MicroRNA-423-5p Mediates Cocaine-Induced Smooth Muscle Cell Contraction by Targeting Cacna2d2.

Cocaine abuse increases the risk of atherosclerotic cardiovascular disease (CVD) and causes acute coronary syndromes (ACS) and hypertension (HTN). Significant research has explored the role of the sympathetic nervous system mediating the cocaine effects on the cardiovascular (CV) system. However, the response of the sympathetic nervous system alone is insufficient to completely account for the CV consequences seen in cocaine users. In this study, we examined the role of microRNAs (miRNAs) in mediating the effect of cocaine on the CV system. MiRNAs regulate many important biological processes and have been associated with both response to cocaine and CV disease development. Multiple miRNAs have altered expression in the CV system (CVS) upon cocaine exposure. To understand the molecular mechanisms underlying the cocaine response in the CV system, we studied the role of miRNA-423-5p and its target Cacna2d2 in the regulation of intracellular calcium concentration and SMC contractility, a critical factor in the modulation of blood pressure (BP). We used in vivo models to evaluate BP and aortic stiffness. In vitro, cocaine treatment decreased miR-423-5p expression and increased Cacna2d2 expression, which led to elevated intracellular calcium concentrations and increased SMC contractility. Overexpression of miR-423-5p, silencing of its target Cacna2d2, and treatment with a calcium channel blocker reversed the elevated SMC contractility caused by cocaine. In contrast, suppression of miR-423-5p increased the intracellular calcium concentration and SMC contractibility. In vivo, smooth muscle-specific overexpression of miR-423-5p ameliorated the increase in BP and aortic stiffness associated with cocaine use. Thus, miR-423-5p regulates SMC contraction by modulating Cacna2d2 expression increasing intracellular calcium concentrations. Modulation of the miR-423-5p-Cacna2d2-Calcium transport pathway may represent a novel therapeutic strategy to improve cocaine-induced HTN and aortic stiffness.

HIV Promotes Atherosclerosis via Circulating Extracellular Vesicle MicroRNAs.

People living with HIV (PLHIV) are at a higher risk of having cerebrocardiovascular diseases (CVD) compared to HIV negative (HIVneg) individuals. The mechanisms underlying this elevated risk remains elusive. We hypothesize that HIV infection results in modified microRNA (miR) content in plasma extracellular vesicles (EVs), which modulates the functionality of vascular repairing cells, i.e., endothelial colony-forming cells (ECFCs) in humans or lineage negative bone marrow cells (lin- BMCs) in mice, and vascular wall cells. PLHIV (N = 74) have increased atherosclerosis and fewer ECFCs than HIVneg individuals (N = 23). Plasma from PLHIV was fractionated into EVs (HIVposEVs) and plasma depleted of EVs (HIV PLdepEVs). HIVposEVs, but not HIV PLdepEVs or HIVnegEVs (EVs from HIVneg individuals), increased atherosclerosis in apoE-/- mice, which was accompanied by elevated senescence and impaired functionality of arterial cells and lin- BMCs. Small RNA-seq identified EV-miRs overrepresented in HIVposEVs, including let-7b-5p. MSC (mesenchymal stromal cell)-derived tailored EVs (TEVs) loaded with the antagomir for let-7b-5p (miRZip-let-7b) counteracted, while TEVs loaded with let-7b-5p recapitulated the effects of HIVposEVs in vivo. Lin- BMCs overexpressing Hmga2 (a let-7b-5p target gene) lacking the 3'UTR and as such is resistant to miR-mediated regulation showed protection against HIVposEVs-induced changes in lin- BMCs in vitro. Our data provide a mechanism to explain, at least in part, the increased CVD risk seen in PLHIV.

MicroRNA-146a Induces Lineage-Negative Bone Marrow Cell Apoptosis and Senescence by Targeting Polo-Like Kinase 2 Expression.

