Cardiomyocyte Na+ and Ca2+ mishandling drives vicious cycle involving CaMKII, ROS, and ryanodine receptors.

Cardiomyocyte Na+ and Ca2+ mishandling, upregulated Ca2+/calmodulin-dependent kinase II (CaMKII), and increased reactive oxygen species (ROS) are characteristics of various heart diseases, including heart failure (HF), long QT (LQT) syndrome, and catecholaminergic polymorphic ventricular tachycardia (CPVT). These changes may form a vicious cycle of positive feedback to promote cardiac dysfunction and arrhythmias. In HF rabbit cardiomyocytes investigated in this study, the inhibition of CaMKII, late Na+ current (INaL), and leaky ryanodine receptors (RyRs) all attenuated the prolongation and increased short-term variability (STV) of action potential duration (APD), but in age-matched controls these inhibitors had no or minimal effects. In control cardiomyocytes, we enhanced RyR leak (by low [caffeine] plus isoproterenol mimicking CPVT) which markedly increased STV and delayed afterdepolarizations (DADs). These proarrhythmic changes were significantly attenuated by both CaMKII inhibition and mitochondrial ROS scavenging, with a slight synergy with INaL inhibition. Inducing LQT by elevating INaL (by Anemone toxin II, ATX-II) caused markedly prolonged APD, increased STV, and early afterdepolarizations (EADs). Those proarrhythmic ATX-II effects were largely attenuated by mitochondrial ROS scavenging, and partially reduced by inhibition of CaMKII and pathological leaky RyRs using dantrolene. In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) bearing LQT3 mutation SCN5A N406K, dantrolene significantly attenuated cell arrhythmias and APD prolongation. Targeting critical components of the Na+-Ca2+-CaMKII-ROS-INaL arrhythmogenic vicious cycle may exhibit important on-target and also trans-target effects (e.g., INaL and RyR inhibition can alter INaL-mediated LQT3 effects). Incorporating this vicious cycle into therapeutic strategies provides novel integrated insight for treating cardiac arrhythmias and diseases.

CytoSpectre: a tool for spectral analysis of oriented structures on cellular and subcellular levels.

BACKGROUND: Orientation and the degree of isotropy are important in many biological systems such as the sarcomeres of cardiomyocytes and other fibrillar structures of the cytoskeleton. Image based analysis of such structures is often limited to qualitative evaluation by human experts, hampering the throughput, repeatability and reliability of the analyses. Software tools are not readily available for this purpose and the existing methods typically rely at least partly on manual operation. RESULTS: We developed CytoSpectre, an automated tool based on spectral analysis, allowing the quantification of orientation and also size distributions of structures in microscopy images. CytoSpectre utilizes the Fourier transform to estimate the power spectrum of an image and based on the spectrum, computes parameter values describing, among others, the mean orientation, isotropy and size of target structures. The analysis can be further tuned to focus on targets of particular size at cellular or subcellular scales. The software can be operated via a graphical user interface without any programming expertise. We analyzed the performance of CytoSpectre by extensive simulations using artificial images, by benchmarking against FibrilTool and by comparisons with manual measurements performed for real images by a panel of human experts. The software was found to be tolerant against noise and blurring and superior to FibrilTool when analyzing realistic targets with degraded image quality. The analysis of real images indicated general good agreement between computational and manual results while also revealing notable expert-to-expert variation. Moreover, the experiment showed that CytoSpectre can handle images obtained of different cell types using different microscopy techniques. Finally, we studied the effect of mechanical stretching on cardiomyocytes to demonstrate the software in an actual experiment and observed changes in cellular orientation in response to stretching. CONCLUSIONS: CytoSpectre, a versatile, easy-to-use software tool for spectral analysis of microscopy images was developed. The tool is compatible with most 2D images and can be used to analyze targets at different scales. We expect the tool to be useful in diverse applications dealing with structures whose orientation and size distributions are of interest. While designed for the biological field, the software could also be useful in non-biological applications.

Data analytics for cardiac diseases.

