Indo-1 Fluorescence Research Articles

Abstract 1451: Hyperpolarization Induces Differentiation Of Human Cardiomyocyte Progenitor Cells

Background: Recently, we have isolated cardiomyocyte progenitor cells (hCMPCs) from human fetal and adult hearts. These cells differentiate into spontaneously beating cardiomyocytes when stimulated with 5-azacytidine. Subsequent stimulation by TGFβ enhances differentiation efficiency to nearly 100%. The underlying molecular mechanisms mediating this cardiomyogenic differentiation are not understood. In skeletal myoblasts, hyperpolarization-mediated activation of calcineurin signaling is crucial for myogenic differentiation. In hCMPCs, whole-cell patch clamp recordings showed a hyperpolarized membrane potential after stimulation with TGFβ or BMP. We hypothesized that hyperpolarization and calcineurin signaling regulate cardiomyogenic differentiation of hCMPCs after TGFβ stimulation. Methods & Results: To test whether hyperpolarization initiates cardiomyogenic differentiation, hyperpolarization was induced by 1) co-culture of hCMPCs with HEK 293 cells overexpressing a Kir2.1GFP fusion protein (KWGF cells) or 2) culture of hCMPCs overnight in medium containing low potassium concentrations. During co-culture, Lucifer Yellow dye injection in KWGF cells spread to neighboring hCMPCs, indicating cellular coupling. This resulted in stable hyperpolarization in hCMPCs, which could be blocked by addition of the gap junction inhibitor halothane. After two weeks, qPCR analysis revealed increased expression of cardiac sarcomeric genes in the hCMPCs in a dose-dependent manner. Induction of hyperpolarization by culturing hCMPCs with low potassium concentrations also resulted in increased expression of cardiac genes and the formation of spontaneously beating cells. Immunofluorescence staining revealed striated patterns of troponin I and α-actinin. Interestingly, hyperpolarization also increased intracellular calcium levels in hCMPCs, as measured by ratiometric imaging of indo-1 fluorescence, and, subsequently, a time-dependent increase in NFAT-Luciferase reporter activity, indicating activation of the calcineurin pathway. Conclusion: TGFβ and/or BMP-mediated hyperpolarization of hCMPCs induces calcineurin-mediated cardiomyogenic differentiation.

H 2O 2-induced left ventricular dysfunction in isolated working rat hearts is independent of calcium accumulation

Reactive oxygen species (ROS) and intracellular Ca 2+ overload play key roles in myocardial ischemia–reperfusion (IR) injury but the relationships among ROS, Ca 2+ overload and LV mechanical dysfunction remain unclear. We tested the hypothesis that H 2O 2 impairs LV function by causing Ca 2+ overload by increasing late sodium current (I Na), similar to Sea Anemone Toxin II (ATX-II). Diastolic and systolic Ca 2+ concentrations (d[Ca 2+] i and s[Ca 2+] i) were measured by indo-1 fluorescence simultaneously with LV work in isolated working rat hearts. H 2O 2 (100 μM, 30 min) increased d[Ca 2+] i and s[Ca 2+] i. LV work increased transiently then declined to 32% of baseline before recovering to 70%. ATX-II (12 nM, 30 min) caused greater increases in d[Ca 2+] i and s[Ca 2+] i. LV work increased transiently before declining gradually to 17%. Ouabain (80 μM) exerted similar effects to ATX-II. Late I Na inhibitors, lidocaine (10 μM) or R56865 (2 μM), reduced effects of ATX-II on [Ca 2+] i and LV function, but did not alter effects of H 2O 2. The antioxidant, N-(2-mercaptopropionyl)glycine (MPG, 1 mM) prevented H 2O 2-induced LV dysfunction, but did not alter [Ca 2+] i. Paradoxically, further increases in [Ca 2+] i by ATX-II or ouabain, given 10 min after H 2O 2, improved function. The failure of late I Na inhibitors to prevent H 2O 2-induced LV dysfunction, and the ability of MPG to prevent H 2O 2-induced LV dysfunction independent of changes in [Ca 2+] i indicate that impaired contractility is not due to Ca 2+ overload. The ability of further increases in [Ca 2+] i to reverse H 2O 2-induced LV dysfunction suggests that Ca 2+ desensitization is the predominant mechanism of ROS-induced contractile dysfunction.

