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Am J Physiol Heart Circ Physiol 287: H645-H651, 2004. First published March 25, 2004; doi:10.1152/ajpheart.00564.2003
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Aging increases stiffness of cardiac myocytes measured by atomic force microscopy nanoindentation

Samuel C. Lieber,1,2 Nadine Aubry,2 Jayashree Pain,1 Gissela Diaz,1 Song-Jung Kim,1 and Stephen F. Vatner1

1Cardiovascular Research Institute and the Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey–New Jersey Medical School, Newark, 07103; and 2Department of Mechanical Engineering, New Jersey Institute of Technology, Newark, New Jersey 07102

Submitted 15 June 2003 ; accepted in final form 16 March 2004


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It is well established that the aging heart exhibits left ventricular (LV) diastolic dysfunction and changes in mechanical properties, which are thought to be due to alterations in the extracellular matrix. We tested the hypothesis that the mechanical properties of cardiac myocytes significantly change with aging, which could contribute to the global changes in LV diastolic dysfunction. We used atomic force microscopy (AFM), which determines cellular mechanical property changes at nanoscale resolution in myocytes, from young (4 mo) and old (30 mo) male Fischer 344 x Brown Norway F1 hybrid rats. A measure of stiffness, i.e., apparent elastic modulus, was determined by analyzing the relationship between AFM indentation force and depth with the classical infinitesimal strain theory and by modeling the AFM probe as a blunted conical indenter. This is the first study to demonstrate a significant increase (P < 0.01) in the apparent elastic modulus of single, aging cardiac myocytes (from 35.1 ± 0.7, n = 53, to 42.5 ± 1.0 kPa, n = 58), supporting the novel concept that the mechanism mediating LV diastolic dysfunction in aging hearts resides, in part, at the level of the myocyte.

cell mechanics; elastic modulus; heart; nanotechnology

AGING IS MARKED BY A DECLINE in left ventricular (LV) diastolic function in the absence of cardiovascular disease (16). This age-related alteration in diastolic function has been linked to structural changes (7) in which stiffness has been measured in the whole heart, excised papillary muscles, or cardiac muscle fibers (17, 18). However, despite agreement that there is an increase in collagen content in the aging heart and a concomitant alteration in LV diastolic dysfunction, results of ventricular stiffness have been controversial, indicating an increase (23), no change (3, 13), and decrease (11) with age. The inconsistencies in stiffness results led us to question what is occurring at the level of the single LV myocyte. No studies have examined the possibility that aging exerts an effect on the material properties at the single cardiac myocyte level, which is the hypothesis of the current investigation. The use of the atomic force microscope (AFM) and its nanoindentation function makes it possible to test our hypothesis that aging affects the material properties of single cardiac myocytes.

Indentation tests are an established method to determine a specimen's material properties [reviewed by Karduna et al. (14)]. Thus the AFM allows the mechanical properties of living cardiac myocytes to be measured with nanoscale resolution in the transverse direction. We utilized this technology to examine potential changes in the stiffness of individual myocytes from young and old rats. Numerous aging studies (reviewed by Lakatta and Sollott, Ref. 17) have been conducted on Wistar and Fischer 344 rat strains. The Fischer 344 x Brown Norway F1 hybrid rat (F344xBN) used in the current investigation has the advantage of displaying less pathology in other organs (19).


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Variation in the apparent elastic modulus with cell length was studied in 2-mo-old male Sprague-Dawley rats (n = 3) (Charles River Breeding Laboratories). Aging effects were studied in male F344xBN rats obtained from the National Institute on Aging colony. AFM studies were conducted on rats of 4 (n = 4) and 30 (n = 4) mo of age representing young and old rats, respectively (1). Cardiac myocyte contractile and relaxation function and measurement of myocyte general dimensions (length, width) were conducted in a parallel group of male F344xBN rats of 4 (n = 3) and 30 (n = 3) mo of age. The myocyte cross-sectional area was measured in a third group of male F344xBN rats of 4 (n = 3) and 30 (n = 3) mo of age. All procedures and protocols used in the present study were approved by the Animal Research Committee of the University of Medicine and Dentistry of New Jersey—New Jersey Medical School and followed the National Institutes of Health guidelines for the care and use of laboratory animals.

