Local Anesthetic Cardiac Toxicity Is Mediated by Cardiomyocyte Calcium Dynamics.

BACKGROUND: Long-lasting local anesthetic use for perioperative pain control is limited by possible cardiotoxicity (e.g., arrhythmias and contractile depression), potentially leading to cardiac arrest. Off-target cardiac sodium channel blockade is considered the canonical mechanism behind cardiotoxicity; however, it does not fully explain the observed toxicity variability between anesthetics. The authors hypothesize that more cardiotoxic anesthetics (e.g., bupivacaine) differentially perturb other important cardiomyocyte functions (e.g., calcium dynamics), which may be exploited to mitigate drug toxicity. METHODS: The authors investigated the effects of clinically relevant concentrations of racemic bupivacaine, levobupivacaine, or ropivacaine on human stem cell-derived cardiomyocyte tissue function. Contractility, rhythm, electromechanical coupling, field potential profile, and intracellular calcium dynamics were quantified using multielectrode arrays and optical imaging. Calcium flux differences between bupivacaine and ropivacaine were probed with pharmacologic calcium supplementation or blockade. In vitro findings were correlated in vivo using an anesthetic cardiotoxicity rat model (females; n = 5 per group). RESULTS: Bupivacaine more severely dysregulated calcium dynamics than ropivacaine in vitro (e.g., contraction calcium amplitude to 52 ± 11% and calcium-mediated repolarization duration to 122 ± 7% of ropivacaine effects, model estimate ± standard error). Calcium supplementation improved tissue contractility and restored normal beating rhythm (to 101 ± 6%, and 101 ± 26% of control, respectively) for bupivacaine-treated tissues, but not ropivacaine (e.g., contractility at 80 ± 6% of control). Similarly, calcium pretreatment mitigated anesthetic-induced arrhythmias and cardiac depression in rats, improving animal survival for bupivacaine by 8.3 ± 2.4 min, but exacerbating ropivacaine adverse effects (reduced survival by 13.8 ± 3.4 min and time to first arrhythmia by 12.0 ± 2.9 min). Calcium channel blocker nifedipine coadministration with bupivacaine, but not ropivacaine, exacerbated cardiotoxicity, supporting the role of calcium flux in differentiating toxicity. CONCLUSIONS: Our data illustrate differences in calcium dynamics between anesthetics and how calcium may mitigate bupivacaine cardiotoxicity. Moreover, our findings suggest that bupivacaine cardiotoxicity risk may be higher than for ropivacaine in a calcium deficiency context.

A Carbon-Based Biosensing Platform for Simultaneously Measuring the Contraction and Electrophysiology of iPSC-Cardiomyocyte Monolayers.

Heart beating is triggered by the generation and propagation of action potentials through the myocardium, resulting in the synchronous contraction of cardiomyocytes. This process highlights the importance of electrical and mechanical coordination in organ function. Investigating the pathogenesis of heart diseases and potential therapeutic actions in vitro requires biosensing technologies which allow for long-term and simultaneous measurement of the contractility and electrophysiology of cardiomyocytes. However, the adoption of current biosensing approaches for functional measurement of in vitro cardiac models is hampered by low sensitivity, difficulties in achieving multifunctional detection, and costly manufacturing processes. Leveraging carbon-based nanomaterials, we developed a biosensing platform that is capable of performing on-chip and simultaneous measurement of contractility and electrophysiology of human induced pluripotent stem-cell-derived cardiomyocyte (iPSC-CM) monolayers. This platform integrates with a flexible thin-film cantilever embedded with a carbon black (CB)-PDMS strain sensor for high-sensitivity contraction measurement and four pure carbon nanotube (CNT) electrodes for the detection of extracellular field potentials with low electrode impedance. Cardiac functional properties including contractile stress, beating rate, beating rhythm, and extracellular field potential were evaluated to quantify iPSC-CM responses to common cardiotropic agents. In addition, an in vitro model of drug-induced cardiac arrhythmia was established to further validate the platform for disease modeling and drug testing.

Correction: Cardiac regenerative capacity is age- and disease-dependent in childhood heart disease.

[This corrects the article DOI: 10.1371/journal.pone.0200342.].

A microdevice platform for characterizing the effect of mechanical strain magnitudes on the maturation of iPSC-Cardiomyocytes.

