Cat Ventricular Myocytes Research Articles

Reactive oxygen species partially mediate high dose angiotensin II-induced positive inotropic effect in cat ventricular myocytes.

Reactive oxygen species, such as superoxide, are being increasingly recognized as key components of a vast array of signaling pathways. Angiotensin II is a well-recognized stimulus for superoxide production through NADPH oxidase activation and opening of the mitochondrial ATP-sensitive potassium channels (mKATP). A role for this mechanism has been proposed to explain several physiological effects of the peptide. The aim of this study was to evaluate the involvement of this mechanism in the inotropic response to 100nmol/L angiotensin II. Sarcomere shortening and intracellular pH (BCECF-epifluorescence technique) were evaluated in isolated cat ventricular myocytes placed in a perfusion chamber on the stage of an inverted microscope. Myocardial superoxide production was evaluated by the lucigenin quimioluminiscence method. Angiotensin II (100nmol/L) increased~70% sarcomere shortening, effect that was only partially prevented by NADPH oxidase inhibition, mKATP channel blockade or inhibition of the cardiac Na(+)/H(+) exchanger (NHE-1). Moreover, angiotensin II stimulates NHE-1 activity by a NADPH oxidase-dependent mechanism. Myocardial superoxide production was also increased by angiotensin II, and this action was completely prevented either by NADPH oxidase inhibition or mKATP channel blockade. The positive inotropic response to 100nmol/L angiotensin II is due to both ROS/NHE-1 dependent and independent pathways, this being a point of divergence with the signaling previously described to be triggered by lower concentrations of angiotensin II (i.e.: 1nmol/L).

Hypoxic regulation of cardiac Ca2+ channel: possible role of haem oxygenase

Acute and chronic hypoxias are common cardiac diseases that lead often to arrhythmia and impaired contractility. At the cellular level it is unclear whether the suppression of cardiac Ca(2+) channels (Ca(V)1.2) results directly from oxygen deprivation on the channel protein or is mediated by intermediary proteins affecting the channel. To address this question we measured the early effects of hypoxia (5-60 s, P(O(2)) < 5 mmHg) on Ca(2+) current (I(Ca)) and tested the involvement of protein kinase A (PKA) phosphorylation, Ca(2+)/calmodulin-mediated signalling and the haem oxygenase (HO) pathway in the hypoxic regulation of Ca(V)1.2 in rat and cat ventricular myocytes and HEK-293 cells. Hypoxic suppression of ICa) and Ca(2+) transients was significant within 5 s and intensified in the following 50 s, and was reversible. Phosphorylation by cAMP or the phosphatase inhibitor okadaic acid desensitized I(Ca) to hypoxia, while PKA inhibition by H-89 restored the sensitivity of I(Ca) to hypoxia. This phosphorylation effect was specific to Ca(2+), but not Ba(2+) or Na(+), permeating through the channel. CaMKII inhibitory peptide and Bay K8644 reversed the phosphorylation-induced desensitization to hypoxia. Mutation of CAM/CaMKII-binding motifs of the α(1c) subunit of Ca(V)1.2 fully desensitized the Ca(2+) channel to hypoxia. Rapid application of HO inhibitors (zinc protoporphyrin (ZnPP) and tin protoporphyrin (SnPP)) suppressed the channel in a manner similar to acute hypoxia such that: (1) I(Ca) and I(Ba) were suppressed within 5 s of ZnPP application; (2) PKA activation and CaMKII inhibitors desensitized I(Ca), but not I(Ba), to ZnPP; and (3) hypoxia failed to further suppress I(Ca) and I(Ba) in ZnPP-treated myocytes. We propose that the binding of HO to the CaM/CaMKII-specific motifs on Ca(2+) channel may mediate the rapid response of the channel to hypoxia.

Antibodies against the cardiac sodium/bicarbonate co-transporter (NBCe1) as pharmacological tools

Na(+) /HCO(3) (-) co-transport (NBC) regulates intracellular pH (pH(i) ) in the heart. We have studied the electrogenic NBC isoform NBCe1 by examining the effect of functional antibodies to this protein. We generated two antibodies against putative extracellular loop domains 3 (a-L3) and 4 (a-L4) of NBCe1 which recognized NBCe1 on immunoblots and immunostaining experiments. pH(i) was monitored using epi-fluorescence measurements in cat ventricular myocytes. Transport activity of total NBC and of NBCe1 in isolation were evaluated after an ammonium ion-induced acidosis (expressed as H(+) flux, J(H) , in mmol·L(-1) min(-1) at pH(i) 6.8) and during membrane depolarization with high extracellular potassium (potassium pulse, expressed as ΔpH(i) ) respectively. The potassium pulse produced a pH(i) increase of 0.18 ± 0.006 (n= 5), which was reduced by the a-L3 antibody (0.016 ± 0.019). The a-L-3 also decreased J(H) by 50%. Surprisingly, during the potassium pulse, a-L4 induced a higher pH(i) increase than control,(0.25 ± 0.018) whereas the recovery of pH(i) from acidosis was faster (J(H) was almost double the control value). In perforated-patch experiments, a-L3 prolonged and a-L4 shortened action potential duration, consistent with blockade and stimulation of NBCe1-carried anionic current respectively. Both antibodies recognized NBCe1, but they had opposing effects on the function of this transporter, as the a-L3 was inhibitory and the a-L4 was excitatory. These antibodies could be valuable in studies on the pathophysiology of NBCe1 in cardiac tissue, opening a path for their potential clinical use.

