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\begin{document}

\title{Preclinical Evaluation of a Novel Single-Shot Pulsed Field Ablation
System for Pulmonary Vein and Atrial Ablation}



\author[1]{Arash Aryana}%
\author[2]{Sang Yong Ji}%
\author[3]{Cary Hata}%
\author[3]{Alan de la Rama}%
\author[3]{Ken Nguyen}%
\author[3]{Dorin Panescu}%
\affil[1]{Mercy General Hospital}%
\affil[2]{COR Healthcare}%
\affil[3]{CRC EP Inc}%


\vspace{-1em}



  
  \date{May 25, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
\textbf{Introduction} \textbf{:} Pulsed field ablation (PFA) is a
non-thermal ablative strategy that achieves cell death via
electroporation. Herein, we investigated the preclinical safety and
efficacy of PFA using two novel 8-French, 16-electrode spiral
PFA/mapping catheters (ElePulse, CRC EP, Inc). \textbf{M}
\textbf{ethods} \textbf{:} Bipolar PFA (\textgreater{}1.8 kV) was
performed using 30 sec, single-shot, QRS-gated applications. Altogether,
94 atrial structures were ablated in 23 swine, 1 canine, and 1 ovine,
including right and left atria and atrial appendages, pulmonary veins,
and superior and inferior (IVC) vena cavae. We also examined the impact
of PFA on phrenic nerve (14 swine) and on a deviated esophagus after
delivery of PFA from inside the IVC (5 swine). \textbf{Results:} All
applications were single-shot without catheter repositioning. Minimal
microbubbling was observed without significant skeletal muscle
twitching/activation (mean acceleration: 0.05 m/s \textsuperscript{2}).
There was marked reduction in post- versus pre-PFA atrial electrogram
amplitude (0.17\selectlanguage{ngerman}±0.21 mV \emph{vs.} 1.18±1.08 mV; P\textless{}0.0001).
Durable conduction block was demonstrated up to 3 months in all targeted
tissues. Lesions were contiguous and transmural, measuring 25±9 mm x
21±7 mm without any thermal effects. Magnetic resonance, gross, and
histologic examinations of the brain, rete mirabile, and kidneys
revealed no thromboembolism. No acute/long-term phrenic nerve
dysfunction was encountered. Though within 2 hours of ablation,
histologic examinations of the esophagus revealed acute PFA-related
changes in the muscular layer, these completely resolved by 21±5 days.
\textbf{Conclusion} \textbf{:} A novel, single-shot, spiral PFA system
is capable of safely creating large, durable atrial lesions without
significant adverse effects on the phrenic nerve or the esophagus.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Introduction}

Pulsed field ablation (PFA) utilizes intermittent, high-intensity
electric field for short durations (micro- or nanoseconds) to achieve
cell death through irreversible
electroporation.\textsuperscript{1,2,3}Most unique to PFA is its tissue
selectivity.\textsuperscript{4} That is, different tissues have specific
characteristic threshold field strengths that induce
necrosis.\textsuperscript{5} Preclinical and clinical studies have found
that cardiac myocytes in general exhibit a lower threshold for
irreversible electroporation as compared to phrenic nerve (PN) or
esophageal tissue.\textsuperscript{6,7,8,9} In this preclinical study,
we demonstrate the feasibility and safety of two novel, 8-French,
16-electrode, single-shot, bipolar, spiral PFA/mapping catheters (CRC
EP, Inc, Tustin, CA).

\textbf{Methods}

All preclinical experiments were approved by the Institutional Animal
Care and Use Committee at the Sutter Institute for Medical Research
(Sacramento, CA). A total of 23 Yorkshire or Yucatan swine (52--92 kg),
1 canine (27 kg), and 1 ovine (73 kg) were included in this study. Given
that these were initial experiments, randomization and sample size
calculation were not performed.

