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.     

转录因子在心肌肥大中的调控作用

虽然近年来对心力衰竭的治疗有了很大的进步,但其病死率、致残率仍然很高。心肌重构是心力衰竭发展的一个重要病理机制,目前认为抑制心肌重构是预防和治疗心力衰竭的重要手段,而心肌细胞肥大是心力衰竭发展过程中心肌重构的一个主要特征之一。大量研究表明心肌细胞转录因子在心肌细胞肥大过程中起重要调控作用,一些心肌细胞转录因子在受到肥大刺激信号作用后能够被激活并且在心肌肥大过程中起重要作用。该文对几种转录因子在心肌细胞肥大过程中的最新研究状况作一简单介绍。     

Calcineurin and matrix protein expression in cardiac hypertrophy

In the compensatory state of human left ventricular hypertrophy (LVH), the remodeling processes in the extracellular matrix and the role of calcineurin (Cn) are not completely understood. The present work aimed to analyze the expression and activity of matrix metalloproteinases (MMPs), their endogenous inhibitors (TIMPs), and of Cn in patients with compensated LVH. By semiquantitative RT-PCR, Western blotting, and gelatine zymography, we determined mRNA, protein, and/or enzyme activity levels of MMPs, TIMPs, atrial natriuretic peptide (ANP), Cn subunits, and of the modulatory calcineurin-interacting protein (MCIP) 1. Myocardial samples from patients showing severe aortic stenosis, normal ejection fraction, and compensated LVH were compared with autopsy samples from healthy hearts. LVH patients showed upregulation of CnA-β mRNA but downregulation of both CnB-α mRNA and protein. Total Cn activity (as determined through NF-AT phosphorylation and MCIP1 mRNA expression) was unchanged. There were no differences in gene expression and activities of MMP-2, MMP-9, and of TIMPs 1–4 between LVH patients and controls. As expected, ANP mRNA expression was high in LVH patients. We propose a prominent role for CnB in controlling Cn activity in compensated LVH. At this stage of the disease, MMP and TIMP activities are balanced. Drs. Grammer and Bleiziffer contributed equally to the study Returned for 1st revision: 23 February 2006 1. revision received: 8 April 2006     

Contrasting roles of NADPH oxidase isoforms in pressure-overload versus angiotensin II-induced cardiac hypertrophy

Increased production of reactive oxygen species (ROS) is implicated in the development of left ventricular hypertrophy (LVH). Phagocyte-type NADPH oxidases are major cardiovascular sources of ROS, and recent data indicate a pivotal role of a gp91phox-containing NADPH oxidase in angiotensin II (Ang II)-induced LVH. We investigated the role of this oxidase in pressure-overload LVH. gp91phox-/- mice and matched controls underwent chronic Ang II infusion or aortic constriction. Ang II-induced increases in NADPH oxidase activity, atrial natriuretic factor (ANF) expression, and cardiac mass were inhibited in gp91phox-/- mice, whereas aortic constriction-induced increases in cardiac mass and ANF expression were not inhibited. However, aortic constriction increased cardiac NADPH oxidase activity in both gp91phox-/- and wild-type mice. Myocardial expression of an alternative gp91phox isoform, Nox4, was upregulated after aortic constriction in gp91phox-/- mice. The antioxidant, N-acetyl-cysteine, inhibited pressure-overload-induced LVH in both gp91phox-/- and wild-type mice. These data suggest a differential response of the cardiac Nox isoforms, gp91phox and Nox4, to Ang II versus pressure overload.     

Alpha1-adrenergic stimulation induced hypertrophy in protein kinase C down-regulated cultured cardiac myocytes

To examine whether protein kinase C (PKC) activation is essential for the induction of cardiac myocyte hypertrophy caused by alpha1-adrenergic stimulation, we investigated the hypertrophic effect of phenylephrine in PKC down-regulated and non-treated cultured cardiac myocytes obtained from neonatal Sprague-Dawley rat ventricles. The treatment with 10 nmol/L 12-tetra decanoylphorbol-13-acetate (TPA) for more than 2 hours decreased PKC activity by approximately 80% without marked hypertrophy. Phenylephrine increased [14C] phenylalanine (Phe) incorporation in both TPA non-treated and treated cells, 1.54- and 1.71-fold as large as control, respectively. The cell surface area also enlarged in both groups, 1.67- and 1.74-fold, respectively. Thus, phenylephrine induced the similar grade hypertrophy in cultured cardiac myocytes even when PKC was down-regulated. These results suggest that conventional PKC activation may not be essential for mediating myocyte hypertrophy by alpha1-adrenergic stimulation.     

