Single Ventricular Myocytes Research Articles

Regulation of Intracellular Calcium by Bupivacaine Isomers in Cardiac Myocytes from Wistar Rats

In this study we investigated the effects of a racemic mixture of bupivacaine (RS(+/-)bupivacaine) and its isomers (S(-)bupivacaine and R(+)bupivacaine) on the Ca2+ handling by ventricular myocytes from Wistar rats. Single ventricular myocytes were enzymatically isolated and loaded with the fluorescent Ca2+ indicator fura 2-am to estimate intracellular Ca2+ concentration during contraction and relaxation cycles. S(-)bupivacaine (10 muM) significantly increased peak amplitude and the rate of increase of Ca2+ transients in 155% +/- 54% (P < 0.05) and 194% +/- 94% (P < 0.01) of control. However, exposure to R(+)bupivacaine had no effect on either peak amplitude or rate of increase at any concentration tested. Saponin-skinned ventricular fibers were used to investigate the effect of bupivacaine on the intracellular Ca2+ regulation by sarcoplasmic reticulum (SR) and on the Ca2+ sensitivity of contractile system. S(-), R(+), and RS(+/-)bupivacaine induced Ca2+ release from SR (P < 0.01). In SR-disrupted skinned ventricular cells, bupivacaine and its isomers (5 mM) increased the sensitivity of contractile system to Ca(2+). S(-), RS(+/-), and R(+)bupivacaine significantly increased pCa50 from 5.8 +/- 0.1, 5.8 +/- 0.1, and 5.8 +/- 0.1, to 6.1 +/- 0.1 (P < 0.05), 6.0 +/- 0.1 (P < 0.05), and 6.1 +/- 0.1 (P < 0.05). Ca2+ release from SR through RyR2 activation could explain the increase of Ca2+ transients in cardiac cells. Increased intracellular Ca2+ in cardiac myocytes display a stereoselectivity to S(-)bupivacaine.

17β-Estradiol Potentiates the Cardiac Cystic Fibrosis Transmembrane Conductance Regulator Chloride Current in Guinea-pig Ventricular Myocytes

There is a well-characterized membrane chloride current (ICl,cAMP) in the heart that can be activated by beta-adrenergic agonists and is due to expression of the cardiac isoform of the epithelial cystic fibrosis transmembrane conductance regulator (CFTR). We have investigated whether 17beta-estradiol (E2) modulates ICl,cAMP in single ventricular myocytes. Under whole-cell tight-seal voltage-clamp conditions, ICl,cAMP was evoked by exposing cells to 20 nM isoprenaline. On the addition of 30 microM E2, membrane slope conductance, measured at potentials near 0 mV, increased over that induced by isoprenaline alone by 2.46 +/- 0.16 (p < 0.001). The effects of E2 were concentration-dependent and described by a Hill Plot with an EC50 of 8.2 microM and a Hill coefficient of 1.63. The application of membrane-impermeant E2 conjugated to bovine serum albumin (E2-BSA) potentiated isoprenaline-evoked ICl,cAMP by approximately the same degree as that for the equivalent level of free E2. Cell surface binding was observed with confocal microscopy by using BSA-FITC tagged E2. This binding was inhibited by nonlabeled, nonconjugate E2, the specific E2 antagonist ICl 182,780, and incubation of E2coBSA with a specific anti-E2 antibody (E2885). ICl 182,780 (100 microM) significantly reduced the increase in ICl,cAMP evoked by 10 microM E2 to 1.46 +/- 0.10 (p < 0.02). The preincubation of myocytes with the NOS inhibitor N-omega-nitro-arginine (L-NNA, 1 mM) reduced the potentiation of ICl,cAMP by 30 microM E2, to 1.93 +/- 0.06 (p < 0.02), and for 10 microM E2, to 1.32 +/- 0.05 (p < 0.002). E2 also increased ICl,cAMP evoked by bath application of 0.5 microM Forskolin. These experiments demonstrate that, under our experimental conditions, E2 dramatically increases ICl,cAMP in ventricular myocytes by mechanisms involving a contribution by NOS, but that can be only partially accounted for through binding to classical plasma membrane estrogen receptor sites. This potentiation of ICl,cAMP by E2 may play a significant role in the observed clinical actions of E2 on the incidence of cardiac arrhythmias and hypertrophy.

