Prenatal cocaine exposure increases heart susceptibility to ischaemia–reperfusion injury in adult male but not female rats
1 Center for Perinatal Biology, Department of Physiology and Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA 92350, USA
Abstract
The present study tested the hypothesis that prenatal cocaine exposure differentially regulates heart susceptibility to ischaemia–reperfusion (I/R) injury in adult offspring male and female rats. Pregnant rats were administered intraperitoneally either saline or cocaine (15 mg kg–1) twice daily from day 15 to day 21 of gestational age. There were no differences in maternal weight gain and birth weight between the two groups. Hearts were isolated from 2-month-old male and female offspring and were subjected to I/R (25 min/60 min) in a Langendorff preparation. Preischaemic values of left ventricular (LV) function were the same between the saline control and cocaine-treated hearts for both male and female rats. Prenatal cocaine exposure significantly increased I/R-induced myocardial apoptosis and infarct size, and significantly attenuated the postischaemic recovery of LV function in adult male offspring. In contrast, cocaine did not affect I/R-induced injury and postischaemic recovery of LV function in the female hearts. There was a significant decrease in PKC and phospho-PKC levels in LV in the male, but not female, offspring exposed to cocaine before birth. These results suggest that prenatal cocaine exposure causes a sex-specific increase in heart susceptibility to I/R injury in adult male offspring, and the decreased PKC gene expression in the male heart may play an important role.
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Introduction
Cocaine abuse among women of childbearing age is prevalent in the United States. It has been estimated that each year more than 100 000 infants who were exposed prenatally to cocaine are born in this country. Clinical studies and various animal models have shown consistent patterns of subtle changes in neurobehavioural function in young and adult offspring who had been exposed to cocaine prenatally (Spear et al. 1998, 2002). Studies in humans and rodent models of prenatal cocaine exposure have shown plasticity of the developing nervous system in compensating for some cocaine-induced perturbations in the fetus, with a cost of an increased vulnerability to environmental and cognitive demands and stressors in postnatal life (Mayes et al. 1998; Spear et al. 1998, 2002). Interestingly, there are consistent sex differences in adult offspring, with males often being more susceptible than females on a diversity of measures to the effects of prenatal cocaine exposure in the rat (Spear et al. 2002).
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In addition to neurobehavioural effects, offspring born to mothers with a history of cocaine abuse have a high incidence of congenital cardiovascular malformations, including abnormalities of ventricular structure and function, arrhythmias, and intracardiac conduction abnormalities, which persist beyond the period of exposure to cocaine (Van de Bor et al. 1990; Lipshultz et al. 1991; Norris & Hill, 1992; Wiggins, 1992; Mehta et al. 1993; Mone et al. 2004). We have recently demonstrated in a rat model that cocaine exposure during gestation increases apoptosis in fetal heart both in vivo and in cultured cardiomyocytes (Xiao et al. 2000, 2001; Li et al. 2005). In addition, prenatal cocaine exposure caused an increase in apoptosis in neonatal hearts during the first two weeks of postnatal life, and cardiac remodelling with myocyte hypertrophy in the left ventricle (Bae & Zhang, 2005). During the early developmental period, either excessive and/or persistent cardiomyocyte loss through apoptosis has been suggested to lead to a variety of cardiac disease (Haunstetter & Izumo, 1998; James, 1998; Fernandez et al. 2001; Gill et al. 2002). Indeed, our recent studies have demonstrated that, although resting cardiac function shows no difference between animals that were exposed to either saline control or cocaine before birth, prenatal cocaine exposure significantly increases the heart susceptibility to ischaemia and reperfusion injury in adolescent offspring (Bae & Zhang, 2005).
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These findings are consistent with previous epidemiological studies in humans, as well as studies in animals, showing an association of adverse intrauterine environment and an increased risk of hypertension and ischaemic heart disease in later adult life (Barker, 2000; Zhang, 2005). In animal models of intrauterine undernutrition, the sex dichotomy in manifestation of the severity of hypertension in adult offspring has been observed, but the results were conflicting (do Carmo Pinho Franco et al. 2003). In the present study, we tested the hypothesis, in a rat model, that the effect of prenatal cocaine exposure on cardiac vulnerability in adult offspring is dependent on the sex of the animal, with the male heart being more susceptible to increased ischaemia–reperfusion injury induced by prenatal cocaine exposure. Given the important cardioprotective roles of Akt and PKC signalling in the regulation of cardiac ischaemia and reperfusion injury (Camper-Kirby et al. 2001; Chen et al. 2001; Sugden & Clerk, 2001; Murriel & Mochly Rosen, 2003), we also tested the hypothesis that prenatal cocaine exposure differentially regulates the expression of Akt and PKC in hearts of adult male and female offspring.
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Methods
Experimental animals
Time-dated pregnant Sprague-Dawley rats were purchased from Charles River Laboratories (Portage, MI, USA). The rats were randomly divided into two groups (n= 3 for each group): (1) saline control, and (2) cocaine 15 mg kg–1 administered intraperitoneally twice daily at 10.00 h and 16.00 h from day 15 to day 21 of gestational age. The intraperitoneal route closely resembles intranasal administration in human in the kinetics and plasma cocaine levels attained (Javaid & Davis, 1993). No fetal loss in the control and cocaine-treated groups was observed. A total of 39 male and 38 female offspring of 2-month-old rats were studied. Hearts were isolated from male and female offspring for functional studies. All procedures and protocols used in the present study were approved by the Institutional Animal Care and Use Committee of Loma Linda University, and followed the guidelines by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
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Perfused hearts subjected to ischaemia and reperfusion
Hearts were excised rapidly from 2-month-old male and female offspring, and were retrogradely perfused via the aorta in a modified Langendorff apparatus under constant pressure (70 mmHg) with gassed (95% O2–5% CO2) Krebs–Heinseleit buffer (KHB) at 37°C, as previously described (Li et al. 2004). A pressure transducer connected to a saline-filled balloon inserted into the left ventricle (LV) was used to assess ventricular function by measuring the left ventricular pressure (mmHg) and its first derivative (dP/dt). LV end diastolic pressure (LVEDP) was set at approximately 5 mmHg. Although the heart was paced in many other studies, the heart was not paced in the present study because the heart rate was a variable to be measured in the study. Nevertheless, the heart rates were not different between the control and cocaine-treated animals before and after ischaemia. After the baseline recording, hearts were subjected to 25 min of global ischaemia by stopping the perfusion, followed by 60 min of reperfusion. LV functional parameters, LV developed pressure (LVDP), heart rate (HR), dP/dtmax, dP/dtmin, and LVEDP were continuously recorded with an on-line computer. Pulmonary artery effluent was collected as an index of coronary flow (CF).
