Haplodeficiency of activin receptor-like kinase 4 alleviates myocardial infarction-induced cardiac fibrosis and preserves cardiac function
Introduction
Myocardial infarction (MI) is one of the most frequent causes of morbidity and mortality worldwide [1]. Even though the current effective percutaneous coronary intervention and thrombolytic treatment have saved numerous patients' lives, the vast majority of patients who survived MI suffer from heart failure due to adverse cardiac fibrosis (CF) and left ventricular (LV) remodeling [2]. CF is a beneficial repairing process for supporting the necrotic LV wall to avoid rupture during the acute stage of MI. However, during the subacute and chronic stages, abnormal CF, characterized by profound proliferation and deregulated secretory function of the cardiac fibroblasts, inevitably results in an imbalance between synthesis and degradation of extracellular matrix (ECM) and a cardiac dysfunction [3], [4].
It has been well established that TGF-β pathway is implicated in the development of post-MI CF [5]. TGF-β superfamily ligands, including TGF-β, activin, bone morphogenetic protein (BMP) and other related proteins, act as multifunctional growth factors in a variety of tissues. Individual ligand transmits specific signal by binding to its cognate type-II transmembrane serine/threonine kinase receptors. Activated type-II receptors then stimulate formation of a heteromeric receptor complex via distinct combinations of one of the seven type-I receptors (also known as activin receptor-like kinase, ALK; ALK1–7), followed by phosphorylation and activation of ALKs. TGF-β activates ALK1/5, BMP activates ALK1/2/3/6, whereas activin specifically activates ALK4. The activated ALKs then propagate signal through phosphorylation of the pathway-restricted Smads [6], [7]. In contrast to BMP that phosphorylates Smad1/5/8, both TGF-β and activin phosphorylate Smad2/3. Subsequently, phosphorylated Smad2/3 bind to Smad4 and the resultant Smad2/3/4 complex relocates from the cytoplasm into the nucleus, where the complex governs transcription of several hundred genes, including profibrogenic ECM and connective tissue growth factor (CTGF) genes. In cardiac fibroblasts, TGF-β regulates their activation, proliferation, and ECM deposition. Overexpression of TGF-β could exacerbate CF while inhibition of TGF-β could attenuate post-MI progression of LV remodeling and heart failure [5], [8].
Based on previous studies, both TGF-β/ALK1/5 and activin/ALK4 pathways share the same three downstream effectors, Smad2/3/4 [6], [7]. Therefore, it is highly possible that activation of activin/ALK4 pathway could also potentiate post-MI CF. However, in contrast to the above well-studied TGF-β/ALK1/5 pathway, little is known about the role of the activin/ALK4 pathway in the pathogenesis of CF. A series of studies have implicated that activin, like TGF-β, suppressed cell growth and stimulated fibrosis in several non-cardiac organs through up-regulation of CTGF [9], [10], [11], [12]. In the cardiovascular system, elevated serum levels of activin have been reported in patients with heart failure [12]. Furthermore, ALK4 has been found to mediate diverse biological process including cell differentiation, proliferation and migration [13], [14]. Although ALK4 is ubiquitously expressed, it is highly expressed in cardiac ventricular myocytes and is important for embryonic heart development since homozygous knocking-out of the ALK4 gene in mice resulted in embryonic lethality [15], [16], [17]. A recent study reported that upregulated ALK4 expression and activated ALK4/Smad signaling contributed greatly to the development of systemic sclerosis, an illness with remarkable fibrosis in skin [18].
In this regards, we focused on exploring the role of ALK4 in CF underlying different cardiovascular diseases. We reported recently that ALK4 was upregulated in the pressure-overloaded heart and its partial inhibition attenuated pressure overload-induced CF and cardiac dysfunction. However, the role of ALK4 in the pathogenesis of MI-induced CF, which is usually more severe than that induced by pressure-overload, remains unknown. In the present study, we reported the role and underlying mechanism of ALK4 in CF in the mouse MI model. ALK4 is mainly upregulated in the fibroblasts of the border zone of wild-type (WT) mice. ALK4 haplodeficient mice present a significantly alleviated CF in the border zone, with smaller scar sizes, preserved cardiac function, and improved survival rate post-MI. Compared to isolated cardiac fibroblasts from WT mice, cardiac fibroblasts from ALK4+/− mice show a suppressed migration, proliferation and ECM synthesis under hypoxia. Both in vivo and in vitro studies suggested that the effects of ALK4 haplodeficiency in reduction of CF are through a partial inactivation of Smad3/4 pathway but not MAPK cascades. These results revealed the critical role of ALK4 in the pathogenesis of CF, and provided a promising pharmaceutical target for prevention of adverse ventricular remodeling post-MI.
Section snippets
Generation of ALK4 gene knockout mice and MI model
ALK4 gene knockout mice were generated using CRISPR-Cas9 technology, as described previously [19]. The passage and genotyping of the mice were conducted as reported [20]. Due to an early embryonic lethality of homozygous (ALK4−/−) mice, heterozygous (ALK4+/−) mice were used in experiment [17]. All procedures were conducted in compliance with both the Guide for the Animal Care and Use Committee of Xinhua Hospital at SJTU School of Medicine and the guidance for the care and use of experimental
ALK4 expression is upregulated in the infarct border zone in WT mice
To investigate the potential role of ALK4 in post-MI CF, we examined its spatiotemporal expression pattern in the border zone and the remote area from the infarct in mice heart by WB. ALK4 protein levels in the border zone showed no change at day 1 post-MI (1.11 ± 0.01), significantly increased at day 7 (2.15 ± 0.14), peaked at day 14 (4.88 ± 0.29), and then slightly declined at day 28 (4.21 ± 0.11 folds; Fig. 1A and B). Whereas, ALK4 levels in the remote area showed no remarkable changes throughout 28
Discussion
CF plays pivotal roles, both beneficial and detrimental, during the post-MI LV remodeling [3], [4], [31]. The infarct region repaired by fibrosis initially maintains the architectural integrity and protects the LV wall from rupture [32]. However, subsequent interstitial fibrosis developed in non-infarct areas progressively contributes to the increased myocardial stiffness and impairment of the cardiac function [33]. It is well established that TGF-β/ALK5 signaling pathway is the most important
Sources of funding
This work was supported the Natural Science Foundation of China (NSFC) Grant No.81170302 (to Y-P. W.), No.81270258 (to Y-G. L.), No.81370257 (to Y-P. W.), No.81470497 (to J. C.), No.81530015 (to Y-G. L.) and No.81670414 (to Y-P. W.).
Disclosures
None.
Conflict of interest:
The authors declare no conflicts of interest.
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