Bayesian approach enabled objective comparison of multiple human iPSC-derived Cardiomyocytes' Proarrhythmia sensitivities.

The one-size-fits-all approach has been the mainstream in medicine, and the well-defined standards support the development of safe and effective therapies for many years. Advancing technologies, however, enabled precision medicine to treat a targeted patient population (e.g., HER2+ cancer). In safety pharmacology, computational population modeling has been successfully applied in virtual clinical trials to predict drug-induced proarrhythmia risks against a wide range of pseudo cohorts. In the meantime, population modeling in safety pharmacology experiments has been challenging. Here, we used five commercially available human iPSC-derived cardiomyocytes growing in 384-well plates and analyzed the effects of ten potential proarrhythmic compounds with four concentrations on their calcium transients (CaTs). All the cell lines exhibited an expected elongation or shortening of calcium transient duration with various degrees. Depending on compounds inhibiting several ion channels, such as hERG, peak and late sodium and L-type calcium or IKs channels, some of the cell lines exhibited irregular, discontinuous beating that was not predicted by computational simulations. To analyze the shapes of CaTs and irregularities of beat patterns comprehensively, we defined six parameters to characterize compound-induced CaT waveform changes, successfully visualizing the similarities and differences in compound-induced proarrhythmic sensitivities of different cell lines. We applied Bayesian statistics to predict sample populations based on experimental data to overcome the limited number of experimental replicates in high-throughput assays. This process facilitated the principal component analysis to classify compound-induced sensitivities of cell lines objectively. Finally, the association of sensitivities in compound-induced changes between phenotypic parameters and ion channel inhibitions measured using patch clamp recording was analyzed. Successful ranking of compound-induced sensitivity of cell lines was in lined with visual inspection of raw data.

Creating cell-specific computational models of stem cell-derived cardiomyocytes using optical experiments.

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have gained traction as a powerful model in cardiac disease and therapeutics research, since iPSCs are self-renewing and can be derived from healthy and diseased patients without invasive surgery. However, current iPSC-CM differentiation methods produce cardiomyocytes with immature, fetal-like electrophysiological phenotypes, and the variety of maturation protocols in the literature results in phenotypic differences between labs. Heterogeneity of iPSC donor genetic backgrounds contributes to additional phenotypic variability. Several mathematical models of iPSC-CM electrophysiology have been developed to help understand the ionic underpinnings of, and to simulate, various cell responses, but these models individually do not capture the phenotypic variability observed in iPSC-CMs. Here, we tackle these limitations by developing a computational pipeline to calibrate cell preparation-specific iPSC-CM electrophysiological parameters. We used the genetic algorithm (GA), a heuristic parameter calibration method, to tune ion channel parameters in a mathematical model of iPSC-CM physiology. To systematically optimize an experimental protocol that generates sufficient data for parameter calibration, we created simulated datasets by applying various protocols to a population of in silico cells with known conductance variations, and we fitted to those datasets. We found that calibrating models to voltage and calcium transient data under 3 varied experimental conditions, including electrical pacing combined with ion channel blockade and changing buffer ion concentrations, improved model parameter estimates and model predictions of unseen channel block responses. This observation held regardless of whether the fitted data were normalized, suggesting that normalized fluorescence recordings, which are more accessible and higher throughput than patch clamp recordings, could sufficiently inform conductance parameters. Therefore, this computational pipeline can be applied to different iPSC-CM preparations to determine cell line-specific ion channel properties and understand the mechanisms behind variability in perturbation responses.

Aging Model for Analyzing Drug-Induced Proarrhythmia Risks Using Cardiomyocytes Differentiated from Progeria-Patient-Derived Induced Pluripotent Stem Cells.

