Endothelin-1 release during the early phase of reperfusion is a mediator of myocardial reperfusion injury.

PURPOSE: In acute myocardial infarction, left ventricular (LV) unloading reduces endothelin-1 (ET-1) release. We tested that endogenous ET-1 released during acute myocardial infarction might mediate ischemia/reperfusion (I/R) injury by stimulating increased intracellular calcium concentration, [Ca(2+)]i, and apoptosis. METHODS: Rabbits were subjected to 1 h of coronary artery occlusion followed by 3 h of reperfusion. Unloading was initiated 15 min prior to reperfusion and was maintained during reperfusion. The control group was subjected to reperfusion. Animals were treated with ET-1 receptor antagonist BQ123. In parallel, isolated rabbit cardiomyocytes subjected to simulated I/R with or without ET-1 or BQ123, intracellular Ca(2+) and cell death were assessed with flow cytometry. RESULTS: LV unloading prior to reperfusion reduced myocardial ET-1 release at 2 h of reperfusion. Infarct size was reduced in unloaded and BQ123 groups versus controls. LV unloading and BQ123 treatment reduced the percentage of apoptotic cells associated with increases in Bcl-2 protein levels in ischemic regions. BQ123 reduced both ET-1-induced [Ca(2+)]i increase and cell death for myocytes subjected to stimulated I/R. CONCLUSION: We propose that components of reperfusion injury involve ET-1 release which stimulates calcium overload and apoptosis. Intravenous ET-1 receptor blockade prior to reperfusion may be a protective adjunct to reperfusion therapy in acute myocardial infarction patients.

Heparins with reduced anti-coagulant activity reduce myocardial reperfusion injury.

Heparin which is desulfated at the 2-O and 3-O positions (ODSH) has reduced anti-coagulant properties, and reduced interaction with heparin antibodies. Because of the reduced anti-coagulant effect, ODSH can be safely administered to animals and humans intravenously at doses up to 20 mg/kg, resulting in a serum concentration of up to 250µg/ml. Administration of ODSH causes a 35% reduction in infarct size in dogs and pigs subjected to coronary artery occlusion and reperfusion when given 5 min before reperfusion. ODSH has anti-inflamatory effects, manifest as a decrease in neutrophil infiltration into ischemic tissue at high doses, but this effect does not entirely account for the reduction in infarct size. ODSH decreases Na(+) and Ca(2+) loading in isolated cardiac myocytes subjected to simulated ischemia. This effect appears due to an ODSH-induced reduction in an enhanced Na(+) influx via the Na channel in the membrane of cardiac myocyes caused by oxygen radicals generated during ischemia and reperfusion. Reduction in Na(+) influx decreases Ca(2+) loading by reducing Ca2(+) influx via Na/Ca exchange, thus reducing Ca(2+) - dependent reperfusion injury. ODSH does not appear to interact with antibodies to the heparin/platelet factor 4 complex, and does not cause heparin-induced thrombocytopenia. Because of these therapeutic and safety considerations, ODSH would appear to be a promising heparin derivative for prevention of reperfusion injury in humans undergoing thrombolytic or catheter-based reperfusion for acute myocardial infarction. The review article discussed the use of heparin and the discussion of some of the important patents, including: US6489311; US7478358; PCTUS2008070836 and PCTUS2009037836.

Nonanticoagulant heparin reduces myocyte Na+ and Ca2+ loading during simulated ischemia and decreases reperfusion injury.

