Senescence marker protein 30 inhibits angiotensin II-induced cardiac hypertrophy and diastolic dysfunction

Background and objective

Senescence marker protein 30 (SMP30) is assumed to behave as an anti-aging factor. Recently, we have demonstrated that deficiency of SMP30 exacerbates angiotensin II-induced cardiac hypertrophy, dysfunction and remodeling, suggesting that SMP30 may have a protective role in the heart. Thus, this study aimed to test the hypothesis that up-regulation of SMP30 inhibits cardiac adverse remodeling in response to angiotensin II.

Methods

We generated transgenic mice with cardiac-specific overexpression of SMP30 gene using α-myosin heavy chain promoter. Transgenic mice and wild-type littermate mice were subjected to continuous angiotensin II infusion (800 ng/kg/min).

Results

After 14 days, heart weight and left ventricular weight were lower in transgenic mice than in wild-type mice, although blood pressure was similarly elevated during angiotensin II infusion. Cardiac hypertrophy and diastolic dysfunction in response to angiotensin II were prevented in transgenic mice compared with wild-type mice. The degree of cardiac fibrosis by angiotensin II was lower in transgenic mice than in wild-type mice. Angiotensin II-induced generation of superoxide and subsequent cellular senescence were attenuated in transgenic mouse hearts compared with wild-type mice.

Conclusions

Cardiac-specific overexpression of SMP30 inhibited angiotensin II-induced cardiac adverse remodeling. SMP30 has a cardio-protective role with anti-oxidative and anti-aging effects and could be a novel therapeutic target to prevent cardiac hypertrophy and remodeling due to hypertension.     

Polydatin prevents angiotensin II-induced cardiac hypertrophy and myocardial superoxide generation

Our studies and others recently demonstrate that polydatin, a resveratrol glucoside, has antioxidative and cardioprotective effects. This study aims to investigate the direct effects of polydatin on Ang II-induced cardiac hypertrophy to explore the potential role of polydatin in cardioprotection. Our results showed that in primary cultured cardiomyocytes, polydatin blocked Ang II-induced cardiac hypertrophy in a dose-dependent manner, which were associated with reduction in the cell surface area and [³H]leucine incorporation, as well as attenuation of the mRNA expressions of atrial natriuretic factor and β-myosin heavy chain. Furthermore, polydatin prevented rat cardiac hypertrophy induced by Ang II infusion, as assessed by heart weight-to-body weight ratio, cross-sectional area of cardiomyocyte, and gene expression of hypertrophic markers. Further investigation demonstrated that polydatin attenuated the Ang II-induced increase in the reactive oxygen species levels and NADPH oxidase activity in vivo and in vitro. Polydatin also blocked the Ang II-stimulated increases of Nox4 and Nox2 expression in cultured cardiomyocytes and the hearts of Ang II-infused rats. Our results indicate that polydatin has the potential to protect against Ang II-mediated cardiac hypertrophy through suppression of NADPH oxidase activity and superoxide production. These observations may shed new light on the understanding of the cardioprotective effect of polydatin.     

CCR2 mediates the uptake of bone marrow-derived fibroblast precursors in angiotensin II-induced cardiac fibrosis

