Resumen
Adenine Nucleotide Translocator isoforms (ANTs) exchange ADP/ATP across the inner mitochondrial membrane, are also voltage-activated proton channels and regulate mitophagy and apoptosis. The ANT1 isoform predominates in heart and muscle while ANT2 is systemic. Here, we report the creation of Ant mutant mouse myoblast cell lines with normal Ant1 and Ant2 genes, deficient in either Ant1 or Ant2, and deficient in both the Ant1 and Ant2 genes. These cell lines are immortal under permissive conditions (IFN-γ + serum at 32 °C) permitting expansion but return to normal myoblasts that can be differentiated into myotubes at 37 °C. With this system we were able to complement our Ant1 mutant studies by demonstrating that ANT2 is important for myoblast to myotube differentiation and myotube mitochondrial respiration. ANT2 is also important in the regulation of mitochondrial biogenesis and antioxidant defenses. ANT2 is also associated with increased oxidative stress response and modulation for Ca++ sequestration and activation of the mitochondrial permeability transition (mtPTP) pore during cell differentiation.
| Idioma original | English |
|---|---|
| Páginas (desde-hasta) | 312-327 |
| Número de páginas | 16 |
| Publicación | Free Radical Biology and Medicine |
| Volumen | 188 |
| DOI | |
| Estado | Published - ago 1 2022 |
Nota bibliográfica
Publisher Copyright:© 2022 The Authors
Financiación
ANT null cell lines cultured in growth medium containing 25 mM glucose showed significantly reduced proliferative capacity (Fig. 2A&B). They also displayed increased vacuolization, and appeared to be smaller and more spindle-shaped than the trapezoid wild type cells (Fig. 2A&B). When cell lines were grown under low glucose (5 mM) conditions, a significant proportion of the ANT2-deficient and ANT null cells detached from the support matrix within 24 h (Fig. 2A3). Culture in aerobic growth medium (galactose) resulted in growth arrest and cell death of all cell lines, with the earliest cell death detected in cell lines deficient in both isoforms, followed by those deficient in either of the two ANT isoforms (data not shown).During rapid cellular growth and remodeling, mitochondrial and cellular biogenesis requires the careful balance of cellular ATP pools. With the ADP/ATP exchange through ANTs is in principle being reversible [86], cells with restricted or compromised mitochondrial respiration could actively consume cytoplasmic ATP to support mitochondrial pathways essential for cell survival and remodeling. A mitochondrial electroneutral adenine nucleotide carrier (AXP/Mg2+/Pi, APC2) has been suggested to play a role in mitochondrial ATP import under these conditions [25], however, this carrier is cotransport dependent and not specific for ATP. Although we did observe increased protein expression of this carrier in ANT-deficient myoblast cell lines, its upregulation appeared to not significantly contribute to mitochondrial ATP levels.C57BL/6 wild type (I+/+/II+/+), ANT1 wild type, ANT2-floxed (I+/+/IIfl/Y) (I+/II+) and ANT1 deleted, ANT2-floxed mice (I−/-/IIfl/Y) (I-/II+) were crossed into the genetic background of CBA; B10-Tg(H2Kb-tsA58) mice (Charles River, Wilmington, MA; USA) to generate mice homozygous also for the H-2Kb–tsA58 transgene. Offspring was genotyped for the genomic and deleted ANT1 isoform [7], the ANT2 floxed gene [8] as well as for the H-2Kb-tsA58 transgene [86]. In accordance with institutional protocols, myoblasts were isolated from gastrocnemius muscles of hind limbs from 3 month old mice by sequential tissue dissociation with 200 μg/ml liberase blendzyme 3 (Roche Applied Science) and 0.25% Trypsin (Invitrogen). Myogenic cell lines were established by repetitive selective plating on ECL-Matrix (Millipore) in F-12/DMEM (1:1), 20% FCS, 4.5 g/l (25 mM) glucose, 1 ng/ml IFN-γ, 1 ng/ml bFGF, 1 mM glutamate, 3 mM sodium pyruvate, 4 mM glutamine and 50 μg/ml gentamycin, 2.5 μg/ml amphotericin B [48]. Cell lines were detached from substrate with 2 mM EDTA in PBS and numbers of viable cells for reseeding were obtained using a Z-1 CoulterR Particle Counter (Beckman Coulter). After 2 weeks in culture, antibiotics in culture medium were discontinued. For specific experiments, complete growth medium (GM) was substituted with formulations containing 15% FCS, but only supporting growth on substrates for glycolytic (Gluc: 25 mM glucose and 2 mM glutamine only) or oxidative (Gal, 5 mM galactose, 100 μM inositol, 40 μM deoxythymidine, 100 μM uridine) metabolism (Invitrogen). For differentiation assays, FCS was omitted from GM or replaced with 1% horse serum (Invitrogen) or 1% DMSO (Sigma) as negative differentiation control.Grants awarded to DC Wallace are National Institutes of Health grants NS021328, MH108592, OD010944, and U.S. Department of Defense grants W81XWH2110128 and PR210230. Grants awarded to DC Wallace are National Institutes of Health grants NS021328 , MH108592 , OD010944 , and U.S. Department of Defense grants W81XWH2110128 and PR210230 .
| Financiadores | Número del financiador |
|---|---|
| DMEM | 1:1 |
| ECL-Matrix | |
| Wallace are National Institutes of Health | MH108592, OD010944, NS021328 |
| U.S. Department of Defense | PR210230, W81XWH2110128 |
ASJC Scopus subject areas
- Biochemistry
- Physiology (medical)
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