Mitochondrial adaptations during myocardial hypertrophy induced by abdominal aortic constriction

    Mitochondrial adaptations during myocardial hypertrophy induced by abdominal aortic constriction Zhusong Mei, Xinxing Wang, Weili Liu, Jingbo Gong, Xiujie Gao, Tao Zhang, Fang Xie, Lingjia Qian PII: DOI: Reference:

S1054-8807(14)00057-X doi: 10.1016/j.carpath.2014.05.003 CVP 6775

To appear in:

Cardiovascular Pathology

Received date: Revised date: Accepted date:

25 March 2014 19 May 2014 19 May 2014

Please cite this article as: Mei Zhusong, Wang Xinxing, Liu Weili, Gong Jingbo, Gao Xiujie, Zhang Tao, Xie Fang, Qian Lingjia, Mitochondrial adaptations during myocardial hypertrophy induced by abdominal aortic constriction, Cardiovascular Pathology (2014), doi: 10.1016/j.carpath.2014.05.003

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Mitochondrial adaptations during myocardial hypertrophy induced by abdominal aortic constriction

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Zhusong Mei1, Xinxing Wang1 , Weili Liu1, Jingbo Gong1, Xiujie Gao1, Tao Zhang1, Fang Xie1, Lingjia Qian1

Key laboratory of stress medicine, Institute of Basic Medical Sciences, 27 Taiping Road,

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Author Affiliations

Haidian District, Beijing, China, 100850

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Short title: Mitochondrial adaptations during myocardial hypertrophy

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Corresponding author: Lingjia Qian Tel: (0086)010-66931393, Fax:(0086)010-68213039,

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E-mail: [email protected]

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Summary:

The mitochondria have an important function in myocardial hypertrophy. In

this study, we found that mitochondrial function and biogenesis decreased during myocardial

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hypertrophy, which could be attributed to the decreased content and activity of mitochondrial complex V dimers and complex I.

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Abstract:

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Introduction: Myocardial hypertrophy is an adaptive response of the heart to work overload. Pathological cardiac hypertrophy is usually associated with the ultimate development of cardiac dysfunction and heart failure. The mitochondria have an important function in the development of cardiac hypertrophy. However, mitochondrial adaptations to hypertrophic stimulus remain

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ambiguous.

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Methods: A rat model of myocardial hypertrophy was established using abdominal aortic constriction. The expression of mitochondrial complexes was evaluated through electrophoresis

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using blue-native and blue-native/SDS. The enzyme activity of mitochondrial complexes was

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detected through in-gel activity. Results: Mitochondrial function and biogenesis decreased in hypertrophied myocardium. The content and activity of mitochondrial complex V dimers and complex I significantly decreased during hypertrophy, as well as those of the α, β, B, and D chains of the complex V dimers. However, the content and activity of mitochondrial complex V oligomers and complexes II, III, and IV did not change. Conclusions: The decreased content and activity of complex V dimers and complex I caused the decline in mitochondrial function and biogenesis during cardiac hypertrophy.

Key words: 2

myocardial hypertrophy, mitochondrial dysfunction; mitochondrial

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complex Ⅴ dimers; mitochondrial complex Ⅴ oligomers

Conflict of interest:

None declared. All authors disclose any commercial

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association or other arrangement that might pose or imply a conflict of interest in connection with the paper

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1. Introduction

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Various stimuli may cause different types of myocardial hypertrophy, which is an adaptive response of the heart to work overload. The mitochondria have important functions in all types of cardiac hypertrophy because of their involvement in energy generation. During cardiac hypertrophy, the expression of mitochondrial proteins is significantly changed, which then affects

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the proteome phenotype and function of the mitochondria [1,2]. Mitochondrial dysfunction

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decreases energy generation and increases cellular reactive oxygen species (ROS) levels, as well as stimulates the myocardium to undergo hypertrophy and contribute to the transformation from

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pathological hypertrophy to heart failure [2,3]. Although the mitochondria have important

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functions in the development of cardiac hypertrophy, their adaptations to hypertrophic stimulus remain ambiguous.

