Perinatal versus adult loss of ULK1 and ULK2 distinctly influences cardiac autophagy and function

Abstract

Impairments in macroautophagy/autophagy, which degrades dysfunctional organelles as well as long-lived and aggregate proteins, are associated with several cardiomyopathies; however, the regulation of cardiac autophagy remains insufficiently understood. In this regard, ULK1 and ULK2 are thought to play primarily redundant roles in autophagy initiation, but whether their function is developmentally determined, potentially having an impact on cardiac integrity and function remains unknown. Here, we demonstrate that perinatal loss of ULK1 or ULK2 in cardiomyocytes (cU1-KO and cU2-KO mice, respectively) enhances basal autophagy without altering autophagy machinery content while preserving cardiac function. This increased basal autophagy is dependent on the remaining ULK protein given that perinatal loss of both ULK1 and ULK2 in cU1/2-DKO mice impaired autophagy causing age-related cardiomyopathy and reduced survival. Conversely, adult loss of cardiac ULK1, but not of ULK2 (i.e., icU1-KO and icU2-KO mice, respectively), led to a rapidly developing cardiomyopathy, heart failure and early death. icU1-KO mice had impaired autophagy with robust deficits in mitochondrial respiration and ATP synthesis. Trehalose ameliorated autophagy impairments in icU1-KO hearts but did not delay cardiac dysfunction suggesting that ULK1 plays other critical, autophagy-independent, functions in the adult heart. Collectively, these results indicate that cardiac ULK1 and ULK2 are functionally redundant in the developing heart, while ULK1 assumes a more unique, prominent role in the adult heart.Abbreviations: ATG4: autophagy related 4, cysteine peptidase; ATG5: autophagy related 5; ATG7: autophagy related 7; ATG9: autophagy related 9; ATG13: autophagy related 13; CYCS: Cytochrome C; DNM1L, dynamin 1-like; MAP1LC3A: microtubule-associated protein 1 light chain 3 alpha; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MFN1: mitofusin 1; MFN2: mitofusin 2; MT-CO1: mitochondrially encoded cytochrome c oxidase I; MYH: myosin, heavy polypeptide; NBR1: NBR1 autophagy cargo receptor; NDUFA9: NADH:ubiquinone oxidoreductase subunit A9; OPA1: OPA1, mitochondrial dynamin like GTPase; PPARGC1A, peroxisome proliferator activated receptor, gamma, coactivator 1 alpha; SDHA: succinate dehydrogenase complex, subunit A, flavoprotein (Fp); SQSTM1: sequestosome 1; ULK1: unc-51 like kinase 1; ULK2: unc-51 like kinase 2; UQCRC1: ubiquinol-cytochrome c reductase core protein 1.

Keywords: Age-related cardiomyopathy; MAP1LC3; NBR1; SQSTM1; dilated cardiomyopathy; heart failure; mitochondria; mitophagy.

Figure 1.

Perinatal deletion of ULK1 leads to a compensatory increase in basal autophagy. Data were obtained in 10 to 14-wk old mice unless otherwise specified. (A) Constitutive ulk1 deletion in isolated cardiomyocytes of cU1-KO mice. (B) Protein levels of MAP1LC3A-MAP1LC3B (MAP1LC3), SQSTM1, and NBR1 in randomly fed U1 fl/fl and cU1-KO mice. Representative immunoblots (Left) and summary of relative expression (Right) (n = 4–7). (C) Protein levels of MAP1LC3 analyzed after chloroquine administration as shown. Representative immunoblots (Top) and summary of relative expression (Bottom) (n = 5–8). (D) Representative B-mode images and serial cardiac function of U1 fl/fl and cU1-KO mice measured at 10, 21, 36, 60, and 90 weeks of age (n = 9–10). (E) Heart weight:tibia length reveals no overt cardiac hypertrophy (n = 7–8). (F) Representative H&E and Masson’s Trichrome images (Left) (scale: 20 μm) and quantification of fibrotic tissue (Right) (n = 4–6). Data are means ± SEM; *P < 0.05.

Figure 2.

