Minocycline suppresses oxidative stress and attenuates fetal cardiac myocyte apoptosis triggered by in utero cocaine exposure - PubMed (original) (raw)

Minocycline suppresses oxidative stress and attenuates fetal cardiac myocyte apoptosis triggered by in utero cocaine exposure

Indrani Sinha-Hikim et al. Apoptosis. 2011 Jun.

Abstract

This study investigates the molecular mechanisms by which minocycline, a second generation tetracycline, prevents cardiac myocyte death induced by in utero cocaine exposure. Timed mated pregnant Sprague-Dawley (SD) rats received one of the following treatments twice daily from embryonic (E) day 15-21 (E15-E21): (i) intraperitoneal (IP) injections of saline (control); (ii) IP injections of cocaine (15 mg/kg BW); and (iii) IP injections of cocaine + oral administration of 25 mg/kg BW of minocycline. Pups were killed on postnatal day 15 (P15). Additional pregnant dams received twice daily IP injections of cocaine (from E15-E21) + oral administration of a relatively higher (37.5 mg/kg BW) dose of minocycline. Minocycline treatment continued from E15 until the pups were sacrificed on P15. In utero cocaine exposure resulted in an increase in oxidative stress and fetal cardiac myocyte apoptosis through activation of c-Jun-NH(2)-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK)-mediated mitochondria-dependent apoptotic pathway. Continued minocycline treatment from E15 through P15 significantly prevented oxidative stress, kinase activation, perturbation of BAX/BCL-2 ratio, cytochrome c release, caspase activation, and attenuated fetal cardiac myocyte apoptosis after prenatal cocaine exposure. These results demonstrate in vivo cardioprotective effects of minocycline in preventing fetal cardiac myocyte death after prenatal cocaine exposure. Given its proven clinical safety and ability to cross the placental barrier and enter into the fetal circulation, minocycline may be an effective therapy for preventing cardiac consequences of in utero cocaine exposure.

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Figures

Fig. 1

Fig. 1

In situ detection of cardiac myocyte apoptosis detected by TUNEL assay. At P15, compared with controls (a), in which little or no apoptosis is detected, a marked increase in the incidence of cardiac myocyte apoptosis is evident in the ventricles after prenatal cocaine exposure (b). Concomitant administration of minocycline (25 mg/kg BW) from E15 to E21 fails to prevent in utero cocaine exposure-induced activation of cardiac myocyte apoptosis in fetal hearts. Scale bar × 50 μm. c Representative examples of cardiac myocytes stained with a-actinin. Chromatin was stained with DAPI. Scale bar × 15 μm. d Co-staining for caspase 3 (green) and a-actinin (red) shows activation of caspase 3 in cardiac myocytes. Scale bar × 10 μm. e, f The overall morphology of apoptotic cardiac myocytes. Note varying degrees of chromatin condensation and fragmentation typical of apoptosis. Scale bar × 2 μm (e) and 1 μm (f). g Western blot analysis of ventricular lysates shows increased levels of phospho-p38 MAPK (p-p38 MAPK) and phospho-JNK (p-JNK) and active caspase 9 and caspase 3 in neonatal hearts after prenatal cocaine exposure compared with controls. Concomitant administration of minocycline fails to prevent in utero cocaine exposure-induced activation of p38 MAPK, JNK, and caspases. The gels are representative of three pups in each group from one of three separate experiments (n × 10 pups per group). GAPDH in the immunoblot is shown as a loading control. Con, Control; Coc, Cocaine; and Coc + M, Cocaine plus minocycline (Color figure online)

Fig. 2

Fig. 2

Continued minocycline (37.5 mg/kg BW) treatment from E15 up to P15 attenuates fetal cardiac myocyte apoptosis triggered by in utero cocaine exposure. a Compared with control neonates, where no apoptosis is detected, prenatal cocaine administration results in a marked increase in cardiac myocyte apoptosis (b) and that can be effectively prevented by minocycline treatment (c). Scale bar × 50 μm. d Quantitative changes in the incidence of cardiac myocyte apoptosis among various treatment groups. Apoptotic rate was expressed as the percentage of TUNEL positive nuclei per total nuclei (apoptotic plus nonapoptotic nuclei) counted in a unit reference area. Values are the mean ± SEM of five pups per group. Means with unlike superscripts are significantly (P < 0.001) different

