High-Throughput Optical Controlling and Recording Calcium Signal in iPSC-Derived Cardiomyocytes for Toxicity Testing and Phenotypic Drug Screening.

Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.

A protocol for extracting immunolabeled murine cardiomyocytes of high-quality RNA by laser capture microdissection.

We developed a highly efficient, ultrashort immunohistochemistry-laser capture microdissection (IHC-LMD) protocol, which allows microdissection of up to 250 single cardiomyocytes. Before LMD, murine hearts are excised, snap-frozen, and cryosectioned. RNA isolated from LMD material is of high RNA quality, making it usable for gene expression analysis and RNA sequencing. Challenges and limitations of this protocol include visualization of the immunostaining and nuclei DAPI dye on the PEN slides, and timing and speed to limit RNA degradation as much as possible.

The yin and yang of intracellular delivery of amphipathic optical probes using n-butyl charge masking.

Monitoring and manipulation of ionized intracellular calcium concentrations within intact, living cells using optical probes with organic chromophores is a core method for cell physiology. Since all these probes have multiple negative charges, they must be smuggled through the plasma membrane in a transiently neutral form, with intracellular esterases used to deprotect the masked anions. Here we explore the ability of the synthetically easily accessible n-butyl ester protecting group to deliver amphipathic cargoes to the cytosol. We show that the size of the caging chromophore conditions the ability of intracellular probe delivery and esterase charge unmasking.

miR-16-5p Suppression Protects Human Cardiomyocytes against Endoplasmic Reticulum and Oxidative Stress-Induced Injury.

Oxidative stress, defined as the excess production of reactive oxygen species (ROS) relative to antioxidant defense, plays a significant role in the development of cardiovascular diseases. Endoplasmic reticulum (ER) stress has emerged as an important source of ROS and its modulation could be cardioprotective. Previously, we demonstrated that miR-16-5p is enriched in the plasma of ischemic dilated cardiomyopathy (ICM) patients and promotes ER stress-induced apoptosis in cardiomyocytes in vitro. Here, we hypothesize that miR-16-5p might contribute to oxidative stress through ER stress induction and that targeting miR-16-5p may exert a cardioprotective role in ER stress-mediated cardiac injury. Analysis of oxidative markers in the plasma of ICM patients demonstrates that oxidative stress is associated with ICM. Moreover, we confirm that miR-16-5p overexpression promotes oxidative stress in AC16 cardiomyoblasts. We also find that, in response to tunicamycin-induced ER stress, miR-16-5p suppression decreases apoptosis, inflammation and cardiac damage via activating the ATF6-mediated cytoprotective pathway. Finally, ATF6 is identified as a direct target gene of miR-16-5p by dual-luciferase reporter assays. Our results indicate that miR-16-5p promotes ER stress and oxidative stress in cardiac cells through regulating ATF6, suggesting that the inhibition of miR-16-5p has potential as a therapeutic approach to protect the heart against ER and oxidative stress-induced injury.

Single-cell sequencing reveals lineage-specific dynamic genetic regulation of gene expression during human cardiomyocyte differentiation.

Dynamic and temporally specific gene regulatory changes may underlie unexplained genetic associations with complex disease. During a dynamic process such as cellular differentiation, the overall cell type composition of a tissue (or an in vitro culture) and the gene regulatory profile of each cell can both experience significant changes over time. To identify these dynamic effects in high resolution, we collected single-cell RNA-sequencing data over a differentiation time course from induced pluripotent stem cells to cardiomyocytes, sampled at 7 unique time points in 19 human cell lines. We employed a flexible approach to map dynamic eQTLs whose effects vary significantly over the course of bifurcating differentiation trajectories, including many whose effects are specific to one of these two lineages. Our study design allowed us to distinguish true dynamic eQTLs affecting a specific cell lineage from expression changes driven by potentially non-genetic differences between cell lines such as cell composition. Additionally, we used the cell type profiles learned from single-cell data to deconvolve and re-analyze data from matched bulk RNA-seq samples. Using this approach, we were able to identify a large number of novel dynamic eQTLs in single cell data while also attributing dynamic effects in bulk to a particular lineage. Overall, we found that using single cell data to uncover dynamic eQTLs can provide new insight into the gene regulatory changes that occur among heterogeneous cell types during cardiomyocyte differentiation.

LncRNA SOX2-OTinhibitionprotects against myocardialischemia/reperfusion-inducedinjury via themicroRNA-186-5p (miR-186-5p)/Yin Yang 1 (YY1)pathway.

