Effect of caffeine on expression of cardiac myosin heavy chain gene in adult hypothyroid and fetal rats.

Changes in cardiac myosin heavy chain (MHC) gene expression and isozyme transitions have been shown to be caused by developmental changes, hemodynamic overload, or the activity of various hormones. In this study, to examine whether caffeine, which has teratogenic effects on the fetal cardiovascular system, causes the distribution of cardiac MHC phenotype and, if so, to evaluate the mechanisms of the distribution of cardiac MHC phenotype by caffeine, we examined the effects of caffeine, theophylline, and cAMP on the cardiac MHC isoform transitions at the gene and protein levels using hypothyroid adult rats. Furthermore, we examined the expression of alpha- and beta-MHC gene in cardiac muscles of fetuses whose dams had received caffeine. The results showed that caffeine, theophylline, and cAMP caused accumulations of alpha-MHC mRNA and MHC isozyme V1. Furthermore, in the fetal hearts, it was recognized that caffeine induced an accumulation of alpha-MHC gene expression, as was also seen in the dams. However, this effect of caffeine on the heart was stronger in the fetus than in the dam. Intracellular cAMP concentration was increased by the administration of caffeine, theophylline, or cAMP, and the levels showed a positive correlation with those of alpha-MHC mRNA. These results suggest that the induction of alpha-MHC mRNA expression by the administration of caffeine may be induced by an increase in intracellular cAMP concentration.     

Type 2 iodothyronin deiodinase transgene expression in the mouse heart causes cardiac-specific thyrotoxicosis

Type 2 iodothyronine deiodinase (D(2)) catalyzes intracellular 3, 5, 3' triiodothyronine (T(3)) production from thyroxine (T(4)), and its messenger RNA mRNA is highly expressed in human, but not rodent, myocardium. The goal of this study was to identify the effects of D(2) expression in the mouse myocardium on cardiac function and gene expression. We prepared transgenic (TG) mice in which human D(2) expression was driven by the alpha-MHC promoter. Despite high myocardial D(2) activity, myocardial T(3) was, at most, minimally increased in TG myocardium. Although, plasma T(3) and T(4), growth rate as well as the heart weight was not affected by TG expression, there was a significant increase in heart rate of the isolated perfused hearts, from 284 +/-12 to 350 +/- 7 beats/min. This was accompanied by an increase in pacemaker channel (HCN2) but not alpha-MHC or SERCA II messenger RNA levels. Biochemical studies and (31)P-NMR spectroscopy showed significantly lower levels of phosphocreatine and creatine in TG hearts. These results suggest that even mild chronic myocardial thyrotoxicosis, such as may occur in human hyperthyroidism, can cause tachycardia and associated changes in high energy phosphate compounds independent of an increase in SERCA II and alpha-MHC.     

Volume overload hypertrophy of the newborn heart slows the maturation of enzymes involved in the regulation of fatty acid metabolism.

OBJECTIVES: The purpose of this study was to determine the effect of volume overload hypertrophy in the newborn heart on the cardiac enzymes controlling fatty acid metabolism. BACKGROUND: Shortly after birth, a rise in 5'-adenosine monophosphate-activated protein kinase (AMPK) activity results in the phosphorylation and inhibition of acetyl coenzyme A (CoA) carboxylase (ACC), and a decline in myocardial malonyl CoA levels with increased fatty acid oxidation rates. Whether the early onset of hypertrophy in the newborn heart alters this maturational increase in fatty acid oxidation is unknown. METHODS: Newborn piglets underwent endovascular stenting of the ductus arteriosus on day 1 of life with a 4.5-mm diameter stent, resulting in a left to right shunt, and left ventricular (LV) volume loading. Left ventricular and right ventricular samples from fetal, newborn, three-week control and three-week stented animals were compared. RESULTS: Stenting resulted in echocardiographic evidence of volume overload and myocardial hypertrophy. In control animals, left ventricular ACC activity declined from 274 +/- 30 pmol/mg/min on day 1 to 115 +/- 12 after three weeks (p < 0.05), but did not display this maturation drop in hypertrophied hearts, remaining elevated (270 +/- 50 pmol/mg/min, p < 0.05). At three weeks, malonyl CoA levels remained 2.8-fold higher in hypertrophied hearts than in control hearts. In control hearts, LV AMPK activity increased 178% between day 1 and three weeks, whereas in hypertrophied hearts AMPK activity at three weeks was only 71% of control values, due to a significant decrease in expression of the catalytic subunit of AMPK. CONCLUSIONS: Early onset LV volume overload with hypertrophy results in a delay in the normal maturation of fatty acid oxidation in the newborn heart.     

