Measurements of 1,2-diacylglycerol and ceramide in hearts subjected to ischemic preconditioning

An accumulation of recent evidence suggests that the mechanism in ischemic preconditioning (IPC) may involve the activation of protein kinase C (PKC) regulatory pathway. In this study, we examined whether the content of 1,2-diacylglycerol (1,2-DAG) and ceramide, which are intracellular second messengers regulating PKC activity, change during IPC in isolated perfused rat hearts, and whether the observed change in 1,2-DAG is accompanied with alteration in its fatty acid composition. Hearts subjected to IPC, consisting of 5-min transient global ischemia followed by 5-min reperfusion, presented a significant functional recovery during subsequent 40-min reperfusion following 40-min global ischemia compared with non-preconditioned hearts. An increase in 1,2-DAG content was observed in hearts subjected to 5-min transient ischemia compared with non-ischemic control hearts, however this was not seen in hearts harvested after 5-min reperfusion following 5-min ischemia. While fatty acid composition in 1,2-DAG was virtually unchanged in hearts subjected to 5-min ischemia, saturated 1,2-DAG decreased and monounsaturated/polyunsaturated 1,2-DAG increased in hearts reperfused for 5-min following 5-min ischemia compared with the non-ischemic control hearts. Ceramide mass did not change significantly, suggesting that the contribution of ceramide may be small in IPC. These data are in concert with the hypothesis that 1,2-DAG is a second messenger in IPC and the changes in fatty acid composition of 1,2-DAG may add new insight concerning signal transduction pathway in IPC.     

Metabolism of very-low-density lipoprotein and chylomicrons by streptozotocin-induced diabetic rat heart: effects of diabetes and lipoprotein preference

Very-low-density lipoprotein (VLDL) and chylomicrons (CM) are major sources of fatty acid supply to the heart, but little is known about their metabolism in diabetic myocardium. To investigate this, working hearts isolated from control rats and diabetic rats 2 wk following streptozotocin (STZ) injection were perfused with control and diabetic lipoproteins. Analysis of the diabetic lipoproteins showed that both VLDL and CM were altered compared with control lipoproteins; both were smaller and had different apolipoprotein composition. Heparin-releasable lipoprotein lipase (HR-LPL) activity was increased in STZ-induced diabetic hearts, but tissue residual LPL activity was decreased; moreover, diabetic lipoproteins stimulated HR-LPL activity in both diabetic and control hearts. Diabetic hearts oxidized lipoprotein-triacylglycerol (TAG) to a significantly greater extent than controls (>80% compared with deposition as tissue lipid), and the oxidation rate of exogenous lipoprotein-TAG was increased significantly in diabetic hearts regardless of TAG source. Significantly increased intracardiomyocyte TAG accumulation was found in diabetic hearts, although cardiac mechanical function was not inhibited, suggesting that lipotoxicity precedes impaired cardiac performance. Glucose oxidation was significantly decreased in diabetic hearts; additionally, however, diabetic lipoproteins decreased glucose oxidation in diabetic and control hearts. These results demonstrate increased TAG-rich lipoprotein metabolism concomitant with decreased glucose oxidation in type 1 diabetic hearts, and the alterations in cardiac lipoprotein metabolism may be due to the properties of diabetic TAG-rich lipoproteins as well as the diabetic state of the myocardium. These changes were not related to cardiomyopathy at this early stage of diabetes.     

Increased 1,2-diacylglycerol content in myopathic hamster hearts at a prenecrotic stage

1,2-Diacylglycerol (DAG) has been suggested to be a secondary messenger. In this study, we determined the amount of 1,2-DAG in heart tissue from Syrian hamsters with hereditary cardiomyopathy at 30 days (prenecrotic stage) and 90 days of age by thin-layer chromatography with flame ionization detection (TLC-FID). Myocardial triglyceride contents were higher at 30 days of age and lower at 90 days of age compared to the levels in age-matched normal hamsters. Decreases in major species of phospholipids in hearts were observed only at 90 days of age. However, elevated 1,2-DAG content in myopathic hearts was found at 30 days of age, whereas there was no difference between the two groups at 90 days of age. It is suggested that the increase in 1,2-DAG at the prenecrotic stage is involved in the pathogenesis of the cardiomyopathy.     

