Human Heart Slices Research Articles

FgF23 and soluble klotho effects on the myocardium: an ex vivo study

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): German Center for Cardiovascular research Background Fibroblast Growth Factor 23 (FGF23) and soluble Klotho (sKL) play a critical role in mineral homeostasis in the body by acting on the kidney to regulate phosphate levels. Circulating FGF23 levels rise in patients with kidney disease and dysregulation of the FGF23 –sKL- axis is a common phenomenon in uremic cardiomyopathy. Studies have shown that elevated FGF23 levels induce cardiac hypertrophy by targeting cardiac myocytes via FGF receptor isoform 4 (FGFR4), while sKL mitigates these effects and may also exert an independent protective effect on the heart(1,2). Purpose We aimed to study the effects of FGF23 and sKL on myocardial contractility, calcium handling and molecular signaling pathways in the human heart using an ex vivo model of cultured contracting myocardial slices. Methods We obtained human heart slices from explanted hearts from patients undergoing cardiac transplantation at the LMU Klinikum Munich, Germany. The 300 µm thick slices were cultured under continuous stimulation (1 Hz, 3 ms pulse width) with 1 mN preload, and contractile function were recorded continuously. Slices with vehicle, recombinant FGF23 protein (100ug/ml), recombinant sKL protein (200ug/ml) and both FGF23((100ug/ml)+sKL (200ug/ml) were continuously monitored over 10 days. We checked for mRNA expression using RT PCR. Results Treatment of human left ventricular tissue with recombinant FGF23 protein resulted in enhanced mRNA expression of the FGFR4. sKL treatment alone induced downregulation of the FGFR4 gene and mitigated upregulation of FGFR4 in FGF23+ sKL treated slices. Post pause potentiation (PPP), assessed by pausing the stimulation of the slices for 120s and then measuring the relative height of the first beat after resumption of pacing, is an indirect measure of sarcoplasmic reticulum Ca2+ handling capacity. PPP was reduced in FGF23 treated slices and heightened in the slices with sKL compared to vehicle treated ones, indicating that FGF23 and sKL play a role in intracellular Ca2+ handling. Conclusions sKL and FGF23 have opposite effects on human cardiac muscle with sKL mitigating FGF23-induced upregulation of FGFR4. Furthermore, sKL improves, while FGF23 impairs myocardial calcium handling . As altered calcium handling may also be involved in hypertrophic signaling altered calcium handling may provide a means by which FGF23 promotes and sKL inhibits myocardial hypertrophy. sKL is advocated as a promising candidate in exploring it therapeutic role in FGF23 mediated cardiomyopathy. We intend to advance our understanding of sKL-FGF23signaling mechanisms by combining transcriptomic analysis with functional data in this model of myocardial slice culture.

Abstract P1076: Rrad Regulates Cardiomyocyte Cell Cycle Entry By Modulating Calcineurin Activity

Background: Cardiomyocytes (CMs) lost during ischemic cardiac injury cannot be replaced due to their limited proliferative capacity, which leads to progressive heart failure. We and others have shown that reduced CM contractility can promote cell cycle induction; however, the underlying mechanisms are still unclear. Calcium (Ca 2+ ), a key regulator of cardiac contractility, is also an important signal transducer that regulates key cellular processes. Ca 2+ enters CMs through the LTCC, which triggers Ca 2+ release from the sarcoplasmic reticulum to initiate CM contractility. The aim of this study is to understand the role of Ca 2+ signaling during CM cell cycle induction. Methods and Results: We performed a drug screening targeting proteins involved in CM calcium cycling in human iPS-derived cardiac organoids. Changes in Ki-67 expression were assessed as an S-G2/M phase marker. The top hit was Nifedipine, an inhibitor of LTCC. To inhibit the LTCC activity, we overexpressed Ras-Related Associated with Diabetes (RRAD), an endogenous regulator of LTCC activity. RRAD overexpression significantly reduced the intracellular calcium amplitude. RRAD overexpression promoted cell cycle induction, increased CM number, and upregulated cell cycle genes in primary neonatal, adult pig, human heart slices, and human iPS-derived cardiac organoids. Moreover, RRAD overexpression significantly augmented the CM response to other cell cycle stimulators such as Cyclin A2, a combination of CDK4 and cyclin D (2F), or a combination of CDK1, CDK4, cyclin D, and cyclin B (4F). Mechanistically, we found that RRAD overexpression reduced the activity of Ca 2+ dependent serine/threonine phosphatase (Calcineurin), as indicated by a significant reduction in calcineurin phosphatase activity in vitro and the expression levels of the calcineurin downstream effectors such as Rcan1 expression as well as translocation of NFAT and Hoxb13 to the CM nucleus. Conclusion: Disruption of Ca 2+ handling by RRAD overexpression promotes cell cycle induction in CMs through modulation of calcineurin activity. Further work in vivo is needed to test whether RRAD overexpression could improve cardiac function after ischemic injury.

