termed preinitiation complex formation, whereas TFIIH mediates promoter clearance of polymerase II. However, the extent to which PGC-1α is required for proper polymerase II dynamics and whether PGC-1α regulates polymerase II recruitment or clearance has not been defined.
In this study, we used cardiac-specific PGC-1α knockout mice to address the hypotheses that (1) PGC-1α has a regulatory role in polymerase II dynamics and (2) polymerase II is dysregulated in PO-induced heart failure model because of PGC-1α dysregulation. We demonstrate that polymerase II occupancy in metabolic gene promoters was reduced in the failing heart, concurrent with reduced occupancy of PGC-1α at those promoters. Cardiac-specific deletion of PGC-1α was sufficient to reduce polymerase II promoter occupancy. Conversely, overexpression of PGC-1α promoted the recruitment of polymerase II to the promoters of metabolic genes in cultured cardiomyocytes and in vitro reconstitution systems. Taken altogether, these results suggest that reduced promoter occupancy of PGC-1α is a mechanism responsible for reduced polymerase II recruitment leading to metabolic disturbances in the failing heart.
Data, Material, and Code Disclosure
PGC-1α-cKO (cardiac-specific PGC-1α knockout) mice can be distributed on material transfer agreement from original sources of PGC-1αflox/flox and αMHC-Cre mice. On request to the corresponding author, plasmid and virus vectors can be distributed unless otherwise any conflicts noticed.
The conditional knockout line of PGC-1αflox/flox mice with a C57BL/6 background was obtained from the Jackson Laboratory.10 The PGC-1αflox/flox mice crossed with cardiac-specific Cre transgenic mice (αMHC-Cre). All procedures involving animals were performed in accordance with protocols approved by Rutgers Biomedical and Health Sciences.
Statistical comparisons were made using Kaplan-Meier log-rank test and a nonparametric Mann-Whitney U test with GraphPad Prism 8.1. P<0.05 was defined as statistically significant. All error bars represent SEM.
Recruitment of Polymerase II to Metabolic Gene Promoters Is Reduced in the Failing Heart
Genome-wide polymerase II localization provides insight into the mechanism responsible for gene expression changes. PO induced by transverse aortic constriction is an animal model of heart failure in which surgical constriction of the aorta induces PO in the left ventricle (LV), leading to downregulation of metabolic genes and cardiac dysfunction in mice. To clarify how polymerase II is regulated in metabolic genes under PO conditions, polymerase II occupancy was examined with chromatin immunoprecipitation-sequencing (ChIP-seq). We focused on PPAR and ERR target genes because their regulation is through coactivator PGC-1α. PPAR target genes are enriched in fatty acid metabolism, including uptake, transport, and oxidation (Figure 1A), whereas ERR target genes are enriched in the mitochondrial Krebs cycle and electron transport chain (Figure 1B). The ChIP-seq analysis revealed that polymerase II occupancy was reduced in 26 out of the 29 gene promoters under PO conditions (blue arrows). In addition, an increase in polymerase II occupancy flanking the transcription termination site was observed in 3 genes: Fabp3, Cycs, and Idh3b indicating impairment of transcriptional termination. The occupancy of polymerase II on Fabp3 and Cycs also increased in the gene body, suggesting impairment of polymerase II release from the transcription termination site. The specific impairment of polymerase II promoter clearance was not observed in any of these genes, which was presumably evidenced by either no change or increased polymerase II occupancy in the promoter but reduced polymerase II occupancy in the gene body. These results suggest that inhibition of promoter recruitment is a major form of polymerase II dysregulation in the PGC-1α target metabolic genes under PO conditions.