Role of beta 1-adrenoceptors in hypertensive cardiac hypertrophy

Experimental and clinical evidence indicates that cardiac hypertrophy in systemic hypertension may not simply result from the mechanical stress of increased afterload. Several lines of evidence suggest that sympathetic nervous influence stimulates cardiac growth. A previous study indicated that sympathetic tone may be important in the two-kidney, one-clip model of renovascular hypertension. Hence, we investigated the role of cardiac beta-receptors by testing the effects of the cardioselective beta-receptor blocker, atenolol, on regression and prevention of ventricular hypertrophy in this model. Renal hypertensive rats were assigned to a 'prevention' and a 'reversal' protocol, receiving the drug before or after the development of hypertension and cardiac hypertrophy. Untreated control animals developed severe hypertension (205 +/- 9 mmHg) and marked cardiac hypertrophy (heart weight/body weight ratio: 3.86 +/- 0.23 mg/g) when compared to sham-operated controls (129 +/- 1 mmHg and 2.38 +/- 0.06 mg/g, respectively). Atenolol (440 mg/kg per day) failed to prevent or reverse hypertension (213 +/- 5 and 194 +/- 11 mmHg) or cardiac hypertrophy (4.10 +/- 0.39 and 3.51 +/- 0.25 mg/g, respectively). Effective beta-blockade was verified by significantly lower heart rates in treated animals (382 +/- 10 and 368 +/- 9 beats/min, respectively) than untreated controls (486 +/- 28 beats/min; P less than 0.01). Similarly, plasma renin activity returned to baseline in atenolol-treated animals. Cardiac catecholamines were markedly decreased in hypertrophied hearts (significant only for norepinephrine) and remained unaffected by atenolol treatment. However, both the prevention and reversal protocol strikingly reduced mortality in hypertensive animals (0 and 14%, respectively, versus 57%; P less than 0.02).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dual roles of an Arabidopsis ESCRT component FREE1 in regulating vacuolar protein transport and autophagic degradation

Protein turnover can be achieved via the lysosome/vacuole and the autophagic degradation pathways. Evidence has accumulated revealing that efficient autophagic degradation requires functional endosomal sorting complex required for transport (ESCRT) machinery. However, the interplay between the ESCRT machinery and the autophagy regulator remains unclear. Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), a recently identified plant-specific ESCRT component essential for multivesicular body (MVB) biogenesis and plant growth, plays roles both in vacuolar protein transport and autophagic degradation. FREE1 also regulates vacuole biogenesis in both seeds and vegetative cells of Arabidopsis. Additionally, FREE1 interacts directly with a unique plant autophagy regulator SH3 DOMAIN-CONTAINING PROTEIN2 and associates with the PI3K complex, to regulate the autophagic degradation in plants. Thus, FREE1 plays multiple functional roles in vacuolar protein trafficking and organelle biogenesis as well as in autophagic degradation via a previously unidentified regulatory mechanism of cross-talk between the ESCRT machinery and autophagy process.The endosomal–lysosomal/vacuolar pathway is the primary catabolic system of eukaryotic cells that degrades extracellular and intracellular materials. Membrane proteins destined for degradation, such as misfolded proteins or endocytosed receptors, become tagged by ubiquitin for further sorting to the endosomal–lysosomal/vacuolar system for degradation (1). During this process, an evolutionarily conserved machinery called endosomal sorting complex required for transport (ESCRT), is responsible for sorting these ubiquitinated cargos into the intraluminal vesicles (ILVs) of prevacuolar compartments/multivesicular bodies (PVCs/MVBs), which subsequently fuse with vacuoles/lysosomes to deliver their contents into the lumen for proteolytic degradation (2, 3). Malfunction of the assembly or dissociation of the ESCRT machinery disrupts MVB formation and thus results in the accumulation of ubiquitinated membrane cargos (4, 5).Macroautophagy (hereafter as autophagy) is another highly conserved catabolic process, which converges on the endosomal–lysosomal/vacuolar pathway to deliver aberrant organelles, long-lived proteins, and protein aggregates to the lysosome/vacuole via a unique structure termed the “autophagosome” (6). Morphologically different from MVBs, autophagosomes are characterized by a double membrane structure, which is initiated from the phagophore assembly site/preautophagosome site (PAS) (7). The proteins or organelles to be degraded are encapsulated by autophagosomes that fuse either directly with the vacuole/lysosome or with endosomes like MVBs for expansion/maturation to form amphisomes, which then fuse with vacuole/lysosome for degradation. A number of conserved autophagy-related gene (ATG) proteins have been identified as participating in the autophagy pathway in eukaryotic cells (8).Even though it is generally accepted that at least one population of developing autophagosomes fuses with late endosomal compartments before their fusion with lysosomes, little is known about the functional relationship between the autophagy and endocytic pathways. New light has been thrown onto this situation through the discovery that ESCRT is also involved in autophagy. More and more studies on nematodes, flies, mammals, and even on plants provide evidence to support the conclusion that the inactivation of ESCRT machinery causes an accumulation of autophagosomes (9–13). Different models, such as the induction of autophagy or the disruption of autophagosome–endosome/lysosome fusion, have been proposed to explain the observation that autophagosomes accumulate in ESCRT-depleted cells (14, 15). However, there are no studies to give a direct link between ESCRT machinery and autophagy regulators.Here, we show that FYVE domain protein required for endosomal sorting 1 (FREE1), which represents a recently identified and unique plant ESCRT component essential for MVB biogenesis (5), plays a crucial role in vacuolar protein transport and vacuole biogenesis. In addition, FREE1 directly interacts with SH3 DOMAIN-CONTAINING PROTEIN2 (SH3P2), a unique regulator in plant autophagy (16), to manipulate the autophagosome–vacuole fusion and finally autophagic degradation in plants. Our studies have thus unveiled a previously unidentified regulatory mechanism for direct cross-talk between the ESCRT machinery and autophagy process.     

