BACKGROUND AND AIMS:LMNA-related dilated cardiomyopathy (LMNA-DCM) is a progressive genetic disorder characterized by conduction disease, malignant arrhythmias, myocardial fibrosis, and heart failure. Although LMNA mutations have traditionally been associated with cardiomyocyte-intrinsic defects, the mechanisms driving fibrotic remodelling remain incompletely understood. METHODS:Spatial transcriptomics and integrated single-nuclei multiomics were performed on explanted human LMNA-DCM hearts to define endothelial transcriptional and epigenomic states associated with fibrosis. Patient-specific induced pluripotent stem cell-derived endothelial cells, engineered cardiac organoids, and the LMNAH222P/H222P mouse model were used to investigate RUNX1-mediated endothelial-to-mesenchymal transition (EndoMT). Genetic and pharmacological RUNX1 inhibition strategies were evaluated in vitro and in vivo. RESULTS:Endothelial populations exhibiting EndoMT-associated transcriptional and epigenomic signatures were identified in human LMNA-DCM hearts. LMNA induced pluripotent stem cell-derived endothelial cells demonstrated endothelial dysfunction, mesenchymal gene activation, and epigenetic activation of RUNX1 following loss of LMNA-mediated repression. Genetic RUNX1 deletion restored endothelial identity, reversed EndoMT-associated transcriptional programmes, and normalized chromatin accessibility at endothelial regulatory loci. In multicellular cardiac organoids, endothelial RUNX1 activation impaired endothelial-cardiomyocyte signalling and cardiomyocyte contractile function, whereas endothelial-specific RUNX1 deletion restored endothelial and myocardial function. Pharmacological RUNX1 inhibition with Ro24-7429 similarly improved endothelial and cardiomyocyte function in vitro and reduced myocardial fibrosis while preserving cardiac function in LMNAH222P/H222P mice, including after disease onset. CONCLUSIONS:RUNX1-driven EndoMT represents a central mechanism linking LMNA mutations to fibrotic remodelling in LMNA cardiomyopathy. These findings support endothelial transcriptional reprogramming and RUNX1 signalling as potential therapeutic targets in fibrotic cardiomyopathy.

AIMS:Heart failure is marked by suppression of fatty acid oxidation (FAO) and mitochondrial ATP production gene expression. While transcriptional downregulation via PGC-1 and PPARα/ERRα has been well documented, the involvement of the general transcriptional machinery remains insufficiently understood. Distinct from its salutary role in many cardiac conditions, endogenous Sirt1 negatively affects cardiac function during pressure overload (PO). This study investigates how Sirt1 modulates preinitiation complex (PIC) assembly and RNA polymerase II (Pol II) recruitment during pathological PO. METHODS AND RESULTS:Cardiac-specific Sirt1 knock-out (Sirt1 cKO) mice were subjected to PO. Interaction between Sirt1 and Sub1, a protein recruiting GTFs to the gene promoter, was assessed with co-immunoprecipitation, protein pull-down and molecular docking. Pol II recruitment was evaluated with ChIP-sequencing and -qPCR analyses. The binding affinity of Sirt1 and GTF to the Sub1 GTF binding domain was assessed by immunoprecipitation and protein pull-down assays. Unbiased ChIP-sequencing and -qPCR analyses showed that Pol II binding to metabolic gene promoters was downregulated during PO, which was reversed in Sirt1 cKO mice. PO upregulated Sirt1 in the heart and increased its binding to Sub1, which interacts PPARα and ERRα, but not NF-kB. The Sirt1 binding to Sub1 competitively displaced the interaction between Sub1 and GTFs, thereby inhibiting the PIC formation at the Sub1-PPARα/ERRα complex. CONCLUSIONS:Sirt1 impairs metabolic gene transcription during PO by competitively inhibiting Sub1-mediated recruitment of GTFs and PIC formation. This repression of general transcriptional machinery contributes to the metabolic disturbance and may represent a maladaptive component of the heart failure phenotype.

