J Mol Cell Cardiol 29, 1037–1043 (1997)

Effects of Calcium Channel Antagonistson Ca2+ Transients in Rat and CanineCardiomyocytesJames Hensley, George E. Billman, J. David Johnson, Charlene M. Hohl andRuth A. AltschuldDepartments of Medical Biochemistry and Physiology, The Ohio State University College of Medicine,Columbus, OH 43210–1218, USA

(Received 3 October 1996, accepted in revised form 27 November 1997)

J. H, G. E. B, J. D. J, C. M. H R. A. A. Effects of Calcium Channel Antagonistson Ca2+ Transients in Rat and Canine Cardiomyocytes. Journal of Molecular and Cellular Cardiology (1997) 29,1037–1043. First-generation Ca2+ channel antagonists depress myocardial contractility, but many of the newerCa2+ channel blockers have a high degree of “vascular selectivity”. This study compares the effects of the Ca2+

antagonists felodipine, amlodipine, mibefradil, verapamil and nifedipine, and the Ca2+ channel agonist, (S)(−)-Bay K-8644 on Ca2+ transient amplitudes in fura-2/AM-loaded rat and canine ventricular cardiomyocytes. At10−11 and 10−10 , felodipine increased [Ca2+]i transient amplitudes by 10–25% in field-stimulated fura-2-loadedcells from both species while at 10−6 it depressed [Ca2+]i transients by 80%. Mibefradil increased [Ca2+]i

transient amplitudes by 16% at 10−11 and 10−10 and decreased the transients by 25% at 10−6 . The calciumchannel agonist, (S)(−)-Bay K-8644 increased [Ca2+]i transient amplitudes at 10−10–10−6 (maximally 37% at10−7 ) but depressed [Ca2+]i transients by 10% at 10−5 M. Nifedipine was inhibitory at all concentrations tested(10−11–10−6 ) in canine myocytes, but in rat cells, 10−10 nifedipine increased [Ca2+]i transient amplitudes by37%. All concentrations of verapamil and amlodipine (10−11–10−6 ) depressed [Ca2+]i transients in both ratand canine myocytes. We conclude that: (1) felodipine and mibefradil may be positive rather than negativeinotropes at low concentrations, which are therapeutically relevant; and (2) low concentrations of nifedipinemay have a positive inotropic effect in the rat but not the dog heart. 1997 Academic Press Limited

K W: Canine myocytes; Rat myocytes; Free calcium transients; Mibefradil, Verapamil; Nifedipine;Felodipine, Bay K 8644, Calcium channel antagonists.

and the chemically novel Ca2+ antagonist, mi-Introductionbefradil, have improved vascular selectivity andcould afford salutary reductions in afterload withoutCa2+ channel antagonists are potent vasodilators

widely used for the treatment of hypertension and negative inotropy (Clozel et al., 1989; Osterriederand Holck, 1989; Perez-Vizcaino et al., 1993;angina pectoris (Opie, 1989). However, many Ca2+

blockers depress myocardial contractility and there- Packer et al., 1996).Establishing whether or not Ca2+ channel an-fore must be used with caution in patients with

poor cardiac function (Walsh, 1987; Multicenter tagonists directly affect myocardial contractility isnot entirely straightforward, however. Negative ino-diltiazem post-infarction trial research group, 1988;

Boden et al., 1991; Hansen, 1991; Furberg et al., tropic effects in vivo can be masked by reflex neuro-humoral responses. In addition, the binding of Ca2+1995). A new generation of dihydropyridine Ca2+

channel antagonists, including felodipine, am- antagonists to Ca2+ channels is often voltage de-pendent, being one or more orders of magnitudelodipine, isradipine, nicardipine and nisoldipine,

Please address all correspondence to: Ruth A. Altschuld, Department of Medical Biochemistry, 333 Hamilton Hall, 1645 Neil Avenue,Columbus, OH 43210–1218, USA.

