(A) Acute concentration-dependent effect of carvedilol (CV) on resting and isoproterenol-stimulated hemodynamic in normal conscious dogs. Isoproterenol (0.80 µg·kg^-1·min^–1 intravenously) was used to produce inotropic response. Data represent mean ± SD obtained from 4 dogs. (B) Representative M-mode echocardiograms. Note that left ventricular (LV) end-diastolic and -systolic diameters were each smaller in the CV-treated dog than in the CV-untreated (–) dog (both 4-week paced). HR = heart rate; LVPSP = left ventricular peak-systolic pressure; +dP/dt = peak (+) dP/dt of LV pressure. *p < 0.05; **p < 0.01 versus CV (–).

(A) Monobromobimane (mBB) fluorescence intensity of the ryanodine receptor (RyR)2 (corresponding to the content of free thiols) in the presence or absence of 100 µmol/l 3-morpholinosydnonimine (SIN-1). The relative content of free thiols in the RyR2 was obtained as the ratio of fluorescence intensity of the mBB to protein abundance of RyR2 and then expressed as a percentage of that in normal sarcoplasmic reticulum. (B) Acute concentration effect of carvedilol (CV) on SIN-1 (100 µmol/l)–induced decrease in mBB fluorescence intensity of the RyR2. (C) Acute effect of CV (100 µmol/l) on the mBB fluorescence intensity of the RyR2 in the presence or absence of SIN-1 (100 µmol/l) and/or DPc10 (a synthetic peptide corresponding to Gly²⁴⁶⁰-Pro²⁴⁹⁵ of RyR) (30 µmol/l).

(A) Representative time courses of Ca²⁺ uptake and the ensuing Ca²⁺ leak from SR vesicles in the presence or absence of 30 µmol/l FK506 and/or 100 µmol/l carvedilol (CV) (left). Summarized data of Ca²⁺ leak in normal, CV-untreated, and CV-treated sarcoplasmic reticulum (SR) vesicles (right). (B) Protein kinase A phosphorylation level of RyR2. (C) The amount of RyR2-bound FKBP12.6. To see the direct acute effect of CV on phosphorylation level of RyR2-pSer²⁸⁰⁸ or amount of RyR2-bound FKBP 12.6 in failing SR vesicles, the SR vesicles were mixed with CV (100 µmol/l) for 30 min and then centrifuged, followed by immunoprecipitation and Western blotting. 4W-pacing = pacing at 250 beats/min for 4 weeks; ATP = adenosine triphosphate; other abbreviations as in Figure 2.

(A, top) Representative time courses of Ca²⁺ uptake and Ca²⁺ leak from SR vesicles in presence of SIN-1 (100 µmol/l), CV (100 µmol/l), or DPc10 (30 µmol/l). Summarized data of Ca²⁺ leak (bottom). (B) Stern-Volmer plots of the fluorescence quenching data with QSY-bovine serum albumin (BSA). The top panel shows the effect of SIN-1 (100 µmol/l) in the presence or absence of CV (100 µmol/l) or CV (100 µmol/l) plus DPc10 (30 µmol/l) in normal SR. Bottom panel shows Stern-Volmer plots of QSY-BSA fluorescence quenching data of normal, failing (CV-untreated/paced), and CV-treated/paced groups. (C) Effect of SIN-1 (100 µmol/l) on phosphorylation level of RyR2-pSer²⁸⁰⁸ (top) or amount of RyR2-bound FKBP12.6 (bottom), in the presence of CV (100 µmol/l) or DPc10 (30 µmol/l). Before immunoprecipitation of RyR, SR vesicles were mixed with CV or DPc10 for 30 min and then centrifuged, followed by Western blotting. Abbreviations as in Figures 2 and 3.

(A) Representative Western blot of sarcoplasmic reticulum (SR) Ca²⁺-ATPase (top panel) and the densitometric analysis of the Western blot (bottom panel). (B) The rate of Ca²⁺ uptake. (C) Representative Western blot of Ser¹⁶-phosphorylated phospholamban (p-PLB) and total PLB (t-PLB) (top panel) and the densitometric analysis of the Western blot (bottom panel). The p-PLB values (sum of pentamer and monomer, in arbitrary units) were normalized with respect to the t-PLB values (sum of pentamer and monomer, in arbitrary units). Data are presented as means ± SD. Abbreviations as in Figure 3.

(A) Dose-dependent effect of CV on cell shortening in normal and failing (4W-paced) cardiomyocytes. (B and C) Effect of forskolin (500 nmol/l) on cell shortening and Ca²⁺ transient in normal (B) and failing cardiomyocytes (C), in the presence of CV (30 nmol/l, incubated for 12 h) or DPc10-incorporation. (D) Estimated SR Ca²⁺ content by caffeine (20 mmol/l) application in the presence of CV (30 nmol/l, incubated for 12 h) or DPc10-incorporation. (E) 2',7'-dichlorofluorescin diacetate (DCFH-DA) fluorescence images in normal and failing cardiomyocytes in presence of CV (30 nmol/l, incubated for 12 h) or DPc10-incorporation. Confocal microscopy clearly detects the DCFH-DA fluorescence signal indicated as green in failing cardiomyocytes. Cell surface membrane was fluorescently labeled as red by wheat germ agglutinin (Alexa Fluor 633 conjugate; Molecular Probes). Abbreviations as in Figure 3.