Cardiac-Specific PGC-1α Deletion Induces Failing Heart Phenotypes
To test the role of PGC-1α in polymerase II dynamics in metabolic gene promoters, we used PGC-1α-cKO mice generated with PGC-1αflox/flox and αMHC-Cre mice. To investigate the extent to which PGC-1α in cardiomyocytes maintains cardiac function and metabolic gene expression, we first characterized cardiac phenotypes and metabolic gene expression in the PGC-1α-cKO mice. As shown in Figure 2A, the hearts of PGC-1α-cKO mice were enlarged compared with control hearts. Echocardiographic measurements demonstrated that cardiac systolic dysfunction and increased LV diameters (LV end-diastolic dimension [mm]: control: 3.27; PGC-1α-cKO: 4.25, P<0.0001, LV end-systolic dimension [mm]: control; 2.13, PGC-1α-cKO; 3.18, P<0.0001), and a trend of reduced wall thicknesses were also observed in PGC-1α-cKO mice, indicating an LV dilation (Figure 2B through 2D). Gene expressional analyses showed decreased expression level of Atp2a2 (P=0.0006), and increased expression levels of Nppa (P=0.0104) and Nppb (P=0.0011), indicating cardiac hypertrophy and failure in PGC-1α-cKO mice (Figure 2E). Histological analyses showed increases in apoptotic cell death (P=0.0159), fibrosis (P=0.0079), and cellular hypertrophy (P=0.0079) in the PGC-1α-cKO mice under basal conditions (Figure 2F through 2H). To investigate the extent to which loss of PGC-1α negatively regulates cardiac function in the adult phase, PGC-1α was reexpressed in 6- to 11-week-old PGC-1α-cKO mice with an adeno-associated virus vector (AAV). Cardiac contractile dysfunction and impaired PGC-1α target gene expression observed in PGC-1α-cKO mice were normalized after 3 weeks of AAV transduction (fractional shortening %: control: 23.9, PGC-1α; 38.2, P=0.0043) (Figure 2I and 2J). In addition, cardiac dilation characterized by reduced wall thickness and increased LV diameters tended to be normalized by AAV-PGC-1α (LV end-diastolic dimension [mm]; control: 4.41, AAV-PGC-1α: 3.82; P<0.017, LV end-systolic dimension [mm]: control: 3.37; AAV-PGC-1α: 2.37; P=0.0087; Table 1). These results suggest that PGC-1α ablation in the adult heart resulted in mild heart failure under basal conditions.
|Cre BL (6)||Cre PO (6)||Wild BL (17)||Wild PO (17)||cKO BL (9)||cKO PO (7)||cKO-AAV-Con (5)||cKO-AAV-PGC-1α (6)|
|DSEP WT, mm||0.71±0.12||0.91±0.06||0.83±0.13||0.93±0.14||0.80±0.15||0.77±0.17‡||0.77±0.04||0.81±0.20|
|DP WT, mm||0.77±0.22||0.77±0.10||0.88±0.17||0.89±0.13||0.68±0.09¶||0.64±0.12*||0.73±0.12||0.85±0.11§|
|SSEP WT, mm||0.99±0.19||1.06±0.16||1.14±0.28||1.17±0.26||1.00±0.23||0.83±0.17*||0.92±0.07||1.18±0.16‖|
|SP WT, mm||0.90±0.29||0.86±0.13||1.01±0.17||1.10±0.16||0.89±0.13||0.72±0.26*||0.95±0.21||1.18±0.20§|
PGC-1α-cKO Mice Are Susceptible to PO
Under the PO conditions, PGC-1α-cKO mice showed a high mortality rate (median survival: 29 days, P=0.0002), compared with the control mice (Figure 3A). Therefore, gross and echocardiographic measurements were performed after 2 weeks of PO (Figure 3B). As shown in Figure 3C, PO induced cardiac contractile dysfunction, evidenced by reduced fractional shortening in wild-type and αMHC-Cre mice.However, PGC-1α-cKO mice showed contractile dysfunction even without PO (fractional shortening [%]; control: 35.1; PGC-1α-cKO: 25.1; P=0.0002), which was further exacerbated under PO conditions (fractional shortening [%]; control: 24.0; PGC-1α-cKO: 8.5; P<0.0001). Echocardiographic data are summarized in Table 1. PO-induced lung congestion, an index of heart failure measured as lung weight/tibia length ratio (mg/mm) was increased in PGC-1α-cKO mice (control: 13.43; PGC-1α-cKO: 22.65; P=0.0016; Figure 3D). PO-induced cardiac hypertrophy measured as heart weight/tibia length ratio (mg/mm) was not significantly changed in PGC-1α-cKO mice (control: 10.08; PGC-1α-cKO: 10.39; P=0.88; Figure 3E). These results suggest that cardiac-specific PGC-1α deletion exacerbates PO-induced failing heart phenotypes. Results of organ weight measurements are summarized in Table 2. The metabolic genes involved in fatty acid metabolism (Figure 3F), Krebs cycle (Figure 3G), and mitochondrial electron transport (Figure 3H) were downregulated in the wild-type mice in response to PO and were downregulated in the PGC-1α-cKO mice even without PO. Fatty acid utilization activity was impaired in the PGC-1α-cKO mice under both basal and PO conditions (Figure 3I). These results suggest that PGC-1α in cardiomyocytes plays an important role in cardiac energetics in both basal and PO conditions.
