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J Appl Physiol 103: 664-672, 2007. First published May 24, 2007; doi:10.1152/japplphysiol.00118.2007
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Altered biomechanical properties of carotid arteries in two mouse models of muscular dystrophy

W. W. Dye,1 R. L. Gleason,2 E. Wilson,3 and J. D. Humphrey1

1Department of Biomedical Engineering and M. E. DeBakey Institute, Texas A&M University; 2Woodruff School of Mechanical Engineering and Coulter Department of Biomedical Engineering, Georgia Tech, Atlanta, Georgia; and 3Department of Systems Biology and Translational Medicine, Texas A&M Health Science Center, College Station, Texas

Submitted 25 January 2007 ; accepted in final form 15 May 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Muscular dystrophy is characterized by skeletal muscle weakness and wasting, but little is known about possible alterations to the vasculature. Many muscular dystrophies are caused by a defective dystrophin-glycoprotein complex (DGC), which plays an important role in mechanotransduction and maintenance of structural integrity in muscle cells. The DGC is a group of membrane-associated proteins, including dystrophin and sarcoglycan-{delta}, that helps connect the cytoskeleton of muscle cells to the extracellular matrix. In this paper, mice lacking genes encoding dystrophin (mdx) or sarcoglycan-{delta} (sgcd–/–) were studied to detect possible alterations to vascular wall mechanics. Pressure-diameter and axial force-length tests were performed on common carotid arteries from mdx, sgcd–/–, and wild-type mice in active (basal) and passive smooth muscle states, and functional responses to three vasoactive compounds were determined at constant pressure and length. Apparent biomechanical differences included the following: mdx and sgcd–/– arteries had decreased distensibilities in pressure-diameter tests, with mdx arteries exhibiting elevated circumferential stresses, and mdx and sgcd–/– arteries generated elevated axial loads and stresses in axial force-length tests. Interestingly, however, mdx and sgcd–/– arteries also had significantly lower in vivo axial stretches than did the wild type. Accounting for this possible adaptation largely eliminated the apparent differences in circumferential and axial stiffness, thus suggesting that loss of DGC proteins may induce adaptive biomechanical changes that can maintain overall wall mechanics in response to normal loads. Nevertheless, there remains a need to understand better possible vascular adaptations in response to sustained altered loads in patients with muscular dystrophy.

vascular remodeling; dystrophin; sarcoglycan-{delta}; axial stress; biomechanics

MUSCULAR DYSTROPHY IS CAUSED by genetic defects in components of the dystrophin-glycoprotein complex (DGC) (6). This complex links the actin cytoskeleton of muscle cells to the adjacent extracellular matrix (ECM) (13) and is responsible for distributing mechanical stresses on these cells to maintain structural integrity during contraction and relaxation (28, 31). Absence of DGC proteins could have important implications for arteries, which consist largely of an abundant ECM with embedded smooth muscle cells (SMCs) that possess DGCs and are subjected to changing circumferential and axial stresses (21, 34). Indeed, altered biomechanical properties of the vasculature in individuals with muscular dystrophies could contribute to the progression of the skeletal muscle disease as well as diverse diseases related to altered flow to end organs (24).

Biomechanical and functional properties of common carotid arteries from dystrophin-deficient (mdx) and sarcoglycan-{delta}-deficient (sgcd–/–) mice were compared with those of wild-type controls (C57Bl/6J) to elucidate muscular dystrophy-related abnormalities. Mice lacking these two particular DGC proteins were chosen because deficiencies in either dystrophin or sarcoglycan-{delta} cause severe muscular dystrophies in humans (Duchenne's muscular dystrophy and limb-girdle muscular dystrophy, respectively) and are associated with vascular abnormalities in mice. For example, carotid and mesenteric arteries in mdx mice exhibit a reduced flow-induced dilation response (24, 25), and coronary arteries in sgcd–/– mice display abnormal vascular constrictions (7). In this study, a computer-controlled perfused organ-culture system (14) was used to measure and control luminal pressure and axial stretch while measuring associated changes in vessel outer diameter and axial force to elucidate possible biomechanical abnormalities in mdx and sgcd–/– arteries. Results revealed significant differences in arteries of mice lacking proteins associated with severe human muscular dystrophies.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Arterial isolation and setup for biaxial testing.   Wild-type and mdx mice were obtained from Jackson Laboratories (Bar Harbor, ME), whereas breeding pairs of sgcd–/– mice were obtained from the laboratory of Dr. K. P. Campbell (University of Iowa, Iowa City, IA), and their offspring were used for experiments. All animals were maintained by the Texas A&M University Laboratory Animals Resources and Research Program and used in accordance with University Laboratory Animal Care Committee (ULACC) guidelines and with ULACC approval. Male wild-type (C57Bl/6J), mdx barrier AX6 (on a C57Bl/10SnJ background), and sgcd–/– (on a C57Bl/6J background) mice, between 8 and 12 wk old, were anesthetized with an injection of pentobarbital sodium (50 mg/kg ip) containing heparin (1,000 U/kg ip), and the left and right common carotid arteries were excised quickly. Left carotids were placed in warm DMEM (Invitrogen), supplemented with 10% heat-inactivated fetal bovine serum (Hyclone) as well as 1,000 U/l penicillin and 1,000 g/l streptomycin (Invitrogen), and mounted on perfusion glass cannulae at both ends by using 6-0 suture.

