•  Tromondae K. Feaster, Nicole Feric, Isabella Pallotta, Akshay Narkar, Maura Casciola, Michael P. Graziano, Roozbeh Aschar-Sobbi and Ksenia Blinova

Acute effects of cardiac contractility modulation stimulation in conventional 2D and 3D human induced pluripotent stem cell-derived cardiomyocyte models

Published in Frontiers in Physiology, Volume 13 - 2022

Abstract

Car­diac con­trac­til­i­ty mod­u­la­tion (CCM) is a med­ical device ther­a­py where­by non-exci­ta­to­ry elec­tri­cal stim­u­la­tions are deliv­ered to the myocardi­um dur­ing the absolute refrac­to­ry peri­od to enhance car­diac func­tion. We pre­vi­ous­ly eval­u­at­ed the effects of the stan­dard CCM pulse para­me­ters in iso­lat­ed rab­bit ven­tric­u­lar car­diomy­ocytes and 2D human induced pluripo­tent stem cell-derived car­diomy­ocyte (hiP­SC-CM) mono­lay­ers, on flex­i­ble sub­strate. In the present study, we sought to extend these results to human 3D micro­phys­i­o­log­i­cal sys­tems to devel­op a robust mod­el to eval­u­ate var­i­ous clin­i­cal CCM pulse para­me­ters in vit­ro. HiP­SC-CMs were stud­ied in con­ven­tion­al 2D mono­lay­er for­mat, on stiff sub­strate (i.e., glass), and as 3D human engi­neered car­diac tis­sues (ECTs). Car­diac con­trac­tile prop­er­ties were eval­u­at­ed by video (i.e., pix­el) and force-based analy­sis. CCM puls­es were assessed at vary­ing elec­tri­cal dos­es’ using a com­mer­cial pulse gen­er­a­tor. A robust CCM con­trac­tile response was observed for 3D ECTs. Under com­pa­ra­ble con­di­tions, con­ven­tion­al 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, dis­played no con­trac­tile response. 3D ECTs dis­played enhanced con­trac­tile prop­er­ties includ­ing increased con­trac­tion ampli­tude (i.e., force), and accel­er­at­ed con­trac­tion and relax­ation slopes under stan­dard acute CCM stim­u­la­tion. More­over, 3D ECTs dis­played enhanced con­trac­til­i­ty in a CCM pulse para­me­ter-depen­dent man­ner by adjust­ment of CCM pulse delay, dura­tion, ampli­tude, and num­ber rel­a­tive to base­line. The observed acute effects sub­sided when the CCM stim­u­la­tion was stopped and grad­u­al­ly returned to base­line. These data rep­re­sent the first study of CCM in 3D hiP­SC-CM mod­els and pro­vide a non­clin­i­cal tool to assess var­i­ous CCM device sig­nals in 3D human car­diac tis­sues pri­or to in vivo ani­mal stud­ies. More­over, this work pro­vides a foun­da­tion to eval­u­ate the effects of addi­tion­al car­diac med­ical devices in 3D ECTs.

1 Intro­duc­tion

Con­ven­tion­al 2D mono­lay­er human induced pluripo­tent stem cell-derived car­diomy­ocytes (hiP­SC-CMs), on stiff sub­strate, have been demon­strat­ed to be use­ful for the eval­u­a­tion of drugs and oth­er chem­i­cal com­pounds (Bli­no­va et al., 2018; Bur­nett et al., 2021; Yang et al., 2022). How­ev­er, such mod­els may not be appro­pri­ate for med­ical device opti­miza­tion or safe­ty assess­ment where induced effects are intrin­si­cal­ly reliant on more com­plex cul­ture con­di­tions, which enable a 3D lev­el tis­sue response (e.g., trans­mur­al lesion for­ma­tion or phys­i­o­log­i­cal effects). Human 3D car­diac micro­phys­i­o­log­i­cal sys­tems, includ­ing hiP­SC-CM engi­neered car­diac tis­sues (ECTs), are gain­ing sig­nif­i­cant inter­est for car­diac safe­ty phar­ma­col­o­gy assess­ment as more biotech­nol­o­gy com­pa­nies explore and adapt this tech­nol­o­gy (Mey­er et al., 2019; Majid et al., 2020; Qu et al., 2020). The 3D ECT mod­el has been estab­lished for the detec­tion of known and nov­el inotrop­ic com­pounds as well as dis­ease mod­el­ing (Fer­ic et al., 2019; Veld­huizen et al., 2019; Qu et al., 2020). Yet, the cur­rent gold-stan­dard, con­ven­tion­al 2D mono­lay­er hiP­SC-CMs plat­ed on stiff sub­strate (i.e., glass/​plastic) for 7 – 14 days, remains pop­u­lar on account of its reduced cost, high-through­put capa­bil­i­ties, tech­ni­cal ease, and numer­ous stan­dard­ized plate-based appli­ca­tions. The move to more com­plex hiP­SC-CM based mod­els begs the ques­tion whether stan­dard con­ven­tion­al 2D mono­lay­er hiP­SC-CM mod­els, on stiff sub­strate, are suf­fi­cient to eval­u­ate var­i­ous car­diac con­trac­til­i­ty mod­u­la­tion (CCM) elec­tro­phys­i­o­log­i­cal sig­nals. Here, we do not intend to improve or devel­op new hiP­SC-CM based mod­els. Rather, we set out to char­ac­ter­ize the poten­tial util­i­ty of hiP­SC-CM mod­els to eval­u­ate CCM device sig­nals by com­par­ing the per­for­mance of two well-estab­lished com­mer­cial­ly-avail­able mod­els. As such, the work described here will focus on the com­par­i­son of the con­trac­tile response of con­ven­tion­al 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, and 3D ECTs to CCM.

CCM is a med­ical device ther­a­py where­in non-exci­ta­to­ry elec­tri­cal stim­u­la­tions are deliv­ered to the myocardi­um dur­ing the absolute refrac­to­ry peri­od (Camp­bell et al., 2020; Feast­er et al., 2021). The first CCM device, an implantable med­ical device with con­tact leads placed in the myocardi­um, was approved in the U.S. in 2019 to treat heart fail­ure (HF) patients (NYHA III), with a left ven­tric­u­lar ejec­tion frac­tion rang­ing from 25 to 45% (Camp­bell et al., 2020; FDA 2019). Car­diac elec­tro­phys­i­o­log­i­cal med­ical devices, includ­ing CCM and car­diac resyn­chro­niza­tion ther­a­py (CRT), have been devel­oped to treat HF patients resis­tant to tra­di­tion­al phar­ma­cother­a­pies. While CRT is the first-line treat­ment for HF patients dis­play­ing an abnor­mal sinus rhythm and a pro­longed QRS dura­tion, a sig­nif­i­cant pop­u­la­tion of HF patients (e.g., 60% – 70%) present with nor­mal sinus rhythm or QRS dura­tion. CCM is indi­cat­ed for such patients with pro­longed QRS who are not eli­gi­ble for CRT. Con­se­quent­ly, there is a sig­nif­i­cant gap for viable treat­ment options for this pop­u­la­tion and CCM is her­ald­ed as a poten­tial solu­tion (Camp­bell et al., 2020; Feast­er et al., 2021). As a result, nov­el CCM devices are expect­ed to be devel­oped to address addi­tion­al device func­tion­al­i­ties and patient populations.