Oestrogen action on the myocardium in vivo: specific and permissive for angiotensin-converting enzyme inhibition

OBJECTIVES : In contrast to the vasculature, it remains unclear whether oestrogens also directly affect the myocardium. In this study, we addressed basic questions regarding oestrogen effects on the myocardium, including specificity, pathophysiological relevance and potential clinical implications, with a special focus on interactions between oestrogen and angiotensin-converting enzyme (ACE) inhibitors in an established in-vivo model of cardiac hypertrophy. METHODS AND RESULTS : Female spontaneously hypertensive rats (SHR) were ovarectomized (OVX) or sham-operated and treated with 17beta-oestradiol (2 microg/kg per day subcutaneously), the oestrogen receptor antagonist ZM-182780 (250 microg/kg per day subcutaneously) and the ACE-inhibitor moexipril (10 mg/kg per day orally) alone or in combination for 3 months. Hormone replacement restored physiological oestradiol serum levels and prevented uterus atrophy. Whereas moexipril alone was ineffective in OVX rats, substitution of oestradiol restored the beneficial effect of moexipril on systolic blood pressure (-30 +/- 5 mmHg) and relative heart weight (-11 +/- 3%) in OVX rats. Oestradiol upregulated alpha-myosin heavy chain (MHC) mRNA (+37 +/- 7%) and protein expression (+43 +/- 6%) in spite of increased blood pressure in OVX rats. Simultaneous treatment with oestradiol plus moexipril most effectively shifted the ratio of alpha-/beta-MHC mRNA and protein expression towards alpha-MHC in OVX animals. Oestradiol (10 nmol/l) also upregulated alpha-MHC mRNA and protein in cultured cardiac myocytes. The oestrogen receptor antagonist ZM-182780 significantly inhibited the observed oestrogen effects. CONCLUSIONS : Oestrogen replacement is permissive for the beneficial effects of ACE-inhibition in female SHR rats. Oestrogen effects on the myocardium in vivo are specific (i.e. oestrogen receptor mediated) because they are inhibited by a pure oestrogen receptor antagonist and occur at physiological hormone levels.     

Modification of myosin gene expression by imidapril in failing heart due to myocardial infarction

The beneficial effects of imidapril, an angiotensin converting enzyme (ACE) inhibitor were investigated on changes in myofibrillar ATPase as well as myosin heavy chain (MHC) content and gene expression due to myocardial infarction (MI). Three weeks after occluding the left coronary artery, rats were treated with or without imidapril (1 mg/kg/day), for 4 weeks. The infarcted hearts exhibited depressed rates of left ventricular (LV) pressure development (57+/-2.4% reduction) and pressure decay (55.5+/-1.6% reduction). LV myofibrillar Ca(2+) ATPase activity, unlike that in the right ventricle (RV), was decreased in the infarcted animals compared with controls (6.8+/-0.4 vs 10.3+/-0.6 micromol Pi/mg/hr). MHC alpha-isoform contents were decreased by 47 and 41% whereas those of MHC beta-isoform were increased by 823 and 1200% in the LV and RV due to MI, respectively. MHC alpha-isoform mRNA levels were decreased by 55 and 35% whereas those for MHC beta-isoform were increased by 50 and 30% in the infarcted LV and RV, respectively. Imidapril treatment partially prevented the changes due to MI in LV function (rate of pressure development, 24+/-2.3% reduction and rate of pressure decay, 14+/-1.8% reduction), myofibrillar Ca(2+) ATPase activity (8.2+/-0.7 micromol Pi/mg/hr), MHC protein content (alpha-MHC, 24% reduction and beta-MHC, 525% increase) and MHC gene expression (alpha-MHC, 18% reduction and beta-MHC, 15% increase). The results suggest that the beneficial effects of ACE inhibition on the failing heart are associated with improvements in myofibrillar ATPase activities as well as prevention of changes in MHC isozyme protein contents and their gene expression.     

