Effects of Constitutive Overexpression of Insulin-Like Growth Factor-1 on the Mechanical Characteristics and Molecular Properties of Ventricular Myocytes

From the Department of Medicine, New York Medical College (G.R., A.M., B.L., P.L., P.A.), Valhalla, NY; the Department of Medicine, Montefiore Medical Center and Albert Einstein College of Medicine (A.M., E.H.S.), New York, NY; and the Department of Physiology and Biophysics, University of Tennessee College of Medicine (P.A.H.), Memphis.

	Abstract

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Abstract—Recently, insulin-like growth factor-1 (IGF-1) has been claimed to positively influence the cardiac performance of the decompensated heart. On this basis, the effects of constitutive overexpression of IGF-1 on the mechanical behavior of myocytes were examined in transgenic mice in which the cDNA for the human IGF-1B was placed under the control of a rat -myosin heavy chain promoter. In mice heterozygous for the transgene and in nontransgenic littermates at 2.5 months of age, the alterations in Ca²⁺ sensitivity of tension development, unloaded shortening velocity, and sarcomere compliance were measured in skinned myocytes. The quantities and state of phosphorylation of myofilament proteins in these enzymatically dissociated ventricular myocytes were also examined. The overexpression of IGF-1 was characterized by a nearly 15% reduction in myofilament isometric tension at submaximum Ca²⁺ levels in the physiological range, whereas developed tension at maximum activation was unchanged. In contrast, unloaded velocity of shortening was increased 39% in myocytes from transgenic mice. Moreover, resting tension in these cells was reduced by 24% to 33%. Myocytes from nontransgenic mice pretreated with IGF-1 failed to reveal changes in myofilament Ca²⁺ sensitivity and unloaded velocity of shortening. The quantities of C protein, troponin I, and myosin light chain-2 were comparable in transgenic and nontransgenic mice, but their endogenous state of phosphorylation increased 117%, 100%, and 100%, respectively. Troponin T content was not altered, and myosin isozymes were essentially 100% V₁ in both groups of mice. In conclusion, constitutive overexpression of IGF-1 may influence positively the performance of myocytes by enhancing shortening velocity and cellular compliance.

Key Words: insulin-like growth factor-1 • transgenic mouse • isometric tension • myofilament protein phosphorylation

	Introduction

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Insulin-like growth factor-1 belongs to the insulin family of peptides and acts as a growth factor in many tissues and tumors.¹ In neonatal cardiac myocytes in culture, IGF-1 activates DNA synthesis^{2 3} and the expression of myofilament proteins,⁴ which are consistent with a hyperplastic and hypertrophic response of the cells. However, long-term cultures of adult myocytes react to IGF-1 by increasing only the formation of myofibrils in the cytoplasm⁵ ; IGF-1 upregulates the rate of protein accumulation, enhancing anabolic and inhibiting catabolic pathways in the cells.⁶ Moreover, an upregulation of IGF-1 mRNA occurs in vivo in pressure-overload hypertrophy,⁷ and the density of IGF-1 receptors is increased in the decompensated heart.⁸ These adaptations have been associated with myocyte hypertrophy. In this regard, the exogenous administration of IGF-1 has been claimed to improve cardiac hemodynamics by increasing the extent of viable myocardium in the diseased heart.⁹ Conversely, in the presence of ventricular failure produced by coronary artery constriction and diffuse tissue damage¹⁰ or by coronary artery occlusion and acute myocardial infarction,¹¹ there is an activation of the myocyte IGF-1–IGF-1 receptor autocrine system that is concurrent with the reentry of myocytes into the cell cycle, DNA replication, nuclear mitotic division, and cell proliferation.^{12 13} Although the growth-promoting effects of IGF-1 on myocytes remain to be defined, the possibility has been raised that IGF-1 may affect the functional behavior of the normal and overloaded myocardium by altering the mechanical properties of the cells.^{14 15 16} Contrasting results have been reported on the beneficial consequences of GH or IGF-1 administration on the physiology of myocytes,^{17 18} most likely reflecting the difficulty of discriminating in vivo the influence of this growth factor on the heart from that on other organs¹ and the circulatory system^{19 20} in particular. Moreover, changes in myocyte performance were not complemented with the analysis of the biochemical characteristics of regulatory proteins and myofilament protein subunits, which are responsible for mechanical modifications.²¹ These are significant issues to establish whether IGF-1 may positively influence myocyte contractility, supporting its application as a therapeutic agent in the failing heart. For this purpose, heterozygous transgenic mice in which the cDNA for the human IGF-1B was placed under the control of a rat -MHC promoter were used.²² The mechanical properties of myocytes in these mice, designated as FVB.Igf+/-, were compared with those of cells in wild-type littermates. These determinations were obtained after chemical removal of the sarcolemma under controlled Ca²⁺ and pH conditions. Such a preparation has allowed the evaluation of myofilament isometric tension as a function of [Ca²⁺], unloaded velocity of shortening, and sarcomere compliance. Additionally, the quantities and state of phosphorylation of myofilament proteins underlying the mechanical characteristics of the cells were examined to identify the molecular basis of IGF-1–mediated myocyte function. Since the plasma level of IGF-1 in FVB.Igf+/- mice is increased,²² myocytes from nontransgenic littermates were exposed to a concentration of IGF-1 comparable to that present in vivo in transgenic animals, and the mechanical characteristics of these cells were established. This intervention was introduced before the application of the detergent.

	Materials and Methods

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Animals
Experiments were carried out in male FVB.Igf+/- and wild-type littermates at 2.5 months of age developed in our laboratory and maintained in the Department of Comparative Medicine at New York Medical College. These animal groups included 39 FVB.Igf+/- and 43 nontransgenic littermates. For cell mechanics, the number of animals used was 23 FVB.Igf+/- and 38 wild-type mice. Corresponding values for biochemistry of myofilament proteins were 20 transgenic and 19 nontransgenic mice.

Myocyte Preparations
Transgenic and nontransgenic mice were anesthetized with chloral hydrate (400 mg/kg IP) and injected with heparin (500 U IP). Subsequently, hearts were rapidly excised, and myocytes were enzymatically dissociated.^{11 12 22} Hearts were cannulated with a PE-50 catheter for retrograde perfusion through the aorta. The solutions were supplements of modified commercial MEM Eagle Joklik (J.R.H. Biosciences). HEPES-MEM contained 117 mmol/L NaCl, 5.7 mmol/L KCl, 4.4 mmol/L NaHCO₃, 1.5 mmol/L KH₂PO₄, 17 mmol/L MgCl₂, 21.1 mmol/L HEPES, 11.7 mmol/L glucose, amino acids and vitamins, 2 mmol/L L-glutamine, 10 mmol/L taurine, and 20 mU/mL insulin and was adjusted to pH 7.2 with NaOH. This solution is 292 mOsm, isosmolar with rodent serum. Resuspension medium was HEPES-MEM supplemented with 0.5% BSA. The cell isolation procedure consisted of three main steps: (1) Ca²⁺-free perfusion, (2) mechanical tissue dissociation, and (3) separation of intact cells.

