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Am J Physiol Heart Circ Physiol 294: H2231-H2241, 2008. First published March 14, 2008; doi:10.1152/ajpheart.91515.2007
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A direct test of the hypothesis that increased microtubule network density contributes to contractile dysfunction of the hypertrophied heart

Guangmao Cheng,1 Michael R. Zile,1 Masaru Takahashi,1 Catalin F. Baicu,1 D. Dirk Bonnema,1 Fernando Cabral,2 Donald R. Menick,1 and George Cooper, 4th1

1Cardiology Division, Gazes Cardiac Research Institute, Medical University of South Carolina, and the Department of Veterans Affairs Medical Center, Charleston, South Carolina; and 2Department of Integrative Biology and Pharmacology, University of Texas Medical School, Houston, Texas

Submitted 21 December 2007 ; accepted in final form 11 March 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Contractile dysfunction in pressure overload-hypertrophied myocardium has been attributed in part to the increased density of a stabilized cardiocyte microtubule network. The present study, the first to employ wild-type and mutant tubulin transgenes in a living animal, directly addresses this microtubule hypothesis by defining the contractile mechanics of the normal and hypertrophied left ventricle (LV) and its constituent cardiocytes from transgenic mice having cardiac-restricted replacement of native β4-tubulin with β1-tubulin mutants that had been selected for their effects on microtubule stability and thus microtubule network density. In each case, the replacement of cardiac β4-tubulin with mutant hemagglutinin-tagged β1-tubulin was well tolerated in vivo. When LVs in intact mice and cardiocytes from these same LVs were examined in terms of contractile mechanics, baseline function was reduced in mice with genetically hyperstabilized microtubules, and hypertrophy-related contractile dysfunction was exacerbated. However, in mice with genetically hypostabilized cardiac microtubules, hypertrophy-related contractile dysfunction was ameliorated. Thus, in direct support of the microtubule hypothesis, we show here that cardiocyte microtubule network density, as an isolated variable, is inversely related to contractile function in vivo and in vitro, and microtubule instability rescues most of the contractile dysfunction seen in pressure overload-hypertrophied myocardium.

molecular biology; hypertrophy; heart failure

IN 1993, WE DISCOVERED that in myocardium hypertrophying in response to pressure but not volume overloading there is a persistent increase in microtubule network density that is associated with contractile dysfunction (42). In the years since then, as reviewed elsewhere (12), we have extended this observation to both cardiac ventricles in multiple species at the levels of sarcomere, cell, and tissue in vitro and to the intact heart in vivo. Before attempting to next move from association to causality, we first reduced the number of variables, as also reviewed elsewhere (13), by establishing the following particulars. First, microtubule depolymerization by either chemical (colchicine) or physical (hypothermia) means removes this viscous impediment to contractile function from hypertrophied myocardium, whereas microtubule hyperpolymerization by either chemical (taxol) or physical (deuterium oxide) means imposes this same viscous impediment to contractile function on normal myocardium. Second, microtubule depolymerization increases neither resting nor peak-activated calcium levels in normal or hypertrophied cardiocytes, neither does it increase either cyclic AMP levels, peak-activated calcium levels, or the rate of rise or fall of intracellular calcium in normal or hypertrophied myocardium. Third, any potential positive inotropic effect on myocardial contraction that might result from {alpha}β-tubulin heterodimers acting as a functional analog of G proteins was excluded by the replication of the colchicine effect by that of vincristine, where both agents depolymerize microtubules and normalize the contractile dysfunction of hypertrophied myocardium, but the {alpha}β-tubulin heterodimer concentration is increased by the former agent and decreased by the latter. Finally, the shift during even very substantial murine cardiac hypertrophy from {alpha}-myosin heavy chain to a greater proportion of the slower ATPase Vmax β-myosin heavy chain has little if any independent effect on cardiac or cardiocyte contractile or constitutive properties.

Given these data, it seemed reasonable to conclude that the structural abnormality of the extramyofilament cytoskeleton and the functional abnormality of the myofilament cytoskeleton are linked via a direct cause-and-effect relationship. However, it must be recognized that this linkage was made in a biological setting wherein confounding variables are especially likely to be present, i.e., working muscle cells living within a high wall stress cardiac ventricle that are continuously exposed to multiple growth signals and metabolic abnormalities (11). The direct test of this hypothesized causality that was the purpose of the present study would thus require that cardiocyte microtubule network density be altered as an isolated variable in a normal biological setting.

