JACC: BASIC TO TRANSLATIONAL SCIENCE

VOL. 8, NO. 3, 2023

ª 2023 THE AUTHORS. PUBLISHED BY ELSEVIER ON BEHALF OF THE AMERICANCOLLEGE OF CARDIOLOGY FOUNDATION. THIS IS AN OPEN ACCESS ARTICLE UNDER
THE CC BY LICENSE (http://creativecommons.org/licenses/by/4.0/).

ORIGINAL RESEARCH - PRECLINICAL

Beneﬁcial Effects of Low-Intensity Pulsed
Ultrasound Therapy on Right Ventricular
Dysfunction in Animal Models
Takashi Nakata, MD,a Tomohiko Shindo, MD, PHD,a Kenta Ito, MD, PHD,a Kumiko Eguchi, MD, PHD,aYuto Monma, MD, PHD,a Sadamitsu Ichijo, MD, PHD,a Rie Ryoke, PHD,b Wakako Satoh, BA,c
Kazunori Kumasaka, BA,c Haruka Sato, MD, PHD,c Ryo Kurosawa, MD, PHD,a Kimio Satoh, MD, PHD,aRyuta Kawashima, MD, PHD,b Masahito Miura, MD, PHD,c Hiroshi Kanai, PHD,d,e Satoshi Yasuda, MD, PHD,aHiroaki Shimokawa, MD, PHDa,f

VISUAL ABSTRACTHIGHLIGHTS eNOS is downregulated in the RV ofpatients with RVF.
 eNOS and its downstream signals are
downregulated in a mouse model of PAB
and a rat model of RVF with Sugen/hypoxia.
 eNOS -/- mice show the worsening of RV
function compared with WT mice after the
PAB procedure.
 LIPUS therapy ameliorates RV
dysfunction in both animal models
associated with activation of eNOS.
 The beneﬁcial effects of LIPUS therapy
are absent in eNOS -/- mice.
 LIPUS therapy may be a possible, novel,and nonpharmacologic therapy for RVF
through eNOS activation.

Nakata T, et al. J Am Coll Cardiol Basic Trans Science. 2023;8(3):283–297.

ISSN 2452-302X

https://doi.org/10.1016/j.jacbts.2022.08.010

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LIPUS Ameliorates Right Ventricular Dysfunction

ABBREVIATIONSSUMMARY

AND ACRONYMS[Ca2þ]i = intracellular Ca2D

Right ventricular failure (RVF) is a leading cause of death in patients with pulmonary hypertension; however,concentration

effective treatment remains to be developed. We have developed low-intensity pulsed ultrasound therapy forcGMP = cyclic guanosine

cardiovascular diseases. In this study, we demonstrated that the expression of endothelial nitric oxide synthasemonophosphate(eNOS) in RVF patients was downregulated and that eNOS expression and its downstream pathway were

CO = cardiac output

ameliorated through eNOS activation in 2 animal models of RVF. These results indicate that eNOS is an

CMR = cardiac magnetic

important therapeutic target of RVF, for which low-intensity pulsed ultrasound therapy is a promising therapy

resonancefor patients with RVF. (J Am Coll Cardiol Basic Trans Science 2023;8:283–297) © 2023 The Authors. Published

dP/dt = rate of rise of left

by Elsevier on behalf of the American College of Cardiology Foundation. This is an open access article under the CC

ventricular pressure

BY license (http://creativecommons.org/licenses/by/4.0/).

eNOS = endothelial nitricoxide synthase

LIPUS = low-intensity pulsed
ultrasound

LV = left ventricular
NO = nitric oxideRight ventricular failure (RVF) is a

We previously demonstrated that low-intensity

clinical syndrome mainly caused bypulsedincreased right ventricle (RV) after-

anti-inﬂammatory

ultrasound(LIPUS)
effectshas

angiogenic

throughits

andmechano-load and is characterized by reduced RV ejec-transduction mechanism, and its clinical application

bandingtion fraction (RVEF) and dilated RV.1 Patients

is widely expected as a noninvasive therapeutic op-

PAH = pulmonary arterial

with RVF show systemic venous congestion,

tion for cardiovascular diseases.9 We previously re-

hypertension

leading to systemic organ dysfunction andported that LIPUS therapy induces angiogenesis in

PKC = protein kinase C

peripheral edema, the pathophysiology ofischemic heart tissue, ameliorating cardiac dysfunc-

PKG = protein kinase G

which appears to be different from thattion in a porcine model of chronic myocardial

PLN = phospholambanof left ventricular (LV) failure.1 RVF is one

ischemia 10 and a mouse model of myocardial infarc-

RV = right ventricleof the most important prognostic factors intion.11 Moreover, we reported that LIPUS therapy
ameliorates LV dysfunction in pressure-overloaded

PAB = pulmonary arterial

RVEF = right ventricularpatients with pulmonary arterial hyperten-ejection fraction

sion (PAH).2,3 It has been reported that 5-

LV by inducing angiogenesis12 and also diastolic

RVF = right ventricular failureyear survival in patients with reduced RVEF

dysfunction in diabetic mice with diastolic LV

RVFAC = right ventricular(<25%) is 47.1%, whereas in those with pre-dysfunction through enhancement of the endothelial

fractional area change

served RVEF ($25%) it is 70.5%.4 Because

nitric oxide synthase (eNOS)–nitric oxide (NO)–cyclic

SERCA = sarcoplasmic

the RV function of PAH patients predicts

guanosine monophosphate (cGMP)–protein kinase G

the prognosis independently of the severity(PKG) pathway.13 eNOS is one of the 3 NOS isoforms,of PAH, RVF itself is an important therapeu-

generating NO from L-arginine and molecular oxygentic target for PAH. 5 In addition, RVF isas substrates. 14 eNOS is mainly expressed in endo-

involved in adverse outcomes of patientsthelial cells but also in cardiomyocytes to a lesser

with LV heart failure with preserved ejection

extent, playing a pivotal role in the regulation of

fraction and patients who have an LV assist

vascular tone and maintenance of endothelial func-

device implanted.6,7 Although a number of pathoge-tion.15 Indeed, the NO signaling pathway plays an

netic factors, such as ischemia, inﬂammation, meta-

important protective role in the cardiovascular sys-

bolic abnormality, and ﬁbrosis, have been proposed

tem. 16,17

reticulum Ca2D-ATPasesGC = soluble guanylate
cyclaseTAPSE = tricuspid annularplane systolic excursion

WT = wild type8

NO

regulates

cardiovascularfunctionfor RVF, the detailed mechanisms still remain to bethrough activating soluble guanylate cyclase (sGC)

fully elucidated. Indeed, there is an urgent need toand its downstream stimulation of cGMP and PKG. 18

develop a robust therapy for RVF.

