MicroRNA-328 as a regulator of cardiac hypertrophy

Cardiac hypertrophy is a primary predictor of progressive heart disease that often results in heart failure. Growing evidence has demonstrated that microRNAs (miRNAs) play a critical role in regulating cardiac hypertrophy. This study was designed to evaluate the effect of miR-328 on cardiac hypertrophy and the potential molecular mechanisms. We found that transgenic overexpression of miR-328 in the heart induced cardiac hypertrophy in mice, which was accompanied by reduced SERCA2a level increased intracellular calcium concentration and calcineurin protein level, and enhanced NFATc3 nuclear translocation. However, normalization of miR-328 level by its antisense chemically modified with locked nucleic acid (LNA-antimiR-328) reversed the changes. Forced expression of miR-328 resulted in cardiomyocyte hypertrophy in cultured neonatal rat ventricular cells, which was accompanied by downregulation of SERCA2a expression and activation of the calcineurin/NFATc3 signaling pathway. These changes were abolished by LNA-antimiR-328. We validated the SERCA2a as a direct target for miR-328. MiR-328 expression was upregulated in cardiomyocyte treated with isoproterenol (ISO) to induce hypertrophy; while knockdown of miR-328 attenuated the hypertrophic responses. The level of miR-328 was significantly elevated in a mouse model of hypertrophy by thoracic aortic banding (TAC). Consistently, SERCA2a was downregulated, whereas calcineurin were upregulated, and NFATc3 nuclear translocation was enhanced. In contrast, hypertrophy in these mice was significantly alleviated when treated with miR-328 antisense. MiR-328 promotes cardiac hypertrophy by targeting SERCA2a. Our study therefore uncovered a novel molecular mechanism for cardiac hypertrophy and indicated miR-328 as a potential therapeutic target for this cardiac condition.     

拉西地平对CTGF诱导大鼠心肌细胞肥大的影响及其与ERK1/2的关系

目的 观察拉西地平对结缔组织生长因子（CTGF）诱导大鼠心肌细胞肥大的作用,探讨其作用与细胞外信号调节激酶1/2（ERK1/2）的关系。方法 以培养的新生SD大鼠的心肌细胞为实验模型,用图象分析法、3H-亮氨酸掺入法、考马斯亮蓝染色、蛋白免疫印迹法（Western blot）等,评价拉西地平对CTGF诱导心肌细胞肥大的抑制作用及其与ERK1/2的关系。结果 ①以50 ng/L的CTGF作用24 h后,心肌细胞表面积为（1 812.52±168.73）μm2,与对照组（689.31±96.58）μm2比较显著增加（P<0.01）;5、10、25及50 μmol/L拉西地平干预组的心肌细胞表面积分别为（1 476.52±156.73）μm2、（1 120.39±149.68）μm2、（926.10±101.44）μm2及（739.81±91.55）μm2,与CTGF组比较呈浓度依赖性降低（P<0.01）。②以50 ng/L的CTGF作用24 h后,心肌细胞的3H-亮氨酸掺入率为（2 368.72±122.45）cpm/孔,与对照组（950.26±89.43）cpm/孔比较显著增加（P<0.01）。 5、10、25及50 μmol/L拉西地平干预组的心肌细胞的3H-亮氨酸掺入率,分别为（2023.12±106.15）cpm/孔、（1629.15±103.46）cpm/孔、（1302.19±98.53）cpm/孔及（1 055.72±90.96）cpm/孔,与CTGF组比较呈浓度依赖性降低（P<0.01）。③以50 ng/L的CTGF作用24 h后,心肌细胞蛋白的含量为（1.692±0.203）ng/细胞,与对照组（0.622±0.068）ng/细胞比较显著增加（P<0.01）;5、10、25及50 μmol/L拉西地平干预组的心肌细胞蛋白的含量分别为（1.269±0.167）ng/细胞、（0.923±0.119）ng/细胞、（0.766±0.085）ng/细胞及（0.682±0.063）ng/细胞,与CTGF组比较呈浓度依赖性降低（P<0.01）。④50 ng/L CTGF组的心肌细胞p-ERK1/2表达强度明显高于对照组,5 μmol/L、10 μmol/L、25 μmol/L、50 μmol/L拉西地平干预组心肌细胞p-ERK1/2的表达强度与CTGF组比较呈浓度依赖性降低。心肌细胞t-ERK1/2的表达在各组之间没有明显的差异。结论 拉西地平可抑制CTGF诱导的心肌细胞肥大,其作用机制可能与ERK1/2磷酸化有关。     

PICOT is a critical regulator of cardiac hypertrophy and cardiomyocyte contractility

