Downregulation of ferritin heavy chain increases labile iron pool, oxidative stress and cell death in cardiomyocytes

Ferritin heavy chain (FHC) protein was significantly reduced in murine failing hearts following left coronary ligation or thoracic transverse aortic constriction. The mRNA expression of FHC was not significantly altered in failing hearts, compared to that in control sham-operated hearts. Prussian blue staining revealed spotty iron depositions in myocardial infarct failing hearts. Oxidative stress was enhanced in the myocardial infarct failing hearts, as evidenced by increases in 4-hydroxy-2-nonenal and 8-hydroxy-2′-deoxyguanosine immunoreactivity. To clarify the functional significance of FHC downregulation in hearts, we infected rat neonatal cardiomyocytes with adenoviral vector expressing short hairpin RNA targeted to FHC (Ad-FHC-RNAi). The downregulation of FHC induced a reduction in the viability of cardiomyocytes. The relative number of iron deposition-, 4-hydroxy-2-nonenal- or 8-hydroxy-2′-deoxyguanosine-positive cardiomyocytes was significantly higher in Ad-FHC-RNAi-infected cardiomyocytes than in control vector-infected cardiomyocytes. Treatment of Ad-FHC-RNAi-infected cardiomyocytes with desferrioxamine, an iron chelator, significantly reduced the number of iron, 4-hydroxy-2-nonenal or 8-hydroxy-2′-deoxyguanosine-positive cells, and increased viability. In addition, treatment with N-acetyl cysteine, an antioxidant, significantly reduced the number of 4-hydroxy-2-nonenal- or 8-hydroxy-2′-deoxyguanosine-positive cells. Reduced viability in Ad-FHC-RNAi-infected cardiomyocytes was significantly improved with N-acetyl cysteine treatment. These findings indicate that excessive free iron and the resultant enhanced oxidative stress caused by downregulation of FHC lead to cardiomyocyte death. The decrease in FHC expression in failing hearts may play an important role in the pathogenesis of heart failure.     

Metallothionein-I/II null cardiomyocytes are sensitive to Fusarium mycotoxin butenolide-induced cytotoxicity and oxidative DNA damage

Previous studies revealed butenolide (BUT), a Fusarium mycotoxin distributes extensively, induced myocardial oxidative damage, which could be abated by antioxidants such as glutathione. Metallothionein (MT) has proved to attenuate several oxidative cardiomyopathies via its potent antioxidant property. The present study is therefore undertaken to investigate the protective potential of the endogenous expression of MT against BUT-induced myocardial toxicity. Primary cultures of neonatal cardiomyocytes from MT-I/II null mice along with the corresponding wild-type mice will be utilized to determine the possible mechanistic properties of MT. BUT treatment to the cardiomyocytes evoked significant cytotoxicity as evidenced by morphological changes and concentration- and time-dependent reductions in cell viability. Additionally, BUT treatment remarkably increased reactive oxygen species (ROS) production in the cardiomyocytes of both MT-I/II null and wild-type mice. As a result, noticeable DNA damage in both cardiomyocytes was detected by alkaline comet assay. Furthermore, the comparison between the MT-I/II null and wild-type cardiomyocytes indicated that ROS production in the cardiomyocytes from the MT-I/II null mice was higher than from wild-type mice. DNA damage as evaluated by percentage of comet tail DNA, tail length and tail moment was more severe in the MT-l/II null cardiomyocytes than in wild-type myocytes. And in agreement with those results mentioned above, the MT-l/II null cardiomyocytes were more sensitive to BUT-induced cytotoxicity than wild-type cardiomyocytes. Taken together, these findings clearly show that basal MT can efficiently attenuate BUT-induced cytotoxic injuries in cardiomyocytes via the inhibition of intracellular ROS production, and associated DNA damage.     

