This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kagaya, Y. Articles by Lorell, B. H. Search for Related Content PubMed PubMed Citation Articles by Kagaya, Y. Articles by Lorell, B. H.

(Circulation. 1996;94:2915-2922.)
© 1996 American Heart Association, Inc.

Articles

Long-term Angiotensin-Converting Enzyme Inhibition With Fosinopril Improves Depressed Responsiveness to Ca²⁺ in Myocytes From Aortic-Banded Rats

Yutaka Kagaya, MD; Roger J. Hajjar, MD; Judith K. Gwathmey, PhD; William H. Barry, MD; Beverly H. Lorell, MD

the Charles A. Dana Research Institute and the Harvard-Thorndike Laboratory of Beth Israel Hospital and the Department of Medicine (Cardiovascular Division), Beth Israel Hospital and Harvard Medical School, Boston, Mass, and the Division of Cardiology, Department of Medicine, University of Utah School of Medicine, Salt Lake City (W.H.B.).

Correspondence to Beverly H. Lorell, MD, Cardiovascular Division, Beth Israel Hospital, 330 Brookline Ave, Boston, MA 02215.

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background We have previously shown that long-term ACE inhibition with fosinopril prolongs survival and improves ventricular function despite persistent severe left ventricular pressure overload in ascending aortic–banded rats with left ventricular hypertrophy during the transition from compensation to failure.

Methods and Results To study the cellular mechanism of the effects of long-term ACE inhibition on the modification of the transition to failure in pressure-overload hypertrophy, we measured simultaneous intracellular Ca²⁺ transients and myocyte shortening in isolated left ventricular myocytes from fosinopril-treated aortic-banded rats (n=9), untreated aortic-banded rats (n=9), and normal age-matched control rats (n=10). Fosinopril therapy was begun 6 weeks after banding and was continued until week 21 after banding, when the animals were killed. Collagenase-dissociated myocytes loaded with indo 1-AM were paced at 3 Hz at 36°C and superfused at [Ca²⁺]_o of 0.6, 1.2, and 3.0 mmol/L. In myocytes from untreated aortic-banded rats, peak systolic [Ca²⁺]_i was higher than in control myocytes, and the relationship between myocyte shortening and [Ca²⁺]_i was depressed relative to control myocytes, implicating impaired responsiveness to Ca²⁺. Long-term fosinopril treatment improved both myocyte shortening and the relationship of shortening to [Ca²⁺]_i (P<.05 versus myocytes from untreated aortic-banded rats). Maximal Ca²⁺-activated force was depressed in chemically skinned left ventricular fibers from untreated aortic-banded hypertrophied rats relative to age-matched controls but not in the fosinopril-treated aortic-banded rats.

Conclusions Long-term ACE inhibition improves responsiveness to Ca²⁺ in the presence of normalization of maximal Ca²⁺-activated force in aortic-banded rats subjected to persistent pressure overload. This may contribute to the favorable effects whereby ACE inhibition modifies the transition from compensated hypertrophy to failure.

Key Words: hypertrophy • heart failure • myocytes • calcium • ventricles

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Angiotensin-converting enzyme inhibitors improve survival and cardiac function both in patients with severe congestive heart failure and in patients with asymptomatic left ventricular dysfunction.^{1 2 3} In rats and humans after long-term myocardial infarction, ACE inhibition has been shown to attenuate the deterioration in left ventricular function and prolong survival.^{4 5} ACE inhibitors also regress left ventricular hypertrophy both in humans with systemic hypertension^{6 7} and in rats with pressure overload.^{8 9} Recent studies from our laboratory^{10 11} showed that long-term treatment with the ACE inhibitor fosinopril prolongs survival and improves cardiac function despite severe persistent left ventricular pressure overload in rats with ascending aortic banding, suggesting that the favorable effects of fosinopril may be related to inhibition of cardiac ACE rather than systemic hemodynamic mechanisms. However, the cellular mechanisms that contribute to the favorable effects of long-term ACE inhibition in pressure-overload hypertrophy and prevent the transition to cardiac failure are still uncertain.

To clarify the cellular mechanisms of the effects of long-term ACE inhibition in pressure-overload hypertrophy independent of changes in cardiac load and geometry, we studied simultaneous intracellular [Ca²⁺]_i transients and cell shortening in isolated myocytes loaded with indo 1-AM from ascending aortic-banded rats randomized to treatment for 15 weeks with the ACE inhibitor fosinopril or no drug and age-matched controls. The relationship between free Ca²⁺ and developed force was also studied in chemically skinned fibers from the three groups. The results of this study suggest that myocyte responsiveness to [Ca²⁺]_i is impaired in myocytes from aortic-banded rats and that this is associated with the depression of maximal myofibrillar Ca²⁺-activated force. Long-term treatment with the ACE inhibitor fosinopril improves the responsiveness to Ca²⁺ in myocytes from rats with long-term pressure overload in association with the normalization of maximal myofibrillar Ca²⁺-activated force.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Preparation of Aortic-Banded Rats
Weanling male Wistar rats were obtained from the Charles River Breeding Laboratories (Wilmington, Del). As previously described by our laboratory,^{10 11 12 13 14} aortic banding was produced at the age of 3 to 4 weeks (body weight, 75 to 100 g) by a stainless steel clip of 0.6-mm ID placed on the ascending aorta via a thoracic incision. Age-matched control rats underwent a sham operation. The rats were fed normal rat chow and water ad libitum. As described in our previous report,¹⁰ 6 weeks after the surgery, the aortic-banded rats were randomized to receive treatment with fosinopril 50 mg·kg^-1·d^-1, which was added to the drinking water, or no treatment. The sham-operated rats received no drug treatment and served as age-matched controls. Drug or no drug treatment was continued for 15 weeks, from week 6 to week 21 after banding. This treatment period was selected because it encompasses the transition from compensated hypertrophy to early failure in this model.^{10 11 12}

