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Characterization of mitochondria isolated from normal and ischemic hearts in rats utilizing atomic force microscopy
Micron 42 (2011) 299–304
Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Characterization of mitochondria isolated from normal and ischemic hearts in rats utilizing atomic force microscopy Gi-Ja Lee a,b , Su-Jin Chae a,b , Jae Hoon Jeong c,d , So-Ra Lee e , Sang-Jin Ha e , Youngmi Kim Pak c,d , Weon Kim e , Hun-Kuk Park a,b,∗ a
Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea Healthcare Industry Research Institute, Kyung Hee University, Seoul 130-701, Republic of Korea c Department of Physiology, College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea d Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea e Department of Internal Medicine, Kyung Hee University Medical Center, Seoul 130-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 19 June 2010 Received in revised form 3 September 2010 Accepted 4 September 2010 Keywords: Atomic force microscopy Mitochondria Myocardial infarction Swelling Force–distance curve
a b s t r a c t Mitochondria play critical roles in both the life and the death of cardiac myocytes. Various factors, such as the loss of ATP synthesis and increase of ATP hydrolysis, impairment in ionic homeostasis, formation of reactive oxygen species (ROS), and release of proapoptotic proteins are related to the generation of irreversible damage. It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli. However, there is a controversy about whether or not the mitochondria, indeed, swell during apoptosis. The major advantages of atomic force microscopy (AFM) over conventional optical and electron microscopes for bio-imaging include the fact that no special coating and vacuum are required and imaging can be done in all environments—air, vacuum or aqueous conditions. In addition, AFM force–distance curve measurements have become a fundamental tool in the fields of surface chemistry, biochemistry, and material science. In this study, we used AFM to observe the morphological and property changes in heart mitochondria that were isolated from a rat myocardial infarction model. From the shape parameters of the mitochondria in the AFM topographic image, it seemed that myocardial infarction caused the mitochondrial swelling. Also, the results of force–distance measurements showed that the adhesion force of heart mitochondria was significantly decreased by myocardial in infarction. Therefore, we suggested that myocardial infarction might be the cause of mitochondrial swelling and the changes in outer membrane of heart mitochondria. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Cardiac mitochondrial dysfunction plays an important role in the pathology of myocardial infarction. Mitochondrial dysfunction gives rise to irreversible damage in cell viability through the following events: loss of adenosine triphosphate (ATP) synthesis and increase of ATP hydrolysis, impairment in ionic homeostasis, formation of reactive oxygen species (ROS) and release of proapoptotic proteins (Lisa and Bernardi, 2006). Changes in mitochondrial morphology, such as swelling and condensation have been associated with a wide range of important biological functions and pathologies (Wilson et al., 2005). In particular, it has been widely known that mitochondrial swelling is one
∗ Corresponding author at: Department of Biomedical Engineering, College of Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 0290; fax: +82 2 6008 5535. E-mail address: [email protected] (H.-K. Park). 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.09.002
of the most important indicators of the opening of the mitochondrial permeability transition (MPT) pore. The opening of channels or swelling sufficient to rupture the outer mitochondrial membrane may cause the release of cytochrome c, which, in turn, leads to necrotic or apoptotic cell death (Green and Reed, 1998). However, the supporting evidence for the mitochondrial swelling theory is based mainly on indirect observations using specific inhibitors of the PT pore or PT pore opening agent (Bradham et al., 1998; Kroemer et al., 1997; Narita et al., 1998; Pastorino et al., 1999). In addition, there is a controversy about whether or not the mitochondria indeed swell during apoptosis. In vitro assessment of mitochondrial swelling is widely performed in suspensions of isolated mitochondria by measuring light scatter (Wilson et al., 2005), and in situ fluorescence microscopic observations for the detection of the temporal dynamics of mitochondrial swelling in living specimens are often presented without quantification (Shalbuyeva et al., 2006). Gerencser et al. (2008) reported a novel quantitative in situ single-cell assay of mitochondrial swelling based on standard wide-field or confocal
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fluorescence microscopy. As mitochondrial swelling is an ultrastructural change, quantitative analysis for morphological changes of mitochondria requires high-resolution microscopic methods, such as electron microscopy (EM) or atomic force microscopy (AFM). And previous studies about mitochondrial swelling have been limited to in vitro assessment utilizing cells and simple observation without quantification. The major advantages of AFM over conventional optical and electron microscopy for bioimaging include the lack of a requirement for special coatings or vacuum and the ability to perform imaging in any environment including air, vacuum or aqueous conditions. AFM has become an important medical and biological tool for the non-invasive imaging of cells and materials since its invention by Binnig et al. (1986). Although AFM was originally used to obtain surface topography, it can also precisely measure interactions between its probe tip and the sample surface, and can be used for force–distance curve measurements, which are a fundamental tool in surface chemistry, biochemistry, and material science (Lee et al., 2010; Xu et al., 2007). The slope of an extended half in a force–distance curve is used to determine the stiffness of materials (Bushell et al., 1999; Velegol and Logan, 2002; Volle et al., 2008), and the retracted half in a force–distance curve is used to determine the adhesion force (Yan et al., 2009). In this study, we investigated quantitatively the morphological changes in rat heart mitochondria that were induced by ischemic stimuli utilizing AFM. We also simultaneously examined the nano-mechanical changes in rat heart mitochondria by myocardial infarction using force–distance curve measurements. 