Microtubule stabilization in pressure overload cardiac hypertrophy

Abstract

Increased microtubule density, for which microtubule stabilization is one potential mechanism, causes contractile dysfunction in cardiac hypertrophy. After microtubule assembly, alpha-tubulin undergoes two, likely sequential, time-dependent posttranslational changes: reversible carboxy-terminal detyrosination (Tyr-tubulin left and right arrow Glu-tubulin) and then irreversible deglutamination (Glu-tubulin --> Delta2-tubulin), such that Glu- and Delta2-tubulin are markers for long-lived, stable microtubules. Therefore, we generated antibodies for Tyr-, Glu-, and Delta2-tubulin and used them for staining of right and left ventricular cardiocytes from control cats and cats with right ventricular hypertrophy. Tyr- tubulin microtubule staining was equal in right and left ventricular cardiocytes of control cats, but Glu-tubulin and Delta2-tubulin staining were insignificant, i.e., the microtubules were labile. However, Glu- and Delta2-tubulin were conspicuous in microtubules of right ventricular cardiocytes from pressure overloaded cats, i.e., the microtubules were stable. This finding was confirmed in terms of increased microtubule drug and cold stability in the hypertrophied cells. In further studies, we found an increase in a microtubule binding protein, microtubule-associated protein 4, on both mRNA and protein levels in pressure-hypertrophied myocardium. Thus, microtubule stabilization, likely facilitated by binding of a microtubule-associated protein, may be a mechanism for the increased microtubule density characteristic of pressure overload cardiac hypertrophy.

Figure 1

Specificity of the antibodies to posttranslationally modified isoforms of α-tubulin. Immunoblot analysis. A 100-ng sample of a single α-tubulin isoform was loaded on each lane before 12.5% SDS-PAGE, where lane 1, Tyr-tubulin; lane 2, Glu-tubulin; and lane 3, Δ2-tubulin. The blot in A was then probed with anti– Tyr-tubulin antibody, the blot in B was probed with anti–Glu-tubulin antibody, and the blot in C was probed with anti–Δ2-tubulin antibody.

Figure 2

Taxol-induced microtubule stabilization in normal cardiocytes. Immunofluorescence confocal micrographs of posttranslationally modified α-tubulin isoforms in the microtubules of cardiocytes from a normal feline heart after exposure to 10⁻⁵ M taxol for 0 (A), 0.5 (B), 1.0 (C), and 2.0 h (D). As indicated, the antibodies employed were, from above down, anti–Tyr-tubulin antibody, anti–Glu-tubulin antibody, and anti–Δ2-tubulin antibody. Each micrograph is a single 0.7-μm confocal section taken at the level of the nuclei. Double-staining of cells exposed to taxol for 2 h both with the β-tubulin antibody and with either the Tyr-tubulin, Glu-tubulin, or Δ2-tubulin antibodies showed that the latter three antibodies decorated microtubules exclusively (data not shown). Bar, 25 μm.

Figure 3

Microtubule stability in pressure overload- hypertrophied myocardium: Immunofluorescence confocal micrographs of posttranslationally modified α-tubulin isoforms in RV and LV cardiocyte microtubules from a feline heart 2 wk after RV pressure overloading. The anti–Tyr-tubulin antibody was used in A, the anti–Glu-tubulin antibody was used in B, and the anti–Δ2-tubulin antibody was used in C. Each micrograph is a single 0.7-μm confocal section taken at the level of the nuclei. Bar, 25 μm.

Figure 4

Densitometric analysis of microtubule stability in pressure overload-hypertrophied myocardium. β-tubulin and posttranslationally modified α-tubulin isoforms in microtubules of RVs and LVs from the same feline hearts. Control cats were compared with cats RV pressure overloaded 2 wk earlier via pulmonary artery banding. Four blots were prepared for each heart; the microtubule fractions from the RV and LV (20 μg protein/ lane) were loaded on two lanes, and four concentrations of the appropriate protein standard (7.5–60.0 ng/lane) were loaded on the remaining lanes before 12.5% SDS-PAGE; the blot was then probed with the corresponding antibody. For the protein standards, the linear relationship between optical density and protein loaded had a correlation coefficient ⩾0.98 in each instance. Of the total α-tubulin, Tyr-tubulin comprised 92.6 ± 2.1%, Glu-tubulin was 4.6 ± 1.4%, and Δ2-tubulin was 2.8 ± 0.7%. Statistical comparisons were by one-way ANOVA followed by Scheffé's S procedure, where n = number of cats in each group.*P < 0.01 for difference within a category from the control group value by Scheffé's S procedure.  ^‡ P < 0.01 for difference from the β-tubulin and Tyr-tubulin values within the hypertrophy group by Scheffé's S procedure.

