Normal Tyrode Solution Research Articles

Melatonin receptor blockade slows ventricular conduction by IK1 downregulation

Abstract Background Melatonin has been reported to prevent life-threatening arrhythmias in myocardial ischemia and reperfusion. The recent study from our group demonstrated that the antiarrhythmic action of melatonin was associated with enhanced recovery of resting membrane potential (RMP) and shortened activation time delay in the affected myocardium. This effect was independent of melatonin antioxidative properties, which raises a question about the involvement of melatonin receptor-dependent signaling in the electrophysiological action of melatonin. The aim of this study was to evaluate a role of MT1/MT2 melatonin receptors in the regulation of RMP and ventricular conduction velocity (CV). Methods Anesthetized male rats underwent coronary occlusion (5 min) and reperfusion (5 min). 5 min before ischemia induction the animals were intravenously injected with saline (control, n=7), melatonin (4 mg/kg, n=8), luzindole (MT1/MT2 receptor blocker, 0.4 mg/kg, n=6), and melatonin/luzindole combination (4 and 0.4 mg/kg, respectively, n=7). CV was measured in baseline and reperfusion by epicardial 64-lead mapping under epicardial pacing. In the enzymatically isolated ventricular myocytes (n=20 cells), voltage- and current-clamp modes of whole cell patch-clamp were used for measurements of IK1 and INa currents, and RMP, respectively. Melatonin (10 uM), luzindole (1 uM) and their combination were added to the bath solution. Results Luzindole application decreased baseline longitudinal CV as compared to placebo (52±3 vs 42±2 cm/s, p=0.028, respectively). Paired comparisons demonstrated that longitudinal CV decreased at reperfusion vs baseline in the control group (35±2 vs 53±6 cm/s, p=0.018) and in the luzindole group (33±4 vs 38±3 cm/s, p=0.039), whereas no significant differences were observed in the melatonin and melatonin/luzindole groups (40±5 vs 51±3 cm/s, p=0.114 and 39±3 vs 43±3 cm/s, p=0.498, respectively). Neither melatonin nor luzindole changed INa current characteristics (Figure, panels a and b). The average amplitude of IK1 current was 2.08±0.92 at −50 mV and −16.11±4.70 pA/pF at −120 mV in the normal Tyrode solution. Luzindole significantly reduced IK1 amplitude to 0.82±0.48 (p=0.028) and −5.6±1.6 (p=0.008) pA/pF at −50 and −120 mV, respectively. Addition of melatonin to the luzindole-containing solution partly abolished the luzindole effect (Figure, panel c). Luzindole also reduced RMP from −63±10 mV to −37±12 mV (p=0.013) and caused an increase in APD100 duration from 35±6 ms to 170±43 ms (p=0.003) (Figure, panel d). Conclusion Thus, MT1/MT2 receptor block with luzindole did not affect INa but led to the down-regulation of IK1 and depolarization of RMP. These effects can reduce accessibility of sodium channels and be the basis for the observed luzindole-dependent CV slowing. The obtained data support the significance of melatonin receptor-dependent signaling in the realization of antiarrhythmic effects of melatonin. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Russian Science Foundation

Selective activation of adrenoceptors potentiates IKs current in pulmonary vein cardiomyocytes through the protein kinase A and C signaling pathways

Delayed rectifier K+ current (IKs) is a key contributor to repolarization of action potentials. This study investigated the mechanisms underlying the adrenoceptor-induced potentiation of IKs in pulmonary vein cardiomyocytes (PVC). PVC were isolated from guinea pig pulmonary vein. The action potentials and IKs current were recorded using perforated and conventional whole-cell patch-clamp techniques. The expression of IKs was examined using immunocytochemistry and Western blotting. KCNQ1, a IKs pore-forming protein was detected as a signal band approximately 100 kDa in size, and its immunofluorescence signal was found to be mainly localized on the cell membrane. The IKs current in PVC was markedly enhanced by both β1- and β2-adrenoceptor stimulation with a negative voltage shift in the current activation, although the potentiation was more effectively induced by β2-adrenoceptor stimulation than β1-adrenoceptor stimulation. Both β-adrenoceptor-mediated increases in IKs were attenuated by treatment with the adenylyl cyclase (AC) inhibitor or protein kinase A (PKA) inhibitor. Furthermore, the IKs current was increased by α1-adrenoceptor agonist but attenuated by the protein kinase C (PKC) inhibitor. PVC exhibited action potentials in normal Tyrode solution which was slightly reduced by HMR-1556 a selective IKs blocker. However, HMR-1556 markedly reduced the β-adrenoceptor-potentiated firing rate. The stimulatory effects of β- and α1-adrenoceptor on IKs in PVC are mediated via the PKA and PKC signal pathways. HMR-1556 effectively reduced the firing rate under β-adrenoceptor activation, suggesting that the functional role of IKs might increase during sympathetic excitation under in vivo conditions.

