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Mitochondrial calcium uniporter inhibition provides cardioprotection in pressure overload-induced heart failure through autophagy enhancement
Accepted Manuscript Mitochondrial calcium uniporter inhibition provides cardioprotection in pressure overload-induced heart failure through autophagy enhancement
Ziqing Yu, Ruizhen Chen, Minghui Li, Yong Yu, Yixiu Liang, Fei Han, Shengmei Qin, Xueying Chen, Yangang Su, Junbo Ge PII: DOI: Reference:
S0167-5273(17)37641-6 doi:10.1016/j.ijcard.2018.05.054 IJCA 26467
To appear in: Received date: Revised date: Accepted date:
10 December 2017 10 May 2018 17 May 2018
Please cite this article as: Ziqing Yu, Ruizhen Chen, Minghui Li, Yong Yu, Yixiu Liang, Fei Han, Shengmei Qin, Xueying Chen, Yangang Su, Junbo Ge , Mitochondrial calcium uniporter inhibition provides cardioprotection in pressure overload-induced heart failure through autophagy enhancement. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ijca(2017), doi:10.1016/ j.ijcard.2018.05.054
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ACCEPTED MANUSCRIPT Mitochondrial Calcium Uniporter Inhibition Provides Cardioprotection in Pressure Overload-Induced Heart Failure through Autophagy Enhancement Ziqing Yuab†, MD, PhD; Ruizhen Chenac†, MD, PhD; Minghui Liac, MD, PhD; Yong Yuac, MD, PhD; Yixiu Liangab, MD, PhD; Fei Hanab, MD, PhD; Shengmei Qina, MD, PhD; Xueying Chena, MD, PhD; Yangang Sua*, MD, PhD; Junbo Gea*, MD, PhD
a: Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital,
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Fudan University, Shanghai 200032, PR China b: Shanghai Medical College, Fudan University, Shanghai 200032, PR China
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c: Department of Cardiovascular Diseases, Key Laboratory of Viral Heart Diseases, Ministry of Public Health, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan
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University, Shanghai 200032, PR China
†: Equal contribution *: Correspondence to:
Email: [email protected]
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Yangang Su*, MD, PhD, FHRS, FEHRA
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Conflicts of interest: None.
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Professor of Medicine/Cardiology
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Junbo Ge*, MD, PhD, FESC, FACC, FSCAI
Department of Cardiology, Zhongshan Hospital, Fudan University; and Shanghai Institute of Cardiovascular Diseases
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180 Fenglin Road, Shanghai 200032, PR China Tel: 86-21-64041990 ext 2745
Fax: 86-21-64223006
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Email: [email protected]
Fundings
This study was supported by National Natural Science Foundation of China (Grant No: 81671934, 81521001, 81400280 and 31570904) and Science and Technology Commission of Shanghai Municipality (Grant No: 17140902400).
Competing interests The authors declare that they have no competing interest.
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ACCEPTED MANUSCRIPT Abstract: Background: HF incurs high disease burden, and the effectiveness of known HF treatments is unsatisfactory. Therefore, seeking novel therapeutic target of HF is important. The present study aimed to investigate the role of the mitochondrial calcium uniporter (MCU) and its relationship with autophagy in overload-induced heart failure (HF).
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Methods and results: In both early-stage and end-stage of pressure overload-induced HF, MCU appeared up-regulated along with heart enlargement, increased microtubule-associated proteins
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1A/1B light chain 3B (LC3B) II/I ratio and autophagosome content, damaged cardiac function, and ventricular asynchrony. However, sequestosome-1 (SQSTM1/p62) level decreased indicating
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blockaded autophagic flux. Seven-week administration of MCU inhibitor ruthenium red improved cardiac function and mitigated its pathological change. MCU inhibition maintained mitochondrial
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integrity, increased LC3B II/I ratio, up-regulated Parkin and Pink1, and down-regulated SQSTM1/p62. MCU inhibition also alleviated ventricular asynchrony of HF, and this might be
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related to connexin-43 up-regulation. In vitro study validated intervention on MCU leading to elevation of autophagy and mitophagy. MCU inhibition could partly prevent from excessive
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cellular enlargement induced by isoprenaline.
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Conclusions: In summary, MCU inhibition played an important role in pressure overload-induced heart failure through autophagy and mitophagy enhancement, and intervention on MCU offered cardioprotective effects. To our knowledge, the role of MCU in HF and its relationship with
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autophagy and mitophagy are firstly disclosed. Moreover, our study suggests that MCU inhibition could be explored as a novel therapeutic concept in HF.
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Key words: mitochondrial calcium uniporter; heart failure; transverse aortic constriction; autophagy; ventricular asynchrony
ACCEPTED MANUSCRIPT Introduction Nowadays, heart failure (HF) has become one of the most predominant disease burdens worldwide, and its mortality and morbidity rates remain high. Current HF therapeutic strategy includes guideline-directed medical therapy and cardiac device therapy, e.g. cardiac resynchronization therapy and implantable cardioverter defibrillator[1, 2], etc., however, their efficacy are limited.
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Given the inadequate effectiveness of the current therapeutic targets, it’s necessary to identify novel therapeutic targets.
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Mitochondria, the energy factory of cells, accounts for a large proportion in cardiomyocytes[3]. Therefore, mitochondrial disorder is usually associated with heart diseases. The maintenance of
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myocardial performance is dependent on normal mitochondrial function[3, 4]. Cardiac contraction and relaxation are ATP-consuming and calcium dependent. In the process of ATP synthesis,
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calcium plays an important role by activating key enzyme in redox reaction and taking part in the constitution of complexes of ECT[5]. Although cytosolic calcium can easily pass through the outer
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mitochondrial membrane (OMM) via voltage-dependent anion channel (VDAC), the inner mitochondrial membrane (IMM) is highly calcium-selective. The mitochondrial calcium uniporter
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(MCU) synthesized in cytosol is localized to IMM. As a pore-forming structural protein, MCU is
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responsible for the majority of calcium uptake[6]. Consequently, MCU determines calcium concentration in mitochondrial matrix, and in turn affects mitochondrial energetic metabolism. Since mitochondrial calcium overload induces cellular injury, MCU also influences cell viability
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and even leads to cell death. Herein we hypothesized that MCU could be a potential therapeutic target of HF. In the present study, we investigated the effect of the MCU inhibitor or
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RNAi-mediated MCU depletion on cardiac function in an overload-induced HF model.
Materials and methods Animal and HF model Male C57BL/6 mice with ages of 8-10 weeks and weights of ±30 g were studied, and overload-induced HF models were established by transverse aortic constriction (TAC) method (see supplementary methods). Ruthenium red (RR) purchased from Sigma is administrated 1 week after the surgery. Animals were divided into 3 groups: sham+NS, TAC+NS, and TAC+RR (n=20 in each group). After TAC surgery, animals are randomly assigned to TAC+NS group or TAC+RR
ACCEPTED MANUSCRIPT group. This animal study was approved by the Ethics Committee of Zhongshan Hospital affiliated to Fudan University.
Echocardiography and electrocardiography Electrocardiograph was recorded and the QRS duration was measured using biological signal
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collecting/processing system (RM6240, Chengdu Instrument Factory, China); Regular echocardiograph and two-dimension speckle tracking imaging (2D-STI) were acquired by using a
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32–55 MHz linear array transducer via Vevo 2100 ultrasound system (VisualSonics Inc., Canada). Murine models were fixed in supine position and anesthetized by isoflurane (4% for induction, 2%
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for maintenance). The following left ventricular measurements were performed: end-systolic diameter (LVESD), end-systolic volume (LVESV), end-diastolic diameter (LVEDD), end-diastolic
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volume (LVEDD), ejection fraction (LVEF), fractional shortening (FS), and ventricular wall thickness on the M-mode at parasternal long-axis view. With a speckle-tracking algorithm, strain
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analysis was quantified by radial and longitudinal description. The ventricular wall asynchrony was evaluated by the standard deviation of time to peak myocardial systolic velocity of the 6 LV
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segments (Ts-SD6), strain, and strain rate (SR) as reported before[7, 8]. All echocardiographic
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image measurements were calculated over 3 consecutive cardiac cycles and then averaged.
Positron emission tomography
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Positron emission tomography (PET) scan was performed to acquire the standardized uptake value (SUV) via MadicLab PET system (Madic Technology Co., Ltd, China)[9] to detect the survival
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myocardium and assess its viability (see supplementary methods).