\textbf{CRC EP ablation catheter and system.}

PFA was performed using one of the two bipolar, non-irrigated, spiral
PFA catheters (ElePulse, CRC EP, Inc) and a custom programmable PFA
generator (CRC EP, Inc). The catheters consist of an 8-French,
16-electrode, bidirectional, 25-mm or 30-mm design with radiopaque tips,
intended for single-shot ablation, with the larger of the two catheters
(30-mm) containing 4 distal mapping electrodes. \textbf{Figure
1,}illustrates the design of the two PFA/mapping catheters and the
ablation system. Briefly, the catheters are inserted through a
commercially-available 8.5-French, long introducer. The system delivers
biphasic, microsecond-wide pulses with amplitudes in excess of 1.8 kV.
The generator allows for PFA using individually-selected electrodes or
simultaneously using all 16 electrodes. With the exception of the first
4 consecutive animals, PFA pulses were synchronized with the cardiac
cycle through QRS gating using a cardiac trigger monitor (Ivy Biomedical
Mode 7600, Ivy Biomedical Systems, Inc, Branford, CT), to avoid energy
delivery during the vulnerable period.

\textbf{Preclinical protocols.}

After an overnight fast, all swine were anesthetized, intubated, and
ventilated mechanically with 1.0 FiO\textsubscript{2} air. The animals
were pretreated with ceftiofur (360 mg) on the day before and with
gentamicin (160 mg) on the day of procedure. General anesthesia was
induced by tiletamine (350 mg) and maintained using inhalational
isoflurane (2.5\%). A decapolar diagnostic electrophysiology catheter
(Response, Abbott, Chicago, IL) was inserted into the coronary sinus for
left atrial (LA) recording and pacing via the right internal jugular
vein. Following percutaneous femoral venous access and systemic
anticoagulation, a single transseptal puncture was performed for LA
access. An 8.5-French deflectable introducer (Agilis, Abbott) was used
for mapping and ablation under intracardiac echocardiographic (ViewMate,
Abbott) guidance. Single-shot, QRS-gated, bipolar PFA (\textgreater{}1.8
kV) was performed using 30 sec applications. The targeted structures
included the right atrium (RA), RA appendage (RAA), right superior PV,
right inferior PV, left PV (also known as left inferior common PV), LA
appendage, LA posterior wall, and superior (SVC) and inferior (IVC) vena
cavae. Pre- and post-PFA intracardiac electrograms, pacing thresholds,
and electrical isolation/conduction block were assessed. In addition,
detailed pre- and post-PFA voltage maps were created using a
high-density mapping catheter (Advisor HD Grid, Abbott) in all swine
(bipolar voltage cutoff \textless{}0.1 mV). Upon completion of the
study, the animals were euthanized. In addition to pre- versus post-PFA
electrogram amplitudes, pacing thresholds, conduction block, and 3D
voltage mapping (EnSite, Abbott), PFA lesions were also analyzed by
necropsy and histology.

\textbf{Skeletal muscle activation studies.}

The intensity of skeletal muscle activation was quantified by measuring
the absolute acceleration of muscular contractions. Briefly, a
smartphone (Galaxy S6, Samsung Electronics, Suwon-si, South Korea) was
secured to the animal's thorax. The Phyphox smartphone application
(Physical Phone Experiments, RWTH Aachen University, Aachen, Germany)
was utilized which uses an integrated acceleration sensor. Acceleration
was measured on the x, y, and z coordinates and computed in absolute
values. This allowed for assessment of acceleration resulting from
contractions triggered by PN stimulation as well as contractions
triggered during PFA energy delivery. Meanwhile, we specifically also
tested the PFA system in the ovine (n=1), as it is widely-believed that
this animal model may be more sensitive to skeletal muscle activation.
Lastly, in one animal an intravenous paralytic agent (succinylcholine 1
mg/kg) was administered to confirm PN-mediated loss of skeletal muscle
contraction during PFA. In other words, acceleration measurements were
recorded without and with administration of paralytics with the catheter
positioned at the same location and using the same PFA energy
parameters.

\textbf{PN safety studies.}

We specifically assessed the safety of PFA with regard to the PN in 14
swine (66\selectlanguage{ngerman}±13 kg). PFA was intentionally performed at anatomical
locations exhibiting low-output PN pacing capture using the PFA
catheter, such as the SVC, the RAA and the LAA. PN function was
evaluated through pacing capture, pre- versus post-PFA, acutely and
during follow-up (up to 3 months post-ablation). In addition, the PNs
were closely examined in all animals at necropsy and histology.