Lack of human non­clin­i­cal mod­els to eval­u­ate car­diac med­ical device safe­ty and effec­tive­ness cur­rent­ly hin­ders the reg­u­la­to­ry review process and pro­duces a sig­nif­i­cant bur­den on ani­mal mod­els (Har­ris et al., 2013; Strauss and Bli­no­va, 2017; Feast­er et al., 2021). Addi­tion­al­ly, the direct effects of var­i­ous CCM stim­u­la­tion para­me­ters on human car­diomy­ocyte phys­i­ol­o­gy remains poor­ly under­stood. Pre­vi­ous stud­ies have pro­vid­ed impor­tant insight into our under­stand­ing of CCM but are hin­dered due to species dif­fer­ences in car­diomy­ocyte biol­o­gy. HiP­SC-CMs are gain­ing inter­est for dis­ease mod­el­ing, drug devel­op­ment, and safe­ty phar­ma­col­o­gy. How­ev­er, their applic­a­bil­i­ty for car­diac med­ical device assess­ment has not been thor­ough­ly vet­ted. HiP­SC-CMs are a use­ful in vit­ro mod­el to assess the mol­e­c­u­lar and func­tion­al effects of CCM on human car­diac tis­sue (Feast­er et al., 2021). How­ev­er, con­trac­tile stud­ies in hiP­SC-CMs have been lim­it­ed as a result of imma­ture con­trac­tile prop­er­ties when plat­ed as con­ven­tion­al 2D mono­lay­ers on stiff sub­strates (i.e., glass or plas­tic) (Feast­er et al., 2021; Korner et al., 2021; Huethorst et al., 2022; Narkar et al., 2022). We pre­vi­ous­ly demon­strat­ed that 2D mono­lay­er hiP­SC-CMs, on flex­i­ble sub­strate, dis­played increased con­trac­tion and cal­ci­um han­dling prop­er­ties when acute­ly stim­u­lat­ed with stan­dard CCM pulse. While sig­nif­i­cant, these effects were tran­sient and atten­u­at­ed rel­a­tive to that of tra­di­tion­al pap­il­lary mus­cle mod­els (e.g., rab­bit) (Brunck­horst et al., 2006). Of the vari­ety of cul­ture con­di­tions test­ed, we dis­cov­ered that the 2D mono­lay­er hiP­SC-CM mod­el required the com­bi­na­tion of both a sub­max­i­mal extra­cel­lu­lar Ca con­cen­tra­tion [0.5 mM] and a flex­i­ble sub­strate to elu­ci­date the CCM response (Feast­er et al., 2021). Using these con­di­tions, we demon­strat­ed con­trac­tile ampli­tude and kinet­ic enhance­ment and cal­ci­um depen­dance (Feast­er et al., 2021). How­ev­er, it is impor­tant to high­light that at a phys­i­o­log­i­cal extra­cel­lu­lar Ca con­cen­tra­tion (e.g., ∼2 mM) there was no CCM con­trac­tile response (Feast­er et al., 2021). To date, most hiP­SC-CM stud­ies rely on the stan­dard con­ven­tion­al 2D mono­lay­er on stiff sub­strate mod­el. In such mod­els, con­trac­tile prop­er­ties are lim­it­ed as a result of vari­able mor­phol­o­gy, the lack of a dom­i­nant axis of myofib­ril align­ment (Feast­er et al., 2015), and a per­ceived glass stiff­ness in the GPa range (Ribeiro et al., 2015). This is con­trary to 2D hiP­SC-CM mono­lay­ers, on flex­i­ble sub­strate (i.e., Matrigel Mat­tress), which have an elas­tic mod­u­lus of approx­i­mate­ly 5.8 kPa rep­re­sent­ing a phys­i­o­log­i­cal­ly rel­e­vant range for myocardi­um (i.e., 4.0 – 46.2 kPa) (Sun et al., 2012; Ribeiro et al., 2015).

Here, we extend these stud­ies to acute CCM eval­u­a­tion in 3D ECTs at phys­i­o­log­i­cal extra­cel­lu­lar Ca con­cen­tra­tions. There is a sig­nif­i­cant need to under­stand the chron­ic effects of CCM (e.g., hours to days) on human biol­o­gy but as a first step the acute effects must be char­ac­ter­ized in a stan­dard­ized human-based mod­el (Narkar et al., 2022). We demon­strate that when cul­tured as 3D ECTs, hiP­SC-CMs respond to acute elec­tri­cal stim­u­la­tion mim­ic­k­ing the stan­dard CCM sig­nal by an increase in con­trac­tile force. More­over, unlike 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, 3D ECTs dis­play a robust con­trac­tile response to var­i­ous CCM stim­u­la­tion sig­nals. We fur­ther eval­u­at­ed the com­plete range of clin­i­cal CCM pulse para­me­ters in 3D ECTs and estab­lished a para­me­ter-depen­dent con­trac­tile response while con­ven­tion­al 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, remained unaf­fect­ed for each para­me­ter test­ed. To the best of our knowl­edge, this is the first 3D hiP­SC-CM study to elu­ci­date the acute effects of CCM and may pro­vide impor­tant insights on the effects of vary­ing CCM pulse para­me­ters at the bench, ahead of in vivo stud­ies. This may improve deci­sion mak­ing and sup­port safe­ty or effec­tive­ness stud­ies for future CCM devices. Here, we estab­lish a stan­dard­ized 3D ECT-based method to quan­ti­fy and opti­mize acute CCM effects in vit­ro.

2 Mate­ri­als and methods

2.1 2D human iPSC-CM deriva­tion and culture

Cry­op­re­served hiP­SC-CMs (iCell Car­diomy­ocytes2 01434, R1017 Fuji­film Cel­lu­lar Dynam­ics, Inc.) were thawed and plat­ed, as pre­vi­ous­ly described, accord­ing to the manufacturer’s instruc­tion (Ma et al., 2011; Bli­no­va et al., 2017; Bli­no­va et al., 2018; Bli­no­va et al., 2019). All hiP­SC-CMs used in this study were derived from the same hiP­SC line, which was repro­grammed from fibrob­last donor tis­sue, iso­lat­ed from an appar­ent­ly healthy nor­mal Cau­casian female, <18 years old (Ma et al., 2011; Feast­er et al., 2021). Briefly, 116,000 viable cells were plat­ed per well of a 48-well glass bot­tom plate (Mat­Tek) P48G1.5 – 6‑F on Matrigel (1:60), 356,230 Corn­ing (Feast­er et al., 2015; Hwang et al., 2015). iCell Car­diomy­ocytes Main­te­nance Medi­um (#M1003, Fuji­film Cel­lu­lar Dynam­ic, Inc.) was changed every 48 h there­after and cells were allowed to recov­er from cry­op­reser­va­tion for 7 days at 37°C before exper­i­ments were performed.

2.2 3D human iPSC-CM Biowire™ II tis­sue generation

Engi­neered car­diac tis­sues (ECTs) were gen­er­at­ed as pre­vi­ous­ly described (Fer­ic et al., 2019; Qu et al., 2020). Briefly 100,000 hiP­SC-CMs (iCell Car­diomy­ocytes2 01434, R1017 Fuji­film Cel­lu­lar Dynam­ics, Inc.) and 10,000 nor­mal human ven­tric­u­lar car­diac fibrob­lasts (Lon­za) were embed­ded in a hydro­gel of fib­rin (Sig­ma-Aldrich), col­la­gen (Sig­ma-Aldrich) and Matrigel (Corn­ing). Each well of the Biowire II plat­form was seed­ed with cell/​hydrogel sus­pen­sion and exposed to a 7‑week elec­tri­cal con­di­tion­ing stim­u­la­tion pro­to­col before exper­i­ments were per­formed. A total of three 3D ECTs were used for each con­trac­tion exper­i­ment, with repeat­ed mea­sure­ments tak­en after base­line was estab­lished. The aver­age cross-sec­tion­al area for the Biowire II plat­form is 0.066 ± 0.001 mm2 this area was used to cal­cu­late stress (Fer­ic et al., 2019). Dur­ing exper­i­ments, 3D ECTs were super­fused with Tyrode’s solu­tion as described below.