Angiotensin II plays an important role in the development of cardiac hypertrophy and fibrosis, but the underlying cellular and molecular mechanisms are not completely understood. Recent studies have shown that bone marrow-derived fibroblast precursors are involved in the pathogenesis of cardiac fibrosis. Since bone marrow-derived fibroblast precursors express chemokine receptor, CCR2, we tested the hypothesis that CCR2 mediates the recruitment of fibroblast precursors into the heart, causing angiotensin II-induced cardiac fibrosis. Wild-type and CCR2 knockout mice were infused with angiotensin II at 1,500 ng·kg(-1)·min(-1). Angiotensin II treatment resulted in elevated blood pressure and cardiac hypertrophy that were not significantly different between wild-type and CCR2 knockout mice. Angiotensin II treatment of wild-type mice caused prominent cardiac fibrosis and accumulation of bone marrow-derived fibroblast precursors expressing the hematopoietic markers, CD34 and CD45, and the mesenchymal marker, collagen I. However, angiotensin II-induced cardiac fibrosis and accumulation of bone marrow-derived fibroblast precursors in the heart were abrogated in CCR2 knockout mice. Furthermore, angiotensin II treatment of wild-type mice increased the levels of collagen I, fibronectin, and α-smooth muscle actin in the heart, whereas these changes were not observed in the heart of angiotensin II-treated CCR2 knockout mice. Functional studies revealed that the reduction of cardiac fibrosis led to an impairment of cardiac systolic function and left ventricular dilatation in angiotensin II-treated CCR2 knockout mice. Our data demonstrate that CCR2 plays a pivotal role in the pathogenesis of angiotensin II-induced cardiac fibrosis through regulation of bone marrow-derived fibroblast precursors.     

Human paraoxonase gene cluster overexpression alleviates angiotensin II-induced cardiac hypertrophy in mice

Cardiac hypertrophy is the strongest predictor of the development of heart failure, and anti-hypertrophic treatment holds the key to improving the clinical syndrome and increasing the survival rates for heart failure. The paraoxonase (PON) gene cluster (PC) protects against atherosclerosis and coronary artery diseases. However, the role of PC in the heart is largely unknown. To evaluate the roles of PC in cardiac hypertrophy, transgenic mice carrying the intact human PON1, PON2, and PON3 genes and their flanking sequences were studied. We demonstrated that the PC transgene (PC-Tg) protected mice from cardiac hypertrophy induced by Ang II; these mice had reduced heart weight/body weight ratios, decreased left ventricular wall thicknesses and increased fractional shortening compared with wild-type (WT) control. The same protective tendency was also observed with an Apoe^-/- background. Mechanically, PC-Tg normalized the disequilibrium of matrix metalloproteinases (MMPs)/tissue inhibitors of MMPs (TIMPs) in hypertrophic hearts, which might contribute to the protective role of PC-Tg in cardiac fibrosis and, thus, protect against cardiac remodeling. Taken together, our results identify a novel anti-hypertrophic role for the PON gene cluster, suggesting a possible strategy for the treatment of cardiac hypertrophy through elevating the levels of the PON gene family.     

钙调神经磷酸酶依赖的信号通路参与血管紧张素Ⅱ刺激的心肌细胞肥大

本研究观察了钙调神经磷酸酶依赖的信号通路在血管紧张素Ⅱ诱导的大鼠心肌细胞肥大中的作用。在ＡｎｇⅡ刺激的大鼠心肌细胞肥大模型上,应用环孢素Ａ（ＣｓＡ）阻断ＣａＮ通路,观察心肌细胞＾３Ｈ－亮氨酸掺入,ＣａＮ,ＭＡＰＫ及ＰＫＣ活性的变化。结果表明,ＡｎｇⅡ（１０＾－７ｍｏｌ／Ｌ）刺激大鼠心肌细胞＾３Ｈ－亮氨酸掺入较对照组增高４６％（Ｐ〈０．０１）,ＣｓＡ（０．５－５μｇ／ｍｌ）可以浓度依赖性方式抑制Ａｎ     

Knockdown of farnesylpyrophosphate synthase prevents angiotensin II-mediated cardiac hypertrophy

