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Role of microtubules versus myosin heavy chain isoforms in contractile dysfunction of hypertrophied murine cardiocytes

¹Gazes Cardiac Research Institute, Cardiology Division, Medical University of South Carolina, and Department of Veterans Affairs Medical Center, Charleston, South Carolina 29401; and ²Division of Molecular Cardiovascular Biology, Children's Hospital Research Foundation, Cincinnati, Ohio 45229

Submitted 24 July 2002 ; accepted in final form 9 May 2003

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX DISCLOSURES REFERENCES

 
In large mammals there is a correlation between microtubule network densification and contractile dysfunction in severe pressure-overload hypertrophy. In small mammals there is a similar correlation for the shift to -myosin heavy chain (MHC), a MHC isoform having a slower ATPase V_max. In this study, murine left ventricular (LV) pressure overload invoked both mechanisms: microtubule network densification and -MHC expression. Cardiac -MHC was also augmented without altering tubulin levels by two load-independent means, chemical thyroidectomy and transgenesis. In hypertrophy, contractile function of the LV and its cardiocytes decreased proportionally; microtubule depolymerization restored normal cellular contraction. In hypothyroid mice having a complete shift from -MHC to -MHC, contractile function of the LV and its cardiocytes also decreased, but microtubule depolymerization had no effect on cellular contraction. In transgenic mice having a cardiac -MHC increase similar to that in hypertrophy, contractile function of the LV and its cardiocytes was normal, and microtubule depolymerization had no effect. Thus, although both mechanisms may cause contractile dysfunction, for the extent of MHC isoform switching seen even in severe murine LV pressure-overload hypertrophy, microtubule network densification appears to have the more important role.

left ventricle; heart failure; cytoskeleton; contractile proteins; sarcomeres

If a long-term load increase is neither too severe initially nor indefinitely progressive, cardiac stress is renormalized and compensated hypertrophy ensues. But hypertrophic compensation is often abrogated by progressively abnormal contractile performance per unit mass of myocardium, even when function at the organ level is maintained initially by the mass increase itself. That is, even when hypertrophy is appropriate to the load imposed, specific phenotypic changes occurring during this growth response may render compensation imperfect, such that congestive heart failure ensues.

This study focuses on two such phenotypic changes during severe pressure-overload cardiac hypertrophy that result from transcriptionally driven alterations either of the extramyofilament or of the myofilament cardiocyte cytoskeleton. Each serves as an example of deleterious concomitants to hypertrophy that may frustrate this compensatory growth response to very specific load alterations.

The first such alteration, increased microtubule network density that imposes a viscous load on the activated sarcomere, is based on transcriptional upregulation both of the ₁-tubulin member of the -tubulin multigene family and of microtubule-associated protein 4 (6). The second such alteration, decreased shortening and relaxation velocity of the activated sarcomere, is based on transcriptional upregulation of -myosin heavy chain (MHC), a MHC isoform having a relatively low ATPase V_max (32).

The initial impetus for this study was the need to ascertain whether the extramyofilament cytoskeletal phenotype seen in severe pressure-overload hypertrophy of the right (RV) and left (LV) ventricles of several larger mammalian species (6), including humans (39), obtains in the mouse, such that mechanistic studies in which load alterations could be imposed on engineered cardiac cytoskeletal genetic backgrounds would be possible. However, because it has been thought that the decrease in the ratio of -MHC to -MHC has a major role in the contractile dysfunction seen in rodent cardiac hypertrophy (32) that would contribute to an unknown extent to any microtubule-based contractile dysfunction, and because this has been thought to be important in human heart disease as well (19, 26), we elected to assess simultaneously both extramyofilament and myofilament cytoskeletal alterations in the setting of murine LV pressure-overload hypertrophy.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX DISCLOSURES REFERENCES

 
Animal Models

All animal usage was under protocols approved by the institutional animal care committee in accordance with National Institutes of Health guidelines. Six groups of mice were used in these experiments: 1) control mice from the C57BL/6 and FVB/N strains; 2) LV pressure-overloaded mice from the C57BL/6 strain; 3) hypothyroid mice from the C57BL/6 strain; and 4) two groups of transgenic mice from the FVB/N strain.

The intent behind using LV pressure-overloaded mice from the C57BL/6 strain was to increase the expression of both tubulin and -MHC via severe pressure-overload hypertrophy produced by transverse aortic constriction. These mice were anesthetized with ketamine (50 mg/kg ip) and xylazine (2.5 mg/kg ip), intubated, and placed on a respirator. The aortic arch was exposed surgically and tightly constricted between the origins of the two carotid arteries with a 7-0 silk suture tied over both the aorta and a juxtaposed 27-gauge needle, causing complete occlusion of the aorta. The needle was withdrawn, resulting in a severely stenotic aortic lumen, and the animals were allowed to recover for 4–8 wk (12).

The intent behind using hypothyroid mice from the C57BL/6 strain was to increase the percentage of cardiac -MHC via a thyroxine-dependent, pressure overload-independent mechanism. These mice were fed an iodine-deficient diet containing 0.15% 6-propyl-2-thiouracil (PTU; diet TD 97061, Harlan Teklad) for 4 wk (28).

The intent behind using transgenic mice from the FVB/N strain was to increase the percentage of cardiac -MHC via a genetic rather than pressure overload-dependent mechanism. The full-length mouse -MHC cDNA was made with a partial cDNA isolated from a mouse embryoid body library and a series of PCR reactions. The full-length product was sequenced multiple times in both directions, and all errors were corrected by site-specific mutagenesis with PCR. The full-length product contained only the coding regions, and it was subsequently linked to the mouse -MHC promoter. The final construct, containing the exon-intron organization of the -MHC promoter linked to the entire -MHC cDNA, was digested free of vector sequence with NotI, purified from agarose, and used to generate transgenic mice (31). Founder mice were identified by PCR and confirmed by genomic Southern blots. Multiple -MHC mouse lines were made; we retained those that had homogeneous transgene expression in all ventricular cardiocytes by immunofluorescence microscopy of transmural frozen sections of the LV myocardium stained with a -MHC antibody.

The transgenic mouse lines so selected were found to homogeneously express -MHC in the LV at either 25 ± 1% or 47 ± 2% of total MHC. Two lines having each of these two levels of -MHC expression were retained for these experiments, and the results found for a given level of -MHC expression were verified in members of the second strain having that level of -MHC expression in each case. Because the primary purpose of creating these transgenic mice was to mimic any effects of the level of cardiac -MHC expression produced by hemodynamic pressure overloading, the results reported here for transgenic mice focus on the 25% cardiac -MHC mice unless otherwise specified.