Ca²⁺-Free Perfusion

Blood washout and collagenase (selected type I, Worthington Biochemical Corp) perfusion of the heart were carried out at 37°C with HEPES-MEM gassed with 85% O₂ and 15% N₂.

Mechanical Tissue Dissociation

After removing the heart from the cannula, the ventricles were minced. Collagenase-perfused tissue was subsequently shaken in resuspension medium containing collagenase. Supernatant cell suspensions were washed and resuspended in resuspension medium.

Separation of Intact Cells

Intact cells were enriched by centrifugation and discarding the supernatant. This procedure was repeated three or four times in each preparation in order to remove nonmyocyte cells, cell debris, and the residual collagenase. Each centrifugation was performed at 30g for 3 minutes. Intact cells were recovered from the pellet and washed, and smears were made to control the purity of the preparation. Rectangular rod-shaped cells constituted 35% to 40% of all myocytes. To obtain skinned myocytes, the myocyte membrane was removed by incubating the cells for 6 minutes at 22°C in a Ca²⁺-free relaxing solution containing 1 mmol/L Mg²⁺, 100 mmol/L KCl, 2 mmol/L EGTA, 4 mmol/L ATP, 10 mmol/L imidazole, and 0.3% Triton X-100 (Pierce) at a pH of 7.0. This procedure preserves myofilament proteins in a given phosphorylation state by rapidly removing soluble and membrane-bound kinases and phosphatases. The skinned cells were washed three times in fresh relaxing solution and stored on ice until use. All solutions had a final ionic strength of 180 mmol/L. Additionally, myocytes obtained from nontransgenic littermates were exposed to 60 ng/mL recombinant human IGF-1 (Genzyme Diagnostic) for a period of 15 minutes at 37°C before chemical removal of the sarcolemma. A similar aliquot of the same myocyte preparation was not treated with IGF-1 and was used as a control. The dose of IGF-1 used corresponds to its plasma concentration in FVB.Igf+/- mice.²²

Experimental Apparatus and Myocyte Attachment
Single myocytes were attached via glass micropipettes to a piezoelectric translator and force transducer on the stage of an inverted microscope (Olympus-CK-2, Olympus Optical). Both devices were mounted on a three-way micromanipulator (Zeiss). To attach the myocyte, the tips of the micropipettes were coated with Great Stuff foam (Insta-Foam) and gently placed on the ends of the myocyte in solution. The micropipettes with the cell attached were lifted to the center of the drop of relaxing solution. Force was measured by a microforce transducer (model 403, Cambridge Technology). The piezoelectric translator (model 173, Physik Institute) was controlled by a 0- to 1000-V power supply (model BOP 1000 mol/L, KEPCO) providing length changes from 0 to 45 µm in <1 millisecond. An analog-to-digital/digital-to-analog conversion board (NB-MIO-16XL-18 µs, National Instruments) was installed in an IBM-compatible personal computer, triggering the piezoelectric power supply and recording the resulting force transducer signal. Computer acquisition of the results and data analysis were carried out by the use of a computer program generated in LABVIEW software (National Instruments). Myocytes were observed and recorded with a Panasonic WV-BD400 video camera connected to the Olympus microscope. Sarcomere length, myocyte length between attachment pipettes, and cell width were assessed during myocyte relaxation and activation through Java video-analysis software (Jandel Scientific). Cell width and depth were measured after each experiment by detaching one pipette and rotating the cell by 90° with the second pipette.²³ The calculation of myocyte cross-sectional area was based on the assumption of an elliptical cross section.^{23 24} For cells in which only width measurements were available, depth was considered to correspond to 84% of the width. This assumption was based on the results obtained in the other cell preparations.

Solutions for Myocyte Mechanics
Cardiac myocytes were activated by rapid changes in bathing solutions containing free Ca²⁺ ranging between pCa 9.0 and pCa 4.5 (pCa=-log [Ca²⁺]). The solutions at pH 7.0 contained 7 mmol/L EGTA, 1 mmol/L Mg²⁺, 20 mmol/L imidazole, 4.42 mmol/L ATP, and 14.5 mmol/L creatine phosphate, along with various free [Ca²⁺] levels and KCl to adjust the ionic strength; [KCl] levels in the solutions were adjusted according to the free [Ca²⁺] to make the final ionic strength 180 mmol/L. The pCa of the relaxing solution was 9.0. The apparent stability constant for Ca²⁺-EGTA was corrected to 22°C and an ionic strength of 180 mmol/L. The computer program of Fabiato²⁵ was used to calculate the concentrations of each metal, ligand, and metal-ligand complex on the basis of the stability constants listed by Godt and Lindley.²⁶

Tension-pCa Relationship
After attachment of a myocyte to the apparatus, sarcomere length was initially set at 2.3 µm and was monitored during activation. If sarcomere length varied by >0.2 µm between the relaxed and maximally activated conditions, the cells were considered too compliant and were discarded. For a typical sequence of tension measurements, the myocyte was activated first in a pCa 4.5 solution and subsequently contracted at randomly chosen submaximum pCa values. Myocytes were then reactivated at pCa 4.5 to assess any decline in the performance of the cell. Active tension was calculated as the difference between the actual level and that in the relaxing solution. For the tension-pCa relationship, the active tension at a submaximum activating level of Ca²⁺ (P) was expressed as a fraction of the maximum active tension (Po) under the same condition (P/Po). The force with maximum activation at pCa 4.5 was compared at the beginning and at the end of the changes in pCa. If the final force was <80% of initial force, the data were excluded from the final analysis. The tension-pCa relationship was determined with eight distinct levels of pCa: 6.2, 6.0, 5.8, 5.7, 5.5, 5.3, 5.0, and 4.5. At each solution change, the resting tension was determined. The Ca²⁺ sensitivity of tension and degree of cooperative activation, as judged by the steepness of the tension-pCa relationship, was assessed from the tension-pCa relationship.²⁷ The data were analyzed by least squares regression with the Hill equation log [Pr/(1-Pr)]=n_H (log[Ca²⁺]+k), where Pr is the relative active tension, n_H is the Hill coefficient, and k is the intercept of the fitted line with the x-axis, which corresponds to the pCa at half-maximum isometric tension (pCa₅₀). With the use of constants derived from the Hill equation, tension-pCa curves were fitted by computer with the equation

Maximum Velocity of Unloaded Shortening
V_max in cardiac myocytes was determined by a modification in the slack-test method of Edman.²⁸ Cardiac myocytes were maximally activated with Ca²⁺ at pCa 4.5. Once a stable tension was achieved, the cells were rapidly shortened in progressive steps from an initial sarcomere length of 2.3 µm, and the time required to take up the slack was measured from the beginning of the length step to the onset of tension redevelopment. The maximum step size imposed was such that cells were not allowed to shorten to a sarcomere length of <1.8 µm, at which point interference from restoring forces was likely to occur. The tension tracings for 200 milliseconds before and 300 milliseconds after the slack step were stored on computer hard drive for later analysis. The time from release to where tension first redeveloped was determined by hand fitting a line through the tension baseline and determining its intersection with a line drawn through the initial portion of the tension rise.²⁴ Length change (L) as a fraction of the initial cell length was plotted relative to the time (t) of unloaded shortening. The slope of this plot (L/t), which is the velocity, was determined by linear regression and recorded as V_max in cell lengths per second. The mean±SD values were calculated from each individual plot. Each individual plot was remeasured to obtain a normalization between curves. Criteria for discarding cells because of excessive compliance or loss of tension were as described above.

