Cardiovascular Research (2021) 117, 1325–1338
doi:10.1093/cvr/cvaa221

Yuto Monma1, Tomohiko Shindo1, Kumiko Eguchi1, Ryo Kurosawa 1, Yuta Kagaya1,
Yosuke Ikumi1, Sadamitsu Ichijo 1, Takashi Nakata1, Satoshi Miyata 2,
Ayana Matsumoto3, Haruka Sato3, Masahito Miura3, Hiroshi Kanai 4,5, andHiroaki Shimokawa 1*
1
Department of Cardiovascular Medicine, Tohoku University Graduate School of Medicine, 1-1 Seiryo-machi, Aoba-ku, Sendai 980-8574, Japan; 2Department of Evidence-Based
Cardiovascular Medicine, Tohoku University Graduate School of Medicine, Sendai, Japan; 3Department of Clinical Physiology, Tohoku University Graduate School of Medicine, Sendai,
Japan; 4Department of Electronic Engineering, Tohoku University Graduate School of Engineering, Sendai, Japan; and 5Division of Biomedical Measurements and Diagnostics, Tohoku
University Graduate School of Biomedical Engineering, Sendai, Japan

Received 9 April 2020; revised 30 May 2020; editorial decision 26 June 2020; accepted 14 July 2020; online publish-ahead-of-print 19 July 2020

Aims

Heart failure with preserved left ventricular ejection fraction (HFpEF) is a serious health problem worldwide, as noeffective therapy is yet available. We have previously demonstrated that our low-intensity pulsed ultrasound(LIPUS) therapy is effective and safe for angina and dementia. In this study, we aimed to examine whether the
LIPUS therapy also ameliorates cardiac diastolic dysfunction in mice.

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Methods andTwelve-week-old obese diabetic mice (db/db) and their control littermates (db/þ) were treated with either the
results
LIPUS therapy [1.875 MHz, 32 cycles, Ispta (spatial peak temporal average intensity) 117–162 mW/cm2, 0.25 W/
cm2] or placebo procedure two times a week for 4 weeks. At 20-week-old, transthoracic echocardiography and invasive haemodynamic analysis showed that cardiac diastolic function parameters, such as e0 , E/e0 , end-diastolic pressure–volume relationship, Tau, and dP/dt min, were all deteriorated in placebo-treated db/db mice compared withdb/þ mice, while systolic function was preserved. Importantly, these cardiac diastolic function parameters were significantly ameliorated in the LIPUS-treated db/db mice. We also measured the force (F) and intracellular Ca2þ([Ca2þ]i) in trabeculae dissected from ventricles. We found that relaxation time and [Ca2þ]i decay (Tau) were prolonged during electrically stimulated twitch contractions in db/db mice, both of which were significantly ameliorated
in the LIPUS-treated db/db mice, indicating that the LIPUS therapy also improves relaxation properties at tissuelevel. Functionally, exercise capacity was also improved in the LIPUS-treated db/db mice. Histologically, db/db micedisplayed progressed cardiomyocyte hypertrophy and myocardial interstitial fibrosis, while those changes were significantly suppressed in the LIPUS-treated db/db mice. Mechanistically, western blot showed that the endothelial nitric oxide synthase (eNOS)-nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway and Ca2þ-handling molecules
were up-regulated in the LIPUS-treated heart.

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Conclusions
These results indicate that the LIPUS therapy ameliorates cardiac diastolic dysfunction in db/db mice through improvement of eNOS-NO-cGMP-PKG pathway and cardiomyocyte Ca2þ-handling system, suggesting its potential
usefulness for the treatment of HFpEF patients.
䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏 䊏*Corresponding author. Tel: þ81 22 717 7152; fax: þ81 22 717 7156, E-mail: shimo@cardio.med.tohoku.ac.jp
C The Author(s) 2020. For permissions, please email: journals.permissions@oup.com.
Published on behalf of the European Society of Cardiology. All rights reserved. V

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Low-intensity pulsed ultrasound ameliorates
cardiac diastolic dysfunction in mice: a possible
novel therapy for heart failure with preserved left
ventricular ejection fraction

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Y. Monma et al.

Graphical Abstract

1. Introduction
Heart failure (HF) is a major cause of death in developed countries, andthe number of patients has been increasing worldwide.1,2 Especially, theprevalence of HF with preserved left ventricular (LV) ejection fraction(LVEF) (HFpEF) has been rapidly increasing worldwide, accounting for
more than 50% of all HF patients.1,2 For decades, effective treatmentsthat improve HF with reduced LVEF have been developed. However, despite a number of large-scale clinical trials, no effective treatment to improve HFpEF is yet available. Indeed, HFpEF is a serious health problem
worldwide.
Diastolic dysfunction is one of the most important pathophysiologicalfactors of HFpEF.3 HFpEF patients have elevated LV filling pressure andsubsequent pulmonary hypertension, which is caused by abnormal LV diastolic function. In addition, epidemiological evidence suggests that diastolic dysfunction is present and progresses in severity before HF
symptoms arise and that the severity of diastolic dysfunction is associated with an increased risk of symptomatic HF and mortality.4 Thus, diastolic dysfunction plays an important role in the development andprogress of HFpEF.
Recently, it has been proposed that vascular endothelial dysfunction
and subsequent impairment of the endothelial nitric oxide synthase(eNOS)-nitric oxide (NO)-cGMP-protein kinase G (PKG) pathway underlie the pathophysiology of HFpEF, associated with comorbidities,
such as hypertension, diabetes mellitus (DM), obesity, chronic kidneydisease (CKD), and ageing.5,6 Thus, the eNOS-NO-cGMP-PKG pathway
could be a novel and promising therapeutic target for HFpEF.5,7,8
Low-intensity pulsed ultrasound (LIPUS) is a low power ultrasoundwith a certain pulse waveform.9 In a series of studies, we demonstrated
that LIPUS directly stimulates vascular endothelial cells and enhances theexpression of eNOS and other angiogenic factors, improving myocardial..
.. ischaemia, and cardiac function in animal models of ischaemic heart dis.. ease and hypertensive heart disease.10–12 We also have recently demon..
.. strated that our LIPUS therapy is effective and safe in mouse models of.. dementia, where up-regulation of eNOS plays a central role.13 Clinical..
.. trials are underway to evaluate the efficacy and safety of our LIPUS treat..
.. ment in patients with severe angina and those with mild cognitive impair.. ment in Japan. Based on these findings, we hypothesized that our LIPUS..
.. therapy is also effective for improving LV diastolic dysfunction. Thus, in.. the present study, we examined whether our LIPUS therapy also amelio..
.. rates diastolic dysfunction in a mouse model of HFpEF associated with.. Type 2 DM, and if so, to elucidate the mechanisms involved.
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.. 2. Methods..
.. A detailed description of the methods is provided in the Supplementary..
.. material online.
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.. 2.1 Animal experiments..
.. All animal experiments were performed along with the NIH guidelines.. (Guide for the care and use of laboratory animals) and were conducted..
.. in accordance with the protocols approved by the Institutional.. Committee for Use and Care of Laboratory Animals of Tohoku..
.. University (No. 2017-Idou-214). Leprdb/db mice (db/db mice), a leptin.. receptor-deficient model of obesity and Type 2 diabetes, and their non..
.. diabetic lean heterozygous Leprdb/þ littermates (db/þ mice) were pur..
.. chased from Charles River Laboratories (BKS.Cg-Dock7mþ/þ Leprdb/.. J).14 The animals were randomly assigned to the groups with the LIPUS..
.. therapy (db/db-LIPUS group: n = 42 and db/þ-LIPUS group: n = 10) or.. the groups with placebo procedure (db/db-control group: n = 42 and..
. db/þ-control group: n = 17). Some animals (db/db-control group:

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Keywords
LIPUS • Diastolic function • Ca2þ-handling system • Non-invasive therapy • eNOS-NO-cGMP-PKG pathway

LIPUS ameliorates cardiac diastolic dysfunction

1327

n = 20, db/db-LIPUS group: n = 20, db/þ-control group: n = 6, db/þLIPUS group: n = 6) were used for echocardiography examination, invasive haemodynamic analysis, exercise testing, blood tests, and histologicalanalysis. Some animals were used for western blot analysis (db/db-control group: n = 10, db/db-LIPUS group: n = 10, db/þ-control group: n = 4,
db/þ-LIPUS group: n = 4), measurement of force and intracellular Ca2þin trabeculae (db/db-control group: n = 5, db/db-LIPUS group: n = 5),
and Langendorff experiments (db/þ-control group: n = 7, db/db-control
group: n = 7, db/db-LIPUS group: n = 7). Every three to six mice wereseparated in the cages where the temperature (22 C) and humidity(60%) were kept stable, and they were fed with food and water supplied
at ad libitum.

2.2 The LIPUS therapy
For the LIPUS therapy, a diagnostic ultrasound device (Prosound a10;
HITACHI, Ltd., Tokyo, Japan) whose treatment conditions could be
modified by software application was used.11,12 The probe used for thetreatment was a sector-type probe commercially available as a diagnostic
device for humans. Based on our previous studies,11,12 we performed
the LIPUS therapy under the following conditions; frequency 1.875 MHz,pulse repetition frequency 4.90 kHz, number of cycles 32, voltage applied to each transducer element 17.67 volts (V), Ispta (spatial peak temporal average intensity) 117–162 mW/cm2, and the power of the LIPUS
was 0.25 W/cm2 (Figure 1A).11,12 The beam of therapeutic ultrasoundwas irradiated as sector shape and focused at 6 cm depth. For safety, toprevent the temperature rise of the ultrasound probe, the voltage applied to each transducer element was controlled to keep the level lower
than the upper limit of acoustic output standards (<720 mW/cm2) for diagnostic ultrasound devices (US Food and Drug Administration’s Track.. 3 Limits). In order to irradiate the heart with stable sound field, LIPUS..
.. was applied to mice through an agar phantom gel (Figure 1B). LIPUS was.. applied to the heart through the chest wall under light anaesthesia with..
.. inhaled isoflurane (0.5–1.0%). The animals in the LIPUS group were sub..
.. jected to the LIPUS therapy two times a week for 4 weeks from 12- to.. 16-weeks-old (Figure 1C). The animals in the control group underwent..
.. the same procedures including anaesthesia but without the LIPUS ther.. apy (Figure 1C).
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.. 2.3 Transthoracic echocardiography..
.. Transthoracic echocardiography was performed under light anaesthesia.. with inhaled isoflurane (0.4–1.5%) at 12, 16, and 20-weeks-old, using..
.. Vevo 2100 (Visual Sonics, Ontario, Canada) as previously reported.15.. Heart rate during echocardiographic study was maintained in the range..
.. of 500600 b.p.m. for M-mode and B-mode, respectively and 450550.. b.p.m. for Doppler studies.15..
..
.. 2.4 Invasive pressure–volume analysis..
.. Invasive haemodynamic measurements were performed in anaesthetized..
.. mice with inhaled isoflurane (0.4–1.5%) and mechanical ventilation at 20.. weeks-old. After thoracotomy, a 4-electrode pressure conductance..
.. catheter (1.4 F, SPR-839, Millar Instruments, Houston, TX, USA) was.. inserted into the LV through the apex. After stabilization, steady-state..
.. measurements were recorded. Subsequently, LV preload was decreased.. by occlusion of the inferior vena cava for 5–10 s to obtain the slope of..
.. the LV end-diastolic pressure–volume relationship (EDPVR) (PowerLab/.. 4SP, AD Instruments, Lab Chart 7 Pro Software, Castle Hill, Australia).14..
.. All data were analysed using the PowerLab data acquisition system (AD.. Instruments) and averaged over 10 sequential beats.

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Figure 1 Study protocol. (A) The setting of the LIPUS therapy. (B) Schematic drawing showing the LIPUS application in a mouse heart. (C)Experimental protocol.

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2.5 Blood pressure measurement
Blood pressure was measured at 12-, 16-, and 20-weeks-old with thetail-cuff system (Muromachi Kikai Co, Ltd, MK-2000ST NP-NIBP
Monitor, Tokyo, Japan) without anaesthesia.

