cardiocyte cytoskeleton in hypertrophied myocardium

Heart Failure Reviews, 5, 187±201, 2000

# 2000 Kluwer Academic Publishers. Manufactured in The Netherlands

Cardiocyte Cytoskeleton in Hypertrophied Myocardium

George Cooper, IV

From the Gazes Cardiac Research Institute, Medical University of

South Carolina and the Department of Veterans Affairs Medical

Center, Charleston, SC

Abstract. The Frank-Starling mechanism, by whichload directly regulates muscle length and thus perfor-mance, is the means by which the mechanics and ener-getics of cardiac muscle are regulated on a beat-to-beatbasis. When this short-term compensation for in-creased load is insuf®cient, the long-term compensa-tion of cardiac hypertrophy ensues. The simplest andmost direct mechanism for load regulation of cardiacmass would obtain if an analog of the short-termFrank-Starling mechanism of functional regulationoperated in the long-term time domain of mass regula-tion; that is, if heart muscle were able to directlytransduce increased load into growth. It is now clearthat load does indeed serve as a direct regulator ofcardiac mass in the adult. Cardiac hypertrophy, at thelevels of intact animal, isolated tissue, and culturedcells, is a direct response of the adult mammaliancardiocyte to increased load, modi®ed by but withoutthe requisite involvement of factors external to the cell.The extent to which such hypertrophy is compensatoryis critically dependent on the type of hemodynamicoverload that serves as the hypertrophic stimulus.Thus, cardiac hypertrophy is not intrinsically mal-adaptive; rather, it is the nature of the inducing loadrather than hypertrophy itself that is responsible forthe frequent deterioration of initially compensatoryhypertrophy into the congestive heart failure state.As one example reviewed here of this load speci®cityof maladaptation, increased microtubule networkdensity is a persistent feature of severely pressureoverloaded, hypertrophied and failing myocardiumwhich imposes a viscous load on active myo®lamentsduring contraction.

Key Words. heart failure, hypertrophy, cytoskeletonmicrotubules

Load and length regulation of the properties ofthe heart represents a remarkably simple anddirect biological response both to the input path-way and to the output function of this organ. Thismechanism is used by the heart in response to itsdynamic loading environment both for short-termadaptation of cardiac mechanics and energetics toinstantaneous load alterations [1] and for long-term adaptation of cardiac mass to sustained loadalterations [2]. Taken together, these autoregula-tory mechanisms represent highly conservativestrategies for preserving cardiovascular homeo-

stasis in the setting of short-term physiologicaland long-term pathological challenges [3].

If a long-term load increase is neither toosevere initially nor inde®nitely progressive,cardiac stress is re-normalized, and compensatedhypertrophy ensues. But hypertrophic compensa-tion is often abrogated by progressively abnormalcontractile performance per unit mass of myocar-dium, even when function at the organ level ismaintained initially by the mass increase itself.That is, even when hypertrophy is appropriate tothe load imposed, speci®c phenotypic changesoccurring during this growth response mayrender compensation imperfect, such that conges-tive heart failure ensues. This fact, and the factthat the presence and nature of the deleteriousphenotypic changes in hypertrophied myocar-dium are critically dependent on the type ofhemodynamic load imposed, mandate thatcardiac hypertrophy must be understood on themost basic level as a growth process if the causesof heart failure following pathological hemody-namic overloads are to be fully understood.

This review will focus on one such phenotypicchange during cardiac hypertrophy that resultsfrom a particular alteration of the cardiocyteextra-myo®lament cytoskeleton. As such, itserves as a single example out of the many nowbeing explored of the deleterious concomitants tohypertrophy that may frustrate this compensa-tory growth response to very speci®c load altera-tions. I will begin this discussion of cardiocytemicrotubules in pressure overload hypertrophywith a description of the phenomenology inprogressively more complex biological systemsand then explore its mechanisms in progressivelysimpler biological systems.

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This work was supported by National Institutes of HealthProgram Project Grant HL-48788 and by research funds fromthe Department of Veterans Affairs.

Address for correspondence: George Cooper, IV, M.D., GazesCardiac Research Institute, P.O. Box 250773, Medical Univer-sity of South Carolina, 114 Doughty Street, Charleston, SC29403. E-mail: [email protected]

The Extra-Myo®lament Cytoskeleton inCardiac Hypertrophy

This work had its impetus in my ®rst two studiesas a postdoctoral fellow, where I found that on thelevel of isolated right ventricular tissue, and foran equivalent degree and duration of hypertro-phy, volume overloading results in entirelynormal cardiac contraction and energetics [4],while pressure overloading results in distinctlyabnormal cardiac contraction and energetics [5].On the level of isolated right ventricular cardio-cytes, we later found for the same model of rightventricular pressure overload that the contractiledefect seen in isolated tissue is duplicated whencharacterized as sarcomere shortening in themuscle cell [6]. Thus, it was the nature of theinducing stress rather than hypertrophy itselfthat caused the qualitative defects of myocar-dium hypertrophying in response to a pressureoverload, and the contractile defect, at least,resides in the cardiocyte.

While we and others had advanced a numberof potential causes for the functional abnormal-ities of hypertrophied myocardium [2], with aleading candidate being altered calcium metabo-lism [5,7], none had accounted fully and convin-cingly either for the contractile abnormalities ofhypertrophied myocardium when present on thetissue or the cellular level or, more particularly,for the dichotomy noted above between pressureversus volume overload-induced hypertrophy ofthe right ventricle.

This dichotomy may be considered fruitfullywithin the context of the general thesis that loadis the primary variable responsible for regulatingcardiocyte properties. That is, cardiocyte struc-ture, composition, and function each responddynamically to the full potential spectrum ofimposed loads, with deviations either below orabove normal loading causing rapid but reversiblechanges in each of these properties [8]. Indeed, thecardiocyte itself is competent to respond directly toload in terms of RNA and protein synthesis rates[9±11]. However, we had found neither qualitativenor quantitative differences between hypertro-phied cardiocytes from our models of pressure-overloaded versus volume-overloaded right ventri-cles when de®ned in terms of standard ultrastruc-ture [12], yet the contractile defect was expressedquite clearly in the pressure-overloaded cell. Itherefore thought it reasonable to seek the causefor this contractile defect in one or more intracel-lular structures which might 1) appear or increasein response to load, 2) discriminate between thestimuli of stress, or pressure loading versus strain,or volume loading, 3) not be obvious ultrastructu-rally, and 4) be plausibly thought to have thepotential for interfering with sarcomere motion.

