Acute aortocaval fistula: role of low perfusion pressure and subendocardial remodeling on left ventricular function

ORIG INAL ART ICLE

Acute aortocaval fistula: role of low perfusion pressure andsubendocardial remodeling on left ventricular functionFl�avia R. R. Mazzo*, Clovis de Carvalho Frimm*, Ana Iochabel S. Moretti†, Maria C. Guido† andMarcia K. Koike**Laboratory of Medical Investigation, LIM-51, Department of Clinical Medicine, University of S~ao Paulo Medical School, S~aoPaulo, Brazil and †Heart Institute (InCor), University of Sao Paulo Medical School, S~ao Paulo, Brazil

INTERNATIONAL

JOURNAL OF

EXPERIMENTAL

PATHOLOGY

doi: 10.1111/iep.12025

Received for publication: 14 August2012Accepted for publication: 5 March2013

Correspondence:Marcia Kiyomi KoikeLaboratory of Medical InvestigationLIM-51Department of Clinical MedicineUniversity of S~ao Paulo MedicalSchoolS~ao PauloBrazilTel.: +55 11 3061-8485Fax: +55 11 3061-7170E-mail: [email protected]

SUMMARY

The experimental model of aortocaval fistula is a useful model of cardiac hypertro-

phy in response to volume overload. In the present study it has been used to investi-

gate the pathologic subendocardial remodeling associated with the development of

heart failure during the early phases (day 1, 3, and 7) following volume overload.

Compared with sham treated rats, aortocaval fistula rats showed lower systemic

blood pressure and higher left ventricular end-diastolic pressure This resulted in

lower coronary driving pressure and left ventricular systolic and diastolic dysfunc-

tion. Signs of myocyte necrosis, leukocyte cell infiltration, fibroplasia and collagen

deposition appeared sequentially in the subendocardium where remodeling was more

prominent than in the non-subendocardium. Accordingly, increased levels of TNF-

alpha, IL-1 beta, and IL-6, and enhanced MMP-2 activity were all found in the sub-

endocardium of rats with coronary driving pressure �60 mmHg. The coronary

driving pressure was inversely correlated with MMP-2 activity in subendocardium in

all time-points studied, and blood flow in this region showed positive correlation

with systolic and diastolic function at day 7. Thus the predominant subendocardial

remodeling that occurs in response to low myocardial perfusion pressure during the

acute phases of aortocaval fistula contributes to early left ventricular dysfunction.

Keywords

aortocaval fistula, coronary perfusion, cytokines, fibrosis, metalloproteinases,

subendocardium

The remodelling of the heart is associated with increased

cardiovascular morbidity and mortality (Fedak et al. 2005).

It has been characterized by enhanced gene and protein

expression resulting in changes of cardiac geometry, shape

and function (Cohn et al. 2000).

Surgical aortocaval fistula represents an experimental

model of cardiac hypertrophy in response to volume over-

load (Garcia & Diebold 1990). In this study it has been

employed in rats to investigate the pathophysiologic mecha-

nisms associated with the development of heart failure dur-

ing the early phases following volume overload. Although

distinct in fundamental structural cardiovascular aspects, we

believe aortocaval fistula mimics the acute volume overload

to the left ventricle that occurs in emergency clinical entities

such as acute aortic valve insufficiency and myocardial

infarction.

The increase in preload gives rise to the development of

the eccentric type of cardiac hypertrophy with sarcomere

lengthening and cardiac chamber enlargement (Olivetti et al.

2000). According to Laplace′s law, the increase in chamber

dimensions corresponds to a proportional elevation in sys-

tolic wall stress. The physiologic adaptation consists of wall

thickening to counteract the elevated systolic wall stress,

thus preserving systolic function. Since myocardial hypertro-

phy is not developed sufficiently to compensate uniformly,

chamber enlargement proves to be out of proportion to wall

thickening. This results in unbalanced systolic wall stress

with elevated oxygen consumption and cardiac dysfunction

(Grossman et al. 1975).

Previously, it has been recognized that myocardial remod-

elling is accompanied by extracellular matrix changes char-

acterized mainly by interstitial fibrosis (Weber 2004).

© 2013 The Authors.

178 International Journal of Experimental Pathology © 2013 International Journal of Experimental Pathology

Int. J. Exp. Path. (2013), 94, 178–187

Furthermore, myocardial fibrosis was shown to be associated

with cardiac dysfunction and heart failure (Weber & Brilla

1991). The contributing role of myocardial fibrosis in the

development of pathologic hypertrophy has been demon-

strated by clinical findings showing a poor cardiovascular

outcome (Brilla et al. 2003). The presence of myocardial

fibrosis has been disputed in aortocaval fistula (Dolgilevich

et al. 2001; Cox et al. 2004), but seems obvious when left

ventricular systolic dysfunction is present (Dolgilevich et al.

