Background Low aerobic fitness and obesity are associated with atherosclerosis, and thereforegreatly increase the risk of cardiovascular disease (CVD) and early death. It has long been known that atherosclerosis my begin early in life. Despite this fact, it remains unknown how obesity and aerobic fitness early in life influence the risks of atherosclerosis, CVD and death. Furthermore, it is unknown whether high aerobic fitness can compensate for the risks associated with obesity, and how genetic confounding affects the relationshipsof aerobic fitness with CVD and all-cause mortality. Thus, the main aims of this thesis were to investigate the associations of aerobic fitness in late adolescence with myocardial infarction (Study I), stroke (Study II) and all-cause mortality (Study III), and how genetic confounding influences the relationshipsof aerobic fitness with CVD, diabetes and death (Study IV). Methods The study population comprised up to1.3 million men who participated in mandatory Swedish military conscription. During conscription, all conscripts underwent highly standardized tests to assess aerobic fitness, body mass index, blood pressure and cognitive function. A physician also examined all conscripts. Data on subjects’ diagnoses, death and socioeconomic status during follow-up were retrieved using record linkage. Subjects were subsequently followed until the study endpoint, date of death or date of any outcome of interest. Associations between baseline variables and the risks of adverse outcomes were assessed using Cox’s proportional hazard models. Genetic confounding of the relationships between aerobic fitness and diabetes, CVD and death was assessed using a twin population and a paired logistic regression model. Results In Study I, low aerobic fitness at conscription was associated with an increased risk of myocardial infarction (MI) during follow-up (hazard ratio [HR] 0.82 per standard deviation increase). Similarly, in Study II, high aerobic fitness reduced the risk of stroke (HR 0.84 for ischemic stroke, HR 0.82 for hemorrhagic stroke; P < 0.001 for all), and obesity was associated with an increased risk of stroke (HR 1.15 for ischemic stroke, HR 1.18 for hemorrhagic stroke; P < 0.001 for all). In Study III, high aerobic fitness was also associated with reduced all-cause mortality later in life (HR 0.49, P < 0.001). High aerobic fitness exerted the strongest protection against death from substance and alcohol abuse, suicide and trauma (HRs 0.20, 0.41 and 0.52, respectively; P < 0.001 for all). Obese individuals with aerobic fitness were at higher risk of MI and all-cause mortality than were normal-weight individuals with low fitness (Studies I and III). In Study IV, fit twins had no reduced risk of CVD or death during follow-up compared with their unfit twin siblings (odds ratio 1.11, 95% confidence interval 0.88–1.40), regardless of how large the difference in fitness was. However, the fitter twins were protected against diabetes during follow-up. Conclusions Already early in life, aerobic fitness is a strong predictor of CVD and all-cause mortality later in life. In contrast to the “fat but fit” hypothesis, it seems that high aerobic fitness cannot fully compensate for the risks associated with obesity. The associationsof aerobic fitness with CVD and all-cause mortality appear to be mediated by genetic factors. Together, these findings have implications for the view of aerobic fitness as a causal risk factor for CVD and early death.

Atherosclerosis is complex inflammatory disease of the arterial wall with progressive accumulation of lipids and narrowing of the vessel. Increasing evidence suggest that inflammation plays an important role in plaque stability and often accelerate cardiovascular events such as myocardial infarction (MI). Among the vast number of inflammatory cytokines, IL-1β is known to be a key modulator in vessel wall inflammation and acceleration of the atherosclerotic process. The biologically active IL-1β is regulated by a multiprotein complex known as the NLRP3 inflammasome complex. In this thesis, we have focused on polymorphisms in the NLRP3 and CARD8 genes and their possible association to atherosclerosis and/or MI. We have also investigated the expression of inflammasome components NLRP3 and CARD8 in atherosclerosis and the role of genetic variants for the expression of these genes. The expression of NLRP3, CARD8, ASC, caspase-1, IL-1β, and IL-18 were found significantly upregulated in atherosclerotic lesions compared to normal arteries. Human carotid plaques not only express the NLRP3 inflammasome, but also release IL-1β upon exposure to lipopolysaccharide (LPS), adenosine triphosphate (ATP) and cholesterol crystals, which suggest NLRP3 inflammasome activation in human atherosclerotic lesions. Also, CARD8 was found to be important in the regulation of several inflammatory markers in endothelial cells, like RANTES, IP10 and ICAM-1. We further assessed the potential association of a CARD8 polymorphism and polymorphisms located downstream of the NLRP3 gene to the risk of MI in two independent Swedish cohorts. The CARD8 variant exhibited no association to risk of MI in either of the two cohorts. Some of the minor alleles of NLRP3 variants were associated with increased IL-1β levels and to NLRP3 mRNA levels in peripheral blood monocytic cells (PBMC). Taken together, the present thesis shows that NLRP3 inflammasome activation and increased expression of CARD8 in the atherosclerotic plaque might be possible contributors to the enhanced inflammatory response and leukocyte infiltration in the pathophysiology of atherosclerosis.