Role of reactive oxygen species (ROS) in angiotensin II-induced stimulation of the cardiac Na+/HCO3− cotransport

The sarcolemmal Na+/HCO3− cotransporter (NBC) plays an important role in intracellular pH (pHi) regulation in the heart. In the present work we studied, in isolated cat ventricular myocytes, the role of Angiotensin II (Ang II) and reactive oxygen species (ROS) production as potential activators of the NBC. pHi was measured in single cells in a medium with HCO3− using the fluorescent pH indicator BCECF. The NH4+ pulse method was used to induce an intracellular acid load and the acid efflux (JH) in the presence of the Na+/H+ exchanger blocker HOE642 (10 μM) was calculated as indicator of NBC activity. The following JH data are presented at pHi of 6.8 (⁎ and # indicate p<0.05 after ANOVA vs. control and Ang II, respectively). The basal JH (1.03±0.12 mM/min, n=11) was significantly increased in the presence of 100 nM Ang II (1.70±0.15 mM/min, n=8⁎). This effect of Ang II was abolished when we added to the extracellular solution 2 mM MPG (ROS scavenger; 0.80±0.08 mM/min, n=11#), 300 μM apocynin (NADPH oxidase blocker; 0.80±0.13 mM/min, n=6#), 500 μM 5-hydroxidecanoate (mitochondrial ATP dependent K+ channel, mKATP, blocker; 0.97±0.21 mM/min, n=9#), or the inhibitor of the MAP kinase ERK pathway U0126 (10 μM; 0.56±0.18 mM/min, n=6#). We also determined the phosphorylation of ERK during the first min of acidosis and we detected that Ang II significantly enhanced the ERK phosphorylation levels, an effect that was cancelled by scavenging ROS with MPG. In conclusion, we propose that Ang II enhances the production of ROS through the activation of the NADPH oxidase, which in turn triggers mKATP opening and mitochondrial ROS production (“ROS-induced ROS-release mechanism”). Finally, these mitochondrial ROS stimulate the ERK pathway, leading to the activation of the NBC.

L-arginine and Tetrahydrobiopterin Inhibit Mitochondrial Permeability Transition Pore by Preventing ROS Formation by Mitochondrial Nitric Oxide Synthase

Background: The functional role of mitochondrial nitric oxide synthase (mtNOS) in heart has remained a matter of debate. Methods: We used laser scanning confocal microscopy in combination with fluorescent dyes to characterize mitochondrial NO and ROS production and the permeability transition pore (PTP) activity in permeabilized cat ventricular myocytes. Results: Stimulation of mitochondrial calcium uptake resulted in a dose-dependent increase in mitochondrial NO production when L-arginine, a substrate for mtNOS, was present. The potential contribution of the caveolae-located eNOS and SR-targeted nNOS was ruled out based on the fact that disruption of caveolae with methyl-βeτα-cyclodextrin or prevention of SR uptake with thapsigargin did not affect calcium-induced NO production. Collapsing the mitochondrial membrane potential, blocking the mitochondrial calcium uniporter and respiratory chain abolished mitochondrial NO production. In the absence of L-arginine, calcium-induced NO production was significantly decreased; however an increased ROS production was observed. Inhibition of mitochondrial arginase (which limits L-arginine availiability) resulted in 50% inhibition of calcium-induced ROS production. Both mitochondrial NO and ROS production were blocked by the nNOS inhibitor (4S)-N-(4-amino-5[aminoethyl]aminopentyl]-N′-nitroguanidine and the calmodulin antagonist W-7, while the eNOS inhibitor L-NIO or the iNOS inhibitor 1400W had no effect. The superoxide dismutase mimetic MnTBAP abolished calcium-induced ROS generation and increased NO production threefold. In the absence of L-arginine, mitochondrial calcium uptake induced opening of the mitochondrial PTP, which was blocked by cyclosporin A, MnTBAP and reversed by L-arginine. The essential mtNOS co-factor tetrahydrobiopterin also inhibited mitochondrial ROS generation and PTP opening at a concentration of 100 μM, while 10 μM tetrahydrobiopterin had no effect. Conclusion: Our data demonstrate the importance of L-arginine and tetrahydrobiopterin for the regulation of mitochondrial oxidative stress and modulation of PTP opening by mtNOS.

