Relaxation abnormalities in single cardiac myocytes from renovascular hypertensive rats RAO V. YELAMARTY, RUSSELL L. MOORE, FRANCIS ANNE M. SEMANCHICK, AND JOSEPH Y. CHEUNG

T. S. YU, MARYBETH

ELENSKY,

Departments of Medicine and of Cellular and Molecular Physiology, Milton S. Hershey Medical The Pennsylvania State University, Hershey 17033; and Department of Electrical Engineering, The Pennsylvania State University, University Park, Pennsylvania 16802 Yelamarty, Rao V., Russell L. Moore, Francis T. S. Yu, MaryBeth Elensky, Anne M. Semanchick, and Joseph Y. Cheung. Relaxation abnormalities in single cardiac myocytes from renovascular hypertensive rats. Am. J. Physiol. 262 (Cell Physiol. 31): C980-C990, 1992.-In myocardial hypertrophy secondary to renovascular hypertension, the rate of intracellular Ca*+ concentration decline during relaxation in paced left ventricular (LV) myocytes isolated from hypertensive (Hyp) rats is much slower compared with that from normotensive (Sham) rats. By use of a novel liquid-crystal televisionbased optical-digital processor capable of performing on-line real-time Fourier transformation and the striated pattern (similar to l-dimensional diffraction grating) of cardiac muscle cells, sarcomere shortening and relaxation velocities were measured in single Hyp and Sham myocytes 18 h after isolation. There were no differences in resting sarcomere length, percent of maximal shortening, time to peak shortening, and average sarcomere shortening velocity between Sham and Hyp cardiac cells. In contrast, average sarcomere relaxation velocity and half-relaxation time were significantly prolonged in Hyp myocytes. Contractile differences between Sham and Hyp myocytes detected by the optical-digital processor are confirmed by an independent method of video tracking of whole cell length changes during excitation-contraction. Despite the fact that freshly isolated myocytes contract more rigorously than 18-hold myocytes, the relaxation abnormality was still observed in freshly isolated Hyp myocytes, suggesting impaired relaxation is an intrinsic property of Hyp myocytes rather than changes brought about by short-term culture. We postulate that reduced sarcomere relaxation velocity is a direct consequence of impaired Ca*+ sequestration-extrusion during relaxation in Hyp myocytes and may be responsible for diastolic dysfunction in hypertensive hypertrophic myocardium at the cellular level. diastolic dysfunction; intracellular calcium; digital video imaging; Fourier transformation; spatial light modulator; cardiac hypertrophy; sarcomere dynamics; cell shortening HALLMARK OF hypertensive hypertrophic cardiomyopathy is decreased diastolic compliance (22, 33). Although tissue environmental factors such as relative subendocardial ischemia during exertion (9)) altered chamber geometry, interstitial fibrosis (33)) and muscle hypertrophy may all be important contributing factors to myocardial contractile failure, pathological changes at the cellular and subcellular levels may also have profound consequences to contractile function. In a rat model of renovascular hypertension, we (24) have recently demonstrated that during excitation-contraction (EC) coupling, cytosolic Ca2+transients in single cardiac myocytes are prolonged compared with myocytes isolated from control hearts. Our results are consistent with known altered activities in sarcolemmal Ca2+-adenosinetriphosphatase and Na+-Ca2+ exchange (1) and diminished sarTHE

C980

0363-6143/92

$2.00 Copyright

Center,

coplasmic reticulum Ca2+ uptake (21) in hypertensive hypertrophied hearts. Because intracellular Ca2’ concentration ([ Ca2+]i) changes during an EC cycle occupy a central role in cardiac contraction-relaxation, one tempting hypothesis is that the impaired relaxation in myocytes from hypertensive rats are the direct consequence of abnormalities in the control of [Ca2+]i during EC. The current study was undertaken to investigate whether the contractile properties of myocytes isolated from normotensive (Sham) and hypertensive (Hyp) rats are different. A novel liquid-crystal television (LCTV) based optical-digital processor was developed to measure sarcomere shortening and relengthening velocities during an EC cycle. In addition, video tracking of whole cell length changes during EC was used as an independent method to assessmyocyte contractile behavior. We found that sarcomere relaxation was significantly impaired in myocytes isolated from renovascular hypertensive rat hearts in agreement with our previous [Ca2+]i transient data. Impaired relaxation was observed in both freshly isolated (2 h) Hyp myocytes as well as cells placed in culture for 18 h. Our results provide a cellular (vs. myocardial) basis for diastolic dysfunction in left ventricular (LV) hypertrophy from chronic hypertension. MATERIALS

AND

METHODS

Isolation of Cardiac Myocytes and Renovascular

Hypertensive

From Control Rats

Renovascular hypertension (Goldblatt 2 kidney, 1 clip; Hyp) was induced in male Sprague-Dawley rats (150-200 g) as described by Moore et al. (24). Normotensive rats underwent the same operation except that the left renal artery was not constricted (Sham). Eight to ten weeks after surgery, cardiac myocytes were isolated from the LV by successive perfusion with collagenase and hyaluronidase as described by Cheung et al. (6, 7) and modified by Moore et al. (24). Our previous experience indicates that this period of recovery postsurgery is sufficient to allow development of LV hypertrophy, shift of myosin isoenzymes, reduction in density of dihydropyridine binding sites, and alterations of [Ca*+]; dynamics during an EC cycle in myocytes (24). Freshly isolated myocytes were seeded on laminin-coated cover slips and used within 2 h of isolation or cultured in medium 199 (1.25 mM Ca*+) with 4% heatinactivated fetal calf serum for 18 h before experiments (8,24). Determination

of Myocyte

Size

Myocytes adherent on cover slips were viewed through an Zeiss x10/0.25 NA objective so as to project the entire length of the myocytes on the video screen. Video images of myocytes were captured by our digital video-imaging system described in detail previously (8, 35). Lengths and widths of myocytes were

