Intracellular Calcium Transient Amplitude Research Articles

Nonclinical evaluation of chronic cardiac contractility modulation on 3D human engineered cardiac tissues.

Cardiac contractility modulation (CCM) is a medical device-based therapy delivering non-excitatory electrical stimulations to the heart to enhance cardiac function in heart failure (HF) patients. The lack of human in vitro tools to assess CCM hinders our understanding of CCM mechanisms of action. Here, we introduce a novel chronic (i.e., 2-day) in vitro CCMassay to evaluate the effects of CCM in a human 3D microphysiological system consisting of engineered cardiac tissues (ECTs). Cryopreserved human induced pluripotent stem cell-derived cardiomyocytes were used to generate 3D ECTs. The ECTs were cultured, incorporating human primary ventricular cardiac fibroblasts and a fibrin-based gel. Electrical stimulation was applied using two separate pulse generators for the CCM group and control group. Contractile properties and intracellular calcium were measured, and a cardiac gene quantitative PCR screen was conducted. Chronic CCM increased contraction amplitude and duration, enhanced intracellular calcium transient amplitude, and altered gene expression related to HF (i.e., natriuretic peptide B, NPPB) and excitation-contraction coupling (i.e., sodium-calcium exchanger, SLC8). These data represent the first study of chronic CCM in a 3D ECT model, providing a nonclinical tool to assess the effects of cardiac electrophysiology medical device signals complementing in vivo animal studies. The methodology established a standardized 3D ECT-based in vitro testbed for chronic CCM, allowing evaluation of physiological and molecular effects on human cardiac tissues.

Understanding the cardiac toxicity of the anthropogenic pollutant phenanthrene on the freshwater indicator species, the brown trout (Salmo trutta): From whole heart to cardiomyocytes

Freshwater systems are faced with a myriad of stressors including geomorphological alterations, nutrient overloading and pollution. Previous studies in marine fish showed polyaromatic hydrocarbons (PAHs) to be cardiotoxic. However, the cardiotoxicity of anthropogenic pollutants in freshwater fishes is unclear and has not been examined across multiple levels of cardiac organization. Here we investigated the effect of phenanthrene (Phe), a pervasive anthropogenic pollutant on a sentinel freshwater species, the brown trout (Salmo trutta). We first examined the electrical activity of the whole heart and found prolongation (∼8.6%) of the QT interval (time between ventricular depolarization and repolarization) of the electrocardiogram (ECG) and prolongation (∼13.2%) of the monophasic action potential duration (MAPD) following ascending doses of Phe. At the tissue level, Phe significantly reduced trabecular force generation by ∼24% at concentration 15 μM and above, suggesting Phe reduces cellular calcium cycling. This finding was supported by florescent microscopy showing a reduction (∼39%) in the intracellular calcium transient amplitude following Phe exposure in isolated brown trout ventricular myocytes. Single-cell electrophysiology was used to reveal the mechanism underlying contractile and electrical dysfunction following Phe exposure. A Phe-dependent reduction (∼38%) in the L-type Ca2+ current accounts, at least in part, for the lowered Ca2+ transient and force production. Prolongation of the MAPD and QT interval was explained by a reduction (∼70%) in the repolarising delayed rectifier K+ current following Phe exposure. Taken together, our study shows a direct impact of Phe across multiple levels of cardiac organization in a key freshwater salmonid.

Pulsed electromagnetic fields promote the proliferation and differentiation of osteoblasts by reinforcing intracellular calcium transients.