OBJECTIVE: Lineage-negative bone marrow cells (lin- BMCs) are enriched in endothelial progenitor cells and mediate vascular repair. Aging-associated senescence and apoptosis result in reduced number and functionality of lin- BMCs, impairing their prorepair capacity. The molecular mechanisms underlying lin- BMC senescence and apoptosis are poorly understood. MicroRNAs (miRNAs) regulate many important biological processes. The identification of miRNA-mRNA networks that modulate the health and functionality of lin- BMCs is a critical step in understanding the process of vascular repair. The aim of this study was to characterize the role of the miR-146a-Polo-like kinase 2 (Plk2) network in regulating lin- BMC senescence, apoptosis, and their angiogenic capability. APPROACH AND RESULTS: Transcriptome analysis in lin- BMCs isolated from young and aged wild-type and ApoE-/- (apolipoprotein E) mice showed a significant age-associated increase in miR-146a expression. In silico analysis, expression study and Luciferase reporter assay established Plk2 as a direct target of miR-146a. miR-146a overexpression in young lin- BMCs inhibited Plk2 expression, resulting in increased senescence and apoptosis, via p16Ink4a/p19Arf and p53, respectively, as well as impaired angiogenic capacity in vitro and in vivo. Conversely, suppression of miR-146a in aged lin- BMCs increased Plk2 expression and rejuvenated lin- BMCs, resulting in decreased senescence and apoptosis, leading to improved angiogenesis. CONCLUSIONS: (1) miR-146a regulates lin- BMC senescence and apoptosis by suppressing Plk2 expression that, in turn, activates p16Ink4a/p19Arf and p53 and (2) modulation of miR-146a or its target Plk2 may represent a potential therapeutic intervention to improve lin- BMC-mediated angiogenesis and vascular repair.

A cardiac myocyte-restricted Lin28/let-7 regulatory axis promotes hypoxia-mediated apoptosis by inducing the AKT signaling suppressor PIK3IP1.

RATIONALE: The let-7 family of microRNAs (miRs) regulates critical cell functions, including survival signaling, differentiation, metabolic control and glucose utilization. These functions may be important during myocardial ischemia. MiR-let-7 expression is under tight temporal and spatial control through multiple redundant mechanisms that may be stage-, isoform- and tissue-specific. OBJECTIVE: To determine the mechanisms and functional consequences of miR-let-7 regulation by hypoxia in the heart. METHODS AND RESULTS: MiR-let-7a, -7c and -7g were downregulated in the adult mouse heart early after coronary occlusion, and in neonatal rat ventricular myocytes subjected to hypoxia. Let-7 repression did not require glucose depletion, and occurred at a post-transcriptional level. Hypoxia also induced the RNA binding protein Lin28, a negative regulator of let-7. Hypoxia ineither induced Lin28 nor repressed miR-let-7 in cardiac fibroblasts. Both changes were abrogated by treatment with the histone deacetylase inhibitor trichostatin A. Restoration of let-7g to hypoxic myocytes and to ischemia-reperfused mouse hearts in vivo via lentiviral transduction potentiated the hypoxia-induced phosphorylation and activation of Akt, and prevented hypoxia-dependent caspase activation and death. Mechanistically, phosphatidyl inositol 3-kinase interacting protein 1 (Pik3ip1), a negative regulator of PI3K, was identified as a novel target of miR-let-7 by a crosslinking technique showing that miR-let-7g specifically targets Pik3ip1 to the cardiac myocyte Argonaute complex RISC. Finally, in non-failing and failing human myocardium, we found specific inverse relationships between Lin28 and miR-let-7g, and between miR-let-7g and PIK3IP1. CONCLUSION: A conserved hypoxia-responsive Lin28-miR-let-7-Pik3ip1 regulatory axis is specific to cardiac myocytes and promotes apoptosis during myocardial ischemic injury.

Lin28a protects against diabetic cardiomyopathy via the PKA/ROCK2 pathway.