2. Materials and methods 2.1. Animals Six male Sprague–Dawley rats (200–300 g) were used for the experiment after 1 week of acclimation under standard laboratory conditions at 22 ± 2 ◦ C, constant humidity, and photoperiod (12 h light–dark cycle). Commercial rat chow and water were provided ad libitum. The experiment was performed in accordance with the “Guide for the Care and Use of Laboratory Animals,” prepared by the Institute of Laboratory Animal Resources and with prior approval by the Animal Experimentation Committee of the Kyung Hee University School of Medicine. 2.2. Induction of myocardial infarction Rats were anesthetized by an intraperitoneal injection (0.9 ml/100 g body weight) of a mixture of ketamine (50 mg/ml) and xylene (20 mg/ml) at the ratio of 6.25:1. Following tracheotomy, rats were connected to a ventilator, and the respiratory rate was adjusted to obtain an arterial pH of 7.35–7.45. Rats were randomly divided into two groups: control group (n = 3) and myocardial infarction group (n = 3). Myocardial infarction was induced by the permanent occlusion of the left anterior descending (LAD) coronary artery, as described previously (Lygate et al., 2003). The LAD was ligated at 2 mm from the origin, using a 5-0 proline suture. In sham-operated control rats, the same procedure was followed, but the ligation suture was not placed in the heart. Studies commenced 3 days after permanent myocardial infarction (Fishbein et al., 1978). The area of myocardial infarction was confirmed by eye and optical microscope, as shown in Fig. 1(a). 2.3. Mitochondria isolation Subcellular fractions of nuclei or mitochondria were isolated by differential centrifugation from normal and ischemic rat hearts,
Fig. 1. (a) The optical microscope image of a heart at 3 days after permanent myocardial infarction. (b) The purity results of mitochondria isolated from normal and ischemic hearts by Western blot analysis (Crude mito: mitochondrial fraction isolated via the differential centrifugation method; Pure mito: mitochondrial fraction isolated by ultracentrifugation method; Nuclei: nucleus fraction). Nucleic markers for HDAC and PARP were expressed only in the nucleus, and mitochondrial markers for Hsp60 and Tfam were detected only in the mitochondrial fraction. Cytosol markers for SOD-1 and -tubulin were not presented in either the nucleus or the mitochondrial fraction. The Western blotting analysis signifies that the mitochondria were clearly isolated with no cytosol contamination.
as described previously (Choi et al., 2005). Highly enriched mitochondria were obtained by additional ultra-centrifugation using 30–50% (1.1 and 1.6 g/ml) OptiprepTM density gradient media (Sigma–Aldrich, St. Louis, MO, USA). The purity of the mitochondria was confirmed by Western blot analysis using anti-HDAC (Abcam Inc., Cambridge, MA), anti-poly(ADP-ribose) polymerase (PARP) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Hsp60 (Santa Cruz Biotechnology), anti-mtTFA (Santa Cruz Biotechnology), antiSOD1 (Santa Cruz Biotechnology, CA, USA), and anti-beta-tubulin (Abcam Inc., Cambridge, MA, USA) antibodies, which are markers for nuclei, mitochondria, and cytoplasm, respectively, as shown in Fig. 1(b). Proteins (30 g) were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane (Schleicher & Schuell BioScience, Inc., Keene, NH, USA). The membrane was incubated with primary antibody overnight at 4 ◦ C. HRP-conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA, USA) followed by ECL (Amersham Biosciences Inc., Piscataway, NJ, USA) were used for detection. 2.4. AFM measurements The mitochondrial solution was diluted with adsorption buffer (10 mM Tris–HCl (pH 7.2), 150 mM KCl, 25 mM MgCl2 ) and dropped onto a fresh mica surface. The prepared samples were briefly air-dried at room temperature and immediately imaged by AFM. Imaging was performed using the non-contact mode of NANOS N8 NEOS (Bruker, Herzogenrath, Germany) equipped with a 42.5 m × 42.5 m × 4 m XYZ scanner and two Zeiss optical microscopes (Epiplan 200× and 500×). External noise was eliminated by placing the AFM on an active vibration isolation table (Table Stable Ltd., Surface Imaging Systems, Herzogenrath, Germany) inside a passive vibration isolation table (Pucotech, Seoul, Republic of Korea). The mitochondria on mica
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were scanned at a resolution of 512 × 512 pixels, with a scan rate of 0.8 line/s. Force–distance curve measurements were performed by the reflex-coated silicon cantilevers for the contact mode (PR-CO, Surface Imaging Systems, Germany) which had a spring constant of 0.2 N/m. The mitochondrial force data were obtained at locations with similar heights to avoid edge effects. The shape parameters of the mitochondria, including their areas, perimeters, lengths, breadths, and aspect ratios, were measured from the topographic images using the Scanning Probe Imaging Processor (SPIPTM , Image Metrology, Hørsholm, Denmark). Fifty mitochondria were selected and their shape parameters measured in each specimen, and 30 sites of mitochondria were selected for the force–distance measurements. The adhesion force was calculated using the SPIPTM software from the retraction process of the force–distance curve, and the stiffness was obtained from the slope of the linear region of the extension curve (Yan et al., 2009). 2.5. Statistics The results of the shape parameters and adhesion forces were expressed as the mean ± standard deviations (SD). A statistical analysis was performed to compare the ultrastructural and nanomechanical changes between the normal and ischemic heart mitochondria using a two-tailed Student’s t-test. P-values less than 0.05 were regarded as statistically significant.
