p53-Mediated Repression of the PGC1A (PPARG Coactivator 1α) and APLNR (Apelin Receptor) Signaling Pathways Limits Fatty Acid Oxidation Energetics: Implications for Cardio-oncology

Bruno Saleme, Subhash K Das, Yongneng Zhang, Aristeidis E Boukouris, Maria Areli Lorenzana Carrillo, Juan Jovel, Cory S Wagg, Gary D Lopaschuk, Evangelos D Michelakis, Gopinath Sutendra, Bruno Saleme, Subhash K Das, Yongneng Zhang, Aristeidis E Boukouris, Maria Areli Lorenzana Carrillo, Juan Jovel, Cory S Wagg, Gary D Lopaschuk, Evangelos D Michelakis, Gopinath Sutendra

No abstract available

Keywords: cardiac metabolism; cardiotoxicity; doxorubicin; p53.

Conflict of interest statement

None.

Figures

Figure 1. p53‐Mediated repression of the PGC1A (PPARG coactivator 1α) and APLNR (apelin receptor) signaling pathways limit fatty acid oxidation (FAO) energetics, and this can be therapeutically prevented in doxorubicin (DXR)–induced cardiotoxicity by the tetrameric PKM2 –stabilizing drug TEPP‐46.

Statistical analysis was performed on SPSS v24.0 (IBM Corp; Somers). Kruskal–Wallis test was used to compare ≥2 independent samples, and the Mann–Whitney U test was used for comparisons between 2 groups, unless otherwise specified. Significance was considered at P<0.05. A, Experimental design for unbiased RNA sequencing platform. Athymic male nude mice were xenotransplanted with human lung tumors, as described previously. 2 Mice were then allocated to receive either vehicle or a cumulative dose of 20 mg/kg of DXR (weekly, intraperitoneally) and/or concurrent daily gavage of TEPP‐46 at 100 mg/kg for 5 weeks. Once cardiac dysfunction was confirmed (shown in Saleme et al 2 ), cardiomyocytes were isolated, RNA was extracted, and RNA sequencing was performed. B, Heat map showing the RNA‐sequencing results from isolated cardiomyocytes from control‐ and DXR‐treated mice. Libraries were prepared using TrueSeq V2 and sequenced on HiSeq 2500 and aligned to GRCm38 using Kallisto. Differential expression analysis was conducted with the DESeq2 R package, and transcript abundance differences with corrected P<0.05 were considered differentially expressed (n=3 for control and n=4 for DXR‐treated mice). Gene variability shown as a percentage of standard deviation over fold change for each gene is shown (right). Gene names for the 4 different pathways are shown to the right of each representative row. C, Experimental design for TP53 (tumor protein p53) overexpression using adenovirus infection of isolated adult mouse cardiomyocytes. The TP53 adenovirus coexpresses GFP. Adenovirus for GFP was used as a control. D, Quantitative reverse transcriptase polymerase chain reaction (qRT‐PCR) for the TP53 overexpression experiment shows the mRNA levels for Pgc1A, Aplnr, and Apln (apelin). In addition, 18S was used as a loading control. Mean data are shown, and each individual experiment (n=9) is shown as an individual yellow circle. *P<0.05 compared with adenovirus (Ad) GFP (green fluorescent protein). E, qRT‐PCR for the PPARGC1A (also known as PGC1A) overexpression experiment shows the mRNA levels for Pgc1A, CD36, Aplnr, and Apln. In addition, 18S was used as a loading control. Mean data are shown, and each individual experiment for Aplnr and Apln (n=9) and for Pgc1A and CD36 (n=6) is shown as an individual yellow circle. *P<0.05 compared with adenovirus (Ad) GFP. F, Disuccinimidyl suberate cross‐linking of isolated adult mouse cardiomyocytes treated with TEPP‐46 shows the levels of tetrameric PKM2 (T.PKM2), using immunoblots. Actin (ACTB) was used as a loading control. G, Schematic showing that p53 activation decreases Aplnr mRNA levels, which subsequently suppresses FAO rates, and mitochondrial respiration. H, Cardiac differentiation of induced pluripotent stem cells (iPSCs) is shown by the loss of stem cell markers (NANOG, OCT4) and the acquisition of cardiac markers (cardiac troponin T [cTNT]). Furthermore, similar to adult cardiomyocytes, which have a high PKM1/PKM2 ratio, 2 PKM1 levels continue to increase, whereas PKM2 levels correspondingly decrease during the differentiation time frame from 1 to 40 days, using immunoblots. ACTB was used as a loading control. I, Immunofluorescence staining of a human iPSC‐derived cardiomyocyte (day 40) showing cTNT (cytosolic) in red and PKM2 (cytosolic and nuclear; shown by white arrow) in green (left). The mRNA levels are shown for APLNR on day 40 of human iPSC‐derived cardiomyocytes treated with DXR (150 nmol/L) and TEPP‐46 (50 µmol/L) for 24 hours, using qRT‐PCR. In addition,18S was used as a loading control. Mean data and individual experiments (yellow circles) are shown. *P<0.05 compared with control, #P<0.05 compared with DXR (right). J through L, qRT‐PCR or immunoblots show the mRNA expression (J) or protein levels (K and L), respectively, of PGC1A, APLN, and APLNR from the myocardial tissue of mice treated with TEPP‐46 (100 mg/kg, daily gavage), DXR (20 mg/kg cumulative dose), or DXR+TEPP‐46, as described previously. 2 β2M and ACTB were used as housekeeping genes for qRT‐PCR and immunoblots, respectively. Mean data and individual animal data (yellow circles) are shown. *P<0.05 compared with control, #P<0.05 compared with DXR. Immunoblots of analyzed data in (K) are shown in (L). M, The working heart model allows for the measurement of glucose and palmitate oxidation rates. Isolated hearts are perfused with radiolabeled glucose or palmitate, and radiolabeled H2O or CO2 provides a relative rate for palmitate oxidation or glucose oxidation, respectively. N and O, DXR treatment in vivo decreases palmitate oxidation (N), but not glucose oxidation (O), compared with control‐treated animals, and this is prevented by cotreatment with TEPP‐46, using the isolated working heart system (n=5 animals per group for all groups except TEPP‐46 [n=3 animals]). *P<0.05 compared with control‐treated animals, #P<0.05 compared with DXR‐treated animals). P, TEPP‐46 prevents a DXR‐induced decrease in oxygen consumption rates in isolated mouse cardiomyocytes, as assessed by Seahorse (n=3 different experiments; *P<0.05 compared with baseline respiration, #P<0.05 compared with DXR; values are expressed as mean±SEM). AM indicates antimycin A; FCCP, carbonyl cyanide‐4‐(trifluoromethoxy)phenylhydrazone; OM, oligomycin; and RT, rotenone.