Autophagy-Related Protein Expression Paper Example

Abstract

The chronic ischemic heart disease, IHD, is a heart disorder caused by a cholesterol blockage hindering the flow of blood, oxygen and nutrients into the blocked tissues. It is a common disease affecting Americans and individuals all over the world. The IHD alters the homeostasis of the body by triggering an upregulation of autophagy. To clearly demonstrate this, the research utilizes a number of tests, among them the Western Blot, and PicroSirius staining and the findings presented via a PET imaging. The statistical analysis uses the ANOVA analytical design to draw a correlation and variation among the various human heart tissues under observation. The PET display reveals four images, that of the normal, slightly reduced, highly reduced and absent FDG uptake. The graphs on the mitochondrial-related and autophagy-related protein expression show equal protein express across the four tissue images. The tissues with normal and slightly reduced FDG uptake have less dense collagen, while those with highly reduced and absent FDG have dense collagen as revealed after the Picrosirius staining. These differences present the conditions that cause ischemic heart disease.

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Keywords: ischemic heart disease, autophagy, mitochondria, collagen, PET imaging, western blot analysis, picrosirius staining, formalin-fixed paraffin embedded (FPE) human tissue.

Preface

The work presented in this bachelor project has been carried out in Research Laboratory for Biochemical Pathology at Aarhus University Hospital from January 2018 to August 2018, under the supervision of Postdoctoral researcher Andreas Buch Moller and Professor Niels Jessen. In this project, the analysis focuses on the autophagy-related protein expression and fibrosis in human hearts from transplantation recipients.

Chronic ischemic heart disease (IHD) is the most common cause of heart failure in the Western world. Heart failure (HF), also known as congestive heart failure, is a leading cause of death worldwide. HF means that the heart isn't pumping blood throughout the body as efficiently as it should. The latest statistics carried out by American Heart Association's 2017 Heart Disease and Stroke Statistics Update, show that the number of people living with heart failure in the United States has increased from 5.7 million, in 2009-2012, to about 6.5 million, in 2011-2014. It is estimated that this number will increase by 46 per cent by 2030, which means that more than 8 million people will be diagnosed with heart failure. It would be desirable to lessen the number of adults diagnosed with HF.

It would, therefore, be of interest to investigate the impact of IHD on a human heart. In this project, I have used Western Blot, and PicroSirius staining to examine how mitochondria and autophagy-related protein expression and fibrosis is regulated in IHD.

During the researcher's work in Research Laboratory for Biochemical Pathology, the acquired practical experience with different experimental methods were used to address molecular and morphological questions: the following questions have been answered:

1. Is fibrosis in ischemic areas of the heart increased in patients with IHD?

2. How is mitochondria-related protein expression in ischemic regions in heart affected in patients with IHD?

3. How is the autophagy-related protein expression in ischemic regions of the heart affected in patients with IHD?

Hypothesis

The researcher hypothesized that areas with slightly and highly reduced glucose uptake are characterized by decreased expression of mitochondrial proteins and accumulation of fibrosis, and that this is associated with altered expression of autophagy related protein.

Introduction

Heart failure

The heart is a muscular organ located in the inferior mediastinum which is responsible for pumping of blood throughout the body via the blood vessels by repeated, rhythmic contractions.The heart consists of three layers: The epicardium, the myocardium, and the endocardium. The myocardium, which is composed of cardiac muscle, is covered by a layer of connecting tissue, the endocardium, on the inside. On the outside the myocardium is covered by the epicardium. The epicardium is visceral layer of the pericardium, which surrounds the heart in the mediastinum.

The right atrium receives deoxygenated systemic venous return from the inferior and superior vena cava. The left atrium receives oxygenated blood from the lungs through the pulmonary circulation. Both atria operate as passive reservoirs more than as mechanical pumps. The blood flows from the atria to the ventricles through the atrioventricular valves. From the right ventricle, the deoxygenated blood is pumped to the pulmonary circulation through the pulmonary valve, and from the left ventricle the oxygenated blood is pumped to the systemic circulation through the aortic valve. The cardiac cycle consists of two phases, the systole, where the ventricles contract, and the diastole, where the ventricles relax.

