Journal of the American Heart Association

ORIGINAL RESEARCH

Anti-Inflammatory and Antiarrhythmic

Effects of Beta Blocker in a Rat Model of
Rheumatoid Arthritis
Ting-Tse Lin , MD, PhD*; Yen-Ling Sung , PhD*; Jhen-Yang Syu , MS; Kia-Yuan Lin , MS;
Hung-Jui Hsu , MS; Min-Tsun Liao , MD, PhD; Yen-Bin Liu , MD, PhD; Shien-Fong Lin , PhD

BACKGROUND: Patients with rheumatoid arthritis are at twice the risk of ventricular arrhythmia and sudden cardiac death as the
general population. We hypothesize that β-blocker treatment of rheumatoid arthritis is antiarrhythmic by producing synergistic
anticatecholaminergic and anti-inflammatory effects.

METHODS AND RESULTS: Collagen-induced arthritis (CIA) was induced in Lewis rats by immunization with type II collagen in
Freund’s incomplete adjuvant. The treatment with propranolol (4 mg/kg) started on the first day of immunization. We evaluated
the ventricular vulnerability to ventricular arrhythmia using in vivo programmed stimulation and performed ex vivo optical map-
ping to measure the electrical remodeling of the heart. The ventricular tissue was further processed for immunohistochemical
staining and protein array analysis. The assessment of ventricular vulnerability showed that the number and duration of the
induced ventricular arrhythmia episodes were increased in CIA rats, which were improved with propranolol treatment. The
sympathovagal index and the plasma level of catecholamines significantly increased in CIA rats, whereas the use of proprano-
lol attenuated sympathetic hyperactivity. In the optical mapping study, electrical remodeling, characterized by prolonged ac-
tion potential duration, slow conduction velocity, and steepened action-potential duration restitution, were noted in CIA rats
and reversed in the propranolol-treatment group. The propranolol treatment was associated with decreases in paw thickness,
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fewer inflammatory cell infiltrations in the heart, reduced levels of cardiac inflammatory cytokines, and less cardiac fibrosis as
compared with the CIA group.

CONCLUSIONS: CIA increased ventricular arrhythmia vulnerability through sympathetic hyperinnervation and proarrhythmic ven-
tricular electrophysiological remodeling. Treatment with propranolol in CIA rats was both anti-inflammatory and antiarrhythmic.

Key Words: propranolol ■ rheumatoid arthritis ■ sympathetic hyperinnervation ■ sympathovagal index ■ ventricular arrhythmia

P
atients with rheumatoid arthritis (RA) have a 2-fold electrical instability. One of the possible mechanisms
risk of sudden cardiac death or ventricular arrhyth- is the chronic activation of inflammatory cytokines in
mia (VA) compared with the general population.1–3 RA, which may elicit deleterious increases in the sym-
Arthritis could boost the immunoinflammatory process pathetic neural outflow.5 In joint tissue, the production
of atherosclerosis; as a result, it could contribute to the of TNF-α (tumor necrosis factor-alpha) and interleukin-1
incidence of myocardial infarction and heart failure.4 beta (IL-1β) from macrophages could be activated by
Notably, proinflammatory cytokines could acceler- β adrenoceptors.6 Cardiac macrophages mediating
ate coronary atherosclerosis and mediate myocardial the production of IL-1β could cause prolongation of the

Correspondence to: Shien-Fong Lin, PhD, Institute of Biomedical Engineering, College of Electrical and Computer Engineering, National Chiao Tung
University, Hsinchu, Taiwan, 1001 Ta-Hsueh Road, Hsinchu, Taiwan 300. E-mail: [email protected] and Yen-Bin Liu, MD, PhD, Division of Cardiology,
Department of Internal Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan. E-mail: [email protected]
Supplementary Materials for this article are available at https://www.ahajo​urnals.org/doi/suppl/​10.1161/JAHA.120.016084
*Dr Ting-Tse Lin and Dr Sung contributed equally to this work.
For Sources of Funding and Disclosures, see page 11.
© 2020 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley. This is an open access article under the terms of the Creative
Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use
is non-commercial and no modifications or adaptations are made.
JAHA is available at: www.ahajournals.org/journal/jaha