Cardiac dysfunction caused by myocardium-specific expression of a mutant thyroid hormone receptor

Thyroid hormone deficiency has profound effects on the cardiovascular system, resulting in decreased cardiac contractility, adrenergic responsiveness, and vascular volume and increased peripheral vascular resistance. To determine the importance of direct cardiac effects in the genesis of hypothyroid cardiac dysfunction, the cardiac myocyte was specifically targeted with a mutant thyroid hormone receptor (TR)-beta (Delta337T-TR-beta(1)) driven by the alpha-myosin heavy chain (alpha-MHC) gene promoter. As a control in these experiments, a wild-type (Wt) TR-beta(1) was also targeted to the heart by using the same promoter. Transgenic mice expressing the mutant TR displayed an mRNA expression pattern consistent with cardiac hypothyroidism, even though their peripheral thyroid hormone levels were normal. When these animals were rendered hypothyroid or thyrotoxic, mRNA expression of MHC isoforms remained unchanged in the hearts of the Delta337T transgenic animals, in contrast to Wt controls or transgenic animals expressing Wt TR-beta(1), which exhibited the expected changes in steady-state MHC mRNA levels. Studies in Langendorff heart preparations from mutant TR-beta(1) transgenic animals revealed evidence of heart failure with a significant reduction in +dP/dT, -dP/dT, and force-frequency responses compared with values in Wt controls and transgenic mice overexpressing the Wt TR-beta(1). In contrast, in vivo measures of cardiac performance were similar between Wt and mutant animals, indicating that the diminished performance of the mutant transgenic heart in vitro was compensated for by other mechanisms in vivo. This is the first demonstration indicating that isolated cardiac hypothyroidism causes cardiac dysfunction in the absence of changes in the adrenergic or peripheral vascular system.     

Thrombospondin-2 is essential for myocardial matrix integrity: increased expression identifies failure-prone cardiac hypertrophy

Cardiac hypertrophy can lead to heart failure (HF), but it is unpredictable which hypertrophied myocardium will progress to HF. We surmised that apart from hypertrophy-related genes, failure-related genes are expressed before the onset of failure, permitting molecular prediction of HF. Hearts from hypertensive homozygous renin-overexpressing (Ren-2) rats that had progressed to early HF were compared by microarray analysis to Ren-2 rats that had remained compensated. To identify which HF-related genes preceded failure, cardiac biopsy specimens were taken during compensated hypertrophy and we then monitored whether the rat progressed to HF or remained compensated. Among 48 genes overexpressed in failing hearts, we focused on thrombospondin-2 (TSP2). TSP2 was selectively overexpressed only in biopsy specimens from rats that later progressed to HF. Moreover, expression of TSP2 was increased in human hypertrophied hearts with decreased (0.19+/-0.01) versus normal ejection fraction (0.11+/-0.03 [arbitrary units]; P<0.05). Angiotensin II induced fatal cardiac rupture in 70% of TSP2 knockout mice, with cardiac failure in the surviving mice; this was not seen in wild-type mice. In TSP2 knockout mice, angiotensin II increased matrix metalloproteinase (MMP)-2 and MMP-9 activity by 120% and 390% compared with wild-type mice (P<0.05). In conclusion, we identify TSP2 as a crucial regulator of the integrity of the cardiac matrix that is necessary for the myocardium to cope with increased loading and that may function by its regulation of MMP activity. This suggests that expression of TSP2 marks an early-stage molecular program that is activated uniquely in hypertrophied hearts that are prone to fail.     

