Single Rat Ventricular Myocytes Research Articles

Effect of Carbenoxolone on Arrhythmogenesis in Rat Ventricular Muscle.

Connexin43 (Cx43) is a major connexin that forms gap junction (GJ) channels in the heart and is also present in the cell membrane as unopposed/non-junctional hemichannels and in the inner mitochondrial membrane. By using carbenoxolone (CBX), a blocker of Cx43, the effect of the blockade of Cx43 on Ca(2+)waves and triggered arrhythmias in the myocardium with non-uniform contraction was examined. Trabeculae were obtained from rat hearts. Force, [Ca(2+)]i, and the diffusion coefficient were measured. Non-uniform contraction was produced with a 2,3-butanedione monoxime jet. Ca(2+)waves were induced by electrical stimulation. Inducibility of arrhythmias was estimated based on the minimal [Ca(2+)]oat which arrhythmias were induced. The Ca(2+)spark rate was measured in isolated single rat ventricular myocytes. CBX reduced the GJ permeability, whereas it did not change force and [Ca(2+)]itransients. CBX increased the Ca(2+)leak from the sarcoplasmic reticulum in trabeculae and increased the Ca(2+)spark rate in isolated single myocytes. CBX increased the velocity of Ca(2+)waves and further increased the inducibility of arrhythmias. Modulation of mitochondrial KATPchannels by diazoxide, cromakalim and 5-hydroxydecanoic acid affected the inducibility of arrhythmias increased by CBX. These results suggest that in diseased hearts, Cx43 plays an important role in the occurrence of triggered arrhythmias, probably under the modulation of mitochondrial KATPchannels.

Effect of reduction in ryanodine receptor calcium leak on arrhythmogenesis in rat ventricular myocytes

BackgroundCardiomyocytes in heart failure have increased ryanodine receptor (RyR) open probability (po) leading to calcium leak from the sarcoplasmic reticulum. This process results in calcium waves, delayed after-depolarisations, and subsequent ventricular arrhythmias. However, this leak will decrease the calcium content of the sarcoplasmic reticulum which will in turn decrease the occurrence of waves. We therefore hypothesised that with extreme RyR leak (such as in end-stage heart failure) the calcium content of the sarcoplasmic reticulum might be too low to sustain calcium waves and that part correction of severe leak could lead to a resurgence of calcium waves. MethodsWe used single rat ventricular myocytes, voltage clamped using the perforated patch clamp technique. Cells were stimulated at 0·5 Hz with 100 ms duration depolarising pulses from −40 to 0 mV. Cytosolic calcium concentration was measured with the Fluo-4 AM indicator. Calcium waves were induced by the application of 1 μmol/L isoproterenol in raised external calcium concentration (2 or 3 mM). Severe RyR leak was induced with caffeine (1–2 mM), and then tetracaine (50–100 μM) was co-applied to reduce RyR po. FindingsIsoproterenol increased the amplitude of the systolic calcium transient in all the myocytes studied and induced calcium waves in 26 (87%) of 30 myocytes. Application of 1 mM caffeine abolished calcium waves in 11 (65%) of 17 of these myocytes and reduced the frequency of waves in the remaining six cells. Application of 2 mM caffeine abolished calcium waves in all nine myocytes. Subsequent addition of tetracaine (50 μM) led to a re-emergence or increased frequency of waves in nine (53%) of 17 myocytes whereas 100 μM tetracaine re-established calcium waves in 14 (93%) of 15 myocytes (50 vs 100 μM tetracaine, Fisher's exact test p=0·02). InterpretationInduction of severe calcium leak depleted the calcium content of the sarcoplasmic reticulum below the level required for calcium waves. Inhibition of RyR leak could be a useful therapeutic strategy in heart failure to improve systolic contraction as well as relaxation. However, this strategy might also be pro-arrhythmogenic since part correction of leak can replenish the calcium content of the sarcoplasmic reticulum above the level required for initiation of calcium waves. FundingBritish Heart Foundation.

