Tropoelastin Improves Post-Infarct Cardiac Function.

BACKGROUND: Myocardial infarction (MI) is among the leading causes of death worldwide. Following MI, necrotic cardiomyocytes are replaced by a stiff collagen-rich scar. Compared to collagen, the extracellular matrix protein elastin has high elasticity and may have more favorable properties within the cardiac scar. We sought to improve post-MI healing by introducing tropoelastin, the soluble subunit of elastin, to alter scar mechanics early after MI. METHODS AND RESULTS: We developed an ultrasound-guided direct intramyocardial injection method to administer tropoelastin directly into the left ventricular anterior wall of rats subjected to induced MI. Experimental groups included shams and infarcted rats injected with either PBS vehicle control or tropoelastin. Compared to vehicle treated controls, echocardiography assessments showed tropoelastin significantly improved left ventricular ejection fraction (64.7±4.4% versus 46.0±3.1% control) and reduced left ventricular dyssynchrony (11.4±3.5 ms versus 31.1±5.8 ms control) 28 days post-MI. Additionally, tropoelastin reduced post-MI scar size (8.9±1.5% versus 20.9±2.7% control) and increased scar elastin (22±5.8% versus 6.2±1.5% control) as determined by histological assessments. RNA sequencing (RNAseq) analyses of rat infarcts showed that tropoelastin injection increased genes associated with elastic fiber formation 7 days post-MI and reduced genes associated with immune response 11 days post-MI. To show translational relevance, we performed immunohistochemical analyses on human ischemic heart disease cardiac samples and showed an increase in tropoelastin within fibrotic areas. Using RNA-seq we also demonstrated the tropoelastin gene ELN is upregulated in human ischemic heart disease and during human cardiac fibroblast-myofibroblast differentiation. Furthermore, we showed by immunocytochemistry that human cardiac fibroblast synthesize increased elastin in direct response to tropoelastin treatment. CONCLUSIONS: We demonstrate for the first time that purified human tropoelastin can significantly repair the infarcted heart in a rodent model of MI and that human cardiac fibroblast synthesize elastin. Since human cardiac fibroblasts are primarily responsible for post-MI scar synthesis, our findings suggest exciting future clinical translation options designed to therapeutically manipulate this synthesis.

Ghrelin mediated cardioprotection using in vitro models of oxidative stress.

Ghrelin is commonly known as the 'hunger hormone' due to its role in stimulating food intake in humans. However, the roles of ghrelin extend beyond regulating hunger. Our aim was to investigate the ability of ghrelin to protect against hydrogen peroxide (H2O2), a reactive oxygen species commonly associated with cardiac injury. An in vitro model of oxidative stress was developed using H2O2 injured H9c2 cells. Despite lentiviral ghrelin overexpression, H9c2 cell viability and mitochondrial function were not protected following H2O2 injury. We found that H9c2 cells lack expression of the preproghrelin cleavage enzyme prohormone convertase 1 (encoded by PCSK1), required to convert ghrelin to its active form. In contrast, we found that primary rat cardiomyocytes do express PCSK1 and were protected from H2O2 injury by lentiviral ghrelin overexpression. In conclusion, we have shown that ghrelin expression can protect primary rat cardiomyocytes against H2O2, though this effect was not observed in other cell types tested.

Overexpression of multidrug resistance-associated protein 1 protects against cardiotoxicity by augmenting the doxorubicin efflux from cardiomyocytes.

Doxorubicin is a commonly used anti-cancer drug used in treating a variety of malignancies. However, a major adverse effect is cardiotoxicity, which is dose dependent and can be either acute or chronic. Doxorubicin causes injury by DNA damage, the formation of free reactive oxygen radicals and induction of apoptosis. Our aim is to induce expression of the multidrug resistance-associated protein 1 (MRP1) in cardiomyocytes derived from human iPS cells (hiPSC-CM), to determine whether this will allow cells to effectively remove doxorubicin and confer cardioprotection. We generated a lentivirus vector encoding MRP1 (LV.MRP1) and validated its function in HEK293T cells and stem cell-derived cardiomyocytes (hiPSC-CM) by quantitative PCR and western blot analysis. The activity of the overexpressed MRP1 was also tested, by quantifying the amount of fluorescent dye exported from the cell by the transporter. We demonstrated reduced dye sequestration in cells overexpressing MRP1. Finally, we demonstrated that hiPSC-CM transduced with LV.MRP1 were protected against doxorubicin injury. In conclusion, we have shown that we can successfully overexpress MRP1 protein in hiPSC-CM, with functional transporter activity leading to protection against doxorubicin-induced toxicity.

