Prenatal cocaine exposure increases heart susceptibility to ischaemia-reperfusion injury in adult male but not female rats

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 PKCepsilon and phospho-PKCepsilon 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 PKCepsilon gene expression in the male heart may play an important role.

Figure 1. Effect of cocaine on maternal body weight gain

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).

Figure 2. Effect of prenatal cocaine on postischaemic recovery of LV function in male offspring

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 × 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).

Figure 3. Effect of prenatal cocaine on postischaemic recovery of LV function in female offspring

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 × 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).

Figure 4. Effect of prenatal cocaine on ischaemia–reperfusion-induced myocardial infarction in male and female offspring

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).

Figure 5. Effect of prenatal cocaine on ischaemia–reperfusion-induced DNA fragmentation in male and female offspring

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).

Figure 6. Effect of prenatal cocaine on PKCɛ protein levels in LV in male and female offspring

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 PKCɛ 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).

Figure 7. Effect of prenatal cocaine on phospho-PKCɛ protein levels in LV in male and female offspring

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-PKCɛ 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).