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Eur J Cardiothorac Surg 2002;21:224-231
© 2002 Elsevier Science NL

Diltiazem during reperfusion preserves high energy phosphates by protection of mitochondrial integrity

A. Krönera, R. Seitelbergera, J. Schirnhoferb, O. Berneckerb, R. Mallingerc, S. Hallströmb, M. Plonera, B.K. Podessera*

a Department of Cardiothoracic Surgery, AKH Wien, Währinger Gürtel 18-20, 1090 Vienna, Austria
b Ludwig Boltzmann Institute for Heart Research, Vienna, Austria
c Department of Histology II, University of Vienna, Vienna, Austria

Received 16 July 2001; received in revised form 12 November 2001; accepted 14 November 2001.

* Corresponding author. Tel.: +43-1-404-005-620; fax: +43-1-404-005-640
e-mail: bruno.podesser{at}akh-wien.ac.at


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: This study evaluates the effects of diltiazem administered during reperfusion on hemodynamic, metabolic, and ultrastructural postischemic outcome. Methods: Hearts of 38 adult White New Zealand rabbits underwent 60 min of global cold ischemia followed by 40 min of reperfusion in an erythrocyte perfused isolated working heart model. Hearts were randomly assigned to four groups and received diltiazem (0.1, 0.25, and 0.5 µmol/l) during reperfusion only, or served as control. Results: The postischemic time courses of heart rate, aortic flow, and external stroke work clearly reflected the dose-dependent negative chronotropic and inotropic efficacy of diltiazem in the two higher concentrations. High energy phosphates (HEP) determined from myocardial biopsies taken after 40 min of reperfusion were significantly better preserved in all treatment groups compared to control hearts. Similarly ultrastructural grading of mitochondria and myofilaments revealed a significant reduction of reperfusion injury in hearts that received diltiazem compared to control. Conclusions: Diltiazem protects mitochondrial integrity and function, thereby preserving myocardial HEP levels. Only low dose diltiazem (0.1 µmol/l) during reperfusion combines both, optimal mitochondrial preservation with minimal changes in hemodynamics.

Key Words: Reperfusion • Mitochondria • Diltiazem • Isolated Working heart • Rabbit


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
A number of previous studies, including our own clinical data, have reported that administration of diltiazem prior or during ischemia reduces infarct size and protects against reperfusion injury [1–3]. Recently, also administration of low dose diltiazem during reperfusion only showed promising experimental results [4,5]. The beneficial effects of diltiazem on ischemic myocardium are generally thought to be due to coronary artery vasodilatation and a decrease of heart rate as well as cardiac contractility [6]. The net result of these effects is a reduction in the imbalance between oxygen supply and demand in acutely ischemic myocardium, through a simultaneous increase in collateral blood flow (mediated by coronary vasodilatation) and a reduction in oxygen consumption (secondary to a decrease in heart rate, contractility, and afterload).

In addition, diltiazem has been shown to decrease infarct size even more than that attributable to reperfusion alone by a mechanism other than altered coronary blood flow or myocardial work [7,8]. It has become apparent that free oxygen radical generation and calcium overload contribute to the development of reperfusion injury [9,10]. Some data suggest that calcium antagonists protect the lipid fraction within the cell membrane against the toxic effects of free oxygen radicals [11,12]. Lipid peroxidation is considered a major mechanism of oxygen radical toxicity, thereby altering membrane permeability [9,13]. Calcium antagonists also prevent calcium-overload [14,15] in reperfused hearts via their effect on the slow L-type calcium channel. This mechanism again influences the formation of oxygen-derived free radicals and lipid peroxidation [9,10].

The purpose of the present study was to assess the possibility that the protective effects of diltiazem are due to its membrane protective properties at the level of ultrastructure, in addition to its known anti-ischemic and anti-arrhythmic properties. This thesis was tested by administering diltiazem during reperfusion only. Hemodynamic, energetic, and ultrastructural postischemic outcome was analyzed. All experiments were performed in an isolated, erythrocyte perfused working heart.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Animal care
This study was approved by the Ethical Committee of the University of Vienna and by the Ministry of Science, Republic of Austria. All animals received humane care in compliance with the European Convention on Animal Care and was approved by their institutional ethics committee.

