Caloric restriction does not alter effects of aging in cardiac side population cells

Abstract

The aged heart displays a loss of cardiomyocyte number and function, possibly due to the senescence and decreased regenerative potential that has been observed in some cardiac progenitor cells. An important cardiac progenitor that has not been studied in the context of aging is the cardiac side population (CSP) cell. To address this, flow cytometry-assisted cell sorting was used to isolate CSP cells from adult (6-10 months old) and aged (24-32 months old) C57Bl/6 mice that were fed either a control diet or an anti-aging diet (caloric restriction, CR). Aging caused a 2.3-fold increase in the total number of CSP cells and a 3.2-fold increase in the cardiomyogenic sca1(+)/CD31(-) subpopulation. Aging did not affect markers of proliferation or senescence, including telomerase activity and expression of cell cycle genes, in sca1(+)/CD31(-) CSP cells. In contrast, the aged cells had reduced expression of genes associated with differentiation, including smooth muscle actin and cardiac muscle actin (5.1- and 3.2-fold, respectively). None of these age effects were altered by CR diet. Therefore, it appears that the manner in which CSP cells age is distinct from the aging of post-mitotic tissue (and perhaps other progenitor cells) that can often be attenuated by CR.

Fig. 1

Effect of aging and CR on CSP cell abundance and composition. a Representative flow cytometry plot used to distinguish cardiac side population (CSP) from cardiac main population (CMP) cells based on the efflux of Hoechst dye in lin⁻ non-cardiomyocytes of adult and aged AL mice. ATP-binding cassette transporter activity is confirmed by inhibition with verapamil. b Aging increased CSP cell number (p < 0.0001), but CR had no effect (n = 6–8 per group). c Representative flow cytometry plots illustrating the division of CSP cells into sub-populations based on expression of sca1 and CD31 in an adult mouse heart. d Aging increased abundance of sca1⁺/CD31⁻ CSP cells (p < 0.0001), but CR had no effect (n = 6–8 per group). Data are expressed as mean ± SE. Asterisk denotes significant effect of age

Fig. 2

Effect of aging and CR on markers of proliferation and senescence in sca1⁺/CD31⁻ CSP cells. a RNA was isolated from flow cytometry-isolated sca1⁺/CD31⁻ CSP cells, and gene expression was analyzed by real-time RT–PCR gene panel. Data are expressed as mean delta Ct (=Ct sample − Ct average of housekeeping genes) ± SD; n = 4 per group. Gene expression did not differ between groups. b Telomerase activity in flow cytometry-isolated CSP cells was determined by quantitative real-time PCR and expressed as mean cycle threshold (Ct) ± SE; n = 4 per group. Activity was not different between groups

Fig. 3

Effect of aging and CR on sca1⁺/CD31⁻ CSP cell gene expression. RNA was collected from flow cytometry-isolated sca1⁺/CD31⁻ CSP cells and CMP cells, and gene expression was analyzed by real-time RT–PCR gene panel (n = 4 per group). a aged AL CSP vs. adult AL CSP, b aged CR CSP vs. aged AL CSP, and c adult AL CSP vs. adult AL CMP. Genes to the far left (downregulation) or far right (upregulation) of the bold vertical lines on the volcano plot indicate a fold change greater than 2-fold; genes above the bold horizontal line indicate a significant fold change (q < 0.05)