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Abstract The epicardium constitutes an untapped reservoir for cardiac regeneration. Upon heart injury, the adult epicardium re-activates, leading to epithelial-to-mesenchymal transition (EMT), migration, and differentiation. While interesting mechanistic and therapeutic findings arose from lower vertebrates and rodent models, the introduction of an experimental system representative of large mammals would undoubtedly facilitate translational advancements. Here, we apply innovative protocols to obtain living 3D organotypic epicardial slices from porcine hearts, encompassing the epicardial/myocardial interface. In culture, our slices preserve the in vivo architecture and functionality, presenting a continuous epicardium overlaying a healthy and connected myocardium. Upon thymosin β4 treatment of the slices, the epicardial cells become activated, upregulating epicardial and EMT genes, resulting in epicardial cell mobilization and differentiation into epicardial-derived mesenchymal cells. Our 3D organotypic model enables to investigate the reparative potential of the adult epicardium, offering an advanced tool to explore ex vivo the complex 3D interactions occurring within the native heart environment.

Thin living tissue slices have recently emerged as a new tissue model for cardiac electrophysiological research. Slices can be produced from human cardiac tissue, in addition to small and large mammalian hearts, representing a powerful in vitro model system for preclinical and translational heart research. In the present protocol, we describe a detailed mouse heart transverse slicing and optical imaging methodology. The use of this technology for high-throughput optical imaging allows study of electrophysiology of murine hearts in an organotypic pseudo two-dimensional model. The slices are cut at right angles to the long axis of the heart, permitting robust interrogation of transmembrane potential (Vm) and calcium transients (CaT) throughout the entire heart with exceptional regional precision. This approach enables the use of a series of slices prepared from the ventricles to measure Vm and CaT with high temporal and spatial resolution, allowing (i) comparison of successive slices which form a stack representing the original geometry of the heart; (ii) profiling of transmural and regional gradients in Vm and CaT in the ventricle; (iii) characterization of transmural and regional profiles of action potential and CaT alternans under stress (e.g., high frequency pacing or β-adrenergic stimulation) or pathological conditions (e.g., hypertrophy). Thus, the protocol described here provides a powerful platform for innovative research on electrical and calcium handling heterogeneity within the heart. It can be also combined with optogenetic technology to carry out optical stimulation; aiding studies of cellular Vm and CaT in a cell type specific manner.

Abstract Cell therapy is a potential novel treatment for cardiac regeneration and numerous studies have attempted to transplant cells to regenerate the myocardium lost during myocardial infarction. To date, only minimal improvements to cardiac function have been reported. This is likely to be the result of low cell retention and survival following transplantation. This study aimed to improve the delivery and engraftment of viable cells by using an injectable microcarrier that provides an implantable, biodegradable substrate for attachment and growth of cardiomyocytes derived from induced pluripotent stem cells (iPSC). We describe the fabrication and characterisation of Thermally Induced Phase Separation (TIPS) microcarriers and their surface modification to enable iPSC‐derived cardiomyocyte attachment in xeno‐free conditions is described. The selected formulation resulted in iPSC attachment, expansion, and retention of pluripotent phenotype. Differentiation of iPSC into cardiomyocytes on the microcarriers is investigated in comparison with culture on 2D tissue culture plastic surfaces. Microcarrier culture is shown to support culture of a mature cardiomyocyte phenotype, be compatible with injectable delivery, and reduce anoikis. The findings from this study demonstrate that TIPS microcarriers provide a supporting matrix for culturing iPSC and iPSC‐derived cardiomyocytes in vitro and are suitable as an injectable cell‐substrate for cardiac regeneration.

