QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.107888v1 319/3/1307    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mueller, E. E. Articles by Howlett, S. E. Search for Related Content PubMed PubMed Citation Articles by Mueller, E. E. Articles by Howlett, S. E.

CARDIOVASCULAR

Protein Kinase A-Mediated Phosphorylation Contributes to Enhanced Contraction Observed in Mice That Overexpress -Adrenergic Receptor Kinase-1

Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada

Received May 15, 2006; accepted August 31, 2006.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Transgenic mice with cardiac specific overexpression of -adrenergic receptor kinase-1 (ARK-1) exhibit reduced contractility in the presence of adrenergic stimulation. However, whether contractility is altered in the absence of exogenous agonist is not clear. Effects of ARK-1 overexpression on contraction were examined in mouse ventricular myocytes, studied at 37°C, in the absence of adrenergic stimulation. In myocytes voltage-clamped with microelectrodes (18–26 M; 2.7 M KCl) to minimize intracellular dialysis, contractions were significantly larger in ARK-1 cells than in wild-type myocytes. In contrast, when cells were dialyzed with patch pipette solution (1–3 M; 0 mM NaCl, 70 mM KCl, 70 mM potassium aspartate, 4 mM MgATP, 1 mM MgCl₂, 2.5 mM KH₂PO₄, 0.12 mM CaCl₂, 0.5 mM EGTA, and 10 mM HEPES), the extent of cell shortening was similar in wild-type and ARK-1 myocytes. Furthermore, when cells were dialyzed with solutions that contained phosphodiesterase-sensitive sodium-cAMP (50 µM), the extent of cell shortening was similar in wild-type and ARK-1 myocytes. However, when patch solutions were supplemented with phosphodiesterase-resistant 8-bromo-cAMP (50 µM), contractions were larger in ARK-1 than wild-type cells. This difference was eliminated by the protein kinase A inhibitor N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89). Interestingly, Ca²⁺ current amplitudes and inactivation rates were similar in ARK-1 and wild-type cells in all experiments. These results suggest components of the adenylyl cyclase-protein kinase A pathway are sensitized by chronically increased ARK-1 activity, which may augment contractile function in the absence of exogenous agonist. Thus, changes in contractile function in myocytes from failing hearts may reflect, in part, effects of chronic up-regulation of ARK-1 on the cAMP-protein kinase A pathway.

Desensitization of ARs is mediated by phosphorylation of the receptors by G protein-coupled receptor kinases (GRKs) and second messengers. GRKs mediate homologous desensitization; thus, their kinase activity is dependent on agonist occupancy (Lohse et al., 1990). GRKs are a family of serine/threonine kinases (Penela et al., 2006). At present, seven isoforms have been identified, known as GRK1 through 7 (Penela et al., 2006). GRK2, also known as -adrenergic receptor kinase-1 (ARK-1), is the predominant isoform expressed in the heart (Hausdorff et al., 1990). ARK-1 is expressed in both myocyte and nonmyocyte cardiac cells (Penela et al., 2006). Interestingly, ARK-1 expression and activity are increased in the failing heart (Ungerer et al., 1993). This up-regulation of ARK-1 increases phosphorylation of activated ARs, which leads to desensitization, and down-regulation of the receptors (Post et al., 1999). This dampens the AR signaling cascade, which in turn alters Ca²⁺ cycling, and impairs cardiac contractile function (Rockman et al., 2002).

To investigate the role of ARK-1 in the modulation of cardiac contractility, a number of studies have examined the effects of ARK-1 overexpression on cardiac function in a transgenic mouse model (Koch et al., 1995; Rockman et al., 1996; Chen et al., 1998). Overexpression of ARK-1 in these mice is driven by the myosin heavy chain promoter (Koch et al., 1995). This leads to a 3- to 5-fold overexpression of ARK-1 in cardiac myocytes, which is similar to the levels seen in heart failure (Koch et al., 1995). However, ARK-1 is overexpressed only in myocytes in this model and may therefore not reflect the overall role of ARK-1 overexpression in heart failure (Penela et al., 2006). In vivo studies have shown that isoproterenol-stimulated increases in heart rate and contractility are significantly reduced in ARK-1 mice compared with wild-type controls (Koch et al., 1995; Rockman et al., 1996; Chen et al., 1998). In addition, isoproterenol-stimulated adenylyl cyclase activity is reduced in sarcolemmal membranes from ARK-1 mice compared with wild-type animals (Koch et al., 1995). Interestingly, adenylyl cyclase activity also is reduced in sarcolemmal membranes from ARK-1 mice in the absence of isoproterenol (Koch et al., 1995). This suggests that chronic overexpression of ARK-1 may alter the adenylyl cyclase-protein kinase A pathway in the heart, and thereby alter cardiac function even in the absence of agonist. However, little is known about the effects of ARK-1 overexpression on intrinsic myocyte function.

We hypothesized that alterations in the cAMP-protein kinase A pathway in ARK-1 ventricular myocytes alter cardiac contractile function in the absence of exogenous AR agonist. The objectives of this study were to 1) compare the extent of cell shortening and magnitude of L-type Ca²⁺ current (I_Ca-L) in myocytes from ARK-1 and wild-type mice when cells were voltage-clamped with high-resistance microelectrodes to minimize intracellular dialysis with pipette solutions; 2) determine whether the extent of cell shortening and magnitude of I_Ca-L differed between ARK-1 and wild-type myocytes when cells were dialyzed with patch pipette solutions in the absence and presence of various analogs of cAMP; and 3) determine whether differences between ARK-1 and wild-type myocytes were mediated by protein kinase A. All experiments were conducted in regularly paced ventricular myocytes, at physiological temperature (37°C), in the absence of exogenous AR stimulation.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Experimental Animals. Experiments were performed on cardiac ventricular myocytes isolated from approximately 6-month-old male and female ARK-1 mice and wild-type littermates. The age ranges used were similar in the wild-type and ARK-1 groups, as shown in Table 1. In addition, there was no significant difference in weight between the wild-type and ARK-1 mice (Table 1). The initial breeding pairs were made up of female wild-type (B6SJLF1/J) and male ARK-1 [B6SJL-TgN(BARK12)12Wjk] mice obtained from The Jackson Laboratory (Bar Harbor, ME). The breeding colony was maintained by breeding ARK-1 animals with wild-type littermates. ARK-1 mice are hemizygous for the transgene. Therefore, transgenic mice were identified by genotyping with a protocol provided by The Jackson Laboratory (www.jax.org/jaxmice/micetech). All experiments respected the guidelines stated in the Canadian Council on Animal Care (1980, 1984) and were approved by the Dalhousie University Committee on Animal Care.