2.3 Elec­tri­cal field (CCM) stimulation

2D and 3D hiP­SC-CM mod­els were stim­u­lat­ed with com­mer­cial pulse gen­er­a­tors: sin­gle chan­nel (Mod­el 4100, A‑M Sys­tems, Sequim, WA) and mul­ti-chan­nel (Mod­el 3800, A‑M Sys­tems, Sequim, WA). For 2D hiP­SC-CMs, cus­tom plat­inum elec­trodes (inter elec­trode dis­tance 2.0 mm, and width 1.0 mm) com­pat­i­ble with stan­dard 48-well glass bot­tom plates (Mat­Tek), were placed in each well sequen­tial­ly, as pre­vi­ous­ly described (Feast­er et al., 2021). For 3D ECTs, a 600 µl cham­ber fab­ri­cat­ed with par­al­lel plat­inum elec­trodes (inter elec­trode dis­tance 1.2 mm, and width 2.0 mm) was used for each tis­sue. In this con­fig­u­ra­tion, the tis­sues are in-line between the par­al­lel elec­trodes. For both mod­els, pac­ing (i.e., base­line) and CCM elec­tri­cal puls­es were deliv­ered through these plat­inum elec­trodes, result­ing in field stim­u­la­tion as pre­vi­ous­ly described (Bli­no­va et al., 2014) (Fig­ure 1C). At base­line, cells were paced at 1.5 times the cap­ture thresh­old using monopha­sic square wave puls­es. 2D hiP­SC-CMs were paced at 1 Hz (2 ms pac­ing pulse dura­tion) and approx­i­mate­ly 14 V/​cm 3D ECTs were paced at 1 Hz (2 ms pac­ing pulse dura­tion) and approx­i­mate­ly 10 V/​cm. In both mod­els, CCM stim­u­la­tion was deliv­ered as 1 to 3 bipha­sic puls­es, 4.5 – 7 ms phase dura­tion, 1 – 10 V pulse ampli­tude, and an inter­phase inter­val of zero. The range for the delay test­ed, 3 – 160 ms, was defined as the inter­val between the end of the base­line pac­ing pulse and the begin­ning of the CCM pulse. All para­me­ters eval­u­at­ed (i.e., pulse delay, pulse dura­tion, pulse ampli­tude, and pulse num­ber) were deter­mined based on the clin­i­cal device para­me­ter range (Impulse­Dy­nam­ics, 2018; Impulse­Dy­nam­ics, 2019; Camp­bell et al., 2020) (Sup­ple­men­tary Table S1).

FIGURE 1. 2D and 3D hiPSC-CM CCM models (A) Effect of standard clinical CCM signal on conventional 2D (stiff substrate) and (B) 3D ECT hiPSC-CM models. Representative contraction recordings before CCM (Baseline), during CCM (7.5 V) and after CCM (recovery) in 2D hiPSC-CM monolayer and 3D ECT. 3D ECTs displaying enhanced CCM-induced force. White arrow indicates edge of 3D ECT where force transducer was connected (C) Baseline (Control) pacing waveform (Top) and standard biphasic CCM waveform (i.e., two biphasic pulse, 7.5 V, 5.14 ms duration, 30 ms delay) (Bottom). (D) Schematic contractility waveform depicting key contractile parameters evaluated. Contraction slope and relaxation slope were calculated as maximum and minimum of the time derivative of the contractility amplitude respectively. White square indicates region of interest of 2D monolayer.

2.4 Mea­sure­ment of con­trac­tile properties

A con­trac­til­i­ty plat­form and soft­ware (Cel­lOP­TIQ, Clyde Bio­sciences), based on video pix­el dis­place­ment, was used to mea­sure 2D hiP­SC-CM con­trac­til­i­ty (Sala et al., 2018; Saleem et al., 2020b; Huethorst et al., 2022). 2D hiP­SC-CMs were imaged direct­ly in plates by an invert­ed flu­o­res­cence micro­scope (Zeiss) using a ×40 objec­tive. For 2D hiP­SC-CMs, a region of inter­est (ROI) was select­ed near the cen­ter of the well and kept con­stant through­out the exper­i­ment. A cam­era con­nect­ed to the front port of the micro­scope was used for con­trac­tion acqui­si­tion. Tem­per­a­ture, 37°C, and 5% CO2 were main­tained by an envi­ron­men­tal con­trol cham­ber (OKO­LAB, Inc.). For 3D ECTs, con­trac­tile force mea­sure­ments were obtained using a force trans­duc­er. Tis­sues were placed in a tis­sue bath (1 cm × 5 cm, 600 μl) and the poly­mer wires on one end of the tis­sue were cut and attached to a force trans­duc­er (AE801, Kro­nex Tech­nolo­gies, Oak­land, CA) using a stain­less steel wire fash­ioned into a bas­ket. The oth­er end of the tis­sue was immo­bi­lized using stain­less steel wires attached to a micro­ma­nip­u­la­tor (Sup­ple­men­tary Fig­ure S1A; Sup­ple­men­tary Video S1). The AE801 is a sil­i­con-based strain gauge with two piezore­sis­tive ele­ments. The AE801 was con­nect­ed to a Wheat­stone bridge ampli­fi­er in half bridge mode (Trans­bridge 4M, WPI, Sara­so­ta, FL) which con­verts the resis­tance changes in the strain gauge to a volt­age sig­nal. The AE801 was pre-cal­i­brat­ed pri­or to exper­i­men­ta­tion with known weights and a rela­tion­ship between volt­age and force of 104.4 μN/​mV was used to con­vert the volt­age record­ing to force mea­sure­ments. Sig­nals were dig­i­tized using the Digi­da­ta 1322 A and record­ed at 10 kHz with Axo­scope Soft­ware (Mol­e­c­u­lar Devices, San Jose, CA). Tis­sues were con­stant­ly super­fused in the tis­sue bath with Tyrode’s solu­tion at 4 ml/​min and the tem­per­a­ture in the bath was main­tained at 37°C.

All exper­i­ments were per­formed in Tyrode’s solu­tion con­tain­ing (in mmol/​L): CaCl2 1.8, NaCl 134, KCl 5.4, MgCl2 1, glu­cose 10, and HEP­ES 10, pH adjust­ed to 7.4 with NaOH at 37 °C. To eval­u­ate extra­cel­lu­lar Ca con­cen­tra­tion effects on CCM, we adjust­ed total extra­cel­lu­lar Ca range from 0.5 to 4 mM (Feast­er et al., 2015; Feast­er et al., 2021). For each exper­i­men­tal group, a min­i­mum 5 s record­ing was tak­en and ana­lyzed, as pre­vi­ous­ly described (Feast­er et al., 2015; Feast­er et al., 2021). The con­trac­tile base­line was estab­lished by allow­ing equi­li­bra­tion to a steady state before mea­sure­ments. Con­trac­tile prop­er­ties, includ­ing con­trac­tion ampli­tude or force, time to peak (the time from 10% peak height to the peak of con­trac­tion), time to base­line at 90% (the time from peak of con­trac­tion to base­line 90% of peak height), con­trac­tion dura­tion at 50%, CD 50% (the time from 50% peak height to peak of con­trac­tion and from peak to 50% peak height of relax­ation), con­trac­tion slope, and relax­ation slope were eval­u­at­ed (Fig­ure 1D, Sup­ple­men­tary Table S2) Con­trac­tion slope and relax­ation slope were cal­cu­lat­ed as the max­i­mum and min­i­mum time deriv­a­tive of the con­trac­til­i­ty ampli­tude, respectively.

