The mechanism of ammonia production and the effect of mechanical work load on proteolysis and amino acid catabolism in isolated perfused rat heart.
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ABSTRACT:
SUBMITTER: Takala T
PROVIDER: S-EPMC1162333 | biostudies-other | 1980 Oct
REPOSITORIES: biostudies-other
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Project description:The Langendorff-perfused model is a powerful tool to study biological responses in the isolated heart in the absence of confounders. The model has been adapted recently to enable study of the isolated mouse heart and the effects of genetic manipulation. Unfortunately, the small size and fragility of the mouse heart pose significant challenges, limiting application of the Langendorff model to the study of adult mice. Cardiac development is a complex and dynamic process that is incompletely understood. Thus, establishing an isolated-perfused heart model in the newborn mouse would be an important and necessary advance. Here we present a method to successfully cannulate and perfuse the isolated newborn murine heart. We describe the basic and fundamental physiological characteristics of the ex-vivo retrograde-perfused beating neonatal heart in wild-type C57Bl/6 male mice. Our approach will enable future study of the physiological and pharmacological responses of the isolated immature murine heart to enhance knowledge of how developmental cardiac biology impacts health and disease.•The Langendorff model is a powerful tool to study the heart without confounders.•An isolated-perfused newborn murine heart model has yet to be established.•We demonstrate the first successful isolated neonatal murine heart preparation.
| S-EPMC7492986 | biostudies-literature
Project description:Metabolic effects of increased mechanical work were studied by comparing isolated pumping rat hearts perfused by the atrial-filling technique with aortic-perfused non-pumping hearts perfused by the technique of Langendorff. The initial medium usually contained glucose (11 mm) and palmitate (0.6 mm bound to 0.1 mm albumin). During increased heart work (comparing pumping with non-pumping hearts) the uptake of oxygen and glucose increased threefold, but that of free fatty acids was unchanged. Tissue contents of alpha-oxoglutarate, NH4+, malate, lactate, pyruvate and Pi rose with increased heart work, but contents of ATP, phosphocreatine and citrate fell. Ketone bodies were produced with a ratio of beta-hydroxybutyrate/acetoacetate of about 3:1 in both pumping and non-pumping hearts but with higher net production rates in non-pumping hearts. When ketone bodies were added in relatively high concentrations (total 4 mm) to a glucose (11 mm) medium the medium, ratios of beta-hydroxybutyrate/acetoacetate were not steady even after 60 min of perfusion. The validity of calculating mitochondrial free NAD+/NADH ratios from the tissue contents of the reactants of the glutamate dehydrogenase system or the beta-hydroxybutyrate dehydrogenase system is assessed. The activities of these enzymes are considerably less in the rat heart than in the rat liver, introducing reservations into the application to the heart of the principles used by Williamson et al. (1967) for calculation of mitochondrial free NAD+/NADH ratios of liver mitochondria...
| S-EPMC1165557 | biostudies-other
Project description:Pharmacological preconditioning (PC) and postconditioning (PoC), for example, by treatment with the ?2-adrenoreceptor agonist Dexmedetomidine (Dex), protects hearts from ischemia-reperfusion (I/R) injury in experimental studies, however, translation into the clinical setting has been challenging. Acute hyperglycemia adversely affects the outcome of patients with myocardial infarction. Additionally, it also blocks cardioprotection by multiple pharmacological agents. Therefore, we investigated the possible influence of acute hyperglycemia on Dexmedetomidine-induced pre- and postconditioning. Experiments were performed on the hearts of male Wistar rats, which were randomized into 7 groups, placed in an isolated Langendorff system and perfused with Krebs-Henseleit buffer. All hearts underwent 33 min of global ischemia, followed by 60 min of reperfusion. Control (Con) hearts received Krebs-Henseleit buffer (Con KHB), glucose (Con HG) or mannitol (Con NG) as vehicle only. Hearts exposed to hyperglycemia (HG) received KHB, containing 11 mmol/L glucose (an elevated, but commonly used glucose concentration for Langendorff perfused hearts) resulting in a total