MICU1 Alleviates Diabetic Cardiomyopathy Through Mitochondrial Ca2+-dependent Antioxidant ...

52 Pages·2017·3.31 MB·English

Page 1 of 52 Diabetes
Short Title: MICU1 and diabetic cardiomyopathy
Full Title: MICU1 alleviates diabetic cardiomyopathy through mitochondrial
Ca2+-dependent antioxidant response

Lele Ji1#, Fengzhou Liu2#, Zhe Jing3#, Qichao Huang1, Ya zhao1, Haiyan Cao1, Jun Li4,
Chun Yin1, Jinliang Xing1*, Fei Li2*
1State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic
Medicine, Fourth Military Medical University, Xi’an, 710032, China
2Department of Cardiology, Xijing Hospital, Fourth Military Medical University,
Xi’an, 710032, China
3Department of Cardiology, General Hospital of Lanzhou Military Area Command,
Lanzhou, 730050, China.
4Department of Physiology, Fourth Military Medical University, Xi’an, 710032, China

#Lele Ji, Fengzhou Liu and Zhe Jing contribute equally to this work.

*Correspondence to: Dr Jinliang Xing, State Key Laboratory of Cancer Biology and
Experimental Teaching Center of Basic Medicine, Fourth Military Medical University;
169 Changle West Road, Xi’an 710032, China. Tel: [phone]; Fax:
[phone]; E-mail: [email protected]. Or correspondence to: Dr Fei Li,
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an,
710032, China. Tel: [phone]; Fax: [phone]; E-mail:
[email protected].
Total words: 4467
Number of tables and figures: 8 figures and 0 table
Diabetes Publish Ahead of Print, published online March 14, 2017
Diabetes Page 2 of 52
MICU1 alleviates diabetic cardiomyopathy through mitochondrial
Ca2+-dependent antioxidant response

Lele Ji1#, Fengzhou Liu2#, Zhe Jing3#, Qichao Huang1, Ya zhao1, Haiyan Cao1, Jun Li4,
Chun Yin1, Jinliang Xing1*, Fei Li2*

1State Key Laboratory of Cancer Biology and Experimental Teaching Center of Basic
Medicine, Fourth Military Medical University, Xi’an, 710032, China
2Department of Cardiology, Xijing Hospital, Fourth Military Medical University,
Xi’an, 710032, China
3Department of Cardiology, General Hospital of Lanzhou Military Area Command,
Lanzhou, 730050, China.
4Department of Physiology, Fourth Military Medical University, Xi’an, 710032, China

#Lele Ji, Fengzhou Liu and Zhe Jing contribute equally to this work.

*Correspondence to: Dr Jinliang Xing, State Key Laboratory of Cancer Biology and
Experimental Teaching Center of Basic Medicine, Fourth Military Medical University;
169 Changle West Road, Xi’an 710032, China. Tel: [phone]; Fax:
[phone]; E-mail: [email protected]. Or correspondence to: Dr Fei Li,
Department of Cardiology, Xijing Hospital, Fourth Military Medical University, Xi’an,
710032, China. Tel: [phone]; Fax: [phone]; E-mail:
[email protected].
Page 3 of 52 Diabetes
Abstract
Diabetic cardiomyopathy is a major cause of mortality in diabetic patients, but
specific strategies for prevention or treatment of diabetic cardiomyopathy have not
been clarified yet. MICU1 is a key regulator of mitochondria Ca2+ uptake, which
plays important roles in regulating mitochondrial oxidative phosphorylation and redox
balance. However, to date, the significance of MICU1 in diabetic hearts has never
been investigated. Here, we demonstrated that MICU1 was downregulated in db/db
mouse heart, which contributes to myocardial apoptosis in diabetes. Importantly, the
reconstitution of MICU1 in diabetic hearts significantly inhibited the development of
diabetic cardiomyopathy as evidenced by enhanced cardiac function, reduced cardiac
hypertrophy and myocardial fibrosis in db/db mice. Moreover, our in vitro data
showed that the reconstitution of MICU1 inhibited the apoptosis of cardiomyocytes
induced by high glucose and high fat through increasing mitochondrial Ca2+ uptake
and subsequently activating the antioxidant system. Finally, our results indicated that
hyperglycemia and hyperlipidemia induced the downregulation of MICU1 via
inhibiting Sp1 expression in diabetic cardiomyocytes. Collectively, our findings
provide the first direct evidence that upregulated MICU1 preserves cardiac function in
diabetic db/db mice, suggesting that increasing the expression or activity of MICU1
may be a pharmacological approach to ameliorate cardiomyopathy in diabetes.

