Biology subjects

Burridge, P. W.

Publications and source records attributed to Burridge, P. W..

6 recordsLinked to original sources

bioRxiv2024

Human induced pluripotent stem cell-derived cardiomyocytes to study inflammation-induced diastolic dysfunction

Heart failure with preserved ejection fraction (HFpEF) is commonly found in persons living with HIV (PLWH) even when antiretroviral therapy (ART) suppresses HIV viremia. However, studying this condition has been challenging because an appropriate animal model is not available. In this paper, we studied calcium transient in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in culture to simulate the cardiomyocyte relaxation defect noted in of PLWH and HFpEF and to assess whether various drugs have an effect. We show that treatment of hiPSC-CMs with inflammatory cytokines (such as interferon-{gamma} or TNF-) impair their Ca2+ uptake into sarcoplasmic reticulum and that SGLT2 inhibitors, clinically proven as effective for HFpEF, reverse this effect. Additionally, treatment with mitochondrial antioxidants (like mito-Tempo) and certain antiretrovirals resulted in the reversal of the effects of these cytokines on calcium transient. Finally, incubation of hiPSC-CMs with serum from HIV patients with and without diastolic dysfunction did not alter their Ca2+-decay time, indicating that the exposure to the serum of these patients is not sufficient to induce the decrease in Ca2+ uptake in vitro. Together, our results indicate that hiPSC-CMs can be used as a model to study molecular mechanisms of inflammation-mediated abnormal cardiomyocyte relaxation and screen for potential new interventions.

molecular biology↗

bioRxiv2023

A Novel Transcription Factor Combination for Direct Reprogramming to a Spontaneously Contracting Human Cardiomyocyte-like State

The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4,960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/532629v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@17d6539org.highwire.dtl.DTLVardef@180a0b8org.highwire.dtl.DTLVardef@1b7cb46org.highwire.dtl.DTLVardef@182618b_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIUsing network-based algorithm Mogrify, acoustic liquid handling, and high-content kinetic imaging cytometry we screened the effect of 4,960 unique transcription factor combinations. C_LIO_LIUsing 24 patient-specific human fibroblast samples we identified the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination. C_LIO_LIMST cocktail results in reprogrammed cells with spontaneous contraction, cardiomyocyte-like calcium transients, and expression of cardiomyocyte associated genes. C_LI

cell biology↗

bioRxiv2022

Recombinant annexin A6 promotes membrane repair in a stem cell derived-cardiomyocyte model of dystrophic cardiomyopathy

Heart failure is a major source of mortality in Duchenne muscular dystrophy (DMD). DMD arises from mutations that ablate expression of the protein dystrophin, which render the plasma membrane unusually fragile and prone to disruption. In DMD patients, repeated mechanical stress leads to membrane damage and cardiomyocyte loss. Induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) offer the opportunity to study specific mutations in the context of a human cell, but these models can be improved by adding physiologic stressors. We modeled the primary defect underlying DMD by applying equibiaxial mechanical strain to DMD iPSC-CMs. DMD iPSC-CMs demonstrated an increased susceptibility to equibiaxial strain after 2 hours at 10% strain relative to healthy control cells, measured as increased lactate dehydrogenase (LDH) release. After 24 hours, both DMD and healthy control iPSC-CMs showed evidence of injury with release of LDH and cardiac troponin T. We exposed iPSC-CMs to recombinant annexin A6, a protein resealing agent, and found reduced LDH and troponin release in DMD and control iPSC-CMs that had been subjected to 24 hour strain at 10%. We used aptamer protein profiling of media collected from DMD and control iPSC-CMs and compared these results to serum protein profiling from DMD patients. We found a strong correlation between the proteins in DMD patient serum and media from DMD iPSC-CMs subjected to mechanical stress. By developing an injury assay that specifically targets an underlying mechanism of injury seen in DMD-related cardiomyopathy, we demonstrated the potential therapeutic efficacy of the protein membrane resealer, recombinant annexin A6, for the treatment of DMD-related cardiomyopathy and general cardiac injury.

bioengineering↗

bioRxiv2020

GS-967 and Eleclazine Block Sodium Channels in Human Induced Pluripotent Stem Cell-derived Cardiomyocytes

