© 1998 American Heart Association, Inc.
Editorial |
Microtubules in Cardiac Hypertrophy
A Mechanical Role in Decompensation?
Correspondence to Henk E.D.J. ter Keurs, MD, PhD, FRCPC, Department of Medicine, Health Sciences Centre, University of Calgary, 3300 Hospital Drive, NW, Calgary AB T2N 4N1, Canada. E-mail Henk{at}cvr.ucalgary.ca
Key Words: heart failure • myocardial contraction • cytoskeleton • microtubule
Cardiac function relies on the ability of the myocytes to develop force and shorten, as well as on the geometry of the heart, which translates shortening into stroke volume and force per cell into pressure in the lumen of the chambers. The requirements of circulation are met by adjustment of heart rate and power output by the myocytes. The heart adapts acutely to a hemodynamic load by increasing myocyte force output in response to stretch due to filling at elevated end-diastolic pressure. The acute response of the contractile system underlying Starling's law of the heart is followed more gradually, but still remarkably rapidly,1 by growth of the myocytes. Whereas stretch causes longitudinal growth by apposition of sarcomeres in series, increased systolic stress generated by the myocytes induces growth of myofibrils with a larger cross-sectional area and, consequently, cells with a larger diameter are produced. The latter response provides a feedback mechanism that keeps systolic force of individual cross-bridges constant. Within limits, these responses allow stable adaptation of cardiac function to the hemodynamic requirements of the body. When the requirements of circulation are still not met, the syndrome of heart failure becomes manifest when short-term control systems have to be activated constantly to maintain pump function that equals (nearly) the circulatory demands. In the process of growth, cells express novel protein isoforms with a character that depends on a host of stimuli2 so the myocyte phenotype alters substantially during development of hypertrophy and progression to heart failure.3 The effect of growth on the function of myocyte organelles serving energy turnover, excitation-contraction coupling, and contraction has been studied extensively with an impressive arsenal of structural-functional and molecular-biological techniques. The effect of structures that mediate growth, such as the microtubular network that mediates transport of material needed for construction of new cell structures, on functioning of the myocardium has been studied,4 although less extensively.
Dr. Cooper and colleagues have provided insights into the role of the microtubules in feline cardiac hypertrophy in recent years.5 Their studies have led to the suggestion that the presence of the microtubular component of the cytoskeleton may increase the load on the cardiac myocyte6 and therefore perpetuate the stimulus to hypertrophy and, ultimately, cardiac failure. These studies have stimulated considerable interest in the role(s) that the microtubular network may play. This issue has become controversial because not all studies that have investigated the role of the microtubules have come to the same conclusions. Several factors probably deserve more attention to resolve the debate regarding when the microtubular network is important and whether the effects of the microtubular network outweigh or approach those of changes in excitation-contraction coupling.
The study of Tagawa et al in this issue of Circulation Research7 investigates contraction of myocytes taken from canine left ventricle (LV) with hypertrophy due to progressive experimental stenosis of the ascending aorta. Some dogs appeared able to generate an aortic gradient of 160 mm Hg after stepwise resistance increase of the aortic occluder over the course of 8 weeks, whereas others failed to do so. As Cooper's group had shown earlier,8 it is possible to foresee which dogs will not be able to generate the gradient and which will. The dogs that failed to generate the large aortic gradient had a LV weight to body weight ratio that was 20% less compared with the dogs that succeeded, even at the start of the experimental protocol. These dogs were not only less capable of cardiac growth but were also handicapped at the outset of the study, because the stress required from their LV wall was higher than in the other group at the first moment the occluder was placed. Consistent with the inability for growth, additional hypertrophy of the LV of these dogs practically ceased after the fourth week, at an aortic gradient of 80 mm Hg, in contrast to the LV of the other dogs, which continued to grow. As a result, systolic stress development in the ventricular wall that failed to cope with the aortic stenosis was significantly higher compared with the LV that could cope with the increased systolic pressures. The hypertrophic response appeared to be complete within 2 weeks after each increase of the aortic stenosis. Myocytes, isolated either at 4 weeks or 1 week after the last hemodynamic evaluation at the end of the study, showed no increase in the density of the microtubular network nor in the density of the precursors of tubulin, as long as the dogs could cope with the aortic stenosis. This contrasted the findings in myocytes from the LV of animals that failed to do so in which total tubulin levels increased, particularly polymerized ß-tubulin.
