Manipulation of death pathways in desmin-related cardiomyopathy

Abstract

Rationale: Transgenic mice with cardiac specific overexpression of mutated alphaB-crystallin (CryAB(R120G)) display Desmin-related myopathy (DRM) with dilated cardiomyopathy and heart failure. Our previous studies showed the presence of progressive mitochondrial abnormalities and activation of apoptotic cell death in CryAB(R120G) transgenic hearts. However, the role of mitochondrial dysfunction and apoptosis in the overall course of the disease was unclear.

Objective: We tested the hypothesis that prevention of apoptosis would ameliorate CryAB(R120G) pathology and decrease morbidity.

Methods and results: We crossed CryAB(R120G) mice to transgenic mice with cardiac specific overexpression of Bcl-2. Sustained Bcl-2 overexpression in CryAB(R120G) hearts prolonged CryAB(R120G) transgenic mice survival by 20%. This was associated with decreased mitochondrial abnormalities, restoration of cardiac function, prevention of cardiac hypertrophy, and attenuation of apoptosis. CryAB(R120G) misfolded protein aggregation was significantly reduced in the double transgenic. However, inhibition of apoptotic signaling resulted in the upregulation of autophagy and alternative death pathways, the net result being increased necrosis.

Conclusion: Although Bcl-2 overexpression prolonged life in this DRM model, in the absence of apoptosis, another death pathway was activated.

Figure 1

A, Western blots showing expression levels in the single and double transgenic mice. When placed in the FVB/N background, the Bcl-2 transgene drove expression levels of between 5- (heterozygote) and 10-fold (homozygote, that is, two copies of the transgene) greater than present in NTG mice, with those levels maintained throughout the adult states (4 months and 6 month levels are shown). Placement of the Bcl-2 transgene in the CryAB^R120G background did not affect expression of either CryAB or Bcl-2. B, Kaplan-Meier curves for NTG, CryAB^R120G, and CryAB^R120G/Bcl-2 mice. C, Cardiac hypertrophy from 2 to 6 months in CryAB^R120G/Bcl-2 mice compared to CryAB^R120G. *P<0.05 versus NTG; ^P<0.05 CryAB^R120G.

Figure 2

A, Ca²⁺-induced swelling of mitochondria from NTG, CryAB^R120G and CryAB^R120G/Bcl-2 hearts. Mitochondria from 2 month old CryAB^R120G hearts were noticeably swollen while mitochondria derived from NTG and CryAB^R120G/Bcl-2 hearts appeared normal. B, Complex I activity in 2 month old NTG, CryAB^R120G, and CryAB^R120G/Bcl-2 hearts. C, Quantification of TUNEL-positive cells in NTG, CryAB^R120G, and CryAB^R120G/Bcl-2 hearts at 2 and 6 months of age CM; cardiomyocytes. D, activation of caspase-3 (17 kD form, arrow) in TG, but not in CryAB^R120G/Bcl-2 hearts; E, The release of cytochrome c from mitochondria to the cytosol was determined by Western blotting in 5-month-old littermate NTG, CryAB^R120G, and CryAB^R120G/Bcl-2 hearts. Shown are representative Western blots next to the group data for cytoplasmic cytochrome c levels normalized to tubulin (not shown).

Figure 3

Overexpression of Bcl-2 significantly reduced the accumulation of misfolded proteins in CryAB^R120G hearts. A, Electron microscopy images of protein aggregates from 5-month-old CryAB^R120G and CryAB^R120G/Bcl-2 hearts. B, Immunofluorescence staining for CryAB (green) with troponin I counterstaining (red) shows decreased aggregate levels. C, Time-dependent quantification of CryAB aggregates using MetaMorph software shows a decreased slope for aggregate accumulation in CryAB^R120G/Bcl-2 hearts. *P<0.05 versus CryAB^R120G.

Figure 4

Autophagy in CryAB^R120G and CryAB^R120G/Bcl-2 mice. A, Representative Western Blot images and quantification for Beclin 1 in 6-month-old CryAB^R120G and CryAB^R120G/Bcl-2 hearts. B, Appearance of LC3II as a marker of autophagy. Western Blots were probed with anti-LC3 antibody. Tubulin was used as a loading control, n=4.

Figure 5

EM analyses of CryAB^R120G and CryAB^R120G/Bcl-2 transgenic hearts. Heart were harvested at 5 months and subjected to TEM analyses. Fields were randomly selected from multiple samples from each experimental cohort and viewed at 20,000× or 40,000× (panel D) magnification. A, Typical morphology observed in a 5 month, CryAB^R120G ventricle midwall. Type 1 (*) and Type 2 (†) aggregates and the frequent abnormal mitochondria showing cristae lysis and abnormal internal membrane whorls (arrows) are readily apparent. B-D, Three fields from sections derived from the CryAB^R120G/Bcl-2 transgenic hearts. Amphisomes (arrowheads), autolysosomes (al) and lysosomes (ly) were invariably present.

Figure 6

Immunofluorescence (IF) for GFP-LC3 and ImageStream (IS) analyses of autophagic activity. A, Representative images of individual infected cells obtained using fluorescent microscope and the ImageStream cytometer. Rat neonatal cardiomyocytes expressing GFP-tagged LC3 only (Control; Ctrl) or in combination with Bcl-2, CryAB^R120G (R120G) as well as R120G and Bcl-2 (R120G/Bcl-2) together in the absence and presence of 10 μM 3-MA. B, quantification of % of cells with punctate LC3 using IDEAS software. For each cell, the punctate LC3 was differentiated from the diffused signal and the number of spots calculated. *P<0.005 versus NTG, *^P<0.005 versus CryAB^R120G.

Figure 7

A, Evans blue permeability of cardiomyocytes derived from CryAB^R120G and CryAB^R120G/Bcl-2 hearts. Sections were counterstained with phalloidin (green) and visualized by fluorescent microscopy at magnifications of ×10 and ×40. B, Quantification of EBD-positive cells. *P<0.005 versus CryAB^R120G. C, Protein derived from CryAB^R120G and CryAB^R120G/Bcl-2 hearts were electrophoresed in SDS-PAGE, Western blotted and probed with anti-RIP1 and anti-tubulin antibodies. D, Quantification of RIP1 levels using ImageJ software, *P<0.005 versus NTG, ^P<0.005 versus CryAB^R120G.