|Wild BL (26)||Wild PO (18)||cKO BL (23)||cKO PO (12)|
PGC-1α Is Not Significantly Downregulated Under PO Conditions
PGC-1α-cKO mice even in the absence of PO showed phenotypic similarity to the PO model in the wild-type mice, such as impaired contractile function and downregulation of PGC-1α target genes. The phenotypic similarity may be caused by downregulation of PGC-1α in the PO model and PGC-1α-cKO mice. To test this, we examined the expression levels of all 3 splice variants of PGC-1α mRNA that encode a protein and excluded the variants subjected to non-sense-mediated decay. As shown in Figure 4A and 4B, an authentic PGC-1α (PGC-1α 203) tended to decrease in PO conditions, but it did not attain a statistical significance (P=0.505). In addition, the other splice variants including PGC-1α 205 and PGC-1α 202 tended to increase in PO conditions. Thus, PGC-1α mRNAs do not significantly decrease in PO conditions. Notably, we found that a region of PGC-1α mRNA corresponding to the floxed exons (exons 3 to 5) was significantly downregulated in PGC-1α-cKO mice (basal: P=0.0007 and PO: P=0.007), but the other regions were not. Thus, artificial mRNA of PGC-1α lacking floxed exons is likely expressed in PGC-1α-cKO mice. To test whether PGC-1α protein is downregulated under PO conditions, Western blot analyses were performed. Because the molecular weight of PGC-1α varies from 80 to 120 kDa among the literature, we first determined the PGC-1α-specific signal in the heart lysate. While we tested several antibodies, 3 of them detected PGC-1α exogenously expressed in HEK293 cells, which was ≈120 kDa (Figure 4C). The signal at 120 kDa was detected in the heart lysate, which was partly but significantly reduced in the PGC-1α-cKO mice, suggesting that the signal at 120 kDa represents PGC-1α (Figure 4D). Then, we investigated whether the PGC-1α protein at 120 kDa is downregulated under PO conditions. As shown in Figure 4E, the protein expression of PGC-1α, detected by all 3 antibodies, was not significantly changed in the PO model. Similarly, we did not observe significant changes in the protein expression of PGC-1α in human failing hearts (relative band intensity: recipients: 1; donors: 1.06, P=0.62; Figure 4F). Taken together, our results suggest that downregulation of PGC-1α is not necessary for the downregulation of PGC-1α target genes in the failing heart.
