In vitro model to study the effects of matrix stiffening on Ca²⁺ handling and myofilament function in isolated adult rat cardiomyocytes

Abstract

Key points This paper describes a novel model that allows exploration of matrix‐induced cardiomyocyte adaptations independent of the passive effect of matrix rigidity on cardiomyocyte function.Detachment of adult cardiomyocytes from the matrix enables the study of matrix effects on cell shortening, Ca2+ handling and myofilament function.Cell shortening and Ca2+ handling are altered in cardiomyocytes cultured for 24 h on a stiff matrix.Matrix stiffness‐impaired cardiomyocyte contractility is reversed upon normalization of extracellular stiffness.Matrix stiffness‐induced reduction in unloaded shortening is more pronounced in cardiomyocytes isolated from obese ZSF1 rats with heart failure with preserved ejection fraction compared to lean ZSF1 rats. AbstractExtracellular matrix (ECM) stiffening is a key element of cardiac disease. Increased rigidity of the ECM passively inhibits cardiac contraction, but if and how matrix stiffening also actively alters cardiomyocyte contractility is incompletely understood. In vitro models designed to study cardiomyocyte–matrix interaction lack the possibility to separate passive inhibition by a stiff matrix from active matrix‐induced alterations of cardiomyocyte properties. Here we introduce a novel experimental model that allows exploration of cardiomyocyte functional alterations in response to matrix stiffening. Adult rat cardiomyocytes were cultured for 24 h on matrices of tuneable stiffness representing the healthy and the diseased heart and detached from their matrix before functional measurements. We demonstrate that matrix stiffening, independent of passive inhibition, reduces cell shortening and Ca2+ handling but does not alter myofilament‐generated force. Additionally, detachment of adult cultured cardiomyocytes allowed the transfer of cells from one matrix to another. This revealed that stiffness‐induced cardiomyocyte changes are reversed when matrix stiffness is normalized. These matrix stiffness‐induced changes in cardiomyocyte function could not be explained by adaptation in the microtubules. Additionally, cardiomyocytes isolated from stiff hearts of the obese ZSF1 rat model of heart failure with preserved ejection fraction show more pronounced reduction in unloaded shortening in response to matrix stiffening. Taken together, we introduce a method that allows evaluation of the influence of ECM properties on cardiomyocyte function separate from the passive inhibitory component of a stiff matrix. As such, it adds an important and physiologically relevant tool to investigate the functional consequences of cardiomyocyte–matrix interactions.

Extracellular matrix stiffness affects contractility in adult rat cardiomyocytes: implications for dynamic nitric oxide signalling and calcium handling

“…In a previous study (Galie et al 2013), adult rat CMs plated on 255 kPa substrates demonstrated stiffness-specific transcription patterns of actinin, integrins and vinculin. These CMs demonstrated modest decreases in shortening and relaxation rates relative to those at lower stiffnesses more representative of nativ physiology, although not nearly to the degree of the CMs from stiff substrates in the study by van Deel et al (2017). In the latter study, CMs plated on 100 kPa for 48 h and then detached tended to decrease by ca 25% in Ca 2+ amplitudes; this was not mirrored when the CMs remained adhered to their substrate of similarly superphysiological stiffness for Ca 2+ measurement (Galie et al 2013).…”

“…Regardless of these difficulties, with further development this research will certainly inspire improvements to the system in question and lead to greater physiological understanding. van Deel et al (2017) have demonstrated robust, viable CMs that can endure significant handling, as well as a model of dynamic mechanical influence which may prove vital in further investigation of CM contractile physiology and pathology.…”

Decellularized Extracellular Matrices and Cardiac Differentiation: Study on Human Amniotic Fluid-Stem Cells

“…It is therefore probable that CMs need a more complex environment than 2D or 3D scaffolds enriched in cardioprotective factors to attach and survive after dissociation [ 41 ]. In addition, Van Deel et al showed that the commonly used enzymatic detaching methods, such as Trypsin, Detachin, and Accutase, are lethal to adult CMs, altering the integrity of the cells [ 42 ]. Thus, the failure of CMs obtained from hiPSCs and Cardiopoietic AF cells to attach onto dECMs was probably due to the fact that, after enzymatic digestion, the CMs were not able to recreate cell–cell and cell–scaffold connections.…”

End‐diastolic force pre‐activates cardiomyocytes and determines contractile force: role of titin and calcium

“…Adult rat cardiomyocytes were isolated from WT (N = 7) and RBM20 HET (N = 7) male and female rats, weighing ß300 g as described before (Helmes et al 2016;van Deel et al 2017). Anaesthesia was induced in rats by 8% sevoflurane and maintained with 1-2.5% sevoflurane.…”

“…From a small piece of left ventricle, cardiomyocytes were isolated as described previously (van der Velden et al 1998). We improved the cell gluing process by using shellac (wax-free Sigma-Aldrich, St Louis, MO, USA, 78471; 0.07 mg ml −1 70% ethanol), instead of the usual silicone glue.…”