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Am J Physiol Heart Circ Physiol 281: H637-H646, 2001;
0363-6135/01 $5.00
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Vol. 281, Issue 2, H637-H646, August 2001

alpha 1-Adrenoceptor-Gq-RhoA signaling is upregulated to increase myofibrillar Ca2+ sensitivity in failing hearts

Nobuhiro Suematsu1, Shinji Satoh2, Shintaro Kinugawa1, Hiroyuki Tsutsui1, Shunji Hayashidani1, Ryo Nakamura1, Kensuke Egashira1, Naoki Makino2, and Akira Takeshita1

1 Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582; and 2 Department of Bioclimatology and Medicine, Medical Institute of Bioregulation, Kyushu University, Beppu 874-0838, Japan


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

alpha 1-Adrenergic stimulation, coupled to Gq, has been shown to promote heart failure. However, the role of alpha 1-adrenergic signaling in the regulation of myocardial contractility in failing myocardium is still poorly understood. To investigate this, we observed 1) the effect of phenylephrine on myofibrillar Ca2+ sensitivity in alpha -toxin-skinned cardiomyocytes, and 2) protein expression of Gq, RhoA, and myosin light chain phosphorylation using tachypacing-induced canine failing hearts. Phenylephrine significantly increased myofibrillar Ca2+ sensitivity in failing but not in normal cardiomyocytes. Whereas Y-27632 (Rho kinase inhibitor) blocked the phenylephrine-induced Ca2+ sensitization in the failing myocytes, calphostin C (protein kinase C inhibitor) had no effect on Ca2+ sensitization. The protein expression of Galpha q and RhoA and the phosphorylation level of regulatory myosin light chain significantly increased in the failing myocardium. Our results suggest that alpha 1-adrenoceptor-Gq signaling is upregulated in the failing myocardium to increase the myofibrillar Ca2+ sensitivity mainly through the RhoA-Rho kinase pathway rather than through the protein kinase C pathway.

adrenergic agonists; cardiomyopathy; contractile proteins; G proteins; protein phosphorylation


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

IN HEART FAILURE, neurohormonal factors including adrenergic stimulating agonists modify myocardial morphology and function to compensate for myocardial dysfunction. Cellular mechanisms inducing changes in intracellular signaling pathways activated by these neurohormonal factors have been intensively investigated. Many lines of evidence indicate that the beta -adrenergic signaling system is impaired due to downregulation of beta -adrenoceptors and uncoupling between the beta -adrenoceptors and stimulatory GTP-binding protein Gs in the failing myocardium (8). This is considered to be a major mechanism for blunted inotropic and chronotropic responses to beta -adrenergic stimulation in the failing myocardium.

On the other hand, the role of alpha -adrenergic signaling in cardiac muscle is still not well understood, especially in heart failure. Under physiological conditions, stimulation of cardiomyocytes by alpha 1-adrenergic receptor agonists is known to cause a positive inotropic effect in which intracellular mechanisms have been proposed to be related with phosphatidylinositol turnover coupled to the Gq class of GTP-binding protein. Recent accumulating evidence has demonstrated that alpha 1-adrenergic stimulation initiates pathological changes in cardiomyocytes, which ultimately lead to heart failure. In cultured neonatal cardiomyocytes, alpha 1-adrenergic stimulation by norepinephrine induces hypertrophy via the Gq-mediated pathway as estimated by increased cell size, organization of actin elements, and expression of fetal genes (24, 26). Furthermore, other neurohormonal factors, such as angiotensin II, which activate myocardial receptors coupled to Gq, also induce hypertrophy in cultured cardiomyocytes (33). In an in vivo animal model, transgenic mice overexpressing Galpha q developed ventricular hypertrophy and dilation, which led to the development of decompensated heart failure (1, 29). Expression of Galpha q was shown to increase in the failing myocardium of a myocardial infarction model in rats (22). Another line of evidence has demonstrated that intracellular GTPase RhoA is located downstream of Gq and that the signaling pathway of Gq-RhoA regulates myocardial morphological changes (2, 34). Overexpression of RhoA results in contractile failure coupled with dilation of the left ventricle (LV) (35).

Therefore, it is strongly suggested that Gq-RhoA signaling plays an important role in the development of heart failure with contractile dysfunction. However, the role of this pathway with respect to regulation of myocardial contractility in heart failure has yet to be elucidated. The present study was conducted to investigate changes in the regulatory mechanisms of myocardial contractility through alpha 1-adrenoceptor-Gq-RhoA-mediated signaling in failing canine hearts. The results demonstrate that this pathway is upregulated to cause an increase in myofibrillar Ca2+ sensitivity, which might partly contribute to contractile dysfunction of the failing myocardium.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Preparation of Animal Models and In Vivo Function Studies

Heart failure was induced in seven adult mongrel dogs (15-25 kg body wt) by rapid ventricular pacing as reported previously (right ventricular pacing of 240 beats/min for 4-6 wk) (42). Control dogs (n = 7) were treated in an identical manner as dogs with heart failure but without pacing. On the final day of the in vivo study, pacing was stopped 5 h before the end of the study, and LV function was assessed by echocardiography and catheterization under general anesthesia with pentobarbital sodium given intravenously (30 mg/kg) as described previously (42). The heart rate at the time of the in vivo study was 150 ± 10 beats/min in normal hearts and 138 ± 10 beats/min in failing hearts, respectively (n = 7, no difference). The protocols were approved by the Committee on the Ethics of Animal Experiments, Kyushu University, and were conducted in conformity with the "Guiding Principles for Research Involving Animals and Human Beings" approved by the American Physiological Society.

