Mitochondrial ferritin protects the murine myocardium from acute exhaustive exercise injury

Mitochondrial ferritin (FtMt) is a mitochondrially localized protein possessing ferroxidase activity and the ability to store iron. FtMt overexpression in cultured cells protects against oxidative damage by sequestering redox-active, intracellular iron. Here, we found that acute exhaustive exercise significantly increases FtMt expression in the murine heart. FtMt gene disruption decreased the exhaustion exercise time and altered heart morphology with severe cardiac mitochondrial injury and fibril disorganization. The number of apoptotic cells as well as the levels of apoptosis-related proteins was increased in the FtMt−/− mice, though the ATP levels did not change significantly. Concomitant to the above was a high ‘uncommitted' iron level found in the FtMt−/− group when exposed to acute exhaustion exercise. As a result of the increase in catalytic metal, reactive oxygen species were generated, leading to oxidative damage of cellular components. Taken together, our results show that the absence of FtMt, which is highly expressed in the heart, increases the sensitivity of mitochondria to cardiac injury via oxidative stress.

expressed in the testis, particularly in the spermatocytes, and also in the heart, kidney, Purkinje cells, [12][13][14] and some other neurons, 15,16 and generally in cells with high respiratory activity. Importantly, the FtMt transcript does not contain a functional iron responsive element (IRE), 17 and thus, FtMt expression is not post-transcriptionally controlled by intracellular iron levels in the same way as that of H-ferrtin. The tissue distribution pattern suggests that the major role of FtMt may be to protect mitochondria from iron-dependent oxidative damage. 18,19 Our previous studies have shown that FtMt exerts a neuroprotective effect against 6-hydroxydopamine (6-OHDA)-induced dopaminergic cell damage 18 and that FtMt is involved in the pathology of AD where it may have a neuroprotective role by reducing oxidative stress. 19 Recent research suggests FtMt may have a protective role in a number of mild pathophysiological conditions in the heart. 20 Because FtMt is highly expressed in cardiac muscle, we hypothesize that the protein may protect mitochondria against oxidative damage from injury induced by acute exhaustive exercise (AEE), which is commonly used as an oxidative stress model. 21 To examine the role of FtMt in the myocardium, we subjected age-matched (10-to 12-week-old) FtMt +/+ and FtMt − / − mice to AEE and evaluated the resultant myocardial injury. We found that AEE induced an increase in intracellular 'uncommitted' iron, accompanied by oxidative stress and activation of the caspase cascade. These effects were exacerbated by FtMt deficiency, leading to increased myocardial injury and apoptosis.

AEE induces FtMt expression in the murine myocardium.
FtMt is critical for maintaining intracellular iron homeostasis in multiple cell types and organs. 22 We performed immunoblotting to test whether FtMt is expressed in the mouse myocardium after AEE. As shown in Figures 1a and b, we found that FtMt expression indeed significantly increased in the heart. We also measured FtMt mRNA levels in the mouse myocardium using the quantitative real-time PCR method. AEE treatment significantly increased FtMt mRNA expression in the mouse hearts ( Figure 1c).
FtMt gene disruption affects myocardial structure and function. Time to exhaustion is a measure of athletic capacity. The time to exhaustion in FtMt − / − mice was decreased when compared with FtMt +/+ mice (Figure 2a). To examine the structural differences between normal-and 1 FtMt-deficient cardiomyocytes, we performed transmission electron microscopy in FtMt +/+ and FtMt − / − cardiomyocytes of untreated mice or those subjected to AEE (Figure 2b). In the hearts of the latter, we observed significant mitochondrial vacuolation. Importantly, fibril organization was comparable between the FtMt +/+ and FtMt +/+ AEE groups. In contrast, the hearts of FtMt − / − mice exhibited mitochondria with slightly disrupted cristae, after experiencing AEE. In addition, the tissue structural damage was more severe with condensation and fragmentation of most myofibrils in some fields. Furthermore, mitochondrial damage was more evident in zones in which the cristae were absent. Functional changes (Figure 2c) accompanied the observed structural damage, when we assayed the ATP content of the cardiac tissue in the four groups, we observed a significant decrease in FtMt − / − mice subjected to AEE.