Nerve growth factor transfer from cardiomyocytes to innervating sympathetic neurons activates TrkA receptors at the neuro-cardiac junction.

Sympathetic neurons densely innervate the myocardium with non‐random topology and establish structured contacts (i.e. neuro‐cardiac junctions, NCJ) with cardiomyocytes, allowing synaptic intercellular communication. Establishment of heart innervation is regulated by molecular mediators released by myocardial cells. The mechanisms underlying maintenance of cardiac innervation in the fully developed heart, are, however, less clear. Notably, several cardiac diseases, primarily affecting cardiomyocytes, are associated with sympathetic denervation, supporting the hypothesis that retrograde ‘cardiomyocyte‐to‐sympathetic neuron’ communication is essential for heart cellular homeostasis. We aimed to determine whether cardiomyocytes provide nerve growth factor (NGF) to sympathetic neurons, and the role of the NCJ in supporting such retrograde neurotrophic signalling. Immunofluorescence on murine and human heart slices shows that NGF and its receptor, tropomyosin‐receptor‐kinase‐A, accumulate, respectively, in the pre‐ and post‐junctional sides of the NCJ. Confocal immunofluorescence, scanning ion conductance microscopy and molecular analyses, in co‐cultures, demonstrate that cardiomyocytes feed NGF to sympathetic neurons, and that this mechanism requires a stable intercellular contact at the NCJ. Consistently, cardiac fibroblasts, devoid of NCJ, are unable to sustain SN viability. ELISA assay and competition binding experiments suggest that this depends on the NCJ being an insulated microenvironment, characterized by high [NGF]. In further support, real‐time imaging of tropomyosin‐receptor‐kinase‐A vesicle movements demonstrate that efficiency of neurotrophic signalling parallels the maturation of such structured intercellular contacts. Altogether, our results demonstrate the mechanisms which link sympathetic neuron survival to neurotrophin release by directly innervated cardiomyocytes, conceptualizing sympathetic neurons as cardiomyocyte‐driven heart drivers.Key points CMs are the cell source of nerve growth factor (NGF), required to sustain innervating cardiac SNs;NCJ is the place of the intimate liaison, between SNs and CMs, allowing on the one hand neurons to peremptorily control CM activity, and on the other, CMs to adequately sustain the contacting, ever‐changing, neuronal actuators;alterations in NCJ integrity may compromise the efficiency of ‘CM‐to‐SN’ signalling, thus representing a potentially novel mechanism of sympathetic denervation in cardiac diseases.

