Aldehyde Dehydrogenase 1 isoforms ALDH1A1 and ALDH1A3 are essential for myogenic differentiation

Satellite cells (SC) constitute the stem cell population of skeletal muscle tissue and are determinants for myogenesis. Aldehyde Dehydrogenase 1 (ALDH1) enzymatic activity correlates with myogenic properties of SCs and, recently, we could show co-localization of its isoforms ALDH1A1 and ALDH1A3 in SCs of human skeletal muscle. ALDH1 is not only the pacemaker enzyme in retinoic acid signaling and differentiation, but also protecting cell maintenance against oxidative stress products. However, the molecular mechanism of ALDH1 in SC activation and regulation of myogenesis has not yet been characterized. Method Human RH30 and murine C2C12 myoblast cell lines were investigated in regard of ALDH1A1 and ALDH1A1 expression in myogenesis using Western Blot, Immunouorescence and Aldeuor Assay. study, we investigated the myogenic behavior of human RH30 and murine C2C12 muscle cells with regard to ALDH1 activity and function. Increased ALDH1 activity and high protein levels of its isoforms ALDH1A1 and ALDH1A3, respectively, could be demonstrated in differentiated cells. Chemical inhibition of ALDH1 enzymatic activity caused accumulated protein levels of ALDH1A1 and ALDH1A3 and induced differentiation. These results were conrmed by recombinant overexpression of ALDH1A1 and ALDH1A3, respectively, that also lead to myotube formation, and genomic knock-out cells showed decient differentiation. Recovery of differentiation ability in knock-out models could be restored by transfection of recombinant ALDH1A1 and ALDH1A3, respectively. We used human RH30 and murine C2C12 myoblast cell lines, which are widely accepted as an appropriate model system for the investigation of satellite cell biology.


Abstract
Background Satellite cells (SC) constitute the stem cell population of skeletal muscle tissue and are determinants for myogenesis. Aldehyde Dehydrogenase 1 (ALDH1) enzymatic activity correlates with myogenic properties of SCs and, recently, we could show co-localization of its isoforms ALDH1A1 and ALDH1A3 in SCs of human skeletal muscle. ALDH1 is not only the pacemaker enzyme in retinoic acid signaling and differentiation, but also protecting cell maintenance against oxidative stress products. However, the molecular mechanism of ALDH1 in SC activation and regulation of myogenesis has not yet been characterized. Method Human RH30 and murine C2C12 myoblast cell lines were investigated in regard of ALDH1A1 and ALDH1A1 expression in myogenesis using Western Blot, Immuno uorescence and Alde uor Assay.

Results
Here, we show, that isoforms ALDH1A1 and ALDH1A3 are pivotal factors in the process of myogenic differentiation, since ALDH1A1 knock-out and ALDH1A3 knock-out, respectively, impaired differentiation potential. Recombinant re-expression of ALDH1A1 and ALDH1A3, respectively, in corresponding ALDH1isoform knock-out cells recovered their differentiation potential. Most interestingly, the chemical inhibition of enzymatic activity by disul ram leads to ALDH1A1 and ALDH1A3 protein upregulation and subsequent myogenic differentiation.

Conclusion
Our ndings indicate that ALDH1A1 and ALDH1A3 proteins are important for myogenic differentiation and, therefore, seem to be essential activators and regulators of SCs.

