JMCC Mitsuy αmα etα'l.

Left ventricular mechanical and energetic changes in long-term isoproterenol-induced

hypertrophied hearts of SERCA2a transgenic rats

Shinichi Mitsuyama 1

へDaisuke Takeshita 1

，a， Koji Obata 1

へGuo-Xing Zhang 1

，2， Miyako Takaki 1

lDepar 防lent of Physiology II， Nara Medical University School of Medicine ， 840 Shijo ・cho ，

Kashihara ， Nara 634 ・8521 ，Japan

2Department of Physiology ， Medical College of Soochow University ， Dushu Lake Campus ，

Suzhou Industrial Park ， Suzhou 215123 ラP.R. China

aτnese authors con 仕ibuted equally to this wor k.

Author contributions:

Miyako Takaki designed and supervised the present sωdy. Shinichi Mitsuyama ， Daisuke

Takeshita ， Koji Obata and Miyako Takaki conducted experiments and obtained the data. Koji

Obata and Guo-Xing Zhang established the transgenic rats. Shinichi Mitsuyama and Miyako

Takaki 組 alyzed the data and drafted the manuscrip t.

Running Head: Cardiac hyper 仕ophy 加 SERCA2a transgenic rats

Corresponding author: Pro 王Miyako Takaki ， Ph.D.

Dep ぽ加lents of Physiology 11 and Molecular Pathology ， Nara Medical University School of

Medicine ， 840 Shijo ・cho ，Kashihara ， Nara 634-8521 ， Japan

Email: mtakaki@naramed ・u.ac.jp ，Tel: +81 ・744 ・29・8829 ラFax: 十81・744 ・23・4696

JMCC Mitsuyam αet α1. 2

Abstract

Overexpression of cardiac sar ∞plasmic reticulum Ca2+ -ATPas 巴(SERCA2a) has been suggested as

as回旬gic intervention 伽 cardiac 白il町巴. However ， its benefit in wild-type (WT) 凶 swith normal

SERCA2a 1ev 巴ls seems to be small. To investigate whether it would be beneficial in a cardiac

failure mode1 with down-regulat 怠dSERCA2a 1evels ， we made a cardiac hyper 仕ophy model using

isoprotereno1 infusion (1. 2 mg kg. 1 day.l for 1 or 4 w田ks; TG-IS01w and TG)S04W ，

respective1y) in SERCA2a transgenic (TG) 削 sand compared these rats with littermate WT rats

that underwent the same treatments (WT-ISOlw and WT・.IS04w). We ana1yzed the 1eft

ventricu1ar (L V) mechanoenergetics inthe excised hear :t using our origina1 cross-circu1ation

system. The downward shift of curvilinear L V end-systolic pressur かv01ume re1ations (ESPVRs)

observed in WT-IS04w rats was ab01ished 也TG-IS04w rats. The s10pe and V02 intercept of the

V02 (myocardia1 oxygen consumption per beat)-PVA (systolic pressure-vo1ume area: tota1

mechanica1 energy per beat) 1inear re1ation did not differ 泊 anv oftheεroups. 百lemost 加lportant

白lding was a significan t1y smaller O2 cost of L V co曲四ti1ity in the TG-IS04w group ， which

means that 1ess O2 is needed to exert the same LV contracti1ity ， compared with the other groups.

百le increased ratio of SERCA2a/phosph01amban r凶lmed to the 1eve1 ofせle WT-con 仕01 group

on1y in fue TG-IS04w group. Longer- 匂四1up-regu1ation of mitochondria1 仕組scription factor A

for genes of mitochondria1 enzymes producing ATP was observed in TG rats. In con c1usion ，

10nger-term overexpression of SERCA2a wi11 be beneficia1 in the present cardiac fai1ure mo白l

wifu down-regu1ated SERCA2a 1eve1s.

Key words: heart fai1ure ， ci+ handling ， oxygen consumption ， excitation. ‘contraction coupl 加g

JMCC Mitsuyama etα!/. 3

1. Introducfion

Heart failure is one of the m吋or causes of mortality and mo巾idity. It can be the result of

various diseases ， and cardiac hypertrophy is a major risk factor for the development of heart

failure. Hype 抗rophy in江ially compensates for a lack of cardiac function but ultimately leads to

heart failure [1]. Meanwhile ， calcium (Ca 2+) handling is closely related to the

contraction-relaxation cycle of the myocardium and to cardiac function [2， 3]. Cardiac

sarcoplasmic reticulum Ca2+回ATPase (SERCA2a) plays a key role in Ca2+ homeostasis [2].

Cardiac dysfunction is related to defective sarcoplasmic reticulum Ca2+ uptake caused by a

SERCA2a deficiency [2， 4-6]. Accordingly ， overexpression of SERCA2a has been suggested as a

strategic therapeutic approach for heart failure ， and many studies have been reported on its use [4，

6-15].

Previous reports have shown improved myocardial function and 加lproved energetic

consumption as a result of a gene transfer of SERCA2a under short-term conditions [8， 9， 12， 13].

However ， one report showed that excessive SERCAla expression caused a decrease in myocyte

shortening in normal rabbit hearts [14]. The effect of a high basal level of SERCA2a has been

assessed using 廿ansgenic SERCA2a-overexpressing rodents [7， 10， 11， 15・17]. However ， little is

known about the effects of a high basal SERCA2a level on whole 回heart mechanics and energetics ，

and there have been no deta i!ed quantitative analyses of changes in Ca2十

handling in

excitation-contraction (E圃C)coupling at a whole-heart level.

To assess the effect of longer-term overexpression of SERCA2a ， we recently established

SERCA2a 廿ansgenic Wistar rats and evaluated the left ven 甘icular (L V) mechanics and energetics

in an excised ， blood-perfused whole-heart preparation with a cross-circulation system [18]. 百1I s

system is designed to perform deta i!ed quantitative analyses of changes in the efficiency of

cross-bridge cycling and Ca2十

handling in E-C coupling at a whole-heart level through indirect

measurements [19]. However ， there were no distinct beneficial effects on LV mechanics or

energetics in normal SERCA2a transgenic rats ， although the tolerance to high Ca2+ w出 greater

than that found in normallittermate wild 句perats [18].

百le aim of the present study was to investigate whether longer-term SERCA2a

overexpression would be beneficial in a cardiac failure model with down-regulated SERCA2a

JMCC Mitsuy αmα 芭tα'1. 4

levels [20]. We previously analyzed a short 却 rm (3 days and 1 week) isoproterenol-induced

cardiac compensatory hypertrophy model in normal Wistar rats [21]. In this short-term

hypertrophy model ， changes in LV mechanical work and energetics were minimal [21]. and

analyzed L V mechanics and energ 巴tics (various kinds of oxygen consumption ， such as

consumption for Ca2+ handling) for L V con 仕action and relaxation 加 these models by using our

original cross-circulation system. 百le functioning of an A TPase such as SERCA2a is highly

dependent on the supply of A TP. Therefore ， A TP production in mitochondria is closely related to

Ca2+ handling in cardiac function [22].

2. Methods

This investigation conformed to the “Guide for the Care and Use of Laboratory An耐lals"

published by the National Institutes of Health (NIH Publication No. 85・23，revised 1996) and was

approved by the Animal Care and Use Committee ofNara Medical University.

2.1. Animal models

We established a strain of SERCA2a ・overexpressing transgenic (TG) Wistar rats as previously

reported in detail [15 ， 18]. Male littermate wild 吋pe (WT) Wistar rats (n = 27) and male TG rats

(n = 21) weighing 325 土 38 g to 403 土26g (Table 1) approximately 12 weeks in age wぽeused in

the present study. Each group was random1y divided into 3 subgroups: vehicle group (contro1) ，

1-week isoproterenol (ISO)-infused group (IS01w) and 4-week ISO ・infused group (IS04w). ISO

(1. 2 mg kg'lday'l for 1 or 4 weeks) was subcutaneously administered using an osmotic minipump

(model 1003D or 2001 ， Alzet; Durect ， Cupertino ， CA) implanted in the neck under pentobarb 社al

sodium (50 mg kg. 1 ip) 叩 esthesia. Each osmotic minipump was removed more than 2 h before the

exper 加lent as previously reported [21 ，23].

