Regulation Of Intracellular Calcium Homeostasis In Early Developing Cardiac Myocytes

Objective : In adult myocardium, excitation-contraction coupling is criticallyregulated by sarcoplasmic reticulum (SR) Ca²⁺ release via type 2 ryanodine receptor(RyR2), but generally it is believed that SR-function is rudimentary in the fetal heartand in embryonic stem (ES) cell derived cardiomyocytes (ESCMs), a possible sourcefor cell replacement therapies. The objectives are to observed the development of thebasal intracellular Ca²⁺ concentration ([Ca²⁺]i) and Ca²⁺ transients during cardiacdevelopment;to determine the crucial role of SR in the regulation of Ca²⁺ homeostasisand cell contraction in early developing cardiomyocytes;to identify the role of themain sarcolemmal Ca²⁺ channels/transporter in the modulation of Ca²⁺ homeostasis andto analysis the development changes of the mechanisms in maintaining Ca²⁺homeostasis during cardiomyocytes development. At the same time, to examinewhether ES cell derived cardiomyocytes can act as the donor cell for the celltransplantation based on their characteristics of the function..Methods: After established the cultivation and proliferation of ES cells, we inducedES cells differentiate into cardiomyocytes by hanging-drop method and isolated thoseESCMs at different differentiation stages. We systematically compared the dynamics of[Ca²⁺]i transients and its relationship to cardiomyocyte contraction in cellsdifferentiated from wild-type (RyR2^+/+) and type 2 ryanodine receptor (RyR2) deficient(RyR2^-/-) ES cell lines both with time of in vitro differentiation and followingpharmacological intervention.Results: With time of RyR2+/+ ESCM differentiation, SR function developedprogressively with characteristics of increased basal [Ca²⁺]i with an enhancedfrequency and amplitude and a progressive decrease in the duration of Ca²⁺ transientsthat were inhibited by ryanodine and thapsigargin. These functional traits correlatedwith SR Ca2+ load, the mRNA expression of RyR2, Sarcoplasmic/endoplasmicreticulum Ca2+ ATPase (SERCA2a), phospholamban, and the delayed expression ofcalsequestrin. RyR2-/-ESCMs, comparatively, demonstrated a significantly prolongedtime-to-peak and reduced frequency of Ca2+ transients and contractions. β-adrenergicstimulation of RyR2+/+ ESCMs increased the frequency and amplitude of Ca2+transients in a differentiation-dependent manner, but β-adrenergic responses were muchweaker in RyR2-/-ESCMs. In early developing ESCMs, the type 2 inositol triphosphatereceptor (IP3R2) inhibitor 2-APB inhibited the amplitude of Ca2+ transient and itsinhibitory effect decreased with cardiac development.ESCMs basal [Ca2+]i increased dramatically when in the Na+ free solution, whichinhibit Na+/Ca2+ exchanger (NCX) to exclude Ca2+ from cytoplasm, but Na+ freesolution didn't inhibit ESCMs Ca2+ transients until late differentiation stage. The largerL-type Ca2+ current density was recorded in RyR2-/-ESCMs compared to RyR2+/+ cells;and nifedipine with low concentration (1 μmol/L) inhibited ESCM Ca2+ transientssignificantly and its inhibitory effect decreased slightly with cardiomyocytedevelopment.Conclusions: We demonstrate that the basal [Ca2+]i increase with the stronger andquicker Ca2+ transients in ESCMs. SR load and RyR2 are essential for upstroke andfrequency of Ca2+ transients, and consequently contraction even at an earlydevelopmental stage and that RyR2 and SR are important for β-adrenergic receptorstimulation in these cells. With time of differentiation, the contributions of the SR andRyR2 to the contractile properties of ESCMs continue to develop. The Ca2+ influxthrough sarcolemmal L-type Ca2+ channel is the critical signaling but its contribution tothe Ca2+ transients decreases with the differentiation. ESCMs therefore have thepotential, at least in vitro, to form a functional SR that can actively contribute toexcitation-contraction coupling mechanisms – a critical cellular function necessary foreventual therapeutic viability. These findings provide important new insights into thefunctional properties of ESCMs and their therapeutic potential.