Characterization of LV Contractile Function in Intact Animals

Simultaneous echocardiography and cardiac catheterization were performed in mice anesthetized as described in Animal Models (24, 30). In addition, phenylephrine was used to measure contractile reserve. This was first done in 14 control C57BL/6 mice to establish a normal relationship. After baseline measurements, changes in LV function were assessed via the fractional shortening vs. systolic wall stress relationship during phenylephrine (3.0 µg/kg iv) infusion. Linear regression analysis was performed with two data points from each mouse, and 95% prediction intervals above and below this mean relationship were calculated and plotted. Data from the other groups of mice were plotted with reference to this normal relationship.

For catheterization, limb lead needle electrodes were used for continual electrocardiographic monitoring. Nasal oxygen was delivered at 2 l/min to mice on a heated rodent surgical table. A 1.4-French Mikro-Tip catheter transducer (Millar Instruments, Houston, TX) was introduced into the right carotid artery and advanced into the ascending aorta and then into the LV. Heart rate and systolic and diastolic pressures were continuously monitored and recorded during the study.

For echocardiography, a 15-MHz transducer (Sonos 5500, Agilent Technologies, Andover, MA) was placed on a layer of acoustic coupling gel applied to the hemithorax. Measurements of LV dimension and posterior wall thickness were made at end systole and end diastole according to the leading edge convention of the American Society of Echocardiography. Three to six beats were averaged for each measurement. Fractional shortening, systolic wall stress, and LV mass were calculated as we have described previously (30) with standard equations.

Murine Cardiocyte Isolation

Mice (25–35 g) were anesthetized as described in Animal Models and heparinized (200 IU ip). After we scaled down our enzymatic perfusion method (20) to accommodate the murine heart, and with mouse-specific methods described elsewhere (17, 18, 25), the hearts were rapidly excised, cannulated via the aorta, and perfused in Langendorff mode at 80 cmH₂O pressure and 37°C. Hearts were initially perfused for 5 min with 1.8 mM Ca²⁺ Tyrode solution containing (in mM) 137 NaCl, 5.4 KCl, 1.8 CaCl₂, 0.5 MgCl₂, 10 HEPES, and 10 glucose at pH 7.4 and then in Ca²⁺-free buffer containing 0.05% collagenase D (Boehringer-Mannheim, Indianapolis, IN) and 0.01% protease XIV (Sigma, St. Louis, MO). After the hearts were palpably flaccid at 6–8 min, the digestion solution was washed out with Ca²⁺-free Tyrode solution for 30 s. The hearts were removed from the cannula, and the LV including septum was separated, minced, and gently agitated, allowing cardiocytes to be dispersed in the Ca²⁺-free Tyrode solution. After 15 min, the cardiocytes were resuspended in Tyrode buffer with gradually increasing Ca²⁺ concentration ([Ca²⁺]) in steps from 0.06 to 0.24 to 0.60 to 1.20 mM Ca²⁺ at room temperature. They were maintained thereafter and studied at 32°C in 1.2 mM Ca²⁺ oxygenated Krebs-Henseleit buffer at pH 7.4.

Sarcomere and Cellular Contractile Function and Ca²⁺ Transients in Unloaded LV Cardiocytes

Sarcomere mechanics. This was measured with our laser diffraction technique (16) during steady-state 0.4-Hz contractions both at baseline and after microtubule depolymerization (35) via a 1-h exposure to either colchicine (10 µM) or vincristine (12 µM).

Cardiocyte mechanics. This was measured with the IonOptix system (17, 18, 25). Cardiocytes were viewed with an inverted microscope; the cell image, collected by a x40 objective lens, was diverted to the side port of the microscope and transmitted to a video camera (MyoCam; IonOptix, Milton, MA). Cardiocyte length and contraction amplitude were recorded in real time with a video edge detector and specialized data acquisition software (SoftEdge and IonWizard; IonOptix). The camera was adapted to acquire images at a 240-Hz frame rate and a cell length measurement time resolution of 4.2 ms. The signal-to-noise ratios were significantly improved by averaging 10 sequential runs. Calibration of the system was accomplished with a micrometer and the data acquisition software.

Cardiocyte Ca²⁺ transients. Ca²⁺ transients were also measured with the IonOptix system (17, 18, 25). Freshly dissociated LV cardiocytes were incubated in a solution containing 1.5 µM membrane-permeant fura 2-AM (Molecular Probes, Eugene, OR) for 10 min at room temperature. After the fura 2-AM in the loading solution was washed out, an additional 40 min was allowed for the deesterification of the fura 2 ester in the cells. Probenecid (0.5 mM) was included throughout this procedure to prevent leakage of fura 2 from the cells. The extent of loading of fura 2 into noncytosolic compartments was tested by the MnCl₂ quenching method (22). The results indicated that >90% of the recorded fura 2 fluorescence emanated from the cytosolic space. Because fura 2 is a Ca²⁺ chelator and is known to buffer cytosolic Ca²⁺, and this Ca²⁺ buffering may affect cytosolic Ca²⁺ concentrations and cardiocyte function, we selected a fura 2 loading concentration that did not affect cell shortening by comparing the percent cell shortening with and without fura 2 loading. In addition, the fura 2-loaded cardiocytes were required to have consistent fura 2-dependent fluorescence (the ratio of 360-nm excited fura 2 fluorescence to 360-nm excited cell autofluorescence was always 4–5). Cytosolic Ca²⁺ transients were measured with a dual-fluorescence calcium ion sensing system (IonOptix). The fura 2-loaded cardiocytes were excited at 360 ± 6.5 and 380 ± 6.5 nm. Emission fluorescence was measured at 510 ± 15 nm. The fluorescence ratio F₃₆₀/F₃₈₀ was independent of intracellular fura 2 concentration, cell geometry, and excitation light intensity and thus reflected intracellular [Ca²⁺] ([Ca²⁺]_i).