Becausethis

NO-sGC-cGMP-PKG

pathway

From the aDepartment of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan; bInstitute ofDevelopment, Aging and Cancer, Tohoku University Graduate School of Medicine, Sendai, Japan; cDepartment of Clinical Physiology, Health Science, Tohoku University Graduate School of Medicine, Sendai, Japan; dDepartment of Electronic Engineering,
Graduate School of Engineering, Tohoku University, Sendai, Japan; eDivision of Biomedical Measurements and Diagnostics,
Graduate School of Biomedical Engineering, Tohoku University, Sendai, Japan; and the fInternational University of Health andWelfare, Narita, Japan.
The authors attest they are in compliance with human studies committees and animal welfare regulations of the authors’
institutions and Food and Drug Administration guidelines, including patient consent where appropriate. For more information,
visit the Author Center.
Manuscript received June 16, 2022; revised manuscript received August 17, 2022, accepted August 17, 2022.

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LIPUS Ameliorates Right Ventricular Dysfunction

second messenger system that mediates vasodilation
and ameliorates cardiomyocyte remodeling,

19(with 5-minute intervals).11-13 Two weeks after LIPUS

target-therapy, echocardiography and invasive hemody-ing the pathway has attracted much attention as

namic measurements were performed, and after thesea possible therapeutic target for heart failure. 20

examinations, they were sacriﬁced by blood loss.

Although it has been reported that targeting the

RAT MODEL OF SUGEN/HYPOXIA-INDUCED PULMONARY

pathway

HYPERTENSION. Adult

improved

cardiomyocyte

hypertrophy,

interstitial ﬁbrosis, and calcium handling in pressureoverloaded LV in animals,21,22 the role of the pathway

male

Sprague-Dawleyratsweighing 180 to 220 g were purchased from Charles
River Laboratories. Rats were subcutaneously injec-

in the pathogenesis of RVF remains to be fully

ted with an inhibitor of vascular endothelial growthelucidated.

factor, SU5416 (Sigma) 20 mg/kg, and were exposed to

We previously demonstrated that RV function issigniﬁcantly deteriorated in eNOS-deﬁcient (eNOS-/-)

hypoxia

(10%

O2)

for

3

weeks

as

previously

described.27 Two weeks after hypoxia, rats received

mice when exposed to chronic hypoxia in vivo, sug-

LIPUS therapy 2 times/week for 3 weeks. Three weeks

gesting the importance of eNOS in the pathogenesis

after LIPUS therapy, RV function was measured, andof RVF. 23 Although several pharmacologic agents that
stimulate the sGC-cGMP-PKG pathway are available
for PAH patients,24 they have systemic side effects
such as hypotension because of their systemic vasodilator effect. On the other hand, our LIPUS therapy
locally activates the eNOS-NO pathway in the heart,which is an advantage over pharmacologic agents. In
the present study, we thus examined whether theeNOS-NO-sGC-cGMP-PKG pathway is impaired in RVF
and if so, whether LIPUS therapy is effective and safe
in animal models of RVF in vivo.

METHODS

rats were sacriﬁced by blood loss.
STATISTICAL ANALYSIS. Results are shown as me-dian (IQR). Differences between the 2 groups wereanalyzed using a Mann-Whitney U test. Data frommultiple groups were analyzed using analysis of variance followed by Tukey’s post hoc test for multiple
pairwise comparisons for normally distributed samples with equal variances. The normality of the distributions

was

conﬁrmedbythe

Shapiro-Wilk

normality test, and the equality of the variances wastested by Bartlett’s test. Pearson’s correlation coefﬁcient was used to determine the association between 2
continuous variables. Survival analysis was performed

A detailed description of the methods is provided inby the Kaplan-Meier method, and between-group dif-the Supplemental Appendix online.

ferences in survival were tested by the log-rank

LIPUS THERAPY. For LIPUS therapy, we used a(Mantel-Cox) test (GraphPad Prism [GraphPad Soft-multifunction generator (WF1974, NF Corporation

ware Inc]). Differences with a P value of <0.05 were

Yokohama) with a bipolar ampliﬁer (BA4825, NF

considered to be statistically signiﬁcant.

Corporation Yokohama) and a planar ultrasoundprobe (Honda Electronics Co).25 LIPUS therapy was

RESULTS

performed in the following conditions: center frequency: 1.875 MHz; pulse repetition frequency: 2.74

eNOS

kHz; number of cycles: 32 (17-ms burst length); spatial

BIOPSY SAMPLES OF PATIENTS WITH DILATEDEXPRESSIONIN

RVENDOMYOCARDIAL

peak temporal average intensity: 117-162 mW/cm 2;

CARDIOMYOPATHY AND THOSE WITH PAH. The

and power of LIPUS: 0.25 MPa.11-13 LIPUS therapy was

eNOS-positive area in the RV was signiﬁcantly

applied to animals through an agar phantom gel,decreased in the patients with RVF vs those without itwhich is adjusted at a 6-cm depth where the beam of(Figures 1A and 1B). We also examined caveolin-1 as an

the ultrasound becomes stable.11-13

endothelial cell marker and regulator of eNOS and

MOUSE MODEL OF PULMONARY ARTERY BANDING.

found that the caveolin-1–positive area was compara-

Male wild-type (WT) mice (10 weeks old; 23  28 g inble among the 3 groups (Figures 1A and 1B). There wasbody weight) were obtained from Charles Rivers Lab-no signiﬁcant correlation between caveolin-1–positive

oratories. eNOS -/- mice were originally provided by P.

area and eNOS-positive area (Figure 1C). In contrast,

Huang (Harvard Medical School). The present mousethere was a positive correlation between eNOS

model of pulmonary artery banding (PAB) was previ-

expression and tricuspid annular plane systolic

ously described in detail.