GS-967 and eleclazine (GS-6615) are novel sodium channel inhibitors exhibiting antiarrhythmic effects in various in vitro and in vivo models. The antiarrhythmic mechanism has been attributed to preferential suppression of late sodium current (INaL). Here, we took advantage of a throughput automated electrophysiology platform (SyncroPatch 768PE) to investigate the molecular pharmacology of GS-967 and eleclazine on peak sodium current (INaP) recorded from human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes. We compared GS-967 and eleclazine to the antiarrhythmic drug lidocaine, the prototype INaL inhibitor ranolazine, and the slow inactivation enhancing drug lacosamide. In human induced pluripotent stem cell-derived cardiomyocytes, GS-967 and eleclazine caused a reduction of INaP in a frequency-dependent manner consistent with use-dependent block (UDB). GS-967 and eleclazine had similar efficacy but evoked more potent UDB of INaP (IC50=0.07 and 0.6 M, respectively) than ranolazine (7.8 M), lidocaine (133.5 M) and lacosamide (158.5 M). In addition, GS-967 and eleclazine exerted more potent effects on slow inactivation and recovery from inactivation compared to the other sodium channel blocking drugs we tested. The greater UDB potency of GS-967 and eleclazine was attributed to the significantly higher association rates (KON) and moderate unbinding rate (KOFF) of these two compounds with sodium channels. We propose that substantial UDB contributes to the observed antiarrhythmic efficacy of GS-967 and eleclazine. SIGNIFICANCE STATEMENTWe investigated the molecular pharmacology of GS-967 and eleclazine on sodium channels in human induced pluripotent stem cell derived cardiomyocytes using a high throughput automated electrophysiology platform. Sodium channel inhibition by GS-967 and eleclazine has unique features including accelerating the onset of slow inactivation and impairing recovery from inactivation. These effects combined with rapid binding and moderate unbinding kinetics explain potent use-dependent block, which we propose contributes to their observed antiarrhythmic efficacy.

pharmacology and toxicology↗

bioRxiv2019

Dissociation of disease phenotype and allele silencing in hypertrophic cardiomyopathy

Allele-specific RNA silencing has been shown to be an effective therapeutic treatment in a number of diseases, including neurodegenerative disorders. Studies of allele-specific silencing in hypertrophic cardiomyopathy to date have focused on mouse models of disease. Here, we investigate two methods of allele-specific silencing, short hairpin RNA (shRNA) and antisense oligonucleotide (ASO) silencing, using a human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) model of disease. We used cellular micropatterning devices with traction force microscopy and automated video analysis to examine each strategys effects on contractile defects underlying disease. We find that shRNA silencing ameliorates contractile phenotypes of disease, reducing disease-associated increases in cardiomyocyte velocity, force, and power. We find that ASO silencing, while better able to target and knockdown a specific disease-associated allele, showed more modest improvements in contractile phenotypes. We find a dissociation between allelic-specificity and functional improvements between the two tested therapeutic strategies, suggesting a more complex method of allelic control underlying HCM-associated transcripts.\n\nAuthor summaryAllele-specific silencing, whereby a therapeutic molecule is used to lower the expression of just one of the two copies or alleles of a gene, may be a potential therapeutic strategy in diseases caused by a single mutation. In this paper, we examine two such strategies in hypertrophic cardiomyopathy, a disease characterized by an overgrowth of the left-ventricular heart muscle as well as contractile dysfunction. We used a human cell model of disease, creating induced pluripotent stem cell derived cardiomyocytes from a patient with HCM caused by a single base pair change in just one allele of the gene MYH7. We used two strategies to silence the disease-associated copy of MYH7, both focused on reducing RNA expression from the mutated allele, as well as state-of-the-art biophysical techniques for measuring contractility. We found that one silencing strategy, which reduced expression of both the disease-associated and the healthy alleles of MYH7, showed great improvements in contractility between treated and untreated cells. Our second strategy, which silenced only the disease-associated copy of MYH7, showed more modest improvements in contractility. This suggests that the disease mechanism underlying this type of hypertrophic cardiomyopathy may be more complex than just presence or absence of the mutated RNA.

genetics↗

bioRxiv2018

Are these cardiomyocytes? Protocol development reveals impact of sample preparation on the accuracy of identifying cardiomyocytes by flow cytometry

Modern differentiation protocols enable efficient, yet imperfect, differentiation of human pluripotent stem cells into cardiomyocytes (hPSC-CM). As the number of laboratories and studies implementing this technology expands, the accurate assessment of cell identity in differentiation cultures is paramount to well-defined studies that can be replicated among laboratories. While flow cytometry is apt for routine assessment, a standardized protocol for assessing cardiomyocyte identity in hPSC-CM cultures has not yet been established. To address this gap, the current study leveraged targeted mass spectrometry to confirm the presence of troponin proteins in hPSC-CM and systematically evaluated multiple anti-troponin antibodies and sample preparation protocols for their suitability in assessing cardiomyocyte identity. Results demonstrate challenges of interpreting data generated by published methods and informed the development of a robust protocol for routine assessment of hPSC-CM. Overall, the new data, workflow for evaluating fit-for-purpose use of antibodies, and standardized protocol described here should benefit investigators new to this field as well as those with expertise in hPSC-CM differentiation.

developmental biology↗