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3. Results 3.1. AFM imaging of heart mitochondria isolated from normal and ischemic rats Initially, the purity of mitochondria was confirmed by Western blot analysis using markers for nuclei, mitochondria, and cytoplasm, respectively. As shown in Fig. 1(b), the Western blotting analysis represented that the mitochondria were clearly isolated with no cytosol contamination. We observed AFM topographical images of 5 m × 5 m to get an initial impression of the morphological differences between normal and infracted mitochondria. As shown in Fig. 2, normal mitochondria showed a homogeneous distribution with similar sizes and shapes. However, irregularities in the shapes and sizes of mitochondria were observed in those of ischemic rats. To observe the changes in the surfaces of the mitochondria, we utilized the magnified phase images of 1 m × 1 m in normal and ischemic mitochondria. As shown in Fig. 3, the surfaces of normal mitochondria looked smooth and had integrity. However, the surfaces of ischemic mitochondria became rugged. Some debris could be observed around the apical ends of the mitochondrial membranes and the outer membranes had collapsed. To characterize the morphological changes in mitochondria by myocardial infarction, the particle analysis module was used for the detection and quantification of mitochondria. Normal mitochon-
Fig. 2. Representative AFM topographic images (5 m × 5 m) of mitochondria isolated from normal (a) and ischemic (b) hearts. The number and perimeter distribution of the mitochondria (c) were obtained from images (a) and (b).
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Fig. 3. Representative AFM phase images (1 m × 1 m) of mitochondria isolated from normal (a) and ischemic (b) hearts.
dria appeared ellipsoidal (mean axial ratio of 1.33 ± 0.14) with a mean area of 4019 ± 1215 nm2 (determined from 50 mitochondria from three different preparations). AFM height and width measurements revealed mitochondria to be unexpectedly flattened, presumably caused by adsorption to the mica surface (Wagner et al., 2003). As compared to the corresponding normal values, ischemic mitochondria showed a significant increase in all parameters, as shown in Table 1. The mean axial ratio and area were 1.14 ± 0.09 and 37,859 ± 32,381 nm2 (n = 50, p < 0.0001 vs. control, respectively). Ultrastructural analysis of mitochondria utilizing AFM demonstrated that myocardial infarction resulted in significant increase in heart mitochondrial size, as compared with that of normal mitochondria.
3.2. The changes in biomechanical property of mitochondria by myocardial infarction The force–distance curve measurements were performed to investigate the change in the adhesive force of mitochondria affected by myocardial infarction. Fig. 4 presents representative force–distance curves of mitochondria isolated from normal and ischemic hearts in rats. As shown in Fig. 5, the adhesion force of ischemic heart mitochondria significantly decreased to 19.56 ± 1.08 nN (n = 30, p < 0.0001), as compared to normal ones with an adhesion force of 27.64 ± 0.88 nN. Also, the stiffnesses calculated from the slopes of the approach curves of normal and ischemic heart mitochondria were 0.205 ± 0.007 and 0.211 ± 0.011 nN/nm (p < 0.05), respec-
Table 1 The shape parameter analysis for the morphological changes in mitochondria due to myocardial infarction. Parameters
Normal heart mitochondria
Area (nm2 ) Perimeter (nm) Length (nm) Breadth (nm) Aspect ratio
4019 235.26 84.68 63.96 1.33
± ± ± ± ±
1215 33.76 12.71 10.35 0.14
Ischemic heart mitochondria 37,859 671.75 221.83 196.65 1.14
± ± ± ± ±
32,381 269.59 84.07 80.15 0.09
p-Value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
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Fig. 4. Representative results of force–distance measurements of mitochondria isolated from normal (a) and ischemic (b) hearts.
tively. It seemed that the ischemic mitochondria were rather stiffer than normal ones. 4. Discussion Mitochondria not only play an important role in myocardial energy metabolism and calcium homeostasis, but are also responsible for the production of reactive oxygen species (Gustafsson and Gottlieb, 2008). In particular, it has been known that induction of MPT during ischemia–reperfusion injury is a critical event that triggers both necrotic and apoptotic cell death (Keith and Bhatnagar, 2010). Nevertheless, the role of the mitochondria in myocardial ischemia-induced injury is complex, and the signal transduction mechanisms that regulate MPT and its downstream consequences remain unclear. It is well known that permanent ischemia causes a loss of matrix density, and this is associated with mitochondrial swelling (Schild et al., 2003). It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli (Sergey et al., 2008). The most widely recognized biochemical features of apoptosis during ischemic stimuli are the