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Measurement of myocardial infarct size
Myocardial infarct size was measured as previously described (Li et al. 2003). Briefly, at the end of reperfusion, left ventricles were collected, cut into four slices, incubated with 1% triphenyltetrazolium chloride (TTC) solution for 15 min at 37°C, and immersed in formalin for 30 min. Each slice was then photographed (Kodak digital camera) separately, and the areas of myocardial infarction (MI) in each slice were analysed by computerized planimetry (Image-Pro Plus), corrected for the tissue weight, summed for each heart, and expressed as a percentage of the total LV weight.
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Measurement of DNA fragmentation by ELISA
DNA fragmentation was quantified by specific determination of cytosolic mononucleosomes and oligonucleosomes using a commercial quantitative sandwich enzyme-linked immunosorbent assay kit (Boehringer Mannheim) as previously described (Piot et al. 1997; Bae et al. 2003). Briefly, tissue samples were obtained from left ventricles after 60-min reperfusion, and put into 500 μl lysis buffer supplied in the kit. Samples were disintegrated by tissue grinder and incubated for 30 min at room temperature. After centrifugation at 200 g for 10 min, the supernatant (cytosolic fraction) was further diluted 40-fold in phosphate-buffered saline (PBS), and used as the antigen source in the sandwich ELISA. The values of absorbance were measured at 405 nm/490 nm and the background value of the immunoassay was subtracted.
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Western blot analysis
Left ventricles were isolated from 2-month-old male and female offspring, and protein levels of PKC, phospho-PKC (pSer729), Akt, and phospho-Akt (pSer473) were determined with Western blot analysis. Briefly, proteins were separated on 10% SDS-PAGE. The gel was then transferred to nitrocellulose membranes, and incubated with the primary antibodies for PKC (Santa Cruz Biotechnology; Santa Cruz, CA, USA), phospho-PKC (Upstate Biotechnology; Lake Placid, NY, USA), Akt, and phospho-Akt (Sigma; St Louis, MO, USA), respectively. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham, Arlington Heights, IL, USA). Proteins were then visualized with an enhanced chemiluminescence detection system. Results were quantified with Kodak Electrophoresis Documentation and Analysis System and Kodak 1D Image Analysis Software. Actin was used to assess equal loading. Average actin density was determined first from all lanes in the film, and the fractional actin density for each lane was then calculated. PKC and Akt density were corrected by the fraction of the average actin for each lane in the film.
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Statistical analysis
Data were expressed as means ±S.E.M. Statistical significance (P < 0.05) was determined by two-way ANOVA or Student's t test, where appropriate.
Results
Effect of cocaine on maternal weight gain and offspring body weight
As shown in Fig. 1, maternal body weight gain was not significantly affected during the period of cocaine treatment. In addition, birth weight (6.6 ± 0.2 versus 6.3 ± 0.1, n= 3 litters, P > 0.05) and litter size (12.3 ± 1.7 versus 13.3 ± 1.5, n= 3, P > 0.05) were not significantly different between the control and cocaine-treated animals. Furthermore, at 2 months of age in both male and female offspring, body weight, LV weight, and the LV weight-to-body weight ratio were the same between the control and treatment groups (Table 1).
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Time-dated pregnant Sprague-Dawley rats received either saline as control or cocaine (30 mg kg–1 day–1) from day 15 to 21 of gestational age. Data were analysed by two-way ANOVA with gestational age as one factor and cocaine treatment as the other (n= 3).
Effect of prenatal cocaine on LV function and postischaemic recovery
Using the Langendorff preparation, LV function was assessed in isolated hearts from 2-month-old adult rats that were exposed to either saline control or cocaine before birth. As shown in Table 2, there were no significant differences in LVDP, HR, dP/dtmax, dP/dtmin and coronary flow at baseline levels between control and cocaine-treated groups in either male or female offspring. Figure 2 shows the effect of 25 min ischaemia followed by 60 min reperfusion on LV function in male offspring. Myocardial contraction was completely eliminated during 25 min ischaemia, but gradually resumed when perfusion was restored. Compared with the saline control, there were significant decreases in postischaemic recovery of LVDP, dP/dtmax, dP/dtmin and pressure-rate product (PRP), in animals exposed to cocaine before birth. However, postischaemic recovery of coronary flow and HR was not significantly different between the two groups. In contrast to the findings in the male offspring, prenatal cocaine exposure showed no effect on postischaemic recovery of LV function in the female offspring (Fig. 3).
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Hearts were obtained from 2-month-old male offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age, and were subjected to 25 min of ischaemia and 60 min of reperfusion in the Langendorff preparation. LVDP, left ventricular developed pressure; HR, heart rate; PRP, pressure rate product (LVDP x HR); CF, coronary flow. Data were analysed by two-way ANOVA with ischaemia–reperfusion as one factor and cocaine treatment as the other. The asterisk (*) indicates a significant difference (P < 0.05) from control for the entire curve (n= 8).
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Hearts were obtained from 2-month-old female offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age, and were subjected to 25 min of ischaemia and 60 min of reperfusion in the Langendorff preparation. LVDP, left ventricular developed pressure; HR, heart rate; PRP, pressure rate product (LVDP x HR); CF, coronary flow. Data were analysed by two-way ANOVA with ischaemia–reperfusion as one factor and cocaine treatment as the other (n= 7).
