CLIC4 localizes mitochondria-associated membranes and mediates cardioprotection

Confocal imaging of cytoplasmic Ca+ as previously described (17) In short, fresh adult mouse cardiomyocytes (54) are loaded into Tyrode solution containing 1 mm Ca for 10 min ^{two+ at room temperature} , then wash Tyrode's solution containing 2 mM Ca for 10 min ^{in two+} cardiomyocyte perfusion of Tyrode's solution containing 2 mM Ca ^{for 10} min during RT recording
.
Fluo-3am is excited at 488 nm and fluorescence emission is at a wavelength of 500-530 nm, collected in line scan mode at
200 Hz.
The pacemaker rate of cardiomyocytes is 0.
5 Hz
.
β adrenergic agonist ISO is used
at 100 nm.
At the end of the experiment, caffeine was used at 10 mm to evaluate the SR-Ca ^II+ content
.
The cytosolic calcium ^di+ transient amplitude and caffeine transient amplitude are expressed as ΔF/F₀, where F0 is the basic fluorescence and ΔF=F? The_F0SERCa2a rate is calculated by fitting a single index of caffeine-induced calcium to ^{a two}+ instantaneous, followed by a two-component exponent fitted cytoplasmic ^Catwo+ Transient with fixed first τ
.

Briefly, newly isolated cardiomyocytes are resuspended in cardiomyocyte culture medium [M199, containing 5% BSA, BDM (1mol/ml), and 1× penicillin/streptomycin
] with calcium di+ (54).
For calcium measurements, adult cardiomyocytes are loaded with Fura-2AM (0.
5 μM) (20) 20 min
prior to data acquisition.
Data collection was performed using an IonOptix system (IonOptix) connected to a Motic AE31 microscope, displayed on a 40× objective and stimulated
at 20 V at 1 Hz.
Changes in intracellular calcium ^II+ were monitored using Fura-2 double-excited (340/380 nm) single emission (510 nm) ratio imaging
.
Measured
after baseline and ISO treatment (100 nM).

Wt CD1 and customer 4?^/? As previously described, mice obtained from indoor rearing (12 to 15 weeks) undergo Langendorff perfusion (12).
Briefly, cut the heart from the mouse and place it in ice-cold Krebs-Henseleit buffer (KHB) (118 mM NaCl, 4.
7 mM KCl, 1.
2 mM KH) 2 personnel _{officer 4,1.
2} mM MgSO ₄25 mM NaHCO _tri 11.
1 mM d-glucose and 2 mM CaCl2 ).
Aortic catheterization, retrograde perfusion of the heart on a Langendorff device, KHB blistered ₂–5% carbon monoxide2 cardiac perfusion at 95%O for 15 min, followed by ischemia for 25 min, and reperfusion at 37°C for 2 h
。 For the sham control group, the heart is perfused continuously at 37 °C for 160 min
.
In some cases, the heart is treated for 10 min
after reperfusion with DMSO (vehicle control) or IAA-94 (50 μM).
To evaluate colorectal cancer, the heart is ischemic for 25 minutes and perfused for 20 minutes
.
After reperfusion, the heart is treated to evaluate mitochondrial colorectal cancer or stained with TTC to assess infarct area
.

Perform IR injury on mice using the classical method and the new method described above to induce myocardial IR injury (23).
LCA is ligated for 40 min and then perfused for 24 h
.
When not using a ventilator, the operation time is completed
within 2 minutes.
Briefly, a small skin incision was made on the left chest and then a pouch suture
was done.
After dissection, the pectoralis minor muscle contracts, revealing the fourth intercostal space
.
A small incision was made between the fourth intercostal space and the heart was gently "ejected"
.
Suture the descending left coronary artery and tie a suture
2-3 mm from the beginning of the left coronary artery with 6-0 silk thread.
After ligation, the heart is placed back into the chest cavity and the air
is artificially expelled before the muscles and skin close.
The inner needle end of the suture is cut short, and the other end of the suture remains outside the chest and is 0.
8 cm
long.
After 40 minutes of ischemia, gently and smoothly pull the long end of the suture to release the synovium and reperfuse
the heart.
The classic in vivo IR injury method is to anesthetize mice with 3% (v/v) isoflurane, intubate with a 20-gauge intravenous catheter, and use O2 and 2% (v/v) isoflurane
.
After dissection, the coronary arteries are sutured in a similar
way to non-classical IR injury methods.
Ischemia is continuously ventilated for 40 minutes, and the incision is covered
with saline-soaked gauze.
After the ischemic phase is over, the chest cavity
is closed after release.
The sham group performed the same surgery, but did not block the LCA
.
Echocardiographic imaging
of mice at basal level and 24 h after reperfusion.
For chronic myocardial ischemic injury, reperfusion is not performed, but ligation is continued for 8 weeks
.

