In an intact heart, adjacent cells influence adult cardiomyocytes. With the method of isolation and cultivation of adult cardiomyocytes, a precise investigation of the behavior of these cells under specific treatments and environments is possible. This manuscript presents a protocol for successful isolation and cultivation of adult rat ventricular cardiomyocytes (ARVC). The rat is sacrificed by cervical dislocation under deep anesthesia. Then, the heart is extracted and the aorta is uncovered. Subsequently, perfusion on the Langendorff perfusion system with calcium depletion and collagenase treatment is performed. Afterwards, ventricular tissue gets minced, re-circulated, and filtered, followed by three centrifugation steps with gradual addition of CaCl2 until physiological calcium concentration is reached. ARVC are plated on cell culture dishes. After refreshing the cell culture medium, ARVC can be cultivated for up to six days without changing the serum-containing culture medium. Isolation of ARVC is a calcium sensitive process. Small changes in the intracellular calcium concentration cause a decrease in the quality and viability of the isolated cells. Freshly isolated ARVC are rod shaped. Within the first days of cultivation they lose the rod-shaped morphology and form pseudopodia-like structures (spreading). During this morphological formation ARVC initially degrade their contractile elements followed by a reformation through actin stress fibers and de novo sarcomerogenesis. After one week of cultivation, most ARVC show a widespread appearance with a clearly detectable cross striation. This process is sensitive to intracellular calcium concentration, as treatment with ionomycin attenuates spreading. Key markers in this process of de- and re-differentiation are β-myosin heavy chain (β-MHC), oncostatin M (OSM), and swiprosin-1 (EFHD2). Recent studies have suggested that cardiac re- and de-differentiation occurring under culture conditions mimics features seen in vivo during cardiac remodeling. Therefore, isolation and cultivation of ARVC play a key role in understanding the biology of cardiomyocytes.

BackgroundRecent findings suggest that adult terminally differentiated cardiomyocytes adapt to stress by cellular de- and redifferentiation. In the present study we tested the hypothesis that swiprosin-1 is a key player in this process. Furthermore, the relationship between swiprosin-1 and β-adrenoceptor coupling was analyzed.MethodsIn order to study the function of swiprosin-1 in adult rat ventricular cardiomyocytes (ARVC) they were isolated and cultured in a medium containing 20% fetal calf serum (FCS). Changes in cell morphology of ARVC during cultivation were quantified by light and confocal laser scan microscopy. Small interfering RNA (siRNA) was used to reduce the expression of swiprosin-1. The impact of calcium on swiprosin-1 dependent processes was investigated with Bapta-AM. Immunoblot techniques and qRT-PCR were performed to measure mRNA and protein expression.ResultsIn culture, ARVC first lost their contractile elements, which was followed by a formation of pseudopodia-like structures (spreading). Swiprosin-1 was detected in ARVC at all time points. However, swiprosin-1 expression was increased when ARVC started to spread. Reduction of swiprosin-1 expression with siRNA delayed ARVC spreading. Similarly, Bapta-AM attenuated swiprosin-1 expression and spreading of ARVC. Furthermore, swiprosin-1 expression correlated with the expression of G protein-coupled receptor kinase 2 (GRK2). Moreover, silencing of swiprosin-1 was associated with a down regulation of GRK2 and caused a sensitization of β-adrenergic receptors.ConclusionSwiprosin-1 is required for ARVC to adapt to culture conditions. Additionally, it seems to be involved in the desensitization of β-adrenergic receptors. Assuming that ARVC adapt to cardiac stress in a similar way, swiprosin-1 may play a key role in cardiac remodeling.

Isolated adult rat ventricular cardiomyocytes (ARVC) adapt to the two-dimensional surface of culture dishes once they are isolated from the three-dimensional heart tissue. This process mimics aspects of cardiac adaptation to pressure overload and requires an initial breakdown of sarcomeric structures. The present study therefore aimed to identify key steps in this remodeling process. ARVC were cultured under serum-free or serum-supplemented conditions and their sizes and shapes were analyzed as well as apoptosis and the ability to disintegrate their sarcomeres. ARVC require serum-factors in order to adapt to cell culture conditions. More ARVC survived if they were able to breakdown their sarcomeres and mononucleated ARVC, which were smaller than binucleated ARVC, had a better chance to adapt. During the early phase of adaptation, proteasome subunit low molecular weight protein (LMP)-2 was induced. Inhibition of LMP-2 up-regulation by siRNA attenuated the process of successful adaptation. In vivo, LMP-2 was induced in the left ventricle of spontaneously hypertensive rats during the early phase of adaptation to pressure overload. In conclusion, the data suggest that breakdown of pre-existing sarcomeres is optimized by induction of LMP-2 and that it is required for cardiac remodeling processes, for example, occurring during pressure overload.

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