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. 2018 Oct;36(5):e12462.
doi: 10.1111/1755-5922.12462. Epub 2018 Aug 22.

TGR5 activation induces cytoprotective changes in the heart and improves myocardial adaptability to physiologic, inotropic, and pressure-induced stress in mice

Zeena Eblimit  1 , Sundararajah Thevananther  2 , Saul J Karpen  3 , Heinrich Taegtmeyer  4 , David D Moore  5 , Luciano Adorini  6 , Daniel J Penny  7 , Moreshwar S Desai  1
Affiliations

TGR5 activation induces cytoprotective changes in the heart and improves myocardial adaptability to physiologic, inotropic, and pressure-induced stress in mice

Zeena Eblimit et al. Cardiovasc Ther. 2018 Oct.

Abstract

Introduction: Administration of cholic acid, or its synthetic derivative, 6-alpha-ethyl-23(S)-methylcholic acid (INT-777), activates the membrane GPCR, TGR5, influences whole body metabolism, reduces atherosclerosis, and benefits the cardiovascular physiology in mice. Direct effects of TGR5 agonists, and the role for TGR5, on myocardial cell biology and stress response are unknown.

Methods: Mice were fed chow supplemented with 0.5% cholic acid (CA) or 0.025% INT-777, a specific TGR5 agonist, or regular chow for 3 weeks. Anthropometric, biochemical, physiologic (electrocardiography and echocardiography), and molecular analysis was performed at baseline. CA and INT-777 fed mice were challenged with acute exercise-induced stress, acute catecholamine-induced stress, and hemodynamic stress induced by transverse aortic constriction (TAC) for a period of 8 weeks. In separate experiments, mice born with constitutive deletion of TGR5 in cardiomyocytes (CM-TGR5del ) were exposed to exercise, inotropic, and TAC-induced stress.

Results: Administration of CA and INT-777 supplemented diets upregulated TGR5 expression and activated Akt, PKA, and ERK1/2 in the heart. CA and INT-777 fed mice showed improved exercise tolerance, improved sensitivity to catecholamine and attenuation in pathologic remodeling of the heart under hemodynamic stress. In contrast, CM-TGR5del showed poor response to exercise and catecholamine challenge as well as higher mortality and signs of accelerated cardiomyopathy under hemodynamic stress.

Conclusions: Bile acids, specifically TGR5 agonists, induce cytoprotective changes in the heart and improve myocardial response to physiologic, inotropic, and hemodynamic stress in mice. TGR5 plays a critical role in myocardial adaptability, and TGR5 activation may represent a potentially attractive treatment option in heart failure.

Keywords: INT-777; TGR5; cholic acid; myocardial adaptation; preconditioning.

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Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

