Download Myocardial Mechanics and Collagen Structure in the Osteogenesis

Myocardial Mechanics and Collagen Structure in the
Osteogenesis Imperfecta Murine (oim)
Sara M. Weis, Jeffrey L. Emery, K. David Becker, Daniel J. McBride, Jr,
Jeffrey H. Omens, Andrew D. McCulloch
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Abstract—Because the amount and structure of type I collagen are thought to affect the mechanics of ventricular
myocardium, we investigated myocardial collagen structure and passive mechanical function in the osteogenesis
imperfecta murine (oim) model of pro-␣2(I) collagen deficiency, previously shown to have less collagen and impaired
biomechanics in tendon and bone. Compared with wild-type littermates, homozygous oim hearts exhibited 35% lower
collagen area fraction (P⬍0.05), 38% lower collagen fiber number density (P⬍0.05), and 42% smaller collagen fiber
diameter (P⬍0.05). Compared with wild-type, oim left ventricular (LV) collagen concentration was 45% lower
(P⬍0.0001) and nonreducible pyridinoline cross-link concentration was 22% higher (P⬍0.03). Mean LV volume during
passive inflation from 0 to 30 mm Hg in isolated hearts was 1.4-fold larger for oim than wild-type (P⫽NS). Uniaxial
stress-strain relations in resting right ventricular papillary muscles exhibited 60% greater strains (P⬍0.01), 90% higher
compliance (P⫽0.05), and 64% higher nonlinearity (P⬍0.05) in oim. Mean opening angle, after relief of residual
stresses in resting LV myocardium, was 121⫾9 degrees in oim compared with 45⫾4 degrees in wild-type (P⬍0.0001).
Mean myofiber angle in oim was 23⫾8 degrees greater than wild-type (P⬍0.02). Decreased myocardial collagen
diameter and amount in oim is associated with significantly decreased fiber and chamber stiffness despite modestly
increased collagen cross-linking. Altered myofiber angles and residual stress may be beneficial adaptations to these
mechanical alterations to maintain uniformity of transmural fiber strain. In addition to supporting and organizing
myocytes, myocardial collagen contributes directly to ventricular stiffness at high and low loads and can influence
stress-free state and myofiber architecture. (Circ Res. 2000;87:663-669.)
Key Words: heart 䡲 ventricle 䡲 collagen 䡲 stiffness 䡲 residual stress
C
ollagen fibers in ventricular myocardium are thought to
contribute tensile stiffness,1 especially at higher cavity
volumes,2 while maintaining the architecture of myocytes, myofiber bundles, and sheets.3 The cardiac collagen extracellular
matrix consists of ⬇85% type I collagen, arranged into a
hierarchy of fibers. Large coiled perimysial collagen fibers
provide tensile stiffness,4 whereas smaller endomysial fibers
surrounding and interconnecting individual myocytes are
thought to prevent myocyte slippage3 and maintain unloaded
ventricular geometry.5
Many cardiac disorders are associated with an accumulation, depletion, or restructuring of the collagen matrix.6
Correlating mechanical alterations with changes in the size,
amount, and structure of collagen fibers in these pathologies
has provided insight into the functional role of myocardial
collagen.7,8 Indeed, most of our knowledge of the structural
contribution of collagen to ventricular wall mechanics has
been deduced either from comparative studies using animal
models of complex, multifactoral pathologies7–9 or from
tissue preparations in which collagen has been proteolytically
degraded5,10 or synthesis has been inhibited.11
Human osteogenesis imperfecta (OI) is a heritable disease,
resulting from deletions, insertions, or exon splice errors in
the genes encoding type I collagen pro-␣1 and pro-␣2 chains.
In most cases, the mutation is unknown and diagnosis is made
by clinical assessment of symptoms, which include bone
fragility, defective skeletal development, smaller stature, and
blue sclera. Few data are available regarding implications of
OI on cardiac mechanics or structure, although clinical
descriptions of aortic dissection,12 left ventricular (LV) rupture,13 and aortic or mitral valve incompetence14 have been
reported in OI patients.
