Investigation into the effects of viral-mediated Ang-(1-9) delivery on cardiac function and remodelling in a mouse model of myocardial infarction

Coronary heart disease (CHD) leading to myocardial infarction (MI) is the primary cause of morbidity and mortality globally. Following an MI, a number of structural and functional changes to the myocardium occur, known as cardiac remodelling. Initially, these changes are adaptive. Inflammation and deposition of extracellular matrix (ECM) components and collagen occurs in order to form the scar tissue, and in response to the rise in wall stress cardiomyocytes undergo adaptive hypertrophy in order to maintain contractile performance of the heart. There are also alterations in the electrical properties of the heart, including dysregulation of a variety of Ca2+-handling proteins. In the long term these processes become maladaptive. Reactive fibrosis stiffens the ventricular wall, eccentric hypertrophy contributes to expansion of the left ventricle (LV) and the electrical changes lead to reduced contraction and increased propensity to arrhythmia, all of which can contribute to the development of heart failure (HF) and the possibility of sudden cardiac death (SCD). Dysregulation of the renin-angiotensin system (RAS) is one of the factors responsible for driving these adaptive and maladaptive remodelling processes. The main effector peptide of the RAS, Angiotensin II (Ang II), acting via the Angiotensin type 1 receptor (AT1R) mediates the majority of the maladaptive changes which occur post-MI. The counter-regulatory axis of the RAS has been found to counteract many of deleterious effects associated with Ang II signalling. The peptide Ang-(1-7), signalling via Mas, has been found to exert anti-fibrotic and anti-hypertrophic effects and improve LV function post-MI. Less is known about the peptide Ang-(1-9), however there is evidence that it too is able to exert anti-hypertrophic and anti-fibrotic effects post-MI, however improvements on cardiac function have not been previously demonstrated. Therefore, the main aim of this thesis was to investigate the therapeutic potential of Ang-(1-9) via a gene transfer approach on adverse remodelling in a mouse model of MI, with a focus on cardiac functional parameters. First, the mouse model of MI was established and characterised for adverse structural and functional remodelling parameters. Haemodynamic and functional measurements using echocardiography and pressure-volume (PV) loops demonstrated a reduction in contractility and ejection fraction (EF) following MI. This was also associated with an increase in concentric cardiomyocyte hypertrophy, fibrosis and collagen deposition. Moreover, alterations in expression of cardiac AT1R, Angiotensin type 2 receptor (AT2R), Angiotensin converting enzyme (ACE) and Angiotensin converting enzyme 2 (ACE2) were detected. Following characterisation, this model was utilised to assess the effects of viral-mediated Ang-(1-9) delivery. Initially, an adenoviral vector (Ad) expressing a biological peptide pump enabling the synthetic production of Ang-(1-9) [RAdAng-(1-9)] was utilised in order to assess the effects of the peptide following MI. Efficient transduction of the healthy myocardium following MI was demonstrated using direct intramyocardial Ad injection. Initially, it was found that administration of RAdAng-(1-9) reduced the mortality rate associated with the MI procedure, with a reduction in deaths from unknown causes and cardiac rupture. Functional cardiac parameters were monitored using echocardiography for a 4 week period, with RAdAng-(1-9) administration found to be associated with increased LV fractional shortening (FS) compared to MI controls from 1 to 4 wks. PV loop measurements confirmed this improved function, with increased end systolic pressure (ESP) and normalised EF found in RAdAng-(1-9) administered animals. Post-mortem analysis and histology demonstrated an anti-hypertrophic effect of RAdAng-(1-9) delivery, with reduced heart weight and cardiomyocyte thickness in MI animals over-expressing Ang-(1-9). An anti-fibrotic effects was also evident, with a reduction in total LV fibrosis demonstrated, primarily due to reduced collagen I expression. Next, an adeno-associated virus serotype 9 (AAV9) vector expressing the Ang-(1-9) fusion protein expression cassette [AAVAng-(1-9)] was utilised in the same mouse MI model in order to assess the effects of Ang-(1-9) 8 wks post-MI. Global transduction of the healthy myocardium in MI hearts using a single tail vein injection of AAV9 was demonstrated, with transgene expression detectable as early as 1 wk post-MI. Similarly to the previous study, AAVAng-(1-9) delivery demonstrated a reduction in the incidence of cardiac rupture following MI. Echocardiography also demonstrated improvements in cardiac contractility, with increased FS evident from 1 to 8 wks post-MI in AAVAng-(1-9) transduced animals. Again, PV loop measurements found that AAVAng-(1-9) increased ESP and normalised EF. Moreover, CO was significantly elevated in Ang-(1-9) expressing animals. Due to the more advanced 8 wk time-point, a reduction in LV stiffness was detectable in AAVAng-(1-9) animals compared to controls as measured by the end diastolic pressure-volume relationship (EDPVR). In contrast to RAdAng-(1-9) administration at 4 wks post-MI, no anti-hypertrophic effect was detectable, with an increase in heart weight and cardiomyocyte thickness found in AAVAng-(1-9) animals equivalent to control MI groups. However, AAVAng-(1-9) administration was associated with a reduction in total fibrosis in MI animals, which was attributable to reduced collagen I expression. Moreover, gene expression analysis in MI animals found AT2R expression was elevated in the myocardium of AAVAng-(1-9) administered animals, which had not previously been identified. Finally, in order to attempt to elucidate the mechanism of action of Ang-(1-9), single cardiomyocyte Ca2+-handling measurements were utilised in order to assess its effects on Ca2+-handling protein function. Pre-treatment of isolated mouse cardiomyocytes with 1 µM Ang-(1-9) increased cardiomyocyte Ca2+-transient amplitude and shortening compared to control, equivalent to the effects seen using 1 µM of Ang II. However, only Ang II induced an increase in spontaneous rises in intracellular Ca2+. Ang-(1-9) pre-treatment was also associated with increased sarcoplasmic reticulum (SR) Ca2+ content. Overall the findings from this thesis have demonstrated for the first time that Ang-(1-9) exerts beneficial effects on cardiac function post-MI, which may in part be due to modulation of cardiomyocyte Ca2+-handling. These findings provide the impetus to further investigate the potential of Ang-(1-9) as a possible therapeutic agent to prevent progression of adverse cardiac remodelling and HF post-MI