2.2. Echocardiography

Transthoracic echocardiography was performed for each of the 6 groups. In the ISO-infused

groups ， the measurement was performed 2 h after the removal ofthe osmotic minipump [20]. The

following parameters were measured and calculated using an Aplio SSA-700A (Toshiba ， Tokyo ，

Japan) equipped with a 12・MHzul 回 sound probe (PL T・1202S) under light sedation: L V intemal

dimensions at systole and diastole (LVIDs and LVIDd ， respectively) ， interventricular sep 旬m

JMCC Mitsuyama et al. 5

thickness at diastole (IVST の， L V posterior wall thickness at diastole (L VPWTd) ， L V吋ection

企action (EF) ， and LV fractional shortening (FS). M-mode recordings were performed at the level

of the papillary muscles.

2.3. Surgical preparation

Analyses of L V mechanoenergetics were performed on the 巴xcised cross-circulated heart

preparations as reported previously [19]. In each experiment ， we used 3 rats: one WT or TG rat

was used as a heart donor ， and 2 retired breeder male Wistar rats weighing 450-650 g were used as

blood supplier and metabolic supporter rats ， respectively. All rats were anesthetized with

pentobarb 社alsodium (50 mg kg同1ip) ， mtubated ， ventilated ， and heparinized (1，000 units iv). After

a median stemotomy ， the blood was ex紅acted via the L V apex of a blood supplier rat. This blood

was used for primmg the cross-circulation tubing. The bilateral common carotid arteries and right

extemal jugular vem of the metabolic supporter 捌 were cannulated and connected to the arterial

and venous cross-circulation 旬bing ，respectively. 百le anesthetic level of the metabolic supporter

rat was maintained at a constant level via additional continuous infusion of pentobarbital sodium at

7.5 mg h-1 by monitoring the systemic arterial pressure and heart rate. Consequently ， the anesthetic

level ofthe donor heart was constantly maintained. 百lebrachiocephalic artery was cannulated and

then the right ventricle (RV) was cannulated via the superior vena cava in the heart donor rat and

both the cannulas were connected to the 釘terial and venous cross-circulation tubing from the

supporter rat.百 lebeating heart supported by cross-circulation was then excised from the chest of

the heart donor rat [24]. Coronary perfusion ofthe excised heart was never interrupted during this

preparation and the excised heart was maintamed at 360

C.

A thin latex balloon (balloon material volume ， 0.08 ml) was inserted into the L V via the left

政 ium and connected to a pressure transducer (Li fe Kit DX-312 ， Nihon Kohden ， Tokyo ， Japan)

and a 0.5 ・ml precision glass syringe with fme scales (min 泊lum scale: 0.005 ml). L V volume

(L VV) was changed by adjustmg the mtra-balloon water volume with the syrmge m 0.025 ・ml

steps between 0.08 ml and 0.23 ml (Fig. 2A幽 1).

Systolic un町essed volume (V 0) was determined by即時the balloon to批 level where peak

isovolumi 比cpr伐es鉛su町re久， and hence p戸，ressure. ヴoh如umロme aぽrea (σPVA; see Dαωtα An α1かysi ，お的S吟)， was zero. 百le

sum of the intraballoon water volume and balloon material volume was used as an initial estimate

JMCC Mitsuy αma eta l. 6

of Vo. Vo was then fmally determined as the volume-axis intercept of the best-fit end-systolic

pressure (ESP)-volume relation (ESPVR ー). We obtained the best-fit ESPVR with the equation

ESP=A{l-exp[-B(V-V o)]} by means of the least-squares method (Delta-Graph ， DeltaPoint;

Mont 巴rey ，CA) on a personal compute r. We also obtained the best-fit end-diastolic pressure

(EDP)-volume relation (EDPVR) with 批判uation EDP=A'{exp[B'(V-V o')]-l}. The

correlation coefficients ofthe best-fit ESPVRs were higher than 0.98.

The heart rate was constantly maintained by electrical pacing of the right atrium at 240 beats

per minute (bpm) ， and the epicardial electrocardiogram was recorded. In our previous study using

the ISO-induced hype 町ophied model ， pacing at 300 bpm caused L V incomplete relaxation [21].

This causes myocardial ischemia and generates lactate. In the present cross-circulation system ，

oxidative phosphory lation exdusively generates A TP using oxygen ， and thus ， we measured

myocardial oxygen consumption. PV A can accurately express mechanical work when L V

complete relaxation occurs. 明弓len this is not the case ， PV A would be underestimated. The

systemic arterial blood pressure of the supporter rat served as the coron 訂yperfusion pressure

(90-130 mmHg). Arterial pH， P02， and PC0 2 of the supporter rat were maintained within their

physiological ranges with supplemental O2 and sodium bicarbonate.

2.4. Oxygen consumption

Myocardial O2 consumption was obtained as the product of coronary flow and coronary

arteriovenous O2 content difference (A V02D). It was divided by pacing rate to obtain O2

consumption per beat (V0 2). Total coronary blood flow was continuously measured with an

electromagnetic flowmeter (MFV -3100 ， Nihon Kohden ， Tokyo ， Japan) placed in the middle of白e

venous drainage tubing from the RV. 百le L V thebesian flow was negligible. The coronary

A V02D was continuously measured by passing all the arterial and venous cross-circulation blood

through the cuvettes of a custom-made oximeter (PW A・200S ，Shoe Technica; Chiba ， Japan) as

previously reported in detail [25 ， 26](Fig. 3A-1). 百le mean concen 仕ation of hemoglobin in the

perfused blood was 10.5 土0.9 mg dr1.

2.5. Data anα加is

We attempted to fit experimentally obtained LV pressure 開volume data to the exponential

equations to describe ESPVR and EDPVR. PV A was defmed as the area circumscribed by the

JMCC Mitsuy αmα etαrl. 7

curvilinear best-fit ESPVR ， EDPVR ， and the systolic portions of the ventricular トVtrajectories.

PV A was normalized by L V mass to 1 g. On the basis of our previous work with in vitro and in

situ rat hearts ， we calculated the mean ESP at midrange LV volume (mLVV)(ESP mLVV ) and PVA

at mLVV (PVA mLVV ) to assess LV mechanical work and energetics [19].

As shown previously [19 ヲ 21] ，the V02・PVA relationship was linear in the rat L V. Its slope

represents the O2 cost of PVA ， and its V02 intercept represents the PVA-independent V02・The

PV A-dependent V02 is consumed by myosin ATPase for cross-bridge cycling. The

PV A-independent V02 is composed of O2 consumption for Ca2+ handling in excitation-contraction

(E-C) coupling and basal metabolism. The RV was kept collapsed by continuous hydrostatic

drainage so that the RV PV A， and hence PV A-dependent V02， were assumed to be negligible [25 ，

26]. The RV component ofPVA ・independent V02 was sub 仕acted 企omthe total V02 to yield L V

V02・百le L V (including the sepωm) and the RV were weighed for normalization ofLVV. 百lese

data are summarized in Table 1.

2.6.E 存leriment protocols

When we changed L VV by adjusting the intra-balloon water volume with the syringe between

o ml and 0.23 ml (more than 5 different volumes) ， LV pressure (LVP) ラV02and PV A data during

isovolumic contractions were simultaneously obtained at each L V volume (volume-loading run:

vol-run)(Figs. 2A-1 and 3A同 1). A食erthe vol-run ， a Ca2+ -induced ino 甘opic run (Ca 2+ ino 同.run) w出

performed at mLVV (0.16 ml = 0.08 ml [Vo] plus 0.08 ml [a halfvalue between the minimum and

max 加lum water volume infused into the balloon]) by intracoronary infusionof 1% CaClz solution.

