Phospholamban-to-SERCA2 ratio controls the force-frequency relationship.
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The force-frequency relationship (FFR) describes the frequency-dependent potentiation of cardiac contractility. The interaction of the sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (SERCA2) with its inhibitory protein phospholamban (PLB) might be involved in the control of the FFR. The FFR was analyzed in two systems in which the PLB-to-SERCA2 ratio was modulated. Adult rabbit cardiac myocytes were transduced with adenovirus encoding for SERCA2, PLB, and beta-galactosidase (control). After 3 days, the relative PLB/SERCA2 values were significantly different between groups (SERCA2, 0.5; control, 1.0; PLB, 4.5). SERCA2 overexpression shortened relaxation by 23% relative to control, whereas PLB prolonged relaxation by 39% and reduced contractility by 47% (0.1 Hz). When the stimulation frequency was increased to 1.5 Hz, myocyte contractility was increased by 30% in control myocytes. PLB-overexpressing myocytes showed an augmented positive FFR (+78%), whereas SERCA2-transduced myocytes displayed a negative FFR (-15%). A more negative FFR was also found in papillary muscles from SERCA2 transgenic mice. These findings demonstrate that the ratio of phospholamban to SERCA2 is an important component in the control of the FFR. (+info)
TAFII250, Egr-1, and D-type cyclin expression in mice and neonatal rat cardiomyocytes treated with doxorubicin.
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Differential display identified that gene fragment HA220 homologous to the transcriptional activator factor II 250 (TAFII250) gene, or CCG1, was increased in hypertrophied rodent heart. To determine whether TAFII250 gene expression is modified after cardiac damage, we measured TAFII250 expression in vivo in mouse hearts after injection of the cardiotoxic agent doxorubicin (DXR) and in vitro in DXR-treated isolated rat neonatal cardiomyocytes. In vivo atrial natriuretic factor (ANF), beta-myosin heavy chain (beta-MHC), Egr-1, and TAFII250 expression increased with dose and time after a single DXR injection, but only ANF and beta-MHC expression were increased after multiple injections. After DXR treatment of neonatal cardiomyocytes we found decreased ANF, alpha-MHC, Egr-1, and TAFII250 expression. Expression of the TAFII250-regulated genes, the D-type cyclins, was increased after a single injection in adult mice and was decreased in DXR-treated cardiomyocytes. Thus expression of Erg-1, TAFII250, and the D-type cyclins is modulated after cardiotoxic damage in adult and neonatal heart. (+info)
alpha1-Adrenergic stimulation of FGF-2 promoter in cardiac myocytes and in adult transgenic mouse hearts.
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Fibroblast growth factor (FGF-2), a mitogenic, angiogenic, and cardioprotective agent, is reported to be released from the postnatal heart by a mechanism of transient remodeling of the sarcolemma during contraction. This release can be increased with adrenergic stimulation. RNA blotting was used to assess whether FGF-2 synthesis in neonatal rat cardiomyocytes might also be regulated by adrenergic stimulation. FGF-2 RNA levels were increased after treatment with norepinephrine for 6 h or with the alpha-adrenergic agonist phenylephrine for 48 h. To assess an effect on transcription, neonatal rat cardiomyocytes were transfected with a hybrid rat FGF-2 promoter/luciferase gene (-1058FGFp.luc) and treated with norepinephrine or phenylephrine for 6 or 48 h, respectively. FGF-2 promoter activity was increased two- to sevenfold in an alpha1-specific manner. Putative phenylephrine-responsive elements (PEREs) were identified at positions -780 and -761 relative to a major transcription initiation site. However, deletion analysis of -1058FGFp.luc showed that the phenylephrine response was independent of the putative PEREs, cell contraction, and Ca2+ influx. In transgenic mice expressing -1058FGFp.luc, a significant three- to sevenfold stimulation of FGF-2 promoter activity was detected in the hearts of two independent lines 6 h after intraperitoneal administration of phenylephrine (50 mg/kg). This increase was still apparent at 24 h but was not detected at 48 h posttreatment. Analysis of FGF-2 mRNA in normal mouse hearts revealed accumulation of the 6.1-kb transcript at 24 h. Control of local FGF-2 synthesis at the transcriptional level through adrenergic stimulation may be important in the response to injury as well as in the maintenance of a healthy myocardium. (+info)
Effects of beta2-adrenergic stimulation on single-channel gating of rat cardiac L-type Ca2+ channels.
