HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 288/2/H695    most recent 00783.2004v1 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 ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Haddad, G. E. Articles by Blackwell, K. N. Search for Related Content PubMed PubMed Citation Articles by Haddad, G. E. Articles by Blackwell, K. N.

Regulation of atrial contraction by PKA and PKC during development and regression of eccentric cardiac hypertrophy

Department of Physiology and Biophysics, College of Medicine, Howard University, Washington, DC

Submitted 3 August 2004 ; accepted in final form 8 October 2004

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
ANG II plays a major role in development of cardiac hypertrophy through its AT₁ receptor subtype, whereas angiotensin-converting enzyme (ACE) inhibitors are effective in reversing effects of ANG II on the heart. The objective of this study was to investigate the role of PKA and PKC in the contractile response of atrial tissue during development and ACE inhibitor-induced regression of eccentric hypertrophy induced by aortocaval shunt. At 1 wk after surgery, sham and shunt rats were divided into captopril-treated and untreated groups for 2 wk. Then isometric contraction was assessed by electrical stimulation of isolated rat left atrial preparations superfused with Tyrode solution in the presence or absence of specific inhibitors KT-5720 (for PKA) and Ro-32-0432 (for PKC) and high Ca²⁺. Peak tension developed was greater in shunt than in sham hearts. However, when expressed relative to tissue mass, hypertrophied muscle showed weaker contraction than muscle from sham rats. In sham rats, peak tension developed was more affected by PKC than by PKA inhibition, whereas this differential effect was reduced in the hypertrophied heart. Treatment of shunt rats with captopril regressed left atrial hypertrophy by 67% and restored PKC-PKA differential responsiveness toward sham levels. In the hypertrophied left atria, there was an increase in the velocity of contraction and relaxation that was not evident when expressed in specific relative terms. Treatment with ACE inhibitor increased the specific velocity of contraction, as well as its PKC sensitivity, in shunt rats. We conclude that ACE inhibition during eccentric cardiac hypertrophy produces a negative trophic and a positive inotropic effect, mainly through a PKC-dependent mechanism.

angiotensin-converting enzyme; atrium; muscle; contraction

On the other hand, AT₁ receptor subtype is positively coupled to phospholipase C and negatively coupled to adenylate cyclase. Early during development of eccentric cardiac hypertrophy, PKA activity was elevated and correlated with the force-generating capacity of the heart (24), which is greatly influenced by intracellular Ca²⁺ homeostasis. Recently, it was reported that ANG II augmented cAMP formation by Ca²⁺-dependent activation of adenylate cyclase (28). On the other hand, binding of ANG II to AT₁ receptors has been shown to activate the Ca²⁺-dependent PKC (for review see Ref. 39). It has been demonstrated that ANG II induces an increase in inositol phosphate production that was mediated through tyrosine phosphorylation (13). Accordingly, the expression of PKC- mRNA was elevated with cardiac hypertrophy and suppressed after regression of cardiac hypertrophy with ACE inhibitor treatment (22). The PKA and PKC signaling pathways are Ca²⁺ dependent and have been shown to regulate Ca²⁺ homeostasis in the heart (16, 31, 35, 44), which would affect myocardial contractility.

Knowing that during eccentric cardiac hypertrophy the atria play an important role in ventricular filling and the renin-angiotensin system affects myocardial contractility through ANG II-dependent signaling, we hypothesize that PKA and PKC play a crucial role in defining the force of contraction of the atrium. Thus the present study was designed specifically to investigate the role of PKA and PKC in the contractile response of atrial tissue during the development and the ACE inhibitor-induced regression of eccentric hypertrophy.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Experimental Animals and Groups

All protocols and animal care were approved and performed according to the Institutional Animal Care and Use Committee at Howard University. Adult male Sprague-Dawley Rats (300 ± 85 g body wt; Charles Rivers Breeding Laboratories, Wilmington, MA) were housed in a temperature-controlled room with a 12:12-h light-dark cycle and fed a standard chow diet throughout their stay in the Animal Section of the College of Medicine.

Animals were separated into two groups: sham-operated rats [control (sham) group] and aortocaval shunt-induced eccentric hypertrophy rats [experimental (shunt) group]. At 1 wk after the surgery, each group was divided into captopril-treated and untreated groups to allow the development of eccentric cardiac hypertrophy. Captopril was administered to the treated groups in the drinking water at 500 mg·l^–1·day^–1 for 2 wk to assess regression of cardiac hypertrophy by ACE inhibitors. The sham and shunt rats drank similar amounts of water during the 2-wk treatment period: 38.73 ± 5.96 and 39.61 ± 5.31 ml/day, respectively. With 500 mg/ml captopril, this breaks down to captopril intake of 19.80 and 19.36 mg/day, respectively.

Surgical Procedure

The aortocaval shunt was produced by the method described by Garcia and Diebold (12). Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and a midline incision was made to expose the abdominal aorta and vena cava. A bulldog vascular clamp was placed across the aorta, proximal to the area of the puncture. The aorta was punctured with a disposable 18-gauge needle, and the common wall between the aorta and inferior vena cava was grasped through the incision, creating a fistula between the two vessels below the renal arteries. The needle was withdrawn and sealed with a drop of cyanoacrylate glue. The abdominal incision was sutured with Biosyn monofilament synthetic absorbable sutures. The shunts were produced under sterile surgical conditions. Sham-operated rats, serving as the control, underwent the same surgical procedure, except the aorta and inferior vena cava were not punctured. Immediately after the procedure, the animals were given 1 ml of sterile saline to prevent dehydration from any blood loss and allowed to recover from surgery in warmed conditions.

Hemodynamic Measurements

Blood pressure measurements were recorded to rule out the development of hypertension and possible development of high-pressure concentric hypertrophy as well as the effects of captopril in the treated groups. Systolic blood pressure was measured by the tail-cuff method without anesthesia. Animals were placed in a specialized rat restrainer, and blood pressure was measured using a rat tail blood pressure system (model RTBP1001, Kent Scientific, Litchfield, CT) and recorded by a digital oscilloscope (model 310, Nicolet) for subsequent analysis. Blood pressures and body weights were recorded in sham and shunt rats 1, 5, 9, 13, 17, and 21 days after the surgery.

