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Augmentation of Left Ventricular Wall Thickness with Alginate
Hydrogel Implants Improves Left Ventricular Function and Prevents
Progressive Remodeling in Dogs with Chronic Heart Failure
SUPPLEMENTAL MATERIAL
METHODS
Experimental Model
The study was approved by Henry Ford Health System Institutional Animal Care and Use
Committee. The canine model of chronic HF used in this study was previously described
in detail (1). In this study, 15 mongrel dogs, weighing between 20 to 25 kg, underwent
coronary microembolizations to produce HF. Embolizations were performed 1 to 2
weeks apart and were discontinued when LV ejection fraction (EF), was <30%. All
procedures were performed during cardiac catheterization under general anesthesia and
sterile conditions. Animals were sedated with intravenous hydromorphone hydrochloride
(0.22 mg/kg) and diazepam (0.17 mg/kg) and a plane of anesthesia was maintained with
1-2% isoflurane.
Alginate Preparation
The two component Alginate Selfgel Formulation hydrogel (Algisyl-LVRTM) was
supplied by LoneStar Heart, Inc. (Laguna Hills, CA). Because alginates are essentially
inert with no known biologic activities, the US Food and Drug Administration has given
Algisyl-LVR a “device” designation. The Na+-Alginate component was supplied as a
sterile aqueous solution with 4.6% mannitol. The Ca2+-Alginate component consisted of
water insoluble particles that were suspended in a sterile 4.6% mannitol solution. The
two liquid components were mixed by transfer back and forth between two syringes
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immediately before use. The final mixture was combined in one syringe for
intramyocardial injections.
Study Protocol
Dogs were randomized to treatment with AHI (n=9) or to sham-operated control (n=6) a
minimum of 2 weeks after the last coronary microembolization. All dogs underwent a
cardiac catheterization to record pre-treatment hemodynamic, ventriculographic,
echocardiographic and Doppler measures.
Immediately after the catheterization, a
midline sternotomy was performed, the pericardium was opened, and the heart cradled in
the pericardial sac. Injections of AHI or normal saline as a control were made directly
into the LV wall using a 21 gauge needle. A single line of 7 injections, 0.25-0.35 ml each
of AHI were administered in the active treatment group and 7 injections of 0.25-0.35 ml
each of normal saline were administered in the control group. In both groups, injections
were made ~1.0 to 1.5 cm apart circumferentially along the LV mid-ventricular level
halfway between the apex and base starting at the antero-septal groove and ending at the
postero-septal groove (Fig. 1). Needle penetration into the LV myocardium was made at
approximately 45o angle to the epicardial surface and delivery of AHI or saline was made
at mid-thickness of the LV wall. After treatment, the chest was closed and the dogs
maintained for 17 weeks. All hemodynamic, ventriculographic, echocardiographic and
Doppler measurements were repeated at 2, 6, 12 and 17 weeks. End-systolic and enddiastolic wall thickness of the anterior and posterior LV walls was measured from
echocardiograms obtained at pre-treatment and at 17 weeks. The dogs were euthanized at
the end of the study and the heart removed and prepared for histology.
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Ventriculographic Measurements
Left ventriculograms (30o right anterior oblique projection) were recorded on digital
media at 30 frame/sec during the injection of 20 ml of contrast material (RENO-M-60
Squibb, Princeton, NJ). Correction for image magnification was made with a radiopaque
calibrated grid placed at the level of the LV. LV end systolic (ESV) and end diastolic
(EDV) volumes and EF were calculated from LV silhouettes (2) and stroke volume (SV)
was calculated as the difference between EDV and ESV and the ratio of SV to end
diastolic pressure (EDP) was calculated. Global LV shape, a measure of LV sphericity,
was quantified from angiographic silhouettes based upon the ratio of the major-to-minor
axes at end-systole (end systolic sphericity index; ESSI) (3). All ventriculographic
analyses were performed by a blinded reader.
Echocardiographic and Doppler Measurements
Echocardiographic and Doppler studies were performed using a General Electric VIVIDultrasound system. LV end-diastolic thickness of the anterior and posterior LV wall were
calculated from LV short-axis 2-dimesional echocardiograms. LV end-diastolic
circumferential wall stress (EDWS) was calculated as previously described (4).
Transmitral inflow velocity was measured using pulsed-wave Doppler. The velocity
waveforms were used to calculate deceleration time of early mitral inflow velocity (DT),
and index of LV diastolic function. The presence and severity of functional mitral
regurgitation (MR) was calculated from color flow Doppler as the ratio of the regurgitant
jet area to area of the left atrium (5). All echocardiographic and Doppler analyses were
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performed by a blinded reader.
LV Pressure-Volume Loops
Left ventricular pressure-volume (P-V) loops were measured using a Millar Instruments
MPVS Ultra system (Houston, TX) in conjunction with a pressure-conductance catheter
positioned within the LV cavity.
P-V loops generated during a transient balloon
occlusion of the inferior Vena Cava were used to assess the slope of the end-systolic
pressure volume relation (ESPVR), a measure of load-independent contractility (6). P-V
loops were obtained at pre-treatment (PRE) and post-treatment (17 weeks) (POST).
Ambulatory ECG Holter Monitoring
A 24 hour ambulatory ECG Holter monitoring study was performed at all study timepoints and full disclosure obtained. Holters were used to quantify the total number of
single premature beats (PVC’s), couplets, triplets, and episodes of sustained and nonsustained ventricular tachycardia (VT).
Histomorphometric Measurements
At autopsy, 3 transverse slices (approximately 3 mm thick) were obtained one each from
basal, middle and apical thirds of the LV. A mid-ventricular slice that incorporated the
sites of Alginate injections was not used in any of the histomorphometric analyses.
