Dr Patrizia Camelliti

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.

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.

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 Mn 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 Mn 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 Mn -based contrast agents open the possibility of using MEMRI for direct evaluation of myocardial viability early after ischemic onset in patients.

Heart diseases are currently the leading cause of death worldwide. The ability to create cardiovascular tissue has numerous applications in understanding tissue development, disease progression, pharmacological testing, bio‐actuators, and transplantation; yet current cardiovascular tissue engineering (CTE) methods are limited. However, there have been emerging developments in the bioelectronics field, with the creation of biomimetic devices that can intimately interact with cardiac cells, provide monitoring capabilities, and regulate tissue formation. Combining bioelectronics with cardiac tissue engineering can overcome current limitations and produce physiologically relevant tissue that can be used in various areas of cardiovascular research and medicine. This review highlights the recent advances in cardiovascular‐based bioelectronics. First, cardiac tissue engineering and the potential of bioelectronic therapies for cardiovascular diseases are discussed. Second, advantageous bioelectronic materials for CTE and implantation and their properties are reviewed. Third, several representative cardiovascular tissue‐bioelectronic interface models and the beneficial functions that bioelectronics can demonstrate in in vitro and in vivo applications are explored. Finally, the prospects and remaining challenges for clinical application are discussed. This review highlights recent advances in cardiovascular‐based bioelectronics. Cardiac tissue engineering and the potential of bioelectronic therapies for cardiovascular diseases are discussed, followed by advantageous bioelectronic materials for cardiovascular tissue engineering/implantation and their properties. Representative cardiovascular tissue‐bioelectronic interface models and their beneficial functions are also explored. Finally, prospects and remaining challenges are discussed for clinical applications.

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.

Traditionally, cardiac electrophysiology has focused on myocytes—the heart muscle cells that generate action potentials and are electrically excitable. Nonmyocytes within the heart are often considered barriers to action potential propagation. Typically defined as extracellular matrix–producing cells, cardiac fibroblasts are one of the largest nonmyocyte cardiac populations. However, they have been recognized to be important for maintaining normal cardiac function and for mediating cardiac remodeling during pathology, when their number substantially increases (1). The presence of electrical coupling between fibroblasts and myocytes has been established in vitro and more recently demonstrated in situ (2–4). But definitive in vivo evidence has been lacking. On page 1480 of this issue, Wang et al. (5) report that fibroblasts and myocytes are electrically coupled in living mice. This finding could transform the understanding of cardiac connectivity and arrhythmogenesis, with profound implications for the management of heart disease patients.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to remuscularize infarcted hearts but their arrhythmogenicity remains an obstacle to safe transplantation. Myofbroblasts are the predominant cell-type in the infarcted myocardium but their impact on transplanted hiPSC-CMs remains poorly defned. Here, we investigate the efect of myofbroblasts on hiPSC-CMs electrophysiology and Ca2+ handling using optical mapping of advanced human cell coculture systems mimicking cell–cell interaction modalities. Human myofbroblasts altered the electrophysiology and Ca2+ handling of hiPSC-CMs and downregulated mRNAs encoding voltage channels (KV4.3, KV11.1 and Kir6.2) and SERCA2a calcium pump. Interleukin-6 was elevated in the presence of myofbroblasts and direct stimulation of hiPSC-CMs with exogenous interleukin-6 recapitulated the paracrine efects of myofbroblasts. Blocking interleukin-6 reduced the efects of myofbroblasts only in the absence of physical contact between cell-types. Myofbroblast-specifc connexin43 knockdown reduced functional changes in contact cocultures only when combined with interleukin-6 blockade. This provides the frst in-depth investigation into how human myofbroblasts modulate hiPSC-CMs function, identifying interleukin-6 and connexin43 as paracrine- and contact-mediators respectively, and highlighting their potential as targets for reducing arrhythmic risk in cardiac cell therapy

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 hours 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.

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

Simple Summary Reactive oxygen species (ROS) are chemically active oxygen-containing molecules and overproduction of ROS can cause oxidative damage to cells and tissues in the body. Oxidative damage to brain cells can not only cause lesions to the brain but also lead to disorders in peripheral organs under the control of the corresponding brain centres, such as the urinary bladder. A unique class of enzymes that produce ROS are the special oxidising enzymes called "Nox" enzymes. These are the body's only enzymes that can be selectively controlled without affecting normal cell activity. Therefore, Nox enzymes are considered to be a drug target. Whether Nox exists in the brain centres that control urination has not been examined. We investigated whether the type 2 Nox enzyme-Nox 2 exists in the brain urination control centre and whether such a Nox enzyme is functional. Our results show that the brain urination control centre has Nox 2 proteins, and the Nox enzyme produces a significant amount of ROS, higher than heart tissue, suggesting the importance of Nox-associated ROS production in physiology and pathology. These findings lay the groundwork for future investigation into Nox 2 and the associated oxidative damage in brain urination control centres and consequent bladder abnormalities. 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.