Oxidative phosphorylation (OXPHOS) is the major metabolic pathway in which the mitochondria generate adenosine triphosphate (ATP). OXPHOS is carried out by the electron transport chain, which consists of a series of protein complexes located in the mitochondrial inner membrane. This research aimed to determine the mitochondrial adaptations and investigate their associated mitochondrial complexes during AAC-induced myocardial hypertrophy.

2. Methods 2.1 Construction of the rat model of myocardial hypertrophy 3

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Abdominal aortic constriction (AAC) was performed as previously described [4]. Young male Wistar rats weighing 180 g to 200 g were anaesthetized. After opening the abdomen, the

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suprarenal abdominal aorta was released from the connective tissue and a bent 7-gauge needle was placed next to the abdominal aorta. The suture was securely tied around the needle and the aorta.

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After ligation, the needle was quickly removed. Sham-operated rats underwent the same

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intervention, except that the aorta was not ligated. After echocardiographic analysis at six weeks after AAC, the rats were sacrificed through cervical dislocation and the hearts were removed and weighed promptly. The experimental procedures performed in the rats conformed to the principles of the Laboratory Animal Care published by the US National Institutes of Health (NIH publication

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No. 86-23, revised 1985) and approved by the Animal Subjects Committee of Institute of Basic

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Medical Sciences, Beijing, China (Approval No. 2013-D-2312). 2.2 Mitochondrial function analysis

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The mitochondria were isolated as previously described [5]. Intracellular ATP level and

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H+-ATPase activity were detected using an ATP assay Kit (Beyotime). Mitochondrial membrane potential was determined using Rhodamine123 (Beyotime). All assays were performed according to the manufacturer’s instructions. The details are provided as supplementary data. 2.3 Measurement of mitochondrial DNA(mtDNA) content by real-time PCR Total heart DNA was isolated using a DNeasy tissue kit (Qiagen 69504). DNA Master SYBR green (Roche, Palo Alto, CA) was used for real-time PCR. The content of mtDNA was determined by co-amplifying the mt D-loop and the nuclear-encoded β-actin gene through real-time PCR. The primers and cycling conditions used were previously described by Branda et al. [6]. 2.4 Blue Native(BN) gel electrophoresis and in-gel activity 4

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BN gel electrophoresis and in-gel activity were performed as previously described [7]. Mitochondrial pellets were solubilized using dodecylmaltoside. After incubation on ice for 30 min,

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the samples were centrifuged at 14,000 rpm for 5 min, and the supernatant was collected for BN gel electrophoresis. After electrophoresis, the vertical lane was cut and washed with deionized

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water. For complex I in-gel activity, the gels were incubated for several minutes at room

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temperature with the following solutions: 2 mM Tris–HCl, 0.1 mg/mL NADH, and 2.5 mg/mL nitrotetrazoliumblue (pH 7.4). For complex V in-gel activity, the vertical lane was cut and washed with deionized water and incubated at room temperature with the following solutions: 35 mM Tris, 270 mM glycine, 14 mM MgSO4, 0.2% Pb(NO3)2, and 8 mM ATP (pH 7.8). For BN/SDS

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two-dimensional gel electrophoresis, the vertical gel was rotated through 90°, placed on a glass

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plate, and incubated with a dissociating solution (1% SDS and 1% 2-mercaptoethanol) for 1 h at

electrophoresis.

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room temperature. The gel was rinsed briefly with water prior to the two-dimensional SDS

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2.5 Western blot

The tissues were lysed, subjected to SDS-PAGE, and transferred to PVDF membranes. The blots were probed with specific antibodies. Equal protein loading was confirmed by probing the membrane with an anti-GAPDH antibody. 2.6 Statistical Analysis Data were expressed as mean ± SEM of a minimum of three independent experiments. Comparison of the data was performed using Student t-test between two means. P < 0.05 was considered statistically significant.

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3. Results 3.1 Mitochondrial function and biogenesis were impaired during myocardial

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hypertrophy

We established a rat model of hypertension-induced cardiac hypertrophy using AAC. At six

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weeks after AAC, the mean arterial blood pressure markedly increased (Figure 1B I). The gross

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morphology and the left ventricle transverse sections (HE staining) of the heart showed that myocardial enlargement was more pronounced in the rats subjected to AAC than those in sham operation (Figure 1 A). Western blot analysis showed that β-MHC expression significantly increased at six weeks after AAC (Figure 1 C). Echocardiographic assessment demonstrated an

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evident increase in the anterior and posterior wall of the left ventricle at six weeks after AAC

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(Figure 1 B). These results suggested that the rats subjected to AAC developed an evident myocardial hypertrophy after six weeks.