J Am Heart Assoc. 2020;9:e016084. DOI: 10.1161/JAHA.120.0160841

 Lin et al Synergistic Effects of Propranolol in RA

corresponding authors on reasonable request. All sup-

CLINICAL PERSPECTIVE porting data are provided within the article and its on-
line supplementary files.
What Is New?
• The present study showed that rheumatoid Animal Subjects
arthritis increased susceptibility to ventricular
arrhythmia by ventricular remodeling, includ- The present study was performed in accordance
ing increased action potential duration and with the Guide for the Care and Use of Laboratory
alternans, significant fibrosis, and heterogene- Animals9and was approved by the Institutional Animal
ous sympathetic hyperinnervation, whereas Care and Use Committee at National Chiao Tung
treatment with β blockers reversed the proar- University (Hsinchu, Taiwan; NCTU-IACUC-106060).
rhythmic ventricular remodeling and reduced At 8 weeks of age, female Lewis rats (weight 210±10 g)
vulnerability to ventricular arrhythmia. were randomly divided into 3 groups: normal control
(CON) rats, rats with collagen-induced arthritis (CIA),
What Are the Clinical Implications? and rats with collagen-induced arthritis treated with
• Patients with rheumatoid arthritis, who receive propranolol (CIA-PRO).
treatment with a β blocker, experience syner-
gistic anticatecholaminergic and anti-inflamma-
tory effects, which reduce the risk of ventricular Collagen-Induced Arthritis
arrhythmia and further cardiovascular events. Type II collagen was dissolved in 0.05  mol/L acetic
acid and emulsified with an equal volume of incomplete
Freund’s adjuvant.10–12 Rats were injected with 2 mg/kg
collagen-incomplete Freund’s adjuvant at the base of
Nonstandard Abbreviations and Acronyms the tail (day 0). To ensure a high incidence and sever-
ity of arthritis, a boost injection (1 mg/kg) was given on
APD action potential duration
day 7 after the initial immunization in the same manner
CIA collagen-induced arthritis
(Figure  1A).13 The severity of arthritis can be evaluated
CV conduction velocity
with a clinical score from 0 to 4 depending on paw in-
HR heart rate flammation. The normal condition is 0, apparent red-
HRV heart rate variability
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ness and swelling of the ankle is 1, moderate redness

IHR intrinsic heart rate and swelling of the ankle or the wrist is 2, severe redness
IL-1β interleukin-1 beta and swelling of the entire paw including digits is 3, and
PCL pacing-cycle length maximally inflamed limb involving multiple joints is 4.14
RA rheumatoid arthritis
SVI sympathovagal index Propranolol Administration
TH tyrosine hydroxylase Propranolol was administered intragastrically using a
TNF-α tumor necrosis factor-alpha rodent feeding tube with a dose of 1 mg/kg at day 1,
VA ventricular arrhythmia then 3 times daily until day 30 of the experiment.

myocardial action-potential duration (APD), a decrease

Heart Rate Variability Analysis
in potassium current, and an increase in calcium sparks The single-lead ambulatory ECG was recorded for
in cardiomyocytes—all of which contribute to the elec- 30 minutes every 2 days.15 The standard deviation (SD)
trical remodeling underlying the VA propensity in mice of N-N intervals and the root mean square of succes-
with diabetes mellitus.7 Treatment with the β blocker, sive differences of N-N interval were calculated as the
propranolol could reduce the gene expression of TNF-α time-domain measures of heart rate variability (HRV).
in mice with viral myocarditis.8 In this study, we hypoth- In the HRV spectral analysis, the low-frequency power
esize that the anticatecholaminergic and anti-inflam- (0.2–0.8 Hz) and high-frequency power (>0.8 Hz) were
matory effects of propranolol are associated with the derived from the sum of the area within a specific
attenuation of autonomic imbalance and increased ven- frequency range under the power spectrum density
tricular arrhythmogenicity in a rat model of RA. curve of the entire 5-minute segment.

Sympathetic, Vagal Tone, and Intrinsic

METHODS Heart Rate
Our data, analytic methods, and study materials are The basal heart rate (HR) was recorded for 10 minutes,
available for the reproduction of our findings from the and methylatropine (3  mg/mL) was injected into the

J Am Heart Assoc. 2020;9:e016084. DOI: 10.1161/JAHA.120.0160842

 Lin et al Synergistic Effects of Propranolol in RA

Figure 1.  Lewis rats with collagen-induced arthritis.

A, Timeline of the experiment’s design and the treatment progress of the CIA rats. B, Inflammation of hind paw. C, Effects of weight
gain and loss (n=6). D, The thickness of CIA hind paw (n=6). E, Clinical score of CIA symptoms (n=6). Data are shown as mean with
±SD. *P<0.05 and ***P<0.001 vs CON group, †P<0.05 and †††P<0.001 vs CIA group. CIA indicates collagen-induced arthritis group;
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CIA-PRO, CIA rats treated with propranolol; and CON, control group.

jugular vein by catheter. HR recording was continued for in a warm bath at 37°C. The heart was stained with Di-
10 minutes. Propranolol (4 mg/kg) was injected 10 min- 4-ANEPPS and blebbistatin to promote observation
utes after the methylatropine injection, and the intrinsic of the electrical response to electrical stimulation at a
heart rate (IHR) was evaluated. After 1 day, the sequence constant pacing rate. The heart was illuminated with
of drug treatment was reversed. In addition, the sympa- a green light-emitting diode (wavelength 505±20 nm).
thovagal index (SVI) was defined as basal HR divided The induced fluorescence was collected through a
by IHR, indicating the autonomic balance of the heart.16 600-nm long-pass filter by a CMOS (complementary
metal oxide semiconductor) camera (SciMedia, Costa
In Vivo Ventricular Arrhythmia Induction Mesa, CA). The digitized fluorescence images were
then transferred to a computer for further analysis.17,18
Animals were anesthetized by an injection of 50 mg/kg
of Zoletil (Virbac, Carros, France) and a tracheotomy
was performed to assist breathing after the thora- Protein Array
cotomy during programmed electric stimulation. The The expression of cytokines in heart tissue, includ-
programmed electric-stimulation protocol, designed ing TNF-α and IL-1β, was quantified by RayBio Rat
as 10 stimuli of S1, was delivered, followed by an extra Inflammation Array 1 (Raybiotech, Inc, Norcross, GA;
stimulus S2, starting at a coupling interval of 70  ms catalog no.: QAR-INF-1).
with 5-ms decrements. VA episodes were calculated The plasma expression of epinephrine and nor-
by 10 stimuli per rat and VA duration was defined as epinephrine were detected using commercial en-
the duration of VA in each episode. zyme-linked immunosorbent assay kits (Abnova,
Taipei, Taiwan; catalog no.: KA1877).
Optical Mapping and Study Protocol
We performed high-speed optical mapping on Immunohistochemical Studies
Langendorff-perfused rat hearts. The rat heart was iso- For the analysis of immune cells and nerve forma-
lated immediately and perfused with Tyrode’s solution tion, myocardial sections (20  μm) were fixed with