Gene therapy with Angiotensin-(1-9) preserves left ventricular systolic function after myocardial infarction

BACKGROUND: Angiotensin-(1-9) [Ang-(1-9)] is a novel peptide of the counter-regulatory axis of the renin angiotensin system previously demonstrated to have therapeutic potential in hypertensive cardiomyopathy when administered via osmotic minipump in mice. Here, we investigate whether gene transfer of Ang-(1-9) is cardioprotective in a murine model of myocardial infarction (MI). OBJECTIVES: To evaluate effects of Ang-(1-9) gene therapy on myocardial structural and functional remodeling post infarction. METHODS: C57BL/6 mice underwent permanent left anterior descending coronary artery ligation and cardiac function was assessed using echocardiography for 8 weeks followed by a terminal measurement of left ventricular (LV) pressure-volume loops. Ang-(1-9) was delivered by adeno-associated viral vector via single tail vein injection immediately following induction of MI. Direct effects of Ang-(1-9) on cardiomyocyte excitation–contraction coupling and cardiac contraction were evaluated in isolated mouse and human cardiomyocytes and in an ex vivo Langendorff perfused whole heart model. RESULTS: Gene delivery of Ang-(1-9) significantly reduced sudden cardiac death post-MI. Pressure–volume measurements revealed complete restoration of end systolic pressure, ejection fraction, end systolic volume and the end diastolic pressure–volume relationship by Ang-(1-9) treatment. Stroke volume and cardiac output were significantly increased versus sham. Histological analysis revealed only mild effects on cardiac hypertrophy and fibrosis, but a significant increase in scar thickness. Direct assessment of Ang-(1-9) on isolated cardiomyocytes demonstrated a positive inotropic effect via increasing calcium transient amplitude and increasing contractility. Ang-(1-9) increased contraction in the Langendorff model through a protein kinase A-dependent mechanism. CONCLUSIONS: Our novel findings show that Ang-(1-9) gene therapy preserves LV systolic function post-MI, restoring cardiac function. Furthermore, Ang-(1-9) has a direct effect on cardiomyocyte 3 calcium handling through a protein kinase A-dependent mechanism. These data highlight Ang-(1-9) gene therapy as a potential new strategy in the context of MI

