Guinea Pig Papillary Muscle Preparations Research Articles

Mechanism of hypoxic preconditionin in guinea pig papillary muscles

Brief ischemia or hypoxia has been found to protect the heart against susbsequent long-lasting ischemia and to improve contractile dysfunction as well to reduce cell necrosis and the incidence of lethal arrhythmias. This phenomenon, termed preconditioning (PC) has been demonstrated in different species. However, little is known about PC in guinea pigs. Moreover, electrophysiological changes underlying protection have not been studied so far in conjuntion with force recovery in a setting of PC. The aim of the study was to study PC in a guinea pig papillary muscle, using recovery of contractility after long hypoxic challenge as the main end-point of protection, and to investigate concominant electrophysiological alterations. In guinea pig papillary muscle preparations contracting isometrically (paced at 2 Hz), transmembrane action potentials (AP) and developed force (DF) were recorded by conventional microelectrode technique and a force tranducer. In addition, effective refractory periods (ERP) were determined. Hypoxia was induced by superfusion with 100% N2 (pO2 < 5 kPa) and pacing at 3,3 Hz. In the control group, long hypoxia lasted for 45 min and was followed by 30 min reoxygenation. In the PC group, muscles were subjected to 5 min hypoxia followed by 10 min recovery prior to sustained hypoxia/reoxygenation. Results: Long hypoxia induced a similar depression of DF in both, PC and control groups. However, a loss of contractile activity occured earlier in the PC group. AP duration and ERP decreased faster and were significantly shorter after PC. Upon reoxygenation, preconditioned muscles showed significantly better recovery of function (DF 86% of prehypoxic value vs. 36% in controls; p < 0,05). AP and ERP were completely restored in both, PC and control groups. Guinea pig papillary muscle can be preconditioned with a brief hypoxic challenge against contractile dysfunction upon long-lasting hypoxia/reoxygenation. Shortening of AP and loss of contractility occured more quickly during hypoxia and may participate in the protective effect of preconditioning. Possible mechanisms might involve facilitated opening of KATP-dependent channels.

Studies on cardiac ingredients of plants. XIII: Chemical modification of gitoxin to cardiotonic compounds without vascular effect.

Nitrated gitoxins (4) and bufotoxin homologues with various lengths of alkyl chain at C-3 of the steroid nucleus (10) were prepared from gitoxin (1). The pharmacological activities of the resulting compounds (4 and 10) were evaluated by measurement of inhibitory effect on NA+, K(+)-adenosine triphosphatase (ATPase) prepared from dog kidney, positive inotropic effect (PIE) on isolated guinea-pig papillary muscle preparations, and the effect on smooth muscle using the mesenteric artery from spontaneously hypertensive rats. Most of the compounds showed a smaller contractile effect on the arterial muscle. Among these compounds, gitoxin 3"-nitrate (4g) exhibited the most desirable biological activities, such as PIE comparable to that of 1, 1.25 times wider concentration-dependent range than 1, and lack of contractile activity on vascular muscle.

The risk of myocardial stunning is decreased concentration-dependently by K ATP channel activation with nicorandil before high K + cardioplegia