2.5 Sta­tis­ti­cal analysis

All sta­tis­ti­cal analy­ses were per­formed using Graph­Pad Prism 8 soft­ware (Prism 8, Graph­Pad Soft­ware, CA). Dif­fer­ences among the groups are pre­sent­ed as mean ± stan­dard error of the mean (SEM). Dif­fer­ences were assessed as fold-change rel­a­tive to base­line (i.e., pac­ing only) using a Two-Way ANO­VA. Results were con­sid­ered sta­tis­ti­cal­ly sig­nif­i­cant if the p-val­ue was less than 0.05, adjust­ed by Tukey cor­rec­tion for mul­ti­ple comparisons.

3 Results

3.1 Com­par­i­son of 2D and 3D human CCM models

Com­mer­cial­ly avail­able, cry­op­re­served hiP­SC-CMs were eval­u­at­ed as con­ven­tion­al 2D mono­lay­ers, on stiff sub­strate (i.e., glass) (Feast­er et al., 2015; Wang et al., 2018), and as 3D ECTs (Fig­ure 1A,B). For con­sis­ten­cy and phys­i­o­log­i­cal rel­e­vance, an extra­cel­lu­lar cal­ci­um con­cen­tra­tion of 1.8 mM was used for both mod­els. We first eval­u­at­ed the effects of the stan­dard acute CCM stim­u­la­tion para­me­ters in both hiP­SC-CM mod­els: two bipha­sic puls­es, 7.5 V, 5.14 ms dura­tion, and 30 ms delay (Fig­ure 1C) (Stix et al., 2004; Kuschyk et al., 2017; Tint et al., 2019; Mas­toris et al., 2021; Ami­raslanov et al., 2022). Sig­nif­i­cant­ly enhanced con­trac­tile prop­er­ties were observed in 3D ECTs includ­ing increased ampli­tude (i.e., force) and accel­er­at­ed con­trac­tion and relax­ation slopes (Fig­ure 1B) (Table. 1). Con­ven­tion­al 2D hiP­SC-CM mono­lay­ers, dis­played no CCM-induced con­trac­tile response (Fig­ure 1A) (Table. 1). Here­in, we mea­sure the acute effects of var­i­ous clin­i­cal CCM dos­es” (Fig­ure 2) (Sup­ple­men­tary Table S1) on car­diac con­trac­tile prop­er­ties (e.g., ampli­tude, time to peak and con­trac­tion slope) (Fig­ure 1D) com­pared to that of base­line (i.e., stan­dard field stim­u­la­tion pac­ing, 1 Hz) (Fig­ure 1C), to deter­mine which mod­el can best eval­u­ate the effects of var­i­ous CCM sig­nals in vit­ro.

TABLE 1. Stan­dard CCM sig­nal con­trac­tile characterization.

FIGURE 2. CCM Pulse Waveforms Evaluated. Waveforms depicting CCM Parameters Tested (A) Pulse Delay 1–160 ms, additional parameters fixed at duration 5.14 ms, amplitude 7.5 V and pulse number 2. (B) Pulse Duration 4.5–7 ms, additional parameters fixed at delay 30 ms, amplitude 7.5 V and pulse number 2 (C) Pulse Amplitude 1–10 V, additional parameters fixed at delay 30 ms, duration 5.14 ms and pulse number 2. (D) Pulse Number 1 to 3 Pulses, additional parameters fixed at delay 30 ms, duration 5.14 ms and amplitude 7.5 V. Pulse waveforms not to scale (ImpulseDynamics, 2018; ImpulseDynamics, 2019).

3.2 CCM is sen­si­tive to extra­cel­lu­lar cal­ci­um mod­u­la­tion in 3D ECTs

To deter­mine the CCM depen­dance on extra­cel­lu­lar Ca con­cen­tra­tion, we eval­u­at­ed the effects of CCM as a func­tion of increas­ing lev­els of extra­cel­lu­lar cal­ci­um con­cen­tra­tion from 0.5 to 4 mM. Using the stan­dard CCM stim­u­la­tion set­tings (Fig­ure 1C), 3D ECTs dis­played sig­nif­i­cant­ly enhanced con­trac­tile ampli­tude (Fig­ure 3B,C) rel­a­tive to base­line at 0.5, 1 and 2 mM Ca. 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, dis­played no CCM-induced response at any Ca con­cen­tra­tion test­ed rel­a­tive to base­line (Fig­ures 2A,C). Con­sis­tent with pre­vi­ous stud­ies, the 3D ECT response to CCM was blunt­ed at high­er extra­cel­lu­lar cal­ci­um con­cen­tra­tions (Burk­hoff et al., 2001; Brunck­horst et al., 2006; Feast­er et al., 2021) (Fig­ures 3B,C). The CCM-induced increase in 3D ECT con­trac­tile force was sig­nif­i­cant­ly more pro­nounced when extra­cel­lu­lar cal­ci­um con­cen­tra­tion was low­ered from 4 to 2 mM. These results sug­gest that the CCM effects on con­trac­tile force in 3D ECTs, includ­ing increased ampli­tude and accel­er­at­ed con­trac­tion and relax­ation slopes, are depen­dent on the extra­cel­lu­lar free Ca concentration.

FIGURE 3. Effect of Extracellular Ca Modulation on CCM Response in 2D and 3D models (A) Representative contraction traces for baseline (i.e., field stimulation pacing, 1 Hz). and CCM for 2D hiPSC-CMs monolayer and (B) 3D ECTs. CCM was applied as the standard biphasic CCM waveform (i.e., two biphasic pulse, 7.5 V, 5.14 ms duration, 30 ms delay). hiPSC-CMs were exposed to increasing concentrations of extracellular Ca [Cao] 0.5–4 mM (C) Summary data graphs. Data are mean ± SEM. n = 3 – 6 per group. *p < 0.05 (3D vs. BL), #p < 0.05 (3D vs. 3D), +p < 0.05 (2D vs. BL).

3.3 CCM pulse delay enhances con­trac­tile properties

We next eval­u­at­ed the effects of vary­ing the dura­tion of the delay between pac­ing and CCM stim­u­la­tion on 2D and 3D hiP­SC-CM mod­els (Fig­ure 2A). The range of delays test­ed was com­pa­ra­ble to that of the clin­i­cal device (i.e., 3 – 160 ms). 3D ECTs exposed acute­ly to clin­i­cal­ly rel­e­vant CCM pulse para­me­ters exhib­it­ed enhanced car­diac con­trac­til­i­ty that sub­sided grad­u­al­ly when the CCM sig­nal was elim­i­nat­ed. Specif­i­cal­ly, 3D ECTs dis­played enhanced con­trac­tile prop­er­ties as a func­tion of CCM delay tim­ing includ­ing increased force ampli­tude and accel­er­at­ed con­trac­tion and relax­ation slopes (Fig­ures 4B,C). Enhanced con­trac­tile force was observed with ≥30 ms delay. At the short­est delay test­ed (3 ms), 3D ECTs dis­played a neg­a­tive inotrop­ic response that was reversed in a time depen­dent-man­ner as the pulse delay time increased into the refrac­to­ry peri­od. At the longest delay test­ed (160 ms), 3D ECTs dis­played an increased con­trac­tion ampli­tude of 115.8 ± 11.4 µN (1.75 ± 0.17 mN/​mm2) (Sup­ple­men­tary Table S3). Like­wise, con­trac­tion and relax­ation slopes were also slowed at 3 ms but accel­er­at­ed in a time depen­dent man­ner as the pulse delay time increased. At the longer delays test­ed (120 – 160 ms), there was a sig­nif­i­cant pro­lon­ga­tion of the con­trac­tion dura­tion 50% and time to base­line 90%. For each CCM pulse delay inves­ti­gat­ed, 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, dis­played a neg­li­gi­ble CCM-induced response (Fig­ures 4A,C). Tak­en togeth­er these data demon­strate that vary­ing the CCM pulse delay of the acute CCM stim­u­la­tion affects human 3D ECT con­trac­tile prop­er­ties in vit­ro while 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, have a neg­li­gi­ble con­trac­tile response.

