Time-Dependent Changes in Matrix Metalloproteinase

Activity and Expression During the Progression of

Congestive Heart Failure
Relation to Ventricular and Myocyte Function
Francis G. Spinale, Mytsi L. Coker, Chadwick V. Thomas, Jennifer D. Walker,
Rupak Mukherjee, Latha Hebbar

AbstractThe development of congestive heart failure (CHF) is associated with left ventricular (LV) dilation and myocardial
remodeling. However, fundamental mechanisms that contribute to this remodeling process with the progression of CHF
remain unclear. The matrix metalloproteinases (MMPs) have been demonstrated to play a significant role in tissue
remodeling in a number of pathological processes. The present project tested the hypothesis that the LV dilation and
remodeling during the progression of CHF is associated with early changes in MMP expression and zymographic activity.
LV and myocyte function, collagen content, and MMP expression and zymographic activity were serially measured during
the progression of CHF caused by pacing-induced supraventricular tachycardia (SVT) in pigs. After 7 days of SVT, LV
end-diastolic dimension and myocyte length both increased by 15% from control values, and LV fractional shortening fell
by 20%. At the level of the myocyte, percent shortening fell by 16% after 7 days of SVT, with no change in the steady-state
velocity of shortening. Longer durations of SVT caused progressive LV dilation, LV pump failure, and myocyte contractile
dysfunction. Specifically, 21 days of SVT resulted in a .50% increase in LV dimension, a 56% fall in LV fractional
shortening, and a 33% decline in myocyte velocity of shortening. The decline in LV and myocyte function with 21 days
of SVT was accompanied by signs and symptoms of CHF. Thus, SVT causes time-dependent changes in LV geometry and
function and the subsequent development of CHF. LV myocardial collagen content and confluence fell by .25% after 7
days of SVT and were accompanied by an 80% increase in LV myocardial MMP zymographic activity against the substrate
gelatin. After 14 days of SVT, total LV myocardial collagen content was reduced by 24%, and LV myocardial MMP
zymographic activity increased by .100% from control values. Interstitial collagenase (MMP-1), stromelysin (MMP-3),
and 72-kD gelatinase (MMP-2) were increased by '2-fold after 7 days of SVT. LV MMP zymographic activity and
abundance remained elevated with longer durations of SVT. The results of the present study demonstrated that in this
model of CHF, early changes in LV myocardial MMP zymographic activity and protein levels occurred with the initiation
and progression of LV dilation and dysfunction. These findings suggest that an early contributory mechanism for the
initiation of LV remodeling that occurred in this model of developing CHF is enhanced expression and potentially
increased activity of LV myocardial MMPs. (Circ Res. 1998;82:482-495.)
Key Words: heart failure n metalloproteinase n myocardial remodeling

A n important event in the progression to CHF is LV

dilation and subsequent pump dysfunction.13 These past
clinical observations suggest that LV remodeling is an impor-
tant initiating event in the transition to severe CHF. However,
the cellular and molecular mechanisms that play a direct
contributory role in the initiation and progression of changes
in LV geometry and function during the development of the
CHF process are unknown. Chronic pacing-induced
tachycardia in animals causes well-defined, predictable, and
progressive LV dilation, contractile dysfunction, and neuro-
hormonal system activation.4 15 These functional and neuro-
hormonal changes are similar to the clinical spectrum of
CHF.13,16,17 Therefore, this chronic pacing model may provide
an opportunity to identify the early and contributory events
responsible for the progression of LV dilation and dysfunction
that occurs with CHF. It has been demonstrated that the
structure and composition of the fibrillar collagen matrix,
which provides structural integrity of adjoining myocytes,18 21
are significantly altered in pacing-induced CHF.7,9,1215,22 Fur-
thermore, we have recently reported that concomitant ACE
inhibition with chronic tachycardia preserved myocardial col-
lagen content and reduced the LV dilation associated with this
disease process.9 These past studies provide indirect evidence
that changes in the myocardial collagen matrix contribute to

Received September 3, 1996; accepted December 15, 1997.

From the Division of Cardiothoracic Surgery, Medical University of South Carolina, Charleston, SC.
Correspondence to Francis G. Spinale, MD, PhD, Division of Cardiothoracic Surgery, Room 418 CSB, 171 Ashley Ave, Medical University of South
Carolina, Charleston, SC 29425.
1998 American Heart Association, Inc.

482
 Spinale et al
Selected Abbreviations and Acronyms
ACE 5 angiotensin-converting enzyme
CHF 5 congestive heart failure
LV 5 left ventricle (ventricular)
MMP 5 matrix metalloproteinase
PMA 5 phorbol 12-myristate 13-acetate
PMSF 5 phenylmethylsulfonyl fluoride
SVT 5 pacing-induced supraventricular tachycardia
TIMP 5 tissue inhibitor of MMP
the LV dilation and subsequent dysfunction that occur in this
rapid-pacing model of CHF. Accordingly, the overall goal of
this project was to define the time-dependent structural and
molecular events that occur within the extracellular matrix
during the progression of pacing-induced CHF.
The MMPs constitute an important enzyme system that has
been demonstrated to contribute to the tissue remodeling
process.2330 The MMPs are a class of zinc-dependent enzymes
that have a high specificity for components of the extracellular
matrix.2330 Increased expression and activity of MMPs have
been identified in pathological processes such as tumor metas-
tases and rheumatoid arthritis.24,28,29 Increased MMP expression
and activity have been identified in atherosclerotic lesions31 and
have been implicated in atheroma formation and plaque
rupture.30 Increased myocardial MMP activity has also been
reported with the development of severe CHF, such as in
cardiomyopathic disease.3234 However, whether increased
MMP zymographic activity and abundance are early events in
the evolution of the CHF process remains unexplored. Ac-
cordingly, the present study was designed to determine the
relationship of time-dependent changes in MMP expression
and zymographic activity to LV and myocyte function and
geometry during the progression of CHF.
Materials and Methods
The present project used a model of chronic SVT in pigs that has been
shown clearly in previous studies to produce signs and symptoms of
CHF after 3 weeks of SVT.5 8 LV geometry and function, neurohor-
monal system activity, myocyte contractility, myocardial collagen
content and structure, and MMP expression and activity were
measured with each week of SVT.
Model of Pacing CHF and Experimental Design
Thirty Yorkshire pigs (20 to 25 kg, male) were chronically instru-
mented in order to measure LV function and arterial blood pressure
while they were conscious. The pigs were anesthetized with isoflurane
(3%, 1.5 L/min) and nitrous oxide (0.5 L/min), intubated with a
cuffed endotracheal tube, and ventilated at a flow rate of 22 mL z kg21 z
min21 and a respiratory rate of 15/min. The left internal carotid artery
was exposed, and a catheter was connected to a vascular access port
(model GPV, 9F, Access Technologies), advanced to the aortic arch,
and sutured in place. The access port was buried in a subcutaneous
pocket over the thoracolumbar fascia. Through a left thoracotomy, a
shielded stimulating electrode was sutured onto the left atrium,
connected to a modified programmable pacemaker (model 8329,
Medtronic, Inc), and buried in a subcutaneous pocket. The pericar-
dium was left open, the thoracotomy was closed, and the pleural space
was evacuated of air. The animals were allowed a recovery period of
7 to 10 days. Six pigs each were then randomly assigned to undergo
7, 14, or 21 days of SVT or to serve as sham-operated controls. In six
pigs, measurements were obtained throughout the 21-day pacing
protocol in order to determine temporal changes with respect to in
483
vivo indices of LV contractile performance. Thus, for indices of LV
size and function and neurohormonal activity, observations were
performed on 12 pigs at baseline and with each week of SVT. With
respect to LV myocyte function, collagen biochemistry, morphome-
try, and MMP zymographic activity, complete studies were performed
on six pigs in the control state and with each week of SVT. All animals
used in the present study were treated and cared for in accordance
with the National Institutes of Healths Guide for the Care and Use of
Laboratory Animals (National Research Council, 1985, NIH publica-
tion 86 23).
LV Function and Plasma Collection
Indices of LV pump function were obtained from simultaneously
recorded pressure and echocardiographic measurements previously
described by this laboratory.59 Briefly, the animals were sedated with
diazepam (20 mg PO, Valium, Hoffmann-La Roche) and placed in a
custom-designed sling that allowed the animal to rest comfortably.
