ß3-Adrenergic regulation of L-type Ca²âº current and force of contraction in human ventricle.

ß3-Adrenergic receptor (ß3-AR) is expressed in human atrial and ventricular tissues. Recently, we have demonstrated that it was involved in the activation of L-type Ca(2+) current (I(Ca,L)) in human atrial myocytes and the force of contraction of human atrial trabeculae. In the present study, we examined the effect of ß3-AR agonist CGP12177 which also is a ß1-AR/ß2-AR antagonist on I(Ca,L) in human ventricular myocytes (HVMs) and the force of contraction of human ventricular trabeculae. CGP12177 stimulated I(Ca,L) in HVMs with high potency but much lower efficacy than isoprenaline. The ß3-AR antagonist L-748,337 inhibited the effect of CGP12177. CGP12177 and L748,337 competed selectively on ß3-ARs because L748,337 had no effect on isoprenaline-induced stimulation of I(Ca,L), while CGP12177 completely blocked the effect of isoprenaline. The activation of ß3-ARs by CGP12177 does not involve the activation of Gi proteins because CGP12177 had no effect on forskolin-induced stimulation of I(Ca,L). CGP12177 had no effect on the force of contraction of human ventricular trabeculae. L-NMMA, an inhibitor of NO synthase, and IBMX, a nonselective inhibitor of phosphodiesterases, did not potentiate the effect of CGP12177 either on contraction of human ventricular trabeculae or on I(Ca,L) in HVMs. We conclude that in human ventricles ß3-AR activation has no inotropic effect, while it slightly increases I(Ca,L). In contrast to human atrium, the activation of ß3-ARs in human ventricle is not accompanied by increased activity of phosphodiesterases.

Characterization of Mg²âº-regulated TRPM7-like current in human atrial myocytes.

BACKGROUND: TRPM7 (Transient Receptor Potential of the Melastatin subfamily) proteins are highly expressed in the heart, however, electrophysiological studies, demonstrating and characterizing these channels in human cardiomyocytes, are missing. METHODS: We have used the patch clamp technique to characterize the biophysical properties of TRPM7 channel in human myocytes isolated from right atria small chunks obtained from 116 patients in sinus rhythm during coronary artery and valvular surgery. Under whole-cell voltage-clamp, with Ca²âº and K⁺ channels blocked, currents were generated by symmetrical voltage ramp commands to potentials between -120 and +80 mV, from a holding potential of -80 mV. RESULTS: We demonstrate that activated native current has dual control by intracellular Mg²âº (free-Mg²âº or ATP-bound form), and shows up- or down-regulation by its low or high levels, respectively, displaying outward rectification in physiological extracellular medium. High extracellular Mg²âº and Ca²âº block the outward current, while Gd³âº, SpM4⁺, 2-APB, and carvacrol inhibit both (inward and outward) currents. Besides, divalents also permeate the channel, and the efficacy sequence, at 20 mM, was Mg²âº>Ni²âº>Ca²âº>Ba²âº>Cd²âº for decreasing outward and Ni²âº>Mg²âº>Ba²âº≥Ca²âº>Cd²âº for increasing inward currents. The defined current bears many characteristics of heterologously expressed or native TRPM7 current, and allowed us to propose that current under study is TRPM7-like. However, the time of beginning and time to peak as well steady state magnitude (range from 1.21 to 11.63 pA/pF, n(cells/patients) = 136/77) of induced TRPM7-like current in atrial myocytes from different patients showed a large variability, while from the same sample of human atria all these parameters were very homogenous. We present new information that TRPM7-like current in human myocytes is less sensitive to Mg²âº. In addition, in some myocytes (from 24 out of 77 patients) that current was already up-regulated at membrane rupture. CONCLUSIONS: This study provides the first electrophysiological description of TRPM7-like current in native human atrial myocytes. Less sensitivity to intracellular Mg²âº suggests for channel operation under physiological conditions. The TRPM7-like current up-regulation indicates the pathophysiological evidence of that current in human heart.

Effect of inhibitors of mitochondrial respiratory chain complexes on the electromechanical activity in human myocardium.