Pulsed electromagnetic fields (PEMF) can be used to treat bone-related diseases, but the underlying mechanism remains unclear, especially the process by which PEMFs initiate biological effects. In this study, we demonstrated the effects of PEMF on proliferation and differentiation of osteoblasts using the model of calcium transients induced by high extracellular calcium. Our results showed that PEMF can increase both the percentage of responding cells and amplitude of intracellular calcium transients induced by high extracellular calcium stimulation. Compared with corresponding extracellular calcium levels, PEMF stimulation increased proliferation and differentiation of osteoblasts and related gene expressions, such as insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), which can be completely abolished by BAPTA-AM. Moreover, PEMF did not affect proliferation and differentiation of osteoblasts if no intracellular calcium transient was present in osteoblasts during PEMF exposure. Our results revealed that PEMF affects osteoblast proliferation and differentiation through enhanced intracellular calcium transients, which provided a cue to treat bone-related diseases with PEMF. Bioelectromagnetics. 38:541-549, 2017. © 2017 Wiley Periodicals, Inc.

Circulating Histone Concentrations Differentially Affect the Predominance of Left or Right Ventricular Dysfunction in Critical Illness.

Cardiac complications are common in critical illness and associated with grave consequences. In this setting, elevated circulating histone levels have been linked to cardiac injury and dysfunction in experimental models and patients with sepsis. The mechanisms underlying histone-induced cardiotoxicity and the functional consequences on left ventricle and right ventricle remain unclear. This study aims to examine dose-dependent effects of circulating histones on left ventricle and right ventricle function at clinically relevant concentrations. Prospective laboratory study with in vitro and in vivo investigations. University research laboratory. Twelve-week old male C57BL/6N mice. Cultured cardiomyocytes were incubated with clinically relevant histone concentrations, and a histone infusion mouse model was also used with hemodynamic changes characterized by echocardiography and left ventricle/right ventricle catheter-derived variables. Circulating histones and cardiac troponin levels were obtained from serial blood samples. IV histone infusion caused time-dependent cardiac troponin elevation to indicate cardiac injury. At moderate sublethal histone doses (30 mg/kg), left ventricular contractile dysfunction was the prominent abnormality with reduced ejection fraction and prolonged relaxation time. At high doses (≥ 60 mg/kg), pulmonary vascular obstruction induced right ventricular pressure increase and dilatation, but left ventricular end-diastolic volume improved because of reduced blood return from the lungs. Mechanistically, histones induced profound calcium influx and overload in cultured cardiomyocytes with dose-dependent detrimental effects on intracellular calcium transient amplitude, contractility, and rhythm, suggesting that histones directly affect cardiomyocyte function adversely. However, increasing histone-induced neutrophil congestion, neutrophil extracellular trap formation, and thrombosis in the pulmonary microvasculature culminated in right ventricular dysfunction. Antihistone antibody treatment abrogated histone cardiotoxicity. Circulating histones significantly compromise left ventricular and right ventricular function through different mechanisms that are dependent on histone concentrations. This provides a translational basis to explain and target the spectral manifestations of cardiac dysfunction in critical illness.

Cardioprotective Effect of Ranolazine in the Process of Ischemia-reperfusion in Adult Rat Cardiomyocytes

Introduction and objectivesRanolazine is used as a complementary treatment for angina in symptomatic patients who are inadequately controlled with first-line antianginal therapies. Ranolazine inhibits sodium voltage-dependent channels, suggesting their possible involvement in the reperfusion process by preventing the sodium and calcium overload that occurs during ischemia. In this study, we characterized the effect of ranolazine on calcium homeostasis in isolated adult cardiac myocytes from rats subjected to a simulated ischemia and reperfusion protocol. MethodsThe effects of ranolazine on changes in intracellular calcium concentration were evaluated at different times using field electrostimulation. The study of intracellular calcium was performed using microfluorimetry with the fluorescent indicator, Fura-2, and by confocal microscopy with the indicator, Fluo-3. ResultsWe found that cardiomyocytes subjected to ischemia-reperfusion showed an increase in the diastolic calcium concentration and a decrease in the amplitude of intracellular calcium transients. The application of ranolazine during ischemia significantly improved intracellular calcium handling, preventing intracellular calcium overload, decreasing the diastolic calcium concentration, increasing the sarcoplasmic reticulum calcium load, and preserving the amplitude of the intracellular calcium transient, which was reflected by successful recovery in the process of excitation-contraction coupling during reperfusion. However, these effects of ranolazine did not occur when it was applied during reperfusion or when applied in both ischemia and reperfusion. ConclusionsRanolazine shows beneficial effects in cardiomyocytes exposed to ischemia/reperfusion but only when applied during ischemia. This effect is achieved through its improvement of calcium handling during ischemia.