The pacemaker was deactivated (SVT groups only). All studies were
performed after a 30-minute acclimation period with the animals in
the conscious state and without additional use of sedation. The
vascular access port was entered using a 12-gauge Huber needle
(Access Technologies), and basal resting arterial pressure and heart rate
were recorded. Pressures from the fluid-filled aortic catheter were
obtained using an externally calibrated transducer (Statham P23ID,
Gould). Two-dimensional and M-mode echocardiographic studies
(ATL Ultramark 7, 3.5-MHz transducer) were used to image the LV
from a right parasternal approach. Echocardiographic data were
measured with the use of American Society of Echocardiography
criteria, including the leading-edge convention. The two-dimensional
parasternal long-axis view of the LV was first recorded in order to
precisely define the LV long axis and papillary muscles. A perpendic-
ular view with respect to the LV long axis was then obtained in order
to obtain the two-dimensional parasternal short-axis view. LV short-
axis two-dimensional and M-mode echocardiographic recordings
were then obtained. The LV dimensions were performed from the
septum to the posterior LV free wall with the cursor directed between
the papillary muscles. LV end-diastolic and end-systolic dimension,
LV end-systolic and end-diastolic wall thickness, and fractional short-
ening were computed from the two-dimensional targeted M-mode
recordings.8,10,35 Peak circumferential global average wall stress was
computed using a spherical model of reference8,12: s(g/cm2)5[PD/4
hours(11h/D)]31.36, where P is aortic systolic pressure, D is
minor-axis dimension at end diastole, and h is wall thickness. After
steady-state LV function measurements, 35 mL of blood was drawn
from the arterial access port into chilled tubes containing EDTA (1.5
mg/mL). The blood samples were immediately centrifuged (2000g, 10
minutes, 4C), and the plasma was decanted into separate tubes, frozen
in a dry ice/methanol bath, and stored at 280C until the time of
assay.
LV fractional shortening was measured after incremental increases
in LV myocardial wall stress8,36 38 in five pigs in the normal state and
with each week of SVT. Simultaneous recordings of two-dimensional
targeted M-mode echocardiograms and aortic pressure were obtained
by a phenylephrine infusion started at a rate of 1.3 mg z kg21 z min21
and increased in such a manner as to obtain three to six isochronal LV
peak circumferential wall stress versus shortening points for each pig
with each week of SVT. This approach for measuring the LV
shortening-stress relation has been described previously by this labo-
ratory and others.8,36 38 Determination of the LV shortening-afterload
relation was chosen, since it provides a relative index of LV contractile
performance and does not require theoretical muscle models and
development of LV pressure-volume loops.39,40 However, this ap-
proach may not completely describe in vivo LV myocardial contractile
performance; accordingly, the LV systolic stiffness constant was
determined using methods described previously.41,42 Briefly, isochronal
LV wall stress-strain values were obtained under ambient conditions
and after phenylephrine infusion. The LV myocardial stiffness constant
k, was determined from the following exponential relationship:
s5Cekln(1/h), where s is wall stress and ln(1/h) is the natural logarithm
of the reciprocal of LV wall thickness. This in vivo index of LV
484 Metalloproteinases and Heart Failure
myocardial performance has been shown previously to be unaffected
by changes in LV loading conditions and volumes.41,42
Terminal Study: Myocardial Sampling and
Myocyte Isolation
After the final set of LV function measurements and plasma collection,
the animals were anesthetized as described in the preceding section, a
sternotomy was performed, and the heart was quickly extirpated and
placed in a phosphate-buffered ice slush. The great vessels, atria, and
right ventricle were carefully trimmed away, and the LV was weighed.
The region of the LV free wall incorporating the circumflex artery
(535 cm) was excised and prepared for myocyte isolation. The region
of the LV free wall composing the left anterior descending artery (335
cm) was cannulated and prepared for perfusion fixation. The posterior
region of the LV free wall (333 cm) was snap-frozen in liquid
nitrogen for subsequent biochemical analysis of collagen content and
MMP zymographic activity and expression.
Myocytes were isolated from the LV free wall using methods
described by this laboratory previously.57,9 Briefly, the left circumflex
coronary artery was perfused with a collagenase solution (0.5 mg/mL,
Worthington, type II; 146 U/mg) for 35 minutes. The tissue was then
minced into 2-mm sections and gently agitated. After 15 minutes, the
supernatant was removed and filtered, and the cells were allowed to
settle. The myocyte pellet was then resuspended in DMEM/F-12
(GIBCO Laboratories). The number of viable myocytes was counted
at 3100 magnification using a hemocytometer (Reichert-Jung, Cam-
bridge Instruments Inc) and resuspended to a final concentration of
(53104 cells/mL). Viable myocytes were defined as those cells that
maintained a rod shape and were quiescent in culture. By use of this
myocyte isolation method, a high yield (8565%) of viable myocytes
was obtained in all LV preparations used in the present study. Viable
myocytes were defined as those cells that retained a rod shape, were
calcium tolerant, responded to electrical stimulation, and excluded
trypan blue.
Neurohormonal Measurements
The plasma samples were assayed for renin activity, endothelin
concentration, and catecholamine levels. Plasma renin activity was
determined by computing angiotensin I production using a radioim-
munoassay (NEA-026, New England Nuclear). The interassay varia-
tion for the measurement of plasma renin activity was 15%. For the
endothelin assays, the plasma was first eluted over a cation exchange
column (C-18 Sep-Pak, Waters Associates) and then dried by vacuum
centrifugation. The samples were reconstituted in 0.02 mol/L borate
buffer, and a high-sensitivity radioimmunoassay was performed
(RPA545, Amersham). The recovery from the extraction procedure
was 7565%, which was based on plasma-spiked standards (4 to 20
fmol/mL). The interassay variation was 10%, and the intra-assay
variation was 9% for the endothelin radioimmunoassay procedure.
Plasma norepinephrine and epinephrine were measured using HPLC
and were normalized to picograms per milliliter of plasma. All assays
were performed in duplicate.
LV Myocyte Contractile Function
Isolated myocyte function was examined as previously reported by this
laboratory.57,9 Briefly, a thermostatically controlled chamber (37C)
containing a volume of 2.5 mL and two stimulating platinum
electrodes was used to image the isolated myocytes on an inverted
microscope (Axiovert IM35, Zeiss Inc). A 320 long-working-
distance Hoffmann modulation contrast objective (Modulation Optics
Inc) was used to image the myocytes. Myocyte contractions were
elicited by field-stimulating the tissue chamber at 1 Hz (S11 stimula-
tor, Grass Instruments) using current pulses of 5-millisecond duration
and voltages 10% above contraction threshold. Myocyte motion
signals were captured and input through an edge-detector system
(Crescent Electronics). The distance between the left and right
myocyte edges was converted into a voltage signal, digitized, and
input to a computer (No. 80386, model ZBV2526, Zenith Data
Systems) for analysis. Parameters computed from the digitized con-
traction profiles include percent shortening, velocity of shortening,
velocity of relengthening, time to peak contraction, and duration of
contraction. In addition to basal measurements of contractility, myo-
cyte function was determined after b-adrenergic receptor stimulation
with 25 nmol/L isoproterenol.
LV Myocardial Structure
The LV section for microscopic analysis was perfused with a buffered
sodium cacodylate solution containing 2% paraformaldehyde and 2%
glutaraldehyde solution (pH 7.4, 325 mOsm) for 20 minutes using a
perfusion pressure of 100 mm Hg.9,12 LV myocardial samples were
then prepared for scanning electron microscopy and light microscopic
examination. For scanning electron microscopy, perfusion-fixed LV
myocardial samples were flash-frozen in liquid nitrogen and freeze-
fractured.9,12,13,15 The freeze-fractured samples (0.2530.25 cm) were
then dehydrated and critical point dried (Ladd Research Industries).
The samples were mounted on 10310-mm stubs using conductive
adhesive tape (Scotch commercial tape, 3M Inc) and sputter-coated
with gold (Hummer II, Technics). The sections were examined in a
JOEL JSM-25S scanning electron microscope at an accelerating
voltage of 15 kV. LV samples were prepared in triplicate, and 10
photomicrographs of the extracellular space were obtained from each
sample at a final magnification of 34000. These photomicrographs
were coded and evaluated using the following semiquantitative scale
developed by Bishop et al22: 1, an absent collagen weave; 2, reduced
collagen distribution; 3, normal collagen density and distribution; 4,
increased collagen density; and 5, significantly increased collagen
density and distribution. The categorical grading system was per-
formed in a blinded fashion in which the photomicrograph codes were
not broken until the completion of the study.
