Estimation of systolic and diastolic free intracellular Ca2+ by titration of Ca2+ buffering in the ferret heart.
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ABSTRACT:
SUBMITTER: Kirschenlohr HL
PROVIDER: S-EPMC1220864 | biostudies-other | 2000 Mar
REPOSITORIES: biostudies-other
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Project description:Imaging with Ca(2+)-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca(2+) signaling. The spatiotemporal evolution of intracellular free Ca(2+) observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca(2+) buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca(2+) imaging data, and can be exploited to investigate endogenous Ca(2+) buffer properties. Here we extended the use of Ca(2+) fluorometry in the characterization of Ca(2+) binding molecules within cells, building on a method of titration of intracellular Ca(2+) binding sites in situ with measured amounts of Ca(2+) entering through voltage-gated Ca(2+) channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca(2+) binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca(2+) indicator fluo-8. Focusing on data sets well described by a single endogenous Ca(2+) buffer and dye, this method yielded estimates of the endogenous buffer concentration and Kd, the dye Kd, and the fraction of Ca(2+) inaccessible cellular volume. The in situ Kd of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca(2+)-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca(2+) titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca(2+) binding species. This method offers a versatile approach to the study of endogenous Ca(2+) binding molecules in their physiological milieu.
| S-EPMC4314461 | biostudies-literature
Project description:For the heart to function as a pump, intracellular calcium concentration ([Ca2+ ]i ) must increase during systole to activate contraction and then fall, during diastole, to allow the myofilaments to relax and the heart to refill with blood. The present study investigates the control of diastolic [Ca2+ ]i in rat ventricular myocytes. We show that diastolic [Ca2+ ]i is increased by manoeuvres that decrease sarcoplasmic reticulum function. This is accompanied by a decrease of systolic [Ca2+ ]i such that the time-averaged [Ca2+ ]i remains constant. We report that diastolic [Ca2+ ]i is controlled by the balance between Ca2+ entry and Ca2+ efflux during systole. The results of the present study identify a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+ ]i .The intracellular Ca concentration ([Ca2+ ]i ) must be sufficently low in diastole so that the ventricle is relaxed and can refill with blood. Interference with this will impair relaxation. The factors responsible for regulation of diastolic [Ca2+ ]i , in particular the relative roles of the sarcoplasmic reticulum (SR) and surface membrane, are unclear. We investigated the effects on diastolic [Ca2+ ]i that result from the changes of Ca cycling known to occur in heart failure. Experiments were performed using Fluo-3 in voltage clamped rat ventricular myocytes. Increasing stimulation frequency increased diastolic [Ca2+ ]i . This increase of [Ca2+ ]i was larger when SR function was impaired either by making the ryanodine receptor leaky (with caffeine or ryanodine) or by decreasing sarco/endoplasmic reticulum Ca-ATPase activity with thapsigargin. The increase of diastolic [Ca2+ ]i produced by interfering with the SR was accompanied by a decrease of the amplitude of the systolic Ca transient, such that there was no change of time-averaged [Ca2+ ]i . Time-averaged [Ca2+ ]i was increased by β-adrenergic stimulation with isoprenaline and increased in a saturating manner with increased stimulation frequency; average [Ca2+ ]i was a linear function of Ca entry per unit time. Diastolic and time-averaged [Ca2+ ]i were decreased by decreasing the L-type Ca current (with 50 μm cadmium chloride). We conclude that diastolic [Ca2+ ]i is controlled by the balance between Ca entry and efflux during systole. Furthermore, manoeuvres that decrease the amplitude of the Ca transient (without decreasing Ca influx) will therefore increase diastolic [Ca2+ ]i . This identifies a novel mechanism by which changes of the amplitude of the systolic Ca transient control diastolic [Ca2+ ]i .
