Electrophysiological effects of homocysteine in isolated rat right ventricular papillary muscles and left atria

General Pharmacology 32 (1999) 439–443

Electrophysiological effects of homocysteine in isolated rat right ventricular papillary muscles and left atria Pal Pacher a, Zoltan Ungvari b, Valeria Kecskemeti a,* a

Department of Pharmacology, Semmelweis University of Medicine, Nagyvarad ter 4, P.O. Box 370, 1445 Budapest, Hungary b Department of Pathophysiology, Semmelweis University of Medicine, Budapest, Hungary Manuscript received April 7, 1998; accepted manuscript July 7, 1998

Abstract There is clinical and epidemiological evidence that elevated plasma homocysteine (Hcy) levels are associated with increased myocardial infarction mortality; however, very little is known about Hcy’s direct cardiac effects. Thus, we aimed to characterize the cellular elecrophysiologic effects of Hcy, a sulfur-containing amino acid in isolated rat hearts. A conventional microelectrode technique was used in left atria and right ventricular papillary muscles. At concentrations higher than 1026 M, Hcy significantly decreased the maximum rate of rise of the depolarization phase (Vmax) in both cardiac preparations in a dose-dependent manner. Hcy at 1024–5 3 1024 M concentrations increased the action potential duration (APD) at late stages of repolarization (at 75% and 90% of APD) both in atria and in ventricles. There was a slight decrease in action potential amplitude in ventricular papillary muscles and atria at concentrations higher that 1025 M. The resting membrane potential and the early repolarization phase (APD25 and APD50) remained unchanged in every preparation studied at all concentrations of Hcy administered. The present data suggest that homocysteine may decrease the Na1 channel activity in in vitro cardiac preparations.  1999 Elsevier Science Inc. All rights reserved. Keywords: Cardiac action potential; Electrophysiology; Homocysteine; Rat

In the past years, homocysteine (Hcy, a sulfur-containing amino acid, an intermediary product in methionine metabolism) has attracted much attention. Once synthesized, homocysteine may undergo remethylation to methionine in a reaction catalyzed by methylenetetrahydrofolate homocysteine methyltransferase (methionine synthetase), which uses methyltetrahydrofolate as a methyl donor and cobalamin as an essential cofactor (Finkelstein, 1990). Alternatively, homocysteine can enter the transsulfuration pathway when cysteine synthesis is required or in the presence of excess methionine. In this pathway, homocysteine first condenses with serine to form cystathione in a rate-limiting reaction catalyzed by cystathione-b-synthase and requiring pyridoxal 59-phosphate; cystathione-g-lyase then catalyzes the hydrolysis of cystathione to yield a-ketobuthyrate and cysteine in another reaction requiring pyridoxal 59-phosphate (Finkelstein, 1990; Loscalzo, 1996).

* Corresponding author. Tel./Fax: 36-1-2104400; E-mail: kecsval@ net.sote.hu.

The link between hyperhomocysteinemia and vascular disease was first described in the severe inherited form in the sixties (Gibson et al., 1964; McCully, 1969; Shimke et al., 1965). Persons homozygous for a deficiency of cystathione-b-synthase are rare (1:200,000 births) and develop severe, frequently fatal atherothrombotic vascular disease in childhood and adolescence (Malinow, 1990). Mild hyperhomocysteinemia corresponds to genetic (heterozygous deficit in cystathioneb-synthase or homozygous deficit in thermolabile methylenetetrahydrofolate reductase) or to environmental factors (dietary or iatrogenic decrease in folate, vitamin B6, or vitamin B12) (Freyburger et al., 1997; Robinson et al., 1995; Selhub et al., 1993). In the general population, hyperhomocysteinemia has proved to be much more common than originally believed, with an estimated prevalence of 1:70 (Boers et al., 1985). More recently, “mild” hyperhomocysteinemia was recognized as a risk factor for premature arterial disease, including peripheral arterial occlusion, thrombotic stroke, and myocardial infarction (Boers et al., 1985; Kang et al., 1992). Moreover, there is increasing evidence that homocysteine may affect the coagulation