Optimizing electrophysiology studies to prevent sudden cardiac death after myocardial infarction.

AIMS: This study assessed associations of minimum final extrastimulus coupling interval utilized within electrophysiology study (EPS) after myocardial infarction (MI) and possible site of origin of induced ventricular tachycardia (VT) with long-term occurrence of spontaneous ventricular tachyarrhythmia and long-term survival. METHODS AND RESULTS: This prospective study recruited consecutive patients with left ventricular ejection fraction (LVEF) ≤ 40% who underwent EPS days 3-5 after MI between 2004 and 2017. Positive EPS was defined as sustained monomorphic VT cycle length ≥200 ms for ≥10 s or shorter duration if haemodynamic compromise occurred. Each of the four extrastimuli was shortened by 10 ms at a time, until it failed to capture the ventricle (ventricular refractoriness) or induced ventricular tachyarrhythmia. Outcomes included spontaneous ventricular tachyarrhythmia occurrence and all-cause mortality. Shorter coupling interval length of final extrastimulus that induced VT was associated with higher risk of spontaneous ventricular tachyarrhythmia (P < 0.001). Significantly higher rates of spontaneous ventricular tachyarrhythmia (65.2% vs. 23.2%; P < 0.001) were observed for final coupling interval at EPS <200 ms vs. >200 ms. Right bundle branch block (RBBB) morphology of induced VT, with possible site of origin from the left ventricle, was associated with all-cause mortality [hazard ratio (HR) 3.2, P = 0.044] and a composite of spontaneous ventricular tachyarrhythmia recurrence or mortality (HR 1.8, P = 0.043). CONCLUSION: Ventricular tachycardia induced with shorter coupling intervals was associated with higher risk of spontaneous ventricular tachyarrhythymia on follow-up, indicating that the final extrastimulus coupling interval at EPS early after MI should be determined by ventricular refractoriness. Induced VT with possible origin from left ventricle was associated with increased risk of spontaneous ventricular tachyarrhythmia recurrence or death.

Emergent Lag Phase in Flux-Regulation Models of Bacterial Growth.

Lag phase is observed in bacterial growth during a sudden change in conditions: growth is inhibited whilst cells adapt to the environment. Bi-phasic, or diauxic growth is commonly exhibited by many species. In the presence of two sugars, cells initially grow by consuming the preferred sugar then undergo a lag phase before resuming growth on the second. Biomass increase is characterised by a diauxic growth curve: exponential growth followed by a period of no growth before a second exponential growth. Recent literature lacks a complete dynamic description, artificially modelling lag phase and employing non-physical representations of precursor pools. Here, we formulate a rational mechanistic model based on flux-regulation/proteome partitioning with a finite precursor pool that reveals core mechanisms in a compact form. Unlike earlier systems, the characteristic dynamics emerge as part of the solution, including the lag phase. Focussing on growth of Escherichia coli on a glucose-lactose mixture we show results accurately reproduce experiments. We show that for a single strain of E. coli, diauxic growth leads to optimised biomass yields. However, intriguingly, for two competing strains diauxic growth is not always the best strategy. Our description can be generalised to model multiple different microorganisms and investigate competition between species/strains.

Guideline-based audit of the hospital management of heart failure with reduced ejection fraction.

