Stanniocalcin1 is a key mediator of amyloidogenic light chain induced cardiotoxicity

Abstract

Immunoglobulin light chain (LC) amyloidosis (AL) results from overproduction of circulating amyloidogenic LC proteins and subsequent amyloid fibril deposition in organs. Mortality in AL amyloidosis patients is highly associated with a rapidly progressive AL cardiomyopathy, marked by profound impairment of diastolic and systolic cardiac function and significant early mortality. While myocardial fibril deposition contributes to the severe diastolic dysfunction seen in AL cardiomyopathy patients, the degree of fibril deposition has not been found to correlate with prognosis. Previously, we and others showed a direct cardiotoxic effect of amyloidogenic LC proteins (AL-LC), which may contribute to the pathophysiology and mortality observed in AL cardiomyopathy patients. However, the mechanisms underlying AL-LC related cardiotoxicity remain unknown. Mammalian stanniocalcin1 (STC1) is associated with a number of cellular processes including oxidative stress and cell death. Herein, we find that STC1 expression is elevated in cardiac tissue from AL cardiomyopathy patients, and is induced in isolated cardiomyocytes in response to AL-LC, but not non-amyloidogenic LC. STC1 overexpression in vitro recapitulates the pathophysiology of AL-LC mediated cardiotoxicity, with increased ROS production, contractile dysfunction and cell death. Overexpression of STC1 in vivo results in significant cardiac dysfunction and cell death. Genetic silencing of STC1 prevents AL-LC induced cardiotoxicity in cardiomyocytes and protects against AL-LC induced cell death and early mortality in zebrafish. The cardiotoxic effects of STC1 appears to be mediated via mitochondrial dysfunction as indicated by loss of mitochondrial membrane potential, ROS production and increased mitochondrial calcium levels. Collectively, this work identifies STC1 as a critical determinant of AL-LC cardiotoxicity.

Fig. 1

STC1 is upregulated in patients with AL cardiomyopathy and in response to AL-LC stimulation. a STC1 gene expression (normalized to 18S) and b STC1 protein expression relative to GAPDH were upregulated in AL cardiomyopathy patients (N=6) as compared to non-failing control patients (N=5). AL-LC triggers c STC1 gene and d, e protein expression in isolated cardiomyocytes exposed to AL-LC for 2 hour and 24 hours, respectively, relative to those exposed to Con-LC or Veh. f STC1 was upregulated early with AL-LC obtained from 5 independent patients with amyloid cardiomyopathy as compared to Veh or Con-LC. * p<0.05 between indicated groups.

Fig. 2

AL-LC induction of STC1 is p38MAPK dependent in vitro and in vivo. a AL-LC induced upregulation of STC1 protein expression in cardiomyocytes was abolished in the presence of p38MAPK inhibitor, SB203580 (5 μmol/L). b AL-LC triggered upregulation of cardiac STC1 protein expression was prevented in p38MAPK dominate negative (DN-TG) mice. * p<0.05, AL-LC vs. vehicle or Con-LC, # p<0.05 AL-LC with vs. without SB203580 or AL-LC infused wild type mice vs. DN-TG mice. N=3 for each group.

Fig. 3

STC1 affects mitochondrial calcium homeostasis and function. a Subcellular localization of STC1 in cardiomyocytes was determined using immunohistochemical staining against DAPI (blue), STC1 (red), mitochondrial COX4 (green), and the myofilament protein, ACTININ (white). b Merged images of immunohistochemical staining indicates that STC1 is co-localized to the mitochondria and nucleus. c Line scanning analysis demonstrates close overlap of spectral profiles for the red fluorophore (STC1) and green fluorophore (COX4), confirming the mitochondrial localization of STC1. d Mitochondrial membrane potential is decreased in cardiomyocytes exposed to 200 ng/mL STC1 for 24 hours as determined by TMRE, a mitochondrial membrane potential sensitive fluorescence dye. e AL-LC induced ROS was prevented in cardiomyocytes treated with a mitochondrial targeted ROS scavenger, MitoTempo. f Representative DCFDA fluorescence images with corresponding bright field images on right. g Representative fluorescent images of Rhod2 (red) and mitotracker (green), suggesting increased mitochondrial calcium in response to 200 ng/mL STC1 stimulation for 24 hours. h Summarized data of mitochondrial calcium levels. * p<0.05 between indicated groups.

Fig. 4

STC1 induces contractile dysfunction and cell death in cardiomyocytes. Adenoviral mediated overexpression of STC1 in cardiomyocytes (Adeno-STC1) decreased a cellular contractile function (% cell shortening) and b intracellular calcium transient amplitude as well as increased cell death determined using c Bax/Bcl2 ratio and d TUNEL assay relative to control (Adeno-GFP). Representative tracing of cell shortening and calcium transient are shown in the upper panels of a and b, respectively. N=3 for each group. * p<0.05 between indicated groups

Fig. 5

Overexpression of STC1 induces contractile dysfunction and cell death in vivo. a Time course of the experiments. STC1 or anti-sense control mRNA was injected into zebrafish at day 0 (one-cell stage). STC1 b gene and c protein expression measured at day 3 post mRNA injection were increased in STC1 mRNA injected zebrafish. STC1mRNA injection in zebrafish also altered cardiac function as demonstrated by the decreased d stroke volume and e cardiac output without change in f heart rate. g Representative images demonstrating pericardial edema with STC1 expression in zebrafish. Scale bar represents 400 μm. h Representative images of isolated zebrafish hearts stained with DAPI (blue) for nuclei, the cardiac specific marker, MF-20 (green) and TUNEL (red). i Quantification of TUNEL positive nuclei in cardiomyocytes. Scale bar=20 μm. * p<0.05 between indicated groups. (N=12 for cardiac function analysis and N=6 for TUNEL assay)

Fig. 6

STC1 is required for AL-LC induced dysfunction and cell death. Isolated cardiomyocytes were infected with STC1 shRNA adenovirus or scrambled shRNA adenovirus 24 hours prior to AL-LC or vehicle exposure. a Adenoviral mediated STC1 shRNA in cardiomyocytes effectively prevent the upregulation of STC1 in response to AL-LC. AL-LC triggered b cellular dysfunction, c impaired calcium homeostasis and d cell death were attenuated in Adeno-STC1-shRNA infected cardiomyocytes. N=3 per condition. *, # p<0.05 between indicated groups.

Fig. 7

Antagonism of STC1 protects against AL-LC induced toxicity in a zebrafish model. AL-LC or Con-LC was introduced via venous injection at 48 hpf, at a final concentration of 100 μg/mL. E3 water injection served as Veh control. a Immunoblotting analysis for STC1 protein expression in zebrafish following venous injection of AL-LC but not Con-LC or Veh 3 days post-injection. b Time line of the experiments conducted. Cardiac contractile function decreased in AL-LC injected zebrafish as shown by the decreased c stroke volume and d cardiac output. AL-LC injection also increased e. cell death as determined by TUNEL assay. f Representative images with nuclei stained with DAPI (blue) and TUNEL positive cells (red). AL-LC induced cardiac dysfunction, cell death and mortality were prevented in STC1 MO injected zebrafish (c-g). h STC1 protein knockdown via STC1 MO, as assessed using immunoblotting at 5 days post MO injection Scale bar=20 μm. (N=12 per group for cardiac function analysis, N=6 hearts per group for TUNEL analysis, N=55-70 per group for survival analysis. *, # p<0.05 between indicated groups.