Summary Our prior work demonstrated that lowering dietary branched-chain amino acids (BCAAs) improves cardiac outcomes during pressure overload-induced stress. Here, we identify isoleucine restriction (IleR) as the key driver of this effect. Dietary isoleucine restriction induces hypophagia and weight loss, recapitulating the effects of caloric restriction (CR). Although it does not prevent the initial development of left ventricular hypertrophy, it halts its progression and the decline in ejection fraction compared with controls. This is associated with preservation of electron transport chain (ETC) gene expression, cristae structure, NAD + /NADH levels, and mitochondrial respiratory capacity in cardiomyocytes, which is recapitulated by CR. Mechanistically, both IleR and CR diets increase Foxo3 expression, thereby blocking the decline in expression of its target ETC and mitochondrial genome-encoded genes. Consequently, this improves mitochondrial respiratory capacity and reduces cardiac fibrosis. We conclude that restricting dietary isoleucine improves cardiac health by increasing Foxo3 expression and mitochondrial function via a cell-autonomous mechanism and by reducing caloric intake.

Klf9 is a cardiac-enriched transcription factor of the Krüppel-like factor (Klf) family. Klf9 levels decrease during cardiac hypertrophy; however, no studies have examined its transcriptional targets or role in the progression of hypertrophy. Here, we report genome-wide differential Klf9 occupancy during cardiac hypertrophy, with a predominant enrichment at the metabolic gene promoters. Further, using conditional Klf9 knock-in mice subjected to pressure overload for 1 or 2 weeks, we show that restoring Klf9 expression initially inhibits hypertrophy but later leads to early-onset heart failure. We conclude that a decrease in Klf9 is required for metabolic adaptations that support the development of compensatory hypertrophy.

Adaptation of gene expression is the earliest response during work overload to maintain cardiac homeostasis and function. We reported a novel function of Krüppel-like factor (Klf) 9 in mediating metabolic adaptations in response to Dexamethasone in cardiomyocytes. Klf9 expression decreases in hearts undergoing cardiac hypertrophy and failure, suggesting that differential regulation of Klf9 could be contributing to the pathogenesis. Here, we present the characterization of a conditional (αMHC-Cre) Klf9 knock-in (Klf9KI) mouse. Constitutive expression of exogenous Klf9 results in spontaneous cardiac dysfunction and the onset of failure by 8 weeks of age, and an early mortality by 12-14 weeks, suggesting that Klf9 dysregulation is sufficient for maladaptation. Transcriptome data from 1-week-old Klf9KI hearts show dysregulation of genes involved in lipid, carbohydrate, and glutathione metabolism, and transcripts of transport, transcription, and motor proteins. At 4 weeks and 8 weeks, we observe a differential expression of genes involved in innate immunity and extracellular matrix, along with metabolic and contractile signaling. These data correlate with the untargeted metabolome analysis, showing dysregulation in metabolites of lipid and fatty acids, glutathione, purine and pyrimidines, and sucrose metabolic pathways. Functional data in Klf9KI hearts show a decrease in mitochondrial ETC complex activity and ATP production, and an increase in ROS. Structural mitochondrial defects include distortion and sparse cristae in Klf9KI vs. Wt-Cre hearts. In conclusion, we show that Klf9 is critical for metabolic adaptations in postnatal heart development. Constitutive expression of Klf9 results in metabolic maladaptation, which precipitates dysfunction, early onset of heart failure, and death.

We reported that Krüppel-like factor 9 (Klf9) regulates metabolic adaptations in neonatal cardiomyocytes. In this study, we characterize conditional Klf9 knock-in (Klf9KI) mice with 3-4-fold increase in Klf9 expression when crossed with αMHC-Cre. Our data shows that Klf9KI mice develop spontaneous hypertrophy by 4wks, followed by cardiac dysfunction and failure by 8wks (EF=24 ±1%; FS=11±0.5%), and early mortality by 12-14 wks. Transcriptome analysis in 1wk, 4wk and 8wks old hearts shows that the number of differentially expressed genes increases over time, with significant changes observed in 108 genes at 1 wk, and further dysregulation of 949 genes and 1247 genes at 4wks and 8wks, respectively. Consistent with Klf9's predicted role as a transcriptional repressor, 64.81% of differentially expressed genes are downregulated at 1wk. Functional annotation reveals a dysregulation of mostly metabolic genes including those involved in lipid metabolism (Bdh1), glutathione pathways (Gstk1), and carbohydrate metabolism (Fbp2). By 4wks and 8wks, along with dysregulation of metabolic pathways, we observe upregulation of genes involved in apoptosis (Bcl2), innate immunity (Tlr genes), and extracellular matrix remodeling (Col8a1). Untargeted metabolomics at 4wks identifies significant alterations in 125 tissue metabolites [VIP >1.5, ILog2FCI ≥0.5, p<0.05]). Pathway enrichment analysis shows Arachidonic acid, Lipid and Fatty acids metabolism and biosynthesis, Sucrose and Glutathione metabolism as top enriched metabolite sets. These findings correlate with dysregulated genes identified in 4wks old Klf9KI mice (RNA-seq). To assess mitochondrial function, we measured mitochondrial electron transport chain complex activities in Klf9KI and Wt-Cre mice at 2 and 4wks. Basal mitochondrial respiration decreases at both time points, while complex II (succinate) and IV (ascorbate +TMPD) activities decline significantly by 4wks. A 3.5-fold increase in ROS production is observed in these hearts, consistent with a decline in mitochondrial function. In conclusion, constitutive expression of Klf9 disrupts transcriptional and metabolic homeostasis, which precipitates spontaneous progressive hypertrophy, dysfunction, and early onset of heart failure.