Perinatal deletion of ULK2 leads to a compensatory increase in basal autophagy. All data was obtained in 10- to 14-wk-old mice unless otherwise specified. (A) Constitutive ULK2 deletion in isolated cardiomyocytes of cU2-KO mice. (B) Protein levels of MAP1LC3A-MAP1LC3B (MAP1LC3), SQSTM1, and NBR1 in randomly fed U2 fl/fl and cU2-KO mice. Representative immunoblots (Left) and quantification of relative expression (Right) (n = 4–7). (C) Protein levels of MAP1LC3 analyzed after chloroquine administration. Representative immunoblots (Top) and summary of relative expression (Bottom) (n = 6–10). (D) Representative B-mode images and serial cardiac function of U2 fl/fl and cULK2-KO mice measured at 10, 38, 64, and 86 weeks of age (n = 5–13). (E) Heart weight:tibia length reveals mild cardiac hypertrophy in cU2-KO mice (n = 5–6). (F) Representative H&E and Masson’s Trichrome images (Left) (scale: 20 μm) and quantification of fibrotic tissue (Right) (n = 4–6). Data are means ± SEM; *P < 0.05.

Figure 3.

Perinatal deletion of ULK1 and ULK2 impairs basal autophagy and leads to age-associated cardiomyopathy. All data was obtained in 10 to14-wk old mice unless otherwise specified. (A) Constitutive ULK1 and ULK2 deletion in whole heart lysates and protein levels of MAP1LC3A-MAP1LC3B (MAP1LC3), SQSTM1, and NBR1 in cU1/2-DKO mice. Representative immunoblots (Left) and quantification (Right) (n = 4–12). (B) Protein levels of MAP1LC3 analyzed after chloroquine administration. Representative immunoblots (Top) and quantification of relative expression (Bottom) (n = 4–12). (C) Heart weight:tibia length reveals overt cardiac hypertrophy in cU1/2-DKO mice (n = 4–5). (D) Survival analysis of U1/2 fl/fl and cU1/2-DKO mice (n = 7–10). (E) Representative H&E, Masson’s Trichrome, and WGA/DAPI images (Left) and quantification (scale: 20 μm) of fibrotic tissue and cardiomyocyte area (Right) (n = 4–6). (F) Representative B-mode images and serial cardiac function of U1/2 fl/fl and cU1/2-DKO mice measured at 11, 33, 59, 69, and 83 weeks (n = 5–6 for U1/2 fl/fl throughout, and n = 3–6 for cU1/2-DKO). Data are means ± SEM; *P < 0.05.

Figure 4.

Adult deletion of ULK1, but not of ULK2, leads to a lethal, rapidly developing dilated cardiomyopathy and mitochondrial dysfunction. Data were obtained in 9-wk old mice 5–8 days after initial tamoxifen injection, unless otherwise specified. (A) Knockdown of ULK1 from isolated cardiomyocytes of icU1-KO mice. (B) Knockdown of ULK2 from isolated cardiomyocytes of icU2-KO mice. (C) Survival analysis of U1 fl/fl and icU1-KO mice (n = 13–14). (D) Survival analysis of U2 fl/fl and icU2-KO mice (n = 12–14). (E) Heart weight:tibia length reveals cardiac hypertrophy in icU1-KO mice (n = 7–15). (F) Heart weight:tibia length reveals no cardiac hypertrophy in icU2-KO mice (n = 5–8). (G) Representative left ventricle images from U1 fl/fl and icU1-KO mice. (H) Representative left ventricle images from U2 fl/fl and icU2-KO mice. (I) Echocardiographs of icU1-KO mice. Representative B-mode images and cardiac function of U1 fl/fl and icU1-KO mice (n = 7–10). (J) Echocardiographs of icU2-KO mice. Representative B-Mode Images (Top) and cardiac function (Bottom) (n = 6–10). (K) Representative H&E, Masson’s Trichrome, WGA/DAPI images (Right) and quantification of fibrotic tissue and cardiomyocyte area (Left). (L) Representative immunoblots of MAP1LC3A-MAP1LC3B (MAP1LC3), SQSTM1, and NBR1 (Left), and quantification of protein expression of MAP1LC3, SQSTM1, and NBR1 in icU1-KO hearts and icU2-KO hearts (Right) (n = 6–8). Data are means ± SEM; *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 5.

