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\begin{document}

\title{Injectable conductive hydrogel restores conduction through ablated
myocardium}



\author[1]{Martin van Zyl}%
\author[2]{Dawn Pedrotty}%
\author[3]{Erdem Karabalut}%
\author[3]{Volodymyr Kuzmenko}%
\author[3]{Sanna Sämfors}%
\author[4]{Chris Livia}%
\author[1]{Vaibhav Vaidya}%
\author[4]{Alan Sugrue}%
\author[1]{Christopher McLeod}%
\author[4]{Atta Behfar}%
\author[1]{Samuel Asirvatham}%
\author[3]{Paul Gatenholm}%
\author[1]{Suraj Kapa}%
\affil[1]{Mayo Clinic}%
\affil[2]{Hospital of the University of Pennsylvania}%
\affil[3]{Chalmers University of Technology}%
\affil[4]{Mayo Clinic Rochester}%


\vspace{-1em}



  
  \date{May 21, 2024}


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\selectlanguage{english}
\begin{abstract}
Abstract Introduction: Therapies for substrate-related arrhythmias
include ablation or drugs targeted at altering conductive properties or
disruption of slow zones in heterogeneous myocardium. Conductive
compounds such as carbon nanotubes may provide a novel personalizable
therapy for arrhythmia treatment by allowing tissue homogenization.
Methods: A nanocellulose-carbon nanotube conductive hydrogel was
developed to have conduction properties similar to normal myocardium. Ex
vivo perfused canine hearts were studied. Electroanatomic activation
mapping of the epicardial surface was performed at baseline, after
radiofrequency ablation, and after uniform needle injections of the
conductive hydrogel through the injured tissue. Gross histology was used
to assess distribution of conductive hydrogel in the tissue. Results:
The conductive hydrogel viscosity was optimized to decrease with
increasing shear rate to allow expression through a syringe. The DC
conductivity under aqueous conduction was 4.3\selectlanguage{ngerman}·10-1 S/cm. In 4 canine
hearts, when compared to the homogeneous baseline conduction, isochronal
maps demonstrated sequential myocardial activation with a shift in
direction of activation to surround the edges of the ablated region.
After injection of conductive hydrogel, isochrones demonstrated
conduction through the ablated tissue with activation similar to
baseline in all 4 hearts. Gross specimen examination demonstrated
retention of the hydrogel within the tissue. Conclusions: This
proof-of-concept study demonstrates that conductive hydrogel can be
injected into acutely disrupted myocardium to restore conduction. Future
experiments should focus on evaluating long-term retention and
biocompatibility of the hydrogel through in vivo experimentation.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Condensed Abstract}

A novel conductive hydrogel was developed as a method to treat
substrate-based arrhythmias by homogenizing conduction through disrupted
myocardium. In \emph{ex vivo} canine hearts, the hydrogel was injected
evenly through ablated regions of myocardium. Electroanatomic activation
mapping of the epicardial surface demonstrated a shift in conduction to
surround edges following ablation of the region. Following injection of
the conductive hydrogel, conduction improved through the ablated region
approximating baseline in all 4 hearts. This proof-of-concept study
demonstrates that conductive hydrogel can be injected into acutely
disrupted myocardium to restore conduction.

\textbf{Abbreviations List}

CNT = carbon nanotube

DC = direct current

EAM = electroanatomic mapping

NFC = nanofibrillated cellulose

SD = standard deviation



\textbf{Introduction}

Arrhythmias in the structurally abnormal heart may be precipitated by
heterogeneous conduction through regions of fibrosis or scar. In cases
of myocardial infarction, the mechanism for arrhythmogenesis is usually
due to the presence of viable myocardium interspersed amidst scarred
tissue that leads to reentry or enhanced automaticity. Currently,
methods to prevent arrhythmia focus on impacting these slow, viable
zones through use of either antiarrhythmic medications that alter
activation characteristics or ablation to destroy (ie, homogenize) the
tissue region. However, both approaches carry significant limitations
either due to medication side effects, including the potential for
proarrhythmia, or creation of new scar. Furthermore, existing technology
is limited by an inability to completely eliminate all arrhythmia
circuits (eg, due to circuits deep in the mid-myocardial region).

