MOESM1 of MicroRNA regulation of unfolded protein response transcription factor XBP1 in the progression of cardiac hypertrophy and heart failure in vivo

Additional file 1. Real-time PCR analysis of the ratio of miR-30a*/miR-214 in mice heart and VEGF and EDEM mRNA in siRNA-XBP1treated H9C2 cells and representative immunostaining of CD31 in hearts are presented

β-AR blockers protected cardiomyocytes against ER stress-mediated apoptosis.

<p>(A) β-AR blockers suppressed activation of caspase-12 in H9c2(2–1) cells. Cells were treated with TG (5 µmol/L) or TM (5 µg/ml) with or without metoprolol (10 µmol/L) or propranolol (10 µmol/L) for 12 hours. Cell lysates were then immunoblotted for caspase-12. Caspase-12 cleavage was normalized to β-actin. *P<0.05 vs. control, #P<0.05 vs. TM, & P<0.05 vs. TG. (B) and (C) Hochst-positive cells (%). H9c(2–1) cells were pretreated with metoprolol (10 µmol/L, 1 h) or propranolol (10 µmol/L, 1 h), then exposed to TG (5 µmol/L, 24 h) or TM (5 µg/ml, 24 h) before staining with Hochst33258 as indicated. Hochst-positive cells are expressed as a percentage of the number of total cells. **P<0.001 vs. control. # P<0.05 vs. TG or TM. (D) Anexin V-positive cells (%). H9c(2–1) cells were pretreated with metoprolol (10 µmol/L, 1 h) or propranolol (10 µmol/L, 1 h), then exposed to TG (5 µmol/L, 24 h) before staining with Anexin V/PI as indicated. Analyze by flow cytometry immediately after incubation. Anexin V-positive cells are expressed as a percentage of the number of total cells. *P<0.05 vs. control. #P<0.05 vs. TG.</p

Hemodynamic parameters in AAC rats and AAC rats with β-AR blocker treatment.

<p>HR, heart rate; LVEDP, left ventricular end diastolic pressure; Ea, arterial elastance; dP/dtmax, maximal slope of systolic pressure increment; dP/dt min maximal slope of diastolic pressure decrement; meto, metoprolol; prop, propranolol. Data are mean ± s.d. * P<0.05 versus control; †P<0.05 vs. AAC.</p

β-AR blockers suppressed ER stress in hypertrophic and failing hearts of rats.

<p>(A) ER chaperone GRP78 and spliced XBP-1 were increased in AAC rats (1or 4 or 8 weeks after operation), and metoprolol treatment (30 mg/kg/d for 8 weeks) reduced the epression of GRP78 and spliced XBP-1. Proteins were normalized to β-actin. (B) GRP78 was increased in AAC rats (4 weeks), and propranolol treatment (30 mg/kg/d for 4 weeks) reduced the epression of GRP78. Protein was normalized to β-actin. (C) Immunohistochemical analyses of rats’ hearts and number of GRP78, KDEL and CHOP-positive cells per mm². Scale bar, 40 µm. For (A) to (C), *P<0.05 vs. sham, #P<0.05 vs. AAC. (D) GRP78 and spliced XBP-1 was increased in Iso rats, and metoprolol treatment (30 mg/kg/d for 2 weeks) reduced the epression of GRP78 and spliced XBP-1. Proteins were normalized to β-actin. *P<0.05 vs. control, #P<0.05 vs. Iso.</p

β-AR blockers suppressed ER stress-mediated apoptosis in cardiac hypertrophy and failure.

<p>(A) and (B) CHOP was increased in AAC rats (4 or 8 weeks after operation), and metoprolol (30 mg/kg/d for 4 or 8 weeks)or propranolol (30 mg/kg/d for 4 or 8 weeks) treatment reduced expression of CHOP. CHOP was normalized to β-actin. (C) Representative images of TUNEL showing cardiac myocytes apoptosis and quantitative analysis of TUNEL-positive myocardial cells in rats. Nuclei of normal cells are blue, and nuclei of apoptosis cells (TUNEL-positive cells) are brown.</p

Hemodynamic parameters in Iso rats and Iso rats with β-AR blocker treatment.

<p>HR, heart rate; PES, end-systolic pressure; PED, end-diastolic pressure; dP/dtmax, maximal slope of systolic pressure increment; dP/dt min maximal slope of diastolic pressure decrement; Iso, isoproterenol; meto, metoprolol. Data are mean ± s.d. *P<0.05 vs. control.</p

β-AR blockers attenuated cardiac hypertrophy and improved the function of failing hearts

<p>. (A) to (C) showed β-AR blockers attenuated cardiac hypertrophy in rats induced by abdominal aortic constriction (AAC). (A) Representative images of hearts. Scale, 5 mm. (B) Ratio of heart weight to body weight. *P<0.05 vs. sham. (C) HE staining of rat hearts and cardiomyocyte cross-sectional diameter (µm). Scale bar, 50 µm. *P<0.05 vs. sham, #P<0.05 vs. AAC. (D) and (E) showed β-AR blocker attenuated Iso-induced cardiac hypertrophy in rats. (D) Ratio of heart weight to body weight. *P<0.05 vs. control. (E) HE staining of rat hearts and cardiomyocyte cross-sectional diameter (µm). Scale bar, 50 µm. Iso, isoproterenol. *P<0.05 vs. control, #P<0.05 vs. Iso.</p

Induction of ER Stress in Human Heart Failure.

<p>ER stress markers including phosphorylated eIF2α (p-eIF2α), phosphorylated PERK (p-PERK), GRP78 and CHOP and apoptosis marker phosphorylated c-Jun (p-c-Jun), were examined in the heart samples from patients with heart failure. (A) ER stress was increased in heart transplant recipients’ failing hearts compared with normal heart. DCM, dilated cardiomyopathy. (B) p1-p9, heart samples of 9 patients undergoing mitral valve replacement; N1 and N2, normal human hearts. Proteins were normalized to β-actin. *<i>P</i><0.05 vs. normal hearts.</p

β-AR blockers suppressed overactivation of Calmodulin Kinase II in rat failing hearts.

<p>(A) Metoprolol alleviated ER stress and overactivation of CaMKII induced by tunicamycin (TM). Cells were pretreated with KN93 (KN, 0.5 µmol/L or 2 µmol/L), metoprolol (M, 10 µmol/L or 20 µmol/L) for 1 hour, and exposed to TM (5 µg/ml) for 24 hours. Cell lysates were then immunoblotted for phosphorylated CaMKII, phosphorylated PERK, GRP78 and CHOP, which were all normalized to β-actin. *P<0.05 vs. control, #P<0.05 vs. TM. (B) Metoprolol suppressed the overactivation of CaMKII in AAC rats. *P<0.05 vs. sham, #P<0.05 vs. AAC.</p

Clinical characteristics of patients with mitral valve replacement.

<p>MI, mitral insufficiency; MS, mitral stenosis; AF, atrial fibrillation; TI, tricuspid insufficiency; MVP, mitral valve prolapse; N/A, not applicable.</p