Nov 11, 2024
Obesity-induced Nox2 activation prolongs cardiac repolarization protocal
Reserved DOI:
10.17504/protocols.io.81wgbrp7olpk/v1
- Bin Li1,
- Yating Chen1,
- Maoxiang Zhao2,
- zhijie chen3,
- zhuhui lin3,
- jie liu2,
- xueping wang4,
- jiancheng zhang3,
- yang li2
- 1Chinese PLA Medical School, Chinese PLA General Hospital, Beijing, China;
- 2Senior Department of Cardiology, the Sixth Medical Center of PLA General Hospital, Beijing, China;
- 3Department of Cardiology, Fujian Provincial Hospital, Provincial Clinical Medicine College of Fujian Medical University, Fuzhou, China;
- 4Medical Innovation Research Department of PLA General Hospital, Beijing, China
Protocol Citation: Bin Li, Yating Chen, Maoxiang Zhao, zhijie chen, zhuhui lin, jie liu, xueping wang, jiancheng zhang, yang li 2024. Obesity-induced Nox2 activation prolongs cardiac repolarization protocal. protocols.io https://protocols.io/view/obesity-induced-nox2-activation-prolongs-cardiac-r-drfz53p6
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: November 09, 2024
Last Modified: November 11, 2024
Protocol Integer ID: 111833
Abstract
We investigated the effect of Nox2 on cardiac repolarization without compromising its expression and function in other systems using mice with conditional cardiac-specific deletions of Nox2 (knockout [KO]).Wild-type, KO, and Flox littermate mice were randomized to either the control or high-fat diet (HFD) groups. Surface electrocardiograms were recorded to analyze repolarization in vivo. Whole-cell patch-clamp techniques were used to evaluate the electrophysiological phenotype of isolated myocytes in vitro. Western blotting was performed to assess protein and gene expression levels.
Body compositions of mice
Body compositions of mice
Power on
First, turn on the power switch of the nuclear magnetic analyzer. Minisec consists of two parts, a magnetic box m-box (with permanent magnets inside) above the table and an electric box e-box below. The switch of the nuclear magnetic analyzer is located next to the fan at the back of the e-box in the electrical box.
Heating up
There are two indicator lights on each of Minispec's e-box and m-box.
The power indicator light on the left side of the e-box will turn yellow when the switch is pressed; On the right side of the e-box is the indicator light for the instrument connection software. This indicator light will only turn on after the software is opened and the device is connected normally on the computer. On the left side of the m-box is the connection status indicator light, which lights up when the m-box and e-box are connected normally; On the right side of the m-box is the magnet temperature indicator light, which is initially red and gradually turns orange as the temperature slowly rises. It will only turn green when the temperature stabilizes at 37 ℃ (which takes about 4 hours).
Detection
After the temperature indicator light of the instrument turns yellow, click on the Bruker minisspec Plus icon on the desktop, enter the software, and log in with your username.
The instrument must undergo daily testing before use. Click on "Daily Testing" and follow the instructions to insert the rapeseed sample. If the daily testing passes, the instrument will automatically jump to the main operating interface. The effective time for each daily test is 24 hours.
Click on 'Measure', enter the sample batch or select an existing sample batch from the drop-down menu.
Select the corresponding model name from the drop-down menu, which is the standard curve.
Enter the sample name.
Place the mouse into the sample tube and press down the white pressure rod until the mouse is no longer active. And insert the sample tube into the probe until the bottom touches the stopper.
Click on 'Measure' and quickly remove the mouse and put it back into the cage after the measurement is completed.
Click on 'View Results' and select the sample batch and time range for the required data.
There is a choice to export to EXCEL or other formats below.
During the normal use phase of the instrument, only the computer software and computer need to be turned off, and the instrument power does not need to be turned off. If not used for a long time, the instrument power can be turned off.
hematoxylin and eosin (H&E) staining and Masson’s trichrome staining
hematoxylin and eosin (H&E) staining and Masson’s trichrome staining
Hematoxylin and eosin (H&E) staining and Masson’s trichrome staining
To conduct histological analysis, we euthanize the mice under deep anesthesia with isoflurane. Their limbs were fixed on a foam console using a needle, and water was sprayed on their chest with a watering can to
minimize hair loss. The chest of each mouse was then cut open along the "V" shape of the xiphoid process. We bluntly separated the pericardial tissue, incised the blood vessels from the base of the heart, promptly remove the heart, and gently rinsed with physiological saline.
Fresh heart tissue samples were taken and fixed in 4% paraformaldehyde. Gradient dehydration, wax immersion. Remove the organization from the three wax and begin embedding it.
After undergoing gradient dehydration, the embedding process commenced, and the tissue samples were cut longitudinally at 5 μ m for hematoxylin and eosin (H&E) staining or Masson’s trichrome staining.
The tissue samples were examined under a microscope and analyzed using the Image J program
(NIH).
Echocardiograms
Echocardiograms
Preparation of mice before ultrasound
Need to depilate the mice in advance (depilatory cream, cotton swabs, depilators)
Item preparation: bring anesthesia isoflurane, tape, coupling agent, and hand towel.