Regenerative approaches for enhancing conduction through regions of
myocardial scar, preventing the fibrotic remodeling that is thought to
contribute to arrhythmogenesis, or reversing the damage done from an
infarct or other injurious events reflect new approaches to treatment of
patients with myocardial disease(1-4). Use of stem cells or biologically
compatible, synthetic compounds have been studied extensively, although
with greater focus on effects on myocardial contractility and
ventricular function rather than arrhythmogenicity. Certain compounds,
such as carbon nanotubes, have been studied in a variety of biological
approaches, including as a means of potentially enhancing myocardial
health(5-11). One promising aspect of carbon nanotubes is that they are
highly conductive. We and others have previously shown that applying 3D
printed patches composed of carbon nanotubes intermixed with
biocompatible compounds could restore conduction across the surface of a
previously injured region(12,13). We sought to evaluate whether an
injectable, conductive carbon nanotube-based hydrogel would impact
conduction properties through a region of iatrogenically disrupted
(ablated) myocardium.

\textbf{Methods}

The protocol was approved by the Institutional Animal Care and Use
Committee at Mayo Clinic in Rochester, MN.

\emph{Conductive hydrogel preparation and evaluation}

Single-walled carbon nanotubes (CNTs; 76.9 mg, P3, 3-6-wt\% R-COOH;
Carbon Solutions Inc., Riverside, CA, USA) with a bundle diameter of
\textasciitilde{}4--5 nm and a bundle length of \textasciitilde{}1 \selectlanguage{greek}µ\selectlanguage{english}m
were purified with nitric acid and added to an aqueous solution of a
nonionic, noncytotoxic surfactant Pluronic F127 BioReagent (10 mL,
0.1-wt\%; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany). After heating
for 8 hours at 70\selectlanguage{ngerman}°C, the mixture was sonicated for 16 hours creating a
0.77-wt\% dispersion of CNTs. Nanofibrillated cellulose (NFC; 2 g\selectlanguage{english}[?]2 ml,
3-wt\%, carboxymethylated nanocellulose, charge density
\textasciitilde{}515 \selectlanguage{greek}μ\selectlanguage{english}eq/g; Innventia AB, Stockholm, Sweden) was
Electron Beam sterilized and then combined with 2 ml of the dispersion
using a SpeedMixer\textsuperscript{TM} (FlackTek Inc., Landrum, SC, USA)
at 2000 rpm twice for 2 minutes each. The result was a homogeneous
hydrogel with dry matter of 20-wt\% and NFC:CNT dry weight ratio of 4:1.
Our method for assessing conductivity of the hydrogel has been described
previously(12).

\emph{Rheology}

The rheological properties of the conductive hydrogel were analyzed
using the Discovery HR-2 rheometer (TA Instruments, UK) with a Peltier
plate. The measurements were performed at 25 \selectlanguage{ngerman}°C, and the samples were
allowed to reach equilibrium temperature for 60 s prior to each
measurement. An aluminum plate-plate (20 mm, gap = 500 \selectlanguage{greek}μ\selectlanguage{english}m) was used and
the shear viscosity was evaluated by increasing the shear rate from 0.1
to 1000 s\textsuperscript{-1} at 25 \selectlanguage{ngerman}°C.