Reduced Occupancy of PGC-1α in Metabolic Gene Promoters in the PO Model
It is generally thought that recruitment of PGC-1α to target gene promoters is essential for PGC-1α-induced transcriptional activation. Because PGC-1α was not significantly downregulated in the failing heart in mice and humans, we hypothesized that PGC-1α dissociates from the target gene promoters in the failing heart. To test this, we performed ChIP assays on wild-type and PGC-1α-cKO mice under PO conditions. We chose PPAR and ERR target gene promoters containing their binding sequences (PPRE [PPAR response element] and ERRE [ERR response element]) nearby the transcription start site, such as Mcad, Idh3a, Sdha, and Atp5k (Figure 5A). As shown in Figure 5B, PGC-1α occupancy in these promoters was significantly reduced in the PO conditions (relative promoter occupancy of PGC-1α in PGC-1α-cKO: control mice defined as 1; Mcad 0.37, P=0.037; Idh3a 0.38, P=0.032; Sdha 0.36, P=0.029; and Atp5k 0.49, P=0.038). As expected, PGC-1α occupancy was significantly reduced in PGC-1α-cKO mice (Figure 5C). These results suggest that PGC-1α dissociates from target gene promoters in the failing heart. To investigate whether reduced promoter occupancy of PGC-1α is accompanied by the reduced polymerase II occupancy, ChIP assays were performed with anti-polymerase II antibody. Polymerase II occupancy in these promoters was also reduced in the PO model (relative promoter occupancy of polymerase II in transverse aortic constriction mice: control mice defined as 1; Mcad 0.48, P=0.0095; Idh3a 0.38, P=0.0095; Sdha 0.36, P=0.0022; and Atp5k 0.54, P=0.0022) and in PGC-1α-cKO mice (relative promoter occupancy of polymerase II in PGC-1α-cKO mice: control mice defined as 1; Mcad 0.65, P=0.035; Idh3a 0.43, P=0.029; Sdha 0.57, P=0.038; and Atp5k 0.62, P=0.0303; Figure 5D). Taken altogether, these results suggest that PGC-1α dissociates from target gene promoters in the failing heart, which is accompanied by reduced polymerase II recruitment.
PGC-1α Promotes the Recruitment of Polymerase II to PGC-1α Target Gene Promoters
Based on our observations, we hypothesized that PGC-1α promotes polymerase II recruitment, and therefore, the reduced PGC-1α promoter occupancy leads to a reduction in the polymerase II recruitment in the failing heart. To test if PGC-1α promotes polymerase II recruitment, we exogenously expressed PGC-1α in primary cultured cardiomyocytes. As shown in Figure 6A, overexpression of PGC-1α in cardiomyocytes promoted the recruitment of polymerase II to PGC-1α target gene promoters, such as Mcad (P=0.029) and Idh3a (P=0.029). Consistent with this result, PGC-1α upregulated Mcad (P=0.0022) and Idh3a (P=0.0022; Figure 6B). These results suggest that PGC-1α-induced polymerase II recruitment is associated with the target gene induction. To verify this result, we performed in vitro DNA binding assays using biotin-labeled DNA comprising 380 bp of the Idh3a promoter containing ERREs and transcription start site (Figure 6C). The promoter was incubated with cell extract as a source of polymerase II and general transcriptional machineries. Polymerase II recruitment to the Idh3a promoter was partly reduced by PGC-1α knockdown in primary cultured cardiomyocytes (relative promoter binding of polymerase II: control: 1; PGC-1α knockdown: 0.48, P=0.029; Figure 6D). Conversely, the polymerase II recruitment was enhanced by overexpression of PGC-1α in HEK293 cells (relative promoter binding of polymerase II: control: 1; PGC-1α overexpression: 2.3, P=0.0079; Figure 6E). However, Flag-PGC-1α (Flag-tagged PGC-1α) did not interact with polymerase II in cardiomyocytes, whereas a direct binding of Flag-PGC-1α to ERRα was detected (Figure 6F). Thus, PGC-1α does not directly bind to polymerase II. To investigate whether PGC-1αs ability to bind to promoters declines in heart failure development, we performed in vitro DNA binding assays with Flag-PGC-1α purified from cardiomyocytes treated with phenylephrine that mimics a pathological consequence of heart failure in cultured cardiomyocytes. PGC-1α from phenylephrine treated cells showed a lesser binding ability to the promoter (relative promoter binding of PGC-1α: control cells: 1; phenylephrine-treated cells: 0.53, P=0.0286). These results suggest that PGC-1α promotes polymerase II recruitment and that the reduced PGC-1α occupancy of its target gene promoters results in a reduction of polymerase II recruitment in the failing heart.
Polymerase II Regulation During Heart Failure Development
The failing heart is known as an energy-starved heart, in association with global downregulation of genes involved in fatty acid metabolism and mitochondrial ATP production.11 Polymerase II plays a central role in transcription and, therefore, gene expression. Importantly, however, whether and how polymerase II is dysregulated and is involved in the downregulation of metabolic genes in the failing heart has not been defined. Although impairment of any process of polymerase II dynamics could result in transcriptional suppression, we here show that inhibited promoter recruitment is a major form of polymerase II dysregulation in metabolic genes. Notably, however, impairment of polymerase II transcriptional termination was also observed in several PGC-1α target genes. Taken together, although inhibited promoter recruitment is the major form of polymerase II dysregulation, the other regulation may concurrently take place. Further studies are needed to clarify how polymerase II dynamics are regulated in the failing heart.