2.6 Exercise testing

2.7 Measurement of force and intracellularCa2þ in mouse trabeculae
After the mice were adequately anaesthetized with intraperitoneal injection of butorphanol tartrate (5.0 mg/kg), midazolam (4.0 mg/kg), andmedetomidine chloride (0.3 mg/kg),16 according to the Guide for thecare and use of laboratory animals, the mouse heart was excised, coronary arteries were immediately perfused via the aorta with a HEPES buffered solution with 15 mmol/L KCl. The trabeculae were dissected fromthe endocardial region of the right ventricle and mounted on an inverted
microscope (Nikon, Japan) between a force transducer and a micromanipulator in a bath superfused with HEPES solution containing 5 mM KCl
as described previously.17,18 Force was measured using a silicon strain
gauge (model AE-801, SenSoNor, Horten, Norway). Intracellular Ca2þlevel was measured using microinjected fura-2 and a photomultipliertube (PMT; E1341 with a C1556 socket, Hamamatsu, Japan).17–19 The
trabeculae were electrically stimulated with parallel platinum electrodes
at 0.5 Hz (22 C, extracellular Ca2þ = 2.0 and 4.0 mM).18,19

2.8 Biochemical analysis
Serum levels of blood glucose and insulin were measured using a glucometer and an insulin ELISA kit [Mouse Insulin ELISA kit (#10-1247-01),
Mercodia AB, Uppsala, Sweden], respectively. Serum B-type natriuretic
peptide (BNP) levels were measured using a BNP ELIZA kit [ELISA Kit
for Brain Natriuretic Peptide (CEA541Ca), Cloud-Clone Corp., TX,
USA]. Western blot analysis was performed by standard methods.

2.9 Histological analysis
Excised hearts were fixed with 4% formalin for histological and immunohistochemical examinations. The tissue specimens were embedded inparaffin and sliced at 3-mm in thickness. The sections were stained byhaematoxylin & eosin (H&E) and Masson-trichrome (MT); the latter wasused to assess LV myocardial fibrosis. CD31 immunostaining was used
to assess myocardial capillary density. The hypoxic regions in the heart
were evaluated by hypoxyprobe-1 kit (HP1-100Kit, Hypoxyprobe Inc.,
MA, USA), according to the manufacturer’s instructions. Semiquantitative analysis regarding the extent of hypoxyprobe was evaluatedfor each image by using the following scale; 0 = none, 1 = slight,
2 = moderate, and 3 = high, as previously described.12 In all analysis,
three sections were assessed per animals.

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2.10 Indirect NO measurement
We performed indirect measurement of NO, using the OxiSelectNitrite Assay Kit (Cell Biolabs, San Diego, CA, USA) based on the conversion of nitrate to nitrite by nitrate reductase followed by quantification of nitrate after the Griess reaction. NOx (NO2 þ NO3) level wasmeasured according to the Product Manual.

2.11 Langendorff experiments
Langendorff experiments were performed as previously described.20–22
Mice were pretreated intraperitoneally with heparin (500 units) and10 min later, they were sacrificed with intraperitoneal injection of pentobarbital sodium (50 mg/kg). The hearts were excised and placed into icecold Krebs-Henseleit buffer (KHB) to be arrested. After all extra-aortic
tissues were removed, the ascending aorta was cannulated with a 21guage blunted needle and tied up with a thread. Then, the heart was retrogradely perfused at a constant flow of 2.0 mL/min by the Langendorff
apparatus (Model IPH-W2, Primetech Corporation, Tokyo, Japan) withwarmed KHB bubbled with 95% O2 and 5% CO2. After a 10-min stabilization period, the heart was paced at 400 b.p.m. and then perfused at aconstant pressure of 80 mmHg. Coronary flow was continuously measured with a flowmeter using an ultrasonic flow probe (FLSC-01,
Primetech Corporation, Tokyo, Japan) and was analysed by a computerbased analysis system in LabChart 7.0 software. Bradykinin (BK, 10-6 mol/L) and sodium nitroprusside (SNP, 10-5 mol/L) were used to evaluate
endothelium-dependent and -independent vasodilator responses, respectively. Coronary flow rate was corrected by heart weight. Baseline
coronary flow was defined as coronary flow rate before administrationof vasodilator agonists. Increases in coronary flow were calculated as thedifference between maximum coronary flow in response to vasodilatorsand baseline coronary flow (Supplementary material online, Figure S8A).

2.12 Statistical analysis
Results are shown as mean ± standard deviation for all experiments.
Differences between two groups were analysed using an unpaired twotailed Student’s t-test or Wilcoxon rank sum test as appropriate. Data
from multiple groups were analysed using analysis of variance followed
by Tukey’s multiple comparison for normally distributed samples withequal variances. The normality of the distributions was confirmed by the
Shapiro’s normality test, and the equality of the variances was tested by
the Bartlett test. For comparisons of the groups with skewed distributions and unequal variances, we applied non-parametric multiple contrast test procedure proposed by previous reports23 (GraphPad Prism
Software Inc., San Diego, CA, USA and R version 3.6.3). Differences witha P-value <0.05 were considered to be statistically significant.

3. Results
3.1 db/db mice
Body weight was greater in db/db mice than in db/þ mice by more than
two-fold, whereas no significant difference was noted between db/dbcontrol and db/db-LIPUS mice (Supplementary material online, Figures
S1A and S3A). During the experiment, systolic blood pressure (BP) anddiastolic BP tended to elevate in both db/db-control and db/db-LIPUS
mice compared with db/þ mice. However, there was no statistical difference in systolic or diastolic BP between db/db-control and db/db-LIPUS
mice (Supplementary material online, Figure S1B and C).

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Exercise capacity of mice was examined using a 6-lane rodent treadmill
system (MK-680C, Muromachi Kikai, Japan) at 12-, 16-, and 20-weeksold. After 10 min of acclimatization running at a low speed (1 m/min), exercise testing was performed by progressively increasing speed (speed
steps of 1 m/min every 2 min). Maximal speed and running distance were
determined when the mouse left the treadmill and remained on a shock
pad for 5 s. Exercise capacity was expressed as work load in Jules (J), calculated by multiply running distance by body weight. All animals were
given water and standard rodent feed ad libitum pre- and post-exercise.