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release of cytochrome c from the mitochondria and the activation of a class of cysteine proteases, called caspase, including caspase3, caspase-8 and caspase-9 (Zhang et al., 2010). However, there is some controversy about whether or not the mitochondria, indeed, swell during apoptosis. As simple optical transmission measurements are sensitive to changes in mitochondrial morphology in preparations of isolated mitochondria, they have been used for many years (Mourant et al., 1998, 2001, 2002). Wilson et al. (2005) reported the oxidative stress-induced changes in mitochondrial morphology in suspensions of EMT6 mouse mammary carcinoma cells and in mitochondria isolated from rabbit liver using angularly resolved light scattering measurements. Gerencser et al. (2008) introduced a novel morphometric technique to quantify the relative diameter of mitochondria labeled by targeted fluorescent proteins. However, there are few reports on the shape changes and swelling of mitochondria isolated from the heart after myocardial infarction utilizing optical measurements, let alone AFM. In this study, we used AFM to observe the morphological and mechanical changes in heart mitochondria which were isolated from a rat myocardial infarction model. Firstly, the morphological differences in the mitochondria between normal and ischemic hearts were quantitatively evaluated using particle shape analysis on AFM topographic images. As a result, it seemed that myocardial infarction resulted in mitochondrial swelling. Several studies have reported on the relationship between mitochondrial swelling and cell death (Carreira et al., 2008; Zhang et al., 2010). He et al. (2010) revealed that, when mitochondria in the cells were damaged more seriously, there were obviously swollen mitochondria and mitochondria in which cristae had almost perfectly disappeared and more vacuolar mitochondria were found. Secondly, force–distance curve measurements were used to investigate the changes in adhesion forces of swollen mitochondria affected by myocardial infarction. Many studies have reported that AFM is a powerful tool to detect the changes in the morphology and biomechanical properties of cells (Cai et al., 2009; Guck et al., 2005; Lee et al., 2010; Suresh, 2007). Wu et al. (2009) reported that the changes in cell volume occurred mainly due to the collapse of red blood cells, and the curves of adhesive force showed the dramatic alterations in the viscoelasticity of red blood cells. In addition, Yan et al. (2009) showed using force–distance measurement that the biomechanical properties of keratocytes changed significantly after culturing on different substrates. However, there have been no studies on the nano-mechanical properties of mitochondria utilizing AFM. The mitochondrial outer membrane consists of a relatively simple phospholipid bilayer containing proteins such as porins. Therefore, we inferred that the changes in the adhesion force of mitochondria might be related to destruction of the highly dense mitochondrial outer membrane by swelling. Additionally, it
Fig. 5. Quantitative analysis of (a) adhesion force and (b) stiffness of normal and ischemic heart mitochondria. († p < 0.05, ‡ p < 0.0001).
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seemed that ischemic stimuli might induce the stiffening of mitochondria. In future studies, we will examine the morphological and mechanical changes in rat heart mitochondria by drugs which were known for protection of myocardial tissue from myocardial ischemia–reperfusion injury. 5. Conclusions This is the first study using AFM to investigate the morphological and nano-mechanical changes in the isolated rat heart mitochondria after myocardial infarction. From the particle analysis of AFM topographic images, we quantitatively evaluated structural changes in heart mitochondria induced by myocardial infarction. Furthermore, the viscoelastic change in ischemic heart mitochondria was revealed by force–distance analysis. Therefore, we suggest that myocardial infarction might be the cause of mitochondrial swelling and the changes in the adhesion force and stiffness of mitochondria. Acknowledgement This study was supported by Bio R&D program through the national research foundation of Korea funded by the Ministry of Education, Science and Technology (2010-0019912 and 20090084844). References Binnig, G., Quate, C.F., Gerber, C., 1986. Atomic force microscope. Phys. Rev. Lett. 56, 930–933. Bradham, C.A., Qian, T., Streetz, K., Trautwein, C., Brenner, D.A., Lemasters, J.J., 1998. The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release. Mol. Cell. Biol. 18, 6353–6364. Bushell, G.R., Cahill, C., Clarke, F.M., Gibson, C.T., Myhra, S., Watson, G.S., 1999. Imaging and force–distance analysis of human fibroblasts in vitro by atomic force microscopy. Cytometry 36, 254–264. Cai, X., Gao, S., Cai, J., 2009. Artesunate induced morphological and mechanical changes of Jurkat cell studied by AFM. Scanning 31, 83–89. Carreira, R.S., Monteiro, P., Kowaltowski, A.J., Gonc¸alves, L.M., Providência, L.A., 2008. Nicorandil protects cardiac mitochondria against permeability transition induced by ischemia–reperfusion. J. Bioenerg. Biomembr. 40, 95–102. Choi, Y.S., Ryu, B.K., Min, H.K., Lee, S.W., Pak, Y.K., 2005. Analysis of proteome bound to D-loop region of mitochondrial DNA by DNA-linked affinity chromatography and reverse-phase liquid chromatography/tandem mass spectrometry. Ann. N.Y. Acad. Sci. 1042, 88–100. Fishbein, M.C., Maclean, D., Maroko, P.R., 1978. Experimental myocardial infarction in the rat: qualitative and quantitative changes during pathologic evolution. Am. J. Pathol. 90, 57–70. Gerencser, A.A., Doczi, J., Torocsik, B., Bossy-Wetzel, E., Adam-Vizi, V., 2008. Mitochondrial swelling measurement in situ by optimized spatial filtering: astrocyte–neuron differences. Biophys. J. 95, 2583–2598. Green, D.R., Reed, J.C., 1998. Mitochondria and apoptosis. Science 281, 1309–1312. Guck, J., Schinkinger, S., Lincoln, B., Wottawah, F., Ebert, S., Romeyke, M., Lenz, D., Erickson, H.M., Ananthakrishnan, R., Mitchell, D., Käs, J., Ulvick, S., Bilby, C., 2005. Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. Biophys. J. 88, 3689–3698. Gustafsson, A.B., Gottlieb, R.A., 2008. Heart mitochondria: gates of life and death. Cardiovasc. Res. 77, 334–343.
