Antibodies used for western-blot.

<p>Primary antibodies were diluted in <i>TBS-T</i>, excepted for Giα₂ protein detection which was diluted in 5% non-fat dry milk in <i>TBS-T</i>. All secondary antibodies were diluted in 1% non-fat dry milk in <i>TBS-T.</i></p

β₂-AR expression and function are increased in Dox-CM hearts.

<p>A. Representative β₂-AR immunoblotting at days 35 (d35) and 70 (d70). B. β₂-AR protein quantification at day 35. C. β₂-AR protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to the corresponding GAPDH band intensity was calculated. D. Cardiac inotropic (dP/dt_max), E. lusitropic (dP/dt_min) and F. chronotropic (HR) effects of increasing concentrations of isoproterenol (10⁻⁹ to 10⁻⁴ M) in presence of 10⁻⁶ M of CGP-20712A a β₁-AR antagonist and 10⁻⁶ M of L-748,337 a β₃-AR antagonist were evaluated on isolated perfused hearts. B: Baseline; B+A; Baseline the in presence of antagonists. **: <i>P</i><0.001 <i>vs</i> Ctrl; Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.</p

Representative images obtained by echocardiography at day 35 in Ctrl (A) and Dox-CM (B) rats.

<p>Images were obtained with a short axis view of a two-dimensionally directed M-mode. Ctrl: control, dAWth: diastolic anterior wall thickness, Dox-CM: Doxorubicin-induced cardiomyopathy, dPWth: diastolic posterior wall thickness, LVEDD: left ventricular end diastolic diameter, LVESD: left ventricular end systolic diameter, sAWth: systolic anterior wall thickness, sPWth: systolic posterior wall thickness.</p

Impaired β₁-AR expression in Dox-CM hearts was not associated to an impaired cardiac β₁-AR function.

<p>A. Representative β₁-AR immunoblotting at days 35 (d35) and 70 (d70). B. β₁-AR protein quantification at day 35. C. β₁-AR protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to the corresponding GAPDH band intensity was calculated. D. Cardiac inotropic (dP/dt_max), E. lusitropic (dP/dt_min) and F. chronotropic (HR) effects of increasing concentrations of isoproterenol (10⁻⁹ to 10⁻⁵ M) in the presence of 10⁻⁶ M ICI-118,551 a β₂-AR antagonist and 10⁻⁶ M of L-748,337 a β₃-AR antagonist were evaluated on isolated perfused hearts. B: Baseline; B+A; Baseline in presence of antagonists. **: <i>P</i><0.001 <i>vs</i> Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.</p

<i>In vivo</i> cardiac parameters obtained by LV catheterization in Ctrl and Dox-CM rats, at d35.

<p>Ctrl: control; Dox-CM: Doxorubicin-induced cardiomyopathy; dP/dt_max: left ventricular contraction velocity; dP/dt_min: left ventricular relaxation velocity; LV: left ventricle; LVEDP: left ventricular end diastolic pressure; Tau: index of left ventricular relaxation constant. Values are means ± sem. *: <i>P</i><0.05 <i>vs</i> Ctrl.</p

Gi protein expression and involvement in β₂-AR cardiac contractility.

<p><b>A.</b> Representative Giα₂ immunoblotting at days 35 (d35) and 70 (d70). <b>B.</b> Giα₂ protein quantification at day 35. <b>C.</b> Giα₂ protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to the corresponding GAPDH band intensity was calculated. <b>D.</b> Cardiac inotropic (dP/dt_max), <b>E.</b> lusitropic (dP/dt_min) and <b>F.</b> chronotropic (HR) effects of increasing concentrations of isoproterenol (10⁻⁹ to 10⁻⁴ M) in the presence of 10⁻⁶ M of CGP-20712A and 10⁻⁶ M of L-748,337 were evaluated on isolated perfused hearts pre-treated or not with 4 µg.L⁻¹ of pertussis toxin (PTX) a Gi protein inhibitor. B: Baseline; B+A; Baseline in the presence of antagonists. *: <i>P</i><0.05 <i>vs</i> Ctrl; **: <i>P</i><0.001 <i>vs</i> Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.</p

Gsα protein expression and forskolin response.

<p><b>A.</b> Representative Gsα immunoblotting at days 35 (d35) and 70 (d70). <b>B.</b> Gsα protein quantification at day 35. <b>C.</b> Gsα protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to the corresponding GAPDH band intensity was calculated. <b>D.</b> Cardiac inotropic (dP/dt_max), <b>E.</b> lusitropic (dP/dt_min) and <b>F.</b> chronotropic (HR) effects of increasing concentrations of the adenylyl cyclase activator forskolin (3.10⁻⁸ to 10⁻⁵ M) were evaluated on isolated perfused hearts. *: <i>P</i><0.05 <i>vs</i> Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.</p

<i>In vivo</i> cardiac parameters obtained by echocardiography-Doppler in Ctrl and Dox-CM rats.

<p>Ctrl are showed in empty bar and Dox-CM in full bar. Values are means ± sem *: <i>P</i><0.05 <i>vs</i> respective Ctrl **: <i>P</i><0.001 <i>vs</i> respective Ctrl. d35: day 35, d70: day 70, Dox-CM: Doxorubicin-induced cardiomyopathy, dPWth: diastolic posterior wall thickness; E: E wave velocity; Ea: peak velocity of basal and lateral walls in early diastole; IVRT: isovolumic relaxation time; LV: left ventricle; LVEF: left ventricular ejection fraction; LVEV: left ventricular end volume; LVSF: left ventricular shortening fraction; Sa: peak velocity of basal and lateral walls in systole.</p

β₃-AR expression and function in Dox-CM hearts.

<p>A. Representative β₃-AR immunoblotting at days 35 (d35) and 70 (d70). B. β₃-AR protein quantification at day 35. C. β₃-AR protein quantification at day 70. Protein levels were quantified using Amersham ImageQuant RT-ECL camera (GE Healthcare). The band signals were assessed by densitometry with ImageQuant TL software (GE Healthcare) and a ratio to the corresponding GAPDH band intensity was calculated. D. Cardiac inotropic (dP/dt_max), E. lusitropic (dP/dt_min) and F. chronotropic (HR) effects of increasing concentrations of SR 58611A (10⁻⁹ to 10⁻⁵ M) were evaluated on isolated perfused hearts. **: <i>P</i><0.001 <i>vs</i> Ctrl. Ctrl: control, Dox-CM: Doxorubicin-induced cardiomyopathy.</p

Cardiac morphological parameters in Ctrl and Dox-CM rats, at d35 and d70.

<p>Ctrl: control; Dox-CM: Doxorubicin-induced cardiomyopathy; LV: left ventricle; wt: weight. Values are means ± sem. *: <i>P</i><0.05 <i>vs</i> respective Ctrl. **: <i>P</i><0.001 <i>vs</i> respective Ctrl.</p