Robust, Automated Analysis of Electrophysiology in Induced Pluripotent Stem Cell-Derived Micro-Heart Muscle for Drug Toxicity

Oguntuyo, Kasoorelope; Schuftan, David; Guo, Junjun; Simmons, Daniel W.; Bhagavan, Druv; Moreno, Jonathan D.; Kang, Po Wei; Miller, Evan W.; Silva, Jonathan R.; Huebsch, Nathaniel

doi:10.1089/ten.tec.2022.0053

Cited by 7 publications

(9 citation statements)

References 37 publications

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“…Videos were imaged at 200–450 fps. The MATLAB Bio-Formats package was used along with custom MATLAB software 41 to calculate waveform parameters. To calculate conduction velocity, a modified version of the open-source MATLAB software Rhythm 1.0 developed by Laugher et al was used.…”

Section: Methodsmentioning

confidence: 99%

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Engineered tissue geometry and Plakophilin-2 regulate electrophysiology of human iPSC-derived cardiomyocytes

Simmons,

Malayath,

Schuftan

et al. 2024

APL Bioengineering

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Engineered heart tissues have been created to study cardiac biology and disease in a setting that more closely mimics in vivo heart muscle than 2D monolayer culture. Previously published studies suggest that geometrically anisotropic micro-environments are crucial for inducing “in vivo like” physiology from immature cardiomyocytes. We hypothesized that the degree of cardiomyocyte alignment and prestress within engineered tissues is regulated by tissue geometry and, subsequently, drives electrophysiological development. Thus, we studied the effects of tissue geometry on electrophysiology of micro-heart muscle arrays (μHM) engineered from human induced pluripotent stem cells (iPSCs). Elongated tissue geometries elicited cardiomyocyte shape and electrophysiology changes led to adaptations that yielded increased calcium intake during each contraction cycle. Strikingly, pharmacologic studies revealed that a threshold of prestress and/or cellular alignment is required for sodium channel function, whereas L-type calcium and rapidly rectifying potassium channels were largely insensitive to these changes. Concurrently, tissue elongation upregulated sodium channel (NaV1.5) and gap junction (Connexin 43, Cx43) protein expression. Based on these observations, we leveraged elongated μHM to study the impact of loss-of-function mutation in Plakophilin 2 (PKP2), a desmosome protein implicated in arrhythmogenic disease. Within μHM, PKP2 knockout cardiomyocytes had cellular morphology similar to what was observed in isogenic controls. However, PKP2−/− tissues exhibited lower conduction velocity and no functional sodium current. PKP2 knockout μHM exhibited geometrically linked upregulation of sodium channel but not Cx43, suggesting that post-translational mechanisms, including a lack of ion channel-gap junction communication, may underlie the lower conduction velocity observed in tissues harboring this genetic defect. Altogether, these observations demonstrate that simple, scalable micro-tissue systems can provide the physiologic stresses necessary to induce electrical remodeling of iPS-CM to enable studies on the electrophysiologic consequences of disease-associated genomic variants.

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Section: Methodsmentioning

confidence: 99%

“…In our prior study, we observed no substantial difference in beat rate correction by Fridericia's formula vs other formulas used for iPS-CM and clinical ECG. 41 …”

Section: Methodsmentioning

confidence: 99%

Engineered tissue geometry and Plakophilin-2 regulate electrophysiology of human iPSC-derived cardiomyocytes

Simmons,

Malayath,

Schuftan

et al. 2024

APL Bioengineering

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“…Calcium transients were captured by imaging endogenously expressed GcaMP6f in the GFP channel (4). Videos was analyzed via custom build code; intensity profile and kinetics parameters were automatically extracted (84). For calcium transient dynamics, initial baseline to 80% of the intensity was measured as UPD80, plateau region of more than 80% intensity was measured, decay times from end of the plateau to 50 and 75% decay of intensity was characterized.…”

Section: Action Potential and Calcium Measurementsmentioning

confidence: 99%

Mechanical Resistance to Micro-Heart Tissue Contractility unveils early Structural and Functional Pathology in iPSC Models of Hypertrophic Cardiomyopathy

