Adverse effects of hydroxychloroquine and azithromycin on contractility and arrhythmogenicity revealed by human engineered cardiac tissues.

The coronavirus disease 2019 (COVID-19) outbreak caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has become a global pandemic as declared by World Health Organization (WHO). In the absence of an effective treatment, different drugs with unknown effectiveness, including antimalarial hydroxychloroquine (HCQ), with or without concurrent administration with azithromycin (AZM), have been tested for treating COVID-19 patients with developed pneumonia. However, the efficacy and safety of HCQ and/or AZM have been questioned by recent clinical reports. Direct effects of these drugs on the human heart remain very poorly defined. To better understand the mechanisms of action of HCQ +/- AZM, we employed bioengineered human ventricular cardiac tissue strip (hvCTS) and anisotropic sheet (hvCAS) assays, made with human pluripotent stem cell (hPSC)-derived ventricular cardiomyocytes (hvCMs), which have been designed for measuring cardiac contractility and electrophysiology, respectively. Our hvCTS experiments showed that AZM induced a dose-dependent negative inotropic effect which could be aggravated by HCQ; electrophysiologically, as revealed by the hvCAS platform, AZM prolonged action potentials and induced spiral wave formations. Collectively, our data were consistent with reported clinical risks of HCQ and AZM on QTc prolongation/ventricular arrhythmias and development of heart failure. In conclusion, our study exposed the risks of HCQ/AZM administration while providing mechanistic insights for their toxicity. Our bioengineered human cardiac tissue constructs therefore provide a useful platform for screening cardiac safety and efficacy when developing therapeutics against COVID-19.

Cardiac small-conductance calcium-activated potassium channels in health and disease.

Small-conductance Ca2+-activated K+ (SK, KCa2) channels are encoded by KCNN genes, including KCNN1, 2, and 3. The channels play critical roles in the regulation of cardiac excitability and are gated solely by beat-to-beat changes in intracellular Ca2+. The family of SK channels consists of three members with differential sensitivity to apamin. All three isoforms are expressed in human hearts. Studies over the past two decades have provided evidence to substantiate the pivotal roles of SK channels, not only in healthy heart but also with diseases including atrial fibrillation (AF), ventricular arrhythmia, and heart failure (HF). SK channels are prominently expressed in atrial myocytes and pacemaking cells, compared to ventricular cells. However, the channels are significantly upregulated in ventricular myocytes in HF and pulmonary veins in AF models. Interests in cardiac SK channels are further fueled by recent studies suggesting the possible roles of SK channels in human AF. Therefore, SK channel may represent a novel therapeutic target for atrial arrhythmias. Furthermore, SK channel function is significantly altered by human calmodulin (CaM) mutations, linked to life-threatening arrhythmia syndromes. The current review will summarize recent progress in our understanding of cardiac SK channels and the roles of SK channels in the heart in health and disease.

An intrinsic, label-free signal for identifying stem cell-derived cardiomyocyte subtype.

Human-induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have many promising applications, including the regeneration of injured heart muscles, cardiovascular disease modeling, and drug cardiotoxicity screening. Current differentiation protocols yield a heterogeneous cell population that includes pluripotent stem cells and different cardiac subtypes (pacemaking and contractile cells). The ability to purify these cells and obtain well-defined, controlled cell compositions is important for many downstream applications; however, there is currently no established and reliable method to identify hiPSC-derived cardiomyocytes and their subtypes. Here, we demonstrate that second harmonic generation (SHG) signals generated directly from the myosin rod bundles can be a label-free, intrinsic optical marker for identifying hiPSC-derived cardiomyocytes. A direct correlation between SHG signal intensity and cardiac subtype is observed, with pacemaker-like cells typically exhibiting ~70% less signal strength than atrial- and ventricular-like cardiomyocytes. These findings suggest that pacemaker-like cells can be separated from the heterogeneous population by choosing an SHG intensity threshold criteria. This work lays the foundation for developing an SHG-based high-throughput flow sorter for purifying hiPSC-derived cardiomyocytes and their subtypes.

