IRE1α Mediates the Hypertrophic Growth of Cardiomyocytes Through Facilitating the Formation of Initiation Complex to Promote the Translation of TOP-Motif Transcripts.

BACKGROUND: Cardiomyocyte growth is coupled with active protein synthesis, which is one of the basic biological processes in living cells. However, it is unclear whether the unfolded protein response transducers and effectors directly take part in the control of protein synthesis. The connection between critical functions of the unfolded protein response in cellular physiology and requirements of multiple processes for cell growth prompted us to investigate the role of the unfolded protein response in cell growth and underlying molecular mechanisms. METHODS: Cardiomyocyte-specific inositol-requiring enzyme 1α (IRE1α) knockout and overexpression mouse models were generated to explore its function in vivo. Neonatal rat ventricular myocytes were isolated and cultured to evaluate the role of IRE1α in cardiomyocyte growth in vitro. Mass spectrometry was conducted to identify novel interacting proteins of IRE1α. Ribosome sequencing and polysome profiling were performed to determine the molecular basis for the function of IRE1α in translational control. RESULTS: We show that IRE1α is required for cell growth in neonatal rat ventricular myocytes under prohypertrophy treatment and in HEK293 cells in response to serum stimulation. At the molecular level, IRE1α directly interacts with eIF4G and eIF3, 2 critical components of the translation initiation complex. We demonstrate that IRE1α facilitates the formation of the translation initiation complex around the endoplasmic reticulum and preferentially initiates the translation of transcripts with 5' terminal oligopyrimidine motifs. We then reveal that IRE1α plays an important role in determining the selectivity and translation of these transcripts. We next show that IRE1α stimulates the translation of epidermal growth factor receptor through an unannotated terminal oligopyrimidine motif in its 5' untranslated region. We further demonstrate a physiological role of IRE1α-governed protein translation by showing that IRE1α is essential for cardiomyocyte growth and cardiac functional maintenance under hemodynamic stress in vivo. CONCLUSIONS: These studies suggest a noncanonical, essential role of IRE1α in orchestrating protein synthesis, which may have important implications in cardiac hypertrophy in response to pressure overload and general cell growth under other physiological and pathological conditions.

Active in vivo translocation of the Methanosarcina mazei Gö1 Casposon.

Casposons are transposable elements containing the CRISPR associated gene Cas1solo. Identified in many archaeal genomes, casposons are discussed as the origin of CRISPR-Cas systems due to their proposed Cas1solo-dependent translocation. However, apart from bioinformatic approaches and the demonstration of Cas1solo integrase and endonuclease activity in vitro, casposon transposition has not yet been shown in vivo. Here, we report on active casposon translocations in Methanosarcina mazei Gö1 using two independent experimental approaches. First, mini-casposons, consisting of a R6Kγ origin and two antibiotic resistance cassettes, flanked by target site duplications (TSDs) and terminal inverted repeats (TIRs), were generated, and shown to actively translocate from a suicide plasmid and integrate into the chromosomal MetMaz-C1 TSD IS1a. Second, casposon excision activity was confirmed in a long-term evolution experiment using a Cas1solo overexpression strain in comparison to an empty vector control under four different treatments (native, high temperature, high salt, mitomycin C) to study stress-induced translocation. Analysis of genomic DNA using a nested qPCR approach provided clear evidence of casposon activity in single cells and revealed significantly different casposon excision frequencies between treatments and strains. Our results, providing the first experimental evidence for in vivo casposon activity are summarized in a modified hypothetical translocation model.

Activation of Autophagic Flux Blunts Cardiac Ischemia/Reperfusion Injury.

[Figure: see text].

Cooperative Binding of ETS2 and NFAT Links Erk1/2 and Calcineurin Signaling in the Pathogenesis of Cardiac Hypertrophy.

