Animal and human heart specimen
Adult Balb/c mice and C57BL/6 mice (8 weeks) were obtained from Vital River (Charles River Laboratories) and μMT mice (Balb/c background) were a gift from Professor Zhihai Qin (Institute of Biophysics, Chinese Academy of Sciences). Mice were perfused with PBS for 5 minutes, and the heart was removed. Conditions of animal housing and all experimental procedures were conducted under institutional guidelines provided by the Institutional Animal Care and Use Committee of China.
human heart autopsy specimen was a gift from Qingyuan Liao (Guilin Medical University) and human iPS-CM was a gift from Feng Lan (Anzhen hospital).
HL-1 myocardial cell culture
HL-1, an adult mouse atrial myocardial cell line, was a gift from Dr. W.C.Claycomb (Louisiana State University, New Orleans, LA) 25. HL-1 cells were cultured in Claycomb medium (Sigma-Aldrich) supplemented with 10% FBS (HyClone), 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, and 0.1 mM norepinephrine (Sigma-Aldrich) at 37°C under 5% CO2 in 0.02% gelatin coated flasks containing 0.5% fibronectin (Sigma-Aldrich).
Isolation and culture of neonatal mouse ventricular myocytes
Ventricular cardiomyocytes were isolated from the left ventricles of neonatal mice using enzymatic digestions containing 0.08% trypsin (Gibco BRL) and 0.1% collagenase (Sigma-Aldrich) for 8 min at 37 °C. Isolated cardiomyocytes were re-suspended in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin and then plated at a density of 6 × 105/dish and cultured for 1.5 h. The supernatant was subsequently collected and centrifuged at 500g for 10 min and resuspended in 5 ml culture medium containing 100 mmol/L 5’BrdU (Sigma-Aldrich) and plated in 35 mm culture dishes for 48 h.
Isolation of adult mouse ventricular myocytes and single cardiomyocyte dissociation.
Ventricular myocytes were isolated from the Langendorff-perfused hearts of adult C57BL/6 mice. Briefly, the mice were anaesthetized with pentobarbital and the heart was removed and mounted on the Langendorff apparatus and perfused with Ca2+ -free Tyrode’s solution to eliminate the blood. Then the heart was perfused with Tyrode’s solution containing 0.67mg/ml of collagenase type II (Worthington) for 30 min for digestion. When the heart became flaccid, remove the ventricle and cut into small pieces and then pelleted by centrifugation at 500rpm for 1 min at room temperature and resuspended in Tyrode’s solution. The calcium concentration was then gradually increased to 500mM over 80 min. The supernatant was discarded and the pellet was resuspended in PBS containing 1mg/ml BSA. Cardiomyocyte single cells were manually picked under the microscope by mouth pipette. To make sure that only single cells were collected, the solution was visually inspected under the microscope and was discarded if multiple cells were observed. Volume of liquid was kept below 0.5 μl. Cells were then transferred to a 0.2 ml thin-wall PCR tube containing lysate buffer.
RNA extraction and cDNA synthesis
Myocardial single cell RNA extraction and cDNA synthesis were carried out according to Tang’s methods as described 26,27. In briefly, myocardial single cells were picked by capillary pipette and reverse transcription was then performed directly on the whole cell lysate. After this, the free primers were removed by ExoSAP-IT and a poly(A) tail was added to the 3’ end of the first-strand cDNA by Terminal Deoxynucleotidyl Transferase. Then the single cell cDNAs were amplified following PCR program: 95 °C for 3 min, then 27 cycles of 95 °C for 30 sec, 67 °C for 1 min, and 72 °C for 6 min (+ 6 sec each cycle). After this step, all cDNAs have been amplified. The single cell cDNA PCR product can be saved at −80 °C for 2 months.
Total RNA was extracted from frozen heart tissue as well as HL-1 cells by using Trizol Reagent (Invitrogen). Reverse transcription (RT) was carried out with the First Stand cDNA Synthesis Kit (Thermo Scientific) according to the manufacturer’s protocol.
PCR amplification and DNA sequencing
After the cDNA was obtained, specific primers were used to amplify the Igk, CD20, Myh6, Myh7, cTnT, cTnI and GAPDH. The sequences of all PCR primers are listed in Supplementary Table 1. Touchdown PCR was performed for amplification. PCR products were cloned into a pGEM-T Easy Vector (Promega). These clones were analyzed by the Sanger method using an ABI 3100 Genetic Analyzer (Applied Biosystems). The sequences of VkJk were compared with those in the BLAST and Immunogenetics databases to identify the best matches for germline gene segments and VJ junctions. All VkJk sequences were shown in supplementary table 2.
The DNA concentration of eluted PCR products were measured and 146 ng of DNA was pooled for sequencing. The following sequencing using the 2 × 250 bp Illumina MiSeq platform were performed by Novogene Corporation (Beijing, China). Single cell of WT mice (n=17) and mMT mice (n=3) was analyzed. The usage of Vk was shown in supplementary table 3.
The sequence for Igk siRNA was as follows: siRNA-1: GAAGATTGATGGCAGTGAA. siRNA-2: GAACGACATAACAGCTATA. These were used to transfect HL-1 cells with the Lipofectamine 3000 kit (Invitrogen) according to the manual; a mock group was transfected with control siRNA. Cells were harvested 24 h and 48 h after transfection for Igk mRNA and protein detection.