Abstract 11204: Emulating the Cardiac Mechanical and Humoral Cues to Prolong Human Heart Slice Culture

Introduction: Drug induced cardiotoxicity is a major cause of market withdrawal. The limited availability of human heart tissue has led to inaccurate interpretations of cardiac related drug effects. Our group had developed a culture system for pig/human heart slices that is functionally and structurally viable 6 days under electric stimulation and enriched media and could reliably demonstrate the subacute toxic effect of anti-cancer therapeutics. However, by day 10, the slices underwent cardiomyocyte dedifferentiation and fibrotic remodeling, making them inadequate for long-term cardiotoxicity testing. Aims: Including physiological mechanical and humoral cues within the culture system can prolong the viability of the human heart tissue in culture. Methods and Results: Following small molecule screening, we found that incorporation of 100 nM Tri-iodo-thyronine (T3) and 1 μM Dexamethasone (Dex) into our culture media preserved microscopic structure for 12 days in pig and human slices. However, RNA sequencing revealed a significant down regulation in cardiac contractile genes as early as day 8 in culture. To address this, we developed a novel bioengineered Cardiac Tissue Culture Model (CTCM) that can electro-mechanically stimulate heart slices with physiological preload and afterload during each cardiac cycle (Figure 1). Transcriptionally, combining CTCM with T3/Dex treatment, CTCM system was able to maintain the cardiac contractile gene expression at the same levels as the fresh hear tissue. Functionally, heart slices cultured in our CTCM with T3/Dex treatment, completely preserved structural viability and metabolic activity for 12 days. Conclusion: This study demonstrates a new physiological culture system for human heart slices that provides physiological mechanical and humoral cues for maintaining viability and functionality of the heart slices. CTCM may be an ideal platform for chronic cardiotoxicity testing.

SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes

AimsCoronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as a global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality. It is unclear whether cardiac injury is caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here, we investigate whether cardiomyocytes are permissive for SARS-CoV-2 infection.Methods and resultsTwo strains of SARS-CoV-2 infected human induced pluripotent stem cell-derived cardiomyocytes (iPS-CMs) as demonstrated by detection of intracellular double-stranded viral RNA and viral spike glycoprotein expression. Increasing concentrations of viral RNA are detected in supernatants of infected cardiomyocytes, which induced infections in Caco-2 cell lines, documenting productive infections. SARS-COV-2 infection and induced cytotoxic and proapoptotic effects associated with it abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signalling, apoptosis, and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a 3D cardiosphere tissue model. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. Coronavirus particles were further observed in cardiomyocytes of a patient with COVID-19. Infection of iPS-CMs was dependent on cathepsins and angiotensin-converting enzyme 2 (ACE2), and was blocked by remdesivir.ConclusionsThis study demonstrates that SARS-CoV-2 infects cardiomyocytes in vitro in an ACE2- and cathepsin-dependent manner. SARS-CoV-2 infection of cardiomyocytes is inhibited by the antiviral drug remdesivir.Translational PerspectiveAlthough this study cannot address whether cardiac injury and dysfunction in COVID-19 patients is caused by direct infection of cardiomyocytes, the demonstration of direct cardiotoxicity in cardiomyocytes, organ mimics, human heart slices and human hearts warrants the further monitoring of cardiotoxic effects in COVID-19 patients.

Slicing and Culturing Pig Hearts under Physiological Conditions.

Many novel drugs fail in clinical studies due to cardiotoxic side effects as the currently available in vitro assays and in vivo animal models poorly predict human cardiac liabilities, posing a multi-billion-dollar burden on the pharmaceutical industry. Hence, there is a worldwide unmet medical need for better approaches to identify drug cardiotoxicity before undertaking costly and time consuming 'first in man' trials. Currently, only immature cardiac cells (human induced pluripotent stem cell-derived cardiomyocytes [hiPSC-CMs]) are used to test therapeutic efficiency and drug toxicity as they are the only human cardiac cells that can be cultured for prolonged periods required to test drug efficacy and toxicity. However, a single cell type cannot replicate the phenotype of the complex 3D heart tissue which is formed of multiple cell types. Importantly, the effect of drugs needs to be tested on adult cardiomyocytes, which have different characteristics and toxicity responses compared to immature hiPSC-CMs. Culturing human heart slices is a promising model of intact human myocardium. This technology provides access to a complete multicellular system that mimics the human heart tissue and reflects the physiological or pathological conditions of the human myocardium. Recently, through optimization of the culture media components and the culture conditions to include continuous electrical stimulation at 1.2 Hz and intermittent oxygenation of the culture medium, we developed a new culture system setup that preserves viability and functionality of human and pig heart slices for 6 days in culture. In the current protocol, we are detailing the method for slicing and culturing pig heart as an example. The same protocol is used to culture slices from human, dog, sheep, or cat hearts. This culture system has the potential to become a powerful predictive human in situ model for acute cardiotoxicity testing that closes the gap between preclinical and clinical testing results.

