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1 Department of Pharmacology2 Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, China
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(Received 26 October 2004;
accepted after revision 11 February 2005; first published online 11 February 2005)
Corresponding author W.-J. Zang: Department of Pharmacology, School of Medicine, Xi'an Jiaotong University, Xi'an, 710061, People's Republic of China. Email: zwj{at}mail.xjtu.edu.cn
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Introduction |
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Ischaemia inhibits aerobic oxidation in myocytes, and the energy supplied by anaerobic glycolysis is insufficient to meet the energy demands of the tissue. This may result in metabolic abnormalities, such as depletion of ATP, lactate accumulation and acidification of the extracellular environment (Neely & Feuvray, 1990). These internal environmental conditions can be induced by an ischaemia-mimetic solution. The ischaemia-mimetic formulations used in cardiac tissue and single cardiomyocyte preparations (Lukas & Ferrier, 1986; Esumi et al. 1991; Cordeiro et al. 1994, 1995; Seki & MacLeod, 1995; Maddaford et al. 1999; Narayan et al. 2001), which are based on Tyrode solution, can produce hypoxia, glucose deficiency, acidosis, lactate accumulation and hyperosmosis, thus allowing researchers to mimic the pathophysiological state of ischaemia. These ischaemia-mimetic formulations have diverse ingredients and dosages. For example, O2 can be driven out by adding N2 (Lukas & Ferrier, 1986) or Na2S2O4 (Seki & MacLeod, 1995) to simulate hypoxia, metabolic inhibitors are used to maintain normal pH values (Seki & MacLeod, 1995), and with yet other formulations severe acidity can be maintained (Maddaford et al. 1999); some formulae include lactate (Cordeiro et al. 1995), others do not (Narayan et al. 2001). Furthermore, in some studies the concentration of sodium ions might be strictly controlled to maintain iso-osmia with normal Tyrode solution (e.g. Esumi et al. 1991), whereas in others this factor may not be considered (e.g. Cordeiro et al. 1994). In other words, differences in the key ingredients of ischaemia-mimetic solutions can be used to determine the conditions that induce cardiac ischaemic injury. This study compared the effects of the following key environmental conditions that can be induced by ischaemia-mimetic solutions: hypoxia, glucose deficiency, acidosis, lactate accumulation and hyperosmosis.
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Methods |
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Adult Sprague–Dawley rats of both sexes, supplied by the Experimental Animal Center of Xi'an Jiaotong University, China, and weighing 250–300 g were used in accordance with the Guidelines on the Care and Use of Laboratory Animals issued by the Chinese Council on Animal Research and the Guidelines of Animal Care. The study was approved by the ethical committee of Xi'an Jiaotong University. Ventricular myocytes were isolated enzymatically from rats using a conventional method (Zang et al. 1993), as follows. Heparinized rats were anaesthetized with sodium pentobarbitone (17 mg kg–1, I.P.; Zang et al. 2003). The heart was rapidly excised, placed in ice-cold Ca2+-free modified Tyrode solution (in mmol l–1: NaCl, 143; KCl, 5.4; MgCl2, 0.5; NaH2PO4, 0.33; Hepes, 5.0; and glucose, 5.0; pH titrated to 7.35 with NaOH), cannulated and then perfused retrogradely with Ca2+-free Tyrode solution via the aorta on a Langendorf perfusion apparatus for about 5 min until spontaneous contractions ceased. The heart was then perfused with Ca2+-free Tyrode solution containing 0.7 mg ml–1 collagenase (type I, Sigma, St Louis, MO, USA) and 1 mg ml–1 bovine serum albumin (Sigma) for 20 min. Finally, the enzymes were washed out with a high-K+, low-Cl– solution (KB solution, in mmol l–1: KCl, 25; taurine, 20; L-glutamic acid, 70; KH2PO4, 10; MgCl2, 3; EGTA, 0.5; glucose, 10; and Hepes, 10; pH 7.35) for 5 min. All of the solutions used during perfusion were bubbled with 100% O2 and maintained at 37°C. Following perfusion, the ventricle was placed in a beaker filled with KB solution and then minced. The ventricular cells were dispersed by shaking the beaker gently and the undigested tissue was removed by filtration through a 250 µm nylon mesh. The cells were kept in Tyrode solution (in mmol l–1: NaCl, 136.9; KCl, 5.4; MgCl2, 0.5; NaH2PO4, 0.33; Hepes, 5.0; glucose, 10.0; and CaCl2, 1.8; pH 7.4; bubbled with 100% O2) at room temperature for at least 1 h before use.
