Targeting Rab7-Rilp Mediated Microlipophagy Alleviates Lipid Toxicity in Diabetic Cardiomyopathy

Abstract

Diabetic cardiomyopathy (DbCM) is characterized by diastolic dysfunction, which progresses into heart failure and aberrant electrophysiology in diabetic patients. Dyslipidemia in type 2 diabetic patients leads to the accumulation of lipid droplets (LDs) in cardiomyocytes and results in lipid toxicity which has been suggested to drive DbCM. It is aimed to explore potential pathways that may boost LDs degradation in DbCM and restore cardiac function. LDs accumulation resulted in an increase in lipid toxicity in DbCM hearts is confirmed. Microlipophagy pathway, rather than traditional macrolipophagy, is activated in DbCM hearts. RNA-Seq data and Rab7-CKO mice implicate that Rab7 is a major modulator of the microlipophagy pathway. Mechanistically, Rab7 is phosphorylated at Tyrosine 183, which allows the recruitment of Rab-interacting lysosome protein (Rilp) to proceed LDs degradation by lysosome. Treating DbCM mice with Rab7 activator ML-098 enhanced Rilp level and rescued the observed cardiac dysfunction. Overall, Rab7-Rilp-mediated microlipophagy may be a promising target in the treatment of lipid toxicity in DbCM is suggested.

Keywords: diabetic cardiomyopathy; lipid toxicity; lipophagy.

Figure 1

Presence of lipid toxicity in DbCM. A) A flowchart describing the establishment of DbCM mice model. B) Changes in blood glucose level measured after injection of α‐d‐glucose on mice and quantification of areas under curve (n = 4 per group). C) Changes in blood glucose level measured after injection of insulin on mice and quantification of areas under curve (n = 3 per group). D) Recorded body weight of DbCM mice (n = 3–5 per group). E) Random blood glucose of DbCM mice (n = 5‐6 per group). F) Plasma insulin level of DbCM mice (n = 3 per group). G) Representative micrographs of m‐mode, doppler, and tissue doppler echocardiography. H) Evaluation of diastolic function by E/e’ (n = 6 per group). I) Analysis on the thickness of left ventricular diastolic anterior wall (LVAW:d) (n = 6 per group). J) Evaluation of systolic function by left ventricular ejection function (n = 6 per group). K) Representative micrographs of Masson's trichrome staining in heart sections of mice and quantification on fibrotic areas from Masson's trichrome staining (n = 5 per group). L) Representative micrographs of Oil‐Red staining in DbCM cryosection and quantification of Oil‐Red Staining Area (%) (n = 5 per group). M) Triglyceride content in DbCM mice hearts (n = 3 per group). N) Representative micrographs of immunofluorescence staining of Lipi‐Red to label LDs in mice heart cryosection and quantification of Lipi‐Red staining area (n = 5 per group). The cell nuclei were stained with DAPI (blue) and cardiomyocytes were stained with cardiac troponin T (cTnT, magenta). O) Representative micrographs of immunofluorescence staining of Lipi‐Red to label LDs in human heart cryosection. The cell nuclei were stained with DAPI (blue) and cardiomyocytes were stained with cardiac troponin T (cTnT, magenta). The Student's t‐test was used to analyze the differences between two groups and the data are expressed as mean ± SD, ns: no significance, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 and ^**** p < 0.0001).

Figure 2

Induction of macroautophagy does not prevent LDs accumulation in DbCM. A) GO analysis on enrichments of differentially expressed genes (DEGs). B) Top upregulated and downregulated DEGs associated with autophagy. C) Representative blots, relative intensity and quantitative analysis of immunoblots analysis of LC3B in Langendroff AMVMs (n = 6 per group). D) Representative micrographs of immunofluorescence double‐staining of LC3 to label autophagosome and Lipi‐Red (Red) to label LDs, and quantification of Lipi‐Red colocalizing LysoTracker, results were presented as Pearson’ R (n = 4‐6 per group). The cell nuclei were stained with DAPI (blue) and cardiomyocytes were stained with cardiac troponin T (cTnT, magenta). E) Representative blots, relative intensity, and quantitative analysis of immunoblots analysis of Ulk1 and Atg5 in Langendroff AMVMs (n = 5‐6 per group). F) Representative micrographs of immunofluorescence double‐staining of Lipi‐Red (red) to label LDs and Atg5 (green), and quantification of Lipi‐Red colocalizing Atg5, results were presented as Pearson’ R (at least n = 30 from 3 individual tests per group). The cell nuclei were stained with DAPI (blue). G) Representative micrographs of immunofluorescence double‐staining of Lipi‐Red (red) to label LDs and Ulk1 (green), and quantification of Lipi‐Red colocalizing Ulk1, results were presented as Pearson’ R (at least n = 30 from 3 individual tests per group). The cell nuclei were stained with DAPI (blue). H) Representative micrographs of immunofluorescence staining of Lipi‐Red (red) to label LDs and quantification of Lipi‐Red staining area (at least n = 40 from three individual tests per group). The cell nuclei were stained with DAPI (blue). I) Quantification of relative triglyceride content in NMCMs after 3‐MA treatment (n = 3 per group). The Student's t‐test was used to analyze the differences between two groups and the data are expressed as mean ± SD, ns: no significance and ^** p < 0.01).

