Synthesis rates of protein in the Langendorff-perfused rat heart in the presence and absence of insulin, and in the working heart.
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ABSTRACT:
SUBMITTER: Sender PM
PROVIDER: S-EPMC1177625 | biostudies-other | 1973 Mar
REPOSITORIES: biostudies-other
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Project description:Insulin exerts positive inotropic effects on cardiac muscle; however, the relationship between cardiac contractility and phosphoinositol-3-kinase/Akt (PI3K/Akt) activation remains unclear. We hypothesized that the positive inotropic effects of insulin are dose-dependent and mediated via the PI3K/Akt pathway in isolated normal rat hearts. The Institutional Animal Investigation Committee approved the use of hearts excised from rats under pentobarbital anesthesia. The hearts were perfused at a constant pressure using the Langendorff technique. After stabilization (baseline), the hearts were randomly divided into the following four insulin (Ins) groups: 1) Ins0 (0 IU/L), 2) Ins0.5 (0.5 IU/L), 3) Ins5 (5 IU/L), and 4) Ins50 (50 IU/L) (n = 8 in each group). To clarify the role of the PI3K/Akt pathway in insulin-dependent inotropic effects, we also treated the insulin groups with the PI3K inhibitor wortmannin (InsW): 5) InsW0 (0 IU/L), 6) InsW0.5 (0.5 IU/L), 7) InsW5 (5 IU/L), and 8) InsW50 (50 IU/L). Hearts were perfused with Krebs–Henseleit buffer solution with or without wortmannin for 10 min, followed by 20 min perfusion with the solution containing each concentration of insulin. The data were recorded as the maximum left ventricular derivative of pressure development (LV dP/dt max). Myocardial p-Akt levels were measured at 3 min, 5 min, and at the end of the perfusion. In the Ins groups, LV dP/dt max in Ins5 and Ins50 increased by 14% and 48%, respectively, 3 min after insulin perfusion compared with the baseline. Tachyphylaxis was observed after 10 min in the Ins5 and Ins50 treatment groups. Wortmannin partially inhibited the positive inotropic effect of insulin; although insulin enhanced p-Akt levels at all time points compared with the control group, this increase was suppressed in the presence of wortmannin. The positive inotropic effect of insulin is dose-dependent and consistent with Akt activation. This effect mediated by high doses of insulin on cardiac tissue was temporary and caused tachyphylaxis, potentially triggered by Akt overactivation, which leads beta 1 deactivation.
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Project description:Autofluorescence spectroscopy is a promising label-free approach to characterize biological samples with demonstrated potential to report structural and biochemical alterations in tissues in a number of clinical applications. We report a characterization of the ex vivo autofluorescence fingerprint of cardiac tissue, exploiting a Langendorff-perfused isolated rat heart model to induce physiological insults to the heart, with a view to understanding how metabolic alterations affect the autofluorescence signals. Changes in the autofluorescence intensity and lifetime signatures associated with reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized during oxygen- or glucose-depletion protocols. Results suggest that both NAD(P)H and FAD autofluorescence intensity and lifetime parameters are sensitive to changes in the metabolic state of the heart owing to oxygen deprivation. We also observed changes in NAD(P)H fluorescence intensity and FAD lifetime parameter on reperfusion of oxygen, which might provide information on reperfusion injury, and permanent tissue damage or changes to the tissue during recovery from oxygen deprivation. We found that changes in the autofluorescence signature following glucose-depletion are, in general, less pronounced, and most clearly visible in NAD(P)H related parameters. Overall, the results reported in this investigation can serve as baseline for future investigations of cardiac tissue involving autofluorescence measurements.
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Project description:Spontaneous Raman spectroscopy, which senses changes in cellular contents of reduced cytochrome c, could be a powerful tool for label-free evaluation of ischemic hearts. However, undetermined is whether it is applicable to evaluation of myocardial viability in ischemic hearts. To address this issue, we investigated sequential changes in Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart before and during ligation of the left coronary artery and its subsequent release and re-ligation. Under 532-nm wavelength excitation, the Raman peak intensity of reduced cytochrome c at 747 cm-1 increased quickly after the coronary ligation, and reached a quasi-steady state within 30 min. Subsequent reperfusion of the heart after a short-term (30-min) ligation that simulates reversible conditions resulted in quick recovery of the peak intensity to the baseline. Further re-ligation resulted in resurgence of the peak intensity to nearly the identical value to the first ischemia value. In contrast, reperfusion after prolonged (120-min) ligation that assumes irreversible states resulted in incomplete recovery of the peak intensity, and re-ligation resulted in inadequate resurgence. Electron microscopic observations confirmed the spectral findings. Together, the Raman spectroscopic measurement for cytochrome c could be applicable to evaluation of viability of the ischemic myocardium without labeling.