Protocols
The ischaemia-mimetic behaviour of single cardio-myocytes was simulated using a modified ischaemia-mimetic solution (Cordeiro et al. 1994; in mmol l–1: NaCl, 135; KCl, 5.4; MgCl2, 0.5; NaH2PO4, 0.33; Hepes, 5.0; CaCl2, 1.8; and Na+-lactate, 20; pH 6.80; bubbled with 100% N2 for >45 min before the experiment was started; this reduced the oxygen tension by 75%). Coverslips to which cardiomyocytes were adhered were placed in a flow-through (1 ml min–1, 25°C) perfusion chamber (the surface of which was surrounded with N2 to prevent reoxygenation of the bath ischaemia-mimetic solution) that was positioned on the stage of an inverted microscope and the chamber was continuously superfused with Tyrode solution. Cells were stimulated electrically with an external electrical field at 5 ms and 0.5 Hz by two Pt electrodes connected to a MyoPacer field stimulator (IonOptix Corporation, Milton, MA, USA). The polarity of the stimulating electrodes was reversed periodically to avoid the potential build up of electrolysis byproducts (Wold et al. 2001). Fields of myocytes were chosen at random. Cells were perfused with the ischaemia-mimetic solution for 15 min and then with Tyrode solution to simulate reperfusion.
The experiments were divided into six groups according to the key factors contained in the ischaemia-mimetic solution. The modified ischaemia-mimetic solution described above was designated as solution A (control solution). One or other of the key factors for inducing hypoxia, glucose deficiency, acidosis, lactate accumulation or hyperosmosis was then removed from the formula for solution A to produce the remaining five solutions: solution B (hypoxia-free solution), for which solution A was bubbled with 100% O2; solution C (acidosis-free solution), for which the pH of solution A was titrated to 7.40; solution D (glucose-containing solution), for which 10 mmol l–1 glucose was added to solution A; solution E (lactate-free solution), for which Na+-lactate was removed from solution A; and solution F (isosmotic solution), for which the amount of NaCl in solution A was lowered to 117 mmol l–1. The characteristics of the solutions were therefore as follows [solution (modification)]: A (control); B (100% O2); C (pH 7.4); D (+10 mM glucose); E (– Na+-lactate); and F (117 mM NaCl).
The effects of ischaemia-mimetic solutions containing the following concentrations of lactate on the contractility of the cardiomyocytes were then determined: 1.25, 2.5, 5, 10 and 20 mmol l–1.
Myocyte shortening and lengthening measurements
Cardiomyocytes were viewed with the aid of an inverted microscope (Olympus X-70, Olympus Optical, Tokyo, Japan) and imaged using an IonOptix MyoCam camera (IonOptix Corporation, Milton, MA, USA; Zang et al. 2005). Myocyte motion was measured using a video-based edge-detection system (IonOptix Corporation). Changes in cell length between shortening and lengthening were quantified using the following parameters: peak shortening (PS, representing the amplitude of myocyte contraction) and cell length (resting cell length). Data were analysed using IonOptix software.
Statistical analysis
Results are expressed as means ± S.E.M. Student's t test and one-factor analysis of variance were used to evaluate statistical significance, as appropriate. A probability value of P < 0.05 was considered indicative of statistical significance.
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Results |
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Cells were first superfused with Tyrode solution for 6 min to establish a steady state. Ischaemia was then simulated by exposing myocytes to the ischaemia-mimetic solution for 15 min.
