AJP - Heart AJP: Cell Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 294: H1135-H1144, 2008. First published January 4, 2008; doi:10.1152/ajpheart.01275.2007
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Videos
Right arrow All Versions of this Article:
294/3/H1135    most recent
01275.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Scholz, D.
Right arrow Articles by Cooper, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scholz, D.
Right arrow Articles by Cooper, G., IV

Microtubule-dependent distribution of mRNA in adult cardiocytes

Dimitri Scholz, Catalin F. Baicu, William J. Tuxworth, Lin Xu, Harinath Kasiganesan, Donald R. Menick, and George Cooper, IV

Gazes Cardiac Research Institute, Cardiology Division, Medical University of South Carolina and Department of Veterans Affairs Medical Center, Charleston, South Carolina

Submitted 31 October 2007 ; accepted in final form 2 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Synthesis of myofibrillar proteins in the diffusion-restricted adult cardiocyte requires microtubule-based active transport of mRNAs as part of messenger ribonucleoprotein particles (mRNPs) to translation sites adjacent to nascent myofibrils. This is especially important for compensatory hypertrophy in response to hemodynamic overloading. The hypothesis tested here is that excessive microtubule decoration by microtubule-associated protein 4 (MAP4) after cardiac pressure overloading could disrupt mRNP transport and thus hypertrophic growth. MAP4-overexpressing and pressure-overload hypertrophied adult feline cardiocytes were infected with an adenovirus encoding zipcode-binding protein 1-enhanced yellow fluorescent protein fusion protein, which is incorporated into mRNPs, to allow imaging of these particles. Speed and distance of particle movement were measured via time-lapse microscopy. Microtubule depolymerization was used to study microtubule-based transport and distribution of mRNPs. Protein synthesis was assessed as radioautographic incorporation of [3H]phenylalanine. After microtubule depolymerization, mRNPs persist only perinuclearly and apparent mRNP production and protein synthesis decrease. Reestablishing microtubules restores mRNP production and transport as well as protein synthesis. MAP4 overdecoration of microtubules via adenovirus infection in vitro or following pressure overloading in vivo reduces the speed and average distance of mRNP movement. Thus cardiocyte microtubules are required for mRNP transport and structural protein synthesis, and MAP4 decoration of microtubules, whether directly imposed or accompanying pressure-overload hypertrophy, causes disruption of mRNP transport and protein synthesis. The dense, highly MAP4-decorated microtubule network seen in severe pressure-overload hypertrophy both may cause contractile dysfunction and, perhaps even more importantly, may prevent a fully compensatory growth response to hemodynamic overloading.

molecular biology; hypertrophy; heart failure

WHILE THERE IS NOW a large body of knowledge describing the cardiac hypertrophic process, much less is known about the basic mechanisms underlying the transition from compensated cardiac hypertrophy to decompensated failure. In broad qualitative terms, this transition must eventually occur when abnormalities of contractile and/or constitutive properties are progressive and thus cause systolic and/or diastolic heart failure, respectively. While multiple abnormalities are responsible for each of these two types of decompensation, a relatively recently described contributor to both systolic and diastolic dysfunction that has been of interest in this laboratory is the formation of a dense, stable, heavily microtubule-associated protein 4 (MAP4)-decorated microtubule network in the pressure-overload hypertrophied cardiocyte (10, 11).

A very different but frequently synergistic cause of the transition to heart failure is a quantitative defect in the hypertrophy process itself. That is, compensatory hypertrophy ordinarily proceeds in response to a hemodynamic overload until the load stimulus is abated via a renormalization of stress per unit mass of myocardium (8). But if the load either initially or eventually exceeds the inherent growth capacity of the terminally differentiated cardiocyte in terms of renormalizing stress (9), then heart failure must ensue even if the myocardium is qualitatively normal. Indeed, there are important clinical situations in which high ventricular wall stress and heart failure occur after hemodynamic overloading but well before the biological limits of cardiocyte hypertrophy are reached (21, 25). Examples studied in this laboratory both in patients and in animal models of human disease include mitral regurgitation (53) and aortic stenosis (40, 56).

The present study had its origin in our study of canine aortic stenosis (50). In that study of chronic progressive pressure overload of the left ventricle (LV), we found that mongrel dogs broke into two groups as the load was increased (Figs. 1 and 2 in Ref. 50): one group developed extensive hypertrophy while retaining normal LV wall stress and contractile function; the LVs of the other group initially hypertrophied at the same rate as those of the first group but then stopped growing and developed high LV wall stress and very depressed contractile function. The first group retained a normal cardiocyte microtubule network throughout, but the second group developed a dense microtubule network coincident with the arrest of the hypertrophic response to increasing load (50).


Figure 1
View larger version (136K):
[in this window]
[in a new window]

  		Fig. 1. Microtubule network structure in normal cardiocytes. Frozen sections of feline myocardium were labeled with a monoclonal anti-{alpha}-tubulin antibody (B-5-1-2) followed by anti-mouse antiserum coupled with Alexa Fluor 488 (green) to show the microtubules in A, C, and E, counterstained with actin-binding phalloidin coupled with Alexa Fluor 568 (red) in B and D to show their relationship to the myofibrils, or counterstained with a polyclonal anti-{gamma}-tubulin antibody (AB11321) followed by anti-rabbit antiserum coupled with Alexa Fluor 647 (red) in F to show the microtubule organizing centers (MTOCs) as overlapping areas of immunoreactivity (yellow). A–C and F are longitudinal and D and E are transverse optical sections obtained by laser confocal microscopy. Images were acquired with a Zeiss LSM510 confocal microscope using 488-nm, 543-nm, and 633-nm laser beams (objective x63/1.40). Scale bars, 10 µm.

 

Figure 2
View larger version (75K):
[in this window]
[in a new window]

  		Fig. 2. Microtubule dependence of messenger ribonucleoprotein particle (mRNP) distribution. Normal freshly isolated adult feline cardiocytes infected with adenovirus encoding fluorescent zipcode binding protein 1 (ZBP-1)-enhanced yellow fluorescent protein (EYFP) fusion protein (AdZBP-1-EYFP) 12 h earlier (A) were treated with 2 µM nocodazole for 18 h and then fixed at 0 (B), 1 (C), 2 (D), 3 (E), or 4 (F) h after nocodazole washout. The green fluorescence of the expressed ZBP-1-EYFP protein and the red fluorescence of the anti-{alpha}-tubulin antibody (B-5-1-2) allowed detection of mRNPs and microtubules, respectively. Images were acquired with a Zeiss LSM510 confocal microscope using 514-nm and 633-nm laser beams (objective x63/1.40). Scale bars, 10 µm.

 
The possibility that these two associated variables might actually be causally related only became apparent later, with the discovery that hypertrophic microtubule network densification was caused by upregulation not only of {alpha}- and β-tubulin (37, 49) but also of MAP4 (42), a protein that decorates and stabilizes microtubules (42, 51). This discovery assumed significance in the context of the concurrently emerging understanding of the central role of microtubules and their associated families of kinesin and dynein motor proteins in the transport and localization of macromolecules and organelles to appropriate subcellular locations (1, 17, 18). The transport of mRNA as a constituent of messenger ribonucleoprotein particles (mRNPs) is an especially important example of this process, which has been studied predominantly in neurons (24) but is probably a universal property of eukaryotic cells. Of special interest to us, this active transport mechanism is essential for protein synthesis in fully differentiated striated muscle cells such as adult cardiocytes (40), since the dense packing of their contractile and energetic organelles severely restricts the macromolecular diffusion seen in other cells (15).