Background: Drug-induced opening of the adenosine triphosphate-sensitive potassium channel (K ATP) during hypoxia and/or ischemia, achieved significant myocardial protection in several in vitro and in vivo models. Pretreatment with K ATP openers simulated preconditioning and thus enhanced recovery from ischemia. We have demonstrated that the risk of hypoxia-induced myocardial stunning is reversed by K ATP activation with 1 mmol/l nicorandil before cold cardioplegic arrest. Whether lower concentrations were effective is not known. Methods: In guinea pig papillary muscle preparations contracting isometrically (driven at 1600 ms cycle), nicorandil was superfused (15 min) either 1 μmol/l ( n = 4), 30 μmol/l ( n = 4), 100 μmol/l ( n = 4), or 1 mmol/l ( n = 8) in Tyrode's solution (oxygen content 16 ml/l, 37 °C, 5 ml/min). Controls were superfused with saline (Tyrode's solution; n = 8). A group containing vehicle (DMSO 1%, n = 8) was also studied. In four preparations the K ATP channel blocker glibenclamide 1 μmol/l was given before nicorandil 1 mmol/l. Then, long-lasting (120 min) but moderately hypoxic (oxygen content 5 ml/l: 31% of Tyrode's solution) superfusion with hypothermic (20 °C) high K + (16 mmol/l) cardioplegic solution (5 ml/min) was performed. Recovery of contractility was evaluated after further 60 min of reoxygenation with Tyrode's solution based on DT TPT (developed tension divided by time to peak tension) as percent of prehypoxia basal values (% DT TPT60 ). DT TPT was also studied following 15 min of inotropic stimulation with dobutamine 10 μmol/l (% DT TPT75 ). To assess the risk of stunning, we used a multivariate linear model by all possible subsets analysis (BMDP-9R) aimed at predicting both % DT TPT60 and % DT TPT75 (as continuous dependent variables). Results: During cardioplegia induction, time to arrest (TTA) was (mean ± S.D.) 103 ± 48s in control preparations which had poor recovery of contractility (stunning) after reoxygenation (% DT TPT60 : 71 ± 20%; % DT TPT75 : 443 ± 272%). Nicorandil (1 μmol/l-1 mmol/l) abbreviated TTA concentration-dependently (163 ± 74, 149 ± 103, 82 ± 20, and 56 ± 27s) and improved both % DT TPT60 (63 ± 9, 78 ± 17, 87 ± 13, and 98 ± 11%) and % DT TPT75 (587 ± 333, 619 ± 107, 971 ± 301, and 666 ± 400%). Glibenclamide reversed the effects of nicorandil 1 mmol/l (TTA: 165 ± 30 s, P < 0.01; % DT TPT60 : 43 ± 12, P < 0.01; % DT TPT75 : 272 ± 147, P < 0.05). Multivariate prediction of myocardial stunning at both 60 and 75 min reoxygenation showed that nicorandil (30 μmol/l–1 mmol/l) was a significant ( P < 0.001) protectant whereas glibenclamide was a significant risk factor ( P = 0.009). It is unclear whether negative inotropic effects of nicorandil (% DT TPT at the end of pretreatment) was mechanistically related to reduced risk of stunning since contribution was seen only to predict % DT TPT75 ( t = −3.24, P = 0.003) whereas a positive association was observed with % DT TPT60 ( t = 1.89, P = 0.068). Conclusion: Pretreatment with nicorandil concentration-dependently enhanced the cardioprotective effect of hypothermic high K + cardioplegia. The risk of myocardial stunning was decreased by K ATP opening with nicorandil and increased by K ATP block with glibenclamide. Inotropic stimulation with dobutamine might unravel the role of negative inotropic effect of K ATP opening as a contributory factor to explain the efficacy of nicorandil in our model.

Pretreatment with the adenosine triphosphate-sensitive potassium channel opener nicorandil and improved myocardial protection during high-potassium ca rdioplegic hypoxia

