Mechanical ventilation regulated the differentiation and proliferation of diaphragm satellite cells

Beijing Key Laboratory of Basic Research with Traditional Chinese Medicine on Infectious Diseases, Beijing Hospital of Traditional Chinese Medicine, Capital Medical University, Beijing Institute of Traditional Chinese Medicine, 100010 Beijing, China, Meakins-Christie Laboratories and Translational Research in Respiratory Diseases Program, McGill University Health Centre and Research Institute, Montreal, QC H4A 3J1, Canada


Abstract
Aim: This study aimed to determine the effect of mechanical ventilation (MV) on the differentiation and proliferation of diaphragm satellite cells. Methods: Diaphragm satellite cells were isolated from C57 mice receiving 6 h of MV with optimized magnetic-activated cell sorting (MACS) approach. The cells were stained with BrdU or antibody for differentiation marker MYH3. The expression of MyoD and myogenin was detected by realtime PCR. Results: Diaphragm satellite cells were successfully isolated from mice by using MACS with a set of optimized parameters. About 1.5 × 10 5 cells could be harvested from a diaphragm. Upon MV, the proliferation rate of diaphragm satellite cells was decreased from 88.74% to 81.92%, while the differentiation rate was increased from 17.94% to 27.58%, compared to controls. Moreover, the expression of MyoD and myogenin were significantly upregulated upon MV. Conclusions: We established a practical method to purify diaphragm satellite cells, and demonstrated that MV regulated the differentiation and proliferation of diaphragm satellite cells.

Introduction
Mechanical ventilation (MV) is known to cause damage to diaphragm muscle [1]. Especially, weaning from MV is challenging for the recovery after serious illness. Moreover, weaning failure is associated with life-threatening complications, leading to increased morbidity and mortality [2]. Accumulating evidence has indicated that prolonged MV longer than one week leads to respiratory muscle weakness because of contractile dysfunction and atrophy, especially in the diaphragm [3][4][5][6]. Diaphragm dysfunction plays a key role in weaning failure from MV, and such a condition is named as ventilator-induced diaphragmatic dysfunction (VIDD) [7].
Diaphragm satellite cell prolifearion is a promising driving force to reverse VIDD. Satellite cells are usually mitotically quiescent but can be activated upon injury, leading to the production of myogenic precursor cells or myoblasts to promote muscle regeneration [8]. Stem cells are known to have great potential of differentiation and regeneration [9,10]. Satellite cells are the primary myogenic cells that contribute to skeletal muscle regeneration after muscle injury [11,12]. Myogenic cells in dystrophic muscle show accelerated differentiation [13]. However, it remains unknown whether such a phenomenon occurs in diaphragm muscle.
Previous study reported that pro-myogenic factors or the transplantation of muscle satellite cells (MuSCs) can enhance skeletal muscle regeneration [14]. Several protocols have been published to isolate MuSCs from skeletal muscle [15,16]. However, the diaphragm is distinct from other skeletal muscles because it is activated constantly to drive ventilation and diaphragm satellite cells are myogenic cells with potential for proliferation and differentiation [17]. Therefore, it is difficult to isolate diaphragm satellite cells because of the challenge to preserve the quiescence state of diaphragm satellite cells during the isolaton. To overcome the problem, in this study we established an optimized method to purify diaphragm satellite cells, and determined the effects of MV on the differentiation and proliferation of satellite cells.

MV intervention
Male C57 mice (10-12 weeks of age) were housed under standard conditions, and divided randomly in two groups: control group and MV group (n = 6). Mice in control group were fasted for 6 h with free access to water. Mice in MV group underwent 6 h of controlled MV as described previously [18].

Isolation of diaphragm satellite cells
All the animals were sacrificed using CO 2 asphyxia method. Diaphragm muscles were dissected, and blood, fat, and tendons were removed. The pure diaphragm muscles were maintained in F12 medium, and chopped into a fine slurry until it could pass through a 10-mL pipette freely. The fine slurry was transferred into a 15-mL Falcon tube and digested with trypsin at 37 • C for 35 min with gentle agitation. Finally, the digested muscle suspension was filtered through an 80-micron nylon cloth and centrifuged at 1800 rpm (688 g) for 10 min at 4 • C. The cells in the supernantnats were resuspended and labeled with the specific antibody at 4 • C for 15 min. MS column was placed on magnetic rack, followed by pre-equilibration using PBS containing 1% FBS. The cell suspension was mixed with the column, and after centrifugation the cell pellet was resuspended at a density of 10 6 cells per 80 µL buffer, and mixed with 20 µL anti-integrin α7 microbeads, followed by incubation at 4 • C for 15 min. Another MS column was placed on magnetic rack, followed by pre-equilibration. Labeled cells were loaded onto the column. The column was then taken from magnetic field and washed by 1 mL PBS. The diaphragm satellite cells were collected and seeded into 12-well plates precoated with Matrigel. The cells were cultured for 4 days at 37 • C.

BrdU staining
The cells were stained with 0.03 mg/mL BrdU (Cat # B5002, Sigma, St. Louis, MO, USA) for 24 h, and sequentially incubated with acetone and 1.5 M HCl. Next, the cells were fixed with 1:5000 Hoechest and covered using coveslips, the cells were observed under Zeiss M2 fluorescence microscope. The proliferation of satellite cells was determined according to the intensity of BrdU staining.

