Acid-sensing ion channel 1a blockade reduces myocardial injury in rodent models of myocardial infarction

Abstract

Myocardial infarction (MI) caused by ischemia-reperfusion injury (IRI) is the leading risk factor for heart failure. The sodium-hydrogen exchange (NHE) inhibitor, cariporide, is the only cardioprotective drug to reach Phase 3 clinical trials, but it caused cerebrovascular side effects.1 There remain no clinically approved drugs that block cardiomyocyte cell death during acute IRI.2 While blockade of acid-sensing ion channel 1a (ASIC1a) is known to protect the brain3 and heart4 against ischemic injury, the cardioprotective effect of ASIC1a inhibition after the onset of ischemia during MI remains unknown. We tested this using a mouse model involving 40 min ischemia induced by ligation of the left anterior descending (LAD) coronary artery followed by reperfusion (Figure 1A). Hi1a (1 mg/kg) was administered (intravenous bolus) 5 min prior to ligation (Pre: pre-treatment), 5 min after onset of ischemia (EP: early post-treatment), or 5 min prior to reperfusion (LP: late post-treatment) (Figure 1A). Twenty-four hours post-MI, myocardial viability was assessed as a fraction of the area at risk (AAR) (Figure 1B). While all groups had comparable AAR, Hi1a treatment reduced infarct size with equal efficacy at all timepoints (Figure 1C and D). Dose-escalation analysis in the LP condition revealed a minimum effective dose of 0.5 mg/kg (Figure 1C and D). In a long-term follow-up study, 0.5 mg/kg Hi1a in the LP model prevented cardiac dysfunction associated with MI, as evidenced by increased ejection fraction at 1 and 4 weeks (Figure 1E). Due to mild injuries, we observed small but significant benefits in systolic volume but no differences in diastolic volume with Hi1a treatment (Figure 1F and G). Cardioprotection during MI by blockade of ASIC1a. (A–G) Murine MI study with endpoints of acute viability and 4-week functional recovery. (A) Surgical procedure depicting thoracotomy with 40 min LAD occlusion and Hi1a administration (i.v.) 5 min prior to ligation (Pre: pre-treatment), 5 min after the onset of ischemia (EP: early post-treatment), or 5 min prior to reperfusion (LP: late post-treatment). Created wtih BioRender.com. (B and C) Analysis of viability and AAR via Evan’s Blue injection followed by TTC staining in hearts collected 24 h post-MI (n = 5–6 per group). (B) Example images depicting viable (red) and infarcted (white) tissue, and region outside AAR (blue). (C) Quantification of AAR normalized to left ventricle (LV) area. (D) Infarct area (normalized to AAR). (E–G) Analysis of echocardiograms (n = 8–10 per group) collected at baseline, and at 1 and 4 weeks post-MI: (E) ejection fraction, (F) left ventricular end-systolic volume index (LVESVI), and (G) left ventricular end-diastolic volume index (LVEDVI). (H) PET imaging in healthy mice injected with radiolabeled 124I-Hi1a (1.9–2.6 MBq or 0.36–0.5 mg/kg) (n = 2). (I and J) Analysis of Hi1a-AF700 and RA-Hi1a-AF700 myocardial distribution after i.v. injection (LP) in uninjured mouse hearts (H: healthy), after 40 min ischemia (I), or after 40 min ischemia plus 5 min reperfusion (IR) (n = 4–5 per group). (I) Representative images taken on Li-COR Odyssey Bioanalyzer of whole hearts (left) and 1 mm sections tiling from base to apex of the heart (right). (J) Quantification of mean pixel intensity (MPI) of Hi1a-AF700 or RA-Hi1a-AF700 signal in 1 mm sections (normalized to internal background signal). (K) Confocal images of cryosection from representative 1 mm tissue slice from IR group (top: remote region; bottom: ischemic region) depicting Hi1a-AF700 (green) with immunofluorescence for ASIC1a (red) and DAPI (white). Yellow arrows indicate regions of peptide and ASIC1a co-localization. Scale bar = 100 µm. (L) Representative confocal images of immunofluorescence staining for Asic1a (green) and DAPI (blue) in mouse myocytes treated with pH 6.0 media and Hi1a-Atto565 (red). Scale bar = 10 µm. (M) Maximum Intensity Projection Z-stacks in Hi1a-Atto565 and RA-Hi1a-Atto565 stained mouse myocytes incubated with either pH 7.4 or pH 6.0 media for 40 min before fixation. Scale bar = 10 µm. (N) Quantification of Hi1a-Atto565 and RA-Hi1a-Atto565 in mouse myocytes incubated with either pH 7.4 or pH 6.0 media. Each symbol represents one myocyte. n = 10–11 myocytes obtained from three separate mice (3–4 myocytes/mouse). (O) Eurofins Safety44 screen data showing Hi1a (10 µM) binding or enzyme inhibition effect calculated as a percent inhibition of the binding of a ligand specific for each target or control enzyme activity. (P and Q) Analysis of heart rhythm (P) and systolic blood pressure (SBP) (Q) in healthy rats 1 h after bolus injection of Hi1a. (R–T) Rat MI study with 90 min LAD ligation and Hi1a or cariporide (1 mg/kg) administered 5 min before reperfusion via external jugular vein injection (EJV) (MI groups: n > 10; sham: n = 6). Functional analysis performed via echocardiography at 1 week post-MI used to determine (R) ejection fraction, (S) LVESVI, and (T) LVEDVI. All data are mean ± SEM. Statistical significance determined using one-way (panels C, D, and R–T) or two-way (panels E–G) repeated measures ANOVA with Dunnet post hoc test relative to control group (MI + veh), or two-way ANOVA between all groups with Tukey post hoc test (panel J) (*P < .05; **P < .01; ***P < .001, ****P < .0001) We next evaluated the biodistribution of Hi1a using dynamic positron emission tomography (PET). After injection of radiolabeled 124I-Hi1a in healthy animals, ∼4% of the signal localized to the heart (a surrogate for circulating Hi1a) for up to 90 min (Figure 1H). To evaluate tissue penetrance of Hi1a in healthy vs. injured myocardium, fluorescently labeled peptide (Hi1a-AF700) was administered to uninjured or infarcted animals (LP) with tissues collected after ischemia (I) or 5 min post-reperfusion (IR). While minimal Hi1a was detected in healthy (H) or ischemic hearts, Hi1a was detected throughout the re-perfused left ventricle (Figure 1I and J). A reduced/alkylated version of Hi1a (RA-Hi1a-AF700) with no ASIC1a inhibitory activity showed no penetrance in healthy or injured myocardium (Figure 1I and J). Immunofluorescence of ischemic hearts revealed co-localization of ASIC1a protein and Hi1a in the infarct region compared to remote or healthy tissue suggesting on-target binding of Hi1a to ASIC1a in ischemic myocardium (Figure 1K). We therefore tested the pH-dependency of Hi1a binding to adult myocytes. Primary mouse myocytes (C57Bl/6J) were cultured in physiologic (pH 7.4) or acidic (pH 6.0) conditions for 40 min and incubated with Hi1a-Atto565 or RA-Hi1a-Atto565. We use transmitted light to select cardiomyocytes for imaging based on their striated appearance. These results show significantly higher puncta in myocytes incubated with Hi1a under acidic conditions compared to all other groups (Figure 1L–N). These data agree with observations that ASIC1a traffics to the membrane under acidosis.5 Following EU-CARDIOPROTECTION COST Action committee recommendations,6 we contracted independent analysis of Hi1a safety and efficacy studies. An industry-standard screen revealed that Hi1a does not bind to any of 44 common drug off-targets7 with the exception of PDE3A, COX1, and COX2 (data provided for positive hits, Figure 1O). Since these enzymes are intracellular, they are unlikely to be safety concerns since Hi1a is a large, cell-impermeant peptide. We also performed in vivo safety and efficacy studies with an FDA-compliant contract research organization, PharmaLegacy. During a 1 h window post-injection across a 100-fold dose range (0.1–10 mg/kg), Hi1a caused no adverse effects on heart rhythm or blood pressure compared to vehicle-treated animals (Figure 1P and Q). Next, we induced severe, transmural infarcts involving a 90 min LAD occlusion in rats and delivered Hi1a or cariporide (1 mg/kg) 5 min prior to reperfusion by external jugular vein injection (EJV) to model treatment prior to stent placement in MI patients. Echocardiographic analysis of hearts at 1 week post-MI revealed that Hi1a treatment significantly improved cardiac contractility and reduced systolic and diastolic volumes to levels not significantly different from sham animals (Figure 1R–T). Importantly, Hi1a performed as well as the benchmark cardioprotective drug, cariporide, in all parameters of organ structure and function. Cardiovascular disease (CVD) is the leading cause of death globally,8 with MI accounting for the majority of CVD-related deaths (∼9 million annually; ∼16% of global mortality).8 This study demonstrates that the ASIC1a inhibitor, Hi1a, is a first-in-class drug candidate that is compatible with clinical treatment scenarios for patients experiencing severe ischemic injury. While NHE inhibitors were cardioprotective, they failed clinically due to cerebrovascular side effects.1 Hi1a is equally cardioprotective but it does not induce adverse effects in rodents.4,8 Consistent with EU-CARDIOPROTECTION guidelines,6 future studies should evaluate Hi1a in co-morbid and large animal MI models to de-risk the path to clinical translation. We thank Darryl Whitehead and Rajiv Bhalla for their support. Infensa Bioscience is developing ASIC1a inhibitors for commercial purposes. G.F.K., N.J.P., and M.L.S. are co-founders and equity holders in Infensa Bioscience. L.D.R. and G.F.K. are inventors on US patents 10,485,847 and 10,881,712 which are licensed to Infensa Bioscience. The data underlying this article are available in the article. This study was supported by The Lott by Golden Casket and University of Queensland (Strategic Funding Research Initiative to N.J.P., G.F.K., and J.F.F.), National Heart Foundation of Australia (grants 101889 and 106721 to N.J.P.), Australian Research Council (DP190102072 to W.G.T.), and the National Health and Medical Research Council (Australia) (APP2000178 to N.J.P., G.F.K., and J.F.F., APP2002857 to N.J.P. and G.F.K.; and Medical Research Future Fund APP2007625 to N.J.P. and G.F.K.). All animal studies performed in house were carried out in accordance with the Australian National Health and Medical Research Council guidelines and the Guide for the Care and Use of Laboratory animals (US National Institutes of Health). All procedures were approved by The University of Queensland (UQ) Animal Ethics Committee under the following protocols: IMB/171/18 or 2021/AE000999 (MI surgery experiments and echocardiography) and QBI/059/13/ARC/NHMRC (biodistribution experiments with radioiodinated Hi1a). Rat myocardial infarct studies were performed by PharmaLegacy Laboratories (Shanghai, China). The test article was configured to the required concentration using 0.1% BSA/saline solution. Pathogen-free male Sprague-Dawley rats (210–230 g) were from Shanghai Jihui Biotechnology Co. Ltd. All surgical procedures were approved by the PharmaLegacy’s Institutional Animal Care and Use Committee (IACUC). Not applicable.

Topics & Concepts