Figure 3

Microlipophagy dominates LDs degradation in DbCM. A) Representative micrographs of immunofluorescence double‐staining of Lipi‐Red (red) to label LDs and Lamp1 (green) to label lysosome, and quantification of Lipi‐Red colocalizing Lamp1, results were presented as Pearson’ R (n = 6 per group). The cell nuclei were stained with DAPI (blue) and cardiomyocytes were stained with cardiac troponin T (cTnT, magenta). B) Live‐cell confocal imaging of Ctrl and HGPA treated NMCMs incubated Lipi‐Red (red) to label LDs and Lysotracker (green) to label lysosome, and quantification of Lipi‐Red colocalizing LysoTracker, results were presented as Pearson’ R (at least 30 cells per group from three individual tests). Cell nuclei were labeled with Hoechst 33342 and arrows indicate examples of Lipi‐Red overlapping LysoTracker. C) Confocal micrographs using the same assay conditions as in (B) after 3‐MA treatment, and quantification of Lipi‐Red colocalizing LysoTracker, results were presented as Pearson’ R. (at least 30 cells per group from three individual tests). Cell nuclei were labeled with Hoechst 33 342 and arrows indicate examples of Lipi‐Red overlapping LysoTracker. D) Confocal micrographs after Rapamycin treatment using the same assay conditions as in (B) and quantification of Lipi‐Red colocalizing LysoTracker, results were presented as Pearson’ R (at least 30 cells per group from three individual tests). Cell nuclei were labeled with Hoechst 33342 and arrows indicate examples of Lipi‐Red overlapping LysoTracker. E) Quantification of Lipi‐Red Staining Area (%) in Ctrl, HGPA, and HGPA+Rapamycin treated NMCMs (at least 40 cells from three individual tests per group f). F) Quantification of relative triglyceride content in NMCMs after Rapamycin treatment (n = 3 per group). G) Representative micrograph and quantitative analysis of intracellular reactive oxygen species (ROS) level in NMCMs after Rapamycin treatment (at least n = 20 from 3 individual tests per group). H) Representative blots, relative intensity, and quantitative analysis of immunoblots analysis of Atf6 and Ire1α in Rapamycin treated NMCMs (n = 6 per group). I) Relative contraction velocity of NMCMs after Rapamycin treatment (at least 30 cells per group from 3 individual tests). J) Representative micrographs of immunofluorescence double‐staining of Lipi‐Red (red) to label LDs and Lamp1 (green) to label lysosome in human heart section. The cell nuclei were stained with DAPI (blue) and cardiomyocytes were stained with cardiac troponin T (cTnT, magenta). K) Confocal micrographs using the same assay conditions as in (B), presenting a gradual fusion of LDs to lysosome from 1–6. L) Transmission electron microscopy (TEM) micrographs from DbCM mice heart. LDs refers to lipid droplets. Lys refers to lysosome. Sequence 1–5 indicate a fusion process between LDs and lysosome. M) Zoomed TEM micrographs from J. Arrows indicates that there are no double‐membrane structures exist. The Student's t‐test was used to analyze the differences between two groups and the data are expressed as mean ± SD, ^* p < 0.05, ^** p < 0.01, and ^**** p < 0.0001).