Cardiac stem cells (CSCs), isolated from heart tissue explants and expanded via the formation of cardiospheres (Csp), are a promising candidate for cell therapy to prevent heart failure following myocardial infarction. To allow early administration to patients, isolation and expansion of CSCs must be performed in the shortest time possible. Hence, this project aimed to optimize culture conditions and characterize the cardiac explant-derived cells (EDCs), Csp and Csp-derived cells (CDCs) produced. Rat neonatal EDCs contained 4-7% c-kit⁺ cells, measured using flow cytometry. Optimal Csp growth conditions were determined, such that plating 3 x 10^4 EDCs per well of a 24-well plate coated with 16.7 µg/ml poly-D-lysine, in CGM containing 7% serum, improved Csp production and generated 1.5 x 10^7 CDCs in 16 days, a sufficient number for cell therapy. The CDCs expressed the stemness markers; c-kit, Oct3/4, SOX2, and Klf-4, and the cardiac differentiation markers; GATA4 and Nkx2.5. The therapeutic effect of CDCs may be limited by the low, 3 ± 0.1%, c-kit⁺ cell numbers. To increase c-kit⁺ cells in CDCs, an alternate culture method for Csp and different extracellular matrices (ECM) for cell expansion were tested. The hanging drop culture method produced Csp with higher levels of c-kit⁺ cells (9 ± 2%) than poly-D-lysine-coated and low-bind culture dishes. Of five ECM tested, collagen IV was found to enhance EDC migration and CDC proliferation, and produced 11 ± 0.4% c-kit⁺ cells, with Csp cultured in hanging drops. Intramyocardial injection of CDCs improved left ventricular ejection fractions of infarcted rat hearts by 9% and prevented the peri-infarct wall from thinning, measured in vivo using MRI over 16 weeks. To improve cell tracking using MRI, two MR positive contrast agents, gadolinium-DTPA and gadonanotubes were tested. Gd-DTPA had low sensitivity after labelling (1.4 x 10^5 cells/mm2); whereas gadonanotubes did not provide positive contrast at 11.7 T. Thus, neither contrast agent could be used for cell tracking using high magnetic field. In conclusion, CDCs were an effective source of stem cells that could be used for heart repair, although cells could not be tracked using positive MR contrast.

Background: Acute myocardial infarction (AMI) is a major public health concern. There are limited recent national-level population-based epidemiological data on AMI in England. As a result, the current burden of disease is difficult to quantify. Aim: This thesis addresses gaps in knowledge on AMI in England. It aims to provide a comprehensive analysis of AMI epidemiology over the last decade. Methods: This is a population-based study using person-linked routine hospital and mortality data for England for the period from 1 April 1998 to 31 March 2008. Main outcome measures include: trends in event rate, case fatality, and mortality for AMI, as well as trends in characteristics of, and hospital care for, the AMI patient population between 1999 and 2007; rates of occurrence and case fatality for first and recurrent AMI in 2007; and five-year survival and risk of a second AMI for 2003 to 2007. Results: Total age-standardised AMI mortality rate fell by around half, while the age-standardised event rate and case fatality rate each declined by around one third between 1999 and 2007. Approximately half of the decline in AMI mortality was attributed to a decline in event rate and half to improved survival. During the 2000s, the hospitalised AMI patient population became increasingly elderly, presented with more comorbidities, underwent more revascularisation procedures, and spent less time in hospital. In 2007, approximately 90,000 AMIs occurred in England, of which around one third were fatal, one in seven were reinfarctions, and three quarters were AMIs in those aged 65 years and older. Among 30-day survivors of a first AMI, around one in three men and one in four women died within five years, and about one in eight men and one in six women experienced a second AMI in the same time period. Conclusions: There have been substantial improvements in AMI occurrence, survival, and mortality over the last decade in England. This was driven by improvements in prevention and acute medical treatment. The results in this thesis emphasise the importance of both.