Preparation of LV myocytes. Cardiac myocytes were prepared from male F344xBN rats and Sprague-Dawley rats, respectively, as previously described (12, 15). In brief, the heart was rapidly excised and perfused with basic solution composed of MEM (Joklik’s modification, Cat. No. M0519; Sigma), 5 mM taurine, 2 mM creatine, 5 mM HEPES, 5 mM NaHCO3, 20 units insulin, and 1% Penn Strep containing 75 U/ml each of collagenase 1 and 2 (Worthington Biochemical; Freehold, NJ) at 37°C. All solutions were continuously bubbled with 95% O2-5% CO2 at 37°C. The digestion was stopped by adding MEM solution containing 0.3 mM CaCl2 and 6% BSA for 10 min. In every 10-min period, the supernatant was removed, and MEM solution was added stepwise with CaCl2 concentration (0.5 and 1.0 mM). Myocytes were washed twice with culture medium. Young rat myocyte isolations resulted in a yield of ~70%, whereas isolations in old rats resulted in ~40% yield.

Measurement of contractile and relaxation function. Myocytes were transferred to a warmed (37°C) and continuously perfused cell chamber located on an inverted microscope stage (Nikon; Melville, NY). The chamber was perfused with physiological buffer containing (in mM) 120 NaCl, 2.6 KCl, 1.2 MgCl2, 1.2 KH2PO4, 11 glucose, 5 HEPES, 25 NaHCO3, 2 taurine, 1 pyruvate, and 1 CaCl2. Myocyte contraction was induced at 1 Hz by platinum field electrodes placed in the cell chamber and attached to a stimulator (model S48; Grass Instruments, Quincy, MA). Cell images were continuously monitored through a x20 objective lens (Nikon) and transmitted to a charge-coupled device (CCD) video camera (TM-640; Pulnix, Mountain View, CA). The output from the CCD camera was displayed on a video monitor (model PVM-135; Sony, New York, NY). Myocyte length was measured by using a video motion edge detector (model VED103; Crescent Electronics, Sandy, UT), and the data were acquired at 240 images/s. The %contraction was calculated from the length data, where myocyte length was calibrated with a hemocytometer grid placed on the microscope stage.

Morphology. Measurements of myocyte length and width were made from photomicrographs of the isolated myocytes and a computerized image-analysis system (Scion, Frederick, MD) (Table 1). Dimensions were calibrated with a hemocytometer grid placed on the microscope stage. A separate method was used to measure the myocyte cross-sectional area in LV tissue fixed in formalin involving the MetaMorph image system software (Universal Imaging, West Chester, PA). Myocyte outlines were apparent after silver staining methacrylate-embedded sections (1 µm thick) that were obtained midway between the LV base and apex. Traces of ~100 myocyte outlines were obtained in the LV of each animal (Table 1).


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  		Table 1. Morphometric characteristics of isolated left ventricular myocytes and cross-sectional areas of formalin-fixed myocardial sections from young and old male F344xBN rats

