Sod2 overexpression preserves myoblast mitochondrial mass and function, but not muscle mass with aging Sukkyoo Lee, 1,2 Holly Van Remmen 3,4 and Marie Csete 1,2 1 Department of Anesthesiology, Emory University School of Medicine, Atlanta, GA, USA 2 Program in Biochemistry, Cell and Developmental Biology, Emory University School of Medicine, Atlanta, GA, USA 3 Department of Cellular and Structural Biology and Barshop Institute for Longevity and Aging Studies, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA 4 South Texas Veterans Health Care System, San Antonio, TX, USA Summary Mice lacking superoxide dismutase-2 (SOD2 or MnSOD) die during embryonic or early neonatal development, with diffuse superoxide-induced mitochondrial damage. Although stem and progenitor cells are exquisitely sensi- tive to oxidant stress, they have not been well studied in MnSOD2-manipulated mouse models. Patterns of prolif- eration and differentiation of cultured myoblasts (muscle progenitor cells), PI3-Akt signaling during differentiation, and the maintenance of mitochondrial mass with aging using myoblasts from young (3–4 week old) and aged (27–29 months old) MnSOD2-overexpressing (Sod2-Tg) and heterozygote (Sod2 + ⁄ ) ) mice were characterized by us. Overexpression of MnSOD2 in myoblasts had a pro- tective effect on mitochondrial DNA abundance and some aspects of mitochondrial function with aging, and preservation of differentiation potential. Sod2 deficiency resulted in defective signaling in the PI3-Akt pathway, specifically impaired phosphorylation of Akt at Ser473 and Thr308 in young myoblasts, and decreased differenti- ation potential. Compared with young myoblasts, aged myoblast Akt was constitutively phosphorylated, unre- sponsive to mitogen signaling, and indifferent to MnSOD2 levels. These data suggest that specific sites in the PI3K-Akt pathway are more sensitive to increased superoxide levels than to the increased hydrogen perox- ide levels generated in Sod2-transgenic myoblasts. In wild-type myoblasts, aging was associated with signifi- cant loss of mitochondrial DNA relative to chromosomal DNA, but MnSOD2 overexpression was associated with maintained myoblast mitochondrial DNA with aging. Key words: hydrogen peroxide; mitochondria; muscle aging; myoblast; PI3 kinase-Akt signaling; superoxide. Introduction Superoxide dismutase-2 (SOD2 or MnSOD) is a mitochondrial antioxidant enzyme that reduces superoxide anion (O 2 ) ) into hydrogen peroxide (H 2 O 2 ) and oxygen in the mitochondrial matrix. MnSOD2 is essential, as homozygous Sod2-knockout mice do not survive embryonic development (Li et al., 1995) or die shortly after birth (Huang et al., 2001) depending on genetic background. This lethality is associated with multi- system pathological abnormalities including dilated cardiomy- opathy (Li et al., 1995; Huang et al., 2001), severe anemia, and degeneration of central nervous system neurons (Lebovitz et al., 1996). Heterozygous Sod2 knockout mice have increased susceptibility to seizures (Liang & Patel, 2004) and reduced survival to superimposed severe oxidant stress (Asikainen et al., 2002). These animals show diffuse increases in oxidant damage at the level of lipid peroxidation (Strass- burger et al., 2005), proteins (Friedman et al., 2004), and compromised mitochondrial function (Melov et al., 1999; Van Remmen et al., 1999, 2001; Kokoszka et al., 2001; Strass- burger et al., 2005). However, the reduced mitochondrial function of Sod2 + ⁄ ) animals is managed by compensatory antioxidant responses with aging, so that there is not a com- pounded oxidant damage with aging and loss of the enzyme (Mansouri et al., 2006), and the animals have a normal life- span. Overexpression of Sod2 in mice leads to enhanced mito- chondrial function (Silva et al., 2005), and protection from various oxidant stressors including reperfusion injury (Suzuki et al., 2002), but no increase in lifespan. Generally, studies of muscle in mice genetically manipu- lated for Sod2 expression have been done at the level of whole muscle, and the effects of Sod2 expression on muscle progenitor cell populations have not been studied. Skeletal muscle contains a resident stem cell population, the satellite cell, located between the basal lamina and plasma mem- brane of the muscle fiber (Mauro, 1961). Satellite cells are generally quiescent, but enter the cell cycle and become myoblasts when muscle is damaged. Myoblasts are the cellu- lar source of muscle regeneration throughout life. This tran- sient amplifying population of skeletal muscle proliferates as single cells, then fuses and forms myotubes in vitro and inte- grates into myofibers in vivo in response to appropriate dif- ferentiation cues. The numbers and capability of myoblasts to fuse and differentiate to myofibers generally decrease with chronic oxidative stress or aging (Lee et al., 2006; Shefer et al., 2006; Collins et al., 2007; Verdijk et al., 2007) Correspondence Marie Csete, California Institute for Regenerative Medicine, 210 King Street, San Francisco, CA 94107, USA. Tel.: 415 396 9106; fax: 415 396 9141; e-mail: mcsete@cirm.ca.gov Accepted for publication 21 March 2009 296 ª 2009 The Authors Journal compilation ª Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland 2009 Aging Cell (2009) 8, pp296–310 Doi: 10.1111/j.1474-9726.2009.00477.x Aging Cell