Introduction

Modern high performance fighters (F15, F16) are capable of producing acceleration as high as +9 Gz sustained for 15–45 s, which may exceed human physiological tolerance [1]. High +Gz stress may challenge the pilot’s physical ability, and the effect of high sustained +Gz stress on the heart has long been a concern [2, 3]. Whinnery et al. [4] reported that high sustained +Gz stress could cause cardiac dysfunction, for example shortened QT interval, in healthy male subjects. Martin et al. found a statistically significant association between pulmonic insufficiency and exposure to high +Gz stress in pilots versus non-pilots. They also reported a greater incidence of tricuspid regurgitation and concurrent pulmonic insufficiency and tricuspid regurgitation in the pilot group [5]. Because it is unethical to modify heart pathophysiology in humans, most studies have used animal centrifuge models to examine the pathophysiological effects of high sustained +Gz stress. Burton and Mackenzie showed that high sustained +Gz stress could cause cardiac subendocardial hemorrhage and minor cardiomyopathy in miniature swine [6]. Increased harm to the heart under conditions of high sustained positive acceleration had also been identified in studies using rat models. It can affect cardiac function, reducing early diastolic myocardial velocity and systolic myocardial velocity in both ventricles and left ventricular systolic pressure [7, 8]; it also induced metabolic disorder of catecholamines and energy in the hearts of rats [9, 10]. It can also damage myocardial structure. Zhang et al. and Zheng et al. reported that high sustained +Gz exposure caused obvious edema of myocardial and endothelial cells, myofibril disorder, injuries of mitochondria and nucleus, and changes of several myocardial enzymes in rats [11, 12]. Although the harmful effect of high sustained positive acceleration on the heart is well known, there is limited knowledge of how high +Gz stress is related to cardiac injury. Previous studies have suggested that cardiac injury may be related to redundant reactive oxygen species (ROS) produced as a result of ischemia and/or hypoxia during high +Gz stress [2, 13].

Mitochondria are the major source of intracellular ROS [14]; they can, however, also be the primary target of ROS that initiate lipid peroxidation and eventually lead to cell death by apoptosis and necrosis. Under physiological conditions, low levels of ROS are required for normal cell function, for example cell signaling, and redundant ROS are, in vivo, reduced by a network of antioxidant defense mechanisms [15]. Under pathological conditions, however, because of elevated ROS production, these mechanisms may fail to eliminate the large amount of ROS generated, which might therefore cause oxidative stress and cellular damage [16]. Thus, mitochondria are important both as an ROS producer and as a sensitive ROS target. Abundant ROS inside the cells will eventually result in mitochondria dysfunction and activate the mitochondria-dependent apoptotic pathway [17].

In the work discussed in this paper we used the animal centrifuge model to investigate the effect of exposure to +Gz on myocardial mitochondrial ultrastructure, respiratory function, and antioxidant capacity in the heart of the rat, to elucidate the mechanisms of cardiac injury.

Materials and methods

Animals

Thirty male Sprague–Dawley rats, weighing 280 ± 20 g, obtained from Weitong Lihua Experimental Animal Technology (Beijing, China), were randomly assigned to three groups: blank control group, stress control group, and high +Gz stress group. Rats were housed individually in an animal room for 3 days, and were starved for 12 h before the experiment to reduce the effects of endogenous substrates, for example myocardial tissue glycogen and fat, on the experimental results. All experiments and procedures performed in this study were reviewed and approved by the Animal Care and Use Committee of the Institute of Aviation Medicine, Air Force.

Exposure of animals to acceleration

The animal centrifuge was 2 m in radius and was capable of producing a range of gravity from +1 to +15 Gz, with an onset rate of 0.1–6 Gz/s. Each rat was put into a 15 cm × 5 cm × 3 cm plexiglass box which was clamped to the centrifuge arm with the head of the rat facing the axis of the centrifuge for +Gz orientation. Rats in the high +Gz stress group were exposed to +10 Gz for 5 min as reported elsewhere [18]. The onset/offset rate was +1 G/s. Stress control rats were placed on the arms of centrifuge and underwent a process similar to that described above, but were not exposed to acceleration. No experiments were conducted on the blank control group.

