Abstract
In the 1970’s, data indicated that the diabetic heart exhibits dysfunction and increases the risk of developing heart failure 2- to 5 fold independent of other common risk factors. We now know that diabetic heart dysfunction is multifactorial including dysregulation or dysfunction of several different mechanisms, such as the sympathetic nervous system, fibrosis, filling volumes and pressures, accumulation of oxidative stress, apoptosis, calcium handling and more. Unfortunately, there is still no treatment specific to diabetic heart dysfunction.
In other aetiologies of heart failure, myofilament protein structure and function are altered and may prove to be an adequate target for normalizing the dysfunctional contraction and relaxation parameters seen in the diabetic heart. The calcium sensitivity of myofilaments can be either increased or reduced in the failing heart and often corresponds with altered post-translational modifications (PTM) of an important regulatory myofilament protein, cardiac troponin I (cTnI). Although there are a few studies testing the calcium sensitivity of diabetic myofilaments, none have investigated both calcium sensitivity and PTM within the same model of diabetes. Lastly, it is well-established that regular exercise benefits individuals with diabetes and increases cardiac reserve, but more work is necessary to establish how exercise may target the diabetic heart at the cellular level, potentially identifying aspects of the diabetic heart that are reversible and feasible to target with novel therapies. Exercise has been shown to alter the calcium sensitivity of healthy myofilaments, but has yet to be tested in a trained, diabetic model.
The Zucker Diabetic Fatty (ZDF) rat was used in this thesis based upon its ability to model the type 2 diabetic phenotype: hyperglycaemia, hyperinsulinemia and hyperlipidaemia. Twelve- and 20-week-old ZDF rats were used in Chapter 4 to test the time course of hyperglycaemia on myofilament function. Calcium sensitivity (pCa50), or the calcium concentration necessary to generate 50% of relative tension, was measured by attaching a permeated cell to a torque motor and force transducer. Total phosphorylation of key myofilament proteins was determined using the phospho-staining technique of ProQ Diamond gel stain followed by Sypro Ruby gel stain to normalize to the total protein present. Site-specific cTnI phosphorylation and total O-GlcNAcylation was determined using immunoblots. pCa50 was increased in diabetic ZDF rat hearts compared to non-diabetic ZDF rats, both at 12 and 20 weeks of age. Total cTnI and phospho-sites Serine (Ser)23/24 on cTnI were reduced in the diabetic ZDF heart. There was no difference in protein O GlcNAcylation.
In Chapter 5, I tested the effects of endurance training on calcium sensitivity and functionally relevant PTM on diabetic myofilaments. ZDF rats were obtained at 11 weeks of age and randomized to either a sedentary (SED) or training (TR) intervention. The TR group underwent an 8-week treadmill running protocol that was progressive, increasing in time and speed. pCa50 was measured with permeated cells as mentioned previously. The plantaris muscle citrate synthase activity was increased in both TR groups, indicating a significant effect of the treadmill protocol. pCa50 was increased in TR groups and, as seen above, pCa50 was also increased in diabetic ZDF hearts. The total and site specific phosphorylation of key myofilament proteins were affected by the diabetic phenotype, but not by the intervention. Training greatly reduced fasted blood glucose levels, but there was no difference in the amount of O GlcNAcylation occurring in the TR myocardium compared to the SED myocardium. In conclusion, the diabetic cardiomyocytes were capable of increasing pCa50 in response to training, as previously shown in the non-diabetic heart, but PTM that commonly affect the diabetic heart, such as phosphorylation and O-GlcNAcylation, were not altered by training.