Calcium signalling of human pluripotent stem cell-derived cardiomyocytes

Abstract

Loss of cardiomyocytes (CMs), which lack the innate ability to regenerate, due to ageing or pathophysiological conditions (e.g. myocardial infarction or MI) is generally considered irreversible, and can lead to conditions from cardiac arrhythmias to heart failure. Human (h) pluripotent stem cells (PSCs), including embryonic stem cells (ESC) and induced pluripotent stem cells (iPSCs), can self-renew while maintaining their pluripotency to differentiate into all cell types, including CMs. Therefore, hPSCs provide a potential unlimited ex vivo source of human CMs for disease modelling, drug discovery, cardiotoxicity screening and cell-based heart therapies. As a fundamental property of working CMs, Ca(2+) signalling and its role in excitation-contraction coupling are well described. However, the biology of these processes in hPSC-CMs is just becoming understood. Here we review what is known about the immature Ca(2+)-handling properties of hPSC-CMs, at the levels of global transients and sparks, and the underlying molecular basis in relation to the development of various in vitro approaches to drive their maturation.

Figure 1. Schematic comparison of Ca²⁺ signalling pathways in adult and hPSC-derived cardiomyocytes

In both adult (A) and hPSC-CMs (B), Ca²⁺ entry via I_CaL triggers Ca²⁺ release from SR via RyRs, leading to the rise of Ca²⁺ transient; the subsequent decay is similarly accomplished by Ca²⁺ reuptake and extrusion via SERCA and NCX, respectively. The smaller amplitude and slower kinetics and null inotropic response of hPSC-CMs compared to adult can be attributed to the following differences: (1) lack of junction (JCTN) and triadin (TRDN) to facilitate RyR function in hPSC-CMs; (2) lack of calsequestrin (CSQ) for SR Ca buffering (rather, calreticulin (CALR) is expressed in hPSC-CMs); (3) lack of phospholamban (PLB) for sarcoplasmic–endoplasmic reticulum Ca²⁺-ATPase (SERCA) modulation; (4) lower SERCA and RyR expression in hPSC-CMs; (5) lack of T-tubules which contributes to a U-shape of Ca²⁺ propagation wavefront.

Figure 2

Action potentials of ventricular, atrial and pacemaker cardiomyocytes derived from HES2 (A) and H1 hESCs (C). The corresponding pie charts (B and D). Adapted from Moore et al. (2008).

Figure 3. Functional yet immature Ca²⁺ handling in hESC-CMs compared with fetal ventricular CMs (FLV-CMs) and adult ventricular CMs (ALV-CMs)

A, caffeine-induced Ca²⁺ transient in HES2-CMs, H1-CMs and FLV-CMs. B, percentage of respective CMs that are sensitive to caffeine treatment. C, Ca²⁺-handling protein expression profile in HES2-CMs, H1-CMs, FLV-CMs and ALV-CMs. D, immunostaining of ryanodine receptors in HES2-CMs, H1-CMs and FLV-CMs. Adapted from Liu et al. (2007).

Figure 4. Absence of T-tubules in hESC-CMs

Di-8-ANEPPS staining shows no intracellular fluorescent spots in hESC-CMs (A) compared with adult cardiomyocytes (C). B and D, Atomic force microscopy imaging reveals no T-tubules, present as regularly spaced pores, in hESC-CMs. E, lack of T-tubules results in a non-uniform, U-shaped Ca²⁺ transient. Adapted from Lieu et al. (2009).

Figure 5. CSQ overexpression increases the amplitude of caffeine-induced Ca²⁺ transient

A, representative caffeine-induced Ca²⁺ transient tracings for Ad-GFP, Ad-CSQ and Ad-CSQΔ transduced hESC-CMs. B, bar graphs of amplitude. *P < 0.05. Adapted from Liu et al. (2009).

Figure 6. Adenovirus-mediated Kir2.1 overexpression led to hESC-CMs maturation

A, representative tracings of human embryonic stem cell-derived cardiomyocytes produced by directed differentiation protocol (ddhESC-CMs), showing action potentials (APs) of spontaneously firing (left) and quiescent (right) ventricular CMs. Arrow indicates phase 4-like depolarization. B, APs of Ad-Kir2.1-transduced ventricular ddhESC-CMs. I_k1 in inset. The phase 4-like depolarization was eliminated (arrow) by Ad-Kir2.1 transduction (left). The percentage of quiescent ventricular ddhESC-CMs increased significantly to 100% after Ad-Kir2.1 transduction (middle). Resting membrane potentials (RMPs) of Ad-Kir2.1-transduced ventricular ddhESC-CMs became significantly hyperpolarized relative to control (right). C, after Ad-Kir2.1 transduction, the mRNA expression of contractile elements is significantly reduced relative to control hESC-CMs. D, both electrically conditioned atrial and ventricular ddhESC-CMs harboured action potentials without phase 4-depolarization (left). More hyperpolarized resting membrane potential (RMP) was shown (right). E, caffeine-elicited Ca²⁺ transients from control (blue) and electrically conditioned (red) ddhESC-CMs, and their averaged peak amplitude were also presented in bar graph. Adapted from Lieu et al. (2012).