Isoform switching from SM-B to SM-A myosin results in decreased contractility and altered expression of thin filament regulatory proteins

Gopal J. Babu,1 Gail J. Pyne,2 Yingbi Zhou,1 Chris Okwuchukuasanya,3 Joseph E. Brayden,3 George Osol,4 Richard J. Paul,3 Robert B. Low,4 and Muthu Periasamy1

1Department of Physiology and Cell Biology, College of Medicine and Public Health, Ohio State University, Columbus 43210; 2Department of Molecular and Cellular Physiology, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267; and 3Department of Pharmacology and 4Department of Molecular Physiology and Biophysics, University of Vermont, Burlington, Vermont 05405

Submitted 16 January 2004 ; accepted in final form 7 May 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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We previously generated an isoform-specific gene knockout mouse in which SM-B myosin is permanently replaced by SM-A myosin. In this study, we examined the effects of SM-B myosin loss on the contractile properties of vascular smooth muscle, specifically peripheral mesenteric vessels and aorta. The absence of SM-B myosin leads to decreased velocity of shortening and increased isometric force generation in mesenteric vessels. Surprisingly, the same changes occur in aorta, which contains little or no SM-B myosin in wild-type animals. Calponin and activated mitogen-activated protein kinase expression is increased and caldesmon expression is decreased in aorta, as well as in bladder. Light chain-17b isoform (LC17b) expression is increased in aorta. These results suggest that the presence or absence of SM-B myosin is a critical determinant of smooth muscle contraction and that its loss leads to additional changes in thin filament regulatory proteins.

aorta; mesenteric vessels; calponin; caldesmon


SMOOTH MUSCLE MYOSIN heavy chains are produced by alternative splicing of a single gene (4). The four known isoforms, SM1A, SM1B, SM2A, and SM2B, are combinations of one of two heavy chains containing different COOH termini (SM1 and SM2) with (SM-B) or without (SM-A) a seven-amino acid insert in the myosin head (4). The expression of these isoforms is regulated in both a developmental and a tissue-specific manner (37). It also has been demonstrated that SM-B has unique contractile properties characterized by its support of a high velocity of shortening (8, 20, 21, 30).

SM-B is the predominant myosin in phasic smooth muscle tissues, including bladder and intestine. This isoform is abundant in neonatal aorta but is replaced by SM-A myosin in the adult (37). Small muscular arteries (e.g., femoral and saphenous arteries; Ref. 8) and arterioles (36) acquire and continue to express substantial amounts of the SM-B isoform in the adult. The expression pattern of SM-B myosin is altered in different pathophysiological conditions (9, 19, 31, 36). However, the functional significance of myosin isoform diversity in smooth muscle physiology is poorly understood.

To determine the role of SM-B myosin in vivo, we generated a mouse model in which the SM-B myosin isoform is replaced by SM-A myosin (3). This results in a significant decrease in velocity of muscle shortening in bladder and mesenteric resistance vessels (3). However, the absence of SM-B myosin does not affect survival, cause any overt smooth muscle pathology, or result in detectable changes in cardiovascular function (3). These results prompted us to hypothesize that loss of SM-B myosin is compensated for by changes in the expression of other thin filament regulatory proteins. To test this hypothesis, we expanded our studies of the contractile properties of aorta and mesenteric vessels to include force generation. We also examined the expression of myosin light chain isoforms and the thin filament proteins calponin and caldesmon, given their potential role in any adaptation that might have occurred. Our results suggest that altered expression of calponin and caldesmon may be involved in the adaptive response required to compensate for the loss of SM-B myosin.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Generation of SM-B-null mice. The generation of SM-B myosin isoform-specific knockout mice was described previously (3). Mice were killed by exposure to rising concentrations of CO2 in accordance with the ethical treatment of animals under a protocol approved by the Ohio State University; or by cervical dislocation and thoracotomy after isoflurane anesthesia according to a similarly approved protocol at the University of Vermont. The mice used for the studies described here were F3 generations of both sexes and between 18 and 25 wk old.

