Gender differences in cardiac function during early remodeling after acute myocardial infarction in mice

Life Sciences 75 (2004) 2181 – 2192 www.elsevier.com/locate/lifescie

Gender differences in cardiac function during early remodeling after acute myocardial infarction in mice Maria A. Cavasin, Zhenyin Tao, Shreevidya Menon, Xiao-Ping Yang* Hypertension and Vascular Research Division, Henry Ford Health System, Henry Ford Hospital, 2799 West Grand Blvd, Detroit, MI 48202, USA Received 4 February 2004; accepted 9 April 2004

Abstract There are conflicting data about gender differences in cardiac function after myocardial infarction (MI), including cardiac rupture and mortality. Using a mouse model of MI, we recently found that the cardiac rupture rate during the first week after MI was significantly lower in females than in males, suggesting that females have attenuated structural remodeling. Thus in this study, we attempted to determine whether: a) females have attenuated remodeling and faster healing during the early phase post-MI, and b) females have better cardiac function and outcome during the chronic phase compared to males. MI was induced in 12-week-old male and female C57BL/6J mice. Signs of early remodeling, including cardiac rupture, infarct expansion, inflammatory response, and collagen deposition, were studied during the first 2 weeks post-MI. Left ventricular remodeling and function were followed for 12 weeks post-MI. We found that males had a higher rate of cardiac rupture, occurring mainly at 3 to 5 days of MI and associated with a higher infarct expansion index. Neutrophil infiltration at the infarct border was more pronounced in males than females during the first days of MI, which were also characterized by increased MMP activity. However, the number of infiltrating macrophages was significantly higher in females at day 4. During the chronic phase post-MI, males had significantly poorer LV function, more prominent dilatation and significant myocyte hypertrophy compared to females. In conclusion, males have delayed myocardial healing, resulting in cardiac rupture, and the survivors have poorer cardiac function and pronounced maladaptive remodeling, whereas females show a better outcome during the development of HF. D 2004 Elsevier Inc. All rights reserved. Keywords: Heart failure; Cardiac function; Rupture; Remodeling

* Corresponding author. Tel.: +1 313 916 2103; fax: +1 313 916 1479. E-mail address: [email protected] (X.-P. Yang). 0024-3205/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2004.04.024

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Introduction It is well known that premenopausal women have a lower risk of cardiovascular disease than men of the same age; however, this advantage disappears after menopause. There are conflicting data about gender differences after myocardial infarction, even after adjusting for age and other risk factors. Some studies showed better survival in women (Brett and Madans, 1995; Heer et al., 2002), whereas others reported higher mortality in women due to more severe MI (Marrugat et al., 1998). With regard to the adaptive response of the heart during the development of heart failure, previous animal studies suggested the possibility of gender differences in cardiac and myocyte remodeling (Gardner et al., 2002; Tamura et al., 1999); however, the mechanisms by which genderrelated hormones may affect remodeling and evolution post-MI remain unclear. It is crucial to identify potential gender-related differences in young individuals whose ovaries are still functioning. This would be impossible in humans because young women rarely have heart attacks. The use of experimental animals to study gender-related differences after MI has the great advantage of normalizing age and risk factors. We previously showed that cardiac rupture during the first week after MI was significantly higher in male than in female mice (Cavasin et al., 2000), suggesting that females have attenuated structural remodeling and faster healing during the acute phase post-MI, and this may result in a better outcome compared to males. Therefore, we attempted to determine whether: a) females have attenuated remodeling and faster healing during the early phase post-MI, and b) females have better cardiac function and outcome during the chronic phase compared to males.

Methods Animals Male and female C57BL/6J mice 12 weeks of age were obtained from Jackson Laboratories (Bar Harbor, ME). Animals were housed in an air-conditioned room with a 12-hour dark/light cycle and given standard mouse chow with free access to tap water. All surgical procedures were conducted under sodium pentobarbital anesthesia (50 mg/kg i.p.). The study was approved by the Institutional Animal Care and Use Committee (IACUC) of Henry Ford Health System. Induction of myocardial infarction MI was induced by coronary artery ligation as described previously (Cavasin et al., 2000; Yang et al., 2001). Briefly, a left thoracotomy was performed via the fourth intercostal space; the lungs were retracted to expose the heart, the pericardium was opened and the left anterior descending coronary artery (LAD) was ligated with 8-0 silk. Acute myocardial ischemia was considered successful when the anterior wall of the LV turned pale and obvious ST segment elevation was observed on the electrocardiogram. Sham MI surgery was performed as described above, passing the suture without ligating the artery. Mice were kept on a heating pad until they recovered.

