Mathematical modelling of the calcium-left ventricular pressure relationship in the intact diabetic rat heart.

AIM: The objective was to characterize cross-bridge kinetics from the cytoplasmic calcium ion concentration ([Ca2+](i)) and the left ventricular pressure (LVP) in the early-stage diabetic rat heart under baseline conditions and upon beta-adrenergic stimulation. METHODS: Four weeks after the induction of diabetes in rats by the injection of streptozotocin, the hearts were perfused according to Langendorff, and [Ca2+](i) was obtained by epifluorescence measurements using Indo-1 AM. [Ca2+](i) and LVP were measured simultaneously at a temporal resolution of 200 Hz. The input/output relationship between the Ca2+ and the pressure transients was described by a mathematical model representing the chemical binding of Ca2+ to troponin C on the actin myofilament (TnCA), and the subsequent cooperative force-producing cross-bridge formation of the Ca2+-TnCA complex with myosin. The kinetic parameters of this model were evaluated using a numerical optimization algorithm to fit the model equations to the experimental data. beta-adrenergic stimulation of the hearts with increasing doses of isoproterenol allowed quantification of the model parameters over an extended dynamic range, because isoproterenol administration increased developed pressure, heart rate, as well as [Ca2+](i) amplitude in a dose-dependent manner. RESULTS: Model analysis of the experimental data indicates that beta-adrenergic stimulation of healthy hearts resulted in a decreased sensitivity of TnCA for Ca2+, increased rates of cross-bridge cycling and decreased cooperativity. By contrast, the responses in cross-bridge kinetic parameters to isoproterenol stimulation were blunted in the 4-week diabetic heart. CONCLUSION: We conclude from our modelling results that myocardial cross-bridge cycling is impaired at the early stage of diabetes.

Identification of a switching model of calcium cycling in isolated rat hearts.

So far, the processes involved in regulation of intracellular calcium (Ca/sub i//sup 2+/) in cardiomyocytes have been mainly studied through biochemical and isolated cell analysis. Here, we present a novel technique to model and identify cardiac Ca/sub i//sup 2+/-cycling under physiologically relevant conditions in the intact beating heart. Ca/sub i//sup 2+/ was measured using fluorescence techniques in ex vivo perfused rat hearts. For analysis, we developed a parametric mathematical model, switching between active and inactive calcium release. The kinetic parameters of the two submodes of the model were computed using a recently developed technique from hybrid system identification. Application of the method to control and isoproterenol-stimulated hearts resulted in parameter values within a physiologically reliable range.

Differences in electrophysiological and contractile properties of mammalian cardiac tissues bathed in bicarbonate - and HEPES-buffered solutions.

AIM: The aim of this study was to compare the action potential configuration, contractility, intracellular Ca2+ and H+ concentrations in mammalian cardiac tissues bathed with Krebs and Tyrode solutions at 37 degrees C. RESULTS: In Langendorff-perfused guinea-pig hearts, loaded with the fluorescent Ca2+-indicator Fura-2, or H+-sensitive dye carboxy-SNARF, shifts from Krebs to Tyrode solution caused intra-cellular acidification, increased diastolic pressure and [Ca2+]i, decreased systolic pressure and [Ca2+]i, leading to a reduction in the amplitude of [Ca2+]i transients and pulse pressure. Contractility was also depressed in canine ventricular trabeculae when transferred from Krebs to Tyrode solution. Shifts from Krebs to Tyrode solution increased the duration of action potentials in multicellular cardiac preparations excised from canine and rabbit hearts but not in isolated cardiomyocytes. All these changes in action potential morphology, contractility, [Ca2+]i and [H+]i were readily reversible by addition of 26 mmol L(-1) bicarbonate to Tyrode solution. Effects of dofetilide and CsCl, both blockers of the delayed rectifier K current, on action potential duration were compared in Krebs and Tyrode solutions. Dofetilide lengthened rabbit ventricular action potentials in a significantly greater extent in Tyrode than in Krebs solution. Exposure of canine Purkinje fibres to CsCl evoked early after depolarizations within 40 min in all preparations incubated with Tyrode solution, but not in those bathed with Krebs solution. CONCLUSION: It is concluded that the marked differences in action potential morphology, [Ca2+]i, [H+]i and contractility observed between preparations bathed with Krebs and Tyrode solutions are more likely attributable to differences in the intracellular buffering capacities of the two media.

