Isolated Heart Cells Research Articles

Insulin sensitivity in the aged heart is improved by down-regulation of KAT7 in vivo and in vitro

ABSTRACT Insulin has an important regulatory effect on the heart, and the important regulatory effect of insulin on the heart is the regulation of substrate utilization. Studies have shown that aging is closely related to insulin resistance, and aging is thought to be one of the underlying causes of insulin resistance. Additionally, chronic inflammation is a major risk factor for aging and aging-related diseases. How to delay or reverse insulin resistance caused by aging is an important scientific problem. In the current study, we used cardiomyocyte cell lines and isolated heart cells as an in vitro model, and aged mice as in vivo model to study the effect of KAT7 on insulin resistance, and results showed that knockdown or inhibiting KAT7 can significantly increase the insulin sensitivity in vivo and in vitro. In addition, the knockdown of KAT7 could reduce inflammation and oxidative stress caused by aging. These findings indicate that KAT7 can be used as one of the potential targets for the treatment of insulin resistance caused by aging.

To the Mechanism of Adrenaline Damage to the Heart Tissue and the Mechanism of Cardioprotection by Neonatal, Xenogenic, Cardiac Cells. Dynamics of Creatine Phosphate, Lactate and Malondialdehyde

The development of disturbances in energy processes and damaging free radical reactions in various pathological processes, including cardiovascular diseases, are interrelated and lead to a significant deterioration in the course of diseases. Aim of the study . Research of the dynamics of malondialdehyde, creatine phosphate and lactate in the cardiac tissue of rats under experimental adrenaline stress and during its correction with neonatal, xenogenic, cardiac cells. Methods . The experiment was carried out on outbred male rats. Adrenaline damage to the heart was simulated by a single subcutaneous injection of 0.1% adrenaline solution at a dose of 0.5 mg per 100 g of body weight. The first group (37 rats) was injected subcutaneously with adrenaline, the second group (41 rats) – adrenaline and isolated heart cells of a newborn rabbit at a dose of 500 000. The third group included 6 healthy rats. Results . It was found that a spike in the level of malondialdehyde and, accordingly, the activation of free radical processes in adrenaline damage to the heart, occurred during the restructuring of the energy of the heart cell from intense glycolysis to the restoration of active mitochondrial ATP synthesis (which corresponded to the end of the depletion of lactate and creatine phosphate, and the beginning of the restoration of their content in heart cells). The dynamics of MDA sensitively reflected both the activity and the inhibition of oxidative processes in mitochondria, which manifested itself, respectively, both in the form of peaks and in a low level of MDA and corresponded to the interpretation of the dynamics of lactate and creatine phosphate. In the cardiac tissue of rats with transplantation of neonatal, xenogenic cardiac cells, the accumulation of lactate in the early stages of the experiment decreased, the subsequent depletion of the cellular reserves of creatine phosphate and lactate was inhibited, the period of inhibition (non-increase) in MDA was shorter, the subsequent increase in MDA was more moderate than in control animals. Conclusion . The data obtained indicate that neonatal cardiac cells are able to limit the disturbance of aerobic and of anaerobic processes in the heart tissue and promote the restoration of mitochondrial energy processes. Moreover, they contribute to a more efficient restoration of mitochondrial ATP synthesis, accompanied by a smaller burst of damaging free radical processes.

Activated macrophages as a feeder layer for growth of resident cardiac progenitor cells.

The adult heart contains a population of cardiac progenitor cells (CPCs). Growing and collecting an adequate number of CPCs demands complex culture media containing growth factors. Since activated macrophages secrete many growth factors, we investigated if activated isolated heart cells seeded on a feeder layer of activated peritoneal macrophages (PM) could result in CPCs and if these, in turn, could exert cardioprotection in rats with myocardial infarction (MI). Heart cells of inbred Wistar rats were isolated by collagenase digestion and cultured on PM obtained 72h after intraperitoneal injection of 12ml thioglycollate. Cells (1×10(6)) exhibiting CPC phenotype (immunohistochemistry) were injected in the periphery of rat MI 10min after coronary artery occlusion. Control rats received vehicle. Three weeks later, left ventricular (LV) function (echocardiogram) was assessed, animals were euthanized and the hearts removed for histological studies. Five to six days after seeding heart cells on PM, spherical clusters composed of small bright and spherical cells expressing mostly c-Kit and Sca-1 antigens were apparent. After explant, those clusters developed cobblestone-like monolayers that expressed smooth muscle actin and sarcomeric actin and were successfully transferred for more than ten passages. When injected in the MI periphery, many of them survived at 21days after coronary ligature, improved LV ejection fraction and decreased scar size as compared with control rats. CPC-derived cells with cardiocyte and smooth muscle phenotypes can be successfully grown on a feeder layer of activated syngeneic PM. These cells decreased scar size and improved heart function in rats with MI.