Sarcomere and Cellular Contractile Function of Externally Loaded Cardiocytes

Sarcomere mechanics. We used our technique (16) for characterizing sarcomere mechanics during variably afterloaded contractions of isolated cardiocytes via defined alterations of superfusate viscosity, where force is imposed on unfettered cardiocytes as a series of known viscous loads that provide resistance to cardiocyte shape changes during contraction. The resultant viscosity-velocity relationship is closely analogous to the classic force-velocity relationship used to characterize isolated linear cardiac muscle. This was done here by increasing the viscosity of the superfusate (32°C, 1.2 mM Ca²⁺ oxygenated Krebs-Henseleit buffer, pH 7.4) in graded, reproducible steps from 1 through 400 cP by the addition of biologically inert high-molecular-weight methylcellulose, which alters neither superfusate osmolarity nor resting sarcomere length.

Cardiocyte mechanics. The methods that we have used before (38, 41) to characterize the constitutive properties of isolated cardiocytes were modified for the present purpose of examining the effects of variable afterloading on cardiocyte mechanics. The basis for and the details of this technique are given in the APPENDIX. In essence, the cardiocytes were cast into a series of collagen gels having known and increasing viscosity and stiffness, such that the cardiocytes were subjected to a graded increase in afterload while whole cell mechanics were defined.

Northern blots, Western blots, and immunofluorescence microscopy. Total RNA was extracted from fresh myocardium and Northern blotted (27) with ³²P-end-labeled -MHC (51-mer) and -MHC (54-mer) oligonucleotide probes. Each blot was normalized by stripping and reprobing with a psoralen-biotin-conjugated GAPDH probe (Ambion). For immunoblots, freshly isolated cardiocytes or myocardium was homogenized, and the protein extract was subjected to PAGE, transferred to polyvinylidene difluoride membranes, and immunoblotted with antibodies to -MHC [A4.951, American Type Culture Collection (ATCC); 1:4,000], -MHC (BA-G5, ATCC; 1:8,000), -tubulin (DM1B, InnoGenex; 1:3,000), or GAPDH (6C5, Research Diagnostics; 1:8,000). Confocal micrographs were obtained (35) with antibodies to -tubulin (B-5-1-2; Sigma; 1:200) or to -MHC (NOQ7.5.4D, Sigma; 1:100).

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX DISCLOSURES REFERENCES

 
Characteristics of the Animal Models

The major features of the animal models used in this study are summarized in Table 1 and Fig. 1. Most notably, when examined in vivo only the aortic-banded mice demonstrated either LV hypertrophy (ratio of LV weight to body weight) or dimensional (LV end-diastolic diameter) and functional (stress vs. shortening) markers of LV contractile failure. But because these data were obtained via simultaneous echocardiography and cardiac catheterization in anesthetized animals, we were concerned that subtle effects on cardiac function of the rather modest level of -MHC expression in the transgenic 25% -MHC mice that we wished to compare directly to the aortic stenosis mice might have been obscured in this setting. However, echocardiograms obtained in conscious FVB/N control and FVB/N transgenic 25% -MHC mice having heart rates of 630–670 beats per minute (bpm) showed no difference between these two groups in terms of indexes measuring the extent and velocity of LV myocardial shortening. Thus fractional shortening was 48.9 ± 0.6% in control vs. 47.5 ± 0.7% in transgenic 25% -MHC mice, and mean velocity of circumferential fiber shortening (V_cf) was 0.94 ± 0.03 s^–1 in control vs. 0.91 ± 0.02 s^–1 in transgenic 25% -MHC mice. In contrast, but as would be expected (11), a complete switch from -MHC to -MHC in the conscious hypothyroid PTU-treated mice had a clear effect on the echocardiographic velocity of LV myocardial shortening, with V_cf being reduced to 0.44 ± 0.01 s^–1 in these mice, although their conscious heart rate was only 438 ± 8 bpm.

View larger version (25K): [in this window] [in a new window]   Fig. 1. Left ventricular (LV) function via simultaneous echocardiography and cardiac catheterization for the groups of mice used in this study. The control and -myosin heavy chain (MHC) transgenic groups were characterized at baseline and after phenylephrine stimulation. The solid and parallel dashed lines in both A and B define the relationship between midwall fractional shortening and systolic wall stress, as well as its 95% prediction interval, calculated with a least-squares linear regression analysis for 14 normal C57BL/6 mice. A: individual values for the mice used in this study. B: summary data for these same mice when grouped as shown. PTU, 6-propyl-2-thiouracil.

Expression of -Tubulin

Data on expression of -tubulin, in terms of protein level and microtubule network structure, are given in Fig. 2. Only LV myocardium from the aortic-banded mice demonstrated an increase in either -tubulin concentration or cardiocyte microtubule network density.

View larger version (57K): [in this window] [in a new window]   Fig. 2. Assessment of tubulin protein levels and microtubule network density. The experimental groups are the same as those in Fig. 1. The example immunoblot (left top) was prepared with an isoform-nonselective -tubulin antibody (DM1B); 10 µg of total protein/lane from LV cardiocytes from 2 mice from each of the 4 indicated groups was loaded. For the confocal micrographs (right), an antibody to -tubulin (B-5-1-2) was used; the scale bar represents 25 µm. For the summary densitometric total -tubulin immunoblot analysis (left bottom), values are means ± SE, and n = no. of mice. Statistical comparison was by 1-way analysis of variance, followed by Scheffé's post hoc test. *P < 0.01 for difference from the control group.

Expression of -MHC and -MHC

Data on expression of -MHC and -MHC at the protein level are given in Fig. 3. There was essentially no -MHC in LV myocardium from control mice and no -MHC in LV myocardium from PTU-treated mice. The aortic band group homogeneously expressed -MHC in the LV at 15 ± 3% of total MHC; the first transgenic strain homogeneously expressed -MHC in the LV at 25 ± 1% of total MHC; and the second transgenic strain homogeneously expressed -MHC in the LV at 47 ± 2% of total MHC. Thus LV myocardium from aortic-banded mice and transgenic 25% -MHC mice had fairly similar levels of -MHC protein expression. These data on the mRNA level for the control and aortic-banded groups of mice are given in Fig. 4. When normalized to GAPDH expression, -MHC mRNA was almost undetectable in LV myocardium from control mice and was 14% of total MHC mRNA in aortic-banded mice.