FIGURE 4. Effect of CCM pulse delay on 2D and 3D hiPSC-CM contractile properties (A) Representative contraction traces for baseline (i.e., field stimulation pacing, 1 Hz). and CCM for 2D monolayer hiPSC-CMs and (B) 3D ECTs (C) Summary data graphs. Data are mean ± SEM. n = 3–6 per group. *p < 0.05 (3D vs. BL), #p < 0.05 (3D vs. 3D), +p < 0.05 (2D vs. BL).

3.4 CCM pulse dura­tion increas­es con­trac­tile properties

To assess the acute effects of CCM pulse dura­tion time on human car­diomy­ocyte con­trac­til­i­ty, we eval­u­at­ed var­i­ous clin­i­cal CCM pulse dura­tions from 4.5 to 7 ms (Fig­ure 2B). 3D ECTs dis­played a sig­nif­i­cant­ly increased inotrop­ic response as a func­tion of increas­ing CCM pulse dura­tion rel­a­tive to base­line (Fig­ures 5B,C). Sim­i­lar­ly, con­trac­tion and relax­ation slopes were enhanced as CCM pulse dura­tion increased (Fig­ures 5B,C). At the longest dura­tion test­ed (7 ms), 3D ECTs dis­played increased con­trac­tion ampli­tude of 80.8 ± 9.2 µN (1.22 ± 0.14 mN/​mm2) (Sup­ple­men­tary Table S3). Vary­ing the CCM pulse dura­tion result­ed in the widen­ing of the con­trac­tion dura­tion 50% at ≥ 5.14 ms. 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, dis­played no CCM-induced response at any of the pulse dura­tions test­ed (Fig­ures 5A,C). 3D ECT effects remained for the entire dura­tion of CCM stim­u­la­tion and returned to base­line when the CCM sig­nal was elim­i­nat­ed. These results sug­gest that increas­ing the CCM pulse dura­tion induces a dose” depen­dent increase in the con­trac­tile prop­er­ties of 3D ECTs.

FIGURE 5. Effect of CCM pulse duration on 2D and 3D hiPSC-CM contractile properties (A) Representative contraction traces for baseline (i.e., field stimulation pacing, 1 Hz). and CCM for 2D monolayer hiPSC-CMs and (B) 3D ECTs (C) Summary data graphs. Data are mean ± SEM. n = 3–6 per group. *p < 0.05 (3D vs. BL), #p < 0.05 (3D vs. 3D), +p < 0.05 (2D vs. BL).

3.5 CCM pulse ampli­tude mod­u­lates con­trac­tile properties

Next, we inves­ti­gat­ed the depen­dence of human car­diac con­trac­tile prop­er­ties on acute CCM ampli­tude (i.e., volt­age), from 1 to 10 V, in 2D and 3D hiP­SC-CMs mod­els (Fig­ure 2C). HiP­SC-CMs were stim­u­lat­ed with var­i­ous CCM ampli­tudes. We found increas­ing the CCM ampli­tude result­ed in a sig­nif­i­cant­ly increased con­trac­tion ampli­tude in 3D ECT rel­a­tive to base­line (Fig­ures 6B,C). Addi­tion­al­ly, con­trac­tion and relax­ation slopes were accel­er­at­ed, and con­trac­tion dura­tion 50% widened at high­er volt­ages in 3D ECTs. At the high­est ampli­tude test­ed (10 V), 3D ECTs dis­played increased con­trac­tion ampli­tude of 111.8 ± 5.8 µN (1.69 ± 0.08 mN/​mm2) (Sup­ple­men­tary Table S3), where­as 2D hiP­SC-CMs dis­played no CCM-induced response (Fig­ures 6A,C). These results sug­gest that increas­ing the CCM ampli­tude pro­duces a volt­age-depen­dent increase in con­trac­tile force and accel­er­at­ed con­trac­tion and relax­ation slopes in 3D ECTs.

FIGURE 6. Effect of CCM pulse amplitude on 2D and 3D hiPSC-CM contractile properties (A) Representative contraction traces for baseline (i.e., field stimulation pacing, 1 Hz). and CCM for 2D hiPSC-CMs monolayer and (B) 3D ECTs (C) Summary data graphs. Data are mean ± SEM. n = 3–6 per group. *p < 0.05 (3D vs. BL), #p < 0.05 (3D vs. 3D), +p < 0.05 (2D vs. BL).

3.6 CCM pulse num­ber aug­ments 3D ECT con­trac­tile properties

To inves­ti­gate the effect of pulse num­ber on the 2D and 3D hiP­SC-CMs mod­els, pulse num­ber was increased from 1 to 3 puls­es. 3D ECTs dis­played a CCM pulse num­ber depen­dent (Fig­ure 2D) increase in con­trac­tion ampli­tude from 1 to 3 puls­es (Fig­ures 7B,C). Addi­tion­al­ly, we observed sig­nif­i­cant­ly accel­er­at­ed con­trac­tion and relax­ation slopes and con­trac­tion dura­tion 50% pro­lon­ga­tion as a func­tion of CCM pulse num­ber (Fig­ures 7B,C). At the high­est pulse num­ber test­ed (3 puls­es), 3D ECTs dis­played an increased con­trac­tion ampli­tude of 76.6 ± 5.2 µN (1.16 ± 0.08 mN/​mm2) (Sup­ple­men­tary Table S3). On the oth­er hand, 2D hiP­SC-CMs dis­played a neg­li­gi­ble response to vary­ing the num­ber of CCM puls­es (Fig­ures 7A,C). These results demon­strate that 3D ECTs respond to increased CCM pulse num­ber by increased peak con­trac­tile force and accel­er­at­ed con­trac­tion and relax­ation slopes.

FIGURE 7. Effect of CCM pulse number on 2D and 3D hiPSC-CM contractile properties (A) Representative contraction traces for baseline (i.e., field stimulation pacing, 1 Hz). and CCM for 2D monolayer hiPSC-CMs and (B) 3D ECTs (C) Summary data graphs. Data are mean ± SEM. n = 3–6 per group. *p < 0.05 (3D vs. BL), #p < 0.05 (3D vs. 3D), +p < 0.05 (2D vs. BL).