Concomitant accumulation of intracellular free calcium and arachidonic acid in the ischemic-reperfused rat heart.

This study was designed to elucidate the relationship between enhanced cytoplasmic calcium levels (Ca2+i) and membrane phospholipid degradation, a key step in the loss of cellular integrity during cardiac ischemia/reperfusion-induced damage. Isolated rat hearts were subjected to 15 min ischemia followed by 30 min reperfusion. Ca2+i was estimated by the Indo-1 fluorescence ratio technique. Degradation of membrane phospholipids as indicated by the increase of tissue arachidonic acid content was assessed in tissue samples taken from the myocardium at various points of the ischemia/reperfusion period. The hemodynamic parameters showed almost complete recovery during reperfusion. Fluorescence ratio increased significantly during ischemia, but showed a considerable heart-to-heart variation during reperfusion. Based upon the type of change of fluorescence ratio during reperfusion, the hearts were allotted to two separate subgroups. Normalization of fluorescence ratio was associated with low post-ischemic arachidonic acid levels. In contrast, elevated fluorescence ratio coincided with enhanced arachidonic acid levels. This observation is suggestive for a relationship between the Ca2+-related fluorescence ratio and arachidonic acid accumulation probably due to a calcium-mediated stimulation of phospholipase A2.

Ischemia/reperfusion-induced changes in intracellular free Ca2+ levels in rat skeletal muscle fibers--an in vivo study.

Accumulation of intracellular free calcium (Ca2+i) may play an essential role in the ischemia/reperfusion injury of skeletal muscle. Although it has been shown that Ca2+i levels significantly increase during ischemia/reperfusion, it is still a matter of debate whether Ca2+i increases during ischemia alone. It was the aim of this study to monitor the in vivo Ca2+i levels in the rat spinotrapezius muscle during ischemia of varying duration and reperfusion, using a ratiometric fluorescence technique, and to investigate the relationship between the postischemic flow patterns and Ca2+i, if any. The muscle was loaded with Indo-1/AM and imaged by a cooled digital camera. Pre- and postischemic tissue perfusion was assessed by means of an analogue camera. Our results show that short-term ischemia (5, 15 and 30 min) and subsequent reperfusion (60 min) does not alter Ca2+i homeostasis and that tissue perfusion promptly recovers after the insult. One or two hours of ischemia resulted in changes in Ca2+i levels, varying from preparation to preparation; increases in some and no changes in others. In these preparations three distinct flow patterns - normal, compromised and no-reflow - could be distinguished during the 60-min reperfusion. Our main conclusion is that in skeletal muscle Ca2+i levels may increase, the increase probably depending on the muscle fiber type exposed.

A novel model for the in vivo monitoring of uterine microcirculation and intracellular free calcium changes in rat.

The aim of this work was to develop a model to study the microcirculation and relative levels of intracellular free calcium in the myometrium of pregnant rats. On Day 21 of gestation a lobe of uterus was prepared free, flipped over, and mounted in a superfusion chamber leaving the radix and thereby the innervation and circulation intact. RBC velocity and arteriolar diameters were determined by means of intravital video microscopy before and after stimulation (norepinephrine). To study intracellular free calcium changes, the fluorescent dye Indo-1 AM was added to the superfusate in the chamber. Fluorescence images were recorded and ratios of the images collected at 400 and 506 nm were calculated and changes thereof were assumed to represent intracellular free calcium changes. RBC velocity and arteriolar diameter did not change for at least 1 h, while the response to norepinephrine was similar at the beginning of the experiment and after 120 min. In four separate interventions, the uterus was challenged with 5 x 10(-4) IU/ml oxytocin, 4.5 mM calcium, 5 x 10(-4) IU/ml oxytocin with 4.5 mM calcium, and 5 microM ionomycin, resulting in an increase of the 400/506 nm ratio of 27, 31, 76, and 103%, respectively, representing a relative increase in intracellular free calcium. This novel in vivo model is suitable for monitoring intracellular free calcium changes and to record RBC velocities and blood vessel diameters in the myometrium of pregnant rats.

