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[(be)21(fo)15.7(re)-329.4(in)18.4(cu)18.3(ba)21.4(ti)15(ng)-330.3(a)13.1(t)-341.9(37)]TJ
/F6 1 Tf
10.0787 0 TD
()Tj
/F3 1 Tf
.3857 0 TD
[(C)-341.6(t)12.2(o)-344.2(i)0(n)18.4(d)0(u)20.9(c)0(e)-328(c)0(o)16.9(l)0(l)18.2(a)13.1(ge)14.7(n)-338.5(p)0(o)21.2(l)0(y)17.3(m)0(e)21.5(r)0(i)14.7(z)13.1(at)19(io)19(n.)]TJ
-10.4644 -1.2203 TD
[(Th)18.9(e)-271.8(a)13.1(pp)16.3(ro)21.8(pr)16.9(ia)22.2(te)-265.9(m)15.1(e)0(d)16.1(i)0(a)-256(w)0(e)17.4(r)0(e)-259.8(t)0(h)15.1(e)12.8(n)-275.3(a)13.1(dd)19.3(e)12.8(d)-274.9(t)12.2(o)-268.3(ea)19.6(ch)-261.9(su)16.3(bs)19.6(t)12.2(r)0(a)18.8(t)0(e)18.6(.)-268.5(By)]TJ
T*
[(us)16.3(in)18.4(g)-466(a)-467.4(li)18.2(ve)18.3(/d)19.3(e)12.8(a)0(d)-457.8(c)0(e)19.8(l)0(l)-462.3(v)0(i)21(a)0(b)21.4(i)0(l)18.2(i)0(t)15(y)-466(a)13.1(s)0(s)16.3(a)13.1(y)-472.3(\()12.2(In)19.8(vi)14.7(t)12.2(r)0(o)15.5(g)14.6(en)15.7(\))12.2(,)-470.8(we)-456.8(es)17.8(ti)15(-)]TJ
0 -1.2266 TD
[(ma)21.9(te)18.6(d)-274.9(c)13.4(el)15.5(l)-269.1(v)0(i)21(a)0(b)21.4(i)0(l)18.2(i)0(t)15(y)-270(ov)21.8(er)-259.8(ti)15(me)21.5(.)-268.5(T)0(h)18.9(e)-271.8(av)18.7(er)18.4(ag)21.4(e)-271.8(r)0(a)18.8(t)12.2(io)-265.5(of)-262.5(li)24.5(vi)14.7(ng)-260.7(c)13.4(e)0(l)15.5(l)0(s)]TJ
0 -1.2203 TD
[(af)19(te)18.6(r)-291.6(s)0(e)17.8(e)12.8(di)18.8(ng)-279.7(in)-285.1(t)12.2(h)0(e)]TJ
/F5 1 Tf
8.6118 0 TD
(m)Tj
/F3 1 Tf
.5438 0 TD
[(TU)20.9(Gs)-284.6(is)-276.7(of)-281.5(64)19.9(%)]TJ
/F6 1 Tf
6.9046 0 TD
()Tj
/F3 1 Tf
.7082 0 TD
[(9)13.1(%)0(,)-282.8(s)0(t)17.2(a)13.1(ti)15(st)23.5(ic)16.2(a)13.1(l)0(l)18.2(y)-288.9(si)14.1(m)15.1(i)0(l)18.2(a)0(r)]TJ
-16.7684 -1.2203 TD
[(to)-414.2(t)12.2(h)0(e)-414.3(v)0(i)21(a)0(b)15(i)0(l)24.6(i)0(t)15(y)-421.7(ri)21.1(gh)17.5(t)-417.8(a)0(f)19(t)0(e)18.6(r)-424.3(is)20.4(ol)19(at)19(io)19(n)-420.7(\()0(6)19(9)0(%)]TJ
/F6 1 Tf
18.2353 0 TD
()Tj
/F3 1 Tf
.7082 0 TD
[(11)19.9(%\))16.9(,)-420.2(a)0(n)16.1(d)-420.3(sl)20.4(ow)20.9(ly)]TJ
-18.9434 -1.2266 TD
[(de)16.1(c)13.4(r)0(e)18.4(a)0(s)18.1(e)12.8(s)-323.8(t)0(o)-319.4(4)13.1(6%)]TJ
/F6 1 Tf
7.5559 0 TD
()Tj
/F3 1 Tf
.7082 0 TD
[(13)19.9(%)-324.1(a)0(f)19(t)12.2(er)-316.7(7)-322(d)0(a)16.5(y)14.6(s)-323.8(o)0(f)-319.4(c)13.4(ul)20.4(tu)17.1(re)18.4(.)]TJ
/F10 1 Tf
-8.264 -2.4406 TD
[(Force)-332.3(quantication)]TJ