View this table: [in this window] [in a new window]   TABLE 1 Characteristics of wild-type and ARK-1 mice Values represent the mean ± S.E.M. The number in parentheses represents the number of animals (age, weight) or myocytes (cell length, cell capacitance).

Cell Isolation. Mice were anesthetized with an intraperitoneal injection of pentobarbital sodium (200–300 mg/kg), coinjected with heparin (100 U) to inhibit blood coagulation. The thoracic cavity was opened with a parasternal incision. The aorta was incised, cannulated in situ, and perfused at 2.2 ml/min for 10 min with oxygenated, Ca²⁺-free solution of the following composition: 130 mM NaCl, 5 mM KCl, 25 mM HEPES, 0.33 mM NaH₂PO₄, 1 mM MgCl₂, 20 mM glucose, 3 mM sodium pyruvate, and 1 mM lactic acid (pH 7.4 with NaOH). The solution was maintained at 37°C with a heated circulating water bath. Then, the hearts were perfused with the Ca²⁺-free solution supplemented with 50 µM Ca²⁺, 24 mg/30 ml of collagenase (type I; Worthington, Biochemicals, Freehold, NJ), 10 mg/30 ml of neutral protease dispase II, and 1 mg/ml trypsin. After approximately 10 min, the ventricles were excised, minced, and stored at room temperature until use in a high K⁺ solution containing 80 mM KOH, 50 mM glutamic acid, 30 mM KCl, 30 mM KH₂PO₄, 20 mM taurine, 10 mM HEPES, 10 mM glucose, 3 mM MgSO₄, and 0.5 mM EGTA (pH 7.4 with KOH).

Data Acquisition. Isolated myocytes were placed on the stage of an inverted microscope and allowed to settle for approximately 10 min. Then, the cells were superfused at 3 ml/min with a standard buffer solution of the following composition: 145 mM NaCl, 10 mM glucose, 10 mM HEPES, 4 mM KCl, 1 mM CaCl₂, and 1 mM MgCl₂ (pH 7.4 with NaOH). This solution was supplemented with 0.3 mM lidocaine and 4 mM 4-aminopyridine to block Na⁺ and K⁺ currents, respectively. All experiments were conducted at 37°C.

In experiments designed to minimize intracellular dialysis with the electrode filling solutions, cells were voltage-clamped with high-resistance microelectrodes (18–26 M; filled with 2.7 M KCl). In other experiments, cells were voltage-clamped with patch pipettes (1–3 M) filled with 0 mM NaCl, 70 mM KCl, 70 mM potassium aspartate, 4 mM MgATP, 1 mM MgCl₂, 2.5 mM KH₂PO₄, 0.12 mM CaCl₂, 0.5 mM EGTA, and 10 mM HEPES (pH 7.2). In some experiments, this pipette filling solution was supplemented with either 50 µM 8-bromo-cAMP or 50 µM sodium-cAMP. In all cases, discontinuous single-electrode voltage clamp (5–8 kHz) was conducted with pCLAMP software, version 8.0 (Molecular Devices, Sunnyvale, CA) and an Axoclamp-2B amplifier (Molecular Devices). Unloaded cell shortening was recorded with a video edge detector (Crescent Electronics, Sandy, UT) and video camera (model TM-640; Pulnix America, Inc., Sunnyvale, CA) operating at 120 Hz. In some experiments, the protein kinase inhibitor H89 (5 µM) was added to the extracellular buffer. In these experiments, the control extracellular solution contained the same concentration of DMSO as used in the H89 studies as a solvent control.

Several voltage-clamp protocols were used. In all cases, voltage-clamp test steps were preceded by five 200-ms conditioning pulses from the holding potential of –80 to 0 mV to provide a consistent history of activation. Cells were then repolarized to –40 mV for 500 ms. In some experiments, complete contraction-voltage and current-voltage relations were elicited by a series of 250-ms depolarizing steps from –40 mV to potentials between –30 and +60 mV, in increments of 10 mV. However, a single 250-ms test step from –40 to 0 mV was used to elicit peak inward I_Ca-L and contraction in experiments with H89. A single test step was used in these experiments because preliminary experiments showed that the concentration of DMSO used to dissolve the H89 caused contractions to run down during lengthy voltage-clamp protocols.

Data Analysis. All analyses were performed with pCLAMP software. The extent of cell shortening was measured as the difference between the peak contraction and a baseline immediately before cell shortening. The velocity of shortening was determined by dividing the amplitude of contraction by the time to peak contraction and is expressed in micrometers per second. The velocity of relengthening was determined by dividing one-half the amplitude of contraction by the time-to-half relaxation and also is expressed in micrometers per second. I_Ca-L amplitude was measured as the difference between the peak inward current and a reference point at the end of the voltage step. Time constants for inactivation of I_Ca-L were measured with Clampfit 8.1 (Axon Instruments). The fast (_f) and slow (_s) time constants of inactivation for I_Ca-L were determined by fitting the inactivation phase of I_Ca-L with an exponential function with two exponential components. Cell membrane area was determined by integrating the capacitive transient with pCLAMP software. I_Ca-L was normalized by cell capacitance and is expressed as current density. Cell length was used to normalize the contraction data.