Krüppel-like factors (Klfs) regulate cellular processes, including metabolism, differentiation, and proliferation, which have implications in development and diseases of the heart. We reported Klf9’s role in transcriptional control of metabolic genes and the GR-Klf9 axis in metabolic adaptation in response to Dexamethasone in neonatal myocytes. Here, we examine the role of Klf9 in adult hearts undergoing pressure overload-induced hypertrophy. Our RNA polymerase II (pol II) ChIPSeq data from mouse hearts undergoing transverse aortic constriction (TAC) induced hypertrophy show reduced pol II occupancy across the Klf9 gene compared to sham hearts, suggesting reduced transcription. Consistently, we observe reduced Klf9 transcript and protein levels in TAC hearts compared to sham, indicating decreased Klf9 expression. Klf9-ChIPSeq identified 5238 genes with Klf9 binding, with 2250 with MaxTag ≥50. 51% of the genes showed decreased Klf9 genomic binding in TAC vs Sham hearts. KEGG pathway showed metabolic pathways on top of the list with 131 genes, including those involved in carbon metabolism, insulin signaling, fatty acid degradation, glycolysis/gluconeogenesis, amino acid, and nucleotide metabolism. Conversely, 22% of genes showed increased Klf9 binding, mostly those involved in innate immunity, apoptosis, mitochondrial organization, and autophagy. Further, we subjected inducible conditional Klf9 knock-in (Klf9KI) mice to sham/TAC operations for 2 weeks, followed by functional and molecular analysis. Contrary to our hypothesis, restoring Klf9 levels with TAC showed systolic dysfunction in Klf9KI-TAC (%EF-38.65±1.9,%FS-18.34±0.77) compared to Wt-TAC (%EF-53.88±3.33,%FS-27.03±1.96) hearts, with no significant change in LV mass within the TAC groups (75.66±13.73 vs. 78.7±8.71). Interestingly, our preliminary data examining gene expression showed a significant increase in hypertrophy markers, Mhy7 (bMHC) and NPPB (BNP), and a decrease in Hmgcs2 (Klf9 target from ChIPSeq) in Klf9KI mice within sham and TAC groups. We conclude that Klf9 expression is critical for metabolic homeostasis, and dysregulated Klf9 levels are sufficient for transcriptional changes and with stress can precipitate early cardiac dysfunction and failure.

Tyrosine kinase inhibitors (TKIs) have improved cancer outcomes but are limited by cardiovascular toxicity, most notably hypertension and heart failure. The underlying mechanisms remain poorly understood, hindering the development of protective strategies. Here, we investigated the role of endothelial mechanotransduction in mediating vascular and cardiac injury caused by the vascular endothelial growth factor receptor-targeting TKI sunitinib. Using patient-specific induced pluripotent stem cell-derived endothelial cells (iPSC-ECs) and a mouse model of TKI-induced hypertension, we identified down-regulation of piezo-type mechanosensitive ion channel component 1 (PIEZO1), a mechanically activated ion channel, as a driver of endothelial dysfunction. Restoring PIEZO1 expression, either pharmacologically with Yoda1, a selective agonist, or through inducible overexpression in iPSC-ECs, reversed sunitinib-induced endothelial dysfunction and mitigated its hypertensive effects, providing both mechanistic and genetic validation of PIEZO1's protective role against vascular toxicity. In mice, cotreatment with sunitinib and Yoda1 prevented the long-term cardiac dysfunction observed after sunitinib exposure and normalized elevations in circulating cardiac stress biomarkers. Single-nucleus multiomic profiling of mouse hearts revealed that sunitinib exposure activated chromatin remodeling and fibrogenic programs, which were reversed with PIEZO1 activation. Human engineered cardiac organoids further demonstrated that sunitinib impaired cardiomyocyte function only in the presence of endothelial cells, confirming a role for disrupted endothelial-cardiomyocyte cross-talk in TKI cardiotoxicity. Together, these findings identify endothelial PIEZO1 as a mediator of TKI-induced hypertension and cardiac dysfunction and highlight PIEZO1 activation as a potential therapeutic strategy for protecting cardiovascular health during cancer therapy.