We knew from our earlier work that in the heart the fetal-predominant β1-tubulin isoform rather than the adult-predominant β4-tubulin isoform is transcriptionally upregulated during cardiac hypertrophy (30, 33), although increased tubulin per se rather than this isoform shift is what contributes to hypertrophy-related microtubule network densification (41). We also knew that the substitution of β1-tubulin for β4-tubulin in the adult cardiocyte in vitro or in the adult heart in vivo has no effect on either the microtubule network density and stability or cardiac structure and function (41). Therefore, to alter intrinsic cardiocyte microtubule stability as an isolated variable, we elected to replace via cardiac-restricted transgenesis β4-tubulin with hemagglutinin (HA)-tagged wild-type β1-tubulin or mutant β1-tubulins that conferred either increased or decreased microtubule stability. We then tested the effects of these two β1-tubulin mutations first on structure in terms of microtubule network density and second on function in terms of cardiac contraction at the levels of cardiocyte sarcomeres in vitro and the heart of the intact animal in vivo. Finally, to come full circle, we tested in mice with pressure overload-induced cardiac hypertrophy whether the β1-tubulin mutation that produces hyperstable microtubules worsens hypertrophy-induced contractile dysfunction and whether the β1-tubulin mutation that produces hypostable microtubules ameliorates hypertrophy-induced contractile dysfunction. Our data provide compelling new evidence in support of the hypothesis that microtubule network densification, whether in the normal or in the hypertrophied heart, is itself sufficient to cause contractile dysfunction.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Production of transgenic mouse lines having cardiac-restricted expression of HA-tagged wild-type or mutant β1-tubulin genes. We had shown earlier in Chinese hamster ovary (CHO) cells that transfected HA-tagged β1-tubulin behaves identically to endogenous β-tubulin in terms of assembly into microtubules, as well as stability and microtubule-associated protein affinity of these microtubules once formed (17). We then imposed directional selection on dividing CHO cells by exposing them to an agent that hyperpolymerizes microtubules. This environmental pressure resulted in the survival of CHO cells expressing a mutant β1-tubulin (β1-L217R, position 217 leucine->arginine), producing hypostable microtubules (18). Similarly, an agent that depolymerizes microtubules was used to generate via directional selection CHO cells expressing a mutant β1-tubulin (β1-D45Y, position 45 aspartate->tyrosine), producing hyperstable microtubules (19).

Wild-type β1-tubulin and these mutant β1-tubulins were expressed in the hearts of transgenic mice; β4-tubulin is normally the only β-tubulin gene expressed in the adult heart (30). The cDNAs encoding hamster β1-WT, β1-L217R, and β1-D45Y were modified to express a nine amino acid HA tag at the COOH terminus of each construct (6). Site-directed mutagenesis was used to remove the NotI site immediately 3' of the HA tag and to introduce a SalI site just 3' of the stop codon for each of the constructs. All constructs were verified by double-stranded sequencing, and the HA-tagged β1-WT, β1-L217R, and β1-D45Y cDNAs were then subcloned into the SalI site of the murine {alpha}-myosin heavy chain promoter vector. The orientation of the insert was determined by sequencing. The constructs were digested with NotI, and the 7.5-kb fragment containing the {alpha}-myosin heavy chain promoter, the β1-tubulins, and the 3' untranslated region and polyadenylation site of the human growth hormone cDNA free of vector sequence were gel purified. Pronuclear microinjection and implantation in the FVB/N mouse strain were performed by the Medical University of South Carolina Transgenic Mouse Facility. The founder mice were identified using PCR and confirmed by genomic Southern blot analysis using DNA obtained from tail clips. Stable transgenic lines were generated by breeding the founder mice with nontransgenic littermates. The offspring were screened by PCR, where third generation or later adult mice heterozygous for the transgene were used for all studies. All animal usage was under protocols approved by the Institutional Animal Care Committee in accordance with the National Institutes of Health (NIH) Guidelines.

Induction of left ventricle hypertrophy. Pressure overload hypertrophy of the murine left ventricle (LV) was produced by transverse aortic constriction (TAC), just as our laboratory has described in detail elsewhere (23). After the mice were anesthetized with ketamine (50 mg/kg ip) and xylazine (2.5 mg/kg ip), they were intubated and placed on a respirator. The aortic arch was exposed surgically and tightly constricted between the origins of the two carotid arteries via a 7-0 silk suture tied over both the aorta and a juxtaposed 30-gauge needle, causing complete occlusion of the aorta. The needle was then withdrawn, resulting in a severely stenotic aortic lumen, and the animals were allowed to recover for 4 wk.

Characterization of LV structure and contractile function in intact mice. These in vivo measurements were made using techniques described by our laboratory in an earlier study (23). The data were obtained in isoflurane-anesthetized mice via echocardiography using a 15-MHz transducer (Sonos 5500; Agilent, Santa Clara, CA) placed on a layer of acoustic coupling gel applied to the hemithorax. Three to six beats were averaged for each measurement. LV dimensions and wall thickness were measured at end systole and end diastole using the American Society of Echocardiography criteria (32). LV wall thickness was derived as the mean of interventricular septal thickness and LV posterior wall thickness. LV mass was determined using standard methods (10, 39). LV volume was determined by using Simpson's method of disks (35). The ejection fraction was calculated as 100 x (LV end-diastolic volume – LV end-systolic volume)/LV end-diastolic volume. Fractional shortening was calculated as 100 x (LV end-diastolic dimension – LV end-systolic dimension)/LV end-diastolic dimension. The transverse aortic band gradient was calculated via a modified Bernoulli equation: {Delta}Pressure = 4 x (Vpeak)2, where Vpeak is the peak continuous wave Doppler velocity at the band site (15). For each transgenic mouse strain, LV mass was measured at autopsy in a subset of control and TAC mice; in no case was there a difference from echocardiographic LV mass by Student's t-test.

