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Cardiovascular Research 1999 44(1):67-80; doi:10.1016/S0008-6363(99)00180-7
© 1999 by European Society of Cardiology
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Copyright © 1999, European Society of Cardiology

Effects of the calcium channel antagonist mibefradil on haemodynamic parameters and myocardial Ca2+-handling in infarct-induced heart failure in rats

Steffen Sandmanna, Jiang-Yong Minb, Achim Meissnerb and Thomas Ungera,*

aInstitute of Pharmacology, Christian-Albrechts-University of Kiel, Kiel, Germany
bDepartment of Cardiology, Christian-Albrechts-University of Kiel, Kiel, Germany

* Corresponding author. Tel.: +49-431-597-3501; fax: +49-431-597-3522 th.unger{at}pharmakologie.uni-kiel.de

Received 4 February 1999; accepted 11 May 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: Abnormal intracellular Ca2+-handling has been implicated in the pathogenesis of contractile dysfunction and arrhythmias in failing hearts. Calcium channel antagonists (CCA) have been proposed for the prevention of cardiac events after myocardial infarction (MI). Recent studies suggest that the blockade of T-type Ca2+-channels induced a heart rate reduction without negative inotropic effects. We investigated the effects of the preferentially T-channel blocking CCA, mibefradil, on haemodynamic parameters and intramyocardial Ca2+-handling and contractility in the early and late period after MI. Methods: MI was induced by permanent ligation of the left coronary artery in male normotensive Wistar rats. Animals were divided in sham-operated and placebo- or mibefradil-treated MI rats. Placebo or Mibefradil treatment (10 mg/kg/d via gastric gavage) was started 7 days prior to MI-induction. Haemodynamic and intramyocardial Ca2+ measurements were performed 1, 3, 7 and 42 days after surgery. At these time points, mean arterial blood pressure (MAP), heart rate (HR), left ventricular enddiastolic pressure (LVEDP) and cardiac contractility (dP/dtmax) were measured in conscious rats. After haemodynamic measurements, the left ventricular papillary muscle was separated to determine developed tension (DT), time to peak tension (TPT) and systolic and diastolic free intracellular Ca2+ concentrations ([Ca2+]i) using the Ca2+ indicator aequorin. Dose-response curves after extracellular isoproterenol- or Ca2+-stimulation were recorded. Results: In the early (1–3 days) period after MI, MAP and dP/dtmax were decreased and LVEDP and HR were increased in placebo-treated MI rats. Mibefradil treatment increased MAP and dP/dtmax and decreased LVEDP and HR in infarcted rats. In the papillary muscle of placebo-treated rats, MI induced a decrease in DT and an increase in TPT and in diastolic and systolic [Ca2+]i. DT of placebo-treated MI rats showed a reduced reactivity after isoproterenol- or Ca2+-stimulation. After mibefradil treatment DT was increased and TPT was reduced in the late period (7–42 days) after MI, and diastolic and systolic [Ca2+]i were decreased in the early period after MI (1–3 days). The inotropic response to β-adrenergic or extracellular Ca2+-stimulation was markedly improved by mibefradil 7 and 42 days after MI. Conclusion: We conclude, that mibefradil improves cardiac function, protects the myocardium against ischemia-induced Ca2+-overload and increases β-adrenergic responsiveness in chronically failing rat hearts.

KEYWORDS Intramyocardial Ca2+-handling; T-type Ca2+-channel blockade; Mibefradil; Myocardial infarction; Rat


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
The cellular mechanisms responsible for contractile dysfunction in congestive heart failure have not been clearly elucidated. Because of the importance of Ca2+ in modulating excitation-contracting coupling in the myocyte, there has been much interest in the factors that regulate intracellular handling of free ionized calcium ([Ca2+]i). An abnormal [Ca2+]i-handling has been described in different disease states of the heart associated with cardiac hypertrophy [1] and during ischemia [2,3]. An increase in myocardial [Ca2+]i-concentration has been proposed to be the major mediator of the structural deterioration of the myocardium leading to cardiac necrosis [4] and to be implicated in the pathogenesis of contractile dysfunction and arrhythmias in failing hearts [5]. Some experimental and clinical studies reported a Ca2+-overload in ventricular myocytes of hypertrophied and failing hearts which was related to an increased Ca2+-influx through the Ca2+-channels as well as a reduced Ca2+-reuptake by the sarcoplasmatic reticulum (SR) [6,7]. Less is known about alterations of myocardial [Ca2+]i in correlation to the systolic and diastolic contractile dysfunction of the myocardium in the acute and chronic state after permanent coronary occlusion.

Calcium channel antagonists (CCA) have been proposed for the prevention of cardiac events after myocardial infarction (MI) because of their inhibiting action on the slow Ca2+-inward current through L-type Ca2+-channels [8]. However, corresponding to the pharmacological profile of the three main classes of CCA, e.g. dihydropyridine, phenylalkylamine and benzothiazepine, some of these compounds have been shown to exert adverse side effects such as negative inotropic and dromotropic actions on the myocardium [9,10], neurohormonal activation [11] and arrhythmias [12] which often limit their use in the therapy of patients with congestive heart failure [13,14]. Recent studies suggest that T-type Ca2+-channels also play an important role in the structural and functional changes during hypertrophy and heart failure. Evidence has been presented that this channel type is developmentally regulated and that its expression varies with cardiac cell type [15,16]. Since T-type Ca2+-channels are predominantly located in pacemaker cells of the sino-atrial node and in Purkinje fibers [17,18] of adult hearts, the blockade of this channel induces a heart rate reduction [19] without negative inotropic effects [20]. Recently, T-type Ca2+-channels have been found reexpressed in post-MI remodeled myocardium [21], in genetic cardiomyopathy [22] and during hypertrophic processes [23].

Mibefradil is a tetrazolium CCA featuring an inhibition of both, the L- and T-type Ca2+-channels, with a higher selectivity for T-type Ca2+-channels with respect to L-type Ca2+-channels [24]. Mibefradil has preferential coronary vasodilative effects and, in animal experiments, this compound has been shown to increase coronary blood flow during ischemia induced by lowering coronary perfusion pressure in dogs [25]. Pharmacological and clinical evidence has been presented that mibefradil does not exert negative inotropic actions in isolated rat hearts [26] and in patients with stable angina pectoris [27]. These data indicate that the blockade of T-type Ca2+-channels might be potentially beneficial in the therapy of patients with heart failure especially when left ventricular dysfunction occurs.

The aim of the present study was to characterize the effects of mibefradil on cardiac function and intramyocardial Ca2+-handling in the acute (1 and 3d) and chronic (7 and 42d) state of cardiac failure after permanent coronary ligation in rats. Mibefradil treatment was started 7d prior to induction of MI. Haemodynamic measurements were performed in conscious animals, and functional properties of the left ventricular papillary muscle were determined by injecting the bioluminescent photoprotein aequorin that permits parallel recording of changes in the myocardial [Ca2+]i-concentration and the corresponding contractile response to extracellular Ca2+- and β-adrenergic stimulation on a beat-to-beat basis.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
Male normotensive Wistar rats (Charles River Viga GmbH, Sulzfeld, Germany) initially weighing 230–270 g were used in the study. All experiments were performed in accordance with the German law on animal protection, version 1993. The animals were housed individually at controlled temperature and humidity under a 12 h light/dark cycle, and had free access to a standard diet (Altromin®, Altromin International, Lage-Lippe, Germany) and to drinking water. Rats were divided randomly into nine groups: 1) sham-operated; 2–5) placebo-treated infarcted; and 6–9) mibefradil-treated infarcted animals. Measurements of haemodynamic parameters and functional properties of the papillary muscle were performed 1, 3, 7 and 42 days after surgery according to the study design as indicated in Fig. 1. After one week in single cages, rats in group 1 underwent a sham operation. In the remainder of rats (groups 2–9), myocardial infarction (MI) was induced by permanent ligation of the left coronary artery. At various times according to the design protocol, haemodynamic studies were performed in conscious rats 12 h after implantation of catheters in the femoral artery and in the left ventricle (LV). Following these measurements, the rats were sacrificed, and the hearts were excised to prepare the left ventricular posterior papillary muscle for functional studies of the noninfarcted myocardium. The number of animals per group was 6–8.


