Atmospheric oxygen tension slows myoblast proliferation via mitochondrial activation.

Stephanie Duguez, William Duddy, Viola Gnocchi, James Bowe, Sherry Dadgar, Terence A Partridge

Research output: Contribution to journalArticle

Abstract

BACKGROUNDMitochondrial activity inhibits proliferation and is required for differentiation of myoblasts. Myoblast proliferation is also inhibited by the ~20% oxygen level used in standard tissue culture. We hypothesize that mitochondrial activity would be greater at hyperoxia (20% O(2)) relative to more physiological oxygen (5% O(2)).METHODOLOGY/PRINCIPAL FINDINGSMurine primary myoblasts from isolated myofibres and conditionally immortalized H-2K myoblasts were cultured at 5% and 20% oxygen. Proliferation, assayed by cell counts, EdU labeling, and CFSE dilution, was slower at 20% oxygen. Expression of MyoD in primary myoblasts was delayed at 20% oxygen, but myogenicity, as measured by fusion index, was slightly higher. FACS-based measurement of mitochondrial activity indicators and luminometric measurement of ATP levels revealed that mitochondria exhibited greater membrane potential and higher levels of Reactive Oxygen Species (ROS) at 20% oxygen with concomitant elevation of intracellular ATP. Mitochondrial mass was unaffected. Low concentrations of CCCP, a respiratory chain uncoupler, and Oligomycin A, an ATP synthase inhibitor, each increased the rate of myoblast proliferation. ROS were investigated as a potential mechanism of mitochondrial retrograde signaling, but scavenging of ROS levels by N-acetyl-cysteine (NAC) or α-Phenyl-N-tert-butylnitrone (PBN) did not rescue the suppressed rate of cell division in hyperoxic conditions, suggesting other pathways. Primary myoblasts from older mice showed a slower proliferation than those from younger adult mice at 20% oxygen but no difference at 5% oxygen.CONCLUSIONS/SIGNIFICANCEThese results implicate mitochondrial regulation as a mechanistic explanation for myoblast response to oxygen tension. The rescue of proliferation rate in myoblasts of aged mice by 5% oxygen suggests a major artefactual component to age-related decline of satellite cell proliferation in standard tissue culture at 20% oxygen. It lends weight to the idea that these age-related changes result at least in part from environmental factors rather than characteristics intrinsic to the satellite cell.
LanguageEnglish
Pagese43853
JournalPLoS ONE
Volume7
Issue number8
DOIs
Publication statusPublished - 24 Aug 2012

Fingerprint

myoblasts
Myoblasts
Chemical activation
Oxygen
oxygen
reactive oxygen species
Reactive Oxygen Species
Tissue culture
Adenosine Triphosphate
tissue culture
mice
Acetylcysteine
Carbonyl Cyanide m-Chlorophenyl Hydrazone
Satellites
oligomycin
hyperoxia
acetylcysteine
Hyperoxia
H-transporting ATP synthase
Mitochondria

Keywords

  • Low Oxygen
  • Muscle Stem Cells
  • cell cyle
  • mitochondria

Cite this

Duguez, Stephanie ; Duddy, William ; Gnocchi, Viola ; Bowe, James ; Dadgar, Sherry ; Partridge, Terence A. / Atmospheric oxygen tension slows myoblast proliferation via mitochondrial activation. In: PLoS ONE. 2012 ; Vol. 7, No. 8. pp. e43853.
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title = "Atmospheric oxygen tension slows myoblast proliferation via mitochondrial activation.",
abstract = "BACKGROUNDMitochondrial activity inhibits proliferation and is required for differentiation of myoblasts. Myoblast proliferation is also inhibited by the ~20{\%} oxygen level used in standard tissue culture. We hypothesize that mitochondrial activity would be greater at hyperoxia (20{\%} O(2)) relative to more physiological oxygen (5{\%} O(2)).METHODOLOGY/PRINCIPAL FINDINGSMurine primary myoblasts from isolated myofibres and conditionally immortalized H-2K myoblasts were cultured at 5{\%} and 20{\%} oxygen. Proliferation, assayed by cell counts, EdU labeling, and CFSE dilution, was slower at 20{\%} oxygen. Expression of MyoD in primary myoblasts was delayed at 20{\%} oxygen, but myogenicity, as measured by fusion index, was slightly higher. FACS-based measurement of mitochondrial activity indicators and luminometric measurement of ATP levels revealed that mitochondria exhibited greater membrane potential and higher levels of Reactive Oxygen Species (ROS) at 20{\%} oxygen with concomitant elevation of intracellular ATP. Mitochondrial mass was unaffected. Low concentrations of CCCP, a respiratory chain uncoupler, and Oligomycin A, an ATP synthase inhibitor, each increased the rate of myoblast proliferation. ROS were investigated as a potential mechanism of mitochondrial retrograde signaling, but scavenging of ROS levels by N-acetyl-cysteine (NAC) or α-Phenyl-N-tert-butylnitrone (PBN) did not rescue the suppressed rate of cell division in hyperoxic conditions, suggesting other pathways. Primary myoblasts from older mice showed a slower proliferation than those from younger adult mice at 20{\%} oxygen but no difference at 5{\%} oxygen.CONCLUSIONS/SIGNIFICANCEThese results implicate mitochondrial regulation as a mechanistic explanation for myoblast response to oxygen tension. The rescue of proliferation rate in myoblasts of aged mice by 5{\%} oxygen suggests a major artefactual component to age-related decline of satellite cell proliferation in standard tissue culture at 20{\%} oxygen. It lends weight to the idea that these age-related changes result at least in part from environmental factors rather than characteristics intrinsic to the satellite cell.",
keywords = "Low Oxygen, Muscle Stem Cells, cell cyle, mitochondria",
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Atmospheric oxygen tension slows myoblast proliferation via mitochondrial activation. / Duguez, Stephanie; Duddy, William; Gnocchi, Viola; Bowe, James; Dadgar, Sherry; Partridge, Terence A.

