The process of LV remodeling after MI is characterized by a series of progressive molecular, cellular, and extracellular matrix (ECM) changes [5, 31]. LV remodeling after permanent LAD leads to infarct expansion, hypertrophy of non-infarcted area, increased collagen accumulation in the infarcted and non-infarcted areas; all together, the changes lead to progressive dilatation and ultimately to impaired LV physiology  and eventual progression to chronic heart failure . However, this response is variable between individuals within the same sex and between sexes and can determine the extent of heart affected when it comes to molecular, physiological, and structural changes . Reports from clinical studies suggests that after MI, women develop less adverse LV remodeling than men with better preservation of LV size and function [33, 34], which is widely attributed to their different sex hormones [25, 33, 35, 36]. In animal models of MI, the literature shows inconsistency. While some studies reported worse cardiac remodeling in male than in female rodents [37-40], others did not show any differences between sexes [21, 22, 41]. Since male and female sexes have different LV mass and volume, their response to the same extent of ischemic injury is expected to show discrepancy and lead to confusion in interpretation. Each sex was compared with the same sex, and direct comparison between males and females, except for inflammatory marker fold change, was avoided in the present study.
Previous studies using male rat models of MI showed that smoke exposure is associated with worse cardiac function and/or cardiac dilatation [42-44]. In accordance with these studies, we observed that males exposed to smoke had lower LVEF, more pronounced increases in LVEDV and LVESV than CS-naïve males at day 7 post-MI. The gap between these two groups at day 1 widened at day 7. It is worth mentioning that in order to eliminate any confounding factor such as temporal remodeling within the 24 hours following MI, study design included only mice with comparable infarct size at day 1. Thus, smoking is the sole factor of discrepancy found in remodeled hearts in male sex at day 7 post-MI. Our observations on EF indicate that smoking is altering the overall contractility independent of infarct size as early as day 1. Interestingly, CS-exposed female groups showed no differences with regard to LVEDV, LVESV and LVEF compared to CS-naïve females either at day 1 or at day 7 post-MI, although LV adverse remodeling was in progress through 7 days. These findings indicate that CS exposure worsens the progression of cardiac dysfunction post-MI in male sex only and in a significant manner.
Since the LV base remains the main part of the heart generating force of contraction, assessment of this region can provide important information. In fact, given the comparable LVEF prior to MI surgery between the male groups regardless of smoke exposure, significantly lower fractional area change of LV base in CS-exposed MI males indicated that CS compromised the compensatory function of the remaining tissue and uncovered the malfunctioning tissue upon facing the hemodynamic stress that existed post-MI. This finding suggested that, alongside promoting inflammation, CS compromises LV function at day 1 and represents a unique and important factor implicated in worsened cardiac remodeling in CS-exposed MI male mice. Distinctively, female groups did not differ in fractional area change of LV base at day 1. The described discrepancy existed at an early-stage post-MI between sexes and could be related to discrepancies in contractility or electrophysiology, both of which are well known targets for estrogen .
Seven days post-MI, increased LV size is accompanied by increased cross-sectional area of myocytes in both the peri-infarct and remote areas [46-49]. Having greater LV mass at the onset of MI and progressive increase in LV mass post-MI are associated with an increased incidence of adverse clinical outcomes [50, 51]. Our results show that MI male mice manifested an increase in their LV mass, but only CS-exposed ones reached statistical significance at day 7 post-MI compared to control male group. Although smoking infarcted males had greater LV mass than non-smoking ones, this result was not significant. Lack of statistical significance might perhaps be attributed to relatively small number of mice in male groups. As for female sex, LV mass significantly increased in both MI groups irrespective of smoke exposure when compared to control group. Similarly, infarct size is strongly correlated with worse outcomes after transmural myocardial infraction in both sexes [52-54]. At 7 days post-MI, infarct size was significantly high only in CS-exposed MI male mice compared to their CS naïve counterparts. However, no significant difference was observed in female sex at day 7 post-MI. This indicates that infarct expansion occurred in males, but not females.