2001; Guido et al. 2007). Previously we have demonstrated

that the accumulation of excess collagen fibres occurs in

chronic aortocaval fistula within the subendocardium predo-

minantly (Guido et al. 2007). Also it has been recognized

that extracellular matrix must be remodelled by the activa-

tion of metalloproteinase (MMP) to allow cardiac chamber

to enlarge (Brower & Janicki 2001). Oxidative stress (Cox

et al. 2002), leucocyte migration (Kolpakov et al. 2009),

degranulation of mast cells (Brower et al. 2002), release of

cytokines (Cox et al. 2002; Guido et al. 2007) may all

potentially activate myocardial MMPs. The inflammatory

response and MMP activation have been suggested to result

from increased preload (Brower et al. 2002). However, in

previous aortocaval fistula experiments, we showed that left

ventricular end-diastolic pressure, an index of preload, had

no significant correlation with MMPs activation in contrast

to low myocardial perfusion pressure which substantially

contributed to left ventricular subendocardial fibrosis (Guido

et al. 2007). These findings suggested that, in addition to

volume overload, the heart has to respond to a chronic is-

chaemic process resulting in fiber loss, inflammation and

fibrosis, all occurring predominantly in the subendocardial

region of the left ventricle. Accordingly, in rats with myo-

cardial infarction, also an experimental model of volume

overload, we found myocyte necrosis, inflammatory cell

infiltration, fibroplasia and reparative fibrosis within the

subendocardial layer of the non-infarcted left ventricle (de

Carvalho Frimm et al. 2003; Koike et al. 2005). Subendo-

cardial fibrosis was significantly and independently related

to left ventricular dilatation and contributed to the develop-

ment of left ventricular dysfunction (Koike et al. 2007).

We hypothesize that the ability of cardiac adaptation to vol-

ume overload may be restricted by ischaemic myocyte losses.

This seems more likely to occur in the acute phases post-aor-

tocaval fistula when left ventricular end-diastolic pressure

increases concomitantly to systemic blood pressure decrease.

The early pathologic changes following aortocaval fistula la

have not been investigated yet. As long as our hypothesis is

correct, we anticipate the development of ischaemia, myocyte

necrosis, inflammation, MMPs activation, all predominantly

occurring in the subendocardial region of the left ventricle and

potentially contributing to cardiac dysfunction.

Objective

To investigate myocardial perfusion and cardiac structural

and functional changes during the early phases post-aorto-

caval fistula.

Methods

Male Wistar rats, weighing 300–350 g, were used. Animals

were divided into two groups undergoing either aortocaval

fistula creation or sham surgical treatment and submitted to

hemodynamic studies Morphometry was performed on 10%

formalin fixed tissue. For geometric determination of left ven-

tricular enlargement the heart was fixed under a perfusion

pressure corresponding to the in vivo systemic diastolic blood

pressure determined at sacrifice. Small samples of liver and

lung tissue were weighed before and after 48 h storage at

50 °C. The water content (%) of these tissue samples was cal-

culated as the percentage difference between wet and dry

weights of each organ and used as an estimate of heart failure.

The following experimental groups were constituted:

� Eighteen sham and 25 aortocaval fistula animals were

submitted to hemodynamic measurements at day 1;

among them, eight sham and 11 aortocaval fistula had

morphometric studies and nine sham and 14 aortocaval

fistula geometric studies.

� Ten sham and 11 aortocaval fistula animals were submit-

ted to hemodynamic measurements at day 3; among

them, five sham and 11 aortocaval fistula had morpho-

metric studies and seven sham and 11 aortocaval fistula

geometric studies

� Eighteen sham and 27 aortocaval fistula animals were

submitted to hemodynamic measurements at day 7;

among them, eight sham and 14 aortocaval fistula had

morphometric studies and eight sham and 13 aortocaval

fistula geometric studies.

� Seven sham and 11 aortocaval fistula animals were submit-

ted to measurements of myocardial blood flow at day 7.

� Four aortocaval fistula animals with coronary driving

pressure � 60 mmHg were used to measure tissue inter-

leukin levels and myeloperoxidase activity (MPO) and

compared with six non-operated control rats at days 1, 3

and 7 to avoid the inflammatory response caused by sur-

gery itself.

� Four aortocaval fistula animals with coronary driving

pressure � 60 mmHg and four aortocaval fistula animals

with coronary driving pressure >60 mmHg were submit-

ted to the measurement of MMP-2 activity at days 1, 3

and 7 and compared with a non-operated control rat

devoid of inflammation.

Ethical approval

All procedures were performed in accordance with the

norms of the Brazilian College of Animal Research. The

study protocol was approved by the ethics committee of the

Medical School, Sao Paulo University (Cappesq-HCFMUSP,

protocol number 1071/08).

Experimental model

Surgical procedures were performed using a previously

described and modified technique (Garcia & Diebold 1990).