Among various cardiac safety concerns, proarrhythmia risks, including QT prolongation leading to Torsade de Pointes, is one of major cause for drugs being withdrawn (~45% 1975-2007). Preclinical study requires the evaluation of proarrhythmia using in silico, in vitro, and/or animal models. Considering that the primary consumers of prescription drugs are elderly patients, applications of "aging-in-a-dish" models would be appropriate for screening proarrhythmia risks. However, acquiring such models, including cardiomyocytes (CMs) derived from induced pluripotent stem cells (iPSCs), presents extensive challenges. We proposed the hypothesis that CMs differentiated from iPSCs derived from Hutchinson-Gilford progeria syndrome (HGPS, progeria) patients, an ultra-rare premature aging syndrome, can mimic the phenotypes of aging CMs. Our objective, therefore, was to examine this hypothesis by analyzing the response of 11 reference compounds utilized by the Food and Drug Administration (FDA)'s Comprehensive in vitro Proarrhythmia Assay (CiPA) using progeria and control CMs. As a sensitive surrogate marker of modulating cardiac excitation-contraction coupling, we evaluated drug-induced changes in calcium transient (CaT). We observed that the 80% CaT peak duration in the progeria CMs (0.98 ± 0.04 s) was significantly longer than that of control CMs (0.70 ± 0.05 s). Furthermore, when the progeria CMs were subjected to four doses of 11 compounds from low-, intermediate-, and high-risk categories, they demonstrated greater arrhythmia susceptibility than control cells, as shown through six-parameter CaT profile analyses. We also employed the regression analysis established by CiPA to classify the 11 reference compounds and compared proarrhythmia susceptibilities between the progeria and control CMs. This analysis revealed a greater proarrhythmia susceptibility in the progeria CMs compared to the control CMs. Interestingly, in both CMs, the compounds categorized as low risk did not exceed the safety risk threshold of 0.8. In conclusion, our study demonstrates increased proarrhythmia sensitivity in progeria CMs when tested with reference compounds. Future studies are needed to analyze underlying mechanisms and further validate our findings using a larger array of reference compounds.

A model for positive feedback control of the transformation of fibroblasts to myofibroblasts.

The phenotypic conversion of normal fibroblasts to myofibroblasts is central to normal wound healing and to pathological fibrosis that can occur in the heart and many other tissues. The transformation occurs in two stages. The first stage is driven mainly by mechanical changes such as increased stiffness of the heart due to hypertension and cellular contractility. The second stage requires both increasing stiffness and biochemical factors such as the growth factor, TGFß. As more and more cells convert from weakly contractile fibroblasts to strongly contractile myofibroblasts, the stiffness of the ventricular muscle increases. We propose a simple model for the establishment of non-equilibrium steady states with different compositions of fibroblasts and myofibroblasts. Under some conditions a positive feedback loop resulting from the increasing stiffness caused by increasing numbers of myofibroblasts can produce a bifurcation between steady states with low and high myofibroblast content. We illustrate the large mechanical differences between normal fibroblasts and myofibroblasts with measurements in engineered tissue constructs.

Genetic and Tissue Engineering Approaches to Modeling the Mechanics of Human Heart Failure for Drug Discovery.

Heart failure is the leading cause of death in the western world and as such, there is a great need for new therapies. Heart failure has a variable presentation in patients and a complex etiology; however, it is fundamentally a condition that affects the mechanics of cardiac contraction, preventing the heart from generating sufficient cardiac output under normal operating pressures. One of the major issues hindering the development of new therapies has been difficulties in developing appropriate in vitro model systems of human heart failure that recapitulate the essential changes in cardiac mechanics seen in the disease. Recent advances in stem cell technologies, genetic engineering, and tissue engineering have the potential to revolutionize our ability to model and study heart failure in vitro. Here, we review how these technologies are being applied to develop personalized models of heart failure and discover novel therapeutics.

Calcium Transient Assays for Compound Screening with Human iPSC-derived Cardiomyocytes: Evaluating New Tools.

Calcium (Ca2+) plays a central role in regulating many biological processes in the cell from muscle contraction to neurotransmitter release. The need for reliable fluorescent calcium indicator dyes is of vast importance for studying many aspects of cell biology as well as screening compounds using phenotypic high throughput assays. We have assessed two of the latest generation of calcium indicator dyes, FLIPR Calcium 6 and Cal-520 AM for studying calcium transients (CaTs) in induced pluripotent stem cell (iPSC) -derived human cardiomyocytes. FLIPR Calcium 6 and Cal-520 dyes both displayed robust CaTs with a high signal-to-noise ratio (SNR) and were non-toxic to the cells. The analysis showed that CaT amplitudes were stable between measurements, but CaT duration was more variable and tended to increase between reads. Two methods were compared for drug-screening hit-selection; difference in average (unstandardized) and standardized difference. The unstandardized difference was better for assessing CaT amplitude, whereas standardized difference was equal to or better for assessing CaT duration. In summary, FLIPR Calcium 6 and Cal-520 are suitable dyes for drug-screening using iPSC-derived human cardiomyocytes.

High-Throughput Phenotyping of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes and Neurons Using Electric Field Stimulation and High-Speed Fluorescence Imaging.