Apoptosis is increased in cardiomyocytes of FtMt − / − mice. We used the TUNEL method to detect apoptosis after exposing mice to AEE (Figure 3a). The number of apoptotic cells (Figure 3b) in the FtMt +/+ AEE group was about four times greater than that in the FtMt +/+ group, whereas the amount of apoptotic cells in the FtMt − / − AEE group was more than three-fold that in the FtMt +/+ AEE group. There was also a marked increase in the FtMt − / − AEE group compared with the FtMt − / − group. Together, these results suggest that the presence of FtMt protects against AEE-induced apoptosis.
Proteins of the Bcl-2 family include critical regulators of programmed cell death. Bcl-2 has the role of promoting cell survival. Increased expression of the pro-apoptotic protein, Bax, can promote cell death by activating elements of the caspase pathway, 23 especially caspase-3. 24 Thus, Bcl-2/Bax levels are widely used as a proxy for the degree of apoptosis. 18 The activation of MAPK is often seen in oxidative stressinduced cell death. 25 MAPK (a.k.a., p38 MAP kinase or P38) is activated by phosphorylation; a high P-P38/P38 ratio can simultaneously promote Bax expression and decrease Bcl-2 levels. AEE significantly increased the P-P38/P38 ratio (Figures 3c and d) with the expected, downstream consequences of decreased ratios of Bcl-2 to Bax (Figures 3e and f) and elevated levels of cleaved caspase-3 (Figures 3g and h). Out of all the tested groups, the largest changes in the Bcl-2/Bax ratios and cleaved caspase-3 levels were observed in the FtMt − / − AEE mice.
Calcium levels increased in the FtMt − / − mouse myocardium. Calcium activates various signal transduction pathways, including those that mediate apoptosis, so it is no surprise that dysregulated calcium metabolism can induce apoptosis. In fact, elevated unsequestered calcium levels can be an early indicator of apoptosis. 26 We utilized synchrotron radiation X-ray fluorescence (SR-XRF) to directly detect the calcium distribution in the myocardia of FtMt +/+ and FtMt − / − mice with or without exposure to acute oxidative stress. Our data show ( Figure 4) that calcium levels increased in the myocardia of all groups after AEE, but was especially elevated in the FtMt − / − AEE group. These results demonstrate that aberrant calcium metabolism caused by AEE may be activating apoptosis the in the myocardia.
Mice deficient in FtMt possess elevated myocardial oxidative stress. To study whether increased levels of oxidative stress are responsible for the increased oxidative damage and apoptosis observed in the myocardium, we examined the antioxidant capacity in each group. Superoxide dismutase (SOD) is an antioxidant enzyme that can mitigate the accumulation of ROS, whereas malondialdehyde (MDA) is a consequence of excess ROS and whose levels are commonly viewed as an indicator of lipid peroxidation. Compared with the FtMt +/+ group, the SOD activity ( Figure 5a) was lower while the levels of MDA ( Figure 5b) were increased in the other three groups. In addition, the lowest SOD activity and the highest level of MDA both appeared in the myocardium of the FtMt − / − AEE group. The level of MDA in the FtMt − / − AEE group was about four-fold the level of that in the FtMt +/+ group, whereas the amount of MDA in the FtMt − / − AEE group was about three-fold greater than that of the FtMt +/+ AEE group. We also found that the decreases in catalase, NAD(P)H:quinone oxidoreductase 1, and heme oxygenase 1 mRNA were greater between the FtMt − / − AEE and FtMt − / − groups, compared with those between the FtMt +/+ AEE and FtMt +/+ groups (Supplementary Figure 2). These findings are congruent with dramatically increased oxidative stress in the myocardium of FtMt − / − mice subjected to AEE.