 
AFM. Indentation tests were conducted with a Digital Instruments MultiMode AFM, Nanoscope IIIa control system, fluid cell, and a "J" scanner (Digital Instruments; Santa Barbara, CA) (Fig. 1). After isolation, myocytes were plated on customized (15 mm diameter) tissue culture-treated petri dishes (Becton Dickinson, Bedford, MA) coated with 20 µg/ml of laminin (Sigma, St. Louis, MO). Myocytes were studied within 6 h from the time of isolation. When the cells were not analyzed with the AFM, they were kept in a CO2 water-jacketed incubator (Thermo Forma model 3110 series) that kept the cells in a 37°C-5% CO2 humidified environment. AFM experiments were conducted by placing the myocytes plated on the 15-mm diameter petri dishes into the AFM Multimode head. The cells were studied in a 37°C culture media environment by using the AFM fluid cell at room temperature. All AFM measurements were conducted within 1 h after insertion into the AFM head. Etched silicon nitride probes (NP-20) were used in these experiments. The NP-20 probe used had a tip radius of 20–60 nm and a cone angle of 35° and was attached to triangular cantilevers 200 µm in length with a spring constant of 0.06 N/m (Digital Instruments; Santa Barbara, CA). The same NP-20 probe cantilever was used throughout the experiments to avoid the variation of the cantilever spring constant with different AFM probes. The NP-20 sensitivity was determined consistently near 50 nm/V. The indentation protocol involved no scanning in the horizontal (xy) plane. The cantilever was positioned directly above the surface of an immobilized myocyte with the aid of a x30 magnification eyepiece. Proper force curves were obtained in the force calibration mode by setting the z-scan start at +220 volts with the z-scan size set at +440 volts and a low frequency of 0.6 µm/s, which was found to minimize not only hysteresis (Fig. 2) but also drag force and to maximize the number of force curves that could be captured (5, 20). Hysteresis was calculated as previously described (5) by subtracting the area under the curves for retraction (Aret) from extension (Aext), which represents the energy dissipated into the cell from the indentation of the AFM tip. This area was normalized by dividing the hysteresis value by the input energy, Aext. The apparent elastic modulus was found to be reproducible at and below that frequency. Force curves were taken at three positions along the middle of the longitudinal axis, as in previous work (20). The effects of replicate indentation in the same location were studied to verify that the cell behavior and material properties did not change with repeated indentations as shown in previous studies (5, 20).



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  		Fig. 1. A: digital Instruments MultiMode atomic force microscope (AFM), fluid cell, and a "J" scanner. B: schematic of the nanoindentation procedure with the Digital Instruments MultiMode AFM. The Nanoscope IIIa control system (A) signals the AFM head (B) to operate in contact mode and directs the movement of the piezoelectric xyz "J" scanner (C) in the z-direction and not in the xy-plane. The sample (D) is indented by the AFM probe (E). The deflection of the AFM cantilever (F) is monitored by emitting a laser beam (G) from a source (H) that after deflecting off the AFM cantilever (F) is directed by a mirror (I) into a position-sensitive photodetector (J). The measurements are sent through the Detector Electronics (K) returning to the Nanoscope IIIa control system (A) for data collection. C: schematic (not to scale) of the nanoindentation of a section of the myocyte sample with a blunted AFM probe. Boundary effects and nonlinearities in AFM measurements are avoided, because the ratio (~161) of the actual myocyte thickness (~15 µm) to indentation depth (~93 nm) greatly exceeds the value of 5, and the ratio (~375) of the myocyte thickness (~15 µm) to indenter radius (~40 nm) is greater than the accepted value of 10. Moreover, in-plane boundary effects are avoided, because the ratio (~2,900) of myocyte length (~120 µm) to indenter size (~40 nm) and the ratio (~750) of myocyte width (~30 µm) to indenter size (~40 nm) are much greater than the accepted value of 15 (14).

 


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  		Fig. 2. The selected probe velocity (0.6 µm/s) minimized hysteresis shown in the figure plotting the ratio of the normalized hysteresis (NH) to the operating velocity's normalized hysteresis (OVNH) with respect to probe velocity.

