Abstract
The myofilament protein, cardiac troponin I (cTnI), is a critical regulatory protein in the contraction and relaxation of cardiac muscle, linking calcium to bind to the thin filament. It has become apparent that myofilament proteins and intracellular calcium availability have an effect on the dynamic modulation on contractile function. The phosphorylation of cTnI at specific serine and threonine residue sites is an important physiological mechanism for altered myofilament functional properties and may play a significant role in the contractile dysfunction observed in diabetes. It is possible that exercise might reverse myofilament dysfunction in the diabetic heart by altering cTnI phosphorylation levels. However, whether exercise alters cardiac myofilament proteins in diabetes is unexplored.
This study aimed to investigate the role of cTnI in the diabetic heart and determine the effects diabetes and exercise might have on calcium sensitivity and phosphorylation of the myofilament protein, cTnI. I hypothesised that myofilament phosphorylation and calcium sensitivity are dysregulated in diabetes, which could be mitigated by exercise training.
Left ventricular tissue –from the Zucker diabetic fatty rat, a model of type 2 diabetes, was used to measure total cTnI expression and levels of phosphorylation within cTnI. Measurements were taken from four groups: non-diabetic sedentary, diabetic sedentary, non-diabetic trained, and diabetic trained. We measured myofilament calcium sensitivity via single-cell contractility experiments and quantified the level of phosphorylation of cTnI via Western blot experiments. Rats were randomized to either eight weeks of treadmill running or a sedentary intervention. This combination of experiments allowed us to determine whether the effects of exercise could reverse expected changes in cTnI phosphorylation measured in diabetic tissue.
Western blot experiments from 12-week old tissue illustrated levels of cTnI phosphorylation at serine 150 were decreased in diabetic animals compared to non-diabetic littermates (P < 0.05, n = 5). In diabetic samples, phosphorylation at serine 23/24 was increased (P < 0.05, n = 10), however phosphorylation at threonine 143 (P = 0.21, n = 5) showed an increasing trend but did not reach significance. Single-cell contractility experiments revealed a leftward shift in the calcium-binding curve of diabetic trained cardiomyocytes, indicating an increase in calcium sensitivity compared to non-diabetic trained cardiomyocytes; however, the results were not significant. Immunoblots from 20-week old tissue revealed that cTnI phosphorylation at serine 23/24 (P < 0.05, n = 6) and threonine 143 (P < 0.05, n = 6) were significantly decreased in trained diabetics compared to trained non-diabetics, corresponding to an increase in calcium sensitivity.
Our results demonstrate that diabetes dysregulates phosphorylation of the myofilament protein, cTnI. Whereby, this relationship was altered in trained diabetic rats, showing exercise reversed the levels of phosphorylation observed at 12-weeks of age. This thesis has provided a deeper insight into the critical role that cTnI phosphorylation (at numerous amino acid residues) plays in the altering of single-cell functionality in the diabetic heart and its ability to be reversed, at least in part, by exercise.