View larger version (56K): [in this window] [in a new window]   Fig. 3. Assessment of protein levels for MHC isoforms. The experimental groups are the same as those in Fig. 1. For the example immunoblots in A, prepared with antibodies to -MHC (A4.951) or -MHC (BA-G5) as indicated, 10 µg of total protein/lane isolated from LV cardiocytes from 2 mice from each of the 4 indicated groups was loaded. For the quantitation in B, because of differing antibody affinities, 2 µg of LV cardiocyte protein/sample was resolved on SDS-PAGE and silver stained; an example is shown in the inset, where the sample sources were as follows: lane 1, control; lane 2, aortic band; lane 3, 25% -MHC transgenic; lane 4, PTU. Immunoblotting of an identically prepared gel established that the upper band is -MHC, and the lower band is -MHC. For the summary densitometric analysis of silver-stained gels, values are means ± SE, and n = no. of mice. Statistical comparisons were by 1-way analysis of variance followed by Scheffé's post hoc test. *P < 0.01 for difference for that variable from the control group.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Assessment of mRNA levels for MHC isoforms. A: example Northern blots of total RNA obtained from 2 normal C57BL/6 mice (lanes 1 and 2) and 2 aortic-banded C57BL/6 mice (lanes 3 and 4). For -MHC, 2 µg of RNA was loaded in each lane. For -MHC, 8 µg of RNA was loaded in lanes 1 and 2, and 2 µg of RNA was loaded in lanes 3 and 4. For the bottom pair of blots, the same membranes were stripped and reprobed with a mouse GAPDH probe. B: summary densitometric analysis of -MHC and -MHC Northern blots. Values are means ± SE, and n = no. of mice. Statistical comparisons between the two groups were by Student's unpaired t-test. *P < 0.01 for difference for that variable from the control group.

Sarcomere Mechanics

Both the example and the summary data in Fig. 5 show that during sequential sampling of the same LV cardiocytes before and after colchicine treatment, microtubule depolymerization was without significant effect on sarcomere motion and its time derivative in cardiocytes from control, transgenic 25% -MHC, and PTU-treated mice, where the latter group exhibited significant abnormalities in both of these contractile indexes at both time points. However, in cardiocytes from the aortic-banded mice, the initial contractile abnormalities were reversed after colchicine-induced microtubule depolymerization. The photomicrographic insets in Fig. 5 show for the transgenic 25% -MHC mice that -MHC is homogeneously expressed both in the LV myocardium and in all of the constituent cardiocytes. For the aortic-banded mice, there was a similar overall LV myocardial level of -MHC expression, but with a greater level in the higher-wall stress subendocardium than in the lower-wall stress subepicardium; it would follow that, as seen here, there was some variation in the expression level of -MHC in individual LV cardiocytes. As was the case for the immunoblots in Fig. 3, -MHC expression was undetectable in control myocardium and cardiocytes.

View larger version (40K): [in this window] [in a new window]   Fig. 5. Effect of colchicine-induced microtubule depolymerization on sarcomere mechanics. The extent (A) and velocity (B) of sarcomere shortening are shown for LV cardiocytes from the same experimental groups as those in Fig. 1; the same cells were studied before and after colchicine treatment. The 3 left panels are examples, and the right panels show summary data, where n = number of mice. Data for control FVB/N were the same as those for the control C57BL/6 mice shown here. Only cells that remained quiescent when not stimulated throughout the study protocol were included (5 cells/mouse). Resting sarcomere length, ranging in these groups from 1.83 ± 0.01 to 1.86 ± 0.01 µm, did not differ among the groups. The photomicrographic insets show LV myocardium stained with a -MHC antibody (NOQ7.5.4D; top) and the same isolated cells via modulation contrast optics and via -MHC antibody staining (bottom) (scale bars represent 400 µm). For the summary sarcomere mechanics data values are means ± SE. Statistical comparisons among groups were by 1-way analysis of variance, followed by Scheffé's post hoc test; within a group they were by Student's paired t-test. *P < 0.01 for difference from the control group either before or after colchicine treatment. P < 0.01 for difference from pretreatment value within a group.

Because, as shown in Fig. 6, colchicine-induced microtubule depolymerization results in an increase in -tubulin heterodimer concentration, and because it has been suggested that this increase in free tubulin has a positive inotropic effect (10), we elected to study sarcomere mechanics before and after microtubule depolymerization by vincristine, which effectively removes soluble -tubulin heterodimers from the cytoplasm via paracrystal formation (9). The example and summary data in Fig. 7 show that during sequential sampling of the same LV cardiocytes before and after vincristine treatment, microtubule depolymerization did not have a significant effect on sarcomere motion and its time derivative in cardiocytes from control mice. In cardiocytes from aortic-banded mice, however, vincristine-induced microtubule depolymerization restored the initial contractile abnormalities to values not significantly different from control values. It should be noted that, as shown in Fig. 6, after comparable reductions in microtubule network density caused by colchicine or by vincristine, -tubulin heterodimer concentration was increased by the former agent and greatly decreased by the latter agent.

View larger version (77K): [in this window] [in a new window]   Fig. 6. Effects of colchicine and vincristine on the microtubules of normal cardiocytes. The confocal micrographs (top) were prepared with an -tubulin antibody (B-5-1-2), and the immunoblots (bottom) were prepared with an isoform-common -tubulin antibody (DM1B). For the immunoblots, the free -tubulin heterodimer fraction (lanes 1) was separated from the insoluble tubulin fraction (lanes 2) (27, 35). For vincristine-treated cells, the insoluble fraction presumably consists largely of tubulin paracrystals rather than microtubules (9). Scale bar represents 25 µm.

View larger version (28K): [in this window] [in a new window]   Fig. 7. Effect of vincristine-induced microtubule depolymerization on sarcomere mechanics. The extent (A) and velocity (B) of sarcomere shortening are shown; the format is the same as that in Fig. 5. The same cells were studied before and after vincristine treatment. Resting sarcomere length (1.85 ± 0.01 µm in control and 1.84 ± 0.01 µm in aortic band) did not differ between the 2 groups. For the summary sarcomere mechanics, data values are means ± SE. Statistical comparisons between the 2 groups were by Student's unpaired t-test; within a group they were by Student's paired t-test. *P < 0.01 for difference from the control group either before or after vincristine treatment. P < 0.01 for difference from pretreatment value within a group.