Figure 1
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  		Fig. 1 Time-table of experimental protocol (study design).

 
2.1 Treatment
Placebo (0.9% saline p.o.) and mibefradil (10 mg/kg/d p.o.) treatment was started one week prior (7d pre) to induction of MI. Treatment was given daily via gastric gavage adjusted to the individual body weight of each rat. Mibefradil was dissolved in water at a concentration of 10 mg/ml. The drug was kindly provided by Hoffmann-La Roche AG (Grenzach-Wyhlen, Germany).

In preliminary experiments, 7 day pretreatment with the dose of 10 mg/kg/d mibefradil via gastric gavage did not significantly influence mean arterial blood pressure (MAP) or heart rate (HR) in infarcted animals. In these experiments, MAP and HR were measured before the induction of MI and during the following 48 h via chronically implanted femoral artery catheters [28].

2.2 Induction of myocardial infarction (MI)
Rats were anaesthetized with ether to cannulate the tail vein for intravenous injections of methohexital-sodium (10 mg/kg) (Brevimytal-Na®, German Lilly GmbH, Gießen, Germany). After shaving and disinfection of the chest, the animals were intubated and artificially ventilated with room air (50 hubs/min, 200 mm H2O, 1.5 ml/hub). The ECG was monitored continuously during surgery. A left thoracotomy was started by incising the skin 2 cm parallel to the third and fourth rib. The pectoral muscles were dislocated to expose the ribs and an incision was made at the fourth intercostal space to insert a rib-spreading chest retractor. After anterior pericardectomy, the heart was exposed. The left coronary artery was then permanently ligated intrathoracally using sterile 6–0 suture material (Ethibond, Ethicon, Norderstedt, Germany) under a stereo-microscope. Successful ligation of the coronary artery was verified by the occurrence of arrhythmias in the ECG and, visually, by observing the color changes of the ischemic area. In rats that underwent sham surgery, ligation was placed beside the coronary artery. The thoracic cavity was closed during respiration hold. At the end of the operation procedure, analgesia was induced by a subcutaneous injection of buprenorphine-HCl (0.2 mg/kg) (Sigma-Aldrich Chemie, Steinheim, Germany).

2.3 Implantation of femoral arterial and left ventricular catheters
At the different time points according to the study design (1d, 3d, 7d and 42d after MI), rats were anaesthetized with ether for chronic implantation of arterial and left ventricular catheters. In these experiments, the right femoral artery was cannulated by inserting a polypropylene tube (PP50, Portex, London, UK) which was then tunneled under the skin and anchored at the posterior neck region of the rat. Then, the LV was cannulated using a specially constructed pig-tail catheter consisting of a PP10 tube 60 mm in length welded to a 350 mm length of PP50 tube [29]. The pig-tail at the end of the PP10 portion was inserted into the right carotid artery and advanced into the LV via the ascending aorta. During cannulation, the catheter was connected to a transducer and blood pressure monitor to verify the position of the tip of the catheter. The LV was considered to be reached when the pulse pressure had a typical LV-configuration. The PP50 portion was then exteriorized at the nape beside the arterial catheter and both catheters were fixed in the neck.

2.4 Haemodynamic measurements
In preliminary experiments, the femoral artery was cannulated to measure MAP and HR during surgery and during the 48 h after induction of MI or sham operation to determine the effect of different mibefradil doses on peripheral haemodynamic parameters.

One day, 3d, 7d and 42d after induction of MI, the haemodynamic measurements in the experimental groups were performed 12 h after implantation of the catheters in conscious rats as described previously [29]. The femoral artery and LV-catheter were connected to the pressure transducers (DTX/Plus, Spectramed Inc., Oxnard, CA, USA). MAP, HR and left ventricular pressure (LVP) were obtained using two pressure processors (Gould Inc., Valley View, OH, USA). The output signals were recorded on a pen recorder (Gould Series 2000, Gould Inc., OH, USA) and analyzed by a computer-based system MEGA [30] at a rate of 800 Hz. The computer program calculated the left ventricular enddiastolic pressure (LVEDP) and the maximum positive change in the left ventricular pressure signal (dP/dtmax) (1000 mmHg/s). The last parameter was considered as a marker of the myocardial contractility. Since ECG was not recorded in this part of study, LVEDP was measured at the point where the slope of the ventricular pressure signal changed from the slowly to the rapidly increasing portion [31]. This point has been shown to be closely linked to the R wave of the ECG and to represent LVEDP in rats [29]. Rats were given time to adjust to the recording procedures for 30 min before MAP, HR and LVP were recorded. LVEDP and dP/dtmax were calculated off-line from the LVP signal using the MEGA program. MAP, HR, LVEDP and dP/dtmax were averaged over 5 min periods to be used in the statistical analysis.

2.5 Preparation of left ventricular papillary muscle
After haemodynamic measurements, rats were sacrificed during chloral hydrate anaesthesia (40 mg/kg i.p.). The chest was opened at the sternum and the hearts were rapidly excised and placed in a dissecting chamber containing a modified Krebs-Henseleit solution (NaCl 120 mM, KCl 5.9 mM, dextrose 5.5 mM, NaHCO3 2.5 mM, NaH2PO4 12 mM, MgCl2 1.2 mM, CaCl2 1.0 mM, pH 7.4) bubbled with carbogen (95% O2/5% CO2). The right ventricle (RV) was dissected at room temperature and an incision into the LV at the septum was done to carefully separate the left ventricular posterior papillary muscle. As previously demonstrated, this muscle is not infarcted after ligation of the left coronary artery and can be used for functional studies of the noninfarcted myocardium of rats with MI [32]. RV and LV were weighed, normalized to the body weight (BW) and used as index of hypertrophy. The muscular end of the papillary muscle was fixed to a muscle holder with a spring clip, and the tendinous end was tied to a 6–0 silk suture (Ethibond, Ethicon, Norderstedt, Germany) and connected to a strain-gauge tension transducer (Type 372, Hugo Sachs Elektronics, Freiburg, Germany). The muscle was then mounted in a 50 ml tissue bath containing modified Krebs-Henseleit solution. The solution was continuously bubbled with carbogen at 30°C. Stimulation of the muscle was elicited by 5 ms square wave pulses at 0.33 Hz and 7–10 mV via a punctate platinum electrode at the base of the organ. The voltage was set to 10% above the threshold. After a 30 min equilibration period, the muscle contracted isometrically and was carefully stretched to the length at which maximal tension occurred. At this length, the following isometric contraction parameters of the left ventricular papillary muscle were measured and documented on a pen recorder (Gould Instruments, Cleveland, OH, USA): stimulation-induced developed tension (DT), time to peak tension (TPT, time from the beginning of the contraction to peak tension), and time to 50% relaxation (RT50, time from peak tension to 50% of relaxation). At the end of the experiments, the muscle was blotted and weighed. The cross-sectional area (CSA) was determined from muscle length (ML) and weight (W) by assuming a uniform cross section and a specific gravity of 1.05 according the formula CSA=ML/Wx1.05.