Morphological examination The hearts were promptly obtained, fixed in formalin, embedded in paraffin, sliced into 5-μm-thick sections, and stained by Hematoxylin-Eosin or Masson’s trichrome method; Immunohistochemistry was performed by the avidin–biotin–peroxidase complex method. After rehydration and microwave antigen retrieval, tissue slices are incubated with MCU antibodies (1:200, orb317655, biorbyt) or connexin-43 antibodies (1:1000, ab11370, abcam) at 4 °C overnight and incubated with secondary antibodies (K5007, Dako, Denmark) at 37 °C for 30 min.
ACCEPTED MANUSCRIPT Finally, 3,3’-diaminobenzidine staining and Mayer’s hematoxylin counterstaining were used for developing. Negative control without the primary antibodies was included to rule out false-negative/positive; Preparing the transmission electron microscope (TEM) sample, cardiac tissue was fixed in 2.5% glutaraldehyde, dehydrated by gradient ethanol, embedded with pure acetone, solidified in oven, sliced, and stained with gold. TEM image was acquired by an
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electro-microscopic system (FEI Tecnai G2 Spirit).
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Western blots (WB)
Tissue homogenate, cytosolic lysates, or mitochondrial lysates were mixed with RIPA lysis buffer
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with protease inhibitor cocktail. Proteins were acquired by ultracentrifugation, determined by BCA method, separated on a 12% SDS-PAGE, and transferred to polyvinylidene difluoride membranes.
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After dilution and blocking, membranes were incubated with primary antibodies of MCU (1:1000, 14997, CST), atrial natriuretic peptide (ANP) (1:500, abcam), microtubule-associated proteins
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1A/1B light chain 3B (LC3B) (1:1000, 2775, CST), sequestosome-1 (SQSTM1/p62) (1:1000, 5114, CST), Beclin-1 (1:1000, 3495, CST), connexin-43 (1:8000, ab11370, abcam), Parkin
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(1:1000, 4211, CST), or Pink1 (1:1000, ab23707, abcam) at 4°C overnight. Incubated with
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rabbit-radish peroxidase-conjugated secondary antibody, the binding reactions in the membranes were detected by enhanced chemiluminescence assays.
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Cardiac tissue mitochondria isolation and mitochondrial membrane potential measurement The protocol was followed as published before[10]. Mitochondrial membrane potential (MMP)
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was measured by JC-1 assay kit. JC-1 polymer with red color indicates polarized MMP. JC-1 monomer with green color indicates depolarized MMP. The Change of MMP was assessed by fluorescence intensity ratio of green color/red color (see supplementary methods).
Cell culture, siRNA plasmid construction, and, transfection In vitro validation of the effect of MCU was carried out by MCU inhibitor RR and siRNA induced MCU ablation on H9c2 cardiomyoblasts, purchased from the cell bank of Chinese Academy of Science. Cells were cultured in DMEM medium with fetal bovine serum (Gemini), treated with RR of three concentrations (5 μM, 50 μM, and 500 μM), and incubated for 48h or 72h. The
ACCEPTED MANUSCRIPT scramble
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(GGACAATTTTTGAATCGTCTC)
or
the
interfering
sequence
(GCTTGTCATTAATGACTTAAC), connecting with a green fluorescent protein (GFP) gene was integrated and amplified in Escherichia coli bacteria system (Clontech). H9c2 cells were transfected with plasmid through lipofectamine 2000 transfection kit (Thermofisher Scientific) for 48h and 72h. Successful transfection was verified by GFP under fluorescence microscope investigation, and MCU knock-down was verified by WB-analysis. HL-1 cell line originating
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from mouse cardiomyocyte with treatment of 10-5 M isoprenaline for 72h was used to testify the
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effect of MCU inhibition by 50 μM Ru360 (sc-222265, Santa Cruz). The difference of the cellular
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area was compared, and the autophagy and mitophagy were evaluated afterwards.
Statistical analysis
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Continuous variables were presented as mean±standard error of mean. Inter-group difference was assessed by Student’s t test or one-way analysis of variance (ANOVA) via SPSS software 19.0. A p
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value of less than 0.05 was regarded as significant.
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Results
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Differential expression of MCU and autophagy associated protein in overload induced cardiac hypertrophy and heart failure
Animal models establishment was confirmed by heart mass index (heart weight/body weight ratio
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and heart weight/tibia length ratio), echocardiography, and the ANP protein expression. Hearts were heavier in the TAC group at different stages (supplementary figure 1A and 1B). Remarkable
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cardiac hypertrophy was observed at 2 weeks of TAC with slightly affected LVEF. However, LVEDD in the TAC group was smaller, indicating abnormal diastolic function in the early phase. LVEF further decreased at four weeks after TAC. Decompensated HF developed at 8 weeks with severely deteriorated LVEF and enlarged intra-ventricular diameter (Figure 2A-F and supplementary table 1). Pathological tests confirmed various levels of fibrosis caused by TAC (Figure 1). Total MCU was up-regulated and reached the summit (p=0.001) at 2 weeks after TAC (Figure 2G-I). Meanwhile, autophagy associated protein expression was detected as well. LC3B-II/I ratio increased steadily after TAC, indicating autophagosome formation. However, the substrate of
ACCEPTED MANUSCRIPT autophagy SQSTMl/p62 in TAC mice was significantly higher than that in the control group (p<0.05). Additionally, Beclin-1’s expression was depressed in TAC mice (p<0.01)，indicating inhibited formation of autolysosomes and autophagic flux.
MCU expression in isolated mitochondria and cytosol
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Furthermore, 8 weeks of TAC was chosen for MCU expression analysis in isolated mitochondria and cytosol. Cellular constituent separation was proved by mitochondrial marker COX IV and
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cytosolic marker GAPDH (Figure 2J). Mitochondrial MCU level was higher in the TAC-induced HF group (p=0.001). In the sham group, MCU was apparently expressed in mitochondrial
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integrity, while it was hardly seen in cytosol. In the HF group, mitochondrial MCU was outstandingly elevated (p<0.01). Interestingly, MCU quantity in cytosol was different from each
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other in comparison between sham group and HF group. Cytosolic MCU level was dramatically elevated in the HF group (p=0.004). These results together revealed that MCU only localized in
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the mitochondria rather than the cytosol in physiological condition. But in TAC-induced HF, both
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mitochondrial MCU and cytosolic MCU were up-regulated.
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MCU inhibition preserving cardiac function and reducing mortality RR was introduced as an inhibitor of MCU, and RR of two concentrations (1.25mg·kg-1·d-1 and 2.5mg·kg-1·d-1) were intraperitonealy injected to the mice to find a better concentration for MCU
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inhibition. Preliminary experiment with 8-week observation showed the higher concentration presented better cardiac function (Supplementary figure 2). Consequently, RR of 2.5mg·kg-1·d-1
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was chosen, and the RR administration protocol was shown in supplementary figure 3. Compared with normal saline (NS) treated TAC group, RR treatment showed significant improvement of cardiac function (LVEF: 60±1 vs. 27±1.4 %, p<0.001; LVEDD: 5.3±0.1 vs. 3.8±0.09, p<0.001; LVESD: 4.6±0.1 vs. 2.6±0.04, p<0.001). Within 1 week of TAC, there was no difference of mortality between TAC+RR and TAC+NS (HR=0.83, 95% CI: 0.22-3.33), while significantly lower death rate was found in TAC+RR group (HR=0.45, 95% CI: 0.18-0.91) at 8 weeks (Figure 3C). MCU inhibition reduced heart size, and alleviated cardiac fibrosis (Figure 1). Lower HW/BWR and HW/TLR proved decreased heart mass in TAC+RR group (supplementary figure 1C and 1D). Compared to TAC group, LC3B II/I ratio, Parkin, and Pink1 increased in TAC+RR
ACCEPTED MANUSCRIPT group (Figure 3C), indicating RR treatment could induce both elevation of autophagy and mitophagy in vivo.
Inhibition of MCU improving myocardial viability, protecting mitochondrial integrity, and maintaining mitochondrial membrane potential in vivo After 8 weeks of TAC, mice were starved overnight and then received PET scanning (Figure 3D).
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FDG than the NS treated TAC
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0.01). However, the RR treated TAC group had higher SUV of
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TAC+NS group showed a significant decrease in SUV of 18FDG compared to the sham group (p <
group (p < 0.05). These results revealed higher proportion of viable myocardia survival from TAC
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in RR treated mice. Moreover, TAC-induced morphological change of mitochondria, e.g. cristae edema and disruption, was evidently alleviated in the mice treated with RR (Figure 3B, yellow
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arrowhead). Besides, rupture of myofilament was more serious in the NS treated mice than that in RR treated mice after TAC (red arrowhead). In consistent with the expression of LC3B, Parkin,
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and Pink1, the number of autophagosomes (black arrowhead) and mitophagosomes (blue arrowhead) increased in TAC+RR group. Semi-quantitative analysis of the mitochondrial
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area/number ratio showed that TAC+NS group had a higher value than TAC+RR group, indicating
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mitigation of mitochondrial swell by RR. Furthermore, mitochondrial membrane potential (MMP) was measured by JC-1 kit to assess mitochondrial integrity (Figure 3E). Higher monomer (green) / polymer (red) ratio was detected in TAC+NS group, indicating dissipated MMP compared with
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TAC+RR group (1.39±0.1 vs. 0.92±0.08, p<0.001).
