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.

Cardiovascular Risk Factors and Disparities in Management of Embolic Stroke: A Western Sydney Perspective.

BACKGROUND: Ischaemic stroke remains one of the leading causes of death and disability worldwide. The population of Western Sydney has a unique demographic with lower socioeconomic status and a culturally and linguistically diverse population. This study aims to investigate the demographics and cardiovascular risk factors of patients in Western Sydney, focusing on the prevalence and profile of cardioembolic (CE) strokes and embolic strokes of undetermined source (ESUS). METHOD: Prospective data were collected in 463 patients with ischaemic stroke presenting to a tertiary centre in Western Sydney, who underwent predischarge transthoracic echocardiography. Patients with haemorrhagic strokes or unclear stroke diagnosis were excluded. Analysis of stroke subtype (CE, ESUS, or non-embolic) and clinical characteristics was performed based on age, gender, and prior atrial fibrillation (AF) prevalence. RESULTS: Of the 463 patients, 147 (32%) had CE strokes, and 147 (32%) had ESUS. Cardioembolic (CE) strokes were associated with older age (≥65 years) and a history of congestive cardiac failure. Older patients had higher rates of hypertension, ischaemic heart disease, AF, and congestive heart failure. History of AF was present in 67 patients (14.5%); however, only 51% received anticoagulation before admission despite a low bleeding risk. The transthoracic echocardiography characteristics of ESUS/non-embolic strokes differed from those of CE strokes; 20% of patients with ESUS had an enlarged left atrium, suggesting a subset of patients with ESUS with a left atrial myopathy. CONCLUSIONS: Patients with ischaemic stroke in Western Sydney have a high prevalence of cardiovascular risk factors which were often undertreated. Half of the patients with prior AF did not receive anticoagulation despite low bleeding risk, indicating a gap in optimal stroke prevention. There were distinct echocardiographic characteristics among stroke subtypes. Further analysis of left atrium parameters may provide greater insights into the pathogenesis and prevention of embolic strokes.

Focal Anticoagulation by Somatic Gene Transfer: Towards Preventing Cardioembolic Stroke.

Cardioembolic stroke (CS) has emerged as a leading cause of ischaemic stroke (IS); distinguished by thrombi embolising to the brain from cardiac origins; most often from the left atrial appendage (LAA). Contemporary therapeutic options are largely dependent on systemic anticoagulation as a blanket preventative strategy, yet this does not represent a nuanced or personalised solution. Contraindications to systemic anticoagulation create significant unmedicated and high-risk cohorts, leaving these patients at risk of significant morbidity and mortality. Atrial appendage occlusion devices are increasingly used to mitigate stroke risk from thrombi emerging from the LAA in patients ineligible for oral anticoagulants (OACs). Their use, however, is not without risk or significant cost, and does not address the underlying aetiology of thrombosis and CS. Viral vector-based gene therapy has emerged as a novel strategy to target a spectrum of haemostatic disorders, achieving success through the adeno-associated virus (AAV) based therapy of haemophilia. Yet, thrombotic disorders, such as CS, have had limited exploration within the realm of AAV gene therapy approaches-presenting a gap in the literature and an opportunity for further research. Gene therapy has the potential to directly address the cause of CS by localised targeting of the molecular remodelling that serves to promote thrombosis.

In Search of Adeno-Associated Virus Vectors With Enhanced Cardiac Tropism for Gene Therapy.

Globally, adeno-associated virus (AAV) vectors have been increasingly used for clinical gene therapy trials. In Australia, AAV-based gene therapy is available for hereditary diseases such as retinal dystrophy or spinal muscular atrophy 1 (SMA1). Many preclinical studies have used AAV vectors for gene therapy in models of cardiac disease with outcomes of varying translational potential. However, major barriers to effective and safe therapeutic gene delivery to the human heart remain to be overcome. These include tropism, efficient gene transfer, mitigating off-target gene delivery and avoidance of the host immune response. Developing such an enhanced AAV vector for cardiac gene therapy is of great interest to the field of advanced cardiac therapeutics. In this review, we provide an overview of the approaches currently being employed in the search for cardiac cell-specific AAV capsids, ranging from natural AAVs selected as a result of infection and latency in the heart, to the use of cutting-edge molecular techniques to engineer and select AAVs specific for cardiac cells with the use of high-throughput methods.