Immunohistochemistry. Immunohistochemical staining of aorta and mesenteric vessels from wild-type (WT) and smb–/– mice was performed exactly as described previously (23).

Western blot analysis. Muscle proteins were extracted as described previously (9). Briefly, bladder, aorta, or mesenteric vessel tissues from WT and SM-B-null mice were cleaned of connective tissues and powdered in liquid nitrogen. The powdered tissue was homogenized in a buffer containing (in mM) 50 Tris·Cl, pH 6.8, and 10 dithiothreitol with 1% SDS and 10% glycerol. The homogenate was boiled for 5 min, and the supernatant containing myofibrillar proteins was clarified by centrifugation at 13,000 g for 20 min. Protein estimation was done by a modified Lowry method and also quantitated by scanning densitometry of the Coomassie blue-stained gel.

Western blotting was carried out as described previously (3). Briefly, total proteins isolated from bladder, aorta, and mesenteric vessels were separated on 8% for SM1 and SM2, 10% for smooth muscle {alpha}-actin, light chain-20 isoform (LC20), calponin, caldesmon, and extracellular signal-regulated kinase (ERK)1/2, or 15% for light chain-17 isoform (LC17) SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane. Membranes were probed with the following primary antibodies: rabbit polyclonal anti-SM1, rabbit polyclonal anti-SM2 (3), mouse monoclonal anti-calponin, mouse monoclonal anti-{alpha}-smooth muscle actin, and mouse monoclonal anti-LC20 (all from Sigma), mouse monoclonal anti-caldesmon (a kind gift from Dr. Michael P. Walsh, University of Calgary, Calgary, AB, Canada), mouse monoclonal anti-LC17 (a kind gift from Dr. Kathy Trybus, University of Vermont), and rabbit monoclonal anti-phospho-p44/42 mitogen-activated protein kinase (MAPK; New England Biolabs). Signals were detected by Super Signal West Dura substrate (Pierce), quantitated by densitometry, and analyzed with Image software (version 6.1, National Institutes of Health). Smooth muscle {alpha}-actin was used as an internal control to normalize the loading of blots.

Isometric force measurements in aorta. Aortic rings of 5-mm width were dissected with a dissecting microscope and cleaned of loose fat and connective tissue, and endothelium was removed. Rings were mounted between a fixed stainless steel post and a force transducer, the output of which was monitored with a computer-based data collection system (BioPac). The mounted aorta was equilibrated in physiological saline solution (PSS; in mM: 116 NaCl, 5.4 KCl, 1.2 MgSO4, 2.5 NaHCO3, 2.4 CaCl2, 0.5 NaH2PO4, and 11 glucose) and maintained at 37°C and aerated with 95% O2-5% CO2 throughout the course of the experiment. Force measurements were performed as described previously (1). The rings were mounted in a 15-ml organ bath and allowed to equilibrate at 37°C for 1 h, during which the tension was adjusted to 50 mN. Data were acquired with BioPac instrumentation and analyzed with the accompanying AcqKnowledge software. Phenylephrine (10–5-10–7 M) concentration-response curves were obtained in a cumulative fashion. Force was normalized to cross-sectional area, using tissue weight and circumference, by the approximation A = 2 x wet wt/circumference.

Force-velocity measurements. Segments of aorta prepared as described above were equilibrated in MOPS-PSS (in mM: 140 NaCl, 4.7 KCl, 1.2 NaH2PO4, 20 MOPS, 0.02 EDTA, 1.2 MgSO4, 2.5 CaCl2, and 11.1 glucose, pH 7.4) at 37°C for 15 min. MOPS-PSS was used in this case to avoid noise attributed to bubbling with 5% CO2, required for PSS. Passive tension was adjusted to a force that was calculated to be equivalent to 100 mmHg. This was previously shown to place the muscle at a length for optimal force development (1). Force-velocity relationships for each aorta were measured as described previously (28). Briefly, aortas were stimulated (50 mM KCl), and after steady-state isometric force was developed, ramp shortenings for a duration of 500 ms at 1-min intervals were imposed with an ergometer (Aurora Scientific). Six different velocities were imposed, and the force maintained at 200 ms was recorded and used for constructing force-velocity relations. Similar measurements were made under unstimulated conditions, and these forces were subtracted from the total tension to create the active force-velocity relations. These data were fit to the hyperbolic Hill equation with Origin software for each aorta. The fitted parameters, maximum isometric force, maximum velocity, and curvature parameter for each aorta were averaged and reported as means ± SE.