Y. Monma et al.

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LIPUS ameliorates cardiac diastolic dysfunction

3.2 Lipus therapy ameliorates cardiac
diastolic dysfunction in db/db mice
Echocardiographic study showed that, in both db/db-control and db/dbLIPUS mice, LV wall was slightly thicker than in db/þ-control mice, although no difference was noted between the two db/db mouse groups(Supplementary material online, Figure S2B and C). Also, there was no significant difference in LV end-diastolic dimension (LVDd) among the fourgroups (Supplementary material online, Figure S2D). Regarding of calculated LV mass, there was a trend of increase, but not significant, in db/dbcontrol group compared with db/þ-control mice. On the other hand,
this change was tend to be suppressed in db/db-LIPUS mice comparedwith db/db-control mice (Supplementary material online, Figure S2E).
Left atrial dimension was significantly larger in both db/db mice than in
db/þ-control mice, but no difference was noted between db/db-control
and db/db-LIPUS mice (Supplementary material online, Figure S2F).
Regarding the parameters of cardiac systolic functions, there was no

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significant difference in LVEF or LV fractional shortening among the fourgroups, suggesting that cardiac systolic function was preserved in db/db
mice (Supplementary material online, Figure 2G and H). In contrast, db/
db-control mice showed lower e0 and higher E/e0 than db/þ-control
mice, indicating impaired cardiac diastolic function. Importantly, e0 and E/
e0 were significantly ameliorated in db/db-LIPUS mice (Figure 2A and B).
For further detailed examinations of cardiac functions, we also performed invasive haemodynamic evaluations. Consistent with the resultsof echocardiography, db/db-control mice, when compared with db/þmice, showed diastolic dysfunction characterized by elevated LV enddiastolic pressure (LVEDP), prolonged tau, reduced negative dP/dt, andincreased slope of EDPVR (Figure 2C and D). Despite marked diastolic
dysfunction in db/db-control mice, conventional systolic function parameters, such as stroke work, cardiac output, and positive dP/dt, were comparable among the four groups (Supplementary material online, Figure
4A, B, and F). Importantly, the LIPUS therapy markedly improved

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Figure 2 LIPUS therapy significantly improves LV diastolic dysfunction in db/db mice. (A) Representative echocardiographic mitral inflow pattern with
Doppler imaging (scale bars, 25 ms and 10 mm/s) and septal mitral annular velocity with tissue Doppler imaging (scale bars, 20 ms and 200 mm/s) at 20weeks-old. (B) Graphs showing the time-course of E/A ratio, mitral e0 , and E/e0 ratio. (C) Representative LV pressure–volume loops with vena cava occlusion at 20-weeks-old. (db/þ-control: n = 6, db/þ-LIPUS: n = 6, db/db-control: n = 15, db/db-LIPUS: n = 15). (D) Graphs showing end-diastolic pressure(EDP), time constant of LV pressure decay (tau), dP/dt min, and slope of the end-diastolic pressure–volume relationship (EDPVR) (db/þ-control: n = 6,
db/þ-LIPUS: n = 6, db/db-control: n = 15, db/db-LIPUS: n = 15). Results are expressed as mean ± SD. Comparisons of parameters were performed withANOVA followed by Tukey’s test for multiple comparisons, or non-parametric multiple comparison test. Differences with a P-value <0.05 were considered to be statistically significant.

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Y. Monma et al.

diastolic function parameters, such as EDP, tau, negative dP/dt, and theslope of EDPVR (Figure 2C and D).

3.3 Lipus therapy improves exercise
tolerance and decreases serum levels of
BNP in db/db mice
Although the treadmill running distance was significantly shorter in db/db
mice than in db/þ mice, work load was not significantly different amongfour groups at 12-weeks-old (Figure 3A and Supplementary material online, Figure S5A). Throughout the experimental period, work load
remained unchanged in db/þ mice (Figure 3A). However, work load was
reduced with ageing in db/db mice, which suggested reduced exercise
capacity (Figure 3A). At 16- and 20-weeks-old, the distance and work
load was significantly larger in db/db-LIPUS mice than in db/db-control
mice, indicating the improvement of exercise capacity (Figure 3B). In addition, regarding of serum BNP levels, db/db mice tended to increase thandb/þ mice (Figure 3C). On the other hand, serum BNP levels were significantly lower in in db/db-LIPUS group (111.9 ± 36.8 pg/mL) than db/dbcontrol mice (152.4 ± 89.7) at 20-weeks-old in the comparison with unpaired Student’s t-test (P < 0.05).

..
.. 3.4 Lipus therapy suppresses myocardial.. hypertrophy and interstitial fibrosis..
.. Histologically, in db/db mice, heart weight (HW), and HW/tibial length..
.. (TL) ratio were significantly greater compared with db/þ-control mice.
.. In db/db-LIPUS mice, HW and HW/TL ratio significantly decreased com..
.. pared with db/db-control mice (Supplementary material online, Figure.. S3B and C). Regarding glucose tolerance, db/db mice showed hypergly..
.. caemia and hyperinsulinaemia, but there was no difference between db/..
.. db-control and db/db-LIPUS mice (Supplementary material online, Figure.. S3E and F). H&E staining showed that myocardial cross-sectional area..
.. (CSA) was significantly increased in db/db-control mice compared with.. db/þ-control mice (Figure 4A and B). Importantly, in db/db-LIPUS mice,..
.. CSA significantly decreased compared with db/db-control mice (Figure.. 4A and B), indicating that the LIPUS therapy suppressed myocardial hy..
.. pertrophy in those mice. Masson trichrome staining showed that myo..
.. cardial interstitial fibrosis was advanced in db/db-control mice compared.. with db/þ-control mice (Figure 4A and C) and was also slightly but signifi..
.. cantly suppressed in db/db-LIPUS mice compared with db/db-control.. mice, suggesting that the LIPUS therapy suppressed the fibrotic remodel..
. ling in those mice (Figure 4A and C).

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Figure 3 LIPUS improves exercise tolerance and decreases serum BNP levels in db/db mice. (A) Graphs showing the time-course of exercise capacity(work load) measured by treadmill test from 12- to 20-weeks-old (db/þ-control, n = 6; db/þ-LIPU, n = 6; db/db-control, n = 15; db/db-LIPUS, n = 15).
(B) In the LIPUS group, work load was fairly preserved compared with the control group at 16- and 20-weeks-old (db/db-control, n = 15; db/db-LIPUS, n= 15). (C) Graph showing the serum levels of BNP at 20-weeks-old (db/þ-control: n = 7, db/þ-LIPUS: n = 7, db/db-control: n = 20, db/db-LIPUS: n = 20).
Results are expressed as mean ± SD. Comparisons of parameters were performed with unpaired Student’s t-test, or Wilcoxon rank sum test.
Differences with a P-value <0.05 were considered to be statistically significant.

LIPUS ameliorates cardiac diastolic dysfunction

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stained with haematoxylin–eosin (H&E, upper, scale bars, 100 lm) and Masson-trichrome (MT, lower, scale bars, 1 mm) at 20-weeks-old. (B)Quantitative analysis of cross-sectional area (H&E staining). (C) Quantitative analysis of myocardial fibrosis area (MT staining) (db/þ-control: n = 6, db/þLIPUS: n = 6, db/db-control: n = 15, db/db-LIPUS: n = 15). Results are expressed as mean ± SD. Comparisons of parameters were performed with oneway ANOVA followed by Tukey’s multiple comparisons, or non-parametric multiple comparison test. Differences with a P-value <0.05 were considered
to be statistically significant.