Sample preparation for transmission electron microscopy and quantitative analysis of myocardial mitochondria morphology

After exposure to acceleration for 4 h, rats were anesthetized and the hearts were quickly removed. Part of the left ventricle tissue was cut into small pieces (approx. 1 mm3).These were then rapidly fixed with 5 % glutaraldehyde and then fixed again with 1 % osmic acid. The fixed tissue were dehydrated with acetone and embedded in epoxy resin 812. Ultrathin sections, approximately 30–50 nm, were cut with ultramicrotome (PowerTome-PC, RMC, USA), mounted on copper grids, and stained with uranyl acetate and lead citrate. The grids were examined by transmission electron microscopy (model H-75000-2; Hitachi, Japan). One piece of section was prepared for each animal, and five photographs magnified 12,500 times were obtained randomly between the microvilli and nucleus for each section.

The photographs were used as input for a CM2000B pathology image-analysis system (Beijing University of Aeronautics and Astronautics, Beijing, China) for quantitative analysis of myocardial mitochondria morphology, and measurement of volume density, surface density, average volume, average surface area, and numerical density of the mitochondria.

Isolation of myocardial mitochondria

Myocardial mitochondria were isolated by differential centrifugation as described elsewhere [19], with slight modifications. Briefly, 1 g fresh left ventricle tissue was placed in ice-cold isolation medium (50 mM sucrose, 200 mM mannitol, 5 mM KH2PO4, 1 mM EGTA, 5 mM Mops, and 0.2 % BSA, pH 7.3), minced to small pieces (approx. 1 mm3), then homogenized for 10 s by use of a homogenizer (Pro200; ProScientific, USA) in 5 ml isolation buffer. The samples were then diluted to 15 ml with isolation buffer and centrifuged at 480g for 10 min. The supernatant, containing the mitochondrial fraction, was further centrifuged at 10,000g for 10 min at 4 °C. Mitochondria were washed and resuspended in isolation medium. Protein concentration was determined by use of a BCA Protein Assay Kit (Bio-Rad, USA) then adjusted to 10–20 mg/ml.

Measurement of myocardial mitochondrial respiratory function

Mitochondrial respiration was measured by determining oxygen consumption by use of a biological oxygen monitor (Model 5300; YSI, USA) equipped with a Clark oxygen electrode. The oxygen consumption studies were conducted at 30 °C in respiration medium (25 mM sucrose, 75 mM mannitol, 95 mM KCl, 5 mM KH2PO4, 20 mM Tris–HCl, 1 mM EGTA, pH 7.4), containing 5 mmol/L sodium succinate as respiratory substrate. Approximately 0.5 mg mitochondrial protein was pre-incubated in the oxygen electrode in a total volume of 1 ml, with substrates, for 2 min. State 3 respiration (ST3) was induced by the addition of adenosine diphosphate (ADP) (1 mM). Approximately 3 min later, ST3 was terminated and state 4 respiration (ST4) was detected. The rates of oxygen consumption during State 4 and State 3 were expressed as nanomoles of oxygen consumed per milligram mitochondrial protein per minute. The ratio of State 3 to State 4 mitochondrial respiration was used to calculate the respiratory control ratio (RCR = ST3/ST4).

Determination of antioxidant levels and lipid peroxidation products in myocardial mitochondria

Isolated mitochondria were disrupted by ultrasonication and protein concentration was determined by use of a BCA Protein Assay Kit (Bio-Rad). The activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), and the concentration of malondialdehyde (MDA) in myocardial mitochondria were measured by use of commercial assay kits (Nangjin Jiancheng Bioengineering Institute, China) in accordance with the manufacturer’s instructions. The nitrite method was used to determine SOD activity; absorbance was measured at 550 nm. CAT activity was measured at 405 nm, in accordance with Góth [20], by use of an assay kit. GSH-Px activity was measured at 412 nm by quantifying the rate of oxidation of reduced GSH to oxidized glutathione. The thiobarbituric acid coloration method was used to determine MDA concentration; absorbance was measured at 532 nm.