The present study uses a mouse model of OI, the OI murine
(oim), to investigate structure-function relationships in the
heart. The oim model of moderate OI results from a guanine
deletion on the Cola-2 gene encoding the pro-␣2(I) chain of
type I collagen,15 causing a loss of functional ␣2(I) chains in
homozygous oim mice. The oim mouse exhibits many symptoms similar to human OI, namely defective skeletal development, smaller stature, and skeletal fragility.15
Recently, several mouse models of OI have been used to
investigate effects of collagen mutations on structure and
Received August 14, 2000; revision received September 7, 2000; accepted September 7, 2000.
From the Departments of Bioengineering (S.M.W., J.L.E., J.H.O., A.D.M.) and School of Medicine (K.D.B., J.H.O.), University of California, San Diego, La
Jolla, Calif, and Division of Geriatric Medicine and Gerontology (D.J.M.), Johns Hopkins School of Medicine, Baltimore, Md.
Correspondence to Andrew D. McCulloch, PhD, University of California, San Diego, Department of Bioengineering, 9500 Gilman Dr, La Jolla, CA
92093-0412. E-mail [email protected].
© 2000 American Heart Association, Inc.
Circulation Research is available at http://www.circresaha.org
663
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Circulation Research
October 13, 2000
mechanics in bone and tendon. Tail tendon in the oim model
had ⬇60% less collagen and ⬇40% lower tensile strength
than wild-type controls,16 and femur strength was reduced in
heterozygous17 and homozygous oim.18 Experiments in several other strains of mice harboring OI-type mutations19,20
suggest a general decrease in collagen but no consistent
changes in elastic stiffness.
We tested the hypothesis that the Cola-2 mutation in the oim
model is associated with altered myocardial collagen structure
and content and consequent alterations in ventricular mechanics.
We studied hearts from wild-type (⫹/⫹), heterozygous (⫹/⫺),
and homozygous oim (⫺/⫺) mice. Our results suggest smaller,
less abundant perimysial collagen fibers and lower passive
uniaxial and ventricular stiffness in oim are accompanied by
altered transmural myofiber angle distribution and increased
residual stresses, which may be beneficial adaptive responses.
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Materials and Methods
Characteristics of oim, Heterozygous (HET), and Wild-Type Mice
WT (n⫽31)
HET (n⫽37)
oim (n⫽31)
23
21
18
8
16
13
All studies
Male, No.
Female, No.
Age, wk
13⫾0.4
16⫾0.3
16⫾0.7*
BW, g
27⫾0.9
26⫾0.8
21⫾0.5*
HW, g
0.17⫾0.005
0.16⫾0.005
0.15⫾0.004*
100⫻HW/BW
0.66⫾0.02
0.62⫾0.02
0.69⫾0.02
Heart rate, bpm
440⫾67
䡠䡠䡠
429⫾61
Body weight, g
26⫾4
䡠䡠䡠
26⫾4
Diastolic LV internal
diameter, mm
3.6⫾0.3
䡠䡠䡠
3.8⫾0.5
Systolic LV internal
diameter, mm
2.4⫾0.3
䡠䡠䡠
2.5⫾0.6
In vivo (echo)
Genotyping
Septal wall
thickness, mm
0.75⫾0.09
䡠䡠䡠
0.85⫾0.07**
The oim model15 is available from the Jackson Laboratory (Bar
Harbor, Maine). A polymerase chain reaction method21 was used to
determine mouse genotype.
Posterior wall
thickness, mm
0.73⫾0.11
䡠䡠䡠
0.83⫾0.08**
Septal h/r
0.42⫾0.06
䡠䡠䡠
0.46⫾0.09
Echocardiography
Posterior h/r
0.41⫾0.07
䡠䡠䡠
0.45⫾0.10
Transthoracic two-dimensional M-mode and Doppler echocardiography was performed on intact mice1 under intraperitoneal avertin
anesthesia (2.5%, 15 to 17 ␮L/g).