At the end of all experiments, wet weights of the heart chambers and total heart weight were recorded to assess the level of cardiac hypertrophy by measuring the heart weight-to-body weight ratio for the sham and shunt groups.

Multicellular Preparation Setup and Experimental Procedure

Tissue isolation and recording. Animals were killed by decapitation under pentobarbital sodium sedation (50 mg/kg ip). The left atrial muscle was isolated and mounted vertically in a Radnoti tissue organ bath system, with one end attached to the organ bearer with field stimulation electrodes and the other end attached to an isometric force transducer (model FTO3, Grass, Quincy, MA). The transducer was mounted on a micromanipulator, so that fine steps to adjust the resting tension could elongate the muscle. All experiments were carried out at 1 g of resting tension. The force transducer was connected to a amplifier (model 7P1G, Grass) and a data-recording system (model 79E, Grass) to record the tension developed. The signal was fed simultaneously into a differentiator amplifier (model 7P20C, Grass) to record the first derivatives of the active tension developed. The two signals were fed into a digital oscilloscope (model 310, Nicolet) and stored on disk for analysis.

Preparations were stimulated via electrical field stimulation at a basic frequency of 0.75 Hz at 1.5 times threshold voltage and stimulus duration of 3.5 ms. At this frequency, preparations can be studied for long periods (3–4 h) without noticeable deterioration.

Multicellular contraction studies. The isolated preparation was superfused in the tissue bath with Tyrode solution (in mM: 137 NaCl, 5.4 KCl, 2.7 CaCl₂, 0.5 MgCl₂, 11.9 NaHCO₃, 0.45 NaH₂PO₄, and 5.55 glucose) at 5 ml/min. The superfusate was continuously aerated with 95% O₂-5% CO₂, and pH was adjusted to 7.4 ± 0.03 with NaOH or HCl. Temperature was kept constant at 31 ± 0.5°C in the tissue bath by means of a thermostatically controlled water jacket surrounding the tissue bath.

At the start of the experimental procedure, the muscular preparations were left to equilibrate to a steady state for 60 min or until the resting tension remained constant. At the end of the equilibration period, control contractions were recorded. The superfusate was then changed sequentially to Tyrode solution supplemented with PKA inhibitor (KT-5720, 10^–7 M), high Ca²⁺ (5.14 mM), normal Tyrode solution, PKC inhibitor (Ro-32-0432, 10^–7 M), high Ca²⁺ (5.14 mM), and then normal Tyrode solution. The peak tension developed (PTD) and first derivatives of contraction and relaxation were recorded after 5, 10, and 15 min in each solution.

KT-5720 (Calbiochem, San Francisco, CA) is a potent specific cell-permeable inhibitor of PKA. It does not significantly affect the activity of other protein kinases, especially PKC, PKG, and myosin light chain kinase. Therefore, KT-5720 was chosen to study the effect of PKA inhibition in cardiac tissue. Ro-32-0432 (Calbiochem) is a selective cell-permeable PKC inhibitor. It is even highly selective for the Ca²⁺-dependent PKC isoforms (e.g., PKC- and PKC-) over the Ca²⁺-independent PKC isoforms (e.g., PKC- and PKC-). The PKC isoforms inhibited by Ro-32-0432 have been implicated in various models of cardiac hypertrophy, with the Ca²⁺-dependent isoforms showing a greater association with contractile function of the heart. Thus this PKC inhibitor seems to be the best choice for determining PKC alterations of inotropic activity during cardiac hypertrophy.

Statistical Methods

Values are means ± SE. All statistical analysis was performed using SigmaStat software and verified using Microsoft Excel and Prism software, which gave the same result. Paired Student’s t-test was used to compare data before and after drug treatment of the same animal group. Because the inhibitors’ effects were not tested in a cumulative manner but, rather, an equilibrium control period (Tyrode) preceded the addition of each inhibitor, one-way ANOVA was used for significance when the effects of one drug were compared in two different animal groups among the four groups of animals: sham (control), shunt (hypertrophy), sham + captopril (experimental control), and shunt + captopril (regression). Furthermore, there were no statistical differences between the equilibrium periods tested in one protocol. Statistical significance was assumed for P 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Physiological Parameters Before and After ACE Inhibitor Treatment

Figure 1 shows the blood pressure profile of sham and shunt groups during the 3 wk after surgery in the presence and absence of the ACE inhibitor captopril. At 3 wk after surgery, there was no significant difference in the blood pressure profile between the untreated and treated sham (106.0 ± 2.5 and 101.8 ± 3.1 mmHg, respectively) and shunt (109.2 ± 1.1 and 110.0 ± 4.3 mmHg, respectively) groups. Table 1 summarizes the level of eccentric cardiac hypertrophy and its regression by captopril within 3 wk after surgery by measures of tissue weight relative to body weight. The body weight did not differ in all groups, whereas the absolute wet weights of the whole heart (1.08 ± 0.04 vs. 1.43 ± 0.04 g), left atria (19 ± 1 vs. 39 ± 3 mg), right atria (52 ± 2 vs. 98 ± 4 mg), left ventricles (684 ± 22 vs. 919 ± 25 mg), and right ventricles (146 ± 14 vs. 291 ± 26 mg) were significantly lower in sham than in shunt rats. Thus, as shown in Table 1, this represents an increase of 34, 104, 90, 36, and 59% in the respective relative weights of the whole heart, left atria, right atria, left ventricles, and right ventricles.

View larger version (28K): [in this window] [in a new window]   Fig. 1. Blood pressure profile of sham and volume overload-induced cardiac hypertrophied rats with and without captopril treatment: blood pressure changes during the first 3 wk after surgery in captopril-treated and untreated sham and shunt-operated rats. Values are means ± SE; n = 10.