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Inhibition of MCU improving ventricular synchronization and narrowing QRS duration QRS duration of ECG correlates to synchronous motion of ventricular wall, and widening QRS duration indicated cardiac asynchrony[1]. ECG was acquired to measure the QRS duration to evaluate the cardiac synchronization. After 8 weeks, TAC+NS mice presented remarkably broadened QRS duration compared to sham+NS mice, while QRS duration of TAC+RR mice was significantly shorter than that of TAC+NS mice (8.8±0.3 vs. 11.7±0.5 ms, p<0.001). Additionally, 2D-STI showed markedly asynchronous ventricular wall motion of TAC+NS mice, while RR could partly correct cardiac asynchrony (Figure 3A). Compared to sham+NS group, dispersion of six-segment ventricular motion assessed by Ts-SD6 was greater in TAC+NS group (5.2±0.5 vs.
ACCEPTED MANUSCRIPT 28.9±4 ms, p=0.006). Nevertheless, RR dramatically reduced this dispersion in HF (18.6±0.9 vs. 28.9±4 ms, p=0.041). TAC+RR group had higher absolute value of radial strain, radial strain rate, longitudinal strain, and longitudinal strain rate compared to TAC+NS group. Connexin-43 plays a key role of cardiac conduction and intercellular communication[11], and its abnormal expression was regarded as critical mechanism of cardiac asynchrony[12, 13]. The Connexin-43 expression
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decreased in TAC+NS group (p<0.001). However, TAC+RR group had higher level of connexin-43 compared to TAC+NS group (p=0.006). Besides, what should be noteworthy was
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that MCU level of TAC+NS group and TAC+RR group was similar (p=0.96), revealing RR
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treatment in vivo didn’t influence MCU expression in cardiac tissue (Figure 3C).
Intervention of MCU prompting autophagy and mitophagy enhancement in vitro
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Three doses of RR (5 μM, 50 μM, and 500 μM) were applied to H9c2 cell line hatched for 48h to investigate the influence of MCU inhibition on autophagy. A dose-dependent autophagy
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enhancement was found. LC3-B II/I ratio was gradually increased, and SQSTMl/p62 expression tended to be down-regulated when the RR concentration elevated. However, RR didn’t affect
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MCU expression since non-significant difference of its expression was detected, which was
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consistent with the in vivo results (Figure 4A). Furthermore, siRNA plasmid transfection of H9c2 cell consolidated the conception that MCU depletion dramatically elevated the autophagic level. The transfection efficiency of GFP-exprssed and mcu-interfered siRNA plasmid in H9c2 cell was
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over 70%, and the interfering sequence (GCTTGTCATTAATGACTTAAC) was effective enough to relegate MCU (p=0.004). MCU-ablation increased LC3B II/I ratio and decreased SQSTMl/p62
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compared to vehicle-control cell and scramble-control cell (Figure 4B). Furthermore, in HL-1 cells, the treatment of isoprenaline for 72h was enough to induce cellular enlargement, while MCU inhibition could partly prevent them from excessively increasing in size induced by the stimulus (Figure 4C). MCU inhibition elevated LC3B II/I ratio and decreased SQSTM1/p62’s level (Figure 4D), suggesting strengthened autophagy. Moreover, Ru360 could up-regulate Parkin and Pink1’s level which were indicators of enhanced mitophagy[14-16].
Discussion In the present study, pressure-overload-induced heart disease manifested as different phenotype
ACCEPTED MANUSCRIPT from compensatory cardiac hypertrophy to decompensatory HF at different phase. MCU up-regulation was proved in this pathological process. Besides, we further examined MCU expression in human heart tissue acquired from heart transplant surgery and a canine HF model, and MCU was proved to be elevated in both conditions (data not shown). These results suggested that MCU play an important role in the initiation and progression of HF. Autophagy was impeded in accompany with MCU’s changes. Mitochondria isolation analysis indicated that MCU was
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hardly seen in cytosol with normal cardiac workload, while mitochondrial MCU’s up-regulation
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and cytosolic MCU’s accumulation emerged in HF. MCU inhibitor RR protected from aggravation of pressure-overload-induced HF and significantly improve the survival rate. SUV of
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FDG was
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higher in RR-treated group revealing more survival of cardiomyocytes and cell viability. RR could preserve mitochondrial integrity and promote autophagy in vivo. Compared to HF group, cardiac
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asynchrony was relieved, and Connexin-43 expression was elevated in RR group. RR or mcu-siRNA could independently increase autophagosome formation and prompt autophagic flux
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in vitro. To our knowledge, this study firstly disclosed the important role of MCU in improving cardiac function and correcting cardiac asynchrony. MCU inhibition recovered autophagic flux in
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HF. Findings from the present study provided a novel therapeutic target of HF.
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MCU as a pore-forming structure of a complex was predominantly responsible for calcium entrance[17]. MCU had high conductance of calcium, and its activation was dependent to cytosolic calcium concentration. Its spatial proximity to sarcoplasmic reticulum renders a
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micro-domain with high enough calcium concentration. And hence, regional and focal elevation of cytosolic calcium was competent enough to activate the MCU channel[3]. Excitation-contraction
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coupling mediates cytosolic calcium transient periodically, and persistent pressure overload would induce high level calcium and overactivation of MCU[4]. Recently, Zaglia et al reported that MCU is elevated in physiological and pathological cardiac hypertrophy[18], suggesting important roles of MCU on compensation and de-compensation of cardiac function. RR is a specific MCU inhibitor, and previous studies confirmed RR protected hearts from ischemia-reperfusion injury (IRI) in in-situ animal and ex-situ Langendorff-perfusion heart[19, 20]. Mice with D261Q/E264Q mutations presented dominant negative MCU (DN-MCU) phenotype, resulting in low calcium affinity and different metabolic profile from wild-type. DN-MCU mutation significantly decreased ROS production and preserved MMP in IRI[4]. MCU regulated mitochondrial bioenergetics
ACCEPTED MANUSCRIPT metabolism[21] and stress response, also known as “fight or flight” reaction[22]. MCU activation promoted pulmonary fibrosis which could be improved by MCU inhibition[23]. Similarly, our results suggested MCU inhibition notably mitigated cardiac fibrosis. Nonetheless, a universally embryonic MCU knock-out mouse failed to offer protection from TAC[24]. Since MCU had close relationship with energetic metabolism, embryonic and universal MCU knock-out could lead to
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other compensatory mechanism to replace MCU. Thus, subsequent studies should establish an inducible and conditional MCU knock-out model.