BACKGROUND: Heart failure is a major burden in Australia in terms of morbidity, mortality and healthcare expenditure. Multiple evidence-based therapies are recommended for heart failure with reduced ejection fraction (HFrEF), but data on physician adherence to therapy guidelines are limited. AIM: To compare use of HFrEF therapies against current evidence-based guidelines in an Australian hospital inpatient population. METHODS: A retrospective review of patients admitted with a principal diagnosis of HFrEF across six metropolitan hospitals in Sydney, Australia, between January 2015 and June 2016. Use of medical and device therapies was compared with guideline recommendations using individual patient indications/contraindications. Readmission and mortality data were collected for a 1-year period following the admission. RESULTS: Of the 1028 HFrEF patients identified, 39 were being managed with palliative intent, leaving 989 patients for the primary analysis. Use of beta-blockers (87.7% actual use/93.6% recommended use) and diuretics (88.4%/99.3%) was high among eligible patients. There were large evidence-practice gaps for angiotensin-converting enzyme inhibitors/angiotensin II receptor blockers (ACEI/ARB; 66.4%/89.0%) and aldosterone antagonists (41.0%/77.1%). In absolute terms, use of these therapies each increased by over 11% from admission. Ivabradine (11.5%/21.2%), automated internal cardiac defibrillators (29.5%/66.1%) and cardiac resynchronisation therapy (13.1%/28.7%) were used in a minority of eligible patients. Over the 1-year follow-up period, the mortality rate was 14.8%, and 44.2% of patients were readmitted to hospital at least once. CONCLUSION: Hospitalisation is a key mechanism for initiation of HFrEF therapies. The large evidence-practice gaps for ACEI/ARB and aldosterone antagonists represent potential avenues for improved HFrEF management.

Implementation of anaerobic digestion for valorizing the organic fraction of municipal solid waste in developing countries: Technical insights from a systematic review.

Anaerobic digestion (AD) as a waste management strategy for the organic fraction of municipal waste (OFMSW) has received attention in developed countries for several decades, leading to the development of large-scale plants. In contrast, AD of OFMSW has only recently drawn attention in developing countries. This systematic review was carried out to investigate the implementation of AD to treat the OFMSW in developing countries, focusing on assessing pilot and full-scale AD plants reported in the last ten years. Studies that met the selection criteria were analyzed and data regarding operating parameters, feedstock characteristics, and biogas, digestate, and energy production were extracted. As outlined in this systematic review, AD plants located in developing countries are mostly one-stage mesophilic systems that treat OFMSW via mono-digestion, almost exclusively with the aim of producing electrical energy. Based on the analysis done throughout this systematic review, it was noted that there is a large difference in the maturity level of AD systems between developing and developed countries, mainly due to the economic capacity of developed countries to invest in sustainable waste management systems. However, the number of AD plants reported in scientific papers is significantly lower than the number of installed AD systems. Research articles regarding large-scale implementation of AD to treat OFMSW in developed countries were analyzed and compared with developing countries. This comparison identified practices used in plants in developed countries that could be utilized in the large-scale implementation and success of AD in developing countries. These practices include exploiting potential products with high market-values, forming partnerships with local industries to use industrial wastes as co-substrates, and exploring different biological and physical pretreatment technologies. Additionally, the analysis of capital and operational costs of AD plants showed that costs tend to be higher for developing countries due to their need to import of materials and equipment from developed countries. Technical, economical, and political challenges for the implementation of AD at a large-scale in developing countries are highlighted.

Therapeutic Angiogenesis Using Growth Factors After Myocardial Infarction: From Recombinant Proteins to Gene Therapies and Beyond.

Ischaemic heart disease is the primary cause of death worldwide with myocardial infarction (MI) contributing to significant morbidity and mortality. The human heart has a limited capacity to regenerate and the significant loss of cardiomyocytes after MI can overwhelm this limited innate regenerative capability. This is in part compensated for by the creation of collagen-rich scar tissue. Therapeutic angiogenesis is an exciting prospect that can assist cardiac regeneration after MI with various approaches having been explored. This review will focus on results from clinical growth factor trials, and the lack of clinical translation. Inconsistencies in results from these may be due to heterogeneity within patient selection and an incomplete understanding of therapeutic differences between isoforms of active agents. The technology used has also evolved with recombinant protein and, subsequently, gene therapy being utilised. Innovative therapeutic designs, such as combinatorial therapies, might help to resolve these issues in the future.