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Contents lists available at ScienceDirect
Micron journal homepage: www.elsevier.com/locate/micron
Characterization of mitochondria isolated from normal and ischemic hearts in rats utilizing atomic force microscopy Gi-Ja Lee a,b , Su-Jin Chae a,b , Jae Hoon Jeong c,d , So-Ra Lee e , Sang-Jin Ha e , Youngmi Kim Pak c,d , Weon Kim e , Hun-Kuk Park a,b,∗ a
Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea Healthcare Industry Research Institute, Kyung Hee University, Seoul 130-701, Republic of Korea c Department of Physiology, College of Medicine, Kyung Hee University, Seoul 130-701, Republic of Korea d Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul 130-701, Republic of Korea e Department of Internal Medicine, Kyung Hee University Medical Center, Seoul 130-701, Republic of Korea b
a r t i c l e
i n f o
Article history: Received 19 June 2010 Received in revised form 3 September 2010 Accepted 4 September 2010 Keywords: Atomic force microscopy Mitochondria Myocardial infarction Swelling Force–distance curve
a b s t r a c t Mitochondria play critical roles in both the life and the death of cardiac myocytes. Various factors, such as the loss of ATP synthesis and increase of ATP hydrolysis, impairment in ionic homeostasis, formation of reactive oxygen species (ROS), and release of proapoptotic proteins are related to the generation of irreversible damage. It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli. However, there is a controversy about whether or not the mitochondria, indeed, swell during apoptosis. The major advantages of atomic force microscopy (AFM) over conventional optical and electron microscopes for bio-imaging include the fact that no special coating and vacuum are required and imaging can be done in all environments—air, vacuum or aqueous conditions. In addition, AFM force–distance curve measurements have become a fundamental tool in the fields of surface chemistry, biochemistry, and material science. In this study, we used AFM to observe the morphological and property changes in heart mitochondria that were isolated from a rat myocardial infarction model. From the shape parameters of the mitochondria in the AFM topographic image, it seemed that myocardial infarction caused the mitochondrial swelling. Also, the results of force–distance measurements showed that the adhesion force of heart mitochondria was significantly decreased by myocardial in infarction. Therefore, we suggested that myocardial infarction might be the cause of mitochondrial swelling and the changes in outer membrane of heart mitochondria. © 2010 Elsevier Ltd. All rights reserved.
1. Introduction Cardiac mitochondrial dysfunction plays an important role in the pathology of myocardial infarction. Mitochondrial dysfunction gives rise to irreversible damage in cell viability through the following events: loss of adenosine triphosphate (ATP) synthesis and increase of ATP hydrolysis, impairment in ionic homeostasis, formation of reactive oxygen species (ROS) and release of proapoptotic proteins (Lisa and Bernardi, 2006). Changes in mitochondrial morphology, such as swelling and condensation have been associated with a wide range of important biological functions and pathologies (Wilson et al., 2005). In particular, it has been widely known that mitochondrial swelling is one
∗ Corresponding author at: Department of Biomedical Engineering, College of Medicine, Kyung Hee University, 1 Hoegi-dong, Dongdaemun-gu, Seoul 130-701, Republic of Korea. Tel.: +82 2 961 0290; fax: +82 2 6008 5535. E-mail address: [email protected] (H.-K. Park). 0968-4328/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.micron.2010.09.002
of the most important indicators of the opening of the mitochondrial permeability transition (MPT) pore. The opening of channels or swelling sufficient to rupture the outer mitochondrial membrane may cause the release of cytochrome c, which, in turn, leads to necrotic or apoptotic cell death (Green and Reed, 1998). However, the supporting evidence for the mitochondrial swelling theory is based mainly on indirect observations using specific inhibitors of the PT pore or PT pore opening agent (Bradham et al., 1998; Kroemer et al., 1997; Narita et al., 1998; Pastorino et al., 1999). In addition, there is a controversy about whether or not the mitochondria indeed swell during apoptosis. In vitro assessment of mitochondrial swelling is widely performed in suspensions of isolated mitochondria by measuring light scatter (Wilson et al., 2005), and in situ fluorescence microscopic observations for the detection of the temporal dynamics of mitochondrial swelling in living specimens are often presented without quantification (Shalbuyeva et al., 2006). Gerencser et al. (2008) reported a novel quantitative in situ single-cell assay of mitochondrial swelling based on standard wide-field or confocal
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fluorescence microscopy. As mitochondrial swelling is an ultrastructural change, quantitative analysis for morphological changes of mitochondria requires high-resolution microscopic methods, such as electron microscopy (EM) or atomic force microscopy (AFM). And previous studies about mitochondrial swelling have been limited to in vitro assessment utilizing cells and simple observation without quantification. The major advantages of AFM over conventional optical and electron microscopy for bioimaging include the lack of a requirement for special coatings or vacuum and the ability to perform imaging in any environment including air, vacuum or aqueous conditions. AFM has become an important medical and biological tool for the non-invasive imaging of cells and materials since its invention by Binnig et al. (1986). Although AFM was originally used to obtain surface topography, it can also precisely measure interactions between its probe tip and the sample surface, and can be used for force–distance curve measurements, which are a fundamental tool in surface chemistry, biochemistry, and material science (Lee et al., 2010; Xu et al., 2007). The slope of an extended half in a force–distance curve is used to determine the stiffness of materials (Bushell et al., 1999; Velegol and Logan, 2002; Volle et al., 2008), and the retracted half in a force–distance curve is used to determine the adhesion force (Yan et al., 2009). In this study, we investigated quantitatively the morphological changes in rat heart mitochondria that were induced by ischemic stimuli utilizing AFM. We also simultaneously examined the nano-mechanical changes in rat heart mitochondria by myocardial infarction using force–distance curve measurements. 