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Acute phase after MI. Experimental protocol a) To study gender differences during the acute phase after MI, mice of both genders were randomly killed at 1, 2, 4, 7, and 14 days after LAD ligation. Half were used to measure infarct size, infarct expansion index (IEI), inflammatory cell infiltration at the infarct border and hydroxyproline content; the other half was used to determine matrix metalloproteinase-2 (MMP-2) and matrix metalloproteinase-9 (MMP-9) activity by zymography. Hearts were removed and weighed. The LV was sectioned transversely into 4 slices, 3 of which were rapidly frozen in isopentane and stored at
70 8C for determination of MI size, IEI and infiltrating inflammatory cells, and the remaining piece was used to assess collagen content by hydroxyproline assay. To calculate MI size and IEI, 6-Am sections were stained with Gomori trichrome. The circumference of the entire endocardium and epicardium, thickness and length of the infarcted portion as well as area of the LV cavity and total LV were determined using computer-assisted planimetry (Sigma Scan, Jandel). The infarcted portion of the LV was expressed as a percentage of the total circumference (Liu et al., 1997a). IEI, a parameter that assesses acute dilatation and thinning of the infarct, was calculated as follows: IEI ¼

LV cavity area Non-infarct septal wall thickness  Total LV area Infarct LV free wall thickness

Measurement of infiltrating neutrophils and macrophages A 6-Am section from each slice was stained with hematoxylin and eosin. Four points at the infarct border were observed under a light microscope (Nikon Labophot; 400), and neutrophils and macrophages were counted in each section. Average numbers were calculated using data from all three slices. Neutrophils are distinguished from macrophages based on their morphological characteristics (Junqueira and Carneiro, 1983; Madri, 1990). In general, neutrophils are 6–8 Am in diameter with abundant pink cytoplasm and a blue nucleus consisting of 2–5 sausage-shaped lobes on H & E staining. Macrophages are oval and measure 9–12 Am in diameter, with dark pink cytoplasm. The nucleus of macrophages is oval or kidney-shaped and generally eccentrically placed. Hydroxyproline assay Collagen content of the LV was determined by hydroxyproline assay (Cleutjens et al., 1995). The entire LV sample was used, including the infarcted portion. Tissue was dried and weighed, then homogenized in 0.1 mol/l NaCl and 5 mmol/l NaHCO3, washed 5 times with the same solution, and hydrolyzed with 6 N HCl for 16 hours at 1108C. Samples were filtered and vacuum-dried, then dissolved in distilled water. Hydroxyproline content was determined using a colorimetric assay and a standard curve of 0 to 5 Ag hydroxyproline. Data were expressed as Ag collagen per mg dry weight, assuming that collagen contains an average of 13.5% hydroxyproline. Zymographic assay The LV free wall, including the infarct, was homogenized in 400 Al of an ice-extraction buffer containing cacodylic acid (10 mM), NaCl (0.15 M), ZnCl (20 mM), NaN3 (1.5 mM), 0.01% Triton X-

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100 ( pH 5.0) and proteinase inhibitor (Complete, Roche) for zymography of MMP-2 and MMP-9 (Spinale et al., 1998). Homogenates were centrifuged for 10 min at 48C and 14,000 rpm, and protein concentration in the supernatant was determined by a standard protein assay. An aliquot containing 60 Ag protein was diluted in 4 sample buffer, loaded onto a 10% zymographic gel (Invitrogen) and run at 100V for 90 min at room temperature. Gels were incubated in renaturing buffer (2.5% Triton X-100) for 30 min and washed in dH2O for 10 min and developing buffer (50 mM Tris pH 8.0, 5 mM CaCl2, 0.2 M NaCl, 0.02% Brij-35) for 30 min, then changed to fresh developing buffer for 16 hours at 378C. The developing buffer was discarded and gels stained with 0.5% Coomassie blue solution (5% acetic acid, 10% methanol) for 30–60 min, then de-stained with 5% acetic acid and 10% methanol until sharp bands were visualized. 1 ng human MMP-2 and MMP-9 was used as a positive control. The gels were scanned with an imaging densitometer (Bio-Rad, Model GS-670) and digitized by Molecular Analyst software (Bio-Rad). Chronic phase after MI. Experimental protocol b) To study gender differences during the chronic phase of MI, another group of mice of both genders was followed for 12 weeks after induction of MI. Cardiac function, LV dimensions, systolic blood pressure (SBP), myocyte cross-sectional area and collagen fraction were assessed. Cardiac function and systolic blood pressure Transthoracic echocardiography was performed in conscious mice before MI was induced and 4, 8 and 12 weeks after MI as described previously (Yang et al., 1999). LV shortening fraction (SF), ejection fraction (EF), and systolic and diastolic dimensions (LVDs and LVDd) were calculated from the echocardiograms. SBP was measured at the end of the study in awake mice, using a noninvasive computerized tail-cuff system (Visitech BP-2000, Apex, NC) (Alfie et al., 1997). After echocardiography, mice were anesthetized and the hearts removed and weighed. The LV was sectioned transversely into 3 slices, rapidly frozen in isopentane and stored at
708C for determination of interstitial collagen fraction (ICF) and cardiomyocyte cross-sectional area (MCSA) as described previously (Liu et al., 1997a; Liu et al., 1997b).