UNLABELLED: THE AIM OF THE STUDY was to investigate the effect of inhibitors of mitochondrial respiratory chain complexes I, III, and IV on the electromechanical activity in human myocardium. MATERIAL AND METHODS: The experiments were performed on the human myocardial strips obtained from patients with heart failure (NYHA class III or IV) using a conventional method of registration of myocardial electromechanical activity. Under the perfusion with physiological Tyrode solution (control), contraction force (P) was 0.94±0.12 mN (n=16), relaxation time (t50) was 173.38±5.03 ms (n=15), action potential durations measured at 50% (AP50) and 90% (AP90) repolarization were 248.96±13.38 ms and 398.59±17.93 ms, respectively (n=13). RESULTS: The inhibition of respiratory chain complex I by rotenone (3 × 10⁻5 M, the highest concentration applied) decreased contraction force of human myocardium to 48.99%±14.74% (n=3) (P<0.05); AP50, to 81.34%±15.81%; and AP90, to 87.28%±7.25% (n=3) (P>0.05) of control level, while relaxation time and resting tension remained almost unchanged. Antimycin A, an inhibitor of complex III, applied at the highest concentration (3 × 10⁻4 M) reduced P to 41.66%±8.8% (n=5) (P<0.001) and marginally increased t50 and decreased the durations of AP. Anoxia (3 mM Na2S2O4) that inhibits the activity of complex IV reduced the contraction force to 9.23%±3.56% (n=6) (P<0.001), AP50 and AP90 to 65.46%±9.95% and 71.07%±8.39% (n=5) (P<0.05) of control level, respectively; furthermore, the resting tension augmented (contracture developed). CONCLUSIONS: Our results show that the inhibition of respiratory chain complex IV had the strongest inhibitory effect on the electromechanical activity of failing human myocardium.

[I(KACh) inhibitor 2-[(4-methylphenyl)sulfonylcarbamido]-1-(4-nitrobenzyl)pyridinium bromide (2-AP27) is a muscarinic M(2) receptor antagonist]. / I(KACh) slopiklis 2-[(4-metilfenil)sufonilkarbamido]-1-(4-nitrobenzil) piridinio bromidas (2-AP27) yra muskarino M(2) receptoriu antagonistas.

Aminopyridines are known to inhibit acetylcholine-activated K(+) current (I(KACh)) in cardiac myocytes. The aim of this study was to examine the effect of 2-aminopyridine sulfonylcarbamide derivative 2-AP27 on isoprenaline-stimulated L-type Ca(2+) current (I(CaL)) and to identify whether 2-AP27 acts via blocking of muscarinic M(2)-receptors in frog cardiomyocytes. The whole-cell configuration of the patch-clamp technique was used to record I(CaL) in enzymatically isolated cardiac myocytes. Isoprenaline (0.1 microM), an agonist of beta(1)-beta(2)-adrenoreceptors, stimulated the I(CaL) up to 475+/-61% (n=4) (P<0.05) vs. control. Then, in the first series of experiments, carbachol (0.01 microM), an agonist of M(2 )muscarinic receptors, reduced the stimulatory effect of isoprenaline to 42+/-15% vs. isoprenaline alone. 2-AP27 (100 microM) alone completely abolished the inhibitory effect of carbachol on isoprenaline-stimulated I(CaL), which recovered to 95+/-5.8% of the effect of isoprenaline. In the second series of experiments, adenosine (1microM), an agonist of A(1)-adenosine receptors, reduced the stimulatory effect of isoprenaline on I(CaL) to 56+/-10% (n=3) (P<0.05). Then 2-AP27 (100 microM) applied in the presence of adenosine, had no effect on I(CaL), which remained at 51+/-7.9% (n=3) (P<0.05) of the effect of isoprenaline. These results suggest that 2-AP27, a new derivative of 2-AP, containing 4-toluolsulfonylcarbamide instead of amino group and quaternizated nitrogen by 4-nitrobenzylbromide in pyridine ring, is acting as an antagonist of muscarinic M(2) receptors in frog ventricular myocytes.