Trends in Modifiable Risk Factors Amongst First Presentation ST Elevation Myocardial Infarction Patients in a Large Longitudinal Registry.

BACKGROUND: Recent studies suggest that the risk factor profile of patients presenting with ST elevation myocardial infarction (STEMI) is changing. AIM: The aim is to determine if there has been a shift of cardiovascular risk factors to cardiometabolic causes in the first presentation STEMI population. METHOD: We analysed data from a STEMI registry from a large tertiary referral percutaneous coronary intervention centre to determine the prevalence and trends of the modifiable risk factors of hypertension, diabetes, smoking and hypercholesterolaemia. PARTICIPANTS: Consecutive first presentation STEMI patients between January 2006 to December 2018. RESULTS: Among the 2,366 patients included (mean age 59, SD 12.66, 80% male) the common risk factors were hypertension (47%), hypercholesterolaemia (47%) current smoking (42%) and diabetes (27%). Over the 13 years, patients with diabetes (20% to 26%, OR 1.09 per year, CI 1.06-1.11, p<0.001) and patients with no modifiable risk factors increased (9% to 17%, OR 1.08, CI 1.04-1.11, p<0.001). Concurrently there was a fall in prevalence of hypercholesterolaemia, (47% to 37%, OR 0.94 per year, CI 0.92-0.96, p<0.001) and smoking (44% to 41%, OR 0.94, CI 0.92-0.96, p<0.001) but no significant change in rates of hypertension (53% to 49%, OR 0.99, CI 0.97-1.01, p=0.25). CONCLUSION: The risk factor profile of first presentation STEMI has changed over time with a reduction in smoking and a concurrent rise in patients with no traditional risk factors. This suggests the mechanism of STEMI may be changing and further investigation of potential causal factors is warranted for the prevention and management of cardiovascular disease.

Cardiac Cell Therapy with Pluripotent Stem Cell-Derived Cardiomyocytes: What Has Been Done and What Remains to Do?

PURPOSE OF REVIEW: Exciting pre-clinical data presents pluripotent stem cell-derived cardiomyocytes (PSC-CM) as a novel therapeutic prospect following myocardial infarction, and worldwide clinical trials are imminent. However, despite notable advances, several challenges remain. Here, we review PSC-CM pre-clinical studies, identifying key translational hurdles. We further discuss cell production and characterization strategies, identifying markers that may help generate cells which overcome these barriers. RECENT FINDINGS: PSC-CMs can robustly repopulate infarcted myocardium with functional, force generating cardiomyocytes. However, current differentiation protocols produce immature and heterogenous cardiomyocytes, creating related issues such as arrhythmogenicity, immunogenicity and poor engraftment. Recent efforts have enhanced our understanding of cardiovascular developmental biology. This knowledge may help implement novel differentiation or gene editing strategies that could overcome these limitations. PSC-CMs are an exciting therapeutic prospect. Despite substantial recent advances, limitations of the technology remain. However, with our continued and increasing biological understanding, these issues are addressable, with several worldwide clinical trials anticipated in the coming years.

Recombinant Adeno-Associated Viral Vector-Mediated Gene Transfer of hTBX18 Generates Pacemaker Cells from Ventricular Cardiomyocytes.

Sinoatrial node dysfunction can manifest as bradycardia, leading to symptoms of syncope and sudden cardiac death. Electronic pacemakers are the current standard of care but are limited due to a lack of biological chronotropic control, cost of revision surgeries, and risk of lead- and device-related complications. We therefore aimed to develop a biological alternative to electronic devices by using a clinically relevant gene therapy vector to demonstrate conversion of cardiomyocytes into sinoatrial node-like cells in an in vitro context. Neonatal rat ventricular myocytes were transduced with recombinant adeno-associated virus vector 6 encoding either hTBX18 or green fluorescent protein and maintained for 3 weeks. At the endpoint, qPCR, Western blot analysis and immunocytochemistry were used to assess for reprogramming into pacemaker cells. Cell morphology and Arclight action potentials were imaged via confocal microscopy. Compared to GFP, hTBX18-transduced cells showed that hTBX18, HCN4 and Cx45 were upregulated. Cx43 was significantly downregulated, while sarcomeric α-actinin remained unchanged. Cardiomyocytes transduced with hTBX18 acquired the tapering morphology of native pacemaker cells, as compared to the block-like, striated appearance of ventricular cardiomyocytes. Analysis of the action potentials showed phase 4 depolarization and a significant decrease in the APD50 of the hTBX18-transduced cells. We have demonstrated that rAAV-hTBX18 gene transfer to ventricular myocytes results in morphological, molecular, physiological, and functional changes, recapitulating the pacemaker phenotype in an in vitro setting. The generation of these induced pacemaker-like cells using a clinically relevant vector opens new prospects for biological pacemaker development.

