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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 to predict 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 in silico datasets by simulating various protocols applied to a population of models with known conductance variations, and then fitted parameters to those datasets. We found that calibrating 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 also held when 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. Author summary: Many drug treatments or environmental factors can trigger cardiac arrhythmias, which are dangerous and often unpredictable. Human cardiomyocytes derived from donor stem cells have proven to be a promising model for studying these events, but variability in donor genetic background and cell maturation methods, as well as overall immaturity of stem cell-derived cardiomyocytes relative to the adult heart, have hindered reproducibility and reliability of these studies. Mathematical models of these cells can aid in understanding the underlying electrophysiological contributors to this variability, but determining these models' parameters for multiple cell preparations is challenging. In this study, we tackle these limitations by developing a computational method to simultaneously estimate multiple model parameters using data from imaging-based experiments, which can be easily scaled to rapidly characterize multiple cell lines. This method can generate many personalized models of individual cell preparations, improving drug response predictions and revealing specific differences in electrophysiological properties that contribute to variability in cardiac maturity and arrhythmia susceptibility. [ABSTRACT FROM AUTHOR]

During reperfusion, the interplay between excess reactive oxygen species (ROS) production, mitochondrial [Ca.sup.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 ([DELTA][[PSI].sub.m]) is an underlying mechanism of protection by anesthetic-induced preconditioning (APC) with isoflurane, specifically addressing the interplay between ROS. [Ca.sup.2+], and mPTP opening. The magnitude of APC-induced decrease in [DELTA][[PSI].sub.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 [DELTA][[PSI].sub.m]. In cardiomyocytes, [DELTA][[PSI].sub.m], ROS, mPTP opening, and cytosolic and mitochondrial [Ca.sup.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.sup.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.sup.2+] uptake. However, in stressed cardiomyocytes, a similar decrease in [DELTA][[PSI].sub.m] attenuated both cytosolic and mitochondrial [Ca.sup.2+] accumulation. In conclusion, a partial decrease in [DELTA][[PSI].sub.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 [DELTA][[PSI].sub.m] primarily attenuates mitochondrial ROS production, with consequential decrease in mitochondrial [Ca.sup.2+] uptake. cardioprotection; oxidative stress; mitochondria; reactive oxygen species doi: 10.1152/ajpcell.00006.2010.

Mechanical stress on the heart can lead to crucially different outcomes. Exercise is beneficial because it causes heart muscle cells to enlarge (hypertrophy). Chronic hypertension also causes hypertrophy, but in addition it causes an excessive increase in fibroblasts and extracellular matrix (fibrosis), death of cardiomyocytes and ultimately heart failure. Recent research shows that stimulation of physiological (beneficial) hypertrophy involves several signaling pathways, including those mediated by protein kinase B (also known as Akt) and the extracellular-signal-regulated kinases 1 and 2 (ERK1/2). Hypertension, [beta]-adrenergic stimulation and agonists such as angiotensin II (Ang II) activate not only ERK1/2 but also p38 and the Jun N-terminal kinase (JNK), leading to pathological heart remodeling. Despite this progress, the mechanisms that activate fibroblasts to cause fibrosis and those that differentiate between exercise and hypertension to produce physiological and pathological responses, respectively, remain to be established.

Thus far, determining the relative contribution of [Ca.sup.2+]/calmodulin-dependent myosin light chain kinase (MLCK) and [Ca.sup.2+]-independent Rho-kinase pathways to myosin II activation and contraction has been difficult. In this study, we characterize the role of Rho-kinase in a rat embryo fibroblast cell line (REF-52), which contains no detectable MLCK. No endogenous MLCK could be detected in REF-52 cells by either Western or Northern blot analysis. In the presence or absence of [Ca.sup.2+], thrombin or lysophosphatidic acid (LPA) increased RhoA activity and Rho-kinase activity, correlating with isometric tension development and myosin II regulatory light chain (RLC) phosphorylation. Resting tension is associated with a basal phosphorylation of 0.31 [+ or -] 0.02 mol P[O.sub.4]/mol RLC, whereas upon LPA or thrombin treatment myosin II RLC phosphorylation increases to 1.08 [+ or -] 0.05 and 0.82 [+ or -] 0.05 tool P[O.sub.4]/mol RLC, respectively, within 2.5 min. [Ca.sup.2+] chelation has minimal effect on the kinetics and magnitude of isometric tension development and RLC phosphorylation. Treatment of REF-52 cells with the Rho-kinase-specific inhibitor Y-27632 abolished thrombin- and LPA-stimulated contraction and RLC phosphorylation. These results suggest that Rho-kinase is sufficient to activate myosin II motor activity and contraction in REF-52 cells. myosin light chain kinase; RhoA; myosin II regulatory light chain phosphorylation

Bio-artificial tissues are being developed as replacements for damaged biologic tissues. Their mechanical properties are critical for load bearing applications. Current testing protocols for bio-artificial tissues vary widely and often do not consider viscoelasticity. Uniaxial stretch tests were performed on fibroblast populated collagen matrices (FPCMs) to determine the influence of specific test protocols on the mechanical behavior. The peak force, hysteresis and shape of the force-stretch curve are affected by the stretch rate, rest period, stretch amplitude and the number and magnitude of preconditioning cycles. [DOI: 10.1115/1.1614818]