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Effect of prenatal cocaine on I/R-induced myocardial infarction and apoptosis
The infarct size of LV at the end of 60 min reperfusion after 25 min ischaemia is shown in Fig. 4. Ischaemia and reperfusion caused LV myocardial infarction in both male and female offspring. Prenatal cocaine treatment significantly increased infarct size in the heart of male, but not female, offspring (Fig. 4). Apoptosis in the heart was assessed by quantitative determination of fragmented DNA into mononucleosomes and oligonucleosomes determined by an ELISA specific for cytosolic histone bound DNA. Consistent with the finding of myocardial infarction, prenatal cocaine exposure significantly increased ischaemia and reperfusion-induced DNA fragmentation in left ventricle from the male, but not female, offspring (Fig. 5).
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Hearts were obtained from 2-month-old male and female offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age, and were subjected to 25 min of ischaemia and 60 min of reperfusion in the Langendorff preparation. Left ventricles were collected at the end of reperfusion, and myocardial infarct size was determined with 1% TTC staining and expressed as a percentage of the total left ventricular weight. The colour image represents one of four tissue sections obtained from each left ventricle. The summed infarct sizes, expressed as a percentage of the total left ventricular weight for the control and prenatal cocaine-treated male and female offspring, are shown below. Data were analysed by t test. *P < 0.05, cocaine versus control (n= 5–7).
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Hearts were obtained from 2-month-old male and female offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age, and were subjected to 25 min of ischaemia and 60 min of reperfusion in the Langendorff preparation. Left ventricles were collected at the end of reperfusion, and DNA fragmentation was determined using an ELISA kit as described in Methods. Data were analysed by t test. *P < 0.05, cocaine versus control (n= 4–6).
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Effect of prenatal cocaine on PKC and Akt expression in LV
Prenatal cocaine exposure significantly decreased protein levels of PKC by 59% and phospho-PKC by 75% in LV of the male offspring, but had no effect on protein levels of either PKC or phospho-PKC in the female LV (Figs 6 and 7). In contrast, prenatal cocaine treatment had no effect on protein levels of Akt or phospho-Akt in LV of either male or female offspring (data not shown).
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Left ventricles were obtained from hearts of 2-month-old male and female offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age. Protein levels of PKC were determined using Western blot analyses. Proteins were visualized with an enhanced chemiluminescence detection system, and were quantified with KODAK Electrophoresis Documentation and Analysis System and KODAK 1D Image Analysis Software. Data were analysed by t test. *P < 0.05, cocaine versus control (n= 4).
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Left ventricles were obtained from hearts of 2-month-old male and female offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age. Protein levels of phospho-PKC were determined using Western blot analyses. Proteins were visualized with an enhanced chemiluminescence detection system, and were quantified with KODAK Electrophoresis Documentation and Analysis System and KODAK 1D Image Analysis Software. Data were analysed by t test. *P < 0.05, cocaine versus control (n= 3–4).
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Discussion
The present study demonstrated that prenatal cocaine exposure increased heart susceptibility to ischaemia and reperfusion injury in adult male, but not female, offspring rats. Previous studies have shown that chronic daily doses of cocaine from 10 to 40 mg kg–1 during rat gestation produces plasma cocaine levels in the human use range (Spear et al. 1989; Javaid & Davis, 1993), suggesting a comparable model in the present study. In addition, kinetics of cocaine via the intraperitoneal route closely resemble those from intranasal administration in humans (Javaid & Davis, 1993). In the present study, maternal intraperitoneal cocaine administration had no effect on maternal weight gain and birth weight. Previously, we showed that subcutaneous cocaine administration to pregnant rats resulted in no change in maternal body weight, but a modest decrease in birth weight (Bae & Zhang, 2005). This may be due to the difference in the route of cocaine administration. Consistent with the present study, it has been shown that chronic intravenous maternal administration of cocaine in the rat has no significant effect on maternal body weight gain and birth weight (Iso et al. 2000).
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The finding that fetal cocaine exposure caused an increase in cardiac vulnerability in adult offspring is consistent with previous epidemiological studies in humans, as well as studies in animals, showing an association of adverse intrauterine environment and an increased risk of hypertension and ischaemic heart disease in later adult life (Barker, 2000; Zhang, 2005). However, fetal growth restriction was not observed in the present study. This is different from previous models of fetal undernutrition with significant fetal growth restriction, suggesting multiple mechanisms of in utero programming. In the present study, hearts of male offspring rats that had been exposed to cocaine before birth showed similar basal LV function to those from control animals, suggesting that prenatal cocaine exposure did not influence contractility in the resting heart. Similar findings have been obtained in our recent studies in adult rats that were exposed to hypoxia before birth (Li et al. 2003). Because the hearts were perfused at a constant pressure, and the end-diastolic pressure was set at approximately 5 mmHg, the present study was not subject to differences in afterload, preload, or endogenous sympathetic tone.
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Previously, we demonstrated in a rat model that cocaine exposure increases apoptosis in the fetal heart both in vivo and in cultured cardiomyocytes (Xiao et al. 2000, 2001; Li et al. 2005). Given that cardiomyocytes are highly differentiated cells and rarely replicate after birth, inappropriate cardiomyocyte loss through apoptosis during the early developmental period is likely to result in a permanent reduction in the number of functioning units in the myocardium. However, the developing heart may show remarkable plasticity in compensating for the loss of cardiomyocytes by an increase in myocyte size for the remaining cells (Bae et al. 2003; Li et al. 2004; Bae & Zhang, 2005). Although myocyte hypertrophy may compensate for the loss of myocytes and maintain cardiac function at the resting level, there appears to be a cost of this compensation: an increase in ischaemic vulnerability of the heart. In agreement with the present findings, previous studies of a rodent model of prenatal cocaine exposure clearly demonstrated that in adult offspring, although neurobehavioural function might appear normal under basal test conditions, deficits emerged when the animals were unable to mount appropriate responses to stressors and other environmental, cognitive or pharmacological challenges (Spear et al. 1998, 2002). This has also been demonstrated in humans (Mayes et al. 1998). Similarly, a stress test with a jet stream of high-pressure air significantly increased blood pressure in adult rats that were hypoxic during prenatal life, but had no effect in the control rats (Peyronnet et al. 2002). Taken together, these previous and our present studies support the notion that ‘it is possible and perhaps common for an organ to be programmed and then vulnerable for life without evidence until a late-life stressor challenges its adaptive capabilities’ (Thornburg, 2003).