Cardiac malformations and ventricular remodeling due to heart diseases result in compromised cardiac function, eventually leading to heart failure. In this study, we examine the role of cardiac Wolf-Hirschhorn Syndrome candidate 2 (Whsc2), also known as Negative elongation factor A (NELFA), one of the genes encoded in the WHS critical region. The Wolf-Hirschhorn Syndrome is a contiguous genetic disorder due to microdeletions in the critical region, with clinical manifestations of neurological defects frequently associated with congenital malformations, including cardiac defects. NelfA has been implicated in RNA polymerase II (pol II) pausing, suggesting a role in pol II-dependent gene transcription. We previously reported an early onset of heart failure with the acute knockdown of NelfA in hearts undergoing pressure overload-induced cardiac hypertrophy. Here, we characterize a mouse model with cardiomyocyte-specific loss of NelfA function, in which these mice develop spontaneous cardiomyopathy at 2 months of age and exhibit early mortality by 3 months, suggesting a critical role for postnatal NelfA in the heart. Interactome data show that chromatin-bound NelfA interacts with proteins involved in chromatin remodeling (Trim28) and pre-mRNA processing (Adrph1l), along with expected binding partners like RNA pol II, Supt5, and other Nelf proteins. Examination of genomic occupancy of these NelfA-associated proteins in the NelfA knockout (KO) hearts reveals a disassembly of the NelfA nucleated complex at promoters of cardiac-enriched genes, including cytoskeletal and metabolic genes. This deconstruction of the NelfA-dependent complex results in the inhibited expression of these essential genes during postnatal cardiac development, leading to a cardiac contractile and metabolic crisis that precipitates dilated cardiomyopathy.

Glucocorticoids through activation of the Glucocorticoid receptor (GR) play an essential role in cellular homeostasis during physiological variations and in response to stress. Our genomic GR binding and transcriptome data from Dexamethasone (Dex) treated cardiomyocytes showed an early differential regulation of mostly transcription factors, followed by sequential change in genes involved in downstream functional pathways. We examined the role of Krüppel-like factor 9 (Klf9), an early direct target of GR in cardiomyocytes. Klf9-ChIPseq identified 2150 genes that showed an increase in Klf9 binding in response to Dex. Transcriptome analysis of Dex treated cardiomyocytes with or without knockdown of Klf9 revealed differential regulation of 1777 genes, of which a reversal in expression is seen in 1640 genes with knockdown of Klf9 compared to Dex. Conversely, only 137 (∼8%) genes show further dysregulation in expression with siKLf9, as seen with Dex treated cardiomyocytes. Functional annotation identified genes of metabolic pathways on the top of differentially expressed genes, including those involved in glycolysis and oxidative phosphorylation. Knockdown of Klf9 in cardiomyocytes inhibited Dex induced increase in glycolytic function and mitochondrial spare respiratory capacity, as measured by glycolysis and mito stress tests, respectively. Thus, we conclude that cyclic, diurnal GR activation, through Klf9 -dependent feedforward signaling plays a central role in maintaining cellular homeostasis through metabolic adaptations in cardiomyocytes.