\textbf{Esophageal safety studies.}

To assess the safety of PFA on the esophagus, PFA was performed from
within the IVC toward a deviated esophagus deflected toward the ablation
catheter in 5 swine, as described in a previously-reported
protocol.\textsuperscript{9} Briefly, following systemic
anticoagulation, the PFA catheter was inserted through an 8.5-French
deflectable introducer (Agilis, Abbott). Next, the esophagus was
intubated using an esophageal balloon retractor device (DV8, Manual
Surgical Sciences Inc, Salt Lake City, UT) under fluoroscopy. The distal
esophageal lumen was anatomically delineated by administering iodinated
contrast medium through an open port in the esophageal retractor device.
The esophageal retractor was then inflated with a mixture of saline and
contrast for fluoroscopic visualization and rotated to physically
deviate the esophagus toward the PFA catheter placed inside the IVC. The
deflectable sheath was used to ensure forceful contact between the
ablation catheter and the esophagus. Orthogonal fluoroscopic views were
used to confirm the immediate proximity of the deviated esophagus to the
ablative portion of the catheter within the IVC \textbf{(Figure
2).}Whenever possible, the PFA catheter was also used to mechanically
displace luminal contrast within the esophagus to further confirm its
proximity to this structure. PFA was then delivered using the spiral
catheter at areas intentionally contacting the deviated esophagus.
Post-mortem, the esophagus was removed in its entirety, carefully
inspected and photographed. The lesions created within the IVC and the
esophagus were analyzed by gross and histologic examination. Esophageal
sections were evaluated for alterations within the normal architecture
as well as inflammation, necrosis, or fibrosis.

\textbf{Assessment for thromboembolization.}

\textbf{Systemic embolization.}

In all the animals, the brain, the kidneys, and the liver were carefully
removed at necropsy and thoroughly examined for signs and evidence of
thromboembolism. If any gross abnormalities were detected, further
histologic examinations were performed.

\textbf{The rete mirabile model.}

The swine rete mirabile model was further used to investigate the
potential embolic effects of PFA using the current
system.\textsuperscript{10} As such, the rete mirabile was meticulously
analyzed grossly and microscopically in 6 swine that received multiple
PFA applications to the RAA, right superior PV, right inferior PV, left
PV, LAA, and the LA posterior wall.

\textbf{Magnetic resonance imaging (MRI).}

In 3 swine, brain T2-weighted MRI scanning was also performed at
baseline and at 1 week post-PFA for comparison to assess for cerebral
emboli. MRI scanning was performed using a Philips MR 5300 Scanner
(Philips, Eindhoven, Netherlands) and the images carefully analyzed for
abnormalities by a radiologist.

\textbf{Follow-up studies and histologic analysis.}

Two swine, 1 canine, and 1 ovine were euthanized within 4 h of PFA to
investigate the acute effects of tissue ablation. Sixteen animals were
survived for 3--4 weeks and 3 swine for 3 months. In all animals,
high-density 3D voltage mapping (Advisor HD Grid, Abbott) was performed
post-PFA and again repeated during follow-up on the day of sacrifice
(range: 3 weeks to 3 months) to reassess lesion durability. Ablation
efficacy was further validated by the analysis of intracardiac
electrograms, pacing thresholds, and electrical isolation/conduction
block. At necropsy, all cardiac chambers were carefully examined and all
lesions were identified and photographed. Detailed analysis of each
treatment site was performed and measurements were taken to determine
the dimensions of the ablation lesions. In addition, all tissues were
fixed in formalin, processed, and stained with hematoxylin and eosin and
elastin Masson trichrome.

\textbf{Statistical methods}

The data are represented as mean ± standard deviation. Continuous
variables were analyzed using a two-sample \emph{t} test. All P-values
were two-sided and a P-value of \textless{}0.05 was considered
statistically significant. The analyses were conducted using Stata 14
(StataCorp LP, College Station, TX).