Positive Inotropic Effects of ATP Released via the Maxi-Anion Channel in Langendorff-Perfused Mouse Hearts Subjected to Ischemia-Reperfusion.

The organic anion transporter SLCO2A1 constitutes an essential core component of the ATP-conductive large-conductance anion (Maxi-Cl) channel. Our previous experiments using Langendorff-perfused mouse hearts showed that the Maxi-Cl channel contributes largely to the release of ATP into the coronary effluent observed during 10-min reperfusion following a short period (6 min) of oxygen-glucose deprivation. The present study examined the effect of endogenous ATP released via Maxi-Cl channels on the left ventricular contractile function of Langendorff-perfused mouse hearts, using a fluid-filled balloon connected to a pressure transducer. After the initial 30-min stabilization period, the heart was then perfused with oxygen-glucose-deprived Tyrode solution for 6 min, which was followed by a 10-min perfusion with oxygenated normal Tyrode solution in the absence and presence of an ATP-hydrolyzing enzyme, apyrase, and/or an adenosine A1 receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX). In the absence of apyrase and DPCPX, the left ventricular developed pressure (LVDP) decreased from a baseline value of 72.3 ± 7.1 to 57.5 ± 5.5 mmHg (n = 4) at the end of 6-min perfusion with oxygen-glucose-deprived Tyrode solution, which was followed by a transient increase to 108.5 ± 16.5 mmHg during subsequent perfusion with oxygenated normal Tyrode solution. However, in the presence of apyrase and DPCPX, the LVDP decreased to the same degree during 6-min perfusion with oxygen-glucose-deprived Tyrode solution, but failed to exhibit a transient increase during a subsequent perfusion with oxygenated normal Tyrode solution. These results strongly suggest that endogenous ATP released through Maxi-Cl channels contributes to the development of transient positive inotropy observed during reperfusion after short-period hypoxia/ischemia in the heart.

Abstract 14416: Mechano-arrhythmogenicity is Enhanced in Late Repolarisation During Acute Ischemia by a Calcium- and TRPA1-dependent Mechanism

Introduction: Altered tissue mechanics in acute regional ischemia contribute to arrhythmias by a mechano-sensitive, Ca 2+ -dependent mechanism. This is facilitated by uncoupling of voltage-Ca 2+ dynamics, creating a vulnerable period (VP) in late repolarisation for stretch-induced arrhythmias (‘mechano-arrhythmogenicity’). However, cellular mechanisms driving mechano-arrhythmogenicity in acute ischemia are unknown. Objective: Define cellular mechanisms of mechano-arrhythmogenicity in the VP during acute ischemia in rabbit ventricular myocytes. Methods: Rabbit (♀, NZW) LV myocytes were transiently stretched (8-16% change in sarcomere length, 100 ms) during diastole or the VP in normal Tyrode (NT) or simulated ischemia (SI) solution (hyperkalemia, acidosis, metabolic inhibition). Drugs were used to buffer Ca 2+ (BAPTA), stabilise RyR (dantrolene), block mechano-sensitive TRPA1 channels (HC-030031), or block (DPI) or increase (bi-product of di-4-ANBDQPQ excitation) ROS production. Voltage-Ca 2+ was simultaneously monitored with fluorescent dyes (di-4-ANBDQPQ, Fluo-5F) and a single camera-optical splitter system. Results: SI shortened AP duration (APD NT =384 vs APD SI =219ms; p <0.0001) more than Ca 2+ transient duration (CaTD NT =424 vs CaTD SI =357 ms; p <0.0001) and increased the length of the VP (=CaTD-APD; VP NT =54 vs VP SI =146ms; n =50 cells for N NT =6 and N SI =14 rabbits ; p <0.0001). Mechano-arrhythmogenicity (single ectopy and complex sustained activity) was increased in SI compared to NT, but only for stretch in the VP (7 vs 1% of stretches; n =50, N =6 each ; p <0.005), and arrhythmias in the VP were proportionally more complex than those that occurred with stretch in diastole (100 vs 69%; n =50, N =6; p <0.05). Arrhythmia incidence in the VP during SI was reduced by BAPTA (2% of stretches; p <0.05), HC-030031 (1%; p <0.005), and DPI (2%; p <0.05), while dantrolene had no effect ( n =50, N =6 each). Fluorescence imaging during SI further increased mechano-arrhythmogenicity in both the VP and diastole (29 and 14%; n =42, N =4; p <0.05). Conclusions: Acute ischemia enhances cellular mechano-arrhythmogenicity specifically in the VP through a mechanism involving Ca 2+ , ROS, and TRPA1, suggesting potential targets for anti-arrhythmic therapy.

Ischemia Enhances the Acute Stretch-Induced Increase in Calcium Spark Rate in Ventricular Myocytes.

