Rag GTPases are cardioprotective by regulating lysosomal function

Abstract

The Rag family proteins are Ras-like small GTPases that have a critical role in amino-acid-stimulated mTORC1 activation by recruiting mTORC1 to lysosome. Despite progress in the mechanistic understanding of Rag GTPases in mTORC1 activation, little is known about the physiological function of Rag GTPases in vivo. Here we show that loss of RagA and RagB (RagA/B) in cardiomyocytes results in hypertrophic cardiomyopathy and phenocopies lysosomal storage diseases, although mTORC1 activity is not substantially impaired in vivo. We demonstrate that despite upregulation of lysosomal protein expression by constitutive activation of the transcription factor EB (TFEB) in RagA/B knockout mouse embryonic fibroblasts, lysosomal acidification is compromised owing to decreased v-ATPase level in the lysosome fraction. Our study uncovers RagA/B GTPases as key regulators of lysosomal function and cardiac protection.

Figure 1. Loss of RagA and RagB causes cardiomegaly and premature death

(A) RagA and RagB protein levels are decreased in RagA/B cKO hearts. Heart tissue lysates were prepared from control or RagA/B cKO mice and analyzed by immunoblotting. S.E, short exposure; L.E, long exposure. (B) Representative images of whole mount hearts (4 months old), and H&E stained longitudinal and transverse sections of the hearts from 4-month and 1-year old mice, respectively. Ruler represents 1 mm per interval. (C) Heart/Body weight index. Heart weight was divided by body weight. Values represent the mean ± SD of data (n=8 per group). P=0.02, Wilcoxon rank sum test. (D) Survival curve of control and RagA/B cKO mice. P <0.00001, Mantel-Cox (logrank) test. (E) Lung/body weight index. Lung weight was divided by body weight of the mouse. Values represent the mean ± SD of data (n=8 per group). (F) Liver/body weight index. Liver weight was divided by body weight of the mouse. Values represent the mean ± SD of data (n=8 per group).

Figure 2. Cardiac hypertrophy in RagA/B cKO mice

(A) Images of wheat germ agglutinin (WGA)-stained heart tissues. Representative images from control and RagA/B cKO tissues are shown. Scale bars, 100 μm. (B) Cross sectional area of cardiomyocytes. The area of cardiomyocytes from (A) was measured using the ImageJ software. Values represent the mean ± SD of data (n=15 per group). (C) Quantification of cell proliferation in heart tissues. Longitudinal sections of whole mount of hearts were stained with phsopho-Histone3 (p-H3) antibody to detect proliferating cardiomyocytes. The p-H3 positive cells in a whole longitudinal tissue section were counted. Values represent the mean ± SD of data (n=3 per group). (D) Quantification of apoptotic cell death in heart tissue. The serial sections of tissues from (C) were examined for apoptotic cell death by immunohistochemical staining using antibody that binds to cleaved (active) caspase-3. The active caspase-3 positive cells in a whole longitudinal tissue section were counted. Values represent the mean ± SD of data (n=5 per group). P values were determined by Wilcoxon rank sum test.

Figure 3. RagA or RagB single conditional KO in heart does not cause cardiac hypertrophy

(A) Representative images of whole mount hearts. Hearts were isolated from Mck-Cre/+;RagA flox/flox (2 months old) or Mck-Cre/+;RagB flox/flox (3 months old) mice, and the size of heart was compared with control littermates. (B) Heart/Body weight index. Heart weight was divided by body weight. Values represent the mean ± SD of data (n=3 per group). P values were determined by signed rank sum test. (C) Immunoblot analysis using heart tissue lysates. Tissue lysates from control, RagA, or RagB single cKO hearts were analyzed by immunoblotting using the indicated antibodies.

Figure 4. Characterization of the cardiac hypertrophy in RagA/B cKO mice

(A) Images of H&E (top, 2 month-old mice) or Masson's trichrome (bottom, 1 year-old mice) stained heart tissues. Arrows and arrowheads point enlarged nuclei and vacuole formation, respectively. Representative images are shown. Scale bars, 50 μm. (B) Molecular markers of cardiac remodeling in RagA/B cKO hearts. mRNA levels of indicated genes were determined by quantitative RT-PCR. Values represent the mean ± SD of data (n=4 per group). (C) Images of echocardiography. Representative images of tracing are shown. (D) Measurements of echocardiography and parameters of hemodynamic analyses. LVEDD, left ventricular end-diastolic diameter; SWTd, diastolic septal wall thickness; PWTd, diastolic posterior wall thickness; E/A ratio, early to late ventricular filling velocity; FS, fractional shortening; LVESS, left ventricular end-systolic stress; dP/dt max and min, the rate of left ventricle pressure rise in systole and diastole, respectively;.Ees, end-systolic elastance; EDPVR, end-diastolic pressure-volume relation slope. Values represent the mean ± S.E of data (n=4 or 5 per group). * P < 0.05, Wilcoxon rank sum test.