Passive Length-Tension Relationship
The passive length-tension relationship was determined in relaxing solution at pCa 9.0 by increasing the sarcomere length from 1.9 to 2.8 µm in 0.1- to 0.2-µm increments. Isometric tension in the absence of Ca²⁺ was measured at each sarcomere length. Measurements of tension were standardized by calculating the cell cross-sectional area.^{23 24} Subsequently, these values were plotted individually and reanalyzed to obtain a normalization between curves for the statistical treatment of the data.

Biochemical Measurements
When possible, myocytes isolated from each heart were divided in two groups, one for phosphorylation assays and the second for pyrophosphate gels and immunoelectrophoretic analysis. The n values for each determination corresponding to the number of hearts is indicated in "Results."

Phosphorylation Studies
Phosphorylation of cardiac myofibrillar proteins in ventricular myocytes by [³²P]P_i was performed as follows: ventricular myocytes were suspended in 2 mL MEM containing 1 nmol/L calyculin A (Sigma) (5x10⁵ cells/mL) and incubated with 50 µCi of [³²P]P_i for 30 minutes at room temperature. After incubation with [³²P]P_i, the myocytes were rapidly washed with physiological saline at 4°C (2 mL, three times) in the presence of various protease inhibitors (0.1 mmol/L pepstatin, 0.1 mmol/L leupeptin, 0.2 mmol/L phenylmethylsulfonyl fluoride, and 1 µg/mL aprotinin) and protein phosphatase inhibitors (0.1 mmol/L sodium orthovanadate and 1 nmol/L calyculin A). Subsequently, the myocytes were sonicated in 0.5 mL of 20 mmol/L Tris-Cl (pH 7.5), 2 mmol/L EDTA, 2 mmol/L EGTA, 6 mmol/L mercaptoethanol, 0.01% Triton X-100, 0.1 mmol/L sodium vanadate, 20 mmol/L NaF, and other protease inhibitors. Proteins were determined by the colorimetric biuret method or Bio-Rad protein assay. To determine the incorporation of ³²P, samples were prepared for SDS-PAGE and autoradiography. Different protein preparations were monitored on SDS-PAGE 12% slab gels. For autoradiography, the gels were dried and developed on X-OMAT film (Eastman Kodak) after 24 to 48 hours.²³

Immunoblot Analysis
Myocyte samples were subjected to 12% SDS-PAGE and transferred onto nitrocellulose membranes. Prestained molecular weight markers were also electrophoresed and transferred onto the same gels. Membranes were washed in Tris-buffered saline+0.05% Tween 20 buffer and incubated in 7% dry milk for 1 hour to saturate nonspecific protein-binding sites. The nitrocellulose membrane was probed with a monoclonal antibody against TnI (TNI-1). This monoclonal antibody has been shown to recognize adult cardiac TnI.²³ In addition, cardiac TnT was quantified on the nitrocellulose membrane by using TnT antibody (JLT-12, Sigma). MLC-2 was identified and measured with a monoclonal antibody (M4401, Sigma Chemical). After the blots were washed, they were bound with anti-IgG alkaline phosphatase–conjugated secondary antibody (Bio-Rad). Western blots were developed by an enhanced chemiluminescence kit (Amersham).

Pyrophosphate Gels
Myosin isoenzymes were analyzed on crude myosin preparations from cardiac myocytes and intact tissue by PAGE with sodium pyrophosphate under nondissociating conditions at 2°C.²⁹ Five to seven micrograms of myosin extracts was layered on each gel and run at a constant voltage gradient of 14 V/cm for 20 to 22 hours. The gels were then stained with Coomassie blue, destained, and scanned densitometrically.

Statistical Analysis
All measurements are presented at mean±SD computed from the average results obtained from each mouse. Comparisons between values were performed with a two-tailed unpaired Student's t test, and values of P<.05 were considered to be significant. Quantification of the amounts of TnI, TnT, and MLC-2 was performed by densitometric analysis using Bio-Rad Gel Doc 100 image analyzer and Molecular Analyst software. The state of phosphorylation of these proteins was also evaluated in this manner. Because all measurements could not be collected in every mouse, n values for each determination are listed in the text or in the legend to each figure.

	Results

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Myocyte Characteristics
The mechanical properties of 153 myocytes isolated from 27 wild-type littermates and 23 transgenic mice were analyzed. Fig 1 shows the photomicrographs of two individual myocytes from a nontransgenic littermate and a transgenic mouse in relaxing solution at pCa 9.0 and during maximum activation at pCa 4.5. Sarcomere length at pCa 9.0 was 2.31±0.05 µm (n=91) and 2.32±0.05 µm (n=62) in myocytes from nontransgenic and FVB.Igf+/- mice. Corresponding values at pCa 4.5 were 2.24±0.07 µm and 2.24±0.06 µm. This indicated low end compliance. Similarly, activation did not affect segment length and cell width. The average segment length and cell width were 69±17 µm and 34±7 µm in wild-type littermates and 72±14 µm and 32±6 µm in FVB.Igf+/- mice.

View larger version (108K): [in this window] [in a new window]   Figure 1. Photomicrographs of two ventricular myocytes (treated with Triton X-100) obtained from a nontransgenic mouse (A and B) and an FVB.Igf+/- mouse (C and D). A and C, Myocytes under relaxing conditions at pCa 9.0. Sarcomere lengths in these preparations were 2.34 µm (A) and 2.34 µm (C). B and D, The same myocytes during maximum activation of pCa 4.5. Sarcomere lengths were 2.26 µm (B) and 2.24 µm (D).

The 45 cells used to establish the myocyte tension-pCa relationship and V_max from FVB.Igf+/- mice and littermates were all capable of maintaining maximum tension throughout the protocol. Cells that did not retain 80% of their force-generating ability at the end of the experiment were excluded. Maximum final active tension, recorded during the determination of the tension-pCa relationship, was 88±10% of the initial value in myocytes (n=14) from nontransgenic mice and 94±9% in myocytes (n=11) from FVB.Igf+/- mice. Maximum final active tension obtained during the assessment of V_max was 85±7% of the initial value in myocytes (n=10) from nontransgenic mice and 82±7% in myocytes (n=10) from transgenic mice. Essentially identical characteristics were obtained in 37 myocytes from nontransgenic littermates, 19 treated and 18 nontreated with IGF-1, before the chemical removal of the sarcolemma. This sampling was collected from an additional group of 11 nontransgenic mice.