Fig. 3

Fig. 3

Continued minocycline treatment from E15 up to P15 prevents in utero cocaine exposure-induced oxidative stress and activation of p38 MAPK and JNK. Ventricles from pups born to cocaine-treated pregnant dams exhibit significantly greater oxidative stress, as evidenced by low (P < 0.01) GSH/GSSG ratio (a) and increased (P < 0.001) 4-HNE levels (b) compared to those of control neonates. Minocycline treatment significantly prevented in utero cocaine exposure-induced oxidative stress in fetal hearts. Values are the mean ± SEM of ten pups per group. Means with unlike superscripts are significantly different. EIA reveals a significant (P < 0.005) increase in both phospho-JNK (c) and phopsho-p38 MAPK (d) levels in fetal hearts after prenatal cocaine exposure when compared with control neonates, which could be significantly (P < 0.005) prevented by minocycline treatment. Values are the mean ± SEM of ten pups per group. Means with unlike superscripts are significantly different

Fig. 4

Fig. 4

a Western blot analysis of ventricular lysates shows significantly (P < 0.05) increased levels of phospho-p38 MAPK and phospho-JNK in neonates after prenatal cocaine exposure compared with control neonates. Minocycline treatment significantly (P < 0.05) prevents such in utero cocaine exposure-induced activation of these kinases. However, in utero cocaine exposure had no effect on ERK activation. The gels are representative of two neonates in each group from one of three separate experiments (n × 10 pups per group). GAPDH in the immunoblot is shown as a loading control. b Double-immunofluorescence staining for TUNEL (green) and phospho-p38 MAPK (red) from pups born to cocaine-treated mothers show activation of p38 MAPK in fetal cardiac myocyte undergoing apoptosis (shown as yellow). Scale bar × 25 μm (Color figure online)

Fig. 5

Fig. 5

In utero cocaine exposure-induced activation of p38-MAPK and JNK is further associated with a significant (P < 0.05) increase in BAX expression (BCL-2 levels remain unaltered) and active caspases 9 and 3 levels (P < 0.001) as detected by immunoblotting. Minocycline treatment significantly (P < 0.05) up-regulates BCL-2 levels, suppresses prenatal cocaine exposure-induced changes in the BAX levels and prevents (P < 0.001) activation of caspases in fetal hearts. The gels are representative of two neonates in each group from one of three separate experiments (n × 10 pups per group). GAPDH in the immunoblot is shown as a loading control

Fig. 6

Fig. 6

Minocycline treatment from E15 up to P15 suppresses in utero cocaine exposure-induced cytochrome c release from mitochondria. Ventricular lysates from neonates were fractionated into cytosolic and mitochondrial fractions and analyzed by Western blotting. a Representative Western blots of mitochondrial (MITO) and cytosolic fractions of neonates show the presence of COX IV only in mitochondrial fractions but not in cytosolic fractions. The absence of COX IV in the cytosolic fractions confirmed that the cytosolic preparations were free of mitochondrial contamination. b Representative Western blots of cytosolic fractions from various treatment groups show accumulation of cytochrome c in ventricles of neonates born to cocaine-treated moms and that can be significantly (P < 0.05) suppressed by minocycline treatment. No cytochrome c is detected in the cytosol of ventricles from control neonates. The gels are representative of two neonates in each group from one of three separate experiments. Actin in the immunoblot is shown as a loading control. c Densitometric analysis shows significant (P < 0.05) inhibition of cocaine-induced cytochrome c release after minocycline treatment

Fig. 7

Fig. 7

Key signaling pathways involved in minocycline-mediated protection of cardiac myocytes from apoptosis trigged by in utero cocaine exposure. Increased oxidative stress generated in response to prenatal cocaine exposure can result in activation of both JNK and p38 MAPK in fetal hearts. It is likely that activation of these signaling cascades promote fetal cardiac myocyte apoptosis through perturbation of the BAX/BCL-2 rheostat and subsequent activation of cytochrome c-mediated death pathway. Minocycline seems to be working at various steps of the signal transduction pathways involving suppression of oxidative stress, activation of JNK and p38 MAPK, perturbation of the BAX/BCL-2 ratio, cytochrome c release, and activation of caspases 9 and 3

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