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 Lin et al Synergistic Effects of Propranolol in RA

acetone for 15  minutes at −20°C (tyrosine hydroxy- swelling and increased clinical scores, but mildly re-
lase [TH]) or 5% paraformaldehyde for 10 minutes at lieved in the late course of inflammation (Figure 1D and
room temperature (CD45 cells). Sections were incu- 1E).
bated with primary antibodies against TH (Millipore,
Burlington, MA; catalog no.: AB152) and CD45 cells Sympathetic Predominance Determined
(Abcam, Cambridge, UK; catalog no.: AB23910)
by HRV and SVI
overnight at 4°C. For the investigation of myocardial
The effects of double pharmacological vagal and
fibrosis, hearts were fixed overnight in 4% paraform-
sympathetic blockade on HR in the 3 groups are
aldehyde and embedded in paraffin. Tissue sections
depicted in Figure 2. The IHR was significantly de-
were stained with Masson trichrome. The density of
creased in the CIA group versus that of the CON
the stained areas was determined by Image-Pro Plus
rats (CIA: 293.94 [33.96], CON: 348.85 [49.62],
software (Media Cybernetics, Rockville, MD). Each
P<0.001), but the IHR was significantly increased
slide was examined under a microscope with ×40
after administration of propranolol (CIA-PRO: 334.59
objectives to select 3 fields with the highest density
[27.57], P<0.001; Figure 2A). Moreover, the SVI sug-
in each section.
gested that a sympathetic autonomic component
was predominant in the determination of the basal
Statistical Analysis HR in CIA rats, which was relieved by the treat-
Comparisons of data between each pair of 3 groups ment with propranolol (CON: 0.98 [0.08], CIA: 1.29
were performed by using the Mann–Whitney U test [0.22], CIA-PRO: 1.12 [0.12], P<0.001; Figure  2B).
when the data were lacking normal distribution, or Additionally, in the 4 weeks of HRV analysis, mean
by unpaired Student t test. Overall, the 3 groups HR and low-frequency and low-frequency/high-
were also compared by using the Kruskal–Wallis test frequency power were significantly increased in
when the data were lacking normal distribution, or by the CIA group (Table  1 and Table  S1; P<0.05). In
1-way ANOVA. A P<0.05 was considered statistically the fourth week, compared with the CON-group
significant. All data were reported as the median with rates, a reduced root mean square of successive
25th to 75th interquartile ranges except HRV, weight, differences of N-N interval and deceleration capac-
paw thickness, clinical score, APD70 (APD at 70% of ity were noted in the CIA rats; these findings were
repolarization), and conduction velocity (CV), which consistent with findings from our previous study
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were presented as mean±SD. Restitution curve (Table  1; P<0.05).19 Moreover, the plasma level of
and maximum slope were calculated by OriginLab epinephrine was significantly increased in the CIA
(OriginLab Corp, Northampton, MA) and GraphPad rats versus the CON rats (CIA: 1.25 [0.19], CON:
Prism 7 software (GraphPad Software, Inc, La Jolla, 1.07 [0.12]; P<0.01). Conversely, norepinephrine
CA). was significantly reduced in the CIA group versus
the CON and CIA-PRO groups (CIA: 0.30 [0.51],
CON: 1.90 [2.06], CIA-PRO: 2.96 [1.46], P<0.01), as
RESULTS shown in Figure 2C and 2D. The differences in epi-
nephrine levels among the groups were in accord-
Joint Inflammation Induced by Collagen- ance with the differences of SVI among the groups.
Induced Arthritis Hence, the use of propranolol appears to attenuate
Severe joint inflammation was induced by CIA after sympathetic hyperinnervation in RA.
4 weeks (Figure 1B). In the CIA group, there was a sig-
nificant decrease in body weight (P<0.05; Figure 1C). In
the 3 groups, there were significant differences at day Changes in VA Inducibility and
15 and day 20 (P<0.05). Paw swelling was profound in Electrophysiological Parameters
CIA rats from day 14 to day 21 (Figure 1D). In addition, VA was induced by the S1- to S2-stimulation proto-
there were significant differences among the 3 groups col described in the Methods section. The time course
at day 15 and day 20 (P<0.05). The clinical scores of of VA induction among the 3 groups is shown in
the CIA rats were also higher after immunization than Figure 3A. To analyze VA reproducibility, the probability
those of the CON rats, indicating that the symptom of of the arrhythmias was calculated as the number of
arthritis was induced to exhibit paralysis and maximally events of VA after 10 S1- to S2-induction episodes for
inflamed limbs with the involvement of multiple joints each rat. The probability of VA was significantly differ-
(Figure 1E). There were significant differences among ent between the CIA and CON groups (CIA: 2.00 [1.00],
the 3 groups from day 10 to day 25 (P<0.05). Of note, CON: 0.00 [0.00]; P<0.01), and was also decreased by
in the CIA-PRO group, the inflammation of joints was propranolol treatment (CIA-PRO: 0.5 [1.00], P<0.01;
similar to that of the CIA rats, complicated with paw Figure  3B). Furthermore, we observed a significantly

