Association of OR6A2 Expression on Monocytes With Inflammation and Major Adverse Cardiovascular Events in Myocardial Ischemia-Reperfusion Injury
Conditions: Myocardial Ischemia-Reperfusion Injury
Sponsor: Southeast University, China

 
1
 
 
STUDY PROTOCOL 
 
 
Official Title: Association of OR6A2 Expression on Monocytes with Inflammation and Major 
Adverse Cardiovascular Events in Myocardial Ischemia-Reperfusion Injury 
Principal Department: Department of Cardiology, Zhongda Hospital, Southeast University 
Institutional Address/Postcode: 83 Dingjiaqiao, Hunan Road Subdistrict, Gulou District, 
Nanjing 210009, Jiangsu Province, P.R.China 
Administrative Authority: Department of Cardiology, Zhongda Hospital, Southeast University 
                           
 
 
Version Number: 1.03 
Version Date: April 20, 2019 
 
 
 
 
Zhongda Hospital, Southeast University 
 
 
 
 
 
 
 

 
2
Study Protocol 
1. Rationale 
Early reperfusion therapy following acute myocardial infarction (AMI) is crucial for salvaging 
ischemic myocardium. However, concomitant myocardial ischemia-reperfusion (IR) injury 
significantly compromises clinical outcomes [1,2]. Studies indicate that the pathological core of 
myocardial IR injury lies in a self-amplifying interplay between oxidative stress and 
inflammation: mitochondrial fission-fusion imbalance triggers explosive release of reactive 
oxygen species (ROS), while aberrant activation of myeloid immune cells (e.g., 
monocytes/macrophages) synergistically propagates cardiomyocyte oxidative damage and pro-
inflammatory milieu [3,4]. The hyperinflammatory response elicited by myocardial IR injury is 
strongly associated with left ventricular remodeling and adverse cardiovascular outcomes [5–9]. 
Recent clinical trials validate anti-inflammatory interventions: the CANTOS trial demonstrated 
that IL-1β neutralization reduces major adverse cardiovascular events (MACEs) by 15–17%, and 
low-dose colchicine achieves 23% MACE risk reduction via NLRP3 inflammasome inhibition 
[10–12]. 
During early IR injury, monocytes infiltrating the infarct zone primarily derive from the spleen 
and bone marrow [13,14]. These infiltrating monocytes differentiate into monocyte-derived 
macrophages, which act as pivotal mediators of myocardial injury progression through secretion 
of IL-1α/β and other pro-inflammatory cytokines. Consequently, targeted modulation of 
inflammatory polarization and oxidative stress in infiltrating monocytes may represent a novel 
therapeutic avenue for mitigating myocardial IR injury. Nevertheless, current anti-inflammatory 
strategies face substantial challenges, notably limited target specificity and safety concerns. 
Emerging research reveals expanding roles of olfactory receptors (ORs) in non-olfactory 
pathologies. OR6A2—a specific receptor for octanal—activates G protein-coupled signaling to 
promote ROS generation and cytokine release in macrophages, thereby driving atherosclerotic 
plaque progression [15]. However, the precise molecular mechanisms linking octanal-OR6A2 
signaling to oxidative stress activation, along with its clinical implications in myocardial IR 
injury, remain elusive. 
Existing evidence identifies mitochondrial dynamics dysregulation as central to ROS burst [16–

