Evaluation of tissue-engineered human acellular vessels as a Blalock-Taussig-Thomas shunt in a juvenile primate model.

Objectives: Palliative treatment of cyanotic congenital heart disease (CCHD) uses systemic-to-pulmonary conduits, often a modified Blalock-Taussig-Thomas shunt (mBTTs). Expanded polytetrafluoroethylene (ePTFE) mBTTs have associated risks for thrombosis and infection. The Human Acellular Vessel (HAV) (Humacyte, Inc) is a decellularized tissue-engineered blood vessel currently in clinical trials in adults for vascular trauma, peripheral artery disease, and end-stage renal disease requiring hemodialysis. In addition to restoring blood flow, the engineered HAV demonstrates the capacity for host cellular remodeling into native-like vasculature. Here we report preclinical evaluation of a small-diameter (3.5 mm) HAV as a mBTTs in a non-human primate model. Methods: We implanted 3.5 mm HAVs as right subclavian artery to pulmonary artery mBTTs in non-immunosuppressed juvenile rhesus macaques (n = 5). HAV patency, structure, and blood flow were assessed by postoperative imaging from 1 week to 6 months. Histology of HAVs and surrounding tissues was performed. Results: Surgical procedures were well tolerated, with satisfactory anastomoses, showing feasibility of using the 3.5 mm HAV as a mBTTs. All macaques had some immunological reactivity to the human extracellular matrix, as expected in this xenogeneic model. HAV mBTTs remained patent for up to 6 months in animals, exhibiting mild immunoreactivity. Two macaques displaying more severe immunoreactivity to the human HAV material developed midgraft dilatation without bleeding or rupture. HAV repopulation by host cells expressing smooth muscle and endothelial markers was observed in all animals. Conclusions: These findings may support use of 3.5 mm HAVs as mBTTs in CCHD and potentially other pediatric vascular indications.

Biological mechanisms of infection resistance in tissue engineered blood vessels compared to synthetic expanded polytetrafluoroethylene grafts.

Objective: Synthetic expanded polytetrafluoroethylene (ePTFE) grafts are known to be susceptible to bacterial infection. Results from preclinical and clinical studies of bioengineered human acellular vessels (HAVs) have shown relatively low rates of infection. This study evaluates the interactions of human neutrophils and bacteria with ePTFE and HAV vascular conduits to determine whether there is a correlation between neutrophil-conduit interactions and observed differences of their infectivity in vivo. Methods: A phase III comparative clinical study between investigational HAVs (n = 177) and commercial ePTFE grafts (n = 178) used for hemodialysis access (ClinicalTrials.gov Identifier: NCT02644941) was evaluated for conduit infection rates followed by histological analyses of HAV and ePTFE tissue explants. The clinical histopathology of HAV and ePTFE conduits reported to be infected was compared with immunohistochemistry of explanted materials from a preclinical model of bacterial contamination. Mechanistic in vitro studies were then conducted using isolated human neutrophils seeded directly onto HAV and ePTFE materials to analyze neutrophil viability, morphology, and function. Results: Clinical trial results showed that the HAV had a significantly lower (0.93%; P = .0413) infection rate than that of ePTFE (4.54%). Histological analysis of sections from infected grafts explanted approximately 1 year after implantation revealed gram-positive bacteria near cannulation sites. Immunohistochemistry of HAV and ePTFE implanted in a well-controlled rodent infection model suggested that the ePTFE matrix permitted bacterial infiltration and colonization but may be inaccessible to neutrophils. In the same model, the HAV showed host recellularization and lacked detectable bacteria at the 2-week explant. In vitro results demonstrated that the viability of human neutrophils decreased significantly upon exposure to ePTFE, which was associated with neutrophil elastase release in the absence of bacteria. In contrast, neutrophils exposed to the HAV material retained high viability and native morphology. Cocultures of neutrophils and Staphylococcus aureus on the conduit materials demonstrated that neutrophils were more effective at ensnaring and degrading bacteria on the HAV than on ePTFE. Conclusions: The HAV material seems to demonstrate a resistance to bacterial infection. This infection resistance is likely due to the HAV's native-like material composition, which may be more biocompatible with host neutrophils than synthetic vascular graft material.