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Cardiac L-type Ca2+ channels can be stimulated by activation of beta2-adrenoceptors. We intended to determine how the gating behavior at the single-channel level (cell-attached configuration) is affected after selective stimulation of beta2-adrenoceptors. Rat cardiomyocytes were exposed to zinterol, a beta2-agonist (n = 7), isoproterenol (n = 6), a nonselective agonist, 8-bromo-cAMP (n = 6), and a combination of isoproterenol and ICI-118551 (n = 8), a selective beta2-receptor antagonist, or isoproterenol and CGP-20712A, a beta1-selective antagonist (n = 7). In all groups the ensemble-average current and the availability of the channels to open on depolarization were increased in a similar fashion. In addition, the open probability (Po) within active sweeps was elevated. However, zinterol exerted this effect in a unique manner. It elevated Po not by shortening closed times but solely by reducing active sweeps with very low Po and a short burst duration. All zinterol effects were abolished by ICI-118551 (n = 5) and mimicked by isoproterenol plus CGP-20712A (n = 7). We conclude that beta2-adrenoceptor activation of L-type channels differs qualitatively from the classical cAMP-dependent mechanism. (+info)
Cyclosporin A treatment alters characteristics of Ca2+-release channel in cardiac sarcoplasmic reticulum.
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Chronic treatment with cyclosporin A (CsA) has been reported (H. S. Banijamali, M. H. ter Keurs, L. C. Paul, and H. E. ter Keurs. Cardiovasc. Res. 27: 1845-1854, 1993; I. Kingma, E. Harmsen, H. E. ter Keurs, H. Benediktsson, and L. C. Paul. Int. J. Cardiol. 31: 15-22, 1991) to induce reversible alterations of contractile properties in rat hearts. To define the molecular mechanisms underlying the physiological alterations, the Ca2+-release channel (CRC) and Ca2+-ATPase from sarcoplasmic reticulum in rats were examined. Ryanodine binding to whole homogenates of rat hearts shows time- and dose-dependent alterations in CRC properties by CsA. On 3 wk of treatment with 15 mg CsA. kg body wt-1. day-1, 1) maximal ryanodine binding (Bmax) decreased, 2) the dissociation constant of ryanodine (Kd) increased, 3) caffeine sensitivity of CRC increased, and 4) ruthenium red sensitivity of CRC decreased. On the other hand, Bmax and Kd of ryanodine binding in rat skeletal muscles were not changed. Ryanodine-sensitive oxalate-supported Ca2+ uptake in whole homogenates was lower in CsA-treated rat hearts than in control hearts, whereas total Ca2+ uptake in the presence of 500 M ryanodine was not changed. Functional experiments with rapamycin and Western blot analysis suggest that the CsA-induced alteration of ryanodine binding is due at least in part to an upregulation of calcineurin. The heart muscle-specific alterations of CRC could be responsible for the previously reported contractile changes of CsA-treated rat hearts. (+info)
Effects of AT1 receptor blockade after myocardial infarct on myocardial fibrosis, stiffness, and contractility.
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Angiotensin II type 1 (AT1) receptor blockade attenuates myocardial fibrosis after myocardial infarction (MI). However, whether inhibition of fibrosis by AT1 receptor blockade influences myocardial stiffness and contractility is unknown. We measured left ventricular (LV) hemodynamics, papillary muscle function, and myocardial stiffness and fibrosis in rats randomized to losartan or placebo 1 day after MI and treated subsequently for 8 wk. Losartan decreased LV and right ventricular weights as well as mean aortic and LV systolic pressures in sham and MI rats. LV end-diastolic pressure increased after MI and was decreased with losartan. Maximal developed tension and peak rate of tension rise and decline were decreased in MI vs. sham rats. Interstitial fibrosis developed after MI and was prevented in losartan-treated MI rats. The development of abnormal myocardial stiffness after MI was prevented by losartan. After MI, AT1 receptor blockade prevents an abnormal increase in myocardial collagen content. This effect was associated with a normalization of passive myocardial stiffness. (+info)
Myocardial creatine kinase kinetics in hearts with postinfarction left ventricular remodeling.