Natural Alkaloid Murrayafoline-A Sensitizes Ca2+ Release Sites via Adenylate Cyclase and Protein Kinase C Pathway in Rat Ventricular Myocytes

A naturally found alkaloid murrayafoline-A (Mu-A, 1-methoxy-3-methylcarbazole) acutely increases ventricle cell contractility partly via enhancement of L-type Ca2+ current. In the present study,we explored the effects of Mu-A on elementary Ca2+ release events (“Ca2+ sparks”) in cardiac myocytes and possible molecular mechanisms underlying the effect of Mu-A. Rapid two-dimensional confocal Ca2+ imaging and image analysis were used to assess spontaneous Ca2+ sparks in single adult rat ventricular myocytes. The frequency of Ca2+ sparks was dose-dependently increased by Mu-A. The duration and the size of single sparks were also significantly increased by Mu-A. The sarcoplasmic reticulum (SR) Ca2+ loading, measured as caffeine-induced Ca2+ transients, was increased by Mu-A. Neither SR Ca2+ clamping nor removal of external Ca2+ and Na+ affects the Mu-A-mediated enhancement in Ca2+ sparks. In contrast, prolongation and enlargement of individual Ca2+ sparks in Mu-A were attenuated by matching SR Ca2+ content to the control level. Higher propensity of Ca2+ sparks in the presence of Mu-A was not suppressed by the protein kinase A inhibitor, KT5720, or by the calmodulin-dependent protein kinase II inhibitor, KN93, whereas it was strongly reduced by the inhibitors of adenylate cyclase (MDL-12,330A) and protein kinase C (GF109203X). Our data suggest that Mu-A sensitizes ryanodine receptor (RyR) Ca2+ release channels via specialized signaling pathway involving adenylate cyclase and protein kinase C, and that larger SR Ca2+ loading in Mu-A may also play a role in enhanced Ca2+ release through the RyRs.

Role of Na+–H+ exchange in the modulation of L-type Ca2+ current during fluid pressure in rat ventricular myocytes

Application of fluid pressure (FP) using pressurized fluid flow suppresses the L-type Ca2+ current through both enhancement of Ca2+ release and intracellular acidosis in ventricular myocytes. As FP-induced intracellular acidosis is more severe during the inhibition of Na+–H+ exchange (NHE), we examined the possible role of NHE in the regulation of ICa during FP exposure using HOE642 (cariporide), a specific NHE inhibitor. A flow of pressurized (∼16dyn/cm2) fluid was applied onto single rat ventricular myocytes, and the ICa was monitored using a whole-cell patch-clamp under HEPES-buffered conditions. In cells pre-exposed to FP, additional treatment with HOE642 dose-dependently suppressed the ICa (IC50=0.97±0.12μM) without altering current–voltage relationships and inactivation time constants. In contrast, the ICa in control cells was not altered by HOE642. The HOE642 induced a left shift in the steady-state inactivation curve. The suppressive effect of HOE642 on the ICa under FP was not altered by intracellular high Ca2+ buffering. Replacement of external Cl− with aspartate to inhibit the Cl−-dependent acid loader eliminated the inhibitory effect of HOE642 on ICa. These results suggest that NHE may attenuate FP-induced ICa suppression by preventing intracellular H+ accumulation in rat ventricular myocytes and that NHE activity may not be involved in the Ca2+-dependent inhibition of the ICa during FP exposure.

Effects of astragaloside IV on L-type calcium channel currents in adult rat ventricular myocytes

Astragaloside IV (AGS-IV), a traditional Chinese herb, has been found to have potent cardioprotective effects. However, the relevant mechanisms still remain unclear. In the present study, using standard whole-cell patch clamp recording technique, we identified a novel functional role of AGS-IV in regulating L-type calcium channel currents (L-currents) in adult rat ventricular myocytes. Our results showed that AGS-IV exerted inhibitory effects on L-currents of the single adult rat ventricular myocytes. This L-current inhibitory action of AGS-IV was concentration-dependent. The current density at +10 mV was reduced by about 48.7% after exposure of the cells to AGS-IV (1 µM), from the control value of 13.7±1.7 to 7.6±1.2 pA/pF. AGS-IV did not markedly affect the activation of L-currents but significantly shifted the inactivation curve to the left. Pretreatment of cells with H89, a protein kinase A (PKA) inhibitor, completely abolished AGS-IV-induced L-current inhibition. Similar results were obtained by another PKA inhibitor PKI 5-24. Taken together, these results suggested that AGS-IV inhibited L-currents via a PKA-dependent pathway in rat ventricular myocytes, which could contribute to its cardioprotective effects. Key words: Astragaloside IV, L-type calcium channels, ventricular myocytes, patch clamp.