FtMt gene disruption results in altered myocardial iron metabolism after AEE. Transferrin receptor 1 (TfR1) and divalent metal transport 1 (DMT1, of which there are two variants, +IRE and -IRE) are major players in iron uptake, whereas ferroportin 1 (FPN1) is the only known cellular iron exporter. Ferritin, comprising H-ferritin and L-ferritin subunits, can sequester and store iron in a safe yet accessible form. 27 By immunoblot analysis, we observed that AEE decreases the expression of both ferritin subunits (H-and L-ferritin) (Figures 6e and f) in the myocardium, but increases TfR1 expression (Figures 6a and b). However, there is no difference in the expression of Fpn1, DMT1+IRE, or DMT1-IRE in any group (Figures 6a-d).
We next utilized SR-XRF to directly detect the iron distribution and content in the myocardium of the four groups. Our data (Figures 7a and b) show that iron indeed accumulated in both the FtMt +/+ AEE and FtMt − / − AEE groups. As elevated, 'uncommitted' iron is able to directly generate ROS through Fenton chemistry, our results show that aberrant iron metabolism may be involved in the oxidative stress brought about by AEE.

Discussion
Mitochondrially generated ROS are involved in a myriad of signaling and damaging pathways in different tissues. 28 In  29 FtMt is expected to protect the mitochondria from the accumulation of ROS; a hypothesis that has been confirmed by in vitro studies of cells that overexpress FtMt. 19,30 Our previous experiments 18,19 confirm FtMt has a protective role in cells by reducing oxidative damage, whereas recent research from others suggests FtMt may have a protective role under a number of not particularly severe pathophysiological conditions in the heart. 22 However, the mechanism of the protective role of FtMt in the heart had not been revealed.
We used an AEE-induced oxidative stress model of cardiac injury in the present study. 21 Our results show that AEE elevates FtMt protein levels in the mouse myocardium, providing a clue that FtMt may have an important role in maintaining myocardial function.
Previous studies have indicated AEE can give rise to oxidative stress. 21,31 To evaluate the possible protective activity of FtMt in this context, we measured the time to exhaustion of FtMt +/+ and FtMt − / − mice and found that the wild-type mice could exercise longer. Histological examination by electron microscopy revealed morphological abnormalities, including mitochondrial vacuolation, in hearts after mice of both genotypes were subjected to AEE. Strikingly, AEEaggravated FtMt − / − hearts showed considerable damage to mitochondrial cristae as well as myofibril disarrangement in some areas of the heart. Biochemical assays of heart functionality revealed decreased ATP content, indicating mitochondrial dysfunction. Together, these results show that FtMt has an important role in maintaining cardiac structure and function.
Apoptosis can be induced by oxidative stress in a caspasemediated pathway. 32 Our data show that apoptosis was induced by AEE in both FtMt +/+ and FtMt − / − mice, whereas FtMt − / − mice exhibited a slightly increased amount of apoptosis without the exercise. Importantly, the FtMt − / − mice had a relatively high level of myocardial apoptosis after AEE. We also observed a reduction in the Bcl-2/Bax protein ratio, an enhanced P-P38/P38 ratio and an increase in caspase-3 protein in the myocardium after AEE. All three of these effects were exacerbated in the FtMt knockout mice. Taken together, our results strongly suggest that a bona fide apoptotic signal transduction pathway is initiated following AEE.
How does acute oxidative stress induce apoptosis and damage in the heart? Our results suggest AEE leads to the generation of ROS and subsequent oxidative damage through increased lipid peroxidation and the release of intracellular Ca 2+ , which is a pro-apoptotic factor. In addition, previous studies have shown that FtMt knockdown induces oxidative damage and stimulates apoptosis in the hippocampus after treatment with Aβ. 19 Why did the FtMt gene knockout aggravate the oxidative injury? Metals, such as iron, copper, and zinc, have been shown to be key factors in biochemical reactions that produce free radicals, which lead to the peroxidation of cellular lipids and cellular injury or apoptosis. 33 Our results show that, the total iron of myocardia was increased after AEE, FtMt gene knockout aggravated iron overloading, which may be the reason of oxidative injury. Serum iron was also found to increase in the FtMt − / − AEE group (Supplementary Figure 3), suggesting that AEE also has an effect on cells that release iron, such as enterocytes and macrophages of the reticuloendothelial system.