Data analysis Data are expressed as mean F SE. Mortality and cardiac rupture rate were analyzed using a chi-square test. All other parameters were analyzed using StudentTs t-test. p b 0.05 was considered significant.

Results Acute phase post-MI Mortality during the first week post-MI was significantly higher in males than in females. The major cause of death was cardiac rupture, which generally occurred 3–5 days after MI (Fig. 1A); thereafter mortality was low and similar in both genders.

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Fig. 1. A) Mortality and cardiac rupture during the first week after MI. Black bars overlapping open bars represent only the percentage of deaths due to cardiac rupture, compared to the total number of deaths. B) Infarct expansion index during the first two weeks post-MI.

Infarct expansion and inflammatory cell infiltration at the infarct border IEI was measured at 1, 2, 4, 7 and 14 days after MI (Fig. 1B). Males tended to have a higher IEI than females, though it did not reach significance. Fig. 2 shows the number of neutrophils and macrophages infiltrating the infarct border at 1, 2, 4, 7 and 14 days after MI. Males had a significantly higher number of neutrophils than females at day 1 and a significantly lower number of macrophages at day 4. MMP activity and collagen content in the total LV Table 1 shows MI size, MMP-2 and MMP-9 activity and LV collagen content measured by the hydroxyproline method at 1, 2, 4, 7 and 14 days after MI in both genders. Infarct size and collagen content were similar in males and females at each time point. Both MMP-2 and MMP-9 activity were slightly higher in males than in females at all time points, but only MMP-2 reached statistical significance by 7 days.

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Fig. 2. Infiltrating neutrophils (top) and macrophages (bottom) at the infarct border during the first two weeks post-MI.

Chronic phase post-MI Cardiac morphology and SBP Table 2 shows body weight, SBP, cardiac morphology, and MI size in females and males with and without MI. In non-infarcted controls, males and females had similar values for SBP and atrial, LV and heart weight corrected for body weight. In both genders, MI significantly increased atrial, LV and heart weight compared to non-infarcted controls; however, SBP only decreased significantly in females. There were no significant differences in atrial, LV and heart weight between males and females with MI. EF, SF and LV dimensions After MI, EF and SF decreased significantly and remained low thereafter in both genders; however, this decrease was significantly more pronounced in males than in females (Fig. 3). In both genders, LVDs and LVDd increased significantly after MI; however, post-MI LV dimensions were significantly

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Table 1 MI Size, MMP-2 and MMP-9 Activity and LV Collagen Content at 1, 2, 4, 7 and 14 Days after MI Days after MI Sham MI 1 day 2 days 4 days 7 days 14 days

females (n = 8) males (n = 8) females (n = 6) males (n = 6) females (n = 7) males (n = 7) females (n = 6) males (n = 6) females (n = 7) males (n = 7) females (n = 5) males (n = 5)

MI size (%)

MMP-2 (Arbitrary Units)

MMP-9 (Arbitrary Units)

Collagen (mg/g dry LV)

NA NA 43.52 38.57 42.57 39.55 41.37 37.33 38.64 42.45 36.71 40.20

0.130 0.075 0.145 0.203 0.269 0.327 0.707 0.783 0.742 1.042 0.763 0.730

0.180 0.122 0.511 0.739 0.778 0.762 0.596 0.671 0.246 0.306 0.206 0.168

7.08 F 7.24 F 8.35 F 7.93 F 8.10 F 6.99 F 11.24 F 11.31 F 19.87 F 20.67 F 22.92 F 29.70 F

F F F F F F F F F F

2.98 3.10 3.81 2.72 3.38 3.30 2.72 3.84 2.52 2.67

F F F F F F F F F F F F

0.04 0.02 0.03 0.04 0.03 0.07 0.11 0.28 0.08 0.15* 0.10 0.11

F F F F F F F F F F F F

0.05 0.02 0.07 0.17 0.03 0.08 0.10 0.13 0.07 0.12 0.05 0.04

0.49 0.37 0.47 0.15 0.56 0.73 0.70 1.20 2.58 1.29 3.08 4.67

MI, myocardial infarction; MMP, matrix metalloproteinase; LV, left ventricle. * p b 0.05 vs females.