0 1992 the American

Physiological

Society

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determined from the video images, using an Ealing high-resolution test target as calibration standard. Measurement of Sarcomere Mechanics by Hybrid Optical-Digital Processor

Theory. Laser light diffraction has been widely used to measure sarcomere lengths of striated muscle preparation (4, 32). The regular cross-striation pattern of the muscle cell can be represented by a homogenous one-dimensional grating, which after undergoing Fourier transform gives rise to a series of welldefined diffraction spots. The distance between the zero- and first-order diffraction spots is inversely proportional to the length of repeating units in the grating, i.e., sarcomere length. The theory of Fourier transform of one-dimensional grating and the principles underlying the Fourier transform properties of optical lenses have been previously described (36). Impkmentation. LCTVASASPATIALLIGHTMODULATOR. To display the striated cell image as a transparent object in real time, a LCTV was used as a spatial light modulator (SLM). Because of its electronic addressable ability, LCTV has been widely used as an elctrooptical interface to address optical images into the object plane (37). Our black-and-white LCTV (Realistic 16-156) consists of a two-dimensional mosaic of raster-scanned liquid crystals (LC) cells, each of which is capable of modulating the coherent or incoherent light transmitted through it. The modulation is based on the rotation of the polarized light transmitted through each LC cell, which consists of spiral-structured 90” twisted nematic crystals sealed between two transparent electrodes and glass plates. The LC cell is sandwiched between a pair of polarizers having their axes of polarization states oriented in parallel (Fig. 1A ). The LC rotates the plane of polarization by 90” when no electrical field is applied, and hence no light is transmitted through the second polarizer (Fig. 1B). When a sufficient electrical field is applied to the electrodes, the orientation of the LC is twisted

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back and the incoming beam is allowed to pass through the LC cell (Fig. 123). Voltage to each LC cell is provided through the horizontal and vertical lines and is determined by two factors. The brightness control of the LCTV provides the standard bias voltage across each LC cell, which allows the transmission level of the entire TV screen to be uniformly varied. In addition, the input video signal furnishes the varied signal voltage. The analog video signal is converted to a four-bit digital signal before being applied across each pixel. Thus the varying signal voltage applied to each LC cell modulates the transmission of each cell, and hence the LCTV acts as a SLM with 16 gray-level capability. To operate the LCTV as a SLM, the stock LCTV was modified as follows. The diffuser and plastic windows were removed, and the hinge was modified to allow it to open 90’. The LCTV was then immersed in a liquid gate filled with mineral oil to remove the phase imperfections across the plane of LCTV. The modified LCTV was used for experiments to measure sarcomere dynamics. HYBRID OPTICAL-DIGITAL PROCESSOR. The assembly of the hybrid optical-digital processor is schematically shown in Fig. 2. Analog video signal from the intensified charge-coupled device (ICCD) camera in our previously described digital videoimaging system (8, 35) was either digitized on-line (for cell size measurements) with an IBM PC/AT computer containing an EPIX digitizing-display video board or routed to the modified LCTV. The image displayed on the LCTV was back-illuminated by a collimated coherent beam derived from a 5-mW HeNe laser (Melles Griot, Irvine, CA) through a spatial filter (SF) and a lens (Ll) of focal length 40 cm (Fig. 2). The area illuminated by the laser beam corresponded to a 7.5 x 4 pm2 region of the myocyte so that four to five sarcomeres were studied at any given time. To improve the contrast of the image displayed on LCTV, the intensity of the collimated beam was varied with a graduated neutral density filter (A), and the

4

A Fig. 1. Operating principle of liquid-crystal television (LCTV). A: simplified structure of a cell in LCTV. B: applied electrical field affects orientation of liquid crystal and transmittance of light through cell.

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upon triggering the digitizing board. Because the complete acquisition lasted -1 s, the time-dependent changesof the Fourier spectra of zero- and first-order diffraction spotsof the contracting muscle cell could be captured for the entire contraction cycle. Digitized Fourier spectra were either displayed on a high-resolution RGB monitor (Sony PVM 1271Q)in real time or directed to a peripheral hard disk drive for permanent storage. Calibration of Optical-Digital Processor

A

SF

Ll

La-v

Fig. 2. Schematic of hybrid optical-digital COInpOnentS are given in MATERIALS AND

I2

M

processor. Details of various METHODS.

brightness control of the LCTV was adjusted. The high-contrast striated image so obtained on the LCTV was Fourier transformed by positioning the LCTV at the front focal plane of a secondlens (L2, focal length 25 cm). An OlympusDPlanapo x20/0.8 NA microscopeobjective (M) was placed at the back focal plane of lens L2 to magnify the Fourier spectra of zeroand first-order diffraction spots derived from the striated cell image on LCTV. The intensity distribution of the selected diffraction spotswasrecordedby a secondunintensified chargecoupleddevice(CCD) camera(Fairchild 3000F,Sunnyvale, CA) operating in the noninterlaced mode. To reduce the intensity of zero-order diffraction spot, a thin strip of a 40% neutral density filter wasplaced at the expected position of the zeroorder diffraction spot on the CCD camerafaceplate. Stimulation of IsolatedMyocytes Myocytes (18 h) adherent to cover slips and bathed in medium199(1.25mM Ca*+)were mounted in a Dvorak-Stotler chamber and placed on the thermoregulated stage (37°C) of a Zeiss IM35 inverted microscope. The phase-contrast image (Zeiss Neofluar ~100/1.30 NA) of a selectedmyocyte at rest wasinitally captured by the ICCD camera,digitized, and stored in the computer. This digitized image (Fig. 5) wasusedlater in the day-to-day calibration of the hybrid optical-digital processor. The ICCD camera was rotated along its optical axis to optimize vertical alignment of the musclecell striation image on the LCTV. Myocytes were stimulated to contract at 0.2 Hz by field stimulation (6, 24) with biphasicpulsesthat were 3 ms in duration; voltage acrossthe platinum electrodesdetermined in air was 10V. To ensurea steady-statedegreeof Ca2+loading in sarcoplasmicreticulum (2) and minimize the effects of negative treppe phenomenon(17,31) observedin rat myocytes, only the 10th contraction was recordedand analyzed. Approximately four to six myocytes on each cover slip were studied. It wasfound that when the experimental period waslimited to 10 min for each cover slip, pH of medium 199 did not detectably changefrom the starting pH value of 7.40. The lo-min limitation wasstrictly adheredto, sinceit is well known that acidosis severely affects cardiac musclecontractile activity (34). Data Acquisition A dual-channel field stimulator (GrassS88 stimulator) was usedto deliver the depolarizingpulseat 0.2 Hz via one channel. The secondchannel was used to deliver a 5-V DC pulse to trigger the EPIX digitizing board (8 bits/pixel) 100 ms before the 10th depolarizingpulse. With a l-MB video board memory and custom-written software, a sequenceof 60 individual noninterlaced frames (16.7-ms duration each) could be acquired