Introduction: In ventricular myocytes, spontaneous release of calcium (Ca2+) from the sarcoplasmic reticulum via ryanodine receptors (“Ca2+ sparks”) is acutely increased by stretch, due to a stretch-induced increase of reactive oxygen species (ROS). In acute regional ischemia there is stretch of ischemic tissue, along with an increase in Ca2+ spark rate and ROS production, each of which has been implicated in arrhythmogenesis. Yet, whether there is an impact of ischemia on the stretch-induced increase in Ca2+ sparks and ROS has not been investigated. We hypothesized that ischemia would enhance the increase of Ca2+ sparks and ROS that occurs with stretch.Methods: Isolated ventricular myocytes from mice (male, C57BL/6J) were loaded with fluorescent dye to detect Ca2+ sparks (4.6 μM Fluo-4, 10 min) or ROS (1 μM DCF, 20 min), exposed to normal Tyrode (NT) or simulated ischemia (SI) solution (hyperkalemia [15 mM potassium], acidosis [6.5 pH], and metabolic inhibition [1 mM sodium cyanide, 20 mM 2-deoxyglucose]), and subjected to sustained stretch by the carbon fiber technique (~10% increase in sarcomere length, 15 s). Ca2+ spark rate and rate of ROS production were measured by confocal microscopy.Results: Baseline Ca2+ spark rate was greater in SI (2.54 ± 0.11 sparks·s−1·100 μm−2; n = 103 cells, N = 10 mice) than NT (0.29 ± 0.05 sparks·s−1·100 μm−2; n = 33 cells, N = 9 mice; p < 0.0001). Stretch resulted in an acute increase in Ca2+ spark rate in both SI (3.03 ± 0.13 sparks·s−1·100 μm−2; p < 0.0001) and NT (0.49 ± 0.07 sparks·s−1·100 μm−2; p < 0.0001), with the increase in SI being greater than NT (+0.49 ± 0.04 vs. +0.20 ± 0.04 sparks·s−1·100 μm−2; p < 0.0001). Baseline rate of ROS production was also greater in SI (1.01 ± 0.01 normalized slope; n = 11, N = 8 mice) than NT (0.98 ± 0.01 normalized slope; n = 12, N = 4 mice; p < 0.05), but there was an acute increase with stretch only in SI (+12.5 ± 2.6%; p < 0.001).Conclusion: Ischemia enhances the stretch-induced increase of Ca2+ sparks in ventricular myocytes, with an associated enhancement of stretch-induced ROS production. This effect may be important for premature excitation and/or in the development of an arrhythmogenic substrate in acute regional ischemia.

Efficacy, potency and kinetics of activation process of Kv7.4 and Kv7.5 channel with novel pharmacological activators targeted for Lower Urinary Tract Syndrome (LUTS)

Among several types of Kv7 (KCNQ) voltage gated potassium channel family, Kv7.4 (KCNQ4) and Kv7.5 (KCNQ5) channels show prominent expression in smooth muscle, cardiac tissues and vasculature. In general, activation of the channel would mediate strong efflux of potassium and lead membrane potential to hyperpolarization, though the extent may depend on established potassium concentration gradient and membrane potential. Once membrane potential is hyperpolarized, it would decrease the excitability of excitable cells. Decreased excitability may reduce calcium spikes and Ca‐CaM mediated activation of myosin light chain kinase (MLCK) in smooth muscle cells. Recently, it was reported that Kv7.4 channels are highly expressed in corpus cavernosum smooth muscle cells and Kv7.5 channels in prostate smooth muscle cells, respectively. Since smooth muscle relaxation of corpus cavernosum and smooth muscle tension in prostate are in the center of pathophysiology of erectile dysfunction and LUTS, we examined the effect of novel Kv7.4 and Kv7.5 activators to the channel activation process.In HEK293 cells expressing human Kv7.4 or Kv7.5 channels, we performed whole‐cell patch clamp technique with two different voltage protocols in order to measure specific activity of the channel. For kinetic analysis of the activation process, voltage steps from –100 mV to +100 mV with 10 mV of step interval was applied. For each step, pulse duration was 3 seconds for activation process of Kv7.4 and Kv7.5 channels is rather slow. Intersweep duration was 10 seconds and holding potential was –80 mV. For measurement of time‐dependent action of the drug, 10 seconds single step with +50 mV was applied in every 30s with holding potential of –80 mV. The composition of internal solution is as following; 120 KCl, 5.37 CaCl2, 1.75 MgCl2, 10 EGTA, 10 HEPES, 4 ATP, 0.2 GTP. pH was adjusted to 7.3 using KOH and [Ca2+]free = 100 nM. Normal tyrode solution was used for external solution and drugs stocked with DMSO was diluted to corresponding concentrations with the Normal tyrode solution.We have found that URO‐K10, a novel activator, increases not only maximum conductance of the channel but also time constant of activation gate. In comparison to positive control, ML‐213, a well‐known Kv7.4 channel activator, URO‐K10 showed significant supremacy. EC50 of URO‐K10 was 0.304 ± 0.058 μM for Kv7.4 channel which is smaller than EC50 of ML‐213 (0.8 ± 0.3 μM). URO‐K10 not only increased the maximum conductance of the channel but also left‐shifted activation curve in terms of Boltzmann distribution. Half‐maximal voltage (V1/2) of Kv7.4 channel without any of the drugs was −43.03 mV whereas V1/2 of URO‐K10 activated channel was −67.65 mV, which is near the resting membrane potential of smooth muscle cells. ML‐213 activated channels showed V1/2 of −51.91 mV.Support or Funding InformationThis work was supported by the National Research Foundation of Korea, which is funded by the Ministry of Science, ICT (Information &amp; Communication Technology), and Future Planning (MSIP) of the Korean Government (2018R1A41023822 to I. So) and by the Education and Research Encouragement Fund of Seoul National University Hospital (I. So). Jung Eun Lee was supported by the BK plus program from the MSIP.