Data are presented as means ± S.E.M. When two groups were compared, differences between means were assessed by an unpaired Student's t test. Differences between mean contraction-voltage and current-voltage relationships for ARK-1 and wild-type mice were analyzed by two-way repeated measures analysis of variance. SigmaStat software, version 2.0 (Systat Software, Inc., San Jose, CA) was used for all statistical analyses. No more than two myocytes from the same heart were included in any one data set.

Chemicals. Pentobarbital sodium, trypsin, lidocaine, HEPES, EGTA, MgCl₂, 4-aminopyridine, H89, 8-bromo-cAMP, sodium-cAMP, EDTA, sodium-pyruvate, lactic acid, L-glutamic acid, taurine, and Mg-ATP were purchased from Sigma-Aldrich Canada (Oakville, ON, Canada). DMSO was purchased from Invitrogen Canada, Inc. (Burlington, ON, Canada). The dispase II was purchased from Roche Diagnostics (Laval, QC, Canada). Heparin was purchased from Organon (Toronto, ON, Canada). Type I collagenase was purchased from Worthington Biochemicals. All other chemicals were purchased from BDH (Toronto, ON, Canada). H89 was dissolved in DMSO; the concentration of DMSO in the extracellular buffer was 0.025%. All other chemicals were dissolved in deionized water.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Ventricular cell size and/or membrane area is altered in myocytes from certain strains of transgenic mice (Yatani et al., 1999; Knollmann et al., 2000; Olsson et al., 2004). Therefore, we measured cell length and cell capacitance to determine whether there were differences in these parameters between ARK-1 and wild-type mice. There was no significant difference in mean cell length between myocytes from wild-type and ARK-1 mice (Table 1). However, cell capacitance was significantly greater in myocytes from ARK-1 mice than in myocytes from wild-type mice (Table 1). Because cell capacitance is proportional to cell membrane area, these results suggest that sarcolemmal area is increased in ARK-1 myocytes. Therefore, in all subsequent experiments, measurements of I_Ca-L were normalized to cell capacitance to account for differences in cell surface area. We also normalized all contraction data to cell length.

In the first series of experiments, we compared the extent of cell shortening and I_Ca-L in wild-type and ARK-1 cells impaled with high-resistance microelectrodes. High-resistance microelectrodes were used in these studies to minimize cell dialysis and distortion of differences in cytosolic composition between wild-type and ARK-1 myocytes. In these experiments, cells were voltage-clamped with 250-ms test steps from –40 mV to potentials between –40 and +60 mV, delivered in increments of 10 mV, to elicit contractions and I_Ca-L (Fig. 1, top). The test step was preceded by five 200-ms conditioning pulses from –80 to 0 mV to provide comparable activation histories in both wild-type and ARK-1 cells. Figure 1, A and B, shows representative contractions and I_Ca-L elicited by a test step from –40 to –10 mV recorded from wild-type and ARK-1 myocytes, respectively. The extent of cell shortening was larger in the ARK-1 myocyte than the wild-type cell, although amplitudes of I_Ca-L were similar in the two cells. The extent of cell shortening and I_Ca-L were plotted as a function of voltage to determine contraction-voltage and current-voltage relations, respectively. The mean contraction-voltage curves were bell-shaped in both wild-type and ARK-1 myocytes (Fig. 1C). However, contractions at the peak of the curve were significantly larger in ARK-1 myocytes than in wild-type cells (Fig. 1C). Figure 1D compares mean current-voltage curves in wild-type and ARK-1 myocytes. Mean amplitudes of I_Ca-L did not differ between wild-type and ARK-1 myocytes at any membrane potential examined (Fig. 1D). We also compared the inactivation rates for I_Ca-L in wild-type and ARK-1 myocytes (Table 2). Results showed that _f and _s time constants of inactivation for I_Ca-L were similar in wild-type and ARK-1 cells (Table 2). We also determined and compared the velocity of shortening and the velocity of relengthening of contractions in wild-type and ARK-1 myocytes. Results showed that there were no significant differences in the rates of contraction and relaxation between wild-type and ARK-1 myocytes (Fig. 1, E and F). Thus, contraction is enhanced in ARK-1 myocytes compared with wild-type controls in the absence of exogenous agonist.

View larger version (24K): [in this window] [in a new window]   Fig. 1. The extent of cell shortening was larger in ARK-1 myocytes than in wild-type cells, when cells were voltage-clamped with high-resistance electrodes to minimize intracellular dialysis with pipette solutions. Cells were impaled with high-resistance microelectrodes. The voltage-clamp protocol is shown at the top. A and B, representative contractions (top) and ICa-L (bottom) recorded from wild-type and ARK-1 myocytes in response to a voltage step from –40 to –10 mV. Contraction was larger in the ARK-1 cell than in the wild-type cell. C, contraction amplitudes were significantly larger near the peak of the contraction-voltage curve in ARK-1 myocytes compared with wild-type cells. D, current-voltage relationships were similar in wild-type and ARK-1 myocytes. E and F, mean velocities of shortening and relengthening were determined for contractions elicited by a step from –40 to 0 mV. The rates of contraction and relaxation were similar in wild-type and ARK-1 myocytes when intracellular dialysis with pipette solutions was minimal. n = 12 wild-type and n = 7 ARK-1 myocytes. *, p < 0.05, denotes significantly different from wild type.