Role of PP2C in cardiac lipid accumulation in obese rodents and its prevention by troglitazone

In obese rodents, excess myocardial lipid accumulation (lipotoxicity) of myocardium may cause cardiomyopathy that in the obese Zucker diabetic fatty (ZDF) fa/fa rat can be prevented by treatment with troglitazone (TGZ). To determine the underlying mechanisms, we measured total 5'-AMP-activated kinase (AMPK) protein and its activated, phosphorylated form, P-AMPK. P-AMPK was significantly reduced in both ZDF fa/fa rat and ob/ob mouse hearts compared with lean, wild-type controls. TGZ treatment of obese ZDF rats, which lowered cardiac lipid content, increased P-AMPK. Expression of protein phosphatase 2C (PP2C), which inactivates AMPK activity by dephosphorylation, was increased in untreated ZDF fa/fa rat hearts, but fell with TGZ treatment, suggesting that PP2C can influence AMPK activity. In cultured myocardiocytes, fatty acids reduced P-AMPK, suggesting a feed-forward effect of lipid overload. Our findings highlight a role of PP2C and AMPK in the derangements of cardiac lipid metabolism in obesity and provide new insights as to the mechanisms of the liporegulatory disorder leading to lipotoxic cardiomyopathy.     

The Critical Role of Astragalus Polysaccharides for the Improvement of PPRA��-Mediated Lipotoxicity in Diabetic Cardiomyopathy

Background

Obesity-related diabetes mellitus leads to increased myocardial uptake and oxidation of fatty acids, resulting in a form of cardiac dysfunction referred to as lipotoxic cardiomyopathy. We have shown previously that Astragalus polysaccharides (APS) administration was sufficient to improve the systemic metabolic disorder and cardiac dysfunction in diabetic models.

Methodology/Principal Findings

To investigate the precise role of APS therapy in the pathogenesis of myocardial lipotoxity in diabetes, db/db diabetic mice and myosin heavy chain (MHC)- peroxisome proliferator-activated receptor (PPAR) α mice were characterized and administrated with or without APS with C57 wide- type mice as normal control. APS treatment strikingly improved the myocyte triacylglyceride accumulation and cardiac dysfunction in both db/db mice and MHC-PPARα mice, with the normalization of energy metabolic derangements in both db/db diabetic hearts and MHC-PPARα hearts. Consistently, the activation of PPARα target genes involved in myocardial fatty acid uptake and oxidation in both db/db diabetic hearts and MHC-PPARα hearts was reciprocally repressed by APS administration, while PPARα-mediated suppression of genes involved in glucose utilization of both diabetic hearts and MHC-PPARα hearts was reversed by treatment with APS.

Conclusions

We conclude that APS therapy could prevent the development of diabetic cardiomyopathy through a mechanism mainly dependent on the cardiac PPARα-mediated regulatory pathways.     

Lack of effect of oral L-carnitine treatment on lipid metabolism and cardiac function in chronically diabetic rats

L-Carnitine is necessary for the transfer of long-chain fatty acids into the mitochondrial matrix where energy production occurs. In the absence of L-carnitine, the accumulation of free fatty acids and related intermediates could produce myocardial subcellular alterations and cardiac dysfunction. Diabetic hearts have a deficiency in the total carnitine pool and develop cardiac dysfunction. This suggested that carnitine therapy may ameliorate alteration in cardiac contractile performance seen during diabetes. In this study, heart function was studied in streptozotocin diabetic rats given L-carnitine orally. Oral L-carnitine treatment (50-250 mg.kg-1.day-1) of 1- and 3-week diabetic rats increased plasma free and total carnitine and decreased plasma acyl carnitine levels. In both groups, myocardial total carnitine levels were increased. However, L-carnitine (200 mg.kg-1.day-1) treatment of diabetic rats for 6 weeks had no effect on plasma carnitine levels. Similarly, plasma lipids remained elevated whereas cardiac function was still depressed. These studies suggest that in the chronically diabetic rat, the route of administration of L-carnitine is an important factor in determining an effect.     