22. Creation of a Biological Pacemaker by Cell Fusion

As an alternative strategy to electronic pacemakers or to gene therapy/stem cell approaches, we explored the feasibility of converting ventricular myocytes into pacemakers by cell fusion. The idea is to create chemically-induced heterokaryons between ventricular myocytes and syngeneic fibroblasts engineered to express pacemaker ion channels, HCN1. In order to examine fusion events, guinea pig (gp) fibroblasts stably-expressing HCN1 channels encoding the pacemaker current, If, were fused with freshly-isolated guinea-pig ventricular myocytes using polyethylene glycol (PEG 1500). Within 3 minutes of dehydration and rehydration, the gp fibroblasts with GFP as a reporter fused with ventricular myocytes as verified by the sudden introduction of GFP fluorescence into the myocytes. Current-clamp of fused myocyte-fibroblast cells exhibited spontaneous action potentials with a slow phase-4 depolarization. Subsequent voltage-clamp recordings with 2 mM external Ba2+ to block IK1 revealed the heterologously expressed If, which was not detectable either in ventricular myocytes alone or in myocytes fused with control fibroblasts expressing only GFP. Equipped with these data, we focally-injected the HCN1-expressing gp fibroblasts suspended in 40% PEG 1500 into the apex of gp heart. Electrocardiograms taken 3-6 days after the injection revealed ectopic ventricular beats that were identical in polarity and similar in morphology to those recorded during bipolar pace-mapping of the apex in the same animal (n = 5 of 13). These ectopic beats were not observed in animals injected with control fibroblasts (n = 7). Furthermore, single ventricular myocytes were isolated from the site of cell-injection to measure the amount of pacemaker currents from the heterokaryons. Freshly isolated heterokaryons formed by fusion between myocytes and fibroblasts expressing HCN1-GFP expressed pacemaker current density of -12 ± 2 pA/pF at -130 mV (n=9), which is comparable to the reported values of pacemaker density in sinoatrial nodal cells. Control heterokaryons formed by fusion between myocytes and fibroblasts expressing GFP alone did not exhibit significant amount of inward currents (-0.2 ± 0.5 pA/pF at -130 mV, n=7). The membrane capacitances from the GFP-positive heterokaryons were significantly larger than the GFP-negative myocytes (124 ± 14 pF, n=9, and 97 ± 8 pF, n=15, respectively), providing a strong evidence for in vivo fusion events. In order to examine the possibility of cell-cell communication via gap junction proteins between fibroblasts and myocytes, immunocytochemistry against Connexin 43 was performed. Evidence of Connexin 43 was present in gp myocytes but absent in gp fibroblasts suggesting that the If which is responsible for the pacemaker activity has likely been generated from the fused myocyte-fibroblast cells exclusively rather than electrotonic coupling between myocytes and fibroblasts. Unlike previous biological pacemakers, the present approach is independent of cell-cell coupling and can be implemented with autologous, nonviral gene therapy using ubiquitous adult cells.

Selective modulation of L-type calcium current by magnesium lithospermate B in guinea-pig ventricular myocytes

Magnesium lithospermate B (MLB) is the main water-soluble principle of Salviae Miltiorrhizae Radix (also called as ‘Danshen’ in the traditional Chinese medicine) for the treatment of cardiovascular diseases. MLB was found to possess a variety of pharmacological actions. However, it is unclear whether and how MLB affects the cardiac ion channels. In the present study, the effects of MLB on the voltage-activated ionic currents were investigated in single ventricular myocytes of adult guinea pigs. MLB reversibly inhibited L-type Ca 2+ current ( I Ca,L). The inhibition was use-dependent and voltage-dependent (the IC 50 value of MLB was 30 μM and 393 μM, respectively, at the holding potential of − 50 mV and − 100 mV). In the presence of 100 μM MLB, both the activation and steady-state inactivation curves of I Ca,L were markedly shifted to hyperpolarizing membrane potentials, whereas the time course of recovery of I Ca,L from inactivation was not altered. MLB up to 300 μM had no significant effect on the fast-inactivating Na + current ( I Na), delayed rectifier K + current ( I K) and inward rectifier K + current ( I K1). The results suggest that the voltage-dependent Ca 2+ antagonistic effect of MLB work in concert with its antioxidant action for attenuating heart ischemic injury.

