Skip Navigation

Cardiovascular Research 1997 35(1):113-119; doi:10.1016/S0008-6363(97)00104-1
© 1997 by European Society of Cardiology
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Zuurbier, C. J
Right arrow Articles by van Beek, J. H.G.M
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zuurbier, C. J
Right arrow Articles by van Beek, J. H.G.M
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

Copyright © 1997, European Society of Cardiology

Undiminished mitochondrial function during stunning in rabbit heart at 28°C

Coert J Zuurbiera,b,* and Johannes H.G.M van Beeka

aLaboratory for Physiology, Institute for Cardiovascular Research, Vrije Universiteit (ICaR-VU), van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands
bCenter for Bioengineering, University of Washington, Box 357962, Seattle, WA 98195-7962, USA

* Corresponding author. Tel.: +1 (206) 685-2840; fax: +1 (206) 685-2651; e-mail: coert@nsr.bioeng.washington.edu

Received 13 August 1996; accepted 9 April 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: To investigate effect of brief ischemia on mitochondrial function in intact myocardium, rather than in isolated mitochondria. Methods: The mitochondrial response was characterized by the mean response time (tmito) of cardiac mitochondrial O2 consumption to steps in heart rate. Isolated isovolumic rabbit hearts were perfused at 28°C with a constant flow of Tyrode solution containing 11 mM glucose. O2 consumption and tmito were determined before ischemia and after 25 min of no-flow global ischemia during which hearts were either paced (I+P, n = 8) or unpaced (I–P, n = 8). A non-ischemic control group (n = 8) was also examined. Results: At 20 min reperfusion, developed left ventricular pressure (DLVP) after I+P was decreased to 47±3% (mean±s.e.m.; P<0.05) of control DLVP without significant changes in venous creatine kinase efflux, indicating contractile stunning. In contrast, complete contractile recovery was observed after I–P. Before ischemia, tmito was 11.2±0.6 and 14.9±0.7 s for heart rate steps from 60 to 70 and from 60 to 120 beats/min, respectively. The tmito was lower (P<0.05) for the corresponding downward steps (10.5±0.6 and 12.4±0.6 s, respectively). An increase (P<0.05) in tmito was observed in the course of the experiment for upward (1.2±0.3 s) and downward steps (1.4±0.3 s), but the change was similar after ischemia to that in time-matched controls (P >0.05, both for I–P and I+P vs. control). Oxygen consumption, compared at fixed levels of the ratexpressure product, was unchanged after ischemia (P >0.05, for both I–P and I+P vs. controls), suggesting undiminished efficiency of mitochondrial ATP production. Conclusions: Twenty-five minutes ischemia does not affect mitochondrial function in rabbit hearts at 28°C, even when contractile stunning resulted.

KEYWORDS Ischemia; Stunning; Pacing; Mitochondrial function, 28°C; Rabbit, heart


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
It is well known that short periods of ischemia which cause no cell necrosis may still depress the contractile function of the heart to a considerable degree. This phenomenon has been called ‘contractile stunning of the heart’ [1, 2], and may result from Ca2+ overload and/or free radical generation (see [3]for review). Reduction of energy supply by the mitochondria was considered not to be the cause of the contractile dysfunction [3]. Indeed, mitochondria isolated from stunned myocardium show an undiminished maximal rate of oxidative phosphorylation [4]. However, extrapolation of results from isolated mitochondria to intact tissue might be misleading, for reasons including possible selectivity of the isolation procedure (most of the mitochondria are lost), the unavoidable disruption of the interface between mitochondria and cytosol [5]and because the regulation of mitochondrial O2 consumption through signals in the cytoplasm, including Ca2+, cannot be studied. Thus, the question whether stunning affects mitochondrial function in the heart in vivo remains to be answered.

Reasons for suspecting that mitochondrial function is affected during stunning are: (1) the Ca2+ overload which may be a cause of stunning may also induce the so-called permeability transition pore in mitochondria [6]which in turn might affect inner membrane permeability; (2) during reperfusion following ischemia the intramitochondrial Ca2+ concentration is elevated and it is known that Ca2+ regulates mitochondrial metabolism [7]; (3) mitochondrial creatine kinase activity, assayed in vitro, is already significantly diminished after very brief ischemia [8], perhaps by reactive oxygen species, and this enzyme is thought to play a key role in the regulation of mitochondrial ATP synthesis [5].