The cannulated left arteries were submerged in a bath of fresh DMEM within the chamber of the biaxial testing device (14). Flow was initiated intraluminally and adventitially from two reservoirs containing supplemented DMEM. An axial load cell was attached via the distal cannula and the vessel was imaged via a charge-coupled device camera. Measurements were first recorded in the unloaded configuration (pressure P = 0 mmHg and axial load f = 0 g), including unloaded diameter D and overall unloaded axial length L where the vessel is just straight. Next, the vessel was stretched axially and pressurized slowly (mean intraluminal flow Q = 0.05 ml/min) to conditions thought to be near in vivo (axial stretch {lambda}z = 1.8 times the unloaded length, P = 100 mmHg, and temperature T between 32–38°C) (9), at which it remained for 30 min. Following equilibration, intraluminal flow was stopped and the vessel was subjected to two initial cycles of preconditioning at the highest loads to be prescribed during testing, that is, P cycled from 0 to 160 mmHg at {lambda}z = 1.95.

Mechanical and functional testing.   Mechanical testing consisted of two protocols, pressure-diameter (P-d) and force-length (f-{ell}) tests. Before the first P-d test, the vessel was depressurized and unloaded to {lambda}z = 1.0 to record the unloaded length and diameter relative to the preconditioned state; this minimized differences in excision-related strain histories between specimens. For P-d tests, the vessel was separately held at three extensions, {lambda}z = 1.65, 1.80, and 1.95, while P was cycled three times from 0 to 160 mmHg. For f-{ell} tests, the vessel was separately held at three pressures, P = 60, 100, and 140 mmHg, while {lambda}z was cycled two times from the length at which the vessel first bent to {lambda}z = 1.95. At the conclusion of P-d and f-{ell} testing, the vessel was unloaded, values of L and D were verified, and the axial force value, f, was zeroed if needed. Inner and outer diameters were measured by manually adjusting video calipers at 10 configurations: P = 0 mmHg and {lambda}z = 1.0, 1.2, 1.4, and 1.65; P = 40 and {lambda}z = 1.4, 1.65, and 1.8; P = 80 mmHg and {lambda}z = 1.65 and 1.8; and P = 100 mmHg and {lambda}z = 1.8.

The vessel was then brought to {lambda}z = 1.65 and P = 80 mmHg (without flow) for 15 min to equilibrate for function testing. At the end of this second equilibration period, the adventitial flow was stopped and a different stimulant was added to the bath every 15 min: first, phenylephrine (PE), at a final concentration of 10–5 M, which tests the contractile ability of SMCs; second, carbamylcholine chloride (CCh), at 10–5 M, which tests endothelial-dependent SMC relaxation; and third, sodium nitroprusside (SNP), at 10–4 M, which induces endothelial-independent SMC dilation. Verifying function after the mechanical testing ensured that no significant smooth muscle or endothelial damage had occurred. Finally, after another 15 min, the bath was emptied and refilled with warm HBSS (Invitrogen) without calcium and magnesium, but with 2 mM EGTA and 10 µM SNP to relax fully the SMCs; the diameter was recorded until it stabilized, typically within 15 min. Repeated mechanical tests in this calcium-free HBSS plus EGTA provided data in a final fully passive state. The P-d and f-{ell} tests were repeated as described above, the only difference being that two, not three, pressure cycles were performed at each axial stretch.

Histology.   At the end of biomechanical and functional testing, vessels were removed from the device, fixed in 4% paraformaldehyde in an unloaded state for 1 h, then immersed in a cryoprotectant (30% sucrose) overnight. Vessels were then mounted in optimum cutting temperature medium (Tissue Tek) in isopentane cooled with liquid nitrogen and stored at –20°C. Frozen samples were sectioned at 5 or 7 µm and stained with hematoxylin and eosin (H&E), Verhoeff van Gieson (VVG), or picrosirius red (PSR) for visualization of wall dimensions, elastin, and collagen, respectively.