Effects of prolactin on ventricular myocyte shortening and calcium transport in the streptozotocin-induced diabetic rat

The physiological role of prolactin (PRL) in the heart, and in particular the diabetic heart, are largely unknown. The effects of PRL on ventricular myocyte shortening and Ca2+ transport in the streptozotocin (STZ) – induced diabetic and in age-matched control rats were investigated. PRL receptor protein, myocyte shortening, intracellular [Ca2+], L-type Ca2+ current were measured by Western blot, cell imaging, fluorescence photometry and whole-cell patch-clamp techniques, respectively. Compared to normal Tyrode solution (NT), PRL (50 ng/ml) significantly (p < 0.05) increased the amplitude of shortening in myocytes from control (7.43 ± 0.38 vs. 9.68 ± 0.46 %) and diabetic (6.57 ± 0.24 vs. 8.91 ± 0.44 %) heart (n = 44–49 cells). Compared to NT, PRL (50 ng/ml) significantly increased the amplitude of Ca2+ transients in myocytes from control (0.084 ± 0.004 vs. 0.115 ± 0.007 Fura-2 ratio units) and diabetic (0.087 ± 0.007 vs. 0.112 ± 0.006 Fura-2 ratio units) heart (n = 36–50 cells). PRL did not significantly alter the amplitude of caffeine-evoked Ca2+ transients however, PRL significantly increased the fractional release of Ca2+ in myocytes from control (21 %) and diabetic (14 %) and heart. The rate of Ca2+ transient recovery following PRL treatment was significantly increased in myocytes from diabetic and control heart. Amplitude of L-type Ca2+ current was not significantly altered by diabetes or by PRL. PRL increased the amplitude of shortening and Ca2+ transients in myocytes from control and diabetic heart. Increased fractional release of sarcoplasmic reticulum Ca2+ may partly underlie the positive inotropic effects of PRL in ventricular myocytes from control and STZ-induced diabetic rat.

Role of TRPC6 on Single Cell Mechanics in Mouse Cardiomyocytes

An increase in preload induces a biphasic response in the heart. The short-term increase in preload rapidly augments contractile force owing to the Frank-Starling mechanism (FSM) that accelerates Ca2+ sensitivity of the contraction protein. A further long-term increase in preload for several minutes causes the increase in [Ca2+]i, leading to slow force response to stretch (SFR), a further increase in the contractile force. The authors’ previous study identified the involvement of TRPC3 and TRPC6, mechanosensitive non-selective cation channels, in SFR. However, the participation of TRPC3 and TRPC6 in FSM remains unclear. To clarify the role of TRPC3 and TRPC6 in FSM, cardiomyocytes were enzymatically isolated from either wild-type (WT), TRPC3 knockout (KO), or TRPC6 KO mice. Both cell ends were held by two pairs of carbon fibers attached to both upper and bottom cell surfaces to apply stretch to the cells. The cells were paced at 4 Hz and superfused in normal Tyrode solution at 37 °C. Cell length and active/passive force calculated from carbon fiber bending were recorded in six different preload conditions. The effect of each genetic deletion on cellular contractility and elastance was assessed by the slope of end-systolic force-length relation curve (ESFLR) and end-diastolic force-length relation curve (EDFLR). The slope of ESFLR was significantly steeper in TRPC6 KO mice than in WT mice, though it remained unchanged in TRPC3 KO mice (0.47 ± 0.1; 0.35 ± 0.14; 0.36 ± 0.14 mN∙mm−2∙μm−1). Meanwhile, the slope of EDFLR was not significantly different between WT, TRPC3 KO, and TRPC6 KO mice (0.16 ± 0.046; 0.19 ± 0.11; 0.19 ± 0.13 mN∙mm−2∙μm−1). These results suggest that TRPC6 regulates the contractile property via modulating the increase in the contractile force controlled by FSM, while TRPC3 is not involved in FSM.

Evaluation of the relaxant effect of levetiracetam on isolated rat duodenum.