FIGURE 1
FIGURE 1
CA induces cytoprotective changes in the heart: (A) Hearts of mice fed CA for 3 wk demonstrate activation of cytoprotective survival kinases such as Akt, ERK, and PKA. Note ~2× increase in phosphorylated Akt, ERK, and PKA (activation) when divided by their unphosphorylated band. GAPDH band shows equal loading of samples. (B) shows key cytoprotective genes (HO-1, HSP 72, 27, and 90) as well as eNOS are upregulated in the CA fed hearts compared to chow fed. Note upregulation of GLUT-1 which determines basal glucose uptake and downregulation of PDK4, which negatively regulates glucose oxidation in CA fed mouse hearts. n = 5 per group; Results: Mean ± SD; *P < 0.05; Stats: t test
FIGURE 2
FIGURE 2
CA improves chronotropic response to catecholamine and enhances whole body oxygen consumption during exercise: (A) shows change in heart rate after intraperitoneal injection of 20 mcg/kg of single dose of isoprenaline. Note significant higher response to catecholamine as evidenced by a higher change in heart rate postisoprenaline. (B) denotes time taken by the mouse to get exhausted on the treadmill. Although the exhaustion times were comparable, there was a significant increase in VO2 (oxygen consumed-a surrogate marker for cardiac output at peak of exercise and decrease in respiratory exchange ratio during peak exercise in CA fed mice compared to chow fed mice (C)). n = 5 per group; Results: Mean ± SD for A and B, SEM for C; *P < 0.05; Stats: t test for A and B. Mann–Whitney across time-points for C
FIGURE 3
FIGURE 3
CA attenuates transverse aortic constriction (TAC) induced hypertrophy and contractile failure: (A) shows M-Mode 2DE images of chow fed and CA fed hearts post- TAC. Note attenuation in contractile dysfunction in CA fed post- TAC hearts. (B) shows serial shortening fractions (%FS) of C57BL6 mice either fed CHOW or 0.5% cholic acid (CA) supplemented chow, when challenged with TAC or SHAM. Note significant attenuation of %FS in CA fed TAC (CA + TAC) mice compared to CHOW+ TAC mice. (n = 5– 10/grp; Stats: Multiple comparison ANOVA; *P < 0.05 compared to all groups; # P < 0.05 vs SHAM, Results: Mean ± SEM). (C) denotes quantification of heart weights indexed to body weight and tibial length at end of 8 wk. Note attenuation in TAC induced pathologic hypertrophy of hearts in CA fed mice (n = 5/sham groups, 10/TAC group); Stats: ANOVA with Tukey’s post hoc comparison between groups; *P < 0.05 when CHOW + TAC was compared to all groups; ^P < 0.05 when CA + TAC is compared to SHAM groups and CHOW + TAC group. vs SHAM, Results: Mean ± SEM). (D) shows images of Ser 473 phospho- AKT, total AKT, Thr, Ser 256 and Ser 319 phospho-FOXO-1 and total FOXO-1. Activation (phosphorylation) of AKT, and inhibition (phosphorylation) of FOXO-1 was evaluated by dividing the phosphorylated to unphosphorylated forms of AKT and FOXO- 1, respectively. GAPDH blot is depicted to show equal loading. CA+TAC mice show enhanced activation of AKT and increased inhibition of FOXO-1 than CHOW+TAC mice. Also note significantly increased inhibition of FOXO-1 in the CA + SHAM mice, compared to CHOW+SHAM.(Results: Mean ± SEM; *P < 0.05 when CA + TAC was compared to all groups; ^P < 0.05 when CA+SHAM is compared to all groups, Stats: ANOVA with Tukey’s post hoc). (E) denotes BNP, βMYH7 RNA along with βMHC:αMHC ratio as measured by QRTPCR, in the 4 experimental groups. RNA has been standardized to 18S subunit and expressed as fold change compared to CHOW fed SHAM mice. Note attenuation of BNP, βMYH7 and βMHC:αMHC ratio at RNA level in the CA + TAC group (#P < 0.05 comparing CA+TAC with CHOW+TAC and SHAM groups; *P < 0.05 compared to all groups). Also note downregulation of PDK4 (*P < 0.05 when CA fed groups are compared to CHOW fed groups), and downregulation of UCP3 (*P < 0.05 when CA+TAC is compared to rest of the groups) (n = 5 per group; Results: Mean ± SD; Stats: ANOVA with Tukey’s post hoc)
FIGURE 4
FIGURE 4
INT-777 induces cytoprotective changes in the hearts at a molecular level, improves exercise tolerance, chronotropic response to catecholamine, and attenuates TAC-induced contractile failure. Hearts of mice fed INT-777 showed activation (phosphorylation) of AKT, ERK, and PKA (A) These are pro-survival kinases and are part of the Reperfusion Injury Salvage Kinase (RISK) pathway. Bar graph shows phosphorylation (activation), with GAPDH as loading control and compared to chow (fold activation) (n = 3–5; Statistics: t test, *P < 0.05). (B) shows images of Ser473 phospho-Akt, total Akt, Thr 32/34 phospho-FOXO1 and total FOXO1. Activation (phosphorylation) of Akt and inhibition (phosphorylation) of FOXO1 was evaluated with densitometry by dividing the phosphorylated