Data analysis.   We analyzed data only from functional vessels (n = 6 wild-type, n = 5 mdx, and n = 6 sgcd–/–), signified by at least a 30% contraction in response to PE (relative to the final passive diameter in calcium-free HBSS) and at least a 15% dilation (relative to the contraction caused by PE) in response to CCh. Overall, 71% of vessels tested retained endothelial and smooth muscle function following biomechanical testing. Axial force, axial stretch, luminal pressure, and outer diameter were collected at 4 Hz during all tests. Separate measurements of inner and outer diameters allowed the volume of the vessel wall to be estimated as

Formula
where a is the inner radius and b the outer radius; values at the aforementioned 10 different states were averaged to give V. Assuming incompressibility, this mean volume plus online values of outer diameter allowed the luminal radius a and wall thickness h to be computed at all states, namely

Formula
This, in turn, allowed calculation of the mean axial and circumferential Cauchy stresses ({sigma}z and {sigma}{theta}, respectively) at every test point, namely

Formula
The axial stretch ratio was calculated as

Formula
whereas the circumferential stretch ratio was calculated by using midwall radii:

Formula
The current midwall radius rmid was calculated as

Formula
whereas the unloaded midwall radius Rmid was calculated as

Formula
where B is the outer radius in the unloaded configuration and A can be calculated from wall volume V. Interpolating between test points allowed the loaded dimensions, axial force, pressure, and axial and circumferential stresses to be analyzed by one-factor ANOVA for P < 0.05, with means separation using Bonferroni's post hoc test. Body weights of the mice as well as mean wall volumes and unloaded dimensions of the arteries were similarly analyzed for P < 0.05. For the P-d protocol, outer diameter and axial force were compared between pressures of 2 and 140 mmHg, and circumferential stretch was compared for values between 0.5 and 1.4 (these ranges of independent variables were common for all vessels, thus enabling statistical comparison). For the f-{ell} protocol, axial force and axial stress were compared from the lowest common axial stretch up to an axial stretch of 1.95.

Another comparison was enabled by the f-{ell} data. It is known that arteries are extended in vivo well beyond their unloaded length and that the axial force tends to remain constant over a range of pressures at this in vivo axial stretch (21). One way to estimate the in vivo axial stretch ({lambda}z in vivo, the in vivo length normalized to the unloaded length) of arteries isolated from the body is to find the so-called crossover point in f-{ell} data collected at multiple constant pressures (3, 35, 37). We compared force-length curves at 60, 100, and 140 mmHg to find intersection points between each pair of curves (calculated by interpolating between the two closest axial stretches), and the three intersection points were averaged to estimate that vessels' in vivo axial stretch {lambda}z in vivo and corresponding axial load fin vivo. We performed an ANOVA on the {lambda}z in vivo values for P < 0.05 with means separation using Bonferroni's post hoc test.

Finally, outer diameters measured in response to various stimulants in the functional tests were normalized with respect to the fully passive diameter (Ca2+ free with EGTA) and compared by ANOVA for P < 0.05, with means separation using Bonferroni's post hoc test. Comparisons were made between all three mouse genotypes, or groups, for all data analyses.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Functional responses.   Arteries from all three groups retained smooth muscle and endothelial function (Table 1) and exhibited a similar partial tone and vasoactive capacity following mechanical testing (Fig. 1). Relative to individual fully passive diameters, the initial diameters at {lambda}z = 1.65 were 88% for the wild-type, 86% for the mdx, and 85% for the sgcd–/– mice, thus indicating a "basal tone" between 12 and 15%. After dosing with PE, wild-type arteries constricted further to 67% of their fully passive diameter, mdx arteries constricted to 66%, and sgcd–/– constricted significantly more than the mdx and wild-type arteries, to 62% of their fully passive diameter. Otherwise, the three groups of vessels did not display any significant differences in response to PE. All groups had similar vasodilatory responses to CCh, with the wild-type dilating to 84% and the mdx and sgcd–/– dilating to 82% of their fully passive diameter. In response to SNP, the wild-type arteries dilated to 97% of their final diameter whereas the mdx and sgcd–/– dilated to 95%.


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  		Table 1. Summary of mouse and vessel properties for wild-type, mdx, and sgcd–/– mice

 

Figure 1
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  		Fig. 1. Representative functional response of carotids from all 3 groups at P = 80 mmHg and {lambda}z = 1.65. Phenylephrine (PE) was used to test smooth muscle contractility, carbamylcholine chloride (CCh) to test endothelial-dependent smooth muscle relaxation, and sodium nitroprusside (SNP) to test endothelial-independent smooth muscle relaxation. A calcium-free salt solution (Ca2+ HBSS) plus a calcium chelator (EGTA) was used to relax smooth muscle cells completely.