Fractional shortening, %
33⫾8
䡠䡠䡠
34⫾7
Aortic ejection time, ms
57⫾8
䡠䡠䡠
53⫾5
0.58⫾0.12
䡠䡠䡠
0.64⫾0.15
2.2⫾0.08
2.2⫾0.06
2.0⫾0.10
Acute Surgery
All experimental protocols were approved by the University of
California, San Diego Animal Subjects Committee. A total of 62
male and 37 female 11- to 16-week-old mice were anesthetized
intraperitoneally using 100 mg/kg ketamine and 8 mg/kg xylazine.
Hearts were arrested in diastole with hyperkalemic cardioplegic
solution containing 30 mmol/L 2,3-butanedione monoxime (BDM)
to prevent muscle contracture.22
Biochemistry
Left ventricles were isolated and lyophilized, and dry weights were
recorded. Hydroxyproline23 and nonreducible collagen cross-link
concentrations were determined.24
Velocity of circumferential
fiber shortening,
diameter/s
In vitro
Posterior wall
thickness, mm
LV external diameter, mm
Posterior h/r
5.8⫾0.13
5.5⫾0.07
4.9⫾0.19*
0.75⫾0.027
0.81⫾0.019
0.80⫾0.016
Smaller HW and BW were measured for oim compared with HET and WT, but
HW/BW ratio was conserved. Significant differences were found for septal and
posterior wall thickness, but LV wall thickness–radius ratios (h/r) were similar
between groups.
*P⬍0.01 for WT vs oim; **P⬍0.05 for WT vs oim.
Histology
Hearts were fixed, processed with graded alcohols, embedded in
paraffin, sectioned 10 ␮m or 15 ␮m thick, and stained with
picrosirius red. To enhance contrast between myocytes and collagen,
15-␮m-thick sections for confocal microscopy were treated with
phosphomolybdic acid.25 Sections were imaged using a laser scanning confocal system (BioRad MRC 1024) with a ⫻60 objective.
Collagen area fraction and fiber number density were measured.4
Average collagen fiber diameter was calculated from 7 to 10
measurements along each fiber.
Myofiber Angle and Zero-Stress State
Myofiber angle was measured from stained sections of paraffinembedded hearts fixed at zero pressure. Myofiber angle was defined
as the angle between the myofibers and a reference line parallel to
the equatorial LV axis.26 To ensure this reference was identical for
all hearts, care was taken to align the axis between the apex and the
junction between the mitral and aortic valves along a straight edge
before making the equatorial slice. There were no differences
observed for heart shape between genotypes that may have led to
systematic errors in obtaining a slice from a consistent location
between hearts. Residual strain was estimated by measuring the
resulting angle from a radial cut through the LV free wall in the
presence of a low calcium buffer containing BDM.27 Images of each
isolated slice were acquired to measure diameter and wall thickness.
Papillary Muscle Uniaxial Testing
Suture was tied to the distal end of a right ventricular (RV) papillary
muscle and attached to a 1-g capacitive force transducer for constant
muscle stretch at 3 mm/min. Surface marker motion in the muscle
central portion was recorded, and two-dimensional finite strain was
computed from video images.28 Fiber stress was computed as force
per unit cross-sectional area. After 3 preconditioning runs, loading
consisted of slow stretch to 10 mN/mm2. Perfusate was replaced with
cardioplegic solution containing 2 mmol/L calcium and no BDM to
confirm specimen contractile viability when field-stimulated by
platinum electrodes.
Isolated Heart Inflation
A pressure transducer was used to measure intraballoon pressures for
a LV balloon with 5 ␮L empty volume. After 3 preconditioning runs,
a volume infusion pump slowly infused volume to a maximum
pressure of 30 mm Hg.22
Weis et al
Myocardial Mechanics in Osteogenesis Imperfecta
665
Figure 1. Representative laser scanning
confocal micrographs of LV tissue stained
with picrosirius red. Perimysial collagen
fibers, which are aligned parallel to the myocytes, appear smaller and less abundant in
tissue from the oim heart compared with the
wild-type heart.