IGF-1 and Isometric Tension Development
Maximum isometric tension was 16.8±8.2 mN/mm² in myocytes constitutively overexpressing IGF-1, and this value was essentially identical in myocytes from nontransgenic littermates (16.4±9.0 mN/mm²). Fig 2A illustrates the cumulative tension-pCa curves of ventricular myocytes isolated from wild-type and FVB.Igf+/- mice. Compared with nontransgenic myocytes, myocytes isolated from FVB.Igf+/- mice exhibited a decrease in myofilament Ca²⁺ sensitivity of tension development. pCa₅₀ was 5.95±0.09 in myocytes from nontransgenic mice and 5.82±0.18 in myocytes from transgenic mice. This difference was statistically significant (P<.03). The Hill coefficient was unaffected by overexpression of IGF-1; the steepness of the isometric pCa relationship did not differ between the two groups of cells. The values of this parameter were 1.6±0.6 and 1.5±0.3 in myocytes from nontransgenic and transgenic mice, respectively. The derivations of pCa₅₀ and the Hill coefficient are illustrated in the inset of Fig 2A.

View larger version (18K): [in this window] [in a new window]   Figure 2. A, Cumulative tension-pCa plots of ventricular myocytes isolated from nontransgenic mice (, n=14) and FVB.Igf+/- mice (, n=11). P/Po indicates active tension at a submaximum level expressed as a fraction of maximum active tension under the same condition. Values are mean±SD. Inset, Hill transformation of tension-pCa plots. pCa values at half-maximum activation (pCa50) and Hill coefficients for these relationships were 5.95±0.09 and 1.6±0.6, respectively, for nontransgenic littermates and 5.82±0.18 and 1.5±0.3, respectively, for FVB.Igf+/- mice. Pr indicates relative tension. B, Cumulative tension-pCa plots of ventricular myocytes isolated from nontransgenic mice not treated (, n=8) and treated with IGF-1 (, n=9). Values are mean±SD. Inset, Hill transformation of tension-pCa plots. pCa values at pCa50 and Hill coefficients for these relationships were 5.94±0.08 and 1.7±0.6, respectively, for nontreated myocytes and 5.95±0.11 and 1.6±0.5, respectively, for IGF-1–treated myocytes.

Fig 2B illustrates the effects of IGF-1 on myocytes obtained from nontransgenic littermates. Compared with no treatment, treatment with IGF-1 did not change the tension-pCa curve. None of the small differences at various [Ca²⁺] levels were significant. pCa₅₀ was 5.94±0.08 in myocytes not exposed to IGF-1 and 5.95±0.11 in IGF-1–treated cells. The Hill coefficient was also comparable in the two groups of myocytes. The dose of IGF-1 used corresponded to the plasma concentration of this growth factor in FVB.Igf+/- mice.²²

IGF-1 and V_max
Fig 3 shows fast time-base recordings of tension from myocytes using the slack-test protocol at maximum activation. The traces collected from cells in relaxing solution at pCa 9.0 were superimposed on those obtained from the same cells after maximum activation in a solution of pCa 4.5. The slack-test protocol was used to establish the linear relationship between length changes and duration of unloaded shortening in each cell. Only myocytes with r >.91 were included in the study.

View larger version (14K): [in this window] [in a new window]   Figure 3. Superimposed tension-vs-time traces during slack-test protocols in a myocyte from a nontransgenic mouse (A through C) and a myocyte from an FVB.Igf+/- mouse (D through F) during maximum activation (pCa 4.5) and while in relaxing solution (pCa 9.0). Recordings were vertically expanded so that plateau of tension and point of initial release are not shown. Time point of tension redevelopment was determined by intersection of a line fitted by hand through baseline and a line fitted through initial part of tension redevelopment. Recordings of the cell from the nontransgenic mouse had a sarcomere length of 2.30 µm, segmental cell length of 88.7 µm, and cell width of 36.8 µm. Recordings of the cell from the FVB.Igf+/- mouse had a sarcomere length of 2.34 µm, segmental cell length of 80.5 µm, and cell width of 31.1 µm. Shortening velocity at maximum activation (pCa 4.5) was 2.13 CL/s for the nontransgenic myocyte and 3.25 CL/s for the transgenic myocyte. L indicates change in length.

Myocytes constitutively overexpressing IGF-1 showed increments in slack-step size that were characterized by smaller increases in time to tension redevelopment than those observed in cells from nontransgenic animals. This phenomenon resulted in an increase in the steepness of the slope of the slack-test plot. Fig 4A depicts the average linear relationships between the slack step and duration of unloaded shortening at maximum activation in myocytes isolated from wild-type and FVB.Igf+/- mice. The slope of the slack plot, V_max, increased 39% in myocytes overexpressing IGF-1, and this difference was statistically significant (P<.02). The average results, corresponding to 10 myocytes in each group of mice, gave values of unloaded shortening velocity of 2.27±0.83 and 3.16±0.69 CL/s in the nontransgenic and transgenic groups, respectively. Sarcomere length was measured directly during the development of isometric tension. The range of sarcomere length shortening during the slack test was calculated on the assumption that the decreases in length change were proportional to the changes in sarcomere length. Sarcomere shortening varied from 2.22 to 1.66 µm. Fig 4B illustrates the influence of IGF-1 treatment on myocytes collected from nontransgenic littermates. V_max did not vary as a result of IGF-1 exposure. The average results, reflecting 10 cells in each group, were 2.15±0.97 CL/s in the absence of IGF-1 and 2.00±0.85 CL/s in the presence of IGF-1.

View larger version (13K): [in this window] [in a new window]   Figure 4. A, Linear plots of average length changes, expressed as a fraction of cell length (CL) vs duration of unloaded shortening during maximum activation at pCa 4.5 for ventricular myocytes isolated from nontransgenic (, n=10) and FVB.Igf+/- (, n=10) mice. Values are mean±SD. Shortening velocities were 2.27±0.83 CL/s in nontransgenic myocytes and 3.15±0.69 CL/s in transgenic myocytes. B, Linear plots of average length changes, expressed as a fraction of CL vs duration of unloaded shortening during maximum activation at pCa 4.5 for myocytes from nontransgenic mice not treated (, n=10) and treated with IGF-1 (, n=10). Values are mean±SD. Shortening velocities were 2.15±0.97 CL/s in nontreated myocytes and 2.00±0.85 CL/s in IGF-1–treated cells.

IGF-1 and Passive Length-Tension Relationship
Resting tension was evaluated by progressively increasing sarcomere length from an initial value of 1.7 to 1.8 µm to a value of 2.9 to 3.1 µm in a solution of pCa 9.0. The final sarcomere length exceeded the physiological range. Fig 5 illustrates the procedure used for the evaluation of resting tension in a myocyte isolated from a FVB.Igf+/- mouse. Sarcomere length was varied from 1.9 to 3.1 µm. Resting tension was obtained by shortening segment length 20% to 30% at each preset sarcomere length. The length-tension relationships of myocytes isolated from wild-type littermates and transgenic mice are shown in Fig 6. In comparison with myocytes (n=11) isolated from wild-type littermates, this relationship was shifted downward in myocytes (n=10) collected from FVB.Igf+/- mice at sarcomere lengths of 2.1 µm. This parameter decreased 29% (P<.02), 33% (P<.005), 33% (P<.004), 32% (P<.003), 32% (P<.003), 30% (P<.003), 27% (P<.006), and 24% (P<.01) at 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, and 2.8 µm, respectively.

