Monophasic and Biphasic Electrical Stimulation Induces a Precardiac Differentiation in Progenitor Cells Isolated from Human Heart

Abstract

Electrical stimulation (ES) of cells has been shown to induce a variety of responses, such as cytoskeleton rearrangements, migration, proliferation, and differentiation. In this study, we have investigated whether monophasic and biphasic pulsed ES could exert any effect on the proliferation and differentiation of human cardiac progenitor cells (hCPCs) isolated from human heart fragments. Cells were cultured under continuous exposure to monophasic or biphasic ES with fixed cycles for 1 or 3 days. Results indicate that neither stimulation protocol affected cell viability, while the cell shape became more elongated and reoriented more perpendicular to the electric field direction. Moreover, the biphasic ES clearly induced the upregulation of early cardiac transcription factors, MEF2D, GATA-4, and Nkx2.5, as well as the de novo expression of the late cardiac sarcomeric proteins, troponin T, cardiac alpha actinin, and SERCA 2a. Both treatments increased the expression of connexin 43 and its relocation to the cell membrane, but biphasic ES was faster and more effective. Finally, when hCPCs were exposed to both monophasic and biphasic ES, they expressed de novo the mRNA of the voltage-dependent calcium channel Cav 3.1(α1G) subunit, which is peculiar of the developing heart. Taken together, these results show that ES alone is able to set the conditions for early differentiation of adult hCPCs toward a cardiac phenotype.

Electrical and mechanical stimulation of cardiac cells and tissue constructs

“…Electrical stimulation of a diverse array of 3-D constructs created using a variety of different biomaterials has led to the development of bioreactors and stimulation regimes which assist in stem cell differentiation towards a myocyte phenotype as well as improved cardiac cell function in vitro [184,315,316,339–344]. Many biomaterials commonly used for in vitro cell culture are not electrically conductive, thus necessitating external methods for achieving synchronous contraction across a 3D tissue.…”

“…1B, action potentials and calcium handling are directly related to contraction and therefore, generation of a synchronously contracting construct requires intercellular communication, as shown by its role in stem cell differentiation and CM maturation [344, 358,359]. Many investigators have utilized electrical cues to promote intercellular communication and CM maturation.…”

“…Stimulated hiPSC biowire constructs showed increased myofibril ultra-structural organization (demonstrated by α-actinin and cTnT staining and imaging of desmosomes) increased conduction velocity and maximum capture rate, along with decreased excitation threshold and improved electrophysiological and Ca 2+ handling properties as shown by response to epinephrine [176]. In another study, a bioreactor was designed to deliver either monophasic (5 V for 2 ms, 1 Hz) or biphasic (+2.5 V for 1 ms, −2.5 V for 1 ms, 1 Hz) electrical pulses with tunable amplitude, pulse width, and frequency to human cardiac progenitors cultured on gelatin coated glass slides [344,361]. After 72 hours, biphasic electrical stimulation resulted in upregulation of early cardiac transcription factors (MEF2D, GATA-4, Nkx2.5), greater expression of late cardiac sarcomeric proteins (cTnT, cardiac α-actinin, and SERCA2a), and earlier expression of Cx43 compared to monophasic electrical stimulation [344].…”

“…In another study, a bioreactor was designed to deliver either monophasic (5 V for 2 ms, 1 Hz) or biphasic (+2.5 V for 1 ms, −2.5 V for 1 ms, 1 Hz) electrical pulses with tunable amplitude, pulse width, and frequency to human cardiac progenitors cultured on gelatin coated glass slides [344,361]. After 72 hours, biphasic electrical stimulation resulted in upregulation of early cardiac transcription factors (MEF2D, GATA-4, Nkx2.5), greater expression of late cardiac sarcomeric proteins (cTnT, cardiac α-actinin, and SERCA2a), and earlier expression of Cx43 compared to monophasic electrical stimulation [344]. Taken together, these results demonstrated that biphasic stimulation achieved cardiac differentiation more effectively than monophasic electrical stimulation.…”

Making it stick: chasing the optimal stem cells for cardiac regeneration

“…Mechanotransduction through stretch-activated channels on stem cells can better translate cardiac environmental stiffness, tensile or shear and properly tune temporal and spatial differentiation patterns of both human CPCs and ESCs ex vivo and in vivo [79,81]. Furthermore, electric stimulation such as mono- and biphasic electrical pulses mediate human CPC expression of the calcium channel Cav1.3 [82]. Cell culture strategies to support synergism between biochemical and mechanical perturbations will help foster stem cells to an environment closer to the native myocardium, facilitate engraftment and encourage cardiogenic commitment.…”