HDL 3B and 3C reduce ischemia and reperfusion injury and preserve the systolic function of the left ventricle after myocardial infarction by a mechanism dependent on nitric oxide

Author(s)

Vitor Wilson de Moura Virginio

• Document type: Doctoral Thesis
• Press: Campinas, SP.
• Institution: Universidade Estadual de Campinas (UNICAMP). Faculdade de Ciências Médicas
• Defense date: 2019-09-11

Examining board members

Andrei Carvalho Sposito; Licio Augusto Velloso; Fabiana Hanna Rached; Marisa Passarelli; Rodrigo Bueno de Oliveira

• Advisor: Andrei Carvalho Sposito

Abstract

The pandemic of ischemic heart disease mainly manifested in the form of myocardial infarction (MI) is associated with a high mortality and morbidity rate in the world population. During MI, even with rapid intervention to restore coronary flow, about half the size of the remaining lesion is due to ischemia and reperfusion (I/R) injury. Several therapeutic strategies have been studied to reduce the damage caused by I/R injury, however, no effective therapy has been demonstrated. Based on the cardioprotective effects of high-density lipoprotein (HDL) particles such as anti-inflammatory, antioxidant, antiapoptotic activity, as well as its important regulation on vascular homeostasis, make HDL a potential candidate for attenuation I/R injury. HDL particles can be divided into five subclasses: 2B and 2A (larger and rich in lipids), 3A, 3B and 3C (smaller and rich in proteins). However, it is not clear until now which HDL subclass would yield the most benefits in the I/R injury. The aim of this study was to evaluate which HDL subclass could result in greater protection after MI, in relation to infarct size, left ventricular (LV) systolic function, and coronary flow. Similarly, we sought to evaluate the role of nitric oxide (NO) synthesis, as well as to explore the cell pathways directly involved in the protection triggered by HDL/NO and mitochondrial protection. The present hypothesis was verified with total and subclass HDL samples isolated from 15 healthy volunteers (26 ± 5 years old). HDL was tested in an I/R model in hearts isolated from Wistar rats perfused in the Langendorff system. The hearts were submitted to 35 minutes of ischemia and 90 minutes of regional myocardial reperfusion. The HDL was infused at very first minutes of reperfusion. The same approach was tested in vitro separately on endothelial and cardiomyocytes cells in the hypoxia-reoxygenation scenario. NO biosynthesis was evaluated ex vivo and in vitro by chemiluminescence, and cardioprotection pathways were evidenced by immunoblotting. Mitochondrial respiration was characterized by the rate of oxygen uptake Our results demonstrated that total HDL was able to decrease -24% (p<0.01) infarct size and improve systolic function during reperfusion. We found that the smallest HDL, subclasses 3C and 3B, were more efficient in reducing infarct size (-30%, p<0.001; -25%, p<0.01, respectively) and increasing viability of cardiomyocytes (three times; p=0.023) in the hypoxia-reoxygenation scenario. HDL 3C and 3B also improved LV systolic function during reperfusion and reduced coronary resistance compared to control and other subclasses. In addition, we have shown that NO plays a key role in cardioprotection after ischemia and that the HDL 3C and 3B were the most effective phenotypes in stimulating increased NO bioavailability in coronary effluent (240%, p<0.0001 and 204%, p<0.0001), in the extracellular environment of human coronary endothelial cells (330%, p<0.0001) and cardiomyocytes (390%, p<0.0001). In endothelial cells, this NO increase by subclasses 3C and 3B were via S1P1 and S1P3 receptors in order of magnitude, confirmed by eNOS activation. Also, in myocardial tissue submitted to I/R and cardiomyocytes in the hypoxia-reoxygenation scenario, the HDL 3B and 3C pool activated the Akt/GSK3? cardioprotective pathway involved in mitochondrial protection (109%, p=0.038 and 90%, p<0.0001). Finally, we demonstrate that the HDL 3B and 3C pool increase the mitochondrial respiratory function at state 3 (60%, p<0.01), maximal respiration (55%, p<0.001) and that NO synthesis is required for such a benefit. Our study concluded that HDL 3C and 3B phenotypes were able to attenuate I/R injury by reducing infarct size and improving hemodynamic function. Part of these benefits were due to: (i) the ability to stimulate NO bioavailability in coronary effluent, endothelial cells and cardiomyocytes; (ii) activation of the Akt/GSK3? cardioprotection pathway responsible for the reduction of cell death; (iii) and finally, by increased mitochondrial respiration function (AU)

• FAPESP's process: 14/26136-9 - Investigation of the myocardial infarction impact regarding high-density lipoprotein effect on endothelial nitric oxide and endothelin-1 production
• Grantee: Vitor Wilson de Moura Virginio
• Support Opportunities: Scholarships in Brazil - Doctorate