Simultaneous Measurement of [Ca²⁺]_i and Cell Motion
Left ventricular myocytes were prepared by a modification of the methods of Capogrossi et al¹⁵ as reported from our laboratory.^{16 17} [Ca²⁺]_i was measured with the Ca²⁺-sensitive fluorescence indicator indo 1-AM¹⁸ (Molecular Probes, Inc) prepared by modification of the method of duBell et al¹⁹ and Ikenouchi et al.²⁰ First, 10 mL of FCS was mixed with 234 µL of 0.25 Pluronic F 127 (BASF Wyandotte Corp) dissolved in DMSO. Then 1 mL of 1 mmol/L indo 1-AM in DMSO was added to 9 mL of FCS-Pluronic F 127 mixture, sonicated, and divided into 400-µL aliquots, which were stored at -20°C. Myocytes were attached on coverslips with cell adhesive (Cell-Tak, Collaborative Research, Inc) and loaded with 5 µmol/L indo 1-AM in HEPES-buffered solution at room temperature for 30 minutes. The coverslip was rinsed with indo 1-AM–free buffer solution and placed in a flow-through heated (36°C) cell superfusion chamber on the stage of an inverted microscope (Nikon). The instrumentation for fluorescence measurement has been described in detail elsewhere.^{16 17 20} The excitation source was a high-pressure mercury arc lamp, which provides an intense emission peak at 360 nm. Further selection of this excitation was made with narrow-bandwidth interference filters. The excitation beam was chopped at 360 Hz to reduce bleaching, and the myocyte was illuminated via epifluorescent optics with a Fluor x40 objective lens (Nikon). The fluorescent light was collected by the objective lens and transmitted to a custom-modified spectrofluorimeter (FM-1000, Rincon Scientific Instruments) for simultaneous measurement of both 400- and 500-nm wavelengths with two separate photomultiplier tubes. The spectrofluorimeter provided analog signals representing the fluorescence intensity at both wavelengths and the ratio of emitted fluorescence (F400/500 nm). The subtraction of background autofluorescence was done by offsetting the photomultiplier tube outputs during the measurement of fluorescence from an unloaded myocyte at the beginning of each experiment. An adjustable iris was used to restrict the optical image to only one myocyte of interest in each experiment to minimize background fluorescence from other myocytes. The image of the beating myocyte was obtained by illumination via the 50-W standard microscope light source passed through a 645-nm sharp-cut filter. This wavelength was long enough not to interfere with the fluorescence detection at 400 and 500 nm. The motion of the myocyte was monitored with a solid-state camera (GP-CD60, Panasonic) and a custom-modified video detector system²¹ (Crescent Electronics). The analog output of the cell motion signal was monitored and recorded continuously with the analog signal of the [Ca²⁺]_i-sensitive fluorescence ratio (F400/500 nm). Two platinum electrodes placed in the bathing fluid were connected to a stimulator (SD9G, Grass Instruments) and used to stimulate the myocyte at 3 Hz with 3-ms pulses. We have recently documented the stability of both cell motion and [Ca²⁺]_i-sensitive fluorescence of normal and hypertrophied myocytes subjected to electrical stimulation of 3 Hz for 15 minutes.¹⁷ Because indo 1 can buffer cytosolic Ca²⁺, we have also previously examined the effects of indo 1 loading on the amplitude of cell shortening in control and hypertrophied myocytes.¹⁷ In the presence of indo 1, cell shortening was slightly decreased to a similar extent in both control and hypertrophied myocytes (-13.6% and -11.5%, P=NS), indicating that any Ca²⁺-buffering effect of indo 1 is similar between control and hypertrophied myocytes. To determine the absolute values of peak-systolic and end-diastolic [Ca²⁺]_i in hypertrophied and control myocytes, we performed calibration studies by a modification of the methods of Cheung et al²² and Borzak et al,²³ which have been described in detail elsewhere.¹⁷

Total numbers of hearts used for the isolation of myocytes in the present experiments were 9 from sham-operated rats, 13 from aortic-banded rats, and 9 from aortic-banded rats treated long-term with fosinopril. The yields of viable myocytes, which were defined as the percentage of rod-shaped myocytes with clear striations and exclusion of trypan blue, were 60% to 70% in control myocytes and 50% to 60% in hypertrophied myocytes. To prevent bias when we selected a myocyte to be analyzed among myocytes in a microscopic field, we chose a rod-shaped myocyte with very clear striation, without any spontaneous cell motion oscillations at a pacing rate of 3 Hz. This selection is against the potential bias to select excessively vigorous myocytes rather than depressed myocytes. One to three experiments were performed in sequence from separate coverslips of myocytes isolated from one heart.

Experimental Protocols
The myocytes from the untreated aortic-banded rats, fosinopril-treated aortic-banded rats, and age-matched controls were superfused with oxygenated HEPES-buffered solution of the following composition (mmol/L): NaCl 137, KCl 3.7, MgCl₂ 0.5, HEPES (free acid) 4, CaCl₂ 0.6, glucose 5.6, acetate 5.0, and probenecid 0.5, with a final pH of 7.40. Probenecid, a blocker of organic anion transport, was added because it has been shown to inhibit secretion of indo 1 free acid from loaded cells in HEPES buffer of physiological temperature.²⁴ The myocytes were paced at 3 Hz at 36°C. After baseline data were recorded, the cells were superfused with oxygenated HEPES-buffered solution containing increasing concentrations of perfusate Ca²⁺ (0.6, 1.2, and 3.0 mmol/L). The analog cell motion signals and the F400/500 nm analog signals were recorded simultaneously in a steady state after 4 to 5 minutes of superfusion at each Ca²⁺ concentration. To calculate the absolute value of [Ca²⁺]_i, the F400/500 values were converted to [Ca²⁺]_i by use of the calibration procedure previously described.¹⁷

To determine whether depressed responsiveness to Ca²⁺ is present in hypertrophied myocytes from young aortic-banded rats before the transition to failure, this protocol was also performed in myocytes dissociated from hearts 9 to 10 weeks after aortic banding (n=4) and age-matched controls (n=6). The temperature, pH, and pacing rate were the same as those in the above protocol.

Effects of Isoproterenol in Hypertrophied Myocytes
To investigate whether the response of [Ca²⁺]_i and cell shortening to isoproterenol differs in hypertrophied versus control myocytes and whether long-term fosinopril therapy modifies this response, myocytes loaded with indo 1 from untreated rats 21 weeks after aortic banding (n=6), fosinopril-treated aortic-banded rats (n=7), and age-matched controls (n=4) were perfused with the same HEPES-buffered solution with [Ca²⁺] of 1.2 mmol/L and paced at 3 Hz. After baseline cell shortening and [Ca²⁺]_i transients were recorded, myocytes were superfused with buffer solution supplemented with 5x10^-8 mol/L and 10^-6 mol/L isoproterenol. Data were recorded in a steady state after 5 minutes of superfusion at each isoproterenol concentration.