Suppression of L-type Ca 2+ current by fluid pressure in rat ventricular myocytes: Possible role of Cl −–OH − exchange

The application of fluid pressure (FP) in ventricular myocytes using pressurized fluid flow inhibits L-type Ca 2+ current (I Ca), with approximately 80% of this effect coming through the enhancement of Ca 2+ releases from the sarcoplasmic reticulum. In the present study, we explored the remaining mechanisms for the inhibition of I Ca by FP. Since FP significantly increases H + concentration and H + is known to inhibit I Ca, we examined whether pH regulation plays a role in the inhibitory effect by FP on I Ca. A flow of pressurized (∼16.3 dyne/cm 2) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes for which the I Ca was monitored using whole-cell patch-clamp under HEPES-buffered conditions. Extracellular application of the alkalizing agent, NH 4Cl (20 mM), enhanced I Ca by ∼34% in the control conditions while increasing I Ca significantly less (by ∼21%) in FP-pretreated myocytes, suggesting an inhibition of the effect of NH 4Cl on I Ca possibly by FP-induced acidosis. Application of DIDS (4,4′-diisothiocyanatostilbene-2,2′-disulphonic acid, 500 μM), which blocks Cl - – HCO 3 - exchange but not Cl −–OH − exchange, did not alter the inhibitory effect of FP on I Ca. Replacement of external Cl − with aspartate attenuated the inhibitory effect of FP on I Ca. In highly Ca 2+-buffered cells, where Ca 2+-dependent inhibition of I Ca was minimized, the external Cl − removal eliminated the inhibitory effect of FP on I Ca. These results suggest that the decrease of I Ca in the presence of FP is at least partly caused by intracellular acidosis via activation of Cl −–OH − exchange in rat ventricular myocytes.

Influences and mechanism of verapamil on ischemia/reperfusion injury in cardiomyocytes of streptozotocin-induced diabetes mellitus rats

To investigate the effects and mechanism of verapamil preventing ischemia/reperfusion (I/R) injury by cardiac performance intracellular free [Ca(2+)](i) and L-type calcium current (I(Ca-L)) in cardiomyocytes of diabetes mellitus rats. Diabetic rats were streptozotocin-induced and received verapamil (8 mg×kg(-1)×d(-1)) from 6 - 14 weeks old. The in vitro heart models of I/R rats were randomly divided into normal control group diabetes group, verapamil control group. the changes of heart functions were observed through a Langendorff-perfusion system. The fluorescence intensity of intracellular Ca(2+) was detected with Fluo-3/AM loading by laser scanning confocal microscope. I(Ca-L) was recorded by the whole-cell technique of patch clamp in enzymatically dissociated single rat ventricular myocytes. (1) In verapamil diabetes group, the values of left ventricular developed pressure [(91.3 ± 4.6) mm Hg], diastolic end pressure [(1535 ± 280) mm Hg], the maximum rising rates of left ventricular pressure [(5833 ± 256) mm Hg/s] and coronary arterial flow [(13.7 ± 0.9) ml/min] were all significantly increased, and the maximum dropping rates of left ventricular pressure [(3504 ± 319) mm Hg/s] was obviously decreased (compared with diabetes group, P < 0.01, respectively). (2) The fluorescence intensities of intracellular free Ca(2+)[(155.6 ± 10.9) nmol/L] in verapamil diabetes group were significantly reduced compared with diabetes group (245.2 ± 17.5 nmol/L, P < 0.01). (3) When clamp voltage was -20mV, I(Ca-L) was (-6.81 ± 0.76) pA/pF in verapamil diabetes group (compared with normal group (-8.17 ± 2.07) pA/pF, P < 0.05, and with diabetes group (-3.21 ± 0.54) pA/pF, P < 0.01, and with verapamil control group (-7.14 ± 2.17) pA/pF, P > 0.05). The current-voltage curve was changed to the lower position with -20mV of peak clamp potential in verapamil diabetes group compared with diabetes group. A poor heart function is closely correlated with a rising [Ca(2+)]i and a declining I(Ca-L) associated with I/R injury in diabetic rats hearts. Along-term verapamil therapy may significantly improve the severe cardiac impairment. The mechanism is probably attributed to the fact that verapamil can adjust I(Ca-L) influx, normalize the balance of intercellular [Ca(2+)]i, and block the Ca(2+) overload trigger by the effects of Ca(2+)-induced Ca(2+) release in diabetic cardiomyocytes.

