Force production in mechanically isolated cardiac myocytes from human ventricular muscle tissue

Abstract-The specific and selective proteolysis of cardiac troponin I (cTnI) has been proposed to play a key role in human ischemic myocardial disease, including stunning and acute pressure overload. In this study, the functional implications of cTnI proteolysis were investigated in human cardiac tissue for the first time. The predominant human cTnI degradation product (cTnI ) and full-length cTnI were expressed in Escherichia coli, purified, reconstituted with the other cardiac troponin subunits, troponin T and C, and subsequently exchanged into human cardiac myofibrils and permeabilized cardiomyocytes isolated from healthy donor hearts. Maximal isometric force and kinetic parameters were measured in myofibrils, using rapid solution switching, whereas force development was measured in single cardiomyocytes at various calcium concentrations, at sarcomere lengths of 1.9 and 2.2 m, and after treatment with the catalytic subunit of protein kinase A (PKA) to mimic ␤-adrenergic stimulation. One-dimensional gel electrophoresis, Western immunoblotting, and 3D imaging revealed that approximately 50% of endogenous cTnI had been homogeneously replaced by cTnI in both myofibrils and cardiomyocytes. Maximal tension was not affected, whereas the rates of force activation and redevelopment as well as relaxation kinetics were slowed down. Ca 2ϩ sensitivity of the contractile apparatus was increased in preparations containing cTnI (pCa 50 : 5.73Ϯ0.03 versus 5.52Ϯ0.03 for cTnI and full-length cTnI, respectively). The sarcomere length dependency of force development and the desensitizing effect of PKA were preserved in cTnI 1-192 -exchanged cardiomyocytes. These results indicate that degradation of cTnI in human myocardium may impair diastolic function, whereas systolic function is largely preserved.

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“…Single myofibrils, bundles of a few myofibrils or single cardiac myocytes were prepared from these biopsies, as described previously. 18,19 …”

Section: Isolation Of Human Myofibrils and Cardiomyocytesmentioning

confidence: 99%

Impaired Diastolic Function After Exchange of Endogenous Troponin I With C-Terminal Truncated Troponin I in Human Cardiac Muscle

Narolska

Piroddi

Belus

et al. 2006

Circulation Research

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confidence: 99%

“…34 In contrast, forces of intact heart muscles can reach 40 to 80 mN/mm 2 . 75 The lower forces in engineered cardiac constructs are mainly explained by lower cardiac myocyte density, lower sarcomere volume fraction, and the overall lower level of cardiac myocyte differentiation. 74 Cardiac 3D constructs from chickens, rats, mice, and humans show a positive inotropic and chronotropic response to β-adrenergic stimulation, which is sensitive to the muscarinic agonist carbachol.…”

mentioning

confidence: 99%

Cardiac Tissue Engineering

Hirt

Hansen

Eschenhagen

2014

Circulation Research

366

294

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Abstract:The engineering of 3-dimensional (3D) heart muscles has undergone exciting progress for the past decade.Profound advances in human stem cell biology and technology, tissue engineering and material sciences, as well as prevascularization and in vitro assay technologies make the first clinical application of engineered cardiac tissues a realistic option and predict that cardiac tissue engineering techniques will find widespread use in the preclinical research and drug development in the near future. Tasks that need to be solved for this purpose include standardization of human myocyte production protocols, establishment of simple methods for the in vitro vascularization of 3D constructs and better maturation of myocytes, and, finally, thorough definition of the predictive value of these methods for preclinical safety pharmacology. The present article gives an overview of the present state of the art, bottlenecks, and perspectives of cardiac tissue engineering for cardiac repair and in vitro testing. ( Jeffrey Robbins, EditorOriginal received May 24, 2013; revision received July 12, 2013; accepted July 15, 2013. In November 2013, the average time from submission to first decision for all original research papers submitted to Circulation Research was 14.6 days.From the Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany; and DZHK (German Centre for Cardiovascular Research), partner site Hamburg/Kiel/Lübeck, Hamburg, Germany.Correspondence to Thomas Eschenhagen, Department of Experimental Pharmacology and Toxicology, University Medical Center Hamburg-Eppendorf, Martinistraße 52, 20246 Hamburg, Germany. E-mail t.eschenhagen@uke.de by guest on May 9, 2018 http://circres.ahajournals.org/ Downloaded from Hirt et al Cardiac Tissue Engineering 355gyratory shaking of embryonic chicken cardiac myocytes in an Erlenmeyer flask induced the formation of spontaneously beating spheroids with improved functionality when compared with standard 2D-cultured myocytes. This selfassembly is still used in variations to generate cardiac microspheres. 5 Current cardiac tissue engineering methods embark on this endogenous capacity and use engineering techniques to make 3D constructs of the desired size, geometry, and orientation (Table). Hydrogel TechniqueThe hydrogel method has been pioneered in the 1980s as an advanced culture method for fibroblasts and skeletal muscle cells 6 and gave rise to the first successful cardiac tissue.7 It needs 3 factors: solutions of gelling natural products, such as collagen I, matrigel, fibrin, or mixtures of them; casting molds; and anchoring constructs. The hydrogel entraps cells in a 3D space during gelling, the mold gives the 3D form, and the anchors allow the growing tissue to fix and to develop mechanical tension between ≥2 anchor points. Important aspects of this technique are that the naturally occurring hydrogels stimulate cells to spread and form intercellular connections and that the cells compress the gel, reduce the w...

