Understanding altered intrinsic heart rate in type 2 diabetes
Heart rate (HR) is generated by sinoatrial node (SAN) intrinsic pacemaking and modulated by autonomic innervation. Within the SAN, intrinsic (ex vivo) HR is determined by the mutual entrainment of the sarcolemmal voltage membrane (Vm) and intracellular Ca2+ clocks. The Vm clock involves membrane ion channels, such as the hyperpolarisation-activated cyclic nucleotide-gated channel 4 (HCN4), transient type (T-type) and long-lasting type (L-type) Ca2+ channels and the ion transporter Na+-Ca2+ exchanger 1 (NCX1). The Ca2+ clock primarily involves the intracellular Ca2+ store, the sarcoplasmic reticulum (SR), and the Ca2+ release protein the ryanodine receptor 2 (RyR2), the Ca2+ uptake protein the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) and its regulator phospholamban. Conduction of the AP within the SAN occurs via the coupling protein connexin 45 (cx45). Additionally, the presence of the non-neuronal cardiac intrinsic cholinergic system within cardiomyocytes suggests it might also be present in the SAN cardiomyocytes and have the capacity to modulate intrinsic HR. Disruption of HR control occurs in patients and animal models with type 2 diabetes (DM). Interestingly, in the DM Zucker Diabetic Fatty (ZDF) rats, intrinsic HR was significantly decreased compared to non-diabetic (nDM) controls. This suggests DM impairs the intrinsic ability of the SAN to generate a normal HR. Therefore, the overall aim of this research was to investigate whether the decreased intrinsic HR in DM was due to changes in the Vm and / or Ca2+ clocks, cx45 and / or increased non-neuronal intrinsic cholinergic system activity. The SAN / hearts of 19 – 22 week-old nDM and DM ZDF rats were used to investigate protein expression of the key SAN clock, cx45 and cholinergic proteins via western blotting, intrinsic HR contributions from HCN4, SERCA2a and muscarinic type 2 (M2) receptor via Langendorff, and SAN cellular / tissue morphology via immunohistochemistry. For the Vm clock, a significant increase in HCN4 (nDM 0.83 ± 0.07 versus DM 1.67 ± 0.19, p<0.05) and NCX1 (nDM 1.74 ± 0.32 versus DM 3.83 ± 0.81, p<0.05) protein expression was found in DM. For the Ca2+ clock, a significant increase in phospholamban (nDM 0.96 ± 0.06 versus DM 1.51 ± 0.18, p<0.05), with no change to SERCA2a protein expression (nDM 2.77 ± 0.55 versus DM 3.33 ± 0.38, p>0.05) or SERCA2a to phospholamban ratio (nDM 2.97 ± 0.68 versus DM 2.37 ± 0.34, p>0.05) was found in DM. A significant increase in the M2 receptor expression (nDM 1.14 ± 0.18 versus DM 3.14 ± 0.80, p<0.01) was also found in DM. The functional effects on intrinsic HR were investigated by increasing ivabradine (HCN4 inhibitor), external Ca2+ ([Ca2+]o) and carbachol (M2 stimulus) to challenge HCN4 and SERCA2a, and determine cholinergic responsiveness respectively in DM. Ivabradine reduced intrinsic HR in nDM but not DM (interaction p<0.05), [Ca2+]o decreased intrinsic HR in DM but not nDM (interaction p<0.05), and carbachol decreased intrinsic HR in nDM and DM to equal measure (interaction p>0.05). For immunohistochemistry, no difference in cellular / tissue distribution of key SAN clock, cx45 or cholinergic proteins was observed, or in the levels of fibrosis (p>0.05) and fat (p>0.05) within the DM SAN. Collectively, this study presents novel mechanisms that are altered in pacemaking in the type 2 DM SAN. From this research, I conclude, the lower intrinsic HR in DM is, in part, a result of changes to both the Vm and Ca2+ clock due to non-functional HCN4 channels and compromised SERCA2a activity that would prolong diastolic depolarisation and repolarisation respectively.
Advisor: Jones, Peter; Lamberts, Regis
Degree Name: Doctor of Philosophy
Degree Discipline: Physiology
Publisher: University of Otago
Keywords: Type 2 diabetes; Sinoatrial node; Intrinsic heart rate; Voltage membrane clock; Calcium clock
Research Type: Thesis