Frozen sections of heart tissues, primary cultured cardiomyocytes, and HL-1 cells were fixed with acetone, and blocked with 5% FBS at room temperature for 30 minutes. The cells were then stained overnight at 4 °C with the following primary antibodies: rabbit anti-mouse Igk (1:50; Proteintech), rabbit anti-mouse Cx43 (1:100; Proteintech), rabbit anti-mouse N-cadherin (1:100; Proteintech), rabbit anti-mouse Plakoglobin (1:100, Proteintech) were added at 4°C overnight. Then, the cells were washed with PBS and incubated for 1 h with the respective secondary antibodies conjugated to 5 (1:1000; Abcam), Alexa Fluor 568 (1:1000; Abcam) or Alexa Fluor 594 (1:1000; Abcam) after which the cells were stained with Hoechst (1:10000; Sigma) for 5min at room temperature.
Western blot analysis
Total cellular protein was extracted from heart tissue and HL-1 cells using RIPA lysis buffer (Beyotime) containing a protease inhibitor cocktail. The samples were subsequently separated by 12.5% SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then incubated with the following primary antibodies: Biotin-labeled goat anti-mouse Igk (1:1000; SouthernBiotech), rabbit anti-mouse Cx43 (1:1000; Proteintech), rabbit anti-mouse N-cadherin (1:1000; Proteintech), rabbit anti Desmoplakin (1:1000, Proteintech), GAPDH (1:1000, ZSGB-Bio) and b-actin (1:1000, ZSGB-Bio). The secondary antibodies conjugated with HRP (ZSGB-Bio) were applied at a concentration of 1:10,000 and visualized using with ECL chemiluminescence (GE). Western Blots were quantified by densitometry using ImageJ software.
Generation of conditional cardiac-specific Igk-knockout mice
To generate inducible cardiac-specific Igk cKO mice, transgenic mice expressing a tamoxifen-inducible Cre recombinase protein under the control of the α-myosin heavy chain promoter, αMHC/MerCreMer mice (MCM) were intercrossed with Igkflox/flox mice (obtained from Shanghai Biomodel Organism Science & Technology Development Co., Ltd) in a C57BL/6 genetic background. To induce Cre recombination, 8- to 12-week-old male aMHC/MerCreMer; Igkflox/flox (Cre+;Igkf/f) were treated with 80mg/kg of tamoxifen (Sigma) via intraperitoneal injection once a day for 4 consecutive days. aMHC-Cre-; Igkf/f mice, aMHC-Cre+ mice with tamoxifen age- and sex-matched littermates were utilized as control mice. Conditions of animal housing and all experimental procedures were conducted under institutional guidelines provided by the Institutional Animal Care and Use Committee of China.
Construction of adenovirus
The full-length cDNA of mouse IgkV17-121/J2/C was inserted into adeno-associated virus serotype 9 vector-cTnT (AAV9-cTnT) to construct the AAV9-cTnT-Igk virus (produced by Likeli, Beijing, China). 5x1011vg of AAV9-cTnT-Igk or AAV9-CMV-GFP were injected intravenously into 10-week-old cardiac Igk knockout mice (αMHC-Cre+ Igkfl/fl), while the same volume of AAV9-CMV-GFP was injected intravenously into αMHC-Cre+ mice. Four weeks after injection, tamoxifen was injected to induce Igk gene knock down. Western blot of myocardial tissues was performed to confirm the re-expression efficiency.
Systolic blood pressure analysis
Systolic blood pressure was measured in conscious mice using BP-98A Blood Pressure Analysis System (Softtron). Briefly, animals were placed in a plastic chamber maintained at 37°C, and a cuff with a pneumatic pulse sensor was attached to the tail. we measured systolic blood pressure five times per animal and recorded the mean overall blood pressure.
To evaluate left ventricular function and dimension, transthoracic two-dimensional echocardiography was performed on mice sedated with 1.5% isoflurane using a Visual Sonics Vevo 770 ultrasound system (Visual Sonics) equipped with a 17.5-MHz linear array transducer. M-mode tracings in the parasternal short axis view were used to measure left ventricular posterior wall thicknesses (LVPW) and left ventricular inner dimension (LVID) at end-diastole, which were used to calculate left ventricular fractional shortening (FS) and the ejection fraction (EF) according to standard formulas.
Sarcomere shortening analysis
Isolated cardiomyocytes were placed on the experimental chamber of an inverted microscope (Olympus) with permanent perfusion of Tyrode solution containing 500mM Ca2+ heated to 37°C. Myocytes were electrically stimulated (15V, 0.5hz) and sarcomere shortening was detected using a video-based detection system (IonOptix) on intact myocytes. Six twitches per myocyte were collected for each sample (n =2). Signal averaged data were analyzed to determine resting sarcomere length, peak shortening normalized for resting sarcomere length (percent peak height), time to peak shortening (TTP).
Surface electrocardiogram (ECG) recordings were performed on mice anaesthetized with 1–1.5% pentobarbital sodium. Needle electrodes were used and placed in the conventional lead II position. A differential amplifier amplified the signals in the bandwidth of 0.1–1000 Hz, and signals were filtered using an adaptive 60 Hz filter. The signals were digitized at 3000 Hz, and analyzed using ECG Analysis Software (QRS phenotyping, Calgary, Canada).
Electron microscope analysis
The heart tissues fixed with 3% glutaraldehyde were chopped into 1 mm2 × 1 mm pieces. Then, the tissues were rinsed, dehydrated, embedded, sliced, stained, and subsequently investigated under a transmission electron microscope (TEM).
All data are presented as the means±s.e.m. Statistical analyses were performed using Prism Software. The statistical significance of the difference between two sets of data was assessed using an unpaired t-test. Statistical significance was defined as P <0.05. * P< 0.05, **P< 0.01, ***P< 0.001.
This study includes no data deposited in external repositories