When stimulated, the failing cells shortened substantially slower
(45%) and less frequently (40%) than control cells (in HEPES;
[Ca2+]o=2.5 mmol/L
at 37°C). In contrast, myocytes isolated from hearts that could cope
with the hemodynamic challenge shortened slightly more
often (
10%) and at a similar velocity as control cells. The
difference between contraction of failing cells and control cells
disappeared in 45 minutes after treatment with colchicine (1
µmol/L). The study by Tagawa et al suggested that failure to cope
with the aortic stenosis was accompanied by increased
systolic stress development in the LV wall. At the cellular
level, these features of failure were accompanied by changes in the
cardiac myocyte, leading to an increased opposing force that impeded
shortening of the cell below slack length. That the
impediment was relieved by colchicine suggests that the polymerized
microtubular network is responsible for the opposing force. Taken
together, the data suggest that elevated wall stress is responsible for
an increase in the density of the microtubular network, which reduces
shortening of the isolated myocyte. Therefore, the authors have
confirmed the results of previous studies performed in the
cat.5
Myocyte Shortening and Viscoelastic Cytoskeletal Forces
Is the effect of the microtubuli sufficient to act like a
substantial mechanical load itself and, if so, does that effect
outweigh changes in excitation-contraction coupling? Previous work by
Cooper's group has suggested that the increased internal load caused
by the microtubular network is primarily viscous (almost 4-fold
increase) rather than elastic (almost 2-fold increase). The conclusions
were derived from ingenious studies of the viscoelastic properties of
myocytes using magnetic beads adherent to the surface of the cells and
magnetic twisting cytometry.6 Nonetheless, these
observations still require additional corroboration at the level of
intact cardiac muscle to allow for extrapolations as to the function of
the intact heart and predictions as to the contribution of the
microtubuli to the development of decompensated hypertrophy
or heart failure. The elasticity of normal myocytes has been shown to
be generated predominantly by titin, the third filament of the
sarcomere.9 The force generated by myocytes on
shortening is 350 µg/µm of sarcomere, shortening below slack
length (1.86 µm), or 100 µg at the minimal length of
sarcomere, occurring in intact cardiac
cells.10 11 The resultant opposing force is
significant compared with active force development by maximally
activated cardiac muscle, which amounts to 1 mg per cell (for a
cell diameter of 15 µm, this is equivalent to a force per
cross-sectional area of cardiac muscle of 60
mN/mm2 observed at a sarcomere length [SL] of
2.15 µm).12 It follows from the force-SL
relationships at different levels of activation13
that the effect of the microtubules on myocyte shortening in the study
of Tagawa et al can, indeed, be explained by doubling the stiffness of
the cellular elastic elements that oppose shortening (assuming that the
level of myocyte activation is half maximal, as would be expected in
HEPES-buffered saline at
[Ca2+]o=2.5 mmol/L
at 37°C). Microtubule-associated proteins (MAPs) may play a role in
this increased stiffness because they have been implied both in the
stabilization of the cardiac microtubular
network14 and in increased microtubular
rigidity.15
The mechanical consequences of these cytoskeletal alterations are of interest and predict changes in the passive and active stress-strain relationships of the myocardium that warrant additional studies with intact preparations. Increased cytoskeletal stiffness should cause a proportional increase of the parallel elastic force of myocytes. In addition, the intercept of the relationship between active force development and SL should shift to a higher SL, but the active force developed at the SL above slack length should be unaffected. The effect of the microtubular network will be partly masked in intact cardiac muscle by the presence of collagen in an extracellular matrix because the total opposing force generated by intracellular and extracellular structures together is four times higher than that of the intracellular structures alone.16 Nevertheless, the effect of the microtubular network would be to reduce the degree of shortening of myocardial cells below slack length and, therefore, modify the contribution of ventricular suction to ventricular filling. These predictions can be tested in isolated muscle or by studying the diastolic and end-systolic pressure-volume relationships of the LV in dogs after a similar hemodynamic challenge. Walsh's group17 has reported that tubulin protein density was not increased in guinea pigs with aortic banding, despite increased tubulin gene expression, especially (20-fold) when failure resulted from the aortic banding. This study did not investigate the polymerization status of tubulin, but one might expect a high-density microtubular network to exist in the animals with failure. The study of the pressure development in Langendorff hearts showed neither an effect of colchicine exposure on developed pressure nor on dP/dtmax. However, whether the microtubular network had modified the passive properties of these hearts cannot be judged from the published data. One would not expect the Langendorff heart to contract below slack length; hence, opposing forces cannot be seen, but measurement of the end-diastolic pressure volume relationship (EDPVR) might reveal whether the compliance of the ventricle is colchicine-sensitive. If the EDPVR would be unchanged, one would surmise that the guinea pig is a poor builder of microtubular scaffolding.