Mitochondrial function, morphology, and biogenesis in icU1-KO hearts. (A) Pyruvate-malate and palmitoyl-carnitine-malate driven oxygen flux (n = 4). (B) Pyruvate-malate and palmitoyl-carnitine-malate driven ATP production (n = 3–4). (C) Representative immunoblots of subunits of the electron transport chain (Left) and quantification (Right) (n = 6). (D) Representative TEM images of mitochondrial structure denoting a mitochondrion with aberrant and/or disrupted cristae structure (arrowhead) and remnants of a cristae structure devoid of outer mitochondrial membrane (arrow) (Top Left). Quantification of mitochondrial content in icU1-KO hearts expressed as a percentage of total area in TEM images (n = 4) (Top Right). Percentage distribution of mitochondria according to area and length assessed from TEM images (n = 3–4) (Bottom). (E) Mitochondrial DNA content quantification (n = 7). (F) Relative mRNA expression of Ppargc1a (n = 3–7). (G) Representative immunoblots of CYCS and TUBA1A in mitochondrial and cytosolic fractions from U1 fl/fl and icU1-KO hearts (Left). Representative immunoblots of DNM1L, MFN1, MFN2, and OPA1 (normalized to SDHA), and p-DNM1L (s616):DNM1L in enriched mitochondrial fractions from U1 fl/fl and icU1-KO hearts (Middle) and quantification (Right) (n = 3–4). Data are means ± SEM; *P < 0.05 and **P < 0.01.

Figure 6.

Adult deletion of ULK1 rapidly impairs cardiac autophagy. Data were obtained in 9-wk old mice 4 days after initial tamoxifen injection, unless otherwise specified. (A) Protein levels of MAP1LC3A-MAP1LC3B (MAP1LC3) analyzed after chloroquine administration. Representative immunoblots (Left) and quantification of relative expression (Right) (n = 5–6). (B) Representative TEM images (8000x magnification) of U1fl/fl and cU1-KO hearts treated with vehicle (VEH) or chloroquine (CQ) (Top). Red arrowheads indicate autophagosomes and red arrows indicate autolysosomes. Quantification of autophagosomes and autolysosomes relative to U1 fl/fl treated with vehicle (Bottom) (n = 4). (C) Representative TEM images (3x zoom of original 2000x magnification) of U1 fl/fl and icU1-KO hearts treated with vehicle (VEH) or chloroquine (CQ) (scale bar: 1 μm). Red arrows indicate mitophagosomes. Quantification of mitophagosomes relative to U1 fl/fl treated with vehicle (Bottom) (n = 4). Data are means ± SEM; *P < 0.05, **P < 0.01 and ***P < 0.001.

Figure 7.

Trehalose administration restores impaired autophagic flux but does not prevent heart failure in icULK1 KO mice. (A) Autophagy flux measurements of vehicle (Veh) and trehalose (Tre) treated WT hearts. Representative immunoblots of MAP1LC3A-MAP1LC3B (MAP1LC3) and GAPDH (Top), quantification of changes in MAP1LC3-II:GAPDH and MAP1LC3-II:MAP1LC3-I and magnitude of change in MAP1LC3-II upon chloroquine (CQ) treatment (Bottom) (n = 3–4). (B) Representative immunoblots of MAP1LC3, SQSTM1, and ubiquitin (Left) and quantification (Right) in U1 fl/fl, icU1-KO, and icU1-KO+Tre mice 5–7 days after the first tamoxifen injection (n = 5–6). (C) Heart weight:tibia length of U1 fl/fl, icU1-KO, and icU1-KO+Tre mice (n = 4–6). (D) Cardiac function assessed from echocardiographs of U1 fl/fl, icU1-KO, and icU1-KO+Tre mice (n = 6–14). Data are means ± SEM; *P < 0.05, **P < 0.01 and ****P < 0.0001.

Figure 8.

Differential consequences of perinatal vs. adult loss of ULK1 and/or ULK2 on cardiac autophagy and function. Perinatal deletion of ULK1 or ULK2 enhances basal cardiac autophagy. This adaptation requires the preserved expression of the remaining ULK (i.e., ULK1 in cU2-KO and ULK2 in cU1-KO) because perinatal loss of both ULK1 and ULK2 leads to impaired autophagy in the adult heart, age-related cardiomyopathy and early death. However, adult loss of cardiac ULK1 (i.e., in icU1-KO mice) rapidly impairs autophagy leading to dilated cardiomyopathy and heart failure, whereas adult loss of cardiac Ulk2 (i.e., in icU2-KO mice) does not impair autophagy or cardiac function. Attenuation of impaired cardiac autophagy following adult ULK1 loss (with trehalose; not depicted) does not delay cardiac dysfunction suggesting that ULK1 plays other important roles in the heart. Collectively, these findings indicate that ULK1 and ULK2 redundantly modulate autophagy and cardiac function early in development, but not in the adult unstressed heart.