| 1. |
- Gennebäck, Nina, 1982-
(författare)
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Cardiac hypertrophy : transcription patterns, hypertrophic progression and extracellular signalling
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: The aim of this thesis was to study transcription patterns and extracellular signalling of the hypertrophic heart to better understand the mechanisms initiating, controlling and maintaining cardiac hypertrophy.Cardiac hypertrophy is a risk factor for cardiovascular morbidity and mortality. Hypertrophy of the myocardium is a state, independent of underlying disease, where the myocardium strives to compensate for an increased workload. This remodelling of the heart includes physiological changes induced by a changed gene expression, alteration of the extracellular matrix and diverse cell-to-cell signalling.Shedding microvesicles and exosomes are membrane released vesicles derived from the plasma membrane, which can mediate messages between cells and induce various cell-related processes in target cells.Methods and materials: Two different microarray studies on different materials were performed. In the first study, cardiac myectomies from 8 patients with hypertrophic obstructive cardiomyopathy (HOCM) and 5 controls without cardiac disease were used. In the second study, myocardial tissue from 6 aorta ligated and 6 sham operated (controls) rats at three different time points (1, 6 and 42 days post-surgically) were analysed. To reveal differences in gene expression the materials were analyzed with Illumina whole genome microarray and multivariate data analysis (PCA and OPLS-DA).Cultured cardiomyocytes (HL-1) were incubated with and without growth factors (TGF-β2 or PDGF BB). Microvesicles and exosomes were collected and isolated after differential centrifugations and ultracentrifugations of the cell culture medium. The microvesicles and exosomes were characterized with dynamic light scattering (DLS), flow cytometry, western blot, electron microscopy and Illumina whole genome microarray.Results: The two different microarray studies revealed differentially expressed gene transcripts and groups of transcripts. When comparing HOCM patients to controls significant down-regulation of the MYH6 gene transcript and two immediate early genes (IEGs, EGR1 and FOS), as well as significant up-regulation of the ACE2, JAK2 and HDAC5 gene transcripts were found. In the rat model, 5 gene groups showed interesting clustering after multivariate data analysis (OPLS-DA) associated with the hypertrophic development: “Atherosclerosis”, “ECM and adhesion molecules”, “Fatty acid metabolism”, “Glucose metabolism” and “Mitochondria”.The shedding microvesicles were rounded vesicles, 40-300 nm in size and surrounded by a bilayered membrane. Chromosomal DNA sequences were identified in the microvesicles. The microvesicles could be taken up by fibroblasts resulting in an altered gene expression in the fibroblasts. The exosomes from cultured cardiomyocytes (incubated with TGF-β2 or PDGF BB) had an average diameter of 50-80 nm, similar to the unstimulated control exosomes. A large, for all cardiomyocyte derived exosomes, common pool of mRNA seems stable and a smaller pool varied in mRNA content according to treatment of the cardiomyocyte. Of the common mRNA about 14% were ribosomal, 14% were of unknown locus and 5% connected to the function of the mitochondria.Conclusions: The microarray studies showed that transcriptional regulation at a stable stage of the hypertrophic development is a balance of pro and anti hypertrophic mechanisms and that diverse gene groups are differently regulated at different time points in the hypertrophic progression.OPLS-DA is a very useful and powerful tool when analyzing gene expression data, especially in finding clusters of gene groups not seen with traditional statistics.The extracellular vesicle studies suggests that microvesicles and exosomes released from cardiomyocytes contain DNA and can be involved in events in target cells by facilitating an array of processes including gene expression changes. Different treatment of the cardiomyocyte influence the content of the exosome produced, indicating that the signal function of the exosome might vary according to the state of the cardiomyocyte.
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| 2. |
- Hellman, Urban, 1966-
(författare)
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About hyaluronan in the hypertrophic heart : studies on coordinated regulation of extracellular matrix signalling
- 2010
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- 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.