Angiotensin-(1-7) and angiotensin-(1-9): function in cardiac and vascular remodeling

The renin angiotensin system (RAS) is integral to cardiovascular physiology, however, dysregulation of this system largely contributes to the pathophysiology of cardiovascular disease (CVD). It is well established that angiotensin II (Ang II), the main effector of the RAS, engages the angiotensin type 1 receptor and promotes cell growth, proliferation, migration and oxidative stress, all processes which contribute to remodeling of the heart and vasculature, ultimately leading to the development and progression of various CVDs including heart failure and atherosclerosis. The counter-regulatory axis of the RAS, which is centered on the actions of angiotensin converting enzyme 2 (ACE2) and the resultant production of angiotensin-(1-7) (Ang-(1-7) from Ang II, antagonizes the actions of Ang II via the receptor Mas, thereby providing a protective role in CVD. More recently, another ACE2 metabolite, Ang-(1-9), has been reported to be a biologically active peptide within the counter-regulatory axis of the RAS. This review will discuss the role of the counter-regulatory RAS peptides, Ang-(1-7) and Ang-(1-9) in the cardiovascular system, with a focus on their effects in remodeling of the heart and vasculature

Runx1 deficiency protects against adverse cardiac remodeling following myocardial infarction

Background: Myocardial infarction (MI) is a leading cause of heart failure and death worldwide. Preservation of contractile function and protection against adverse changes in ventricular architecture (cardiac remodeling) are key factors to limiting progression of this condition to heart failure. Consequently, new therapeutic targets are urgently required to achieve this aim. Expression of the Runx1 transcription factor is increased in adult cardiomyocytes after MI; however, the functional role of Runx1 in the heart is unknown. Methods: To address this question, we have generated a novel tamoxifen-inducible cardiomyocyte-specific Runx1-deficient mouse. Mice were subjected to MI by means of coronary artery ligation. Cardiac remodeling and contractile function were assessed extensively at the whole-heart, cardiomyocyte, and molecular levels. Results: Runx1-deficient mice were protected against adverse cardiac remodeling after MI, maintaining ventricular wall thickness and contractile function. Furthermore, these mice lacked eccentric hypertrophy, and their cardiomyocytes exhibited markedly improved calcium handling. At the mechanistic level, these effects were achieved through increased phosphorylation of phospholamban by protein kinase A and relief of sarco/endoplasmic reticulum Ca2+-ATPase inhibition. Enhanced sarco/endoplasmic reticulum Ca2+-ATPase activity in Runx1-deficient mice increased sarcoplasmic reticulum calcium content and sarcoplasmic reticulum–mediated calcium release, preserving cardiomyocyte contraction after MI. Conclusions: Our data identified Runx1 as a novel therapeutic target with translational potential to counteract the effects of adverse cardiac remodeling, thereby improving survival and quality of life among patients with MI

Basic science232. Certolizumab pegol prevents pro-inflammatory alterations in endothelial cell function

Background: Cardiovascular disease is a major comorbidity of rheumatoid arthritis (RA) and a leading cause of death. Chronic systemic inflammation involving tumour necrosis factor alpha (TNF) could contribute to endothelial activation and atherogenesis. A number of anti-TNF therapies are in current use for the treatment of RA, including certolizumab pegol (CZP), (Cimzia ®; UCB, Belgium). Anti-TNF therapy has been associated with reduced clinical cardiovascular disease risk and ameliorated vascular function in RA patients. However, the specific effects of TNF inhibitors on endothelial cell function are largely unknown. Our aim was to investigate the mechanisms underpinning CZP effects on TNF-activated human endothelial cells. Methods: Human aortic endothelial cells (HAoECs) were cultured in vitro and exposed to a) TNF alone, b) TNF plus CZP, or c) neither agent. Microarray analysis was used to examine the transcriptional profile of cells treated for 6 hrs and quantitative polymerase chain reaction (qPCR) analysed gene expression at 1, 3, 6 and 24 hrs. NF-κB localization and IκB degradation were investigated using immunocytochemistry, high content analysis and western blotting. Flow cytometry was conducted to detect microparticle release from HAoECs. Results: Transcriptional profiling revealed that while TNF alone had strong effects on endothelial gene expression, TNF and CZP in combination produced a global gene expression pattern similar to untreated control. The two most highly up-regulated genes in response to TNF treatment were adhesion molecules E-selectin and VCAM-1 (q 0.2 compared to control; p > 0.05 compared to TNF alone). The NF-κB pathway was confirmed as a downstream target of TNF-induced HAoEC activation, via nuclear translocation of NF-κB and degradation of IκB, effects which were abolished by treatment with CZP. In addition, flow cytometry detected an increased production of endothelial microparticles in TNF-activated HAoECs, which was prevented by treatment with CZP. Conclusions: We have found at a cellular level that a clinically available TNF inhibitor, CZP reduces the expression of adhesion molecule expression, and prevents TNF-induced activation of the NF-κB pathway. Furthermore, CZP prevents the production of microparticles by activated endothelial cells. This could be central to the prevention of inflammatory environments underlying these conditions and measurement of microparticles has potential as a novel prognostic marker for future cardiovascular events in this patient group. Disclosure statement: Y.A. received a research grant from UCB. I.B. received a research grant from UCB. S.H. received a research grant from UCB. All other authors have declared no conflicts of interes