Bakgrund: Hjärt- och kärlsjukdomar är en vanlig dödsorsak i världen och i Sverige. Hjärtinfarkt orsakas av ateroskleros som i sin tur leder till syrebrist i hjärtmuskulaturen. Symtombilden är individuell och symtom som bröstsmärta, illamående och andningspåverkan kan förekomma. Förekomsten av att drabbas av hjärt- och kärlsjukdom skiljer sig mellan män och kvinnor. Syfte: Syftet med denna litteraturstudie var att beskriva och jämföra kvinnor och mäns symtombild vid hjärtinfarkt samt att granska undersökningsgrupper i de valda studierna. Metod: En beskrivande litteraturstudie. 13 kvantitativa artiklar valdes ut till den föreliggande litteraturstudien. Huvudresultat: Bröstsmärta var det vanligaste och mest förekommande symtomet hos både män och kvinnor vid hjärtinfarkt. Kvinnor beskrev mer ryggsmärta, smärta lokaliserat till skulderbladen och nacksmärta. Män rapporterade mer smärta i höger sida av bröstkorgen och arm. Illamående, andningsbesvär och svaghetskänsla visade sig mer förekommande hos kvinnor medan män presenterade mer svettningar. Tydliga skillnader fanns i antal rapporterade symtom i samband med hjärtinfarkt, kvinnor rapporterade fler än män. Slutsats: Det framkom både likheter och skillnader i män och kvinnors symtombild vid hjärtinfarkt, symtomen var varierande och individuella. För att sjuksköterskan ska kunna tillgodose patientens individuella omvårdnadsbehov krävs kunskap om hjärtinfarktens varierande symtombild. Detta är viktigt för att i god tid kunna handla korrekt för att förhindra komplikationer och bestående men hos patienter som drabbas av hjärtinfarkt. Mer forskning inom området skulle öka kunskapen hos sjuksköterskan och bidra till en bättre omvårdnad för patienter som drabbas av hjärtinfarkt. / Background: Cardiovascular disease is a common cause of death in the world and in Sweden. Myocardial infarction is caused by atherosclerosis, which in turn leads to hypoxia in the heart muscles. The symptoms are individual and symptoms such as chest pain, nausea and respiratory effects may occur. The existence of suffering from cardiovascular disease differ between men and women. Purpose: The purpose of this study was to describe and compare women's and men's symptoms of myocardial infarction and to review the study groups in the selected studies. Method: A descriptive literature. 13 quantitative articles were selected to the present literaturestudy. Main Results: Chest pain was the most common symptom in both men and women with myocardial infarction. Women described more frequent back pain, pain localized to the shoulder blades and neck pain. Men reported more pain in the right side of the chest and arm. Nausea, dyspnea and weakness proved to be more common in women, while men presented more sweating. Distinct differences were found in the number of reported symptoms associated with a myocardial infarction, women reported more symptoms than men. Conclusion: The litteraturstudy revealed both similarities and differences in men's and women's symptoms of myocardial infarction, symptoms were variable and individual. For the nurse to meet individual patient care needs requires knowledge of myocardial infarctions varying symptoms. This is important in order to act properly and in an early stage to prevent complications and permanent damage in patients who suffer from myocardial infarction. More research in this area would empower the nurse and contribute to better care for patients who suffer from myocardial infarction.