2.2. Experimental design and heart preparation
The isolated, red cell perfused working heart model (Hugo Sachs Electronics, Freiburg, Germany) has been described in detail recently by Podesser et al. [16]. Hearts, excised from 38 adult White New Zealand rabbits (2950±200 g), were randomly assigned to four groups. Twenty-nine hearts received diltiazem (Dilzem®, Gödecke AG, Freiburg, Germany) during reperfusion in three different concentrations (0.1, 0.25, and 0.5 µmol/l), nine hearts served as control.

Anesthesia was accomplished with ketamine-hydrochloride (5 mg/kg) and propofol (20 mg/kg) administered by the ear vein and was preceded by a 300 IU/kg bolus of heparin i.v. to prevent coagulation. After bilateral thoracotomy, the beating heart was quickly excised, immersed into ice-cold saline, weighed, and mounted on the already perfused aortic cannula of the working heart apparatus.

2.3. Perfusion media and experimental protocol
The perfusate consisted of a Krebs–Henseleit buffer based suspension of purified bovine erythrocytes (hct, 30%), oxygenated with low gas (75% N2; 20% O2; 5% CO2) to provide a constant pO2of 100±10 mmHg. Composition of the buffer was as follows (in mmol/l): NaCl, 118; KCl, 4.7; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.2; NaEDTA, 0.5; NaHCO3, 25; glucose: 11.1; insulin, 2.5 IU/l; bovine albumin, 0.2 g/dl. Blood cardioplegia according to Buckberg (Buckberg Kardioplegie, Köhler Chemie, Alsbach Hänlein, Germany) was prepared freshly from a crystalloid stock solution and the erythrocyte-enriched buffer in a 1:4, resulting in a hematokrit of 25% for the cardioplegic solution, similar to the clinical scenario. Composition of the cold induction and re-infusion stock solution was as follows (in mmol/l): NaCl, 40,3; KCl, 87,28; Trometamol, 43,63; citrat, 4,5; Na-Citrat, 26; NaH2PO4, 4,7; glucose 924,2. Composition of the hot-shot stock solution was as follows (in mmol/l): NaCl, 38,9; KCl, 37; Trometamol, 66,5; Na-glutamat. 70,4; Na-aspartat, 69,8; citrat, 17,3; Na-Citrat, 99,1; NaH2PO4, 17,8; glucose 1022,6. The perfusion was performed according to a standardized protocol Fig. 1 : 15 min of Langendorff mode (LD) at constant pressure (70 mmHg) were followed by 25 min of working heart mode (WH) to assess preischemic baseline values. The following cardioplegic arrest (CP, 60 min) was induced with cold blood cardioplegia (30 ml at 10°C). Cardiac arrest was maintained with two re-infusions (10 ml at10°C) after 20 and 40 min of ischemia, and terminated with the ‘hot-shot’ (20 ml at 37°C) to resuscitate cardiac function. Diltiazem was added to the ‘hot-shot’ and to the perfusate, to be available from the very first minute of reperfusion onward. The protocol for the ensuing reperfusion period corresponded to the preischemic sequence (rLD 15 min and rWH 25 min, respectively).



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  		Fig. 1. Experimental protocol. LD, preischemic Langendorff mode; WH, preischemic working heart mode; rLD, Langendorff mode during reperfusion; rWH, working heart mode during reperfusion; Ind, induction of cardioplegia; Re, re-infusions. Diltiazem was added to the ‘hot-shot’ and to the perfusate, to be available from the very first minute of reperfusion onward.

 
2.4. Hemodynamic Measurements
Hemodynamic data were registered continuously and monitored by an on-line data acquisition system (P. Hellige, Freiburg, Germany). Working with constant pressure, preload was kept between 7–9 mmHg, afterload was fixed at 70 mmHg. Aortic and left atrial pressure (AP and LAP) were obtained by two pressure transducers (Statham P23XL, Spectramed Inc., USA), connected to side arms of their respective cannulas, while two electromagnetic flow probes (Flowmeter Narcomatic RT-500, Narco Biosystems, USA) measured the aortic and left atrial flow (AF and LAF). Tiny suction electrodes (Hugo Sachs Electroniks, Freiburg, Germany) placed on the epicardial surface permitted the registration of a two-lead ECG for evaluation of heart rate.