BACKGROUND: Cardiac hypertrophy is an independent risk factor for heart failure, a leading cause of morbidity and mortality globally. The calcineurin/NFAT (nuclear factor of activated T cells) pathway and the MAPK (mitogen-activated protein kinase)/Erk (extracellular signal-regulated kinase) pathway contribute to the pathogenesis of cardiac hypertrophy as an interdependent network of signaling cascades. How these pathways interact remains unclear and few direct targets responsible for the prohypertrophic role of NFAT have been described. METHODS: By engineering cardiomyocyte-specific ETS2 (a member of the E26 transformation-specific sequence [ETS] domain family) knockout mice, we investigated the role of ETS2 in cardiac hypertrophy. Primary cardiomyocytes were used to evaluate ETS2 function in cell growth. RESULTS: ETS2 is phosphorylated and activated by Erk1/2 on hypertrophic stimulation in both mouse (n=3) and human heart samples (n=8 to 19). Conditional deletion of ETS2 in mouse cardiomyocytes protects against pressure overload-induced cardiac hypertrophy (n=6 to 11). Silencing of ETS2 in the hearts of calcineurin transgenic mice significantly attenuates hypertrophic growth and contractile dysfunction (n=8). As a transcription factor, ETS2 is capable of binding to the promoters of hypertrophic marker genes, such as ANP, BNP, and Rcan1.4 (n=4). We report that ETS2 forms a complex with NFAT to stimulate transcriptional activity through increased NFAT binding to the promoters of at least 2 hypertrophy-stimulated genes: Rcan1.4 and microRNA-223 (=n4 to 6). Suppression of microRNA-223 in cardiomyocytes inhibits calcineurin-mediated cardiac hypertrophy (n=6), revealing microRNA-223 as a novel prohypertrophic target of the calcineurin/NFAT and Erk1/2-ETS2 pathways. CONCLUSIONS: Our findings point to a critical role for ETS2 in calcineurin/NFAT pathway-driven cardiac hypertrophy and unveil a previously unknown molecular connection between the Erk1/2 activation of ETS2 and expression of NFAT/ETS2 target genes.

Xbp1s-FoxO1 axis governs lipid accumulation and contractile performance in heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.

Epigenetic Reader BRD4 (Bromodomain-Containing Protein 4) Governs Nucleus-Encoded Mitochondrial Transcriptome to Regulate Cardiac Function.

BACKGROUND: BET (bromodomain and extraterminal) epigenetic reader proteins, in particular BRD4 (bromodomain-containing protein 4), have emerged as potential therapeutic targets in a number of pathological conditions, including cancer and cardiovascular disease. Small-molecule BET protein inhibitors such as JQ1 have demonstrated efficacy in reversing cardiac hypertrophy and heart failure in preclinical models. Yet, genetic studies elucidating the biology of BET proteins in the heart have not been conducted to validate pharmacological findings and to unveil potential pharmacological side effects. METHODS: By engineering a cardiomyocyte-specific BRD4 knockout mouse, we investigated the role of BRD4 in cardiac pathophysiology. We performed functional, transcriptomic, and mitochondrial analyses to evaluate BRD4 function in developing and mature hearts. RESULTS: Unlike pharmacological inhibition, loss of BRD4 protein triggered progressive declines in myocardial function, culminating in dilated cardiomyopathy. Transcriptome analysis of BRD4 knockout mouse heart tissue identified early and specific disruption of genes essential to mitochondrial energy production and homeostasis. Functional analysis of isolated mitochondria from these hearts confirmed that BRD4 ablation triggered significant changes in mitochondrial electron transport chain protein expression and activity. Computational analysis identified candidate transcription factors participating in the BRD4-regulated transcriptome. In particular, estrogen-related receptor α, a key nuclear receptor in metabolic gene regulation, was enriched in promoters of BRD4-regulated mitochondrial genes. CONCLUSIONS: In aggregate, we describe a previously unrecognized role for BRD4 in regulating cardiomyocyte mitochondrial homeostasis, observing that its function is indispensable to the maintenance of normal cardiac function.

Lactate Dehydrogenase A Governs Cardiac Hypertrophic Growth in Response to Hemodynamic Stress.

The heart manifests hypertrophic growth in response to high blood pressure, which may decompensate and progress to heart failure under persistent stress. Metabolic remodeling is an early event in this process. However, its role remains to be fully characterized. Here, we show that lactate dehydrogenase A (LDHA), a critical glycolytic enzyme, is elevated in the heart in response to hemodynamic stress. Cardiomyocyte-restricted deletion of LDHA leads to defective cardiac hypertrophic growth and heart failure by pressure overload. Silencing of LDHA in cultured cardiomyocytes suppresses cell growth from pro-hypertrophic stimulation in vitro, while overexpression of LDHA is sufficient to drive cardiomyocyte growth. Furthermore, we find that lactate is capable of rescuing the growth defect from LDHA knockdown. Mechanistically, lactate stabilizes NDRG3 (N-myc downregulated gene family 3) and stimulates ERK (extracellular signal-regulated kinase). Our results together suggest that the LDHA/NDRG3 axis may play a critical role in adaptive cardiomyocyte growth in response to hemodynamic stress.

