Gut-Derived Metabolite Indole-3-Propionic Acid Modulates Mitochondrial Function in Cardiomyocytes and Alters Cardiac Function - PubMed (original) (raw)
Gut-Derived Metabolite Indole-3-Propionic Acid Modulates Mitochondrial Function in Cardiomyocytes and Alters Cardiac Function
Maren Gesper et al. Front Med (Lausanne). 2021.
Abstract
Background: The gut microbiome has been linked to the onset of cardiometabolic diseases, in part facilitated through gut microbiota-dependent metabolites such as trimethylamine-_N_-oxide. However, molecular pathways associated to heart failure mediated by microbial metabolites remain largely elusive. Mitochondria play a pivotal role in cellular energy metabolism and mitochondrial dysfunction has been associated to heart failure pathogenesis. Aim of the current study was to evaluate the impact of gut-derived metabolites on mitochondrial function in cardiomyocytes via an in vitro screening approach. Methods: Based on a systematic Medline research, 25 microbial metabolites were identified and screened for their metabolic impact with a focus on mitochondrial respiration in HL-1 cardiomyocytes. Oxygen consumption rate in response to different modulators of the respiratory chain were measured by a live-cell metabolic assay platform. For one of the identified metabolites, indole-3-propionic acid, studies on specific mitochondrial complexes, cytochrome c, fatty acid oxidation, mitochondrial membrane potential, and reactive oxygen species production were performed. Mitochondrial function in response to this metabolite was further tested in human hepatic and endothelial cells. Additionally, the effect of indole-3-propionic acid on cardiac function was studied in isolated perfused hearts of C57BL/6J mice. Results: Among the metabolites examined, microbial tryptophan derivative indole-3-propionic acid could be identified as a modulator of mitochondrial function in cardiomyocytes. While acute treatment induced enhancement of maximal mitochondrial respiration (+21.5 ± 7.8%, p < 0.05), chronic exposure led to mitochondrial dysfunction (-18.9 ± 9.1%; p < 0.001) in cardiomyocytes. The latter effect of indole-3-propionic acids could also be observed in human hepatic and endothelial cells. In isolated perfused mouse hearts, indole-3-propionic acid was dose-dependently able to improve cardiac contractility from +26.8 ± 11.6% (p < 0.05) at 1 μM up to +93.6 ± 14.4% (p < 0.001) at 100 μM. Our mechanistic studies on indole-3-propionic acids suggest potential involvement of fatty acid oxidation in HL-1 cardiomyocytes. Conclusion: Our data indicate a direct impact of microbial metabolites on cardiac physiology. Gut-derived metabolite indole-3-propionic acid was identified as mitochondrial modulator in cardiomyocytes and altered cardiac function in an ex vivo mouse model.
Keywords: cardiomyocyte physiology; gut microbiota; heart failure; microbial metabolites; mitochondrial dysfunction.
Copyright © 2021 Gesper, Nonnast, Kumowski, Stoehr, Schuett, Marx and Kappel.
Conflict of interest statement
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Figures
Figure 1
Effects of gut-derived metabolites on mitochondrial respiration in HL-1 cardiomyocytes. (A) Flowchart of mitochondrial stress testing by Seahorse Flux Analyzer. Oligomycin and FCCP were simultaneously added to the cells (stressed OCR). (B) Effects of gut-derived metabolites on mitochondrial function in HL-1 cardiomyocytes after 24 h incubation with substances. Basal oxygen consumption rate (OCR) of control cells was defined as 1.0. Basal OCR is the sum of three baseline measurements. All metabolites were displayed in relation to control or their vehicle (DMSO/ethanol). All results were verified in two independent replicates with N = 5/group. Data were shown as mean. *p < 0.05 and **p < 0.01 by 2-way ANOVA with Dunnett post-hoc test. The black arrow indicates the most prominent effect on mitochondrial function of tested metabolites.
Figure 2
Effects of indole-3-propionic acid (IPA) on HL-1 cardiomyocytes. (A) Cell viability and cytotoxic effects of IPA after 24 h of treatment in HL-1 cardiomyocytes by resazurin-based assay. RFU, relative fluorescence units. N = 4/group, mean ± SD, 1-way ANOVA with post-hoc test. (B) Cell proliferation was measured by BrdU assay after 24, 48, and 72 h treatment with IPA. RLU, relative light units. N = 5/group, mean ± SEM, 1-way ANOVA with post-hoc test. (C,D) Measurement of the metabolic function by Seahorse Flux Analyzer in HL-1 cardiomyocytes after incubation with IPA for 24 h. (C) Changes in oxygen consumption rate (OCR) over time. N = 5/group. Two-way ANOVA with Dunnett post-hoc test. Data are mean ± SEM. (D) Metabolic phenotypes including OCR and extracellular acidification rate (ECAR) under basal (opened cycle) and stressed (filled cycle) conditions. Data were shown as mean ± SEM with N = 5/group. *p < 0.05, **p < 0.01, and ****p < 0.0001.