Isolation of Cardiac Myocytes

Single cardiomyocytes were isolated enzymatically from the LV myocardium as described previously (42). A wedge of the LV free wall was dissected from the heart and was perfused through a branch of the left circumflex coronary artery with a nominally Ca2+-free buffer of the following composition (in mM): 140 NaCl, 4.8 KCl, 2.4 MgSO4, 1.2 NaH2PO4, 2.5 NaHCO3, 12 HEPES, and 12.5 glucose warmed to 37°C and gassed with 100% O2 at pH 7.4. To dissociate the myocytes, the perfusate was changed to a buffer solution containing type II collagenase (60 U/ml, Wako Pure Chemical; Tokyo, Japan), and the perfusion was continued with a constant mean perfusion pressure of 80 mmHg until the heart became flaccid. After the perfusion was completed, the LV myocardium was minced with scissors in a fresh collagenase-containing buffer with 3% bovine serum albumin and was agitated for 5 min in the same buffer. The isolated myocytes were harvested by drawing off the supernatant in which they were suspended and then filtering them through a 210-µm nylon mesh. The harvested single myocytes were thereafter kept in a nominally Ca2+-free solution at 4°C for 1 h before being used in the tension study.

Solutions and Reagents

The relaxing and activating solutions for the skinned cells were prepared similarly as described previously (37). The relaxing solution was composed of (in mM) 110 potassium-methanesulfonate, 5 Mg(MS)2, 5 Na2ATP, 10 creatine phosphate, 4 EGTA, and 20 piperazine-N,N'-bis(2-ethanesulfonic acid), and pH was adjusted to 7.1 with KOH at 25°C. The activating solutions of the desired Ca2+ concentrations (pCa7 to pCa4.5) were prepared by adding the appropriate amounts of Ca(MS)2 to the relaxing solution at 0.2 M ionic strength using a computer program.

Skinning Procedures and Force Measurement

Force measurement of alpha -toxin-skinned isolated cardiomyocytes was performed as described previously (37) using the same relaxing and activating solutions. Single myocytes were skinned with 250 U/ml alpha -toxin (Sigma; St. Louis, MO) for 10 min in the relaxing solution. The cell image was visualized on a television monitor through a charged-coupled device (CCD) camera (model XC-77, Sony; Tokyo, Japan). The cell size and sarcomere length were measured by a computer-assisted scale (model SVS 3000, Showa Electric; Fukuoka, Japan). The length of 10-20 sarcomeres in an uniform section of the cell was measured, and the average sarcomere length was calculated. Initial resting sarcomere length was set at 2.02 ± 0.02 µm in normal and 1.99 ± 0.02 µm in dogs with heart failure, respectively (no difference, n = 23 each total cell number used in the tension study). The skinned cells were then cumulatively activated by various concentrated Ca2+ from pCa7 to pCa4.5 in the absence (first activation at baseline) or presence (second activation at 10-min intervals) of phenylephrine plus GTP in the presence of 1 µM propranolol to block potential involvement of beta -adrenergic stimulation (40). The sarcomere length was monitored during Ca2+ activation on a TV monitor through a CCD camera. If the sarcomere length at <= pCa6 varied by >0.2 µm, the cells were discarded (40). To determine pCa-tension relationships, 1-2 cells from each dog were used and data were analyzed by the least-squares regression method according to the Hill equation (using Prism 3.0, GraphPad Software; San Diego, CA). With the use of the measured values of cell width, each maximum tension was expressed as force/cross-sectional area (CSA), assuming the thickness of the cells to be 60% of their width (12, 32). Force/CSA, Hill coefficients, and pCa50 values were compared in the absence and presence of phenylephrine plus GTP.

SDS-PAGE and Western Blot Analysis

After intravenous administration of a lethal dose of pentobarbital sodium, hearts were quickly removed and LV myocardial tissues were snap-frozen in liquid nitrogen. The tissues were kept at -80°C until biochemical assay.