Figure 5. Defective autophagy flux in RagA/B cKO hearts and MEFs

(A) Immunoblot analysis using heart tissue lysates. Tissue lysates from control or RagA/B cKO hearts were analyzed by immunoblotting using the indicated antibodies. (B) Immunohistochemical analysis of heart tissues. Heart tissue sections were analyzed by immunohistochemistry using the indicated antibodies. Representative images are shown. Scale bars, 200 μm (◅S6S^235/236 and p62); 50 μm (LAMP1). (C) Transmission electron microscopy of heart tissues. The ultrastructure of control and RagA/B cKO hearts was examined by TEM. Arrows and arrowheads indicate accumulation of AVi (initial autophagic vacuoles) and AVd (late autophagic vacuoles/autolysosomes), respectively. Asterisks point the electron-lucent spaces. Scale bars, 1 μm (top panel); 200 nm (bottom panel). (D) Immunoblot analysis of control and RagA/B KO MEFs. Cells were cultured in a nutrient rich condition, and then cell lysates were analyzed by immunoblotting with the indicated antibodies. (E) Quantification of immunoblot data. Levels of p62 and LC3II were determined by densitometry. Values represent the mean ± SD of data (n=3). *P < 0.05, Wilcoxon rank sum test. (F) Immunoblot analysis of autophagy flux. Cells were cultured in a nutrient rich medium, and then incubated in HBSS to induce autophagy. For the replenished samples, cells were re-incubated for another 90 min in a nutrient rich medium after 90 min of starvation. A set of control cells was cultured in the presence of bafilomycin A₁ (100 nM). Cell lysates were prepared at the indicated time points and analyzed by immunoblotting. CON, control MEFs; KO, RagA/B KO MEFs; Baf, bafilomycin A₁.

Figure 6. Altered lysosomal protein expression in RagA/B KO MEFs

(A) Nuclear localization of TFEB in RagA/B KO MEFs. TFEB-GFP expressing control or RagA/B KO MEFs were cultured either in a nutrient rich medium or in HBSS for 2 hours, and then cells were fixed to examine the localization of TFEB-GFP proteins and immuno-stained with phospho-S6^S235/236 antibody. Representative images are shown. Scale bars, 50 μm. (B) Quantitative RT-PCR of v-ATPase subunits and lysosomal membrane proteins. Total RNAs were prepared from control, 2 hour-fasted control, or RagA/B KO MEFs, and the expression levels of indicated genes were determined. (C) Immunoblot analysis of v-ATPase subunits and lysosomal membrane proteins. The levels of lysosomal membrane proteins and v-ATPase subunits were compared by immunoblotting. (D) Quantification of immunoblot analysis by densitometry. (E) The levels of v-ATPase subunits in lysosome fraction. Cell lysates were fractionated, and the amounts of v-ATPase subunits in each fraction were compared between control and RagA/B KO MEFs. The loading amount of cell lysates was normalized with ATP6V1D, and the percentage of the v-ATPase subunit level in lysosome fraction was determined by densitometric analysis. (F) Comparison of the lysosomal v-ATPase level in TFEB^S142A expressing cells. The amounts of LAMP2, ATP6V1B2, and ATP6V1D in total or lysosomal fraction of GFP- TFEB^S142A expressing cells were compared with control and RagA/B KO MEFs. For the lysosomal fraction, the protein loading amounts were normalized with LAMP2 levels. C, control MEFs; K, RagA/B KO MEFs; TOT, total; CYT, cytosolic fraction; LYS, lysosome fraction. All values represent the mean ± SD of data (n=3 per group). *P < 0.05, Wilcoxon rank sum test.

Figure 7. Restored autophagy flux and lysosomal v-ATPase localization in RagA/B KO MEFs by RagA^WT expression, but not by Rheb^WT or Rheb^S16H expression

(A) Restoration of autophagy flux in RagA/B KO MEFs by reintroducing RagA^WT. RagA/B KO MEFs were infected with wild-type RagA (RagA^WT) expressing retroviruses, and then autophagy flux was examined by immunoblotting. (B) Immunoblot analysis of TFEB target gene expression. The expression levels of LAMP2, ATP6V1B2, and ATP6V1D were restored by RagA^WT expression in the RagA/B KO MEFs. (C) Immunoblot analysis of lysosomal v-ATPase subunits. Cells were fractionated, and the levels of v-ATPase subunits in total, cytoplasm, and lysosomal fraction were examined by immunoblotting. The protein loading amounts were normalized with LAMP2 protein levels. (D) Rheb^WT or Rheb^S16H expression substantially restores mTORC1 activity in RagA/B KO MEFs. Stable cell lines were generated by infecting RagA/B KO MEFs with Rheb^WT or Rheb^S16H encoding retroviruses, and then cell lysates were analyzed by immunoblotting. (E) Rheb^WT or Rheb^S16H does not restore lysosomal v-ATPase localization in RagA/B KO MEFs. Cells were fractionated, and the lysates were analyzed by immunoblotting. (F) Immunoblot analysis of autophagy flux. Cells were cultured in a nutrient rich medium followed by starvation in HBSS for 90 min, and autophagy flux markers were examined by immunoblotting. (G) Nutrient deprivation or RagA^TN expression in control MEFs does not alter lysosomal v-ATPase localization. Starved control MEFs (in HBSS for 2 hrs) or RagA^TN expressing control MEFs were fractionated and analyzed by immunoblotting. CON, control MEFs; KO, RagA/B KO MEFs; A^WT, RagA wild-type; TOT, total; CYT, cytoplasm; LYS, lysosome fraction.

