Phenotypic consequences of β₁-tubulin expression and MAP4 decoration of microtubules in adult cardiocytes

Abstract

.-In pressure-overload cardiac hypertrophy, microtubule network densification is one cause of contractile dysfunction. Cardiac transcriptional upregulation of ␤ 1-tubulin rather than the constitutive ␤ 4-tubulin and of microtubuleassociated protein (MAP)4 accompanies hypertrophy, with extensive microtubule decoration by MAP4. Because MAP4 stabilizes microtubules, and because the isoform-variable carboxy terminus of ␤-tubulin binds to MAP4, we wished to determine whether one or both of these proteins has etiologic significance for cardiac microtubule network densification. Recombinant adenoviruses encoding ␤ 1-tubulin, ␤4-tubulin, and MAP4 were used to infect isolated cardiocytes. Overexpressed MAP4 caused a shift of tubulin dimers to the polymerized fraction and formation of a dense, stable microtubule network. Overexpressed ␤ 1-or ␤4-tubulin had neither any independent effect on these variables nor any effect additive to that of simultaneously overexpressed MAP4. Results from transgenic mice with cardiac overexpression of ␤ 1-tubulin or MAP4 were confirmatory, but unlike the effects of brief adenovirus-mediated MAP4 overexpression in isolated cardiocytes, MAP4 transgenic hearts showed a marked increase in total ␣-and ␤-tubulin. Thus MAP4 overexpression caused increased tubulin expression, formation of stable microtubules, and altered microtubule network properties, such that MAP4 upregulation may be one cause for the dense, stable microtubule network characteristic of pressure-overloaded, hypertrophied cardiocytes. myocardium; hypertrophy; adenovirus ONE REASON for the contractile dysfunction characteristic of severe pressure-overload cardiac hypertrophy is increased density of the cellular microtubule network (41,42). This imposes a viscous load on active myofilaments, such that structural damping via intracellular frictional dissipation impedes sarcomere shortening (38). This is accompanied by persistent increases in ␣-and ␤-tubulin on both the mRNA and protein levels (37). Thus increased synthesis of ␣␤-tubulin heterodimers is one cause for the greater cardiac microtubule network density. However, given that the ␣␤-tubulin heterodimer-microtubule system is in dynamic equilibrium, enhanced microtubule stability is a potentially synergistic second mechanism for microtubule network densification. Indeed, we (34) found that the microtubules of pressure-hypertrophied cardiocytes demonstrate markedly enhanced stability, in which microtubule stabilization begins very shortly after cardiac pressure overloading and persists indefinitely thereafter.We then turned to two possible bases for this microtubule stabilization. The first of these was the role of microtubule-associated proteins (MAPs), which are known to regulate microtubule stability (11,23,26,28,44). Here, we focused on MAP4, the dominant structural MAP of the heart (29, 34), and found on both the mRNA and protein levels striking MAP4 upregulation in hypertrophied cardiocytes (34). This was associated with extensive microtubule decoration with MAP4, which wa...

Designing Heart Performance by Gene Transfer

“…Adenoviral-mediated overexpression of these proteins in normal adult cardiac myocytes revealed that MAP4 is sufficient for these myocytes to develop microtubular structure similar to that observed in myocytes isolated from pressure-overloaded hearts. In contrast, adenoviralmediated expression of either ␤ 4 -or ␤ 1 -tubulin had no significant effect on microtubule assembly (864). The increased density of microtubules present within pressureoverloaded myocytes resulted in a significant increase in the viscous load placed on the myofilaments.…”

Cytoskeletal role in protection of the failing heart by β-adrenergic blockade

“…Myocardial protein fractions were prepared as before (50). For the total tubulin fraction, the myocardium was homogenized in 1% SDS buffer containing (in mM) 10 Tris·HCl (pH 7.4), 0.5 DTT, and 1 Na 3VO4, boiled for 5 min, and centrifuged at 16,000 g, 4°C for 10 min; this supernatant was saved as the total protein fraction.…”

Cytoskeletal networks and the regulation of cardiac contractility: microtubules, hypertrophy, and cardiac dysfunction

“…The most definitive solution, however, would be to close the loop and address the microtubule hypothesis directly in a context apart from hemodynamic alterations in vivo or microtubule alterations in vitro. On the basis of some of our other work defining the basic causes of microtubule network densification in cardiac hypertrophy (21,23,28), I very recently tested, via a collaboration with F. Cabral and D. R. Menick, the effects of ␤ 1 -tubulin mutants that had been selected for their effects on microtubule stability and then expressed in the hearts of transgenic mice (4). We found that when intrinsic microtubule stability was increased as an isolated variable, the contractile defects characteristic of pressure-overloaded hypertrophied myocardium were reproduced.…”

“…Instead, active transport is required for this purpose, and microtubules supply this function: the dynein family of motor proteins moves these cargoes along microtubules toward the cell center, and the kinesin family of motor proteins moves these cargoes along microtubules from the nuclei toward the cell periphery. In cardiac hypertrophy, the microtubules not only proliferate but also become heavily decorated by microtubule-associated protein-4, the predominant cardiac microtubule-associated protein (23,28). One consequence of this is that the normal microtubule transport function is sterically inhibited by the presence of microtubule-associated protein-4 on these transport tracks, and as one example that may help explain ␤-adrenergic receptor downregulation in cardiac hypertrophy and failure, we have shown that activated G proteincoupled receptors fail to be recycled properly to the cell membrane (2,3).…”