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| 3. |
- Henein, Mark, 1960-
(författare)
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Left atrial function in health and disease
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The Objectives of this thesis are:1) To study possible atrial interaction in patients with right and left ventricular outflow tract obstruction due to significant pulmonary (PS) and aortic valve stenosis (AS), respectively.2) To assess left atrial (LA) intrinsic myocardial function and its relationship to indirect measures of left ventricular (LV) filling pressures in patients with paroxysmal atrial fibrillation (PAF).3) To test the hypothesis that the LA function is affected in patients with pulmonary arterial hypertension (PAH).4) To test the hypothesis that raised LA pressure as shown by pulmonary capillary wedge pressure (PCWP) correlates with severity of LA intrinsic systolic function.We conducted 4 studies to achieve the objective sabove.Study IMethods:We studied 41 PS patients (age 36±10 year) and 41 AS patients (age 35 ± 12 year) and compared them with 27 controls (age 30 ± 7 year). RV and LV filling were recorded by conventional PW Doppler. Biventricular segmental function was studied using the PW tissue Doppler imaging (TDI) and M mode techniques.Results:The 2 patient groups had similar degree of ventricular outflow tract obstruction. In the pressureoverloaded ventricle, global systolic function was preserved but long axis function was impaired.Patients had higher peak late filling (Awave)and TDI late diastolic (a’) velocities recorded in the disease free ventricles despite having similar peak early filling velocities (E wave), E wave deceleration time and E/e’ ratios were not different from controls (p>0.05 for all). The accentuation of atrial activity (A wave) was moderately correlated with the degree of contra lateral ventricular outflow tract obstruction (p<0.001 for both).Conclusion:In the pressure overloaded ventricle long axis function is more sensitive than global function in revealing myocardial dysfunction. The increased contra lateral atrial systolic activity suggests an evidence for atrial interaction in the form of ‘Cross Talk’.Study IIMethods:Twentyfive PAF patients (age 68±7 year, 10 males) with Doppler signs of raised filling pressures were studied using speckle tracking echocardiography and compared with 21 controls. LA segmental longitudinal strain (S), strain rate (SR) and myocardial velocities during atrial systole were measured as were LA longitudinal and transverse diameters. Markers of LV filling pressures were E/A andE/e’.Results:LA longitudinal diameter was larger in patients (5.5±0.6 vs. 4.8±0.6cm,p<0.01) and global LAS and SR were reduced (p<0.05 for both) and correlated with E/A (r=0.52 and r=0.43, p<0.05 for both). LA segmental S and SR were uniformly reduced compared with controls (p<0.05 for all) and also correlated with E/A (p<0.05 for all). LA myocardial velocities (TDI) were highest at the annular level and lowest at the rear in both patients and controls (p<0.01 for all), with the absolute values at each level not different between groups. Myocardial velocities negatively correlated with E/A at the annular level only in patients (septal: r=0.52; lateral: r=0.62, p<0.01 for both).Conclusion:In PAF patients, LA systolic function is suppressed and is directly related to the raised filling pressures. While intrinsic global and segmental function can reproducibly be studied by S and SR, myocardial velocities reflect only regional motion. These findings provide a sound explanation to the known beneficial effect of vasodilators in PAF patients.Study IIIMethods:We studied LA size and reservoir function in 35 patients (age 63 ± 15 years, 16 male) with idiopathic PAH using speckle tracking echocardiography who also underwent right heart catheterization simultaneously to assess pulmonary artery systolic pressure, and compared them with 27 age and gender normal controls.Results:In PAH patients, LA longitudinal diameter was not different from controls but transverse diameter was reduced (3.0 ± 0.6 vs. 3.7 ± 0.5cm, p<0.001). LA lateral wall strain rate (SR) during LV systole (atrial reservoir function was reduced at annular (p<0.001) and mid cavity (p<0.01) levels as were septal segments (p<0.03, for both) compared to controls. Opposite to controls, the two LA walls responded differently to right heart pressures. Lateral SR inversely correlated with pulmonary artery systolic pressure (PASP) (annular: r=0.45, p<0.005 and midcavity: r=0.43, p<0.01), but not with right atrial pressure (RAP). In contrast, septal SR inversely correlated with RAP (annular: r=0.39, p=0.02 and midcavity: r=0.38, p=0.03) but not with PASP.Conclusion:In patients with PAH, LA reservoir function is significantly impaired showing reduced myocardial strain rate properties. In addition,segmental function differs in their response to raised right heart pressures with the septal wall related to right atrial pressure and lateral wall related to the PASP. These findings