The positive inotropic effect of endothelin-1 is mediated by mitochondrial reactive oxygen species

We have previously demonstrated the participation of reactive oxygen species (ROS) in the positive inotropic effect of a physiological concentration of Angiotensin II (Ang II, 1 nM). The objective of the present work was to evaluate the role and source of ROS generation in the positive inotropic effect produced by an equipotent concentration of endothelin-1 (ET-1, 0.4 nM). Isolated cat ventricular myocytes were used to measure sarcomere shortening with a video-camera, superoxide anion ( O 2 −) with chemiluminescence, and ROS production and intracellular pH (pH i) with epifluorescence. The ET-1-induced positive inotropic effect (40.4 ± 3.1%, n = 10, p < 0.05) was associated to an increase in ROS production (105 ± 29 fluorescence units above control, n = 6, p < 0.05). ET-1 also induced an increase in O 2 − production that was inhibited by the NADPH oxidase blocker, apocynin, and by the blockers of mitochondrial ATP-sensitive K + channels (mK ATP), glibenclamide and 5 hydroxydecanoic acid. The ET-1-induced positive inotropic effect was inhibited by apocynin (0.3 mM; 6.3 ± 6.6%, n = 13), glibenclamide (50 μM; 8.8 ± 3.5%, n = 6), 5 hydroxydecanoic acid (500 μM; 14.1 ± 8.1, n = 9), and by scavenging ROS with MPG (2 mM; 0.92 ± 5.6%, n = 8). ET-1 enhanced proton efflux (J H ) carried by the Na +/H + exchanger (NHE) after an acid load, effect that was blocked by MPG. Consistently, the ET-induced positive inotropic effect was also inhibited by the NHE selective blocker HOE642 (5 μM; 9.37 ± 6.07%, n = 7). The data show that the effect of a concentration of ET-1 that induces an increase in contractility of about 40% is totally mediated by an intracellular pathway triggered by mitochondrial ROS formation and stimulation of the NHE.

Ginsenoside Re suppresses electromechanical alternans in cat and human cardiomyocytes

Ginseng botanicals are increasingly used as complementary or alternative medicines for a variety of cardiovascular diseases, yet little is known about their cellular actions in cardiac muscle. Electromechanical alternans (EMA) is a proarrhythmic cardiac abnormality that results from disturbances of intracellular Ca(2+) homeostasis. This study sought to determine whether a purified ginsenoside extract of ginseng, Re, exerts effects to suppress EMA and to gain insight into its mechanism of action. Alternans was induced by electrically pacing cardiomyocytes at room temperature. Re (> or = 10 nM) reversibly suppressed EMA recorded from cat ventricular and atrial myocytes and Langendorff-perfused cat hearts. In cat ventricular myocytes, Re reversibly suppressed intracellular Ca(2+) concentration ([Ca(2+)](i)) transient alternans. Re exerted no significant effects on baseline action potential configuration or sarcolemmal L-type Ca(2+) current (I(Ca,L)), Na(+) current, or total K(+) conductance. In human atrial myocytes, Re suppressed mechanical alternans and exerted no effect on I(Ca,L). In cat ventricular myocytes, Re increased [Ca(2+)](i) transient amplitude and decreased sarcoplasmic reticulum (SR) Ca(2+) content, resulting in an increase in fractional SR Ca(2+) release. In SR microsomes isolated from cat ventricles, Re had no effect on SR Ca(2+) uptake. Re increased the open probability of ryanodine receptors (RyRs), i.e., SR Ca(2+)-release channels, isolated from cat ventricles and incorporated into planar lipid bilayers. We concluded that ginsenoside Re suppresses EMA in cat atrial and ventricular myocytes, cat ventricular muscle, and human atrial myocytes. The effects of Re are not mediated via actions on sarcolemmal ion channels or action potential configuration. Re acts via a subcellular mechanism to enhance the opening of RyRs and thereby overcome the impaired SR Ca(2+) release underlying EMA.