Anesthetize mice with a gas mixture of isoflurane.
The parasternal long axis is imaged using B-mode imaging, and the papillary muscle level is obtained when the scanning head is rotated clockwise by 90 °. Obtain the wall thickness of the left ventricle during end diastolic and systolic phases using an M-shaped cursor. Record M-mode images of diastolic left ventricular posterior wall thickness (LVPW; d) and diastolic left ventricular anterior wall thickness (LVAW; d) from three consecutive cardiac cycles.
The parasternal long axis view of the left ventricle can be used for assessing left ventricular volume and function in rodents. Collecting data requires both recording and displaying electrocardiograms, as well as recording respiratory movements, in order to obtain more accurate data.
Until the apex is pointed, the apex is at the same level as the outflow tract, and the outflow tract is unobstructed, the standard parasternal long axis section is found. In order to obtain the best M-Mode ultrasound image, the LV must be in the horizontal direction, that is, the apex of the heart and the outflow tract must be at the same level. Place the M-Mode ultrasound sampling line at the posterior edge of the papillary muscle and collect data. Note that if the apex of the heart is at an angle to the outflow tract, M-Mode ultrasound measurements may increase.
The parasternal short axis section of the left ventricle (papillary muscle level) is commonly used for measuring parameters such as left ventricular wall thickness and left ventricular systolic function.
Rotate the probe clockwise by 90 ° in the parasternal long axis section and adjust the Y-axis to find the maximum section of the left ventricular cavity, which is the parasternal short axis section.
Relevant indicators for testing
LVID: Left ventricular diameter
LVIDs: Left ventricular systolic diameter
LVIDd: Left ventricular diastolic diameter
LVESD: Left ventricular end systolic diameter
LVEDD: Left ventricular end diastolic diameter
IVSD: interventricular end diastolic thickness
LVPWs: Left ventricular posterior wall thickness during systole
LVPWd: Left ventricular posterior wall thickness diastolic phase
Electrocardiographic recordings and analysis
Electrocardiographic recordings and analysis
The ECG was recorded for 5 minutes at a rate of 1 kHz using needle electrodes placed subcutaneously in anesthetized mice under isoflurane anesthesia.
The PowerLab system (ADInstruments Ltd., Australia) was used to calculate the heart rate.
labchart→OK→ECG mouse→Setup→stimulator
The ECG intervals were measured manually from the recordings with minimal artifacts using LabChart 8 software, and the electrocardiographic parameters were calculated.
Cardiomyocyte isolation
Cardiomyocyte isolation
A Langendorff perfusion system was used to isolate ventricular myocytes.
20min heparinization
Under a stereomicroscope, retrograde catheterization of the aorta was performed, and the aorta was suspended on a pillow. The aorta was ligated with thread 0, and a 1ml syringe was used to flush the remaining blood in the heart and coronary arteries with the mother liquor. Then, it was transferred to the Langendorff perfusion device. It should be noted that the transfer from cervical dislocation to perfusion device should be controlled within 5 minutes, preferably within 3 minutes, otherwise it may affect the activity of myocardial cells due to cellular hypoxia.
Infuse the mother liquor , and observe that the blood in the atrial appendage, cardiac cavity, and coronary artery is washed clean. Stop the peristaltic pump and replace the digestion solutionfor perfusion digestion. Use a dropper to collect the digestion solution for 1 minute in a 37 ℃ water bath for later use. First, perfuse with digestive fluid for 2 minutes, leaving 12.5ml remaining. Then circulate the digestive fluid . Observe if the heart color becomes lighter, elasticity decreases, and the edges are unclear. Stop the perfusion and use ophthalmic forceps to pick up the left and right atrial appendage, place it in a culture dish, drip the digestive fluid from the previous water bath, use ophthalmic forceps to tear up the tissue, gently blow the tip of the gun until it becomes cloudy, be careful not to produce bubbles, and try to blow until there is no block tissue.
Drop 1ml of 10% FBS (Ca2+final concentration of 40umol/l) to terminate digestion, use a pipette tip to remove tissue blocks, transfer the remaining liquid to the remaining 10% FBS, centrifuge at 1000 r/min for 20s700r/min for 30-40 seconds; Alternatively, physical settling can be used to wait for the cells to settle to the bottom. Discard the supernatant (aspirate with a dropper, leaving 0.5-1.0 ml of supernatant containing cells), then add 2ml of 5% FBS (Ca2+final concentration of 40 umol/l) and gently blow evenly with a dropper. Pour the remaining 5% FBS into a centrifuge tube.
Whole-cell patch-clamp recordings
Whole-cell patch-clamp recordings
The amplifier of MultiClamp 700B (Axon Instruments, USA) was used to measure membrane currents. Digidata 1440A (Axon Instruments) was used as the output, and pCLAMP 10.7 was used as the software control.
Generally,the series resistance was 2–3 MΩ, with an 80% compensation. The trials were conducted at room temperature. A voltage clamp was used to measure K+ currents. Current amplitudes were divided by myocyte membrane capacitance to calculate current densities.