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.

Electrophysiologic and electroanatomic characterization of ventricular arrhythmias in non-compaction cardiomyopathy: A systematic review.

BACKGROUND: Non-compaction cardiomyopathy (NCCM) is a form of structural heart disease prone to ventricular arrhythmias (VAs) and sudden cardiac death. Non-compacted myocardium may harbor VA substrate, though some reports suggest otherwise. OBJECTIVE: This study aimed to characterize the electrophysiologic (EP) features of VA in NCCM. METHODS: We performed a systematic review of case reports, case series, and observational studies. RESULTS: One hundred and thirty-five cases of NCCM from studies between 2000 and 2020 were included. Mean age was 34 ± 20 years, mean left ventricular (LV) ejection fraction was 42 ± 15% with two cases having late gadolinium enhancement on magnetic resonance imaging. The LV apex was the most common non-compacted segment (86%); 10% involved the right ventricle (RV). Antiarrhythmic failure was documented in 16 cases, of which 50% failed more than one agent. Only 23% of monomorphic VAs localized to regions of non-compaction on electrocardiogram. Most frequently, VAs localized to the RV outflow tract (n = 21), posterior fascicle (n = 19), and anterolateral LV apex (n = 9). All cases with apical exits arose from the non-compacted myocardium. On EPS, 83% of sustained VTs were due to re-entry, 17% due to focal mechanism. Catheter ablation was performed in 39 cases, with 7 requiring more than 1 procedure. Acute VA non-inducibility was achieved in 82% and VA-free survival was reported in 85% over a mean follow-up of 24 months. CONCLUSION: The majority of VAs in NCCM arise remotely from non-compacted myocardium, and non-re-entrant mechanism seen in ~1/5th of sustained VTs. Catheter ablation outcomes appear favorable. Further study is needed to understand the pathophysiology of VA in NCCM.

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.

Ventricular Tachycardia Storm Ablation With Pre-Emptive Circulatory Support by Extracorporeal Membrane Oxygenation: Australian Experience.

BACKGROUND: Veno-arterial extracorporeal membrane oxygenation (VA-ECMO) can provide circulatory support in high-risk patients undergoing drug refractory ventricular tachycardia (VT) ablation procedures. We report experience using VA-ECMO in a pre-emptive approach for high-risk patients with VT storm and previously ineffective ablation procedures. METHODS AND RESULTS: Four (4) patients with drug refractory ventricular tachycardia (mean age 61±3 years; left ventricular ejection fraction 21±5%) presenting for VT ablation had pre-emptive VA-ECMO. All patients during current admission had VT storm. Pre-ablation, 22 total monomorphic VTs (cycle length 402±69 ms) were induced or spontaneously observed (median of 4, IQR25-75% 1-6). At the end of the procedure, 86% of all inducible VTs were rendered non-inducible. Median hospitalisation following VA-ECMO supported ablation was 5 days (IQR25-75% 3-12). During follow-up (median 138 days [IQR25-75% 57-277]), VT recurred in one patient as an isolated episode reverted by anti-tachycardia pacing. There was a 99% reduction in VT burden post ablation. One (1) patient died of cardiogenic shock within 24 hours whilst still on VA-ECMO, all other patients were successfully weaned off support and discharged. Two (2) patients underwent cardiac transplantation at 199 and 512 days post ablation following implantation of ventricular assist devices for worsening heart failure. CONCLUSIONS: The pre-emptive use of VA-ECMO for high-risk patients undergoing catheter ablation for VT storm was found to be effective in maintaining haemodynamic status, and allowing successful mapping and catheter ablation for VT.

Catheter ablation of ventricular arrhythmia guided by a high-density grid catheter.