胆固醇酯转运蛋白在家兔心脏肥大中表达的实验研究

目的 复制异丙肾上腺素致家兔心脏肥大模型,考察胆固醇酯转运蛋白（CETP）的表达变化。 方法 将雄性新西兰兔随机分为正常组和模型组。模型组采用腹腔注射异丙肾上腺素15 mg/(kg·d)（分早晚2次进行）建立家兔心脏肥大模型,正常组腹腔注射等体积的生理盐水,连续14 d后,取血、心脏和肺脏,分离左、右心室,考察脏器指数;HE染色观察心肌组织形态学改变;RT-PCR法检测心脏肥大相关胚胎基因ANP、β-MHC mRNA表达;检测血清TG、TC、LDL-C及HDL-C的含量;检测左心室CETP的mRNA水平、蛋白表达及含量。 结果 与正常组比较,模型组家兔心脏质量指数（HW/BW）和左心室质量指数（LVW/BW）显著增加（P<0.05）,右心室质量指数（RVW/BW）和肺脏质量指数（LW/BW）有增加的趋势,左心室心肌细胞体积明显增大,间质增宽,左心室ANP、β-MHC的mRNA表达显著上调（P<0.05）。与正常组比较,模型组家兔血清TG含量显著升高（P<0.05）,TC、LDL-C水平呈升高趋势,HDL-C水平呈下降趋势,模型组家兔左心室组织中CETP mRNA水平和蛋白表达均显著上调（P<0.05）,CETP含量显著增加（P<0.05）。 结论 在异丙肾上腺素诱导的家兔心脏肥大模型中,CETP表达上调,但其是否与心脏肥大有关还有待进一步深入研究。本研究结果为后续研究CETP与心脏肥大的关系提供了理想动物模型。     

Upregulation of TRPC1 in the development of cardiac hypertrophy

The importance of Ca(2+) entry in the cardiac hypertrophic response is well documented, but the actual Ca(2+) entry channels remain unknown. Transient receptor potential (TRP) proteins are thought to form either homo- or heteromeric Ca(2+) entry channels that are involved in the proliferation and differentiation of various cells. The purpose of this study was to explore the potential involvement of TRP channels in the development of cardiac hypertrophy. The mRNA and protein expression of several TRP channel subunits were evaluated using hearts from abdominal aortic-banded (AAB) rats. Although TRPs C1, C3, C5, and C6 were constitutively expressed, only TRPC1 expression was significantly increased in the hearts of AAB rats compared to sham-operated rats. Using primary cultures of neonatal rat cardiomyocytes, we detected increases in the expression of TRPC1, brain natriuretic peptide (BNP), and atrial natriuretic factor (ANF), as well as increases in store-operated Ca(2+) entry (SOCE) and cell surface area, following endothelin-1 (ET-1) treatment. Silencing of the TRPC1 gene via small interfering RNA (siRNA) attenuated SOCE and prevented ET-1-, angiotensin-II (AT II)-, and phenylephrine (PE)-induced cardiac hypertrophy. In HEK 293T cells, overexpression of TRPC1 augmented SOCE, leading to an increase in nuclear factor of activated T cells (NFAT) promoter activity, while co-transfection with dominant-negative forms of TRPC1 suppressed it. In conclusion, TRPC1 functions in Ca(2+) influx, and its upregulation is involved in the development of cardiac hypertrophy; moreover, it plays an important role in the regulation of the signaling pathways that govern cardiac hypertrophy. These findings establish TRPC1 as a functionally important regulator of cardiac hypertrophy.     