The hypothesis then tested was that the micro-tubule component of the cytoskeleton is such anintracellular structure, which in excess mightcontribute to the contractile abnormalities ofcardiocytes hypertrophying in response to a pres-sure overload. The basis for this hypothesis, interms of the four criteria set forth above, is asfollows. First, there is evidence in taxa as diverseas plants [13] and both invertebrate [14] andvertebrate [15] animals that microtubules formalong stress axes and bear intracellular andtranscellular loads, suggesting selective polymer-ization or organization in response to theirmechanical environment. Second, for a linearsteady-state polymer such as the ab-tubulinheterodimerÐmicrotubule system, thermody-namics dictate that the critical subunit concen-tration for assembly is lowered and polymerstability is enhanced by an extending, or stressforce [16], and there is experimental evidencethat this obtains for microtubules in growingaxons [17]. Third, microtubules are not obviousultrastructurally in mature striated muscleabsent special efforts. Fourth, given the intimatecardiac myo®brillar investment by microtubules[18], there is a clear opportunity for an excess ofthis polymer to interfere with sarcomere motion.

Our ®rst test of this hypothesis [19,20]employed cardiocytes from both the functionallyabnormal pressure overload-hypertrophied felineright ventricle, where cardiocytes from the leftventricle served as a same-animal normallyloaded control, as well as cardiocytes from thefunctionally normal volume-overloaded andequivalently hypertrophied feline right ventricle,which served as a control for any effects of hyper-trophy itself. The major ®ndings were that micro-tubules are selectively and persistently increasedin right ventricular cardiocytes hypertrophyingin response to pressure but not volume overload-ing, that this alteration is responsible both for thereduced extent and velocity of sarcomere short-ening in externally unloaded cells and for thereduced extent of sarcomere and cellular short-ening in externally loaded cells, that thesecontractile defects are reproduced in normalcells when microtubule hyperpolymerization isinduced by a chemical or physical as opposed toa pathophysiological stimulus, and that thesecontractile defects are speci®c to the microtubulecomponent of the cytoskeleton. As such, thissingle molecular defect could well be explicativenot only of abnormal sarcomere and cellularmotion during contraction but also of the linkedcontractile and energetic abnormalities which Ihad found [5] to be characteristic of pressureoverload cardiac hypertrophy.

Of note, increased microtubules, as well as freeab-tubulin heterodimers, were present as soon as

188 Cooper

hypertrophy was fully established and werepersistent thereafter during the state of compen-sated hypertrophy. Thus, two major questions offunctional signi®cance followed from these initialobservations: ®rst, do both the increased micro-tubule network density and the associatedcontractile defects observed in compensated pres-sure overload right ventricular hypertrophypersist during, and thus potentially contributeto, the eventual development of right heart fail-ure in this model when the pressure overload issevere; second, are the more marked contractiledefects seen at this later stage also fully rever-sible with microtubule depolymerization? That is,while the initial ®ndings had some intrinsicbiological interest, their practical implicationswould likely be based on the answers to thesetwo questions, since our long-standing goal oflearning the causes of the transition from initi-ally compensatory pressure overload cardiachypertrophy to decompensated cardiac failureconstituted the relevant clinical rationale forthese studies. A study employing more severefeline right ventricular pressure overloadingleading to overt right heart failure answeredboth of these questions in the af®rmative [21].Thus, alterations in microtubule network densityand sarcomere mechanics were a persistent andprogressive feature of the hypertrophied andthen failing cat right ventricle, and the ratio ofpolymerized to free tubulin was selectivelyincreased in the failing right ventricle. In addi-tion, the mechanical defect, while more severe infailing than in hypertrophied but non-failingright ventricles, was normalized by microtubuledepolymerization. Because we found persistentand progressive increases both in microtubulesand in the functional consequence of aberrantsarcomere mechanics during the transition fromhypertrophy to failure when right ventricularpressure overloading was severe, we concludedthat this cytoskeletal abnormality may wellcontribute to the contractile dysfunction charac-teristic of pressure overload-induced right heartfailure.

The next logical step in establishing the signif-icance of this cytoskeletal alteration was to askwhether correction of the cellular contractilestate by microtubule depolymerization correctsthe myocardial contractile state when examinedat the more highly integrated and complex levelof muscle tissue. This rather straightforward goalwas made more compelling by the fact that themyocardial contractile defects characteristic ofchronic pressure overload hypertrophy may bedue to changes intrinsic or extrinsic to the cardi-ocyte, such that it was entirely possible that,despite the dramatic amelioration of contractiledysfunction seen at the level of isolated cardio-

cytes, microtubule depolymerization might wellnot correct the myocardial contractile dysfunc-tion which results from severe chronic rightventricular pressure overload hypertrophy. Wetherefore, using the same right ventricular pres-sure overload model as that used for the cardio-cyte studies discussed above, examined papillarymuscles removed from these and from normalfeline right ventricles [22]. Indirect immuno¯uor-escence confocal microscopy demonstratedincreased microtubule network density that wasselective for hypertrophied muscles, with colchi-cine causing complete microtubule depolymeriza-tion in both control and hypertrophied papillarymuscles. As in cardiocytes, microtubule depoly-merization normalized myocardial contractilityin pressure overload-hypertrophied papillarymuscles but did not alter contractility in controlpapillary muscles. Also as in cardiocytes, micro-tubule hyperpolymerization in normal papillarymuscles reproduced the contractile dysfunctionseen with pressure overload hypertrophy. Weconcluded, therefore, that excess microtubulenetwork density is equally important to cellularand to myocardial contractile dysfunction in thesetting of severe chronic pressure overload-induced cardiac hypertrophy.

The work reviewed to this point is restricted tocells and tissue isolated from hypertrophied rightventricles. Two important issues, however, couldnot be addressed in this setting. First, the rele-vance of these data to the pressure-overloadedleft ventricle of a large animal with an adultonset, progressive and pathological left ventricu-lar afterload imposition similar to that found inhuman disease was undetermined. Second, therelationship of these cytoskeletal abnormalitiesto the mechanical environment of the cardiocytein vivo was unknown, since characterization ofright ventricular mass, function and wall stress,especially in a small animal, is infeasible.Further, since functional and physical ventricu-lar and cellular sampling over time is similarlyinfeasible in the right ventricle of a small animal,it was unknown whether increased microtubulenetwork density is a characteristic of pressureoverload hypertrophy per se, or whether insteadit has a speci®c relationship to progressivechanges in the mechanical environment of thecardiocyte as myocardial stress loading increases.Given these issues, as well as the predominantimportance of the left ventricle in clinical patho-physiology, we next devised a model of progres-sive pressure overload of the canine left ventriclefor the purpose of addressing these issues [23].The two design criteria for this model, both ofwhich were met, were a progressive and readilycontrollable increase in afterload to a levelcomparable to that causing signi®cant clinical

Cytoskeleton In Cardiac Hypertrophy 189

pathophysiology in humans, and a resultantincrease in left ventricular mass comparable tothat known to be associated with ventriculardysfunction in the setting of clinical left ventri-cular pressure overload.