The accurate measurementof grating spacing is possible only when the relative positions of zero- and first-order diffraction spotsare known. To determine changesin sarcomere lengthsaccurately, the processorwascalibrated with an Ealing high-resolution test target with spacingsranging from 1.25 to 3.16pm. The distance (D, pixels) between the centroidal locations (28) of the two first-order diffraction spotson both sides of the zero-order diffraction spotwasplotted againstthe grating spacing (pm; Fig. 3). It can be appreciated that although the overall calibration curve appearscurvilinear, over the range of expected sarcomerelengths (1.2-2.2 pm) the relationship between the distancebetweenthe two first-order diffraction spots and grating spacingis linear. This linear region of the calibration curve is thus usedfor sarcomerelength determinations. In addition, from the slopeof the calibration curve, it is theoretically possibleto detect sarcomerelength changesas small as 0.02 pm. Experience with our hybrid optical-digital processorindicatesthat although the slope(m) of the calibration curve ( y = mx + c) remainedconstant from day-to-day, the y-intercept (c) varied primarily as a result of adjustment of the standard bias voltage to the LCTV to obtain the maximal contrast of striated cell images.Thus a family of calibration curves was obtained with the same m but different c. We emphasize that this variation in c from myocyte to myocyte does not affect the calculation of sarcomereshortening and relengthening velocities, since with differentiation of both sides of the equation with respect to time, the constant c drops out. In other words, changesin sarcomerelength (dx/dt) are directly proportional to changesin distancebetweenfirst-order diffraction spots(dy/ dt).

n

n

O t;

0 TEST

1

TARGET

2

3

CALIBRATION,

4

5

MICRONS

tFig. 3. Calibration of hybrid optical-digital processor. Ealing highresolution test target is used as standard grating. Distance between left and right lst-order diffraction spots is plotted against calibrated target distance. Results of 2 separate calibrations are shown. Only linear portion (1.2-2.2 Mm) was used in calibration of sarcomere lengths.

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The average resting sarcomere length was determined from the distance histogram (Fig. 4A) of the white light video image of the myocyte (Fig. 5) using an Ealing high-resolution test target as standard. Our experience with the Bausch and Lomb stage micrometer suggests that spacings between etchings on the micrometer are of sufficient variations to preclude its use as an accurate standard.

2

___-______-_____

2000

d El500

---------------J TV

$1000

t

F

I

4 d 500 c

6 5 c’)

I

I

0’ 0

20

40

60

X-DISTANCE,

c3000 iz iTi $2400

80

100

120

PIXELS

B

i1800 X

iT ,120o I

c

k 600 5 3 0

0

0

20

40 X-DISTANCE,

60

80

100

120

PIXELS

Fig. 4. Improvement in image contrast by LCTV. A: variations of cumulative pixel intensity (20 rows summed/pixel distance) of digitized video image (Fig. 5, top) along long axis of isolated myocyte (X100/ 1.30 NA objective). B: boxed area in A is displayed on LCTV (40 rows summed/pixel distance on LCTV). Note 330% improvement in shadow cast of LCTV image.

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Data Analysis

The recorded time-dependent changes of the Fourier spectra during myocyte contraction were transferred to a VAX 6310 computer for image processing, determination of centroidal locations of first-order diffraction spots, and calculation of sarcomere shortening and relaxation velocities. The sarcomere length in each successive frame (16.7-ms duration, total ~60 frames) captured during myocyte contraction was determined from the corresponding Fourier spectra (Fig. 6) and plotted as a function of time (Fig. 7). Data (Fig. 7) were initially subjected to cubic-spline data smoothing (10) using IMSL Math Library subroutine ICSSCU. The first derivative (velocity) was then calculated by DCSEVU subroutine. To estimate sarcomere shortening velocity, the location of the peak contraction frame (minimum sarcomere length) was first identified. The velocities (1st derivatives) from the seventh frame from initiation of data acquisition (corresponding to time electrical depolarization was applied) to peak contraction frame were averaged to arrive at average sarcomere shortening velocity. Similarily, relaxation velocities at each frame after peak contraction frame were averaged until the frame with half the peak contraction magnitude was encountered. Measurement of Myocyte Shortening by Video Tracking and Edge-Detection