concentration of 22 mmol/L glucose throughout the whole experiment. To ensure comparable osmolarity with HG conditions, normoglycemic (NG) hearts received mannitol in addition to KHB. Hearts were treated with 3 nM Dexmedetomidine (Dex) before (DexPC) or after ischemia (DexPoC), under hyperglycemic or normoglycemic conditions. Infarct size was determined by triphenyltetrazoliumchloride staining. Acute hyperglycemia had no impact on infarct size compared to the control group with KHB (Con HG: 56 ± 9% ns vs. Con KHB: 56 ± 7%). DexPC reduced infarct size despite elevated glucose levels (DexPC HG: 35 ± 3%, p < 0.05 vs. Con HG). However, treatment with Dex during reperfusion showed no infarct size reduction under hyperglycemic conditions (DexPoC HG: 57 ± 9%, ns vs. Con HG). In contrast, hearts treated with mannitol demonstrated a significant decrease in infarct size compared to the control group (Con NG: 37 ± 3%, p < 0.05 vs. Con KHB). The combination of Dex and mannitol presents exactly opposite results to hearts treated with hyperglycemia. While DexPC completely abrogates infarct reduction through mannitol treatment (DexPC NG: 55 ± 7%, p < 0.05 vs. Con NG), DexPoC had no impact on mannitol-induced infarct size reduction (DexPoC NG: 38 ± 4%, ns vs. Con NG). Acute hyperglycemia inhibits DexPoC, while it has no impact on DexPC. Treatment with mannitol induces cardioprotection. Application of Dex during reperfusion does not influence mannitol-induced infarct size reduction, however, administering Dex before ischemia interferes with mannitol-induced cardioprotection.
| S-EPMC7290666 | biostudies-literature
Project description:Clinical trials infusing Bone Marrow Cells (BMCs) into injured hearts have produced measureable improvements in cardiac performance, but were insufficient to improve patient outcomes. Low engraftment rates are cited as probable contributor to limited improvements. To understand the mechanisms that control myocardial engraftment of BMCs following ischemia-reperfusion injury, in isolated-perfused mouse hearts, stop-flow ischemia was followed by variable-duration reperfusion (0-60 min) before addition of labeled syngenic BMCs to the perfusate. After a buffer-only wash, the heart was disaggregated. Retained BMCs (digest) and infused BMCs (aliquot) were compared by flow cytometry for c-kit and CD45 expression to determine the proportion of cell subtypes engrafted versus delivered (selectivity ratio). In these studies, a time-dependent selective retention of c-kit(+) cells was apparent starting at 30 min of reperfusion, at which time c-kit(+)/CD45(+) BMCs showed a selectivity ratio of 18 ± 2 (versus 2 ± 1 in sham-ischemic controls). To study the underlying mechanism for this selective retention, neutralizing antibodies for P-selectin or L-selectin were infused into the heart preparation and incubated with BMCs prior to BMC infusion. Blocking P-selectin in ischemic hearts ablated selectivity for c-kit(+)/CD45(+) BMCs at 30 min reperfusion (selectivity ratio of 3 ± 1) while selectivity persisted in the presence of L-selectin neutralization (selectivity ratio of 17 ± 2). To corroborate this finding, a parallel plate flow chamber was used to study capture and rolling dynamics of purified c-kit(+) versus c-kit- BMCs on various selectin molecules. C-kit(+) BMCs interacted weakly with L-selectin substrates (0.03 ± 0.01% adhered) but adhered strongly to P-selectin (0.28±0.04% adhered). C-kit- BMCs showed intermediate binding regardless of substrate (0.18 ± 0.04% adhered on L-selectin versus 0.17 ± 0.04% adhered on P-selectin). Myocardial ischemia-reperfusion stress induces selective engraftment of c-kit(+) bone marrow progenitor cells via P-selectin activation.
| S-EPMC3246079 | biostudies-literature
Project description:Although mechanical stress is known to profoundly influence the composition and structure of the extracellular matrix (ECM), the mechanisms by which this regulation occurs remain poorly understood. We used a single-molecule magnetic tweezers assay to study the effect of force on collagen proteolysis by matrix metalloproteinase-1 (MMP-1). Here we show that the application of ∼10 pN in extensional force causes an ∼100-fold increase in proteolysis rates. Our results support a mechanistic model in which the collagen triple helix unwinds prior to proteolysis. The data and resulting model predict that biologically relevant forces may increase localized ECM proteolysis, suggesting a possible role for mechanical force in the regulation of ECM remodeling.
