The role of calcium calmodulin dependent protein kinase II in type 2 diabetic heart dysfunction

Type 2 diabetes is a well-established risk factor for cardiovascular disease, with the landmark Framingham study in the 1970’s reporting the incidence of heart failure is significantly higher in diabetic (DM) patients compared to non-diabetic (nDM) patients. However the underlying mechanisms that link type 2 diabetes with heart failure are still unclear. Recent evidence has shown that diabetic patients and mouse models of diabetes have an up-regulation of calmodulin-dependent protein kinase delta (CaMKIIδ) activity. CaMKIIδ is a multifunctional serine-threonine kinase expressed as four different genes that generate four isoforms (αβγδ) and a number of splice variants. Within the myocardium δ is the predominant form, whilst a modest expression of γ has also been shown. Upon CaMKIIδ activation is involved in coordination of the ion channels and Ca2+ handling proteins involved in excitation-contraction and excitation-transcription coupling in the myocardium. Excessive CaMKIIδ activation in the myocardium has been linked with reduced myocardial performance due to disturbed Ca2+ handling. However this mechanism of reduced cardiac function and the involvement of CaMKIIδ has not been investigated in the diabetic heart. Therefore, this thesis set out to investigate the role of CaMKIIδ in the type 2 diabetic heart and the potential therapeutic role for CaMKIIδ inhibitors. To investigate whether CaMKIIδ activation is increased prior to systolic dysfunction in the type 2 diabetic heart western blots were carried out to measure the activation levels of CaMKIIδ in leptin-receptor-deficient 20-week-old male non-diabetic and type 2 diabetic Zucker diabetic fatty (ZDF) rats. Western blots confirmed that there was no change in expression of CaMKIIδ (0.95 ± 0.08 vs 1.15 ± 0.10, nDM (n=8) vs DM (n=8) but CaMKIIδ phosphorylation was significantly increased in the DM animals (0.77 ± 0.13 vs 1.40 ± 0.26) indicating that CaMKIIδ activation was increased in the type 2 diabetic ZDF heart prior to systolic dysfunction. The next step was to measure contraction and relaxation in the heart and the effects of CaMKIIδ inhibitors, KN93 and AIP. To do this small cardiac muscles (trabeculae) were isolated from the right ventricle free wall and stimulated at various frequencies. It was shown that the diabetic trabeculae had a reduced contractile force and speed of contraction and relaxation, but CaMKIIδ inhibition with both KN93 and AIP was able to reverse the impairments in contractility. To investigate the mechanisms behind the reduced contractility in the type 2 diabetic trabeculae and the effects of CaMKIIδ inhibition, cardiomyocytes were isolated from 18-19 week old male nDM (n=6) and DM (n=6) ZDF rats and loaded with Fluo-4AM and viewed under a confocal microscope to allow for the measurement of intracellular Ca2+ during and between transients. Measurements were completed in the presence of the CaMKIIδ inhibitors KN93 and AIP. It was observed that there was no alteration in Ca2+ homeostasis in the diabetic and non-diabetic cells during transients but it was altered between transients, as demonstrated by an increase in Ca2+ waves. CaMKIIδ inhibition with KN93 and AIP was able reduce this marker of pro-arrhythmic behaviour. To further interrogate the potential of CaMKIIδ inhibition as a therapeutic measure in the clinic the effects of chronic inhibition must be investigated. To do this a mouse model of type 2 diabetes (db/db) (n=8) alongside nDM control mice (n=11) were injected with KN93 (10 μmol/kg) every other day from the age of 14 to 18 weeks of age. At 18 weeks of age right ventricle tissue was taken for the measurement of cardiac fibrosis using Masson’s Trichrome stain. It was found that there was no difference in fibrotic remodelling between the DM and nDM right ventricular tissue (1.01% vs 1.05%), and also no effect of the CaMKIIδ inhibitor, KN93, on cardiac fibrosis (nDM= 1.24% vs DM= 0.93 %). Therefore indicating that chronic CaMKIIδ inhibition has no detrimental effects on inducing cardiac fibrosis in either the nDM or DM heart. Therefore the results from this study identify CaMKIIδ as a novel mediator of cardiac contractility in the type 2 diabetic heart, pre systolic dysfunction. The findings have suggested that altered Ca2+ handling may not be the only mechanism to attribute to for the reducing contractility in the diabetic heart, as Ca2+ homeostasis was not altered during transients. However pro-arrhythmic behaviour was increased in the isolated diabetic cardiac muscle and cardiomyocytes, which CaMKIIδ inhibition was able to reduce. Therefore providing evidence of the potential therapeutic role of CaMKIIδ, which can be built upon to further investigate its efficacy in the clinic