2.6 Measurement of Aequorin Light Signals
Aequorin (Friday Harbor Laboratories, WA, USA) was loaded into the muscle preparation by macroinjection technique described by Kihara and Morgan [33] to measure intracellular diastolic and systolic Ca2+-concentration. Briefly, the stimulation of the muscle was stopped and the preparation was raised from the tissue bath to inject 1–2 µl of the aequorin solution (1 mg/ml) at the base of the papillary muscle under the epimysium with a short-glass micropipette. The muscle was then returned into the bath and after an equilibration period of 60 min when a steady state was reached, stimulation was restarted (5 ms, 33 Hz, 7–10 mV). At this time point, aequorin has been demonstrated to be localized in the myocytes [34]. Aequorin light signals were detected with a light-collecting apparatus (Photomultiplier PM28B, Thorn EMI Electronic Tubes, Rockaway, NJ, USA) and converted to a voltage signal [35]. Analog signals from the isometric force transducer and photometer were recorded with a chart-strip recorder (Model 56-1 x 40-006158, Gould Instrument Systems Inc., Cleveland, OH, USA) and also stored on videotape (Model HR-J400U, JVC Company of America, NJ, USA). To improve the signal-to-noise ratio, 8–64 steady-state light signals and isometric twitches were averaged with a digital oscilloscope (Model 5460113, 100 MHz, Hewlett-Packard, Böblingen, Germany) for quantitative measurements [36]. Physiological parameters of the papillary muscle derived from the aequorin light signals were time to peak light (TPL) and time from peak light to 50% fall in the light signal (RL50).

2.7 Quantitation of the intracellular Ca2+-signal
The free intracellular concentration of calcium ([Ca2+]i) was estimated by normalizing the recorded light signal during isometric twitches (L) by the maximal amount of light emitted after the lysis of the muscle membranes (Lmax) at the end of the experiments with a 5% solution of the detergent Triton X-100 in phosphate-free physiological salt solution containing 50 mM Ca2+. The normalized light signal was then converted to [Ca2+]i using an in-vitro calibration curve of the following formula [33]:

Formula
KR=4.5x106/M and KTR=130.0 are model constants obtained from a nonlinear regression analysis with iteration.

2.8 Dose-response-curve to extracellular Ca2+- and isoproterenol-stimulation
The concentration-effect-curves for extracellular Ca2+ and isoproterenol were determined by the method of cumulative addition. For these experiments, the tissue bath was changed to remove phosphate from the bath, to avoid a possible precipitation at higher concentration of extracellular Ca2+ ([Ca2+]e). Steady-state conditions were observed for at least 30 min after the intracellular aequorin light signal had stabilized. After measurement of baseline parameters, the steady-state response to the step-wise increase of [Ca2+]e-concentration (0.5, 1.0, 2.0, 3.0, 4.0 mM) in the tissue bath was recorded at the plateau of inotropic response which was reached after 10 min. The bath solution was then replaced with modified Krebs-Henseleit solution containing 1.0 mM [Ca2+]e. Subsequently, isoproterenol was added step-wise (10–7, 10–6, 10–5, 10–4 M) to determine inotropic responses to β-adrenergic stimulation. Light signals and isometric contractions were measured 10 min after each dose of isoproterenol.

2.9 Statistical analysis
Statistical evaluation of obtained haemodynamic and morphometric data was performed using one-way analysis of variance with repeated measures (ANOVA). Means shown to be different between individual groups were compared using the post-hoc Student’s t-test and were considered significant at p<0.05. Data were expressed as mean±standard error of the mean (SEM).


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Haemodynamics
Mean arterial blood pressure (MAP) was reduced in all infarcted groups (placebo- and mibefradil-treated MI rats) in the early (1–3d) and late (7–42d) period after induction of MI compared to the sham-operated group. In contrast to placebo-treated MI rats, mibefradil-pretreated MI rats showed a time-dependent increase in MAP from the early to the late period after MI. Forty two days after induction of MI, MAP in this group was significantly higher than in placebo-treated MI animals (Fig. 2, panel A). In the early period (1–3d) after MI-induction, heart rate (HR) of placebo-treated and 7d pre MI mibefradil-treated MI groups was significantly increased compared to the sham-operated group. However, animals of the mibefradil-pretreated MI group showed a significantly lower HR compared to the placebo-treated MI group at day 1 after induction of MI (Fig. 2, panel B).


Figure 2
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  		Fig. 2 Mean arterial blood pressure (MAP) (panel A) and heart rate (HR) (panel B) measured 1d, 3d, 7d and 42d after MI. Animals are subjected to sham surgery (Sham, white columns), placebo-treated infarcted animals (Placebo-MI, black columns) and mibefradil-treated infarcted animals (Mibefradil 7d pre, striped columns). Data represent mean±SEM, n=6–8. #significant versus sham (p<0.05), *significant versus placebo-MI (p<0.05).

 
Time-dependent changes in left ventricular enddiastolic pressure (LVEDP) and in myocardial contractility (dP/dtmax) are displayed in Fig. 3. LVEDP of placebo-treated, infarcted animals was increased at all measured time points (1d, 3d, 7d and 42d post MI) when compared to sham-operated, noninfarcted animals. Mibefradil-pretreated MI animals had significantly reduced LVEDP values at day 42 post MI compared to placebo-treated MI animals. At this time point, LVEDP of 7d pre MI mibefradil-treated MI animals was not different from LVEDP of sham-operated animals (Fig. 3, panel A). Cardiac contractility (dP/dtmax) was reduced in all four placebo-treated MI groups (1d, 3d, 7d and 42d post MI) compared to sham-operated animals. In contrast, dP/dtmax measured 42 days after induction of MI was significantly higher in mibefradil-pretreated MI groups than in those of the placebo-treated MI group and did not differ from dP/dtmax of the sham-operated group (Fig. 3, panel B).


Figure 3
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  		Fig. 3 Left ventricular enddiastolic pressure (LVEDP) (panel A) and cardiac contractility (dP/dtmax) (panel B) measured 1d, 3d, 7d and 42d after MI. Animals are subjected to sham surgery (Sham, white columns), placebo-treated infarcted animals (Placebo-MI, black columns) and mibefradil-treated infarcted animals (Mibefradil 7d pre, striped columns). Data represent mean±SEM, n=6–8. #significant versus sham (p<0.05), *significant versus placebo-MI (p<0.05).

 
3.2 Contractility of the left ventricular papillary muscle
The developed tension (DT) of papillary muscles of all infarcted animals (placebo- and mibefradil-treated) was significantly reduced at all measured time points (1d, 3d, 7d and 42d post MI) after extracellular Ca2+- and isoproterenol-stimulation, compared to animals that underwent sham surgery (Fig. 4). Compared to baseline values, graded increases in extracellular Ca2+ or isoproterenol concentrations resulted in a significant increase in DT of papillary muscles from sham-operated animals. In contrast, an increase in papillary muscle DT after extracellular stimulation was only observed 42d post MI in mibefradil-pretreated MI animals at Ca2+ concentrations up to 1.0 mM and isoproterenol concentration up to 10–5 M.