Next, we determined whether EC coupling was altered in ventricular myocytes isolated from ARK-1 mice when cells were dialyzed with a standard potassium-based patch pipette solution (Fig. 2). The experimental protocol is shown at the top of the figure. Figure 2, A and B, shows representative contractions and currents recorded from a wild-type and ARK-1 myocyte, respectively. Contractions and I_Ca-L were similar in the two cell types. We next determined whether the voltage dependence of contraction and I_Ca-L differed in wild-type and ARK-1 myocytes when cells were dialyzed with pipettes containing a standard potassium-based solution. Mean amplitudes of cell shortening were similar in wild-type and ARK-1 cells over a wide range of voltages (Fig. 2C). Figure 2D compares mean current-voltage relations in wild-type and ARK-1 myocytes. Mean amplitudes of I_Ca-L did not differ between wild-type and ARK-1 myocytes until the voltage exceeded +40 mV. At membrane potentials more positive than +40 mV, I_Ca-L was larger in ARK-1 cells than in wild-type cells (Fig. 2D). However, the _f and _s time constants of inactivation of I_Ca-L were similar in wild-type and ARK-1 cells dialyzed with standard pipette solution (Table 2). The velocities of shortening and relengthening also were compared in wild-type and ARK-1 myocytes dialyzed with standard pipette solution. Results showed that the rates of contraction and relaxation were similar in wild-type and ARK-1 myocytes (Fig. 2, E and F). These experiments demonstrate that the increase in the extent of cell shortening in ARK-1 myocytes is abolished when ARK-1 and wild-type cells are dialyzed with the same potassium-based patch pipette solution. These observations suggest that a factor or factors required to increase contraction amplitude in ARK-1 cells may be removed by intracellular dialysis with pipette solutions.

View larger version (23K): [in this window] [in a new window]   Fig. 2. The extent of cell shortening and magnitudes of ICa-L were similar in ARK-1 and wild-type myocytes when cells were dialyzed with pipettes filled with standard potassium-based solution. The voltage-clamp protocol is shown at the top. A and B, representative contractions and ICa-L recorded from wild-type and ARK-1 cells in response to a voltage step from –40 to 0 mV. C, contraction-voltage relationships were similar in wild-type and ARK-1 myocytes. D, current-voltage relationships showed that the magnitude of ICa-L was similar in wild-type and ARK-1 myocytes, except for a small increase in the current in ARK-1 cells at +50 and +60 mV. E and F, the average velocities of cell shortening and relengthening in response to a voltage step from –40 to 0 mV were similar in wild-type and ARK-1 myocytes dialyzed with standard pipette solution. n = 10 wild-type myocytes and n = 17 ARK-1 myocytes. *, p < 0.05, denotes significantly different from wild type.

Previous studies have shown that dialysis with pipette solutions can disrupt the adenylyl cyclase-protein kinase A pathway and alter contractile function and that inclusion of cAMP in pipette solutions can restore contractile function in ventricular myocytes (Ferrier et al., 1998; Ferrier and Howlett, 2003). Therefore, we next determined whether inclusion of cAMP in patch pipette solutions would restore the differences in contraction observed in undialyzed cells between wild-type and ARK-1 myocytes. Cells were voltage-clamped with pipettes that contained the phosphodiesterase-resistant analog of cAMP, 8-bromo-cAMP (50 µM; Meyer and Miller, 1974). Figure 3, A and B, shows representative contractions and I_Ca-L recorded from wild-type and ARK-1 myocytes in the presence of 50 µM 8-bromo-cAMP in the pipette. The contraction was larger in the ARK-1 cell compared with the wild-type myocyte, whereas I_Ca-L was similar in the two cells. Contraction-voltage and current-voltage relations in wild-type and ARK-1 cells dialyzed with 8-bromo-cAMP are shown in Fig. 3, C and D. The extent of cell shortening was significantly increased in ARK-1 myocytes compared with wild-type myocytes at voltages between –20 and +20 mV (Fig. 3C). In contrast, amplitudes of I_Ca-L were similar in wild-type and ARK-1 cells at all membrane potentials examined (Fig. 3D), and the time constants of inactivation of I_Ca-L were similar in the two groups (Table 2). The velocities of cell shortening and relengthening in wild-type and ARK-1 myocytes also were examined in these experiments. As shown in Fig. 3, E and F, the rates of contraction and relaxation were similar in wild-type and ARK-1 myocytes. These results show that contraction amplitudes were significantly larger in ARK-1 cells compared with wild-type cells when 8-bromo-cAMP was included in the pipette solution.

View larger version (23K): [in this window] [in a new window]   Fig. 3. In cells dialyzed with a phosphodiesterase-resistant analog of cAMP, the extent of cell shortening was greater in ARK-1 myocytes than in wild-type myocytes. Cells were dialyzed with 50 µM 8-bromo-cAMP. The voltage-clamp protocol is shown at the top. A and B, representative recordings of contractions and ICa-L from wild-type and ARK-1 myocytes initiated by a voltage step from –40 to 0 mV. Contraction was larger in the ARK-1 cell compared with the wild-type cell. C, the peak of the contraction-voltage relationship was significantly larger in ARK-1 compared with wild-type myocytes. D, current-voltage relationships were similar in wild-type and ARK-1 myocytes. E and F, mean velocities of shortening and velocities of relengthening elicited by a voltage step from –40 to 0 mV did not differ between wild-type and ARK-1 myocytes when cells were dialyzed with 8-bromo-cAMP. n = 8 wild-type and 10 ARK-1 myocytes. *, p < 0.05, denotes significantly different from wild type.

We also determined whether differences in contraction between ARK-1 and wild-type myocytes were present when cells were dialyzed with the phosphodiesterase-sensitive analog of cAMP, sodium-cAMP (50 µM; Meyer and Miller, 1974). Figure 4, A and B, shows representative contractions and I_Ca-L recorded from wild-type and ARK-1 myocytes. In these examples, magnitudes of contractions and I_Ca-L were similar in wild-type and ARK-1 myocytes dialyzed with sodium-cAMP (Fig. 4, A and B). We compared the extent of cell shortening and magnitude of I_Ca-L between wild-type and ARK-1 myocytes dialyzed with sodium-cAMP over a range of membrane potentials (Fig. 4, C and D). Mean contraction-voltage and current-voltage relations were similar in the two groups (Fig. 4, C and D). Furthermore, the time constants of inactivation of I_Ca-L (Table 2) as well as the rates of contraction and relaxation were similar in wild-type and ARK-1 myocytes (Fig. 4, E and F). Thus, differences in the extent of cell shortening between wild-type and ARK-1 myocytes were observed only when cells were dialyzed with solution that contained a phosphodiesterase-resistant analog of cAMP or when high-resistance electrodes were used to minimize cell dialysis.