0022–2828/97/031037+07 $25.00/0 mc960348 1997 Academic Press Limited

J. Hensley et al.1038

more effective when the sarcolemma is depolarized with 95% O2–5% CO2-saturated buffer containing(in m): 121 NaCl, 4.85 KCl, 1.2 MgSO4, 1.2(Sun and Triggle, 1995). Because cardiac myocytes

depolarize and repolarize with each heartbeat, one KH2PO4, 1 CaCl2, 25 NaHCO3, 5 sodium pyruvate.Cells were superfused with each drug concentrationcannot easily predict from ligand binding to mem-

brane fragments the concentration dependence for for 8 min and then field stimulated at 0.2 Hz (caninecells) or 0.5 Hz (rat cells). Experiments were carriedCa2+ antagonist effects on functioning cardiac Ca2+

channels. There are also species differences in myo- out in the dark and all solutions were wrapped inaluminum foil because some Ca2+ channel an-cyte electrophysiology that influence the effects of

individual Ca2+ channel antagonists (Godfraind et tagonists are light sensitive. Stock solutions of ver-apamil and Bay K 8644 were prepared in absoluteal., 1992).

In the present study, we have used electrical ethanol. Stock solutions of nifedipine, felodipine,and amlodipine were prepared in dimethyl sulf-field stimulation of isolated adult rat and canine

ventricular cardiomyocytes to compare the direct oxide. Stock solutions of mibefradil were preparedin distilled water.cardiac effects of the Ca2+ channel antagonists

felodipine, mibefradil (Ro 40-5967), verapamil, ni- Steady-state fura-2 fluorescence data were col-lected at 30 points per second with a PTI Filterscan.fedipine and amlodipine, and the Ca2+ channel

agonist, (S)(−)-Bay K 8644, on intracellular free Sixteen consecutive steady-state [Ca2+]i transientswere signal averaged for each experimental con-Ca2+ ([Ca2+]i) transients. Isolated myocytes elim-

inate concerns about complex in vivo neurohumoral dition. The [Ca2+]i-dependent fluorescence ratio sig-nals were not calibrated because 10–30% of theresponses. Canine cells were investigated because

they closely resemble those from human hearts, fura-2 free anion partitions into the mitochondria(Davis et al., 1987). This precludes accurate es-and in vivo canine experiments have been widely

used for mechanistic studies of the cardiac effects timation of cytosolic free Ca2+ ion concentrations.However, changes in the amplitudes of intracellularof Ca2+ channel antagonists. Rat myocytes were

investigated because studies of the chronic effects fura-2 ratio transients reflect accurately changes incytosolic Ca2+ transient amplitudes. When cytosolicof Ca2+ channel antagonists are often conducted

in spontaneously hypertensive rats (Brunner et al., [Ca2+]i indicators are selectively quenched withMn2+ in rat myocytes, the cells continue to twitch1991; Lonsberry et al., 1992; Lemmer et al., 1994;

Bohm et al., 1995) and in the closely related spon- in response to electrical stimulation, but the Ca2+-sensitive mitochondrial signal does not exhibit beattaneously hypertensive, heart failure prone SHHF/

Mccfacp rat (Radin et al., 1993; McCune et al., 1995). to beat oscillations, indicating little contribution towhole cell [Ca2+]i transients (Miyata et al., 1991).

Data were analyzed using single factor analysisof variance followed by the Scheffe test (Dawson-Materials and Methods Saunders and Trapp, 1990).

Isolated myocytes

ResultsVentricular myocytes were isolated from adult maleSprague–Dawley rats and adult mongrel dogs of Figure 1 shows typical signal-averaged [Ca2+]i tran-either sex using previously described collagenase sients for canine myocytes incubated with varying con-perfusion methods (Wimsatt et al., 1990; Hohl and centrations of felodopine and nifedipine. In Figure 2 weAltschuld, 1991). present dose–response curves for the effects of the Ca2+

channel antagonists and agonist on relative [Ca2+]i

transient amplitudes in rat and canine myocytes. Fel-odipine (a) significantly augmented [Ca2+]i transientFura-2 AM measurementsamplitudes at low concentrations (10−11–10−9 ) incanine cells but became inhibitory as the concentrationCells were loaded with 2 l fura-2/AM for 5 min

followed by a 45–60-min post-incubation at room was raised. Low concentrations of felodipine also in-creased average Ca2+ transient amplitudes in rat cells,temperature to ensure complete hydrolysis of the

acetoxymethyl ester groups and generate the Ca2+- but when the dose–response curves were subjected toanalysis of variance (ANOVA), the only statisticallysensitive fura-2 free anion. Cells were loaded into

a plexiglass superfusion chamber and allowed to significant effect was that observed at 10−6 . By con-trast, the new non-dihydropyridine Ca2+ channel an-attach to the bottom glass coverslip. After 10 min,

superfusion at 2 ml/min, 37°C, pH 7.4, was begun tagonist, mibefradil, elicited nearly identical responses

