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Abnormal Myocardial Contraction in α1A– and α1B–adrenoceptor double-knockout mice
Journal of Molecular and Cellular Cardiology 35 (2003) 1207–1216 www.elsevier.com/locate/yjmcc
Original Article
Abnormal Myocardial Contraction in a1A– and a1B–adrenoceptor double-knockout mice Diana T. McCloskey a,b, Lynne Turnbull a,b, Philip Swigart a,b, Timothy D. O’Connell a,b, Paul C. Simpson a,b, Anthony J. Baker a,b,* b
a Departments of Medicine and Radiology, Cardiovascular Research Institute, University of California, San Francisco, CA, USA Cardiology Division (111C), Veterans Affairs Medical Center, University of California, 4150 Clement Street, San Francisco, CA 94121, USA
Received 9 May 2003; accepted 16 June 2003
Abstract We used double-knockout mice (ABKO) lacking both predominant myocardial a1–adrenergic receptor (AR) subtypes (a1A and a1B) to determine if a1–ARs are required for normal myocardial contraction. Langendorff-perfused ABKO hearts had higher developed pressure than wild type (WT) hearts (123 ± 3 mmHg n = 22 vs. 103 ± 3 mmHg, n = 38, P < 0.001). Acutely inhibiting a1–ARs in WT hearts with prazosin did not increase pressure, suggesting that the increased pressure of ABKO hearts was mediated by long-term trophic effects on contraction rather than direct regulatory effects of a1–AR removal. Similar to perfused hearts, ABKO ventricular trabeculae had higher submaximal force at 2 mM extracellular [Ca2+] than WT (11.4 ± 1.7 vs. 6.9 ± 0.6 mN/mm2, n = 8, P < 0.05); however, the peaks of fura-2 Ca2+ transients were not different (0.79 ± 0.11 vs. 0.75 ± 0.16 µM, n = 10–12, P > 0.05), suggesting ABKO myocardium had increased myofilament Ca2+–sensitivity. This conclusion was supported by measuring the Ca2+–force relationship using tetanization. Increased myofilament Ca2+–sensitivity was not explained by intracellular pH, which did not differ between ABKO and WT (7.41 ± 0.01 vs. 7.39 ± 0.02, n = 4–6, P > 0.05; from BCECF fluorescence). However, ABKO displayed impaired troponin I phosphorylation, which may have played a role. In contrast to increased submaximal force, ABKO trabeculae had lower maximal force than WT at high extracellular [Ca2+] (29.6 ± 1.9 vs. 37.6 ± 1.4 mN/mm2, n = 7, P < 0.01). However, peak cytosolic [Ca2+] was not different (1.13 ± 0.15 vs. 1.19 ± 0.04 µM, n = 6–7, P > 0.05), suggesting ABKO myocardium had impaired myofilament function. Finally, ABKO myocardium had decreased responsiveness to b-AR stimulation. We conclude: a1–ARs are required for normal myocardial contraction; a1–ARs mediate long-term trophic effects on contraction; loss of a1–AR function causes some of the functional abnormalities that are also found in heart failure. © 2003 Elsevier Ltd. All rights reserved. Keywords: a1–adrenergic receptor; Langendorff; Trabeculae; Ca2+; pH; ABKO; Myofilament Ca2+–sensitivity; b-adrenergic receptor
1. Introduction Several studies suggest that a1–adrenergic receptors (ARs) play a trophic role in regulating both myocardial growth during development and the growth response to hypertropic stimuli [1,2]. In the myocardium, a1–ARs exist in two predominant subtypes, a1A and a1B. To investigate the trophic role of a1–AR subtypes, a1–AR subtype-knockout mice were generated [3,4]. Heart size was normal in singleknockout mice lacking the gene for the a1A–AR or the a1B–AR subtype [3,4]. Therefore, a double-knockout mouse
* Corresponding author. Tel.: +1-415-221-4810x4790; fax: +1-415-750-6950. E-mail address: [email protected] (A.J. Baker). © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0022-2828(03)00227-X
lacking both a1A–AR and a1B–AR subtypes (ABKO) was developed to investigate if a1–ARs are required for normal myocardial growth. Recent studies have shown that ABKO mice had smaller hearts suggesting a1–ARs are required for normal cardiac growth [5]. In addition to a trophic role, a1–ARs also have a functional role in regulating myocardial contraction. The goal of this study was to determine if a1–ARs are required for normal myocardial function. Previously, the baseline function of right ventricular (RV) trabeculae from a1A–AR-knockout mice was not different from wild type (WT) [6]; moreover, baseline blood pressure and heart rate of a1B–AR knockout were similar to WT [4]. Therefore, here we determined the effect of knockout of both a1–AR subtypes on myocardial function using ABKO mice.