Immunofluorescence
The cells were fixed in 4% PFA, washed and then incubated with primary antibody for MYH3 (Cat # sc-53091, Santa Cruz Biotech, Santa Cruz, CA, USA) and rabbit anti-mouse secondary antibody Alexa Fluor 488 (Cat #A11059, Invitrogen, Carlsbad, CA, USA). Next, cells were fixed with 1:5000 Hoechest and covered using coverslips. The cells were observed under Zeiss M2 fluorescence microscope.

Real-time PCR
Total RNA was isolated from satellite cells using TRIzol regent (Cat #15596018, Invitrogen, Carlsbad, CA, USA) and RT-PCR was performed using iScript reverse transcription supermix for RT-qPCR (Cat # 1708840, Bio-Rad, Hercules, CA, USA). HPRT1 was selected as the housekeeping gene. The primer sequences were as follows: The parameters were denaturation at 94 • C for 10 min, and then melting at 94 • C for 30 s and annealing at 60 • C for 1 min (40 cycles). The relative expression of target genes was calculated using 2 −∆∆Ct method.

Statistical analysis
The data were shown as mean ± SD, and an independent sample t-test was used to analyze the variables. p < 0.05 indicated significant difference.

MV inhibited the proliferation of diaphragm satellite cells
Using MACS, 1.5 × 10 5 diaphragm satellite cells could be isolated from a diaphragm using the optimized parameters. We found that the morphology of satellite cells in control group was normal with active proliferation. However, the proliferation of satellite cells in MV group was significantly reduced, exhibiting apparent disintegration (Fig. 1A,B). BrdU staining showed that the number of positively stained cells was higher in control group than in MV group (Fig. 1C,D). Furthermore, we counted the numbers of cells stained positive for proliferation in randomly selected areas in control group and MV group (Fig. 1E,F). We found that the proliferation ratio of diaphragm satellite cells was decreased from 88.74% to 81.92% by MV (p < 0.05) (Fig. 1G). Collectively, these data confirmed that MV reduced the proliferation of diaphragm satellite cells.

MV promoted the differentiation of diaphragm satellite cells
All satellite cells were detected with the antibody for differentiation marker MYH3. We found that the diaphragm satellite cells in the control group exhibited spindle-shaped morphology, and the nucleus was distributed orderly in fiber bundles. However, the satellite cells of the MV group were not spindle-shaped, and the nucleus was irregular and scattered, indicating that the cells of the MV group tended to differentiate ( Fig. 2A-D). Furthermore, we counted the numbers of cells stained positive for differentiation in randomly selected areas in control group and MV group (Fig. 2E,F). We found that the differentiation ratio was increased from 17.94% to 27.58% by MV (p < 0.05) (Fig. 2G). Collectviely, these data confirmed that MV promoted the differentiation of diaphragm satellite cells.

MV upregulated MyoD and myogenin expression in diaphragm satellite cells
MyoD and myogenin are markers of muscle cell differentiation. Real-time PCR analysis showed that MyoD and myogenin mRNA levels were significantly higher in diaphragm satellite cells from MV group compared to control group (Fig. 3). These results supported that MV promoted the differentiation of diaphragm satellite cells.

Discussion
Currently, no reliable approach is available to isolate diaphragm satellite cells, which has been a challenge for further investigation on diaphragm satellite cells. In present study, we developed a method to isolate diaphragm satellite cells with MACS. Based on parameter optimization, we successfully established a stable method to obtain diaphragm satellite cells. The muscle weight of a single diaphragm from C57 mouse was about 150 mg, and 1.5 × 10 5 satellite cells could be harvested from a diaphragm.
MV is a life-saving supportive therapy for patients with respiratory failure, and has been associated with some complications such as infection, barotrauma, tracheal injury, and ventilator-induced lung injury [19]. A previous study indicated that diaphragmatic strength and endurance played critical role in successfully weaning patients from MV [20]. MV can cause sudden interruption of diaphragmatic contractile activity [21]. Taken together, the mechanisms underlying MV-induced diaphragm damage may involve oxidative stress, muscle atrophy and muscle fiber remodeling, but need further investigations.
In present study, we speculated that the differentiation and proliferation of diaphragm satellite cells played a critical role in weaning difficulties from MV. It is known that in adult muscle, satellite cells are myogenic progenitors [13]. It has been reported that the atrophy of the diaphragm was faster compared with other skeletal muscles under adverse stimulation [18]. Interestingly, myogenic cells from the dystrophic mouse showed accelerated differentiation [13]. In present study, we found that diaphragm satellite cells tended to differentiate after 6 h of MV. This result was consistent with the reduced diaphragmatic flexibility, disuse atrophy, and dysfunction after MV.
Among them, MyoD can regulate skeletal muscle differentiation vai the interaction with chromatin modifying complexes [22]. Myogenin is a target of MyoD and directs satellite cell regeneration to skeletal muscle [23]. In present study, we confirmed that MyoD and myogenin expression in diaphragm satellite cells significantly increased after 6 h of MV. These findings might help explain diaphragm cell differentiation tendency and diaphragm dysfunction after MV.

Conclusions
In conclusion, we developed a feasible and efficient approach to isolate diaphragm stem cells through optimized MACS and demonstrated that diaphragm stem cells tended to differentiate after MV, accompanied by increased expression of MyoD and myogenin.

Author contributions
JD performed experiments, conducted data analysis, and drew the manuscript. QL (Qian Li), FL and SB took part in the animal experiments and contributed to the tests. QL (Qingquan Liu) provided financial support.

Acknowledgment
This work was performed at Mcgill University Health Centre. The authors are grateful to Basil Petrof for scientific advice.