Fam64a is a novel cell cycle promoter of hypoxic fetal cardiomyocytes in mice

Fetal cardiomyocytes actively proliferate to form the primitive heart in utero in mammals, but they stop dividing shortly after birth. The identification of essential molecules maintaining this active cardiomyocyte proliferation is indispensable for potential adult heart regeneration. A recent study has shown that this proliferation depends on a low fetal oxygen condition before the onset of breathing at birth. We have established an isolation protocol for mouse fetal cardiomyocytes, performed under strict low oxygen conditions to mimic the intrauterine environment, that gives the highest proliferative activities thus far reported. Oxygen exposure during isolation/culture markedly inhibited cell division and repressed cell cycle-promoting genes, and subsequent genome-wide analysis identified Fam64a as a novel regulatory molecule. Fam64a was abundantly expressed in hypoxic fetal cardiomyocyte nuclei, but this expression was drastically repressed by oxygen exposure, and in postnatal cardiomyocytes following the onset of breathing and the resulting elevation of oxygen tension. Fam64a knockdown inhibited and its overexpression enhanced cardiomyocyte proliferation. Expression of a non-degradable Fam64a mutant suggested that optimum Fam64a expression and subsequent degradation by anaphase-promoting complex/cyclosome (APC/C) during the metaphase-to-anaphase transition are required for fetal cardiomyocyte division. We propose that Fam64a is a novel cell cycle promoter of hypoxic fetal cardiomyocytes in mice.


Supplementary Methods
All animal procedures were approved by the Institutional Animal Care and Use Committee at the Kawasaki Medical School. All experiments were performed in accordance with relevant guidelines and regulations of Kawasaki Medical School.

Low O 2 isolation and culture protocol.
To mimic intrauterine low O 2 tension (20-25 mmHg; 2.6-3.2% O 2 ) during isolation, all solutions were preconditioned to 2-3% O 2 by nitrogen gas bubbling and were then kept until further use. The working space, including the CO 2 incubator and a boxed bench (AS-600P; As-One, Japan) was also kept strictly in the same low O 2 conditions by nitrogen or argon gas loading. Under this condition, primary CMs were isolated from ventricles of fetal mice (embryonic day, E12-E18) bred on a C57BL/6 background by gentle trypsin treatment, using a modification of the protocol used for neonatal rat hearts 1 . Pregnant mice were euthanized with an overdose of Sevofrane. Delivered fetuses were decapitated, and ventricles were rapidly excised, cut into small pieces, and washed once with PBS followed by gentle shaking. The tissues were then digested four times with 0.06% trypsin and 0.24 mmol/L EDTA in PBS for 10 min at 37°C, again with gentle agitation. Cell suspensions were then resuspended in DMEM with 5% FBS and cultured for 45 min to exclude non-CMs (mostly fibroblasts), which preferentially adhered to the plates. Supernatants containing CMs were filtered through a 100 µm cell strainer (Falcon ™ , BD Biosciences, NJ, USA), and the cell pellets were resuspended in fresh DMEM with 5% FBS, and plated onto fibronectin-coated or non-coated culture vessels. This protocol took ~3h and consistently yielded >95% pure CMs with a few contaminating fibroblasts, which were evaluated by FACS as positive sarcomeric α-actinin expression in CMs (Fig.   1a). Isolated CMs were subsequently cultured under low (2-3%) or high (21%) O 2 conditions in a multi-gas incubator (APM-30DR, Astec, Japan) at 37 °C with 5% CO 2 . In this study, low or high O  conditions refer to 2-3% or 21% O  tension, respectively. For neonatal mice (postnatal day, P1-P3) and in some experiments using fetuses or where indicated, isolation was conducted under high O 2 conditions (ambient air) without the special procedures described above.

CM proliferation analysis by FACS.
fCMs isolated under low O 2 conditions were subsequently cultured under low or high O 2 conditions for 96h. At the start and end of the culture, total cell numbers were manually counted, and the proportions of CMs and non-CMs in the same sample were analyzed by FACS. This allowed us to determine the absolute number of each cell type (CMs and non-CMs) at the start and end of the culture. For FACS analyses, trypsinized cells were fixed with 2% paraformaldehyde, permeabilized with 0.5% Tween-20, blocked with 5% BSA, and labelled with primary antibody for sarcomeric α-actinin (clone EA-53, Sigma-Aldrich, MO, USA) followed by secondary Alexa 488-conjugated goat anti-mouse IgG (Thermo-Fisher, MA, USA). Negative control samples were labelled with Alexa 488-conjugated mouse IgG 1  isotype control antibody (eBioscience, CA, USA). Cells were analyzed with a BD FACSCalibur ™ (BD Biosciences, Singapore) as described 2 ; α-actinin-positive cells were regarded as CMs and α-actinin-negative cells as non-CMs.