Expression of Galpha q, RhoA, and myofibrillar proteins. The ventricular tissues from normal dogs and dogs with heart failure were homogenized in 40 mM Tris (pH 7.4) containing 1 mM phenylmethylsulfonyl fluoride, 10% Triton-X, and 1% glycerol. Western blot analysis was performed to quantify the level of Galpha q and RhoA expression using the methods of Ju et al. (22) and Aoki et al. (2) with some modifications. Samples were centrifuged at 700 g for 5 min at 4°C to remove unbroken cells and nuclei followed by centrifugation at 5,000 g for 10 min at 4°C. A part of the supernatant was subjected to 10% SDS-PAGE for RhoA assay by Western blotting and 12.5% SDS-PAGE for myofibrillar proteins assay by Coomassie blue staining. The rest of the supernatant was further subjected to centrifugation at 48,000 g for 20 min at 4°C, and the obtained membrane pellet was subjected to 10% SDS-PAGE for Galpha q assay. Prestained molecular weight markers and the 20-µg (myofibrils and Galpha q) and 60-µg (RhoA) proteins from the samples were separated on SDS-PAGE as described by Laemmli (25). Separated proteins were transferred onto a 0.45-µm polyvinylidene difluoride membrane. The membrane was blocked with 5% skim milk at 4°C overnight and was probed with the primary antibodies Galpha q (Chemicon International) or RhoA (Santa Cruz Biotechnology; Santa Cruz, CA). Horseradish peroxidase-labeled anti-rabbit IgG (for Galpha q) and anti-mouse IgG (for RhoA) were diluted 1:10,000 in a solution of Tris-buffered saline containing Tween 20 and used as the secondary antibodies. Galpha q and RhoA were visualized using an enhanced chemiluminescence kit (Amersham Pharmacia Biotech).

Phosphorylation of regulatory myosin light chain. Phosphorylation of regulatory myosin light chain (RMLC-P) was analyzed by charged gel electrophoresis as described by Hathaway and Haeberle (17). Proteins in the ventricular tissue kept in liquid nitrogen were precipitated by 10% trichloroacetic acid, washed with acetone containing 10 mM of dithiothreitol, and resuspended in 8 M of urea sample buffer. The samples (30 µg protein each) were separated on 10% acrylamide-glycerol gels and probed with primary anti-myosin light chain (MLC) mouse monoclonal antibody (Sigma) and secondary anti-mouse IgM antibody as described in the above section. The extent of RMLC-P was expressed as a density ratio of RMLC-P/RMLC-P + unphosphorylated RMLC (RMLC-UP).

Statistical Analysis

The measured values were expressed as means ± SE. The Hill coefficients and pCa50 values obtained in the absence and presence of stimulation were compared using one-way ANOVA for repeated measures. The protein expression of Galpha q and RhoA, amounts of myofibrillar proteins, and phosphorylation level of RMLC were compared using Student's t-test for unpaired values. A value of P < 0.05 was considered statistically significant.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of alpha 1-Adrenergic Stimulation on pCa-Tension Relationships

pCa-tension relationships were quantified in alpha -toxin-skinned single cardiomyocytes from normal dogs and dogs with heart failure. Thirteen cells were studied from seven dogs. Twelve cells came from six dogs (2 cells from each dog), and one cell came from the remaining one dog. Figure 1 and Table 1 summarize changes in the pCa-tension relationships in the absence and presence of 10 µM phenylephrine plus 100 µM GTP when each cell was treated as a separate sample. At baseline, there was no significant difference in pCa50 values between normal and failing cardiomyocytes (6.01 in normal vs. 6.04 in failure; Table 1). Stimulation by phenylephrine plus GTP significantly increased pCa50 from 6.04 to 6.13 (P < 0.05) in failing but not in normal cardiomyocytes (6.01 to 6.07). The pCa-tension curve thus shifted to the left only in the failing cardiomyocytes (Fig. 1, A and B). There was no significant difference in the pCa50 values between the two groups in the presence of phenylephrine plus GTP (6.07 in normal vs. 6.13 in failure). The maximum tension evoked at pCa 4.5 (force/CSA in Table 1) was not different between the normal and failing cardiomyocytes, and phenylephrine did not change either the maximum tension or Hill coefficients.


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  		Fig. 1.   Normalized pCa-tension relationships in the absence or presence of 10 µM phenylephrine plus 100 µM GTP obtained from alpha -toxin-skinned single cardiomyocytes. Maximum tension in each activation was defined as 100%, whereas submaximal tension was expressed as a fraction of the maximum tension. Normal (A) and failing (B) cardiomyocytes (n = 13 each are shown).


                              
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  		Table 1.   Effects of phenylephrine on pCa-tension relationships

When each dog was treated as a separate sample, there was no significant difference in the baseline myofibrillar Ca2+ sensitivity between the two groups (pCa50, 6.01 ± 0.05 in normal vs. 6.00 ± 0.03 in failure, n = 7 each). Whereas phenylephrine + GTP induced no significant shift in the Ca2+ sensitivity of the normal cardiomyocytes (pCa50, 6.01 ± 0.05 to 6.09 ± 0.06), there still existed a significant increase in the Ca2+ sensitivity by phenylephrine + GTP in the failing cardiomyocytes (pCa50, 6.00 ± 0.03 to 6.14 ± 0.04, P < 0.05). However, when the myofibrillar Ca2+ sensitivity was compared between the normal and failing cardiomyocytes in the presence of phenylephrine + GTP, there was no significant difference between the two groups (pCa50, 6.09 ± 0.06 in normal vs. 6.14 ± 0.04 in failure).

