Abstract Body (Do not enter title and authors here): Background: Myocardial ischemia-reperfusion injury (IRI) arises by abrupt myocardial blood flow restoration after ischemia leading to cardiomyocyte dysfunction and death. Mechanisms of myocardial IRI involve an interplay of metabolic dysfunction, including branched-chain amino acid (BCAAs) imbalance, and mitochondrial dysfunction, resulting in oxidative stress, inflammation, and cardiomyocyte death. Our lab was the first to demonstrate the cardioprotective effects of intralipid (ILP), a safe lipid emulsion, against myocardial IRI in rodents; However, underlying mechanisms remain unclear.
Research Hypothesis: We hypothesize that ILP protects IRI by restoring the depletion of BCAAs, thereby attenuating inflammation, oxidative stress, and apoptosis.
Methods: Male rats were subjected to sham or IRI by LAD ligation for 30 min followed by 180 min of reperfusion. The IRI group received either a single bolus of ILP (20%, 5ml/kg body weight) or saline at the onset of reperfusion. RNA seq and LC Mass spectrometry were performed on left ventricle (LV). In vitro, H9C2 cardiac myoblasts were exposed to 3h of hypoxia followed by 6h of reoxygenation, with or without 20% ILP during reoxygenation. Seahorse assessed H9C2 mitochondrial function and RT-PCR quantified inflammatory and apoptotic markers expression.
Results: LV transcriptomic analysis revealed that IRI significantly downregulated oxidative phosphorylation and fatty acid oxidation, while upregulating glycolysis, hypoxia, inflammation, and apoptosis pathways compared to sham. LV from ILP-treated rats exhibited similar pathway dynamics to levels observed in sham rats. In vitro, ILP administration during reoxygenation significantly enhanced ATP production and oxygen consumption in H9C2 cells compared to controls. Also, ILP-treated H9C2 cells exhibited upregulated expression of respective anti-apoptotic and antioxidant markers Bcl-2 and Sod2, downregulated expression of pro-apoptotic marker Bax, and pro-inflammatory TNA-α. Metabolomic profiling revealed that BCAAs including valine, leucine, and isoleucine were among the downregulated LV metabolites by IRI, but upregulated by ILP treatment upon IRI, with significant changes observed specifically for valine.
Conclusion(s): Together, our data suggest that ILP administration at the onset of myocardial reperfusion preserves reduced BCAAs levels caused by IRI and improves mitochondrial function, leading to attenuated cardiomyocyte inflammation, apoptosis and oxidative stress.