Advancing physiological maturation in human induced pluripotent stem cell-derived cardiac muscle by gene editing an inducible adult troponin isoform switch

Abstract

Advancing maturation of stem cell-derived cardiac muscle represents a major barrier to progress in cardiac regenerative medicine. Cardiac muscle maturation involves a myriad of gene, protein, and cell-based transitions, spanning across all aspects of cardiac muscle form and function. We focused here on a key developmentally controlled transition in the cardiac sarcomere, the functional unit of the heart. Using a gene-editing platform, human induced pluripotent stem cell (hiPSCs) were engineered with a druginducible expression cassette driving the adult cardiac troponin I (cTnI) regulatory isoform, a transition shown to be a rate-limiting step in advancing sarcomeric maturation of hiPSC cardiac muscle (hiPSC-CM) toward the adult state. Findings show that induction of the adult cTnI isoform resulted in the physiological acquisition of adult-like cardiac contractile function in hiPSC-CMs in vitro. Specifically, cTnI induction accelerated relaxation kinetics at baseline conditions, a result independent of alterations in the kinetics of the intracellular Ca 2+ transient. In comparison, isogenic unedited hiPSC-CMs had no cTnI induction and no change in relaxation function. Temporal control of adult cTnI isoform induction did not alter other developmentally regulated sarcomere transitions, including myosin heavy chain isoform expression, nor did it affect expression of SERCA2a or phospholamban. Taken together, precision genetic targeting of sarcomere maturation via inducible TnI isoform switching enables physiologically relevant adult myocardium-like contractile adaptations that are essential for beat-to-beat modulation of adult human heart performance. These findings have relevance to hiPSC-CM structure-function and drug-discovery studies in vitro, as well as for potential future clinical applications of physiologically optimized hiPSC-CM in cardiac regeneration/ repair.

Myofibrillar Structural Variability Underlies Contractile Function in Stem Cell-Derived Cardiomyocytes

“…Finally, contractile function may also vary due to cell-cell differences in transcriptional programs independent from myofibrillar abundance, such as sarcomere gene transcript variant / isoform expression (e.g. TNNI3) and metabolic capacity (Birket et al, 2015;Correia et al, 2017;Wheelwright et al, 2020).…”

Myofibrillar Structural Variability Underlies Contractile Function in Stem Cell-Derived Cardiomyocytes

“…Actual intracellular relationships among myofilament organization and contractile output are likely more complex. Finally, contractile function may also vary due to cell-cell differences in transcriptional programs independent from myofibrillar abundance, such as sarcomere gene transcript variant/isoform expression (e.g., TNNI3) and metabolic capacity ( Birket et al., 2015 ; Correia et al., 2017 ; Wheelwright et al., 2020 ).…”

Effects of Sarcomere Activators and Inhibitors Targeting Myosin Cross-Bridges on Ca2+-Activation of Mature and Immature Mouse Cardiac Myofilaments

“…The effect of Mava and OM to modify Ca 2+force relations of skinned fiber preparations encouraged development to the next stage eventually leading to studies in myocytes, hearts, animal models, and human safety trials (Green et al, 2016;Morgan et al, 2010). Our data are also relevant to the use of immature cardiac myocytes, often expressing ssTnI, derived from human inducible pluripotent stem cells and proposed for use in drug development and diagnosis (Wheelwright et al, 2020) (Andrysiak et al, 2021).…”