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We found that ABKO myocardium displayed significant functional abnormalities that included increased Ca2+–sensitivity, decreased maximal force generation, and decreased responsiveness to b-AR stimulation. We also found that the ABKO phenotype could not be mimicked by inhibition of a1–ARs in WT using prazosin. These findings suggest that a1–ARs are required for normal myocardial contraction; secondly, the contraction abnormalities of ABKO myocardium are not solely due to acute regulatory signaling effects from loss of a1–ARs but also involve long-term effects on cardiac function.
2. Methods 2.1. ␣1–AR-knockout mouse Double-knockout mice lacking both a1A–ARs and a1B– ARs (ABKO) were generated by crossing single knockouts as recently described [5]. Control mice consisted of WT littermates. Male and female mice (25–30 g) were used. 2.2. Langendorff-perfused heart preparations As previously described [7], hearts were mounted on a Langendorff perfusion apparatus (Radnoti constant pressure non-recirculating system, Radnoti Glass Technology Inc, Monrovia, CA). Briefly, hearts were retrogradely perfused (at a perfusion pressure of 70 mmHg) with Krebs–Henseleit solution ([Ca2+] 2.5 mM, 37 °C, pH 7.4). Hearts were electrically paced at a rate of 6 Hz and left ventricular (LV) pressure was monitored by placing into the LV a fluid-filled balloon that was coupled to a pressure transducer. The balloon volume was adjusted to achieve a diastolic pressure of 10 mmHg. Potentially, differences in diastolic stiffness between ABKO and WT myocardium could result in setting of different diastolic sarcomere lengths. Therefore, we also used RV trabeculae where diastolic sarcomere length was set. 2.3. RV trabeculae As previously described [6] mouse trabeculae were isolated from the right ventricle, mounted in a muscle chamber, set to a diastolic sarcomere length of 2.1 µm, superfused with Krebs–Henseleit solution at 22 °C, electrically stimulated (0.5 Hz) and measurements were made of force, cytosolic [Ca2+] ([Ca2+]c), and intracellular pH (pHi). The width and thickness of trabeculae from WT mice (192 ± 23 and 101 ± 15 µm, respectively, n = 17) were not statistically different (P > 0.05) from those of ABKO mice (221 ± 32 and 105 ± 18 µm, respectively, n = 13). 2.4. Steady-state tetanization Ryanodine and cyclopiazonic acid (CPA) were used to enable steady-state tetanization as previously described [8,9]. After 30 min of exposure to ryanodine (1 µM) and CPA
(100 µM), tetanization was induced briefly (~4–8 s) by stimulating trabeculae at 10 Hz. The extracellular [Ca2+] was varied (1–20 mM) to obtain different steady-state levels of force and [Ca2+]c. 2.5. ␣1– and b-AR stimulation a1–ARs were stimulated with the non-subtype selective agonist phenylephrine [6,10] (10 µM) added to the Krebs– Henseleit solution containing 2 mM extracellular [Ca2+]. This agonist dose was sufficient to produce a maximal response. The b-antagonist timolol (10 µM) was present >15 min before and throughout phenylephrine stimulation. The b-AR-agonist isoproterenol was added in a dose-dependent manner to the superfusate. For these experiments low extracellular [Ca2+] was used (0.3 mM) to avoid saturation of the force response at the higher doses of isoproterenol. Forskolin (20 µM) was used to stimulate adenylyl cyclase directly. 