Requirement of Rac1 in the development of cardiac hypertrophy

The development of cardiac hypertrophy is mediated, in part, by increase in NADPH oxidase activity and myocardial oxidative stress. The Rho GTPase, Rac, regulates NADPH oxidase activity through interaction with gp91(phox) and p67(phox) (in which "phox" is phagocyte oxidase). However, it is not known which Rac isoform mediates this effect in the heart. Here we show that Rac1 is critical for generating oxidative stress and producing cardiac hypertrophy in the adult heart. The Rac1 gene was temporally and specifically deleted in adult mouse cardiomyocytes (c-Rac1(-/-)). Compared with wild-type or Rac1 heterozygous mice, the hearts of c-Rac1(-/-) mice showed decreased gp91(phox) and p67(phox) interaction, NADPH oxidase activity, and myocardial oxidative stress in response to angiotensin II (400 ng/kg per day for 2 weeks) stimulation. This result correlated with decreased myocardial hypertrophy. These results indicate that Rac1 is critical for the hypertrophic response in the heart and suggest that therapies which target myocardial Rac1 may be beneficial in the treatment of cardiac hypertrophy.     

小G蛋白Rad基因对心肌肥大的影响

目的研究Rad基因对心肌细胞肥大的影响,为心肌肥大早期干预提供理论基础。方法针对Rad基因序列构建Rad过表达和低表达腺病毒载体,体外培养乳鼠心肌细胞,分为8组:空白对照组,Ad-GFP组,Ad-RNA干扰(RNAi)组,Ad-Rad组,肥大模型组(心肌营养素1诱导),肥大+Ad-GFP组,肥大+Ad-RNAi组,肥大+Ad-Rad组,48h后检测Rad、心房利钠因子(ANF)mRNA和蛋白表达水平。结果肥大模型组ANF蛋白表达较空白对照组明显升高;肥大+Ad-Rad组ANF蛋白表达较肥大+Ad-GFP组明显降低,肥大+Ad-RNAi组ANF蛋白表达较肥大+Ad-GFP组明显升高(P<0.05)。肥大模型组Rad mRNA和蛋白表达较空白对照组明显降低;Ad-Rad组Rad mRNA和蛋白表达较Ad-GFP组明显增加,Ad-RNAi组Rad mRNA和蛋白表达较Ad-GFP组明显降低(P<0.05)。结论 Rad基因可抑制在细胞水平心肌营养素1诱导的心肌细胞肥大反应。     

Emerging roles of SIRT1 deacetylase in regulating cardiomyocyte survival and hypertrophy

Calorie restriction is considered to be the best environmental intervention providing health benefits to mammals. The underlying mechanism of this intervention seems to be controlled by a group of NAD-dependent deacetylases, collectively called sirtuins. In mammals, there are seven sirtuin analogs, SIRT1-SIRT7. The founding member of this family, SIRT1, is shown to protect cardiomyocytes from apoptosis and age-dependent degeneration in a dose dependent manner—protecting cells at low doses but showing detrimental effects at high doses. Studies performed with overexpression or knockdown of SIRT1 indicated that, although it protects cells from oxidative stress and ischemia-reperfusion injury, it promotes hypertrophy of cardiomyocytes. Activation of endogenous SIRT1 by resveratrol also displayed pro-survival and pro-hypertrophic activity of SIRT1. In this article, we review recent findings documenting the role of SIRT1 in regulating cardiac myocyte growth and survival under stress, and the proposed mechanism behind its cardioprotective effects. We also briefly discuss two other sirtuin analogs which have been shown to have cardioprotective effects. This article is part of a special issue entitled "Key Signaling Molecules in Hypertrophy and Heart Failure".