Manipulating L-type calcium channels in cardiomyocytes using split-intein protein transsplicing

Manipulating expression of large genes (>6 kb) in adult cardiomyocytes is challenging because these cells are only efficiently transduced by viral vectors with a 4-7 kb packaging capacity. This limitation impedes understanding structure-function mechanisms of important proteins in heart. L-type calcium channels (LTCCs) regulate diverse facets of cardiac physiology including excitation-contraction coupling, excitability, and gene expression. Many important questions about how LTCCs mediate such multidimensional signaling are best resolved by manipulating expression of the 6.6 kb pore-forming α1C-subunit in adult cardiomyocytes. Here, we use split-intein-mediated protein transsplicing to reconstitute LTCC α1C-subunit from two distinct halves, overcoming the difficulty of expressing full-length α1C in cardiomyocytes. Split-intein-tagged α1C fragments encoding dihydropyridine-resistant channels were incorporated into adenovirus and reconstituted in cardiomyocytes. Similar to endogenous LTCCs, recombinant channels targeted to dyads, triggered Ca(2+) transients, associated with caveolin-3, and supported β-adrenergic regulation of excitation-contraction coupling. This approach lowers a longstanding technical hurdle to manipulating large proteins in cardiomyocytes.

Abstract 225: The Mitochondria-localizing Peptide Bendavia Reduces Cardiac Injury By Targeting Cardiolipin

Bendavia is a cardioprotective mitochondria-targeting peptide that is currently being utilized in the EMBRACE-STEMI clinical trial for acute coronary syndromes (ACS). Across a variety of pre-clinical models examining ACS and heart failure, administration of Bendavia reduces infarction and coronary “no-reflow”, improves function, preserves mitochondrial energetics, and lowers ROS-dependent cell death. Despite the clear effects in mitigating reperfusion injury, Bendavia’s molecular targets and underlying mechanisms are not fully understood. The objective of these studies was to determine Bendavia’s molecular mechanism of action and pharmacokinetic profile. In diabetic animals, Bendavia treatment reversed the decline in 18:2 cardiolipin composition observed in diabetic heart. In vivo (i.v.) administration of 3H-Bendavia showed tissue distribution that correlated with tissue mitochondrial volume density. 3H-Bendavia levels in heart mitochondria peaked 1 hour after treatment, and by 24 hours there was no discernible 3H-Bendavia in the myocardium. Biologic processing of Bendavia results in two major metabolites, and neither of these metabolites showed cardioprotective efficacy, suggesting that the cardioprotection is unique to the intact, tetra-peptide. In isolated heart cells, 3H-Bendavia uptake was found to be independent of mitochondrial membrane potential, and respiratory studies revealed no discernible uncoupling activity. We then tested the hypothesis that Bendavia selectively targeted cardiolipin biophysical organization by synthesizing lipid vesicles of differing phospholipid composition. Fluorescence quenching and electrostatically sensitive probes revealed Bendavia preferentially associated with cardiolipin-enriched lipid vesicles. We modeled changes in inner membrane lipids during cardiac ischemia/reperfusion. Bendavia augmented membrane microviscosity in proportion to the cardiolipin content. As a number of disease states (heart failure, diabetes, ischemia/reperfusion) are characterized by low membrane microviscosity, Bendavia’s putative ability to restore the lipid microenvironment represents a novel paradigm for preserving tissue bioenergetics.

Cardiac structural changes and electrical remodeling in a thiamine-deficiency model in rats

