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. 2018 Apr 27;9(6):613.
doi: 10.1038/s41419-018-0573-2.

Cardiac fibrosis can be attenuated by blocking the activity of transglutaminase 2 using a selective small-molecule inhibitor

Zhuo Wang  1 , Daniel J Stuckey  2 , Colin E Murdoch  3 , Patrizia Camelliti  4 , Gregory Y H Lip  5 , Martin Griffin  6
Affiliations

Cardiac fibrosis can be attenuated by blocking the activity of transglutaminase 2 using a selective small-molecule inhibitor

Zhuo Wang et al. Cell Death Dis. .

Abstract

Cardiac fibrosis is implicit in all forms of heart disease but there are no effective treatments. In this report, we investigate the role of the multi-functional enzyme Transglutaminase 2 (TG2) in cardiac fibrosis and assess its potential as a therapeutic target. Here we describe the use a highly selective TG2 small-molecule inhibitor to test the efficacy of TG2 inhibition as an anti-fibrotic therapy for heart failure employing two different in vivo models of cardiac fibrosis: Progressively induced interstitial cardiac fibrosis by pressure overload using angiotensin II infusion: Acutely induced focal cardiac fibrosis through myocardial infarction by ligation of the left anterior descending coronary artery (AMI model). In the AMI model, in vivo MRI showed that the TG2 inhibitor 1-155 significantly reduced infarct size by over 50% and reduced post-infarct remodelling at 20 days post insult. In both models, Sirius red staining for collagen deposition and levels of the TG2-mediated protein crosslink ε(γ-glutamyl)lysine were significantly reduced. No cardiac rupture or obvious signs of toxicity were observed. To provide a molecular mechanism for TG2 involvement in cardiac fibrosis, we show that both TGFβ1-induced transition of cardiofibroblasts into myofibroblast-like cells and TGFβ1-induced EndMT, together with matrix deposition, can be attenuated by the TG2 selective inhibitor 1-155, suggesting a new role for TG2 in regulating TGFβ1 signalling in addition to its role in latent TGFβ1 activation. In conclusion, TG2 has a role in cardiac fibrosis through activation of myofibroblasts and matrix deposition. TG2 inhibition using a selective small-molecule inhibitor can attenuate cardiac fibrosis.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. In vivo assessment of TG2 inhibition on AngII-induced cardiac fibrosis.
AngII 1.1 mg/kg/day in 50% DMSO in PBS pH 7.4 was used to induce cardiac fibrosis for 2 weeks with or without TG2 inhibitor (25 mg/kg/day) via a subcutaneously implanted mini-pump (Alzet1002). a Representative images of heart sections from the inhibitor and non-inhibitor-treated animals showing collagen staining using Picro-Sirius red. Bar = 100 µm. b Averaged data of Picro-Sirius red/collagen staining in heart sections from control and TG2 inhibitor 1–155 treated animals. c TG2-mediated ε(γ-glutamyl)lysine crosslink (XL) formation in the AngII and AngII+ 1–155 treated animals. d–f Systolic blood pressure (SBP) (d), heart rate (e), and heart weight (HW, wet weight, mg) against body weight (BW, g). f were obtained after 2 weeks of treatment with 1–155. Data are means ± SE. n = 5/4. *p < 0.05
Fig. 2
Fig. 2. In vivo assessment of cardiac structure, function and viability in a mouse AMI model following TG2 inhibition.
MRI was performed 6 h after myocardial infarction, prior to mini-pump implantation, and again at 20 days. a Representative end systolic frames of cine-MRI acquisitions along with matching late gadolinium-enhanced MRI (LGE) acquisitions used for assessment of infarct size. Red lines define the edges of the hyper-enhanced infarct region. b Left ventricular masses, end diastolic volumes and end systolic volumes increased from day 0 to day 20 in the control group, but there was no significant increase in these parameters in the treatment group. Total infarct size and infarct size as a percentage of left ventricular mass was significantly lower in the treatment group compared with controls at 20 days. *P < 0.05 compared with day 0; #P < 0.05 compared with control. Bar = 2 mm
Fig. 3
Fig. 3. Assessment of collagen and TG2-mediated ε-(γ-glutamyl)-lysine in the AMI mouse model following TG2 inhibition.
a Representative images of Picro-Sirius red/collagen-stained heart sections from the AMI mouse model following treatment with or without TG2 inhibitor 1–155. b Averaged data of Picro-Sirius red/collagen staining in the remote myocardium showing interstitial collagen deposition is significantly reduced by 1–155. c 1–155 reduces the TG2-mediated ε-(γ-glutamyl)-lysine crosslink (XL) in the 1–155 treated mice. Data are means ± SE. n = 5/7. *p < 0.05. **p < 0.005
Fig. 4
Fig. 4. The effect of TG2 inhibition on TGFβ1-induced cardiofibroblast to myofibroblast transition and collagen deposition in human cardiofibroblasts.
a Representative western blots (n = 3) showing p-Smad2/3 activation, αSMA and FN and FN deposition and the inhibition of these parameters using TG2 inhibitor 1–155 (2.5 µM) in 72 h TGFβ1-treated cardiofibroblasts at the concentrations shown. Matrix FN was measured according to the procedures described in the 'Materials and methods'. b Representative western blot (n = 3) showing the effect of 72 h TGFβ1 treatment in cardiofibroblasts showing increased TG2 expression and increased TG2 at the cell surface and ECM and inhibition of this by TG2 inhibitor 1–155. c Representative western blot (n = 3) of TG2 in the syndecan-4 immunocomplex from co-IP carried out as described in 'Materials and methods'. d Immunofluorescence detection of collagen I in cardiofibroblasts treated with 1 ng/ml TGFβ1 over 5 days with and without the TG2 inhibitor at 2.5 µM undertaken as described in the 'Materials and methods'. Bar = 25 µm
Fig. 5
Fig. 5. The importance of TG2 on TGFβ1-mediated EndMT.
a Representative western blot (n = 3) showing p-Smad2/3 activation, VE-cadherin and FN expression, and FN deposition into the matrix in HUVECs treated with TGFβ1 in the presence or absence of t TG2 inhibitor 1–155. b The V2a AngioKit co-culture model was used as described in the 'Materials and methods' to study the effects of TGFβ1 on endothelial tubule formation. Representative images show the inhibitory effects of TGFβ1 on tubule formation at the concentration shown over 12 days. c Representative western blot (n = 3) showing Smad2/3 activation in mouse TG2+/+ and TG2−/− microvascular ECs following TGFβ1 treatment for 72 h at the concentrations shown. d Representative western blot (n = 3) showing the effects of thioredoxin treatment on HUVECs showing Smad2/3 activation, expression of FN, VE-Cadherin and TG2
Fig. 6
Fig. 6. Schematic showing the role of TG2 in cardiac fibrosis and the TG2/TGFβ1 positive feedback mechanism indicating its importance in the progression of cardiac fibrosis.
a How hemodynamic or oxidative stress and hypoxia can lead to an inflammatory response causing increased amounts of extracellular TGFβ1 leading to increased amounts of TG2 and increased numbers of myofibroblast leading to the deposition of a highly TG2 crosslinked fibrotic matrix. b The functional relationship between TG2 in the progression of fibrosis. The arrow shows how inactivation of TG2 can block this vicious cycle in fibrosis development
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