Prevention of diabetes-induced myocardial dysfunction in rats by methyl palmoxirate and triiodothyronine treatment

Diabetes results in myocardial functional alterations which are accompanied by a depression of biochemical parameters such as myosin ATPase and calcium uptake in the sarcoplasmic reticulum. Methyl palmoxirate, a fatty acid analog, is reported to decrease circulating glucose levels by inhibiting fatty acid metabolism, thus forcing carbohydrate utilization. In the present study, we attempted to prevent streptozotocin diabetes-induced myocardial alterations in the rat. Using the isolated working heart preparation, we observed a depression of myocardial function in rats 6 weeks after the induction of diabetes, which was characterized by the inability of these hearts to develop left ventricular pressures and rates of ventricular contraction and relaxation as well as control hearts at higher left atrial filling pressures. Methyl palmoxirate treatment (25 mg kg-1 day-1 po daily) was unable to control diabetes-induced changes in plasma glucose, triglycerides, insulin, and total lipids. Also, the functional depression seen in diabetic rat hearts was present despite the treatment. However, depression of calcium uptake and elevation of long chain acyl carnitines seen in sarcoplasmic reticulum (SR) prepared from diabetic rat hearts could be prevented by the treatment. As triiodothyronine (T3) treatment has been shown to normalize depression of cardiac myosin ATPase in diabetic rats, we repeated the study using a combination of T3 (30 micrograms kg-1 day-1 sc daily) and methyl palmoxirate. While diabetic rats treated with T3 alone did not show significant improvement of myocardial function when compared with untreated diabetics, the function of those treated with both T3 and methyl palmoxirate was not significantly different from that in control rat hearts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metoprolol improves cardiac function and modulates cardiac metabolism in the streptozotocin-diabetic rat

The effects of diabetes on heart function may be initiated or compounded by the exaggerated reliance of the diabetic heart on fatty acids and ketones as metabolic fuels. beta-Blocking agents such as metoprolol have been proposed to inhibit fatty acid oxidation. We hypothesized that metoprolol would improve cardiac function by inhibiting fatty acid oxidation and promoting a compensatory increase in glucose utilization. We measured ex vivo cardiac function and substrate utilization after chronic metoprolol treatment and acute metoprolol perfusion. Chronic metoprolol treatment attenuated the development of cardiac dysfunction in streptozotocin (STZ)-diabetic rats. After chronic treatment with metoprolol, palmitate oxidation was increased in control hearts but decreased in diabetic hearts without affecting myocardial energetics. Acute treatment with metoprolol during heart perfusions led to reduced rates of palmitate oxidation, stimulation of glucose oxidation, and increased tissue ATP levels. Metoprolol lowered malonyl-CoA levels in control hearts only, but no changes in acetyl-CoA carboxylase phosphorylation or AMP-activated protein kinase activity were observed. Both acute metoprolol perfusion and chronic in vivo metoprolol treatment led to decreased maximum activity and decreased sensitivity of carnitine palmitoyltransferase I to malonyl-CoA. Metoprolol also increased sarco(endo)plasmic reticulum Ca(2+)-ATPase expression and prevented the reexpression of atrial natriuretic peptide in diabetic hearts. These data demonstrate that metoprolol ameliorates diabetic cardiomyopathy and inhibits fatty acid oxidation in streptozotocin-induced diabetes. Since malonyl-CoA levels are not increased, the reduction in total carnitine palmitoyltransferase I activity is the most likely factor to explain the decrease in fatty acid oxidation. The metabolism changes occur in parallel with changes in gene expression.     