 
The indentation force (F) was calculated by using Hooke's law (F = k{delta}), where k and {delta} denote the cantilever's spring constant and the cantilever's measured deflection, respectively (see, e.g., Ref. 20). The indentation depth (d) was calculated from the difference in the z-movement of the piezo and the deflection of the cantilever (5, 6, 20). The general equation for the total force exerted by an indenter for infinitesimal indentation, according to classical infinitesimal strain theory (CIST) with a blunted conical indenter, is given (6) as

(1)
where

(2)

Here, E refers to the apparent elastic modulus, {nu} denotes the Poisson ratio, and d is the indentation depth. The Poisson ratio is assumed to be 0.5 for cells (22). The function of {phi}(d) used in this work is that corresponding to a blunted conical indenter shape with tip angle 2{alpha} ({alpha} = 35°) and radius r (r = 40 nm). Its analytic expression has been previously reported (6). The elastic properties are defined as an "apparent" elastic modulus, because there are viscous contributions within the cellular response (6). The apparent elastic modulus was determined by plotting the extension force in equation 1 above as a function of the coefficient {Omega} and by identifying the resulting slope with the apparent elastic modulus. The cardiac myocyte apparent elastic modulus was found to be constant (up to 2%) at three positions on the cell's longitudinal line close to the center, avoiding boundary effects. All data reported here were obtained at a probe speed of 0.6 µm/s and analyzed at the depth of 93.2 ± 0.1 nm (n = 111) from the approaching part of the force curve. To verify that nonlinearities associated with finite thickness effects are avoided, we measured the adult cardiac myocyte thickness by confocal microscopy [14.5 ± 0.7 µm, in good agreement with published data (9)] and found that the myocyte thickness-to-indentation depth ratio far exceeds the accepted value necessary to avoid nonlinearities (14). Moreover, the indentation depth (~93 nm) and probe size (~40 nm) were sufficiently minimal to prevent the underlying substrate properties and boundary effects from affecting indentation results. As noted in Table 2, there were differences in the number of myocytes that could be studied successfully from each animal with variations from animal to animal but not between old and young.


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  		Table 2. Average apparent elastic modulus of young and old F344xBN rats

 
Geometry independence of apparent elastic modulus. Hypertrophy is known to be characteristic of aging; however, the apparent elastic modulus is a material property measure and should be independent of geometry. To prove that this is the case, we conducted an experiment on 2-mo-old Sprague-Dawley rats (n = 33 cells from 3 animals). The apparent elastic modulus was determined as described in AFM and myocyte morphology was determined by conducting photomicroscopic measurements with the AFM Multimode's x30 magnification eyepiece and image analysis software (Scion Corp.). Myocyte morphology (length, width) was calibrated by using the AFM cantilever known length (200 µm).

Statistical analysis. All data are presented here as means ± SE and the statistical significance was determined by calculating a probability value (P) with Student's t-test. Values of P < 0.05 were considered to be significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Myocyte contractile and relaxation function. Figure 3 shows representative contraction/relaxation recordings at baseline in young and old rats with the average data shown in Fig. 3. A significant (P < 0.01) decrease in myocyte contraction was found from young (5.4%) to old (3.4%). The relaxation function as assessed by the time required for 70% relaxation was also increased in old myocytes (159.7 ms) compared with young myocytes (84.9 ms).



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  		Fig. 3. A: photomicrograph of a representative myocyte used for morphological and contractile measurements obtained through a x40 objective lens (Nikon), charge-coupled device (CCD) video camera (TM-640; Pulnix, Mountain View, CA), and video capture system. B: representative contraction/relaxation recording at baseline in young and old rats demonstrating a difference in myocyte contraction and relaxation with aging (C). Age affects myocyte contraction and relaxation, where the %contraction data show a significant (*P < 0.01) decrease, left (38.3%), and a significant (*P < 0.01) increase, right (88.2%), in the time for the myocyte to return from the contracted state to 70% of its starting length.

 
Myocyte morphology. A significant increase (P < 0.01) was seen in length from young (113.7 ± 1.5 µm; n = 64) to old (120.0 ± 1.8 µm; n = 70) and also an increase in width from young (28.2 ± 0.5 µm; n = 64) to old (31.5 ± 0.7 µm; n = 70). A significant 22% increase (P < 0.01) was also seen in the cross-sectional area from young (249 ± 9.3 µm2) to old (305 ± 12.7 µm2).