Human induced pluripotent stem cell line with genetically encoded fluorescent voltage indicator generated via CRISPR for action potential assessment post-cardiogenesis.

Genetically encoded fluorescent voltage indicators, such as ArcLight, have been used to report action potentials (APs) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). However, the ArcLight expression, in all cases, relied on a high number of lentiviral vector-mediated random genome integrations (8-12 copy/cell), raising concerns such as gene disruption and alteration of global and local gene expression, as well as loss or silencing of reporter genes after differentiation. Here, we report the use of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 nuclease technique to develop a hiPSC line stably expressing ArcLight from the AAVS1 safe harbor locus. The hiPSC line retained proliferative ability with a growth rate similar to its parental strain. Optical recording with conventional epifluorescence microscopy allowed the detection of APs as early as 21 days postdifferentiation, and could be repeatedly monitored for at least 5 months. Moreover, quantification and analysis of the APs of ArcLight-CMs identified two distinctive subtypes: a group with high frequency of spontaneous APs of small amplitudes that were pacemaker-like CMs and a group with low frequency of automaticity and large amplitudes that resembled the working CMs. Compared with FluoVolt voltage-sensitive dye, although dimmer, the ArcLight reporter exhibited better optical performance in terms of phototoxicity and photostability with comparable sensitivities and signal-to-noise ratios. The hiPSC line with targeted ArcLight engineering design represents a useful tool for studying cardiac development or hiPSC-derived cardiac disease models and drug testing.

Model Systems for Addressing Mechanism of Arrhythmogenesis in Cardiac Repair.

PURPOSE OF REVIEW: Cardiac cell-based therapy represents a promising approach for cardiac repair. However, one of the main challenges is cardiac arrhythmias associated with stem cell transplantation. The current review summarizes the recent progress in model systems for addressing mechanisms of arrhythmogenesis in cardiac repair. RECENT FINDINGS: Animal models have been extensively developed for mechanistic studies of cardiac arrhythmogenesis. Advances in human induced pluripotent stem cells (hiPSCs), patient-specific disease models, tissue engineering, and gene editing have greatly enhanced our ability to probe the mechanistic bases of cardiac arrhythmias. Additionally, recent development in multiscale computational studies and machine learning provides yet another powerful tool to quantitatively decipher the mechanisms of cardiac arrhythmias. Advancing efforts towards the integrations of experimental and computational studies are critical to gain insights into novel mitigation strategies for cardiac arrhythmias in cell-based therapy.

Multimodal SHG-2PF Imaging of Microdomain Ca2+-Contraction Coupling in Live Cardiac Myocytes.

RATIONALE: Cardiac myocyte contraction is caused by Ca(2+) binding to troponin C, which triggers the cross-bridge power stroke and myofilament sliding in sarcomeres. Synchronized Ca(2+) release causes whole cell contraction and is readily observable with current microscopy techniques. However, it is unknown whether localized Ca(2+) release, such as Ca(2+) sparks and waves, can cause local sarcomere contraction. Contemporary imaging methods fall short of measuring microdomain Ca(2+)-contraction coupling in live cardiac myocytes. OBJECTIVE: To develop a method for imaging sarcomere level Ca(2+)-contraction coupling in healthy and disease model cardiac myocytes. METHODS AND RESULTS: Freshly isolated cardiac myocytes were loaded with the Ca(2+)-indicator fluo-4. A confocal microscope equipped with a femtosecond-pulsed near-infrared laser was used to simultaneously excite second harmonic generation from A-bands of myofibrils and 2-photon fluorescence from fluo-4. Ca(2+) signals and sarcomere strain correlated in space and time with short delays. Furthermore, Ca(2+) sparks and waves caused contractions in subcellular microdomains, revealing a previously underappreciated role for these events in generating subcellular strain during diastole. Ca(2+) activity and sarcomere strain were also imaged in paced cardiac myocytes under mechanical load, revealing spontaneous Ca(2+) waves and correlated local contraction in pressure-overload-induced cardiomyopathy. CONCLUSIONS: Multimodal second harmonic generation 2-photon fluorescence microscopy enables the simultaneous observation of Ca(2+) release and mechanical strain at the subsarcomere level in living cardiac myocytes. The method benefits from the label-free nature of second harmonic generation, which allows A-bands to be imaged independently of T-tubule morphology and simultaneously with Ca(2+) indicators. Second harmonic generation 2-photon fluorescence imaging is widely applicable to the study of Ca(2+)-contraction coupling and mechanochemotransduction in both health and disease.