Effects of Colchicine on Cell Shortening in Hypertrophied Myocytes
To investigate whether cytoskeletal microtubules contribute to the depressed cell shortening in myocytes from untreated aortic-banded rats, a separate experiment was performed in unloaded hypertrophied myocytes (n=4) superfused with HEPES-buffered solution and paced at 3 Hz. After baseline cell shortening was recorded, cells were superfused with solution supplemented with 10^-6 mol/L colchicine for 80 minutes.^{25 26} Myocytes were paced at 3 Hz, and measurements of cell shortening were repeated.

Skinned Fiber Preparations
Trabeculae carneae from additional rat left ventricles from the three groups were dissected and attached to a muscle holder at one end and to a force transducer at the other end in a bath containing Krebs-Henseleit buffer at 22°C. The diameters of the trabeculae from the control (n=8), untreated aortic-banded (n=8), and fosinopril-treated aortic banded (n=8) rats were 0.20±0.04, 0.22±0.08, and 0.15±0.03 mm, respectively, whereas muscle lengths were 2.75±0.32, 2.60±0.38, and 2.48±0.44 mm, respectively. The muscles were stretched to L_max and chemically skinned as described previously in an EGTA-buffered solution containing 500 µg/mL of saponin.^{27 28 29 30} The muscles were activated with solutions containing increasing concentrations of Ca²⁺ (calculated with the program described by Fabiato and Fabiato³¹ ). The force versus [Ca²⁺] relationships were fitted by the modified Hill equation: F=F_max{[Ca²⁺]ⁿ/([Ca²⁺]ⁿ+[Ca²⁺]_50%ⁿ)}x100, where F is developed force, F_max is the maximum force developed at pCa of 4.0, n is the Hill coefficient, [Ca²⁺] is the Ca²⁺ concentration, and [Ca²⁺]_50% is the Ca²⁺ concentration required for 50% activation. Activation curves were fitted individually to the Hill equation. The mean of the Hill parameters generated in each group was tested for statistical significance by ANOVA.

Statistical Analysis
All values are expressed as the mean±SEM. The statistical analysis of differences between the groups was done by ANOVA comparison or ANOVA for repeated measures where appropriate and Fisher's exact test for post hoc analyses. Statistical significance was accepted at the level of P<.05.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Table 1 reports the end-diastolic cell length, time course of shortening and relaxation, and the time course of the [Ca²⁺]_i transients in the three groups under the experimental conditions of stimulation at 3 Hz and 36°C. End-diastolic cell length was significantly increased in myocytes from aortic-banded rats 21 weeks after banding compared with controls, whereas end-diastolic cell length was similar in myocytes from aortic-banded and fosinopril-treated aortic-banded rats. The fractional cell shortening was depressed and the time to peak shortening and time to half relengthening were prolonged in myocytes from the untreated aortic-banded rats relative to controls. Long-term ACE inhibition with fosinopril was associated with significant improvement in cell shortening, the time to peak shortening, and the time to half relengthening in myocytes from treated aortic-banded rats compared with untreated aortic-banded rats. None of the myocytes from the aortic-banded rats exhibited the biphasic time course of markedly aberrant Ca²⁺ decay that has been observed in myocardium from end-stage failing human hearts studied under hypothermic conditions.³²

View this table: [in this window] [in a new window]   Table 1. Characteristics of Myocyte Function and Intracellular Ca2+

Representative tracings from the myocytes from the aortic-banded rats randomized for 15 weeks of treatment with fosinopril or no drug and age-matched controls are shown in Fig 1. Fig 2 shows the relationship between fractional myocyte shortening and peak systolic [Ca²⁺]_i (top) and between myocyte shortening and the [Ca²⁺]_i transient (bottom). There was no difference in end-diastolic [Ca²⁺]_i between the three groups. Peak systolic [Ca²⁺]_i was increased in myocytes from the untreated aortic-banded rats compared with control myocytes. In contrast, peak systolic [Ca²⁺]_i was significantly lower in myocytes from the fosinopril-treated aortic-banded rats relative to untreated aortic-banded rats and similar to the control myocytes. The relationship between cell shortening and peak systolic [Ca²⁺]_i was shifted downward and to the right in the myocytes from the untreated aortic-banded rats compared with the control myocytes (P<.05). In contrast, in the myocytes from fosinopril-treated aortic-banded rats, the relationship between cell shortening and peak systolic [Ca²⁺]_i was shifted toward that of the control myocytes and significantly different from the myocytes from the untreated aortic-banded rats (P<.01).

View larger version (18K): [in this window] [in a new window]   Figure 1. Representative traces of myocytes from control sham-operated rats (Sham, top), untreated aortic-banded rats (LVH, middle), and aortic-banded rats treated long-term with fosinopril (FosLVH, bottom) in response to three different perfusate Ca2+ concentrations. Upper trace displays cell motion, and lower trace displays [Ca2+]i-sensitive fluorescence transient. Tracings are displayed with the convention that systolic cell shortening is shown as an upward deflection. Myocytes from aortic-banded rats were studied 21 weeks after banding.

View larger version (16K): [in this window] [in a new window]   Figure 2. Top, Relationship between peak systolic [Ca2+]i and myocyte shortening (% baseline) during exposure to three different perfusate Ca2+ concentrations of 0.5, 1.2, and 3.0 mmol/L. Bottom, Relationship between myocyte shortening and [Ca2+]i transient, ie, [Ca2+]i. Abbreviations as in Fig 1.

We also wished to determine whether this impairment of the relationship of cell shortening to Ca²⁺ that was present in myocytes from the untreated aortic-banded rats at the stage of transition to failure was also present at an earlier stage after banding. Fig 3 shows the relationship between cell shortening and peak systolic [Ca²⁺]_i (top) and cell shortening and the [Ca²⁺]_i transient (bottom) in myocytes from age-matched controls and aortic-banded rats killed 9 to 10 weeks after banding at the earlier stage of compensated hypertrophy when we have shown that left ventricular ejection fraction and chamber volume are normal.^{10 11 12} The relationship between cell shortening and peak systolic [Ca²⁺]_i was shifted slightly down and to the right in myocytes from aortic-banded rats early after banding compared with their age-matched controls. As illustrated in Fig 3, marked depression of cell shortening relative to [Ca²⁺]_i was present at the later stage 21 weeks after banding compared with 10 to 11 weeks after banding.