Efecto cardioprotector de la ranolazina en el proceso de isquemia-reperfusión en cardiomiocitos de rata adultos

Introduction and objectivesRanolazine is used as a complementary treatment for angina in symptomatic patients who are inadequately controlled with first-line antianginal therapies. Ranolazine inhibits sodium voltage-dependent channels, suggesting their possible involvement in the reperfusion process by preventing the sodium and calcium overload that occurs during ischemia. In this study, we characterized the effect of ranolazine on calcium homeostasis in isolated adult cardiac myocytes from rats subjected to a simulated ischemia and reperfusion protocol. MethodsThe effects of ranolazine on changes in intracellular calcium concentration were evaluated at different times using field electrostimulation. The study of intracellular calcium was performed using microfluorimetry with the fluorescent indicator, Fura-2, and by confocal microscopy with the indicator, Fluo-3. ResultsWe found that cardiomyocytes subjected to ischemia-reperfusion showed an increase in the diastolic calcium concentration and a decrease in the amplitude of intracellular calcium transients. The application of ranolazine during ischemia significantly improved intracellular calcium handling, preventing intracellular calcium overload, decreasing the diastolic calcium concentration, increasing the sarcoplasmic reticulum calcium load, and preserving the amplitude of the intracellular calcium transient, which was reflected by successful recovery in the process of excitation-contraction coupling during reperfusion. However, these effects of ranolazine did not occur when it was applied during reperfusion or when applied in both ischemia and reperfusion. ConclusionsRanolazine shows beneficial effects in cardiomyocytes exposed to ischemia/reperfusion but only when applied during ischemia. This effect is achieved through its improvement of calcium handling during ischemia.Full English text available from:www.revespcardiol.org/en

Circulating Pneumolysin Is a Potent Inducer of Cardiac Injury during Pneumococcal Infection.

Streptococcus pneumoniae accounts for more deaths worldwide than any other single pathogen through diverse disease manifestations including pneumonia, sepsis and meningitis. Life-threatening acute cardiac complications are more common in pneumococcal infection compared to other bacterial infections. Distinctively, these arise despite effective antibiotic therapy. Here, we describe a novel mechanism of myocardial injury, which is triggered and sustained by circulating pneumolysin (PLY). Using a mouse model of invasive pneumococcal disease (IPD), we demonstrate that wild type PLY-expressing pneumococci but not PLY-deficient mutants induced elevation of circulating cardiac troponins (cTns), well-recognized biomarkers of cardiac injury. Furthermore, elevated cTn levels linearly correlated with pneumococcal blood counts (r=0.688, p=0.001) and levels were significantly higher in non-surviving than in surviving mice. These cTn levels were significantly reduced by administration of PLY-sequestering liposomes. Intravenous injection of purified PLY, but not a non-pore forming mutant (PdB), induced substantial increase in cardiac troponins to suggest that the pore-forming activity of circulating PLY is essential for myocardial injury in vivo. Purified PLY and PLY-expressing pneumococci also caused myocardial inflammatory changes but apoptosis was not detected. Exposure of cultured cardiomyocytes to PLY-expressing pneumococci caused dose-dependent cardiomyocyte contractile dysfunction and death, which was exacerbated by further PLY release following antibiotic treatment. We found that high PLY doses induced extensive cardiomyocyte lysis, but more interestingly, sub-lytic PLY concentrations triggered profound calcium influx and overload with subsequent membrane depolarization and progressive reduction in intracellular calcium transient amplitude, a key determinant of contractile force. This was coupled to activation of signalling pathways commonly associated with cardiac dysfunction in clinical and experimental sepsis and ultimately resulted in depressed cardiomyocyte contractile performance along with rhythm disturbance. Our study proposes a detailed molecular mechanism of pneumococcal toxin-induced cardiac injury and highlights the major translational potential of targeting circulating PLY to protect against cardiac complications during pneumococcal infections.