The use of human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) as an in vitro model of the heart is limited by their structurally and functionally immature phenotypes. During heart development, mechanical stimuli from in vivo microenvironments are known to regulate cardiomyocyte gene expression and maturation. Accordingly, protocols for culturing iPSC-CMs have recently incorporated mechanical or electromechanical stimulation to induce cellular maturation in vitro; however, the response of iPSC-CMs to different mechanical strain magnitudes is unknown, and existing techniques lack the capability to dynamically measure changes to iPSC-CM contractility in situ as maturation progresses. We developed a microdevice platform which applies cyclical strains of varying magnitudes (5%, 10%, 15% and 20%) to a monolayer of iPSC-CMs, coincidentally measuring contractile stress during mechanical stimulation using fluorescent nanobeads embedded in the microdevice's suspended membrane. Cyclic strain was found to induce circumferential cell alignment on the actuated membranes. In situ contractility measurements revealed that cyclic stimulation gradually increased cardiomyocyte contractility during a 10-day culture period. The contractile stress of iPSC-CM monolayers was found to increase with a higher strain magnitude and plateaued at 15% strain. Cardiomyocyte contractility positively correlated with the elongation of sarcomeres and an increased expression of ß-myosin heavy chain (MYH7) in a strain magnitude-dependent manner, illustrating how mechanical stress can be optimized for the phenotypic and proteomic maturation of the cells. iPSC-CMs with improved maturity have the potential to create a more accurate heart model in vitro for applications in disease modeling and therapeutic discovery.

Cardiac regenerative capacity is age- and disease-dependent in childhood heart disease.

OBJECTIVE: We sought to define the intrinsic stem cell capacity in pediatric heart lesions, and the effects of diagnosis and of age, in order to inform evidence-based use of potential autologous stem cell sources for regenerative medicine therapy. METHODS: Ventricular explants derived from patients with hypoplastic left heart syndrome (HLHS), tetralogy of Fallot (TF), dilated cardiomyopathy (DCM) and ventricular septal defect (VSD) were analyzed following standard in vitro culture conditions, which yielded cardiospheres (C-spheres), indicative of endogenous stem cell capacity. C-sphere counts generated per 5 mm3 tissue explant and the presence of cardiac progenitor cells were correlated to patient age, diagnosis and echocardiographic function. RESULTS: Cardiac explants from patients less than one year of age with TF and DCM robustly generated c-kit- and/or vimentin-positive cardiac mesenchymal cells (CMCs), populating spontaneously forming C-spheres. Beyond one year of age, there was a marked reduction or absence of cardiac explant-derivable cardiac stem cell content in patients with TF, VSD and DCM. Stem cell content in HLHS and DCM strongly correlated to the echocardiographic function in the corresponding ventricular chamber, with better echocardiographic function correlating to a more robust regenerative cellular content. CONCLUSIONS: We conclude that autologous cardiomyogenic potential in pediatric heart lesions is robust during the first year of life and uniformly declines thereafter. Depletion of stem cell content occurs at an earlier age in HLHS with the onset of ventricular failure in a chamber-specific pattern that correlates directly to ventricular dysfunction. These data suggest that regenerative therapies using autologous cellular sources should be implemented in the neonatal period before the potentially rapid onset of single ventricle failure in HLHS or the evolution of biventricular failure in DCM.

Microdevice Platform for Continuous Measurement of Contractility, Beating Rate, and Beating Rhythm of Human-Induced Pluripotent Stem Cell-Cardiomyocytes inside a Controlled Incubator Environment.

The heart completes a complex set of tasks, including the initiation or propagation of an electrical signal with regularity (proper heart rate and rhythm) and generating sufficient force of contraction (contractility). Probing mechanisms of heart diseases and quantifying drug efficacies demand a platform that is capable of continuous operation inside a cell incubator for long-term measurement of cardiomyocyte (CM) monolayers. Here, we report a microdevice array that is capable of performing continuous, long-term (14 days) measurement of contractility, beating rate, and beating rhythm in a monolayer of human-induced pluripotent stem cell-CMs (hiPSC-CMs). The device consists of a deformable membrane with embedded carbon nanotube (CNT)-based strain sensors. Contraction of the hiPSC-CMs seeded on the membrane induces electrical resistance change of the CNT strain sensor. Continuously reading the sensor signals revealed that hiPSC-CMs started to beat from day 2 and plateaued on day 5. Average contractile stress generated by a monolayer of hiPSC-CMs was determined to be 2.34 ± 0.041 kPa with a beating rate of 1.17 ± 0.068 Hz. The device arrays were also used to perform comprehensive measurement of the beating rate, rhythm, and contractility of the hiPSC-CMs and quantify the cell responses to different concentrations of agonists and antagonists, which altered the average contractile stress to the range of 1.15 ± 0.13 to 3.96 ± 0.53 kPa. The continuous measurement capability of the device arrays also enabled the generation of Poincaré plots for revealing subtle changes in the beating rhythm of hiPSC-CMs under different drug treatments.