In: PLoS ONE, Vol. 7, No. 8, 24.08.2012, p. e43853.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Atmospheric oxygen tension slows myoblast proliferation via mitochondrial activation.

AU - Duguez, Stephanie

AU - Duddy, William

AU - Gnocchi, Viola

AU - Bowe, James

AU - Dadgar, Sherry

AU - Partridge, Terence A

PY - 2012/8/24

Y1 - 2012/8/24

N2 - BACKGROUNDMitochondrial activity inhibits proliferation and is required for differentiation of myoblasts. Myoblast proliferation is also inhibited by the ~20% oxygen level used in standard tissue culture. We hypothesize that mitochondrial activity would be greater at hyperoxia (20% O(2)) relative to more physiological oxygen (5% O(2)).METHODOLOGY/PRINCIPAL FINDINGSMurine primary myoblasts from isolated myofibres and conditionally immortalized H-2K myoblasts were cultured at 5% and 20% oxygen. Proliferation, assayed by cell counts, EdU labeling, and CFSE dilution, was slower at 20% oxygen. Expression of MyoD in primary myoblasts was delayed at 20% oxygen, but myogenicity, as measured by fusion index, was slightly higher. FACS-based measurement of mitochondrial activity indicators and luminometric measurement of ATP levels revealed that mitochondria exhibited greater membrane potential and higher levels of Reactive Oxygen Species (ROS) at 20% oxygen with concomitant elevation of intracellular ATP. Mitochondrial mass was unaffected. Low concentrations of CCCP, a respiratory chain uncoupler, and Oligomycin A, an ATP synthase inhibitor, each increased the rate of myoblast proliferation. ROS were investigated as a potential mechanism of mitochondrial retrograde signaling, but scavenging of ROS levels by N-acetyl-cysteine (NAC) or α-Phenyl-N-tert-butylnitrone (PBN) did not rescue the suppressed rate of cell division in hyperoxic conditions, suggesting other pathways. Primary myoblasts from older mice showed a slower proliferation than those from younger adult mice at 20% oxygen but no difference at 5% oxygen.CONCLUSIONS/SIGNIFICANCEThese results implicate mitochondrial regulation as a mechanistic explanation for myoblast response to oxygen tension. The rescue of proliferation rate in myoblasts of aged mice by 5% oxygen suggests a major artefactual component to age-related decline of satellite cell proliferation in standard tissue culture at 20% oxygen. It lends weight to the idea that these age-related changes result at least in part from environmental factors rather than characteristics intrinsic to the satellite cell.

AB - BACKGROUNDMitochondrial activity inhibits proliferation and is required for differentiation of myoblasts. Myoblast proliferation is also inhibited by the ~20% oxygen level used in standard tissue culture. We hypothesize that mitochondrial activity would be greater at hyperoxia (20% O(2)) relative to more physiological oxygen (5% O(2)).METHODOLOGY/PRINCIPAL FINDINGSMurine primary myoblasts from isolated myofibres and conditionally immortalized H-2K myoblasts were cultured at 5% and 20% oxygen. Proliferation, assayed by cell counts, EdU labeling, and CFSE dilution, was slower at 20% oxygen. Expression of MyoD in primary myoblasts was delayed at 20% oxygen, but myogenicity, as measured by fusion index, was slightly higher. FACS-based measurement of mitochondrial activity indicators and luminometric measurement of ATP levels revealed that mitochondria exhibited greater membrane potential and higher levels of Reactive Oxygen Species (ROS) at 20% oxygen with concomitant elevation of intracellular ATP. Mitochondrial mass was unaffected. Low concentrations of CCCP, a respiratory chain uncoupler, and Oligomycin A, an ATP synthase inhibitor, each increased the rate of myoblast proliferation. ROS were investigated as a potential mechanism of mitochondrial retrograde signaling, but scavenging of ROS levels by N-acetyl-cysteine (NAC) or α-Phenyl-N-tert-butylnitrone (PBN) did not rescue the suppressed rate of cell division in hyperoxic conditions, suggesting other pathways. Primary myoblasts from older mice showed a slower proliferation than those from younger adult mice at 20% oxygen but no difference at 5% oxygen.CONCLUSIONS/SIGNIFICANCEThese results implicate mitochondrial regulation as a mechanistic explanation for myoblast response to oxygen tension. The rescue of proliferation rate in myoblasts of aged mice by 5% oxygen suggests a major artefactual component to age-related decline of satellite cell proliferation in standard tissue culture at 20% oxygen. It lends weight to the idea that these age-related changes result at least in part from environmental factors rather than characteristics intrinsic to the satellite cell.

KW - Low Oxygen

KW - Muscle Stem Cells

KW - cell cyle

KW - mitochondria

U2 - 10.1371/journal.pone.0043853

DO - 10.1371/journal.pone.0043853

M3 - Article

VL - 7

SP - e43853

JO - PLoS ONE

T2 - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 8

ER -