It therefore seemed quite possible that microtubule-based mRNP transport to the sites of protein translation adjacent to nascent sarcomeres is essential for the generation of additional myofibrils and other structures during cardiac hypertrophy. In addition, newly emerging data were showing that the motion of motor proteins and their cargoes such as mRNPs along microtubules is saltatory rather than continuous and that MAP4 microtubule decoration such as we see in cardiac hypertrophy inhibits net vectorial cargo movement via a steric inhibition of motor-microtubule interactions rather than because of MAP4-induced microtubule stabilization (2, 46). We then began our own exploration of microtubule-based transport by looking at the effects of MAP4 microtubule decoration on G protein-coupled receptor trafficking, first with adenoviral or transgenic MAP4 overexpression in neuroblastoma cells (3) and isolated cardiocytes (4) and then in hypertrophied cardiocytes (4). We found that in each case receptor transport was inhibited by excess MAP4.

To gain insight into the causes of hypertrophic growth cessation and functional deterioration found in our earlier study (50) discussed above, in the present study we asked whether increased cardiocyte MAP4 expression and microtubule decoration, whether via gene transfer into normal cells or intrinsically as part of the cardiocyte hypertrophic response, inhibits mRNP transport and the synthesis of proteins at a distance from the nucleus. We did this by imaging living cardiocytes infected with an adenovirus encoding a fluorescent zipcode binding protein 1 (ZBP-1)-enhanced yellow fluorescent protein (EYFP) fusion protein, since ZBP-1 binds to β-actin mRNA and translocates through the cytoplasm as a component of mRNPs to the active polysomes (12). The data show that MAP4 microtubule decoration in normal and hypertrophied cardiocytes does in fact inhibit mRNP transport and apparent protein synthesis in the cell periphery.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Experimental animals and cell isolation. All animal usage was under protocols approved by the institutional animal care committee in accordance with National Institutes of Health guidelines. Pressure-overload hypertrophy of the feline right ventricle (RV) was induced as described previously (5) by constriction of the pulmonary artery with a 3.2-mm internal diameter band (PAB). The characteristics of these PAB cats and control cats are given in Table 1. All operative procedures were carried out under full surgical anesthesia, consisting initially of ketamine HCl (11 mg/kg im) and acepromazine maleate (0.3 mg/kg im); after intubation and respiratory support, the cats were given nitrous oxide (0.5 l/min) and 3% isoflurane (1.5 l/min) supplemented with 100% O2. RV and LV cardiocytes were isolated separately (7, 33) and maintained for 1 h before further usage at 37°C in 2.5 mM Ca2+ mitogen-free M199 medium at pH 7.4 in order to remove the fibroblasts. Only cells that were quiescent under these conditions were studied. For immunofluorescence studies the cells were immersion fixed in freshly prepared 4% formaldehyde for 10 min at 37° C and permeabilized in 0.1% Triton X-100; for live cell studies they were cultured in Piper's medium at a density of 5 x 105 cells in 22-mm-diameter laminin-coated Willco Wells dishes (32).


View this table:
[in this window]
[in a new window]

  		Table 1. Characteristics of feline pulmonary artery band model

 
Construction of adenoviruses. As before, we used the AdEasy system to generate recombinant adenovirus plasmids (22, 44). The ZBP-1-EYFP cDNA construct (44) (a generous gift from S. Hüttelmaier and R. Singer, Albert Einstein College of Medicine, Bronx, NY) was cloned into the cytomegalovirus shuttle vector. The resultant plasmid was linearized, and homologous recombination was carried out by transformation of Escherichia coli strain BJ5183 with the PmeI-digested adenoviral backbone plasmid pAdEasy-1. Recombinant adenoviral DNA was digested with PacI and transfected into HEK-293 cells. The ZBP-1-EYFP adenovirus (AdZBP-1-EYFP) was plaque purified and amplified, and the titer was determined by the Gazes Adenoviral Core, which also made the previously described (51) adenovirus encoding MAP4 (AdMAP4). On day 1 of culture, the cardiocytes were infected with a multiplicity of infection (MOI) of 1.5 for 12 h. When more than one adenoviral construct was used to infect cells, experiments were carried out to ensure that there was no significant competition for infection between the constructs at the MOIs used. Cardiocyte infection with an MOI of 1 resulted in >85% infection as estimated by fluorescence-based detection of ZBP-1-EYFP expression or antibody-based detection of MAP4 expression. Infection with an adenovirus encoding β-galactosidase (Adβ-gal) was used as a control, where the histochemical X-Gal reaction (31) was used to define transfection efficiency.

Frozen sections of perfusion-fixed myocardium. With the full surgical anesthesia described above, the heart was perfusion fixed in diastole for 30 min with freshly prepared 1% formaldehyde as described previously (6). The fixed heart was excised, soaked overnight in 20% sucrose, and snap frozen in methylbutane at –130°C. Cryosections (10 µm thick) were prepared with a Leica Cryocut 1800 cryostat.

Cardiocyte immunofluorescent labeling. Cardiocytes were immersion fixed in freshly prepared 4% formaldehyde for 10 min at 37°C and permeabilized in 0.1% Triton X-100. Remaining aldehyde groups were blocked by 10 mM glycine for 10 min, and the cells were labeled with specific antibodies followed by antiserum tagged with the fluorochromes Alexa Fluor 488 or 647 (Molecular Probes, Eugene, OR). A mouse monoclonal antibody against {alpha}-tubulin (clone B-5-1-2) was purchased from Sigma-Aldrich, and a rabbit polyclonal antibody against {gamma}-tubulin (AB11321) was purchased from Abcam.

Microscopy and time-lapse photography. For imaging fixed cells, we used a Zeiss LSM510META microscope equipped with a 30-mW argon laser (458, 477, 488, and 514 nm), a 1-mW helium-neon laser (543 nm), a 5-mW helium-neon laser (633 nm), and a Plan-Apochromat x63/1.40 DIC objective to obtain high-resolution images. For time-lapse imaging of live cardiocytes, we used an Olympus IX71 fluorescence microscope equipped with a 100-W mercury lamp, two 10-window Chroma filter wheels, a 37°C climatized chamber containing 5% CO2, a Plan-Apochromat x60/1.42 objective, and a high-speed 1.3-megapixel Hamamatsu video camera. A filter configuration designed for EYFP (exciter S500/20, emitter D535/30) enabled selective ZBP-1-EYFP detection. Picture sequences of 100 frames were captured with an exposure time of 100 ms and an interval of 5 s between frames. The movement of fluorescent particles was measured with Olympus SlideBook 4.1 software. Only particles moving in a straight line in at least three frames and over a distance of >1 µm were considered to be moving via active transport and thus measured. Spontaneously contracting and light-damaged cells were excluded.