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The finding of sex dichotomy in manifestation of increased cardiac vulnerability in adult offspring following prenatal cocaine exposure is intriguing, and is consistent with previous findings. In rodent models of prenatal cocaine exposure, it has been demonstrated that male offspring are more susceptible than females on a diversity of measures of neurobehavioural function in response to environmental and cognitive demands and stressors (Grimm & Frieder, 1985; Molina et al. 1994; Goodwin et al. 1995; Heyser et al. 1995; Spear et al. 1998; Wood & Spear, 1998; Spear et al. 2002). The sex dichotomy has also been observed in animal models of intrauterine undernutrition. Although the results are conflicting, especially in models of severe nutrient restriction during pregnancy, it has been generally accepted that the severity of hypertension in adult offspring is greater in males than in females (do Carmo Pinho Franco et al. 2003).
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It is likely that multiple mechanisms are involved in the sex-specific effect of prenatal cocaine exposure. In the present study, we found that protein levels of PKC and phospho-PKC in the left ventricle were significantly decreased only in male offspring following prenatal cocaine treatment. In contrast, Akt and phospho-Akt levels were not affected in either male or female offspring. Both Akt and PKC are downstream targets of phosphatidylinositol 3-kinase, and play an important role of cardioprotection in the regulation of cardiac ischaemia and reperfusion (Camper-Kirby et al. 2001; Chen et al. 2001; Sugden & Clerk, 2001; Murriel & Mochly Rosen, 2003). The present results suggest that Akt may not be involved in prenatal cocaine-mediated increase in heart susceptibility to ischaemia and reperfusion in adult offspring. The finding of decreased PKC and phospho-PKC levels in adult male offspring is intriguing, and suggests that prenatal cocaine may cause in utero programming of the PKC gene expression pattern leading to a down-regulation of PKC expression in the male heart. It has been demonstrated that PKC isozyme expression is controlled by distinct mechanisms that are regulated differently during development (Rybin & Steinberg, 1994). Fetal rat heart expresses multiple PKC isozymes, , , , and . In the adult heart, , , and isozymes decline markedly, and , and isozymes are found primarily in non-myocyte elements of the heart. PKC expression shows only modest age-dependent differences, and is the most abundantly expressed PKC isozyme in the adult rat heart (Bogoyevitch et al. 1993; Rybin & Steinberg, 1994). This is consistent with many studies, demonstrating an important cardioprotective role of PKC in the adult heart (Chen et al. 2001; Ping et al. 2001; Inagaki et al. 2003; Murriel & Mochly Rosen, 2003). In agreement with the present finding, studies in PC12 cells demonstrated that cocaine treatment selectively down-regulated the expression of PKC, among other isozymes (Onaivi et al. 1998). In addition, PKC protein levels were found decreased in the heart of 2-month-old male rats that had been exposed to hypoxia before birth (Li et al. 2004).
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Given the importance of PKC in cardioprotection against ischaemia and reperfusion injury, the cocaine-mediated decrease in PKC levels observed in the present study is likely to have functional significance. A recent study in a PKC knock-out mouse model has demonstrated that PKC expression is not required for normal cardiac function under physiological conditions, but PKC activation is necessary and sufficient for acute cardioprotection in cardiac ischaemia and reperfusion (Gray et al. 2003). The cardioprotective effect of PKC is proposed to be inhibition of apoptosis and hence reduction of myocardial infarction following ischaemia and reperfusion (Heidkamp et al. 2001; Liu et al. 2001, 2002; Shizukuda & Buttrick, 2002; Murriel & Mochly Rosen, 2003; Sparagna et al. 2004). In the present study, we found a significant increase in apoptosis in the heart following ischaemia and reperfusion in the male offspring that were exposed to cocaine prenatally. This is consistent with the increased myocardial infarction observed in the male heart. Different methods have been used to measure apoptosis in tissues. In our recent studies, apoptosis in isolated rat hearts subjected to ischaemia and reperfusion has been demonstrated using multiple methods including DNA fragmentation, apoptotic nuclear morphology, cleaved active form of caspase-3, and caspase-3 activity (Li et al. 2003, 2004). Ischaemia and reperfusion-mediated cell death in the heart occurs through both necrosis and apoptosis (Haunstetter & Izumo, 1998; Gill et al. 2002). Many studies have clearly demonstrated that apoptosis plays an important role in ischaemia and reperfusion-induced myocardial injury (Yaoita et al. 1998; Ma et al. 1999; Okamura et al. 2000; Yue et al. 2000; Condorelli et al. 2001).
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In conclusion, we have demonstrated for the first time, to our knowledge, in a rat model, that prenatal cocaine exposure produces a sex-specific increase in heart susceptibility to ischaemia and reperfusion injury in adult male offspring, by increasing myocardial infarct size and decreasing postischaemic recovery of left ventricular function. Although the mechanisms underlying this sex-dependent effect in the adult heart are likely to be multiplex, the present study suggests that cocaine exposure during fetal development may cause in utero programming of the PKC gene expression pattern, which may have lifelong importance in cardiac function, and may play an important role in the increased susceptibility of the heart to ischaemia and reperfusion injury in male adult offspring. Given that DNA methylation plays a critical role in epigenetic modification of gene expression, and methylation in promotor regions is generally associated with transcription repression, future studies of the potential effect of cocaine on DNA methylation in the PKC gene in the heart are warranted.