Intestinal Permeation Enhancers for Oral Delivery of Macromolecules: A Comparison between Salcaprozate Sodium (SNAC) and Sodium Caprate (C10)

Salcaprozate sodium (SNAC) and sodium caprate (C10) are two of the most advanced intestinal permeation enhancers (PEs) that have been tested in clinical trials for oral delivery of macromolecules. Their effects on intestinal epithelia were studied for over 30 years, yet there is still debate over their mechanisms of action. C10 acts via openings of epithelial tight junctions and/or membrane perturbation, while for decades SNAC was thought to increase passive transcellular permeation across small intestinal epithelia based on increased lipophilicity arising from non-covalent macromolecule complexation. More recently, an additional mechanism for SNAC associated with a pH-elevating, monomer-inducing, and pepsin-inhibiting effect in the stomach for oral delivery of semaglutide was advocated. Comparing the two surfactants, we found equivocal evidence for discrete mechanisms at the level of epithelial interactions in the small intestine, especially at the high doses used in vivo. Evidence that one agent is more efficacious compared to the other is not convincing, with tablets containing these PEs inducing single-digit highly variable increases in oral bioavailability of payloads in human trials, although this may be adequate for potent macromolecules. Regarding safety, SNAC has generally regarded as safe (GRAS) status and is Food and Drug Administration (FDA)-approved as a medical food (Eligen®-Vitamin B12, Emisphere, Roseland, NJ, USA), whereas C10 has a long history of use in man, and has food additive status. Evidence for co-absorption of microorganisms in the presence of either SNAC or C10 has not emerged from clinical trials to date, and long-term effects from repeat dosing beyond six months have yet to be assessed. Since there are no obvious scientific reasons to prefer SNAC over C10 in orally delivering a poorly permeable macromolecule, then formulation, manufacturing, and commercial considerations are the key drivers in decision-making.European Commission Horizon 2020Science Foundation IrelandSANOFI-AVENTIS Recherche & DevelopmentFrench National Agency of Research and TechnologyCÚRAM Center for Medical Device

Association between prior physical activity and 30-day prognostic following a myocardial infarction