/F3 1 Tf
1.1128 -1.8336 TD
[(For)-463.1(quantifying)-465.2(microtissue)-467(forces,)-462.6(brighteld)-462.6(and)-461.1(uo-)]TJ
-1.1128 -1.2203 TD
[(rescence)-395.7(images)-385.6(were)-394.1(taken)-387.1(at)-392(20)-163.4(Hz)-391.5(within)-395.3(each)-387.8(template,)]TJ
0 -1.2266 TD
[(using)-241.6(a)-233.5(Photometrics)-247.5(Evolve)-238.3(EMCCD)-237.5(camera)-236.7(\(Photometrics\),)]TJ
0 -1.2203 TD
[(and)-372.6(an)-363.3(A-Plan)-376.4(10)]TJ
/F6 1 Tf
8.0617 0 TD
()Tj
/F3 1 Tf
1.0875 0 TD
[(objective)-370(on)-372.9(a)-366.2(Nikon)-368.2(Eclipse)-372.3(Ti)-366.9(\(Nikon)]TJ
-9.1493 -1.2203 TD
[(Instruments,)-362.7(Inc.\))-362.3(equipped)-362.6(with)-363.1(a)-359.9(live)-355.5(cell)-360.3(incubator.)-366.1(Only)]TJ
0 -1.2266 TD
[(tissues)-350.6(that)-351.6(were)-349.9(uniformly)-352.8(anchored)-353.4(to)-351(the)-351.5(tips)-348.1(of)-351(the)-351.5(can-)]TJ
0 -1.2203 TD
[(tilevers)-402.1(were)-394.1(included)-396.4(in)-398.9(the)-395.8(analysis.)-398.8(The)-398.3(displacement)-398.5(of)]TJ
T*
[(uorescent)-290.3(microbeads)-283.9(at)-278.2(the)-288.3(top)-282.8(of)-281.5(the)-288.3(cantilevers)-284.2(was)-280.7(then)]TJ
0 -1.2266 TD
[(tracked)-639.6(with)-635(using)-633.7(the)-636(SpotTracker)-642.9(plug-in)]TJ
6.7246 0 0 5.9768 247.9747 280.4597 Tm
(31)Tj
8.9663 0 0 8.9663 260.4472 276.4345 Tm
[(in)-632.9(ImageJ)]TJ
-22.1302 -1.2203 TD
[(\(National)-335.6(Institutes)-343.1(of)-332(Health\).)]TJ
/F10 1 Tf
0 -2.4406 TD
[(Electrical)-337.5(stimulation)]TJ
/F3 1 Tf
1.1128 -1.8336 TD
[(Electrical)-272(stimulation)-270.4(was)-268(started)-270.9(3)-271.4(days)-267.5(after)-266.6(cell)-271.8(seeding,)]TJ
-1.1128 -1.2266 TD
[(by)-502(placing)-501.9(two)-504.4(carbon)-503.3(electrodes)-510.3(\(1/4)-502(in)-500.1(diameter;)-507(Ladd)]TJ
0 -1.2203 TD
[(Research)-381.3(Industries\))-378(on)-379.2(the)-376.8(sides)-375.8(of)-382.6(the)-376.8(samples)-377.5(\(separated)]TJ
T*
[(by)-343.9(2)-170.2(cm\),)-341.7(connected)-352.8(through)-345.3(platinum)-345.8(wires)-348.5(to)-344.7(a)-347.3(stimulator)]TJ
0 -1.2266 TD
[(as)-614.1(described)-609.4(previously.)]TJ
6.7246 0 0 5.9768 163.1622 187.3133 Tm
(21)Tj
8.9663 0 0 8.9663 175.4078 183.2881 Tm
[(Voltage)-614.1(threshold)-612.8(\(VT\))-613.8(is)-611.8(the)]TJ
-12.6458 -1.2203 TD
