Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 281-284

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 281-284

NMR Study of Damage on Isolated Perfused Rat Heart Exposed to Ischemia and Hypoxia

LUO Xuechun , YAN Yongbin,   ZHANG Riqing, WANG Xiaoyin†,   FAN Lili ††

State Key Laboratory of Biomembrane and Membrane Biotechnology,  Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China;
†Institute of China-Japan Clinical Medicine, Beijing 100029, China;
††Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing 100050, China

Received: 2000-11-08

Code Number: ts01083

Abstract:   

Myocardial ischemia is the most common and primary cause of myocardium damage. Numerous conventional techniques and methods have been developed for ischemia and reperfusion studies. However, because of damage to the heart sample, most of these techniques can not be used to continuously monitor the full dynamic course of the myocardial metabolic pathway. The nuclear magnetic resonnance (NMR) surface coil technique, which overcomes the limitations of conventional instrumentation, can be used to quantitatively study every stage of the perfused heart (especially after perfusion stoppage) continuously, dynamically, and without damage under normal or designed physiological conditions at the molecular level. In this paper,    ³¹P -NMR was used to study the effects of ischemia and hypoxia on isolated perfused hearts. The results show that complete hypoxia caused more severe functional damage to the myocardial cells than complete ischemia.

Key  words:  ischemia; hypoxia; isolated perfused rat heart

Introduction   

Myocardial ischemia is the most common and primary cause of myocardium damage. A rapid restoration of the heart blood supply after injury is very important for retaining its function. However, after a certain period of time, resuming the blood supply may aggravate the damage to the cardiac muscle.  Therefore, the mechanisms of myocardial ischemia and reperfusion damage must be studied to provide important data to reduce or prevent reperfusion damage in clinical treatment of ischemic heart diseases.

Numerous conventional techniques and methods have been developed for ischemia and reperfusion studies. However, because of damage to the heart sample, most of these techniques can not be used to continuously monitor the full dynamic course of the myocardial metabolic pathway. The nuclear magnetic resonance (NMR) surface coil technique, which overcomes the limitations of conventional instrumentation, can be used to quantitatively study every stage of the perfused heart (especially after perfusion stoppage) continuously, dynamically and without any damage under normal or designed physiological conditions at the molecular level[1-3]. The NMR surface coil technique was used to measure ³¹P  spectra for different preparations at various time intervals. Since the myocardial ³¹P  spectra contains information about energy conversions, i.e., information about structures, locations, and concentrations of phosphorous-containing metabolic products (ATP, ADP, PCr, NAD, and Pi) and the cellular pH, the  ischemia/reperfusion mechanism can be studied from the view point of energy metabolism.

As demonstrated by Cave's experiment[4] , after pretreatment with a 5-min ischemia and then a 5-min reperfusion, an isolated rat heart can then sustain a 30-min ischemia. However, when the 30-min heart perfusion was replaced by a 25-min hypoxia perfusion, the protection due to the pretreatment was lost. The effect of hypoxia and oxygen restoration were studied here using NMR. This paper will emphasize the effects of ischemia and hypoxia on the isolated perfused heart.

1 Materials and Methods  

1.1 Isolated perfused rat hearts

Experimental animal: healthy male Wistar rat, body mass 270-330 grams.Perfusion solution: Krebs-Henseleit (K-H) buffer saturated with 95% O₂ -5% CO₂ (V/V), components (mmol/L, pH~7.4): NaCl 118, KCl 4.7, CaCl₂ 2.5, MgSO₄ ·7H₂O 1.2, NaHCO₃  24.88 , KH₂PO₄ 1.2 and glucouse 11. The hypoxia perfusion solution was saturated with 95% N2-5% CO₂ (V/V), which completely expels oxygen from the K-H buffer and maintains a constant pH.

1.2 NMR measurements

The ³¹P  surface radio field (RF) coil is a two-turn planar copper-coil (12 mm in diameter) connected to variable capacitors for fine tuning to the ³¹P  resonance frequency of  202.24  MHz and for matching of the coil impedance to that of the receiver. The excised hearts from male Wistar rats were quickly mounted through their aorta onto a Langendorff perfusion system and placed in a 20-mm-diameter polyethylene tube placed in the middle of the surface coil probe. Perfusate was administered at a pressure of 70 cm of water. The ³¹P  NMR spectra were recorded without proton decoupling at 202.458 MHz and with a spectral width of 8064 Hz using 4K data points. 600 free induction decays were accumulated with 0.246 s delay between pulses (90° pulse was 16 ms). The resulting time-domain data was smoothed by an exponential line broadening of 20 Hz before performing the Fourier transformation of the raw data. The peaks characteristic of Pi, PCr and the phosphate groups of adenosine were identified. The chemical shifts were referred to the resonance position of PCr.  

1.3 Experimental protocol

Six rats were used in the experiment and were randomly divided into two groups, Fig.1.

(1) Hypoxia group: K-H buffer equilibrated with oxygen for 20 min ® a 40-min hypoxia perfusion ® a 30-min reperfusion with oxygen.

(2) Ischemia group: K-H buffers are equilibrated with oxygen for 20 min ® a 40-min perfusion suspension ® a 30-min reperfusion with oxygen.

2 Results and Discussion  

Five minutes fast ³¹P  spectra were measured to monitor the rapid change of the 5 min ischemia or the 5 min hypoxia. There were 3 rats in each group in the experiments. The results showed that the ³¹P  spectra of all 3 rats in each group evolved in a consistent manner. Therefore, we only provide a typical ³¹P  spectrum for every stage of the heart perfusion from one of the rats. The assignments of the spectra were conducted as described earlier[5, 6].

