Cardiomyocyte-Restricted Low Density Lipoprotein Receptor-Related Protein 6 (LRP6) Deletion Leads to Lethal Dilated Cardiomyopathy Partly Through Drp1 Signaling

Figure 1. LRP6 expression in heart tissues. (A) Western blot analysis of LRP6 and LRP5 expression in human heart tissues. 3 rejected healthy donor hearts (control) and 3 end-stage heart failure patients with dilated cardiomyopathy (DCM) were studied. ** p<0.01 vs. control group. (B) Immunofluorescence staining of LRP6 expression in left ventricular tissue from C57BL/6 mice. Green: LRP6; blue: nucleus. Scale bars, 10 μm.

Figure 2. Cardiomyocyte-specific deletion of LRP6 causes cardiac dysfunction. (A) Schematic presentation of experimental strategy in vivo. (B) Western blot analysis of LRP6 expression in heart tissue from MCM/LRP6fl/fl mice after treatment with diluent or tamoxifen for 3 days. ** p<0.001 vs. diluent group (n=3/group). (C) Survival proportion was analyzed in MCM mice and MCM/LRP6fl/fl mice for 60 days following three-day-treatment with tamoxifen. n=14/MCM/LRP6fl/fl group, n=6/MCM group. (D) Echocardiographic analysis of fraction shortening (FS) in MCM or MCM/LRP6fl/fl injected with tamoxifen at different time-points during the experimental process. n=7-14/MCM/LRP6fl/fl group, n=6/MCM group. (E) Representative M-Mode echocardiographs. (F-G) Fraction shortening (FS) and left ventricular cavity diastolic dimension (LVID.d) were analyzed by echocardiography. n=6-8/group. (H-K) dp/dt, -dp/dt, left ventricular systolic pressure (LVSP) and left ventricular end diastolic pressure (LVEDP) were determined by hemodynamic analysis. n=8/group. (L) Representative photographs of gross hearts. (M) Hematoxylin-eosin (HE) stains of longitudinal heart sections. Scale bars, 1 mm. n=3-8/group. (N) Histological…

Figure 3. Cardiomyocyte-specific deletion of LRP6 impairs mitochondrial function. (A) Representative electron microscopy images of hearts from MCM/LRP6fl/flor MCM at day 2 after 3-day consecutive injection with tamoxifen. Scale bars: 5 μm, n=3/group. (B) Relative cardiac ATP production in MCM or MCM/LRP6fl/flat day 2 after 3-day consecutive injection with tamoxifen or diluent. *p<0.05 vs. diluent-injected-MCM/LRP6fl/fl. n=3-6/group. (C) Mitochondrial membrane potential assessment with JC-1 staining. Mitochondria were freshly isolated from heart tissue of mice as in B. *p<0.05; vs. diluent-injected-MCM/LRP6fl/fl. n=4-8/group. (D) Mitochondrial swelling induced by Ca2+ (50 μM). Left panel: each data curve represents the average of 3 individual measurements. Right panel: the decrease in optical density at 520 nm at 10 min induced by Ca2+. *p<0.05 vs. MCM group. n=3/group. (E-G) Mitochondrial respiratory chain complex I, II/III, and IV activity, respectively. The activity in mitochondria from diluent-injected MCM hearts or MCM/LRP6fl/fl ones is expressed as 1. *p<0.05, **p<0.01 vs. diluent-injected mice. n=3/group. (H-J) GC-FID/MS analysis of fatty acid composition in heart tissue from…

Figure 4. Cardiomyocyte-specific deletion of LRP6 inhibits autophagic degradation. (A) Western blot analysis of LC3-II, PPARα, PPARδ, PGC1α and LC3b (LC3-I: the upper band; LC3-II: the down band) in heart tissue from diluent or tamoxifen-injected MCM or MCM/LRP6fl/fl mice (B) Western blot analysis of LRP6, p-Drp1, Drp1, p-mTOR, mTOR, p-AMPK, AMPK, PINK1 in heart tissue from the same mice as in (A). (C) Western blot analysis of p-Drp1, Drp1, PINK1, VDAC1, Found1, Parkin, Tom22, expression in cardiac mitochondria in tamoxifen-injected MCM or MCM/LRP6fl/fl mice. *p<0.05, **p<0.01vs. diluent-injected MCM/LRP6fl/fl (n=3-4/group). Analysis of hearts or cardiac mitochondria from MCM/LRP6fl/flor MCM at day 1 after 3-day consecutive injection with tamoxifen or diluent.