The Rho guanosine triphosphatases (Rho GTPases) family, including RhoA, plays an important role in angiotensin II (Ang II)-mediated cardiac hypertrophy. Farnesylpyrophosphate synthase (FPPS)-catalyzed isoprenoid intermediates are vital for activation of RhoA. The present study was designed to investigate the role of FPPS in myocardial hypertrophy mediated with Ang II. First, we demonstrated that FPPS expression was elevated both in cultured neonatal cardiomyocytes (NCMs) following Ang II treatment and in the hypertrophic myocardium of 18-week-old spontaneously hypertensive rats (SHRs). Then, the importance of FPPS was assessed by RNA interference (RNAi) against FPPS in NCMs. Successful FPPS silencing in NCMs completely inhibited the hypertrophy marker genes of β-myosin heavy chain (β-MHC) and brain natriuretic peptide (BNP), as well as cell surface area. Furthermore, FPPS knockdown prevented elevated RhoA activity compared with non-silenced controls. Similarly, increased-phosphorylation of p-38 and c-Jun N-terminal kinase (JNK) mitogen-activated protein kinases (MAPK) by Ang II was attenuated. In vivo gene transfer also attenuated hypertrophic responses as indexed by left ventricular weight/body weight (LVW/BW), heart weight/body weight (HW/BW), and echocardiography, as well as expression of β-MHC and BNP mRNA in SHRs. In conclusion, FPPS with RhoA associated p-38 and JNK MAPK signaling might play an important role in Ang II-induced cardiac hypertrophy.     

一氧化氮在血管紧张素Ⅱ诱导心肌细胞肥大中的作用

在培养新生大鼠心肌细胞上,探讨一氧化氮（ＮＯ）在血管紧张素Ⅱ诱导的心肌细胞以大中的作用。结果显示,血管紧张素Ⅱ可使心肌细胞蛋白质含量显著增加,心肌细胞一氧化氮合酶（ＮＯＳ）活性和培养液ＮＯ浓度明显降低。血管紧张素Ⅱ可明显降低心肌细胞ｅＮＯＳｍＲＮＡ水平。Ｓａｒａｌｓｉｎ和百日咳毒素（ＰＴＸ）可抑制血管紧张素Ⅱ诱导的蛋白质含量增加、心肌细胞ＮＯＳ活性减弱和培养液ＮＯ浓度降低。硝普钠提高心肌细胞培养     

Interleukin-6 family of cytokines mediate angiotensin II-induced cardiac hypertrophy in rodent cardiomyocytes

This study was designed to investigate whether angiotensin II induces the interleukin (IL)-6 family of cytokines in cardiac fibroblasts and, if so, whether these cytokines can augment cardiac hypertrophy. Angiotensin II increased IL-6, leukemia inhibitory factor (LIF) and cardiotrophin-1 mRNA by 6.5-, 10.2-, and 2.0-fold, respectively, but did not affect IL-11, ciliary neurotrophic factor, or oncostatin M in cardiac fibroblasts. Enzyme-linked immunosorbent assay revealed that angiotensin II-stimulated conditioned medium from cardiac fibroblasts contained 9.3 ng/ml IL-6 at 24 h, which was 24-fold higher than the control. It phosphorylated gp130 and STAT3 in cardiomyocytes, which was reduced with RX435 (anti-gp130 blocking antibody). It increased [(3)H]phenylalanine uptake and cell area by 44% and 86% in cardiomyocytes compared with mock medium. RX435 suppressed these increases by 26% and 38%, while TAK044 (endothelin-A/B-R blocker) suppressed them by 52% and 52%, respectively. Antisense oligonucleotides against LIF and cardiotrophin-1 blocked their up-regulation, and attenuated the conditioned medium-induced increase in [(3)H]phenylalanine uptake by 21% and 13%, respectively. The combination of antisense oligonucleotides to LIF and cardiotrophin-1 decreased their uptake by 33%. These results indicated that angiotensin II induced IL-6, LIF, and cardiotrophin-1 in cardiac fibroblasts, and that these cytokines, particularly LIF and cardiotrophin-1, activated gp130-linked signaling and contributed to angiotensin II-induced cardiomyocyte hypertrophy.     