Electrophysiology of excitable cells, including muscle cells and neurons, has been measured by making direct contact with a single cell using a micropipette electrode. To increase the assay throughput, optical devices such as microscopes and microplate readers have been used to analyze electrophysiology of multiple cells. We have established a high-throughput (HTP) analysis of action potentials (APs) in highly enriched motor neurons and cardiomyocytes (CMs) that are differentiated from human induced pluripotent stem cells (iPSCs). A multichannel electric field stimulation (EFS) device enabled the ability to electrically stimulate cells and measure dynamic changes in APs of excitable cells ultra-rapidly (>100 data points per second) by imaging entire 96-well plates. We found that the activities of both neurons and CMs and their response to EFS and chemicals are readily discerned by our fluorescence imaging-based HTP phenotyping assay. The latest generation of calcium (Ca2+) indicator dyes, FLIPR Calcium 6 and Cal-520, with the HTP device enables physiological analysis of human iPSC-derived samples highlighting its potential application for understanding disease mechanisms and discovering new therapeutic treatments.

Computational modeling for cardiac safety pharmacology analysis: Contribution of fibroblasts.

INTRODUCTION: Drug-induced proarrhythmic potential is an important regulatory criterion in safety pharmacology. The application of in silico approaches to predict proarrhythmic potential of new compounds is under consideration as part of future guidelines. Current approaches simulate the electrophysiology of a single human adult ventricular cardiomyocyte. However, drug-induced proarrhythmic potential can be different when cardiomyocytes are surrounded by non-muscle cells. Incorporating fibroblasts in models of myocardium is important particularly for predicting a drugs cardiac liability in the aging population - a growing population who take more medications and exhibit increased cardiac fibrosis. In this study, we used computational models to investigate the effects of fibroblast coupling on the electrophysiology and response to drugs of cardiomyocytes. METHODS: A computational model of cardiomyocyte electrophysiology and ion handling (O'Hara, Virag, Varro, & Rudy, 2011) is coupled to a passive model of fibroblast electrophysiology to test the effects of three compounds that block cardiomyocyte ion channels. Results are compared to model results without fibroblast coupling to see how fibroblasts affect cardiomyocyte action potential duration at 90% repolarization (APD90) and propensity for early after depolarization (EAD). RESULTS: Simulation results show changes in cardiomyocyte APD90 with increasing concentration of three drugs that affect cardiac function (dofetilide, vardenafil and nebivolol) when no fibroblasts are coupled to the cardiomyocyte. Coupling fibroblasts to cardiomyocytes markedly shortens APD90. Moreover, increasing the number of fibroblasts can augment the shortening effect. DISCUSSION: Coupling cardiomyocytes and fibroblasts are predicted to decrease proarrhythmic susceptibility under dofetilide, vardenafil and nebivolol block. However, this result is sensitive to parameters which define the electrophysiological function of the fibroblast. Fibroblast membrane capacitance and conductance (CFB and GFB) have less of an effect on APD90 than the fibroblast resting membrane potential (EFB). This study suggests that in both theoretical models and experimental tissue constructs that represent cardiac tissue, both cardiomyocytes and non-muscle cells should be considered when testing cardiac pharmacological agents.

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System.

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.

Improving Cardiac Action Potential Measurements: 2D and 3D Cell Culture.

Progress in the development of assays for measuring cardiac action potential is crucial for the discovery of drugs for treating cardiac disease and assessing cardiotoxicity. Recently, high-throughput methods for assessing action potential using induced pluripotent stem cell (iPSC) derived cardiomyocytes in both two-dimensional monolayer cultures and three-dimensional tissues have been developed. We describe an improved method for assessing cardiac action potential using an ultra-fast cost-effective plate reader with commercially available dyes. Our methods improve dramatically the detection of the fluorescence signal from these dyes and make way for the development of more high-throughput methods for cardiac drug discovery and cardiotoxicity.

The Stagnant Adaptation of Defined and Xeno-Free Culture of iPSCs in Academia.

Pluripotent Stem Cells were originally derived and cultured using a feeder layer of cells. Movements have been undertaken to transition from this method to one more defined, high-throughput, and without xenogenic factors. Tremendous research has been done in this area and many products have been developed, however, based on our analysis of recent publications in stem cell related journals many in academia are still using older methods like a feeder layer. In this short communication, we discuss the feasibility of transitioning to defined, xeno-free methods, how a standardized method could improve the field and industry, and that a study bringing together multiple institutions comparing culture methods could be done to evaluate the efficacy of these new methods.