BACKGROUND: Lin28a enhances glucose uptake and insulin-sensitivity. However, the role of Lin28a on experimental diabetic cardiomyopathy (DCM) is not well understood. We investigated the potential role and mechanism ofLin28a in diabetes-induced myocardial dysfunction in mice. METHODS: Diabetes was induced by intraperitoneal (i.p.) injections of Streptozocin (STZ) in mice. Animals were randomized to be treated with lentivirus carrying Lin28a siRNA or Lin28a cDNA. Cardiac function, cardiomyocyte autophagy, apoptosis and mitochondria morphology in diabetic mice were compared between groups. The target proteins of Lin28a were examined by western blot analysis. RESULTS: Lin28a levels were markedly reduced in the cardiac tissue compared to the control mice. Lin28a overexpression significantly improved left ventricular ejection fraction (LVEF), promoted autophagy, decreased myocardial apoptotic index and alleviated mitochondria cristae destruction in diabetic mice. Lin28a knockdown exacerbated diabetic injury as evidenced by decreased LVEF, increased apoptotic index and aggravated mitochondria cristae destruction. Interestingly, pretreatment with a PKA inhibitor, N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide], di-HCl Salt (H89) abolished the beneficial effects of Lin28a overexpression. RhoA-expression and ROCK2-expression were decreased in vivo after Lin28a overexpression, while Lin28a knockdown increased the expression of RhoA and ROCK2 in diabetic mice. CONCLUSIONS: Lin28a protects against DCM through PKA/ROCK2 dependent pathway. Lin28a might serve as a potential therapeutic target for the treatment of the patients with DCM.

Erratum to: PI3K/mTOR inhibition can impair tumor invasion and metastasis in vivo despite a lack of antiproliferative action in vitro: implications for targeted therapy.

Erratum to: Breast Cancer Res Treat (2013),138:369­381,DOI 10.1007/s10549-012-2389-6. In the original publication of the article, the Fig. 4c and d were published erroneously. The revised Fig. 4 is given in this erratum.

MicroRNA-10A* and MicroRNA-21 modulate endothelial progenitor cell senescence via suppressing high-mobility group A2.

RATIONALE: Endothelial progenitor cells (EPCs) contribute to the regeneration of endothelium. Aging-associated senescence results in reduced number and function of EPCs, potentially contributing to increased cardiac risk, reduced angiogenic capacity, and impaired cardiac repair effectiveness. The mechanisms underlying EPC senescence are unknown. Increasing evidence supports the role of microRNAs in regulating cellular senescence. OBJECTIVE: We aimed to determine whether microRNAs regulated EPC senescence and, if so, what the underlying mechanisms are. METHODS AND RESULTS: To map the microRNA/gene expression signatures of EPC senescence, we performed microRNA profiling and microarray analysis in lineage-negative bone marrow cells from young and aged wild-type and apolipoprotein E-deficient mice. We identified 2 microRNAs, microRNA-10A* (miR-10A*), and miR-21, and their common target gene Hmga2 as critical regulators for EPC senescence. Overexpression of miR-10A* and miR-21 in young EPCs suppressed Hmga2 expression, caused EPC senescence, as evidenced by senescence-associated ß-galactosidase upregulation, decreased self-renewal potential, increased p16(Ink4a)/p19(Arf) expression, and resulted in impaired EPC angiogenesis in vitro and in vivo, resembling EPCs derived from aged mice. In contrast, suppression of miR-10A* and miR-21 in aged EPCs increased Hmga2 expression, rejuvenated EPCs, resulting in decreased senescence-associated ß-galactosidase expression, increased self-renewal potential, decreased p16(Ink4a)/p19(Arf) expression, and improved EPC angiogenesis in vitro and in vivo. Importantly, these phenotypic changes were rescued by miRNA-resistant Hmga2 cDNA overexpression. CONCLUSIONS: miR-10A* and miR-21 regulate EPC senescence via suppressing Hmga2 expression and modulation of microRNAs may represent a potential therapeutic intervention in improving EPC-mediated angiogenesis and vascular repair.

Synergistic induction of miR-126 by hypoxia and HDAC inhibitors in cardiac myocytes.

