Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Coppini, Raffaele; Ferrantini, Cecila; Aiazzi, Alessandro; Mazzoni, Luca; Sartiani, Laura; Mugelli, Alessandro; Poggesi, Corrado; Cerbai, Elisabetta

doi:10.3791/51116-v

Cited by 6 publications

(4 citation statements)

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“…These primary sources directly represent the situation in the human heart. However, when isolating cardiomyocytes, tissues need to be immersed into a collagenase-containing bath as quickly as possible, since snap-frozen or fixated tissue is unsuitable for cardiomyocyte isolation [15]. A major drawback of this collagenase treatment to digest the heart tissue is the low yield.…”

Section: Primary Cardiac Tissuementioning

confidence: 99%

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance

Geraets

Chanda

Tienen

et al. 2018

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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Patients with type 2 diabetes (T2D) and/or insulin resistance (IR) have an increased risk for the development of heart failure (HF). Evidence indicates that this increased risk is linked to an altered cardiac substrate preference of the insulin resistant heart, which shifts from a balanced utilization of glucose and long-chain fatty acids (FAs) towards an almost complete reliance on FAs as main fuel source. This shift leads to a loss of endosomal proton pump activity and increased cardiac fat accumulation, which eventually triggers cardiac dysfunction. In this review, we describe the advantages and disadvantages of currently used in vitro models to study the underlying mechanism of IR-induced HF and provide insight into a human in vitro model: human embryonic stem cell-derived cardiomyocytes (hESC-CMs). Using functional metabolic assays we demonstrate that, similar to rodent studies, hESC-CMs subjected to 16h of high palmitate (HP) treatment develop the main features of IR, i.e., decreased insulin-stimulated glucose and FA uptake, as well as loss of endosomal acidification and insulin signaling. Taken together, these data propose that HP-treated hESC-CMs are a promising in vitro model of lipid overload-induced IR for further research into the underlying mechanism of cardiac IR and for identifying new pharmacological agents and therapeutic strategies. This article is part of a Special issue entitled Cardiac adaptations to obesity, diabetes and insulin resistance, edited by Professors Jan F.C. Glatz, Jason R.B. Dyck and Christine Des Rosiers.

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Section: Primary Cardiac Tissuementioning

confidence: 99%

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance

Geraets

Chanda

Tienen

et al. 2018

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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“…However, the similarity of iPSC-derived CMs in morphology and function with fatal cells restrained its applications in late-onset diseases [ 30 ]. To date, the commonly used methods for isolating primary CMs have been enzymatic bulk digestion and the Langendorff method [ 30 , 31 ], both suffering from relatively low cell yield and the requirement of tissue integrity. By effectively combining tissue slicing and enzymatic digestion, the isolation approaches here robustly improved cell viability [ 32 ], preserving ionic reactions of CMs with intact morphology and function.…”

Section: Resultsmentioning

confidence: 99%

Mass Spectrometry Imaging-Based Single-Cell Lipidomics Profiles Metabolic Signatures of Heart Failure

Ren

Chen

et al. 2023

Research

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Heart failure (HF), leading as one of the main causes of mortality, has become a serious public health issue with high prevalence around the world. Single cardiomyocyte (CM) metabolomics promises to revolutionize the understanding of HF pathogenesis since the metabolic remodeling in the human hearts plays a vital role in the disease progression. Unfortunately, current metabolic analysis is often limited by the dynamic features of metabolites and the critical needs for high-quality isolated CMs. Here, high-quality CMs were directly isolated from transgenic HF mice biopsies and further employed in the cellular metabolic analysis. The lipids landscape in individual CMs was profiled with a delayed extraction mode in time-of-flight secondary ion mass spectrometry. Specific metabolic signatures were identified to distinguish HF CMs from the control subjects, presenting as possible single-cell biomarkers. The spatial distributions of these signatures were imaged in single cells, and those were further found to be strongly associated with lipoprotein metabolism, transmembrane transport, and signal transduction. Taken together, we systematically studied the lipid metabolism of single CMs with a mass spectrometry imaging method, which directly benefited the identification of HF-associated signatures and a deeper understanding of HF-related metabolic pathways.

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“…After collection of surgical cardiac samples, trabeculae were cut for mechanical experiments, single cardiomyocytes isolated as previously described 21 and the remaining tissue used for skinned strips, myofibril, and proteomic analysis.…”

Section: Methodsmentioning

confidence: 99%

Slower Calcium Handling Balances Faster Cross-Bridge Cycling in Human MYBPC3 HCM

Pioner

Vitale

Steczina

et al. 2023

Circulation Research

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RATIONALE The pathogenesis of MYBPC3-associated hypertrophic cardiomyopathy is still unresolved. We exploited a large and well-characterized patient population carrying the MYBPC3-c.772G>A variant (p. Glu258Lys, E258K) to provide translational insight based on studies on surgical myectomy samples, human-induced pluripotent stem cell (hiPSC)-cardiomyocytes and engineered heart tissues. OBJECTIVE To gain insights into the pathogenic mechanisms driven by the MYBPC3-c.772G>A mutation using a comprehensive investigation of human disease models. METHODS AND RESULTS Haplotype analysis revealed MYBPC3-c.772G>A as a founder mutation in Tuscany. The mutation leads to reduced cMyBP-C (cardiac myosin binding protein-C) expression, supporting haploinsufficiency as the main primary disease mechanism. Functional perturbations were studied in left ventricular samples from 4 patients who underwent myectomy, as well as in human hiPSC-cardiomyocytes and engineered heart tissues harboring c.772G>A, compared with samples from nonfailing nonhypertrophic surgical patients and hiPSC lines from healthy controls. Mechanical studies in single myofibrils and permeabilized muscle strips highlighted faster cross-bridge cycling, and higher energy cost of tension generation. A novel approach based on tissue clearing and advanced optical microscopy supported the idea that the sarcomere energetics dysfunction is intrinsically related with the reduction in cMyBP-C. Studies in single cardiomyocytes (native and hiPSC-derived), intact trabeculae and hiPSC-engineered heart tissues revealed prolonged action potentials, slowerCa 2+ transients and preserved twitch duration, suggesting that the slower excitation-contraction coupling counterbalanced the faster sarcomere kinetics. This conclusion was strengthened by in silico simulations. Of note, the results from patient tissues and hiPSC-derived models obtained from the same patients were essentially the same, supporting the use of hiPSC-models for hypertrophic cardiomyopathy studies. CONCLUSIONS Hypertrophic cardiomyopathy–related MYBPC3 -c.772G>A mutation invariably impairs sarcomere energetics and cross-bridge cycling. Compensatory electrophysiological changes (eg, reduced potassium channel expression) appear to preserve twitch contraction parameters, but may expose patients to greater arrhythmic propensity and disease progression. Therapeutic approaches correcting the primary sarcomeric defects may prevent secondary cardiomyocyte remodeling.

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Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cited by 6 publications

References 0 publications

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance

Human embryonic stem cell-derived cardiomyocytes as an in vitro model to study cardiac insulin resistance

Mass Spectrometry Imaging-Based Single-Cell Lipidomics Profiles Metabolic Signatures of Heart Failure

Slower Calcium Handling Balances Faster Cross-Bridge Cycling in Human MYBPC3 HCM

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