The infusion rate of Ca2+ was increased gradually until we observed a decrease in ESP or

arrhythmia due to Ca2十

overload. To obtain steady-state data ， every data point was measured 3 min

after changing the L VV or infusion rate of Ca2+.

Finally ， to me出ure the basal metabolic O2 consumption ， cardiac arrest was induced by

in回 coronary infusion ofKCl (0.5 M) at 5-10 ml h-1 as previously reported [18]. All data were

measured and sampled at 1証12 for 5-10 s and averaged using a PowerLab unit and Chart

software (AD Instruments ， Bella Vista ， NSW ， Australia).

2.7. Oxygen cωt 01 L V contractility

We obtained the specific best-fit curve for the ESP at Vo (0 mmHg) and the observed ESP mLVV

JMCC Mitsuyam αet al. 8

with the best 回fit ESPVR function in the con 位。1vo1 圃run by the 1east squares method ， and we

calcu1ated PVA mLVV during Ca2+ infusion using this specific best 同日tcurve function on a persona1

computer as previous1y reported [26]. The V02・♂V A 1inear re1ation during Ca2+ infusion was

shifted upward in paralle1 with the contro1 V02・PVA re1ation. Based on this parallelism ， the 1in 巴s

of VOrPVA 1inear re1ationships at different Ca2+ infusion rates at mLVV were drawn in paralle1

to the con 仕01 VOrPVA r巴1ation 1ine as described previous1y [25 ， 27]. The gradually increased

V02・intercept va1ues (PV A・independentV0 2va1ues) ofthe 1ines proportiona1 to the e吐mnced LV

contractility by Ca2+ were obtained by this procedure. Our proposed index for LV contracti1ity ，

equiva1ent maxima1 e1astance at mL VV (eEmaXmL vv) ， was calcu1ated 企'om a triangu1ar area

equiva1ent to PVA mLVV [19 ，26伺]. The O2 c∞os幻toぱfLVc ∞ont仕racωti 出lit勿ywa部sthe slop 伊eoぱft白he relationship

be凶れ何N問巴巴叩nPVA- 恒de叩p巴end由e釘叩n凶1I V02 組 deEma 侃xm此LVV (i. e.， V02 usedfor Ca2+ handling in E・Ccoupling

per unit changes in L V contractility).

2.8. Analyses of one-be α!t L V pressur ・e・time cu.門 eby single logistic function

To evaluate the L V end 同diastolic relaxation rate or lusi 仕opy ，we analyzed the logistic time

constant (T L)企omthe respective b邸 t・fit functions to a one-beat L V pressure-time curve at mL VV

during re1axation with our proposed single logistic function [28 ， 29].

2.9. Reαrl-time quantit αrtive reverse transcription (R1)-polymer αse chα'in reαction (PCR)

百le messenger RNA (mRNA) expression levels of SERCA2a ， mitochondrial transcription

factors A (TF AM) and B2 (TFB2M) ， and glyceraldehyde-3 ・phosphate dehydrogenase (GAPDH)

were determined by RT-PCR as previously described [22]. RT-PCR was performed using SYBR

Green technology on a StepOnePlus™ (Applied Biosystems ， Foster City ， CA). Tota1 RNA was

isolated from 企ozen tissue samples by a spin protocol using the SV Total RNA Isolation System

(Promega ， Madison ， WI). Complementary DNA (cDNA) w出 synthesized from 0.5 μg of the total

RNA using a ReverTra Ace ⑧ qPCR RT kit (Toyobo ， Osaka ， Japan). The cDNA w出 initially

denatured at 950C for 30 s and then amplified by PCR for 40 cycles (denaturation at 950C for 10 s，

annea1ing at 550C品r10 s， and extension at 720C for 45 s) using Thunderbird™ SYBR@ qPCR

Mix (Toyobo). The results were normalized to GAPDH expression.

2.10. Poly αcrylamide gel electrophoresis and western blots for SERCA2 α， phosphol αmbαn (PLB) ，

phospho-SerJ 6PLB (P-PLB) ， Na+/Ca 2キ自changer 1 (NCX1) ， and TF AM

JMCC Mitsuyam αetα!l. 9

Membrane proteins were isolated 企om the L V wall of each frozen heart. The frozen hearts

were homogenized and centrifuged at 1，000 x g for 10 min.τ'he supema 旬nts were centrifuged at

100 ，000 x g for 60 min at 40C. The 100 ，000 x g pellets were cellular membrane fractions and were

used for the immunoblotting ofSERCA2a ， monomeric PLB ， p-PLB ， NCX1 ， and TFAM [21 ， 22].

Equal amounts ofthe membrane proteins (10μg lane 勺were separated on SDS-polyacrylamide

gels (10% for SERCA2; 15% for PLB ， p-PLB ，組d TF AM) in a minigel apparatus

(Mini-PROTEAN II， Bio-Rad) and transferred to polyvinyliodene difluoride membranes. The

membranes were blocked (4% Block Ace ， Dainippon Pharmaceutical Co. ， Osaka ， Japan) and then

incubated with anti-SERCA2a antibody (1:1000 dilution; Affmi ザ Bio Reagents ， Golden ， CO) ，

anti 中LB antibody (1:2000 dilution; Upstate Biotechnology ， Lake Placid ， NY) ， anti-p-PLB (Ser 16)

antibody (1: 1000 dilution; Upstate Biotechnology) ， anti-NCXl antibody (1:200 dilution; a

generous gift from Dr. Iwamoto ， Fukuoka University) ， and anti-TFAM antibody (1:1000 dilution;

Cell Signaling Technology ， Danvers ， MA). The antigens were detected by the luminescence

me仕lO d(ECL Westem blotting detection kit ， GE Healthcare UK Ltd.， Amersham Place ， Little

Chalfont ， England) with peroxidase 闘linked anti-mouse IgG (1:2000 dilution) or peroxidase-linked

anti-rabbit IgG (1:2000 or 1:5000 dilution). 百le amounts of membrane proteins were determined

to obtain the linear response ofthe ECL-immunoblo t. After immunoblotting ， the fihn was scanned ，

and the intensity of the bands was calculated by N旧in1 age analysis [18 ，21]. 百lenrotein ratio of

SERCA2aIPLB ‘p-PLBIPLB and TFA M/N CXl for each sample was expressed as a proportion of

the mean value obtained for the WT contro l.

2.11. Histological α!nalysis

百le hearts of all rats 加 the WT and TG groups were excised and fixed with 3.7%

paraformaldehyde in phosphate-buffered saline ， embedded in paraffin ， cut into 6・flm slices ， and

stained with Masson' s trichrome (MT) to detect the fibrosis. 百leL V collagen volume 企action was

calculated by counting the computerized pixels in a digital in1 age of the collagen area stained by

民1T.

2.12. Statistics

Multiple comparisons were performed by one-way analysis of variance (ANOVA) with

post-hoc Tukey's test ， Bonferroni's test ， or Games-Howell's tes t. A value of Pく 0.05 was

JMCC Mitsuy αmαetαrl. 10

considered statistically significan t. All data are expressed as the mean 土 S.D.

3.Re 喧ults

3.1. Bo砂weights and heart weights

Body weights (BW) and RV weights (RV W) did not significantly differ among all the WT and

TG rat groups. The ratios of heart weight (HW) to BW (HW/BW) in the IS01w and IS04w

groups were significantly (P < 0.05) 1arger than those of the contro1s in both the WT and TG rats.