In terms of the two issues noted above whichformed the basis for this study [24], considerableinsight was gained in each case. First, the samemicrotubule-based cardiocyte contractile defectfound in the pressure-overloaded feline rightventricle, where with ®xed afterloading wallstress is probably continuously elevated fromthe outset, was also found in the pressure-over-loaded canine left ventricle. However, this studyallowed us to assign considerably more speci®cityto this ®nding. In this canine model we were ableto sample left ventricular mechanical and geome-trical properties, on the organ and on the cellularlevels, throughout the development of left ventri-cular hypertrophy both of a degree relevant tohuman disease and in response to a pattern ofprogressive afterloading that is also relevant tohuman disease. In the context of consideringcompensatory hypertrophy to be that in whichmyocardial mass increases so as to maintainnormal ventricular wall stress, we found thatthose animals whose capacity for left ventriculargrowth was such that normal left ventricular wallstress was sustained despite a very high aorticpressure gradient retained normal ventricularand cellular contractile function. Further, theyexhibited increases neither in tubulin protein norin the density of the cytoskeletal microtubulenetwork. Indeed, a unique and unanticipated®nding was that cellular contractile function, interms of the extent of sarcomere shortening, wasnormal in left ventricular biopsy specimensobtained at the mid-point of the hypertrophicgrowth response in this group but was signi®-cantly increased at ®nal study, despite the factthat left ventricular mass had doubled. This®nding raises the provocative possibility that asyet unknown compensatory mechanisms may beinvoked on the cellular level when pressure over-load cardiac hypertrophy becomes substantialthat may have a signi®cant and heretofore un-recognized role in the maintenance of normalcontractile function on the organ level, i.e., func-tionally compensatory hypertrophy.

The ®ndings for the group of dogs which devel-oped left ventricular failure were both quitedifferent from those seen in the group of dogswhich maintained compensated left ventricularhypertrophy and quite similar to those seen incats with right ventricular pressure overload-induced hypertrophy with associated right heartfailure. The hallmark which distinguished thisgroup of failure dogs from the group of compen-sated hypertrophy dogs was a conspicuous break-

down of the left ventricular growth response toprogressive afterloading. That is, through thetime of biopsy, when only part of the ®nal after-load increase had been achieved, these dogsshowed progressive increases in left ventricularmass in response to progressive increases in leftventricular afterload, albeit to lesser values thanthose for the compensated hypertrophy groupand with the development of early abnormalitiesof left ventricular stress and function. Further,normal contractile function was maintained atthis time in cardiocytes from these left ventricles,and no abnormalities of tubulin or microtubuleswere apparent. However, after the time of biopsythere was no further compensatory growthresponse of the left ventricle to further afterload-ing in these dogs. As a direct consequence, therewas a striking increase in left ventricular meansystolic stress. Of greatest pathophysiologicalconsequence, however, there was also a strikingdeterioration of left ventricular contractile func-tion, which was mimicked by, and may well havehad its basis in, a parallel striking deteriorationof sarcomere contractile mechanics in cardiocytesfrom these same left ventricles. These observa-tions, and their contrast with those made in thegroup of dogs with compensated hypertrophy, arelent particular cogency by the facts that in manycases the same dogs were sampled longitudinallyin both groups, and the ventricular, cellular, andbiochemical data were all gathered from the sameanimals.

Just as in the feline right ventricle, whereinsevere pressure overloading caused right ventri-cular failure, these dogs from the left ventricularfailure group demonstrated a profound, butcolchicine-reversible depression of sarcomeremechanics that was associated with remarkableincreases both in free and polymerized tubulinand in the density of the cellular microtubulenetwork. We concluded from these data thatincreased microtubule network density, and itsfunctional consequences, are properties of bothright and left ventricular pressure overloadhypertrophy; this cytoskeletal alteration isneither species-speci®c nor chamber-speci®c.But data from the canine model, where ventricu-lar mechanics could be de®ned and where longi-tudinal sampling was possible, showed clearlythat this is not a ubiquitous feature of pressureoverload cardiac hypertrophy. Instead, it has aspeci®c association with increased ventricularwall stress, which in turn appears duringprogressive pressure overloading when, and inthose animals wherein, the hypertrophic res-ponse to load is exhausted.

We next carried this description of thephenomenology of microtubule alterations inpressure overload cardiac hypertrophy to the

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level of the heart in the intact organism [25],since a logical extension of the question askedin the previous study was whether ®ndingsderived from cells and tissue in vitro are applic-able to the left ventricle in vivo of a largemammal having a clinically relevant form ofpressure overload hypertrophy and failure. Themodel used for this purpose [23] was the same asthat used for the studies of left ventricular cardi-ocytes [24] reviewed above. In dogs in whichgradual stenosis of the ascending aorta hadcaused severe left ventricular pressure overload-ing with contractile dysfunction, left ventricularperformance was measured at baseline and onehour after intravenous administration of colchi-cine. Cardiocytes obtained by biopsy before andafter in vivo colchicine administration wereexamined in tandem. Our major ®nding wasthat colchicine, in our hands a non-inotropicagent, caused microtubule depolymerization andrestored left ventricular contractile function bothin vivo and in vitro. Our major conclusion wasthat contractile dysfunction due to increasedmicrotubule network density improves aftercolchicine-induced microtubule depolymerizationin vivo.

In order to substantiate this conclusion weneeded to be certain that contractile dysfunctionwas actually present, that function improved,and that changes in the microtubules were causa-tive of this improvement. It was clear that aftereight weeks of pressure overload these canine leftventricles exhibited contractile dysfunction. Ejec-tion performance either at the endocardium or atthe midwall was reduced much more than waspredicted from afterload excess alone. Indeed,when afterload was returned to normal byremoval of the aortic gradient, ejection perfor-mance gauged by ejection fraction, by meannormalized systolic ejection rate, or by midwallshortening rate was still quite depressed.However, colchicine returned ejection perfor-mance toward or to normal. Our previous studiesreviewed above had shown that colchicine doesnot exert a direct positive inotropic effect eitheron normal cardiocytes or on hypertrophied cardi-ocytes exhibiting normal contractile function anddensity of the microtubule network. In fact, incontrol animals in vivo colchicine tended todepress left ventricular performance. It was ourinference that in the pressure-overloadedanimals this negative inotropic effect was morethan overridden by the positive effect on myo®la-ment loading of microtubule depolymerization.Further, the data obtained in the parallel invitro portion of this study were very compelling.At a time when contractility was depressed invivo, contractility of cardiocytes taken from thesame ventricle was also depressed. Increased

microtubule network density was present inthese cardiocytes, and its reduction by in vitrocolchicine returned the function of the cardio-cytes to normal. Hours later, after colchicinehad been administered in vivo and ventricularfunction had returned to normal, the function ofcardiocytes from these same left ventricles hadalso returned to normal, and increased microtu-bule network density was no longer present. Asexpected, additional in vitro colchicine adminis-tration caused no further improvement in cardi-ocyte function. Finally, controls intended toparallel those employed in the earlier studies ofcardiocytes and myocardium yielded equivalentresults: in vivo microtubule depolymerization bylow temperature had the same ameliorative effecton depressed contractility in severe left ventricu-lar hypertrophy as did colchicine, and in vivomicrotubule hyperpolymerization in normalanimals recreated, as shown in Figure 1, theleft ventricular contractile dysfunction character-istic of severe pressure overload hypertrophy.

