Autophagy is an adaptive response in desmin-related cardiomyopathy

Abstract

A missense mutation in the alphaB-crystallin (CryAB) gene triggers a severe form of desmin-related cardiomyopathy (DRCM) characterized by accumulation of misfolded proteins. We hypothesized that autophagy increases in response to protein aggregates and that this autophagic activity is adaptive. Mutant CryAB (CryAB(R120G)) triggered a >2-fold increase in cardiomyocyte autophagic activity, and blunting autophagy increased the rate of aggregate accumulation and the abundance of insoluble CryAB(R120G)-associated aggregates. Cardiomyocyte-restricted overexpression of CryAB(R120G) in mice induced intracellular aggregate accumulation and systolic heart failure by 12 months. As early as 2 months (well before the earliest declines in cardiac function), we detected robust autophagic activity. To test the functional significance of autophagic activation, we crossed CryAB(R120G) mice with animals harboring heterozygous inactivation of beclin 1, a gene required for autophagy. Blunting autophagy in vivo dramatically hastened heart failure progression with a 3-fold increase in interstitial fibrosis, greater accumulation of polyubiquitinated proteins, larger and more extensive intracellular aggregates, accelerated ventricular dysfunction, and early mortality. This study reports activation of autophagy in DRCM. Further, our findings point to autophagy as an adaptive response in this proteotoxic form of heart disease.

Fig. 1.

CryAB^R120G expression is a potent activator of cardiomyocyte autophagy. (A) Representative low (×5,000; Upper) and high (×20,000; Lower) magnification images of NRVMs 5 days after expression of CryAB^WT or CryAB^R120G. Aggresomes are evident in CryAB^R120G-expressing cells (asterisks) as are extensive perinuclear autophagosomes (arrows). (B) Despite extensive induction of autophagy, there is no appreciable change in cell viability 5 days postinfection. (C) NRVMs were transiently transfected with a GFP-LC3 construct and then infected with WT or mutant CryAB. Twenty-four hours later, autophagy was quantified as the number of punctate-positive cells divided by the total number of GFP+ cells. (D) Representative images of NRVMs (two examples of each) infected with CryAB and processed for mTOR immunocytochemistry. mTOR is distributed throughout the cytoplasm in NRVMs expressing WT CryAB. In contrast, CryAB^R120G triggered formation of perinuclear aggregates that stain for both mTOR and crystallin.

Fig. 2.

Autophagic activity serves to clear CryAB^R120G-induced protein aggregates. (A) Representative images depicting NRVMs subjected to 5 days of CryAB^R120G expression, demonstrating robust perinuclear aggresome formation. Blunting of autophagy with 3MA (5 mM) dramatically increased aggresome size. (B) Blunting autophagy increased the amount of insoluble CryAB in cells expressing mutant, but not WT, protein. (C) Dose–response relationship between CryAB^WT or CryAB^R120G expression and accumulation of soluble and insoluble protein. (D) CryAB^R120G-induced aggregate accumulation is accelerated in the setting of blunted autophagy, suggesting that autophagic pathways function to clear the aggregates.

Fig. 3.

Accelerated heart failure and early mortality in αMHC-CryAB^R120G;beclin 1^+/− mice. (A) Cardiac function was monitored by serial echocardiography in nonsedated animals. αMHC-CryAB^R120G animals developed late-onset heart failure with the first signs of functional decline appearing at 9 months and development of heart failure by 12 months of age. In contrast, αMHC-CryAB^R120G;beclin 1^+/− mice manifested an accelerated disease course, with early signs of functional decline apparent at 6 months and terminal heart failure at 9 months. (B) Representative M-mode echocardiograms recorded in 9-month-old animals. (C) Declines in systolic performance are caused primarily by progressive increases in left ventricular internal dimension at end systole (LVIDs), with little change seen in end-diastolic diameter (LVIDd). By 12 months of age, the CryAB^R120G;Beclin 1^+/− animals had experienced 100% mortality. (D) Accumulation of RCAN1, indicative of calcineurin activation, as observed in αMHC-CryAB^R120G hearts at 9 months of age. RCAN1 levels were substantially higher in αMHC-CryAB^R120G;beclin 1^{+/ −} hearts.