Figure 4

Rab7‐Rilp axis mediate microlipophagy in DbCM. A) All DEGs that were associated with LDs within the group Ctrl and HGPA from RNA‐Seq analysis. B) Flowchart representing the analysis of DEGs. Among all 16 276 DEGs, there are 245 genes associated with autophagy and 77 associated with LDs, while only 6 DEGs are associated with both. C) Expressions of statistically significant DEGs that were associated both with autophagy and LDs. D) Confocal micrographs of human heart samples stained with Rab7 (Green) and Lipi‐Red (Red). The cell nuclei were stained with DAPI (blue) and cardiomyocytes were stained with cardiac troponin T (cTnT, magenta). E) Representative micrographs of immunofluorescence staining of Rab7 (green) and Lipi‐Red (red) to label LDs in DbCM mice heart and quantification of Lipi‐Red colocalizing Rab7. Results were presented as Pearson’ R (n = 5 per group). The cell nuclei were stained with DAPI (blue) and cardiomyocytes were stained with cardiac troponin T (cTnT, magenta). F) Representative blots, relative intensity, and quantitative analysis of immunoblots analysis of Rab7 and Rilp in DbCM AMVMs (n = 6 per group). G) Representative blots of GST interacted Rab7 (Q67L) in HL‐1 cells transfected GST‐Rilp. H) Relative mRNA expression of GST‐Rilp in HL‐1 cells transfected with GST‐Rilp. I) Representative blots of Rab7 immunoprecipitated Rilp in Ctrl and HGPA group. The Student's t‐test was used to analyze the differences between two groups and the data are expressed as mean ± SD, ^** p < 0.01, ^*** p < 0.001 and ^**** p < 0.0001).

Figure 5

Microlipophagy can be manipulated by targeting Rab7‐Rilp axis. A) Representative blot of Rab7 immunoprecipitated Rilp after treatment of CID‐1067700. B) Live‐cell confocal imaging of Ctrl, HGPA, and HGPA + CID‐1067700 treated NMCMs incubated Lipi‐Red (red) to label LDs and Lysotracker (green) to label lysosome, and quantification of Lipi‐Red colocalizing LysoTracker, results were presented as Pearson’ R (at least 30 cells from three individual tests per group). Cell nuclei were labeled with Hoechst 33342 and arrows indicate examples of Lipi‐Red overlapping LysoTracker. C) Quantification of Lipi‐Red Staining Area (%) in Ctrl, HGPA, and HGPA+CID‐1067700 treated NMCMs (at least 30 cells per group from three individual tests). D) Quantification of relative triglyceride content in NMCMs after CID‐1067700 treatment (n = 3 per group). E) Relative contraction velocity of Ctrl, HGPA, and HGPA+CID‐1067700 incubated NMCMs (at least 30 cells per group from three individual tests). F) Relative mRNA expression of Rilp in HL‐1 cells transfected with siRilp. G Representative micrographs of immunofluorescence staining of Lysotracker (Green) and Lipi‐Red (Red) in cells transfected with siRilp, and quantification of Lipi‐Red colocalizing LysoTracker, results were presented as Pearson’ R (at least 30 cells per group from 3 individual tests). H) Quantification of Lipi‐Red Staining Area (%) in Ctrl, HGPA, HGPA with or without siRilp transfected NMCMs (at least 30 cells per group from 3 individual tests). I) Quantification of relative triglyceride content in NMCMs after siRilp transfection (n = 3 per group). J) Relative contraction velocity of Ctrl, HGPA, HGPA with or without siRilp transfected treated in NMCMs (at least 30 cells per group from 3 individual tests). K) Representative blot of Rab7 immunoprecipitated Rilp after treatment of ML‐098. L) Live‐cell confocal imaging of Ctrl, HGPA, and HGPA + ML‐098 treated NMCMs incubated Lipi‐Red (red) to label LDs and Lysotracker (green) to label lysosome and quantification of Lipi‐Red colocalizing LysoTracker, results were presented as Pearson’ R. Cell nuclei were labeled with Hoechst 33 342, arrows indicate examples of Lipi‐Red overlapping LysoTracker. M Quantification of Lipi‐Red Staining Area (%) in Ctrl, HGPA, and HGPA+ML‐098 treated NMCMs (at least 30 cells per group from 3 individual tests). N) Quantification of relative triglyceride content in NMCMs after ML‐098 treatment (n = 3 per group). O) Relative contraction velocity of NMCMs after ML‐098 treatment (at least 30 cells per group from 3 individual tests). P) Representative blot of Rab7 immunoprecipitated Rilp after treatment of lambda‐PPase. Q) Structure of Rab7 with Serine 17, Serine 72, and Tyrosine 183 highlighted. R) Representative blot of FLAG‐Rab7 immunoprecipitated HA‐Rilp after mutations of the targeted amino acid. The Student's t‐test was used to analyze the differences between two groups and the data are expressed as mean ± SD, ns: no significance, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 and ^**** p < 0.0001).

