Animal and Ethical Statement

The present study used eight to ten-week-old male Sprague-Dawley rats (Charles River), weighing 200±20 g, which were obtained from the Experimental Animal Center of the Kunming Medical University (Kunming, Yunnan, China) and were housed in the Laboratory Animal Center of the Kunming Medical University. Animal experimental protocols were approved and performed according to the guidelines of the Institutional Medical Experimental Animal Care Committee of the Kunming Medical University. Guidelines for Laboratory Animal Care and Safety from the United States National Institutes of Health (Bethesda, Maryland, USA) were also followed.

Reagents

Triphenyl tetrazolium chloride (TTC) was obtained from the Sigma-Aldrich Corporation (Missouri, USA). Kits for detecting lactate dehydrogenase (LDH), creatine kinase (CK), and troponin I (cTnI) were purchased from AU Clinical Chemistry Systems of the Beckman Coulter Inc. company (California, USA). The kit for detecting melatonin was purchased from the Abcam Inc. company (ab213978, Abcam, Cambridge, Massachusetts, USA).

Equipment

The geomagnetic experimental platform consists of four partial compositions, including a metal shielding experiment cage, radiation antenna, programmable signal generator, and control computer. The signal generator controlled by the main control computer can produce below 50 Hz and an arbitrary combination of signal spectra, especially analog Schumann resonances and geomagnetic burst, in the space of electromagnetic radiation (Figure 1). Patent No.: ZL201520208744.2.

Fig. 1 - The structure drawing of geomagnetic experimental platform.

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Fig. 1 - The structure drawing of geomagnetic experimental platform.

Animal Grouping and Treatment

The male rats were randomly divided into the geomagnetic shielding place, geomagnetic radiation place, and normal place (rats living in general environment without intervention). The rats in each place were divided into sham operation group and operation group, being nine rats in each group. The shield+sham group, radiation+sham group, normal+sham group, and normal+operation group were the control group. The shield+operation group and radiation+operation group were the experimental group.

MI/RI Protocol

The MI/RI model was generated and utilized as previously described^[4,5]. Rats were anesthetized using an intraperitoneal injection of 3.6% chloral hydrate (1 ml/kg) and ventilated isoflurane gas via tracheal intubation, with an RWD rodent respirator. Body temperature was maintained at 37ºC via a heated operating table. MI/RI surgery was performed using the following procedure: the heart was exteriorized by a left thoracic incision, and a slipknot (6-0 silk) was placed and ligated around the left anterior descending (LAD) coronary artery. The slipknot was released after 30 min of ischemia, and after that the animal received 24 hours of reperfusion. Ischemia was confirmed by noting the change in color of myocardial tissue in the ischemic area, and reperfusion was achieved by loosening the knot. The MI/RI rats were placed in different places for 24 hours of reperfusion, and then their physiological and biochemical indicators were tested.

Myocardial Infarct Sizes

Following reperfusion, the heart was then excised, both atria and the right ventricle were removed, and the left ventricle was cut into five equal slices to create cross sections from apex to base. The slices were separated into normal zone and area at risk, both followed by incubation in 1% TTC to measure the viability of myocardial tissue. Viable tissue stained red, while nonviable tissue remained unstained or was white (Figure 2A). Infarct size as a percentage of area at risk was determined gravimetrically.

Fig. 2 - Heart physiological indexes. A) results of TTC staining of the myocardium of rats; B) The infarct areas of the left ventricular tissue were measured by TTC staining (n=6); C, D, and E) the left ventricular pressures were recorded by electrophysiolograph (n=9).
LEVDP=left ventricular end-diastolic pressure; LVDP=left ventricular developed pressure; TTC=triphenyl tetrazolium chloride