Geometry independence of apparent elastic modulus. Figure 4 shows there was no significant difference in the apparent elastic modulus: 32.4 ± 2.5, 31.8 ± 0.7, 35.1 ± 2.1, and 30.8 ± 1.7 kPa determined from myocytes of lengths 71.2 ± 2.4 (n = 6), 89.8 ± 1.3 (n = 11), 104.3 ± 1.0 (n = 6), and 131.5 ± 2.8 µm (n = 10), respectively, with an average modulus value of 32.3 ± 0.7 kPa.



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  		Fig. 4. Study demonstrating that the apparent elastic modulus is a material property essentially independent of geometry where myocytes isolated from 2-mo-old Sprague-Dawley rats (n = 33 cells from 3 animals) showed no significant difference (P > 0.05) in the apparent elastic modulus: 32.4 ± 2.5, 31.8 ± 0.7, 35.1 ± 2.1, and 30.8 ± 1.7 kPa determined from myocytes of length 71.2 ± 2.4 (n = 6), 89.8 ± 1.3 (n = 11), 104.3 ± 1.0 µm (n = 6), and 131.5 ± 2.8 µm (n = 10), respectively.

 
AFM measured effect of age on mechanical properties. A force-indentation graph (Fig. 5A) was plotted for single cardiac myocytes. The linear regression fit of force as a function of the coefficient (Fig. 5B) gives correlation coefficient (R2) values near 1 for the myocytes of young and old F344xBN. The slope of this linear regression fit is analyzed as the apparent elastic modulus of the young and old myocyte cells. The cardiac myocyte apparent elastic modulus did not vary significantly among different animals in their respective age group, and the lowest apparent elastic modulus from the old group was significantly higher (P < 0.01) than the highest apparent elastic modulus from the young group. Therefore, the apparent elastic moduli of myocytes from animals in their respective young and old age groups were grouped for statistical purposes. The average apparent elastic modulus value reported is an average of the results obtained with all myocytes collected from rats in the respective young (4 animals, n = 53 cells) and old (4 animals, n = 58 cells) F344xBN myocytes. The AFM data (Table 2) show a significant difference (P < 0.01) in the apparent elastic modulus of young vs. old rats: 35.1 ± 0.7 (n = 53) and 42.5 ± 1.0 kPa (n = 58), respectively (Fig. 6). The AFM data show no significant difference in the effects of replicate indentation in the same cellular location on young and old cardiac myocytes, respectively (Fig. 7A), and the data show no significant difference in measurements taken over a 6-h period in both young and old cardiac myocytes, respectively (Fig. 7B). The ratio of old myocyte normalized hysteresis to young myocyte normalized hysteresis was 0.90, indicating a 10% change in hysteresis with aging.



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  		Fig. 5. A: sample force-indentation plot on young and old F344xBN myocytes. B: representative sample plot of the linear regression fit of the force as a function of the coefficient ({Omega}) for young and old F344xBN myocytes. The force indentation data fit the classical infinitesimal strain theory well with the AFM probe modeled as a blunted conical indenter (R2 values near 1). The slope of the linear regression fit is then identified with the apparent elastic modulus of the young and old myocyte cells.

 


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  		Fig. 6. The cardiac myocyte apparent elastic modulus did not vary significantly among different animals in their respective age group (**P > 0.05). AFM data show a significant difference (*P < 0.01) in the apparent elastic modulus of young and senescent rats: 35.1 ± 0.7 (n = 53) and 42.5 ± 1.0 kPa (n = 58), respectively, demonstrating a 21% increase with age.

