Human myofibroblasts increase the arrhythmogenic potential of human induced pluripotent stem cell-derived cardiomyocytes.

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. Myofibroblasts are the predominant cell-type in the infarcted myocardium but their impact on transplanted hiPSC-CMs remains poorly defined. Here, we investigate the effect of myofibroblasts on hiPSC-CMs electrophysiology and Ca2+ handling using optical mapping of advanced human cell coculture systems mimicking cell-cell interaction modalities. Human myofibroblasts 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 myofibroblasts and direct stimulation of hiPSC-CMs with exogenous interleukin-6 recapitulated the paracrine effects of myofibroblasts. Blocking interleukin-6 reduced the effects of myofibroblasts only in the absence of physical contact between cell-types. Myofibroblast-specific connexin43 knockdown reduced functional changes in contact cocultures only when combined with interleukin-6 blockade. This provides the first in-depth investigation into how human myofibroblasts 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.

Emerging Bioelectronic Strategies for Cardiovascular Tissue Engineering and Implantation.

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.

Electrotonic coupling of excitable and nonexcitable cells in the heart revealed by optogenetics.

Electrophysiological studies of excitable organs usually focus on action potential (AP)-generating cells, whereas nonexcitable cells are generally considered as barriers to electrical conduction. Whether nonexcitable cells may modulate excitable cell function or even contribute to AP conduction via direct electrotonic coupling to AP-generating cells is unresolved in the heart: such coupling is present in vitro, but conclusive evidence in situ is lacking. We used genetically encoded voltage-sensitive fluorescent protein 2.3 (VSFP2.3) to monitor transmembrane potential in either myocytes or nonmyocytes of murine hearts. We confirm that VSFP2.3 allows measurement of cell type-specific electrical activity. We show that VSFP2.3, expressed solely in nonmyocytes, can report cardiomyocyte AP-like signals at the border of healed cryoinjuries. Using EM-based tomographic reconstruction, we further discovered tunneling nanotube connections between myocytes and nonmyocytes in cardiac scar border tissue. Our results provide direct electrophysiological evidence of heterocellular electrotonic coupling in native myocardium and identify tunneling nanotubes as a possible substrate for electrical cell coupling that may be in addition to previously discovered connexins at sites of myocyte-nonmyocyte contact in the heart. These findings call for reevaluation of cardiac nonmyocyte roles in electrical connectivity of the heterocellular heart.

Transverse cardiac slicing and optical imaging for analysis of transmural gradients in membrane potential and Ca2+ transients in murine heart.

KEY POINTS: A robust cardiac slicing approach was developed for optical mapping of transmural gradients in transmembrane potential (Vm ) and intracellular Ca2+ transient (CaT) of murine heart. Significant transmural gradients in Vm and CaT were observed in the left ventricle. Frequency-dependent action potentials and CaT alternans were observed in all ventricular regions with rapid pacing, with significantly greater incidence in the endocardium than epicardium. The observations demonstrate the feasibility of our new approach to cardiac slicing for systematic analysis of intrinsic transmural and regional gradients in Vm and CaT. ABSTRACT: 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.

Role of Non-Myocyte Gap Junctions and Connexin Hemichannels in Cardiovascular Health and Disease: Novel Therapeutic Targets?

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.

Prolongation of atrio-ventricular node conduction in a rabbit model of ischaemic cardiomyopathy: Role of fibrosis and connexin remodelling.

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 the 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 protein (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.

Injectable biodegradable microcarriers for iPSC expansion and cardiomyocyte differentiation.

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.

Cellular coupling in the heart.

Fibroblasts in scar tissue elicit myocyte excitation and promote arrhythmia in mouse hearts.

A novel method to extend viability and functionality of living heart slices.

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.

Hydroxychloroquine and azithromycin alter the contractility of living porcine heart slices.

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.

Myofibroblast senescence promotes arrhythmogenic remodeling in the aged infarcted rabbit 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.

Pip5 transduction peptides direct high efficiency oligonucleotide-mediated dystrophin exon skipping in heart and phenotypic correction in mdx mice.

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.

Identification of the NADPH Oxidase (Nox) Subtype and the Source of Superoxide Production in the Micturition Centre.

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.

Porcine Organotypic Epicardial Slice Protocol: A Tool for the Study of Epicardium in Cardiovascular Research.

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.

Adult human heart slices are a multicellular system suitable for electrophysiological and pharmacological studies.

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.

Axial stretch of rat single ventricular cardiomyocytes causes an acute and transient increase in Ca2+ spark rate.

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.

Myocardial Viability Imaging using Manganese-Enhanced MRI in the First Hours after Myocardial Infarction.

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.

In vitro evaluation of novel antisense oligonucleotides is predictive of in vivo exon skipping activity for Duchenne muscular dystrophy.

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.

DEP-Dots for 3D cell culture: low-cost, high-repeatability, effective 3D cell culture in multiple gel systems.

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 in the electrophysiological membrane properties of the adherent cell type suggest this parameter plays an important role in the differences found in the 3D drug response.

Tissue slices from adult mammalian hearts as a model for pharmacological drug testing.

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.