Levetiracetam (LEV) is an approved drug for the treatment of some epileptic disorders. With few and controversial reports addressing its possible pharmacodynamic interactions, the current study aimed at studying the effect of LEV on isolated rat duodenal strips to enlighten its possible intestinal adverse effects using the isolated smooth muscle strips of rat duodenum. LEV showed a dose-dependent inhibition in KCl (80 mm)-induced contractions in normal Tyrode's solution. Moreover, preincubation with LEV (3 mm) in K+ -rich/Ca2+ -free medium led to a significant decrease in the maximum contractions (Emax ) coupled to a right shift of the cumulative CaCl2 concentration curves implying a possible Ca2+ channel blocking potential. In addition, LEV exhibited a typical noncompetitive inhibition in the cumulative carbachol concentration curves evidenced as a decrease in Emax without the alteration of EC50 , thus eliminating any possible role of the muscarinic receptors in the relaxant effect. To rule out other possible relaxant mechanisms, tests were conveyed in Tyrode's solution containing either 100 μm l-NAME or 10 μm glimepiride to test the possible relaxant roles exhibited by nitric oxide (NO) and KATP channel opening, respectively. None of the tested pathways was involved in LEV-mediated relaxation. Taken altogether, the results of the current study entail that LEV might exert a relaxant effect on intestinal smooth muscles through blocking L-type voltage-operated calcium channels, but not involving either NO release or KATP channel opening.

MPS 11-05 nNOS regulation of myofilament Ca2+ desensitization mediates metabolic substrates-induced susceptibility of cardiac arrhythmias in normal and hypertensive rats

Objective: Metabolic syndrome is the precursor for cardiovascular complications, such as ventricular arrhythmias (sudden cardiac death), heart failure and stroke. Here, we test metabolic substrates’ regulation of cardiac excitation-contraction coupling and the roles of endothelial and neuronal nitric oxide synthases (eNOS, nNOS) in left ventricular (LV) myocytes from sham and angiotensin (Ang II)-induced hypertensive rats. Design and Method: LV myocyte contraction and Ca2+ transients were assessed by using a video-sarcomere detection system or with Fura-2AM (2 mM, IonOptix Cop, field stimulation at 2 Hz, 361). Fatty acids and other metabolic substrates (oleic acid 200 mM, palmitic acid 100 mM, linoleic acid 100 mM, lactate 1 mM, pyruvate 100 mM and carnitine 50 mM) were supplemented (termed nutrition full, NF solution) to the normal tyrode (NT) solution. Results: NF increased LV myocyte contraction and Ca2+ transients in sham and hypertension. B-adrenergic stimulation with isoprenaline (ISO) increased myocyte contraction in both groups. Notably, ISO induced delayed-aftercontraction (DAC) and spontaneous Ca2+ transients (sCa) in NF and the frequency of DAC was ∼3 fold higher in hypertension. Diastolic and peak [Ca2+]i were greater in NF in hypertension, suggesting the role of intracellular Ca2+. eNOS phosphorylation was significantly reduced by NF in both groups, therefore, reduced NO availability might be responsible for the arrhythmogenic effects of NF. Inhibition of NOS with L-NAME increased DAC and sCa in sham but reduced these parameters in hypertension. Such effects were mimicked by nNOS inhibitor, SMTC, suggesting the role of nNOS. Mechanistically, NF significantly reduced myofilament Ca2+ sensitivity (Myo-Ca-Sen) in both groups. SMTC increased Myo-Ca-Sen in sham with NF + ISO but significantly reduced it with NF + ISO in hypertension. Conclusions: Our results demonstrate that nNOS prevents NF-induced arrhythmias in normal heart but assists arrhythmogenesis in hypertension via its contrasting roles in Myo-Ca-Sen.

Effect of Ethanol Exposure on Slow Wave Activity and Smooth Muscle Contraction in the Rat Small Intestine.

Ethanol ingestion causes a variety of gastrointestinal disturbances including motility alterations. Slow wave propagation coordinates gastrointestinal motility, and abnormal slow wave activity is thought to contribute to motility disorders. To date, however, little is known about the effect of acute ethanol on motility disturbances associated with slow wave activity. To investigate the effect of ethanol on small intestine slow wave activity. Segments (3-5 cm long) were isolated from the rat duodenum, jejunum, and ileum and mounted in an organ bath superfused with a normal Tyrode solution or with 1, 3, or 5% ethanol containing Tyrode. The electrical activities were recorded using an array of 121 extracellular electrodes, and motility recordings were performed using a digital video camera. The frequency and amplitude of slow wave activity were not altered at 1, 3, or 5% ethanol concentrations, but a significant drop in velocity was found at 3 and 5% ethanol. Furthermore, inexcitable areas appeared in a dose-dependent manner. Slow wave was sometimes also seen to propagate in a circular fashion, thereby describing a reentrant loop. Finally, in all duodenal, jejunal, and ileal segments, ethanol inhibited contractions and became fully quiescent at 3-5%. These studies for the first time demonstrate that ethanol significantly inhibits slow wave and spike activity in a dose-dependent manner and could also initiate reentrant activities. Intestinal contractions were also inhibited in a dose-dependent manner. In conclusion, ethanol inhibits both slow wave activity and motor activity to cause ethanol-induced intestinal disturbances.