We hypothesized that pretreatment with the potassium channel opener nicorandil might enhance myocardial protection achieved by cold (20 degrees C) high-potassium (16 mmol/L) cardioplegia (5 ml/min) during long-duration (120 minutes) myocardial hypoxia (average oxygen content 5.4 ml/dl). We tested a 15-minute infusion of nicorandil (1 mmol/L) given only before (group A, n = 8) or before and during cardioplegia (group B, n = 8) in guinea pig papillary muscle preparations contracting isometrically while stimulated (4 mA, 2 msec) at 1600 msec cycle length. Nicorandil was significantly negative inotropic before cardioplegia and shortened significantly action potential duration. During cardioplegia, time to arrest of contraction was shortened from 145 +/- 28 seconds (mean +/- standard error) in the vehicle group (dimethyl sulfoxide 1:100; n = 8) to 56 +/- 10 seconds (p < 0.02) and 68 +/- 5 seconds (p < 0.05) in groups A and B, respectively. Recovery of developed tension at 60 minutes of normothermic reoxygenation (expressed as percent of prehypoxia basal value) was ameliorated from 54% +/- 6% (vehicle group) to 92% +/- 4% (group A, p < 0.01) and to 119% +/- 19% (group B, p < 0.01). The specific potassium channel blocker glibenclamide (glib: 1 mumol/L, n = 8) prolonged action potential duration and was without effect on time to arrest. On reoxygenation, the glib group had prolonged time to half relaxation (versus group A, p < 0.02) and the worst percent developed tension at 60 minutes (40% +/- 4%). In the overall study, time to arrest and percent developed tension at 60 minutes were inversely correlated (r = -0.45, p < 0.01). Arrhythmias were never observed. Multivariate analysis showed that pretreatment with nicorandil (with or without drug adjunct to cardioplegic solution) was a significant factor (r2 = 0.65, p = 0.0001) to influence reoxygenation-mediated recovery of mechanical function. Neither the negative inotropic effect of nicorandil before cardioplegia nor its abbreviating action on time to arrest during cardioplegia was contributory to explain recovery of function on reoxygenation. In subgroup analysis, negative inotropism and the shortening of action potential duration were contributory factors. These data suggest that nicorandil pretreatment activates potassium channels and enhances the myocardial protection provided by cold cardioplegia an effect, which is evident after a long hypoxic period, late on reoxygenation.

Studies on Cardiac Ingredients of Plants. XI. Synthesis of New Bufotoxin Homologues Utilizing Scillarenin (the Genuine Aglycone of Proscillaridin), and Their Biological Activities.

New bufotoxin homologues (3) with various lengths of alkyl chain including longer ones than a suberoyl group at C-3 of the steroid nucleus were prepared from proscillaridin (1) via its genuine aglycone, scillarenin (2), in excellent yield. In the course of preparation, we established conditions for efficient enzymatic glycolysis of 1 to give 2 quantitatively. The pharmacological activities of the resulting bufotoxin homologues (3) were evaluated by measurement of the effect on smooth muscle using the mesenteric artery from spontaneously hypertensive rats, inhibitory effect on Na+, K+-adenosine triphosphatase (ATPase) prepared from dog kidney, and positive inotropic effect (PIE) on isolated guinea-pig papillary muscle preparations. The bufotoxin homologues (3) showed only slight contraction of vascular muscle followed by a small relaxation, in addition to the high Na+, K+-ATPase inhibitory activity and PIE comparable to those of clinically used ouabain, digoxin, and digitoxin. Those bufotoxin homologues (3) may be useful as cardiac agents with a minimal vascular contractile effect.

Studies on cardiac ingredients of plants. X. Preparation of nitrates of tetrahydroproscillaridin and their pharmacological activities

To reduce the vascular contracting effect of the hydrogenated cardiac glycosides, 20-(R)- and 20-(S)-tetrahydroproscillaridins (THPs, 1a, 1b), and to extend the concentration-dependent range, mono- and dinitrates of THPs were prepared. The pharmacological activities of the nitrates of THP were evaluated by use of isolated guinea-pig papillary muscle preparations and Na+,K(+)-adenosine triphosphatase preparations from dog kidney. Furthermore, the effect for smooth muscle was examined using the helical strips isolated from 13-week-old spontaneously hypertensive rat. The positive inotropic effects of mononitrates (11a, 11b, 2a, 2b, 8a, and 8b) were more potent than those of THPs. Nitration of the sugar moiety in THPs resulted in a vascular relaxing effect unobserved in the case of THPs.

Studies on Cardiac Ingredients of Plants. VII. Chemical Transformation of Proscillaridin by Means of the Diels-Alder Reaction and Biological Activities of Its Derivatives.