4 Dis­cus­sion

4.1 2D and 3D hiP­SC-CM CCM models

In this study, we estab­lish a robust in vit­ro method to quan­ti­fy and opti­mize the effect of var­i­ous CCM stim­u­la­tion para­me­ters in 3D ECTs to improve device devel­op­ers deci­sion-mak­ing capa­bil­i­ties. CCM is a car­diac ther­a­py approved for HF patients with reduced ejec­tion frac­tion. How­ev­er, the ways in which var­i­ous CCM sig­nal para­me­ters affect human car­diomy­ocyte con­trac­tile prop­er­ties have not been com­plete­ly defined in vit­ro. Video-based and force-based analy­ses were used to quan­ti­fy the effects of a range (i.e., dos­es”) of clin­i­cal elec­tri­cal CCM para­me­ters on human car­diomy­ocyte con­trac­til­i­ty (e.g., force) in con­ven­tion­al 2D hiP­SC-CMs, on stiff sub­strate, and 3D hiP­SC-CM (ECTs) mod­els. We demon­strat­ed that 3D ECTs respond­ed to var­i­ous CCM sig­nals with an increased peak con­trac­tile force in a pulse para­me­ter-depen­dent man­ner. More­over, to our knowl­edge, we have for the first time quan­ti­fied the acute effects of CCM puls­es on a 3D micro­phys­i­o­log­i­cal sys­tem com­prised of mul­ti­ple car­diac cell types, specif­i­cal­ly car­diomy­ocytes and car­diac fibrob­lasts. Here, uti­liz­ing a 3D ECT mod­el, we demon­strate a sus­tained aug­ment­ed CCM-induced con­trac­til­i­ty response, at phys­i­o­log­i­cal Ca con­cen­tra­tions (1.8 mM). On the oth­er hand, stan­dard 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, that were cul­tured for 7 days dis­play no con­trac­tile response to CCM. The dis­crep­an­cy between 2D mono­lay­er hiP­SC-CMs mod­els and 3D ECTs is like­ly the result of elec­tri­cal con­di­tion­ing, time in cul­ture, uni­ax­i­al ten­sion, and the con­tri­bu­tion of non-car­diomy­ocytes (i.e., car­diac fibrob­last) (Eng et al., 2016; Ronald­son-Bouchard et al., 2018).

4.2 Var­i­ous CCM elec­tri­cal sig­nals mod­u­late 3D ECT function

Clin­i­cal­ly CCM has been asso­ci­at­ed with reduced HF hos­pi­tal­iza­tion and improved qual­i­ty of life (Camp­bell et al., 2020). Sev­er­al stud­ies have demon­strat­ed an increased ejec­tion frac­tion and accel­er­at­ed dP/​dtmax (i.e., max­i­mum sys­tolic ups­lope) with CCM treat­ment (Pap­pone et al., 2002; Lawo et al., 2005; Nagele et al., 2008). How­ev­er, how var­i­ous CCM para­me­ters affect human car­diomy­ocyte biol­o­gy is large­ly unknown. To under­stand how dif­fer­ent CCM sig­nal para­me­ters affect car­diac con­trac­til­i­ty, we inves­ti­gat­ed the effects of var­i­ous CCM pulse sig­nals on human car­diac func­tion in vit­ro. We found that var­i­ous CCM para­me­ters (ampli­tude, dura­tion, delay, and pulse num­ber) enhanced the con­trac­tile force of 3D ECTs in a para­me­ter-depen­dent man­ner. Specif­i­cal­ly, we demon­strate a para­me­ter-depen­dent accel­er­a­tion of con­trac­tion and relax­ation slopes that is con­sis­tent with an accel­er­at­ed dP/​dtmax. Addi­tion­al­ly, we observed a pulse num­ber-depen­dent increase in con­trac­tion ampli­tude in 3D ECTs. By set­ting the total pulse dura­tion time to a fixed inter­val, we demon­strat­ed that these effects were like­ly the result of an increase in total dura­tion time rather than absolute num­ber of CCM puls­es (Sup­ple­men­tary Fig­ure S2). This is con­sis­tent with a com­pa­ra­ble elec­tric field (E) for 1 to 3 CCM puls­es when total dura­tion was fixed. One poten­tial expla­na­tion of this could be pro­longed chan­nel acti­va­tion by L‑type cal­ci­um chan­nels or intra­cel­lu­lar SR cal­ci­um stores as the total CCM stim­u­la­tion dura­tion is increased. When eval­u­at­ing CCM pulse delay we found the short­est delay between pac­ing and CCM pulse inves­ti­gat­ed (3 ms) result­ed in reduced con­trac­tile ampli­tude, and a pulse delay of 120 ms induced a pro­lon­ga­tion of con­trac­tion dura­tion 50% and time to base­line 90%. Con­verse­ly, we did not inves­ti­gate the effects of CCM pulse delay on action poten­tial mor­phol­o­gy, or if this trans­lates to pro­lon­ga­tion or tri­an­gu­lar­iza­tion of the action poten­tial, as it was out­side the scope of this work. How­ev­er, it is impor­tant to note that sig­nif­i­cant pro­lon­ga­tion or tri­an­gu­lar­iza­tion of the action poten­tial may indi­cate a pos­si­ble safe­ty lia­bil­i­ty (Bli­no­va et al., 2018; Gin­tant et al., 2020) or a proar­rhyth­mic sub­strate. We have pre­vi­ous­ly inves­ti­gat­ed patient-spe­cif­ic respons­es in 2D hiP­SC-CMs, set­ting the stage for such a com­par­i­son in 3D ECTs. We com­pared clin­i­cal drug con­cen­tra­tion-depen­dent QT pro­lon­ga­tion with in vit­ro drug con­cen­tra­tion-depen­dent action poten­tial dura­tion pro­lon­ga­tion (Bli­no­va et al., 2019). In the future, 3D ECT data may be lever­aged to bet­ter under­stand CCM in patient-spe­cif­ic pop­u­la­tions or cohorts using dis­ease-spe­cif­ic mod­els (e.g., clin­i­cal tri­al in a dish) (Strauss and Bli­no­va, 2017; Fer­mi­ni et al., 2018; Bli­no­va et al., 2019), and to iden­ti­fy poten­tial CCM super respon­ders (Al-Gham­di et al., 2017; Hes­sel­son et al., 2022) before ther­a­py is need­ed. Like­wise, these data pro­vide an in vit­ro tool to opti­mize CCM para­me­ters and tai­lor said para­me­ters to an indi­vid­ual patient using patient-spe­cif­ic 3D ECTs fol­low­ing clin­i­cal presentation.

