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Crucial role of the sarcoplasmic reticulum in the developmental regulation of Ca²⁺ transients and contraction in cardiomyocytes derived from embryonic stem cells

Ji-Dong Fu^*, Jun Li^*, David Tweedie, Hui-Mei Yu^*, Le Chen^*, Rong Wang^*, Daniel R. Riordon, Sheryl A. Brugh, Shi-Qiang Wang, Kenneth R. Boheler and Huang-Tian Yang^*,1

^* Laboratory of Molecular Cardiology of Institute of Health Sciences, Shanghai Institutes for Biological Sciences (SIBS), Chinese Academy of Sciences (CAS) & Shanghai Jiao Tong University School of Medicine; Key Laboratory of Stem Cell Biology, SIBS; Graduate School of the CAS, Shanghai, China;
Laboratory of Cardiovascular Science, National Institute on Aging, Baltimore, Maryland, USA; and
National Key Laboratory of Biomembrane and Membrane Biotechnology, College of Life Sciences, Peking University, Beijing, China

¹Correspondence: Laboratory of Molecular Cardiology, Institute of Health Sciences, SIBS, CAS & Shanghai Jiao Tong University School of Medicine, 225 Chong Qing Nan Rd., #1 Bulg. of Institute of Health Sciences, Shanghai, 200025, China. E-mail: htyang{at}sibs.ac.cn

SPECIFIC AIM

In adult myocardium, excitation-contraction (EC) coupling is critically regulated by sarcoplasmic reticulum (SR) Ca²⁺ release via type 2 ryanodine receptor (RyR2), but it is unknown whether SR Ca²⁺ release directly contributes to or regulates contraction in embryonic stem (ES) cell-derived cardiomyocytes (ESCMs), a possible source for cell transplantation therapies. The aim of this study was to test the hypothesis that ES cell-derived cardiomyocytes develop a functional SR that actively regulates the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) transients, an essential event in EC coupling, and subsequent contractions during early cardiac differentiation. For this purpose, we used wild-type (RyR2^+/+) and RyR2 null (RyR2^–/–) ESCMs to determine at what stage of cardiomyogenesis the SR is capable of Ca²⁺ uptake and release of sufficient magnitude to regulate cardiomyocyte contractile activity, and at what point these functional parameters can be modulated by external stimulation like ß-adrenergic receptor agonists.

PRINCIPAL FINDINGS

1. Dynamics of Ca²⁺ transients during differentiation of ESCMS
Spontaneous Ca²⁺ transients were determined in isolated single RyR2^+/+ ESCMs at early (EDS, 7+24d); intermediate (IDS, 7+68d), and late differentiation stages (LDS, 7+1114d) (Fig. 1 ). From EDS to LDS, basal [Ca²⁺]_i of ESCMs rose significantly from 94 ± 7 nmol/L to 135 ± 11 nmol/L (Fig. 1B , P<0.01), and the amplitude of Ca²⁺ transients was significantly higher in LDS compared with EDS and IDS ESCMs (Fig. 1C ). The frequency of Ca²⁺ transients increased with differentiation, and was 2.3-fold higher in LDS than in EDS (Fig. 1D ). In parallel, a significant increase in the maximum upstroke velocity (V_max) of Ca²⁺ transients was detected from EDS to LDS ESCMs (Fig. 1E ). The time-to-peak and half-decay time of Ca²⁺ transients also shortened with differentiation (Fig. 1F, G ). These results demonstrated that during in vitro cardiomyogenesis, Ca²⁺ transients develop into a rapid process characterized at later stages by a higher spontaneous frequency, higher amplitude, and shortened duration, similar to that reported in adult cardiomyocytes. These functional traits directly correlated with the gene expression of major SR handling proteins RyR2, SR Ca²⁺ ATPase (SERCA2a), phospholamban (PLB), and calsequestrin.

View larger version (40K): [in this window] [in a new window]   Figure 1. Dynamics of spontaneous Ca2+ transients in RyR2+/+ and RyR2–/– cardiomyocytes differentiation. A) Representative tracings of Ca2+ transients in RyR2+/+ and RyR2–/– ESCMs. B–G) comparison of the basal [Ca2+]i (B), amplitude (C), frequency (D), maximum upstroke velocity (Vmax, E), the time-to-90% of maximum amplitude (time-to-peak, F), and the time to 50% decay of the maximum amplitude (T-50%decay, G) of Ca2+ transients (CaTs) in RyR2+/+ and RyR2–/– cells at EDS, IDS, and LDS. n = 34, 40, and 33 in RyR2+/+ cells, and 20, 40, and 21 in RyR2–/– cells, at EDS, IDS, LDS respectively. *P < 0.05, **P < 0.01, vs. corresponding EDS values. #P < 0.05 vs. corresponding IDS values. P < 0.01, RyR2–/– vs. corresponding RyR2+/+ values.