Murine cardiocyte isolation and contractile function. Murine cardiocytes were isolated as before (9) from adult mice of random sex. The mice were anesthetized with ketamine (100 mg/kg ip) and xylazine (5 mg/kg ip), heparinized (200 IU ip), intubated, and placed on a respirator. The hearts were rapidly excised, cannulated via the aorta, and perfused at 37°C in Langendorff mode at 80 cmH2O pressure. The hearts were initially perfused for 5 min with 1.8 mM Ca2+-Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl2, 0.5 MgCl2, 10 HEPES, and 10 glucose at pH 7.4 and then in Ca2+-free buffer for 5 min. This was followed by perfusion with Ca2+-free buffer containing 0.05% collagenase D (Boehringer-Mannheim, Indianapolis, IN) and 0.01% protease XIV (Sigma, St. Louis, MO). After the hearts were palpably flaccid at 6–8 min, the digestion solution was washed out with Ca2+-free Tyrode solution for 30 s. The hearts were removed from the cannula, and the LV including septum was separated, minced, and gently agitated, allowing cardiocytes to be dispersed into the Ca2+-free Tyrode solution. After 15 min, the cardiocytes were resuspended at 25°C in oxygenated 0.1 mM Ca2+-Tyrode buffer at pH 7.4. The [Ca2+] was gradually increased to 1.2 mM over a 45-min period at 25°C.

Cardiocyte contractile function was defined in terms of sarcomere mechanics, measured as before (41) with our laser diffraction technique during steady-state 0.4-Hz contractions at 32°C in 1.2 mM Ca2+ oxygenated Krebs-Henseleit buffer at pH 7.4.

Immunoblots. Myocardial protein fractions were prepared as before (41). For the total protein fraction, the myocardium was homogenized in 1% SDS buffer containing (in mM) 10 Tris·HCl (pH 7.4), 0.5 DTT, and 1 Na3VO4 and centrifuged at 16,000 g, 25°C for 10 min. The supernatant was boiled for 5 min and centrifuged at 16,000 g, 4°C for 10 min; this supernatant was saved as the total protein fraction. For immunoblotting, an equal amount of protein (bicinchoninic acid assay) was loaded in each lane. For the free tubulin heterodimer and polymerized tubulin microtubule fractions, the myocardium was homogenized in a microtubule stabilization buffer containing 50% glycerol, 5% DMSO, 10 mM Na2HPO4, 0.5 mM EGTA, and 0.5 mM MgSO4 and centrifuged at 100,000 g, 25°C for 20 min. The supernatant was saved as the heterodimer fraction. The pellet was resuspended in 1% SDS buffer, boiled for 10 min, and centrifuged at 16,000 g, 4°C for 10 min. This supernatant was saved as the microtubule fraction. For immunoblotting, to compare the amount of tubulin in the heterodimer with the microtubule fraction of a given sample, an equal proportion of the two fractions from the same sample was applied. For between-sample comparisons, an equal amount of protein from each (bicinchoninic acid assay) was applied. The blots were incubated for 1 h with the primary antibody. After the blots were incubated with peroxidase-labeled secondary antibody, specific protein bands were detected by using avidin-biotinylated horseradish peroxidase in conjunction with enhanced chemiluminescence (Perkin-Elmer, Boston, MA). To derive semiquantitative data from these blots, NIH ImageJ software was used to provide background-corrected integrated optical density values from scanned images of very lightly exposed films.

Colchicine administration to intact mice. To cause cardiac microtubule depolymerization in vivo, colchicine (Sigma) was administered intraperitoneally. To first determine the optimal drug dosage and exposure duration in normal mice, we injected 0.00, 0.25, 0.50, or 1.00 mg/kg ip of colchicine in 0.9% normal saline at 0, 4, 8, or 12 h before death. The hearts were then excised; half of each heart was used to prepare the free tubulin heterodimer and polymerized tubulin microtubule fractions as described above under Immunoblots, and the other half of each heart was used for total tubulin isolation as standard SDS lysates. We found that essentially complete microtubule depolymerization had occurred at 4 h after drug administration with a colchicine dosage of 0.50 mg/kg. Furthermore, although we had been concerned that the increase in free tubulin heterodimer concentration would lead to a cotranslational reduction in {alpha}- and β-tubulin mRNA stability and thus total {alpha}- and β-tubulin concentration (3, 30), we did not see this until well after the 4 h drug-death interval. We therefore used colchicine at a dosage of 0.50 mg/kg ip given 4 h before death in these studies.