J Am Heart Assoc. 2020;9:e016084. DOI: 10.1161/JAHA.120.0160844

 Lin et al Synergistic Effects of Propranolol in RA

Figure 2.  The measurement of the sympathovagal index imbalance.

A, The intrinsic heart rate demonstrates the net autonomic nerve activities after heart-rate responses to
methylatropine and propranolol (n=6). B, The sympathovagal index expresses the autonomic balance
of the heart (n=6). C, Sympathetic overactivity is presented as a high level of epinephrine expression in
CIA rats (n=6). D, Treatment with propranolol increases the plasma level of norepinephrine and balances
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the autonomic dysfunction (n=6). Data are shown as the median with 25th to 75th interquartile ranges. In
the 3 groups, there are revealing significant differences in intrinsic heart rate, sympathovagal index, and
epinephrine and norepinephrine expression (all P<0.05). **P<0.01 and ***P<0.001 vs CON group, ††P<0.01
and †††P<0.001 vs CIA group. CIA indicates collagen-induced arthritis group; CIA-PRO, CIA rats treated
with propranolol; and CON, control group.

longer duration of VA in CIA versus CIA-PRO rats, re- shows the APD restitution curves. The APD restitu-
spectively (P<0.001; Figure 3C). Our finding shows that tion slope in CIA rats was constantly higher than in
the vulnerability to VA was increased in CIA rats, and CON rats from slow (250 ms) to fast (120 ms) PCLs.
that treatment with propranolol was associated with a The maximum slope of APD restitution was also sig-
reduction in VA inducibility. nificantly increased in the CIA rats as compared with
Figure  4A shows APDs obtained with differ- CON rats (CIA: 1.46 [1.77], CON: 0.28 [0.12]; P<0.01),
ent pacing-cycle lengths (PCLs) of the 3 groups. but the maximum slope was lower than 1 in CIA-PRO
Compared with CON rats, the APD70 of the CIA and rats (CIA: 0.50 [0.23], P<0.05), meaning that treat-
CIA-PRO rats was significantly prolonged at all PCLs ment with propranolol could reduce VA vulnerability
(P<0.05). After propranolol administration, the APD70 (Figures 3 and 4E). The aforementioned parameters
was shorter in the CIA rats at PCLs of 250, 180, and of the CIA-PRO rats were improved with the propran-
140 ms (P<0.05; Figure 4B). Furthermore, the CV was olol treatment, showing less APD prolongation and
decreased in the CIA rats at all PCLs as opposed restoration of CV heterogeneity as compared with
to the CON rats (Figure  4C). Although the CV was CIA rats.
not significantly different between the CIA and CIA-
PRO rats, the phase maps showed that the elec-
tric propagations were gradually delayed in the CIA Increased Expression of Inflammatory
group (Figure  5A) Also, enhanced heterogeneity of Cytokines in Myocardium
myocardial conduction and severe cardiac instability Because of the arthritis-induced systemic inflamma-
were found in CIA rats, but administration of propran- tion, the serum levels of TNF-α and IL-1β were signifi-
olol could restore the heterogeneous conduction as cantly elevated in CIA and CIA-PRO rats compared
shown in the isochrone maps (Figure 5B). Figure 4D with CON rats, but there was no difference between

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 Lin et al Synergistic Effects of Propranolol in RA

Table 1.  Effects of Propranolol on HRV Parameters in the Fourth Week

ANOVA
Parameters CON CIA CIA-PRO P Value

Linear HRV parameters

Mean HR, 1/min 414.47±11.3 489.98±9.3*** 423.72±23.7††† <0.001
SDNN, ms 4.76±1.1 4.31±1.0 4.65±0.9 0.34
RMSSD, ms 3.68±0.7 2.40±0.0* 4.20±1.3†† 0.03
LF (n.u) 55.09±4.1 66.64±5.7*** 49.37±2.0††† 0.002
HF (n.u) 39.75±3.9 41.77±5.6 50.40±3.5**† 0.02
LF/HF 1.24±0.2 1.80±0.6* 0.98±0.1†† 0.03
Nonlinear HRV parameters