Regional Oxidative Stress Disrupts the Normal Propagation of Voltage Waves and Promotes Reentry in Monolayers of Cardiac Myocytes

It has been suggested that the oxidative stress due to ischemic injury plays an important role in arrhythmogenesis. Reactive oxygen species can activate the mitochondrial inner membrane anion channel causing mitochondrial depolarization, which can lead to arrhythmias as we previously showed using experimental and computational models. In particular, our previous experiments showed that coverslip-induced ischemia leads to oxidation of GSH/GSSG couple. Here, we study the effects of regional depletion of GSH pool on mitochondrial function and propagation of the voltage wave in monolayers of cardiac myocytes. Neonatal rat ventricular myocytes were isolated and cultured on fibronectin-coated plastic coverslips (D = 2.1 cm) for 5-7 days. Mitochondrial inner membrane potential was observed using potentiometric fluorescent dye, TMRM, in the dequench mode (2 μM). Optical mapping was used to record the changes in sarcolemmal membrane potential using voltage-sensitive fluorescent dye, di-4-ANEPPS (5 μM), as the voltage waves elicited by a stimulation pulse train, propagated through the monolayer. A thiol oxidizing agent, diamide (1 mM) was locally perfused in the center part (D = 0.5 cm) of the monolayer, while the rest of the coverslip received normal Tyrode's solution. The resulting GSH oxidation led to mitochondrial depolarization (left figure). Action potential amplitude decrease was followed by complete inexcitability in 20 minutes and reentry occurred in 2 out of 3 monolayers (right figure). There was no incidence of reentry in control. The results emphasize the role of oxidative stress in mitochondrial dysfunction and the following post-ischemic arrhythmias. This work was supported by NIH's R01HL105216 and R37HL54598 (B.O'R.) and AHA's 14POST20000018 (S.S.).

Cardiotoxic Electrophysiological Effects of the Herbicide Roundup(®) in Rat and Rabbit Ventricular Myocardium In Vitro.

Roundup (R), a glyphosate (G)-based herbicide (GBH), containing unknown adjuvants is widely dispersed around the world. Used principally by farmers, intoxications have increasingly been reported. We have studied R effects (containing 36% of G) on right ventricular tissues (male Sprague-Dawley rats, up to 20,000ppm and female New Zealand rabbits, at 25 and 50ppm), to investigate R cardiac electrophysiological actions in vitro. We tested the reduced Ca(++) intracellular uptake mechanism as one potential cause of the electrical abnormalities after GBH superfusion, using the Na(+)/K(+)-ATPase inhibitor ouabain or the 1,4-dihydropyridine L-type calcium channel agonist BAY K 8644 which increases I Ca. R concentrations were selected based on human blood ranges found after acute intoxication. The study showed dose-dependent V max, APD50 and APD90 variations during 45min of R superfusion. At the highest concentrations tested, there was a high incidence of conduction blocks, and 30-min washout with normal Tyrode solution did not restore excitability. We also observed an increased incidence of arrhythmias at different doses of R. Ouabain and BAY K 8644 prevented V max decrease, APD90 increase and the cardiac inexcitability induced by R 50ppm. Glyphosate alone (18 and 180ppm) had no significant electrophysiological effects. Thus, the action potential prolonging effect of R pointing to I Ca interference might explain both conduction blocks and proarrhythmia in vitro. These mechanisms may well be causative of QT prolongation, atrioventricular conduction blocks and arrhythmias in man after GBH acute intoxications as reported in retrospective hospital records.

Beat-to-beat cycle length variability of spontaneously beating guinea pig sinoatrial cells: relative contributions of the membrane and calcium clocks.

The heartbeat arises rhythmically in the sino-atrial node (SAN) and then spreads regularly throughout the heart. The molecular mechanism underlying SAN rhythm has been attributed by recent studies to the interplay between two clocks, one involving the hyperpolarization activated cation current If (the membrane clock), and the second attributable to activation of the electrogenic NaCa exchanger by spontaneous sarcoplasmic releases of calcium (the calcium clock). Both mechanisms contain, in principle, sources of beat-to-beat cycle length variability, which can determine the intrinsic variability of SAN firing and, in turn, contribute to the heart rate variability. In this work we have recorded long sequences of action potentials from patch clamped guinea pig SAN cells (SANCs) perfused, in turn, with normal Tyrode solution, with the If inhibitor ivabradine (3 µM), then back to normal Tyrode, and again with the ryanodine channels inhibitor ryanodine (3 µM). We have found that, together with the expected increase in beating cycle length (+25%), the application of ivabradine brought about a significant and dramatic increase in beat-to-beat cycle length variability (+50%). Despite the similar effect on firing rate, ryanodine did not modify significantly beat-to-beat cycle length variability. Acetylcholine was also applied and led to a 131% increase of beating cycle length, with only a 70% increase in beat-to-beat cycle length variability. We conclude that the main source of inter-beat variability of SANCs firing rate is related to the mechanism of the calcium clock, whereas the membrane clock seems to act in stabilizing rate. Accordingly, when the membrane clock is silenced by application of ivabradine, stochastic variations of the calcium clock are free to make SANCs beating rhythm more variable.