GW24-e1896 Effects of cyclovirobuxin-D on intracellular Ca2+ levels in isolated ventricular myocytes of diabetic rats

ObjectivesTo explore the effect of cyclovirobuxin-D(CVB-D) on intracellular calcium in electrically stimulated ventricular myocytes of diabetic ratsMethodsDiabetes was induced in male SD rats, using a single injection of alloxan into...

08 Ras-Association Domain Family 1 Isoform A (RASSF1A) is a Novel Regulator of TNF-alpha Signalling in Cardiomyocytes

Tumour necrosis factor-alpha; (TNF-alpha) plays key roles in the pathogenesis of heart failure. Cardiomyocytes express the TNF-alpha; receptor (TNFR), however, the mechanism of TNF-alpha; signal transmission in cardiomyocytes is not completely understood. Recent studies showed that in cancer cells TNFR is regulated by Ras-association domain family 1 isoformA (RASSF1A). Therefore, we investigated whether RASSF1A modulates TNF-alpha; signalling in cardiomyocytes. We used RASSF1A knockout (KO) mice and wild type (WT) controls and stimulated them with TNF-alpha; (10µg/kg i.v.). In WT mice acute treatment with low dose of TNF-alpha increased cardiac contractility and intracellular calcium transient amplitude, which is consistent with previously published data (Circulation 2004; 109:406-411). However, KO mice showed a blunted contractile response following acute TNF-alpha treatment as indicated by the change in end systolic elastance (in vivo) and intracellular calcium transient amplitude (isolated adult cardiomyocytes). We also found that RASSF1A formed a molecular complex with TNF-alpha; receptor in cardiomyocytes and this interaction was essential in the recruitment of TRADD and TRAF2, the major downstream effectors of TNF-alpha; signalling. By mapping the interaction domain we found that the C-terminal region of RASSF1A was responsible for the formation of TNF-alpha; receptor complex. Furthermore, using an adenoviral-mediate d shRNA construct we found that cardiomyocytes lacking RASSF1A exhibited reduced activation of NFkB, a downstream target of TNF-alpha. Overall, our data indicate an essential role of RASSF1A in regulating TNF-alpha; signalling in cardiomyocytes, with RASSF1A being key in the formation of TNF receptor complex and in the signal transmission to the downstream targets.

Effects of cyclovirobuxin-D on intracellular Ca2+ levels in isolated ventricular myocytes of diabetic rats

ObjectiveTo explore the effect of cyclovirobuxin-D (CVB-D) on intracellular calcium in electrically stimulated ventricular myocytes of diabetic rats.MethodsDiabetes was induced in male SD rats, using a single injection of alloxan...

Ca 2+ ‐independent positive molecular inotropy for failing rabbit and human cardiac muscle by α‐myosin motor gene transfer

Current inotropic therapies used to increase cardiac contractility of the failing heart center on increasing the amount of calcium available for contraction, but their long-term use is associated with increased mortality due to fatal arrhythmias. Thus, there is a need to develop and explore novel inotropic therapies that can act via calcium-independent mechanisms. The purpose of this study was to determine whether fast alpha-myosin molecular motor gene transfer can confer calcium-independent positive inotropy in slow beta-myosin-dominant rabbit and human failing ventricular myocytes. To this end, we generated a recombinant adenovirus (AdMYH6) to deliver the full-length human alpha-myosin gene to adult rabbit and human cardiac myocytes in vitro. Fast alpha-myosin motor expression was determined by Western blotting and immunocytochemical analysis and confocal imaging. In experiments using electrically stimulated myocytes from ischemic failing hearts, AdMYH6 increased the contractile amplitude of failing human [23.9+/-7.8 nm (n=10) vs. AdMYH6 amplitude 78.4+/-16.5 nm (n=6)] and rabbit myocytes. The intracellular calcium transient amplitude was not altered. Control experiments included the use of a green fluorescent protein or a beta-myosin heavy chain adenovirus. Our data provide evidence for a novel form of calcium-independent positive inotropy in failing cardiac myocytes by fast alpha-myosin motor protein gene transfer.