In addition ， the ratios of L V weights (L VW) to BW (L VW/BW) were significant1y (P < 0.05)

larger in the IS01 w and IS04w groups than in the WT-control ， which indicated ISO-induced

cardiac hyper 廿ophy. 百le ratio ofLVWIBW in the TG-IS04w group was significantly (P < 0.05)

smaller than that in the TG-IS01w group but not significantly different 合om that in the

TG回control and WT-IS04w groups ， indicating a 阿武ial protection from hyper 加 phy in the

TG・IS04w group (Table 1).

The summarized data of heart rate (HR) and blood pressure are shown in Table 2. 百le HR of

IS01w and IS04w were significantly lower than that of control in both WT and TG， indicating

that ISO decre 出 edHR. Systolic blood pr郎 sure (SBP) ， mean blood pressure (MBP) ， and diastolic

blood press 町巴 (DBP) were significantly higher in the TG-IS04w group than in the others ， but not

significantly different from those in WT-IS04w. 百lis result suggests the possibility that the

significantly higher in vivo SBP and DBP in the TG-IS04w group are not related to the

overexpression of SERCA2a but are instead related to other unknown factors.

3.2. Echocardiograp 砂

Summarized echocardiographic data are shown in Figure 1. The functional parameters ， L VIDd ，

and LVIDs of the WT-IS04w group were significantly (P < 0.05) higher than those of the

WT-IS01w group ， whereas those ofthe TG-IS04w group were not further increased from those of

the TG-IS01 w group (Fig. 1A， B)， indicating a partial improvement of L V dysfunction in the TG

rats.

The wall thickness parameters ， IVSTd and LVPWTd ， ofthe IS01w and IS04w in WT and TG

groups were significantly (P < 0.05) larger than those of each control group. 百leIVSTd -but not

L VPWTd-was significantly (P < 0.05) lower in the IS04w group than that 泊 those of the ISO 1 w

JMCC Mitsuy αmα et α!l. 11

WT and TG groups (Fig. 1 C， D). Hence ， it is uncertain whether the f3-adrenergic receptor inducing

cardiac hyper 仕ophy was down-regulated between 1SO 1 w and 1S04w.

Two other functional parameters ， EF and FS， were significantly decreased in the 1S04w group

compared with the 1SO 1 w group in WTヲ but not in TG (Fig. lE， F)， which indicates a partial

improvement of L V dysfunction in the TG rats.

3.3. LVmech αnicalwork

Representative ESPVRs and EDPVRs for all WT and TG rat groups are shown in Figure 2A.

The ESPVR of the WT同1S04w group (blue solid curve in Fig. 2A) was shifted downward

compared to the other groups. The ESPVR ofthe TG-1S04w group (blue dashed curve in Fig. 2A)

was not shifted compared to the other groups. Summarized data of L V mechanics are shown in

Table 3. The mean ESP mLVV and PVA mLVV ofthe WT-1S04w group were significantly (P < 0.05)

lower than those ofthe WT-control group ， but the mean ESP mLVV and PVA mLVV ofthe TG-1S04w

group were not decreased 企omthose ofthe TG-control group. The mean percentages ofESP mLVV

and PVA mLVV normalized to control (=100%) were significantly (P く 0.05) smaller in the

WT圃1S04w group (Figure 2B and 2C) but not in the TG-1S04w group. Moreover ， the mean

percentage of PVA mLVV was significantly (P < 0.05) larger in the TG圃1S04w group 出an in the

WT-1S04w group (Figure 2C). 1n the 1S04w group ， mechanical work was improved by TG.

3.4. VOrPVA rel αtions

Figure 3A shows each representative V02・PVA relation in all groups ， as obtained by the L V

volume loading protocol (Fig. 3A・1). All of仕leVOrPV A relations could be superimposed. Hence ，

there were no significant differences in廿lemean slopes and V02 intercepts ofV0 2・PVA relations

and ， thus ， no significant differences in O2 consumptions per minute for basal metabolism and E-C

coupling among all groups (Fig. 3B-E).

3.5. 。め!gen cost 01 L V contr αctili 砂

Figure 4A shows that the representative PVA-independent V02 and eEmaxmLVV relations in all

groups are 1凶ear. 百le slope ofthe relation appeared to be lower in the TG-IS04w group than in

the other groups. The slope is defined as the O2 costs of L V con 廿actility: V02 used for Ca2+

handling in E・Ccoupling per unit change in L V contract 出ty. The mean O2 cost ofLV con 廿actility

in吐le TG・IS04w group was significantly (P < 0.05) smaller than those in the other groups (Fig.

且臥vfCC Mitsuy αmaet αl. 12

4B).

3.6. Logistic time const αnt(T r)

Figure 5A and B show L V pressure-t 凶 ecurves at mL VV norma1ized to peak pressure in the

WT and TG groups ， respectively. 百le normalized systolic L V pressure-t 泊le curves could be

superimposed. We obtained T L企om each end-diastolic pressure-t 加le curve. The mean T L of

ISO 1 w and 1S04w were significantly (P < 0.05) larger than that the WT control groups (Fig. 5C).

The mean TL was significantly (P<0.05) smaller in all TG groups than in the WT-1S01w and

WT-1S04w groups (Fig. 5C). Each representative raw LV pressure 同time curve at mL VV in the

WT and TG groups showed a similar systolic pressure-time curve ， although the L V peak pressure

differed between the groups (Fig. 5D).

3.7. Western blotsfor SERCA2a ， PLB ， p-PLB ，αndNCXl

Figure 6A shows representative immunoblotting of SERCA2a ， PLB ， p-PLB ， and NCX1 in each

WT and TG group. The mean fold change in the SERCA2 a!P LB ratio in the TG-control group was

significantly (P く 0.05) increased compared to that 泊the WT-con 位01 group (Fig. 6B). Although

the mean fold change in 8ERCA2a/PLB ratio gradually decreased in each WT and TG group over

the I80 infusion period ， the level in the TG-1804w group was significantly (P く 0.05) higher than

that in the WT-I804w group and was maintained at the level ofthe WT同control group (Fig. 6B).

百le protein expressions of PLB and p-PLB gradually decreased over the 180 infusion period in

the WT and TG groups in a similar manner ラand hence ， there were no differences in the mean fold

change in社le p-PLBIPLB ratio among all groups (Fig. 6C). The protein level of NCX1 was not

significantly different 創nong all 白egroups (Fig. 6D).

3.8. RT-PCRfor mRNA ofSERCA2a ， TFA M， and TFB2M in comparison with GAPDH

百le expression level of each mRNA of SERCA2a ， TF AM， and TFB2M in comparison with

GAPDH in the WT group was unchanged (Fig. 7A-C). 1n contrast ， the mean expression levels of

SERCA2a mRNA in all TG groups were significantly (P < 0.05) higher than those in all WT

groups (Fig. 7 A). 百le mean expression level of each mRNA of TF AM and TFB2M in all TG

groups tended to increase but not significantly (Fig. 7B， C).

3.9. Western blotsfor TFAM

Figure 7D shows representative immunoblotting of TF AM in each WT and TG group. The

JMCC Mits 句7αmα etα'1. 13

mean[ 起立ofTFAM 担CXl protein expression in the WT group was not significantly differen t. In

con 回 st，the me加盟jQ臼 fTFA MlN C茎1protein 堅E盟鐙担旦 in all TG groups were significantly

(P く0.05) higher than that ofthe WT-ISOlw and WT-IS04w groups (Fig.7D).

3.10. Fibrosis αnα !lysis

The ratio of fibrotic area to whole tissue area in L V was significantly (P く 0.05) increased in

the ISOlw and IS04w groups compared with that in the WT and TG control groups ， but no

differences were found between the ISO 1 w and IS04w groups or between the WT and TG groups

(Fig.8).