DFAα1 0.76±0.2 0.33±0.1*** 0.70±0.1†† <0.001

DFAα2 0.99±0.3 0.78±0.1 0.73±0.3 0.13

†††
DC, ms 10.50±1.6 6.75±2.4* 14.49±2.1* <0.001
AC, ms −8.32±1.7 −4.95±1.6** −11.38±1.8*††† <0.001

Data are shown as means±SD. An unpaired Student t test was performed for each pair of 3 groups. AC indicates acceleration capacity; CIA, collagen-
induced arthritis; CIA-PRO, collagen-induced arthritis treated with propranolol; CON, normal control; DC, deceleration capacity; DFA, detrended fluctuation
analysis; HF, high frequency; HR, heart rate; HRV, heart-rate variability; LF, low frequency; n.u., normalized units; RMSSD, the root mean square of successive
differences of N-N interval; and SDNN, the standard deviation of N-N intervals.
*
P<0.05, **P<0.01, and ***P<0.001 vs CON group.
†
P<0.05, ††P<0.01, and †††P<0.001 vs CIA group.

CIA and CIA-PRO rats (data not shown). With respect (protein gene product 9.5), which is a known neu-
to the expression of inflammatory cytokines in the left ronal marker. We found that the sympathetic neurons
ventricle (LV) of rat hearts, the TNF-α (CIA: 181518.75 were overactive in CIA rats and that the administra-
[56236.35], CON: 87504.86 [13814.64]; P<0.01) and tion of propranolol was able to reduce the hyperin-
IL-1β (CIA: 995.06 [550.03], CON: 674.76 [110.25]; nervation (Figure S1). We further examined the level
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P<0.05) levels in the CIA rats were significantly el- of ventricular fibrosis among the 3 groups, and found
evated versus the CON rats (Figure  5). However, the increased fibrosis in the CIA group (CIA: 4.61 [1.95],
TNF-α (CIA-PRO: 112688.11 [30150.53]; P≤0.05) and CON: 0.54 [0.19]; P<0.001), but the fibrosis dramati-
IL-1β (CIA-PRO: 659.11 [76.66], P<0.05) levels in CIA- cally decreased after administration of propranolol
PRO rats were significantly reduced when compared (CIA-PRO: 0.76 [0.12], P<0.001; Figure 7C).
with CIA rats (Figure 6).

Infiltration With Inflammatory Cells and

Cardiac Fibrosis DISCUSSION
Because arthritis triggers a systemic inflamma- Our study is the first to demonstrate the interaction
tory response, we examined the number of CD45- between autonomic function and arrhythmogenic
positive cells in the myocardium of the CIA rats cardiac electrophysiology during highly inflamma-
and the CON rats. As expected, arthritis induced a tory stages in RA. CIA rats exhibited severe arthri-
prominent and long-lasting myocardial infiltration of tis, sympathetic hyperinnervation, and increased
inflammatory cells (CIA: 0.15 [0.08], CON: 0.03 [0.04]; ventricular arrhythmogenicity, which was charac-
P<0.001) in the CIA rats, but this was significantly terized by prolonged APD, slow CV, and steepened
reduced in the CIA-PRO rats (CIA-PRO: 0.08 [0.04], APD restitution. As a result, the vulnerability to VA
P<0.001; Figure  7A). Immunohistochemical staining increased in CIA rats. The possible mechanism un-
showed that sympathetic nerves, immunopositive to derlying the proarrhythmic electrical remodeling may
TH (the sympathetic nerve marker), were more abun- be explained by the sympathetic hyperinnervation,
dant in rats with CIA than in the controls (CIA: 0.06 increased infiltration of inflammatory cells, elevated
[0.07], CON: 0.01 [0.02]; P<0.001). After the propran- inflammatory cytokines, and a higher proportion of
olol treatment, the severity of sympathetic hyperin- fibrosis in the LV of rat hearts with CIA. On the other
nervation was alleviated in CIA-PRO rats (CIA-PRO: hand, we observed that the levels of TNF-α and IL-1β
0.08 [0.04], P<0.01; Figure 7B). To confirm the spe- in the LV were reduced, and the SVI imbalance was
cific expression of the sympathetic nerve, cardiac attenuated in CIA-PRO rats. These findings suggest
tissues were double-stained with TH and PGP9.5 that there might be synergic anticatecholaminergic

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 Lin et al Synergistic Effects of Propranolol in RA
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Figure 3.  Probability and duration of induced VA episodes.

A, Examples of VA induction attempts in the 3 groups. B, The number of VA episodes was increased
in CIA rats (n=6). In a comparison of the 3 groups, there are significant differences (P=0.02). C, The
overall duration of VA was significantly decreased in CIA-PRO rats (n=6). Overall comparison also
shows a significant difference (P=0.004). Data are shown as the median with 25th to 75th interquartile
ranges. **P<0.01 and ***P<0.001 vs CON group, ††P<0.01 and †††P<0.001 vs CIA group. CIA indicates
collagen-induced arthritis group; CIA-PRO, CIA rats treated with propranolol; CON, control group; and
VA, ventricular arrhythmia.

and anti-inflammatory effects of β blockers, which impact of a neuro-immune interaction in cardiac tis-
protect the heart from VA in rats with CIA. To our sue on ventricular arrhythmogenesis and the protec-
knowledge, this study is the first to demonstrate the tive effect of β blockers in RA.