Exclusion Criteria
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None of the 15 hearts were excluded from the opening angle study
for contracture, indicated by ⬎20 degree change in angle after 20
minutes. Of 25 hearts studied for ventricular tissue mechanics, 6
were rejected for incompatible balloon size for ventricular dimensions and 5 were excluded for tissue contracture, evidenced as
firmness to touch. Of 66 hearts studied for passive uniaxial stretch,
39 were excluded: 30 because of observation of muscles with
geometry not suitable for our experiment (many RV papillary
muscles exhibited a conical geometry rather than cylindrical and
often were attached to the septal wall by an extensive network of
trabeculae), 3 for insufficient muscle contraction after the experiment, and 6 for experimental difficulties.
Statistical Analyses
Results presented reflect mean⫾SE per group; 1- or 2-way ANOVA
was performed with Bonferroni-Dunn post hoc comparisons.
An expanded Materials and Methods section can be found in an
online data supplement available at http://www.circresaha.org.
Results
The characteristics of all mice are summarized in the Table. The
oim group exhibited an average smaller heart weight (HW) and
body weight (BW), but ratio of HW to BW was not statistically
different than in wild-type mice.
Despite advances in mouse echocardiography, the signal-tonoise ratio is still not high. We acknowledge that limited
resolution may prevent detection of small differences between
genotypes. Echocardiograms showed no significant differences
in heart rate or LV systolic and diastolic diameters (Table).
End-diastolic septal and posterior wall thickness was 12% larger
in oim than wild-type mice (P⬍0.05), and the end-diastolic LV
internal diameter tended to be larger in oim mice (P⫽NS), but
ratios of wall thickness to LV radius were similar between
groups. Systolic function was not different between genotypes.
Images of the entire heart were recorded on video, and
diameter at the equator and distance from apex to base were
measured to estimate LV volume (tissue⫹cavity) using a prolate
spheroidal model. Estimated LV volume was not statistically
different between groups (wild-type: 150⫾13 ␮m, oim:
128⫾9 ␮m; P⫽0.14), but there was a strong correlation between
estimated LV volume and HW. Because this estimated LV
volume accounts for both LV tissue mass as well as LV cavity
capacity, it was used to normalize for heart size in the isolated
LV inflation experiment rather than using HW or initial volume
alone. Images of equatorial sections from the opening angle
experiment were acquired to measure dimensions. Hearts in the
oim group had a lower mean outer diameter than in the wild-type
group (P⬍0.0001) (Table). However, ratio of wall thickness to
radius and gross morphology for oim were not significantly
different compared with wild-type and heterozygous animals.
Histology
Large coiled perimysial collagen fibers were detected readily in
wild-type tissue but were thinner and less abundant in heterozygous and homozygous oim tissue (Figure 1). Compared with
wild-type, collagen area fraction (Figure 2) was 35% lower in
oim hearts (P⬍0.05) but not different in heterozygous hearts
(P⫽0.70). Collagen fiber number density (Figure 2) was 38%
lower in oim compared with wild-type hearts (P⬍0.05), but not
significantly different from heterozygous hearts (P⫽0.66). The
distribution of perimysial collagen fiber diameters (Figure 3)
was more homogeneous for oim compared with wild-type.
Wild-type perimysial collagen consisted of fiber diameters
ranging from 0.5 to 2.3 ␮m, whereas most oim fiber diameters
were ⬍1 ␮m. We also measured collagen characteristics in
several RV papillary muscles, but because of their small size
(⬍1 mm in length), it was difficult to obtain histological sections
parallel to the muscle long axis. From the samples of RV
papillary muscle tissue studied, we observed effects of the oim
mutation consistent with those seen in the LV (data not shown).
Biochemistry
Collagen amount (Figure 4), measured as hydroxyproline content per dry weight, was 45% less in oim compared with
wild-type mice (P⬍0.0001). The nonreducible collagen crosslink concentration (Figure 4), measured as [mol pyridoxamine/
mol collagen], was 22% higher in oim compared with wild-type
mice (P⬍0.03).