Living heart slices have recently emerged as a powerful experimental model for fundamental cardiac research. By retaining the structure and function of the native myocardium while maintaining the simplicity of cell culture models, heart slices can be easily employed in electrophysiological, pharmacological, biochemical, and structural investigations. One single heart yields many slices (>20 slices for rodents, >100 slices for porcine or human hearts), however due to the low throughput of most assays and rapid slice degeneration within 24 h of preparation, many slices remain unused and are discarded at the end of the preparation day. Here we present a novel method to extend viability and functionality of living heart slices, enabling their use in experiments over several consecutive days following preparation. By combining hypothermic conditions with inhibition of myosin II ATPase using 2,3-butanedione monoxime (BDM), slices prepared from the left ventricle of porcine hearts remain viable and exhibit preserved contractile function and morphology for up to 6 days. Electrophysiological function was also confirmed over the 6 days by extracellular field potentials recordings. This simple method not only maximizes the use of slices prepared from one single heart, thus reducing the number of animals required, but also increases data reproducibility by allowing multiple electrophysiological, pharmacological, biochemical, and structural studies to be performed from the same heart.

Progressive tissue remodeling after myocardial infarction (MI) promotes cardiac arrhythmias. This process is well studied in young animals, but little is known about pro-arrhythmic changes in aged animals. Senescent cells accumulate with age and accelerate age-associated diseases. Senescent cells interfere with cardiac function and outcome post-MI with age, but studies have not been performed in larger animals, and the mechanisms are unknown. Specifically, age-associated changes in timecourse of senescence and related changes in inflammation and fibrosis are not well understood. Additionally, the cellular and systemic role of senescence and its inflammatory milieu in influencing arrhythmogenesis with age is not clear, particularly in large animal models with cardiac electrophysiology more similar to humans than previously studied animal models. Here, we investigated the role of senescence in regulating inflammation, fibrosis, and arrhythmogenesis in young and aged infarcted rabbits. Aged rabbits exhibited increased peri-procedural mortality and arrhythmogenic electrophysiological remodeling at the infarct border zone (IBZ) compared to young rabbits. Studies of the aged infarct zone revealed persistent myofibroblast senescence and increased inflammatory signaling over a 12-week timecourse. Senescent IBZ myofibroblasts in aged rabbits appear to be coupled to myocytes, and our computational modeling showed that senescent myofibroblast-cardiomyocyte coupling prolongs action potential duration (APD) and facilitates conduction block permissive of arrhythmias. Aged infarcted human ventricles show levels of senescence consistent with aged rabbits, and senescent myofibroblasts also couple to IBZ myocytes. Our findings suggest that therapeutic interventions targeting senescent cells may mitigate arrhythmias post-MI with age.

The cardiotoxicity risk of hydroxychloroquine (HCQ) and azithromycin (AZM) has been the subject of intensive research triggered by safety concerns in COVID-19 patients. HCQ and AZM have been associated with QT interval prolongation and drug-induced arrhythmias, however other cardiotoxicity mechanisms remain largely unexplored. Our group has pioneered the living heart slice preparation, an ex-vivo platform that maintains native cardiac tissue architecture and physiological electrical and contractile properties. Here, we evaluated the cardiotoxic effect of HCQ and AZM applied alone or in combination on cardiac contractility by measuring contractile force and contraction kinetics in heart slices prepared from porcine hearts. Our results show that clinically relevant concentrations of HCQ monotherapy (1–10 µM) reduced contractile force and contraction kinetics in porcine slices in a dose-dependent manner. However, AZM monotherapy decreased contractile force and contraction kinetics only at higher concentrations (30 µM). Combination of HCQ and AZM induced a dose-dependent effect similar to HCQ alone. Furthermore, pre-treating porcine heart slices with the L-type calcium channel agonist Bay K8644 prevented the effect of both drugs, while administration of Bay K8644 after drugs interventions largely reversed the effects, suggesting a mechanism involving inhibition of L-type calcium channels. These findings indicate that HCQ and AZM alter cardiac function beyond QT prolongation with significant contractile dysfunction in intact cardiac tissue. Our porcine heart slices provide a powerful platform to investigate mechanisms of drug cardiotoxicity.

