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of the biohybrid stingray (6 days) ( 3 ) and the
skeletal muscle-based biohybrid actuator
(7 days) ( 40 )], equivalent to 38 million beats
(Fig. 5, A and B, and movies S23 and S24). Fur-
ther, its locomotion could also be controlled
by external optogenetic stimulation (movie
S24) throughout this time. The autonomously
swimming biohybrid fish also increased mus-
cle contraction amplitude, maximum swim-
ming speed, and muscle coordination for the
first month before maintaining its swim-
ming performance over 108 days (Fig. 5C).
By contrast, biohybrid fish equipped with
single-layered muscle showed deteriorating
tail-beat amplitude within the first month (Fig.
5C and movie S25). These data demonstrate
the potential of muscular bilayer systems and
mechanoelectrical signaling as a means to pro-
mote maturation of in vitro muscle tissues.

Discussion

We integrated two functional design features
of the heart—mechanoelectrical signaling and
automaticity—into a biohybrid platform and
recreated an autonomously actuating car-
diac muscular system in the form of a bio-
hybrid fish. This fish is a closed-loop system in
which muscle contraction–induced bending
is used as a feedback input to the endoge-
nous mechanosensors—stretch-activated ion
channels—in the muscles. These channels
respond to this feedback input and induce
muscle activation and contraction, producing
self-sustainable rhythmic BCF propulsion. The
self-driven spontaneous contractions in our
muscular bilayer induced coordinated global
tissue-level contractions with comparable effi-
ciencies to wild-type fish. Alternatively, inte-
grated optogenetic control enabled overriding
of internal control mechanisms to stop and con-
trol asynchronous muscle contractions. There
are few, if any, closed-loop mechanical fish
robots that are free-swimming; fish robots also
typically require numerous actuators and sen-
sors to control fin movements, and these are
difficult to engineer at smaller size scales (mil-
limeters to centimeters scale) ( 41 ). However,
integration of the cardiac activation system as
an embedded mechanism of both sensing and
control enabled the generation of fishlike loco-
motion at such smaller scales ( 42 ). The use of
biological muscle actuators with intrinsic closed-
loop control simplifies the construction com-
pared with current mechanical robotic systems
and provides control beyond existing biohy-
brid systems.
Additionally, our muscular bilayer con-
struct provides a platform for studying tissue-
level cardiac biophysics. We demonstrate that
dynamic axial stretching can induce excita-
tions and contractions on a beat-by-beat basis
in engineered human stem cell–derived CM
tissues by contributing to antagonistic mus-
cle contractions. We found that antagonistic

contractions are sensitive to streptomycin

and Gd3+, which indicates that mechano-

electrical signaling by means of stretch-activated

ion channels is one of the essential mecha-

nisms that mediate antagonistic contractions.

Notably, in normal myocardium where CMs

are mechanically and electrically coupled,

mechanoelectrical signaling contributes to

synchronizing local ventricular repolarization

and protects against cell-to-cell repolariza-

tions and contractile heterogeneities across

the heart ( 43 ). By contrast, in our muscular

bilayer where antagonistic muscle pairs are

mechanically coupled yet electrically decou-

pled across sides, mechanoelectrical signaling

generates stretch-induced depolarizations

on a beat-by-beat basis. The stretch-induced

excitations and contractions were also ob-

served in quiescent single CMs and in a rest-

ing ventricular myocardium ( 10 ), but these

observations were restricted to the ectopic

responses of CMs to acute mechanical stimu-

lation, which induced re-entrant arrhythmias.

Our muscular bilayer construct is the first to

demonstrate that the mechanoelectrical sig-

naling of CMs could induce self-sustaining

muscle excitations and contractions for ex-

tended periods (108 days, equivalent to 38 mil-

lion beats). These findings are aligned with

the growing appreciation for cardiac stretch-

activated channels and mechanoelectrical sig-

naling mechanisms as targets of heart rhythm

management ( 10 , 44 ). The longevity of the

autonomously moving fish system also raises

the question of whether a feedback between

repetitive electrical and mechanical activity

and the regulation of its molecular elements

through altered gene expression or other basic

cellular processes is correlated.

The G-node, an isolated cluster of cells con-

nected through a single conducting exit path-

way, initiated spontaneous activation waves

by reducing the impedance between source

and sink. G-node integration improved locomo-

tion speeds by enhancing the pacing frequency.

This increased frequency in the presence of

the G-node is reminiscent of entrainment in

re-entry cycles in which the focus shortens the

re-entry cycle ( 45 ). Another possible under-

lying mechanism of the increased frequency

is that the G-node produced regular contrac-

tions and consequently induced stronger

and more rapid contractions of the muscular

bilayer, which could enhance the dynamics

of antagonistic, asynchronous muscle contrac-

tions. The G-node functionality as a node of

automaticity in the biohybrid fish suggests

that, functionally, a pacemaker may be de-

fined by its geometry and source-sink relation-

ships as well as its ion channel expression.

Taken together, the technology described

here may represent foundational work toward

the goal of creating autonomous systems ca-

pable of homeostatic regulation and adaptive

behavioral control. Our results suggest an op-

portunity to revisit long-standing assumptions

of how the heart works in biomimetic systems,

which may allow a more granular analysis of

structure-function relationships in cardiovas-

cular physiology.

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ACKNOWLEDGMENTS

We thank M. Rosnach for photography and illustrations.

Funding:this work was funded by the Harvard Paulson School

of Engineering and Applied Sciences, the Wyss Institute for

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