In the case of in vivo IR injury, 24 h after reperfusion, the mouse is anesthetized, the thoracic cavity is opened, and ligaments around the LCA are re-ligated by previous ligation
.
Aortic injection of 2% (w/v) Evans blue dye 20 μl
.
The heart is quickly removed, frozen, and sliced into thick sections
perpendicular to the long axis of the heart.
The unstained fraction is defined as AAR
.
Sections are incubated with 1% TTC (PBS preparation) for 15 min at room temperature, fixed with 4% (w/v) paraformaldehyde (PFA) for 5 min, and imaged
with Leica S9i.
Myocardial infarction area (pallor) is quantified by ImageJ, expressed
as a percentage of infarct area on AAR.
AAR is calculated as AAR as a percentage
of the total area of the left ventricle.
The heart after ex vivo ischemia-reperfusion injury is frozen and TTC stained to calculate the infarct area
.

High-frequency, high-resolution ultrasound imaging systems for echocardiography (Vevo 2100 and Vevo 3100 imaging systems, FUJIFILM Visual Sonics Inc.
, Toronto, Canada).

For WT and customer 4?^/? A high-frequency sensor probe (VisualSonics MS400, frequency range 18 to 38 MHz, VisualSonics MX550D, frequency range 25 to 55 MHz, FUJIFILM Visual Sonics Inc.
, Toronto) was used
.
Simply put, anesthetize the mouse 2 heart rate with 1.
5-2% (v/v) of isoflurane and O mixture to maintain between 400~450 beats per minute
.
M and B images were acquired along the long axis of the parasternum to measure cardiac function
.
Analyze images
using Vevo LAB 3.
1.
1 analysis software.

Different cell components obtained during MAM and ultrapure mitochondrial preparation are resuspended in RIPA buffer and cultured for 1 h
at 4 °C with continuous shaking.
From wt and customer 4?^/? Mice or heart sections from the marginal zone after IR injury are crushed in RIPA buffer and homogenized to prepare cardiac lysate
.
Flash freeze the lysate in liquid nitrogen and, once thawed, incubate at 4 °C and shake continuously for 1 h
.
After 1 h, centrifuge the samples at 12,000 g for 20 min at 4 °C, collect the supernatant and load it onto SDS-polyacrylamide gel electrophoresis at 4 to 20% (w/v) and transfer to a
nitrocellulose membrane.
The protein load was confirmed
with carmine S staining.
Block the membrane with LI-COR blocking buffer and rinse with Tris-buffered saline (TBS), then incubate overnight
at 4 °C with various major antibodies.
The main antibodies used are GRP78/BIP (ab21685), CLIC4 (sc-135739), CLIC1 (sc-374202), adenosine triphosphate (ATP) synthase (ab14748), MCU (14997), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118), mitomycin 2 (ab-205236), ACSL4 (PA5-27137), Inhibin (ab-10198), cytochrome c (pa1-12250), and cyclophilin D (AP1035).

After three washes with TTBS (20 mM Tris buffer saline containing 0.
05% Tween-20) for 10 min, culture with anti-rabbit/mouse secondary antibodies bound to IR680/IR800 for 1 h at room temperature, followed by three more washes with TTBS for 5 min
.
Observe the signal
using an infrared fluorescence system (Bio-Rad).
The intensity of the bands was quantified using ImageJ and normalized to the loading protein
using the Lichun red S signal of different subcellular components.
In some cases, the protein amount is normalized to the corresponding GAPDH control
.

