Knockout of Eva1a leads to rapid development of heart failure by impairing autophagy

Abstract

EVA1A (Eva-1 homologue A) is a novel lysosome and endoplasmic reticulum-associated protein that can regulate cell autophagy and apoptosis. Eva1a is expressed in the myocardium, but its function in myocytes has not yet been investigated. Therefore, we generated inducible, cardiomyocyte-specific Eva1a knockout mice with an aim to determine the role of Eva1a in cardiac remodelling in the adult heart. Data from experiments showed that loss of Eva1a in the adult heart increased cardiac fibrosis, promoted cardiac hypertrophy, and led to cardiomyopathy and death. Further investigation suggested that this effect was associated with impaired autophagy and increased apoptosis in Eva1a knockout hearts. Moreover, knockout of Eva1a activated Mtor signalling and the subsequent inhibition of autophagy. In addition, Eva1a knockout hearts showed disorganized sarcomere structure and mitochondrial misalignment and aggregation, leading to the lack of ATP generation. Collectively, these data demonstrated that Eva1a improves cardiac function and inhibits cardiac hypertrophy and fibrosis by increasing autophagy. In conclusion, our results demonstrated that Eva1a may have an important role in maintaining cardiac homeostasis.

Figure 1

Characterization of Eva1a expression in Eva1a^f/f and Eva1a-deficient mice. (a) Representative RT-PCR results showing endogenous Eva1a expression in different tissues from wild-type mice. (b) Representative RT-PCR results showing endogenous Eva1a expression in ISO-induced cardiac remodelling. (c) Scheme to generate Eva1a-deficient mice. (d) Southern blot analysis to detect Eva1a expression in the heart in Eva1a^f/f and Eva1a-deficient mice. (e) Representative RT-PCR results showing endogenous Eva1a expression in different tissues from Eva1a^f/f and Eva1a-deficient mice. (f) Eva1a expression was detected by immunofluorescence assay

Figure 2

Effects of Eva1a-deficiency on cardiac function and structure. (a) Kaplan–Meyer survival curve (***P<0.0001, n=10). (b) Representative M-mode echocardiography images showing left ventricular wall thickness and systolic function in different mice. (c) Ejection fraction (EF) was significantly lower in Eva1a-deficient mice than in the other mice (*P<0.05, n=8). (d) Echocardiographic analysis revealed enlarged left ventricular diastolic dimension (LVID-d) was significantly higher in Eva1a-deficient mice than in the other mice (*P<0.05, n=8). (e) Diastolic left ventricular wall thickness (LVPW-d) did not significantly differ between Eva1a-deficient mice and the other groups (n=8)

Figure 3

Effects of Eva1a deficiency on cardiac hypertrophy. (a) The hearts of Eva1a deficient mice were enlarged on gross morphology. Haematoxylin and eosin (H&E) staining of heart sections. Scale bar=1 mm or 50 μm. (b) The ratio of heart weight to body weight (HW/BW) did not significantly differ between Eva1a-deficient mice and the other mice (**P<0.01, n=9). (c) The ratio of lung weight to body weight (LW/BW) was significantly different between Eva1a-deficient mice and the other mice (*P<0.05, n=9). (d) Measurements of two-dimensional cardiomyocyte cross-sectional areas (*P<0.05, n=4). (e and f) Analysis of hypertrophy markers Bnp (e) and Anp (f) by qRT-PCR (*P<0.05, **P<0.01, n=5)

Figure 4

Effects of Eva1a deficiency on cardiac fibrosis. (a) Representative micrographs of picrosirius red-stained sections of the ventricle. Red areas represent collagen. Scale bar=2 mm or 50 μm. (b) Quantification of cardiac interstitial collagen content in picrosirius red-stained sections. Results are expressed as the ratio of collagen area to heart area (***P<0.001, n=4). (c–f) Analysis of fibrosis markers Ctgf (c), Collagen I (d), Collagen III (e), and Fibronectin (f) by qRT-PCR (*P<0.05, **P<0.01, n=6)

Figure 5

Assessment of autophagy and apoptosis in Eva1a-deficient mice. (a) Representative western blot analysis of Lc3b and Sqstm1 in heart extracts obtained from different groups. (b and c) Densitometric analysis of Sqstm1 and Lc3b-II (*P<0.05, **P<0.01, n=4). (d) Representative images of TUNEL staining (green) and Hoechst staining (blue) of nuclei in cryosectioned heart tissue. (e) Quantification of positive cells displaying terminal deoxynucleotidyl transferase-mediated dUDP nick-end labelling (TUNEL) staining. (f) Ultrastructural images reveal the presence of swollen mitochondria and lipid accumulation in Eva1a-deficient mice. (g) Detection of ATP levels (*P<0.05, n=3)

Figure 6

Eva1a deficiency increases the Mtor signalling pathway. (a) Representative western blot analysis of Mtor, Rps6kb1, Ulk1, Erk, and Akt, and their phosphorylated forms in heart extracts obtained from different groups of mice. (b–g) Densitometric analysis of p-Mtor, p-Rps6kb1, p-Erk, and p-Akt (*P<0.05, n=3)