4. Discussion

百le present study revealed that (1) L V systolic function was altered in the WT -IS04w group

but was preserved in the TG・IS04w by analyzing ESPVR ， ESP mLVV ， and PVAιvv and that (2)

LV diastolic function was altered in the WT-ISOlw and WT-IS04w groups but was preserved in

the TG-ISOlw and TG-IS04w groups by analyzing the logistic time constan t. Furthermore ， the

me組 slope and mean V02 intercept of the V02・PVA relationships in each of the 6 groups were

unchanged under 240 ・bpm pacing ， which is consistent with our previous study [21]. 百lis result

indicated iliat the mean oxygen consumption for Ca2+ hand1ing in E・Ccoupling per minute and ilie

mean oxygen consumption for basal metabolism per minute did not differ among all ilie groups.

百lemost interesting fmding in the present study was the smaller O2 cost of L V contractility

in 仕le IS04w group ilian in the contro1 and ISO 1 w TG groups ， a1iliough the O2 costs of L V

con 仕acti1i 勿inWT rats were not different among the contro1 ， ISO 1 w， and IS04w groups.

4.1. LV mechanical function

We have previous1y reported a detailed ana1ysis of a short-term ISO-induced hype 町ophy

mode1 in WT rats [21]. A1iliough these model hearts showed significant decreases of SERCA2a

expression ， they were in a compensatory phase ， and hence ， their L V mechanica1 work and

energetics (except diastolic dysfunction) were unchanged compared with norma1 WT rats at

240 ゐpmpacing [21]. In the present study ， we made short-term and 1ong-term ISO-infused models.

A1though we have already reported 1onger-term ISO ・infused mode1s ， ilie cardiac performance was

eva1uated on1y by echocardiography [20]. 百le present study is the frrst to ana1yze the detai1ed

JMCC Mitsuy αmaet αl. 14

mechanoenergetics oflong-term ISO ・infused models.

The ESPVR was unchanged in the WT-ISOlw group but shi 丘eddownward in the WT同IS04w

group.τnus ラ decreases in mean ESP mLVV and PVA mLVV were demonstrated in the WT-IS04w

group ， indicating systolic dysfunction. Th巴normal systolic function in the WT -ISO 1 w group was

consistent with our previous study [20]. The significantly longer mean TL of the WT園田01wand

WT-IS04w groups indicated diastolic dys 白nction. Taken together ， the initial L V diastolic

dysfunction without systolic dysfunction and the subsequent L V systolic and diastolic dysfunction

presented here is similar to the disease progression of concentric L V hypertrophy in human [1].

The systolic and diastolic function could be impaired by the deterioration of Ca2+ handling [2，

3]. SERCA2a plays a key role in the Ca2+ handling of E・C coupling [2]. 百le ratio of

SERCA2 a!PLB in the TG-IS04w group was maintained at tlle same level as that of the

WT圃control group. Therefore ， TG-IS04w could be prevented 企om 加pairing the systolic and

diastolic function.

4.2. Smaller O2 cost 01 LV contractili ち!in TG・lS04w

Recently ， Pinz et al. demonstrated that overexpression of SERCA2a enhances myocardial

mechanical 白nction in a normal model during inotropic stimulation ， but tllat this advantage is not

maintained in an L V hypertrophy model 加which the energetic status is compromised [16]. 百ley

also suggested tlle possibility that enぽgy supply might be a limiting factor for the bene 五tof

SERCA overexpression in hypertrophied hearts [16]. In the present s旬dy，this possibility could be

excluded because the lactate production indicating ischemia was not observed in the TG-IS04w

group (data not shown). 百le smaller O2 cost of LV contract i1i ty in the TG-IS04w group than in

the other groups could not be explained by this mechanism [16].

百le functioning of ATPases like SERCA2a is highly dependent on the supply of ATP.

Therefore ， ATP production is tightly related to ci+ handling in cardiac function. Watanabe et al.

[22] demons 廿ated that mitochondrial TF AM and TFB2M coordinate to regulate the 仕anscription

of genes for mitochondrial enzymes that produce ATP and for SERCA2a ， which consumes ATP.

In con 仕ast ，Niwano et al. [3Q] reported that after SERCA2 gene 仕組sfer into the failing heart ，

mRNA levels of enzymes contributing to the energy supply in mitochondria recovered to norτnal

level ， which led to elevated production of ATP. The present result indicated that the ratio of

JMCC Mitsz のほ'mαetα!l. 15

TFAM 担巳茎1in each TG group was significantly larger than that in the WT -1 w and WT・4w

g盟盟主(see Fig. 7D). Accordingly ， ATP production might have increased in the TG group. In the

TG-IS04w group ， the ratio of SERCA2 a1PLB was maintained at the level of that in the

WT-control group ， (i.e. ， it did not increase)(see Fig. 6B). Accordingly ， ATP production relatively

dominates over ATP consumption only in the TG-IS04w group. This may contribute to the

smaller O2 cost of L V contractility in the TG回目04w group (see Fig. 4).

Furthermore ， in the present s同dy，we used 240 ・bpm pacing for the IS01w and IS04w hearis

due to the relaxation dysfunction. In contrast ， the smaller O2 cost of L V contractility has been

reported in normal hearts that overexpress SERCA2a 2 to 3 days after the infection with an

adenovirus carrying SERCA2a under 300 ゐpm pacing [12]. The experimental models and

conditions used were definitely different 企omours ， leading to the different outcome.

4.3. VOrPVA relation

Because the ESPVR of the WT-IS04w group was shifted downward 企om those of the other

groups ， each PV A at any L VV decreased. Each V02 at each L VV decreased proportionally to each

PV A. However ，由eslope and the V02 intercept of the V02・PVA relation were almost unchanged

among a1l6 groups ， including WT-IS04w. 百leslope ofthe V02-PVA relation reflects the ratio of

chemomechanical energy transduction efficiency of the contractile machinery [19]. 百le V02

泊tercept represents PVA-independent V02・百lIsis composed ofthe oxygen consumption for Ca2+

handling in E・Ccoupling and for basal metabolism [19]. 百lese components were also unchanged

in all 6 groups.

Nevertheless ， the systolic and diastolic dys 白nction observed in the WT回IS04w group was

greatly improved in the TG・IS04w group ， and the beneficial smaller oxygen cost of LV

contractility w出 observed in the TG-IS04w group.

4.4. Comparison 01 ex vivo (in vi. か0) d，αtα with in vivo data

In the present report ， the data 企om the in vivo study are shown in Figure 1 and Table 2. 百le

results obtained 企oman in vivo study are 企equently complicated and different 企omthose obtained

企oman ex vivo study [31 ， 3~] because various integrative control mechanisms can 向nction in vivo.

The result obtained from the ex vivo study is relatively simple ， but experimental conditions can

sometimes be different 企om those in 仕le in vivo study. The present ex vivo study gave us

JMCC MitSl 勺lam αetαl. 16

information about mechanica1 work and energetics ， but the in vivo study cou1d not give us such

information. Integrative studies ， both in vivo and ex vivo ， are needed to mak 巴the resu1t beneficia1

for clinical therapy.

4.5. Functionallimitations

The HV.刀 BW and LV/BW ofthe TG-IS04w group were significant1y greater than those ofthe

WT同co耐 01 (ratio: 2.89 vs 2.52 and 2.29 vs 2.02). Furthermore ， the HW/BW and LVIBW ofthe

TG-IS04w group were not significantly different 企om those of the WT・.IS04w group. 1t seems

1ikely that the increased expression of SERCA2a did not prevent hypertrophic remodeling.

EF and FS remained significantly depressed in the TG-1S04w compared to the WT control ，

although the trend was 加lproved. 1t seems like1y that the increased expression of SERCA2a did

not preserve EF or FS with IS0 tre a:加len t.

The ratios of SERCA2 a!P LB in the TG-con 加 1and TG- IS0 1 w groups were significantly higher

than in the TG-1S04w group ， but the mechanica1 parameters (ESP mLVV and PVA 叫 VV) were

unchanged. Overexpression of SERCA2a was not s泊中lyreflected in mechanical parameters.