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Project description:Ischemia postconditioning (IPo) is a promising strategy in reducing myocardial ischemia reperfusion (I/R) injury (MIRI), but its specific molecular mechanism is incompletely understood. Langendorff-perfused isolated rat hearts were subjected to global I/R and received IPo in the absence or presence of the mitochondrial ATP-sensitive potassium channel (mitoKATP) blocker 5-hydroxydecanoate (5-HD). Myocardial mitochondria were extracted and mitochondrial comparative proteomics was analyzed. IPo significantly reduces post-ischemic myocardial infarction and improved cardiac function in I/R rat hearts, while 5-HD basically cancelled IPo's myocardial protective effect. Joint application of two-dimensional polyacrylamide gel electrophoresis (2DE) and MALDI-TOF MS identified eight differentially expressed proteins between groups. Expression of cardiac succinate dehydrogenase (ubiquinone) flavoprotein subunit (SDHA) increased more than two-fold after I/R, while IPo led to overexpression of dihydrolipoyl dehydrogenase (DLD), NADH dehydrogenase (ubiquinone) flavoprotein 1 and isoform CRA_b (NDUFV1). When the mitoKATP was blocked, MICOS complex subunit Mic60 (IMMT) and Stress-70 protein (Grp75) were over expressed, while DLDH, ATPase subunit A (ATPA) and rCG44606 were decreased. Seven of the differential proteins belong to electron transport chain (ETC) or metabolism regulating proteins, and five of them were induced by closing mitoKATP in I/R hearts. We thus conclude that IPo's myocardial protective effect relies on energy homeostasis regulation. DLD, SDHA, NDUFV1, Grp75, ATPA and rCG44606 may contribute to IPo's cardial protective effect.
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Project description:1. The metabolic pattern of [U-(14)C]glucose in the isolated rat heart has been studied, with both retrograde aortic (Langendorff) and atrially (working) perfused preparations in the presence and absence of insulin, in normal animals, animals rendered insulin-deficient (by injection of anti-insulin serum 1hr. before excision of the heart) and animals rendered diabetic by streptozotocin injection 7 days before use. 2. Radioautochromatograms of heart extracts show that the pattern of glucose metabolism in heart muscle is more complex than in diaphragm muscle. In addition to (14)CO(2), glycogen, oligosaccharides, phosphorylated sugars and lactate (the main metabolites formed from [(14)C]glucose in diaphragm muscle), (14)C label from [(14)C]glucose appears in heart muscle in glutamate, glutamine, aspartate and alanine, and in tricarboxylic acid-cycle intermediates. 3. By a quantitative scanning technique of two-dimensional chromatograms it was found that a mechanical work load stimulates glucose metabolism, increasing by a factor of 2-3 incorporation of (14)C into all the metabolites mentioned above except lactate and phosphorylated sugars, into which (14)C incorporation is in fact diminished; (14)CO(2) production is equally stimulated. 4. Addition of insulin to the perfusion fluid of the working heart causes increases in (14)C incorporation, by a factor of about 1.5 into (14)CO(2), by a factor of about 3-5 into glycogen, lactate and phosphorylated sugars, by a factor of about 2-3 into glutamate and tricarboxylic acid-cycle intermediates and by a factor of about 0.5 into aspartate, whereas incorporation into alanine and glutamine is not affected. The effect of a work load on the pattern of glucose metabolism is thus different from that of insulin. 5. Increasing the concentration of glucose in the perfusion fluid from 1 to 20mm leads to changes of the pattern of glucose metabolism different from that brought about by insulin. (14)CO(2) production steadily increases whereas [(14)C]lactate and glycogen production levels off at 10mm-glucose, at values well below those reached in the presence of insulin. 6. In Langendorff hearts of animals rendered insulin-deficient by anti-insulin serum or streptozotocin, glucose uptake, formation of (14)CO(2) and [(14)C]lactate, and (14)C incorporation into glycogen and oligosaccharides are decreased. In insulin-deficient working hearts, however, glucose uptake and (14)CO(2) production are normal, whereas incorporation of (14)C into glycogen and [(14)C]lactate production are greatly decreased. 7. Insulin added to the perfusion fluid restores (14)C incorporation from glucose into (14)CO(2), glycogen and lactate in the Langendorff heart from animals rendered insulin-deficient by anti-insulin serum; in hearts from streptozotocin-diabetic animals addition of insulin restores (14)C incorporation into glycogen and lactate, but (14)CO(2) production remains about 50% below normal. 8. The bearing of these results on the problem of the mode of action of insulin is discussed.