The Diels-Alder reactions of a cardiac glycoside, proscillaridin (1), with some dienophiles were investigated. The reaction of 1 with alkenes such as methyl vinyl ketone and methyl acrylate afforded 3-oxo-2-oxabicyclo[2.2.2]oct-7-enes (2-5) and para-substituted benzene derivatives (6 and 7), while 1 reacted with alkynes (3-butyn-2-one, methyl propiolate) to yield para- or meta-substituted benzene derivatives (6-9). The biological activities of the resulting derivatives were evaluated by the use of isolated guinea-pig papillary muscle preparations and Na+,K(+)-adenosine triphosphatase (ATPase) preparation from dog kidney. Among the proscillaridin derivatives, compounds 4 and 7 moderately inhibited Na+,K(+)-ATPase activity. Furthermore, the concentration range of 7 over which its positive inotropic effect on guinea-pig papillary muscle preparations, increased from 5% to 95% of maximum was broader than that of 1, i.e., concentration dependency was maintained over a greater range of concentration.

Studies on cardiac ingredients of plants. VIII. Preparation of nitrates of proscillaridin and their pharmacological activities

To reduce the vascular contracting effect of the cardiac glycoside, proscillaridin (1), all kinds of its nitrates were prepared by utilizing effectively an isopropylidene function as a protective group. The pharmacological activities of proscillaridin nitrates were evaluated by the use of isolated guinea-pig papillary muscle preparations and Na+,K(+)-adenosine triphosphatase preparations from the dog kidney. Furthermore, the effect for smooth muscle using the helical strips isolated from 13-week old spontaneously hypertensive rat was examined. The positive inotropic effects and Na+, K(+)-adenosine triphosphatase inhibition activities of mononitrates (6, 9, 15) and dinitrates (3, 4, 5) were a little less potent than 1, but those of trinitrate (2) were much reduced. Every nitrate did not exhibited a vascular contracting effect but a relaxing effect. Among them, the vascular relaxing effects of 2',3'-dinitrate (3) and 2',4'-dinitrate (4) were more potent than those of the other nitrates.

In-vitro and in-vivo electrophysiologic effects of encainide

The intracellular electrophysiologic effects of encainide (E) and its main metabolite, O-desmethyl-encainide (ODE), were studied in guinea-pig papillary muscle preparations and related to the in-vivo electrophysiologic effects observed after intravenous (IV) infusion of E in 11 patients undergoing electrophysiologic study (EPS). At equipotent concentrations of E and ODE, frequency-dependent reductions in Vmax studied at pacing rates of 30-180 beats/min ranged from -11.5% to -53%, with maximum reductions of -53% and -47%, respectively at the highest frequency. The kinetics of onset of use-dependent Vmax reduction were slower for ODE than for E at each studied pacing rate. The kinetics of total recovery from use-dependent block were still slower (120 seconds for E and 300 seconds for ODE at a 90 beats/min pacing rate). These in-vitro electrophysiologic data could explain the marked alterations in intraventricular and atrioventricular conduction observed in humans 60 minutes after IV administration of 1 mg/kg of E over a 15-minute period. The QRS, PA, AH, and HV intervals were significantly increased (p less than 0.01) and the Wenckebach cycle length was increased by 8% (p less than 0.05). Blood pressure, RR, QT, CSNRT, ESACT, ERP, and FRP did not vary significantly. The HV interval was already increased 2 minutes after drug administration, while AH was not increased until 15 minutes after drug administration. There was a positive correlation between the increase of the AH interval and the blood level of ODE.

Inotropic Activities of Imidazopyridines

A series of 2-substituted 1H-imidazo[4,5-b]pyridines and the isomeric 1H-imidazo[4,5-c]pyridine derivatives has been prepared and evaluated as inotropic agents. The 1H-imidazo-[4,5-b] derivatives were found to be consistently more potent than their isomers in the [4,5-c] series in isolated guinea pig papillary muscle preparations. Structure-activity relationships and the species-dependence of inotropic potencies are discussed.