2. Materials and methods 2.1. Animals Six male Sprague–Dawley rats (200–300 g) were used for the experiment after 1 week of acclimation under standard laboratory conditions at 22 ± 2 ◦ C, constant humidity, and photoperiod (12 h light–dark cycle). Commercial rat chow and water were provided ad libitum. The experiment was performed in accordance with the “Guide for the Care and Use of Laboratory Animals,” prepared by the Institute of Laboratory Animal Resources and with prior approval by the Animal Experimentation Committee of the Kyung Hee University School of Medicine. 2.2. Induction of myocardial infarction Rats were anesthetized by an intraperitoneal injection (0.9 ml/100 g body weight) of a mixture of ketamine (50 mg/ml) and xylene (20 mg/ml) at the ratio of 6.25:1. Following tracheotomy, rats were connected to a ventilator, and the respiratory rate was adjusted to obtain an arterial pH of 7.35–7.45. Rats were randomly divided into two groups: control group (n = 3) and myocardial infarction group (n = 3). Myocardial infarction was induced by the permanent occlusion of the left anterior descending (LAD) coronary artery, as described previously (Lygate et al., 2003). The LAD was ligated at 2 mm from the origin, using a 5-0 proline suture. In sham-operated control rats, the same procedure was followed, but the ligation suture was not placed in the heart. Studies commenced 3 days after permanent myocardial infarction (Fishbein et al., 1978). The area of myocardial infarction was confirmed by eye and optical microscope, as shown in Fig. 1(a). 2.3. Mitochondria isolation Subcellular fractions of nuclei or mitochondria were isolated by differential centrifugation from normal and ischemic rat hearts,
Fig. 1. (a) The optical microscope image of a heart at 3 days after permanent myocardial infarction. (b) The purity results of mitochondria isolated from normal and ischemic hearts by Western blot analysis (Crude mito: mitochondrial fraction isolated via the differential centrifugation method; Pure mito: mitochondrial fraction isolated by ultracentrifugation method; Nuclei: nucleus fraction). Nucleic markers for HDAC and PARP were expressed only in the nucleus, and mitochondrial markers for Hsp60 and Tfam were detected only in the mitochondrial fraction. Cytosol markers for SOD-1 and -tubulin were not presented in either the nucleus or the mitochondrial fraction. The Western blotting analysis signifies that the mitochondria were clearly isolated with no cytosol contamination.
as described previously (Choi et al., 2005). Highly enriched mitochondria were obtained by additional ultra-centrifugation using 30–50% (1.1 and 1.6 g/ml) OptiprepTM density gradient media (Sigma–Aldrich, St. Louis, MO, USA). The purity of the mitochondria was confirmed by Western blot analysis using anti-HDAC (Abcam Inc., Cambridge, MA), anti-poly(ADP-ribose) polymerase (PARP) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-Hsp60 (Santa Cruz Biotechnology), anti-mtTFA (Santa Cruz Biotechnology), antiSOD1 (Santa Cruz Biotechnology, CA, USA), and anti-beta-tubulin (Abcam Inc., Cambridge, MA, USA) antibodies, which are markers for nuclei, mitochondria, and cytoplasm, respectively, as shown in Fig. 1(b). Proteins (30 g) were separated by 12% SDS-PAGE and transferred onto nitrocellulose membrane (Schleicher & Schuell BioScience, Inc., Keene, NH, USA). The membrane was incubated with primary antibody overnight at 4 ◦ C. HRP-conjugated secondary antibodies (Cell Signaling Technology, Beverly, MA, USA) followed by ECL (Amersham Biosciences Inc., Piscataway, NJ, USA) were used for detection. 2.4. AFM measurements The mitochondrial solution was diluted with adsorption buffer (10 mM Tris–HCl (pH 7.2), 150 mM KCl, 25 mM MgCl2 ) and dropped onto a fresh mica surface. The prepared samples were briefly air-dried at room temperature and immediately imaged by AFM. Imaging was performed using the non-contact mode of NANOS N8 NEOS (Bruker, Herzogenrath, Germany) equipped with a 42.5 m × 42.5 m × 4 m XYZ scanner and two Zeiss optical microscopes (Epiplan 200× and 500×). External noise was eliminated by placing the AFM on an active vibration isolation table (Table Stable Ltd., Surface Imaging Systems, Herzogenrath, Germany) inside a passive vibration isolation table (Pucotech, Seoul, Republic of Korea). The mitochondria on mica
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were scanned at a resolution of 512 × 512 pixels, with a scan rate of 0.8 line/s. Force–distance curve measurements were performed by the reflex-coated silicon cantilevers for the contact mode (PR-CO, Surface Imaging Systems, Germany) which had a spring constant of 0.2 N/m. The mitochondrial force data were obtained at locations with similar heights to avoid edge effects. The shape parameters of the mitochondria, including their areas, perimeters, lengths, breadths, and aspect ratios, were measured from the topographic images using the Scanning Probe Imaging Processor (SPIPTM , Image Metrology, Hørsholm, Denmark). Fifty mitochondria were selected and their shape parameters measured in each specimen, and 30 sites of mitochondria were selected for the force–distance measurements. The adhesion force was calculated using the SPIPTM software from the retraction process of the force–distance curve, and the stiffness was obtained from the slope of the linear region of the extension curve (Yan et al., 2009). 2.5. Statistics The results of the shape parameters and adhesion forces were expressed as the mean ± standard deviations (SD). A statistical analysis was performed to compare the ultrastructural and nanomechanical changes between the normal and ischemic heart mitochondria using a two-tailed Student’s t-test. P-values less than 0.05 were regarded as statistically significant.