26

One week after the sur-

excursion (TAPSE)/RV fractional area change (RVFAC),

gery, mice in the treatment group received LIPUS

but no correlation with LV function (Figure 1D).

therapy under general anesthesia with inhaled iso-

IMPORTANCE

ﬂurane (0.5%-1.0%) 3 times/week for 2 weeks. Irradi-

SIGNALING PATHWAY IN RV DYSFUNCTION. To

ation time of LIPUS therapy was 20 minutes for 3 times

conﬁrm the signiﬁcance of the eNOS-NO-sGC-cGMP-

OF

THE

eNOS-NO-sGC-cGMP-PKG

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F I G U R E 1 Reduced eNOS Expression in Right Ventricular Endomyocardial Biopsy Samples From Patients With Dilated Cardiomyopathy and Those

With PAH

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LIPUS Ameliorates Right Ventricular Dysfunction

PKG signaling pathway in the pathogenesis of RVF,

decreased after PAB (Supplemental Figure 2A). In

we ﬁrst evaluated physiologic function and molecular

contrast, weights of the RV and lungs were compa-

regulation in the mouse model of PAB in vivo. One

rable between WT and eNOS -/- mice after PAB

week after the PAB operation, we found that pulmo-(Supplemental Figure 2B).

nary arterial velocity was signiﬁcantly increased, RV
function was signiﬁcantly decreased, and the RV wassigniﬁcantly hypertrophied and dilated compared
with

the

sham-operatedgroup(Supplemental

Figures 1A to 1C). Although eNOS phosphorylation at
Thr495 was signiﬁcantly increased, that at Ser1177
was signiﬁcantly decreased in PAB mice comparedwith sham-operated mice (Supplemental Figures 1D
and 1E), indicating that eNOS activity was signiﬁcantly decreased in pressure-overloaded RVs. Moreover, expression of protein kinase C (PKC) was
signiﬁcantly increased, and downstream signals of
the eNOS-NO-sGC-cGMP-PKG pathway were alsosigniﬁcantly decreased in PAB mice compared withsham-operated mice (Supplemental Figures 1D and
1E). Importantly, there was a strong correlation between PKC and phosphorylated eNOS at Thr495 andbetween phosphorylated eNOS at Ser1177 and thedownstream signals (Supplemental Figure 1F). To
further elucidate the importance of eNOS in RVF, we
examined the effects of PAB in eNOS-/- mice
(Figure 2A). eNOS -/- mice without PAB showed normal
cardiac function in both ventricles and showednormal body weight and weights of the RV, lungs, andliver (Figures 2C to 2F, Supplemental Figures 2A and
2B). In contrast, in eNOS-/- mice with PAB, RV function

andsurvivalrate

signiﬁcantly

worsened

compared with WT mice with PAB (Figures 2B, 2E, and
2F). Histologic examination showed that myocardial
hypertrophy was signiﬁcantly increased and capillarydensity was signiﬁcantly decreased in eNOS -/- PAB
mice compared with WT PAB mice, whereas intersti-

LIPUS THERAPY AMELIORATES RV DYSFUNCTION IN
PAB

MICE. Next,

weexamined

whether

LIPUS

therapy is effective for RVF in PAB mice (Figures 3A
and 3B). Two weeks after LIPUS therapy, when pulmonary arterial velocity was comparable between the
PAB-LIPUS and PAB-placebo groups, echocardiography showed that TAPSE, right ventricular dilated
diameter, and cardiac index were all signiﬁcantly
improved in the former compared with the latter(Figures 3C and 3D). In contrast, there was no signiﬁcant difference in body weight, blood pressure, heartrate, organ weight, or survival rate between the 2groups (Supplemental Figures 3A to 3C). Hemodynamic evaluation showed that RV end-diastolicpressure was signiﬁcantly decreased in the PABLIPUS group vs the PAB-placebo group and thatstroke work, stroke volume, cardiac output (CO), andpositive rate of rise of LV pressure (dP/dt) weresigniﬁcantly increased in the PAB-LIPUS group vs the
PAB-placebo group (Figures 3E and 3F, Supplemental
Figure 4B). Other RV function markers were comparable between the 2 groups (Supplemental Figures 4A
and 4B). There was no difference in RV systolicpressure between the 2 groups, indicating that the 2groups had a similar extent of RV pressure overload(Figure 3F). LIPUS therapy signiﬁcantly ameliorated
myocardial

hypertrophy

andinterstitialﬁbrosiscompared with the PAB-placebo group (Figures 4A
and 4B). In contrast, there was no difference in
capillarydensity

between

the

2

PAB

groups(Supplemental Figures 5A and 5B).

tial ﬁbrosis was comparable between the 2 groups

LIPUS THERAPY ACTIVATES THE eNOS-NO-cGMP-PKG(Figures 2C and 2D). Macroscopic liver congestion was

SIGNALING

noted in eNOS-/- PAB mice, and liver weight was

extent of eNOS phosphorylation at Ser1177 was

PATHWAY. After

LIPUS

therapy,thesigniﬁcantly higher in eNOS -/- PAB mice than in WT

signiﬁcantly increased in PAB-LIPUS mice compared

PAB mice at 5 weeks after PAB (Figures 2C and 2D). In

with PAB-placebo mice (Figures 4C and 4D), whereas

the present study, although eNOS-/- mice showedthat at Thr495 and the expression of PKC were com-slightly elevated blood pressure as previously re-parableported,28 their blood pressure was signiﬁcantly

