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 (MI) onset occurs with greater incidence during the early morning hours in humans. However, whether MI 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 (WT) mice were subjected to ischemia/reperfusion (I/R; 45 minutes ischemia followed by one day or one month reperfusion) at distinct times of the day, using the closed-chest left anterior descending coronary artery occlusion model. Following one day of reperfusion, hearts subjected to ischemia at the sleep-to-wake transition (ZT12) resulted in 3.5-fold increases in infarct size compared to hearts subjected to ischemia at the wake-to-sleep transition (ZT0). Following one 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 CCM mice) attenuated/abolished time-of-day variations in I/R outcomes observed in WT hearts. Investigation of Akt and GSK-3β in WT 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.

Introduction

Numerous aspects of cardiovascular physiology and pathophysiology demonstrate circadian rhythms.¹ In humans, heart rate, blood pressure, and cardiac output all increase in the early hours of the morning, as does the onset of adverse cardiac events, such as myocardial infarction (MI).^{2, 3} These rhythms have been attributed primarily to time-of-day oscillations in neurohumoral influences, such as sympathetic or autonomic stimulation.^{3, 4} While extracardiac factors undoubtedly play critical roles in modulation of cardiovascular function/dysfunction, increasing evidence suggests that intrinsic factors, such as cell-autonomous circadian clocks, likely contribute.¹

Circadian clocks are transcriptionally-based molecular mechanisms, composed of positive and negative feedback loops, with a free-running period of approximately 24 hours.⁵ This mechanism allows the cell to anticipate alterations in environmental stimuli, through time-of-day-dependent modulation of cellular responsiveness to extrinsic factors.⁵ Circadian clocks have been identified/characterized in multiple cardiovascular-relevant cell types, including cardiomyocytes, vascular smooth muscle cells, and endothelial cells.^6–8 Ubiquitous genetic ablation of circadian clock function markedly influences multiple cardiovascular parameters, including heart rate and blood pressure.⁹ We have recently utilized a Cardiomyocyte-specific circadian Clock Mutant (CCM) mouse to reveal regulation of myocardial gene expression, β-adrenergic responsiveness, metabolism, heart rate, and cardiac power by this mechanism.^{10, 11}

Although circadian rhythms in MI onset are well established, time-of-day oscillations in myocardial ischemia/reperfusion (I/R) tolerance have not been reported. Given that the cardiomyocyte circadian clock influences myocardial physiology^{1, 11}, and that this mechanism becomes rapidly inactivated following an ischemic event¹², we hypothesized that the cardiomyocyte circadian clock modulates I/R tolerance in a time-of-day-dependent manner.

Methods

Wild-type (WT) and CCM mice were housed under controlled conditions in a 12-hr light/12-hr dark cycle (lights on at ZT0, lights off at ZT12). The closed-chest left anterior descending coronary artery occlusion model of I/R was utilized (45 minutes ischemia); sham animals were subjected to identical surgical procedures, without coronary occlusion.¹³ Following one day of reperfusion, TTC staining was performed for measurement of infarct size; area at risk was determined by Evan's Blue staining. Echocardiographic (M-mode) assessment of cardiac function and histological analyses were performed following one month of reperfusion. Hearts from a separate set of mice not having undergone surgical intervention were isolated at distinct times of the day for gene and protein expression analyses. See Supplemental Material for detailed methods.

Results

Consistent with loss of cardiomyocyte circadian clock function, diurnal oscillations in clock (bmal1; Fig 1A) and clock output (dbp, e4bp4; Fig 1B/C) gene expression were significantly attenuated in CCM hearts.

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.

Following one day of reperfusion, WT hearts subjected to I/R at ZT12 exhibited an infarct size 3.5-fold greater than at ZT0 (p<0.05; Fig 2A and Online Fig 1A). ZT12 infarct size was approximately 21%, consistent with previous studies utilizing the murine closed-chest model.¹³ Area at risk did not vary between groups (Online Fig 1B). Following identification of the peak and trough in infarct size (i.e., ZT12 and ZT0, respectively), these two time points were utilized for long-term studies (i.e., one month reperfusion).

