A combination of an iron chelator with an antioxidant exerts greater efficacy on cardioprotection than monotherapy in iron-overload thalassemic mice

Many recent studies have shown that antioxidant compounds decrease cardiac oxidative stress, decrease cardiac iron deposition, and improve cardiac dysfunction in iron-overload induced cardiomyopathy in animal models. Interestingly, a therapy including the combination of the iron chelator deferiprone (DFP) plus the antioxidant N-acetylcysteine (NAC) has been shown to significantly decrease oxidative stress and restore heart and brain function in iron-overloaded rats. However, the cardioprotective effects of this combined DFP and NAC treatment in thalassemic mice have not been investigated. We hypothesised that the combination of DFP and NAC exerts better cardioprotection than monotherapy via decreasing cardiac iron accumulation, oxidative stress, and apoptosis in thalassemic mice. The iron-overload condition was induced in heterozygous β^KO HT and wild-type mice by instigating high iron diet consumption (FE) for three months. Then, iron chelator DFP (75?mg/kg/day twice a day), antioxidant NAC (100?mg/kg/day once a day), and combined DFP plus NAC were fed via oral gavage for one month with continuous iron feeding. Left ventricular (LV) function, heart rate variability (HRV), apoptosis, and cardiac iron accumulation were determined. Chronic iron-overload in mice led to increased cardiac iron deposition, oxidative stress, apoptosis, and impaired LV function and HRV. Although DFP and NAC showed similar cardioprotective efficacy, combined DFP plus NAC exerted greater efficacy in reducing both cardiac iron deposition and cellular apoptosis than monotherapy. In conclusion, combined iron chelator and NAC treatment exert the greatest cardioprotective efficacy when compared with either of the monotherapies in iron-overload thalassemic mice.     

Continuing Treatment with Salvia miltiorrhiza Injection Attenuates Myocardial Fibrosis in Chronic Iron-Overloaded Mice

Iron overload cardiomyopathy results from iron accumulation in the myocardium that is closely linked to iron-mediated myocardial fibrosis. Salvia miltiorrhiza (SM, also known as Danshen), a traditional Chinese medicinal herb, has been widely used for hundreds of years to treat cardiovascular diseases. Here, we investigated the effect and potential mechanism of SM on myocardial fibrosis induced by chronic iron overload (CIO) in mice. Kunming male mice (8 weeks old) were randomized to six groups of 10 animals each: control (CONT), CIO, low-dose SM (L-SM), high-dose SM (H-SM), verapamil (VRP) and deferoxamine (DFO) groups. Normal saline was injected in the CONT group. Mice in the other five groups were treated with iron dextran at 50 mg/kg per day intraperitoneally for 7 weeks, and those in the latter four groups also received corresponding daily treatments, including 3 g/kg or 6 g/kg of SM, 100 mg/kg of VRP, or 100 mg/kg of DFO. The iron deposition was estimated histologically using Prussian blue staining. Myocardial fibrosis was determined by Masson’s trichrome staining and hydroxyproline (Hyp) quantitative assay. Superoxide dismutase (SOD) activity, malondialdehyde (MDA) content and protein expression levels of type I collagen (COL I), type I collagen (COL III), transforming growth factor-β1 (TGF-β₁) and matrix metalloproteinase-9 (MMP-9) were analyzed to investigate the mechanisms underlying the effects of SM against iron-overloaded fibrosis. Treatment of chronic iron-overloaded mice with SM dose-dependently reduced iron deposition levels, fibrotic area percentage, Hyp content, expression levels of COL I and COL III, as well as upregulated the expression of TGF- β₁ and MMP-9 proteins in the heart. Moreover, SM treatment decreased MDA content and increased SOD activity. In conclusion, SM exerted activities against cardiac fibrosis induced by CIO, which may be attributed to its inhibition of iron deposition, as well as collagen metabolism and oxidative stress.     