The purpose of the present study was to investigate whether brief ischemia affects the mitochondria by obtaining a direct estimate of the mitochondrial response in the isolated heart, rather than by studying isolated mitochondria. To this end we determined the mean response time of mitochondrial oxygen consumption to a step in cardiac work load, tmito [9], and cardiac O2 consumption in relation to contractile performance in isolated rabbit hearts at 28°C. The tmito is proportional to the potential depletion of high-energy phosphates, mainly of phosphocreatine (PCr), during rapid increases in ATP hydrolysis induced by steps in heart rate [10]. A large tmito is caused by low mitochondrial oxidative capacity and/or delay of energetic signals between sites of ATP hydrolysis (myofibrils and ion pumps) and the mitochondria [5, 10, 11]. In the Discussion (Section 4) we explain why a lower than physiological temperature was chosen. To dissociate ischemia from post-ischemic contractile dysfunction, we either continued electrical stimulation during the ischemic intervention, so that contractile stunning resulted, or we stopped electrical stimulation, resulting in fast contractile recovery after ischemia [12], and studied the effect of ischemia on tmito in both groups.

In the present study we found that brief ischemia at 28°C did not change the time course of the mitochondrial oxidative response to steps in work load or O2 consumption at fixed levels of cardiac contractile performance, even when ischemia resulted in contractile stunning.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Heart perfusion
New Zealand White rabbits of both sexes, weighing 2.9±0.4 kg (s.d.), were anesthetized with intramuscularly administered 10 mg/kg fluanisone and 0.3 mg/kg fentanyl citrate (Hypnorm, Janssen Pharmaceutica), supplemented with pentobarbital sodium (10 mg/kg intravenously). After opening the thorax and intravenous heparin injection (2500 IU), the aorta was cannulated in situ and perfusion started prior to excision of the heart. The hearts were perfused according to Langendorff at 28°C with Tyrode solution containing (in mM): 128.3 NaCl, 4.7 KCl, 1.36 CaCl2, 1.05 MgCl2, 20.2 NaHCO3, 0.42 NaH2PO4 and 11.0 glucose, gassed with 95% O2 and 5% CO2. Adenosine (10–5 M) was added to the perfusate to obtain maximal vasodilation, so that changes in coronary resistance cannot disturb our determination of the response time of O2 consumption (see below). The right atrium was closed by ligation of the caval veins to ensure that coronary venous effluent left the heart via the right ventricle and the pulmonary artery. The Thebesian venous effluent was drained from the left ventricular lumen by a cannula pierced through the apex. To measure contractile performance, a water-filled latex balloon was inserted into the left ventricle and connected to a Statham P23 Db pressure transducer (Statham Instruments, Oxnard, CA, USA). The atrioventricular node was destroyed by crushing, resulting in a spontaneous heart rate below 60 beats/min. Hearts were then electrically stimulated via two electrodes on the right ventricle. Details of the preparation have been described previously [9, 13]. The investigation conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985).

2.2 Experimental procedures
End-diastolic pressure was set at 5 mmHg by inflating the LV balloon. The hearts were pump-perfused at constant flow throughout the experiment after setting pump speed to obtain an initial perfusion pressure of 80 mmHg. Coronary perfusion pressure was measured just above the aortic cannula with a second Statham P23 Db pressure transducer. Coronary O2 tensions in arterial inflow and venous outflow were continuously measured with two Clark-type electrodes, which were calibrated before and after the experiment. Oxygen concentrations were calculated by multiplying the O2 tension with the O2 solubility of 1.53 µmol O2.liter Tyrode–1·mmHg–1 at 28°C. Oxygen uptake was calculated by multiplying the arterial-to-venous O2 concentration difference by perfusate flow. Data were registered on a multichannel recorder and simultaneously digitized on a personal computer.