Long noncoding RNAs (lncRNAs) exert essential effects in regulating myocardial ischemia/reperfusion (MI/R)-induced injury. This work intended to explore the functions of lncRNA SOX2-OT and its regulatory mechanism within MI/R-induced injury. In this study, gene expression was determined by RT-qPCR. Western blotting was applied for the detection of protein levels. Pro-inflammatory cytokine concentrations, cardiomyocyte viability, and apoptosis were detected via ELISA, CCK-8 and flow cytometry. In the in vitro model, SOX2-OT and YY1 were both upregulated, while miR-186-5p was downregulated. SOX2-OT knockdown attenuated oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cardiomyocyte dysregulation through relieving inflammation, promoting proliferation, and reducing apoptosis in OGD/R-treated H2C9 cells. SOX2-OT positively regulated YY1 expression via miR-186-5p. Moreover, miR-186-5p inhibition or YY1 upregulation abolished the effects of SOX2-OT blocking on the inflammatory responses, proliferation, and apoptosis of OGD/R-challenged H2C9 cells. In conclusion, our results, for the first time, demonstrated that SOX2-OT inhibition attenuated MI/R injury in vitro via regulating the miR-186-5p/YY1 axis, offering potential therapeutic targets for MI/R injury treatment.

Exercise downregulates HIPK2 and HIPK2 inhibition protects against myocardial infarction.

BACKGROUND: Exercise can protect myocardial infarction (MI) and downregulate cardiac Homeodomain-Interacting Protein Kinase 2 (HIPK2). However, the role of HIPK2 in MI is unclear. METHODS: HIPK2-/- mice and miR-222-/- rats, HIPK2 inhibitor (PKI1H) and adeno-associated virus serotype 9 (AAV9) carrying miR-222 were applied in the study. Animals were subjected to running, swimming, acute MI or post-MI remodeling. HIPK2 inhibition and P53 activator were used in neonatal rat cardiomyocytes (NRCMs) and human embryonic stem cell-derived cardiomyocytes (hESC-CMs) subjected to oxygen glucose deprivation/reperfusion (OGD/R). Serum miR-222 levels were analyzed in healthy people and MI patients that were survival or readmitted to the hospital and/or died. FINDINGS: Cardiac HIPK2 protein levels were reduced by exercise while increased in MI. In vitro, HIPK2 suppression by lentiviral vectors or inhibitor prevented apoptosis induced by OGD/R in NRCMs and hESC-CMs. HIPK2 inhibitor-treated mice and HIPK2-/- mice reduced infarct size after acute MI, and preserved cardiac function in MI remodeling. Mechanistically, protective effect against apoptosis by HIPK2 suppression was reversed by P53 activators. Furthermore, increasing levels of miR-222, targeting HIPK2, protected post-MI cardiac dysfunction, whereas cardiac dysfunction post-MI was aggravated in miR-222-/- rats. Moreover, serum miR-222 levels were significantly reduced in MI patients, as well as in MI patients that were readmitted to the hospital and/or died compared to those not. INTERPRETATION: Exercise-induced HIPK2 suppression attenuates cardiomyocytes apoptosis and protects MI by decreasing P-P53. Inhibition of HIPK2 represents a potential novel therapeutic intervention for MI. FUNDING: This work was supported by the grants from National Key Research and Development Project (2018YFE0113500 to JJ Xiao), National Natural Science Foundation of China (82020108002, 81722008, and 81911540486 to JJ Xiao, 81400647 to MJ Xu, 81800265 to YJ Liang), Innovation Program of Shanghai Municipal Education Commission (2017-01-07-00-09-E00042 to JJ Xiao), the grant from Science and Technology Commission of Shanghai Municipality (18410722200 and 17010500100 to JJ Xiao), the "Dawn" Program of Shanghai Education Commission (19SG34 to JJ Xiao), Shanghai Sailing Program (21YF1413200 to QL Zhou). JS is supported by Horizon2020 ERC-2016-COG EVICARE (725229).

Subcellular Localization of Connexin 26 in Cardiomyocytes and in Cardiomyocyte-Derived Extracellular Vesicles.

Connexins (Cxs) are a family of membrane-spanning proteins, expressed in vertebrates and named according to their molecular weight. They are involved in tissue homeostasis, and they function by acting at several communication levels. Cardiac Cxs are responsible for regular heart function and, among them, Cx26 and Cx43 are widely expressed throughout the heart. Cx26 is present in vessels, as well as in cardiomyocytes, and its localization is scattered all over the cell aside from at the intercalated discs as is the case for the other cardiac Cxs. However, having been found in cardiomyocytes only recently, both its subcellular localization and its functional characterization in cardiomyocytes remain poorly understood. Therefore, in this study we aimed to obtain further data on the localization of Cx26 at the subcellular level. Our TEM immunogold analyses were performed on rat heart ventricles and differentiated H9c2 cardiac cell sections as well as on differentiated H9c2 derived extracellular vesicles. The results confirmed the absence of Cx26 at intercalated discs and showed the presence of Cx26 at the level of different subcellular compartments. The peculiar localization at the level of extracellular vesicles suggested a specific role for cardiac Cx26 in inter-cellular communication in an independent gap junction manner.

