Unlocking Personalized Biomedicine and Drug Discovery with Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes: Fit for Purpose or Forever Elusive?

In recent decades, drug development costs have increased by approximately a hundredfold, and yet about 1 in 7 licensed drugs are withdrawn from the market, often due to cardiotoxicity. This review considers whether technologies using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) could complement existing assays to improve discovery and safety while reducing socioeconomic costs and assisting with regulatory guidelines on cardiac safety assessments. We draw on lessons from our own work to suggest a panel of 12 drugs that will be useful in testing the suitability of hiPSC-CM platforms to evaluate contractility. We review issues, including maturity versus complexity, consistency, quality, and cost, while considering a potential need to incorporate auxiliary approaches to compensate for limitations in hiPSC-CM technology. We give examples on how coupling hiPSC-CM technologies with Cas9/CRISPR genome engineering is starting to be used to personalize diagnosis, stratify risk, provide mechanistic insights, and identify new pathogenic variants for cardiovascular disease.

Altered contractility, Ca2+ transients, and cell morphology seen in a patient-specific iPSC-CM model of Ebstein's anomaly with left ventricular noncompaction.

Patients with two congenital heart diseases (CHDs), Ebstein's anomaly (EA) and left ventricular noncompaction (LVNC), suffer higher morbidity than either CHD alone. The genetic etiology and pathogenesis of combined EA/LVNC remain largely unknown. We investigated a familial EA/LVNC case associated with a variant (p.R237C) in the gene encoding Kelch-like protein 26 (KLHL26) by differentiating induced pluripotent stem cells (iPSCs) generated from affected and unaffected family members into cardiomyocytes (iPSC-CMs) and assessing iPSC-CM morphology, function, gene expression, and protein abundance. Compared with unaffected iPSC-CMs, CMs containing the KLHL26 (p.R237C) variant exhibited aberrant morphology including distended endo(sarco)plasmic reticulum (ER/SR) and dysmorphic mitochondria and aberrant function that included decreased contractions per minute, altered calcium transients, and increased proliferation. Pathway enrichment analyses based on RNASeq data indicated that the "structural constituent of muscle" pathway was suppressed, whereas the "ER lumen" pathway was activated. Taken together, these findings suggest that iPSC-CMs containing this KLHL26 (p.R237C) variant develop dysregulated ER/SR, calcium signaling, contractility, and proliferation.NEW & NOTEWORTHY We demonstrate here that iPSCs derived from patients with Ebstein's anomaly and left ventricular noncompaction, when differentiated into cardiomyocytes, display significant structural and functional changes that offer insight into disease pathogenesis, including altered ER/SR and mitochondrial morphology, contractility, and calcium signaling.

Tyrosine 308 is necessary for ligand-directed Gs protein-biased signaling of ß2-adrenoceptor.

Interaction of a given G protein-coupled receptor to multiple different G proteins is a widespread phenomenon. For instance, ß2-adrenoceptor (ß2-AR) couples dually to Gs and Gi proteins. Previous studies have shown that cAMP-dependent protein kinase (PKA)-mediated phosphorylation of ß2-AR causes a switch in receptor coupling from Gs to Gi. More recent studies have demonstrated that phosphorylation of ß2-AR by G protein-coupled receptor kinases, particularly GRK2, markedly enhances the Gi coupling. We have previously shown that although most ß2-AR agonists cause both Gs and Gi activation, (R,R')-fenoterol preferentially activates ß2-AR-Gs signaling. However, the structural basis for this functional selectivity remains elusive. Here, using docking simulation and site-directed mutagenesis, we defined Tyr-308 as the key amino acid residue on ß2-AR essential for Gs-biased signaling. Following stimulation with a ß2-AR-Gs-biased agonist (R,R')-4'-aminofenoterol, the Gi disruptor pertussis toxin produced no effects on the receptor-mediated ERK phosphorylation in HEK293 cells nor on the contractile response in cardiomyocytes expressing the wild-type ß2-AR. Interestingly, Y308F substitution on ß2-AR enabled (R,R')-4'-aminofenoterol to activate Gi and to produce these responses in a pertussis toxin-sensitive manner without altering ß2-AR phosphorylation by PKA or G protein-coupled receptor kinases. These results indicate that, in addition to the phosphorylation status, the intrinsic structural feature of ß2-AR plays a crucial role in the receptor coupling selectivity to G proteins. We conclude that specific interactions between the ligand and the Tyr-308 residue of ß2-AR stabilize receptor conformations favoring the receptor-Gs protein coupling and subsequently result in Gs-biased agonism.

RBPMS regulates cardiomyocyte contraction and cardiac function through RNA alternative splicing.