0306-3623/99/$–see front matter  1999 Elsevier Science Inc. All rights reserved. PII: S0306-3623(98)00213-4

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system and the resistance of the endothelium to thrombosis (Malinow, 1994), and it may interfere with the vasodilator and antithrombotic functions of nitric oxide (Stamler and Slivka, 1996). Among patients with cerebrovascular, coronary, or peripheral atherosclerosis, hyperhomocysteinemia is found in as many as 40% of them (Clarke et al., 1991). Inspite of the abundant clinical and epidemiological studies showing that elevated plasma homocysteine levels are associated with increased myocardial infarction mortality (Arnesen et al., 1995; Malinow et al., 1996; Nygard et al., 1997; Schmitz et al., 1996; Stampfer et al., 1992; Verhoef et al., 1996), very little is known about direct cardiac effects of homocysteine. Thus, we aimed to evaluate the direct cardiac electrophysiological effects of 1026–5 3 1024 M Hcy on action potential (AP) characteristics in isolated left atria and right ventricular papillary muscles of rats.

The tip of the microelectrodes was positioned on a region of the preparation where movement was minimal during contraction. To avoid moment-to-moment variation in action potential shape, the microelectrode was kept in a single cell for a minimum of 25 min before measurements were made, although it often remained in a single cell for periods of 2 h or more. The following criteria were used to select the action potentials: (1) resting membrane potential more negative than 270 mV, (2) overshoot exceeding 115 mV, and (3) absence of mechanical artifacts. The technique was described previously in detail (Kecskemeti and Braquet, 1992). Homocysteine was obtained from Sigma Chemicals (USA). All the other substances used were of analytical grade (Reanal, Hungary; Sigma and RBI, USA). The data of the action potential parameters were expressed as the mean 6 SEM. Statistical significance was determined by using Student’s t-test for paired data. Values of p less than 0.05 were considered significant.

1. Materials and methods Principles of laboratory animal care met the standards set by the U.S. National Institutes of Health and Ethical Committee of Semmelweis University of Medicine. Male Wistar (n 5 12) rats weighting 180–220 g were used for all studies. Subsequent to cervical dislocation, the hearts were removed by a median sternotomy, and the left atria and right ventricular papillary muscles were rapidly excised and placed in a tissue chamber, pinned, and superfused (flow rate: 6–8 ml/min) with oxygenated standard (95% O2/5% CO2) modified Krebs solution (32 6 0.48C, pH 7.4) (in mmol/l: 137 NaCl, 4.0 KCl, 1.8 CaCl2, 1.05 MgCl2, 11.9 NaHCO3, 0.42 NaH2PO4, 5.5 glucose). The preparations were equilibrated for at least 2–3 h before action potentials were recorded. Preparations showing spontaneous activity were discarded. The organs were stimulated by rectangular pulses (1 Hz, 1-ms duration and intensity twice the stimulus threshold) through bipolar platinum electrodes. Stimuli were applied to the preparations through a stimulusisolation unit. Transmembrane potentials were recorded with conventional glass microelectrodes (resistance 7– 18 MOhm; Clark Ltd., England), filled with 3 M KCl, with the use of an assembly of preamplifier-amplifier coupled to the Intrasys computer-analyzing system (Experimetria Ltd., Budapest). Action potential curves were fed into an analog-digital converter card interface fitted to an IBM-computer system. The following parameters were calculated by Intrasys 2.1 computer program: resting membrane potential (RP: mV), action potential amplitude (APA: mV), maximum rate of rise of depolarization phase (Vmax: V/s), and the duration of action potential measured at 25%, 50%, 75%, and 90% of repolarization (APD25, APD50, APD75, APD90: ms). To minimize mechanical artifacts in the recorded action potentials, care was taken to correctly align preparations so as to reduce lateral and torsional movements.