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Insufficient activation of autophagy leads to delayed elimination and remarkable accumulation of cellular debris and harmful metabolic products in HF, thus enhancing autophagy could be a
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potential therapy target of HF[25]. Shirakabe, et al. found up-regulation of autophagy emerged in early phase of pressure-overload, which was, however, followed by continuously depressed
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autophagy[26]. LC3B and SQSTM1/p62 were recommended as indispensable markers of autophagy[27]. In this study, although the LC3B II/I ratio increased, reduction of SQSTM1/p62
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degradation revealed an obstructed autophagic flux and depressed total autophagy. A recent study found autophagic flux was impeded in HF, and exercise intervention could evidently improve
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cardiac function through reestablishing autophagy[28]. Likewise, our findings supported the
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conception of weakened autophagy in HF. Besides, it suggested an important role of MCU inhibition in preventing from HF and restoring autophagy. Damaged autophagy aggravated HF[29], and intact autophagic flux was related to calcium homeostasis and mitochondria
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integrity[26, 28].Therefore, MCU inhibition mitigated HF partly through restoring autophagic flux. On the other hand, MCU also closely correlated with the mitophagy. From our data, mitophagy
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remained at low level, while MCU inhibitors could elevate mitophagy. Damaged mitochondria were degraded by a specialized process: mitophagy. Evidence suggested mitophagy should be important for cellular homeostasis, and dampened mitophagy might lead to inadequate removal of dysfunctional mitochondria[14, 16]. In recent studies, mitophagy was found to be impaired in HF, and ways to enhance mitophagy were believed to protect cardiac function[15]. Consistent with the results relating to total cellular homogenates, MCU increased in mitochondria isolated from failing heart. Interestingly, cytosolic MCU, which was scarcely seen in the heart with normal workload, increased in overload-induced HF. This difference suggested stunted MCU transportation from cytosol to mitochondria and cytosolic MCU accumulation. SLC25A as one of
ACCEPTED MANUSCRIPT the solute carrier family 25 proteins participated in the transportation of some mitochondrial membrane proteins synthesized in cytosol[30]. It was reported that SLC25A23 interacted with MCU, and SLC25A23 knockdown remarkably attenuated mitochondrial calcium influx[31]. We speculated MCU expression up-regulated as a compensatory mechanism in response to cardiac overload. However, the number of MCU transporters was limited, and sustained cardiac overload
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induced MCU increase would ultimately saturate its transporters and thus leading to cytosolic MCU accumulation. The specific mechanism of MCU transportation from cytosol to mitochondria
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had never been confirmed before. Moreover, it remains unclear whether abnormal accumulation of MCU in cytosol would be harmful, and more researches in this field are needed. Connexin-43
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expression was proved to be down-regulated in HF[32, 33], and decreased connexin-43 contributed to cardiac asynchrony[34-36]. Prolonged QRS duration indicated asynchronous
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ventricular motion[1], and it was related to connexin-43 decrease as well. In this study, cardiac asynchrony, prolonged QRS duration, and decreased myocardial connexin-43 were proved in TAC
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induced HF, while RR treatment improved cardiac synchronization, shortened QRS duration, and increased connexin-43 expression. Connexin-43 was reported to be expressed in mitochondria[32,
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37]. However, whether MCU had an interaction with connexin-43 was not tested yet.
Limitations
Although our study testified the protective effects of MCU inhibition, the inhibitor’s
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pharmacological specificity remained disputable. It was reported RR could act on L-type calcium channel[38]. Consequently, an extra head-to-head comparison of the effects of RR and amlodipine,
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a well-known L-type calcium channel inhibitor, on 8-week-TAC mice was implemented. The result showed amlodipine failed to prevent from cardiac dilation and dysfunction (supplementary figure 4). Nevertheless, an inducible and conditional MCU-knockout mouse should be established in future study. In this study, changes of connexin-43 were thought to be related to the improvement of cardiac conduction. However, instead of the change of the quantity, lateralization and phosphorylation of connexin43 also influenced its function. Therefore, it is worthy of further investigation of the mechanism of the connexin43 in heart failure and ventricular asynchrony.
ACCEPTED MANUSCRIPT Conclusion Autophagy was dampened in HF, meanwhile, outstanding MCU was up-regulated in response to cardiac overload. Cytosolic MCU accumulation occurred in the TAC group instead of the sham group. Furthermore, MCU inhibition dramatically decreased total mortality and morbidity, improved cardiac function, and maintained mitochondrial integrity. MCU inhibition increased connexin-43 expression and alleviated cardiac asynchrony. Besides, RR treatment in vivo could
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partly restore autophagy and mitophagy. In vitro experiment confirmed MCU’s intervention
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promoted autophagy and mitophagy. In summary, MCU played an important role in pressure overload-induced HF through autophagy and mitophagy alteration. The intervention on MCU
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offered cardioprotective effects. To our knowledge, our study firstly disclosed the role of MCU in HF and its relationship with autophagy and mitophagy. Moreover, this study threw lights on MCU
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as a potential therapeutic target of HF.
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calcium channels. J Pharmacol Exp Ther 289:1447-1453 doi:
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Figure legends: Figure 1. Gross samples and histological analyses
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The heart size tended to increase along with the elongated duration of pressure overload. At 2 weeks of TAC, a remarkable myocardial hypertrophy was observed with mild cardiac fibrosis; At 4 weeks hypertrophy continued and the cavity was enlarged. Meanwhile moderate cardiac fibrosis occurred; At 8 weeks, the typical decompensated HF characterized with outstanding cardiac dilation, thickened wall, and severe fibrosis was observed. However, pressure-overloaded hearts receiving RR treatment were partly immune from cardiac dilation. With 400× magnification, the CSA and fibrosis area were larger in TAC groups, while RR could partly prevent from cardiomyocyte enlargement and cardiac fibrosis (n=5 in each group). Scale bars = 2mm for gross appearance and mid-transverse section, scale bars = 50μm for cardiomyocyte size of left ventricle. *: p < 0.01 vs. sham. †: p < 0.01 vs. TAC8w. TAC: transverse aortic constriction; CSA: cross-sectional area; RR: ruthenium red.
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Figure 2. Cardiac function and expression of protein at different phases of TAC
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Panel A-F showed the echocardiological evaluation (n = 10 in each group). Red arrowhead indicates the acoustic shadow of ligation around the constrictive aortic arch before the initiation of left carotid artery. Heart rates (HR) between different groups showed no difference, and influence of HR on echocardiographic results could be neglected. Echocardiographic test showed no difference of left ventricular ejection fraction (LVEF) between sham and TAC at 2 weeks, while LVEF decreased afterwards. At 2 weeks, there were no difference of left ventricular end-diastolic/systolic diameter (LVEDD and LVESD) between sham and TAC, while LVEDD and LVESD both increased afterwards. And this phenomenon was consistent with the clinical concept of LVEF-preserved HF and LVEF-reduced HF; Panel G-I showed differential expression of atrial natriuretic peptide (ANP), mitochondrial calcium uniporter (MCU), microtubule-associated protein 1 Light Chain 3 beta (LC3B), SQSTM1/p62, Beclin-1, and GAPDH (white bar referring to sham group, black bar referring to TAC group, n = 6 in each group). ANP, MCU, and LC3B II/I ratio increased, while Beclin-1 decreased throughout the three phases. Panel G revealed the differential expression of MCU in mitochondria and cytosol (n = 5 in each group). Isolated mitochondrial MCU expression was consistent with the results of total tissue protein. At baseline, MCU was hardly seen in cytosol, but it emerged in cytosol after 8 weeks of TAC. Figure 3. The effect of MCU inhibition on pressure-overloaded heart Panel A showed in situ differential expression of MCU and connexin-43 by immunohistochemistry (n = 5 in each group). Besides, cardiac strain curve at long-axis view and electrocardiograph could be seen (n = 10 in each group); Panel B showed the difference of mitochondrial area/number ratio among 3 groups (white referring to sham group, black bar referring to TAC+NS group, and grey bar referring to TAC+RR group, n = 6 in each group); Panel C showed differential expression of MCU, connexin-43, and autophagy and mitophagy related protein (white bar referring to sham group, black bar referring to TAC+NS group, and grey bar referring to TAC+RR group, n = 6 in each group). Survival analysis included 2 parts, short period (7 days) and long period (56 days). There was no difference in death rate within 1 week, while
ACCEPTED MANUSCRIPT TAC+RR group had lower mortality than TAC+NS group (black line referring to sham group, n = 20; red line referring to TAC+NS group, n = 40; blue line referring to TAC+RR group, n = 40); Panel D showed the difference of 18FDG metabolism assessed by positron emission tomography (PET) scan (n = 5 in each group), and the result was presented as standardized uptake value (SUV); Panel E showed mitochondrial membrane potential measured by JC-1 kit (n = 5 in each group); Panel F revealed the difference of cardiac function evaluated by echocardiograph among groups (n = 10 in each group). NS: normal saline; FDG: fluorodeoxyglucose.
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Figure 4. In vitro validation of MCU in correlation with autophagy and mitophagy Panel A showed 3 concentrations of RR influenced H9c2 cell autophagy at 48h and 72h respectively (white bar referring to control cell, light grey bar referring to 5 nM RR treatment, dark grey bar referring to 50 nM RR treatment, and black bar referring to 500 nM RR treatment, n = 6 in each group); Panel B disclosed a similar result through GFP expressed siRNA plasmid transfection induced MCU knock-down in H9c2 cells (white bar referring to blank vector control, grey bar referring to scrambleRNA control, and black bar referring to siRNA interference, n = 6 in each group); Panel C showed the difference of the HL-1 cell size in three groups to testify the effect of MCU inhibition by 50 μM Ru360 in HL-1 cells with treatment of 10-5 M isoprenaline for 72h (scale bars = 50μm; n = 6 in each groups; NS: normal saline; Iso: isoprenaline); Panel D indicated the evaluation of the change of autophagy and mitophagy in different groups (n = 6 in each groups).
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Mitochondrial calcium uniporter (MCU) inhibition played an important role in pressure overload-induced heart failure through autophagy and mitophagy enhancement.
Intervention on MCU offered cardioprotective effects: improving cardiac function, correcting ventricular asynchrony, and preventing from excessive enlargement of cardiomyocytes.