Secoisolariciresinol Diglucoside Abrogates Oxidative Stress-Induced Damage in Cardiac Iron Overload Condition

Cardiac iron overload is directly associated with cardiac dysfunction and can ultimately lead to heart failure. This study examined the effect of secoisolariciresinol diglucoside (SDG), a component of flaxseed, on iron overload induced cardiac damage by evaluating oxidative stress, inflammation and apoptosis in H9c2 cardiomyocytes. Cells were incubated with 50 μ5M iron for 24 hours and/or a 24 hour pre-treatment of 500 μ M SDG. Cardiac iron overload resulted in increased oxidative stress and gene expression of the inflammatory mediators tumor necrosis factor-α, interleukin-10 and interferon γ, as well as matrix metalloproteinases-2 and -9. Increased apoptosis was evident by increased active caspase 3/7 activity and increased protein expression of Forkhead box O3a, caspase 3 and Bax. Cardiac iron overload also resulted in increased protein expression of p70S6 Kinase 1 and decreased expression of AMP-activated protein kinase. Pre-treatment with SDG abrogated the iron-induced increases in oxidative stress, inflammation and apoptosis, as well as the increased p70S6 Kinase 1 and decreased AMP-activated protein kinase expression. The decrease in superoxide dismutase activity by iron treatment was prevented by pre-treatment with SDG in the presence of iron. Based on these findings we conclude that SDG was cytoprotective in an in vitro model of iron overload induced redox-inflammatory damage, suggesting a novel potential role for SDG in cardiac iron overload.     

ICS II protects against cardiac hypertrophy by regulating metabolic remodelling,not by inhibiting autophagy

Cardiac hypertrophy is characterized by a shift in metabolic substrate utilization. Therefore, the regulation of ketone body uptake and metabolism may have beneficial effects on heart injuries that induce cardiac remodelling. In this study, we investigated whether icariside II (ICS II) protects against cardiac hypertrophy in mice and cardiomyocytes. To create cardiac hypertrophy animal and cell models, mice were subjected to transverse aortic constriction (TAC), and embryonic rat cardiomyocytes (H9C2) were stimulated with angiotensin II, a neurohumoral stressor. Both the in vivo and in vitro results suggest that ICS II treatment ameliorated pressure overload–induced cardiac hypertrophy and preserved heart function. In addition, apoptosis and oxidative stress were reduced in the presence of ICS II. Moreover, ICS II inhibited excess autophagy in TAC-induced hearts and angiotensin II–stimulated cardiomyocytes. Mechanistically, we found that ICS II administration regulated SIRT3 expression in cardiac remodelling. SIRT3 activation increased ketone body transportation and utilization. Collectively, our data show that ICS II attenuated cardiac hypertrophy by modulating ketone body and fatty acid metabolism, and that this was likely due to the activation of the SIRT3-AMPK pathway. ICS II treatment may provide a new therapeutic strategy for improving myocardial metabolism in cardiac hypertrophy and heart failure.     

降香通过抗氧化应激改善后负荷增加型心衰小鼠的心脏功能

目的:研究降香对后负荷增加引起的的心脏功能下降的保护作用及其机制。方法:雄性C57小鼠30只,随机分为三组,分别给予假手术(sham)、主动脉弓结扎(Transverse aortic constriction,TAC)手术和主动脉弓结扎手术降香治疗(TAC+DO)处理。通过灌胃给药4周,随后超声检测心脏功能、四腔心切片观察心肌重构,RT-PCR检测左心室αMHC、βMHC的m RNA表达、相应试剂盒心肌总抗氧化能力(TAOC)和丙二醇(MDA)含量。结果:同sham组相比,TAC组射血分数(EF),αMHC m RNA水平和TOAC均显著降低,且左室舒张末内径(LVIDd)、左室舒张期后壁厚度(LVPWd)、左室质量(LV mass)、心肌质量/胫骨长度(HW/TL)及β及β度、MDA均显著增加。同TAC组相比,DO组射血分数(EF),αMHC m RNA水平和TOAC均显著增加,且左室舒张末内径(LVIDd)、舒张末室间隔厚度(IVSd)、左室质量(LV mass)、心肌质量/胫骨长度(HW/TL)及βMHC、MDA均显著下降。在离体培养的心肌细胞,H_2O_2可显著增加细胞内ROS含量,给予降香或TEMPOL处理均可减轻H_2O_2诱导的氧化应激并增加心肌细胞存活率。结论:降香可通过降低氧化应激抑制线粒体分裂并改善后负荷增加型心衰的心脏功能。     