Dynamics Technique

Myocytes (2 and 18 h) adherent to cover slips and bathed in medium 199 were viewed with our previously described digital video-imaging system (8,35) using an Olympus Dplanapo x20/ 0.8 NA objective. The ICCD camera was rotated along its optical axis so that the long axis of the on-line real-time video image of the myocyte was aligned along the horizontal axis of the RGB monitor. Thus, with each stimulated contraction, the cell borders moved horizontally. Myocytes were stimulated with a protocol identical to that used for measurement of sarcomere mechanics described above (0.2 Hz, 37°C). The 10th contraction was captured as a sequence of 60 individual noninterlaced frames (16.7 ms each). To accurately detect cell borders in each individual digitized frame (Fig. 8A), an edge detection algorithm based on digital image-processing techniques (14) was applied off-line to each individual digitized frame. Briefly, the pixel intensities of four rows of pixels at each horizontal (n-axis) pixel location were accumulated to improve the signal-to-noise ratio and plotted as a function of X distance (Fig. 8B, top). A gradient operator X,) was then applied to identify the cell boundwn=xn+laries (Fig. 8B, bottom). The difference between pixel locations that correspond to the left and right edges (cell boundaries) was taken as the cell length. X distance in pixels was converted to microns with an Ealing high-resolution test target. To calculate whole cell shortening and relengthening velocities, data were first normalized to resting cell length (Fig. 9). The frame with the shortest cell length was identified. Starting at the seventh frame, normalized cell lengths were subjected to piecewise linear regression over four consecutive frames to derive velocity at each time point. The maximal values for cell shortening and relengthening velocities are reported in Table 3. Materials

All chemicals used were of reagent grade. Optical lenses used in the construction of the optical-digital processor were purchased from Melles Griot (Irvine, CA). Fig. 5. Fourier transform spectra of isolated myocyte. Top: digitized video image of an isolated myocyte; xl00 objective. Bottom: 0- and lstorder diffraction spots of Fourier spectra of myocyte image projected on LCTV. Intensity of O-order diffraction spot was attenuated as describedin MATERIALS AND METHODS.

Statistics

All results are expressed as means + SE. Significance was determined by Student’s two-sample t test. P < 0.05 was taken to be statistically significant.

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xels

Fig. 6. Time course of lst-order diffraction spots during contraction of isolated myocyte. Three-dimensional plot is shown: z-axis, intensity; y-axis, time in noninterlaced videoframe (16.7 ms) units; x-axis, distance (pixels) between O-order (left) and lst-order (right) diffraction spots. For clarity, only right lstorder diffraction spot is shown.

i9102 ifi a JO0

the current study, the widths of myocytes isolated from Hyp hearts were larger than those isolated from Sham hearts, but the lengths did not differ (Table 1). By modeling isolated cardiac cells as a perfect cyclinder, and ignoring T-tubule invaginations, the volumes of myocytes from Hyp and Sham hearts can be approximated and are shown in Table 1. It can be appreciated that LV myocytes from Hyp hearts had indeed undergone hypertrophy 8-10 wk after induction of hypertension.

r

Es 2 98

0 ii

a 96

Improvement 0

200

400

600

800

1000

MILLISECONDS Fig. 7. Sarcomere dynamics during contraction of normotensive (Sham) and hypertensive (Hyp) myocytes. Cells were cultured for 18 h before experiments. At 100 ms, depolarizing pulse is applied. q , Sham; A, Hyp. Note longer sarcomere relaxation time in Hyp myocyte. RESULTS

Renovascular Hypertension-Induced Cardiac Myocyte Hypertrophy

In a previous study, we reported that 8-10 wk after induction of renovasuclar hypertension myocytes isolated from the hypertrophied LV had cellular biochemical changes consistent with cellular hypertrophy (24). In

of Contrast

in Striated

Image by LCTV

The accuracy of sarcomere length measurement throughout the EC cycle in part depends on the shape and intensity of first-order diffraction spots. The intensity of first-order diffraction spots, in turn, depends on the contrast of the striated cell image. Thus it is important to maximize the contrast of striations in real time before performing the optical Fourier transform operation. Factors that limit the contrast of the striated cell image include defocused light from neighboring planes and scattering from multiple lens elements. Figure 4A shows the intensity profile of a digitized (256 gray scale/pixel) white light video image (as captured by the ICCD camera) along the long axis of a cardiac myocyte. The peaks and valleys represent I and A bands, respectively, of the sarcomeres. The contrast of the image can be estimated to be 0.176. In contrast, the

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Table 1. Cell size measurements Length, pm Width, pm Volume, pm3 No. of obs

of isolated myocytes

Sham

HYP

116.521.8

122.2rt4.6

22.2kO.8

27.1+1.3*

48,269&3,411

79,818+11,349*

56

36

Values are means * SE. Sham, normotensive rats; Hyp, hypertensive rats. * Sham vs. Hyp, P < 0.05 (unpaired Student’s t test).

Table 2. Sarcomere ventricular

mechanics

in left

myocytes Sham

k

600

Sarcomere length, pm %Shortening Time to peak shortening, ms Half-relaxation time, ms Avg shortening velocity, pm/s Max shortening velocity, pm/s Avg relaxation velocity, pm/s Max relaxation velocity, pm/s No. of obs

-

-200 0

50

100

150

X-DISTANCE,

200

250

PIXELS

Fig. 8. Whole myocyte shortening and edge detection algorithm. A: digitized video image of an isolated myocyte (x20 objective). B, top: variations of cumulative pixel intensity (4 rows summed/X pixel distance) of digitized video image along long axis of isolated myocyte; bottom, after application of gradient operator, X, = X, +i - X,, resulting pixel intensity profile is plotted against X distance in pixels. Arrows point to edges (cell boundaries) of myocyte.