Instead, an adjacent slice taken from one side of the injected region was used in the
analyses. From each slice, transmural tissue blocks were obtained and embedded in
paraffin blocks. Transmural tissue blocks were also obtained from the free wall segment
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of additional slices, mounted on cork using Tissue-Tek embedding medium, rapidly
frozen
in
isopentane
pre-cooled
in
liquid
nitrogen,
and
stored
at
–70oC.
Histomorphometric measurements for the volume fraction of replacement fibrosis
(VFRF), volume fraction of interstitial fibrosis (VFIF), myocyte cross-sectional area
(MCSA), a measure of cardiomyocyte hypertrophy, capillary density (CD), and oxygen
diffusion distance (ODD), defined as half the distance between two adjoining capillaries,
were performed as previously described (7). The same histomorphometric measures
were also assessed in LV tissue from 6 normal dogs for comparison.
RESULTS
Ambulatory 24 hour Holters showed no significant differences between groups with
respect to minimum, average and maximum heart rate and frequency and severity of
ventricular arrhythmias (Table 1).
There was no difference in volume fraction of replacement fibrosis between groups
(Table 2). Volume fraction of interstitial fibrosis, cardiomyocyte cross-sectional area,
and oxygen diffusion distance decreased significantly in AHI-treated dogs compared to
controls and capillary density increased (Table 2).
REFERENCES
1.
Sabbah HN, Stein PD, Kono T, Gheorghiade M, Levine TB, Jafri S, et al. A
canine model of chronic heart failure produced by multiple sequential coronary
microembolizations. Am J Physiol. 1991;260:H1379-84.
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2.
Sabbah HN, Shimoyama H, Kono T, Gupta RC, Sharov VG, Scicli G, et al.
Effects of long-term monotherapy with enalapril, metoprolol, and digoxin on the
progression of left ventricular dysfunction and dilation in dogs with reduced ejection
fraction. Circulation. 1994;89:2852-9.
3.
Sabbah HN, Kono T, Stein PD, Mancini GB, Goldstein S. Left ventricular shape
changes during the course of evolving heart failure. Am J Physiol. 1992;263:H266-70.
4.
W G, DS B. Pressure measurements. In: DS B, editor. Cardiac Catheterization,
Angiography, and Interventions: Lippincott and Williams; 2006. p. 133-47.
5.
Kono T, Sabbah HN, Rosman H, Alam M, Stein PD, Goldstein S. Left atrial
contribution to ventricular filling during the course of evolving heart failure. Circulation.
1992;86:1317-22.
6.
Saavedra WF, Tunin RS, Paolocci N, Mishima T, Suzuki G, Emala CW, et al.
Reverse remodeling and enhanced adrenergic reserve from passive external support in
experimental dilated heart failure. J Am Coll Cardiol. 2002;39:2069-76.
7.
Sabbah HN, Stanley WC, Sharov VG, Mishima T, Tanimura M, Benedict CR, et
al. Effects of dopamine beta-hydroxylase inhibition with nepicastat on the progression of
left ventricular dysfunction and remodeling in dogs with chronic heart failure.
Circulation. 2000;102:1990-5.
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Table 1
Ambulatory 24 Hours ECG Holter Monitoring Studies Obtained at Post-Treatment (17
weeks) in Control Dogs (n=6) and Dogs Treated with Alginate Hydrogel Implants (AHI)
(n=8)
CONTROL
AHI
P-Value
Minimum
HR
(beats/min)
Average
HR
(beats/min)
Maximum
HR
(beats/min)
Total
VPBs*
Total
Couplets
Total
Triplets
Total
VT
Runs
47 ± 3
48 ± 3
0.80
84 ± 4
80 ± 5
0.60
185 ± 6
165 ± 9
0.11
34 ± 23
339 ± 318
0.43
7±6
24 ± 18
0.45
2±2
1±0
0.44
1±0
0±0
0.11
Table Legend: HR = heart rate; VPBs = ventricular premature beats; VT = ventricular
tachycardia; ECG - electrocardiogram. P-Value = Probability value. * One AHI-treated
dog had a high number of single ventricular premature beats (Total VPBs) compared to
all of the other treated and Control dogs. The exact reason for this elevated ectopic
activity is uncertain. However, the animal model of chronic heart failure used in this
study manifests considerable ectopic activity and this type of frequent ventricular
premature beats has been observed in previous studies. Data are shown as Mean ± SEM.
Table 2
Histomorphometric Measures Obtained at Post-Treatment in Control Dogs (n=6) and
Dogs Treated With Alginate Hydrogel Implants (AHI) (n=8)
CONTROL
AHI
P-Value
VFRF (%)
14.1 ± 0.9
12.3 ± 0.8
0.18
VFIF (%)
15.3 ± 1.1
11.4 ± 0.9
0.016
CD
1638 ± 83
2485 ± 109
0.001
ODD (μm)
15.8 ± 0.6
10.1 ± 0.3
0.001
MCSA (μm2)
677 ± 33
471 ± 12
0.001
Table Legend: CD = capillary density or number of capillaries per mm 2; MCSA =
myocyte cross-sectional area; ODD = oxygen diffusion distance; VFIF = volume fraction
of interstitial fibrosis; VFRF = volume fraction of replacement fibrosis. Data are shown
as Mean ± SEM. P-Value = Probability value.
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FIGURE 1
PRE
POST (17 weeks)
Figure Legend: Left ventricular pressure-volume loops generated during balloon
occlusion of the inferior vena cava in a dog before delivery of AHI (PRE) and after
delivery of AHI (POST).
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