Partial Inhibition of Sarcoplasmic Reticulum Ca Release Evokes Long-Lasting Ca Release Events in Ventricular Myocytes: Role of Luminal Ca in Termination of Ca Release

In cardiac myocytes, local sarcoplasmic reticulum (SR) Ca depletion during Ca sparks is believed to play an important role in the termination of SR Ca release. We tested whether decreasing the rate of SR Ca depletion by partially inhibiting SR Ca release channels (ryanodine receptors) delays Ca spark termination. In permeabilized cat ventricular myocytes, 0.7 mM tetracaine caused almost complete Ca spark inhibition followed by a recovery significantly below control level. The recovery was associated with increased SR Ca load and increased Ca spark duration. Additionally, SR Ca release events lasting several hundred milliseconds occurred consistently. These events had a significantly lower initial Ca release flux followed by a stable plateau, indicating delayed release termination and maintained SR Ca load. Increasing SR Ca load (without inhibiting SR Ca release rate) or decreasing SR Ca release rate (without increasing SR Ca load) both induced only a small increase in spark duration. These results show that the combination of decreased release flux and increased SR Ca load has synergistic effects and exerts major changes on the termination of Ca release events. Long-lasting Ca release events may originate from highly interconnected release junctions where Ca diffusion from neighboring sites partially compensates Ca depletion, thereby delaying SR Ca-dependent termination. Eventually, these events terminate by luminal Ca-independent mechanisms, such as inactivation, adaptation, or stochastic attrition.

Signalling mechanisms in contraction‐mediated stimulation of intracellular NO production in cat ventricular myocytes

In this study we sought to determine whether contractile activity has a role as a signalling mechanism in the activation of intracellular nitric oxide (NO(i)) production induced by electrical stimulation of cat ventricular myocytes. Field stimulation (FS) of single ventricular myocytes elicited frequency-dependent increases in NO(i) that were blocked by the calmodulin (CaM) inhibitor 10 microM W-7 and partially inhibited by the phosphatidylinositol 3'-kinase (PI-(3)K) inhibitor 10 microMm LY294002. Increasing extracellular [Ca(2+)] caused a concentration-dependent increase in FS-induced NO(i) that was partially inhibited by LY294002. The negative inotropic agents BDM (5 mm) or blebbistatin (10 microM) decreased cell shortening and NO(i) production without concomitant changes in L-type Ca(2+) current (I(Ca,L)) or [Ca(2+)](i) transients. The positive inotropic agents EMD 57033 or CGP 48506 (1 microM) increased cell shortening and NO(i) production without concomitant changes in I(Ca,L) or [Ca(2+)](i) transients. FS-induced NO(i) production was decreased in myocytes infected (100 multiplicity of viral infection (MOI); 24 h) with a replication-deficient adenovirus expressing a dominant-negative mutant of protein kinase B (Akt) compared with cells infected with a control adenovirus expressing beta-galactosidase. FS-induced NO(i) was partially inhibited by either endothelial (eNOS) or neuronal nitric oxide synthase (nNOS) inhibitors and completely blocked by simultaneous exposure to both. FS-induced [Ca(2+)](i) transients were increased by the nNOS inhibitor nNOS-I (0.24 microM), decreased by the eNOS inhibitor L-NIO (1 microM) and unchanged by exposure to both inhibitors. We conclude that in cat ventricular myocytes, FS-induced NO(i) production requires both Ca(2+)-dependent CaM signalling and Ca(2+)-independent PI-(3)K-Akt signalling activated by contractile activity. FS activates NO(i) production from both eNOS and nNOS, and each source of NO(i) exerts opposing effects on [Ca(2+)](i) transient amplitude. These findings are important for understanding the regulation of NO(i) signalling in the normal and mechanically failing heart.

The electrogenic Na+/HCO3− cotransport modulates resting membrane potential and action potential duration in cat ventricular myocytes

Perforated whole-cell configuration of patch clamp was used to determine the contribution of the electrogenic Na+/HCO3- cotransport (NBC) on the shape of the action potential in cat ventricular myocytes. Switching from Hepes to HCO3- buffer at constant extracellular pH (pH(o)) hyperpolarized resting membrane potential (RMP) by 2.67 +/- 0.42 mV (n = 9, P < 0.05). The duration of action potential measured at 50% of repolarization time (APD50) was 35.8 +/- 6.8% shorter in the presence of HCO3- than in its absence (n = 9, P < 0.05). The anion blocker SITS prevented and reversed the HCO3- -induced hyperpolarization and shortening of APD. In addition, no HCO3- -induced hyperpolarization and APD shortening was observed in the absence of extracellular Na+. Quasi-steady-state currents were evoked by 8 s duration voltage-clamped ramps ranging from -130 to +30 mV. A novel component of SITS-sensitive current was observed in the presence of HCO3-. The HCO3- -sensitive current reversed at -87 +/- 5 mV (n = 7), a value close to the expected reversal potential of an electrogenic Na+/HCO3- cotransport with a HCO3-:Na+ stoichiometry ratio of 2: 1. The above results allow us to conclude that the cardiac electrogenic Na+/HCO3- cotransport has a relevant influence on RMP and APD of cat ventricular cells.