2. Results Concentration-dependent effects of homocysteine on action potential parameters were examined in six right ventricular papillary muscle and six left atrial preparations paced at a constant cycle length of 1000 ms. After stabilization of action potential parameters, homocysteine was applied in a cumulative manner (for 25 min at each concentration). The results are shown in Tables 1 and 2 and Fig. 1. The parameters of control action potentials (Tables 1 and 2) were close to those found in earlier reports (Gristwood and Rothaul, 1988; Watanabe et al., 1983). In both right ventricular papillary muscles and left atria 1025–5 3 1024 M Hcy significantly decreased the maximum rate of rise of depolarization phase (Vmax) in a dose-dependent manner (Tables 1 and 2; Fig. 1). Hcy at concentrations of 1024–5 3 1024 M significantly increased the action potential duration (APD) at a late stage of repolarization (at 75% and 90% of APD) in both atria and ventricles. There was a slight but significant decrease in action potential amplitude (APA) in ventricular papillary muscles at 1025–5 3 1024 M concentrations. In left atria, it was noted only when 1024–5 3 1024 M Hcy was applied. The resting membrane potential and the early repolarization phase (APD25 and APD50) remained unchanged in every preparation studied at all concentrations of Hcy administered (Tables 1 and 2).

3. Discussion It is well known that the time course of repolarization depends on the function of several ion channels that have different properties, depending on species and situation in the heart (Reuter, 1984). The rat ven-

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Table 1 Effects of Hcy on action potential parameters in rat right ventricular papillary muscle paced at constant cycle length of 1000 ms n56

RP (mV)

Control 1026 M Hcy p, 1025 M Hcy p, 1024 M Hcy p, 5 3 1024 M Hcy p, Washout p,

282.1 281.1 N.S. 280.8 N.S. 281.3 N.S. 281.5 N.S. 281.0 N.S.

APA (mV) 6 0.4 6 0.5 6 0.4 6 0.3 6 0.4 6 0.6

109.3 108.5 N.S. 107.2 0.05 106.2 0.01 104.8 0.01 107.9 N.S.

6 0.7 6 0.5 6 0.5 6 0.6 6 0.7 6 0.6

APD25 (ms)

APD50 (ms)

7.2 6 7.0 6 N.S. 7.4 6 N.S. 6.9 6 N.S. 6.8 6 N.S. 7.0 6 N.S.

12.6 6 12.4 6 N.S. 13.0 6 N.S. 12.8 6 N.S. 13.5 6 N.S. 13.3 6 N.S.

0.4 0.3 0.3 0.4 0.5 0.4

APD75 (ms) 0.5 0.4 0.5 0.4 0.5 0.5

22.6 6 22.7 6 N.S. 22.6 6 N.S. 23.3 6 N.S. 24.2 6 0.05 23.4 6 N.S.

APD90 (ms) 0.4 0.3 0.4 0.5 0.4 0.5

42.4 6 42.1 6 N.S. 42.0 6 N.S. 46.4 6 0.05 46.9 6 0.01 44.2 6 N.S.

Vmax (V/s) 0.8 0.5 0.6 1.0 0.9 1.1

230.9 226.3 N.S. 205.7 0.001 190.0 0.001 181.0 0.001 220.0 N.S.

6 2.6 6 2.3 6 2.8 6 3.0 6 4.6 6 4.9

Abbreviations: RP, resting membrane potential; APA, action potential amplitude; Vmax, maximum velocity of action potential upstroke; APD25,50,75 and APD90, action potential duration measured at 25, 50, 75, and 90% of repolarization; N.S., not significant. Homocysteine (1, 10, 100, and 500 mmol/l) was superfused for 25 min each; washout with drug-free bathing solution lasted for 60 min. Mean 6 SEM values are given; Student’s t-test for paired data was applied to determine statistical differences from control values.