HDAC inhibitors are under clinical development for the treatment of hypertrophic cardiomyopathy and heart failure although the mechanisms of protection are incompletely understood. Micro-RNA 126, an endothelium-specific miR has been assigned essential developmental roles in the heart by activating survival kinases ERK1/2 and Akt and increasing pro-angiogenic signaling. Here we provide the first evidence that hypoxia and HDAC inhibitors selectively and synergistically stimulate expression of miR-126 in cardiac myocytes. MiR-126 expression was increased 1.7-fold (p<0.05) after 1h of hypoxic exposure and this was further enhanced to 3.0-fold (p<0.01) by simultaneously blocking HDAC with the pan-HDAC inhibitor Tricostatin A (TSA). TSA alone did not increase miR-126. In parallel, hypoxia and TSA synergistically increased p-ERK and p-Akt without effecting VEGF-A level. Knockdown of miR-126 with si-RNA eliminated inductions of p-ERK and p-Akt by hypoxia, whereas miR-126 overexpression mimicked hypoxia and amplified p-ERK and p-Akt in parallel with miR-126. The results suggest that miR-126 is a hypoxia-inducible target of HAT/HDAC and its activation in cardiac myocytes may contribute to cardioprotection by activating cell survival and pro-angiogenic pathways selectively during ischemia.

PI3K/mTOR inhibition can impair tumor invasion and metastasis in vivo despite a lack of antiproliferative action in vitro: implications for targeted therapy.

Oncogenic PI3K/mTOR activation is frequently observed in human cancers and activates cell motility via p27 phosphorylations at T157 and T198. Here we explored the potential for a novel PI3K/mTOR inhibitor to inhibit tumor invasion and metastasis. An MDA-MB-231 breast cancer line variant, MDA-MB-231-1833, with high metastatic bone tropism, was treated with a novel catalytic PI3K/mTOR inhibitor, PF-04691502, at nM doses that did not impair proliferation. Effects on tumor cell motility, invasion, p27 phosphorylation, localization, and bone metastatic outgrowth were assayed. MDA-MB-231-1833 showed increased PI3K/mTOR activation, high levels of cytoplasmic p27pT157pT198 and increased cell motility and invasion in vitro versus parental. PF-04691502 treatment, at a dose that did not affect proliferation, reduced total and cytoplasmic p27, decreased p27pT157pT198 and restored cell motility and invasion to levels seen in MDA-MB-231. p27 knockdown in MDA-MB-231-1833 phenocopied PI3K/mTOR inhibition, whilst overexpression of the phosphomimetic mutant p27T157DT198D caused resistance to the anti-invasive effects of PF-04691502. Pre-treatment of MDA-MB-231-1833 with PF-04691502 significantly impaired metastatic tumor formation in vivo, despite lack of antiproliferative effects in culture and little effect on primary orthotopic tumor growth. A further link between cytoplasmic p27 and metastasis was provided by a study of primary human breast cancers which showed cytoplasmic p27 is associated with increased lymph nodal metastasis and reduced survival. Novel PI3K/mTOR inhibitors may oppose tumor metastasis independent of their growth inhibitory effects, providing a rationale for clinical investigation of PI3K/mTOR inhibitors in settings to prevent micrometastasis. In primary human breast cancers, cytoplasmic p27 is associated with worse outcomes and increased nodal metastasis, and may prove useful as a marker of both PI3K/mTOR activation and PI3K/mTOR inhibitor efficacy.

c-Jun N-terminal kinase (JNK-1) confers protection against brief but not extended ischemia during acute myocardial infarction.