2.3 Experimental protocol for response time experiments
Following 30 min of equilibration, the hearts were paced at a basal heart rate of 60 beats/min and a series of tests were executed over the next 30 min in fixed order:

1. Application of steps in heart rate: stepping up from 60 beats/min to 70, 80, 100 and 120 beats/min and back to 60 beats/min in each case; the time course of the venous O2 tension was measured to characterize the dynamic response of O2 uptake to a range of cardiac work load elevations.
2. Downward step in arterial O2 tension by ~6% and back to baseline, at 60 beats/min; from the venous response to these steps the transport time of O2 in the heart was calculated to correct the venous response to the heart rate steps (see test 1) for transport delay (see below).
3. Sudden start of constant arterial infusion of the intravascular indicator Evans Blue dye which was bound to albumin, at heart rate 60 beats/min; from the venous dye concentration response to this step the intravascular transport time was calculated, using indicator-dilution theory; this intervention was also necessary, in addition to test 2, for correction of the venous O2 response to heart rate steps for transport delay [9, 13].

Values determined during this period before ischemia will be referred to as ‘pre-intervention’. Subsequently, during the next 25 min different interventions were applied to 3 groups: in the first ischemic group flow was completely stopped for 25 min and pacing was also stopped (I–P hearts; n = 8); in the second ischemic group flow was completely stopped but pacing was continued at 60 beats/min (I+P hearts; n = 8); a non-ischemic control group (n = 8) was continuously perfused and paced at 60 beats/min. During this 25 min period, the volume of the balloon in the left ventricle was left unchanged.

Flow was gradually increased to the pre-ischemic flow rate within the first minute of reperfusion. Between 20 and 50 min of reperfusion the tests 1–3 described above were repeated, and values determined in this period will be referred to as ‘post-intervention’. In the control group measurements were taken at the same times as in I+P and I–P, and are also termed ‘post-intervention values’ for the post-intervention time point, although controls were merely continuously perfused. Because the recovery of mechanical function had not plateaued after 50 min of reperfusion, 4 of the 8 hearts in the I+P group were reperfused for an additional 50 min (total of 100 min reperfusion time) to follow contractile recovery.

2.4 Calculation of the mean response time of mitochondrial oxygen consumption
The speed at which mitochondrial O2 consumption and ATP production adapt to a change in cardiac ATP consumption is characterized by the mean response time, tmito [9]. The venous response time (tv) is determined from the time course of the venous O2 tension to a step in heart rate. Subsequently, the mean transport time necessary for diffusion and vascular transport of O2 between the mitochondria and the O2 electrode, ttransport, is subtracted from tv [9]:


Formula 1

(1)

Thus, the true response time of oxidative phosphorylation at the level of the mitochondria (tmito) is obtained. The ttransport can be calculated from the venous response to small step changes in arterial O2 tension, provided that these do not cause a change in O2 consumption. The transit time of the intravascular indicator (see test 3 above) is also taken into account in this calculation [9, 13].

The tmito obtained from Eq. (1)Formula was further corrected for the deviation of the contractile performance from an ideal step change during the steps in heart rate. The ratexpressure product of heart rate (1/period between beats) and systolic pressure (RPP) is used as an index of metabolic demand on a beat-by-beat basis [11, 13]. Immediately after an upward step in heart rate a fast increase in systolic pressure is found, followed by a slow secondary decrease before a stable level of systolic pressure is reached. Thus, RPP reaches a steady state after an initial overshoot [11, 12]. The mean response time for RPP (tRPP) is defined as:


Formula 2

(2)
analogous to the definition of tmito [13]. The tRPP tends to be negative because {Delta}RPP(t), the change in RPP from the level before the heart rate step, is usually larger immediately after the heart rate step than {Delta}RPP({infty}), the change in RPP between the steady states before and after the step in heart rate. As described previously, we also apply a small correction for the change (termed ‘initial deflection’) in venous O2 tension caused by the fast, transitory increase in venous outflow immediately after the step in heart rate [9, 13]. The tmito given in this paper has been corrected for both tRPP and the initial deflection, so that tmito may be considered to be the true response time of mitochondrial O2 consumption to rapid changes in cardiac contractile performance.