4.3 Com­par­i­son of in vit­ro, ex vivo, and in vivo CCM studies

Pre­vi­ous non­clin­i­cal CCM stud­ies are chal­leng­ing to cor­re­late with each oth­er because they apply a vari­ety of CCM para­me­ters, mod­els, and species (Feast­er et al., 2021). Con­se­quent­ly, there are con­flict­ing reports of the effects of CCM on human CM con­trac­til­i­ty (Camp­bell et al., 2020; Burk­hoff et al., 2001; Win­ter et al., 2014). Like­wise, clin­i­cal trans­la­tion of such results is com­plex. Despite this, the con­sen­sus is that CCM stim­u­la­tion increas­es con­trac­til­i­ty, cal­ci­um han­dling, and enhances gene expres­sion with­out neg­a­tive­ly affect­ing mito­chon­dr­i­al func­tion. One in vit­ro study using an iso­lat­ed rab­bit pap­il­lary mus­cle mod­el demon­strat­ed enhanced con­trac­til­i­ty in a man­ner depen­dent on the CCM pulse para­me­ters (e.g., ampli­tude polar­i­ty) (Brunck­horst et al., 2006). How­ev­er, this study used non­clin­i­cal CCM pulse para­me­ters and a non­hu­man ani­mal mod­el. We pre­vi­ous­ly demon­strat­ed CCM induced enhanced con­trac­til­i­ty and cal­ci­um han­dling using in vit­ro iso­lat­ed rab­bit CMs and 2D hiP­SC-CMs, on flex­i­ble sub­strate (Bli­no­va et al., 2014; Feast­er et al., 2021). Still, the max­i­mum response was tran­sient in these two stud­ies. In 2D hiP­SC-CMs, on flex­i­ble sub­strate, a sub­max­i­mal extra­cel­lu­lar Ca con­cen­tra­tion of 0.5 mM was nec­es­sary to reveal the CCM con­trac­tile response. Here, our goal was to use a phys­i­o­log­i­cal extra­cel­lu­lar cal­ci­um con­cen­tra­tion of 1.8 mM. How­ev­er, in the pres­ence of sub­max­i­mal extra­cel­lu­lar cal­ci­um con­cen­tra­tions, 3D ECTs main­tained a supe­ri­or con­trac­tile response rel­a­tive to 2D hiP­SC-CMs, on flex­i­ble sub­strate (Sup­ple­men­tary Fig­ure S3). Sev­er­al ex vivo whole heart fer­ret mod­els demon­strat­ed that CCM induced increased force and cal­ci­um han­dling as well as cal­ci­um depen­dance (Burk­hoff et al., 2001; Mohri et al., 2002; Mohri et al., 2003). Sim­i­lar­ly, an ex vivo whole heart rab­bit mod­el demon­strat­ed increased con­trac­tion and short­ened monopha­sic action poten­tial dura­tion along with a depen­dance on β‑adrenergic sig­nal­ing (Win­ter et al., 2014; Win­ter et al., 2011). We pre­vi­ous­ly demon­strat­ed, in an ex vivo whole rat heart mod­el, that CCM enhanced left ven­tric­u­lar pres­sure and β‑adrenergic sig­nal­ing con­tributed to the CCM response (Bli­no­va et al., 2014). A mul­ti­tude of in vivo canine mod­els (i.e., fail­ing heart) demon­strate improved left ven­tric­u­lar func­tion (enhanced ejec­tion frac­tion) induced by CCM. While these sem­i­nal papers pro­vide impor­tant insight into the effects and mech­a­nisms of CCM, these stud­ies are cost­ly, time con­sum­ing, and rely heav­i­ly on large and small ani­mal mod­els (Sab­bah et al., 2001; Mohri et al., 2002; Mori­ta et al., 2003). As such, a robust human-based mod­el to repro­ducibly eval­u­ate CCM sig­nals in vit­ro is need­ed to aid the devel­op­ment of nov­el devices and under­stand the effects of var­i­ous sig­nals on human car­diomy­ocyte biol­o­gy. In this work we demon­strate the first non­clin­i­cal CCM study using the approved clin­i­cal range of CCM para­me­ters (Camp­bell et al., 2020) in a human micro­phys­i­o­log­i­cal sys­tem (3D ECTs). Although oth­er meth­ods, such as tra­di­tion­al pap­il­lary mus­cle mod­els (e.g., rab­bit) and in vivo canine mod­els are amenable to CCM assess­ment, our 3D ECT method enables CCM eval­u­a­tion in human car­diac tis­sue, ahead of cost­ly ani­mal test­ing, sig­nif­i­cant­ly assist­ing the 3Rs ini­tia­tive (Schecht­man, 2002). This 3D ECT CCM method can serve as a foun­da­tion for the devel­op­ment and opti­miza­tion of nov­el car­diac med­ical devices and can be mul­ti­plexed to eval­u­ate CCM effects on addi­tion­al car­diac exci­ta­tion-con­trac­tion cou­pling (E‑C) read­outs includ­ing elec­tro­phys­i­ol­o­gy (i.e., action poten­tial) and cal­ci­um handling.

4.4 Study limitations

We rec­og­nize there are inher­ent dif­fer­ences in 2D and 3D mod­els includ­ing time in cul­ture and inclu­sion of car­diac fibrob­lasts. To ensure the most accu­rate com­par­i­son of con­ven­tion­al 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, to 3D ECTs, exper­i­men­tal con­di­tions were uni­fied in a num­ber of impor­tant ways includ­ing: 1) selec­tion of a phys­i­o­log­i­cal extra­cel­lu­lar cal­ci­um con­cen­tra­tion of 1.8 mM (Yee, 2008), an accept­ed stan­dard for in vit­ro exper­i­ments (Saleem et al., 2020a; Bar­toluc­ci et al., 2020; Tsan et al., 2021); 2) we used com­mer­cial­ly avail­able hiP­SC-CMs from the same man­u­fac­ture in both 2D and 3D mod­els as well as com­pa­ra­ble cell num­bers for each 2D well and 3D ECT; 3) plat­inum elec­trodes were used in both sys­tems to lim­it cor­ro­sion poten­tial; 4) both 2D and 3D exper­i­ments were con­duct­ed at a phys­i­o­log­i­cal tem­per­a­ture. How­ev­er, our study has sev­er­al lim­i­ta­tions. For exam­ple, con­ven­tion­al 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, dis­play sev­er­al fea­tures of imma­ture car­diomy­ocytes includ­ing spon­ta­neous beat­ing. As a stan­dard, con­ven­tion­al 2D hiP­SC-CMs are rou­tine­ly cul­tured for 7 – 14 days with­out elec­tri­cal con­di­tion­ing ahead of exper­i­ments. How­ev­er, there are fre­quent­ly stim­u­lat­ed dur­ing exper­i­men­ta­tion to elim­i­nate poten­tial rate-depen­dent effects (Patel et al., 2019). 3D ECTs were cul­tured for approx­i­mate­ly 7 weeks with the addi­tion of elec­tri­cal con­di­tion­ing. As such, we can­not exclude the impact of long-term cul­ture or elec­tri­cal con­di­tion­ing on the 3D ECT CCM response. While tech­ni­cal­ly fea­si­ble, cul­tur­ing 2D mono­lay­ers for 7 weeks is not triv­ial and the nature of the 2D envi­ron­ment does not pro­vide the opti­mal con­di­tions for long-term pac­ing. In con­ven­tion­al 2D cul­ture, unsta­ble extra­cel­lu­lar matrix and mono­lay­er integri­ty are of prime con­cern. On the oth­er hand, 3D ECTs ben­e­fit from sup­port­ing cells and a sta­ble 3D envi­ron­ment, as the cells are embed­ded in the extra­cel­lu­lar matrix gel. 3D ECTs rep­re­sent a func­tion­al­ly enhanced hiP­SC-CM mod­el with intact iso­pro­terenol-induced pos­i­tive inotropy, a pos­i­tive force-fre­quen­cy, and post-rest poten­ti­a­tion, which like­ly aug­ment­ed the CCM con­trac­tile response and enabled the shift in the exper­i­men­tal con­di­tions to a phys­i­o­log­ic extra­cel­lu­lar Ca con­cen­tra­tion (Fer­ic et al., 2019). Direct force mea­sure­ments were used for 3D ECTs for the eval­u­a­tion of con­trac­tion ampli­tude. In con­ven­tion­al 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate, video-based pix­el dis­place­ment was used to mea­sure con­trac­tile prop­er­ties due to a lack of cel­lu­lar anisotropy and the lim­it­ed cel­lu­lar move­ment (i.e., short­en­ing) of the mod­el. While both con­trac­tion ampli­tude mea­sure­ments (i.e., force and dis­place­ment) typ­i­cal­ly have a syn­er­gis­tic rela­tion­ship, it is con­ceiv­able for con­trac­tile force to increase while cel­lu­lar move­ment reduces or remains neu­tral as is the case for iso­met­ric con­trac­tion forces. Both 2D and 3D mod­els lack a neu­ronal com­po­nent nec­es­sary to elu­ci­date the con­tri­bu­tion of sym­pa­thet­ic stim­u­la­tion through car­diac gan­glion. Toward this goal, we are active­ly inves­ti­gat­ing the con­tri­bu­tion of hiP­SC-neu­rons to the CCM response (Narkar et al., 2022). More­over, the con­tri­bu­tion of non-car­diomy­ocytes (i.e., car­diac fibrob­last) can­not be over­looked as well as the mixed pop­u­la­tion of hiP­SC-CMs from each car­diac sub­type (i.e., ven­tric­u­lar, atri­al, and nodal) rep­re­sent­ed in both 2D and 3D mod­els used here. Addi­tion­al­ly, the com­mer­cial hiP­SC-CMs used here rep­re­sent an appar­ent­ly healthy’ car­diac mod­el where­as CCM is indi­cat­ed for HF patients. These mod­els will be extend­ed to dis­eased back­grounds includ­ing HF, DCM, and HCM. How­ev­er, as a first step in this direc­tion, demon­stra­tion of a CCM response on healthy cells is required. Clin­i­cal trans­la­tion and the cor­re­la­tion of these data with human patient out­comes is of sig­nif­i­cant inter­est but is cur­rent­ly hin­dered due to lim­it­ed access to human clin­i­cal data. In this study we focused pri­mar­i­ly on the cur­rent­ly approved clin­i­cal range of CCM para­me­ters. As such, we did not inves­ti­gate min­i­mum and max­i­mum response. The CCM para­me­ter range eval­u­at­ed here was select­ed to span that of the clin­i­cal device capa­bil­i­ties where applic­a­ble. Exper­i­ments to test com­bi­na­tions of the most promis­ing para­me­ters that yield max­i­mal con­trac­tile response with min­i­mal patho­log­i­cal con­se­quences are ongo­ing in our lab­o­ra­to­ries. Clin­i­cal­ly the ben­e­fi­cial effects of CCM are achieved fol­low­ing pro­longed stim­u­la­tion and have been sug­gest­ed to be relat­ed to slow tis­sue remod­el­ing which is out­side of the scope of this acute study (But­ter et al., 2008).