Brief periods of ischemia do not damage the heart and can actually protect against reperfusion injury caused by extended ischemia. It is not known what causes the transition from protection to irreversible damage as ischemia progresses. c-Jun N-terminal kinase-1 (JNK-1) is a stress-regulated kinase that is activated by reactive oxygen and thought to promote injury during severe acute myocardial infarction. Because some reports suggest that JNK-1 can also be protective, we hypothesized that the function of JNK-1 depends on the metabolic state of the heart at the time of reperfusion, a condition that changes progressively with duration of ischemia. Mice treated with JNK-1 inhibitors or transgenic mice wherein the JNK-1 gene was ablated were subjected to 5 or 20 min of ischemia followed by reperfusion. When JNK-1 was inactive, ischemia of only 5 min duration caused massive apoptosis, infarction, and negative remodeling that was equivalent to or greater than extended ischemia. Conversely, when ischemia was extended JNK-1 inactivation was protective. Mechanisms of the JNK-1 switch in function were investigated in vivo and in cultured cardiac myocytes. In vitro there was a comparable switch in the function of JNK-1 from protective when ATP levels were maintained during hypoxia to injurious when reoxygenation followed glucose and ATP depletion. Both apoptotic and necrotic death pathways were affected and responded reciprocally to JNK-1 inhibitors. JNK-1 differentially regulated Akt phosphorylation of the regulatory sites Ser-473 and Thr-450 and the catalytic Thr-308 site in vivo. The studies define a novel role for JNK-1 as a conditional survival kinase that protects the heart against brief but not protracted ischemia.

Auto-acetylation stabilizes p300 in cardiac myocytes during acute oxidative stress, promoting STAT3 accumulation and cell survival.

The nuclear acetyltransferase p300 is rapidly and stably induced in the heart during hemodynamic stress, but the mechanism of this induction is unknown. To determine the role of oxidative stress in p300 induction, we exposed neonatal rat cardiac myocytes to doxorubicin (DOX, 1 µM) or its vehicle, and monitored p300 protein content and stability for 24 h. Levels of p300 rose substantially within 1 h and remained elevated for at least 24 h, while p300 transcript levels declined. In the presence of cycloheximide, the estimated half-life of p300 in control cells was approximately 4.5 h, typical of an immediate-early response protein. DOX treatment prolonged p300 t(1/2) to >24 h, indicating that the sharp rise in p300 levels was attributable to rapid protein stabilization. p300 stabilization was entirely due to an increase in acetylated p300 species with greatly enhanced resistance to proteasomal degradation. The half-life of p300 was dependent on its acetyltransferase activity, falling in the presence of p300 inhibitors curcumin and anacardic acid, and increasing with histone deacetylase (HDAC) inhibition. At the same time, acetyl-STAT3, phospho-STAT3-(Tyr 705) and -(Ser 727) increased, together with a prolongation of STAT3 half-life. SiRNA-mediated p300 knockdown abrogated all of these effects, and strongly enhanced DOX-mediated myocyte apoptosis. We conclude that DOX induces an acute amplification of p300 levels through auto-acetylation and stabilization. In turn, elevated p300 provides a key defense against acute oxidative stress in cardiac myocytes by acetylation, activation, and stabilization of STAT3. Our results suggest that HDAC inhibitors could potentially reduce acute anthracycline-mediated cardiotoxicity by promoting p300 auto-acetylation.

MiR-126-HMGB1-HIF-1 Axis Regulates Endothelial Cell Inflammation during Exposure to Hypoxia-Acidosis.

Crosstalk between molecular regulators miR-126, hypoxia-inducible factor 1-alpha (HIF-1-α), and high-mobility group box-1 (HMGB1) contributes to the regulation of inflammation and angiogenesis in multiple physiological and pathophysiological settings. Here, we present evidence of an overriding role for miR-126 in the regulation of HMGB1 and its downstream proinflammatory effectors in endothelial cells subjected to hypoxia with concurrent acidosis (H/A). Methods. Primary mouse endothelial cells (PMEC) were exposed to hypoxia or H/A to simulate short or chronic low-flow ischemia, respectively. RT-qPCR quantified mRNA transcripts, and proteins were measured by western blot. ROS were quantified by fluorogenic ELISA and luciferase reporter assays employed to confirm an active miR-126 target in the HMGB1 3'UTR. Results. Enhanced expression of miR-126 in PMECs cultured under neutral hypoxia was suppressed under H/A, whereas the HMGB1 expression increased sequentially under both conditions. Enhanced expression of HMGB1 and downstream inflammation markers was blocked by the premiR-126 overexpression and optimized by antagomiR. Compared with neutral hypoxia, H/A suppressed the HIF-1α expression independently of miR-126. The results show that HMGB1 and downstream effectors are optimally induced by H/A relative to neutral hypoxia via crosstalk between hypoxia signaling, miR-126, and HIF-1α, whereas B-cell lymphoma 2(Bcl2), a HIF-1α, and miR-126 regulated gene expressed optimally under neutral hypoxia. Conclusion. Inflammatory responses of ECs to H/A are dynamically regulated by the combined actions of hypoxia, miR-126, and HIF-1α on the master regulator HMGB1. The findings may be relevant to vascular diseases including atherosclerotic occlusion and interiors of plaque where coexisting hypoxia and acidosis promote inflammation as a defining etiology.