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Abstract
The present study tested the hypothesis that prenatal cocaine exposure differentially regulates heart susceptibility to ischaemia–reperfusion (I/R) injury in adult offspring male and female rats. Pregnant rats were administered intraperitoneally either saline or cocaine (15 mg kg–1) twice daily from day 15 to day 21 of gestational age. There were no differences in maternal weight gain and birth weight between the two groups. Hearts were isolated from 2-month-old male and female offspring and were subjected to I/R (25 min/60 min) in a Langendorff preparation. Preischaemic values of left ventricular (LV) function were the same between the saline control and cocaine-treated hearts for both male and female rats. Prenatal cocaine exposure significantly increased I/R-induced myocardial apoptosis and infarct size, and significantly attenuated the postischaemic recovery of LV function in adult male offspring. In contrast, cocaine did not affect I/R-induced injury and postischaemic recovery of LV function in the female hearts. There was a significant decrease in PKC and phospho-PKC levels in LV in the male, but not female, offspring exposed to cocaine before birth. These results suggest that prenatal cocaine exposure causes a sex-specific increase in heart susceptibility to I/R injury in adult male offspring, and the decreased PKC gene expression in the male heart may play an important role.
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Introduction
Cocaine abuse among women of childbearing age is prevalent in the United States. It has been estimated that each year more than 100 000 infants who were exposed prenatally to cocaine are born in this country. Clinical studies and various animal models have shown consistent patterns of subtle changes in neurobehavioural function in young and adult offspring who had been exposed to cocaine prenatally (Spear et al. 1998, 2002). Studies in humans and rodent models of prenatal cocaine exposure have shown plasticity of the developing nervous system in compensating for some cocaine-induced perturbations in the fetus, with a cost of an increased vulnerability to environmental and cognitive demands and stressors in postnatal life (Mayes et al. 1998; Spear et al. 1998, 2002). Interestingly, there are consistent sex differences in adult offspring, with males often being more susceptible than females on a diversity of measures to the effects of prenatal cocaine exposure in the rat (Spear et al. 2002).
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In addition to neurobehavioural effects, offspring born to mothers with a history of cocaine abuse have a high incidence of congenital cardiovascular malformations, including abnormalities of ventricular structure and function, arrhythmias, and intracardiac conduction abnormalities, which persist beyond the period of exposure to cocaine (Van de Bor et al. 1990; Lipshultz et al. 1991; Norris & Hill, 1992; Wiggins, 1992; Mehta et al. 1993; Mone et al. 2004). We have recently demonstrated in a rat model that cocaine exposure during gestation increases apoptosis in fetal heart both in vivo and in cultured cardiomyocytes (Xiao et al. 2000, 2001; Li et al. 2005). In addition, prenatal cocaine exposure caused an increase in apoptosis in neonatal hearts during the first two weeks of postnatal life, and cardiac remodelling with myocyte hypertrophy in the left ventricle (Bae & Zhang, 2005). During the early developmental period, either excessive and/or persistent cardiomyocyte loss through apoptosis has been suggested to lead to a variety of cardiac disease (Haunstetter & Izumo, 1998; James, 1998; Fernandez et al. 2001; Gill et al. 2002). Indeed, our recent studies have demonstrated that, although resting cardiac function shows no difference between animals that were exposed to either saline control or cocaine before birth, prenatal cocaine exposure significantly increases the heart susceptibility to ischaemia and reperfusion injury in adolescent offspring (Bae & Zhang, 2005).
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These findings are consistent with previous epidemiological studies in humans, as well as studies in animals, showing an association of adverse intrauterine environment and an increased risk of hypertension and ischaemic heart disease in later adult life (Barker, 2000; Zhang, 2005). In animal models of intrauterine undernutrition, the sex dichotomy in manifestation of the severity of hypertension in adult offspring has been observed, but the results were conflicting (do Carmo Pinho Franco et al. 2003). In the present study, we tested the hypothesis, in a rat model, that the effect of prenatal cocaine exposure on cardiac vulnerability in adult offspring is dependent on the sex of the animal, with the male heart being more susceptible to increased ischaemia–reperfusion injury induced by prenatal cocaine exposure. Given the important cardioprotective roles of Akt and PKC signalling in the regulation of cardiac ischaemia and reperfusion injury (Camper-Kirby et al. 2001; Chen et al. 2001; Sugden & Clerk, 2001; Murriel & Mochly Rosen, 2003), we also tested the hypothesis that prenatal cocaine exposure differentially regulates the expression of Akt and PKC in hearts of adult male and female offspring.
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Methods
Experimental animals
Time-dated pregnant Sprague-Dawley rats were purchased from Charles River Laboratories (Portage, MI, USA). The rats were randomly divided into two groups (n= 3 for each group): (1) saline control, and (2) cocaine 15 mg kg–1 administered intraperitoneally twice daily at 10.00 h and 16.00 h from day 15 to day 21 of gestational age. The intraperitoneal route closely resembles intranasal administration in human in the kinetics and plasma cocaine levels attained (Javaid & Davis, 1993). No fetal loss in the control and cocaine-treated groups was observed. A total of 39 male and 38 female offspring of 2-month-old rats were studied. Hearts were isolated from male and female offspring for functional studies. All procedures and protocols used in the present study were approved by the Institutional Animal Care and Use Committee of Loma Linda University, and followed the guidelines by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
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Perfused hearts subjected to ischaemia and reperfusion
Hearts were excised rapidly from 2-month-old male and female offspring, and were retrogradely perfused via the aorta in a modified Langendorff apparatus under constant pressure (70 mmHg) with gassed (95% O2–5% CO2) Krebs–Heinseleit buffer (KHB) at 37°C, as previously described (Li et al. 2004). A pressure transducer connected to a saline-filled balloon inserted into the left ventricle (LV) was used to assess ventricular function by measuring the left ventricular pressure (mmHg) and its first derivative (dP/dt). LV end diastolic pressure (LVEDP) was set at approximately 5 mmHg. Although the heart was paced in many other studies, the heart was not paced in the present study because the heart rate was a variable to be measured in the study. Nevertheless, the heart rates were not different between the control and cocaine-treated animals before and after ischaemia. After the baseline recording, hearts were subjected to 25 min of global ischaemia by stopping the perfusion, followed by 60 min of reperfusion. LV functional parameters, LV developed pressure (LVDP), heart rate (HR), dP/dtmax, dP/dtmin, and LVEDP were continuously recorded with an on-line computer. Pulmonary artery effluent was collected as an index of coronary flow (CF).
