mESC culture and mESC-CM differentiation
mESCs from D3 line (ATCC, Manassas, VA, USA) were cultured as previously described [25, 28,29,30]. Briefly, mESCs were grown on Gamma-ray-irradiated mouse embryonic fibroblasts (MEFs), with Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) containing 15% heat-inactivated fetal bovine serum (FBS) (Hyclone, GE Healthcare, South Logan, UT, USA), 2 mM L-glutamine (Invitrogen), 0.1 mM non-essential amino acids (NEAA) (Invitrogen), 1% v/v penicillin-streptomycin (Invitrogen), 0.1 mM β-mercaptoethanol (Invitrogen), and 1000 U/mL leukemia inhibitory factor (LIF) (Chemicon, Millipore, Billerica, MA, USA) to maintain the undifferentiated status of mESCs. The medium was changed every day and the cells were passaged every other day. Differentiation was conducted through embryoid body (EB) method. Briefly, on day 0, cells were dissociated with 0.05% trypsin (Invitrogen) and resuspended in the medium without LIF. The cells were plated on a dish pre-coated with 0.1% gelatin (Sigma, St. Louis, Missouri, USA) and placed in the incubator for 30 min, allowing separation of mESCs from MEFs. mESCs suspending in the medium were collected and made into hanging drops, with each drop containing 20 μL medium and 800 mESCs. EBs were washed down to Petri dishes on day 2, maintained in suspension in 10 mL medium. On day 7, EBs were attached to dishes pre-coated with 0.1% gelatin and checked daily for the appearance of beating phenotypes.
Isolation of mESC-CMs
Isolation of mESC-CMs was performed as previously described [25, 30]. Briefly, beating regions of EBs were dissected out and minced into small pieces with a 27-G needle under a dissection microscope. The EB pieces were centrifuged at 1000×g to remove medium and washed with pre-chilled PBS, then dissociated with 1 mg/mL collagenase B (Roche Diagnostics, Basel, Switzerland) in NB buffer containing 120 mM NaCl, 5.4 mM KCl, 5 mM MgSO4, 5 mM Na pyruvate, 20 mM glucose, 20 mM taurine, 10 mM HEPES, and 30 μMCaCl2, and pH was adjusted to 6.9 by NaOH. Dissociation was conducted at 37 °C for 20 min with gentle shaking. Collagenase B was then removed by centrifugation at 1000×g and 1 mL KB buffer was added to resuspend the cells. KB buffer was made of 85 mM KCl, 30 mM K2HPO4, 5 mM MgSO4, 1 mM EGTA, 5 mM pyruvic acid, 5 mM creatine, 20 mM taurine, 20 mM glucose, and 87 μM of Na-ATP (Sigma), and pH was adjusted to 7.2 by NaOH. The cells were further dissociated by gentle pipetting. Thereafter, they were plated on glass coverslips or confocal dishes for further use.
Isolation of neonatal rat ventricular myocytes (NRVMs)
This study was approved by the Animal Experimentation Ethics Committee, the Chinese University of Hong Kong (17-006-MIS) and conformed to Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 80-23, revised 2011). Neonatal male rat pups (1–2 days postnatal) were sacrificed and the hearts were cut out. Atria were removed and ventricles of were cut into small pieces and washed in pre-chilled PBS on ice to remove blood. Ventricles were digested using 0.5 mg/mL collagenase type II (17101015, Gibco, Thermo) with gentle agitation. The digestion was ceased by adding DF-20 medium containing DMEM/F12 medium (11320033, Gibco, Thermo) and 20% FBS (Gibco, Thermo). Dissociated NRVMs were centrifuged at 1500 rpm, 4 °C for 10 min. The supernatant was discarded, and the cells were resuspended into plating medium containing DMEM/F12 medium, 5% FBS, 10% horse serum (26050088, Gibco, Thermo), and 1% v/v penicillin-streptomycin. The cells were plated to dishes or glass slides for further experiments.