0.96 0.97 .-P : r 0

0.96

5

0.95

ii s .-

0.94

2 I;:

0.93 0.92 0.91

0

100

200

300

400

500

600

700

Milliseconds Fig. 9. Myocyte shortening dynamics during contraction of Sham and Hyp myocytes. Cells were used within 2 h of isolation. At 100 ms, depolarizing pulse is applied. q , Sham (n = 59 cells); A, Hyp (n = 86 cells.

shadow cast of the same myocyte displayed on LCTV screen (Fig. 4B) had an estimated contrast of 0.758. The improvement in contrast (330%) of the LCTV image is mainly due to adjustment of bias voltage of the LCTV, so that the dark A bands have minimal intensity values,

1.871fO.01

1.84+0.01

8.K%O.O6

6.71kO.55

174f6 162+8

171f8 208+12*

0.82kO.06

0.67kO.05

1.87kO.14

1.34+0.14*

0.65zkO.07

0.36+0.04*

1.67kO.14 69

1.18+0.12* 52

Values are means + SE. Sham, normotensive; Hyp, hypertensive. Half-relaxation time was calculated from the time elapsed between peak contraction and when half the peak contraction magnitude was attained. Sarcomere shortening and relaxation velocities are corrected for 6.6% underestimation error (see RESULTS). * Sham vs. Hyp, P < 0.05 (unpaired Student’s t test).

and the limited number of available gray levels in LCTV that truncates the cell image to 16 gray cells, thus effectively increasing the contrast. Accuracy of Optical-Digital of Sarcomere Shortening

O.QS

HYP

Processor Velocity

in Determination

An uniform alternating dark-white bar pattern (length of repeat units 2.12 pm, contrast 0.2) was generated by the EPIX digitizing-display video board to serve as the ideal striated myocyte image and displayed on the LCTV screen. To simulate image formation, the bar pattern was convolved with the in-focus point spread function of our digital video-imaging system (35) and projected onto the LCTV. The distance between the two first-order diffraction spots of the Fourier spectra was measured. To simulate sarcomere contraction from 2.0 to 1.53 pm, width of white bar was progressively decreased (2%/ videoframe or 16.7 ms) while width of dark bar remained constant. The corresponding Fourier spectra of this ideal cell during rest and contraction were recorded and analyzed. The theoretical shortening velocity was 1.20 pm/ s, and the measured velocity was 1.12 pm/s. This 6.6% underestimate error has been corrected for values of sarcomere mechanics (Table 2). Sarcomere Mechanics of Single Cardiac from Sham and Hyp Hearts

Myocytes

The typical time courses of sarcomere length changes in two isolated myocytes cultured for 18 h and externally paced to contract are shown in Fig. 7. At 100 ms, a depolarizing pulse was delivered to the cell. Peak contraction was reached 183 ms after depolarization for Sham and 217 ms for Hyp myocytes. In addition, maxi-

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ma1 shortening was similar between Sham (6.3%) and Hyp (4.5%) myocytes. The major difference appears to be delayed sarcomere relaxation in the Hyp myocyte in that the time taken for the sarcomere to relax back to half the peak contraction amplitude (half-relaxation time) was 214 vs. 104 ms for the Sham myocyte. This is also reflected in the slower average relaxation velocity (0.22 pm/s) in the Hyp myocyte compared with the Sham myocyte (0.56 pm/s). The composite results of myocytes isolated from nine Sham and five Hyp hearts are shown in Table 2. It should be noted that similar results are obtained for average relaxation velocities if one chooses to average from peak contraction down to an arbitrarily defined 98.5% of resting sarcomere length (0.72 t 0.07 pm/s for Sham and 0.45 & 0.05 pm/s for Hyp myocytes) rather than back to 50% of peak contraction amplitude (0.65 t 0.07 pm/s for Sham vs. 0.36 t 0.04 pm/s for Hyp myocytes) . Cell Contraction Mechanics of Single Cardiac from Sham and Hyp Hearts

Myocytes

The time courses of cell length changes in freshly isolated myocytes (2 h) externally paced to contract are shown in Fig. 9. Similar to the results obtained with the optical-digital processor in cultured myocytes (Fig. 7)) cardiac cells freshly isolated from Hyp hearts displayed impaired relaxation (Table 3). This suggests that relaxation abnormalities in Hyp myocytes are a consequence of cell hypertrophy (Table 1; Ref. 24) from chronic renovascular hypertension rather than culturing for 18 h. In freshly isolated cells, maximal cell shortening velocity also appears to be slower in Hyp myocytes (Table 3). This difference in maximal shortening velocities between Sham and Hyp myocytes is also detectable after 18 h in culture (Table 2). Effects of short-term (18 h) culture on myocyte contraction mechanics. Cell shortening dynamics were measured

in 59 freshly isolated (2 h) and 31 Sham myocytes cultured for 18 h. Fractional cell length at peak contraction was significantly longer in 18-h myocytes (0.917 t 0.007) compared with 2-h cells (0.898 t 0.006; P 5 0.05). Time to peak shortening was also prolonged in 18-h myocytes (147 & 10 vs. 124 t 5 ms; P 5 0.03), as was the halfrelaxation time (137 t 7 vs. 90 t 8 ms; P s 0.001). Maximal shortening velocity was faster in freshly isolated myocytes (1.41 t 0.09 vs. 0.99 t 0.09 cell length/s; P 5 O.OOl), as was maximal relaxation velocity (1.29 t Table 3. Contractile ventricular

mechanics

of left

myocytes Sham

Normalized cell length at peak contraction Time to peak shortening, ms Half-relaxation time, ms Max shortening velocity, cell length/s Max relaxation velocity, cell length/s No. of obs

WP

0.898t0.006

0.913t0.005*

124t5 9Ok8 1.41t0.09

149t6* 114s* l.OSt0.06*

1.29t0.09

0.91t0.05'

59

86

Values are means & SE. Sham, normotensive; Hyp, hypertensive. * Sham vs. Hvp, P c 0.05 (unpaired Student’s t test).