Following the ®rst, and to me astonishingobservation of the striking effect of microtubuledepolymerization on the contractile dysfunctionof hypertrophied feline cardiocytes, it had beenmy intent to move in terms of phenomenologyfrom cell, to tissue, to intact animal, and ®nally toman if the data validated the microtubulehypothesis at each step along the way. We havenow completed a study [26] that constitutes the®nal link in this chain. This study was designedto address the question of whether increasedcardiac microtubules are characteristic of pres-sure overload hypertrophy in man. To this end,patients with symptomatic aortic stenosis andcontrol patients without aortic stenosis werestudied. No patient had aortic insuf®ciency,signi®cant coronary artery disease, or abnormalsegmental left ventricular wall motion. Leftventricular function was assessed by echocardio-graphy and cardiac catheterization before aorticvalve replacement. Left ventricular biopsiesobtained at surgery were separated into freeand polymerized tubulin fractions before analy-sis. Midwall left ventricular fractional shorteningversus mean left ventricular wall stress in theaortic stenosis patients was compared to that innormal patients. We found that in those patientswith aortic stenosis having an increase in theconcentration of myocardial microtubule protein,there is a corresponding decrease in ventricularfunction as measured by midwall fractionalshortening, associated with signi®cantly increas-ing left ventricular wall stress. In the clinicalsetting, a modality having an acceptable thera-peutic index does not exist for removing themicrotubules from interphase cells, such thatwe could not establish in these patients, as we


Fig. 1. Effects of parenteral taxol on feline cardiac microtubules and contractile function in vivo. For the immunoblots andventriculograms, the left panels are from the control state, and the right panels are from the post-taxol state; the bottom panelsummarizes LV contractile function with and without taxol. For the immunoblots, lane 1 represents free b-tubulin, and lane 2represents polymerized b-tubulin. The blot on the left is from LV myocardium of a normal cat three hours after beginning theintravenous infusion of vehicle alone, and the blot on the right was obtained three hours after beginning the intravenous infusion ofthe same volume of vehicle containing 25 mg=kg of taxol. For each ventriculogram, the single end-systolic frame was superimposedon the single end-diastolic frame, with the two frames aligned at the position of the aortic valve. The bottom panel shows the effectson LV contractile function assessed by the relationship of mean normalized systolic ejection rate to mean systolic wall stress ofvehicle alone in one cat and vehicle containing taxol in another cat; the modi®ed midwall mean velocity of circumferential ®bershortening (cmmVcf ) versus mean systolic wall stress relationship was con®rmatory (data not shown). Data are shown at baseline

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had done in experimental models, a direct cause-and-effect relationship between increased myo-cardial microtubules and decreased myocardialcontractile function. Nonetheless, these data doallow us to conclude that in man, as in animalmodels of chronic, severe pressure overloadhypertrophy, myocardial dysfunction is asso-ciated with an increase in the density of thecardiocyte microtubule network, such that thiscytoskeletal alteration may be one mechanismcausative of the development of clinical myocar-dial failure in this speci®c hemodynamic setting.

Mechanisms of Microtubule NetworkDensi®cation in Cardiac Hypertrophy

Turning from phenomena to mechanismsrequired a clear de®nition of what was beingstudied in biophysical terms. That is, what arethe mechanical and metabolic loads imposedduring contraction by increased microtubulenetwork density in the hypertrophied heart?Since we had found in both normal and hyper-trophied cells that the mechanical couplingbetween sarcomere and sarcolemma is unalteredby microtubule depolymerization, even in thepresence of an extracellular load [20], we testedthe hypothesis that the microtubule component ofthe cytoskeleton imposes an intracellular loadthat physically resists sarcomere shorteningrather than altering the structure or function ofthe sarcomere itself [27]. An expected conse-quence of this impediment to sarcomere motionwould be an increase in cardiac energy require-ments for a given amount of mechanical output.Thus, the mechanical and energetic abnormal-ities in pressure overload cardiac hypertrophywould be causally linked via increased internalloading of the contractile cytoskeleton, such thatmechanical energy generated within the sarco-meres is lost through an immediate conversion ofmechanical energy to heat energy, i.e., anincrease in apparent viscosity, instead of beingconserved within cellular elastic elements aspotential energy.

We therefore measured cytoskeletal stiffnessand apparent viscosity in isolated cardiocytes viamagnetic twisting cytometry, a technique bywhich magnetically induced force is applieddirectly to the cytoskeleton through integrin-coupled ferromagnetic beads coated with RGDpeptide. Measurements were again made inright and left ventricular cardiocytes from catswith right ventricular hypertrophy induced by

pulmonary artery banding. In hypertrophiedright ventricular cardiocytes, both the apparentviscosity and the stiffness of the cytoskeletonwere found to be greater than in normal leftventricular cardiocytes, and the increase inapparent viscosity was considerably more pro-nounced than the increase in stiffness. Microtu-bule depolymerization in hypertrophied cardio-cytes, either with or without the opportunity fordiastolic myo®lament activation, normalized bothviscosity and stiffness. Furthermore, microtubulehyperpolymerization in normal cardiocytescaused cytoskeletal viscosity and stiffness tomimic those of hypertrophied cells. These ®nd-ings were not due to differences between the twogroups of cells in terms of their af®nity for extra-cellular matrix protein ligands. Finally, whenthese observations in quiescent cardiocytes wereextended to contracting cardiocytes, the micro-tubule-based viscous impediment to sarcomeremotion was found to be exhibited only at highersarcomere shortening rates.

It is important to note here that rotationalshear stresses were applied to the cytoskeletonvia speci®c integrin receptors. Thus, it would beexpected that the stiffness of the extra-myo®la-ment cytoskeletal network as measured wouldnot necessarily re¯ect either the stiffness of theindividual ®laments in a one-to-one fashion orthat of the cell as a whole. Further, the dampingparameter that we used here, i.e., apparentviscosity, does not represent intracellular ¯uidviscosity. Rather, the changes in apparent vis-cosity observed in hypertrophied cardiocytesappear to represent structural damping attribu-table to the microtubule component of the cyto-skeleton. In these terms, apparent viscositymight well re¯ect a process of intracellular fric-tional dissipation that impedes cellular short-ening. Hence, one might expect this impedingeffect, as observed here experimentally, tobecome more pronounced at higher rates ofsarcomere motion, and one would also expecton this basis the loss of energetic ef®ciencyduring contraction seen in my earlier workwith this model [5]. Thus, as hypothesized to bethe case, this study showed that the micro-tubule-dependent biophysical effect is one ofimposing a primarily viscous intracellular loadon the shortening sarcomeres. Again assuggested initially, this single molecular defectcould well be explicative not only of abnormalsarcomere and cellular motion during contrac-tion but also of the linked contractile and ener-

(1) and three hours later after administering vehicle or taxol (2). The `taxol' cat in this bottom panel is the same as the cat whoseventriculograms are shown before and after taxol in the middle panels and whose LV immunoblot is shown in the upper right panel.The taxol results were replicated in a second cat (data not shown).


getic abnormalities characteristic of pressureoverload cardiac hypertrophy.