FoxO1-Dio2 signaling axis governs cardiomyocyte thyroid hormone metabolism and hypertrophic growth.

Forkhead box O (FoxO) proteins and thyroid hormone (TH) have well established roles in cardiovascular morphogenesis and remodeling. However, specific role(s) of individual FoxO family members in stress-induced growth and remodeling of cardiomyocytes remains unknown. Here, we report that FoxO1, but not FoxO3, activity is essential for reciprocal regulation of types II and III iodothyronine deiodinases (Dio2 and Dio3, respectively), key enzymes involved in intracellular TH metabolism. We further show that Dio2 is a direct transcriptional target of FoxO1, and the FoxO1-Dio2 axis governs TH-induced hypertrophic growth of neonatal cardiomyocytes in vitro and in vivo. Utilizing transverse aortic constriction as a model of hemodynamic stress in wild-type and cardiomyocyte-restricted FoxO1 knockout mice, we unveil an essential role for the FoxO1-Dio2 axis in afterload-induced pathological cardiac remodeling and activation of TRα1. These findings demonstrate a previously unrecognized FoxO1-Dio2 signaling axis in stress-induced cardiomyocyte growth and remodeling and intracellular TH homeostasis.

Chronic activation of hexosamine biosynthesis in the heart triggers pathological cardiac remodeling.

The hexosamine biosynthetic pathway (HBP) plays critical roles in nutrient sensing, stress response, and cell growth. However, its contribution to cardiac hypertrophic growth and heart failure remains incompletely understood. Here, we show that the HBP is induced in cardiomyocytes during hypertrophic growth. Overexpression of Gfat1 (glutamine:fructose-6-phosphate amidotransferase 1), the rate-limiting enzyme of HBP, promotes cardiomyocyte growth. On the other hand, Gfat1 inhibition significantly blunts phenylephrine-induced hypertrophic growth in cultured cardiomyocytes. Moreover, cardiac-specific overexpression of Gfat1 exacerbates pressure overload-induced cardiac hypertrophy, fibrosis, and cardiac dysfunction. Conversely, deletion of Gfat1 in cardiomyocytes attenuates pathological cardiac remodeling in response to pressure overload. Mechanistically, persistent upregulation of the HBP triggers decompensated hypertrophy through activation of mTOR while Gfat1 deficiency shows cardioprotection and a concomitant decrease in mTOR activity. Taken together, our results reveal that chronic upregulation of the HBP under hemodynamic stress induces pathological cardiac hypertrophy and heart failure through persistent activation of mTOR.

Spliced X-box Binding Protein 1 Stimulates Adaptive Growth Through Activation of mTOR.

BACKGROUND: The unfolded protein response plays versatile roles in physiology and pathophysiology. Its connection to cell growth, however, remains elusive. Here, we sought to define the role of unfolded protein response in the regulation of cardiomyocyte growth in the heart. METHODS: We used both gain- and loss-of-function approaches to genetically manipulate XBP1s (spliced X-box binding protein 1), the most conserved signaling branch of the unfolded protein response, in the heart. In addition, primary cardiomyocyte culture was used to address the role of XBP1s in cell growth in a cell-autonomous manner. RESULTS: We found that XBP1s expression is reduced in both human and rodent cardiac tissues under heart failure. Furthermore, deficiency of XBP1s leads to decompensation and exacerbation of heart failure progression under pressure overload. On the other hand, cardiac-restricted overexpression of XBP1s prevents the development of cardiac dysfunction. Mechanistically, we found that XBP1s stimulates adaptive cardiac growth through activation of the mechanistic target of rapamycin signaling, which is mediated via FKBP11 (FK506-binding protein 11), a novel transcriptional target of XBP1s. Moreover, silencing of FKBP11 significantly diminishes XBP1s-induced mechanistic target of rapamycin activation and adaptive cell growth. CONCLUSIONS: Our results reveal a critical role of the XBP1s-FKBP11-mechanistic target of rapamycin axis in coupling of the unfolded protein response and cardiac cell growth regulation.