2.5 Myocardial economy
To investigate myocardial economy, the O2 consumption at fixed RPP levels (6, 8, 10 and 12x103 mmHg/min) was estimated using a linear regression model for interpolation. For the I+P group only the 6x103 mmHg/min RPP level was estimated, because the depression of contractile function resulted in lack of overlap between pre- and post-intervention RPP values. Even some extrapolation was necessary for I+P (see Section 3), which is justified because extrapolation was over a very small range and the relation between O2 consumption and RPP is linear ([14], and present study).

2.6 Experimental protocol for creatine kinase leakage experiments
In 10 additional experiments it was examined whether myocardial necrosis occurs during our ischemia protocols. To this end, creatine kinase activity was determined in samples [15]of coronary venous effluent collected regularly during perfusion. This was done for control (n = 4), I+P (n = 4) and I–P (n = 2) groups. These groups underwent the same perfusion and pacing conditions as described above, except that tests 2 and 3 (steps in arterial O2 tension and intravascular indicator) were omitted. All 4 control hearts in the creatine kinase protocol were followed equally long as the 4 I+P hearts from the response time protocol with 100 min reperfusion (see above) to serve as non-ischemic controls for the latter.

2.7 Statistics
Data are presented as mean±s.e.m., except where indicated otherwise. Two-way analysis of variance (ANOVA) for repeated measurements was used to analyze the effect of interventions on diastolic pressure and ratexpressure product over the course of reperfusion, and to analyze changes in response times, O2 consumption levels at fixed RPP levels and changes in various other variables between pre-intervention and post-intervention measurements, before and after ischemia or at time-matched measurement points [16]. One-way ANOVA was performed for comparison among groups of coronary flow, wet and dry weight of the hearts, total creatine kinase release, and changes in perfusion pressure. If a significant overall effect (P<0.05) was found, the ANOVA was followed by multiple planned comparisons, using Bonferroni correction [16].


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Response time experiments
No differences in coronary flow (overall mean of all 3 groups: 6.8±0.3 ml·min–1·g–1 wet weight), wet (11.0±1.2 g) and dry weight (1.7±0.2 g) of the hearts were observed among the 3 groups (P >0.05). Perfusion pressure did not change during the course of the experiment in any of the 3 groups (P >0.05).

During the 25 min intervention period, left ventricular diastolic pressure remained constant for all 3 groups, and an increase (P<0.001, vs control) was only observed transitorily around 5 min reperfusion for the I+P group (Fig. 1). Heart rate during ischemia for the unpaced I–P group decreased from an average of 15 beats/min for the first 5 min, to 7 beats/min for 5–10 min, to 2 beats/min for 10–15 min, and after 15 min of ischemia no heart beat was discernible; in contrast, in the paced hearts contractile activity continued for the whole ischemic period. The RPP at every time point during ischemia was always lower for the I–P group than for the I+P group (P<0.001). As a consequence, the time-averaged RPP during 25 min ischemia was 7 times larger (P<0.001) for the I+P group (874±57 mmHg/min) than for the I–P group (128±37 mmHg/min). This indicates a much larger energy turnover during ischemia with pacing than without pacing.


Figure 1
View larger version (18K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 1 The left ventricular end-diastolic pressure (Pdia) and the ratexpressure product (RPP, normalized to 100% for the value at t = 0 min) as function of time for control hearts, for hearts subjected to ischemia without pacing (I–P) and for ischemia with pacing (I+P). The pace rate was 60 beats/min, except during flow stop in I–P. The 25 min ischemic period, given by the bar, starts at t = 0 min. Values were read every 10 min before t = 0, every min during first 5 min of ischemia, and then every 5 min during ischemia, at first min of reperfusion, and every 5 min for first 20 min reperfusion and then every 10 min for remaining 30 min of reperfusion. Values are mean±s.e.m. (n = 8 hearts for each group). *Difference (P<0.05) for I+P vs. I–P and at the same time also for I+P vs. control. #I–P differs (P<0.05) from control group.