Figure 3
Mitochondrial characterization of HL-1 cardiomyocytes in response to treatment with indole-3-propionic acid (IPA). (A) Schematic representation of real-time measurement of mitochondrial function by Seahorse Flux Analyzer. Oligomycin, FCCP and rotenone/antimycin A (rot/AA) were injected sequentially with determination of basal respiration, ATP production, proton leak, maximum respiration including spare capacity and non-mitochondrial respiration. (B) Schematic overview on the mitochondrial respiratory chain with points of action of different inhibitors. (C,D) Mitochondrial stress testing of HL-1 cardiomyocytes after 24 h incubation with IPA. (C) Changes in oxygen consumption rate (OCR) over time in response to different inhibitors. Data were shown as mean ± SEM with N = 5/group. (D) Changes in basal respiration, ATP production, proton leak, maximal respiration, spare capacity, and non-mitochondrial respiration after 24 h incubation with IPA. For calculation, the last measurement points at basal and the first measurement points after injection of the respiratory chain inhibitors were used. Data were shown as mean ± SEM with N = 5/group. (E,F) Mitochondrial stress testing of HL-1 cardiomyocytes after incubation of IPA at different time points (24 h, 6 h, 2 h, 30 min and direct injection). (E) Changes in OCR over time in response to different inhibitors and IPA 1 mM. Data were shown as mean ± SEM with N = 5/group. (F) Changes in OCR over time in response to different inhibitors and IPA 10 μM. Data were shown as mean ± SEM with N = 5/group. For all data, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, 2-way ANOVA with Dunnett post-hoc test.
Figure 4
(A) Schematic overview of experimental procedure of Langendorff model. (B) Serial injections of buffer, dobutamine, and IPA with intermittent washing steps in the Langendorff mouse model show dose-depended effects of IPA on left ventricle developed pressure (LVdp). Dobutamine (1 mg/ml, 1:256) was administered as positive control. (C) Percentage difference of LVdp in relation to baseline/wash between buffer, IPA, and wash. All data were shown as mean ± SD with N = 7/group. *p < 0.05, **p < 0.01, and ***p < 0.001 by 1-way ANOVA.
Figure 5
Mitochondrial characterization in human hepatic cell line (Huh7) and Human Umbilical Vein Endothelial Cells (HUVEC) in response to 1 mM indole-3-propionic acid (IPA) after 24 h. (A,B) Mitochondrial stress testing in Huh7 after 1 mM IPA treatment for 24 h. (A) Changes of oxygen consumption rate (OCR) over time in response to respiratory chain inhibitors. Data were shown as mean ± SEM with N = 5/group. (B) Changes in basal respiration, ATP production, proton leak, maximal respiration, spare capacity and non-mitochondrial respiration after 24 h incubation of 1 mM IPA. For calculation, the last measurement points at basal and the first measurement points after injection of the respiratory chain inhibitors were used. Data were shown as mean ± SEM with N = 5/group. (C,D) Mitochondrial stress testing in HUVEC after 1 mM IPA treatment for 24 h. (C) Changes of OCR over time in response to respiratory chain inhibitors. Data were shown as mean ± SEM with N = 5/group. (D) Changes in basal respiration, ATP production, proton leak, maximal respiration, spare capacity and non-mitochondrial respiration after 24 h incubation of 1 mM IPA. Data were shown as mean ± SEM with N = 5/group. Rot/AA, combination of the inhibitors rotenone and antimycin A. *p < 0.05 and **p < 0.01, 2-way ANOVA with Dunnett post-hoc test.
Figure 6
Detailed characterization of mitochondrial function in response to indole-3-propionic acid (IPA) in HL-1 cardiomyocytes. (A) Schematic representation of respiratory chain including substrates for detailed analysis of respiratory chain complexes. TMPD: N,N,N_′,N_′-Tetrametyhl-_p_-phenylenediamine. (B) Measurement of oxygen consumption rate (OCR) over time in response to substrates of respiratory chain complexes injected sequentially. Rot, rotenone; AA, antimycin A. (C) Mitochondrial stress testing in permeabilized HL-1 cardiomyocytes including palmitoyl-L-carnitine in response to 1 mM IPA (24 h). (D) Mitochondrial stress testing in permeabilized HL-1 cardiomyocytes including octanoyl-L-carnitine in response to 1 mM IPA (24 h). (E) Addition of exogenous cytochrome c to permeabilized HL-cardiomyocytes after treatment with IPA (24 h). Measurement of OCR over time. Sap, saponin; suc, succinate; rot, rotenone; cyt c, cytochrome c. (B–E) Data were shown as mean ± SEM with N = 5/group by 2-way ANOVA with Dunnett post-hoc test. (F) Mitochondrial membrane potential was measured by TMRE staining in HL-1 cardiomyocytes after treatment with IPA (24 h). 20 μM FCCP for 10 min was used as positive control. RFU: relative fluorescence units. Data were shown as mean ± SD with N = 4–5/group by 1-way ANOVA with post-hoc test. (G) Measurement of ROS production and hydroxyl radicals by H2DCFDA assay in HL-1 cardiomyocytes. HL-1 cardiomyocytes were treated with 1 mM IPA for 24 h and 30 min., 1 mM iron(II) sulfate for 30 min was used as positive control. Data were shown as mean ± SD with N = 4/group by 1-way ANOVA with post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.
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