HF is a condition triggered when the heart's ability to pump blood is insufficient. HF does not mean that the heart has stopped or is about to stop working. Basically, it means that the heart cannot keep up with its own workload. To compensate for this the heart enlarges by stretching and the muscle mass of the heart increases as the contractile cells of the heart gets bigger. This increases the overall output of the heart. HF can result in swelling (edema): swelling in legs, ankles and other parts of the body (MFMER 2018). HF can also affect the kidneys and their ability to dispose sodium and water, and this can also lead to swelling.

Generally, it is the left ventricle, the main pumping chamber of the heart that is involved in heart failure but in some cases, both the left ventricle and the ventricle could be affected.

Chronic Ischemic heart disease (IHD)IHD is caused by oxygen deficiency in the heart muscle. The deficiency is caused by progressive building up of plaque, cholesterol and adipose inside of the coronary arteries (this process is also known as atherosclerosis), resulting in less oxygen being delivered to heart muscle. The ischemic areas of the human heart are characterized by different morphological changes: accumulation of fibrous tissue, and dysfunctional mitochondria. Several studies also suggest that autophagy is down regulated: a process that degrade and recycle cellular components

MitochondriaMitochondria are fundamental for normal cell physiology and survival. This is especially important for cells with a demand of high energy, as the hearts myocytes. The mitochondria make up approximately 30 % of the total cell volume in the heart and generate enormous number of ATP through oxidative phosphorylation, to maintain the hearts contractile function. Mitochondria are also the primary source of reactive oxygen species (ROS). ROS contributes to mitochondrial dysfunction, cardiomyocytes death and heart failure (Tong & Sadoshima 2016). To protect against this, mitochondrion has evolved well-coordinated control mechanism, such as mitochondrial autophagy (mitophagy).

Several studies have shown that in ischemic heart tissue the capacity of oxidative phosphorylation of mitochondria is decreased which has an impact on the energetic state of the heart. This project will examine different mitochondrial proteins.

Fibroses and collagen synthesis in patients with heart failureA study performed in 2004, showed that myocardial fibrosis and collagen type I synthesis and deposition is increased in the myocardium of the heart muscle. The study also proposed that increased myocardial fibrosis and collagen synthesis contributes to the development of HF. For patients with hypertension, an excess of myocardial collagen results from the uncoupling between increased syntheses and decreased or unchanged degradation of type I collagen fibres. .

Autophagy

Autophagy is also called, a "self-eating" process. It plays a vital role under oxidative stress, reduced supply of nutrients and hypoxia, which is also characterized by ischemic areas of the heart. Dysfunctional organelles such as mitochondria, endoplasmic reticulum (ER) and Golgi apparatus is degraded by autophagy. And reactive species such as ROS are prevented from releasing into the cells. Autophagy also induces apoptosis. There are at least three different types of autophagy described and possibly more: macro-autophagy (referred to as autophagy), micro-autophagy and chaperone mediated autophagy. The process macro-autophagy is described in this project.

The initial step of autophagy is formation of a double membrane that surrounds the degrading components in the cytoplasm and a phagophore is formed (see figure 1a). The double membrane can be generated by Golgi complex, endosomes, ER, mitochondria and the plasma membrane. Initiation process is mediated by ULK-complex which is inhibited by the protein mTOR.

The next step is nucleation, which is mediated by Beclin-1. The double membrane elongates and fuses at the edges to form a double-membrane vesicle, called autophagosome. Different protein mediates the elongation process. Hereafter the autophagosomes undergo a maturation process. An important protein in maturation of autophagosomes is LC3B. LC3B is found in the cytosol as LC3B-1. Under maturation of autophagosomes LC3B-1 binds to phosphatidylethanolamine to form LC3B-11 (LC3B-phosphatidylethanolamine conjugate). LC3B-II is then recruited to the autophagosomal membranes. LC3B-II correlates with the amount of autophagosomes in a tissue-sample.

Autophagosome fuses with lysosomes to form autolysosomes. Intra-autophagosomal components are degraded by lysosomal hydrolyses (Kang et al 2011). Several studies have suggested that autophagy may play an important role in ischemic heart. Different autophagy protein has been investigated in this project. Materials and MethodMaterials

Patients

The material used in this project is formalin-fixed paraffin embedded (FFPE) human heart tissue, obtained from 6 patients with IHD. All patients gave written approval to participate in the study. The principles of the Helsinki Declaration approve the study.

PET imaging of gl...