[(minimum)-390.2(voltage)-384.4(at)-385.7(which)-390.6(the)-389.5(CMTs)-389(are)-385.8(observed)-385.8(to)-388.9(beat)]TJ
T*
[(synchronously)-386.1(under)-381.4(electrical)-382.7(eld)-376.2(stimulation)-384.2(at)-379.4(1)-170.2(H)0(z)-378.9(us-)]TJ
0 -1.2266 TD
[(ing)-599.4(1)-163.9(m)0(s)-599.6(square)-601.6(biphasic)-598(pulses.)-599.7(Maximum)-601.2(capture)-596.9(rate)]TJ
0 -1.2203 TD
[(\()-82.7(MCR\))-541.1(is)-529.6(dened)-537(as)-538.3(the)-534.9(maximum)-536.7(pacing)-536.3(frequency)-538.6(for)]TJ
T*
[(synchronous)-574.2(contractions)-573.3(at)]TJ
/F5 1 Tf
13.4615 0 TD
(*)Tj
/F3 1 Tf
.999 0 TD
[(20%)-563.4(over)-573.2(VT.)-565.2(Continuous)]TJ
-14.4605 -1.2203 TD
[(stimulation)-295.7(was)-293.3(achieved)-293.8(by)-293.4(using)-292.2(biphasic)-294.5(square)-291.7(pulses)-293.3(of)]TJ
0 -1.2267 TD
[(1)-170.2(m)0(s)-327.7(a)0(t)-335.1(6)-334.6(V/cm)-331.5(and)-334.7(0.2)-166.3(Hz.)]TJ
6.7246 0 0 5.9768 171.8362 110.5512 Tm
(22)Tj
/F10 1 Tf
8.9663 0 0 8.9663 62.022 84.6992 Tm
[(Immunouorescence)-331.3(and)-335.7(image)-334.3(analysis)]TJ
/F3 1 Tf
1.1128 -1.8336 TD
[(Microtissues)-564.4(were)-564.9(xed)-558.2(with)-565.4(4%)-557.6(paraformaldehyde)-569.3(in)]TJ
-1.1128 -1.2267 TD
[(PBS,)-252.4(permeabilized)-250.3(with)-255.6(0.2%)-243.8(Triton)-254.1(X-100)-249.8(in)-253.5(PBS,)-246(incubated)]TJ
27.9978 74.5598 TD
[(with)-375.7(antibodies)-375.9(against)-378.9(troponin)-378.8(T)-369.7(\(Thermo)-374.5(Scientic\),)-381(and)]TJ
0 -1.2203 TD
[(detected)-347.4(with)-337.8(uorophore-conjugated,)-344.7(isotype-specic,)-347.3(anti-)]TJ
0 -1.2266 TD
[(IgG)-397.3(antibodies)-394.9(and)-391.6(counterstained)-398.4(with)-394.7(DAPI)-392(\(Invitrogen\).)]TJ
0 -1.2203 TD
[(Alignment)-409.6(was)-400.8(quantied)-404.3(from)-399.8(DAPI)-404.6(images)-398.3(by)-400.9(tting)-401(el-)]TJ
T*
[(lipses)-257.5(with)-249.3(ImageJ.)-253.5(Nuclei)-254.2(were)-255(considered)-250.7(aligned)-257.6(when)-248.6(the)]TJ
0 -1.2266 TD
[(angle)-295.3(between)-300.4(their)-292.5(long)-292.3(axis)-291(and)-296.7(the)]TJ
/F4 1 Tf
16.8379 0 TD
(x)Tj
/F3 1 Tf
.4995 0 TD
[(-axis)-291.5(\(the)-295.1(axis)-297.4(joining)]TJ
-17.3374 -1.2203 TD
[(the)-351.5(two)-346.3(cantilevers\))-347.9(was)-343.9(less)-347.5(than)-348.2(20)]TJ
/F6 1 Tf
16.4522 0 TD
()Tj
/F3 1 Tf
.3983 0 TD
[(.)-344.4(Cross-sectional)-349.2(areas)]TJ