Glucocorticoids (GCs) through activation of the Glucocorticoid receptor (GR) plays an essential role in cellular homeostasis during physiological variations and in response to stress. GC-GR signaling is involved in regulating several cellular processes including metabolism, circadian rhythm and inflammation for diurnal adaptations. Our genomic GR binding (ChIP) and transcriptome (RNAseq) data from Dexamethasone (Dex) treatment in cardiomyocytes show an early (1hr) differential regulation of mostly transcription factors, followed by sequential change in downstream signaling pathways (6-12hr). Here, we examine the role of an early direct target of GR in cardiomyocytes, Krüppel-like factor 9 (Klf9) in metabolic homeostasis. Our Klf9-ChIPseq identified 4100 genes with change in promoter Klf9 binding in response to Dex. Functional annotation of these genes lists metabolic pathway on the top of KEGG pathway, along with genes regulating transcription and survival. Interestingly, integration of GR and Klf9 data show overlapping targets (1181), suggesting that Klf9 could be serving as feedback regulator for these genes. Further, our transcriptome analysis of Dex treated cardiomyocytes with knockdown of Klf9 reveal differential regulation of 1777 genes (Dex+siKLf9 vs Dex+siLUC), of which a reversal in expression is seen in 1640 (92%) genes with siKlf9 vs. Dex. Conversely, only 137 (8%) genes show further dysregulation in expression with siKLf9 as Dex. Gene ontology of these 1640 genes show metabolic pathway on the top, including genes involved in glycolysis and oxidative phosphorylation. Conversely, mostly immuno-regulatory genes are among those 137 genes. Expectedly, knockdown of Klf9 in cardiomyocytes inhibits Dex induced increase in glycolysis and glycolytic capacity by 24%, and spare respiratory capacity by 50%, as measured by ECAR and OCR with glycolysis and mito stress tests, respectively. Thus, we conclude that cyclic, diurnal GC mediated GR activation, through Klf9 -dependent feedforward signaling plays a central role in maintaining cellular metabolic homeostasis. With hypertrophic stress a decrease in cardiac GR and dysregulated Klf9 signaling might result in metabolic crisis, contributing to mitochondrial and cardiac dysfunction

Identification of branched-chain amino acid (BCAA) oxidation enzymes in the nucleus led us to predict that they are a source of the propionyl-CoA that is utilized for histone propionylation and, thereby, regulate gene expression. To investigate the effects of BCAAs on the development of cardiac hypertrophy and failure, we applied pressure overload on the heart in mice maintained on a diet with standard levels of BCAAs (BCAA control) versus a BCAA-free diet. The former was associated with an increase in histone H3K23-propionyl (H3K23Pr) at the promoters of upregulated genes (e.g., cell signaling and extracellular matrix genes) and a decrease at the promoters of downregulated genes (e.g., electron transfer complex [ETC I-V] and metabolic genes). Intriguingly, the BCAA-free diet tempered the increases in promoter H3K23Pr, thus reducing collagen gene expression and fibrosis during cardiac hypertrophy. Conversely, the BCAA-free diet inhibited the reductions in promoter H3K23Pr and abolished the downregulation of ETC I-V subunits, enhanced mitochondrial respiration, and curbed the progression of cardiac hypertrophy. Thus, lowering the intake of BCAAs reduced pressure overload-induced changes in histone propionylation-dependent gene expression in the heart, which retarded the development of cardiomyopathy.

Adaptation of gene expression is one of the most fundamental response of cardiomyocytes to hypertrophic stimuli. G3bp1, an RNA binding protein with site-specific endoribonuclease activity regulates the processing of pre-miR-1 stem-loop, and thus levels of cardiomyocyte -enriched mature miR-1. Here, we examine the role of G3bp1 in regulating gene expression in quiescent cardiomyocytes and those undergoing growth-factor induced hypertrophy. Further, we determine if these changes are facilitated through G3bp1-mediated regulation of miR-1 in these cardiomyocytes. Using isolated cardiomyocytes with knockdown of endogenous G3bp1, we performed high throughput RNA sequencing to determine the change in cardiac transcriptome. Then, using gain and loss of function approach for both, G3bp1 and miR-1, alone or in combination we examine the G3bp1-miR-1 signaling in regulating gene expression and Endothelin (ET-1) -induced cardiomyocyte hypertrophy. We show that knockdown of endogenous G3bp1 results in inhibition of genes involved in calcium handling, cardiac muscle contraction, action potential and sarcomeric structure. In addition, there is inhibition of genes that contribute to hypertrophic and dilated cardiomyopathy development. Conversely, an increase is seen in genes that negatively regulate the Hippo signaling, like Rassf1 and Arrdc3, along with inflammatory genes of TGF-β and TNF pathways. Knockdown of G3bp1 restricts ET-1 induced cardiomyocyte hypertrophy. Interestingly, concurrent silencing of G3bp1 and miR-1 rescues the change in gene expression and inhibition of hypertrophy seen with knockdown of G3bp1 alone. Similarly, expression of exogenous G3bp1 reverses the miR-1 induced inhibition of gene expression. Intriguingly, expression of Gfp tagged G3bp1 results in perinuclear accumulations of G3bp1-Gfp, resembling Stress Granules. Based on our results, we conclude that G3bp1 through its regulation of mature miR-1 levels plays a critical role in regulating the expression of essential cardiac-enriched genes and those involved in development of cardiomyocyte hypertrophy.