Ca2+ Antagonists and Cardiomyocytes 1039

mibefradil and felodipine, did not depress [Ca2+]i

transient amplitudes at therapeutically relevantconcentrations in isolated rat or canine ventricularcardiomyocytes. In fact, low concentrations of thesedrugs had slight stimulatory effects, which reachedstatistical significance for canine cells treated withfelodipine and for both rat and canine cells treatedwith mibefradil. Second, there are species differencesin the responses of field stimulated adult mam-malian ventricular cardiomyocytes to some Ca2+

channel antagonists, especially nifedipine.Our data with felodipine are in agreement with

other studies demonstrating a positive inotropiceffect of this Ca2+ channel blocker in dogs. In vivo15

5

Felodipine

Time (s)

Flu

ores

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ce r

atio

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0/38

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C

studies by Cheng et al. (1994) have shown thatfelodipine (but not nifedipine or amlodipine) exhibitsFigure 1 Typical signal-averaged [Ca2+]i transients froma significant positive inotropic effect in the presencea canine myocyte superfused with 0, 10−9 and 10−6

felodipine and a second canine myocyte superfused with of autonomic blockade in canine myocardium. Fur-0, 10−9 and 10−6 nifedipine. The transients have ther, Pettersson et al. (1987) have shown a positivebeen superimposed to illustrate drug effects on the [Ca2+]i inotropic effect with felodipine in the isovolumicallytransient amplitudes.

contracting canine heart. Our studies suggest thatthe positive inotropic effects reported by these in-vestigators are due to an elevation of peak systolicin rat and canine myocytes (b). Mibefradil had a small[Ca2+]i. Positive inotropic effects of mibefradil havebut significant stimulatory effect at low concentrationsnot previously been described, but most in vitro(10−11–10−10 ) and at 10−6 was the weakest neg-studies have investigated higher drug con-ative inotrope of any of the Ca2+ channel antagonistscentrations. In vivo, mibefradil is reported only toinvestigated.be devoid of negative inotropic effects (Clozel et al.,Verapamil depressed [Ca2+]i transient amplitudes1991; Portegies et al., 1991).over a wide concentration range (10−11–10−6 ) in

As expected, the first generation Ca2+ channelboth rat and canine myocytes (c) and the canineantagonist, verapamil, depressed [Ca2+]i transientcells were significantly more sensitive to the negativeamplitudes in both rat and canine myocytesinotropic effects of the drug. Like verapamil, am-throughout the concentration range investigated.lodipine depressed [Ca2+]i transient amplitudesThis phenylalkylamine has minimal vascular se-throughout the concentration range examined, butlectivity and is capable of precipitating congestivethere was a minimal species difference (d). The effectsheart failure when myocardial contractility is other-of nifedipine, on the other hand, were strongly spe-wise compromised (Walsh, 1987; Godfraind et al.,cies dependent (e). Nifedipine gave a biphasic dose–1992). The inhibitory effects of nifedipine on [Ca2+]iresponse curve for rat myocytes, significantlytransient amplitudes in canine myocytes were alsoaugmenting [Ca2+]i transient amplitudes atpredictable on the basis of its negative inotropic10−11–10−9 but inhibiting [Ca2+]i transients ateffects demonstrated in vivo in both awake andhigher concentrations. By contrast, in canine myo-anesthetized dogs (Cheng et al., 1994; Pagel et al.,cytes, nifedipine had only an inhibitory effect. Like1994).felodipine, mibefradil, and nifedipine (in the rat), the

The augmentation of rat myocyte [Ca2+]i tran-Ca2+ channel agonist, (S)(−)-Bay K 8644, also gavesients by 10−11–10−9 nifedipine was un-biphasic dose–response curves (f) with maximalanticipated, but it might explain the oppositestimulation at 10−7 and 20% inhibition at 10−5 .cardiac effects of nifedipine and verapamil observedUnlike felodipine and nifedipine, however, the effectsin the SHHF/Mcc-facp rat. Antihypertensive dosesof (S)(−)-Bay K 8644 on Ca2+ transient amplitudesof nifedipine cause a marked regression of cardiacin rat and canine myocytes were nearly identical.hypertrophy in these heart failure-prone, obese,hypertensive animals (Radin et al., 1993), butcomparable blood pressure reduction with ver-Discussionapamil exacerbates cardiac hypertrophy and pre-cipitates decompensation and premature deathThere are two novel and important results of this

study. First, the newer Ca2+ channel antagonists, from congestive heart failure (Park et al., 1996).