International Journal of Experimental Pathology, 2013, 94, 178–187

Subendocardial remodeling in aortocaval fistula 179

Briefly, under anaesthesia with 10% chloral hydrate

(330 mg/kg, i.p.) (Zausinger et al. 2002) and positive

pressure ventilation (Rodent Ventilator, model 683; Har-

vard, South Natick, MA, USA), a median laparotomy was

performed, and the abdominal aorta and inferior vena cava

were identified and isolated from neighbouring structures

below the emergence of the renal arteries. Arterial and

venous blood flows were briefly interrupted with vascular

clamps to allow the insertion of an 18-G needle into the

aorta. The needle was advanced carefully, and the posterior

aortic wall was punctured three to four times aiming at the

vena cava wall. After removing the needle, homoeostasis

was conducted with local application of superglue. The

abdominal wall was closed, and the animals were returned

to their cages after full recovery. Fistula patency was

assessed daily using a paediatric stethoscope. Mortality rate

was 22% at day 1, 20% at day 3 and 24% at day 7.

Haemodynamics

Under general anaesthesia, rats underwent systemic and left

ventricular haemodynamic measurements. Briefly, a 0.5 mm

polyvinyl polychloride catheter was inserted through the

right carotid artery into the ascending aorta and advanced

into the left ventricle. Another similar catheter was inserted

into the right jugular vein and advanced to the right

atrium. Both catheters were connected to pressure transduc-

ers coupled to a calibrated preamplifier (General Purpose

Amplifier 4 model 2; Stemtech Inc., Wiscosin, USA). Pres-

sure tracings were recorded and analysed using a computer-

ized system processor (Windaq AT/Codas; Dataq

Instruments, Akron, OH, USA). Once complete stabiliza-

tion of the pressure curves was achieved, haemodynamic

measurements were taken. Computed values correspond to

the average of beat-to-beat measurements of each haemody-

namic parameter were recorded continuously over a 10-

minute period.

The following parameters were measured: systemic sys-

tolic and diastolic blood pressure (mmHg), right atrial mean

pressure (mmHg), left ventricular systolic and end-diastolic

pressure (mmHg) and maximum rates of increase and

decrease of left ventricular pressure (+dP/dt and �dP/dt,

respectively, mmHg/s). Coronary driving pressure was calcu-

lated as the difference between aortic pressure and left ven-

tricular pressure at end-diastole and used as an estimation

of myocardial perfusion pressure (Cross et al. 1961).

Morphometry

Immediately after the haemodynamic study, rats were killed

using an anaesthetic overdose, and the heart was arrested in

irreversible diastole using 100 mM/l cadmium chloride solu-

tion (Capasso et al. 1992). The heart was removed, cleansed

and relative heart weight index (g/Kg) determined. Subse-

quently, the right atrium and the left atrium were separately

dissected from the ventricles, and relative weight index (g/

Kg) of each was calculated.

Following fixation, a 1–2 mm transverse slice of the heart,

including both ventricles, was obtained at the equatorial

plane, embedded in paraffin and cut into 5-lm sections. Tis-

sue sections stained with haematoxylin and eosin, and Sirius

red underwent morphometric and geometric studies using an

image analysis system (Nikon Image System Elements AR

3.1, Japan).

The areas corresponding to the interventricular septum,

the left ventricular free wall, and the left ventricular total

cavity were determined separately using a telemacro lens

(Carl Zeiss, Vario Tessar, 2.8–5.8/5.35–21.4, Oberkochen,

Germany). The appropriateness of eccentric left ventricular

hypertrophy was calculated by the ratio between the areas

of interventricular septum plus left ventricular free wall and

the entire area of the left ventricle including walls and

chamber cavity.

Left ventricular hypertrophy was examined using haemat-

oxylin and eosin stained sections under 9 1000 magnifica-

tion. Myocyte diameter (lm) was measured around oval and

central nuclei of longitudinally displayed myocytes.

Myocyte necrosis, inflammatory cell infiltration, fibropla-

sia and left ventricular fibrosis were examined separately

taking into account two regions: the subendocardium, corre-

sponding to the inner one-third of the left ventricular wall,

and the non-subendocardium, corresponding to the remain-

ing outer two-thirds. The inner third of the left ventricle has

been previously shown to be the most susceptible myocar-

dial region to ischaemia during low perfusion events (Buck-

berg et al. 1972).

Myocyte necrosis (cells/mm2) was examined in haemat-

oxylin and eosin stained tissue sections under 9 1000 mag-

nification. Nuclear pyknosis and karyolysis as well as

cytoplasmatic changes including vacuolization, contraction

bands and hypereosinophilia were taken into account alto-

gether (Kumar et al. 2012).

Leucocyte infiltration (cells/mm2) was examined in hae-

matoxylin and eosin stained sections under 9 1000 magnifi-

cation. Inflammatory cells were identified by nuclear and

cytoplasmic typical aspects. Cells with morphological char-

acteristics of fibroblasts, cardiomyocytes, endothelial vascu-

lar cells and smooth muscle cells were excluded (Gartner &

Hiatt 2009).

Fibroplasia was examined by measuring positive alpha-

smooth muscle actin myofibroblasts (cells/mm2) detected by

immunohistochemistry under 9 1000 magnification. The tis-

sue sections were incubated with a 1:800 dilution of the

mouse antihuman alpha-smooth muscle actin antibody

(Sigma Aldrich, St. Louis, USA). Sections were then incubated

with labelled streptavidin-biotin-peroxidase kit (Dako Cyto-

mation LSAB+ System-HRP, California, USA) and diam-

inobenzidine for colour development. Finally, the sections

were faintly counterstained with haematoxylin. Vascular

smooth muscle cells with intense staining were used as posi-

tive controls. Negative controls were obtained by omitting the

primary antibody and using a non-immune bovine serum.

