title: The Role of Inducible Nitric Oxide Synthase (iNOS) in Human Myocardial Infarction
Creators: name: Wilmes, Verena
Publisher: name: UNSPECIFIED
Year: 2023

description: Cardiovascular diseases (CVDs), which include disorders of the heart and its blood vessels, such as myocardial infarction (MI), are the leading cause of death globally. During MI necrosis of myocardial cells leads to inflammation and fibrosis. Cardiac scarring and subsequently adverse remodeling, negatively impact myocardial healing and may lead to further disease states after MI, such as hypertension, heart failure, and death. While elevated levels of cardiac-specific troponins (cTnT and cTnI), as well as cardiac specific creatine kinase (CK-MB) are central biomarkers for the diagnosis of MI, interleukins, natriuretic peptides, matrix metallopeptidases (MMPs) and noncoding RNAs are currently discussed as biomarkers for adverse cardiac remodeling after MI. Nevertheless, a thorough understanding of the molecular processes during MI may lead to further biomarkers or improved therapy options. Inducible nitric oxide synthase (iNOS) is one of the three NOS isoforms that produce nitric oxide (NO). It is induced by stimuli such as inflammation and hypoxia and has been described in patients with end-stage heart failure, cardiomyopathy and ischemic heart disease. In murine and rabbit models of MI increased iNOS expression is suggested to contribute to left ventricular dysfunction, heart failure progression, myocardial injury and extent of infarct size, even late after ischemia and reperfusion. These detrimental effects are due to the reaction of iNOS derived NO with superoxide anion (O2-), which leads to reactive intermediates that can nitrate, nitrosate or oxidize their biological environment. This oxidative nitrosative stress promotes DNA damage, suppresion of DNA-repair enzymes and post-translational modifications of proteins. Specifically, peroxynitrite (ONOO-) is formed by the reaction of NO with O2- and is a strong oxidant causing nitrosylation of proteins and the formation of nitrotyrosine. Further peroxynitrite inactivates anti-proteases and activates MMPs, that are known for degrading and remodeling extracellular matrix under physiological and pathological conditions, leading to left ventricular remodeling after MI. Hence, understanding the role of iNOS in MI may lead to improved therapy options and to the development of biomarkers for the progression of heart failure after MI. In the present thesis the role of iNOS in human MI is thoroughly examined for the first time. It includes genetic and cellular approaches to determine the iNOS expression and factors influencing iNOS expression in human MI tissue. Further, methods of measuring iNOS derived oxidative stress are established. Especially, the present work emphasizes the differences in iNOS expression between animal models of MI and human MI. The first study focused on examining iNOS mRNA and protein levels in postmortem human MI hearts. A significant increase of iNOS mRNA in infarcted and non-infarcted tissue was detected, in comparison to healthy controls. Further, significantly increased iNOS protein levels were found in infarcted and non-infarcted regions. Interestingly, iNOS was predominantly found in “M2” resident macrophages and to a smaller amount in “M1” inflammatory macrophages. Accordingly, the significant increase in iNOS protein in infarcted regions was seen in the “M2” resident macrophages. In cardiomyocytes no iNOS protein expression was detected. A distance analysis between iNOS+ and nitrotyrosine (NT)+ cells revealed that NT+ cells peak within 10-15 µm of iNOS, suggesting a dependence and a range of activity by iNOS produced NO. The second study examined microRNAs as posttranscriptional regulators of the iNOS expression. The expression of miR-939, miR-21 and miR-30e was measured and correlated with iNOS mRNA and protein levels in postmortem human MI hearts. miR-939 is known to decrease cytokine induced iNOS protein expression and NO synthesis in human hepatocytes. miR-21 has its strongest expression in cardiac macrophages in mice, where it is the strongest expressed miRNA among all cardiac miRNAs. The function of miR-21 in fibroblasts comprises of regulating fibroblast proliferation and fibrosis. miR-30e is described as downregulated in hypoxic cardiomyocytes and MI tissue of rats to protect the myocardium via enhancing cardiomyocyte viability and inhibiting apoptosis. The results revealed significant upregulation of miR-939, miR-21 and miR-30e in infarcted and non-infarcted regions of postmortem human MI hearts in comparison to healthy controls. Correlation analysis showed a significant correlation between miR-939 expression and the iNOS mRNA expression in the control group and the infarcted regions, which was more pronounced in the controls. A massive iNOS activation might exceed the capability of miR-939 to keep its expression in balance. On the other hand, miR-30e and miR-21 did not seem to influence cardiac iNOS levels in MI or macrophage polarization. In the third study, the iNOS gene NOS2 was genotyped for the (CCTTT)n repeat polymorphism in the promoter region, the rs2779249, G&gt;T single nucleotid polymorphism (SNP) -1026 basepairs (bp) before transcription start and the rs2297518, C&gt;T SNP at exon 16. These polymorphisms are known to influence the iNOS expression and subsequently oxidative stress in atrial fibrilliation and hypertension amongst others. In the MI group we found an accumulation of NOS2 risk polymorphisms, such as long (CCTTT)n repeat polymorphisms or the T alleles at rs2779249, G&gt;T and rs2297518, C&gt;T in contrast to healthy controls. Additionally, we detected an increase in iNOS protein levels, in the control and MI group, when NOS2 risk polymorphisms were present. Serum levels of malondialdehyde (MDA), a marker of oxidative stress, of creatinine, an independent risk factor for and after MI and of nitrate/nitrite (NOx), which are known the reflect the activity of iNOS, were increased in the MI group in comparison to the controls. Further, in the MI group homozygous carriers of the long (CCTTT)n repeat had increased serum MDA levels in comparison to heterozygote carriers or carriers of short (CCTTT)n repeats. Homozygous carriers of the T allele at rs2779249, G&gt;T in the MI group showed increased serum levels of nitrate and creatinine. This suggests a connection between serum markers and NOS2 risk polymorphism in MI, however, no direct influence of risk polymorphisms on cardiac iNOS levels was observed. In conclusion the present thesis revealed increased iNOS mRNA expression in postmortem human infarction hearts and increased iNOS protein production. Interestingly, iNOS is predominantly expressed in “M2”-resident macrophages and upon MI, the iNOS production is even more increased in these macrophages. Since NT+ cells peak within 10-15µm of iNOS+ cells, this could represent an activity range of iNOS and subsequent oxidative stress production within that perimeter. miR-939 seems to regulate the iNOS expression posttranscriptional, however, upon MI this regulation is not efficiently to keep the iNOS mRNA level at bay. Risk polymorphisms in the NOS2 gene accumulate in the MI group and seem to enhance iNOS protein expression. This may lead to increased oxidative stress and adverse cardiac remodeling after MI. The seen increased serum levels of MDA, creatinine and NOx in the MI group could not be correlated with cardiac iNOS mRNA and protein levels. However, long (CCTTT)n repeats and the homozygous T-allele at rs2779249, G&gt;T seem to influence serum MDA, creatinine and nitrate level in the MI group. The role of iNOS in MI seems complex, however we could reveal increased oxidative stress upon MI, by increased iNOS production. Hence, increased iNOS expression and activity due to certain risk polymorphisms may impair the healing process after MI, lead to adverse remodeling and further heart diseases such as heart failure.