Figure 4
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  		Fig. 4 Effects of increasing extracellular Ca2+-concentrations (panel A) and of increasing extracellular isoproterenol-concentrations (panel B) on developed tension (DT) measured 1d, 3d, 7d and 42d after MI. Animals are subjected to sham surgery (Sham, cross marker), placebo-treated infarcted animals (Placebo-MI, closed marker) and mibefradil-treated infarcted animals (7d pre MI Mib, opened marker). Data represent mean±SEM, n=6–8. #significant versus sham (p<0.05), *significant versus placebo-MI (p<0.05).

 
The inotropic response to extracellular Ca2+-stimulation showed that DT of placebo-treated MI animals was highest at 1d post MI compared to 3d, 7d and 42d post MI. In contrast, isometric contractions were improved in all mibefradil-pretreated MI animals. Developed papillary muscle tension of these animals was higher than DT of placebo-treated MI animals at all time points measured after Ca2+-stimulation. In contrast to placebo-treated MI animals, highest DT values after inotropic stimulation were measured 42d post MI in the papillary muscles from mibefradil-pretreated MI animals (Fig. 4, panel A). After β-adrenergic stimulation induced by isoproterenol, the contraction force of placebo-treated MI animals reduced from the early (1–3d) to the late (7–42d) period after MI so that values of DT 7d and 42d post MI were lower than under basal conditions (not statistically significant). In contrast, isoproterenol-stimulation induced an increase in DT of mibefradil-pretreated MI groups which was significantly elevated 42d after induction of MI at concentrations of 10–5 and 10–4 M compared to basal conditions (Fig. 4, panel B). During recording of dose-response curves for extracellular Ca2+- and isoproterenol-stimulation, aftercontractions and corresponding afterglimmers of the aequorin light signal were observed in all placebo-treated but not in mibefradil-pretreated MI animals (data not shown).

The time to peak tension (TPT) in papillary muscles from placebo-treated MI animals was significantly higher in the early period (1–3d) after MI than in those from sham-operated animals and was further increased in the late period (7–42d) after MI under basal conditions. In contrast, TPT of mibefradil-pretreated MI animals decreased from the early to the late period after MI so that TPT of these animals was significantly lower 7d and 42d post MI compared to the contraction time of placebo-treated MI animals. Forty two days post MI, TPT of mibefradil-pretreated MI animals was not significantly different any more from that of sham-operated animals (Table 1). After extracellular Ca2+-stimulation, TPT of all animals (sham-operated and infarcted) was not significantly different at any time from TPT measured under basal conditions. After extracellular isoproterenol-stimulation, TPT was reduced in all animals (sham-operated and infarcted) at all time points measured compared to basal conditions. Mibefradil-pretreated MI animals showed a significantly reduced TPT after extracellular Ca2+- and isoproterenol-stimulation in the late period (7–42d) after MI compared to placebo-treated MI animals (Table 1).


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  		Table 1 Functional parameters of the left ventricular papillary muscle 1d, 3d, 7d and 42d after myocardial infarction or sham surgerya

 
Time from peak tension to 50% of relaxation (RT50) of all infarcted animals (placebo- and mibefradil-treated) was not significantly different from of sham-operated animals under basal conditions (Table 1). Whereas extracellular Ca2+-stimulation did not influence RT50 in all animals at any time point compared to basal conditions, β-adrenergic stimulation induced a shortening in RT50 compared to basal conditions in sham-operated and infarcted animals at day 1, 3, 7 and 42 after induction of MI. At day 42 post MI placebo- and mibefradil-treated animals had significantly higher values of RT50 after isoproterenol-stimulation compared to sham-operated animals (Table 1).

3.3 Intracellular Ca2+-concentration of the left ventricular papillary muscle
The systolic intracellular calcium concentration ([Ca2+]i) of papillary muscles of placebo-treated MI animals was increased at all measured time points (1d, 3d, 7d and 42d post MI) compared to sham-operated animals. Highest values of systolic [Ca2+]i were observed in the early period (1–3d) after MI. In papillary muscles from mibefradil-pretreated MI animals, the increase of systolic [Ca2+]i in the early period after MI was diminished so that systolic [Ca2+]i of these animals was significantly lowered 1d and 3d post MI compared to placebo-treated MI animals. After extracellular Ca2+-stimulation, systolic [Ca2+]i of sham-operated animals were increased at all measured time points compared to basal conditions, whereas systolic [Ca2+]i of all infarcted animals (placebo-treated and mibefradil-pretreated) were unchanged. Extracellular isoproterenol-stimulation induced an increase in systolic [Ca2+]i of sham-operated and mibefradil-pretreated MI animals at all measured time points compared to basal conditions, but not in placebo-treated MI animals (Fig. 5, panels A–D).


Figure 5
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  		Fig. 5 Intracellular systolic calcium concentration ([Ca2+]i) at basal conditions (white columns) and after extracellular Ca2+-stimulation (black columns) or isoproterenol-stimulation (striped columns) measured 1d (panel A), 3d (panel B), 7d (panel C) and 42d (panel D) after MI. Animals are subjected to sham surgery (Sham), placebo-treated infarcted animals (Placebo-MI) and mibefradil-treated infarcted animals (Mibefradil-MI). Data represent mean±SEM, n=6–8. §significant versus basal (p<0.05), #significant versus sham (p<0.05), *significant versus placebo-MI (p<0.05).

 
The diastolic intracellular calcium concentration ([Ca2+]i) of placebo-treated MI animals was increased in the early period (1–3d) after MI under basal conditions as well as after extracellular Ca2+- and isoproterenol-stimulation when compared to those of sham-operated animals. Diastolic [Ca2+]i of mibefradil-pretreated MI animals was not increased at days 1, 3, 7 and 42 post MI compared to sham-operated animals and was reduced 1d and 3d post MI under basal conditions and after extracellular Ca2+- and isoproterenol-stimulation compared to placebo-treated MI animals. After extracellular Ca2+-stimulation, diastolic [Ca2+]i of placebo-treated MI animals but not of mibefradil-pretreated MI animals, was increased in the early period after MI compared to basal conditions. In all groups (sham-operated, placebo-treated and mibefradil-pretreated) diastolic [Ca2+]i was increased in the early as well as in the late period after MI following isoproterenol-stimulation (Fig. 6, panels A–D).


Figure 6
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  		Fig. 6 Intracellular diastolic calcium concentration ([Ca2+]i) at basal conditions (white columns) and after extracellular Ca2+-stimulation (black columns) or isoproterenol-stimulation (striped columns) measured 1d (panel A), 3d (panel B), 7d (panel C) and 42d (panel D) after MI. Animals are subjected to sham surgery (Sham), placebo-treated infarcted animals (Placebo-MI) and mibefradil-treated infarcted animals (Mibefradil-MI). Data represent mean±SEM, n=6–8. §significant versus basal (p<0.05), #significant versus sham (p<0.05), *significant versus placebo-MI (p<0.05).

 
No significant differences in time to peak aequorin light signal (TPL) and time from peak light signal to 50% fall in the light signal (RL50) were recorded at any time between groups.