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Measurement of myocardial infarct size
Myocardial infarct size was measured as previously described (Li et al. 2003). Briefly, at the end of reperfusion, left ventricles were collected, cut into four slices, incubated with 1% triphenyltetrazolium chloride (TTC) solution for 15 min at 37°C, and immersed in formalin for 30 min. Each slice was then photographed (Kodak digital camera) separately, and the areas of myocardial infarction (MI) in each slice were analysed by computerized planimetry (Image-Pro Plus), corrected for the tissue weight, summed for each heart, and expressed as a percentage of the total LV weight.
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Measurement of DNA fragmentation by ELISA
DNA fragmentation was quantified by specific determination of cytosolic mononucleosomes and oligonucleosomes using a commercial quantitative sandwich enzyme-linked immunosorbent assay kit (Boehringer Mannheim) as previously described (Piot et al. 1997; Bae et al. 2003). Briefly, tissue samples were obtained from left ventricles after 60-min reperfusion, and put into 500 μl lysis buffer supplied in the kit. Samples were disintegrated by tissue grinder and incubated for 30 min at room temperature. After centrifugation at 200 g for 10 min, the supernatant (cytosolic fraction) was further diluted 40-fold in phosphate-buffered saline (PBS), and used as the antigen source in the sandwich ELISA. The values of absorbance were measured at 405 nm/490 nm and the background value of the immunoassay was subtracted.
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Western blot analysis
Left ventricles were isolated from 2-month-old male and female offspring, and protein levels of PKC, phospho-PKC (pSer729), Akt, and phospho-Akt (pSer473) were determined with Western blot analysis. Briefly, proteins were separated on 10% SDS-PAGE. The gel was then transferred to nitrocellulose membranes, and incubated with the primary antibodies for PKC (Santa Cruz Biotechnology; Santa Cruz, CA, USA), phospho-PKC (Upstate Biotechnology; Lake Placid, NY, USA), Akt, and phospho-Akt (Sigma; St Louis, MO, USA), respectively. After washing, membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham, Arlington Heights, IL, USA). Proteins were then visualized with an enhanced chemiluminescence detection system. Results were quantified with Kodak Electrophoresis Documentation and Analysis System and Kodak 1D Image Analysis Software. Actin was used to assess equal loading. Average actin density was determined first from all lanes in the film, and the fractional actin density for each lane was then calculated. PKC and Akt density were corrected by the fraction of the average actin for each lane in the film.
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Statistical analysis
Data were expressed as means ±S.E.M. Statistical significance (P < 0.05) was determined by two-way ANOVA or Student's t test, where appropriate.
Results
Effect of cocaine on maternal weight gain and offspring body weight
As shown in Fig. 1, maternal body weight gain was not significantly affected during the period of cocaine treatment. In addition, birth weight (6.6 ± 0.2 versus 6.3 ± 0.1, n= 3 litters, P > 0.05) and litter size (12.3 ± 1.7 versus 13.3 ± 1.5, n= 3, P > 0.05) were not significantly different between the control and cocaine-treated animals. Furthermore, at 2 months of age in both male and female offspring, body weight, LV weight, and the LV weight-to-body weight ratio were the same between the control and treatment groups (Table 1).
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Time-dated pregnant Sprague-Dawley rats received either saline as control or cocaine (30 mg kg–1 day–1) from day 15 to 21 of gestational age. Data were analysed by two-way ANOVA with gestational age as one factor and cocaine treatment as the other (n= 3).
Effect of prenatal cocaine on LV function and postischaemic recovery
Using the Langendorff preparation, LV function was assessed in isolated hearts from 2-month-old adult rats that were exposed to either saline control or cocaine before birth. As shown in Table 2, there were no significant differences in LVDP, HR, dP/dtmax, dP/dtmin and coronary flow at baseline levels between control and cocaine-treated groups in either male or female offspring. Figure 2 shows the effect of 25 min ischaemia followed by 60 min reperfusion on LV function in male offspring. Myocardial contraction was completely eliminated during 25 min ischaemia, but gradually resumed when perfusion was restored. Compared with the saline control, there were significant decreases in postischaemic recovery of LVDP, dP/dtmax, dP/dtmin and pressure-rate product (PRP), in animals exposed to cocaine before birth. However, postischaemic recovery of coronary flow and HR was not significantly different between the two groups. In contrast to the findings in the male offspring, prenatal cocaine exposure showed no effect on postischaemic recovery of LV function in the female offspring (Fig. 3).
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Hearts were obtained from 2-month-old male offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age, and were subjected to 25 min of ischaemia and 60 min of reperfusion in the Langendorff preparation. LVDP, left ventricular developed pressure; HR, heart rate; PRP, pressure rate product (LVDP x HR); CF, coronary flow. Data were analysed by two-way ANOVA with ischaemia–reperfusion as one factor and cocaine treatment as the other. The asterisk (*) indicates a significant difference (P < 0.05) from control for the entire curve (n= 8).
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Hearts were obtained from 2-month-old female offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age, and were subjected to 25 min of ischaemia and 60 min of reperfusion in the Langendorff preparation. LVDP, left ventricular developed pressure; HR, heart rate; PRP, pressure rate product (LVDP x HR); CF, coronary flow. Data were analysed by two-way ANOVA with ischaemia–reperfusion as one factor and cocaine treatment as the other (n= 7).
