Human embryonic stem cell-derived cardiomyocytes as a pharmacological preparation: beta-adrenoceptor responses

Nadire N. Ali*, Marta Brito-Martins*, Philip A. Poole-Wilson and Sian E. Harding. National Heart and Lung Institute, Imperial College London. (*joint first author)

The cardiac field has always lacked a stable model system for investigation of effects on agents on contractile function; primary neonatal myocytes retain beating for only a few weeks in culture, and adult myocytes are stable for just days. In contrast, the embryonic stem cell derived cardiomyocytes (ESCM) can continue to exhibit contractile activity for months. They can also be obtained from human lines, increasing the relevance of results to the final clinical objective. To be a useful pharmacological preparation, responses to agents should be stable and reproducible. The present study investigates these properties for the beta-adrenoceptor system, and compares responses to those in adult myocytes.

ESCM were generated from human line H7 (obtained in a collaborative project with Geron, CA). Beating areas were identified between days 9 and 15, and were plated on 0.9 cm coverslips, which could be transferred between culture dishes and a superfusion chamber on the stage of a Nikon TE200 microscope. ESCM were then superfused with physiological medium containing 2 mM Ca2+ at 37°C. ESCM were either allowed to beat spontaneously, when effects on pacemaker frequency were under investigation, or paced when amplitude of contraction was to be studied. To monitor contraction, beating areas were seeded with charcoal to allow tracking of movement of clusters of cells by a video-edge detection system. Isoprenaline (Iso) was superfused for 10 min with wash periods of 1 h between challenges. Antagonists, where present, were superfused for 15-20 min before as well as during agonist exposure.

The mixed β1 - and β2 AR agonist Iso increased beating rate from 42.0 ± 4.1 to 81.8 ± 6.3 beats per min (bpm) (n=27, P<0.001). There was a significant increase in both basal rate (r=0.57, P<0.01) and Iso response (r=0.61, P<0.001) with day of differentiation from 12-41 days. Responses to Iso were not always maintained during prolonged exposure, so non-cumulative concentration-response curves were constructed. The half-maximal (EC50) value for Iso was 9.6nM (CI 7.4-12.5nM). For comparison, adult human ventricular myocytes increased contraction amplitude with Iso with an EC50 of 1.16 nM in younger (<40 years) or 2.76nM in older adults without heart failure. Iso challenge was repeated in the presence of CGP20172A (300nM), a selective antagonist of β1 -adrenoceptors ( β1 AR). The beat frequency in the presence of 1μM Iso was reduced from 90.1 ± 7.9 to 43.0 ± 5.7 bpm (P<0.001), but remained above the basal value of 33.0 ± 5.3 bpm (P<0.01, n=7), indicating a β1 AR-independent component. The β2 AR antagonist ICI 118,551 (50nM) reduced the Iso response from 98.5 ± 13.9 to 73.6 ± 11.1 bpm (P<0.02, n=8). Two consecutive Iso challenges without antagonist gave values of 80.6 ± 13.3 and 79.3 ± 14.1 bpm respectively (NSD, n=7). A small β2 AR component was therefore evident in the human ESCM: this tended to increase with developmental day in the range 14 to 41 days.

Human ESCM are comparable to adult human ventricular myocytes in that Iso responses are mediated by both b 1 - and b 2 ARs. The human ESCM can be used as a preparation for determining subtype-specific responses to agonists.