Heparin desulfated at the 2-O and 3-O positions (ODSH) decreases canine myocardial reperfusion injury. We hypothesized that this occurs from effects on ion channels rather than solely from anti-inflammatory activities, as previously proposed. We studied closed-chest pigs with balloon left anterior descending coronary artery occlusion (75-min) and reperfusion (3-h). ODSH effects on [Na(+)](i) (Na Green) and [Ca(2+)](i) (Fluo-3) were measured by flow cytometry in rabbit ventricular myocytes after 45-min of simulated ischemia [metabolic inhibition with 2 mM cyanide, 0 glucose, 37 degrees C, pacing at 0.5 Hz; i.e., pacing-metabolic inhibition (PMI)]. Na(+)/Ca(2+) exchange (NCX) activity and Na(+) channel function were assessed by voltage clamping. ODSH (15 mg/kg) 5 min before reperfusion significantly decreased myocardial necrosis, but neutrophil influx into reperfused myocardium was not consistently reduced. ODSH (100 microg/ml) reduced [Na(+)](i) and [Ca(2+)](i) during PMI. The NCX inhibitor KB-R7943 (10 microM) or the late Na(+) current (I(Na-L)) inhibitor ranolazine (10 microM) reduced [Ca(2+)](i) during PMI and prevented effects of ODSH on Ca(2+) loading. ODSH also reduced the increase in Na(+) loading in paced myocytes caused by 10 nM sea anemone toxin II, a selective activator of I(Na-L). ODSH directly stimulated NCX and reduced I(Na-L). These results suggest that in the intact heart ODSH reduces Na(+) influx during early reperfusion, when I(Na-L) is activated by a burst of reactive oxygen production. This reduces Na(+) overload and thus Ca(2+) influx via NCX. Stimulation of Ca(2+) extrusion via NCX later after reperfusion may also reduce myocyte Ca(2+) loading and decrease infarct size.

Direct toxic effects of aqueous extract of cigarette smoke on cardiac myocytes at clinically relevant concentrations.

AIMS: Our goal was to determine if clinically relevant concentrations of aqueous extract of cigarette smoke (CSE) have direct deleterious effects on ventricular myocytes during simulated ischemia, and to investigate the mechanisms involved. METHODS: CSE was prepared with a smoking chamber. Ischemia was simulated by metabolic inhibition (MI) with cyanide (CN) and 0 glucose. Adult rabbit and mouse ventricular myocyte [Ca(2+)](i) was measured by flow cytometry using fluo-3. Mitochondrial [Ca(2+)] was measured with confocal microscopy, and Rhod-2 fluorescence. The mitochondrial permeability transition (MPT) was detected by TMRM fluorescence and myocyte contracture. Myocyte oxidative stress was quantified by dichlorofluorescein (DCF) fluorescence with confocal microscopy. RESULTS: CSE 0.1% increased myocyte contracture caused by MI. The nicotine concentration (HPLC) in 0.1% CSE was 15 ng/ml, similar to that in humans after smoking cigarettes. CSE 0.1% increased mitochondrial Ca(2+) uptake, and increased the susceptibility of mitochondria to the MPT. CSE 0.1% increased DCF fluorescence in isolated myocytes, and increased [Ca(2+)](i) in paced myocytes exposed to 2.0 mM CN, 0 glucose (P-MI). These effects were inhibited by the superoxide scavenger Tiron. The effect of CSE on [Ca(2+)](i) during P-MI was also prevented by ranolazine. CONCLUSIONS: CSE in clinically relevant concentrations increases myocyte [Ca(2+)](i) during simulated ischemia, and increases myocyte susceptibility to the MPT. These effects appear to be mediated at least in part by oxidative radicals in CSE, and likely contribute to the effects of cigarette smoke to increase myocardial infarct size, and to decrease angina threshold.

Left ventricular unloading before reperfusion reduces endothelin-1 release and calcium overload in porcine myocardial infarction.