Immunofluorescence confocal microscopy. To visualize native and transgenic β-tubulins in cardiac tissue, hearts were perfusion fixed as before (41). For tissue fixation, we used a periodate-lysine-paraformaldehyde fixative containing (in mM) 10 NaIO4, 75 lysine, and 37.5 phosphate buffer and 2% paraformaldehyde perfused through the coronary arteries at a pressure of 60 cmH2O, 37°C for 1 h. The fixed tissue was cut into small pieces, placed in 100 mM phosphate-buffered 10% sucrose for 4 h, 15% sucrose for 4 h, and 20% sucrose overnight. It was then embedded in tissue-freezing medium (Triangle Biomedical Sciences, Durham, NC), frozen in liquid nitrogen, and sectioned at 8 µm with a cryostat microtome, mounted on glass slides, and air dried. After incubation with 1% Triton X-100 at 25°C for 10 min, tissue sections were treated in the same way as described below for cardiocytes.

To visualize the appearance and density of the cardiocyte microtubule network, freshly isolated LV cardiocytes were sedimented as before at 1 g for 45 min (9) onto laminin-coated coverslips. Laminin-adherent cardiocytes were extracted for 1 min in a microtubule-stabilizing buffer of (in mM) 2 EGTA, 0.1 EDTA, 1 MgSO4, and 100 MES (pH 6.75) containing 1% Triton X-100, rinsed three times in the same buffer with no detergent, and fixed in 3.7% formaldehyde in this buffer for 30 min, all at 25°C. After each coverslip was blocked with 10% donkey serum in 0.1 M glycine and 0.05 M PBS for 30 min at 25°C, the cells were incubated at 4°C overnight in a 1:100 dilution of primary antibody in PBS. After being washed three times with PBS, the cells were incubated at 25°C for 2 h in a 1:50 dilution of fluorescein-conjugated secondary antibody in 2% normal donkey serum in PBS. Optical sections (0.1 µm) were acquired using a confocal microscope (Fluoview Scanning Laser Biological Microscope BX50WI system; Olympus, Center Valley, PA) equipped with two lasers (Ar 488 and Kr 568) and a PlanApo x60 numerical aperature 1.40 oil immersion objective. Fluoview version 1.26 software (Olympus) was used for image acquisition from the microscope. Adobe Photoshop 7.0 software was used for superimposing the laser channels and for cropping and rotating images. To derive semiquantitative data from cardiocyte micrographs, the lasso tool in Adobe Photoshop 7.0 software was used to outline the cell boundary, and the mean pixel intensity within this boundary was then determined using the histogram tool.

Data analysis. Data were expressed as means ± SE; any statistical comparisons are specified in the legends of the table and of the individual figures.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cardiac expression and phenotypic effects of wild-type or mutant β1-tubulins. For the mice used in this study, the murine {alpha}-myosin heavy chain promoter directed the cardiac-restricted expression of wild-type β1-tubulin (β1-WT), an increased microtubule stability β1-tubulin mutation at position 45 that changed aspartate to tyrosine (β1-D45Y), or a decreased microtubule stability β1-tubulin mutation at position 217 that changed leucine to arginine (β1-L217R). Since much of our hypothesis testing was to be based on assays of isolated LV cardiocytes, we first needed to select transgenic mouse lines that expressed the HA-tagged wild-type or mutant β1-tubulins homogeneously throughout the LV myocardium rather than in a mosaic pattern. Figure 1 shows that this was accomplished, since for each transgene there is a complete penetrance of expression in all LV cardiocytes. These mouse lines were then used for all subsequent experiments.


Figure 1
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  		Fig. 1. Confocal micrographs of frozen sections of mouse left ventricles (LVs). The myocardial samples are from FVB/N wild-type (WT; A), β1-WT (B), β1-D45Y stable microtubule mutant (C), and β1-L217R unstable microtubule mutant (D). A hemagglutinin (HA) tag antibody (12CA5; green) was used, which binds to β1-tubulin in the cells expressing each of the 3 transgenes, as well as an isoform-nonselective {alpha}-tubulin antibody (B-5-1-2; red). The yellow signal shows colocalization of these proteins. Scale bar = 200 µm.

 
We next determined the effects of these transgenically expressed proteins on cardiocyte microtubule morphology and stability, both intrinsically and in response to colchicine-induced microtubule depolymerization. Two variables were of interest: β4-tubulin versus β1-tubulin expression and microtubule stability-altering mutations of the latter gene.

With respect to β-tubulin isoform expression, we have used our isoform-specific β-tubulin peptide antibodies to show in the rat (30) that although β1-tubulin is the greatly predominant member of the β-tubulin multigene family expressed in the embryonic heart, the expressed isoform shifts almost entirely to β4-tubulin in the normal adult heart, and we have also found this to be true in the mouse (unpublished data). We further showed (41) that the substitution of β1-tubulin for the constitutive β4-tubulin of adult feline cardiocytes in vitro has no phenotypic effect in terms of microtubule network density or stability. We show here (Fig. 2) that this also holds true for the adult murine heart in vivo; that is, the micrographs of cardiocytes isolated from normal β4-tubulin-predominant FVB/N murine hearts and those from the β1-tubulin-predominant hearts of the β1-WT transgenic mouse line show a comparable microtubule network density before (Fig. 2A vs. 2C) and after (Fig. 2B vs. 2D) parenteral colchicine.