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3. Results 3.1. AFM imaging of heart mitochondria isolated from normal and ischemic rats Initially, the purity of mitochondria was confirmed by Western blot analysis using markers for nuclei, mitochondria, and cytoplasm, respectively. As shown in Fig. 1(b), the Western blotting analysis represented that the mitochondria were clearly isolated with no cytosol contamination. We observed AFM topographical images of 5 m × 5 m to get an initial impression of the morphological differences between normal and infracted mitochondria. As shown in Fig. 2, normal mitochondria showed a homogeneous distribution with similar sizes and shapes. However, irregularities in the shapes and sizes of mitochondria were observed in those of ischemic rats. To observe the changes in the surfaces of the mitochondria, we utilized the magnified phase images of 1 m × 1 m in normal and ischemic mitochondria. As shown in Fig. 3, the surfaces of normal mitochondria looked smooth and had integrity. However, the surfaces of ischemic mitochondria became rugged. Some debris could be observed around the apical ends of the mitochondrial membranes and the outer membranes had collapsed. To characterize the morphological changes in mitochondria by myocardial infarction, the particle analysis module was used for the detection and quantification of mitochondria. Normal mitochon-
Fig. 2. Representative AFM topographic images (5 m × 5 m) of mitochondria isolated from normal (a) and ischemic (b) hearts. The number and perimeter distribution of the mitochondria (c) were obtained from images (a) and (b).
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Fig. 3. Representative AFM phase images (1 m × 1 m) of mitochondria isolated from normal (a) and ischemic (b) hearts.
dria appeared ellipsoidal (mean axial ratio of 1.33 ± 0.14) with a mean area of 4019 ± 1215 nm2 (determined from 50 mitochondria from three different preparations). AFM height and width measurements revealed mitochondria to be unexpectedly flattened, presumably caused by adsorption to the mica surface (Wagner et al., 2003). As compared to the corresponding normal values, ischemic mitochondria showed a significant increase in all parameters, as shown in Table 1. The mean axial ratio and area were 1.14 ± 0.09 and 37,859 ± 32,381 nm2 (n = 50, p < 0.0001 vs. control, respectively). Ultrastructural analysis of mitochondria utilizing AFM demonstrated that myocardial infarction resulted in significant increase in heart mitochondrial size, as compared with that of normal mitochondria.
3.2. The changes in biomechanical property of mitochondria by myocardial infarction The force–distance curve measurements were performed to investigate the change in the adhesive force of mitochondria affected by myocardial infarction. Fig. 4 presents representative force–distance curves of mitochondria isolated from normal and ischemic hearts in rats. As shown in Fig. 5, the adhesion force of ischemic heart mitochondria significantly decreased to 19.56 ± 1.08 nN (n = 30, p < 0.0001), as compared to normal ones with an adhesion force of 27.64 ± 0.88 nN. Also, the stiffnesses calculated from the slopes of the approach curves of normal and ischemic heart mitochondria were 0.205 ± 0.007 and 0.211 ± 0.011 nN/nm (p < 0.05), respec-
Table 1 The shape parameter analysis for the morphological changes in mitochondria due to myocardial infarction. Parameters
Normal heart mitochondria
Area (nm2 ) Perimeter (nm) Length (nm) Breadth (nm) Aspect ratio
4019 235.26 84.68 63.96 1.33
± ± ± ± ±
1215 33.76 12.71 10.35 0.14
Ischemic heart mitochondria 37,859 671.75 221.83 196.65 1.14
± ± ± ± ±
32,381 269.59 84.07 80.15 0.09
p-Value <0.0001 <0.0001 <0.0001 <0.0001 <0.0001
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Fig. 4. Representative results of force–distance measurements of mitochondria isolated from normal (a) and ischemic (b) hearts.
tively. It seemed that the ischemic mitochondria were rather stiffer than normal ones. 4. Discussion Mitochondria not only play an important role in myocardial energy metabolism and calcium homeostasis, but are also responsible for the production of reactive oxygen species (Gustafsson and Gottlieb, 2008). In particular, it has been known that induction of MPT during ischemia–reperfusion injury is a critical event that triggers both necrotic and apoptotic cell death (Keith and Bhatnagar, 2010). Nevertheless, the role of the mitochondria in myocardial ischemia-induced injury is complex, and the signal transduction mechanisms that regulate MPT and its downstream consequences remain unclear. It is well known that permanent ischemia causes a loss of matrix density, and this is associated with mitochondrial swelling (Schild et al., 2003). It has been proposed that the release of cytochrome c is caused by a swelling of the mitochondrial matrix triggered by the apoptotic stimuli (Sergey et al., 2008). The most widely recognized biochemical features of apoptosis during ischemic stimuli are the