Left Atrial Contraction During Development of Eccentric Hypertrophy

Modulation of left atrial PTD of sham and shunt rats by PKA, PKC, and high Ca²⁺ is presented in Fig. 2. In the left atrium of sham-operated rats, peak tension induced by electrical stimulation (Fig. 2A) was reduced from 267.93 ± 16.87 to 243.83 ± 17.01 mg (P < 0.01) by the PKA inhibitor KT-5720 (10^–7 M). Increasing the extracellular Ca²⁺ concentration to 5.14 mM significantly augmented PTD to 408.48 ± 49.16 mg (P < 0.01). On the other hand, addition of the PKC inhibitor Ro-32-0432 (10^–7 M) reduced the peak tension to 198.63 ± 18.45 mg (P < 0.01), which was elevated to 367.35 ± 53.46 mg (P < 0.01) by high extracellular Ca²⁺ solution.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Alterations of left atrial tension by PKA, PKC, and high Ca2+. A and B: effects of KT-5720 (KT, 10–7 M), high Ca2+ (5.14 mM), and Ro-32-0432 (Ro, 10–7 M) on peak tension developed (PTD) and specific PTD by left atrial (LA) muscle from untreated sham and shunt-operated rats. Effect of each protein kinase inhibitor was assessed after an equilibration period with Tyrode solution. Subsequent to each protein kinase inhibition, muscle perfusion was switched to high Ca2+ solution. C and D: percent changes in peak tension of left atria from untreated sham and shunt-operated rats due to PKA and PKC inhibition and extracellular Ca2+ elevation. Values are means ± SE; n = 8. *P < 0.05 compared with preceding Tyrode in sham. +P < 0.05 compared with preceding Tyrode in shunt. #P < 0.05 vs. sham; xP < 0.05 Ro vs. KT.

Relative left atrial weight is a measure of the level of left atrial hypertrophy. During cardiac hypertrophy, muscular dysfunction at the unit level may be mitigated by increased muscle mass, whereby normalization would definitely unveil its proper functional state. Thus, expressing the PTD in relation to the relative left atrial weight is a measurement of the specific PTD value, which is independent of the degree of tissue mass. In that sense, specific PTD is an interesting approach when inotropy of shunt and sham rats is compared. Accordingly, in control conditions (Fig. 2B), the PTD was higher (P < 0.05), whereas the specific PTD was lower (38.96 ± 2.75 vs. 49.56 ± 3.84 mg/relative left atrial wt, P < 0.05) in shunt than in sham rats. High extracellular Ca²⁺ similarly increased PTD in sham and shunt rats (51.08 ± 0.13 and 49.24 ± 0.08%, respectively), with a tendency for higher specific PTD in sham than in shunt atrial muscle that did not reach statistical significance (58.02 ± 4.84 vs. 77.05 ± 12.12 mg/relative left atrial wt, respectively).

Figure 2C shows the percent change in PTD of the left atrium due to PKA inhibitor (10^–7 M) and PKC inhibitor (10^–7 M) in sham and shunt rats. In the control sham group, PKC inhibition was 2.3-fold more effective than PKA inhibition in reducing PTD (7.94 ± 0.02 vs. 17.76 ± 0.04%, P < 0.01). However, in shunt rats, reductions of PTD induced by PKA and PKC inhibition were not significantly different. On the other hand, the percent change in PTD induced by elevation of extracellular Ca²⁺ (Fig. 2D) was not significantly different between sham and shunt rats.

Left Atrial Velocity of Contraction During Development of Eccentric Hypertrophy

Modulation of left atrial velocity of contraction (+dT/dt) of sham and shunt rats by PKA, PKC, and high Ca²⁺ is presented in Fig. 3. In sham rats, +dT/dt of the left atrium was not significantly affected by PKA inhibition (Fig. 3A). Elevation of extracellular Ca²⁺ resulted in an increase in +dT/dt to 597.06 ± 78.70 mg/s (P < 0.01). On the other hand, PKC inhibition reduced +dT/dt (429.05 ± 29.53 and 396.82 ± 41.13 mg/s with Tyrode solution and Ro-32-0432, respectively). Subsequently, switching to high Ca²⁺ increased +dT/dt to 582.55 ± 86.52 mg/s (P < 0.05). On the other hand, +dT/dt in the shunt group was reduced by PKC inhibition (715.45 ± 71.92 vs. 622.80 ± 83.49 mg/s, P < 0.01), with a slight effect of KT-5720. Specific +dT/dt (Fig. 3B) did not differ between sham and shunt groups (80.34 ± 9.70 and 74.06 ± 6.69 mg·s^–1·relative LA wt^–1, respectively), although the absolute +dT/dt was higher in the shunt than in the sham group (425.35 ± 23.62 vs. 682.71 ± 76.76 mg/s, P < 0.01; Fig. 3A). High extracellular Ca²⁺ was more effective in increasing +dT/dt in the shunt than in the sham rats (963.97 ± 70.42 vs. 597.06 ± 78.70 mg·s^–1·relative LA wt^–1, P < 0.01); however, specific +dT/dt was similar for both groups (114.34 ± 22.78 and 105.83 ± 8.45 mg·s^–1·relative LA wt^–1). This may indicate that factors that affect +dT/dt at the myofibrillar level may not be challenged in the shunt animals, although the eccentric hypertrophy of the heart is producing a higher +dT/dt at the tissue level.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Changes in left atrial velocity of contraction (+dT/dt) by PKA, PKC, and high Ca2+. A and B: effects of KT-5720 (10–7 M), high Ca2+ (5.14 mM), and Ro-32-0432 (10–7 M) on +dT/dt and specific +dT/dt of left atrial muscle from untreated sham and shunt-operated rats. Effect of each protein kinase inhibitor was assessed after equilibration with Tyrode solution. Subsequent to each protein kinase inhibition, muscle perfusion was switched to high-Ca2+ solution. C and D: percent changes in +dT/dt of left atria from untreated sham and shunt-operated rats after PKA and PKC inhibition and extracellular Ca2+ elevation. Values are means ± SE; n = 8. *P < 0.05 compared with preceding Tyrode in sham. +P < 0.05 compared with preceding Tyrode in shunt. #P < 0.05 vs. sham; xP < 0.05 Ro vs. KT.

