INTRODUCTION

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CARDIAC MUSCLES rapidly increase their mass in response to imposed mechanical load. Accelerated rates of total protein synthesis in excess of the rate of protein degradation result in hypertrophic growth of the myocardium (18, 20). Previous in vivo studies showed that the increased hemodynamic load is a primary regulator of cardiac mass (7). Other in vitro studies suggested that the stretching of cultured cardiac muscle cells (myocytes) increases the rate of protein synthesis (14, 31). Together with acceleration in the synthesis of general cellular protein, the changes in gene programming characterized by the induction of a specific set of proteins, such as -myosin heavy chain (MHC), -skeletal actin, and atrial natriuretic peptide, have been recognized as the main features of the myocardial response to mechanical load (41). Therefore, these features are thought to represent the qualitative and quantitative alterations in the myocardium under mechanical overload and may be a hallmark of the hypertrophic response that is observed as early as 48-72 h of pressure overload and normally precedes protein accumulation (16, 19). However, the mechanisms responsible for these molecular alterations remain obscure.

Along with this rapid and transient gene programming, an increase in the microtubule density streaming away from the nuclei towards cytoplasm is observed in actively hypertrophying and hypertrophied cardiac myocytes (11, 27, 34). Microtubules have been demonstrated to be associated with membranous organelles such as ribosomes and Golgi apparatus (3, 11, 28), which suggests that they might play an important role in the protein synthesis in adult cardiac myocytes (33). In addition to these structural features of microtubules, previous studies in vitro have suggested that microtubule cytoskeleton is involved in cellular functions such as protein synthesis, including the transcription of mRNAs and their transport within the cytoplasm (5, 17, 42) and positioning of intracellular organelles (2). Moreover, microtubules could respond to the variations in environmental conditions by their nature of dynamic arrangement and may act as mechanotransducers in muscle cells (22). These lines of evidence led us to hypothesize that microtubules are involved in the initiation of pressure-overload cardiac hypertrophy and that the disruption of microtubule organization could inhibit early molecular events such as the reinduction of fetal contractile protein genes as well as the increase in protein synthesis capacity. However, the exact relation between these molecular changes and microtubule integrity has not been clearly defined. In fact, Sadoshima et al. (32) showed that cytoskeletal constituents were not associated with the hypertrophic responses in a stretch model of cultured neonatal myocytes. With the use of functional muscle, the extent and significance to which microtubule integrity is essential to this process have not yet been defined.

In this study, we examined whether the inhibition of microtubule function lessened the expression of the immediate early gene c-fos and fetal contractile protein genes including -MHC and -skeletal actin as well as the increase of total cardiac RNA content, using an adult rat model of abdominal aortic constriction. To inhibit microtubule function in vivo, we treated the animals with colchicine, which binds to tubulin and dissociates the microtubule network into tubulin subunits (4). Our previous studies demonstrated that in vitro exposure of isolated cardiac myocytes to colchicine induced loss of microtubules from cytoplasm in ~1 h (12, 40). In this study, we confirmed the specificity and efficacy of in vivo colchicine treatment in myocyte microtubules by using both Western blot analysis and immunofluorescent micrographs. To exclude the possibility that the effects observed by colchicine were unrelated to microtubules, we further performed the same experiments using animals in which microtubule function was inhibited by different means. For this purpose, we chose taxol and deuterium oxide (²H₂O), both of which produce nonfunctioning microtubules when administered to rats in vivo (38). Taxol lowers the critical concentration of -tubulin heterodimers required to form microtubules, leading to the hyperpolymerization of existing microtubules as well as the formation of abnormally aberrant, nonfunctional microtubules (35). Similarly, ²H₂O reduces the critical concentration of -tubulin heterodimers required to form microtubules by rapidly and reversibly strengthening hydrophobic interactions of tubulin molecules (13). An advantage of the use of ²H₂O is that it allows us to exclude the possibility of nonspecific toxic effects of colchicine or taxol on cellular physiological function. Importantly, even though the mechanisms of action on microtubules are different among colchicine, taxol, and ²H₂O, they inhibit microtubular function.

	METHODS

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In vivo left ventricular pressure-overload model. Male Wistar rats were obtained from an established colony of the Animal Research Institution of Kyushu University School of Medicine. The abdominal aortic constriction model of left ventricular (LV) pressure overload was created in the rat according to methods described previously (12), with some modifications. Briefly, under anesthesia with an intraperitoneal administration of pentobarbital sodium (50 mg/kg), a 5-mm segment of the abdominal aorta was exposed through a midline abdominal incision. The aorta was tied firmly over a rigid steel wire (0.65-mm external diameter) placed against the free wall. The wire was then removed, leaving a constriction equal to the outer diameter of the wire. These animals were identified as aortic-constricted (AoC) rats. We also performed sham operations in control rats, using procedures identical to those for AoC rats except for hemodynamic interventions (sham-operated rats). The abdomen was closed surgically, and the animals were allowed to recover for 3 days.