OBJECTIVES: Myocardial infarction leads to extensive changes in the organization of cardiac myocytes and fibroblasts, and changes in gap junction protein expression. In the immediate period following ischemia, reperfusion causes hypercontraction, spreading the necrotic lesion. Further progressive infarction continues over several weeks. In reperfusion injury, the nonspecific gap junction channel uncoupler heptanol limits necrosis. We hypothesize that gap junction coupling and fibroblast invasion provide a substrate for progressive infarction via a gap junction mediated bystander effect. METHODS: A sheep coronary occlusion infarct model was used with samples collected at 12, 24 and 48 h, and 6, 12 and 30 d (days) post-infarction. Immunohistochemical labelling of gap junction connexins Cx40, Cx43, and Cx45 was combined with cell-specific markers for fibroblasts (anti-vimentin) and myocytes (anti-myomesin). Double and triple immunolabelling and confocal microscopy were used to follow changes in cardiac myocyte morphology, fibroblast content and gap junction expression after myocardial infarction. Gap junction protein levels and fibroblast numbers were quantified. RESULTS: Within 12 h of ischemia, myocyte viability is impaired within small islands in the ischemic region. These islands spread and fuse into larger infarct zones until 12 d post-infarction. Thereafter, surviving myocytes within the infarct and in the border-zone appear to become stabilized. Distant from the infarct, continuing myocyte disruption is regularly observed, even after 30 d. Cx43 becomes redistributed from intercalated discs to the lateral surface of structurally compromised myocytes within 12 d. Cx45 expressing fibroblasts infiltrate the damaged region within 24 h, becoming most numerous at 6-12 d post-infarction, with peak Cx45 levels at 6 d. Later, Cx43 expressing fibroblasts are observed, and the related Cx43 label increases over the 30 d observation period, even though fibroblast numbers decline after 12 d. Cx40 was only seen in vascular endothelium. CONCLUSIONS: Progressive infarction, identified by myocyte sarcomere disruption and subsequent cell loss, occurs in parallel with fibroblast invasion and gap junction remodeling. Two fibroblast phenotypes occur within infarcts, expressing either Cx43 or Cx45. Coupled fibroblasts may play a number of roles in tissue remodeling following myocardial infarction, including provision of a possible substrate for progressive infarction via a gap junction mediated bystander effect.

Conduction abnormalities are frequently associated with cardiac disease, though the mechanisms underlying the commonly associated increases in PQ interval are not known. This study uses a chronic left ventricular (LV) apex myocardial infarction (MI) model in the rabbit to create significant left ventricular dysfunction (LVD) 8weeks post-MI. In vivo studies established that PQ interval increases by approximately 7ms (10%) with no significant change in average heart rate. Optical mapping of isolated Langendorff perfused rabbit hearts recapitulated this result; time to earliest activation of the LV was increased by 14ms (16%) in the LVD group. Intra-atrial and LV transmural conduction times were not altered in the LVD group. Isolated AVN preparations from the LVD group demonstrated a significantly longer conduction time (by approximately 20ms) between atrial and His electrograms than sham controls across a range of pacing cycle lengths. This difference was accompanied by increased effective refractory period and Wenckebach cycle length, suggesting significantly altered AVN electrophysiology post-MI. The AVN origin of abnormality was further highlighted by optical mapping of the isolated AVN. Immunohistochemistry of AVN preparations revealed increased fibrosis and gap junction proteins (connexin43 and 40) remodelling in the AVN of LVD animals compared to sham. A significant increase in myocyte-non-myocyte connexin co-localization was also observed after LVD. These changes may increase the electrotonic load experienced by AVN muscle cells and contribute to slowed conduction velocity within the AVN.

Cardiomyocytes form a conducting network that is assumed to be electrically isolated from nonmyocytes in vivo. In cell culture, however, cardiac fibroblasts can contribute to the spread of excitation via functional gap junctions with cardiomyocytes. To assess the ability of fibroblasts to form gap junctions in vivo, we combine in situ detection of connexins in rabbit sinoatrial node (a tissue that is particularly rich in fibroblasts) with identification of myocytes and fibroblasts using immunohistochemical labeling and confocal microscopy. We distinguish two spatially distinct fibroblast populations expressing different connexins: fibroblasts surrounded by other fibroblasts preferentially express connexin40, whereas fibroblasts that are intermingled with myocytes largely express connexin45. Functionality of homogeneous and heterogeneous cell coupling was investigated by dye transfer in sinoatrial node tissue explants. These studies reveal spread of Lucifer yellow, predominantly along extended threads of interconnected fibroblasts (probably via connexin40), and occasionally between neighboring fibroblasts and myocytes (probably via connexin45). Our findings show that cardiac fibroblasts form a coupled network of cells, which may be functionally linked to myocytes in rabbit SAN.

Electrical correlates of the physiological state of a cell, such as membrane conductance and capacitance, as well as cytoplasm conductivity, contain vital information about cellular function, ion transport across the membrane, and propagation of electrical signals. They are, however, difficult to measure; gold-standard techniques are typically unable to measure more than a few cells per day, making widespread adoption difficult and limiting statistical reproducibility. We have developed a dielectrophoretic platform using a disposable 3D electrode geometry that accurately (r2 > 0.99) measures mean electrical properties of populations of ~20,000 cells, by taking parallel ensemble measurements of cells at 20 frequencies up to 45 MHz, in (typically) ten seconds. This allows acquisition of ultra-high-resolution (100-point) DEP spectra in under two minutes. Data acquired from a wide range of cells – from platelets to large cardiac cells - benchmark well with patch-clamp-data. These advantages are collectively demonstrated in a longitudinal (same-animal) study of rapidly-changing phenomena such as ultradian (2–3 hour) rhythmicity in whole blood samples of the common vole (Microtus arvalis), taken from 10 µl tail-nick blood samples and avoiding sacrifice of the animal that is typically required in these studies.