Left Atrial –dT/dt During Development of Eccentric Hypertrophy

Modulation of left atrial velocity of relaxation (–dT/dt) of sham and shunt rats by PKA, PKC, and high Ca²⁺ is presented in Fig. 4. PKA inhibition reduced –dT/dt of the left atrium (Fig. 4A) only in the shunt group (from 581.40 ± 74.81 to 567.70 ± 75.60 mg/s, P < 0.05). High extracellular Ca²⁺ increased –dT/dt by 42.77 ± 0.15% (P < 0.01) in sham rats and by 50.06 ± 0.08% (P < 0.01) in shunt rats. On the other hand, PKC inhibition reduced –dT/dt to 346.31 ± 45.05 mg/s (P < 0.01) in sham rats and to 534.76 ± 78.62 mg/s (P < 0.01) in shunt rats. High-Ca²⁺ Tyrode solution increased –dT/dt to 517.88 ± 43.55 mg/s (P < 0.01) in sham rats and to 796.06 ± 78.94 mg/s (P < 0.01) in shunt rats.

View larger version (22K): [in this window] [in a new window]   Fig. 4. Changes in left atrial velocity of relaxation (–dT/dt) by PKA, PKC, and high Ca2+. A and B: effects of KT-5720 (10–7 M), high Ca2+ (5.14 mM), and Ro-32-0432 (10–7 M) on –dT/dt and specific –dT/dt of left atrial muscle from untreated sham and shunt-operated rats. Effect of each protein kinase inhibitor was assessed after equilibration with Tyrode solution. Subsequent to each protein kinase inhibition, muscle perfusion was switched to high-Ca2+ solution. C and D: percent changes in –dT/dt of left atria from untreated sham and shunt-operated rats after PKA and PKC inhibition and extracellular Ca2+ elevation. Values are means ± SE; n = 8. *P < 0.05 compared with preceding Tyrode in sham. +P < 0.05 compared with preceding Tyrode in shunt. #P < 0.05 vs. sham; xP < 0.05 Ro vs. KT.

Figure 4C represents the percent change in –dT/dt of the left atrium due to PKA inhibitor (10^–7 M) and PKC inhibitor (10^–7 M) in sham and shunt rats. PKA inhibition did not produce a significant reduction in either group. However, PKC inhibition significantly reduced –dT/dt in the shunt group (P < 0.05). In parallel, high extracellular Ca²⁺ increased –dT/dt more in the shunt than in the sham group (543.51 ± 42.60 vs. 854.57 ± 82.88 mg/s, P < 0.05), but specific –dT/dt or percent change in –dT/dt was increased to similar levels in both groups (Fig. 4D).

Changes in Left Atrial Contraction After Regression of Eccentric Hypertrophy by ACE Inhibitor

Inhibition of ACE reduces circulating as well as local tissue ANG II concentrations, which attenuates the activation of ANG II-dependent signaling pathways, such as those related to PKA and PKC. Thus modulation of the inotropic activity by these second messengers would depend on their relative contribution to muscular contractility. Figure 5 shows that treatment of shunt rats with ACE inhibitor improved their specific PTD to a level comparable to that of the untreated sham control rats (61.31 ± 9.91 and 49.56 ± 3.84 mg/relative left atrial wt, respectively), which is greater than that of the shunt rats (38.42 ± 5.82, P < 0.05). In addition and similar to the untreated sham rats, captopril-treated shunt rats showed an enhanced response to elevated extracellular Ca²⁺ with respect to the untreated shunts (P < 0.05). Interestingly, this improved contractility in the captopril-treated hypertrophied hearts was associated with a restoration of the differential influence of PKA and PKC on PTD (2.78 ± 0.04 and 22.42 ± 0.07% for PKA and PKC, respectively, P < 0.01), as in control untreated sham rats. On the other hand, there was no significant difference in the effects of PKA and PKC inhibition or high extracellular Ca²⁺ on PTD between treated and untreated sham rat hearts.

View larger version (24K): [in this window] [in a new window]   Fig. 5. Alterations of left atrial tension by PKA, PKC, and high Ca2+ after treatment with angiotensin-converting enzyme (ACE) inhibitor. A and B: effects of KT-5720 (10–7 M), high Ca2+ (5.14 mM), and Ro-32-0432 (10–7 M) on PTD and specific PTD by left atrial muscle from captopril-treated sham and shunt-operated rats. Effect of each protein kinase inhibitor was assessed after equilibration with Tyrode solution. Subsequent to each protein kinase inhibition, muscle perfusion was switched to high-Ca2+ solution. C and D: percent changes in peak tension of left atria from captopril-treated sham and shunt-operated rats due to PKA and PKC inhibition and extracellular Ca2+ elevation. Values are means ± SE; n = 6. *P < 0.05 vs. preceding Tyrode in sham. +P < 0.05 vs. preceding Tyrode in shunt. #P < 0.05 vs. sham; xP < 0.05 Ro vs. KT.

With respect to the untreated shunt-operated rats, captopril treatment significantly improved the specific +dT/dt by 37.41 ± 2.58% (P < 0.01) and its response to elevated extracellular Ca²⁺ by 46.67% ± 2.88 (P < 0.01) in shunt rat hearts (Fig. 6). In addition, +dT/dt was reduced by PKC inhibition (20.87 ± 0.04%, P < 0.05) but not by PKA inhibition in the captopril-treated shunt rat hearts.