3.4 Morphological parameters of the heart
Body weight (BW) of placebo-treated and mibefradil-pretreated MI animals was increased 42d after induction of MI compared to BW of sham-operated animals. Additionally, right ventricular weight (RVW) and left ventricular weight (LVW) as well as RVW and LVW normalized to body weight (RVW/BW, LVW/BW) were increased in placebo-treated and mibefradil-pretreated MI animals 42d post MI. At day 42 post MI, LVW and LVW/BW of mibefradil-pretreated infarcted animals were significantly reduced when compared to placebo-treated MI animals (Table 2)


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  		Table 2 Morphological parameters of the heart 1d, 3d, 7d and 42d after myocardial infarction or sham surgerya

 
Placebo-treated and mibefradil-pretreated MI animals showed an increase in cross-sectional area (CSA) of the papillary muscle 42 days post MI compared to sham-operated animals. At day 42, papillary muscle hypertrophy of infarcted animals was reduced by 20% after chronic mibefradil treatment compared to placebo treatment (Table 2).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
The purpose of this investigation was to examine the effects of pretreatment with the calcium channel antagonist (CCA), mibefradil, on cardiac function and intramyocardial Ca2+-handling in the acute (1 and 3d) and chronic (7 and 42d) state of cardiac failure after myocardial infarction (MI).

The results of the haemodynamic measurements show an increased enddiastolic pressure (LVEDP) and a reduced myocardial contractility (dP/dtmax) especially on day 1 and day 3 after MI in animals of the placebo-treated MI groups. At these time points, the changes of LVEDP and dP/dtmax were accompanied by an increase in heart rate (HR) and a reduction of mean arterial blood pressure (MAP). A greater deterioration of cardiac and peripheral haemodynamic parameters in the acute than in the chronic phase after MI have previously been demonstrated in MI-induced cardiac failure in rats [37]. In contrast, LVEDP was lowered and dP/dtmax and MAP were raised in the late phase (42d) after induction of MI in mibefradil-pretreated MI rats compared to placebo-treated MI animals. Indeed, LVEDP and dP/dtmax of mibefradil-pretreated MI rats were not significantly different from sham-operated animals. The observed effect of mibefradil on cardiac function of infarcted animals can be partly attributed to the afterload as well as to the preload reduction [38]. The infarct size-limiting property of mibefradil in the rat model of MI-induced heart failure might also contribute to the improved cardiac function [28]. The lack of a negative inotropic effect of mibefradil in our experiments is in agreement with results of other studies showing that mibefradil had no adverse effect on contractile function in several rat models of heart failure, whereas verapamil [19,20], diltiazem [39], and nifedipine [9,40] depressed cardiac function in these models.

Some of the dihydropyridine CCA stimulate the sympathetic nervous system which is reflected by an elevation of HR as well as of circulating adrenaline and noradrenaline levels [41,42]. In the present study, mibefradil pretreatment abolished the MI-induced stimulation of sympathetic nervous system in the early period (1d) post MI as evidenced by a significant reduction of HR in these animals compared to placebo-treated MI rats. A HR-reducing action and the prevention of ischemia-induced sympathetic activation and reflex tachycardia by mibefradil have been demonstrated in several experimental studies in rats and dogs [19,25,43]. These findings could be partly explained by the fact that cardiac T-type Ca2+-channels, preferentially blocked by mibefradil [24], are located primarily in the sinus node [18]. It is possible that the prevention of sympathetic activation in the early phase after MI lowered the oxygen demand of the contracting heart and improved cardiac oxygen supply due to prolonged diastolic relaxation. Additionally, mibefradil pretreatment might protect the contractile force of the myocardium in the early period after MI by increasing oxygen supply due to an improved myocardial perfusion via the marked coronary vasodilatory action of the drug [20,44].

4.1 Functional properties of left ventricular papillary muscle after myocardial infarction
The results of measurements of the myocardial functional parameters show that in left ventricular papillary muscles from placebo-treated MI rats the developed tension (DT) was reduced and the time to peak tension (TPT) was prolonged in the early and late period after MI compared to sham-operated rats. Intracellular diastolic and systolic Ca2+-concentration ([Ca2+]i) were elevated 1d and 3d post MI in these animals. The decreased DT and the prolonged TPT of papillary muscles from infarcted rat hearts in spite of an increase in systolic [Ca2+]i, points to abnormalities in the structure and function of the intramyocardial contracting filaments. Several experiments on rats with cardiac failure demonstrated that the contractile dysfunction of the failing myocardium was associated with a reduced responsiveness of the myofilaments to an increase in [Ca2+]i [45], an increase in the "slow activity" V3 myosin isoform [46,47], a reduction in the actomyosin-ATPase-activity [48] and an increase in troponin I activity [49,50]. An accumulation of inorganic phosphates (Pinorg) and of H+ in the noninfarcted myocardium has also been discussed to be responsible for the lowered myofilament contractility [51,52]. An intramyocardial increase in Pinorg induced a decrease in the number of cross linkages between actin and myosin filaments during the contracting phase [53], whereas intracellular acidosis inhibited the binding of Ca2+ to troponin C [54].

The early elevation in diastolic and systolic [Ca2+]i indicates abnormalities in the release and/or uptake of Ca2+ by the sarcoplasmatic reticulum (SR) and increased Ca2+-currents into the myocytes after MI. Experiments performed in cultivated heart muscle cells demonstrated a reduced Ca2+-reuptake by the SR during metabolic inhibition [55], which was explained by a reduced expression of the SR Ca2+-ATPase [56]. Additionally, ischemia-dependent alterations of the membrane potential induced an increase in the Ca2+-transient into the myocytes through Ca2+-channels [57]. An elevation in diastolic [Ca2+]i results in an impaired diastolic relaxation associated with an prolongation of TPT [58,59]. Several clinical studies reported an impaired diastolic function of failing hearts dependent on the diastolic [Ca2+]i-level [60,61].

After extracellular Ca2+- or isoproterenol-stimulation, left ventricular papillary muscles from placebo-treated MI animals showed a prolonged relaxation time (RT50) and an increased myocardial [Ca2+]i compared to sham-operated animals. The myocardial contractile reactivity of these animals decreased from the early to the late period after MI. Interestingly, placebo-treated MI animals showed aftercontractions and corresponding aequorin light signal afterglimmers during extracellular stimulation. The aftercontractions and afterglimmers of infarcted rats have been interpreted as a SR dysfunction associated with spontaneous Ca2+-releases by the SR [49,62]. The rise in [Ca2+]i observed after the stimulation of cardiac β-adrenergic receptors by isoproterenol has been explained by an increased Ca2+-transient into the myocytes through the Ca2+-channels [49] via intracellular cAMP-dependent protein kinase A (PKA) mediated phosphorylation of the Ca2+-channels [63]. The prolongation of RT50 by isoproterenol in placebo-treated MI animals was based on a lowered reuptake of Ca2+ by the SR [63]. Several experimental and clinical studies demonstrated that failing hearts exhibited a lowered number of β-adrenergic receptors which was associated with a dissociation of inotropic and lusitropic response to β-agonists [64,65].

In the late period after MI, the lowered contractility of the myocardium of infarcted animals could be partly explained by alterations of the extracellular matrix. In our experiments, 42 days after induction of MI a cardiac hypertrophy was present in placebo-treated MI rats evidenced by an increase in left (LVW) and right (RVW) ventricular weight as well as an increase in the cross-sectional area (CSA) of the papillary muscles (Table 2). Results of several studies demonstrated that the progressive development of cardiac fibrosis is accompanied by the accumulation of collagen type I and III in the interstitium associated with an elevated stiffness of the noninfarcted hypertrophied myocardium [66,67], and is responsible for the deteriorated DT of the papillary muscle in the chronic state of MI-induced heart failure.

4.2 Effects of mibefradil on the function of left ventricular papillary muscle
In contrast to placebo-treated MI rats, left ventricular papillary muscles from mibefradil-pretreated rats showed an increase in DT and a shortening in TPT and RT50 in the late period (7–42d) as well as a reduction of diastolic and systolic [Ca2+]i of the noninfarcted myocardium in the early period (1–3d) after induction of MI. The contractile responsiveness of the papillary muscle from these animals after extracellular Ca2+- or isoproterenol-stimulation increased from the early to the late period after MI.