Light-microscopic examination was performed on the perfusion-
fixed LV myocardium in order to determine myocyte cross-sectional
area, the percent area occupied by extracellular space, and the
connectivity of the extracellular network.12,15 For examination of the
extracellular matrix, LV sections were stained using the picrosirius
histochemical technique.14,21,43 The stained LV sections were then
digitized at a final magnification of 3320 and analyzed using an image
analysis system (Zeiss/Kontron, IBAS). The percent area of extracel-
lular staining was computed from 15 random fields within the
midmyocardium in order to exclude large epicardial arteries and veins
and any cutting or compression artifact. The integrity or continuity of
the collagen network was examined in these same fields by using a grid
pattern of 100-mm horizontal and vertical lines.44 The percentage of
collagen profiles intersecting this grid was computed and was used as
an index of the integrity of the collagen latticework.12,15
For determination of myocyte cross-sectional area, full-thickness
perfusion-fixed LV myocardial sections were stained with hematox-
ylin and eosin. These sections were imaged using an epifluorescence
illuminator with a rhodamine filter at a magnification of 31000.
Myocytes in a cross-sectional orientation were digitized and analyzed
using the previously described image analysis system. Only those
myocytes in which the nucleus was centrally located within the cell
were digitized and analyzed in order to ensure uniformity for the
measurement of cross-sectional area.
LV myocardial collagen content was also determined by a biochem-
ical assay for hydroxyproline using methods well described previous-
ly.12 Briefly, the LV midmyocardial sections were weighed and
lyophilized. The sections were then hydrolyzed and measured spec-
trophotometrically (550 nm) after reaction with Ehrlichs reagent.12 A
conversion factor of 7.46 was used to convert the final hydroxyproline
values to total collagen values. All measurements were performed in
duplicate and expressed as collagen content in milligrams per gram wet
weight of LV myocardium.
LV Zymographic MMP Activity
After a stringent washing in ice-cold saline, the LV myocardial samples
were homogenized (three 30-second bursts) in 5 mL of an ice-cold
extraction buffer (1:3 wt/vol) containing cacodylic acid (10 mmol/L),
NaCl (0.15 mol/L), ZnCl (20 mmol/L), NaN3 (1.5 mmol/L), and
0.01% Triton X-100 (pH 5.0). The maintenance of a low pH and
temperature prevented proteolytic activation during the extraction
process. The homogenate was then centrifuged (4C, 10 minutes,
 Spinale et al
1200g), and the supernatant was decanted and saved on ice. The pellet
was resuspended in extraction buffer, and the procedure was repeated
in triplicate. The samples were then raised to a pH of 7.6 using Tris
buffer and concentrated using an Amicon B-15 concentrator (Amicon
Inc) at 4C. Final protein concentration of the myocardial extracts was
determined using a standardized colorimetric assay (Bio-Rad protein
assay). These extracts were aliquoted, immediately flash-frozen using
liquid nitrogen, and stored at 280C until the time of assay.
The myocardial extracts were directly loaded onto electrophoretic
gels (SDS-PAGE) containing gelatin (0.5 mg/mL, Sigma Chemical
Co).28,3234,45,46 A homogeneous impregnation of this MMP substrate
into the gels was facilitated by constant stirring and heating to 45C
before casting. The myocardial extracts at a final protein content of 4
mg were loaded onto the gels using a 3:1 sample buffer (10% SDS, 4%
sucrose, 0.25 mol/L Tris-Cl, and 0.1% bromophenol blue, pH 6.8).
The gels were run at 15 mA/gel through the stacking phase (4%) and
at 20 mA/gel for the separating phase (10%), maintaining a running
buffer temperature of 4C. After SDS-PAGE, the gels were washed
twice in 2.5% Triton X-100 for 30 minutes each, rinsed twice in PBS,
and incubated for 3 hours in a substrate buffer at 37C (50 mmol/L
Tris-Cl, 5 mmol/L CaCl2, 0.5% Brij-35, and 0.02% NaN3, pH 8).
After incubation, the gels were stained using 0.1% amido black,
destained in water, digitized, and analyzed as described in the
following paragraph. In order to provide a means of comparison with
respect to the zymographic activity obtained from the present study,
samples were collected from the cell culture medium of the human
fibrosarcoma HT 1080 cell line (American Type Culture Collection)
as described previously.28,45 Briefly, HT 1080 cells were grown to
confluence in DMEM with 10% fetal calf serum and then incubated
for 24 hours in serum-free medium containing 1 mmol/L insulin and
5 mg/L transferrin. After which, the cell cultures were incubated for
24 hours in the presence and absence of 100 ng/mL of PMA (Sigma).45
Treatment of this cell culture system with a phorbol ester has been
demonstrated previously to increase MMP zymographic activity.28,45
After this incubation period, the cell culture medium was drawn off,
concentrated, and subjected to zymography.
The zymograms were digitized using a Kodak DCS 420 digital
camera (Kodak Inc), which provides high resolution (150031000
pixels) and consistent exposure control between scans. The proteolytic
regions for each sample were determined by quantitative image
analysis (Gel-Pro Analyzer, Media Cybernetics). A 3-pixel-wide
profile was constructed along the long axis of each lane and plotted as
a two-dimensional array with line intensity on the y-axis and
molecular weight on the x-axis. The peaks that correspond to
proteolytic zones were summated by two-dimensional integrated
optical density (OD) as follows: OD(x,y)51/{2log
[intensity(x,y)2black reference/incident light2black reference]}.
These two-dimensional integrated OD values were converted to pixel
values on the basis of internal standardization with bacterial collage-
nase (0.1 to 1 mg/mL, Worthington, type II; 146 U/mg). Zymo-
graphic analysis was performed in control and weekly SVT samples on
the same gel using identical protein concentrations.
LV Myocardial MMP Abundance
Relative abundance of MMPs was examined in LV myocardial
extracts using standard immunoblotting procedures.26 28 Before im-
munoblotting, the LV myocardial extracts were eluted over an anion
exchange column as previously described.47 Briefly, myocardial ex-
tracts were chromatographed on a DEAE column (Bio-Rad Labora-
tories) in 20 mmol/L Tris-HCl and 10 mmol/L CaCl2, containing
0.02% NaN3, in which stepwise pH changes were performed in order
to elute the MMPs of interest. Fractions (1 mL) were collected at pH
9.0 for MMP-1 and at pH 7.5 for MMP-2 and -3. This chromato-
graphic procedure was optimized for these MMP species in prelimi-
nary immunoblotting studies. The LV myocardial extracts were
lyophilized and reconstituted in electrophoresis buffer (0.1 mol/L
Tris-HCl and 0.2 mol/L dithiothreitol, pH 6.8, containing 4% SDS
and 0.01% bromophenol blue). LV extracts (3.0 mg) were then loaded
on an 8% SDS-polyacrylamide gel and separated at 40 mA in 0.02
mol/L Tris-base and 0.2 mol/L glycine, pH 6.8, containing 0.1%
SDS. The separated proteins were transferred at 100 V to a nitrocel-
485
lulose membrane (Trans-blot transfer medium, 0.45 mm, Bio-Rad
Laboratories) in 0.025 mol/L Tris-base and 0.2 mol/L glycine, pH
8.2, containing 20% methanol (vol/vol).48,49 Membranes were blocked
with 0.2 mol/L Tris-base and 1.4 mol/L NaCl, pH 7.6, containing 5%
powdered goat milk, 0.1% Tween 20, and 0.02% NaN3. After they
were washed with 0.2 mol/L Tris-base and 1.4 mol/L NaCl, pH 7.6,
containing 0.1% Tween 20, membranes were incubated overnight at
4C in monoclonal antibodies corresponding to MMP-1, -2, or -3
(1.0 mg/mL, Oncogene Research Products). The antibody for
MMP-1 (clone 411E5) was a mouse monoclonal generated by
immunizing mice with the oligopeptide corresponding to residues 332
to 350 of human MMP-1. The antibody for MMP-2 was a mouse
monoclonal antibody generated by immunizing mice with the oli-
gopeptide corresponding to residues 524 to 539 of human MMP-2
(clone 425D11). The antibody for MMP-3 was a mouse monoclonal
antibody generated by immunizing mice with human pro-MMP-3
purified from the conditioned media of rheumatoid synovial fibro-
blasts. The primary antisera were diluted in 0.2 mol/L Tris-base and
1.4 mol/L NaCl, pH 7.6, containing 1% powdered goat milk, 0.1%
Tween 20, 0.08% BSA, 13% DMEM/F-12 cell culture medium
(GIBCO Life Technologies), and 0.02% NaN3. After a stringent
washing, the membranes were incubated for 1 hour in horseradish
peroxidase conjugated goat anti-mouse antibody (1:5000 dilution,
Bio-Rad Laboratories). The membranes were washed again, and the
horseradish peroxidase conjugated secondary antibody was activated
with peracid and luminol (ECL Western blotting detection reagents,
Amersham Life Science). The luminescent signal was detected by
exposure to x-ray film (Eastman Kodak Co) for exactly 5 minutes.