Comparison of microsomal prostaglandin E synthase-1 deletion and COX-2 inhibition in acute cardiac ischemia in mice.

The aim of the present study was to compare the effects of genetic mPGES-1 loss and COX-2 inhibition on myocardial damage after coronary occlusion. mPGES-1(-/-) mice and their wild-type littermates were injected with vehicle or COX-2 inhibitor (celecoxib), and 30min later the left coronary artery was surgically occluded. At 24h, myocardial infarct (MI) volume was measured histologically. Post-MI survival was reduced in WT mice receiving celecoxib (12/20) compared with vehicle-treated controls (12/12) or the loss of mPGES-1 (13/13) together with increased phosphokinase (CPK) and cardiac troponin-I release. Endogenous mPGES-1 expression was unchanged by ischemia in WT mice and absent in mPGES-1(-/-) hearts. COX-2 expression was markedly increased at 24h after MI in WT hearts; this upregulation was largely attenuated in mPGES-1(-/-) mice. We conclude that loss of mPGES-1 prevents the upregulation of COX-2 after myocardial infarct, and in contrast to inhibition of COX-2, does not increase ischemic myocardial damage.

MicroRNA-25 Protects Smooth Muscle Cells against Corticosterone-Induced Apoptosis.

BACKGROUND AND AIMS: Vascular smooth muscle cells (VSMCs) are central components of atherosclerotic plaque. Loss of VSMCs through apoptotic cell death can cause fibrous cap thinning, necrotic core formation, and calcification that may destabilize plaque. Elevated glucocorticoid levels caused by psychological stress promote VSMC apoptosis and can exacerbate atherosclerosis in mice and humans. Changes in the levels of antiapoptosis microRNA-25 (miR-25) have been linked with heart disease, inflammation, VSMC phenotype, oxidative stress, and apoptosis. Here, we investigated the pathways and mechanisms of glucocorticoid-induced apoptosis of mouse VSMCs and the protective role of miR-25. METHODS: Primary mouse VSMCs were cultured +/- corticosterone for 48 h. Apoptosis, ROS, apoptotic protein activities, miR-25, MOAP1, a miR-25 target, and p70S6 kinase were quantified at intervals. The roles of miR-25 were assessed by treating cells with lenti-pre-miR-25 and anti-miR-25. RESULTS: VSMC apoptosis, caspase-3 activity, and Bax were increased by corticosterone, and cell death was paralleled by marked loss of miR-25. Protection was conferred by pre-miR-25 and exacerbated by anti-miR-25. Pre-miR-25 conferred reduced expression of the proapoptotic protein MOAP1, and the protective effects of pre-miR-25 were abrogated by overexpressing MOAP1. The antiapoptotic effects of miR-25 were paralleled by inhibition of the p70S6K pathway, a convergence target for the survival signaling pathways, and protection by pre-miR-25 was abrogated by the p70S6k inhibitor rapamycin. CONCLUSIONS: MicroRNA-25 blocks corticosterone-induced VSMC apoptosis by targeting MOAP1 and the p70S6k pathway. Therapeutic manipulation of miR-25 may reduce atherosclerosis and unstable plaque formation associated with chronic stress.