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 Lin et al Synergistic Effects of Propranolol in RA
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Figure 4.  Prolongation of APD and arrhythmogenic ventricular substrate in CIA rats.
A, The left ventricles of the CIA rats have longer APDs than the CON rats. B, APDs measured in 70% repolarization with different
PCLs (n=6). C, CV in the CIA rats at different PCLs (n=6). D, APD restitution curve and (E) maximum slope (n=6). Data are shown as
mean with ±SD, expect maximum slope, which are shown as the median with 25th to 75th interquartile ranges. The overall 3-group
comparison shows a significant difference in maximum slope (P=0.02). *P<0.05, and **P<0.01 vs CON group, †P<0.05, and ††P<0.01 vs
CIA group. APD indicates action potential duration; CIA, collagen-induced arthritis group; CIA-PRO, CIA rats treated with propranolol;
CON, control group; CV, conduction velocity; and PCL, pacing-cycle length.

Inflammation and Ventricular Arrhythmia decrease the transient outward current and delayed
in Rheumatoid Arthritis rectifier potassium channel, resulting in prolonged
APD.21,22 Perfused hearts from transgenic mice over-
Although the underlying mechanisms accounting for expressing TNF-α exhibited a prolonged APD and
the proarrhythmogenic substrate in RA are intricate, a re-entrant VA.23 In addition, human recombinant IL-1
leading role seems to be played by chronic systemic prolonged APD by enhancing an L-type calcium cur-
inflammatory activation.20 Many basic studies have rent in guinea pig ventricular cells.24 Inflammasome
demonstrated significant direct effects of inflamma- activation in cardiac macrophages mediates the
tory cytokines on cardiac electrophysiology. Previous production of IL-1β in mice with diabetes mellitus.
studies have also shown that increased TNF-α could IL-1β causes the prolongation of APD, and induces a

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 Lin et al Synergistic Effects of Propranolol in RA

Figure 5.  Schematic and effect of propranolol on ventricular CV and electrical propagation in CIA.
A, Consequent phase maps of electrical propagation in the left ventricle. B, Isochronal map was fragmented
in CIA rats, but it was recognizable by the administration of propranolol. CIA indicates collagen-induced
arthritis group; CIA-PRO, CIA rats treated with propranolol; and CON, control group.
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decrease in potassium current and an increase in cal- by inducing autonomic dysfunction. Many basic and
cium sparks in cardiomyocytes, which are changes clinical studies have demonstrated that inflammatory
that underlie diabetes mellitus-induced VA.7 The clini- cytokines increase the sympathetic outflow by tar-
cal evidence that inflammatory cytokine levels corre- geting the autonomic centers of the brain.20,26 Local
late with the QTc interval in patients with RA strongly inflammation is also detected by vagal and sensory
indicates that in vivo these mechanisms are of crucial nerve fibers with express receptors for inflammatory
importance.25 Systemic inflammation could also in- mediators like IL-1β. An afferent signal is generated
directly produce proarrhythmogenic changes in RA and transmitted to the brain, which in turn leads to

Figure 6.  The measurement of inflammatory cytokines in cardiac tissue.

Relative expression of TNF-α (A) and IL-1β (B) in the left ventricle (n=6). There are significant differences
among the 3 groups in the expression of TNF-α (P=0.003) and IL-1β (P=0.03). Data are shown as the
median with 25th to 75th interquartile ranges. *P<0.05, and **P<0.01 vs CON group, †P<0.05 vs the CIA
group. CIA indicates collagen-induced arthritis group; CIA-PRO, CIA rats treated with propranolol; CON,
control group; IL-1β, interleukin-1 beta; and TNF-α, tumor necrosis factor-alpha.

J Am Heart Assoc. 2020;9:e016084. DOI: 10.1161/JAHA.120.0160849

 Lin et al Synergistic Effects of Propranolol in RA

Figure 7.  Effect of collagen-induced arthritis on ventricular fibrosis and sympathetic nerve density and infiltration of
lymphocytes.
A, Examples of left ventricle tissue from the 3 groups of rats stained with anti-CD45 antibody. There is increased infiltration of CD45-
Downloaded from http://ahajournals.org by on December 5, 2021

positive lymphocytes in CIA rats compared with CON and CIA-PRO rats (n=6). B, Example of ventricular immunostaining of tyrosine
hydroxylase-positive nerves. Compared with CON rats, there is increased sympathetic nerve density in CIA and CIA-PRO rats. Of
note, there was a reduced proportion of tyrosine hydroxylase-positive nerves in the CIA-PRO rats vs the CIA rats (n=6). C, Examples
of fibrosis percentage in the 3 groups using Masson’s trichrome staining. Increased fibrosis of CIA rats was noted (n=6). Overall, the
3 groups have significant differences (P<0.001). Data are shown as the median with 25th to 75th interquartile ranges. ***P<0.001 vs
CON group, ††P<0.01 and †††P<0.001 vs CIA group. CIA indicates collagen-induced arthritis group; CIA-PRO, CIA rats treated with
propranolol; and CON, control group.