 
At 5 min of reperfusion the RPP for the I–P group was not different from control (P >0.05), demonstrating complete recovery of RPP by 5 min for the I–P group. Recovery was not complete for the I+P group by 50 min of reperfusion (P<0.001, vs control). The RPP for the I+P group was always smaller (P<0.001) during 50 min reperfusion than the RPP of the I–P group. Prolonged reperfusion of 4 I+P hearts showed that between 70 and 100 min of reperfusion RPP remained stable at 82±3% of the pre-ischemic RPP value, significantly smaller (P<0.001) than in 4 controls (from ‘Creatine kinase leakage protocol’, see Section 2.6above) where, at the same time point, RPP was 101±4% of the value at the pre-intervention measurement (data not shown). Thus, there is depression of cardiac contractility in the I+P group compared to control up to 100 min of reperfusion.

Following the small stepwise decrease in arterial O2 tension (by 6.4±1.4%) no change in O2 consumption was found, indicating that O2 supply is not limiting [13]. Table 1 shows the pre-intervention and post-intervention transport times of O2 between mitochondria and venous O2 electrode for the 3 groups. The ttransport increased slightly from pre- to post-intervention, but this increase was identical in ischemic and control groups (P >0.05).


View this table:
[in this window]
[in a new window]

  		Table 1 Mean transport time ttransport (mean±s.e.m., n = 8 for each group) derived from the arterial O2 concentration step

 
The pre-intervention tRPP was –0.4 and –3.1 s for steps from 60–70 and 60–140 beats/min, respectively, and the initial deflection time of the venous O2 tension was –0.06 s and –4.0 s, respectively, for these steps. No changes in tRPP and in the initial deflection were observed during the experiment for all 3 groups (P >0.05). The tmito (corrected for tRPP and initial deflection) as function of heart rate is presented in Fig. 2. The tmito was lower (P = 0.003) for downward steps than for the upward steps in heart rate in all 3 groups. The tmito increased (P<0.001) with heart rate, both in the downward and the upward step panels of Fig. 2, as also reported earlier [10]. A small increase (P = 0.002 for upward step, P = 0.013 for downward step) of tmito between pre- and post-intervention was found in all ischemic and control groups. This change in tmito during the experiment was the same for all heart rates (P >0.05). However, the change in tmito after ischemia was not different from the change in time-matched controls (P >0.05, both for I–P and I+P). We conclude that tmito increases gradually during the perfusion experiment, but that tmito is not specifically affected by the ischemia.


Figure 2
View larger version (19K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 2 The mitochondrial response time, tmito, as function of test heart rate for control hearts and for I–P (ischemia without pacing) and I+P (ischemia with pacing) hearts. Pre-intervention (open symbols) and post-intervention values (closed symbols) are given. The post-intervention value means after ischemia and reperfusion for the I–P and I+P groups, but after continuous perfusion for the time-matched control group. The tmito had been corrected for oxygen transport delay, for the contribution of the initial deflection and for the response time of ratexpressure product, tRPP. The tmito for upward and downward steps in heart rate are different (P<0.05) and are given separately. The heart was always paced up to the test heart rate from 60 beats/min, and then back again to 60 beats/min from the test heart rate. Mean±s.e.m. (n = 8 for each group) given. *P<0.05 vs. upward step.

 
No increase (P >0.05) in O2 consumption at fixed RPP levels was found between pre- and post-intervention measurements (Fig. 3), and no differences (P >0.05) among the 3 groups in the change in oxygen consumption at fixed RPP levels were observed. This indicates that myocardial economy is not changed after 25 min ischemia.


Figure 3
View larger version (14K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 3 Myocardial oxygen consumption as function of ratexpressure product (RPP) for control, ischemia without pacing (I–P) and ischemia with pacing (I+P) hearts. For each heart rate the RPP and O2 consumption values were averaged. Pre-intervention (open symbols) and post-intervention values (filled symbols) are shown; see Fig. 2 for details. Mean±s.e.m. (n = 8 each group) given. dw = dry weight.

 
3.2 Creatine kinase leakage experiments
During the separate experiments on venous creatine kinase leakage, a similar mechanical performance was found to that during the response time experiments: at 50 min of reperfusion the RPP for control, I–P and I+P was 104±4, 93±7 and 75±3%, respectively, of the pre-intervention value, whereas the corresponding mean values for the response time experiments were 98±3, 93±3 and 73±2%, respectively. The total cellular creatine kinase release was not significantly different (P >0.05) among the 3 groups (Fig. 4), indicating that 25 min ischemia, either with or without pacing, did not result in cell necrosis. On average 40±5 U/gdw creatine kinase was released, which represents less than 1% of total tissue content, given that rabbit heart contains about 5000–6000 U/gdw creatine kinase [17].