 
3
19]. Our preliminary work demonstrates that octanal stimulation triggers mitochondrial 
homeostasis dysregulation in monocytes via OR6A2 activation, manifesting as aberrant fission, 
membrane depolarization, and pathological accumulation of mitochondrial ROS (mtROS) [20–
22]. Excessive mtROS and mitochondrial fragments cooperatively act as "Signal 2" to activate 
the NLRP3 inflammasome, initiating IL-1α/β secretion. These findings implicate OR6A2 as a 
critical molecular nexus connecting immunometabolic dysregulation to myocardial IR injury, 
though clinical validation and translational potential warrant urgent exploration. 
This clinical cohort study addresses three pivotal questions: (1) Whether plasma octanal levels 
and monocyte OR6A2 expression demonstrate specific alterations in myocardial IR injury 
patients; (2) Whether these biomarkers correlate with oxidative stress, inflammatory phenotypes, 
and MACEs risk; (3) Whether OR6A2 expression patterns across monocyte subsets (classical 
CD14⁺CD16⁻, intermediate CD14⁺CD16⁺, non-classical CD14⁻CD16⁺) hold prognostic value. 
Integrating multi-omics profiling with long-term follow-up data, this research will systematically 
characterize clinicopathological features of octanal-OR6A2 signaling in myocardial IR injury, 
advancing novel biomarker discovery and precision-targeted therapeutic strategies. 
References: 
[1] IBANEZ B, JAMES S, AGEWALL S, et al. 2017 ESC Guidelines for the management of 
acute myocardial infarction in patients presenting with ST-segment elevation: The Task Force 
for the management of acute myocardial infarction in patients presenting with ST-segment 
elevation of the European Society of Cardiology (ESC) [J]. Eur Heart J, 2018, 39(2): 119-77. 
[2] FRANK A, BONNEY M, BONNEY S, et al. Myocardial ischemia reperfusion injury: from 
basic science to clinical bedside [J]. Semin Cardiothorac Vasc Anesth, 2012, 16(3): 123-32. 
[3] YANPISET P, MANEECHOTE C, SRIWICHAIIN S, et al. Gasdermin D-mediated 
pyroptosis in myocardial ischemia and reperfusion injury: Cumulative evidence for future 
cardioprotective strategies [J]. Acta Pharm Sin B, 2023, 13(1): 29-53. 
[4] CHEN L, SHI D, GUO M. The roles of PKC-delta and PKC-epsilon in myocardial 
ischemia/reperfusion injury [J]. Pharmacol Res, 2021, 170: 105716. 
[5] DUTTA P, COURTIES G, WEI Y, et al. Myocardial infarction accelerates atherosclerosis 
[J]. Nature, 2012, 487(7407): 325-9. 
[6] DUTTA P, SAGER H B, STENGEL K R, et al. Myocardial Infarction Activates CCR2(+) 

 
4
Hematopoietic Stem and Progenitor Cells [J]. Cell Stem Cell, 2015, 16(5): 477-87. 
[7] FRANTZ S, NAHRENDORF M. Cardiac macrophages and their role in ischaemic heart 
disease [J]. Cardiovasc Res, 2014, 102(2): 240-8. 
[8] LEUSCHNER F, RAUCH P J, UENO T, et al. Rapid monocyte kinetics in acute myocardial 
infarction are sustained by extramedullary monocytopoiesis [J]. J Exp Med, 2012, 209(1): 123-
37. 
[9] SHI C, PAMER E G. Monocyte recruitment during infection and inflammation [J]. Nat Rev 
Immunol, 2011, 11(11): 762-74. 
[10] RIDKER P M, EVERETT B M, THUREN T, et al. Antiinflammatory Therapy with 
Canakinumab for Atherosclerotic Disease [J]. N Engl J Med, 2017, 377(12): 1119-31. 
[11] RIDKER P M, DEVALARAJA M, BAERES F M M, et al. IL-6 inhibition with ziltivekimab 
in patients at high atherosclerotic risk (RESCUE): a double-blind, randomised, placebo-
controlled, phase 2 trial [J]. Lancet, 2021, 397(10289): 2060-9. 
[12] NELSON K, FUSTER V, RIDKER P M. Low-Dose Colchicine for Secondary Prevention 
of Coronary Artery Disease: JACC Review Topic of the Week [J]. J Am Coll Cardiol, 2023, 82(7): 
648-60. 
[13] PRABHU S D, FRANGOGIANNIS N G. The Biological Basis for Cardiac Repair After 
Myocardial Infarction: From Inflammation to Fibrosis [J]. Circ Res, 2016, 119(1): 91-112. 
[14] MARINKOVIC G, KOENIS D S, DE CAMP L, et al. S100A9 Links Inflammation and 
Repair in Myocardial Infarction [J]. Circ Res, 2020, 127(5): 664-76. 
[15] ORECCHIONI M, KOBIYAMA K, WINKELS H, et al. Olfactory receptor 2 in vascular 
macrophages drives atherosclerosis by NLRP3-dependent IL-1 production [J]. Science, 2022, 
375(6577): 214-21. 
[16] JIN Q, LI R, HU N, et al. DUSP1 alleviates cardiac ischemia/reperfusion injury by 
suppressing the Mff-required mitochondrial fission and Bnip3-related mitophagy via the JNK 
pathways [J]. Redox Biol, 2018, 14: 576-87. 
[17] CAI C, GUO Z, CHANG X, et al. Empagliflozin attenuates cardiac microvascular 
ischemia/reperfusion through activating the AMPKalpha1/ULK1/FUNDC1/mitophagy pathway 
[J]. Redox Biol, 2022, 52: 102288. 
[18] ZOU R, SHI W, QIU J, et al. Empagliflozin attenuates cardiac microvascular 
ischemia/reperfusion injury through improving mitochondrial homeostasis [J]. Cardiovasc 
Diabetol, 2022, 21(1): 106. 