An in vivo model for studying the dynamics of intracellular free calcium changes in slow- and fast-twitch muscle fibres.

The understanding of the regulation of the free cytosolic [Ca2+] ([Ca2+]i) in skeletal muscle is hampered by the lack of techniques for quantifying free [Ca2+]i in muscle fibres in situ. We describe a model for studying the dynamics of free [Ca2+]i in the fast-twitch extensor digitorum longus (EDL) and the slow-twitch soleus (SOL) muscles of the rat in vivo using caffeine superfusion to induce changes in free [Ca2+]i. We assumed that differences in sensitivity between the two muscle types for this substance reflect differences in intracellular Ca2+ handling in the fibres of which these muscles consist. The Indo-1 ratiometric method, using intravital microscopy with incident light, was adapted to measure free [Ca2+]i in vivo. Fluorescence images were collected by means of a digital camera. Caffeine superfusion at 37 degrees C for 2 min, at concentrations of 1, 2, 5, 10 or 20 mmol/l, induced a concentration-dependent increase in free [Ca2+]i and revealed differences in caffeine sensitivity between the muscle types, with the SOL being more sensitive. In a separate set of experiments the contracture threshold, as assessed by topical application of caffeine, was determined in both muscle types. EDL had a higher threshold for developing contracture than SOL. These finding are in agreement with previous in vitro studies. We may conclude that the dynamics of free [Ca2+]i can be assessed reliably in intact mammalian muscle in vivo.

Quantitative assessment of [Ca2+]i levels in rat skeletal muscle in vivo.

Intracellular free Ca2+ concentration ([Ca2+]i) plays an essential role in physiological regulatory processes and common pathological conditions. Better understanding of these phenomena is still hampered by problems encountered in the quantitative assessment of [Ca2+]i changes, especially in blood-perfused organs. This study demonstrates that the ratiometric fluorescence technique can be adapted for quantitative in vivo [Ca2+]i determinations. The rat spinotrapezius muscle was topically loaded with indo 1-AM and imaged by a cooled digital camera. Ratio images were calculated in small regions (100 micrometers x 100 micrometers) practically devoid of large vessels in the resting state, after 30 min of ischemia, 20 min of reperfusion, or ionomycin or manganate treatments. When we assumed an average [Ca2+]i of 100 nM in the resting blood-perfused muscle, ischemia increased [Ca2+]i to approximately 200 nM. During reperfusion [Ca2+]i decreased to approximately 140 nM. Ionomycin induced an increase in [Ca2+]i to well above 750 nM. Manganate reduced Ca2+-dependent fluorescence to virtually zero. Our main conclusion is that changes in [Ca2+]i can be monitored and quantitatively determined in vivo.

Can the Indo-1 fluorescence approach measure brain intracellular calcium in vivo? A multiparametric study of cerebrocortical anoxia and ischemia.

Indo-1 fluorescence was used to monitor intracellular calcium levels in the cat brain in vivo, using the approach proposed by Uematsu et al. [Uematsu D., Greenberg J. H., Reivich M., Karp A. In vivo measurement of cytosolic free calcium during cerebral ischemia and reperfusion. Ann Neurol 1988; 24: 420-428]. In addition, extracellular calcium and potassium levels, NADH redox state, electrocorticogram (ECoG), DC potential and relative cerebral blood flow were monitored simultaneously. Changes in the Indo-1 fluorescence ratio F400/F506 were monitored during anoxia, reversible ischemia and irreversible ischemia. Although these perturbations resulted in the expected changes in extracellular calcium and potassium levels, NADH redox state, ECoG and other physiological parameters, they did not result in significant increases in the F400/F506 ratio. The apparent insensitivity of the in vivo Indo-1 approach is due to the difficulty in obtaining accurate fluorescence signals from Indo-1 in the brain. Two reasons for this difficulty appear to be problems in loading Indo-1 into the brain, and problems in correcting Indo-1 fluorescence signals for changes in NADH fluorescence and changes in absorption of intrinsic chromophores. Under the conditions of our in vivo cat experiments, Indo-1 fluorescence is not a viable approach for measuring changes in cerebral intracellular calcium levels.