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This study examined whether alterations in myocardial creatine kinase (CK) kinetics and high-energy phosphate (HEP) levels occur in postinfarction left ventricular remodeling (LVR). Myocardial HEP and CK kinetics were examined in 19 pigs 6 wk after myocardial infarction was produced by left circumflex coronary artery ligation, and the results were compared with those from 9 normal pigs. Blood flow (microspheres), oxygen consumption (MVO2), HEP levels [31P magnetic resonance spectroscopy (MRS)], and CK kinetics (31P MRS) were measured in myocardium remote from the infarct under basal conditions and during dobutamine infusion (20 micrograms. kg-1. min-1 iv). Six of the pigs with LVR had overt congestive heart failure (CHF) at the time of study. Under basal conditions, creatine phosphate (CrP)-to-ATP ratios were lower in all transmural layers of hearts with CHF and in the subendocardium of LVR hearts than in normal hearts (P < 0.05). Myocardial ATP (biopsy) was significantly decreased in hearts with CHF. The CK forward rate constant was lower (P < 0.05) in the CHF group (0.21 +/- 0.03 s-1) than in LVR (0.38 +/- 0.04 s-1) or normal groups (0.41 +/- 0.03 s-1); CK forward flux rates in CHF, LVR, and normal groups were 6.4 +/- 2.3, 14.3 +/- 2.1, and 20.3 +/- 2.4 micromol. g-1. s-1, respectively (P < 0.05, CHF vs. LVR and LVR vs. normal). Dobutamine caused doubling of the rate-pressure product in the LVR and normal groups, whereas CHF hearts failed to respond to dobutamine. CK flux rates did not change during dobutamine in any group. The ratios of CK flux to ATP synthesis (from MVO2) under baseline conditions were 10.9 +/- 1.2, 8. 03 +/- 0.9, and 3.86 +/- 0.5 for normal, LVR, and CHF hearts, respectively (each P < 0.05); during dobutamine, this ratio decreased to 3.73 +/- 0.5, 2.58 +/- 0.4, and 2.78 +/- 0.5, respectively (P = not significant among groups). These data demonstrate that CK flux rates are decreased in hearts with postinfarction LVR, but this change does not limit the response to dobutamine. In hearts with end-stage CHF, the changes in HEP and CK flux are more marked. These changes could contribute to the decreased responsiveness of these hearts to dobutamine. (+info)
Formation of 4-hydroxy-2-nonenal-modified proteins in ischemic rat heart.
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4-Hydroxy-2-nonenal (HNE) is a major lipid peroxidation product formed during oxidative stress. Because of its reactivity with nucleophilic compounds, particularly metabolites and proteins containing thiol groups, HNE is cytotoxic. The aim of this study was to assess the extent and time course for the formation of HNE-modified proteins during ischemia and ischemia plus reperfusion in isolated rat hearts. With an antibody to HNE-Cys/His/Lys and densitometry of Western blots, we quantified the amount of HNE-protein adduct in the heart. By taking biopsies from single hearts (n = 5) at various times (0, 5, 10, 15, 20, 35, and 40 min) after onset of zero-flow global ischemia, we showed a progressive, time-dependent increase (which peaked after 30 min) in HNE-mediated modification of a discrete number of proteins. In studies with individual hearts (n = 4/group), control aerobic perfusion (70 min) resulted in a very low level (296 arbitrary units) of HNE-protein adduct formation; by contrast, after 30-min ischemia HNE-adduct content increased by >50-fold (15,356 units, P < 0.05). In other studies (n = 4/group), administration of N-(2-mercaptopropionyl)glycine (MPG, 1 mM) to the heart for 5 min immediately before 30-min ischemia reduced HNE-protein adduct formation during ischemia by approximately 75%. In studies (n = 4/group) that included reperfusion of hearts after 5, 10, 15, or 30 min of ischemia, there was no further increase in the extent of HNE-protein adduct formation over that seen with ischemia alone. Similarly, in experiments with MPG, reperfusion did not significantly influence the tissue content of HNE-protein adduct. Western immunoblot results were confirmed in studies using in situ immunofluorescent localization of HNE-protein in cryosections. In conclusion, ischemia causes a major increase in HNE-protein adduct that would be expected to reflect a toxic sequence of events that might act to compromise tissue survival during ischemia and recovery on reperfusion. (+info)