Modeling Cardiovascular Diseases with hiPSC-Derived Cardiomyocytes in 2D and 3D Cultures

In the last decade, the generation of cardiac disease models based on human-induced pluripotent stem cells (hiPSCs) has become of common use, providing new opportunities to overcome the lack of appropriate cardiac models. Although much progress has been made toward the generation of hiPSC-derived cardiomyocytes (hiPS-CMs), several lines of evidence indicate that two-dimensional (2D) cell culturing presents significant limitations, including hiPS-CMs immaturity and the absence of interaction between dierent cell types and the extracellular matrix. More recently, new advances in bioengineering and co-culture systems have allowed the generation of three-dimensional (3D) constructs based on hiPSC-derived cells. Within these systems, biochemical and physical stimuli influence the maturation of hiPS-CMs, which can show structural and functional properties more similar to those present in adult cardiomyocytes. In this review, we describe the latest advances in 2D- and 3D-hiPSC technology for cardiac disease mechanisms investigation, drug development, and therapeutic studies

PKCα regulates the hypertrophic growth of cardiomyocytes through extracellular signal–regulated kinase1/2 (ERK1/2)

Members of the protein kinase C (PKC) isozyme family are important signal transducers in virtually every mammalian cell type. Within the heart, PKC isozymes are thought to participate in a signaling network that programs developmental and pathological cardiomyocyte hypertrophic growth. To investigate the function of PKC signaling in regulating cardiomyocyte growth, adenoviral-mediated gene transfer of wild-type and dominant negative mutants of PKCα, βII, δ, and ɛ (only wild-type ζ) was performed in cultured neonatal rat cardiomyocytes. Overexpression of wild-type PKCα, βII, δ, and ɛ revealed distinct subcellular localizations upon activation suggesting unique functions of each isozyme in cardiomyocytes. Indeed, overexpression of wild-type PKCα, but not βII, δ, ɛ, or ζ induced hypertrophic growth of cardiomyocytes characterized by increased cell surface area, increased [3H]-leucine incorporation, and increased expression of the hypertrophic marker gene atrial natriuretic factor. In contrast, expression of dominant negative PKCα, βII, δ, and ɛ revealed a necessary role for PKCα as a mediator of agonist-induced cardiomyocyte hypertrophy, whereas dominant negative PKCɛ reduced cellular viability. A mechanism whereby PKCα might regulate hypertrophy was suggested by the observations that wild-type PKCα induced extracellular signal–regulated kinase1/2 (ERK1/2), that dominant negative PKCα inhibited PMA-induced ERK1/2 activation, and that dominant negative MEK1 (up-stream of ERK1/2) inhibited wild-type PKCα–induced hypertrophic growth. These results implicate PKCα as a necessary mediator of cardiomyocyte hypertrophic growth, in part, through a ERK1/2-dependent signaling pathway

Cardiovascular extracellular microRNAs: emerging diagnostic markers and mechanisms of cell-to-cell RNA communication

Cardiovascular diseases are a leading cause of morbidity and mortality in Western societies. It is now well established that microRNAs (miRNAs) are determinant regulators in various medical conditions including cardiovascular diseases. The recent discovery that miRNAs, while associated with different carriers, can be exported out of the cell, has triggered a renewed interest to analyze the potential to use extracellular miRNAs as tools for diagnostic and therapeutic studies. Circulating miRNAs in biological fluids present a technological advantage compared to current diagnostic tools by virtue of their remarkable stability and relative ease of detection rendering them ideal tools for non-invasive and rapid diagnosis. Extracellular miRNAs also represent a novel form of inter-cellular communication by transferring genetic information from a donor cell to a recipient cell. This review briefly summarizes recent insights in the origin, function and diagnostic potential of extracellular miRNAs by focusing on a select number of cardiovascular diseases

MEK1 Inhibits Cardiac PPARα Activity by Direct Interaction and Prevents Its Nuclear Localization

BACKGROUND: The response of the postnatal heart to growth and stress stimuli includes activation of a network of signal transduction cascades, including the stress activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK) and the extracellular signal-regulated kinase (ERK1/2) pathways. In response to increased workload, the mitogen-activated protein kinase kinase (MAPKK) MEK1 has been shown to be active. Studies embarking on mitogen-activated protein kinase (MAPK) signaling cascades in the heart have indicated peroxisome-proliferators activated-receptors (PPARs) as downstream effectors that can be regulated by this signaling cascade. Despite the importance of PPARα in controlling cardiac metabolism, little is known about the relationship between MAPK signaling and cardiac PPARα signaling. METHODOLOGY/PRINCIPAL FINDING: Using co-immunoprecipitation and immunofluorescence approaches we show a complex formation of PPARα with MEK1 and not with ERK1/2. Binding of PPARα to MEK1 is mediated via a LXXLL motif and results in translocation from the nucleus towards the cytoplasm, hereby disabling the transcriptional activity of PPARα. Mice subjected to voluntary running-wheel exercise showed increased cardiac MEK1 activation and complex formation with PPARα, subsequently resulting in reduced PPARα activity. Inhibition of MEK1, using U0126, blunted this effect. CONCLUSION: Here we show that activation of the MEK1-ERK1/2 pathway leads to specific inhibition of PPARα transcriptional activity. Furthermore we show that this inhibitory effect is mediated by MEK1, and not by its downstream effector kinase ERK1/2, through a mechanism involving direct binding to PPARα and subsequent stimulation of PPARα export from the nucleus