Having established the existence, in biologi-cally relevant systems, and the effects, in realphysical terms, of increased microtubule networkdensity in pressure overload cardiac hypertrophy,one could begin to think usefully about its causes.Two questions were addressed in our ®rst suchstudy of causality [28]. First, is the increasedmicrotubule network density a direct result ofload or instead a concomitant of the hypertrophyprocess? That is, while both theory [16] andexperiments [29] suggested that an extendingforce might rapidly shift the dynamic equilibriumbetween free and polymerized tubulin towardsthe polymerized form, we did not know whetherdirect load input into the pressure-overloadedcardiocyte occasions an immediate increase inmicrotubule network density or whether addi-tional and=or alternative mechanisms mightcause a more gradual increase in microtubulenetwork density during the hypertrophic growthprocess. In this context, it should be noted thatthere must be speci®city associated with pressureinput per se, since as reviewed above an equiva-lent degree and duration of hypertrophy inresponse to a volume overload results neither incardiocyte contractile dysfunction nor in micro-tubule changes. Second, since the cotranslationalnegative feedback control by both a- and b-tubu-lin of their own synthesis rates should down-regulate a- and b-tubulin expression, why arethe increases in microtubules, and especially infree tubulin, persistent? That is, it is establishedin other biological contexts that mRNA half-lifeand thus mRNA concentration for both a-tubulinand b-tubulin each decrease as the concentrationof the respective protein in the cytoplasmincreases [30]. Given what we now knew aboutfree tubulin levels in cardiac hypertrophy, if themyocardial concentration of a-tubulin and b-tubulin mRNA were found to be increasedduring and following hypertrophic cardiacgrowth in response to a pressure overload, itwould suggest that additional and=or alternativemechanisms must be responsible for the controlof tubulin synthesis in the speci®c context ofpressure overload cardiac hypertrophy. Further,if tubulin mRNA and protein levels were found tobe elevated concurrently, it would allow us toaddress this second question in terms of thecontrol of tubulin mRNA expression in cardiachypertrophy.

Again using the feline right ventricular pres-sure overload system, we found that while loadmodulation of the set point of the tubulin±micro-tubule equilibrium might be partially responsiblefor the induction and persistence of increasedmicrotubule network density, alterations in

microtubule network density and sarcomeremechanics are not an immediate consequence ofpressure overloading, but instead appear inparallel with the load-induced increase in cardiacmass. Of potential mechanistic importance, boththese changes and increases in tubulin poly A�mRNA and protein coexist inde®nitely after anew higher steady-state of right ventricularmass is reached. Because we found persistentincreases both in microtubules and in theirbiosynthetic precursors in pressure overload-hypertrophied myocardium, I concluded that themechanisms for this cytoskeletal abnormalitymust be sought through studies ®rst of thecontrol of microtubule stability and second ofthe control of tubulin synthesis.

Our study [31] of the ®rst of these two controlmechanisms was based on the ®nding that micro-tubule network density increases only afterhypertrophic growth is initiated, which suggestedmicrotubule stabilization as an attractive candi-date explanation for this phenomenon. Toexplore this hypothesis, we took advantage ofthe fact that the a-tubulin moiety of the ab-tubulin heterodimer, once assembled into amicrotubule, undergoes two posttranslationalmodi®cations, such that the prevalence in micro-tubules of the ®rst and then the second of thesemodi®ed forms of a-tubulin serves as a clockindicating microtubule age. The ®rst of thesemodi®cations is a reversible carboxy-terminaldetyrosination by tubulin carboxypeptidase andretyrosination by tubulin tyrosine ligase, and thesecond modi®cation is irreversible removal of asecond carboxy-terminal amino acid. Thus, accu-mulation of these post-translationally modi®edforms of a-tubulin in microtubules provides amarker for the stability of these polymers. Toexploit these markers of microtubule stability,we prepared antibodies to the native form andto each of the two modi®ed forms of a-tubulin andexamined the prevalence of each in the micro-tubules of cardiocytes and in the total protein ofmyocardium from normally loaded control andpressure overload-hypertrophied feline rightventricles. Staining for the native form of a-tubu-lin in microtubules was equal to right and leftventricular cardiocytes of control cats, but stain-ing for the post-translationally modi®ed forms ofa-tubulin was insigni®cant, i.e., the microtubuleswere labile. However, staining for the post-trans-lationally modi®ed forms of a-tubulin wasconspicuous in microtubules of right ventricularcardiocytes from pressure overloaded cats, i.e.,the microtubules were stable. This ®nding wascon®rmed in terms of increased microtubule drugand cold stability in the hypertrophied cells. Iconcluded that the microtubules of pressure over-load-hypertrophied cardiocytes do, in fact,

194 Cooper

demonstrate markedly enhanced stability,wherein microtubule stabilization begins veryshortly after cardiac pressure overloading andpersists inde®nitely thereafter.

We then focused in this same study [31] on thebasis for this microtubule stabilization. Sinceother data in this study indicated that neitherincreased microtubule nucleation nor decreasedmicrotubule breakdown appear to be operativemechanisms for the microtubule network densi-®cation characteristic of pressure overloadcardiac hypertrophy, I turned to extra-micro-tubule factors which might stabilize cardio-cyte microtubules. Here, microtubule-associatedprotein 4 (MAP 4) was both an obvious candidatein that it is the predominant non-motor MAP ofcardiac muscle [32] and a logical candidate inthat its functional role has been thought to be thestabilization of microtubules during the inter-phase portion of the cell cycle [33]. We foundthat indeed there is a clear increase in MAP 4protein in the pressure overload-hypertrophiedright ventricle, that this protein is preferentiallylocalized to the microtubule fraction by immuno-blotting, and that MAP 4 co-localizes with micro-tubules micrographically. In fact, this increase inMAP 4 was considerably greater than theincrease in tubulin in hypertrophied myocar-dium. Further, MAP 4 mRNA increases as earlyas four hours after right ventricular pressureoverloading, with this increase persistingthroughout the hypertrophy process, whileincreased MAP 4 protein is seen as early as twodays after RV pressure overloading, with thisincrease, as is the case for microtubule densi®-cation and microtubule-related cardiocytecontractile dysfunction, persisting inde®nitelythereafter. Of note, MAP 4 upregulation occursearlier after myocardial pressure loading thandoes tubulin upregulation. Thus, while a directcause-and-effect relationship was not estab-lished, MAP 4 stabilization of the cardiocytemicrotubule array would appear to be a logicalcandidate etiology for the increased microtubulenetwork density, and thereby the functionalconsequence of impaired cardiocyte contractilefunction, characteristic of pressure overloadcardiac hypertrophy.