Expression of Galpha q and RhoA in the LV Myocardium

Protein expression of Galpha q and RhoA in the LV myocardium was assessed by Western blot analysis. Actin contents evaluated by SDS-PAGE were similar between the normal and failing myocardium (Fig. 2A, bottom lanes), indicating that the amount of major myofibrillar protein was unchanged in the failing myocardium. On the other hand, the protein expression of Galpha q in the membrane fraction, when normalized to that in the normal myocardium, increased significantly (1.6-fold, P < 0.05) in the failing myocardium compared with that in the normal myocardium (Fig. 2B).


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  		Fig. 2.   Western blot analysis of Galpha q expression in the membrane fraction of the normal and failing myocardium. A: representative Western blot showing specific bands for 42-kDa Galpha q. Lanes 1 and 2 are from the failing myocardium, and lanes 3 and 4 are from the normal myocardium. An equal amount of sample (20 µg protein) was applied in each lane. Coomassie blue-stained SDS-PAGE gel of actin protein is shown in the bottom lanes. B: quantified data of Galpha q protein expression in the normal and failing myocardium. The mean density value of Galpha q in the normal myocardium was defined as 1.0 (n = 6). *P < 0.05.

Similarly, total RhoA expression in the fraction containing both membrane and soluble parts also increased significantly (2.4-fold, P < 0.01) in the failing but not in the normal myocardium (Fig. 3).


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  		Fig. 3.   Western blot analysis of RhoA expression in the fraction containing membrane and soluble parts of the normal and failing myocardium. A: representative Western blot showing specific bands for 21-kDa RhoA. Lanes 1-3 are from the failing myocardium, and lanes 4-6 are from the normal myocardium. An equal amount of samples (60 µg protein) was applied in each lane. Coomassie blue-stained SDS-PAGE gel of actin protein is shown in the bottom lanes. B: quantified data of RhoA protein expression in the normal and failing myocardium. The mean density value of RhoA in the normal myocardium was defined as 1.0 (n = 4). dagger P < 0.01.

Effects of Y-27632 and Calphostin C on Ca2+-Induced Force in Skinned Single Cardiomyocytes

RhoA has been shown to partially translocate from the soluble to the membrane fraction when activated (4). To evaluate the functional activity of Rho kinase (ROCK), a target enzyme of RhoA (28), we studied the effect of Y-27632, a selective inhibitor of ROCK (46), on the phenylephrine-induced enhancement of Ca2+-activated force in alpha -toxin-skinned cardiomyocytes. Figure 4 shows the effect of Y-27632 on the phenylephrine-induced Ca2+ sensitization of pCa6-induced contraction in failing cardiomyocytes. pCa6-induced force remained constant for 10 min in four independent cells (not shown). Phenylephrine (10 µM) plus 100 µM GTP gradually increased the pCa6-activated force (Fig. 4A), and this effect was almost blocked by 10 µM Y-27632 (Fig. 4B). Y-27632 did not affect the Ca2+-activated force itself in four independent cells (not shown). Figure 4C summarizes the effects of Y-27632 on the phenylephrine-induced Ca2+ sensitization in the failing cardiomyocytes. Phenylephrine + GTP enhanced pCa6-activated force by 64.3 ± 9.0% (P < 0.01 vs. baseline, n = 5 cardiomyocytes), and this enhancement was significantly (111.4 ± 5.3%, P < 0.05 vs. phenylephrine + GTP) inhibited by 10 µM Y-27632.


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  		Fig. 4.   Effect of Y-27632 on phenylephrine-induced enhancement of Ca2+-activated force in alpha -toxin-skinned cardiomyocytes from the failing hearts. A: representative tension recording activated at pCa6. After Ca2+-activated force reached a plateau, 10 µM phenylephrine plus 100 µM GTP was applied (arrow). B: same application as in A was repeated in the presence of 10 µM Y-27632. C: quantified data of the effects of Y-27632 on the phenylephrine-induced Ca2+ sensitization in the failing cardiomyocytes (n = 5). Open bar, baseline force at pCa6 was defined as 100%; solid bar, pCa6-activated force in the presence of phenylephrine plus GTP; hatched bar, pCa6-activated force in the presence of phenylephrine plus GTP after treatment with 10 µM Y-27632. *P < 0.05; dagger P < 0.01.