Acute Administration of Fish Oil Inhibits Triggered Activity in Isolated Myocytes From Rabbits and Patients With Heart Failure

Fish oil reduces sudden death in patients with prior myocardial infarction. Sudden death in heart failure may be due to triggered activity based on disturbed calcium handling. We hypothesized that superfusion with omega3-polyunsaturated fatty acids (omega3-PUFAs) from fish inhibits triggered activity in heart failure. Ventricular myocytes were isolated from explanted hearts of rabbits with volume- and pressure-overload-induced heart failure and of patients with end-stage heart failure. Membrane potentials (patch-clamp technique) and intracellular calcium (indo-1 fluorescence) were recorded after 5 minutes of superfusion with Tyrode's solution (control), omega-9 monounsaturated fatty acid oleic acid (20 micromol/L), or omega3-PUFAs (docosahexaenoic acid or eicosapentaenoic acid 20 micromol/L). omega3-PUFAs shortened the action potential at low stimulation frequencies and caused an approximately 25% decrease in diastolic and systolic calcium (all P<0.05). Subsequently, noradrenalin and rapid pacing were used to evoke triggered activity, delayed afterdepolarizations, and calcium aftertransients. omega3-PUFAs abolished triggered activity and reduced the number of delayed afterdepolarizations and calcium aftertransients compared with control and oleic acid. Omega3-PUFAs reduced action potential shortening and intracellular calcium elevation in response to noradrenalin. Results from human myocytes were in accordance with the findings obtained in rabbit myocytes. Superfusion with omega3-PUFAs from fish inhibits triggered arrhythmias in myocytes from rabbits and patients with heart failure by lowering intracellular calcium and reducing the response to noradrenalin.

Interaction between bovine serum albumin and Indo-1 using fluorescence spectroscopic method

This work attempts to calculate the binding-site number using fluorescence spectroscopic method with bovine serum albumin (BSA) and Indo-1 as protein and ligand models, respectively. The method for calculating the binding-site number in BSA for Indo-1 was developed based on the relationships between changes in Indo-1 fluorescence intensity and the analytical concentration of BSA. The interaction between BSA with Indo-1 was investigated comprehensively using fluorescence techniques as well as fluorescence resonance energy transfer, and the thermodynamic parameters were calculated according to the effect of enthalpy on temperature. Three binding sites in BSA for Indo-1 were revealed, and the distances from Trp212 in BSA to the three binding sites were 2.93, 2.57 and 2.40 nm, respectively. It was also proven that Indo-1 embedded into the three hydrophobic cavities of BSA by hydrophobic association. This paper provides a reference on calculating the binding-site number in proteins for ligands and studying their interactions by fluorescence spectroscopic methods. In fluorescent quenching experiments, fluorescence changes were automatically recorded in real time by combining the Microlab 500 Series Dispenser and PTI fluorescence apparatus.

Altered Ca2+handling and myofilament desensitization underlie cardiomyocyte performance in normothermic and hyperthermic heat-acclimated rat hearts

Heat acclimation (AC) improves cardiac mechanical and metabolic performance. Using cardiomyocytes and isolated hearts from 30-day and 2-day acclimated rats (AC and AC-2d, 34 degrees C), we characterized cellular contractile mechanisms under normothermic (37 degrees C) and hyperthermic (39-42 degrees C) conditions. To determine contractile responses, Ca2+ transients (Ca2+ T), sarcoplasmic reticulum (SR) Ca2+ pool size (fura-2/indo-1 fluorescence), force generation [amplitude systolic motion (ASM)], L-type Ca2+ channels [dihydropyridine receptor (DHPR)], ryanodine receptors (RyRs), and total (PLBt) and phosphorylated phospholamban [serine phosphorylated (PLBs) and theonine phosphorylated (PLBtr)] proteins and transcripts were measured (Western blot, RT-PCR). Cardiac mechanical performance was measured using a Langendorff system. We demonstrated that AC and AC-2d increased Ca2+ T amplitude (148% and 147%, respectively) and twitch force (180% and 130%, respectively) and desensitized myofilaments, as indicated by a rightward shift in the ASM-Ca2+ relationships, despite no change in SR Ca2+ pool size. Hence, generation of higher Ca2+ T underlies greater force development in AC and AC-2d myocytes. In isolated hearts, ryanodine administration eliminated differences between AC and control (C) hearts, implying an important role for RyRs in that acclimation phase. Increased expression of DHPR and RyRs, and decreased PLBs/PLBt in AC hearts only, suggest that different pathways increase force generation in the AC-2d vs. AC myocytes. At basal beating rates, hyperthermia (39-41 degrees C) enhanced pressure generation in AC hearts. C hearts failed to restitute pressure beyond 39 degrees C. Increased beating frequency produced negative inotropic response. In C cardiomyocytes, hyperthermia elevated basal cytosolic Ca2+ and tension, Ca2+ T, and ASM. AC myocytes enhanced Ca2+ T but showed myofilament desensitization, suggesting its involvement in cardiac protection against hyperthermia. Collectively, both Ca2+ turnover and myofilament responsiveness are important adaptive acclimatory targets during normothermic and hyperthermic conditions.