Same-Single-Cell Analysis of Pacemaker-Specific Markers in Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte Subtypes Classified by Electrophysiology.

Insights into the expression of pacemaker-specific markers in human induced pluripotent stem cell (hiPSC)-derived cardiomyocyte subtypes can facilitate the enrichment and track differentiation and maturation of hiPSC-derived pacemaker-like cardiomyocytes. To date, no study has directly assessed gene expression in each pacemaker-, atria-, and ventricular-like cardiomyocyte subtype derived from hiPSCs since currently the subtypes of these immature cardiomyocytes can only be identified by action potential profiles. Traditional acquisition of action potentials using patch-clamp recordings renders the cells unviable for subsequent analysis. We circumvented these issues by acquiring the action potential profile of a single cell optically followed by assessment of protein expression through immunostaining in that same cell. Our same-single-cell analysis for the first time revealed expression of proposed pacemaker-specific markers-hyperpolarization-activated cyclic nucleotide-modulated (HCN)4 channel and Islet (Isl)1-at the protein level in all three hiPSC-derived cardiomyocyte subtypes. HCN4 expression was found to be higher in pacemaker-like hiPSC-derived cardiomyocytes than atrial- and ventricular-like subtypes but its downregulation over time in all subtypes diminished the differences. Isl1 expression in pacemaker-like hiPSC-derived cardiomyocytes was initially not statistically different than the contractile subtypes but did become statistically higher than ventricular-like cells with time. Our observations suggest that although HCN4 and Isl1 are differentially expressed in hiPSC-derived pacemaker-like relative to ventricular-like cardiomyocytes, these markers alone are insufficient in identifying hiPSC-derived pacemaker-like cardiomyocytes. Stem Cells 2016;34:2670-2680.

Single-cell RNA sequencing reveals maturation trajectory in human pluripotent stem cell-derived cardiomyocytes in engineered tissues.

Cardiac in vitro models have become increasingly obtainable and affordable with the optimization of human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) differentiation. However, these CMs are immature compared to their in vivo counterparts. Here we study the cellular phenotype of hPSC-CMs by comparing their single-cell gene expression and functional profiles in three engineered cardiac tissue configurations: human ventricular (hv) cardiac anisotropic sheet, cardiac tissue strip, and cardiac organoid chamber (hvCOC), with spontaneously aggregated 3D cardiac spheroids (CS) as control. The CM maturity was found to increase with increasing levels of complexity of the engineered tissues from CS to hvCOC. The contractile components are the first function to mature, followed by electrophysiology and oxidative metabolism. Notably, the 2D tissue constructs show a higher cellular organization whereas metabolic maturity preferentially increases in the 3D constructs. We conclude that the tissue engineering models resembling configurations of native tissues may be reliable for drug screening or disease modeling.

The sinoatrial node extracellular matrix promotes pacemaker phenotype and protects automaticity in engineered heart tissues from cyclic strain.