beta3-adrenergic receptor activation increases human atrial tissue contractility and stimulates the L-type Ca2+ current.

beta3-adrenergic receptor (beta3-AR) activation produces a negative inotropic effect in human ventricles. Here we explored the role of beta3-AR in the human atrium. Unexpectedly, beta3-AR activation increased human atrial tissue contractility and stimulated the L-type Ca2+ channel current (I Ca,L) in isolated human atrial myocytes (HAMs). Right atrial tissue specimens were obtained from 57 patients undergoing heart surgery for congenital defects, coronary artery diseases, valve replacement, or heart transplantation. The I(Ca,L) and isometric contraction were recorded using a whole-cell patch-clamp technique and a mechanoelectrical force transducer. Two selective beta3-AR agonists, SR58611 and BRL37344, and a beta3-AR partial agonist, CGP12177, stimulated I(Ca,L) in HAMs with nanomolar potency and a 60%-90% efficacy compared with isoprenaline. The beta3-AR agonists also increased contractility but with a much lower efficacy (approximately 10%) than isoprenaline. The beta3-AR antagonist L-748,337, beta1-/beta2-AR antagonist nadolol, and beta1-/beta2-/beta3-AR antagonist bupranolol were used to confirm the involvement of beta3-ARs (and not beta1-/beta2-ARs) in these effects. The beta3-AR effects involved the cAMP/PKA pathway, since the PKA inhibitor H89 blocked I(Ca,L) stimulation and the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) strongly increased the positive inotropic effect. Therefore, unlike in ventricular tissue, beta3-ARs are positively coupled to L-type Ca2+ channels and contractility in human atrial tissues through a cAMP-dependent pathway.

[Anticholinergic effect of 2-aminopyridine and its sulfonylcarbamide derivatives on electromechanical activity in guinea pig atrium]. / 2-aminopiridino ir jo sulfonilkarbamidiniu analogu anticholinerginis poveikis juru kiaulytes priesirdziu elektromechaniniam aktyvumui.

The aim of the study was to investigate an action of 2-aminopyridine and its new sulfonylcarbamide derivatives 2-AP21, 2-AP22, 2-AP26, and 2-AP27 (10(-5)-10(-3) M) on carbachol-induced shortening of action potential duration and reduction of contraction force in guinea pig atrial muscles. Experiments were carried out using a standard method of myocardium electromechanical activity registration. Under control conditions (perfusion of atrial strips with Tyrode solution), an average of action potential duration, measured at 90% (AP90) and 50% (AP50) of repolarization, were 112.32+/-6.07 ms and 50.21+/-3.25 ms, (n=19), respectively, and contraction force was of 1.42+/-0.28 mN (n=20). Carbachol (10(-6)M), an agonist of muscarinic acetylcholine receptor and activator of K(Ach) channels, markedly decreased AP90 to 35.31+/-4.21%, AP50--to 26.42+/-2.66% (n=19) (P<0.001), and contraction force--to 24.23+/-2.0% (n=20) (P<0.001) vs. control. Modification of 2-aminopyridine structure by replacing 2-amino group by 4-toluolsulfonylcarbamide fragment and quaternization of nitrogen in pyridine ring increased anticholinergic effect on action potential duration and contraction force. According to their maximal prolongation of AP at 90% of repolarization, all new drugs ranked as follows: 2-AP27>>2-AP26>2-AP22> or =2-AP>2-AP21. 2-aminopyridine derivative 2-AP27, containing 4-toluolsulfonylcarbamide fragment and 4-nitrobenzyl radical at quaternized nitrogen of the pyridine, had the most potent anticholinergic effect on AP90 (936.60+/-178.23%). 2-AP22 and 2-AP26 (containing methyl or allyl radicals at quaternized nitrogen of the pyridine, respectively) showed a much weaker anticholinergic effect (231.39+/-28.48% and 318.25+/-63.81%, respectively). The weakest anticholinergic effect (63.59+/-34.38%) was induced by 2-aminopyridine derivative 2-AP21, which had non-quaternized nitrogen of the pyridine.