Activation of α1B-adrenoceptors alleviates ischemia/reperfusion injury by limitation of mitochondrial Ca2+ overload in cardiomyocytes

Activation of alpha(1)-adrenergic receptors (alpha(1)-ARs) mimics ischemic preconditioning (IP). However, the subtypes of alpha(1)-ARs involved and the protective mechanisms are not entirely clear. Here we tested the hypothesis that preservation of mitochondrial integrity, in particular, Ca(2+) homeostasis via the epsilon isoform of protein kinase C (PKCepsilon) and mitoK(ATP) channels, may underlie the basis of alpha(1B)-AR-triggered cardioprotection. Indo-1 fluorescence in adult rat cardiomyocytes was used as an index of cytosolic ([Ca(2+)](c)) or mitochondrial free Ca(2+) concentration ([Ca(2+)](m)), and cell shortening was measured simultaneously. Cells were subjected to 20 min of simulated ischemia followed by 30 min of reperfusion (I/R). Activation of a(1)-ARs by phenylephrine significantly decreased I/R-induced [Ca(2+)](c) and [Ca(2+)](m) overload, mitochondrial cytochrome c release and ATP reduction, and improved Ca(2+) transients and cell shortening. These protective effects were markedly inhibited by blockade of alpha(1B)-AR (chloroethylclonidine) but not alpha(1A)-AR (5'-methylurapidil) or alpha(1D)-AR (BMY 7378). Moreover, phenylephrine-afforded protection on the [Ca(2+)](m), [Ca(2+)](c), and cell shortening was lost when mitoK(ATP) channels were inhibited with 5-hydroxydecanoate and PKCepsilon with PKCepsilon V(1-2). However, PKCepsilon V(1-2) did not affect the mitoK(ATP) channel opener diazoxide-induced protection on these parameters. These findings indicate that phenylephrine-induced protection on [Ca(2+)](m) homeostasis is mediated by selective activation of alpha(1B)-AR via mitoK(ATP) channel opening and PKCepsilon activation. Mitochondrial function appears to be a determinant of [Ca(2+)](c) and contractile function during I/R injury.

Improved mitochondrial Ca 2+ handling and functional recovery after ischemia reperfusion injury in hearts from old vs. young guinea pigs

We hypothesized that aging-induced oxidative damage to mitochondria would result in mitochondrial [Ca2+] (m[Ca2+]) overload, depressed contractile function, and decreased NADH after ischemia reperfusion (IR) injury in old vs. young guinea pig hearts. Unpaced hearts (n=7 each) from young (< 2 mo) and old (> 24 mo) animals were perfused with Krebs-Ringer’s solution (2.1 mM [Ca2+]) at 37°C. Left ventricular pressure (LVP, mmHg) was recorded with a saline filled latex balloon and transducer. Simultaneously, m[Ca2+] (nM) was measured ratiometrically by indo-1 fluorescence with a fiber optic probe on the LV free wall. Hearts were subjected to 30 min global ischemia and 60 min reperfusion (RP). All data ± SEM, p<0.05 vs. old. We found: No difference at baseline in developed LVP (systolic-diastolic), diastolic LVP, or m[Ca2+] in old vs. young hearts. Developed LVP was higher at RP10 and RP60 in old (57±11 and 55±10) vs. young (29±2 and 32±2) hearts. Diastolic LVP was lower at RP10 and RP60 in old (9±2 and 7±2) vs. young (20±2 and 14±2) hearts. m[Ca2+] was lower at RP10 and RP 60 in old (129±2 and 122±2) vs. young (293±23 and 234±15) hearts. Contrary to our initial hypothesis, old hearts showed reduced m[Ca2+] loading and improved mechanical function after IR injury compared to young hearts. There were no differences in redox state due to age. Aging associated protection may be linked to better collateral vessel formation, better preservation of contractile apparatus, or preconditioning mechanisms from prior stress. (AHA, NIH)

Sarcoplasmic ATP-sensitive potassium channel blocker HMR1098 protects the ischemic heart: Implication of calcium, complex I, reactive oxygen species and mitochondrial ATP-sensitive potassium channel