4.7. Conclusion

百le present study indicated that longer 回term SERCA2a overexpression improved L V systolic

and diastolic 命sおnction ‘inthe excised heart ‘and the O2 cost of LV contractility (i.e. ，社le V02

used for Ca2+ handling in E・Ccoupling per unit change in L V corr 回 ctility) in a 10ng 聞term

isoprotereno1-induced cardiac failure rat mode1 with a down-regulated SERCA2a leve l. In

conclusion ， target 加gfor SERCA2a may be a potential therapeutic s廿ategy for chronic

hyper 廿ophic heart failure.

Acknowledgments

This work was supported in part by grants-in 帽aid N0.22790216 for Scientific Research from the

Minis 廿yof Education ， Cul 印re，Sports ， Science and Technology of Japan. The authors thank for a

generous gift of anti 国NCXl antibody to Dr. T. Iwamoto ， Fukuoka University ， Japan.

Disclosures

JMCC Mitsuy αmaet αl. 17

None

Author contributions

Miyako Takaki designed and supervised the present study. Shinichi Mitsuyama ， Daisuke

Takeshita ， Koji Obata and 恥1iyako Takaki conducted experiments and obtained the data. Koji

Obata and Guo-Xing Zhang established the transgenic rats. Shinichi Mitsuyama and Miyako

Takaki analyzed the data and dra 武edthe manuscrip t.

JMCC Mitsuyama et al. 18

References

[1] Milani RV， Drazner MH， Lavie CJ， Morin DP， Ventura HO. Progression 企om concentric left

ventricular hypertrophy and normal ejection 仕action to left ventricular dysfunction. Am J

Cardio12011; 108: 992 ・6.

[2] Bers DM. Calcium fluxes involved in control of cardiac myocyte contraction. Circ Res 2000;

87:275 ・81.

[3] Gwathmey JK， Copelas L， MacKinnon R， Schoen FJ， Feldman MD， Grossman W， et al.

Abnormal in仕acellular Ca2十

handling in myocardium from patients with end-stage heart

failure. Circ Res 1987; 61: 70-6.

[4] Arai M， Matsui H， Periasamy M. Sarcoplasmic reticulum gene expression in cardiac

hypertrophy and heart failure. Circ Res 1994; 74: 555-64.

[5] Bers DM， Eisner DA， Valdivia HH Sarcoplasmic reticulum Ca2+ and heart failure: roles of

diastolic leak and Ca2+ transport. Circ Res 2003; 93: 487 ・90.

[6] Mercadier JJ， Lompre AM， Duc P， Boheler KR， Fraysse JB， Wisnewsky C， et al. Altered

sarcoplasmic reticulum Ca2+-ATPase gene expression in the human ven 甘ic1eduring

end-stage heart failure. J Clin Invest 1990; 85: 305-9.

[7] Baker DL， Hashirnoto K， Grupp IL， Ji Y， Reed T， Loukianov E， et al. Targeted overexpression

of the SR Ca2+ -A TPase increases cardiac contractility in transgenic mouse hearts. Circ Res

1998;83: 1205 ・14.

[8] del Monte F， Wi1l iams E， Lebeche D， Schmidt U， Rosenzweig A， Gwathmey JK， et al.

Improvement in survival and cardiac metabolism after gene transfer of sarcoplasmic

reticulum Ca2+-ATPase in a rat model ofheart failure. Circulation 2001; 104: 1424 ・9.

[9] Giordano FJ， He H， McDounough P， Meyer M， Sayen MR， Dillmann "眉.

Adenovirus-mediated gene transfer reconstitutes depressed SR Ca2+ -ATPase levels and

shortens prolonged cardiac myocyte Ca2+ transien t. Circulation 1997; 96: 400 ・3.

[10] Maier LS， Wah トSchott C， Hom W， Weichert S， Pagel C， Wagner S， et al. Increased SR Ca2+

cycling con 仕ibutes to irnproved contractile performance in SERCA2a-overexpressing

仕ansgenic rats. Cardiovasc Res 2005; 67: 636-46.

JMCC Mitsuy αmα et αrl. 19

[11] Muller OJ， Lange M， Rattunde H， Lorenzen HP， Muller M， Frey N， et al. Transgenic rat

hearts overexpressing SERCA2a show improved contractility under baseline conditions and

pressure overload. Cardiovasc Res 2003; 59: 380 ・9.

[12] Sakata S， Lebeche D， Sakata N， Sakata Y， Chemaly ER， Liang LF， et al. Targeted gene

transfer increases contractility and decreases oxygen cost of contractility in normal rat hearts.

Am J Physiol Heart Circ Physio12007; 292: H2356-63.

[13] Sakata S， Lebeche D， Sakata Y， Sakata N， Chemaly ER， Liang L， et al. Transcoronary gene

transfer of SERCA2a increases coronary blood flow and decreases cardiomyocyte size in a

type 2 diabetic rat mode l. Am J Physiol Heart Circ Physio12007; 292: H1204-7.

[14] Teucher N， Prestle J， Seidler T， Currie S， Elliott EB， Reynolds DF， et al. Excessive

sarcoplasmic/endoplasmic reticulum Ca2+-AτPase expression causes increased sarcoplasmic

reticulum Ca2+ uptake but decreases myocyte shortening. Circulation 2004; 110: 3553-9.

[15] Vetter R， Rehfeld U， Reissfelder C， Weis W， Wagner KD， Gunther J， et al. Transgenic

overexpression of the sarcoplasmic reticulum Ca2+ ATPase improves reticular Ca2+ handling

in normal and diabetic rat he紅白. FASEB J 2002; 16: 1657 ・9.

[16] Pinz 1， Tian R， Belke D， Swanson E， Dillmann W， Ingwall JS. Compromised myocardial

energetics in hypertrophied mouse hearts diminish the beneficial effect of overexpressing

SERCA2a. J Biol Chem 2011; 286: 10163 ・8.

[17] Vetter R， Rehfeld U， Reissfelder C， Fechner H， Seppet E， Kreutz R. Decreased cardiac

SERCA2 expression ， SR Ca uptake ， and contractile function in hypothyroidism are

attenuated in SERCA2 overexpressing transgenic rats. Am J Physiol Heart Circ Physiol

2011; 300: H943 ・50.

[18] Zhang GX， Obata K， Takeshita D， Mitsuyama S， Nakash 加1a T， Kikuta A， et al. Evaluation of

left ven 仕icular mechanical work and energetics of normal hearts in SERCA2a transgenic rats.

J Physiol Sci 2012; 62: 22ト31.

[19] Takaki M. Left ven 仕icular mechanoenergetics in small animals. Jpn J Physiol 2004; 54:

175 幽207.

[20] Takeshita D， Sh出 izu J， Kitagawa Y， Yamashita D， Tohne K， Nakajima-Takenaka C， et al.

Isoproterenol-induced hypertrophied rat hearts: does short-term trea 加lent co汀espond to

JMCC Mitsuy αmα etα'l. 20

1ong-term tr切なnent? J Physio1 Sci 2008; 58: 179-88.

[21] Nakajima-Takenaka C， Zhang GX， Obata K， Tohne K，民1atsuyoshi H， Nagai Y， et al. Lef 王

ven 仕icu1ar function of isoprotereno1-induced hyper 仕ophied rat hearts perfused with b1ood:

mechanica1 work and energetics. Am J Physio1 Heart Circ Physio12009; 297: H1736-43.

[22] Watanabe A， Arai M， Koitabashi N， Niwano K， Ohyama Y， Yamada Y， et al. Mitochondria1

transcription factors TF AM and TFB2M regu1ate Serca2 gene transcription. Cardiovasc Res

2011; 90: 57・67.

[23] Shibata M， Takeshita D， Obata K， Mitsuyama S， Ito H， Zhang G-X ， et al. NHE-1 participates

in isoprotereno1-induced downregu1ation of SERCA2a and deve10pment of cardiac

remode1ing in rat hearts. Am J Physio1 Heart Circ Physio12011; 301: H2154-60.