Meet the First Authors.

Meet the First Authors.

Abstract 208: Reliable Biomimetic Culture System for Pig and Human Heart Slices

Rationale: Heart failure is the number one killer and drug induced cardiotoxicity is a major cause of market withdrawal. The lack of availability of culture systems for human heart tissue that is functionally and structurally viable for more than 24 hours is a limiting factor in validation of novel heart failure therapies as well as reliable cardiotoxicity testing. Therefore, there is an urgent need to develop a reliable system for culturing human heart tissue for testing drug efficacy and toxicity. Objective: To develop a reliable method to culture pig and human heart slices under full physiological conditions for a period of time sufficient to test therapeutic efficacy and acute drug toxicity. Methods and Results: Here we describe a novel biomimetic culture system that maintains full viability and functionality of human and pig heart slices (300 μm thickness) for 6 days in culture through optimization of the medium and culture conditions with continuous electrical stimulation at 1.2 Hz and oxygenation of the medium. Functional viability of these slices over 6 days was confirmed by assessing their calcium homeostasis, twitch force generation, and response to β-adrenergic stimulation. Temporal transcriptome analysis using RNAseq at day 2, 6, and 10 in culture confirmed overall maintenance of normal gene expression for up to 6 days, while over 500 transcripts were differentially regulated after 10 days. Electron microscopy demonstrated intact mitochondria and Z-disc ultra-structures after 6 days in culture under our optimized conditions. This biomimetic culture system was successful in keeping human heart slices completely viable and functionally and structurally intact for 6 days in culture. We also used this system to demonstrate the effects of a novel gene therapy approach in human heart slices. Conclusions: We have developed and optimized a reliable and easily reproducible culture system for pig and human heart slices as a platform for testing the efficacy of novel heart failure therapeutics as well as reliable testing of cardiotoxicity in a 3D heart model.

Physiological Biomimetic Culture System for Pig and Human Heart Slices.

Preclinical testing of cardiotoxicity and efficacy of novel heart failure therapies faces a major limitation: the lack of an in situ culture system that emulates the complexity of human heart tissue and maintains viability and functionality for a prolonged time. To develop a reliable, easily reproducible, medium-throughput method to culture pig and human heart slices under physiological conditions for a prolonged period of time. Here, we describe a novel, medium-throughput biomimetic culture system that maintains viability and functionality of human and pig heart slices (300 µm thickness) for 6 days in culture. We optimized the medium and culture conditions with continuous electrical stimulation at 1.2 Hz and oxygenation of the medium. Functional viability of these slices over 6 days was confirmed by assessing their calcium homeostasis, twitch force generation, and response to β-adrenergic stimulation. Temporal transcriptome analysis using RNAseq at day 2, 6, and 10 in culture confirmed overall maintenance of normal gene expression for up to 6 days, while over 500 transcripts were differentially regulated after 10 days. Electron microscopy demonstrated intact mitochondria and Z-disc ultra-structures after 6 days in culture under our optimized conditions. This biomimetic culture system was successful in keeping human heart slices completely viable and functionally and structurally intact for 6 days in culture. We also used this system to demonstrate the effects of a novel gene therapy approach in human heart slices. Furthermore, this culture system enabled the assessment of contraction and relaxation kinetics on isolated single myofibrils from heart slices after culture. We have developed and optimized a reliable medium-throughput culture system for pig and human heart slices as a platform for testing the efficacy of novel heart failure therapeutics and reliable testing of cardiotoxicity in a 3-dimensional heart model.