Modulation of intracellular calcium transient in response to β-adrenoceptor stimulation in the hearts of 4-wk-old rats during simulated weightlessness

Modulation of intracellular calcium ([Ca(2+)](i)) transient in response to beta-adrenoceptor stimulation in the hearts of hindlimb unweighted (HLU) rats during simulated weightlessness has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness. Effects of simulated microgravity on beta-adrenoceptor responsiveness were then studied. Mean arterial blood pressure, left ventricular pressure (LVP), systolic function [maximum positive change in pressure over time (+dP/dt(max))], and diastolic function [maximum negative change in pressure over time (-dP/dt(max))] were monitored during the in vivo experiment. beta-Adrenoceptor density was quantitated by radioactive ligand binding. Single rat ventricular myocyte was obtained by enzymatic dissociation method. +/-dP/dt(max), myocyte contraction, intracellular [Ca(2+)](i) transient, and L-type calcium current in response to beta-adrenoceptor stimulation with isoproterenol were measured. Compared with the control group, no significant changes were found in heart weight, body weight, and mean arterial blood pressure, whereas LVP and +/-dP/dt(max) were significantly reduced. LVP and +/-dP/dt(max) were significantly attenuated in the HLU group in response to isoproterenol administration. In the in vitro study, the beta-adrenoceptor density was unchanged. Effects of isoproterenol on electrically induced single-cell contraction and [Ca(2+)](i) transient in myocytes of ventricles in HLU rats were significantly attenuated. The enhanced L-type Ca(2+) current elicited by isoproterenol in cardiomyocytes was significantly decreased in the HLU group. The above results indicate that impaired function of L-type Ca(2+) current and decreased [Ca(2+)](i) transient cause the depressed responsiveness of the beta-adrenoceptor stimulation, which may be partially responsible for the depression of cardiac function.

Urotensin II Activates Sarcolemmal Na+/H+ Exchanger in Adult Rat Ventricular Myocytes

Our aims in the present study were (1) to determine the effects of urotensin II (UT-II) on the sarcolemmal Na/H exchanger (NHE1) activity, and (2) to investigate possible kinase pathways for UT-II-mediated NHE1 stimulation. In single rat ventricular myocytes (n = 5-10/group) loaded with the pH-sensitive fluoroprobe carboxy-seminaphthorhodafluor-1, acid efflux rates (JH) were determined as an index of NHE1 activity by rate of recovery of intracellular pH (pHi) from NH4Cl-induced acidosis and the intrinsic buffering power. Phosphorylation of extracellular signal-regulated kinase (ERK), a key kinase of NHE1 activation, was determined by Western blot analysis. JH increased by 31%-71% relative to control in the presence of 100 nmol/L UT-II at pHi range of 6.6-7.0. Stimulation of NHE1 activity by UT-II was abolished by inhibitors of phospholipase C, protein kinase C, and ERK kinase; 2-nitro-4-carboxyphenil-N,N-diphenilcarbamate at 100 micromol/L, GF109203X at 300 nmol/L, and PD98059 at 50 micromol/L, respectively. Moreover, UT-II at 100 nmol/L produced a significant increase in cellular ERK1/2 phosphorylation, which was also inhibited by those inhibitors. Our study was the first to demonstrate that UT-II activates the cardiac sarcolemmal NHE1 and that the phenomenon may involve, at least in part, the phospholipase C-protein kinase C-ERK pathway.

Three‐dimensional high‐resolution imaging of cardiac proteins to construct models of intracellular Ca2+ signalling in rat ventricular myocytes

Quantitative understanding of the Ca(2+) handling in cardiac ventricular myocytes requires accurate knowledge of cardiac ultrastructure and protein distribution. We have therefore developed high-resolution imaging and analysis approaches to measure the three-dimensional distribution of immunolabelled proteins with confocal microscopy. Labelling of single rat cardiac myocytes with an antibody to the Z-line marker alpha-actinin revealed a complex architecture of sarcomere misalignment across single cells. Double immunolabelling was used to relate the Z-line structure to the distribution of ryanodine receptors (RyRs, the intracellular Ca(2+) release channels) and the transverse tubular system. Both RyR and transverse tubular system distributions exhibited frequent dislocations from the simple planar geometry generally assumed in existing mathematical models. To investigate potential effects of these irregularities on Ca(2+) dynamics, we determined the three-dimensional distribution of RyR clusters within an extended section of a single rat ventricular myocyte to construct a model of stochastic Ca(2+) dynamics with a measured Ca(2+) release unit (CRU) distribution. Calculations with this model were compared with a second model in which all CRUs were placed on flat planes. The model with a realistic CRU distribution supported Ca(2+) waves that spread axially along the cell at velocities of approximately 50 mum s(-1). By contrast, in the model with planar CRU distribution the axial wave spread was slowed roughly twofold and wave propagation often nearly faltered. These results demonstrate that spatial features of the CRU distribution on multiple length scales may significantly affect intracellular Ca(2+) dynamics and must be captured in detailed mechanistic models to achieve quantitative as well as qualitative insight.