Positive controls for MMP-2 and -3 were included in all immunoblots
and were obtained from human epithelial and fibroblast cell lines
(AG771 and AG770, respectively, Chemicon International Inc). Cell
culture medium from a PMA-stimulated HT 1080 fibrosarcoma cell
line45,50 was also used as a positive control for immunoblotting.
Prestained molecular weight markers (Bio-Rad Laboratories) were
used to ensure adequate protein separation and transfer. The intensity
of the signal was analyzed as described in the previous paragraph and
normalized to control values.
Data Analysis
Indices of LV and myocyte function, collagen structure and compo-
sition, and MMP zymographic activity and expression were compared
with each week of SVT using multivariate ANOVA. If the ANOVA
revealed significant differences, pairwise tests of individual group
means were compared using Bonferroni probabilities. The LV short-
ening-stress data obtained at each week of pacing were fit to a
polynomial regression model. Comparisons of the coefficients ob-
tained from the LV shortening-stress relation were compared using the
t distribution. For comparisons of neurohormonal profiles, the Stu-
dent-Newman-Keuls test was used. With respect to the myocyte
function data, each pig was considered a complete block. Thus, the
numbers of myocytes studied from each pig were considered repeated
observations within each block. The summary statistics include the
number of myocytes studied from each group; however, all statistical
comparisons were performed on a per pig (block) basis. The categor-
ical scores obtained from the scanning electron micrographs were
compared between groups using x2 analysis. All statistical procedures
were performed using the BMDP statistical software package (BMDP
Statistical Software Inc). Results are presented as mean6SEM. Values
of P,.05 were considered to be statistically significant.
Results
All of the pigs that were assigned to undergo chronic SVT
successfully completed the protocol. The animals that under-
went 3 weeks of chronic SVT exhibited signs and symptoms of
CHF, which included ascites, tachypnea, and hepatomegaly.
LV Function and Neurohormonal Profiles With
the Progression of SVT-Induced CHF
Weekly changes in LV function with SVT are summarized in
Table 1. All measurements were performed with the pace-
486 Metalloproteinases and Heart Failure

TABLE 1. Serial Changes in LV Function and Neurohormonal Profiles With Chronic SVT
SVT

Nonpaced Control 7 Days 14 Days 21 Days

LV function
Heart rate, bpm 11462 14064 15964* 17164*
End-diastolic dimension, cm 3.360.1 3.960.1* 4.460.2* 5.360.1*
Fractional shortening, % 4462 3562* 2562* 1462*
LVED wall thickness, mm 7.960.1 7.360.3* 6.360.4* 4.960.2*
Mean aortic pressure, mm Hg 9462 9362 9162 7764*
LV peak stress, g/cm2 6467 10369* 162617* 279627*
LV/BW, g/kg 3.260.2 3.060.1 3.260.1 3.160.2
Plasma neurohormones
Norepinephrine, pg/mL 3276107 12406522* 18646460* 32816574*
Endothelin, fmol/mL 2.660.2 5.961.5 7.960.7* 10.161.3*
Renin, ng z mL21 z h21 3.760.5 6.661.4 8.261.6* 16.261.3*
Sample size, n 12 12 12 12
LVED indicates LV end-diastolic; LV/BW, ratio of LV mass to body weight. Values are mean6SEM.
*P,.05 vs nonpaced control; P,.05 vs SVT 7-day value.

maker deactivated and with the animals in the conscious state.
After 7 days of chronic SVT, LV end-diastolic dimension in-
creased, wall thickness was reduced, and LV peak systolic wall
stress was increased. These changes in LV geometry after 7 days of
SVT were associated with a decline in LV fractional shortening.
After 14 days of SVT, the basal resting heart rate was increased
from control levels. After 14 days of SVT, LV end-diastolic
dimension and peak wall stress were significantly increased, and
wall thickness was significantly reduced from both control and
7-day SVT values. The changes in LV geometry and wall stress
that occurred after 14 days of SVT were associated with a
significant decline in LV fractional shortening compared with
control and 7-day SVT values. After 21 days of SVT, a further
increase in LV end-diastolic dimension and wall stress was
observed, along with a continued decline in LV fractional short-
ening. After 21 days of SVT, mean aortic pressure was signifi-
cantly reduced. LV mass did not significantly change at any time
point with chronic SVT. Thus, time-dependent changes in LV
pump function and geometry occurred during the development
of SVT-induced CHF.
In order to more carefully examine LV myocardial perfor-
mance, the LV ejection-afterload relation was serially measured
under normal conditions and with each week of SVT, and the
results from these studies are shown in Fig 1. In normal
conscious pigs, the LV fractional shortening fell in a propor-
tional manner with increased LV wall stress, and this curvilin-
ear relation is consistent with the force-velocity relation
obtained in human and animal subjects.36 40 Since this relation
was curvilinear, the isochronal LV fractional shorteningwall
stress points were subjected to polynomial regression. The
results from the regression analysis with each week of SVT are
shown in Table 2. After 7 days of SVT, the isochronal LV
fractional shorteningwall stress points fell below the control
curvilinear relation, but the regression coefficients were not
different from control values. However, with longer durations
of SVT, this relation shifted down and to the right, with a
significant change in the regression coefficients computed from
this relationship. In addition to the LV shortening-stress
relation, LV myocardial performance was also examined
through computing the LV systolic stiffness constant with each
week of SVT (Fig 2). After 7 days of SVT, the LV systolic
stiffness constant was similar to control values. However, after
2 and 3 weeks of SVT, the LV systolic stiffness constant was
significantly lower than control values. Thus, using either the
Figure 1. The LV ejection-afterload relation was serially mea-
sured under normal conditions and with each week of SVT
through a graded phenylephrine infusion.8 In normal conscious
pigs (control), LV fractional shortening fell in a proportional man-
ner with increased LV wall stress, and this curvilinear relation is
consistent with past reports.36 40 The isochronal LV fractional
shorteningwall stress points were subjected to polynomial
regression, and the results from this analysis are summarized in
Table 2. The polynomial regression line (solid line) was plotted
for each week of SVT as well as the 95% confidence interval for
the control state (dashed lines). After 1 week of SVT, the iso-
chronal LV fractional shorteningwall stress points fell within the
normal confidence interval, and the regression coefficients were
not different from control values. However, with longer durations
of SVT, this relation shifted down and to the right, with a signifi-
cant change in the regression coefficients computed from this
relationship. These findings suggest that a significant fall in LV
contractile performance occurred after 2 weeks of SVT.
 Spinale et al
TABLE 2. LV Shortening-Afterload Relation With Chronic SVT Polynomial
Regression Coefficients
Nonpaced Control 7 Days
23 23 23
a 0.43e 60.23e 0.50e 60.33e
b 20.2260.06 20.3160.11
c 59.9863.63 59.9167.81
Polynomial regression was used: y5ax 1bx1c (n55 for each week of SVT). Values are mean6SEM.
2
*P,.05 vs nonpaced control; P,.05 vs SVT 7-day value.
stress-shortening relation or the systolic stiffness constant as
indices of LV myocardial performance, a significant and
time-dependent fall in LV myocardial function was observed
during the progression of SVT-induced CHF.
In light of the fact that activation of several neurohormonal
systems commonly occurs during the development of CHF,
weekly changes in neurohormonal profiles with each week of
SVT were determined and are summarized in Table 1. Plasma
norepinephrine increased by 3-fold from control values after 7
days of SVT and increased another 2-fold from these elevated
values after 21 days of SVT. Plasma endothelin levels signifi-
cantly increased after 14 days of SVT and increased further after
21 days of SVT. Similarly, plasma renin activity significantly
increased by over 120% after 14 days of SVT and by over 250%
after 21 days of SVT compared with control values. Thus,
consistent with clinical forms of severe CHF,16,17 the develop-
ment and progression of SVT-induced CHF was associated
with increased neurohormonal system activity.