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Fig. 2 - Heart physiological indexes. A) results of TTC staining of the myocardium of rats; B) The infarct areas of the left ventricular tissue were measured by TTC staining (n=6); C, D, and E) the left ventricular pressures were recorded by electrophysiolograph (n=9).
LEVDP=left ventricular end-diastolic pressure; LVDP=left ventricular developed pressure; TTC=triphenyl tetrazolium chloride

Left Ventricular Pressure Test

Cardiac function was measured by left ventricular cannulation. Rats were fasted for 12 hours and anesthetized with 3.6% chloral hydrate (1 ml/kg). The left common carotid artery was isolated, then the left ventricular developed pressure (LVDP), left ventricular end-diastolic pressure (LVEDP), and ± dP/dtmax were recorded by electrophysiolograph (BIOPAC 150).

Serum Biochemistry Test

The blood was collected from the abdominal aorta artery catheter and added to procoagulant tube to centrifuge for 3000 min at 4ºC. The serum was stored in an ultra-low temperature refrigerator at -20ºC. The automatic blood biochemical analyzer (Beckman AU480, USA) detect cTnI, CK, and LDH in the blood with specific reagents from AU Clinical Chemistry Systems.

Melatonin Test

The serum level of melatonin was detected by using the enzyme-linked immunosorbent assay (ELISA) kit according to the manufacturer’s instructions. The results were determined spectrophotometrically at 450 nm.

Statistical Analysis

All statistical analyses were performed using the Statistical Package for the Social Sciences (SPSS) software, version 19 (SPSS Inc., Chicago, Illinois, USA). The data were presented as MEDIAN ± INTERQUARTILE RANGE and were compared using Kruskal-Wallis test. P<0.05 was considered statistically significant.

Myocardial Infarction Area

The results showed that there was no significant difference in the myocardial infarction area between the shielding + operation group, radiation + operation group, and normal+operation group (P>0.05), but there was significant difference in infarct size between the sham group and operation group (P<0.05) (Figure 2B).

Left Ventricular Pressure

The results showed that LVDP in the shield+operation group and normal+operation group were higher than in the radiation+operation group (P<0.05) (Figure 2C). There was no significant difference in LEVDP in the shielding+operation group and radiation+operation group (P>0.05), but in the shielding+operation group it was higher than in the normal+operation group (P<0.05) (Figure 2D). The ± dP/dtmax of the left ventricle in the shielding+operation group and normal+operation group were higher than that in the radiation+operation group(P<0.05) (Figure 2E).

Troponin I

The results showed that cTnI in the radiation+operation group was higher than in the shielding+operation group, and in the operation group it was higher than in the sham group (P<0.05) (Figure 3A).

Fig. 3 - Serum biochemical indexes. A, B, and C). The cTnI, CK, and LDH were measured by automatic blood biochemical analyzer (n=3), which are specific indicators of myocardial infarction; D) melatonin was measured by ELISA kit, which protects the myocardial tissue (n=4).
CK=creatine kinase; cTnI=troponin I; ELISA=enzyme-linked immunosorbent assay; LDH=lactate dehydrogenase

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Fig. 3 - Serum biochemical indexes. A, B, and C). The cTnI, CK, and LDH were measured by automatic blood biochemical analyzer (n=3), which are specific indicators of myocardial infarction; D) melatonin was measured by ELISA kit, which protects the myocardial tissue (n=4).
CK=creatine kinase; cTnI=troponin I; ELISA=enzyme-linked immunosorbent assay; LDH=lactate dehydrogenase

Creatine Kinase

The results showed that CK in the shielding+operation group was lower than in the radiation+operation group and normal+operation group (P<0.05) (Figure 3B).

Lactate Dehydrogenase

The results showed that LDH in the shielding+operation group had no significant difference from the radiation+operation group (P>0.05), but LDH in the shielding+operation group and radiation+operation group were lower than in the normal+operation group (P<0.05) (Figure 3C).

Melatonin

The concentrations of melatonin in the radiation+operation group were significantly lower than in the shielding+operation group and normal+operation group (P<0.05) (Figure 3D).