Cardiocyte Mechanics and Ca²⁺ Levels and Transients

We also wanted to evaluate the potential importance of altered calcium metabolism to these contractile function data, either as a causative abnormality in the aortic-banded mice or as a compensatory mechanism in the -MHC transgenic mice. Thus there are three important features of the example and summary data in Fig. 8. First, as was the case for sarcomere mechanics, the contractile behavior of LV cardiocytes from control mice, and the simultaneously measured Ca²⁺ levels and transients, were unaffected by colchicine treatment. Second, the initially abnormal contractile behavior of LV cardiocytes from aortic-banded mice was normalized after colchicine treatment. Third, the abnormal Ca²⁺ levels and transients seen in these cardiocytes were unaffected by colchicine treatment.

View larger version (25K): [in this window] [in a new window]   Fig. 8. Effects of colchicine-induced microtubule depolymerization on cardiocyte mechanics and intracellular Ca2+ concentration ([Ca2+]i). The extent and velocity of cellular shortening (A) as well as [Ca2+]i levels and transients (B) are shown for the same LV cardiocytes from C57BL/6 control mice (n = 8) and C57BL/6 aortic-banded mice (n = 6). The left panels give examples, and the right panels show summary data, where n = number of cells. For the summary data, values are means ± SE. Statistical comparisons between the 2 groups were by Student's unpaired t-test; within a group they were by Student's paired t-test. *P < 0.01 for difference from the control group either before or after colchicine treatment. P < 0.01 for difference from pretreatment value within a group.

These data identified the primary role of microtubule network densification as opposed to abnormal calcium metabolism in the LV contractile dysfunction of these aortic-banded mice. However, in further considering the apparent lack of an effect on contractile function of -MHC expression at the level of 15–25% of total MHC, we wondered whether the normal behavior of the -MHC transgenic mice in terms of LV function in vivo and sarcomere mechanics of isolated LV cardiocytes in vitro might be the result of a change in excitation-contraction coupling compensatory for any mechanical effects of the expression of this protein, such that decrements in the velocity and extent of shortening resulting solely from a decreased ATPase V_max might be obscured. Therefore, we repeated the protocol used to generate the data in Fig. 8 with LV cardiocytes from FVB/N control and transgenic 25% -MHC mice. No difference between these two groups was found: The cardiocyte shortening extent was 0.094 ± 0.004 vs. 0.095 ± 0.002 cell lengths, the cardiocyte shortening velocity was 1.88 ± 0.12 vs. 1.84 ± 0.09 cell lengths/s, peak [Ca²⁺]_i was 395 ± 30 vs. 376 ± 13 nM, and resting [Ca²⁺]_i was 148 ± 14 vs. 143 ± 11 nM (control vs. transgenic 25% -MHC mice).

Externally Loaded Contractions

Because it is possible that unloaded shortening might not be the most sensitive mechanical setting for detecting changes in contractile function dependent on MHC ATPase V_max, we elected to examine the interplay of these two factors during variably loaded cardiocyte contractions in terms of both sarcomere and whole cell mechanics. As seen in Fig. 9, there was no selective effect of increasing the percentage of -MHC from 0% to 25% to 47% on sarcomere mechanics during variably afterloaded contractions. That is, similar decrements in the extent and velocity of sarcomere shortening with increasing cardiocyte viscous loading were seen for all three percentages of -MHC. Similarly, as seen in Fig. 10, increasing the percentage of -MHC from 0% to 25% to 47% had no significant effect on cellular mechanics as the viscosity and stiffness of the external load were increased.

View larger version (31K): [in this window] [in a new window]   Fig. 9. Effect of viscous external loading on sarcomere mechanics in control murine cardiocytes (0% -MHC) and in cardiocytes from transgenic mice expressing 25% or 47% -MHC. Left: an example in a control cardiocyte of the decrements in the extent (A) and velocity (B) of sarcomere shortening induced by progressive increases in superfusate viscosity. Right: summary data for the effects of increasing superfusate viscosity on sarcomere mechanics in cardiocytes from the 3 groups noted above, where n = number of mice (3 cells/mouse were studied). There was no statistical difference at any external load among the 3 groups for either the extent or the velocity of sarcomere shortening.

View larger version (34K): [in this window] [in a new window]   Fig. 10. Effect of external loading via gel embedment on cellular mechanics in control murine cardiocytes (0% -MHC) and in cardiocytes from transgenic mice expressing 25% or 47% -MHC. A: an example in control cardiocytes of the decrements in the extent (top) and velocity (bottom) of cellular shortening induced by progressive increases in gel stiffness and viscosity. B: summary data for the effects of increasing gel density on cellular mechanics in cardiocytes from the 3 groups noted above, where n = number of mice (4 cells/mouse were studied). There was no statistical difference at any external load among the 3 groups for either the extent or the velocity of cellular shortening.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX DISCLOSURES REFERENCES

 
The intent of this study was to address two questions about the properties of severely pressure-overloaded myocardium. First, do the microtubule network densification and associated functional consequences seen in several larger mammals (6, 39) also obtain in the mouse? The answer to this question is specifically important to us for our ongoing work using pressure-overloaded transgenic mice to uncover the mechanisms responsible for this cytoskeletal change. It is important more generally for a variety of other studies of hemodynamically overloaded murine models, in which it is necessary to know whether this cytoskeletal alteration might be having unanticipated effects on the cardiac properties under investigation. Second, does a reduction in the ratio of -MHC to -MHC to the extent reported with pressure-overload hypertrophy in mammals ranging in size from mice (32) to humans (19, 26) contribute to a significant degree to the attendant contractile dysfunction? Although in our own work (36, 37) we have not found a myosin isoform shift in two large-animal models of severe pressure-overload cardiac hypertrophy, the answer to this question is important for the same two reasons that are pertinent to the first question, in that it is essential to know the functional significance of this potentially confounding variable when studying other properties of hypertrophied myocardium, especially in hemodynamically overloaded transgenic mice.

The murine pressure-overload model generated here to answer these questions exhibited very substantial LV hypertrophy (Table 1), of a degree quite similar to that seen in our LV and RV pressure-overload hypertrophy models in several larger species (6). This is an important point. A critical consideration when designing, studying, and drawing inferences from animal models of human hypertrophic cardiac disease is the long-established fact that only a twofold or greater mass increase is regularly associated with pathological LV hypertrophy in response to pressure overloading in the clinical setting (14, 21). Pertinent to this point in terms of the murine surgical model of human disease generated for the present study is the fact that we do not find microtubule network densification in humans with pressure-overload LV hypertrophy until the load is severe enough to cause marked abnormalities in the LV stress-shortening relationship (39), similar to that shown in Fig. 1 for our murine LV pressure-overload model.