Concurrent micro- to macro-cardiac electrophysiology in myocyte cultures and human heart slices

The contact cardiac electrogram is derived from the extracellular manifestation of cellular action potentials and cell-to-cell communication. It is used to guide catheter based clinical procedures. Theoretically, the contact electrogram and the cellular action potential are directly related, and should change in conjunction with each other during arrhythmogenesis, however there is currently no methodology by which to concurrently record both electrograms and action potentials in the same preparation for direct validation of their relationships and their direct mechanistic links. We report a novel dual modality apparatus for concurrent electrogram and cellular action potential recording at a single cell level within multicellular preparations. We further demonstrate the capabilities of this system to validate the direct link between these two modalities of voltage recordings.

Isoproterenol effects evaluated in heart slices of human and rat in comparison to rat heart in vivo

Human response to isoproterenol induced cardiac injury was evaluated by gene and protein pathway changes in human heart slices, and compared to rat heart slices and rat heart in vivo. Isoproterenol (10 and 100μM) altered human and rat heart slice markers of oxidative stress (ATP and GSH) at 24h. In this in vivo rat study (0.5mg/kg), serum troponin concentrations increased with lesion severity, minimal to mild necrosis at 24 and 48h. In the rat and the human heart, isoproterenol altered pathways for apoptosis/necrosis, stress/energy, inflammation, and remodeling/fibrosis. The rat and human heart slices were in an apoptotic phase, while the in vivo rat heart exhibited necrosis histologically and further progression of tissue remodeling. In human heart slices genes for several heat shock 70kD members were altered, indicative of stress to mitigate apoptosis. The stress response included alterations in energy utilization, fatty acid processing, and the up-regulation of inducible nitric oxide synthase, a marker of increased oxidative stress in both species. Inflammation markers linked with remodeling included IL-1α, Il-1β, IL-6 and TNFα in both species. Tissue remodeling changes in both species included increases in the TIMP proteins, inhibitors of matrix degradation, the gene/protein of IL-4 linked with cardiac fibrosis, and the gene Ccl7 a chemokine that induces collagen synthesis, and Reg3b a growth factor for cardiac repair. This study demonstrates that the initial human heart slice response to isoproterenol cardiac injury results in apoptosis, stress/energy status, inflammation and tissue remodeling at concentrations similar to that in rat heart slices.

Adult human heart slices are a multicellular system suitable for electrophysiological and pharmacological studies

Electrophysiological and pharmacological data from the human heart are limited due to the absence of simple but representative experimental model systems of human myocardium. The aim of this study was to establish and characterise adult human myocardial slices from small patients' heart biopsies as a simple, reproducible and relevant preparation suitable for the study of human cardiac tissue at the multicellular level. Vibratome-cut myocardial slices were prepared from left ventricular biopsies obtained from end-stage heart failure patients undergoing heart transplant or ventricular assist device implantation, and from hearts of normal dogs. Multiple slices were prepared from each biopsy. Regular contractility was observed at a range of stimulation frequencies (0.1–2 Hz), and stable electrical activity, monitored using multi-electrode arrays (MEA), was maintained for at least 8 h from slice preparation. ATP/ADP and phosphocreatine/creatine ratios were comparable to intact organ values, and morphology and gap junction distribution were representative of native myocardium. MEA recordings showed that field potential duration (FPD) and conduction velocity (CV) in human and dog slices were similar to the values previously reported for papillary muscles, ventricular wedges and whole hearts. Longitudinal CV was significantly faster than transversal CV, with an anisotropic ratio of 3:1 for human and 2.3:1 for dog slices. Importantly, slices responded to the application of E-4031, chromanol and 4-aminopyridine, three potassium channel blockers known to affect action potential duration, with an increase in FPD. We conclude that viable myocardial slices with preserved structural, biochemical and electrophysiological properties can be prepared from adult human and canine heart biopsies and offer a novel preparation suitable for the study of heart failure and drug screening.