The role of chemokine receptor 9/chemokine ligand 25 signaling: From immune cells to cancer cells.

Chemokine ligand 25 (CCL25) and chemokine receptor 9 (CCR9) are important regulators of migration, proliferation and apoptosis in leukocytes and cancer cells. Blocking of the CCR9/CCL25 signal has been demonstrated to be a potential novel cancer therapy. Research into CCR9 and CCL25 has revealed their associated upstream and downstream signaling pathways; CCR9 is regulated by several immunological factors, including NOTCH, interleukin 2, interleukin 4 and retinoic acid. NOTCH in particular, has been revealed to be a crucial upstream regulator of CCR9. Furthermore, proteins including matrix metalloproteinases, P-glycoprotein, Ezrin/Radixin/Moesin and Livin are regulated via phosphatidylinositol-3 kinase/protein kinase B, which are in turn stimulated by CCR9/CCL25. This is a review of the current literature on the functions and signaling pathways of CCR9/CCL25.

Cocaine Exposure Increases Blood Pressure and Aortic Stiffness via the miR-30c-5p-Malic Enzyme 1-Reactive Oxygen Species Pathway.

Cocaine abuse increases the risk of cardiovascular mortality and morbidity; however, the underlying molecular mechanisms remain elusive. By using a mouse model for cocaine abuse/use, we found that repeated cocaine injection led to increased blood pressure and aortic stiffness in mice associated with elevated levels of reactive oxygen species (ROS) in the aortas, a phenomenon similar to that observed in hypertensive humans. This ROS elevation was correlated with downregulation of Me1 (malic enzyme 1), an important redox molecule that counteracts ROS generation, and upregulation of microRNA (miR)-30c-5p that targets Me1 expression by directly binding to its 3'UTR (untranslated region). Remarkably, lentivirus-mediated overexpression of miR-30c-5p in aortic smooth muscle cells recapitulated the effect of cocaine on Me1 suppression, which in turn led to ROS elevation. Moreover, in vivo silencing of miR-30c-5p in smooth muscle cells resulted in Me1 upregulation, ROS reduction, and significantly suppressed cocaine-induced increases in blood pressure and aortic stiffness-a similar effect to that produced by treatment with the antioxidant N-acetyl cysteine. Discovery of this novel cocaine-↑miR-30c-5p-↓Me1-↑ROS pathway provides a potential new therapeutic avenue for treatment of cocaine abuse-related cardiovascular disease.

Regulation of Bnip3 death pathways by calcium, phosphorylation, and hypoxia-reoxygenation.

Bnip3 is a proapoptotic member of the Bcl-2 family of death-regulating proteins that promote the intrinsic pathway of programmed cell death. The Bnip3 death program requires membrane insertion through an N-terminal transmembrane domain that directs the protein to mitochondrial and endoplasmic reticular (ER) membranes. We have reported that simulated ischemia induces transcription of the Bnip3 gene, and Bnip3 protein is stabilized by acidosis. Bnip3 programmed death is atypical, with features of both apoptosis and necrosis. Here we demonstrate that hypoxia-reoxygenation and agents that activate protein kinase C, including calcium ionophore, phorbol 12-myristate 13-acetate, and okadaic acid, also induce Bnip3. The molecular size of Bnip3 predicted from the amino acid sequence is 21.5 kDa, but the protein typically migrates in SDS-PAGE as a 31-kDa monomer and 60-kDa dimer. Treatment of cell extracts containing Bnip3 with phosphatase yielded a series of rapidly migrating species, the smallest of which corresponded with the theoretic molecular size of Bnip3. Conversely, treatment of cells with okadaic acid eliminated the rapidly migrating species, suggesting that Bnip3 phosphorylation is a dynamic process. Elevated levels of the phosphoprotein correlated with initiation of Bnip3-dependent death, whereas the dephosphorylated species correlated with extreme acidosis.

Reversal of pathological cardiac hypertrophy via the MEF2-coregulator interface.