The epicardium is a single layer of epithelial (mesothelial) cells that covers the entire heart surface, but whose function in adult mammals is poorly understood. Defining the role of epicardial cells during homeostasis, growth and injury has potential to provide new treatment strategies for human diseases that result in heart failure, due to extensive loss of viable cardiac tissue. We hypothesized that epicardial cells contribute to repair as transplantable progenitor cells for cellular regeneration and as a source of secreted growth factors for cell protection after myocardial infarction. Adult epicardial cells were prospectively isolated as uncommitted epithelial cells using epithelial-specific beta-4 integrin (CD104). These cells underwent epithelial to mesenchymal transformation in culture to generate epicardial cell derivatives (EPDCs). We demonstrate that the C-terminal peptide from Connective Tissue Growth Factor (CTGF-D4), when combined with insulin, effectively primes EPDCs for robust cardiac engraftment in rats and contributes to improvement in cardiac function at one month after MI. Furthermore, we define a signaling axis comprised of CTGF-D4, low density lipoprotein receptor-related protein 6 (LRP6), sex determining region Y-box 9 (Sox9) and Endothelin Receptor B (ETBR) that controls several key processes that impact EPDC graft success: cell survival, proliferation and migration. Interestingly, conditional deletion of ETBR using epicardial-specific transgenic mice prevented epicardial cell proliferation and migration into myocardium after MI. We therefore observed a congruence in the signals and signaling pathways that control the proliferation and migration of endogenous EPDCs after MI and EPDCs that can be generated in cell culture and grafted back to the heart. To gain additional insight into the cellular contribution of the epicardium, we utilized a non-injurious running exercise model to evaluate epicardial activity as a consequence of cardiac hypertrophy (i.e. myocardial growth model). We employed an inducible lineage-tracing system to specifically label and track epicardial cells by GFP expression. Prolonged exercise resulted in a significant number of GFP-positive proliferating epicardial cells and epicardial-derived GFP-positive endothelial cells and few GFP-positive smooth muscle cells in the heart. These observations highlight the cellular plasticity of the adult epicardium and its function as a cardiac progenitor cell niche, maintaining a source of replacement cells. To investigate the paracrine properties of adult epicardial cells for their role in cell protection after MI and reperfusion, human epicardial cells were isolated from donor atrial tissue explants. We predicted that medium conditioned by cultured epicardial cells (EPI CdM) contained secreted reparative factors that would promote endothelial cell survival. Administration of EPI CdM promoted endothelial cell survival in culture and in vivo, 24 hours after ischemia-reperfusion injury. By screening EPI CdM, we detected protein complexes containing hepatocyte growth factor (HGF) with polyclonal IgG that imparted vascular protection in vivo in a manner similar to EPI CdM. Overall, the studies presented here illustrate the unique biology of epicardial cells, their signaling networks, and their contribution to cardiac cell protection and regeneration. Importantly, these properties have the potential to be exploited in translational applications for cardiac repair.

Several studies have shown cellular replacement therapy as a treatment strategy of myocardial infarction but results have been limited. Therefore, enhancing the therapeutic potential of stem cells injected into ischemic microenvironments by novel preconditioning (PC) techniques is critical for improving cellular therapy. Recent studies have shown that inhibition of phosphodiesterase-5 (PDE-5) is a powerful strategy to precondition the heart and cardiomyocytes against ischemia/reperfusion injury. We therefore tested the hypothesis that inhibition of PDE-5 with sildenafil (Viagra®) or selective knockdown with a silencing vector in adipose derived stem cells (ASCs) would improve their survival after ischemia/reoxygenation in vitro and enhance cardiac function following myocardial implantation in vivo. ASCs were treated with sildenafil or infected with PDE-5 silencing vector shRNA (shRNAPDE-5). The cells were subjected to simulated ischemia (SI) and reoxygenation (RO). Both sildenafil and shRNAPDE-5 significantly reduced cell injury, as shown by improved viability, decreased lactate dehydrogenase, and apoptosis. The preconditioned ASCs also demonstrated an increase in the release of growth factors including VEGF, b-FGF, and IGF. The protective effect against SI/RO injury was abolished by inhibition of protein kinase G (PKG) using both a pharmacological inhibitor and selective knockdown with shRNAPKG1α suggesting a PKG-mediated mechanism. To show the effect of preconditioned ASCs in vivo, adult male CD-1 mice underwent myocardial infarction (MI) by occlusion of the left descending coronary artery, followed by direct injection of PBS (control), non-preconditioned ASCs, or preconditioned ASCs (4x105) ASCs into the left ventricle (LV). Preconditioned ASC-treated hearts showed consistently superior cardiac function by all measures as compared with PBS and non-preconditioned ASCs after 4 weeks of treatment. Post-mortem histological analysis demonstrated that preconditioned ASC-treated mice had significantly reduced fibrosis, increased vascular density and reduced resident myocyte apoptosis as compared to mice receiving non-preconditioned ASCs or PBS. VEGF, b-FGF, and Ang-1 were also significantly elevated 4 weeks after cell therapy with preconditioned ASCs. Our data suggests that genetic or pharmacological inhibition of PDE-5 is a powerful new approach to improve stem cell therapy following myocardial infarction.

Administration of small interfering RNA (siRNA) specific for prolyl-4 hydroxylase-2 (PHD2) results in PHD2 inhibition, Hypoxia Inducible Factor-I (HIF-1) activation, and cardioprotection versus Ischemia Reperfusion (IR). This study observes the effects of siRNA-mediated PHD2 inhibition on the distribution of cardioprotective proteins by immunofluorescence and basic histology. Fifteen mice were divided into 5 groups: PHD2 Control, Non-Targeting scramble (NTS) Control, IR Control, PHD2 IR, and NTS IR. Histologically, tissue damage was reduced dramatically in the PHD2 IR group compared to the NTS IR and IR control groups. From confocal images, total fluorescent pixels and intensities were quantified. The PHD2 IR group yielded the highest pixel quantity and intensity for HIF-1 and possessed increased pixels and intensity for Inducible Nitric Oxide Synthase, another cardioprotective protein. These results further demonstrate the cardioprotection and HIF-1 activation conferred by PHD2 siRNA administration and supports its role as a potential therapy to alleviate cardiac IR injury.