 


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  		Fig. 7. A: cardiac myocyte apparent elastic modulus did not vary significantly [not significant (NS)] between replicate indentations on the same cellular location in the young (n = 9 cells) and old (n = 10 cells) rats, while still showing a significant difference (P < 0.01) in the apparent elastic modulus of young and senescent rats. B: cardiac myocyte apparent elastic moduli did not vary significantly (NS) over the time period (0–2, 2–4, and 4–6 h) during which they were analyzed. However, significant differences (P < 0.01) in the apparent elastic modulus of myocytes from young and senescent rats were observed at each time period.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Most studies on aging have demonstrated LV diastolic dysfunction (16). The implication from these studies is that the mechanical dysfunction observed with aging is due to changes in the extracellular matrix, which could be attributed to increases in collagen (17). The underlying hypothesis of our investigation was that mechanical changes in the myocytes per se could participate in the LV diastolic dysfunction due to aging. We chose to use the male F344xBN rat model (19) and found that the increase in cell size and decrease in isolated myocyte contraction-relaxation data are consistent with previous studies (4, 8, 17, 26) in other aging male rat strains, which have been conducted previously in male animals. This present study was also conducted in LV myocytes from male hearts, because aging-induced LV diastolic dysfunction is more pronounced in men than women (16, 21).

To test our hypothesis that the material properties of cardiac myocytes change with aging in the rat model, we performed indentation tests with an AFM, which has been used to measure the viscoelastic response of different cell types (e.g., endothelial, platelets) with typical cellular apparent elastic modulus values between 1 and 200 kPa as described previously (20, 25). The AFM technique has been well documented in its ability to measure cytoskeleton components (25). The AFM indentation measurement is thus registering changes in the myocyte sarcolemma, sarcomeric skeleton, and general cytoskeleton proteins (tubulin, desmin, actin). The force indentation graph (Fig. 5A) compares favorably to previously published theoretical and experimental curves for biological cell material (6, 20). For the same indentation depth, the cantilever deflection was greater on old F344xBN myocytes than on the young F344xBN myocytes, indicating that on average a larger force is necessary to indent the myocyte surface of old F344xBN rats compared with that of young F344xBN rats (Fig. 5A). Therefore, before deriving a measure of stiffness (apparent elastic modulus) from the force-indentation relationship, we demonstrated qualitatively that the stiffness of aged cardiac myocytes is greater than young ones. The force indentation data fit CIST well, with the AFM probe modeled as a blunted conical indenter (Fig. 5B).

Results of the present investigation demonstrate that the apparent elastic modulus of isolated cardiac myocytes increases significantly (P < 0.01) with advanced age (Fig. 6). This increase was found to be ~21% from 35.1 ± 0.7 (n = 53) to 42.5 ± 1.0 kPa (n = 58). The apparent elastic modulus was determined by analyzing the relationship between AFM indentation force and depth with the accepted and widely used CIST approach and modeling the AFM probe as a blunted conical indenter, which has been shown to be a more accurate representation of the AFM probe shape (6, 20). Cardiac myocytes behave in a viscoelastic manner, and when they are indented by the AFM tip, energy is dissipated into the cell (hysteresis). To accurately determine an apparent elastic modulus of the myocyte, we minimized the energy dissipated into the cell by selecting a tip speed (0.6 µm/s) after which the hysteresis remained essentially constant while also maximizing the number of force curves that could be captured. Because minimal hysteresis still exists, we cannot conclude that our measurement reflects a purely elastic modulus but rather an apparent elastic modulus, which takes into account the viscoelastic nature of the myocyte. Analysis of the effects of age on normalized hysteresis shows a significant 10% decrease in the energy dissipated into the myocyte cell with aging. A decrease in the energy dissipated by the cell implies that the viscous component of the cell's mechanical properties has decreased with aging.

