Effects Of Calorie Restriction On The Expression Of SIRTs In Cardiomyocytes

Objective:1. To investigate the cellular localization of SIRTs (SIRT1-7) both in rat cardiac tissues and H9c2 cells;2. To study the effects of calorie restriction on the expression of SIRTs in cardiomyocytes.Methods:Twenty 3-month-old SD rats of both sexes were randomized to either control group (n=10) or CR group (n=10), with five males and five females in each group. Rats in control group were fed ad libitum (AL), while rats in CR group were fed by 60% of AL. After 3 weeks, twenty rats were weighed and killed. Hearts were flushed with 0.9% normal saline solution before taken. The hearts were then sectioned into three parts, the middle part of the heart were used for immunohistochemistry, for detecting the cellular localization of SIRTs (SIRT1-7) in rats cardiac tissues. The same area of the heart from each rat including dual atriums and dual ventricles were used for immunohistochemistry. The rest parts of hearts were used for Western blot to investigate the protein levels of SIRTs.The H9c2 embryonic rat heart-derived cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 100ml/L fetal bovine serum (FBS) and 4.5g/L glucose. The cells were trypsinized, plated (10 000-12 000cells/cm2) in culture dishes, then were randomly divided into two groups after culturing for 48 h, (1) normal control group(Con): without any treatment; (2) calorie restriction group (CR) : cells were incubated in DMEM containing 1g/L glucose. Both groups were incubated in 5% CO2 at 37℃for 24 h. Immunohistochemistry examination was used to determine the cellular localization of SIRTs (SIRT1-7) in H9c2 cells. The expressions of SIRT1-7 mRNA were detected by RT-PCR. Results:1. The differences in body weight between AL group and CR group was not significant before the study protocol. However, after maintenance on a CR diet for three weeks, the body weight (BW) and the heart mass index (HMI=Heart weight/BW) were significantly lower in the CR group when compared with the AL group (p<0.05).2. Each of the seven SIRT proteins was detected in cardiac tissues of SD rats both in AL group and CR group, evidenced by immunohistochemistry. SIRT1 is ubiquitous, as which was distributed in nucleus and cytoplasm. SIRT2, 3, 4, 5 were detected primarily in cytoplasm. SIRT6 and SIRT7 are mainly concentrated in the nucleus.3. A similar expression pattern of SIRT1-7 was observed from immunohistological study in H9c2 cells. Our results show that SIRT1, 3, 4, 5, 7 in H9c2 cells share the same distribution characteristics as in rat heart tissues. SIRT1 is localized both in nucleus and cytoplasm. SIRT3, 4, 5 were detected mainly in cytoplasm while SIRT7 was distributed in nucleus. Intriguingly, high density of SIRT2 in nucleus was observed while SIRT6 was detected primarily in cytoplasm.4. To explore the possible effects of CR on the expression of sirtuins in rat hearts, twenty SD 3-month-old rats were fed either AL or on a CR regimen for 3 weeks. We investigated whether protein levels of SIRT1-7 change after CR. Western blot analysis showed that CR caused SIRT1, 2, 3, 4, 7 statistically significant up-regulations (p<0.05), while the expression of SIRT5, 6 were unaffected.5. The expression pattern of SIRT1-7 mRNA consists with the results of protein, which further support the results mentioned above. H9c2 cells were cultured either in CR group (4.5g/L glucose) or in control group (1g/L glucose) for 24h separately. RT-PCR revealed that the expression of SIRT1, 2, 3, 4, 7 mRNA significantly increased in CR group compared with control group (p<0.05), but SIRT5, 6 were unaffected. Conclusion:1. SIRTs are expressed in the cardiomyocytes both in vivo and in vitro; SIRT1 is distributed in nucleus and cytoplasm, mainly in nucleus. SIRT2, 3, 4, 5 were detected primarily in cytoplasm. SIRT6 and SIRT7 are mainly concentrated in the nucleus.2. Calorie restriction has effects on cardiomyocytes by increasing the expression of SIRT1, 2, 3, 4, 7.