Protein kinase C, another important intracellular signaling molecule, is also activated by Gq-coupled agonists. We therefore studied the role of protein kinase C in the phenylephrine-induced Ca2+ sensitization in skinned cardiomyocytes. Figure 5 shows representative tension recordings demonstrating the effect of calphostin C, a selective inhibitor of protein kinase C (45), on agonist-induced Ca2+ sensitization of pCa6-induced contraction. Figure 5A, left, is a representative tension recording showing that pCa6-activated force was increased by 1 µM phorbol 12-myristate 13-acetate (PMA), a direct protein kinase C activator, in three independent normal cardiomyocytes (138.5 ± 3.8% of pCa6-force, P < 0.05). In Fig. 5A, right, this Ca2+- sensitizing effect was inhibited by 0.5 µM calphostin C (111.6 ± 2.6% of pCa6-force, P < 0.05 vs. PMA alone). On the other hand, the pCa6-activated force increased by 10 µM phenylephrine plus 100 µM GTP (Fig. 5B, left) was not affected by 0.5 µM calphostin C in a failing cardiomyocyte (Fig. 5B, right). Calphostin C itself had no effect on the Ca2+-activated force in four independent normal cells (not shown). Figure 5C summarizes the effects of calphostin C on the phenylephrine-induced Ca2+ sensitization in the failing cardiomyocytes. Unlike Y-27632, the increased Ca2+-activated force by phenylephrine plus GTP (134.3 ± 12.0%, P < 0.05 vs. baseline, n = 5) was not inhibited by 0.5 µM calphostin C (132.9 ± 9.2% of baseline).


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  		Fig. 5.   Effect of calphostin C on phorbol 12-myristate 13-acetate (PMA)- and phenylephrine-induced enhancement of the Ca2+-activated force in alpha -toxin-skinned cardiomyocytes. A: left, after the Ca2+-activated force reached a plateau, 1 µM PMA was applied in a normal cardiomyocyte. Right, same application as in left was repeated in the presence of 0.5 µM calphostin C. B: left, after the Ca2+-activated force reached a plateau, 10 µM phenylephrine plus 100 µM GTP was applied in a failing cardiomyocyte (arrow). Right, same application as in left was repeated in the presence of 0.5 µM calphostin C. C: quantified data of the effects of calphostin C on the phenylephrine-induced Ca2+ sensitization in the failing cardiomyocytes (n = 5). Open bar, baseline force at pCa6 was defined as 100%; solid bar, pCa6-activated force in presence of phenylephrine plus GTP; hatched bar, pCa6-activated force in the presence of phenylephrine plus GTP after treatment with 0.5 µM calphostin C. *P < 0.05.

Changes in Myofibrillar Proteins

Figure 6 shows the comparison of the amounts of myofibrillar proteins separated by SDS-PAGE. With respect to the major myofibrillar proteins (tropomyosin, troponin I, and MLCs), there were no significant differences of the protein contents between the normal and failing myocardium, when normalized to actin content to correct for small variations in the total protein load of each sample (actin content was unchanged in the failing heart as shown in Figs. 2 and 3).


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  		Fig. 6.   Myofibrillar proteins separated on Coomassie blue-stained SDS-PAGE. A: representative 12.5% SDS-PAGE of left ventricular myocardium from normal dogs and dogs with heart failure. MW, molecular weight (mass); Std, standard; MHC, myosin heavy chain; TnT, troponin T; Tm, tropomyosin; TnI, troponin I; MLC1, essential myosin light chain; MLC2, regulatory myosin light chain. B: quantified data of myofibrillar protein contents. Open bars, normal myocardium; solid bars, failing myocardium. Each gel density was normalized to that of the actin band in each dog (n = 5 each).

Figure 7 demonstrates changes in the phosphorylation level of RMLC. RMLC-P was clearly separated from RMLC-UP in both groups (Fig. 7A). As shown in Fig. 7B, the phosphorylation level was significantly higher in the failing myocardium than in the normal myocardium (phosphorylation ratio of 0.44 ± 0.01 in normal to 0.66 ± 0.06 in failure, P < 0.01).


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  		Fig. 7.   Regulatory myosin light chain (RMLC) phosphorylation detected by charged gel electrophoresis. A: representative bands of unphosphorylated (RMLC-UP) and phosphorylated RMLC (RMLC-P). B: quantified data of the extent of RMLC phosphorylation. Open bar, normal myocardium; solid bar, failing myocardium (n = 5 each). The extent of RMLC phosphorylation was expressed as a density ratio as described in MATERIALS AND METHODS. dagger P < 0.01.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study demonstrated 1) functional evidence showing that phenylephrine induced a small but significant increase in myofibrillar Ca2+ sensitivity, which was inhibited by a ROCK inhibitor only in the failing cardiomyocytes, and 2) biochemical evidence showing that protein expression of Galpha q and RhoA was increased in the failing myocardium compared with that in the normal myocardium in association with an increase in the RMLC phosphorylation. These results suggest that alpha 1-adrenergic stimulation causes an increase in the myofibrillar Ca2+ sensitivity mainly through the upregulated Gq-RhoA-ROCK signaling pathway in the failing myocardium.