3.5 Effects of the LIPUS therapy on
capillary density and coronary blood flow
Recently, coronary microcirculatory dysfunction is recognized to play animportant role in the pathophysiology of HFpEF.7,24 It has been proposed that microvascular rarefaction and microvascular endothelial dysfunction could impair oxygen supply to the myocardium and promote
myocardial hypertrophy and fibrosis, resulting in cardiac diastolic dysfunction.25 Thus, we examined whether cardiac diastolic dysfunction in
db/db mice is associated with myocardial ischaemia and/or with alternation in coronary microcirculation, and if so, whether the LIPUS therapy
could improve it. Histological analysis showed that there was no significant difference in capillary density among the four groups(Supplementary material online, Figure S7A and B). We further examinedthe extent of myocardial ischaemia by hypoxyprobe staining and foundno significant difference among the four groups (Supplementary material
online, Figure S7A and C). These results indicated that db/db mice did notdisplay myocardial microvascular rarefaction or myocardial ischaemia
and that LIPUS did not affect them.
In addition, in order to examine the effects of the LIPUS therapy on
coronary microcirculation, we performed Langendorff experiments, in.. which we measured coronary blood flow in response to endothelium..
.. dependent and -independent vasodilators. At baseline, there was no sig..
.. nificant difference in coronary blood flow between db/þ mice and db/db.. mice and LIPUS did not have significant effect on coronary blood flow in..
.. db/db mice (Supplementary material online, Figure S8B). However,.. endothelium-dependent increase in coronary flow in response to BK..
.. tended to be decreased in db/db-control mice compared with db/þ.. mice, which was ameliorated in db/db-LIPUS group (Supplementary ma..
.. terial online, Figure S8B). In contrast, endothelium-independent increase..
.. in coronary flow in response to SNP was comparable among the three.. groups (Supplementary material online, Figure S8C).
..
..
.. 3.6 Lipus therapy improves relaxation..
.. properties of isolated myocardial tissue in..
.. db/db mice..
.. It was previously reported that Ca2þ handling system is impaired in dia.. betic heart with resultant impairment of relaxation capacities at both..
.. myocardial tissue and cardiomyocyte levels.26 To evaluate the effects of.. LIPUS on myocardial relaxation capacities and Ca2þ flux at a tissue level,..
. we examined force and intracellular Ca2þ concentration ([Ca2þ]i) in the

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Figure 4 LIPUS therapy attenuates cardiomyocyte hypertrophy and interstitial fibrosis in db/db mice. (A) Representative images of myocardial tissues

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trabeculae dissected from the right ventricle during electrical stimulation.
There was no difference in developed force, peak [Ca2þ]i, or positive
peak derivative forces (dF/dt max) between db/db-control and db/dbLIPUS mice, indicating that LIPUS had no effect on myocardial contractility (Supplementary material online, Figure S6A–C). In contrast, negative
peak derivative force (dF/dt min) and relaxation time to 50% relaxation(RT50%) were significantly increased in db/db-LIPUS mice comparedwith db/db-control mice group, indicating that the LIPUS therapy improved relaxation ability of diabetic myocardial tissue (Figure 6C and D).
Consistently, the trabeculae from db/db-LIPUS mice showed reduced
time constant of [Ca2þ]i decay compared with db/db-control mice
(Figure 6E), whereas there was no significant difference in diastolic[Ca2þ]i (Supplementary material online, Figure S6E). These results indicate that the LIPUS therapy ameliorates myocardial relaxation property
but not contractility.

3.7 Effects of the LIPUS therapy
on Ca2þ-handling proteins
In order to elucidate the molecular mechanisms of the beneficial effectsof LIPUS on relaxation properties and Ca2þ cycling at myocardial tissue..
.. level, we examined the protein expressions and activity of Ca2þ-handling.. molecules in the myocardium, including sarcoplasmic reticulum Ca2þ..
.. ATPase (SERCA2a) and phospholamban (PLN). The expression level of.. phosphorylated PLN at Ser16, but not that at Thr17, was significantly..
.. lower in db/db-control than in db/þ-control mice, and the phosphoryla..
.. tion level tended to be restored in db/db-LIPUS mice (Figure 6A–C). In.. contrast, there was no difference in the expression of PLN among the..
.. four groups (Figure 6D). Additionally, the expression of SERCA2a was.. significantly up-regulated in db/db-LIPUS mice compared with db/db..
.. control mice (Figure 6E). The ratio of SERCA/PLN was also significantly.. increased in db/db-LIPUS mice compared with db/db-control mice..
.. (Figure 6F).
..
..
.. 3.8 Effects of the LIPUS therapy on the..
.. expression and phosphorylation of..
.. sarcomeric proteins.. Recent studies have shown that the expression and function of sarco..
.. meric proteins are closely related to myocardial diastolic function.27 In.. order to examine the effects of the LIPUS therapy on sarcomeric pro..
. teins such as TnI and MyBP-C, we examined the expression and

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Figure 5 LIPUS therapy improves relaxation properties of isolated myocardial tissue in vitro. (A and B) Representative tracings of force and intracellularCa2þ ([Ca2þ]i) by 0.5 Hz electrical stimulation (22 C, extracellular Ca2þ = 4.0 mmol/L) in the trabeculae dissected from the right ventricles at 20-weeksold. (C–E) Graph showing relaxation rate (dF/dt min), relaxation time to 50% relaxation (RT50%), and time constant of [Ca2þ]i decay (n = 4) trabeculae
per group (one trabeculae per one mouse). Results are expressed as mean ± SD. Comparisons of parameters were performed with unpaired Student’s ttest, or Wilcoxon rank sum test. Differences with a P-value <0.05 were considered to be statistically significant.

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LIPUS ameliorates cardiac diastolic dysfunction

lamban (PLN) phosphorylation (Ser16/Thr17), total PLN, SERCA2a, and SERCA2a/PLN ratio in mice (db/þ-control: n = 3, db/þ-LIPUS: n = 3, db/dbcontrol: n = 9, db/db-LIPUS: n = 9). Results are expressed as mean ± SD. Comparisons of parameters were performed with one-way ANOVA followed
by Tukey’s multiple comparisons, or non-parametric multiple comparison test. Differences with a P-value <0.05 were considered to be statisticallysignificant.

phosphorylation of those proteins. There was no significant difference inthe expression of TnI nor MyBP-C among the four groups(Supplementary material online, Figure S10B and D). Regarding the phosphorylation, there was no difference in the expression level of phosphorylated TnI among the four groups (Supplementary material online, Figure
S10C). In addition, although the expression level of phosphorylated
MyBP-C tended to increase in db/db mice compared with db/þ mice,
there was no significant difference among the four groups(Supplementary material online, Figure S10D).

