Low cardiac lipolysis reduces mitochondrial fission and prevents lipotoxic heart dysfunction in Perilipin 5 mutant mice

Figure 1. Overexpression of Plin5-S155A reduces lipolysis in H9c2 cardiac cells. (A) Confocal life cell imaging in cells overexpressing GFP-tagged Plin5 or Plin5-S155A upon incubation with 400 μM oleic acid (OA) and lipid staining using LipidTox™. Bar: 10 μm. (B) Incorporation of radioactivity into TAG upon incubation with OA and 3H-labelled OA for 20 h (n = 3). (C) Measurement of (FA) release into the medium after incubation with cold and 3H-labelled OA. (D) FA release upon incubation with 20 μM forskolin and 0.5 mM IBMX. (E) Released CO2 and production of acid soluble metabolites (ASM) after incubation with 14C-palmitic acid for 90 min (n = 4). (F) Oxygen consumption rate (OCR) during sequential addition of oligomycin, FCCP, and antimycin A, as indicated. Data are shown as means ± SEM. Statistical significance determined by unpaired Student’s t-test (*P < 0.05; **P > 0.01; ***P < 0.001).

Figure 2. Cardiomyocyte-specific overexpression of Plin5-S155A provokes cardiac steatosis and increases ceramide and DAG levels. (A) Plin5 protein levels in cardiac homogenates of Wt and Plin5-S155A mice and in LD fractions of Plin5-S155A mice. (B) Heart to body weight ratio (n = 6–7) and heart images. (C) Oil red O staining (ORO) of cardiac tissue sections (Scale bar: 200 μm) and cardiac tissue TAG and TC levels (n = 4–5). Targeted lipidomic analyses of ceramide (D) and DAG species (E) in CM of Wt and Plin5-S155A mice (n = 5). (F) Representative histological images of heart sections stained with Haematoxylin and eosin (H&E) and Masson’s trichrome (M.T.) (Scale bar: H&E 200 μm; M.T. 100 μm). (G) Cardiac mRNA expression levels of genes associated with cardiac fibrosis and hypertrophy (Col1a1, Col3a1, Bnp, Tgfb) and inflammation (Cd11c, F4/80, IL-6) determined by RT-qPCR with 36b4 as reference gene (n = 5–6). Data are presented as means ± SEM. Statistical significance was tested using unpaired Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001).

Figure 3. Plin5-S155A divergently affects cardiac lipolysis compared to Plin5 and displays changes in the Plin5 phosphorylation pattern. (A) Cardiac mRNA expression levels of Atgl, Cgi-58, G0s2, and Hsl determined by RT-qPCR with 36b4 as reference gene. Data are presented as means ± SEM (n = 5–6). Statistical significance determined by unpaired Student’s t-test (**P < 0.01; ***P < 0.001). (B) Protein levels of ATGL, CGI-58, and HSL in cardiac homogenates. (C) TAG hydrolase activities in cardiac lysates from Wt and Plin5-S155A mice (n = 5 per genotype) applying a micellar TAG substrate. Recombinant CGI-58 or Atglistatin (Ai) was added for stimulation or inhibition of ATGL activity (*P < 0.05; ***P < 0.001 vs. Wt; #P < 0.05; ###P < 0.001 vs. DMSO conditions). (D) Cardiac LDs incubated with COS-7 cell lysates enriched with LacZ (basal), ATGL, ATGLþCGI-58, or HSL in the absence or presence of recombinant PKA (-PKA/þPKA) (n = 5). Data are shown as means ± SEM. Statistical significance was tested using unpaired Student’s t-test (**P < 0.01; ***P < 0.001 vs. basal levels; #P < 0.05; ##P < 0.01; ###P < 0.001 vs. LDs without PKA). (E) Cardiac Plin5-phosphosites determined by Q-TOF MS. Values were…

Figure 4. Cardiac Plin5-S155A overexpression decreases FAO and affects amino acid homeostasis without changes in overall energy production. (A) Cardiac mRNA levels of Ppara and target genes of fasted mice determined by RT-qPCR using 36b4 as reference gene (n = 4 –6). (B) Mitochondrial CPT1 protein levels in CM of Wt and Plin5-S155A mice using CoxIV as loading control (n = 3). (C) Measurement of 14CO2 release and the production of ASM in mitochondria preparations from CM of Wt and Plin5-S155A mice (n = 5). (D) Cardiac metabolites in fasted Wt and Plin5-S155A mice quantified by NMR spectroscopy (n = 5). (E) pAMPK and total AMPK protein levels in cardiac homogenates from fasted mice. (F) Oxygen consumption rates (OCR) in cardiac homogenates from fasted mice measured with Oroboros under basal conditions (Routine), after addition of pyruvate and ADP (OXPHOS CI), and after addition of oligomycin (LEAK). Data are presented as means ± SEM. Statistical significance was determined by unpaired Student’s t-test. (*P <0.05; **P <0.01; ***P <0.001).

Figure 5. Cardiac Plin5-S155A overexpression reduces mitochondrial fission. (A) Mitochondria size range analysed from electron microscopy images of heart sections (Analysis of 41 and 48 images from Wt and Plin5-S155A mice, respectively). (B) Representative transmission electron microscopy images of CM slides from Wt and Plin5-S155A mice. (C) Cardiac mRNA levels of genes involved in mitochondrial fusion (Mfn1, Mfn2, Opa1) and fission (Drp1, Fis1) analysed by RT-qPCR using 36b4 as reference gene (n = 6). (D) Mfn1 and Fis1 corresponding peptide levels in CM from fasted mice quantified by MS proteomics analysis (n = 5–6). (E) Cardiac phospho-Mff (S146) levels quantified by phosphoproteome analyses applying Q-TOF MS (n = 5–6) and normalized to total Mff levels determined by western blot analyses. Protein levels of Drp1 and Mfn2 in total homogenates and mitochondrial fractions of Wt, Plin5-S155A (F), ATGL-ko (G), and CM-ATGL mice (H) using GAPDH (homogenate) and CoxIV (mitochondria) as loading controls. Data are presented as means ± SEM. Statistical significance was determined by unpaired Student’s t-test. For (phospho)proteomics, ANOVA with subsequent multiple testing correction by…

Figure 6. Increased cardiac expression of ROS defense genes in Plin5-S155A mice. (A) Cardiac MDA levels were quantified in fasted mice using a colorimetric kit (Wt: n = 8, Plin5-S155A: n = 5). (B) Non-esterified thiol groups determined by the Ellman’s reaction in CM of fasted mice (n = 4). (C) CM glutathione levels determined by MS analysis in fasted mice (n = 5). (D) Cardiac mRNA expression of Gsta1/2, Gclc, Gss, and Gpx4 using 36b4 as reference gene (n = 6). (E) Catalase expression in CM of Wt and Plin5-S155A mice determined by western blot and RT-qPCR (n = 6). Data are presented as means ± SEM. Statistical significance was determined by unpaired Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001). (F) Scheme depicting the impact of Plin5-S155A or Plin5 overexpression on lipolysis, mitochondrial fission, and ROS defense.