Parallel TXNIP‐ and TLR2‐ Dependent Pathways Synergistically Mediate Saturated Fatty Acid‐Induced Apoptosis in Cardiomyocytes

Abstract

BACKGROUNDSaturated fatty acids (SFA) induce apoptosis in many cell types in vitro, and high SFA diet increases apoptotic cardiomyocytes, but mechanism remains unclear. We had previously demonstrated that p38α mitogen activated protein kinase (MAPK)–dependent signaling mediates cardiomyocyte apoptosis induced by palmitic acid (PA), a SFA. More recently, direct binding of thioredoxin interacting protein (TXNIP) and p38 MAPK has been demonstrated. SFA has been shown to bind to and activate Toll‐like receptor‐2 (TLR2), as well as the TLR4 accessory protein, MD2, triggering inflammation, oxidative stress, and cell death. We hypothesized that parallel TXNIP‐, and TLR‐ dependent pathways, both triggered by SFA, synergistically mediate apoptosis in cardiomyocytes.METHODS AND RESULTSA human cardiomyocyte line, AC16, was used in the study. TXNIP was inhibited by pre‐treating (24hours) cells with SBI‐477, a chemical inhibitor of MondoA/ChREBP. TXNIP knockdown was achieved with siRNA. TXNIP was overexpressed by transfecting with TXNIP plasmid, then 48 h later treated with TLR2 ligand, peptidoglycan (PGN) or TLR4 ligand, lipopolysaccharide (LPS), for an additional 2–3 days. TXNIP expression was determined by Western blotting of whole cell lysates. Apoptosis was measured by flow cytometry using Annexin V/PI staining.PA increased TXNIP protein levels dose‐dependently (1.5‐ and 2.3‐fold increase at 150 uM and 300 uM PA respectively) (ANOVA p<0.01, n=3). PA did not affect MondoA and ChREBP protein expression but increased nuclear translocation 2‐fold. TXNIP inhibition reduced PA‐induced apoptosis by 79% (p<0.05; n=3). TXNIP knockdown reduced PA‐induced cardiomyocyte apoptosis by 61% (p<0.01; n=7).TXNIP overexpression for 24 h increased apoptosis 1.8‐fold compared to control plasmid (PCDNA), but this was attenuated by siRNA knockdown of p38α or c‐fos, 59% and 84% respectively. TXNIP overexpression for 4–5 days markedly increased apoptosis rate to 4.0‐fold. The combined stressors of transfection with control plasmid and TLR ligand increased cell death rate 3.2 and 4.6‐fold for PGN and LPS respectively, compared to PCDNA + Vehicle. TXNIP overexpression followed by PGN increased apoptosis rate 8‐fold, whereas LPS reduced it to 2.7‐fold.CONCLUSIONSThis study in a human cardiomyocyte line demonstrated that (1) PA increased TXNIP levels, (2) TXNIP inhibition or knockdown attenuated PA‐induced apoptosis, (3) TXNIP overexpression increased apoptosis, (4) TXNIP overexpression combined with a TLR2 ligand, PGN, synergistically amplified the apoptosis rate, whereas LPS, a TLR4 ligand did not. These findings suggest that TXNIP‐dependent pathway synergistically combine with TLR2‐dependent signaling, but not TLR4‐, to amplify apoptosis, and suggest a mechanism by which elevated plasma SFA levels may lead to development of lipotoxic cardiomyopathy.Support or Funding InformationThis work has been funded by VA VISN 18 New Investigator Grant (to C.O.), CSR&D 5I01CX000598 grant (to P.R.), and VA Merit Grant BLRD I01BX007080 (to R.M.). The study was supported by VA Employment and Carl T. Hayden Medical Research Foundation. K.T. is supported by the M. Lowell Edwards Endowment.