To assess mechanical cardiac functioning, the primary hemodynamic readings served for computing the following parameters: coronary flow (CF) was calculated by subtracting AF from cardiac output (CO). The latter in our closed perfusion system was identical to the volume entering the heart and therefore determined to be LAF. To underline the vasodilatative capacities we expressed CF as a percentage of AF (%). In order to see the dose-dependent negative chronotropic effect of diltiazem, hearts were not paced. To determine the inotropic state, we used the external stroke work (ESW=COxAP/HR) and the AF, the latter independent of HR.

Postischemic hemodynamic recovery was expressed as percentage of preischemic baseline values, which were derived from five consecutive preischemic measurements.

2.5. High energy phosphates
Immediately at the end of the postischemic rWH period freeze-clamped biopsies of the apical myocardium were taken. The specimens were stored in liquid nitrogen until further treatment. High energy phosphates (HEP) such as phosphocreatine (PCr) and adenine nucleotides (ATP, ADP, AMP) were analyzed by high performance liquid chromatography (HPLC) as described in detail recently [17]. After sample preparation the absorbance of the column effluent was monitored simultaneously at 214 nm (PCr) and 254 nm (adenine nucleotides) with a diode array detector module 168 (System Gold®-Beckmann Instruments, Inc. San Ramon, CA, USA).

The energy charge (EC), regarded as a sensitive parameter for the purin metabolism, was calculated according to the following formula:

2.6. Histological processing
After having removed the biochemical sample, two myocardial specimens were carefully harvested from the septal and free wall of the left ventricle and fixed with a mixture of 4% paraformaldehyde and 1% glutaraldehyde in a 0.1 M cacodylate buffer. Histologic processing was performed using standard techniques [18]. Transmission electron microscopy (JEM-1200 EX, Jeol, Japan) at magnifications of x30,000 (two microscopic fields for each heart) and x45,000 (three microscopic fields for each heart) was used to determine myofilamental and mitochondrial damage, respectively. This evaluation was conducted by two independent histologists, which were blinded as to treatment versus control. Ultrastructural damage was classified semiquantitatively according to the following standardized scheme with respect to mitochondria and myofilaments as reported by Schaper et al. [19]:

Ischemia-reperfusion injury score
Mitochondrial damage:
Grade 0: no visible damage, normal matrix granules
Grade 1: loss of matrix granules, light clearing of matrix
Grade 2: moderate clearing of matrix, moderate swelling, partial fragmentation of cristae
Grade 3: severe clearing, severe swelling, loss of cristae
Grade 3a:amorphous dense granules
Myofilamental damage:
Grade 0: no visible damage
Grade 1: Z lines not in register (Z lines of adjacent myofibrills not aligned)
Grade 2: enlarged I bands
Grade 3: disintegration.

2.7. Statistical analysis
For statistical analysis all data were expressed as mean±standard deviation. To compare groups an ANOVA was performed. Post hoc tests were performed using the Tukey test on a SPSS 8.0 software package. For not normal distributed data a logarithmic transformation was necessary.

Ordinal parameters (ultrastructural grades) were transformed for MANOVA by Fechner's marginal normalization. Ultrastructural data show differences between control and treatment groups, reflecting the distribution of the ischemic damage within a group in toto. Data were considered significant at a P-value <0.05.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. Hemodynamic data
Baseline hemodynamics, presented in Table 1, demonstrated comparable preischemic conditions in all parameters, without significant differences. As expected HR decreased significantly with increasing concentrations of diltiazem (0.25 and 0.5 µmol/l vs. 0.1 µmol/l diltiazem and control; P<0.05, Fig. 2, panel A ). Additionally, in the two higher concentrations diltiazem revealed also its known negative inotropic effects resulting in decreased postischemic recovery of AF (Fig. 2, panel B). Postischemic recovery of ESW was significantly decreased in the high dose group compared to the other groups (P<0.01) (Fig. 2, panel C). Only low dose diltiazem had no negative inotropic and chronotropicc effects.