\emph{Ex vivo heart preparation}

A total of 4 mongrel dogs (25-40 kg in weight) were placed under deep
sedation with isoflurane inhalational anesthesia. A left lateral
thoracotomy allowed visualization of the heart and great vessels.
Therapeutic heparinization was confirmed (activated clotting time
\textgreater{} 300 s) after which the aorta was cannulated to allow
cardioplegia solution to be administered in a retrograde fashion. After
asystole was achieved, cardiectomy was performed and the heart was
immediately cooled to 4°C. A large animal Langendorff perfusion
apparatus (Radnoti, Covina, CA, USA) was used to perfuse the
heart\emph{ex vivo} in a warmed basin (\emph{Figure 1} )(14). The hearts
were perfused with a modified Krebs\selectlanguage{english}-Henseleit solution which was
oxygenated with 95\% O2 and buffered to 5\% CO2(15). The perfusate
solution consisted of the following: NaCl (118 mmol/L),
NaHCO\textsubscript{3} (25 mmol/L), mannitol (16 mmol/L), d-glucose (11
mmol/L), KCl (4.7 mmol/L), sodium pyruvate (2.3 mmol/L),
CaCl\textsubscript{2} (2.0 mmol/L),
KH\textsubscript{2}PO\textsubscript{4} (1.18 mmol/L),
MgSO\textsubscript{4} (1.17 mmol/L), and human insulin (10 U/L; Novolin;
Novo Nordisk, Plainsboro, NJ). The perfusate temperature was maintained
at 37degC+-1degC.

\emph{Ablation and hydrogel injection in beating heart and approach to
cardiac mapping}

Upon establishing the \emph{ex vivo} suspension and stable function of
the heart, patches were attached around the basin to permit \emph{ex
vivo} electroanatomical mapping (EAM; CARTO3 system, Biosense Webster,
Diamond Bar, CA, USA). A 3.5 mm tip irrigated ablation catheter
(ThermoCool Navistar; Biosense Webster, Diamond Bar, CA, USA) was used
to perform both mapping and focal ablation. First, during normal sinus
rhythm, point-by-point mapping of the epicardial surface of the lateral
left ventricle was performed. Each point was adjudicated to establish an
appropriate timing reference. After this, a point with homogeneous
conduction and voltage was selected within the mapped region as a target
for ablation at 30 Watts for 1 minute with normal saline irrigation at
30 ml/min. We then remapped the region including within the scar region
and around in regions of normal tissue. The hydrogel was then injected
using an 18-gauge needle by sequentially injecting circumferentially
around the area of ablated myocardium by pushing the needle into the
tissue and slowly injecting the hydrogel as it was withdrawn with each
injection. Injections were then performed in perpendicular lines spaced
evenly 1 mm apart through the center of the lesion in a grid pattern.
After this, remapping was done of the region with again careful
attention to annotation of individual points. The time between injection
and remapping was within minutes. All mapping was done during sinus
rhythm.

\emph{Gross specimen evaluation}

Gross examination of the regions of injection was undertaken by first
dissecting out the entire transmural region where ablation was
performed. We then did two orthogonal incisions through the center of
the lesion region to be able to evaluate the myocardium throughout the
ablated region. Presence of the conductive hydrogel was based on the
gross appearance throughout the region given that the black appearance
of the hydrogel is otherwise obvious against the surrounding myocardial
tissue. No specific quantification was done of the amount of hydrogel
retained within the volume of ablated tissue.

\emph{Statistical approach}

Mean and SD were calculated based on data from 5 samples for analysis of
mechanical hydrogel properties. Statistical significance was determined
based on an \selectlanguage{greek}α \selectlanguage{english}\textless{} 0.05. All statistics were performed in JMP Pro
11 (SAS Institute, Cary, NC, USA).

\textbf{Results}

\emph{Characterization of conductive hydrogel}

Rheological measurements of the shear viscosity of the conductive
hydrogel showed that the hydrogel had good shear thinning properties
(\emph{Figure 2A} ), i.e. becomes more liquid when a shear force is
applied. This allows for ejection from a needle while still keeping its
form after it is extruded from the needle head (\emph{Figure 2B} ). To
evaluate gel formation ability, a solution of 100 mM
CaCl\textsubscript{2} was added to the extruded hydrogel. The hydrogel
exhibited a gelling behavior and after five minutes the hydrogel was
able to be lifted using tweezers (\emph{Figure 2C} ). This indicated
that the conductive hydrogel can crosslink with the presence of divalent
cations to form a stable gel.