| S-EPMC5556151 | biostudies-other
Project description:RATIONALE:The development of a refractory period for Ca2+ spark initiation after Ca2+ release in cardiac myocytes should inhibit further Ca2+ release during the action potential plateau. However, Ca2+ release sites that did not initially activate or which have prematurely recovered from refractoriness might release Ca2+ later during the action potential and alter the cell-wide Ca2+ transient. OBJECTIVE:To investigate the possibility of late Ca2+ spark (LCS) activity in intact isolated cardiac myocytes using fast confocal line scanning with improved confocality and signal to noise. METHODS AND RESULTS:We recorded Ca2+ transients from cardiac ventricular myocytes isolated from rabbit hearts. Action potentials were produced by electric stimulation, and rapid solution changes were used to modify the L-type Ca2+ current. After the upstroke of the Ca2+ transient, LCSs were detected which had increased amplitude compared with diastolic Ca2+ sparks. LCS are triggered by both L-type Ca2+ channel activity during the action potential plateau, as well as by the increase of cytosolic Ca2+ associated with the Ca2+ transient itself. Importantly, a mismatch between sarcoplasmic reticulum load and L-type Ca2+ trigger can increase the number of LCS. The likelihood of triggering an LCS also depends on recovery from refractoriness that appears after prior activation. Consequences of LCS include a reduced rate of decline of the Ca2+ transient and, if frequent, formation of microscopic propagating Ca2+ release events (Ca2+ ripples). Ca2+ ripples resemble Ca2+ waves in terms of local propagation velocity but spread for only a short distance because of limited regeneration. CONCLUSIONS:These new types of Ca2+ signaling behavior extend our understanding of Ca2+-mediated signaling. LCS may provide an arrhythmogenic substrate by slowing the Ca2+ transient decline, as well as by amplifying maintained Ca2+ current effects on intracellular Ca2+ and consequently Na+/Ca2+ exchange current.
| S-EPMC5796647 | biostudies-literature
Project description:Fast synchronous neurotransmitter release at the presynaptic active zone is triggered by local Ca(2+) signals, which are confined in their spatiotemporal extent by endogenous Ca(2+) buffers. However, it remains elusive how rapid and reliable Ca(2+) signaling can be sustained during repetitive release. Here, we established quantitative two-photon Ca(2+) imaging in cerebellar mossy fiber boutons, which fire at exceptionally high rates. We show that endogenous fixed buffers have a surprisingly low Ca(2+)-binding ratio (? 15) and low affinity, whereas mobile buffers have high affinity. Experimentally constrained modeling revealed that the low endogenous buffering promotes fast clearance of Ca(2+) from the active zone during repetitive firing. Measuring Ca(2+) signals at different distances from active zones with ultra-high-resolution confirmed our model predictions. Our results lead to the concept that reduced Ca(2+) buffering enables fast active zone Ca(2+) signaling, suggesting that the strength of endogenous Ca(2+) buffering limits the rate of synchronous synaptic transmission.
| S-EPMC4466756 | biostudies-literature
Project description:The measurement of cardiac Ca(2+) transients using spectroscopic Ca(2+) indicators is significantly affected by the buffering properties of the indicators. The aim of the present study was to construct a model of cardiac Ca(2+) buffering that satisfied the kinetic constraints imposed by the maximum attainable rates of cardiac contraction and relaxation on the Ca(2+) dissociation rate constants and which would account for the observed effects of (19)F-NMR indicators on the cardiac Ca(2+) transient in the Langendorff-perfused ferret heart. It is generally assumed that the Ca(2+) dependency of myofibril activation in cardiac myocytes is mediated by a single Ca(2+)-binding site on troponin C. A model based on 1:1 Ca(2+) binding to the myofilaments, however, was unable to reproduce our experimental data, but a model in which we assumed ATP-dependent co-operative Ca(2+) binding to the myofilaments was able to reproduce these data. This model was used to calculate the concentration and dissociation constant of the ATP-independent myofilament Ca(2+) binding, giving 58 and 2.0 microM respectively. In addition to reproducing our experimental data on the concentration of free Ca(2+) ions in the cytoplasm ([Ca(2+)](i)), the resulting Ca(2+) and ATP affinities given by fitting of the model also provided good predictions of the Ca(2+) dependence of the myofibrillar ATPase activity measured under in vitro conditions. Solutions to the model also indicate that the Ca(2+) mobilized during each beat remains unchanged in the presence of the additional buffering load from Ca(2+) indicators. The new model was used to estimate the extent of perturbation of the Ca(2+) transient caused by different concentrations of indicators. As little as 10 microM of a Ca(2+) indicator with a dissociation constant of 200 nM will cause a 20% reduction in peak-systolic [Ca(2+)](i) and 30 microM will cause approx. 50% reduction in the peak-systolic [Ca(2+)](i) in a heart paced at 1.0 Hz.