Pluripotent stem cell-derived mesenchymal stromal cells improve cardiac function and vascularity after myocardial infarction.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have been shown to improve cardiac function after injury and are the subject of ongoing clinical trials. In this study, the authors tested the cardiac regenerative potential of an induced pluripotent stem cell-derived MSC (iPSC-MSC) population (Cymerus MSCs) in a rat model of myocardial ischemia-reperfusion (I/R). Furthermore, the authors compared this efficacy with bone marrow-derived MSCs (BM-MSCs), which are the predominant cell type in clinical trials. METHODS: Four days after myocardial I/R injury, rats were randomly assigned to (i) a Cymerus MSC group (n = 15), (ii) a BM-MSC group (n = 15) or (iii) a vehicle control group (n = 14). For cell-treated animals, a total of 5 × 106 cells were injected at three sites within the infarcted left ventricular (LV) wall. RESULTS: One month after cell transplantation, Cymerus MSCs improved LV function (assessed by echocardiography) compared with vehicle and BM-MSCs. Interestingly, Cymerus MSCs enhanced angiogenesis without sustained engraftment or significant impact on infarct scar size. Suggesting safety, Cymerus MSCs had no effect on inducible tachycardia or the ventricular scar heterogeneity that provides a substrate for cardiac re-entrant circuits. CONCLUSIONS: The authors here demonstrate that intra-myocardial administration of iPSC-MSCs (Cymerus MSCs) provide better therapeutic effects compared with conventional BM-MSCs in a rodent model of myocardial I/R. Because of its manufacturing scalability, iPSC-MSC therapy offers an exciting opportunity for an "off-the-shelf" stem cell therapy for cardiac repair.

Chromosomal location of the crown rust resistance gene Pc98 in cultivated oat (Avena sativa L.).

KEY MESSAGE: SNP loci linked to the crown rust resistance gene Pc98 were identified by linkage analysis and KASP assays were developed for marker-assisted selection in breeding programs. Crown rust is among the most damaging diseases of oat and is caused by Puccinia coronata var. avenae f. sp. avenae (Urban and Marková) (Pca). Host resistance is the preferred method to prevent crown rust epidemics. Pc98 is a race-specific, seedling crown rust resistance gene obtained from the wild oat Avena sterilis accession CAV 1979 that is effective at all growth stages of oat. Virulence to Pc98 has been very low in the Pca populations that have been tested. The objectives of this study were to develop SNP markers linked to Pc98 for use in marker-assisted selection and to locate Pc98 on the oat consensus map. The Pc98 gene was mapped using F2:3 populations developed from the crosses Pc98/Bingo and Pc98/Kasztan, where Pc98 is a single-gene line carrying Pc98. Both populations were evaluated in seedling inoculation experiments. Pc98 was mapped relative to Kompetitive Allele-Specific PCR SNP markers in both populations, placing Pc98 on the Mrg20 linkage group of the consensus map. Pc98 was bracketed by two SNP markers GMI_ES22_c3052_382_kom399 and GMI_ES14_lrc18344_662_kom398 in the Pc98/Bingo mapping population with genetic distances of 0.9 cM and 0.3 cM, respectively. Pc98 co-segregated with four SNP markers in the Pc98/Kasztan population, and the closest flanking markers were GMI_DS_LB_6017_kom367 and avgbs2_153634.1.59_kom410 with genetic distances of 0.7 cM and 0.3 cM, respectively. Two SNP loci defined a haplotype that accurately predicted Pc98 status in a diverse group of oat germplasm, which will be valuable for marker-assisted selection of Pc98 in breeding of new oat cultivars.