Modulation of Myocardial Mitochondrial VDAC/ MitoKATP Channels in Sepsis

In the present study, we tested the hypothesis that norepinephrine by modulating myocardial voltage‐dependent anion channel (VDAC)/mitoKATP channels can affect the release of cytochrome C and regulate intrinsic apoptotic cascade. Male Sprague‐Dawley rats (350‐400g) were made septic using 200mg/kg cecal inoculum, ip. Sham animals received 5% dextrose water, ip. VDAC mRNA and protein expression was determined in the myocardium and isolated single adult rat ventricular myocytes (ARVMs) obtained from sham and septic rats. In addition, mitochondria and cytosolic fractions were isolated from ARVM treated with norepinephrine (NE, 10ìmoles) in the presence and absence of 5‐Hydroxydecanoic acid (5HD; 100ìmoles), a selective mitoKATP channel blocker. Sepsis produced a significant down‐regulation of VDAC mRNA and protein along with upregulation of Bax in both myocardium and ARVMs. Pretreatment of septic ARVMs with 5HD blocked NE‐induced decrease in VDAC expression and the release of cytochrome C. These results suggest that sepsis downregulates myocardial VDAC expression. It appears that NE modulates mitoKATP channels and increases the release of cytochrome C, which may play a crucial role in sepsis‐induced cardiomyocyte dysfunction.

Dofetilide Enhances the Contractility of Rat Ventricular Myocytes via Augmentation of Na+–Ca2+ Exchange

Dofetilide (DOF), a novel Class III antiarrhythmic drug, prolongs the action potential duration (APD) and shows a positive inotropic effect in guinea pig papillary muscle. The present experiments were designed to study the positive inotropic effect of DOF on rat ventricle and explore its possible mechanism(s). Hearts from male Wistar rats (260-320 g) were divided into five groups and perfused in Langendorff mode. Ventricular myocytes were enzymatically isolated from male Wistar rats. Whole-cell voltage-clamping technique was used to test the Na(+)-Ca(2+) exchange (NCE) current (I(NCX)); Calcium transients and cell shortening provoked by field stimulation or using calcium current command waveform were observed synchronously with an ionic imaging system. DOF (0.03-1.0 microM) increased left ventricular function in isolated rat hearts in a concentration-dependent manner. DOF (0.03-1.0 microM) also concentration-dependently increased both inward and outward I (NCX) in isolated rat ventricular cells. The EC(50) values of DOF were 0.149 microM for the inward I(NCX) and 0.249 microM for outward I(NCX), respectively. DOF 0.2 microM significantly enhanced Ca(2+) transient and cell shortening in single rat ventricular myocytes driven by field electric stimulation. When the patch clamp system was connected to the ionic imaging system, Ca(2+) current (I(Ca)), Ca(2+) transient and cell shortening amplitude in a same cell were recorded synchronously. Application of DOF 0.2 microM had no effect on I(Ca), but significantly increased Ca(2+) transient and cell shortening. NCX inhibitor KB-R7943 0.6 microM significantly depressed the effects of DOF on Ca(2+) transient and cell shortening. We conclude that DOF enhanced contractility of rat ventricular myocytes. The enhancement of NCE may be involved in the positive inotropic action of DOF.

Modulation of β-adrenoceptor signaling in the hearts of 4-wk simulated weightlessness rats

The modulation of beta-adrenoceptor signaling in the hearts of hindlimb unweighting (HU) simulated weightlessness rats has not been reported. In the present study, we adopted the rat tail suspension for 4 wk to simulate weightlessness; then the effects of simulated microgravity on beta-adrenoceptor signaling were studied. Mean arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dP/dtmax), and diastolic function (-dP/dtmax) were monitored in the course of the in vivo experiment. Single rat ventricular myocyte was obtained by the enzymatic dissociation method. Hemodynamics, myocyte contraction, and cAMP production in response to beta-adrenoceptor stimulation with isoproterenol or adenylyl cyclase stimulation with forskolin were measured, and Gs protein was also determined. Compared with the control group, no significant changes were found in heart weight, body weight and ABP, while LVP and +/-dP/dtmax were significantly reduced. The ABP decrease, LVP increase, and +/-dP/dtmax in response to isoproterenol administration were significantly attenuated in the HU group. The effects of isoproterenol on electrically induced single-cell contraction and cAMP production in myocytes of ventricles in the HU rats were significantly attenuated. The biologically active isoform, Gsalpha (45 kDa) in the heart, was unchanged. Both the increased electrically induced contraction and cAMP production in response to forskolin were also significantly attenuated in the simulated weightlessness rats. Above results indicated that impaired function of adenylyl cyclase causes beta-adrenoceptor desensitization, which may be partly responsible for the depression of cardiac function.