Figure 2 shows the typical ³¹P  spectra for each stage in both the ischemia group and the hypoxia group. In the ischemia group, the perfusion was stopped after 20 min so the cardiac muscle could not obtain a continuous energy supply from the blood. The PCr and ATP levels in the myocardial cells dropped rapidly after the perfusion was stopped.  Twenty minutes after the perfusion was stopped, the PCr peaks were completely gone in all 3 rat hearts, while the ATP peaks was greatly reduced and the Pi peaks were significantly increased with the Pi chemical shift moved toward the upper field (PCr side, acidic). After resuming the perfusion for 10 min, the PCr peaks reappeared with considerable size in all 3 rat hearts, while the ATP and the Pi peaks moved toward their equilibrium positions. After another 20 min of reperfusion, the PCr and Pi peaks returned to their equilibrium levels and the Pi chemical shift moved back to its original position, indicating recovery of the cellular pH. There was only partial recovery of the ATP peaks.

In contrast, in the hypoxia group, the cardiac muscle had ample energy supply except for oxygen. Throughout the hypoxic period, the PCr and the ATP peaks continuously decreased while the Pi peaks increased in the myocardium of all 3 rats. However, the PCr peaks did not completely disappear as in the ischemia group. After reperfusion with restored oxygen, the Pi peaks increased significantly but did not return to their equilibrium level. The PCr and Pi peaks also partially recovered. No significant movement of the Pi chemical shift was observed in the experiment, indicating no substantial change of the pH value during the perfusion process.

The  hypoxia group had no accumulation of metabolic products in the intercellular plasma because of the rinsing effect of the hypoxic perfusion. Therefore, the pH value remained constant in the hypoxic period. The experimental results showed that since the intra-and intercellular pH values stayed constant in the myocardial hypoxia period, the hypoxia damage was not caused by a drastic change of the intra-and intercellular pH gradient as occurred with the ischemia damage. In the hypoxia perfusion, the continuous reduction of the PCr and ATP levels demonstrated the sustained effect of hypoxia damage on the energy metabolism.

Although the myocardial cells had ample energy supply during the hypoxia perfusion, the mitochondria pathway of energy generation by glucose oxidation and phosphoinsitide degradation was mostly inhibited so the energy (ATP) was mainly produced by glycolysis. This process generated large amounts of lactic acid and H⁺. The H⁺ accumulated inside the cell and created a transmembrane pH gradient. This gradient activated the Na⁺-H⁺ exchange protein and Na⁺-HCO₃^-

3 transportation to alleviate the acid poisoning. However, a substantial amount of Na⁺ was built up inside the cell while the H⁺ was expelled which activated the Na⁺-K⁺ ATPase and, consequently, used more energy to eliminate the Na⁺ on the myocardial membrane. The ATP degradation subsequently enhanced the glycolysis, generating more H⁺ and the cell fell into adverse cycles.  When Na⁺-K⁺ ATPase activity was inadequate to effectively eliminate the accumulated Na⁺, the increasing intracellular Na⁺ would reverse the Na⁺-Ca²⁺  transport through the myocardial membrane which lead to calcium overloading in the myocardial cell. In addition, the H⁺ concentration increased because of the Ca²⁺ -H⁺ interaction which further stimulated the Na⁺-H⁺ exchange. This process drastically elevated the intracellular calcium concentration which resulted in calcium overloading damage. Calcium overloading can cause severe damage to the cell function and structure.

In the ischemia group, the lack of myocardial blood lead to the accumulation of metabolic products in the intercellular plasma, causing a significant drop in the ATP supply and a considerable decrease in the intra-and intercellular pH. Although the decrease in pH indirectly enhanced the calcium influx, the ATP reduction and intercellular pH decrease inhibited the Na⁺-Ca²⁺  exchange. Thus, hypoxia caused more severe damage to the myocardial function than ischemia. Therefore, in the hypoxia perfusion process, hypoxic damage had sustained effects on the energy metabolism. In the 40 min  damage test, complete hypoxia perfusion caused more severe functional damage to the myocardial cells than complete ischemia perfusion. The experiments also demonstrated that the NMR surface coil technique can provide very valuable information in dynamic studies.

References

1. Buser P T, Wikman-Coffelt J, Wu S T, et al. Post ischemic recovery of mechanical performance and energy metabolism in the presence of left ventricular hypertrophy: A  ³¹P  MRS study. Circ Res, 1990, 66: 735-746.
2. Headrick J P. Ischemic preconditioning - bioenergetic and metabolic changes and the role of endogenous adenosine. J Mol Cell Cardiol, 1996, 28: 1227-1240.
3. Sharoni R, Olivson A, Chandra M, et al. A ³¹P  study of preconditioned isolated perfused rat heart exposed to intermittent ischemia. Magn Reson Med, 1996, 36: 66-71.
4. Cave A C. Preconditioning induced protection against post-ischemic contractive dysfunction: characteristics and mechanisms. J Mol Cell Cardial, 1995, 27:   969-979.
5. Gupta R K. NMR Spectroscopy of Cells and Organisms. CRC Press Inc, New York, 1987.
6. Barlaban R S. The application of nuclear magnetic resonance to the study of cellular physiology. Am J Physiol, 1984, 246: C10-C19.

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