 
Morphology.   Table 1 shows that body weight and gross vessel dimensions were also very similar (i.e., not significantly different) across the three groups. Based on measurements in the testing device, mean unloaded outer diameter and wall thickness were 394 and 72 µm, respectively. Despite inevitable shrinkage of histological samples, overall similarities across the three groups were confirmed in H&E-, VVG-, and PSR-stained sections (Fig. 2), including a qualitatively similar media consisting of two to three musculoelastic fascicles or layers and adventitia consisting of abundant collagen. Quantitative image analysis suggested, however, that the percentage of the wall occupied by the media (mean ± SD) was less in both the mdx (0.32 ± 0.09) and the sgcd–/– (0.34 ± 0.07) mice compared with wild-type controls (0.39 ± 0.08), thus implying a greater percentage of adventitia and hence fibrillar collagen in the two mouse models of muscular dystrophy. These trends did not reach statistical significance, however, likely because of the relatively small sample size. Although an overall increased collagen-to-elastin ratio in unloaded sections may suggest a possible increase in passive stiffness, this must be evaluated directly because stiffness at physiological loads also depends on collagen undulation, orientation, and cross-linking, which cannot all be measured directly in cross sections since most adventitial collagen is oriented axially or obliquely relative to the axial direction (21).


Figure 2
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  		Fig. 2. Representative cross sections of carotid arteries from wild-type (left column), mdx (middle column), and sgcd–/– (right column) mice. Shown are hematoxylin and eosin (H&E)-stained (top row) and Verhoeff van Gieson (VVG)-stained (middle row) sections viewed under normal light as well as picrosirius red (PSR)-stained (bottom row) sections viewed under circularly polarized light. Despite similar overall morphological features and wall thickness, the percentage of the wall occupied by the media appeared to be less in both the mdx (0.32 ± 0.09) and the sgcd–/– (0.34 ± 0.07) mice compared with wild-type controls (0.39 ± 0.08), thus suggesting more adventitia and adventitial collagen in the 2 mouse models of muscular dystrophy.

 
Biaxial mechanical responses.   The general mechanical response of all three groups of carotids to biaxial testing was similar to that reported for other mouse carotids tested in our laboratory (FVB/N) (14) and elsewhere (C57-B110, C57Bl/6J, and 129S6) (15, 24, 36). In pressure-diameter tests, the arteries initially distended substantially in response to increasing pressure but stiffened at higher pressures. The sudden stiffening was more apparent in passive testing because the active P-d curves displayed a slightly more linear response due to partial SMC contraction. Vessels from the three groups responded similarly for pressure cycles at low ({lambda}z = 1.65) and mid ({lambda}z = 1.80) axial stretches; there were no significant differences between the wild-type and either the mdx or the sgcd–/– outer diameters at these stretches and pressures. Differences between groups were found at the high stretch ({lambda}z = 1.95) and mid to high pressures (Fig. 3A), however, because diameters were significantly smaller in the active and passive tests for both mdx and sgcd–/– compared with wild-type.


Figure 3
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  		Fig. 3. Mean passive (Ca2+ free) mechanical data. A: P-d curves at the high ({lambda}z = 1.95) axial stretch. The mdx diameters became significantly smaller (P < 0.05) than those of the wild type over the pressure range P = 92–140 mmHg in the active state (not shown) and at P = 66–140 in the passive state (indicated by the *M->). The sgcd–/– diameters were significantly smaller for the pressure range P = 102–140 in the active state (not shown) and P = 74–140 in the passive state (indicated by the *S->). B: circumferential stress-stretch curves at the high ({lambda}z = 1.95) axial stretch. There was a range in the passive state ({lambda}{theta} = 0.860–1.145), indicated by *M, for which mdx stresses were significantly greater (P < 0.05) than wild-type values. There was a small range in the passive (0.890–0.920) state for which mdx stresses were significantly greater than sgcd–/– stresses, indicated by *S-M. Although there are visible trends in the data, the variance of the stresses at high stretches prevented statistical differences from reaching significance. C: axial stress-stretch curves at the high (P = 140 mmHg) pressure. The mdx stresses became significantly greater (P < 0.05) than those of the wild type over the axial extension range {lambda}z = 1.77–1.95 in the active state (indicated by *M->) and at {lambda}z = 1.83–1.95 in the passive state (not shown). The sgcd–/– stresses were significantly greater for the pressure range {lambda}z = 1.83–1.95 in the active state (indicated by *S->) and {lambda}z = 1.77–1.95 in the passive state (not shown).

 
Circumferential stress-strain data showed that only mdx stresses were significantly different from those of the other two groups. The mdx circumferential stresses {sigma}{theta} were increased significantly compared with wild-type stresses for a small range of circumferential stretches for the low and mid axial stretches and over a broader range at the high axial stretch. There was also a range of circumferential stretches in active and passive states at which the mdx circumferential stress was significantly greater than the sgcd–/– circumferential stress (Fig. 3B).

