Adoptive passive transfer of rabbit beta1-adrenoceptor peptide immune cardiomyopathy into the Rag2-/- mouse: participation of the ER stress

Abstract

Auto-antibodies against the beta(1)-adrenoceptors are present in 30-40% of patients with dilated cardiomyopathy. Recently, a synthetic peptide corresponding to a sequence of the second extracellular loop of the human beta(1)-adrenoceptor (beta(1)-EC(II)) has been shown to produce endoplasmic reticulum (ER) stress, myocyte apoptosis and cardiomyopathy in immunized rabbits. To study the direct cardiac effects of anti-beta(1)-EC(II) antibody in intact animals and if they are mediated via beta(1)-adrenoceptor stimulation, we administered IgG purified from beta(1)-EC(II)-immunized rabbits to recombination activating gene 2 knock-out (Rag2(-/-)) mice every 2 weeks with and without metoprolol treatment. Serial echocardiography and cardiac catheterization showed that beta(1)-EC(II) IgG reduced cardiac systolic function after 3 months. This was associated with increase in heart weight, myocyte apoptosis, activation of caspase-3, -9 and -12, and increased ER stress as evidenced by upregulation of GRP78 and CHOP and cleavage of ATF6. The Rag2(-/-) mice also exhibited increased phosphorylation of CaMKII and p38 MAPK. Metoprolol administration, which attenuated the phosphorylation of CaMKII and p38 MAPK, reduced the ER stress, caspase activation and cell death. Finally, we employed the small-interfering RNA technology to reduce caspase-12 in cultured rat cardiomyocytes. This reduced not only the increase of cleaved caspase-12 but also of the number of myocyte apoptosis produced by beta(1)-EC(II) IgG. Thus, we conclude that ER stress plays an important role in cell death and cardiac dysfunction in beta(1)-EC(II) IgG cardiomyopathy, and the effects of beta(1)-EC(II) IgG are mediated via the beta(1)-adrenergic receptor.

Figure 1

Effects of β₁-EC_II immunoglobulin G (IgG) and metoprolol on cardiomyocyte apoptosis, and activation of caspase-9 and -3 in Rag2^−/− mice. Representative Western blots are shown for caspase-9 and -3. The optical density is expressed in arbitrary units normalized against a control sample. Bars denote SEM. N=6−8. *P<0.05, compared to Control. †P<0.05, compared to β₁-EC_II IgG.

Figure 2

Effects of β₁-EC_II IgG and metoprolol on CaMKII and p-CaMKII in Rag2^−/− mice. The optical density is expressed in arbitrary units normalized against a control sample. Representative Western blots shows that unlike p-CaMKII, CaMKII was not affected by β₁-EC_II IgG. Bars denote SEM. N=6. *P<0.05, compared to Control. †P<0.05, compared to β₁-EC_II IgG.

Figure 3

Effects of β₁-EC_II IgG and metoprolol on MAPKs in Rag2^−/− mice. Representative Western blots are shown on the left panel. The group data are shown in the bar graphs. The optical density is expressed in arbitrary units normalized against a control sample. Bars denote SEM. N=6. *P<0.05, compared to Control. †P<0.05, compared to β₁-EC_II IgG.

Figure 4

Effects of β₁-EC_II IgG and metoprolol on GRP78, CHOP and ATF6 proteins in Rag2^−/− mice. Representative Western blots are shown on the left panel. Anti-actin was used to show equal loading. β₁-EC_II IgG treatment caused cleavage of p90ATF6. Group data are shown in the bar graphs. Bars denote SEM. N=6. *P<0.05, compared to Control. †P<0.05, compared to β₁-EC_II IgG.

Figure 5

Effects of β₁-EC_II IgG and metoprolol on GRP78 and CHOP mRNA in Rag2^−/− mice. GAPDH was used as a marker for equal loading. Group data in arbitrary optical density units are shown in the bar graphs. Bars denote SEM. N=6. *P<0.05, compared to Control. †P<0.05, compared to β₁-EC_II IgG.

Figure 6

Effects of β₁-EC_II IgG and metoprolol on the processing of caspase-12 in Rag2^−/− mice. The representative Western blot shows the increased cleavage of caspase-12 by β₁-EC_II IgG, and reduction of the increased cleavage by metoprolol. Group data in arbitrary optical density units for the cleaved caspase-12 are shown in the bargraph. Bars denote SEM. N=6. *P<0.05, compared to Control. †P<0.05, compared to β₁-EC_II IgG.

Figure 7

Effects of β₁-EC_II IgG and metoprolol on Akt and GSK3β phosphorylation in Rag2^−/− mice. Representative Western blots are shown on the left panel. Group data in arbitrary optical density units for p-Akt and p-GSK3β are shown in the bargraphs. Bars denote SEM. N=6. *P<0.05, compared to Control. †P<0.05, compared to β₁-EC_II IgG.

Figure 8

Effects of caspase-12 siRNA on β₁-EC_II–induced increases of p38 MAPK, CHOP, cleaved caspase-12 and cell apoptosis in cultured neonatal rat cardiomyocytes. Representative Western blots are shown for p-p38 MAPK, CHOP, and cleaved caspase-12. Group data are shown in arbitrary optical density units. Bars denote SEM. N=6. *P<0.05, compared to Control. †P<0.05, compared to β₁-EC_II IgG without siRNA.