About hyaluronan in the hypertrophic heart : studies on coordinated regulation of extracellular matrix signalling

Background. Myocardial hypertrophy is a risk factor for cardiovascular morbidity and mortality. Independent of underlying disease, the cardiac muscle strives in different ways to compensate for an increased workload. This remodelling of the heart includes changes in the extracellular matrix which will affect systolic and diastolic cardiac function. Furthermore, signal transduction, molecular diffusion and microcirculation will be affected in the hypertrophic process. One important extracellular component is the glycosaminoglycan hyaluronan. It has been shown to play a major role in other conditions that feature cellular growth and proliferation, such as wound healing and malignancies. The aim of this thesis was to investigate hyaluronan and its role in both an experimental rat model of cardiac hypertrophy as well as in cultured mouse cardiomyocytes and fibroblasts. Methods. Cardiac hypertrophy was induced in rats by aortic ligation. Hyaluronan concentration was measured and expression of genes coding for hyaluronan synthases were quantified after 1, 6 and 42 days after operation, in cardiac tissue from the left ventricular wall. Localization of hyaluronan and its receptor CD44 was studied histochemically. Hyaluronan synthesis was correlated to gene transcription using microarray gene expression analysis. Cultures of cardiomyocytes and fibroblasts were stimulated with growth factors. Hyaluronan concentration was measured and expression of genes coding for hyaluronan synthases were detected. Hyaluronan size was measured and crosstalk between cardiomyocytes and fibroblasts was investigated. Results. Increased concentration of hyaluronan in hypertrophied cardiac tissue was observed together with an up-regulation of two hyaluronan synthase genes. Hyaluronan was detected in the myocardium and in the adventitia of cardiac arteries whereas CD44 staining was mainly found in and around the adventitia. Hyaluronan synthesis correlated to the expression of genes, regulated by transcription factors known to initiate cardiac hypertrophy. Stimulation of cardiomyocytes by PDGF-BB induced synthesis of hyaluronan. Cardiomyocytes also secreted a factor into culture media that after transfer to fibroblasts initiated an increased synthesis of hyaluronan. When stimulated with hyaluronan of different sizes, a change in cardiomyocyte gene expression was observed. Different growth factors induced production of different sizes of hyaluronan in fibroblasts. The main synthase detected was hyaluronan synthase-2. Cardiomyocytes were also shown to secrete microvesicles containing both DNA and RNA. Isolated microvesicles incubated with fibroblasts were observed by confocal microscopy to be internalized into fibroblasts. Altered gene expression was observed in microvesicle stimulated fibroblasts. Conclusion. This study shows that increased hyaluronan synthesis in cardiac tissue during hypertrophic development is a part of the extracellular matrix remodelling. Cell cultures revealed the ability of cardiomyocytes to both synthesize hyaluronan and to convey signals to fibroblasts, causing them to increase hyaluronan synthesis. Cardiomyocytes are likely to express receptors for hyaluronan, which mediate intracellular signalling causing the observed altered gene expression in cardiomyocytes stimulated with hyaluronan. This demonstrates the extensive involvement of hyaluronan in cardiac hypertrophy