Evaluation of vascular repair by tissue-engineered human acellular vessels or expanded polytetrafluoroethylene grafts in a porcine model of limb ischemia and reperfusion.

BACKGROUND: This study evaluated performance of a tissue-engineered human acellular vessel (HAV) in a porcine model of acute vascular injury and ischemia. The HAV is an engineered blood vessel consisted of human vascular extracellular matrix proteins. Limb reperfusion and vascular outcomes of the HAV were compared with those from synthetic expanded polytetrafluoroethylene (ePTFE) grafts. METHODS: Thirty-six pigs were randomly assigned to four treatment groups, receiving either the HAV or a PTFE graft following a hind limb ischemia period of either 0 or 6 hours. All grafts were 3-cm-long interposition 6-mm diameter grafts implanted within the right iliac artery. Animals were not immunosuppressed and followed for up to 28 days after surgery. Assessments performed preoperatively and postoperatively included evaluation of graft patency, hind limb function, and biochemical markers of tissue ischemia or reperfusion injury. Histological analysis was performed on explants to assess host cell responses. RESULTS: Postoperative gait assessment and biochemical analysis confirmed that ischemia and reperfusion injury were caused by 6-hour ischemia, regardless of vascular graft type. Hind limb function and tissue damage biomarkers improved in all groups postoperatively. Final patency rates at postoperative day 28 were higher for HAV than for ePTFE graft in both the 0-hour (HAV, 85.7%; ePTFE, 66.7%) and 6-hour (HAV, 100%; ePTFE, 75%) ischemia groups, but these differences were not statistically significant. Histological analyses identified some intimal hyperplasia and host reactivity to the xenogeneic HAV and also to the synthetic ePTFE graft. Positive host integration and vascular cell infiltration were identified in HAV but not ePTFE explants. CONCLUSION: Based on the functional performance and the histologic profile of explanted HAVs, this study supports further investigation to evaluate long-term performance of the HAV when used to repair traumatic vascular injuries.

Arterial reconstruction with human bioengineered acellular blood vessels in patients with peripheral arterial disease.

OBJECTIVE: Vascular conduit is essential for arterial reconstruction for a number of conditions, including trauma and atherosclerotic occlusive disease. We have developed a tissue-engineered human acellular vessel (HAV) that can be manufactured, stored on site at hospitals, and be immediately available for arterial vascular reconstruction. Although the HAV is acellular when implanted, extensive preclinical and clinical testing has demonstrated that the HAV subsequently repopulates with the recipient's own vascular cells. We report a first-in-man clinical experience using the HAV for arterial reconstruction in patients with symptomatic peripheral arterial disease. METHODS: HAVs were manufactured using human vascular smooth muscle cells grown on a biodegradable scaffold. After the establishment of adequate cell growth and extracellular matrix deposition, the vessels were decellularized to remove human cellular antigens. Manufactured vessels were implanted in 20 patients with symptomatic peripheral arterial disease as above-knee, femoral-to-popliteal arterial bypass conduits. After HAV implantation, all patients were assessed for safety, HAV durability, freedom from conduit infection, and bypass patency for 2 years. RESULTS: Twenty HAVs were placed in the arterial, above-knee, femoral-to-popliteal position in patients with rest pain (n = 3) or symptomatic claudication (n = 17). All HAVs functioned as intended and had no evidence of structural failure or rejection by the recipient. No acute HAV infections were reported, but three surgical site infections were documented during the study period. Three non-HAV-related deaths were reported. One vessel developed a pseudoaneurysm after suspected iatrogenic injury during a balloon thrombectomy. No amputations of the HAV implanted limb occurred over the 2-year period, and no HAV infections were reported in approximately 34 patient-years of continuous patient follow-up. CONCLUSIONS: Human tissue engineered blood vessels can be manufactured and readily available for peripheral arterial bypass surgery. Early clinical experience with these vessels, in the arterial position, suggest that they are safe, have acceptable patency, a low incidence of infection, and do not require the harvest of autologous vein or any cells from the recipient. Histologic examination of tissue biopsies revealed vascular remodeling and repopulation by host cells. This first-in-man arterial bypass study supports the continued development of human tissue engineered blood vessels for arterial reconstruction, and potential future expansion to clinical indications including vascular trauma and repair of other size-appropriate peripheral arteries.