Measurement of protein synthesis. The rate of total protein synthesis was measured by radiolabeling as described previously (28), with a few minor modifications. Cardiocytes were radiolabeled for 4 h with 0.4 mM [3H]Phe (7 µCi/ml), a concentration that facilitates the rapid expansion of the intracellular Phe pool and the equilibration of Phe-tRNA-specific radioactivity with the specific radioactivity of Phe in the culture medium. After the labeling period, the cardiocytes were rinsed three times with PBS containing 10 mM Phe and scraped in protein isolation buffer (1x SSC-0.25% SDS). The protein was precipitated by adding HClO4 to a final concentration of 7% and washed 6 times with 7% HClO4. The protein was solubilized in 0.3 N NaOH at 37°C for 1 h. Radioactivity was counted, and the protein concentration was measured. The rate of protein synthesis (nmol Phe·g protein–1·h–1) was calculated by dividing the incorporation of Phe into protein by the specific radioactivity of Phe in the culture medium.

Radioautography. To assess the overall rate of spread of all newly synthesized RNA species into the cytoplasm, radioautography was conducted as described previously (14), but adapted to cardiocytes. Briefly, cells were cultured on laminin-coated coverslips in 35-mm culture dishes. [3H]uridine (10 µCi/ml) was added to the culture medium. Cells isolated from the RV and LV of a cat 2 wk after PAB were incubated for 1, 2, 3, 4, 6, 9, and 12 h, fixed with formaldehyde, dried in a series of alcohols, and coated with Kodak NTB2 photographic emulsion at 42°C in the dark. After a 14-day exposure, the emulsion was developed in Kodak D-19 developer as recommended by the manufacturer, the nuclei were counterstained with DAPI, and the slides were mounted in Mowiol polyvinyl alcohol.

Electron microscopy. Perfusion-fixed 1-mm3 myocardial samples removed by sharp dissection from the RV free wall were postfixed in 3% glutaraldehyde followed by 1% OsO4, dehydrated in alcohols, and embedded in Epon. Ultrathin 70-nm sections were prepared with a Reichert-Jung Ultracut microtome, stained for contrast with uranyl acetate and lead citrate, and photographed with a JEOL LSEM 1010 electron microscope.

Videos of cardiocyte microtubule-based transport. Videos illustrating the microtubule-based movement of ZBP-1-EYFP mRNPs in AdZBP-1-EYFP-infected living cardiocytes were made of a normal cardiocyte (Fig. 5, supplemental video Control.mov), a normal cardiocyte preinfected with Adβ-gal (Fig. 5, supplemental video β-Gal.mov), a normal cardiocyte preinfected with AdMAP4 (Fig. 5, supplemental video MAP4.mov), a hypertrophied RV cardiocyte from a PAB cat (Fig. 5, supplemental video PAB.mov), and a normal cardiocyte pretreated with colchicine (supplemental video Colchicine.mov). These can be found in the online data supplement to this article.


Figure 5
View larger version (112K):
[in this window]
[in a new window]

  		Fig. 5. Illustration of effects of MAP4 microtubule decoration on mRNP transport. Freshly isolated adult feline cardiocytes from normal or hypertrophied RVs were cultured for 24 h at 37°C; they were stimulated to contract at 1 Hz throughout the experimental protocol. Normal cells were either untreated (Control) or infected with Adβ-gal or AdMAP4 just before beginning stimulation; hypertrophied RV cells (PAB) were untreated. At 24 h all groups were infected with AdZBP-1-EYFP for a further 24 h to label the mRNPs. The infection efficiency with AdZBP-1-EYFP was found in preliminary experiments to be 90%. For each type of cardiocyte, the same cell was imaged sequentially, with the time between images indicated. Note that for the PAB cell longer intervals were used. To help with visual identification, mRNPs that were seen to exhibit saltatory movement were labeled red with Olympus Slidebook 4.1 software. A particle that showed consistent stepwise vectorial movement during several frames is indicated by yellow arrows in the Control and β-Gal cardiocytes. For this figure and the accompanying online videos, images were acquired with an Olympus IX71 fluorescence microscope, objective x60/1.42. Scale bar, 10 µm. Online videos: there is an supplemental online video for each of the 4 panels in this figure and for a cardiocyte pretreated with colchicine to depolymerize the microtubules. Directed stepwise movement of ZBP-1-EYFP-containing mRNPs is evident in the control (supplemental video Control.mov) and Adβ-gal-infected (supplemental video β-Gal.mov) cells, but only saltatory dithering of these particles in place is seen in the AdMAP4-infected (supplemental video MAP4.mov) and hypertrophied PAB (supplemental video PAB.mov) cardiocytes. In the colchicine-treated (supplemental video Colchicine.mov) cardiocyte, very few mRNPs are seen, and they are immobile near the nuclear envelope. For each video, 100 time-lapse frames were captured at a rate of 1 frame every 5 s. A QuickTime movie was made from each image sequence at a frame rate of 10 frames/s. The diffuse green background color represents autofluorescence of ZBP-1-EYFP, while the green particles represent ZBP-1-EYFP-containing mRNPs. Moving particles were labeled blue to enhance visual identification with Olympus SlideBook 4.1 software. A time recording and scale bar is included in each of the videos, which can be found in the online data supplement.

 
Data analysis. Values are means ± SE. In Table 1, statistical comparisons were by unpaired Student's t-test. In Figs. 1–8, statistical comparisons were by one-way ANOVA followed by a Dunnett's post hoc pairwise test for comparisons to control. A level of P < 0.05 was considered significant.


Figure 8
View larger version (62K):
[in this window]
[in a new window]

  		Fig. 8. Myocardial hypertrophic response to hemodynamic loading. A: electron micrograph of a feline RV pressure overloaded for 1 day; we do not see active cardiocyte nucleoli such as this in normal myocardium. B: tubulin-stained microtubule tracks (green) along which the mRNPs from the nucleus must be transported to the phalloidin-stained myofibrils (red). C, adapted from our earlier study of progressive canine left ventricle (LV) afterloading (50), shows ventriculographic LV mass on the left y-axis and pressure gradient across a proximal aortic band on the right y-axis. The gradient was initiated at time 0 and increased every 2 wk thereafter, as indicated on the x-axis. Scale bars, 5 µm.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cardiocyte microtubule network structure. Cardiocyte microtubules are not chaotically dispersed but instead extend throughout the cell and are oriented both along and across the cardiocyte long axis (Fig. 1, A, C, and E). Figure 1, B and D, in which the myofibrils are also stained, show that the microtubules envelop each myofibril extensively. Figure 1F, in which both the {alpha}-tubulin of the microtubules and the {gamma}-tubulin of the microtubule organizing centers (MTOCs) are stained, shows that unlike the single MTOC found in almost all other interphase cells cardiocytes have a series of {gamma}-tubulin-positive MTOCs dispersed along the nuclear envelopes from which the microtubules extend throughout the cell from sites contiguous to the nuclear pores through which mRNP transport occurs (26).