Inhibition of microtubule function. To depolymerize cardiac microtubules and inhibit their function, colchicine (1 mg/kg body wt) was administered to the rats via intraperitoneal injection on the next day after surgery. Control animals received saline only. The efficacy and specificity of colchicine treatment for myocyte microtubules were defined by both Western blot analysis and immunofluorescent micrograph analysis of tubulin as described previously (12, 40). Briefly, a 250-mg sample of LV myocardial tissue was homogenized in microtubule stabilizing buffer and was centrifuged at 100,000 g at 25°C for 10 min. The supernatants were saved as the free tubulin fractions, and the pellets were resuspended at 0°C in microtubule depolymerizing buffer. After 1 h at 0°C, they were centrifuged at 100,000 g at 4°C for 15 min, and the supernatants were saved as polymerized tubulin fractions. For the subsequent 8- to 16%-gradient SDS-polyacrylamide gel electrophoresis, samples were transferred and probed with a monoclonal antibody to -tubulin (Amersham International). Quantification of Western blots was obtained by the integrated optical density increase over background density in a rectangular region of interest. Film exposures were kept short enough to avoid saturating the emulsion. Within a given experiment, the mean signal value in the control group was defined as 1, and the signal density for colchicine-treated rats was calculated as a ratio with the mean value obtained from the vehicle-treated control rats within the same blot. For immunofluorescent micrographic analysis of tubulin, the frozen sections (6-µm thick, 20°C) were treated with phosphate buffer containing 0.1% Triton X-100 for 30 min for permeabilization. After the sections were blocked with 10% normal goat serum, they were incubated with the monoclonal antibody to tubulin overnight at 4°C. After several washes in buffer, they were incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG for 1 h at room temperature.

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Experimental protocol. At the time of the final study (i.e., 3 days after the surgery), the rats were anesthetized as previously described and the left common carotid artery was cannulated using a stiff, fluid-filled catheter attached to a strain gauge for the measurement of arterial pressure. Only animals with systolic arterial pressure >160 mmHg were used as AoC rats in this study. After hemodynamic measurements, the hearts were quickly removed. The atria and great vessels were trimmed away, and the right ventricle (free wall) and the LV (free wall + septum) were separated and weighed. Myocardial tissue specimens, obtained from LV free wall, were quickly frozen and stored at 80°C for subsequent molecular and biochemical analysis. Experiments were coded so that surgery and data analysis were performed without knowledge of treatment group. All procedures and animal care were reviewed and approved by the Committee on Ethics of Animal Experiments, Faculty of Medicine, Kyushu University, and conducted according to the Guidelines for Animal Experiments of the Faculty of Medicine, Kyushu University, and Law No. 150 and Notification No. 6 of the Japanese Government.

Northern blot analysis. To determine the expression of molecular markers of the early hypertrophic gene program by Northern blot analysis, frozen LV myocardial tissue (300 mg) was homogenized with a homogenizer in a solution containing 4 M guanidinium thiocyanate and total RNA was isolated according to the methods of Chomczynski and Sacchi (6) with some modifications (25). RNA was quantified by absorbance at 260 nm, and the integrity was determined by examining the 28S and 18S rRNA bands in ethidium bromide-stained agarose gels visualized under ultraviolet (UV) light. Poly(A)⁺-containing RNA was enriched via oligo(dT)₃₀-Latex [Oligotex-dt30(Super), Japan Synthetic Rubber; Ref. 15]. Poly(A)⁺-containing mRNA (2-3 µg) was denatured (65°C, 15 min) and size fractionated by electrophoresis on 1% (wt/vol) agarose gels under denaturing conditions. RNA was transferred to nylon membranes (Hybond N⁺; Amersham International) and immobilized by UV irradiation. Hybridization with cDNA probes [2 × 10⁶ counts per minute (cpm)/ml] was performed overnight at 42°C in buffer containing 50% formamide, 5× SSPE (0.9 M NaCl, 0.05 M sodium phosphate), 5× Denhardt's solution (0.2% polyvinylpyrrolidone, 0.2% bovine serum albumin, and 0.2% Ficoll), 0.5% SDS, and 50 µg/ml denatured salmon sperm DNA. Probes for rat cDNA c-fos, -MHC, -skeletal actin, and chicken glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were radioactively labeled using a random-prime DNA labeling kit (Boehringer Mannheim). [³²P]dCTP (DuPont-NEN) was included in the reaction mixture to obtain a specific activity of 5-20 × 10⁸ cpm/µg DNA. Blots were washed in 0.2× standard saline citrate (SSC)-0.1% SDS (55°C, 90 min) and 0.2× SSC-0.1% SDS (42°C, 1 h). All membranes were exposed at 80°C for varying time periods to X-Omat X-ray film (Eastman Kodak) using intensifier screens. Quantification of Northern blots was obtained by the integrated optical density increase over background density in a rectangular region of interest. Data are expressed as the densitometric intensity of the hybridization signals for mRNA levels of interest relative to the consistently expressed GAPDH mRNA to avoid variations in sample loading and blotting efficiency of RNA.

