KCl-arrested Hearts Research Articles

Rhodamine 800 as a near-infrared fluorescent deposition flow tracer in rodent hearts

We investigated the use of a near-infrared (NIR) fluorescent dye, Rhodamine 800 (Rhod800, λ(exc) = 693 nm, λ(em) > 720 nm) as a flow-dependent molecular tracer for NIR spectroscopy and high-resolution cardiac imaging. Rhod800 accumulates in isolated mitochondria in proportion to the mitochondrial membrane potential (ΔΨ). However, in the intact myocardium, Rhod800 binding is ΔΨ-independent. Rat hearts were perfused in a Langendorff mode with Krebs-Henseleit buffer containing 45-nM Rhod800 at normal (100%), increased (150%), or reduced (50%) baseline coronary flow (CF) per gram, for 30 to 60 min. In a different group of hearts, the left anterior descending artery (LAD) was occluded prior to Rhod800 infusion to create a flow deficit area. Rhod800 deposition was analyzed by: 1. absorbance spectroscopy kinetics in the Rhod800-perfused hearts, 2. Rhod800 absorbance and fluorescence imaging in the short-axis heart slices, and 3. dynamic epicardial/subepicardial fluorescence imaging of Rhod800 in KCl-arrested hearts, with a spatial resolution of ∼ 200 μm. Rhod800 deposition was proportional to the perfusate volume (CF and perfusion time) and there was no Rhod800 loss during the washout period. In the LAD-ligated hearts, Rhod800 fluorescence was missing from the no-flow, LAD-dependent endocardial and epicardial/subepicardial area. We concluded that Rhod800 can be used as a deposition flow tracer for dynamic cardiac imaging.

Sarcolemmal and mitochondrial effects of a K ATP opener, P-1075, in “polarized” and “depolarized” Langendorff-perfused rat hearts

We investigated consequences of cardiac arrest on sarcolemmal and mitochondrial effects of ATP-sensitive potassium channel (K ATP) opener, P-1075, in Langendorff-perfused rat hearts. Depolarised cardiac arrest (24.7 mM KCl) did not affect glibenclamide-sensitive twofold activation of rubidium efflux by P-1075 (5 μM) from rubidium-loaded hearts, but eliminated uncoupling produced by P-1075 in beating hearts: 40% depletion of phosphocreatine and ATP, 50% increase in oxygen consumption, and reduction of cytochrome c oxidase. Depolarized cardiac arrest by calcium channel blocker, verapamil (5 μM), also prevented uncoupling. Lack of P-1075 mitochondrial effects in depolarized hearts was not due to changes in phosphorylation potential, because 2,4-dintrophenol (10 μM) reversed the [PCr]/[Cr] increase and P i decrease, characteristic of KCl-arrest, but did not restore uncoupling. In agreement with this conclusion, pyruvate (5 mM) increased [PCr]/[Cr] and decreased P i, but did not prevent uncoupling in beating hearts. A decrease in mean [Ca 2+] in KCl-arrested hearts could not account for lack of P-1075 mitochondrial effects, because calcium channel opener, S-(−)-Bay K8644 (50 nM), and beta-agonist, isoproterenol (0.5 μM), did not facilitate uncoupling. In contrast, in adenosine (1 mM)-arrested hearts (polarized arrest), P-1075 caused 40% phosphocreatine and ATP depletion. In isolated rat liver mitochondria, P-1075 (20 μM) decreased mitochondrial membrane potential (Δ Ψ) by approximately 14 mV (demonstrated by redistribution of Δ Ψ-sensitive dye, rhodamine 800) in a glibenclamide-sensitive manner. We concluded that cell membrane depolarization does not prevent activation of sarcolemmal K ATP by P-1075, but it plays a role in mitochondrial uncoupling effects of P-1075.

Effect of passive myocardium on the compliance of porcine coronary arteries.