Mechanical measurements in mesenteric vessels. The mechanical properties of mesenteric vessel were measured as previously described (5). Briefly, mice were euthanized, and small mesenteric arteries (130–200 µm internal diameter) were removed and placed in cold PSS (in mM: 119 NaCl, 4.7 KCl, 24 NaHCO3, 0.2 KH2PO4, 1.1 EDTA, 1.2 MgSO4, 1.6 CaCl2, and 10.6 glucose, pH 7.4). Arterial segments (2 mm in length) were then mounted in a resistance artery myograph and perfused with warmed (37°C), gassed (95% O2-5% CO2, pH 7.4) PSS. Arteries were then stretched to a preload of 300-mg force, which in preliminary experiments was determined to be the optimal preload for maximal force development. After 30-min equilibration, the arteries were contracted by exposure to bath solution containing 10 µM phenylephrine and 120 mM K+ (equimolar substitution of KCl for NaCl). The rate of isometric contraction velocity was calculated as the time required to reach half-maximal contraction, to confirm prior results (3). Vessel length and media thickness of the arterial segments were measured directly in the mounted vessels by phase-contrast microscopy (Nikon TMS microscope). Media stress was calculated as F/2L x M, where F is maximum active force (mg), L is segment length (mm), and M is media thickness (mm).

Statistical analysis. Data are presented as means ± SE or means ± SD of at least three independent experiments. Statistical analysis was performed with two-tailed ANOVA. Significance was assigned at P < 0.05.


    RESULTS
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 METHODS
 RESULTS
 DISCUSSION
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SM-B distribution in aorta and mesenteric vessels. We used immunohistochemistry to evaluate SM-B myosin expression in mesenteric vessels and aortas of WT and SM-B knockout mice (Fig. 1). Aorta, which contains very low amounts of SM-B myosin (37), showed negative staining (Fig. 1E) for this isoform in the WT animals. The pattern of SM-B-specific staining was heterogeneous in mesenteric vessels of WT animals, in that the smooth muscle layer of the vessels was not uniformly reactive (Fig. 1B). This was reported previously for small blood vessels (23) as well as for bladder (17) and cultured bladder myocytes (2). Staining was negative in mesenteric vessels from smb–/– mice (Fig. 1C). The negative staining observed in the SM-B knockout animals represents the critical negative control that also rules out the possible existence of other (nonmuscle) myosins that might contain the insert.



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Fig. 1. Immunohistochemical analysis of SM-B myosin expression. Mesenteric vessels: A: routine hematoxylin and eosin stain. Bar = 10 µm. B: vessel from wild-type (WT) animal immunostained for SM-B. Arrows indicate SM-B-specific immunostaining. C: vessel from smb–/– animal immunostained for SM-B myosin, showing complete lack of SM-B reactivity. Original magnifications x400. L, lumen. Aorta: D: routine hematoxylin and eosin stain. Bar = 20 µm (original magnification, x200). Arrows show borders of smooth muscle tissue (lumen at top). E: aorta from WT animal immunostained for SM-B, showing lack of SM-B reactivity. F: aorta from smb–/– animal immunostained for SM-B myosin, again showing lack of SM-B reactivity.

 
Loss of SM-B alters mechanical properties of mesenteric vessels. We confirmed the decrease in shortening velocity in mesenteric vessels that we reported previously and in which three different measures had been used (Fig. 2; Ref. 3). Here we report for the first time that both maximal force and wall stress are increased in vessels from smb–/– mice (Fig. 2). There was a very good positive correlation between force and rate of isometric contraction velocity in WT vessels that contain both SM-A and SM-B (r = 0.98) but a much poorer correlation for vessels from smb–/– mice containing only SM-A (r = 0.35).