The axial forces required to maintain constant the axial stretch during P-d tests (Fig. 4) were similar for all three groups at the low axial stretch of 1.65. At the mid stretch of 1.80, however, mdx axial forces were significantly greater than those of wild type at high pressures. At the high stretch of 1.95, mdx axial forces were significantly greater than those of wild type over the whole pressure range, whereas the sgcd–/– forces were greater at high pressures. A particularly interesting finding was that the axial force response at the high axial stretch of 1.95 was fairly constant for the wild type but increased greatly for the mdx and sgcd–/– vessels; in contrast, the axial force responses were fairly constant for the mdx and sgcd–/– vessels at the mid axial stretch of 1.80 whereas the axial force decreased for the wild-type vessels at this stretch. Comparing all axial force responses during pressurization for the three groups at the three axial stretches (Fig. 4) revealed that the wild-type force response at the axial stretch of 1.95 was similar to the mdx and sgcd–/– force responses at the axial stretch of 1.80. Because the axial force remains constant over a range of pressures when the axial stretch is near the in vivo value (21), whereas it increases at axial stretches greater than in vivo and decreases at axial stretches less than in vivo, this suggested that the in vivo axial stretch was greater for wild-type mice than for mdx and sgcd–/– mice.


Figure 4
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  		Fig. 4. Mean passive axial force response during P-d testing for all mouse groups at all 3 fixed axial stretches, which are indicated on the right axis. Note the similarity between the wild-type response at the high axial stretch ({lambda}z = 1.95) and the mdx and sgcd–/– responses at the mid axial stretch ({lambda}z = 1.80). The importance of this observation is revealed in Fig. 5. Note, too, that the wild-type response at the mid axial stretch ({lambda}z = 1.80) was also similar to the mdx and sgcd–/– curves at the low axial stretch ({lambda}z = 1.65)

 
Results from the axial force-length tests also showed significant differences between the three groups, especially at the high pressure (140 mmHg) and higher axial stretches. That is, the axial force corresponding to axial stretches for the mdx and sgcd–/– vessels was not significantly greater than for the wild type at the low pressure (60 mmHg), except that the mdx value at very high axial stretches was significantly greater than that for the wild type in the active and passive tests. At the mid pressure (100 mmHg), however, the mdx and sgcd–/– axial forces were significantly higher than those for the wild type for a range of axial stretches in the active and passive states. At the high pressure (140 mmHg), the mdx and sgcd–/– axial forces were also significantly higher than those for the wild type at midrange axial stretches in both active and passive states.

The axial stress-stretch data were similar to the axial force-length data with the exception of subtle differences. There were few significant differences between groups in the axial stress-stretch data at the low pressure in active tests. At the mid pressure, there were significant differences between sgcd–/– and mdx values compared with wild type, with a larger range of significant differences in the passive than the active state. At the high pressure, there was a range of axial stretches for both mdx and sgcd–/– at which the axial stresses were significantly higher than for the wild type (Fig. 3C). These differences were again more pronounced in passive tests. We then estimated the in vivo axial stretch ({lambda}z in vivo) by calculating the intersection point between the three force-length curves for each vessel (measured at the low, mid, and high P set points as shown in Fig. 5A) and found that wild-type vessels had a significantly higher in vivo axial stretch (e.g., 1.84) compared with mdx (1.71) and sgcd–/– (1.73). Values for the passive and active in vivo axial stretches {lambda}z in vivo are included in Table 1 for each mouse type. The axial load required to maintain the in vivo axial stretch was also significantly lower for mdx and sgcd–/– vessels than for the wild type (Fig. 5B).


Figure 5
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  		Fig. 5. A: estimation of the in vivo axial stretch ({lambda}z in vivo). Intersections among the 3 force-length curves for each vessel (measured at low, mid, and high P set points) were used to estimate the in vivo axial stretch, that is, the axial stretch at which axial load remains constant during pressurization, and the corresponding in vivo axial load sustained at that axial stretch for each mouse type. B: axial stretch {lambda}z and axial force values (with standard errors) where the average axial f-{ell} curves for each mouse type, measured at 3 different pressure set points, intersect in the active (basal tone) and passive (Ca2+ free) states. *Significant differences of the mdx and sgcd–/– values from the wild-type (P < 0.05), with Bonferroni's test for means separation.