The Positive Inotropic Effect of Pimobendan Involves Stereospecific Increases in the Calcium Sensitivity of Cardiac Myofilaments

We have studied the effect of pimobendan (UD-CG 1 15 BS) on the electrical, mechanical, and biochemical activity of intact and detergent-skinned preparations of cardiac muscle. Racemic pimobendan increased the contractile force of guinea pig papillary muscle preparations and this positive inotropic action was associated with potentiation of the Ca:2+-dependent slow action potentials (APs). However, in the presence of 25 mM [K]o and maximally activating concentrations of isoproterenol, isometric force was increased further by addition of 50 μM pimobendan with no effect on the slow action potential. Experiments with chemically skinned heart μscle fibers showed that pimobendan. in a dose-dependent manner, increased active tension developed at submaximally activating concentrations of Ca The tension-cost (unit increase in ATPase rate/unit increase in force) was unchanged in the presence of pimobendan. Force-pCa and ATPase-pCa relations of skinned fiber preparations contracting isometrically were shifted to the left by 0.15–0.20 pCa units in the presence of 50 μ.M pimobendan. The mechanism for this effect was shown to be an increase in the Ca affinity of the regulatory binding sites of troponin C (TNC). These effects are due mainly to the /optical isomer of pimobendan. Addition of either the d or / isomer of pimobendan to preparations. maximally stiμlated by 1 μM isoproterenol, did not affect the slow AP parameters, but did increase contractile force to 124; of control by the d isomer and to 184“; of control by the / isomer. The Ca:2+-sensitizing effect of/-pimobendan on skinned liber preparations was substantially greater than that of the d isomer. Our results indicate that the Ca2+-sensitizing action of pimobendan (a) is due mainly to the / isomer, (b) involves direct effects on the Ca:2+-binding to TNC. and (c) involves specific interactions with domains of the regulatory protein complex

Cardiotonic agents 4. Dimaprit analogues as potential cardiovascular selective H 2-agonists

Several derivatives of dimaprit including carbon chain substitution, nitrogen substitution and isothiourea replacements were prepared and tested for inotropic activity in guinea pig papillary muscle preparations. Two derivatives showed moderate positive inotropic effects which were mediated by the H 2-histaminergic pathway.

Studies on cardiac ingredients of plants. V. Chemical transformation of proscillaridin and biological activities of derivatives.

In order to investigate the relationship between the chemical structures and the biological activities of proscillaridin (1) and related compounds, transformations of the lactone (1) into the lactams (2a-i) with monoalkylamines, into 1, 4-cycloadducts (3, 5) with dimethyl acetylenedicarboxylate, and into epoxides (6-8) with tris(acetylacetonate)iron(III)-H2O2 have been under-taken. Further, alkoxyalkylation of the teriary C14β-hydroxy group was carried out with alkoxyalkyl halides. The biological activities of the resulting proscillaridin derivatives were studied by the use of isolated guinea-pig papillary muscle preparations and an Na+, K+-adenosine triphosphatase preparation from dog kidney.Although the activities of proscillaridin derivatives were less potent than that of 1, compounds 2a, 2g, 3 and 9 showed slightly expanded concentration ranges of positive inotropic effect development on guinea-pig papillary muscle preparations.A significant correlation was obtained between the van der Waals volumes (Vw) and pIC50 values of 1 and 2a-h (r=-0.09, p<0.01).

Electrophysiological effects of bunaftine, an antiarrhythmic drug, on action potential characteristics in ventricular muscle preparations

The electrophysiological effects of bunaftine were studied using the glass microelectrode technique. Bunaftine at 1, 5 and 10 mg/l produced a concentration-dependent depression of the maximum rate of rise of the action potential in guinea pig papillary muscle preparations without affecting the resting membrane potential and the action potential amplitude. The action potential duration (APD50, APD90) was significantly prolonged by the treatment with 1 and 5 mg/l bunaftine, while it was not changed by the treatment with 10 mg/l. The absolute refractory period (ARP) was also prolonged dose-dependently by the treatment of bunaftine; It was excessively prolonged (177% of control) at the concentration of 10 mg/l. Disopyramide produced similar electrophysiological changes to those of bunaftine except for the prolongation of ARP. ARP/APD90 ratio was significantly increased by bunaftine, but not by disopyramide. These electrophysiological effects of bunaftine and disopyramide observed in guinea pig ventricular preparations were similarly found in canine ventricular muscle preparations. These results indicate that the electrophysiological characteristics of bunaftine are similar to those of disopyramide in that the most prominent effect was the prolongation of ARP.

Evidence that oxmetidine inhibits transmembrane-calcium flux in cardiac and vascular tissue.