Figure 6

Rab7‐CKO mice present attenuated lipophagy and severe lipid toxicity. A) Representative image, relative intensity, and quantification of immunoblots analyzing Rab7 in WT and Rab7‐CKO mice respectively establishing DbCM model (n = 3 per group). B) Recorded body weight changes of DbCM mice (n = 5 per group). C) Random blood glucose of DbCM mice (n = 5‐6 per group). D) Representative micrographs of immunofluorescence double‐staining of Lipi‐Red (red) to label LDs and Lamp1 (green) to label lysosome, and quantification of Lipi‐Red colocalizing Lamp1, results were presented as Pearson’ R (n = 6 per group). The cell nuclei were stained with DAPI (blue) and cardiomyocytes were stained with cTnT (magenta). Arrows indicate examples of Lipi‐Red overlapping Lamp1. E) Quantification of Lipi‐Red Staining Area (%) (n = 5‐6 per group) in WT and Rab7‐CKO mice. F) Representative micrographs of Oil‐Red staining in DbCM cryosection and quantification of Oil‐Red Staining Area (%) (n = 4‐6 per group). G) Quantification of triglyceride content in DbCM hearts (n = 3 per group). H) Evaluation of systolic function by left ventricular ejection function (n = 6 per group). I) Evaluation of diastolic function by E/e’ (n = 6 per group). J) Representative micrographs of m‐mode, doppler, and tissue doppler echocardiography. K) Evaluation of left ventricular diastolic anterior wall thickness (LVAW:d) (n = 6 per group). Evaluation on L) percentage of shortening, M) maximum systolic velocity, and N) maximum diastolic velocity in Langendorff‐isolated adult mouse cardiomyocytes (AMCMs) (at least 90 cells from three individual mice per group). The Student's t‐test was used to analyze the differences between two groups and the data are expressed as mean ± SD, ns: no significance, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001 and ^**** p < 0.0001).

Figure 7

Activating Rab7 by ML‐098 improves cardiac function in DbCM mice. A) A flowchart describing the establishment of DbCM mice model and injection of ML‐098. B) Random blood glucose of DbCM mice (n = 5‐6 per group). C) Representative micrographs of m‐mode, doppler, and tissue doppler echocardiography. D) Evaluation of diastolic function by E/e’ (n = 6 per group). E) Evaluation of E/A waves ratio. (n = 5 per group). F) Analysis on thickness of left ventricular diastolic anterior wall (LVAW:d) (n = 6 per group). G) Evaluation on global longitudinal strain (GLS) (n = 6 per group). H) Evaluation of systolic function by left ventricular ejection function (n = 6 per group). I) Analysis on thickness of left ventricular diastolic posterior wall (LVPW:d) (n = 6 per group). J) Representative micrographs of Masson's trichrome staining in heart sections of mice and quantification on fibrotic areas (n = 3 per group). K) Representative micrographs of immunofluorescence double‐staining of Lipi‐Red (red) to label LDs and Lamp1 (green) to label lysosome and quantification of Lipi‐Red colocalizing Lamp1, and quantification of Lipi‐Red colocalizing Lamp1, results were presented as Pearson’ R (n = 6 per group). The cell nuclei were stained with DAPI (blue) and cardiomyocytes were stained with cardiac troponin T (cTnT, magenta). I) Representative micrographs of immunofluorescence staining of Lipi‐Red to label LDs in mice heart cryosection, and quantification of Lipi‐Red Staining Area (%) in (I) (n = 6 per group). The cell nuclei were stained with DAPI (blue) and cardiomyocytes were stained with cardiac troponin T (cTnT, magenta). M) Representative micrographs of Oil‐Red staining in DbCM cryosection and quantification of Oil‐Red Staining Area (%) (n = 8 per group). N) Quantification of triglyceride content in DbCM hearts (n = 3 per group). O) Representative blots, relative intensity, and quantitative analysis of immunoblots analysis of Rilp and Rab7 in Langendroff AMVMs from groups Ctrl, DbCM, and DbCM treated ML‐098. (n = 6 per group). The Student's t‐test was used to analyze the differences between two groups and the data are expressed as mean ± SD, ns: no significance, ^* p < 0.05, ^** p < 0.01, ^*** p < 0.001, and ^**** p < 0.0001).