[24] Yoshikawa Y， Zhang Q茎， Obata K， Ohga Y， Matsuyoshi H， Taniguchi S， et al.

Cardioprotective effects of a nove1 ca1pain inhibitor SNJ ・1945 for reperfusion injury after

cardiop1egic cardiac arres t. Am J Physio1 Heart Circ Physio12010; 298: H643 ・51.

[25] Hata Y， Sakamoto T， Hosogi S， Ohe T， Suga H， Takaki M. Linear 02 use 剛pressure-vo1ume

area re1ation 企omcurved end-systo1ic pressure-volume relation ofthe blood-perfused rat left

ven 仕icle. Jpn J Physio11998; 48: 197 ・204.

[26] Tsuji T， Ohga Y， Yoshikawa Y， Sakata S， Kohzuki H， Misawa H， et al. New index for oxygen

cost ofcorr 仕actility from curved end-systolic pressure-vo1ume re1ations in cross-circu1ated rat

hearts. Jpn J Physi011999; 49: 513 之O.

[27] Sakata S， Ohga Y， Abe T， Tabayashi N， Kobayashi S， Tsuji T， et al. No dependency of a new

index for oxygen cost of left ven 廿icu1ar contr ・actility on heart rates in the b100d-perfused

excised rat heart. Jpn J Physi01 2001; 51: 177 ・85.

[28] Matsubara H， Araki J， Takaki M， Nakagawa ST， Suga H. Logistic characterization of 1e丘

ven 廿icular isov01umic pressure-time curve. Jpn J Physio11995; 45: 535 回52.

[29] Matsubara H， Takaki M， Yas 吐lara S， Araki J， Suga H. Logistic time constant of isovo1umic

re1axation pressure 同time curve in the canine 1eft ven 廿icle. Better altemative to exponentia1

time constan t. Circu1ation 1995; 92: 2318 同26.

[3Q] Niwano K， Arai M， Koitabashi N， Watanabe A， Ikeda Y， Miyoshi H， et al. Lentivira1

vector-mediated SERCA2 gene 仕ansfer protects against heart failure and 1eft ven 甘icu1ar

JMCC Mitsuyama et al. 21

remodeling after myocardial infarction in rats. Mol Ther 2008; 16: 1026 ・32.

[31] Huke S， Prasad V， Nieman ML， Nattamai KJ， Grupp IL， Lorenz JN， et al. Altered dose

response to s-agonists in SERCA1-expressing hearts ex vivo and in vivo. Am J Physiol Heart

Circ Physio12002; 283: H958 剛長丘

[3~] O'Donnell JM， Fields A， Xu X， Chowdhury SA， Geenen DL， J Bi. Limit 巴dfunctional and

metabolic improvements in hypertrophic and healthy hearts expressing the skeletal muscle

isoform of SERCA1 by adenoviral gene transfer in vivo. Am J Physiol Heart Circ Physiol

2008; 295: H2483-94.

JMCC Mits 句7αmaet α'1. 22

Figure Legends

Fig. 1 Echocardiographic parameters

The mean va1ues of L V interna1 dimensions at diasto1e (L VIDd)(A) and systo1e (L VIDs )(B) ，

interventricu1ar septum thickness at diasto1e (IVSTd)(C) ， 1e食 ventricu1ar (L V) posterior wall

thickness at diasto1e (L VPWTd) (D)， ejection fraction (EF)(E) and fractiona1 shorteningσS)(F) in

each wild-type (WT; n=9 each) and SERCA2a-transgenic rat group (TG; n=7 each) infused with

none (Contro1; solid co1umn) ，トweek (ISO 1 w; gray co1umn) and 4哨 eek isoprotereno1 (IS04w;

open co1umn) are shown. *， P < 0.05 vs. WT-Contro 1. t P < 0.05 vs. WT同ISOlw. t， P < 0.05 vs.

WT同IS04w. g， P < 0.05 vs. TG・Con 位01. ~， P < 0.05 vs. TG・ISOlw

Fig. 2 Representative LV end-systolic pressure-vo1ume re1ations (ESPVRs) and end-diasto1ic

pressure-vo1ume re1ations (EDPVRs)(A) and mechanica1 indices (B， C)

A・1: Representative simu1taneous tracings of 1eft ventricu1ar (L V) isovo1umic pressure and L V

vo1ume during vo1ume-1oading run between 0.08 (Vo) m1 and 0.23 (Vo + 0.15) m1. From the data

by this protoco1 ， a sing1e set ofESPVR and EDPVR is obtainab1e.

B: 百le mean percentage normalized to con 仕01 (=100%) in end-systolic pressure (ESP) at

midrange LV vo1ume (mLVV)(= ESP mLVV ) in each WT (n=9 each) and TG rat group (n=7 each).

C: 百leme阻 percentage normalized to con 廿01 (=100%) in systolic pressure-v01ume 訂ea(PVA: a

tota1 mechanica1 energy per beat) at mLVV (= PV AmLVV ) in each WT (n=9 each) and TG rat group

(n=7 each). 大P< 0.05 vs. WT・Co甜 01.t， P < 0.05 vs. WT-IS04w.

Fig.3 Representative L V oxygen consumption per beat (V0 2)-PV A re1ations (A) and summary of

mechanoenergetic indices (B-E)

A -1: Representative simu1taneous 仕acings of 1eft vell 仕icu1ar (L V) isovo1umic pressure ， mean

coronary flow ， coronary arteriovenous O2 content difference (A V02D)， and LV vo1ume during

vo1ume 同10ading run bewteen 0.08 (Vo) m1 and 0.25 (VO + 0.15) ml. From the data by this protoco1 ，

a sing1e V02・PVA re1ation is obtainab1e. The mean slopes (B) and V02 intercepts (C) of the

V02・PVA re1ations ， and oxygen consumption per minute for basa1 metabolism (D) and

JMCC Mitsuyama et al 23

excitation-con 仕action (E同C) coupling (E) are shown in each WT (n=9 each) and TG group (n=7

each). 百lere was no sign 出cant difference among all groups.

Fig. 4 Representative PVA-independent VOTequivalent maximal elastance (eEmax) at mLVV (=

eEmaxmLVV) relations (A) and the mean slopes ofthe relations (B) in the 6 groups.

The slope ofthe relation denotes oxygen cost of eEmaXmLVV (=LV contractility). 百lemean oxygen

costs of L V contractility are shown in each WT (n=5-6) and TG group (n=5 ・8). *， P < 0.05 vs.

W下Contro l.t， P < 0.05 vs. WT-ISOlw. ~， P < 0.05 vs. TG・Contro l. ~， Pく 0.05 vs. TG耐IS01w.

Fig. 5 Representative normalized LV pressure-time curves at mL VV in each WT (A) and TG (B)

group and the mean logistic time constants (Matsubara et al.， Circulation 1995; 92: 2318 ・26)(C)

From end-diastolic pressure-time curves within the frame (A， B)， we obtained the mean logistic

time constants (C) in each WT (n=9 each) and TG group (n=7 each). D: Raw pressure-time curves

in each WT and TG group. Raw systolic pressure-t 凶ecurves were almost superimposable ，

indicating similar con 仕action r彼自. *， P < 0.05 vs. WT・-Con 廿ol.t， P < 0.05 vs. WT-IS01w. t， P <

0.05 vs. WT-IS04w.