Figure 5. Cardiomyocytes-specific deletion of LRP6 inhibits TFEB nuclear translocation. (A) Western blot analysis of TFEB and β-catenin expression in nucleus, cytosol and tissue lysates of hearts from diluent-injected or tamoxifen-injected MCM/LRP6fl/fl. *p<0.05 vs. diluent-injected MCM/LRP6fl/fl. n=4/group. (B) Western blot analysis of nuclear TFEB expression in hearts from tamoxifen-injected MCM/LRP6fl/fl with pre-injection of DMSO or Midvi-1. *p<0.05 vs. DMSO group; n=6/group. Analysis of hearts from MCM/LRP6fl/flat day 1 after 3-day consecutive injection with tamoxifen or diluent.

Figure 6. Drp1 inhibitor attenuates the autophagic degradation and mitochondrial dysfunction induced by LRP6 deletion in cardiomyocytes. (A) Electron microscopy images (scale bars, 1 μm), of hearts from tamoxifen-injected MCM/LRP6fl/fl treated with mdivi-1 or the same volume of DMSO. n=3/group. (B) Western blot analysis of LRP6, p-Drp1, Drp1, p-mTOR, mTOR, and P62 in heart tissue from diluent or tamoxifen-injected MCM/LRP6fl/flmice treated with mdivi-1 or the same volume of DMSO. (C) Western blot analysis of PPARα, PPARδ and LC3b in heart tissue from diluent or tamoxifen-injected MCM/LRP6fl/flmice treated with mdivi-1 or the same volume of DMSO. n=3-6/group. (D-E) Cardiac ATP production and mitochondrial membrane potential were analyzed in mice as in (B). n=4/group. *p<0.05; **p<0.01; ***p<0.001 vs. DMSO group; #p<0.05, ##p<0.01, ###p<0.001 vs. tamoxifen+DMSO group.

Figure 7. Drp1 inhibitor improves cardiac dysfunction induced by LRP6 deletion in cardiomyocyte. (A) Survival proportion was analyzed in MCM/LRP6fl/fl mice treated with mdivi-1 or DMSO within 7 days after tamoxifen injection. n=12/group. (B) Representative M-Mode echocardiographs from diluent or tamoxifen-injected MCM/LRP6fl/fl mice treated with DMSO or mdivi-1. (C-D) Echocardiographic analysis of LVID.d and FS in mice from (A). n=3-6/group. (E-H) Hemodynamic analysis of dp/dtmax, -dp/dtmax, LVSP and LVEDP in mice from (A). n=6-8/group. (I) HE staining of longitudinal heart sections from mice from (A). n=3-6/group. (J) HW/BW was analyzed in mice from (A). n=3-6/group *p<0.05; **p<0.01 vs. DMSO group. #p<0.05, ##p<0.01 vs. tamoxifen+DMSO group.

Figure 8. A proposed model for cardiac-specific deletion of LRP6 induced cardiac dysfunction. Tamoxifen-inducible cardiac-specific LRP6 knockout promotes Drp1 phosphorylation at ser616 and its translocation to mitochondria, which leads to mitochondrial fragmentation and autophagosome formation. Meanwhile, LRP6 deletion-induced phosphorylation of Drp1 also enhances p-mTOR level, which inhibits TFEB nuclear translocation by decreasing TFEB phosphorylation. TFEB-induced autophagy genes as well as fatty acids metabolism genes PPARs are suppressed, resulting in the inhibition of mitochondrial degradation and fatty acids oxidation, which contribute to cardiomyocyte death and cardiac dysfunction.