View larger version (144K): [in this window] [in a new window]   Figure 5. Photomicrographs of a ventricular myocyte (treated with Triton X-100) obtained from an FVB.Igf+/- mouse. Passive tension was recorded from a cell illustrated here at sarcomere lengths ranging from 1.9 to 2.95 µm while in relaxing solution (pCa 9.0).

View larger version (17K): [in this window] [in a new window]   Figure 6. Length-tension relationships of ventricular myocytes isolated from nontransgenic (, n=11) and transgenic (, n=10) mice. Determinations were performed in relaxing solution (pCa 9.0). Compared with myocytes from nontransgenic mice, the passive length-tension curve was shifted downward in myocytes from FVB.Igf+/- mice at sarcomere lengths of 2.1 µm. *P<.05 vs corresponding result in nontransgenic myocytes.

IGF-1 and Myofilament Proteins
The myofibrillar protein composition of ventricular myocytes was analyzed by SDS-PAGE. Coomassie blue–stained gels showed that there was no change in the quantity of C protein in cells isolated from FVB.Igf+/- and nontransgenic littermates (Fig 7; nontransgenic, OD=0.22±0.02, n=7; transgenic, OD=0.20±0.04, n=8; P=NS). Western blots of myocytes from both groups of mice did not show any difference in cardiac TnI isozymes (nontransgenic, OD=0.52±0.09, n=9; transgenic, OD=0.55±0.05, n=7; P=NS) and cardiac TnT isozymes (nontransgenic, OD=0.37±0.08, n=8; transgenic, OD=0.38±0.11, n=8; P=NS) (Fig 8A). In addition, the quantity of MLC-2 protein was measured (Fig 8B; nontransgenic, OD=0.47±0.16, n=6; transgenic, OD=0.45±0.15, n=7; P=NS). Conversely, phosphorylation of C protein, TnI, and MLC-2 increased in myocyte overexpression of IGF-1 (Fig 9). Although some variability in the characteristic of loading may be noted in terms of MHC, actin was much more constant and was used as an internal marker. Moreover, the differences in MHC in the samples were significantly less than the increases in the state of phosphorylation of the myofibrillar proteins. Additionally, in an attempt to minimize the effects of various preparations in the quantitative analysis, samples from nontransgenic mice were always assayed with samples from transgenic mice. In comparison with nontransgenic mice, the degree of phosphorylation of C protein increased 117% in transgenic mice (nontransgenic, 6±2, n=8; transgenic, 13±5, n=8; P<.003). Moreover, TnI phosphorylation was 100% higher in FVB.Igf+/- mice (nontransgenic, 1.3±0.3, n=8; transgenic, 2.6±1.1, n=8; P<.006), and MLC-2 phosphorylation was 100% greater in transgenic mice (nontransgenic, 1.2±0.2, n=8; transgenic, 2.4±1.4, n=8; P<.01). Nondenaturing pyrophosphate gels demonstrated that the V₁ MHC isoform was the only detectable myosin isozyme in cardiac tissue and in ventricular myocytes (nontransgenic, V₁=100%, n=8; transgenic, V₁=100%, n=8).

View larger version (40K): [in this window] [in a new window]   Figure 7. Coomassie blue–stained SDS-PAGE of myofibrillar proteins from myocytes isolated from transgenic (lanes 1 to 3) and nontransgenic (lanes 4 and 5) mice. High magnification of the top region of the gel to illustrate the constancy in the quantity of C protein. MW indicates molecular weight markers.

View larger version (42K): [in this window] [in a new window]   Figure 8. A, Western blots of TnI and TnT. Myofibrillar proteins from nontransgenic (lanes 1 to 4) and FVB.Igf+/- (lanes 5 to 8) mice were separated by 12% SDS-PAGE and detected on nitrocellulose membranes by immunoblottings with an anti-TnI monoclonal antibody and an anti-cardiac TnT monoclonal antibody. B, Western blot of MCL-2. Myofibrillar proteins from transgenic (lanes 1 to 3) and nontransgenic (lanes 4 and 5) mice. MW indicates molecular weight markers. C indicates C protein. Protein loading is illustrated by Coomassie blue staining in the lower panel.

View larger version (82K): [in this window] [in a new window]   Figure 9. Coomassie blue–stained SDS-PAGE of myofibrillar proteins (A) and corresponding [-32P]orthophosphate autoradiograph (B). Lanes 1 to 4 correspond to myocytes isolated from nontransgenic mice; lanes 5 to 8 correspond to myocytes collected from transgenic mice. Myocytes from each group were incubated with [-32P]Pi for 30 minutes at room temperature. Phosphorylated proteins were analyzed by SDS-PAGE followed by autoradiography. Major myofibrillar proteins were identified by Coomassie blue staining as MHC, C protein (C), actin, TnT, TnI, MLC-1, and MLC-2. MW indicates molecular weight markers. The inset shows a shorter exposure of the same autoradiograph that illustrates better the level of phosphorylation of C protein.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
IGF-1 and Myocyte Mechanics
In the last few years, several experimental studies have attempted to characterize the effects of exogenous administration of GH and/or IGF-1 on the hemodynamics of the normal and pathological heart. With the exception of one observation,¹⁸ this form of therapeutic intervention ameliorates ventricular performance in the absence^{16 17} or presence^{19 20} of an increased load. This beneficial impact of IGF-1 has been shown also in humans under physiological conditions³⁰ and ventricular dysfunction and failure.³¹ GH and IGF-1 have been claimed to improve cardiac function by enhancing myocardial contractility and decreasing peripheral vascular resistance.^{15 16 17 19 20} In vitro preparations have raised the possibility that GH and IGF-1 increase myofilament Ca²⁺ sensitivity and decrease the velocity of shortening.^{15 17} These cellular adaptations have been considered the consequences of defects in Ca²⁺ handling³² and changes in the relative proportion of myosin isozymes with an increase in the V₃ isoform.¹⁵ However, not all studies are in agreement.¹⁴

One of the difficulties in interpreting the influence of GH or IGF-1 administration in vivo on the heart concerns the distinction between systemic and local effects. Moreover, it is impossible to establish in vivo whether the functional changes of the myocardium are related exclusively to the growth factor or are dependent, at least in part, on its impact on the other organs and the circulation. In the present study, myocytes constitutively overexpressing IGF-1 were used, and their mechanical properties were measured in skinned cells under controlled Ca²⁺, pH, and temperature. IGF-1 overexpression was coupled with a decrease in myofilament isometric tension as a function of [Ca²⁺]_i and an increase in V_max and compliance at sarcomere length in the physiological range and longer. These latter two cellular modifications may be significant in the failing heart,^{33 34} in which an enhanced response of the myocardium to diastolic Laplace overloading may improve cardiac pump function. IGF-1 may ameliorate ventricular performance via a more effective Frank-Starling relation. The reduction in myofilament responsiveness to Ca²⁺ may attenuate the energy requirements of the decompensated heart. However, this phenomenon may negatively affect the impaired myocardium^{23 35} in view of the decrease in cytosolic systolic [Ca²⁺]_i with failure.^{36 37}