Figure 2
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  		Fig. 2. Effects of β1-tubulin mutants on cardiac microtubule stability. These samples are from the LVs of mice that were given either vehicle or colchicine, 0.5 mg/kg ip, at 4 h before death. For the micrographs, the left vertical bar (A, C, E, and G) shows cardiocytes from vehicle-treated mice, and the right vertical bar (B, D, F, and H) shows cardiocytes from colchicine-treated mice. An isoform-nonselective {alpha}-tubulin antibody (B-5-1-2) was used. The horizontal pairs of cells from top to bottom are FVB/N WT (A and B), β1-WT (C and D), β1-D45Y stable microtubule mutant (E and F), and β1-L217R unstable microtubule mutant (G and H). The number inset in each micrograph gives the mean pixel intensity (white level) within the boundary of each cardiocyte. Scale bar = 20 µm. For the myocardial immunoblot, an isoform-nonselective β-tubulin antibody (DM1B) was used. For each set of 4 lanes, the first 2 are from untreated mice, and the second 2 are from mice treated for 4 h with colchicine. Within each set of 4 lanes, lanes 1 and 3 are free tubulin and lanes 2 and 4 are polymerized tubulin. Since an isoform-common β-tubulin antibody was used, the bottom band is endogenous β4-tubulin, and the top band is the protein from the transgene that runs higher because of the HA tag. Note that the behavior of β4-tubulin, which in the transgenic strains is from cardiac interstitial cells wherein the cardiocyte-specific {alpha}-myosin heavy chain promoter-driven transgenes are not expressed, is comparable in all 4 types of mice. The table was prepared from integrated optical density measurements of lightly exposed blots (n = 5 experiments); the β4-tubulin bands were measured in the FVB/N mice, and the β1-tubulin bands were measured in the transgenic mice. Statistical comparisons among groups at baseline or after colchicine were by one-way ANOVA followed by the Bonferroni/Dunn post hoc test. *P < 0.05 for difference from the FVB/N value at baseline or after colchicine.

 
With respect to microtubule stability-altering β1-tubulin mutations, we show here (Fig. 2) that just as in the CHO cells wherein these mutants were generated (4, 18, 19), the two HA-tagged β1-tubulin mutants have the intended effects on microtubule network density and stability in vivo; that is, for the microtubule-stabilizing β1-D45Y mutant, LV cardiocyte microtubule network density is greater than that in FVB/N control and β1-WT cells both before (Fig. 2E vs. 2A and 2C) and after (Fig. 2F vs. 2B and 2D) parenteral colchicine. In contrast, for the microtubule-destabilizing β1-L217R mutant, LV cardiocyte microtubule network density is less than that in FVB/N control and β1-WT cells both before (Fig. 2G vs. 2A and 2C) and after (Fig. 2H vs. 2B and 2D) parenteral colchicine. Thus the behavior of the β1-WT transgenic microtubules resembles that of the β4-tubulin FVB/N wild-type; the β1-D45Y transgenic microtubules are more stable than either, and the β1-L217R transgenic microtubules are less stable. The accompanying β-tubulin immunoblot prepared from the LVs of identically treated mice confirms the immunofluorescence data, i.e., FVB/N wild-type and β1-WT transgenic animals have similar levels of polymerized myocardial tubulin before and after parenteral colchicine. In contrast, when compared with both wild-type controls, the β1-D45Y animals have a higher amount of polymerized tubulin that is colchicine stable, whereas the β1-L217R animals have a lower amount of polymerized tubulin that is colchicine labile.

Of interest, the in vivo effects on the microtubule stability of replacing the native cardiac β4-tubulin with each of these three transgenic β1-tubulins are remarkably similar to the in vitro effects seen in the CHO cells in which the β1-tubulin mutants were generated. Thus if the numerical data at the bottom of Fig. 2 for the baseline ratio of free/polymerized myocardial β-tubulin are expressed as the percentage of tubulin that is polymerized, these values are ~33% for β1-WT, ~49% for β1-D45Y, and ~23% for β1-L217R. These values correspond quite well to the original CHO cell values (4, 18, 19), such that there is a notable conservation of the effects of these mutations on tubulin assembly across species, tissues, and in vitro versus in vivo cellular environments.

To interpret our subsequent studies of the effects of these tubulin transgenes, with or without superimposed parenteral colchicine, on the contractile function of the normal and hypertrophied heart, we next examined the effects of transgene expression on steady-state tubulin levels. Figure 3, the result of one of several studies done using the same LV samples as those used to prepare the data in Fig. 2, shows that the amount of both {alpha}- and β-tubulin protein is very similar in all four types of mice, both before and after 4 h of parenteral colchicine treatment, and the level of transgene expression is the same in the three transgenic strains. The GAPDH blot shows that protein loading was equal throughout.


Figure 3
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  		Fig. 3. Effects of transgene expression on levels of myocardial {alpha}- and β-tubulin. These samples were prepared as total protein in 1% SDS buffer from the same homogenized murine LVs used for the immunoblot in Fig. 2. For each pair of lanes, lane 1 is from an untreated mouse and lane 2 is from a colchicine-treated mouse. From top to bottom, the identically loaded immunoblots were probed with primary antibodies to β-tubulin (DM1B), {alpha}-tubulin (B-5-1-2), HA tag (12CA5), or GAPDH (6C5). Just as in Fig. 2, in the top blot here using an isoform-common β-tubulin antibody, the HA-tagged β1-tubulins run higher than the endogenous β4-tubulin. There was no statistically significant effect of the transgenes on these parameters (n = 5 experiments).