PICOT (PKC-interacting cousin of thioredoxin) was previously shown to inhibit the development of cardiac hypertrophy, concomitant with an increase in cardiomyocyte contractility. To explore the physiological function of PICOT in the hearts, we generated a PICOT-deficient mouse line by using a gene trap approach. PICOT^−/− mice were embryonic lethal indicating that PICOT plays an essential role during embryogenesis, whereas PICOT^+/− mice were viable with no apparent morphological defects. The PICOT protein levels were reduced by about 50% in the hearts of PICOT^+/− mice. Significantly exacerbated cardiac hypertrophy was induced by pressure overload in PICOT^+/− mice relative to that seen in wild type littermates. In line with this observation, calcineurin-NFAT signaling was greatly enhanced by pressure overload in the hearts of PICOT^+/− mice. Cardiomyocytes from PICOT^+/− mice exhibited significantly reduced contractility, which may be due in part to hypophosphorylation of phospholamban and reduced SERCA activity. These data indicate that the precise PICOT protein level significantly affects the process of cardiac hypertrophy and cardiomyocyte contractility. We suggest that PICOT plays as a critical negative regulator of cardiac hypertrophy and a positive inotropic regulator.     

Genetic inhibition of cardiac ERK1/2 promotes stress-induced apoptosis and heart failure but has no effect on hypertrophy in vivo

MAPK signaling pathways function as critical regulators of cellular differentiation, proliferation, stress responsiveness, and apoptosis. One branch of the MAPK signaling pathway that culminates in ERK1/2 activation is hypothesized to regulate the growth and adaptation of the heart to both physiologic and pathologic stimuli, given its known activation in response to virtually every stress- and agonist-induced hypertrophic stimulus examined to date. Here we investigated the requirement of ERK1/2 signaling in mediating the cardiac hypertrophic growth response in Erk1(-/-) and Erk2(+/-) mice, as well as in transgenic mice with inducible expression of an ERK1/2-inactivating phosphatase in the heart, dual-specificity phosphatase 6. Although inducible expression of dual-specificity phosphatase 6 in the heart eliminated ERK1/2 phosphorylation at baseline and after stimulation without affecting any other MAPK, it did not diminish the hypertrophic response to pressure overload stimulation, neuroendocrine agonist infusion, or exercise. Similarly, Erk1(-/-) and Erk2(+/-) mice showed no reduction in pathologic or physiologic stimulus-induced cardiac growth in vivo. However, blockade or deletion of cardiac ERK1/2 did predispose the heart to decompensation and failure after long-term pressure overload in conjunction with an increase in myocyte TUNEL. Thus, ERK1/2 signaling is not required for mediating physiologic or pathologic cardiac hypertrophy in vivo, although it does play a protective role in response to pathologic stimuli.     

KMUP-1 inhibits hypertension-induced left ventricular hypertrophy through regulation of nitric oxide synthases,ERK1/2, and calcineurin

Hypertension can induce left ventricular hypertrophy (LVH), and the nitric oxide (NO) pathway plays an important role in the pathogenesis of cardiac hypertrophy. This study aimed to examine whether KMUP-1, a novel xanthine-based derivative, could inhibit LVH in spontaneously hypertensive rats (SHRs) and to investigate potential mechanisms underlying its antihypertrophic effects. Two groups of animals with chronic or subacute LVH were treated. In the chronic LVH group, KMUP-1 (10 or 30 mg/kg/d orally) was administered for 28 days to both normotensive rats and SHRs. In the subacute LVH group, KMUP-1 (0.5 mg/kg/d intraperitoneally) or sildenafil (0.7 mg/kg/d intraperitoneally) was administered for 10 days with or without co-treatment with the nitric oxide synthase (NOS) inhibitor N-omega-nitro-l-arginine (L-NNA; 20 mg/L orally). After treatment, the effects of KMUP-1 or sildenafil on hypertension, cardiac hypertrophy, survival, expression of the NO/soluble guanylate cyclase (sGC)/protein kinase G (NO/sGC/PKG) pathway in the aorta andleft ventricle, and calcineurin A/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the left ventricle were examined. In the chronic LVH group, the SHRs developed hypertension with LVH over the 28 days. KMUP-1 attenuated the hypertension and LVH, increased survival rate, enhanced endothelial NOS/cyclic guanosine monophosphate/PKG (eNOS/cGMP/PKG) and decreased inducible NOS (iNOS) expression in the aorta and left ventricle of the SHRs. In the subacute LVH group, both KMUP-1 and sildenafil administered for 10 days attenuated the LVH in SHRs, with enhanced eNOS/cGMP/PKG and suppressed iNOS/calcineurin A/ERK1/2 expression in the left ventricle. In addition, both KMUP-1 and sildenafil attenuated L-NNA-induced LVH. KMUP-1 inhibition of hypertension-induced LVH with associated upregulation of eNOS, downregulation of iNOS in both the aorta and left ventricle, and attenuation of calcineurin A and ERK1/2 signaling in the left ventricle.     