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  		Table 1. Preischemic hemodynamic baseline valuesa

 


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  		Fig. 2. (Panel A) Postischemic recovery (mean ± SEM) of heart rate; the time-points define the time (min) elapsed in working heart mode during reperfusion (rWH); rHR3: ANOVA, P=0.0013; ##P=0.008 vs. dil 0.1, P=0.031 vs. control, ++P=0.011 vs. dil 0.1, P=0,044 vs. control; rHR5: ANOVA, P=0.0005; ## P=0.026 vs. dil 0.1, P=0.036 vs. control, ++P=0.003 vs. dil 0.1, P=0,005 vs. control; rHR10: ANOVA, P=0.0005; ##P=0.016 vs. dil 0.1, P=0.034 vs. control, ++P=0.003 vs. dil 0.1, P=0.008 vs. control; rHR25: ANOVA, P=0.0012; ##P=0.018 vs. dil 0.1, P=0.029 vs. control, ++P=0.01 vs. dil 0.1, P=0.016 vs. control. (Panel B) Postischemic recovery (mean ± SEM) of aortic flow; the time-points define the time (min) elapsed in working heart mode during reperfusion (rWH); rAF3: ANOVA, P<0.0001; ##P=0.047 vs. dil 0.1, P=0.042 vs. control; ++P<0.001 vs. dil 0.1, P=0.029 vs. dil 0.25, P<0.001 vs. control; rAF5: ANOVA, P<0.0001; ++P<0.001 vs. dil 0.1, P=0.001 vs. dil 0.25, P<0.001 vs. control; rAF10: ANOVA, P<0.0001; ++P<0.001 vs. dil 0.1, P<0.001 vs. dil 25, P<0.001 vs. control; rAF25: ANOVA, P<0.0001; ++P<0.001 vs. dil 0.1, P=0.001 vs. dil 25, P<0.001 vs. control. (Panel C) Postischemic recovery (mean ± SEM) of external stroke work; the time-points define the time (min) elapsed in working heart mode during reperfusion (rWH); rESW3: ANOVA, P<0.0001; ++P<0.001 vs. dil 0.1, P=0.013 vs. dil 25, P<0.001 vs. control; rESW5: ANOVA, P<0.0001; ++P<0.001 vs. dil 0.1, P=0.001 vs. dil 25, P<0.001 vs. control; rESW10: ANOVA, P<0.0001; ++P<0.001 vs. dil 0.1, P<0.001 vs. dil 25, P<0.001 vs. control; rESW25: ANOVA, P<0.0001; ++P<0.001 vs. dil 0.1, P=0.001 vs. dil 25, P<0.001 vs. control. (Panel D) Values of CF, expressed as percentage of AF; the time-points define the time (min) elapsed in working heart mode during reperfusion (rWH); rCF/AF3: ANOVA, P=0.02; ++P=0.022 vs. dil 0.1; rCF/AF5: ANOVA, P=0.009; ++P=0.013 vs. dil 0.1, P=0.032 vs. control; rCF/AF10: ANOVA, P=0.005; ++P=0.008 vs. dil 0.1, P=0.018 vs. control; rCF/AF25: ANOVA, P=0.0013; ++P=0.015 vs. dil 0.1, P=0.07 vs. control.

 
Interestingly, the measurements of CF did not significantly differ between the treatment and control groups. However, when CF was expressed as % of AF, the vasodilatative capacity was clearly visible in the high-dose treatment group (Fig. 2, panel D).

3.2. High energy phosphates
Table 2 depicts myocardial HEP determined at the end of reperfusion. Mean levels of PCr and ATP were consistently elevated in all treatment groups compared to control. In diltiazem 0.25 and diltiazem 0.5 these differences were proven significant regarding PCr concentrations (P<0.05 vs. control). ADP was significantly lower in diltiazem 0.25, AMP was significant lower in all treatment groups compared to control (P<0.05 and P<0.01, respectively). Also, EC reflects these differences and unmasked a superior preservation of the total energetic reserve concerning the adenine nucleotide pool at the end of reperfusion in all treatment groups compared to control. Preischemic reference data on HEP (µmol/g wet weight) from six rabbits, obtained after 15 min of LD and 25 min of WH in the same experimental model, were in good correlation (PCr, 3.10±0.36; ATP, 2.19±0.09; ADP, 0.55±0.01; AMP, 0.06±0.01; EC, 0.88±0.003).