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Project description:New findingsWhat is the central question of this study? Rate-pressure product (RPP) is commonly used as an index of cardiac 'effort'. In canine and human hearts (which have a positive force-frequency relationship), RPP is linearly correlated with oxygen consumption and has therefore been widely adopted as a species-independent index of cardiac work. However, given that isolated rodent hearts demonstrate a negative force-frequency relationship, its use in this model requires validation. What is the main finding and its importance? Despite its widespread use, RPP is not correlated with oxygen consumption (or cardiac 'effort') in the Langendorff-perfused isolated rat heart. This lack of correlation was also evident when perfusions included a range of metabolic substrates, insulin or ?-adrenoceptor stimulation. Langendorff perfusion of hearts isolated from rats and mice has been used extensively for physiological, pharmacological and biochemical studies. The ability to phenotype these hearts reliably is, therefore, essential. One of the commonly used indices of function is rate-pressure product (RPP); a rather ill-defined index of 'work' or, more correctly, 'effort'. Rate-pressure product, as originally described in dog or human hearts, was shown to be correlated with myocardial oxygen consumption (MV?O2). Despite its widespread use, the application of this index to rat or mouse hearts (which, unlike the dog or human, have a negative force-frequency relationship) has not been characterized. The aim of this study was to examine the relationship between RPP and MV?O2 in Langendorff-perfused rat hearts. Paced hearts (300-750 beats min(-1)) were perfused either with Krebs-Henseleit (KH) buffer (11 mm glucose) or with buffer supplemented with metabolic substrates and insulin. The arteriovenous oxygen consumption (MV?O2) was recorded. Metabolic status was assessed using (31) P magnetic resonance spectroscopy and lactate efflux. Experiments were repeated in the presence of isoprenaline and in unpaced hearts where heart rate was increased by cumulative isoprenaline challenge. In KH buffer-perfused hearts, MV?O2 increased with increasing heart rate, but given that left ventricular developed pressure decreased with increases in rate, RPP was not correlated with MV?O2, lactate production or phosphocreatine/ATP ratio. Although the provision of substrates or ?-adrenoceptor stimulation changed the shape of the RPP-MV?O2 relationship, neither intervention resulted in a positive correlation between RPP and oxygen consumption. Rate-pressure product is therefore an unreliable index of oxygen consumption or 'cardiac effort' in the isolated rat heart.
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Project description:We investigated the effect of an increase in the left-atrial filling pressure on the rate of left-atrial protein synthesis in the left-side-perfused working rat heart preparation of Taegtmeyer, Hems & Krebs [(1980) Biochem. J. 186, 701-711]. An increase in filling pressure (preload) at a constant aortic pressure (afterload) increased both the intra-atrial pressure and the atrial stroke volume. The aortic pressure (afterload) was held constant. An increase in filling pressure from 5 to 20 cmH2O at an aortic pressure of 70 cmH2O, or an increase in filling pressure of 7.5 to 20 cmH2O at an aortic pressure of 100 cmH2O, significantly stimulated the rates of left-atrial protein synthesis by 30-40%. The stimulation was observed when the rates of protein synthesis were expressed relative to either protein or RNA content. Since perfusate entering the right atrium from the coronary circulation left that atrium passively, the rate of protein synthesis in this compartment can be used as an internal control. Rates of right-atrial protein synthesis were similar to those in the left atria exposed to the lower filling pressures and were unaffected by the increases in left-atrial filling pressure. We suggest that the acute effects of increased left-atrial filling pressure on protein synthesis in that compartment may be important in the development of left-atrial hypertrophy. This condition is seen in patients who have raised pulmonary venous pressures in, for example, mitral stenosis.
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