Relationship between the myofibrillar ATPase activity of human biopsy material and hemodynamic parameters

The myofibrillar ATPase activity and pyrophosphate gel electrophoretic pattern of native myosin of fresh human left ventricular papillary muscles were examined in 52 cases of mitral valve replacement. The myofibrillar ATPase activity of hypertrophied myocardium did not differ from that of non-hypertrophied myocardium (mean +/- SD, 36.2 +/- 8.7 vs 31.8 +/- 8.6 nmolPi/mg/min, ns) and there was no significant difference in myofibrillar ATPase activity as a function of left ventricular enddiastolic pressure. Pyrophosphate gel electrophoresis of myosin revealed the presence of two components. It is questionable whether the component of higher electrophoretic mobility (approximately 25-35% in concentration) is identical with rat ventricular myosin VM-1 because an increase in this component seems to correlate with a decrease of myofibrillar ATPase activity, its concentration was significantly higher in the hearts with left ventricular hypertrophy, high enddiastolic pressure, high aortic pressure or low cardiac index. From these results, it is not necessarily clear whether hemodynamic overload in valvular heart diseases can alter left ventricular myofibrillar ATPase activity, but it can be said that the overload influences the concentration of the two components of native myosin revealed by pyrophosphate gel electrophoresis.     

Mechanical unloading improves intracellular Ca2+ regulation in rats with doxorubicin-induced cardiomyopathy

OBJECTIVES: We sought to assess whether mechanical unloading has beneficial effects on cardiomyocytes from doxorubicin-induced cardiomyopathy in rats. BACKGROUND: Mechanical unloading by a left ventricular assist device (LVAD) improves the cardiac function of terminal heart failure in humans. However, previous animal studies have failed to demonstrate beneficial effects of mechanical unloading in the myocardium. METHODS: The effects of mechanical unloading by heterotopic abdominal heart transplantation were evaluated in the myocardium from doxorubicin-treated rats by analyzing the intracellular free calcium level ([Ca(2+)](i)) and the levels of intracellular Ca(2+)-regulatory proteins. RESULTS: In doxorubicin-treated rats, the duration of cell shortening and [Ca(2+)](i) transients in cardiomyocytes was prolonged (432 +/- 28.2% of control in 50% relaxation time; 184 +/- 10.5% of control in [Ca(2+)](i) 50% decay time). Such prolonged time courses significantly recovered after mechanical unloading (114 +/- 10.4% of control in 50% relaxation time; 114 +/- 5.8% of control in 50% decay time). These effects were accompanied by an increase in sarcoplasmic reticulum Ca(2+) ATPase (SERCA2a) protein levels (0.97 +/- 0.05 in unloaded hearts vs. 0.41+/- 0.09 in non-unloaded hearts). The levels of other intracellular Ca(2+)-regulatory proteins (phospholamban and ryanodine receptor) were not altered after mechanical unloading in doxorubicin-treated hearts. These parameters in unloaded hearts without doxorubicin treatment were similar to normal hearts. CONCLUSIONS: Mechanical unloading increases functional sarcoplasmic reticulum Ca(2+) ATPase and improves [Ca(2+)](i) handling and contractility in rats with doxorubicin-induced cardiomyopathy. These beneficial effects of mechanical unloading were not observed in normal hearts.     

Altered myocardial contractility and energetics in hypertrophied myocardium

Alterations of myocardial contractility and energetics were examined in cardiac hypertrophy induced by different types of cardiac overload. Myocardial contractility was estimated by isometric contraction of isolated left ventricular papillary muscles. Myocardial energetics were assessed from ventricular myosin isoenzyme patterns obtained by pyrophosphate gel electrophoresis. Cardiac hypertrophy was induced by endurance swim-training and sustained pressure-overload by abdominal aortic constriction or volume-overload created by an arteriovenous shunt. In swim-trained hypertrophied myocardium, isometric developed tension (T) and dT/dtmax showed a tendency to increase and response of dT/dtmax to isoproterenol increased significantly as compared with sedentary rats. Training shifted the left ventricular myosin isoenzyme pattern toward VM-1, which has the highest ATPase activity. In pressure- or volume-overloaded myocardium, dT/dtmax decreased significantly and mechanical response to isoproterenol also decreased (or tended to decrease in volume-overloaded hearts) as compared with the respective sham-operated controls. In pressure- or volume-overloaded hearts, left ventricular myosin isoenzyme pattern shifted toward VM-3, which has the lowest ATPase activity. These results indicate that alterations in myocardial contractility, mechanical catecholamine responsiveness and myocardial energetics in hypertrophied myocardium do not always display the same trend, but are greatly influenced by the causes or duration of cardiac overload.     