Erythropoietin attenuates hypertrophy of neonatal rat cardiac myocytes induced by angiotensin‐II in vitro

Objective: Erythropoietin (EPO) is a haematopoietic hormone that has been confirmed as a novel cardioprotective agent. In this study, we test the hypothesis that EPO inhibits angiotensin‐II (Ang‐II)‐induced hypertrophy in cultured neonatal rat cardiomyocytes. Material and methods: Cultured neonatal rat cardiomyocytes were used to evaluate the effects of EPO on Ang‐II‐induced hypertrophy in vitro. The surface area and mRNA expression of atrial natriuretic (ANF) myocytes were employed to detect cardiac hypertrophy. A phosphatidylinositol 3′‐kinase (PI3K) inhibitor LY294002 and an endothelial nitric oxide synthase (eNOS) inhibitor l‐NAME were also employed to detect the underlying mechanism of EPO. Intracellular signal molecules, such as Akt (PKB), phosphorylated Akt, eNOS and transforming growth factor‐β1 (TGF‐β1) protein expression were determined by Western blot. Nitric oxide (NO) levels in the supernatant of cultured cardiomyocytes were assayed using an NO assay kit. Results: The results indicate that EPO significantly attenuates Ang‐II‐induced hypertrophy shown as inhibition of increases in cell surface area and ANF mRNA levels. NO production was also increased proportionally in the EPO‐treated group. EPO enhanced Akt activation and eNOS protein expression, whereas LY294002 or l‐NAME partially abolished the anti‐hypertrophic effect of EPO, accompanied by a decrease in Akt activation, eNOS protein expression and/or a reduction of NO production. EPO also down‐regulated the protein expression of TGF‐β1. Conclusion: We conclude that EPO attenuates cardiac hypertrophy via activation of the PI3K‐Akt‐eNOS‐NO pathway and the down‐regulation of TGF‐β1.     

Expression of cTnI-R145G affects shortening properties of adult rat cardiomyocytes

The R145G amino acid exchange in the inhibitory subunit (cTnI) of cardiac troponin, which regulates muscle contraction, is related to familial hypertrophic cardiomyopathy. Information on its impact on contractility of adult cardiomyocytes is scarce. We studied shortening of adult rat cardiomyocytes before and during ss-adrenergic stimulation using adenovirus-driven expression of human cTnI-wild type (wt) and cTnI-R145G. Baseline sarcomere shortening was significantly decreased by cTnI-R145G expression. Upon ss-adrenergic stimulation using isoproterenol (ISO), nearly identical amplitudes of shortening were obtained with cells expressing cTnI-R145G and control cardiomyocytes (native and cTnI-wt). However, rates of shortening and relengthening were depressed in cTnI-R145G-expressing cells but were comparable to those of control cells upon addition of forskolin or ISO and ICI118,551. This indicates that cTnI-R145G expression influences the response to ss-adrenergic stimulation dependent on the receptor subtype.     

Isolation and preparation of single mouse cardiomyocytes for confocal microscopy

Myofibril degeneration is a common hallmark of cardiomyopathies leading to cardiac failure. However, information regarding myofibrillar organization in histologic analysis of sectioned tissue is limited by both large size and variable orientation of the cardiomyocytes. In order to observe individual cardiomyocytes from a single mouse heart, we have developed a new procedure for trapping dissociated cardiomyocytes on a polyester mesh. Cells can then be visualized by fluorescence confocal microscopy and three-dimensional reconstruction of cells can be performed without distortion of intracellular organization resulting from cytofugation. Since the cells are trapped between the mesh and coverslip, the mesh is not seen in reconstructed images. Sarcomeric protein organization in cardiomyocytes can now be conveniently studied using this procedure. Furthermore, individual atrial or ventricular cardiomyocytes are easily identified in the preparation and decreased expression of -myosin heavy chain protein expression is apparent in propythiouracil (PTU) treated mice. This procedure will be a valuable asset in the analysis of intracellular changes occurring within the hearts of cardiomyopathic mice.     