The ability of constitutively expressed IGF-1 to modify the mechanical behavior of myocytes has no precedent. Myocyte cell volume does not differ in FVB.Igf+/- mice and nontransgenic littermates,²² excluding the impact of cellular hypertrophy on myocyte mechanics. Additionally, ventricular function is comparable in these two groups of animals. The attenuation in myofilament Ca²⁺ sensitivity and the enhanced velocity and compliance characteristics of myocytes overexpressing IGF-1 tend to resemble the effects of ß-adrenergic stimulation and protein kinase A phosphorylation of myofilament proteins on myocyte contractility.^{24 38} Although the possibility has been raised that activation of protein kinase C isoforms may have similar consequences on myocytes,³⁹ opposite observations have been reported as well.⁴⁰ Mice with only 30% IGF-1 are characterized by increased adenylyl cyclase activity,^{41 42} suggesting that IGF-1 and the ß-adrenergic system may complement each other to sustain myocyte performance. Whether the overexpression of IGF-1 in FVB.Igf+/- mice is associated with a downregulation of the ß-adrenergic effector pathway in myocytes remains to be determined.

IGF-1 and Myofilament Proteins
The troponin complex comprises Ca²⁺-binding (TnC), ATPase- inhibiting (TnI), and tropomyosin-binding (TnT) subunits. After an increase in intracellular free Ca²⁺, this cation binds to TnC, triggering contraction; the affinity of TnI for TnC increases, causing a conformational change in troponin, which allows the interaction between actin and myosin.²¹ TnI phosphorylation is coupled with a loss in myofilament Ca²⁺ sensitivity and an attenuation in actomyosin ATPase activity.^{43 44} Such an effect on phosphorylation of TnI has been shown by protein kinase A activation,^{21 38} whereas different results have been reported after stimulation of protein kinase C.^{40 45 46} Alterations in the relative proportion of TnT isoforms in the myocardium may affect myofilament isometric tension as a function of [Ca²⁺],^{47 48} and TnT phosphorylation may decrease force generation.⁴⁹ The results here, documenting an increase in TnI phosphorylation, provide the molecular basis for the reduction in myofilament Ca²⁺ sensitivity of tension in myocytes overexpressing IGF-1. Moreover, MLC-2 contents did not vary in this animal group. Conversely, MLC-2 subunit phosphorylation was enhanced, and this adaptation may have upregulated the myofilament responsiveness to Ca²⁺,^{50 51} reducing, at least in part, the consequences of TnI phosphorylation.

The state of phosphorylation of MLC-2 and C protein have been claimed to modify the velocity of cell shortening.^{52 53 54} However, this relationship has not been confirmed in all studies.³⁸ Although changes in the relative proportion of V₁ and V₃ myosin isozymes have been correlated with changes in V_max,^{21 38} transgenic mice and their nontransgenic littermates showed 100% V₁. Whether phosphorylation of C protein and/or MLC-2 alone constitute the molecular counterpart of increased V_max in FVB.Igf+/- remains to be established. These factors were capable of offsetting the negative effect of TnI phosphorylation on unloaded myocyte shortening.²³ The preservation of TnI quantity and increased state of phosphorylation can be expected to reflect a faster dissociation of Ca²⁺ from TnC, speeding relaxation.⁵⁵

It is noteworthy to emphasize that the results obtained here are in contrast with recent observations on the acute effects of IGF-1 on the mechanics of isolated rat papillary muscle.⁵⁶ The addition of the growth factor was reported to increase the force development of the myocardium, whereas a decrease in the myofilament responsiveness to physiological levels of Ca²⁺ was detected in skinned myocytes of FVB.Igf+/- mice. Moreover, maximum force was unchanged in myocytes from transgenic animals. An increase in free cytosolic Ca²⁺ was noted in the early investigation,⁵⁶ but no biochemical measurements were performed to characterize the changes in the quantities and state of phosphorylation of regulatory proteins or other components of the myofibrillar assembly. Therefore, the molecular bases of the claimed inotropic action of IGF-1 in the papillary muscle remain to be analyzed. Conversely, these determinations were performed in the present study and were found to be consistent with the mechanical behavior of myocytes overexpressing IGF-1. Surprisingly, the unloaded velocity of shortening and the compliance properties of the myocardium were not evaluated in IGF-1–treated papillary muscles, leaving unclear whether these critical aspects of contractility are influenced by the local administration of IGF-1. Finally, difficulties exist in comparing information on skinned myocytes with that derived from myocardial tissue in which the impact of the interstitium, sarcolemmal membrane, and surface receptors on myocardial performance cannot be discriminated.

Limitations of the Study
There are several limitations in the present investigation that have to be acknowledged. The myocyte preparation yielded an average of 35% to 40% of rectangular rod-shaped cells, which is significantly lower than that commonly obtained in rats.^{11 12} This may reflect a preferential selection of myocytes and has to be considered in the interpretation of the results. However, difficulties exist in obtaining viable myocytes in excess of 50% in mice.⁵⁷ In addition, left and right ventricular myocytes were not examined separately. The overexpression of IGF-1 in myocytes was associated with an increase in circulating levels of the growth factor, which may have affected the myocardium chronically.²² The absolute values of force development in mouse myocytes were lower than those reported in rats.^{15 23 35 38} Conversely, similar differences have been found in papillary muscle preparations from rats and mice.^{58 59}

	Selected Abbreviations and Acronyms

 

CL/s = cell lengths per second GH = growth hormone IGF-1 = insulin-like growth factor-1 MHC = myosin heavy chain MLC = myosin light chain OD = optical density TnC, TnI, TnT = troponin C, I, and T Vmax = maximum velocity of unloaded shortening

	Acknowledgments

 
This study was supported by grants HL-38132, HL-39902, PO1-HL-43023, and AG-15756 from the National Institutes of Health and by Grants-in-Aid (No. 950321 and No. 95264321) from the American Heart Association. The expert technical assistance of Maria Feliciano is greatly appreciated.

	Footnotes

 
Reprint requests to Piero Anversa, MD, Department of Medicine, Vosburgh Pavilion, Room 302, New York Medical College, Valhalla, NY 10595.