Simultaneous measurement of cerebral oxygen consumption and blood flow using 17O and 19F magnetic resonance imaging.

17O and 19F magnetic resonance (MR) imaging were used to determine simultaneously the concentrations of H2 17O and CHF3 in 0.8-cc voxels in the cat brain during inhalation of a gas mixture containing both 17O2 and CHF3. The arterial time course of CHF3 was determined by "on-line" mass spectrometer detection of expired CHF3, and the arterial time course of H2 17O was determined by 17O MR analysis of arterial samples withdrawn during the inhalation period. The brain data and the arterial data for the two tracers were combined to calculate the cerebral oxygen consumption (CMRO2) and the CBF. The protocol was repeated on seven cats, using pentobarbital anesthesia. The average values of CMRO2 and CBF for a 0.8-cc voxel in the parietal cortex were 1.5 +/- 0.5 mmol kg-1 min-1 and 38 +/- 15 ml 100 g-1 min-1, respectively. In individual animals the average uncertainty in CMRO2 and CBF, calculated from Monte Carlo approaches, was +/- 9%.

Determination of cerebral oxygen consumption and blood flow by magnetic resonance imaging.

Simultaneous 17O and 19F magnetic resonance imaging were used to determine the concentrations of H2(17)O and CHF3 in 0.8 cc voxels in the cat brain during in- and exhalation of a gas mixture containing both 17O2 and CHF3. The arterial time course of H2(17)O was determined by 17O MR analysis of arterial samples withdrawn during the inhalation period and the arterial time concentration of CHF3. The brain data and the arterial data for the two tracers were used to calculate the cerebral oxygen consumption (CMRO2) and the cerebral blood flow (CBF). The average values of CMRO2 and CBF for a 0.8 cc voxel in the parietal cortex were 1.5 +/- 0.5 mmol/kg/min and 38 +/- 15 ml/100g/min, respectively. 17O/19F MR imaging approach has the potential to image CMRO2 and CBF simultaneously in humans and might become a strong diagnostic tool.

19F magnetic resonance imaging of cerebral blood flow with 0.4-cc resolution.

19F magnetic resonance imaging techniques were used to determine "wash-in" and "wash-out" curves of the inert, diffusible gas CHF3 from 0.4-cc voxels in the cat brain, and mass spectrometer gas detection was used to determine the CHF3 concentration in expired air. These two sets of data were used to calculate cerebral blood flow values in the 0.4-cc voxels, and the blood flow images were registered with high-resolution 1H magnetic resonance images. Data were collected both during the wash-in and wash-out phases of the experiment, but the two sets of data were analyzed separately to obtain independent estimates of the blood flow during the two phases, i.e., Qin and Qout. Repeated determinations of cerebral blood flow images were performed in individual animals, and the entire protocol was repeated on five different animals. The average values of Qin and Qout for a typical 0.4-cc voxel in the parietal cortex were 83 ml 100 g-1 min-1 and 72 ml 100 g-1 min-1, respectively. Monte Carlo calculations utilizing the noise in the 19F NMR signal from this voxel predict an average standard deviation for Qin and Qout of +/- 10%. The average standard deviation for repeated measurements (in the same animal) of Qin and Qout in this voxel was +/- 14%. We conclude that 19F magnetic resonance imaging approaches have the potential to image cerebral blood flow in humans.