 
Normalization to in vivo axial stretch.   Given the observation that the axial force-pressure data was similar between the wild type at high axial stretch and the mdx and sgcd–/– at mid axial stretch, additional comparisons were warranted. For the P-d test, we calculated the stretch ratio

Formula
for the high ({lambda}z = 1.95), mid ({lambda}z = 1.80), and low ({lambda}z = 1.65) axial stretches used during testing. We found that for the active and passive P-d tests, the wild-type vessels experienced a small {Lambda} (~1.03 for the active and ~1.06 for the passive) at the high axial stretch, which was very close to that experienced by the mdx (~1.03 active, ~1.05 passive) and sgcd–/– (~1.01 active, ~1.04 passive) vessels at the mid axial stretch. We then compared, at the same pressures, the wild-type outer diameters at the high axial stretch of 1.95 to the mdx and sgcd–/– values at the mid axial stretch of 1.8 and found no significant differences (P < 0.05) in either the active or passive tests (Fig. 6A). There were likewise no significant differences between the circumferential stress-stretch data measured at the high axial stretch for the wild type and the mid axial stretch for the mdx and sgcd–/– (Fig. 6B). Finally, for f-{ell} tests, we compared axial loads and stresses at common values of the stretch ratio {Lambda}. We found that this way of comparing the data further reduced the number of apparent differences between the wild-type and the mdx and sgcd–/– vessels. For example, in active or passive states at the high pressure of 140 mmHg, there were no significant differences between groups in either the axial load or the axial stress once we took the in vivo axial stretch into account (Fig. 6C). At the pressures of 60 and 100 mmHg, there were also no significant differences between the sgcd–/– and wild type for axial load or axial stress values. Indeed, there were only significant differences between the mdx and wild type over a small range of stretches at the mid pressure and over a slightly larger range of stretches at the low pressure for both the axial load and axial stress (not shown).


Figure 6
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  		Fig. 6. Passive mechanical data for each mouse type normalized to the individual estimated axial in vivo stretch. There are no longer any significant differences between the responses of the mdx, sgcd–/–, and wild-type arteries in either the active or passive state (P < 0.05). A: P-d curves at the high ({lambda}z = 1.95) axial stretch for the wild-type and at the mid ({lambda}z = 1.80) axial stretch for the mdx and sgcd–/–. Comparing the results from these 2 different axial stretch allows us to see the common change in diameter with pressurization for all 3 groups of vessels when stretched to {Lambda} of ~1.05 of their in vivo axial stretch. B: circumferential stress-stretch curves at the high ({lambda}z = 1.95) axial stretch for the wild-type and at the mid ({lambda}z = 1.80) axial stretch for the mdx and sgcd–/– arteries (with all 3 groups stretched to {Lambda} of ~1.05 of their in vivo axial stretch). C: axial stress-stretch curves, with the axial stretch normalized to the in vivo axial stretch, at the high (P = 1 40 mmHg) pressure set point.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Differences in biaxial mechanical behavior appeared between wild-type control carotid arteries and those from both mdx mice lacking dystrophin (as in the human disease Duchenne's muscular dystrophy) and sgcd–/– mice lacking sarcoglycan-{delta} (as in human limb-girdle muscular dystrophy) when compared under the same conditions. Specifically, mdx and sgcd–/– carotid arteries distended less than wild-type arteries, similar to a prior report (24), and mdx arteries exhibited higher mean circumferential stresses in standard pressure-diameter testing. Similarly, mdx and sgcd–/– arteries exhibited higher axial forces and mean axial stresses during standard axial force-length testing at extensions of 1.80 and 1.95 compared with wild-type arteries. Together, these findings suggested an apparently greater circumferential and axial stiffness in the two mouse models of muscular dystrophy, seemingly consistent with greater adventitial collagen despite an overall comparable wall thickness (Table 1, Fig. 2).

One might first think to explain the apparently stiffer behaviors of the mdx and sgcd–/– arteries in terms of a phenotypic modulation by the medial SMCs from more of a contractile to more of a synthetic phenotype. SMC phenotype is intimately associated with the surrounding ECM (21). Linkages between SMCs and ECM (specifically, to laminin, fibronectin, collagen, and glycosaminoglycans) are formed by transmembrane proteins called integrins, but also through syndecans, cell adhesion molecules, and the DGC (27). {alpha}-Dystroglycan is the component of the DGC that connects muscle cells to the ECM by binding to laminin. This, in turn, signals SMCs to take on a contractile phenotype, as characterized by expression of {alpha}-smooth muscle actin and presence of structural myofilaments (13, 19). Binding to fibronectin, on the other hand, tends to be associated with vascular SMCs of the synthetic phenotype, as in early development and injury responses (33). Although a phenotypic modulation may have occurred during development, Table 1 and Figs. 1 and 2 show that medial (but not adventitial) morphology and overall functional responses were similar for all three groups, consistent with prior findings by others (24), thus suggesting that SMC phenotype was similar at the time of testing. A similar finding for mdx mice was reported by Loufrani et al. (25), who reported endothelial, not smooth muscle, differences in mdx mice.