The aim of this study was to investigate the effects of HMR1098, a selective blocker of sarcolemmal ATP-sensitive potassium channel (sarcK ATP), in Langendorff-perfused rat hearts submitted to ischemia and reperfusion. The recovery of heart hemodynamic and mitochondrial function, studied on skinned fibers, was analyzed after 30-min global ischemia followed by 20-min reperfusion. Infarct size was quantified on a regional ischemia model after 2-h reperfusion. We report that the perfusion of 10 μM HMR1098 before ischemia, delays the onset of ischemic contracture, improves recovery of cardiac function upon reperfusion, preserves the mitochondrial architecture, and finally decreases infarct size. This HMR1098-induced cardioprotection is prevented by 1 mM 2-mercaptopropionylglycine, an antioxidant, and by 100 nM nifedipine, an L-type calcium channel blocker. Concomitantly, it is shown that HMR1098 perfusion induces (i) a transient and specific inhibition of the respiratory chain complex I and, (ii) an increase in the averaged intracellular calcium concentration probed by the in situ measurement of indo-1 fluorescence. Finally, all the beneficial effects of HMR1098 were strongly inhibited by 5-hydroxydecanoate and abolished by glibenclamide, two mitoK ATP blockers. This study demonstrates that the HMR1098-induced cardioprotection occurs indirectly through extracellular calcium influx, respiratory chain complex inhibition, reactive oxygen species production and mitoK ATP opening. Taken together, these data suggest that a functional interaction between sarcK ATP and mitoK ATP exists in isolated rat heart ischemia model, which is mediated by extracellular calcium influx.

Abstract 580: Skeletal Myoblasts and Bone Marrow-Derived Cells Enhance the Contractile Function of Isolated Failing Cardiomyocytes in Co-culture

Cell transplantation has been shown to enhance ventricular function in heart failure. However, the low numbers of surviving transplanted cells can not explain the functional myocardial improvement directly, and the cellular mechanisms responsible remain unknown. We hypothesized that transplanted cells influence the native myocardium by paracrine mechanisms, and investigated the in vitro effects of bone marrow mononuclear cells or skeletal myoblasts on neighboring cardiomyocytes isolated from a rat model of heart failure. Myocardial infarction was induced in adult female Sprague-Dawley rats by left coronary artery ligation. Left ventricular ejection fraction after 3 weeks was 35.5 ± 2.0%. Isolated left ventricular myocytes were cultured (density 5.2 cells/mm 2 ) on their own (Control) or with either bone marrow mononuclear cells (BM, density 52 cells/mm 2 ) or skeletal myoblasts (SK, density 5.2 cells/mm 2 ). After 48 hours of culture with SK or BM cardiomyocyte relaxation speed was increased (time to 50% relaxation, in ms - Control 34 ± 6 [ 28 ]; BM 18 ± 3 [ 16 ] p &lt; 0.05 vs Control; SK 13 ± 2 [ 20 ] p &lt; 0.05 vs Control). The decay time course of the Ca 2+ transient, assessed by indo-1 fluorescence, was also hastened by SK (time to 50% decay, in ms – HF 22 ± 2 [ 29 ]; BM 18 ± 2 [ 17 ] ns ; SK 13 ± 1 [ 17 ] p &lt; 0.05 vs Control). SK also increased the contraction amplitude compared to Control (sarcomere shortening, in μm - HF 0.046 ± 0.006 [ 28 ]; BM 0.055 ± 0.01 [ 16 ] ns ; SK 0.091 ± 0.014 [ 20 ] p &lt; 0.05 vs Control). We conclude that bone marrow mononuclear cells and skeletal myoblasts enhance cardiomyocyte function in co-culture, demonstrating that paracrine mechanisms are involved. This may account for the functional improvement seen after cell transplantation.

Ca2+Flux and Signaling Implications by Nicotinic Acetylcholine Receptors in Rat Medial Habenula

The fraction of inward current carried by Ca(2+) (FCa(2+)) through nicotinic acetylcholine receptors (nAChRs) on acutely isolated rat medial habenula (MHb) neurons was calculated from experiments that simultaneously monitored agonist-induced membrane currents and intracellular [Ca(2+)], measured with patch-clamp and indo-1 fluorescence, respectively. In physiological concentrations of extracellular Ca(2+) (2 mM) at -50 mV, the percentage of current carried by Ca(2+) was determined to be roughly 3-4%, which is in close agreement with measurements from other heteromeric nicotinic receptors expressed in peripheral tissue. Among factors that may have affected this measurement, such as Ca(2+) influx through voltage-gated Ca(2+) channels, the concentration of intracellular Ca(2+) buffer, and Ca(2+) sequestration and release from intracellular stores, only Ca(2+) uptake by mitochondria was shown to confound the analysis. Furthermore, we find that because of the high density of nAChRs on MHb cells, low concentrations of ACh (10 microM) and its hydrolysis product, choline (1 mM), can significantly elevate intracellular Ca(2+). Moreover, during persistent activation of nAChRs, the level of intracellular Ca(2+) is proportional to its extracellular concentration in the physiological range. Together, these findings support the suggestion that nAChRs may be capable of sensing low concentrations of diffusely released neurotransmitter and, in addition, transfer information about ongoing local synaptic activity by changes in extracellular Ca(2+).