Figure 8. Increased TFEB activity in RagA/B KO MEFs and RagA/B cKO hearts

(A) p62 mRNA levels were upregulated in RagA/B KO MEFs and TFEB^S142A expressing control MEFs. Total RNAs were prepared from cells and p62 mRNA levels were quantified using qPCR. Values represent mean ± SD of data (n=4 per group). *P value=0.021. (B) Immunoblot analysis of p62 degradation by starvation. p62 levels were substantially decreased in control and TFEB^S142A expressing control MEFs by starvation (in HBSS for 90min), but not in RagA/B KO MEFs. (C) Immunohistochemical analyses of heart tissues using TFEB antibody. Top panel, low magnification view shows uneven distribution of TFEB expression in the control hearts (scale bars=500μm). Middle panel, high magnification view of left ventricular wall areas (scale bars=50μm). Bottom panel, inter-ventricular septum areas (scale bars=50μm). (D) Quantification of TFEB positive nuclei. The number of TFEB positive nuclei were divided by total number of nuclei per microscopic field. Values represent mean ± SD of data (n=5 per group). (E) Immunobot analysis of heart tissue lysates. TFEB and its target ATP6V1D levels were upregulated in RagA/B cKO hearts compared with controls. (F) Quantitative RT-PCR analyses of TFEB target genes from heart tissues. Total RNAs were prepared from heart tissues, and mRNA levels of TFEB target genes, CD63 and p62 were quantified by qPCR. Values represent mean ± SD of data (n=4 per group). P values were determined by Wilcoxon rank sum test.

Figure 9. Lysosomal acidification is compromised in RagA/B KO MEFs

(A) Lysosomal pH measurement in live cells. Cells were fed with OG541-conjugated dextran for overnight followed by serum-starvation for 1 hour, and then trypsinized for spectrofluorimetric measurement. Values represent the mean ± SD of data (n=3 per group). *P < 0.05, Wilcoxon rank sum test. (B) Immunoblot analysis of cathepsin D maturation in lysosome fraction. Lysosome fractions from control and RagA/B KO MEFs were analyzed by immunoblotting using the indicated antibodies. The amount of protein loading was normalized with LAMP1 protein level. Arrowhead indicates the matured form of cathepsin D. (C) Sialidase A treatment increases mobility of LAMP2 on SDS-PAGE. Cell lysates were incubated with sialidase A for 3hrs followed by SDS-PAGE and immunoblotting. (D) Cathepsin D maturation in GFP-TFEB^S142A expressing cells. The amounts of matured cathepsin D in lysosomal fraction of GFP-TFEB^S142A expressing cells were compared with control and RagA/B KO MEFs using immunoblotting. The amount of protein loading was normalized with LAMP2 protein level. Arrowhead indicates the matured form of cathepsin D. (E) A compromised lysosomal acidification capacity in RagA/B KO MEFs. Cells were fed with a pH sensitive fluorescent dye (OG541)-conjugated dextran for overnight, and then starved in a serum-free medium for 2 hours. v-ATPase-mediated acidification of lysosome-enriched fraction was measured in the absence or in the presence of bafilomycin A₁ (100nM) in vitro. The amount of lysosome fraction was normalized with LAMP2 protein level. Each point represents the mean ± S.E of three 30-second intervals from three independent measurements. CON, control MEFs; KO, RagA/B KO MEFs; Baf, bafilomycin A₁.

Figure 10. Cardiac hypertrophy in RagA/B cKO mice exhibits the features of lysosomal storage diseases

(A) Periodic acid-Schiff staining of heart tissues. Representative images are shown. Scale bars, 50 μm. (B) Biochemical measurement of glycogen level in heart tissues. Glycogen levels were determined using an enzyme-based colorimetric assay kit. Values represent the mean ± SD of data (n=4 per group). *P < 0.03, Wilcoxon rank sum test. (C) Immunoblot analysis of glycogen synthase-1 in heart. Tissue lysates were prepared from control and RagA/B cKO hearts, and levels of glycogen synthase and phosphoglycogen synthase were investigated by immunoblotting. (D) Glycogen phosphorylase activity assay. Using an enzyme-based assay, activity of glycogen phosphorylase in heart tissue was determined by measuring NADPH production in the reaction. Each point represents the mean ± SD of data (n=4 per group). (E) Accumulation of ubiquitinated proteins in RagA/B cKO hearts. Levels of ubiquitinated proteins in heart tissues were examined by immunoblotting.