Metabolism of VLDL is increased in streptozotocin-induced diabetic rat hearts

In streptozotocin (STZ)-induced diabetic rats, we previously showed an increased heparin-releasable (luminal) lipoprotein lipase (LPL) activity from perfused hearts. To study the effect of this enlarged LPL pool on triglyceride (TG)-rich lipoproteins, we examined the metabolism of very-low-density lipoprotein (VLDL) perfused through control and diabetic hearts. Diabetic rats had elevated TG levels compared with control. However, fasting for 16 h abolished this difference. When the plasma lipoprotein fraction of density <1.006 g/ml from fasted control and diabetic rats was incubated in vitro with purified bovine or rat LPL, VLDL from diabetic animals was hydrolyzed as proficiently as VLDL from control animals. Post-heparin plasma lipolytic activity was comparable in control and diabetic animals. However, perfusion of control and diabetic rats with heparinase indicated that diabetic hearts had larger amounts of LPL bound to heparan sulfate proteoglycan-binding sites. [(3)H]VLDL obtained from control rats, when recirculated through the isolated heart, disappeared at a significantly faster rate from diabetic than from control rat hearts. This increased VLDL-TG hydrolysis was essentially abolished by prior perfusion of the diabetic heart with heparin, implicating LPL in this process. These findings suggest that the enlarged LPL pool in the diabetic heart is present at a functionally relevant location (at the capillary lumen) and is capable of hydrolyzing VLDL. This could increase the delivery of free fatty acid to the heart, and the resultant metabolic changes could induce the subsequent cardiomyopathy that is observed in the chronic diabetic rat.     

Diabetic heart and kidney exhibit increased resistance to lipid peroxidation

Alloxan-diabetic rats and age-matched controls were killed after 6 weeks of diabetes; heart and kidneys were removed and assayed for thiobarbituric acid-reactive substances (TBARS), lipid hydroperoxides, lipid phosphorus, total fatty acid composition and glutathione. Tissue homogenates from a second group of diabetic and control rats were incubated in oxygen-saturated buffer with and without the free radical generating system Fe2+/ascorbate (0.1/1.0 mM) and were assayed for lipid peroxidation. Diabetic hearts contained markedly lower levels of TBARS and lipid hydroperoxides (40% and 18%, respectively) than control hearts, whereas differences in TBARS were less pronounced in kidneys (9%). Incubation of homogenates of both organs in the presence or absence of Fe2+/ascorbate for up to 2 h yielded significantly lower levels of TBARS and lipid hydroperoxides with diabetic tissue. Diabetic hearts and kidneys contained higher levels of glutathione (28% and 13% over controls) and both diabetic tissues showed much higher linoleate/arachidonate ratios than did the controls (9.86 vs. 2.56 for heart, 2.01 vs. 0.86 for kidney). We conclude that diabetic tissues develop enhanced defense systems against oxidative stress and we assume tha the lower levels of arachidonate contribute to their resistance to lipid peroxidation as well.     

Gender differences in myosin heavy chain-beta and phosphorylated phospholamban in diabetic rat hearts

The objective of this study was to determine whether a gender difference exists in myosin heavy chain (MHC) isoform or sarcoplasmic reticulum protein levels in diabetic rat hearts. As is the case with normal rodent hearts, all four chambers of the control rat hearts expressed almost 100% MHC-alpha. In 6-wk diabetic rats, MHC-beta expression in ventricles of males was significantly greater (78 +/- 7%) than in females (50 +/- 5%). The cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) protein level was decreased and the phospholamban (PLB) protein level was increased in the left ventricle of diabetic rats, but there was no difference between male and female diabetic rats. The phosphorylated PLB level was decreased more in male than in female diabetic rats. Insulin treatment completely normalized blood glucose level, cardiac SERCA2a and PLB protein levels, and the decrease in MHC-beta levels in both male and female diabetic rats. Insulin treatment completely normalized serum insulin and almost completely normalized phosphorylation of PLB at serine 16 in male diabetic rats. Although insulin treatment completely normalized serum insulin levels in male diabetic rats, in females it only partially normalized serum insulin levels. Also, insulin treatment almost completely normalized phosphorylation of PLB at threonine 17 in female diabetic rats; however, the increase was significantly greater than that identified for insulin-treated male diabetic rats. We conclude that higher levels of MHC-beta and dephosphorylated PLB may contribute to more contractile dysfunction in male than in female diabetic rat hearts, and that phosphorylation of PLB at threonine 17 is more responsive to insulin in female diabetic rat hearts.     

Energy metabolism and myocardial ultrastructure in autoimmune cardiomyopathy

Alterations in the heart energy metabolism, early defects in cardiomyocyte sarcolemma and heart resistance to ischemic damage have been investigated in experimental autoimmune cardiomyopathy. Systolic and diastolic pressures were registered and the speed of ischemic contracture development was determined on the isolated perfused rat hearts. Oxidative phosphorylation parameters, macroenergetic phosphate levels were determined. The ultrastructure and cell membrane permeability to lanthanum were studied. The results obtained have shown that the hearts of rats with autoimmune cardiomyopathy revealed decreased macroenergetic phosphate levels: ATP level was 22% lower and Pcr was 45% lower. Resistance of cardiomyopathic hearts to ischemic stress significantly reduced, cell membrane permeability was distorted. The above changes are believed to be due to incompetent myocardial hypertrophy and Ca overload.     