 
5
[19] NIU X, ZHANG J, HU S, et al. lncRNA Oip5-as1 inhibits excessive mitochondrial fission 
in myocardial ischemia/reperfusion injury by modulating DRP1 phosphorylation [J]. Cell Mol 
Biol Lett, 2024, 29(1): 72. 
[20] SWANSON K V, DENG M, TING J P. The NLRP3 inflammasome: molecular activation 
and regulation to therapeutics [J]. Nat Rev Immunol, 2019, 19(8): 477-89. 
[21] TSUCHIYA K, HOSOJIMA S, HARA H, et al. Gasdermin D mediates the maturation and 
release of IL-1alpha downstream of inflammasomes [J]. Cell Rep, 2021, 34(12): 108887. 
[22] YARON J R, GANGARAJU S, RAO M Y, et al. K(+) regulates Ca(2+) to drive 
inflammasome signaling: dynamic visualization of ion flux in live cells [J]. Cell Death Dis, 2015, 
6(10): e1954. 
2. Research Content 
This project investigates the "Association of OR6A2 Expression on Monocytes with 
Inflammation and Major Adverse Cardiovascular Events in Myocardial Ischemia-Reperfusion 
Injury." 
2.1. Study Objectives 
Primary Aim: Elucidate the clinical relevance of octanal-OR6A2 signaling in myocardial IR 
injury and its prognostic value for MACEs. 
Specific Objectives: 
(1) Characterize pathobiomarker profiles in IR injury: 
• 
Plasma octanal dynamics (LC/MS/MS) 
• 
OR6A2 expression kinetics on monocyte subsets: 
o Classical (CD14⁺CD16⁻) 
o Intermediate (CD14⁺CD16⁺) 
o Non-classical (CD14⁻CD16⁺) 
• 
Correlations with oxidative stress (mtROS, MDA, H₂O₂) and inflammation (IL-1α/β, 
CRP) 
(2) Define prognostic utility: 
• 
Association of baseline OR6A2⁺ monocyte subsets with 44-month MACEs 
• 
Predictive value of plasma octanal levels for 44-month MACEs 
(3) Validate translational potential: 