Background
Satellite cells (SC) constitute the stem cell population of skeletal muscle tissue and are the resource for muscle growth and regeneration. SC action is induced by various stimuli, such as signaling for adaptive growth after physical activity and for regenerative processes after insult, in ammation or oxidative stress (Forcina et al., 2019).
Markers for quiescent and activated SCs have been established in experimental studies (Cobb, 2013;Shen et al., 2003), that allow the tracing of proliferation and differentiation processes. Although the in uence of environmental stimuli on myogenic growth and myotube formation has been addressed in several studies (Mukund & Subramaniam, 2020;Pownall et al., 2002), the molecular mechanism of SC activators and regulators has yet not been fully understood.
The enzyme Aldehyde Dehydrogenase 1, in particular its isoforms ALDH1A1 and ALDH1A3, has been identi ed as an interesting actor in SC activation and myogenic differentiation. Recently, we were able to demonstrate the co-localization of isoforms ALDH1A1 and ALDH1A3 in SCs of human skeletal muscle tissue (Rihani et al., unpublished data). ALDH1 oxidizes retinaldehyde into retinoic acid (RA) in the vitamin A signaling pathway and regulates differentiation (Wang et al., 2010, Soprano et al., 2007. In addition, ALDH1 is detoxifying aldehydes resulting from oxidative stress and protects cells against mitochondria damage and apoptosis (Schieber & Chandel, 2014). ALDH1 enzymatic activity has been identi ed as a hallmark of myoblast subpopulations with high myogenic capacity and resistance against oxidative stress (Vella et al., 2011).
These ndings indicate that ALDH1 seems to play an important role in SC maintenance and myogenesis, since it coordinates antioxidative cell protection and the regulation of differentiation by RA signaling. For this reason, detailed analysis of ALDH1 function in myogenic processes would be interesting.
Here we show, that isoforms ALDH1A1 and ALDH1A3 are key factors in the process of myogenic differentiation, since ALDH1A1 knock-out and ALDH1A3 knock-out, respectively, lost their differentiation potential. Recombinant re-expression of ALDH1A1 and ALDH1A3, respectively, in ALDH1-isoform knockout cells recovered myogenic differentiation. Most interestingly, the chemical inhibition of enzymatic activity leads to ALDH1A1 and ALDH1A3 protein upregulation and consequently to myogenic differentiation.

Cell culture
Human RH30 and murine C2C12 myoblast cell lines were purchased from American Type Culture Collection (ATCC) and cultivated in antibiotic-free standard high-glucose medium without pyruvate (Life Technologies, Darmstadt, Germany). Both cell lines showed irreversible myotube formation by serumwithdrawal (10% fetal bovine serum versus 2% horse serum). For microscopic analysis an inverted microscope (Nikon, Düsseldorf, Germany; TS100) was used and captures were taken with a NIS-Elements Camera (Nikon, Düsseldorf, Germany). For chemical inhibition of ALDH1 enzymatic activity Disul ram (DSF; Sigma, Munich, Germany) was applied with a concentration of 3 µM of DSF.

Western Analysis
Samples were separated in SDS-PAGE and subsequently transferred to PVDF-membranes. To prevent unspeci c binding of rst antibodies the membrane was incubated for 45 minutes in 5% blocking-buffer (roti-block and milk, respectively). Subsequently, primary antibodies were adjusted as described above and incubated overnight at 4 °C, rolling. Anti-Vinculin was included for loading control. HRP-conjugated secondary antibodies were applied as indicated above and visualized by ECL substrate following the manufacturer's recommendations. Image documentation was performed using AI680 Imager (GE Healthcare Life Science, Munich, Germany).

Immuno uorescence
Cells were cultivated and treated on µ-slides (Ibidi GmbH, Gräfel ng, Germany) for immuno uorescent staining. Slides were washed with PBS and cells were xed in 4% formalin for 20 minutes at room temperature. Blocking buffer provided with donkey-serum was added and incubated for 30 minutes. After washing, primary antibody, diluted in blocking buffer with additive donkey serum, was applied overnight at 4 °C. Secondary antibody was added in a dilution as indicated with an incubation time of 40 minutes.
Nucleic counterstaining with DAPI was conducted for 10 minutes. Samples were mounted with Aqua Poly/Mount (PolySciences, Hirschberg an der Bergstrasse, Germany). Immuno uorescence was examined with a Carl Zeiss Axioplan uorescence microscope (Zeiss, Munich, Germany; HBO 100 Upright Fluorescence Microscope). Captures were conducted with an AxioCam and AxioVision 4.8 software.
Alde uor assay Enzymatic ALDH-activity analysis (Alde uor assay) was performed according to the manufacturer`s guidelines (Stem Cell Technologies, Cologne, Germany). Cells were incubated for 30 minutes at 37 °C in Alde uor assay buffer containing ALDH substrate bodipy-aminoacetaldheyde (1 µmol/l per 1.000.000 cells). As negative control, a fraction of each cell sample incubated under identical conditions with additional ALDH inhibitor diethylaminobenzaldehyde (DEAB). Flow cytometer Calibur (BD Biosciences, Heidelberg, Germany) was applied ALDH-positive population detection.
Genomic ALDH1-isoform knockout using CRISPR-Cas9 In C2C12 and RH30 genomic knockout of ALDH1A1 or ALDH1A3 isoforms was conducted following the protocol of Ran et al., 2013 and Wu et al., 2020. Oligonucleotides for consecutive single guide RNA (sgRNA) generation were designed using the online-tool at Massachusetts Institute of Technology (https://zlab.bio/guide-design-resources). Plasmid pSpCas9(BB)-2A-GFP (PX458) with GFP-insert for uorescence activated cell sorting (FACS) was a gift from Feng Zhang (Addgene plasmid # 48138). In order to achieve high puri cation, single cell colony cloning was performed on the next day. After two weeks ALDH1A1 knock out (1A1 ko) and ALDH1A3 knock out (1A3 ko), respectively, was con rmed in cells grown from single colonies.