Automatic detection of adult cardiomyocyte for high throughput measurements of calcium and contractility.

Simultaneous calcium and contractility measurements on isolated adult cardiomyocytes have been the gold standard for the last decades to study cardiac (patho)physiology. However, the throughput of this system is low which limits the number of compounds that can be tested per animal. We developed instrumentation and software that can automatically find adult cardiomyocytes. Cells are detected based on the cell boundary using a Sobel-filter to find the edge information in the field of view. Separately, we detected motion by calculating the variance of intensity for each pixel in the frame through time. Additionally, it detects the best region for calcium and contractility measurements. A sensitivity of 0.66 ± 0.08 and a precision of 0.82 ± 0.03 was reached using our cell finding algorithm. The percentage of cells that were found and had good contractility measurements was 90 ± 10%. In addition, the average time between 2 cardiomyocyte calcium and contractility measurements decreased from 93.5 ± 80.2 to 15.6 ± 8.0 seconds using our software and microscope. This drastically increases throughput and provides a higher statistical reliability when performing adult cardiomyocyte functional experiments.

Single-Cardiomyocyte RNA Sequencing to Dissect the Molecular Pathophysiology of the Heart.

Heart failure is caused by a complicated pathogenic process and has a poor prognosis. Quality of life is often impaired due to repeated hospitalization. Integrative analysis of the morphological, physiological, and molecular profiles of cardiomyocytes, which are responsible mainly for heart contraction, may lead to a deeper understanding of the pathogenesis of heart failure. However, unlike other types of cells, cardiomyocytes are relatively large, vulnerable to stress, and difficult to use for single-cell analysis. With some ingenuity, we have established a single-cardiomyocyte analysis pipeline. Here, we describe the procedure for single-cell RNA sequencing of adult mouse cardiomyocytes from isolation to analysis.

RNA-Sequencing Analysis of Differentially Expressed Genes in Human iPSC-Derived Cardiomyocytes.

RNA sequencing profiles and characterizes cell and tissue samples, giving important insights into molecular mechanisms. Such data is imperative for cardiomyocytes derived from induced pluripotent stem cells (iPSC-CMs) and used in related translational and basic research. Here we provide reliable protocols to extract differentially expressed genes in iPSC-CMs with RNA sequencing.

Analysis of Transcriptional Profiling of Chamber-Specific Human Cardiac Myocytes Derived from Pluripotent Stem Cells.

Differentiation protocols to direct cell fate decision from pluripotent stem cells to cardiac myocytes normally achieve high purity and quality of cells. Nonetheless, the highly specialized anatomy of the heart enables the possibility that acquisition of terminal somatic differentiation from pluripotency might imply heterogeneity of non-desire cell lineages. Directed cardiac differentiation empowers differentiation of pool of cells commonly reported to contain different proportions of ventricular, atrial, and nodal-like cells. RNA sequencing (RNA-Seq) allows a precise transcriptional profiling, ensuring a quality checking of the cell identity our protocol has derived as a main outcome. Here we describe a workflow methodology on how to adapt RNA sequencing analysis for integration into the R analysis pipeline in order to characterize chamber-specific gene signatures of the major cardiac lineages of myocytes in the heart.

Calibration and measurement of mitochondrial pH in intact adult rat cardiomyocytes.

Mitochondrial pH is a vital parameter of the mitochondrial environment, which determines the rate of many mitochondrial functions, including metabolism, membrane potential, fate, etc. Abnormal mitochondrial pH is always closely related to the health status of cells. Analyzing mitochondrial pH can serve as a proxy for mitochondrial and cellular function. This protocol describes the use of SNARF-1 AM, a pH-sensitive fluorophore, to measure mitochondrial pH. This protocol details the steps to evaluate mitochondrial pH in live adult cardiomyocytes using confocal microscopy. The protocol can be adapted to other adherent cell types. For complete details on the use and execution of this protocol, please refer to Wei-LaPierre et al. (2013).

Mimicking cardiac tissue complexity through physical cues: A review on cardiac tissue engineering approaches.

Cardiovascular diseases are the number one killer in the world.1,2 Currently, there are no clinical treatments to regenerate damaged cardiac tissue, leaving patients to develop further life-threatening cardiac complications. Cardiac tissue has multiple functional demands including vascularization, contraction, and conduction that require many synergic components to properly work. Most of these functions are a direct result of the cardiac tissue structure and composition, and, for this reason, tissue engineering strongly proposed to develop substitute engineered heart tissues (EHTs). EHTs usually have combined pluripotent stem cells and supporting scaffolds with the final aim to repair or replace the damaged native tissue. However, as simple as this idea is, indeed, it resulted, after many attempts in the field, to be very challenging. Without design complexity, EHTs remain unable to mature fully and integrate into surrounding heart tissue resulting in minimal in vivo effects.3 Lately, there has been a growing body of evidence that a complex, multifunctional approach through implementing scaffold designs, cellularization, and molecular release appears to be essential in the development of a functional cardiac EHTs.4-6 This review covers the advancements in EHTs developments focusing on how to integrate contraction, conduction, and vascularization mimics and how combinations have resulted in improved designs thus warranting further investigation to develop a clinically applicable treatment.

