Attenuation of myocardial injury in mice with functional deletion of the circadian rhythm gene mPer2

Abstract

Variations in circadian rhythms are evident in the incidence of cardiovascular disease, and the risk of cardiovascular events increases when rhythms are disrupted. The suprachiasmatic nucleus is the central circadian pacemaker that regulates the daily rhythm of peripheral organs. Diurnal rhythms have more recently been shown to exist in myocardial tissue and are involved in metabolism and contractile function. Thus we sought to determine whether the functional deletion of the circadian rhythm mouse periodic gene 2 (mPer2) would protect the heart against ischemic injury. Nonreperfused myocardial infarction was induced in anesthetized, ventilated C57 (n = 17) and mPer2 mutant (mPer2-M; n = 15) mice via permanent ligation of the left anterior descending coronary artery. At 4 days post-myocardial infarction, we observed a 43% reduction of infarct area in mPer2-M mice compared with wild-type mice. This is coincident with 25% less macrophage infiltration, 43% higher capillary density, 17% increase in hypertrophy, and 15% less cardiomyocyte apoptosis in the infarct zone. Also, matrix metalloproteinase-9 was expressed in inflammatory cells in both groups, but total protein was 40% higher in wild-type mice, whereas it was not elevated in mPer2-M mice in response to injury. The functional deletion of the mPer2 gene reduces the severity of myocardial infarct injury by limiting the inflammatory response, reducing apoptosis, and inducing cardiomyocyte hypertrophy, thus preserving cardiac function. These findings collectively imply that the disruption of the circadian clock gene mPer2 is protective. Understanding the interactions between circadian rhythm genes and cardiovascular disease may provide insights into potential preventative and therapeutic strategies for susceptible populations.

Fig. 1.

Mouse periodic gene 2-mutant (mPer2-M) hearts have reduced infarct area at 4 days post-myocardial infarction (post-MI). Left: representative histology of wild-type (WT; top, left) and mPer2-M (top, right) control mouse hearts and WT (bottom, left) and mPer2-M (bottom, right) hearts 4 days after chronic MI. Infarct area (in mm²) was 43% smaller in mPer2-M hearts 4 days post-MI (P < 0.05). Arrows point to granulation tissue; asterisks indicate necrosis. RV, right ventricle; LV, left ventricle. Right: *P < 0.05.

Fig. 2.

Right: reduced cardiomyocyte apoptosis (A) and increased myocyte cross-sectional area (B) in mPer2-M mouse hearts 4 days after chronic infarction are shown. Left: there were 15% less terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL)-positive apoptotic cardiomyocytes observed in mPer2-M mice (top, right) compared with WT (top, left; *P < 0.05). The average myocyte cross-sectional area (in μm²; †P < 0.001) was increased in mPer2-M mice (bottom, right) vs. WT (bottom, left).

Fig. 3.

Pan-leukocyte marker CD45 staining showed reduced CD45-positive inflammatory cells in mPer2-M mice (A) compared with WT mice (B) at 4 days after infarction (C; *P < 0.05). KO, knockout. There was a significant increase in vessel density in mPer2-M (E) hearts compared with WT hearts (D) at 4 days post-MI (F; †P < 0.01). Fibroblast density was also increased in mPer2-M (H) compared with WT (G) mouse hearts at 4 days post-MI (I; *P < 0.05).

Fig. 4.

Matrix metalloproteinase-9 (MMP-9) expression in inflammatory cells is reduced in mPer2-M hearts 4 days (4D) after chronic infarction. MMP-9 is expressed in inflammatory cells in mPer2-M hearts (right) compared with WT (left) at 4 days after infarction. A representative Western blot (bottom) for MMP-9 shows that there is no difference between uninjured WT and mPer2-M hearts. In contrast to the increased expression observed in WT hearts at 4 days post-MI, there is no change in MMP-9 expression in mPer2-M hearts at 4 days post-MI.

Fig. 5.

mPer2-M mice showed improved LV pressure, end-systolic pressure-volume relationship (ESPVR), and preload recruitable stroke work (PRSW) 4 days after infarction. ESPVR: slope of the best fit line through the end-systolic pressure-volume points in the series of pressure-volume loops. With linear fit, all slopes have regressions r > 0.94. PRSW: r > 0.95 for each of the stroke work/end-diastolic volume slopes.