I thought that it seemed especially unlikelythat upregulation of two functionally relatedproteins, in the setting of cardiocyte hypertrophicgrowth wherein changes in the cellular molecularphenotype are minimal, is fortuitous. Thus, apartfrom the direct relationship of MAP 4 to micro-tubule stability, what linkage might exist? Andspeci®cally, why are MAP 4 and tubulin upregu-lation coordinate, and how does their joint up-regulation relate to greater microtubule networkdensity? Here, it is of interest that in the course

of neuronal development there is coordinateregulation of the expression of the several neuro-nal MAP isoforms as well as of the multiple a-and b-tubulin isoforms [34]. Thus, one can spec-ulate that with hypertrophic cardiac growth re-initiation in speci®c hemodynamic settings, theremay be coordinate upregulation of both MAP andtubulin genes as well as coordinate growth-related changes in tubulin isoform expression.In this context, it is notable that while for themost part the a-tubulin and b-tubulin isoformsare functionally equivalent in terms of coassem-bly into microtubules [35], it is the carboxy-term-inal isoform-variable domain of these tubulinsthat appears to be most important to MAP bind-ing kinetics and, thereby, to microtubule stability[36,37]. As an example, again in neuronalsystems, a change in the proportion of the threemajor b-tubulin isoforms has a substantial effecton microtubule stability in these cells [38,39],such that variation in the ratio of expressed b-tubulin isoforms might represent a means bywhich the stability of the cellular microtubulearray is regulated in response to differing physio-logical input.

In view of the ®ndings in this last study [31] ofthe interrelated developmental regulation oftubulin and MAP 4 genes, and of the potentialfor differing b-tubulin isoforms to alter microtu-bule stability either directly via differing intrinsicproperties or indirectly via differing MAP 4 af®-nities, I thought that it would be of considerableinterest to determine whether there are isoform-speci®c changes in expression of the b-tubulinmultigene family in the pressure overload-hyper-trophied cardiocyte. Such was the case [40]. Wefound that while in normal feline hearts b-4tubulin expression is greatly preponderant, inthe hypertrophied right ventricle, despite themarked increase in both free and polymerizedisoform-common b-tubulin, there was littlechange in b-4 tubulin. Rather, the increased b-tubulin was accounted for by marked increases infree and polymerized b-2 tubulin and especially b-1 tubulin. When expression of b-tubulin isoformtranscripts was then examined, we found that thepattern of b-tubulin isoform expression on themRNA level mimicked that seen on the proteinlevel, and further data showed that increased b-1and b-2 tubulin protein in hypertrophied myocar-dium resulted from transcriptional upregulationof these genes.

A central goal of this study [40] was to deter-mine whether, if upregulation of speci®c b-tubu-lin isoforms during cardiac hypertrophy werediscovered, such changes in gene expressionhave functional signi®cance. It was thus of singu-lar interest that solely for b-1 tubulin in thehypertrophied right ventricle we found a dispro-


portionate increase in the microtubule-assembledpool and especially in stable microtubules. Thatis, since stable microtubules are resistant to cold-induced depolymerization, we measured b-1, b-2,and b-4 tubulin in the cold-stable cytoskeletalfraction of normal and hypertrophied myocar-dium and cardiocytes. We found that while theproportion of b-2 or b-4 tubulin in this fraction isvery low for normal or hypertrophied myocar-dium, a signi®cant proportion of b-1 tubulin isfound in the cold-stable cytoskeletal fraction, andthis was more pronounced for the hypertrophiedright ventricle. Further, confocal micrographsshowed that the microtubule array of the hyper-trophied cardiocyte is more cold-stable that of thecontrol cardiocyte, that microtubules of thehypertrophied cardiocyte incorporate more b-1tubulin than those of the control cardiocyte, andthat this latter ®nding is especially pronouncedin the cold-stable microtubules of the hyper-trophied cell. These ®ndings were con®rmed bydensitometric analysis of tubulin isoform immu-noblots, which showed ®rst in normal right andleft ventricles a greater proportion of b-1 tubulinin the cold-stable microtubule fraction and seconda selective further shift solely of b-1 tubulin tothis fraction in the hypertrophied right ventricle.

Apart from the intrinsic interest of these obser-vations, the major impetus for this study was toascertain their bases in terms of the augmentedtubulin quantity and microtubule stability whichwe ®nd in the pressure overload-hypertrophiedheart. While our ®nding of MAP 4 upregulationin cardiac hypertrophy may well be important tothe latter phenomenon, it does not directlyexplain the former. Thus, the possibility thatincreased expression of one or more members ofthe b-tubulin multigene family might explainboth the greater quantity of tubulin and thegreater microtubule stability, either directly viadiffering intrinsic properties or indirectly viadiffering MAP 4 af®nities, was quite intriguing.The data in this study indeed showed that whileexpression of the predominant cardiac b-tubulinisoform was but little affected, there was markedupregulation of two ordinarily minor cardiac b-tubulin isoforms. In addition to the possibilitythat this explains augmented tubulin quantity,selective localization of the b-1 tubulin isoform tostable microtubules may also explain augmentedmicrotubule stability.

Perspective

Because the studies reviewed here broached anovel mechanism in the study of cardiac hyper-trophy, a design prerequisite was the inclusion ofcontrols for as many variables as possible; theformal goal was to meet the test of Koch's postu-