5 Con­clu­sion

This work lays the foun­da­tion for an in vit­ro CCM para­me­ter eval­u­a­tion tool and may sup­port safe­ty or effec­tive­ness stud­ies for future CCM devices as well as oth­er car­diac elec­tro­phys­i­o­log­i­cal med­ical devices in gen­er­al. Here, we demon­strate sev­er­al impor­tant find­ings. 1) 3D ECTs respond to acute clin­i­cal CCM stim­u­la­tion para­me­ters at phys­i­o­log­i­cal Ca con­cen­tra­tions. 2) 3D ECTs respond to the changes in var­i­ous clin­i­cal CCM stim­u­la­tion para­me­ters (i.e., pulse delay, pulse dura­tion, pulse ampli­tude, and pulse num­ber), as a func­tion of each para­me­ter, by an increase in con­trac­tile force. This pro­vides a non­clin­i­cal mod­el to test and opti­mize var­i­ous non-exci­ta­to­ry elec­tri­cal sig­nal para­me­ters and com­bi­na­tions. 3) 3D ECTs dis­play accel­er­at­ed con­trac­tion and relax­ation slopes when stim­u­lat­ed with CCM, which is con­sis­tent with an accel­er­at­ed dP/​dtmax and may be ben­e­fi­cial in the con­text of sys­tolic or dias­tolic dys­func­tion. 4) Con­ven­tion­al 2D mono­lay­er hiP­SC-CMs, on stiff sub­strate (e.g., glass/​plastic), and cul­tured for 7 days do not respond to CCM. 5) CCM pulse num­ber had a neg­li­gi­ble effect on con­trac­tile response in human car­diomy­ocytes unlike the total pulse dura­tion time, which was the dri­ving fac­tor for enhanced con­trac­tile force in sit­u­a­tions where pulse num­ber was increased. 6) In 3D ECTs, the CCM response is sen­si­tive to changes in the extra­cel­lu­lar Ca con­cen­tra­tion result­ing in a blunt­ed effect at high­er con­cen­tra­tions (i.e., 4 mM). Tak­en togeth­er, the cur­rent study demon­strat­ed that the 3D ECT mod­el can reca­pit­u­late CCM-induced con­trac­til­i­ty increase, con­sis­tent with the mod­el being pre­dic­tive of the effects of elec­tro­phys­i­o­log­i­cal stim­u­la­tion on human tis­sue. Thus, there is a sig­nif­i­cant need to eval­u­ate the effects of addi­tion­al car­diac elec­tro­phys­i­o­log­i­cal med­ical devices (e.g., abla­tion, CRT, or ICD) in human mod­els such as 3D ECTs. Toward that goal, we are active­ly eval­u­at­ing car­diac med­ical devices in a vari­ety of nov­el 3D in vit­ro hiP­SC mod­els to address reg­u­la­to­ry sci­ence knowl­edge gaps.

Data avail­abil­i­ty statement

The raw data sup­port­ing the con­clu­sion of this arti­cle will be made avail­able by the authors, with­out undue reservation.

Author con­tri­bu­tions

TF, RA‑S, and KB. con­ceived and designed research. TF and RA‑S per­formed exper­i­ments and analysed data. TF, RA‑S, and KB inter­pret­ed results of exper­i­ments. MC and TF estab­lished stim­u­lat­ing elec­trodes and exper­i­men­tal set­up for 2D study. TF, RA‑S, and KB. draft­ed man­u­script; NF, MC, and AN edit­ed and revised the man­u­script. TF, NF, IP, AN, MC, MG, RA‑S, and KB approved final ver­sion of manuscript.

Fund­ing

The study was fund­ed through the U.S. Food and Drug Admin­is­tra­tion, Office of Sci­ence and Engi­neer­ing Laboratories.

Con­flict of interest

NF, IP, MG, and RA‐S were employed by TARA Biosys­tems at the time of the study. Cer­tain aspects of the study were per­formed by TARA Biosys­tems, includ­ing: study design, col­lec­tion, analy­sis, inter­pre­ta­tion of data, the writ­ing of this man­u­script. TARA Biosys­tems was acquired by Valo Health dur­ing the prepa­ra­tion of this man­u­script. NF, IP, MG, and RA‐S are now employed by Valo Health.

The remain­ing authors declare that the research was con­duct­ed in the absence of any com­mer­cial or finan­cial rela­tion­ships that could be con­strued as a poten­tial con­flict of interest.

Publisher’s note

All claims expressed in this arti­cle are sole­ly those of the authors and do not nec­es­sar­i­ly rep­re­sent those of their affil­i­at­ed orga­ni­za­tions, or those of the pub­lish­er, the edi­tors and the review­ers. Any prod­uct that may be eval­u­at­ed in this arti­cle, or claim that may be made by its man­u­fac­tur­er, is not guar­an­teed or endorsed by the publisher.

Author Dis­claimer

This arti­cle reflects the views of the authors and should not be con­strued to rep­re­sent the US Food and Drug Administration’s views or poli­cies. The men­tion of com­mer­cial prod­ucts, their sources, or their use in con­nec­tion with mate­r­i­al report­ed here­in is not to be con­strued as either an actu­al or implied endorse­ment of such prod­ucts by the Depart­ment of Health and Human Services.