View larger version (23K): [in this window] [in a new window]   Fig. 4. In cells dialyzed with phosphodiesterase-sensitive sodium-cAMP, the extent of cell shortening was similar in wild-type and ARK-1 cells. Cells were dialyzed with 50 µM sodium-cAMP. The voltage-clamp protocol is shown at the top. A and B, representative recordings of contractions and ICa-L initiated by a test step from –40 to 0 mV were similar in wild-type and ARK-1 myocytes. Contraction-voltage (C) and current-voltage (D) relationships were similar in wild-type and ARK-1 myocytes when cells were dialyzed with sodium-cAMP. E and F, average velocities of shortening and relengthening initiated by a voltage step from –40 to 0 mV were similar in wild-type and ARK-1 myocytes dialyzed with sodium-cAMP. n = 6 wild-type and n = 4 ARK-1 myocytes.

The difference in the extent of cell shortening between ARK-1 and wild-type cells was observed with the same concentration of 8-bromo-cAMP in the pipette (e.g., 50 µM). Thus, the increase in the extent of cell shortening in ARK-1 cells is not likely to be due to differences in cAMP levels between wild-type and ARK-1 cells. However, it is possible that a component or components of the cAMP/protein kinase A pathway might be sensitized by chronic overexpression of ARK-1. Therefore, we next determined whether enhanced protein kinase A-mediated phosphorylation might contribute to differences between wild-type and ARK-1 myocytes. In these experiments, cells were dialyzed with 8-bromo-cAMP and superfused with buffer in the absence and presence of the protein kinase A inhibitor H89 (Bassani et al., 1995; Ferrier et al., 1998). The experimental protocol involved a test step from –40 to 0 mV delivered after a train of conditioning pulses as shown at the top of Fig. 5. Figure 5A shows representative examples of cell shortening and I_Ca-L recorded from wild-type and ARK-1 myocytes in the absence and presence of 5 µM H89. The contraction was larger in the ARK-1 cell than in the wild-type cell in the absence of H89. However, H89 abolished the difference between the wild-type and ARK-1 myocytes (Fig. 5A). Mean amplitudes of cell shortening were significantly larger in ARK-1 myocytes compared with wild-type cells, although magnitudes of I_Ca-L were similar in the two groups (Fig. 5B). H89 abolished the increase in contraction amplitude in ARK-1 myocytes, but it had no effect on amplitudes of I_Ca-L in the two groups (Fig. 5C). Furthermore, the time constants of inactivation for I_Ca-L were similar in wild-type and ARK-1 myocytes in the presence and absence of H89 (Table 2). Velocities of cell shortening and relengthening also were compared in wild-type and ARK-1 myocytes in the absence and presence of H89. The rates of contraction and relaxation also were similar in wild-type and ARK-1 myocytes in the absence and presence of H89 (Fig. 5, D and E).

View larger version (23K): [in this window] [in a new window]   Fig. 5. The protein kinase A inhibitor H89 abolished the difference in extent of cell shortening between wild-type and ARK-1 cells. Cells were dialyzed with patch pipettes filled with 50 µM 8-bromo-cAMP. The voltage-clamp protocol is shown at the top of the figure. A, representative recordings of contractions and ICa-L initiated by a test step from –40 to 0 mV. The extent of cell shortening was greater in the ARK-1 cell compared with the wild-type myocyte in the absence of H89. However, this difference was not observed in the presence of H89. B, contractions were significantly increased in ARK-1 cells compared with wild-type cells under control conditions. In contrast, amplitudes of ICa-L were similar in the two groups. C, when cells were superfused with 5 µM H89, the increase in contraction amplitude in ARK-1 myocytes was abolished. D and E, the mean velocities of shortening and relengthening did not differ between wild-type and ARK-1 myocytes in the absence or presence of H89. n = 3 to 10 myocytes per group. *, p < 0.05 denotes significantly different from wild type.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
We determined whether alterations in the cAMP-protein kinase A pathway in ARK-1 myocytes might alter contractile function in the absence of exogenous AR agonist. When high-resistance microelectrodes were used to minimize intracellular dialysis, contractions were larger in ARK-1 than in wild-type cells. However, when cells were dialyzed with pipettes filled with standard solution or a phosphodiesterase-sensitive analog of cAMP, the extent of cell shortening was similar in wild-type and ARK-1 myocytes. In contrast, when cells were dialyzed with solutions that contained a phosphodiesterase-resistant analog of cAMP, contractions were larger in ARK-1 than in wild-type cells. However, the amplitudes and rates of inactivation of I_Ca-L were similar in ARK-1 and wild-type cells in all experiments, and rates of contraction and relaxation did not differ between the two groups. Interestingly, the protein kinase A inhibitor H89 abolished differences between ARK-1 and wild-type myocytes. These results demonstrate that contractile function is augmented in ARK-1 myocytes when the intracellular milieu is not disrupted by dialysis with pipette solutions, or when phosphorylation pathways are preserved with phosphodiesterase-resistant cAMP. Furthermore, these results suggest that protein kinase A-mediated phosphorylation contributes to enhanced contractile function in myocytes that chronically overexpress ARK-1.

