Protein aggregates and novel presenilin gene variants in idiopathic dilated cardiomyopathy

Abstract

Background: Heart failure is a debilitating condition resulting in severe disability and death. In a subset of cases, clustered as idiopathic dilated cardiomyopathy (iDCM), the origin of heart failure is unknown. In the brain of patients with dementia, proteinaceous aggregates and abnormal oligomeric assemblies of beta-amyloid impair cell function and lead to cell death.

Methods and results: We have similarly characterized fibrillar and oligomeric assemblies in the hearts of iDCM patients, pointing to abnormal protein aggregation as a determinant of iDCM. We also showed that oligomers alter myocyte Ca(2+) homeostasis. Additionally, we have identified 2 new sequence variants in the presenilin-1 (PSEN1) gene promoter leading to reduced gene and protein expression. We also show that presenilin-1 coimmunoprecipitates with SERCA2a.

Conclusions: On the basis of these findings, we propose that 2 mechanisms may link protein aggregation and cardiac function: oligomer-induced changes on Ca(2+) handling and a direct effect of PSEN1 sequence variants on excitation-contraction coupling protein function.

Figure 1

A) Congo-red staining of a case of senile amyloid (a, b) 2 non failing control (c, d) and 3 cases of iDCM (e–h) B) Confocal microscopy of the Thioflavin-S staining of iDCM (a, b), control (c, d) and AL-amyloid cardiomyopathy (e, f). Panels a, d, e shows the sections under direct light, panels b, d, f show the sections under fluorescence light. The AL-amyloid cardiomyopathy shows the distribution of the staining in the interstitial space whereas in the iDCM the staining occupies also the cytosol of the myocytes. C) Electron micrographs of super-thin sections from AL-amyloid cardiomyopathy (a, b), iDCM left ventricle (c, d). Higher magnification of the fibrillar aggregates from AL-amyloidosis (b) and iDCM (d). The insert in panel d shows collagen fibers from the same heart for comparison. D) Electron micrographs of myocardial diagnostic biopsies from iDCM patients showing extracellular aggregates (plaques) (panel a–c, Panel b shows a higher magnification of panel a) and intracellular fibrils (tangles) (panel d–g). Panels e and g are higher magnifications of panels d and f respectively.

Figure 1

A) Congo-red staining of a case of senile amyloid (a, b) 2 non failing control (c, d) and 3 cases of iDCM (e–h) B) Confocal microscopy of the Thioflavin-S staining of iDCM (a, b), control (c, d) and AL-amyloid cardiomyopathy (e, f). Panels a, d, e shows the sections under direct light, panels b, d, f show the sections under fluorescence light. The AL-amyloid cardiomyopathy shows the distribution of the staining in the interstitial space whereas in the iDCM the staining occupies also the cytosol of the myocytes. C) Electron micrographs of super-thin sections from AL-amyloid cardiomyopathy (a, b), iDCM left ventricle (c, d). Higher magnification of the fibrillar aggregates from AL-amyloidosis (b) and iDCM (d). The insert in panel d shows collagen fibers from the same heart for comparison. D) Electron micrographs of myocardial diagnostic biopsies from iDCM patients showing extracellular aggregates (plaques) (panel a–c, Panel b shows a higher magnification of panel a) and intracellular fibrils (tangles) (panel d–g). Panels e and g are higher magnifications of panels d and f respectively.

Figure 1

A) Congo-red staining of a case of senile amyloid (a, b) 2 non failing control (c, d) and 3 cases of iDCM (e–h) B) Confocal microscopy of the Thioflavin-S staining of iDCM (a, b), control (c, d) and AL-amyloid cardiomyopathy (e, f). Panels a, d, e shows the sections under direct light, panels b, d, f show the sections under fluorescence light. The AL-amyloid cardiomyopathy shows the distribution of the staining in the interstitial space whereas in the iDCM the staining occupies also the cytosol of the myocytes. C) Electron micrographs of super-thin sections from AL-amyloid cardiomyopathy (a, b), iDCM left ventricle (c, d). Higher magnification of the fibrillar aggregates from AL-amyloidosis (b) and iDCM (d). The insert in panel d shows collagen fibers from the same heart for comparison. D) Electron micrographs of myocardial diagnostic biopsies from iDCM patients showing extracellular aggregates (plaques) (panel a–c, Panel b shows a higher magnification of panel a) and intracellular fibrils (tangles) (panel d–g). Panels e and g are higher magnifications of panels d and f respectively.

Figure 1

A) Congo-red staining of a case of senile amyloid (a, b) 2 non failing control (c, d) and 3 cases of iDCM (e–h) B) Confocal microscopy of the Thioflavin-S staining of iDCM (a, b), control (c, d) and AL-amyloid cardiomyopathy (e, f). Panels a, d, e shows the sections under direct light, panels b, d, f show the sections under fluorescence light. The AL-amyloid cardiomyopathy shows the distribution of the staining in the interstitial space whereas in the iDCM the staining occupies also the cytosol of the myocytes. C) Electron micrographs of super-thin sections from AL-amyloid cardiomyopathy (a, b), iDCM left ventricle (c, d). Higher magnification of the fibrillar aggregates from AL-amyloidosis (b) and iDCM (d). The insert in panel d shows collagen fibers from the same heart for comparison. D) Electron micrographs of myocardial diagnostic biopsies from iDCM patients showing extracellular aggregates (plaques) (panel a–c, Panel b shows a higher magnification of panel a) and intracellular fibrils (tangles) (panel d–g). Panels e and g are higher magnifications of panels d and f respectively.