The composite material-like extracellular matrix (ECM) in the sinoatrial node (SAN) supports the native pacemaking cardiomyocytes (PCMs). To test the roles of SAN ECM in the PCM phenotype and function, we engineered reconstructed-SAN heart tissues (rSANHTs) by recellularizing porcine SAN ECMs with hiPSC-derived PCMs. The hiPSC-PCMs in rSANHTs self-organized into clusters resembling the native SAN and displayed higher expression of pacemaker-specific genes and a faster automaticity compared with PCMs in reconstructed-left ventricular heart tissues (rLVHTs). To test the protective nature of SAN ECMs under strain, rSANHTs and rLVHTs were transplanted onto the murine thoracic diaphragm to undergo constant cyclic strain. All strained-rSANHTs preserved automaticity, whereas 66% of strained-rLVHTs lost their automaticity. In contrast to the strained-rLVHTs, PCMs in strained-rSANHTs maintained high expression of key pacemaker genes (HCN4, TBX3, and TBX18). These findings highlight the promotive and protective roles of the composite SAN ECM and provide valuable insights for pacemaking tissue engineering.

Engineered human pluripotent stem cell-derived cardiac cells and tissues for electrophysiological studies.

Human cardiomyocytes (CMs) do not proliferate in culture and are difficult to obtain for practical reasons. As such, our understanding of the mechanisms that underlie the physiological and pathophysiological development of the human heart is mostly extrapolated from studies of the mouse and other animal models or heterologus expression of defective gene product(s) in non-human cells. Although these studies provided numerous important insights, much of the exact behavior in human cells remains unexplored given that significant species differences exist. With the derivation of human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSCs) from patients with underlying heart disease, a source of human CMs for disease modeling, cardiotoxicity screening and drug discovery is now available. In this review, we focus our discussion on the use of hESC/ iPSC-derived cardiac cells and tissues for studying various heart rhythm disorders and the associated pro-arrhythmogenic properties in relation to advancements in electrophysiology and tissue engineering.

Cardiac tissue engineering using human stem cell-derived cardiomyocytes for disease modeling and drug discovery.

Cardiovascular disease (CVD) is the most prevalent health problem in the world, and the high mortality rate associated with irreversibly injured heart muscle motivates an urgent need for the development of novel therapies to treat damaged myocardium. Recently, human engineered cardiac tissues (hECT) have been created using cardiomyocytes derived from human embryonic stem cells and human induced pluripotent stem cells. Although a healthy adult phenotype remains elusive, such hECT display structural and functional properties that recapitulate key aspects of natural human myocardium, including dose related responses to compounds with known chronotropic, inotropic and arrhythmogenic effects. Thus, hECT offer the advantage over traditional in vitro culture models of providing a biomimetic 3D environment for the study of myocardial physiopathology, and may be used to generate preclinical models for the development and screening of therapies for CVD.

Ouabain facilitates cardiac differentiation of mouse embryonic stem cells through ERK1/2 pathway.

AIM: To investigate the effects of the cardiotonic steroid, ouabain, on cardiac differentiation of murine embyronic stem cells (mESCs). METHODS: Cardiac differentiation of murine ESCs was enhanced by standard hanging drop method in the presence of ouabain (20 µmol/L) for 7 d. The dissociated ES derived cardiomyocytes were examined by flow cytometry, RT-PCR and confocal calcium imaging. RESULTS: Compared with control, mESCs treated with ouabain (20 µmol/L) yielded a significantly higher percentage of cardiomyocytes, and significantly increased expression of a panel of cardiac markers including Nkx 2.5, α-MHC, and ß-MHC. The α1 and 2- isoforms Na(+)/K(+)-ATPase, on which ouabain acted, were also increased in mESCs during differentiation. Among the three MAPKs involved in the cardiac hypertrophy pathway, ouabain enhanced ERK1/2 activation. Blockage of the Erk1/2 pathway by U0126 (10 µmol/L) inhibited cardiac differentiation while ouabain (20 µmol/L) rescued the effect. Interestingly, the expression of calcium handling proteins, including ryanodine receptor (RyR2) and sacroplasmic recticulum Ca(2+) ATPase (SERCA2a) was also upregulated in ouabain-treated mESCs. ESC-derived cardiomyocyes (CM) treated with ouabain appeared to have more mature calcium handling. As demonstrated by confocal Ca(2+) imaging, cardiomyocytes isolated from ouabain-treated mESCs exhibited higher maximum upstroke velocity (P<0.01) and maximum decay velocity (P<0.05), as well as a higher amplitude of caffeine induced Ca(2+) transient (P<0.05), suggesting more mature sarcoplasmic reticulum (SR). CONCLUSION: Ouabain induces cardiac differentiation and maturation of mESC-derived cardiomyocytes via activation of Erk1/2 and more mature SR for calcium handling.