lates [41]. Thus, in the initial studies of cardio-cytes from the feline right ventricle [19,20],controls for the pathophysiological modelemployed, i.e., hypertrophied cardiocytes fromthe pressure-overloaded right ventricle, consistedboth of non-hypertrophied cardiocytes from thenormally loaded same-animal left ventricle andequivalently hypertrophied cardiocytes from thevolume overloaded feline right ventricle which isknown to exhibit normal contractile function [4].In neither control were the linked cytoskeletaland contractile defects present. Controls for theexperimental interventions employed wereperhaps inherent in their multiplicity, but theywere carefully selected. To avoid making ourconclusions dependent on the usage of a singlemodality such as colchicine for microtubule de-polymerization, and because any drug has unin-tended and likely unrecognized secondary effects,we also depolymerized microtubules via thephysical means of low temperature, whichwould be anticipated to have very differentsecondary effects from those of a chemicalagent, and replicated the effects seen with colchi-cine. To determine whether excess microtubulenetwork density was itself responsible for thecontractile dysfunction seen in cardiocytes hyper-trophying in response to a pressure overload,excess microtubule polymerization was causedto occur in normal cells by two independentmeans. First, taxol was used [42] to hyperpoly-merize the microtubules of normal cardiocytes;the contractile dysfunction characteristic of pres-sure overload-hypertrophied cardiocytes wasreproduced. Second, and for the same reasonthat a physical in addition to a chemical agentwas used to depolymerize microtubules, deuter-ium oxide was used [43] and found to replicatethe effects of taxol. Therefore, in accordance withKoch's ®rst and second postulates, the microtu-bule increase is absent in even extensive cardiachypertrophy so long as contractile functionremains normal; rather, increased microtubulesdevelop solely in dysfunctional hypertrophiedmuscle and only at the time that such dysfunc-tion appears, at which time microtubule depoly-merization restores normal contractile function.And, in accordance with Koch's third postulate,chemical and physical agents which increasemicrotubule network density independent ofhemodynamic input were shown to reproducethe linked contractile and cytoskeletal abnormal-ities seen in severe pressure overload cardiachypertrophy. In our subsequent studies at thelevels of right ventricular cardiac tissue in vitro[22] and left ventricular cardiac cells in vitro [24]and myocardium in vivo (Figure 1) [25], thesecontrols were replicated, to the extent permittedby each preparation, with equivalent results.

196 Cooper

Despite these control studies, however, themultiple roles of microtubules in interphasecells must raise some concern about the effectsof microtubule depolymerization on propertiesother than cytoplasmic viscosity. One potentialconcern, given that microtubule-based transportis important for a number of intracellular trans-port processes, including that of activated b-adrenergic receptors [44], is any inotropic conse-quence of altered b-adrenergic receptor activity.However, this concern is not applicable to ourprevious in vitro studies where b-adrenergicinput was absent, and it was obviated in our invivo work by the usage of b-receptor blockade. Amore substantive concern, in view of the fact thatwe see a positive inotropic effect of microtubuledepolymerization in pressure overload-hypertro-phied cardiocytes and myocardium, is calciumhomeostasis. Here, our work provides directevidence in both normal and hypertrophiedhearts, and at the levels of the cardiocyte cyto-skeleton itself [27] and of the whole cell [45], thatextra-myo®lament microtubule-based viscousdamping is unaltered by changes in [Ca��i. Butmight inotropic state be altered by microtubuledepolymerization and=or by the agents that areused to cause it in a manner independent of thedecrease in apparent viscosity, expressed as fric-tional dissipation, that we ®nd when the exces-sive microtubules are removed from pressureoverload-hypertrophied cells or tissue [27]? Onestudy has shown that microtubule depolymeriza-tion by colchicine causes the time that the L-typecalcium channel in embryonic chick cardiocytesspends in the closed state to increase, and taxoldoes the opposite [46], such that colchicine shouldhave if anything a negative inotropic effect innormal cardiocytes, and there are other data [47]showing that calcium release from intracellularstores is decreased by colchicine. It has also beenshown in adult cardiocytes that neither colchicinenor taxol has a direct effect on the L-type calciumchannel in terms of voltage-dependent para-meters [48]. But quite recently, and in contrast,it was reported that colchicine increases ICa

current density and the [Ca��i transient inadult cardiocytes [49]. However, the positedexplanation for these effects, that ab-tubulinheterodimers act as a functional analog of Gproteins to activate adenyl cyclase when theirconcentration is increased by colchicine, is dif®-cult to accept, since other data in this same studyshow that taxol, which markedly reduces thecardiac ab-tubulin heterodimer concentration[22], is without effect on these same calciumvariables. In our own work we ®nd that withhypertrophy there is, along with an increase inmicrotubules, a very signi®cant and persistentincrease in free ab-tubulin heterodimer concen-

tration [19], yet in contrast to what this mechan-ism would predict if it has functional signi®cance,there is a marked decrement rather than incre-ment in contractile function. Further, we ®ndthat colchicine increases neither resting norpeak activated calcium levels in normal or hyper-trophied cardiocytes [20], and colchicineincreases neither cyclic AMP levels, peak acti-vated calcium levels, nor the rate of rise or fall ofintracellular calcium in normal or hypertrophiedmyocardium [22]. Most pertinent, however, toany consideration of potentially direct inotropiceffects of microtubule depolymerization oncontractile function is the fact that while weconsistently ®nd only a 5±10% increase in theextent and velocity of shortening of sarcomeres,cells, and tissue from normal hearts, as well of asthe normal heart itself in vivo after microtubuledepolymerization by any means, we ®nd a muchgreater response to purposive inotropic interven-tions. Thus, while the relevant studies reachcon¯icting conclusions, colchicine may well havesubtle effects on calcium homeostasis, but thefunctional signi®cance of such ®ndings [49] inthe context of the extensive changes in cardiacmechanics caused by microtubule depolymeriza-tion in severely pressure overload-hypertrophiedmyocardium is open to quite substantial ques-tion.

Again because the studies reviewed herebroached a novel mechanism in the study ofcardiac hypertrophy, it is not surprising thatthe place of this cytoskeletal alteration in theoverall spectrum of pathophysiological changesin hypertrophied myocardium is still beingsettled. In the process of putting this newmechanism in context, it is very important tonote that the data from this laboratory showunequivocally that this cardiac cytoskeletalresponse to a hemodynamic overload is depen-dent on the type of ventricular loading, beingabsent in eccentric volume overload and presentin concentric pressure overload, and it is alsodependent on the extent of hypertrophy, the ageof the animal in which it is imposed, the durationof hypertrophy, and whether the hypertrophicresponse is suf®cient to normalize ventricularwall stress. Microtubule hyperpolymerization,therefore, cannot be viewed as the only mechan-ism causing the development of contractiledysfunction in hemodynamically overloaded,hypertrophied and failing myocardium. Forexample, as we have reported [21], the abnorm-alities in cellular systolic function which mayoccur in chronic left ventricular pressure over-load hypertrophy with normal or subnormal wallstress in the juvenile mammal are not accompa-nied by an increase in the microtubule portion ofthe cytoskeleton and are not corrected by altering


the microtubule polymerization state. This situa-tion is consistent with rather than in oppositionto ®ndings [50] in chronic progressive pressureoverload of the juvenile left ventricle, where,although ventricular mechanics were not charac-terized, the left ventricular mass and pressuredata would strongly suggest a relatively low leftventricular systolic wall stress, and microtubuleswere not found to play a role in the cardiocytecontractile dysfunction. In contrast, as found inour work, with substantial, ®xed pressure over-loading of the adult right ventricle, wall stress isprobably elevated from the outset, thus explicat-ing our ®ndings of persistent cytoskeletal andcontractile abnormalities for right ventricularpressure overloading within the context providedby our data from the adult left ventricular show-ing the crucial role of increased wall stress.