Steroidogenic acute regulatory protein-binding protein cloned by a yeast two-hybrid system

Steroidogenic acute regulatory (StAR) protein plays a key role in the transport of cholesterol from the outer mitochondrial membrane to the inner membrane. A StAR mutant protein lacking the first 62 amino acids (N-62 StAR protein) has been reported to be as effective as wild-type StAR protein. In the present study, we examined the mechanism by which StAR protein stimulates steroidogenesis. A Gal4-based yeast two-hybrid system was used to identify proteins interacting with N-62 StAR protein. Nine positive clones were obtained from screening 1 x 106 clones. The results of pull-down assays and mammalian two-hybrid assays confirmed interaction between N-62 StAR protein and the clone 4 translated product. The clone 4 translated product was named StAR-binding protein (SBP). We prepared an expression plasmid (pSBP) by inserting SBP cDNA into the pTarget vector. After cotransfection with the human cytochrome P450scc system, StAR expression vector, and pSBP, the amount of pregnenolone produced by COS-1 cells was increased. The amount of steroid hormones produced by steroidogenic cells subjected to small interfering RNA treatment was less than that produced by control cells. In conclusion, SBP binds StAR protein in cells and enhances the ability of StAR protein to promote syntheses of steroid hormones.     

Androgen receptor gene knockout male mice exhibit impaired cardiac growth and exacerbation of angiotensin II-induced cardiac fibrosis

Androgen has anabolic effects on cardiac myocytes and has been shown to enhance left ventricular enlargement and function. However, the physiological and patho-physiological roles of androgen in cardiac growth and cardiac stress-induced remodeling remains unclear. We aimed to clarify whether the androgen-nuclear androgen receptor (AR) system contributes to the cardiac growth and angiotensin II (Ang II)-stimulated cardiac remodeling by using systemic AR-null male mice. AR knock-out (ARKO) male mice, at 25 weeks of age, and age-matched wild-type (WT) male mice were treated with or without Ang II stimulation (2.0 mg/kg/day) for 2 weeks. ARKO mice with or without Ang II stimulation showed a significant reduction in the heart-to-body weight ratio compared with those of WT mice. In addition, echocardiographic analysis demonstrated impairments of both the concentric hypertrophic response and left ventricular function in Ang II-stimulated ARKO mice. Western blot analysis of the myocardium revealed that activation of extracellular signal-regulated kinases (ERK) 1/2 and ERK5 by Ang II stimulation were lower in ARKO mice than those of WT mice. Ang II stimulation caused more prominent cardiac fibrosis in ARKO mice than in WT mice with enhanced expression of types I and III collagen and transforming growth factor-beta1 genes and with increased Smad2 activation. These results suggest that, in male mice, the androgen-AR system participates in normal cardiac growth and modulates cardiac adaptive hypertrophy and fibrosis during the process of cardiac remodeling under hypertrophic stress.     

Poly(ADP-ribose) polymerase-1-deficient mice are protected from angiotensin II-induced cardiac hypertrophy

Poly(ADP-ribose) polymerase-1 (PARP), a chromatin-bound enzyme, is activated by cell oxidative stress. Because oxidative stress is also considered a main component of angiotensin II-mediated cell signaling, it was postulated that PARP could be a downstream target of angiotensin II-induced signaling leading to cardiac hypertrophy. To determine a role of PARP in angiotensin II-induced hypertrophy, we infused angiotensin II into wild-type (PARP(+/+)) and PARP-deficient mice. Angiotensin II infusion significantly increased heart weight-to-tibia length ratio, myocyte cross-sectional area, and interstitial fibrosis in PARP(+/+) but not in PARP(-/-) mice. To confirm these results, we analyzed the effect of angiotensin II in primary cultures of cardiomyocytes. When compared with PARP(-/-) cardiomyocytes, angiotensin II (1 microM) treatment significantly increased protein synthesis in PARP(+/+) myocytes, as measured by (3)H-leucine incorporation into total cell protein. Angiotensin II-mediated hypertrophy of myocytes was accompanied with increased poly-ADP-ribosylation of nuclear proteins and depletion of cellular NAD content. When cells were treated with cell death-inducing doses of angiotensin II (10-20 microM), robust myocyte cell death was observed in PARP(+/+) but not in PARP(-/-) myocytes. This type of cell death was blocked by repletion of cellular NAD levels as well as by activation of the longevity factor Sir2alpha deacetylase, indicating that PARP induction and subsequent depletion of NAD levels are the sequence of events causing angiotensin II-mediated cardiomyocyte cell death. In conclusion, these results demonstrate that PARP is a nuclear integrator of angiotensin II-mediated cell signaling contributing to cardiac hypertrophy and suggest that this could be a novel therapeutic target for the management of heart failure.     