Collagen volume fraction (%) was determined in Sirius

red-stained tissue sections under 9 580 magnification


180 F. R. R. Mazzo et al.

(Junqueira et al. 1979). The collagen volume fraction was

calculated as the per cent of red-stained connective tissue

areas per total myocardium, excluding perivascular areas

(de Carvalho Frimm et al. 1997).

For myocyte necrosis and leucocyte cell infiltration, a

total amount of 15 microscopic fields were examined in

each subendocardium and non-subendocardium regions. For

collagen volume fraction, a total amount of 20 microscopic

fields were examined in each subendocardium and non-sub-

endocardium regions. For myocyte hypertrophy, a total

amount of 20 fields of the interventricular septum were

examined.

Myocardial blood flow

To estimate myocardial blood flow, the colour microsphere

method was used (Hakkinen et al. 1995; De Angelis et al.

2005). Briefly, rats were anesthetized, and the femoral and

carotid arteries were catheterized as already described. The

femoral artery catheter was advanced into the aorta and the

carotid artery catheter into the left ventricle to withdraw

blood and to administer 180 ll of 300,000 yellow micro-

spheres in suspension respectively (Dye-Trak CM; Triton

Technology, USA).

After removing the heart, the left ventricle was weighed

separately. Left ventricular subendocardial and non-suben-

docardial regions were dissected apart and, in addition to a

blood sample, were processed to calculate regional myocar-

dial blood flow.

MPO activity, Interleukin expression, and MMPs activ-

ity. After the sacrifice, the hearts were rapidly removed,

and a slice corresponding to the medium one-third of the

left ventricular myocardium was cut off at the equatorial

plane. For each analysis, tissue samples were obtained from

two distinct myocardial regions carefully trimmed away: the

inner subendocardial one-third and the outer non-subendo-

cardial left ventricular wall. All samples were snap-frozen in

liquid nitrogen.

For MPO activity, each of the two distinct left ventricular

tissue samples was homogenized on 0.5% hexadecyltrime-

thylammonium bromide in 10 mM/l 3-N-morpholinopro-

panesulphonic acid and centrifuged at 15,000 g for 40 min

(Soriano et al. 2002). An aliquot of the supernatant was

mixed with 1.6 mM/l tetramethylbenzidine and 1 mM/l

hydrogen peroxide. Activity was measured spectrophotomet-

rically as the change in absorbance at 650 nm at 37 °C,using a Spectramax microplate reader (Molecular Devices,

Sunny-vale, CA, USA). Results are expressed as mU MPO

activity/mg protein determined by the Bradford protein

assay (Bio-Rad).

For interleukin and MMPs analysis, the two distinct left

ventricular tissue samples were grounded in ice-cold RIPA

buffer (20 mM/l Tris-HCl pH 7.5, 0.5% deoxycholic acid,

0.1% SDS, 150 mM/l NaCl, 2 mM/l sodium pyrophosphate

and 20 mM/l NaF) containing 1% Triton X-100 and agi-

tated for 1 h at 4 °C. Upon completion of the extraction

incubation, samples were centrifuged (4 °C, 15 min,

10,000 g). Supernatant samples were divided in aliquots and

stored at �80 °C. Protein concentrations were determined

by the Bradford protein assay (Bio-Rad).

Tumour necrosis factor-alpha, interleukin-1beta, interleu-

kin-6 and interleukin-10 were measured in each of the two

distinct left ventricular samples by ELISA, according to the

manufacturer’s instructions (R&D Systems, Minneapolis,

MN, USA) (Deten et al. 2002).

Gelatin zymography was performed for evaluation of

MMPs activity using non-reducing, non-denaturating SDS-

PAGE. For this analysis, equal amounts of protein from

each sample were mixed with non-reducing Laemmli SDS

sample buffer and electrophorized at 20 °C in a 10% poly-

acrylamide gel containing 1 mg/ml gelatin. Later, gels were

washed for 1 h in 2% Triton X-100 to remove the SDS and

allow enzyme renaturation. Gels were then placed in enzyme

activation buffer (50 mM/l Tris-HCl, pH 7.4, 200 mM/L

NaCl, 5 mM/L CaCl2 and 0.02% NaN3; all from Sigma (St

Louis, MO, USA) for 18 h at 37 °C. Gels were then stained

with 0.1% Coomassie brilliant blue, and the activity of the

MMPs bands was quantified by densitometry (ImageQuant

LAS4000, GE Healthcare Bio-Sciences, Uppsala, Sweden).

Zymograms showed two lytic bands corresponding to gelati-

nase A (MMP-2) pro-enzyme (68 kDa) and activated

(62 kDa) forms. To normalize our results, subendocardial

and non-subendocardial left ventricular extracts from a single

non-operated rat were used. Percentage activities from left

ventricular tissue samples belonging to the same group were

averaged (Brower et al. 2002).