Cytosolic energy reserves determine the effect of glycolytic sugar phosphates on sarcoplasmic reticulum Ca2+ release in cat ventricular myocytes

Localization of glycolytic enzymes in close proximity to Ca(2+) transport systems of the sarcoplasmic reticulum (SR) in cardiac cells suggests an important functional role for glycolysis in intracellular [Ca(2+)] regulation and, consequently, excitation-contraction coupling. Here, we investigated the mechanisms of regulation of SR Ca(2+) release by glycolytic sugar phosphate intermediates in cat ventricular myocytes. Experiments with permeabilized myocytes revealed that with normal cytosolic energy reserves (mm: ATP 5, ADP 0.01, phosphocreatine (CrP) 10) fructose-1,6-bisphosphate (FBP; 1 mm) and fructose-6-phosphate (F6P; 1 mm) caused a transient increase of Ca(2+) spark frequency by 62 and 42%, respectively. This effect of sugar phosphates was associated with a 13% decrease in SR Ca(2+) load. Pretreatment of the cells with an inhibitor of glycolysis, iodoacetate (IAA; 0.5 mm), did not prevent the effects of FBP and F6P on Ca(2+) sparks. Recording of single ryanodine receptor (RyR) channel activity indicated that FBP and F6P significantly increased RyR open probability. Reduction of cytosolic energy reserves decreased Ca(2+) spark activity. Increasing [ADP] to 0.4 mm or removal of CrP ([ATP] was kept constant) caused a slowly developing decrease of Ca(2+) spark frequency by 29 and 42%, respectively. Changing [ADP] and [CrP] simultaneously decreased Ca(2+) spark frequency by 66%. This inhibition of Ca(2+) sparks was associated with a 40% decrease in SR Ca(2+) load. The subsequent addition of FBP (1 mm) partially restored Ca(2+) spark frequency and SR Ca(2+) load. This recovery of Ca(2+) sparks was blocked completely by IAA. These data suggest that at physiological ATP, ADP and CrP levels accumulation of sugar phosphates from glycolysis can stimulate SR Ca(2+) release. This effect does not require the activity of downstream glycolytic enzymes, but rather is the result of direct activation of RyRs. However, under conditions associated with depletion of cellular energy reserves (e.g. myocardial ischaemia), ATP generated from glycolysis may play an important role in maintaining myocardial Ca(2+) homeostasis by improving SR Ca(2+) uptake.

The inotropic effect of cardioactive glycosides in ventricular myocytes requires Na+–Ca2+ exchanger function

Glycoside-induced cardiac inotropy has traditionally been attributed to direct Na(+)-K(+)-ATPase inhibition, causing increased intracellular [Na(+)] and consequent Ca(2+) gain via the Na(+)-Ca(2+) exchanger (NCX). However, recent studies suggested alternative mechanisms of glycoside-induced inotropy: (1) direct activation of sarcoplasmic reticulum Ca(2+) release channels (ryanodine receptors; RyRs); (2) increased Ca(2+) selectivity of Na(+) channels (slip-mode conductance); and (3) other signal transduction pathways. None of these proposed mechanisms requires NCX or an altered [Na(+)] gradient. Here we tested the ability of ouabain (OUA, 3 microm), digoxin (DIG, 20 microm) or acetylstrophanthidin (ACS, 4 microm) to alter Ca(2+) transients in completely Na(+)-free conditions in intact ferret and cat ventricular myocytes. We also tested whether OUA directly activates RyRs in permeabilized cat myocytes (measuring Ca(2+) sparks by confocal microscopy). In intact ferret myocytes (stimulated at 0.2 Hz), DIG and ACS enhanced Ca(2+) transients and cell shortening during twitches, as expected. However, prior depletion of [Na(+)](i) (in Na(+)-free, Ca(2+)-free solution) and in Na(+)-free solution (replaced by Li(+)) the inotropic effects of DIG and ACS were completely prevented. In voltage-clamped cat myocytes, OUA increased Ca(2+) transients by 48 +/- 4% but OUA had no effect in Na(+)-depleted cells (replaced by N-methyl-d-glucamine). In permeabilized cat myocytes, OUA did not change Ca(2+) spark frequency, amplitude or spatial spread (although spark duration was slightly prolonged). We conclude that the acute inotropic effects of DIG, ACS and OUA (and the effects on RyRs) depend on the presence of Na(+) and a functional NCX in ferret and cat myocytes (rather than alternate Na(+)-independent mechanisms).