LV Myocyte Geometry and Contractile
Performance With Progression of
SVT-Induced CHF
Since LV dilation occurred in the absence of hypertrophy with
the development of SVT-induced CHF, significant myocardial
remodeling must have occurred. Accordingly, isolated LV
Figure 2. The LV systolic stiffness constant was computed for
with each week of SVT. This constant was determined from the
exponential relationship between LV wall stress and the natural
logarithm of the reciprocal of wall thickness.41,42 This relationship
was obtained with each week of SVT through a graded phenyl-
ephrine infusion.8 The LV systolic stiffness constant significantly
decreased with 2 and 3 weeks of SVT. These observations sug-
gest that significant defects in LV myocardial contractile perfor-
mance occurred with 14 days of SVT. Time 0 represents control
values. *P,.05 vs day 0; 1P,.05 vs 7 days of SVT.
487
SVT
14 Days 21 Days
23 23 22
20.12e 60.23e * 20.19e2360.17e23*
20.1060.82 0.46e2260.08
41.77670.18 26.5568.95
myocyte geometry was examined with each week of SVT.
Myocyte cross-sectional area was determined from .700
myocyte profiles from each group, and this analysis resulted in
an approximate gaussian distribution for this parameter. After 7
days of chronic SVT, myocyte cross-sectional area was un-
changed from control values (35664 versus 36365 mm2,
P..70). However, after 14 and 21 days of SVT, myocyte
cross-sectional area significantly decreased (30564 and
28264 mm2, respectively) from control values (P,.05).
Steady-state myocyte contractile function for controls and
after each week of SVT are shown in Table 3. Resting
myocyte length was increased after 7 days of SVT and
significantly increased from this value after 21 days of SVT.
After 7 days of SVT, myocyte steady-state percent shortening
was reduced from control values, but shortening velocity was
unchanged. After 14 and 21 days of SVT, steady-state myocyte
contractile performance was significantly reduced from both
control and 7-day SVT values. Specifically, after 14 days of
SVT, steady-state percentage and velocity of shortening fell by
25% from control values. After 21 days of chronic SVT,
myocyte percentage and velocity of shortening was reduced by
over 30% from control values. Myocyte contractile function
was also examined after b-adrenergic receptor stimulation with
the nonselective b-receptor agonist isoproterenol. The results
from this portion of the study are summarized in Table 3.
Myocyte b-adrenergic responsiveness was reduced after 7 days
of SVT. The reduced myocyte b-adrenergic response contin-
ued to deteriorate with longer durations of SVT. For example,
compared with control values, myocyte velocity of shortening
was 15% lower after 7 days of SVT and 28% lower after 14 days
of SVT and was reduced by .50% after 21 days of SVT. Thus,
the progression of SVT-induced CHF was accompanied by
significant changes in isolated LV myocyte geometry, contrac-
tility, and inotropic responsiveness.
LV Myocardial Collagen With Progression of
SVT-Induced CHF
Fibrillar collagen structure and composition were examined
during the development of SVT-induced CHF, and the results
from this analysis are summarized in Fig 3. Morphometric
analysis of picrosirius-stained LV myocardial sections revealed
a significant reduction in the confluence, or connectivity, of
the collagen matrix after 7 days of SVT. The confluent nature
of the collagen weave continued to decline with longer
durations of SVT. Representative scanning electron micro-
graphs taken of control myocardium and of myocardial samples
taken after 7, 14, and 21 days of SVT are shown in Fig 4. In
488 Metalloproteinases and Heart Failure

TABLE 3. Serial Changes in Isolated Myocyte Contractile

Performance With Chronic SVT
Isoproterenol
Baseline (25 nmol/L)
Resting length, mm
Nonpaced control 13762 13563
SVT
7 Days 16068* 14766
14 Days 16962* 16262*
21 Days 17564* 17366*
Percent shortening, % Figure 3. Quantitative morphometry was performed using picro-
Nonpaced control 4.460.2 10.660.8 sirius staining on full-thickness LV myocardial sections. A signifi-
cant reduction in the confluence, or connectivity, of the collagen
SVT
weave was observed with 7 days of SVT and continued to
7 Days 3.760.1* 7.960.3* decline with longer durations of SVT. Collagen content, deter-
14 Days 2.660.2* 6.060.6* mined by hydroxyproline assay, fell in a time-dependent manner
with SVT. Time 0 represents control values. *P,.05 vs day 0;
21 Days 2.360.2* 4.260.5* 1P,.05 vs 7 days of SVT.
Shortening velocity, mm/s
Nonpaced control 48.262.9 173.2611.3 this value after 21 days of SVT (1.860.2, P,.05). In order to
SVT more carefully quantify biochemical changes in LV myocardial
7 Days 45.963.2 145.2612.7*
collagen, analysis of hydroxyproline was performed on LV
midmyocardial samples and converted to collagen values (Fig
14 Days 36.762.9* 122.7613.6*
3). LV myocardial collagen content fell in a time-dependent
21 Days 33.962.2* 82.469.7*
fashion with SVT, and a significant fall from control values was
Relengthening velocity, mm/s observed after 7 days of SVT. Therefore, the significant LV
Nonpaced control 50.562.8 173.9613.7 dilation that occurred during the progression of SVT-induced
SVT CHF was paralleled by changes in LV myocardial collagen
7 Days 45.361.9 121.1611.7* structure and content.
14 Days 38.563.1* 101.3611.3*
21 Days 35.062.5* 67.6611.2*
LV Myocardial Zymographic Activity and MMP
Abundance With Progression of
Number of myocytes
SVT-Induced CHF
Nonpaced control 317 107 In order to examine whether changes in LV myocardial
SVT collagen degradative processes occurred during the progression
7 Days 533 404 of SVT-induced CHF, MMP zymographic activity and rela-
14 Days 647 499 tive abundance were examined from LV myocardial extracts
21 Days 242 141 with each week of SVT. A representative zymogram from
The number of myocytes studied in each group is shown for reference purposes
control LV myocardium and after each week of SVT using
only. Statistical analysis was performed with each animal serving as the treatment gelatin as a proteolytic substrate is shown in Fig 5. In control
block (n56 pigs in each group). Values are mean6SEM. LV myocardium, zymographic activity could be identified
*P,.05 vs nonpaced control; P,.05 vs SVT 7-day value; and P,.05 vs between the 70- and 50-kD region on the basis of calibrated
baseline. molecular weight markers, which were included in each
zymogram. After 7 days of SVT, zymographic activity ap-
control myocardium, a fine weave of collagen was observed peared increased from time 0 control values and remained
within the interstitial space. After 7 days of SVT, this collagen elevated with longer durations of SVT. The LV myocardial
weave surrounding individual myocytes appeared significantly gelatin zymograms were subjected to densitometric analysis in
disrupted. After 14 days of SVT, the fine fibrillar nature of the order to determine total proteolytic activity. LV myocardial
collagen weave could not be readily appreciated, and the MMP gelatinolytic activity increased after 7 days of SVT
interstitial spaces between adjacent myocytes appeared devoid compared with control values (6765 versus 36612 pixels,
of collagen fibrils. After 21 days of SVT, significant disruption P,.05) and remained increased from control values at 14 days
and dissolution of the collagen matrix could be readily ob- (7466 pixels, P,.05) and 21 days (6365 pixels, P,.05) of
served in the majority of LV myocardial samples. Semiquan- SVT. In order to provide an internal reference, cell culture
titative grading of these scanning electron micrographs re- medium (2 mg total protein) from HT 1080 cells incubated for
vealed a pattern similar to that observed at the light- 24 hours in the and absence and presence of 100 ng/mL of
microscopic level using computer-assisted morphometry. PMA were included in all zymograms (Fig 5). Incubation with
Specifically, after 7 days of SVT, the grade of the fibrillar PMA significantly increased zymographic activity in the HT
collagen weave was reduced from control values (2.460.3 1080 cell culture medium and is consistent with past re-
versus 3.360.1, respectively; P,.05) and significantly fell from ports.28,45,50,51 From the PMA-treated HT 1080 cell culture
 Spinale et al
Figure 4. By use of scanning electron microscopy, a fine fibril-
lar collagen weave was observed within the interstitium of con-
trol LV myocardium and evenly surrounded individual myocytes.