The apparent elastic modulus determined for young cardiac myocytes using AFM nanoindentation are concordant with data derived from studies in isolated rat cardiocytes with a variety of other techniques. However, no previous measurements exist in old cardiac myocytes to compare with our data. Our apparent elastic modulus value of 35.1 ± 0.7 kPa (n = 53) on young (4 mo old) cardiac myocytes compares favorably to Granzier's stiffness value (32 kPa) obtained by studying the stress versus strain relationship on skinned rodent cardiocytes by attaching glass microneedles with urethane foam and applying a tensile force (10, 24) and Brady's stiffness measure (26 kPa) found in skinned rodent cardiocytes held between double micropipettes by suction and barnacle cement (2). Differences in our value compared with published results could be attributed to the fact that our myocytes are not skinned and maintain their membrane, where the removal of the membrane could have an effect on cardiac myocyte viscoelasticity. The measurement technique used could also affect viscoelasticity values, where we measured viscoelasticity in the transverse direction as opposed to previous studies that measured viscoelasticity in the tangential direction through application of a tensile force.

Experimental limitations.

The myocytes studied by AFM were not electrically stimulated to contract and therefore remained essentially motionless during nanoindentation. However, the myocytes could not be considered completely passive, because of the effect of cross-bridge cycling. We found that the variation in stiffness with location prevalent on other cell types (e.g., embryonic cardiocytes) measured with the AFM was negligible with adult mammalian cardiac myocytes (see also Ref. 20). Moreover, the boundary effects that would influence results of AFM indentation on smaller cells are not relevant in our experimental design, because of the AFM probe's nanosize with respect to the myocyte's microsize. Therefore, boundary effects such as the interaction among the myocyte laminin receptors and laminin-coated substrate and the material properties of the substrate itself do not affect the indentation measurements. AFM indentation may also affect the material properties of the cell; however, we proved that replicate indentation in one cellular location does not significantly change the apparent elastic modulus (Fig. 7A) as has also been shown by others (20). AFM measurements were conducted within a 6-h period from the time of isolation. The variation in the time at which AFM measurements were conducted could affect the apparent elastic modulus measured; however, we proved that this was not the case in both young and old myocytes by displaying similar data from the AFM over a 6-h period (Fig. 7B). It is also possible that the size of the cell per se may affect the apparent elastic modulus measurement. This is particularly important, because aging causes myocyte hypertrophy (17), which was demonstrated in the present study. However, the apparent elastic modulus is a material property reflecting the extent to which cardiac myocytes deform after application of a stress and under ideal conditions should be independent of the cell's geometry. We proved this by verifying that the modulus determined by AFM nanoindentation is independent of geometry (Fig. 4). Nonetheless, the age-related increase in the myocyte apparent elastic modulus could be a result of hypertrophy (18). Regardless, this would not impact the conclusions of the present study, which proposes that changes in material properties of aging myocytes could contribute to the altered properties of the aging heart. Another limitation of this study as well as most other studies examining myocyte function is that the yield of healthy myocytes is less in the old than the young heart. However, as noted above, there is very little difference in myocyte function in cells from young rats over a broad range of cell size. Future investigations in the field need not only examine alterations in collagen/elastin, but also the mechanisms involved in changing the myocyte's resilience and stiffness, and finally potential differences due to gender.

In summary, this is the first study to use the AFM to examine either a disease state or change in physiological function in cardiac myocytes. The findings indicate that the altered LV diastolic function in the whole heart resulting from age may not only be due to structural changes in the heart, but also due to changes occurring at the single myocyte level, independent of the extra cellular matrix.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants AG-14121, HL-59139, HL-69020, and HL-33107 and the New Jersey Commission on Science and Technology Grant 01-2042-007-25. The authors also acknowledge the financial support of the W. M. Keck Foundation, which allowed the establishment of the W. M. Keck Laboratory at the New Jersey Institute of Technology.


   ACKNOWLEDGMENTS
 
The technical assistance provided by Digital Instruments (Santa Barbara, CA) and the technical staff of the W. M. Keck Laboratory at New Jersey Institute of Technology are gratefully acknowledged.


   FOOTNOTES
 

Address for reprint requests and other correspondence: S. F. Vatner, Dept. of Cell Biology and Molecular Medicine, MSB G-609, Univ. of Medicine and Dentistry of New Jersey–New Jersey Medical School, PO Box 1709, Newark, NJ 07101-1709 (E-mail: vatnersf{at}umdnj.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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