Functional improvement and maturation of rat and human engineered heart tissue by chronic electrical stimulation

Abstract

Spontaneously beating engineered heart tissue (EHT) represents an advanced in vitro model for drug testing and disease modeling, but cardiomyocytes in EHTs are less mature and generate lower forces than in the adult heart. We devised a novel pacing system integrated in a setup for videooptical recording of EHT contractile function over time and investigated whether sustained electrical field stimulation improved EHT properties. EHTs were generated from neonatal rat heart cells (rEHT, n=96) or human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hEHT, n=19). Pacing with biphasic pulses was initiated on day 4 of culture. REHT continuously paced for 16-18 days at 0.5Hz developed 2.2× higher forces than nonstimulated rEHT. This was reflected by higher cardiomyocyte density in the center of EHTs, increased connexin-43 abundance as investigated by two-photon microscopy and remarkably improved sarcomere ultrastructure including regular M-bands. Further signs of tissue maturation include a rightward shift (to more physiological values) of the Ca(2+)-response curve, increased force response to isoprenaline and decreased spontaneous beating activity. Human EHTs stimulated at 2Hz in the first week and 1.5Hz thereafter developed 1.5× higher forces than nonstimulated hEHT on day 14, an ameliorated muscular network of longitudinally oriented cardiomyocytes and a higher cytoplasm-to-nucleus ratio. Taken together, continuous pacing improved structural and functional properties of rEHTs and hEHTs to an unprecedented level. Electrical stimulation appears to be an important step toward the generation of fully mature EHT.

Integrating cardiomyocytes from human pluripotent stem cells in safety pharmacology: has the time come?

“…Defined culture conditions (Burridge et al , 2014; Ribeiro et al , 2015a, 2015b) can be used to improve the maturation of hPSC‐CMs to reveal hidden disease phenotypes (Birket et al , 2015) or to drive differentiation to chamber‐specific cell populations (Devalla et al , 2015), and drug testing might benefit from their standardization. Exogenous stimuli, such as adjusted pacing frequency (Chan et al , 2013), 3D‐microenvironments (Zhao et al , 1999; Nunes et al , 2013; Hirt et al , 2014; Eder et al , 2016) and heterotypic cell co‐culture (Robertson et al , 2013) may all contribute to cell maturation, increasing the expression of ion channels and improving functional properties. Recent advances in electrophysiology (Meijer van Putten et al , 2015) and cell biology (Vaidyanathan et al , 2016) allow the introduction of exogenous I K1 conductance in hiPSC‐CMs, although neither single nor combined approaches were successful in mimicking adult maturation state entirely, in particular of Ca 2 + ‐handling, signalling and compartmentalization, mainly due to their lack of T‐tubules (Kane and Terracciano, 2015).…”

An epigenetic perspective on the failing heart and pluripotent-derived-cardiomyocytes for cell replacement therapy

“…Further, observations of automaticity, a relative depolarized resting potential, and delayed action potential upstroke suggest immature contractile behavior (BritoMartins et al, 2008;Porrello et al, 2011). Some evidence indicates immature CMs may have improved survivability post-engraftment and accompanies intriguing ideas of inculture biomechanical force training and endothelial cell coculture to coax further maturation (Reinecke et al, 1999;Caspi et al, 2007b;Sartiani et al, 2007;Rajala et al, 2011;Tulloch et al, 2011;Chong et al, 2014;Hirt et al, 2014); but the hypertrophic and arrythmogenic capacity that is inherent to heterogeneous and immature CMs is a major concern (Zhang et al, 2002b;Ng et al, 2010;Papait and Condorelli, 2010). For example, though nodal cells may be essential for creating biologic pacemakers, transplant of such protracted pacemaker activity with distinct neurohormonal response characteristics could intensify the already heightened arrhythmia risk following MI (Zhang et al, 2002b;Yi et al, 2009).…”

Conductive interpenetrating networks of polypyrrole and polycaprolactone encourage electrophysiological development of cardiac cells

“…Application of exogenous electrical pacing to condition engineered heart tissue has been shown to improve Cx43 density and organization [33]. Exogenous electrical pacing has also been demonstrated to influence electrical and calcium wave propagation and may also be implicated in reduced heterogeneity of engineered heart tissue.…”