Received November 5, 1997; accepted January 5, 1998.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Le Roith D. Insulin-like growth factors. N Engl J Med. 1997;336:633–640.[Free Full Text]
2. Kardami E. Stimulation and inhibition of cardiac myocyte proliferation in vitro. Mol Cell Biochem. 1990;92:129–135.
3. Kajstura J, Cheng W, Reiss K, Anversa P. The IGF-1-IGF-1 receptor system modulates myocyte proliferation but not myocyte cellular hypertrophy in vitro. Exp Cell Res. 1994;215:273–283.[Medline] [Order article via Infotrieve]
4. Ito H, Hiroe M, Hirata Y, Tsujino M, Adachi S, Schichiri M, Koike A, Nogami A, Marumo F. Insulin-like growth factor 1 induces hypertrophy with enhanced expression of muscle specific genes in cultured rat cardiomyocytes. Circulation. 1993;87:1715–1721.[Abstract/Free Full Text]
5. Donath MY, Azpf J, Eppenberger-Eberhardt M, Froesch EF, Eppenberger HM. Insulin-like growth factor 1 stimulates myofibril development and decreases smooth muscle -actin of adult cardiomyocytes. Proc Natl Acad Sci U S A. 1994;91:1686–1690.[Abstract/Free Full Text]
6. Decker RS, Cook MG, Behnke-Barclay M, Decker ML. Some growth factors stimulate cultured adult rabbit ventricular myocyte hypertrophy in the absence of mechanical loading. Circ Res. 1995;77:544–555.[Abstract/Free Full Text]
7. Donohue TJ, Dworkin LD, Lango MN, Fliegner K, Lango RP, Benstein JA, Slater WR, Catanese VM. Induction of myocardial insulin-like growth factor-1 gene expression in left ventricular hypertrophy. Circulation. 1994;89:799–809.[Abstract/Free Full Text]
8. Toyozaki T, Hiroe M, Hasumi M, Horie T, Hosoda S, Tsushima T, Sekiguchi M. Insulin-like growth factor 1 receptors in human cardiac myocytes and their relation to myocardial hypertrophy. Jpn Circ J. 1993;57:1120–1127.[Medline] [Order article via Infotrieve]
9. Duerr RL, Huang S, Miraliakbar HR, Clark R, Chien KR, Ross J Jr. Insulin-like growth factor-1 enhances ventricular hypertrophy and function during the onset of experimental cardiac failure. J Clin Invest. 1995;95:619–627.
10. Anversa P, Zhang X, Li P, Capasso JM. Chronic coronary artery constriction leads to moderate myocyte loss and left ventricular dysfunction and failure in rats. J Clin Invest. 1992;89:618–629.
11. Cheng W, Reiss K, Li P, Chun MJ, Kajstura J, Olivetti G, Anversa P. Aging does not affect the activation of the myocyte insulin-like growth factor-1 autocrine system after infarction and ventricular failure in Fischer 344 rats. Circ Res. 1996;78:536–546.[Abstract/Free Full Text]
12. Kajstura J, Zhang X, Reiss K, Szoke E, Li P, Lagrasta C, Cheng W, Darzynkiewicz Z, Olivetti G, Anversa P. Myocyte cellular hyperplasia and myocyte cellular hypertrophy contribute to chronic ventricular remodeling in coronary artery narrowing–induced cardiomyopathy in rats. Circ Res. 1994;74:383–400.[Abstract/Free Full Text]
13. Anversa P, Kajstura J, Cheng W, Reiss K, Cigola E, Olivetti G. Insulin-like growth factor-1 and myocyte growth: the danger of a dogma, II: induced myocardial growth: pathologic hypertrophy. Cardiovasc Res. 1996;32:484–495.[Medline] [Order article via Infotrieve]
14. Timsit J, Riou B, Bertherat J, Wisnewsky C, Kato NS, Weisberg AS, Lubetzki J, Lecarpentier Y, Winegrad S, Mercadier J-J. Effects of chronic growth hormone hypersecretion on intrinsic contractility, energetics, isomyosin pattern, and myosin adenosine triphosphatase activity of rat left ventricle. J Clin Invest. 1990;86:507–515.
15. Mayoux E, Ventura-Clapier R, Timsit J, Béhar-Cohen F, Hoffmann C, Mercadier J-J. Mechanical properties of rat cardiac skinned fibers are altered by chronic growth hormone hypersecretion. Circ Res. 1993;72:57–64.[Abstract/Free Full Text]
16. Cittadini A, Strömer H, Katz SE, Clark R, Moses AC, Morgan JP, Douglas PS. Differential cardiac effects of growth hormone and insulin-like growth factor-1 in the rat: a combined in vivo and in vitro evaluation. Circulation. 1996;93:800–809.[Abstract/Free Full Text]
17. Strömer H, Cittadini A, Douglas PS, Morgan JP. Exogenously administered growth hormone and insulin-like growth factor-1 alter intracellular Ca²⁺ handling and enhance cardiac performance: in vitro evaluation in the isolated isovolumic buffer–perfused rat heart. Circ Res.. 1996;79:227–236.[Abstract/Free Full Text]
18. Shen Y-T, Wiedmann RT, Lynch JJ, Grossman W, Johnson RG. GH replacement fails to improve ventricular function in hypophysectomized rats with myocardial infarction. Am J Physiol. 1996;271:H1721–H1727.[Abstract/Free Full Text]
19. Yang R, Bunting S, Gillett N, Clark R, Jin H. Growth hormone improves cardiac performance in experimental heart failure. Circulation. 1995;92:262–267.[Abstract/Free Full Text]
20. Duerr RL, McKirnan MD, Gim RD, Clark RG, Chien KR, Ross J Jr. Cardiovascular effects of insulin-like growth factor-1 and growth hormone in chronic left ventricular failure in rat. Circulation. 1996;93:2188–2196.[Abstract/Free Full Text]
21. Solaro J, Van Eyk J. Altered interactions among thin filament proteins modulate cardiac function. J Mol Cell Cardiol. 1996;28:217–230.[Medline] [Order article via Infotrieve]
22. Reiss K, Cheng W, Ferber A, Kajstura J, Li P, Li B, Olivetti G, Homcy CJ, Baserga R, Anversa P. Overexpression of insulin-like growth factor-1 in the heart is coupled with myocyte proliferation in transgenic mice. Proc Natl Acad Sci U S A. 1996;93:8630–8635.[Abstract/Free Full Text]
23. Li P, Hofmann PA, Li B, Malhotra A, Cheng W, Sonnenblick EH, Meggs LG, Anversa P. Myocardial infarction alters myofilament calcium sensitivity and mechanical behavior of myocytes. Am J Physiol. 1997;272:H360–H370.[Abstract/Free Full Text]
24. Strang KT, Sweitzer NK, Greaser ML, Moss RL. ß-Adrenergic receptor stimulation increases unloaded shortening velocity of skinned single ventricular myocytes from rats. Circ Res. 1994;74:542–549.[Abstract/Free Full Text]
25. Fabiato A. Computer programs for calculating total from specific free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 1988;157:378–417.[Medline] [Order article via Infotrieve]
26. Godt RE, Lindley BD. Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog. J Gen Physiol. 1982;80:279–297.[Abstract/Free Full Text]
27. Shiner JS, Solaro RJ. The Hill coefficient for the Ca²⁺-activation of striated muscle contraction. Biophys J. 1984;46:541–543.[Abstract/Free Full Text]
28. Edman KAP. The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibers. J Physiol (Lond).. 1979;291:143–150.[Abstract/Free Full Text]