Figure 5

Microtubule stability in pressure overload-hypertrophied myocardium: Immunofluorescence confocal micrographs, using the DM1B anti–β-tubulin antibody, of RV and LV cardiocyte microtubules during exposure to nocodazole (A–H) or low temperature (I–L). Cardiocytes from a feline heart 2 wk after RV pressure overloading were exposed to 0.3 μM nocodazole for 0 min (A and B), 30 min (C and D), 60 min (E and F), or 90 min (G and H) or they were exposed to a temperature of 0°C for 0 (I and J) or 60 min (K and L) before fixation. The cardiocytes on the left (A, C, E, G, I, and K) are from the RV, and the cardiocytes on the right (B, D, F, H, J, and L) are from the LV. Of interest, after 90 min of nocodazole exposure the majority of the residual microtubules of RV but not LV cardiocytes was found by double-staining to be decorated by both the β-tubulin and either the Glu-tubulin or Δ2-tubulin antibodies (data not shown). Each micrograph is a single 0.7-μm confocal section taken at the level of the nuclei. Bar, 25 μm.

Figure 6

Microtubule stability in volume overload-hypertrophied RV myocardium. Immunofluorescence confocal micrographs of posttranslationally modified α-tubulin isoforms in microtubules of RV cardiocytes from a feline heart 2 wk after RV volume overloading. Anti–Tyr-tubulin antibody was used in A, anti–Glu-tubulin antibody was used in B, and anti–Δ2-tubulin antibody was used in C. Each micrograph is a single 0.7-μm confocal section taken at the level of the nuclei. Bar, 25 μm.

Figure 7

MAP 4 protein in pressure overload-hypertrophied RV and control LV myocardium. Immunoblot and immunofluorescence confocal micrographic analysis of MAP 4. The immunoblot in A was prepared from a normal feline heart, and the immunoblot in B was prepared from a feline heart 2 wk after RV pressure overloading. For both panels, lanes 1 and 2 were prepared as the free tubulin fraction, lanes 3 and 4 were prepared as the polymerized tubulin fraction, and lanes 5 and 6 were prepared as the total tubulin fraction. Lanes 1, 3, and 5 of the immunoblots are from the RV, and lanes 2, 4, and 6 of the immunoblots are from the LV. Each blot was probed with anti–MAP 4 antibody. C is a micrograph of a cardiocyte from a normal feline heart, and D is a micrograph of a RV cardiocyte from a feline heart 2 wk after RV pressure overloading. These cardiocytes were double-stained for β-tubulin (red) and MAP 4 (green), where the β-tubulin and MAP 4 primary antibodies were followed by species-specific fluorochrome- conjugated secondary antibodies. Each micrograph is a single 0.7-μm confocal section taken at the level of the nuclei.

Figure 8

Upregulation of β-tubulin versus MAP 4 protein in pressure overload-hypertrophied myocardium. Immunoblot analysis. The samples for each lane were prepared as the total tubulin fraction, and equal protein loading was employed for the RV and LV samples. For β-tubulin, the DM1B antibody used here recognizes all β-tubulin isoforms. While both β-tubulin and MAP 4 were greater in the hypertrophied RV than in the same-animal normally loaded LV, densitometric analysis of immunoblots from 4 such cats RV pressure overloaded 2 wk earlier via pulmonary artery banding showed that the RV/LV ratio of MAP 4 was 3.0 ± 0.5-fold greater than the RV/LV ratio of β-tubulin.

Figure 9

Time course of MAP 4 mRNA and protein upregulation in control LV and pressure overload-hypertrophied RV myocardium. Northern blot and immunoblot analysis. (A and B) At the indicated times after RV pressure overloading, poly(A)⁺ RNA was prepared from the RV and LV of each heart, and 1 μg was loaded on each lane. The blot in A was probed for MAP 4, and the blot in B, which was used to verify equal RV versus LV loading at each time point, was probed for constitutively expressed glyceraldehyde-3-phosphate dehydrogenase. (C and D) At the indicated times after RV pressure overloading, the total tubulin protein fraction was prepared from the RV and LV of each heart, and 40 μg was loaded on each lane before probing with anti–MAP 4 antibody. Note that the time points chosen for the mRNA and protein blots do not coincide.