The MIC-KEY button vesicostomy: a superior alternative for suprapubic drainage?

OBJECTIVES: To evaluate the MIC-KEY button vesicostomy as an alternative to indwelling suprapubic catheters (SPCs) for bladder drainage in adults. PATIENTS AND METHODS: Phase II pilot study prospectively evaluating patients with indwelling SPCs that were converted to MIC-KEY buttons, or cystoscopic-guided de novo insertion, between November 2014 and February 2019. In all, 15 patients (14 female, one male) had indwelling SPCs that had conversion or attempted conversion to MIC-KEY button, and one (male) had a cystoscopic-guided de novo insertion with a history of previous suprapubic catheterisation. The mean (range) age was 44.2 (13-73) years. Catheter-related quality-of-life (C-IQoL) questionnaire data were collected at baseline and 3 months. RESULTS: Two patients had attempted conversion but were abandoned perioperatively due to sizing issues and insertion difficulties, respectively. Three patients were subsequently converted back to a SPC; due to button sizing (18 days), leaking (3 months), and recurrent infection (13 months). The remaining 11 patients have remained well with continued drainage via the MIC-KEY button; mean (range) duration since conversion was 34.2 (5-105) months. The C-IQoL score improved 3 months after insertion, from 50.0 to 75.4. Changes were performed dependent on patient's personalised management, typically every 3 months, under local or general anaesthetic. CONCLUSION: The MIC-KEY button is a safe alternative to SPC drainage in adults in the short- to medium-term, in a selected cohort.

Human embryonic-stem-cell-derived cardiomyocytes regenerate non-human primate hearts.

Pluripotent stem cells provide a potential solution to current epidemic rates of heart failure by providing human cardiomyocytes to support heart regeneration. Studies of human embryonic-stem-cell-derived cardiomyocytes (hESC-CMs) in small-animal models have shown favourable effects of this treatment. However, it remains unknown whether clinical-scale hESC-CM transplantation is feasible, safe or can provide sufficient myocardial regeneration. Here we show that hESC-CMs can be produced at a clinical scale (more than one billion cells per batch) and cryopreserved with good viability. Using a non-human primate model of myocardial ischaemia followed by reperfusion, we show that cryopreservation and intra-myocardial delivery of one billion hESC-CMs generates extensive remuscularization of the infarcted heart. The hESC-CMs showed progressive but incomplete maturation over a 3-month period. Grafts were perfused by host vasculature, and electromechanical junctions between graft and host myocytes were present within 2 weeks of engraftment. Importantly, grafts showed regular calcium transients that were synchronized to the host electrocardiogram, indicating electromechanical coupling. In contrast to small-animal models, non-fatal ventricular arrhythmias were observed in hESC-CM-engrafted primates. Thus, hESC-CMs can remuscularize substantial amounts of the infarcted monkey heart. Comparable remuscularization of a human heart should be possible, but potential arrhythmic complications need to be overcome.

Mapping of the Oat Crown Rust Resistance Gene Pc39 Relative to Single Nucleotide Polymorphism Markers.