Oxmetidine, at concentrations in excess of 1 X 10(-6)M, caused concentration-dependent negative inotropic and chronotropic responses in guinea-pig isolated heart preparations. Oxmetidine, at concentrations in excess of 1 X 10(-5)M, caused negative inotropic responses in guinea-pig papillary muscle preparations. The negative inotropic responses to oxmetidine were associated with shortening of the plateau phase of the action potential. Verapamil and nifedipine caused similar shortening of the plateau phase of the action potential at equivalent negative inotropic concentrations indicating that oxmetidine may also act as a calcium antagonist. In preparations partially depolarized by raising extracellular K+ concentration, oxmetidine also exhibited negative inotropic activity and reduced the calcium-dependent action potential. However, unlike verapamil and nifedipine, oxmetidine did not show voltage-dependent activity. Oxmetidine, at concentrations in excess of 1 X 10(-5)M, inhibited Ca2+-dependent contractions of dog saphenous vein preparations and inhibited 45Ca2+-uptake into veins depolarized by high extracellular K+. In vivo, these calcium antagonist actions of oxmetidine were demonstrated by vasodilatation, reduction in blood pressure, bradycardia and reduced cardiac output in anaesthetized cats. Oxmetidine, at concentrations of 1 X 10(-5)M and above, shows properties consistent with inhibition of transmembrane Ca2+ flux. This action can be distinguished from other calcium antagonists as the effects of oxmetidine are not voltage-dependent.

Negative inotropic effects of aldosterone antagonists in isolated human and guinea-pig ventricular heart muscle

The effects of K+-canrenoate (Aldactone pro inj.) and its metabolite canrenone on isometric force of contraction were measured in isolated guinea-pig and human papillary muscle preparations driven electrically at a frequency of 1 Hz. In guinea-pig hearts both substances exerted a concentration-dependent negative inotropic effect; the IC50 of K+-canrenoate and canrenone were 129 +/- 22 mumol l-1 (n = 5) and 85 +/- 11 mumol l-1 (n = 12), respectively. At the maximally tested concentration canrenone (250 mumol l-1) and K+-canrenoate (1,000 mumol l-1) reduced force of contraction by 68 +/- 4% (n = 12) and 83 +/- 3% (n = 5), respectively. The negative inotropic effects of canrenone and K+-canrenoate were not affected by 10 mumol l-1 atropine. The negative inotropic effect of canrenone was also not affected by 14 mumol l-1 aldosterone, but canrenone (10 mumol l-1 diminished the maximal positive inotropic effect of dihydro-ouabain from 554 +/- 75% (n = 4) to 269 +/- 39% (n = 4) of the predrug value. In human heart muscles K+-canrenoate and canrenone also exerted a concentration-dependent negative inotropic effect. K+-canrenoate (1,000 mumol l-1) and canrenone (250 mumol l-1) reduced force of contraction by 57 +/- 7% (n = 8) and 67 +/- 2% (n = 6), respectively. A positive inotropic effect of both substances was never observed. It is concluded that the improvement of cardiac performance after application of aldosterone antagonists observed in patients cannot be explained by a direct effect on the heart. K+-canrenoate and canrenone are devoid of any direct cardiotonic action. Instead, K+-canrenoate and canrenone have direct negative inotropic effects at high concentrations.

Interval-dependent effects of small antiarrhythmic drugs on excitability of guinea-pig myocardium

Myocardial depressant actions of seven antiarrhythmic drugs have been studied in vitro in guinea-pig papillary muscle preparations, using upstroke velocity of action potentials as a measure of myocardial excitability. Drug-induced depression of upstroke velocity is shown to be greater after an action potential than before; upstroke velocity recovers from such use-induced depression with widely varying half-times ranging from a tenth of a second to several seconds, depending upon the drug. Lipophilic drug structures having small molecular weights (below 250) all appear to display the short half-times required to give these drugs unique capabilities to block selectively extrasystoles and other high frequency arrhythmias in myocardium. Larger drug structures, or those having very low lipid solubility such as procainamide, will block excitability in a much less frequency-dependent or interval-dependent manner.