Pure NFC as well as NFC/CNT hydrogel were demonstrated to have
well-defined dynamic yield stress (G'=G''; 217 and 106 Pa, respectively)
with transition from elastic to viscous behavior. DC conductivity under
aqueous conduction was 4.3\selectlanguage{ngerman}·10\textsuperscript{-1} S/cm in the
homogeneous dispersion of 20 wt\% CNT in NFC hydrogel.

\emph{Mapping pre-ablation, post-ablation, and post-injection}

In 4 \emph{ex vivo} perfused beating canine hearts, a detailed EAM was
obtained of the epicardial lateral left ventricle during sinus rhythm at
baseline, following ablation, and again following injection of the
conductive hydrogel (\emph{Figure 3} ). In all 4 animals (100\%), we
were able to show a disruption of homogeneous baseline conduction by
ablation. Following injection of conductive hydrogel, a bridge of
improved conduction was demonstrated across the ablated region in all 4
animals (100\%).

By placing the mapping catheter on the same site of the muscle in 1
animal, we were able to demonstrate a double potential indicative of
far-field and near-field bipolar activation at baseline likely
suggestive of endocardial to epicardial activation. Local activation was
supported by the steepest negative deflection in the unipolar signal.
Far-field and near-field activation appeared to separate with a small
isoelectric interval following ablation. Following CNT hydrogel
injection, the bipolar signals reapproximated and appeared similar to
baseline (\emph{Figure 4} ).

\emph{Gross specimen evaluation of myocardium post-injection}

We performed gross examination of the myocardial specimen following
ablation and CNT hydrogel injection. The hydrogel could be seen
circumferentially around the ablated lesion at the points where the
needle entered the epicardium. Orthogonal transection showed that most
of the hydrogel was retained within the ablated tissue. Some hydrogel
did extrude from the lesion into areas of nonablated myocardium
(\emph{Figure 5} ).

\textbf{Discussion}

An injectable carbon nanotube hydrogel was developed by our group with
enhanced conductive properties that retain conductivity in aqueous
environments. Furthermore, we have demonstrated that injection of such a
hydrogel into iatrogenic regions of myocardial injury can enhance
conduction through these regions. These data provide support that such a
conductive material may impact conduction characteristics of myocardium.
They also lay the foundation for future work to enhance understanding of
the utility of such an approach and the potential impact on
arrhythmogenesis.

\emph{Future needs of injectable preparations}

The conductive hydrogel preparation in our study represented an initial
evaluation of a carbon nanotube complex that was made to be expressible
through a syringe and needle. However, materials properties need to be
considered in the context of the specific use case in which they are
applied. In the case of injectable materials, they should liquify upon
applying a specific pressure for easy flow out of a container (eg, a
syringe) and then immediately solidify upon expression out into whatever
material they will be contained in (eg, within myocardial tissue). In
the context of our study, while some conductive hydrogel was retained in
the myocardium as seen on gross examination, some loss of hydrogel also
occurred during injection. We performed the experiments \emph{ex vivo}in
a still beating heart with an epicardial injection approach. However,
any such injectable material, if delivered endocardially, will have to
be optimized to prevent any material loss that could then embolize.
Furthermore, the amount injected and retained needs to be predictable
and thus further research to evaluate the minimum quantity of material
needed to impact conduction will be necessary.

Prior studies have looked at complexing material with alginate or other
substances to optimize properties for injection and retention in
tissue(16,17). However, most of these studies focused on alginate as a
material scaffold that could optimize structural integrity of the tissue
or as a delivery tool for stem cells, drugs, or other materials. The
impact of complexing other substances with carbon nanotubes and similar
conductive materials will need to be considered in the context of their
impact on conductivity of the material itself as well as the impact on
the material-myocardial interface in terms of cell-to-material-to-cell
interaction. Also, whether commercially available trans-endocardial
injection catheters such as the MyoStar catheter (Biosense Webster,
Diamond Bar, CA, USA) will be able to accommodate the conductive
hydrogel, will need to be further evaluated. Thus, while our work
demonstrates proof of concept, these limitations need to be considered
prior to \emph{in vivo} studies, particularly if endocardial injections
are performed.