Epigenetic Assays in Purified Cardiomyocyte Nuclei.

The adult mammalian heart's potential for regeneration is very inefficient. Importantly, adult mammalian cardiomyocytes (CMs) are characterized as a cell population with very limited mitotic potential. Conversely, the neonatal mouse heart possesses a brief, yet robust, regenerative capacity within the first week of life. Cell type-specific enrichment procedures are essential for characterizing the full spectrum of epigenomic landscapes and gene regulatory networks deployed by mammalian CMs. In this chapter, we describe a protocol useful for purifying CM nuclei from mammalian cardiac tissue. Furthermore, we detail a low-input procedure suitable for the parallel genome-wide profiling of chromatin accessibility, histone modifications, and transcription factor-binding sites. The CM nuclei purified using this process are suitable for multi-omic profiling approaches.

Genetic Lineage Tracing of Non-cardiomyocytes in Mice.

Genetic lineage tracing is accomplished using bi-transgenic mice, where one allele is altered to express Cre recombinase, and another allele encodes a Cre-dependent genetic reporter protein. Once Cre is activated (constitutive or in response to tamoxifen), the marker gene-expressing cells become indelibly labeled by the reporter protein. Therefore, daughter cells derived from labeled cells are permanently labeled even if the marker gene that drove Cre recombinase expression is no longer expressed in these cells. This system is commonly used to label putative progenitor cells and determine the fate of their progeny. Here, we describe the use of c-kit-based genetic lineage-tracing mouse line as an example and discuss caveats for performing these types of experiments.

miR­449a­5p suppresses CDK6 expression to inhibit cardiomyocyte proliferation.

Induction of cardiomyocyte (CM) proliferation is a promising approach for cardiac regeneration following myocardial injury. MicroRNAs (miRs) have been reported to regulate CM proliferation. In particular, miR­449a­5p has been identified to be associated with CM proliferation in previous high throughput functional screening data. However, whether miR­449a­5p regulates CM proliferation has not been thoroughly investigated. This study aimed to explore whether miR­449a­5p modulates CM proliferation and to identify the molecular mechanism via which miR­449a­5p regulates CM proliferation. The current study demonstrated that miR­449a­5p expression levels were significantly increased during heart development. Furthermore, the results suggested that miR­449a­5p mimic inhibited CM proliferation in vitro as determined via immunofluorescence for ki67 and histone H3 phosphorylated at serine 10 (pH3), as well as the numbers of CMs. However, miR­449a­5p knockdown promoted CM proliferation. CDK6 was identified as a direct target gene of miR­449a­5p, and CDK6 mRNA and protein expression was suppressed by miR­449a­5p. Moreover, CDK6 gain­of­function increased CM proliferation. Overexpression of CDK6 also blocked the inhibitory effect of miR­449a­5p on CM proliferation, indicating that CDK6 was a functional target of miR­449a­5p in CM proliferation. In conclusion, miR­449a­5p inhibited CM proliferation by targeting CDK6, which provides a potential molecular target for preventing myocardial injury.

Biophysically detailed mathematical models of multiscale cardiac active mechanics.

We propose four novel mathematical models, describing the microscopic mechanisms of force generation in the cardiac muscle tissue, which are suitable for multiscale numerical simulations of cardiac electromechanics. Such models are based on a biophysically accurate representation of the regulatory and contractile proteins in the sarcomeres. Our models, unlike most of the sarcomere dynamics models that are available in the literature and that feature a comparable richness of detail, do not require the time-consuming Monte Carlo method for their numerical approximation. Conversely, the models that we propose only require the solution of a system of PDEs and/or ODEs (the most reduced of the four only involving 20 ODEs), thus entailing a significant computational efficiency. By focusing on the two models that feature the best trade-off between detail of description and identifiability of parameters, we propose a pipeline to calibrate such parameters starting from experimental measurements available in literature. Thanks to this pipeline, we calibrate these models for room-temperature rat and for body-temperature human cells. We show, by means of numerical simulations, that the proposed models correctly predict the main features of force generation, including the steady-state force-calcium and force-length relationships, the length-dependent prolongation of twitches and increase of peak force, the force-velocity relationship. Moreover, they correctly reproduce the Frank-Starling effect, when employed in multiscale 3D numerical simulation of cardiac electromechanics.