Dependence of mRNP transport on intact microtubules. After infection of cardiocytes with AdZBP-1-EYFP, discrete fluorescent particles became visible in live and fixed cells. These particles likely correspond to the fusion protein bound to mRNPs. Immune labeling using an anti-{alpha}-tubulin antibody revealed strong colocalization of mRNPs with microtubules (Fig. 2A). To probe the functional relationship between the microtubule network and mRNP distribution, we reversibly disrupted microtubules with 2 µM nocodazole. The microtubules disappeared by 10 min after the addition of nocodazole and remained absent as long as nocodazole was present in the culture medium (Fig. 2B). No effect of microtubule depolymerization on the localization of preexisting mRNPs was apparent immediately after nocodazole was added, but when cardiocytes were treated with nocodazole for 18 h, mRNPs that presumably were labeled with newly synthesized ZBP-1-EYFP were localized predominantly in the perinuclear region just after drug washout (Fig. 2B). At later times after nocodazole washout, the microtubules repolymerized over 1–4 h, and as seen in Fig. 2, C–F, there was a concurrent redistribution of ZBP-1-EYFP-labeled mRNPs into the cardiocyte periphery, presumably reflecting the recovery of microtubule-based mRNP transport.

Dependence of protein synthesis on intact microtubules. To determine the extent to which net cardiocyte protein synthesis is mediated by the microtubule network, 2 µM nocodazole was used to depolymerize the microtubules for 24 h and protein synthesis was then measured at fixed time points during microtubule repolymerization following nocodazole washout. For this series of experiments, adult cardiocytes were electrically stimulated to contract at a frequency of 1 Hz in order to maintain a reasonably constant basal protein synthesis rate (Fig. 7 in Ref. 29). Figure 3 shows that the rate of protein synthesis was quite significantly reduced to 60% of control in cardiocytes whose microtubules were fully depolymerized. Importantly, removal of nocodazole from the culture medium resulted in repolymerization of the microtubules (Fig. 2) and a return of the rate of protein synthesis to the control level (Fig. 3). Thus at least 40% of protein synthesis in fully differentiated adult cardiocytes depends on an intact microtubule network, and it is possible that this value is an underestimation, since some portion of the residual 60% of total protein synthesis utilized microtubule-delivered material that was already in place.


Figure 7
View larger version (75K):
[in this window]
[in a new window]

  		Fig. 7. RNA distribution into the cytoplasm of normal and hypertrophied cardiocytes: [3H]uridine radioautography. Freshly isolated cardiocytes from the 2 groups were exposed to [3H]uridine beginning at 0 h, and the cells were fixed at the indicated times thereafter. Radiolabel incorporated into newly synthesized RNA is shown by the dense black grains. In the Control cells the labeling spread rapidly from the nucleus throughout the cytoplasm, while in the hypertrophied PAB cells this process was markedly delayed. Images were acquired with an Olympus IX71 fluorescence microscope, objective x60/1.42, and Olympus Slidebook 4.1 software. Scale bar, 10 µm.

 

Figure 3
View larger version (16K):
[in this window]
[in a new window]

  		Fig. 3. Microtubule dependence of cardiocyte protein synthesis. Normal freshly isolated adult feline cardiocytes were cultured at 37°C while stimulated to contract at 1 Hz for 24 h. They then were either untreated for 24 h (Control) or treated with 2 µM nocodazole for 24 h (Nocodazole). These conditions were maintained for both groups of cardiocytes during a subsequent 4-h period of radiolabeling with 0.4 mM [3H]Phe (7 µCi/ml). Nocodazole Washout #1 cardiocytes were treated the same as the Nocodazole cells except that nocodazole was removed from the culture medium at the beginning of the 4-h radiolabeling period. Nocodazole Washout#2 cardiocytes were also treated the same as the Nocodazole cardiocytes except that nocodazole was removed from the culture medium at 3 h before the 4-h radiolabeling period, such that protein synthesis was measured after the microtubule network was reestablished. *P < 0.05 for a difference from the Control group.

 
Both to control for any nonspecific effects of nocodazole and to control for any effects stemming from the possibility that {alpha}β-tubulin heterodimers might act as a functional analog of G proteins when the free tubulin concentration is increased by microtubule depolymerization (20), we repeated the study shown in the first two bars of Fig. 3 using 1 µM colchicine or 12 µM vincristine to depolymerize the microtubules as described previously (27). Complete microtubule depolymerization followed nocodazole, colchicine, or vincristine treatment (27); however, {alpha}β-tubulin heterodimer concentration increased comparably after nocodazole or colchicine but fell to essentially zero after vincristine because of tubulin paracrystal formation (27). For the nocodazole data in Fig. 3, the protein synthesis rate was 58.9 ± 4.9% of control, for colchicine this value was 54.8 ± 1.2% of control, and for vincristine it was 60.5 ± 1.5% of control [P = not significant (n.s.) vs. control by unpaired Student's t-test for n = 4 in each case]. Lumicolchicine (10 µM) was without effect on microtubules or protein synthesis.

Dependence of ZBP-1-EYFP protein synthesis and distribution on adenovirus-mediated or hypertrophy-induced MAP4 decoration of microtubules. As shown in detail previously (42, 51) and illustrated here in Fig. 4, top (inset), there is a striking decoration of almost all of the cellular microtubules by MAP4 in cardiocytes infected by AdMAP4 and in cardiocytes from PAB cats with severe pressure-overload RV hypertrophy (Table 1). In cardiocytes from control cats (data not shown) and in cardiocytes infected with Adβ-gal there is very little coincident tubulin and MAP4 staining. For Fig. 4, top and bottom, the cardiocytes were fixed at 16 h after AdZBP-1-EYFP infection. In Fig. 4, top, it is seen that the total EYFP fluorescence signal, which we take to be a function of ZBP-1-EYFP fusion protein synthesis, is strongly dependent on an intact microtubule network that is largely free of bound MAP4, since in the cardiocyte groups with MAP4-decorated or absent microtubules this total EYFP fluorescence is only ~15% of control. As in the controls done for Fig. 3 for any nonspecific drug effects and for any effects from the possibility that {alpha}β-tubulin heterodimers might act as a functional analog of G proteins when the free tubulin concentration is increased by microtubule depolymerization by colchicine (20), we repeated the study in Fig. 4, top, using 1 µM colchicine, 2 µM nocodazole, or 12 µM vincristine to depolymerize the microtubules. Cardiocyte ZBP-1 mean fluorescence as a percentage of control was 13.9 ± 0.8%, 14.0 ± 0.6%, and 16.5 ± 1.2%, respectively (P = n.s. by 1-way ANOVA followed by Student-Newman-Keuls post hoc test for n = 25 in each case), and lumicolchicine was again without effect.