Bioengineered human acellular vessels recellularize and evolve into living blood vessels after human implantation.

Traditional vascular grafts constructed from synthetic polymers or cadaveric human or animal tissues support the clinical need for readily available blood vessels, but often come with associated risks. Histopathological evaluation of these materials has shown adverse host cellular reactions and/or mechanical degradation due to insufficient or inappropriate matrix remodeling. We developed an investigational bioengineered human acellular vessel (HAV), which is currently being studied as a hemodialysis conduit in patients with end-stage renal disease. In rare cases, small samples of HAV were recovered during routine surgical interventions and used to examine the temporal and spatial pattern of the host cell response to the HAV after implantation, from 16 to 200 weeks. We observed a substantial influx of alpha smooth muscle actin (αSMA)-expressing cells into the HAV that progressively matured and circumferentially aligned in the HAV wall. These cells were supported by microvasculature initially formed by CD34+/CD31+ cells in the neoadventitia and later maintained by CD34-/CD31+ endothelial cells in the media and lumen of the HAV. Nestin+ progenitor cells differentiated into either αSMA+ or CD31+ cells and may contribute to early recellularization and self-repair of the HAV. A mesenchymal stem cell-like CD90+ progenitor cell population increased in number with duration of implantation. Our results suggest that host myogenic, endothelial, and progenitor cell repopulation of HAVs transforms these previously acellular vessels into functional multilayered living tissues that maintain blood transport and exhibit self-healing after cannulation injury, effectively rendering these vessels like the patient's own blood vessel.

Generation and customization of biosynthetic excitable tissues for electrophysiological studies and cell-based therapies.

We describe a two-stage protocol to generate electrically excitable and actively conducting cell networks with stable and customizable electrophysiological phenotypes. Using this method, we have engineered monoclonally derived excitable tissues as a robust and reproducible platform to investigate how specific ion channels and mutations affect action potential (AP) shape and conduction. In the first stage of the protocol, we combine computational modeling, site-directed mutagenesis, and electrophysiological techniques to derive optimal sets of mammalian and/or prokaryotic ion channels that produce specific AP shape and conduction characteristics. In the second stage of the protocol, selected ion channels are stably expressed in unexcitable human cells by means of viral or nonviral delivery, followed by flow cytometry or antibiotic selection to purify the desired phenotype. This protocol can be used with traditional heterologous expression systems or primary excitable cells, and application of this method to primary fibroblasts may enable an alternative approach to cardiac cell therapy. Compared with existing methods, this protocol generates a well-defined, relatively homogeneous electrophysiological phenotype of excitable cells that facilitates experimental and computational studies of AP conduction and can decrease arrhythmogenic risk upon cell transplantation. Although basic cell culture and molecular biology techniques are sufficient to generate excitable tissues using the described protocol, experience with patch-clamp techniques is required to characterize and optimize derived cell populations.

Susceptibility of ePTFE vascular grafts and bioengineered human acellular vessels to infection.

BACKGROUND: Synthetic expanded polytetrafluorethylene (ePTFE) grafts are routinely used for vascular repair and reconstruction but prone to sustained bacterial infections. Investigational bioengineered human acellular vessels (HAVs) have shown clinical success and may confer lower susceptibility to infection. Here we directly compared the susceptibility of ePTFE grafts and HAV to bacterial contamination in a preclinical model of infection. MATERIALS AND METHODS: Sections (1 cm2) of ePTFE (n = 42) or HAV (n = 42) were inserted within bilateral subcutaneous pockets on the dorsum of rats and inoculated with Staphylococcus aureus (107 CFU/0.25 mL) or Escherichia coli (108 CFU/0.25 mL) before wound closure. Two weeks later, the implant sites were scored for abscess formation and explanted materials were halved for quantification of microbial recovery and histological analyses. RESULTS: The ePTFE implants had significantly higher abscess formation scores for both S. aureus and E. coli inoculations compared to that of HAV. In addition, significantly more bacteria were recovered from explanted ePTFE compared to HAV. Gram staining of explanted tissue sections revealed interstitial bacterial contamination within ePTFE, whereas no bacteria were identified in HAV tissue sections. Numerous CD45+ leukocytes, predominantly neutrophils, were found surrounding the ePTFE implants but minimal intact neutrophils were observed within the ePTFE matrix. The host cells surrounding and infiltrating the HAV explants were primarily nonleukocytes (CD45-). CONCLUSIONS: In an established animal model of infection, HAV was significantly less susceptible to bacterial colonization and abscess formation than ePTFE. The preclinical findings presented in this manuscript, combined with previously published clinical observations, suggest that bioengineered HAV may exhibit low rates of infection.