Effects of electroacupuncture at Neiquan (PC6) on protein expressions of Raf-1,ERK 1/2,and p-ERK 1/2 of Rats with myocardial hypertrophy

正Objective To observe the effects of electroacupuncture(EA)at Neiguan(PC6)on protein expressions of proto-oncogene serine/threonine-protein kinase-1(Raf-1),phospho-extracellular signal-regulated kinase 1/2(p-ERK 1/2),and extracellular signal-regulated kinase1/2(ERK 1/2)in rats with myocardial hypertrophy.Methods Totally 40 healthy Sprague-Dawley(SD)rats of clean grade were divided into 4 groups according to random digit table,i.e.,the normal group,the model     

Anti-beta1-adrenergic receptor autoantibodies are potent stimulators of the ERK1/2 pathway in cardiac cells

OBJECTIVE: Antibodies specific for the beta1-adrenergic receptor (beta1AR) are highly prevalent in patients with idiopathic dilated cardiomyopathy (DCM) and known to contribute to the pathogenesis of heart failure, though the precise molecular mechanisms involved are largely unknown. METHODS: We have explored the effects of beta(1)AR autoantibodies obtained from DCM patients on extracellular signal-regulated kinase (ERK) activation in murine cardiomyocytes. RESULTS: We find that human beta(1)AR autoantibodies potently stimulate ERK1/2 in cardiac cells by using signalling pathways different from those triggered by the classic beta-agonist isoproterenol, also leading to a different pattern of activated ERK subcellular localization. The extent of ERK stimulation by endogenous cardiac beta(1)AR is markedly enhanced in the presence of both beta(1)AR-autoantibodies and isoproterenol. Interestingly, beta(1)AR-autoantibody-mediated ERK activation is not blocked by some betaAR antagonists used in the treatment of heart failure. CONCLUSIONS: Our results suggest that these antibodies elicit a distinct beta(1)AR active conformation that would lead to the engagement of signaling effectors different from those recruited by classic beta-agonists, a finding that could lead to better understanding of DCM pathogenesis and aid in designing diagnostic and therapeutic strategies.     

BMK1/ERK5 is a novel regulator of angiogenesis by destabilizing hypoxia inducible factor 1alpha

Big MAP kinase 1 (BMK1 or ERK5) is a key mediator of endothelial cell (EC) function as shown by impaired embryonic angiogenesis and vascular collapse in BMK1 knockout mice. Hypoxia inducible factor 1alpha (HIF1alpha), a potent mediator of angiogenesis, is positively regulated by the MAP kinases, ERK1/2. Because BMK1 deficiency is associated with impaired angiogenesis we hypothesized that BMK1 might regulate HIF1alpha. To test this hypothesis, bovine lung microvascular ECs (BLMECs) were transfected with HIF1alpha and BMK1 cDNAs, and stimulated by hypoxia. HIF1alpha activity was measured by a reporter gene assay in which luciferase expression was driven by HIF1alpha activation. Hypoxia (1% O2, 24 hours) stimulated HIF1alpha activity by 5.1+/-0.6 fold. In the presence of dominant negative (DN)-BMK1, which inhibited BMK1 activity, hypoxia induced HIF1alpha activity was enhanced significantly to 6.4+/-0.4 fold. BMK1 activation by constitutively active (CA)-MEK5 inhibited HIF1alpha activity by 46+/-4%, suggesting BMK1 functions as a negative regulator of HIF1alpha activation. Activation of BMK1 reduced HIF1alpha protein levels. Ubiquitination inhibitors (30 micromol/L ALLN, 2 micromol/L lactacystin, or 100 nmol/L MG132) reduced the BMK1-mediated effect on HIF1alpha expression by >80%, suggesting that BMK1 stimulated HIF1alpha proteolysis. The negative effect of BMK1 on HIF1alpha was functionally important because transfection with CA-MEK5 significantly decreased EC migration by 68+/-10%, and inhibited angiogenesis (in vitro Matrigel assay) by 76+/-7%. In summary, BMK1 is a novel negative regulator of HIF1alpha and angiogenesis by increasing HIF1alpha ubiquitination and inhibiting HIF1alpha activity in endothelial cells.     

Ghrelin induces cardiac lineage differentiation of human embryonic stem cells through ERK1/2 pathway

Background

Ghrelin, an endogenous ligand for growth hormone secretagogue receptor (GHS-R), shows cardioprotective activity and regulates the differentiation of several mesoderm-derived cells, including myocytes, adipocytes and osteoblasts. The effect of ghrelin on cardiogenesis and its underlying mechanism, however, have not been studied in detail.