To distinguish Ito from IK,slow, 50μM 4-aminopyridine (4-AP) was added to the mixture. Furthermore, 0.1 mM CdCl2 was used to inhibit the Ca2+ current, and 10 μM tetrodotoxin (TTX) and 25 ms of depolarization to –40 mV
were used to block the inward Na+ current.
IK1 was sensitive to 300 μM Ba2+. A protocol of 600 ms of test pulses ranging from −140 to +20 mV was used to measure IK1. To record APs, TTX and CdCl2 were omitted. APs were recorded after injections of a depolarizing current at a frequency of 1 Hz.
For IK recording, the external solution in the patch electrode contained 140 NaCl, 1 CaCl2, 1 MgCl2, 4 KCl, 10 HEPES, and 5 mM glucose, with the pH adjusted to 7.3–7.4 with NaOH. The internal solution contained 130 KCl,
1 MgCl2, 5 EGTA, 10 HEPES, 10 glucose, and 5 mM Na2ATP, with the pH adjusted to 7.2–7.3 with KOH.
For AP recording, the external solution in the patch electrode contained 140 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 5 mM glucose, and 4 mM KCl, with the pH adjusted to 7.35 with CsOH. The internal solution contained 120 mM K-aspartate, 20 mM KCl, 1 mM MgCl2, 4 mM Na2ATP, 10 mM HEPES, 10 mM glucose, and 0.1 mM GTP, with the pH adjusted to 7.25–7.35 with CsOH.
Western blotting
Western blotting
Tissue protein extraction
Pre cool RIPA protein extraction reagent and add protease inhibitor (phosphorylated protein requires the addition of phosphatase inhibitor). Add 0.1M PMSF mother liquor before protein extraction, with a final PMSF concentration of 1mM. Weigh the tissue in a ratio of weight: lysis buffer volume=1:9 and add the lysis buffer. Use a Fluka electric tissue homogenizer at 15000rpm for homogenization, with 10 seconds each time and an interval of 10 seconds. Perform homogenization three times. During homogenization, the EP tube needs to be immersed in an ice water mixture for cooling. After homogenization, incubate on ice for 20 minutes, centrifuge at 4 degrees, 13000rpm for 20 minutes. After centrifugation, take the supernatant, package and store it for testing.
Pre cool RIPA protein extraction reagent and add protease inhibitor. Add 0.1M PMSF mother liquor before protein extraction, with a final PMSF concentration of 1mM. Cell counting was performed by adding 300ul of lysis buffer with a cell count of 1 × 107, and thoroughly suspending the cells by blowing them with a pipette tip. After completion, the cells were incubated on ice for 20 minutes, centrifuged at 4 degrees Celsius at 13000rpm for 20 minutes. After centrifugation, take the supernatant, package and store it for testing.
BCA protein quantification method
Prepare BCA working solution A: B=50:1, dilute each extracted BSA standard. Dilute the sample with PBS.
Sample: BCA working solution=1:8. After mixing, incubate at 37 ° C for 30 minutes or at room temperature for 60 minutes. Use an enzyme-linked immunosorbent assay (ELISA) reader with a 570nm wavelength filter to read the OD value.
Protein concentration adjustment
Adjust the protein concentration with RIPA and add 5 x reduction sample buffer to achieve a final sample concentration of 4mg/ml. Boil and denature for 5 minutes.
Objective protein WB formal experiment
According to the molecular weight of the target protein, prepare a 12% or 8% separation gel and concentrate the gel at a concentration of 5%.
Sample loading amount of protein to be tested: 20ug/well
Electrophoretic conditions: Constant pressure of 90V for concentrated gel, about 20 minutes; Separate the gel at a constant pressure of 160V and determine the electrophoresis stop time by pre staining protein markers.
Wet transfer method, film transfer conditions: 300mA constant current; 0.45um pore size NC membrane, with a transfer time of 12% separation gel for 1 hour or 8% separation gel for 2 hours. After the membrane transfer is completed, the Lichun Red staining reagent is used to stain the membrane, observe the transfer effect, and mark the swimming lane at the same time.
Closure: Fully immerse the membrane in 3% BSA-TBST and gently shake at room temperature for 30 minutes.
Primary antibody incubation: Dilute the primary antibody with 3% BSA-TBST, incubate at room temperature for 10 minutes, and let it sit overnight at 4 ℃.Anti-KV4.2 (APC-023), anti-KChIP2 (APC-142), anti-Kir2.1 (APC-026), and anti-KV1.5 (APC-004, Alomone); anti-Nox2 (Proteintech); and anti-tubulin (Immunoway) were used.
The next day, remove the membrane from 4 degrees and incubate at room temperature for 30 minutes. Membrane washing: TBST membrane washing 5 times, each time for 3 minutes.
Secondary antibody incubation: Dilute the secondary antibody with 5% skim milk powder TBST, goat anti rabbit IgG (H+L) HRP or goat anti mouse IgG (H+L) HRP, 1:10000, and shake gently at room temperature for 40 minutes. Membrane washing: TBST membrane washing 6 times, each time for 3 minutes.
After ECL is added to the film, it reacts for 3-5 minutes. The film is exposed for 10 seconds to 5 minutes (the exposure time varies with different light intensities), developed for 2 minutes, and fixed.