tricular AP has a unique repolarization phase that is referred to as the early phase (APD25,50) and late phase (APD75,90) (Clark et al., 1993; Nobe et al., 1990; Watanabe et al., 1983). The early phase is determined by a combination of the decaying slow inward current (Isi) or calcium current (ICa) and different potassium currents, especially the transient outward current (Ito) (Mitchell et al., 1984; Nobe et al., 1990) and the very slowly inactivating K1 current (Iss) (Apkon and Nerbonne, 1991). The late phase is primarily attributed to the inward currents arising from electrogenic Na1-Ca21 exchange (INa/Ca) (Mitchell et al., 1984; Nobe et al., 1990). In the rat, action potentials from atria and the underlying ionic currents are similar to those from ventricles; however, there are differences, especially in the outward K1 current(s) and its characteristics (Apkon and Nerbonne, 1991; Boyle and Nerbonne, 1991, 1992). Although the identity of the impaired mechanism(s)

cannot be determined from the present experiments, our result demonstrates that Hcy depresses Vmax (an indirect indicator of the fast Na1 channel activity) and, to some extent, the APA and prolongs APD at 75 and 90% of repolarization in both right ventricles and left atria in a concentration-dependent manner. Although this paper did not address the exact ionic mechanisms that determine the former effects of Hcy, the inhibition of Vmax, the decrease in APA, and the prolongation of late repolarization could be a result of the inhibition of the fast Na1 channel, on the one hand, and alteration of the inward currents arising from electrogenic Na1-Ca21 exchange (INa/Ca), on the other. To clarify the mechanisms and the nature of these electrophysiologic effects clearly, much further ionic current measurements are required. As plasma Hcy concentrations in hyperhomocysteinemic patients are close to those in our experiments, our results may have clinical relevance.

Table 2 Effects of Hcy on action potential parameters in rat left atrium paced at constant cycle length of 1000 ms n56

RP (mV)

Control 1026 M Hcy p, 1025 M Hcy p, 1024 M Hcy p, 5 3 1024 M Hcy p, Washout p,

278.3 278.7 N.S. 277.9 N.S. 278.2 N.S. 277.8 N.S. 277.9 N.S.

APA (mV) 6 0.5 6 0.3 6 0.4 6 0.3 6 0.3 6 0.5

107.2 107.0 N.S. 106.1 N.S. 105.0 0.05 104.2 0.01 105.8 N.S.

6 0.5 6 0.4 6 0.3 6 0.4 6 0.5 6 0.5

APD25 (ms)

APD50 (ms)

7.0 6 6.7 6 N.S. 7.2 6 N.S. 6.8 6 N.S. 7.4 6 N.S. 7.2 6 N.S.

15.2 6 14.8 6 N.S. 15.5 6 N.S. 15.0 6 N.S. 13.3 6 N.S. 15.4 6 N.S.

0.4 0.3 0.4 0.5 0.6 0.5

APD75 (ms) 0.5 0.4 0.5 0.4 0.5 0.6

36.2 6 36.3 6 N.S. 36.8 6 N.S. 39.0 6 0.05 40.4 6 0.05 37.5 6 N.S.

APD90 (ms) 0.6 0.4 0.6 0.8 0.9 0.9

66.2 6 67.2 6 N.S. 67.4 6 N.S. 71.6 6 0.01 73.7 6 0.01 68.5 6 N.S.

Vmax (V/s) 0.9 0.8 0.7 0.9 1.2 1.0

215.1 212.9 N.S. 206.6 0.05 197.8 0.001 181.6 0.001 207.3 N.S.

6 1.8 6 1.6 6 2.2 6 2.6 6 3.3 6 3.8

Abbreviations: RP, resting membrane potential; APA, action potential amplitude; Vmax, maximum velocity of action potential upstroke; APD25,50,75 and APD90, action potential duration measured at 25, 50, 75, and 90% of repolarization; N.S., not significant. Homocysteine (1, 10, 100, and 500 mmol/l) was superfused for 25 min each; washout with drug-free bathing solution lasted for 60 min. Mean 6 SEM values are given; Student’s t-test for paired data was applied to determine statistical differences from control values.

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Fig. 1. Effect of Hcy on action potential duration measured at 75 and 90% of repolazition (APD75 and APD90) and maximal rate of rise of depolarization phase (Vmax) in rat right ventricular papillary muscles (A) and left atria (B). WO, washout. The changes expressed as a percentage of baseline values 6 SEM. Baseline values are shown in Tables 1 and 2. Values of p less than * 0.05, ** 0.01, and $ 0.001 were considered significant.

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