-16.8506 -1.2203 TD
[(were)-482.7(estimated)-479.9(from)]TJ
/F4 1 Tf
9.9712 0 TD
(z)Tj
/F3 1 Tf
.4363 0 TD
[(-stack)-479.7(images)-474.1(obtained)-478.6(with)-483.2(a)-473.7(4)0(0)]TJ
/F6 1 Tf
15.5417 0 TD
()Tj
/F3 1 Tf
-25.9492 -1.2266 TD
[(water-immersion)-398.6(objective)-395.3(attached)-397.6(to)-395.3(a)-391.5(laser)-390.7(scanning)-397.6(mi-)]TJ
0 -1.2203 TD
[(croscope)-342.5(Zeiss)-332.8(LSM)-335.4(510)-333.6(\(Carl)-333.2(Zeiss,)-335.7(Inc.\).)]TJ
/F10 1 Tf
0 -2.4406 TD
[(Statistical)-342.4(comparisons)]TJ
/F3 1 Tf
1.1128 -1.8336 TD
[(Statistical)-351.6(comparisons)-348.6(were)-349.9(based)-349.6(on)-347.6(an)-344.3(analysis)-351.6(of)-344.7(var-)]TJ
-1.1128 -1.2266 TD
[(iance)-618.9(for)-617.3(pairwise)-612.2(comparisons)-620.5(\()]TJ
/F4 1 Tf
15.7757 0 TD
(p)Tj
/F6 1 Tf
.6702 0 TD
(<)Tj
/F3 1 Tf
.7145 0 TD
[(0.05)-614.8(was)-615.8(considered)]TJ
-17.1604 -1.2203 TD
(signicant\).)Tj
/F7 1 Tf
0 -3.054 TD
(Results)Tj
/F10 1 Tf
0 -1.8336 TD
[(Generation)-335.7(of)-336.7(CMTs)]TJ
/F3 1 Tf
1.1128 -1.8336 TD
[(To)-410.4(generate)-406.5(microscale)-412.1(constructs)-408.7(of)-407.9(cardiac)-409.5(cells)-405.9(within)]TJ
-1.1128 -1.2203 TD
[(collagen/brin)-508.6(3D)-506.7(matrices,)-511(we)-501(microfabricated)-507.8(arrays)-505.7(of)]TJ
0 -1.2266 TD
[(wells)-427.2(within)-426.9(a)-423.1(PDMS)-423.7(mold.)-420.8(The)-423.6(mold)-424.2(was)-419.8(immersed)-427.1(in)-424.2(a)]TJ
0 -1.2203 TD
[(suspension)-436.9(of)-426.9(cells)-431.2(and)-429.5(reconstitution)-434.3(mixture)-429.3(\(liquid)-430.8(neu-)]TJ
T*
[(tralized)-408.5(collagen)-408.4(I)-400.5(and)-404.2(brinogen\))-405.4(and)-404.2(the)-408.4(entire)-406.2(assembly)]TJ
0 -1.2266 TD
[(was)-457.7(centrifuged)-465.3(to)-458.5(drive)-456.8(the)-459(cells)-456.5(into)-459.1(the)-459(micropatterned)]TJ
0 -1.2203 TD
[(wells.)-487(Excess)-481.2(collagen/brinogen)-487.5(and)-486.4(cells)-481.8(were)-482.7(removed)]TJ
T*
[(and)-372.6(the)-370.5(remaining)-376.3(constructs)-377.1(were)-368.9(polymerized.)-373.2(Over)-371.1(time)]TJ
0 -1.2266 TD
[(of)-382.6(cultivation,)-388.2(the)-383.1(cells)-386.9(spread)-378.8(inside)-387.7(the)-383.1(matrix,)-379.9(form)-387.2(cell)]TJ
0 -1.2203 TD
[(cell)-499.4(contacts,)-489.9(and)-492.8(spontaneously)-499.7(compact)-493.4(the)-496.9(matrix)-490.8(over)]TJ