The epicardium has recently gained interest in the cardiovascular field due to its capacity to support heart regeneration after ischemic injury. Models to study the epicardium of large animals in vitro are limited and mainly based on epicardial cell isolation/differentiation from stem cells, followed by 2D cells culture. In this method paper, we describe the procedure to obtain and culture 3D organotypic heart slices presenting an intact epicardium, as a novel model to study the epicardial physiology and activation. Epicardial slices are obtained from porcine hearts using a high-precision vibratome and retain a healthy epicardial layer embedded in its native extracellular environment and connected with other cardiac cells (cardiomyocytes, fibroblasts, vascular cells etc.). Epicardial slices can be cultured for 72 h, providing an ideal model for studying the epicardium physiology or perform pharmacological interventions/gene therapy approaches. We also report on methods to assesses the viability and composition of the epicardial slices, and evaluate their architecture in 3D through tissue decoloration. Finally, we present a potential application for a nanomaterial-based gene transfer method for tracking of epicardial cells within the slice. Crucially, given the similarity in morphology and physiology of porcine heart with its human counterpart, our system provides a platform for translational research while providing a clinically relevant and ethical alternative to the use of small animals in this type of research.

Abstract Immune checkpoint inhibitors (ICIs) have made a breakthrough in the treatment of different types of tumors, leading to improvement in survival, even in patients with advanced cancers. Despite the good clinical results, a certain percentage of patients do not respond to this kind of immunotherapy. In addition, in a fraction of nonresponder patients, which can vary from 4 to 29% according to different studies, a paradoxical boost in tumor growth after ICI administration was observed: a completely unpredictable novel pattern of cancer progression defined as hyperprogressive disease. Since this clinical phenomenon has only been recently described, a universally accepted clinical definition is lacking, and major efforts have been made to uncover the biological bases underlying hyperprogressive disease. The lines of research pursued so far have focused their attention on the study of the immune tumor microenvironment or on the analysis of intrinsic genomic characteristics of cancer cells producing data that allowed us to formulate several hypotheses to explain this detrimental effect related to ICI therapy. The aim of this review is to summarize the most important works that, to date, provide important insights that are useful in understanding the mechanistic causes of hyperprogressive disease.

Abstract Early measurements of tissue viability after myocardial infarction (MI) are essential for accurate diagnosis and treatment planning but are challenging to obtain. Here, manganese, a calcium analogue and clinically approved magnetic resonance imaging (MRI) contrast agent, is used as an imaging biomarker of myocardial viability in the first hours after experimental MI. Safe Mn2+ dosing is confirmed by measuring in vitro beating rates, calcium transients, and action potentials in cardiomyocytes, and in vivo heart rates and cardiac contractility in mice. Quantitative T1 mapping‐manganese‐enhanced MRI (MEMRI) reveals elevated and increasing Mn2+ uptake in viable myocardium remote from the infarct, suggesting MEMRI offers a quantitative biomarker of cardiac inotropy. MEMRI evaluation of infarct size at 1 h, 1 and 14 days after MI quantifies myocardial viability earlier than the current gold‐standard technique, late‐gadolinium‐enhanced MRI. These data, coupled with the re‐emergence of clinical Mn2+‐based contrast agents open the possibility of using MEMRI for direct evaluation of myocardial viability early after ischemic onset in patients.