29. D'Albis A, Pantoloni C, Rechet JJ. An electrophoretic study of native myosin isoenzymes and of their subunit content. Eur J Biochem. 1979;99:261–272.[Medline] [Order article via Infotrieve]
30. Thuesen L, Christiansen JS, Sørensen KE, Jørgensen JOL, Ørskov H, Henningsen P. Increased myocardial contractility following growth hormone administration in normal man. Dan Med Bull. 1988;35:193–196.[Medline] [Order article via Infotrieve]
31. Fazio S, Sabatini D, Capaldo B, Vigorito C, Giordano A, Guida R, Pardo F, Biondi B, Saccà L. A preliminary study of growth hormone in the treatment of dilated cardiomyopathy. N Engl J Med. 1996;334:809–814.[Abstract/Free Full Text]
32. Xu X, Best PM. Decreased transient outward K⁺ current in ventricular myocytes from acromegalic rats. Am J Physiol. 1991;260:H935–H942.[Abstract/Free Full Text]
33. Katz AM. Cell death in the failing heart: role of an unnatural growth response to overload. Clin Cardiol. 1995;18(suppl IV):IV-36–IV-44.
34. Cohn JN. The management of chronic heart failure. N Engl J Med. 1996;335:490–498.[Free Full Text]
35. Hofmann PA, Miller WP, Moss RL. Altered calcium sensitivity of isometric tension in myocyte-sized preparations of porcine postischemic stunned myocardium. Circ Res. 1993;72:50–56.[Abstract/Free Full Text]
36. Capasso JM, Li P, Anversa P. Cytosolic calcium transients in myocytes isolated from rats with ischemic heart failure. Am J Physiol. 1993;265:H1953–H1964.[Abstract/Free Full Text]
37. Li P, Park C, Micheletti R, Li B, Cheng W, Sonnenblick EH, Anversa P, Bianchi G. Myocyte performance during evolution of myocardial infarction in rats: effects of propionyl-L-carnitine. Am J Physiol. 1995;268:H1702–H1713.[Abstract/Free Full Text]
38. Moss RL. Ca²⁺ regulation of mechanical properties of striated muscle: mechanistic studies using extraction and replacement of regulatory proteins. Circ Res. 1992;70:865–884.[Abstract/Free Full Text]
39. Noland TA Jr, Guo X, Raynor RL, Jedeama NM, Avery-Hart-Fullard V, Solaro RJ, Kuo JF. Cardiac troponin I mutants: phosphorylation by protein kinases C and A and regulation of Ca²⁺-stimulated MgATPase of reconstituted actomyosin S-1. J Biol Chem. 1995;270:25445–25454.[Abstract/Free Full Text]
40. Lester JW, Gannaway KF, Reardon RA, Koon LD, Hofmann PA. Effects of adenosine and protein kinase C stimulation on mechanical properties of rat cardiac myocytes. Am J Physiol. 1996;271:H1778–H1785.[Abstract/Free Full Text]
41. Lembo G, Rockman HA, Hunter JJ, Steinmetz H, Koch WJ, Ma L, Prinz MP, Ross J Jr, Chien KR, Powell-Braxton L. Elevated blood pressure and enhanced myocardial contractility in mice with severe IGF-1 deficiency. J Clin Invest. 1996;98:2648–2655.[Medline] [Order article via Infotrieve]
42. Ishikawa Y, Homcy CJ. The adenylyl cyclases as integrators of transmembrane signal transduction. Circ Res. 1997;80:297–304.[Free Full Text]
43. Garvey JL, Kranias EG, Solaro RJ. Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts. Biochem J. 1988;249:709–714.[Medline] [Order article via Infotrieve]
44. Hofmann PA, Lang JH III. Effects of phosphorylation of troponin I and C protein on isometric tension and velocity of unloaded shortening in skinned single cardiac myocytes from rats. Circ Res. 1994;74:718–726.[Abstract/Free Full Text]
45. Puceat M, Clement O, Lechene P, Pelosin JM, Ventura-Clapier R, Vassort G. Neurohormonal control of calcium sensitivity of myofilaments in rat single heart cells. Circ Res. 1990;67:517–524.[Abstract/Free Full Text]
46. Gwathmey JK, Hajjar RJ. Effect of protein kinase C activation on sarcoplasmic reticulum function and apparent myofibrillar Ca²⁺ sensitivity in intact and skinned muscles from normal and diseased human myocardium. Circ Res. 1990;67:744–752.[Abstract/Free Full Text]
47. Anderson PAW, Malouf NN, Oakeley AE, Pagani ED, Allen PD. Troponin T isoform expression in humans: a comparison among normal and failing adult heart, fetal heart, and adult and fetal skeletal muscle. Circ Res. 1991;69:1226–1233.[Abstract/Free Full Text]
48. Nassar R, Malouf NN, Kelly MB, Oakeley AE, Anderson PAW. Force-pCa relation and troponin T isoforms of rabbit myocardium. Circ Res. 1991;69:1470–1475.[Abstract/Free Full Text]
49. Noland TA Jr, Kuo JF. Protein kinase C phosphorylation of cardiac troponin I or troponin T inhibits Ca²⁺-stimulated actomyosin MgATPase activity. J Biol Chem. 1991;266:4974–4978.[Abstract/Free Full Text]
50. Venema RC, Raynor RL, Noland TA Jr, Kuo JF. Role of protein kinase C in the phosphorylation of cardiac myosin light chain 2. Biochem J. 1993;294:401–406.
51. Damron DS, Darvish A, Murphy L, Sweet W, Moravec CS, Bond M. Arachidonic acid-dependent phosphorylation of troponin I and myosin light chain 2 in cardiac myocytes. Circ Res. 1995;76:1011–1019.[Abstract/Free Full Text]
52. Hartzell HC. Effects of phosphorylated and unphosphorylated C-protein on cardiac actomyosin ATPase. J Mol Biol. 1985;186:185–195.[Medline] [Order article via Infotrieve]
53. Metzger JM, Greaser ML, Moss RL. Variations in cross-bridge attachment rate and tension with phosphorylation of myosin in mammalian skinned skeletal muscle fibers. J Gen Physiol. 1989;93:855–883.[Abstract/Free Full Text]
54. Noland TA Jr, Kuo JF. Phosphorylation of cardiac myosin light chain 2 by protein kinase C and myosin light chain kinase increases Ca²⁺-stimulated actomyosin MgATPase activity. Biochem Biophys Res Commun. 1993;193:254–260.[Medline] [Order article via Infotrieve]
55. Zhang R, Zhao J, Mandveno A, Potter JD. Cardiac troponin I phosphorylation increases the rate of cardiac muscle relaxation. Circ Res. 1995;76:1028–1035.[Abstract/Free Full Text]
56. Freestone NS, Ribaric S, Mason WT. The effect of insulin-like growth factor-1 on adult rat cardiac contractility. Mol Cell Biochem. 1996;163/164:223–229.
57. Wolska BM, Solaro RJ. Method for isolation of adult mouse cardiac myocytes for studies of contraction and microfluorimetry. Am J Physiol. 1996;271:H1250–H1255.[Abstract/Free Full Text]
58. Capasso JM, Robinson TF, Anversa P. Alterations in collagen cross-linking impair myocardial contractility in the mouse heart. Circ Res. 1989;65:1657–1664.[Abstract/Free Full Text]
59. Capasso JM, Li P, Zhang X, Meggs LG, Anversa P. Alterations in Ang II responsiveness in left and right myocardium after infarction-induced heart failure in rats. Am J Physiol. 1993;264:H2056–H2067.[Abstract/Free Full Text]