Nitrosative stress drives heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome with high morbidity and mortality for which there are no evidence-based therapies. Here we report that concomitant metabolic and hypertensive stress in mice-elicited by a combination of high-fat diet and inhibition of constitutive nitric oxide synthase using Nω-nitro-L-arginine methyl ester (L-NAME)-recapitulates the numerous systemic and cardiovascular features of HFpEF in humans. Expression of one of the unfolded protein response effectors, the spliced form of X-box-binding protein 1 (XBP1s), was reduced in the myocardium of our rodent model and in humans with HFpEF. Mechanistically, the decrease in XBP1s resulted from increased activity of inducible nitric oxide synthase (iNOS) and S-nitrosylation of the endonuclease inositol-requiring protein 1α (IRE1α), culminating in defective XBP1 splicing. Pharmacological or genetic suppression of iNOS, or cardiomyocyte-restricted overexpression of XBP1s, each ameliorated the HFpEF phenotype. We report that iNOS-driven dysregulation of the IRE1α-XBP1 pathway is a crucial mechanism of cardiomyocyte dysfunction in HFpEF.

Fibroblast Primary Cilia Are Required for Cardiac Fibrosis.

BACKGROUND: The primary cilium is a singular cellular structure that extends from the surface of many cell types and plays crucial roles in vertebrate development, including that of the heart. Whereas ciliated cells have been described in developing heart, a role for primary cilia in adult heart has not been reported. This, coupled with the fact that mutations in genes coding for multiple ciliary proteins underlie polycystic kidney disease, a disorder with numerous cardiovascular manifestations, prompted us to identify cells in adult heart harboring a primary cilium and to determine whether primary cilia play a role in disease-related remodeling. METHODS: Histological analysis of cardiac tissues from C57BL/6 mouse embryos, neonatal mice, and adult mice was performed to evaluate for primary cilia. Three injury models (apical resection, ischemia/reperfusion, and myocardial infarction) were used to identify the location and cell type of ciliated cells with the use of antibodies specific for cilia (acetylated tubulin, γ-tubulin, polycystin [PC] 1, PC2, and KIF3A), fibroblasts (vimentin, α-smooth muscle actin, and fibroblast-specific protein-1), and cardiomyocytes (α-actinin and troponin I). A similar approach was used to assess for primary cilia in infarcted human myocardial tissue. We studied mice silenced exclusively in myofibroblasts for PC1 and evaluated the role of PC1 in fibrogenesis in adult rat fibroblasts and myofibroblasts. RESULTS: We identified primary cilia in mouse, rat, and human heart, specifically and exclusively in cardiac fibroblasts. Ciliated fibroblasts are enriched in areas of myocardial injury. Transforming growth factor ß-1 signaling and SMAD3 activation were impaired in fibroblasts depleted of the primary cilium. Extracellular matrix protein levels and contractile function were also impaired. In vivo, depletion of PC1 in activated fibroblasts after myocardial infarction impaired the remodeling response. CONCLUSIONS: Fibroblasts in the neonatal and adult heart harbor a primary cilium. This organelle and its requisite signaling protein, PC1, are required for critical elements of fibrogenesis, including transforming growth factor ß-1-SMAD3 activation, production of extracellular matrix proteins, and cell contractility. Together, these findings point to a pivotal role of this organelle, and PC1, in disease-related pathological cardiac remodeling and suggest that some of the cardiovascular manifestations of autosomal dominant polycystic kidney disease derive directly from myocardium-autonomous abnormalities.

Endoplasmic Reticulum Chaperone GRP78 Protects Heart From Ischemia/Reperfusion Injury Through Akt Activation.

RATIONALE: Restoration of coronary artery blood flow is the most effective means of ameliorating myocardial damage triggered by ischemic heart disease. However, coronary reperfusion elicits an increment of additional injury to the myocardium. Accumulating evidence indicates that the unfolded protein response (UPR) in cardiomyocytes is activated by ischemia/reperfusion (I/R) injury. Xbp1s (spliced X-box binding protein 1), the most highly conserved branch of the unfolded protein response, is protective in response to cardiac I/R injury. GRP78 (78 kDa glucose-regulated protein), a master regulator of the UPR and an Xbp1s target, is upregulated after I/R. However, its role in the protective response of Xbp1s during I/R remains largely undefined. OBJECTIVE: To elucidate the role of GRP78 in the cardiomyocyte response to I/R using both in vitro and in vivo approaches. METHODS AND RESULTS: Simulated I/R injury to cultured neonatal rat ventricular myocytes induced apoptotic cell death and strong activation of the UPR and GRP78. Overexpression of GRP78 in neonatal rat ventricular myocytes significantly protected myocytes from I/R-induced cell death. Furthermore, cardiomyocyte-specific overexpression of GRP78 ameliorated I/R damage to the heart in vivo. Exploration of underlying mechanisms revealed that GRP78 mitigates cellular damage by suppressing the accumulation of reactive oxygen species. We go on to show that the GRP78-mediated cytoprotective response involves plasma membrane translocation of GRP78 and interaction with PI3 kinase, culminating in stimulation of Akt. This response is required as inhibition of the Akt pathway significantly blunted the antioxidant activity and cardioprotective effects of GRP78. CONCLUSIONS: I/R induction of GRP78 in cardiomyocytes stimulates Akt signaling and protects against oxidative stress, which together protect cells from I/R damage.