Role of PGC-1α in Downregulation of PGC-1α Target Genes in Heart Failure
Downregulation of PGC-1α was originally thought to be a mechanism responsible for downregulation of PGC-1α target genes in the PO-induced heart failure model because PGC-1α mRNA is downregulated.2 However, diverse outcomes of PGC-1α mRNA expressional changes have been reported, which include upregulation and no change.4,12,13 We here observed that the mRNA was not significantly downregulated after 2 weeks of transverse aortic constriction in control mice. Because all control mice used for testing PGC-1α mRNA levels were PGC-1αflox/flox mice, we should be aware of the possibility in which the flox insertion may disrupt a critical element that mediates PO-induced downregulation. About PGC-1α protein levels, it was reported that PGC-1α protein was significantly downregulated in the PO model,4 although the same group later reported no changes in PGC-1α protein in the PO model.14 No changes in PGC-1α protein were also reported by another group.6 Our data also showed that there were no changes in PGC-1α protein in the PO model and in human failing hearts. Thus, it is unlikely that downregulation of PGC-1α is essentially required for downregulation of PGC-1α target genes in the failing heart. In this study, we showed that PGC-1α dissociated from the promoters in the failing heart, which could be because of a posttranslational modification or conformational changes of PGC-1α. In addition, it partially explains the downregulation of PGC-1α target genes without changing PGC-1α expression. Although the mechanism by which PGC-1α dissociates from the promoter is different between the PGC-1α-cKO and PO model, PGC-1α-cKO may mimic pathological consequences triggered by promoter dissociation of PGC-1α in the PO model. This could account for the phenotypic similarity between the PGC-1α-cKO and PO model. The mechanism by which PGC-1α dissociates from the promoters should be further investigated. In addition, the ChIP-seq analysis of PGC-1α in the PO model would be an intriguing future study. Unfortunately, the H300 anti-PGC-1α antibody qualified for ChIP assay is no longer commercially available, thus we could not conduct the ChIP-seq analysis in this study.
Immunoblot Analysis of PGC-1α
While the molecular weight of PGC-1α varies from 80 to 120 kDa among the literature, we showed that the signal at 120 kDa corresponded to a full-length PGC-1α. The signal at 120 kDa was partly reduced in PGC-1α-cKO mice. The persistent signal in PGC-1α-cKO mice is possibly because of the following 3 reasons. First, Cre-induced deletion is not fully achieved. Second, there is PGC-1α protein derived from noncardiomyocytes such as fibroblasts. Indeed, persistent expression of PGC1α mRNA corresponding to even in the floxed region was detected in PGC-1α-cKO hearts (Figure 4A and 4B). Third, there may be a nonspecific signal that overlaps with the specific signal. Notably, the signal at 120 kDa was a minor band detected by all 3 PGC-1α antibodies, whereas they detected major bands at a different molecular weight. This characteristic outcome may account for the varied PGC-1α molecular weight among the literature. Namely, investigators may interpret that a major signal is a PGC-1α specific band. Besides the band at 120 kDa, the others may be splice variants or PGC-1α homologs. The signal above the 120 kDa detected by AB3243 anti-PGC-1α antibody may be PGC-1β or PPRC1 (PGC-1 related protein 1) because of the homology with the antigen peptide and their molecular weight. In addition, several bands below the 120 kDa detected by all 3 antibodies may be a PGC-1α 205 splice variant which encodes ≈100 amino acids shorter than the full length. However, the relative expression levels of PGC-1α 205 mRNA were ≈10 000× lower than the full length (Figure 4B). Therefore, these signals may represent a posttranslational modification such as cleavage rather than the PGC-1α 205 splice variant. Importantly, none of these signals below 120 kDa were significantly downregulated in the PO model (data not shown). Thus, even if these signals are derived from PGC-1α, they are not significantly downregulated in the PO model.