Transmural differences in respiratory capacity across the rat left ventricle in health, aging, and streptozotocin-induced diabetes mellitus: evidence that mitochondrial dysfunction begins in the subepicardium

In diabetic cardiomyopathy, ventricular dysfunction occurs in the absence of hypertension or atherosclerosis and is accompanied by altered myocardial substrate utilization and depressed mitochondrial respiration. It is not known if mitochondrial function differs across the left ventricular (LV) wall in diabetes. In the healthy heart, the inner subendocardial region demonstrates higher rates of blood flow, oxygen consumption, and ATP turnover compared with the outer subepicardial region, but published transmural respirometric measurements have not demonstrated differences. We aim to measure mitochondrial function in Wistar rat LV to determine the effects of age, streptozotocin-diabetes, and LV layer. High-resolution respirometry measured indexes of respiration in saponin-skinned fibers dissected from the LV subendocardium and subepicardium of 3-mo-old rats after 1 mo of streptozotocin-induced diabetes and 4-mo-old rats following 2 mo of diabetes. Heart rate and heartbeat duration were measured under isoflurane-anesthesia using a fetal-Doppler, and transmission electron microscopy was employed to observe ultrastructural differences. Heart rate decreased with age and diabetes, whereas heartbeat duration increased with diabetes. While there were no transmural respirational differences in young healthy rat hearts, both myocardial layers showed a respiratory depression with age (30-40%). In 1-mo diabetic rat hearts only subepicardial respiration was depressed, whereas after 2 mo diabetes, respiration in subendocardial and subepicardial layers was depressed and showed elevated leak (state 2) respiration. These data provide evidence that mitochondrial dysfunction is first detectable in the subepicardium of diabetic rat LV, whereas there are measureable changes in LV mitochondria after only 4 mo of aging.     

Heart failure progression is accelerated following myocardial infarction in type 2 diabetic rats

Clinical studies have shown a greater incidence of myocardial infarction in diabetic patients, and following an infarction, diabetes is associated with an increased risk for the development of left ventricular (LV) dysfunction and heart failure. The goal of this study was to determine if the progression of heart failure following myocardial infarction in type 2 diabetic (T2D) rats is accelerated compared with nondiabetic rats. Male nondiabetic Wistar-Kyoto (WKY) and T2D Goto-Kakizaki (GK) rats underwent coronary artery ligation or sham surgery to induce heart failure. Postligation (8 and 20 wk), two-dimensional echocardiography and LV pressure measurements were made. Heart failure progression, as assessed by enhanced LV remodeling and contractile dysfunction, was accelerated 8 wk postligation in the T2D animals. LV remodeling was evident from increased end-diastolic and end-systolic diameters and areas in the GK compared with the WKY infarcted group. Furthermore, enhanced LV contractile dysfunction was evident from a greater deterioration in fractional shortening and enhanced myocardial performance index (an index of global LV dysfunction) in the GK infarcted group. This accelerated progression was accompanied by greater increases in atrial natriuretic factor and skeletal alpha-actin (gene markers of heart failure and hypertrophy) mRNA levels in GK infarcted hearts. Despite similar decreases in metabolic gene expression (i.e., peroxisome proliferator-activated receptor-alpha-regulated genes associated with fatty acid oxidation) between infarcted WKY and GK rat hearts, myocardial triglyceride levels were elevated in the GK hearts only. These results, demonstrating enhanced remodeling and LV dysfunction 8 wk postligation provide evidence of an accelerated progression of heart failure in T2D rats.     