Engineering physiologically controlled pacemaker cells with lentiviral HCN4 gene transfer

Research on biological pacemakers for the heart has so far mainly focused on short-term gene and cell therapies. To develop a clinically relevant biological pacemaker, long-term function and incorporation of autonomic modulation are crucial. Lentiviral vectors can mediate long-term gene expression, while isoform 4 of the Hyperpolarization-activated Cyclic Nucleotide-gated channel (encoded by HCN4) contributes to pacemaker function and responds maximally to cAMP, the second messenger in autonomic modulation. Action potential (AP) properties and pacemaker current (I(f)) were studied in single neonatal rat ventricular myocytes that overexpressed HCN4 after lentiviral gene transduction. Autonomic responsiveness and cycle length stability were studied using extracellular electrograms of confluent cultured monolayers. Perforated patch-clamp experiments demonstrated that HCN4-transduced single cardiac myocytes exhibited a 10-fold higher I(f) than non-transduced single myocytes, along with slow diastolic depolarization, comparable to pacemaker cells of the sinoatrial node, the dominant native pacemaker. HCN4-transduced monolayers exhibited a 47% increase in beating rate, compared to controls. Upon addition of DBcAMP, HCN4-transduced monolayers had beating rates which were 54% faster than baseline and significantly more regular than controls. Lentiviral vectors efficiently transduce cardiac myocytes and mediate functional gene expression. Because HCN4-transduced myocytes demonstrate an increase in spontaneous beating rate and responsiveness to autonomic modulation, this approach may be useful to create a biological pacemaker.

Fluid pressure modulates L-type Ca2+ channel via enhancement of Ca2+-induced Ca2+ release in rat ventricular myocytes

This study examines whether fluid pressure (FP) modulates the L-type Ca(2+) channel in cardiomyocytes and investigates the underlying cellular mechanism(s) involved. A flow of pressurized (approximately 16 dyn/cm(2)) fluid, identical to that bathing the myocytes, was applied onto single rat ventricular myocytes using a microperfusion method. The Ca(2+) current (I(Ca)) and cytosolic Ca(2+) signals were measured using a whole cell patch-clamp and confocal imaging, respectively. It was found that the FP reversibly suppressed I(Ca) (by 25%) without altering the current-voltage relationships, and it accelerated the inactivation of I(Ca). The level of I(Ca) suppression by FP depended on the level and duration of pressure. The Ba(2+) current through the Ca(2+) channel was only slightly decreased by the FP (5%), suggesting an indirect inhibition of the Ca(2+) channel during FP stimulation. The cytosolic Ca(2+) transients and the basal Ca(2+) in field-stimulated ventricular myocytes were significantly increased by the FP. The effects of the FP on the I(Ca) and on the Ca(2+) transient were resistant to the stretch-activated channel inhibitors, GsMTx-4 and streptomycin. Dialysis of myocytes with high concentrations of BAPTA, the Ca(2+) buffer, eliminated the FP-induced acceleration of I(Ca) inactivation and reduced the inhibitory effect of the FP on I(Ca) by approximately 80%. Ryanodine and thapsigargin, abolishing sarcoplasmic reticulum Ca(2+) release, eliminated the accelerating effect of FP on the I(Ca) inactivation, and they reduced the inhibitory effect of FP on the I(Ca). These results suggest that the fluid pressure indirectly suppresses the Ca(2+) channel by enhancing the Ca(2+)-induced intracellular Ca(2+) release in rat ventricular myocytes.