Short term triiodo-L-thyronine treatment inhibits cardiac myocyte apoptosis in border area after myocardial infarction in rats

Thyroid hormone (TH) levels decline after a myocardial infarction (MI). Treatment with TH has been shown to improve left ventricular (LV) function in MI and other cardiovascular diseases, but the mechanisms are not clear. We have previously shown that TH can prevent myocyte apoptosis via Akt signaling in cultured neonatal rat cardiomyocytes. In this study, the effects of triiodo-l-thyronine (T3) on LV function and myocyte apoptosis after MI was examined in rats. After surgery, MI rats were treated with T3 for 3 days. Compared with sham-operated rats, MI rats showed significantly increased LV chamber dimension during systole and decreased LV function. T3 treatment increased LV ± dP/dt but did not change LV chamber dimensions. MI rats also showed significantly increased myocyte apoptosis in the border area as assessed by DNA laddering and TUNEL assay. T3 treatment decreased the amount of DNA laddering, and reduced TUNEL positive myocytes in the border area, which was associated with phosphorylation of Akt at serine 473. These results suggest that T3 can protect myocytes against ischemia-induced apoptosis, which may be mediated by Akt signaling.     

circRNA_100395通过结合miR-31-5p抑制心肌细胞肥大相关基因的表达

目的:探讨环状RNA(circRNA)_100395对心肌细胞肥大表型的影响及其作用机制.方法:通过RT-qPCR检测健康器官捐献者(n=8)与心衰患者(n=14)心肌组织中circRNA_100395及其宿主基因Kelch蛋白样家族成员20(KLHL20)的表达水平.原代分离、培养乳小鼠心室肌细胞(NMVCs),分别...     

体外定向诱导大鼠骨髓基质干细胞向心肌细胞分化的实验研究

目的：研究骨髓基质干细胞体外向心肌细胞分化的能力。方法：通过密度梯度离心和贴壁培养法分离大鼠骨髓基质干细胞,首先将第3代细胞用5-氮胞苷诱导24h,传至4代时再次诱导24h,当培养细胞汇合并形成肌管时传代;出现跳动细胞时用免疫细胞化学方法检测心肌特异性肌钙蛋白T(cTnT)的表达。结果：原代细胞首先形成细胞集落,10d后集落间接近汇合,传代细胞体积变大,5—7d传代1次,两次诱导后细胞呈梭形,约15d后细胞汇合并出现肌管,再传代时,细胞出现跳动。免疫细胞化学显示细胞表达cTnT。结论：骨髓基质干细胞在体外能够诱导分化为心肌细胞。     

黄芪甲苷对过氧化氢致损心肌细胞的保护作用及机制研究

目的 探讨黄芪甲苷对过氧化氢致损心肌细胞的保护作用及机制.方法 选取新生Wistar 大鼠3只,体外分离大鼠原代心肌细胞,按0.6×106/ml密度接种至75 cm2培养瓶培养,待80%融合后予0.5 mmol/L过氧化氢培养基培养4 h,分别加入100、10、1、0.1 μmol/L黄芪甲苷培养基6 ml培养24 h;设立对照组,仅加入等量普通培养基.观察心肌细胞存活率,乳酸脱氢醇(LDH)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-Px)酶活性情况,bax和bcl-2基因的表达,bax和bcl-2蛋白的表达情况.结果 与不同浓度黄芪甲苷共培养24 h的过氧化氢致损心肌细胞的成活率(0.1、1、10、100 μmol/L黄芪甲苷组分别为48%、55%、57%、52%)均明显高于对照组(30%),(均P＜0.01);黄芪甲苷可降低H2O2致损心肌细胞内的bax mRNA和蛋白的表达,提高bcl-2 mRNA和蛋白的表达.结论 黄芪甲苷对过氧化氢致损心肌细胞有保护作用,其机制可能是通过调节bax/cbl-2基因和蛋白的表达实现的.

