Parabolic, Flight-Induced, Acute Hypergravity and Microgravity Effects on the Beating Rate of Human Cardiomyocytes

Abstract

Functional studies of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hCMs) under different gravity conditions contribute to aerospace medical research. To study the effects of altered gravity on hCMs, we exposed them to acute hypergravity and microgravity phases in the presence and absence of the β-adrenoceptor isoprenalin (ISO), L-type Ca²⁺ channel (LTCC) agonist Bay-K8644, or LTCC blocker nifedipine, and monitored their beating rate (BR). These logistically demanding experiments were executed during the 66th Parabolic Flight Campaign of the European Space Agency. The hCM cultures were exposed to 31 alternating hypergravity, microgravity, and hypergravity phases, each lasting 20-22 s. During the parabolic flight experiment, BR and cell viability were monitored using the xCELLigence real-time cell analyzer Cardio Instrument^®. Corresponding experiments were performed on the ground (1 g), using an identical set-up. Our results showed that BR continuously increased during the parabolic flight, reaching a 40% maximal increase after 15 parabolas, compared with the pre-parabolic (1 g) phase. However, in the presence of the LTCC blocker nifedipine, no change in BR was observed, even after 31 parabolas. We surmise that the parabola-mediated increase in BR was induced by the LTCC blocker. Moreover, the increase in BR induced by ISO and Bay-K8644 during the pre-parabola phase was further elevated by 20% after 25 parabolas. This additional effect reflects the positive impact of the parabolas in the absence of both agonists. Our study suggests that acute alterations of gravity significantly increase the BR of hCMs via the LTCC.

Keywords: Bay-K8644; Isoprenaline; L-type Ca2+ channels; adrenoceptor agonist; cardiomyocytes; human induced pluripotent stem cells; hypergravity; microgravity.

Figure 1

Experimental scheme for parabolic flight experiments carried out during the 66th Parabolic Flight Campaign of the European Space Agency. (A) Schematic of the instruments used during parabolic flight experiments. (B) Schematic showing flight maneuvers and time points before parabola P0 and after P30.

Figure 2

Analysis of the beating rate (BR) of human cardiomyocytes (hCMs) during parabolic flights. (A) Representative BR pattern of hCMs at a timepoint 15 min (1 g) before starting the first parabola (P0) and after 31 parabolas (P30). (B) Data from three independent parabolic flight experiments (each executed in triplicate), as well as data collected from the 1 g ground experiment carried out in parallel. The BR values were normalized by setting the control pre-parabola flight or the ground control value to 100% (mean ± SEM; n = 9, * p < 0.05 for control versus parabolic flight experiment values). (C) Parallel monitoring of the cell viability expressed as cell index (CI) values. The CI values were normalized by setting the control values to 1. (D) Representative BR patterns of hCMs treated on the ground (1 g) with nifedipine (1 µM) 40 min before starting P0 and after P30. In this case, no BR was observed before P0 or after P30 because of complete inhibition of the L-Type Ca²⁺ Channel. SEM, standard error of the mean.

Figure 3

Analysis of the beating rate (BR) of human cardiomyocytes (hCMs) stimulated with isoprenalin (ISO; 1 µM) during the parabolic flight experiments (A) Upper panel: Representative BR pattern of unstimulated hCMs recorded 15 min (1 g) prior to the first parabola (P0); Middle panel: Representative BR pattern of hCMs when stimulated with agonists 40 min prior to the parabolic flight and monitored 15 min before starting parabola P0. Lower panel: Representative BR pattern of hCMs measured after 31 parabolas. (B) Data from three independent parabolic flight experiments, each executed in triplicate, as well as data collected from the 1 g ground experiment carried out in parallel. The results were normalized by setting the ISO-induced BR of the ground control experiment prior to P0 to 100% (mean ± SEM, n = 9, * p < 0.05 for the ISO control versus the ISO parabolic flight values). (C) Parallel monitoring of the cell viability expressed as the cell index (CI) value. The CI values were normalized by setting the control values to 1. SEM, standard error of the mean.

Figure 4

Analysis of the beating rate (BR) of human cardiomyocytes (hCMs) stimulated with Bay-K8644 (Bay-K; 1 µM) during the parabolic flights experiments (A) Upper panel: Representative BR pattern of unstimulated hCMs recorded 15 min (1 g) prior to the first parabola (P0); Middle panel: Representative BR pattern of hCMs when stimulated with agonists 40 min prior to P0 and monitored 15 min before starting parabola P0. Lower panel: Representative BR pattern of hCMs stimulated 40 min prior to P0 and monitored again after 31 parabolas. (B) Data from three independent parabolic flight experiments, each executed in triplicate, as well as data collected from 1 g ground experiments carried out in parallel. The BR values were normalized by setting the Bay-K-induced BR of the ground control experiments prior to P0 to 100% (mean ± SEM, n = 9, * p < 0.05 for the ISO control values versus the Bay-K parabolic flight values). (C) Parallel monitoring of the cell viability expressed as the cell index (CI). The CI values were normalized by setting the control values to 1. SEM, standard error of the mean.