Figure 1 shows a rapid-scan image of myocyte contraction illustrating that the PS of myocytes decreased during ischaemia and then recovered to exceed the pre-ischaemia value during the early phase of reperfusion. PS then decreased again during the later stage of reperfusion. The PS at 13 equidistant time points, each separated by 3 min, is shown in Fig. 1B. Points 4–8 occurred in the ischaemic phase. The maximum inhibitory effect occurred after 3 min of exposure to solution A (point 4), at which point PS had decreased to 10.46 ± 5.40% of the pre-ischaemia value (Fig. 1A). The inhibitory effect of solution A on PS diminished during the exposure (point 5, 27.28 ± 11.45%; point 6, 39.84 ± 15.11%; point 7, 42.99 ± 13.98%; point 8, 46.97 ± 13.81%; all higher than at point 4, P < 0.05). During the early period of reperfusion, PS increased rapidly to 139.76 ± 27.53% of the pre-ischaemia value (P < 0.05, point 9 versus point 3). Thereafter, the PS decreased to 28.25 ± 11.14% (point 11), 9.52 ±4.57% (point 12) and 7.67 ± 3.49% (point 13) of the pre-ischaemia value, which indicates that the contractility of the cardiomyocytes was severely reduced by the reperfusion.
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Figure 2 compares the effects of solutions A, B, D, and F on PS at the same time points as in Fig. 1B. The effects of these solutions on PS were similar (P > 0.05; Fig. 2A, n = 6). Figure 2B shows that the maximum ischaemic effects of solutions A (control solution) and C (acidosis-free solution) were similar, but the PS for solution C was higher than that for solution A during ischaemia and reperfusion (P < 0.05, n = 6, points 5–8 and 11–13). Furthermore, it is shown that the maximum ischaemic effect of solution E occurred late, and that PS decreased to 59.86 ± 12.52% of the pre-ischaemia value (n = 6). The PS of solution E was stronger than that of solution A at points 4 and 11–13 (P < 0.05). PS exhibited no rebound during the reperfusion period (P > 0.05 versus pre-ischaemia). Thus, the effect of solution E (lactate-free solution) was significantly weaker than that of solution A (control solution).
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Figure 3 shows that ventricular cells went into contracture during reperfusion following superfusion with solutions A, B, C, D and F, but not with solution E (P < 0.05, n = 6, versus pre-ischaemia). During the later phase of reperfusion, solutions A, B, D and F had similar effects on cell length (59.64 ± 8.91%, 52.56 ± 10.76%, 44.94 ± 9.74% and 64.24 ± 5.51% of the pre-ischaemia value, respectively; P > 0.05). However, cell length shortened to 77.8 ± 2.45% of the pre-ischaemia value in solution C, indicating that the contracture-inducing effect of solution C was weaker than that of solution A (P < 0.05). Solution E had no statistically significant effect on cell length (P > 0.05 versus pre-ischaemia). The effects were similar with those on PS.
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Figure 4A shows, on a slow time base, the effect of increasing the concentration of lactate on cell contraction. Reducing the lactate concentration decreased the inhibition exerted by the ischaemia-mimetic solution, and increased the recovery of cell length and PS during the later stage of reperfusion.
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‘Recovery of PS’ on Fig. 4B compares the recovery of cardiomyocyte PS during the later stage of reperfusion. During this stage, the recovery of cardiomyocyte PS in the presence of lactate concentrations of 1.25, 2.5, 5, 10 and 20 mmol l–1 was 94.00 ± 1.89, 86.68 ± 3.61, 67.00 ± 5.40, 32.14 ± 2.44 and 11.04 ± 5.21% of the pre-ischaemia value (control), respectively (n = 6). The effects of 1.25 and 2.5 mmol l–1 lactate were similar (P > 0.05), whereas those of 5, 10 and 20 mmol l–1 lactate were individually significantly different from those of the other four lactate concentrations (P < 0.05).