[Effects of ATP-regulated potassium channels modulators, N-(2-pyridyl)-N'-(4-toluol) sulfonylcarbamides, on electromechanical activity in guinea pig myocardium]. / ATF-reguliuojamu kalio kanalu moduliatoriu, N-(2-piridil)-N'-(4-toluol) sulfonilkarbamidu itaka juru kiaulytes miokardo elektromechaniniam aktyvumui.

The aim of the study was to investigate the effects of 2-aminopyridine (2-AP) and its new sulfonylcarbamide derivatives AP21, AP22, AP26, and AP27 (10(-(5))-10(-(3)) M) on pinacidil (5x10(-(5)) M), an activator of K(ATP) channels, induced shortening of action potential duration and reduction of contraction force in guinea pig papillary muscles. Experiments were carried out using a standard method of myocardium electromechanical activity registration. Under control conditions (perfusion of papillary muscles with Tyrode solution), an average of action potential duration (APD), measured at 90% (APD(90)) and 50% (APD(50)) of repolarization, were 211.78+/-8.6 ms and 173.22+/-8.3 ms (n=18), respectively, and contraction force was 1.77+/-0.36 mN (n=18). Pinacidil markedly decreased APD(90) to 58.16+/-4.4%, APD(50) - to 52.51+/-4.85% (n=18), and contraction force - to 30.45+/-4.06% (n=18), (p<0.001) vs. control. 2-aminopyridine and its sulfonylcarbamide derivative AP22 (with 4-toluolsulfonylcarbamide fragment and methyl iodide-quaternized nitrogen of the pyridine ring) had no effect on the pinacidil-induced shortening of action potential and reduction of contraction force. 2-aminopyridine derivatives, AP21 (with 4-toluolsulfonylcarbamide fragment) and AP26 (with allyl bromide-quaternized nitrogen of the pyridine ring), showed a weak effect on pinacidil-induced shortening of action potential duration. The 2-aminopyridine derivative AP27 (with the 4-toluolsulfonylcarbamide fragment where nitrogen of the pyridine ring was quaternized by 4-nitrobenzyl bromide) had the most potent stimulatory effect on action potential duration and contraction force, which were reduced by pinacidil.

Effect of hyperosmolarity on beta2-adrenergic stimulation in human atrium.

The purpose of the present study was to determine whether extracellular osmotic pressure modulates beta2-adrenergic stimulation of the contraction force and L-type Ca2+ current in human atrial myocytes. Experiments were performed on human atrial trabeculae and myocytes isolated from the right atrium. We have studied the effect of salbutamol (SAL), a beta2-adrenoceptor agonist, on peak tension (P), time to half peak tension (tc), time to half relaxation (tr), resting tension (contracture) (C) and L-type calcium current (ICaL) under isosmotic (345 mOsm) and hyperosmotic (525 mOsm in experiments for P, and 405 mOsm for ICaL) conditions. Salbutamol (10(-8)/10(-5) M) added to the control solution increased P, with a half-stimulation constant EC50=(2.7+/-0.6)x10(-8) M (p<0.05) and the maximal stimulation of contraction force Pmax=180.6+/-45.8% (n=10). The time to half peak and time to half relaxation were reduced by salbutamol to 89.1+/-2.7% and 78.8+/-4.1% (n=8), respectively. Mannitol (180 mM) added to the Tyrode solution decreased Pmax to 34+/-5.5%; tc, tr and C were increased to 126.3+/-7.2%, 158.3+/-22.6% (n=4) and 0.54+/-0.22 (n=5) of the control level, respectively. Under these hyperosmotic conditions the same concentrations of SAL increased P with a half-stimulation constant EC50=(6.4+/-2.6)x10(-7) M and Pmax=57.2+/-12.6% (n=4). The tc and tr were reduced by 23.2+/-5.9% and 53.1+/-19.4% (n=4), respectively (as compared to mannitol). There was no significant effect of salbutamol on the resting tension induced by mannitol. Under isosmotic conditions salbutamol (10(-9)/10(-6) M) increased ICaL with an EC50 value of (2.9+/-0.9)x10(-9) M and Emax 182.3+/-19.8% (n=4). In hyperosmotic solutions the EC50 and Emax for ICaL were (1.2+/-0.5)x10(-8) M and 217.2+/-70.5%, respectively (n=5). These results indicated that hyperosmolarity reduced the effect of beta2-adrenergic stimulation on human atrial cells.