Monitoring of intracellular free calcium in perfused rat liver.

Fluorescent calcium indicators have been widely used to assess cytoplasmic calcium concentration in cells. To examine the role of calcium ions on different physiological functions (e.g. in case of liver; bile secretion, glucose metabolism, etc.) there is a need for whole organ studies. We have developed a technique to estimate intracellular free calcium changes in perfused rat liver. Krebs-Henseleit perfused livers were loaded with 7 microM or 35 microM Indo-1/AM. An area 3 mm in diameter and approximately 300 microns in depth was illuminated at 340 nm. Fluorescence was monitored with photomultiplier tubes at 3 wavelengths (400 nm for Ca-bound dye, 504 nm for free dye and 464 nm for NADH). The viability of liver preparations was assessed by measurement of the concentrations of lactate dehydrogenase and alanine aminotransferase in the effluent. Loading of the livers with 7 microM Indo-1/AM via the portal vein resulted in a 5-fold increase of fluorescence at 400 nm. However the dye 'leaked' out of the liver with a half-time of 18 min. Probenecid (a specific anion carrier blocker) inhibited loss of dye in a dose dependent fashion (2.5-10 mM). Transient calcium elevations were observed in response to vasopressin (5-50 nM) at physiological levels, ethanol (0.3-0.8 M) and the calcium ionophore, ionomycin. Certain limitations were apparent with this approach: (1) it was necessary to use an anion carrier blocker to maintain a relatively steady dye concentration; (2) endogenous NADH fluorescence interfered with the calcium signal; and (3) absolute values of calcium concentration could not be determined.

In vivo 17O NMR study of rat brain during 17O2 inhalation.

In vivo 17O NMR has been used to monitor the H2(17)O concentration in rat brain during inhalation of 17O2. The results are discussed in terms of oxygen consumption in the brain and recirculation into the brain of H2(17)O produced in other organs.

In vivo measurement of cerebral oxygen consumption and blood flow using 17O magnetic resonance imaging.

We used 17O NMR imaging techniques to measure the H2(17)O concentration in a 0.8-ml voxel in the cat brain following injection of an arterial bolus of enriched H2(17)O and during inhalation of enriched 17O2. We also measured the H2(17)O concentration in arterial blood during 17O2 inhalation. The data from the first measurement were used to calculate the blood flow in the voxel. The data from all three measurements were combined to calculate the oxygen consumption in the voxel. The values of cerebral blood flow and oxygen consumption calculated with 17O NMR techniques agree reasonably well with values calculated for a similar region of the cat brain using autoradiographic techniques.

Cerebral blood flow and metabolic rate in the conscious, freely moving rat: the effects of hypercapnia, and acute ethanol administration.

We propose a simple method that can be used to measure cerebral blood flow (CBF), cerebral oxygen consumption (CMRO2), and cerebral glucose consumption (CMRglu) in the conscious, freely moving rat. The method is based on the classical Kety-Schmidt approach, and uses a chronic cannula in the confluens sinuum. We tested the method by investigating the response of CBF, CMRO2, and CMRglu to hypercapnia and used the approach to investigate the effects of acute alcohol administration. Severe hypercapnia (PaCO2 approximately 80 mmHg) increased the CBF by a factor of 3.5, decreased the CMRO2 by 30%, and had no significant effect on the CMRglu. Under normocapnic conditions moderate blood alcohol levels (100-200 mg%) caused no significant effects on CBF, CMRO2, or CMRglu, but high blood alcohol levels (250-400 mg%) decreased all three parameters by approximately 25%. Under hypercapnic conditions high blood alcohol levels had no effect on CBF, CMRO2, and CMRglu.

Measurement of cerebral blood flow by volume-selective 19F NMR spectroscopy.

A stimulated echo sequence was used to obtain 19F NMR spectra from within a 4-ml voxel in a cat brain. The time dependence of the 19F NMR signal from an inert gas (CHF3) was used to calculate the blood flow in the voxel. The position of the voxel was selected using a 1H MR image.