As seen in Table 1 and Fig. 1, only the aortic-banded mice exhibited major LV contractile dysfunction in vivo although the PTU-treated mice had a modest (P = 0.02) decrease in fractional shortening. The aortic-banded animals were also the only mice in which LV tubulin increased, with concomitant microtubule network densification (Fig. 2). Thus, absent hypertrophy, a decreased -MHC-to--MHC ratio, whether effected hormonally or transgenically (Fig. 3), was not associated with major contractile abnormalities in vivo until, as in the PTU-treated mice, the percentage of -MHC was very high. A further important point to note in Fig. 3 is that the transgenic line that we selected for study homogeneously expressed -MHC in the LV (Fig. 5) at a level close to that seen in the aortic-banded animals. A second transgenic line, which homogeneously expressed -MHC in the LV at 47 ± 2% of total MHC, showed contractile function very similar both to that of the two control groups and to that of the 25% -MHC line.

In vitro, only the aortic-banded mice and the PTU-treated mice exhibited contractile dysfunction in terms of sarcomere mechanics in isolated LV cardiocytes (Figs. 5 and 7), and only the aortic-banded mice showed an increased density of the cardiocyte microtubule network (Fig. 2). In the aortic-banded mice, but in no other group, microtubule depolymerization by colchicine (Fig. 6) affected contractile function (Fig. 5), and for these mice the maximum extent and velocity of sarcomere shortening were restored to normal. This is the same microtubule-dependent effect that we have reported previously (6) for unloaded contractions of isolated cardiocytes and for loaded contractions of in vitro tissue and in vivo myocardium in the pressure-overloaded RV and LV of several species. In contrast, this change in MHC isoform composition was without an independent effect on sarcomere mechanics in the aortic-banded or the 25% or 47% -MHC transgenic mice. Only in the PTU-treated mice, in which -MHC constituted essentially all of the MHC, was there a depression of contractile function. This contractile abnormality was due to the effective replacement of -MHC by -MHC and/or to multiple other myocardial effects of the hypothyroid state (1), but it is clear both that an increase in -MHC from 0% to 15–25% or to 47% of total MHC does not cause contractile dysfunction without hypertrophy and that the contractile dysfunction with hypertrophy is not caused by the altered MHC composition of the myocardium.

Thus it would appear that increased microtubule network density, and not altered MHC isoform expression, is an independent variable responsible for contractile dysfunction in substantial pressure-overload cardiac hypertrophy. We have attributed (33) this contractile dysfunction to increased cytoplasmic viscosity caused by microtubule network densification. However, this conclusion is based in part on experimental interventions that cause microtubule depolymerization. This, in turn, raises concerns about experimental artifacts, both because of possible unanticipated effects of the resultant increase in free -tubulin heterodimer concentration and because of the disruption of normal microtubule function in the homeostasis of interphase cells. It has, for instance, been reported that colchicine increases depolarizing L-type Ca²⁺ channel current density and the [Ca²⁺]_i transient in adult cardiocytes (10), in which -tubulin heterodimers would act as a functional analog of G proteins to activate adenyl cyclase when their concentration is increased by colchicine. However, other data in that same study show that taxol, which markedly reduces the cardiac -tubulin heterodimer concentration (40), is without effect on these same calcium variables, and we found (35) that there is with hypertrophy, along with an increase in microtubules, a significant and persistent increase in free -tubulin heterodimer concentration; however, in contrast to what this mechanism would predict if it has functional significance, there is a marked decrement rather than increment in contractile function. Furthermore, it has since been shown by others in intact neonatal (23) and adult (2) cardiocytes that colchicine and tubulin dimers are without effect either on Ca²⁺ signaling or on contractile function. Most pertinent, however, to any consideration of potentially direct inotropic effects of microtubule depolymerization on contractile function is the fact that although we consistently find (6) only a 5–10% increase in the extent and velocity of shortening of sarcomeres, cells, and tissue from normal hearts, as well of as the normal heart itself in vivo after microtubule depolymerization by any means, we find a much greater response in normal and hypertrophied hearts to purposive inotropic inputs.

Nonetheless, we elected to address directly these possibilities, consequent to microtubule depolymerization, of inotropic effects of free -tubulin heterodimers and of increased [Ca²⁺]_i. The data in Fig. 7 show that microtubule depolymerization via vincristine, which as shown in Fig. 6 decreases the soluble -tubulin heterodimer concentration, duplicates the mechanical effects on cardiocytes from aortic-banded mice of microtubule depolymerization via colchicine, which increases the soluble -tubulin heterodimer concentration. A positive inotropic effect of -tubulin heterodimers independent of the direct physical consequences of microtubule depolymerization as an explanation for the ameliorative effects of this intervention on the contractile dysfunction of pressure-hypertrophied myocardium is therefore excluded. The data in Fig. 8 show that, as expected (7, 15), there are distinct abnormalities of calcium kinetics in pressure-hypertrophied cardiocytes. However, although colchicine-induced microtubule depolymerization returns cellular contractile function to normal, it has no effect on [Ca²⁺]_i, and the abnormal calcium kinetics are not of sufficient degree to prevent this normalization of contractile function.

Role of MHC Isoforms in Cardiac Contractile Function

In the context of a large body of work reporting the effects of MHC isoform shifts on contractile function, and especially that in very unambiguous in vitro motility assays (13), this aspect of the present results was unexpected, because we found no real effect of this variable when increasing -MHC from 0% through 47% of total MHC, whether hemodynamically or transgenically. Thus we are not sure why in an earlier study of aortic-banded mice expressing -MHC at 15% of total MHC there was a quite unusual dissociation between decreased velocity but not extent of cellular shortening (8); these two mechanical variables changed in concert in the aortic stenosis mice reported here and for a variety of cardiac pathophysiological states in other species reviewed elsewhere (3–6). We are also not sure why in another study of mice expressing cardiac -MHC at 12% of total MHC there was a modest decrease in the rate of pressure rise in isolated hearts (34), but absent data defining intraventricular volumes as well as peak and developed pressures it is difficult to assess this single index in terms of defining ventricular function. Nor are we entirely sure why in the present study there is, in contrast, no measurable effect of an increase in -MHC on in vitro and in vivo contractile function. Nonetheless, the data in Figs. 9 and 10 argue clearly against any masking of such an effect by external loading during contraction in vitro, and it should be noted that the echocardiographic data given here were obtained during physiological loading in vivo.