View larger version (20K): [in this window] [in a new window]   Figure 3. Top, Relationship between peak systolic [Ca2+]i and amplitude of cell shortening in myocytes from aortic-banded rats at an early stage of hypertrophy (9 to 10 weeks after banding) and their age-matched controls and aortic-banded rats 21 weeks after banding and their age-matched controls. Bottom, Relationship between myocyte shortening and [Ca2+]i transient, ie, [Ca2+]i. Abbreviations as in Fig 1.

To determine whether the depressed relationship between cell shortening and peak systolic [Ca²⁺]_i in the myocytes from the untreated failing aortic-banded rats 21 weeks after banding is related to increased microtubule density, experiments were performed using the strategy of Tsutsui et al,²⁵ in which myocytes were exposed to colchicine to depolymerize the intracellular microtubules. Fig 4 illustrates that exposure to colchicine promoted no improvement in cell shortening in myocytes from the untreated aortic-banded rats.

View larger version (14K): [in this window] [in a new window]   Figure 4. Effects of 80 minutes of exposure to colchicine on myocyte shortening in myocytes from rats 21 weeks after aortic banding. Exposure to colchicine to depolymerize intracellular microtubules did not enhance the amplitude of cell shortening. Abbreviation as in Fig 1.

To determine whether the differences in the relationship between cell shortening and peak systolic [Ca²⁺]_i among the groups were associated with differences in myocyte responsiveness to the ß-adrenergic stimulation, the response of cell shortening and peak systolic [Ca²⁺]_i to isoproterenol was studied. Compared with baseline, myocyte shortening significantly increased in response to isoproterenol in the control myocytes (Fig 5, top). Although cell shortening was significantly greater at baseline in the absence of isoproterenol in the myocytes from fosinopril-treated aortic-banded rats compared with untreated aortic-banded rats, isoproterenol caused no further increase in cell shortening in the myocytes from untreated aortic-banded rats or fosinopril-treated aortic-banded rats. In contrast, peak systolic [Ca²⁺]_i increased in response to isoproterenol in all three groups, and there was no difference in the magnitude of this response (Fig 5, bottom).

View larger version (16K): [in this window] [in a new window]   Figure 5. Effects of isoproterenol on amplitude of cell shortening (top) and change in cell shortening versus change in [Ca2+]i (bottom). Abbreviations as in Fig 1.

To directly examine myofilament Ca²⁺ sensitivity, the relationship between free Ca²⁺ and force was directly measured in chemically skinned fiber preparations from the control, untreated aortic-banded, and fosinopril-treated aortic-banded rats. The activation range of skinned fibers from the three groups was 10^-7 to 10^-4 mol/L, as shown in Fig 6, top. Table 2 depicts the Hill parameters from the force-pCa relationships. There were no significant differences in the sensitivity of the myofilaments to Ca²⁺ among the three groups. However, the maximal Ca²⁺-activated force (F_max) was significantly depressed in the aortic-banded group relative to the control group, whereas maximal Ca²⁺-activated force was not depressed in the fosinopril-treated aortic-banded group (Fig 6, bottom).

View larger version (17K): [in this window] [in a new window]   Figure 6. Top, Force-pCa relationships normalized to maximal Ca2+-activated force from chemically skinned myofibers from control rats (CON), aortic-banded rats (LVH), and aortic-banded rats treated long-term with fosinopril (FOS). There was no significant difference between the groups. Bottom, Absolute force-pCa relationships from each experimental group. FOS group was significantly different from control and LVH groups (P<.05, ANOVA).

View this table: [in this window] [in a new window]   Table 2. Parameters of the Force-Ca2+ Relationship in Skinned Fiber Preparations

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates that in myocytes from rats with long-term pressure overload due to ascending aortic banding that were studied at a stage of transition to failure, peak systolic [Ca²⁺]_i is increased, whereas the relationship between cell shortening and [Ca²⁺]_i is depressed relative to myocytes from age-matched control rats. Long-term treatment with the ACE inhibitor fosinopril in aortic-banded rats is associated with a reduction in the elevation of intracellular Ca²⁺ and improvement in the relationship between cell shortening and [Ca²⁺]_i relative to myocytes from untreated aortic-banded rats. This observation may elucidate a cellular mechanism that contributes to the beneficial effects of long-term ACE inhibition in pressure-overload hypertrophy and heart failure.^{1 2 3 4 5 10 11}

We have previously characterized the natural history and the molecular phenotype of the model of pressure-overload hypertrophy in the ascending aortic-banded rat in detail.^{10 11 12 13} For a period until 10 to 12 weeks after banding, the model is characterized by concentric left ventricular hypertrophy with normal left ventricular ejection fraction and chamber volume in vivo and preserved force generation per gram of left ventricular mass. At a later stage, through 20 to 22 weeks after banding, the animals undergo a transition to failure characterized by premature mortality, wet lungs, a 45% to 50% increase in left ventricular mass, and impairment of force generation per gram relative to age-matched controls.^{10 12} At this stage, in vivo echocardiography studies demonstrate the development of a mild depression of left ventricular ejection fraction and increase in end-systolic volume that differs from the severe left ventricular dilatation and depression of ejection fraction that characterizes the end-stage failing human heart at the time of cardiac transplantation.¹¹ Quantitative morphometry and biochemical studies have shown that at this stage of transition to failure, the hypertrophied hearts are not characterized by a significant increase in the volume fraction of noncollagen interstitial matrix, interstitial fibrillar collagen, or hydroxyproline relative to age-matched controls.¹² Thus, a major strength of this model is that it permits the study of an early stage of the development of heart failure before the development of extensive collagen deposition, which is not available for study with tissues from end-stage human failing hearts obtained at cardiac transplantation or models such as the failing spontaneously hypertensive rat.³³ Changes in left ventricular gene expression that recapitulate the fetal cardiac gene program are present early after aortic banding,³⁴ including an enhanced expression of ß-myosin heavy chain and -skeletal actin as well as cardiac paracrine genes, including atrial natriuretic factor and ACE.^{12 13 14} In contrast, alterations in the expression of Ca²⁺ regulatory genes, including reduced levels of sarcoplasmic reticulum Ca²⁺-ATPase mRNA,^{35 36} are not present until the late period of transition to failure.¹² In a recent study, we showed that long-term treatment with the ACE inhibitor fosinopril from week 6 to week 21 after banding in this model markedly improved survival and preserved left ventricular function in vivo, although the treated and untreated animals had persistent and comparable severe elevation of left ventricular systolic pressure.^{10 11} In addition, we showed that isolated normothermic hearts exhibited enhanced force development per gram in response to increasing levels of perfusate Ca²⁺ in the fosinopril-treated aortic-banded rats relative to untreated aortic-banded rats.¹⁰

