As a cardiovascular disease with global prevalence, heart failure is gaining more and more importance. Nevertheless, the pathogenesis of the syndrome and its pathomechanisms remain poorly understood. Current cardiovascular research is mainly based on a variety of animal models. In recent years, animal-free alternatives have emerged with the use of cardiomyocytes derived from induced pluripotent stem cells (iPSC). The development of cell cultures that model cardiomyocytes of heart failure patients is a major challenge. One approach is to induce hypertrophy by treatment with endothelin-1 (ET-1). To date, this treatment has been shown to result in cell enlargement, expression of fetal genes, and reduction of cell contractility. However, detailed characterization of the mechanical and functional properties of the cells has been lacking.

We aimed to investigate cell mechanics as well as the contractile behavior of iPSC derived cardiomyocytes treated with ET-1 in order to establish a cell culture model for the study of heart failure.

Cardiomyocytes² (FUJIFILM Cellular Dynamics, Madison, WI, USA) were treated with ET-1 for 24 and 48 hours, respectively. Immediately after treatment, contraction of the cells was recorded by phase-contrast microscopy for 24 hours. Cell mechanics were examined by real-time deformability cytometry (RT-DC). Gene expression of relevant targets was assessed using the NanoString (Seattle, WA, USA) panel for Cardiovascular Disease. Cytoskeletal markers were then selected and examined in more detail by immunofluorescence microscopy.

First, cell hypertrophy was confirmed both microscopically and by RT-DC in comparison to untreated control cells (352 µm² vs. 330 µm² after 24 h, p ≤ 0.05, n=3). Analysis of the contractile behavior showed an initial decrease during the first 24 h but then, in the further course, an increase in the amplitude of contraction (6.12 vs. 4.22 after 48 h, p ≤ 0.001, n=3). The reverse was true for cardiomyocyte beating frequency (58 bpm vs. 32.3 bpm acute, p ≤ 0.05, and 19.7 bpm vs. 30 bpm after 48 h, p ≤ 0.05, n=3). However, hypertrophy induction had a particularly strong effect on cell relaxation: the amplitude of relaxation in hypertrophic cells was only about 50 % of that in control cells (2.08 vs. 4.40 after 48 h, p ≤ 0.001, n=3). In addition, the duration of relaxation was significantly increased (0.62 ms vs. 0.35 ms after 48 h, p ≤ 0.001, n=3). Investigations of cell mechanics revealed an increased Young’s modulus (0.75 kPa vs. 0.57 kPa after 24 h, p ≤ 0.001, n=3) indicating that hypertrophy leads to cardiomyocyte stiffening.

The results show that treatment of cardiomyocytes with ET-1 not only causes hypertrophy of the cells, but also induces changes at the mechanical and the functional level that resemble the properties of cells during heart failure. Particularly evident is the decrease in relaxation and increased stiffness of the cells. Both in combination show great similarity to the diastolic dysfunction that occurs in heart failure patients with preserved ejection fraction. Therefore, characterization of this model of hypertrophic cardiomyocytes after ET-1 treatment has allowed us to establish it as a cell culture model for heart failure. By using this model, animal experiments can be reduced and refined by including intensive preliminary studies in hypertrophic cardiomyocytes.