\emph{Anti-arrhythmic or proarrhythmic?}

As with any treatment (eg, ablation or drugs) targeting regions of
heterogeneous conduction, therapies meant to alter local conduction
should be considered in light of their potential for proarrhythmia. We
propose injecting conductive materials into regions of damaged
myocardium as a means of ``normalizing'' conduction. However, it should
be noted that we did not account in our study for conduction
endocardially or mid-myocardially. Furthermore, while conduction
post-nanotube injection seemed similar to baseline, it cannot be assumed
to be precisely the same. Use of omnipolar smaller electrodes or optical
mapping may allow for enhanced understanding of local conduction
direction. While globally it appears that conduction follows the same
path as baseline, heterogeneity due to disarray of conduction or
conduction abnormalities only revealed with a different wavefront of
activation may result in continued arrhythmic potential, as seen in
studies of ventricular mapping for substrate characterization.

It is also possible that with chronic retention in tissue, myocardial
fibrosis or immune reaction may occur that may alter the tissue-material
interface. Furthermore, conductivity may change over time that may
result in proarrhythmia in the long-term even if activation appeared to
be normalized acutely. Many of these concerns may be clarified in
chronic \emph{in vivo} studies. However, animal-based studies may never
fully recapitulate human tissue reactions. Furthermore, how integration
varies in different types of substrate, whether related to myocardial
ischemia and infarction, inflammation-mediated injury, genetic
abnormalities, or other causes, needs to be considered.

\emph{Comparison to prior work on conductive materials}

It is important to consider these findings in light of prior data on the
impact of conductive materials on cell-cell interaction in the heart. We
have previously reported data on the use of 3D-printed patches to
restore conduction across disrupted areas of myocardial activation(12).
This has similarly been demonstrated by others. However, scar and
arrhythmogenic circuits are often multi-dimensional and not simply
reflected along a single surface. Thus, identifying ways to enhance
conduction not just along the surface (whether endocardially or
epicardially) but transmurally may be critical when developing a
solution that may be antiarrhythmic. Each will have theoretical benefits
and limitations -- a patch may be less invasive, reversible, and easier
to apply with a lower risk of complications due to lack of need to
inject directly into myocardial tissue. However, injection approaches
may more directly address the conduction heterogeneity attributable to
3-dimensional complexity that is likely seen in most cases of
scar-related arrhythmia.

Furthermore, prior work has suggested that other materials aside from
carbon nanotubes may allow for enhanced conduction between otherwise
electrically disconnected myocytes. We did not directly compare the
effects of our material with these other materials. However, most work
has been on creating a surface over which cells are placed and then
evaluating conduction rather than methods that may facilitate enhanced
conduction transmurally. Determination of the optimal conductive
materials based on chronic maintenance of conduction and
biocompatibility will require further study.

\emph{Mechanisms of cell-material interaction}

One of the key questions when using materials to impact cell to cell
interaction is the mechanism that facilitates the cell-material-cell
interaction. It is well established that conductive materials can be
used to sense electrical activity from a cell and stimulate cells (eg,
with pacemakers). However, such ability is often dependent on
considerations of source/sink and other factors. The fact is that the
electrical impulse propagated by a conductive material from a cell that
is activated to a distant cell is not well understood and will require
further study.