Figures 5C and 5D). Furthermore, LIPUS therapy

betweenthe2groups(Supplemental

F I G U R E 1 Continued(A) Representative images of right ventricular myocardial tissues stained for eNOS and caveolin-1 (scale bars: 100 mm for both). (B) Quantitative analysis of
the positive area of eNOS and caveolin-1. (C) Graphs showing the correlation between the positive area of caveolin-1 and eNOS. (D) Graphs showing thecorrelation between the positive area of eNOS and TAPSE, RVFAC, RVSP, mPAP, PCWP, and LVEF. Results are expressed as mean  SD. *P < 0.05.
Comparisons of parameters were performed with analysis of variance followed by Tukey’s post hoc test for multiple pairwise comparisons. Pearson’s
correlation coefﬁcient was used to determine the association between 2 continuous variables. Cav-1 ¼ caveolin-1; DCM ¼ dilated cardiomyopathy;
eNOS ¼ endothelial nitric oxide synthase; mPAP ¼ mean pulmonary arterial pressure; PAH ¼ pulmonary arterial hypertension; PCWP ¼ pulmonary artery
wedge pressure; RVF ¼ right ventricular failure; RVFAC ¼ right ventricular fractional area change; RVSP ¼ right ventricular systolic pressure;
TAPSE ¼ tricuspid annular plane systolic excursion.

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F I G U R E 2 eNOS Plays a Crucial Role in Mice With Pulmonary Artery Banding

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LIPUS Ameliorates Right Ventricular Dysfunction

signiﬁcantly upregulated the expression levels ofwith PAB, LIPUS therapy had no effect on survival

downstream molecules, such as sGC a and PKG Ia in

rate, echocardiography or hemodynamic data, cardiomyocytethat LIPUS therapy improves RV functions via acti-

capillary density (Figures 5B to 5F). Furthermore, thevating the eNOS-NO-cGMP-PKG signaling pathway.

effects of LIPUS on the downstream molecules of theThen, we examined the upstream molecules that are

eNOS-NO-cGMP-PKG signaling pathway were absent

known to phosphorylate eNOS at Ser1177. 29 Phos-in eNOS -/- mice (Figures 5G and 5H). Thus, eNOS isphorylation of Akt was signiﬁcantly increased in PAB-

crucial for the beneﬁcial effects of LIPUS therapy in

LIPUS

the PAB model.

micecompared

with

PAB-placebo

mice

hypertrophy,

interstitialﬁbrosis,PAB mice (Figures 4C and 4D). These results suggest

or(Figures 4C and 4D), whereas phosphorylation of

THE EFFECTS OF LIPUS THERAPY TO OTHER PARTS

adenosine monophosphate-activated protein kinase

OF THE HEART IN PAB MICE. We additionally exam-(AMPK) and expression of Ca 2þ/calmodulin-depen-

ined protein expression of the whole LV at 1 week

dent protein kinase II (CaMKII) were comparable be-after the PAB procedure with and without LIPUS

tween the 2 groups (Supplemental Figures 5C and 5D).

therapy. Interestingly, the expression of total eNOS

Consistently, we found a positive correlation between

was signiﬁcantly decreased in the LV of PAB mice, but

the extent of phosphorylated eNOS at Ser1177 andphosphorylation of eNOS and the expression of thesGC a , PKGI a , and phosphorylated Akt (Figure 4E),

downstream signaling molecules were comparable

whereas there was no correlation between the extent

betweenof phosphorylated eNOS at Ser1177 and phosphory-

Figures 7A and 7B), and LV systolic function was

sham

and

PAB

mice

(Supplemental

lated AMPK, total AMPK, or CaMKII (Supplemental

unaffected after PAB (Figure 2F). Because LIPUS

Figure 5E). The extent of Akt phosphorylation wastherapy was applied to the whole heart, we also

comparable between sham-operated mice and pla-

examined the protein expression of the whole LV af-

cebo treatment mice (Supplemental Figures 5F andter LIPUS therapy and found no difference in LV

5G). In addition, we performed indirect measure-function or protein expression levels of the LV

ment of NO by measuring NO x (NO 2 þ NO3 ) levels in
the myocardium. Although PAB mice showed lower
levels of NOx compared with WT mice, there was no
signiﬁcant difference between PAB-placebo and PABLIPUS mice (Supplemental Figure 6A). Then, we
measuredcGMPlevelsinmyocardial

tissue.

Interestingly, myocardial cGMP levels were signiﬁcantly

increased

in

PAB

mice

comparedwith

WT mice, and there was no signiﬁcant difference
between

PAB-placebo

and

PAB-LIPUS

mice(Supplemental Figure 6B).

(Figure 3D, Supplemental Figures 7C and 7D).
LIPUS THERAPY AMELIORATES RV DYSFUNCTION IN
SU/HX RATS. To further conﬁrm the beneﬁcial ef-

fects of LIPUS therapy on RVF, we next addressed thisissue using another rat model with Sugen/hypoxia(Su/Hx)-induced PAH. SU/Hx rats showed a signiﬁcant reduction in body weight compared with control
rats but showed normal blood pressure and heart rate
(Supplemental Figures 8A and 8B). In a treadmill
exercise

test,

SU/Hx

rats

showedsigniﬁcantly

reduced exercise tolerance compared with control

CRUCIAL ROLES OF eNOS IN THE THERAPEUTIC

rats (Supplemental Figure 8C). There was no differ-

EFFECTS OF THE LIPUS THERAPY. To conﬁrm theence in exercise tolerance after LIPUS therapyrole of eNOS in the beneﬁcial effects of LIPUS therapy(Supplemental Figure 8C). RV and lung weights were

on RVF, we examined the effects of LIPUS therapy ingreater in SU/Hx rats than in control rats, whereas noeNOS-/- mice with PAB (Figure 5A). In eNOS-/- micesigniﬁcant difference was noted between SU/Hx-