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Fig. 2. Mechanical measurements on mesenteric vessels. A: force; B: wall stress; C: time to half peak concentration. Shown are means ± SE (n = 6). *P < 0.05.

 
Expression of contractile proteins is not altered in smb–/–mesenteric vessels. Quantitation of SM1, SM2, smooth muscle {alpha}-actin, LC20, and LC17 protein levels by Western blot analysis demonstrated that expression levels of SM1 and SM2 myosin heavy chains and light chain isoforms are not changed in the smb–/– mesenteric vessels (Fig. 3) as was found previously for bladder (3).



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Fig. 3. Expression of myosin heavy and light chain isoforms and {alpha}-actin in mesenteric vessels. Three different concentrations (1–3 µg) of total protein extracted from WT, smb+/–, and smb–/– mouse mesenteric vessels were resolved on SDS-PAGE and immunoblotted with anti-SM1, -SM2, -light chain-20 (LC20), or -light chain-17 (LC17) or {alpha}-actin antibody. Expression of these proteins is not altered in SM-B-null mesenteric vessels. Data presented are representative of 2 independent experiments.

 
Loss of SM-B myosin decreases shortening velocity and increases force generation in aorta. Our results demonstrating that loss of SM-B myosin alters the mechanical properties of mesenteric resistance vessels caused us to ask whether changes also occurred in conducting vessels as well, specifically in aorta (6). As seen in Fig. 4A, smb–/– aorta generated significantly more peak force than WT aorta in response to increasing concentrations of KCl (13.9 ± 0.9 and 11.7 ± 0.4 mN/mm2, respectively; n = 7, P < 0.05). The maximal force generated when stimulated with phenylephrine (3 µM) was also greater in the smb–/– aorta (18.6 ± 1.5 and 29.7 ± 2.5 mN/mm2 for WT and smb–/– mice, respectively; Fig. 4B; n = 7, P < 0.05). The sensitivity of smb–/– aorta to KCl depolarization was not significantly changed from WT, based on the ED50 values for the KCl concentration-response curve (Fig. 4A; WT = 21.3 ± 1.8 mM and smb–/– = 24.3 ± 0.3 mM). However, the sensitivity of smb–/– aorta to phenylephrine stimulation was significantly higher (Fig. 4B; WT ED50 = 82.5 ± 21.7 nM and smb–/– ED50 = 46.7 ± 2.6 nM; P < 0.05).



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Fig. 4. Isometric force generation in aorta. Force development by WT and smb–/– mouse aortic rings in response to cumulative additions of KCl (A) or phenylephrine (B). *P < 0.05 (ANOVA).

 
We next determined the maximal velocity of shortening. Aortic tissues were equilibrated at 10 mN of resting tension, and the force response to imposed shortening of varying speeds was determined. The force-velocity relation shows a steep drop in force with even the slowest imposed velocity (Fig. 5). In contrast to skeletal muscle, the relation is not hyperbolic (as the fitted lines indicate) but tends to flatten out with higher velocities. Importantly, for any imposed velocity, the maintained force was significantly less in the knockout tissue. In other words, the loss of the seven-amino acid insert leads to slower speeds at any given load. These results demonstrate that loss of SM-B myosin decreases the velocity of shortening in aorta. Results for aorta were qualitatively similar to what we observed for mesenteric vessels with respect to both shortening velocity and force. Reduced velocity, therefore, is a response seen for all smooth muscle tissues that have been studied (3).



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Fig. 5. Force-velocity relationship for aortic rings. Error bars show SD (n = 4). WT aorta was able to maintain a higher force than smb–/– aorta in response to imposed shortening velocities at varying speeds. KO, knockout; Lo, muscle length for optimal force development.

 
Effects of SM-B loss on expression of SM1 and SM2 myosin, smooth muscle {alpha}-actin, and myosin light chains in aorta. Figure 6 shows that the levels of SM1 and SM2 myosin in smb+/– and smb–/– aorta are the same as for WT aorta. This also is true for {alpha}-actin. These results are similar to what was found for mesenteric vessels (above) and bladder (3). LC20 expression was unaltered in the SM-B-null aorta (Fig. 7A). The ratio of LC17a to LC17b isoforms, however, was decreased because of relative upregulation of LC17b (Fig. 7B).



