Structural and functional plasticity in long-term cultures of adult ventricular myocytes

Abstract

Cultured heart cells have long been valuable for characterizing biological mechanism and disease pathogenesis. However, these preparations have limitations, relating to immaturity in key properties like excitation-contraction coupling and β-adrenergic stimulation. Progressive attenuation of the latter is intimately related to pathogenesis and therapy in heart failure. Highly valuable would be a long-term culture system that emulates the structural and functional changes that accompany disease and development, while concurrently permitting ready access to underlying molecular events. Accordingly, we here produce functional monolayers of adult guinea-pig ventricular myocytes (aGPVMs) that can be maintained in long-term culture for several weeks. At baseline, these monolayers exhibit considerable myofibrillar organization and a significant contribution of sarcoplasmic reticular (SR) Ca2+ release to global Ca2+ transients. In terms of electrical signaling, these monolayers support propagated electrical activity and manifest monophasic restitution of action-potential duration and conduction velocity. Intriguingly, β-adrenergic stimulation increases chronotropy but not inotropy, indicating selective maintenance of β-adrenergic signaling. It is interesting that this overall phenotypic profile is not fixed, but can be readily enhanced by chronic electrical stimulation of cultures. This simple environmental cue significantly enhances myofibrillar organization as well as β-adrenergic sensitivity. In particular, the chronotropic response increased, and an inotropic effect now emerges, mimicking a reversal of the progression seen in heart failure. Thus, these aGPVM monolayer cultures offer a valuable platform for clarifying long elusive features of β-adrenergic signaling and its plasticity.

The Effects of Swiprosin-1 on the Formation of Pseudopodia-Like Structures and β-Adrenoceptor Coupling in Cultured Adult Rat Ventricular Cardiomyocytes

“…Experiments with ARVC in culture showed a change in the responsiveness of β 1 - and β 2 - adrenoceptors [17–19]. As swiprosin-1 significantly affected the adaptation to culture conditions during which also changes in β-adrenoceptor coupling were observed, we correlated the expression of swiprosin-1 in vitro with genes known to interfere with β-adrenoceptors.…”

“…As a control molecule that has already been identified to be required in the process of spreading, oncostatin M was investigated [4]. Additionally, former studies have shown a reduction of β-adrenoceptor responsiveness under the same culture conditions that induce spreading of cardiomyocytes [17–19]. Therefore, we correlated swiprosin-1 expression with genes known to interfere with β-adrenoceptor-coupling.…”

Calmodulin mutations associated with long QT syndrome prevent inactivation of cardiac L-type Ca2+ currents and promote proarrhythmic behavior in ventricular myocytes

“…Hearts were excised after guinea pigs were anesthetized with pentobarbital (35 mg/kg, intraperitoneal injection). Single ventricular myocytes were isolated from both ventricles according to a published protocol [18] and plated on glass coverslips coated with laminin (20 μg/ml overnight at 4 °C). Cells were transduced with adenovirus carrying wild-type or mutant CaM upon plating in the presence of M199 medium supplemented with 20% fetal bovine serum.…”

Human iPSC-derived cardiomyocytes and tissue engineering strategies for disease modeling and drug screening

“…MEAs permit recording of electrical field potentials from cardiac monolayers similar to whole heart analysis by electrocardiography (Clements and Thomas, 2014, Navarrete, Liang, 2013, Riedel et al, 2014), whereas the use of either genetically encoded or exogenously applied voltage-sensitive dyes enable real-time monitoring of action potential propagation through cultured cells (Kijlstra, Hu, 2015). Ca 2+ sensitive dyes have also been employed for analysis of cardiac function (Burridge, Thompson, 2011, Joshi-Mukherjee et al, 2013). However, it should be noted that Ca 2+ dyes do not directly report electrophysiological endpoints, and are actually used as a surrogate for direct voltage measurements in most cases.…”