OBJECTIVES: The aim of this study was to test the hypothesis that after an acute myocardial infarction, endothelin-1 release with subsequent calcium overload is a mediator of myocardial reperfusion injury, which can be inhibited, in part, by left ventricular unloading immediately before reperfusion. We recently have reported that left ventricular unloading before reperfusion reduces infarct size after acute myocardial infarction. However, the biologic mechanisms of infarct salvage in unloaded hearts subjected to ischemia/reperfusion remain undefined. METHODS: Twelve pigs were subjected to 1 hour of left anterior descending coronary artery occlusion followed by 4 hours of reperfusion. A left ventricular assist device was initiated 15 minutes before reperfusion and maintained during reperfusion (assist device group, n = 6). A control group (n = 6) was subjected to reperfusion alone. Infarct size, endothelin-1 plasma levels, intracellular calcium levels, and apoptosis were analyzed in both groups. RESULTS: At reperfusion, left ventricular unloading significantly decreased left ventricular end-diastolic and end-systolic pressures. Infarct size, expressed as a percentage of zone at risk, was also significantly reduced by 54% in the group with the left ventricular assist device compared with controls. Support with a left ventricular assist device reduced endothelin-1 release from the heart at 15 minutes, 30 minutes, and 1 hour of reperfusion. Myocardial release of endothelin-1 was significantly correlated with infarct size at 15 minutes of reperfusion (r = 0.79; P = .008). Left ventricular unloading caused a significant reduction of calcium overload and of the percentage of apoptotic cells in the ischemic region. CONCLUSION: Our findings suggest that endothelin-1 release and calcium overload are important mediators of reperfusion injury and that they can be significantly reduced by left ventricular unloading before coronary artery reperfusion during myocardial infarction.

Ranolazine inhibits an oxidative stress-induced increase in myocyte sodium and calcium loading during simulated-demand ischemia.

Ranolazine inhibits the late Na current and is proposed to reduce angina by decreasing [Na]i during ischemia, thereby reducing Ca influx via Na/Ca exchange (NCX). We sought to test this hypothesis and to determine whether oxidative stress during simulated-demand ischemia activates the late Na current. We measured [Ca]i and [Na]i in rabbit ventricular myocytes by flow cytometry during metabolic inhibition (MI) with 2 mM cyanide and 0 mM glucose at 37 degrees C plus pacing (P) at 0.5 Hz (P-MI), and in P-MI + 1, 10, or 50 microM ranolazine. In the clinically relevant concentration range (1-10 microM), ranolazine decreased Na and Ca loading and the development of myocyte contracture. P-MI caused an increase in fluorescence of the oxidative radical probe CM-H2DCFDA, which was inhibited by the radical scavenger Tiron 20 mM. The NCX inhibitor KB-R7943 (10 microM) and Tiron 20 mM reduced the rise in [Ca]i during P-MI and eliminated the effect of 10 microM ranolazine on [Ca]i. These results indicate that oxidative stress increases the late Na current during MI. Inhibition of the resulting increase in Na and Ca loading and contracture seems to account for the observed antiischemia effects of ranolazine.

Human alpha B-crystallin mutation causes oxido-reductive stress and protein aggregation cardiomyopathy in mice.

The autosomal dominant mutation in the human alphaB-crystallin gene inducing a R120G amino acid exchange causes a multisystem, protein aggregation disease including cardiomyopathy. The pathogenesis of cardiomyopathy in this mutant (hR120GCryAB) is poorly understood. Here, we show that transgenic mice overexpressing cardiac-specific hR120GCryAB recapitulate the cardiomyopathy in humans and find that the mice are under reductive stress. The myopathic hearts show an increased recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is due to the augmented expression and enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, and glutathione peroxidase. The intercross of hR120GCryAB cardiomyopathic animals with mice with reduced G6PD levels rescues the progeny from cardiac hypertrophy and protein aggregation. These findings demonstrate that dysregulation of G6PD activity is necessary and sufficient for maladaptive reductive stress and suggest a novel therapeutic target for abrogating R120GCryAB cardiomyopathy and heart failure in humans.

Activation of coagulation during routine diagnostic coronary angiography.

