Sex alters thyroid hormone's effect on protein O-GlcNAcylation in the aged mouse heart

The aging heart undergoes physiological changes, many of which are sex dependent and encompass differential responses to cardiac stress. However, much about the molecular changes that occur within the aging heart is still unknown. Thyroid hormone (TH) and the posttranslational modification O-GlcNAcylation (O-GlcNAc) are independently known to regulate cardiac function; therefore, we tested the hypothesis that TH disorders affect cardiac protein O-GlcNAcylation in aged hearts.

We treated male and female 18-22 month-old aged C57BL/6 mice to create euthyroid, hypothyroid, or hyperthyroid states. Western blots and RT-qPCR from cardiac tissue were used to determine changes in global O-GlcNAc levels along with key regulatory proteins in the O-GlcNAcylation process. Immunoprecipitation and western blotting compared global O-GlcNAc changes to differences on an individual protein. We found increased total O-GlcNAc levels for female hypo- and hyperthyroid mice and male hyperthyroid mice compared to sex-matched euthyroid hearts, with no change for male hypothyroid mice. TH's O-GlcNAc effect on female mice appears heart specific as liver O-GlcNAc levels were unchanged. The proteins regulating O-GlcNAcylation also demonstrated sex differences. Female hyperthyroid mice had increased protein expression of the O-GlcNAc regulatory proteins GFAT 1, GFAT 2, and OGT, whereas the hyperthyroid male mice showed decreased expression for the regulatory protein OGA. The hypothyroid female mice had increased protein expression for OGT and NAGK, whereas the hypothyroid male mice showed increased protein expression for NAGK alone. Interestingly, the directional changes in these protein levels did not match RNA transcription. We further found O-GlcNAc levels of the mitochondrial thiolase protein ACAA2 diverged from global O-GlcNAc changes. ACAA2 was hyper O-GlcNAcylated in the female hypothyroid group and hypo O-GlcNAcylated in the male hyperthyroid group whereas there was no change in female hyperthyroid or male hypothyroid.

Protein O-GlcNAcylation is potentially an important mechanism whereby TH perturbations affect the aged heart. We found sex influences O-GlcNAc regulation, global O-GlcNAc levels, and O-GlcNAc protein specificity in response to thyroid hormone perturbations. Our results also suggest the changes in cardiac O-GlcNAc levels are not solely due to TH transcriptional regulation of key O-GlcNAc regulatory enzymes.