In some previous clinical studies, the smoker showed better prognosis than non-smoker [27, 55, 56]. A plausible explanation of the “smoker’s paradox” is that smokers were on average 14 years younger than non-smokers and had fewer atherosclerotic risk factors and comorbidities . A large study involving pooled analysis of 18 randomized controlled trials with up to 5 year follow up clearly demonstrated that after multivariable adjustment for potential confounders, smoking is a strong independent predictor of death, cardiac death, MI, stent thrombosis, and target lesion failure . A recent clinical study, however, reported that smoking status has no impact on infarct size, while sex does. Female patients with ST-elevation myocardial infarction showed smaller myocardium at risk, smaller infarct size, and larger myocardial salvage index . Our study differs from the above clinical studies in many ways. Firstly, the mice in our study (irrespective of sex and smoke exposure) were in the same age range and free of comorbidities, unlike clinical studies. Secondly, we selected mice having comparable infarct size at day 1 in order to show the impact of smoking throughout 7 days of cardiac remodeling. Thirdly, while the patients in the clinical studies received reperfusion treatment, the mice in our study underwent permeant coronary artery ligation. Altogether, our study revealed that unlike clinical studies, CS status has worse impact on left ventricular remolding post MI, especially in males. Female sex alleviated the worsen impact of CS post-MI, at least for the first 7 days of cardiac remodeling post MI.
ECM composition fractions and percentages are in constant change post-MI with, net ECM accumulation the result of synthesis minus degradation [60, 61]. A good example is the collagen-rich reparative scar that determines an early surge of collagen deposition from around day 5 post-MI in order to replace the massive loss of cardiomyocytes in the infarcted area . This has been documented experimentally in infarcted rats, in which collagen III gene expression is initiated 2 days post-MI and continues to increase up to 21 days post-MI. However, collagen I gene expression starts to increase 4 days post-MI, peaks at 7 days post-MI, and stabilizes by 21 days post-MI . In our study, CS-exposed MI male mice presented greater collagen accumulation in both infarcted and peri-infarcted areas than CS-naïve MI male mice at day 7 post-MI (Fig 7). However, an experimental study with a male rat model of MI showed comparable results regarding collagen content in CS-exposed and CS-naïve groups . On the other hand in post-MI female groups no statistically significant differences were seen in interstitial collagen density in both infarcted and peri-infarct area between CS-exposed and CS-naïve females at day 7 post-MI. This might be attributed to the role of estrogen in other organs to suppress collagen accumulation [64, 65]. An in vitro study showed that 17β-estradiol along with its metabolites, as well as progesterone, inhibit cardiac fibroblast growth . This notion has been supported by in vivo studies in which ovariectomized rats with MI showed more intense cardiac collagen accumulation than intact rats with MI . Experimental studies with estrogen receptors also provide further information regarding the impact of estrogen in cardiac fibrosis [68, 69]. The anti-fibrotic property of estrogen was previously associated with its inhibitor action on angiotensin II and endothelin-1, which promote fibrosis in part by inducing TGFβ1 production . This can partially explain the differences in response to CS exposure between MI males and females in our present study.
Pulmonary congestion as a result of LV dysfunction is a common clinical manifestation of dilated LV, which can cause RV dysfunction and remodeling. However, RV function can deteriorate even in the absence of pulmonary hypertension and alteration in RV afterload in case of acute MI involving the left ventricle . Furthermore, it was observed that RV remodeling, even spared from initial ischemic damage, involves cardiac remodeling that is originated from LV post-MI [72-74]. In the present study, we observed significantly increased RV mass only in CS-exposed MI male group compared to CS-naïve MI and control groups. However, CS-exposed MI male mice had significantly larger RV area than CS-naïve MI males, whereas no difference was found between female groups. This finding suggests dilatation of RV rather than hypertrophy, which is usually a consequence of increased pulmonary artery pressure. In line with this, pulmonary congestion did not differ between groups, indicating that development of RV remodeling was independent of pulmonary congestion. Of note, chronic exposure to CS can cause pulmonary vascular resistance and cor pulmonale, which refers to RV enlargement resulting from pulmonary hypertension . Given the duration of CS exposure in the present study, development of cor pulmonale and its implication in RV remodeling are less likely.