Statistical Analysis. Data are expressed as the mean � SD.

Normal distribution and equality of variances were tested.

Student′s t-test or Mann–Whitney rank test were used for

morphometric and hemodynamic comparisons, and one-

way repeated-measures ANOVA, complemented by Bonfer-

roni′s test, was used for myocardial blood flow, MPO, in-

terleukins and MMP-2 comparisons between groups, taking

into account subendocardial and non-subendocardial

regions.

Linear regression was used to test the potential relation-

ships between MMP-2 activity and coronary driving pres-

sure, between coronary driving pressure and myocardial

blood flow, and between myocardial blood flow and left

ventricular function. Statistical significance was established

as P < 0.05. Analyses were performed using Sigma Stat Sta-

tistical Software (version 3.1; Jandel Scientific Software, San

Rafael, CA, USA) and SAS software (Statistical Analysis

System, version 9.1; SAS Institute Inc., Cary, NC, USA).

Results

Haemodynamics

Haemodynamic results corresponding to the three follow-up

periods are depicted in Table 1. Compared with sham, aor-

tocaval fistula groups had significantly lower systemic blood



pressure and higher left ventricular end-diastolic pressure

resulting in coronary driving pressure 48% lower at day 1,

52% lower at day 3 and 56% lower at day 7. Also,

right atrial mean pressure was comparatively elevated in

aortocaval fistula groups. Left ventricular systolic dysfunc-

tion occurred at the three follow-up periods and left ventric-

ular diastolic dysfunction at days 1 and 7.

Morphometry

The results regarding the morphometric study are shown in

Table 2. Heart failure was evidenced by significantly higher

lung percentage of water content found in aortocaval fistula

rats at the three time points assessed. Heart failure was also

demonstrated to occur according to Davidoff′s criteria

(Davidoff et al. 2004). The ratio between lung and body

weight was higher (> mean + 2 SD) in aortocaval fistula

than in sham (day 1: 6.2 vs. 5.6, day 3: 6.8 vs. 5.5, and day

7: 7.3 vs. 7.1 respectively).

Left ventricular hypertrophy was evidenced by increased

myocyte diameter at days 3 and 7. The calculated ratio

between interventricular septum plus left ventricular free

wall area and total left ventricular area including the cham-

ber cavity showed that hypertrophy was inappropriate.

Myocytes with signs of necrosis were found at days 1 and

3 in the subendocardium where they outnumbered those in

the non-subendocardium by fivefold.

Leucocyte infiltration was evident throughout all acute

phases, but was more pronounced during the two earlier

periods in the subendocardial region where inflammatory

cells outnumbered those in the non-subendocardium by two-

fold (Figure 1).

Table 1 Haemodynamic measurements at days 1, 3 and 7 following aortocaval fistula

Group sham1 ACF1 sham3 ACF3 sham7 ACF7

SBP (mmHg) 116 � 6 88 � 17† 121 � 4 83 � 28† 122 � 6 96 � 21†

DBP (mmHg) 84 � 13 51 � 19* 92 � 5 46 � 21† 93 � 6 66 � 21†

CDP (mmHg) 77 � 14 37 � 18† 86 � 5 29 � 20† 86 � 6 48 � 19†

RAP (mmHg) 4.6 � 1.8 7.1 � 2.9* 5.6 � 2.3 8.8 � 2.1* 4.7 � 2.5 9.1 � 2.8*LVSP (mmHg) 122 � 15 106 � 14* 115 � 12 104 � 11 129 � 13 109 � 15*LVEDP (mmHg) 7 � 3 15 � 6† 7 � 3 18 � 3* 7 � 3 18 � 5†

+dP/dt (mmHg/s) 7898 � 4045 5828 � 2262† 5594 � 571 4553 � 983* 7924 � 2317 4564 � 2044*�dP/dt (mmHg/s) 6626 � 1717 4728 � 863† 5309 � 1325 4528 � 803 6435 � 1302 4750 � 1442*

ACF, aortocaval fistula; 1, day 1; 3, day 3; 7, day 7; SBP, systolic blood pressure; DBP, diastolic blood pressure; CDP, coronary driving pres-

sure; RAP, mean right atrial pressure; LVSP, left ventricular systolic pressure; LVEDP, left ventricular end-diastolic pressure; +dP/dt, maxi-

mum rate of increase of left ventricular pressure; �dP/dt, maximum rate of decrease of left ventricular pressure.

*P < 0.05 vs. sham (Student’s t-test).†P < 0.05 vs sham (MannWhitney test).