Integration of rapid cytosolic Ca2+ signals by mitochondria in cat ventricular myocytes

Decoding of fast cytosolic Ca(2+) concentration ([Ca(2+)](i)) transients by mitochondria was studied in permeabilized cat ventricular myocytes. Mitochondrial [Ca(2+)] ([Ca(2+)](m)) was measured with fluo-3 trapped inside mitochondria after removal of cytosolic indicator by plasma membrane permeabilization with digitonin. Elevation of extramitochondrial [Ca(2+)] ([Ca(2+)](em)) to >0.5 microM resulted in a [Ca(2+)](em)-dependent increase in the rate of mitochondrial Ca(2+) accumulation ([Ca(2+)](em) resulting in half-maximal rate of Ca(2+) accumulation = 4.4 microM) via Ca(2+) uniporter. Ca(2+) uptake was sensitive to the Ca(2+) uniporter blocker ruthenium red and the protonophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone and depended on inorganic phosphate concentration. The rates of [Ca(2+)](m) increase and recovery were dependent on the extramitochondrial [Na(+)] ([Na(+)](em)) due to Ca(2+) extrusion via mitochondrial Na(+)/Ca(2+) exchanger. The maximal rate of Ca(2+) extrusion was observed with [Na(+)](em) in the range of 20-40 mM. Rapid switching (0.25-1 Hz) of [Ca(2+)](em) between 0 and 100 microM simulated rapid beat-to-beat changes in [Ca(2+)](i) (with [Ca(2+)](i) transient duration of 100-500 ms). No [Ca(2+)](m) oscillations were observed, either under conditions of maximal rate of Ca(2+) uptake (100 microM [Ca(2+)](em), 0 [Na(+)](em)) or with maximal rate of Ca(2+) removal (0 [Ca(2+)](em), 40 mM [Na(+)](em)). The slow frequency-dependent increase of [Ca(2+)](m) argues against a rapid transmission of Ca(2+) signals between cytosol and mitochondria on a beat-to-beat basis in the heart. [Ca(2+)](m) changes elicited by continuous or pulsatile exposure to elevated [Ca(2+)](em) showed no difference in mitochondrial Ca(2+) uptake. Thus in cardiac myocytes fast [Ca(2+)](i) transients are integrated by mitochondrial Ca(2+) transport systems, resulting in a frequency-dependent net mitochondrial Ca(2+) accumulation.

The D3-dopaminergic Agonist 7-hydroxy-dipropylaminotetralin (7-OH-DPAT) Increases Cardiac Action Potential Duration and Blocks Human Ether-a-go-go-related Gene K+ Channel

The D3-dopaminergic agonist (+/-) 7-hydroxy-dipropylaminotetralin (7-OH-DPAT) prolonged cycle length and action potential duration, depolarized maximum diastolic potential, and reduced the upstroke velocity of the action potential of rabbit sinoatrial node cells. These effects were not mediated by D3-dopaminergic receptors. In cat Purkinje fibers, the drug increased action potential duration. In voltage-clamped cat ventricular myocytes, 7-OH-DPAT blocked the rapid component of the delayed rectifier potassium current, IKr. This effect was corroborated in experiments studying the effect of the drug on human Ether-a-go-go-related Gene channels expressed in Xenopus oocytes and in HEK293 cells. We conclude that the direct electrophysiologic effects of 7-OH-DPAT on cardiac tissues are caused by the blockade of the rapid component of the delayed rectifier potassium current, IKr.

Biosensors to Measure Inositol 1,4,5-Trisphosphate Concentration in Living Cells with Spatiotemporal Resolution

Phosphoinositides participate in many signaling cascades via phospholipase C stimulation, which hydrolyzes phosphatidylinositol 4,5-bisphosphate, producing second messengers diacylglycerol and inositol 1,4,5-trisphosphate (InsP3). Destructive chemical approaches required to measure [InsP3] limit spatiotemporal understanding of subcellular InsP3 signaling. We constructed novel fluorescence resonance energy transfer-based InsP3 biosensors called FIRE (fluorescent InsP3-responsive element) by fusing plasmids encoding the InsP3-binding domain of InsP3 receptors (types 1-3) between cyan fluorescent protein and yellow fluorescent protein sequences. FIRE was expressed and characterized in COS-1 cells, cultured neonatal cardiac myocytes, and incorporated into an adenoviral vector for expression in adult cardiac ventricular myocytes. FIRE-1 exhibits an approximately 11% increase in the fluorescence ratio (F530/F480) at saturating [InsP3] (apparent K(d) = 31.3 +/- 6.7 nm InsP3). In COS-1 cells, neonatal rat cardiac myocytes and adult cat ventricular myocytes FIRE-1 exhibited comparable dynamic range and a 10% increase in donor (cyan fluorescent protein) fluorescence upon bleach of yellow fluorescent protein, indicative of fluorescence resonance energy transfer. In FIRE-1 expressing ventricular myocytes endothelin-1, phenylephrine, and angiotensin II all produced rapid and spatially resolved increases in [InsP3] using confocal microscopy (with free [InsP3] rising to approximately 30 nm). Local entry of intracellular InsP3 via membrane rupture by a patch pipette (containing InsP3)in myocytes expressing FIRE-1 allowed detailed spatiotemporal monitoring of intracellular InsP3 diffusion. Both endothelin-1-induced and direct InsP3 application (via pipette rupture) revealed that InsP3 diffusion into the nucleus occurs with a delay and blunted rise of [InsP3] versus cytosolic [InsP3]. These new biosensors allow studying InsP3 dynamics at high temporal and spatial resolution that will be powerful in under-standing InsP3 signaling in intact cells.

