Introduction: The healthy heart displays a circadian rhythm in which heart rate and blood pressure decrease overnight. However, this rhythm is blunted, absent or even reversed in the diabetic heart, which is a crucial risk factor for the development of cardiovascular disease. The origin of the circadian rhythm in the heart and its dysregulation, has not yet been fully elucidated, but changes in autonomic neural control have been implicated.

Objective: Determine changes in circadian cardiac autonomic responsiveness in diabetes.

Methods: We investigated autonomic regulation in 20-week old male Zucker type 2 Diabetic Fatty rats (DM) and their non-diabetic littermates (ND) at two timepoints, the start of the inactive and the active period (Zeitgeber times 3 and 15, respectively). Autonomic responsiveness (noradrenaline 0.5µM, acetylcholine 1µM) was assessed in the Langendorff-perfused isolated heart preparation, following pentobarbital anaesthesia (80mg/kg i.p.) (2-way RM ANOVA). Sinoatrial node (SAN) and left ventricle were dissected from naïve ND and DM ZDF for assessment of protein expression via Western Blot (2-way ANOVA). Brains, post-fixed in paraformaldehyde (4%), were immunostained for quantification of activated sympathetic cells (c-fos and tyrosine-hydroxylase co-expression) (2-way ANOVA). Data are presented as mean±SEM.

Results: We observed diurnal variation in cardiac sympathetic responsiveness (noradrenaline; ΔDevP ND: ZT3 25.7±13.4, ZT15 32.6±11.9; DM: ZT3 6.3±10.1, ZT15 32.5±7.9 ΔmmHg, n=8-9, p<0.05). Conversely, lower parasympathetic responsiveness was associated with diabetes (acetylcholine; ΔHR ND: ZT3 -93.5±21.6, ZT15 -88.0±15.4; DM: ZT3 -66.4±11.5, ZT15 -72.3±13.5 Δbpm, n=4-10, p<0.05). While no significant difference in the expression of cardiac beta-adrenergic (β₁, β₂) or muscarinic (M₂) autonomic receptors was found, circadian rhythms were observed in the expression of calcium handling proteins (SERCA2a, PLB), which were higher during the active period (ZT15). CLOCK protein levels were also lower in the SAN in DM (ND: ZT3 0.5±0.1, ZT15 0.6±0.1; DM: ZT3 0.4±0.1, ZT15 0.3±0.02, n=5-6, p<0.05). Activation of sympathetic cells was higher in DM in the nucleus tractus solitarius (p<0.001), and significantly higher both in DM and during the active period (ZT15) in the rostral ventrolateral medulla (p<0.001).

Conclusions: Physiological circadian signalling was primarily associated with sympathetic regulation, while we uncover changes in parasympathetic responsiveness in diabetes that might signal an underestimated therapeutic target. Increased activation of sympathoregulatory brain regions might contribute to sympathetic overactivation as well as disrupted circadian rhythms in diabetes.