J. Hensley et al.1040

–6

150

0C

Nifedipine log[M]

50

125

100

75

25

–10 –8

(e)

*

*

*

**

*

*

*

*

*

–6

150

0C

(S)(–)-Bay K 8644 log[M]

50

125

100

75

25

–10 –8

(f)

**

*

*

*

*

–6

150

0C

Verapamil log[M]

Per

cen

tage

of

con

trol

am

plit

ude

50

125

100

75

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–10 –8

(c)

**

**

*

*

** *

*

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–6

150

0C

Amlodipine log[M]

50

125

100

75

25

–10 –8

(d)

**

**

*

*

**

**

* *

–6

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0C

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50

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75

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(a)*

*

*

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*

–6

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Mibefradil log[M]

50

125

100

75

25

–10 –8

(b)

* *

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* *

**

*

*

*

*

*

Ca2+ Antagonists and Cardiomyocytes 1041

The deleterious response to verapamil was as- et al., 1989). Inhibition of [Ca2+]i transients in bothspecies at high concentrations of (S)(−)-Bay Ksumed to be associated with its negative inotropic

effects, but this hypothesis had been difficult to 8644 is in agreement with previous reports (Kass,1987; Bechem and Hoffmann, 1993). Effects ofreconcile with the positive response to nifedipine

in the same animal model. The present de- mibefradil were also the same in rat and caninemyocytes. Nevertheless, the effects of mibefradil onmonstration of a positive rather than negative

inotropic effect of low concentrations of nifedipine inward Ca2+ currents in guinea-pig myocytes arestrongly influenced by the experimentally imposedin the rat could explain this apparent discrepancy.

This hypothesis is currently being tested using in holding potential (Liang-min and Osterrieder,1991). At a membrane holding potential ofvivo echocardiography of SHHF/Mcc-facp rats

treated with antihypertensive doses of nifedipine −80 mV, the IC50 is 12 l, a value comparable tothat shown in Figure 2 for field-stimulated rat andor verapamil.

The large species difference between the responses canine myocytes. However, the IC50 declines>50-fold when the holding potential is brought to aof rat and canine myocytes to nifedipine was espe-

cially striking, but significant species differences moderately depolarized value of −50 mV (Liang-min and Osterrieder, 1991). Thus, while mibefradilwere also observed with verapamil and felodipine.

Each of these compounds was more effective in may be insensitive to action potential duration, itis extraordinarily sensitive to resting membranereducing [Ca2+]i transients in canine than in rat

myocytes. It is not clear whether these differences potential. Bezprozvanny and Tsien (1995) con-cluded that the strong voltage dependency of mi-can be attributed to structural differences in the

-type Ca2+ channels. However, dihydropyridine befradil most likely results from a preferentialbinding to the open and inactivated state of the -binding to isolated cardiac sarcolemmal fragments

is very similar across species (Janis et al., 1987) and type Ca2+ channel.The fact that dihydropyridines can have opposingexhibits a complex voltage dependency (Sanguinetti

and Kass, 1984; Godfraind et al., 1992). Resting effects on myocardial contractility depending onconcentration was first reported by Thomas et al.membrane potentials in the rat and canine myo-

cytes prepared in our laboratory are both close to (1984). The stimulatory effect at low drug con-centrations is probably analogous to the increases−80 mV, but the rat cells have an abbreviated

action potential (APD50=20–30 ms, Li et al., 1989). in -type Ca2+ current seen after a brief exposureof isolated myocytes to D600 (McDonald et al.,Thus, even though the rat myocytes in the present

study were paced more rapidly than the canine 1989). In that study, increases in -type Ca2+ cur-rent were attributed to a stimulation of single Ca2+cells, to reflect the more rapid in vivo heart rate of

the rat, the brief action potential could have limited channel currents with longer openings and fewerblanks.the time available for depolarization-induced Ca2+

antagonist binding to the rat myocyte Ca2+ chan- In the present study, we used the pure stimulatoryS)(−) enantiomer of Bay K 8644 (Ferrante et al.,nels. By extension, the prolonged canine action

potential (APD50=300–400 ms; Freeman and Li, 1989) and mibefradil exists as a single enantiomer(Clozel et al., 1991). Thus, the dual effects on Ca2+1991) could have favored the antagonistic form of

binding of some of the voltage-sensitive Ca2+ chan- transient amplitudes cannot be attributed to the useof racemic compounds where different enantiomersnel modulators. Thus, action potential duration

may be an important factor in determining the have differing effects on Ca2+ channels. Based onour results and those of others (for a review, seeeffects of varied concentrations of nifedipine, fel-

odipine and verapamil on cardiac [Ca2+]i transients. McDonald et al., 1989) a dual effect of compoundsthat bind to cardiac Ca2+ channels appears to be aEffects of the Ca2+ channel activator, (S)(−)-Bay