Involvement of TRPC in the Slow Force Response Observed in Mouse Ventricular Myocytes

When cardiac muscle is stretched persistently, the contractile force slowly increases over several minutes. This phenomenon is termed by a slow force response (SFR). The SFR is due to a stretch-induced increase in Ca2+ influx, however, underlying mechanism remains unclear. The transient receptor potential, canonical (TRPC) channels are mechano-sensitive, non-selective cation channels. We hypothesized that activation of TRPC channels is involved in stretch-induced increase in Ca2+ influx in SFR. To test this hypothesis, mouse cardiac myocytes were isolated with collagenase, and a pair of computer-controlled piezo-positioned carbon fibers was attached to each cell-end. Cells were paced at 1 Hz and superfused in normal Tyrode solution. Passive and active forces were calculated from carbon fiber bending. Stretch was applied by moving carbon fiber positions to achieve 3-10 % increase in sarcomere length and maintained for 200 to 300 seconds to obtain SFR. At the plateu of SFR, the contractile force increased to 115.7 ± 3.7% (n = 16) of the value immediately after the stretch. Inhibition of TRPC channel with BTP-2 (10 μM) significantly reduced the magnitude of SFR (103.5 ± 1.0%, n = 8). It has been reported that activation of angiotensin II type 1 (AT1) receptor induces Ca2+ influx via TRPC channels. To reveal whether TRPC channels are activated secondarily by AT1 receptor activated by strech, we tested the effect of Olmesartan, an AT1 receptor inhibitor, in SFR. Olmesartan (10 μM) also reduced the magnitude of SFR (101.1 ± 2.4%, n = 8). These results suggest that stretch-induced activation of AT1 receptors leads to increase in Ca2+ influx via TRPC channels in SFR.

Effect of azelnidipine and amlodipine on single cell mechanics in mouse cardiomyocytes

Azelnidipine and amlodipine are dihydropyridine-type Ca2+ channel blockers for the treatment of hypertension. Although these drugs have high vasoselectivity and small negative inotropic effects in vivo, little is known regarding their direct effects on cellular contractility without humoral regulation or the additive effects of these drugs with other antihypertensive drugs on myocardial contractility. To investigate the effects of Ca2+ channel blockers on single cell mechanics, mouse cardiomyocytes were enzymatically isolated, and a pair of carbon fibers was attached to opposite cell-ends to stretch the cells. Cells were paced at 4Hz superfused in normal Tyrode solution at 37°C. Cell length and active/passive force calculated from carbon fiber bending were recorded in 6 different preload conditions. Slopes of end-systolic force—length relation curves (maximum elastance) were measured as an index of contractility before and after drugs were administered. Azelnidipine at 10nM and 100nM did not change maximum elastance, while amlodipine at 100nM did decrease maximum elastance. The combination of RNH-6270 (active form of angiotensin II receptor blocker, olmesartan, 10nM) and either amlodipine (10nM) or azelnidipine (10nM) did not affect maximum elastance. Although both amlodipine and azelnidipine can be used safely at therapeutically relevant concentrations even in combination with olmesartan, the present results suggest that azelnidipine has a less negative inotropic action compared to amlodipine.

The angiotensin receptor blocker and PPAR-γ agonist, telmisartan, delays inactivation of voltage-gated sodium channel in rat heart: novel mechanism of drug action

Telmisartan is an angiotensin II receptor blocker and partial peroxisome proliferator-activated receptor gamma agonist that modulates the renin-angiotensin-aldosterone system. It is used primarily to manage hypertension, diabetic nephropathy, and congestive heart failure. Recent studies have reported that myocardial infarction (MI) has occurred in telmisartan-treated patients. The purpose of the study was to investigate the specific conditions and underlying mechanisms that may result in telmisartan-induced MI. We evaluated the effect of telmisartan on whole hearts, cardiomyocytes, and cardiac sarcolemmal ion channels. Hearts of 8-week-old male Sprague-Dawley rats were perfused with 3, 10, 30, or 100μM telmisartan or losartan or with normal Tyrode's solution (control) for 3h. We found that telmisartan induced myocardial infarction, with an infarct size of 21% of the total at 30μM (P < 0.0001) and 63% of the total area at 100μM (P < 0.001). Telmisartan also induced cardiac dysfunction (e.g., decreased heart rate, diminished coronary flow, hypercontracture, and arrhythmia). Confocal microscopy demonstrated that 30μM telmisartan significantly elevated the intracellular Ca(2+) level, leading to hypercontracture and cell death. Patch clamp analysis of isolated cardiomyocytes revealed that telmisartan induced Na(+) overload by slowing the inactivation of voltage-gated Na(+) current (I (Na)), activating the reverse mode of Na(+)-Ca(2+) exchanger activity, and causing Ca(2+) overload. Telmisartan significantly delayed the inactivation of the voltage-gated Na(+) channel, causing cytosolic Na(+) overload, prolonged action potential duration, and subsequent Ca(2+) overload. Above 30μM, telmisartan may potentially cause cardiac cell death and MI.

