Sex differences in ischaemia reperfusion injury in the rat heart: role of specific chemokines and Toll-like receptors

Background & Aim: The incidence of acute myocardial infarction (AMI) in humans is significantly less in females compared to age-matched males. Animal models show that the injury caused by myocardial I/R (simulating AMI) is less in females compared to males. The response to I/R includes the activation and recruitment of circulating cells, initiated by several pathways including release of specific chemokines and stimulation of Toll-like receptors (TLRs). Whether disparate regulation of these pathways within the vasculature underlies sex-differences in myocardial injury is unknown.

Methods: Isolated hearts from male and female Wistar rats (8-10 week, 260-300g) were perfused in vitro with Krebs buffer (pH7.4, 37°C, gassed with 95% O2/5% CO2) at constant flow rate (10ml/min). Following equilibration (20min), hearts were subjected to 30min global ischaemia and 2h reperfusion or perfused for 2.5h (control hearts). Myocardial function was assessed by measuring the following parameters: coronary perfusion pressure (CPP), left ventricular developed pressure (LVDP), left ventricular end diastolic pressure (LVEDP) and heart rate. After reperfusion, the area of infarction was determined using nitroblue tetrazolium dye or the left ventricle was snap frozen for mRNA analysis. Real-time qPCR, using SYBR Green, was performed to quantify mRNA expression of TLR2, TLR4, and chemokines CCL2 (MCP1) and CX3CL1 (fractalkine). Level of mRNA was expressed relative to expression of 18S and compared to basal expression in non-infarcted male hearts.

Results: I/R-induced infarct size was smaller (P<0.01, n = 6) in female compared to male rats (36±0.9% vs. 47±0.6%, respectively). After I/R, LVDP was greater (at 2.5h: 51±2.3 mmHg vs. 31±2.5mmHg; P<0.05, n = 15) in hearts of female rats compared to male rats. Similarly, the rise in CPP and LVEDP following I/R was less (P<0.05, n = 15) in hearts of females (at 2.5h CPP: 131±3.0 mmHg and LVEDP: 15±1.7 mmHg) compared to males (CPP: 161±2.6 mmHg and LVEDP: 26±1.9 mmHg). Pre-ischaemic heart rate was significantly lower (P<0.05, n = 15) in hearts of female compared to male animals (277±6.6 bpm v. 300±6.8 bpm, respectively) but was not affected by I/R in either sex (P>0.05, n = 15). Data is expressed as mean±SEM and analyzed by a Student t-test.

There was no significant sex-difference in mRNA expression of CCL2, CX3CL1, TLR2, or TLR4 in control hearts (n = 7). I/R had no effect on expression of CCL2, CX3CL1 or TLR2 in female hearts but elevated (P<0.01, n = 9) expression in male hearts (9.6 ±0.6, 8.1±0.9 and 3.6±0.4-fold respectively). I/R elevated expression TLR4 in both sexes but the induction was (P<0.05) less in female hearts (male: 4.4 ±0.3 fold vs. female: 2.4 ± 0.1 fold). Data is expressed as mean±SEM and analyzed using one-way ANOVA.

Conclusion: This study shows that myocardial I/R causes substantially less injury and impairment of contractile function in hearts of female compared to male rats. This difference is associated with significantly less expression of CCL2, CX3CL1, TLR2 and TLR4, which are key regulators of myocardial inflammation and tissue injury. The disparate regulation of chemokines and TLRs between the sexes may contribute to decreased leukocyte recruitment and impairment of vascular function in female hearts.

SM is funded by RAB Barts & The London PhD studentship and RSS by a Wellcome Trust Career Development Fellowship.