Baculovirus-mediated protein expression in CMs.
Baculovirus that effectively infects mammalian cells was obtained by expressing vesicular stomatitis virus G-protein (VSVG) on the virus envelope 3 . The virus was produced by Bac-to-Bac system (Invitrogen) using the modified donor vector (pFastBac1-VSVG-CMV-WPRE) that was constructed as follows. Cytomegalovirus (CMV) promoter was amplified by PCR with primers

Time-lapse imaging analysis of CM cell division.
Isolated fCMs were placed on the stage of the microscopic live cell analyzer (JuLI FL; NanoEnTek, Korea), which was accommodated in a multi-gas incubator. Time-lapse imaging of fCM division under low or high O 2 conditions was recorded at 10 min intervals by phase contrast microscopy, which was initiated at 9-11h after plating, and continued for ~20 h. This time frame was optimal to image fCM division. Complete fCM division events, in which mitosis was followed by cytokinesis, resulting in the generation of two daughter cells (examples shown in Fig. 2a-b), were manually counted and defined as the percentage of total fCMs in the imaging field. A few contaminating non-CMs (mostly fibroblasts) were easily excluded by their morphology, smaller cell size, and active migration. In addition, post-imaging samples were fixed with 4% paraformaldehyde and immunostained for sarcomeric α-actinin to confirm that the dividing cells were unequivocally CMs (Fig. 2b, c). For baculovirus-transduced fCMs, time-lapse experiments were performed with inverted fluorescence microscope (BZ-X710, Keyence, Japan) equipped with a stage incubation system (INUG2-KIW, Tokai Hit, Japan) or confocal microscope (Fluoview ™ FV1000; Olympus, Japan) equipped with a stage incubation system (Chamlide TC, Live Cell Instrument, Korea).

DNA microarray.
For the comparison between neonates and fetuses (array #1 , Fig 3a) conditions for 48h and then total RNA was obtained, also using the RNeasy Plus Mini Kit (QIAGEN).

Immunoblotting.
Hearts were collected from mice and snap frozen in liquid nitrogen. Minced tissues were homogenized using a Kinematica ™ Polytron ™ homogenizer (PT1600E; Fisher Scientific, MA, USA) in lysis buffer (10 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl, 0.5 mmol/L EDTA, 10 mmol/L NaF, and 0.5% Triton X-100), RIPA buffer (Thermo-Fisher), or M-PER buffer (Thermo-Fisher) in the presence of protease and phosphatase inhibitor cocktail (Thermo-Fisher or Roche, Basel, CH). For cultured CMs, harvested cell pellets were lysed in the same buffer and processed as was done for heart tissues. Lysates were centrifuged at 14,000×g and supernatants were used as the whole protein extract.

Chromatin immunoprecipitation (ChIP) assay.
ChIP assays were performed using EpiScope ChIP Kit (Takara Bio) as per the manufacturer's protocol using HEK293T/17 cells or fetal mouse heart tissues. HEK293T/17 cells were crosslinked with 1% formaldehyde for 5 min at room temperature. Fetal mouse hearts were crosslinked with 1-1.8% formaldehyde for 2-3h at room temperature and homogenized. Samples were then sonicated with Bioruptor (Cosmo Bio, Japan) and subjected to IP using antibodies against HIF-1α (Santa Cruz), HIF-2α (Novus Biologicals, CO, USA) or normal mouse IgG (Santa Cruz). Purified DNA was quantified using SYBR Green real-time PCR system (Takara Bio). Six primers for humans and five primers for mice were designed to include putative Hif-1α binding sites (5'-GCGTG-3') ( Supplementary Fig 4d for  Luciferase activity was measured using LightSwitch ™ luciferase assay reagent (SwitchGear Genomics) as per the manufacturer's protocol. Luciferase activities of housekeeping gene ribosomal protein L10 (RPL10) and empty vector were used as positive reference and negative background control, respectively.

Statistics.
All data were expressed as mean plus or minus standard error of the mean (SEM