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MCU inhibition could be explored as a novel therapeutic concept in heart failure treatment.
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Ziqing Yu, Ruizhen Chen, Minghui Li, Yong Yu, Yixiu Liang, Fei Han, Shengmei Qin, Xueying Chen, Yangang Su, Junbo Ge PII: DOI: Reference:
S0167-5273(17)37641-6 doi:10.1016/j.ijcard.2018.05.054 IJCA 26467
To appear in: Received date: Revised date: Accepted date:
10 December 2017 10 May 2018 17 May 2018
Please cite this article as: Ziqing Yu, Ruizhen Chen, Minghui Li, Yong Yu, Yixiu Liang, Fei Han, Shengmei Qin, Xueying Chen, Yangang Su, Junbo Ge , Mitochondrial calcium uniporter inhibition provides cardioprotection in pressure overload-induced heart failure through autophagy enhancement. The address for the corresponding author was captured as affiliation for all authors. Please check if appropriate. Ijca(2017), doi:10.1016/ j.ijcard.2018.05.054
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ACCEPTED MANUSCRIPT Mitochondrial Calcium Uniporter Inhibition Provides Cardioprotection in Pressure Overload-Induced Heart Failure through Autophagy Enhancement Ziqing Yuab†, MD, PhD; Ruizhen Chenac†, MD, PhD; Minghui Liac, MD, PhD; Yong Yuac, MD, PhD; Yixiu Liangab, MD, PhD; Fei Hanab, MD, PhD; Shengmei Qina, MD, PhD; Xueying Chena, MD, PhD; Yangang Sua*, MD, PhD; Junbo Gea*, MD, PhD
a: Department of Cardiology, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital,
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Fudan University, Shanghai 200032, PR China b: Shanghai Medical College, Fudan University, Shanghai 200032, PR China
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c: Department of Cardiovascular Diseases, Key Laboratory of Viral Heart Diseases, Ministry of Public Health, Shanghai Institute of Cardiovascular Diseases, Zhongshan Hospital, Fudan
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University, Shanghai 200032, PR China
†: Equal contribution *: Correspondence to:
Email: [email protected]
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Yangang Su*, MD, PhD, FHRS, FEHRA
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Conflicts of interest: None.
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Professor of Medicine/Cardiology
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Junbo Ge*, MD, PhD, FESC, FACC, FSCAI
Department of Cardiology, Zhongshan Hospital, Fudan University; and Shanghai Institute of Cardiovascular Diseases
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180 Fenglin Road, Shanghai 200032, PR China Tel: 86-21-64041990 ext 2745
Fax: 86-21-64223006
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Email: [email protected]
Fundings
This study was supported by National Natural Science Foundation of China (Grant No: 81671934, 81521001, 81400280 and 31570904) and Science and Technology Commission of Shanghai Municipality (Grant No: 17140902400).
Competing interests The authors declare that they have no competing interest.
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ACCEPTED MANUSCRIPT Abstract: Background: HF incurs high disease burden, and the effectiveness of known HF treatments is unsatisfactory. Therefore, seeking novel therapeutic target of HF is important. The present study aimed to investigate the role of the mitochondrial calcium uniporter (MCU) and its relationship with autophagy in overload-induced heart failure (HF).
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Methods and results: In both early-stage and end-stage of pressure overload-induced HF, MCU appeared up-regulated along with heart enlargement, increased microtubule-associated proteins
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1A/1B light chain 3B (LC3B) II/I ratio and autophagosome content, damaged cardiac function, and ventricular asynchrony. However, sequestosome-1 (SQSTM1/p62) level decreased indicating
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blockaded autophagic flux. Seven-week administration of MCU inhibitor ruthenium red improved cardiac function and mitigated its pathological change. MCU inhibition maintained mitochondrial
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integrity, increased LC3B II/I ratio, up-regulated Parkin and Pink1, and down-regulated SQSTM1/p62. MCU inhibition also alleviated ventricular asynchrony of HF, and this might be
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related to connexin-43 up-regulation. In vitro study validated intervention on MCU leading to elevation of autophagy and mitophagy. MCU inhibition could partly prevent from excessive
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cellular enlargement induced by isoprenaline.
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Conclusions: In summary, MCU inhibition played an important role in pressure overload-induced heart failure through autophagy and mitophagy enhancement, and intervention on MCU offered cardioprotective effects. To our knowledge, the role of MCU in HF and its relationship with
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autophagy and mitophagy are firstly disclosed. Moreover, our study suggests that MCU inhibition could be explored as a novel therapeutic concept in HF.
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Key words: mitochondrial calcium uniporter; heart failure; transverse aortic constriction; autophagy; ventricular asynchrony
ACCEPTED MANUSCRIPT Introduction Nowadays, heart failure (HF) has become one of the most predominant disease burdens worldwide, and its mortality and morbidity rates remain high. Current HF therapeutic strategy includes guideline-directed medical therapy and cardiac device therapy, e.g. cardiac resynchronization therapy and implantable cardioverter defibrillator[1, 2], etc., however, their efficacy are limited.
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Given the inadequate effectiveness of the current therapeutic targets, it’s necessary to identify novel therapeutic targets.
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Mitochondria, the energy factory of cells, accounts for a large proportion in cardiomyocytes[3]. Therefore, mitochondrial disorder is usually associated with heart diseases. The maintenance of
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myocardial performance is dependent on normal mitochondrial function[3, 4]. Cardiac contraction and relaxation are ATP-consuming and calcium dependent. In the process of ATP synthesis,
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calcium plays an important role by activating key enzyme in redox reaction and taking part in the constitution of complexes of ECT[5]. Although cytosolic calcium can easily pass through the outer
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mitochondrial membrane (OMM) via voltage-dependent anion channel (VDAC), the inner mitochondrial membrane (IMM) is highly calcium-selective. The mitochondrial calcium uniporter
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(MCU) synthesized in cytosol is localized to IMM. As a pore-forming structural protein, MCU is
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responsible for the majority of calcium uptake[6]. Consequently, MCU determines calcium concentration in mitochondrial matrix, and in turn affects mitochondrial energetic metabolism. Since mitochondrial calcium overload induces cellular injury, MCU also influences cell viability
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and even leads to cell death. Herein we hypothesized that MCU could be a potential therapeutic target of HF. In the present study, we investigated the effect of the MCU inhibitor or
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RNAi-mediated MCU depletion on cardiac function in an overload-induced HF model.
Materials and methods Animal and HF model Male C57BL/6 mice with ages of 8-10 weeks and weights of ±30 g were studied, and overload-induced HF models were established by transverse aortic constriction (TAC) method (see supplementary methods). Ruthenium red (RR) purchased from Sigma is administrated 1 week after the surgery. Animals were divided into 3 groups: sham+NS, TAC+NS, and TAC+RR (n=20 in each group). After TAC surgery, animals are randomly assigned to TAC+NS group or TAC+RR
ACCEPTED MANUSCRIPT group. This animal study was approved by the Ethics Committee of Zhongshan Hospital affiliated to Fudan University.
Echocardiography and electrocardiography Electrocardiograph was recorded and the QRS duration was measured using biological signal
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collecting/processing system (RM6240, Chengdu Instrument Factory, China); Regular echocardiograph and two-dimension speckle tracking imaging (2D-STI) were acquired by using a
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32–55 MHz linear array transducer via Vevo 2100 ultrasound system (VisualSonics Inc., Canada). Murine models were fixed in supine position and anesthetized by isoflurane (4% for induction, 2%
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for maintenance). The following left ventricular measurements were performed: end-systolic diameter (LVESD), end-systolic volume (LVESV), end-diastolic diameter (LVEDD), end-diastolic
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volume (LVEDD), ejection fraction (LVEF), fractional shortening (FS), and ventricular wall thickness on the M-mode at parasternal long-axis view. With a speckle-tracking algorithm, strain
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analysis was quantified by radial and longitudinal description. The ventricular wall asynchrony was evaluated by the standard deviation of time to peak myocardial systolic velocity of the 6 LV
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segments (Ts-SD6), strain, and strain rate (SR) as reported before[7, 8]. All echocardiographic
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image measurements were calculated over 3 consecutive cardiac cycles and then averaged.
Positron emission tomography
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Positron emission tomography (PET) scan was performed to acquire the standardized uptake value (SUV) via MadicLab PET system (Madic Technology Co., Ltd, China)[9] to detect the survival
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myocardium and assess its viability (see supplementary methods).