T*
[(several)-473.5(days)-469.8(\(Fig.)-471(1A\).)-465.9(Two)-469(T-shaped)-473.5(cantilevers)-473.8(incorpo-)]TJ
0 -1.2266 TD
[(rated)-471.5(within)-477.5(each)-470(template)-470.6(anchor)-470.6(the)-471.7(contracting)-478.8(matrix,)]TJ
0 -1.2203 TD
[(constraining)-341.8(the)-338.9(contraction)-344.4(of)-332(the)-338.9(collagen/brin)-344.2(matrix)-332.7(to)]TJ
T*
[(form)-298.7(a)-290.4(linear)-295.1(band)-294.8(that)-288.4(spanned)-296.4(across)-295.7(the)-294.6(top)-295.4(of)-294.1(the)-294.6(pair)-289.6(of)]TJ
T*
[(cantilevers.)-325(This)-314.7(resulted)-313.7(in)-316.7(a)-315.7(large)-317.9(array)-314.7(of)-313.1(CMTs)-313.1(anchored)]TJ
0 -1.2266 TD
[(to)-395.3(the)-389.5(tips)-392.4(of)-395.3(the)-389.5(cantilevers)-398(per)-387.7(substrate.)-398.1(The)-392(spring)-394.1(con-)]TJ
0 -1.2203 TD
[(stant)-409.8(of)-414.2(each)-406.7(cantilever)-409.3(could)-408.3(be)-409(controlled)-411.4(by)-407.2(altering)-407.5(the)]TJ
T*
[(ratio)-620.3(between)-616.6(PDMS)-619.7(and)-612.9(curing)-619.7(agent.)-617.6(We)-618.6(used)-612.6(linear)]TJ
0 -1.2266 TD
[(bending)-489.9(theory)-479.5(and)-486.4(experimental)-486.1(measurements)-483.4(to)-483.8(report)]TJ
0 -1.2203 TD
[(the)-484.3(load-displacement)-483.6(relationship)-485(for)-478.2(two)-479.1(different)-486.1(ratios)]TJ
T*
[(PDMS/curing)-407.7(agent,)-408.9(leading)-403(to)-401.6(measured)-401.6(spring)-406.7(constants)]TJ
0 -1.2266 TD
[(of)-414.2(0.45)-406.1(and)-410.6(0.20)]TJ
/F5 1 Tf
7.4357 0 TD
(m)Tj
/F3 1 Tf
.5564 0 TD
(N/)Tj
/F5 1 Tf
1.4227 0 TD
(m)Tj
/F3 1 Tf
.5564 0 TD
[(m.)-405.1(These)-412.2(spring)-413(constants)-416.6(were)-413.1(then)]TJ
-9.9712 -1.2203 TD
[(used)-644.2(to)-641.9(link)-647.2(the)-642.4(measured)-641.9(cantilever)-649.6(deections)-642.5(to)-641.9(the)]TJ
T*
[(amount)-340.2(of)-332(force)-338.2(generated)-333.7(by)-337.6(CMTs)-332.1(\(Fig.)-331.9(1B\).)]TJ
1.1128 -1.2203 TD
[(After)-515.1(cell)-518.4(seeding,)-511.6(at)-518.5(day)-512.8(0,)-508.3(the)-515.9(collagen/brin)-514.9(matrix)]TJ
-1.1128 -1.2266 TD
[(contained)-388.3(evenly)-378.6(distributed,)-385(amorphous,)-380.4(round)-384.3(heart)-383.3(cells)]TJ
0 -1.2203 TD
[(\(Fig.)-363.5(1A\).)-364.7(Over)-358.5(time,)-364.8(cells)-361.6(elongated,)-361.3(aligned)-365(along)-361.4(the)-364.2(axis)]TJ