View larger version (22K): [in this window] [in a new window]   Fig. 6. Changes in left atrial +dT/dt by PKA, PKC, and high Ca2+ after treatment with ACE inhibitor. A and B: effects of KT-5720 (10–7 M), high Ca2+ (5.14 mM), and Ro-32-0432 (10–7 M) on +dT/dt or specific +dT/dt of left atrial muscle from captopril-treated sham and shunt-operated rats. Effect of each protein kinase inhibitor was assessed after equilibration with Tyrode solution. Subsequent to each protein kinase inhibition, muscle perfusion was switched to high-Ca2+ solution. C and D: percent changes in +dT/dt of left atria from captopril-treated sham and shunt-operated rats after PKA and PKC inhibition and extracellular Ca2+ elevation. Values are means ± SE; n = 6. *P < 0.05 vs. preceding Tyrode in sham. +P < 0.05 vs. preceding Tyrode in shunt. #P < 0.05 vs. sham; xP < 0.05 Ro vs. KT.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Changes in left atrial –dT/dt by PKA, PKC, and high Ca2+ after treatment with ACE inhibitor. A and B: effects of KT-5720 (10–7 M), high Ca2+ (5.14 mM), and Ro-32-0432 (10–7 M) on –dT/dt and specific –dT/dt of left atrial muscle from captopril-treated sham and shunt-operated rats. Effect of each protein kinase inhibitor was assessed after equilibration with Tyrode solution. Subsequent to each protein kinase inhibition, muscle perfusion was switched to high-Ca2+ solution. C and D: percent changes in –dT/dt of left atria from captopril-treated sham and shunt-operated rats after PKA and PKC inhibition and extracellular Ca2+ elevation. Values are means ± SE; n = 6. *P < 0.05 vs. preceding Tyrode in sham. +P < 0.05 vs. preceding Tyrode in shunt. #P < 0.05 vs. sham; xP < 0.05 Ro vs. KT.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
In the present study, we have found that 2 wk of treatment with captopril prevented the development of eccentric cardiac hypertrophy by 80%, whereas the left atrial and ventricular hypertrophy regressed by 80 and 50%, respectively. This represents a regression from a moderate to a low level of cardiac hypertrophy (27). Similar findings were reported previously showing regression of eccentric hypertrophy by ACE inhibitors (17, 34), specifically in the atria. The aortocaval shunt did not induce a change in the systolic blood pressure, which was not affected by captopril treatment. In the sham rats, left atrial PTD was significantly more affected by PKC than by PKA inhibition. At the same concentration, PKC inhibition produced a greater response than PKA inhibition in the sham rats; this difference in the atrial response to protein kinase inhibitors was significantly reduced in rats with aortocaval shunt. In addition, the force of contraction produced by the hypertrophied left atrium was greater than that of the sham-operated controls. In contrast, the specific tension developed per relative left atrial weight was significantly weaker in the hypertrophied than in the sham control, in agreement with reduced contraction amplitude at the level of the myofiber (26). In the atrial tissue, elevation of subsarcolemmal Ca²⁺ by influx of Ca²⁺ through the slow Ca²⁺ channels (I_Ca,L) establishes the cytosolic Ca²⁺ gradient necessary to initiate the regenerative and propagating Ca²⁺-induced Ca²⁺ release from nonjunctional sarcoplasmic reticulum to generate a contraction (26, 33). Effectively, at high Ca²⁺, the specific PTD by the hypertrophied left atrium tended to be weaker than that by the sham controls. Therefore, these data may indicate a functional inotropic defect at the basic contractile unit (myofiber) level in shunt rats that is compensated for by the hypertrophy of the heart tissue to maintain a normal cardiac function (compensated phase). Treatment with ACE inhibitor effectively reduced the aortocaval shunt-induced structural changes and restored the differential in protein kinase influence on contractility. The tension developed in the captopril-treated shunt-operated rat hearts reverted to a predominantly PKC-dependent process, as in the sham hearts. Thus the ACE inhibitor showed opposite, but disproportionate, effects on the PKA- and PKC-induced inotropic activity of the eccentric hypertrophied heart, with significant enhancement of the latter. Concurrently, captopril treatment restored the strength of the specific PTD in the shunt hearts to levels in the sham hearts, under normal as well as elevated extracellular Ca²⁺. This may suggest that treatment of eccentric cardiac hypertrophy with ACE inhibitor improves the inotropic activity of the left atrium mainly through a Ca²⁺-dependent PKC mechanism. The effect of ACE inhibitors on PKC is still controversial because of differences in species or model used and various experimental designs (1, 4, 36). Nevertheless, it has been shown that chronic captopril treatment increases PKC activity in the rat (1, 45), which may improve myocardial contractility and intracellular Ca²⁺ transients (2, 30). On the other hand, it has been shown that treatment with ACE inhibitor does not significantly affect PKA level or activity in the heart (7, 19, 29), which is consistent with our findings.

In the hypertrophied left atria, there is an increase in the +dT/dt and –dT/dt that was not evident when expressed in specific relative terms. Treatment with ACE inhibitor increased the specific +dT/dt in the shunt-operated rat heart. In addition, our data show that, in contrast to the sham heart, the left atrial +dT/dt in the hypertrophied heart is mainly PKC sensitive. Captopril treatment of shunt-operated rats increased the atrial +dT/dt along with its PKC sensitivity. Therefore, the increase in +dT/dt in the hypertrophied left atrial muscle seems to be mediated mainly through a PKC-dependent mechanism. This observation is consistent with earlier findings that PKC improves myofilament Ca²⁺ sensitivity (41), decreases the Ca²⁺ requirement of the contractile machinery (31), and increases activation of the slow Ca²⁺ channels, which are main determinants of +dT/dt (2, 35). Furthermore, it has been reported that ACE inhibitor treatment increases junctional conductance through activation of PKC (10), but not PKA (9), suggesting an improved contractility due to enhanced synchronization and excitation-contraction coupling of the myocardial tissue. On the other hand, –dT/dt shows a stronger dependency on PKC than on PKA in both groups, which was more significant in the hypertrophied heart. ACE inhibitor treatment improved –dT/dt and its PKC, but not PKA, sensitivity. This is in agreement with the recent findings (43) showing an increase in the relaxation rate of the adult rat myocytes through a PKC-dependent phosphorylation of troponin I, which is conducive for an increase in the rate of Ca²⁺ dissociation from the myofilament.