FKBP12.6 overexpression does not protect against remodelling after myocardial infarction

Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca(2+) handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca(2+)](i) transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca(2+) content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca(2+) content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca(2+)](i) transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca(2+) content and Na(+)- Ca(2+) exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 μm)(-1) s(-1) in Tg MI versus 1.6 ± 0.2 sparks (100 μm)(-1) s(-1) in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted.

Local β-Adrenergic Stimulation Overcomes Source-Sink Mismatch to Generate Focal Arrhythmia

β-Adrenergic receptor stimulation produces sarcoplasmic reticulum Ca(2+) overload and delayed afterdepolarizations in isolated ventricular myocytes. How delayed afterdepolarizations are synchronized to overcome the source-sink mismatch and produce focal arrhythmia in the intact heart remains unknown. To determine whether local β-adrenergic receptor stimulation produces spatiotemporal synchronization of delayed afterdepolarizations and to examine the effects of tissue geometry and cell-cell coupling on the induction of focal arrhythmia. Simultaneous optical mapping of transmembrane potential and Ca(2+) transients was performed in normal rabbit hearts during subepicardial injections (50 μL) of norepinephrine (NE) or control (normal Tyrode's solution). Local NE produced premature ventricular complexes (PVCs) from the injection site that were dose-dependent (low-dose [30-60 μmol/L], 0.45±0.62 PVCs per injection; high-dose [125-250 μmol/L], 1.33±1.46 PVCs per injection; P<0.0001) and were inhibited by propranolol. NE-induced PVCs exhibited abnormal voltage-Ca(2+) delay at the initiation site and were inhibited by either sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase inhibition or reduced perfusate [Ca(2+)], which indicates a Ca(2+)-mediated mechanism. NE-induced PVCs were more common at right ventricular than at left ventricular sites (1.48±1.50 versus 0.55±0.89, P<0.01), and this was unchanged after chemical ablation of endocardial Purkinje fibers, which suggests that source-sink interactions may contribute to the greater propensity to right ventricular PVCs. Partial gap junction uncoupling with carbenoxolone (25 μmol/L) increased focal activity (2.18±1.43 versus 1.33±1.46 PVCs per injection, P<0.05), which further supports source-sink balance as a critical mediator of Ca(2+)-induced PVCs. These data provide the first experimental demonstration that localized β-adrenergic receptor stimulation produces spatiotemporal synchronization of sarcoplasmic reticulum Ca(2+) overload and release in the intact heart and highlight the critical nature of source-sink balance in initiating focal arrhythmias.

Blocking effect of salvianolic acid a on calcium channels in isolated rat ventricular myocytes

To study the effect of salvianolic acid A (SAA) on L-type calcium current (I-CaL) in isolated ventricular myocytes of Sprague-Dawley rats. SAA powder was dissolved in normal Tyrode's solution to reach the concentrations of 1, 10, 100, and 1000 μmol/L. The traditional whole-cell patch-clamp recording technique was employed to evaluate the effects of SAA on I-CaL in single ventricular myocytes which were prepared by Langendorff perfusion apparatus from Sprague-Dawley rats. SAA (1, 10, 100, and 1000 μmol/L) inhibited I-CaL peak value by 16.23%±1.3% (n=6, P<0.05), 22.9%±3.6% (n=6, P<0.05), 53.4%±3.0% (n=8, P<0.01), and 62.26%±2.9% (n=6, P<0.01), respectively. SAA reversibly inhibited I-CaL in a dose-dependent manner and with a half-blocking concentration (IC(50)) of 38.3 μmol/L. SAA at 100 μmol/L elevated the I-V curve obviously, and shifted the half-active voltage (V(0.5)) from (-15.78±0.86) mV to (-11.24 ±0.77) mV (n=6, P<0.05) and the slope (K) from 5.33±0.74 to 4.35±0.74 (n=6, P>0.05). However, it did not alter the shapes of I-V curve, steady-state inactivation curve, or recovery from inactivation curve. SAA inhibited I-CaL in a dose-dependent manner. It shifted the steady-state activation curve to a more positive voltage, which indicated that the drug affected the activated state of calcium channels, and suggested that the Ca(2+) antagonistic effect of SAA be beneficial in the treatment of myocardial ischemia reperfusion injury.