Neonatal cardiomyocytes were hypoxic for 6 hours and reoxygenated for 12 hours
.
After culturing newborn cardiomyocytes in a 35 mm glass-bottom dish or coverslip coated with 0.
1% (w/v) gelatin for 4 days, the cells were placed in a modular humidified hypoxic chamber (Biospherix, C127) and 1% (v/v)O2,5% (v/v) carbon monoxide2 at 37 °C Equilibrium_N2 is placed in DMEM free of glucose and FBS for 6 h
.
After hypoxia, cells are re-fused in DMEM containing glucose (4 g/l) and 20% (v/v) FBS and cultured for 12 h
.
Cells after reperfusion are stained with JC1 to assess membrane potential, 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) detects ROS, and TUNEL detects cell death
.
Image analysis uses ImageJ
.

Isolated coarse mitochondria, adult or neonatal cardiomyocytes, HEK transfected cells, and suckling mouse cardiomyocytes subjected to normoxia/hour injury, fixed with 4% (w/v) PFA for 10 min (12) as described in the previous section Then, coarse mitochondria, cardiomyocytes, and HEK cells are infiltrated with 0.
5% (v/v) tritonx-100 for 10 min, followed by 30 min
with 10% normal goat serum (NGS).
Samples are then incubated overnight
at 4 °C with various specific primary antibodies [diluted in PBS containing 1% (w/v) NGS and 0.
1% (v/v) tritonx-100].
After incubation with the main antibodies GRP78/BIP (ab21685), CLIC4 (sc-135739), CLIC1 (sc-374202), ATP synthase (ab14748), mitomycin 2 (ab-205236), ACSL4 (PA5-27137), inhibition protein (ab-10198), and cytochrome c (PA1-12250).
Samples were washed (three times) with PBS containing 0.
1% (v/v) Triton X-100 and incubated for 1 h at RT with secondary antibodies against rat/rabbit Alexa Fluor 488 (2 μg/ml) and anti-rabbit/mouse ATTO647N (1 μg/ml), 6-diamino-2-phenylindole dihydrochloride (DAPI) (nuclear label: 1:10000), with Mowiol mounted 4-88 or Extended Gold (molecular probe).

Cells are imaged with Nikon A1R high-resolution confocal microscope and median filtering (8).
ImageJ is used to calculate the percentage of the
colocalization index.

ROS generation
was measured using chloromethyl 22′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Life Technologies, C26827).
Neonatal cardiomyocytes after reperfusion are stained with DCFDA (10 μM) in phenol-red free conventional DMEM for 30 min, cells are washed with phenol-red free DMEM after staining, and imaged
at excitation and emission wavelengths of 485/535 nm with Nikon A1R confocal microscope.
Cells in the normobic state were used as controls and treated
similarly.
The fluorescence intensity is quantified
with ImageJ.
The multiple change in ROS production relative to normoxy is represented
by a bar chart.

Neonatal cardiomyocytes after HR injury or in normal oxygen (control) are fixed with 4% (w/v) PFA for 10 min, infiltrated with 0.
25% (v/v) Triton X-100 for 20 min, and stained
with TUNEL using the TUNEL assay kit as per the manufacturer's instructions (Thermo Fisher Scientific).
Staining the nuclei
with DAPI counterstaining.
With a Nikon-A1R confocal microscope, the excitation emission wavelengths were 488 and 530 nm, respectively, to obtain fluorescence images
.

From wt and customer 4?^/? Mice are washed in PBS, minced, and homogenized in buffer A (70 mM sucrose, 210 mM mannitol, 1 mM EDTA, and 50 mM tris HCl (pH 7.
4)) using a Potter-Elvehjem homogenizer
(seven strokes).
Centrifuge the homogenate at 2500 gfor 5 min
.
Collect the supernatant and centrifuge at 12,000 g to resuspend the pellet containing coarse mitochondria in 55 μl Buffer B [70 mM sucrose, 210 mM mannitol, 0.
1 mM EDTA, and 50 mM tris HCl (pH 7.
4)].

Extramitochondrial calcium
is detected with a fluorescence spectrophotometer (Hitachi F-2710 or Hitachi F-7100).
In the presence or absence of CsA, add calcium green-5N, hexapotassium salt (2.
5 μM) (Thermo Fisher Scientific, extract 10 μl from a 500 μM stock) to CRC buffer [150 mM sucrose, 50 mM KCl, 2 mM KH2 personnel officer].
_4,5 mM succinic acid and 20 mM tris-HCl (pH 7.
4)], and fluorescence (500 nm excitation and 530 nm emission)
are measured.
After 30 s, add 50 μl of the isolated mitochondrial sample
.
After another 90 sec, and every 60 sec thereafter, 5 μM CaCl2 pulses are added until a rapid increase in fluorescence indicates mitochondrial death
.
Perform protein estimation
of mitochondria using the Bio-Rad kit according to the manufacturer's instructions.
Fluorescence values are normalized according to protein concentration and calcium required to induce mitochondrial death in different experimental groups
is compared.