Endothelin-1 Stimulates the Na + /Ca 2+ Exchanger Reverse Mode Through Intracellular Na + (Na + i )–Dependent and Na + i -Independent Pathways

This study aimed to explore the signaling pathways involved in the positive inotropic effect (PIE) of low doses of endothelin-1 (ET-1). Cat papillary muscles were used for force and intracellular Na(+) concentration (Na(+)(i)) measurements, and isolated cat ventricular myocytes for patch-clamp experiments. ET-1 (5 nmol/L) induced a PIE and an associated increase in Na(+)(i) that were abolished by Na(+)/H(+) exchanger (NHE) inhibition with HOE642. Reverse-mode Na(+)/Ca(2+) exchanger (NCX) blockade with KB-R7943 reversed the ET-1-induced PIE. These results suggest that the ET-1-induced PIE is totally attributable to the NHE-mediated Na(+)(i) increase. However, an additional direct stimulating effect of ET-1 on NCX after the necessary increase in Na(+)(i) could occur. Thus, the ET-1-induced increase in Na(+)(i) and contractility was compared with that induced by partial inhibition of the Na(+)/K(+) ATPase by lowering extracellular K(+) (K(+)(o)). For a given Na(+)(i), ET-1 induced a greater PIE than low K(+)(o). In the presence of HOE642 and after increasing contractility and Na(+)(i) by low K(+)(o), ET-1 induced an additional PIE that was reversed by KB-R7943 or the protein kinase C (PKC) inhibitor chelerythrine. ET-1 increased the NCX current and negatively shifted the NCX reversal potential (E(NCX)). HOE642 attenuated the increase in NCX outward current and abolished the E(NCX) shift. These results indicate that whereas the NHE-mediated ET-1-induced increase in Na(+)(i) seems to be mandatory to drive NCX in reverse and enhance contractility, Na(+)(i)-independent and PKC-dependent NCX stimulation appears to additionally contribute to the PIE. However, it is important to stress that the latter can only occur after the primary participation of the former.

Ouabain increases sarcoplasmic reticulum calcium release in cardiac myocytes.

The inotropic and toxic effects of cardiac glycosides are thought to be related to their ability to inhibit the Na,K-ATPase. We examined the effects of ouabain and its analogs on sarcoplasmic reticulum (SR) Ca(2+) release in intact cat ventricular myocytes under Na(+)-free conditions and in myocytes in which the sarcolemma was permeabilized using saponin so that cytoplasmic ionic composition was fixed by the bath solutions. We also compared ouabain actions in cat myocytes to those in rat myocytes because the latter is considered to be a glycoside-insensitive species. In intact cat myocytes (Na(+)-free conditions), spontaneous Ca(2+) sparks were prolonged and frequency, amplitude and width were reduced by exposure to ouabain (3 microM). Nearly identical results were obtained with its analogs dihydroouabain or ouabagenin (10 microM). The frequency of spontaneous Ca(2+) waves was also reduced by ouabain. In contrast, ouabain (100 microM) had negligible effects on sparks and waves in rat myocytes in Na(+)-free conditions, consistent with the decreased sensitivity to cardiac glycosides observed in this species. In cat myocytes permeabilized with saponin (0.01%), ouabain (>or=50 nM) decreased spark frequency and increased background SR Ca(2+) leak only when the SR was well loaded (free [Ca(2+)] = 275 nM) and not when SR load was low (free [Ca(2+)] = 50 nM). Similar effects were observed in rat myocytes only when ouabain concentration was 1 microM. These results suggest that the cellular actions of cardiac glycosides may include a direct effect on SR Ca(2+) release, possibly through activation of SR Ca(2+) release channels (ryanodine receptors). In addition, these results are consistent with the idea that direct activation of SR Ca(2+) release is dependent on the extent of SR Ca(2+) load, with elevated load increasing sensitivity of the channel release mechanism to activation by glycoside.

Effect of cardiac glycosides on action potential characteristics and contractility in cat ventricular myocytes: role of calcium overload.