AIMS: RNA binding proteins play essential roles in mediating RNA splicing and are key post-transcriptional regulators in the heart. Our recent study demonstrated that RBPMS (RNA binding protein with multiple splicing) is crucial for cardiac development through modulating mRNA splicing, but little is known about its functions in the adult heart. In this study, we aim to characterize the post-natal cardiac function of Rbpms and its mechanism of action. METHODS AND RESULTS: We generated a cardiac-specific knockout mouse line and found that cardiac-specific loss of Rbpms caused severe cardiomyocyte contractile defects, leading to dilated cardiomyopathy and early lethality in adult mice. We showed by proximity-dependent biotin identification assay and mass spectrometry that RBPMS associates with spliceosome factors and other RNA binding proteins, such as RBM20, that are important in cardiac function. We performed paired-end RNA sequencing and RT-PCR and found that RBPMS regulates mRNA alternative splicing of genes associated with sarcomere structure and function, such as Ttn, Pdlim5, and Nexn, generating new protein isoforms. Using a minigene splicing reporter assay, we determined that RBPMS regulates target gene splicing through recognizing tandem intronic CAC motifs. We also showed that RBPMS knockdown in human induced pluripotent stem cell-derived cardiomyocytes impaired cardiomyocyte contraction. CONCLUSION: This study identifies RBPMS as an important regulator of cardiomyocyte contraction and cardiac function by modulating sarcomeric gene alternative splicing.

Strengthening cardiac therapy pipelines using human pluripotent stem cell-derived cardiomyocytes.

Advances in hiPSC isolation and reprogramming and hPSC-CM differentiation have prompted their therapeutic application and utilization for evaluating potential cardiovascular safety liabilities. In this perspective, we showcase key efforts toward the large-scale production of hiPSC-CMs, implementation of hiPSC-CMs in industry settings, and recent clinical applications of this technology. The key observations are a need for traceable gender and ethnically diverse hiPSC lines, approaches to reduce cost of scale-up, accessible clinical trial datasets, and transparent guidelines surrounding the safety and efficacy of hiPSC-based therapies.

A cell-based framework for modeling cardiac mechanics.

Cardiomyocytes are the functional building blocks of the heart-yet most models developed to simulate cardiac mechanics do not represent the individual cells and their surrounding matrix. Instead, they work on a homogenized tissue level, assuming that cellular and subcellular structures and processes scale uniformly. Here we present a mathematical and numerical framework for exploring tissue-level cardiac mechanics on a microscale given an explicit three-dimensional geometrical representation of cells embedded in a matrix. We defined a mathematical model over such a geometry and parametrized our model using publicly available data from tissue stretching and shearing experiments. We then used the model to explore mechanical differences between the extracellular and the intracellular space. Through sensitivity analysis, we found the stiffness in the extracellular matrix to be most important for the intracellular stress values under contraction. Strain and stress values were observed to follow a normal-tangential pattern concentrated along the membrane, with substantial spatial variations both under contraction and stretching. We also examined how it scales to larger size simulations, considering multicellular domains. Our work extends existing continuum models, providing a new geometrical-based framework for exploring complex cell-cell and cell-matrix interactions.

Is Zn2+ the new Ca2+ for TRPC6 channels in the myocardium?

Transient Receptor Potential (TRP) channels are nonselective cation channels, which are mainly permeable to Ca2+ and Na+ but many of them are also permeable to Zn2+. In a new elegant study, a Zn2+-dependent pathway involving the TRP member TRPC6 and α1- as well as ß-adrenoceptors (AR) was dissected in rodent myocytes. Norepinephrine-mediated activation of α1-AR induces Zn2+ influx through TRPC6 channels, which reinforces ß-AR-mediated positive inotropy and may help patients with heart failure. This work encourages a closer look at the consequences of Zn2+ permeation through TRP channels in human health and disease.

Cardiovascular Functions of Ena/VASP Proteins: Past, Present and Beyond.

Actin binding proteins are of crucial importance for the spatiotemporal regulation of actin cytoskeletal dynamics, thereby mediating a tremendous range of cellular processes. Since their initial discovery more than 30 years ago, the enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family has evolved as one of the most fascinating and versatile family of actin regulating proteins. The proteins directly enhance actin filament assembly, but they also organize higher order actin networks and link kinase signaling pathways to actin filament assembly. Thereby, Ena/VASP proteins regulate dynamic cellular processes ranging from membrane protrusions and trafficking, and cell-cell and cell-matrix adhesions, to the generation of mechanical tension and contractile force. Important insights have been gained into the physiological functions of Ena/VASP proteins in platelets, leukocytes, endothelial cells, smooth muscle cells and cardiomyocytes. In this review, we summarize the unique and redundant functions of Ena/VASP proteins in cardiovascular cells and discuss the underlying molecular mechanisms.

Membranes for Modelling Cardiac Tissue Stiffness In Vitro Based on Poly(trimethylene carbonate) and Poly(ethylene glycol) Polymers.

