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Aberrant epigenetic transcriptional regulation is linked to metastasis, a primary cause of cancer-related death. Dissecting the epigenetic mechanisms controlling metastatic progression may uncover important insights to tumor biology and potential therapeutic targets. Here, we investigated the role of the SIN3A histone deacetylase 1 and 2 (SIN3A–HDAC1/2) complex in cancer metastasis. Using a mouse model of melanoma metastasis, we found that the SIN3A–HDAC1/2 transcription repressor complex silences BMP6 expression, causing increased metastatic dissemination and tumor growth via suppression of BMP6-activated SMAD5 signaling. We further discovered that FAM83G/PAWS1, a downstream effector of BMP6–SMAD5 signaling, contributes critically to metastatic progression by promoting actin-dependent cytoskeletal dynamics and cell migration. Pharmacologic inhibition of the SIN3A–HDAC1/2 complex reduced the numbers of melanoma cells in the circulation and inhibited metastatic tumor growth by inducing disseminated cell dormancy, highlighting the SIN3A–HDAC1/2 repressor complex as a potential therapeutic target for blocking cancer metastasis.Implications:This study identifies the novel molecular links in the metastatic progression to target cytoskeletal dynamics in melanoma and identifies the SIN3A–HDAC1/2 complex and FAM83G/PAWS1 as potential targets for melanoma adjuvant therapy.

Aims Thioredoxin 1 (Trx1) is an evolutionarily conserved oxidoreductase that cleaves disulphide bonds in oxidized substrate proteins such as mechanistic target of rapamycin (mTOR) and maintains nuclear-encoded mitochondrial gene expression. The cardioprotective effect of Trx1 has been demonstrated via cardiac-specific overexpression of Trx1 and dominant negative Trx1. However, the pathophysiological role of endogenous Trx1 has not been defined with a loss-of-function model. To address this, we have generated cardiac-specific Trx1 knockout (Trx1cKO) mice. Methods and results Trx1cKO mice were viable but died with a median survival age of 25.5 days. They developed heart failure, evidenced by contractile dysfunction, hypertrophy, and increased fibrosis and apoptotic cell death. Multiple markers consistently indicated increased oxidative stress and RNA-sequencing revealed downregulation of genes involved in energy production in Trx1cKO mice. Mitochondrial morphological abnormality was evident in these mice. Although heterozygous Trx1cKO mice did not show any significant baseline phenotype, pressure-overload-induced cardiac dysfunction, and downregulation of metabolic genes were exacerbated in these mice. mTOR was more oxidized and phosphorylation of mTOR substrates such as S6K and 4EBP1 was impaired in Trx1cKO mice. In cultured cardiomyocytes, Trx1 knockdown inhibited mitochondrial respiration and metabolic gene promoter activity, suggesting that Trx1 maintains mitochondrial function in a cell autonomous manner. Importantly, mTOR-C1483F, an oxidation-resistant mutation, prevented Trx1 knockdown-induced mTOR oxidation and inhibition and attenuated suppression of metabolic gene promoter activity. Conclusion Endogenous Trx1 is essential for maintaining cardiac function and metabolism, partly through mTOR regulation via Cys1483.

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.

Cardiovascular disease (CVD) remains the leading cause of death globally, and heart failure is a major component of CVD-related morbidity and mortality. The development of cardiac hypertrophy in response to hemodynamic overload is initially considered to be beneficial; however, this adaptive response is limited and, in the presence of prolonged stress, will transition to heart failure. Yes-associated protein (YAP), the central downstream effector of the Hippo signaling pathway, regulates proliferation and survival in mammalian cells. Our previous work demonstrated that cardiac-specific loss of YAP leads to increased cardiomyocyte (CM) apoptosis and impaired CM hypertrophy during chronic myocardial infarction (MI) in the mouse heart. Because of its documented cardioprotective effects, we sought to determine the importance of YAP in response to acute pressure overload (PO). Our results indicate that endogenous YAP is activated in the heart during acute PO. YAP activation that depended upon RhoA was also observed in CMs subjected to cyclic stretch. To examine the function of endogenous YAP during acute PO, Yap(+/)(flox);Cre(α-MHC) (YAP-CHKO) and Yap(+/)(flox) mice were subjected to transverse aortic constriction (TAC). We found that YAP-CHKO mice had attenuated cardiac hypertrophy and significant increases in CM apoptosis and fibrosis that correlated with worsened cardiac function after 1 week of TAC. Loss of CM YAP also impaired activation of the cardioprotective kinase Akt, which may underlie the YAP-CHKO phenotype. Together, these data indicate a prohypertrophic, prosurvival function of endogenous YAP and suggest a critical role for CM YAP in the adaptive response to acute PO.