F I G U R E 2 Continued(A) Experimental protocol. (B) Kaplan-Meier curve of WT mice and eNOS-/- mice after PAB (**P < 0.01, log-rank test). (C) Representative images of RV
myocardial tissues stained with hematoxylin-eosin (scale bars: 100 mm), Masson’s trichrome (scale bars: 100 mm), CD31 (scale bars: 100 mm), andmacroscopic image of the liver (scale bars: 1 cm). (D) Quantitative analysis of CSA (HE), myocardial ﬁbrosis (MT), CD31-positive cells, and liver weight (WT
sham: n ¼ 5; eNOS-/- sham: n ¼ 4; WT PAB: n ¼ 16; eNOS-/- PAB: n ¼ 14). (E) Representative echocardiography images of parasternal short-axis view
(upper) at 1 week and (lower) at 5 weeks after PAB. The arrowhead represents pericardial effusion. (F) Graphs showing the time course of TAPSE, cardiac
index, RVDd, RVWT, and LVEF evaluated with echocardiography. Results are expressed as mean  SD. Comparisons of parameters were performed
with analysis of variance followed by Tukey’s post hoc test for multiple pairwise comparisons. *P < 0.05, **P < 0.01, ***P < 0.001 vs WT sham group;†P < 0.05, ††P < 0.01, †††P < 0.001 vs eNOS-/- sham group #P < 0.05, ##P < 0.01, ###P < 0.001 vs WT PAB group. BW ¼ body weight;

CSA ¼ cross-sectional area; LIPUS ¼ low-intensity pulsed ultrasound; HE ¼ hematoxylin and eosin; LVEF ¼ left ventricular ejection fraction;
MT ¼ Masson’s trichrome; PAB ¼ pulmonary artery banding; RV ¼ right ventricle; RVDd ¼ right ventricular diastolic diameter; RVWT ¼ right ventricularwall thickness; TAPSE ¼ tricuspid annular plane systolic excursion; TTE ¼ transthoracic echocardiography; w ¼ weeks; WT ¼ wild type; other abbreviations
as in Figure 1.

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placeboand

LIPUS Ameliorates Right Ventricular Dysfunction(Supplemental

comparable between the 2 groups (Supplemental

Figure 8D). Liver weight was comparable among the

SU/Hx-LIPUS

rats

Figures 11G and 11H), indicating that LIPUS therapy

3 groups (Supplemental Figure 8D). There was no

had no effects on pulmonary vascular occlusion in thissigniﬁcant difference in survival rate between SU/Hx-

SU/Hx model of PAH.

placebo

LIPUS

and

SU/Hx-LIPUS

rats(Supplemental

THERAPY

IMPROVES
2D

CA2D

MYOCARDIAL

Figure 8E). Echocardiography examination showed

HANDLING AND CA

that LIPUS therapy signiﬁcantly improved TAPSE,IN SU/HX RATS. To evaluate the effects of LIPUS-RELATED PROTEIN EXPRESSIONS

right ventricular dilated diameter, CO, and RVFAC

therapy at the cardiomyocyte level, we examined(Supplemental Figures 9C, 9D, and 10A). There wasforce and intracellular Ca 2þ concentration ([Ca2þ]i) inno signiﬁcant difference in LVEF or right ventricular

the trabeculae dissected from the RV during electricalwall thickness between the 2 groups (Supplemental

stimulation. There was no difference in developed

Figure 10A). In the cardiac magnetic resonanceforce between the 2 groups. In the SU/Hx-LIPUS rats,(CMR) study, LIPUS therapy signiﬁcantly ameliorated

positive peak derivative force (dF/dt max) and nega-

RV dysfunction in SU/Hx rats, including reduction intive peak derivative force (dF/dt min) were signiﬁ-right ventricular end diastolic volume and right ven-cantly improved compared with SU/Hx-placebo rats

tricular end systolic volume and improvement of(Supplemental Figures 12A and 12B). Consistently, theRVEF (Supplemental Figures 9E and 9F). There wastrabeculae from the SU/Hx-LIPUS group showedno signiﬁcant difference in RV mass, left ventricularreduced time constant of [Ca 2þ]i decay comparedend diastolic volume and left ventricular end systolicwith SU/Hx-placebo group, whereas there was novolume, LVEF, or LV mass between SU/Hx-placebosigniﬁcantdifferenceindiastolic[Ca 2þ]iand SU/Hx-LIPUS rats (Supplemental Figure 10B).

(Supplemental Figures 12C and 12D). These results

Invasive hemodynamic evaluation showed that LIPUS

indicate that LIPUS therapy ameliorates myocardial

therapy signiﬁcantly improved RV end-diastolic pres-systolic and diastolic functions at the cardiomyocyte

sure, CO, right ventricular end systolic volume, andlevel. Then, we examined the expressions of Ca2þRVEF in SU/Hx rats (Supplemental Figures 9G and 9H).

handling–related

In contrast, positive dP/dt and negative dP/dt wereexpression level of phosphorylated phospholamban

unaffected

by

LIPUS

therapymoleculesinRV

tissue.

The(Supplemental(PLN) at Ser16 was signiﬁcantly restored in SU/Hx-

Figure 10C). Importantly, there was no difference in

LIPUS rats (Supplemental Figures 12E and 12F). In

RV systolic pressure, mean pulmonary arterial pres-contrast, there was no difference in the expression ofsure, pulmonary vascular resistance, or effective

total phosphorylated PLN at Thr17 or total PLN amongarterial elastance between the SU/Hx-placebo and SU/

the 3 groups (Supplemental Figures 12E and 12F).