Morphological examination The hearts were promptly obtained, fixed in formalin, embedded in paraffin, sliced into 5-μm-thick sections, and stained by Hematoxylin-Eosin or Masson’s trichrome method; Immunohistochemistry was performed by the avidin–biotin–peroxidase complex method. After rehydration and microwave antigen retrieval, tissue slices are incubated with MCU antibodies (1:200, orb317655, biorbyt) or connexin-43 antibodies (1:1000, ab11370, abcam) at 4 °C overnight and incubated with secondary antibodies (K5007, Dako, Denmark) at 37 °C for 30 min.
ACCEPTED MANUSCRIPT Finally, 3,3’-diaminobenzidine staining and Mayer’s hematoxylin counterstaining were used for developing. Negative control without the primary antibodies was included to rule out false-negative/positive; Preparing the transmission electron microscope (TEM) sample, cardiac tissue was fixed in 2.5% glutaraldehyde, dehydrated by gradient ethanol, embedded with pure acetone, solidified in oven, sliced, and stained with gold. TEM image was acquired by an
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electro-microscopic system (FEI Tecnai G2 Spirit).
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Western blots (WB)
Tissue homogenate, cytosolic lysates, or mitochondrial lysates were mixed with RIPA lysis buffer
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with protease inhibitor cocktail. Proteins were acquired by ultracentrifugation, determined by BCA method, separated on a 12% SDS-PAGE, and transferred to polyvinylidene difluoride membranes.
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After dilution and blocking, membranes were incubated with primary antibodies of MCU (1:1000, 14997, CST), atrial natriuretic peptide (ANP) (1:500, abcam), microtubule-associated proteins
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1A/1B light chain 3B (LC3B) (1:1000, 2775, CST), sequestosome-1 (SQSTM1/p62) (1:1000, 5114, CST), Beclin-1 (1:1000, 3495, CST), connexin-43 (1:8000, ab11370, abcam), Parkin
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(1:1000, 4211, CST), or Pink1 (1:1000, ab23707, abcam) at 4°C overnight. Incubated with
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rabbit-radish peroxidase-conjugated secondary antibody, the binding reactions in the membranes were detected by enhanced chemiluminescence assays.
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Cardiac tissue mitochondria isolation and mitochondrial membrane potential measurement The protocol was followed as published before[10]. Mitochondrial membrane potential (MMP)
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was measured by JC-1 assay kit. JC-1 polymer with red color indicates polarized MMP. JC-1 monomer with green color indicates depolarized MMP. The Change of MMP was assessed by fluorescence intensity ratio of green color/red color (see supplementary methods).
Cell culture, siRNA plasmid construction, and, transfection In vitro validation of the effect of MCU was carried out by MCU inhibitor RR and siRNA induced MCU ablation on H9c2 cardiomyoblasts, purchased from the cell bank of Chinese Academy of Science. Cells were cultured in DMEM medium with fetal bovine serum (Gemini), treated with RR of three concentrations (5 μM, 50 μM, and 500 μM), and incubated for 48h or 72h. The
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(GCTTGTCATTAATGACTTAAC), connecting with a green fluorescent protein (GFP) gene was integrated and amplified in Escherichia coli bacteria system (Clontech). H9c2 cells were transfected with plasmid through lipofectamine 2000 transfection kit (Thermofisher Scientific) for 48h and 72h. Successful transfection was verified by GFP under fluorescence microscope investigation, and MCU knock-down was verified by WB-analysis. HL-1 cell line originating
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from mouse cardiomyocyte with treatment of 10-5 M isoprenaline for 72h was used to testify the
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effect of MCU inhibition by 50 μM Ru360 (sc-222265, Santa Cruz). The difference of the cellular
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area was compared, and the autophagy and mitophagy were evaluated afterwards.
Statistical analysis
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Continuous variables were presented as mean±standard error of mean. Inter-group difference was assessed by Student’s t test or one-way analysis of variance (ANOVA) via SPSS software 19.0. A p
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value of less than 0.05 was regarded as significant.
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Results
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Differential expression of MCU and autophagy associated protein in overload induced cardiac hypertrophy and heart failure
Animal models establishment was confirmed by heart mass index (heart weight/body weight ratio
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and heart weight/tibia length ratio), echocardiography, and the ANP protein expression. Hearts were heavier in the TAC group at different stages (supplementary figure 1A and 1B). Remarkable
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cardiac hypertrophy was observed at 2 weeks of TAC with slightly affected LVEF. However, LVEDD in the TAC group was smaller, indicating abnormal diastolic function in the early phase. LVEF further decreased at four weeks after TAC. Decompensated HF developed at 8 weeks with severely deteriorated LVEF and enlarged intra-ventricular diameter (Figure 2A-F and supplementary table 1). Pathological tests confirmed various levels of fibrosis caused by TAC (Figure 1). Total MCU was up-regulated and reached the summit (p=0.001) at 2 weeks after TAC (Figure 2G-I). Meanwhile, autophagy associated protein expression was detected as well. LC3B-II/I ratio increased steadily after TAC, indicating autophagosome formation. However, the substrate of
ACCEPTED MANUSCRIPT autophagy SQSTMl/p62 in TAC mice was significantly higher than that in the control group (p<0.05). Additionally, Beclin-1’s expression was depressed in TAC mice (p<0.01)，indicating inhibited formation of autolysosomes and autophagic flux.
MCU expression in isolated mitochondria and cytosol
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Furthermore, 8 weeks of TAC was chosen for MCU expression analysis in isolated mitochondria and cytosol. Cellular constituent separation was proved by mitochondrial marker COX IV and
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cytosolic marker GAPDH (Figure 2J). Mitochondrial MCU level was higher in the TAC-induced HF group (p=0.001). In the sham group, MCU was apparently expressed in mitochondrial
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integrity, while it was hardly seen in cytosol. In the HF group, mitochondrial MCU was outstandingly elevated (p<0.01). Interestingly, MCU quantity in cytosol was different from each
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other in comparison between sham group and HF group. Cytosolic MCU level was dramatically elevated in the HF group (p=0.004). These results together revealed that MCU only localized in
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the mitochondria rather than the cytosol in physiological condition. But in TAC-induced HF, both
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mitochondrial MCU and cytosolic MCU were up-regulated.
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MCU inhibition preserving cardiac function and reducing mortality RR was introduced as an inhibitor of MCU, and RR of two concentrations (1.25mg·kg-1·d-1 and 2.5mg·kg-1·d-1) were intraperitonealy injected to the mice to find a better concentration for MCU
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inhibition. Preliminary experiment with 8-week observation showed the higher concentration presented better cardiac function (Supplementary figure 2). Consequently, RR of 2.5mg·kg-1·d-1
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was chosen, and the RR administration protocol was shown in supplementary figure 3. Compared with normal saline (NS) treated TAC group, RR treatment showed significant improvement of cardiac function (LVEF: 60±1 vs. 27±1.4 %, p<0.001; LVEDD: 5.3±0.1 vs. 3.8±0.09, p<0.001; LVESD: 4.6±0.1 vs. 2.6±0.04, p<0.001). Within 1 week of TAC, there was no difference of mortality between TAC+RR and TAC+NS (HR=0.83, 95% CI: 0.22-3.33), while significantly lower death rate was found in TAC+RR group (HR=0.45, 95% CI: 0.18-0.91) at 8 weeks (Figure 3C). MCU inhibition reduced heart size, and alleviated cardiac fibrosis (Figure 1). Lower HW/BWR and HW/TLR proved decreased heart mass in TAC+RR group (supplementary figure 1C and 1D). Compared to TAC group, LC3B II/I ratio, Parkin, and Pink1 increased in TAC+RR
ACCEPTED MANUSCRIPT group (Figure 3C), indicating RR treatment could induce both elevation of autophagy and mitophagy in vivo.
Inhibition of MCU improving myocardial viability, protecting mitochondrial integrity, and maintaining mitochondrial membrane potential in vivo After 8 weeks of TAC, mice were starved overnight and then received PET scanning (Figure 3D).
18
FDG than the NS treated TAC
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0.01). However, the RR treated TAC group had higher SUV of
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TAC+NS group showed a significant decrease in SUV of 18FDG compared to the sham group (p <
group (p < 0.05). These results revealed higher proportion of viable myocardia survival from TAC
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in RR treated mice. Moreover, TAC-induced morphological change of mitochondria, e.g. cristae edema and disruption, was evidently alleviated in the mice treated with RR (Figure 3B, yellow
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arrowhead). Besides, rupture of myofilament was more serious in the NS treated mice than that in RR treated mice after TAC (red arrowhead). In consistent with the expression of LC3B, Parkin,
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and Pink1, the number of autophagosomes (black arrowhead) and mitophagosomes (blue arrowhead) increased in TAC+RR group. Semi-quantitative analysis of the mitochondrial
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area/number ratio showed that TAC+NS group had a higher value than TAC+RR group, indicating
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mitigation of mitochondrial swell by RR. Furthermore, mitochondrial membrane potential (MMP) was measured by JC-1 kit to assess mitochondrial integrity (Figure 3E). Higher monomer (green) / polymer (red) ratio was detected in TAC+NS group, indicating dissipated MMP compared with
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TAC+RR group (1.39±0.1 vs. 0.92±0.08, p<0.001).