Figure 4
View larger version (54K):
[in this window]
[in a new window]

  		Fig. 4. Effects of microtubule-associated protein 4 (MAP4) microtubule decoration induced by AdMAP4 infection or pressure-overload hypertrophy on the synthesis and distribution of ZBP-1. Normal freshly isolated adult feline cardiocytes were either untreated (Control), infected with Adβ-gal or AdMAP4, or treated continuously with 1 µM colchicine. One day later these 4 groups of cells, as well as hypertrophied cardiocytes isolated 1 day earlier from the (RV) ventricle of a pulmonary artery banded (PAB) cat, were infected with AdZBP-1-EYFP for 16 h and fixed, and the nuclei were counterstained with DAPI. The extensive microtubule decoration by MAP4 in AdMAP4-infected normal cardiocytes and hypertrophied PAB cardiocytes compared with Adβ-gal-infected cells is shown in the confocal micrograph insets, top, in which the microtubules were labeled red with a monoclonal anti-{alpha}-tubulin antibody (B-5-1-2) and MAP4 was labeled green with our rabbit polyclonal MAP4 antibody (51) so that MAP4-decorated microtubules are yellow. Top: ZBP-1 synthesis. With the DAPI channel on the Olympus IX71 fluorescence microscope to identify the cardiocytes, the specified numbers of cells from each group were randomly selected and photographed with the EYFP channel; the total EYFP-dependent fluorescence after background subtraction was quantified with Olympus Slidebook 4.1 software. These results were confirmed in 3 independent experiments. Bottom: ZBP-1 distribution. Photomicrographs show the location of AdZBP-1-EYFP-containing mRNPs at 16 h after AdZBP-1-EYFP infection of each of the specified groups of cardiocytes. These mRNPs are the punctate black dots. Images were acquired with a Zeiss LSM510 confocal microscope using 514-nm and 633-nm laser beams (objective x63/1.40). Scale bars, 10 µm. *P < 0.05 for a difference from the Control group.

 
In Fig. 4, bottom, it is seen that the centrifugal distribution of EYFP fluorescent mRNPs at a fixed time after AdZBP-1-EYFP infection is also strongly dependent on an intact microtubule network that is largely free of MAP4. That is, the discrete black particles are scattered throughout the cytoplasm in the control and β-gal cardiocytes, but they are largely confined to the immediate perinuclear area in the MAP4, PAB, and colchicine cardiocytes. The most important point of this figure is that hypertrophy-dependent MAP4 microtubule decoration has the same quantitative and qualitative effects on the synthesis and transport of a mRNP-associated protein as does a primary intervention designed to have a selective effect only on MAP4 expression.

Dependence of mRNP transport on adenovirus-mediated or hypertrophy-induced MAP4 decoration of microtubules. The intrinsic autofluorescence of EYFP allowed AdZBP-1-EYFP-infected cardiocytes to be used to visualize the movement of mRNPs containing the ZBP-1-EYFP fusion protein. Thus the data in Fig. 4 show that movement of ZBP-1 mRNPs away from the nucleus is obligatorily dependent on an intact microtubule network and is inhibited by MAP4 microtubule decoration. The data in Figs. 5 and 6 show that the rate and extent of this movement is inversely related to MAP4 decoration of these microtubules. Figure 5 and the online supplemental videos for each of the specified conditions provide examples of this effect. While the great majority of the fluorescent particles in all of the cells examined exhibited only short-range randomly directed saltatory movement, as we and others have observed before (44, 46), in the Control and β-gal cells particles could be readily identified that exhibited long-range progressive stepwise vectorial movement. However, in the MAP4 and PAB cells it was very difficult to identify such vectorial particle movement; rather, the great majority of the particles oscillated in place. These contrasting effects of "bare" vs. decorated microtubules on mRNP transport are even more apparent in the online videos, and in the colchicine video, as might be expected from Fig. 4, bottom, there is no apparent mRNP movement at all.


Figure 6
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 6. Quantitation of effects of MAP4 microtubule decoration on mRNP transport. The experimental conditions used here were the same as those used for the cardiocytes in Fig. 5. Time-lapse images were captured with an exposure time of 100 ms at 24 h after Ad-ZBP1-EYFP infection; the number of cells in each experimental group is specified. To avoid cell damage, observation time could not exceed 2–5 min or 100 frames. Because of this limited observation time, several cardiocyte isolations were required for each group of RV cardiocytes; the total number of cardiocytes for each group is indicated. *P < 0.05 for a difference from the Control distance; {dagger}P < 0.05 for a difference from the Control velocity.

 
Figure 6 provides quantitative information describing the effects of these same experimental conditions on microtubule-based ZBP-1-EYFP mRNP movement. For this purpose, time-lapse images with an exposure time of 100 ms were acquired from isolated RV cardiocytes on the day after AdZBP-1-EYFP infection. Because for an individual cardiocyte the observation time could not exceed 2–5 min or 100 frames without fluorescence quenching or cell damage, several cardiocyte isolations were required for each group of cells specified in Fig. 6. It should be noted that in the PAB cats used here and described in Table 1 there is severe RV hypertrophy of an extent that leads in this model to right heart failure; such animals have a marked expression of the abnormal microtubule phenotype (48). While most mRNPs exhibited no net vectorial movement during this observation period, a small fraction of particles did move in this manner, just as has been described for other cell types (44, 46). Each such directed movement (defined as being ≥1.0 µm) was recorded and measured. Figure 5 and the online videos show that in control and Adβ-gal cardiocytes mRNPs tend to move parallel to the cellular long axis over a distance of up to 20 µm, while in the AdMAP4 and PAB cardiocytes the movement is slow and irregular. Figure 6 shows that mean particle velocity and distance traveled were comparable in the control and Adβ-gal-infected cells, but both of these measures were reduced about twofold in the AdMAP4-infected and PAB cardiocytes whose microtubules were extensively decorated by MAP4. Analysis of the distribution of both velocity and distance showed that in all groups short and slow movement occurred equally, but in AdMAP4 and PAB cells the fast and long-distance movements were rare. Cardiocytes with no microtubules (pretreated for 45 min with 1 µM colchicine) served as a negative control: no net movement was detected in these cardiocytes. Of interest, while what is being described here is discontinuous rather than continuous motion, Fig. 6 has the form of a phase plane diagram describing viscosity, such that the net effect of MAP4 interference with microtubule-based motor protein transport resembles a viscous impediment to vectorial motion.

Kinetics of total RNA distribution in normal and hypertrophied cardiocytes. While [3H]uridine radioautography such as that shown in Fig. 7 labels all newly synthesized RNA indiscriminately, and the majority of this is ribosomal RNA rather than mRNA, this method does allow us to illustrate here at the indicated times after labeling how slow the movement of the RNA components of the protein synthesis machinery into the sites of structural protein synthesis is in hypertrophied as opposed to normal cardiocytes. That is, the initial nuclear labeling becomes dispersed throughout the cytoplasm by 9 h in the control cells, but the label tends to remain in the nuclear region indefinitely in the PAB cells. Furthermore, while we cannot distinguish here between ribosomal and messenger RNA, both are often transported as part of mRNPs (16). This process is apparently entirely microtubule dependent, since the [3H]uridine label did not move away from the nucleus at all in colchicine-treated cells, even after 12 h (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Localization of cellular protein synthesis and microtubules. There are three spatial categories of cellular protein synthesis (16). For the first, the majority of mRNAs encoding soluble proteins are targeted to ribosomes and translated in the cytoplasm in a location-independent manner (19). For the second, mRNAs encoding secretory proteins have their translation halted and are transported in concert with the ribosome to the endoplasmic reticulum so that translation can be coupled with import of the protein into that structure (55). For the third, mRNAs encoding a number of structural proteins are targeted to specific regions of the cell where they undergo localized translation (1). These mRNAs contain elements, usually located in their 3'-untranslated region, that bind to RNA-binding proteins that direct mRNA targeting. Translation of transported mRNAs is repressed during transport, and the repression is removed after arrival at the proper location (1).

