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Novel GSK-3β Inhibitor Neopetroside A Protects Against Murine Myocardial Ischemia/Reperfusion Injury


. 2022 Sep 28;7(11):1102-1116.


doi: 10.1016/j.jacbts.2022.05.004.


eCollection 2022 Nov.

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Hyoung Kyu Kim et al.


JACC Basic Transl Sci.


.

Abstract

Recent trends suggest novel natural compounds as promising treatments for cardiovascular disease. The authors examined how neopetroside A, a natural pyridine nucleoside containing an α-glycoside bond, regulates mitochondrial metabolism and heart function and investigated its cardioprotective role against ischemia/reperfusion injury. Neopetroside A treatment maintained cardiac hemodynamic status and mitochondrial respiration capacity and significantly prevented cardiac fibrosis in murine models. These effects can be attributed to preserved cellular and mitochondrial function caused by the inhibition of glycogen synthase kinase-3 beta, which regulates the ratio of nicotinamide adenine dinucleotide to nicotinamide adenine dinucleotide, reduced, through activation of the nuclear factor erythroid 2-related factor 2/NAD(P)H quinone oxidoreductase 1 axis in a phosphorylation-independent manner.


Keywords:

ATP, adenosine triphosphate; GSK-3, glycogen synthase kinase–3; GSK-3β inhibition; I/R, ischemia/reperfusion; MI, myocardial infarction; NAD+, nicotinamide adenine dinucleotide; NADH, nicotinamide adenine dinucleotide, reduced; NPS A; NPS A, neopetroside A; Nqo1, NAD(P)H:quinone oxidoreductase 1; Nrf2, nuclear factor erythroid 2–related factor 2; OCR, oxygen consumption rate; ischemia/reperfusion injury; mPTP, mitochondrial permeability transition pore; mTOR, mammalian target of rapamycin; marine pyridine α-nucleoside; mitochondria.

Conflict of interest statement

This work was supported by the Basic Science Research Program (NRF-2020R1A4A1018943 and NRF-2018R1A2A3074998) through the National Research Foundation of Korea, funded by the Ministry of Education and the Ministry of Science and ICT. The synthesis of NPS A was supported by the Russian Science Foundation (grant 19-73-30017). The authors have reported that they have no relationships relevant to the contents of this paper to disclose.

Figures



Graphical abstract


Figure 1


Figure 1

NPS A Is Not Toxic in Vitro and in Vivo (A) Chemical structure of neopetroside A (NPS A). (B) Stability of NPS A in vivo following intravenous administration, measured at the indicated time points. Data represent the average of 3 independent experiments. (C) Effects of increasing concentrations (0-300 μmol/L) of NPS A on H9c2 cell viability. Four independent in vitro experiments were performed. (D, E) Single-cell contractility assay in the presence and absence of 3 μmol/L NPS A. Data represent the average of 3 independent experiments. Data were analyzed using 1-way analysis of variance followed by the Bonferroni multiple-comparisons test (C) and Student’s t-test (D).


Figure 2


Figure 2

NPS A Elevates Glycolysis, OCR, and Metabolic Processes in Rat Cardiomyoblast H9c2 Cells (A) Left: Extracellular acidification rate (ECAR) output of H9c2 cells and response to 10 mmol/L glucose (G), 1 μmol/L oligomycin (O), and 10 mmol/L 2-deoxyglucose (2DG). Right: Effects of increasing concentrations of neopetroside A (NPS A) on glycolysis, glycolytic capacity, and glycolytic reserve on the basis of data derived from the left panel. Four independent in vitro experiments were performed. #P < 0.05 vs control cells. (B) Left: Representative graph of oxygen consumption rate (OCR) of H9c2 cells in response to 1 μmol/L O, 1 μmol/L carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP), and 1 μmol/L rotenone with 1 μmol/L antimycin (R/A). Right: Effects of increasing concentrations of NPS A on mitochondrial function parameters (adenosine triphosphate [ATP]–linked respiration, proton leakage, mitochondrial oxygen maximal capacity, and mitochondrial OCR [mtOCR]) on the basis of data derived from the left panel. Four independent in vitro experiments were performed. #P < 0.05 vs control cells. (C) ATP levels after treatment with NPS A for the indicated times. Data represent the average of 4 independent experiments. (D) OCR (y-axis) against ECAR (x-axis) according to concentration of NPS A. Four independent in vitro experiments were performed. #P < 0.05 vs control cells. (E) OCR measurement in isolated cardiac mitochondria. Three independent in vitro experiments were performed. Data were analyzed using 1-way analysis of variance followed by the Bonferroni multiple-comparisons test (A to D) and Student’s t-test (E). ADP = adenosine diphosphate; ATPase = adenosine triphosphatase; G/M = glutamate-maleate; S = succinate.