Physiological Role of alpha 1-Adrenergic Stimulation in Cardiomyocytes

Stimulation of normal cardiomyocytes by alpha 1-adrenergic receptor agonists has been known to induce a positive inotropic effect in rats and rabbits (9). One of the proposed intracellular mechanisms of the positive inotropism by the alpha 1-adrenergic stimulation is a direct modification of the contractile proteins to induce an increase in myofibrillar Ca2+ sensitivity. However, some ambiguity still remains regarding the role of alpha 1-adrenoceptor stimulation in the regulation of the myofibrillar Ca2+ sensitivity. Endoh and Blinks (10) suggested that alpha 1-adrenergic stimulation causes an increase in Ca2+ sensitivity in a study using the simultaneous recording of force and Ca2+ transient in intact muscle strips from rabbits. Whereas Puceat et al. (32) also reported that alpha 1-adrenergic stimulation increased the Ca2+ sensitivity in Triton-skinned single rat cardiac cells, Strang and Moss (40) reported no change in the Ca2+ sensitivity using similar preparations in rats. Even though the reason for these discrepancies is not clear, some intracellular factors coupled to the receptors on the plasma membrane may have been lost after triton skinning. Using a beta -escin-skinned preparation, in which receptor-intracellular coupling is preserved, we (37) previously reported that alpha 1-adrenoceptor stimulation induced an increase in the myofibrillar Ca2+ sensitivity through the GTP-binding protein-mediated mechanisms in rats. However, in the present study, phenylephrine did not change the Ca2+ sensitivity in normal dogs (Fig. 1 and Table 1). This may be due to species differences, because the myocardial density of alpha 1-adrenergic receptor is lower in dogs than in rats, as reported by Endoh et al. (11).

Role of alpha 1-Adrenergic Stimulation in Heart Failure

Recent progress in the understanding of the pathophysiology of heart failure has revealed that Gq-mediated signaling plays a major role in the progression of myocardial hypertrophy, which contributes to the development of heart failure. Agonists coupled to Gq, such as phenylephrine and angiotensin II, induced myocardial cell hypertrophy in cultured neonatal cardiomyocytes (24, 26, 33). Expression of activated Galpha q is sufficient to induce cardiac hypertrophy based on the findings of genetically manipulated animal models. Overexpression of Galpha q induces myocardial hypertrophy or apoptosis associated with decompensated contractile failure in mice (1, 29). Furthermore, in animal disease models, Galpha q expression is increased in the viable, border, and scar tissues after myocardial infarction in rats (22). Thus enhanced Galpha q expression may play an important role in scar remodeling as well as in cardiac hypertrophy.

Another line of evidence has demonstrated that the intracellular small GTP-binding protein RhoA is located downstream of Gq and that RhoA signaling is also associated with the progression of heart failure. Angiotensin II-induced myocardial cell hypertrophy requires the activation of RhoA (2). Signaling mediated by alpha 1-adrenergic receptor and Gq also requires RhoA activation in cardiomyocytes to induce myofibril organization (34). Cardiac-specific overexpression of RhoA induces contractile dysfunction associated with LV dilation in mice (35). Thus it is strongly suggested that the signaling from Gq to RhoA is a mutual pathway to develop myocardial hypertrophy leading to decompensated heart failure associated with contractile dysfunction.

In the tachypacing-induced heart failure model used here, LV hypertrophy did not develop but LV dilation did occur, just as in human dilated cardiomyopathy. The role of alpha 1-adrenoceptor signaling is not clear in this failure model. In the present study, we showed that alpha 1-adrenoceptor signaling was upregulated to increase myofibrillar Ca2+ sensitivity in heart failure characterized by LV dilation with impaired contraction and relaxation. Regarding pathophysiological implications, the increased Ca2+ sensitivity induced by alpha 1-adrenergic stimulation may work partly as a compensation mechanism for contractile dysfunction as suggested by Wolff et al. (47).

There are still conflicting results regarding changes in the myofibrillar Ca2+ sensitivity in the failing heart. Previous reports using skinned preparations suggest that the Ca2+ sensitivity increases in human dilated cardiomyopathy (47), whereas it decreases in rat myocardial infarction (27) and rat pressure overloaded failure (13) and remains unchanged in turkey dilated cardiomyopathy (15) and human dilated cardiomyopathy (7, 16). Hajjar et al. (16) reported that myofibrillar Ca2+ sensitivity did not differ between nonfailing and failing human hearts at pH 7.1 but was increased in the failing myocardium at pH 6.8 and 6.9 compared with that in the nonfailing myocardium. The former result is consistent with our observation in the tension study performed at pH 7.1. Regarding the tachypacing-induced canine heart failure model used in our study, the myofibrillar Ca2+ sensitivity was unchanged from a previous in vivo study (18) or already increased at the basal state in a study using triton-skinned cardiomyocytes (48). Using alpha -toxin-skinned cardiomyocytes in which receptor-intracellular coupling is well preserved, we showed that the Ca2+ sensitivity did not change at the unstimulated state but increased on stimulation by upregulated alpha 1-adrenoceptor-Gq-Rho signaling. Differences in heart failure models, muscle preparations, and experimental methods may have contributed to these discrepancies. Although there was no significant difference in the pCa50 values between the normal and failing hearts in the presence of phenylephrine plus GTP (Table 1), this might be because the basal Ca2+ sensitivity in the failing cardiomyocytes was slightly higher than that in the normal cardiomyocytes. It would be rather important that the degree of changes in myofibrillar Ca2+ sensitivity is significantly larger on the stimulation by alpha 1-adrenergic stimulation in the failing heart.