| S-EPMC1220865 | biostudies-other
Project description:We elucidate the role of late Na+ current (INaL) for diastolic intracellular Ca2+ (DCa) accumulation in chronic heart failure (HF). HF was induced in 19 dogs by multiple coronary artery microembolizations; 6 normal dogs served as control. Ca2+ transients were recorded in field-paced (0.25 or 1.5 Hz) fluo-4-loaded ventricular myocytes (VM). INaL and action potentials were recorded by patch-clamp. Failing VM, but not normal VM, exhibited (1) prolonged action potentials and Ca2+ transients at 0.25 Hz, (2) substantial DCa accumulation at 1.5 Hz, and (3) spontaneous Ca2+ releases, which occurred after 1.5 Hz stimulation trains in ~31% cases. Selective INaL blocker ranolazine (10 microM) or the prototypical Na+ channel blocker tetrodotoxin (2 microM) reversibly improved function of failing VM. The DCa accumulation and the beneficial effect of INaL blockade were reproduced in silico using an excitation-contraction coupling model. We conclude that INaL contributes to diastolic Ca2+ accumulation and spontaneous Ca2+ release in HF.
| S-EPMC2891122 | biostudies-literature
Project description:Ischemic heart disease (IHD) is the leading cause of death and chronic disability in the world. IHD affects both the systolic and diastolic function of the heart which progressively leads to heart failure; a structural and functional impairment of filling or ejection of blood from the heart. In this study, the progression of systolic and diastolic dysfunction characterized according to their echocardiographic parameters including left ventricular ejection fraction (EF), grades of diastolic dysfunction and ratio between early mitral inflow velocity and mitral annular early diastolic velocity (E/e'), were correlated with differential regulation of various metals in patients sera samples (n?=?62) using inductive coupled plasma-mass spectrometry (ICP-MS). Chromium, nickel and selenium were found significant (p?<?0.05) in patients having EF?<?45% compared with EF?>?45%. In patients with systolic dysfunction (EF?<?45%), the level of selenium was decreased while the level of chromium and nickel was increased compared to patients with EF?>?45%. Selenium level was also decreased significantly (p?<?0.05) in grade 1A and 2 patients that are considered as higher grades of diastole dysfunction in comparison to grade 0-1. Overall, selenium deficiency was identified in both systolic and diastolic dysfunctions of IHD patients corresponding to the progression of disease that could be related to many metabolic and translational pathways specifically which involve selenoproteins.
| S-EPMC7434915 | biostudies-literature
Project description:AimsCardiac miR-132 activation leads to adverse remodelling and pathological hypertrophy. CDR132L is a synthetic lead-optimized oligonucleotide inhibitor with proven preclinical efficacy and safety in heart failure (HF) early after myocardial infarction (MI), and recently completed clinical evaluation in a Phase 1b study (NCT04045405). The aim of the current study was to assess safety and efficacy of CDR132L in a clinically relevant large animal (pig) model of chronic heart failure following MI.Methods and resultsIn a chronic model of post-MI HF, slow-growing pigs underwent 90 min left anterior descending artery occlusion followed by reperfusion. Animals were randomized and treatment started 1-month post-MI. Monthly intravenous (IV) treatments of CDR132L over 3 or 5 months (3× or 5×) were applied in a blinded randomized placebo-controlled fashion. Efficacy was evaluated based on serial magnetic resonance imaging, haemodynamic, and biomarker analyses. The treatment regime provided sufficient tissue exposure and CDR132L was well tolerated. Overall, CDR132L treatment significantly improved cardiac function and reversed cardiac remodelling. In addition to the systolic recovery, diastolic function was also ameliorated in this chronic model of HF.ConclusionMonthly repeated dosing of CDR132L is safe and adequate to provide clinically relevant exposure and therapeutic efficacy in a model of chronic post-MI HF. CDR132L thus should be explored as treatment for the broad area of chronic heart failure.