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We detected a change in mitochondrial function and biogenesis after AAC. The ATP content

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of the rats after sham operation and AAC was 0.75 ± 0.056 μM and 0.58 ± 0.085 μM, respectively (Figure 2B). The H+-ATPase activity of the sham group was 721±28.6, whereas that of the AAC group was 563 ± 50.4 (Figure 2C). We also detected the changes in membrane permeability transition (MPT) during hypertrophy. The MPT of the rats during hypertrophy decreased by 21% (Figure 2A). These results demonstrated that mitochondrial function declined during myocardial hypertrophy. The mtDNA content, an important marker of mitochondrial proliferation, was determined using real-time PCR. As shown in Figure 2D, the mtDNA content significantly decreased during AAC-induced myocardial hypertrophy. Moreover, PGC-1α functioned as a major regulator of mitochondrial biogenesis. The expression of PGC-1α decreased during hypertrophy, 6

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and the expression of mitochondrial protein Cox IV and ATP synthase β subunit decreased accordingly. These results demonstrated that mitochondrial biogenesis decreased during

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hypertrophy. 3.2 Changes in mitochondrial complexes during hypertrophy

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The results of BN gel electrophoresis showed that the protein expression of mitochondrial

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complex I and complex V dimers decreased during hypertrophy, whereas that of mitochondrial complex III and complex V oligomers remained the same (Figures 3A, B, and C). The enzyme activity of mitochondrial complexes was evaluated using in-gel activity. The enzyme activity of mitochondrial complex I and complex V dimers decreased, whereas that of mitochondrial complex

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V oligomers and complexes II, III, and IV did not change (Figures 3D and E; Supplementary

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Figure 2).

3.3 The subunits of mitochondrial complex V dimers decreased during hypertrophy,

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whereas the subunits of mitochondrial complex V oligomers remained unchanged

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BN/SDS two-dimensional gel electrophoresis was used to determine the changes in the expression of the subunits of mitochondrial complex V dimers and oligomers. The first-dimension BN-PAGE was performed under non-denaturing condition. Under this condition, the assembly of OXPHOS complexes and protein–protein interactions was retained. In the second-dimension SDS-PAGE, the protein complex was completely denatured by SDS and the subunits of the protein complex were dissociated through protein mass. The results showed that the α, β, B, and D chains of complex V synthase dimers significantly decreased during hypertrophy. The α, β, and B chains of complex V oligomers increased, but D chain did not change during hypertrophy. These results provided a new finding that the decline of ATPase synthase during hypertrophy was mainly 7

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due to the reduction in mitochondrial complex V dimers.

4. Discussion

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Mitochondrial dysfunction has been proven to contribute to many diseases from major metabolic disorders, including obesity, insulin resistance, and type 2 diabetes, to cardiovascular

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disease, cancer, and aging-associated neurodegenerative diseases [8,9]. In our research,

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mitochondrial function and biogenesis decreased during myocardial hypertrophy, which possibly resulted from the decrease in the activity and content of mitochondrial complex V dimers and complex I.

Mitochondrial complex I is the first enzyme in the mitochondrial electron transport chain.

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This complex catalyzes the transfer of electrons from NADH to coenzyme Q and functions as a

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major source of ROS. Mitochondrial complex V, known as ATPase synthase, is an enzyme that provides energy for the cells to use through ATP synthesis. The decrease of complex V activity

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and complex I could directly decrease the production of ATP and increase the intracellular ROS

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levels [10,11]. ATP synthase is organized in dimers and higher oligomers in the mitochondria. The dimerization of mitochondrial complex V has important functions in mitochondrial biogenesis. Moreover, an angle is formed when two monomers of complex V form a dimerization. This angle bends the inner mitochondrial membrane, which causes its protrusions in the matrix and eventually generates mitochondrial cristae. The absence of complex V dimerization affects the formation of mitochondrial cristae and results in onion-like structure in yeast [12]. Moreover, the dimerization of complex V generates a proton trap by forming a positive curvature that could facilitate ATP synthesis [13]. Thus, the reduction of mitochondrial complex V dimers could result in the decline of ATP production and affect the formation of mitochondrial cristae, which could 8