We demonstrate a simple, accurate and versatile method to manipulate Parylene C, a material widely known for its high biocompatibility, and transform it to a substrate that can effectively control the cellular microenvironment and consequently affect the morphology and function of the cells in vitro. The Parylene C scaffolds are fabricated by selectively increasing the material's surface water affinity through lithography and oxygen plasma treatment, providing free bonds for attachment of hydrophilic biomolecules. The micro-engineered constructs were tested as culture scaffolds for rat ventricular fibroblasts and neonatal myocytes (NRVM), toward modeling the unique anisotropic architecture of native cardiac tissue. The scaffolds induced the patterning of extracellular matrix compounds and therefore of the cells, which demonstrated substantial alignment compared to typical unstructured cultures. Ca(2+) cycling properties of the NRVM measured at rates of stimulation 0.5-2 Hz were significantly modified with a shorter time to peak and time to 90% decay, and a larger fluorescence amplitude (p < 0.001). The proposed technique is compatible with standard cell culturing protocols and exhibits long-term pattern durability. Moreover, it allows the integration of monitoring modalities into the micro-engineered substrates for a comprehensive interrogation of physiological parameters.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CM) have been widely proposed as in vitro models of myocardial physiology and disease. A significant obstacle, however, is their immature phenotype. We hypothesised that Ca(2+) cycling of iPSC-CM is influenced by culture conditions and can be manipulated to obtain a more mature cellular behaviour. To test this hypothesis we seeded iPSC-CM onto fibronectin coated microgrooved polydimethylsiloxane (PDMS) scaffolds fabricated using photolithography, or onto unstructured PDMS membrane. After two weeks in culture, the structure and function of iPSC-CM were studied. PDMS microgrooved culture substrates brought about cellular alignment (p < 0.0001) and more organised sarcomere. The Ca(2+) cycling properties of iPSC-CM cultured on these substrates were significantly altered with a shorter time to peak amplitude (p = 0.0002 at 1 Hz), and more organised sarcoplasmic reticulum (SR) Ca(2+) release in response to caffeine (p < 0.0001), suggesting improved SR Ca(2+) cycling. These changes were not associated with modifications in gene expression. Whilst structured tissue culture may make iPSC-CM more representative of adult myocardium, further construct development and characterisation is required to optimise iPSC-CM as a model of adult myocardium.

Cardiac fibroblasts influence cardiomyocyte structure and function through direct physical interaction and/or by the secretion of soluble factors. A role for cardiac fibroblasts in cardiomyopathies has been proposed but clear mechanisms are still lacking. This in vitro study set out to characterise the influence of cardiac fibroblasts from patients with dilated cardiomyopathy on cardiomyocyte Ca2+ cycling, a fundamental mechanism of cardiac function universally altered in cardiac disease. Human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) were cultured with human DCM ventricular fibroblasts at a ratio of 2:1 (120,000 fibroblasts to 60,000 iPS-CMs) for 24 hours in three groups: iPS-CMs with fibroblast conditioned medium, co-cultured in transwells to allow bi-directional paracrine communication but prevent physical contact, and iPS-CMs in direct contact with fibroblasts. iPS-CMs alone were used as a baseline. iPS-CMs were field-stimulated at 1Hz and calcium transients were recorded optically. TGF-β, a cytokine previously shown to be important in fibroblast-myocyte interaction, was measured in culture supernate using an ELISA assay. Versus iPS-CMs alone, Ca2+ transient amplitude was reduced in the conditioned medium group but increased in co-culture (p

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.

Cardiac fibrosis is implicit in all forms of heart disease but there are no effective treatments. In this report, we investigate the role of the multi-functional enzyme Transglutaminase 2 (TG2) in cardiac fibrosis and assess its potential as a therapeutic target. Here we describe the use a highly selective TG2 small-molecule inhibitor to test the efficacy of TG2 inhibition as an anti-fibrotic therapy for heart failure employing two different in vivo models of cardiac fibrosis: Progressively induced interstitial cardiac fibrosis by pressure overload using angiotensin II infusion: Acutely induced focal cardiac fibrosis through myocardial infarction by ligation of the left anterior descending coronary artery (AMI model). In the AMI model, in vivo MRI showed that the TG2 inhibitor 1–155 significantly reduced infarct size by over 50% and reduced post-infarct remodelling at 20 days post insult. In both models, Sirius red staining for collagen deposition and levels of the TG2-mediated protein crosslink ε(γ-glutamyl)lysine were significantly reduced. No cardiac rupture or obvious signs of toxicity were observed. To provide a molecular mechanism for TG2 involvement in cardiac fibrosis, we show that both TGFβ1-induced transition of cardiofibroblasts into myofibroblast-like cells and TGFβ1-induced EndMT, together with matrix deposition, can be attenuated by the TG2 selective inhibitor 1–155, suggesting a new role for TG2 in regulating TGFβ1 signalling in addition to its role in latent TGFβ1 activation. In conclusion, TG2 has a role in cardiac fibrosis through activation of myofibroblasts and matrix deposition. TG2 inhibition using a selective small-molecule inhibitor can attenuate cardiac fibrosis.

The spontaneously hypertensive rat (SHR) is a well-characterised model for studies of hypertension and atrial arrhythmias but little is known about the electrophysiological properties of the left ventricle (LV) and their relation with ventricular arrhythmias in the development of this disease. To investigate the mechanisms behind electrophysiological abnormalities in the LV we used myocardial slices which allow the investigation of functional and structural properties in the same tissue location. Myocardial slices (300μm thick) were prepared from young (3months) and old (20months) SHR and age-matched control LVs. Slices were point-stimulated and analysed using a multi-electrode array system; longitudinal conduction velocity (CVL) was measured. CVL was unchanged between the young and old control groups. However, CVL was significantly reduced in the old SHR group compared to the corresponding age-matched control and young SHR groups (20months: 27±2 cm/s, n=29 slices/3 hearts vs control 39±4 cm/s, n=22 slices/4hearts and 3months: 37±3 cm/s, n=18 slices/3 hearts; p

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.