Aims Thiamine is an important cofactor present in many biochemical reactions, and its deprivation can lead to heart dysfunction. Little is known about the influence of thiamine deprivation on the electrophysiological behavior of the isolated heart cells and information about thiamine deficiency in heart morphology is controversial. Thus, we decided to investigate the major repolarizing conductances and their influence in the action potential (AP) waveform as well as the changes in the heart structure in a set of thiamine deficiency in rats. Main methods Using the patch-clamp technique, we investigated inward ( I K1) and outward K + currents ( I to), T-type and L-type Ca 2+ currents and APs. To evaluate heart morphology we used hematoxylin and eosin in transversal heart sections. Key findings Thiamine deficiency caused a marked decrease in left ventricle thickness, cardiomyocyte number, cell length and width, and membrane capacitance. When evaluating I to we did not find difference in current amplitude; however an acceleration of I to inactivation was observed. I K1 showed a reduction in the amplitude and slope conductance, which implicated a less negative resting membrane potential in cardiac myocytes isolated from thiamine-deficient rats. We did not find any difference in L-type Ca 2+ current density. T-type Ca 2+ current was not observed. In addition, we did not observe significant changes in AP repolarization. Significance Based on our study we can conclude that thiamine deficiency causes heart hypotrophy and not heart hypertrophy. Moreover, we provided evidence that there is no major electrical remodeling during thiamine deficiency, a feature of heart failure models.

Cross-Functioning between the Extraneuronal Monoamine Transporter and Multidrug Resistance Protein 1 in the Uptake of Adrenaline and Export of 5-(Glutathion-S-yl)adrenaline in Rat Cardiomyocytes

Isolated heart cells are highly susceptible to the toxicity of catecholamine oxidation products, namely, to catecholamine-glutathione adducts. Although cellular uptake and/or efflux of these products may constitute a crucial step, the knowledge about the involvement of transporters is still very scarce. This work aimed to contribute to the characterization of membrane transport mechanisms, namely, extraneuronal monoamine transporter (EMT), the multidrug resistant protein 1 (MRP1), and P-glycoprotein (P-gp) in freshly isolated cardiomyocytes from adult rats. These transporters may be accountable for uptake and/or efflux of adrenaline and an adrenaline oxidation product, 5-(glutathion-S-yl)adrenaline, in cardiomyocyte suspensions. Our results showed that 5-(glutathion-S-yl)adrenaline efflux was mediated by MRP1. Additionally, we demonstrated that the adduct formation occurs within the cardiomyocytes, since EMT inhibition reduced the intracellular adduct levels. The classical uptake2 transport in rat myocardial cells was inhibited by the typical EMT inhibitor, corticosterone, and surprisingly was also inhibited by low concentrations of another drug, a well-known P-gp inhibitor, GF120918. The P-gp activity was absent in the cells since P-gp-mediated efflux of quinidine was not blocked by GF120918. In conclusion, this work showed that freshly isolated cardiomyocytes from adult rats constitute a good model for the study of catecholamines and catecholamines metabolites membrane transport. The cardiomyocytes maintain EMT and MRP1 fully active, and these transporters contribute to the formation and efflux of 5-(glutathion-S-yl)adrenaline. In the present experimental conditions, P-gp activity is absent in the isolated cardiomyocytes.

The W105G and W99G Sorcin Mutants Demonstrate the Role of the D Helix in the Ca2+-Dependent Interaction with Annexin VII and the Cardiac Ryanodine Receptor

Sorcin, a 21.6 kDa two-domain penta-EF-hand (PEF) protein, when activated by Ca(2+) binding, interacts with target proteins in a largely uncharacterized process. The two physiological EF-hands EF3 and EF2 do not belong to a structural pair but are connected by the D helix. To establish whether this helix is instrumental in sorcin activation, two D helix residues were mutated: W105, located near EF3 and involved in a network of interactions, and W99, located near EF2 and facing solvent, were substituted with glycine. Neither mutation alters calcium affinity. The interaction of the W105G and W99G mutants with annexin VII and the cardiac ryanodine receptor (RyR2), requiring the sorcin N-terminal and C-terminal domain, respectively, was studied. Surface plasmon resonance experiments show that binding of annexin VII to W99G occurs at the same Ca(2+) concentration as that of the wild type, whereas W105G requires a significantly higher Ca(2+) concentration. Ca(2+) spark activity of isolated heart cells monitors the sorcin-RyR2 interaction and is unaltered by W105G but is reduced equally by W99G and the wild type. Thus, substitution of W105, via disruption of the network of D helix interactions, affects the capacity of sorcin to recognize and interact with either target at physiological Ca(2+) concentrations, while mutation of solvent-facing W99 has little effect. The D helix appears to amplify the localized structural changes that occur at EF3 upon Ca(2+) binding and thereby trigger a structural rearrangement that enables interaction of sorcin with its molecular targets. The same activation process may apply to other PEF proteins in view of the D helix conservation.

Cardiovascular News

Cardiovascular News

Reprogramming of vascular smooth muscle alpha-actin gene expression as an early indicator of dysfunctional remodeling following heart transplant.