suggest an evidence for atrial interaction in PAH, which is likely to have significant impact on LV performance.Study IVMethods:We studied 46 patients, mean age 61 ± 13 years, 17 males, of various etiologies with exertional breathlessness who underwent right heart catheterization and simultaneous transthoracic Doppler echocardiography using spectral, tissue Doppler and speckle tracking echocardiography techniques for assessing LA structure and function.Results:PCWP correlated with direct measurements of LA structure and function: LA volume (r= 0.43, p<0.01), LA global systolic strain rate (r=0.79, p<0.001) and to a lesser extent with LA systolic filling fraction (r=0.52, p<0.001). PCWP also correlated with indirect measures of LA pressure: LV E/A (r=0.66, p<0.001), E wave deceleration time (r=0.54, p<0.001), lateral E/e’ (r=0.49, p<0.001) and LV isovolumic relaxation time (r=0.36, p<0.01). LA strain rate was 78% sensitive and 84% specific in identifying patients with PCWP>15 mmHg, having accurately predicted PCWP in 63% of the cases.Conclusion:PCWP correlates with LA intrinsic systolic function and to a much lesser degree with indirect Doppler measures of raised LV filling pressures. These findings should have significant clinical implications in identifying breathless patients with raised LA pressure.
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| 4. |
- Ramzy Guirguis, Ihab, 1963-
(författare)
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Insights into the effect of myocardial revascularisation on electrical and mechanical cardiac function
- 2012
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Background: Acute coronary syndrome is known for its effect on cardiac function and can lead to impaired segmental and even global myocardial function. Evidence exists that myocardial revascularisation whether pharmacological, interventional or surgical results in improvement of systolic and diastolic left ventricular (LV) function, particularly that of the long axis which represents the sub-endocardial function, known as the most sensitive layer to ischaemia. Objective: We sought to gain more insight into the early effect of pharmacological and interventional myocardial revascularisation on various aspects of cardiac function including endocrine, electrical, segmental, twist, right ventricular (RV) and left atrial (LA) function. In particular, we aimed to assess the response of ventricular electromechanical function to thrombolysis and its relationship with peptides levels. We also investigated the behaviour of RV function in the setting of LV inferior myocardial infarction (IMI) during the acute insult and early recovery. In addition, we aimed to assess in detail LA electrical and mechanical function in such patients. Finally, we studied the early effect of surgical revascularisation on the LV mechanics using the recent novel of speckle tracking echocardiography technology to assess rotation, twist and torsion and the strain deformation parameters as a tool of identifying global ventricular function. Methods: We used conventionally Doppler echocardiographic transthoracic techniques including M-mode, 2-Dimentional, myocardial tissue Doppler, and speckle tracking techniques. Commercially available SPSS as a software was used for statistical analysis. Results: 1-The elevated peptide levels at 7 days post-myocardial infarction correlated with the reduced mechanical activity of the adjacent non-infarcted segment thus making natriuretic peptides related to failure of compensatory hyperdynamic activity of the non-infarcted area rather than the injured myocardial segments. 2-RV segmental and global functions were impaired in acute IMI, and recovered in 87% of patients following thrombolysis. In the absence of clear evidence for RV infarction the disturbances in the remaining 13% may represent stunned myocardium with its known delayed recovery. 3-LA electromechanical function was impaired in acute inferior STEMI and improved after thrombolysis. The partial functional recovery suggests either reversible ischaemic pathology or a response to a non-compliant LV segment. The residual LA electromechanical and pump dysfunction suggest intrinsic pathology, likely to be ischaemic in origin. 4-LV function was maintained in a group of patients with multivessel coronary artery disease who underwent coronary artery bypass graft (CABG) surgery. Surgical myocardial revascularisation did not result in any early detectable change in the three functional components of the myocardium, including twist and torsion, as opposite to conventional percutaneous coronary intervention (PCI). Conclusion: The studied different materials in this thesis provide significant knowledge on various aspects of acute ischaemic cardiac pathology and early effect of revascularisation. The use of non-invasive imaging, particularly echocardiography with its different modalities, in studying such patients should offer immediate thorough bed-side assessment and assist in offering optimum management.
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