AIMS: The role of anticoagulation during percutaneous coronary intervention has been well established. However, the role of anticoagulation during diagnostic coronary angiography remains unclear. Prothrombin fragment1+2 (PF1+2) and D-dimer (DD) have been reported to be useful in evaluating thrombotic phenomena. This study was designed to determine whether activation of coagulation occurs during diagnostic coronary angiography as measured by DD and PF1+2. METHODS AND RESULTS: Patients not on anticoagulation (except for aspirin) and with no documented coagulopathy undergoing elective diagnostic coronary angiography were enrolled in this prospective study. Blood samples for DD and PF1+2 were obtained serially after the femoral arterial sheath was placed. Peripheral venous blood was drawn along with an initial arterial blood sample from the sheath; thereafter, arterial blood samples from the sheath were obtained every 10 minutes for a maximum of 60 minutes or until the procedure was completed or when anticoagulation was initiated. A final venous sample was drawn at the end of the procedure. The data were analysed in time interval correlation to the DD and PF1+2 level.Forty-two patients were enrolled in this study, 15 were female (35%). There were 25 (59%) patients with diabetes. The mean fluoroscopic time was 8.8+/-7.81 minutes and the average time for the procedure was 29+/-22.70 minutes. There were 192 blood samples analysed. 67% of patient completed the procedure within 20 minutes and 91% within 30 minutes. Mean venous PF1+2 level was 0.20 nmol/L at baseline and 0.39 nmol/L (p=0.06) at the final interval, while the mean arterial PF1+2 level was significantly elevated. There was an increase of 0.2 nmol/L of arterial PF1+2 every 10 minutes (p<0.001). Mean venous DD at baseline and final levels were 0.41 ug/mL and 0.45 ug/mL respectively (p=0.68). There was a significant change in arterial DD with an increase of 0.02ug/ml every 10 minutes (p=0.023). CONCLUSIONS: In diagnostic coronary angiography, there is an early rise in PF1+2 levels in blood drawn through the arterial sheath suggesting that the procedure triggers local activation of coagulation that is not observed systemically. Prophylactic anticoagulation may not be necessary in stable patients without other known risk factors who will be undergoing elective diagnostic coronary angiography for less than 30 minutes. For procedures that are prolonged, or anticipated to be prolonged greater than 30 minutes, it may be advisable to administer anticoagulation to prevent thrombus formation. These findings may not be pertinent to patients with thrombophilia.

Na"Fuzzy space": does it exist, and is it important in ischemic injury?

Work is reviewed which provides evidence for the presence of a subsarcolemmal microdomain, in which the [Na(+)] is influenced by Na(+) influx via the Na(+) channel and the activity of the Na pump. The sarcolemma adjacent to this microdomain, which has been referred to as "Na(+) fuzzy space," appears to include Na-Ca exchangers, and thus alterations of [Na(+)] in this space may influence Ca(2+) influx and efflux, and thus Ca(2+) loading, more directly than do alterations of bulk cytoplasmic [Na(+)]. The degree of Ca(2+) loading is an important determinant of contractility in heart muscle, and therefore alterations in fuzzy space [Na(+)] may be important in mediating the positive inotropic effects of Na pump inhibitors, such as digitalis, and the negative inotropic effects of Na(+) channel blockers such as disopyramide. During ischemia, myocyte Ca(2+) overload can contribute to ATP depletion by activation of Ca(2+)-dependent ATPases and by induction of the mitochondrial permeability transition, and thus is an important contributor to myocyte dysfunction and injury. Hypoxia associated with ischemia can induce Na(+) influx via a persistent opening of the Na(+) channel. It is likely that this Na(+) influx elevates [Na(+)] in the fuzzy space, and thus increases Ca(2+) influx via Na-Ca exchange, and increases Ca(2+) loading. Inhibition of the persistent Na(+) current with a resulting decrease in Ca(2+) loading by drugs such as tetrodotoxin (TTX) and ranolazine may be a mechanism by which these agents produce a cardio-protective effect during ischemia.

CRYAB and HSPB2 deficiency increases myocyte mitochondrial permeability transition and mitochondrial calcium uptake.