Electrical correlates of the physiological state of a cell, such as membrane conductance and capacitance, as well as cytoplasm conductivity, contain vital information about cellular function, ion transport across the membrane, and propagation of electrical signals. They are, however, difficult to measure; gold-standard techniques are typically unable to measure more than a few cells per day, making widespread adoption difficult and limiting statistical reproducibility. We have developed a dielectrophoretic platform using a disposable 3D electrode geometry that accurately (r2>0.99) measures mean electrical properties of populations of ~20,000 cells, by taking parallel ensemble measurements of cells at 20 frequencies up to 45 MHz, in (typically) ten seconds. This allows acquisition of ultra-high-resolution (100-point) DEP spectra in under two minutes. Data acquired from a wide range of cells – from platelets to large cardiac cells - benchmark well with patch-clamp-data. These advantages are collectively demonstrated in a longitudinal (same-animal) study of rapidly-changing phenomena such as ultradian (2-3 hour) rhythmicity in whole blood samples of the common vole (Microtus arvalis), taken from 10 µl tail-nick blood samples and avoiding sacrifice of the animal that is typically required in these studies.

Translation of novel therapies from bench to bedside is hampered by profound disparities between animal and human genetics and physiology. The ability to test for efficacy and cardiotoxicity in a clinically relevant human model system would enable more rapid therapy development. We have developed a preclinical platform for validation of new therapies in human heart tissue using organotypic slices isolated from donor and endstage failing hearts. A major advantage of the slices when compared with human iPSderived cardiomyocytes is that native tissue architecture and extracellular matrix are preserved, thereby allowing investigation of multi-cellular physiology in normal or diseased myocardium. To validate this model, we used optical mapping of transmembrane potential and calcium transients. We found that normal human electrophysiology is preserved in slice preparations when compared with intact hearts, including slices obtained from the region of the sinus node. Physiology is maintained in slices during culture, enabling testing the acute and chronic effects of pharmacological, gene, cell, optogenetic, device, and other therapies. This methodology offers a powerful high-throughput platform for assessing the physiological response of the human heart to disease and novel putative therapies.

Crosstalk between cardiomyocytes and fibroblasts in physiological conditions and during disease remains poorly defined. Previous studies have shown that fibroblasts and myocytes interact via paracrine communication, but several experimental confounding factors, including the use of immature myocytes and the induction of alpha-smooth muscle actin (α-SMA) expression in fibroblasts by prolonged culture, have hindered our understanding of this phenomenon. We hypothesize that fibroblasts and myofibroblasts differentially affect cardiomyocytes viability, volume, and Ca(2+) handling via soluble mediators. More specifically here: (i) we compare the effects of freshly isolated fibroblasts and cultured fibroblasts from normal rat hearts on adult cardiomyocytes; (ii) we compare the effects of (freshly isolated) normal fibroblasts and myofibroblasts from pressure-overloaded hearts; and (iii) we study the contribution of TGF-β and the importance of the crosstalk between the two cell types.

Cardiac myocytes and fibroblasts form extensive networks in the heart, with numerous anatomical contacts between cells. Fibroblasts, obligatory components of the extracellular matrix, represent the majority of cells in the normal heart, and their number increases with aging and during disease. The myocyte network, coupled by gap junctions, is generally believed to be electrically isolated from fibroblasts in vivo. In culture, however, the heterogeneous cell types form functional gap junctions, which can provide a substrate for electrical coupling of distant myocytes, interconnected by fibroblasts only. Whether similar behavior occurs in vivo has been the subject of considerable debate. Recent electrophysiological, immunohistochemical, and dye-coupling data confirmed the presence of direct electrical coupling between the two cell types in normal cardiac tissue (sinoatrial node), and it has been suggested that similar interactions may occur in post-infarct scar tissue. Such heterogeneous cell coupling could have major implications on in vivo electrical impulse conduction and the transport of small molecules or ions in both the normal and pathological myocardium. This review illustrates that it would be wrong to adhere to a scenario of functional integration of the heart that does not allow for a potential active contribution of non-myocytes to cardiac electrophysiology, and proposes to focus further research on the relevance of non-myocytes for cardiac structure and function.

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.

Cardiac fibroblasts are known to modulate cardiomyocyte structure and function through soluble mediators. While less studied, the ability of cardiomyocytes to influence fibroblast properties, such as proliferation and activation, is likely to be of equal importance, as myocytes are known to produce a number of soluble factors to which fibroblasts are sensitive. Further, whether disease alters this myocyte-fibroblast cross talk is an important question to address.

We describe here a new in vitro protocol for structuring cardiac cell cultures to mimic important aspects of the in vivo ventricular myocardial phenotype by controlling the location and mechanical environment of cultured cells. Microlithography is used to engineer microstructured silicon metal wafers. Those are used to fabricate either microgrooved silicone membranes or silicone molds for microfluidic application of extracellular matrix proteins onto elastic membranes (involving flow control at micrometer resolution). The physically or microfluidically structured membranes serve as a cell culture growth substrate that supports cell alignment and allows the application of stretch. The latter is achieved with a stretching device that can deliver isotropic or anisotropic stretch. Neonatal ventricular cardiomyocytes, grown on these micropatterned membranes, develop an in vivo-like morphology with regular sarcomeric patterns. The entire process from fabrication of the micropatterned silicon metal wafers to casting of silicone molds, microfluidic patterning and cell isolation and seeding takes approximately 7 days.