 
Note especially that the β-tubulin blot in Fig. 3, employing an isoform-common β-tubulin antibody, also shows that in contrast to most overexpression studies, there is very little overexpression at the protein level of the three β1-tubulin transgenes relative to the expression level of native β4-tubulin. This is because, regardless of the mRNA level, the amount of a given tubulin monomer is tightly regulated by two factors. First, newly synthesized {alpha}-tubulin or β-tubulin that is not immediately assembled into an {alpha}β-tubulin heterodimer is rapidly degraded (7, 34, 38), and the {alpha}-tubulin blot in Fig. 3 shows that there was no change in {alpha}-tubulin levels in these transgenic mice. Second, the cotranslational regulation of β-tubulin mRNA stability via the NH2-terminal portion of the nascent polypeptide provides an autoregulatory negative feedback loop (3) that tightly controls the concentration of cellular β-tubulin. Thus the net effect of transgene expression for each of the three β1-tubulins, despite the fact that each is being driven by the {alpha}-myosin heavy chain promoter, is the replacement of endogenous β4-tubulin with the transgenic β1-tubulin at near endogenous levels for total β-tubulin.

Because microtubule stability in response to exogenous short-term poisons does not necessarily reflect long-term intrinsic behavior, we next looked at time-dependent {alpha}-tubulin posttranslational modifications in the three strains of transgenic mice. To do this, we took advantage of the fact that after {alpha}β-tubulin heterodimer assembly into microtubules, the {alpha}-tubulin moiety undergoes two sequential time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin{leftrightarrow}Glu-tubulin) and then irreversible deglutamination (Glu-tubulin->{Delta}2-tubulin), such that Glu- and {Delta}2-tubulin are markers for long-lived, stable microtubules (8, 31). Therefore, we have generated antibodies for Tyr-, Glu-, and {Delta}2-tubulin and used them as markers of intrinsic cardiocyte microtubule stability (33), since in contrast to the situation in nervous tissue and testis, essentially all muscle {alpha}-tubulin participates in this cycle of posttranslational modifications (1). Figure 4 shows that when compared with β1-WT myocardium, the β1-D45Y tubulin mutant has a greater proportion of {alpha}-tubulin in the Glu- and {Delta}2-tubulin fractions, whereas the β1-L217R tubulin mutant has a lesser proportion of {alpha}-tubulin in the Glu- and {Delta}2-tubulin fractions. Thus, in the β1-D45Y mutant intrinsic myocardial microtubule, stability is greater than that in β1-WT, but in the β1-L217R mutant intrinsic myocardial microtubule, stability is less than that in β1-WT.


Figure 4
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  		Fig. 4. Effects of transgene expression on intrinsic microtubule stability. These are immunoblots of total protein prepared in 1% SDS buffer from homogenized LVs of untreated mice. The antibodies used for each of the top 3 blots (Ref. 33) and the transgenic mouse strains are specified. An identical amount of protein was loaded for each blot in each lane, and this was confirmed in the bottom blot probed with a GAPDH antibody (6C5). The table was prepared from integrated optical density measurements of lightly exposed blots (n = 3 experiments).

 
Effects of LV hypertrophy on hearts expressing wild-type or mutant β1-tubulins. The most important features of pressure overload hypertrophy induction via TAC in the three transgenic mouse strains are given in Table 1. We have found that the TAC duration chosen allows a new steady-state LV mass to be reached (29), even for the 70–80% LV hypertrophy employed in the present study. Furthermore, the degree of LV pressure overloading, the extent of resultant LV hypertrophy, and the attendant perioperative mortality are clearly relevant to clinically significant pressure overloading of the LV by aortic stenosis (25).


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  		Table 1. Murine LV pressure overloading

 
Figure 5 shows first that at baseline the expression of the stable microtubule β1-D45Y mutant causes a shift of LV tubulin into the polymerized fraction, whereas the expression of the unstable microtubule β1-L217R mutant causes a shift of LV tubulin out of the polymerized fraction. After TAC-induced LV hypertrophy, there is the same shift of cardiac tubulin into the microtubule fraction for β1-WT transgenic mice that we see in normal mice (23). Of note, however, in the β1-D45Y mutant mice, this shift is accentuated, whereas in the L217R mutant mice this shift is attenuated.


Figure 5
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  		Fig. 5. Effects of pressure overload hypertrophy on the levels of free and polymerized myocardial β-tubulin. The samples used for these immunoblots are from the LVs of the 3 strains of transgenic mice either in the baseline control state or at 1 mo after transverse aortic constriction (TAC). An isoform-nonselective β-tubulin antibody (DM1B) was used. For each pair of lanes, lane 1 is free tubulin and lane 2 is polymerized tubulin. The table was prepared from integrated optical density measurements of lightly exposed blots (n = 4 experiments).