Possible Mechanisms of an Increase in Myofibrillar Ca2+ Sensitivity in Heart Failure

Calderone et al. (5) reported that the density of alpha 1-adrenergic receptor was unchanged in the failing myocardium in tachypacing-induced canine heart failure. Together with our results, these findings suggest mechanisms that increase the myofibrillar Ca2+ sensitivity of failing cardiomyocytes lie downstream of the alpha 1-adrenergic receptors.

The molecular mechanisms of the increase in the myofibrillar Ca2+ sensitivity through alpha 1-adrenergic stimulation in cardiomyocytes are not known in detail. The phosphatidylinositol turnover activated by phospholipase C coupled to Gq has been suggested to be involved in this Ca2+ sensitization. In the present study, the protein expression of Galpha q and RhoA in the failing myocardial tissues increased and phenylephrine-induced sensitization of Ca2+-activated force was blocked by a specific ROCK inhibitor, Y-27632 (Fig. 4). This result suggests that the upregulated RhoA-ROCK pathway causes the myofibrillar Ca2+ sensitization by alpha 1-adrenergic stimulation in the failing heart.

Recent reports have demonstrated that the RhoA-ROCK pathway plays an important role in the regulation of morphological changes of cardiomyocytes via phosphorylation of RMLC. Aoki et al. (3) reported that RMLC phosphorylation through myosin light chain kinase (MLCK) activated by phenylephrine and angiotensin II induces myofibril formation in cultured cardiomyocytes. This signaling is dependent on the RhoA-ROCK pathway (2, 34). Accumulating evidence suggests that RMLC plays a definite role in the regulation of myofibrillar Ca2+ sensitivity in cardiac muscle, just as in smooth muscle. RMLC phosphorylation increases the Ca2+ sensitivity in skinned striated muscle preparations (41). In transgenic mice overexpressing nonphosphorylatable RMLC, there was no shift of the myofibrillar Ca2+ sensitivity after treatment with MLCK, whereas the Ca2+ sensitivity increased depending on the treatment with MLCK in nontransgenic mice (36). These transgenic mice also showed enlargement of cardiac chambers. These results suggest that the phosphorylation of RMLC plays an important role in maintaining normal cardiac contractile function and morphology.

In the present study, the level of RMLC phosphorylation was increased in frozen samples from the failing hearts. Because the myocardial tissues in our study were quickly fixed with liquid nitrogen just after their removal from beating hearts, these tissues were thought to remain at least partially stimulated by intrinsic catecholamines. Spinale et al. (39) reported that the level of total catecholamines was increased 1.7-fold in a canine model of tachypacing-induced heart failure. The increased RMLC phosphorylation observed in our study thus could be induced by upregulated Gq-RhoA-ROCK signaling to cause the phenylephrine-induced sensitization of Ca2+-activated force. However, there could exist several other factors to influence RMLC phosphorylation. First, it could be possible that pacing itself induces RMLC phosphorylation, although little is known about the effect of pacing on RMLC phosphorylation. We think that in our study, the RMLC phosphorylation by pacing itself may contribute only partly to the total level of RMLC phosphorylation in the failing myocardium based on the following reasons. On the day of the study, pacing was stopped 5 h before the hearts were excised, and there was no significant difference of the heart rate at the time of in vivo study between the two groups (150 ± 10 beats/min in normal vs. 138 ± 10 beats/min in failure). Although we did not investigate the time course of the dephosphorylation of RMLC after pacing off, the level of the RMLC phosphorylation would be expected to decrease to some extent by the time the hearts were excised. In addition, Fitzsimons et al. (14) reported the effect of heart rate on RMLC phosphorylation in rats. When the heart rate was increased by treadmill exercise from 361 to 531 beats/min, the RMLC phosphorylation level was increased from 33.3 to 39.5% (1.19-fold). Isoproterenol infusion also caused an increase in RMLC phosphorylation up to 41.1% (1.23-fold), associated with an increase in heart rate up to 583 beats/min. These reported levels of the phosphorylation are less than those observed in the present study (from 44 to 66%, 1.5-fold increase). Silver et al. (38) also reported that pacing at 126 beats/min induced only 0.4 mol phosphate/mol RMLC in isolated perfused rabbit hearts.