Time course of the in vivo effects of thyroid hormone on cardiac gene expression.

The rate of response to thyroid hormone on cardiac growth, heart rate, and the relative changes in messenger RNA (mRNA) coding for alpha- and beta-myosin heavy chain (MHC), slow sarcoplasmic reticulum calcium-adenosine triphosphatase, and thyroid hormone receptors in ventricular tissue of hypothyroid rats was investigated. Hypothyroid rats had significantly smaller hearts, with slower heart rates and expressed no alpha-MHC mRNA as analyzed by an S1 nuclease protection assay when compared to euthyroid animals that expressed 79% alpha-MHC. Twelve hours after treating hypothyroid rats with 20 micrograms of L-T4, detectable levels of alpha-MHC mRNA were present and the shift to alpha-MHC mRNA was complete by 72 h of treatment. Northern blot analysis showed that hypothyroidism resulted in a 60% decrease in the level of sarcoplasmic reticulum calcium-adenosine triphosphatase mRNA which increased after 12 h of T4 administration and was 2.5-fold (P less than 0.05) greater than euthyroid levels after 72 h. In contrast, thyroid hormone receptor mRNA levels measured in poly(A)+ RNA were elevated in hypothyroid rats and decreased to euthyroid levels within 24 h after thyroid hormone treatment. These changes in cardiac gene expression occurred simultaneously with changes in both cardiac size and heart rate. The current studies characterize the coordinated changes and the time course for gene expression that occur in the hypothyroid heart after acute T4 administration.     

Function of the sarcoplasmic reticulum and expression of its Ca2(+)-ATPase gene in pressure overload-induced cardiac hypertrophy in the rat

The reduction in Ca2+ concentration during diastole and relaxation occurs differently in normal hearts and in hypertrophied hearts secondary to pressure overload. We have studied some possible molecular mechanisms underlying these differences by examining the function of the sarcoplasmic reticulum and the expression of the gene encoding its Ca2(+)-ATPase in rat hearts with mild and severe compensatory hypertrophy induced by abdominal aortic constriction. Twelve sham-operated rats and 31 operated rats were studied 1 month after surgery. Eighteen animals exhibited mild hypertrophy (left ventricular wt/body wt less than 2.6) and 13 animals severe hypertrophy (left ventricular wt/body wt greater than 2.6). During hypertrophy we observed a decline in the function of the sarcoplasmic reticulum as assessed by the oxalate-stimulated Ca2+ uptake of homogenates of the left ventricle. Values decreased from 12.1 +/- 1.2 nmol Ca2+/mg protein/min in sham-operated rats to 9.1 +/- 1.5 and 6.7 +/- 1.1 in rats with mild and severe hypertrophy, respectively (p less than 0.001 and p less than 0.001, respectively, vs. shams). This decrease was accompanied by a parallel reduction in the number of functionally active CA2(+)-ATPase molecules, as determined by the level of Ca2(+)-dependent phosphorylated intermediate: 58.8 +/- 7.4 and 48.1 +/- 13.5 pmol P/mg protein in mild and severe hypertrophy, respectively, compared with 69.7 +/- 8.2 in shams (p less than 0.05 and p less than 0.01, respectively, vs. shams). Using S1 nuclease mapping, we observed that the Ca2(+)-ATPase messenger RNA (mRNA) from sham-operated and hypertrophied hearts was identical. Finally, the relative level of expression of the Ca2(+)-ATPase gene was studied by dot blot analysis at both the mRNA and protein levels using complementary DNA clones and a monoclonal antibody specific to the sarcoplasmic reticulum Ca2(+)-ATPase. In mild hypertrophy, the concentrations of Ca2(+)-ATPase mRNA and protein in the left ventricle were unchanged when compared with shams (mRNA, 93.8 +/- 10.6% vs. sham, NS; protein, 105.5 +/- 14% vs. sham, NS). in severe hypertrophy, the concentration of Ca2(+)-ATPase mRNA decreased to 68.7 +/- 12.9% and that of protein to 80.1 +/- 15.5% (p less than 0.001 and p less than 0.05, respectively), whereas the total amount of mRNA and enzyme per left ventricle was either unchanged or slightly increased. The slow velocity of relaxation of severely hypertrophied heart can be at least partially explained by the absence of an increase in the expression of the Ca2(+)-ATPase gene and by the relative diminution in the density of the Ca2+ pumps.(ABSTRACT TRUNCATED AT 400 WORDS)     