Recombinant overexpression of ALDH1-isoforms
For recombinant ALDH1A1 or ALDH1A3 overexpression plasmids containing GFP-tagged open reading frame of ALDH1A1 and ALDH1A3, respectively, were purchased (OriGENE, Herford, Germany; MG208039, MG222097, respectively). Transfection was performed using 2 µg of DNA and Lipofectamine 3000 reagent (Thermo Fisher Scienti c, Munich, Germany) following the manufacturer's recommendations. Empty GFP-vector transfection served as control. Transfected cells were selected by G418-treatment (Sigma, Munich, Germany), subsequently protein expression was con rmed by Western blotting using antibodies against ALDH1A1-/ALDH1A3 and GFP as indicated before.

Morphological alteration in myogenesis
Proliferating murine C2C12 and human RH30 myoblasts showed a characteristic roundish shape with small protrusions (Fig. 1. A left). Myotube formation was induced by serum-withdrawal and the typical multinucleic elongated shape could be observed within 6 days Enzymatic ALDH1 activity in myogenesis was analyzed using Alde uor assay (data not shown). Both cell lines demonstrated an increase of ALDH1 enzyme activity of 31.9% in differentiated C2C12 and 43.7% in differentiated RH30. We next inhibited generic ALDH activity by chemical inhibition using Disul ram (DSF) in proliferating C2C12 and RH30 cells. Against our expectation, we observed myotube formation within 4 days of treatment despite no serum withdrawal was applied ( Fig. 1. A middle). Western Analysis showed comparable up-regulation of ALDH1A1 and ALDH1A3 protein levels in differentiated C2C12 and RH30 cells after DSF treatment and serum withdrawal (Fig. 2. A). In addition, differentiated cells of serum-withdrawal and DSF-treatment demonstrated in immuno uorescent staining increased immunoreaction to antibodies directed against ALDH1A1 and ALDH1A3 (Fig. 3.).

Expression of ALDH1A1 and ALDH1A3 in differentiation
Since ALDH1 protein levels seemed to be important for myotube formation, we then transfected C2C12 and RH30 cells with ALDH1A1-GFP and ALDH1A3-GFP fusion vectors. Transfected clones showed a strong overexpression of the fusion products by Western analysis (Fig. 2. B) and, most importantly, spontaneously differentiated without serum withdrawal ( Fig. 1. B). In contrast, recombinant knock-out by CRISPR-Cas9 of ALDH1A1 and ALDH1A3, respectively, in C2C12 and RH30 completely inhibited myotube formation in the standard paradigm of serum withdrawal ( Fig. 1. C, 4. A, Fig. 5.). Consecutive Alde uor Assay demonstrated 1% ALDH1 activity in knock-out cells.
Rescue by re-transfection of ALDH1a1 and ALDH1a3 in knockout cells In order to con rm the functional role of ALDH1 in differentiation, we re-expressed ALDH1A1 and ALDH1A3 proteins by transfection using ALDH1A1-GFP and ALDH1A3-GFP fusion vectors in corresponding knock-out cells. Successful expression of proteins could be demonstrated by Western blots (Fig. 4. B upper line). As expected, the re-expression of ALDH1A1 and ALDH1A3 rescued the defective differentiation phenotype of the knock-outs. Re-transfected knock-out cells showed spontaneous differentiation by morphology ( Fig. 1. D) and Western analysis (Fig. 4. B lower line).

