Cardiac multiscale bioimaging: from nano- through micro- to mesoscales.

Cardiac multiscale bioimaging is an emerging field that aims to provide a comprehensive understanding of the heart and its functions at various levels, from the molecular to the entire organ. It combines both physiologically and clinically relevant dimensions: from nano- and micrometer resolution imaging based on vibrational spectroscopy and high-resolution microscopy to assess molecular processes in cardiac cells and myocardial tissue, to mesoscale structural investigations to improve the understanding of cardiac (patho)physiology. Tailored super-resolution deep microscopy with advanced proteomic methods and hands-on experience are thus strategically combined to improve the quality of cardiovascular research and support future medical decision-making by gaining additional biomolecular information for translational and diagnostic applications

Stem Cell Research on Cardiology

Even today, cardiovascular diseases are the main cause of death worldwide, and therapeutic approaches are very restricted. Due to the limited regenerative capabilities of terminally differentiated cardiomyocytes post injury, new strategies to treat cardiac patients are urgently needed. Post myocardial injury, resident fibroblasts begin to generate the extracellular matrix, resulting in fibrosis, and finally, cardiac failure. Recently, preclinical investigations and clinical trials raised hope in stem cell-based approaches, to be an effective therapy option for these diseases. So far, several types of stem cells have been identified to be promising candidates to be applied for treatment: cardiac progenitor cells, bone marrow derived stem cells, embryonic and induced pluripotent stem cells, as well as their descendants. Furthermore, the innovative techniques of direct cardiac reprogramming of cells offered promising options for cardiovascular research, in vitro and in vivo. Hereby, the investigation of underlying and associated mechanisms triggering the therapeutic effects of stem cell application is of particular importance to improve approaches for heart patients. This Special Issue of Cells provides the latest update in the rapidly developing field of regenerative medicine in cardiology

Characterisation of cardiosphere derived cells :investigating hypoxic pre-conditioning on pro-angiogenic properties and tracking the cardiac fibroblast component

PhD ThesisCoronary heart disease is still the leading cause of death in the UK, despite significant advances in clinical treatments. Stem cell transplantation has the potential to improve cardiac function and patient outcome, but optimal cell types, cell preparation methods and cell delivery routes are yet to be established. The heart contains a small population of progenitor cells that, in culture, contribute to spontaneously formed spheroids known as cardiospheres (Csphs). Following further culture, Csphs give rise to cardiosphere derived cells (CDCs). Both Csphs and CDCs show paracrine benefit including neovascularisation in myocardial ischaemia, leading to improvement in heart function. The aims of this project were to use mouse models to (i) investigate the effect of hypoxic preconditioning on the pro-angiogenic potential of CDCs and (ii) characterise the contribution of cardiac fibroblasts (CFs) to CDCs. I used Col1a2-CreERT;Rosa26-STOP-YFP mice to track YFP-expressing CFs in myocardial tissue and in CDC culture. Co-staining experiments showed only partial overlap of YFP with other CF markers (vimentin and Fsp1) in heart tissue, which may be due to the heterogeneity of CFs and/or incomplete activation of YFP in CFs. I showed that CF-derived cells exist in all stages of CDC culture, and a small subset of these cells also expressed the stem cell markers Sca-1 or cKit, suggesting CF derived cells may contribute to the progenitor cell population. My results showed that preconditioning CDCs with 3%O2 enhances cell outgrowth from heart explants and promotes expression of stem cell and pro-angiogenic markers. I then assessed the pro-angiogenic potential of CDCs in vivo using a sub-dermal matrigel plug assay and showed that CDCs alone have limited pro-angiogenic potential. However, 3%O2 preconditioning of CDCs significantly enhances this process. Further research will increase our understanding of CDC-mediated angiogenesis and improve clinical therapies for MI patients

Targeting hydrogen sulfide breakdown for regulation of myocardial injury and repair