Mechanism by Which PGC-1α Stimulates Transcription
Although PGC-1α is a well-investigated transcriptional coactivator, the mechanism by which PGC-1α stimulates transcription remains elusive. PGC-1α recruits histone acetyltransferases and chromatin remodeling complexes to the target gene promoters, thereby stimulating transcription.15,16 However, the extent to which PGC-1α is essential for their recruitment has not been investigated with a loss of PGC-1α model. We here show that PGC-1α is essential for polymerase II recruitment in a subset of genes, although significant binding of PGC-1α to polymerase II was not observed. Thus, it is likely that PGC-1α indirectly interacts with polymerase II. PGC-1α may promote polymerase II recruitment through recruitment of histone acetyltransferases and chromatin remodeling complexes. Whether recruitment of histone acetyltransferases and chromatin remodeling complexes is inhibited in the loss of PGC-1α model would be an important investigation. Notably, we show that PGC-1α promotes polymerase II recruitment with in vitro DNA binding assays. Because we used naked DNA without histones, PGC-1α recruits polymerase II independently of histone acetyltransferases and chromatin remodeling complexes in the in vitro DNA binding system. PGC-1α may regulate polymerase II dynamics through the general transcription factors and mediators. The mechanism by which PGC-1α promotes polymerase II recruitment should be further investigated.
Discrepancy of Cardiac Phenotypes in PGC-1α Knockout Mouse Lines
Previous studies investigated the role of PGC-1α in cardiac function in 2 independent lines of global PGC-1α knockout mice. One group used a global PGC-1α KO line to show cardiac systolic dysfunction under basal conditions, whereas the other group which used the other line, did not show any significant baseline phenotypes.2 Interestingly, the PGC-1α knockout line originally showing cardiac systolic dysfunction did not show any baseline phenotypes, when these mice were characterized by the other investigators at another institute.3 It is also reported that cardiac-specific PGC-1α knockout mice do not show any significant baseline phenotypes.17 Notably, the cardiac-specific PGC-1α mouse line previously reported is essentially the same as the PGC-1α-cKO mice used by this study because we used the identical PGC-1αflox/flox and αMHC-Cre lines. Contrary to the previous report, we observed that the PGC-1α-cKO mouse line showed a certain degree of failing heart phenotypes under basal conditions. Despite the phenotypic variation in the PGC-1α knockout lines under basal conditions, the deteriorated cardiac dysfunction under PO conditions has been commonly observed in the 2 independent global PGC-1α knockout mice.2,3 We here verified the deteriorated cardiac dysfunction under PO conditions in the PGC-1α-cKO mice. Taken altogether, loss of PGC-1α exacerbates PO-induced failing heart phenotypes regardless global or cardiac-specific deletion, while whether or not a loss of PGC-1α spontaneously develops heart failure under basal conditions may depend on genetic background or housing conditions.
In summary, impaired promoter recruitment is a major form of polymerase II dysregulation in PGC-1α target genes in the failing heart, which is associated with downregulation of the target genes and metabolic disturbances. Additionally, we observe PGC-1α positively regulates the promoter recruitment of polymerase II. Last, we find PGC-1α dissociates from the target gene promoters in the failing heart, which could be a mechanism responsible for the impaired promoter recruitment of polymerase II.
We thank Christopher D. Brady for critical reading of the article.
Sources of Funding
This work was supported in part by the American Heart Association (AHA) Scientist Developmental Grant 12SDG11890014 (Dr Oka), Grant in Aid 17GRNT33440031 (Dr Oka), New Jersey Health Foundation research grants (PC56-16 and PC80-17) (Dr Oka), an AHA student scholarship (Dr Schesing), the Glorney-Raisbeck Medical Student Grant (Dr Schesing), Ministry of Science and Technology Taiwan NSC102-2628-B075-002-MY3 (Dr Hsu), Foundation Leducq Transatlantic Networks 15CBD04 (Dr Sadoshima), and US Public Health Service Grant HL067724, HL091469, HL138720, HL112330, and AG23039 (Dr Sadoshima).
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