3.9 Lipus therapy up-regulates the eNOSNO-cGMP-PKG signalling pathway
To further elucidate the underlying molecular mechanisms of the beneficial effects of the LIPUS therapy on cardiac diastolic functions, we performed Western blotting for several proteins. The LIPUS therapy had
no effects on the eNOS-NO-cGMP-PKG signalling pathway in nondiabetic mice (Figure 7A–F). Importantly, the LIPUS therapy significantly
increased myocardial level of phosphorylated eNOS in db/db-LIPUS
mice than in db/db-control mice (Figure 7C). Furthermore, the LIPUS
therapy significantly up-regulated the expression of the downstream

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molecules, such as soluble guanylate cyclase (sGC) and PKG I, in db/db
mice (Figure 7D–F). In addition, we performed indirect measurement of
NO by measuring NOx (NO2þNO3) level in the myocardium.
Although db/db mice showed lower levels of NOx level compared withdb/þ mice, there was no significant difference between db/db-LIPUS
mice and db/db-control mice (Supplementary material online, Figure S9).

4. Discussion
The novel findings of the present study were that the LIPUS therapy
markedly improved cardiac diastolic dysfunction and exercise tolerance
without any adverse effects in obese db/db mice with Type 2 DM, forwhich enhancement of the eNOS-NO-cGMP-PKG signalling pathway
and improvement of myocardial Ca2þ-handling proteins are involved(Graphical abstract). To the best of our knowledge, this is the first reportdemonstrating that the LIPUS therapy is effective and safe to improve
cardiac diastolic function in a mouse model of DM/obesity-related
HFpEF. The present results suggest that the LIPUS therapy is a promising,novel, and non-invasive therapy for cardiac diastolic dysfunction in
HFpEF patients.

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Figure 6 LIPUS therapy improves the expressions of Ca2þ handling-related proteins. (A–E) Representative western blot and quantification of phospho-

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Y. Monma et al.

4.1 Cardiac diastolic dysfunction in db/db
mice
HFpEF is closely associated with LV diastolic dysfunction, which can becaused by increased LV stiffness due to hypertrophy and interstitial fibrosis and abnormal LV relaxation due to impaired calcium cycling.3 HFpEF
patients often have various comorbidities with endothelial dysfunction,
where DM is one of the most frequent comorbidities.28 Indeed, it has
been reported that about 45% of HFpEF patients have DM, with highest
prevalence noted in those with new-onset HFpEF.2,29 It also has beenreported that DM itself has a close correlation with cardiac diastolic dysfunction in diabetic patients.30 In addition, DM is associated with adverse
cardiovascular outcomes in HFpEF patients.31 Thus, the pathophysiologyof DM-associated HFpEF remains to be fully elucidated for developmentof a novel therapeutic strategy.
Metabolic stress due to DM and obesity is thought to be one of the
major mechanisms underlying HFpEF pathophysiology.6 db/db mice arean established animal model of obese-related Type 2 DM due to leptin
receptor mutations.32 They exhibit LV diastolic dysfunction accompanied by LV hypertrophy and fibrosis and preserved LV systolic function.14,33 Since such features are also noted in patients with HFpEF anddiabetic patients, db/db mice are widely used as a useful animal model of.. HFpEF.32 In the present study with db/db mice, we also observed the..
.. typical phenotypes of cardiac diastolic dysfunction, including significant..
.. increase in LV stiffness and prolonged relaxation time in pressure–vol.. ume analysis, accompanied by morphological changes, such as myocar..
.. dial hypertrophy and interstitial fibrosis, while cardiac systolic.. performance was preserved. In addition, we observed a reduced exer..
.. cise tolerance in db/db mice, which is closely related to dyspnoea on ex..
.. ertion, the cardinal HFpEF symptom.34..
..
.. 4.2 Lipus therapy improved cardiac..
.. diastolic function and exercise capacity.. In the present study, the LIPUS therapy ameliorated cardiac diastolic dys..
.. function as confirmed by echocardiography and invasive hemodynamic.. evaluation. Furthermore, at tissue level, we were able to demonstrate..
.. that LIPUS improved myocardial relaxation property. Such improvement..
.. of LV diastolic performance would contribute to decreases in LV filling.. pressure and serum BNP levels without decrease in systemic blood..
.. pressure.
..
More importantly, we showed that the improvement of cardiac func..
.. tion as described above has led to the improvement of exercise toler.. ance. Patients with HFpEF are characterized by reduced ‘reserve

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Figure 7 LIPUS therapy activates the eNOS-NO-cGMP-PKG signalling pathway. (A–F) Representative western blot and quantification of eNOS, phosphorylaed eNOS at Ser1177, soluble guanylated cyclase a (sGCa), sGCb, and protein kinase G I in the mouse heart at 16-weeks-old. (db/þ-control: n =3, db/þ-LIPUS: n = 3, db/db-control: n = 9, db/db-LIPUS: n = 9) Results are expressed as mean ± SD. Comparisons of parameters were performed with
one-way ANOVA followed by Tukey’s multiple comparisons, or non-parametric multiple comparison test. Differences with a P-value <0.05 were considered to be statistically significant.

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LIPUS ameliorates cardiac diastolic dysfunction

capacity’.3,34 Indeed, although they are often asymptomatic at rest, undersome stress conditions (e.g. exercise), LV filling pressure and pulmonary
venous pressure increase, leading to HF symptoms (e.g. shortness ofbreath). Thus, the evaluation of exercise capacity is important in the evaluation of HFpEF patients.34 In the present study, the LIPUS therapy improved exercise tolerance of db/db mice, indicating its potential
usefulness for HFpEF patients.