Myocytes, while giving rise to the bulk volume of normal cardiac muscle, form a "minority cell population" in the heart compared with nonmyocytes, chiefly fibroblasts. The heterogeneous cell types show very intimate spatial interrelation in situ, with virtually every myocyte in the mammalian heart bordering to 1 or more fibroblasts. Nonetheless, gap junction coupling in the heart is traditionally assumed to occur exclusively between myocytes. Yet, both freshly isolated cells and cell cultures have unambiguously shown functional heterogeneous myocyte-fibroblast coupling (mainly via connexin 43). Such coupling is sufficient, in vitro, to synchronize spontaneous beating in distant myocytes, connected over distances of up to 300 microm by fibroblasts only. More recently, functional myocyte-fibroblast coupling (via connexin 45) has been demonstrated in situ for sinoatrial node pacemaker tissue, and preliminary immunohistochemical data suggest that myocyte-fibroblast coupling may be present in postinfarct scar tissue. The functional relevance of such heterogeneous coupling for cardiac electrophysiology is only starting to emerge and has thus far mainly been assessed in theoretical studies. According to this research, fibroblasts may affect the origin and spread of excitation in several ways above and beyond formation of "passive" barriers that obstruct electrical conduction. Thus, fibroblasts may act as current sinks, contributing to the formation of unidirectional block or to the delay in atrioventricular conduction. Via short-range interaction, fibroblasts may help to smooth out propagating wave fronts, in particular in the sinoatrial node and in the cross-sheet direction of healthy ventricular myocardium, 2 tissues that might otherwise be expected to show fragmented conduction patterns. As long-distance communication lines, fibroblasts may bridge posttransplantation or ischemic scar tissue, with beneficial or detrimental effects on organ function (depending on the relation to normal conduction patterns), and explain the recruitment of myocyte islands embedded in fibrotic scar tissue. The inherent mechanosensitivity of cardiac fibroblasts could, furthermore, allow them to play a sensory role and to affect cardiac electrophysiology via mechanoelectric feedback. This article reviews the currently available experimental and theoretical evidence on the previous scenarios, and highlights areas for further research.

Cardiac fibroblasts form one of the largest cell populations, in terms of cell numbers, in the heart. They contribute to structural, biochemical, mechanical and electrical properties of the myocardium. Nonetheless, they are often disregarded by in vivo and in vitro studies into cardiac function. This review summarizes our understanding of fibroblast origin and identity, their structural organization and role in myocardial architecture, as well as functional aspects related to cell signalling and electro-mechanical function in the heart.

The majority of Na+ channels in the heart are composed of the tetrodotoxin (TTX)-resistant (KD, 2-6 microm) Nav1.5 isoform; however, recently it has been shown that TTX-sensitive (KD, 1-10 nm) neuronal Na+ channel isoforms (Nav1.1, Nav1.3 and Nav1.6) are also present and functionally important in the myocytes of the ventricles and the sinoatrial (SA) node. In the present study, in mouse SA node pacemaker cells, we investigated Na+ currents under physiological conditions and the expression of cardiac and neuronal Na+ channel isoforms. We identified two distinct Na+ current components, TTX resistant and TTX sensitive. At 37 degrees C, TTX-resistant iNa and TTX-sensitive iNa started to activate at approximately -70 and approximately -60 mV, and peaked at -30 and -10 mV, with a current density of 22 +/- 3 and 18 +/- 1 pA pF(-1), respectively. TTX-sensitive iNa inactivated at more positive potentials as compared to TTX-resistant iNa. Using action potential clamp, TTX-sensitive iNa was observed to activate late during the pacemaker potential. Using immunocytochemistry and confocal microscopy, different distributions of the TTX-resistant cardiac isoform, Nav1.5, and the TTX-sensitive neuronal isoform, Nav1.1, were observed: Nav1.5 was absent from the centre of the SA node, but present in the periphery of the SA node, whereas Nav1.1 was present throughout the SA node. Nanomolar concentrations (10 or 100 nm) of TTX, which block TTX-sensitive iNa, slowed pacemaking in both intact SA node preparations and isolated SA node cells without a significant effect on SA node conduction. In contrast, micromolar concentrations (1-30 microm) of TTX, which block TTX-resistant iNa as well as TTX-sensitive iNa, slowed both pacemaking and SA node conduction. It is concluded that two Na+ channel isoforms are important for the functioning of the SA node: neuronal (putative Nav1.1) and cardiac Nav1.5 isoforms are involved in pacemaking, although the cardiac Nav1.5 isoform alone is involved in the propagation of the action potential from the SA node to the surrounding atrial muscle.

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.

BACKGROUND: Targeted splice modulation of pre-mRNA transcripts by antisense oligonucleotides (AOs) can correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) arises as a result of mutations that interrupt the open-reading frame in the DMD gene encoding dystrophin such that dystrophin protein is absent, leading to fatal muscle degeneration. AOs have been shown to correct this dystrophin defect via exon skipping to yield functional dystrophin protein in animal models of DMD and also in DMD patients via intramuscular administration. To advance this therapeutic method requires increased exon skipping efficiency via an optimized AO sequence, backbone chemistry and additional modifications, and the improvement of methods for evaluating AO efficacy. METHODS: In the present study, we establish the conditions for rapid in vitro AO screening in H(2)K muscle cells, in which we evaluate the exon skipping properties of a number of known and novel AO chemistries [2'-O-methyl, peptide nucleic acid, phosphorodiamidate morpholino (PMO)] and their peptide-conjugated derivatives and correlate their in vitro and in vivo exon skipping activities. RESULTS: The present study demonstrates that using AO concentrations of 300 nM with analysis at a single time-point of 24 h post-transfection allowed the effective in vitro screening of AO compounds to yield data predictive of in vivo exon skipping efficacy. Peptide-conjugated PMO AOs provided the highest in vitro activity. We also show for the first time that the feasibility of rapid AO screening extends to primary cardiomyocytes. CONCLUSIONS: In vitro screening of different AOs within the same chemical class is a reliable method for predicting the in vivo exon skipping efficiency of AOs for DMD.