‘Recovery of cell length’ in Fig. 4B compares the recovery of cell length of the cardiomyocytes during the later stage of reperfusion. During this stage, cell length at lactate concentrations of 1.25, 2.5, 5, 10 and 20 mmol l–1 recovered to 98.69 ± 0.43, 97.36 ± 0.62, 91.89 ± 1.23, 80.74 ± 3.56 and 59.72 ± 8.93% of the pre-ischaemia value (control), respectively (n = 6). The effects of 1.25, 2.5 and 5 mmol l–1 lactate were similar (P > 0.05), as were those of 5 and 10 mmol l–1 lactate (P > 0.05). However, the effects of 20 mmol l–1 lactate were significantly different from those of the other four lactate concentrations (P < 0.05).
These results indicate that lactate had a concentration-dependent effect on cardiomyocyte contractility.
The effects of all of the solutions on maximal/minimal shortening rate (±dl/dt) values were very similar to those on PS (data not shown).
The results show that: (1) the solutions containing lactate severely reduced the contractility of the cardiomyocytes, but their effect on cell contraction was not significantly different; (2) the effect of the solution without acidosis was weaker than that of the conventional ischaemia-mimetic solution; (3) the solution lacking lactate produced the least depression of cell contractility; and (4) lactate has a concentration-dependent inhibitory effect on cardiomyocyte contractility.
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Discussion |
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The effects of different key factors of the ischaemia-mimetic solution
Many types of ischaemia-mimetic Tyrode solution are available, which may or may not include a hyperkalaemic factor (Stambaugh et al. 1997; Dougherty et al. 1998; Narayan et al. 2001; Yan et al. 2001). We tested a hyperkalaemic ischaemia-mimetic solution on several cells in a preliminary experiment and found the effects to be similar to those of a normokalaemic ischaemia-mimetic solution. Both solutions effectively and severely depressed mechanical function to a similar degree. We therefore chose to exclude this factor from further study.
Solution E, which was the only one in which lactate was omitted, had the smallest effect on the ischaemia-mimetic-induced inhibition of cell contractility and it induced little contracture during perfusion. This suggests that lactate is one of the main factors responsible for cardiac ischaemic injury and that it can severely reduce the contractility of cardiomyocytes, in agreement with the findings of Maddaford et al. (1999).
Of the other solutions (all of which contained lactate), the ischaemic effect of solution C was the weakest. This suggests that acidosis also has an impact on cardiomyocyte contractility, but the effect is weaker than that of lactate. Acidosis was excluded by superfusion with solution C, in which the extracellular H+ concentration was approximately 25% of that in solution A (pH 7.4 versus 6.8, based on the definition of pH value). This reduction in H+ concentration could reduce the acidosis-induced inhibition of contractility and improve the contractility of ischaemic myocytes. During reperfusion, the reduction in H+ concentration would also cause a reduction in Na+–Ca2+ exchange, leading to a reduced inflow of Ca2+. This would in turn result in a reduction in Ca2+ overload and cell damage.
There was little difference between the ischaemic effect of solution A and that of solutions B, D and F. This leads us to several hypotheses. First, hypoxia was absent from solution B, but this had little effect on the ischaemia relative to solution A, suggesting that normal aerobic respiration was not possible in myocytes during ischaemia even though O2 was supplied. A possible explanation for this is that the mitochondria were damaged or the energy source was insufficient. Second, the ischaemic effect of solution D (which excluded glucose deficiency) was also similar to that of solution A, suggesting that the ischaemic injury was not prevented by this particular nutrient substance. During ischaemia, glycogen became the primary substrate for anaerobic glycolysis as an energy supply for the cardiomyocytes (Vander Heide et al. 1996). However, the utilization of energy substrate was so limited that the added glucose appeared superfluous, which suggests that the energy utilization of myocytes was blocked even during the earlier stage of ischaemia. Third, solution F, in which the osmotic pressure was close to that of normal Tyrode solution, had a similar ischaemic effect to that of solution A, suggesting that hyperosmosis is not a prerequisite of ischaemic injury. These results indicate that hypoxia itself, glucose deficiency, and an increased osmotic pressure have little impact on the contractility of cardiomyocytes. Instead, acidosis and lactate accumulation, which are consequences of anaerobic glycolysis, are responsible for lethal ischaemic cardiomyocyte injury.
