Cardioprotective GLP-1 metabolite prevents ischemic cardiac injury by inhibiting mitochondrial trifunctional protein-α
M. Ahsan Siraj, … , Peter Backx, Mansoor Husain
M. Ahsan Siraj, … , Peter Backx, Mansoor Husain
Published January 27, 2020
Citation Information: J Clin Invest. 2020;130(3):1392-1404. https://doi.org/10.1172/JCI99934.
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Research Article Cardiology Metabolism

Cardioprotective GLP-1 metabolite prevents ischemic cardiac injury by inhibiting mitochondrial trifunctional protein-α

Abstract

Mechanisms mediating the cardioprotective actions of glucagon-like peptide 1 (GLP-1) were unknown. Here, we show in both ex vivo and in vivo models of ischemic injury that treatment with GLP-1(28–36), a neutral endopeptidase–generated (NEP-generated) metabolite of GLP-1, was as cardioprotective as GLP-1 and was abolished by scrambling its amino acid sequence. GLP-1(28–36) enters human coronary artery endothelial cells (caECs) through macropinocytosis and acts directly on mouse and human coronary artery smooth muscle cells (caSMCs) and caECs, resulting in soluble adenylyl cyclase Adcy10–dependent (sAC-dependent) increases in cAMP, activation of protein kinase A, and cytoprotection from oxidative injury. GLP-1(28–36) modulates sAC by increasing intracellular ATP levels, with accompanying cAMP accumulation lost in sAC–/– cells. We identify mitochondrial trifunctional protein-α (MTPα) as a binding partner of GLP-1(28–36) and demonstrate that the ability of GLP-1(28–36) to shift substrate utilization from oxygen-consuming fatty acid metabolism toward oxygen-sparing glycolysis and glucose oxidation and to increase cAMP levels is dependent on MTPα. NEP inhibition with sacubitril blunted the ability of GLP-1 to increase cAMP levels in coronary vascular cells in vitro. GLP-1(28–36) is a small peptide that targets novel molecular (MTPα and sAC) and cellular (caSMC and caEC) mechanisms in myocardial ischemic injury.

Authors

M. Ahsan Siraj, Dhanwantee Mundil, Sanja Beca, Abdul Momen, Eric A. Shikatani, Talat Afroze, Xuetao Sun, Ying Liu, Siavash Ghaffari, Warren Lee, Michael B. Wheeler, Gordon Keller, Peter Backx, Mansoor Husain

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Figure 2

The cardioprotective effects of GLP-1(28–36) are sAC dependent.

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The cardioprotective effects of GLP-1(28–36) are sAC dependent. (A) Repr...
(A) Representative Western blot of 50-kDa sAC isoform expression in WT mouse lysates of whole heart (Hrt), LV, right ventricle (RV), atria (Atr), septum (Sep), and brain (Br, negative control). GAPDH was used as a loading control (n = 3).(B) cAMP levels in coronary effluents were measured at timed intervals during an initial 10-minute perfusion of WT hearts with either 6 nM GLP-1(28–36), scrambled(28–36) control (Scram), or buffer-only control (Cntl), or with 0.3 nM GLP-1, and were normalized to coronary flow (mL/min/g heart weight). (C) Schematic of IRI protocol and (D) percentage of LVDP recovery of isolated WT mouse hearts with the sAC inhibitor KH7 (20 μmol/L) or the tmAC inhibitor Ddox (50 μmol/L) prior to perfusion with buffer only (Cntl), scrambled(28–36), GLP-1(28–36), or GLP-1 (n = 3/group). (E) Quantification of infarct size as a percentage of the AAR on day 2 after in vivo IRI in WT (sAC+/+) mice (n = 8/group, gray bars) and sAC–/– littermates (n = 8–9/group, white bars). (F) Intracellular cAMP accumulation after treatment with GLP-1(28–36) in caSMCs isolated from sAC–/– mice (white bars) as compared with sAC+/+ littermates (gray bars). (G) Pretreatment with 100 nM GLP-1(28–36) prior to oxidative stress injury with H2O2 as measured by LDH release in sAC–/– caSMCs (white bars) versus sAC+/+ control caSMCs (gray bars). (H) Intracellular cAMP accumulation in human caSMCs treated with the sAC inhibitor KH7 (25 μM, dark gray bars), the tmAC inhibitor Ddox (50 μM, white bars), or PBS (light gray bars) for 3 hours followed by a 15-minute treatment with GLP-1(28–36), scrambled control, IPE, or forskolin. Intracellular cAMP accumulation was normalized to the total cellular protein concentration. (I) cAMP levels in cell lysates were determined following pretreatment with 100 nM each of GLP-1(28–36), scrambled control, or forskolin in siRNA-treated cells (white bars) versus the scrambled siRNA control (gray bars) (n = 3/group for all cAMP and LDH assays, each in triplicate). Data represent the mean ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001 versus the corresponding control by 2-way ANOVA with Bonferroni’s post hoc test. Forsk, forskolin.