There is increasing evidence that cardiac glycosides act through mechanisms distinct from inhibition of the sodium pump but which may contribute to their cardiac actions. To more fully define differences between agents indicative of multiple sites of action, we studied changes in contractility and action potential (AP) configuration in cat ventricular myocytes produced by six cardiac glycosides (ouabain, ouabagenin, dihydroouabain, actodigin, digoxin, and resibufogenin). AP shortening was observed only with ouabain and actodigin. There was extensive inotropic variability between agents, with some giving full inotropic effects before automaticity occurred whereas others produced minimal inotropy before toxicity. AP shortening was not a result of alterations in calcium current or the inward rectifier potassium current, but correlated with an increase in steady-state outward current (Iss), which was sensitive to KB-R7943, a Na+-Ca2+ exchange (NCX) inhibitor. Interestingly, Iss was observed following exposure to ouabain and dihydroouabain, suggesting that an additional mechanism is operative with dihydroouabain that prevents AP shortening. Further investigation into differences in inotropy between ouabagenin, dihydroouabain and ouabain revealed almost identical responses under AP voltage clamp. Thus all agents appear to act on the sodium pump and thereby secondarily increase the outward reverse mode NCX current, but the extent of AP duration shortening and positive inotropy elicited by each agent is limited by development of their toxic actions. The quantitative differences between cardiac glycosides suggest that mechanisms independent of sodium pump inhibition may result from an altered threshold for calcium overload possibly involving direct or indirect effects on calcium release from the sarcoplasmic reticulum.

Na(+)-Ca2+ exchange function underlying contraction frequency inotropy in the cat myocardium.

In most mammalian species, an increase in stimulation frequency (ISF) produces an increase in contractility (treppe phenomenon), which results from larger Ca2+ transients at higher frequencies, due to an increase in sarcoplasmic reticulum Ca2+ load and release. The present study attempts to elucidate the contribution of the Na(+)-Ca2+ exchanger (NCX) to this phenomenon. Isolated cat ventricular myocytes, loaded with [Ca2+]i- and [Na+]i-sensitive probes, were used to determine whether the contribution of the NCX to the positive inotropic effect of ISF is due to an increase in Ca2+ influx (reverse mode) and/or a decrease in Ca2+ efflux (forward mode) via the NCX, due to frequency-induced [Na+]i elevation, or whether it was due to the reduced time for the NCX to extrude Ca2+. The results showed that the positive intropic effect produced by ISF was temporally dissociated from the increase in [Na+]i and was not modified by KB-R7943 (1 or 5 microM), a specific blocker of the reverse mode of the NCX. Whereas the ISF from 10 to 30 beats min(-1) (bpm) did not affect the forward mode of the NCX (assessed by the time to half-relaxation of the caffeine-induced Ca2+ transient), the ISF to 50 bpm produced a significant reduction of the activity of the forward mode of the NCX, which occurred in association with an increase in [Na+]i (from 4.33+/-0.40 to 7.25+/-0.50 mM). However, both changes became significant well after the maximal positive inotropic effect had been reached. In contrast, the positive inotropic effect produced by ISF from 10 to 50 bpm was associated with an increase in diastolic [Ca2+]i, which occurred in spite of a significant increase in the relaxation rate and at a time at which no increases in [Na+]i were detected. The contribution of the NCX to stimulus frequency inotropy would therefore depend on a decrease in NCX-mediated Ca2+ efflux due to the reduced diastolic interval between beats and not on [Na+]i-dependent mechanisms.

Positive inotropic effects of ouabain in isolated cat ventricular myocytes in sodium-free conditions.

The inotropic and toxic effects of cardiac steroids are thought to result from Na(+)-K(+)-ATPase inhibition, with elevated intracellular Na(+)(Na)causing increased intracellular Ca(2+)(Ca) via Na-Ca exchange. We studied the effects of ouabain on cat ventricular myocytes in Na(+)-free conditions where the exchanger is inhibited. Cell shortening and Ca transients (with fluo 4-AM fluorescence) were measured under voltage clamp during exposure to Na(+)-free solutions [LiCl or N-methyl-D-glucamine (NMDG) replacement]. Ouabain enhanced contractility by 121 +/- 55% at 1 micromol/l (n = 11) and 476 +/- 159% at 3 micromol/l (n = 8) (means +/- SE). Ca transient amplitude was also increased. The inotropic effects of ouabain were retained even after pretreatment with saxitoxin (5 micromol/l) or changing the holding potential to -40 mV (to inactivate Na(+) current). Similar results were obtained with both Li(+) and NMDG replacement and in the absence of external K(+), indicating that ouabain produced positive inotropy in the absence of functional Na-Ca exchange and Na(+)-K(+)-ATPase activity. In contrast, ouabain had no inotropic response in rat ventricular myocytes (10-100 micromol/l). Finally, ouabain reversibly increased Ca(2+) overload toxicity by accelerating the rate of spontaneous aftercontractions (n = 13). These results suggest that the cellular effects of ouabain on the heart may include actions independent of Na(+)-K(+)-ATPase inhibition, Na-Ca exchange, and changes in Na.