According to a widely discussed hypothesis, the beneficial effects of CCA in the ischemic heart are related to the inhibition of Ca2+-entry through the Ca2+-channels into the myocytes. However, evidence has been presented that T-type Ca2+-channels are overexpressed in failing hearts [23], and that the entry of Ca2+ through these channels induced a Ca2+-release by the SR in ventricular myocytes of guinea pig hearts [68]. Additionally, in myocytes of cardiomyopathic hamsters the density of T-type Ca2+-channel currents was increased, and abnormal channel activity and inactivation kinetics were observed, whereas the density of L-type Ca2+-channel currents and activity were constant [22]. These data indicate that this channel type contributes to the regulation of intracellular signaling mechanisms especially during pathological stages. Thus, the observed reduction of diastolic and systolic [Ca2+]i by mibefradil in the early period after MI seems to be attributed to the inhibition of the Ca2+-entry through L- and/or T-type Ca2+-channels, and might be more pronounced in the failing heart which is related to an increased expression and activity of T-type Ca2+-channels.

Since the intracellular Ca2+-concentration of the rat myocardium is determined by the SR [69] the effect of mibefradil on contraction and relaxation time is related to an improved Ca2+-handling of the SR. In the literature, several directions have been published that CCA can stabilize the function of the SR during pathological stages [70]. In our experiments, mibefradil-pretreatment abolished aftercontractions and afterglimmers of papillary muscles of infarcted animals after extracellular stimulation, indicating that mibefradil prevented the spontaneous release of Ca2+ by the SR. The reduction of systolic [Ca2+]i by mibefradil in infarcted animals might reduce the desensibilisation of the contractile myofilaments and thereby improved reactivity of these filaments to Ca2+ with an increased DT of papillary muscles from these rats.

The observed effects of mibefradil on contractile force in the late period after MI might be related to the prevention of cardiac interstitial fibrosis by the compound [71,72]. As shown in the present study, mibefradil-pretreated MI animals had a significantly lowered LVW and papillary muscle CSA at day 42 post MI compared to placebo-treated MI animals. An antiproliferative effect of mibefradil in rat arteries has already been reported [73]. Evidence has been presented that T-type Ca2+-channels can promote cell growth and proliferation [17,74], and that this channel type contributes to remodeling processes in the failing heart [22,23]. Based on these findings, it is tempting to speculate that mibefradil, through to its T-channel blocking property, acted antihypertrophic and antiproliferative and reduced post-MI cardiac hypertrophy. It is also possible that the reduction of LVEDP by mibefradil reduced the left ventricular wall stress, and thereby reduced cardiac fibrosis and improved contractile force of the noninfarcted myocardium in the late period after MI. Additionally, the prevention of ischemia-induced increase in HR by mibefradil reduced chronic expose of the noninfarcted myocardium to stress factors such as catecholamines and angiotensin II (Ang II). These hormones have been demonstrated to contribute in the development of cardiac fibrosis, in the downregulation of β-receptors and in the loss of myocytes during heart failure [75–77].

In conclusion, the present study demonstrated that the preferentially T-type Ca2+ channel blocking agent, mibefradil, when treatment was started 7d prior to induction of MI, improved functional properties and contractile reactivity to external stimulation of the noninfarcted myocardium in the acute phase after ischemia whereas haemodynamic parameters were greatest improved in the chronic state of heart failure. The effects of mibefradil on the acute ischemic myocardium might be related to the protection of ischemia-induced intracellular Ca2+ overload and the prevention of desensibilisation of the contractile myofilaments to Ca2+. Several features of selective T-type Ca2+-channel blockade may have contributed to mibefradil’s beneficial action on the ischemic heart including improvement of coronary perfusion, and the lack of negative inotropy and sympathetic activation. In the chronic failing heart the inhibition of growth effects mediated through this particular Ca2+-channel and the reduction of cardiac remodeling explained the cardioprotective effects of mibefradil in MI-induced heart failure.

Time for primary review 32 days.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

