Amyloidogenic light chains induce cardiomyocyte contractile dysfunction and apoptosis via a non-canonical p38alpha MAPK pathway

Abstract

Patients with primary (AL) cardiac amyloidosis suffer from progressive cardiomyopathy with a median survival of less than 8 months and a 5-year survival of <10%. Contributing to this poor prognosis is the fact that these patients generally do not tolerate standard heart failure therapies. The molecular mechanisms underlying this deadly form of heart disease remain unclear. Although interstitial amyloid fibril deposition of Ig light chain proteins is a major cause of cardiac dysfunction in AL cardiac amyloidosis, we have previously shown that amyloid precursor proteins directly impair cardiac function at the cellular and isolated organ levels, independent of fibril formation. In this study, we report that amyloidogenic light chain (AL-LC) proteins provoke oxidative stress, cellular dysfunction, and apoptosis in isolated adult cardiomyocytes through activation of p38 mitogen-activated protein kinase (MAPK). AL-LC-induced p38 activation was found to be independent of the upstream MAPK kinase, MKK3/6, and instead depends upon transforming growth factor-beta-activated protein kinase-1 binding protein-1 (TAB1)-mediated p38alpha MAPK autophosphorylation. Treatment of cardiomyocytes with SB203580, a selective p38 MAPK inhibitor, significantly attenuated AL-LC-induced oxidative stress, cellular dysfunction, and apoptosis. Our data provide a unique mechanistic insight into the pathogenesis of AL-LC cardiac toxicity and suggest that TAB1-mediated p38alpha MAPK autophosphorylation may serve as an important event leading to cardiac dysfunction and subsequent heart failure.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

AL-LC activates p38 MAPK in isolated adult rat cardiomyocytes. (A) Immunoblots showing phospho- and total p38, JNK, and ERK MAPKs in isolated cardiomyocytes incubated with vehicle, Con-LC (20 μg/mL), or AL-LC (20 μg/mL) for 15 min. Bar graphs summarize the data from three independent experiments, with vehicle control set as 1-fold. *, P < 0.05 vs. vehicle; #, P < 0.05 vs. Con-LC. (B) Phosphorylation of p38 in cardiomyocytes under the same treatment conditions as above for designated times. Immunoblots are representatives of two separate experiments. (C) Bar graph summarizing the ratio of phospho- to total p38 expression in cardiomyocytes incubated for 15 min with Con-LC and AL-LC isolated from two myeloma patients (code no. : 96–100 and 00–161) and eight AL patients (code no.: 00–131, 00–127, 01–003, 99–145, 00–112, 01–091, 02–131, and 01–052), respectively, with vehicle control set as 1-fold. The experiments were repeated three times. Detailed information for the human samples is summarized in Table S1.

Fig. 2.

Pharmacological inhibition of p38 MAPK activity prevents AL-LC–induced oxidant stress and contractile dysfunction in isolated adult cardiomyocytes. Inhibition of p38 MAPK with SB203580 (5 μM) attenuated AL-LC–induced reactive oxygen species (ROS) production as determined by DCF fluorescence (A) and contractile dysfunction indicated by the decreased percent cell shortening (%CS) as measured by edge detection methodology (B). Bar-graph data are from three independent experiments. For B, data are means ± SE from three independent experiments, with each individual experiment average values taken from 8 to 12 cell measurements for each group. Representative tracings of single cell shortening at respective conditions are displayed above the bar graph. *, P < 0.05 vs. vehicle and #, P < 0.05 vs. Con-LC in the presence or absence of SB203580; †, P < 0.05 vs. AL-LC in the absence of SB203580.

Fig. 3.