Figure 4
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Fig. 4 Total cellular creatine kinase release in control (n = 4), ischemia without pacing (I–P, n = 2) and ischemia with pacing (I+P, n = 4) groups. CK was determined at start of ischemia at t = 0, at 15 min (for control only), in the first minute after start of reperfusion at t = 25 min, and at 30, 40, 50, 60 and 75 min of reperfusion and CK release (perfusion flow times venous CK concentration) was numerically summed over the period from t = 0–75 min. Mean±s.e.m. given. No differences among ischemic and control groups were found (P >0.05). dw = dry weight.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
4.1 Mitochondrial function after brief ischemia
We found that the response time of mitochondrial O2 consumption to heart rate steps was not affected by a short period of ischemia, whether ischemia resulted in mechanical dysfunction or not. This finding, together with the unchanged economy of myocardial O2 consumption, indicates normal mitochondrial function in stunned intact myocardium at 28°C. Heat rate measurements at 20°C in right ventricular papillary muscles excised from rabbit hearts showed that the time course of the decay of recovery heat development after a series of contractions was not slower after 40 min anoxia resulting in stunning [18], suggesting that the dynamic response of oxidative phosphorylation was also not affected during stunning at 20°C. In studies on mitochondria isolated from stunned hearts an undiminished state 3 oxidation rate had been observed [4, 19]. Indeed, intact mitochondrial function helps explain why stunned hearts can still show high and undiminished levels of phosphocreatine [20]and high contractile reserve revealed by strong inotropic stimulation [21].

After ischemic insults leading to diastolic contracture, an increased O2 consumption relative to work load has been found [22], and the regulation of oxidative phosphorylation was shifted from a normal regime, not limited by ADP and inorganic phosphate (Pi), to limitation by ADP and Pi [23]. Both types of ischemic insults applied in the present study were relatively mild, in the sense that diastolic contracture during ischemia was not observed. This may explain why in the present study O2 consumption was not increased if examined at a certain fixed contractile work load. The relative mildness of the ischemic insult may have prevented the previously observed changes in mitochondrial inner membrane permeability after 30 min ischemia and reperfusion [6]. Effects of inner membrane permeability transitions should be further investigated. Alterations in mitochondrial function and O2 consumption relative to work load are clearly not a prerequisite for stunning, despite the reported correlation between in vitro mitochondrial creatine kinase activity and contractile dysfunction after brief ischemia [8].

4.2 Methodological considerations
The response time of O2 consumption can only be determined in the isolated heart. The adequacy of O2 supply in the isolated saline-perfused heart at 37°C is under debate [24–26]. Improving the oxygen-carrying capacity of the perfusate could ameliorate possible inadequate O2 supply. In about 20 pilot experiments we examined the stability of isolated heart preparations at 37°C using perfusate containing intermolecularly cross-linked hemoglobin to improve O2 supply. Unfortunately, perfusion pressure increased steadily during cell-free hemoglobin perfusion and we were unable to obtain a stable preparation for longer than 30 min. We have shown that the isolated rabbit heart preparation perfused with erythrocyte suspensions is also not sufficiently stable [27]. Therefore, we chose to perform this study at 28°C, because adequacy of oxygenation at this temperature has been shown [13]. Adequacy of oxygenation was tested in all hearts in the present study by applying a small reduction of the arterial O2 concentration [26], which showed no limitation of O2 consumption by supply. The regulation of mitochondrial oxidative function might depend on the temperature. For example, tmito increases with increasing heart rate at 28°C ([10], and present study), whereas this was not observed at 37°C [11]. The response time increased by 110% per 10°C decrease in temperature [13]. Extrapolations to 37°C should therefore be made with caution, and predictions derived from the present study require corroboration by experiments at 37°C.

4.3 Summary
We have shown that a 25 min period of ischemia at 28°C followed by reperfusion caused no change in mitochondrial function in the heart. Even if cardiac contractile function was depressed upon reperfusion, as a result of continuous pacing of the heart during the ischemia, this was not associated with changes in mitochondrial function. Mitochondrial function appears remarkably resistant to deterioration during ischemia and reperfusion and may be affected at a later stage of ischemia than contractile function.