K 8644, were nearly identical in rat and canine relatively common occurrence that can be over-looked when a narrow range of drug concentrationsmyocytes, consistent with the observation that

binding of this dihydropyridine is not influenced by is used (see Perez-Vizcaino et al., 1993, for example).Recent site-directed mutagenesis studies of -typemembrane potential in cardiac myocytes (Ferrante

Figure 2 Cumulative dose–response curves for: (a) felodipine; (b) mibefradil; (c) verapamil; (d) amlodipine; (e) nifedipine;and (f) (S)-Bay K-8644 in rat (Χ) and canine (Φ) myocytes. [Ca2+]i transient amplitudes were expressed as percentageof control for each myocyte prior to data analysis. All data are mean±... for 6–10 myocytes in each group. (Thestandard error bars for mibefradil are smaller than the symbols at some points.) ∗ Indicates that the [Ca2+]i transientamplitude was significantly different from its respective control. Statistically significant species differences were observedat 10−8 felodipine, 10−11–10−8 verapamil, 10−8 amlodipine and 10−11–10−7 nifedipine.

J. Hensley et al.1042

40–5967, a novel calcium antagonist, on myocardialCa channels have defined the IVS6 segment of thefunction during ischemia induced by lowering cor-a1 subunit as being critical for the agonist andonary perfusion pressure in dogs: comparison with

antagonist effects of dihydropyridines (Schuster et verapamil. J Cardiovasc Pharmacol 14: 713–721.al., 1996). Three amino acids Tyr1485, Met1486 C J-P, O W, K CH, W

HA, S B, T R, H F, S R, Eand Ile1493 are required for high affinity block byH, 1991. Ro 40–5967: a new nondihydropyridinedihydropyridines, whereas only the first two aminocalcium antagonist. Cardiovasc Drug Rev 9: 4–17.acids, Tyr1485 and Met1486 are required for stimu-

D MH, A RA, J DW, B GP, 1987.lation of the Ca channel by Bay K 8644. Because Estimation of intramitochondrial pCa and pH by fura-of the similarity of agonist and antagonist binding 2 and 2,7 biscarboxyethyl-5(6)-carboxy-fluorescein

(BCECF) fluorescence. Biochem Biophys Res Communsites, it is not surprising that one compound can149: 40–45.exhibit both agonist and antagonist effects de-

D-S B, T RG, 1990. Basic and Clinicalpending on concentration. Three amino acids ad-Biostatistics. Norwalk, CT Appleton and Lange.

jacent to that for the dihydropyridine block are F J, L E, R A, T DJ, 1989.required for high affinity block by the phenyl- Binding of a 1,4-dihydropyridine calcium channel ac-

tivator, (−)S-Bay K 8644, to cardiac preparations.alkylamines, but mutation of neither the di-Biochem Biophys Res Commun 158: 149–154.hydropyridine nor the phenylalkylamine sites

F LC, L Q, 1991. Effects of halothane on delayedaffects the affinity of the channel for mibefradil.afterdepolarization and calcium transients in dog vent-

Other pharmacologically relevant regulatory site(s) ricular myocytes exposed to isoproterenol. An-must also exist on -type Ca channels (Schuster et esthesiology 74: 146–154.

F CD, P BM, M JV, 1995. Nifedipine:al., 1996).dose-related increases in mortality in patients withcoronary heart disease. Circulation 92: 1326–1331.

G T, S S, D C, V B, D R,S JC, 1992. Selectivity of calcium ant-Acknowledgementagonists in the human cardiovascular system basedon in vitro studies. J Cardiovasc Pharmacol 20: S34–S41.These studies were supported in part by NIH grants

H JF, D S G V M-HL36240 and HL48835, and DK-33727, and by , 1991. Treatment with verapamilgifts from Merck and Hoffmann LaRoche. during and after an acute myocardial infarction: a

review based upon the Danish verapamil infarctiontrials I and II. J Cardiovasc Pharmacol 18 (Suppl. 6)S20–S25.

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