Quantification of total cardiac RNA, DNA and protein. Total RNA content was quantitated using frozen LV myocardial tissue according to the Munro-Fleck assay (23) with minor modifications (30). Myocardial tissue was homogenized in 1× SSC, and the homogenate was precipitated with 4 M perchloric acid (PCA) and centrifuged at 15,000 g for 15 min. The pellet was washed three times with 0.5 M PCA and then hydrolyzed in 0.3 M sodium hydroxide at 37°C. The solution was reprecipitated with the addition of 4 M PCA to a final concentration of 1 M and was centrifuged at 15,000 g for 15 min. The absorbance of the supernatant was measured at 260 nm to obtain total RNA concentration. The remaining pellet was washed three times in 0.2 M PCA and dissolved in a solution (pH 7.0) containing 1× SSC and 0.25% SDS at 37°C. This solution was then used to determine DNA and protein per gram of tissue. The DNA concentration was determined by a fluorometric assay using Hoechst dye 33258 (Sigma Chemical) at an excitation wavelength of 360 nm and an emission wavelength of 450 nm and compared with a standard curve of calf thymus DNA. The protein content was determined by bicinchoninic acid assay (Pierce) using bovine serum albumin as the standards.

Statistical analysis. Data are expressed as means ± SE. Paired data were compared by Student's t-test. Body weight, blood pressure, heart weight, RNA, DNA, protein per gram of tissue, and the RNA-to-DNA ratio were compared by using one-way ANOVA followed by Bonferroni's test for multiple comparisons. Differences were considered statistically significant at P < 0.05.

	RESULTS

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Characteristics of experimental groups. As shown in Table 1, body weights before surgery were comparable among the four groups of rats. Surgical procedure decreased body weight only in AoC rats. In vivo administration of colchicine for 3 days did not produce a further loss of body weight [30 ± 6 for AoC + vehicle vs. 23 ± 9 g for AoC + colchicine; P = not significant (NS)]. Systolic arterial blood pressure 3 days after aortic constriction was significantly elevated in both AoC + vehicle and AoC + colchicine rats compared with that in sham-operated rats, and, importantly, there were no significant differences in systolic arterial pressure between these two groups of rats (185 ± 8 vs. 177 ± 4 mmHg; P = NS). Similarly, in vivo treatment of rats with taxol or ²H₂O produced no significant loss of body weight and did not affect systolic arterial pressure in AoC rats (data not shown).

Effects of colchicine on microtubules. Fig. 1A represents Western blot analysis showing the time-dependent changes of both free and polymerized tubulin fractions after in vivo administration of colchicine. At baseline, most of the tubulin was in the free fraction in sham-operated control rats. After 2 h, the polymerized fraction of tubulin was substantially diminished and microtubules remained depolymerized until 48 h after injection to sham-operated rats (Fig. 1, B-D). Polymerization of tubulin into microtubules was inhibited by in vivo administration of colchicine also in AoC rats (Fig. 1A). Immunofluorescence micrographs demonstrated that microtubules, which ran along and encircled the myofibrillar bundles as the longitudinal and transverse filaments in normal myocytes (arrowheads in Fig. 2A), were almost absent from the cytoplasm after the treatment with colchicine (Fig. 2B). We also confirmed no apparent morphological changes in other cytoskeletal elements including actin or desmin within cardiac myocytes from colchicine-treated rats (data not shown).

