(20f) An Optically Controlled Microphysiological System for the Heart-Brain Axis

An Optically Controlled Microphysiological System for the
Heart-Brain Axis

Jonathan Soucy¹, Tess Torregrosa¹,
Abigail Koppes¹, Nasim Annabi^1,2, Ryan Koppes¹

¹Chemical
Engineering, Northeastern University, Boston, MA

²Harvard-MIT Division of Health Sciences and
Technology, Massachusetts Institute of Technology, Cambridge, MA Introduction:

While, there are more than
700,000 people in the United States suffer a heart attack annually, the
underlying cellular mechanisms that lead to cardiac dysfunction remains poorly
understood¹. Previously, animal models have been used to investigate
cardiovascular disease, but because of their inherent complexities,
variability, and failure to translate to human physiology, in vitro
alternatives must be developed². Towards this goal, we aim to
recapitulate the human cardiac sympathovagal balance in vitro to study
the underlying cellular changes of cardiac tissue in response to ANS
modulation.  

In vivo, the heart
responds to environmental changes by controlling cardiac output via the ANS. In
the case of deteriorating cardiac function, the sympathetic nervous system
(SNS) will innervate the cardiac tissue, while parasympathetic nervous system
(PSNS) will withdraw to maintain resting heart rate³. However, as
the branches of the ANS work to restore cardiac output, the overall balance of
neural input leans towards the sympathetic. This understanding of the cardiac
ANS pathophysiology has led to development of beta-blocker therapies to inhibit
SNS activity. Conversely, the role of the PSNS in heart failure not completely
understood and is an area of ongoing research. Preliminary cardiac ANS studies
have shown that increased cardiac PSNS activity can reduced risk of
arrhythmias, potentially leading to vagal stimulation as a new therapeutic
strategy⁴. Yet, mimicking this imbalance in vitro for investigating
the underlying cellular mechanisms has not been demonstrated.

Towards developing innervated muscle organ systems on
a chip, microfluidic devices have been used for compartmentalized co-culture of
skeletal muscle cells with neurons⁵, but only recently have applied
to cardiac systems². These neural cardiac co-culture devices can be
improved upon by culturing cardiomyocytes (CM) in a physiologically relevant
three-dimensional (3D) environment as well as the addition of means for
real-time, bi-directional control/output of neural and cardiac function.
Optogenetics allows for optical control of specific cell populations via
genetic modification with light-sensitive ion channels, microbial rhodopsins⁶.
In contrast to electrical stimulation, optogenetics enables temporally precise
excitatory and inhibitory control of neural activity in distinct cell types
expressing light-gated ion channels and pumps, such as channelrhodopsin 2
(ChR2) and halorhodopsin (NpHR)⁷. Furthermore, optogenetics is
compatible with simultaneous neural recording due to the decoupling of
electrical and optical signals. Independent, multiplexing modulation of
multiple cell types can be accomplished with opsins possessing distinct
absorption spectra such as channelrhodopsin and Chrimson⁸.
Therefore, there is are advantages for utilizing an optogenetic toolbox to
control SNS and PSNS activity independently without concern for confounding, non-specific
stimulation or confounding influence on monitored electrical activity.

Here, we aim to design a custom
chip to mimic an in vivo cardiac ANS and allow for optical control of
neural activity. In vitro models provide numerous benefits over their in
vivo counterparts by reducing variability, lowering cost, and by allowing
for the inclusion of knockout transgenic cell lines used to study specific
genes. Developing a functional model of the cardiac ANS will allow for an
improved fundamental understanding of the relationship between the cholinergic
PSNS neurons, the adrenergic SNS neurons. Additionally, by studying the
cellular responses to physiological relevant changes, new therapeutic targets
may be identified to treat cardiovascular disease.

Methods:

A custom microfluidic chip made of PDMS with three
independent channels, separated by microposts for capillary separation of the
central cardiac laden 3D hydrogel (Fig 1). This microfluidic chip will be
mounted onto a 60-channel multielectrode array (MEA, Multichannel Systems) to
enable both the stimulation and monitoring each cell’s electrical potential.

Pure populations of CM and cardiac fibroblast (CF)
from the heart, cholinergic neurons from the intrinsic cardiac ganglia, and
adrenergic neurons from the superior cervical ganglia from neonatal rat pups
will be isolated for the in vitro platform. Using isolated cells, we will
encapsulate varying ratios of CM and CF within a photocrosslinkable gelatin
based hydrogel, gelatin methacrylate (GelMA)⁹in situ using a
prototype chip design and in micropatterned gel channels. We used an in-house
photocrosslinking system with 405nm LEDs to polymerize GelMA in the presence of
lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP, BioBots). The use of
this system resulted in CM viability significantly greater than systems using
UV light; however, beating was only observed when both CM and CF were present
within the gel. 

Light sensitive neuron populations will be produced
via transfection with adeno-associated viruses carrying either Chronos-GFP or
ChrimsonR-dTomato (UNC Vector Core), to allow for independent optical control
of SNS or PSNS populations. Transfection will be validated via positive
expression of appropriate fluorescent reporter protein. 

In addition to monitoring electrical activity, to
enable quantification of cell beating activity, we developed a custom MATLAB
code to calculate beating on a cell-by-cell basis using video microscopy.
Furthermore, we developed a novel method to quantify the synchronicity of CM
beating (Fig. 1B), based on an algorithm originally developed to investigate
neuronal activity¹⁰. 

Results
and Discussion:

We have successfully fabricated the PDMS microfluidic
platform on a 64-channel microelectrode array for real-time monitoring of both
neural and muscle populations. Primary populations for cardiomyocytes,
fibroblasts, as well as adrenergic, and cholinergic neurons were successfully
isolated from neonatal rat pups. Independent transfection of the SNS and PSNS
populations was validated with robust expression of fluorescent reporter
proteins in Tyrosine Hydroxylase or Acetyl Choline positive neurons.
Furthermore, seeding of our platform showed spontaneous neurite ingrowth into
the cardiomyocyte laden hydrogel.

Our visual method for quantifying cardiomyocyte
contraction demonstrated good correlation to contraction monitored on the MEA.
Our method was able to detect changes in beating and coordinated contraction
induced via the addition of Isoproterenol, a beta-adrenergic agonist (Fig. 1C).
Further, using our visual quantification method of cardiomyocyte beating over
long-term culture, we observed that the degree of synchrony increased to a
steady value after 5 days. However, for measurements from longer culture times
great than 2 weeks, there were increased arrhythmias. Nevertheless,
immunocytochemistry showed that CM phenotype was maintained throughout the
study.

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