Although arterial wall mechanics is complex (21), salient features can often be appreciated in terms of the three primary stresses that act on the wall: mean wall shear stress, circumferential wall stress, and the often neglected axial wall stress, which tend to govern the deformed inner radius, wall thickness, and axial length, respectively. Given the similarity in undeformed radii and wall thickness across groups (Table 1), which compare favorably to C57Bl/6J controls in Wagenseil et al. (36) as well as the observation from Fig. 3 that the axial force response was similar for wild-type carotids tested at an axial stretch of 1.95 and mdx and sgcd–/– carotids tested at an axial stretch of 1.80, we then focused on the axial behavior.

Van Loon et al. (35) showed 30 years ago that the in vivo axial stretch ratio can be estimated well in vitro via the intersection of axial f-{ell} curves obtained at different pressure set points and noted that the axial force needed to maintain this value of stretch will remain nearly constant over a range of pressures, which is physiologically advantageous as noted by others (12). In particular, they showed that in vivo axial stretches estimated from f-{ell} curves compared extremely well with those measured directly for canine carotid arteries ({lambda}z in vivo = 1.52 ± 0.11 vs. 1.53 ± 0.12, n = 12) as well as for canine femorals, human carotids, and human iliacs. This excellent agreement has been confirmed by many others, as, for example, Weizsacker et al. (37), who reported estimated and measured values of {lambda}z in vivo to be 1.701 and 1.696 for rat carotid arteries, with an n = 6, and Brossollet and Vito (3), who reported estimated and measured in vivo gage lengths to be within 4%, n = 21, for canine saphenous veins. Using a formal stability analysis, Brossollet and Vito suggested further that this unique axial behavior reflects a structural optimization against "longitudinal buckling," which complements numerous physiological roles.

Whereas it is often straightforward to measure the in vivo axial stretch in vessels such as the descending aorta (17), it can be difficult to measure this stretch accurately in small vessels such as the mouse carotid artery wherein neck position may play a role (8, 10, 11) [e.g., see Figs. 1D and 6D in Wagenseil et al. (36), who reported that excised mouse carotids curve upon excision, which complicates strain analysis by yielding a nonaxisymmetric field, but appeared to find values within ~10% of that where the "longitudinal force-pressure relationship... remains relatively constant"]. Hence we relied on the proven in vitro estimation based on axial f-{ell} curves obtained at multiple pressures using a micron-resolution computer-controlled system (14). Our data suggested strongly that mdx and sgcd–/– carotid arteries had significantly reduced in vivo axial stretches compared with wild-type arteries (Fig. 5, A and B). We thus normalized the stretch data using the estimated in vivo axial stretch ratios for all three genotypes and found that this drastically reduced the apparent differences in behavior (cf. Fig. 3, A–C with Fig. 6, A–C). In particular, there were no longer differences, between mdx or sgcd–/– and wild-type, in outer diameters and circumferential stresses measured in the P-d tests or axial forces and stresses measured in the f-{ell} tests. These findings of similar P-d behaviors are consistent with the report of Loufrani et al. (24) for mdx mice. The only significant differences that persisted herein were decreased axial forces during pressurization in active and passive P-d tests for the mdx arteries compared with wild-type (Fig. 7).


Figure 7
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  		Fig. 7. Passive (Ca2+ free) axial force for P-d curves at the high ({lambda}z = 1.95) axial stretch for the wild type and at the mid ({lambda}z = 1.80) axial stretch for the mdx and sgcd–/–. Comparing results from these 2 different axial stretches reveals common changes with pressurization for all 3 groups of vessels when stretched to {Lambda} of ~1.05 of their in vivo axial stretch. The axial force values for the sgcd–/– were decreased but not significantly different from the wild type in either the active or passive state (P < 0.05). The mdx values were significantly decreased from those of the wild type over low pressures in the active state and over a wide range of pressures (P = 2–116 mmHg) in the passive state (indicated by *M).

 
In summary, mdx and sgcd–/– carotid arteries appeared to decrease their in vivo axial stretch, relative to controls, to a level such that their biaxial response to the mechanical loads studied herein was nearly identical to arteries from normal, wild-type mice. In other words, it appears that DGC-deficient arteries, although perhaps having more adventitial collagen, compensated (i.e., adapted) so as to lower the intramural stresses (back to normal values) that would otherwise have been experienced by the SMCs, which in turn could be protective for the more fragile cells (which are susceptible to stress concentrations and thus degeneration). Indeed, such an adaptation would be consistent with similar findings that circumferential and axial stress-strain behaviors are similar for wild-type controls and ELN+/– mouse carotid arteries that contain 30% less elastin and have a significantly lower in vivo axial stretch (36). It is interesting to note, therefore, that large arteries tend to decrease their in vivo stretch during adaptations in hypertension (34), which is also thought to help decrease wall stress back toward a normal, or homeostatic, value that may be nearly equibiaxial in some vessels (21). In maturity, such decreases in axial stretch arise because of axial growth, or lengthening, as evidenced in ex vivo and in vivo studies wherein "overextended" vessels lengthen (5, 15, 18, 22), again appearing to return wall stresses toward normal values (16). In these reports, imposed increases in axial extension were normalized rapidly via cell and matrix rearrangement or turnover without a loss of overall vascular function. For example, in just 2 days, mouse carotid arteries cultured at axial stretches greater or less than their in vivo axial stretch appeared to adapt to the new length and thereby restore their force-pressure response toward its in vivo character (15).