This third category of protein synthesis localization is especially important for determining cellular asymmetry (35), in that directional transport of large protein- and mRNA-containing complexes along microtubules or actin filaments and localized expression of this mRNA allow for the temporal and spatial control of cellular gene expression (47). This process requires an intact cytoskeleton and the formation of motor protein-containing complexes (54). As part of such transport complexes, dedicated RNA-binding proteins bind to specific sequence regions, termed zip code elements, in the cargo mRNA and interact with additional proteins to form a functional mRNP. Such mRNPs are large particles containing transported mRNAs, RNA-binding proteins, motors, and ribosomal subunits (16). Microtubule-based transport of these particles is inherently directional, in that the dynein family of microtubule-associated motor proteins moves cargo toward the minus end of the microtubules at the cell center and the kinesin family of microtubule-associated motor proteins moves cargo toward the plus end of the microtubules at the cell periphery. Data showing that cardiocyte mRNP components are excluded from within the myofibrils but are associated with the extramyofilament cytoskeleton in an extremely diffusion-restricted cytoplasm (40), as well as data showing that microtubule-based transport of cardiocyte β-adrenergic receptors is inhibited by MAP4 microtubule decoration such as we find in cardiac hypertrophy (4), led to the present study.

The presence of microtubules in adult mammalian cardiocytes was first reported more than 30 years ago (38), and their spatial distribution within these cells has been well defined at both the light and electron microscopic levels (41). However, both because these are labile polymers with a half-life in normal cardiocytes of ~30 min (52) and because enzymatic cell isolation is a lengthy process that unloads the cytoskeleton, we thought that before trying to define their normal function we should define their normal structure in freshly fixed tissue sections. Figure 1 shows that myocardial microtubules originate at {gamma}-tubulin-positive MTOCs and extend primarily longitudinally toward the cell periphery while tightly enveloping the myofibrils. These MTOCs are in close spatial and functional relationship with nuclear pores, through which newly formed mRNPs are exported from the nucleus into the cytoplasm (13). Thus microtubules are ideally arranged to serve as tracks for the motor-based transport of ribosomes and mRNAs for structural proteins to the sites of myofibril renewal and/or de novo assembly.

The present data do support the idea that microtubule-based transport is important for the synthesis of cardiocyte structural proteins. Using ZBP-1 as a marker for this mRNP transport process, we found in Fig. 2 that mRNP distribution away from the nucleus is strongly microtubule dependent. As predicted by other data showing that colchicine reduces cardiac protein synthesis (45), Fig. 3 shows that at least 40% of protein synthesis depends on an intact microtubule array, a value that may well underestimate the in vivo value given that these were contracting but unloaded cells studied in vitro. In accordance with the hypothesis for this study and our earlier data showing that cardiocyte β-adrenergic receptor recycling is not inhibited by microtubule stabilization but is inhibited by MAP4 microtubule decoration (4), the data in Figs. 4–6 show that MAP4 microtubule decoration, whether caused by adenovirus-mediated or hypertrophy-induced MAP4 upregulation, strongly inhibits mRNP transport, and Fig. 7 shows that in hypertrophied cardiocytes transport of newly synthesized RNA from the nucleus is markedly slowed. Thus, apart from its deleterious effects on the contractile function of high-wall stress hypertrophied myocardium (10, 11), this dense, MAP4-decorated microtubule network also has strikingly deleterious effects on processes subserving cardiac structural protein synthesis.

Does the hypertrophic response to hemodynamic overloading affect functional compensation? The real significance of this study, and much else, depends to a great extent on the answer to this question. That is, does it really matter, either in the normal adult heart or more especially in the hypertrophying heart, whether the transcytoplasmic transport process that subserves the synthesis and assembly of cardiocyte structural proteins is intact? Since it is clear that cardiac hypertrophy is associated with increased clinical morbidity and mortality (30), a simplistic view would be that this is an inherently maladaptive process. However, any such generalization is immediately invalidated by specific examples to the contrary, for instance, the very extensive physiological cardiac hypertrophy seen in elite athletes such as cyclists while they are actively competing (Fig. 2 in Ref. 34). We would instead suggest that the cardiac hypertrophy that occurs in response to pathological overloads is at least initially inherently compensatory, with the associated increase in morbidity and mortality being caused by 1) the underlying disease process and 2) a variably time-dependent deterioration of the structural and functional properties of the hypertrophied heart leading to some combination of systolic and/or diastolic heart failure.

Figure 8 serves both to illustrate this point and to frame the present study of our model of severe pressure overload of the feline RV (48) that is characterized here in Table 1. The same marked changes in the extramyofilament cytoskeleton, as well as microtubule-dependent contractile defects, occur both here and in severe pressure overload of the canine LV (50). Figure 8A, an electron micrograph of feline myocardium at 1 day after pressure overloading, shows the prominent nucleolus with highly branched nucleolonema characteristic of a cell initiating transcription of the rRNA needed for the greater protein synthesis capacity that is required for cardiac hypertrophy (Fig. 4 in Ref. 36). If these ribosomes and the other components of the mRNPs are transported along the green microtubule tracks into the red myofibrillar sites of sarcomerogenesis shown in Fig. 8B, compensatory hypertrophy proportional to the increasing load, such as that shown by the open circles in Fig. 8C, ensues. If this transport process or some other aspect of this anabolic load response is attenuated, as is shown by the filled circles in Fig. 8C, decompensated failure ensues. Pertinent to the present study is the fact that both LV wall stress and the microtubule network remained normal throughout the earlier canine study (50) in the compensated hypertrophy group, while at the time of biopsy and progressively thereafter a dense microtubule network, increased LV wall stress, and systolic LV myocardial and chamber contractile dysfunction were found in the decompensated failure group. Of interest, this cytoskeletal alteration is similar both to what we (56) have found in patients with compensated vs. decompensated aortic stenosis and to what Schaper and colleagues (23, 43) have reported in patients with severe dilated cardiomyopathy.

Many other factors such as chamber remodeling determine whether and for how long hypertrophy is compensatory. One would expect, for instance, on the basis of the known natural history of clinical aortic stenosis (Fig. 1 in Ref. 39), that the compensated hypertrophy group might well develop diastolic and perhaps eventual systolic heart failure. However, Fig. 8 does make the point that the extent to which hypertrophy maintains normal wall stress in the face of increasing load is a major determinant of whether pathological hypertrophy is initially compensatory. The data in the present study support the concept that this hypertrophic response is dependent on an intact mRNP transport system and that when this is compromised by the dense, MAP4-decorated microtubule network that we see in high-wall stress cardiac hypertrophy (10, 11), this growth process is compromised such that "decompensated failure" ensues. Thus microtubule network alterations may be one cause not only for the contractile dysfunction of hypertrophied myocardium but also for an inadequate cardiac growth response to hemodynamic overloading in terms of an initially compensatory renormalization of wall stress.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by Program Project Grant HL-48788 from the National Heart, Lung, and Blood Institute and by a Department of Veterans Affairs Merit Review Grant.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. Cooper IV, 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
 

