An Alpha-1A Adrenergic Receptor Agonist Prevents Acute Doxorubicin Cardiomyopathy in Male Mice

Figures

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  Fig 1. A61603 potently activates ERK in cultured neonatal rat ventricular myocytes (NRVMs). ERK activation was quantified by immunoblot as the ratio of phospho- to total-ERK (P-ERK/T-ERK) after 10 min with the beta-blocker propranolol (PROP) 200 nM, then 5 min with various α1-agonists or vehicle. Dose-response curves normalized to vehicle+PROP were analyzed by non-linear regression (N = 3–8 independent cultures, each dose in duplicate in each experiment). A61603 blots are shown. Table 1 has Emax and EC50 for each agonist.

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  Fig 2. A61603 activates ERK and protects WT adult mouse ventricular myocytes (AMVMs) from DOX toxicity in vitro, via the α1A. (A) ERK activation in cultured AMVMs was quantified by immunoblot as the ratio of phospho- to total-ERK (P-ERK/T-ERK) after 10 min with the beta-blocker PROP 200 nM, then 5 min with A61603 or vehicle. Dose-response curves normalized to vehicle+PROP were analyzed by non-linear regression (n = 5 cultures from separate hearts, each in duplicate; values are mean ± SE). Emax is significantly different from 1.0 by 95% confidence limits (p<0.05). Representative blots are shown. (B) WT or α1A KO cultured AMVMs were treated overnight with DOX 20 μM and PROP 200 nM, in the presence of varying doses of A61603 or vehicle. Myocyte survival by MTT assay is shown as mean ± SE, and was analyzed by non-linear regression and 2-way ANOVA, relative to PROP alone at 100% and DOX+PROP at 0%; n = 3–8 cultures from separate hearts, each in duplicate or triplicate. WT pEC50 and Emax are shown. Emax is significantly different from 1.0 by 95% confidence limits (p<0.05).

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  Fig 3. A61603 activates ERK in vivo at subpressor dosing. (A) Normal male WT mice age 10–12 w were treated with A61603 (0.01–10 μg/kg/d) or vehicle (100 μM vitamin C) continuously for 7 d via s.c. osmotic mini-pump, and cardiac ERK activation was quantified by immunoblot as the ratio of P-ERK/T-ERK. Results are for the cytosolic fraction of ventricular homogenates, which contained approximately 75% of total- and phospho-ERK. The A61603 dose-response curve was normalized to vehicle, and analyzed by non-linear regression (N = 5 mice per dose, mean ± SE). Emax is significantly different from 1.0 by 95% confidence limits (p<0.05). A blot is shown below. (B) Baseline BP was recorded for 4 d, then A61603 (10 ng/kg/d and 10 μg/kg/d) or vehicle were infused continuously for 7 d by osmotic pump. Systolic BP (upper traces) and diastolic BP (lower traces) are shown during (left) periods of rest (2–3 pm) and (right) periods of activity (12–1 am). Numbers of mice are indicated. A hypertensive dose of NE (2.5 mg/kg/d) was a positive control.

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  Fig 4. Low-dose A61603 increases survival of WT male mice in a doxorubicin (DOX) cardiomyopathy model, but not in α1A-KO mice. (A) Young male WT and α1A-KO mice were randomized to A61603 10 ng/kg/d (A6) or vehicle (100 μM vitamin C, Veh) via s.c. osmotic mini-pump. Immediately after pump implantation, mice were injected with a single dose of DOX 25 mg/kg i.p. Mice were monitored daily, and euthanized for HF per protocol or on day 7. Echo was done prior to treatment in some mice, and on the day of sacrifice in surviving mice. Operators were blinded to genotype and treatment. (B) Kaplan-Meier 7-day survival curves, with numbers of mice indicated.

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  Fig 5. A61603 preserves cardiac function after DOX in WT male mice, but not in α1A-KO mice. Mice given DOX were treated with A61603 or Vehicle as in Fig 4. Values are fractional shortening (FS) by echo, mean ± SE, n = mice, p by one-way ANOVA with Newman-Keuls multiple comparisons test.

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  Fig 6. A61603 preserves hemodynamics in male mice after DOX, via the α1A. Mice given DOX were treated with A61603 or Vehicle as in Fig 4. Shown are (A) HR, (B) SV, and (C) CO, all from echo, for each treatment group and genotype. Values are mean ± SE, n = mice, p by one-way ANOVA with Newman-Keuls multiple comparisons test.

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  Fig 7. A61603 prevents cardiac cell death and myocardial fibrosis after DOX in WT male mice. Male mice were treated with DOX or saline injection, and randomized to A61803 or Vehicle, as in Fig 4. Shown are indices at end of study for (A) Cardiac myocyte necrosis measured by serum CK activity in blood collected by LV puncture, and TUNEL staining for apoptosis in LV sections; and (B) LV myocardial fibrosis by sirius red staining. Number (n) is hearts; p by one-way ANOVA with Newman-Keuls multiple comparisons test.

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  Fig 8. Young female mice are resistant to DOX cardiac toxicity. Female WT mice age 15 weeks were randomized to vehicle or a single dose of DOX 15–40 mg/kg i.p. Mice were monitored daily for 7 days, and euthanized for HF per protocol or on day 7. Echo was done prior to treatment, and on the day of sacrifice. Operators were blinded. The highest DOX dose reduced HR (A) and SV (B), and tended to reduce survival (C), but DOX did not change FS (D). p by one-way ANOVA with Newman-Keuls multiple comparisons test.