In summary, notwithstanding the need to understand better the underlying molecular processes (1, 2, 4, 9, 20), our results affirm that mean axial stress is an important factor driving the development and adaptation of large arteries even though prior emphasis has been on the effects of wall shear stress and circumferential wall stress on vascular mechanobiology. Axial stretches and forces appear to arise in arteries during normal development and to depend strongly on the presence of elastin (10, 12); they are revealed easily when a vessel is freed from periarterial tissue and excised. These axial stretches may be maintained, in part, by connections to periarterial tissues, but they also reflect an underlying local structure that appears to be optimized mechanically (3, 21, 35). We speculate that for mdx and sgcd–/– arteries, the axial stress concentrations felt by the SMCs at particular integrins would be higher at normal axial stretches because of the absence of intact DGCs, which otherwise would help to distribute stresses throughout the cytoskeleton and thereby protect the cell from mechanical damage (28). Consequently, the SMCs appeared to adapt wall structure, perhaps working together with adventitial fibroblasts, to lengthen (not thicken) the vessel locally and thereby lower the axial stresses back to normal values (cf. Figs. 3 and 6). Indeed, this adaptation may be one reason for the near-normal blood pressures and flows in these animals under normal conditions (24, 25). Nevertheless, endothelial production of NO in response to changes in wall shear stress is not normal in mdx carotid and mesenteric arteries (24, 25), hence not all compensatory adaptations are complete. In summary, mdx and sgcd–/– arteries did not appear to adapt in response to altered wall shear or circumferential stresses (cf. Ref. 22), because wall thickness and inner diameters in unloaded and loaded states were not significantly different from the wild type. Rather, growth and remodeling in the arteries of mdx and sgcd–/– mice may have occurred primarily to reduce the axial force and thereby restore axial and circumferential stress toward normal [note: because of axial-circumferential coupling (21), {sigma}{theta} and {sigma}z each vary with {lambda}{theta} and {lambda}z]. There is, therefore, a need to explore better the specific cell mechanics and cell-matrix interactions in these mouse models, particularly in the axial direction. Finally, it is interesting that our estimated values of in vivo carotid axial stretches (1.84 for wild type and 1.72 and 1.73 for mdx and sgcd–/–, respectively) appear to fall in order with those reported for other species, namely 1.70 for rats (37), 1.62 for rabbits (22), 1.53 to 1.62 for canines (8, 11, 35), and 1.19 for humans (35). Why the normal axial stretch increases as animal size decreases has not been addressed but may hold clues with regard to normal biomechanical development and subsequent adaptations.

Closure.   Current research efforts seek to provide a better and longer life for patients with muscular dystrophy. As these patients live longer, however, there is concern that they may suffer from cardiovascular diseases such as atherosclerosis, hypertension, and aneurysms that affect many older people or that they may not enjoy the same benefits from cardiovascular exercise. Hence, if the vasculature of muscular dystrophy patients has compensated only in part for the absence of dystrophin or other DGC proteins, there is a need to determine whether standard exercise or treatment strategies are appropriate or need to be modified accordingly. It is important, therefore, to continue to study the vasculature in the absence of dystrophin, sarcoglycan-{delta}, and other DGC components to understand how the vessel achieves a new functional state without the DGC. Understanding the mechanism of adaptation of blood vessels that lack the DGC should further our understanding of muscular dystrophy and may enable physicians to treat patients more effectively.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
ACKNOWLEDGMENTS
 REFERENCES
 
This research project was supported, in part, by grants from the Muscular Dystrophy Association (MDA 3681) and the National Heart, Lung, and Blood Institute (HL-64372).


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
REFERENCES
 
We gratefully acknowledge the sgcd–/– breeding pairs provided by Professor Kevin P. Campbell, HHMI at the University of Iowa, and we thank Jan Patterson, Anne Taucer, and Lisa Auckland at Texas A&M for expert technical assistance during this study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. D. Humphrey, Dept. of Biomedical Engineering, 337 Zachry Engineering Center, 3120 TAMU, Texas A&M Univ., College Station, TX 77843-3120 (e-mail: jhumphrey{at}tamu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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