Dose-dependent effects of chronic iron burden on heart aldehyde and acyloin production in mice

Iron’s chemical structure and its ability to initiate one-electron reactions are properties that cause it to play a major role in the production and metabolism of oxygen free radicals in biological systems. Oxygen free radicals are conjectured to cause cardiac failure in individuals afflicted with disorders of iron overload. We report on the use of both acyloins and aldehydes as markers of oxidative stress in a murine model of chronic iron-overload cardiomyopathy. Twenty mice were randomized to four treatment groups: (1) control (0.2 mL normal saline ip/mouse/d); (2) 100 mg iron (0.05 mL iron dextran/mouse/d); (3) 200 mg iron (0.1 mL iron dexxtran/mouse/d); (4) 400 mg iron (0.2 mL iron dextran/mouse/d). Significant dose-dependent increases in both total heart aldehyde and total heart acyloin concentrations were found. Furthermore, a significant positive correlation existed between the dose of iron administered and each quantified aldehyde and acyloin found in the heart.     

Effects of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac function and Ca2+ regulation in iron-overloaded thalassemic mice

Although disturbance of cardiac Ca²⁺ regulation is involved in the pathophysiology of iron-overload cardiomyopathy, the obvious mechanisms involved in the dysregulation of iron-induced cardiac Ca²⁺ are unclear. Moreover, the roles of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac intracellular Ca²⁺ transients and Ca²⁺ regulatory proteins in thalassemic mice are still unknown. We tested the hypothesis that treatment with either deferiprone or efonidipine attenuated cardiac Ca²⁺ dysregulation and led to improved left ventricular (LV) function in iron-overloaded thalassemic mice. Wild-type (WT) mice and β-thalassemic (HT) mice were fed with either a normal diet (ND) or a high iron-diet (FE) for 90 days. Then, the FE-fed mice were treated with either deferiprone (75 mg/kg/day) or efonidipine (4 mg/kg/day) for 30 days. ND-fed HT mice had an increase in T-type calcium channels (TTCC) and an increased level of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), compared with ND-fed WT mice. Chronic iron feeding led to an increase in TTCC and expression of SERCA proteins in FE-fed WT mice. Moreover, chronic iron overload led to increased plasma non-transferrin bound iron (NTBI) and cardiac iron deposition, impaired cardiac intracellular Ca²⁺ transients including decreased intracellular Ca²⁺ transient amplitude, rising rate and decay rate, as well as impaired LV function as indicated by a decreased %LV ejection fraction (%LVEF) in both WT and HT mice. Our findings showed that treatment with either deferiprone (DFP) or efonidipine (EFO) showed similar benefits in reducing plasma NTBI and cardiac iron deposition, and improving %LVEF from 84.3 (WT) to 89.3 (DFP) and 89.2 (EFO) treatment; and from 84.2 (HT) to 88.8 (DFP) and 89.5 (EFO) treatment, however there was no improvement in the regulation of cardiac Ca²⁺ in iron-overloaded thalassemic mice. These findings provide the understanding of the effects of these drugs on the iron-overloaded heart in thalassemic mice and suggest that an alternative intervention that could improve calcium regulation under this condition is needed to improve the therapeutic outcome. Moreover, whether the benefits of the TTCC blocker is via its inhibition of the TTCC alone or together with its ability to chelate iron are still unclear and need further investigation.     

Altered glutathione homeostasis in heart augments cardiac lipotoxicity associated with diet-induced obesity in mice