实验性2型糖尿病心肌病大鼠模型的建立与评价

目的建立和评价2型糖尿病心肌病（DC）大鼠模型,探究高糖脂饮食在模型建立中的作用。方法将雄性Wistar大鼠随机分成正常对照组、高糖脂饮食组和高糖脂负荷小剂量STZ组。高糖高脂膳食诱导11周负荷小剂量链脲佐菌素（STZ）（30 mg/kg）腹腔注射建立DC模型,并观察糖代谢、脂代谢和心功能的变化。结果①大鼠经高糖高脂饲料诱导4周后,与正常对照组相比,胆固醇（TCH）和甘油三酯（TG）均显著增高（P〈0.05）,血糖值没有明显变化（P〉0.05）。②大鼠注射30 mg/kg STZ后72 h,血糖水平开始升高,继续以高糖高脂饲料喂养6周后,与正常对照组比较,高糖脂饮食组和高糖脂负荷小剂量STZ组大鼠TG、TCH维持高水平,差异有显著性（P〈0.05）;高糖脂负荷小剂量STZ组大鼠血糖值持续高水平,与正常对照组差异有显著性（P〈0.001）。③心功能测量结果显示,高糖脂饮食组大鼠出现温和的心脏功能异常（左心室收缩压降低,左心室舒张末压升高）;高糖脂负荷小剂量STZ组大鼠左心室收缩和舒张功能均出现异常（LVSP、每搏输出量、心排量降低,LVEDP、左心室最大舒张速率升高）,但以舒张功能异常为主。结论大鼠高糖脂饮食诱导负荷小剂量STZ可建立类似临床症状的2型DC模型,高糖脂饮食在糖脂代谢紊乱和心脏功能损伤过程中有重要作用,结合糖、脂代谢指标和心脏功能指标可以有效简便评价糖尿病心肌病模型。     

阿托伐他汀通过RGS6改善糖尿病心肌病大鼠心功能的作用及其机制研究

目的:探讨阿托伐他汀通过调节RGS6/NAD(P)H氧化酶/活性氧生成通路保护糖尿病心肌病大鼠心功能的药理作用机制。方法:40只6周龄雄性Wistar大鼠按随机数字表法随机分为对照组,糖尿病心肌病模型组,低剂量阿托伐他汀组,高剂量阿托伐他汀组,每组10只。实验过程中动态监测大鼠体质量及血脂水平;实验结束后脉冲多普勒检测各组大鼠心功能指标;组织活性氧检测试剂盒检测心肌组织中活性氧的水平;免疫组化法检测大鼠心肌组织中RGS6的表达;Western blot法检测大鼠心肌组织中RGS6及NAD (P)H氧化酶活性亚单位p47phox和p67phox的水平。结果:与对照组相比,糖尿病心肌病模型大鼠体质量明显减少(P0.01),血脂水平明显升高(P0.01),心脏E/A、LVEF、FS值降低(P0.01),心肌组织活性氧生成明显增多(P0.01),心肌组织RGS6及p47phox、p67phox表达明显上调(P0.01),而不同剂量阿托伐他汀干预均可有效逆转上述指标的改变。结论:阿托伐他汀对糖尿病心肌病大鼠的心脏具有明显保护作用,其机制可能与对RGS6/NAD(P)H氧化酶/活性氧生成通路的抑制有关。     

Altered SR protein expression associated with contractile dysfunction in diabetic rat hearts

The goal of this study was to examine whether alteration of sarcoplasmic reticulum (SR) protein levels is associated with early-onset diastolic and late-onset systolic dysfunction in streptozotocin (STZ)-induced diabetic rat hearts. Four-week diabetic rat hearts exhibited slow relaxation, whereas 6-wk diabetic rat hearts exhibited slow and depressed contraction. Total phospholamban level was increased, and phosphorylated level was decreased in 4- and 6-wk diabetic rat hearts. Sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) protein level was unchanged in 4-wk but decreased in 6-wk diabetic rat hearts. Only the apparent affinity of SR Ca2+ uptake for Ca2+ was decreased in 4-wk diabetic rat hearts, but the apparent affinity and the maximum rate was decreased in 6-wk diabetic rat hearts. Insulin treatment of the diabetic rats normalized SR protein expression and function. It was concluded that an increase in nonphosphorylated phospholamban and a decrease in the apparent affinity of SR Ca2+ pump for Ca2+ are associated with early-onset diastolic dysfunction and decreases in SERCA2 protein level and apparent affinity and maximum velocity of SR Ca2+ pump are associated with late-onset systolic dysfunction in diabetic rats.