Hx-LIPUS groups, indicating that the severity of pul-

Additionally, the expression of sarcoplasmic reticu-

monary hypertension was comparable between the 2lum Ca 2þ-ATPase 2a (SERCA2a) was signiﬁcantly

groups (Supplemental Figures 9H and 10C). Histologic

upregulated in the SU/Hx-LIPUS compared with theanalysis in SU/Hx rats showed that LIPUS therapy

SU/Hx-placebo group (Figures 12E and 12F). The

signiﬁcantly ameliorated cardiomyocyte hypertrophy

expression of total eNOS was signiﬁcantly decreased

and interstitial ﬁbrosis compared with the placebo-in the SU/Hx-placebo group and tended to betreated group (Supplemental Figures 11A to 11D).

increased in the SU/Hx-LIPUS group (Supplemental

There was no signiﬁcant difference in capillary density

Figures 12E and 12F). Interestingly, we found a posi-

between the SU/Hx-placebo and SU/Hx-LIPUS groups

tive correlation between total eNOS expression and(Supplemental Figures 11E and 11F). Microvascular

phosphorylated PLN at Ser16 but not at Thr17 or(<200 m m) vessel occlusion rate in the lung was

SERCA2a (Supplemental Figure 12G).

F I G U R E 3 Continued(A) Experimental protocol. (B) Schematic drawing showing LIPUS application to a mouse heart. (C) Representative echocardiography images of parasternal short-axis
view at (upper) 1 week and (lower) 5 weeks after PAB. (D) Graphs showing the time course of PA velocity, TAPSE, RVDd, cardiac index, and LVEF evaluated withechocardiography. V Representative RV pressure-volume loops. (F) Graphs showing right ventricular systolic pressure, right ventricular end-diastolic pressure, cardiac
output, and dP/dt max evaluated with invasive hemodynamic measurement. Results are expressed as median (IQR). *P < 0.05, **P < 0.01, ***P < 0.001. Comparisonsof parameters were performed with the Mann-Whitney U test. CO ¼ cardiac output; dP/dt ¼ rate of rise of left ventricular pressure; PA Vel ¼ pulmonary arterial
velocity; RVEDP ¼ right ventricular end-diastolic pressure; RVP ¼ right ventricular pressure; WB ¼ Western blot; other abbreviations as in Figures 1 and 2.

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F I G U R E 4 LIPUS Therapy Attenuates Cardiomyocyte Hypertrophy and Interstitial Fibrosis and Activates the eNOS-NO-cGMP-PKG

Signaling Pathway in Mice With PAB

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LIPUS Ameliorates Right Ventricular Dysfunction

DISCUSSION

1 week after the LV pressure overload. These results
indicate that eNOS expression varies depending on

The major ﬁndings of the present study are that: 1)

the phase of RVF. Taken together, we need further

eNOS is downregulated in the RV of patients with RVF

studies to evaluate the time course of eNOS expres-caused by dilated cardiomyopathy (DCM) or PAH; 2)sion, including the hyperacute phase of RV pressure

impaired eNOS function is substantially involved inoverload. In PAB mice, eNOS suppression may be

the pathogenesis of RVF; 3) LIPUS therapy exerts

caused by eNOS phosphorylation at Thr495 as a result

beneﬁcialthroughof sustained PKC upregulation caused by pressure

enhancement of the eNOS-NO-cGMP-PKG pathway inoverload. In addition, eNOS-/- mice with PAB showeda mouse model of pressure-overloaded RVF (PAB

worsened pathologic features of RVF compared withmodel);RV

WT mice with the same procedure. Thus, sustained

dysfunction and myocardial Ca 2þ handling without

suppression of eNOS by phosphorylated PKC at

affecting pulmonary arterial lesions in a rat model ofThr495 and its downstream signaling are highly likely

PAH (SU/Hx model). To the best of our knowledge,

to be involved in the pathogenesis of pressure-

this is the ﬁrst report demonstrating that LIPUS

overloaded

therapy is effective and safe to improve RV dysfunc-

expression of total eNOS was decreased comparedtion in RVF in animals, suggesting that it could be awith that in WT mice. This result may be explained bynovel, nonpharmacologic, and less-invasive therapy

LV diastolic dysfunction induced by compression offor patients with PAH.

enlarged RV.

effects

and

4)

on

RV

LIPUS

dysfunction

therapyameliorates

RV

dysfunction.

In

PAB

mice,

the

THE

LIPUS AS A NOVEL THERAPY FOR RVF. Ser1177 is a

MECHANISM OF PRESSURE OVERLOAD–INDUCED RV

main phosphorylation site for eNOS activation, which

DYSFUNCTION. eNOS plays an important role in theis mediated by several kinases and stimulators. 29 In

IMPORTANCE

OF

eNOS

16,17

DYSFUNCTION

IN

It has been demonstrated

contrast, eNOS phosphorylation at Thr495 suppresses

that eNOS and its downstream effectors of the NO-

eNOS activity, for which PKC is involved. 31 PKC is

sGC-cGMP-PKG pathway improve cardiac inﬂamma-

activated in response to pressure overload, sympa-

tion, hypertrophy, and ﬁbrosis of the LV. 19 However,thetic nerve stimulation, and the renin-angiotensin-

the protective role of eNOS in pressure-overloaded

aldosterone system. 32 Thus, the RV may be more

RV remains to be fully elucidated. In the present

vulnerable to pressure overload than the LV, result-study, we conﬁrmed that eNOS is downregulated ining in sustained activation of PKC and eNOS phos-

the RV of patients with RVF caused by DCM orphorylation at Thr495 in the RV. Because chronic

PAH and SU/Hx rats and that eNOS activity and the

suppression of the eNOS-NO-sGC-cGMP-PKG pathway

NO-sGC-cGMP-PKG pathway were signiﬁcantly sup-may have a detrimental effect on RVF, LIPUS therapy

pressed in PAB mice. There was no statistical differ-could be an effective therapeutic option for thisence in the expression of total eNOS in PAB mice. Thiscondition, as demonstrated in the present study.

difference may be explained by the characteristic of

In the present study, we also demonstrated that

this model; the PAB model used in this study was an

LIPUS therapy improves RV systolic/diastolic func-

acute model of RVF. Liao et al30 reported that thetions and Ca2þ handling of RV cardiomyocytes in

expression of total eNOS in cardiomyocytes was

SU/Hx rats. It has been reported that the expression

signiﬁcantly upregulated at the hyperacute phase inof Ca 2þ-handling proteins, such as SERCA2a and PLN,

the LV pressure overload animal model as a stretch-were impaired in animal models of PAH 33 andinduced response. They also reported that thethat Ca2þ-handling proteins were altered in RV

expression of total eNOS expression normalized atsamples from PAH patients undergoing heart-lungcardiovascular system.