In the present study of isolated paced myocytes studied under normothermic conditions, we demonstrated that peak systolic [Ca²⁺]_i was increased in association with depressed myocyte shortening in myocytes from untreated aortic-banded rats at this stage of transition to failure compared with control myocytes. We did not observe marked prolongation of the Ca²⁺ transient, which has been reported in other studies of myocytes from end-stage failing hearts under hypothermic conditions.³² Since the yield of rod-shaped cells with stable paced contraction from the dissociation process is less in the aortic-banded than in the normal hearts, it is possible that the dissociation process itself selected for "healthier" myocytes from a heterogeneous population in the aortic-banded hearts. Furthermore, shortening of relatively unloaded myocytes was measured in this experiment rather than force. It is interesting that the relationship between cell shortening and [Ca²⁺]_i is shifted slightly but significantly downward even in myocytes from younger rats 9 to 10 weeks after banding at an early stage of "compensated" hypertrophy, when in vivo echocardiography shows preservation of left ventricular ejection fraction and the absence of ventricular dilatation.¹¹ This is consistent with recent observations in humans with pressure-overload hypertrophy, which suggest that favorable changes in cardiac geometry may initially compensate for and mask early intrinsic depression of contractile function.³⁷ These observations indicate that a decreased availability of intracellular activator Ca²⁺ is not the mechanism responsible for the depressed systolic cell shortening observed in this study or the depressed systolic function observed in vivo and in the isolated heart preparation in our previous studies at this early stage of transition to heart failure in this model.^{10 11 13}

These findings are consistent with recent observations by others using alternative techniques to assess intracellular Ca²⁺ regulation. Keung³⁸ reported that the cardiac transmembrane L-type Ca²⁺ current was increased in the Goldblatt renovascular hypertensive rat in which cardiac hypertrophy was present, although myocardial function was not characterized. Litwin and Morgan³⁹ used the luminescent indicator aequorin and showed that peak systolic [Ca²⁺]_i tended to be higher rather than depressed in papillary muscles from the noninfarcted region of rats during the development of postinfarction heart failure in which both depression of left ventricular +dP/dt and elevation of left ventricular end-diastolic pressure were present. Furthermore, in human heart failure, Gwathmey and Hajjar²⁹ showed that peak [Ca²⁺]_i was not different from that in nonfailing muscles. These observations differ from those of Siri et al⁴⁰ and Beuckelmann et al,⁴¹ who reported that peak systolic [Ca²⁺]_i was decreased in myocytes from end-stage guinea pig hearts and end-stage human dilated cardiomyopathy, respectively. These discrepancies may be partially explained by critical differences in the stage of myocardial failure (early failure versus end-stage failure) and in experimental conditions, including hypothermia versus normothermia and depolarization rate of the myocytes. It is of interest that the differences in myocyte shortening relative to [Ca²⁺]_i in the myocytes from the untreated and fosinopril-treated aortic-banded rats are not associated with normalization of the contractile response to ß-adrenergic stimulation, which was depressed in both groups relative to controls.

Altered intracellular microtubule density could potentially account for the decrease in cell shortening in response to Ca²⁺ in the myocytes from the untreated aortic-banded rats in this study. Tsutsui et al^{25 26} reported that impaired contractile function of hypertrophied right ventricular myocytes from cats after pulmonary artery banding was associated with increased intracellular microtubule density, which could be restored to normal by microtubule depolymerization with colchicine. In the present study, the depressed cell shortening in the myocytes from aortic-banded rats did not improve under a similar protocol of exposure to colchicine, and immunoblots did not demonstrate any consistent increase in levels of free, polymerized, or total myocardial ß-tubulin in the hypertrophied left ventricles (George Cooper IV, MD, written communication, December 1995). These data suggest that an increase in the microtubule component of the cardiocyte cytoskeleton is not the predominant mechanism of impaired myocyte shortening in this model.

Role of Shortening Velocity Versus Myofilament Ca²⁺ Activation
In rats with pressure-overload hypertrophy, the replacement of -myosin by ß-myosin results in a decrease in shortening velocity and cross-bridge cycling rate. A decrease in cross-bridge cycling rate has also been observed in a number of animal models of hypertrophy and in human heart failure even without this change in myosin isoforms.^{28 30} A decrease in shortening velocity itself could contribute to the reduced cell shortening observed in the myocytes from the untreated aortic-banded rats. A depressed shortening velocity could also account for the prolongation of time to half relengthening in myocytes from the untreated aortic-banded group due to altered restoring forces. We do not know whether the improvement in myocyte shortening relative to [Ca²⁺]_i observed in the fosinopril-treated group is related to a reversal of ß-myosin to -myosin or a change in thin-filament composition resulting in an increase in cross-bridge cycling rate.^{28 29 30}

Since steady-state contraction more accurately reflects myofilament Ca²⁺ activation than behavior during repetitive twitches,²⁷ we also studied Ca²⁺ activation in chemically skinned left ventricular fibers from the three experimental groups. These experiments showed that the peak myocyte shortening versus peak [Ca²⁺]_i does not precisely reflect the sensitivity of the myofilaments to Ca²⁺, just as the relationship of peak force versus peak [Ca²⁺]_i in multicellular preparations does not reflect the sensitivity of the myofilaments to Ca²⁺.²⁷ In the skinned fiber preparations, the hypertrophied muscles exhibited similar sensitivity of the myofilaments to Ca²⁺ in comparison with the control muscles when the relationship between force and Ca²⁺ was normalized to 100% of maximal Ca²⁺-activated force. This is consistent with findings from other animal models of hypertrophy and failure.^{27 28 29 30 42} Treatment with fosinopril did not significantly change the sensitivity of the myofilaments to Ca²⁺. On the other hand, maximal Ca²⁺-activated force and absolute values of Ca²⁺-activated force were reduced in the myofibers from the untreated aortic-banded rats in comparison with control rats, which was not observed in myofibers from aortic-banded rats treated long-term with fosinopril.