AL-LC activates p38 MAPK via autophosphorylation but not MKK3/6 activation. (A) Immunoblot shows no difference in MKK3/6 phosphorylation among the cardiomyocytes incubated with vehicle, Con-LC (20 μg/mL), or AL-LC (20 μg/mL) for 15 min. (B) AL-LC–induced p38 phosphorylation was inhibited in cardiomyocytes by SB203580 (5 μM) that was added 30 min before the addition of vehicle, Con-LC, or AL-LC. (C) The inhibition of AL-LC–induced p38 phosphorylation was further demonstrated with three more AL-LC isolated from AL patients (code no.: 01–052, 00–112, and 02–131). The experimental conditions were the same as described in B. (D) Co-IP assay on isolated cardiomyocytes incubated with vehicle, Con-LC, or AL-LC for 15 min demonstrated an increased interaction of p38 MAPK and TAB1 after incubation with AL-LC. Cardiomyocyte extract IP with anti-TAB1 antibody was followed by immunoblotting with anti-p38 antibody (Upper) and anti-TAB1 antibody (Lower, served as loading control). Each bar graph data are from three independent experiments. *, P < 0.05 vs. vehicle and #, P < 0.05 vs. Con-LC in the presence or absence of SB203580; †, P < 0.05 vs. AL-LC in the absence of SB203580.

Fig. 4.

AL-LC–induced cellar dysfunction is prevented by adenovirus mediated overexpression of dominant negative p38α but not dominant negative p38β. (A) Immunoblot of total p38 MAPK expression in isolated adult cardiomyocytes infected with adenovirus (MOI of 50) expressing LacZ, dominant negative p38α (p38αDN), or dominant negative p38β (p38βDN). Arrows indicate endogenous p38 (Lower), p38αDN (Middle), and p38βDN (Upper), respectively. (B) Overexpression of p38αDN, but not p38βDN, prevents AL-LC–induced contractile dysfunction in isolated cardiomyocytes as shown by percent cell shorting. Data are means ± SE from three independent experiments, with each individual experiment average values taken from 8 to 12 cell measurements for each group. * and #, P < 0.05 vs. corresponding vehicle and control-LC, respectively; † and ‡, P < 0.05 vs. AL-LC–treated cells overexpressing LacZ and without adenovirus infection, respectively.

Fig. 5.

AL-LC activates programmed cell death through a p38α MAPK-dependent mechanism. (A) Increased apoptosis as determined by TUNEL assay in isolated cardiomyocytes incubated with AL-LC for 48 h relative to vehicle or Con-LC treatment. Coincubation of AL-LC with MnTMPyP (50 μM), a MnSOD mimetic, reduced cellular apoptosis. (B) AL-LC–induced apoptosis was prevented by inhibition of p38 MAPK with SB203580 (5 μM). (C) AL-LC enhanced Bax/Bcl2 expression ratio, which was prevented by treatment with SB203580. (D) AL-LC–induced apoptosis was inhibited by adenovirus-mediated overexpression of dominant negative p38α (p38αDN) but not dominant negative p38β (p38βDN). Bar graph data are means ± SE, each from three independent experiments. * and #, P < 0.05 vs. corresponding vehicle and Con-LC, respectively; †, P < 0.05 vs. AL-LC treated cells without MnTmPyP (A), SB203580 (B and C), as well as with vehicle or LacZ (D). (E) i.v. administration of AL-LC for 7 days increased cardiac tissue Bax/Bcl2 expression ratio in wild-type (WT) mice but not in DNp38αTG mice. n = 3 for each group, *, P < 0.05.

Fig. 6.

Increased apoptosis is observed in explanted human myocardium obtained from patients with primary cardiac amyloidosis as compared to myocardium obtained from nonfailing human hearts. Nonfailing heart (Non-FH) samples (n = 6) were obtained from NDRI and explanted AL heart samples (n = 4) were obtained from patients with AL cardiac amyloidosis undergoing cardiac transplantation. Apoptosis was evaluated by TUNEL assay (A), caspase3/7 activity (B), and ratio of Bax/Bcl2 protein expression and p53 expression normalized with GAPDH (C and D). Corresponding immunoblots of Bax, Bcl2, p53, and GAPDH are shown in C, where GAPDH serves as a loading control. Data are presented as means ± SE. *, P < 0.05 vs. Non-FH.