C-reactive protein mediates CD11b expression in monocytes through the non-receptor tyrosine kinase, Syk, and calcium mobilization but not through cytosolic peroxides

C-Reactive protein (CRP) can modulate integrin surface expression on monocytes following Fcgamma receptor engagement. We have investigated the signal transduction events causing this phenotypic alteration. CRP-induced signalling events were examined in THP-1 and primary monocytes, measuring Syk phosphorylation by Western blotting, intracellular Ca(2+) ([Ca(2+)](i)) by Indo-1 fluorescence and surface expression of CD11b by flow cytometry. Cytosolic peroxides were determined by DCF fluorescence. CRP induced phosphorylation of Syk and an increase in [Ca(2+)](i) both of which were inhibitable by the Syk specific antagonist, piceatannol. Piceatannol also inhibited the CRP-induced increase in surface CD11b. In addition, pre-treatment of primary monoytes with the Ca(2+) mobiliser, thapsigargin, increased CD11b expression; this effect was accentuated in the presence of CRP but was abolished in the presence of the [Ca(2+)](i) chelator, BAPTA. CRP also increased cytosolic peroxide levels; this effect was attenuated by antioxidants (ascorbate, alpha-tocopherol), expression of surface CD11b not being inhibited by antioxidants alone. CRP induces CD11b expression in monocytes through a peroxide independent pathway involving both Syk phosphorylation and [Ca(2+)](i) release.

Lymphoid signal transduction mechanisms linked to cellular prion protein

The normal cellular isoform of the prion protein (PrPC) is a glycosylphosphatidylinositol-anchored cell surface protein that is expressed widely, including in lymphoid cells. We compared lectin-induced mitogenesis and selected cell signaling pathways in splenocytes from wild-type BALB/c mice and Zrch Prnp0/0 (PrP0/0) mice bred on a BALB/c background for more than 10 generations. 3H-thymidine incorporation induced by concanavalin A (Con A) or phytohemagglutinin (PHA) was significantly reduced in PrP0/0 splenocytes, most prominently early in activation (24 and 48 h). Con A activation in PrP0/0 splenocytes was associated with differences in the phosphorylation (P) patterns of protein kinase C (PKC alpha/beta, but not delta) and the PKC downstream effectors p44/42MAPK (mitogen-activated protein kinase). P-PKC and P-MAPK profiles were similar in wild-type and PrP0/0 splenocytes following PMA treatment, indicating that the ability of these 2 enzymes to be phosphorylated is not impaired in the absence of PrPC. Con A-induced calcium fluxes, monitored by indo-1 fluorescence, were equivalent in PrP0/0 and PrP+/+ splenocytes, suggesting that calcium-dependent mechanisms are not directly implicated in the differential phosphorylation patterns or mitotic responses. Our data indicate that PrP0/0 splenocytes display defects in upstream or downstream mechanism(s) that modulate PKCalpha/beta phosphorylation, which in turn affects its capacity to regulate splenocyte mitosis, consistent with a role for PrPC in immune function.

Effects of endotoxic shock on neuronal NOS and calcium transients in rat cardiac myocytes

Objective: The effects of endotoxic shock on transcriptional and translational pattern of nitric oxide synthase isoforms (NOSs) and cytoplasmic calcium were investigated. Methods: Male SD rats injected with lipopolysaccharides or saline were sacrificed after 6 and 20 h. Cardiac myocytes were enzimatically isolated from the excised hearts and evaluated for: (1) expression of constitutive (e and n) and inducible (i) NOSs by RT-PCR; (2) NOSs protein levels by Western blot, enzymatic activities by a radioimmunometric assay and nitric oxide metabolites by spectrophotometry; (3) calcium transients by Indo-1 fluorescence. Results: Increase in iNOS mRNA, and decrease in e and nNOS mRNAs were observed in cardiac myocytes isolated 6 h after LPS injection with recovery to basal levels at 20 h. Significant down-regulation of e and nNOS protein levels ( p < 0.01) and calcium-dependent activity ( p < 0.05) were detected at 20 h. Serum TNF-α increased after 6 and 20 h ( p < 0.05), whereas NO metabolites rose only after 20 h ( p < 0.0001). The diastolic calcium increased 6 h from LPS injection ( p < 0.0001) and remained significantly higher after 20 h. Calcium transients amplitude was not affected by LPS injection. Conclusions: Endotoxic shock stimulates iNOS and down-regulates expression of nNOS in purified cardiac myocytes, but endogenous NO production does not likely affect calcium transients.