Statistical analysis

All results are expressed as mean ± standard deviation (SD). Comparisons among groups were made by one-way analysis of variance (ANOVA) followed by the least-significant-difference (LSD) post-hoc multiple comparisons test. The statistical software package SPSS 16.0 for Windows (SPSS, Chicago, IL, USA) was used. Statistical differences of P < 0.05 were considered to be significant.

Results

Observation of ultrastructure, and quantitative analysis of myocardial mitochondria morphology

Electron microscopy photographs of mitochondria from myocardiocytes of each group are shown in Fig. 1. The ultrastructure of the cardiac muscle from rats of the blank control and stress control groups was identical. Mitochondria of normal size and with well-delineated cristae were distributed among the cardiac muscle. However, myocardial mitochondria from the high +Gz stress group had suffered substantial structural damage; they were substantially swollen, with broken or disrupted cristae or incomplete membranes, and some mitochondria had a condensed or polymorphic matrix. Moreover, deformation of some degenerated mitochondria was apparent as monstrous shapes containing myelin-like figures. This damage was confirmed by quantitative analysis of morphology; the high +Gz stress group was associated with higher surface density, volume density, average volume, and average surface area, and lower numeral density of the mitochondria compared with the blank control and stress control groups (P < 0.01) (Fig. 2).

Fig. 1
figure 1

High +Gz stress impairs the ultrastructure of myocardial mitochondria in rats (bar = 1,000 nm). a Representative transmission electron microscopy photograph from blank control group; b representative transmission electron microscopy photograph from stress control group; c representative transmission electron microscopy photograph from high +Gz stress group. Original magnification ×12,500

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Fig. 2
figure 2

Effects of high +Gz stress on quantitative indicators of the morphology of myocardial mitochondria from rats: a volume density, b surface density, c numerical density, d average volume, e average surface area. Columns and vertical bars represent mean ± SD from 50 different photographs

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Myocardial mitochondrial respiratory function

Results from measurement of the myocardial mitochondrial respiratory function of the rats are shown in Fig. 3, which includes ST3, ST4, and RCR of mitochondria from all groups. High +Gz exposure impaired the myocardial mitochondrial respiratory function of rats. In the high +Gz stress group, ST3 was significantly lower and ST4 was significantly higher than in the blank control group or the stress control group (P < 0.01 or P < 0.05). RCR calculated from ST3 and ST4 respiration rates was also substantially lower in high +Gz-stressed rats (P < 0.01).

Fig. 3
figure 3

Effects of high +Gz stress on respiratory function of myocardial mitochondria in rats. a State 3 respiration (ST3), b state 4 respiration (ST4), c respiratory control ratio (RCR). Columns and vertical bars represent mean ± SD from 10 rats

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Activity of myocardial mitochondrial antioxidant enzymes, and MDA content

Figure 4 illustrates the effect of high +Gz stress on the activity of antioxidant enzymes and on the level of MDA. In comparison with the blank and stress control group, high +Gz stress induced significant down-regulation of SOD, CAT, and GSH-Px activity (P < 0.05) but increased MDA production (P < 0.01). These results suggest that high +Gz stress can reduce the activity of antioxidant enzymes and increase lipid peroxidation.

Fig. 4
figure 4

Effects of high +Gz stress on the activity of antioxidant enzymes and on MDA production by myocardial mitochondria in rats: a catalase (CAT), b superoxide dismutase (SOD), c glutathione peroxidase (GSH-Px), d malondialdehyde (MDA). Columns and vertical bars represent mean ± SD from 10 rats

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Discussion

It has been known for decades that high sustained +Gz can damage the hearts of experimental animals [6, 7]. G-related cardiomyopathy occurs as two general forms of cardiac cell damage: myofibrillar degeneration and frank myocardial necrosis, and both forms of damage can be caused by a great number of humoral, ischemic, mechanical, and stressful stimuli [2, 21–23]. The main harmful factors related to +Gz injury have not been completely clarified. Laughlin [2] proposed it is strongly related to the effects on the heart of sympathetic nervous and hormonal catecholamines. However, most other studies regarded ischemia or hypoxia as the most important aspects of this heart lesion [24, 25]. Because upregulation of the concentration of blood catecholamines or ischemia and/or hypoxia augment ROS production and lead to oxidative stress, oxidative stress was of concern in this study. Interestingly, food and drugs known to reduce oxidative stress also reduce G-related injury of experimental animals [8].