NODAL inhibition promotes differentiation of pacemaker-like cardiomyocytes from human induced pluripotent stem cells.

Directed cardiomyogenesis from human induced pluripotent stem cells (hiPSCs) has been greatly improved in the last decade but directed differentiation to pacemaking cardiomyocytes (CMs) remains incompletely understood. In this study, we demonstrated that inhibition of NODAL signaling by a specific NODAL inhibitor (SB431542) in the cardiac mesoderm differentiation stage downregulated PITX2c, a transcription factor that is known to inhibit the formation of the sinoatrial node in the left atrium during cardiac development. The resulting hiPSC-CMs were smaller in cell size, expressed higher pro-pacemaking transcription factors, TBX3 and TBX18, and exhibited pacemaking-like electrophysiological characteristics compared to control hiPSC-CMs differentiated from established Wnt-based protocol. The pacemaker-like subtype increased up to 2.4-fold in hiPSC-CMs differentiated with the addition of SB431542 relative to the control. Hence, Nodal inhibition in the cardiac mesoderm stage promoted pacemaker-like CM differentiation from hiPSCs. Improving the yield of human pacemaker-like CMs is a critical first step in the development of functional human cell-based biopacemakers.

Suppression of inflammation and fibrosis using soluble epoxide hydrolase inhibitors enhances cardiac stem cell-based therapy.

Stem cell replacement offers a great potential for cardiac regenerative therapy. However, one of the critical barriers to stem cell therapy is a significant loss of transplanted stem cells from ischemia and inflammation in the host environment. Here, we tested the hypothesis that inhibition of the soluble epoxide hydrolase (sEH) enzyme using sEH inhibitors (sEHIs) to decrease inflammation and fibrosis in the host myocardium may increase the survival of the transplanted human induced pluripotent stem cell derived-cardiomyocytes (hiPSC-CMs) in a murine postmyocardial infarction model. A specific sEHI (1-trifluoromethoxyphenyl-3-(1-propionylpiperidine-4-yl)urea [TPPU]) and CRISPR/Cas9 gene editing were used to test the hypothesis. TPPU results in a significant increase in the retention of transplanted cells compared with cell treatment alone. The increase in the retention of hiPSC-CMs translates into an improvement in the fractional shortening and a decrease in adverse remodeling. Mechanistically, we demonstrate a significant decrease in oxidative stress and apoptosis not only in transplanted hiPSC-CMs but also in the host environment. CRISPR/Cas9-mediated gene silencing of the sEH enzyme reduces cleaved caspase-3 in hiPSC-CMs challenged with angiotensin II, suggesting that knockdown of the sEH enzyme protects the hiPSC-CMs from undergoing apoptosis. Our findings demonstrate that suppression of inflammation and fibrosis using an sEHI represents a promising adjuvant to cardiac stem cell-based therapy. Very little is known regarding the role of this class of compounds in stem cell-based therapy. There is consequently an enormous opportunity to uncover a potentially powerful class of compounds, which may be used effectively in the clinical setting.

Facilitated maturation of Ca2+ handling properties of human embryonic stem cell-derived cardiomyocytes by calsequestrin expression.