Background: The benefits of physical activity (PA) in prevention of Myocardial Infarcion (MI) are well established, however few studies correlate the severity of the infarction and complexity of myocardial injury with the PA level of the patients who suffered it. Aim: To evaluate the association between PA level and post-MI prognosis. Methods: A prospective cohort study, nested inside the Catarina Heart Study cohort with patients who had a MI between July (2016) and November (2019). The PA level was measured through the Baecke’s questionnaire in order to try to associate it with myocardial injury severity indicators: Syntax score, left ventricle ejection fraction (LVEF) and combined cardiovascular events (CCE). The analysis was made through the T-Test, Chi-Squared. Results: The study showed lower EB score for: (a) patients who had a CCE within 30 days [4,0(3,3-5,0) e p=0,007] compared to those who had not [4,5(3,8-5,5)], (b) pacientes who had rehospitalization within 30 days [3,7(3,0-4,8)] compared with those who had not [4,5(3,8-5,5) e p<0,001)] and (c) patienst who suffered cardiovascular death [5,2(5,0-7,8) e p=0,012)] compared with those who had not [4,5(3,5-5,5)]. There was no correlation between EB and LVEF, TIMI Frame count or Syntax. Conclusion: This study shows that previous PA to a MI may improve 30-day prognosis, with lesser rehospitalization, cardiovascular death and CCE. Keywords: physical activity; myocardial infarction; coronary artery disease; physical exercise, ischemiaIntrodução: Os benefícios da atividade física (AF) na prevenção do Infarto Agudo do Miocárdio (IAM) são conhecidos, porém poucos estudos correlacionam a gravidade do infarto e a complexidade das lesões coronarianas com o grau de AF prévio dos pacientes que infartam. Objetivo: Avaliar a associação entre o grau de AF e prognóstico pós IAM. Métodos: Estudo de coorte prospectiva, aninhado a Coorte denominada Catarina Heart Study em pacientes com IAM no período de Julho de 2016 a Novembro de 2019. O grau de AF foi avaliado pelo Escore de Baecke (EB) para tentar encontrar associação com dados indicadores de gravidade de lesão miocárdica: escore Syntax, Fração de Ejeção Ventricular Esquerda, TIMI Frame e evento cardiovascular combinado (ECC). A análise prevista foi feita através do Teste de Mann Whitney e correlação de Sperman. Resultados: O estudo evidenciou menor escore de EB para: (a) pacientes que tiveram ECC em 30 dias [4,0(3,3-5,0) e p=0,007] comparado com àqueles que não tiveram [4,5(3,8-5,5)], (b) pacientes que reinternaram em 30 dias [3,7(3,0-4,8)] comparados àqueles que não reinternaram [4,5(3,8-5,5) e p<0,001)] e (c) pacientes que sofreram morte de etiologia cardiovascular [5,2(5,0-7,8) e p=0,012)] comparados àqueles que não tiveram [4,5(3,5-5,5)]. Não foi possível estabelecer correlação entre EB e FEVE, TIMI Frame ou Syntax. Conclusão: O presente estudo evidencia que a AF prévia a um evento de IAM pode melhorar o prognóstico nos primeiros 30 dias, com menos reinternações, mortalidade de etiologia cardiovascular e ECC. Palavras-chave: atividade física; infarto do miocárdio; doença arterial coronariana; exercício físico; isquemi

Intestinal permeation enhancers for oral delivery of macromolecules: A comparison between salcaprozate sodium (SNAC) and sodium caprate (C10)

Salcaprozate sodium (SNAC) and sodium caprate (C10) are two of the most advanced intestinal permeation enhancers (PEs) that have been tested in clinical trials for oral delivery of macromolecules. Their effects on intestinal epithelia were studied for over 30 years, yet there is still debate over their mechanisms of action. C10 acts via openings of epithelial tight junctions and/or membrane perturbation, while for decades SNAC was thought to increase passive transcellular permeation across small intestinal epithelia based on increased lipophilicity arising from noncovalent macromolecule complexation. More recently, an additional mechanism for SNAC associated with a pH-elevating, monomer-inducing, and pepsin-inhibiting effect in the stomach for oral delivery of semaglutide was advocated. Comparing the two surfactants, we found equivocal evidence for discrete mechanisms at the level of epithelial interactions in the small intestine, especially at the high doses used in vivo. Evidence that one agent is more efficacious compared to the other is not convincing, with tablets containing these PEs inducing single-digit highly variable increases in oral bioavailability of payloads in human trials, although this may be adequate for potent macromolecules. Regarding safety, SNAC has generally regarded as safe (GRAS) status and is Food and Drug Administration (FDA)-approved as a medical food (Eligen®-Vitamin B12, Emisphere, Roseland, NJ, USA), whereas C10 has a long history of use in man, and has food additive status. Evidence for co-absorption of microorganisms in the presence of either SNAC or C10 has not emerged from clinical trials to date, and long-term effects from repeat dosing beyond six months have yet to be assessed. Since there are no obvious scientific reasons to prefer SNAC over C10 in orally delivering a poorly permeable macromolecule, then formulation, manufacturing, and commercial considerations are the key drivers in decision-making