T*
[(between)-313.1(the)-307.3(cantilevers,)-312.4(and)-309.4(started)-308.9(to)-306.7(beat)-307.8(as)-310.6(single)-306.9(cells)-311.1(at)]TJ
0 -1.2267 TD
[(days)-558.3(12)-555(at)-550.1(a)-555.9(spontaneous)-561.3(frequency)-557.6(of)-553.3(1.1)]TJ
/F6 1 Tf
20.9478 0 TD
()Tj
/F3 1 Tf
.7208 0 TD
[(0.1)-166.3(Hz.)-552.5(Re-)]TJ
-21.6686 -1.2203 TD
[(modeling)-334.3(and)-328.4(compaction)-332.4(of)-332(the)-332.6(matrix)-326.4(led)-328.9(to)-332(a)-328.3(slower)-332.3(and)]TJ
T*
[(coherent)-838.1(beating,)-834.2(rhythmically)-839.4(deecting)-832.5(the)-838.4(posts)-835.5(at)]TJ
0 -1.2266 TD
(0.5)Tj
/F6 1 Tf
1.4163 0 TD
()Tj
/F3 1 Tf
.7208 0 TD
[(0.1)-166.3(Hz)-328.3(after)-329.8(day)-323.1(3)-328.3(\(Fig.)-325.6(1C\),)-331.9(and)-328.4(to)-325.7(a)-328.3(marked)-327.6(reduction)]TJ
-2.1371 -1.2203 TD
[(of)-515.4(construct)-521.2(size)-516.4(\(nal)-515.7(diameter)-516.7(5070)]TJ
/F5 1 Tf
17.9824 0 TD
(m)Tj
/F3 1 Tf
.5564 0 TD
[(m)-509.7(a)0(t)-518.5(the)-515.9(thinnest)]TJ
-18.5388 -1.2203 TD
[(portion)-375.4(of)-370(the)-364.2(construct)-375.8(at)-366.7(day)-367.3(7\).)-369.6(The)-366.7(beating)-369.7(frequency)-374.2(is)]TJ
0 -1.2266 TD
[(slower)-680.1(than)-677(the)-680.3(beat)-674.5(rate)-677.1(of)-679.8(an)-673.1(intact)-676.9(adult)-678.1(rat)-677.2(heart)]TJ
0 -1.2203 TD
[(\(57)-169.7(Hz\))]TJ
6.7246 0 0 5.9768 345.6566 126.9921 Tm
(10)Tj
8.9663 0 0 8.9663 357.1653 123.0236 Tm
[(or)-528.3(a)-530.6(neonatal)-537.3(rat)-531.8(heart)-528.8(\(46)-169.7(Hz\))]TJ
6.7246 0 0 5.9768 491.0172 126.9921 Tm
(32)Tj
8.9663 0 0 8.9663 502.5258 123.0236 Tm
[(but)-531(close)-531.6(to)]TJ
-21.1312 -1.2203 TD
[(frequencies)-697(measured)-692.4(in)-689.8(centimeter-scale)-692(cardiac)-694(tissues)]TJ
T*
[(generated)-555(from)-551.6(isolated)-551.5(cardiomyocytes.)]TJ
6.7246 0 0 5.9768 480.6991 105.1086 Tm
(5,33)Tj
8.9663 0 0 8.9663 497.4235 101.1401 Tm
[(Over)-548.2(time)-551.6(of)]TJ
-20.5621 -1.2266 TD
[(cultivation,)-464.1(the)-465.3(total)-462(contraction)-464.5(duration)-463.7(\(contraction)-465(and)]TJ
0 -1.2203 TD
[(relaxation\))-262.5(appeared)-258.1(to)-262.5(be)-257.2(roughly)-260.9(stable)-255.7(around)-257.3(0.25)]TJ
/F6 1 Tf
23.6161 0 TD
()Tj
/F3 1 Tf
.7208 0 TD
[(0.01)-165.9(s)]TJ
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