2. SR and RyR2 are essential to the regulation of Ca²⁺ transients in ESCMs
To unequivocally determine whether the SR is a primary source and whether RyR2 contributes to the development of [Ca²⁺]_i dynamics during cardiomyogenesis, Ca²⁺ transients were compared between RyR2^+/+ and RyR2^–/– ESCMs (Fig. 1) . In EDS to LDS RyR2^–/– ESCMs, basal [Ca²⁺]_i and the amplitude of Ca²⁺ transients were similar to those found in RyR2^+/+ cells (Fig. 1A-C ), but the frequency of Ca²⁺ transients was significantly decreased, and increased frequency with the time of differentiation observed in RyR2^+/+ cells could not be seen in RyR2^–/– cells (Fig. 1D ). RyR2^–/– ESCMs also showed a marked decrease in upstroke V_max of the Ca²⁺ transients, accompanied with prolonged time-to-peak (2.4-fold at EDS and 4.2-fold at LDS) and half-decay time (Fig. 1E-G ). Ryanodine (10 µmol/L), a specific inhibitor of RyR2 and thapsigargin (0.5 µmol/L), a SR Ca²⁺ ATPase inhibitor, did not affect basal [Ca²⁺]_i and the dynamics of Ca²⁺ transients in RyR2^–/– cells at any differentiation time, but significantly increased basal [Ca²⁺]_i, reduced the amplitude, frequency, and upstroke V_max, and prolonged the time-to-peak in RyR2^+/+ cells. Exposure of RyR2^+/+ ESCMs to caffeine (10 mmol/L) caused a large Ca²⁺ transient at all differentiation stages, and the amplitude was 2.3-fold higher in LDS cells than that in EDS cells; however, caffeine had no effects on RyR2^–/– ESCMs. These data confirm that the SR and RyR2 are functional even at early differentiation stages and that both play a critical role in regulating the dynamics of Ca²⁺ transients as well as beating rate in ESCMs.

3. Functional SR and RyR2 are important to the ß-adrenergic receptor (AR)-stimulated changes in the Ca²⁺ transients with in vitro differentiation of cardiomyocytes
Because ß-adrenergic stimulation plays a pivotal role in modulating the kinetics of Ca²⁺ transients and increases the amplitude of contraction as well as beating rates in adult cardiomyocytes, isoproterenol (30 nmol/L) was added to RyR2^+/+ and RyR2^–/– ESCMs to determine its effects on SR function in early cardiomyocytes. ß-AR stimulation increased the frequency, amplitude, and upstroke V_max of the spontaneous Ca²⁺ transients in a differentiation-dependent manner in RyR2^+/+ ESCMs, but the observed increases were significantly smaller in RyR2^–/– ESCMs. Isoproterenol prolonged the time-to-peak of Ca²⁺ transients in both RyR2^+/+ and RyR2^–/– ESCMs, but the prolongation was much greater in RyR2^–/– cells at each differentiation stage. These findings demonstrate that ß-adrenergic stimulation modulates Ca²⁺ transient dynamics in ESCMs even in early developmental stages, and that this modulation is correlated with the development of the SR and the function of RyR2.

4. SR and RyR2 affect the contractions even in the early differentiated cardiomyocytes
To correlate the differentiation-dependent changes in SR Ca²⁺ release with the dynamics of cell contraction in RyR2^+/+ and RyR2^–/– ESCMs, the contractile parameters of isolated ESCMs were also determined. Similar to that seen for the Ca²⁺ transients, the amplitude of cell shortening did not differ between RyR2^+/+ and RyR2^–/– ESCMs at any differentiation time; however, RyR2^–/– cells showed a significant prolongation in the time-to-peak and half-decay time of twitch contractions (Fig. 2 ). Neither ryanodine nor thapsigargin inhibited the contractions of RyR2^–/– ESCMs, but both had significant inhibitory effects in RyR2^+/+ cells. Moreover, isoproterenol enhanced the amplitude of contraction in both types of cardiomyocytes, but the response in RyR2^–/– cells was smaller in RyR2^–/– cells. These results establish that Ca²⁺ release from ryanodine-sensitive SR stores is a critical determinant of spontaneous and ß-adrenergic stimulated increases in ESCM contractility.

View larger version (35K): [in this window] [in a new window]   Figure 2. Cell shortening measured simultaneously with Ca2+ transients in RyR2+/+ and RyR2–/– ESMCs (length, 20–30 µm). Representative tracings of cell shortening in IDS RyR2+/+ and RyR2–/– cardiomyocytes, and comparison of the amplitude, the time-to-90% of maximum amplitude (time-to-peak), and half-decay time (T-50%decay) of cell shortening between RyR2+/+ and RyR2–/– cardiomyocytes at EDS, IDS, and LDS. n = 13, 10, 13 in RyR2+/+ cells and 6, 10, 13 in RyR2–/– cells at EDS, IDS and LDS, respectively. *P < 0.05 vs. corresponding EDS values; P < 0.01 vs. corresponding RyR2+/+ values.