Second, phosphatase activity could be altered by the time of the assay, and thus it may be different between the two groups. Little is known about the regulation of phosphatase involved in RMLC phosphorylation in cardiac muscle. If phosphatase activity is increased more in the failing heart, the resultant level of RMLC phosphorylation would be expected to be less in the failing heart than that in the normal heart. On the contrary, if the activity is decreased in the failing heart, RMLC phosphorylation would be increased. In smooth muscle, RhoA is proposed to cause an increase in RMLC phosphorylation mainly through the inhibition of MLC phosphatase by the Rho kinase-catalyzed phosphorylation of the myosin-binding subunit (23). Although little is known concerning the mechanisms of RhoA action in cardiac muscle, it would be significant that the total level of the RMLC phosphorylation is increased in the failing heart whether the phosphatase activity is altered or not.

Third, it would be also possible that beta -adrenergic stimulation causes RMLC phosphorylation activated by the elevated plasma catecholamine levels in vivo. To the best of our knowledge, however, there have been conflicting results concerning RMLC phosphorylation induced by beta -adrenergic stimulation. Fitzsimons et al. (14) reported that isoproterenol infusion in vivo caused an increase in RMLC phosphorylation from 33.3 ± 1.1 to 41.1 ± 1.2% in rats. On the other hand, High and Stull (19) and Silver et al. (38) reported that isoproterenol, the dose of which induced positive inotropy, did not increase RMLC phosphorylation in perfused rabbit hearts (the phosphorylation level reached ~48% vs. basal 44%). Holroyde et al. (20) and Jeacocke and England (21) reported similar results showing that the infusion of 5-10 µM epinephrine did not increase RMLC phosphorylation in perfused rat and rabbit hearts. In addition, the level of the isoproterenol-induced RMLC phosphorylation reported by Fitzsimons et al. (14) was 41.1 ± 1.2% at maximum, which is less than that observed in the present study (66 ± 6%). From these findings, we think that RMLC phosphorylation by beta -adrenergic stimulation, if any, may contribute only partly to the total level of RMLC phosphorylation in the failing myocardium observed in the present study.

Another possible mechanism for the regulation of myofibrillar Ca2+ sensitivity in cardiac muscle has been proposed. Activated Gq protein is known to activate protein kinase C to transfer various biological signals. Protein kinase C phosphorylates both troponin I (6, 30) and RMLC (6, 31). It has been reported that the phosphorylation of serine residue of troponin I causes a reduced Ca2+ sensitivity of the myofibrillar MgATPase (30). On the other hand, phosphorylated RMLC by protein kinase C is associated with an increase in the ATPase activity and the Ca2+ sensitivity of force production (6, 31). In transgenic mice, overexpression of different isoforms of protein kinase C exerts different effects on the myofibrillar Ca2+ sensitivity; i.e., the sensitivity is decreased by protein kinase C-beta (43) and increased by protein kinase C-epsilon (44). At present, therefore, the net effect of protein kinase C would seem to be highly complex in terms of the regulation of myofibrillar Ca2+ sensitivity in cardiac muscle, because it would depend on the differences in the phosphorylation levels of troponin I and RMLC, and protein kinase C isoforms. In the present study, phenylephrine-induced myofibrillar Ca2+ sensitization was not inhibited by calphostin C (Fig. 5, B and C). The concentration of calphostin C used here (0.5 µM) is sufficiently selective for protein kinase C (IC50 for protein kinase C, protein kinase A, and protein kinase G was 0.05 µM, >50 µM, and >25 µM, respectively) (45) and was enough to inhibit PMA-induced Ca2+ sensitization (Fig. 5A). Protein kinase C thus may be considered not to play a major role in the increased myofibrillar Ca2+ sensitivity in the tachypacing-induced failure model, which closely resembles human dilated cardiomyopathy.

On the basis of the present results, alpha 1-adrenoceptor-Gq signaling is augmented in the failing myocardium, thus causing an increased myofibrillar Ca2+ sensitivity. This Ca2+ sensitization seems to be mediated through the RhoA-ROCK pathway rather than through the protein kinase C pathway. The increased Ca2+ sensitivity by the upregulated Gq signaling may be one of the abnormal regulatory mechanisms of contractility in the failing heart.


    ACKNOWLEDGEMENTS

The authors thank Y. Ueda for excellent technical assistance.


    FOOTNOTES

This study was supported in part by Ministry of Education, Science and Culture Grants 08670803 and 09670724 (Japan).

Address for reprint requests and other correspondence: S. Satoh, Dept. of Bioclimatology and Medicine, Medical Institute of Bioregulation, Kyushu Univ., 4546 Tsurumihara, Beppu 874-0838, Japan (E-mail: satoshin{at}tsurumi.beppu.kyushu-u.ac.jp).

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.

Received 8 January 2001; accepted in final form 5 April 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 281(2):H637-H646
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