Cardiomyocytes (CMs) are nonregenerative. Self-renewable pluripotent human embryonic stem cells (hESCs) can differentiate into CMs for cell-based therapies. We recently reported that Ca(2+) handling, crucial to excitation-contraction coupling of hESC-derived CMs (hESC-CMs), is functional but immature. Such immature properties as smaller cytosolic Ca(2+) transient amplitudes, slower kinetics, and reduced Ca(2+) content of sarcoplasmic reticulum (SR) can be attributed to the differential developmental expression profiles of specific Ca(2+) handling and regulatory proteins in hESC-CMs and their adult counterparts. In particular, calsequestrin (CSQ), the most abundant, high-capacity but low-affinity, Ca(2+)-binding protein in the SR that is anchored to the ryanodine receptor, is robustly expressed in adult CMs but completely absent in hESC-CMs. Here we hypothesized that gene transfer of CSQ in hESC-CMs suffices to induce functional improvement of SR. Transduction of hESC-CMs by the recombinant adenovirus Ad-CMV-CSQ-IRES-GFP (Ad-CSQ) significantly increased the transient amplitude, upstroke velocity, and transient decay compared with the control Ad-CMV-GFP (Ad-GFP) and Ad-CMV-CSQDelta-IRES-GFP (Ad-CSQDelta, which mediated the expression of a nonfunctional, truncated version of CSQ) groups. Ad-CSQ increased the SR Ca(2+) content but did not alter L-type Ca(2+) current. Pharmacologically, untransduced wild-type, Ad-GFP-, Ad-CSQDelta-, and Ad-CSQ-transduced hESC-CMs behaved similarly. Whereas ryanodine significantly reduced the Ca(2+) transient amplitude and slowed the upstroke, thapsigargin slowed the decay. Neither triadin nor junctin was affected. We conclude that CSQ expression in hESC-CMs facilitates Ca(2+) handling maturation. Our results shed insights into the suitability of hESC-CMs for therapies and as certain heart disease models for drug screening.

Label-free separation of human embryonic stem cells and their cardiac derivatives using Raman spectroscopy.

Self-renewable, pluripotent human embryonic stem cells (hESCs) can be differentiated into cardiomyocytes (CMs), providing an unlimited source of cells for transplantation therapies. However, unlike certain cell lineages such as hematopoietic cells, CMs lack specific surface markers for convenient identification, physical separation, and enrichment. Identification by immunostaining of cardiac-specific proteins such as troponin requires permeabilization, which renders the cells unviable and nonrecoverable. Ectopic expression of a reporter protein under the transcriptional control of a heart-specific promoter for identifying hESC-derived CMs (hESC-CMs) is useful for research but complicates potential clinical applications. The practical detection and removal of undifferentiated hESCs in a graft, which may lead to tumors, is also critical. Here, we demonstrate a nondestructive, label-free optical method based on Raman scattering to interrogate the intrinsic biochemical signatures of individual hESCs and their cardiac derivatives, allowing cells to be identified and classified. By combination of the Raman spectroscopic data with multivariate statistical analysis, our results indicate that hESCs, human fetal left ventricular CMs, and hESC-CMs can be identified by their intrinsic biochemical characteristics with an accuracy of 96%, 98%, and 66%, respectively. The present study lays the groundwork for developing a systematic and automated method for the noninvasive and label-free sorting of (i) high-quality hESCs for expansion and (ii) ex vivo CMs (derived from embryonic or adult stem cells) for cell-based heart therapies.

Correlation between frataxin expression and contractility revealed by in vitro Friedreich's ataxia cardiac tissue models engineered from human pluripotent stem cells.