The change in protein kinase dependence of +dT/dt is consistent with a major role of the PKC-dependent signaling transduction during eccentric hypertrophy. In addition, it has been shown that the ventricular myosin light chain subunit isoforms VLC1 and VLC2 are ectopically reexpressed and -myosin heavy chain becomes largely replaced by -myosin heavy chain in atrial tissue from volume-overloaded hearts. PKC, but not PKA, phosphorylates VLC to increase the Ca²⁺ sensitivity of force development and ATPase activity (32). This may translate into an increased +dT/dt and –dT/dt of the hypertrophied heart.

The aim of this study is to assess the contribution of PKA and PKC, and not the different subtypes, to atrial contraction; however, recent work by others lends additional support to our data. Accordingly, one PKC isoform has been shown to be involved in regulating cardiac contractility. Adenoviral-mediated expression of wild-type PKC-, but not PKC-, PKC-, PKC-, or PKC-, induced cardiac hypertrophy characterized by increased cell surface area, increased protein synthesis, and increased expression of atrial natriuretic factor. In agreement with our data using Ro-32-0432 as a selective Ca²⁺-dependent PKC inhibitor, experiments with dominant-negative forms of PKC isozymes revealed a necessary role for PKC- as a mediator of agonist-induced cardiac hypertrophy. More recently, observations with PKC- knockout and transgenic mice suggest that PKC- is a fundamental regulator of cardiac contractility and Ca²⁺ handling. Modulation of PKC- activity affects dephosphorylation of phospholamban and alters sarcoplasmic reticulum Ca²⁺ loading and the Ca²⁺ transient. Mechanistically, PKC- directly phosphorylates protein phosphatase inhibitor-1, altering the activity of protein phosphatase-1, which may account for the effects of PKC- on phospholamban phosphorylation (5). This is in agreement with a recent report that heart PKC--deficient mice are hypercontractile, whereas mice overexpressing PKC- are hypocontractile (5).

On the other hand, the specific rates of contraction and relaxation in hypertrophied and normal hearts were not different at normal or elevated extracellular Ca²⁺; nevertheless, they were increased by captopril treatment. Because the slow Ca²⁺ channels are the main mediators of extracellular Ca²⁺ transport to the cytoplasm during the atrial action potential, we may speculate that the slow Ca²⁺ channels are not directly affected during the compensated phase of eccentric cardiac hypertrophy; however, they may be activated by ACE inhibitor treatment through a PKC-dependent mechanism (35).

In summary, we have shown that, in the aortocaval shunt-operated rat atrium, there is a decrease in the specific PTD that is overcome by the hypertrophy of the heart tissue to sustain normal cardiac function in the presence of a volume overload (compensated phase). ACE inhibitor treatment induces regression of eccentric cardiac hypertrophy. In addition, captopril improved peak tension, +dT/dt, and –dT/dt and increased PKC responsiveness of the contractile machinery in the shunt-operated rat heart. We suggest that ACE inhibition during eccentric cardiac hypertrophy produces a negative trophic and a positive inotropic effect through a PKC-dependent mechanism, inasmuch as PKC isozymes may have different and/or opposing effects in the heart (6).

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work is supported by National Institute of General Medical Sciences Grant GM-08016 33S1 and the Mort and Mower Foundation.

	ACKNOWLEDGMENTS

 
This work was carried out in partial fulfillment of the requirement of a Ph.D. degree in Physiology and Biophysics at Howard University (K. N. Blackwell).

	FOOTNOTES

 

Address for reprint requests and other correspondence: G. E. Haddad, Dept. of Physiology and Biophysics, College of Medicine, Howard Univ., 520 W St. NW, Rm. 2418-B, Washington, DC 20059 (E-mail: ghaddad{at}howard.edu)