β-AR Blockers Suppresses ER Stress in Cardiac Hypertrophy and Heart Failure

Long-term β-adrenergic receptor (β-AR) blockade reduces mortality in patients with heart failure. Chronic sympathetic hyperactivity in heart failure causes sustained β-AR activation, and this can deplete Ca(2+) in endoplasmic reticulum (ER) leading to ER stress and subsequent apoptosis. We tested the effect of β-AR blockers on ER stress pathway in experimental model of heart failure.ER chaperones were markedly increased in failing hearts of patients with end-stage heart failure. In Sprague-Dawley rats, cardiac hypertrophy and heart failure was induced by abdominal aortic constriction or isoproterenol subcutaneous injection. Oral β-AR blockers treatment was performed in therapy groups. Cardiac remodeling and left ventricular function were analyzed in rats failing hearts. After 4 or 8 weeks of banding, rats developed cardiac hypertrophy and failure. Cardiac expression of ER chaperones was significantly increased. Similar to the findings above, sustained isoproterenol infusion for 2 weeks induced cardiac hypertrophy and failure with increased ER chaperones and apoptosis in hearts. β-AR blockers treatment markedly attenuated these pathological changes and reduced ER stress and apoptosis in failing hearts. On the other hand, β-AR agonist isoproterenol induced ER stress and apoptosis in cultured cardiomyocytes. β-AR blockers largely prevented ER stress and protected myocytes against apoptosis. And β-AR blockade significantly suppressed the overactivation of CaMKII in isoproterenol-stimulated cardiomyocytes and failing hearts in rats.Our results demonstrated that ER stress occurred in failing hearts and this could be reversed by β-AR blockade. Alleviation of ER stress may be an important mechanism underlying the therapeutic effect of β-AR blockers on heart failure

Resveratrol Improves Survival, Hemodynamics and Energetics in a Rat Model of Hypertension Leading to Heart Failure

Heart failure (HF) is characterized by contractile dysfunction associated with altered energy metabolism. This study was aimed at determining whether resveratrol, a polyphenol known to activate energy metabolism, could be beneficial as a metabolic therapy of HF. Survival, ventricular and vascular function as well as cardiac and skeletal muscle energy metabolism were assessed in a hypertensive model of HF, the Dahl salt-sensitive rat fed with a high-salt diet (HS-NT). Resveratrol (18 mg/kg/day; HS-RSV) was given for 8 weeks after hypertension and cardiac hypertrophy were established (which occurred 3 weeks after salt addition). Resveratrol treatment improved survival (64% in HS-RSV versus 15% in HS-NT, p<0.001), and prevented the 25% reduction in body weight in HS-NT (P<0.001). Moreover, RSV counteracted the development of cardiac dysfunction (fractional shortening −34% in HS-NT) as evaluated by echocardiography, which occurred without regression of hypertension or hypertrophy. Moreover, aortic endothelial dysfunction present in HS-NT was prevented in resveratrol-treated rats. Resveratrol treatment tended to preserve mitochondrial mass and biogenesis and completely protected mitochondrial fatty acid oxidation and PPARα (peroxisome proliferator-activated receptor α) expression. We conclude that resveratrol treatment exerts beneficial protective effects on survival, endothelium–dependent smooth muscle relaxation and cardiac contractile and mitochondrial function, suggesting that resveratrol or metabolic activators could be a relevant therapy in hypertension-induced HF

Apoptosis-Inducing Factor Regulates Skeletal Muscle Progenitor Cell Number and Muscle Phenotype

Apoptosis Inducing Factor (AIF) is a highly conserved, ubiquitous flavoprotein localized in the mitochondrial intermembrane space. In vivo, AIF provides protection against neuronal and cardiomyocyte apoptosis induced by oxidative stress. Conversely in vitro, AIF has been demonstrated to have a pro-apoptotic role upon induction of the mitochondrial death pathway, once AIF translocates to the nucleus where it facilitates chromatin condensation and large scale DNA fragmentation. Given that the aif hypomorphic harlequin (Hq) mutant mouse model displays severe sarcopenia, we examined skeletal muscle from the aif hypomorphic mice in more detail. Adult AIF-deficient skeletal myofibers display oxidative stress and a severe form of atrophy, associated with a loss of myonuclei and a fast to slow fiber type switch, both in “slow” muscles such as soleus, as well as in “fast” muscles such as extensor digitorum longus, most likely resulting from an increase of MEF2 activity. This fiber type switch was conserved in regenerated soleus and EDL muscles of Hq mice subjected to cardiotoxin injection. In addition, muscle regeneration in soleus and EDL muscles of Hq mice was severely delayed. Freshly cultured myofibers, soleus and EDL muscle sections from Hq mice displayed a decreased satellite cell pool, which could be rescued by pretreating aif hypomorphic mice with the manganese-salen free radical scavenger EUK-8. Satellite cell activation seems to be abnormally long in Hq primary culture compared to controls. However, AIF deficiency did not affect myoblast cell proliferation and differentiation. Thus, AIF protects skeletal muscles against oxidative stress-induced damage probably by protecting satellite cells against oxidative stress and maintaining skeletal muscle stem cell number and activation