View larger version (59K): [in this window] [in a new window]   Fig. 1.   Western blot analysis (A) showing time-dependent changes of myocardial free (F) and polymerized (P) tubulins before (Control) and after intraperitoneal administration of colchicine (1 mg/kg body wt) to sham-operated control and aortic-constricted (AoC) rats. MM, molecular mass. Summary data are shown for alterations of myocardial total (B), free (C), and polymerized (D) tubulin at control (n = 8) and 24 (n = 4) and 48 (n = 4) h after colchicine administration to sham-operated control rats. Polymerization of tubulin into microtubules was inhibited by in vivo administration of colchicine in both groups of rats. NS, not significant. ** P < 0.01 vs. control.

View larger version (130K): [in this window] [in a new window]   Fig. 2.   Immunofluorescence micrographs of microtubules in myocytes. For control (A), a network pattern of microtubules is apparent within cell (arrowheads). In rats treated with colchicine (B), microtubules are absent from myocyte. Scale bar, 10 µm.

Effects of microtubule inhibition on hypertrophic gene program. Northern blot analysis demonstrated that c-fos was highly expressed after pressure overloading (9.7-fold increase), which was significantly attenuated by in vivo microtubule inhibition (Fig. 3). Similarly, the -MHC mRNA level was significantly upregulated in AoC rats, which was significantly inhibited by colchicine (Fig. 4). An inactive stereoisomer, lumicolchicine, had no significant effects on -MHC mRNA levels (data not shown). -Skeletal actin was also reexpressed after AoC. In vivo treatment with colchicine tended to suppress the increased expression of -skeletal actin in AoC rats (4.5 ± 1.1-fold increase in AoC vs. 3.1 ± 0.4-fold increase in AoC + colchicine), but the changes were not statistically significant.

View larger version (39K): [in this window] [in a new window]   Fig. 3.   Northern blot analysis (A) of c-fos mRNA (top) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (bottom) in sham-operated control rats (lane 1), AoC rats (lane 2), and AoC rats treated with taxol (lane 3). Summarized data of Northern blot analysis are shown in B for sham-operated control rats (n = 4), AoC rats (n = 4), and AoC rats treated with taxol (n = 6). Increased c-fos mRNA level was significantly inhibited by microtubule inhibition. ** P < 0.01 vs. sham-operated rats. P < 0.01 vs. AoC rats.

View larger version (40K): [in this window] [in a new window]   Fig. 4.   Northern blot analysis (A) of -myosin heavy chain (MHC) mRNA (top) and GAPDH mRNA (bottom) in sham-operated control rats (lane 1), AoC rats (lane 2), and AoC rats treated with colchicine (lanes 3 and 4). Summarized data of Northern blot analysis are shown in B for sham-operated control rats (n = 4), AoC rats (n = 4), and AoC rats treated with colchicine (n = 4). Increased -MHC mRNA was significantly inhibited by colchicine treatment. ** P < 0.01 vs. sham-operated rats. P < 0.01 vs. AoC rats.

Effects of microtubule inhibition on total cardiac RNA per unit tissue weight. After 3 days of pressure overloading, total RNA per unit weight of tissue was significantly increased in AoC rats when compared with the values for sham-operated rats treated with vehicle (Table 1). The RNA-to-DNA ratio was also significantly increased in AoC + vehicle rats compared with sham + vehicle rats. Thus LV pressure overload resulted in the initiation of hypertrophic growth of the myocardium. However, protein and DNA contents were unchanged in AoC + vehicle rats. Moreover, LV weight and the LV weight-to-right ventricle weight ratio were not yet increased after 3 days of pressure overloading. Colchicine treatment (1 mg/kg body wt) significantly inhibited the increase of total cardiac RNA content by 64% (P < 0.01) and the RNA-to-DNA ratio by 85% (P < 0.01) in the AoC + colchicine rats. Furthermore, the treatment of AoC rats with a smaller dose of colchicine (0.7 mg/kg; n = 4) still exerted the same inhibitory effects on the increase of total cardiac RNA content (50% inhibition; P < 0.05) and RNA-to-DNA ratio (62% inhibition; P < 0.05). Colchicine did not alter total protein or DNA content. In contrast, colchicine did not affect any of the measures of the myocardium in the sham-operated rats. Lumicolchicine had no significant effects on total cardiac RNA per unit tissue weight (data not shown).

View larger version (27K): [in this window] [in a new window]   Fig. 5.   RNA-to-DNA ratios in sham-operated and AoC rats treated with taxol (A) and 2H2O (B). RNA-to-DNA ratio was significantly elevated in AoC + vehicle rats, which was completely inhibited by taxol (AoC + taxol) or 2H2O (2H2O + AoC) treatment. ** P < .01.