In the present research we tackled the classification of seven genetic cardiac diseases and control subjects by using an extensive set of machine learning algorithms with their variations from simple K-nearest neighbor searching method to support vector machines. The research was based on calcium transient signals measured from induced pluripotent stem cell-derived cardiomyocytes. All in all, 55 different machine learning alternatives were used to model eight classes by applying the principle of 10-fold crossvalidation with the peak data of 1626 signals. The best classification accuracy of approximately 69% was given by random forests, which can be seen high enough here to show machine learning to be potential for the differentiation of the eight disease classes.

Pneumatic unidirectional cell stretching device for mechanobiological studies of cardiomyocytes.

In this paper, we present a transparent mechanical stimulation device capable of uniaxial stimulation, which is compatible with standard bioanalytical methods used in cellular mechanobiology. We validate the functionality of the uniaxial stimulation system using human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs). The pneumatically controlled device is fabricated from polydimethylsiloxane (PDMS) and provides uniaxial strain and superior optical performance compatible with standard inverted microscopy techniques used for bioanalytics (e.g., fluorescence microscopy and calcium imaging). Therefore, it allows for a continuous investigation of the cell state during stretching experiments. The paper introduces design and fabrication of the device, characterizes the mechanical performance of the device and demonstrates the compatibility with standard bioanalytical analysis tools. Imaging modalities, such as high-resolution live cell phase contrast imaging and video recordings, fluorescent imaging and calcium imaging are possible to perform in the device. Utilizing the different imaging modalities and proposed stretching device, we demonstrate the capability of the device for extensive further studies of hiPSC-CMs. We also demonstrate that sarcomere structures of hiPSC-CMs organize and orient perpendicular to uniaxial strain axis and thus express more maturated nature of cardiomyocytes.

Metabolic Maturation Media Improve Physiological Function of Human iPSC-Derived Cardiomyocytes.

Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) have enormous potential for the study of human cardiac disorders. However, their physiological immaturity severely limits their utility as a model system and their adoption for drug discovery. Here, we describe maturation media designed to provide oxidative substrates adapted to the metabolic needs of human iPSC (hiPSC)-CMs. Compared with conventionally cultured hiPSC-CMs, metabolically matured hiPSC-CMs contract with greater force and show an increased reliance on cardiac sodium (Na+) channels and sarcoplasmic reticulum calcium (Ca2+) cycling. The media enhance the function, long-term survival, and sarcomere structures in engineered heart tissues. Use of the maturation media made it possible to reliably model two genetic cardiac diseases: long QT syndrome type 3 due to a mutation in the cardiac Na+ channel SCN5A and dilated cardiomyopathy due to a mutation in the RNA splicing factor RBM20. The maturation media should increase the fidelity of hiPSC-CMs as disease models.

Polyethylene Terephthalate Textiles Enhance the Structural Maturation of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have the potential to serve as a model for human cardiomyocytes. However, hiPSC-CMs are still considered immature. CMs differentiated from hiPSCs more resemble fetal than adult cardiomyocytes. Putative factors enhancing maturation include in vitro culture duration, culture surface topography, and mechanical, chemical, and electrical stimulation. Stem cell-derived cardiomyocytes are traditionally cultured on glass surfaces coated with extracellular matrix derivatives such as gelatin. hiPSC-CMs are flat and round and their sarcomeres are randomly distributed and unorganized. Morphology can be enhanced by culturing cells on surfaces providing topographical cues to the cells. In this study, a textile based-culturing method used to enhance the maturation status of hiPSC-CMs is presented. Gelatin-coated polyethylene terephthalate (PET)-based textiles were used as the culturing surface for hiPSC-CMs and the effects of the textiles on the maturation status of the hiPSC-CMs were assessed. The hiPSC-CMs were characterized by analyzing their morphology, sarcomere organization, expression of cardiac specific genes, and calcium handling. We show that the topographical cues improve the structure of the hiPSC-CMs in vitro. Human iPSC-CMs grown on PET textiles demonstrated improved structural properties such as rod-shape structure and increased sarcomere orientation.

Simultaneous recordings of action potentials and calcium transients from human induced pluripotent stem cell derived cardiomyocytes.

