Cardiomyocyte maturation: advances in knowledge and implications for regenerative medicine

Abstract

Our knowledge of pluripotent stem cell (PSC) biology has advanced to the point where we now can generate most cells of the human body in the laboratory. PSC-derived cardiomyocytes can be generated routinely with high yield and purity for disease research and drug development, and these cells are now gradually entering the clinical research phase for the testing of heart regeneration therapies. However, a major hurdle for their applications is the immature state of these cardiomyocytes. In this Review, we describe the structural and functional properties of cardiomyocytes and present the current approaches to mature PSC-derived cardiomyocytes. To date, the greatest success in maturation of PSC-derived cardiomyocytes has been with transplantation into the heart in animal models and the engineering of 3D heart tissues with electromechanical conditioning. In conventional 2D cell culture, biophysical stimuli such as mechanical loading, electrical stimulation and nanotopology cues all induce substantial maturation, particularly of the contractile cytoskeleton. Metabolism has emerged as a potent means to control maturation with unexpected effects on electrical and mechanical function. Different interventions induce distinct facets of maturation, suggesting that activating multiple signalling networks might lead to increased maturation. Despite considerable progress, we are still far from being able to generate PSC-derived cardiomyocytes with adult-like phenotypes in vitro. Future progress will come from identifying the developmental drivers of maturation and leveraging them to create more mature cardiomyocytes for research and regenerative medicine.

Fig. 1 |. Cardiomyocyte maturation features.

a | Cardiomyocytes undergo dynamic changes structurally and functionally during the course of maturation in vivo. The features of human pluripotent stem cell-derived cardiomyocytes and the sought-after maturation features of adult-like cardiomyocytes are illustrated in parts b–g. b | Cardiomyocytes undergo an increase in size and become anisotropic, organizing their contractile cytoskeleton and remodelling nuclei, junctions and other organelles, as described below. c | As cardiomyocytes mature, their action potentials change dramatically, including loss of automaticity, acquiring a more negative resting membrane potential (approximately −90 mV), and increases in action potential duration and amplitude. At the tissue level, immature cardiomyocytes have circumferentially distributed gap junctions and, during maturation, the junctions become polarized to intercalated discs at the cell ends, resulting in faster electrical conduction. d | Improved calcium handling in mature cardiomyocytes is mediated by the increased volume and calcium stores of the sarcoplasmic reticulum, the development of T-tubules and the expression and synchrony of calcium-handling proteins for the establishment of excitation–contraction coupling. e | Increases in contractile force with maturation are due to increases in myofibril content, improved alignment of myofibrils with registration of their sarcomeres and the switch in the expression of myofibril protein isoforms. f | Metabolic maturation entails the switch to fatty acid utilization, concurrent with increases in mitochondria number and the expression of the machinery for β-oxidation (β-Ox) and oxidative phosphorylation. g | Immature cardiomyocytes have proliferation potential whereas more mature cells principally undergo hypertrophy (increase in volume) with increasing load. Mature human cardiomyocytes have more DNA content per nucleus than immature cells, whereas rodent cardiomyocytes have increased nuclei number. CPT, carnitine O-palmitoyltransferase; FA-CoA, fatty acyl-CoA ester; mtDNA, mitochondrial DNA; n, haploid content of chromosomes; NCX, sodium–calcium exchanger; RYR2, ryanodine receptor 2; SERCA2a, sarcoplasmic/endoplasmic reticulum calcium ATPase 2a.

Fig. 2 |. Cardiomyocyte electrophysiology.

Action potential recordings from a human pluripotent stem cell (hPSC)-derived cardiomyocyte (part a) and an isolated human primary left ventricular cardiomyocyte (part b). The major ion currents involved in establishing the phases of the cardiomyocyte action potential and the corresponding genes and channels are shown below the tracings. The action potentials were recorded by patch clamp and, given the variability in patch clamp preparation, the magnitude and the duration of the different currents are approximate^,. Both tracings are on the same timescale. Ca_v1.2, voltage-dependent L-type calcium channel; Ca_v3.2, voltage-dependent T-type calcium channel; HCN4, hyperpolarization-activated cyclic nucleotide-gated channel 4; hERG, human Ether-à-go-go-related gene (also known as K_v11.1); I_CaL, L-type calcium channel current; I_CaT, T-type calcium channel current; I_f, pacemaker or funny current; I_K1, inward-rectifier potassium current; I_Kr, rapid delayed-rectifier potassium current; I_Ks, slow delayed-rectifier potassium current; I_Na, sodium current; I_to, transient outward potassium current; K_ir2.1, inward-rectifier potassium channel; K_v4.3, voltage-gated potassium channel; KvLQT1 or K_v7.1, potassium channel; Na_v1.5, sodium channel; pA/pF, picoampere per picofarad. Part a tracing courtesy of C. E. Murry and X. Yang. Part b tracing is reprinted from ReF., CC BY 4.0 (https://creativecommons.org/licenses/by/4.0).

Fig. 3 |. Cell cycle activity in cardiomyocytes.

A high proliferative activity is characteristic of immature cardiomyocytes, whereas mature cells are largely quiescent and polyploid. Critical regulators of cell cycle activity are highlighted. CDK, cyclin-dependent kinase; n, haploid content of chromosomes.