The Amiodarone Derivative KB130015 [2-Methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] Induces an Na+-Dependent Increase of [Ca2+] in Ventricular Myocytes

KB130015 [KB; 2-methyl-3-(3,5-diiodo-4-carboxymethoxybenzyl)benzofuran] is a novel amiodarone derivative designed to retain the antiarrhythmic effects without the side effects. Unlike amiodarone, KB slows Na(+) current inactivation and could, via an increase in [Na(+)](i), potentially lead to Ca(2+) overload. Therefore, we studied the effects of KB on Na(+) and Ca(2+) handling in single pig ventricular myocytes using the whole-cell ruptured patch-clamp technique and K(5)fluo-3 as [Ca(2+)](i) indicator. KB at 10 microM did not prolong action potential duration but slightly increased the early plateau; spontaneous afterdepolarizations were not observed. The amplitude of the [Ca(2+)](i) transient was larger (434.9 +/- 37.2 versus 326.8 +/- 39.8 nM at baseline, n = 13, P < 0.05), and the time to peak [Ca(2+)](i) was prolonged. During voltage-clamp pulses, [Ca(2+)](i) transient peak was also larger (578.1 +/- 98.9 versus 346.4 +/- 52.6 nM at baseline, P < 0.05). Although L-type Ca(2+) current was reduced (by 21.9% at +10 mV, n = 9, P < 0.05), sarcoplasmic reticulum Ca(2+) content was significantly enhanced with KB. Forward Na(+)/Ca(2+) exchange was significantly decreased after KB application, but reverse mode of the Na(+)/Ca(2+) exchanger was significantly larger, suggesting an increase in [Na(+)](i) with KB. This was confirmed by a 2-fold increase of the [Na(+)]-dependent current generated by the Na/K-ATPase (from 0.17 +/- 0.02 to 0.38 +/- 0.06 pA/pF, P < 0.05). In conclusion, as predicted from the slowing of I(Na) inactivation, KB130015 leads to an increase in [Na(+)](i) and consequently in cellular Ca(2+) load. This effect is partially offset by a decrease in I(CaL) resulting in a mild inotropic effect without the signs of Ca(2+) overload and related arrhythmias usually associated with Na(+) channel openers.

Effects of ischaemia‐mimetic factors on isolated rat ventricular myocytes

Conventional ischaemia-mimetic solutions contain several key components for inducing hypoxia, glucose deficiency, acidosis, lactate accumulation and hyperosmosis. The effect of each component on myocyte contractility during cardiac ischaemia was investigated in this study. A video-based edge-detection system was used to monitor single ventricular myocytes isolated from the rat. The effect of each factor was compared by preparing the following ischaemia-mimetic solutions: solution A, containing all of the above-mentioned factors; and solutions B, C, D, E and F, each with one of the factors excluded. The solutions that contained lactate severely reduced the contractility of the cardiomyocytes, but cell contraction did not differ significantly between the cardiomyocytes in these solutions. The effect of the solution without the acidosis-inducing component was weaker than that of the conventional ischaemia-mimetic solution. The solution lacking lactate produced the least depression of cell contractility. Lactate impaired cardiomyocyte contractility in a concentration-dependent manner. Our observations suggest that lactate is the main contributor to cardiac ischaemic injury and that its effects are attributable to acidosis and are concentration dependent. Imposition of hypoxia, glucose deficiency and hyperosmosis had little impact on the cardiomyocytes.