| S-EPMC7813625 | biostudies-literature
Project description:AimsCardiac energy metabolism is centrally involved in heart failure (HF), although the direction of the metabolic alterations is complex and likely dependent on the particular stage of HF progression. Vascular endothelial growth factor B (VEGF-B) has been shown to modulate metabolic processes and to induce physiological cardiac hypertrophy; thus, it could be cardioprotective in the failing myocardium. This study investigates the role of VEGF-B in cardiac proteomic and metabolic adaptation in HF during aldosterone and high-salt hypertensive challenges.Methods and resultsMale rats overexpressing the cardiac-specific VEGF-B transgene (VEGF-B TG) were treated for 3 or 6 weeks with deoxycorticosterone-acetate combined with a high-salt (HS) diet (DOCA + HS) to induce hypertension and cardiac damage. Extensive longitudinal echocardiographic studies of HF progression were conducted, starting at baseline. Sham-treated rats served as controls. To evaluate the metabolic alterations associated with HF, cardiac proteomics by mass spectrometry was performed. Hypertrophic non-treated VEGF-B TG hearts demonstrated high oxygen and adenosine triphosphate (ATP) demand with early onset of diastolic dysfunction. Administration of DOCA + HS to VEGF-B TG rats for 6 weeks amplified the progression from cardiac hypertrophy to HF, with a drastic drop in heart ATP concentration. Dobutamine stress echocardiographic analyses uncovered a significantly impaired systolic reserve. Mechanistically, the hallmark of the failing TG heart was an abnormal energy metabolism with decreased mitochondrial ATP, preceding the attenuated cardiac performance and leading to systolic HF.ConclusionsThis study shows that the VEGF-B TG accelerates metabolic maladaptation which precedes structural cardiomyopathy in experimental hypertension and ultimately leads to systolic HF.
| S-EPMC10318391 | biostudies-literature
Project description:BackgroundChronic heart failure (CHF) is a complex syndrome characterized by a progressive reduction of the left ventricular (LV) contractility, low exercise tolerance, and increased mortality and morbidity. Diastolic dysfunction (DD) of the LV, is a keystone in the pathophysiology of CHF and plays a major role in the progression of most cardiac diseases. Also, it is well estimated that exercise training induces several beneficial effects on patients with CHF.AimTo evaluate the impact of a cardiac rehabilitation program on the DD and LV ejection fraction (EF) in patients with CHF.MethodsThirty-two stable patients with CHF (age: 56 ± 10 years, EF: 32% ± 8%, 88% men) participated in an exercise rehabilitation program. They were randomly assigned to aerobic exercise (AER) or combined aerobic and strength training (COM), based on age and peak oxygen uptake, as stratified randomization criteria. Before and after the program, they underwent a symptom-limited maximal cardiopulmonary exercise testing (CPET) and serial echocardiography evaluation to evaluate peak oxygen uptake (VO2peak), peak workload (Wpeak), DD grade, right ventricular systolic pressure (RVSP), and EF.ResultsThe whole cohort improved VO2peak, and Wpeak, as well as DD grade (P < 0.05). Overall, 9 patients (28.1%) improved DD grade, while 23 (71.9%) remained at the same DD grade; this was a significant difference, considering DD grade at baseline (P < 0.05). In addition, the whole cohort improved RVSP and EF (P < 0.05). Not any between-group differences were observed in the variables assessed (P > 0.05).ConclusionExercise rehabilitation improves indices of diastolic and systolic dysfunction. Exercise protocol was not observed to affect outcomes. These results need to be further investigated in larger samples.
| S-EPMC8462045 | biostudies-literature