Estrogen is required for maintaining the quality of cardiac stem cells

Abstract

Compared to the age-matched men, the incidence of cardiovascular diseases is lower in premenopausal but higher in postmenopausal women, suggesting the cardio-protective role of estrogen in females. Although cardiac stem cells (CSCs) express estrogen receptors, yet the effects of estrogen on CSCs remain unclear. In this study, we investigated the potential role of estrogen in maintaining the quality of CSCs by in vivo and in vitro experiments. For the in vivo study, estrogen deficiency was induced by ovariectomy in 6-weeks-old C57BL/6 female mice, and then randomly given 17β-estradiol (E2) replacements at a low dose (0.01 mg/60 days) and high dose (0.18 mg/60 days), or vehicle treatment. All mice were killed 2 months after treatments, and heart tissues were collected for ex vivo expansion of CSCs. Compared to age-matched healthy controls, estrogen deficiency slightly decreased the yield of CSCs with significantly lower telomerase activity and more DNA damage. Interestingly, E2 replacements at low and high doses significantly increased the yield of CSCs and reversed the quality impairment of CSCs following estrogen deficiency. For the in vitro study, twice-passaged CSCs from the hearts of adult healthy female mice were cultured with the supplement of 0.01, 0.1, and 1 μM E2 in the medium for 3 days. We found that E2 supplement increased c-kit expression, increased proliferative activity, improved telomerase activity, and reduced DNA damage of CSCs in a dose-dependent manner. Our data suggested the potential role of estrogen in maintaining the quality of CSCs, providing new insight into the cardio-protective effects of estrogen.

Fig 1. The outgrowth of cardiac stem cells (CSCs) from the atrial tissues of mice.

The outgrowth of CSCs was evaluated 2 weeks after the initiation of culture. Control: sham-operated mice, Ovx: ovariectomized mice, LE₂: ovariectomized mice supplemented with low dose of 17β-estradiol (0.01 mg/60 days), HE₂: ovariectomized mice supplemented with high dose of 17β-estradiol (0.18 mg/60 days). All data are mean±SD from 3 independent experiments (n = 3 for each group). *P<0.05, **P<0.01.

Fig 2. The expression of c-kit and Ki-67 in cardiac stem cells (CSCs).

Immunostaining was performed to evaluate the expression of c-kit (A) and Ki-67 (B) in twice-passaged CSCs. Representative images (upper) and quantitative data (lower) are shown. The nuclei were labeled with DAPI. Scale bar: 20 μm. Control: sham-operated mice, Ovx: ovariectomized mice, LE₂: ovariectomized mice received a low dose of 17β-estradiol (0.01 mg/60 days), HE₂: ovariectomized mice received a high dose of 17β-estradiol (0.18 mg/60 days). All data are mean±SD from 3 independent experiments (n = 3 for each group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

Fig 3. Dose dependency of the 17β-estradiol supplement on the expression of c-kit and Ki67 in cardiac stem cells (CSCs) from healthy female mice.

The expression of c-kit (A) and Ki-67 (B) in CSCs from healthy female mice were evaluated by immunostaining after 3 days of culture with the supplement of different concentrations of 17β-estradiol in the medium. Representative images (upper) and Quantitative data (lower). The nuclei were labeled with DAPI. Scale bar: 20 μm. Control: sham-operated mice, Ovx: ovariectomized mice, LE₂: ovariectomized mice received a low dose of 17β-estradiol (0.01 mg/60 days), HE₂: ovariectomized mice received a high dose of 17β-estradiol (0.18 mg/60 days). All data are mean±SD from 3 independent experiments (n = 3 for each group). *P<0.05, **P<0.01.

Fig 4. The telomerase activity and DNA damage of cardiac stem cells (CSCs).

The telomerase activity and DNA damage in twice-passaged CSCs were evaluated by immunostaining analysis on the expression of TERT (A) and 53BP1 (B), respectively. Representative images (upper) and quantitative data (lower) are shown. The nuclei were labeled with DAPI. Scale bar: 20 μm. Control: sham-operated mice, Ovx: ovariectomized mice, LE₂: ovariectomized mice received a low dose of 17β-estradiol (0.01 mg/60 days), HE₂: ovariectomized mice received a high dose of 17β-estradiol (0.18 mg/60 days). All data are mean±SD from 3 independent experiments (n = 3 for each group). *P<0.05, ***P<0.001, ****P<0.0001.

Fig 5. Dose dependency of the 17β-estradiol supplement on the telomerase activity and DNA damage in cardiac stem cells (CSCs) from healthy female mice.

The telomerase activity and DNA damage in CSCs were evaluated by immunostaining analysis on the expression of TERT (A) and 53BP1 (B), respectively, after 3 days of culture with the supplement of different concentrations of 17β-estradiol in the medium. Representative images (upper) and quantitative data (lower) are shown. The nuclei were labeled with DAPI. Scale bar: 20 μm. Control: sham-operated mice, Ovx: ovariectomized mice, LE₂: ovariectomized mice received a low dose of 17β-estradiol (0.01 mg/60 days), HE₂: ovariectomized mice received a high dose of 17β-estradiol (0.18 mg/60 days). All data are mean±SD from 3 independent experiments (n = 3 for each group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.