Guo,

Jiang,

Schuftan

et al. 2023

Preprint

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Hypertrophic cardiomyopathy is the most common cause of sudden death in the young. Because the disease exhibits variable penetrance, there are likely nongenetic factors that contribute to the manifestation of the disease phenotype. Clinically, hypertension is a major cause of morbidity and mortality in patients with HCM, suggesting a potential synergistic role for the sarcomeric mutations associated with HCM and mechanical stress on the heart. We developed anin vitrophysiological model to investigate how the afterload that the heart muscle works against during contraction acts together with HCM-linked MYBPC3 mutations to trigger a disease phenotype. Micro-heart muscle arrays (μHM) were engineered from iPSC-derived cardiomyocytes bearing MYBPC3 loss-of-function mutations and challenged to contract against mechanical resistance with substrates stiffnesses ranging from the of embryonic hearts (0.4 kPa) up to the stiffness of fibrotic adult hearts (114 kPa). Whereas MYBPC3+/-iPSC-cardiomyocytes showed little signs of disease pathology in standard 2D culture, μHMs that included components of afterload revealed several hallmarks of HCM, including cellular hypertrophy, impaired contractile energetics, and maladaptive calcium handling. Remarkably, we discovered changes in troponin C and T localization in the MYBPC3+/-μHM that were entirely absent in 2D culture. Pharmacologic studies suggested that excessive Ca2+intake through membrane-embedded channels, rather than sarcoplasmic reticulum Ca2+ATPase (SERCA) dysfunction or Ca2+buffering at myofilaments underlie the observed electrophysiological abnormalities. These results illustrate the power of physiologically relevant engineered tissue models to study inherited disease mechanisms with iPSC technology.

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“…Along with the increasing efforts to fully differentiate CMs from pluripotent stem cells and, hence, derive functional engineered cardiac tissues, research is also focusing on designing and developing devices that can monitor in real-time and thus allow to progressively follow and reconstruct the whole tissue dynamics, also in response to exogenous stimuli. Technical challenges exist in the full thickness analysis but several researchers have developed advanced stimulating/sensing methods and equipment, e.g., microscopy visualization, contractility measurements, mechanical manipulation, and related high-throughput data collection that can easily combine with the engineered tissue models and, therefore, create automated, integrated platforms for robust readout ( Ramade et al, 2014 ; Pointon et al, 2017 ; Sasaki et al, 2018 ; Gracioso Martins et al, 2021 ; Ohya et al, 2021 ; Sasaki et al, 2021 ; Oguntuyo et al, 2022 ). So far, a relatively short time of a few hours or days has been dedicated to observing drug-induced alterations.…”

Section: Unmet Needs and Issues To Be Addressed For Clinical Translationmentioning

confidence: 99%

Advances in the design, generation, and application of tissue-engineered myocardial equivalents

Bernava,

Iop

2023

Front. Bioeng. Biotechnol.

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Due to the limited regenerative ability of cardiomyocytes, the disabling irreversible condition of myocardial failure can only be treated with conservative and temporary therapeutic approaches, not able to repair the damage directly, or with organ transplantation. Among the regenerative strategies, intramyocardial cell injection or intravascular cell infusion should attenuate damage to the myocardium and reduce the risk of heart failure. However, these cell delivery-based therapies suffer from significant drawbacks and have a low success rate. Indeed, cardiac tissue engineering efforts are directed to repair, replace, and regenerate native myocardial tissue function. In a regenerative strategy, biomaterials and biomimetic stimuli play a key role in promoting cell adhesion, proliferation, differentiation, and neo-tissue formation. Thus, appropriate biochemical and biophysical cues should be combined with scaffolds emulating extracellular matrix in order to support cell growth and prompt favorable cardiac microenvironment and tissue regeneration. In this review, we provide an overview of recent developments that occurred in the biomimetic design and fabrication of cardiac scaffolds and patches. Furthermore, we sift in vitro and in situ strategies in several preclinical and clinical applications. Finally, we evaluate the possible use of bioengineered cardiac tissue equivalents as in vitro models for disease studies and drug tests.

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Robust, Automated Analysis of Electrophysiology in Induced Pluripotent Stem Cell-Derived Micro-Heart Muscle for Drug Toxicity

Cited by 7 publications

References 37 publications

Engineered tissue geometry and Plakophilin-2 regulate electrophysiology of human iPSC-derived cardiomyocytes

Engineered tissue geometry and Plakophilin-2 regulate electrophysiology of human iPSC-derived cardiomyocytes

Mechanical Resistance to Micro-Heart Tissue Contractility unveils early Structural and Functional Pathology in iPSC Models of Hypertrophic Cardiomyopathy

Advances in the design, generation, and application of tissue-engineered myocardial equivalents

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