  1. Bassell GJ, Oleynikov Y, Singer RH. The travels of mRNAs through all cells large and small. FASEB J 13: 447–454, 1999.[Free Full Text]
  2. Bulinski JC, McGraw TE, Gruber D, Nguyen HL, Sheetz MP. Overexpression of MAP4 inhibits organelle motility and trafficking in vivo. J Cell Sci 110: 3055–3064, 1997.[Abstract]
  3. Cheng G, Iijima Y, Ishibashi Y, Kuppuswamy D, Cooper G. Inhibition of G protein-coupled receptor trafficking in neuroblastoma cells by MAP4 decoration of microtubules. Am J Physiol Heart Circ Physiol 283: H2379–H2388, 2002.[Abstract/Free Full Text]
  4. Cheng G, Qiao F, Gallien TN, Kuppuswamy D, Cooper G. Inhibition of β-adrenergic receptor trafficking in adult cardiocytes by MAP4 decoration of microtubules. Am J Physiol Heart Circ Physiol 288: H1193–H1202, 2005.[Abstract/Free Full Text]
  5. Cooper G, Satava RM. A method for producing reversible long-term pressure overload of the cat right ventricle. J Appl Physiol 37: 762–764, 1974.[Free Full Text]
  6. Cooper G, Tomanek RJ. Load regulation of the structure, composition, and function of mammalian myocardium. Circ Res 50: 788–798, 1982.[Free Full Text]
  7. Cooper G, Mercer WE, Hoober JK, Gordon PR, Kent RL, Lauva IK, Marino TA. Load regulation of the properties of adult feline cardiocytes: the role of substrate adhesion. Circ Res 58: 692–705, 1986.[Abstract/Free Full Text]
  8. Cooper G. Cardiocyte adaptation to chronically altered load. Annu Rev Physiol 49: 501–518, 1987.[CrossRef][Web of Science][Medline]
  9. Cooper G. Basic determinants of myocardial hypertrophy: a review of molecular mechanisms. Annu Rev Med 48: 13–23, 1997.[CrossRef][Web of Science][Medline]
  10. Cooper G. Cardiocyte cytoskeleton in hypertrophied myocardium. Heart Fail Rev 5: 187–201, 2000.[CrossRef][Medline]
  11. Cooper G. Cytoskeletal networks and the regulation of cardiac contractility: microtubules, hypertrophy, and cardiac dysfunction. Am J Physiol Heart Circ Physiol 291: H1003–H1014, 2006.[Abstract/Free Full Text]
  12. Czaplinski K, Singer RH. Pathways for mRNA localization in the cytoplasm. Trends Biochem Sci 31: 687–693, 2006.[CrossRef][Web of Science][Medline]
  13. Daneholt B. A look at messenger RNP moving through the nuclear pore. Cell 88: 585–588, 1997.[CrossRef][Web of Science][Medline]
  14. Davis L, Banker GA, Steward O. Selective dendritic transport of RNA in hippocampal neurons in culture. Nature 330: 477–479, 1987.[CrossRef][Medline]
  15. Dinh AT, Theofanous T, Mitragotri S. A model for intracellular trafficking of adenoviral vectors. Biophys J 89: 1574–1588, 2005.[CrossRef][Web of Science][Medline]
  16. Elvira G, Wasiak S, Blandford V, Tong XK, Serrano A, Fan X, del Rayo Sanchez-Carbente M, Servant F, Bell AW, Boismenu D, Lacaille JC, McPherson PS, DesGroseillers L, Sossin WS. Characterization of an RNA granule from developing brain. Mol Cell Proteomics 5: 635–651, 2006.[Abstract/Free Full Text]
  17. Farina KL, Singer RH. The nuclear connection in RNA transport and localization. Trends Cell Biol 12: 466–472, 2002.[CrossRef][Web of Science][Medline]
  18. Farina KL, Huttelmaier S, Musunuru K, Darnell R, Singer RH. Two ZBP-1 KH domains facilitate β-actin mRNA localization, granule formation, and cytoskeletal attachment. J Cell Biol 160: 77–87, 2003.[Abstract/Free Full Text]
  19. Gingras AC, Raught B, Sonenberg N. eIF4 initiation factors: effectors of mRNA recruitment to ribosomes and regulators of translation. Annu Rev Biochem 68: 913–963, 1999.[CrossRef][Web of Science][Medline]
  20. Gómez AM, Kerfant BG, Vassort G. Microtubule disruption modulates Ca2+ signalling in rat cardiac myocytes. Circ Res 86: 30–36, 2000.[Abstract/Free Full Text]
  21. Gunther S, Grossman W. Determinants of ventricular function in pressure-overload hypertrophy in man. Circulation 59: 679–688, 1979.[Free Full Text]
  22. He TC, Zhou S, da Costa LT, Yu J, Kinzler KW, Vogelstein B. A simplified system for generating recombinant adenoviruses. Proc Natl Acad Sci USA 95: 2509–2514, 1998.[Abstract/Free Full Text]
  23. Heling A, Zimmermann R, Kostin S, Maeno Y, Hein S, Devaux B, Bauer E, Klovekorn WP, Schlepper M, Schaper W, Schaper J. Increased expression of cytoskeletal, linkage, and extracellular proteins in failing human myocardium. Circ Res 86: 846–853, 2000.[Abstract/Free Full Text]
  24. Hirokawa N. mRNA transport in dendrites: RNA granules, motors, and tracks. J Neurosci 26: 7139–7142, 2006.[Abstract/Free Full Text]
  25. Huber D, Grimm J, Koch R, Krayenbuehl HP. Determinants of ejection performance in aortic stenosis. Circulation 64: 126–134. 1981.[Free Full Text]
  26. Ishibashi Y, Hazen-Martin DJ, Cooper G. Existence and localization of microtubule organizing center (MTOC) in terminally differentiated cardiocytes. J Histochem Cytochem 47: 1646, 1999.[Free Full Text]
  27. Ishibashi Y, Takahashi M, Isomatsu Y, Qiao F, Iijima Y, Shiraishi H, Simsic JM, Baicu CF, Robbins J, Zile MR, Cooper G. Role of microtubules versus myosin heavy chain isoforms in contractile dysfunction of hypertrophied murine cardiocytes. Am J Physiol Heart Circ Physiol 285: H1270–H1285, 2003.[Abstract/Free Full Text]
  28. Ivester CT, Kent RL, Tagawa H, Tsutsui H, Imamura T, Cooper G, McDermott PJ. Electrically stimulated contraction accelerates protein synthesis rates in adult feline cardiocytes. Am J Physiol Heart Circ Physiol 265: H666–H674, 1993.[Abstract/Free Full Text]
  29. Kato S, Ivester CT, Cooper G, Zile MR, McDermott PJ. Growth effects of electrically stimulated contraction on adult feline cardiocytes in primary culture. Am J Physiol Heart Circ Physiol 268: H2495–H2504, 1995.[Abstract/Free Full Text]
  30. Levy D, Garrison RJ, Savage DD, Kannel WB, Castelli WP. Prognostic implications of echocardiographically determined left ventricular mass in the Framingham Heart Study. N Engl J Med 322: 1561–1566, 1990.[Abstract]
  31. Ma W, Rogers K, Zbar B, Schmidt L. Effects of different fixatives on β-galactosidase activity. J Histochem Cytochem 50: 1421–1424, 2002.[Abstract/Free Full Text]