When intracellular dialysis was minimized with high-resistance microelectrodes, contractions were larger in ARK-1 myocytes than wild-type cells. However, when wild-type and ARK-1 cells were dialyzed with identical K⁺-based intracellular solutions, differences in contractions were abolished. This suggests that a component required to increase the extent of cell shortening in ARK-1 myocytes was removed from the cells by dialysis. Because dialysis with pipette solutions can disrupt the cAMP-protein kinase A pathway and affect contractile function in ventricular myocytes (Ferrier et al., 1998; Ferrier and Howlett, 2003), we hypothesized that removal of signaling components important in this pathway might reduce the extent of cell shortening in ARK-1 cells. Therefore, myocytes were dialyzed with pipette solutions supplemented with phosphodiesterase-resistant 8-bromo-cAMP. Under these conditions, contractions were larger in ARK-1 myocytes than in wild-type cells. Because increased cell shortening was observed in ARK-1 myocytes exposed to the same concentration of cAMP as wild-type cells, it is unlikely that increased intracellular cAMP was responsible for differences in contractions between ARK-1 and wild-type myocytes. However, these findings suggest that the cAMP-protein kinase A pathway is involved in the increase in contraction in ARK-1 cells.

Korzick et al. (1997) previously showed that the extent of cell shortening was similar in field-stimulated ARK-1 and wild-type cells, in the absence of exogenous catecholamines. This contrasts with our results, which showed that the extent of cell shortening was greater in undialyzed ARK-1 myocytes. There are several possible reasons for the differences between these two studies. Korzick et al. (1997) used field-stimulated myocytes where contractions were initiated by action potentials. Because action potential characteristics may differ between ARK-1 and wild-type cells, the stimuli used to initiate contraction may differ between the two cell types (Korzick et al., 1997). In contrast, our study used voltage-clamp steps of fixed duration, so identical stimuli were used to initiate contraction in wild-type and ARK-1 cells. In addition, Korzick et al. (1997) conducted their studies at 23°C, whereas our experiments were conducted at 37°C. It is well established that temperature can dramatically alter cardiac contractile function (Bers, 2001; Ferrier et al., 2003). Therefore, methodological differences may explain differences between our study and previous work (Korzick et al., 1997).

We found no difference in the extent of cell shortening between wild-type and ARK-1 myocytes dialyzed with phosphodiesterase-sensitive sodium-cAMP (Meyer and Miller, 1974). This suggests that a reduction in phosphodiesterase activity does not account for the enhanced contractions we observed in ARK-1 myocytes. If phosphodiesterase activity were reduced in ARK-1 myocytes, this would reduce degradation of sodium-cAMP and thereby promote accumulation of intracellular cAMP (Francis et al., 2001). Thus, we would have expected to see an increase in the extent of cell shortening in ARK-1 cells dialyzed with sodium-cAMP compared with wild-type cells. However, we found no differences in contractions between wild-type and ARK-1 myocytes dialyzed with sodium-cAMP. Consequently, a reduction in phosphodiesterase activity in ARK-1 cells is unlikely to be responsible for increased cell shortening.

One explanation for our results is that downstream components of the cAMP-protein kinase A pathway might become sensitized by a chronic increase in ARK-1 activity. We hypothesized that enhanced protein kinase A-mediated phosphorylation might account for differences in contraction between wild-type and ARK-1 myocytes. Indeed, the selective protein kinase A inhibitor H89 (Bassani et al., 1995; Ferrier et al., 1998) abolished the difference in cell shortening between ARK-1 and wild-type myocytes when cells were dialyzed with 8-bromo-cAMP. These results suggest that enhanced protein kinase A-mediated phosphorylation of components important in cardiac EC-coupling can explain the increase in cell shortening in ARK-1 myocytes dialyzed with 8-bromo-cAMP. This explanation also may account for the increase in contractions in undialyzed ARK-1 cells, although this was not addressed here.

The increase in extent of cell shortening in ARK-1 myocytes might be due to changes in the regulation of proteins involved in EC-coupling. L-type Ca²⁺ channels, phospholamban, ryanodine receptors, and protein phosphatase-1 are substrates for protein kinase A-mediated phosphorylation (Yoshida et al., 1992; Kapiloff, 2002; El-Armouche et al., 2003). Protein kinase A-mediated phosphorylation of L-type Ca²⁺ channels increases channel conductance (Striessnig, 1999). This could increase the size of I_Ca-L that triggers SR Ca²⁺ release (Bers, 2001). However, it is unlikely that changes in Ca²⁺ channel phosphorylation increase the extent of cell shortening in ARK-1 myocytes, because we found I_Ca-L amplitudes and inactivation rates were similar in ARK-1 and wild-type myocytes. Nevertheless, protein kinase A-mediated phosphorylation of phospholamban, ryanodine receptors, and/or protein phosphatase-1 might contribute to the increase in contractions in ARK-1 myocytes. Phosphorylation of phospholamban would relieve inhibition of SR Ca²⁺ ATPase, increase the rate of SR Ca²⁺ uptake, and thereby increase SR Ca²⁺ (Frank and Kranias, 2000; Frank et al., 2003). This would increase Ca²⁺ available for release and thus increase contraction in ARK-1 myocytes. However, we found that the rates of relaxation of contraction were similar in wild-type and ARK-1 myocytes. Thus, it is unlikely that increased SR Ca²⁺ uptake accounts for increased contraction in ARK-1 myocytes. Alternatively, phosphorylation of ryanodine receptors increases their open probability and increases SR Ca²⁺ release, which would produce a larger contraction (Marks, 2001; Marks et al., 2002). However, we found that wild-type and ARK-1 myocytes had similar rates of contraction, which makes it unlikely that an increased rate of SR Ca²⁺ release enhances contraction in ARK-1 cells. Finally, phosphorylation of protein phosphatase-1 would reduce dephosphorylation of targets such as phospholamban and/or ryanodine receptors (El-Armouche et al., 2003). Decreases in phosphorylation would amplify the cAMP-protein kinase A cascade and increase the extent of cell shortening in ARK-1 myocytes. Further experiments will be required to distinguish between these possibilities.