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Inhibition of MCU improving ventricular synchronization and narrowing QRS duration QRS duration of ECG correlates to synchronous motion of ventricular wall, and widening QRS duration indicated cardiac asynchrony[1]. ECG was acquired to measure the QRS duration to evaluate the cardiac synchronization. After 8 weeks, TAC+NS mice presented remarkably broadened QRS duration compared to sham+NS mice, while QRS duration of TAC+RR mice was significantly shorter than that of TAC+NS mice (8.8±0.3 vs. 11.7±0.5 ms, p<0.001). Additionally, 2D-STI showed markedly asynchronous ventricular wall motion of TAC+NS mice, while RR could partly correct cardiac asynchrony (Figure 3A). Compared to sham+NS group, dispersion of six-segment ventricular motion assessed by Ts-SD6 was greater in TAC+NS group (5.2±0.5 vs.
ACCEPTED MANUSCRIPT 28.9±4 ms, p=0.006). Nevertheless, RR dramatically reduced this dispersion in HF (18.6±0.9 vs. 28.9±4 ms, p=0.041). TAC+RR group had higher absolute value of radial strain, radial strain rate, longitudinal strain, and longitudinal strain rate compared to TAC+NS group. Connexin-43 plays a key role of cardiac conduction and intercellular communication[11], and its abnormal expression was regarded as critical mechanism of cardiac asynchrony[12, 13]. The Connexin-43 expression
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decreased in TAC+NS group (p<0.001). However, TAC+RR group had higher level of connexin-43 compared to TAC+NS group (p=0.006). Besides, what should be noteworthy was
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that MCU level of TAC+NS group and TAC+RR group was similar (p=0.96), revealing RR
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treatment in vivo didn’t influence MCU expression in cardiac tissue (Figure 3C).
Intervention of MCU prompting autophagy and mitophagy enhancement in vitro
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Three doses of RR (5 μM, 50 μM, and 500 μM) were applied to H9c2 cell line hatched for 48h to investigate the influence of MCU inhibition on autophagy. A dose-dependent autophagy
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enhancement was found. LC3-B II/I ratio was gradually increased, and SQSTMl/p62 expression tended to be down-regulated when the RR concentration elevated. However, RR didn’t affect
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MCU expression since non-significant difference of its expression was detected, which was
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consistent with the in vivo results (Figure 4A). Furthermore, siRNA plasmid transfection of H9c2 cell consolidated the conception that MCU depletion dramatically elevated the autophagic level. The transfection efficiency of GFP-exprssed and mcu-interfered siRNA plasmid in H9c2 cell was
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over 70%, and the interfering sequence (GCTTGTCATTAATGACTTAAC) was effective enough to relegate MCU (p=0.004). MCU-ablation increased LC3B II/I ratio and decreased SQSTMl/p62
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compared to vehicle-control cell and scramble-control cell (Figure 4B). Furthermore, in HL-1 cells, the treatment of isoprenaline for 72h was enough to induce cellular enlargement, while MCU inhibition could partly prevent them from excessively increasing in size induced by the stimulus (Figure 4C). MCU inhibition elevated LC3B II/I ratio and decreased SQSTM1/p62’s level (Figure 4D), suggesting strengthened autophagy. Moreover, Ru360 could up-regulate Parkin and Pink1’s level which were indicators of enhanced mitophagy[14-16].
Discussion In the present study, pressure-overload-induced heart disease manifested as different phenotype
ACCEPTED MANUSCRIPT from compensatory cardiac hypertrophy to decompensatory HF at different phase. MCU up-regulation was proved in this pathological process. Besides, we further examined MCU expression in human heart tissue acquired from heart transplant surgery and a canine HF model, and MCU was proved to be elevated in both conditions (data not shown). These results suggested that MCU play an important role in the initiation and progression of HF. Autophagy was impeded in accompany with MCU’s changes. Mitochondria isolation analysis indicated that MCU was
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hardly seen in cytosol with normal cardiac workload, while mitochondrial MCU’s up-regulation
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and cytosolic MCU’s accumulation emerged in HF. MCU inhibitor RR protected from aggravation of pressure-overload-induced HF and significantly improve the survival rate. SUV of
18
FDG was
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higher in RR-treated group revealing more survival of cardiomyocytes and cell viability. RR could preserve mitochondrial integrity and promote autophagy in vivo. Compared to HF group, cardiac
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asynchrony was relieved, and Connexin-43 expression was elevated in RR group. RR or mcu-siRNA could independently increase autophagosome formation and prompt autophagic flux
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in vitro. To our knowledge, this study firstly disclosed the important role of MCU in improving cardiac function and correcting cardiac asynchrony. MCU inhibition recovered autophagic flux in
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HF. Findings from the present study provided a novel therapeutic target of HF.
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MCU as a pore-forming structure of a complex was predominantly responsible for calcium entrance[17]. MCU had high conductance of calcium, and its activation was dependent to cytosolic calcium concentration. Its spatial proximity to sarcoplasmic reticulum renders a
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micro-domain with high enough calcium concentration. And hence, regional and focal elevation of cytosolic calcium was competent enough to activate the MCU channel[3]. Excitation-contraction
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coupling mediates cytosolic calcium transient periodically, and persistent pressure overload would induce high level calcium and overactivation of MCU[4]. Recently, Zaglia et al reported that MCU is elevated in physiological and pathological cardiac hypertrophy[18], suggesting important roles of MCU on compensation and de-compensation of cardiac function. RR is a specific MCU inhibitor, and previous studies confirmed RR protected hearts from ischemia-reperfusion injury (IRI) in in-situ animal and ex-situ Langendorff-perfusion heart[19, 20]. Mice with D261Q/E264Q mutations presented dominant negative MCU (DN-MCU) phenotype, resulting in low calcium affinity and different metabolic profile from wild-type. DN-MCU mutation significantly decreased ROS production and preserved MMP in IRI[4]. MCU regulated mitochondrial bioenergetics
ACCEPTED MANUSCRIPT metabolism[21] and stress response, also known as “fight or flight” reaction[22]. MCU activation promoted pulmonary fibrosis which could be improved by MCU inhibition[23]. Similarly, our results suggested MCU inhibition notably mitigated cardiac fibrosis. Nonetheless, a universally embryonic MCU knock-out mouse failed to offer protection from TAC[24]. Since MCU had close relationship with energetic metabolism, embryonic and universal MCU knock-out could lead to
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other compensatory mechanism to replace MCU. Thus, subsequent studies should establish an inducible and conditional MCU knock-out model.
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Insufficient activation of autophagy leads to delayed elimination and remarkable accumulation of cellular debris and harmful metabolic products in HF, thus enhancing autophagy could be a
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potential therapy target of HF[25]. Shirakabe, et al. found up-regulation of autophagy emerged in early phase of pressure-overload, which was, however, followed by continuously depressed
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autophagy[26]. LC3B and SQSTM1/p62 were recommended as indispensable markers of autophagy[27]. In this study, although the LC3B II/I ratio increased, reduction of SQSTM1/p62
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degradation revealed an obstructed autophagic flux and depressed total autophagy. A recent study found autophagic flux was impeded in HF, and exercise intervention could evidently improve
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cardiac function through reestablishing autophagy[28]. Likewise, our findings supported the
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conception of weakened autophagy in HF. Besides, it suggested an important role of MCU inhibition in preventing from HF and restoring autophagy. Damaged autophagy aggravated HF[29], and intact autophagic flux was related to calcium homeostasis and mitochondria
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integrity[26, 28].Therefore, MCU inhibition mitigated HF partly through restoring autophagic flux. On the other hand, MCU also closely correlated with the mitophagy. From our data, mitophagy
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remained at low level, while MCU inhibitors could elevate mitophagy. Damaged mitochondria were degraded by a specialized process: mitophagy. Evidence suggested mitophagy should be important for cellular homeostasis, and dampened mitophagy might lead to inadequate removal of dysfunctional mitochondria[14, 16]. In recent studies, mitophagy was found to be impaired in HF, and ways to enhance mitophagy were believed to protect cardiac function[15]. Consistent with the results relating to total cellular homogenates, MCU increased in mitochondria isolated from failing heart. Interestingly, cytosolic MCU, which was scarcely seen in the heart with normal workload, increased in overload-induced HF. This difference suggested stunted MCU transportation from cytosol to mitochondria and cytosolic MCU accumulation. SLC25A as one of
ACCEPTED MANUSCRIPT the solute carrier family 25 proteins participated in the transportation of some mitochondrial membrane proteins synthesized in cytosol[30]. It was reported that SLC25A23 interacted with MCU, and SLC25A23 knockdown remarkably attenuated mitochondrial calcium influx[31]. We speculated MCU expression up-regulated as a compensatory mechanism in response to cardiac overload. However, the number of MCU transporters was limited, and sustained cardiac overload
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induced MCU increase would ultimately saturate its transporters and thus leading to cytosolic MCU accumulation. The specific mechanism of MCU transportation from cytosol to mitochondria
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had never been confirmed before. Moreover, it remains unclear whether abnormal accumulation of MCU in cytosol would be harmful, and more researches in this field are needed. Connexin-43
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expression was proved to be down-regulated in HF[32, 33], and decreased connexin-43 contributed to cardiac asynchrony[34-36]. Prolonged QRS duration indicated asynchronous
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ventricular motion[1], and it was related to connexin-43 decrease as well. In this study, cardiac asynchrony, prolonged QRS duration, and decreased myocardial connexin-43 were proved in TAC
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induced HF, while RR treatment improved cardiac synchronization, shortened QRS duration, and increased connexin-43 expression. Connexin-43 was reported to be expressed in mitochondria[32,
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37]. However, whether MCU had an interaction with connexin-43 was not tested yet.