In summary, our study shows that acute exhaustive stress gives rise to the formation of ROS that initiate lipid peroxidation, protein oxidation, and cytoplasmic Ca 2+ accumulation, eventually leading to myocardial apoptosis and damage. Furthermore, the ROS generated under this stress modality may be potentiated by the accumulation of iron that occurs when cellular iron metabolism is thrown off balance. Our data indicate that normal mice can mitigate oxidative damage by upregulating FtMt protein levels, whereas the absence of FtMt leads to severe heart mitochondria and myofibril damage. The cardiomyocyte mitochondria in FtMt − / − mice are more sensitive to oxidative stress-mediated injury, 20 which likely underlies the decrease in time to exhaustion, possibly due to myocardial damage. In addition, increased IL-6 indicates that an inflammatory reaction may result from FtMt gene inactivation, further aggravating the heart injury by AEE (Supplementary Figure 4). All our data suggest FtMt has a protective role against myocardial injury induced by AEE. Moreover, the results of our study represent the first in-depth evidence for a function of FtMt in vivo and points to FtMt as a potential therapeutic target to protect the heart from oxidative stress-induced cardiomyopathy. Materials and Methods Animals. Originally, the C57BL/6 wild and FtMt-null mice were generated by Dr. M Fleming's group. 34 The mice were bred and genotyped by PCR amplification of genomic DNA and total RNA (Supplementary Figure 1b) Figures 1c and 1d). Mice were housed under conditions controlled for temperature (22°C) and humidity (40%), using a 12 h light (0700 to 1900), 12 h dark (1900 to 0700) cycle. 35 Mice were fed a standard rodent diet and water ad libitum. Age-matched FtMt +/+ and FtMt − / − male mice were subjected to AEE as described below. All procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by the Animal Care and Use Committee of the Hebei Science and Technical Bureau in the PRC.
Antibodies and reagents. The following antibodies and reagents were used: FtMt (generous gift from Sonia Levi, Italy), β-actin (Alpha Diagnostic International, . Reagents for RNA extraction and reverse transcription were purchased from Takara (Dalian, China). Real-time RT-PCR quantification reagents were purchased from Genstar (Beijing, China). Protein expression was assessed by immunoblotting as previously described. 13 Acute exhaustive exercise. FtMt +/+ and FtMt − / − mice at 3 months of age (n = 6-8/group/experiment) were subjected to exercise on a gradient treadmill for 3 consecutive days at a 10°incline (15 min at 10 m/min the first day; 30 min at 14 m/min the second day; 45 min at 17 m/min the third day). On the fourth day, the exercised group ran on the treadmill at an initial speed of 12 m/min and incline 10°for 7 min, then at a speed of 14 m/min for 5 min. The speed was incremented by 2 m/min every 2 min until the mice reached 22 m/min, and then the speed was incremented by 1 m/min every 3 min until the mice reached their maximum velocity. 21 Empirically, we observed that mice, like humans, are not all able to run at the same maximum velocities. Thus we defined the maximum velocity as the highest velocity at which the mouse would continue to run, without falling off the treadmill and being unable to hop back on, despite continued efforts and lack of clear signs of exhaustion. The mice were then run to exhaustion (i.e., run at maximum velocity until they would not remain on the treadmill despite gentle electric shocks). 36,37 Immediately after exhaustion exercise, mice were killed and the left ventricule of hearts excised, processed/frozen in liquid nitrogen and stored at − 80°C for subsequent analysis. Determination of ATP content. The ATP content of heart tissue, from samples stored at − 80°C, was measured by a colorimetric assay using a commercial kit (ATP colorimetric assay kit, Nanjing Jiancheng Bioengineering Institute, Nanjing, China) following the manufacturer's instructions.
Detection of apoptosis. The presence of apoptosis in the myocardium was assessed by the TUNEL method, 38 as previously described. 14 Nuclei were stained with DAPI. The number of TUNEL-DAPI-positive cardiomyocytes was counted using the same quantification method as described previously for counting. 39 The counting area was located in the same position of the myocardium in all groups. For each group, quantification was performed in sections from three different mice, and the data are provided as the number of TUNEL-positive cells compared with that of the control group.
