Differential cardiotoxic/cardioprotective effects of beta-adrenergic receptor subtypes in myocytes and fibroblasts in doxorubicin cardiomyopathy

Abstract

beta-Adrenoceptor (beta-AR) subtypes act through different signaling pathways to regulate cardiac function and remodeling. Previous in vivo data show a markedly enhanced cardiotoxic response to doxorubicin in beta2-/- mice, which is rescued by the additional deletion of the beta1-AR. We determined whether this differential response was myocyte specific by examining the effects of doxorubicin in myocytes and fibroblasts from WT and beta1, beta2 and beta1/beta2-/- mice. Cells were exposed to doxorubicin at 1-50 microM and viability and apoptosis assessed at 6, 24 and 48 h. WT myocytes showed a time and dose-dependent decrease in viability (42% decrease at 1 microM after 24 h). beta2-/- Myocytes showed a greater decrease in viability vs. WT (20.8% less at 6 h; 14% less at 24 h, P<0.05); beta1-/- and beta1/beta2-/- myocytes showed enhanced survival (beta1-/- 11%; beta1/beta2-/- 18% greater than WT, P<0.05). TUNEL staining demonstrated a similar differential susceptibility (WT 26% apoptotic nuclei, beta2-/- 45.9%, beta1/beta2-/- 16.8%, P<0.05). beta2-/- Fibroblasts also showed enhanced toxicity. Pertussis toxin pretreatment of WT cells decreased survival similar to the beta2-/-, suggesting a role for Gi signaling. JNK was differentially activated in beta2-/- myocytes after doxorubicin and its inhibition increased cardiotoxicity. In conclusion, the differential cardioprotective/cardiotoxic effects mediated by beta1 vs. beta2-AR subtypes in knockout mice are recapitulated in myocytes isolated from these mice. beta2-ARs appear to play a cardioprotective role, whereas beta1-ARs a cardiotoxic role.

Fig. 1

Doxorubicin decreases viability in WT cardiomyocytes in a time and dose-dependent manner. Doxorubicin 1, 10 and 50 µM was administered to neonatal cardiomyocytes obtained from WT mice. Viability was assessed at 6 and 24 h by MTT assay. At 6 h viability was decreased only at 50 µM, whereas at 24 h viability was decreased at both 1 and 10 µM and severely decreased at 50 µM. * P < 0.05 vs. baseline.

Fig. 2

Disruption of β1 vs. β2-AR has opposite effects on viability of cardiomyocytes exposed to doxorubicin. Doxorubicin 1, 10 and 50 µM was administered to neonatal cardiomyocytes obtained from WT and β-AR knockout mice. After 6 h viability was assessed by MTT assay. A differential response to doxorubicin was observed in β-AR knockout myocytes: slightly improved survival in β1−/− myocytes, markedly decreased survival in β2−/− myocytes, and markedly improved survival in β1/β2−/− myocytes. *P < 0.05.

Fig. 3

Effect of β-AR disruption on doxorubicin-induced cardiomyocyte apoptosis. TUNEL staining of isolated cardiomyocytes from WT and β-AR knockout mice shows a differential susceptibility to doxorubicin. Cells were exposed to doxorubicin 1 µM for 6 h. (a) WT myocytes without doxorubicin (control), (b) WT myocytes with doxorubicin, (c) β2−/− show increased apoptotic nuclei with doxorubicin, (d) β1/β2−/− myocytes show decreased apoptotic nuclei with doxorubicin.

Fig. 4

Cardiotoxic/cardioprotective effects of β-AR subtypes during exposure of cardiomyocytes to doxorubicin. (a) Changes in cell viability compared to WT. Cardiomyocytes were exposed to 1 µM doxorubicin for 24 h. Myocytes lacking the β2-AR showed increased toxicity; in contrast, myocytes lacking the β1-AR showed enhanced survival. Absence of both receptors (β1/β2−/−), rescued the increased toxicity in the β2−/−. (b) Changes in apoptosis compared with WT. Cardiomyocytes were exposed to 1 µM doxorubicin for 24 h. Myocytes lacking the β2-AR showed increased apoptotic nuclei after doxorubicin compared with WT; in contrast, myocytes lacking both β1 and β2-receptors showed decreased apoptosis compared with both WT and β2−/−. * P < 0.05.

Fig. 5

Differential activation of MAPK in β-AR knockout cardiomyocytes after exposure to doxorubicin. Western blot and graphic representation for phospho-JNK (a), p38 (b), and ERK1/2 (p44/p42) (c, d) showing increased phosphorylation of JNK in β2−/− cardiomyocytes after doxorubicin compared to baseline (B), no significant change in p38 activity, and increase in ERK1/2 (p44/p42) activity compared to WT. † P < 0.05, compared to baseline, * P < 0.05, compared to WT. AU = Arbitrary Units.

Fig. 6

Effect of MAPK inhibitors on doxorubicin-induced toxicity in β2-AR knockout cardiomyocytes. Myocytes derived from β2−/− mice were exposed to MAPK inhibitors: SB203580 10 µM for p38, PD98059 10 µM for ERK and SP600125 10 µM for JNK. Inhibition of both ERK and JNK further decreased viability. † P < 0.05, compared to control, * P < 0.05, compared to doxorubicin-treated cells.