Because mitochondria are the major source of intracellular ROS and can also be the primary organelle damaged by ROS, we focused on alteration of mitochondrial ultrastructure, respiratory function, and antioxidant capacity in the animals after high sustained +Gz exposed. The results showed the mitochondria of +Gz exposed animals swelled, degenerated, and decreased in number, and their cristae broke or disappeared, as reported elsewhere [12]. Swelling of the mitochondria of rats in the high +Gz stress group led to an increase in surface density, volume density, average volume, and average surface area, and to a decrease in numerical density (reduction of the number of mitochondria per unit visual field in electron microscopy). Changes of quantitative indicators of morphology were therefore consistent with electron microscopy observation.

Besides reported ischemia and/or hypoxia [24, 25], cardiac workload also increased among centrifuged rats [10]. Both may lead to depletion of ATP and ADP and impair the respiratory chain in high sustained +Gz exposed animals [26]. Thus, electrons are transferred directly to molecular oxygen to generate more oxygen radicals [27, 28]. Although mitochondrial respiration can be a major source of oxygen radicals, production of these oxygen radicals can also interrupt oxygen delivery to myocardial cells and aggravate depression of mitochondrial respiratory function [29]. Among the five states during the process of mitochondrial respiration, state 3 and state 4 are most important. State 3 is the fast oxidation period in the presence of ADP and respiratory substrate [30]. When ADP is exhausted, mitochondrial respiration enters state 4. Respiration in state 4 can be used to reflect mitochondrial permeability [31]. The ratio of state 3 to state 4 respiratory rates is the RCR. The RCR is a measure of the coupling between substrate oxidation and phosphorylation and basically informs the researcher how intact or how coupled mitochondria are [32]. Alteration of the properties of myocardial mitochondria isolated from high sustained +Gz exposure animals was indicative of functional impairment of oxidative phosphorylation and less efficient utilization of oxygen by high +Gz stress-treated rats. ROS damages the process of oxidative phosphorylation of mitochondria and may reduce, even abolish ATP production. As a consequence, Na+ concentration was increased in mitochondria, which caused mitochondria to absorb much water; this finally resulted in swelling. Mitochondrial respiratory dysfunction may therefore contribute to disruption of its ultrastructure.

In this study, the concentration of MDA, a marker of lipid peroxidation, was shown to be increased in mitochondria from rats from the high +Gz stress group; this is a consequence of increased generation of oxygen radicals. CAT, SOD, and GSH-Px are regarded as the first line of defense of the antioxidant enzyme system against ROS [33]. SOD catalyzes the reaction of superoxide radicals into oxygen and hydrogen peroxide (H2O2). GSH-Px is responsible for removal of a wide range of hydroperoxides, from complex organic hydroperoxides to H2O2. Thus, it may protect membrane lipids, proteins, and nucleic acids from oxidation. The main function of CAT is to degrade H2O2 into H2O and O2 [34]. In our study, the activity of the enzymes SOD, CAT and GSH-Px was reduced in rats from the high +Gz stress group. It is possible the reduced activity of these antioxidant enzymes was because of consumption by ROS, and other reasons. Irrespective of the reason, high +Gz stress-induced damage is a result not only of overproduction of ROS but also reduction of antioxidant enzyme activity. The changes of antioxidant enzymes and MDA in this study were similar to those for ischemia and/or hypoxia rats [35], which could also confirm ischemia or hypoxia as the main cause of high +Gz stress-induced heart lesion.

In conclusion, in this study we examined the integrity of mitochondria and measured a variety of indicators of mitochondrial function in rats suffering high +Gz stress. We showed that high +Gz stress could damage mitochondrial ultrastructure, compromise mitochondrial respiratory function, reduce the activity of antioxidant enzymes, and enhance lipid peroxidation. This research may aid discovery of more potentially harmful effects of high +Gz stress on the human heart, and, subsequently, help to prevent heart injury.