[Anticholinergic effect of procainamide and its sulfonylcarbamide derivatives on electromechanical activity in guinea pig atrium]. / Prokainamido ir jo sulfonilkarbamidiniu analogu anticholinerginis poveikis juru kiaulytes priesirdziu elektromechaniniam aktyvumui.

The aim of the study was to investigate the effect of class IA antiarrhythmic drug procainamide and its new derivatives PA20, PA23, PA28 and PA53 on carbachol-induced action potential duration and contraction force in guinea pig atrial muscles. Experiments were carried out using standard method of registration of myocardium electromechanical activity. Under control conditions (perfusion of atrial strips with Tyrode solution), mean action potential duration measured at 90% (AP90) and 50% (AP50) of repolarization were 92.5+/-4.5 ms and 44.4+/-2.9 ms (n=18), respectively, and contraction force was of 2.7+/-0.5 mN (n=12). Carbachol (10(-6) M), an agonist of muscarinic acetylcholine receptor and activator of K(Ach) channels, markedly decreased AP90 to 32.4+/-2.4%, AP50 - to 25.4+/-2.2% (n=18) (p<0.001) and contraction force - to 24.2+/-5.8% (n=16) (p<0.05), vs. control. Procainamide and its new sulfonylcarbamide derivatives PA20, PA23, PA28 (10(-5)-3x10(-3) M), and PA53 (10(-5)-10(-3) M) reversed the carbachol-induced action potential duration shortening at different extent: procainamide derivative PA20 (N-cyclohexylsulfonylcarbamide fragment is linked up to benzene ring) had the most potent anticholinergic effect on action potential duration and contraction force of guinea pig atrial muscles. N-ethylsulfonylcarbamide fragment in PA23 or permanent positive charge of aliphatic nitrogen in PA28 had a weaker anticholinergic effect (similar to procainamide action) on action potential duration and contraction force than PA20. The weakest anticholinergic effect was induced by procainamide derivative PA53 with isosteric form of sulfonylcarbamide.

[Effect of pyruvate on the dependence of electromechanical activity on stimulation frequency in human myocardium]. / Piruvato itaka zmogaus miokardo elektromechaninio aktyvumo priklausomumui nuo dirginimo daznio.

The aim of the study was to determine the effect of pyruvate, a substrate of energetic metabolism, on the dependence of electromechanical activity on stimulation frequency in human myocardium. The experiments were performed on human ventricle myocardium from patients undergoing cardiac corrective open heart surgery at Clinic of Cardiac Surgery in Kaunas University of Medicine by using standard method of registration of myocardium electromechanical activity. The stimulation frequency of myocardial strips were 0.2, 0.5, 1.0, 1.5, 2.0, 2.5 and 3 Hz, duration of pulses -2-5 ms. In control, i.e. at perfusion of mycoardial strips by Tyrode solution and stimulation frequency 1 Hz, an average of contraction force (P) was 0.94+/-0.18 mN (n=12), half time of relaxation (t(a)) - 178.88+/-9.31 ms (n=12), and action potential duration measured at 50% repolarization level (VP(50)) - 294.75+/-27.95 ms (n=9). Pyruvate (10 mM) increased P to 159.9+/-13.4% (p<0.00), t(a)- 106.9+/-2.2% (n=12) (p<0.001), and VP(50)- 111.1+/-7.4% (n=5) (p<0.05), as compared to control. In the absence of pyruvate values of P, t(a) and VP(50) of myocardial strips were higher at 0.2 Hz and 0.5 Hz and lower at 3 Hz compared with those at 1 Hz of stimulation frequency. Although the dependence of these parameters on stimulation frequency in the presence of pyruvate (10 mM) was not altered but their values were persistently increased at all stimulation frequencies. The present data indicate that pyruvate is an effective inotropic agent, which can improve contractility function in wide-ranging diapason of stimulation frequency in failing human heart.