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Effect of prenatal cocaine on I/R-induced myocardial infarction and apoptosis
The infarct size of LV at the end of 60 min reperfusion after 25 min ischaemia is shown in Fig. 4. Ischaemia and reperfusion caused LV myocardial infarction in both male and female offspring. Prenatal cocaine treatment significantly increased infarct size in the heart of male, but not female, offspring (Fig. 4). Apoptosis in the heart was assessed by quantitative determination of fragmented DNA into mononucleosomes and oligonucleosomes determined by an ELISA specific for cytosolic histone bound DNA. Consistent with the finding of myocardial infarction, prenatal cocaine exposure significantly increased ischaemia and reperfusion-induced DNA fragmentation in left ventricle from the male, but not female, offspring (Fig. 5).
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Hearts were obtained from 2-month-old male and female offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age, and were subjected to 25 min of ischaemia and 60 min of reperfusion in the Langendorff preparation. Left ventricles were collected at the end of reperfusion, and myocardial infarct size was determined with 1% TTC staining and expressed as a percentage of the total left ventricular weight. The colour image represents one of four tissue sections obtained from each left ventricle. The summed infarct sizes, expressed as a percentage of the total left ventricular weight for the control and prenatal cocaine-treated male and female offspring, are shown below. Data were analysed by t test. *P < 0.05, cocaine versus control (n= 5–7).
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Hearts were obtained from 2-month-old male and female offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age, and were subjected to 25 min of ischaemia and 60 min of reperfusion in the Langendorff preparation. Left ventricles were collected at the end of reperfusion, and DNA fragmentation was determined using an ELISA kit as described in Methods. Data were analysed by t test. *P < 0.05, cocaine versus control (n= 4–6).
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Effect of prenatal cocaine on PKC and Akt expression in LV
Prenatal cocaine exposure significantly decreased protein levels of PKC by 59% and phospho-PKC by 75% in LV of the male offspring, but had no effect on protein levels of either PKC or phospho-PKC in the female LV (Figs 6 and 7). In contrast, prenatal cocaine treatment had no effect on protein levels of Akt or phospho-Akt in LV of either male or female offspring (data not shown).
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Left ventricles were obtained from hearts of 2-month-old male and female offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age. Protein levels of PKC were determined using Western blot analyses. Proteins were visualized with an enhanced chemiluminescence detection system, and were quantified with KODAK Electrophoresis Documentation and Analysis System and KODAK 1D Image Analysis Software. Data were analysed by t test. *P < 0.05, cocaine versus control (n= 4).
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Left ventricles were obtained from hearts of 2-month-old male and female offspring that were exposed to either saline control or cocaine (30 mg kg–1 day–1) before birth from day 15 to 21 of gestational age. Protein levels of phospho-PKC were determined using Western blot analyses. Proteins were visualized with an enhanced chemiluminescence detection system, and were quantified with KODAK Electrophoresis Documentation and Analysis System and KODAK 1D Image Analysis Software. Data were analysed by t test. *P < 0.05, cocaine versus control (n= 3–4).
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Discussion
The present study demonstrated that prenatal cocaine exposure increased heart susceptibility to ischaemia and reperfusion injury in adult male, but not female, offspring rats. Previous studies have shown that chronic daily doses of cocaine from 10 to 40 mg kg–1 during rat gestation produces plasma cocaine levels in the human use range (Spear et al. 1989; Javaid & Davis, 1993), suggesting a comparable model in the present study. In addition, kinetics of cocaine via the intraperitoneal route closely resemble those from intranasal administration in humans (Javaid & Davis, 1993). In the present study, maternal intraperitoneal cocaine administration had no effect on maternal weight gain and birth weight. Previously, we showed that subcutaneous cocaine administration to pregnant rats resulted in no change in maternal body weight, but a modest decrease in birth weight (Bae & Zhang, 2005). This may be due to the difference in the route of cocaine administration. Consistent with the present study, it has been shown that chronic intravenous maternal administration of cocaine in the rat has no significant effect on maternal body weight gain and birth weight (Iso et al. 2000).
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The finding that fetal cocaine exposure caused an increase in cardiac vulnerability in adult offspring is consistent with previous epidemiological studies in humans, as well as studies in animals, showing an association of adverse intrauterine environment and an increased risk of hypertension and ischaemic heart disease in later adult life (Barker, 2000; Zhang, 2005). However, fetal growth restriction was not observed in the present study. This is different from previous models of fetal undernutrition with significant fetal growth restriction, suggesting multiple mechanisms of in utero programming. In the present study, hearts of male offspring rats that had been exposed to cocaine before birth showed similar basal LV function to those from control animals, suggesting that prenatal cocaine exposure did not influence contractility in the resting heart. Similar findings have been obtained in our recent studies in adult rats that were exposed to hypoxia before birth (Li et al. 2003). Because the hearts were perfused at a constant pressure, and the end-diastolic pressure was set at approximately 5 mmHg, the present study was not subject to differences in afterload, preload, or endogenous sympathetic tone.
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Previously, we demonstrated in a rat model that cocaine exposure increases apoptosis in the fetal heart both in vivo and in cultured cardiomyocytes (Xiao et al. 2000, 2001; Li et al. 2005). Given that cardiomyocytes are highly differentiated cells and rarely replicate after birth, inappropriate cardiomyocyte loss through apoptosis during the early developmental period is likely to result in a permanent reduction in the number of functioning units in the myocardium. However, the developing heart may show remarkable plasticity in compensating for the loss of cardiomyocytes by an increase in myocyte size for the remaining cells (Bae et al. 2003; Li et al. 2004; Bae & Zhang, 2005). Although myocyte hypertrophy may compensate for the loss of myocytes and maintain cardiac function at the resting level, there appears to be a cost of this compensation: an increase in ischaemic vulnerability of the heart. In agreement with the present findings, previous studies of a rodent model of prenatal cocaine exposure clearly demonstrated that in adult offspring, although neurobehavioural function might appear normal under basal test conditions, deficits emerged when the animals were unable to mount appropriate responses to stressors and other environmental, cognitive or pharmacological challenges (Spear et al. 1998, 2002). This has also been demonstrated in humans (Mayes et al. 1998). Similarly, a stress test with a jet stream of high-pressure air significantly increased blood pressure in adult rats that were hypoxic during prenatal life, but had no effect in the control rats (Peyronnet et al. 2002). Taken together, these previous and our present studies support the notion that ‘it is possible and perhaps common for an organ to be programmed and then vulnerable for life without evidence until a late-life stressor challenges its adaptive capabilities’ (Thornburg, 2003).