We have previously demonstrated that LIPUS stimulates endothelial
cells and enhances eNOS activity.11–13 In endothelial cells, eNOS localizes in the caveolae of the endothelial membrane, which is known to beflow-sensing organelles and converts mechanical stimuli (e.g. blood flowinduced shear stress) into chemical signals.11,35 Cyclic strain induced byultrasonic pulse waves is also sensed by the caveolae, leading to eNOS
activation.11,36 Indeed, in the present study, we demonstrated that the
LIPUS therapy activates eNOS and up-regulates downstream molecules
such as sGC and PKG, indicating that LIPUS activates the eNOS-NOcGMP-PKG pathway.
Cardiac diastolic function is determined by myocardial stiffness associated with myocardial hypertrophy and fibrosis and relaxation ability associated with myocardial Ca2þ flux. The eNOS-NO-cGMP-PKG
pathway is closely related to myocardial stiffness. It was shown that modulation of the sGC-PKG pathway with sildenafil was effective to suppress
myocardial remodelling through inhibition of myocardial hypertrophy
and interstitial fibrosis in vivo.37 Hamdani et al. reported that pharmacological up-regulation of the myocardial cGMP-PKG pathway attenuates
myocardial hypertrophy and fibrosis, leading to significant improvementof cardiac diastolic dysfunction in a mouse model of DM-related
HFpEF.14 In addition, other previous reports showed that the enhancement of the eNOS-NO-cGMP-PKG pathway suppressed myocardial hypertrophy and interstitial fibrosis.14,38,39 In the present study, the LIPUS
therapy suppressed myocardial hypertrophy and interstitial fibrosis, andsubsequently improved LV stiffness in the hearts of db/db mice throughthe enhancement of the eNOS-NO-cGMP-PKG pathway.
On the other hand, the eNOS-NO-cGMP-PKG pathway is closely related to myocardial relaxation ability.40 Active relaxation is determinedby energy-dependent removal of Ca2þ from the cytosol, and the rate ofcytosolic Ca2þ clearance during diastolic phase determines the patternof muscle relaxation.41 This process of dynamic change in cytosolic Ca2þis known to be regulated by Ca2þ handling-related proteins, such as
SERCA2a and PLN.41 Several studies using rodent models of HF demonstrated that restoring the eNOS-NO-cGMP-PKG pathway could improve myocardial Ca2þ handling and its molecular modulation withresultant therapeutic effects on cardiac function.40 It also has beenreported that cardiomyocyte-specific PKG-KO mice showed attenuatedexpression of SERCA2a and PLN and diminished PLN phosphorylation
at Ser16, the target site for PKG activation.42 In the present study, wedemonstrated that the LIPUS therapy improves LV active relaxation
through ameliorating the Ca2þ clearance ability and the relaxation rateof myocardial tissue. Consistently, the expression and activation of
Ca2þ-handling molecules, such as SERCA2a and PLN, were significantly
enhanced along with the activation of the eNOS-NO-cGMP-PKG pathway. In contrast, although sarcomeric proteins (e.g. TnI and MyBP-C) areclosely related to myocardial relaxation properties,27 the LIPUS therapy
had no effect on their expression or phosphorylation. The present study

provides the first evidence that the LIPUS therapy improves Ca2þ handling system and myocardial relaxation property.

4.4 Effects of LIPUS on angiogenesis andcoronary microcrocirculation in db/db
mice
We previously demonstrated in several HF animal models that the cardioprotective effects of the LIPUS therapy are mediated in part throughenhanced angiogenesis.10–12 However, in the present study, no obvious
angiogenic effects were noted. This is probably because capillary density
was not reduced and there was no myocardial ischaemia in db/db heart(Supplementary material online, Figure S7). This finding is consistent with
our previous finding that the LIPUS therapy promotes angiogenesis onlyat the ischaemic myocardium but not at the normal myocardium in
mice.11 These results may represent the unique property of the LIPUS
therapy that enhances an endogenous self-healing ability only in diseased
tissues but not in normal tissues. Indeed, we have repeatedly noted thisunique property of the LIPUS therapy, where increased susceptibility of
diseased tissue to the cell surface molecules (e.g. caveolin-1) may be involved.11–13
In the present study, coronary microvascular endothelial function
tended to be decreased in db/db mice and the LIPUS therapy improved
it. It has been recently proposed that coronary microvascular dysfunction is closely related to cardiac diastolic dysfunction in HFpEF.7 Since
endothelial eNOS system plays an important role in regulating coronary
microcirculation, up-regulated eNOS and its downstream pathway in response to LIPUS could improve microvascular function and ameliorate
cardiac diastolic dysfunction. Indeed, in the present study, we demonstrated that LIPUS up-regulates eNOS and downstream molecules, suchas sGC and PKG, but we were unable to detect significant increase in
NO production in the myocardium (Supplementary material online,
Figure S9). This suggests that other mechanisms could also contribute to
the LIPUS-induced improvement of coronary microcirculation. We previously demonstrated that the eNOS system serves as the NO generating system in relatively large arteries, while it serves as the generating
system of endothelium-dependent hyperpolarization (EDH) in microcirculation43 and that EDH plays a crucial role in coronary microcirculationin vivo.44 Recently, we also have demonstrated that EDH plays importantroles in maintaining cardiac diastolic function in vivo.23 Taken together, it
is conceivable that the beneficial effect of LIPUS on cardiac diastolic dysfunction may be mediated, at least in part, through EDH up-regulation incoronary microcirculation.

4.5 Clinical implications
HFpEF is the most common type of HF and has been emerging as one of
the most serious health problems worldwide.3 Many clinical trials for
HFpEF have failed to establish the effective therapy to improve the prognosis of HFpEF patients.1,3 In the present study, based on our previous
experiences with the LIPUS therapy,10–12 we were able to demonstratethat our LIPUS therapy improves cardiac diastolic dysfunction and exercise tolerance in a mouse model of HFpEF. The power of LIPUS is controlled to keep the level lower below the upper limit of acoustic output
standards (<720 mW/cm2) for diagnostic ultrasound devices (US Foodand Drug Administration’s Track 3 Limits). In addition, since LIPUS directly emits low-intensity ultrasound to the heart from outside of thebody, potential focal or systemic adverse effects should be minimal.
Indeed, in the present study, no adverse effects were noted. In addition,
we have previously demonstrated the efficacy and safety of our LIPUS

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4.3 Mechanism of the beneficial effects of
LIPUS therapy on cardiac diastolic function

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therapy with similar irradiation setting in a large animal (pig) model ofchronic myocardial ischaemia.10 Since HFpEF patients are often have various comorbidities, this minimally invasive feature of the LIPUS therapy
appears to be important, suggesting that the LIPUS therapy could be anovel promising treatment for HFpEF patients.