activation of the sympathetic nervous system.27 On and maintaining the fibrotic process.30 Meanwhile,
the other hand, local inflammation in the joint or heart β-adrenergic-receptor overexpression and an in-
could occur also as a consequence of activation of the creased level of β-adrenergic-receptor agonists,
central sympathetic nervous system through a proin- including epinephrine and norepinephrine, could
flammatory influence on adaptive immune cells, for produce cardiac hypertrophy and fibrosis in vivo.31
example, the recruitment of leukocytes and the induc- Cardiac fibroblasts also have adrenergic receptors,
tion of proinflammatory cytokines.28 Overactivation of and stimulation of the β2-adrenergic receptor leads
the β-adrenergic system results in the induction of an to increased proliferation of human and rodent car-
inflammatory cascade and eventually activates pro- diac fibroblasts.32
duction of IL-1β and IL-18.27 Our study provides evidence linking myocardial
Cardiac fibrosis is an important structural re- inflammation and VA in RA. We observed significant
modeler in RA. It contributes to arrhythmogenesis sympathetic hyperactivity, prolonged APD, decreased
by altering source-sink relationships to create a vul- CV, and altered APD restitution with a steepened max-
nerable substrate, while simultaneously facilitating imal slope in CIA rats as compared with CON rats. A
the emergence of triggers such as depolarization- steepened APD restitution resulted in increased APD
induced premature ventricular complexes, both alternans, promoting breakup of the electrical wave
factors combining synergistically to promote initiation into fibrillation-like states and arrhythmogenesis. As
of reentrant ventricular tachycardia.29 A network of a result, the vulnerability to VA increased in CIA rats.
proinflammatory cytokines (such as TNF-α and IL-1β) This proarrhythmic remodeling was associated with
and cardiac fibroblasts is responsible for initiating sympathetic hyperinnervation, increased infiltration of

J Am Heart Assoc. 2020;9:e016084. DOI: 10.1161/JAHA.120.01608410

 Lin et al Synergistic Effects of Propranolol in RA

inflammatory cells, elevated inflammatory cytokines, fibrosis by directly inhibiting the β-adrenergic recep-
and a higher proportion of fibrosis in the LV of the tor in cardiac fibroblasts and indirectly attenuating the
hearts of rats with CIA. effects of inflammatory cytokines induced by sympa-
thetic overactivity in fibrosis.
Anti-Inflammation and Antiarrhythmic
Effects of β Blockers CONCLUSIONS
A previous clinical study has shown that treatment with
nonselective β blockers is associated with reduced se- We found that RA increased vulnerability to VA by a
verity of systemic inflammation and improved survival broad range of ventricular remodeling, including sym-
of patients with acute-on-chronic liver failure.33 In a pathetic hyperinnervation, prolonged APD, slow CV,
canine model of ischemic cardiomyopathy, the anti- steepened slope of APD restitution, and increased
inflammation effect of a β blocker was shown by de- cardiac fibrosis; thus providing further insight into the
creased levels of myocardial proinflammatory cytokine mechanisms underlying clinical RA-induced VA and
IL-1β, increased myocardial levels of the anti-inflam- sudden death. Furthermore, our results show that the
matory cytokine IL-10, and less myocardial oxidative synergistic anticatecholaminergic and anti-inflamma-
stress, leukocytosis, and fibrosis.34 Local sympathetic tory effects of β blockers could reverse proarrhythmic
denervation by bilateral cervical sympathetic gangli- ventricular remodeling and reduce vulnerability to VA in
onectomy attenuated myocardial inflammation in mice a rat model of RA.
with myocardial infarction.35 Reduced inflammatory
cell infiltration of the LV and reduced levels of proin- ARTICLE INFORMATION
flammatory cytokine TNF-α coinciding with increased Received January 28, 2020; accepted July 21, 2020.
levels of anti-inflammatory molecule IL-10 upon sym-
pathetic denervation were found. Our results are con- Affiliations
From the Department of Internal Medicine, National Taiwan University
sistent with prior studies showing that the levels of Hospital Hsinchu and Biomedical Park Branch, Hsinchu, Taiwan (T.-T.L.,
TNF-α and IL-1β in the LV were reduced, and myocar- M.-T.L.); College of Medicine, National Taiwan University, Taipei, Taiwan
dial infiltration of inflammatory cell and cardiac fibrosis (T.-T.L.); Institute of Biomedical Engineering, College of Electrical and
Computer Engineering (T.-T.L., Y.-L.S., J.-Y.S., K.-Y.L., H.-J.H., S.-F.L.) and
were decreased after propranolol treatment in rats of Department of Electrical and Computer Engineering (Y.-L.S.), National
the CIA-PRO group. There is a reciprocal relationship
Downloaded from http://ahajournals.org by on December 5, 2021