Limitations
Although our study testified the protective effects of MCU inhibition, the inhibitor’s
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pharmacological specificity remained disputable. It was reported RR could act on L-type calcium channel[38]. Consequently, an extra head-to-head comparison of the effects of RR and amlodipine,
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a well-known L-type calcium channel inhibitor, on 8-week-TAC mice was implemented. The result showed amlodipine failed to prevent from cardiac dilation and dysfunction (supplementary figure 4). Nevertheless, an inducible and conditional MCU-knockout mouse should be established in future study. In this study, changes of connexin-43 were thought to be related to the improvement of cardiac conduction. However, instead of the change of the quantity, lateralization and phosphorylation of connexin43 also influenced its function. Therefore, it is worthy of further investigation of the mechanism of the connexin43 in heart failure and ventricular asynchrony.
ACCEPTED MANUSCRIPT Conclusion Autophagy was dampened in HF, meanwhile, outstanding MCU was up-regulated in response to cardiac overload. Cytosolic MCU accumulation occurred in the TAC group instead of the sham group. Furthermore, MCU inhibition dramatically decreased total mortality and morbidity, improved cardiac function, and maintained mitochondrial integrity. MCU inhibition increased connexin-43 expression and alleviated cardiac asynchrony. Besides, RR treatment in vivo could
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partly restore autophagy and mitophagy. In vitro experiment confirmed MCU’s intervention
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promoted autophagy and mitophagy. In summary, MCU played an important role in pressure overload-induced HF through autophagy and mitophagy alteration. The intervention on MCU
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offered cardioprotective effects. To our knowledge, our study firstly disclosed the role of MCU in HF and its relationship with autophagy and mitophagy. Moreover, this study threw lights on MCU
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as a potential therapeutic target of HF.
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Figure legends: Figure 1. Gross samples and histological analyses
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The heart size tended to increase along with the elongated duration of pressure overload. At 2 weeks of TAC, a remarkable myocardial hypertrophy was observed with mild cardiac fibrosis; At 4 weeks hypertrophy continued and the cavity was enlarged. Meanwhile moderate cardiac fibrosis occurred; At 8 weeks, the typical decompensated HF characterized with outstanding cardiac dilation, thickened wall, and severe fibrosis was observed. However, pressure-overloaded hearts receiving RR treatment were partly immune from cardiac dilation. With 400× magnification, the CSA and fibrosis area were larger in TAC groups, while RR could partly prevent from cardiomyocyte enlargement and cardiac fibrosis (n=5 in each group). Scale bars = 2mm for gross appearance and mid-transverse section, scale bars = 50μm for cardiomyocyte size of left ventricle. *: p < 0.01 vs. sham. †: p < 0.01 vs. TAC8w. TAC: transverse aortic constriction; CSA: cross-sectional area; RR: ruthenium red.
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Figure 2. Cardiac function and expression of protein at different phases of TAC
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Panel A-F showed the echocardiological evaluation (n = 10 in each group). Red arrowhead indicates the acoustic shadow of ligation around the constrictive aortic arch before the initiation of left carotid artery. Heart rates (HR) between different groups showed no difference, and influence of HR on echocardiographic results could be neglected. Echocardiographic test showed no difference of left ventricular ejection fraction (LVEF) between sham and TAC at 2 weeks, while LVEF decreased afterwards. At 2 weeks, there were no difference of left ventricular end-diastolic/systolic diameter (LVEDD and LVESD) between sham and TAC, while LVEDD and LVESD both increased afterwards. And this phenomenon was consistent with the clinical concept of LVEF-preserved HF and LVEF-reduced HF; Panel G-I showed differential expression of atrial natriuretic peptide (ANP), mitochondrial calcium uniporter (MCU), microtubule-associated protein 1 Light Chain 3 beta (LC3B), SQSTM1/p62, Beclin-1, and GAPDH (white bar referring to sham group, black bar referring to TAC group, n = 6 in each group). ANP, MCU, and LC3B II/I ratio increased, while Beclin-1 decreased throughout the three phases. Panel G revealed the differential expression of MCU in mitochondria and cytosol (n = 5 in each group). Isolated mitochondrial MCU expression was consistent with the results of total tissue protein. At baseline, MCU was hardly seen in cytosol, but it emerged in cytosol after 8 weeks of TAC. Figure 3. The effect of MCU inhibition on pressure-overloaded heart Panel A showed in situ differential expression of MCU and connexin-43 by immunohistochemistry (n = 5 in each group). Besides, cardiac strain curve at long-axis view and electrocardiograph could be seen (n = 10 in each group); Panel B showed the difference of mitochondrial area/number ratio among 3 groups (white referring to sham group, black bar referring to TAC+NS group, and grey bar referring to TAC+RR group, n = 6 in each group); Panel C showed differential expression of MCU, connexin-43, and autophagy and mitophagy related protein (white bar referring to sham group, black bar referring to TAC+NS group, and grey bar referring to TAC+RR group, n = 6 in each group). Survival analysis included 2 parts, short period (7 days) and long period (56 days). There was no difference in death rate within 1 week, while
ACCEPTED MANUSCRIPT TAC+RR group had lower mortality than TAC+NS group (black line referring to sham group, n = 20; red line referring to TAC+NS group, n = 40; blue line referring to TAC+RR group, n = 40); Panel D showed the difference of 18FDG metabolism assessed by positron emission tomography (PET) scan (n = 5 in each group), and the result was presented as standardized uptake value (SUV); Panel E showed mitochondrial membrane potential measured by JC-1 kit (n = 5 in each group); Panel F revealed the difference of cardiac function evaluated by echocardiograph among groups (n = 10 in each group). NS: normal saline; FDG: fluorodeoxyglucose.
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Figure 4. In vitro validation of MCU in correlation with autophagy and mitophagy Panel A showed 3 concentrations of RR influenced H9c2 cell autophagy at 48h and 72h respectively (white bar referring to control cell, light grey bar referring to 5 nM RR treatment, dark grey bar referring to 50 nM RR treatment, and black bar referring to 500 nM RR treatment, n = 6 in each group); Panel B disclosed a similar result through GFP expressed siRNA plasmid transfection induced MCU knock-down in H9c2 cells (white bar referring to blank vector control, grey bar referring to scrambleRNA control, and black bar referring to siRNA interference, n = 6 in each group); Panel C showed the difference of the HL-1 cell size in three groups to testify the effect of MCU inhibition by 50 μM Ru360 in HL-1 cells with treatment of 10-5 M isoprenaline for 72h (scale bars = 50μm; n = 6 in each groups; NS: normal saline; Iso: isoprenaline); Panel D indicated the evaluation of the change of autophagy and mitophagy in different groups (n = 6 in each groups).
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Mitochondrial calcium uniporter (MCU) inhibition played an important role in pressure overload-induced heart failure through autophagy and mitophagy enhancement.
Intervention on MCU offered cardioprotective effects: improving cardiac function, correcting ventricular asynchrony, and preventing from excessive enlargement of cardiomyocytes.
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MCU inhibition could be explored as a novel therapeutic concept in heart failure treatment.
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