Background— Heart failure is accompanied by changes in cardiac metabolism characterized by reduced fatty acid (FA) utilization. However, the underlying mechanism and its causative involvement in the progression of heart failure are poorly understood. The peroxisome proliferator activated receptor-α (PPARα)/retinoid X receptor (RXR) heterodimer promotes transcription of genes involved in FA metabolism through binding to the PPAR response element, called direct repeat 1 (DR1). Silent information regulator 1 (Sirt1) is a histone deacetylase, which interacts with PPARα. Methods and Results— To investigate the role of PPARα in the impaired FA utilization observed during heart failure, genetically altered mice were subjected to pressure overload. The DNA binding of PPARα, RXRα, and Sirt1 to DR1 was evaluated with oligonucleotide pull-down and chromatin immunoprecipitation assays. Although the binding of PPARα to DR1 was enhanced in response to pressure overload, that of RXRα was attenuated. Sirt1 competes with RXRα to dimerize with PPARα, thereby suppressing FA utilization in the failing heart. DR1 sequence analysis indicated that the typical DR1 sequence favors PPARα/RXRα heterodimerization, whereas the switch from RXRα to Sirt1 takes place on degenerate DR1s. Sirt1 bound to PPARα through a region homologous to the PPARα binding domain in RXRα. A short peptide corresponding to the RXRα domain not only inhibited the interaction between PPARα and Sirt1 but also improved FA metabolism and ameliorated cardiac dysfunction. Conclusions— A change in the heterodimeric partner of PPARα from RXRα to Sirt1 is responsible for the impaired FA metabolism and cardiac dysfunction in the failing heart.

Background— Although methods for generating cardiomyocytes from pluripotent stem cells have been reported, current methods produce heterogeneous mixtures of cardiomyocytes and noncardiomyocyte cells. Here, we report an entirely novel system in which pluripotent stem cell–derived cardiomyocytes are purified by cardiomyocyte-specific molecular beacons (MBs). MBs are nanoscale probes that emit a fluorescence signal when hybridized to target mRNAs. Method and Results— Five MBs targeting mRNAs of either cardiac troponin T or myosin heavy chain 6/7 were generated. Among 5 MBs, an MB that targeted myosin heavy chain 6/7 mRNA (MHC1-MB) identified up to 99% of HL-1 cardiomyocytes, a mouse cardiomyocyte cell line, but Conclusions— We developed a novel cardiomyocyte selection system that allows production of highly purified cardiomyocytes. These purified cardiomyocytes and this system can be valuable for cell therapy and drug discovery.

Background: Human pluripotent stem cell (hPSC)–derived endothelial cells (ECs) have limited clinical utility because of undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Furthermore, we aimed to enhance cell survival, vessel formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel. Methods: We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a glycogen synthase kinase 3β inhibitor. Next, vascular endothelial growth factor, endothelial growth factor, and basic fibroblast growth factor were added for endothelial lineage differentiation, followed by sorting for CDH5 (VE-cadherin). We constructed an extracellular matrix–mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2–degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5 + cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hind-limb ischemia model with laser Doppler perfusion imaging, bioluminescence imaging, real-time reverse transcription–polymerase chain reaction, and histological analysis. Results: The resultant hPSC-derived CDH5 + cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and proangiogenic activities. When injected into ischemic hind limbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared with media-, PA-RGDS–, or human umbilical vein EC–injected groups. However, the group receiving the PA-RGDS–encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC–injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months. Conclusions: We generated highly enriched hPSC-ECs via a clinically compatible system. Furthermore, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS–mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs.

Peroxisome proliferator-activated receptor-α (PPARα), a nuclear receptor, plays an important role in the transcription of genes involved in fatty acid metabolism through heterodimerization with the retinoid x receptor (RXR). The consensus sequence of the PPAR response element (PPRE) is composed of two AGGTCA-like sequences directionally aligned with a single nucleotide spacer. PPARα and RXR bind to the 5’ and 3’ hexad sequences, respectively. However, the precise sequence definition of the PPRE remains obscure, and thus, the consensus sequence currently available remains AGGTCANAGGTCA with unknown redundancy. The vague PPRE sequence definition poses an obstacle to understanding how PPARα regulates fatty acid metabolism. Here we show that, rather than the generally accepted 6-bp sequence, PPARα actually recognized a 12-bp DNA sequence, of which the preferred binding sequence was WAWVTRGGBBAH. Additionally, the optimized RXRα hexad binding sequence was RGKTYA. Thus, the optimal PPARα/RXRα heterodimer binding sequence was WAWVTRGGBBAHRGKTYA. The single nucleotide substitution, which reduces binding of RXRα to DNA, attenuated PPARα-induced transcriptional activation, but this is not always true for PPARα. Using the definition of the PPRE sequence, novel PPREs were successfully identified. Taken altogether, the provided PPRE sequence definition contributes to the understanding of PPARα signaling by identifying PPARα direct target genes with functional PPARα response elements.

Nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme for nicotinamide adenine dinucleotide (NAD+) synthesis, and Sirt1, an NAD+-dependent histone deacetylase, protect the heart against ischemia/reperfusion (I/R). It remains unknown whether Nampt mediates the protective effect of ischemic preconditioning (IPC), whether nicotinamide mononucleotide (NMN, 500 mg/kg), a product of Nampt in the NAD+ salvage pathway, mimics the effect of IPC, or whether caloric restriction (CR) upregulates Nampt and protects the heart through a Sirt1-dependent mechanism. IPC upregulated Nampt protein, and the protective effect of IPC against ischemia (30 minutes) and reperfusion (24 hours) was attenuated at both early and late phases in Nampt +/- mice, suggesting that Nampt plays an essential role in mediating the protective effect of IPC. In order to mimic the effect of Nampt, NMN was administered by intraperitoneal injection. NMN significantly increased the level of NAD+ in the heart at baseline and prevented a decrease in NAD+ during ischemia. NMN protected the heart from I/R injury when it was applied once 30 minutes before ischemia or 4 times just before and during reperfusion, suggesting that exogenous NMN protects the heart from I/R injury in both ischemic and reperfusion phases. The protective effect of NMN was accompanied by decreases in acetylation of FoxO1, but it was not obvious in Sirt1 KO mice, suggesting that the effect of NMN is mediated through activation of Sirt1. Compared to control diet (90% calories), CR (60% calories for 6 weeks) in mice led to a significant reduction in I/R injury, accompanied by upregulation of Nampt. The protective effect of CR against I/R injury was not significant in cardiac-specific Sirt1 KO mice, suggesting that the protective effect of CR is in part mediated through the Nampt-Sirt1 pathway. In conclusion, exogenous application of NMN and CR protects the heart by both mimicking IPC and activating Sirt1.

Background: While various methods for generating cardiomyocytes (CMs) from pluripotent stem cells (PSCs) including both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have been reported, all available methods are only allowed to produce heterogeneous population of CM mixed with non-CM cells. Therefore, strategies to enrich pure CMs for scientific and clinical applications have been highly required. Hence, we developed a novel system in which CMs can be purified by cardiac specific molecular beacons (MBs). MBs are dual-labeled antisense nano-scale probes that emit a fluorescence signal when hybridized to target mRNAs. We hypothesized that MBs targeted to CM specific mRNAs can identify CMs and allow isolation of purified CMs by fluorescence-activated cell sorting (FACS). Methods and results: Five MBs targeting distinct sites on either cardiac troponin T (cTNT) or α/β myosin heavy chain (α/βMHC) were designed and characterized in various cell types. To find the optimal MB that can selectively identifying CMs, each MB was delivered into HL-1 CMs, an immortalized mouse CM cell line, smooth muscle cells, endothelial cells, mouse ESCs and fibroblasts and its specificity was determined by flow cytometry. As a result, two MBs identified MB+ cells up to 98% from HL-1 CMs but lower than 10% of the non-CM cells suggesting these MBs are CM specific. Subsequently, the selected MBs were delivered into both mouse and human PSCs derived CMs and 41 to 49% of the cells were identified as an MB+ population. Interestingly, the rate of MB+ cells was similar to CM quantification determined by cTNT intracellular flow cytometry. Finally, we determined whether cell sorting with cardiac-specific MBs can enrich CMs from the heterogeneous mouse and human PSC cultures and found that ∼97% of MB-based sorted CMs expressed cTNT. These enriched cells were further cultured and their CM identity was verified by immunocytochemistry and qRT-PCR analysis. Ca2+ transient analysis further confirmed that these purified CMs displayed functional CM characteristics Conclusion: Using cardiac specific MBs, we were able to obtain highly purified CMs. These purified CMs and the system can be highly useful for clinical applications as well as drug discovery.