Site-specific Microtubule-associated Protein 4 Dephosphorylation Causes Microtubule Network Densification in Pressure Overload Cardiac Hypertrophy

Abstract

In severe pressure overload-induced cardiac hypertrophy, a dense, stabilized microtubule network forms that interferes with cardiocyte contraction and microtubule-based transport. This is associated with persistent transcriptional up-regulation of cardiac ␣-and ␤-tubulin and microtubule-stabilizing microtubule-associated protein 4 (MAP4). There is also extensive microtubule decoration by MAP4, suggesting greater MAP4 affinity for microtubules. Because the major determinant of this affinity is site-specific MAP4 dephosphorylation, we characterized this in hypertrophied myocardium and then assessed the functional significance of each dephosphorylation site found by mimicking it in normal cardiocytes. We first isolated MAP4 from normal and pressure overload-hypertrophied feline myocardium; volume-overloaded myocardium, which has an equal degree and duration of hypertrophy but normal functional and cytoskeletal properties, served as a control for any nonspecific growth-related effects. After cloning cDNA-encoding feline MAP4 and obtaining its deduced amino acid sequence, we characterized by mass spectrometry any site-specific MAP4 dephosphorylation. Solely in pressure overload-hypertrophied myocardium, we identified striking MAP4 dephosphorylation at Ser-472 in the MAP4 N-terminal projection domain and at Ser-924 and Ser-1056 in the assembly-promoting region of the C-terminal microtubule-binding domain. Site-directed mutagenesis of MAP4 cDNA was then used to switch each serine to non-phosphorylatable alanine. Wild-type and mutated cDNAs were used to construct adenoviruses; microtubule network density, stability, and MAP4 decoration were assessed in normal cardiocytes following an equivalent level of MAP4 expression. The Ser-924 3 Ala MAP4 mutant produced a microtubule phenotype indistinguishable from that seen in pressure overload hypertrophy, such that Ser-924 MAP4 dephosphorylation during pressure overload hypertrophy may be central to this cytoskeletal abnormality.Although many important alterations have been described in the properties of hypertrophied myocardium, the mechanisms responsible for contractile dysfunction and many other maladaptive changes of cardiac muscle cells, or cardiocytes, have yet to be fully defined. Although most research in this area has focused on structural and regulatory changes within the myofilament, it has also been found that changes in the microtubule component of the extra-myofilament cytoskeleton may lead both to contractile dysfunction by increasing the internal resistance to sarcomere motion (1, 2) and to disordered cellular homeostasis by impeding cytoskeleton-based intracellular transport (3-6).Our major original finding was that, in severe pressure overload cardiac hypertrophy with increased ventricular wall stress, there is the early appearance and then the persistence of a dense microtubule network and associated contractile dysfunction (7,8). We have now found several synergistic bases for this dense microtubule network. First, during hypertrophy there is persistent tran...

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“…5 yet further downstream from the data shown in Fig. 4 at MAP4 Ser-924 and Ser-1056 dephosphorylation, since this appears to be the major determinant of MAP4-microtubule affinity and thus microtubule network stability and density (10). It is clear from the data in Fig.…”

“…Example immunoblot above and summary immunoblot data below from the RVs and LVs of control cats and of cats with 4 wk of PAB-induced RV hypertrophy are shown; the animals were either ␤-blocked with propranolol for the final 2 wk or they were not ␤-blocked. A: example immunoblot and summary data prepared using our anti-non-phospho-Ser-924 MAP4 antibody (10). B: example immunoblot and summary data prepared using our anti-non-phospho-Ser-1056 MAP4 antibody (10).…”

“…It was then found that this MAP4 dephosphorylation is, in turn, driven by persistent activation of type 1 and type 2 phosphatases, and especially that of protein phosphatase 2A (PP2A) in pathological hypertrophy (10). Furthermore, this phosphatase activation is apparently driven by ongoing activation of the upstream stress-related kinase p21-activated kinase-1, or Pak1.…”

“…A search for the etiology of this cytoskeletal alteration led to the discovery, again in pathological pressure overloading but not in physiological volume overloading, of site-specific dephosphorylation of MAP4 that drives its microtubule binding and stabilizing function (10); that is, dephosphorylation of the serine in the KXGS motif of the first of four basic pseudorepeats within the MAP4 microtubule binding domain was both found in pathological hypertrophy and phenocopied each of the major features of the pathological microtubule network when introduced genetically into normal cardiomyocytes. It was then found that this MAP4 dephosphorylation is, in turn, driven by persistent activation of type 1 and type 2 phosphatases, and especially that of protein phosphatase 2A (PP2A) in pathological hypertrophy (10).…”

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“…To test this, we generated a MAP4 mutant GFP-MAP4-S914A/S1046A, which prevents MARK family kinase phosphorylation of MAP4 and promotes microtubule assembly (Chinnakkannu et al, 2010). We overexpressed GFP-MAP4-S914A/S1046A in LKB1 addback cells (Fig.…”