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“…Small human cardiac tissue samples were thawed in relaxing solution and cardiomyocytes were mechanically isolated by tissue disruption as described previously (58). Cardiomyocytes were membrane permeabilized by incubation for 5 min in relaxing solution containing 0.5% (vol/vol) Triton X-100 and glued between a force transducer and a piezoelectric motor.…”

Section: Methodsmentioning

confidence: 99%

“…The sarcomere length of the preparation was measured in relaxing solution and adjusted to 2.2 μm. Average sarcomere lengths were determined by means of a spatial Fourier transformation as described previously (58). The force measurements started with a maximal activation at ADP concentration of 10 mM (ADP-F total ).…”

Section: Methodsmentioning

confidence: 99%

ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease

Sequeira

Najafi

Wijnker

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Diastolic dysfunction is general to all idiopathic dilated (IDCM) and hypertrophic cardiomyopathy (HCM) patients. Relaxation deficits may result from increased actin-myosin formation during diastole due to altered tropomyosin position, which blocks myosin binding to actin in the absence of Ca 2+ . We investigated whether ADP-stimulated force development (without Ca 2+ ) can be used to reveal changes in actin-myosin blockade in human cardiomyopathy cardiomyocytes. Cardiac samples from HCM patients, harboring thick-filament (MYH7 mut , MYBPC3 mut ) and thin-filament (TNNT2 mut , TNNI3 mut ) mutations, and IDCM were compared with sarcomere mutation-negative HCM (HCM smn ) and nonfailing donors. Myofilament ADP sensitivity was higher in IDCM and HCM compared with donors, whereas it was lower for MYBPC3. Increased ADP sensitivity in IDCM, HCM smn , and MYH7 mut was caused by low phosphorylation of myofilament proteins, as it was normalized to donors by protein kinase A (PKA) treatment. Troponin exchange experiments in a TNNT2 mut sample corrected the abnormal actin-myosin blockade. In MYBPC3 trunc samples, ADP sensitivity highly correlated with cardiac myosin-binding protein-C (cMyBP-C) protein level. Incubation of cardiomyocytes with cMyBP-C antibody against the actin-binding N-terminal region reduced ADP sensitivity, indicative of cMyBP-C's role in actin-myosin regulation. In the presence of Ca 2+ , ADP increased myofilament force development and sarcomere stiffness. Enhanced sarcomere stiffness in sarcomere mutation-positive HCM samples was irrespective of the phosphorylation background. In conclusion, ADP-stimulated contraction can be used as a tool to study how protein phosphorylation and mutant proteins alter accessibility of myosin binding on actin. In the presence of Ca 2+ , pathologic [ADP] and low PKA-phosphorylation, high actin-myosin formation could contribute to the impaired myocardial relaxation observed in cardiomyopathies.H eart failure (HF) is a syndrome clinically defined as the inability of the heart to sufficiently supply blood to organs and tissues (1). Systolic dysfunction is present in approximately one-half of the HF population, whereas diastolic dysfunction is a common feature in almost all HF patients (2). Moreover, in hypertrophic cardiomyopathy (HCM), which is caused by mutations in genes encoding thinand thick-filament proteins, impaired diastolic function is frequently observed (3). Impaired relaxation of the heart may be caused by high myofilament Ca 2+ sensitivity. This increased sensitivity for Ca 2+ would result in residual myofilament activation at diastolic [Ca 2+ ], which may delay the onset of ventricular relaxation and limit proper filling of the heart. High myofilament Ca 2+ sensitivity has been observed in both acquired and genetic forms of cardiomyopathy (3, 4). In human idiopathic dilated cardiomyopathy (IDCM), high myofilament Ca 2+ sensitivity has been associated with reduced β-adrenergic receptor-mediated phosphorylation by protein kinase A (PKA) (4). Reduced PKA phospho...

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Force production in mechanically isolated cardiac myocytes from human ventricular muscle tissue

Abstract: These measurements indicate that it is possible to study the correlation between mechanical properties and protein composition in small biopsies from human ventricular muscle.

Cited by 77 publications

References 32 publications

Impaired Diastolic Function After Exchange of Endogenous Troponin I With C-Terminal Truncated Troponin I in Human Cardiac Muscle

Impaired Diastolic Function After Exchange of Endogenous Troponin I With C-Terminal Truncated Troponin I in Human Cardiac Muscle

Cardiac Tissue Engineering

ADP-stimulated contraction: A predictor of thin-filament activation in cardiac disease

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