Myofiber Angle
Transmural distribution of myofiber angle from epicardium to
endocardium was shifted positively for oim compared with
Figure 2. Measurements from 15-␮m-thick confocal micrographs of LV sections (n⫽6 for each group). Compared with
wild-type, oim LV tissue contains less perimysial collagen, as
measured by area fraction and fiber number density (P⫽0.05).
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October 13, 2000
Figure 3. Collagen fiber diameter distribution from LV tissue
measured from confocal micrographs. Average oim fiber diameter was 42% smaller than wild-type (P⬍0.05). n⫽4 animals per
genotype, 20 to 30 fibers measured per animal.
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wild-type mice (Figure 5). Myofiber angles for oim were 19 to
25 degrees larger at each depth, averaging 23⫾8 degrees
(P⬍0.02), but slope of myofiber angle–wall depth relation was
not different (wild-type⫽1.1⫾0.1, oim⫽1.0⫾0.1; P⫽0.80). For
heterozygous mice, average myofiber angles were greater than
for wild-type controls but less than for homozygous oim mice.
Zero-Stress State
We measured 2-fold larger opening angles (P⬍0.0001) for oim
compared with wild-type mice (Figure 6). The mean opening
angle for heterozygous mice was intermediate.
Uniaxial Papillary Muscle Stretch
Average RV papillary muscle cross-sectional area (wild-type:
0.07⫾0.01 ␮m, oim 0.07⫾0.01 ␮m; P⫽0.51) and muscle length
(wild-type: 1.0⫾0.06 ␮m, oim: 0.8⫾0.04 ␮m; P⫽0.14) were
not different between groups. Strains were 36% to 40% higher
(Figure 7A) for oim compared with wild-type mice (P⬍0.01).
The slope of the stress-strain relation increased with increasing
stress. Mean slope for oim mice was smaller than for wild-type
mice (P⬍0.05) at each stress level except 10 mN/mm2. A
measure of nonlinearity of the stress-strain relation was calculated as ratio of slope at 10 mN/mm2 to slope at a stress of 0.1
mN/mm2. This ratio was 64% higher for oim than for wild-type
mice: 52⫾16 compared with 19⫾3, respectively (P⬍0.05).
After the passive stretch experiment, most of the muscles
demonstrated contractile behavior characteristic of viable tissue,
Figure 4. Collagen content (hydroxyproline) was 45% less in
oim heart compared with wild-type heart (P⬍0.0001). Collagen
nonreducible cross-links (pyridinoline) were 29% higher in oim
heart compared with wild-type heart (P⬍0.03). n⫽8 animals per
genotype.
Figure 5. Transmural distribution of myofiber angle measured
using light microscopy. Average oim fiber angle is 19 to 25
degrees higher than wild-type at all depths (P⬍0.02). n⫽6 for
heterozygous and homozygous oim, n⫽5 for wild-type.
but 3 (5%) were excluded for poor contraction strength, suggesting that the muscles may have undergone ischemic injury, such
as contracture, which could have affected passive stiffness.
Isolated Heart Mechanics
Compared with wild-type mice, average relative volume
changes (Figure 7B) for oim were 1.4-fold higher during
inflation pressures ranging from 0 to 30 mm Hg (R2⫽0.99, linear
regression). Pressure-volume relation slopes were calculated
from linear interpolation of data in a narrow range surrounding
each pressure step. For pressures 10 to 30 mm Hg, mean slopes
for wild-type mice were 1.35 times higher than for oim
(R2⫽0.78). HW (0.17⫾0.01 versus 0.15⫾0.01 g), absolute
initial volume (5.9⫾0.4 versus 6.1⫾0.4 ␮L), and LV estimated
volume (130⫾6 versus 113⫾6 ␮L) of hearts in this study did not
differ statistically between wild-type and oim mice, respectively.