Plasma‐membrane K ATP channel‐mediated cardioprotection involves posthypoxic reductions in calcium overload and contractile dysfunction: mechanistic insights into cardioplegia

Our recent data demonstrate that activation of pmKATP channels polarizes the membrane of cardiomyocytes and reduces Na+/Ca2+ exchange-mediated Ca2+ overload. However, it is important that these findings be extended into contractile models of hypoxia/reoxygenation injury to further test the notion that pmKATP channel activation affords protection against contractile dysfunction and calcium overload. Single rat heart right ventricular myocytes were enzymatically isolated, and cell contractility and Ca2+ transients in field-stimulated myocytes were measured in a cellular model of metabolic inhibition and reoxygenation. Activation of pmKATP with P-1075 (5 microM) or inhibition of the Na+/Ca2+ exchanger with KB-R7943 (5 microM)reduced reoxygenation-induced diastolic Ca2+ overload and improved the rate and magnitude of posthypoxic contractile recovery during the first few minutes of reoxygenation. Moreover,diastolic Ca2+ overload and posthypoxic contractile dysfunction were aggravated in ventricular myocytes either subjected to specific blockade of pmKATP with HMR1098 (20 microM) or expressing the dominant-negative pmKATP construct Kir6.2(AAA) in the presence of P-1075. Our results suggest that a common mechanism, involving resting membrane potential-modulated increases in diastolic [Ca2+]i, is responsible for the development of contractile dysfunction during reoxygenation following metabolic inhibition. This novel and highly plausible cellular mechanism for pmKATP-mediated cardioprotection may have direct clinical relevance as evidenced by the following findings: a hypokalemic polarizing cardioplegia solution supplemented with the pmKATP opener P-1075 improved Ca2+ homeostasis and recovery of function compared with hyperkalemic depolarizing St. Thomas' cardioplegia following contractile arrest in single ventricular myocytes and working rat hearts. We therefore propose that activation of pmKATP channels improves posthypoxic cardiac function via reductions in abnormal diastolic Ca2+ homeostasis mediated by reverse-mode Na+/Ca2+ exchange.

Modulation of Mg2+ Efflux from Rat Ventricular Myocytes Studied with the Fluorescent Indicator Furaptra

The fluorescent Mg2+ indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350nm was used to estimate the apparent cytoplasmic [Mg2+] ([Mg2+]i). In Ca2+-free extracellular conditions (0.1mM EGTA) at 25°C, [Mg2+]i averaged 0.842±0.019mM. After the cells were loaded with Mg2+ by exposure to high extracellular [Mg2+] ([Mg2+]o), reduction of [Mg2+]o to 1mM (in the presence of extracellular Na+) induced a decrease in [Mg2+]i. The rate of decrease in [Mg2+]i was higher at higher [Mg2+]i, whereas raising [Mg2+]o slowed the decrease in [Mg2+]i with 50% reduction of the rate at ∼10mM [Mg2+]o. Because a part of the furaptra molecules were likely trapped inside intracellular organelles, we assessed possible contribution of the indicator fluorescence emitted from the organelles. When the cell membranes of furaptra-loaded myocytes were permeabilized with saponin (25μg/ml for 5min), furaptra fluorescence intensity at 350-nm excitation decreased to 22%; thus ∼78% of furaptra fluorescence appeared to represent cytoplasmic [Mg2+] ([Mg2+]c), whereas the residual 22% likely represented [Mg2+] in organelles (primarily mitochondria as revealed by fluorescence imaging). [Mg2+] calibrated from the residual furaptra fluorescence ([Mg2+]r) was 0.6–0.7mM in bathing solution [Mg2+] (i.e., [Mg2+]c of the skinned myocytes) of either 0.8mM or 4.0mM, suggesting that [Mg2+]r was lower than and virtually insensitive to [Mg2+]c. We therefore corrected furaptra fluorescence signals measured in intact myocytes for this insensitive fraction of fluorescence to estimate [Mg2+]c. In addition, by utilizing concentration and dissociation constant values of known cytoplasmic Mg2+ buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg2+ flux across the cell membrane. The calculations indicate that, in the presence of extracellular Na+, Mg2+ efflux is markedly activated by [Mg2+]c above the normal basal level (∼0.9mM), with a half-maximal activation of ∼1.9mM [Mg2+]c. We conclude that [Mg2+]c is tightly regulated by an Mg2+ efflux that is dependent on extracellular [Na+].