Cardiac hypertrophy, as a response to hemodynamic stress, is associated with cardiac dysfunction and death, but whether hypertrophy itself represents a pathological process remains unclear. Hypertrophy is driven by changes in myocardial gene expression that require the MEF2 family of DNA-binding transcription factors, as well as the nuclear lysine acetyltransferase p300. Here we used genetic and small-molecule probes to determine the effects of preventing MEF2 acetylation on cardiac adaptation to stress. Both nonacetylatable MEF2 mutants and 8MI, a molecule designed to interfere with MEF2-coregulator binding, prevented hypertrophy in cultured cardiac myocytes. 8MI prevented cardiac hypertrophy in 3 distinct stress models, and reversed established hypertrophy in vivo, associated with normalization of myocardial structure and function. The effects of 8MI were reversible, and did not prevent training effects of swimming. Mechanistically, 8MI blocked stress-induced MEF2 acetylation, nuclear export of class II histone deacetylases HDAC4 and -5, and p300 induction, without impeding HDAC4 phosphorylation. Correspondingly, 8MI transformed the transcriptional response to pressure overload, normalizing almost all 232 genes dysregulated by hemodynamic stress. We conclude that MEF2 acetylation is required for development and maintenance of pathological cardiac hypertrophy, and that blocking MEF2 acetylation can permit recovery from hypertrophy without impairing physiologic adaptation.

Bnip3 Binds and Activates p300: Possible Role in Cardiac Transcription and Myocyte Morphology.

Bnip3 is a hypoxia-regulated member of the Bcl-2 family of proteins that is implicated in apoptosis, programmed necrosis, autophagy and mitophagy. Mitochondria are thought to be the primary targets of Bnip3 although its activities may extend to the ER, cytoplasm, and nucleus. Bnip3 is induced in the heart by ischemia and pressure-overload, and may contribute to cardiomyopathy and heart failure. Only mitochondrial-dependent programmed death actions have been described for Bnip3 in the heart. Here we describe a novel activity of Bnip3 in cultured cardiac myocytes and transgenic mice overexpressing Bnip3 in the heart (Bnip3-TG). In cultured myocytes Bnip3 bound and activated the acetyltransferase p300, increased acetylation of histones and the transcription factor GATA4, and conferred p300 and GATA4-sensitive cellular morphological changes. In intact Bnip3-TG hearts Bnip3 also bound p300 and GATA4 and conferred enhanced GATA4 acetylation. Bnip3-TG mice underwent age-dependent ventricular dilation and heart failure that was partially prevented by p300 inhibition with curcumin. The results suggest that Bnip3 regulates cardiac gene expression and perhaps myocyte morphology by activating nuclear p300 acetyltransferase activity and hyperacetylating histones and p300-selective transcription factors.

Repression of miR-142 by p300 and MAPK is required for survival signalling via gp130 during adaptive hypertrophy.

An increase in cardiac workload, ultimately resulting in hypertrophy, generates oxidative stress and therefore requires the activation of both survival and growth signal pathways. Here, we wanted to characterize the regulators, targets and mechanistic roles of miR-142, a microRNA (miRNA) negatively regulated during hypertrophy. We show that both miRNA-142-3p and -5p are repressed by serum-derived growth factors in cultured cardiac myocytes, in models of cardiac hypertrophy in vivo and in human cardiomyopathic hearts. Levels of miR-142 are inversely related to levels of acetyltransferase p300 and MAPK activity. When present, miR-142 inhibits both survival and growth pathways by directly targeting nodal regulators p300 and gp130. MiR-142 also potently represses multiple components of the NF-κB pathway, preventing cytokine-mediated NO production and blocks translation of α-actinin. Forced expression of miR-142 during hypertrophic growth induced extensive apoptosis and cardiac dysfunction; conversely, loss of miR-142 fully rescued cardiac function in a murine heart failure model. Downregulation of miR-142 is required to enable cytokine-mediated survival signalling during cardiac growth in response to haemodynamic stress and is a critical element of adaptive hypertrophy.