Polycystin-2-dependent control of cardiomyocyte autophagy.

AIMS: Considerable evidence points to critical roles of intracellular Ca2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca2+ homeostasis and autophagy. METHODS AND RESULTS: Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca2+ chelation using BAPTA-AM, whereas removal of extracellular Ca2+ had no effect, pointing to a role of intracellular Ca2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca2+-channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca2+. Furthermore, PC2 ablation was associated with impaired Ca2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca2+ stores. Finally, we provide evidence that Ca2+-mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. CONCLUSION: Together, this study unveils PC2 as a novel regulator of autophagy acting through control of intracellular Ca2+ homeostasis.

Cytosolic DNA Sensing Promotes Macrophage Transformation and Governs Myocardial Ischemic Injury.

BACKGROUND: Myocardium irreversibly injured by ischemic stress must be efficiently repaired to maintain tissue integrity and contractile performance. Macrophages play critical roles in this process. These cells transform across a spectrum of phenotypes to accomplish diverse functions ranging from mediating the initial inflammatory responses that clear damaged tissue to subsequent reparative functions that help rebuild replacement tissue. Although macrophage transformation is crucial to myocardial repair, events governing this transformation are poorly understood. METHODS: Here, we set out to determine whether innate immune responses triggered by cytoplasmic DNA play a role. RESULTS: We report that ischemic myocardial injury, along with the resulting release of nucleic acids, activates the recently described cyclic GMP-AMP synthase-stimulator of interferon genes pathway. Animals lacking cyclic GMP-AMP synthase display significantly improved early survival after myocardial infarction and diminished pathological remodeling, including ventricular rupture, enhanced angiogenesis, and preserved ventricular contractile function. Furthermore, cyclic GMP-AMP synthase loss of function abolishes the induction of key inflammatory programs such as inducible nitric oxide synthase and promotes the transformation of macrophages to a reparative phenotype, which results in enhanced repair and improved hemodynamic performance. CONCLUSIONS: These results reveal, for the first time, that the cytosolic DNA receptor cyclic GMP-AMP synthase functions during cardiac ischemia as a pattern recognition receptor in the sterile immune response. Furthermore, we report that this pathway governs macrophage transformation, thereby regulating postinjury cardiac repair. Because modulators of this pathway are currently in clinical use, our findings raise the prospect of new treatment options to combat ischemic heart disease and its progression to heart failure.

Temporal dynamics of cardiac hypertrophic growth in response to pressure overload.

Hypertension is one of the most important risk factors of heart failure. In response to high blood pressure, the left ventricle manifests hypertrophic growth to ameliorate wall stress, which may progress into decompensation and trigger pathological cardiac remodeling. Despite the clinical importance, the temporal dynamics of pathological cardiac growth remain elusive. Here, we took advantage of the puromycin labeling approach to measure the relative rates of protein synthesis as a way to delineate the temporal regulation of cardiac hypertrophic growth. We first identified the optimal treatment conditions for puromycin in neonatal rat ventricular myocyte culture. We went on to demonstrate that myocyte growth reached its peak rate after 8-10 h of growth stimulation. At the in vivo level, with the use of an acute surgical model of pressure-overload stress, we observed the maximal growth rate to occur at day 7 after surgery. Moreover, RNA sequencing analysis supports that the most profound transcriptomic changes occur during the early phase of hypertrophic growth. Our results therefore suggest that cardiac myocytes mount an immediate growth response in reply to pressure overload followed by a gradual return to basal levels of protein synthesis, highlighting the temporal dynamics of pathological cardiac hypertrophic growth.NEW & NOTEWORTHY We determined the optimal conditions of puromycin incorporation in cardiac myocyte culture. We took advantage of this approach to identify the growth dynamics of cardiac myocytes in vitro. We went further to discover the protein synthesis rate in vivo, which provides novel insights about cardiac temporal growth dynamics in response to pressure overload.

Static mechanical strain induces capillary endothelial cell cycle re-entry and sprouting.

