Abstract
Diabetes induces specific cardiac dysfunction in the absence of heart disease, including impairment of cardiac β-adrenergic responsiveness. The underlying mechanisms of cardiac dysfunction induced by diabetes are unclear. Recent studies suggest that diabetes-induced cardiac dysfunction cannot be fully explained by changes in calcium (Ca²⁺) regulation through sarco/endoplasmic reticulum Ca²⁺ ATPase (SERCA) alone. Another potential, but almost unexplored cause might be alterations in myofilament Ca²⁺ sensitivity. Myofilament Ca²⁺ sensitivity is regulated by post-translational modification through phosphorylation of cardiac troponin I (cTnI) at Ser23/24. However, it is unclear how diabetes affects phosphorylation of cTnI. Moreover, the effects of diabetes on myofilament Ca²⁺ sensitivity and SERCA activity in response to adrenergic stimulation have not been explored. Therefore, this thesis aimed (1) to compare myofilament Ca²⁺ sensitivity, expression of Ca²⁺-handling proteins and SERCA activity in type 2 diabetic heart, and (2) to investigate whether alteration in myofilament Ca²⁺ sensitivity and/or Ca²⁺ handling contributes to reduced adrenergic responsiveness in type 2 diabetic heart.
In my first study, I investigated the effects of diabetes on myofilament Ca²⁺ sensitivity, expression of Ca²⁺-handling proteins, and SERCA activity in type 2 diabetic heart. Left ventricular tissues were obtained from 20-week-old male Zucker type 2 diabetic fatty (ZDF) rats (n = 5) and age-matched non-diabetic control rats (n = 5). Cardiomyocytes isolated from the diabetic and non-diabetic rat tissues were chemically “skinned” to permeabilise and remove all membrane-bound structures. This allowed direct determination of the myofilament properties by manipulating the external environment and activation with buffer solutions containing different Ca²⁺ concentrations. Myofilament Ca²⁺ sensitivity was greater in diabetic cardiomyocytes as indicated by a greater pCa50 when compared to non-diabetic cardiomyocytes. Western blots showed that phosphorylation of cTnI at Ser23/24 was lower in the diabetic heart. SERCA to phospholamban (PLB) ratio and phosphorylation of PLB at Ser16 were lower in the diabetic hearts, but SERCA Ca²⁺ uptake activity was not different between groups. These findings suggest that increased myofilament Ca²⁺ sensitivity, but not SERCA activity is most likely contributing to cardiac dysfunction in type 2 diabetes.
In my second study, I investigated the mechanism of reduced adrenergic responsiveness in the diabetic heart with regard to myofilament Ca²⁺ sensitivity and SERCA activity. Langendorff perfused hearts isolated from 20-week-old male type 2 diabetic ZDF rats and non-diabetic rats were randomised to noradrenaline stimulation or to a time-control group (n = 8/ group). When paced at equal rates (6Hz), the left ventricular developed pressure (LVDP), maximal rate of contraction (+dP/dtmax), and maximal rate of relaxation (-dP/dtmax) were lower by 33.8%, 40.1%, and 39.4%, respectively in the diabetic hearts when compared to the non-diabetic hearts. LVDP, +dP/dtmax and -dP/dtmax increased less across all concentrations of noradrenaline in the diabetic hearts. At the end of the isolated heart experiments, the left ventricular tissues were snap frozen in liquid nitrogen and stored at -80˚C for myofilament Ca²⁺ sensitivity measurement, western blot and SERCA Ca²⁺ uptake activity assay. Noradrenaline stimulation increased phosphorylation of cTnI at Ser23/24, and reduced myofilament Ca²⁺ sensitivity in both groups to a similar extent. SERCA activity was not different between the diabetic and non-diabetic hearts. Noradrenaline stimulation had no effect on SERCA and PLB expression, and increased SERCA activity to a similar extent, in the diabetic and non-diabetic hearts. Interestingly, noradrenaline stimulation increased NCX expression in the diabetic hearts. These findings suggest that impaired adrenergic responsiveness in the diabetic heart was not caused by changes in myofilament Ca²⁺ sensitivity or SERCA activity.
These studies show that cardiac function is impaired in the type 2 diabetic heart. Increased myofilament Ca²⁺ sensitivity, but not changes in SERCA activity may cause cardiac dysfunction in type 2 diabetes. These findings also show depressed cardiac function during adrenergic stimulation in the diabetic heart, but this impairment is not caused by changes in myofilament Ca²⁺ sensitivity or SERCA activity. Increased NCX expression by noradrenaline stimulation suggests that changes in intracellular Ca²⁺ transient during adrenergic stimulation may underlie impaired diabetic heart function. The findings from my thesis highlight the important role of cardiac myofilament in diabetic heart dysfunction, suggesting that the myofilament could potentially be a new therapeutic target for specific cardiac dysfunction induced by diabetes. My thesis did not support the hypothesis that changes in myofilament Ca²⁺ sensitivity or SERCA activity underlie impaired adrenergic responsiveness in the type 2 diabetic heart. Importantly, to our knowledge, this is the first study that found upregulated NCX expression with adrenergic stimulation in the diabetic heart. This provides a new mechanistic insight into the pathophysiological mechanism of diabetic heart dysfunction.