  32. Mallardo M, Deitinghoff A, Muller J, Goetze B, Macchi P, Peters C, Kiebler MA. Isolation and characterization of Staufen-containing ribonucleoprotein particles from rat brain. Proc Natl Acad Sci USA 100: 2100–2105, 2003.[Abstract/Free Full Text]
  33. Mann DL, Kent RL, Cooper G. Load regulation of the properties of adult feline cardiocytes: growth induction by cellular deformation. Circ Res 64: 1079–1090, 1989.[Abstract/Free Full Text]
  34. Maron BJ, Pelliccia A. The heart of trained athletes: cardiac remodeling and the risks of sports, including sudden death. Circulation 114: 1633–1644, 2006.[Free Full Text]
  35. Muller M, Heuck A, Niessing D. Directional mRNA transport in eukaryotes: lessons from yeast. Cell Mol Life Sci 64: 171–180, 2007.[CrossRef][Web of Science][Medline]
  36. Nagatomo Y, Carabello BA, Hamawaki M, Nemoto S, Matsuo T, McDermott PJ. Translational mechanisms accelerate the rate of protein synthesis during canine pressure-overload hypertrophy. Am J Physiol Heart Circ Physiol 277: H2176–H2184, 1999.[Abstract/Free Full Text]
  37. Narishige T, Blade KL, Ishibashi Y, Nagai T, Hamawaki M, Menick DR, Kuppuswamy D, Cooper G. Cardiac hypertrophic and developmental regulation of the β-tubulin multigene family. J Biol Chem 274: 9692–9697, 1999.[Abstract/Free Full Text]
  38. Page E. Tubular systems in Purkinje cells of the cat heart. J Ultrastruct Res 17: 72–83, 1966.[CrossRef][Web of Science]
  39. Ross J Jr, Braunwald E. Aortic stenosis. Circulation 38, Suppl 1: 61–67, 1968.[Medline]
  40. Russell B, Dix DJ. Mechanisms for intracellular distribution of mRNA: in situ hybridization studies in muscle. Am J Physiol Cell Physiol 262: C1–C8, 1992.[Abstract/Free Full Text]
  41. Sætersdal T, Greve G, Dalen H. Associations between β-tubulin and mitochondria in adult isolated heart myocytes as shown by immunofluorescence and immunoelectron microscopy. Histochemistry 95: 1–10, 1990.[CrossRef][Web of Science][Medline]
  42. Sato H, Nagai T, Kuppuswamy D, Narishige T, Koide M, Menick DR, Cooper G. Microtubule stabilization in pressure overload cardiac hypertrophy. J Cell Biol 139: 963–973, 1997.[Abstract/Free Full Text]
  43. Schaper J, Froede R, Hein S, Buck A, Hashizume H, Speiser B, Friedl A, Bleese N. Impairment of the myocardial ultrastructure and changes of the cytoskeleton in dilated cardiomyopathy. Circulation 83: 504–514, 1991.[Abstract/Free Full Text]
  44. Scholz D, McDermott P, Garnovskaya M, Gallien TN, Hüttelmaier S, DeRienzo C, Cooper G. Microtubule-associated protein 4 (MAP4) inhibits microtubule-dependent distribution of mRNA in isolated neonatal cardiocytes. Cardiovasc Res 71: 506–516, 2006.[Abstract/Free Full Text]
  45. Scopacasa BS, Teixeira VP, Franchini KG. Colchicine attenuates left ventricular hypertrophy but preserves cardiac function of aortic-constricted rats. J Appl Physiol 94: 1627–1633, 2003.[Abstract/Free Full Text]
  46. Sheetz MP. Motor and cargo interactions. Eur J Biochem 262: 19–25, 1999.[Web of Science][Medline]
  47. St-Johnston D. Moving messages: the intracellular localization of mRNAs. Nat Rev Mol Cell Biol 6: 363–375, 2005.[CrossRef][Web of Science][Medline]
  48. Tagawa H, Koide M, Sato H, Cooper G. Cytoskeletal role in the contractile dysfunction of cardiocytes from hypertrophied and failing right ventricular myocardium. Proc Assoc Am Physicians 108: 218–229, 1996.[Web of Science][Medline]
  49. Tagawa H, Rozich JD, Narishige T, Kuppuswamy D, Tsutsui H, McDermott PJ, Koide M, Cooper G. Basis for increased microtubules in pressure-hypertrophied cardiocytes. Circulation 93: 1230–1243, 1996.[Abstract/Free Full Text]
  50. Tagawa H, Koide M, Sato H, Zile MR, Carabello BA, Cooper G. Cytoskeletal role in the transition from compensated to decompensated hypertrophy during adult canine left ventricular pressure overloading. Circ Res 82: 751–761, 1998.[Abstract/Free Full Text]
  51. Takahashi M, Shiraishi H, Ishibashi Y, Blade KL, McDermott PJ, Menick DR, Kuppuswamy D, Cooper G. Phenotypic consequences of β1-tubulin expression and MAP4 decoration of microtubules in adult cardiocytes. Am J Physiol Heart Circ Physiol 285: H2072–H2083, 2003.[Abstract/Free Full Text]
  52. Tsutsui H, Ishihara K, Cooper G. Cytoskeletal role in the contractile dysfunction of hypertrophied myocardium. Science 260: 682–687, 1993.[Abstract/Free Full Text]
  53. Urabe Y, Mann DL, Kent RL, Nakano K, Tomanek RJ, Carabello BA, Cooper G. Cellular and ventricular contractile dysfunction in experimental canine mitral regurgitation. Circ Res 70: 131–147, 1992.[Abstract/Free Full Text]
  54. Vale RD. The molecular motor toolbox for intracellular transport. Cell 112: 467–480, 2003.[CrossRef][Web of Science][Medline]
  55. Walter P, Johnson AE. Signal sequence recognition and protein targeting to the endoplasmic reticulum membrane. Annu Rev Cell Biol 10: 87–119, 1994.[CrossRef][Web of Science][Medline]
  56. Zile MR, Green GR, Schuyler GT, Aurigemma GP, Miller DC, Cooper G. Cardiocyte cytoskeleton in patients with left ventricular pressure overload hypertrophy. J Am Coll Cardiol 37: 1080–1084, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
J. T. Fassett, X. Xu, X. Hu, G. Zhu, J. French, Y. Chen, and R. J. Bache
Adenosine regulation of microtubule dynamics in cardiac hypertrophy
Am J Physiol Heart Circ Physiol, August 1, 2009; 297(2): H523 - H532.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
G. Cooper IV
Proliferating cardiac microtubules
Am J Physiol Heart Circ Physiol, August 1, 2009; 297(2): H510 - H511.
[Full Text] [PDF]

Home page
 Am. J. Physiol. Heart Circ. Physiol.Home page
S. Hein, S. Kostin, and J. Schaper
mRNA cargo no longer on time
Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1130 - H1132.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Videos
Right arrow All Versions of this Article:
294/3/H1135    most recent
01275.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (2)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Scholz, D.
Right arrow Articles by Cooper, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Scholz, D.
Right arrow Articles by Cooper, G., IV

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.