 
Figure 6 shows the structural and functional consequences of these alterations in intrinsic microtubule stability in terms of LV mass, geometry, and contraction, first at baseline and then after TAC-induced LV hypertrophy. Figure 6, top and bottom left, shows that at baseline there is a very modest but significant 13.6% increase in LV mass normalized to tibial length in the stabilized microtubule β1-D45Y LVs and a similar but nonsignificant increase in LV wall thickness. The β1-D45Y mice also had at baseline a 15.4% increase in LV mass normalized to body weight and a 14.1% increase in absolute LV mass. This differential effect of the β1-D45Y transgene on LV mass was persistent, since in sham-operated β1-WT, β1-D45Y, and β1-L217R mice studied at the same postoperative interval as the TAC groups in Fig. 6, there was again a significant increase in LV mass, seen only in the β1-D45Y mice, of 13.5%. Given that an increased viscous load during contraction is imposed by a denser microtubule network (40), such as that found in the β1-D45Y myocardium (Fig. 2A vs. 2E), we attribute this mass increase to a load-dependent hypertrophic response. After TAC, LV mass and thickness increase substantially in all three transgenic mouse strains, with the increases once again being especially marked in the β1-D45Y LVs. Figure 6, top and bottom middle, shows that at baseline LV end-diastolic diameter and volume, structural features that we find to be inversely related to murine LV contractile function (23), are both increased in the β1-D45Y LVs. After TAC, LV end-diastolic diameter and volume increase more in the stabilized microtubule β1-D45Y LVs than in either the β1-WT or the destabilized microtubule β1-L217R LVs. Figure 6, top and bottom right, shows that at baseline LV contractile function measured as fractional shortening and ejection fraction is significantly reduced in the β1-D45Y LVs. After TAC, the destabilized microtubule β1-L217R mutation protects against hypertrophy-induced LV contractile dysfunction, whereas the stabilized microtubule β1-D45Y mutation exacerbates hypertrophy-induced LV contractile dysfunction.


Figure 6
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  		Fig. 6. Effects of transgene expression on LV structure and function before and 1 mo after TAC induction of LV hypertrophy. These data, where n = number of mice in each group and values are means ± SE, were obtained by echocardiography. Heart rate during these studies in the 3 groups of isoflurane-anesthetized mice was very similar to that reported elsewhere under these conditions (Ref. 10). The values ranged from 360 ± 6 to 382 ± 9 beats/min at baseline and from 375 ± 11 to 383 ± 7 beats/min post-TAC and did not differ statistically. Statistical comparisons among groups were by one-way ANOVA followed by Scheffé's post hoc test; within a group, they were by Student's paired t-test. *P < 0.01 for difference from the β1-WT group either before or after TAC; {dagger}P < 0.01 for difference from the baseline control value within a group.

 
Turning our attention from the heart to its constituent cardiocytes, Fig. 7A shows that these changes in cardiac mechanics are apparently related specifically to microtubule network density, since cardiocyte contractile function becomes equivalent among the normal β4-tubulin-expressing and transgenic wild-type and mutant β1-tubulin-expressing cells after microtubule depolymerization. Furthermore, as would be expected (23, 40) for contractile function in the setting of the quite modest degree of hypertrophy present in the β1-D45Y LVs, there was no residual hypertrophy-related contractile impairment in the β1-D45Y cardiocytes after the microtubules were removed. Figure 7B then shows the functional consequences of these alterations in intrinsic microtubule stability in terms of LV cardiocyte sarcomere mechanics, first at baseline and then after TAC-induced LV hypertrophy. The data are concordant with those shown for the LVs of the intact animals in Fig. 6; that is, the stabilized microtubule β1-D45Y mutation causes baseline abnormalities of sarcomere motion during contraction and exacerbates hypertrophy-induced dysfunction. In contrast, the destabilized microtubule β1-L217R mutation protects against hypertrophy-induced LV cardiocyte contractile dysfunction.


Figure 7
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  		Fig. 7. Effects of transgene expression on LV cardiocyte sarcomere mechanics. A: the extent and velocity of sarcomere shortening are shown for LV cardiocytes from all 4 experimental groups, where the same cell was studied both at baseline and 30 min later after colchicine-induced microtubule depolymerization (Ref. 42); n = number of cells in each group. Statistical comparisons among groups were by one-way ANOVA followed by the Student-Newman-Keuls post hoc test; within a group, they were by Student's paired t-test. *P < 0.05 for difference from the FVB/N group either before or after colchicine; {dagger}P < 0.05 for difference from the baseline control value within a group. B: the extent and velocity of sarcomere shortening are shown for LV cardiocytes from each of the same 3 experimental groups as those in Fig. 6; cells from these transgenic mouse strains were studied either before or 1 mo after TAC induction of LV hypertrophy; n = number of mice in each group. Statistical comparisons among groups were by one-way ANOVA followed by Scheffé's post hoc test; within a group, they were by Student's paired t-test. *P < 0.01 for difference from the β1-WT group either before or after TAC; {dagger}P < 0.01 for difference from the baseline control value within a group.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Proliferation of microtubules in the cardiac muscle cell hypertrophying in response to a pressure overload appears to impair both systolic (42) and diastolic (46) cardiac function by increasing cytosolic viscous damping (40). Our initial reports showed that this cytoskeletal alteration was entirely absent during even very extensive physiological hypertrophy (42, 43), and as reviewed elsewhere (12, 14), it is not seen universally in pressure overload hypertrophy, especially if myocardial wall stress is not substantially increased. However, further studies from our laboratory, also reviewed elsewhere (12–14), showed that a dense microtubule network stabilized by microtubule-associated protein 4 is present uniformly in the pressure-overloaded, hypertrophied and failing right and left ventricles of multiple species, including humans, if the load is severe enough to cause a sustained increase in systolic stress per unit volume of myocardium. Concurrently, in other laboratories, these basic findings were both confirmed (2, 5, 20, 21, 36, 44, 45) and extended to other abnormal cardiac muscle states (16, 22, 27, 37).