Regulation of systolic [Ca2+]i and cellular Ca2+ flux balance in rat ventricular myocytes by SR Ca2+, L‐type Ca2+ current and diastolic [Ca2+]i

The force-frequency response is an important physiological mechanism regulating cardiac output changes and is accompanied in vivo by beta-adrenergic stimulation. We sought to determine the role of sarcoplasmic reticulum (SR) Ca2+ content and L-type current (ICa-L) in the frequency response of the systolic Ca2+ transient alone and during beta-adrenergic stimulation. Experiments (on single rat ventricular myocytes) were designed to be as physiological as possible. Under current clamp stimulation SR Ca2+ content increased in line with stimulation frequency (1-8 Hz) but the systolic Ca2+ transient was maximal at 6 Hz. Under voltage clamp, increasing frequency decreased both systolic Ca2+ transient and ICa-L. Normalizing peak ICa-L by altering the test potential decreased the Ca2+ transient amplitude less than an equivalent reduction achieved through changes in frequency. This suggests that, in addition to SR Ca2+ content and ICa-L, another factor, possibly refractoriness of Ca2+ release from the SR contributes. Under current clamp, beta-adrenergic stimulation (isoprenaline, 30 nm) increased both the Ca2+ transient and the SR Ca2+ content and removed the dependence of both on frequency. In voltage clamp experiments, beta-adrenergic stimulation still increased SR Ca2+ content yet there was an inverse relation between frequency and Ca2+ transient amplitude and ICa-L. Diastolic [Ca2+]i increased with stimulation frequency and this contributed substantially (69.3 +/- 6% at 8 Hz) to the total Ca2+ efflux from the cell. We conclude that Ca2+ flux balance is maintained by the combination of increased efflux due to elevated diastolic [Ca2+]i and a decrease of influx on IC-L) on each pulse.

Cardioprotective and antiarrhythmic effect of U50,488H in ischemia/reperfusion rat heart

The objective of this study was to investigate the protective effect of U50,488H, a selective kappa-opioid receptor agonist, in the ischemia/reperfusion (I/R) rat and to delineate the underlying mechanism. Rat heart I/R injury was induced by occluding the left anterior descending coronary artery for 45 min and restoring perfusion for 120 min. U50,488H or vehicle was intravenously injected before ischemia. Electrocardiogram, heart rate (HR), arterial blood pressure (ABP), left ventricular pressure (LVP), systolic function (+dp/dtmax), and diastolic function (-dp/dtmax) were monitored in the course of the experiment. Myocardial infarction size was evaluated. Plasma concentrations of cardiac troponin T (cTnT), creatine kinase (CK), and lactate dehydrogenase (LDH) were measured. Single rat ventricular myocyte was obtained by enzymatic dissociation method. The potassium currents (IK) of isolated ventricular myocytes were recorded with the whole-cell configuration of the patch-clamp technique. Compared with the sham control group, no significant change was found in HR, while ABP, LVP and +/-dp/dtmax were significantly reduced in the I/R group. Administration of U50,488H significantly lowered HR in both control and I/R groups. Compared with the vehicle-treated I/R group, administration of U50,488H had no significant effect on I/R-induced reduction in ABP, LVP, and +/-dp/dtmax. However, this treatment significantly reduced the myocardial infarction size, and markedly decreased the contents of plasma cTnT, CK and LDH. During ischemia and reperfusion, the incidence of ventricular arrhythmia in U50,488H-treated rats was significantly reduced. These effects were independent of the bradycardia induced by U50,488H, as the reducing infarct size and antiarrhythmic effect of U50,488H were still observed in animals in which heart rate was kept constant by electrical pacing. U50,488H and BRL-52537 still produced an antiarrhythmic effect when the rat heart was subjected to a shorter ischemic period of 10 min occlusion of coronary artery, which produced no infarction. IK of the myocytes were inhibited by U50,488H in a dose-dependent manner in normal and hypoxic rat ventricular myocytes. However, the effects of U50,488H on IK did not show any significant difference in normal and hypoxic myocytes. The above-described effects of U50,488H were totally blocked by nor-Binaltorphimine, a selective kappa-opioid receptor antagonist. The results suggest that kappa-opioid agonist U50,488H exerts its direct cardioprotective and antiarrhythmic effects against I/R via kappa-opioid receptor, which participates in the regulation of potassium channels in normal and hypoxic ventricular myocytes.