  1. Gwathmey J.K., Morgan J.P. Altered calcium handling in experimental pressure-overload hypertrophy in the ferret. Circ Res (1985) 57:836–843.[Abstract/Free Full Text]
  2. Steenberger C., Murphy E., Levy L., London R.E. Elevation in cytosolic free calcium concentration early in myocardial ischemia in perfused rat heart. Circ Res (1987) 60:700–707.[Abstract/Free Full Text]
  3. Lee H.C., Mohabir R., Smith N., Franz M.R., Clusin W.T. Effects of ischemia on calcium-dependent fluorescence transients in rabbit hearts containing indo 1: Correlation with monophasic action potentials and contraction. Circ (1988) 78:1047–1059.[Abstract/Free Full Text]
  4. Grinwald P.M., Nayler W.G. Calcium entry in the calcium paradox. J Mol Cell Cardiol (1981) 13:867–880.[CrossRef][Web of Science][Medline]
  5. Duncan J.C. Role of intracellular calcium in promoting muscle damage: A strategy for controlling the dystrophic condition. Experientia (1978) 34:1531–1535.[CrossRef][Web of Science][Medline]
  6. Gwathmey J.K., Copleas L., MacKinnon R., et al. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circ Res (1987) 61:70–76.[Abstract/Free Full Text]
  7. Limas C.J., Olivari M.T., Goldenberg I.F., et al. Calcium uptake by cardiac sarcoplasmatic reticulum fractions in human dilated cardiomyopathy. Cardiovasc Res (1987) 21:601–605.[Web of Science][Medline]
  8. Braunwald E. Calcium channel blockers: pharmacologic considerations. Am Heart J (1982) 104:665–671.[CrossRef][Web of Science][Medline]
  9. Walsh R.A., Badke F.R., O’Rourke R.A. Differential effects of systemic and intracoronary calcium channel blocking agents on global and regional left ventricular function in conscious dogs. Am Heart J (1981) 102:341–350.[CrossRef][Web of Science][Medline]
  10. Smith H.J., Goldstein R.A., Griffith J.M., Kent K.M., Epstein S.E. Regional contractility: Selective depression of ischemic myocardium by verapamil. Circulation (1976) 54:629–635.[Abstract/Free Full Text]
  11. Opie L.H. Calcium channel antagonists. Part IV: Side effects and contradictions. Drug interactions and combinations. Cardiovasc Drugs Ther (1988) 2:177–189.[CrossRef][Medline]
  12. Henry P.D. Comparative pharmacology of calcium antagonists: nifedipine, verapamil and diltiazem. Am J Cardiol (1980) 46:1047–1058.[CrossRef][Web of Science][Medline]
  13. Packer M., Kessler P.D., Lee W.H. Calcium channel blockade in the management of severe chronic congestive heart failure: a bridge too far. Circulation (1987) 75(suppl_V):V56–64.[Medline]
  14. Packer M. Pathophysiological and mechanisms underlying the adverse effects of calcium channel-blocking drugs in patients with chronic heart failure. Circulation (1989) 80(suppl.IV):IV59, IV67.
  15. Bean B.P. Two kinds of calcium channels in canine atrial cells. J Gen Physiol (1985) 86:1–30.[Abstract/Free Full Text]
  16. Nilius B., Hess P., Lansman J.B., Tsien R.W. A novel type of cardiac calcium channel in ventricular cells. Nature (1985) 316:443–446.[CrossRef][Medline]
  17. Katz A.M. Calcium channel diversity in the cardiovascular system. JACC (1996) 28(suppl 2):522–529.[Abstract]
  18. Hagiwara N., Irisawa H., Kameyama M. Contribution of two types of calcium currents to the pacemaker potentials of rabbit sino-atrial node cells. J Physiol (1988) 395:233–253.[Abstract/Free Full Text]
  19. Clozel J.P., Veniant M., Osterrieder W. The structurally novel calcium channel blocker RO 40-5967, which binds to the (H3)-desmethoxyverapamil receptor, is devoid of the negative inotropic effects of verapamil in normal and failing rat hearts. Cardiovasc Drugs Ther (1990) 4:731–736.[CrossRef][Web of Science][Medline]
  20. Osterrieder W., Holck M. In vitro pharmacologic profile of RO 40-5967, a novel calcium channel blocker with potent vasodilator but weak inotropic action. J Cardiovasc Pharmacol (1989) 13:754–759.[Web of Science][Medline]
  21. Qin D., Jian P., Boutjdir M., El-Sherif N. Expression of T-type calcium channel current in remodeled hypertrophied left ventricle from adult rat following myocardial infarction (abstr). Circ (1995) 92(suppl I):I587.
  22. Sen L., Smith T.W. T-type Ca2+-channels are abnormal in genetically determined cardiomyopathic hamster hearts. Circ Res (1994) 75(1):149–155.[Abstract/Free Full Text]
  23. Nuss B.H., Houser S.R. T-type calcium current is expressed in hypertrophied adult feline left ventricular myocytes. Circ Res (1993) 73:777–782.[Abstract/Free Full Text]
  24. Mishra S.K., Hermsmeyer K. Selective inhibition of T-Typ calcium channels by RO 40-5967. Circ Res (1994) 75:144–148.[Abstract/Free Full Text]
  25. Clozel J.P., Banken L., Osterrieder W. Effects of RO 40-5967, a novel calcium antagonist, on myocardial function during ischemia induced by lowering coronary perfusion pressure in dogs: comparison with verapamil. J Cardiovasc Pharmacol (1989) 14:713–721.[Web of Science][Medline]
  26. Clozel J.P., Veniant M., Osterrieder W. Effects of RO 40-5967 and verapamil in rats without and with heart failure. Cardiovasc Drugs Ther (1990) 4:731–736.[CrossRef][Web of Science][Medline]
  27. Portegies M.C.M., Schmitt R., Kraaij C.J., et al. Lack of negative inotropic effects of the new calcium channel antagonist RO 40-5967 in patients with stable angina pectoris. J Cardiovasc Pharmacol (1991) 18:746–751.[Web of Science][Medline]
  28. Sandmann S., Spitznagel H., Chung O., et al. Effects of the calcium channel antagonist mibefradil on haemodynamic and morphological parameters in myocardial infarction-induced cardiac failure in rats. Card Res (1998) 39:339–350.[CrossRef]
  29. Stauss H.M., Zhu Y.C., Redlich Th., et al. Angiotensin-converting enzyme inhibition in infarct-induced heart failure in rats: bradykinin versus angiotensin II. J Cardiovasc Risk (1994) 1:255–262.[Medline]
  30. Stauss B., Itoi K., Stauss H., Unger Th. A novel inexpensive computer system to record and analyze hemodynamic data in conscious animals. Eur J Pharmacol (1990) 183:863–864.[CrossRef][Web of Science]
  31. Veniant M., Clozel J.P., Hess P., Wolfgang R. RO 40-5967, in contrast to diltiazem, does not reduce left ventricular contractility in rats with chronic myocardial infarction. J Cardiovasc Pharmacol (1991) 17:277–284.[Web of Science][Medline]
  32. Litwin S.E., Litwin C.M., Raya T.E., Warner A., Goldman S. Contractility and stiffness of noninfarcted myocardium following coronary liagtion in rats: Effects of chronic angiotensin converting enzyme inhibition. Circulation (1991) 83:1082–1087.
  33. Kihara Y., Morgan J.P. A comparative study of three methods for intracellular loading of the calcium indicator aequorin in ferret papillary muscle. Biochem Biophys Res Commun (1989) 162:402–407.[CrossRef][Web of Science][Medline]
  34. Kihara Y., Grossman W., Morgan J.P. Direct measurement of changes in intracellular calcium transients during hypoxia, ischemia, and reperfusion of the intact mammalian heart. Circ Res (1989) 65:1029–1044.[Abstract/Free Full Text]
  35. Blinks J.R. Techniques in Cellular Physiology. Baker P.E., ed. (1982) Shannon: Elsevier/North Holland Science. 1, 126/38. Part III.
  36. Blinks J.R. The heart and cardiovascular system. Fozzard H.A., Haber E., Jennings R.B., Katz A.M., Morgan H.E., eds. (1986) 1. New York: Raven Press. 671–701.
  37. Pfeffer M.A., Pfeffer J.M., Fishbein M.C., et al. Myocardial infarct size and ventricular function in rats. Circ Res (1979) 44(suppl 4):503–512.[Abstract/Free Full Text]
  38. Raya T.E., Gay R.G., Augirre M., Goldmann S. Importance of venodilatation in prevention of left ventricular dilatation after chronic large myocardial infarction in rats: A comparison of captopril and hydralazine. Circ Res (1989) 64(2):330–337.[Abstract/Free Full Text]
  39. Veniant M., Clozel J.P. RO 40-5967, in contrast to diltiazem, does not reduce left ventricular function in rats with and without chronic myocardial infarction (abstr). J Mol Cell Cardiol (1990) suppl 22:S61.
  40. Millard R.W., Lathrop D.A., Grupp G., et al. Differential cardiovascular effects of calcium channel blocking agents: potential mechanisms. Am J Cardiol (1982) 49:499–506.[CrossRef][Web of Science][Medline]