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

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 

1. Amiri F and Garcia R. Renal angiotensin II receptors and protein kinase C in diabetic rats: effects of insulin ACE inhibition. Am J Physiol Renal Physiol 278: F603–F612, 2000.[Abstract/Free Full Text]
2. Baudet S, Weisser J, Janssen AP, Beulich K, Bieligk U, Pieske B, Noireaud J, Janssen PM, Hasenfuss G, and Prestle J. Increased basal contractility of cardiomyocytes overexpressing protein kinase C- and blunted positive inotropic response to endothelin-1. Cardiovasc Res 50: 486–491, 2001.[Abstract/Free Full Text]
3. Bauer P, Regitz-Zagrosek V, Kallisch H, Linz W, Schoelkens B, Hildebrandt AG, and Fleck E. Myocardial angiotensin receptor type 1 gene expression in a rat model of cardiac volume overload. Basic Res Cardiol 92: 139–146, 1997.[CrossRef][ISI][Medline]
4. Braun MU, Szalai P, Strasser RH, and Borst MM. Right ventricular hypertrophy and apoptosis after pulmonary artery banding: regulation of PKC isozymes. Cardiovasc Res 59: 658–667, 2003.[Abstract/Free Full Text]
5. Braz JC, Gregory K, Pathak A, Zhao W, Sahin B, Klevitsky R, Kimball TF, Lorenz JN, Nairn AC, Liggett SB, Bodi I, Wang S, Schwartz A, Lakatta EG, Depaoli-Roach AA, Robbins J, Hewett TR, Bibb JA, Westfall MV, Kranias EG, and Molkentin JD. PKC- regulates cardiac contractility and prospensity toward heart failure. Nat Med 10: 248–254, 2004.[CrossRef][ISI][Medline]
6. Chen L, Hahn H, Wu G, Chen CH, Liron T, Schechtman D, Cavallaro G, Banci L, Guo Y, Bolli R, Dorn GW II, and Mochly-Rosen D. Opposing cardioprotective actions and parallel hypertrophic effects of PKC and PKC. Proc Natl Acad Sci USA 98: 11114–11119, 2001.[Abstract/Free Full Text]
7. D’Amico C, Paterna S, Di Pasquale P, Antona A, Palazzoadriano M, and Licata G. Effect of captopril on lymphocytic -adrenergic receptors in normal and hypoxic conditions. Int J Cardiol 44: 137–143, 1994.[CrossRef][ISI][Medline]
8. Dell’italia LJ, Balcells E, Meng QC, Su X, Schultz D, Bishop SP, Machida N, Straeter-Knowlen IM, Hankes GH, Dillon R, Cartee RE, and Oparil S. Volume-overload cardiac hypertrophy is unaffected by ACE inhibitor treatment in dogs. Am J Physiol Heart Circ Physiol 273: H961–H970, 1997.[Abstract/Free Full Text]
9. De Mello W and Altieri P. Enalapril, an inhibitor of angiotensin-converting enzyme, increases the junctional conductance in isolated heart cell pairs. J Cardiovasc Pharmacol 18: 643–646, 1991.[ISI][Medline]
10. De Mello W and Altieri P. The role of the renin-angiotensin system in the control of cell communication in the heart: effects of enalapril and angiotensin II. J Cardiovasc Pharmacol 20: 643–651, 1992.[ISI][Medline]
11. Fareh J, Touyz RMS, Schiffrin EL, and Thibault G. Endothelin-1 and angiotensin II receptors in cells from rat hypertrophied heart. Receptor regulation and intracellular Ca²⁺ modulation. Circ Res 78: 302–311, 1996.[Abstract/Free Full Text]
12. Garcia R and Diebold S. Simple, rapid, and effective method of producing aortocaval shunts in the rat. Cardiovasc Res 24: 430–432, 1990.[Abstract/Free Full Text]
13. Goutsouliak V and Rabkin SW. Angiotensin II-induced inositol phosphate generation is mediated through tyrosine kinase pathways in cardiomyocytes. Cell Signal 9: 505–512, 1997.[CrossRef][ISI][Medline]
14. Haddad G and Garcia R. Effect of angiotensin converting enzyme two-week inhibition on renal ANG II receptors, and reactivity of renal vessels of the SHR. J Mol Cell Cardiol 29: 813–822, 1997.[CrossRef][ISI][Medline]
15. Haddad G, Saadeh F, Sharaf L, Nahle Z, Abou-Fares M, Haddad R, Bitar K, and Bikhazi A. Alterations in IGF-I binding on cardiac myofibers and capillary endothelium during chronic volume-overload-induced hypertrophy. J Biochem Mol Biol Biophys 3: 65–74, 1999.
16. Haddad G, Sperelakis N, and Bkaily G. Regulation of the calcium slow channel by cyclic GMP-dependent protein kinase in chick heart cells. Mol Cell Biochem 148: 89–94, 1995.[CrossRef][ISI][Medline]
17. Haddad GE, Blackwell N, and Bikhazi A. Regulation of insulin-like growth factor-1 by the renin-angiotensin system during regression of cardiac eccentric hypertrophy through angiotensin-converting enzyme inhibitor and AT₁ antagonist. Can J Physiol Pharmacol 81: 142–149, 2003.[CrossRef][ISI][Medline]
18. Harrap SB, Van der Merwe WM, Griffin SA, MacPherson F, and Lever AF. Brief angiotensin-converting enzyme inhibitor treatment in young spontaneously hypertensive rats reduces blood pressure long-term. Hypertension 16: 603–614, 1990.[Abstract/Free Full Text]
19. Holmer SR, Bruckschlegel G, Schunkert H, Rataj DB, Kromer EP, and Riegger GA. Functional activity and expression of the myocardial postreceptor adenylyl cyclase system in pressure overload hypertrophy in rat. Cardiovasc Res 31: 719–728, 1996.[CrossRef][ISI][Medline]
20. Ishiye M, Umemura K, Uematsu T, and Nakashima M. Effects of losartan, an angiotensin II antagonist, on the development of cardiac hypertrophy due to volume overload. Biol Pharm Bull 18: 700–704, 1995.[ISI][Medline]
21. Iwai N, Shimoike H, and Kinoshita M. Cardiac renin-angiotensin system in the hypertrophied heart. Circulation 92: 2690–2696, 1995.[Abstract/Free Full Text]
22. Kanayama Y, Negoro N, Okamura M, Konishi Y, Nishimura M, Umetani N, Inoue T, and Takeda T. Modulation of protein kinase C in aorta of spontaneously hypertensive rats with enalapril treatment. Osaka City Med J 40: 83–97, 1995.
23. Kim S, Sada T, Mizumo M, Ikeda M, Yano M, Miura K, Yamanaka S, Koike H, and Iwao H. Effects of angiotensin AT₁ receptor antagonist on volume overload-induced cardiac gene expression in rats. Hypertens Res 20: 133–142, 1997.[Medline]
