Slicing and Culturing Pig Hearts under Physiological Conditions

Ou, Qinghui; Abouleisa, Riham; Tang, Xian‐Liang; Juhardeen, Hamzah R; Meki, Moustafa; Miller, Jessica M.; Giridharan, Guruprasad A.; El‐Baz, Ayman; Bolli, Roberto; Mohamed, Tamer

doi:10.3791/60913

Cited by 13 publications

(15 citation statements)

References 20 publications

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“…Then, we further validated the e ciency of this reporter system in detecting mitotic events induced by 4F in hiPS-CMs (Extended Data Fig. 10c-d) and pig heart tissue in situ using our recently developed heart slices culture system 23,24 (Extended Data Fig. 10e-f).…”

Section: Double Reporter To Permanently Label Cardiomyocytes Activatimentioning

confidence: 88%

Transient Cell Cycle Induction in Cardiomyocytes to Treat Ischemic Heart Failure

Abouleisa¹,

Ou²,

Tang³

et al. 2020

Preprint

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The regenerative capacity of the heart after myocardial infarction (MI) is limited. Our previous study showed that ectopic introduction of Cdk1/CyclinB1 and Cdk4/CyclinD1 complexes (4F) promotes cardiomyocyte proliferation in 15-20% of infected cardiomyocytes in vitro and in vivo and improves cardiac function after MI. Here, we aim to identify the necessary reprogramming stages during the forced cardiomyocyte proliferation with 4F on a single cell basis. Also, we aim to start the first preclinical testing to introduce 4F gene therapy as a candidate for the treatment of ischemia-induced heart failure. Temporal bulk and single-cell RNAseq and further biochemical validations of mature hiPS-CMs treated with either LacZ or 4F adenoviruses revealed full cell cycle reprogramming in 15% of the cardiomyocyte population after 48 h post-infection with 4F, which was associated with sarcomere disassembly and metabolic reprogramming. Transient overexpression of 4F, specifically in cardiomyocytes, was achieved using a polycistronic non-integrating lentivirus (NIL) encoding the 4F; each is driven by a TNNT2 promoter (TNNT2-4F-NIL). TNNT2-4F-NIL or control virus was injected intramyocardially one week after MI in rats or pigs. TNNT2-4F-NIL treated animals showed significant improvement in left ventricular ejection fraction and scar size compared with the control virus treated animals four weeks post-injection. In conclusion, the present study provides a mechanistic demonstration of the process of forced cardiomyocyte proliferation and advances the clinical feasibility of this approach by minimizing the oncogenic potential of the cell cycle factors using a novel transient and cardiomyocyte-specific viral construct.

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Section: Double Reporter To Permanently Label Cardiomyocytes Activatimentioning

confidence: 88%

Transient Cell Cycle Induction in Cardiomyocytes to Treat Ischemic Heart Failure

Abouleisa¹,

Ou²,

Tang³

et al. 2020

Preprint

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“…hiPSC-CMs were exposed to a range of Harvesting porcine heart tissue: All animal procedures were in accordance with the institutional guidelines and approved by the University of Louisville Institutional Animal Care and Use Committee. The protocol for harvesting pig hearts has been described in detail 27,34 . Briefly, following deeply anesthetizing the pig with 5% isoflurane, pig heart was quickly excised out and the heart was clamped at the aortic arch and perfused with 1L sterile cardioplegia solution (110 mM NaCl, 1.2 mM CaCl2, 16 mM KCl, 16 mM MgCl2, 10 mM NaHCO3, 5 units/mL heparin, pH to 7.4), then the heart was preserved on an ice-cold cardioplegic solution and immediately transported to the lab on wet ice.…”

Section: Methodsmentioning

confidence: 99%

“…Heart slicing and culturing: Slicing and culturing 300 µm thick heart tissue slices were performed as previously described in 27,34 . A refined oxygenated growth medium was used (Medium 199, 1x ITS supplement, 10% FBS, 5 ng/mL VEGF, 10 ng/mL FGF-basic, and 2x…”

Section: Methodsmentioning

confidence: 99%

Heart Slice Culture System Reliably Demonstrates Clinical Drug-Related Cardiotoxicity

