While diabetes is a multifactorial cardiac insult, and diabetic cardiomyopathy is associated with multiple factors such as oxidative stress, lipotoxicity and mitochondrial dysfunction[55–57], and impaired calcium signaling, O-GlcNAc is emerging as an important signalling mechanism in the development of diabetic cardiomyopathy. Total protein O-GlcNAcylation is chronically elevated in the type 1 and 2 diabetic heart[4, 39], and reducing protein O-GlcNAcylation by adenoviral overexpression of OGA improves cardiac function. Similarly, lowering O-GlcNAc by intensive swim training[43, 44] has been proposed as a mechanism by which exercise benefits the diabetic heart, and exercise lowers both the O-GlcNAc modification of the SP1 transcription factor and the OGT enzyme. O-GlcNAc directly mediates the expression of fetal genes in response to hypertrophic stimuli, and O-GlcNAc modifies mSin3A and HDAC1, which regulate cardiac hypertrophy[20, 58]. Previously, we have shown that exercise lowers the O-GlcNAc modification of the OGT enzyme, and others have shown that exercise lowers O-GlcNAcylation of the SP1 transcription factor. Moderate exercise improves cardiac structure and function in humans with type 2 diabetes[59, 60]; we therefore tested the hypothesis that moderate exercise would reduce O-GlcNAc in the type 2 diabetic heart, and would be associated with changes in the O-GlcNAc modification and activity of the mSin3A/HDAC1/2 transcription factor complex, which regulates hypertrophic genes.
Surprisingly, and in contrast with the previous studies, we found that 4 weeks of moderate treadmill exercise increased total O-GlcNAc in type 2 diabetic db mouse hearts. Also, while the previous studies showed that OGT was also reduced by exercise[43, 44], we found that OGT and OGA expression was elevated in db hearts and did not change with exercise. Such parallel regulation of OGT and OGA expression has been previously reported, and may represent a compensatory relationship between these two opposing enzymes. The difference in our findings may be due to the use of type 2 db mice rather than streptozotocin-induced type 1 diabetic mice, and the use of moderate treadmill exercise rather than more intensive swimming exercise. However, other studies have shown that an upregulation of O-GlcNAc is essential in the cardiac stress response[61, 62], is acutely cardioprotective[63, 64], and is part of a constitutively active cardioprotection mechanism in the diabetic myocardium. Therefore, these data suggest that an increase in cardiac O-GlcNAc in the type 2 diabetic heart may be a beneficial effect of exercise.
In our study, mSin3A immunoprecipitation revealed that exercise increased the O-GlcNAc modification of mSin3A; however, this was not supported by reciprocal O-GlcNAc immunoprecipitation. It is possible that the large amount of protein captured in the O-GlcNAc immunoprecipitation masked the changes in mSin3A O-GlcNAcylation, which we observed in the more specific mSin3A immunoprecipitation. However, these data underscore the importance of verifying changes in O-GlcNAcylation of individual proteins with reciprocal assays, and suggest that moderate changes in protein O-GlcNAcylation – including those in the present study – should be interpreted cautiously and confirmed by additional studies.
Nevertheless, our data do suggest an alternate mechanism for the beneficial effect of exercise on the diabetic heart. Db hearts showed lower protein levels of mSin3A, HDAC1, and HDAC2, and an increased association of mSin3A with REST, independent of exercise. Likewise, mRNA transcript levels of BNP and α-skeletal actin, which are typical markers of cardiac hypertrophy activated by HDAC1/2 that are regulated via REST/mSin3A, were significantly lower in db hearts independent of exercise. The finding that blunted expression of fetal genes in diabetic hearts is not altered by exercise has been shown in previous studies[27, 28]. Therefore, we suggest that the loss of HDAC1/2 and the increased association of the mSin3A corepressor with REST may underlie the blunted expression of fetal genes in the diabetic heart. Further, since the natriuretic peptides are both anti-hypertrophic and cardioprotective[15, 17, 28], we suggest that this mechanism may be responsible for the increased vulnerability of the diabetic heart to stress and heart failure[66, 67].
Although we did not measure the structural or hemodynamic effects of the exercise protocol in db hearts, previous work has shown that the db heart shows similar cardiomyopathy to humans with type 2 diabetes[68, 69], which are improved by exercise[13, 14]. We show additionally that even the low intensity of exercise used in this protocol was sufficient to elevate the expression of cardiac α-actin in C57 hearts (P = 0.050). Cardiac α-actin is a marker of cardiomyocyte differentiation and hypertrophy, and is increased in physiologically hypertrophied hearts after chronic endurance exercise training. Additional transcriptional changes were observed in db hearts, in which the exercise protocol significantly increased the association of mSin3A and OGT with HDAC1 and HDAC2, respectively. Therefore, although the exercise stimulus used in this study did not cause overt changes in cardiac mass, it induced transcriptional events consistent with the early stages of physiological cardiac remodelling.
Finally, these data show a potential interaction between HDAC1 and HDAC2 that has not previously been described in the heart. HDAC1 and HDAC2 regulate cardiac hypertrophy in a similar manner, and HDAC1 deficiency induces HDAC2 expression in embryonic stem cells. In our study, the loss of HDAC1 protein preceded the loss of HDAC2 protein in db hearts, and was similarly associated with an increase in HDAC2 gene expression in db hearts. When HDAC2 deficiency was present at the 4 week time point, we observed an increase in the total activity of class I HDACs in db hearts, which was verified by an increase in the phosphorylation status of HDAC1 at Ser421/423. Phosphorylation at these residues is specifically associated with HDAC1 activity. Therefore, these data suggest that the class I HDACs have compensatory effects on each other’s expression levels and activation by phosphorylation. Further, the reduction in HDAC2 protein levels in db mouse hearts did not occur until 4 weeks, and was associated with overt cardiac hypertrophy. Thus, the loss of HDAC2 in the diabetic heart is associated with the progression of hypertrophy in the diabetic heart, and may be more specifically involved in hypertrophy than HDAC1.