Double knockout (DKO) of the small heat shock proteins CRYAB and HSPB2 increases necrosis and apoptosis induced by ischemia/reperfusion (I/R) in vitro, but the mechanisms involved are unknown. We examined [Ca2+]i during metabolic inhibition (MI) changes in [Ca2+]m induced by exposure to elevated [Ca2+]i, and whether mitochondria in isolated DKO ventricular myocytes (VM) are more susceptible than wild type (WT) to induction of the mitochondrial permeability transition (MPT). The rise in [Ca2+]i in DKO myocytes during metabolic inhibition (MI) was less than in WT, and ouabain caused a greater increase in [Ca2+]m in DKO than in WT. These findings suggested that Ca2+ uptake was increased in mitochondria in DKO myocytes. Measurements of Rhod 2 fluorescence during exposure of permeabilized VM to 1000 nM [Ca2+] for 5 min confirmed that DKO myocytes have enhanced mitochondrial Ca2+ uptake, and this difference between DKO and WT myocyte mitochondria was eliminated by inhibition of NO synthesis. MPT was induced more readily by ouabain, PAO, or TMRM in DKO myocytes than in WT. Thus, Ca2+ uptake by mitochondria is increased in DKO VM by a NO-dependent mechanism. This can predispose to the development of MPT, and increased VM injury during I/R. These findings indicate an important role of CRYAB and/or HSPB2 in mitochondrial function.

Percutaneous coronary interventions with stents in cardiac transplant recipients.

BACKGROUND: Allograft coronary vasculopathy is a major cause of death beyond the first year after cardiac transplantation. The aim of this study was to review our experience with percutaneous coronary intervention (PCI) with stents in cardiac transplant recipients. METHODS: We identified patients who were treated with PCI using stents. Patient characteristics, procedure information and clinical outcomes were assessed for these patients by review of their medical records. We also compared results for those who had bare metal stents vs those who had drug-eluting stents. RESULTS: Forty patients from our program's 865 cardiac transplant recipients received a total of 78 coronary stents. There were 35 males (87.5%) and 5 females (12.5%). The indication for PCI was progressive asymptomatic coronary vasculopathy in 18 patients (45%), angina in 5 (12.5%), acute myocardial infarction (MI) in 4 (10%) and congestive heart failure (CHF) in 6 (15%). Primary success (<50% residual stenosis) was obtained in 71 (91%) of 78 stents. During the mean follow-up of 40.8 +/- 34.5 months, 6 patients died (15%) and 2 (5%) were re-transplanted. There was a lower rate of re-stenosis with drug-eluting stents (2 of 13, 15%) compared with bare metal stents (20 of 65, 31%), although this difference was not statistically significant (p = 0.27). CONCLUSIONS: In cardiac transplant recipients, PCI with stents can be performed with high rates of primary success. Restenosis rates are higher compared with PCI in native coronary arteries. A trend toward less restenosis with drug-eluting stents was observed, which needs to be confirmed in larger studies.

Propagation of Ca2+ release in cardiac myocytes: role of mitochondria.

Factors contributing to "local control" of Ca2+ release in cardiac myocytes are incompletely understood. We induced local release of Ca2+ by regional exposure of mouse atrial and ventricular myocytes to 10mM caffeine for 500 ms using a rapid solution switcher. Propagation of Ca2+ release was imaged by means of a Nipkow confocal microscope, and fluo-3. Under physiologic conditions, a local release of Ca2+ propagated in atrial myocytes, not in ventricular myocytes. Inhibition of SR Ca2+ uptake (500 nM thapsigargin), and of Ca2+ extrusion via Na/Ca exchange (5mM Ni2+), did not result in propagation in ventricular myocytes. The density of mitochondria was greater in ventricular than in atrial myocytes, although the abundance of ryanodine receptors and myofilaments was similar. Partial inhibition of Ca2+ uptake via the mitochondrial Ca2+ uniporter (5 microM Ru360) caused an increase in the [Ca2+]i transient in paced ventricular myocytes, and consistently resulted in propagation of Ca2+ release. This effect of Ru360 did not appear to be due to altered SR Ca2+ content. These data indicate that Ca2+ uptake via the mitochondrial uniporter occurs on a beat-to-beat basis, and may contribute to local control of Ca2+ release. Propagation of Ca2+ release in atrial myocytes may result in part from the relatively low density of mitochondria present.