Effects of Eugenol, an Essential Oil, on the Mechanical and Electrical Activities of Cardiac Muscle

Eugenol (EUG) acts as a calcium antagonist but effects on the contractile proteins could also occur. We investigated inotropic effects of EUG in rat left ventricular papillary muscles, measuring isometric force, time variables, and post rest potentiation and EUG actions on the effects of Ca2+ (0.62 to 2.5 mM) and isoproterenol (5 ng/ml), on myosin ATPase activity and on the calcium currents in single ventricular myocytes. EUG reduced tension and time variables without altering the sarcoplasmic reticulum activity increasing post-pause relative potentiation. Isoproterenol and Ca2+ counteract these negative inotropic effects. Tetanic tension diminished, but not the myosin ATPase activity suggesting an isolated sarcolemmal effect. EUG 0.1 mM decreased the Ca2+ current amplitude in the entire potential range tested and 0.5 mM almost completely blocked this inward current. Results suggested that EUG depresses force without affecting the contractile machinery and its action is the only dependent blockade of the calcium inward current.

Photoperiod-dependent modulation of cardiac excitation contraction coupling in the Siberian hamster

In mammals, changes in photoperiod regulate a diverse array of physiological and behavioral processes, an example of which in the Siberian hamster (Phodopus sungorus) is the expression of bouts of daily torpor following prolonged exposure to a short photoperiod. During torpor, body temperature drops dramatically; however, unlike in nonhibernating or nontorpid species, the myocardium retains the ability to contract and is resistant to the development of arrhythmias. In the present study, we sought to determine whether exposure to a short photoperiod results in alterations to cardiac excitation-contraction coupling, thus potentially enabling the heart to survive periods of low temperature during torpor. Experiments were performed on single ventricular myocytes freshly isolated from the hearts of Siberian hamsters that had been exposed to either 12 wk of short-day lengths (SD) or 12 wk of long-day lengths (LD). In SD-acclimated animals, the amplitude of the systolic Ca(2+) transient was increased (e.g., from 142 +/- 17 nmol/l in LD to 229 +/- 31 nmol/l in SD at 4 Hz; P < 0.001). The increased Ca(2+) transient amplitude in the SD-acclimated animals was not associated with any change in the shape or duration of the action potential. However, sarcoplasmic reticulum Ca(2+) content measured after current-clamp stimulation was increased in the SD-acclimated animals (at 4 Hz, 110 +/- 5 vs. 141 +/- 15 mumol/l, P < 0.05). We propose that short photoperiods reprogram the function of the cardiac sarcoplasmic reticulum, resulting in an increased Ca(2+) content, and that this may be a necessary precursor for maintenance of cardiac function during winter torpor.

The Impact of Isoflurane During Simulated Ischemia/Reoxygenation on Intracellular Calcium, Contractile Function, and Arrhythmia in Ventricular Myocytes

Some of isoflurane's cellular actions, such as interference with intracellular Ca(2+) handling, inhibition of the respiratory chain, and the capability to produce oxygen radicals, could result in impaired cellular function during ischemia/reoxygenation (I/R). We investigated the effects of isoflurane applied during I/R on intracellular Ca(2+), oxygen radical formation, arrhythmic events, and contractile function in rat cardiomyocytes. Single ventricular myocytes were subjected to 30 min of simulated ischemia followed by 30 min of reoxygenation. After baseline measurements, isoflurane-treated cells were exposed to 1 minimum alveolar concentration of isoflurane in air, whereas control cells were exposed to air only. Cytosolic Ca(2+) overload was observed in the isoflurane group (P < 0.05). During ischemia, systolic cell shortening decreased in both groups. In the isoflurane group, arrhythmic events and hypercontracture occurred more often during I/R, and the recovery of contractility during reoxygenation was less marked (P < 0.05). Furthermore, increased oxygen radical generation was detected in isoflurane-treated myocytes during reoxygenation (P < 0.05). Isoflurane given during I/R in this study induced intracellular Ca(2+) accumulation and impaired cell function. These potentially harmful effects were associated with a diminished Ca(2+) clearance and an accelerated oxygen radical production.

Protective effect of carvedilol on abnormality of L-type calcium current induced by oxygen free radical in cardiomyocytes.