Time for primary review 28 days.


   Acknowledgements
 
Supported by the Netherlands Heart Foundation (grant no. 91.120) and an Established Investigator grant to J.H.G.M. van B.


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

  1. Braunwald E, Kloner R.A. The stunned myocardium: Prolonged, postischemic ventricular dysfunction. Circulation (1982) 66:1146–1149.[Abstract/Free Full Text]
  2. Heyndrickx G.R, Millard R.W, McRitchie R.J, Maroko P.R, Vatner S.F. Regional myocardial functional and electrophysiological alterations after brief coronary artery occlusion in conscious dogs. J Clin Invest (1975) 56:978–985.[Web of Science][Medline]
  3. Bolli R. Mechanism of myocardial stunning. Circulation (1990) 82:723–738.[Abstract/Free Full Text]
  4. Flameng W, Andres J, Ferdinande P, Mattheussen M, Van Belle H. Mitochondrial function in myocardial stunning. J Mol Cell Cardiol (1991) 23:1–11.[Web of Science][Medline]
  5. Saks V.A, Khuchua Z.A, Vasilyeva E.V, Belikova O.Y, Kuznetsov A.V. Metabolic compartmentation and substrate channelling in muscle cells. Mol Cell Biochem (1994) 133/134:155–192.
  6. Halestrap AP. Interactions between oxidative stress and calcium overload on mitochondrial function. In: Darley-Usmar V, Schapira AHV, editors. Mitochondria: DNA, protein and disease. London: Portland Press Research Monographs, 1994:113–142.
  7. McCormack J.G, Halestrap A.P, Denton R.M. Role of calcium ions in regulation of mammalian intramitochondrial metabolism. Physiol Rev (1990) 70:391–425.[Free Full Text]
  8. Bittl J.A, Weisfeldt M.L, Jacobus W.E. Creatine kinase of heart mitochondria. The progressive loss of enzyme activity during in vivo ischemia and its correlation to depressed myocardial function. J Biol Chem (1985) 260:208–214.[Abstract/Free Full Text]
  9. Van Beek J.H.G.M, Westerhof N. Response time of cardiac mitochondrial oxygen consumption to heart rate steps. Am J Physiol (1991) 260:H613–H625.[Web of Science][Medline]
  10. Eijgelshoven M.H.J, Hak J.B, Van Beek J.H.G.M, Westerhof N. Adaptation speed of cardiac mitochondrial oxygen consumption decreases with higher heart rate. Am J Physiol (1993) 265:H1893–H1898.[Web of Science][Medline]
  11. Eijgelshoven M.H.J, Van Beek J.H.G.M, Mottet I, Nederhoff M, Van Echteld C.J.A, Westerhof N. Cardiac high-energy phosphates adapt faster than oxygen consumption to changes in heart rate. Circ Res (1994) 75:751–759.[Abstract/Free Full Text]
  12. Dietrich D.L.L, Elzinga G. Energy demand, supply, and utilization in hypoxia, and force recovery after reoxygenation in rabbit heart muscle. Circ Res (1990) 67:1089–1096.[Abstract/Free Full Text]
  13. Hak J.B, Van Beek J.H.G.M, Van Wijhe M.H, Westerhof N. Influence of temperature on the response time of mitochondrial oxygen consumption in isolated rabbit heart. J Physiol (1992) 447:17–31.[Abstract/Free Full Text]
  14. Kobayashi K, Neely J.R. Control of maximum rate of glycolysis in rat cardiac muscle. Circ Res (1979) 44:166–175.[Free Full Text]
  15. Szasz G, Gruber W, Bernt E. Creatine kinase in serum. I. Determination of optimum reaction conditions. Clin Chem (1976) 22:650–656.[Abstract/Free Full Text]
  16. Snedecor GW, Cochran WG. Statistical methods. 8th ed. Ames, Iowa: Iowa State University Press, 1989.
  17. Janier M.F, Vanoverschelde J.L, Bergmann S.R. Ischemic preconditioning stimulates anaerobic glycolysis in the isolated rabbit heart. Am J Physiol (1994) 267:H1353–H1360.[Web of Science][Medline]
  18. Zuurbier C.J, Mast F, Elzinga G, Van Beek J.H.G.M. Mitochondrial function is not decreased in stunned papillary muscle at 20°C. J Moll Cell Cardiol (1997) 29:347–355.[CrossRef][Web of Science][Medline]
