Abstract
Diabetes Mellitus affects a wide range of body systems, with extensive pathological effects on the cardiovascular system. Post-translational modifications (PTMs) are responsible for some of these pathologies, with dysregulations of O-GlcNAcylation and S-Nitrosylation evident throughout diabetic cardiac tissue. This thesis aimed to examine the effect of acute modulation of each PTM in human diabetic cardiac tissue, with the hypothesis that detrimental effects arising from this were partially attributable to downstream effects of these PTMs on Ca2+/Calmodulin dependent protein kinase II (CaMKII). I also aimed to investigate the specific role of the CaMKII C273 residue on the effects of S-Nitrosylation in isolated cardiac tissue.
Using an isolated trabeculae model, I acutely modulated O-GlcNAcylation using the compounds DON (O-GlcNAc inhibitor) and THG (O-GlcNAc potentiator) in diabetic and non-diabetic human cardiac tissue. Unexpectedly, DON had no effects on contractility or arrhythmogenesis when applied to diabetic tissue, but was able to alter Ca2+ handling in the diabetic tissue. DON treatment was significantly detrimental to contractility in non-diabetic tissue, with no effects on arrhythmia. THG treatment was significantly detrimental to contractility regardless of diabetic status, and unexpectedly protected against arrhythmogenesis in non-diabetic tissue. This study highlighted the importance of maintaining homeostasis of O-GlcNAcylation in non-diabetic cardiac tissue, and suggested that the lack of effects of DON in diabetic tissue was due to chronic tissue alterations, such as fibrogenesis.
The same isolated trabeculae model was used to examine the roles of S-Nitrosylation on contractility and arrhythmogenesis in human diabetic and non-diabetic cardiac tissue, along with the impacts of biological sex on these processes. A regulatory mechanism of GSNO (NO donor) and ISO (β-adrenergic agonist) has been previously discovered by our laboratory, which affected arrhythmogenesis via regulation of CaMKII. Obvious sex differences were noted in non-diabetic tissue, with females but not males able to reduce contractile indices with GSNO treatment following ISO. These sex-specific differences appeared to be lost in diabetic female tissue, suggesting that diabetes is affecting the NO signalling cascade that differentiates male and female cardiac function. This study suggested that the diabetic state may alters the contractile and response to GSNO/ISO differently depending on biological sex. These data suggest that alterations in estrogen signalling and downstream effects on NO-signalling, dependent on biological sex, is being altered by the diabetic disease state. Further research is required to confirm these sex-dependent findings, due to the small sample sizes in this study.
Isolated murine papillary muscles were used to examine the specific role of the C273 residue on CaMKII in the NO-signalling mechanisms discussed previously. ECG examinations of mutant C273S mice showed that the C273 S-Nitrosylation residue on CaMKII is important for regulating ventricular conduction, with significant differences between mutant and WT mice. The C273 site was also shown to affect ventricular structure, with alterations in ventricular morphology noted during echocardiography exams in mutant mice. Contrasting with the isolated human trabeculae model, both mice strains showed the ability for GSNO to reverse ISO-induced increases in contractility. GSNO pre-treatment was unable to prevent contractile increases with ISO in either mouse strain. Similar to results previously shown in our laboratory, C273S mice were unable to prevent ISO-induced arrhythmia when pre-treated with GSNO, unlike WT mice which benefited from this protection. These data show the importance of the C273 residue in regulating arrhythmogenesis, but not contractility.
Overall this project showed the impacts of both O-GlcNAcylation and S-Nitrosylation on diabetic cardiac tissue, along with the specific effects of the C273 residue on CaMKII. These findings should inform future clinical treatments to better improve patient outcomes and equity between biological sex.