CONCLUSIONS AND SIGNIFICANCE

In the present study we have addressed the question of whether SR and RyR2 are functional and contribute to the regulation of Ca²⁺ transients and contraction in ES cell-derived cardiomyocytes. To unequivocally determine the role of RyR2, we employed RyR2^+/+ and RyR2^–/– ESCMs, together with pharmacological approaches and gene expression profiles of SR regulatory proteins, and we characterized the dynamics of Ca²⁺ transients and contraction during early cardiomyocyte development. We unequivocally demonstrate that the SR and RyR2 function both in the regulation of Ca²⁺ transients and cell contraction through their critical control on the sequestration and release of Ca²⁺ from and to the cytosol, respectively, even during early cardiomyogenesis (Fig. 3 ).

View larger version (26K): [in this window] [in a new window]   Figure 3. Schematic model of the roles of SR and RyR2 in regulating Ca2+ transients and contraction during cardiomyoycte differentiation. In the early differentiation stage of cardiomyocytes. RyR2-mediated SR Ca2+ release is critical to regulate the rapid upstroke of Ca2+ transients and contraction, although the contraction depends largely on trans-sarcolemmal Ca2+ influx. In the late differentiation stage, SR and RyR2 take more important roles in the regulation of the Ca2+ transient and contraction.

Previous studies suggested that the SR has a limited capacity to load Ca²⁺ and that RyR2 does not act as a functional Ca²⁺ release channel in mediating contraction in cardiomyocytes during early embryonic stage. We demonstrate that the gene expression of the major SR handling proteins (RyR2, SERCA2a, PLB), at a time coincident with or prior to the occurrence of spontaneous contraction of ESCMs, is in line with the expression pattern seen in the heart during early fetal development. Our data with ryanodine and thapsigargin from RyR2^+/+ and RyR2^–/– ESCMs demonstrate that the RyR2 and SERCA2a are essential for upstroke and the diastolic decay of Ca²⁺ transients, which subsequently regulate beating rate and contractile activity via their capacity for the Ca²⁺ release and uptake even in very immature ESCMs. Moreover, the developmental transition of Ca²⁺ transients and the increased SR load in RyR2^+/+ ESCMs indicate that the function of the SR to sequester and release Ca²⁺ increases with time of ESCM differentiation (Fig. 3) . RyR2 and SR are important for ß-AR stimulated increases in the frequency, amplitude, and upstroke of Ca²⁺ transients and contraction even at early differentiation stage.

We observed that the basal [Ca²⁺]_i, the amplitude of Ca²⁺ transients and the amplitude of cell shortening did not differ between RyR2^+/+ and RyR2^–/– ESCMs at any differentiation stage. The data may be interpreted to mean that RyR2-mediated SR Ca²⁺ release is not very important for cellular contraction; however, RyR2^–/– ESCMs showed significantly prolonged time-to-peak and half-decay time of Ca²⁺ transients and twitch contractions at all three differentiation stages examined. Moreover, ryanodine and thapsigargin increased basal [Ca²⁺]_i and reduced the peak of Ca²⁺ transients and cell shortening in RyR2^+/+ cells, but neither had any effect on the Ca²⁺ transients and cell shortening of RyR2^–/– ESCMs. ß-AR stimulation increased the amplitude and time-to-peak of spontaneous Ca²⁺ transients and the amplitude of contraction was significantly smaller in RyR2^–/– ESCMs. Taken together, these data indicate that RyR2-mediated SR Ca²⁺ release is important in maintaining normal contraction via its critical control of Ca²⁺ transients and that RyR2^–/– ESCMs may have developed some compensatory mechanisms to maintain [Ca²⁺]_i. Our observation of RyR2 deficiency accompanied with the increased L-type Ca²⁺ current density in RyR2^–/– ESCMs suggests that sarcolemmal-dependent mechanisms via L-type Ca²⁺ channels may be implicated in maintaining basal and peak levels of [Ca²⁺]_i in RyR2^–/– ESCMs. Our data clearly demonstrate that RyR2-mediated SR Ca²⁺ release is faster than any other mechanism in early embryonic cardiomyocytes (Fig. 1) and is critical and irreplaceable in maintaining normal contractile activity and beating rates and might account for the embryonic lethality seen in RyR2^–/– mice.

In summary, we demonstrate that functional SR and control of RyR2-mediated SR Ca²⁺ release directly contribute to the spontaneous and ß-AR-stimulated beating rates and cellular contraction of ESCMs even at very immature stages of development. ESCMs therefore have the potential, at least in vitro, to form a functional SR that can contribute to EC coupling mechanisms, a critical cellular function necessary for eventual therapeutic viability.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4501fje;

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