Regional variation in cardiac myosin isoforms of female F344 rats during aging.

Myosin heavy chain (MHC) is a major contractile protein of heart muscle consisting of two isoforms in the rat, alpha-MHC that predominates in the hearts of young rats, and beta-MHC that progressively replaces it as the rats age. It was hypothesized that the magnitude of the age-associated decrease in the proportion of cardiac alpha-MHC would be similar in regions of the heart that differed in their initial MHC isoform pattern. MHCs from hearts of female Fischer 344 rats 3, 9, 15, 18, 24, and 27 months of age were separated by gradient gel electrophoresis. Hypertrophy was assessed by indexing regional heart mass to tibial length From 9 through 27 months of age, hypertrophy was 19% and 77% in the left ventricle and left atrial appendage, respectively. There was no significant hypertrophy in either the right ventricular free wall or the right atrial appendage. The proportion of alpha-myosin heavy chain ranged from 86 +/- 1.3% (mean +/- SE) in the right ventricular free wall to 62 +/- 5.8% in left ventricular papillary muscle of 9-month-old rats. In 27-month-old rats, it ranged from 59 +/- 2.7% in the right ventricular free wall to 20 +/- 3.1% in the left ventricular papillary muscle. There was a marked age-associated decrease in the proportion of alpha-myosin heavy chain overall (p <.001) that did not differ significantly among the regions studied (p = .109). These results suggest that the effects of advancing age on the cardiac MHC pattern are independent of age-associated hypertrophy.     

腺相关病毒介导心肌肌浆网Ca2+-ATPase 2a基因转导治疗大鼠慢性心力衰竭

目的 研究腺相关病毒为载体的心肌肌浆网Ca^2＋ -ATPase 2a（sarcoplasmic reticulum Ca^2＋ -ATPase,SERCA2a）基因转导对慢性心力衰竭（HF）大鼠的治疗作用,并探讨其多种可能的机理.方法 采用腹主动脉缩窄术建立HF大鼠模型,应用经腹心包腔内注射术分别将生理盐水、携带eGFP基因和携带SERCA2a基因的重组腺相关病毒导入HF、HF＋EGFP和HF＋SERCA2a组大鼠心脏.于导入30天,检测各组大鼠的心脏功能、SERCA2a蛋白表达和活性;比较HF组和HF＋SERCA2a组大鼠心肌蛋白质组表达的差异;检测各组大鼠心肌肌球蛋白重链（MHC）亚型的表达.结果 HF大鼠心脏内转导入SERCA2a基因30天,心脏收缩和舒张功能达到对照组大鼠水平,并且HF＋SERCA2a组左室重/体重比值显著降低;SERCA2a蛋白表达和活性明显升高至对照组大鼠水平;多种能量代谢酶表达明显增加;α-MHC、β-MHC的表达以及α-MHC/β-MHC恢复至对照组大鼠水平.结论 以重组腺相关病毒2作为载体,SERCA2a基因转导可以增强衰竭心脏的SERCA2a功能,增加心脏能量代谢,纠正MHC亚型的异常表达;在临床方面表现为显著改善心脏收缩和舒张功能,可能能够减轻心脏肥厚等病理性结构改变.