F I G U R E 4 Continued(A) Representative images of myocardial tissues stained with HE (scale bars: 100 mm) and MT (upper scale bars: 1 mm; lower scale bars:
100 mm). (B) Quantitative analysis of CSA (HE) and myocardial ﬁbrosis area (MT). (C, D) Representative Western blot and quantiﬁcation ofeNOS, phosphorylated eNOS at Ser1177, soluble guanylate cyclase a, protein kinase G Ia, Akt and phosphorylated Akt (p-Akt) in the mousewhole RV. (E) Graphs showing the correlation between the expression of phosphorylated eNOS at Ser1177 and sGCa, PKGIa, and phosphorylated Akt. Results are expressed as median (IQR). *P < 0.05, **P < 0.01, ***P < 0.001. Comparisons of parameters were performed
with the Mann-Whitney U test. Pearson’s correlation coefﬁcient was used to determine the association between 2 continuous variables.
cGMP ¼ cyclic guanosine monophosphate; eNOS ¼ endothelial nitric oxide synthase; GAPDH ¼ glyceraldehyde 3-phosphate dehydrogenase;NO ¼ nitric oxide; p- ¼ phosphorylated; PKG ¼ protein kinase G; PKGIa ¼ protein kinase G Ia; sGCa ¼ soluble guanylate cyclase a; t- ¼ total;
other abbreviations as in Figures 1 and 2.

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F I G U R E 5 No Therapeutic Effects of LIPUS Therapy in eNOS -/- PAB Mice

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LIPUS Ameliorates Right Ventricular Dysfunction

transplantation.34 Although we were unable totherapy over these pharmacologic agents is that it isdemonstrate the acute effects of LIPUS therapy in SU/

minimally invasive and safe, acting through the

Hx rats, we demonstrated a positive correlation be-

suppression of eNOS phosphorylation at Thr495 by

tween the expression of total eNOS and that ofenhancing eNOS at Ser1177, the most upstream site

phosphorylated PLN at Ser16. Because Ser16 is theof the pathway. To improve the pressure-overloaded

major phosphorylation site of PLN activation bycondition in RVF, LIPUS therapy is a novel thera-

PKG,35 it is possible that LIPUS therapy improves

peutic approach because it enhances the eNOS-sGC-

myocardial Ca 2þ handling through this PKG-mediatedcGMP-PKG pathway by controlling the activity of

PLN activation. Additionally, LIPUS had no effects on

eNOS. Although we demonstrated eNOS activation

the LV (Supplemental Figures 7C and 7D). As weafter LIPUS therapy, we were unable to directly

previously reported,11 LIPUS therapy has no effects onshow the increase in NO production. Additionally,

healthy tissue.

intracellular

cGMP

LIPUS THERAPY IMPROVES RV FUNCTION IN PAB MICE

increasedthe

BY SWITCHING THE PHOSPHORYLATION SITE OF eNOS

compared with sham-operated mice (Supplemental

AND

Figures 6A and 6B). Consistently, protein expres-

ACTIVATINGTHE

eNOS-NO-sGC-cGMP-PKG

inlevelswere

cardiomyocytes

signiﬁcantly
of

PAB

mice

PATHWAY. In the present study, we demonstrated

sion of PDE5 was signiﬁcantly decreased in the RV ofthat LIPUS therapy improves RV function and ame-

PAB

lioratesinterstitial(Supplemental Figures 1D and 1E). These results may

ﬁbrosis in PAB mice, where the eNOS-sGC-cGMP-

have some discrepancies, which may be explained as

PKG pathway plays a crucial role for the therapeu-

a compensatory response in the acute phase. In thetic effects of LIPUS. eNOS is regulated by phos-present study, we were able to measure myocardial

phorylation at multiple sites in response to humoral,cGMP and PDE5 levels only in the acute phase of

mechanical, and pharmacologic stimuli.16,17 Akt is

RVF. We need to evaluate myocardial cGMP levels in

one of the kinases that phosphorylate eNOS atother phases of RVF and those in other sources (eg,Ser1177, and the activation of Akt is regulated by

the serum). Further studies are needed to elucidate

tyrosine kinases, G-protein–coupled receptors, andother possible mechanisms involved in the thera-

stimulation of cell surface by mechanical forces.36

peutic effects of LIPUS therapy for RVF.

myocardial

hypertrophy

and

mice

comparedwithsham-operatedmice

We previously reported that LIPUS therapy acti-

STUDY LIMITATIONS. First, in the present study, wevates the Akt-eNOS pathway in vivo, 11,12 in whichshowed the decreased expression of eNOS in RVF

LIPUS-induced mechanical stimulation is involved.

patients, but we were unable to show the localiza-

AMPK and CaMKII are also known as kinases thattion of eNOS because myocardial biopsy samples

phosphorylate eNOS at Ser1177; however, they were

were ﬁxed only with formalin. Second, we compared

not involved in the effects of LIPUS therapy in thethe expression of eNOS between DCM patients andpresent study. Taken together, these results suggest

PAH patients. Although a comparison of PAH pa-that in RVF with enhanced Thr495 phosphorylation,

tients with and without RVF would be ideal, in

LIPUS therapy switches the eNOS phosphorylation

clinical practice, myocardial biopsy is not performed

site

resultantin PAH patients without RVF. Third, in the present

enhancement of the eNOS-sGC-cGMP-PKG pathway.