The protective effect of carvedilol on abnormality of L-type calcium current induced by oxygen free radical in single guinea pig ventricular myocytes was studied. Whole-cell patch clamp technique was used to study the effect of H2O2 (0.5 mmol/L) on L-type calcium current in single guinea pig ventricular myocytes and the action of pretreatment with carvedilol (0.5 micromol/L). 0.5 micromol/L carvedilol had no significant effect on ICa,L and its channel dynamics. In the presence of 0.5 mmol/L H2O2, peak current of ICa,L was reduced significantly (P<0.001), the I-V curve of ICa,L was shifted upward, steady-state activation curve and steady-state deactivation curve of ICa,L were shifted left and recovery time of ICa,L was delayed significantly (P<0.001). 0.5 micromol/L carvedilol significantly alleviated the inhibitory effect of H2O2 on ICa,L as compared with that in H2O2 group (P<0.01). In addition, carvedilol reversed the changes of dynamics of ICa,L induced by H2O2. It was concluded that carvedilol could alleviate the abnormality of L-type calcium current induced by oxygen free radical in cardiomyocytes. It shows partly the possible mechanism of the special availability of carvedilol in chronic heart failure.

Novel method for measuring junctional proton permeation in isolated ventricular myocyte cell pairs.

Partial exposure of single ventricular myocytes to membrane-permeant weak acids or bases, using a dual-microperfusion technique, generates large and stable intracellular pH (pHi) gradients. In this study, we have investigated the feasibility of using the technique to estimate junctional proton permeability. This was done by recording the pHi gradient developed across the junctional region of a pair of conjoined ventricular myocytes, isolated enzymically from a guinea pig heart when one of the cells was partially exposed to acetate or ammonium. We show that under HEPES-buffered conditions, the junctional discontinuity in the pHi profile can be used to derive an apparent proton permeability coefficient (PHapp). The mean PHapp obtained was 4.45 +/- 0.21.10(-4) cm/s (n=43) at an average junctional pHi of 7.04 +/- 0.02. In the presence of the junctional inhibitor alpha-glycyrrhetinic acid, exposure of the proximal cell to weak acid or base produced no pHi change in the distal cell, confirming that distal changes were normally caused by acid-base flux through connexons assembled into junctional channels. The validity of the dual-microperfusion method was tested further by using a diffusion-permeation-reaction model for intracellular protons, designed to highlight possible errors in the estimates of PHapp. Our technique for measuring PHapp provides a useful alternative to the previous, more invasive technique of locally loading acid through a cell-attached patch pipette. The technique may provide a simple method for investigating the factors regulating cell-to-cell proton transmission.

Beta-adrenoceptor blockers as agonists: coupling of beta2-adrenoceptors to multiple G-proteins in the failing human heart.

Beta blockers have been shown in clinical trials to improve cardiac function and reduce mortality of heart failure patients. However, these agents require careful titration since they can produce an initial decrease in cardiac output. The authors have recently shown that beta blockers, including some used clinically, can directly depress contraction of myocardium from the failing (but not nonfailing) human heart. This occurs on single ventricular myocytes and is therefore completely independent of any inhibition of endogenous catecholamines. The effect appears to be mediated primarily by the beta2-adrenoceptor (AR) and is dependent on the inhibitory guanine nucleotide binding protein, Gi. Using a transgenic mouse model, as well as adenoviral vectors to overexpress Gi or the human beta2AR in adult myocytes of various species, the authors demonstrate that agents that are blockers for bAR/Gs coupling can be agonists at a beta2AR/Gi-coupled form of the receptor. The negative effect of beta blockers could contribute to the initial cardiodepression that is observed when introducing these agents in heart failure patients. However, in the long term, beta2AR/Gi coupling may enhance the ability of beta blockers to protect and improve the function of the failing heart.

Protective effect of edaravone against hypoxia-reoxygenation injury in rabbit cardiomyocytes.