smaller in females than in males (Fig. 4). There were no differences in EF, SF, LVDs or LVDd between non-infarcted males and females at any time point. Myocyte size and interstitial fibrosis after MI MI significantly increased MCSA and ICF in both genders (Fig. 5). Although both myocyte hypertrophy and interstitial collagen deposition were more pronounced in males, only MSCA was significantly higher in males than in females. There were no gender-related differences in MCSA and ICF among non-infarcted controls.

Discussion A growing body of basic and clinical data points to fundamental gender-related differences in myocardial adaptation, hypertrophy, and performance after MI. Using a mouse model of heart failure Table 2 Body Weight, Systolic Blood Pressure, Cardiac Morphology, and Infarct Size 12 weeks after MI Parameters

FEMALES Sham MI (n = 6)

MALES Sham MI (n = 7)

FEMALES MI (n = 13)

BW (g) SBP (mm Hg) Atria/BW (mg/10g) RV/BW (mg/10g) LV/BW (mg/10g) HW/BW (mg/10g) MI size ( % )

21.6 F 0.7 112.4 F 5.0 4.33 F 0.31 7.84 F 0.27 30.31 F 1.54 42.47 F 1.94 NA

31.9 F 1.1 116.9 F 4.4 3.20 F 0.42 7.38 F 0.45 27.24 F 0.74 37.81 F 0.86 NA

22.9 F 0.2 94.5 F 4.6** 5.71 F 0.36z 9.74 F 0.30z 41.56 F 1.37z 58.69 F 1.73z 31 F 2

MALES MI (n = 10) 28.5 F 0.5 113.6 F 2.8* 5.16 F 0.44z 10.00 F 0.42z 39.16 F 1.94z 54.32 F 2.74z 32 F 3

MI, myocardial infarction; BW, body weight; RV, right ventricle; LV, left ventricle; HW, heart weight. zp b 0.001 and ** p b 0.01 vs sham MI; * p b 0.05 vs MI females.

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Fig. 3. Time course of ejection fraction (EF, left) and shortening fraction (SF, right) in mice with and without MI. zp b 0.01.

secondary to MI, we found that 1) early mortality due to cardiac rupture is significantly higher in males than in females, and this is accompanied by a tendency toward a higher infarct expansion index and increased MMP activity; 2) males have a greater number of neutrophils infiltrating the infarct border at day 1 and a smaller number of macrophages at day 4 than females; and 3) after the onset of heart failure, males have significantly worse LV performance, greater chamber dilatation and more pronounced myocyte hypertrophy compared to females.

Fig. 4. Time course of systolic (LVDs, left) and diastolic left ventricular dimension (LVDd, right) in mice with and without MI. zp b 0.01.

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Fig. 5. Myocyte cross-sectional area (MCSA, left) and interstitial collagen fraction (ICF, right) measured at the end of the study in mice with and without MI.