\textbf{Results}

During this study, 94 structures were ablated in 25 healthy animals
including 23 swine, 1 canine, and 1 ovine. The targeted structures
included: RA (5), LA posterior wall (2), RAA (6), LAA (14), right
superior PV (22), right inferior PV (20), left PV (16), SVC (4) and IVC
(5). Each structure was ablated using 1.1±0.2 PFA applications resulting
in successful ablation/isolation \textbf{(Figure 3).} Real-time
locations of the spiral PFA catheter during ablation were recorded
within the 3D map \textbf{(Figure 4).} Given the advent of QRS gating,
no ventricular arrhythmias were inadvertently triggered during PFA.
Meanwhile, PFA resulted in marked reduction in the post- versus pre-PFA
electrogram amplitudes (0.17±0.21 mV \emph{vs.} 1.18±1.08 mV;
P\textless{}0.0001) as well as a significant increase in the post-
versus pre-PFA pacing threshold at the ablated sites (\textgreater{}15
mA versus 0.6±0.7 mA, P\textless{}0.001) \textbf{(Table 1).} Minimal
microbubbling and no discernable skeletal muscle twitching was observed
during PFA. Though administration of an intravenous paralytic agent
(succinylcholine 1 mg/kg) temporarily abolished all forms of skeletal
muscle twitch/activation; in the absence of a paralytic agent, PFA at
sites adjacent to the right/left PN (\emph{e.g.,} lateral RA or LAA)
commonly resulted in visible PN capture and hemi-diaphragmatic
stimulation. Yet, there was no incidence of diminished PN function
during or post-PFA as confirmed by low-output PN pacing capture using
the PFA catheter. When PN pacing capture was present (\emph{e.g.,} when
targeting the right superior PV), acceleration levels were measured in
the range of 5--9 m/s\textsuperscript{2}. When ablating structures
remote from the right/left PNs (\emph{e.g.,} the left PV), the absolute
mean acceleration was only 0.05 m/s\textsuperscript{2} which was
indiscernible from the background noise level \textbf{(Supplemental
Figure).}

Complete and durable conduction block was demonstrated acutely and up to
3 months post-PFA \textbf{(Figure 5).} All lesions were large and
durable at follow-up (range: 3 weeks to 3 months). Histologically, the
PFA lesions were contiguous and transmural \textbf{(Figure 6),}measuring
25±9 mm x 21±7 mm and 7.6±1.6 mm deep. No evidence of collateral injury
or abnormalities involving the PN \textbf{(Figure 6)}could be detected
in any of the animals, including the 8 swine in which the PNs were
intentionally targeted -- neither acutely, nor during follow-up at 3--4
weeks post-PFA. Additionally, there were no observed cases of vascular
injury. Likewise, gross and microscopic examinations of the brain, the
rete mirabile, the kidneys, the liver, the lungs, and the bronchi
revealed no evidence of embolization or adjacent structure/tissue injury
in any of the animals \textbf{(Figure 7).}Moreover, post- versus pre-PFA
T2-weighted MRI scanning (n=3 swine) did not show any evidence of
embolic events.

In 5 swine (62 ± 12 kg), 14 PFA applications were delivered within the
IVC toward an opposing esophagus, intentionally deviated toward/adjacent
to this structure. All 14 animals completed the follow-up period without
clinical sequelae. Their body weights increased by 15±4\% during
follow-up. All IVC treatments were transmural, measuring 15.6±5.5 mm x
5.9±1.4 mm. Histopathologic examinations revealed acute PFA-related
changes in the esophageal muscular layer which appeared within 2 h of
PFA \textbf{(Figure 8).} However, these changes completely resolved by
21±5 days of follow-up. No other acute or chronic lesions/abnormalities
were detected in the esophageal mucosa or submucosa in any of the 5
animals at 3 weeks of follow-up.

\textbf{Discussion}

PFA is primarily a non-thermal ablative strategy that relies on pulsed
applications of high-intensity electric fields for short durations which
result in cellular and tissue electroporation.\textsuperscript{1} This
phenomenon represents a process whereby the applied electric field
results in the formation of pores within the cell membrane. Depending on
the parameters of the applied electric fields, pore formation can lead
to permeabilization which may be either reversible or irreversible. In
reversible electroporation, cells remain viable and this process
underlies the basis of electrochemotherapy and gene electrotransfer.
Conversely, irreversible electroporation renders cells and tissues
nonviable as a consequence of the programmed cell death cascade.
Irreversible electroporation has recently been revisited in a number of
preclinical and clinical studies with favorable outcomes for the
treatment and ablation of atrial and ventricular
tissues.\textsuperscript{4,5,7,8} Furthermore, irreversible
electroporation can create lesions without tissue heating and is also
believed to be tissue/cell selective enabling preservation of the
adjacent or surrounding structures.