Perhaps most simply, if fast and slow cross bridges are stochastically distributed within the sarcomere when both -MHC and -MHC are expressed, it may be that in terms of net cross bridge, i.e., sarcomere, kinetics the faster bridges mask the effects of the slower bridges over a broad range of loads that impede sarcomere motion until a rather high proportion of the low-ATPase V_max -MHC cross bridges is reached. But because this question deserves to be explored in more detail, we generated Fig. 11 as a discussion point. The present study shows that the velocities of in vivo myocardial and in vitro sarcomere and cardiocyte shortening are not significantly altered by the percentage of -MHC until this exceeds 47%; this is shown for peak sarcomere shortening velocity by the concave solid line in Fig. 11. Here, there is very little decrease in shortening velocity over a -MHC range of 0–47%, but there is a major decrease over the range of 47–100%. These results differ substantially from those of the Warshaw group (13), where even the small increase in -MHC from 0% to 10% caused a major reduction in the velocity of actin sliding over myosin heads in an in vitro assay system (Fig. 11, dashed line). The dashed line in Fig. 11 defines a convex relationship between actin sliding velocity, and the percentage of -MHC, such that from 0% to 20% -MHC there is a marked reduction in velocity, but over a range of 20–100% there is little further effect.

View larger version (22K): [in this window] [in a new window]   Fig. 11. Relationship between the percentage of -MHC and the velocity of myosin-based motion both in an in vitro assay of actin filament motion and in an intact, isolated cardiocyte assay of sarcomere-bounding Z-line motion. The dashed line and closed circles show the convex relationship between actin sliding velocity and percentage of -MHC; it was calculated from Harris et al. (Fig. 3B in Ref. 13). The solid line shows the concave relationship between sarcomere shortening velocity and percentage of -MHC; this curve was fit from data from the 4 indicated experimental groups of the present study.

Although the two sets of data in Fig. 11 do not initially appear congruent, they in fact may well be complementary. However, this only becomes clear when differences in the experimental conditions and resulting differences in the internal and external forces and loads are taken into account. For the actin motility assay, the only forces acting to move the isolated actin filament are produced by actin-myosin cross-bridge formation and release. Thus cross-bridge cycling determines internal force development, and all external viscoelastic and inertial loads are negligible. Although the MHC isoform-based differences in force development are small, because this is the only force present in an essentially unloaded system these small force changes cause readily measurable alterations in actin filament sliding velocity.

However, when the cross bridge is integrated into its native environment, with structural extra-cross-bridge complexity and relative quantity increasing as one progresses from sarcomere to cell to tissue to organ, one must consider, in addition to internal force development by the cross-bridge motor, the opposing external forces and loads. Here, small changes in MHC isoform-based differences in force in the context of large external loads cause no detectable changes in shortening velocity.

To put this in context, there are three major categories of external loads that act to oppose cross-bridge force development. 1) Inertial forces given by the combined mass of actin and myosin head molecules can be considered to be negligible both in the in vitro motility assay and in more complex systems, because for a uniform sliding motion the acceleration becomes null. 2) Viscous drag forces grow to be considerable and cannot be ignored in systems more complex than an in vitro motility assay. Both intracellular and extracellular viscosity components are major external factors opposing contraction. Although extracellular viscosity is the afterload opposing cardiocyte contraction, the intracellular viscosity opposes both actin sliding and myosin head motion. Moreover, the viscous drag forces have amplitudes directly proportional to shortening velocity, to actin sliding velocity, and, furthermore, to myosin head velocity, with significant consequences on the curvature of the corresponding force-velocity relationship. 3) Elastic forces must also be included in the force and load dynamic equilibrium of a more fully integrated system. Both the extracellular elastic forces stored in extracellular matrix molecules such as collagen and elastin and the intracellular elastic forces given by various elastic domains of sarcomeric molecules such as titin represent external forces that coordinate and control the whole cycle of cardiac contraction.

When viscous drag and elastic forces either are not considered or are effectively not present, as in an unloaded in vitro motility assay (Fig. 3B in Ref. 13), the relationship between actin filament velocity and percent -MHC has the convex profile given by the dashed line in Fig. 11, where very small increases in the percent -MHC above zero cause large decreases in actin sliding velocity, and as clearly shown in a recent study (29), increases in the percent -MHC at the higher end of the potential range have very little effect. However, our intact cardiocyte data include both viscous drag and elastic forces. When the action of the sarcomeric motor within this complex loading system is plotted, the concave velocity vs. percent -MHC relationship shown by the solid line in Fig. 11 results, where small changes in percent -MHC over a range of 0–47% do not cause a large decrease in peak shortening velocity, and this only becomes apparent at the higher end of this percentage range.

Taken together in the context of coexistent but competing forces, the two sets of data in Fig. 11 are therefore actually concordant. In the absence of any external forces in vitro, even a small change in internal forces causes a decrease in actin filament velocity. However, for the sarcomere in the living cell and for the whole cell, and by conceptual extension for higher levels of integration of the sarcomeric motor into its living environment, when this change in internal force occurs against the backdrop of counteracting external forces, no significant change in actin velocity is detected. It is only when the change in internal force becomes large, reaching the same order of magnitude as the competing external forces, that a change in internal developed force results in a decrease in shortening velocity such as that shown for the 100% -MHC mice. Thus, when the slower -MHC isoform approaches 100% of the total, Fig. 11 shows that the results of the in vitro motility assay and the present results become quite similar.

In conclusion, the data from this study show that the response of the cardiac myofilament cytoskeleton to pressure overloading in terms of altered MHC isoform expression does not contribute to the associated contractile dysfunction. The data from this and many other studies from this laboratory show that the response of the cardiac extramyofilament cytoskeleton to severe pressure overloading in terms of microtubule network densification does contribute to the associated contractile dysfunction.

	APPENDIX

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX DISCLOSURES REFERENCES

 
Contractile Mechanics of Externally Loaded Cardiocytes

Collagen gel viscoelastic properties. Lyophilized type I bovine collagen was solubilized in acetic acid, mixed with a physiological buffer, and brought to a neutral pH. The collagen solution was poured into a custom-made Teflon-coated mold to create a polymerized gel. The gel was then placed in a custom-designed, computer-controlled stretching device and uniaxially stretched with forces (stresses) sufficient to increase gel length (strain) at a rate of 5 mm/s to a maximum strain of 10–15% of initial gel length without producing plastic deformation of the gel. We have described the basis for these techniques in detail elsewhere (38, 41).