After 7 days of chronic SVT, disruption of collagen fibrils
between adjacent myocytes could be readily observed. With
longer durations of SVT, disruption and dissolution of the colla-
gen weave was observed throughout the LV myocardial sam-
ples. These structural changes were quantified by light micros-
copy and histochemical staining and are summarized in Fig 2. A
semiquantitative grading system was applied to these electron
micrographs, and the results from this analysis are presented in
Results. Original magnification 34000.
experiments, a more rapidly migrating band that likely repre-
sents the activated form of MMP-2 was observed.51 In an
additional series of studies, MMP zymographic activity was
examined in the presence of 10 mmol/L EDTA or 2 mmol/L
of PMSF. Incubation with EDTA inhibited all zymographic
activity, consistent with past reports (data not shown)28; how-
ever, in the presence of 2 mmol/L PMSF, a serine proteinase
inhibitor, zymographic activity was unchanged, consistent
with MMP activity.52,53
In order to determine whether the increased MMP zymo-
graphic activity observed during the progression of SVT-
induced CHF was accompanied by an absolute increase in
MMP abundance, immunoblotting was performed for inter-
stitial collagenase (MMP-1), the 72-kD gelatinase (MMP-2),
and stromelysin (MMP-3). A representative immunoblot for
these specific MMP species with each week of SVT is shown
in Fig 6. In all LV myocardial samples, a distinct immunore-
active band could be localized to the appropriate molecular
weight that corresponded to the specific MMP of interest. The
internal controls included in each immunoblotting procedure
resulted in a strong immunoreactive signal consistent with the
489
Figure 5. Zymographic activity in LV myocardial extracts was
examined in the control condition (time 0) and with each week
of SVT, with gelatin used as a proteolytic substrate. Proteolytic
activity was examined in LV myocardial extracts (4 mg total pro-
tein). In order to provide an internal reference, cell culture media
(2 mg total protein) from HT 1080 cells incubated for 24 hours in
the absence (2) and presence (1) of 100 ng/mL of PMA were
included in all zymograms. Incubation with PMA significantly
increased zymographic activity in the HT 1080 cell culture
media and is consistent with past reports.28,45,50 In the gelatin
zymogram, proteolytic activity was observed in LV myocardial
extracts in the 50- to 90-kD region. This proteolytic activity likely
reflects 92-kD gelatinase, or MMP-9, and the 72-kD gelatinase,
or MMP-2.24,29,51 For reference purposes, the migration of the
MMP-9 and MMP-2 products with respect to the HT 1080 cell
media experiments is indicated on the right. On gelatin zymo-
grams, these MMPs migrate differently under nonreducing con-
ditions than would be predicted from the primary, denatured
sequence.51 The activated form of these MMP species for both
the HT 1080 cell culture media and LV myocardial extracts is
indicated on the right by an asterisk. After 7 days of SVT, zymo-
graphic activity appeared increased from time 0 control values
and remained elevated with longer durations of SVT. Total
zymographic activity for this gelatin substrate was quantified
using densitometric methods and is summarized in Results.
molecular weight for these specific MMP species.2328,30,52,53
After 7 days of SVT, the immunoreactive signals for MMP-1,
MMP-2, and MMP-3 were increased from control levels.
With longer durations of SVT, the relative abundance of
MMP-1 appeared to increase in a time-dependent manner.
Densitometry of the immunoblots was performed, and the
results were normalized to control values (Fig 7). After 7 days
of SVT, the abundance of MMP-1 increased by 150615%
from control values and by 360630% after 21 days of SVT.
The relative abundance of MMP-2 and MMP-3 increased by
2-fold after 7 days of SVT and appeared to plateau with longer
durations of SVT. Thus, increased MMP zymographic activity
was demonstrable in LV myocardial extracts during the pro-
gression of SVT-induced CHF and was accompanied by a
relative increase in the abundance of several MMP species.
Discussion
Structural remodeling of the LV and subsequent changes in LV
geometry are common events in the progression to CHF.13,1820,34
However, fundamental mechanisms that contribute to the LV
remodeling and the temporal relationship that contributes to
changes in myocyte contractile performance with developing
CHF remain unclear. The LV fibrillar collagen matrix ensures
structural integrity of adjoining myocytes, provides the means by
which myocyte shortening is translated into overall LV pump
function, and has been postulated to be essential for maintaining
490 Metalloproteinases and Heart Failure
Figure 6. Immunoblotting was performed on LV myocardial
extracts for interstitial collagenase (MMP-1), the 72-kD gelati-
nase (MMP-3), and stromelysin (MMP-3) taken under the control
condition (time 0) and with each week of SVT. Top, A positive
immunoreactive band that corresponds to the proenzyme form
of MMP-1 was observed at the 52/57-kD region in LV myocar-
dial extracts.24 30 A time-dependent increase in MMP-1 abun-
dance was observed after 7 and 14 days of SVT, with a strong
signal observed after 3 weeks of SVT. In addition to substitution
of nonimmune anti-sera, which abolished all staining, cell cul-
ture medium from a PMA-stimulated HT 1080 fibrosarcoma cell
line45,50 was used as a positive control for immunoblotting and
revealed an immunoreactive band corresponding to MMP-1.
Middle, Immunoblotting for MMP-2 revealed a robust increase
in this MMP species after 7 days of SVT, which remained ele-
vated for longer durations of SVT. After 7 days of SVT, a posi-
tive immunoreactive band for MMP-2 was observed at a lower
molecular weight, which likely reflects an intermediate form of
this MMP species. In addition to substitution of nonimmune
sera that abolished all staining, a positive control for MMP-2
that was obtained from the cell culture media of a fibroblast cell
line (AG770, Chemicon International Inc) was included in all im-
munoblotting procedures. A positive immunoreactive signal was
obtained in these positive control samples at the 72-kD region
that corresponds to MMP-2.2329 Bottom, After 7 days of SVT,
an increase in the density of the immunoreactive band corre-
sponding to MMP-3 was readily observed. This immunoreactive
signal for MMP-3 remained elevated with longer durations of
SVT. A positive control for MMP-3 (1CON) was included in all
immunoblots and was obtained from an human epithelial cell
line (AG771, Chemicon). A strong immunoreactive signal was
detected using this positive control at the 59/57-kD region that
corresponds to MMP-3.2329,47,52 The immunoreactive bands cor-
responding to MMP-1, MMP-2, and MMP-3 were digitized, and
the quantitative results from this analysis are summarized in Fig 7.
alignment of myofibrils within the myocyte through a collagen-
integrin-cytoskeletal-myofibril relation.18 21 A number of past
reports have demonstrated that abnormalities in LV fibrillar
collagen structure and composition occur with changes in LV
geometry and function.7,9,1215,18,21,22,32 The MMPs selectively de-
grade extracellular proteins such as the fibrillar collagens and have
been implicated in directly contributing to tissue remodeling in a
number of pathological processes.2334 The present study was
designed in order to test the hypothesis that an early event in the
progression to CHF is LV remodeling and increased MMP
zymographic activity. Accordingly, the present study measured
time-dependent changes in LV geometry, myocyte contractility,
myocardial collagen content, and MMP zymographic activity and
Figure 7. The relative abundance of specific MMPs was serially
quantified in LV myocardial extracts with SVT. The abundance
of interstitial collagenase, or MMP-1, in LV myocardial samples
increased significantly from control values (time 0) after 7 days
of chronic SVT (P,.05) and increased further with longer peri-
ods of chronic SVT (P,.05). The relative abundance of the
72-kD gelatinase, MMP-2, increased significantly after 7 days of
SVT (P,.05) and appeared to plateau with longer durations of
chronic SVT. The abundance stromelysin, or MMP-3, in LV
myocardial samples increased significantly from control (time 0)
values after 7 days of chronic SVT (P,.05) and appeared to pla-
teau with longer durations chronic SVT. Time 0 represents con-
trol values.
expression during the progression of SVT-induced CHF. The
significant and unique finding of the present study was that a time
course of events in the progression of SVT-induced CHF is LV
dilation and myocyte lengthening, alterations in LV myocardial
collagen structure, and significantly increased MMP zymographic
activity and abundance. These observations suggest that early and
dynamic changes in collagen degradative pathways occur within
the LV myocardial interstitium during the progression of CHF.
LV and Myocyte Function During Progression of
SVT-Induced CHF
This study examined time-dependent changes in LV geometry
and myocyte contractile processes during the development of
SVT-induced CHF. After 1 week of SVT, significant LV
dilation and increased LV peak wall stress accompanied by a fall
in LV fractional shortening occurred. The diminished LV
pump function that occurred early with SVT may be due to
differences in loading conditions, chamber geometry, contrac-
tile performance, or a combination of these determinants.