CS exacerbates ROS production that overwhelms intracellular antioxidant mechanisms. In addition, tobacco smoke contains substantial amounts of ROS and chemicals that weaken antioxidant defense mechanisms, boost inflammatory response, and worsen damage, both in the presence and/or the absence of cardiac morbidities [7, 76, 77]. Numerous studies in the literature revealed that CS exposure enhances gene expression of pro-inflammatory cytokines in the cardiac tissue and causes systemic inflammation by increasing circulating pro-inflammatory cytokines. In accordance with these observations, we documented a significant increase in the mRNA expression of two major pro-inflammatory markers, IL-6 and TNF-α, at day 7 post-MI in MICS male group only. Both IL-6 and TNF-α spike with initial acute inflammation post-MI (day1-day3) and decrease to normal levels during transition to granulation phase post-MI (day-4-day7) . Persistent inflammation could be the attributing factor to the pronounced cardiac deterioration observed in CS-exposed MI male group that was not seen in the female counterparts. In fact, prolonged inflammation following MI is associated with poor prognosis and a high risk of systolic dysfunction . Estrogen well known anti-inflammatory and antioxidant effects might have played a crucial role in this process by curbing the CS-induced prolonged inflammation in MICS female mice [80, 81]). Further studies are warranted to reach a definitive conclusion around estrogen involvement in this process.
Our study has some limitations. First, we did not follow the functional and structural changes for longer than 7 days after MI. Second, we did not assess the role of sex hormones on the effect of CS on cardiac remodeling post-MI, such as performing ovariectomy or castration, which prevents us from being conclusive regarding underlying mechanisms. Third, it might be argued that exposing animals to CS after MI does not reflect what is happening with patients. Continued smoking following an acute coronary syndrome is associated with greater mortality and patients are recommended to stop smoking . Even with counseling, however, smoking relapse remains a significant issue, especially in Lebanon, with 50-60% of patients with an acute coronary syndrome continuing to smoke after discharge from the hospital [83-85]. Fourth, our study design does not permit an assessment of the effect of smoking per se on cardiac function nor can we completely dismiss the possibility that CS exposure may have induced stress that affected the cardiovascular system; however, in our study we used two different sexes that were subjected to the same stressors and thus the only difference between groups was sex. In addition, we did not observe any gross indications that CS exposure induced stress. Fifth, it is possible that the CS protocol induces stress in mice and consequently has an impact on cardiovascular physiology; however, both sexes were subjected to the same CS protocol. Sixth, ischemia-reperfusion was not performed in our study, but will be done in future experiments; however, a significant percentage of MI patients are not reperfused and suffer from permanent occlusion MIs [86, 87]. Finally, CS exposure was performed for 2 weeks before MI and it would be informative to assess longer exposure times. The key goal, however, was to eliminate the risk of CS-induced cardiomyopathy that normally occur with long-term CS exposure and focus solely on acute events. Nonetheless, our study provides novel data on sex-differences in the response of the infarcted heart to CS.
Perspectives and Significance
Associated with significant morbidity and mortality, MI is the most common cause of death among patients with CVD in industrialized countries . CS is a major cause of most CVD, and especially, coronary heart disease for both men and women . However, the difference in extent of LV damage after MI, has not been fundamentally assessed between smoking men and women before. In the present study, it was shown that CS exposure exacerbated both left and right ventricular remodeling only in males at an early stage of post-MI. Females did not display a significant structural and/or functional alteration within 7 days of cardiac remodeling post-MI upon CS exposure. Worsened RV remodeling in males was independent of pulmonary congestion.
Further studies are also required to illuminate the differences between the sexes and the underlying potential mechanisms. Molecular and cellular time continuum of the different MI response phases should be studied more in greater detail between sexes. Understanding which factors contribute to this discrepancy will provide mechanistic insight into how the progression to heart failure is oriented in both sexes and identify new personalized targets to examine in males and females, separately or both similarly.