Desmin-related cardiomyopathy in transgenic mice: a cardiac amyloidosis

Abstract

An R120G missense mutation in the small heat shock protein alpha-B-crystallin (CryAB(R120G)) causes desmin-related cardiomyopathy (DRM). DRM is characterized by the formation of aggregates containing CryAB and desmin, and it can be recapitulated in transgenic mice by cardiac-specific expression of the mutant protein. In this article, we show that expression of CryAB(R120G) leads to the formation of electron-dense bodies characteristic of the DRMs and identify these bodies as aggresomes, which are characteristic of the neurodegenerative diseases. Cardiomyocytes transfected with adenovirus containing CryAB(R120G) establish the necessity and sufficiency of CryAB(R120G) expression for aggresome formation. The commonality of these aggresomes with oligomeric protein aggregates found in the amyloid-related degenerative diseases was corroborated by the presence of high levels of amyloid oligomers that may represent a primary toxic species in the amyloid diseases. These oligomeric amyloid intermediates are present also in cardiomyocytes derived from many human dilated and hypertrophic cardiomyopathies.

Fig. 1.

Immunocytochemical analyses in CryAB- and CryAB^R120G-transfected rat neonatal cardiomyocytes. (a) CryAB and CryAB^R120G distribution in permeabilized rat neonatal cardiomyocytes. Control, empty adenoviral vector. (b) Expression of CryAB^R120G results first in the formation of small aggregates, which then coalesce around the nucleus and whose size increases in a time-dependent manner. (c) Formation of aggregates is microtubule-dependent. After 3 days in culture, 10 μM nocodazole was added.

Fig. 2.

Aggresomal markers in the CryAB^R120G aggregates. The CryAB^R120G-induced aggregates are immunoreactive with antibodies for ubiquitin, HSP25, the endoplasmic reticulum complex marker protein SEC61α, and tubulin. For ubiquitin, HSP25, and SEC61α, actin is stained red (phalloidin). For tubulin, actin is stained green and tubulin is red. Nuclei are stained blue with To-Pro-3. The bottom row shows a ×2 magnification of the perinuclear regions of the row above. Arrowheads indicate immunopositive aggregates. Veh., empty adenovirus vector.

Fig. 3.

CryAB^R120G aggregates show amyloid-like characteristics. (a) At 3 days after initial plating, rat neonatal cardiomyocytes expressing CryAB^R120G contain the characteristic aggregates. Control, empty adenoviral vector. (b) Perinuclear aggregates stain positive for Congo red. The nuclei are also stained. (c) Cultures were treated with Congo red starting at day 1. (d) Congo red staining of these cultures confirmed the lack of amyloid material and the effectiveness of the dye in preventing aggresome formation. CryAB (green) and cardiac TnI (red) are shown in a and c. (e and f) Quantitation of aggregates. The filter retardation assay confirms that Congo red treatment partially prevents aggregate formation. *, P < 0.001; #, P < 0.05.

Fig. 4.

Amyloid oligomer is present in both mouse and human heart failure. (a) Transverse and longitudinal sections derived from the hearts of CryAB^R120G mice show the subcellular distribution of the amyloid oligomer, which correlates closely to the perinuclear location of the aggresomes. (b) Ventricular sections derived from normal human ventricles. (c) Sections derived from a 33-year-old woman suffering from nonobstructive HCM. (d) Ventricular sections derived from a 22-year-old male with idiopathic dilated cardiomyopathy. (e) Higher-magnification picture of ventricular section derived from a 37-year-old woman with nonischemic cardiomyopathy. Anti-oligomer antibody is shown in green, and phalloidin (actin staining) is shown in red.

Fig. 5.

Amyloid oligomer reactivity in human heart failure samples. (a–c) Samples derived from normal hearts. (d) A 50-year-old Caucasian male diagnosed with dilated cardiomyopathy (DCM) 1 year before death. (e) A 33-year-old Caucasian female with HCM. Complete heart block with pacemaker placement is shown. The patient died of an arrhythmia. (f) A 57-year-old African-American female with idiopathic DCM diagnosed 6 years before death. (g) A 37-year-old Caucasian male with idiopathic DCM. The patient died of CHF. (h) A 34-year-old African-American female with nonischemic cardiomyopathy diagnosed 3 years before death from CHF. (i) A 16-year-old Caucasian male diagnosed with idiopathic DCM 2 years before death. The patient died of an arrhythmia. (j) A 45-year-old Caucasian male with family history of HCM was diagnosed as being in the dilated stage of HCM 1.5 years before death. The patient died of CHF. (k) A 22-year-old African-American male with idiopathic DCM diagnosed 6 months before death. The patient died of CHF. (l) A 32-year-old Caucasian male with muscular dystrophy identified tentatively as Becker's. He died of pneumonia with complicating episodes of cardioverted ventricular tachycardia–fibrillation.