INTRODUCTION: Minimal data exist on the Advisor HD Grid (HDG) catheter and the Precision electroanatomic mapping (EAM) system for ventricular arrhythmia (VA) procedures. Using the HDG catheter, the EAM uses the high-density (HD) wave mapping and best duplicate software to compare the maximum peak-to-peak bipolar voltages within a small zone independent of wavefront direction and catheter orientation. This study aimed to summarize the procedural experience for VAs using the HDG catheter. METHODS: Clinical and procedural characteristics of VA ablation procedures were retrospectively reviewed that used the HDG catheter and the Precision EAM over a 12-month period. RESULTS: A total of 22 patients, 18 with sustained ventricular tachycardia and 4 with premature ventricular contractions were included. Clinically indicated left and/or right ventricular (LV, RV, respectively), and aortic maps were created. LV substrate maps (n = 13) used a median 1700 points (interquartile range [IQR]25%-75% , 1427-2412) out of a median 18 573 (IQR25%-75% , 15 713-41 067) total points collected. RV substrate maps (n = 11) used a median 1435 points (IQR25%-75% , 1114-1871) out of a median 16 005 (IQR25%-75% , 11 063-21 405) total points collected. Total point utilization, used vs collected, was 9%. Mean mapping time was 43 ± 17 minutes (substrate, 34 ± 18 minutes; activation/pace mapping, 9 ± 13 minutes). Acute success was achieved in 56 (86%) and short-term success achieved in 16 patients (73%) at a median follow-up of 145 days (IQR25%-75% , 62-273 days). There were no procedural complications. CONCLUSION: HD wave mapping using the novel HDG catheter integrated with the Precision EAM is safe and feasible in VA procedures in the LV, RV, and aorta. Mapping times are consistent with other multielectrode mapping catheters.

Scar nonexcitability using simultaneous pacing for substrate ablation of ventricular tachycardia.

OBJECTIVES: To describe an expedited strategy of simultaneous high-output pacing during radiofrequency ablation to achieve scar homogenization and electrical inexcitability as an approach for substrate ablation for scar-related ventricular tachycardia (VT). BACKGROUND: Scar homogenization with additional testing for electrical inexcitability is known endpoints for catheter ablation, but achieving both can be time consuming. We describe a strategy of simultaneous pacing during radiofrequency ablation to expedite this approach. METHODS AND RESULTS: Ten patients (age 74 ± 6 years; all men, (LV) ejection fraction of 33% ± 8%, ischemic cardiomyopathy, 9; VT storm, 7) underwent scar homogenization with electrical inexcitability to pacing (10 mA, 9 ms pulse width), as well as noninducibility of any VT as an acute procedural endpoint. Thirty-four VTs were inducible in 10 patients with a total of 1127 ablation lesions applied. Median ablation lesions per patient were 97 (interquartile range [IQR]25-75 71-151), and the total ablation time was 49 minutes (IQR25-75 45-56 minutes) with average duration per lesion of 32.2 seconds (IQR25-75 25.8-37.8 seconds). Average power was 33 W (IQR25-75 32-38 W), average contact force was 13 g (IQR25-75 11.9-14.6 g) with a median impedance drop of 9.6 Ω/lesion (IQR25-75 8.1-10.0 Ω). There were no ventricular fibrillation episodes using this strategy. The median procedure time was 246 minutes (IQR25-75 214-293 minutes). Acute procedural success was seen in nine patients with 97% of VTs noninducible. CONCLUSION: Simultaneous ablation with high output pacing to achieve scar inexcitability, when combined with scar homogenization and noninducibility of any VT may be an expeditious, safe, and effective technique for catheter ablation.

Demographic and clinical profile of cardioembolic stroke patients in Western Sydney.