The viscoelastic properties of gels having varying collagen concentrations were determined from the uniaxial stretch experiments described above. Four concentrations of collagen were examined: 0.1%, 0.2%, 0.3%, and 0.5%. The relationship between the force applied to the gel (stress, ) and the resultant change in gel length (strain, ) was examined in each gel.

Gel stiffness was determined as shown in Fig. 12 from this stress vs. strain relationship. This relationship was exponential and was fit by the equation = ae + b, where a and b are constants and is the stiffness index. There was a linear relationship, shown in Fig. 13, between the stiffness index and the collagen concentration of the gel.

View larger version (11K): [in this window] [in a new window]   Fig. 12. Derivation of gel stiffness index. A: example showing data generated by stretching a 0.5% collagen gel; , stiffness index. B: schematic of the exponential stress () vs. strain () relationship. An increase in gel stiffness is reflected in a shift upward and to the left of the stress vs. strain curve, and this in turn increases ; a and b are constants.

View larger version (13K): [in this window] [in a new window]   Fig. 13. Linear relationship of gel viscoelastic properties vs. gel collagen concentration. A: linear relationship characterizing the stiffness index vs. collagen concentration. B: linear relationship characterizing the viscosity index vs. collagen concentration.

Gel viscosity was determined from conservation of energy, E = H + W, where E (change in strain energy), H (heat energy), and W (amount of applied work energy) were calculated from stress vs. strain data obtained during application of the load used to stretch the gel (load) followed by removal of load to allow the gel to return to its resting length (unload). The heat energy, H, was calculated from the hysteresis loop area formed during the loading-unloading cycles, whereas the total change in potential strain energy, E, was derived from the total area under the loading - curve. The gel viscosity index, , was derived as shown in Fig. 14 by examining the amount of heat energy (H, created by stretching the gel and then allowing the gel to return to resting length) normalized by the total variation of internal strain energy (E, created by stretching the gel): = H/E x 100 (%). There was a linear relationship, shown in Fig. 13, between the viscosity constant, or index, and the collagen concentration of the gel.

View larger version (23K): [in this window] [in a new window]   Fig. 14. Derivation of gel viscosity index. A: example showing a loading-unloading cycle of a 0.5% collagen gel; , gel viscosity index. B: schematic of the conservation of energy throughout a stretch-relaxation cycle. The normalized viscosity index is derived from the ratio between hysteresis area (representing energy lost through heat) and the area under the stress vs. strain loading curve (representing the total change in the potential energy level of the system).

Embedding cardiocytes in three-dimensional collagen gels. Murine cardiocytes were isolated with standard coronary arterial collagenase retroperfusion techniques from FVB wild-type mice and two transgenic mouse lines having cardiac-restricted expression of either 25% -MHC or 47% -MHC. The cells were suspended at 37°C in 1.2 mM Ca²⁺ oxygenated Krebs-Henseleit buffer solution at pH 7.4 containing laminin (0.5 µg/ml) and having a collagen concentration of 0.2%, 0.3%, or 0.5% type I bovine collagen. The suspensions were then poured into custom-made 2 x 3-cm chambers, cooled to 32°C to cause the collagen gels to polymerize, and maintained at 32°C in a 95% O₂-5% CO₂ incubator. The gel-embedded cardiocytes were then studied with the IonOptix system described in MATERIALS AND METHODS to assess cardiocyte contractile function.

Contractile properties of embedded cardiocytes. The methods used to measure cell shortening were described in MATERIALS AND METHODS. We showed previously (38) that for such gel-embedded cardiocytes there is <1% difference between gel strain and cell strain under these loading conditions. The embedded cardiocytes were studied at 32°C during electrically stimulated contractions (0.25 Hz, 5-ms pulse width, and voltage 10% above threshold).

	DISCLOSURES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX DISCLOSURES REFERENCES

 
This study was supported by National Heart, Lung, and Blood Institute (NHLBI) Grants HL-60546 and HL-66157 (J. Robbins) and by NHLBI Program Project Grant HL-48788 and by Merit and Research Enhancement Award Program awards from the Research Service of the Department of Veterans Affairs (G. Cooper).

	ACKNOWLEDGMENTS

 
We thank Mary Barnes for excellent technical assistance.

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. Cooper, Gazes Cardiac Research Institute, 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.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION APPENDIX DISCLOSURES REFERENCES

 

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				D. Scholz, C. F. Baicu, W. J. Tuxworth, L. Xu, H. Kasiganesan, D. R. Menick, and G. Cooper IV Microtubule-dependent distribution of mRNA in adult cardiocytes Am J Physiol Heart Circ Physiol, March 1, 2008; 294(3): H1135 - H1144. [Abstract] [Full Text] [PDF]

				G. Cooper IV Cytoskeletal networks and the regulation of cardiac contractility: microtubules, hypertrophy, and cardiac dysfunction Am J Physiol Heart Circ Physiol, September 1, 2006; 291(3): H1003 - H1014. [Abstract] [Full Text] [PDF]

				J. R. Wilding, C. A. Lygate, K. E. Davies, S. Neubauer, and K. Clarke MLP accumulation and remodelling in the infarcted rat heart Eur J Heart Fail, June 1, 2006; 8(4): 343 - 346. [Abstract] [Full Text] [PDF]

				G. Cheng, F. Qiao, T. N. Gallien, D. Kuppuswamy, and G. Cooper IV Inhibition of {beta}-adrenergic receptor trafficking in adult cardiocytes by MAP4 decoration of microtubules Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1193 - H1202. [Abstract] [Full Text] [PDF]

				F. J. Davis, J. B Pillai, M. Gupta, and M. P. Gupta Concurrent opposite effects of trichostatin A, an inhibitor of histone deacetylases, on expression of {alpha}-MHC and cardiac tubulins: implication for gain in cardiac muscle contractility Am J Physiol Heart Circ Physiol, March 1, 2005; 288(3): H1477 - H1490. [Abstract] [Full Text] [PDF]

				H. Chen, X. N. Huang, A. F. R. Stewart, and J. L. Sepulveda Gene expression changes associated with fibronectin-induced cardiac myocyte hypertrophy Physiol Genomics, August 11, 2004; 18(3): 273 - 283. [Abstract] [Full Text] [PDF]

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