Figure 3


Figure 3

NPS A Preserves Decreased LVDP After Global I/R Injury (A, B) Representative recordings of left ventricular (LV) pressure in perfused hearts without (A) or with (B) 3 μmol/L neopetroside A (NPS A) treatment following ischemia. (C, D) LV developed pressure (LVDP) and maximum dP/dt were determined before ischemia and during reperfusion in the presence and absence of NPS A. n = 5 per group. ∗∗∗∗P < 0.0001 vs preischemia in the control, and ###P < 0.001 vs reperfusion in the control. (E, F) Representative photographs and quantitative analysis of rat hearts comparing infarct sizes in animals with and without NPS A treatment after ischemia/reperfusion (I/R) injury. n = 6 per group. Evaluated points for I/R: n = 12 in each group. ∗P < 0.05 vs non–NPS A–treated control. (G) Representative images and quantitative analysis of nontreated and NPS A–treated isolated cardiomyocytes stained with CM-H2-DCFDA during basal conditions or after I/R injury. Control n = 3, I/R n = 4 per group. ∗P < 0.05 vs preischemia in the control, and #P < 0.05 vs reperfusion in the control. In A, B, and G, a and b are zoomed images of the recordings from the representative Figures. Data were analyzed using 2-way analysis of variance followed by the Bonferroni multiple-comparisons test (C, D, Gb) and Student’s t-test (F).


Figure 4


Figure 4

NPS A Protects Against MI (A) In vivo protocol of the 4-week myocardial infarction (MI) model in 8-week-old C57BL/6 mice. Arrows indicate intraperitoneal (i.p.) injection. (B) Survival rates of model animals. Start of the analysis, n = 15 mice per group. ∗∗P < 0.01 vs sham group. (C) Representative heart morphology after MI surgery. Arrows indicate coronary occlusion. (D) Representative photograph of Masson’s trichrome–stained mouse heart (top 2 rows, longitudinal section; bottom 2 rows, cross section; longitudinal section: black scale bar = 5 mm, white scale bar = 40 μm; cross section, black scale bar = 1 mm, white scale bar = 100 μm). (E) Heart weight (HW)/body weight (BW) and (F) HW/tibia length ratios after sham, MI, and MI plus neopetroside A (NPS A) treatment. n = 5 mice in each group. ∗∗P < 0.01, ∗∗∗P < 0.001, and ∗∗∗∗P < 0.0001 vs sham group. (G) Infarct analysis of infarct area on the basis of Figure 3D. n = 4 mice in each group. ∗∗P < 0.01 and ∗∗∗∗P < 0.0001 vs sham group. Data were analyzed using 1-way analysis of variance followed by the Bonferroni multiple-comparisons test (E to G).