Relative abundance of alpha2 Na(+) pump isoform influences Na(+)-Ca(2+) exchanger currents and Ca(2+) transients in mouse ventricular myocytes.

In the mouse, genetic reduction in the Na(+), K(+)-ATPase alpha1 or alpha2 isoforms results in different functional phenotypes: heterozygous alpha2 isolated hearts are hypercontractile, whereas heterozygous alpha1 hearts are hypocontractile. We examined Na(+)/Ca(2+) exchange (NCX) currents in voltage clamped myocytes (pipette [Na(+)]=15 mM) induced by abrupt removal of extracellular Na(+). In wild-type (WT) myocytes, peak exchanger currents were 0.59+/-0.04 pA/pF (mean+/-S.E.M., n=10). In alpha1(+/-) myocytes (alpha2 isoform increased by 54%), NCX current was reduced to 0.33+/-0.05 (n=9, P<0.001) indicating a lower subsarcolemmal [Na(+)]. In alpha2(+/-) myocytes (alpha2 isoform reduced by 54%), the NCX current was increased to 0.89+/-0.11 (n=8, P=0.03). The peak sarcolemmal Na(+) pump currents activated by abrupt increase in [K(+)](o) to 4 mM in voltage clamped myocytes in which the Na(+) pump had been completely inhibited for 5 min by exposure to 0 [K(+)](o) were similar in alpha1(+/-) (0.86+/-0.12, n=10) and alpha2(+/-) myocytes (0.94+/-0.08 pA/pF, n=16), and were slightly but insignificantly reduced relative to WT (1.03+/-0.05, n=24). The fluo-3 [Ca(2+)](i) transient (F/F(o)) in WT myocytes paced at 0.5 Hz was 2.18+/-0.09, n=34, was increased in alpha2(+/-) myocytes (F/F(o)=2.56+/-0.14, n=24, P=0.02), and was decreased in alpha1(+/-) myocytes (F/F(o)=1.93+/-0.08, n=28, P<0.05). Thus the alpha2 isoform rather than the alpha1 appears to influence Na(+)/Ca(2+) exchanger currents [Ca(2+)](i) transients, and contractility. This finding is consistent with the proposal that alpha2 isoform of the Na pump preferentially alters [Na(+)] in a subsarcolemmal micro-domain adjacent to Na(+)/Ca(2+) exchanger molecules and SR Ca(2+) release sites.

Difference in propagation of Ca2+ release in atrial and ventricular myocytes.

Intracellular [Ca2+] ([Ca2+]i) was imaged in atrial and ventricular rat myocytes by means of a high-speed Nipkow confocal microscope. Atrial myocytes with an absent t-tubule system on 8-di- ANEPPS staining showed an initial rise in Ca2+ at the periphery of the cell, which propagated to the interior of the cell. Ventricular myocytes showed a uniform rise in [Ca2+]i after electrical stimulation, consistent with a prominent t-tubular network. In atrial myocytes, there was a much shorter time between the peak of the [Ca2+]i transient and the peak contraction as compared to ventricular myocytes. A regional release of Ca2+ induced by an exposure of one end of the myocyte to caffeine with a rapid solution switcher resulted in a uniform propagation of Ca2+ down the length of the cell in atrial myocytes, but we found no propagation in ventricular myocytes. A staining with rhodamine 123 indicated a much greater density of mitochondria in ventricular myocytes than in atrial myocytes. Thus the atrial myocytes display a lack of "local control" of Ca2+ release, with propagation after the Ca2+ release at the periphery induced by stimulation or at one end of the cell induced by exposure to caffeine. Ventricular myocytes showed the presence of local control, as indicated by an absence of the propagation of a local caffeine-induced Ca2+ transient. We suggest that this finding, as well as a reduced delay between the peak of the [Ca2+]i transient and the peak shortening in atrial myocytes, could be due in part to reduced Ca2+ buffering provided by mitochondria in atrial myocytes as opposed to ventricular myocytes.