1 We examined whether edaravone (Eda), a clinically available radical scavenger, directly protects cardiomyocytes from ischemia/reperfusion (I/R) injury, and whether the timing of its application is critical for protection. 2 Cardioprotective effects of edaravone were tested in the modified cell-pelleting model of ischemia and under exogenous oxidative stress (hydrogen peroxide: H2O2) in isolated adult rabbit ventricular cells. Cell death and reactive oxygen species (ROS) generation were detected using propidium iodide (PI) and DCFH-DA, respectively. These parameters were evaluated objectively using flow cytometory. 3 Hypoxia and reoxygenation aggravated the proportion of dead cells from 32.2+/-1.8% (Baseline) to 51.3+/-2.7% (Control). When 15 microm edaravone was applied either throughout the entire experiment (Through) or only at reoxygenation (Reox), cell death was significantly reduced to 39.9+/-1.8% (P<0.01 vs Control) and 43.3+/-2.5% (P<0.05 vs Control), respectively. In contrast, when edaravone was applied 10 min after reoxygenation, its protective effect disappeared. Cardioprotection by edaravone was more remarkable than that afforded by other free radical scavengers, such as ascorbate and superoxide dismutase (SOD). There is a positive correlation between the cardioprotective effect of edaravone and the extent of ROS reduction. 4 Edaravone blunted the H2O2-induced changes in electrical properties, and significantly prolonged the time to contracture induced by H2O2 in single ventricular myocytes. 5 Taken together, edaravone directly protects cardiomyocytes from I/R injury by attenuating ROS production, even when applied at the time of reoxygenation, suggesting that edaravone could be a potent cardioprotective therapeutic agent against hypoxia-reoxygenation injury.

Swelling-activated chloride current is activated in guinea pig cardiomyocytes from endotoxic shock.

Myocardial swelling occurs during endotoxic shock. The hypothesis that swelling-activated Cl- current (ICl,swell) activates during endotoxic shock was tested. Endotoxic shock was induced by intravenous lipopolysaccharides (10 mg/kg) in guinea pigs. The effects of ICl,swell blockers on the cardiac action potentials in papillary muscles and on the ICl,swell in single ventricular myocytes were tested. Action potential duration (APD) at 90% of repolarization (APD90) was significantly shortened after 5-h endotoxic shock in guinea pig papillary muscles. I(Cl,swell) blockers, 9-anthracene carboxylic acid (9-AC) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), dose-dependently prolonged the shortened APD90. Inducible nitric oxide synthase (iNOS) inhibitors, L-N6-(1-iminoethyl) lysine (L-NIL) and N-[[3-(aminomethyl)phenyl]methyl]-ethanimidamide (1400 W), also prolonged the APD90. Protein kinase C (PKC) activators, 4beta-phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-didecanoate (PDD), also prolonged the APD. The addition of glibenclamide (an ATP-sensitive K+ channel blocker) on top of these ICl,swell blockers hastened the recovery of APD90 compared to the use of ICl,swell blockers alone. Whole-cell voltage-clamp study in single ventricular myocytes from endotoxic shock heart disclosed activation of a DIDS- and 9-AC-sensitive current. These currents displayed outward rectification with reversal potentials similar to the calculated Nernst potential for Cl-. The reversal potentials tracked the ECl closely when the Cl- gradient was changed, suggesting that Cl- was the major charged carrier. We have shown for the first time that ICl,swell activates in guinea pig heart in endotoxic shock. The change in this membrane current, together with the activation of ATP-sensitive K+ current, contributes to the electrophysiological derangement in endotoxic shock.

Intracellular Ca 2+ dynamics and sarcomere length in single ventricular myocytes

The measurements of the sarcomere length in dissociated cardiac ventricular myocytes are discussed using mainly our own experimental data. The striation periodicity of these unloaded cells was found to be that which is to be expected of a myocyte free of the ultrastrucural constraints imposed upon it by the normal syncytial matrix of the ventricular wall. The sarcomere length and [Ca 2+] relationship was consistent as expected from the intact tissue, when it was measured soon after partial rupturing the cell membrane. Miniature fluctuations of individual sarcomere length were demonstrated during rest, which was augmented by the Ca 2+ overload. The [Ca 2+] could be estimated from the measurements of sarcomere length during the positive staircase of contraction. The usefulness of the optical measurement of sarcomere pattern was indicated.

Distinct subcellular localization of different sodium channel alpha and beta subunits in single ventricular myocytes from mouse heart.