Human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CMs) offer a unique in vitro platform to study cardiac diseases, as they recapitulate many disease phenotypes. The membrane potential (Vm) and intracellular calcium (Ca2+) transient (CaT) are usually investigated separately, because incorporating different techniques to acquire both aspects concurrently is challenging. In this study, we recorded Vm and CaT simultaneously to understand the interrelation between these parameters in hiPSC-CMs. For this, we used a conventional patch clamp technique to record Vm, and synchronized this with a Ca2+ imaging system to acquire CaT from same hiPSC-CMs. Our results revealed that the CaT at 90% decay (CaT90) was longer than action potential (AP) duration at 90% repolarization (APD90). In addition, there was also a strong positive correlation between the different parameters of CaT and AP. The majority of delayed after depolarizations (DADs) observed in the Vm recording were also characterized by elevations in the intracellular Ca2+ level, but in some cases no abnormalities were observed in CaT. However, simultaneous fluctuations in CaT were always observed during early after depolarizations (EADs) in Vm In summary, simultaneous recording of Vm and CaT broadens the understanding of the interrelation between Vm and CaT and could be used to elucidate the mechanisms underlying arrhythmia in cardiac disease condition.

Automatic Optimization of an in Silico Model of Human iPSC Derived Cardiomyocytes Recapitulating Calcium Handling Abnormalities.

The growing importance of human induced pluripotent stem cell-derived cardiomyoyctes (hiPSC-CMs), as patient-specific and disease-specific models for studying cellular cardiac electrophysiology or for preliminary cardiotoxicity tests, generated better understanding of hiPSC-CM biophysical mechanisms and great amount of action potential and calcium transient data. In this paper, we propose a new hiPSC-CM in silico model, with particular attention to Ca2+ handling. We used (i) the hiPSC-CM Paci2013 model as starting point, (ii) a new dataset of Ca2+ transient measurements to tune the parameters of the inward and outward Ca2+ fluxes of sarcoplasmic reticulum, and (iii) an automatic parameter optimization to fit action potentials and Ca2+ transients. The Paci2018 model simulates, together with the typical hiPSC-CM spontaneous action potentials, more refined Ca2+ transients and delayed afterdepolarizations-like abnormalities, which the old Paci2013 was not able to predict due to its mathematical formulation. The Paci2018 model was validated against (i) the same current blocking experiments used to validate the Paci2013 model, and (ii) recently published data about effects of different extracellular ionic concentrations. In conclusion, we present a new and more versatile in silico model, which will provide a platform for modeling the effects of drugs or mutations that affect Ca2+ handling in hiPSC-CMs.

Mutation-Specific Phenotypes in hiPSC-Derived Cardiomyocytes Carrying Either Myosin-Binding Protein C Or α-Tropomyosin Mutation for Hypertrophic Cardiomyopathy.

Hypertrophic cardiomyopathy (HCM) is a genetic cardiac disease, which affects the structure of heart muscle tissue. The clinical symptoms include arrhythmias, progressive heart failure, and even sudden cardiac death but the mutation carrier can also be totally asymptomatic. To date, over 1400 mutations have been linked to HCM, mostly in genes encoding for sarcomeric proteins. However, the pathophysiological mechanisms of the disease are still largely unknown. Two founder mutations for HCM in Finland are located in myosin-binding protein C (MYBPC3-Gln1061X) and α-tropomyosin (TPM1-Asp175Asn) genes. We studied the properties of HCM cardiomyocytes (CMs) derived from patient-specific human induced pluripotent stem cells (hiPSCs) carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn mutation. Both types of HCM-CMs displayed pathological phenotype of HCM but, more importantly, we found differences between CMs carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn gene mutation in their cellular size, Ca(2+) handling, and electrophysiological properties, as well as their gene expression profiles. These findings suggest that even though the clinical phenotypes of the patients carrying either MYBPC3-Gln1061X or TPM1-Asp175Asn gene mutation are similar, the genetic background as well as the functional properties on the cellular level might be different, indicating that the pathophysiological mechanisms behind the two mutations would be divergent as well.