Hydrogen sulfide (H2S) is an endogenous gasotransmitter that regulates vascular function and blood pressure, and also protects the heart from injury associated with myocardial infarction (MI). The mitochondrial enzyme thiosulfate sulfurtransferase (TST) has a putative role in the breakdown of H2S but its role in the cardiovascular system is unknown. I hypothesised that TST reduces cardiovascular H2S availability and that inhibiting TST activity may therefore ameliorate cardiovascular pathology. In the heart, TST was expressed by cardiomyocytes and vascular smooth muscle cells. Tst-/- mice all survived to adulthood and had normal cardiac structure and function. Cardiac and hepatic H2S breakdown rates were reduced and H2S levels were higher in the blood of Tst-/- mice. However, in heart tissue, protein levels for the H2S-activated Nrf2 downstream targets, thioredoxin (Trx1) and heme oxygenase-1 (HO-1) were comparable. In contrast, protein levels for the cardiac specific H2S-synthetic enzyme, cystathionine gamma lyase (CSE) was reduced, suggesting a homeostatic negative feedback mechanism to maintain H2S at non-toxic levels. Respiration, measured using an oxygen-sensing electrode was normal in isolated mitochondria from whole Tst-/- compared to control C57BL6 hearts. Endothelial nitric oxide synthase (eNOS) protein expression was lower in Tst-/- hearts, highlighting potential cross talk between H2S and nitric oxide (NO) signalling. TST was expressed in whole aorta homogenates and in isolated endothelial cells from aorta and small intramuscular vessels of the hindlimb from C57BL/6N control mice. Myography and western blotting revealed a greater influence of NO in aorta from Tst-/- mice that was associated with increased phosphorylation of the activating serine1177 residue of eNOS (PeNOSSer1177). NO plays a lesser role in resistance arteries, but in comparison to control vessels, small mesenteric vessels from Tst-/- mice was more reliant on small and intermediate calcium activated potassium channels for relaxation. Tst-/- mice were normotensive, despite this alteration in the regulation of vascular tone. However, metabolic cage experiments identified that Tst-/- mice presented with diuresis, polydipsia, and increased urinary electrolyte excretion of sodium, potassium and chloride, possibly to compensate for increased vascular tone in order to maintain stable blood pressure. To investigate the role of TST in regulating the response to pathological challenge, MI was induced by coronary artery ligation (CAL). In control mice, gene expression of CSE was downregulated by 2 days after CAL, but TST expression was 12-fold increased, suggesting regulation of H2S bioavailability during the acute MI-healing phase. Tst-/- male mice had a 40% greater incidence of cardiac rupture during infarct healing and surviving Tst-/- mice had greater left ventricular dilatation and impaired function compared to controls. Ex vivo, isolated perfused hearts from Tst-/- mice were more susceptible to ischaemia/ reperfusion injury, suggesting an additional role of TST in determining cardiomyocyte susceptibility to injury. In conclusion, these data indicate that cardiovascular H2S bioavailability is regulated through degradation by TST. The data presented here provide evidence for significant tissue specific crosstalk between H2S synthetic and degradative mechanisms and between H2S and other local regulatory mechanisms, including ion channels and NOS. We infer TST has a physiological role in the kidney where its loss leads to changes in renal electrolyte and water handling, although other compensatory mechanisms prevent a change in blood pressure. Under conditions of pathological challenge following MI, loss of TST is detrimental, illustrating its key role in removal of H2S. The data refute the original hypothesis that TST inhibition would be protective against cardiovascular pathology. Further studies in mice with tissue specific deletion of TST are now required to more fully reveal the cardiovascular role of TST

Cell-based therapies for ischemic cardiomyopathy : investigations of intramyocardial retention and safety of high dose intracoronary delivery of c-kit positive cardiac progenitor cells, and therapeutic utility of a novel population of cardiac mesenchymal stem cells expressing stage-specific embryonic antigen-3 (SSEA-3).

Over the last decade attempts at reducing morbidity and mortality of patients with chronic heart failure have been made via the development and implementation of novel cell based therapies. Substantial advances in cell based therapies with indications of efficacy have been shown along with a robust safety profile. Despite these advances, there is a substantial unmet need for novel therapies, specifically addressing repair and regeneration of the damaged or lost myocardium and its vasculature. Accordingly, cardiac cell-based therapies have gained attention. Various cell-types have been utilized, including bone marrow-derived mononuclear cells, bone marrow-derived mesenchymal stem cells, mobilized CD34+ cells, and more recently, cardiosphere-derived cells and cardiac-derived c-kit positive progenitor cells. Early studies have suggested a potential of cell-based therapies to reduce cardiac scar size and to improve cardiac function in patients with ischemic cardiomyopathy. However, variability of results has been observed necessitating improvement of current methodologies related to optimizing the cell type(s), infusion techniques, timing, dosage, acuity related to ischemic injury, and perhaps repeat dosing over time among others, all the while ensuring complete and total patient safety. Accordingly, present efforts and goals of my research are aimed at i.) Optimizing methodologies utilized within the recent phase I clinical trial (SCIPIO) that showed intracoronary infusion of 1 million c-kit positive cardiac progenitor cells was safe with indications of efficacy in cardiac repair, as well as, ii.) Development of a novel cell based approach with a newly discovered cardiac cell type. Within the present dissertation, I explored the impact of coronary stop-flow on cardiac retention of intracoronarily infused c-kit positive cardiac progenitor cells given that balloon inflation in a non-stented coronary artery is inherently dangerous, especially in already damaged hearts. I demonstrate that intracardiac retention with or without stop-flow is equivalent and balloon inflation confers an undue risk to patients. Furthermore, I investigated the safety of intracoronary infusion of 20 million c-kit positive cardiac progenitor cells in pigs, an equivalent dose 40 times larger than was used in the SCIPIO trial. High dose of cells delivered intracoronarily is safe and does not result in myocardial injury or functional deficit. Therefore, larger doses may reasonably be utilized in future clinical trials. Finally, I describe a novel adult cardiac cell type that maintains expression of an embryonic stem cell associated marker, stage-specific embryonic antigen (SSEA)-3, resides within the native adult heart, and can be isolated and utilized for cardiac repair as a cell based therapy