The objective of this study was to determine the effect of passive myocardium on the coronary arteries under distension and compression. To simulate distension and compression, we placed a diastolic-arrested heart in a Lucite box, where both the intravascular pressure and external (box) pressure were varied independently and expressed as a pressure difference (DeltaP = intravascular pressure - box pressure). The DeltaP-cross-sectional area relationship of the first several generations of porcine coronary arteries and the DeltaP-volume relationship of the coronary arterial tree (vessels >0.5 mm in diameter) were determined using a video densitometric technique in the range of +150 to -150 mmHg. The vasodilated left anterior descending (LAD) coronary artery of six KCl-arrested hearts were perfused with iodine and 3% Cab-O-Sil. The intravascular pressure was varied in a triangular pattern, whereas the absolute cross-sectional area of each vessel and the total arterial volume were calculated using video densitometry under different box pressures (0, 50, 100, and 150 mmHg). In the range of positive DeltaP, we found that the compliance of the proximal LAD artery in situ (4.85 +/- 3.8 x 10-3 mm2/mmHg) is smaller than that of the same artery in vitro (16.5 +/- 6 x 10-3 mm2/mmHg; P = 0.009). Hence, the myocardium restricts the compliance of the epicardial artery under distension. In the negative DeltaP range, the LAD artery does not collapse, whereas the same vessel readily collapses when tested in vitro. Hence, we conclude that myocardial tethering prevents collapse of large blood vessel under compression.

Temperature dependence of monovalent cation fluxes in isolated rat hearts: a magnetic resonance study

Ion flux studies were performed on Langendorff-perfused rat hearts using 87Rb, 7Li and 23Na NMR at 36, 20 and 10°C, and at constant extracellular pH (7.40). Using 31P NMR, the intracellular pH was estimated and the high energy phosphate content monitored. Compared to 36°C ( k=0.044±0.015 min −1), our measurements showed incomplete Rb + efflux with a dramatically (5-fold) increased rate constant, k, at 20°C, k=0.238±0.080 min −1. 5 μM glibenclamide, a K ATP-channel inhibitor, completely depressed the hypothermia-activated Rb + efflux at this temperature ( k=0.052±0.018 min −1). 7Li NMR efflux studies on KCl-arrested hearts at 20°C also showed an increase (3-fold) in efflux rate constant: k=0.090±0.003 min −1 relative to its value at 36°C. At 10°C, both Rb + and Li + showed efflux rate constants similar to those observed at 36°C, k=0.071±0.016 min −1 and k=0.050±0.005 min −1, respectively, and the washout was complete. 31P NMR at 36, 20 and 10°C indicated cytosolic alkalinization at pH values of 7.05, 7.21 and 7.40, respectively. The ion transport data could be interpreted in terms of a myocyte model allowing for temperature-dependent changes in transport coefficients. The incomplete efflux of Rb + at 20°C may indicate the existence of a mitochondrial Rb +-pool with a very low Rb + permeability for efflux. These findings correlate with previously observed membrane phase transitions in these systems.

MCI-154, a Ca2+ sensitizer, decreases the oxygen cost of contractility in isolated canine hearts.

An increase in the responsiveness of the contractile machinery to Ca2+ could theoretically enhance the mechanoenergetics of the heart. To clarify this unresolved issue, we studied the effects of MCI-154, a Ca2+ sensitizer, on the mechanoenergetics in terms of the left ventricular contractility index [slope of end-systolic pressure-volume relationship (Emax)] and the relationship between myocardial oxygen consumption (VO2) and left ventricular pressure-volume area in excised cross-circulated canine hearts. MCI-154 increased Emax by 42 +/- 31% (SD), although the slope of the VO2-PVA relationship (an indicator of contractile efficiency) was unchanged by MCI-154. Despite equal increases in Emax, the relative increase in unloaded VO2 (delta VO2/delta Emax) during infusion of MCI-154 was, however, significantly less than that during CaCl2 infusion (0.0016 +/- 0.0018 vs. 0.0059 +/- 0.0054; P < 0.05). By contrast, delta VO2/delta Emax for milrinone was the same as that for CaCl2 (0.0043 +/- 0.0041 vs. 0.0039 +/- 0.0045; P > 0.05). Basal metabolism in KCl-arrested hearts was unchanged by MCI-154, indicating that MCI-154 consumes less energy than CaCl2 for excitation-contraction coupling. These findings suggest that MCI-154 acts energetically as a Ca2+ sensitizer in beating canine whole hearts.

Lithium ion as a probe of Na+ channel activity in isolated rat hearts: a multinuclear NMR study.