This initial \emph{in vivo} study illustrates the safety and efficacy of
a novel, bipolar, single-shot family of PFA catheters for the treatment
and ablation of atrial tissue. The lesion durability and the
histopathologic findings described in this report are highly consistent
with prior reports using different biphasic waveforms delivered with
other multipolar PFA catheters. Accordingly, the PFA catheters
investigated in this study proved capable of creating large, transmural
atrial lesions. The PFA applications were generally single-shot and did
not require catheter repositioning resulting in acute marked reduction
in post- versus pre-PFA electrogram amplitudes. Moreover, all PFA
lesions were found to be completely durable up to 3 months of follow-up
and despite minimal microbubbling observed during ablation, no
discernable embolic events were encountered by MRI or careful histologic
analysis of the brain, the rete mirabile, and the systemic organs.

Also, consistent with prior reports, PFA using the studied catheter
system did not produce any collateral damage to the bronchi or PN, nor
any long-term injuries to a manually-deviated, esophagus when performing
ablations within the adjacent IVC. Though by design, the findings from
such a simulation bear obvious limitations, this model does indeed offer
certain clinical merits. As previously described,\textsuperscript{9} it
involves manual deviation of the esophagus toward/against the PFA
catheter placed transvenously within the IVC, to approximate the
anatomical proximities required to simulate esophageal injury during LA
ablation. The inherent advantage of this model is that it allows for
delivery of the ablative energy over a large area of the esophageal
tissue, within the blood pool as in the case of LA ablation, while
affording placement of multiple ablation lesions for
analysis.\textsuperscript{9} In addition, it represents ablation within
an intra-vascular milieu similar to conventional clinical workflows
allowing for assessment of PFA lesions delivered directly on a
thin-walled structure (\emph{i.e.,} the IVC) similar to the posterior LA
wall. Also, given that the posterior LA wall shares its embryologic
origins with PV tissues\textsuperscript{11} and that the electrical
conductivity of the IVC is similar to that of the posterior wall of the
LA,\textsuperscript{12} these characteristics render this model both
relevant and clinically suitable. Meanwhile, similar to previously
published reports,\textsuperscript{13} the findings from this study
showed evidence of acute, but transient PFA-related changes within the
muscularis layer of the deviated esophagus when delivering PFA
applications in the adjacent IVC, which completely resolved at 3 weeks
of follow-up without any detectable long-term sequelae. Although these
findings appear highly promising, future studies and clinical
investigations are clearly warranted to further validate the safety and
efficacy outcomes reported in this preclinical study.

\textbf{Limitations}

The authors recognize several important limitations in this study.
First, as with all preclinical studies, there are anatomic and
physiologic differences between humans and quadrupeds that may limit
translation of the study results. Second, the tissue specificity of PFA
can be altered with even small changes in the delivery parameters,
including waveform composition and catheter electrode design and
configuration. Therefore, the results described herein cannot be assumed
to be applicable to other PFA systems. Third, the esophageal deviation
model proposed and used in this study represents merely an approximation
of clinical conditions and scenarios. Moreover, the lack of endoscopic
examination of the studied animals could have limited our assessment of
reversible esophageal injury at earlier time points. Finally, the
preclinical findings reported in this study require validation through
clinical studies prior to adoption for clinical use.

\textbf{Conclusions}

In this initial study, we demonstrate that endocardial ablation can be
performed safely and effectively using a novel family of bipolar,
single-shot, spiral PFA/mapping catheters. Furthermore, this PFA system
allows for creation of wide, transmural lesions within the atria and
durable PV isolation without significant skeletal muscle
twitch/activation, thromboembolism, collateral injury to the PN or
bronchi or long-term abnormalities in the adjacent esophagus when
manually deviated to render contiguous with the site of the energy
source.

\textbf{Acknowledgments}

The authors wish to sincerely thank Taylor Spangler, DVM, DACVP for
performing gross and histologic analyses on all the examined tissues.
The authors also wish to thank Keith Pound, MS for his assistance with
3D mapping during this study.

\textbf{Funding}

This study was supported by CRC EP, Inc.

