Keywords

Introduction

Apoptosis is a tightly regulated and energy-requiring mechanism in which cell death follows a programmed sequence of structural changes including cell shrinkage, plasma membrane blebbing, nuclear condensation, DNA fragmentation, formation of apoptotic bodies in the nucleus and inflammation [1,2,3,4,5]. Various cellular stress signals including intracellular Ca2+-overload, alterations in cytokines, loss of growth factors, oxidative stress, functional hypoxia and nitric oxide are considered to participate in the occurrence of apoptosis [6,7,8,9,10,11,12,13,14,15,16]. Apoptosis, in the cell is initiated by two major pathways: (i) extrinsic/death receptor pathway via ligand binding to external death receptors and (ii) intrinsic/mitochondrial pathway near the surface of the mitochondria. These are regulated by pro- and anti-apoptotic protein content including caspases 3 and 9, Bcl-2, Bad as well as BAX, which play a critical role in the development of apoptotic cell death in the myocardium [16,17,18,19,20,21,22].

Since apoptosis causes structural deformities in the myocardium, which has a limited ability for self-renewal, an increase in the rate of cardiomyocyte apoptosis has been shown to profoundly affect cardiac contractility and induce cardiac dysfunction [23,24,25,26,27]. Contribution of this mechanism is now well known in the development of contractile heart failure due to different etiologies such as myocardial infarction [28,29,30,31,32,33,34], ischemia-reperfusion injury [35,36,37,38], pressure overload [21, 39,40,41,42,43,44], volume overload [45,46,47,48,49,50,51,52,53,54], cardiomyopathies (dilated, ischemic, toxic and hypertrophic) [24,25,26, 55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70], hypertension [34, 71, 72], as well as atherosclerosis and restenosis [73,74,75,76,77]. Although cardiac hypertrophy is considered to be an adaptive mechanism for maintaining heart function, the ongoing loss of cardiomyocytes by apoptosis may result in the transition of cardiac hypertrophy to heart failure. In view of the well known differences in the responses of male and female hearts to various stressful situations, it appears that the development of cardiac hypertrophy and heart failure are gender-dependent events. Accordingly, the present article is intended to focus discussion on whether the occurrence of cardiomyocyte apoptosis and alterations of the associated pro-and anti-apoptotic proteins in different types of heart diseases are gender-dependent. In particular, it is planned to discuss the role and mechanisms of cardiomyocyte apoptosis in male and female hearts during the development of cardiac hypertrophy and heart failure upon the induction of volume overload.

Gender Differences of Cardiomyocyte Apoptosis in Heart Disease

It is now well known that the status of cardiac function and the extent of cardiomyocyte apoptosis in healthy males are essentially not different from those in the normal female hearts. However, cardiomyocyte apoptosis and cardiac dysfunction in diverse heart diseases have been found more prevalent in men compared to women [21, 25, 31,32,33,34, 44,45,46, 78,79,80,81,82,83,84,85,86,87,88]. This could be due to the difference in the presence of apoptosis-related genes on the Y chromosomes or the role of estrogen and estrogen receptors in female population [25, 89,90,91,92,93,94]. Also, with aging the prevalence of cardiomyocyte apoptosis activity in human hearts has been found more pronounced in males than females [95,96,97,98]. In this regard, it is noteworthy that, unlike the females, a 4-fold increase in cardiomyocyte apoptosis has been described in the senescent male monkey hearts; this observation has signified the role of aging in gender differences [98].

Cardiomyocyte loss, which is invariably associated with heart failure, has been reported to exhibit gender difference [31, 99]. The extent of cardiomyocyte apoptosis was lower in female failing hearts in various heart diseases such as myocardial infarction [100], volume overload [25], pulmonary hypertension [101], and different cardiomyopathies [25, 88, 102, 103]. Improved cardiac function, and a greater reversibility of heart failure were observed in women with dilated cardiomyopathy [104,105,106]. However, the occurrence of heart failure as well as morbidity and mortality in patients with ≥20 years of age, were found to be equal in both genders [107]. Furthermore, the treatment approaches for heart failure in both males and females are similar except for using angiotensin-converting enzyme inhibitors and angiotensin receptor antagonists in females during pregnancy due to the risk of birth defects [108,109,110,111]. Nonetheless, there are dramatic sex differences with respect to heart dysfunction during the development of heart failure [112]. In comparison to males, females with heart failure have shown improved survival and were found to maintain normal left ventricular size and preserve ejection fraction when challenged with myocardial infarction [55, 93, 113,114,115,116,117,118]; atherosclerotic burden [93, 119]; and ischemia/reperfusion injury [114, 120,121,122,123,124,125,126]. In addition to, common causes including aging, pregnancy, environmental factors and lifestyle changes, there are other pathological influences such as hypertension which have shown sex differences with respect to hormonal imbalances, as well as cardiac remodeling processes, and signal transduction mechanisms for apoptosis [88, 89, 108, 127,128,129,130,131,132].