Table 2 Morphometric measurements at days 1, 3 and 7 following aortocaval fistula

Groups sham1 ACF1 sham3 ACF3 sham7 ACF7

Heart weight (g/Kg) 3.2 � 0.3 3.5 � 0.6 3.6 � 0.1 3.3 � 0.3 3.4 � 0.2 3.2 � 0.8Right atrium (g/Kg) 0.11 � 0.02 0.18 � 0.08* 0.11 � 0.03 0.19 � 0.09* 0.10 � 0.03 0.2 � 0.18*Left atrium (g/Kg) 0.12 � 0.02 0.12 � 0.02 0.10 � 0.02 0.12 � 0.05 0.11 � 0.03 0.13 � 0.03

Lung percentage of water content (%) 76.5 � 4.4 83.7 � 4.0* 79.0 � 2.0 82.8 � 2.0* 78.1 � 2.0 80.7 � 1.0*Liver percentage of water content (%) 75.2 � 0.1 74.7 � 2.0 73.2 � 0.5 74.0 � 3.0 73.8 � 0.4 78.3 � 1.0*IVS + LV free wall area / total LV area 0.77 � 0.04 0.71 � 0.10 0.88 � 0.02 0.71 � 0.15* 0.77 � 0.07 0.60 � 0.12*Myocyte diameter (lm) 10.0 � 0.6 11.0 � 1.4 10.0 � 0.2 14.0 � 0.1* 11.3 � 1.1 14.3 � 1.0*Myocyte necrosis (cells/mm2)SE 114 � 5† 208 � 8*† 82 � 12† 148 � 31*† 34 � 12† 42 � 9†

non-SE 27 � 3 42 � 10* 21 � 3 31 � 6* 17 � 2 21 � 4

Leucocytes (cells/mm2)

SE 48 � 5 84 � 13*† 48 � 10 117 � 13*† 33 � 5 50 � 6*†

non-SE 33 � 4 40 � 6* 41 � 6 65 � 8* 27 � 6 38 � 7*Fibroplasia (cells/mm2)

SE 10.9 � 4.0 14.6 � 5.7 10.8 � 2.6 26.1 � 10.0*† 10.9 � 2.3 47.5 � 12.0*†

non-SE 10.9 � 1.6 10.9 � 2.6 9.8 � 1.6 11.7 � 2.6 10.8 � 2.2 15.3 � 2.5CVF (%)

SE 1.2 � 0.4 1.2 � 0.2† 1.7 � 0.3 5.4 � 0.1*† 1.8 � 0.4 10.3 � 2.3*†

non-SE 1.2 � 0.2 1.7 � 0.5* 1.4 � 0.3 1.8 � 0.1* 1.7 � 0.1 2.8 � 0.7*

ACF, aortocaval fistula; 1, day 1; 3, day 3; 7, day 7; IVS, interventricular septum; LV, left ventricle; CVF, collagen volume fraction.*P < 0.05 vs. sham†P < 0.05 vs. non-SE, Repeated-Measures ANOVA complemented by Bonferroni′s test.



Fibroplasia was evident during the late two periods in the

subendocardium where the number of alpha-actin-positive

myofibroblasts outnumbered those in the non-subendocardi-

um by two- to threefold.

Fibrosis deposition became evident by days 3 and 7

particularly in the subendocardium where collagen volume frac-

tion was threefold greater than in the non-subendocardium.

Myocardial blood flow

The haemodynamic changes of rats submitted to the study

of myocardial blood flow at day 7 did not differ from those

described before. Compared with sham, aortocaval fistula

showed lower myocardial blood flow, particularly within

the subendocardial region (Figure 2). The relationships

examined between coronary driving pressure, blood flow

and left ventricular function are depicted in Figure 3. As

coronary driving pressure was below the lower limit of cor-

onary blood flow auto regulation in all rats examined, a

positive correlation was found between coronary driving

pressure and myocardial blood flow. Furthermore, subendo-

cardial blood flow but not non-subendocardial blood flow

was positively related to both +dP/dt and �dP/dt.

MPO and Interleukins

The coronary driving pressure of aortocaval fistula groups

submitted to the measurement of MPO and interleukins was

below the lowest limit of coronary flow auto regulation.

MPO activity was increased in all aortocaval fistula groups

regardless the left ventricular region examined; interleukin-

1beta levels were increased at day 1 in subendocardium and

non-subendocardium; Tumour necrosis factor-alpha levels

were increased at day 3 in subendocardium; and interleukin-

6 levels were increased at day 7 in subendocardium (Fig-

ure 4). Interleukin-10 did not change in either region and

was comparable between the two groups at the three differ-

ent follow-ups.

MMP-2

MMP-2 activity was measured comparing between aorto-

caval fistula groups with coronary driving pressure either

� 60 mmHg or >60 mmHg (Figure 5). MMP-2 activity of

rats with coronary driving pressure � 60 mmHg was

increased in both subendocardium and non-subendocardium

at days 1 and 7 and only in subendocardium at day 3. There

was an inverse relationship between coronary driving pres-

sure and MMPs activity measured in subendocardium at the

three time points examined.