24. Lavandero S, Cartagena G, Guarda E, Corbalan R, Godoy I, Sapag-Hagar M, and Jalil JE. Changes in cyclic AMP-dependent protein kinase and active stiffness in the rat volume overload model of heart hypertrophy. Cardiovasc Res 27: 1634–1638, 1993.[Abstract/Free Full Text]
25. Lear W, Ruzicka M, and Leenen FH. ACE inhibitors and cardiac ACE mRNA in volume overload-induced cardiac hypertrophy. Am J Physiol Heart Circ Physiol 273: H641–H646, 1997.[Abstract/Free Full Text]
26. McCall E, Kenneth GS, Bassani RA, Shannon TR, Qi M, Samarel A, and Bers DM. Ca flux, contractility, and excitation-contraction coupling in hypertrophic rat ventricular myocytes. Am J Physiol Heart Circ Physiol 274: H1348–H1360, 1998.[Abstract/Free Full Text]
27. Mukherjee R and Spinale FG. L-type calcium channel abundance and function with cardiac hypertrophy and failure: a review. J Mol Cell Cardiol 30: 1899–1916, 1998.[CrossRef][ISI][Medline]
28. Ostrom RS, Naugle JE, Hase M, Gregorian C, Swaney JS, Insel PA, Brunton LL, and Meszaros JG. Angiotensin II enhances adenylyl cyclase signaling via Ca²⁺/calmodulin. G_q-G_s cross-talk regulates collagen production in cardiac fibroblasts. J Biol Chem 278: 24461–24468, 2003.[Abstract/Free Full Text]
29. Pechanova O and Bernatova I. Effect of captopril on cyclic nucleotide concentrations during long-term NO synthase inhibition. Physiol Res 49: 55–63, 2000.[ISI][Medline]
30. Pi Y, Sreekumar R, Huang X, and Walker JW. Positive inotropy mediated by diacylglycerol in rat ventricular myocytes. Circ Res 81: 92–100, 1997.[Abstract/Free Full Text]
31. Pi Y, Zhang D, Kemnitz KR, Wang H, and Walker JW. Protein kinase C and A sites on troponin I regulate myofilament Ca²⁺ sensitivity and ATPase activity in the mouse myocardium. J Physiol 552: 845–857, 2003.[Abstract/Free Full Text]
32. Schaub MC, Hefti MA, Zuellig RA, and Morano I. Modulation of contractility in human cardiac hypertrophy by myosin essential light chain isoforms. Cardiovasc Res 37: 381–404, 1998.[Abstract/Free Full Text]
33. Sheehan KA and Blatter LA. Regulation of junctional and non-junctional sarcoplasmic reticulum calcium release in excitation-contraction coupling in cat atrial myocytes. J Physiol 546: 119–135, 2003.[Abstract/Free Full Text]
34. Shikata C, Takeda A, and Takeda N. Effect of ACE inhibitor and AT₁ receptor antagonist on cardiac hypertrophy. Mol Cell Biochem 248: 197–202, 2003.[CrossRef][ISI][Medline]
35. Sperelakis N, Xiong Z, Haddad G, and Masuda H. Regulation of slow calcium channels of myocardial cells and vascular smooth muscle cells by cyclic nucleotides and phosphorylation. Mol Cell Biochem 140: 103–117, 1994.[CrossRef][ISI][Medline]
36. Takeishi Y, Bhagwat A, Ball NA, Kirkpatrick DL, Periasamy M, and Walsh RA. Effect of angiotensin-converting enzyme inhibition on protein kinase C and SR proteins in heart failure. Am J Physiol Heart Circ Physiol 276: H53–H62, 1999.[Abstract/Free Full Text]
37. Timmermans PB, Duncia JV, Carini DJ, Chiu AT, Wong PC, Wexler RR, and Smith RD. Discovery of losartan, the first angiotensin II receptor antagonist. J Hum Hypertens 9: S3–S18, 1995.
38. Tojo T, Tsunada Y, Nakada S, and Tomoika H. Effects of long-term treatment with non-selective endothelin antagonist, TAK-04, on remodeling of cardiovasculature with sustained volume overload. J Cardiovasc Pharmacol 35: 777–785, 2000.[CrossRef][ISI][Medline]
39. Touyz RM and Berry C. Recent advances in angiotensin II signaling. Braz J Med Biol Res 35: 1001–1015, 2002.[ISI][Medline]
40. Unger T. The role of the renin-angiotensin system in the development of cardiovascular disease. Am J Cardiol 89: 3A–10A, 2002.[ISI][Medline]
41. Venema RC and Kuo JF. Protein kinase C-mediated phosphorylation of troponin I and C-protein in isolated myocardial cells is associated with inhibition of myofibrillar actomyosin Mg-ATPase. J Biol Chem 268: 2705–2711, 1993.[Abstract/Free Full Text]
42. Vulpis V, Lograno MD, Seccia TM, and Pirrelli A. L-type calcium channels modulate the regression of left ventricular hypertrophy after ACE inhibition in genetic hypertension. Pharmacol Res 38: 317–322, 1998.[CrossRef][ISI][Medline]
43. Westfall MV and Borton AR. Role of troponin I phosphorylation in protein kinase C-mediated enhanced contractile performance of rat myocytes. J Biol Chem 278: 33694–33700, 2003.[Abstract/Free Full Text]
44. Wu G, Toyokawa T, Hahn H, and Dorn GW II. Protein kinase C- in pathological myocardial hypertrophy. Analysis by combined transgenic expression of translocation modifiers and G_q. J Biol Chem 275: 29927–29930, 2000.[Abstract/Free Full Text]
45. Yonemochi H, Yasunaga S, Teshima Y, Iwao T, Akiyoshi K, Nakagawa M, Saikawa T, and Ito M. Mechanism of -adrenergic receptor upregulation induced by ACE inhibition in cultured neonatal rat cardiac myocytes: roles of bradykinin and protein kinase C. Circulation 97: 2268–2273, 1998.[Abstract/Free Full Text]

This article has been cited by other articles:

				B. Cat, D. Stuhlmann, H. Steinbrenner, L. Alili, O. Holtkotter, H. Sies, and P. Brenneisen Enhancement of tumor invasion depends on transdifferentiation of skin fibroblasts mediated by reactive oxygen species J. Cell Sci., July 1, 2006; 119(13): 2727 - 2738. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 288/2/H695    most recent 00783.2004v1 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 ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via ISI Web of Science (1) Citing Articles via Google Scholar Google Scholar Articles by Haddad, G. E. Articles by Blackwell, K. N. Search for Related Content PubMed PubMed Citation Articles by Haddad, G. E. Articles by Blackwell, K. N.