A growing number of studies have supported the view regarding gender differences for cardiomyocyte apoptosis in heart failure. Compared to men, a markedly lower extent of cardiomyocytes apoptosis was seen in the women’s failing hearts [133, 134]. In hearts of patients undergoing cardiac transplantation, a significantly lesser degree of apoptosis in female heart was observed in comparison to males, and it has been pointed out that higher cardiomyocyte death in men was associated with earlier onset of heart failure [55]. It was also reported that due to increased extent of cardiomyocyte apoptosis after myocardial infarction [100], men were at much higher risk of worsened myocardial function and cardiac rupture while women showed greater myocardial retrieve [135]. Furthermore, females were found relatively more protected than males in response to prolonged acute coronary ischemia [55, 93, 110]. It may also be noted that there is a rapid progress in the transition from cardiac hypertrophy to heart failure in males compared to females, and cardiomyocyte apoptosis may play a role at the late stage of this transition [58, 63, 136,137,138,139,140,141,142]. The development of heart failure in pathological conditions such as pressure overload [143,144,145,146,147,148,149] and volume overload [25, 150,151,152] have been observed to be slower in females compared to males. These observations indicate sex-specific differences concerning apoptosis in a wide variety of cardiovascular diseases and provide evidence that cardiomyocyte apoptosis may play an important role in determining the gender-dependent differences in the development of heart failure.

Gender Differences of Cardiomyocyte Apoptosis in Heart Failure Due to Volume Overload

Since cardiomyocytes apoptosis plays a significant role in transition from cardiac hypertrophy to heart failure, this section is concerned with discussion on differences in cardiomyocyte apoptosis during the development of cardiac hypertrophy and heart failure induced by volume overload as a consequence of arteriovenous (AV) shunt in male and female rats [25]. Upon induction of volume overload, the degree of cardiac hypertrophy was increased and the cardiac output was unaltered, but the extent of apoptosis was depressed in both male and female hearts at early stage (4 weeks) (Fig. 7.1a). These events were not accompanied by any changes in mRNA levels for both caspase 3 or caspase 9 (Fig. 7.1b, c). On the other hand, at late stage of heart failure (16 weeks), the parameters of cardiac performance were considerably reduced in males hearts, while, these were maintained in female hearts [25]. Compared to the females, the levels of mRNA for caspase 3 and caspase 9 were higher (Fig. 7.1d, e) and extent of apoptosis was significantly greater in the male hearts (Fig. 7.1a). Furthermore, the data for different signaling transduction pathways involved in the regulation of apoptosis at 16 weeks volume overload are shown in Fig. 7.2a–e. The pro-apoptotic Bad protein content was higher in both phosphorylated and non- phosphorylated forms in male failing hearts whereas no change was seen in female volume overload hearts (Fig. 7.2a, b). The anti-apoptotic Bcl-2 protein content was remained unaltered in the non-phosphorylated form in both sexes (Fig. 7.2c) but the level of phosphorylated form was higher in female hearts and lower in male hearts upon the induction of volume overload for 16 weeks (Fig. 7.2d). Furthermore, protein content of pro-apoptotic factor BAX was higher in male failing hearts but was unaltered in the female volume overload heart (Fig. 7.2e). These results have demonstrated that the potential for the occurrence of apoptosis in the female heart is less than the male heart at the hypertrophic as well as the failing stages after the induction of volume overload. It appears that the observed decrease in the extent of apoptosis in both males and females may be due to an adaptive response to volume overload at the early stage of cardiac hypertrophy. However, the male failing heart showed a marked increase in the extent of apoptosis whereas female volume overload heart exhibited a lesser extent of apoptosis 16 weeks post AV shunt. These observations are consistent with the view that there are sex-specific differences in volume overload induced heart failure.

Fig. 7.1
figure 1

Sex-specific changes in apoptosis as well as mRNA levels for caspases 3 and 9 in sham and AV shunt failing (4 weeks and 16 weeks) hearts due to volume overload in male and female rats. Data are taken from our paper—Dent MR, Tappia PS, Dhalla NS. Apoptosis 15: 499–510, 2010. Erratum in: Apoptosis. 16, 757–758, 2011. *_ P < 0.05 versus sham; #_ P < 0.05 versus corresponding value for male

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Fig. 7.2
figure 2

Sex-specific changes in Bad, phosphorylated Bad (p-Bad), Bcl-2, phosphorylated Bcl-2 (p- Bcl-2) and BAX protein content in sham and 16 weeks AV shunt hearts due to volume overload in male and female rats. Data are taken from our paper—Dent MR, Tappia PS, Dhalla NS. Apoptosis 15: 499–510, 2010. Erratum in: Apoptosis. 16, 757–758, 2011. *_ P < 0.05 versus sham; #_ P < 0.05 versus corresponding value for male

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Role of Estrogen in Cardiomyocyte Apoptosis Due to Volume Overload