 
6
• 
Develop risk-stratification models integrating OR6A2 biomarkers 
2.2 Research Design 
(1) Study Site 
Department of Cardiology, Zhongda Hospital, Southeast University 
(2) Study Population 
• 
200 AMI patients (STEMI/NSTEMI) with confirmed myocardial IR injury 
• 
Inclusion: Meeting myocardial IR diagnostic criteria (multimodal assessment) 
• 
Exclusion: active systemic infections, advanced heart failure (NYHA III-IV), acute 
cerebrovascular events, myocarditis, cardiomyopathy, refractory ventricular tachycardia, 
malignancies diagnosed within the preceding 5 years, severe renal insufficiency 
(estimated glomerular filtration rate [eGFR] <30 mL/min/1.73m2 or dialysis dependence), 
and Child-Pugh class C hepatic dysfunction. 
(3) Diagnostic criteria for myocardial ischemia-reperfusion injury 
Myocardial ischemia-reperfusion injury was diagnosed based on clinical evidence meeting both 
temporal and multimodal criteria. The diagnostic window encompassed clinical manifestations 
or objective findings within 24 hours following percutaneous coronary intervention (PCI), 
extendable to 48 hours if symptoms persisted. 
• 
Diagnostic Requirements (≥2 criteria): 
o Symptomatic criteria: Sudden recurrent chest pain post-procedure (excluding in-stent 
thrombosis or re-occlusion); 
o Electrocardiographic evidence: Delayed ST-segment resolution or recurrent ST 
elevation (>1 mm in at least two contiguous leads); New-onset ventricular 
arrhythmias (e.g., frequent premature ventricular complexes, ventricular tachycardia, 
ventricular flutter, ventricular fibrillation accelerated idioventricular rhythm); New-
onset 
bradyarrhythmias 
(e.g., 
sinus 
bradycardia, 
atrioventricular 
block, 
intraventricular conduction delays). 
o Imaging abnormalities: New myocardial edema (manifested by high-signal intensity 
on T2-weighted magnetic resonance imaging [MRI]); Lack of improvement in 
regional wall motion abnormalities; Evidence of microvascular obstruction (e.g., 
perfusion defect on myocardial contrast echocardiography or late gadolinium 
enhancement on MRI). 

 
7
o Biomarker kinetics: Abnormal elevation of cardiac troponin (cTnI/cTnT) or CK-MB 
within 6 hours post-procedure, excluding procedural re-infarction. 
• 
Coronary Flow Validation: 
o Confirmation of successful coronary flow restoration (TIMI flow grade ≥2) to 
exclude ischemia from technical failure or residual stenosis; 
o Post-procedural confirmation of stent patency through coronary angiography or 
computed tomography angiography (CTA) is required to exclude acute thrombosis or 
re-occlusion. 
• 
Exclusion Criteria: 
o Non-ischemic myocardial injury etiologies (e.g., Takotsubo cardiomyopathy, 
myocarditis, toxic/metabolic insults) were systematically excluded. 
(4) Sample Size 
• 
Myocardial IR cohort: n=200 (longitudinal follow-up) 
• 
Power analysis: 80% power (α=0.05) for HR≥2.5 between OR6A2 expression strata 
(5) Recruitment & Group Assignment 
• 
Consecutive enrollment of eligible patients (September 1st, 2019-August 30th, 2027) 
• 
Non-interventional cohort design (observational) 
(6) Ethical Considerations 
• 
Approved by Zhongda Hospital IEC (2020ZDSYLL051-P01) 
• 
Written informed consent obtained pre-enrollment 
2.3 Methodology 
(1) Biosample Collection & Analysis Timeline 
Timepoint 
Assessments 
T0 (Baseline) 
Plasma octanal (LC/MS/MS) 
Flow cytometry: Monocyte subsets + OR6A2 MFI 
Serum: IL-1α/β, MDA, H₂O₂, CRP 
T1 (24h post-PCI) 
Repeat flow cytometry 
Caspase-9/mPTP activity assays 
T2 (Month 3,6,12...44) 
MACEs surveillance 