Chiao Tung University, Hsinchu, Taiwan; and Division of Cardiology,

between inflammation and increased sympathetic Department of Internal Medicine, National Taiwan University Hospital,
Taipei, Taiwan (Y.-B.L.).
activity, with inflammation predisposing to increases
in sympathetic activity and sympathetic overactiv- Sources of Funding
ity worsening inflammation. Treatment with β-blocker This work was supported by the Science and Technology Unit, Ministry of
Health and Welfare, Executive Yuan, Taiwan (grant nos. 106-2314-B-002-
propranolol could block this vicious cycle at both the
046-MY2 and 105-2314-B-002-072); the National Taiwan University Hospital
central sympathetic nerve system and the local car- Hsinchu Branch (grant nos. 107-HCH010, 105-HCH065, and HCH104-073);
diac sympathetic innervation level. We observed CIA and the National Chiao Tung University (grant nos. 104W970 and 105W970).
rats treated with propranolol had attenuated sympa- Disclosures
thetic overactivity and similar IHR as the controls. The None.
reduced level of TH in the cardiac tissue of CIA-PRO
suggested less local distribution of the sympathetic Supplementary Materials
Table S1
nerve compared with the CIA rats. Thus, propranolol Figure S1
decreased sympathetic tone and attenuated local in-
flammation in CIA rats. In addition to the attenuation of
joint swelling of CIA rats after treatment with proprano-
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J Am Heart Assoc. 2020;9:e016084. DOI: 10.1161/JAHA.120.01608412

 Table S1. Effects of propranolol on HRV parameters in the 1st to 3rd weeks.
1st week 2nd week 3rd week
Parameters
CON CIA CIA-PRO CON CIA CIA-PRO CON CIA CIA-PRO
Linear HRV parameters
Mean HR (1/min) 404.59±22.6 458.56±35.8* 402.71±27.9† 409.89±35.8 467.25±26.4* 424.92±50.3 411.53±6.6 471.25±31.8* 435.22±26.3†
SDNN(ms) 4.53±2.5 8.23±1.6 5.92±3.2 3.78±1.6 7.68±5.2 4.37±2.3 3.9±0.9 5.82±2.7 5.60±0.4
RMSSD(ms) 3.03±0.8 3.37±0.7 3.91±1.4 3.18±0.3 3.13±0.6 3.87±1.5 2.37±0.2 2.73±0.6 3.27±0.9
LF(n.u) 61.71±9.3 69.48±5.7 66.70±4.9 55.78±4.4 69.6±1.8** 62.06±0.7*,†† 62.73±11.1 52.12±6.1* 59.00±9.7
HF(n.u) 38.01±9.1 30.34±5.6 33.15±4.9 43.95±4.2 30.24±1.9** 37.63±0.7* ††
,
37.13±11 47.60±6.0* 40.83±9.7
LF/HF 1.76±0.6 2.41±0.7 2.08±0.5 1.3±0.2 2.33±0.2** 1.72±0.1*,†† 1.9±0.9 1.13±0.3* 1.53±0.5
Non-linear HRV parameters
DFAα1 0.62±0.2 0.88±0.2 0.59±0.2 0.86±0.1 0.58±0.1** 0.73±0.1† 0.39±0.1 0.70±0.3 0.65±0.3
DFAα2 0.71±0.4 1.12±0.1 0.8±0.28 0.42±0.55 0.94±0.2 0.57±0.28 0.84±0.1 0.91±0.1 0.95±0.2
Downloaded from http://ahajournals.org by on December 5, 2021

DC(ms) 11.39±2.3 9.50±2.7 9.95±1.1 12.49±2.1 9.2±2.6 13.14±4.1 11.20±2.9 8.60±2.0 12.01±2.5
AC(ms) –9.75±1.7 –7.34±2.6 –7.63±1.0 –9.31±1.7 –7.11±2.3 –10.28±4.5 –9.11±2.9 –6.5±1.9 –9.91±2.4

Data are shown as means ± SD. One-way ANOVA is utilized to analyze the differences among three groups. The results show significant differences in

the respect of mean HR (1st week, p<=0.03, 2nd week, p=0.04 and 3rd week, p=0.03), LF (2nd week, p=0.01 and 3rd week, p=0.03), HF (2nd week,

p=0.003 and 3rd week, p=0.03), LF/HF (2nd week, p=0.003, 3rd week, p=0.02) and DFAα1 (2nd week, p=0.01). *P<0.05, and **P<0.01 versus CON

group, †P<0.05, and ††P<0.01 versus CIA group. HRV, heart rate variability; HR, heart rate; SDNN, the standard deviation of N-N intervals; RMSSD,

the root mean square of successive differences of N-N interval; LF, low frequency; HF, high frequency; DFA, detrended fluctuation analysis; DC,

deceleration capacity; AC, acceleration capacity.

 Figure S1. TH and PGP9.5 immunofluorescense demonstrated co-labeling and decreased

expression in cardiac tissue after propranolol administration.

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Cardiac sections were labeled for TH (green) and PGP9.5 (red) immunofluorescence and

counterstained with DAPI (blue, nuclei). Abbreviations: CON: control group; CIA: collagen-

induced arthrtis group; CIA-PRO, CIA rats treated with propranolol; TH, tyrosine hydroxylase;

PGP9.5, protein gene product 9.5.