A Dynamic Pool of Calcium in Catecholamine Storage Vesicles: EXPLORATION IN LIVING CELLS BY A NOVEL VESICLE-TARGETED CHROMOGRANIN A-AEQUORIN CHIMERIC PHOTOPROTEIN

Chromaffin vesicles contain very high concentration of Ca2+ (approximately 20-40 mM total), compared with approximately 100 nM in the cytosol. Aequorin, a jellyfish photoprotein with Ca(2+)-dependent luminescence, measures [Ca2+] in specific subcellular compartments wherein proteins with organelle-specific trafficking domains are fused in-frame to aequorin. Because of the presence of vesicular trafficking domain within CgA we engineered sorting of an expressed human CgA-Aequorin fusion protein (hCgA-Aeq) into the vesicle compartment as confirmed by sucrose density gradients and confocal immunofluorescent co-localization studies. hCgA-Aeq and cytoplasmic aequorin (Cyto-Aeq) luminescence displayed linear functions of [Ca2+] in vitro, over >5 log10 orders of magnitude (r > 0.99), and down to at least 10(-7) M sensitivity. Calibrating the pH dependence of hCgA-Aeq luminescence allowed estimation of [Ca2+]ves at granule interior pH (approximately 5.5). In the cytoplasm, Cyto-Aeq accurately determined [Ca2+]cyto under both basal ([Ca2+]cyto = 130 +/- 35 nM) and exocytosis-stimulated conditions, confirmed by an independent reference technique (Indo-1 fluorescence). The hCgA-Aeq chimera determined vesicular free [Ca2+]ves = 1.4 +/- 0.3 microM under basal conditions indicating that >99% of granule total Ca2+ is in a "bound" state. The basal free [Ca2+]ves/[Ca2+]cyto ratio was thus approximately 10.8-fold, indicating active, dynamic Ca2+ uptake from cytosol into the granules. Stimulation of exocytotic secretion revealed prompt, dynamic increases in both [Ca2+](ves) and [Ca2+]cyto, and an exponential relation between the two (y = 0.99 x e(1.53x), r = 0.99), reflecting a persistent [Ca2+]ves/[Ca2+]cyto gradient, even during sharp increments of both values. Studies with inhibitors of Ca2+ translocation (Ca(2+)-ATPase), Na+/Ca(+)-exchange, Na+/H(+)-exchange, and vesicle acidification (H(+)-translocating ATPase), documented a role for these four ion transporter classes in accumulation of Ca2+ inside the vesicles.

Ratiometric intracellular calcium imaging in the isolated beating rat heart using indo-1 fluorescence.

Abnormalities in intracellular calcium (Ca(i)(2+)) handling have been implicated as the underlying mechanism in a large number of pathologies in the heart. Study into the relation between Ca(i)(2+) behavior and performance of the whole heart function could provide detailed information into the cellular basis of heart function. In this study we describe an optical ratio imaging setup and an analysis method for the beat-to-beat Ca(i)(2+) videofluorescence images of an indo-1 loaded, isolated Tyrode-perfused beating rat heart. The signal-to-noise ratio and the spatiotemporal resolution (with an optimum of 1 ms and 0.6 mm, respectively) made it possible to register different temporal Ca(i)(2+) transients together with left ventricle pressure changes. The Ca(i)(2+) transients showed that Ca(i)(2+) activation propagates horizontally from left to right during sinus rhythm or from the stimulus site during direct left ventricle stimulation. The indo-1 ratiometric video technique developed allows the imaging of ratio changes of Ca(i)(2+) with a high temporal (1 ms) and spatial (0.6 mm) resolution in the isolated Tyrode-perfused beating rat heart.

Mechanism of activation of the tonic component of contraction in myocytes of guinea pig heart.