Several variables can affect Ca²⁺-activated force in skinned fibers. The response of myofilaments to Ca²⁺ is higher in intact than in skinned fibers.^{42 43} The response of the myofilaments to Ca²⁺ is also exquisitely sensitive to sarcomere length.⁴⁴ Sarcomere length was not directly measured in these studies. However, in the skinned-fiber experiments, muscle length was controlled, and it is unlikely that different distributions of sarcomere lengths between the three groups of animals contributed to differences in the responsiveness of the myofilaments to Ca²⁺. Different models of hypertrophy and failure have exhibited differences in maximal Ca²⁺-activated force. In end-stage human dilated cardiomyopathy and in the turkey model of furazolidone-induced myopathy, maximal Ca²⁺-activated force is similar to controls, whereas it is decreased in the Syrian hamster cardiomyopathy model and in experimental alcoholic cardiomyopathy.⁴⁵ In the present study, the fosinopril-treated group displayed an increase in maximal Ca²⁺-activated force relative to the untreated aortic-banded group. This may be due to an increase in myofibrillar content or to a change in composition of the thin filaments. Troponin T isoforms have been demonstrated to change in human cardiomyopathy with associated changes in myofibrillar ATPase and activation of the myofilaments to Ca²⁺,⁴⁶ and myosin light chain kinase 2, which modifies the actomyosin reaction, has also been shown to be depressed in human cardiomyopathy.⁴⁷

An alternative mechanism is that changes in lateral separation between thick and thin filaments also affect the myofibrillar responsiveness of the myofilaments to Ca²⁺. We have previously reported that myocytes from the untreated aortic-banded rats had markedly increased myocyte diameter, and presumably cell volume, compared with myocytes from the long-term fosinopril-treated aortic-banded rats and the normal rats.¹⁰ Since osmotic compression can increase the myofilament responsiveness to Ca²⁺,⁴⁸ the decrease in maximal Ca²⁺-activated force in the untreated aortic-banded group could be partially due to an increase in lateral separation between the thick and thin filaments, decreasing the likelihood of cross-bridge interactions. Future studies are needed to examine these potential subcellular mechanisms for impaired force development and unloaded myocyte shortening in aortic-banded rats undergoing the transition to failure and their potential modification by long-term ACE inhibition.

	Acknowledgments

 
This work was supported in part by National Heart, Lung, and Blood Institute grants HL-38189 (Dr Lorell) and HL-39091, HL-367797, and RO1-HL-49574 (Dr Gwathmey); by Established Investigatorships of the American Heart Association (Dr Lorell and Dr Gwathmey); and by an educational grant from Bristol-Myers Squibb. We greatly appreciate the contributions of Dr G. Cooper IV and his laboratory regarding assessment of the contribution of intracellular microtubules in this model.

Received January 11, 1996; revision received June 5, 1996; accepted June 6, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. The CONSENSUS Trial Study Group. Effects of enalapril on mortality in severe congestive heart failure: results of the Cooperative North Scandinavian Enalapril Study Group. N Engl J Med. 1987;316:1429-1435.[Abstract]
   
2. The SOLVD Investigators. Effect of enalapril on mortality and the development of heart failure in asymptomatic patients with reduced left ventricular ejection fractions. N Engl J Med. 1992;327:685-691.[Abstract]
   
3. Swedberg K, Held P, Kjekshus J, Rasmussen K, Ryden L, Wedel H, on behalf of the CONSENSUS II Study Group. Effects of the early administration of enalapril on mortality in patients with acute myocardial infarction: results of the Cooperative New Scandinavian Enalapril Survival Study II (CONSENSUS II). N Engl J Med. 1992;327:678-684.[Abstract]
   
4. Pfeffer MA, Braunwald E, Moye LA, Basta L, Brown EJ Jr, Cuddy TE, Davis BR, Geltman EM, Goldman S, Flaker CC, Klein M, Wertheimer JH, Hawkins CM, on behalf of the SAVE Investigators. Effect of captopril on mortality and morbidity in patients with left ventricular dysfunction after myocardial infarction: results of the Survival and Left Ventricular Enlargement Trial. N Engl J Med. 1992;327:669-677.[Abstract]
   
5. Pfeffer JM, Pfeffer MA, Braunwald E. Influence of chronic captopril therapy on the infarcted left ventricle of the rat. Circ Res. 1985;57:84-95.[Abstract/Free Full Text]
   
6. Nakashima Y, Fouad FM, Tarazi RC. Regression of left ventricular hypertrophy from systemic hypertension by enalapril. Am J Cardiol. 1984;53:1044-1049.[Medline] [Order article via Infotrieve]
   
7. Dahlof B, Pennert K, Hansson L. Regression of left ventricular hypertrophy: a meta-analysis. Clin Exp Hypertens. 1992;A14:173-180.
   
8. Kromer EP, Elsner D, Riegger GAJ. Role of neurohormonal systems for pressure induced left ventricular hypertrophy in experimental supravalvular aortic stenosis in rats. Am J Hypertens. 1991;4:521-524.[Medline] [Order article via Infotrieve]
   
9. Linz W, Schoelkens BA, Ganten D. Converting enzyme inhibitor specifically prevents development and induces the regression of cardiac hypertrophy in rats. Clin Exp Hypertens. 1989;11:1325-1350.
   
10. Weinberg EO, Schoen FJ, George D, Kagaya Y, Benedict CR, Lorell BH. Angiotensin-converting enzyme inhibition prolongs survival and modifies the transition to heart failure in rats with pressure overload hypertrophy due to ascending aortic stenosis. Circulation. 1994;90:1410-1422.[Abstract/Free Full Text]
    
11. Litwin SE, Katz SE, Weinberg EO, Lorell BH, Aurigemma GP, Douglas PS. Serial echocardiographic-Doppler assessment of left ventricular geometry and function in rats with pressure-overload hypertrophy: chronic angiotensin-converting enzyme inhibition attenuates the transition to heart failure. Circulation. 1995;91:2642-2654.[Abstract/Free Full Text]
    
12. Feldman A, Weinberg EO, Ray P, Lorell BH. Selective changes in gene expression during compensated hypertrophy and the transition to cardiac decompensation in rats with chronic aortic banding. Circ Res. 1993;73:184-192.[Abstract]
    
13. Schunkert H, Dzau VJ, Tang SS, Hirsch AT, Apstein CS, Lorell BH. Increased rat cardiac angiotensin converting enzyme activity and mRNA expression in pressure overload left ventricular hypertrophy: effects on coronary resistance, contractility, and relaxation. J Clin Invest. 1990;86:1913-1920.
    