Modeling an Excitable Biosynthetic Tissue with Inherent Variability for Paired Computational-Experimental Studies.

To understand how excitable tissues give rise to arrhythmias, it is crucially necessary to understand the electrical dynamics of cells in the context of their environment. Multicellular monolayer cultures have proven useful for investigating arrhythmias and other conduction anomalies, and because of their relatively simple structure, these constructs lend themselves to paired computational studies that often help elucidate mechanisms of the observed behavior. However, tissue cultures of cardiomyocyte monolayers currently require the use of neonatal cells with ionic properties that change rapidly during development and have thus been poorly characterized and modeled to date. Recently, Kirkton and Bursac demonstrated the ability to create biosynthetic excitable tissues from genetically engineered and immortalized HEK293 cells with well-characterized electrical properties and the ability to propagate action potentials. In this study, we developed and validated a computational model of these excitable HEK293 cells (called "Ex293" cells) using existing electrophysiological data and a genetic search algorithm. In order to reproduce not only the mean but also the variability of experimental observations, we examined what sources of variation were required in the computational model. Random cell-to-cell and inter-monolayer variation in both ionic conductances and tissue conductivity was necessary to explain the experimentally observed variability in action potential shape and macroscopic conduction, and the spatial organization of cell-to-cell conductance variation was found to not impact macroscopic behavior; the resulting model accurately reproduces both normal and drug-modified conduction behavior. The development of a computational Ex293 cell and tissue model provides a novel framework to perform paired computational-experimental studies to study normal and abnormal conduction in multidimensional excitable tissue, and the methodology of modeling variation can be applied to models of any excitable cell.

Core Transcription Factors, MicroRNAs, and Small Molecules Drive Transdifferentiation of Human Fibroblasts Towards The Cardiac Cell Lineage.

Transdifferentiation has been described as a novel method for converting human fibroblasts into induced cardiomyocyte-like cells. Such an approach can produce differentiated cells to study physiology or pathophysiology, examine drug interactions or toxicities, and engineer cardiac tissues. Here we describe the transdifferentiation of human dermal fibroblasts towards the cardiac cell lineage via the induced expression of transcription factors GATA4, TBX5, MEF2C, MYOCD, NKX2-5, and delivery of microRNAs miR-1 and miR-133a. Cells undergoing transdifferentiation expressed ACTN2 and TNNT2 and partially organized their cytoskeleton in a cross-striated manner. The conversion process was associated with significant upregulation of a cohort of cardiac-specific genes, activation of pathways associated with muscle contraction and physiology, and downregulation of fibroblastic markers. We used a genetically encoded calcium indicator and readily detected active calcium transients although no spontaneous contractions were observed in transdifferentiated cells. Finally, we determined that inhibition of Janus kinase 1, inhibition of Glycogen synthase kinase 3, or addition of NRG1 significantly enhanced the efficiency of transdifferentiation. Overall, we describe a method for achieving transdifferentiation of human dermal fibroblasts into induced cardiomyocyte-like cells via transcription factor overexpression, microRNA delivery, and molecular pathway manipulation.

Engineering prokaryotic channels for control of mammalian tissue excitability.

The ability to directly enhance electrical excitability of human cells is hampered by the lack of methods to efficiently overexpress large mammalian voltage-gated sodium channels (VGSC). Here we describe the use of small prokaryotic sodium channels (BacNav) to create de novo excitable human tissues and augment impaired action potential conduction in vitro. Lentiviral co-expression of specific BacNav orthologues, an inward-rectifying potassium channel, and connexin-43 in primary human fibroblasts from the heart, skin or brain yields actively conducting cells with customizable electrophysiological phenotypes. Engineered fibroblasts ('E-Fibs') retain stable functional properties following extensive subculture or differentiation into myofibroblasts and rescue conduction slowing in an in vitro model of cardiac interstitial fibrosis. Co-expression of engineered BacNav with endogenous mammalian VGSCs enhances action potential conduction and prevents conduction failure during depolarization by elevated extracellular K+, decoupling or ischaemia. These studies establish the utility of engineered BacNav channels for induction, control and recovery of mammalian tissue excitability.

