Table 1.

In vitro studies demonstrate co-regulated circuits in maturation

Perturbation	Primary response	Secondary effect	Reference
Structural
Anisotropic ECM micropatterning	Myofibril alignment Increased contractile force	Improved anisotropic calcium propagation Improved action potential amplitude and maximum upstroke velocity Primitive t-tubulation Improved basal and maximal respiration, and spare respiratory capacity	(Ribeiro et al. 2015; Lyra-Leite et al. 2017)
3D CM aggregate formation	Improved sarcomeric gene expression and structure	Increased oxidative phosphorylation gene expression Increased TCA cycle flux	(Correia et al. 2018)
Functional
Auxotonic contraction of engineered heart tissue (EHT)	Increased cellular alignment and sarcomeric structure	Improved mitochondrial structure Increased mitochondrial protein content and mass Increased oxidative metabolism	(Ulmer et al. 2018)
Electrical stimulation with increasing intensity	Increased structural, calcium handling, and mature ion channel gene expression Improved calcium handling and contraction force	Improved ultrastructural organization Improved mitochondrial density Increased oxidative phosphorylation gene expression and activity	(Ronaldson-Bouchard et al. 2018)
Metabolic
Differentiation with fatty acids and galactose	Increased oxidative metabolism	Improved transcription of contractile and sarcomeric genes Improved sarcomeric structure and alignment Improved calcium transient velocity Improved fractional shortening and force generation	(Correia et al. 2017)
Palmitate treatment	Increased fatty acid oxidation and oxidative metabolism	Cell cycle inhibition Increased sarcomeric isoform switching	(Mills et al. 2017)
Glucose deprivation following differentiation	Increased mitochondrial structure and oxidative capacity	Increased sarcomere and contractile gene expression Cell cycle inhibition Improved calcium handling dynamics Increased maximal upstroke velocity	(Nakano et al. 2017)
Cell cycle
Mitomycin C treatment	Abrogated Ki67 expression and cell cycle cessation	Increased sarcomere assembly Improved beat rate	(Zhou et al. 2017)

Here, we describe several in vitro studies of myocyte maturation in which one pathway of maturation (e.g. structural, functional, metabolic, cell cycle) was perturbed experimentally. We describe putative primary and secondary effects of the intervention. This table is not an exhaustive list of tissue engineered approaches to improved CM maturation; for more comprehensive reviews on that topic, please see Zhu et al. (2014) and Scuderi & Butcher (2017). Instead, we compile studies in which perturbation of one maturation pathway led to previously undescribed changes to other maturation-related processes, suggesting potential co-regulation between pathways.