The aim of this study was to analyze Na+ fluxes in whole perfused hearts using Li+ as a Na+ congener and 7Li-nuclear magnetic resonance as a detection method. Hearts were equilibrated for 32 min with 15 mM LiCl added to P1-free Krebs-Henseleit buffer (intracellular space (ICS) [Li+] = 21.5 +/- 3.4 mM). Li+ efflux was monitored using a Li(+)-free perfusate. The effects of drugs on Li+ efflux were studied by adding the compounds 4 min prior to initiating Li+ washout. 7Li-NMR spectra were collected every 2 min at 139.95 MHz. Li+ efflux was biphasic with rate constants (k +/- SD, min-1) of 0.5 +/- 0.1 (extracellular) and 0.09 +/- 0.01 (ICS). Li+ efflux from ICS was dependent on heart rate (HR): cardiac arrest produced by 1 mM lidocaine or 20 mM KCl reduced k to 1/3 of its control value (Lidocaine, 0.030 +/- 0.004; KCl, 0.035 +/- 0.003). Increasing concentrations of carbachol (0.2-3.0 microM) caused a gradual decrease in HR and revealed a linear relationship between k and HR. In KCl-arrested hearts the Na+ channel opener veratridine increased k by 60% (10 microM, 0.057 +/- 0.006). Dimethylamiloride did not affect k (10 microM, 0.024 +/- 0.006) in Lidocaine-arrested hearts. Bumetanide (30 microM, 0.094 +/- 0.013), nifedepine (0.33 microM, 0.088 +/- 0.009), Bay K8644 (0.1 microM, 0.080 +/- 0.002), 4-aminopyridine (1.5 mM, 0.076 +/- 0.006) and cromakalim (10 microM, 0.088 +/- 0.006) did not significantly affect either k or HR. Li+ efflux from myocytes in perfused rat heart is mediated mainly by voltage-dependent Na+ channels.

Oxygen Regulation of Energy Metabolism in Isolated Pig Hearts: A Near-IR Spectroscopy Study

Intracellular pO2in cardiac muscle is determined by the balance between its diffusion from vascular (VS) to intracellular space (IS) and uptake by mitochondria. Cessation of mechanical work decreases O2demand and should reduce the O2gradient between VS and IS. In this work, the effects of arterial pO2(pO2a) on myoglobin (Mb) oxygenation, O2uptake and lactate formation rates [V(O2) and V(Lac),μmol/min/g wet weight, respectively] in beating and KCl-arrested pig hearts were compared. pO2awas decreased stepwise (five steps, 10 min/step) from 528±24 to 54±19 mmHg by changing the ratio of O2/CO2(95:5%) to N2/CO2(95:5%) in the gas mixture from 100/0 through 50/50, 25/75, 10/90 to 0/100. While recording mechanical function, visible/near-infrared (near-IR) spectra (400–2500 nm) were acquired from the left ventricular wall every 45 s (32 scans) using a fiber-optic light guide connected to an NIRSystems spectrometer. Relative MbO2in the epicardium + midmural layer and the epicardium was estimated from absorbance changes (second derivative) at 764 and 584 nm, respectively. In beating hearts, a decrease in pO2aresulted in a decrease in MbO2(similar in both layers) and V(O2) (from 1.26±0.15 to 0.22±0.10) and an increase in V(Lac) from 0 to 2.43±0.76. In arrested hearts an increase in V(Lac) (0.55±0.24) was observed only at the lowest pO2aand the Mb oxygenation curve was shifted towards lower vascular pO2[(pO2a+pO2v)/2] by 160 mmHg. Oxygen consumption (oxygen flux) correlated linearly with calculated values of the oxygen gradients between the vascular and cytoplasmic spaces in both beating and arrested hearts. However, the diffusion rate constant was 3.5 times lower in arrested hearts. Conclusions: (1) hypoxia in KCl-arrested hearts results in only moderate activation of anaerobic glycolysis; (2) oxygenation of the epicardial and midmural LV layers is similar; and (3) a large pO2gradient exists between VS and IS in beating and arrested crystalloid-perfused hearts.

Acetylcholine-induced vasoconstrictor response of coronary vessels in rats: a possible contribution of M2 muscarinic receptor activation.

A mechanism by which acetylcholine (ACh) may elicit vasoconstrictor response in coronary vessels was studied in rat hearts perfused at a constant flow rate. In spontaneously beating hearts, bolus injections of ACh and carbachol (CCh) produced biphasic changes in coronary perfusion pressure (CPP): a transient increase at the initial period followed by a sustained decrease. In KCl-arrested hearts, ACh and CCh produced a monophasic increase in CPP, which was attenuated by either removal of endothelial cells by saponin or cyclooxygenase inhibition by diclofenac sodium. In the spontaneously beating heart, ACh-induced vasoconstriction was almost abolished by atropine (0.1 microM) and was markedly attenuated by an M2 antagonist, methoctramine (0.1 microM), but not by an M1 antagonist, pirenzepine (1 microM). Arecaidine propargyl ester (APE), an M2 agonist, produced coronary artery constriction which was attenuated by methoctramine (0.1 microM) but not by pirenzepine (0.1 microM) in both spontaneously beating and KCl-arrested hearts. McN-A-343, an M1 agonist, increased CPP in both beating and KCl-arrested hearts, but to a lesser degree than APE. These results suggest that the release of vasoconstrictor prostaglandins from endothelial cells contributes to the vasoconstrictor response to ACh in perfused rat coronary vessels, and the response to ACh appears to be mediated, in part, via the M2 subtype of muscarinic receptors.