L'infarctus du myocarde (IDM) est l'une des principales causes de morbi-mortalité dans le monde et ce malgré une revascularisation rapide de l’artère coronaire occlue. L'obésité est un facteur de risque majeur pour l’IDM, et concerne 15 % de la population française (enquête ObEpi-Roche/INSERM, 2012). L'objectif de notre étude a été d'étudier l'efficacité de stratégies cardioprotectrices comme l’exercice physique chronique ou l’administration d’un donneur de CO (CORM-3) dans un contexte d’obésité. Un effet cardioprotecteur de l'exercice physique chronique a été démontré sur un modèle d’ischémie-reperfusion chez la souris ob/ob (génétiquement dépourvu en leptine). Cet effet implique l’activation de la voie de signalisation cardioprotectrice RISK associée à une diminution des taux cytosoliques des protéines phosphatases inhibitrices correspondantes PTEN et MK3P ce qui aboutit à une amélioration des fonctions mitochondriales. Ce travail montre la capacité différentielle de stratégies à réduire la taille de l’infarctus en cas d’obésité. Par ailleurs, il souligne l’importance du rôle des mécanismes HCO3- dépendants dans le contrôle du pH intracellulaire à la reperfusion. Ils pourraient représenter une étape clef dans la cardioprotection. / Myocardial infarction (MI) remains the leading cause of morbidity and mortality in the developing countries despite significant therapeutic advances over the last years. Obesity is a major risk factor for coronary heart disease and concern 15% of the French population (ObEpi-Roche/INSERM survey, 2012). The aim of our studie was to investigate the efficacy of cardioprotective strategies such as regular treadmill exercice or CO donor administration (CORM-3) against MI during obesity. In ob/ob mice, regular exercise induces a robust cardioprotection by increasing kinase phosphorylation of RISK pathway, decreasing levels of corresponding phosphatases and improving the resistance of mitochondrial permeability transition pore opening. The present study shows differential capabilities of cardioprotective strategies at reducing myocardial infarct size with obesity. In addition, it underlines the role of HCO3- dependent mechanisms in the control of intracellular pH at reperfusion. They could represent a key step for mediating cardioprotection.

Master of Science / Department of Kinesiology / Timothy I. Musch / Both cardiac and peripheral vasculature dysfunction likely contribute, in part, to elevations in TNF-[alpha] and exercise intolerance in chronic heart failure (CHF). The pharmaceutical TNF-[alpha] synthesis suppressor pentoxifylline (PTX) reduces plasma [TNF-[alpha]] and improves left ventricular (LV) function in CHF rats, but the effects of PTX on skeletal muscle blood flow (BF) and vascular conductance (VC) during exercise are unknown. We tested the hypothesis that PTX would elevate skeletal muscle BF and VC at rest and during submaximal treadmill exercise in CHF rats (coronary artery ligation). CHF rats received i.p. injections of 30 mg·kg[superscript]-[superscript]1·day[superscript]-[superscript]1 of PTX (CHF+PTX, n=13) or saline (CHF, n=8) for 21 days. Mean arterial pressure (MAP) and BF (radiolabeled microsphere infusions) were measured at rest and during treadmill exercise (20 m/min, 5% grade). Myocardial infarct (MI) size was not different between groups (CHF: 37±4, CHF+PTX: 37±3% of LV wall; p>0.05). Resting and exercising MAP was greater in CHF+PTX compared to CHF (p<0.05 for both). At rest, total hindlimb skeletal muscle BF and VC were not different between groups (p>0.05). However, during exercise PTX increased total hindlimb BF (CHF: 83±9, CHF+PTX: 114±8 ml·min[superscript]-[superscript]1·100g[superscript]-[superscript]1, p<0.05) and VC (CHF: 0.75±0.08, CHF+PTX: 0.88±0.06 ml·min[superscript]-[superscript]1·100g[superscript]-[superscript]1·mmHg[superscript]-[superscript]1, p<0.05). Furthermore, exercising BF was increased in 21, and VC in 11, of the 28 individual hindlimb muscles or muscle parts with no apparent fiber-type specificity. Thus, PTX administration augments skeletal muscle BF and VC during locomotory exercise in CHF rats, which carries important therapeutic implications for CHF patients.