4.6 Study limitations..
.. should be paid when extrapolating the present finding to the left ven.. tricle physiology. Eighth, in the present study, we evaluated exercise..
.. tolerance (capacity) using a work load calculated from treadmill run.. ning distance and body weight. However, for more appropriate evalua..
.. tion of exercise capacity, it would be desirable to measure other.. parameters such as maximal oxygen uptake (VO max) and anaerobic
2..
.. threshold (AT) by cardiopulmonary exercise testing (CPX) using an..
.. expired gas analyser. Ninth, since db/db mice had severe obesity with.. body weight about twice as much as control mice, this must be taken..
.. into account in the evaluation of some parameters. For example, in.. echocardiographic and haemodynamic analysis, correction by body..
.. surface area or body weight may be desirable. In addition, regarding..
.. serum BNP levels, the comparison between db/db mice and db/þ.. mice may not be simple because serum BNP levels are known to be..
.. lower in obese mice (e.g. db/db mice), just like in humans, in general.50.. Thus, in the present study, db/db mice may have lower BNP levels in..
.. comparison to the degree of cardiac dysfunction. Tenth, in the mouse.. trabeculae experiment, we used 4.0 mM of extracellular [Ca2þ], which..
.. was relatively higher than that of actual condition. Although we..
.. adopted this preparation in order to make it easier to evaluate the.. property of contraction and relaxation of the myocardium, physiologi..
.. cal conditions have not been completely replicated.
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.. 5. Conclusions..
..
.. In the present study, we were able to demonstrate that the LIPUS ther.. apy ameliorates cardiac diastolic dysfunction in db/db mice through im..
.. provement of the eNOS-NO-cGMP-PKG pathway and cardiomyocyte..
.. Ca2þ-handling system without any side effects. Our results suggested.. that LIPUS may be a promising, new, non-invasive strategy for the treat..
.. ment of HFpEF, for which there is no established therapy available.
.. However, this concept has to be confirmed in humans.
..
..
..
..
.. Data availability..
.. The data underlying this article will be shared on reasonable request to..
.. the corresponding author.
..
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..
..
.. Supplementary material..
.. Supplementary material is available at Cardiovascular Research online.
..
..
..
..
.. Authors’ contributions..
.. Y.M. performed most experiments and wrote the manuscript. T.S. and..
.. H.S. supervised the project and edited the manuscript. K.E., R.K., Y.K.,.. Y.I., S.I., T.N., A.M., H.S., M.M., and H.K. designed or conducted experi..
.. ments. S.M. performed analysing data.
..
..
..
.. Acknowledgements..
.. We appreciate Y. Watanabe, H. Yamashita, and A. Nishihara for excel..
.. lent technical supports.
..
. Conflict of interest: none declared.

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Several limitations should be mentioned for the present study. First, inthe present study, we showed the beneficial effect of the LIPUS therapy only in db/db mice, a mouse model of HFpEF associated withType 2 DM.32 Although DM is one of the most frequent comorbidities
of HFpEF patients, there are a number of other background comorbidities that may contribute to the pathogenesis of HFpEF (e.g. hypertension, CKD, and chronic obstructive pulmonary disease). Thus, itremains to be examined whether our LIPUS therapy is also beneficial
for other types of HFpEF. Second, in the present study, we showed
the effectiveness of the LIPUS therapy in a 4-week treatment protocol.
It is possible that the longer therapeutic period with the LIPUS therapy
would achieve greater efficacy. This issue also remains to be examinedin future studies. Third, in the present study, although we focused on
the eNOS-NO-cGMP-PKG pathway as a main mechanism of the
LIPUS-induced beneficial effects, other mechanisms may also be involved. Especially, regarding the antihypertrophic and antifibrotic effectof the LIPUS, its effects on neurohormonal factors, such as the renin–
angiotensin–aldosterone system (RAAS), may possible.45 Suppressionof the RAAS may have attenuated LV remodelling (hypertrophy and fibrosis) and contributed to the reduction in LVEDP as confirmed byhemodynamic analysis, resulting in a reduction in serum BNP levels.
This point remains be examined in future studies. Fourth, in the present study, although we demonstrated that the LIPUS therapy improved myocardial stiffness associated with suppression of myocardial
hypertrophy and interstitial fibrosis, other factors, such as titin, mayalso be involved. Titin is a protein anchored to the sarcomere Z-linethat serves as a major determinant of myocardial passive tension andstiffness.46 The spring property is defined by the expression ratio of
the cardiac isoforms N2B and N2BA and is modulated by its phosphorylation. It is known that PKG phosphorylates a target site of theN2B element, resulting in reduced passive tension.47 Thus, in thisviewpoint, it is possible that the LIPUS therapy would modulate thetitin property by enhancing the eNOS-NO-cGMP-PKG pathway. This
point remains be examined in future studies. Fifth, although LIPUStreated db/db mice showed some evidence of reduced cardiac load(e.g. decreased LVEDP and lower levels of serum BNP), unexpectedly,
it did not affect left arterial dimension evaluated by echocardiography(Supplementary material online, Figure S2D). This is possibly because
the measurement was done only from parasternal long axis view,which is not necessarily appropriate method for evaluation of left atrial
volume.48 Other measurement modalities such as magnetic resonanceimaging could provide more accurate measurement on left atrial dimension. Sixth, in invasive haemodynamic analysis, an open thorax
preparation was used without fluid replacement. This might have led
to lower performances and lower end-diastolic pressures in mice, although a series of measurements were performed under the same
conditions in all animals. Seventh, in the present study, we demonstrated that LIPUS improves relaxation property at tissue level in theexperiments using mouse trabeculae from the right ventricle.
Although this technique with the right ventricular trabeculae has been
widely used as an established experimental method to evaluate theelectrophysiological properties of myocardial tissue,17–19,49 caution

Y. Monma et al.

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LIPUS ameliorates cardiac diastolic dysfunction

Funding
This research was supported by the Japan Society for the Promotion of
Science (JSPS) (Grant No. 18K15877).

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Translational perspective
Although heart failure with preserved left ventricular ejection fraction (HFpEF) is a serious health problem worldwide, no effective treatment is yet
available. We have previously demonstrated that our low-intensity pulsed ultrasound (LIPUS) therapy is effective and safe in animal models of angina
and dementia. In this study, we examined whether our LIPUS therapy is also effective to improve cardiac diastolic dysfunction in mice. We foundthat the LIPUS therapy ameliorated myocardial structures and Ca2þ-handling proteins, resulting in the improvement of cardiac diastolic functions
and exercise tolerance in a mouse model of HFpEF. These results suggest that the LIPUS therapy is useful for the treatment of HFpEF in humans.

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