B-type natriuretic peptide (BNP) attenuates the L-type calcium current and regulates ventricular myocyte function

A fundamental question in physiology is how hormones regulate the functioning of a cell or organ. It was therefore the aim of this study to investigate the effect(s) of BNP-32 on calcium handling by ventricular myocytes obtained from the rat left ventricle. We specifically tested the hypothesis that BNP-32 decreased the L-type calcium current ( I Ca,L). Perforated patch clamp technique was used to record I Ca,L and action potential (AP) in voltage and current clamp mode, respectively. Myocyte shortening was measured using a photodiode array edge-detection system and intracellular calcium transients were measured by fluorescence photometry. Western blotting was used to determine the relative change in the expression of proteins. At the concentrations tested, BNP-32 significantly decreased cell shortening in a dose-dependent manner; increased the phase II slope of the AP by 53.0%; increased the APD 50 by 16.9%; reduced the I Ca,L amplitude with a 22.9% decrease in the peak amplitude and reduced Ca 2+-dependent inactivation; increased the V 1/2 activation of the L-type calcium channel by 51.1% and decreased V 1/2 inactivation by 31.8%; and, intracellular calcium transient amplitude was significantly decreased by 32.0%, whereas the time to peak amplitude and T 1/2 were both significantly increased by 38.7% and 89.4% respectively. Sarcoplasmic reticulum Ca 2+-ATPase (SERCA2a) protein expression was reduced by BNP-32. These data suggest that BNP-32 regulates ventricular myocyte function by attenuating I Ca,L, altering the AP and reducing SERCA2a activity and/or expression. This study suggests a novel constitutive mechanism for the autocrine action of BNP on the L-type calcium channel in ventricular myocytes.

Dissociation of force decline from calcium decline by preload in isolated rabbit myocardium

It is well known that the rate of intracellular calcium ([Ca2+]i) decline is an important factor governing relaxation in unloaded myocardium. However, it remains unclear to what extent, under near physiological conditions, the intracellular calcium transient amplitude and kinetics contribute to the length-dependent increase in force and increase in duration of relaxation. We hypothesize that myofilament properties rather than calcium transient decline primarily determines the duration of relaxation in adult mammalian myocardium. To test this hypothesis, we simultaneously measured force of contraction and calibrated [Ca2+]i transients in isolated, thin rabbit trabeculae at various lengths at 37 degrees C. Time from peak tension to 50% relaxation (RT50(tension)) increases significantly with length (from 49.8+/-3.4 to 83.8+/-7.4 ms at an [Ca2+]o of 2.5 mM), whereas time from peak calcium to 50% decline (RT50(calcium)) was not prolonged (from 124.8+/-5.3 to 107.7+/-11.4 ms at an [Ca2+]o of 2.5 mM). Analysis of variance revealed that RT50(tension) is significantly correlated with length (P<0.0001). At optimal length, varying the extracellular calcium concentration increased both developed force and calcium transient amplitude, but RT50(tension) remained unchanged (P=0.90), whereas intracellular calcium decline actually accelerated (P<0.05). Thus, an increase in muscle length will result in an increase in both force and duration of relaxation, whereas the latter is not primarily governed by the rate of [Ca2+]i decline.

Galpha(i2) but not Galpha(i3) is required for muscarinic inhibition of contractility and calcium currents in adult cardiomyocytes.