Protease-activated receptor 1 inhibition by SCH79797 attenuates left ventricular remodeling and profibrotic activities of cardiac fibroblasts.

PURPOSE: Fibroblast activity promotes adverse left ventricular (LV) remodeling that underlies the development of ischemic cardiomyopathy. Transforming growth factor-ß (TGF-ß) is a potent stimulus for fibrosis, and the extracellular signal-regulated kinases(ERK) 1/2 pathway also contributes to the fibrotic response. The thrombin receptor, protease-activated receptor 1 (PAR1), has been shown to play an important role in the excessive fibrosis in different tissues. The aim of this study was to investigate the influence of a PAR1 inhibitor, SCH79797, on cardiac fibrosis, tissue stiffness and postinfarction remodeling, and effects of PAR1 inhibition on thrombin-induced TGF-ß and (ERK) 1/2 activities in cardiac fibroblasts. METHODS: We used a rat model of myocardial ischemia-reperfusion injury, isolated cardiac fibroblasts, and 3-dimensional (3D) cardiac tissue models fabricated to ascertain the contribution of PAR1 activation on cardiac fibrosis and LV remodeling. RESULTS: The PAR1 inhibitor attenuated LV dilation and improved LV systolic function of the reperfused myocardium at 28 days. This improvement was associated with a nonsignificant decrease in scar size (%LV) from 23 ± % in the control group (n = 10) to 16% ± 5.5% in the treated group (n = 9; P = .052). In the short term, the PAR1 inhibitor did not rescue infarct size or LV systolic function after 3 days. The PAR1 inhibition abolished thrombin-mediated ERK1/2 phosphorylation, TGF-ß and type I procollagen production, matrix metalloproteinase-2/9 activation, myofibroblasts transformation in vitro, and abrogated the remodeling of 3D tissues induced by chronic thrombin treatment. CONCLUSION: These studies suggest PAR1 inhibition initiated after ischemic injury attenuates adverse LV remodeling through late-stage antifibrotic events.

A method for quantifying mechanical properties of tissue following viral infection.

Viral infection and replication involves the reorganization of the actin network within the host cell. Actin plays a central role in the mechanical properties of cells. We have demonstrated a method to quantify changes in mechanical properties of fabricated model three-dimensional (3D) connective tissue following viral infection. Using this method, we have characterized the impact of infection by the human herpesvirus, cytomegalovirus (HCMV). HCMV is a member of the herpesvirus family and infects a variety of cell types including fibroblasts. In the body, fibroblasts are necessary for maintaining connective tissue and function by creating mechanical force. Using this 3D connective tissue model, we observed that infection disrupted the cell's ability to generate force and reduced the cumulative contractile force of the tissue. The addition of HCMV viral particles in the absence of both viral gene expression and DNA replication was sufficient to disrupt tissue function. We observed that alterations of the mechanical properties are, in part, due to a disruption of the underlying complex actin microfilament network established by the embedded fibroblasts. Finally, we were able to prevent HCMV-mediated disruption of tissue function by the addition of human immune globulin against HCMV. This study demonstrates a method to quantify the impact of viral infection on mechanical properties which are not evident using conventional cell culture systems.

Effects of substrate mechanics on contractility of cardiomyocytes generated from human pluripotent stem cells.

Human pluripotent stem cell (hPSC-) derived cardiomyocytes have potential applications in drug discovery, toxicity testing, developmental studies, and regenerative medicine. Before these cells can be reliably utilized, characterization of their functionality is required to establish their similarity to native cardiomyocytes. We tracked fluorescent beads embedded in 4.4-99.7 kPa polyacrylamide hydrogels beneath contracting neonatal rat cardiomyocytes and cardiomyocytes generated from hPSCs via growth-factor-induced directed differentiation to measure contractile output in response to changes in substrate mechanics. Contraction stress was determined using traction force microscopy, and morphology was characterized by immunocytochemistry for α-actinin and subsequent image analysis. We found that contraction stress of all types of cardiomyocytes increased with substrate stiffness. This effect was not linked to beating rate or morphology. We demonstrated that hPSC-derived cardiomyocyte contractility responded appropriately to isoprenaline and remained stable in culture over a period of 2 months. This study demonstrates that hPSC-derived cardiomyocytes have appropriate functional responses to substrate stiffness and to a pharmaceutical agent, which motivates their use in further applications such as drug evaluation and cardiac therapies.