A beneficial role of cardiac P2X4 receptors in heart failure: rescue of the calsequestrin overexpression model of cardiomyopathy.

The P2X4 purinergic receptor (P2X4R) is a ligand-gated ion channel. Its activation by extracellular ATP results in Ca2+ influx. Transgenic cardiac overexpression of the human P2X4 receptor showed an in vitro phenotype of enhanced basal contractility. The objective here was to determine the in vivo cardiac physiological role of this receptor. Specifically, we tested the hypothesis that this receptor plays an important role in modulating heart failure progression. Transgenic cardiac overexpression of canine calsequestrin (CSQ) showed hypertrophy, heart failure, and premature death. Crossing the P2X4R mouse with the CSQ mouse more than doubled the lifespan (182 +/- 91 days for the binary CSQ/P2X4R mouse, n = 35) of the CSQ mouse (71.3 +/- 25.4 days, n = 50, P < 0.0001). The prolonged survival in the binary CSQ/P2X4R mouse was associated with an improved left ventricular weight-to-body weight ratio and a restored beta-adrenergic responsiveness. The beneficial phenotype of the binary mouse was not associated with any downregulation of the CSQ level but correlated with improved left ventricular developed pressure and +/-dP/dt. The enhanced cardiac performance was manifested in young binary animals and persisted in older animals. The increased contractility likely underlies the survival benefit from P2X4 receptor overexpression. An increased expression or activation of this receptor may represent a new approach in the therapy of heart failure.

Saxitoxin blocks L-type ICa.

Saxitoxin (STX) and tetrodotoxin (TTX) are frequently used to selectively block sodium channels. In this study, we provide evidence that commercial STX also inhibits L-type Ca2+ currents (I(Ca,L)) in adult mouse ventricular myocytes (VMs) and tsA-201 cells that were transiently cotransfected with three calcium channel subunits. We measured inhibition of sodium currents (INa) in mouse VMs, of I(Ca,L) in mouse VM and tsA-201 cells, and intracellular calcium concentration ([Ca2+]i) transients in single mouse VMs. STX or TTX was abruptly applied before the test voltage pulse using a rapid solution switcher device. STX (10 microM; Calbiochem) and TTX (60 microM; Sigma-Aldrich) completely blocked INa in mouse VMs. However, STX at 10 microM also reduced I(Ca,L) in mouse VM by 39% (P < 0.0001; n = 14), whereas TTX at 60 microM had no effect on I(Ca,L). STX (10 microM; Calbiochem) reduced the amplitude of the [Ca2+]i transients in mouse VMs by 36% (P < 0.0001; n = 10). In contrast, TTX (60 microM; Sigma-Aldrich) only reduced the amplitude of the [Ca2+]i transients by 9% (P = 0.003; n = 5). STX (10 microM) obtained from Sigma-Aldrich showed a similar inhibitory effect on I(Ca,L) (33%) (P < 0.0001; n = 5) in mouse VMs. STX (Calbiochem) inhibited the calcium currents of tsA-201 cells in a dose-dependent manner. This inhibition was voltage-independent. The current-voltage relationship of calcium currents in tsA-201 cells was not altered by STX. These results indicate that STX partially blocks L-type Ca2+ channels and thus provide further evidence that its effects are not specific for Na+ channels.

Elective coronary angiography and percutaneous coronary intervention during uninterrupted warfarin therapy.

The management of patients anticoagulated with warfarin and referred for coronary angiography presents a substantial challenge to the physician who must minimize risks of periprocedural hemorrhage and thromboembolism. The aim of this study was to evaluate the feasibility and safety of performing diagnostic coronary angiography and percutaneous coronary intervention during uninterrupted warfarin therapy. Patients treated with warfarin were prospectively identified and enrolled in the study. Nineteen diagnostic cardiac catheterizations and six percutaneous coronary interventions were performed in 23 patients. The mean international normalized ratio was 2.4 +/- 0.5 (range, 1.8-3.5). Hemostasis was achieved with AngioSeal following 21 procedures and with Perclose following 4 procedures. No patient experienced a predefined endpoint. Specifically, no patient experienced procedure-related myocardial infarction, major or minor bleeding. We conclude that cardiac catheterization and percutaneous coronary intervention may be considered in the setting of uninterrupted warfarin therapy.