Despite the increased expenditure of the pharmaceutical industry on research and development, the number of drugs for cardiovascular diseases that reaches the market is decreasing. A major issue is the limited ability of the current in vitro and experimental animal models to accurately mimic human heart disease, which hampers testing of the efficacy of potential cardiac drugs. Moreover, many non-heart-related drugs have severe adverse cardiac effects, which is a major cause of drugs' retraction after approval. A main hurdle of current in vitro models is their inability to mimic the stiffness of in vivo cardiac tissue. For instance, poly(styrene) petri dishes, which are often used in these models, have a Young's modulus in the order of GPa, while the stiffness of healthy human heart tissue is <50 kPa. In pathological conditions, such as scarring and fibrosis, the stiffness of heart tissue is in the >100 kPa range. In this study, we focus on developing new membranes, with a set of properties for mimicry of cardiac tissue stiffness in vitro, based on methacrylate-functionalized macromers and triblock-copolymers of poly(trimethylene carbonate) and poly(ethylene glycol). The new membranes have Young's moduli in the hydrated state ranging from 18 kPa (healthy tissue) to 2.5 MPa (pathological tissue), and are suitable for cell contraction studies using human pluripotent stem-cell-derived cardiomyocytes. The membranes with higher hydrophilicity have low drug adsorption and low Young's moduli and could be suitable for drug screening applications.

Biomechanical Characterization of Human Pluripotent Stem Cell-Derived Cardiomyocytes by Use of Atomic Force Microscopy.

Atomic force microscopy (AFM) is not only a high-resolution imaging technique but also a sensitive tool able to study biomechanical properties of bio-samples (biomolecules, cells) in native conditions-i.e., in buffered solutions (culturing media) and stable temperature (mostly 37 °C). Micromechanical transducers (cantilevers) are often used to map surface stiffness distribution, adhesion forces, and viscoelastic parameters of living cells; however, they can also be used to monitor time course of cardiomyocytes contraction dynamics (e.g. beating rate, relaxation time), together with other biomechanical properties. Here we describe the construction of an AFM-based biosensor setup designed to study the biomechanical properties of cardiomyocyte clusters, through the use of standard uncoated silicon nitride cantilevers. Force-time curves (mechanocardiograms, MCG) are recorded continuously in real time and in the presence of cardiomyocyte-contraction affecting drugs (e.g., isoproterenol, metoprolol) in the medium, under physiological conditions. The average value of contraction force and the beat rate, as basic biomechanical parameters, represent pharmacological indicators of different phenotype features. Robustness, low computational requirements, and optimal spatial sensitivity (detection limit 200 pN, respectively 20 nm displacement) are the main advantages of the presented method.

Atomic force microscopy combined with human pluripotent stem cell derived cardiomyocytes for biomechanical sensing.

Cardiomyocyte contraction and relaxation are important parameters of cardiac function altered in many heart pathologies. Biosensing of these parameters represents an important tool in drug development and disease modeling. Human embryonic stem cells and especially patient specific induced pluripotent stem cell-derived cardiomyocytes are well established as cardiac disease model.. Here, a live stem cell derived embryoid body (EB) based cardiac cell syncytium served as a biorecognition element coupled to the microcantilever probe from atomic force microscope thus providing reliable micromechanical cellular biosensor suitable for whole-day testing. The biosensor was optimized regarding the type of cantilever, temperature and exchange of media; in combination with standardized protocol, it allowed testing of compounds and conditions affecting the biomechanical properties of EB. The studied effectors included calcium , drugs modulating the catecholaminergic fight-or-flight stress response such as the beta-adrenergic blocker metoprolol and the beta-adrenergic agonist isoproterenol. Arrhythmogenic effects were studied using caffeine. Furthermore, with EBs originating from patient's stem cells, this biosensor can help to characterize heart diseases such as dystrophies.

Functional maturation of human pluripotent stem cell derived cardiomyocytes in vitro--correlation between contraction force and electrophysiology.

Cardiomyocytes from human pluripotent stem cells (hPSC-CM) have many potential applications in disease modelling and drug target discovery but their phenotypic similarity to early fetal stages of cardiac development limits their applicability. In this study we compared contraction stresses of hPSC-CM to 2nd trimester human fetal derived cardiomyocytes (hFetal-CM) by imaging displacement of fluorescent beads by single contracting hPSC-CM, aligned by microcontact-printing on polyacrylamide gels. hPSC-CM showed distinctly lower contraction stress than cardiomyocytes isolated from hFetal-CM. To improve maturation of hPSC-CM in vitro we made use of commercial media optimized for cardiomyocyte maturation, which promoted significantly higher contraction stress in hPSC-compared with hFetal-CM. Accordingly, other features of cardiomyocyte maturation were observed, most strikingly increased upstroke velocities and action potential amplitudes, lower resting membrane potentials, improved sarcomeric organization and alterations in cardiac-specific gene expression. Performing contraction force and electrophysiology measurements on individual cardiomyocytes revealed strong correlations between an increase in contraction force and a rise of the upstroke velocity and action potential amplitude and with a decrease in the resting membrane potential. We showed that under standard differentiation conditions hPSC-CM display lower contractile force than primary hFetal-CM and identified conditions under which a commercially available culture medium could induce molecular, morphological and functional maturation of hPSC-CM in vitro. These results are an important contribution for full implementation of hPSC-CM in cardiac disease modelling and drug discovery.