\emph{Limitations}

There are several limitations to our study beyond those noted above.
First, evaluation was done on a limited number of hearts. Thus,
consistency in results across larger sample sizes needs to be evaluated.
Second, these studies reflected acute evaluations of changes in
conduction both post-ablation and post-injection. It is possible that
lesion maturation may occur over a longer period of time, resulting in
dynamic shifts in cardiac activation that may not have been reflected in
the acute state. Third, as stated previously, we could not inject a
predictable amount of hydrogel each time due to materials properties not
permitting appropriate in-tissue retention. Thus, we could not determine
a threshold amount of material that would need to be injected to
facilitate some change in conduction. In addition, the approach to the
testing and mapping (\emph{ex vivo} perfused hearts and epicardial only
mapping) may not necessarily extend to in vivo examples, and, thus,
before further conclusions can be made, \emph{in vivo} acute and chronic
studies will be needed. Finally, we could not evaluate immunogenicity or
potential for chronic maintenance of the perceived conduction changes
due to the acute and \emph{ex vivo} nature of the study.

\textbf{Conclusion} Our study shows that novel injectable conductive
hydrogel can enhance myocardial conduction through regions of tissue
injury. Future studies will need to focus on optimizing rheological,
electrical conductivity, and in-situ gel formation properties of the
conductive hydrogel such that it can be predictably retained within
tissue while maintaining conductive properties. In addition, it will be
important to improve understanding of the effect on local conduction not
just epicardially but transmurally. Finally, establishing both long-term
durability of activation normalization and biocompatibility will be key.

\textbf{Acknowledgments}

EK, VK, SS, PG acknowledge the Knut and Alice Wallenberg Foundation for
funding of The Wallenberg Wood Science Center.

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7. Kharaziha M, Shin SR, Nikkhah M et al. Tough and flexible
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Ghamkhari Nejad G. The cardiac effects of carbon nanotubes in rat.
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\textbf{Figure Legends}

\textbf{Figure 1.} Langendorff apparatus -- The Langendorff apparatus
(A) and \emph{ex vivo} perfused heart in warmed basin (B) are shown.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image1/image1}

\end{center}
\end{figure}

\textbf{Figure 2.} Rheological properties for the conductive hydrogel --
Flow curve showing the shear thinning property of the hydrogel (A).
Image of the conductive hydrogel after ejection from a needle (B).
Conductive hydrogel after 5 minutes of cross-linking in 100 mM
CaCl\textsubscript{2} solution (C).

\textbf{Hosted file}

\verb`image2.emf`  available at \url{https://authorea.com/users/337704/articles/463297-injectable-conductive-hydrogel-restores-conduction-through-ablated-myocardium}

\textbf{Figure 3.} Electroanatomical mapping -- The electroanatomical
propagation map showing activation for the same epicardial area of the
lateral left ventricle in a representative animal. At baseline (A),
homogenous and rapid activation is demonstrated. There is disruption of
conduction as evidenced by clustering of isochrones following ablation
(B). Following injection of conductive hydrogel (C), a bridge of
improved conduction is restored across the ablated myocardium. Red dots
indicate site of ablation, purple dots indicate border zone of abnormal
myocardium as determined by voltage (\textless{}1.5 mV), and block dots
indicate sites of needle entry for hydrogel injection. LAT=local
activation time.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image3/image3}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image4/image4}

\end{center}
\end{figure}

\textbf{Figure 4.} Local activation signals -- The Mapping catheter
location is shown annotated on the electroanatomical map by the orange X
(A). The catheter was placed in the same location at baseline (B),
following ablation (C), and following conductive hydrogel injection (D).
The steepest negative unipolar deflection corresponds with the sharp
near-field bipolar potential suggesting that this point reflects local
activation. A far-field potentials closely precedes the near-field
activation at baseline. Following ablation there is separation with an
isoelectric interval between far-field and near-field potentials.
Following conductive hydrogel injection, the bipolar potentials
approximate -- similar in appearance to the baseline signal.

\textbf{Figure 5.} Hydrogel retention within myocardium -- Gross
evaluation of the myocardium from the epicardial surface shows the pale
ablated tissue and black circumferentially injected conductive hydrogel
(A). Orthogonally transected ablation lesions (B \& C) demonstrate the
hydrogel mostly retained within the ablated lesion, however, some
hydrogel can be seen extruding into regions of unablated myocardium
(arrows).\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/image5/image5}

\end{center}
\end{figure}

\selectlanguage{english}
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