 
8
(2) Core Techniques 
Method 
Target 
Instrument/Kit 
Flow Cytometry 
OR6A2 surface expression 
(CD14/CD16 subsets) 
Attune™ NxT (Thermo) 
LC/MS/MS 
Plasma octanal 
d16-octanal isotope standard 
Western Blot 
Mitochondrial OR6A2 
Invitrogen PA5-102216 
Multiplex ELISA 
IL-1α/β, oxidative stress markers 
Cusabio/Beyotime kits 
(3) Primary endpoint 
• 
MACEs (composite of AMI, cardiac death, stroke, hospitalization for unstable angina, 
or unplanned coronary revascularization) 
(4) Statistical Analysis 
Data Presentation & Normality Assessment: 
• 
Continuous variables are reported as mean ± standard deviation (SD) for normally 
distributed data or median with interquartile range (IQR) for non-normally distributed 
data. Normality was evaluated using the Shapiro-Wilk test. 
• 
Categorical variables are presented as frequencies and percentages. 
 Hypothesis Testing: 
• 
Normally Distributed Data: Parametric tests were used: Student's t-test (two groups) or 
one-way ANOVA with Tukey's post hoc test (≥3 groups). 
• 
Non-Normally Distributed Data: Non-parametric tests were applied: Wilcoxon rank-
sum test (two independent groups) or Kruskal-Wallis H test with Dunn's post hoc 
correction (≥3 groups). 
• 
Categorical Variables: Analyzed using the χ² test or Fisher's exact test (sparse data). 
• 
Correlations: Assessed using Spearman correlation analysis. 
• 
Time-to-Event Data: Survival curves generated via Kaplan-Meier estimation. 
Primary Exposure Analysis (OR6A2 & Octanal): 
• 
OR6A2 median fluorescence intensity (MFI) was analyzed both as a continuous 
variable (per 1000-unit increment) and categorically (dichotomized at the median). 
• 
Plasma octanal concentration was similarly evaluated as a continuous metric and 

 
9
categorically (median-dichotomized). 
• 
Association with MACEs: Quantified using multivariable Cox proportional hazards 
regression, yielding adjusted hazard ratios (HRadj) and 95% confidence intervals (CIs). 
Three sequential adjustment models were employed: 
o Model 1: Age, gender. 
o Model 2: Model 1 + body weight, smoking status, hypertension, diabetes 
mellitus. 
o Model 3: Model 2 + lipid-lowering therapy (with/without PCSK9 inhibitors), 
eGFR, white blood cell count, C-reactive protein (CRP), Creatine Kinase-
Myocardial Band (CK-MB), onset-to-reperfusion time, echocardiographic 
parameters (ejection fraction, interventricular septum thickness, left ventricular 
internal diameter, left ventricular posterior wall thickness), discharge 
medications (aspirin, P2Y12 inhibitors, statins, β-blockers, ACEI/ARB/ARNI, 
aldosterone receptor antagonists). 
• 
Non-Linear Association (Octanal): The relationship between continuous plasma octanal 
levels and MACEs was further examined using multivariable Cox regression with 
restricted cubic spline functions, fully adjusted for Model 3 covariates, allowing 
flexible assessment of potential non-linearity. 
Follow-up & Software: 
• 
Complete 44-month follow-up data were obtained for all participants with no loss to 
follow-up. 
• 
Analyses were performed using SPSS (v25.0), GraphPad Prism (v8.0), and R software 
(v4.2.3). 
Significance: 
• 
Statistical significance was defined as a two-sided p-value < 0.05. 
• 
Significance levels denoted as: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 
2.4 Feasibility & Innovation 
Strengths: 
• 
Novelty: First clinical validation of olfactory receptor (OR6A2) in IR injury prognosis 
• 
Methodological rigor: 
o Dual biomarkers (octanal + cell-type-specific OR6A2) 

 
10
o Advanced statistics (spline-adjusted dose-response models) 
• 
Resources: 
o Hospital’s accredited flow cytometry core (ISO-15189) 
o Established biorepository with 90% follow-up rate 
2.5 Expected Outcomes 
(1) Clinical validation of OR6A2 as a prognostic biomarker for IR injury 
(2) Risk stratification algorithm integrating monocyte subset profiling 
(3) ≥3 high-impact publications (European Heart Journal/Circulation) 
(4) Patent application for OR6A2-targeted diagnostic kits