Contractions of myocytes of guinea pig heart consist of a phasic component relaxing independently on the voltage and a tonic component relaxing upon repolarization. We found previously that Ca(2+) activating the tonic component is released from the sarcoplasmic reticulum. In this study, we analysed the mechanism of activation and maintenance of this release. Experiments were performed at 37 degrees C in ventricular myocytes of guinea pig heart. Voltage-clamped myocytes were stimulated by the pulses of the duration of 300 ms to 15-45 s from the holding potential of -40 to +5 mV. [Ca(2+)](i) was monitored as fluorescence of Indo-1 and contractions were recorded with the TV edge-tracking system. Myocytes responded to the short and long pulses with phasic contraction or Ca(2+) transient followed by the sustained contraction or increase in [Ca(2+)](i). Repolarization brought about relaxation. 10 mmol L(-1) Ni(2+) blocking Na(+)/Ca(2+) exchange superfused during the tonic component increased its amplitude. Superfusion of Ca(2+)-free solution during sustained contraction brought about relaxation both in normal cells and in cells superfused with Ni(2+) despite preserved sarcoplasmic reticulum Ca(2+) content assessed with caffeine spritz. Relaxing effect of Ca(2+)-free solution was not affected by carboxyeosin, a blocker of sarcolemmal Ca(2+)-ATPase. Tonic component of contraction and of Ca(2+) transient was inhibited by 200 micromol L(-1) ryanodine, a blocker of Ca(2+) release channels of the sarcoplasmic reticulum and by 20 micromol L(-1) nifedipine, a blocker of L-type I(Ca). Tonic component of contraction results from Ca(2+) release via the sarcoplasmic reticulum Ca(2+) channels activated by sustained, nifedipine-sensitive and Ni(2+)-insensitive Ca(2+) influx. Alternatively, the SR Ca(2+) release is activated by voltage, the dihydropyridine receptors acting like voltage sensors.

Effect of low [CaCl2] and high [MgCl2] cardioplegia and moderate hypothermic ischemia on myoplasmic [Ca2+] and cardiac function in intact hearts.

Cold cardioplegia (CP) protects against ischemic damage in part by reducing [Ca(2+)](i) overload on reperfusion. Hyperkalemic cardioplegic solutions are widely used in coronary artery bypass procedures, and the specific ionic composition of these solutions may contribute to their variable myocardial protective effects secondary to reduced Ca(2+)(i) loading. We reported previously that CP decreased the rise in cardiac diastolic (dia) [Ca(2+)](i) observed during 4 h cold storage at 3 degrees C in Krebs-Ringer's (KR) solution and decreased dia[Ca(2+)](i) and increased systolic (sys) [Ca(2+)](i) and function on reperfusion after cold storage. Our aim here was to determine if low Ca(2+)(o) and high Mg(2+)(o) adds to the protective effects of high K(+)(o) by decreasing [Ca(2+)](i) during ischemia and reperfusion. We compared effects of 4.5 mM K(+)(o), 2.5 mM Ca(2+)(o) and 2.4 mM Mg(2+)(o) KR solution with a higher K(+)(o) (18 mM), a lower Ca(2+)(o) (1.25 mM) and/or higher Mg(2+)(o) (7.2 mM) CP solutions on cardiac mechanic function and sys and dia[Ca(2+)](i) during and after moderate hypothermic global ischemia (17 degrees C for 4 h) in guinea pig intact hearts isolated by the Langendorff technique. Isovolumetric left ventricular pressure (LVP) was measured with a transducer connected to a fluid-filled balloon placed in the LV and [Ca(2+)](i) was measured using indo-1 fluorescence and a fiberoptic cable placed on the LV free wall. For all CP groups compared to the KR control group after 60 min reperfusion, we observed significant lowering of dia[Ca(2+)](i) by 47%, left ventricular diastolic pressure (diaLVP) by 55%, and infarct size by 43%. We also found significant elevation of sys[Ca(2+)](i) by 25%, d[Ca(2+)](i)/dt(max) and d[Ca(2+)](i)/dt(min) by 33 and 34%, sys-diaLVP by 55%, dLVP/dt(max) and dLVP/dt(min) by 34 and 40%, coronary flow by 31%, cardiac efficiency by 21%, and MVO(2) by 25%. These results indicate that CP reduces myoplasmic Ca(2+) loading and improves mechanical and metabolic function on warm reperfusion compared to KR. However, there were no differences in these indices of Ca(2+)(i) cardiac function or metabolism among any CP group after warm reperfusion with KR solution. Increasing K(+)(o) to produce cardiac arrest was the most cardioprotective effect of CP against ischemia reperfusion injury; lowering Ca(2+)(o) or raising Mg(2+)(o) did not add to this protective effect or additionally alter [Ca(2+)](i).

Na(+)-Ca(2+) exchanger overexpression predisposes to reactive oxygen species-induced injury.

