(447d) Neuro-Cardiac Axis on a Chip: Neural Remodeling of the Cardiac Microenvironment

Neuro-Cardiac Axis on a Chip: Neural Remodeling of the
Cardiac Microenvironment

Jonathan R Soucy,
Tess Torregrosa, Sanjin Hosic,
Sebastian Moreno Arteaga, Abigail N Koppes, Ryan A Koppes

Chemical
Engineering, Northeastern University, Boston, MA

Autonomic nervous system
(ANS) function is vital for maintaining homeostasis in the heart [1], and its
role in cardiac pacing is essential to consider when developing new therapies
for cardiovascular repair. There exists a complex interplay between the heart
and the two branches of the ANS: the parasympathetic nervous system (PSNS) and
sympathetic nervous system (SNS) [2]. In
vivo, the SNS increases the heart rate (HR), while the PSNS lowers the HR [1,
2]. In cases of deteriorating cardiac function, commonly caused by ischemia or
aging, the ANS works to restore cardiac output by promoting SNS
hyperinnervation/activation, and PSNS withdrawal/deactivation [1, 3]. However,
the sympathetic imbalance of neural inputs has often been linked to increased
occurrences of arrythmias and sudden cardiac death [4-6]. This understanding of
the cardiac ANS pathophysiology has led to the prescription of beta-blockers to
inhibit SNS activity [6-8]. Despite these current pharmacological innervations,
there remains no treatment to restore the cardiac ANS homeostasis following
neurocardiac remodeling. Therefore, there is an immediate need to understand
the underlying cellular mechanisms of cardiac innervation to develop new
strategies for restoring ANS balance. Previously, animal models have been used
to investigate cardiac ANS dysfunction, but due to their inherent complexities,
multi-system interactions, and variability, in
vitro alternatives must be developed [9, 10].

Here, we have
developed a novel microfluidic platform for investigating innervation of the
cardiac microenvironment in vitro.
This approach will allow us to recapitulate the cardiac sympathovagal balance in vitro, to study the underlying changes in cellular function for both
neural and cardiac tissues. To mimic the discrete structures of the cardiac ANS
and the physiologically relevant three-dimensional (3D) tissue environment,
neural and cardiac cells populations were compartmentalized within microfluidic
devices and encapsulated within biomimetic polymeric hydrogels, respectively.
Innervation in response to cardiac stimulation and cardiomyocyte function
with/without stimuli will be assessed on-chip. Together, these approaches will
help improve our current understanding of the interactions and remodeling of
the neural-cardiac axis, towards identifying novel therapeutic strategies.

Methods

Custom microfluidic chips to support the
compartmentalization of each ANS population and cardiac cells within a
biomimetic scaffold were fabricated using a new “cut and assembly” method (Figure 1). Acrylic sheets, double sided
adhesives, and a polycarbonate track etched membrane were cut into defined
geometries using a commercial laser engraver system and assembled
layer-by-layer. Cardiac
cells, PSNS neurons, and SNS neurons were isolated from neonatal rat pups as
previously described [11], and cultured in situ within a photocrosslinkable
gelatin-based hydrogel. Innervation of the cardiac microenvironment was
investigated via immunofluorescent imaging at discrete time points throughout
the duration of culture and quantified using commercial neuron tracing software
(Neurolucida®). To investigate how cardiac function
effects innervation, cardiac cells were paced pharmacologically using a either
a beta-adrenergic receptor or calcium channel agonist to increase and decrease
beat rate, respectively. In each case, cardiac beat rate and degree of
coordination was quantified with a custom MATLAB® code to calculate beating on
a cell-by-cell basis using video microscopy.

Results and Discussion

Towards improving our understanding of neural
remodeling in the cardiac microenvironment, we have developed a physiologically
relevant organ-chip model of the cardiac ANS using a novel microfabrication
technique. This microfluidic chip utilizes a phase guide to establish discrete
cell compartmentalization within a contiguous hydrogel in the basal channel,
and polycarbonate membrane to allow for medium diffusion from the apical
channel and mimic circulation. Using this system, on-chip innervation of the
cardiac microenvironment was confirmed via immunostaining (Figure 2). Under these control conditions, we observe trending
differences in cardiomyocyte beating between those innervated by each ANS
neuron population (PSNS vs. SNS: 37.4 ± 6.7 bpm vs. 44.8 ± 7.9 bpm, n = 8, p =
0.0716), that are likely a consequence of spontaneous neural activity.

Using
this cardiac ANS in vitro model, we have investigated if cardiac pacing can be
exploited to promote or inhibit innervation from one or both branches of the
ANS. Towards this goal, we have demonstrated that isoproterenol, a beta-
adrenergic receptor agonist increases cardiomyocyte beat rate on-chip, and FPL
64176, a L-type calcium channel agonist, decreases beating. Ongoing work will
quantify innervation and corelated total neurite extension with the
pharmacological regulation of cardiomyocyte beating. Qualitatively, preliminary
results suggest that addition of isoproterenol induces in a high degree of
sympathetic innervation on-chip after 10 days of culture (Figure 2). As we continue to investigate innervation within the
cardiac microenvironment in response to cardiac stimulus, we will quantify
neurite outgrowth and cardiac function compared to unstimulated controls. The
development, characterization, and systematic perturbation of external stimulus
within this cardiac ANS organ-chip will further our understanding of
neurocardiac remodeling and may lead to the development of novel curative
therapies to restore ANS balance in the heart.

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