Obesity-related cardiac lipid accumulation is associated with increased myocardial oxidative stress. The role of the antioxidant glutathione in cardiac lipotoxicity is unclear. Cystathionine β-synthase (Cbs) catalyzes the first step in the trans-sulfuration of homocysteine to cysteine, which is estimated to provide ～50% of cysteine for hepatic glutathione biosynthesis. As cardiac glutathione is a reflection of the liver glutathione pool, we hypothesize that mice heterozygous for targeted disruption of Cbs (Cbs(+/-)) are more susceptible to obesity-related cardiolipotoxicity because of impaired liver glutathione synthesis. Cbs(+/+) and Cbs(+/-) mice were fed a high fat diet (60% energy) from weaning for 13 weeks to induce obesity and had similar increases in body weight and body fat. This was accompanied by increased hepatic triglyceride but no differences in hepatic glutathione levels compared with mice fed chow. However, Cbs(+/-) mice with diet-induced obesity had greater glucose intolerance and lower total and reduced glutathione levels in the heart, accompanied by lower plasma cysteine levels compared with Cbs(+/+) mice. Higher triglyceride concentrations, increased oxidative stress, and increased markers of apoptosis were also observed in heart from Cbs(+/-) mice with diet-induced obesity compared with Cbs(+/+) mice. This study suggests a novel role for Cbs in maintaining the cardiac glutathione pool and protecting against cardiac lipid accumulation and oxidative stress during diet-induced obesity in mice.     

Cardiac-specific CGI-58 deficiency activates the ER stress pathway to promote heart failure in mice

Excess myocardial triacylglycerol accumulation (i.e., cardiac steatosis) impairs heart function, suggesting that enzymes promoting triacylglycerol metabolism exert essential regulatory effects on heart function. Comparative gene identification 58 (CGI-58) is a key enzyme that promotes the hydrolysis of triglycerides by activating adipose triglyceride lipase and plays a protective role in maintaining heart function. In this study, the effects of CGI-58 on heart function and the underlying mechanism were investigated using cardiac-specific CGI58-knockout mice (CGI-58^cko mice). Echocardiography and pathological staining were performed to detect changes in the structure and function of the heart. Proteomic profiling, immunofluorescent staining, western blotting, and real-time PCR were used to evaluate molecular changes. In CGI-58^cko mice, we detected cardiac hypertrophic remodeling and heart failure associated with excessive cardiac lipid accumulation, ROS production, and decreased expression of regulators of fatty acid metabolism. These changes were markedly attenuated in CGI-58^cko mice injected with rAAV9-CGI58. A quantitative proteomics analysis revealed significant increases in the expression of ER stress-related proteins and decreases in proteins related to fatty acid and amino acid metabolism in the hearts of CGI-58^cko mice. Furthermore, the inhibition of ER stress by the inhibitor 4-PBA improved mitochondrial dysfunction, reduced oxidative stress, and reversed cardiac remodeling and dysfunction in cultured cardiomyocytes or in CGI-58^cko mice. Our results suggested that CGI-58 is essential for the maintenance of heart function by reducing lipid accumulation and ER stress in cardiomyocytes, providing a new therapeutic target for cardiac steatosis and dysfunction.Subject terms: Heart failure, Heart failure     

Differential effects of iron overload on GST isoform expression in mouse liver and kidney and correlation between GSTA4 induction and overproduction of free radicles.

We have investigated the effect of iron overload on the expression of mouse GSTA1, A4, M1, and P1 in liver, the main iron storage site during iron overload, and in kidney. In iron-overloaded animals, mRNA and protein levels of GSTA1, A4, and M1 were increased in liver. In kidney, GSTA4 protein level was also increased while, unexpectedly, GSTA1 and M1 expression was strongly decreased. We showed, by immunohistochemistry, that GSTA4 was more abundant in hepatocytes of periportal areas and in convoluted proximal tubular cells in normal liver and kidney, respectively. In iron-overloaded mice, GSTA4 staining was more intense in cells that preferentially accumulated iron, and conjugation of 4-hydroxynonenal, a specific substrate of GSTA4, was enhanced in both organs. Moreover an acute exposure of primary cultures of mouse hepatocytes to iron-citrate strongly induced oxidative stress and cellular injury and resulted in an increase in GSTA4 expression, while cotreatment with iron-citrate and either desferrioxamine or vitamin E prevented both toxicity and GSTA4 induction. These data demonstrate that GSTA1 and M1 are differentially regulated in liver and kidney while GSTA4 is induced in both organs during iron overload. Moreover, they support the view that iron-induction of GSTA4 is related to an overproduction of free radicals.     

