Reverse remodeling of the extracellular matrix in heart failure after left ventricular mechanical support

This thesis describes a study of the changes in the extracellular matrix (ECM) of the myocardium, in patients with end stage heart failure and during mechanical support (Left Ventricular Assist Device support, LVAD) of the left ventricle. The changes during LVAD support may lead to recovery of the heart. Chapter 1 is a general introduction about heart failure, heart transplantation, and the LVAD, The process of remodeling which leads to heart failure, and of reverse remodeling during LVAD support. A summary is given of the ECM components, and of the role of enzymes, hormones, and cytokines in the myocardial ECM changes in patients before and after LVAD support. Chapter 2 gives an overview of the role of different cytokine polymorphisms in transplant rejection. Cytokines influence each other's function and production. Single nucleotide polymorphism (SNP) in the genes of cytokines may influence the production level. These SNPs have been studied extensively in the context of acute rejection after organ transplantation. A variety of results have been obtained by many different groups. The main focus in most studies has been SNP genotype of the recipient. However, it is becoming increasingly clear that, in addition to the recipient's immune system, the microenvironment of the donor organ does also contribute to cytokine production, and may thereby influence rejection events. Chapter 3 concerns a study of TNF alpha promoter polymorphisms, and their possible contribution to the severity of heart failure and transplant rejection. TNF? plasma levels were measured before heart transplantation, in patients on medication, and in patients supported by a LVAD. TNF alpha plasma levels were high in patients with end stage heart failure on pharmaco-therapy, compared to healthy controls, but were increased in patients which required LVAD support before heart transplantation. This increase seems to correlate with the G allele at position -308. After LVAD support the TNF alpha levels were decreased. The A allele at position -308 (in which A is associated with relatively high levels of TNF alpha) does not correlate with the severity of heart failure and transplant rejection. However, patients having donor hearts with the A-308 polymorphism suffered more rejection episodes than patients receiving hearts with the G allele. Chapter 4 deals with the changes in the fibrillar collagen network in the ECM. The cardiomyocyte size appeared to decreases by 36% in the first 100 days after LVAD support. This was paralleled by an increase in ECM volume. However, the ECM volume decreased after prolonged LVAD support (>365 days). In patients with end-stage heart failure, the degradation of the fibrillar collagens was high before LVAD support, and their synthesis was low compared to the healthy controls. After LVAD support the opposite was observed: increased synthesis of the fibrillar collagens and decreased degradation. Furthermore, we showed that the quality of the fibrillar collagens increases, because more un-denatured (uncoiled) collagen was detected. These results suggest that after LVAD support the heart becomes smaller and the fibrillar collagen network in the ECM improves. Chapter 5 describes the changes in the basement membrane (BM) surrounding the cardiomyocytes. Using quantitative PCR (QPCR), no changes were observed in terms of collagen α-chain mRNA expression . However, immunohistochemistry did disclose a decreased staining pattern of type IV collagen after LVAD support. Using electron microscopy we showed that in healthy control hearts, and in patients with severe end-stage heart failure, the BM is thick and irregular, in contrast to patients after LVAD support. There were close connections between the ECM collagen fibers and the BM. Post-LVAD the BM became more regular and the lamina lucida and lamina densa were compacter and could be clearly discerned then in the healthy control. However, the connection between the BM and the collagen fibers in the ECM appeared to be disturbed. Furthermore, it was investigated whether matrix metalloproteinases (MMP) plays a role in degradation of the BM. MMP-2, which is able to degrade type IV collagen, was high in patients before LVAD support, but was increased even more in patients on LVAD support. Using gel zymography, active MMP expression was increased in these patients and was localized in the BM. This suggests that MMP-2 degrades type IV collagen in the BM during LVAD support. Chapter 6 focuses on the role of neuro-hormone brain natriuretic peptide (BNP) in the remodeling process. BNP is a member of the natriuretic peptide family and is mainly synthesized and secreted in the left cardiac ventricle. BNP is known to have natriuretic, diuretic and vasorelaxant properties,and have antagonistic effects on the renin-angiotensin-aldosteron system; it is increased in patients with end-stage heart failure. Measurement of BNP plasma levels is of prognostic value for the assessment of cardiac morbidity and mortality. We did observe an increase of BNP in patients with end-stage heart failure. After LVAD, the BNP plasma levels were decreased. Immuno-histochemical staining indicated that, apart from cardiomyocytes, infiltrating T-cells are also capable of producing BNP. Chapter 7 describes the role of cathepsin K in the remodelings process. Cathepsin K mRNA and protein expression were high in patients with end stage heart failure before LVAD support. However, after LVAD support the mRNA expression was increased, but the protein level decreased. Immunohistochemistry disclosed that the macrophages in the heart are the main producers of cathepsin K. After LVAD support, their number was decreased. These results indicate that cathepsin K plays an important role in the remodeling process in patients before LVAD support. Chapter 8 is a general discussion of the investigations included in this thesis. Currently, LVADs serve as bridge to heart transplantation and, in some patients, even as bridge to recovery. However, recovery of the heart during LAVD support is generally neither complete nor permanent. In this thesis it is demonstrated that the cardiomyocytes, the ECM volume, and the fibrillar collagen network improves in quality and even almost normalizes. Yet, degradation of the basement membrane and of the connections between the collagens in the BM and the cardiomyocytes did also occur. Such connections are very important for the transmission of force between the cardiomyocytes, and as long as they are not restored, LVAD as a temporary tool for recovery would not appear to be a realistic option