Abstract
The cardiac non-neuronal cholinergic system (cNNCS) is a newly described system where the cardiomyocytes possess the ability to synthesise and release acetylcholine (ACh) that acts in an auto-/paracrine manner to induce cardioprotective effects. In this study, we determined the effects of type-1 diabetes mellitus (T1DM) on the expression of the cNNCS components namely choline acetyltransferase (ChAT), choline transporter-1 (CHT-1: rate-limiting step of ACh synthesis), vesicular acetylcholine transporter (VAChT), and acetylcholinesterase (AChE) which synthesise, release and degrade acetylcholine (ACh) respectively. Secondly, we determine the potential therapeutic effect of activation of the cNNCS in preventing T1DM induced cardiovascular dysfunction. T1DM was chemically induced by injection of low dose streptozotocin (STZ, 50mg/kg, i.p) over five consecutive days in C57Bl6 mice (T1DM; n=6) by our collaborator Prof Yoshihiko Kakinuma at Nippon Medical School, Japan. Age-matched healthy mice served as controls (ND; n=6). To determine the effects of activation of the cNNCS on T1DM, mice with cardiomyocyte-specific overexpression of the ChAT gene in the ventricles were injected with STZ to induce T1DM (ChAT-TG-T1DM; n=6). The western blotting analysis demonstrated that CHT-1 expression in T1DM mice at 16-weeks after STZ injection was significantly decreased. Interestingly, the western blotting analysis in T1DM mice compared to ND mice showed marked impairment in the downstream targets of pro-survival AKT pathway such as pAkt/Akt (activate mammalian target of rapamycin 1/2 to reduce fibrosis) and Bcl-2 (anti-apoptotic protein) at all time points. Further, evaluating apoptosis (determined by TUNEL positive nuclei) and fibrosis (determined by Picro-Sirius staining) showed a significant increase in apoptotic and fibrotic area at all time points. All these consequently resulted in T1DM-induced cardiac dysfunction, as reflected by the manifestation of both diastolic (reduced ejection fraction, increased end-diastolic volume) and systolic dysfunction (increased end-systolic volume). However, at the same time, western blot analysis in ChAT-TG-T1DM mice compared to T1DM mice resulted in a significant increase in CHT-1 along with an increase in pAkt and Bcl-2 expression. Moreover, apoptosis and fibrosis were significantly decreased in ChAT-TG-T1DM mice compared to T1DM mice preventing cardiac dysfunction. In conclusion, our results provide the first evidence that the cNNCS is altered in the T1DM heart and activation of the cNNCS prevented the alterations, to improve cardiac function and reduce apoptosis and fibrosis. Taken together, targeting cNNCS to increase the availability of ACh can be a potential therapeutic intervention, which will prevent the development of cardiac dysfunction in type 1 diabetic heart.