Non-transferrin-bound iron reaches mitochondria by a chelator-inaccessible mechanism: biological and clinical implications

Non-transferrin-bound iron, commonly found in the plasma of iron-overloaded individuals, permeates into cells via pathways independent of the transferrin receptor. This may lead to excessive cellular accumulation of labile iron followed by oxidative damage and eventually organ failure. Mitochondria are the principal destination of iron in cells and a primary site of prooxidant generation, yet their mode of acquisition of iron is poorly understood. Using fluorescent probes sensitive to iron or to reactive oxygen species, targeted to cytosol and/or to mitochondria, we traced the ingress of labile iron into these compartments by fluorescence microscopy and quantitative fluorimetry. We observed that 1) penetration of non-transferrin-bound iron into the cytosol and subsequently into mitochondria occurs with barely detectable delay and 2) loading of the cytosol with high-affinity iron-binding chelators does not abrogate iron uptake into mitochondria. Therefore, a fraction of non-transferrin-bound iron acquired by cells reaches the mitochondria in a nonlabile form. The physiological role of occluded iron transfer might be to confer cells with a "safe and efficient cytosolic iron corridor" to mitochondria. However, such a mechanism might be deleterious in iron-overload conditions, because it could lead to surplus accumulation of iron in these critical organelles. transport; fluorescence; oxidative stress     

Induction of Manganese Superoxide Dismutase by Nuclear Translocation and Activation of SIRT1 Promotes Cell Survival in Chronic Heart Failure

Oxidative stress plays a pivotal role in chronic heart failure. SIRT1, an NAD⁺-dependent histone/protein deacetylase, promotes cell survival under oxidative stress when it is expressed in the nucleus. However, adult cardiomyocytes predominantly express SIRT1 in the cytoplasm, and its function has not been elucidated. The purpose of this study was to investigate the functional role of SIRT1 in the heart and the potential use of SIRT1 in therapy for heart failure. We investigated the subcellular localization of SIRT1 in cardiomyocytes and its impact on cell survival. SIRT1 accumulated in the nucleus of cardiomyocytes in the failing hearts of TO-2 hamsters, postmyocardial infarction rats, and a dilated cardiomyopathy patient but not in control healthy hearts. Nuclear but not cytoplasmic SIRT1-induced manganese superoxide dismutase (Mn-SOD), which was further enhanced by resveratrol, and increased the resistance of C2C12 myoblasts to oxidative stress. Resveratrol''s enhancement of Mn-SOD levels depended on the level of nuclear SIRT1, and it suppressed the cell death induced by antimycin A or angiotensin II. The cell-protective effects of nuclear SIRT1 or resveratrol were canceled by the Mn-SOD small interfering RNA or SIRT1 small interfering RNA. The oral administration of resveratrol to TO-2 hamsters increased Mn-SOD levels in cardiomyocytes, suppressed fibrosis, preserved cardiac function, and significantly improved survival. Thus, Mn-SOD induced by resveratrol via nuclear SIRT1 reduced oxidative stress and participated in cardiomyocyte protection. SIRT1 activators such as resveratrol could be novel therapeutic tools for the treatment of chronic heart failure.     

Cardiac volume overload rapidly induces oxidative stress-mediated myocyte apoptosis and hypertrophy

Oxidative stress stimulates both growth and apoptosis in cardiac myocytes in vitro. We investigated the role of oxidative stress in the initial phases of cardiac remodeling induced in an animal model by volume overload. As plausible candidates for a connection between oxidative stress and cardiomyocyte apoptosis or hypertrophy, we explored the behaviour of two MAPKs, specifically JNK and ERK. At 48 h of overload, the greatest increase in oxidative stress coincided with a peak of cardiomyocyte apoptosis. This was possibly induced through the mitochondrial metabolism, as evidenced by the release of cytochrome c and a significant increase in the active forms of caspase-9 and -3, but not caspase-8. Oxidative stress markers significantly decreased at 96 h of overload, combined with a marked attenuation of apoptosis and the appearance of hypertrophy. The highest levels of JNK and the lowest levels of ERK phosphorylation were observed at 48 h of overload. Conversely, a sharp increase in ERK phosphorylation was detected at 96 h of overload coinciding with the hypertrophic response. Together these results show that oxidative stress is an early and transient event in myocardial volume overload. They suggest that oxidative stress mediates amplitude dependent apoptotic and hypertrophic responses in cardiomyocytes through the selective activation of, respectively, JNK and ERK.     