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  		Table 2. HEP in the left ventricular myocardiuma

 
3.3. Micromorphology
Overall, the assessment by two independent observers yielded a concordance rate of 95%, discordant findings were quoted according to the higher grade. With respect to mitochondrial damage 89% of the hearts in the control group showed plainly visible signs of ischemia-reperfusion injury. In contrast, hearts treated with diltiazem showed a significant reduction of injury compared to control (P<0.001 vs. control; Table 3). The highest degree of protection was obtained in hearts treated with diltiazem 0.5 µmol/l (100% no damage). Also the two other diltiazem groups revealed a high degree of protection (dilti 0.1/83% no damage; dilti 0.25/75% no damage) and no differences were found compared to diltiazem 0.5.


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  		Table 3. Ultrastructural resultsa

 
The results of the analysis of the myofilaments led to the same conclusion. Treatment groups showed superior preservation of myofilamental integrity compared to control (P<0.001 vs. control; Table 3). Representative photomicrographs of mitochondrial and myofilamental damages are depicted in Figs. 3 and 4 .



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  		Fig. 3. Mitochondrial ultrastructure. Representative photomicrographs (magnification: x45,000) depicting the range of observed postischemic damages, graded as defined in text. (A) No discernable damage (grade 0). (B) Light clearing of matrix with loss of granules, assigned to grade 1. (C) Additional swelling and partial fragmentation of cristae, defining grade 2 damage.

 


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  		Fig. 4. Mitochondrial ultrastructure. Representative photomicrographs (magnification: x30,000) depicting the range of observed postischemic damages, graded as defined in text. (A) No discernable damage (grade 0). (B) Z lines not in register, assigned to grade 1. (C) Additional enlarged I bands, defining grade 2 damage.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
Experimental study always raises a question about the validity of clinical conclusions extrapolated from the results. In organ preparations, the artificial isolation from intervening systemic influences constitutes the primary limiting factor. The lack of platelets, leukocytes, and inflammatory cytokines has to be taken into account. However, cardioplegic arrest as a paradigm for global ischemia-reperfusion injury with comparable separation of the heart from systemic variables lends itself for studies using the isolated heart. Using a model of blood-cardioplegia incorporating the ‘hot-shot’ technique closely mimics cardiac surgery. The isolated heart offers major advantages, in particular, by demonstrating pharmacological effects at the organ level. Using the working heart perfusion mode and an erythrocyte-containing perfusate provides an approach to in vivo conditions. The addition of red blood cells to the more commonly used crystalloid perfusion fluid not only affords oxygen carrying capacity and CF auto-regulation within the physiologic range, but also provides crucial biochemical functions, such as buffering of oxygen radicals [16]. A further limitation of this study is, that we did not measure the reactive oxygen species (ROS) directly. However, the close relationship between ROS and ultrastructural damage is well established [13]. Therefore ultrastructural measurements can be assumed as an indirect way to describe reperfusion injury.

The purpose of this study was to examine the hemodynamic, energetic, and ultrastructural changes of diltiazem, when administered during reperfusion only. It is important to stress once more that all hearts underwent the same ischemic burden. The results clearly demonstrate a significant cardioprotective capacity of diltiazem, which is characterized by significant maintenance of HEP and ultrastructural integrity. This can only in part be explained by the known negative inotropic and chronotropic effects of this drug.

The time courses of HR, AF, and ESW clearly reflect the dose-dependent negative chronotropic and inotropic efficacy of diltiazem in the two higher concentrations (0.25 and 0.5 µmol/l) after 40 min of reperfusion. Clinically, the possible negative inotropic- and chronotropic effects of diltiazem when used intraoperatively have caused some concern, especially in patients with left ventricular dysfunction [20]. In the present study low-dose diltiazem (0.1 µmol/l) revealed no significant difference in global LV functions compared to control, indicating no detectable effect on myocardial contractility and heart rate. These findings go on to confirm the results of Grover et al., that diltiazem reduces ischemia-reperfusion injury with the lowest cost in cardiac function compared to other calcium antagonists [8]. The relative increase of CF in the relation to aortic flow demonstrates the well-known vasodilatative capacity, which was significant in the high dose group.