Chronic rejection in cardiac allografts depletes vascular smooth muscle (VSM) alpha-actin from the coronary arterial smooth muscle bed while promoting its abnormal accumulation in cardiomyocytes and myofibroblasts. The objective was to determine if the newly discovered TEF1, MSY1, Puralpha and Purbeta VSM alpha-actin transcriptional reprogramming proteins (TRPs) were associated with development of chronic rejection histopathology in accepted murine cardiac allografts. A mouse heterotopic cardiac transplant model was employed using H2 locus-mismatched mouse strains (DBA/2 or FVB/N to C57BL/6). Recipients were immunosuppressed to promote long-term allograft acceptance and emergence of chronic rejection. Explanted grafts and isolated heart cells were evaluated for changes in the DNA-binding activity and subcellular distribution of VSM alpha-actin transcriptional regulatory proteins. The DNA-binding activity of all four TRPs was high in the developing mouse ventricle, minimal in adult donor hearts and increased substantially within 30 days after transplantation. Immunohistologic analysis revealed nuclear localization of Purbeta and MSY1 particularly in fibrotic areas of the allograft myocardium demonstrating extravascular accumulation of VSM alpha-actin. Cardiomyocytes isolated from adult, non-transplanted mouse hearts not only exhibited less VSM alpha-actin expression and lower levels of TRPs compared to isolated cardiac fibroblasts or neonatal cardiomyocytes, but also contained a novel size variant of the MSY1 protein. Accumulation of TRPs in cardiac allografts, particularly within the fibroblast-enriched myocardial interstitium, was consistent with their potential role in VSM alpha-actin gene reprogramming, fibrosis and dysfunctional remodeling following transplant. These nuclear protein markers could help stage peri-transplant cellular events that precede formation of graft-destructive fibrosis and coronary vasculopathy during chronic rejection.

The study of oxidative stress in freshly isolated Ca 2+-tolerant cardiomyocytes from the adult rat

Cardiotoxicity studies using isolated heart cells are becoming increasingly advocated as a supplement to, and sometimes as a replacement for, whole heart or whole animal experimentation. In fact, the use of isolated cardiomyocytes has the great advantage of enabling mechanistic and comparative studies of compounds, which are directly toxic to cardiomyocytes. Since the 1970s, different procedures have been developed in order to obtain Ca 2+-tolerant cardiomyocytes. The advances in this field will be reviewed and an optimised method to obtain freshly isolated Ca 2+-tolerant cardiomyocytes from the adult rat for use in toxicological studies will be described. With this procedure, a high number of rod-shaped cells can be obtained (6–7×10 6/heart corresponding to 70% of total number of cells). It is also possible to maintain cell viability, glutathione content and enzymatic activity of glutathione reductase (GR), glutathione peroxidase (GPX) and glutathione S-transferase (GST) in acceptable levels for 4 hours. Cardiotoxicity studies performed with isoproterenol (ISO) in the presence of copper and with the model toxic substance tert-butylhydroperoxide ( t-BHP) demonstrate the importance of oxidative stress as a cardiotoxic mechanism elicited by these molecules. The results obtained are also good indicators for future applications of this methodology to other cardiotoxicity studies.

Miniature heart cell force transducer system implemented in MEMS technology.

A fully submersible force transducer system for use with isolated heart cells has been implemented using microelectromechanical systems (MEMS) technology. By using integrated circuit fabrication techniques to make mechanical as well as electrical components, the entire low-mass transducer is only a few cubic millimeters in size and is of higher fidelity (approximately 100 nN and 13.3 kHz in solution) than previously available. When chemically activated, demembranated single cells attached to the device contract and slightly deform a strain gauge whose signal is converted to an amplified electrical output. When integrated with a video microscope, the system is capable of optical determination of contractile protein striation periodicity and simultaneous measurement of heart cell forces in the 100-nN to 50-microN range. The average measured maximal force was Fmax = 5.77 +/- 2.38 microN. Normalizing for the cell's cross-sectional area, Fmax/area was 14.7 +/- 7.7 mN/mm2. Oscillatory stiffness data at frequencies up to 1 kHz has also been recorded from relaxed and contracted cells. This novel MEMS force transducer system permits higher fidelity measurements from cardiac myocytes than available from standard macro-sized transducers.

Mitochondrial NAD(P)H, ADP, oxidative phosphorylation, and contraction in isolated heart cells.