Background1,25-dihydroxyvitamin D3 [1,25(OH)2D3] plays a role in calcium homeostasis but can also exert immunomodulatory effects. In lungs, characterized by a particular immunosuppressive environment primarily due to the presence of alveolar macrophages (AM), 1,25(OH)2D3 has been shown to favor the immune response against pathogens. Here, we explored the ability of aerosolized 1,25(OH)2D3 to locally promote an anti-tumor phenotype in alveolar macrophages (AM) in the treatment of lung metastases.MethodsCytotoxicity assay has been used to assess the capability of AM, in vitro treated of not with 1,25(OH)2D3, to stimulate NK cells. Sulforhodamine B (SRB) assay has been used to assess the effect of 1,25(OH)2D3 on MC-38 and B16 tumor cells in vitro growth. 1,25(OH)2D3 was aerosolized in immunocompetent mouse models to evaluate the effect of local administration of 1,25(OH)2D3 on in vivo growth of MC-38 and B16 tumor cells within lungs and on infiltrating immune cells.ResultsIn vitro incubation of naïve AM with 1,25(OH)2D3 improved their ability to stimulate NK cell cytotoxicity. In vivo aerosolized 1,25(OH)2D3 significantly reduced the metastatic growth of MC-38 colon carcinoma, a tumor histotype that frequently metastasizes to lung in human. Immune infiltrate obtained from digested lungs of 1,25(OH)2D3-treated mice bearing MC-38 metastases revealed an increased expression of MHCII and CD80 on AM and an up-modulation of CD69 expression on effector cells that paralleled a strong increased ability of these cells to kill MC-38 tumor in vitro.ConclusionsTogether, these data show that aerosol delivery can represent a feasible and novel approach to supplement 1,25(OH)2D3 directly to the lungs promoting the activation of local immunity against cancer.

The mdx mouse has proven to be useful in understanding the cardiomyopathy that frequently occurs in muscular dystrophy patients. Here we employed a comprehensive array of clinically relevant in vivo MRI techniques to identify early markers of cardiac dysfunction and follow disease progression in the hearts of mdx mice.Serial measurements of cardiac morphology and function were made in the same group of mdx mice and controls (housed in a non-SPF facility) using MRI at 1, 3, 6, 9 and 12 months after birth. Left ventricular (LV) and right ventricular (RV) systolic and diastolic function, response to dobutamine stress and myocardial fibrosis were assessed. RV dysfunction preceded LV dysfunction, with RV end systolic volumes increased and RV ejection fractions reduced at 3 months of age. LV ejection fractions were reduced at 12 months, compared with controls. An abnormal response to dobutamine stress was identified in the RV of mdx mice as early as 1 month. Late-gadolinium-enhanced MRI identified increased levels of myocardial fibrosis in 6, 9 and 12-month-old mdx mice, the extent of fibrosis correlating with the degree of cardiac remodeling and hypertrophy.MRI could identify cardiac abnormalities in the RV of mdx mice as young as 1 month, and detected myocardial fibrosis at 6 months. We believe these to be the earliest MRI measurements of cardiac function reported for any mice, and the first use of late-gadolinium-enhancement in a mouse model of congenital cardiomyopathy. These techniques offer a sensitive and clinically relevant in vivo method for assessment of cardiomyopathy caused by muscular dystrophy and other diseases.

Inhibitors of apoptosis proteins (IAPs) are validated onco-targets, as their overexpression correlates with cancer onset, progression, diffusion and chemoresistance. IAPs regulate cell death survival pathways, inflammation, and immunity. Targeting IAPs, by impairing their protein–protein interaction surfaces, can affect events occurring at different stages of cancer development.To this purpose, we employed a rational virtual screening approach to identify compounds predicted to interfere with the assembly of pro-survival macromolecular complexes. One of the candidates, FC2, was shown to bind in vitro the BIR1 domains of both XIAP and cIAP2. Moreover, we demonstrated that FC2 can induce cancer cell death as a single agent and, more potently, in combination with the Smac-mimetic SM83 or with the cytokine TNF. FC2 determined a prolonged activation of the NF-κB pathway, accompanied to a stabilization of XIAP-TAB1 complex. This candidate molecule represents a valuable lead compound for the development of a new class of IAP-antagonists for cancer treatment.