	DISCUSSION

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The findings of this study are summarized as follows. First, pressure overloading to the myocardium for 3 days significantly increased c-fos and -MHC mRNA levels, total cardiac RNA content, and RNA-to-DNA ratio but did not affect cardiac DNA or protein per gram of tissue. Second, these pressure overload-induced molecular changes were abolished by the in vivo inhibition of microtubule function using colchicine, taxol, or ²H₂O. Third, the inhibition of microtubules did not affect these molecular indexes in control hearts. These results support the hypothesis that intact microtubules are involved in the early hypertrophic responses of the myocardium during pressure overload.

Early hypertrophic responses in myocardium during pressure overload. One of the most important properties of the myocardium is adaptation to an increased hemodynamic load. When the mechanical load is imposed on the myocardium, two forms of adaptive responses occur: first, the rapid induction of immediate early genes such as c-fos and c-myc and the reexpression of fetal isoforms of contractile proteins such as -MHC and -skeletal actin in small mammals; and second, an overall increase in the rate of protein synthesis and the increase of cardiac mass, i.e., cardiac hypertrophy (8).

Role of microtubules in pressure overload-induced hypertrophic responses. The findings in this study indicate that the selective inhibition of microtubule cytoskeleton resulted in suppression of the upregulation of immediate early genes and reexpression of fetal contractile protein genes as well as increase of protein synthesis capacity induced by the acute pressure overloading to the myocardium. Further support for the concept that normally functioning polymerized microtubules are the critical and essential elements involved in cardiac hypertrophy comes from the finding that both taxol and ²H₂O produced the same inhibitory effects on these molecular events as colchicine, even though the mechanism to inhibit microtubule function is different.

Limitations. Several potential limitations should be acknowledged in this study. First, our results relate to the early molecular changes in response to pressure overload rather than to the alterations in cardiac protein per se that occur over the longer term. It is of extreme importance to examine whether the inhibition of microtubule function can eventually attenuate the increase of myocardial mass. In our preliminary experiments, even a lower dose of colchicine (0.3 mg/kg body wt), which was about one-third of the dose we used in this short-term study, exerted systemic toxic effects in the animals when administered for 2 wk. Therefore, it has not yet been determined whether the long-term inhibition of microtubule function can attenuate the development of cardiac hypertrophy. However, we consider that the microtubule inhibition could attenuate the subsequent increase in cardiac mass when the inhibitors are administered to the animals for a longer period of time, because it has been shown that cardiac hypertrophy nevertheless developed subsequently after aortic constriction when serial changes in RNA content and LV weight were followed in rats (36). Second, we measured total cardiac RNA per unit tissue weight instead of the actual rate of protein synthesis in this study. As we discussed above, an increase in total cardiac RNA is a key regulatory element of protein synthesis and thus could be a conventional index of capacity for protein synthesis. Furthermore, we used total RNA in place of rRNA, which is considered to be valid because previous studies showed that the proportions of different RNA species were not altered in hypertrophied hearts (9) and almost all of total RNA (i.e., 90%) is composed of rRNA (37). Third, we could not exclude the possibility that other cytoskeletal elements, such as actin or desmin, might also contribute to the initiation and progression of cardiac hypertrophy. We carefully confirmed the specificity of pharmacological agents for microtubules by using three different agents, all of which can inhibit microtubule function by different mechanisms, and by using lumicolchicine, an inactive stereoisomer of colchicine. Furthermore, no significant alterations of other cytoskeletal proteins such as actin and desmin filaments were observed in myocytes under pressure overload. Therefore, microtubules should be a major cytoskeletal component that would be involved in the pathogenesis of hypertrophy (27).

Clinical implications. Even though precise reasons for the disparity between in vitro studies by Sadoshima et al. (31) and our in vivo studies in the role of microtubule cytoskeleton in cardiac hypertrophy are not clear, microtubules should play an important role in the initiation of hypertrophy in vivo. In addition to the transcription of fetal contractile protein genes and rRNA, microtubules have been suggested to play an important role in myofibrillogenesis by working as a template for the organization of actin filaments and by forming a lattice to permit the aggregation of myosin filaments and their assembly (26). Thus microtubules also could be involved at posttranslational levels in cardiac hypertrophic responses. Furthermore, strategies to interfere with microtubule function such as vinca alkaloids and taxol have been used with significant success in the treatment of cancer patients (29). Therefore, the present study would offer the possibility of pharmacological as well as molecular approaches to modulate microtubules in the treatment of patients with pathological cardiac hypertrophy.