Analysis of HEP at the end of our experimental protocol provides postischemic steady state levels, as determined by both synthesis and expenditure. All treatment groups consistently showed higher mean concentrations of PCr and ATP than respective controls. In addition the ATP breakdown products (ADP, AMP) which regulate the compensatory pathways (glycogenolysis, glycolysis and the mitochondrial oxidative system) were significantly lower in all treatment groups compared to control [21]. Consequently, the significant lower levels of ADP and AMP in treatments groups can be interpreted as a high rate of rephosphorylation in the well-preserved mitochondria (via the salvage pathway) [21]. In particular, the EC unmasked this superior preservation of the total energetic reserve and showed normal energy stores in all treatment groups compared to control. Comparable results were attained by Takeo et al. using rabbit hearts perfused under hypoxic and reoxygenated conditions. The authors found that a hypoxia induced decline in myocardial HEP and a release of ATP metabolites were effectively depressed by diltiazem and verapamil, administered during hypoxia [22]. Kavanaugh et al. demonstrated that diltiazem provides a protective effect on myocardial high-energy phosphate metabolism during regional ischemia and reperfusion in the rabbit heart [23].

Ultrastructural damage was classified to characterize ischemia-reperfusion injury. A score was adapted from Schaper et al., providing not only a qualitative but also a semiquantitative analysis [19]. The authors were able to standardize ultrastructural changes occurring during ischemia and compared them to function and metabolic data of the heart. They concluded a close correlation between the rate of ultrastructural, functional, and metabolic deterioration at the end of ischemia [19]. In the present study the grading of mitochondrial and myofilamental damage revealed a significant reduction of reperfusion injury in hearts that received diltiazem compared to control. According to Tagami et al. diltiazem is bound to the plasma membrane, is transported through the T-system and may accumulate within the sarcoplasmatic reticulum and mitochondria. The authors demonstrated that the intracellular sites of action of diltiazem are possibly the mitochondria. Our results underline these findings that the drug may inhibit the mitochondria permeability transition pore, thereby preserving ultrastructural integrity [24]. The ultrastructure and the content of the HEP correlated well and reflected the severity of reperfusion injury afflicted upon the myocardium. The evaluated ultrastructural alterations primarily represent membrane disruption. Lipid peroxidation, reflecting the involvement of oxygen free radicals, have been seen as the main cause for this membrane disruption [9,13]. Thus our results could be an indirect evidence of an antioxidant effect of diltiazem. Comparable studies in the literature confirm the favorable preservation of ultrastructure with diltiazem. Zografos and Watts demonstrated improved recovery of contractile function and prevention of mitochondrial swelling, structural grade change, and increase in mitochondrial Ca2+ after global ischemia and reperfusion in rat hearts pretreated with diltiazem [25]. Koller and Bergmann suggested that in the setting of reperfusion the salutary capacity of diltiazem may be mediated by a protection against lipid peroxidation, reducing the malondialdehyd (MAD) content [12].

The mechanism responsible for diltiazem's protective effect during reperfusion may depend on a relative reduction of cellular calcium influx via its effect on the slow L-type calcium channel [14,15]. Accordingly, reduction of intracellular calcium concentrations by diltiazem may reduce free radical generating mechanisms and lipid peroxidation. Alternatively, diltiazem may inhibit iron-dependent lipid peroxidation directly in ventricular myocyte membranes which can be ascribed to its capacity to scavenge or impair oxygen free radical generation [12]. A reasonable concept is that on the one hand formation of free radicals increase cytosolic calcium and on the other hand calcium overload influences the formation of oxygen-derived free radicals and lipid peroxidation. Thus the beneficial action of diltiazem for reperfusion damage may be twofold: first prevention of cytosolic calcium overload and second inhibition of the toxic effects of oxygen-derived free radicals.

In conclusion, the present study provides a comprehensive analysis of the effects of diltiazem administered during reperfusion only with respect to hemodynamics, HEP, and micromorphology. In all treatment groups diltiazem protects mitochondrial integrity and function, thereby preserving myocardial HEP. However, only low dose diltiazem (0.1 µmol/l) during reperfusion combines both, optimal mitochondrial preservation with minimal changes in hemodynamics. These experimental findings emphasize that the cardioprotective effects of diltiazem are not only mediated by its negative inotropic, chronotropic, and vasodilatative capacities, but also are due to its membrane protective effects.


   Acknowledgments
 
We would like to thank Dr R. Bakovic-Alt, Goedecke A.G., Freiburg, Germany for providing Diltiazem. We are grateful to Mrs Scherzer for preparing and cutting semithin and ultrathin sections to analyze ultrastructure.


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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