AIMS: Intracellular pH (pHi), an important modulator of cardiac function, is normally regulated to within narrow limits (7.1-7.2). In adult ventricular cell pairs, localized cellular pHi disturbances are removed by sarcolemmal acid/base transporters, but can also be dissipated (diluted) across gap junctions, aboard mobile buffers such as CO2/HCO3- and histidine-containing dipeptides (HCDPs). In the present work, we test this model of spatial pHi regulation in multicellular strands of neonatal rat ventricular myocytes. METHODS AND RESULTS: We confocally image pHi (intracellular fluorescence emitted from the pH dye carboxy-SNARF-1) in multicellular (>500 microm long, approximately 30 microm wide) cultured strands of electrically coupled, neonatal rat ventricular myocytes. Activity of sarcolemmal Na+/H+ exchange and Na+-HCO3- co-transport resembles that in adult cells. Localized photolytic H+ uncaging from intracellular 2-nitrobenzaldehyde, in the presence of CO2/HCO3- buffer, triggers considerable passive H+ spread along a strand, thus helping to dissipate the acid load. Inhibition of gap junctions (with alpha-glycyrrhetinic acid) truncates the spread, indicating they are conduits for local intracellular H+ flux. Without CO2/HCO3- buffer, longitudinal H+ mobility is reduced by approximately 90%, indicating that intracellular and cell-to-cell H+ flux relies far less on intrinsic mobile buffers (e.g. HCDPs) in neonates than in adults. This is consistent with five-fold lower HCDP levels in neonatal, compared to adult, ventricular tissue, and also with measurements of a lower intrinsic (non-CO2/HCO3-) H+ buffering capacity in neonatal strands compared with freshly isolated adult cells. CONCLUSION: We conclude that mobile buffers and gap junctions are key spatial controllers of pHi in cardiac tissue, helping to maintain a myocardial pHi syncitium. In neonatal tissue, intracellular H+ movement is CO2/HCO3- dependent, while adult tissue relies increasingly on intrinsic dipeptides that provide additional spatial pHi control, appropriate for the developmental increase in myocyte size.

Transmural and regional gradients in membrane potential and Ca2+ transient in the murine heart are largely unexplored. Here, we developed and validated a robust approach which combines transverse ultra‐thin cardiac slices and high resolution optical mapping to enable systematic analysis of transmural and regional gradients in transmembrane potential (Vm) and intracellular Ca2+ transient (CaT) across the entire murine ventricles. The voltage dye RH237 or Ca2+ dye Rhod‐2 AM were loaded through the coronary circulation using a Langendorff perfusion system. Short‐axis slices (300 μm thick) were prepared from the entire ventricles (from the apex to the base) by using a high‐precision vibratome. Action potentials (APs) and CaTs were recorded with optical mapping during steady‐state baseline and rapid pacing. Significant transmural gradients in Vm and CaT were observed in the left ventricle, with longer AP duration (APD50 and APD75) and CaT duration (CaTD50 and CaTD75) in the endocardium compared with that in the epicardium. No significant regional gradients were observed along the apico‐basal axis of the left ventricle. Interventricular gradients were detected with significantly shorter APD50, APD75 and CaTD50 in the right ventricle compared with left ventricle and ventricular septum. During rapid pacing, AP and CaT alternans were observed in most ventricular regions, with significantly greater incidence in the endocardium in comparison with epicardium. In conclusion, these observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in Vm and CaT in murine ventricular tissue.

Cardiac myocytes and fibroblasts are essential elements of myocardial tissue structure and function. In vivo, myocytes constitute the majority of cardiac tissue volume, whereas fibroblasts dominate in numbers. In vitro, cardiac cell cultures are usually designed to exclude fibroblasts, which, because of their maintained proliferative potential, tend to overgrow the myocytes. Recent advances in microstructuring of cultures and cell growth on elastic membranes have greatly enhanced in vitro preservation of tissue properties and offer a novel platform technology for producing more in vivo-like models of myocardium. We used microfluidic techniques to grow two-dimensional structured cardiac tissue models, containing both myocytes and fibroblasts, and characterized cell morphology, distribution, and coupling using immunohistochemical techniques. In vitro findings were compared with in vivo ventricular cyto-architecture. Cardiac myocytes and fibroblasts, cultured on intersecting 30-microm-wide collagen tracks, acquire an in vivo-like phenotype. Their spatial arrangement closely resembles that observed in native tissue: Strands of highly aligned myocytes are surrounded by parallel threads of fibroblasts. In this in vitro system, fibroblasts form contacts with other fibroblasts and myocytes, which can support homogeneous and heterogeneous gap junctional coupling, as observed in vivo. We conclude that structured cocultures of cardiomyocytes and fibroblasts mimic in vivo ventricular tissue organization and provide a novel tool for in vitro research into cardiac electromechanical function.

AIM: Isolated papillary muscles and enzymatically dissociated myocytes of guinea-pig hearts are routinely used for experimental cardiac research. The aim of our study is to investigate adult mammalian ventricular slices as an alternative preparation. METHOD: Vibratome cut ventricular slices (350 microm thick) were examined histologically and with 2-photon microscopy for fibre orientation. Intracellular action potentials were recorded with conventional glass microelectrodes, extracellular potentials were measured with tungsten platinum electrodes and multi-electrode arrays (MEA). RESULTS: Dominant direction of fibre orientation was absent in vertical and horizontal transmural slices, but was longitudinal in tangential slices. Control action potential duration (APD(90), 169.9 +/- 4 ms) and drug effects on this parameter were similar to papillary muscles. The L-type Ca-channel blocker nifedipine shortened APD(90) with a half maximal effective concentration (EC(50)) of 4.5 microM. The I(Kr) blocker E4031 and neuroleptic drug risperidone prolonged APD(90) with EC(50) values of 31 nM and 0.67 microM, respectively. Mapping field potentials on multi-electrode arrays showed uniform spread of excitation with a mean conduction velocity of 0.47 m s(-1). CONCLUSION: Slices from adult mammalian hearts could become a useful routine model for electrophysiological and pharmacological research.