Parasympathetic stimulation of the heart acts through M(2)-muscarinic acetylcholine receptors to regulate ion channel activity and subsequent inotropic status. Although muscarinic signal transduction is mediated via pertussis toxin-sensitive G proteins Galpha(i/o), the specific signal transduction requirements of Galpha(i2) and Galpha(i3) in mediating muscarinic regulated L-type calcium currents (I(Ca, L)), intracellular calcium, and cell contractility remain to be determined. Adult ventricular myocytes were isolated from Galpha(i2)-null mice, Galpha(i3)-null mice, and their wild-type littermates. Cell shortening, intracellular calcium levels, and I(Ca, L) were all measured in response to isoproterenol, a beta-adrenergic receptor agonist, and carbachol, a cholinergic receptor agonist. With isoproterenol stimulation, myocytes from all groups demonstrated a marked increase in calcium currents, correlating with augmented intracellular calcium transient amplitude and cell shortening. Carbachol significantly attenuated the isoproterenol response in wild-type and Galpha(i3)-null cells but had no effect in Galpha(i2)-null cells. This study demonstrates that Galpha(i2), but not Galpha(i3), is required for muscarinic inhibition of the beta-adrenergic response in adult murine ventricular myocytes.

Biphasic effect of bimoclomol on calcium handling in mammalian ventricular myocardium.

1. Concentration-dependent effects of bimoclomol, the novel heat shock protein coinducer, on intracellular calcium transients and contractility were studied in Langendorff-perfused guinea-pig hearts loaded with the fluorescent calcium indicator dye Fura-2. Bimoclomol had a biphasic effect on contractility: both peak left ventricular pressure and the rate of force development significantly increased at a concentration of 10 nM or higher. The maximal effect was observed between 0.1 and 1 microM, and the positive inotropic action disappeared by further increasing the concentration of bimoclomol. The drug increased systolic calcium concentration with a similar concentration-dependence. In contrast, diastolic calcium concentration increased monotonically in the presence of bimoclomol. Thus low concentrations of the drug (10 - 100 nM) increased, whereas high concentrations (10 microM) decreased the amplitude of intracellular calcium transients. 2. Effects of bimoclomol on action potential configuration was studied in isolated canine ventricular myocytes. Action potential duration was increased at low (10 nM), unaffected at intermediate (0.1 - 1 microM) and decreased at high (10 - 100 microM) concentrations of the drug. 3. In single canine sarcoplasmic calcium release channels (ryanodine receptor), incorporated into artificial lipid bilayer, bimoclomol significantly increased the open probability of the channel in the concentration range of 1 - 10 microM. The increased open probability was associated with increased mean open time. The effect of bimoclomol was again biphasic: the open probability decreased below the control level in the presence of 1 mM bimoclomol. 4. Bimoclomol (10 microM - 1 mM) had no significant effect on the rate of calcium uptake into sarcoplasmic reticulum vesicles of the dog, indicating that in vivo calcium reuptake might not substantially be affected by the drug. 5. In conclusion, the positive inotropic action of bimoclomol is likely due to the activation of the sarcoplasmic reticulum calcium release channel in mammalian ventricular myocardium.

Effects of prostaglandin F2 alpha on intracellular pH, intracellular calcium, cell shortening and L-type calcium currents in rat myocytes.