Abstract：

Objective To study the effects of Astragaloside Ⅳ on injured rat cardiomyocytes caused by hydrogen peroxide (H2O2). Methods Three clean Wistar rats were supplied by Experimental Animal Center,Xingjiang medical university. Primary rat cardiomyocytes were isolated in vitro and cultured in 75 cm2 flasks. When cells were at 80% confluence, they were cultured with 0.5 mmol/L H2O2 medium for 4 hours and then treated with 6 ml 100, 10, 1,0. 1 μmol/L Astragaloside Ⅳ medium for24 hours. Cells in the control group were treated with an equal volume of normal medium. Cell survival rate were examined by trypan blue exclusion assay. Enzyme activities of lactate dehydrogenase(LDH), superoxide dismutase(SOD) and glutathione peroxidase(GSH-Px) were assayed by Elisa. The mRNA and protein expression were detected with realtime polymerase clain reaction(PCR) and Westernhlot method separately. Results Astragaloside Ⅳ promoted cardiomyocytes survival rate in a dose-dependent manner, while high concentration showed an inhibition tendency. Astragaloside Ⅳ decreased LDH activity (P ＜0.01 ) but increased SOD and GSH-Px activities (P ＜0.05 ). The results of Realtime PCR and Westernblot showed that Astragaloside Ⅳ could suppress expression of bax mRNA and protein, promote expression of cbl-2 mRNA and protein. Conclusions Astragaloside Ⅳ has protective effects on injured rat cardiomyocytes caused by H2O2, which is probably associated with regulating expression of bax/bcl-2 gene and protein.     

Growth promoting and metabolic activity of the human insulin analogue [Gly,Arg,Arg]insulin (HOE 901) in muscle cells

[Gly^A21,Arg^B31,Arg^B32]insulin (HOE 901) represents a biosynthetic human insulin analogue that, due to its isoelectric point, precipitates at neutral tissue pH leading to a retarded absorption rate and a corresponding longer duration of action. In the present investigation we have evaluated the growth promoting and metabolic activity of this analogue in muscle tissue using exponentially growing H9c2 cardiac myoblasts and adult rat ventricular cardiomyocytes. Equilibrium binding studies of ¹²⁵I-labelled IGF-I (insulin-like growth factor I) to differentiating myoblasts revealed the presence of 7×10³ IGF-I receptors per cell. In contrast, no specific binding of insulin could be detected. Competition binding experiments showed a slightly higher affinity of HOE 901 for the IGF-I receptor when compared to regular human insulin with IC₅₀ (half-inhibitory concentration) values of 70 and 101 nM, respectively. However, the supermitogenic insulin analogue [Asp^B10]insulin competed significantly more efficiently for IGF-I binding (IC₅₀: 44 nM). Maximum growth promoting activity of the peptides was then determined in serum-starved myoblasts by an incubation with the peptides (5×10⁻⁷ M) for 16 h in the presence of [³H]thymidine. [Asp^B10]Insulin produced a stimulation of DNA synthesis (about 3-fold) which was comparable to the effect of IGF-I and significantly (P<0.005) higher than the effect of HOE 901 with the latter being essentially equipotent to native insulin. Comparable results were obtained at lower concentrations of the peptides (10⁻⁹ to 10⁻⁸ M). Metabolic activity of HOE 901 was determined by measuring the dose-dependent stimulation of 3-O-methylglucose transport in adult cardiomyocytes. Maximum transport stimulation was identical for insulin and HOE 901 with EC₅₀ (half-effective concentration) values of 0.7×10⁻¹⁰ and 1.9×10⁻⁹ M, respectively. We concluded that the IGF-I receptor-mediated growth promoting activity of HOE 901 in muscle cells and the maximal metabolic activity of this analogue are not different from those of native human insulin. It is suggested that differential interaction with IGF-I receptors significantly contributes to the action profile of insulin analogues.