To examine the relationship between mitochondrial NADH (NADH(m)) and cardiac work output, NADH(m) and the amplitude and frequency of the contractile response of electrically paced rat heart cells were measured at 25 degrees C. With 5.4 mM glucose plus 2 mM beta-hydroxybutyrate, NADH(m) was reversibly decreased by 23%, and the amplitude of contraction was reversibly decreased by 27% during 4-Hz pacing. With glucose plus 2 mM pyruvate or with 10 mM 2-deoxy-D-glucose, NADH(m) was maintained during rapid pacing, and the contractile amplitude remained high. Phosphocreatine levels decreased with 2-deoxy-D-glucose administration but not with rapid pacing. Respiration increased to meet the increased ATP demand at 30 degrees C. The data suggest that 1) when NADH(m) is decreased during rapid pacing with defined substrates, the amplitude of contraction is decreased; 2) the amplitude of contraction during electrical pacing does not change with rate of pacing when both the ATP and NADH(m) levels are continuously replenished; and 3) the replenishment of NADH(m) during pacing with physiological substrates may be rate-limited by substrate supply to mitochondrial dehydrogenases. During activation of mitochondrial dehydrogenases, or a significant increase in free ADP induced by 2-deoxy-D-glucose, this rate limitation is bypassed or overcome.

Scale-invariant fluctuations at different levels of organization in developing heart cell networks.

In the last few years it has been realized that the intervals of spontaneous spiking events in the intact heart exhibit coexisting scale-invariant count fluctuations and anticorrelated interspike intervals fluctuations. Here, we show experimentally that this feature is an intrinsic property of single isolated heart cells, which is preserved when the cells couple into networks. We present a model explaining this behavior at both the single cell and network levels.

Cardiac organogenesis in vitro: reestablishment of three-dimensional tissue architecture by dissociated neonatal rat ventricular cells.

The mammalian heart does not regenerate in vivo. The heart is, therefore, an excellent candidate for tissue engineering approaches and for the use of biosynthetic devices in the replacement or augmentation of defective tissue. Unfortunately, little is known about the capacity of isolated heart cells to re-establish tissue architectures in vitro. In this study, we examined the possibility that cardiac cells possess a latent organizational potential that is unrealized within the mechanically active tissue but that can be accessed in quiescent environments in culture. In the series of experiments presented here, total cell populations were isolated from neonatal rat ventricles and recombined in rotating bioreactors containing a serum-free medium and surfaces for cell attachment. The extent to which tissue-like structure and contractile function were established was assessed using a combination of morphological, physiological, and biochemical techniques. We found that mixed populations of ventricular cells formed extensive three-dimensional aggregates that were spontaneously and rhythmically contractile and that large aggregates of structurally-organized cells contracted in unison. The cells were differentially distributed in these aggregates and formed architectures that were indistinguishable from those of intact tissue. These architectures arose in the absence of three-dimensional cues from the matrix, and the formation of organotypic structures was apparently driven by the cells themselves. Our observations suggest that cardiac cells possess an innate capacity to re-establish complex, three-dimensional, cardiac organization in vitro. Understanding the basis of this capacity, and harnessing the organizational potential of heart cells, will be critical in the development of tissue homologues for use in basic research and in the engineering of biosynthetic implants for the treatment of cardiac disease.

Anoxia generates rapid and massive opening of KATP channels in ventricular cardiac myocytes.

The aim was to improve the measurement of both the time course and amplitude of anoxia-induced KATP-channel current (IKATP) in isolated heart cells to specify the role of these channels in the time course of K+ accumulation in the ischemic myocardium. Ionic currents in isolated ventricular heart cells of the mouse were measured with a patch clamp technique under normoxic conditions (atmospheric pO2), during wash-out of oxygen, and under anoxic conditions (pO2 < 0.2 mmHg). During the measurement, the actual pO2 in the close proximity of the cell was determined with an optical technique by exciting Pd-meso-tetra(4-carboxyphenyl)porphin with light flashes of 508-570 nm and evaluating the quenching kinetics of the emitted phosphorescence signal at 630-700 nm. These quenching kinetics steeply depend on pO2 and can be evaluated best at pO2 values near 0 mmHg. Out of 28 cells, 23 cells started to develop IKATP at pO2 values between 0 and 0.4 mmHg, i.e. in the range of the level of half maximum activity of the cytochrome oxidase. The remaining five cells developed IKATP between 0.4 and 1.8 mmHg. With respect to the time course, 18 out of 27 cells started to develop IKATP within the first minute after pO2 had decreased to values below 0.2 mmHg. The amplitude of IKATP induced by anoxia and various metabolic inhibitors was large, 29 +/- 12 and 48 +/- 21 nA (+40 mV), respectively. The anoxia-induced IKATP was significantly smaller than IKATP induced by metabolic inhibitors. During the pulses of 50 ms duration to +40 mV, the amplitude of IKATP decayed and, after clamping back to -80 mV, IKATP generated large tail currents. This suggests a notable change in the concentration gradient of K+ ions in the time range of tens of milliseconds. The results in isolated myocytes indicate that KATP channels open sufficiently rapidly after starting anoxia and generate sufficiently large conductance at maintained anoxia to explain both the time course and magnitude of the ischemic K+ accumulation if an appropriate counter-ion flux is available.