It is known that cells grown in 3D are more tolerant to drug treatment than those grown in dispersion but the mechanism for this is still not clear; cells grown in 3D have opportunities to develop inter-cell communication, but are also closely packed which may impede diffusion. In this study we examine methods for dielectrophoresis-based cell aggregation of both suspension and adherent cell lines and compare the effect of various drugs on cells grown in 3D and 2D. Comparing viability of pharmacological interventions on 3D cell clusters against both suspension cells and adherent cells grown in monolayer, as well as against a unicellular organism with no propensity for intracellular communication, we suggest that 3D aggregates of adherent cells, compared to suspension cells, show a substantially different drug response to cells grown in monolayer, which increases as the IC50 is approached. Further, a mathematical model of the system for each agent demonstrates that changes to drug response are due to inherent changes in the system of adherent cells from the 2D to 3D state. Finally, differences within electrophysiological membrane properties of the adherent cell type suggest this parameter plays an important role in the differences found in the 3D drug response.

Sarcomere length (SL) is an important determinant and indicator of cardiac mechanical function; however, techniques for measuring SL in living, intact tissue are limited. Here, we present a technique that uses two-photon microscopy to directly image striations of living cells in cardioplegic conditions, both in situ (Langendorff-perfused rat hearts and ventricular tissue slices, stained with the fluorescent marker di-4-ANEPPS) and in vitro (acutely isolated rat ventricular myocytes). Software was developed to extract SL from two-photon fluorescence image sets while accounting for measurement errors associated with motion artifact in raster-scanned images and uncertainty of the cell angle relative to the imaging plane. Monte-Carlo simulations were used to guide analysis of SL measurements by determining error bounds as a function of measurement path length. The mode of the distribution of SL measurements in resting Langendorff-perfused heart is 1.95 mum (n = 167 measurements from N = 11 hearts) after correction for tissue orientation, which was significantly greater than that in isolated cells (1.71 mum, n = 346, N = 9 isolations) or ventricular slice preparations (1.79 mum, n = 79, N = 3 hearts) under our experimental conditions. Furthermore, we find that edema in arrested Langendorff-perfused heart is associated with a mean SL increase; this occurs as a function of time ex vivo and correlates with tissue volume changes determined by magnetic resonance imaging. Our results highlight that the proposed method can be used to monitor SL in living cells and that different experimental models from the same species may display significantly different SL values under otherwise comparable conditions, which has implications for experiment design, as well as comparison and interpretation of data.

We investigate acute effects of axial stretch, applied by carbon fibers (CFs), on diastolic Ca2+ spark rate in rat isolated cardiomyocytes. CFs were attached either to both cell ends (to maximize the stretched region), or to the center and one end of the cell (to compare responses in stretched and nonstretched half-cells). Sarcomere length was increased by 8.01+/-0.94% in the stretched cell fraction, and time series of XY confocal images were recorded to monitor diastolic Ca2+ spark frequency and dynamics. Whole-cell stretch causes an acute increase of Ca2+ spark rate (to 130.7+/-6.4%) within 5 seconds, followed by a return to near background levels (to 104.4+/-5.1%) within 1 minute of sustained distension. Spark rate increased only in the stretched cell region, without significant differences in spark amplitude, time to peak, and decay time constants of sparks in stretched and nonstretched areas. Block of stretch-activated ion channels (2 micromol/L GsMTx-4), perfusion with Na+/Ca2+-free solution, and block of nitric oxide synthesis (1 mmol/L L-NAME) all had no effect on the stretch-induced acute increase in Ca2+ spark rate. Conversely, interference with cytoskeletal integrity (2 hours of 10 micromol/L colchicine) abolished the response. Subsequent electron microscopic tomography confirmed the close approximation of microtubules with the T-tubular-sarcoplasmic reticulum complex (to within approximately 10(-8)m). In conclusion, axial stretch of rat cardiomyocytes acutely and transiently increases sarcoplasmic reticulum Ca2+ spark rate via a mechanism that is independent of sarcolemmal stretch-activated ion channels, nitric oxide synthesis, or availability of extracellular calcium but that requires cytoskeletal integrity. The potential of microtubule-mediated modulation of ryanodine receptor function warrants further investigation.

Electrophysiological and pharmacological data from the human heart are limited due to the absence of simple but representative experimental model systems of human myocardium. The aim of this study was to establish and characterise adult human myocardial slices from small patients' heart biopsies as a simple, reproducible and relevant preparation suitable for the study of human cardiac tissue at the multicellular level. Vibratome-cut myocardial slices were prepared from left ventricular biopsies obtained from end-stage heart failure patients undergoing heart transplant or ventricular assist device implantation, and from hearts of normal dogs. Multiple slices were prepared from each biopsy. Regular contractility was observed at a range of stimulation frequencies (0.1-2 Hz), and stable electrical activity, monitored using multi-electrode arrays (MEA), was maintained for at least 8 h from slice preparation. ATP/ADP and phosphocreatine/creatine ratios were comparable to intact organ values, and morphology and gap junction distribution were representative of native myocardium. MEA recordings showed that field potential duration (FPD) and conduction velocity (CV) in human and dog slices were similar to the values previously reported for papillary muscles, ventricular wedges and whole hearts. Longitudinal CV was significantly faster than transversal CV, with an anisotropic ratio of 3:1 for human and 2.3:1 for dog slices. Importantly, slices responded to the application of E-4031, chromanol and 4-aminopyridine, three potassium channel blockers known to affect action potential duration, with an increase in FPD. We conclude that viable myocardial slices with preserved structural, biochemical and electrophysiological properties can be prepared from adult human and canine heart biopsies and offer a novel preparation suitable for the study of heart failure and drug screening.