Diffusion of MMPs on the surface of collagen fibrils: the mobile cell surface-collagen substratum interface.

Remodeling of the extracellular matrix catalyzed by MMPs is central to morphogenetic phenomena during development and wound healing as well as in numerous pathologic conditions such as fibrosis and cancer. We have previously demonstrated that secreted MMP-2 is tethered to the cell surface and activated by MT1-MMP/TIMP-2-dependent mechanism. The resulting cell-surface collagenolytic complex (MT1-MMP)(2)/TIMP-2/MMP-2 can initiate (MT1-MMP) and complete (MMP-2) degradation of an underlying collagen fibril. The following question remained: What is the mechanism of substrate recognition involving the two structures of relatively restricted mobility, the cell surface enzymatic complex and a collagen fibril embedded in the ECM? Here we demonstrate that all the components of the complex are capable of processive movement on a surface of the collagen fibril. The mechanism of MT1-MMP movement is a biased diffusion with the bias component dependent on the proteolysis of its substrate, not adenosine triphosphate (ATP) hydrolysis. It is similar to that of the MMP-1 Brownian ratchet we described earlier. In addition, both MMP-2 and MMP-9 as well as their respective complexes with TIMP-1 and -2 are capable of Brownian diffusion on the surface of native collagen fibrils without noticeable dissociation while the dimerization of MMP-9 renders the enzyme immobile. Most instructive is the finding that the inactivation of the enzymatic activity of MT1-MMP has a detectable negative effect on the cell force developed in miniaturized 3D tissue constructs. We propose that the collagenolytic complex (MT1-MMP)(2)/TIMP-2/MMP-2 represents a Mobile Cell Surface-Collagen Substratum Interface. The biological implications of MT1-MMP acting as a molecular ratchet tethered to the cell surface in complex with MMP-2 suggest a new mechanism for the role of spatially regulated peri-cellular proteolysis in cell-matrix interactions.

Hydrogel tissue construct-based high-content compound screening.

Current pharmaceutical compound screening systems rely on cell-based assays to identify therapeutic candidates and potential toxicities. However, cells grown on 2D substrata or in suspension do not exhibit the mechanical or physiological properties of cells in vivo. To address this limitation, the authors developed an in vitro, high-throughput, 3D hydrogel tissue construct (HTC)-based assay system to quantify cell and tissue mechanical properties and multiple parameters of physiology. HTC mechanics was quantified using an automated device, and physiological status was assessed using spectroscopy-based indicators that were read on microplate readers. To demonstrate the application of this system, the authors screened 4 test compounds--rotenone (ROT), cytochalasin D (CD), 2,4-dinitrophenol (DNP), and Rho kinase inhibitor (H-1152)--for their ability to modulate HTC contractility without affecting actin integrity, mitochondrial membrane potential (MMP), or viability. All 4 compounds dose-dependently reduced HTC contractility. However, ROT was toxic, DNP dissipated MMP, and CD reduced both intracellular F-actin and viability. H-1152 was found to be the best candidate compound since it reduced HTC contractility with minimal side effects. The authors propose that their HTC-based assay system can be used to screen for compounds that modulate HTC contractility and assess the underlying physiological mechanism(s) of compound activity and toxicity.

Monitoring mitochondrial electron fluxes using NAD(P)H-flavoprotein fluorometry reveals complex action of isoflurane on cardiomyocytes.

Mitochondrial bioenergetic studies mostly rely on isolated mitochondria thus excluding the regulatory role of other cellular compartments important for the overall mitochondrial function. In intact cardiomyocytes, we followed the dynamics of electron fluxes along specific sites of the electron transport chain (ETC) by simultaneous detection of NAD(P)H and flavoprotein (FP) fluorescence intensities using a laser-scanning confocal microscope. This method was used to delineate the effects of isoflurane, a volatile anesthetic and cardioprotective agent, on the ETC. Comparison to the effects of well-characterized ETC inhibitors and uncoupling agent revealed two distinct effects of isoflurane: uncoupling-induced mitochondrial depolarization and inhibition of ETC at the level of complex I. In correlation, oxygen consumption measurements in cardiomyocytes confirmed a dose-dependent, dual effect of isoflurane, and in isolated mitochondria an obstruction of the ETC primarily at the level of complex I. These effects are likely responsible for the reported mild stimulation of mitochondrial reactive oxygen species (ROS) production required for the cardioprotective effects of isoflurane. In conclusion, isoflurane exhibits complex effects on the ETC in intact cardiomyocytes, altering its electron fluxes, and thereby enhancing ROS production. The NAD(P)H-FP fluorometry is a useful method for exploring the effect of drugs on mitochondria and identifying their specific sites of action within the ETC of intact cardiomyocytes.