Voltage-gated sodium channels composed of pore-forming alpha and auxiliary beta subunits are responsible for the rising phase of the action potential in cardiac muscle, but their localizations have not yet been clearly defined. Immunocytochemical studies show that the principal cardiac alpha subunit isoform Na(v)1.5 and the beta2 subunit are preferentially localized in intercalated disks, identified by immunostaining of connexin 43, the major protein of cardiac gap junctions. The brain alpha subunit isoforms Na(v)1.1, Na(v)1.3, and Na(v)1.6 are preferentially localized with beta1 and beta3 subunits in the transverse tubules, identified by immunostaining of alpha-actinin, a cardiac z-line protein. The beta1 subunit is also present in a small fraction of intercalated disks. The recently cloned beta4 subunit, which closely resembles beta2 in amino acid sequence, is also expressed in ventricular myocytes and is localized in intercalated disks as are beta2 and Na(v)1.5. Our results suggest that the primary sodium channels present in ventricular myocytes are composed of Na(v)1.5 plus beta2 and/or beta4 subunits in intercalated disks and Na(v)1.1, Na(v)1.3, and Na(v)1.6 plus beta1 and/or beta3 subunits in the transverse tubules.

Gender-dependent attenuation of cardiac potassium currents in type 2 diabetic db/db mice.

Single ventricular myocytes were prepared from control db/+ and insulin-resistant diabetic db/db male mice at 6 and 12 weeks of age. Peak and sustained outward potassium currents were measured using whole-cell voltage clamp methods. At 6 weeks currents were fully developed in control and diabetic mice, with no differences in the density of either current. By 12 weeks both currents were significantly attenuated in the diabetic mice, but could be augmented by in vitro incubation with the angiotensin-converting enzyme (ACE) inhibitor quinapril (1 microM, 5-9 h). In cells from female db/db mice (12 weeks of age), K(+) currents were not attenuated and no effects of quinapril were observed. To investigate whether lack of insulin action accounts for these gender differences, cells were also isolated from cardiomyocyte-specific insulin receptor knockout (CIRKO) mice. Both K(+) currents were significantly attenuated in cells from male and female CIRKO mice, and action potentials were significantly prolonged. Incubation with quinapril did not augment K(+) currents. Our results demonstrate that type 2 diabetes is associated with gender-selective attenuation of K(+) currents in cardiomyocytes, which may underlie gender differences in the development of some cardiac arrhythmias. The mechanism for attenuation of K(+) currents in cells from male mice is due, at least in part, to an autocrine effect resulting from activation of a cardiac renin-angiotensin system. Insulin is not involved in these gender differences, since the absence of insulin action in CIRKO mice diminishes K(+) currents in cells from both males and females.

ClC-3-independent, PKC-dependent Activity of Volume-sensitive Cl- Channel in Mouse Ventricular Cardiomyocytes

Volume-sensitive outwardly rectifying (VSOR) Cl^- channels are activated during osmotic swelling and involved in the subsequent volume regulation in most animal cells. To test the hypothesis that the ClC-3 protein is the molecular entity corresponding to the VSOR Cl^- channel in cardiomyocytes, the properties of VSOR Cl^- currents in single ventricular myocytes isolated from ClC-3-deficient (Clcn3^-/-) mice were compared with those of the same currents in ClC-3-expressing wild-type (Clcn3^+/+) and heterozygous (Clcn3^+/-) mice. Basal whole-cell currents recorded under isotonic conditions in ClC-3-deficient and -expressing cells were indistinguishable. The biophysical and pharmacological properties of whole-cell VSOR Cl^- currents in ClC-3-deficient cells were identical in ClC-3-expressing cells. The VSOR Cl^- current density, which is an indicator of the plasmalemmal expression of functional channels, was essentially the same in cells isolated from these 3 types of mice and C57BL/6 mice. Activation of protein kinase C (PKC) by a phorbol ester was found to upregulate VSOR Cl^- currents in ClC-3-deficient and -expressing cardiomyocytes. This effect is opposite to the reported downregulatory effect of PKC activators on ClC-3-associated Cl^- currents. We thus conclude that functional expression of VSOR Cl^- channels in the plasma membrane of mouse cardiomyocytes is independent of the molecular expression of ClC-3.