BACKGROUND: Cardioembolism (CE) contributes to a large proportion of ischaemic stroke. AIMS: To evaluate the demographic and clinical profile of CE stroke in Western Sydney. METHODS: A retrospective analysis of ischaemic stroke patients presenting to Westmead Hospital (January-October 2016) was performed. Strokes were classified by TOAST (Trial of Org 10172 in Acute Stroke Treatment) criteria into different categories. Clinical and demographic data were collected on all stroke patients, and differences between CE and other stroke causes were identified. RESULTS: Two hundred and twenty-eight consecutive patients (70.9 years; 53% male) were identified. By TOAST criteria, 21 (9%) had large-artery atherosclerosis, 94 (41%) CE, 10 (5%) small-vessel disease, 2 (1%) other aetiology and 101 (44%) undetermined aetiology. A significant proportion of CE stroke patients had cardiovascular risk factors including hypertension (66%), hypercholesterolaemia (50%), diabetes (26%) and ischaemic heart disease (28%). The majority (81%) of patients with CE had atrial flutter/flutter. CE stroke, compared with other types of stroke, was more common in females (56 vs 41%, P = 0.022) and patients with CE stroke were more likely to have previous cerebral ischaemia (34 vs 21%, P = 0.026), suggesting increased recurrence in this group. Of the patients with atrial flutter/flutter (n = 56), the majority (87%) had a high CHA2 DS2 -VASC score (≥2); however, a significant proportion (55.4%) were not on anticoagulation. CONCLUSIONS: Cardioembolic stroke remains a significant burden in Western Sydney, and it is likely that a significant proportion may be preventable, as evidenced by the substantial presence of modifiable cardiovascular risk factors, and inadequate anticoagulation of patients with atrial arrhythmias.

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.

Early and long-term outcomes after manual and remote magnetic navigation-guided catheter ablation for ventricular tachycardia.

Aims: Remote magnetic navigation (RMN) is a safe and effective means of performing ventricular tachycardia (VT) ablation. It may have advantages over manual catheter ablation due to ease of manoeuvrability and catheter stability. We sought to compare the safety and efficacy of RMN vs. manual VT ablation. Methods and results: Retrospective study of procedural outcomes of 139 consecutive VT ablation procedures (69 RMN, 70 manual ablation) in 113 patients between 2009 and 2015 was performed. Remote magnetic navigation was associated with overall higher acute procedural success (80% vs. 60%, P = 0.01), with a trend to fewer major complications (3% vs. 9% P = 0.09). Seventy-nine patients were followed up for a median of 17.0 [interquartile range (IQR) 3.0-41.0] months for the RMN group and 15.5 (IQR 6.5-30.0) months for manual ablation group. In the ischaemic cardiomyopathy subgroup, RMN was associated with longer survival from the composite endpoint of VT recurrence leading to defibrillator shock, re-hospitalization or repeat catheter ablation and all-cause mortality; single-procedure adjusted hazard ratio (HR) 0.240 (95% CI 0.070-0.821) P = 0.023, multi-procedure HR 0.170 (95% CI 0.046-0.632) P = 0.002. In patients with implanted defibrillators, multi-procedure VT-free survival was superior with RMN, HR 0.199 (95% CI 0.060-0.657) P = 0.003. Conclusion: Remote magnetic navigation may improve clinical outcomes after catheter ablation of VT in patients with ischaemic cardiomyopathy. Further prospective clinical studies are required to confirm these findings.

Human Connexin40 Mutations Slow Conduction and Increase Propensity for Atrial Fibrillation.

BACKGROUND: Patch clamping studies using non-cardiomyocytes revealed that the human connexin40 mutations P88S, G38D, and A96S are associated with reduced gap junction conductances compared to wild type connexin40 (wtCx40). Their effects within myocytes however are unclear. We aimed to characterise P88S, G38D, and A96S after expression in rat hearts and primary cardiomyocyte cultures. METHODS: Adult Sprague-Dawley rat atria were transduced with a lentivector containing a transgene encoding wtCx40, P88S, G38D, A96S, or eGFP (n=6 per transgene). Electrophysiology studies (EPS) were performed just prior to and 7 days after surgery. Left atria were assessed for connexin expression, mRNA levels, inflammation and fibrosis. Primary cardiomyocyte cultures were also transduced with the abovementioned vectors (n=6 per transgene) and monolayer conduction velocities (CV) and protein expression were assessed at 96hours. RESULTS: At day 7 EPS, P wave and induced atrial fibrillation (AF) durations were significantly longer in the mutant groups when compared to wtCx40 controls (p<0.05). There were no significant differences in inflammation, fibrosis, or heart to body weight ratios. Monolayer CV's were reduced in the A96S group compared to the wtCx40 group. While similar to wtCx40 controls, P88S velocities were reduced compared to eGFP controls. G38D monolayers possessed spontaneous fibrillatory activity and could not be paced. Immunofluorescence revealed that P88S and G38D reduced native connexin43 myocyte coupling while A96S appeared to co-localise with connexin43 in gap junctions. Connexin43 mRNA levels were similar between groups. CONCLUSIONS: The A96S, G38D, and P88S Cx40 mutations slow conduction and increased the propensity for inducible AF.