Analysis of the diffusion of Ras2 in Saccharomyces cerevisiae using fluorescence recovery after photobleaching.

Binding, lateral diffusion and exchange are fundamental dynamic processes involved in protein association with cellular membranes. In this study, we developed numerical simulations of lateral diffusion and exchange of fluorophores in membranes with arbitrary bleach geometry and exchange of the membrane-localized fluorophore with the cytosol during fluorescence recovery after photobleaching (FRAP) experiments. The model simulations were used to design FRAP experiments with varying bleach region sizes on plasma membrane-localized wild-type GFP-Ras2 with a dual lipid anchor and mutant GFP-Ras2C318S with a single lipid anchor in live yeast cells to investigate diffusional mobility and the presence of any exchange processes operating in the time scale of our experiments. Model parameters estimated using data from FRAP experiments with a 1 microm x 1 microm bleach region-of-interest (ROI) and a 0.5 microm x 0.5 microm bleach ROI showed that GFP-Ras2, single or dual lipid modified, diffuses as single species with no evidence of exchange with a cytoplasmic pool. This is the first report of Ras2 mobility in the yeast plasma membrane. The methods developed in this study are generally applicable for studying diffusion and exchange of membrane-associated fluorophores using FRAP on commercial confocal laser scanning microscopes.

Mitochondrial depolarization underlies delay in permeability transition by preconditioning with isoflurane: roles of ROS and Ca2+.

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial Ca(2+) overload, and mitochondrial permeability transition pore (mPTP) opening, as the crucial mechanism of cardiomyocyte injury, remains intriguing. Here, we investigated whether an induction of a partial decrease in mitochondrial membrane potential (DeltaPsi(m)) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS, Ca(2+), and mPTP opening. The magnitude of APC-induced decrease in DeltaPsi(m) was mimicked with the protonophore 2,4-dinitrophenol (DNP), and the addition of pyruvate was used to reverse APC- and DNP-induced decrease in DeltaPsi(m). In cardiomyocytes, DeltaPsi(m), ROS, mPTP opening, and cytosolic and mitochondrial Ca(2+) were measured using confocal microscope, and cardiomyocyte survival was assessed by Trypan blue exclusion. In isolated cardiac mitochondria, antimycin A-induced ROS production and Ca(2+) uptake were determined spectrofluorometrically. In cells exposed to oxidative stress, APC and DNP increased cell survival, delayed mPTP opening, and attenuated ROS production, which was reversed by mitochondrial repolarization with pyruvate. In isolated mitochondria, depolarization by APC and DNP attenuated ROS production, but not Ca(2+) uptake. However, in stressed cardiomyocytes, a similar decrease in DeltaPsi(m) attenuated both cytosolic and mitochondrial Ca(2+) accumulation. In conclusion, a partial decrease in DeltaPsi(m) underlies cardioprotective effects of APC by attenuating excess ROS production, resulting in a delay in mPTP opening and an increase in cell survival. Such decrease in DeltaPsi(m) primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial Ca(2+) uptake.

Activator of G protein signaling 3 promotes epithelial cell proliferation in PKD.

The activation of heterotrimeric G protein signaling is a key feature in the pathophysiology of polycystic kidney diseases (PKD). In this study, we report abnormal overexpression of activator of G protein signaling 3 (AGS3), a receptor-independent regulator of heterotrimeric G proteins, in rodents and humans with both autosomal recessive and autosomal dominant PKD. Increased AGS3 expression correlated with kidney size, which is an index of severity of cystic kidney disease. AGS3 expression localized exclusively to distal tubular segments in both normal and cystic kidneys. Short hairpin RNA-induced knockdown of endogenous AGS3 protein significantly reduced proliferation of cystic renal epithelial cells by 26 +/- 2% (P < 0.001) compared with vehicle-treated and control short hairpin RNA-expressing epithelial cells. In summary, this study suggests a relationship between aberrantly increased AGS3 expression in renal tubular epithelia affected by PKD and epithelial cell proliferation. AGS3 may play a receptor-independent role to regulate Galpha subunit function and control epithelial cell function in PKD.