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also contribute to the decline of mitochondrial function and biogenesis during hypertrophy. Our results showed that the activity of mitochondrial complexes I and V decreased during

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hypertension-induced myocardial hypertrophy, which is consistent with the results of previous studies [3,14]. However, the decrease of mitochondrial complex V was mainly due to the decrease

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of mitochondrial complex V dimers. Tang et al. [15] emphasized that complexes I and V decreased

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during hypertrophy, whereas complexes III and IV remain unchanged, consistent with our results. However, a significant decrease in complex Ⅱ activity was observed, which is different from our results. This finding may be due to the difference in hypertrophic mechanisms between spontaneously hypertensive rats (SHRs) and AAC model. Unlike AAC-induced hypertrophy,

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SHRs follow the progression of pre-hypertension and develop to sustain hypertensive phases, with

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each phase lasting for several weeks. Further study should be endeavored to elucidate the effects of hypertension development in SHRs on the activity of mitochondrial complex II.

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5. Conclusions

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The decreased activity and content of complex V dimers and complex I, rather than the changes in complex V oligomers, caused the decline in mitochondrial function and biogenesis during cardiac hypertrophy.

Figure legends FIGURE 1 Biological characteristics of AAC-induced myocardial hypertrophy. A: Gross morphology of the whole heart and LV transverse sections (HE staining) showing cardiac enlargement in the rats after AAC. B: (I) and (II) Heart weight/body weight ratios and mean arterial blood pressure of the rats subjected to AAC or sham operation; (III) and (IV) Echocardiographic evaluation of the rats subjected to AAC or sham operation. n = 8, *p ≤ 0.05 vs. 9

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same-time sham operation. C: Western blot analysis of the β-MHC expression levels of the rats subjected to AAC or sham procedure.

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FIGURE 2 Mitochondrial function and biogenesis declined during myocardial hypertrophy. A, B, and C: Analysis of ATP content, H+-ATPase activity, and MPT in the rats subjected to AAC

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or sham operation, respectively. n = 8, *p ≤ 0.05 vs. same-time sham operation. D: Measurement

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of the mtDNA content using real-time PCR. n = 8, *p ≤ 0.05 vs. same-time sham operation. E: Western blot analysis of the expression of ATP synthase β Subunit, Cox IV, and PGC-1α during myocardial hypertrophy.

FIGURE 3 Changes in mitochondrial complex during hypertrophy.

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A: A total of 200 μg of heart mitochondria from the rats subjected to AAC or sham procedure were

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separated using one-dimensional BN-PAGE. Eight independent experiments were performed to confirm the results. B: Densitometry analyses of gels for AAC or sham samples. Complexes IV

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and II were not included because of their low signal. C: The relative quantities of complexes I, V,

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and III in the heart mitochondria from sham or AAC rats. n = 8, *p ≤ 0.05 vs. same-time sham operation. D and E: In-gel activity analysis of complexes I and V of the rats subjected to AAC or sham procedure. Eight independent experiments were performed to confirm the results. D-V indicated the mitochondrial complex V dimers; and O-V indicated the mitochondrial complex V oligomers. Figure 4 Changes in the subunits of mitochondrial complex V during hypertrophy. A: A total of 200 μg of the heart mitochondria from the rats subjected to AAC or sham procedure were separated using BN/SDS two-dimensional gel electrophoresis. Eight independent experiments were performed to confirm the results. B and C: The significant changes of the 10

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subunits of mitochondrial complex V dimers and oligomers determined using mass spectra and quantified through densitometry analyses. n = 8, *p ≤ 0.05 vs. same-time sham operation. D-V

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indicated the mitochondrial complex V dimers; and O-V indicated the mitochondrial complex V oligomers.

Bugger H, Schwarzer M, Chen D, Schrepper A, Amorim PA, Schoepe M, Nguyen TD, Mohr

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Funding This work was supported by the National Natural Science Foundation of China

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(grant numbers: 31071022, 81372124); and the Natural Science Foundation of Tianjin,

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China (grant number: 10JCZDJC19300

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