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.

To investigate whether observed diurnal variations in infarct size following one day reperfusion were associated with differences in long-term remodeling, hearts were studied following one month of reperfusion. As predicted, infarct volume and fibrosis increased in WT hearts following I/R at both ZT0 and ZT12 (relative to respective shams; Fig 2B/C and Online Fig 1A). However, these parameters were significantly greater when the ischemic insult was performed at ZT12 versus ZT0. Similarly, histological analysis revealed increased left ventricular end diastolic diameter (LVEDD; a marker of adverse remodeling) only for WT hearts subjected to I/R at ZT12 (Fig 3A). LVEDD measures were reiterated by echocardiographic assessment of contractile function; while all I/R groups exhibited decreased ejection fraction and fractional shortening compared to their sham counterparts, WT hearts that underwent I/R at ZT12 exhibited the greatest depression of contractile function (Fig 3B/C).

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.

Time-of-day-dependent oscillations in I/R tolerance in vivo could be secondary to extrinsic (e.g., neurohumoral factors) and/or intrinsic (e.g., circadian clocks) influences.¹ To investigate the role of the cardiomyocyte circadian clock, CCM mice were utilized. Consistent with temporal suspension of CCM hearts at the dark-to-light phase transition (i.e., ZT0; Fig 1 and reference 11), infarct size in CCM hearts (following one day reperfusion) was similar to that observed for WT hearts at ZT0, independent of the time-of-day (Fig 2A). Absence of time-of-day-dependence in infarct size for CCM hearts was accompanied by attenuation/abolition of diurnal variations in infarct volume, fibrosis, remodeling, and contractile dysfunction following one month reperfusion (Fig 2/3).

Previously published studies in extracardiac tissues suggest that GSK-3β, whose phosphorylation status oscillates over the course of the day in liver, is an integral circadian clock component.¹⁴ Given the known central roles of Akt and GSK-3β in modulating I/R-induced myocardial damage^{15, 16}, we investigated Akt and GSK-3β in WT and CCM hearts. Figure 4A/B shows that the phosphorylation of Akt and GSK-3β oscillate in WT hearts (e.g., 1.8-fold oscillation for PGSK-3β, peaking at ZT0; p<0.05), and that phosphorylation is chronically elevated in CCM hearts. In contrast, total Akt and GSK-3β levels (as well as gsk-3β mRNA) are not different between groups (Online Fig 2/3). Importantly, negative correlations were observed for P-Akt and P-GSK3β levels with infarct size (Fig 4C/D). In contrast, phosphorylation of p70S6 (downstream of Akt) did not correlate with infarct size (Online Fig 4).

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).

Discussion

The purpose of the present study was to determine whether I/R tolerance varies over the course of the day, and if so, to investigate the potential mediation by the cardiomyocyte circadian clock. Here, we expose dramatic time-of-day-dependent variations in I/R tolerance, which are lost following genetic ablation of the cardiomyocyte circadian clock. Initial interrogation of possible molecular links between the cardiomyocyte circadian clock and myocardial I/R tolerance suggest that Akt and/or GSK-3β are potential mediators.

Diurnal variations in the onset of myocardial infarction are well established.³ Humans are at greatest risk for MI in the early hours of the morning, due to a combination of increased shear stress, sympathetic tone, and prothrombolytic factors at this time.^{1, 3} However, whether the myocardium exhibits diurnal variations in I/R tolerance has not been reported. The brain exhibits a time-of-day-dependence in ischemic tolerance.¹⁷ Rats subjected to an ischemic episode at different times of the day exhibit greatest infarct sizes at ZT14, similar to our observations in the mouse heart (greatest infarct size at ZT12; Fig 2). ZT12 corresponds to the sleep-to-wake transition in the nocturnal rodent. As such, diurnal oscillations in the stimulus (i.e., ischemia) and responsiveness (i.e., infarct development) are in phase.