While our ®ndings have been con®rmed byothers both in the rodent [51±54] and in thehuman [55,56] left ventricle, there is one study[57] of the pressure-overloaded rodent left ven-tricle wherein this was not the case. Of note,however, neither hemodynamics nor left ven-tricular wall stress were de®ned here, and the®nding of no increase in microtubules, despite atwenty-fold increase in tubulin mRNA, hasrather questionable relevance in that only aminor pressure overload was imposed, with acorrespondingly minor increase in left ventricu-lar mass in the three animals actually studiedand said to have contractile dysfunction. Thislatter conclusion too is open to serious questionin view of the fact that left ventricular preloadwas unde®ned in comparing the contractile func-tion of normal left ventricles to that of hypertro-phied ventricles, which in this experimentalanalogue of renovascular hypertension, wouldbe expected to have markedly abnormal myocar-dial compliance. Further, this study has sincebeen challenged by the ®nding of a very substan-tial increase in cardiocyte microtubules in theidentical animal model when left ventricularwall stress was measured and found to beincreased [53,54]. In the context of our data,and that of another study [58] showing only atransient increase in cardiocyte microtubulenetwork density after ®xed left ventricular pres-sure overloading which disappeared during thecompensatory hypertrophic growth processwherein, presumably, initially increased leftventricular wall stress was renormalized, theseapparently disparate ®ndings may well be recon-ciled in terms of the speci®c linkage of thesecytoskeletal and contractile abnormalities toincreased ventricular wall stress.

Nonetheless, while the goal here was to reviewthe role of microtubules in the contractiledysfunction of pressure overload-hypertrophied

myocardium, there are clearly a number ofother molecular and cellular mechanisms whichcontribute to the development of contractiledysfunction in this entity [1±3,5]. For instance,in the model of right ventricular pressure over-load which we have used, abnormalities havebeen identi®ed in myocardial energetics, calciumhomeostasis, and the extracellular matrix [1±3].Further, a number of other abnormalities havebeen identi®ed in right ventricular and leftventricular models of pressure overload hypertro-phy, including changes in the myosin heavy chainand myosin light chain isoforms and changes inthe b-adrenergic receptor pathway and in otherreceptor pathways [1±3]. The cytoskeletal changereviewed here is restricted, in ®ndings to date, topressure overload hypertrophy imposed on theadult heart that causes a documented, persistentincrease in ventricular wall stress. It is demon-strably not found, for instance, in ischemicmyocardium [59,60] or in catecholamine-induced[61], tachycardia-induced [62] or genetically-induced [63] cardiomyopathies. Thus, the manyother abnormalities of hypertrophied myocar-dium identi®ed by ourselves and by many otherinvestigators must be responsible for the myocar-dial dysfunction seen in the numerous otherforms of pathological cardiac hypertrophy.Further, when pressure overload hypertrophy orany other cardiomyopathic process reaches anadvanced and irreversible stage, there arealmost certainly multiple changes that areresponsible for the myocardial dysfunctionwhich is present. At such an advanced stage, itwould be quite unlikely indeed that changes inonly one protein would cause all of the myocar-dial dysfunction which is present or that normal-izing changes in that single protein would restorenormal myocardial function.

Future Directions

As is apparent from this review, my recent cyto-skeletal work has focused on two areas: ®rst,de®ning the mechanical effects of microtubulenetwork densi®cation and the hemodynamicsettings in which this new pathophysiologicalmechanism is applicable and second, de®ningthe molecular processes responsible for thesealterations in the microtubule portion of theextra-myo®lament cytoskeleton. Further workin this area now will be devoted to a considerationboth of the effects on microtubule stability ofupregulation of b-1 and b-2 tubulin as seenduring hypertrophy and of the interaction ofMAP 4 with microtubules as a determinant ofincreased microtubule stability. Critical to thesegenetic approaches is the fact that, as shown inFigure 2, we can now work with a murine model

198 Cooper

system in the same way that we have used largeanimal models of human disease heretofore.

I also intend to turn from the direct mechan-ical effects of microtubule network densi®cationon cardiocyte constitutive properties to a consid-eration of the effects of this cytoskeletal altera-tion on unique microtubule-related functions.That is, given the central role of microtubules inlocalizing and regulating cellular constituents, itwould seem quite unlikely that the markedchanges in the microtubule network that I ®ndin the hypertrophied cardiocyte would be withoutmajor speci®c effects on cellular homeostasis;especially interesting here are the potentialeffects of microtubule network densi®cation,MAP 4 upregulation, and especially increasedMAP 4 decoration of microtubules in the contextof hypertrophic cardiac growth. It is known, forinstance, both that increased microtubule decora-tion with MAP 4 inhibits the microtubule inter-actions of each of the major families ofmicrotubule-associated motor proteins, withconsequent inhibition of intracellular microtu-bule-based transport, and that in cells whosemicrotubules are highly decorated with MAP 4,

vesicle transport as well as steady-state vesicleposition are abnormal [64±66]. Given that inaddition to the marked increase in microtubulenetwork density that I ®nd in hypertrophiedmyocardium, MAP 4 is upregulated even moreextensively than microtubules [31], this couldhave at least two major implications for micro-tubule transport-dependent processes in thehemodynamically challenged heart. First, sinceb-adrenergic receptor resensitization followingagonist activation involves receptor internaliza-tion and endosomal transport, b-adrenergicreceptor downregulation in heart failure couldoccur, at least in part, through reduced receptorrecycling after activation and b-arrestin binding.Second, it could also have implications for theef®cacy of the hypertrophic growth response tohemodynamic loading, since such growthrequires microtubule-dependent transport oftranslation-competent mRNAs to intracellularsites at which the translated proteins will beassembled. Here, for instance, because I havefound that MAP 4 upregulation is most strikingin the setting where ventricular wall stress isincreased as the result of a reduced hypertrophic

Fig. 2. Immuno¯uorescence confocal micrographs, using an a-tubulin antibody, of the microtubule array in cardiocytes fromnormal and pressure overload-hyptertrophied murine left ventricles. Hypertrophy was induced by banding the transverse aorta sixweeks prior to sacri®ce. Left ventricular mass was 85% greater than that in normal mice. Left ventricular contractile function asassessed by echocardiography was depressed, as was contractile function as assessed by laser diffraction of cardiocytes from thesame left ventricle. The scale bar represents 25 mm.


growth response to load, the possibility that MAP4 upregulation might have a causative role in thisinadequate growth response is, I think, fascinat-ing. Thus, since in substantial pressure overloadhypertrophy there is a marked increase in micro-tubules, a greater, disproportional increase inMAP 4, and a major increase in microtubuleMAP 4 decoration, it will be interesting to seekthe results of these changes in the context of thenormal roles of these two functionally relatedclasses of proteins.

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cardiocyte cytoskeleton in hypertrophied myocardium

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