Chronic ARK-1 overexpression had little effect on the magnitude or inactivation rates of I_Ca-L. The magnitude of I_Ca-L was increased in ARK-1 compared with wild-type cells when cells were dialyzed with standard solutions, but only at very positive potentials. However, these potentials are much more positive than the plateau of the murine action potential (Fiset et al., 1997). Thus, this increase in I_Ca-L is unlikely to affect upon EC-coupling under physiological conditions. However, in most experiments we found that amplitudes of I_Ca-L were similar in ARK-1 and wild-type cells. This finding was unexpected, as protein kinase A-mediated phosphorylation of the Ca²⁺ channel increases the magnitude of I_Ca-L (Kapiloff, 2002). However, it previously has been shown that protein kinase A regulation of slow outward potassium current requires a macromolecular complex that includes protein kinase A, protein phosphatase-1, and a targeting protein called yotiao (Marx et al., 2002). Therefore, one explanation for our data is that protein kinase A-dependent regulation of the Ca²⁺ channel also requires a macromolecular complex, which is disrupted in ARK-1 myocytes.

Cell capacitance was increased in ARK-1 myocytes compared with wild-type cells, which suggests that cell membrane area is increased in ARK-1 cells. This increase in membrane area does not result from an increase in cell length, because cell length was similar in ARK-1 and wild-type myocytes. However, an increase in membrane surface area in ARK-1 cells could arise from an increase in cell width, cell volume, t-tubule area, and/or caveolae area. Whether chronic ARK-1 overexpression alters one or more of these factors to increase membrane area will require further investigation.

The results of the present investigation have important implications for studies of contractile function in myocytes isolated from models of heart failure. The sympathetic nervous system is activated in heart failure to compensate for diminished contractile function through activation of ARs (Keys and Koch, 2004). This chronic adrenergic stimulation results in up-regulation of ARK-1 in failure (Ungerer et al., 1993). Up-regulation of ARK-1 plays a key role in phosphorylation and desensitization of ARs, which further impairs contractile function in the failing heart (Hausdorff et al., 1990; Post et al., 1999). Our results show that chronic ARK-1 overexpression also may sensitize components of the cAMP-protein kinase A pathway. Thus, changes in contractile function observed in failing myocytes may reflect, at least in part, effects of chronic up-regulation of ARK-1 on components of the cAMP-protein kinase A pathway.

	Acknowledgements

 
We thank Peter Nicholl, Cindy Mapplebeck, and Steve Foster for excellent laboratory technical support and for assistance in preparation of illustrations. We also acknowledge helpful discussions of these results with the late Dr. Gregory R. Ferrier, who died on August 30, 2005.

	Footnotes

 
This work was supported in part by grants from The Canadian Institutes of Health Research and from the Heart and Stroke Foundation of Nova Scotia. S.A.G. was supported by a graduate studentship from The Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.106.107888.

ABBREVIATIONS: SR, sarcoplasmic reticulum; EC, excitation-contraction coupling; AR, -adrenergic receptor; GRK, G protein-coupled receptor; ARK-1, -adrenergic receptor kinase-1; I_Ca-L, L-type calcium current; H89, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride; DMSO, dimethyl sulfoxide; _f, fast time constant of inactivation; _s, slow time constant of inactivation.

Address correspondence to: Dr. Susan Ellen Howlett, Department of Pharmacology, Sir Charles Tupper Medical Bldg., 5850 College St., Halifax, NS B3H 1X5, Canada. E-mail: susan.howlett{at}dal.ca

	References

Top Abstract Materials and Methods Results Discussion References

 

Bassani RA, Mattiazzi A, and Bers DM (1995) CaMKII is responsible for activity-dependent acceleration of relaxation in rat ventricular myocytes. Am J Physiol 268: H703–H712.

Bers DM (2001) Excitation-Contraction Coupling and Cardiac Contractile Force, 2nd ed., Kluwer Academic Publishers, Boston, MA.

Canadian Council on Animal Care (1980) Guide to the Care and Use of Experimental Animals, Vol. 1, Council on Animal Care, Ottawa, ON, Canada.

Canadian Council on Animal Care (1984) Guide to the Care and Use of Experimental Animals, Vol. 2, Council on Animal Care, Ottawa, ON, Canada.

Chen EP, Bittner HB, Akhter SA, Koch WJ, and Davis RD (1998) Myocardial recovery after ischemia and reperfusion injury is significantly impaired in hearts with transgenic overexpression of beta-adrenergic receptor kinase. Circulation 98: II249–II253.

El-Armouche A, Rau T, Zolk O, Ditz D, Pamminger T, Zimmermann WH, Jackel E, Harding SE, Boknik P, Neumann J, et al. (2003) Evidence for protein phosphatase inhibitor-1 playing an amplifier role in beta-adrenergic signaling in cardiac myocytes. FASEB J 17: 437–439.[Abstract/Free Full Text]

Ferrier GR and Howlett SE (2003) Differential effects of phosphodiesterase-sensitive and -resistant analogs of cAMP on initiation of contraction in cardiac ventricular myocytes. J Pharmacol Exp Ther 306: 166–178.[Abstract/Free Full Text]

Ferrier GR, Smith RH, and Howlett SE (2003) Calcium sparks in mouse ventricular myocytes at physiological temperature. Am J Physiol 285: H1495–H1505.