A simple mathematical model and practical approach for evaluating citric acid cycle fluxes in perfused rat hearts by 13C-NMR and 1H-NMR spectroscopy.

We propose a simple mathematical model and a practical approach for evaluating the flux constant and the absolute value of flux in the citric acid cycle in perfused organs by 13C-NMR and 1H-NMR spectroscopy. We demonstrate that 13C-NMR glutamate spectra are independent of the relative sizes of the mitochondrial and cytosolic compartments and the exchange rates of glutamates, unless there is a difference in 13C chemical shifts of glutamate carbons between the two compartments. Wistar rat hearts (five beating and four KCl-arrested hearts) were aerobically perfused with 100% enriched [2-(13)C]acetate and the kinetics of glutamate carbon labeling from perchloric acid extracts were studied at various perfusion times. Under our experimental conditions, the citric acid cycle flux constant, which represents the fraction of glutamate in exchange with the citric acid cycle per unit time, is about 0.350 +/- 0.003 min(-1) for beating hearts and 0.0741 +/- 0.004 min(-1) for KCl-arrested hearts. The absolute values of the citric acid flux for beating hearts and for KCl-arrested hearts are 1.06 +/- 0.06 micromol x min(-1) x mg(-1) and 0.21 +/- 0.02 micromol x min(-1) x g(-1), respectively. The fraction of unlabeled acetate determined from the proton signal of the methyl group is small and essentially the same in beating and arrested hearts (7.4 +/- 1.7% and 8.8 +/- 2.1%, respectively). Thus, the large difference in the Glu C2/C4 between beating and arrested hearts is not due to the important contribution from anaplerotic sources in arrested hearts but simply to a substantial difference in citric acid cycle fluxes. Our model fits the experimental data well, indicating a fast exchange between 2-oxoglutarate and glutamate in the mitochondria of rat hearts. Analysis of the flux constant, calculated from the half-time of glutamate C4 labeling given in the literature, allows for a comparison of the citric acid flux for various working conditions in different animal species.

Function and bioenergetics in isolated perfused trained rat hearts.

To evaluate the resistance of physiologically hypertrophied hearts to hypoxic insult, we quantified the development of functional deficits during hypoxia and reoxygenation in hypertrophied hearts from swim-trained female rats and we correlated this with assessment of high-energy phosphate (HEP) metabolites from simultaneous 31P nuclear magnetic resonance (NMR) measurements. Furthermore, in vivo enzymatic studies were carried out with saturation transfer NMR under well-oxygenated perfusion conditions for both beating and KCl-arrested hearts. Finally, in vitro enzymatic assays were performed. During hypoxia, the trained hearts exhibited improved systolic and diastolic function compared with hearts from sedentary animals. After 16 min of hypoxia, left ventricular (LV) developed pressure fell to 9% of baseline in control hearts but to only 21% of baseline in trained hearts (P < 0.01). LV diastolic function was also improved by training, increasing during hypoxia from a baseline of 10 to 71.0 +/- 3.3 mmHg in control hearts and to 55.3 +/- 4.8 mmHg in trained hearts (P < 0.05). Trained hearts also showed more rapid and complete recovery of function during reoxygenation and greater coronary flow per gram of heart throughout the entire protocol. Functional differences were not accompanied by differences in HEP at baseline; moreover, ATP and phosphocreatine (PCr) loss during hypoxia was similar between control and trained hearts, as was the recovery of PCr during reoxygenation. Saturation transfer experiments showed an increase in the forward creatine kinase (CrK) rate constant in trained hearts of 18% while beating, whereas in vitro enzymatic analysis revealed a 16% increase in the ratio of mitochondrial CrK to citrate synthase activity in LV tissue. Thus the relative preservation of function in hearts from trained rats could not be accounted for by overall HEP levels but may reflect adaptations in the CrK system.