form with its un-phosphorylated form. INT-777 fed mice show a robust activation of Akt and inhibition of FOXO1 compared to chow fed mice. (n = 3–5 per group; Stats: t test). (C) When challenged with acute physiologic stress in the form of treadmill exercise, INT-777 fed mice ran longer (higher time to fatigue) than chow fed mice. (n = 8/group; Statistics: t test; *P < 0.05 when INT-777 is compared to CHOW). (D) When mice were challenged with acute i.p injection of isoprenaline, hearts from INT-777 fed mice showed a stronger chronotropic response compared to chow fed mice. There was no significant difference in the effect of INT-777 on shortening fraction or the cardiac output. (n = 8/group; Statistics: t test; *P < 0.05 pre-isoprenaline vs. post-isoprenaline. Also, * P < 0.05 with change in heart rate (Δ) between CHOW fed and INT-777 fed groups). (E) Figure shows serial shortening fractions (%FS) of C57BL6 mice either fed CHOW or 0.025% INT-777 supplemented diet, when challenged with TAC or SHAM. Note significant attenuation of %FS in INT-777 fed TAC (INT-777 + TAC) mice compared to CHOW+TAC mice. (n = 5–8/grp; Stats: Multiple comparison ANOVA, *P < 0.05 compared to all groups; #P < 0.05vs SHAM, Results: Mean ± SEM). (F) shows INT-777 feeding attenuates key genes involved in TAC induced pathologic remodeling as evidenced by attenuated increase in TAC induced BNP, βMYHC RNA along with βMHC:αMHC ratio in INT-777 fed TAC mice compared with chow fed controls. RNA has been standardized to GAPDH and expressed as fold change compared to chow fed SHAM mice. (*P < 0.05, n = 5–10 per group; Stats: ANOVA with Tukey’s post hoc)
FIGURE 5
FIGURE 5
Cardiomyocyte specific deletion of TGR5 (CM-TGR5del) in mice, demonstrate exercise intolerance, increased mortality and exaggerated contractile dysfunction in response to TAC, compared to littermates: (i) and (ii) shows proof of TGR5 deletion from the whole heart and adult isolated cardiomyocyte in CM-TGR5del mice. (i) shows knockdown of TGR5 from the whole hearts in CM-TGR5del mice at both RNA and protein level using primers and antibodies used and tested by us before. As this is a cardiomyocyte specific deletion, weak bands seen on PCR and Western blots are a result of presence of TGR5 in the vascular endothelium which is not deleted. A ~60% deletion was achieved from the whole heart when quantified objectively by qRTPCR and densitometry (data not shown). [M] is marker, [WT] is whole hearts of TGR5 flox/flox/cre− littermates, deletion (del) shown as arrow [CM-TGR5del hearts], with spleen(S) as positive control and water (W) as negative control. (ii) shows complete deletion of TGR5 from adult cardiomyocytes. We isolated cardiomyocytes from the hearts of adult CM-TGR5del mice and probed them for the presence of TGR5 using primers as before. We found a near complete deletion of TGR5 RNA (arrow). [WT] is whole hearts of TGR5 flox/flox/cre− mice, deletion (del) shown as arrow [CM-TGR5del hearts], water [W] as negative control. When challenged with acute physiologic stress in the form of treadmill exercise, CM- TGR5del mice ran shorter distance (A) and fatigued (B) earlier than WT mice. (n = 5/group; Statistics: t test; *P < 0.05; RESULTS: Mean ± SEM). There was no significance in shortening fraction between the two groups on isoprenaline (C). When CM-TGR5del mice were randomized to SHAM or TAC, increased mortality was noted in CM-TGR5del mice compared to WT mice post-TAC (D). No mortality noted in sham groups. (*P = 0.013 compared to all groups; # P < 0.05 compared to sham (Stats: Mantell-Cox test, n = 3/grp in SHAM and 10/grp in TAC). (E) shows serial analysis of shortening fractions (%FS) as evaluated by ECHOs. Note significant decrease in FS in CM- TGR5del mice post- TAC over 8 wk, starting as early as 2 wk (n = 3–5/grp; Stats: Multiple comparison ANOVA, P < 0.05). (F) shows bar graphs of heart weight/tibial length ratios of WT and CM-TGR5del mice who undergo either SHAM or TAC. CM- TGR5del mice had ~60% higher heart weight/TL ratio suggesting increased hypertrophy compared to WT+TAC mice. (*P < 0.05; ANOVA; Results: Mean ± SD; number of animals = WT + SHAM (n = 3); WT+TAC (n = 7 survived of 10); CM- TGR5del + SHAM (n = 3); CM- TGR5del (n = 6 survived of 20)
FIGURE 6
FIGURE 6
Proposed mechanisms of cardioprotection: Cholic acid and INT-777, acting as TGR5 agonists, activate Akt, ERK and PKA pathways which are cytoprotective and improve myocardial response to stress. Activation of Akt, may inhibit FOXO-1 and suppress PDK4, which improves the metabolic efficiency and adaptability of the heart to catecholamine, exercise, and pressure overload mediated stress
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