Discussion
Our results suggest that the Cola-2 mutation in the oim model
produces significant alterations in the structural and mechanical
properties of ventricular myocardium. Although oim animals are
smaller in general than wild-type animals, HW/BW ratio is
similar and basal LV function in vivo is not significantly altered.
Figure 6. Opening angle represents LV circumferential residual
strain. The oim hearts displayed a 2-fold higher opening angle
compared with wild-type hearts. n⫽5 for each group.
Weis et al
Myocardial Mechanics in Osteogenesis Imperfecta
667
Figure 7. A, Mean stress-strain relations
during passive uniaxial stretch of RV papillary muscle. Strains for oim mice are higher
at all pressures compared with wild-type
mice, indicating lower stiffness under uniaxial tension. B, Pressure-relative volume
change during passive inflation of isolated
hearts. Volumes for oim are higher at all
pressures compared with wild-type, suggesting a softer ventricle under passive
loading.
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The mild increase in LV wall thickness in oim mice may
represent a degree of compensation for reduced collagen, although the ratio of wall thickness to chamber diameter was
comparable with wild-type mice. Important differences between
oim and wild-type mice were manifested as smaller myocardial
collagen fiber area fraction, number density, and diameter.
Hydroxyproline results support a decrease in total LV collagen
content. These alterations were associated with softer passive
uniaxial stress strain and ventricular pressure–volume relations
in oim mice. Unexpected alterations in transmural myofiber
angles, residual strains, and collagen cross-link density in oim
may be adaptations to these alterations in collagen amount and
mechanical properties and could explain the normal cardiac
function and lifespan for oim mice.
Myocardial Collagen Structure
Loss of functional ␣2(I) chains and deposition of ␣1(I) homotrimers are the main structural extracellular matrix alterations in
homozygous oim. The biological effect of the mutation is
probably compounded by decreased deposition of homotrimeric
collagen. Altered myocardial collagen structure and size in oim
are consistent with data published by Misof et al16 reporting an
⬇60% decrease in collagen fiber diameter in the oim tendon.
Both collagen content and structure are important determinants
of passive stiffness. A microstructural model of a single collagen
fiber suggested that myocardial fiber stiffness is affected by
collagen fiber density, tortuosity, and, most of all, diameter.4 The
42% smaller diameter and 38% lower number density measured
for oim would suggest significant differences in LV stress-strain
behavior. Other factors that may potentially influence myocardial stiffness in oim could be altered type III collagen or titin2
concentrations, homotrimeric collagen fibers comprised of three
␣1(I) chains,29 or altered integrin expression.30 Our biochemistry
results suggest a mild increase in nonreducible cross-links,
suggesting that the lower amount of collagen present is more
highly cross-linked than typical myocardial collagen. The increase in cross-links could be a form of compensation for the
compromised tissue stiffness in the oim heart. Without this
increase, the greater material compliance measured in oim may
have been even more pronounced. The mechanism by which
cross-link density affects the stiffness or morphology of the
relatively large perimysial collagen fibers has not been investigated in the myocardium or other tissues. Increased cross-link
density directly affects adjacent collagen fibrils, which pack
together to form large collagen fibers.31 It is unclear how
increased cross-link density would alter the tortuosity, periodic-
ity, or bending modulus of a large coiled perimysial collagen
fiber, for example.
The ratio of collagen types I/III can affect myocardial stiffness
and is altered in some models of heart disease.32 33 It is possible
that there was a compensatory increase in type III collagen in the
oim. Hydroxyproline measurements showed a markedly lower
total collagen concentration in oim myocardium, suggesting that
any changes in type III collagen did not appreciably affect total
collagen. The oim model has also been reported to assemble
homotrimeric collagen I molecules that may also affect collagen
fiber and tissue stiffness.15 Biochemical measurements in the
present study only addressed differences in total collagen between genotypes, whereas the histological measurements only
addressed structural changes in perimysial collagen fibers. A
more complete biochemical investigation, including collagen
type distribution as well as finer levels of histology using
electron microscopy, would help to better characterize the
determinants of altered stiffness in this model. But in light of the
abnormality of type I collagen in the oim and the uncertain
specificity of available polyclonal antibodies against type III
collagen (a highly conserved protein), we elected not to measure
the collagen I/III ratio in the present study.
