Exercise reverses preamyloid oligomer and prolongs survival in alphaB-crystallin-based desmin-related cardiomyopathy

Abstract

The R120G mutation in the small heat shock-like protein alphaB-crystallin (CryAB(R120G)) causes desmin-related myopathy (DRM), which is characterized by the formation of desmin- and CryAB-containing aggregates within muscle fibers. Mice with cardiac-specific overexpression of CryAB(R120G) develop cardiomyopathy at 3 months and die at 6-7 months from heart failure (HF). Previous studies showed that overexpression of CryAB(R120G) results in accumulation of preamyloid oligomer (PAO). PAO is considered to be the cytotoxic entity in many of the protein misfolding-based neurodegenerative diseases. On the basis of data from mouse models of neurodegenerative diseases showing that exercise or environmental enrichment reduces the amyloid oligomer level and improves cognitive ability, we hypothesized that CryAB(R120G)-induced DRM would also respond favorably to prolonged voluntary exercise, reducing HF symptoms and rescuing the mice from premature death. Six months of voluntary exercise in CryAB(R120G) animals resulted in 100% survival at a time when all unexercised mice had died. After 22 weeks of exercise, PAO levels were decreased by 47% compared with the unexercised CryAB(R120G) control mice (P = 0.00001). Although CryAB(R120G) expression led to decreased levels of the metallomembrane endopeptidase neprilysin, normal levels were maintained in the exercised CryAB(R120G) mice, and in vitro loss-of-function and gain-of-function experiments using adenovirus-infected cardiomyocytes confirmed the importance of neprilysin in ameliorating PAO accumulation. The data demonstrate that voluntary exercise slows the progression to HF in the CryAB(R120G) DRM model and that PAO accumulation is mediated, at least in part, by decreased neprilysin activity.

Fig. 1.

Voluntary exercise. (A) Kaplan–Meier curves for exercised (solid line) and unexercised (broken line) CryAB^R120G mice. (B) Quantitative PCR analysis revealed that voluntary exercise significantly attenuated activation of the fetal gene program that is normally observed in the CryAB^R120G TG mice. ∗, significant difference vs. NTG (P < 0.05); ⋀, significant difference vs. unexercised TG (P < 0.05).

Fig. 2.

Long-term voluntary exercise and aggregate accumulation. Mice were aged for 1 month and then divided into the exercised and unexercised groups. (A) Cardiomyocytes were identified by phalloidin (red fluorescence). Anti-CryAB (green fluorescence) staining in the unexercised CryAB^R120G (TG) and exercised CryAB^R120G hearts was used to detect aggresome accumulation. The mice were exercised for 22 weeks. (B) Aggresomes were quantitated by using the filtration assay. (C) PAO levels were determined before the exercise regimen when the mice were 1 month old. PAO levels were then determined after 1 month, 3 months, and 6 months of exercise (2m, 4m, and 6m, respectively). Cardiomyocytes were identified by phalloidin (red fluorescence). PAO was detected by using the A11 antibody (green fluorescence) in the unexercised CryAB^R120G (TG) and exercised CryAB^R120G hearts. (D) Quantitative analysis of the amount of A11 immunoreactivity, expressed as a percentage of total area of each section ± SE. ∗, significant difference vs. unexercised TG (P < 0.005).

Fig. 3.

The effect of long-term voluntary exercise on apoptotic cell death in CryAB^R120G mice. Mice were housed for 6 months. (A) Caspase-3 activation in the nonexercised and exercised mice was determined, as was the cleaved forms of two of its substrates, Rho-associated coiled-coil protein kinase 1 (ROCK-1) and poly-(ADP-ribose)-polymerase (PARP) (46), early in the trial at 2 months. (B) Quantitation of TUNEL-stained nuclei in frozen sections from exercised and unexercised CryAB^R120G hearts. ∗, significant difference vs. unexercised TG (P < 0.005).

Fig. 4.

CryAB^R120G expression decreases neprilysin levels. (A) Semiquantitative RT-PCR analysis of neprilysin mRNA. All data points were taken from cycle 24, which was in the linear range of the reaction. Neprilysin RNA was significantly reduced in both the unexercised and exercised hearts. (B) Time course of neprilysin protein reduction. Shown are the Western blots prepared from the same groups used to derive the data shown in Fig. 2C. The mice were allowed access to the wheels starting at 1 month (1m). (C) Exercise conserves normal neprilysin protein levels. The representative Western blot data and quantitation show significant conservation of normal neprilysin levels in the exercised CryAB^R120G hearts. β-Actin was used as a loading control. Volume is expressed in arbitrary units ± SE. ∗, significant difference vs. NTG (P < 0.001); ⋀, significant difference vs. unexercised TG (P < 0.001); n = 6.

Fig. 5.

Neprilysin plays a causative role in PAO accumulation. (A) Representative Western blot showing neprilysin levels in untreated NRCs and CryAB^R120G-infected NRCs in the presence or absence of thiorphan 24 h after infection. (B) PAO accumulation (green) in CryAB^R120G-infected NRCs 24 h after infection was enhanced by thiorphan. Nuclei (blue) were stained with TO-PRO-3. To confirm that the increased PAO levels were not due to NO production, the NO synthase inhibitor N^G-nitro-l-arginine methyl ester (L-NAME) was also added to selected cultures. (C) Overexpression of neprilysin significantly reduces PAO 3 days after infection. (D and E) Quantitation of PAO-positive areas from B and C, respectively, as a percentage of total NRC area. (F) The effect of overexpression or inhibition of neprilysin on the rate of cardiac cell death as measured by the release of adenylate kinase into the medium. Values shown are the average-fold increase relative to control, nontransfected NRCs for three independent series ± SE. ∗, P < 0.005 vs. control; ⋀, P < 0.005 vs. CryAB^R120G-infected NRCs.