Tagawa et al confirm previous studies that have revealed that the
presence of polymerized microtubules has a substantial effect on
shortening velocity of myocytes. This observation deserves additional
evaluation at a higher level of integration of the
myocardium. It is evident that sarcomere shortening
velocity below slack length in the absence of an external load is
limited by both the intrinsic properties of the cross-bridges and
internal elastic load as well as viscous properties of the
cell.18 However, conclusions about these effects
require knowledge of the level of activation of the cell that
determines the number of activated cross-bridges supporting the
viscoelastic load. We have studied sarcomere dynamics extensively in
isolated cardiac trabeculae, and data from these studies
allow some predictions regarding the effects of the microtubular
network. Unloaded sarcomere shortening velocity in cardiac muscle
composed of V3 isoform of myosin would be
40 µm/s at maximal activation at
37°C.19 The velocity of sarcomere shortening in
the heart in vivo is substantially lower20 ;
hence, the role of viscous forces opposing shortening is commensurably
smaller. It follows from the force-velocity relationship of the cardiac
sarcomere that a 4-fold increase of the viscosity of the "failing"
myocytes compared with control cells (0.06
mN/mm2/µm/s)21 would
reduce unloaded sarcomere shortening velocity to nearly 15 µm/s
at the activation level of the "failing" myocytes in Tagawa's
study. The velocity of sarcomere shortening reported by Tagawa et al is
far lower than this value (<4 µm/s in the control cells and
1.7 µm/s in the "failing" cells at SL=1.85 µm),
probably because sarcomere shortening is measured while activation of
the cell is just beginning at the time of the measurement. Additional
study of the level of activation of the cross-bridges is needed to
evaluate the contribution of viscous or elastic forces that hinder
shortening in these cells. Nevertheless, the effect of colchicine shows
a contribution of microtubule-dependent viscoelastic forces opposing
shortening.
Hypertrophy, Heart Failure, and the Microtubular Network
In the normal heart with physiological loading, the microtubular network is sparse and its mechanical effect is negligible. However, under conditions in which a dense microtubular network is formed, it may have a sufficiently large mechanical effect on cardiac mechanics to be of importance to the development of heart failure. The state of the myocardium is clearly not singular but encompasses a spectrum of cellular phenotypes that fit scenarios ranging from an equilibrium between power output of the heart and circulatory requirements via enhanced growth because the latter two are out of balance to the extreme of failure. So, does one encounter cardiac myocytes in which the microtubules impede shortening during hypertrophy (as suggested in the earlier studies by Cooper's group; see also Reference 2222 ) or in cardiac failure, as is defended in the article presented by Tagawa?