Key words: mitochondrial calcium; MICU1; diabetic cardiomyopathy; antioxidant
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INTRODUCTION
Increased prevalence of Type 2 diabetes is a major threat to human health. It is
estimated that diabetes will affect nearly 366 million adults by the year 2030 (1).
Cumulative evidences reveal that diabetes is associated with structural and functional
abnormalities of the myocardium, leading to diabetic cardiomyopathy. However,
specific strategies for prevention and treatment of cardiomyopathy in diabetic patients
have not been clarified yet. Emerging evidences suggest that mitochondrial
dysfunction may play a critical role in the pathogenesis of diabetic cardiomyopathy
(2). For example, recent data have demonstrated that mitochondria isolated from atrial
cardiomyocytes of diabetic patients exhibit the remarkable respiratory defects and
increased generation of reactive oxygen species (ROS) when compared with those
from non-diabetic control subjects (3; 4). Moreover, it is essential to protect
mitochondria from losing their ability to generate energy and to control their own
ROS emission for the prevention of diabetic cardiomyopathy (5). However, despite
increasing data implicating mitochondrial pathology in diabetic cardiomyopathy, the
mechanism underlying these processes still remains largely unknown.
Mitochondria Ca2+ plays important roles in regulating the intrinsic functions of
the organelle. One of the best characterized functions of mitochondrial Ca2+ is the
control of organelle metabolic activity. For example, physiological increases in
mitochondria Ca2+ lead to the allosteric activation of tricarboxylic acid (TCA) cycle
enzymes including isocitrate dehydrogenase (IDH) and α-ketogluterate
dehydrogenase (α-KGDH), as well as pyruvate dehydrogenase (PDH). The net effect
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of TCA cycle activation is a boost in reduced oxidative phosphorylstion substrate
synthesis (NADH and FADH), the enhanced activity of respiratory chain and a
subsequent increase in ATP synthesis (6). Another key role of mitochondrial Ca2+ is to
modulate oxidative stress by misbalancing ROS generation and ROS detoxification. It
has been reported that mitochondiral Ca2+ regulates cellular antioxidant defense
systems by stimulating NADPH regeneration, which donates electrons to regenerate
GSH from GSSG (7; 8). Interestingly, several studies have reported that reduced
mitochondrial calcium uptake is observed in diabetic rodent hearts (9; 10). However,
the mechanisms by which diabetes impairs mitochondrial calcium handling in
cardiomyocytes remain to be defined.
In the last few years, the discovery of the pore-forming subunit of the
mitochondrial Ca2+ uptake channel (Mitochondrial Calcium Uniporter, MCU) (11; 12)
and its regulatory subunits, termed MICU1 (mitochondrial calcium uptake 1) (13) and
MCUR1 (mitochondrial calcium uniporter regulator 1) (14) has opened new avenues
for the study of mitochondrial Ca2+ homeostasis regulation and its key roles. Specially,
a loss-of-function mutation in MICU1 has been reported to cause human disease
through alterations in mitochondrial Ca2+ handling (15). A previous study has reported
that MICU1, but not MCU mRNA levels were markedly downregulated in human
cardiovascular disease-derived primary endothelial cells (16). However, to our
knowledge, the significance of MICU1 in diabetic hearts, especially in the
pathogenesis of diabetic cardiomyopathy, has never been reported.
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In the present study, we identified the downregulated MICU1 as a contributing
factor to cardiomyocyte apoptosis in diabetic cardiomyopathy. Moreover, MICU1
overexpression effectively alleviated diabetic cardiomyopathy via promoting
mitochondrial Ca2+ uptake to inhibit mitochondrial ROS-mediated apoptosis. These
findings suggest that upregulating MICU1 expression may be a potential therapeutic
strategy in diabetic cardiomyopathy.
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RESEARCH DESIGN AND METHODS
Animals
All experiments were performed in accordance with the National Institutes of
Health Guidelines on the Use of Laboratory Animals and approved by the Fourth
Military Medical University Committee on Animal Care. Leptin receptor-deficient
(db/db) C57BLKS mice and wild-type C57BLKS mice were purchased from
Changzhou Cavens Laboratory Animal Co. Ltd. (Jiangsu, China). The investigators
were blinded to the treatment conditions during experiments.
Neonatal Rat Cardiomyocyte Culture
Primary cardiomyocytes were prepared from neonatal rats as previously
described (17). For the treatment of high glucose and high fat (HGHF), cells were
cultured with DMEM (containing 25 mmol/L glucose and 500 µmol/L saturated FFA
palmitate (16:0) for 24 h.
Knockdown and Forced Expression of Target Genes
MICU1-specific small interfering RNA (siRNA), Sp1-specific siRNA and
scrambled siRNA were designed and synthesized by GenePharma Company
(Shanghai, China). The sequences of siRNAs were provided in Supplementary Table
S1. Transfection of siRNAs was preformed as previously described (17).
Recombinant adenoviruses overexpressing rat MICU1 (NM_199412.1) and Sp1
(NM_012655.2) were respectively constructed by Genechem Company (Shanghai,
China) and HanBio Company (Shanghai, China). Recombinant adenovirus