Uniaxial and Ventricular Chamber Stiffness
Because myofiber and collagen fiber axes coincide with the
papillary muscle long axis,34 uniaxial stretch reveals properties
along the perimysial collagen fiber axis. Although papillary
muscle stiffness was reduced ⬇40% in oim mice, LV chamber
stiffness was only ⬇20% lower than in wild-type mice. This
may indicate that perimysial collagen contributes more to
stiffness in the fiber direction than transverse directions, which
are also strained during filling. In that case, anisotropy in oim
myocardium would also be reduced. The relationship between
collagen fiber tortuosity and strain may also be affected by the
oim mutation, because the smaller average fiber diameter measured in oim may affect the bending and uncoiling of the
perimysial collagen fibers.35 Investigators have used various
experimental models exhibiting alterations in collagen content to
study LV structure-function relationships. Decreased collagen
has been achieved through treating with bacterial collagenase5
and 5,5⬘-dithio-2-nitrobenzoic acid,36 whereas increased collagen has been measured in hypertension,37 pressure-overload
hypertrophy,38 and exercise.39 Changes in myocardial stiffness
for these models were varied, and some differences were
attributed to other factors, such as edema, altered collagen
cross-links, or hypertrophy. Alterations in resting sarcomere
length, integrin expression, collagen cross-linking, adaptive
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Circulation Research
October 13, 2000
mechanisms, or ventricular geometric remodeling may counterbalance the 35% reduction in collagen and 42% smaller fiber
diameter in oim hearts to approximate normal function. Even if
sarcomere lengths in the zero-stress state were not different, the
differences in residual strain would make their transmural
distributions different in the unloaded (zero-pressure) state,40
and this could alter the mechanics of ventricular filling.
Zero-Stress State
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A 2-fold higher opening angle in oim indicates increased
circumferential residual strain gradients in the LV wall, suggesting that decreased collagen causes increased residual stresses. In
the tightskin mouse heart with twice as much type I collagen, the
opening angle was only 7⫾6 degrees.22 These results suggest
that collagen may influence the distribution of residual stresses.
In normal myocardium, residual stress is compressive at the
endocardium and tensile at the epicardium.27 Higher strains may
induce higher residual stresses until circumferential fiber strain
gradients are normalized.41 Minimal transmural gradients of
sarcomere length have been observed in the stress-free state,
whereas large gradients occurred in the unloaded state.40 This
sarcomere length gradient may offset an opposite sarcomere
extension gradient during filling and promote more uniform
end-diastolic sarcomere length distribution and, hence, more
uniform systolic force development. Increasing residual stress
would additionally increase sarcomere length gradient and thus
balance higher stress and strain gradients transmurally. Regardless of mechanism, increased residual strain in oim may be an
adaptation to higher ventricular strains measured during isolated
heart inflation and higher fiber strains measured during uniaxial
stretch, which are consequences of less stiff material properties.