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0.09 vs. 0.92 t 0.10 cell length/s; P 5 0.002). Thus, in general, short-term culture resulted in slowing of cell shortening dynamics, although the same differences in maximal shortening and relaxation velocities between Sham and Hyp myocytes were still detectable after 18 h of culture (Table 2). DISCUSSION

Optical diffraction methods have been used by many investigators to study muscle mechanics since the original report by Ranvier over a century ago (26). Most of the studies utilizing optical diffraction methods were performed in skeletal muscle, particularly in skinned muscle fibers from skeletal muscle (4, 13, 29, 32). Deviations in shape, position, and symmetry of the optical diffraction spectra from those expected of an ideal diffraction grating suggest that, in skeletal muscle fibers, there are many structural “domains” of relatively uniform sarcomere length and striation orientation separated by dislocations and other irregularities in the sarcomere pattern. These elegant studies also point out a potential drawback of laser diffraction studies of thick specimens such as muscle preparations in that in a diffraction experiment, constructive interference of rays scattered from different depths within a thick specimen depends on both the spacing and the tilt of the lattice planes (3). This Bragg-angle phenomenon has two consequences in term of muscle mechanics: 1) the sarcomere length derived from diffraction experiments may not be representative of all the domains within the illuminated area of the fiber; and 2) during muscle contraction, changes in sarcomere length and/or striation tilt can cause apparent discontinuities in the sarcomere length signal, giving rise to pauses in an otherwise smooth shortening pattern (4, 13, 29, 32). This point is particularly relevant to our current study in which our goal is to compare shortening and relaxation velocities of Sham and Hyp myocytes. On the other hand, in intact and tetanized frog skeletal muscle fibers subjected to an abrupt change in load, Granzier et al. (15) measured the resultant sarcomere length changes (the so-called “isotonic velocity transient”) and found that tracings obtained with the optical diffraction method and fiberlength record were similar, suggesting laser diffraction method may be applicable to measurement of dynamic sarcomere length changes under special circumstances. The major difference between previously published optical diffraction methods and our current study is the use of a LCTV as an SLM. Rather than directly illuminating the cardiac myocyte (avg diam 22-27 pm) with He-Ne laser, a thin optical section of the myocyte is projected onto the LCTV screen which is then illuminated. With a 1.30-NA aperture objective the in-focus optical section (depth of field) projected onto the LCTV can be estimated to be 0.11-0.22 pm, corresponding to visible light range of 390-780 nm. Illuminating a thin section rather than the entire cell tends to minimize the Bragg-angle phenomenon (3, 4), because rays scattered from different depths of the thick specimen are largely eliminated. Another advantage of illuminating the projected image rather than the mvocvte directly is avoid-

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ante of laser-induced photodynamic damage to the cell. Kent et al. (19) have reported that continued exposure to a 5mW laser beam for 5 min caused inhibition of cell contraction A thi .rd advantage iS the major improvement in contrast n the sh.adow cast of a myocyte displayed on LCTV screen (Fig. 4B) compared with the digitized video image of the same cell (Fig. 4A). This improvement in contrast is obtained under on-line real-time conditions as opposed to digital-enhancement techniques which are usually performed off-line. Constrast improvement enhances the intensity of first-order diffraction spots, thus increasing the accuracy of sarcomere length determination during myocyte contraction. The major disadvantage of our hybrid optical-digital processor is its relatively slow temporal resolution (16.7 ms) compared with the faster photodiode sensors (1 ms, Ref. 19; 5 ms, Ref. 11). Despite the limited temporal resolution imposed on the system by the RS-170 standard video format, our processor still responds with sufficient speed to capture sarcomere length changes during myocyte contraction (Figs. 6 and 7). Several methodological issues relating to the use of short-term cultured myocytes adherent to cover slips for contraction studies must be adressed before discussion and interpretation of data. The choice of 18-h cultured myocytes rather than freshly isolated cells for part of the current study relates to our previous experience that freshly isolated myocytes are very leaky to the fluorescent Ca2+ indicator fura- (5, 8), rendering the capture and analysis of [ Ca2+]i transients (especially calibration of fura- signals) difficult. The problems of excessive fura- leakiness and difficult calibration were overcome by placing myocytes in short-term culture (8), a strategy that we have successfully employed to demonstrate alterations in [Ca2+]i transients in Hyp myocytes compared with Sham cells (24). Because our ultimate goal is to investigate possible relationships between alterations in [ Ca2+]i-transients and impaired mechanical activity, it seemed logical to study contractile activity of myocytes under the same conditions established for measurement of [ Ca2+]i transients (24). We recognize the possibility that short-term culture may alter morphological and functional properties of myocytes, as reported for adult feline ventricular myocytes placed in culture (25). Indeed, short-term culture of our Sham myocytes for 18 h appears to slow both shortening and relaxation as well as decrease the peak amplitude of cell shortening (see RESULTS). Nevertheless, compared with “age-matched” Sham myocytes, impaired relaxation was observed in both freshly isolated (Table 3) and cultured Hyp myocytes (Table 2), indicating that this aspect of mechanical dysfunction is inherent in Hyp myocytes rather than as a consequence of culture. Another issue concerns the potential effects of cell adhesion to cover slips on measurements of sarcomere mechanics and cell contractions. In feline myocytes, Pollack et al. (25) reported that attachment to laminincoated cover slips resulted in an immediate decrease in extent of cell contraction (9.2 to 5.9% of resting cell length) and rates of cell shortening (0.67 to 0.32 cell length/s) and relaxation (0.52 to 0.28 cell length/s). Published data by Haworth et al. (17) on isolated rat

