Activation of the Amino Acid Response Pathway Blunts the Effects of Cardiac Stress

Abstract

BackgroundThe amino acid response (AAR) is an evolutionarily conserved protective mechanism activated by amino acid deficiency through a key kinase, general control nonderepressible 2. In addition to mobilizing amino acids, the AAR broadly affects gene and protein expression in a variety of pathways and elicits antifibrotic, autophagic, and anti‐inflammatory activities. However, little is known regarding its role in cardiac stress. Our aim was to investigate the effects of halofuginone, a prolyl‐tRNA synthetase inhibitor, on the AAR pathway in cardiac fibroblasts, cardiomyocytes, and in mouse models of cardiac stress and failure.Methods and ResultsConsistent with its ability to inhibit prolyl‐tRNA synthetase, halofuginone elicited a general control nonderepressible 2–dependent activation of the AAR pathway in cardiac fibroblasts as evidenced by activation of known AAR target genes, broad regulation of the transcriptome and proteome, and reversal by l‐proline supplementation. Halofuginone was examined in 3 mouse models of cardiac stress: angiotensin II/phenylephrine, transverse aortic constriction, and acute ischemia reperfusion injury. It activated the AAR pathway in the heart, improved survival, pulmonary congestion, left ventricle remodeling/fibrosis, and left ventricular function, and rescued ischemic myocardium. In human cardiac fibroblasts, halofuginone profoundly reduced collagen deposition in a general control nonderepressible 2–dependent manner and suppressed the extracellular matrix proteome. In human induced pluripotent stem cell–derived cardiomyocytes, halofuginone blocked gene expression associated with endothelin‐1‐mediated activation of pathologic hypertrophy and restored autophagy in a general control nonderepressible 2/eIF2α‐dependent manner.ConclusionsHalofuginone activated the AAR pathway in the heart and attenuated the structural and functional effects of cardiac stress.

EPRS Regulates Proline-rich Pro-fibrotic Protein Synthesis during Cardiac Fibrosis

“…Halo has been used to treat Duchenne Muscular Dystrophy (DMD) (Akashi Therapeutics) by reducing fibrosis and increasing muscle strength in phase II clinical trials. Also, GSK company confirmed its anti-fibrotic and cardiac protective activity in multiple mouse heart failure models 15 . However, the therapeutic mechanism of Halo has only been studied at the transcriptional level in triggering an amino acid starvation response (AAR) and a noncanonical TGF-b signaling pathway 12,15,16 .…”

“…Also, GSK company confirmed its anti-fibrotic and cardiac protective activity in multiple mouse heart failure models 15 . However, the therapeutic mechanism of Halo has only been studied at the transcriptional level in triggering an amino acid starvation response (AAR) and a noncanonical TGF-b signaling pathway 12,15,16 . The direct downstream translational targets of EPRS by Halo inhibition remains unclear.…”

“…( Figure 5E and Table S5). In contrast, the genes in Area 3 (induced at RNA and TE levels) are enriched in general translation factors, including ribosome biogenesis, ribosome (17 cytosolic and 6 mitochondrial ribosome proteins), aminoacyl-tRNA biosynthesis (16 ARSs including Eprs) ( Figure S4D), and amino acid biosynthesis ( Figure 5E), which suggests an activation of a compensatory amino acid starvation response upon EPRS inhibition 12,15 .…”

“…We first performed in silico bioinformatic analyses to uncover conserved PRR genes that bear PP genetic codon motifs across the reviewed human and mouse proteins from Uniprot proteomic sequences 30 at the genome-wide scale (Table S7), including PPG-rich ECM genes such as collagens among many Pro-rich proteins. Next, we overlapped PP motif bearing genes with genes that were downregulated in our transcriptomic and translatomic analyses ( Figure 6B) and proteins that were reduced at the steadystate level in human CFs upon low-dose Halo treatment in the published quantitative proteomic data 15 ( Figure S6A, Table S8). As we expected, 8 collagen genes (i.e., COL1A1/1A2/3A1/5A1/ 6A1/6A2/6A3/12A1) were translationally reduced in mouse fibroblasts, and their steady-state protein level was also decreased in human CFs after Halo treatment.…”

A potent prolyl tRNA synthetase inhibitor antagonizes Chikungunya and Dengue viruses

“…Although inhibiting host translation may lead to toxic effects, halofuginone and similar derivatives have been shown in murine models to inhibit malaria (Herman et al, 2015), reduce cardiac stress (Qin et al, 2017), suppress viral myocarditis (Sun et al, 2016), and improve hepatitis B virus-induced liver inflammation (Zhan et al, 2017). Furthermore, commercial preparations of halofuginone (Stenorol and Halocur) are used in livestock to inhibit parasite growth (Pines and Spector, 2015), and a clinical trial for a slow-release form of halofuginone has been approved by the FDA to treat Duchenne muscular dystrophy (Pines and Spector, 2015).…”