Use of 19F NMR spectroscopy for measurement of cerebral blood flow: a comparative study using microspheres.

19F NMR was used to determine washout curves of an inert, diffusible gas (CHF3) from the cat brain. The cerebral blood flow was estimated from a bi- or tri-phasic fit to the deconvoluted wash-out curve, using the Kety-Schmidt approach. Cerebral blood flow values determined by 19F NMR show the expected responsiveness to alterations in Paco2, but are approximately 28% lower than cerebral blood flow values determined simultaneously by radioactive microsphere techniques. High concentrations of CHF3 have little effect on intracranial pressure, mean arterial blood pressure or Paco2, but cause small changes in the blood flow to certain regions of the brain. We conclude that 19F NMR techniques utilizing low concentrations of CHF3 have potential for the noninvasive measurement of cerebral blood flow.

In vivo myocardial bioenergetics during acute volume and/or pressure loading in a canine model: a 31P NMR study.

The effects of acute volume and/or pressure loading on myocardial metabolic and mechanical function were studied in 13 dogs. Volume loads were applied by shunting the abdominal aorta to the vena cava using polyethylene tubing (5 mm inner diameter). A plastic regulator allowed shunts to be opened or closed. Dogs were heparinized (100 units/kg) to prevent shunts from clotting. To study the effects of pressure loading, a norepinephrine infusion (1 microgram/kg/min) was administered. Mechanical function of the heart was evaluated using heart rate X systolic blood pressure (HR X SBP), cardiac output (CO), pressure X volume work (systolic blood pressure X stroke volume); (P X V), and oxygen consumption (MVO2) to estimate external myocardial work. Metabolic function was evaluated by 31P NMR. Phosphocreatine/adenosine triphosphate (PCr/ATP) ratios were used to estimate the bioenergetic regulation of oxidative phosphorylation during increased work load. HR X SBP, CO, P X V, and MVO2 were correlated with PCr/ATP. Although there was some variability, generally volume loading was associated with an increase in HR X SBP, CO, P X V, and MVO2 accompanied by no change, or small increases or small decreases in PCr/ATP throughout the loading period. These data indicate that the heart bioenergetics are quite stable during volume and/or pressure loading and that 31P spectroscopy methods can document this stability and tight metabolic regulation during in vivo loading conditions.

Correlated in vivo 31P-NMR and NADH fluorometric studies on gerbil brain in graded hypoxia and hyperoxia.

Mitochondrial energy coupling in the gerbil brain was characterized by the relationship between intracellular phosphocreatine (PCr)/inorganic phosphate (Pi), phosphorylation ratio, and the mitochondrial redox state in graded hypoxia. Phosphorus-nuclear magnetic resonance (NMR) spectra of the brain and whole head were taken by surface and saddle coil, respectively. The NADH level of the brain cortex was monitored by in vivo fluororeflectometry. The PCr and Pi of the head and brain did not change between 100 and 10% O2 inhalation. PCr progressively decreased and Pi progressively increased with 6 and 4% 0% inhalation in the head. The PCr/Pi of the brain decreased by 44% at 6% fraction of inhaled oxygen (FIO2) and 57% at 4% FIO2. The ATP level did not change during hypoxia. The calculated phosphorylation ratio of the brain ([PCr] Kck[H+]/[Cr][Pi]) = ([ATP]/[ADP][Pi]) was 4.1 X 10(4) M-1 in normoxia. Hypoxia of increasing severity induced increasing NAD reduction of the brain cortex with 17% NAD reduction at 10% FIO2 when there was no change in phosphorylation ratio. The phosphorylation ratio decreased, i.e., the mitochondria failed to maintain the energy level of the brain when the magnitude of the change in NAD reduction to hypoxia was half of the total redox change between hyperoxia and anoxia. These studies demonstrated the feasibility of combined 31P-NMR and NADH fluorometry measurements on brain in vivo. The observations show similarities between the responses of mitochondrial oxidative phosphorylation to hypoxia in vivo and in vitro.