Cardiomyocyte apoptosis in autoimmune cardiomyopathy: mediated via endoplasmic reticulum stress and exaggerated by norepinephrine

Abstract

-s. Cardiomyocyte apoptosis in autoimmune cardiomyopathy: mediated via endoplasmic reticulum stress and exaggerated by norepinephrine. Am J Physiol Heart Circ Physiol 293: H1636-H1645, 2007. First published June 1, 2007; doi:10.1152 doi:10. /ajpheart.01377.2006 suggests that the autoimmune cardiomyopathy produced by a peptide corresponding to the sequence of the second extracellular loop of the ␤ 1-adrenergic receptor (␤1-ECII) is mediated via a biologically active anti-␤ 1-ECII antibody, but the mechanism linking the antibody to myocyte apoptosis and cardiac dysfunction has not been well elucidated. Since the ␤ 1-ECII autoantibody is a partial ␤1-agonist, we speculate that the cardiomyopathy is produced by the ␤ 1-receptormediated stimulation of the CaMKII-p38 MAPK-ATF6 signaling pathway and endoplasmic reticulum (ER) stress, and that excess norepinephrine (NE) exaggerates the cardiomyopathy. Rabbits were randomized to receive ␤ 1-ECII immunization, sham immunization, NE pellet, or ␤ 1-ECII immunization plus NE pellet for 6 mo. Heart function was measured by echocardiography and catheterization. Myocyte apoptosis was determined by terminal deoxytransferasemediated dUTP nick-end labeling and caspase-3 activity, whereas CaMKII, MAPK family (JNK, p38, ERK), and ER stress signals (ATF6, GRP78, CHOP, caspase-12) were measured by Western blot, immunohistochemistry, and kinase activity assay. ␤ 1-ECII immunization produced progressive LV dilation, systolic dysfunction, and myocyte apoptosis. These changes were associated with activation of GRP78 and CHOP and increased cleavage of caspase-12, as well as increased CaMKII activity, increased phosphorylation of p38 MAPK, and nucleus translocation of cleaved ATF6. NE pellet produced additive effects. In addition, KN-93 and SB 203580 abolished the induction of ER stress and cell apoptosis produced by the ␤ 1-ECII antibody in cultured neonatal cardiomyocytes. Thus ER stress occurs in autoimmune cardiomyopathy induced by ␤ 1-ECII peptide, and this is enhanced by increased NE and caused by activation of the ␤ 1-adrenergic receptor-coupled CaMKII, p38 MAPK, and ATF6 pathway.cardiomyocytes; signal transduction EVIDENCE HAS ACCUMULATED indicating that autoimmunity plays an important role in viral myocarditis and dilated cardiomyopathy (18). Among the various known anti-heart autoantibodies, an antibody activating the human ␤ 1 -adrenergic receptor (␤ 1 -AR) has been best studied. The autoantibody is not only present in 30 -40% of patients with dilated cardiomyopathy (7,20) but also has been shown to predict increased mortality in the patients (30). The importance of the ␤ 1 -AR antibody in heart failure has been established by direct demonstration of dilated cardiomyopathy in animals immunized with a peptide corresponding to the sequence of the second extracellular loop of the ␤ 1 -AR (␤ 1 -EC II ) (8, 21). In addition, the ␤ 1 -EC II antisera obtained from the experimental animals have been shown to be biologically active (28,29), with a proapoptotic action via activation of the...

Adoptive passive transfer of rabbit β1-adrenoceptor peptide immune cardiomyopathy into the Rag2−/− mouse: Participation of the ER stress

“…Also, like azetidine, anti-β 1 -EC II antibody induced ER stress in cardiomyocytes via phosphorylation of CaMKII and p38 MAPK. The apoptotic effects of anti-β 1 -EC II antibody are abolished by specific inhibitors of CaMKII and p38 MAPK (17). Furthermore, we found that inhibition of caspase-12 function by siRNA led to a marked reduction of the apoptotic effect of β 1 -EC II IgG.…”

“…The TUNEL assay was performed on paraffin-embedded myocardial tissue slides using an Apoptosis Detection System (Promega, Madison, WI), as described recently (17). The number of TUNEL-positive cells was measured by counting cells exhibiting green fluorescent nuclei at ×20 power in 10 randomly chosen fields in triplicate sections under an Olympus BX-FLA reflected light fluorescence microscope (Olympus Imaging America Corp., Melville, NY).…”

“…Ventricular muscle was prepared for Western blot analysis as described previously (17). Protein (20 μg) was denatured by heating at 95 °C for 5 min in SDS sample buffer (Cell Signaling Technology, Inc., Danvers, MA), loaded onto 10−12% SDS-polyacrylamide gels, and then transferred electrically to a polyvinylidene fluoride membrane.…”

“…Since the β 1 -adrenergic stimulation has been shown to induce endoplasmic reticulum (ER) stress and cell apoptosis (17) and inhibit the phosphatidylinositol (PI) 3-kinase/Akt survival pathway (18), we also sought to determine the transcriptional and translational induction of 78 kDa glucose regulated protein (GRP78) and C/EBP-homologous protein (CHOP), activation of ATF6, and processing of ER-resident caspase-12, as well as the functional state of Akt and its downstream GSK3β in the β 1 -EC II treated Rag2 −/− mice with and without metoprolol treatment. Finally, to determine the functional importance of caspase-12 in the β 1 -EC II IgG-induced myocyte apoptosis, we carried out experiments in cultured neonatal rat cardiomyocytes using small-interfering RNA (siRNA) technology to inhibit caspase-12 synthesis.…”

“…This dose of metoprolol is well-tolerated by the animals, and is large enough to block the inotropic effect of dobutamine at the end of experiment. β 1 -EC II IgG was purified from the sera of New Zealand White rabbits after 6 months of immunization with a synthetic β 1 -EC II peptide (17), using Montage antibody purification kits with PROSEP-G spin columns (Millipore, Billerica, MA). The IgG fraction is judged highly purified by SDS-PAGE showing only two bands (∼55 kDa and ∼26 kDa) corresponding to the heavy chain and the light chain, respectively.…”