Vascular endothelial cells are known to respond to a range of biochemical and time-varying mechanical cues that can promote blood vessel sprouting termed angiogenesis. It is less understood how these cells respond to sustained (i.e., static) mechanical cues such as the deformation generated by other contractile vascular cells, cues which can change with age and disease state. Here we demonstrate that static tensile strain of 10%, consistent with that exerted by contractile microvascular pericytes, can directly and rapidly induce cell cycle re-entry in growth-arrested microvascular endothelial cell monolayers. S-phase entry in response to this strain correlates with absence of nuclear p27, a cyclin-dependent kinase inhibitor. Furthermore, this modest strain promotes sprouting of endothelial cells, suggesting a novel mechanical 'angiogenic switch'. These findings suggest that static tensile strain can directly stimulate pathological angiogenesis, implying that pericyte absence or death is not necessarily required of endothelial cell re-activation.

Inhibition of class I histone deacetylases blunts cardiac hypertrophy through TSC2-dependent mTOR repression.

Altering chromatin structure through histone posttranslational modifications has emerged as a key driver of transcriptional responses in cells. Modulation of these transcriptional responses by pharmacological inhibition of class I histone deacetylases (HDACs), a group of chromatin remodeling enzymes, has been successful in blocking the growth of some cancer cell types. These inhibitors also attenuate the pathogenesis of pathological cardiac remodeling by blunting and even reversing pathological hypertrophy. The mechanistic target of rapamycin (mTOR) is a critical sensor and regulator of cell growth that, as part of mTOR complex 1 (mTORC1), drives changes in protein synthesis and metabolism in both pathological and physiological hypertrophy. We demonstrated through pharmacological and genetic methods that inhibition of class I HDACs suppressed pathological cardiac hypertrophy through inhibition of mTOR activity. Mice genetically silenced for HDAC1 and HDAC2 had a reduced hypertrophic response to thoracic aortic constriction (TAC) and showed reduced mTOR activity. We determined that the abundance of tuberous sclerosis complex 2 (TSC2), an mTOR inhibitor, was increased through a transcriptional mechanism in cardiomyocytes when class I HDACs were inhibited. In neonatal rat cardiomyocytes, loss of TSC2 abolished HDAC-dependent inhibition of mTOR activity, and increased expression of TSC2 was sufficient to reduce hypertrophy in response to phenylephrine. These findings point to mTOR and TSC2-dependent control of mTOR as critical components of the mechanism by which HDAC inhibitors blunt pathological cardiac growth. These results also suggest a strategy to modulate mTOR activity and facilitate the translational exploitation of HDAC inhibitors in heart disease.

Propranolol targets the contractility of infantile haemangioma-derived pericytes.

BACKGROUND: Propranolol, a ß-adrenergic receptor (AR) antagonist, is an effective treatment for endangering infantile haemangioma (IH). Dramatic fading of cutaneous colour is often seen a short time after initiating propranolol therapy, with accelerated regression of IH blood vessels discerned after weeks to months. OBJECTIVES: To assess a possible role for haemangioma-derived pericytes (HemPericytes) isolated from proliferating and involuting phase tumours in apparent propranolol-induced vasoconstriction. METHODS: HemPericytes were assayed for contractility on a deformable silicone substrate: propranolol (10 µmol L(-1)) restored basal contractile levels in HemPericytes that were relaxed with the AR agonist epinephrine. Small interfering RNA knockdown of ß2-AR blunted this response. HemPericytes and haemangioma-derived endothelial cells were co-implanted subcutaneously in nude mice to form blood vessels; at day 7 after injection, mice were randomized into vehicle and propranolol-treated groups. RESULTS: HemPericytes expressed high levels of ß2-AR mRNA compared with positive control bladder smooth muscle cells. In addition, ß2-AR mRNA levels were relatively high in IH specimens (n = 15) compared with ß1-AR, ß3-AR and α1b-AR. Normal human retinal and placental pericytes were not affected by epinephrine or propranolol in this assay. Propranolol (10 µmol L(-1)) inhibited the proliferation of HemPericytes in vitro, as well as normal pericytes, indicating a nonselective effect in this assay. Contrast-enhanced microultrasonography of the implants after 7 days of treatment showed significantly decreased vascular volume in propranolol-treated animals, but no reduction in vehicle-treated animals. CONCLUSIONS: These findings suggest that the mechanism of propranolol's effect on proliferating IH involves increased pericytic contractility.