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heart cells, however, indicate that cell attachment had no effect on the extent (17.2 vs. 19.4%) or velocity of sarcomere shortening (2.58 vs. 2.47 pm/s) or on the rate of relaxation (2.24 vs. 2.33 pm/s). The difference between these two studies is not obvious but may relate to differences in species, extracellular media, cell isolation techniques, or degree of cell attachment. The peak shortening magnitude of our laminin-attached myocytes of 10% (Table 3) compares favorably with that of unattached guinea pig myocytes (10.8 * 2.1%, Ref. 30), rat myocytes “lightly attached to Petri dish at their midportion” (6.0 t 0.32%, Ref. 12), and unattached feline myocytes (9.2 + 1.2%, Ref. 25). Taken together, our data and those of Haworth et al. (17) suggest that the contractile behavior of rat myocytes is little affected, if at all, by cell attachment. Alternatively, isolated rat myocytes may not attach to laminin as tightly or at as many points as feline myocytes. In any case, some degree of cell attachment is essential for ventricular myocyte survival under culture conditions, since unattached myocytes in culture deteri-. orate and die within several hours after seeding (18). Finally, it should be recalled that myocytes are attached to each other as well as to extracellular matrix in their natural state, and thus the mechanical parameters measured in myocytes adherent to laminin-coated cover slips may more closely reflect contractile behavior in vivo than those obtained in unattached cells. The resting sarcomere length (1.87 t 0.01 pm) of our isolated rat cardiac myocytes is in the range of those determined by Fraunhofer diffraction (1.83 t 0.04 pm; Ref. 17), digitized cell imaging (1.83 t 0.13 pm; Ref. 27), and photomicrographic techniques (1.83 t 0.005 pm; Ref. 12). With respect to sarcomere dynamics, our value for percentage of peak shortening of 8.2 t 0.1% is almost identifical to that reported by Fraticelli et al. (6.0 t 0.3%; Ref. 12), although it is smaller than that reported by Haworth et al. (19.4 t 4.4%; Ref. 17). The apparent discrepancy between our results and those of Haworth et al. (17) is partly due to the fact that the larger peak shortening value relates to first-beat shortening dynamics rather than the steady-state shortening amplitude resulting after several contractions. Indeed, the amplitude of the fifth beat reported by Haworth et al. was in the range of 5% (Fig. 3 in Ref. 17), which is corroborated by our measurements of steady-state amplitude of sarcomere shortening (Table 2). Superficially, our value for sarcomere shortening velocity (0.82 t 0.06 pm/s) is quite slow compared with published values in the literature (2.18 t 0.13 pm/s, Ref. 12; 2.47 t 0.89 pm/s, Ref. 17). We hasten to point out, however, that our values represent the average shortening velocity (see MATERIALS AND METHODS), whereas published values are maximal velocities of shortening. We believe that averaging sarcomere shortening and relaxation velocities during the entire myocyte contraction and relaxation phases may be more representative of sarcomere dynamics than the usual practice of reporting maximal velocities. For comparison, our maximal sarcomere shortening velocity of 1.87 t 0.14 pm/s is within the range of published values (12, 17). A second factor accounting for our apparently slower sarcomere dynamics is that instead of measuring velocities derived from the first beats as was done previously (l7),

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sarcomere velocities derived from subsequent beats with more steady-state contraction amplitudes are reported in this study. Finally, sarcomere mechanics were measured in 18-h cultured myocytes as opposed to freshly isolated cells in previous studies (l&17). We have shown that short-term culture resulted in some 30% decrease in maximal cell shortening velocity in Sham myocytes (see RESULTS). Extrapolating back to freshly isolated conditions, our maximal sarcomere shortening velocity would have been 1.87 x 1.41/0.99 or 2.66 pm/s, in good agreement with previous investigations (12, 17). Similary, our average sarcomere relaxation velocity (0.65 t 0.07 pm/s) appears slow compared with the maximal relengthening velocity of 2.33 t 0.72 pm/s reported by Haworth et al. (17), although our maximal sarcomere relaxation velocity (1.67 -t 0.14 pm/s) is quite comparable, given the differences in contraction amplitudes between the 1st and 10th beats and the differences between freshly isolated and short-term cultured cells. Because our hybrid optical-digital processor, like any optical diffraction technique, only measures average periodicity of sarcomeres within the region illuminated by the laser beam and thus the values obtained for sarcomere mechanics may not be representative for the whole cell, we undertook studies of whole cell shortening by video tracking. Our value for maximal shortening velocity of 1.41 t 0.09 cell length/s (-164 pm/s) in freshly isolated Sham cells is similar to values reported by Fraticelli et al. (162 t 8.9 pm/s; Ref. 12) as well as by Duthinh and Houser in feline cardiomyocytes (0.92 t 0.03 cell length/s; Ref. 11). In addition, the magnitude of peak shortening for our 2-h Sham myocytes (Table 3) is also similar to those reportedly previously (11, 12, 25, 30). More importantly, relaxation abnormalities at the sarcomere level detected by our optical-digital processor (Fig. 7, Table 2) were also evident at the whole cell level as measured with video tracking (Fig. 9, Table 3). Thus the theorectical concern that the optical diffraction method only samples small domains of the myocyte and that the findings may not be generalized to the whole cell, although valid, is probably of little practical significance in view of the excellent agreement between sarcomere dynamics data obtained with optical diffraction (Table 2 and Ref. 17) and those derived from whole cell length tracking (Table 3 and Ref. 12). A major finding of the current study is that cardiac myocytes isolated from rats with chronic renovascular hypertension not only undergo increases in cell size (hypertrophy; Table 1) but also have impaired sarcomere and cell relaxation during an EC cycle (Tables 2 and 3). Impaired sarcomere relaxation and cell relengthening are intrinsic abnormalities in Hyp myocytes rather than changes brought on by cell culture conditions, since the abnormalities were observed in both freshly isolated (Table 3) and short-term cultured myocytes (Table 2). Previous studies on trabeculae carneae isolated from human hypertrophic cardiomyopathic hearts have demonstrated prolonged Ca2+ transient (detected by aequorin) and isometric twitch compared with controls (16). More recent investigations using myocytes isolated from hypertensive hypertrophic guinea pig hearts showed lower maximal fractional shortening and peak velocities