Accordingly, indices of LV myocardial performance and iso-
lated myocyte contractile function were serially examined
during the progression of SVT-induced CHF. After 1 week of
SVT, the LV ejection-afterload relation and the LV systolic
stiffness constant were not significantly reduced from control
values. Using these relatively load-independent indices of LV
myocardial function, the results from the present study suggest
that the reduction in LV pump function that occurred after 1
week of chronic SVT may have not been solely due to changes
in myocardial contractile performance. However, after 1 week
of SVT, plasma catecholamines were increased by 4-fold from
control values and would suggest that significantly increased
LV myocardial sympathetic activation had occurred. Thus, the
in vivo measurements of LV myocardial function that were
obtained in control conditions and after 1 week of SVT were
 Spinale et al
likely performed under different inotropic states. As a result,
inherent defects in LV myocardial contractile function that
may have occurred early in the progression of SVT-induced
CHF would have been difficult to detect. Accordingly, the
present study examined LV isolated myocyte contractile func-
tion after each week of SVT. Through this approach, differ-
ences in external loading conditions and neurohormonal in-
fluences that occurred during the progression of SVT-induced
CHF were removed. After 1 week of SVT, isolated LV
myocyte length was increased and paralleled the LV dilation
that had occurred. The increased LV myocyte length after 1
week of SVT was accompanied by a significant reduction in
myocyte percent shortening. This reduction in myocyte per-
cent shortening was similar to the relative reduction in LV
fractional shortening that occurred after 1 week of SVT.
Although myocyte percent shortening was reduced after 1
week of SVT, myocyte velocity of shortening, which reflects
the rate of actin-myosin crossbridge formation, was not
changed from control values. Taken together, these findings
would suggest that contributory mechanisms for the reduction
in LV pump function that occurred early in the progression of
SVT-induced CHF include changes in both LV and myocyte
geometry and shortening characteristics. However, after 2
weeks of SVT, the continued LV dilation and reduction in LV
pump function were paralleled by diminished capacity of the
LV myocardium to generate contractile force. Furthermore,
these changes in LV geometry and function after 2 weeks of
SVT were associated with diminished steady-state myocyte
contractile function. Consistent with past reports,57 3 weeks of
SVT caused signs and symptoms consistent with severe CHF
and was accompanied by significant LV and myocyte contrac-
tile dysfunction. Past clinical and experimental reports have
documented that changes in LV geometry occur with the
development of LV dysfunction.15,19 However, the time
course of changes in LV geometry and the relationship to
inherent contractile performance are not well understood. The
findings of the present study would suggest that early events in
the progression to LV failure in this model of chronic SVT are
LV and myocyte remodeling and intrinsic defects in contractile
performance.
The present study demonstrated that indices of LV myocar-
dial performance were relatively preserved after 1 week of
SVT. These findings are consistent with a report by Morgan et
al38 in which indices of LV contractility were serially assessed
with chronic pacing in dogs. In their study, rapid atrial pacing
in dogs (with confirmed atrioventricular capture rates of 220 to
260 bpm) was not associated with reduced indices of LV
contractile performance until after 1 week of pacing.38 How-
ever, past studies have reported an early reduction in several
indices of LV contractile function after rapid ventricular
pacing.4,54,55 The divergence between these past reports and the
present study is likely due to methodological differences,
which include the mode and site of pacing, as well as the
conditions under which LV measurements were performed.
Nevertheless, the findings from the present study as well as
these past reports have clearly demonstrated a reduction in LV
contractile function with more prolonged durations of chronic
rapid pacing, irrespective of these methodological differenc-
es.4,6,8,9,13,54,55 The present study builds on these past reports by
491
demonstrating that a potential contributory mechanism for the
diminished LV pump performance that occurs early in the
progression of SVT-induced CHF is LV and myocyte remod-
eling, which is accompanied by significant defects in LV and
myocyte contractile performance.
Although the present study provides evidence that an early
event in the progression of SVT-induced CHF is LV and
myocyte remodeling, it must be recognized that other factors
contribute to the progressive decline in LV pump function and
myocyte contractile performance. After 1 week of SVT,
myocyte length was increased, with no change in steady-state
velocity of shortening. However, these measurements were
performed under ambient conditions in the absence of neuro-
hormonal stimulation or external loading conditions. Accord-
ingly, in an additional series of studies, myocyte function was
examined after b-adrenergic receptor stimulation. Myocyte
b-adrenergic responsiveness was significantly reduced after 1
week of SVT. In the present study and consistent with past
reports, chronic rapid pacing causes an early and sustained
increase in plasma catecholamines.9,10,56 The early increase in
plasma catecholamines with chronic rapid pacing has been
reported to cause defects in b-adrenergicmediated phosphor-
ylation and transduction.56,57 Thus, an early defect in the
progression of pacing-induced CHF appears to be diminished
b-receptor transduction and myocyte inotropic responsiveness.
After 2 weeks of SVT, steady-state myocyte function was
significantly reduced. Abnormalities in a number of processes
responsible for myocyte excitation-contraction have
been identified with the development of pacing-induced
CHF. For example, defects in Ca21 homeostatic mechanisms
have been identified to occur with the development of
tachycardia-induced CHF.58,59 Taken together, these past re-
ports suggest that a number of cellular and intracellular
processes likely contribute to the progression of SVT-induced
CHF. Thus, the present study demonstrated that early events
in the progression of this CHF process include LV and
myocyte remodeling and inherent defects in the capacity of the
myocyte to respond to an inotropic stimulus.
LV Collagen Matrix Remodeling During
Progression of SVT-Induced CHF
In the present study, early changes in fibrillar collagen structure
were observed to occur with chronic SVT and paralleled
changes in LV geometry. The early fall in LV pump perfor-
mance with SVT was accompanied by LV dilation and wall
thinning and by myocyte lengthening. A contributory factor in
these changes in LV and myocyte geometry may have been a
loss of myocardial fibrillar collagen support. Furthermore, the
changes in the myocardial fibrillar collagen matrix that were
observed early in the development of SVT-induced CHF may
have contributed to a loss in the coordination between
myocyte contractile performance and an effective LV ejection.
The collagen matrix has been proposed to provide the support
essential for maintaining alignment of myofibrils within the
myocyte as well as for maintaining myocyte alignment within
the LV free wall.18 21 This laboratory and others have demon-
strated previously that significant alterations in extracellular
myocyte support and basement membrane adhesion capacity
occur with pacing-induced CHF.7,1215,22 The loss of fibrillar
492 Metalloproteinases and Heart Failure
collagen tethering of the myocyte that occurred during the
development of SVT-induced CHF could potentially result in
myocyte lengthening, LV wall thinning, and dilation. In the
present study, the first time point chosen for LV myocardial
collagen and MMP studies was after 1 week of SVT. This time
point was selected since global changes in LV geometry and
pump function had been clearly documented to occur after this
period of chronic rapid pacing.4,9,10 However, Weber et al14
have reported changes in LV myocardial collagen structure
after 24 hours of pacing tachycardia in dogs. In light of the
findings from the present study in which changes in LV
collagen content and structure were temporally related to the
onset of LV dilation and pump dysfunction, future studies
examining LV myocardial collagen structure and MMP activ-
ity at earlier time points in the progression of this CHF process
would be appropriate.
This laboratory has demonstrated previously that concomi-
tant ACE inhibition with chronic rapid pacing reduced the
degree of LV dilation and improved LV myocardial collagen
structure compared with that of untreated animals undergoing
chronic rapid pacing.9 Thus, the reduced LV dilation that was
observed with concomitant ACE inhibition during rapid
pacing may have been due, at least in part, to a preservation of
myocardial collagenmediated extracellular support. Cleavage
of fibrillar collagen molecules by MMPs occurs at specific
peptide lengths and sequences.23,29 The remaining collagen
fragments would not be reflected in the total LV myocardial
hydroxyproline pool but would be evident on structural
analysis. Thus, the present study coupled LV myocardial
hydroxyproline measurements with quantitative histomor-
phometry in order to determine LV fibrillar collagen structure
as well as total abundance. In the present study, early LV
dilation was paralleled by changes in both LV myocardial
structure and hydroxyproline content. These findings suggest
that changes in fibrillar collagen support is an early contribu-
tory mechanism responsible for the LV dilation and diminished
pump function with chronic SVT. However, the present study
did not address whether possible changes in collagen pheno-
types or stability occurred during the development of SVT-
induced CHF. It has been clearly demonstrated that alterations
in myocardial collagen phenotype and cross-linking can occur
in different cardiac pathologies,34,60 which would influence
steady-state myocardial collagen content. Thus, appropriate
future studies would include examination of potential changes
in collagen synthetic pathways, both transcriptional and post-
translational, which occur during the development of SVT-
induced CHF.