Comparison of sarcoplasmic reticulum Ca2+-ATPase function in human, dog, rabbit, and mouse ventricular myocytes.

It has been reported that sarcoplasmic reticulum (SR) Ca(2+) uptake is more rapid in rat than rabbit ventricular myocytes, but little information is available on the relative SR Ca(2+) uptake activity in others species, including humans. We induced Ca(2+) transients with a short caffeine pulse protocol (rapid solution switcher, 10 mM caffeine, 100 ms) in single ventricular myocytes voltage clamped (-80 mV) with pipettes containing 100 microM fluo-3 and nominal 0 Ca(2+), in 0 Na(+)(o)/0 Ca(2+)(o) solution to inhibit Na/Ca exchange. SR in non-paced human, dog, rabbit, and mouse ventricular myocytes could be readily loaded with Ca(2+) under our experimental conditions with a pipette [Ca(2+)] = 100 nM. Resting [Ca(2+)](i) was similar in four types of ventricular myocytes. Activation of the Ca(2+)-release channel with a 100-ms caffeine pulse produced a rise in [caffeine](i) to slightly above 2 mM, the threshold for caffeine activation of Ca(2+) release. This caused a similar initial rate of rise and peak [Ca(2+)](i) in the four types of ventricular myocytes. However, there were significant differences in the duration of the plateau (top 10%) [Ca(2+)](i) transients and the time constant of the [Ca(2+)](i) decline (reflecting activity of the SR Ca(2+)-ATPase), with values for human > dog > rabbit > mouse. In paced myocytes under physiologic conditions, SR Ca(2+) content was greater in mouse than in rabbit myocytes, while peak I(Ca,L) was smaller in mouse. These findings confirm substantial species difference in SR Ca(2+)-ATPase activity, and suggest that the smaller the animal and the more rapid the heart rate, greater the activity of the SR Ca(2+)-ATPase. In addition, it appears that substantial species differences exist in the degree of SR Ca(2+) loading and I(Ca,L) under physiologic conditions.

Ca2+ sparks induced by Na/Ca exchange.

Whether Ca(2+) influx on the Na/Ca exchanger (NCX) can trigger elementary sarcoplasmic reticulum (SR) Ca(2+) release events (Ca(2+) sparks) is controversial. We imaged [Ca(2+)](i) (Nipkow confocal microscope and fluo-3) in left ventricular myocytes isolated from wild type (WT) and transgenic (TG) mice overexpressing NCX 2.5-fold. Sudden activation of Ca(2+) influx via NCX induced by abrupt exposure to "0" [Na(+)](o)/normal [Ca(2+)](o) solution by means of a rapid solution switcher-induced Ca(2+) sparks in NCX TG myocytes in 425+/-17 ms, n=21. The diameter and amplitude (F/F(0)) of these sparks (2.74+/-0.14 microm, F/F(0)=2.16+/-0.06, n=18) were similar to those induced by field stimulation of myocytes in the presence of 20 microM nifedipine (2.70+/-0.10 microm, F/F(0)=1.98+/-0.08, n=17). In WT myocytes no Ca(2+) sparks were observed within the first 600 ms after abrupt removal of extracellular Na. In parallel experiments, voltage clamp current measurements (-80 mV) showed that the Na/Ca exchange current (I(NCX)) began within 60 ms of activation of the switcher, and peaked at 312+/-57 pA in TG myocytes within 300-500 ms. I(Ca,L) in 20 microM nifedipine was 10.3+/-4.3 pA, n=7. These results indicate that Ca(2+) entering the myocyte via NCX can cause Ca(2+) sparks which are similar to those elicited by electrical stimulation. However, Ca(2+) influx on NCX is much less efficient in inducing Ca(2+) sparks than Ca(2+) influx via I(Ca,L).