Steroidogenic acute regulatory protein binds cholesterol and modulates mitochondrial membrane sterol domain dynamics

The steroidogenic acute regulatory protein (StAR) mediates the rate-limiting step of steroidogenesis, delivery of cholesterol to the inner mitochondrial membrane. However, the mechanism whereby cholesterol translocation is accomplished has not been resolved. Recombinant StAR proteins lacking the first N-terminal 62 amino acids comprising the mitochondrial-targeting sequence were used to determine if StAR binds cholesterol and alters mitochondrial membrane cholesterol domains to enhance sterol transfer. First, a fluorescent NBD-cholesterol binding assay revealed 2 sterol binding sites (K(d) values near 32 nm), whereas the inactive A218V N-62 StAR mutant had only a single binding site with 8-fold lower affinity. Second, NBD-cholesterol spectral shifts and fluorescence resonance energy transfer from StAR Trp residues to NBD-cholesterol showed (i) close molecular interaction between these molecules (R(2/3) = 33 A) and (ii) sensitized NBD-cholesterol emission from only one of the two sterol binding sites. Third, circular dichroism showed that cholesterol binding induced a change in StAR secondary structure. Fourth, a fluorescent sterol transfer assay that did not require separation of donor and acceptor mitochondrial membranes demonstrated that StAR enhanced mitochondrial sterol transfer as much as 100-fold and induced/increased the formation of rapidly transferable cholesterol domains in isolated mitochondrial membranes. StAR was 67-fold more effective in transferring cholesterol from mitochondria of steroidogenic MA-10 cells than from human fibroblast mitochondria. In contrast, sterol carrier protein-2 (SCP-2) was only 2.2-fold more effective in mediating sterol transfer from steroidogenic cell mitochondria. Taken together these data showed that StAR is a cholesterol-binding protein, preferentially enhances sterol transfer from steroidogenic cell mitochondria, and interacts with mitochondrial membranes to alter their sterol domain structure and dynamics.     

Steroidogenic acute regulatory protein (StAR), a novel mitochondrial cholesterol transporter

Cholesterol is a vital component of cellular membranes, and is the substrate for biosynthesis of steroids, oxysterols and bile acids. The mechanisms directing the intracellular trafficking of this nearly insoluble molecule have received increased attention through the discovery of the steroidogenic acute regulatory protein (StAR) and similar proteins containing StAR-related lipid transfer (START) domains. StAR can transfer cholesterol between synthetic liposomes in vitro, an activity which appears to correspond to the trans-cytoplasmic transport of cholesterol to mitochondria. However, trans-cytoplasmic cholesterol transport in vivo appears to involve the recently-described protein StarD4, which is expressed in most cells. Steroidogenic cells must also move large amounts of cholesterol from the outer mitochondrial membrane to the first steroidogenic enzyme, which lies on the matrix side of the inner membrane; this action requires StAR. Congenital lipoid adrenal hyperplasia, a rare and severe disorder of human steroidogenesis, results from mutations in StAR, providing a StAR knockout of nature that has provided key insights into its activity. Cell biology experiments show that StAR moves large amounts of cholesterol from the outer to inner mitochondrial membrane, but acts exclusively on the outer membrane. Biophysical data show that only the carboxyl-terminal alpha-helix of StAR interacts with the outer membrane. Spectroscopic data and molecular dynamics simulations show that StAR's interactions with protonated phospholipid head groups on the outer mitochondrial membrane induce a conformational change (molten globule transition) needed for StAR's activity. StAR appears to act in concert with the peripheral benzodiazepine receptor, but the precise itinerary of a cholesterol molecule entering the mitochondrion remains unclear.