BACKGROUND: Friedreich's ataxia (FRDA) is an autosomal recessive disease caused by a non-coding mutation in the first intron of the frataxin (FXN) gene that suppresses its expression. Compensatory hypertrophic cardiomyopathy, dilated cardiomyopathy, and conduction system abnormalities in FRDA lead to cardiomyocyte (CM) death and fibrosis, consequently resulting in heart failure and arrhythmias. Murine models have been developed to study disease pathology in the past two decades; however, differences between human and mouse physiology and metabolism have limited the relevance of animal studies in cardiac disease conditions. To bridge this gap, we aimed to generate species-specific, functional in vitro experimental models of FRDA using 2-dimensional (2D) and 3-dimensional (3D) engineered cardiac tissues from FXN-deficient human pluripotent stem cell-derived ventricular cardiomyocytes (hPSC-hvCMs) and to compare their contractile and electrophysiological properties with healthy tissue constructs. METHODS: Healthy control and FRDA patient-specific hPSC-hvCMs were derived by directed differentiation using a small molecule-based protocol reported previously. We engineered the hvCMs into our established human ventricular cardiac tissue strip (hvCTS) and human ventricular cardiac anisotropic sheet (hvCAS) models, and functional assays were performed on days 7-17 post-tissue fabrication to assess the electrophysiology and contractility of FRDA patient-derived and FXN-knockdown engineered tissues, in comparison with healthy controls. To further validate the disease model, forced expression of FXN was induced in FXN-deficient tissues to test if disease phenotypes could be rescued. RESULTS: Here, we report for the first time the generation of human engineered tissue models of FRDA cardiomyopathy from hPSCs: FXN-deficient hvCTS displayed attenuated developed forces (by 70-80%) compared to healthy controls. High-resolution optical mapping of hvCAS with reduced FXN expression also revealed electrophysiological defects consistent with clinical observations, including action potential duration prolongation and maximum capture frequency reduction. Interestingly, a clear positive correlation between FXN expression and contractility was observed (ρ > 0.9), and restoration of FXN protein levels by lentiviral transduction rescued contractility defects in FXN-deficient hvCTS. CONCLUSIONS: We conclude that human-based in vitro cardiac tissue models of FRDA provide a translational, disease-relevant biomimetic platform for the evaluation of novel therapeutics and to provide insight into FRDA disease progression.

Mechanistic role of I(f) revealed by induction of ventricular automaticity by somatic gene transfer of gating-engineered pacemaker (HCN) channels.

BACKGROUND: Although I(f), encoded by the hyperpolarization-activated cyclic-nucleotide-modulated (HCN) channel gene family, is known to be functionally important in pacing, its mechanistic action is largely inferential and indeed somewhat controversial. To dissect in detail the role of I(f), we investigated the functional consequences of overexpressing in adult guinea pig left ventricular cardiomyocytes (LVCMs) various HCN1 constructs that have been engineered to exhibit different gating properties. METHODS AND RESULTS: We created the recombinant adenoviruses Ad-CMV-GFP-IRES (CGI), Ad-CGI-HCN1, Ad-CGI-HCN1-delta delta delta, and Ad-CGI-HCN1-Ins, which mediate ectopic expression of GFP alone, WT, EVY235-7delta delta delta, and Ins HCN1 channels, respectively; EVY235-7delta delta delta and Ins encode channels in which the S3-S4 linkers have been shortened and lengthened to favor and inhibit opening, respectively. Ad-CGI-HCN1, Ad-CGI-HCN1-delta delta delta, and Ad-CGI-HCN1-Ins, but not control Ad-CGI, transduction of LVCMs led to robust expression of I(f) with comparable densities when fully open (approximately = -22 pA/pF at -140 mV; P>0.05) but distinctive activation profiles (V(1/2) = -70.8+/-0.6, -60.4+/-0.7, and -87.7+/-0.7 mV; P<0.01, respectively). Whereas control (nontransduced or Ad-CGI-transduced) LVCMs were electrically quiescent, automaticity (206+/-16 bpm) was observed exclusively in 61% of Ad-HCN1-delta delta delta-transduced cells that displayed depolarized maximum diastolic potential (-60.6+/-0.5 versus -70.6+/-0.6 mV of resting membrane potential of control cells; P<0.01) and gradual phase 4 depolarization (306+/-32 mV/s) that were typical of genuine nodal cells. Furthermore, spontaneously firing Ad-HCN1-delta delta delta-transduced LVCMs responded positively to adrenergic stimulation (P<0.05) but exhibited neither overdrive excitation nor suppression. In contrast, the remaining 39% of Ad-HCN1-delta delta delta-transduced cells exhibited no spontaneous action potentials; however, a single ventricular action potential associated with a depolarized resting membrane potential and a unique, incomplete "phase 4-like" depolarization that did not lead to subsequent firing could be elicited on simulation. Such an intermediate phenotype, similarly observed in 100% of Ad-CGI-HCN- and Ad-CGI-HCN1-Ins-transduced LVCMs, could be readily reversed by ZD7288, hinting at a direct role of I(f). Correlation analysis revealed the specific biophysical parameters required for I(f) to function as an active membrane potential oscillator. CONCLUSIONS: Our results not only contribute to a better understanding of cardiac pacing but also may advance current efforts that focus primarily on automaticity induction to the next level by enabling bioengineering of central and peripheral cells that make up the native sinoatrial node.