Interaction of octanoate with branched-chain 2-oxo acid oxidation in rat and human muscle in vitro

1. 1. Acetate and butanoate inhibited and hexanoate and octanoate increased the 14CO 2 production from 0.1 mM [1- 14C]-labelled 2-oxoisocaproate (KIC) and 2-oxoisovalerate (KIV) in rat hemidiaphragms. 2. 2. Octanoate increased KIC and KIV oxidation in rat soleus muscle, too, inhibited it in human skeletal muscle and had a divergent effect in rat and human heart slices. 3. 3. In rat hemidiaphragms octanoate primarily affected the process of oxidative decarboxylation. No effect was found on transamination rates of branched-chain amino acids and on the CO 2 production beyond α-decarboxylation. 4. 4. The reverse transamination of branched-chain 2-oxo acids and their incorporation into protein decreased in the presence of octanoate. 5. 5. Octanoate had no effect on KIC and KIV oxidation at higher 2-oxo acid concentrations and in hemidiaphragms from 3-day-starved rats. 6. 6. The observed interactions are discussed and related to regulatory mechanisms, which are known to affect the branched-chain 2-oxo acid dehydrogenase complex.

Degradation of branched-chain amino acids and their derived 2-oxo acids and fatty acids in human and rat heart and skeletal muscle

Studies were made on the oxidation of the branched-chain amino acids, leucine and valine, and their derived 2-oxo acids and branched-chain short-chain fatty acids in intact muscular preparations and homogenates of man and rat. In rat hemidiaphragm transamination rates of the amino acids are higher than oxidative decarboxylation rates and oxidative decarboxylation rates of the 2-oxo acids are higher than those of the corresponding amino acids at the same concentration. The last phenomenon also was observed in rat- and human-heart slices. Transaminase and 2-oxo acid dehydrogenase activities are much higher in homogenates than in intact preparations from the same muscle. Homogenates from heart muscle have generally a higher transaminase and 2-oxo acid dehydrogenase activity than those from skeletal muscle and those from rat muscle have higher activities than those from comparable human muscle. Homogenates from rat heart and liver have similar 2-oxo acid dehydrogenase activities. In homogenates and mitochondria, CO 2 was only produced by oxidative decarboxylation of the branched-chain 2-oxo acids and no CO 2 was produced from the corresponding branched-chain short-chain fatty acids. In all intact muscular preparations the branched-chain 2-oxo acids were oxidized more to completeness and CO 2 production from the coresponding short-chain fatty acids was observed. The further degradation of the branched-chain 2-oxo acids beyond α-decarboxylation appeared to be limited, for 3-methyl-2-oxobutanoate still more than for 4-methyl-2-oxopentanoate.

Binding sites related to ouabain-induced stimulation or inhibition of the sodium pump.

THE concentration of free cardiac glycosides in the blood of patients treated for heart failure ranges between 1 and 5 nM (refs 1–3). Studies on human heart slices have shown that, within this range of concentrations, digitoxin does not inhibit the sodium pump but does stimulate 42K uptake3. Similar observations have been made with isolated guinea pig atria: low concentrations of ouabain (near 1 nM) stimulate 42K uptake whereas higher concentrations inhibit it4. These results are in agreement with recent electrophysiological observations on Purkinje fibres showing that low doses of ouabain produce changes in the K gradient that reflect stimulation of the sodium pump5. It has also been observed that the stimulation of the sodium pump by ouabain is associated with a positive ionotropic effect6. Earlier studies with guinea pig atria4 and other preparations7–9 showed that the binding of ouabain occurs on non-specific and specific sites. The specific binding is fitted by a Langmuir curve with an equilibrium constant close to that estimated by measuring the inhibition of the sodium pump. We report here the existence of a binding with a higher affinity associated with the stimulation of the sodium pump.