In heart failure (HF), the generation of reactive oxygen species (ROS) is enhanced. It was shown that failing cardiac myocytes are more susceptible to ROS-induced damage, possibly due to increased expression of the sarcolemmal Na-Ca exchanger (NCX). We investigated the consequences of increased expression levels of NCX in adult rabbit ventricular cardiomyocytes (via adenovirus-mediated gene transfer, Ad-NCX1-GFP) with respect to tolerance towards ROS. After 48-h incubation, cells were monitored for morphological changes on an inverted microscope. ROS were generated via hydrogen peroxide (H(2)O(2)) (100 micromol/l) and Fe(3+)/nitrilotriacetate (Fe(3+)/NTA, 100/200 micromol/l) for 4 min and cell morphology was followed over 30 min. [Na(+)](i) and [Ca(2+)](i) in native cells were measured using SBFI-AM and Indo1-AM, respectively. In native myocytes, exposure to ROS induced hypercontracture. This was accompanied by a 1.3-fold increase in diastolic Indo1 fluorescence ratio (P<0.05). Overexpression of NCX significantly enhanced development of hypercontracture. After 15 min, the percentage of cells that had undergone hypercontracture (F(hyper)) was 85+/-4% vs. only 44+/-10% in control cells (P<0.05). Inhibition of NCX-mediated Ca(2+) entry with KB-R7943 (5 micromol/l) reduced F(hyper) to 33+/-11% (P<0.05). [Na(+)](i) was increased 2.9-fold 1 min prior to hypercontracture (P<0.05). ROS-induced hypercontracture is due to Ca(2+) entry via NCX which could be triggered by a concomitant substantial increase in [Na(+)](i). Elevated NCX levels predispose to ROS-induced injury, a mechanism likely contributing to myocyte dysfunction and death in heart failure.

Intracellular calcium handling heterogeneities in intact guinea pig hearts.

Regional heterogeneities of ventricular repolarizing currents and their role in arrhythmogenesis have received much attention; however, relatively little is known regarding heterogeneities of intracellular calcium handling. Because repolarization properties and contractile function are heterogeneous from base to apex of the intact heart, we hypothesize that calcium handling is also heterogeneous from base to apex. To test this hypothesis, we developed a novel ratiometric optical mapping system capable of measuring calcium fluorescence of indo-1 at two separate wavelengths from 256 sites simultaneously. With the use of intact Langendorff-perfused guinea pig hearts, ratiometric calcium transients were recorded under normal conditions and during administration of known inotropic agents. Ratiometric calcium transients were insensitive to changes in excitation light intensity and fluorescence over time. Under control conditions, calcium transient amplitude near the apex was significantly larger (60%, P < 0.01) compared with the base. In contrast, calcium transient duration was significantly longer (7.5%, P < 0.03) near the base compared with the apex. During isoproterenol (0.05 microM) and verapamil (2.5 microM) administration, ratiometric calcium transients accurately reflected changes in contractile function, and, the direction of base-to-apex heterogeneities remained unchanged compared with control. Ratiometric optical mapping techniques can be used to accurately quantify heterogeneities of calcium handling in the intact heart. Significant heterogeneities of calcium release and sequestration exist from base to apex of the intact heart. These heterogeneities are consistent with base-to-apex heterogeneities of contraction observed in the intact heart and may play a role in arrhythmogenesis under abnormal conditions.

Lead enters Rcho-1 trophoblastic cells by calcium transport mechanisms and complexes with cytosolic calcium-binding proteins

Within the placenta, a specialized Ca 2+ transport pathway develops in trophoblasts to promote growth of the fetus and hypothetically to enhance fetal uptake of Pb 2+. This hypothesis could not be tested until a method to monitor Pb 2+ influx by indo-1 fluorescence quench became available. We have applied this new method to cultured undifferentiated and differentiated Rcho-1 trophoblastic cells. Pb 2+ concentrations of 1 and 10 μM are equivalent to blood levels of 20 and 200 μg/dl in pregnant women. Over this range, Pb 2+ uptake increased with time and concentration in medium containing 1 mM Ca 2+ but was greater in Ca 2+-omitted solutions. Activation of capacitative Ca 2+ entry (CCE) with thapsigargin, an endoplasmic reticulum (ER) Ca 2+ pump inhibitor, increased Pb 2+ uptake, while inhibition of CCE by La 3+ decreased influx. Parathyroid hormone-related peptide (PTHrP) stimulates the synthesis of Ca 2+-binding proteins (CaBPs), as well as Ca 2+ transporters, during trophoblastic differentiation. Pretreatment for 72 h with PTHrP increased Pb 2+ uptake by undifferentiated Rcho-1 cells but had little effect on the quench in differentiated cells, probably due to their greater content of CaBPs which competed for Pb 2+-binding with indo-1. This competition was most evident in differentiated cells when 1 μM Pb 2+ caused an initial quench, followed by a rise in fluorescence. This rise was not inhibited by thapsigargin, thereby ruling out sequestration into the ER and leaving complexation of Pb 2+ by CaBPs as the most plausible interpretation. We conclude that trophoblasts have the ability to clear Pb 2+ from the maternal circulation and deliver it to the fetus.