Circadian dyssynchronization is classically associated with cardiovascular morbidity and mortality. In humans, shift work significantly increases risk for cardiovascular disease development.¹⁸ Similarly, subjecting cardiomyopathic hamsters to light/dark cycle manipulations augments early mortality.¹⁹ Genetic modulation of circadian clock timing, resulting in subtle circadian dyssychronization accelerates cardiac and renal disease, which is rescued by light/dark cycle-mediated circadian resynchronization.²⁰ In the present study, genetic ablation of the cardiomyocyte circadian clock results in cardioprotection. The apparent discrepancy is likely due to the observation that CCM hearts are temporally suspended at the wake-to-sleep (dark-to-light) phase transition¹¹ (Fig 1), a time of the day at which WT hearts exhibit greatest I/R tolerance. CCM mice are therefore distinct from models wherein circadian dyssynchronization occurs following classic shift work paradigms.

In summary, the current study reveals profound time-of-day-dependence in myocardial I/R tolerance, mediated by the cardiomyocyte circadian clock. Rhythms in I/R-induced myocardial damage/dysfunction are in phase with established rhythms of MI onset. The implications of these observations range from benchside (e.g., time of day consideration for experiments) to bedside (e.g., chronotherapy).

Summary of Significance and Novelty.

The onset of a myocardial infarction exhibits a marked time-of-day-dependence in humans, peaking in the early hours of the morning (i.e., upon awakening). However, what is less clear is whether the time-of-day at which an ischemic event occurs influences the severity of the insult. We therefore investigated whether a normal mouse heart exhibits a time-of-day-dependence in ischemia/reperfusion tolerance. We report that a 3.5-fold difference in the extent of damage (i.e., infarct size) is observed, depending on the time-of-day at which the ischemic event occurs. Poorest tolerance to ischemia/reperfusion is observed at the sleep-to-wake transition. Disruption of the molecular clock specifically within the cardiomyocytes of mice completely abolishes time-of-day oscillations in ischemia/reperfusion tolerance. Our studies therefore clearly show, for the first time, that the heart exhibits a time-of-day-dependence in ischemia/reperfusion tolerance, which is mediated by the cardiomyocyte circadian clock. Poorest tolerance to an ischemic event is at a time-of-day at which myocardial infarctions occur with greatest incidence in humans. Modulation of the cardiomyocyte circadian clock may therefore prove to be a prudent candidate to target for improving ischemia/reperfusion tolerance.

What is known.

• The onset of a myocardial infarction exhibits a marked time-of-day-dependence in humans, peaking in the early hours of the morning (i.e., upon awakening).

• Factors external to the heart (e.g., pro-thrombolytic factors) correlate with time-ofday-dependence in human myocardial infarction onset.

• A molecular mechanism known as the circadian clock has been identified and characterized within human and rodent hearts.

What this article contributes.

• A normal mouse heart exhibits a dramatic time-of-day-dependence in ischemia/reperfusion tolerance.

• Worst ischemia/reperfusion tolerance is observed upon awaking for the rodent (i.e., poorest ischemia/reperfusion tolerance is at the time-of-day for which myocardial infarction incidence is greatest).

• The circadian clock within the cardiomyocyte mediates time-of-day-dependence in cardiac ischemia/reperfusion tolerance.

Non-standard Abbreviations and Acronyms

Akt

v-akt murine thymoma viral oncogene

CCM

Cardiomyocyte-specific circadian clock mutant

E4BP4

E4 promoter binding-protein 4

GAPDH

Glyceraldehyde 3-phoshate dehydrogenase

GSK-3β

Glycogen synthase kinase 3β

I/R

Ischemia/reperfusion

LVEDD

Left ventricular end diastolic dimensions

MI

Myocardial infarction

p70S6K

Ribosomal protein S6 kinase, 70 kDa

TTC

2,3,5-Triphenyltetrazolium chloride

WT

Wild-type

ZT

Zeitgeber time