\textbf{References}

\begin{enumerate}
\tightlist
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\textbf{Table 1.} Pre- versus post-PFA electrogram amplitude and pacing
threshold.\selectlanguage{english}
\begin{longtable}[]{@{}lllllll@{}}
\toprule
\textbf{Structure} & \textbf{Electrogram amplitude, mV} &
\textbf{Electrogram amplitude, mV} & \textbf{Electrogram amplitude, mV}
& \textbf{Pacing threshold, mA} & \textbf{Pacing threshold, mA} &
\textbf{Pacing threshold, mA}\tabularnewline
\midrule
\endhead
& \textbf{Pre-PFA} & \textbf{Post-PFA} & \emph{P}\textbf{-value} &
\textbf{Pre-PFA} & \textbf{Post-PFA} &
\emph{P}\textbf{-value}\tabularnewline
Atrium & 1.52\selectlanguage{ngerman}±1.19 & 0.17\selectlanguage{ngerman}±0.16 & \textless{}0.0001* & 0.5\selectlanguage{ngerman}±0.6 &
\textgreater{}15 & \textless{}0.001*\tabularnewline
Atrial appendage & 2.41\selectlanguage{ngerman}±1.57 & 0.21\selectlanguage{ngerman}±0.15 & \textless{}0.0001* & 0.4\selectlanguage{ngerman}±0.5
& \textgreater{}15 & \textless{}0.001*\tabularnewline
Pulmonary vein & 0.92\selectlanguage{ngerman}±0.79 & 0.15\selectlanguage{ngerman}±0.13 & \textless{}0.0001* & 0.7\selectlanguage{ngerman}±0.4 &
\textgreater{}15 & \textless{}0.001*\tabularnewline
All structures & 1.18\selectlanguage{ngerman}±1.08 & 0.17\selectlanguage{ngerman}±0.21 & \textless{}0.0001* & 0.6\selectlanguage{ngerman}±0.7 &
\textgreater{}15 & \textless{}0.001*\tabularnewline
\bottomrule
\end{longtable}

*Significant \emph{P} -value.

\textbf{Figure Legends}

\textbf{Figure 1. CRC EP PFA catheters and system.} The ElePulse PFA
catheter design consists of an 8-French, 16-electrode, bidirectional,
spiral mapping/ablation catheter intended for single-shot ablation. The
PFA catheters are available in two diameter sizes (25-mm and 30-mm) with
radiopaque tips and inserted through an 8.5-Fr long introducer. The
larger (30-mm) catheter has 4 distal mapping electrodes. The PFA system
delivers microsecond-wide, QRS-gated, bipolar, biphasic, pulsed electric
fields synchronized with the cardiac cycle which allows for ablation
using individually-selected or all electrodes.

\textbf{Figure 2. PFA from inside the inferior vena cava (IVC) toward
the adjacent deviated esophagus. A,} the esophagus was deviated toward
the IVC using an esophageal retractor device (DV8; Manual Surgical
Sciences) as bipolar PFA (1.8--2.5 kV) was performed within the IVC
through a deflectable introducer at areas contacting the deviated
esophagus. \textbf{B,} A 3D map depicts the location of the spiral PFA
catheter within the IVC at the time of ablation. \emph{CS=coronary
sinus; ESO=esophagus; SVC=superior} \emph{vena cava.}

\textbf{Figure 3. Electrical isolation following PFA confirmed by 3D
mapping and intracardiac electrograms.} Shown, are post-PFA 3D voltage
maps with corresponding surface (aV\textsubscript{F}) and intracardiac
electrograms recorded using a high-density mapping catheter placed
initially inside the right superior \textbf{(A)} and subsequently in the
right inferior \textbf{(B)} and left \textbf{(C)} PVs demonstrating
complete electrical isolation of all 3 PVs and a significant portion of
the posterior wall. \emph{CS=coronary sinus recording channels
(proximal, mid and distal); HD=high-density (Advisor HD Grid, Abbott)
mapping catheter recording channels; LAA=left atrial appendage; LPV=left
PV; RIPV=right inferior PV; RSPV=right superior PV.}

\textbf{Figure 4. Pre- and post-PFA voltage maps.} 3D LA map created
pre- \textbf{(A)} and post-PFA \textbf{(B)} demonstrating the locations
of PFA applications as illustrated by the spiral catheter contours on
the 3D maps. \textbf{C,} Surface and intracardiac electrograms recorded
during QRS-gated PFA using the spiral ablation catheter. Also shown, are
3D LA maps created at baseline \textbf{(D)} and at 1 day \textbf{(E)}and
21 days \textbf{(F)} post-PFA. As seen, there is complete and durable
isolation of all 3 PVs and also a segment of the posterior wall.