We have studied the mechanisms underlying the positive inotropic action of prostaglandin F2 alpha (PGF2 alpha) by monitoring intracellular calcium transients, intracellular pH, L-type calcium currents and cell shortening in isolated ventricular myocytes. Rat myocytes were loaded with fura-2AM for intracellular calcium measurements, or BCECF-AM for pH measurements. Cell shortening was recorded using an edge detection system, and L-type calcium currents measured using whole cell patch clamping. PGF2 alpha (3 nmol l-1-3 mumol l-1 increased single myocyte shortening and reduced resting cell length in a concentration-dependent manner. While myocyte shortening was increased by PGF2 alpha, this was not associated with any change in the amplitude of intracellular calcium transients, diastolic calcium, or L-type calcium currents. However, the same myocytes were capable of responding to catecholamines with increases in calcium transient amplitude and L-type calcium currents. PGF2 alpha (3 mumol l-1 caused a reversible rise in intracellular pH of 0.08 +/- 0.01 pH units (n = 5, p < 0.05). The Na(+)-H+ exchanger inhibitor, HOE 694 (10 mumol l-1, abolished the PGF2 alpha-induced rise in pH and the increase in cell shortening. PGF2 alpha-induced increases in cell shortening and intracellular pH were also attenuated by the protein kinase C (PKC) inhibitor, chelerythrine (2 mumol l-1. The positive inotropic action of PGF2 alpha appears to be mediated via activation of the Na(+)-H+ exchanger with the possible involvement of PKC. This suggests that PGF2 alpha-produces intracellular alkalosis, which then sensitizes cardiac myofilaments to calcium.

Localization and regulation of ryanodine receptor in bovine oocytes.

We have previously reported that injection of ryanodine receptor agonists into mature bovine oocytes induces intracellular calcium release, indicative of the existence of ryanodine receptors. In this experiment, further evidence of the ryanodine receptor localization, and developmental regulation in bovine oocytes is presented. The possible physiological significance is also suggested. Using a rabbit antibody against the rabbit cardiac muscle ryanodine receptor, the ryanodine receptor was observed uniformly localized in the periphery of mature bovine oocytes, while a weak and discontinuous signal was observed in the germinal vesicle intact stage of bovine oocytes. As oocytes progress to the metaphase I stage, the ryanodine receptor localization became more intense and continuous, yet not comparable to that observed in the metaphase II oocytes. These modifications correlate with the intracellular calcium responsiveness to ryanodine. A 200-microM injection of ryanodine induces a low intracellular calcium transient in germinal vesicle-stage bovine oocytes, while peaked intracellular calcium transients are subsequently observed in the metaphase II-stage oocytes. However, no significant changes in the amplitude of intracellular calcium transients induced by 250 nM inositol 1,4,5-trisphosphate and 10 microM ionomycin were observed in oocytes at a comparable stage. Fertilization induced a significant decrease in ryanodine receptor signal; similar changes were also observed in oocytes injected with 200 microM ryanodine or incubated with 10 microM ionomycin. However, no changes in ryanodine signal were observed in oocytes injected with vehicle medium. Furthermore, injection of either ryanodine or inositol 1,4,5-trisphosphate induced subsequent pronuclear formation and cleavage. These data indicate that the ryanodine receptor is closely regulated and associated with early cellular changes following fertilization; stimulation of this receptor results in the activation of bovine oocytes, and it is likely that this receptor may play a role at fertilization.

997-86 Intracellular Calcium Transients Underlie Mechanical Restitution in Whole Rat Hearts

Mechanical restitution describes the recovery of myocardial contractility during the interval between heart beats. Although restitution has been widely studied, much of the understanding about restitution of the whole heart has been inferred from isolated muscle studies. We investigated whether results from isolated muscle regarding intracellular calcium transients underlying mechanical restitution could be substantiated in the whole heart. Therefore, we obtained restitution curves for left ventricular pressure and, simultaneously, for intracellular calcium transients in isolated rat hearts (pressure and calcium plotted as a function of extrasystolic intervals). Intracellular calcium transients were assessed by surface fluorometry after Indo-1-loading. Restitution curves were generated by computer-controlled pacing protocols. We found that, similar to isolated muscle, restitution curves for both pressure and intracellular calcium transients in the whole heart followed similar monoexponential functions. Moreover, interval-dependent changes in pressure were strongly correlated to interval-dependent changes in the amplitude of intracellular calcium transients (correlation by linear regression: r = 0.92, P = 0.00). Thus, for the first time, the results from isolated muscle regarding intracellular calcium transients underlying mechanical restitution could be substantiated in the whole heart.