Transmural Myofiber Angle
Myofiber angles in normal hearts,42 disease models,43 between
systole and diastole,44 and from our experience with rodents,
porcine, and canine models show small variation over a wide
range of conditions and between individuals or species. Thus,
the ⫹20-degree shift in transmural myofiber angles in oim hearts
from wild-type mice throughout the LV wall thickness is quite
remarkable. Myofiber angle is an important determinant of
torsion, which results from myocardial anisotropy in systole and
diastole. Both torsion and myofiber angle distribution may act to
reduce transmural strain gradients.45 Mathematical models in
which fiber angles were allowed to redistribute show fiber
angles assume a physiological configuration to minimize transmural strain or fiber strain gradients.46 Changes in fiber orientation hardly affect pressure-volume behavior but significantly
affect distribution of active muscle fiber stress and sarcomere
length.47
The myofiber angle shift in oim hearts may be an adaptation
to altered mechanical properties. Mathematical models have
shown torsional deformation contributes to equalizing transmural fiber length changes by increasing epicardial fiber lengthening and decreasing endocardial fiber lengthening during filling.26,45 Without torsion, fiber strains would be larger at the
endocardium than the epicardium. During systole, twist is
generated by anisotropic muscle fiber contraction. During passive filling, torsion is also the reflection of anisotropic myocardial stiffness,45 which is greater in the fiber direction and can be
partially attributed to alignment of large coiled collagen fibers
with myofibers. The loss of myocardial collagen in oim may be
associated with greater decreased stiffness in the myofiber
direction and material properties that are more isotropic compared with normal. This reduced anisotropy would produce a
consequent loss of twist.45 Yet our data suggest normal epicardial shear strains in oim isolated hearts during passive inflation
(wild-type: ⫺0.015, oim: ⫺0.019). We propose that realignment
of myofibers in oim is an adaptation to restore normal myocardial twisting in the setting of reduced passive anisotropy. This
adaptation serves to neutralize transmural stress and sarcomere
length gradients from endocardium to epicardium.45 Similarly,
lower fiber stiffness would increase end-diastolic volume and
strain and thus increase stress and sarcomere length gradient
between endocardium and epicardium. We propose that oim
hearts develop larger residual strains that compensate for these
higher gradients and, in addition to the realignment of myofibers, promote a normal gradient of stresses through the wall
thickness.45
Alternatively, collagen itself may be required for normal
myofiber patterning. Neonatal cardiac myocytes express in
vivo–like phenotypes and spread parallel to a substrate of
aligned type I collagen, and the cardiac ␣1␤1 integrin complex
can detect and transduce phenotypic and positional information
stored within the structure of the surrounding matrix.48 The oim
␣2(I) mutation may impede normal myofiber development by
interfering with this pathway of signal transduction.
Although this study investigated collagen structure, collagen
biochemistry, fiber stiffness, LV chamber stiffness, myofiber
angle distribution, and zero-stress state, other factors were not
investigated. Integrin expression and titin concentration affect
tissue stiffness but were not measured. If they were increased in
oim, they may partially compensate for loss of type I collagen.
The endomysial collagen structure is undetectable with light or
confocal microscopy; thus, our conclusions and discussion
reflect only differences between genotypes measured for perimysial collagen fibers. The major advantage of our approach is
the ability to relate how ␣2(I) collagen deficiency in the heart
affects more than structure and myocardial stiffness but results in
altered fiber angle morphology and increased residual stress.
In conclusion, we have measured significant structural alterations in the heart resulting from the oim model of ␣2(I) collagen
deficiency. Reductions in passive uniaxial and ventricular chamber stiffness were accompanied by substantially altered myofiber
angles and residual strain, which may be beneficial adaptations.
Our work supports the hypothesis that in addition to providing a
structural role by supporting and organizing myocytes, myocardial collagen directly contributes to mechanical stiffness at high
and low loads and may indirectly influence stress-free states.
The present study serves as the first characterization of the
cardiac phenotype for the oim model of OI. These results can
provide a basis to examine structural and mechanical roles of
collagen in other tissues and can be combined with a diseased
state, such as myocardial ischemia, to investigate how collagen
contributes to cardiac mechanics in health and disease.
Acknowledgments
This research was supported by National Institutes of Health grant
HL41603 and an American Heart Association predoctoral fellow-
Weis et al
ship. We acknowledge the technical assistance of Babak Fazeli. Dr
Nancy Dalton performed the mouse echocardiography, and Dr John
Ross, Jr, assisted with data analysis and interpretation. Dr Fred
Harwood and Dr David Amiel facilitated the collagen
biochemistry assays.
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Myocardial Mechanics and Collagen Structure in the Osteogenesis Imperfecta Murine (
oim)
Sara M. Weis, Jeffrey L. Emery, K. David Becker, Daniel J. McBride, Jr, Jeffrey H. Omens and
Andrew D. McCulloch
Circ Res. 2000;87:663-669
doi: 10.1161/01.RES.87.8.663
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