Spatial profiles of electrical mismatch determine vulnerability to conduction failure across a host-donor cell interface.

BACKGROUND: Electrophysiological mismatch between host cardiomyocytes and donor cells can directly affect the electrical safety of cardiac cell therapies; however, the ability to study host-donor interactions at the microscopic scale in situ is severely limited. We systematically explored how action potential (AP) differences between cardiomyocytes and other excitable cells modulate vulnerability to conduction failure in vitro. METHODS AND RESULTS: AP propagation was optically mapped at 75 µm resolution in micropatterned strands (n=152) in which host neonatal rat ventricular myocytes (AP duration=153.2±2.3 ms, conduction velocity=22.3±0.3 cm/s) seamlessly interfaced with genetically engineered excitable donor cells expressing inward rectifier potassium (Kir2.1) and cardiac sodium (Na(v)1.5) channels with either weak (conduction velocity=3.1±0.1 cm/s) or strong (conduction velocity=22.1±0.4 cm/s) electrical coupling. Selective prolongation of engineered donor cell AP duration (31.9-139.1 ms) by low-dose BaCl2 generated a wide range of host-donor repolarization time (RT) profiles with maximum gradients (∇RT(max)) of 5.5 to 257 ms/mm. During programmed stimulation of donor cells, the vulnerable time window for conduction block across the host-donor interface most strongly correlated with ∇RT(max). Compared with well-coupled donor cells, the interface composed of poorly coupled cells significantly shortened the RT profile width by 19.7% and increased ∇RT(max) and vulnerable time window by 22.2% and 19%, respectively. Flattening the RT profile by perfusion of 50 µmol/L BaCl2 eliminated coupling-induced differences in vulnerability to block. CONCLUSIONS: Our results quantify how the degree of electrical mismatch across a cardiomyocyte-donor cell interface affects vulnerability to conduction block, with important implications for the design of safe cardiac cell and gene therapies.

Transcription factors MYOCD, SRF, Mesp1 and SMARCD3 enhance the cardio-inducing effect of GATA4, TBX5, and MEF2C during direct cellular reprogramming.

Transient overexpression of defined combinations of master regulator genes can effectively induce cellular reprogramming: the acquisition of an alternative predicted phenotype from a differentiated cell lineage. This can be of particular importance in cardiac regenerative medicine wherein the heart lacks the capacity to heal itself, but simultaneously contains a large pool of fibroblasts. In this study we determined the cardio-inducing capacity of ten transcription factors to actuate cellular reprogramming of mouse embryonic fibroblasts into cardiomyocyte-like cells. Overexpression of transcription factors MYOCD and SRF alone or in conjunction with Mesp1 and SMARCD3 enhanced the basal but necessary cardio-inducing effect of the previously reported GATA4, TBX5, and MEF2C. In particular, combinations of five or seven transcription factors enhanced the activation of cardiac reporter vectors, and induced an upregulation of cardiac-specific genes. Global gene expression analysis also demonstrated a significantly greater cardio-inducing effect when the transcription factors MYOCD and SRF were used. Detection of cross-striated cells was highly dependent on the cell culture conditions and was enhanced by the addition of valproic acid and JAK inhibitor. Although we detected Ca(2+) transient oscillations in the reprogrammed cells, we did not detect significant changes in resting membrane potential or spontaneously contracting cells. This study further elucidates the cardio-inducing effect of the transcriptional networks involved in cardiac cellular reprogramming, contributing to the ongoing rational design of a robust protocol required for cardiac regenerative therapies.

Genetic engineering of somatic cells to study and improve cardiac function.