It is generally accepted that the main role of the microtubular network is to act as a railway that allows for the transport of cargo particles between sites that synthesize proteins and lipid particles and target organelles in construction.23 24 The microtubules constitute a dynamic network in the cytoskeleton25 that forms by polymerization and disassembles rapidly by depolymerization. This process allows for rapid modifications of the extent and shape of the microtubular network, even without changes of the pool of available tubulin in the cell. One would expect that the microtubular network would be expressed in increased amounts as long as the myocyte receives a stimulus to grow and would disappear when the stimulus disappears.26 The detailed layout of this network would depend on the nature of the growth. Consequently, the network might differ between longitudinal growth of myocytes by apposition of sarcomeres and sarcoplasmic reticulum after volume loading of the heart compared with the increase of the myocyte cross-sectional area that is typical for pressure overload hypertrophy, such as in Tagawa's study. In this respect, it should be noted that the response of myocytes from pressure-overloaded versus volume-overloaded heart differs significantly.27 It has been shown that the total level of tubulin increases during accelerated growth10 22 ; in addition, the presence of a dense microtubular network has been reported to be nonuniform (5% of the cells in a rat heart), with an increase of the fraction of cells with a dense microtubular network (to 35%) in hearts of animals with an acute hemodynamic load.10 The results of Tagawa et al do not address the question whether the microtubular network exists as long as the driving stimulus for hypertrophy persists, but their data do not rule out that the microtubular network disassembles when the microtubuli have done their work serving the construction sites of cellular growth and systolic LV stress has normalized. Such dynamic behavior of the microtubules would predict that cells isolated shortly after increase of the aortic stenosis would show increased amounts of polymerized microtubules even in the hearts of the dogs that appear able to cope with the stenosis.
Although it may be reasonable to conjecture that the microtubular
network is built up with intense growth, we know little about the
kinetics of synthesis and breakdown of this network. Thus, the actual
life cycle of dense microtubular network is not exactly known but is
probably less than 1 week, as is shown by the response to an acute load
to the ventricle.4 Importantly, this period
included the time needed for the trophic response to the
stimulus.4 Hence, it is possible that the
presence of a dense microtubular network is a fleeting phenomenon
captured in Cooper's studies, which manifests itself with intense
growth (see also Reference 44 ) when the microtubules form such a dense
scaffolding that they hinder cell shortening. When growth is less
intense or is completed in experiments in which the stimulus to growth
is more gradual (Houser's studies30 ) the
number of cells with increased microtubular density is
lower4 and mechanical effect of a less sparse
microtubular network may not be perceptible. Houser's study is one of
the few that reports such a negative finding. (Unfortunately, the
literature usually fails to educate us exhaustively about negative
findings!) Consequently, Houser emphasizes the role of modified
excitation-contraction coupling in changes of myocardial function of
hypertrophied heart. Rappaport's observation that the distribution of
myocytes with a dense microtubular network is
nonuniform4 is of interest here: one might expect
that with growth, 35% of the cells would have a dense microtubular
network and would resist accelerated shortening; the remaining 65%
should shorten more normally. Hence, the variance of the shortening
distribution of myocytes randomly taken from dogs that could not cope
with the hemodynamic stenosis in Tagawa's
study should be larger than in the control dogs. The opposite seems to
have been observed in the study of Tagawa et al.
Tagawa's study seeks to gain insight into the mechanisms underlying heart failure in humans. However, the animals in the group designated as "failures" show no signs of overt heart failure (eg, decreased cardiac output or increased arteriovenous O2 differences). Previous work by the same group had shown that severe pressure overloading of the feline right ventricle induced similar changes in the myocytes from the affected ventricle and in the absence of overt heart failure as well.28 Hence, although the data are convincing that the microtubular network is present as long as excessive stress development has not been corrected, it seems premature to conclude that the mechanical abnormalities encountered in the cells studied by Tagawa et al are typical of the myocardium in overt heart failure. It is possible that the increased internal load owing to changes of the viscoelastic properties of the myocytes is essential in the vicious cycle that leads to development of heart failure. This intriguing possibility requires the fulfillment of several important conditions. From the present data, it is not possible to conclude that these conditions are met. First, the microtubules should persist in cells of failing myocardium. Immunohistochemical studies of ß-tubulin levels have shown a transient increase after aortic banding, returning to normal in 2 weeks.4 Stabilization of polymerized microtubules has been shown to be related to the effect of MAP4,14 but it is not clear from this study how long the microtubular network persists after its assembly. Lastly, an increased microtubular network is indeed found in human explanted heart in heart failure. However, this observation was made when there was extensive myofibrillar disarray29 so the interpretation of this finding is ambiguous.
