Tubulin hyperacetylation is adaptive in cardiac proteotoxicity by promoting autophagy

Abstract

Proteinopathy causes cardiac disease, remodeling, and heart failure but the pathological mechanisms remain obscure. Mutated αB-crystallin (CryAB(R120G)), when expressed only in cardiomyocytes in transgenic (TG) mice, causes desmin-related cardiomyopathy, a protein conformational disorder. The disease is characterized by the accumulation of toxic misfolded protein species that present as perinuclear aggregates known as aggresomes. Previously, we have used the CryAB(R120G) model to determine the underlying processes that result in these pathologic accumulations and to explore potential therapeutic windows that might be used to decrease proteotoxicity. We noted that total ventricular protein is hypoacetylated while hyperacetylation of α-tubulin, a substrate of histone deacetylase 6 (HDAC6) occurs. HDAC6 has critical roles in protein trafficking and autophagy, but its function in the heart is obscure. Here, we test the hypothesis that tubulin acetylation is an adaptive process in cardiomyocytes. By modulating HDAC6 levels and/or activity genetically and pharmacologically, we determined the effects of tubulin acetylation on aggregate formation in CryAB(R120G) cardiomyocytes. Increasing HDAC6 accelerated aggregate formation, whereas siRNA-mediated knockdown or pharmacological inhibition ameliorated the process. HDAC inhibition in vivo induced tubulin hyperacetylation in CryAB(R120G) TG hearts, which prevented aggregate formation and significantly improved cardiac function. HDAC6 inhibition also increased autophagic flux in cardiomyocytes, and increased autophagy in the diseased heart correlated with increased tubulin acetylation, suggesting that autophagy induction might underlie the observed cardioprotection. Taken together, our data suggest a mechanistic link between tubulin hyperacetylation and autophagy induction and points to HDAC6 as a viable therapeutic target in cardiovascular disease.

Keywords: HDAC6; alphaB-crystallin; autophagy; heart; proteotoxicity.

Fig. 1.

Protein acetylation is perturbed in hearts of CryAB^R120G TG mice. (A) Global profile of protein hypoacetylation is observed in CryAB^R120G hearts at 6 mo. Total acetylation is reduced by ∼60%, **P < 0.01. (B) Hyperacetylation of α-tubulin is observed in CryAB^R120G hearts at 6 mo. *P < 0.05, n = 4 per group.

Fig. 2.

Hyperacetylation of α-tubulin occurs despite induction of HDAC6. (A and B) HDAC6 protein levels increase concomitantly with tubulin acetylation. Acetylated tubulin and HDAC6 are both increased. **P < 0.01, n = 4–6 per group. (C) Class IIb HDAC activity is significantly increased. ***P < 0.01, n = 8–10 per group.

Fig. 3.

Tubulin acetylation status influences aggregate formation in CryAB^R120G-expressing cardiomyocytes. (A) HDAC6 overexpression leads to reduced tubulin acetylation in RNCs. n = 4 per group. (B) HDAC6 overexpression increased aggregate formation in CryAB^R120G-infected RNCs. (Scale bar, 50 μm.) n = 50–100 cells per group. (C) HDAC6 overexpression increased levels of insoluble CryAB-positive cellular protein, confirming increased aggregate formation. *P < 0.05, n = 5 per group. (D) siRNA knockdown increased tubulin acetylation. (Scale bar, 25 μm.) (E) Aggregate formation was inhibited in CryAB^R120Ginfected RNCs when HDAC6 was reduced, compared with cells treated with negative control (scrambled) siRNA (Negsi). n = 50–100 cells per group. (F) Reduced HDAC6 reduced aggregate/myocyte ratios compared with CryAB^R120G RNCs transfected with scrambled siRNA. ***P < 0.001. (G and H) Similar effects were seen using a specific HDAC6 inhibitor, tubastatin A. ***P < 0.001. (Scale bar, 25 μm.) n = 50–100 cells per group.

Fig. 4.

SAHA treatment of RNCs promotes tubulin acetylation and reduces aggregates. (A) SAHA induced α-tubulin acetylation, confirming the inhibitory activity toward HDAC6. n = 4 per group. (B and C) SAHA treatment reduced aggregate formation in CryAB^R120G-infected RNCs. ***P < 0.001. (Scale bar, 50 μm.)

Fig. 5.

Modulating tubulin acetylation in vivo in CryAB^R120G TG mice. (A and B) SAHA treatment (p.o.) increased tubulin acetylation after 2 wk of treatment in mice. n = 4 per group. (C) SAHA treatment in CryAB^R120G TG mice resulted in substantial reduction in aggregate formation. (Top) cross-sectional areas; (Bottom) longitudinal views. Area of aggregates from n = 300–380 myocytes was measured, n = 2 hearts per group. (D) Aggregate area per cell was reduced by ∼40%. ***P < 0.001. (Scale bar, 50 μm.) (E and F) SAHA-mediated aggregate reduction led to significantly improved cardiac fractional shortening (FS) and ejection fraction (EF) compared with vehicle-treated controls. *P < 0.05 (G) Cardiac hypertrophy, as assessed by left ventricular mass, was unchanged. n = 5–7 per group; for NTG, n = 3.

Fig. 6.

SAHA induces autophagy in cardiomyocytes and increased autophagy underlies the protective effects. (A) Increased autophagic vacuoles (white arrows) are increased in SAHA-treated hearts and are proximal to aggregates (*). (Scale bar, 2 μm.) (B) SAHA treatment (10 μM) in CryAB^R120G-infected cardiomyocytes increased autophagic flux. n = 4 per group. (C) Tubastatin A treatment (10 μM) confirms autophagy induction is mediated by HDAC6. ***P < 0.001, n = 4 per group.

Fig. 7.

Voluntary exercise increases acetylated tubulin levels. (A and B) Increased acetylated tubulin in CryAB^R120G hearts after voluntary exercise.