  41. Graefe K.H., Ziegler R., Wingender W., Ramsch K.D., Schmitz H. Plasma concentration-response relationship for some cardiovascular effects of dihydropyridines in healthy subjects. Clin Pharmacol Ther (1988) 43(suppl 1):16–22.[Web of Science][Medline]
  42. Corea L., Miele N., Bentivoglio M., et al. Plasma renin activity and plasma epinephrine and norepinephrine in controls and hypertensive patients. Clin Sci (1979) 57:115s–117s.[Medline]
  43. Guth B.D. Reduction of exercise-induced regional contractile dysfunction in dogs using a novel calcium channel blocker (RO 40-5967). Cardiovasc Drugs Ther (1992) 6:167–171.[CrossRef][Web of Science][Medline]
  44. Boulanger C.M., Desta B., Clozel J.P., van Houtte P.M. Chronic treatment with the calcium channel inhibitor RO 40-5967 potentiates endothelium-dependent relaxation in the aorta of the hypertensive salt sensitive Dahl rat. Blood Pressure (1994) 3:193–196.[Medline]
  45. Morgan J.P. Abnormal intracellular modulation of calcium as a major cause of cardiac contractile dysfunction. N Engl J Med (1991) 325(29):625–632.[Web of Science][Medline]
  46. Gay R.C., Graham S., Aguirre M., Goldman S., Morkin E. Effects of 10–12 days treatment with L-thyroxine in rats with myocardial infarction. Am J Physiol (1988) 255:H801–H806.[Web of Science][Medline]
  47. Michel J.B., Lattion A.L., Salzmann J.L., et al. Hormonal and cardiac effects of converting enzyme inhibition in rat myocardial infarction. Circ Res (1988) 62:641–650.[Abstract/Free Full Text]
  48. Fabiato A., Fabiato F. Effects of pH on the myofilaments and the sarcoplasmatic reticulum of skinned cells from cardiac and skeletal muscles. J Physiol (Lond) (1978) 276:233–255.[Abstract/Free Full Text]
  49. Endoh M., Blinks J.R. Action of sympathomimetic amines on the Ca2+ transients and contractions of rabbit myocardium: Reciprocal changes in myofibrillar responsiveness to Ca2+ mediated through {alpha}- and β-adrenoceptors. Circ Res (1988) 62:247–265.[Abstract/Free Full Text]
  50. Puceat M., Clement O., Lechene P., et al. Neurohormonal control of calcium sensitivity of myofilaments in rat single heart cells. Circ Res (1990) 67:517–524.[Abstract/Free Full Text]
  51. Garlick P.B., Radda G.K., Seeley P.J. Studies of acidosis in ischemic heart by phosphorus nuclear magnetic resonance. Biochem J (1979) 184:547–554.[Web of Science][Medline]
  52. Momomura S.I., Ingwall J.S., Parker J.A., et al. The relationship of high energy phosphates, tissue pH and regional blood flow to diastolic distensibility in the ischemic dog myocardium. Circ Res (1985) 57:822–835.[Abstract/Free Full Text]
  53. Kusuoka H., Weisfeldt M.L., Zweier J.L., Jacobus W.E., Marban E. Mechanism of early contractile failure during hypoxia in intact ferret heart: Evidence for modulation of maximal Ca2+-activated force by inorganic phosphate. Circ Res (1986) 59:270–282.[Abstract/Free Full Text]
  54. Blanchard E.M., Solaro R.J. Inhibition of the activation and troponin calcium binding of dog cardiac myofibrils by acidic pH. Circ Res (1984) 55:382–391.[Abstract/Free Full Text]
  55. Suko J., Vogel J.H.K., Chidsey C.A. Intracellular calcium and myocardial contractility. III. Reduced calcium uptake and ATPase of the sarcoplasmatic reticular fraction prepared from chronically failing calf hearts. Circ Res (1970) 27:235–247.[Abstract/Free Full Text]
  56. De la Bastie D., Levitsky D., Rappaport L., et al. Function of the sarcoplasmatic reticulum and expression of its Ca2+-ATPase gene in pressure overload-induced hypertrophy in the rat. Circ Res (1990) 66:554–564.[Abstract/Free Full Text]
  57. Elliott A.C., Smith G.L., Allen D.G. Simultaneous measurements of action potential duration and intracellular ATP in isolated ferret hearts exposed to cyanide. Circ Res (1989) 64:583–591.[Abstract/Free Full Text]
  58. Bourdillon P.D., Poole-Wilson P.A. The effects of verapamil, quiescence and cardioplegia on calcium exchange and mechanical function in ischemic rabbit myocardium. Circ Res (1982) 50:360–368.[Abstract/Free Full Text]
  59. McLaurin L.P., Rolett E.L., Grossman W. Impaired left ventricular relaxation during pacing induced ischemia. Am J Cardiol (1973) 32:751–757.[CrossRef][Web of Science][Medline]
  60. Barry W.H., Brooker J.Z., Alderman E.L., Harrison D.C. Changes in diastolic stiffness and tone of the left ventricle during angina pectoris. Circulation (1974) 49:255–263.[Abstract/Free Full Text]
  61. Sasayama S., Nonogi H., Miyazaki S., et al. Changes in diastolic properties of the regional myocardium during pacing-induced ischemia in human subjects. J Am Coll Cardiol (1985) 5:599–606.[Abstract]
  62. Litwin S.E., Morgan J.P. Captopril enhances intracellular calcium handling and β-adrenergic responsiveness of myocardium from rats with postinfarction failure. Circ Res (1992) 71(4):797–807.[Abstract/Free Full Text]
  63. Katz A. Interplay between inotropic and lusitropic effects of cyclic adenosine monophosphate on the myocardiale cell. Circulation (1990) 82(suppl I):I7–I11.[Medline]
  64. Fowler M.B., Laser J.A., Hopkins G.I., Minobe W., Bristow M.R. Assessment of the beta-adrenergic receptor pathway in the intact failing human heart: progressive receptor down-regulation and subsensitivity to agonist response. Circulation (1986) 74:1290–1302.[Abstract/Free Full Text]
  65. Lakatta E.G., Gerstenblith G., Angell C.S., Shock N.W., Weisfeldt M.L. Diminished inotropic response of aged myocardium to catecholamines. Circ Res (1975) 36:262–269.[Abstract/Free Full Text]
  66. Weber K.T., Janicki J.S., Shroff S.G., et al. Collagen remodeling of the pressure-overloaded, hypertrophied nonhuman primate myocardium. Circ Res (1988) 62:757–765.[Abstract/Free Full Text]
  67. Sen S., Bumpus M. Collagen synthesis in development and reversal of cardiac hypertrophy in spontaneously hypertensive rats. Am J Cardiol (1979) 44:954–958.[CrossRef][Web of Science][Medline]
  68. Sipido K.R., Carmeliet E., van de Werf F. T-type Ca2+ current as a trigger for Ca2+ release from the sarcoplasmatic reticulum in guinea-pig ventricular myocytes. J Physiol (1998) 508(2):439–451.[Abstract/Free Full Text]
  69. Wier W.G. Cytosolic [Ca2+] in mammalian ventricle: dynamic control by cellular process. Ann Rev Physiol (1990) 52:467–485.[CrossRef][Web of Science][Medline]
  70. Zucchi R, Limbruno U, Ronca-Testoni S. et al. Effects of verapamil, gallopamil, diltiazem and nifedipine on sarcoplasmtic reticulum function in rat heart.
  71. Veniant M., Clozel J.P., Heudes D., Banken L., Menard J. Effects of RO 40-5967, a new calcium antagonist, and enalapril on cardiac remodeling in renal hypertensive rats. J Cardiovasc Pharmacol (1993) 21:544–551.[Web of Science][Medline]
  72. Karam H., Heudes D., Bruneval P., et al. Endothelin antagonism in end-organ damage of spontaneously hypertensive rats. Comparison with angiotensin-converting enzyme inhibition and calcium antagonism. Hypertension (1996) 28(3):379–385.[Abstract/Free Full Text]
  73. Schmitt R., Clozel J.P., Iberg N., Bühler F.R. Mibefradil prevents neointima formation after vascular injury in rats. Arterioscler Thromb Vasc Biol (1995) 15:1161–1165.[Abstract/Free Full Text]
  74. Mehrke G., Zong X.G., Flockerzi V., Hofmann F. The calcium-channel-blocker RO 40-5967 blocks differently of T-type and L-type calcium channels. J Pharmacol Exp Ther (1994) 271:1483–1488.[Abstract/Free Full Text]
  75. Bristow M.R., Hershberger R.E., Port J.D., et al. β-adrenergic pathways in nonfailing human and failing human ventricular myocardium. Circ (1990) 82(suppl I):I12–I25.[Medline]
  76. Kubo S.H. Neurohumoral activation and the response to coverting enzyme inhibition in congestive heart failure. Circ (1990) 81(suppl III):III107, III114.
  77. Tan L.B., Jalil J.E., Pick R., Janicki J.S., Weber K.T. Cardiac myocyte necrosis induced by angiotensin II. Circ Res (1991) 69:1185–1195.[Abstract/Free Full Text]

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