Risk of iron overload is decreased in beating heart coronary artery surgery compared to conventional bypass.

Conventional cardiopulmonary bypass surgery (CCPB) increases the iron loading of plasma transferrin often to a state of plasma iron overload, with the presence of low molecular mass iron. Such iron is a potential risk factor for oxidative stress and microbial virulence. Here we assess 'off-pump' coronary artery surgery on the beating heart for changes in plasma iron chemistry. Seventeen patients undergoing cardiac surgery using the 'Octopus' myocardial wall stabilisation device were monitored at five time points for changes in plasma iron chemistry. This group was further divided into those (n=9) who had one- or two- (n=8) vessel grafts, and compared with eight patients undergoing conventional coronary artery surgery. Patients undergoing beating heart surgery had significantly lower levels of total plasma non-haem iron, and a decreased percentage saturation of their transferrin at all time points compared to conventional bypass patients. Plasma iron overload occurred in only one patient undergoing CCPB. Beating heart surgery appears to decrease red blood cell haemolysis, and tissue damage during the operative procedures and thereby significantly decreases the risk of plasma iron overload associated with conventional bypass.     

p38alpha mitogen-activated protein kinase plays a critical role in cardiomyocyte survival but not in cardiac hypertrophic growth in response to pressure overload

The molecular mechanism for the transition from cardiac hypertrophy, an adaptive response to biomechanical stress, to heart failure is poorly understood. The mitogen-activated protein kinase p38alpha is a key component of stress response pathways in various types of cells. In this study, we attempted to explore the in vivo physiological functions of p38alpha in hearts. First, we generated mice with floxed p38alpha alleles and crossbred them with mice expressing the Cre recombinase under the control of the alpha-myosin heavy-chain promoter to obtain cardiac-specific p38alpha knockout mice. These cardiac-specific p38alpha knockout mice were born normally, developed to adulthood, were fertile, exhibited a normal life span, and displayed normal global cardiac structure and function. In response to pressure overload to the left ventricle, they developed significant levels of cardiac hypertrophy, as seen in controls, but also developed cardiac dysfunction and heart dilatation. This abnormal response to pressure overload was accompanied by massive cardiac fibrosis and the appearance of apoptotic cardiomyocytes. These results demonstrate that p38alpha plays a critical role in the cardiomyocyte survival pathway in response to pressure overload, while cardiac hypertrophic growth is unaffected despite its dramatic down-regulation.     

Oxidative stress and redox regulation of phospholipase D in myocardial disease

Oxidative stress may be viewed as an imbalance between reactive oxygen species (ROS) and oxidant production and the state of glutathione redox buffer and antioxidant defense system. Recently, a new paradigm of redox signaling has emerged whereby ROS and oxidants can function as intracellular signaling molecules, where ROS- and oxidant-induced death signal is converted into a survival signal. It is now known that oxidative stress is involved in cardiac hypertrophy and in the pathogenesis of cardiomyopathies, ischemic heart disease and congestive heart failure. Phospholipase D (PLD) is an important signaling enzyme in mammalian cells, including cardiomyocytes. PLD catalyzes the hydrolysis of phosphatidylcholine to produce phosphatidic acid (PA). Two mammalian PLD isozymes, PLD1 and PLD2 have been identified, characterized and cloned. The importance of PA in heart function is evident from its ability to stimulate cardiac sarcolemmal membrane and sarcoplasmic reticular Ca2+-related transport systems and to increase the intracellular concentration of free Ca2+ in adult cardiomyocytes and augment cardiac contractile activity of the normal heart. In addition, PA is also considered an important signal transducer in cardiac hypertrophy. Accordingly, this review discusses a role for redox signaling mediated via PLD in ischemic preconditioning and examines how oxidative stress affects PLD in normal hearts and during different myocardial diseases. In addition, the review provides a comparative account on the regulation of PLD activities in vascular smooth muscle cells under conditions of oxidative stress.