Fig. 6 Representative immunoblotting (A) and the summary ofthe mean protein levels of cardiac

Ca2+ handling proteins (B-D)

百四 mean ratios of sarcoplasmic reticulum Ca2+ATPase (SERCA2a)/phospholamban (PLB)

(n=7 ・II)(B) and the mean ratios ofphosphorylated-Serl 6 PLB (P-PLB)/ PLB (n=5 ・11)(C) and the

mean protein levels of Na+/Ca 2+ exchanger 1 (NCX1) (n=8)(D) are shown in each WT and TG

group. 大P< 0.05 vs. WT・Contro 1.t， P < 0.05 vs. WT-IS01w. t， Pく 0.05 vs. WT-IS04w. ~， P <

0.05 vs. TG同Con仕01.~， P < 0.05 vs. TG-ISOlw

Fig. 7 RT-PCR for mRl'、~A of SERCA2a (A) ， mitochondrial transcription factors A (TF AM)(B)

and B2 (TFB2M)(C) and west 芯mblots for TF AM (D)

RT-PCR for mRNA of SERCA2a ， TFAM ， and TFB2M was compared with glyceraldehyde-

勾hosphate dehydrogenase (GAPDH). IFAM protein level was normalized to NCXl protein

JMCC Mitsuy αmα et al. 24

民盟L百lemean values of RT干CRand westem blots are shown in each WT and TG group (n=5-7

each). 十，Pく0.05 vs. WT-ISOlw. ~:， P < 0.05 vs. WT-IS04w.

Fig. 8 Fibrotic area ratio to whole tissue area in L V in each WT (n= each 6 view 加6rats) and TG

(n= each 6 view in 6 rats) group

The mean fibrotic area ratios in the ISOlw and IS04w groups were significantly higher compared

with those in the con 位。1groups. ヘP< 0.05 vs. WT幽Con 位。1. S， P < 0.05 vs. TG・Contro 1.

Figure 1 Control 叫 IS01w 口ISO 仰

A (mm)

LVIDd

10

OAVO ゐf10AVO

止 す ム す*

5

。WT TG

C (mm) IVSTd

2.0

1.5

1.0

0.5

。E (%)

WT TG

EF

90 *

80

70

60

0十圃関門圃関門

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B (mm)

LVIDs

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4

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2

。WT TG

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1.5

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WT TG

FS

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WT

WT

TG

TG

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Cont ISOlw IS04w

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Table 1. Body

weights and heart

weights

WT

TG

Control IS01w

IS04w

Control IS01w

IS04w

(n=ll) (nニ9)

(n=7) (n=7)

(n=7) (n=7)

BW(g)

428士57.2

407土35.2

415土27.4

393土39.6

373士19.9

395土21.6

LVW(g)

0.863土0.149

0.961土0.086

0.988土0.092

0.808土0.074l

0.970士0.077

0.905士0.065

RVW(g)

0.218土0.040

0.266土0.051

0.251土0.046

0.207土0.024

0.258士0.030

0.237土0.032

HWIBW

(X1O-3 )

2.52土0.185

3.02士0.152*

2.99土0.127*

2.59土0.180H

3.29土0.124*tH

2.89土

0.1l

5*~11

LV¥¥刀BW(X

lO-3)

2.02土0.187

2.37土0.186*

2.38土0.081

* 2.07

土0.157H

2.60士0.126*~

2.29土0.159*

11

RVWIBW

(XlO-3 )

0.508土0.054

0.649土0.082*

0.605土0.089

0.527土0.039t

0.692士0.062*~

0.597土0.062

Values are means

土SD;n， number

ofanimals; WT， wild-type

rats; TG， SERCA2a-transgenic

rats; ISOlw

and IS04w

， isoproterenol (1.2 mg kg-

1 day-l

for 1 week

or 4 weeks

， respectively)同加白sedgroup;

BW， body

weight; LVW， left ventricle

weight; RVW， right

vel町icleweight;

HW， heart

weight. * P < 0.05

vs. WT同Contro

l.

tP < 0.05

vs. WT-ISOlw.lP

く0.05

vs. WT-IS04w.

~Pく

0.05vs. TG-Co

蹴01.

11 Pく0.05

vs. TG品01w. All animals

胸tedwith

ISO for 4 weeks

survived.

Table 2. Heart

rate and blood

pressure

WT

TG

Control ISOlw

IS04w

Control ISOlw

IS04w

(n=12) (n=9)

(n=13) (n=l1)

(n=lO)

(n=15)

Heart Rate

(bpm) 378

土35

325土38*~

329土26*~403

土26

347士35~

336::J:: 37*~

Systolic Blood Pressure

(mmHg)

135土15

132土12

149土11t

142土9

131土14t

158土13*H

11

Mean Blood Pressure

(mmHg)

116土13

115士11

125土10

120土9

114土12

137土

12*↑~11

Diastolic Blood

Pressure (mmHg)

107土13

108士10

113土11

109土9

106土12

126土13*H

11

Values are me

佃S土SD;n， number

of animals;

WT， wild

勺rperats;

TG， SERCA2

かtransgenic

rats; ISOlw

and IS04w

， isoproterenol

(1.2 mg kg-1 day-l

for 1 week

or 4 weeks

， respectively)-infused group;

bpm， beats

per minute;

* Pく0.05

vs. WT

四Con

仕01.↑P< 0.05

vs. WT-ISOlw.

tP < 0.05

vs. WT-IS04w.

~P < 0.05

vs. TG

四Contro

1.

11 Pく0.05

vs. TG

同ISOlw.

Table 3. Variables

of left ventricular

mechanics

WT

TG

Control ISOlw

IS04w

Control ISOlw

IS04w (n=l1)

(n=9) (n=7)

(n=7) (n=7)

(n=7)

ESPVR

A(mmHg)

212土51.9

160土26.4

135士21.8*

265土64.5tt

207土59.7

164土21.29

B (ml勺

15.0士6.03

27.1土9.22*

21.1土4.60

8.90土3.75t~

17.6土7.33

19.2土8.90

Vo(xlO-2 ml g-l)

0.095土0.014

0.083土0.007

0.081土0.008

0.100土0.009tt

0.083土0.0079

0.088土0.006

EDPVR

A' (mmHg)

0.124士0.128

0.276土0.172

0.091土0.071

0.214土0.183

0.175土0.134

0.104土0.100

B' (mr1 )

35.2土14.0

35.2土11.2

34.1土16.8

29.2士9.00

35.3士13.4

38.1士20.0

ESPmLVV (mmHg)

139土36.7

129士26.5

102:1: 16.6*

132土20.2

128土10.8

119土16.6

PVA

mLVV (mmHg

9.11土2.09

7.11土2.00

5.63土1.05*

8.04土1.92

7.15土1.43

7.27土1.28

ml beaf1

ぎ1)

mLVV (mlg勺

0.190土0.028

0.162士0.016*

0.163土0.015

0.199土0.018

↑t

0.166土0.0149

0.178:1:0.014

Values are means

土SD; n， number

of an凶als;WT， wild-type

rats; TG， SERCA2a

間仕ansgenic

rats; ISOlw

and IS04w

， isoproterenol

(1.2 mg kg-1 day-1

for 1 week

or 4 weeks

， respectively)-in

血sedgroup;

ESPVR

， end-systolic

pressure (ESP)-volume

(V) relation;

A and B， parameters

of best-fit

ESPVR

obtained by the

equation: ESP=A{1

ー-exp[-B(V-V

o)]).Each

best-fit curve

w田obtained

by ESP

同Vdata during

left ventricular

(L V) volume-Ioading

run; Vo， volume

加tercept

of

ESPVR

normalized by L V mass

to 1 g; EDPVR，

end-diastolic pressure

四volume

relation; A' and B'， par

創neters

of best-fit

EDPVR

obtained by the equation:

EDP=A'{exp[B'(V-V

o')]-I}; mLVV

， midrange

LV volume;

ESPmLvv， ESP

at mLVV;

PVA

血LVV，systolic

pressure司olume

area at mLVV.

* P < 0.05

vs.

WT

同Contro

1.tP

く0.05vs. WT-ISOlw.

~Pく

0.05vs. WT-IS04w.

9Pく0.05

vs. TG-Con

仕01.