Induced splice modulation of pre-mRNAs shows promise to correct aberrant disease transcripts and restore functional protein and thus has therapeutic potential. Duchenne muscular dystrophy (DMD) results from mutations that disrupt the DMD gene open reading frame causing an absence of dystrophin protein. Antisense oligonucleotide (AO)-mediated exon skipping has been shown to restore functional dystrophin in mdx mice and DMD patients treated intramuscularly in two recent phase 1 clinical trials. Critical to the therapeutic success of AO-based treatment will be the ability to deliver AOs systemically to all affected tissues including the heart. Here, we report identification of a series of transduction peptides (Pip5) as AO conjugates for enhanced systemic and particularly cardiac delivery. One of the lead peptide-AO conjugates, Pip5e-AO, showed highly efficient exon skipping and dystrophin production in mdx mice with complete correction of the aberrant DMD transcript in heart, leading to >50% of the normal level of dystrophin in heart. Mechanistic studies indicated that the enhanced activity of Pip5e-phosphorodiamidate morpholino (PMO) is partly explained by more efficient nuclear delivery. Pip5 series derivatives therefore have significant potential for advancing the development of exon skipping therapies for DMD and may have application for enhanced cardiac delivery of other biotherapeutics.

BACKGROUND: Recent experimental studies have documented that functional gap junctions form between fibroblasts and myocytes, raising the possibility that fibroblasts play roles in cardiac electrophysiology that extend beyond acting as passive electrical insulators. OBJECTIVE: The purpose of this study was to use computational models to investigate how fibroblasts may affect cardiac conduction and vulnerability to reentry under different fibroblast-myocyte coupling conditions and tissue structures. METHODS: Computational models of two-dimensional tissue with fibroblast-myocyte coupling were developed and numerically simulated. Myocytes were modeled by the phase I of the Luo-Rudy model, and fibroblasts were modeled by a passive model. RESULTS: Besides slowing conduction by cardiomyocyte decoupling and electrotonic loading, fibroblast coupling to myocytes elevates myocyte resting membrane potential, causing conduction velocity to first increase and then decrease as fibroblast content increases, until conduction failure occurs. Fibroblast-myocyte coupling can also enhance conduction by connecting uncoupled myocytes. These competing effects of fibroblasts on conduction give rise to different conduction patterns under different fibroblast-myocyte coupling conditions and tissue structures. Elevation of myocyte resting potential due to fibroblast-myocyte coupling slows sodium channel recovery, which extends postrepolarization refractoriness. Owing to this prolongation of the myocyte refractory period, reentry was more readily induced by a premature stimulation in heterogeneous tissue models when fibroblasts were electrotonically coupled to myocytes compared with uncoupled fibroblasts acting as pure passive electrical insulators. CONCLUSIONS: Fibroblasts affect cardiac conduction by acting as obstacles or by creating electrotonic loading and elevating myocyte resting potential. Functional fibroblast-myocyte coupling prolongs the myocyte refractory period, which may facilitate induction of reentry in cardiac tissue with fibrosis.

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 (1-10µM) reduced contractile force and contraction kinetics in porcine slices in a dose-depended manner. However, AZM monotherapy decreased contractile force and contraction kinetics only at higher concentrations (30µM). 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.

OBJECTIVES: (i) to characterize the electrophysiological properties of the slowly activating delayed rectifier potassium current, i(Ks), defined as the 293b-sensitive current, during the action potential (AP) of rabbit sino-atrial node (SAN) pacemaker cells; (ii) to evaluate the contribution of i(Ks) to the pacemaker AP under physiological conditions and during beta-adrenergic stimulation. METHODS: Rabbit SAN pacemaker cells were studied using the perforated patch clamp technique in voltage-, AP- and current-clamp modes. RESULTS: Voltage-clamp findings. Block of i(Ks) by 293b is dose-dependent, with an IC(50) (half block) in rabbit SAN cells of 1.35 microM and an IC(80) (sub-maximal block) of 5 microM. Sub-maximal concentrations of 293b have no significant effects on long-lasting and transient inward calcium currents, i(Ca,L) and i(Ca,T), inward hyperpolarization activated current, i(f), and transient outward current, i(to). AP-clamp experiments. The 293b-sensitive current activates near the peak of the SAN pacemaker action potential, reaches a mean maximal current density of 1.0+/-0.3 pA/pF (n=8, cell capacitances 27 to 62 pF, mean 35+/-4.0 pF) during late repolarization, and inactivates towards the end of repolarization. Additionally, in two smaller cells (cell capacitances 15 and 23 pF), no discernible 293b-sensitive current component was detected. Current-clamp data. In spontaneously beating SAN cells under control conditions, sub-maximal block of i(Ks) by 5 microM 293b has negligible effects on action potential characteristics and does not change average cycle length (n=11). In contrast, after pre-treatment with 10 nM isoprenaline to mimic beta-adrenergic stimulation, cells showed a 293b-induced depolarization of maximum diastolic potential by 2.2+/-1%, a decrease in diastolic depolarization rate by 9.9+/-4%, and a slowing of late action potential repolarization by 28.7+/-10.2%, resulting in a prolongation of spontaneous cycle length by 9.8+/-3.0% (P

Virtual anatomy models show in detail characteristics of the human body systems. These models are based in surface representation of the structures and lack information from the interior of the object. Creating models that represent the surface, the interior of the object and are able to provide pathological information is the current challenge of research in life sciences. We present a method to synthesize realistic three-dimensional organic tissues starting from bidimensional textured multi-channel samples. The method relies on an energy function that measures the difference between the reference texture and the synthesized object, through a distance metric that compares perpendicular neighborhoods in the object to neighborhoods in the sample. When this function is minimized by IRLS, the result is a solid object that resembles the sample at every slice. In some cases, the optimization might be aided by adding the feature distance transform, calculated from a given binary mask. This allows to code large textured areas. Multiple textures can also be provided to the optimization in order to create anistropic textures. We apply our method starting from various micrometric images such as histology images or slices of Synchrotron Radiation Computed Micro-Tomography (SRμCT) images. A major advantage of our method is to extend 2D histological information to a 3D representation. We demonstrate the accuracy of the generated texture by comparing statistical and morphological parameters computed from the synthetic object with those obtained from the real object underlying the reference images.