Figure 5


Figure 5

NPS A Inhibits GSK-3β Kinase Activity in Vitro and Interacts With GSK-3β in Molecular Docking Simulations (A) An in vitro kinase activity screen was performed to determine which kinases were modulated by neopetroside A (NPS A). Three independent in vitro experiments were performed. #P < 0.05 vs control. (B, C) Inhibitory activities of increasing concentrations of NPS A on glycogen synthase kinase–3β (GSK-3β) and mammalian target of rapamycin (mTOR), with half maximal inhibitory concentration (IC50) values. Three independent in vitro experiments were performed. #P < 0.05 vs control. (D) Overlay of the docked NPS A conformation (red) and the crystallographic conformation of I-5 (yellow). Corresponding interactions with GSK-3β are shown in red and yellow dashes, respectively. (E) Surface plasmon resonance (SPR) binding assay for NPS A and GSK-3β. Data were analyzed using 1-way analysis of variance followed by the Bonferroni multiple-comparisons test (A).


Figure 6


Figure 6

NPS A Increases the NAD+/NADH Ratio Through the Nrf2/Nqo1 Pathway In H9c2 Cells (A, B) In vivo and in vitro representative western blots of neopetroside A (NPS A) and SB216763-treated C57BL/6 mice and H9c2 cells, respectively, and their respective quantitative analysis. In vivo (A): n = 5, all groups. In vitro (B): 5 independent in vitro experiments were performed. ∗P < 0.05 vs control and ∗∗P < 0.01 vs control. (C) Nicotinamide adenine dinucleotide (NAD+)/nicotinamide adenine dinucleotide, reduced (NADH) levels as determined with control, nuclear factor erythroid 2–related factor 2 (Nrf2), or NAD(P)H quinone oxidoreductase 1 (Nqo1) small interfering RNA (siRNA) treatment in the presence or absence of 10 μmol/L NPS A or SB216763. Four independent in vitro experiments were performed. ∗P < 0.05 vs control, †P < 0.05 vs siCon + NPS A, and ###P < 0.001 vs siCon + SB216763. (D) Measurement of NAD+/NADH ratio after treatment with SB216763 and/or NPS A. Six independent in vitro experiments were performed. ∗P < 0.05 vs control, ∗∗P < 0.01 vs control, and †P < 0.05 vs NPS A. (E) Basal oxygen consumption rate (OCR) in H9c2 cells following treatment with control, Nrf2, or Nqo1 siRNA in the presence or absence of NPS A. Four independent in vitro experiments were performed. ∗P < 0.05 vs respective controls. (F) Cell viability after simulated ischemia/reperfusion (sI/R) injury in cells treated with control, Nrf2, or Nqo1 siRNA in the presence or absence of NPS A. Five independent in vitro experiments were performed. ∗∗∗∗P < 0.0001 vs siCon and ####P < 0.0001 vs sI/R with NPS A treatment. (G) Proposed mechanism of the protective role of NPS A in I/R injury. Created with BioRender.com. Data were analyzed using 1-way analysis of variance (ANOVA) followed by the Bonferroni multiple-comparisons test (A, B, D), 2-way ANOVA followed by the Bonferroni multiple-comparisons test (C, F), and Student’s t-test (E).

References

    1. Handy D.E., Loscalzo J. Redox regulation of mitochondrial function. Antioxid Redox Signal. 2012;16:1323–1367.



      PMC



      PubMed

    1. Brown D.A., Perry J.B., Allen M.E., et al. Expert consensus document: mitochondrial function as a therapeutic target in heart failure. Nat Rev Cardiol. 2017;14:238–250.



      PMC



      PubMed

    1. Oku N., Matsunaga S., van Soest R.W., Fusetani N. Renieramycin J, a highly cytotoxic tetrahydroisoquinoline alkaloid, from a marine sponge Neopetrosia sp. J Nat Prod. 2003;66:1136–1139.



      PubMed

    1. Shubina L.K., Makarieva T.N., Yashunsky D.V., et al. Pyridine nucleosides neopetrosides A and B from a marine Neopetrosia sp. sponge. Synthesis of neopetroside A and its beta-riboside analogue. J Nat Prod. 2015;78:1383–1389.



      PubMed

    1. Ussher J.R., Jaswal J.S., Lopaschuk G.D. Pyridine nucleotide regulation of cardiac intermediary metabolism. Circ Res. 2012;111:628–641.



      PubMed



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