  19. Piper H.M, Sezer O, Schleyer M, Schwartz P, Hütter J.F, Spieckermann P.G. Development of ischemia-induced damage in defined mitochondrial subpopulations. J Mol Cell Cardiol (1985) 17:885–896.[CrossRef][Web of Science][Medline]
  20. Ambrosio G, Jacobus W.E, Bergman C.A, Weisman H.F, Becker L.C. Preserved high energy phosphate metabolic reserve in globally ‘stunned’ hearts despite reduction of basal ATP content and contractility. J Mol Cell Cardiol (1987) 19:953–964.[Web of Science][Medline]
  21. Ito B.R, Tate H, Kobayashi M, Schaper W. Reversibly injured, postischemic canine myocardium retains normal contractile reserve. Circ Res (1987) 61:834–846.[Abstract/Free Full Text]
  22. Zimmer SD, Bache RJ. Metabolic correlates of reversibly injured myocardium. In: Kloner RA, Przyklenk K, editors. Stunned myocardium, properties, mechanisms, and clinical manifestations. New York: Marcel Dekker, Inc. 1993:41–70.
  23. Zimmer S.D, Ugurbil K, Michurski S.P, Mohanakrishnan P, Ulstadt V, Foker J.E. From AHL. Alterations in oxidative function and respiratory regulation in the post-ischemic myocardium. J Biol Chem (1989) 264:12402–12411.[Abstract/Free Full Text]
  24. Kammermeier H. Isolated (Langendorff) hearts perfused with an aqueous buffer (should) have excess oxygen availability [Invited comment]. Basic Res Cardiol (1994) 89:545–548.[CrossRef][Web of Science][Medline]
  25. Poizat C, Keriel C, Cuchet C. Is oxygen supply sufficient to induce normoxic conditions in isolated rat heart? Basic Res Cardiol (1994) 89:535–544.[CrossRef][Web of Science][Medline]
  26. Van Beek J.H.G.M, Bouma P, Westerhof N. Oxygen uptake in saline-perfused rabbit heart is decreased to a similar extent during reductions in flow and in arterial oxygen concentration. Pflügers Arch (1989) 414:82–88.[CrossRef][Web of Science][Medline]
  27. Hak JB, Biessels PTM, Van Beek JHGM, Bakker JC, Westerhof N. Function of isolated rabbit hearts perfused with erythrocyte suspension is not stable but improvement may be feasible with hemoglobin solution. In: Vaupel P, Zander R, Bruley DF, editors. Oxygen transport to tissue XV. New York: Plenum Press, 1994:291–297.

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
W. D. Gao, T. Dai, and D. Nyhan
Increased cross-bridge cycling rate in stunned myocardium
Am J Physiol Heart Circ Physiol, February 1, 2006; 290(2): H886 - H893.
[Abstract] [Full Text] [PDF]

Home page
 Cardiovasc ResHome page
S. A.I.P. Trines, C. J. Slager, T. A.M. Onderwater, J. M.J. Lamers, P. D. Verdouw, and R. Krams
Oxygen wastage of stunned myocardium in vivo is due to an increased oxygen cost of contractility and a decreased myofibrillar efficiency
Cardiovasc Res, July 1, 2001; 51(1): 122 - 130.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. H. G. M. van Beek, M. H. van Wijhe, M. H. J. Eijgelshoven, and J. B. Hak
Dynamic adaptation of cardiac oxidative phosphorylation is not mediated by simple feedback control
Am J Physiol Heart Circ Physiol, October 1, 1999; 277(4): H1375 - H1384.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow E-letters: Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when E-letters are posted
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Right arrow Disclaimer
Google Scholar
Right arrow Articles by Zuurbier, C. J
Right arrow Articles by van Beek, J. H.G.M
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Zuurbier, C. J
Right arrow Articles by van Beek, J. H.G.M
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?