Figure 2

Immunohistochemical analysis for Aβ of samples from non-failing donor and iDCM (representative) patients showing no difference in the Aβ expression in patients carrying PSEN mutations.

Figure 3

A) Immunohistochemical analysis of a sample from iDCM patient (panel a and b) vs. a non-failing donor (panel c and d). Oligomeric fibrils in red with anti-oligo antibodies (panel a and c) or aggregates with Thioflavin-S (panel b and d). The insert indicates absence of secondary antibody. DAPI stains nuclei in blue. B) Immuno-gold electron micrographs of myocardial diagnostic biopsies from 2 iDCM patients showing the presence of oligomeric fibrils in the myocardium.

Figure 3

A) Immunohistochemical analysis of a sample from iDCM patient (panel a and b) vs. a non-failing donor (panel c and d). Oligomeric fibrils in red with anti-oligo antibodies (panel a and c) or aggregates with Thioflavin-S (panel b and d). The insert indicates absence of secondary antibody. DAPI stains nuclei in blue. B) Immuno-gold electron micrographs of myocardial diagnostic biopsies from 2 iDCM patients showing the presence of oligomeric fibrils in the myocardium.

Figure 4

a) dot-blot of the preparation of oligomers showing the formation of oligomers after 4 days and subsequent hexane exposure of the oligomeric preparation forming fibrils (EM). b) Acute increase in contractility and Ca²⁺ transients in a representative tracing. This particular cell died after 80 sec of exposure. c) Quantitative changes of systolic and diastolic Ca²⁺ and velocity of Ca²⁺ release and reuptake.

Figure 5

A) Presenilin sequencing electropherograms (upper panel). PSEN1–92delC and -21G>A detected in iDCM patients are shown (top panels are sequences from controls). Measurement of the transcriptional activity by luciferase reporter gene assay (lower panel). PSEN1 -92delC variant exhibited 25% of transcriptional activity over the basal expression of PSEN1 promoter with wild-type allele -92C by the measurement of relative luciferase activity. PSEN1 -21A variant reduced the transcriptional activity to 68% compared with the basal expression of PSEN1 promoter with wild-type allele -21G. *p=0.0029. B) Immunoblotting for PS1 in donor controls, iDCM (p=0.01 vs controls) and iDCM with the genetic variations in the PS promoter (indicated by asterisk). PS1 expression normalized by GAPDH in the patients carrying the genetic variations was 0.79±0.18 vs 0.93±25 in the iDCM patients without the genetic variants (the level in the non-failing population was 0.56±0.24) p=0.046 at Kruskal-Wallis test. C) Co-immunoprecipitation of PS and SERCA2a. Panel a: PS detection after immunoprecitpitation with SERCA2a. Panel b: SERCA2a detection after immunoprecitpitation with PS1. Patients carrying the genetic variations are indicated by asterisk.

Figure 5

A) Presenilin sequencing electropherograms (upper panel). PSEN1–92delC and -21G>A detected in iDCM patients are shown (top panels are sequences from controls). Measurement of the transcriptional activity by luciferase reporter gene assay (lower panel). PSEN1 -92delC variant exhibited 25% of transcriptional activity over the basal expression of PSEN1 promoter with wild-type allele -92C by the measurement of relative luciferase activity. PSEN1 -21A variant reduced the transcriptional activity to 68% compared with the basal expression of PSEN1 promoter with wild-type allele -21G. *p=0.0029. B) Immunoblotting for PS1 in donor controls, iDCM (p=0.01 vs controls) and iDCM with the genetic variations in the PS promoter (indicated by asterisk). PS1 expression normalized by GAPDH in the patients carrying the genetic variations was 0.79±0.18 vs 0.93±25 in the iDCM patients without the genetic variants (the level in the non-failing population was 0.56±0.24) p=0.046 at Kruskal-Wallis test. C) Co-immunoprecipitation of PS and SERCA2a. Panel a: PS detection after immunoprecitpitation with SERCA2a. Panel b: SERCA2a detection after immunoprecitpitation with PS1. Patients carrying the genetic variations are indicated by asterisk.

Figure 5

A) Presenilin sequencing electropherograms (upper panel). PSEN1–92delC and -21G>A detected in iDCM patients are shown (top panels are sequences from controls). Measurement of the transcriptional activity by luciferase reporter gene assay (lower panel). PSEN1 -92delC variant exhibited 25% of transcriptional activity over the basal expression of PSEN1 promoter with wild-type allele -92C by the measurement of relative luciferase activity. PSEN1 -21A variant reduced the transcriptional activity to 68% compared with the basal expression of PSEN1 promoter with wild-type allele -21G. *p=0.0029. B) Immunoblotting for PS1 in donor controls, iDCM (p=0.01 vs controls) and iDCM with the genetic variations in the PS promoter (indicated by asterisk). PS1 expression normalized by GAPDH in the patients carrying the genetic variations was 0.79±0.18 vs 0.93±25 in the iDCM patients without the genetic variants (the level in the non-failing population was 0.56±0.24) p=0.046 at Kruskal-Wallis test. C) Co-immunoprecipitation of PS and SERCA2a. Panel a: PS detection after immunoprecitpitation with SERCA2a. Panel b: SERCA2a detection after immunoprecitpitation with PS1. Patients carrying the genetic variations are indicated by asterisk.