study, we used the same condition as in the original

Recently,

been

experiment.10 Further studies are needed to opti-

approved that systemically enhance the sGC-cGMP-

mize the condition of LIPUS therapy for RVF. Fourth,

PKG pathway 37; however, eNOS is not involved inin the present study, we used 2 different animal

their effects. Also, phosphodiesterase (PDE5) in-models of RV pressure overload. Because the path-

hibitors act through enhancement of cGMP down-

ophysiology of each model is different, the detailed

stream of this pathway. The advantage of LIPUS

mechanisms of the beneﬁcial effects of LIPUS

from

Thr495severaltosGC

Ser1177withstimulators

a

have

F I G U R E 5 Continued(A) Experimental protocol. (B) Kaplan-Meier curve of eNOS-/- PAB mice with and without LIPUS therapy (log-rank test). (C) Graphs showing
the time course of TAPSE, cardiac index, RVDd, RVWT, and LVEF evaluated with echocardiography. (D) Graphs showing RVSP, RVEDP, CO, SV,and dP/dt max evaluated with invasive hemodynamic measurement. (E) Representative images of myocardial tissues stained with HE (scalebars: 100 mm), MT (scale bars: 500 mm), and CD31 (scale bars: 100 mm). (F) Quantitative analysis of CSA (HE), myocardial ﬁbrosis area (MT),and CD31-positive cells. (G, H) Representative Western blot and quantiﬁcation of (sGCa, soluble guanylate cyclase b (sGCb), and PKGIa) in
the mouse whole RV. Results are expressed as median (IQR). SV ¼ stroke volume; other abbreviations as in Figures 1 to 4.

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LIPUS Ameliorates Right Ventricular Dysfunction

therapy remain to be fully elucidated. In SU/Hx rats,

CONCLUSIONS

because the acute phase evaluation of the LIPUS
therapy was not performed, we were unable to show

In the present study, we were able to demonstrate

the activation of eNOS and its downstream signalsthat LIPUS therapy ameliorates RVF through acti-

after LIPUS therapy. However, there was a positive

vating the eNOS-NO-cGMP-PKG pathway in PAB micecorrelation between total eNOS and phosphorylated

and improved the cardiomyocyte Ca2þ-handling sys-

PLN at Ser16 and between total eNOS and SERCA2a

tem in SU/Hx rats without any adverse effects. The(Supplemental Figure 12G), which suggests that

present results indicate that LIPUS therapy could be a

LIPUS-induced eNOS upregulation was involved in

novel, nonpharmacologic, and less-invasive therapy

the improvement of RV function in SU/Hx rats via

strategy for the treatment of patients with PAH.

restoring the Ca 2þ-handling system. Additionally, it
wasreported

that

the

eNOS-NO-sGC-cGMP-PKG

pathway possibly activates the Ca 2þ-handling system, and this pathway exerts inhibitory effects on
myocardial hypertrophy and ﬁbrosis as well.35,38
Although the effects of LIPUS therapy on myocardial hypertrophy and ﬁbrosis were relatively small in

ACKNOWLEDGMENTS The

authorsappreciate

Y.

Watanabe, H. Yamashita, and H. Nonaka for their
excellent technical support.

FUNDING SUPPORT AND AUTHOR DISCLOSURESThis research was supported in part by the Japan Agency for Medical
ResearchandDevelopment(grantnos.

JP19lm0203047

andthe present study, LIPUS therapy seems to signiﬁ-

JP19lk1403011). The authors have reported that they have no re-cantly enhance the Ca 2þ-handling system. Further

lationships relevant to the contents of this paper to disclose.

studies are needed to elucidate the detailed mechanisms in the beneﬁcial effects of LIPUS therapy.

ADDRESS

Fifth, the present mouse model with PAB represents

Shimokawa, International University of Health anda mild pulmonary hypertension model because we

Welfare, Narita 286-8686, Japan. E-mail: shimo@

used a 25-gauge needle to create pulmonary arterial

iuhw.ac.jp.

banding.

26

impairedFOR

CORRESPONDENCE:

Dr

Hiroaki

However, RV function was signiﬁcantlybysham-operated

theprocedure

mice(Supplemental

compared
Figurewith
1A).

Additionally, in PAB mice, the results of invasive
hemodynamic analysis were inconsistent with thoseof echocardiography. In general, pressure volume
analysis of the RV in mice is technically difﬁcult
because of the complex shape and small size of the
RV. For this reason, we obtained the accurate datawith CMR in the SU/Hx rat model. Importantly, in
SU/Hx rats, the results of pressure volume analysis
were consistent with those of CMR (Supplemental
Figures 9E and 9F). Sixth, in the present study, weevaluated the extent of RV ischemia by counting
capillary density alone. This issue remains to be
examined in future studies. Seventh, although it has
been reported that inﬂammation is involved in thepathophysiology of PAH and RVF,

39

we did not

examine inﬂammation-related factors in the present
study. The anti-inﬂammatory effects of LIPUS ther-PERSPECTIVESCOMPETENCY IN MEDICAL KNOWLEDGE:
Although RVF is a major cause of death in pulmonary
hypertension patients, no effective treatment is yet
available. In this study, we demonstrated that eNOS
downregulation is involved in the pathophysiology of
RVF and that LIPUS therapy ameliorates RV function
through activating eNOS.
TRANSLATIONAL OUTLOOK: LIPUS therapy,
which is a noninvasive therapeutic application, could
be a promising strategy for RVF patients througheNOS activation without systemic side effects.
Because LIPUS therapy could locally activate eNOS in
the heart, it could be used as an additional option for
RVF patients in addition to established medical
treatment.

apy remain to be examined in future studies.

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KEY WORDS endothelial NO synthase,
LIPUS, noninvasive therapy, pulmonary
hypertension, right ventricular dysfunction

A PP END IX For a supplemental Methods
section as well as a table, ﬁgures, and references, please see the online version of thispaper.

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