Methods

The effects of ghrelin on cardiomyocyte differentiation were tested both in human embryonic stem cells (hESCs) cultured in embryoid body (EB)-based differentiation protocol, and in hESCs transplanted into rat hearts. The signaling mechanisms of ghrelin were further investigated under the EB-based culture condition.

Results

The generation of beating EBs and the expression of cardiac-specific markers including cardiac troponin I (cTnI) and α-myosin heavy chain (α-MHC) were 2 to 3-fold upregulated by ghrelin. Although GHS-R1α protein was expressed in differentiated EBs, the effects of exogenous ghrelin were unchanged by D-[lys³]-GHRP-6, a specific GHS-R1α antagonist. Moreover, des-acyl ghrelin, which does not bind to GHS-R1α, displayed similar effects with ghrelin. Importantly, activation of ERK1/2, but not Akt, was induced by ghrelin in the newly-formed EBs, and the ghrelin-induced effects of cardiomyocyte differentiation were abolished by adding specific ERK1/2 inhibitor PD98059, but not specific PI3K inhibitor Wortmannin. In addition, ghrelin promoted the differentiation of grafted hESCs into Sox9- and Flk1-positive mesodermal/cardiac progenitor cells in rat hearts.

Conclusions

These results suggest that ghrelin induces cardiomyocyte differentiation from hESCs via the activation of the ERK1/2 signaling pathway. Our study, therefore, indicates that using ghrelin may be an effective strategy to promote the differentiation of hESCs into cardiomyocytes.     

Myocyte-enriched calcineurin-interacting protein, MCIP1, inhibits cardiac hypertrophy in vivo

Signaling events controlled by calcineurin promote cardiac hypertrophy, but the degree to which such pathways are required to transduce the effects of various hypertrophic stimuli remains uncertain. In particular, the administration of immunosuppressive drugs that inhibit calcineurin has inconsistent effects in blocking cardiac hypertrophy in various animal models. As an alternative approach to inhibiting calcineurin in the hearts of intact animals, transgenic mice were engineered to overexpress a human cDNA encoding the calcineurin-binding protein, myocyte-enriched calcineurin-interacting protein-1 (hMCIP1) under control of the cardiac-specific, alpha-myosin heavy chain promoter (alpha-MHC). In unstressed mice, forced expression of hMCIP1 resulted in a 5-10% decline in cardiac mass relative to wild-type littermates, but otherwise produced no apparent structural or functional abnormalities. However, cardiac-specific expression of hMCIP1 inhibited cardiac hypertrophy, reinduction of fetal gene expression, and progression to dilated cardiomyopathy that otherwise result from expression of a constitutively active form of calcineurin. Expression of the hMCIP1 transgene also inhibited hypertrophic responses to beta-adrenergic receptor stimulation or exercise training. These results demonstrate that levels of hMCIP1 producing no apparent deleterious effects in cells of the normal heart are sufficient to inhibit several forms of cardiac hypertrophy, and suggest an important role for calcineurin signaling in diverse forms of cardiac hypertrophy. The future development of measures to increase expression or activity of MCIP proteins selectively within the heart may have clinical value for prevention of heart failure.     

Increased afterload induces pathological cardiac hypertrophy: a new in vitro model

Increased afterload results in ‘pathological’ cardiac hypertrophy, the most important risk factor for the development of heart failure. Current in vitro models fall short in deciphering the mechanisms of hypertrophy induced by afterload enhancement. The aim of this study was to develop an experimental model that allows investigating the impact of afterload enhancement (AE) on work-performing heart muscles in vitro. Fibrin-based engineered heart tissue (EHT) was cast between two hollow elastic silicone posts in a 24-well cell culture format. After 2?weeks, the posts were reinforced with metal braces, which markedly increased afterload of the spontaneously beating EHTs. Serum-free, triiodothyronine-, and hydrocortisone-supplemented medium conditions were established to prevent undefined serum effects. Control EHTs were handled identically without reinforcement. Endothelin-1 (ET-1)- or phenylephrine (PE)-stimulated EHTs served as positive control for hypertrophy. Cardiomyocytes in EHTs enlarged by 28.4?% under AE and to a similar extent by ET-1- or PE-stimulation (40.6 or 23.6?%), as determined by dystrophin staining. Cardiomyocyte hypertrophy was accompanied by activation of the fetal gene program, increased glucose consumption, and increased mRNA levels and extracellular deposition of collagen-1. Importantly, afterload-enhanced EHTs exhibited reduced contractile force and impaired diastolic relaxation directly after release of the metal braces. These deleterious effects of afterload enhancement were preventable by endothelin-A, but not endothelin-B receptor blockade. Sustained afterload enhancement of EHTs alone is sufficient to induce pathological cardiac remodeling with reduced contractile function and increased glucose consumption. The model will be useful to investigate novel therapeutic approaches in a simple and fast manner.