14. Schunkert H, Jackson B, Tang SS, Schoen FJ, Smits JFM, Apstein CS, Lorell BH. Distribution and functional significance of cardiac ACE in hypertrophied rat hearts. Circulation. 1993;87:1328-1339.[Abstract/Free Full Text]
    
15. Capogrossi MC, Kort AA, Spurgeon MA, Lakatta EG. Single adult rabbit and rat cardiac myocytes retain the Ca²⁺- and species-dependent systolic and diastolic contractile properties of the intact muscle. J Gen Physiol. 1986;88:589-613.[Abstract/Free Full Text]
    
16. Ikenouchi H, Barry WH, Bridge JHB, Weinberg EO, Apstein CS, Lorell BH. Effects of angiotensin II on contractility, intracellular Ca²⁺, I_Ca and pH: studies in isolated beating hearts and myocytes loaded with the indicator indo 1. J Physiol (Lond). 1994;480:203-215.[Medline] [Order article via Infotrieve]
    
17. Kagaya Y, Weinberg EO, Ito N, Mochizuki T, Barry WH, Lorell BH. Glycolytic inhibition: effects on diastolic relaxation and intracellular calcium handling in hypertrophied rat ventricular myocytes. J Clin Invest. 1995;95:2766-2776.
    
18. Grynkiewicz G, Poenie M, Tsien RY. A new generation of Ca²⁺ indicators with greatly improved fluorescence properties. J Biol Chem. 1985;260:3440-3450.[Abstract/Free Full Text]
    
19. duBell WH, Philips C, Houser SR. A technique for measuring cytosolic free Ca²⁺ with indo-1 in feline myocytes. In: Clark W, ed. Biology of Isolated Adult Cardiac Myocytes. New York, NY: Elsevier Science Publishing Co; 1988:187-201.
    
20. Ikenouchi H, Kohmoto O, McMillan M, Barry WH. The contributions of [Ca²⁺]_i, [Pi]_i, and pH_i to altered diastolic myocyte tone during partial metabolic inhibition. J Clin Invest. 1991;88:55-61.
    
21. Steadman BW, Moore KB, Spitzer KW, Bridge JHB. A video system for measuring motion in contracting heart cells. IEEE Trans Biomed Eng. 1988;35:264-272.[Medline] [Order article via Infotrieve]
    
22. Cheung JY, Tillotson DL, Yelamarty RV, Scaduto RC Jr. Cytosolic free calcium concentration in individual cardiac myocytes in primary culture. Am J Physiol. 1989;256:C1120-C1130.[Abstract/Free Full Text]
    
23. Borzak S, Kelly RA, Kramer BK, Matoba Y, Marsh JD, Reers M. In situ calibration of fura-2 and BCECF fluorescence in adult rat ventricular myocytes. Am J Physiol. 1990;259:H973-H981.[Abstract/Free Full Text]
    
24. Arkhammer P, Nilsson T, Berggren PO. Glucose-stimulated efflux of indo-1 from pancreatic beta-cells is reduced by probenecid. FEBS Lett. 1990;273:182-184.[Medline] [Order article via Infotrieve]
    
25. Tsutsui H, Tagawa H, Kent RL, McCollam PL, Ishihara K, Nagatsu M, Cooper G IV. Role of microtubules in contractile dysfunction of hypertrophied cardiocytes. Circulation. 1994;90:533-555.[Abstract/Free Full Text]
    
26. Tsutsui H, Ishihara K, Cooper G. Cytoskeletal role in the contractile dysfunction of hypertrophied myocardium. Science. 1993;260:682-687.[Abstract/Free Full Text]
    
27. Gwathmey JK, Hajjar RJ. Intracellular calcium related to force development in twitch contraction of mammalian myocardium. Cell Calcium. 1990;11:531-538.[Medline] [Order article via Infotrieve]
    
28. Gwathmey JK, Hajjar RJ. Calcium-activated force in a turkey model of spontaneous dilated cardiomyopathy: adaptive changes in thin myofilament Ca²⁺ regulation with resultant implications on contractile performance. J Mol Cell Cardiol. 1992;24:1459-1470.[Medline] [Order article via Infotrieve]
    
29. Gwathmey JK, Hajjar RJ. Relation between steady-state force and intracellular [Ca²⁺]_i in intact human myocardium: index of myofibrillar response to Ca²⁺. Circulation. 1990;82:1266-1278.[Abstract/Free Full Text]
    
30. Hajjar R, Gwathmey JK. Cross-bridge dynamics in human ventricular myocardium. Circulation. 1992;86:1819-1826.[Abstract/Free Full Text]
    
31. Fabiato A, Fabiato F. Calculator programs for computing the compositions of solutions containing multiple metals and ligands used for experiments in skinned muscle cells. J Physiol Paris. 1979;75:463-505.[Medline] [Order article via Infotrieve]
    
32. Gwathmey JK, Copelas L, MacKinnon R, Schoen F, Feldman M, Grossman W, Morgan JP. Abnormal intracellular calcium handling in myocardium from patients with end-stage heart failure. Circulation. 1987;75:331-339.[Abstract/Free Full Text]
    
33. Boluyt MO, O'Neill L, Meredith AL, Bing OH, Brooks WN, Conrad CH, Crow MT, LaKatta EG. Alterations in cardiac gene expression during the transition from stable hypertrophy to heart failure: marked upregulation of genes encoding extracellular matrix proteins. Circ Res. 1994;75:23-32.[Abstract/Free Full Text]
    
34. Schwartz K, LeCarpentier Y, Martin JL, Lompre AM, Mercardier JJ, Swynghedauw B. Myosin isoenzymic distribution correlates with speed of myocardial contraction. J Mol Cell Cardiol. 1981;13:1071-1075.[Medline] [Order article via Infotrieve]
    
35. De la Bastie D, Levitsky DD, Rappaport L, Mercadier JJ, Marotte F, Wisnewsky C, Brovkovich V, Schwartz K, Lompre A-M. Function of the sarcoplasmic reticulum and expression of its Ca²⁺-ATPase gene in pressure overload induced cardiac hypertrophy in the rat. Circ Res. 1990;66:554-564.[Abstract/Free Full Text]
    
36. Arai M, Alpert NR, MacLennan DH, Barton P, Periasamy M. Alterations in sarcoplasmic reticulum gene expression in human heart failure. Circ Res. 1993;72:463-469.[Abstract/Free Full Text]
    
37. Aurigemma GP, Silver KH, Priest MA, Gaasch WH. Geometric changes allow normal ejection fraction despite depressed myocardial shortening in hypertensive left ventricular hypertrophy. J Am Coll Cardiol. 1995;26:195-202.[Abstract]
    
38. Keung EC. Calcium current is increased in isolated adult myocytes from hypertrophied rat myocardium. Circ Res. 1989;64:753-763.[Abstract/Free Full Text]
    