Neonatal cardiomyocytes under normal and HR injury conditions are stained with JC1 dye (33) in humidified 5% (v/v) CO and kept at 37 °C _{for 30 min in 2} incubators
.
The cells are then washed with phenol red-free DMEM and imaged
with a Nikon A1R confocal microscope.
JC1 dye is an indicator
of mitochondrial membrane potential.
At higher mitochondrial membrane potentials, the JC1 dye aggregates in the mitochondria, forming red J1 aggregates that are emitted
at 590 nm.
At lower membrane potentials, the JC1 dye remains in its green monomer form, emitting
at 530 nm.
The ratio of red and green fluorescence intensity determines the change in
mitochondrial membrane potential.

Human and mouse tissue sections are deparaffinized in xylene, rehydrated with ethanol and PBS, and then extracted
using citrate buffer (Vector Laboratories, #H-3300-250).
Unqualified human heart tissue was obtained with approval and informed consent from an institutional review board, and non-failing heart tissue was procured in partnership with the Ohio Lifeline Organ Procurement Program
.
Sections are incubated at 3% (v/v) H at _3O2 for 10 min at room temperature to inhibit endogenous peroxidase activity and blocked with 5% (w/v) NGS in PBS for 1 h
.
Wheat germ (WGS) was cultured for 1~5min, and each piece was cultured with WGS containing wheat germ for 1~5min
.
After three rinses with PBS, incubate overnight
at 4 °C with anti-CLIC4, anti-suppressin, and anti-cytochrome c antibodies.
After primary antibody incubation, wash sections with 0.
1% (v/v) tritonx-100 containing PBS and then incubate with secondary antibodies
.
After secondary antibody culture, rinse and stain with DAPI (1:10,000 diluent) and then mount
with Mowiol 4-88 or Extended Gold (molecular probe).
Cells are imaged with Nikon A1R high-resolution confocal microscope and median filtering (8).

As mentioned earlier, frozen human heart tissue is subfractionated (32).
Briefly, 150 mg of tissue is homogenized in cytolysis buffer (CLB) buffer [10 mM Hepes, 10 mM NaCl, 1 mM KH2 personnel _{officer 4}5 mM NaHCO _III , 5 mM EDTA, 1 mM Calc Cl 2 0.
5 mM MgCl2 (pH 7.
5) and complete protease inhibitor tablets], and restore isotonnity
by adding 100 μl of 2.
5 M sucrose.
Homogenate at 6,300 g for 10 min
at 4 °C.
Transfer the supernatant to a fresh tube (sup1), resuspend the pellet in 500 μl CLB/0.
25 M sucrose, and recentrifuge at 6,300 g for 5 min
at 4 °C.
Mix the supernatant (sup2) with the previous supernatant sample (sup1) and resuspend the pellet in 1 ml TSE buffer [10 mM tris, 300 mM sucrose, 1 mM EDTA, and 0.
1% (v/v) NP-40 (pH 7.
5)] and homogenize (30 strokes).

Samples are centrifuged at 4,000 °C and kept at 4 °C for 5 min
.
The final particles obtained after homogenization with TSE buffer are then suspended in RIPA buffer and represent the concentrated nuclear fraction
.
Mix the supernatants (sup1 and sup2) at 107,000 g in a Beckmann ultracentrifuge and keep at 4 °C for 30 min
.
The supernatant obtained after the centrifugation step represents the cytosolic fraction
.
The resulting pellet after ultracentrifugation is then suspended in RIPA buffer, representing the membrane and organelle fractions
.
The lysate was treated with Western blotting and probed
with anti-CLIC1 and anti-CLIC4 antibodies.
The expression of CLIC4 and CLIC1 was normalized to the total protein content detected by Li Chun S staining, and quantitative band intensity
was analyzed using ImageJ software.