Endothelial cells (ECs) have emerged as key pathogenic players in cardiac disease due to their proximity with cardiomyocytes. Induced pluripotent stem cells (iPSCs) have been employed to generate ECs. However, it may be more clinically relevant to transdifferentiate fibroblasts into ECs directly without introducing pluripotent or virally driven transcription factors. Here, we present a protocol that describes the direct conversion of human cardiac fibroblasts into ECs by leveraging the innate immune system. Our protocol produces bona fide human ECs with 95%-98% purity by first passage.For complete details on the use and execution of this protocol, please refer to Liu et al. (2020) and Sayed et al. (2015).

Innate immune signaling has recently been shown to play an important role in nuclear reprogramming, by altering the epigenetic landscape and thereby facilitating transcription. However, the mechanisms that link innate immune activation and metabolic regulation in pluripotent stem cells remain poorly defined, particularly with regard to key molecular components. In this study, we show that hypoxia-inducible factor 1α (HIF1α), a central regulator of adaptation to limiting oxygen tension, is an unexpected but crucial regulator of innate immune-mediated nuclear reprogramming. HIF1α is dramatically upregulated as a consequence of Toll-like receptor 3 (TLR3) signaling and is necessary for efficient induction of pluripotency and transdifferentiation. Bioenergetics studies reveal that HIF1α regulates the reconfiguration of innate immune-mediated reprogramming through its well-established role in throwing a glycolytic switch. We believe that results from these studies can help us better understand the influence of immune signaling in tissue regeneration and lead to new therapeutic strategies.

Coordinated functional balance of negative and positive transcription complexes maintain and accommodate gene expression in hearts during quiescent and hypertrophic conditions, respectively. Negative elongation factor (Nelf) complex has been implicated in RNA polymerase II (pol II) pausing, a widespread regulatory transcriptional phenomenon observed across the cardiac genome. Here, we examine the role of NelfA aka, Wolf-Hirschhorn syndrome candidate 2 (Whsc2), a critical component of the negative elongation complex in hearts undergoing pressure-overload induced hypertrophy. Alignment of high-resolution genome-wide occupancy data of NelfA, Pol II, TFIIB and H3k9ac from control and hypertrophied hearts reveal that NelfA associates with active gene promoters. High NelfA occupancy is seen at promoters of essential and cardiac-enriched genes, expressed under both quiescent and hypertrophic conditions. Conversely, de novo NelfA recruitment is observed at inducible gene promoters with pressure overload, accompanied by significant increase in expression of these genes with hypertrophy. Interestingly, change in promoter NelfA levels correlates with the transcript output in hypertrophied hearts compared to Sham, suggesting NelfA might be playing a critical role in the regulation of gene transcription during cardiac hypertrophy. In vivo knockdown of NelfA (siNelfA) in hearts subjected to pressure-overload results in early ventricular dilatation and dysfunction, associated with decrease in expression of inducible and cardiac-enriched genes in siNelfA hypertrophied compared to control hypertrophied hearts. In accordance, in vitro knockdown of NelfA in cardiomyocytes showed no change in promoter pol II, however significant decrease in in-gene and downstream pol II occupancy was observed. These data suggest an inhibited pol II progression in transcribing and inducible genes, which reflects as a decrease in transcript abundance of these genes. These results indicate that promoter NelfA occupancy is essential for pol II -dependent transcription. Therefore, we conclude that NelfA is required for active transcription and gene expression during cardiac hypertrophy.

Background: 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. Conclusions: 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.