Miller

Meki

et al. 2020

Preprint

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AbstractThe limited availability of human heart tissue and its complex cell composition are major limiting factors for reliable testing drug efficacy, toxicity and understanding mechanism. Recently, we developed a functional human and pig heart slice biomimetic culture system that fully preserves the viability and functionality of 300 µm heart slices for 6 days. Here, we tested the reliability of this culture system in delineating the mechanisms of known anti-cancer drugs that cause cardiomyopathy. We tested three anti-cancer drugs (doxorubicin, trastuzumab, and sunitinib) associated with different mechanisms leading to cardiotoxicity at three concentrations and assessed the effect of these drugs on heart slice viability, structure, function and transcriptome. Slices incubated with any of these drugs for 48 h showed significant loss in viability, cardiomyocyte structure and functionality. Mechanistically, RNA sequencing demonstrated a significant downregulation of cardiac genes and upregulation of oxidative response in doxorubicin-treated tissues. Trastuzumab treatment caused major downregulation in cardiac muscle contraction-related genes, consistent with its clinically known direct effect on cardiomyocytes. Interestingly, sunitinib treatment resulted in significant downregulation of angiogenesis-related genes in line with its mechanism of action. Heart slices are not only able to demonstrate the expected toxicity of doxorubicin and trastuzumab similar to hiPS-derived-cardiomyocytes; they are superior in detecting sunitinib cardiotoxicity phenotypes and mechanism in the clinically relevant concentration range, 100 nM – 1 µM. These results indicate that heart slice tissue culture models have the potential to become a reliable platform for testing drug toxicity and mechanism of action.

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“…Since their inception, heart slices have been prepared from several animals as well as human hearts. Detailed protocols for preparing heart slices have been described by our group and others ( Brandenburger et al, 2012 ; Watson et al, 2017 ; Watson et al, 2019 ; Ou et al, 2020 ). While multiple factors control the maturation and regulation of the adult cardiac phenotype, the structural fidelity of heart slices provides cell-cell interactions, transmembrane regulation and maintains standard tissue stiffness.…”

Section: Animal/human-derived Heart Slices To Model Cardiac Physiologmentioning

confidence: 99%

“…Our group has shown that through optimized proprietary culture media, frequent oxygenation, and electrical stimulation, tissue slices can preserve viability, functionality, and cardiomyocyte structure for up to 6 days ( Ou et al, 2019 ; Ou et al, 2020 ). The 300 µm human and pig heart slices were cultured at a physiological rate (1.2 Hz, 72 beats/min) and showed normal viability, gene expression profile, calcium homeostasis, contractility, twitch force generation, and response to ß-adrenergic stimulation over the first six days.…”

Section: Optimization Of Nutrients and Electrical Stimulationmentioning

confidence: 99%

Heart Slices to Model Cardiac Physiology

2021

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Translational research in the cardiovascular field is hampered by the unavailability of cardiac models that can recapitulate organ-level physiology of the myocardium. Outside the body, cardiac tissue undergoes rapid dedifferentiation and maladaptation in culture. There is an ever-growing demand for preclinical platforms that allow for accurate, standardized, long-term, and rapid drug testing. Heart slices is an emerging technology that solves many of the problems with conventional myocardial culture systems. Heart slices are thin (<400 µm) slices of heart tissue from the adult ventricle. Several recent studies using heart slices have shown their ability to maintain the adult phenotype for prolonged periods in a multi cell-type environment. Here, we review the current status of cardiac culture systems and highlight the unique advantages offered by heart slices in the light of recent efforts in developing physiologically relevant heart slice culture systems.

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Slicing and Culturing Pig Hearts under Physiological Conditions

Cited by 13 publications

References 20 publications

Transient Cell Cycle Induction in Cardiomyocytes to Treat Ischemic Heart Failure

Transient Cell Cycle Induction in Cardiomyocytes to Treat Ischemic Heart Failure

Heart Slice Culture System Reliably Demonstrates Clinical Drug-Related Cardiotoxicity

Heart Slices to Model Cardiac Physiology

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