Conclusion
The process of skeletal muscle growth relies on satellite cells (SCs), that constitute the skeletal muscle Previously, the enzyme Aldehyde Dehydrogenase 1 (ALDH1) was associated as regulator of differentiation through retinoic acid (RA) signaling and as scavenger of oxidative stress products. Hence, ALDH1 is not only a functional part of growth behavior, but also a protector of cell maintenance.
Moreover, highest ALDH1 activity was identi ed in a subpopulation of myogenic progenitors with a remarkable myogenic capacity (Vauchez et al., 2009).
In the present study, we investigated the myogenic behavior of human RH30 and murine C2C12 muscle cells with regard to ALDH1 activity and function. Increased ALDH1 activity and high protein levels of its isoforms ALDH1A1 and ALDH1A3, respectively, could be demonstrated in differentiated cells. Chemical inhibition of ALDH1 enzymatic activity caused accumulated protein levels of ALDH1A1 and ALDH1A3 and induced differentiation. These results were con rmed by recombinant overexpression of ALDH1A1 and ALDH1A3, respectively, that also lead to myotube formation, and genomic knock-out cells showed de cient differentiation. Recovery of differentiation ability in knock-out models could be restored by transfection of recombinant ALDH1A1 and ALDH1A3, respectively. We used human RH30 and murine C2C12 myoblast cell lines, which are widely accepted as an appropriate model system for the investigation of satellite cell biology.
Our data are in line with previous studies that demonstrated high ALDH activity in myogenic progenitors (Vauchez et al., 2009;Vella et al., 2011). More recently, several studies showed the importance of ALDH1 for skeletal muscle homeostasis in longevity (Papaconstantinou et al., 2015) and in protection against ageing effects (Etienne et al., 2020). Although these experiments demonstrated the importance of ALDH1 activity in muscle cell preservation (Jean et al., 2011) and myogenesis, functional aspects in differentiation have not been addressed in detail. Importantly, the effect of a functional blockade by chemical inhibition of ALDH1 on cell differentiation could solve the question about the role of its enzymatic activity. We, therefore, inhibited ALDH1 by DSF, but contrary to our expectation RH30 and C2C12 cells showed myogenic differentiation without serum withdrawal. This effect was most likely due to a reactive upregulation of protein levels, since differentiated cells after DSF showed high ALDH1A1 and ALDH1A3. Next, we knocked-out ALDH1A1 and ALDH1A3, respectively, and could demonstrate, that the absence of its proteins blocked differentiation. These results were corroborated by transfection experiments, since ALDH1A1 and ALDH1A3 overexpression by recombinant re-expression in prior knockout cells recovered the differentiation phenotype.
The exact mechanisms involved in ALDH1 transcriptional and post-translational regulation are not characterized in detail. Recently, we showed regulation of ALDH1A3 in glioma cells by autophagy in response to chemotherapy (Wu et al., 2018). Interestingly, protein levels were upregulated in a reactive manner comparable with the results of DSF inhibition in myoblast cells. Moreover, autophagy has been demonstrated as an important biological mechanism in maintaining the regenerative potential of satellite cells (Wen & Klionsky, 2016). Therefore, ALDH1 seems to be a key factor for the understanding of skeletal muscle biology.
Our ndings indicate that ALDH1A1 and ALDH1A3 proteins are important for myogenic differentiation and, therefore, seem to be essential activators and regulators of SCs.

Availability of data and material
The data and material will not be shared, since all items are able to be purchased and reproduced.

Competing interests
The authors declare that they have no competing interests.

Funding Sources
None.

Authors' contributions
Cell cultivation, experimental implementation, data collection and analysis were performed LR, SF, WW and JS. The rst draft of the manuscript was written by LR. All authors commented on previous versions of the manuscript, contributed to the study conception and design, read and approved the nal manuscript.  Re-transfected cells depict spontaneous myotube formation.    Immuno uorescent staining of wildtype C2C12 cells. Immuno uorescence of C2C12 cells in standard media (Ctrl), DSF-treatment and serum-withdrawal for 6 days (Diff) with additional nucleic DAPI staining.