Crown rust, caused by Puccinia coronata f. sp. avenae Eriks. (Pca), is among the most important oat diseases resulting in significant yield losses in many growing regions. A gene-for-gene interaction is well established in this pathosystem and has been exploited by oat breeders to control crown rust. Pc39 is a seedling crown rust resistance gene that has been widely deployed in North American oat breeding. DNA markers are desired to accurately predict the specific Pc genes present in breeding germplasm. The objectives of the study were as follows: (i) to map Pc39 in two recombinant inbred line (RIL) populations (AC Assiniboia/MN841801 and AC Medallion/MN841801) and (ii) to identify single nucleotide polymorphism (SNP) markers for postulation of Pc39 in oat germplasm. Pc39 was mapped to a linkage group consisting of 16 SNP markers, which placed the gene on linkage group Mrg11 (chromosome 1C) of the oat consensus map. Pc39 cosegregated with SNP marker GMI_ES01_c12570_390 in the AC Assiniboia/MN841801 RIL population and was flanked by the SNP markers avgbs_126086.1.41 and GMI_ES15_c276_702, with genetic distances of 1.7 and 0.3 cM, respectively. In the AC Medallion/MN841801 RIL population, similar results were obtained but the genetic distances of the flanking markers were 0.4 and 0.4 cM, respectively. Kompetitive Allele-Specific PCR assays were successfully designed for Pc39-linked SNP loci. Two SNP loci defined a haplotype that accurately predicted Pc39 status in a diverse panel of oat germplasm and will be useful for marker-assisted selection in oat breeding.

Analysis of recombinant adeno-associated viral vector shedding in sheep following intracoronary delivery.

Differences between mouse and human hearts pose a significant limitation to the value of small animal models when predicting vector behavior following recombinant adeno-associated viral (rAAV) vector-mediated cardiac gene therapy. Hence, sheep have been adopted as a preclinical animal, as they better model the anatomy and cardiac physiological processes of humans. There is, however, no comprehensive data on the shedding profile of rAAV in sheep following intracoronary delivery, so as to understand biosafety risks in future preclinical and clinical applications. In this study, sheep received intracoronary delivery of rAAV serotypes 2/6 (2 × 1012 vg), 2/8, and 2/9 (1 × 1013 vg) at doses previously administered in preclinical and clinical trials. This was followed by assessment over 96 h to examine vector shedding in urine, feces, nasal mucus, and saliva samples. Vector genomes were detected via real-time quantitative PCR in urine and feces up to 48 and 72 h post vector delivery, respectively. Of these results, functional vector particles were only detected via a highly sensitive infectious replication assay in feces samples up to 48 h following vector delivery. We conclude that rAAV-mediated gene transfer into sheep hearts results in low-grade shedding of non-functional vector particles for all excreta samples, except in the case of feces, where functional vector particles are present up to 48 h following vector delivery. These results may be used to inform containment and decontamination guidelines for large animal dealings, and to understand the biosafety risks associated with future preclinical and clinical uses of rAAV.

Vitamin D Improves Cardiac Function After Myocardial Infarction Through Modulation of Resident Cardiac Progenitor Cells.

BACKGROUND: Vitamin D has been implicated in the prevention of heart failure. However the underlying mechanism remains unclear. We hypothesised that these effects may be partially mediated by cardiac stem/progenitor cells (CPCs). Therefore, we examined the effects of 1,25-dihydroxyvitamin D3 (1,25D) on cell cycle activity and differentiation of a previously described CPC population called cardiac colony-forming unit fibroblasts (cCFU-Fs). METHODS: cCFU-Fs were isolated from adult male C57Bl/6 mouse hearts using fluorescence-activated cell sorting. The effect of 1,25D on cell proliferation and differentiation were was assessed by colony-forming and fibroblast differentiation assays. Cell cycle was analysed by flow cytometry. Mice with induced myocardial infarction (MI) were treated with 1,25D or vehicle controls and cardiac function assessed by echocardiography. RESULTS: 1,25D dose-dependently increased expression of vitamin D receptor (Vdr) and reduced large colony formation. Addition of 1,25D to cCFU-Fs slowed cell proliferation, promoted cell cycle arrest and decreased expression of pro-fibrotic factors during TGF-ß-induced fibroblast differentiation of cCFU-Fs. After MI, 1,25D-treated mice had less left ventricular wall thinning and significant improvement in left ventricular systolic function compared to vehicle-treated controls. Although no significant changes in myocardial fibrotic area and cardiomyocyte size were noted, treatment with 1,25D significantly inhibited cardiac interstitial cell proliferation after MI. CONCLUSIONS: Vitamin D signalling promotes cardioprotection after myocardial infarction. This may be through modulation of cCFU-F cell cycle. The role of 1,25D and VDR in regulating cardiac stem/progenitor cell function therefore warrants further investigation.