Analysis of ryanodine receptor clusters in rat and human cardiac myocytes

Single rat ventricular myocytes and human ventricle tissue sections were labeled with antibodies against the ryanodine receptor (RyR) and alpha-actinin to examine the 3D distribution of RyRs with confocal microscopy. Image contrast was maximized by refractive index matching and deconvolution. The RyR label formed discrete puncta representing clusters of RyRs or "couplons" around the edges of the myofilaments with a nearest-neighbor spacing of 0.66 +/- 0.06 microm in rat and 0.78 +/- 0.07 microm in human. Each bundle of myofibrils was served by approximately six couplons, which supplied a cross-sectional area of approximately 0.6 microm(2) in rat and approximately 0.8 microm(2) in human. Although the couplons were in reasonable registration with z-lines, there were discontinuities in the longitudinal position of sarcomeres so that dislocations in the order of RyR clusters occurred. There was approximately 53% longitudinal registration of RyR clusters, suggesting a nonrandom placement of couplons around the sarcomere. These data can explain the spherical propagation of Ca(2+) waves and provide quantitative 3D data sets needed for accurate modeling of cardiac Ca(2+)-induced Ca(2+) release. By quantifying labeling intensity in rat ventricular myocytes, a lower limit of 78 RyRs per cluster (on average) was obtained. By modeling the couplon as a disk wrapping around a t-tubule and fitting cluster images, 95% of couplons contained between 120 and 260 RyRs (assuming that RyRs are tight packed with a spacing of 29 nm). Assuming similar labeling efficiency in human, from the fluorescence intensity alone we estimate that human ventricular myocytes contain approximately 30% fewer RyRs per couplon than rat.

Application of blebbistatin as an excitation–contraction uncoupler for electrophysiologic study of rat and rabbit hearts

Application of fluorescence imaging of cardiac electrical activity is limited by motion artifacts and/or side effects of currently available pharmacologic excitation-contraction uncoupling agents. The purpose of this study was to test whether blebbistatin, a recently discovered inhibitor of myosin II isoforms, can be used as an excitation-contraction uncoupler. The specificity and potency of blebbistatin were examined by assaying the effects of blebbistatin on the contraction and basic cardiac electrophysiologic parameters of Langendorff-perfused rabbit hearts, isolated rabbit right ventricle and right atrium, and single rat ventricular myocytes using conventional ECG, surface electrograms, microelectrode recordings, and optical imaging with voltage-sensitive and Ca(2+)-sensitive dyes. Action potential morphology, ECG parameters, cardiac conduction, and refractoriness were determined after perfusion with 0.1-10 microM blebbistatin. Blebbistatin 5-10 microM completely eliminated contraction in all cardiac preparations but did not have any effect on electrical activity, including ECG parameters, atrial and ventricular effective refractory periods, and atrial and ventricular activation patterns. Blebbistatin 10 microM had no effects on action potential morphology in rabbit cardiac tissue. Blebbistatin inhibited single cellular contraction in a dose-dependent manner with half-maximal inhibitory concentration (IC(50)) = 0.43 microM, without altering the morphologies of intracellular calcium transients. The blebbistatin effect was completely reversible by simultaneous washout and photobleaching by ultraviolet light Blebbistatin is a promising novel selective excitation-contraction uncoupler that can be used for optical imaging of cardiac tissues.

Effect of resveratrol on L-type calcium current in rat ventricular myocytes

To study the effect of resveratrol on L-type calcium current (I(Ca-L)) in isolated rat ventricular myocytes and the mechanisms underlying these effects. I(Ca-L) was examined in isolated single rat ventricular myocytes by using the whole cell patch-clamp recording technique. Resveratrol (10-40 micromol/L) reduced the peak amplitude of I(Ca-L) and shifted the current-voltage (I-V) curve upwards in a concentration-dependent manner. Resveratrol (10, 20, 40 micromol/L) decreased the peak amplitude of I(Ca-L) from -14.2+/-1.5 pA/pF to -10.5+/-1.5 pA/pF (P<0.05), -7.5+/-2.4 pA/pF (P<0.01), and -5.2+/-1.2 pA/pF (P<0.01), respectively. Resveratrol (40 micromol/L) shifted the steady-state activation curve of I(Ca-L) to the right and changed the half-activation potential (V0.5) from -19.4+/-0.4 mV to -15.4+/-1.9 mV (P<0.05). Resveratrol at a concentration of 40 micromol/L did not affect the steady-state inactivation curve of I(Ca-L), but did markedly shift the time-dependent recovery curve of I(Ca-L) to the right, and slow down the recovery of I(Ca-L) from inactivation. Sodium orthovanadate (Na(3)VO(4); 1 mmol/L), a potent inhibitor of tyrosine phosphatase, significantly inhibited the effects of resveratrol (P<0.01). Resveratrol inhibited I(Ca-L) mainly by inhibiting the activation of L-type calcium channels and slowing down the recovery of L-type calcium channels from inactivation. This inhibitory effect of resveratrol was mediated by the inhibition of protein tyrosine kinase in rat ventricular myocytes.