Discussion

The present study demonstrated that during the first week

of aortocaval fistula systemic blood pressure decreases and

left ventricular end-diastolic pressure increases resulting in

markedly low coronary driving pressure and in ischaemic

remodelling of the subendocardium. The direct relationship

found between left ventricular subendocardial blood flow

and function suggests that low coronary perfusion pressure

impairs the development of appropriate left ventricular

hypertrophy. The present results are in agreement with

our previous findings showing that subendocardial fibrosis

was associated with left ventricular dysfunction in chronic

Figure 1 Photomicrographs of the subendocardial layer of the left ventricle of rats with aortocaval fistula in HE, Sirius red andsmooth muscle alpha-actin immunohistochemistry-stained tissue sections. Panels a depicts intense cytoplasmatic vacuolization ofmyocytes, as necrosis signs at day 1; in panel b, inflammatory cell infiltration stained with HE at day 3; in panel c, fibroplasiaevidenced by myofibroblasts positively marked for smooth muscle alpha-actin in brown at day 7; and in panel d, Sirius red-stainedpatch of collagen fibres indicative of fibrosis repair at day 7.

Figure 2 Myocardial blood flow of subendocardial (SE, in blackbar) and non-subendocardial (non-SE, in white bar) layers ofthe left ventricle at day 7. ACF, aortocaval fistula. Statisticalanalysis: one-way repeated-measure ANOVA complemented byBonferroni′s test; *P < 0.05 vs. SHAM SE; #P < 0.05 vs.SHAM non-SE; $P < 0.05 vs. ACF non-SE.



aortocaval fistula (Guido et al. 2007). The importance of

decreased perfusion pressure and subendocardial damage to

pathologic remodelling following experimental acute aorto-

caval fistula had not being examined before. We have

observed the development of myocyte necrosis, followed by

inflammation, fibroplasia and fibrosis, all changes found

prominently within the subendocardial region.

Markedly low coronary driving pressure resulted in a cor-

responding deprivation of myocardial perfusion. In fact, val-

ues of coronary driving pressure currently below 60 mmHg

are under the inferior limits of auto regulation of myocar-

dial blood flow (Le et al. 2004). At these levels, driving

pressure directly determines perfusion and explains the posi-

tive relationship found between coronary driving pressure

and myocardial blood flow in the present study. Interest-

ingly, subendocardial hypoperfusion was associated with left

ventricular dysfunction, implying subendocardial ischaemic

damage for the cardiac inability to adapt to acute volume

overload. Indeed, myocyte diameter was enlarged as early as

3 days following aortocaval fistula, but left ventricular wall

to total left ventricular area ratio was reduced, indicating

that the hypertrophy was inappropriate. We suggest this has

occurred as a result of subendocardial ischaemic damage.

The determinant role of local MMPs and leucocyte infil-

tration in the pathophysiological mechanism resulting in

eccentric cardiac remodelling following aortocaval fistula

has been recognized (Kolpakov et al. 2009). This exagger-

ated MMPs activation may result in pathological remodel-

ling by restricting wall thickening of enough magnitude to

counteract chamber enlargement and elevated systolic wall

stress. Accordingly, left ventricular enlargement and dys-

function may be prevented by the inhibition of MMP activ-

ity (Cox et al. 2004; Brower et al. 2007). Previously we

have demonstrated that, in contrast to left ventricular end-

diastolic pressure, low coronary driving pressure and suben-

docardial fibrosis were the only independent determinants of

left ventricular dysfunction. In the present study, we found

an inverse relationship between coronary driving pressure

and MMP-2 activation within the subendocardial region.

MMP-2 activation accompanies myocyte necrosis at day 1,

increases further in association with the inflammatory pro-

cess at day 3 and still remains elevated at day 7. These

results confirm similar findings found during the chronic

phase (Guido et al. 2007) and support the hypothesis that at

least in part, MMPs are activated in response to myocardial

ischaemia that follows the decrease in perfusion pressure.

The determinant role of ischaemia in pathological remodel-

ling may be recognised better by the observation that relat-

ing it to outstanding numbers of myocytes undergoing

necrosis in the subendocardium at day 1. As many as 10%

of myocyte fibres showed necrotic changes, corresponding to

3–4% of the total number of left ventricular myocytes.

Myocyte necrosis also occurred beyond the SE region and

was still present 3 days after aortocaval fistula surgery.

Besides myocyte necrosis, local inflammation is likely to

have contributed to left ventricular dysfunction. The associa-

tion between cytokine levels and pathologic remodelling is

well recognized (Hwang et al. 2001). Elevated tumour

necrosis factor-alpha and interleukin-1beta levels have been

related to the development of myocardial fibrosis, myocyte

hypertrophy, left ventricular enlargement, apoptosis and

cardiac dysfunction (Yndestad et al. 2006). In the present

study, increased tissue levels of tumour necrosis factor-

alpha, interleukin-6, and interleukin-1beta, leucocyte

infiltration and MPO activation occurred predominantly in

the subendocardium.

The first region of the left ventricle to undergo ischaemia

in response to low perfusion pressure is the subendocardium

where, in comparison with the mid wall, there is a paucity

of collateral vessels, the metabolic activity is enhanced, and

oxygen extraction increased (Moss 1968; Weiss & Sinha

1978). Accordingly, the subendocardium is the left ventricu-

lar region showing the greatest myocyte losses following

non-transmural infarcts and the first region affected by

ongoing ischaemia following transmural infarcts (Kuwada

& Takenaka 2000). We have demonstrated using the rat

model of experimental infarction a predominant damage to

the subendocardial non-infarcted region (Koike et al. 2007).

