F344 Rat
Four weeks old F344/jcl female rat (50 g or more) (CLEA Japan, Tokyo, Japan) were used to isolate vibrissa hair follicles. The experimental animals were kept in an animal housing system maintained at 24 ± 1℃, relative humidity of 50-60%, and 14 hours of light 10 hours of dark intervals. The experimental protocol was approved by the Kitasato University School of Medicine Animal Care Committee (No. 2020035). The study was conducted according to the ARRIVE guidelines for the reporting of animal experiments. All methods complied with the guidelines for the proper conduct of animal experiments of the Science Council of Japan.
Isolation and division of rat vibrissa hair follicles.
Isolation and division of rat vibrissa hair follicles were performed as previously reported [22]: For isolation of vibrissa hair follicles from the F344/jcl rats, the animals were anesthetized with a combination of 0.375 mg/kg medetomidine, 2.0 mg/kg midazolam and 2.5 mg/kg butorphanol. The upper lip containing the vibrissa pad was cut, and inner surface was exposed. All vibrissa hair follicles were gently pulled out from the pad, one by one, with fine forceps, under a binocular microscope. The tissues around the isolated hair follicles were removed, and the hair follicles were divided into thirds. All surgical procedures were performed in a sterile environment.
Induction of cardiomyocyte and atrial-myocyte differentiation
The upper third parts of rat vibrissa hair follicles were placed on Matrigel® Matrix (#356231, Corning Incorporated, Corning, NY, USA) culture in 27 mm glass-bottom culture dishes (#BC-SFGD27, Bio Medical Science, Tokyo, Japan), in DMEM (#D6429, Sigma-Aldrich, St. Louis, MO, USA) containing 10% fetal bovine serum (FBS), 50 μg/ml gentamycin (#15750-060, GIBCO, Grand Island, NY, USA), 2 mM L-glutamine (#25030, GIBCO) ,10 mM HEPES (#H0887, Sigma-Aldrich). To induce cardiomyocyte differentiation, isoproterenol (3 µM) (#I6504, Sigma-Aldrich), activin A (10 ng/ml) (#338-AC-010, HumanZyme, Chicago, IL, USA), BMP4 (10 ng/ml) (#120-05ET, HumanZyme,) and bFGF (5 ng/ml) (#GF003, Millipore, Temecula, CA, USA) were added to 10% FBS-DMEM for 7 days. Afterward, to study the effect of CSA on cardiomyocytes differentiation, CSA (1 µg/mL) (#AG-CN2-0079, AdipoGen Life Sciences, San Diego, CA, USA) was added to 10% FBS-DMEM for 14 days. The culture period was 21 days (Fig. 5). Medium was replaced every 3 days. Beating cardiomyocytes and atrial myocytes were recorded with a video camera (VCE-i700T, Shodensha, Osaka, Japan), attached to a microscope CKX53 (Olympus Corporation, Tokyo, Japan).
Immunofluorescence staining
Immunofluorescence staining was performed as previously reported [4]. Primary antibodies used were: anti-cardiac troponin I (cTnI) rabbit polyclonal antibody (1:200, #PAA478Ra01, Cloud-Clone Corp, Houston, TX, USA); anti-MLC-2a mouse monoclonal antibody (1:40, #565496, BD Biosciences, San Jose, CA, USA); anti-c-kit polyclonal antibody (1:200, #bs-0672R, Bioss Antibodies, Woburn, MA, USA). Secondary antibodies used were: goat anti-rabbit IgG conjugated with Alexa Flour® 568 (1:400, #A-21069, Invitrogen, Waltham, MA, USA); goat anti-mouse IgG conjugated with Alexa Fluor® 488 (1:400, #A-11001, Invitrogen). 4’, 6-diamino-2-phenylindole, dihydrochloride (DAPI) (#SE196, DOJINDO, Kumamoto, Japan) was used for counter staining. The images of stained cells were obtained with a LSM 710 microscope (Carl Zeiss, Oberkochen, Germany). The images were analyzed by LSM software ZEN (Carl Zeiss).
Quantitative Polymerase Chain Reaction (qPCR)
Total RNA was extracted from 21day cardiomyocytes using the RNeasy Plus Mini Kit (#74134, Qiagen, Hilden, Germany) and then cDNA were synthesized with QuantiTect® Reverse Transcription (#205311, Qiagen) according to the manufacturer’s instructions. GAPDH was used to normalize gene expressions. Quantitative PCR was performed using the Power SYBR® Green PCR Master mix (#4367659, Applied Biosystems, Waltham, MA, USA) on a CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) and analyzed by the Delta Delta Ct method. Forward and reverse primer sequences are shown in Table. 1.
Ca2+ Imaging
Intracellular calcium imaging was performed as previously reported [5]: The cells were washed with Earle’s balanced salt solution (#14155-063, GIBCO) with 2 mmol CaCl2 at 37℃ and were loaded with the calcium-sensitive dye Fluo 4-AM (1 µM) (#F312, DOJINDO) and AM ester-dissolving reagent Pluronic F-127 (0.04%) (#59004, FUJIFILM Wako, Osaka, Japan) for 20 min at 37°C. Fluo 4-AM fluorescence (excitation at 495 nm and emission at 518 nm) of beating cardiomyocytes was measured with a LSM 710 microscope (Carl Zeiss), were analyzed by LSM soft-ware ZEN Time series (Carl Zeiss).
Transmission Electron Microscopy
Differentiated cells from HAP stem cells were pre-fixed with 2.5% glutaraldehyde in 0.1M sodium cacodylate buffer for 30 min at 4℃, post-fixed with 1% osmium tetroxide for 1 hour at 4℃. A biological specimen was immersed in a solution of 2% uranyl acetate for 15 min an aqueous solution after post-fixation, for en-bloc staining. The specimens were then dehydrated through graded ethanol and embedded in epoxy resin. Ultrathin sections (80 nm), double-stained with 2% uranyl acetate and Reynold's lead citrate, were examined under electron microscopy H-7650 (Hitachi, Tokyo, Japan).
Image processing
Image processing was with image J software (National Institutes of Health, Bethesda, MD, USA) (Rasband, W.S. ImageJ, U. S. National Institutes of Health. Bethesda, MD, USA, 351 Available online: http://imagej.nih.gov/ij/, 1997-2012(accessed on 15 September 2021).). cTnI images were set to threshold from 50 to 255. MLC-2a images were set to threshold from 40 to 255.
Statistical Analysis
The experimental data are expressed as the mean SD. Statistical analyses were performed with the unpaired Student’s t-test. A probability (P) value of p ≤ 0.05 was considered significant.