Quantitative real-time PCR. The relative purity of isolated total RNA was assessed spectrophotometrically and the ratio of A260/A280 nm exceeded 1.9 for all preparations. Total RNA (1 μg) was reverse transcribed in a 20 μl reaction and 1 μl cDNA were then used as the template for real-time PCR with SYBR Green (GenStar). PCR amplification was performed with the BIO-RAD CFX Connect Real-Time System (Hercules, CA, USA) with the following cycling parameters: 95°C for 10 s, followed by 40 cycles of 95°C for 15 s and then 60°C for 1 min. Expression of the target gene was determined by normalizing to the respective β-actin levels. Each amplification was repeated three times and the data were averaged. The forward primer of the FtMt sequence is: 5′-ATTTCCTTCGCCAGTCCC-3′ and the reverse primer of FtMt is 5′-CCGCATTCCCAGTCATCT-3′; the β-actin forward primer sequence is 5′-AGGCCCAGAGCAAGAGAGGTA-3′ and reverse primer sequence is 5′-TCTCCATGTCGTCCCAGTTG-3′.
Measurements of oxidative stress. MDA, a reliable marker of lipid peroxidation, was measured using the MDA kit, according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, China). SODs, enzymes which catalyze the dismutation of superoxide into oxygen and hydrogen peroxide to provide important antioxidant defense in cells performing oxidative metabolism, were measured by a SOD kit according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute, China). The levels of MDA and the SOD activity were determined in each group. The left ventricular region of the heart was homogenized in ice-cold saline. The homogenate was centrifuged at 3000 × g at 4°C for 15 min, and then the supernatant was used to determine SOD activity and MDA levels with a UV-vis spectrophotometer at wavelengths of 550 and 532 nm, respectively.
Synchrotron radiation X-ray fluorescence. Under anesthesia with nembutal, animals were perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer. The myocardium was removed, postfixed for 1.5-4 h and then stored overnight in 30% sucrose. Serial coronal sections were cut at 30 μm intervals on a freezing microtome. Equivalent slices from the same coordinates were fixed onto 3 mm-thick Mylar films (polycarbonate) from each of the four groups of mice. The myocardial samples were clamped between two layers of cellophane, dried at room temperature, and stored in a vacuum desiccator before analysis by SR-XRF. The microdistributions of iron and calcium in mouse myocardial samples were collected in fluorescence mode at room temperature at the Beijing Synchrotron Radiation Facility (BSRF). The continuous synchrotron X-rays were monochromatized by a Si (111) double crystal. The sample was placed at a 45°a ngle to the incident X-ray beam, and X-ray fluorescence was detected with a 50 mm 2 silicon drift detector (Vortex, Hitachi High-Technologies Science America Inc, Northridge, CA, USA) oriented at a 90°angle to the incident beam. A light microscope was coupled to a computer for sample viewing. A monochromatic SR with a photon energy of 10 keV was used to excite the samples. The beam was adjusted with grating to about 38 × 28 μm 2 . The sample platform was moved along the X and Z directions at intervals of 38 and 28 μm, respectively. The XRF signals were collected for up to 2 s at each point. In order to correct the effect of the SR beam flux variation on the signal intensity, the peak areas of Fe and calcium were normalized to the current intensity (I0) in an ionization chamber, which was placed in front of the samples. The peak areas were used for estimating the relative iron and calcium content in the samples. 35 The results were analyzed using Origin 8.0 (Northampton, MA, USA), and iron signals in the tip of the heart were compared among the different groups.
Data analysis. All the data are expressed as the mean ± S.D. The statistical analysis of four groups difference was assessed by a one-way analysis of variance followed by a post hoc test. The student's t-test was performed to compare between the two groups data. Differences were considered significant for Po0.05. All the tests were performed with SPSS21.0 (Armonk, NY, USA).

Conflict of Interest
The authors declare no conflict of interest.