\emph{LAA=left atrial appendage; RIPV=right inferior PV; RSPV=right
superior PV.}

\textbf{Figure 5. Pre- and post-PFA maps of the left atrium and the PVs
with histology at 3 months of follow-up.} 3D maps created
pre-\textbf{(A, B),} immediately post-PFA \textbf{(D, E),} and 3 months
post-PFA \textbf{(G, H),} along with the corresponding intracardiac
electrograms \textbf{(C, F, I)} demonstrating electrical isolation of
the right superior (RSPV) and inferior (RSPV) and left PVs
(LPV).\textbf{J,} Subgross histology illustrating complete,
circumferential and transmural fibrous replacement of the cardiac sleeve
around the LPV.\textbf{K,} Subgross histology of the RSPV demonstrating
full-thickness, circumferential fibrous replacement (blue staining,
yellow outline) of the cardiac sleeve. Moreover, the treatment extended
to involve the myocardium from the posterior wall of the right atrium
and a small branch of the pulmonary artery (PA). \textbf{L,}
Photomicrograph (hematoxylin and eosin) demonstrating acute contraction
band degeneration of cardiac myocytes consistent with PFA.

\textbf{Figure 6. Histology of lesions created using PFA. A,} Sections
of right superior pulmonary vein (RSPV) illustrating a circumferential,
transmural PFA lesion (arrow). A normal phrenic nerve (arrow) following
PFA depicted both on gross \textbf{(B)} and histologic
examinations\textbf{(C)} after deliberately performing PFA adjacent to
this structure. As illustrated, no phrenic nerve injury has occurred and
the nerve is completely intact. \textbf{D} and \textbf{E,} Sections of
RSPV and right inferior pulmonary vein (RIPV) demonstrating
circumferential ablation following PFA. \textbf{F,} Depicts a higher
magnification of\textbf{panel D,} demonstrating an area of transmural
ablation (arrow) around the circumference of the RSPV. \textbf{G,}
Represents a higher magnification of \textbf{panel D,} demonstrating
transmural fibrous replacement of the muscular sleeve around the RSPV
(arrow). \textbf{H,}Histologic representation demonstrating fibrous
replacement of the muscular sleeve (outlined) of the left common
pulmonary vein (LPV).\textbf{I} and \textbf{J,} illustrate higher
magnifications of\textbf{panel H,} showing transmural ablation (arrows)
and replacement fibrosis of the muscular sleeve of the LPV with normal
surrounding epicardial fat and sparing of the nerve and artery.

\textbf{Figure 7. Assessment for embolic events following PFA.} Gross
and histologic examination of the porcine brain \textbf{(A, B),} the
rete mirabile \textbf{(C, D),} and the kidneys \textbf{(G, H)} following
PFA of the atria, the atrial appendages, and the pulmonary veins using
the CRC EP system showed no evidence of embolization. Additionally, MRI
was also performed to radiologically investigate for embolic events. As
shown, compared to baseline/pre-PFA \textbf{(E),} no gross or
radiological abnormalities suggestive of cerebral embolization/events
were encountered in any of the animals on MRI, 1 week
post-PFA\textbf{(F).} \emph{DWI=diffusion-weighted imaging; FLAIR=fluid
attenuated inversion recovery; GRE=gradient recalled echo T2-weighted;
PD=proton density-weighted imaging; T1=T1-weighted.}

\textbf{Figure 8. Histologic examination of the esophagus following PFA
performed within the adjacent IVC. A,} Sections of porcine esophagus
depicting normal tissue without evidence of injury or lesions following
PFA. \textbf{B,} Acute PFA-related changes (outlined) in the esophageal
muscular layer within 2 h of PFA with complete resolution during
follow-up by 3 weeks.

\textbf{Supplemental Figure. Assessment of chest contraction
acceleration during following PFA.} Absolute chest contraction
acceleration recorded during PFA in the left atrium, the PVs, and the
left atrial appendage (LAA). As seen, pulsed field applications
delivered at anatomical locations immediately adjacent to the left and
right phrenic nerves (\emph{e.g.,} right superior PV or deep within the
LAA) was accompanied by prominent phrenic nerve capture, yielding
acceleration levels of 5--9 m/s\textsuperscript{2}. Conversely, PFA
using the same waveform parameters performed at anatomical sites far
enough from phrenic nerves (\emph{e.g.,} left PV or posterior wall)
yielded no measurable chest contractions with an absolute mean
acceleration of 0.05 m/s\textsuperscript{2} indiscernible from
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