Short communication: ischemia/reperfusion tolerance is time-of-day-dependent: mediation by the cardiomyocyte circadian clock

Abstract

Rationale: Cardiovascular physiology and pathophysiology vary dramatically over the course of the day. For example, myocardial infarction onset occurs with greater incidence during the early morning hours in humans. However, whether myocardial infarction tolerance exhibits a time-of-day dependence is unknown.

Objective: To investigate whether time of day of an ischemic insult influences clinically relevant outcomes in mice.

Methods and results: Wild-type mice were subjected to ischemia/reperfusion (I/R) (45 minutes of ischemia followed by 1 day or 1 month of reperfusion) at distinct times of the day, using the closed-chest left anterior descending coronary artery occlusion model. Following 1 day of reperfusion, hearts subjected to ischemia at the sleep-to-wake transition (zeitgeber time [ZT]12) resulted in 3.5-fold increases in infarct size compared to hearts subjected to ischemia at the wake-to-sleep transition (ZT0). Following 1 month of reperfusion, prior ischemic event at ZT12 versus ZT0 resulted in significantly greater infarct volume, fibrosis, and adverse remodeling, as well as greater depression of contractile function. Genetic ablation of the cardiomyocyte circadian clock (termed cardiomyocyte-specific circadian clock mutant [CCM] mice) attenuated/abolished time-of-day variations in I/R outcomes observed in wild-type hearts. Investigation of Akt and glycogen synthase kinase-3beta in wild-type and CCM hearts identified these kinases as potential mechanistic ties between the cardiomyocyte circadian clock and I/R tolerance.

Conclusions: We expose a profound time-of-day dependence for I/R tolerance, which is mediated by the cardiomyocyte circadian clock. Further understanding of I/R tolerance rhythms will potentially provide novel insight regarding the etiology and treatment of ischemia-induced cardiac dysfunction.

Figure 1

bmal1 (A), dbp (B), and e4bp4 (C) mRNA diurnal variations in WT and CCM hearts. Data are shown as means ± SEM for 5–6 hearts per group.

Figure 2

Infarct size (A), volume (B), and fibrosis (C) are dependent on time-of-day of I/R. Infarct size was measured by TTC staining following one day of reperfusion (A). Following one month of reperfusion, H&E (B) and Sirius Red (C) staining was performed to assess infarct volume and fibrosis respectively. Sh: sham. Data are shown as means ± SEM for 6 (A) or 3 (B/C) hearts per group. *P<0.05 Sham vs. I/R within genotype and ZT; $P<0.05 WT vs. CCM within procedure and ZT; #P<0.05 ZT0 vs. ZT12 within procedure and genotype.

Figure 3

LVEDD (A), ejection fraction (B), and fractional shortening (C) are dependent on time-of-day of I/R. All measures were performed following one month of reperfusion. LVEDD was assessed by H&E staining. Ejection fraction and fractional shortening were assessed by echocardiography. Data are shown as means ± SEM for 3 (A) or 6 (B/C) hearts per group. *P<0.05 Sham vs. I/R within genotype and ZT; $P<0.05 WT vs. CCM within procedure and ZT; #P<0.05 ZT0 vs. ZT12 within procedure and genotype; §P=0.056 ZT0 vs. ZT12 within procedure and genotype; ‡P=0.058 ZT0 vs. ZT12 within procedure and genotype.

Figure 4

Akt (A) and GSK-3β (B) phosphoprotein diurnal variations in WT and CCM hearts (normalized to Ran-GTPase). Data are shown as means ± SEM for 5–6 hearts per group. Panels C and D illustrate relationships between P-Akt and P-GSK3β levels with infarct size (TCC assessment).