Role of Cardiac KATP Channels During Anoxia and Ischemia

In isolated heart cells, maintained anoxia causes a transient opening of KATP channels. In the ischemic myocardium, this extra K+ conductance results in a decreased contractility and may be arrhythmogenic. Recent studies provide further evidence that the transient activity of KATP channels during anoxia is correlated with the time course of extracellular K+ accumulation during ischemia.

Confocal imaging of calcium in intact ventricular muscle provides a new view of excitation-contraction coupling

A fundamentally new understanding of cardiac excitation-contraction (E-C) coupling is being developed from recent experimental work using confocal microscopy of single isolated heart cells. In particular, the transient change in intracellular free calcium ion concentration ([Ca2+]i transient) that activates muscle contraction is now viewed as resulting from the spatial and temporal summation of small (∼ 8 μm3), subcellular, stereotyped ‘local [Ca2+]i-transients' or, as they have been called, ‘calcium sparks'. This new understanding may be called ‘local control of E-C coupling'. The relevance to normal heart cell function of ‘local control, theory and the recent confocal data on spontaneous Ca2+ ‘sparks', and on electrically evoked local [Ca2+]i-transients has been unknown however, because the previous studies were all conducted on slack, internally perfused, single, enzymatically dissociated cardiac cells, at room temperature, usually with Cs+ replacing K+, and often in the presence of Ca2-channel blockers. The present work was undertaken to establish whether or not the concepts derived from these studies are in fact relevant to normal cardiac tissue under physiological conditions, by attempting to record local [Ca2+]i-transients, sparks (and Ca2+ waves) in intact, multi-cellular cardiac tissue.

Micro-scale force-transducer system to quantify isolated heart cell contractile characteristics

Abstract A custom-designed microelectromechanical force transducer, with a volume of less than 1 mm3, is being developed to quantify forces generated in isolated cardiac muscle cells. A single heart cell will be attached to flexible, hinged polysilicon plates submerged in a nutrient solution. As the cell contracts, the plates will bend, and the contractile force can be measured based on the known spring constant of the plate and the amount of deflection. Prototype structures have been fabricated and have been mechanically tested. We have demonstrated that living rat heart cells can be attached to polysilicon using a commercial silicone sealant. We have also observed that polysilicon is an inert material when exposed to cardiac cells and their saline environment, and has no detectable effect on the cells themselves.

19F nuclear magnetic resonance studies of free calcium in heart cells

19F nuclear magnetic resonance is used in conjunction with 5,5'-difluoro-1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5FBapta), a fluorinated calcium chelator, to report steady-state intracellular free calcium levels ([Ca2+]i) in populations of resting, quiescent, isolated adult heart cells. 31P nuclear magnetic resonance shows that 5FBapta-loaded cells maintain normal intracellular high-energy phosphates, pH, and free Mg2+. The intracellular free calcium concentration of well perfused, isolated heart cells is 61 +/- 5 nM, measured with 5FBapta, which has a dissociation constant (Kd) for calcium chelation of 500 nM. A similar value is obtained with Quin-MF, another fluorinated calcium chelator with Kd and maximum calcium sensitivity at 80 nM. We find that the steady-state level of intracellular free calcium is increased by decreased extra-cellular sodium concentration, omission of extracellular magnesium, decreased extracellular pH, hyperglycemia, and upon treatment with lead acetate. Further, extracellular ATP caused a large transient increase in [Ca2+]i. Thus, while heart cells maintain a very low level of intracellular free Ca2+, acute alterations in extracellular environment can cause derangement of calcium homeostasis, resulting in measurable increases in [Ca2+]i.