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Fig. 6. Expression of SM1 and SM2 myosin heavy chains in aorta. Equal amounts (1–3 µg) of total protein extracted from WT, smb+/–, and smb–/– mouse aorta were resolved on 8% SDS-PAGE and immunoblotted with anti-rabbit SM1 or SM2 antibody. Expression of SM1 and SM2 myosin heavy chain isoforms is not altered in the SM-B-null aorta. Smooth muscle {alpha}-actin was used an internal control. Data presented are representative of 3 independent experiments.

 


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Fig. 7. Myosin light chain expression in aorta. A: 3 different concentrations (1–3 µg) of total proteins extracted from WT, smb+/–, and smb–/– mouse aorta were resolved on a 10% or 15% SDS-PAGE and immunoprobed with LC20- or LC17-specific antibodies. LC17b isoform is upregulated in smb+/– and smb–/– aorta. Data presented are representative of 3 independent experiments. B: expression of LC17b isoform as % of total LC17 expression in WT and SM-B-null aorta (WT 8 ± 0.3%, smb+/– 13.3 ± 0.1%, and smb–/– 16.1 ± 0.2%).

 
Expression of thin filament regulatory proteins calponin and caldesmon are altered in aorta and bladder from SM-B-null mice. Thin filament-associated proteins such as calponin and caldesmon play important roles in determining force generation in smooth muscle (26). Therefore, it was of interest to determine whether SM-B loss alters expression of these key regulatory proteins. We found that calponin expression is upregulated in both the aorta (smb+/– 1.7 ± 0.06-fold and smb–/– 1.4 ± 0.04-fold; P < 0.05) and bladder (smb+/– 3.3 ± 0.16-fold and smb–/– 2.4 ± 0.07-fold; P < 0.05) from SM-B-null mice (Fig. 8). In contrast, as shown in Fig. 8, caldesmon expression is decreased in both aorta (WT 100%, smb+/– 64.6 ± 54.0%, and smb–/– 55.4 ± 48.8%; P < 0.05) and bladder (WT 100%, smb+/– 89.4 ± 83.4%; and smb–/– 69.0 ± 56.9%; P < 0.05) from SM-B-null mice.



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Fig. 8. Calponin and caldesmon expression. Equal amounts (3, 6, or 9 µg) of total protein extracted from WT, smb+/–, and smb–/– mouse bladder (A) or aorta (B) were resolved on a 10% SDS-PAGE and immunoblotted with anti-calponin or anti-caldesmon antibody. Calponin (CaP) was upregulated and caldesmon (CaD) was downregulated in the knockout mouse. Bar graphs show the relative expression levels of CaP (C) and CaD (D) in aorta and bladder. Smooth muscle {alpha}-actin was used an internal control. Data presented are representative of 3 independent experiments and are means ± SE. *P < 0.05.

 
ERK MAPKs are activated in aorta and bladder from SM-B null mice. It has been shown that activation of ERK, a subfamily of MAPK, can affect force generation in vascular smooth muscle tissues by phosphorylating thin filament regulatory proteins such as caldesmon (7, 12, 15). We found that ERK MAPK phosphorylation was significantly elevated (~1.6-fold; P < 0.05) in both smb–/– aorta and bladder, whereas MAPK levels were unaltered (Fig. 9).



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Fig. 9. Western blot analysis showing extracellular signal regulated kinase (ERK) mitogen-activated protein kinase (MAPK) phosphorylation. Three different concentrations (10, 20, and 30 µg) of total proteins extracted from WT, smb+/–, and smb–/– mouse bladder or aorta were resolved on a 10% SDS-PAGE and immunoblotted with common or phospho-specific p44/42 MAPK antibody. Phosphorylation of 44/42 MAPK was increased in the knockout mice bladder and aorta. The data presented are representative of 3 independent experiments. P-44/42 MAPK, phosphorylated ERK MAPK.