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The finding of sex dichotomy in manifestation of increased cardiac vulnerability in adult offspring following prenatal cocaine exposure is intriguing, and is consistent with previous findings. In rodent models of prenatal cocaine exposure, it has been demonstrated that male offspring are more susceptible than females on a diversity of measures of neurobehavioural function in response to environmental and cognitive demands and stressors (Grimm & Frieder, 1985; Molina et al. 1994; Goodwin et al. 1995; Heyser et al. 1995; Spear et al. 1998; Wood & Spear, 1998; Spear et al. 2002). The sex dichotomy has also been observed in animal models of intrauterine undernutrition. Although the results are conflicting, especially in models of severe nutrient restriction during pregnancy, it has been generally accepted that the severity of hypertension in adult offspring is greater in males than in females (do Carmo Pinho Franco et al. 2003).
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It is likely that multiple mechanisms are involved in the sex-specific effect of prenatal cocaine exposure. In the present study, we found that protein levels of PKC and phospho-PKC in the left ventricle were significantly decreased only in male offspring following prenatal cocaine treatment. In contrast, Akt and phospho-Akt levels were not affected in either male or female offspring. Both Akt and PKC are downstream targets of phosphatidylinositol 3-kinase, and play an important role of cardioprotection in the regulation of cardiac ischaemia and reperfusion (Camper-Kirby et al. 2001; Chen et al. 2001; Sugden & Clerk, 2001; Murriel & Mochly Rosen, 2003). The present results suggest that Akt may not be involved in prenatal cocaine-mediated increase in heart susceptibility to ischaemia and reperfusion in adult offspring. The finding of decreased PKC and phospho-PKC levels in adult male offspring is intriguing, and suggests that prenatal cocaine may cause in utero programming of the PKC gene expression pattern leading to a down-regulation of PKC expression in the male heart. It has been demonstrated that PKC isozyme expression is controlled by distinct mechanisms that are regulated differently during development (Rybin & Steinberg, 1994). Fetal rat heart expresses multiple PKC isozymes, , , , and . In the adult heart, , , and isozymes decline markedly, and , and isozymes are found primarily in non-myocyte elements of the heart. PKC expression shows only modest age-dependent differences, and is the most abundantly expressed PKC isozyme in the adult rat heart (Bogoyevitch et al. 1993; Rybin & Steinberg, 1994). This is consistent with many studies, demonstrating an important cardioprotective role of PKC in the adult heart (Chen et al. 2001; Ping et al. 2001; Inagaki et al. 2003; Murriel & Mochly Rosen, 2003). In agreement with the present finding, studies in PC12 cells demonstrated that cocaine treatment selectively down-regulated the expression of PKC, among other isozymes (Onaivi et al. 1998). In addition, PKC protein levels were found decreased in the heart of 2-month-old male rats that had been exposed to hypoxia before birth (Li et al. 2004).
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Given the importance of PKC in cardioprotection against ischaemia and reperfusion injury, the cocaine-mediated decrease in PKC levels observed in the present study is likely to have functional significance. A recent study in a PKC knock-out mouse model has demonstrated that PKC expression is not required for normal cardiac function under physiological conditions, but PKC activation is necessary and sufficient for acute cardioprotection in cardiac ischaemia and reperfusion (Gray et al. 2003). The cardioprotective effect of PKC is proposed to be inhibition of apoptosis and hence reduction of myocardial infarction following ischaemia and reperfusion (Heidkamp et al. 2001; Liu et al. 2001, 2002; Shizukuda & Buttrick, 2002; Murriel & Mochly Rosen, 2003; Sparagna et al. 2004). In the present study, we found a significant increase in apoptosis in the heart following ischaemia and reperfusion in the male offspring that were exposed to cocaine prenatally. This is consistent with the increased myocardial infarction observed in the male heart. Different methods have been used to measure apoptosis in tissues. In our recent studies, apoptosis in isolated rat hearts subjected to ischaemia and reperfusion has been demonstrated using multiple methods including DNA fragmentation, apoptotic nuclear morphology, cleaved active form of caspase-3, and caspase-3 activity (Li et al. 2003, 2004). Ischaemia and reperfusion-mediated cell death in the heart occurs through both necrosis and apoptosis (Haunstetter & Izumo, 1998; Gill et al. 2002). Many studies have clearly demonstrated that apoptosis plays an important role in ischaemia and reperfusion-induced myocardial injury (Yaoita et al. 1998; Ma et al. 1999; Okamura et al. 2000; Yue et al. 2000; Condorelli et al. 2001).
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In conclusion, we have demonstrated for the first time, to our knowledge, in a rat model, that prenatal cocaine exposure produces a sex-specific increase in heart susceptibility to ischaemia and reperfusion injury in adult male offspring, by increasing myocardial infarct size and decreasing postischaemic recovery of left ventricular function. Although the mechanisms underlying this sex-dependent effect in the adult heart are likely to be multiplex, the present study suggests that cocaine exposure during fetal development may cause in utero programming of the PKC gene expression pattern, which may have lifelong importance in cardiac function, and may play an important role in the increased susceptibility of the heart to ischaemia and reperfusion injury in male adult offspring. Given that DNA methylation plays a critical role in epigenetic modification of gene expression, and methylation in promotor regions is generally associated with transcription repression, future studies of the potential effect of cocaine on DNA methylation in the PKC gene in the heart are warranted.
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