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.

Vascularized Cardiac Spheroids as Novel 3D in vitro Models to Study Cardiac Fibrosis.

Spheroid cultures are among the most explored cellular biomaterials used in cardiovascular research, due to their improved integration of biochemical and physiological features of the heart in a defined architectural three-dimensional microenvironment when compared to monolayer cultures. To further explore the potential use of spheroid cultures for research, we engineered a novel in vitro model of the heart with vascularized cardiac spheroids (VCSs), by coculturing cardiac myocytes, endothelial cells, and fibroblasts isolated from dissociated rat neonatal hearts (aged 1-3 days) in hanging drop cultures. To evaluate the validity of VCSs in recapitulating pathophysiological processes typical of the in vivo heart, such as cardiac fibrosis, we then treated VCSs with transforming growth factor beta 1 (TGFß1), a known profibrotic agent. Our mRNA analysis demonstrated that TGFß1-treated VCSs present elevated levels of expression of connective tissue growth factor, fibronectin, and TGFß1 when compared to control cultures. We demonstrated a dramatic increase in collagen deposition following TGFß1 treatment in VCSs in the PicroSirius Red-stained sections. Doxorubicin, a renowned cardiotoxic and profibrotic agent, triggered apoptosis and disrupted vascular networks in VCSs. Taken together, our findings demonstrate that VCSs are a valid model for the study of the mechanisms involved in cardiac fibrosis, with the potential to be used to investigate novel mechanisms and therapeutics for treating and preventing cardiac fibrosis in vitro.

SUMO1-dependent modulation of SERCA2a in heart failure.

The calcium-transporting ATPase ATP2A2, also known as SERCA2a, is a critical ATPase responsible for Ca(2+) re-uptake during excitation-contraction coupling. Impaired Ca(2+) uptake resulting from decreased expression and reduced activity of SERCA2a is a hallmark of heart failure. Accordingly, restoration of SERCA2a expression by gene transfer has proved to be effective in improving cardiac function in heart-failure patients, as well as in animal models. The small ubiquitin-related modifier (SUMO) can be conjugated to lysine residues of target proteins, and is involved in many cellular processes. Here we show that SERCA2a is SUMOylated at lysines 480 and 585 and that this SUMOylation is essential for preserving SERCA2a ATPase activity and stability in mouse and human cells. The levels of SUMO1 and the SUMOylation of SERCA2a itself were greatly reduced in failing hearts. SUMO1 restitution by adeno-associated-virus-mediated gene delivery maintained the protein abundance of SERCA2a and markedly improved cardiac function in mice with heart failure. This effect was comparable to SERCA2A gene delivery. Moreover, SUMO1 overexpression in isolated cardiomyocytes augmented contractility and accelerated Ca(2+) decay. Transgene-mediated SUMO1 overexpression rescued cardiac dysfunction induced by pressure overload concomitantly with increased SERCA2a function. By contrast, downregulation of SUMO1 using small hairpin RNA (shRNA) accelerated pressure-overload-induced deterioration of cardiac function and was accompanied by decreased SERCA2a function. However, knockdown of SERCA2a resulted in severe contractile dysfunction both in vitro and in vivo, which was not rescued by overexpression of SUMO1. Taken together, our data show that SUMOylation is a critical post-translational modification that regulates SERCA2a function, and provide a platform for the design of novel therapeutic strategies for heart failure.