Nonetheless, it had not been possible to unequivocally attribute contractile dysfunction to microtubule network densification, since these two variables have been linked in abnormal myocardium wherein many structural and regulatory alterations may contribute to changes in myocardial contractile and constitutive properties (11). What was needed, in an analogy to Koch's third postulate for establishing infectious disease causality (26), was to introduce the putative causative organism, i.e., microtubule network densification, into a normal host as a single variable and to see whether the disease, i.e., contractile dysfunction, is reproduced. Although we had done this in a rather crude way by altering microtubule properties with chemical and physical agents that likely had unintended and largely unknown secondary effects (42, 43), the goal here was to do this with a level of precision and specificity that would indicate more directly whether there is a causal linkage of microtubule network densification to cardiac contractile dysfunction.

This goal was accomplished by creating transgenic mice in which the native cardiac β4-tubulin was replaced with HA-tagged β1-tubulin that contained mutations known to either stabilize or destabilize the microtubule network. We had documented previously that, in concert with many other studies reviewed elsewhere (28), there is no functional consequence of replacing the endogenous cardiocyte β4-tubulin with β1-tubulin (30, 41), and as shown in Fig. 3, at the protein level our transgenic mouse lines did not significantly overexpress wild-type or mutant β1-tubulins. Interestingly, the mutations were well tolerated in murine myocardium, and the effects of the mutations on microtubule assembly in the normal heart were quantitatively very similar to those reported for cultured CHO cells (4, 18, 19). Thus, although not the focus of this study, these results do indicate that cultured cells provide a good model for studying microtubule assembly and suggest that the extent of microtubule assembly is relatively constant among species and tissues. It is also worth noting parenthetically that tubulin is an essential and highly conserved protein for which there has previously been only a single report of naturally occurring mutations in mammals (24). This report, along with the ability of mice to tolerate the mutations we introduced, suggests that tubulin mutations in mammals may be more prevalent than previously thought.

The data in Fig. 7A show that the transgenic strategy used here had effects on cardiocyte contraction that were apparently restricted to those caused by microtubule stability alterations, since cardiocyte contractile function became equivalent when the microtubules were depolymerized in the four types of mice that we studied, including the slightly hypertrophied β1-D45Y mice having a 14% increase in LV mass. Indeed, this observation is concordant with our first studies of the cardiac extramyofilament cytoskeleton (42, 43), where removal of the normal microtubule network from control or physiologically hypertrophied cardiocytes results in increases of about 11% in the extent and about 18% in the velocity of unloaded shortening. Although not statistically significant in any particular study, we have always seen this trend in the large number of subsequent studies of normal right and left ventricular cardiocytes from multiple species reviewed elsewhere (12–14). Furthermore, the removal of the dense microtubule network from hypocontractile pathologically hypertrophied cardiocytes, including those from severely pressure overloaded murine LVs having a 155% increase in LV mass (23), restores these measures of contractile function to those of control cells.

The functional studies shown in Figs. 6 and 7B, therefore, the first ever reported using wild-type and mutant tubulin transgenes in a living animal, directly support three conclusions. First, both in intact mice and in isolated murine cardiocytes, there is an inverse relationship between intrinsic microtubule network density and contractile function. Second, and also both in intact mice and in isolated murine cardiocytes, increased intrinsic microtubule network density, apparently as a single variable, exacerbates hypertrophy-related contractile dysfunction. Third, decreased intrinsic microtubule network density, again apparently as a single variable, ameliorates hypertrophy-related contractile dysfunction. Thus microtubule network density is inversely related to cardiac contractile function, and we conclude that the present study provides persuasive evidence that the cardiac microtubule hypothesis linking hypertrophy-related microtubule network densification to contractile dysfunction is valid.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Heart, Lung, and Blood Institute Program Project Grant HL-48788 and a Department of Veterans Affairs Merit Review Grant (to G. Cooper) and by National Cancer Institute Grant CA-085935 (to F. Cabral).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Cooper, Gazes Cardiac Research Inst., PO Box 250773, Medical Univ. of South Carolina, 114 Doughty St., Charleston, SC 29403 (e-mail: cooperge{at}musc.edu)

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


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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