Mouse TRPC7 cDNA  was kindly provided by Professor Yasuo Mori (Kyoto University). For constructing TRPC7 overexpression plasmid Blue-pAdTrack-CMV-TRPC7, restriction endonuclease sites KpnI and NotI were added to 5′- and 3′- end of mTRPC7 cDNA, respectively, by PCR and the cDNA was ligated into the adenoviral shuttle plasmid Blue-pAdTrack-CMV which was modified from pAdTrack-CMV plasmid (Addgene plasmid # 16405). For constructing Tag-TRPC7 overexpression plasmid Blue-pAdTrack-CMV-TagTRPC7 KpnI site plus 3× Flag was added to the 5′- end, 3× hemagglutinin (HA) plus NotI site was added to the 3′- end of mTRPC7 cDNA, the cDNA was then ligated into Blue-pAdTrack-CMV. For constructing TRPC7 knockdown plasmids pAdTrack-U6-shRNA458 and pAdTrack-U6-shRNA459, two sequences of shRNA, shRNA-458 (targeting mouse TRPC7) and shRNA-459 (targeting rat TRPC7), adopted from Genetic Perturbation Platform were synthesized and ligated into the adenoviral shuttle plasmid pAdTrack-U6 at AgeI and XhoI restriction sites. pAdTrack-U6 was modified from pAdTrack plasmid (Addgene plasmid # 16404). The plasmid pAdTrack-U6-shRNAluc harboring shRNA targeting luciferase (shRNA-luc) was constructed as a negative control . Targeting sequences of these shRNAs were shRNA-458: 5′-GCCGAATCAAACTCGCCATTA-3′, shRNA-459: 5′-GCCAACATTGAGACTGAATTT-3′, shRNA-luc: 5′-CCTAAGGTTAAGTCGCCCTCG-3′.
Adenovirus production and infection
Adenovirus was prepared using AdEasy Adenoviral Vector System Kit (Agilent Technologies, Santa Clare, CA, USA) according to the manufacturer’s protocol. Briefly, the resultant adenoviral shuttle plasmids with gene-of-interest were linearized by the digestion of PmeI. The linearized shuttle plasmids were co-transformed with adenoviral backbone plasmid into E. coli BJ5183 cells by electroporation. The bacteria were spread onto kanamycin-containing agarose plates to grow for 24 h. Candidate recombinants which were the smallest colonies on the plates were picked and shaken in 5 mL kanamycin-containing LB medium for 16 h. Plasmids were extracted, and PCR and diagnostic restriction digestion were used to screen for the positive recombinants. Validated plasmids were transformed into E. coli DH5α cells for a further amplification. Finally, the recombinant plasmids were digested with PacI and transfected into HEK-293-AD cells with Lipofectamine 2000 (Invitrogen). Ten days after transfection, the cells were collected to extract adenoviruses by repeated freeze-and-thaw cycles. The viruses were further amplified in HEK-293-AD cells to gain a higher titration. For infection, viruses at 1 multiplicity of infection (MOI) were used for a moderate overexpression, and viruses at 20 MOI were used for a full knockdown. mESC-CMs or NRVMs were infected with viruses for 6 h in normal medium. Assays for overexpression were conducted 2 days after infection, and assays for knockdown were conducted 4 or 8 days after infection.
Confocal Ca2+ imaging
Isolated mESC-CMs on confocal dishes were stained with 1 μM rhod-2 AM (Invitrogen) for 15 min in medium, then washed with pre-warmed Tyrode’s solution containing 1 mM MgCl2, 1.8 mM CaCl2, 5.4 mM KCl, 10 mM glucose, 10 mM HEPES, and 140 mM NaCl, pH 7.2 (adjusted by NaOH). The cells were bathed in Tyrode’s solution. Leica SP8 confocal microscope equipped with 552 nm laser and × 63 oil immersion objective was used for Ca2+ imaging. For the Ca2+ transients (CaTs) recording, the XYT mode was adopted. Images with a size of 16 × 16 pixels were captured in a frequency of 50 Hz for 1 min for each beating mESC-CM. For the LCRs recording, a XT line-scanning mode was used. A line in a length of 512 pixels was used to scan each beating cell in a frequency of 600 Hz for 13 s. ImageJ (NIH, Bethesda, MD, USA) was used to analyze the signal from CaTs images, a customized Python script was developed to analyze the parameters of CaTs. SparkMaster (University of California, Davis, CA, USA) was used for the analysis of LCRs .