Publication of the article by Tagawa et al in this issue of Circulation Research should stimulate additional investigations into the kinetics of synthesis and breakdown of the microtubular network, as well as on the repercussion of the network for the mechanical properties of the myocyte and, more importantly, for the pump function of the intact heart.
Footnotes
The opinions expressed in this editorial are not necessarily those of the editor or of the American Heart Association.
References
1.
Sadoshima J, Jahn L, Takahashi T, Kulik TJ, Izumo
S. Molecular characterization of the stretch-induced adaptation of
cultured cardiac cells. An in vitro model of load-induced cardiac
hypertrophy. J Biol Chem. 1992;267:10551–10560.
2. Wagoner LE, Walsh RA. The cellular pathophysiology of progression to heart failure. Curr Opin Cardiol. 1996;11:237–244.[Medline] [Order article via Infotrieve]
3. Boheler KR, Carrier L, de la Bastie D, Allen PD, Komajda M, Mercadier JJ, Schwartz K. Skeletal actin mRNA increases in the human heart during ontogenic development and is the major isoform of control and failing adult hearts. J Clin Invest. 1991;88:323–330.
4. Rappaport L, Samuel JL. Microtubules in cardiac myocytes. Int Rev Cytol. 1988;113:101–143.[Medline] [Order article via Infotrieve]
5.
Tsutsui H, Ishihara K, Cooper G IV. Cytoskeletal role
in the contractile dysfunction of hypertrophied myocardium.
Science. 1993;260:682–687.
6.
Tagawa H, Wang N, Narishige T, Ingber DE, Zile MR,
Cooper G IV. Cytoskeletal mechanics in pressure-overload cardiac
hypertrophy. Circ Res. 1997;80:281–289.
7.
Tagawa H, Koide M, Sato H, Zile MR, Carabello BA,
Cooper G IV. Cytoskeletal role in the transition from compensated to
decompensated hypertrophy during adult canine left
ventricular pressure overloading. Circ Res. 1998;82:751–761.
8.
Koide M, Nagatsu M, Zile MR, Hamawaki M, Swindle MM,
Keech G, DeFreyte G, Cooper G IV, Carabello BA. Premorbid determinants
of left ventricular dysfunction in a novel model of
gradually induced pressure overload in the adult canine.
Circulation. 1997;95:1601–1610.
9. Granzier HLM, Irving TC. Passive tension in cardiac muscle: contribution of collagen, titin, microtubules, and intermediate filaments [abstract]. Biophys J. 1995;68:1027–1044.[Medline] [Order article via Infotrieve]
10.
Helmes M, Trombitas K, Granzier HLM. Titin develops
restoring force in rat cardiac myocytes. Circ Res. 1996;79:619–626.
11. ter Keurs HEDJ, Iwazumi T. Restoring forces in rat cardiac myocytes and ultra small muscle trabeculae. Biophys J. 1988;53:167a.
12.
Kentish JC, ter Keurs HEDJ, Ricciardi L, Bucx JJJ,
Noble MIM. Comparison between the sarcomere length-force relations of
intact and skinned trabeculae from rat right ventricle.
Circ Res. 1986;58:755–768.
13.
de Tombe PP, ter Keurs HEDJ. Sarcomere dynamics in cat
cardiac trabeculae [abstract]. Circ Res. 1991;68:588–596.
14.
Sato H, Nagai T, Kuppuswamy D, Narishige T, Koide M,
Menick DR, Cooper G IV. Microtubule stabilization in pressure overload
cardiac hypertrophy. J Cell Biol. 1997;139:963–973.
15. Matus A. Stiff microtubules and neuronal morphology. Trends Neurosci. 1994;17:19–22.[Medline] [Order article via Infotrieve]
16. Backx PHM. Force Sarcomere Relation in Cardiac Myocardium [dissertation]. Calgary, Alberta: University of Calgary; 1989.