AIMS: To demonstrate the utility of genetically engineered excitable cells for studies of basic electrophysiology and cardiac cell therapy. METHODS AND RESULTS: 'Zig-zag' networks of neonatal rat ventricular myocytes (NRVMs) were micropatterned onto thin elastomeric films to mimic the slow action potential (AP) conduction found in fibrotic myocardium. Addition of genetically engineered excitable human embryonic kidney cells (HEK-293 cells) ('Ex-293' cells stably expressing Kir2.1, Na(v)1.5, and Cx43 channels) increased both cardiac conduction velocity by 370% and twitch force amplitude by 64%. Furthermore, we stably expressed mutant Na(v)1.5 [A1924T (fast sodium channel mutant (substitution of alanine by threonine at amino acid 1924)] channels with hyperpolarized steady-state activation and showed that, despite a 71.6% reduction in peak I(Na), these cells propagated APs at the same velocity as the wild-type Na(v)1.5-expressing Ex-293 cells. Stable expression of Ca(v)3.3 (T-type voltage-gated calcium) channels in Ex-293 cells (to generate an 'ExCa-293' line) significantly increased their AP duration and reduced repolarization gradients in cocultures of these cells and NRVMs. Additional expression of an optogenetic construct [ChIEF (light-gated Channelrhodopsin mutant)]enabled light-based control of AP firing in ExCa-293 cells. CONCLUSION: We show that, despite being non-contractile, genetically engineered excitable cells can significantly improve both electrical and mechanical function of engineered cardiac tissues in vitro. We further demonstrate the utility of engineered cells for tissue-level studies of basic electrophysiology and cardiac channelopathies. In the future, this novel platform could be utilized in the high-throughput design of new genetically encoded indicators of cell electrical function, validation, and improvement of computer models of AP conduction, and development of novel engineered somatic cell therapies for the treatment of cardiac infarction and arrhythmias.

Fibroblast growth factor homologous factor 13 regulates Na+ channels and conduction velocity in murine hearts.

RATIONALE: Fibroblast growth factor homologous factors (FHFs), a subfamily of fibroblast growth factors (FGFs) that are incapable of functioning as growth factors, are intracellular modulators of Na(+) channels and have been linked to neurodegenerative diseases. Although certain FHFs have been found in embryonic heart, they have not been reported in adult heart, and they have not been shown to regulate endogenous cardiac Na(+) channels or to participate in cardiac pathophysiology. OBJECTIVE: We tested whether FHFs regulate Na(+) channels in murine heart. METHODS AND RESULTS: We demonstrated that isoforms of FGF13 are the predominant FHFs in adult mouse ventricular myocytes. FGF13 binds directly to, and colocalizes with, the Na(V)1.5 Na(+) channel in the sarcolemma of adult mouse ventricular myocytes. Knockdown of FGF13 in adult mouse ventricular myocytes revealed a loss of function of Na(V)1.5-reduced Na(+) current density, decreased Na(+) channel availability, and slowed Na(V)1.5-reduced Na(+) current recovery from inactivation. Cell surface biotinylation experiments showed ≈45% reduction in Na(V)1.5 protein at the sarcolemma after FGF13 knockdown, whereas no changes in whole-cell Na(V)1.5 protein or in mRNA level were observed. Optical imaging in neonatal rat ventricular myocyte monolayers demonstrated slowed conduction velocity and a reduced maximum capture rate after FGF13 knockdown. CONCLUSION: These findings show that FHFs are potent regulators of Na(+) channels in adult ventricular myocytes and suggest that loss-of-function mutations in FHFs may underlie a similar set of cardiac arrhythmias and cardiomyopathies that result from Na(V)1.5 loss-of-function mutations.

Engineering biosynthetic excitable tissues from unexcitable cells for electrophysiological and cell therapy studies.

Patch-clamp recordings in single-cell expression systems have been traditionally used to study the function of ion channels. However, this experimental setting does not enable assessment of tissue-level function such as action potential (AP) conduction. Here we introduce a biosynthetic system that permits studies of both channel activity in single cells and electrical conduction in multicellular networks. We convert unexcitable somatic cells into an autonomous source of electrically excitable and conducting cells by stably expressing only three membrane channels. The specific roles that these expressed channels have on AP shape and conduction are revealed by different pharmacological and pacing protocols. Furthermore, we demonstrate that biosynthetic excitable cells and tissues can repair large conduction defects within primary 2- and 3-dimensional cardiac cell cultures. This approach enables novel studies of ion channel function in a reproducible tissue-level setting and may stimulate the development of new cell-based therapies for excitable tissue repair.

Characterizing functional stem cell-cardiomyocyte interactions.