Despite progressive reduction in short-term mortality from acute MI over the last 30 years, ventricular free wall rupture is still a common complication and cause of death during the first 10 days post-MI (Shapira et al., 1987; Kawano et al., 1994). We found that just like humans, mice can also die of cardiac rupture during the first week post-MI, suggesting that they could represent a good model to study the mechanisms involved in this process and find ways of preventing it. As we showed previously (Cavasin et al., 2000), males had a higher mortality and cardiac rupture rate than females. The gender-related difference in early mortality and rupture may be related to differing mechanisms of infarct healing, tissue repair, degradation of the extracellular matrix (ECM) and/or myocyte slippage, since our data show that a) males had a higher IEI than females at each time point, though it did not reach statistical significance, possibly indicating pronounced LV dilatation and increased wall stress; b) the number of neutrophils infiltrating the infarct border [which are known to secrete MMPs (Lindsey et al., 2001; Nagaoka and Hirota, 2000)] was much higher in males than in females at day 1 and remained high at day 2 post-MI, and this fact together with the tendency for males to have higher MMP-2 and MMP-9 activity during the first week could result in earlier and exaggerated degradation of pre-existing collagen and ECM in males; and c) the number of macrophages infiltrating the infarct border on day 4 was significantly higher in females. Since macrophages can also secrete MMPs, they are precursors of fibroblast infiltration; thus this observation may indicate that in females fibroblasts are infiltrating more efficiently to prompt repair of necrotic tissue. Taking all of these factors together, we may conclude that the gender difference in cardiac rupture could be attributable to premature ECM degradation in males, promoted by the higher number of neutrophils and increased MMP activity, probably causing heightened myocyte slippage as deduced from the higher IEI. In addition, the lower number of macrophages may result in delayed removal of necrotic tissue and scar formation. It has been shown that in male mice genetically lacking MMP-9 or urokinase plasminogen activator (an activator of MMPs), cardiac rupture was dramatically diminished during the first week post-MI (Heymans et al., 1999), suggesting that MMP activation and collagen degradation play a crucial role in the pathogenesis of cardiac rupture. Although we found that mice of both genders had increased MMP activity after MI, in males it tended to be higher at all time points; thus exaggerated ECM degradation combined with increased IEI and delayed infarct healing may

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contribute to increased wall stress and finally cause a significantly higher rate of myocardial disruption in males. During the chronic phase post-MI, we found significant gender-related differences in LV performance and remodeling. After the onset of heart failure, males had a poorer outcome and pronounced maladaptive remodeling compared to females, as indicated by lower EF and SF and higher LVDs, LVDd and MCSA, though cardiac hypertrophy was the same in both genders. Similar results were found with other models of heart failure, such as chronic volume overload and MI in rats (Gardner et al., 2002; Litwin et al., 1999). The mechanisms underlying these differences are not completely understood, but they may be related in part to a) relative concentrations of collagen type I/III, an important determinant of mechanical properties of the myocardium, since estrogen supplementation reportedly prevents increase of collagen type I/III ratio in old female rats, resulting in decreased stiffness (Xu et al., 2003). Although we measured total collagen deposition and found no statistical difference, this may not exclude a difference in collagen type I/III ratio between males and females; and b) myocyte hypertrophy has been shown to be affected by gender, since Guerra et al showed that myocyte volume was much higher in end-stage failing hearts from men than women (Guerra et al., 1999), and we found that myocyte cross-sectional area was significantly higher in males than in females. Another factor that may contribute to the low rate of cardiac rupture in females and their more favorable long-term outcome, as evidenced by higher SF and EF compared to males, is that they may have lower peripheral resistance and cardiac wall stress due to lower blood pressure. It has been shown that blood pressure in men younger than 60 is higher than in women the same age (Burt et al., 1995); male spontaneously hypertensive rats (SHR) and Dahl salt-sensitive rats have higher blood pressure than females, which is reduced by castration in males but is not increased by ovariectomy in females (Reckelhoff et al., 2000; Masubuchi et al., 1982; Rowland and Fregly, 1992; Ouchi et al., 1987). Although systolic blood pressure in our study was only measured at the end of the study, this may be an indication that in females blood pressure decreases during the development of heart failure, averting complications and cardiac distress, or conversely that males lack this ability due to the presence of testosterone. It could be that sexual hormones have vascular or other endocrine effects that indirectly regulate myocardial adaptation. For example, males and females with MI might have differences in LV preload or afterload due to differences in blood volume regulation, venous or arterial tone. It has been reported that estrogen directly relaxes coronary arteries (Williams et al., 1992; Lantin-Hermoso et al., 1997) and restores endothelial function of peripheral resistance arteries in normotensive and hypertensive post-menopausal women (Higashi et al., 2001) via mechanisms involving release of nitric oxide.

Limitations of the study Although we speculate that cardiac rupture in males might be due to premature ECM degradation resulting from earlier neutrophil infiltration, higher MMP activity and impaired myocardial healing during the early healing phase, neither MMP activity nor collagen was studied at the infarct border. In addition, inflammatory cell infiltration and IEI were not measured in mice that died due to rupture, because this event normally occurs during the night, when rodents are highly active. The results of our study are based on the survivors, a fact that could underestimate gender-related differences.

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Conclusion Our study demonstrates that the process of myocardial adaptation after MI differs significantly between male and female mice. During the acute phase of MI, males have delayed myocardial healing, resulting in a higher rate of cardiac rupture; once heart failure has developed, males have poorer cardiac function and pronounced maladaptive remodeling compared to females.

Acknowledgments This work was supported by American Heart Association grant 0030232N.

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