Advances in Electrocardiograms

Electrocardiograms have become one of the most important, and widely used medical tools for diagnosing diseases such as cardiac arrhythmias, conduction disorders, electrolyte imbalances, hypertension, coronary artery disease and myocardial infarction. This book reviews recent advancements in electrocardiography. The four sections of this volume, Cardiac Arrhythmias, Myocardial Infarction, Autonomic Dysregulation and Cardiotoxicology, provide comprehensive reviews of advancements in the clinical applications of electrocardiograms. This book is replete with diagrams, recordings, flow diagrams and algorithms which demonstrate the possible future direction for applying electrocardiography to evaluating the development and progression of cardiac diseases. The chapters in this book describe a number of unique features of electrocardiograms in adult and pediatric patient populations with predilections for cardiac arrhythmias and other electrical abnormalities associated with hypertension, coronary artery disease, myocardial infarction, sleep apnea syndromes, pericarditides, cardiomyopathies and cardiotoxicities, as well as innovative interpretations of electrocardiograms during exercise testing and electrical pacing

The regionality of cardiac beta-2-adrenoceptor signalling

The dysfunction of the apical myocardium is observed following chronic exposure to catecholamines, or after acutely stressful scenarios, in the syndrome of Takotsubo cardiomyopathy. The physiological functions of the β2AR were assessed via measurement of cell shortening; β2AR-cAMP signalling was assessed by FRET microscopy, comparing cells from the apical and basal myocardium. The effects of pre-stimulation of the β2AR with high levels of endogenous catecholamines were investigated to simulate pathological scenarios. The structure of the cellular membranes of cells from different myocardial regions was assessed and manipulated to investigate their role in β2AR signalling. An improved animal model of the regional pathology in Takotsubo was investigated using ovariectomized female rats. Apical cardiomyocytes are sensitized to β2AR stimulation, displaying larger increases in inotropy and lusitropy in comparison to basal cells. This was not due to differences in cAMP levels induced by β2AR within the cell cytosol. Differences were apparent in the amount of cAMP reaching the RII-PKA domains (the main arbiters of cellular inotropy and lusitropy). β2AR-cAMP signalling was discovered to be more persistent in the apical cardiomyocytes. Basal cardiomyocytes were found to have a larger number of caveolae within their membranes, caveolae have been demonstrated to modulate β2AR. Following the disruption of caveolar domains via chelation of cholesterol, apical and basal responses to β2AR stimulation were equalized; inhibition of phosphodiesterase 4 had the same effect. Catecholamine pre-stimulation reduced β2AR responses; adrenaline pre-stimulation exerts a more Gi-dependent desensitization than noradrenaline. Female rats are shown to be relatively protected from the effects of adrenergic overstimulation and ovariectomized female rats suffer acutely high mortality. Cells from different regions of the myocardium may control their receptor signalling in different ways to produce desirable physiological activity. In settings of pathology this phenomena may leave certain regions vulnerable to damage.Open Acces