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release of cytochrome c from the mitochondria and the activation of a class of cysteine proteases, called caspase, including caspase3, caspase-8 and caspase-9 (Zhang et al., 2010). However, there is some controversy about whether or not the mitochondria, indeed, swell during apoptosis. As simple optical transmission measurements are sensitive to changes in mitochondrial morphology in preparations of isolated mitochondria, they have been used for many years (Mourant et al., 1998, 2001, 2002). Wilson et al. (2005) reported the oxidative stress-induced changes in mitochondrial morphology in suspensions of EMT6 mouse mammary carcinoma cells and in mitochondria isolated from rabbit liver using angularly resolved light scattering measurements. Gerencser et al. (2008) introduced a novel morphometric technique to quantify the relative diameter of mitochondria labeled by targeted fluorescent proteins. However, there are few reports on the shape changes and swelling of mitochondria isolated from the heart after myocardial infarction utilizing optical measurements, let alone AFM. In this study, we used AFM to observe the morphological and mechanical changes in heart mitochondria which were isolated from a rat myocardial infarction model. Firstly, the morphological differences in the mitochondria between normal and ischemic hearts were quantitatively evaluated using particle shape analysis on AFM topographic images. As a result, it seemed that myocardial infarction resulted in mitochondrial swelling. Several studies have reported on the relationship between mitochondrial swelling and cell death (Carreira et al., 2008; Zhang et al., 2010). He et al. (2010) revealed that, when mitochondria in the cells were damaged more seriously, there were obviously swollen mitochondria and mitochondria in which cristae had almost perfectly disappeared and more vacuolar mitochondria were found. Secondly, force–distance curve measurements were used to investigate the changes in adhesion forces of swollen mitochondria affected by myocardial infarction. Many studies have reported that AFM is a powerful tool to detect the changes in the morphology and biomechanical properties of cells (Cai et al., 2009; Guck et al., 2005; Lee et al., 2010; Suresh, 2007). Wu et al. (2009) reported that the changes in cell volume occurred mainly due to the collapse of red blood cells, and the curves of adhesive force showed the dramatic alterations in the viscoelasticity of red blood cells. In addition, Yan et al. (2009) showed using force–distance measurement that the biomechanical properties of keratocytes changed significantly after culturing on different substrates. However, there have been no studies on the nano-mechanical properties of mitochondria utilizing AFM. The mitochondrial outer membrane consists of a relatively simple phospholipid bilayer containing proteins such as porins. Therefore, we inferred that the changes in the adhesion force of mitochondria might be related to destruction of the highly dense mitochondrial outer membrane by swelling. Additionally, it
Fig. 5. Quantitative analysis of (a) adhesion force and (b) stiffness of normal and ischemic heart mitochondria. († p < 0.05, ‡ p < 0.0001).
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seemed that ischemic stimuli might induce the stiffening of mitochondria. In future studies, we will examine the morphological and mechanical changes in rat heart mitochondria by drugs which were known for protection of myocardial tissue from myocardial ischemia–reperfusion injury. 5. Conclusions This is the first study using AFM to investigate the morphological and nano-mechanical changes in the isolated rat heart mitochondria after myocardial infarction. From the particle analysis of AFM topographic images, we quantitatively evaluated structural changes in heart mitochondria induced by myocardial infarction. Furthermore, the viscoelastic change in ischemic heart mitochondria was revealed by force–distance analysis. Therefore, we suggest that myocardial infarction might be the cause of mitochondrial swelling and the changes in the adhesion force and stiffness of mitochondria. Acknowledgement This study was supported by Bio R&D program through the national research foundation of Korea funded by the Ministry of Education, Science and Technology (2010-0019912 and 20090084844). References Binnig, G., Quate, C.F., Gerber, C., 1986. Atomic force microscope. Phys. Rev. Lett. 56, 930–933. Bradham, C.A., Qian, T., Streetz, K., Trautwein, C., Brenner, D.A., Lemasters, J.J., 1998. The mitochondrial permeability transition is required for tumor necrosis factor alpha-mediated apoptosis and cytochrome c release. Mol. Cell. Biol. 18, 6353–6364. Bushell, G.R., Cahill, C., Clarke, F.M., Gibson, C.T., Myhra, S., Watson, G.S., 1999. Imaging and force–distance analysis of human fibroblasts in vitro by atomic force microscopy. Cytometry 36, 254–264. Cai, X., Gao, S., Cai, J., 2009. Artesunate induced morphological and mechanical changes of Jurkat cell studied by AFM. Scanning 31, 83–89. Carreira, R.S., Monteiro, P., Kowaltowski, A.J., Gonc¸alves, L.M., Providência, L.A., 2008. Nicorandil protects cardiac mitochondria against permeability transition induced by ischemia–reperfusion. J. Bioenerg. Biomembr. 40, 95–102. Choi, Y.S., Ryu, B.K., Min, H.K., Lee, S.W., Pak, Y.K., 2005. Analysis of proteome bound to D-loop region of mitochondrial DNA by DNA-linked affinity chromatography and reverse-phase liquid chromatography/tandem mass spectrometry. Ann. N.Y. Acad. Sci. 1042, 88–100. Fishbein, M.C., Maclean, D., Maroko, P.R., 1978. Experimental myocardial infarction in the rat: qualitative and quantitative changes during pathologic evolution. Am. J. Pathol. 90, 57–70. Gerencser, A.A., Doczi, J., Torocsik, B., Bossy-Wetzel, E., Adam-Vizi, V., 2008. Mitochondrial swelling measurement in situ by optimized spatial filtering: astrocyte–neuron differences. Biophys. J. 95, 2583–2598. Green, D.R., Reed, J.C., 1998. Mitochondria and apoptosis. Science 281, 1309–1312. Guck, J., Schinkinger, S., Lincoln, B., Wottawah, F., Ebert, S., Romeyke, M., Lenz, D., Erickson, H.M., Ananthakrishnan, R., Mitchell, D., Käs, J., Ulvick, S., Bilby, C., 2005. Optical deformability as an inherent cell marker for testing malignant transformation and metastatic competence. Biophys. J. 88, 3689–3698. Gustafsson, A.B., Gottlieb, R.A., 2008. Heart mitochondria: gates of life and death. Cardiovasc. Res. 77, 334–343.
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