LV MMPs During Progression of
SVT-Induced CHF
An important determinant of collagen degradation is through
the activation of the MMPs, which have high selectivity and
affinity for components of the extracellular matrix.2330 MMPs
are secreted in a proenzyme form and require proteolytic
cleavage for activation.23,26,27,29,52,53 Studies have provided evi-
dence that an important MMP activation process occurs
through a proteolytic cascade that can be initiated by serine
proteases.2330,52,53 One approach for measuring relative MMP
activity in tissue extracts is through the use of zymographic
assays.26 28,31,33,34,45 47,50 53 A significant and sustained increase in
MMP zymographic activity against the proteolytic substrate
gelatin was observed early in the progression of SVT-induced
CHF. Through the use of in vitro assay systems, several past
reports have provided evidence to support the concept that
increased MMP activity may contribute to the development of
LV remodeling.18,3234,61 After 3 hours of coronary occlusion in
the rat, a 2-fold increase in LV collagen protease activity
occurred and was associated with a loss in the fibrillar collagen
weave and transmural LV wall thinning.61 Increased MMP
zymographic activity has been reported to occur as a function
of age in the Syrian cardiomyopathic hamster model.32 More
recently, MMP zymographic activity has been demonstrated to
be significantly increased in human end-stage cardiomyopathic
disease.34 The present study builds on these past reports by
demonstrating that a potential contributory mechanism for the
LV myocardial remodeling that occurs during the progression
of a CHF process may be due to increased MMP activity.
There are a number of species of MMPs that have different
specificities to the fibrillar collagens.2330 In the present study,
proteolytic banding patterns were observed on the zymograms,
suggesting that several species of MMPs likely contributed to
the LV myocardial collagen remodeling during the progression
of SVT-induced CHF. However, the proteolytic patterns
observed with gelatin zymography may not necessarily reflect
different species of MMPs, and quantification of MMP species
based on zymographic activity can be problematic.29,30,53 For
example, Atkinson et al51 demonstrated that in type IV collagen
film assays, MMP-9 and MMP-2 migrated to molecular
weights that differed from the predicted molecular weight on
the basis of primary sequence data. Accordingly, the present
study used immunoblotting techniques in order to examine
whether the increase in LV myocardial zymographic activity
during the progression of SVT-induced CHF was associated
with changes in the relative abundance of specific MMPs. The
results from this portion of the study demonstrated that after 1
week of SVT, the relative abundance of several species of
MMPs was increased. Specifically, a significant increase in LV
myocardial content of interstitial collagenase (MMP-1), the
72-kD gelatinase (MMP-2), and stromelysin (MMP-3) oc-
curred after 1 week of chronic SVT and was temporally related
to the development of LV dilation and reduced myocardial
collagen content.
Although increased MMP-2 abundance was observed in LV
myocardial extracts during the progression of SVT-induced
CHF, LV myocardial zymographic activity, which would
correspond to the activated form of this species of MMP, did
not appear increased. There are several problematic issues
surrounding the in vitro zymographic assays performed in the
present study that prevent direct extrapolation to in vivo LV
myocardial MMP activity. First, it is likely that only a relatively
small proportion of total myocardial MMPs is active at any one
point in time. Second, the zymographic assays were performed
under optimal enzymatic conditions and substrate availability.
Third, an important control point of MMP activity is the
TIMPs.23,24,29,30,45,62,63 These TIMPs form tight complexes with
MMPs and therefore play an important role in overall MMP
enzymatic activity. The MMP assays performed in the present
study could not address whether potential changes in TIMP
 Spinale et al
abundance and/or the stoichiometric relation to specific
MMPs may have occurred during the development of SVT-
induced CHF. Moreover, in the absence of activation, the
overall increase in MMP abundance that was observed to occur
in this model of LV dilation and dysfunction may not neces-
sarily result in increased LV myocardial MMP activity. In light
of the findings of the present study in which increased MMP
zymographic activity was observed to occur early in the
progression of this CHF process, future studies focusing on the
determinants that regulate MMP activity in vivo would be
appropriate.
Using immunoblotting techniques, the present study dem-
onstrated that a significant increase in MMP-3 abundance
occurred early in the progression of SVT-induced CHF. The
early increase in MMP-3 abundance has particular relevance
with respect to collagen degradation and MMP activation
states. MMP-3 has the widest range of substrates and includes
all of the fibrillar collagens as well as components of the
basement membrane.23,24,27,29,53 MMP-3 can activate other
MMPs as well as proenzyme and intermediate forms of
MMP-3 (autoactivation).23,27,29,30 Thus, the increased abun-
dance of MMP-3 that was observed to occur early during the
progression of SVT-induced CHF may have had two impor-
tant consequences. First, early LV myocardial MMP-3 activity
with chronic SVT would result in the cleavage of fibrillar
collagens with subsequent disruption of the collagen weave
surrounding myocytes. Second, the early increase in MMP-3
abundance within the LV myocardium that was observed to
occur during the progression of SVT-induced CHF may have
induced the proteolytic activation of other MMPs within the
LV myocardium.
Chronic pacing-induced tachycardia in animals causes well-
defined, predictable, and progressive LV dilation, contractile
dysfunction, and neurohormonal activation.4 13,38,54 57 Al-
though the etiology of clinical CHF is diverse, a common end
point is LV remodeling and pump dysfunction.13 The SVT
model was chosen for the present study since it provides a
reliable and practical means for identifying early and contrib-
utory events responsible for the LV remodeling and progres-
sion of LV dilation and dysfunction that occur with severe
CHF. However, there are inherent differences in this specific
model of SVT with respect to past studies, which have
employed rapid ventricular pacing to induce the CHF pheno-
type.4,10,11,13,56,57 For example, ventricular pacing in dogs has
been reported previously to result in reduced indices of LV
contractile function, such as peak rate of LV pressure devel-
opment, after 1 week of chronic rapid pacing.4 However, a
heterogeneous pattern of LV contractile performance has been
reported after 1 week of ventricular pacing in dogs.54 In a study
by Scott et al,64 differences in the time-dependent changes in
LV geometry were reported in which rapid pacing was
induced from the atrium versus the ventricle. The present
study used SVT, which preserved normal ventricular activation
patterns and provided for a homogeneous LV myocardial
contraction. Thus, the temporal differences in the onset of LV
contractile dysfunction during the progression of pacing-
induced CHF that were observed in past reports and the
present study were likely due to changes in myocardial
activation sequences, ejection patterns, and filling characteris-
493
tics that occur with rapid ventricular pacing.64 66 Although this
rapid pacing model may serve as a useful tool for the elucida-
tion of the mechanisms of CHF, it must be recognized that any
animal model will not fully represent the complex clinical
spectrum of CHF. Specifically, the changes in LV myocardial
structure that occur with pacing-induced CHF are not similar
to the clinical forms of CHF that are due to chronic ischemia
or hypertensive disease. Thus, extrapolation of the findings
from this project to clinical forms of CHF should be done with
caution. Gunja-Smith et al34 recently reported that in human
idiopathic cardiomyopathic disease, collagen cross-linking was
reduced by 50% and MMP zymographic activity was increased
by 30-fold. This laboratory has demonstrated previously that
SVT-induced CHF was associated with a similar reduction in
collagen cross-linking.15 Therefore, this model of SVT could
provide fundamental temporal and mechanistic information on
MMP activity and expression in the remodeling myocardium.
The findings of the present study provide direct evidence that
robust and early changes in LV myocardial MMPs occur in the
progression of CHF and provide a potential novel pharmaco-
logical target for modulating LV structure and geometry in this
pathological process.
Acknowledgments
This study was supported by National Institutes of Health grants
HL-45024 and HL-56603 (Dr Spinale), an American Heart Associa-
tion Grant-in Aid (Dr Spinale), the Thoracic Surgery Foundation for
Research and Education (Dr Walker), a Medical University of South
Carolina postdoctoral research award (Dr Walker), and a basic research
grant from Pfizer (Dr Spinale). Dr Walker participated in this study as
a Nina S. Braunwald Research Fellow. C.V. Thomas performed this
work as a Medical Student Fellow of the American Heart Association.
Dr Spinale is an Established Investigator of the American Heart
Association. The authors wish to extend their appreciation to Drs
Thomas Borg and Louis Terracio, University of South Carolina, for
their advice and support during the execution of this project. The
technical assistance of Patrick Thomas, Steve Krombach, Julie
Ianninni, Catherine R. Aversa, Maria Webb, and Charles Basler is
gratefully acknowledged.
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