Malignant hypertension in the transgenic Ren-2 rat

The transgenic rat line TGR(mREN2)27 has been previously shown to develop severe hypertension as a consequence of over -expression of the mouse Ren -2 renin gene (Mullins et al 1990). It was observed that an alteration in phenotype occurred in hybrids (HanRen2/Edin --) derived from crossing homozygous Ren -2 transgenic rats with the Edinburgh Sprague -Dawley strain of rats. Investigations into this phenotypic change revealed it to be due to spontaneous development of malignant phase hypertension. Furthermore the incidence of the malignant hypertensive phenotype was altered by the genetic background into which the transgene was introduced.The introduction of this thesis has reviewed the literature on the genetics of human essential hypertension and rat models of genetic hypertension, the role of the kidney in essential hypertension and the pathogenesis of malignant hypertension. Techniques in molecular biology which include transgenesis have been used to investigate the role of individual genes in blood pressure regulation. In this context the literature concerning the transgenic rat line TGR(mREN2)27 was extensively reviewed.The heterozygote cross HanRen2/Edin -- was found to develop malignant phase hypertension within a relatively narrow age range. Seventy -three percent of male and 52% of female HanRen2/Edin -- developed malignant hypertension. In contrast, other heterozygote crosses HanRen2/Han-- and HanRen2/Lew -- had an incidence in males of 18% and 0% and in females of 4% and 0% respectively. Telemetry was used to record blood pressure continuously in unrestrained conscious rats and demonstrated an accelerated rise in blood pressure in rats with clinical features of malignant phase hypertension. Histopathology showed fibrinoid necrosis and myo- intimal proliferation of afferent arterioles and small renal arteries. An associated deterioration in renal function occurred with a rise in plasma urea and creatinine. TGR(mREN2)27 normally have a suppressed renal renin -angiotensin system but in malignant phase affected animals had a significant elevation of plasma renin, angiotensin II and aldosterone. Immunohistochemistry demonstrated increased renin at the site of the afferent arterioles near the vascular poles of glomeruli. Blood films demonstrated a microangiopathic haemolytic anaemia. A genetic basis for the differing incidence of malignant phase between the three heterozygote crosses was further supported by the results of an analytical cross set up to segregate Edinburgh Sprague -Dawley alleles. Results suggested that malignant phase hypertension complicated benign hypertension due to the effects of one or possibly two genetic loci.A further study looked at the role of endothelin in malignant hypertension. Previous investigators had suggested that endothelin may be involved in the pathogenesis of malignant phase hypertension. RNase protection assays demonstrated increased expression of endothelin- 1 mRNA in kidneys from malignant hypertensive rats. Chronic inhibition of endothelin receptors using an oral non -specific endothelin receptor antagonist (Bosentan) did not prevent or reduce the transition from benign to malignant phase hypertension. It would therefore appear that endothelin synthesis occurs in response to the transition to malignant hypertension but it is not a central initiating factor.In conclusion, this is a representative model showing many of the characteristics of malignant phase hypertension in humans. The differing incidence between transgenic Ren -2 crosses appeared to be a consequence of genetic factor(s). This may therefore be another example of a genetic pre -disposition to develop target organ damage from hypertension

Cardioprotective and proangiogenic activities of small extracellular vesicles released by amniotic fluid stem cells

Protection against myocardial ischaemia/reperfusion injury and regeneration of the damaged myocardium are long-sought goals. The use of small extracellular vesicles (sEVs) released by mesenchymal stem cells (MSCs) was shown to be of benefit in the myocardial infarction setting. However, MSCs are frequently harvested from aged or diseased patients and suboptimal sEV isolation methods are used. A subtype of young, foetal MSCs, namely spindle-shaped amniotic fluid stem cells (SS-AFSCs), is known to possess better expansion and functional capacity than its adult counterparts. Here, sEVs released by SS-AFSCs were isolated using size-exclusion chromatography (SEC) – an isolation technique that yields vesicles of superior purity – and their cardioprotective and proangiogenic activities were studied. Firstly, using rat blood plasma, it was demonstrated that SEC isolates higher sEV yields with significantly compromised purity, mostly due to the presence of lipoproteins. To overcome this, a serum-free environment was used for sEVs isolation from SS-AFSC-conditioned medium. Comprehensive characterisation experiments showed that the harvested SS-AFSC sEVs are of high purity. Functionally, SS-AFSC sEVs protected the rat myocardium from ischaemia/reperfusion injury in vivo, but not isolated cardiomyocytes in vitro, indicative of indirect cardioprotective effects. Additionally, SS-AFSC sEVs promoted migration of endothelial cells in vitro and recapitulated the promigratory effects of the SS-AFSC-conditioned medium. Using pharmacological inhibition, it was shown that PI3K pathway, a known player in cell migration, mediates the sEV effects, while a series of potential candidates in the sEV cargo were excluded. Finally, cellular sEV uptake was studied by use of lipophilic dye-labelling experiments. Surprisingly, this commonly used approach was found to be unsuitable for sEV tracking due to non-specific dye retention by non-sEV contaminants. Overall, SEC-isolated SS-AFSC sEVs possess cardioprotective potential manifested only in vivo, and promigratory activity which requires PI3K signalling. These data indicate that SS-AFSC sEVs have multifactorial beneficial effects in a myocardial infarction setting