A versatile multiplexed assay to quantify intracellular ROS and cell viability in 3D on-a-chip models

Reactive oxygen species (ROS) have different properties and biological functions. They contribute to cell signaling and, in excessive amounts, to oxidative stress (OS). Although ROS is pivotal in a wide number of physiological systems and pathophysiological processes, direct quantification in vivo is quite challenging and mainly limited to in vitro studies. Even though advanced in vitro cell culture techniques, like on-a-chip culture, have overcome the lack of crucial in vivo-like physiological aspects in 2D culture, the majority of in vitro ROS quantification studies are generally performed in 2D. Here we report the development, application, and validation of a multiplexed assay to quantify ROS and cell viability in organ-on-a-chip models. The assay utilizes three dyes to stain live cells for ROS, dead cells, and DNA. Confocal images were analyzed to quantify ROS probes and determine the number of nuclei and dead cells. We found that, in contrast to what has been reported with 2D cell culture, on-a-chip models are more prone to scavenge ROS rather than accumulate them. The assay is sensitive enough to distinguish between different phenotypes of endothelial cells (ECs) based on the level of OS to detect higher level in tumor than normal cells. Our results indicate that the use of physiologically relevant models and this assay could help unravelling the mechanisms behind OS and ROS accumulation. A further step could be taken in data analysis by implementing AI in the pipeline to also analyze images for morphological changes to have an even broader view of OS mechanism

Human Engineered Heart Tissue as a Versatile Tool in Basic Research and Preclinical Toxicology

Human embryonic stem cell (hESC) progenies hold great promise as surrogates for human primary cells, particularly if the latter are not available as in the case of cardiomyocytes. However, high content experimental platforms are lacking that allow the function of hESC-derived cardiomyocytes to be studied under relatively physiological and standardized conditions. Here we describe a simple and robust protocol for the generation of fibrin-based human engineered heart tissue (hEHT) in a 24-well format using an unselected population of differentiated human embryonic stem cells containing 30–40% α-actinin-positive cardiac myocytes. Human EHTs started to show coherent contractions 5–10 days after casting, reached regular (mean 0.5 Hz) and strong (mean 100 µN) contractions for up to 8 weeks. They displayed a dense network of longitudinally oriented, interconnected and cross-striated cardiomyocytes. Spontaneous hEHT contractions were analyzed by automated video-optical recording and showed chronotropic responses to calcium and the β-adrenergic agonist isoprenaline. The proarrhythmic compounds E-4031, quinidine, procainamide, cisapride, and sertindole exerted robust, concentration-dependent and reversible decreases in relaxation velocity and irregular beating at concentrations that recapitulate findings in hERG channel assays. In conclusion this study establishes hEHT as a simple in vitro model for heart research

Healthy and diseased placental barrier on-a-chip models suitable for standardized studies

Pathologies associated with uteroplacental hypoxia, such as preeclampsia are among the leading causes of maternal and perinatal morbidity in the world. Its fundamental mechanisms are yet poorly understood due to a lack of good experimental models. Here we report an in vitro model of the placental barrier, based on co-culture of trophoblasts and endothelial cells against a collagen extracellular matrix in a microfluidic platform. The model yields a functional syncytium with barrier properties, polarization, secretion of relevant extracellular membrane components, thinning of the materno-fetal space, hormone secretion, and transporter function. The model is exposed to low oxygen conditions and perfusion flow is modulated to induce a pathological environment. This results in reduced barrier function, hormone secretion, and microvilli as well as an increased nuclei count, characteristics of preeclamptic placentas. The model is implemented in a titer plate-based microfluidic platform fully amenable to high-throughput screening. We thus believe this model could aid mechanistic understanding of preeclampsia and other placental pathologies associated with hypoxia/ischemia, as well as support future development of effective therapies through target and compound screening campaigns.Statement of Significance: The human placenta is a unique organ sustaining fetus growth but is also the source of severe pathologies, such as Preeclampsia. Though leading cause of perinatal mortality in the world, preeclampsia remains untreatable due to a lack of relevant in vitro placenta models. To better understand the pathology, we have developed 3D placental barrier models in a microfluidic device. The platform allows parallel culture of 40 perfused physiological miniaturized placental barriers, comprising a differentiated syncytium and endothelium that have been validated for transporter functions. Exposure to a hypoxic and ischemic environment enabled the mimicking of preeclamptic characteristics in high-throughput, which we believe could lead to a better understanding of the pathology as well as support future effective therapies development.</p