| S-EPMC3320677 | biostudies-literature
Project description:AimsMechanical stimulation (MS) represents a readily available, non-invasive means of pacing the asystolic or bradycardic heart in patients, but benefits of MS at higher heart rates are unclear. Our aim was to assess the maximum rate and sustainability of excitation by MS vs. electrical stimulation (ES) in the isolated heart under normal physiological conditions.Methods and resultsTrains of local MS or ES at rates exceeding intrinsic sinus rhythm (overdrive pacing; lowest pacing rates 2.5±0.5 Hz) were applied to the same mid-left ventricular free-wall site on the epicardium of Langendorff-perfused rabbit hearts. Stimulation rates were progressively increased, with a recovery period of normal sinus rhythm between each stimulation period. Trains of MS caused repeated focal ventricular excitation from the site of stimulation. The maximum rate at which MS achieved 1:1 capture was lower than during ES (4.2±0.2 vs. 5.9±0.2 Hz, respectively). At all overdrive pacing rates for which repetitive MS was possible, 1:1 capture was reversibly lost after a finite number of cycles, even though same-site capture by ES remained possible. The number of MS cycles until loss of capture decreased with rising stimulation rate. If interspersed with ES, the number of MS to failure of capture was lower than for MS only.ConclusionIn this study, we demonstrate that the maximum pacing rate at which MS can be sustained is lower than that for same-site ES in isolated heart, and that, in contrast to ES, the sustainability of successful 1:1 capture by MS is limited. The mechanism(s) of differences in MS vs. ES pacing ability, potentially important for emergency heart rhythm management, are currently unknown, thus warranting further investigation.
| S-EPMC5400084 | biostudies-literature
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.
| S-EPMC6179415 | biostudies-literature
Project description:1. The lipids of the rat heart have been studied with regard to amount, classes present and fatty acid composition of free fatty acids, triglycerides and phospholipids. Myocardial lipid contained 300mumoles of total fatty acid/g. dry wt. of which only 2-4mumoles were free; the remainder was esterified, chiefly as phospholipid. Neutral esters, of which triglyceride was the principal form, made up 15% of the total fatty acids. 2. When normal hearts were perfused with a nutrient-free medium until exhaustion, the triglyceride concentration declined from 43 to 13mumoles/g. dry wt. The content of phospholipids, partial glycerides and cholesteryl esters did not change. When the lipids of the rat heart were labelled with [1-(14)C]palmitate before perfusion with non-nutrient medium, radioactivity disappeared from the triglyceride, diglyceride and free fatty acid fractions, but not from the phospholipid or other ester classes. 3. These experiments support the view that only a small fraction of the total cardiac lipid, principally triglycerides and to a smaller extent diglycerides, is available as a source of fuel in the absence of exogenous substrate.
| S-EPMC1270485 | biostudies-other
Project description:1. Prompted by the finding of markedly differing specific radioactivities of tissue alanine and lactate in isolated rat hearts perfused with [1-14C]pyruvate, a more detailed study on the cytosolic subcompartmentalization of pyruvate was undertaken. Isolated rat hearts were perfused by the once-through Langendorff technique under metabolic and isotopic steady-state conditions but with various routes of radioactive label influx, and the specific radioactivities of pyruvate, lactate and alanine were determined. An enzymic method was devised to determine the specific radioactivity of C-1 of pyruvate. 2. Label introduction as [1-14C]pyruvate resulted in a higher specific radioactivity of tissue alanine and mitochondrial pyruvate than of lactate, and a higher specific radioactivity of perfusate lactate than of tissue lactate. Label introduction as [1-14C]lactate resulted in a roughly similar isotope dilution into the tissue and perfusate pyruvate and the tissue alanine. Label introduction as [3,4-14C]glucose resulted in the same specific radioactivity of tissue lactate and alanine and a roughly similar specific radioactivity of mitochondrial pyruvate. 3. The results can be reconciled with a metabolic model containing two cytosolic functional pyruvate pools. One pool (I) communicates more closely with the glycolytic system, whereas the other (II) communicates with extracellular pyruvate and intracellular alanine. Pool II is in close connection with intramitochondrial pyruvate. The physical identity of the cytosolic subcompartments of pyruvate is discussed.
| S-EPMC1154205 | biostudies-other
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.
| S-EPMC4833194 | biostudies-literature