Despite the progress in traditional pharmacological and organ transplantation therapies, heart failure still afflicts 5.3 million Americans. Since June 2000, stem cell-based approaches for the prevention and treatment of heart failure have been pursued in clinics with great excitement; however, the exact mechanisms of how transplanted cells improve heart function remain elusive. One of the main difficulties in answering these questions is the limited ability to directly access and study interactions between implanted cells and host cardiomyocytes in situ. With the growing number of candidate cell types for potential clinical use, it is becoming increasingly more important to establish standardized, well-controlled in vitro and in situ assays to compare the efficacy and safety of different stem cells in cardiac repair. This article describes recent innovative methodologies to characterize direct functional interactions between stem cells and cardiomyocytes, aimed to facilitate the rational design of future cell-based therapies for heart disease.

Electrotonic loading of anisotropic cardiac monolayers by unexcitable cells depends on connexin type and expression level.

Understanding how electrotonic loading of cardiomyocytes by unexcitable cells alters cardiac impulse conduction may be highly relevant to fibrotic heart disease. In this study, we optically mapped electrical propagation in confluent, aligned neonatal rat cardiac monolayers electrotonically loaded with cardiac fibroblasts, control human embryonic kidney (HEK-293) cells, or HEK-293 cells genetically engineered to overexpress the gap junction proteins connexin-43 or connexin-45. Gap junction expression and function were assessed by immunostaining, immunoblotting, and fluorescence recovery after photobleaching and were correlated with the optically mapped propagation of action potentials. We found that neonatal rat ventricular fibroblasts negative for the myofibroblast marker smooth muscle alpha-actin expressed connexin-45 rather than connexin-43 or connexin-40, weakly coupled to cardiomyocytes, and, without significant depolarization of cardiac resting potential, slowed cardiac conduction to 75% of control only at high (>60%) coverage densities, similar to loading effects found from HEK-293 cells expressing similar levels of connexin-45. In contrast, HEK-293 cells with connexin-43 expression similar to that of cardiomyocytes significantly decreased cardiac conduction velocity and maximum capture rate to as low as 22% and 25% of control values, respectively, while increasing cardiac action potential duration to 212% of control and cardiac resting potential from -71.6 +/- 4.9 mV in controls to -65.0 +/- 3.8 mV. For all unexcitable cell types and coverage densities, velocity anisotropy ratio remained unchanged. Despite the induced conduction slowing, none of the loading cell types increased the proportion of spontaneously active monolayers. These results signify connexin isoform and expression level as important contributors to potential electrical interactions between unexcitable cells and myocytes in cardiac tissue.

Cardiac fibroblast paracrine factors alter impulse conduction and ion channel expression of neonatal rat cardiomyocytes.

AIMS: The pathological proliferation of cardiac fibroblasts (CFs) in response to heart injury results in fibrosis, which correlates with arrhythmia generation and heart failure. Here we systematically examined the effect of fibroblast-derived paracrine factors on electrical propagation in cardiomyocytes. METHODS AND RESULTS: Neonatal rat cardiac monolayers were exposed for 24 h to media conditioned by CFs. Optical mapping, sharp microelectrode recordings, quantitative RT-PCR, and immunostaining were used to assess the changes in the propagation and shape of the action potential and underlying changes in gene and protein expression. The fibroblast paracrine factors produced a 52% reduction in cardiac conduction velocity, a 217% prolongation of action potential duration, a 64% decrease of maximum capture rate, a 21% increase in membrane resting potential, and an 80% decrease of action potential upstroke velocity. These effects were dose dependent and partially reversible with removal of the conditioned media. No fibroblast proliferation, cardiomyocyte apoptosis, or decreased connexin-43 expression, phosphorylation, and function were found in conditioned cardiac cultures. In contrast, the expression of the fast sodium, inward rectifying potassium, and transient outward potassium channels were, respectively, reduced 3.8-, 6.6-fold, and to undetectable levels. The expression of beta-myosin heavy chain increased 17.4-fold. No electrophysiological changes were observed from media conditioned by CFs in the presence of cardiomyocytes. CONCLUSION: Paracrine factors from neonatal CFs alone produced significant electrophysiological changes in neonatal rat cardiomyocytes resembling those found in several cardiac pathologies.