Facilitated maturation of Ca2+ handling properties of human embryonic stem cell-derived cardiomyocytes by calsequestrin expression - PubMed (original) (raw)

Facilitated maturation of Ca2+ handling properties of human embryonic stem cell-derived cardiomyocytes by calsequestrin expression

Jing Liu et al. Am J Physiol Cell Physiol. 2009 Jul.

Abstract

Cardiomyocytes (CMs) are nonregenerative. Self-renewable pluripotent human embryonic stem cells (hESCs) can differentiate into CMs for cell-based therapies. We recently reported that Ca(2+) handling, crucial to excitation-contraction coupling of hESC-derived CMs (hESC-CMs), is functional but immature. Such immature properties as smaller cytosolic Ca(2+) transient amplitudes, slower kinetics, and reduced Ca(2+) content of sarcoplasmic reticulum (SR) can be attributed to the differential developmental expression profiles of specific Ca(2+) handling and regulatory proteins in hESC-CMs and their adult counterparts. In particular, calsequestrin (CSQ), the most abundant, high-capacity but low-affinity, Ca(2+)-binding protein in the SR that is anchored to the ryanodine receptor, is robustly expressed in adult CMs but completely absent in hESC-CMs. Here we hypothesized that gene transfer of CSQ in hESC-CMs suffices to induce functional improvement of SR. Transduction of hESC-CMs by the recombinant adenovirus Ad-CMV-CSQ-IRES-GFP (Ad-CSQ) significantly increased the transient amplitude, upstroke velocity, and transient decay compared with the control Ad-CMV-GFP (Ad-GFP) and Ad-CMV-CSQDelta-IRES-GFP (Ad-CSQDelta, which mediated the expression of a nonfunctional, truncated version of CSQ) groups. Ad-CSQ increased the SR Ca(2+) content but did not alter L-type Ca(2+) current. Pharmacologically, untransduced wild-type, Ad-GFP-, Ad-CSQDelta-, and Ad-CSQ-transduced hESC-CMs behaved similarly. Whereas ryanodine significantly reduced the Ca(2+) transient amplitude and slowed the upstroke, thapsigargin slowed the decay. Neither triadin nor junctin was affected. We conclude that CSQ expression in hESC-CMs facilitates Ca(2+) handling maturation. Our results shed insights into the suitability of hESC-CMs for therapies and as certain heart disease models for drug screening.

PubMed Disclaimer

Figures

Fig. 1.

Relative expression levels of the transcripts of various Ca2+ handling proteins probed by real-time PCR (A) and their representative protein bands from Western blot (B and C) after adenoviral transduction. Cav1.2, L-type Ca2+ channel. ALV, human adult left ventricular cardiomyocytes. Data for Ad-GFP and Ad-CSQ groups were normalized to that in Ad-CSQΔ. Values shown as means ± SE; n = 3. **P < 0.01 vs. Ad-CSQΔ; ##P < 0.01 vs. Ad-GFP.

Fig. 2.

Ad-CSQ transduction increased the caffeine-induced Ca2+ transient peak amplitude. n = 12, 20, and 14 for Ad-GFP, Ad-CSQ, and Ad-CSQΔ, respectively. Values shown as means ± SE; *P < 0.05 vs. Ad-CSQ.

Fig. 3.

Electrically induced Ca2+ transients. A: representative tracings; bar graphs summarizing basal Ca2+ (B), amplitude (C), maximum upstroke velocity (_V_max,upstroke) (D), and maximum decay velocity (_V_max,decay) (E) of transients. Values shown as means ± SE; n = 12, 29, and 15 for Ad-GFP, Ad-CSQ, and Ad-CSQΔ, respectively. *P < 0.05, **P < 0.01 vs. Ad-CSQ.

Fig. 4.

Effects of ryanodine on electrically induced Ca2+ transients. A: representative tracings; amplitude (B)and maximum _V_max,upstroke (C) after ryanodine application normalized to values recorded under control ryanodine-free conditions (dashed line i.e., 100%). n = 5, 8, and 5 for Ad-GFP, Ad-CSQ, and Ad-CSQΔ, respectively. **P < 0.01 vs. dashed line.

Fig. 5.

Effects of thapsigargin on electrically induced Ca2+ transients. A: representative tracings; amplitude (B) and _V_max,decay (C) normalized to values recorded under control thapsigargin-free conditions (dashed line, i.e., 100%); n = 6, 7, and 6 for Ad-GFP, Ad-CSQ, and Ad-CSQΔ, respectively. *P < 0.05, **P < 0.01 vs. dashed line.

Fig. 6.

Voltage-clamp protocol (A) used to investigate the current-voltage (I-V) curve of L-type Ca current (_I_Ca,L, C) in Ad-CSQ and Ad-CSQΔ groups. B: typical tracings of _I_Ca,L; n = 4 for Ad-CSQ and 3 for Ad-CSQΔ. D: membrane capacitance; n = 13 for Ad-CSQ and 10 for Ad-CSQΔ.

Fig. 7.

Typical t-tubule imaging (×60) from Ad-GFP, Ad-CSQ, and Ad-CSQΔ groups. Green, green fluorescent protein (GFP) expression from the successful transduction with either Ad-GFP, Ad-CSQ, or Ad-CSQΔ; Red, di-8-ANEPPS staining of the lipid membrane.

References

1. Beard NA, Casarotto MG, Wei L, Varsanyi M, Laver DR, Dulhunty AF. Regulation of ryanodine receptors by calsequestrin: effect of high luminal Ca2+ and phosphorylation. Biophys J 88: 3444–3454, 2005. - PMC - PubMed
1. Beard NA, Laver DR, Dulhunty AF. Calsequestrin and the calcium release channel of skeletal and cardiac muscle. Progr Biophy Mol Biol 85: 33–69, 2004. - PubMed
1. Beard NA, Sakowska MM, Dulhunty AF, Laver DR. Calsequestrin is an inhibitor of skeletal muscle ryanodine receptor calcium release channels. Biophys J 82: 310–320, 2002. - PMC - PubMed
1. Bers DM Cardiac excitation-contraction coupling. Nature 415: 198–205, 2002. - PubMed
1. Brette F, Orchard C. Resurgence of cardiac t-tubule research. Physiology 22: 167–173, 2007. - PubMed

Facilitated maturation of Ca2+ handling properties of human embryonic stem cell-derived cardiomyocytes by calsequestrin expression - PubMed (original) (raw)