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The major goal of this study was to explore the effects of SM-B loss on the contractile properties of vascular smooth muscle including both resistance (mesentery) and conducting vessels (aorta; Ref. 6). We found in both vessel types that loss of SM-B results in reduced shortening velocity, whereas force generation is increased. The velocity change is similar to what has been found for bladder (3), but the increase in force is opposite.

Although SM-B is present in the mesenteric vessels, the pattern of SM-B expression is heterogeneous, as we (23) and others (2, 10, 17) previously reported, becoming more homogeneous in smaller vessels. Individual cells may contain both head isoforms as heterodimers (10), which have unique mechanical properties (29), although the existence of heterodimers in vivo remains to be determined. This, together with the unique contractile properties of SM-B itself, provides one basis for the changes in contractile properties we observed in these vessels. Adult aorta contains SM-A myosin, although the presence of small amounts of SM-B has not been excluded. Nonetheless, both shortening velocity and force are affected in the smb–/– aorta. It is possible that the absence of SM-B myosin in developing aorta, when it normally is present (37), leads to subsequent functional changes in the adult. Additionally, although the levels are too low to be easily detected, the presence of even very small amounts of SM-B conceivably could have functional consequences. In any case, the reduction in shortening velocity at the tissue level was predictable from studies of purified SM-B myosin (8, 20, 21, 30).

Our previous studies (3) demonstrated that loss of SM-B leads to a decrease in force generation in bladder. Force generation, however, is increased in mesenteric vessels and aorta from smb–/– animals. Clearly, these two smooth muscle types have substantially different functions. Bladder is distinctly phasic, and aorta has a tonic function, whereas mesenteric vessels also have phasiclike activity critical for regulation of blood pressure and distribution (6).

The unexpected changes in force we observed caused us to look for alterations in other contractile and regulatory proteins that might accompany the loss of SM-B (32). Changes we found included changes in the LC17 ratio, the expression of calponin and caldesmon, and the level of activity of the ERK signaling pathway.

Increased expression of LC17b might contribute to the reduction in velocity (16, 24) we observed in aorta. Upregulation of calponin expression would do likewise, although the effect of this change on force remains controversial (13, 27, 34). Increased expression of calponin together with the activation of ERK in the SM-B-null mice suggests that calponin also may be involved in the activation of ERK signaling, again modifying contractile function (18, 22, 25). Decreased caldesmon expression might minimize the loss of velocity (35).

The question arises as to why changes have occurred in aorta, in which there is little or no SM-B in WT animals. The aforementioned argument that SM-B expression during development somehow is an essential determinant of the contractile properties of adult aorta is one possibility that could be explored further by studies of contractile properties in developing aorta in SM-B-knockout animals. However, we prefer the hypothesis that the changes in velocity and force in mesenteric resistance vessels that lack SM-B require an adaptive response in the rest of the vascular system to preserve cardiovascular integrity (3). The combination of changes in caldesmon and calponin in aorta suggests that the adaptive response may involve conversion of aortic smooth muscle to an even more toniclike muscle (11, 13, 14, 33) to match the changes in performance that have occurred more distally. One approach we are now using to examine this issue further is to determine the consequence of knocking out those regulatory proteins singly and in combination with SM-B knockout. In any case, our findings suggest that the altered expression of key regulatory proteins, such as calponin and caldesmon, and the activation of ERK signaling pathway are important physiological adjustments that must be made to compensate for the loss of SM-B myosin.


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 ABSTRACT
 METHODS
 RESULTS
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 GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-38355-15 (to M. Periasamy) and American Heart Association Grant 0365173B (to G. J. Babu).


    ACKNOWLEDGMENTS
 
We thank Kristin Park for her contribution to the immunohistochemical studies.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Periasamy, Dept. of Physiology and Cell Biology, Ohio State Univ. College of Medicine, 304 Hamilton Hall, 1645 Neil Ave., Columbus, OH 43210 (E-mail: periasamy.1{at}osu.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.


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