Ferrier GR, Zhu J, Redondo IM, and Howlett SE (1998) Role of cAMP-dependent protein kinase A in activation of a voltage-sensitive release mechanism for cardiac contraction in guinea-pig myocytes. J Physiol (Lond) 513: 185–201.[Abstract/Free Full Text]

Fiset C, Clark RB, Larsen TS, and Giles WR (1997) A rapidly activating sustained K⁺ current modulates repolarization and excitation-contraction coupling in adult mouse ventricle. J Physiol (Lond) 504: 557–563.[CrossRef][Medline]

Francis SH, Turko IV, and Corbin JD (2001) Cyclic nucleotide phosphodiesterases: relating structure and function. Prog Nucleic Acid Res Mol Biol 65: 1–52.[Medline]

Frank K and Kranias EG (2000) Phospholamban and cardiac contractility. Ann Med 32: 572–578.[Medline]

Frank KF, Bolck B, Erdmann E, and Schwinger RH (2003) Sarcoplasmic reticulum Ca²⁺-ATPase modulates cardiac contraction and relaxation. Cardiovasc Res 57: 20–27.[Abstract/Free Full Text]

Hausdorff WP, Caron MG, and Lefkowitz RJ (1990) Turning off the signal: desensitization of beta-adrenergic receptor function. FASEB J 4: 2881–2889.[Abstract]

Kapiloff MS (2002) Contributions of protein kinase A anchoring proteins to compartmentation of cAMP signaling in the heart. Mol Pharmacol 62: 193–199.[Abstract/Free Full Text]

Keys JR and Koch WJ (2004) The adrenergic pathway and heart failure. Recent Prog Horm Res 59: 13–30.[Abstract/Free Full Text]

Knollmann BC, Knollmann-Ritschel BE, Weissman NJ, Jones LR, and Morad M (2000) Remodelling of ionic currents in hypertrophied and failing hearts of transgenic mice overexpressing calsequestrin. J Physiol (Lond) 525: 483–498.[Abstract/Free Full Text]

Koch WJ, Rockman HA, Samama P, Hamilton RA, Bond RA, Milano CA, and Lefkowitz RJ (1995) Cardiac function in mice overexpressing the beta-adrenergic receptor kinase or a ARK inhibitor. Science (Wash DC) 268: 1350–1353.[Abstract/Free Full Text]

Korzick DH, Xiao RP, Ziman BD, Koch WJ, Lefkowitz RJ, and Lakatta EG (1997) Transgenic manipulation of beta-adrenergic receptor kinase modifies cardiac myocyte contraction to norepinephrine. Am J Physiol 272: H590–H596.

Lohse MJ, Benovic JL, Caron MG, and Lefkowitz RJ (1990) Multiple pathways of rapid beta 2-adrenergic receptor desensitization. Delineation with specific inhibitors. J Biol Chem 265: 3202–3211.[Abstract/Free Full Text]

Marks AR (2001) Ryanodine receptors/calcium release channels in heart failure and sudden cardiac death. J Mol Cell Cardiol 33: 615–624.[CrossRef][Medline]

Marks AR, Marx SO, and Reiken S (2002) Regulation of ryanodine receptors via macromolecular complexes: a novel role for leucine/isoleucine zippers. Trends Cardiovasc Med 12: 166–170.[CrossRef][Medline]

Marx SO, Kurokawa J, Reiken S, Motoike H, D'Armiento J, Marks AR, and Kass RS (2002) Requirement of a macromolecular signaling complex for beta adrenergic receptor modulation of the KCNQ1-KCNE1 potassium channel. Science (Wash DC) 295: 496–499.[Abstract/Free Full Text]

Meyer RB Jr and Miller JP (1974) Analogs of cyclic AMP and cyclic GMP: general methods of synthesis and the relationship of structure to enzymic activity. Life Sci 14: 1019–1040.[CrossRef][Medline]

Olsson MC, Palmer BM, Stauffer BL, Leinwand LA, and Moore RL (2004) Morphological and functional alterations in ventricular myocytes from male transgenic mice with hypertrophic cardiomyopathy. Circ Res 94: 201–207.[Abstract/Free Full Text]

Penela P, Murga C, Ribas C, Tutor AS, Peregrin S, and Mayor F Jr (2006) Mechanisms of regulation of G protein-coupled receptor kinases (GRKs) and cardiovascular disease. Cardiovasc Res 69: 46–56.[CrossRef][Medline]

Pieske B, Maier LS, and Schmidt-Schweda S (2002) Sarcoplasmic reticulum Ca²⁺ load in human heart failure. Basic Res Cardiol 97: I63–I71.

Post SR, Hammond HK, and Insel PA (1999) Beta-adrenergic receptors and receptor signaling in heart failure. Annu Rev Pharmacol Toxicol 39: 343–360.[CrossRef][Medline]

Rockman HA, Choi DJ, Rahman NU, Akhter SA, Lefkowitz RJ, and Koch WJ (1996) Receptor-specific in vivo desensitization by the G protein-coupled receptor kinase-5 in transgenic mice. Proc Natl Acad Sci USA 93: 9954–9959.[Abstract/Free Full Text]

Rockman HA, Koch WJ, and Lefkowitz RJ (2002) Seven-transmembrane-spanning receptors and heart function. Nature (Lond) 415: 206–212.[CrossRef][Medline]

Striessnig J (1999) Pharmacology, structure and function of cardiac L-type Ca²⁺ channels. Cell Physiol Biochem 9: 242–269.[Medline]

Ungerer M, Bohm M, Elce JS, Erdmann E, and Lohse MJ (1993) Altered expression of beta-adrenergic receptor kinase and beta 1-adrenergic receptors in the failing human heart. Circulation 87: 454–463.[Abstract/Free Full Text]

Wallukat G (2002) The beta-adrenergic receptors. Herz 27: 683–690.[CrossRef][Medline]

Yatani A, Frank K, Sako H, Kranias EG, and Dorn GW 2nd (1999) Cardiac-specific overexpression of Gq alters excitation-contraction coupling in isolated cardiac myocytes. J Mol Cell Cardiol 31: 1327–1336.[CrossRef][Medline]

Yoshida A, Takahashi M, Imagawa T, Shigekawa M, Takisawa H, and Nakamura T (1992) Phosphorylation of ryanodine receptors in rat myocytes during beta-adrenergic stimulation. J Biochem (Tokyo) 111: 186–190.[Abstract/Free Full Text]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.106.107888v1 319/3/1307    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Mueller, E. E. Articles by Howlett, S. E. Search for Related Content PubMed PubMed Citation Articles by Mueller, E. E. Articles by Howlett, S. E.