Highly efficient transfection of human induced pluripotent stem cells using magnetic nanoparticles.

PURPOSE: The delivery of transgenes into human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) represents an important tool in cardiac regeneration with potential for clinical applications. Gene transfection is more difficult, however, for hiPSCs and hiPSC-CMs than for somatic cells. Despite improvements in transfection and transduction, the efficiency, cytotoxicity, safety, and cost of these methods remain unsatisfactory. The objective of this study is to examine gene transfection in hiPSCs and hiPSC-CMs using magnetic nanoparticles (NPs). METHODS: Magnetic NPs are unique transfection reagents that form complexes with nucleic acids by ionic interaction. The particles, loaded with nucleic acids, can be guided by a magnetic field to allow their concentration onto the surface of the cell membrane. Subsequent uptake of the loaded particles by the cells allows for high efficiency transfection of the cells with nucleic acids. We developed a new method using magnetic NPs to transfect hiPSCs and hiPSC-CMs. HiPSCs and hiPSC-CMs were cultured and analyzed using confocal microscopy, flow cytometry, and patch clamp recordings to quantify the transfection efficiency and cellular function. RESULTS: We compared the transfection efficiency of hiPSCs with that of human embryonic kidney (HEK 293) cells. We observed that the average efficiency in hiPSCs was 43%±2% compared to 62%±4% in HEK 293 cells. Further analysis of the transfected hiPSCs showed that the differentiation of hiPSCs to hiPSC-CMs was not altered by NPs. Finally, robust transfection of hiPSC-CMs with an efficiency of 18%±2% was obtained. CONCLUSION: The difficult-to-transfect hiPSCs and hiPSC-CMs were efficiently transfected using magnetic NPs. Our study offers a novel approach for transfection of hiPSCs and hiPSC-CMs without the need for viral vector generation.

Beta1 integrin expression pattern in transitional urothelium does not allow for efficient stem cell enrichment as in other epithelia.

With a lack of distinct stem cell markers, isolation of tissue-specific stem cells for tissue engineering and gene therapy is a great challenge. Beta (beta)(1) integrin expression has been used as a way of enriching for putative epithelial stem cells through rapid adhesion to collagen IV or flow cytometry. This is a first report of enrichment of putative urothelial stem cells using rapid adhesion and flow cytometric methods. We assessed our success by determining the clonogenic and proliferative potential of the isolated cells. We demonstrated that enrichment based on beta(1) integrin expression with flow cytometry yields highly clonogenic and proliferative urothelial cells, whereas the rapid adhesion method is not as efficient, possibly because of the unique nature of urothelium, a transitional epithelium, compared to results reported in stratified and columnar epithelia.