17. Collins JF, Pawloski-Dahm C, Davis MG, Ball N, Dorn GW II, Walsh RA. The role of the cytoskeleton in left ventricular pressure overload hypertrophy and failure. J Mol Cell Cardiol. 1996;28:1435–1443.[Medline] [Order article via Infotrieve]
18. Chiu Y-L, Ballou EW, Ford LE. Internal viscoelastic loading in cat papillary muscle. Biophys J. 1982;40:109–120.[Medline] [Order article via Infotrieve]
19.
de Tombe PP, ter Keurs HEDJ. Force and velocity of
sarcomere shortening in trabeculae from rat heart: effects
of temperature. Circ Res. 1990;66:1239–1254.
20.
Rodriguez EK, Hunter WC, Royce MJ, Leppo MK, Douglas
AS, Weisman HF. A method to reconstruct myocardial sarcomere lengths
and orientations at transmural sites in beating canine hearts
[abstract]. Am J Physiol. 1992;263:H293–H306.
21.
de Tombe PP, ter Keurs HEDJ. An internal viscous
element limits unloaded velocity of sarcomere shortening in rat
myocardium. J Physiol (Lond). 1992;454:619–642.
22.
Ishibashi Y, Tsutsui H, Yamamoto S, Takahashi M,
Imanaka-Yoshida K, Yoshida T, Urabe Y, Sugimachi M, Takeshita A. Role
of microtubules in myocyte contractile dysfunction during cardiac
hypertrophy in the rat. Am J Physiol. 1996;271:H1978–H1987.
23.
Hirokawa N. Kinesin and dynein superfamily proteins and
the mechanism of organelle transport. Science. 1998;279:519–526.
24. Ioshii SO, Imanaka-Yoshida K, Yoshida T. Organization of calsequestrin-positive sarcoplasmic reticulum in rat cardiomyocytes in culture. J Cell Physiol. 1994;158:87–96.[Medline] [Order article via Infotrieve]
25.
Fuchs E, Cleveland DW. A structural scaffolding of
intermediate filaments in health and disease. Science. 1998;279:514–519.
26. Samuel JL, Marotte F, Delcayre C, Rappaport L. Microtubule reorganization is related to rate of heart myocyte hypertrophy in rat. Am J Physiol. 1986;251:1118–1125.
27.
Urabe Y, Hamada Y, Spinale F, Carabello BA, Kent RL,
Cooper G IV, Mann DL. Cardiocyte contractile
performance in experimental biventricular
volume-overload hypertrophy. Am J Physiol. 1993;264:H1615–H1623.
28.
Tagawa H, Rozich JD, Tsutsui H, Narishige T, Kuppuswamy
D, Sato H, Koide M, Cooper G IV. Basis for increased microtubules in
pressure-hypertrophied cardiocytes. Circulation. 1996;93:1230–1243.
29. Hein S, Schaper J. The cytoskeleton of cardiomyocytes is altered in the failing human heart. Heart Failure. 1996;12:128–136.
30. Bailey BA, Dipla K, Li S, Houser SR. Cellular basis of contractile derangements of hypertrophied feline ventricular myocytes. J Mol Cell Cardiol. 1997;29:1823–1835.[Medline] [Order article via Infotrieve]
This article has been cited by other articles:
 |
|
 |
|
  X. Yang, P. J I Salas, T. V Pham, B. J Wasserlauf, M. J D Smets, R. J Myerburg, H. Gelband, B. F Hoffman, and A. L Bassett Cytoskeletal actin microfilaments and the transient outward potassium current in hypertrophied rat ventriculocytes J. Physiol., June 1, 2002; 541(2): 411 - 421. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Koide, M. Hamawaki, T. Narishige, H. Sato, S. Nemoto, G. DeFreyte, M. R. Zile, G. Cooper IV, and B. A. Carabello Microtubule Depolymerization Normalizes In Vivo Myocardial Contractile Function in Dogs With Pressure-Overload Left Ventricular Hypertrophy Circulation, August 29, 2000; 102(9): 1045 - 1052. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Hein, S. Kostin, A. Heling, Y. Maeno, and J. Schaper The role of the cytoskeleton in heart failure Cardiovasc Res, January 14, 2000; 45(2): 273 - 278. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||