Several studies have been reported that cardiomyocyte apoptosis is affected by sex hormones such as estrogen, testosterone and progesterone, which exert different actions during the development of heart failure in males and females [25, 92,93,94, 120, 153,154,155,156,157,158,159,160,161,162,163]. The effects of estrogens in the females include the delay in heart failure and improved survival during the pre-menopausal period in addition to slowing down the development of left ventricular hypertrophy [112, 144, 164,165,166,167]. Moreover, in comparison to male heart, the loss of viable cardiomyocytes was found to be markedly low in the healthy aging female heart [141, 145, 167, 168]. These effects in female hearts are believed to be due to estrogens and estrogen receptors signaling, particularly mediated by the estrogen receptor-β [90, 152,153,154, 169,170,171,172]. Since sex-specific differences in cardiovascular systems have been attributed to the consequence of higher levels of estrogen, as well as the presence or absence of other hormones, which modify apoptosis and reduce the risk of heart failure in women [144], estrogen has been suggested to prevent cardiomyocyte apoptosis due to volume overload in females [25].

The extent of cardiomyocyte apoptosis was increased markedly in ovariectomized female rats upon induction of volume overload at 16 weeks (Fig. 7.3a), but was depressed upon treatment of these animals with estrogen (Fig. 7.3a). It was seen that the level of protein content for caspase 3 was increased, whereas, that for caspase 9 remained unchanged due to volume overload in ovariectomized animals. Treatment of ovariectomized volume overload rats with estrogen, protein content for both caspase 3 and caspase 9 was observed to be depressed (Fig. 7.3b, c). Alterations in different signaling transduction pathways involved in the regulation of apoptosis after inducing AV shunt in the ovariectomized rats are depicted in Fig. 7.4a–e. Induction of volume overload in ovariectomized rats were found to reduce protein content for Bcl-2 and phosphorylated Bcl-2 but increased phosphorylated Bad and BAX protein content without any changes in bad protein content (Fig. 7.4). It can also be seen from Fig. 7.4d that protein content for phosphorylated Bcl-2 was increased in sham control ovariectomized rats. Treatment of ovariectomized volume overload rats with estrogen was found to attenuate the ovariectomy-induced changes in these parameters fully or partially (Fig. 7.4). Thus, estrogen may modify both pro-apoptotic and anti-apoptotic regulatory mechanisms and prevent the occurrence of cardiomyocyte apoptosis in females. Accordingly, these observations are consistent with the view that estrogen may play an important role in determining the reduced susceptibility of females to different cardiovascular risk factor.

Fig. 7.3
figure 3

Changes in apoptosis as well as protein content for caspases 3 and 9 in sham and 16 weeks post AV-shunt female rats with or without ovariectomy (OVX) as well as OVX animals treated with estrogen. Data are taken from our paper – Dent MR, Tappia PS, Dhalla NS. Apoptosis 15: 499–510, 2010. Erratum in: Apoptosis. 16, 757–758, 2011. *_ P < 0.05 vs sham; †_ P < 0.05 versus corresponding value for control female; ∂_ P < 0.05 versus corresponding value for OVX animals without 17-β estradiol treatment

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Fig. 7.4
figure 4

Changes in Bad, phosphorylated Bad (p-Bad), Bcl-2, phosphorylated Bcl-2 (p- Bcl-2), and BAX protein content in sham and 16 weeks post AV-shunt female rats with or without ovariectomy (OVX) as well as OVX animals treated with estrogen (16 weeks post AV-shunt). Data are taken from our paper – Dent MR, Tappia PS, Dhalla NS. Apoptosis 15: 499–510, 2010. Erratum in: Apoptosis. 16, 757–758, 2011. *_ P < 0.05 vs sham; †_P < 0.05 versus corresponding value for control female; ∂_P < 0.05 versus corresponding value for OVX animals without 17-β estradiol treatment

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Conclusions

This study has provided evidence that cardiac hypertrophy due to induction of volume overload at early stages in both male and female rats was associated with an equal extent of depressions in cardiomyocyte apoptosis without any changes in the mRNA levels of caspase 3 and 9. However, a marked degree of apoptosis in male hearts, unlike female hearts, was evident in heart failure at late stage of volume overload. These sex-dependent differences in the cardiomyocyte apoptosis in the failing heart were associated elevated levels of mRNA for both caspase 3 and 9. Sex-specific differences in some pro-apoptotic and anti apoptotic parameters such as Bad, BAX and Bcl-2 protein content were also seen in the failing hearts due to volume overload. Volume overload in ovariectomized animals was found to increase the extent of apoptosis in association with increased levels of caspase 3 as well as BAX protein content and decreased protein of phosphorylated Bcl-2. These alterations in ovariectomized volume overload animals were partially or fully prevented by treatment with estrogen. These observations support the view that cardiomyocyte apoptosis and associated heart failure is sex-specific. Furthermore, this study provides evidence for the role of estrogen in reducing the susceptibility of female population to different cardiovascular risk factor.