overexpressing mouse MICU1 (NM_001291442.1) or mitochondrial-targeted
parvalbumin (mito-PV) was generated following the instructions of ViraPower
Adenoviral Expression System (Life Technologies) manufacturer protocol. The
coding sequences of MICU1 and mito-PV were amplified using primers listed in
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Supplementary Table S2. The viral titer was determined using an Adeno-X Rapid
Titer kit (Clontech Laboratories, Mountain View, CA). Cardiomyocytes were infected
with adenovirus at a multiplicity of infection (MOI) of 50 for 2 hours. Subsequently,
cells were cultured in serum-free DMEM medium for an additional 24 h and then
used for further analysis. For adenovirus administration, the mice were anesthetized
with 2% isoflurane, and the heart was exposed. Adenovirus (2×1010 IFU/mL) was
injected into the left ventricular free wall (three sites, 10 µL/site, 30-gauge needle).
The transfection efficiency was evaluated by western blot analyses three days after
adenoviral injection.
Quantitative Reverse Transcription PCR (qRT-PCR), Western Blot and
Immunohistochemistry (IHC)
RNA extraction, complementary DNA synthesis, qRT-PCR reactions were
performed as described previously (18). Primer sequences were provided in
Supplementary Table S3. Mouse heart tissue or primary neonatal cardiomyocytes
were processed for western blot as previously described (19). Immunohistochemical
staining of cardiac sections was carried out as previously described (20).
Histological Analysis
The hearts of mice were fixed in 4% paraformaldehyde (pH 7.4) overnight,
embedded in paraffin, and serially sectioned at a thickness of 5 µm for histological
analysis. Standard hematoxylin and eosin (H&E) staining was carried out following
standard procedures. Cardiac collagen content was assessed via Masson trichrome
staining. Myocyte size was detected by staining with wheat germ agglutinin (red,
Sigma-aldrich).
Cell Apoptosis Assay
Cell apoptosis was determined with a PE Annexin V Apoptosis Detection Kit (BD
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Pharmingen™, 559763) or a FITC Annexin V Apoptosis Detection Kit (BestBio,
BB-4101) following the manufacturers’ instructions. For analysis of apoptosis in heart
tissues, terminal deoxynucleotidyl transferase–mediated dUTP nick-end labeling
(TUNEL) assay (Roche Applied Science, 11684795910) was performed according to
the manufacturer’s protocol. Images of TUNEL and DAPI-stained sections were
obtained by a fluorescence microscope (DM5000B; Leica, Heerbrugg, Switzerland).
Only TUNEL- and DAPI-positive nuclei that were located within heart tissues were
counted as apoptotic nuclei.
Measurement of Mitochondrial Membrane Potential
To measure mitochondrial membrane potential (MMP), cells (1×106/mL) were
incubated with the tetramethyl rhodamine methyl ester (TMRM, Invitrogen) (10 nM)
for 30 min at 37 °C. TMRM images were captured using a confocal laser scanning
microscope (Olympus FluoView™ FV1000, Japan) with excitation at 530 nm and
emission at 573 nm.
Isolation of Mitochondria
Mitochondria were isolated from cardiomyocytes or hearts with the
Mitochondria Isolation Kit (Beyotime, Shanghai, China) according to the
manufacturer’s instructions.
Isolation of Adult Cardiomyocytes
Mouse hearts were excised and then washed in Ca2+-free tyrode solution,
followed by perfusion with buffer containing liberase (Roche Diagnostics) at 37 °C
for 20 min. The left ventricular was subsequently dissociated into single myocytes,
and extracellular Ca2+ was added incrementally back to 1.2 mM. Cardiomyocytes
were placed on glass coverslips coated with laminin (Thermo Fisher Scientific). Cells
were used within 4 hours after isolation.
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Measurement of Mitochondrial [Ca2+]
The fluorescent dye Rhod-2/AM (Invitrogen) was used to monitor mitochondrial
Ca2+ concentration ([Ca2+]) in cardiomyocytes according to the manufacturer’s
instructions. Then, cardiomyocytes were viewed with a confocal laser scanning
microscope (Olympus FluoView™ FV1000, Japan). For the measurement of
mitochondrial Ca2+ uptake, adult cardiomyocytes or neonatal cardiomyocytes were
stimulated by pacing (3 Hz) or histamine (10 µM), respectively. Images were
recorded every 5 seconds using the same confocal imaging system. The endogenous
mitochondrial Ca2+ content was measured in isolated cardiac mitochondria. Briefly,
isolated mitochondria were resuspended in buffers, sonicated and then centrifuged.
The supernatants were recovered and the Ca2+ content in the supernatants was
determined using a Calcium Detection Kit (Abcam).
Detection of Mitochondrial Reactive Oxygen Species (mitoROS)
MitoROS was detected by the fluorescent probe MitoSOX (Invitrogen)
according to the manufacturer’s protocols. Images were captured by laser confocal
microscope (Olympus FluoView™ FV1000, Japan) and ImagePro image analysis
software.
Activity Detection of PDH and α-KGDH
The activity of PDH and α-KGDH was measured using the pyruvate
dehydrogenase enzyme activity microplate assay kit (Abcam) and α-ketoglutarate
dehydrogenase activity assay kit (Biovision) according to the manufacturers'
instructions, respectively.
Detection of Mitochondrial NADH, NADPH Contents and GSH/GSSG Ratio
NADH and NADPH concentrations were measured in lysates from isolated
mitochondria using the NAD/NADH and NADP/NADPH assay kit (Abcam)