Membrane potential and ionic current were measured with the ruptured whole-cell patch clamp using Axopatch 200B amplifier (Molecular Devices, Sunnyvale, CA, USA) and pCLAMP 10.4 software (Molecular Devices) as previously described . Signals were digitized at 10 kHz and filtered at 2 kHz. Microelectrodes (1B150F-4, World Precision Instruments, Sarasota, FL, USA) with resistances of 3–6 MΩ were pulled from P-97 puller (Sutter Instrument, Novato, CA, USA). For the AP recording, cells were bathed in Tyrode’s solution. The pipette solution was composed of 50 mM KCl, 80 mM KAspartate, 1 mM MgCl2, 3 mM MgATP, 10 mM EGTA, and 10 mM HEPES, pH 7.4 (adjusted with KOH). For NCX current (INCX) measurement, cells were bathed in the solution containing 140 mM NaCl, 1.8 mM CaCl2, 1.2 mM MgCl2, 1 μM nifedipine, 20 μM ouabain, 1 μM ryanodine, 10 μM zatebradine, 5.5 mM HEPES, and 11 mM glucose, pH 7.4 (adjusted with NaOH). The pipette solution was composed of 65 mM CsCl, 20 mM NaCl, 5 mM Na2ATP, 1.75 mM CaCl2, 4 mM MgCl2, 10 mM HEPES, 20 mM tetraethylammonium chloride, and 5 mM EGTA, pH 7.2 (adjusted with CsOH). The following ramp-voltage-clamp protocol was used to elicit the INCX: the potential was held at − 40 mV, then gradually increased to + 50 mV, followed by a decline to − 100 mV, finally increased back to + 40 mV, with a ramp rate of 200 mV/500 ms. INCX was indicated by the repolarization limb (from − 50 mV to − 100 mV) of the protocol as documented previously [25, 33]. For the recording of If, cells were bathed in Tyrode solution supplied with 2 mM BaCl2 and 2 mM MmCl2 to suppress K+ and Ca2+ current . The pipette solution contained 10 mM NaCl, 50 mM KCl, 80 mM KOH, 1 mM MgCl2, 10 mM HEPES, and 3 mM MgATP, pH 7.2 (adjusted with KOH). Five millimoles CsCl was applied to the bath solution to inhibit If.
One hundred micrograms proteins were loaded to 7.5% polyacrylamide gel and transferred to 0.45 μm PVDF membranes (Millipore). Membranes were blocked with 5% (w:v) milk for 1 h at room temperature then incubated with primary antibodies at 4 °C overnight. The membranes were washed with TBST three times and incubated with secondary antibody for 1 h at room temperature. Finally, the membranes were developed with Clarity Western ECL Substrate (Bio-Rad) and pictures were taken by ChemiDoc Touch (Bio-Rad). Antibodies used were anti-TRPC7 1:500 (HPA031126, Sigma), anti-β-tubulin 1:1000 (15,115, Cell Signaling, Danvers, Massachusetts, USA), anti-HCN4 1:200 (APC-052, Alomone), anti-Cav1.3 1:100 (ACC-005, Alomone), anti-Cav3.1 1:200 (ACC-021, Alomone), anti-Cav3.2 1:200 (ACC-025, Alomone), anti-RyR2 1:1000 (MA3-916, Invitrogen), anti-SERCA 1:200 (sc-30110, Santa Cruz), anti-IP3R 1:500 (ACC-019, Alomone), anti-p(S2814)RyR2 1:5000 (A010-31, Badrilla), anti-phospholamban (PLN) 1:1000 (A010-14, Badrilla), anti-p(T17) PLN 1:5000 (A010-13, Badrilla), HRP-conjugated goat anti-rabbit secondary antibody 1:5000 (Dako, Zug, Switzerland), and HRP-conjugated goat anti-mouse secondary antibody 1:5000 (Dako).
Drugs and chemical reagents
Ouabain was purchased from the International Laboratory USA (South San Francisco, CA, USA). 2-2-Aminoethoxydipheylborate (2-APB) and thapsigargin were purchased from Tocris (Bristol, UK). Nifedipine, ryanodine, zatebradine, and all chemical reagents used to setup buffer solutions were purchased from Sigma.
Three or more biological repeats were performed for each experiment. Data were presented as mean ± SEM. Data between two groups were compared by unpaired Student’s t-test. Data between three or more groups were compared by one-way analysis of variance (ANOVA) followed by Bonferroni’s multiple comparison tests. P < 0.05 was considered to be statistically significant.