Impairment of the ischaemic myocardium by lactate
During myocardial ischaemia the lactate concentration increases to 5–10 mM (Lee & Allen, 1988), and this is regarded as an indicator of ischaemic injury (Marzouk et al. 2002). Lactate can damage the ischaemic myocardium through three possible mechanisms (Anderson et al. 1990; Maddaford et al. 1999). First, high concentrations of lactate may directly damage ischaemic myocytes. Second, the intracellular acidification due to the inflow of H+ to cardiomyocytes through the lactate–H+ cotransport pathway as a result of lactate accumulation (Poole-Wilson., 1978) might stimulate Na+–H+ exchange. When coupled to Na+–Ca2+ exchange, this would result in increased cytosolic free Ca2+. This would in turn lead to intracellular Ca2+ overload and damage the ischaemic myocardium. Third, lactate accumulation during ischaemia can increase intracellular osmolality by up to 170% of normal (Jennings et al. 1986), therefore causing mild cell swelling during ischaemia and severe cell swelling, further damaging myocytes, during reperfusion when the hypertonic ischaemic tissue is exposed to isotonic arterial blood or perfusate.
There are some contrasting results on lactate-induced myocardial impairment. For example, Geisbuhler & Rovette (1990) found that rat cardiomyocytes exposed to an ischaemia-mimetic solution including lactate did not die. However, their myocytes were not stimulated, whereas the cells should have contracted continuously in the physiological state, and the status between the quiescent and the beating myocytes was not identical (Maddaford et al. 1999). Vander Heide et al. (1996) used glucagons to deplete glycogen in order to reduce lactate accumulation during myocardial ischaemia, and found no relief from myocardial injury. They concluded that lactate was not the key factor underlying ischaemic myocardial injury. However, the depletion of glycogen (by glucagons) caused a reduction in both the energy reserve of the myocardium and lactate; the serious depletion of ischaemic myocardial energy (ATP) is thus also a key factor responsible for depressing heart contractility and inducing ischaemic contracture (Koretsune & Marban, 1990). Hence, it was revealed that lactate was not the only damaging factor. In contrast, our myocytes were motile throughout the experiment and we did not purposely exhaust the myocardial energy reserve. We found that lactate was the key factor responsible for ischaemic myocardial injury and that its effects were concentration dependent.
Our results show that desensitization occurred after the contractility of cardiomyocytes was inhibited to its nadir by the solutions containing lactate, and there was rebound during the earlier period of reperfusion. This represents the universal phenomenon of adaptation to a changing environment. One explanation for this desensitization of the inhibitory effect of lactate involves the timing of the inhibition of aerobic oxidation relative to the commencement of anaerobic glycolysis. Initially, when aerobic oxidation was already being inhibited, anaerobic glycolysis may not have properly commenced. During that time, the energy sources would have been insufficient to meet the energy demands of the tissue, such that the contractility of cardiomyocytes decreased to the nadir. Anaerobic glycolysis subsequently increased, beginning to supply energy and thus resulting in the observed partial recovery of contractility. The ischaemic contracture might be due to the energy reserve of the cardiomyocytes being depleted severely by the rebound when the function of mitochondria had not fully recovered and aerobic oxidation did not start to energize efficiently. This would lead to a depletion of ATP together, after reperfusion, with intracellular Ca2+ overload, resulting in contracture of the ischaemic myocytes.
Ischaemia-mimetic solutions lacking lactate are currently used in some laboratories. Our results demonstrate that such solutions cannot meet the pathophysiological demands of the cells. Our results also have clinical significance, since they suggest that depressing the lactate concentration in the ischaemic myocardium is of therapeutic benefit.
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