Oxidative inflammatory damage to specialised brain centres may lead to dysfunction of their associated peripheral organs, such as the bladder. However, the source of reactive oxygen species (ROS) in specific brain regions that regulate bladder function is poorly understood. Of all ROS-generating enzymes, the NADPH oxidase (Nox) family produces ROS as its sole function and offers an advantage over other enzymes as a drug-targetable molecule to selectively control excessive ROS. We investigated whether the Nox 2 subtype is expressed in the micturition regulatory periaqueductal gray (PAG) and Barrington’s nucleus (pontine micturition centre, PMC) and examined Nox-derived ROS production in these structures. C57BL/6J mice were used; PAG, PMC, cardiac tissue, and aorta were isolated. Western blot determined Nox 2 expression. Lucigenin-enhanced chemiluminescence quantified real-time superoxide production. Western blot experiments demonstrated the presence of Nox 2 in PAG and PMC. There was significant NADPH-dependent superoxide production in both brain tissues, higher than that in cardiac tissue. Superoxide generation in these brain tissues was significantly suppressed by the Nox inhibitor diphenyleneiodonium (DPI) and also reduced by the Nox-2 specific inhibitor GSK2795039, comparable to aorta. These data provide the first evidence for the presence of Nox 2 and Nox-derived ROS production in micturition centres.

Dopo la premessa riguardo la distribuzione in Italia, l’autore riporta l’osservazione tre cavità nido in un edificio nella cittadina di Gambarie, Parco Nazionale dell’Aspromonte.

The heart is a complex organ composed of multiple cell types, including cardiomyocytes and different non-myocyte populations, all working closely together to determine the hearts properties and maintain normal cardiac function. Connexins are abundantly expressed proteins that form plasma membrane hemichannels and gap junctions between cells. Gap junctions are intracellular channels that allow for communication between cells, and in the heart they play a crucial role in cardiac conduction by coupling adjacent cardiomyocytes. Connexins are expressed in both cardiomyocytes and non-myocytes, including cardiac fibroblasts, endothelial cells, and macrophages. Non-myocytes are the largest population of cells in the heart, and therefore it is important to consider what roles connexins, hemichannels, and gap junctions play in these cell types. The aim of this review is to provide insight into connexin-based signalling in non-myocytes during health and disease, and highlight how targeting these proteins could lead to the development of novel therapies. We conclude that connexins in non-myocytes contribute to arrhythmias and adverse ventricular remodelling following myocardial infarction, and are associated with the initiation and development of atherosclerosis. Therefore, therapeutic interventions targeting these connexins represent an exciting new research avenue with great potential.

Endogenous cardiac progenitor cells, expanded from explants via cardiosphere formation, present a promising cell source to prevent heart failure following myocardial infarction. Here we used cine-magnetic resonance imaging (MRI) to track administered cardiosphere-derived cells (CDCs) and to measure changes in cardiac function over four months in the infarcted rat heart.CDCs, cultured from neonatal rat heart, comprised a heterogeneous population including cells expressing the mesenchymal markers CD90 and CD105, the stem cell marker c-kit and the pluripotency markers Sox2, Oct3/4 and Klf-4. CDCs (2 × 10(6)) expressing green fluorescent protein (GFP+) were labelled with fluorescent micron-sized particles of iron oxide (MPIO). Labelled cells were administered to the infarcted rat hearts (n = 7) by intramyocardial injection immediately following reperfusion, then by systemic infusion (4 × 10(6)) 2 days later. A control group (n = 7) was administered cell medium. MR hypointensities caused by the MPIOs were detected at all times and GFP+ cells containing MPIO particles were identified in tissue slices at 16 weeks. At two days after infarction, cardiac function was similar between groups. By 6 weeks, ejection fractions in control hearts had significantly decreased (47 ± 2%), but this was not evident in CDC-treated hearts (56 ± 3%). The significantly higher ejection fractions in the CDC-treated group were maintained for a further 10 weeks. In addition, CDC-treated rat hearts had significantly increased capillary density in the peri-infarct region and lower infarct sizes. MPIO-labelled cells also expressed cardiac troponin I, von Willebrand factor and smooth muscle actin, suggesting their differentiation along the cardiomyocyte lineage and the formation of new blood vessels.CDCs were retained in the infarcted rat heart for 16 weeks and improved cardiac function.