Furthermore, the decrease in coronary driving pressure that

followed acute infarction was the hemodynamic parameter

that best explained subsequent left ventricular enlargement

and dysfunction (de Carvalho Frimm et al. 2003; Koike

et al. 2007). As a matter of fact, in both these two experi-

mental models of acute volume overload, infarction and

(a) (b)

(c) (d)

Figure 3 The relationships found between coronary perfusionpressure and blood flow and between coronary blood flow andleft ventricular function of rats with aortocaval fistula at day 7.Graphs represent the relationships of coronary driving pressure(CDP) with subendocardial (SE) and non-subendocardial (non-SE) blood flow (panels a and b); and of SE blood flow with+dP/dt and –dP/dt (panels c and d). Statistical analysis: linearregression.



aortocaval fistula, our findings indicate that cardiac dysfunc-

tion seems to result more likely from low perfusion pressure

than from increased preload. The limited ability of the myo-

cardium to adapt to acute increases in preload appears lar-

gely a consequence of ischaemic myocyte losses occurring

predominantly in the subendocardium. Remnant myocytes

increase in width but wall thickening turns out to be inap-

propriate to maintain a proportional wall to volume ratio

and to preserve left ventricular function.

The present results indicate that heart failure develops

early after experimental aortocaval fistula as a consequence

of low coronary perfusion pressure that jeopardizes suben-

docardial integrity and limits the ability of the heart to

adapt to volume overload.

Study limitations

We assessed myocyte necrosis in paraffin-embedded tissue

sections stained with haematoxylin and eosin, which may

give rise to artefacts represented mostly by contraction

bands. In fact, both aortocaval fistula and sham animals

showed histological evidence of myocardial contraction

bands. However, other parameters of myocyte necrosis such

as pyknosis, karyolysis, karyorrhexis and eosinophilic cyto-

plasm staining were also taken into account, and for that

reason, we believe that the assessment of myocyte damage

was not biased.

We found signs consistent of some degree of subendocar-

dial damage other than contraction bands in sham rats.

These changes were of little magnitude and did not result in

fibrosis deposition, meaning they have most likely occurred

during sacrifice. Potential causes include factors related to

the experimental procedure and to the anaesthesia. The

impairment of venous return and the decrease in systemic

blood pressure associated with positive pressure ventilation

might have jeopardized subendocardial perfusion to some

extent. The effects of anaesthetic drugs with different hae-

modynamic properties on myocardial perfusion have been

poorly investigated to date and deserve more attention

(Rodrigues et al. 2006).

This study lacks an appropriate estimation of the degree of

volume overload to which the animals were submitted

because the magnitude of fistula shunt was not assessed.

Fistula dimension may be estimated indirectly by pulse

pressure. In contrast to the present findings, pulse pressure

has been shown to be elevated in chronic aortocaval fistula

(Guido et al. 2007). The absence of pulse pressure elevation

in the acute phase post-aortocaval fistula is likely to be due

to the recent surgical trauma. Blood losses and fluid seques-

tration into the third space in response to surgical trauma

Figure 4 Myeloperoxidase activity (MPO) (panel a), interleukin levels (IL-1b and IL-6; panels b and d respectively) and tumournecrosis factor-alpha level (TNF-a; panel c) measured in the subendocardial (SE) and non-subendocardial (non-SE) layers of the leftventricle of rats with aortocaval fistula (ACF) and coronary driving pressure � 60 mmHg at days 1, 3 and 7. Statistical analysis:one-way repeated-measure ANOVA complemented by Bonferroni′s test; *P < 0.05 vs. control SE; †P < 0.05 vs. control non-SE.



may have impaired venous return and reduced systemic

blood pressure.

Systemic volume overload has been well recognized to

represent a hemodynamic burden to both cardiac chambers.

Unfortunately, right ventricular pressure or function were

not determined. In the absence of tricuspid valve dysfunc-

tion, however, right atrial changes indeed indicated the pres-

ence of right ventricular dysfunction. The importance of

right as opposed to left ventricular remodelling in contribut-

ing to heart failure was beyond the scope of this study.

Whether or not similar subendocardial remodelling changes

observed in the left ventricle also occurs in the right ventri-

cle has yet to be investigated.

Clinical implications

The present findings suggest that the adequacy of myocardial

perfusion, particularly to the left ventricular subendocardium,

is expected to be negatively affected by low coronary driving

pressure, which may occur especially in the setting of acute

volume overload and low systemic blood pressure.

Conclusion

Subendocardial ischaemic remodelling in response to low

coronary driving pressure may result in left ventricular dys-

function during the acute phases of aortocaval fistula.

Conflict of interest

There are no conflicts of interest.

Funding source

This work was supported by Fundacao Faculdade de Medi-

cina, University of Sao Paulo, and Conselho Nacional de

Desenvolvimento Cientıfico e Tecnologico (CNPq), Brazil.

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(a) (b) (c)

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Acute aortocaval fistula: role of low perfusion pressure and subendocardial remodeling on left ventricular function