39. Litwin SE, Morgan JP. Captopril enhances intracellular calcium handling and ß-adrenergic responsiveness of myocardium from rats with post-infarction failure. Circ Res. 1992;71:797-807.[Abstract/Free Full Text]
    
40. Siri FM, Krueger J, Nordin C, Ming Z, Aronson RS. Depressed intracellular calcium transient and contraction in myocytes from hypertrophied and failing guinea pig hearts. Am J Physiol. 1991;261:H514-H530.[Abstract/Free Full Text]
    
41. Beuckelmann DJ, Nabauer M, Erdmann E. Intracellular calcium handling in isolated ventricular myocytes from patients with terminal heart failure. Circulation. 1992;85:1046-1055.[Abstract/Free Full Text]
    
42. Gao WD, Backy PH, Azan-Backy M, Marban E. Myofilament Ca²⁺ sensitivity in intact versus skinned rat ventricular muscle. Circ Res. 1994;74:408-415.[Abstract/Free Full Text]
    
43. Gwathmey JK, Hajjar RJ. Relationship between steady-state force and intracellular calcium in intact human myocardium: index of myofibrillar responsiveness to Ca²⁺. Circulation. 1990;82:1266-1278.
    
44. Kentish JC, ter Keurs HE, Ricciardi L, Bucx JJ, Noble MI. Comparison between the sarcomere length-force relations of intact and skinned trabeculae from rat right ventricle: influence of calcium concentrations on these relations. Circ Res. 1986;58:755-768.[Abstract/Free Full Text]
    
45. Gwathmey JK, Davidoff AJ. Experimental aspects of cardiomyopathy. Curr Opin Cardiol. 1993;8:480-495.
    
46. Anderson PAW, Malouf NN, Oakley A, Pagani ED, Allen PD. Troponin T isoform expression in humans: a comparison among normal and failing adult heart and adult and fetal skeletal muscle. Circ Res. 1991;69:1226-1233.[Abstract/Free Full Text]
    
47. Margossian SS, White HD, Caulfield JB, Norton P, Taylor S, Slayter HS. Light chain-2 profile and activity of human ventricular myosin during dilated cardiomyopathy. Circulation. 1992;85:1720-1733.[Abstract/Free Full Text]
    
48. McDonald KS, Moss RL. Osmotic compression of single cardiac myocyte eliminates the reduction in Ca²⁺ sensitivity of tension at short sarcomere length. Circ Res. 1995;77:199-205.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				V. Ciobotaru, M. Heimburger, L. Louedec, C. Heymes, R. Ventura-Clapier, P. Bedossa, B. Escoubet, J.-B. Michel, J.-J. Mercadier, and D. Logeart Effect of Long-Term Heart Rate Reduction by If Current Inhibition on Pressure Overload-Induced Heart Failure in Rats J. Pharmacol. Exp. Ther., January 1, 2008; 324(1): 43 - 49. [Abstract] [Full Text] [PDF]

				R. J. Belin, M. P. Sumandea, E. J. Allen, K. Schoenfelt, H. Wang, R. J. Solaro, and P. P. de Tombe Augmented Protein Kinase C-{alpha}-Induced Myofilament Protein Phosphorylation Contributes to Myofilament Dysfunction in Experimental Congestive Heart Failure Circ. Res., July 20, 2007; 101(2): 195 - 204. [Abstract] [Full Text] [PDF]

				R. J. Belin, M. P. Sumandea, T. Kobayashi, L. A. Walker, V. L. Rundell, D. Urboniene, M. Yuzhakova, S. H. Ruch, D. L. Geenen, R. J. Solaro, et al. Left ventricular myofilament dysfunction in rat experimental hypertrophy and congestive heart failure Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2344 - H2353. [Abstract] [Full Text] [PDF]

				F. Del Monte, K. Butler, W. Boecker, J. K. Gwathmey, and R. J. Hajjar Novel technique of aortic banding followed by gene transfer during hypertrophy and heart failure Physiol Genomics, April 10, 2002; 9(1): 49 - 56. [Abstract] [Full Text] [PDF]

				Z. Su, A. Yao, I. Zubair, K. Sugishita, M. Ritter, F. Li, J. J. Hunter, K. R. Chien, and W. H. Barry Effects of deletion of muscle LIM protein on myocyte function Am J Physiol Heart Circ Physiol, June 1, 2001; 280(6): H2665 - H2673. [Abstract] [Full Text] [PDF]

				S. Y. Boateng, R. U. Naqvi, M. U. Koban, M. H. Yacoub, K. T. MacLeod, and K. R. Boheler Low-dose ramipril treatment improves relaxation and calcium cycling after established cardiac hypertrophy Am J Physiol Heart Circ Physiol, March 1, 2001; 280(3): H1029 - H1038. [Abstract] [Full Text] [PDF]

				W. W. Brooks, O. H. L. Bing, M. O. Boluyt, A. Malhotra, J. P. Morgan, N. Satoh, W. S. Colucci, and C. H. Conrad Altered Inotropic Responsiveness and Gene Expression of Hypertrophied Myocardium With Captopril Hypertension, June 1, 2000; 35(6): 1203 - 1209. [Abstract] [Full Text] [PDF]

				J. Bartunek, E. O. Weinberg, M. Tajima, S. Rohrbach, S. E. Katz, P. S. Douglas, and B. H. Lorell Chronic NG-Nitro-L-Arginine Methyl Ester-Induced Hypertension : Novel Molecular Adaptation to Systolic Load in Absence of Hypertrophy Circulation, February 1, 2000; 101(4): 423 - 429. [Abstract] [Full Text] [PDF]

				M. I. Miyamoto, F. del Monte, U. Schmidt, T. S. DiSalvo, Z. B. Kang, T. Matsui, J. L. Guerrero, J. K. Gwathmey, A. Rosenzweig, and R. J. Hajjar Adenoviral gene transfer of SERCA2a improves left-ventricular function in aortic-banded rats in transition to heart failure PNAS, January 18, 2000; 97(2): 793 - 798. [Abstract] [Full Text] [PDF]

				K. Ito, Y. Kagaya, T. Ishizuka, N. Ito, N. Ishide, and K. Shirato Diacylglycerol delays pHi overshoot after reperfusion and attenuates contracture in isolated, paced myocytes Am J Physiol Heart Circ Physiol, November 1, 1999; 277(5): H1708 - H1717. [Abstract] [Full Text] [PDF]