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of sarcomere shortening and relengthening (30). The results of our current study on hypertensive hypertrophic cardiomyopathic myocytes (Tables 2 and 3) are in agreement with those of Siri et al. (30), despite differences in animal species (rat vs. guinea pig) and method of inducing hypertension in the animal (renovascular vs. aortic banding). In addition, depressed myocyte shortening and relengthening are also observed in a model of right ventricular pressure overload in the cat (23). Taken together, these results as well as ours suggest that, in hypertrophic myocardium secondary to chronic hypertension, contraction abnormalities exist at the cellular level (myocyte), as opposed to tissue (papillary muscle) and organ (intact heart) levels. Thus, in addition to subendocardial ischemia (9), increased interstitial fibrosis (33), and altered chamber geometry, sarcomere and cell relaxation abnormalities may also contribute to the diastolic stiffness observed clinically and experimentally in hypertensive hypertrophied mycardium (22, 33). The mechanism of impaired sarcomere and cell relaxation in Hyp myocytes has not been addressed directly in this study but is unlikely to be a consequence of increases in cell size alone. Duthinh and Houser (11) clearly showed that, in myocytes isolated from normal feline hearts, there was no statistically significant difference in maximal rates of cell shortening and relengthening between small and large myocytes, despite a fourfold difference in the surface area. Thus residence in a setting of chronic renovascular hypertension appears necessary for myocytes to acquire the defect in sarcomere and cell relaxation. Altered Ca2+ sensitivity of the contractile proteins in Hyp myocytes also appears unlikely to be the explanation of impaired sarcomere relaxation, since Kimura et al. (20) have recently shown similar pCa-tension relationships in saponin-skinned muscle fibers derived from normal and pressure-overloaded left ventricles. In a previous study, we (24) have reported altered [ Ca2+]i dynamics in Hyp myocytes during EC. Specifically, peak [ Ca2+]i, +d[ Ca2+];/dt, -d[Ca2+]i/dt, and half time of [Ca2+]i decline were slower in Hyp myocytes, but time to peak [Ca2+]i was similar compared with that in Sham myocytes. Our results are corroborated by a later study, using guinea pig myocytes isolated from chronic aorticbanded hearts (30), in which peak [ Ca2+]i and +d[ Ca”‘]i/ dt were depressed but time to peak [Ca2+]i was not different from normotensive controls. The time required for [Ca2+]i transient to decay to 50% of its peak value was longer in hypertensive heart cells, although the difference did not reach statistical significance in that study (30), in contrast to our finding that half time of [Ca2+]i decline was longer in Hyp myocytes (24). Because [Ca2+]i changes occupy a central role in EC, it is tempting to attribute abnormal sarcomere and cell dynamics to prolonged [Ca”‘] transients in Hyp myocytes. In this light, it is particularly instructive to consider the elegant study of Yue (38) on the relationship between intracellular [Ca”‘] transient and force development during a twitch of isolated feline papillary muscle. The interval from peak [Ca2+]i to peak tension was -100 ms in papillary muscle, similar to what we observed in isolated Sham (mean: 93 ms) and Hyp (mean: 81 ms) myocytes. Our observations that maximal extent of sarcomere and

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cell shortening were quite similar between Sham and Hyp myocytes (Tables 2 and 3) despite a lower peak [Ca”‘]i in Hyp myocytes (24, 30) could also be accommodated by Yue’s model (38). Because the [Ca”‘]i transient is prolonged in Hyp myocytes (24), the total Ca2+time integral is also larger for a give n peak [Ca2+]i. Given similar myofilamen t sensitivity to Ca2+ between Sham and Hyp myocytes (20), the longer [Ca”‘]i transient effectively shifts the peak [Ca2+]i - peak tension curve to the left (38). Thus the same peak tension will be achieved at a lower [ Ca2+]i peak . Because active tension generation in papillary muscle is related to sarcomere shortening at the myocyte level, it is likely that the same line of reasoning can explain the lower peak [ Ca2+]i but similar percentage of maximal sarcomere shortening in Hyp myocytes. With regard to slowed cell shortening and relaxation, it is tempting to attribute these defects to the slower +d[Ca2+]i/dt and -d[Ca2+]i/ dt in Hyp myocytes. In support of this, Andrawis et al. (1) and Kimura et al. (21) have described subnormal Ca2+ transport activities in the sarcolemma and sarcoplasmic reticulum in hypertensive hypertrophied hearts. In summary, we have measured sarcomere dynamics in isolated cardiac myocytes with a novel on-line realtime hybrid digital-optical processor. We found that sarcomere relaxation was impaired in cardiac myocytes isolated from renovascular hypertensive rats. Impaired sarcomere relaxation was manifested at the whole cell level as slower cell rel .engthening velocity. We suggest that slower sarcomere relaxation provides the cellular basis for diastolic stiffness observed in the hypertensive hypertrophied ventricle. In addition, we hypothesize that the abnormal contraction mechanics is a direct consequence of altered [ Ca2+]i dynamics in hypertensive myocytes. The authors thank Tracey Erickson for assistance in the preparation of the manuscript. This work was supported in part by grants-in-aid from the Pennsylvania Affiliate of the American Heart Association (J. Y. Cheung and R. L. Moore), Research Grants from the Whitaker Foundation (J. Y. Cheung and R. V. Yelamarty), National Heart, Lung, and Blood Institute Grants ROl-HL-40306 (R. L. Moore and J. Y. Cheung), ROlHL-41582 (J. Y. Cheung and R. L. Moore), and ROl-HL-44146 (R. L. Moore), US Army Research Office under Contract DAAL03-87-K0147 (F. T. S. Yu), and National Institutes of Health Biomedical Research Support Grant Program, Div. of Research Resources, Grant S07-RR-05680-21. Address for reprint requests: J. Y. Cheung, Div. of Nephrology, Dept. of Medicine, The Milton S. Hershey Medical Center, Hershey, PA 17033. Received 4 February 1991; accepted in final form 13 November

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