2.6. Troponin I phosphorylation assay Troponin I (TnI) phosphorylation was detected using a phospho-specific antibody (cardiac phospho-TnI: Ser23/24) and a total TnI antibody (both were a generous gift from Dr. Qingyuan Ge, Cell Signaling Technology Inc). The phosphoantibody detects endogenous levels of cardiac TnI only when phosphorylated on both Ser23 and Ser24. Thus, the degree of phosphorylation measured will differ from estimates using incorporation of 32P, which detects total phosphorylation at either or both sites. Adult mouse myocytes were isolated, plated at 50,000 cells per 35–mm tissue culture dish, and cultured overnight [11]. Myocytes were treated in duplicate at 18 h, with isoproterenol (1 µM for 10 min) or forskolin (100 µM for 10 min) or dibutyryl cAMP (5 mM for 20 min). After treatment, each dish was harvested by scraping in 75 µl of 1.5 times Laemmli sample buffer [12] supplemented with protease inhibitor cocktail (#1836153 Roche), and phosphatase inhibitor cocktails (P–2850 and P–5726, Sigma). Samples placed in 1.5–ml Eppendorf tubes were boiled for 3 min and 20 µl per lane was run on an 18% SDS-PAGE gel (Bio Rad 345–0023). Gels were transferred to nitrocellulose membranes (Millipore HAHY00010) using wet transfer (25 mM Tris–HCl, 192 mM glycine, 0.1% SDS, and 20% methanol) at 250 mA for 1 h. Membranes were blocked for 1 h at room temperature with Tris-buffered saline (TBS) containing Tween-20 (0.1%) and 5% non-fat dried milk, then washed 3 times for 5 min with TBS/Tween-20 solution. Membranes were incubated overnight at 4 °C in TBS/Tween-20 solution containing BSA (5%) and primary antibody diluted 1:2000. Membranes were washed 3 times for 5 min with TBS/Tween-20 and incubated with anti-rabbit horseradish peroxidase diluted 1:3000 for 1 h at room temperature in TBS/Tween-20 with 5% non-fat dried milk. Membranes were washed 3 times for 5 min in TBS/Tween-20 and 2 times for 5 min in TBS. Proteins were visualized using ECL reagents (Amersham RPN2106) and exposed to X–ray
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Fig. 1. Knockout of a1A– and a1B–ARs caused increased pressure in Langendorff-perfused hearts: (A) representative examples of pressure transients for ABKO (bold) and WT hearts; (B) pooled LV developed pressure (LVDP: systolic–diastolic pressure) (mean ± S.E., n = 22 (ABKO) and 38 (WT); ** P < 0.01).
film and quantified using Molecular Dynamics ImageQuant Version 1.2. 2.7. Materials Timolol maleate, CPA, L–phenylephrine hydrochloride, and Forskolin were from Sigma-Aldrich (St. Louis, MO). Ryanodine was from Calbiochem-Novabiochem Corp. (San Diego, CA). Isoproterenol HCL (Isuprel) was from Abbott Labs (Chicago, IL). Fura-2 and BCECF AM were from Molecular Probes Inc., (Eugene, Oregon). 2.8. Statistical analysis Data are presented as mean ± S.E. Comparisons between WT and ABKO mice were made using Students t–test and one– and two-way repeated measures ANOVA, where values of P < 0.05 were considered statistically significant.
3. Results 3.1. Knockout of ␣1–ARs increased LV pressure We measured the LV pressure in the Langendorffperfused mouse heart at 2.5 mM extracellular [Ca2+]. In Fig. 1A representative superimposed LV pressure transients recorded from WT and ABKO hearts show greater pressure development in ABKO hearts. The pooled data for all experiments (Fig. 1B) show that the increased LV pressure developed in ABKO hearts was statistically significant (P< 0.01). There was no difference (P > 0.05) in LV pressure developed by males vs. females for ABKO (125 ± 4 mmHg, n = 11 vs. 122 ± 4 mmHg, n = 11) or WT (104 ± 4 mmHg, n = 22 vs. 102 ± 4 mmHg, n = 16). The time course of contraction and relaxation for all experiments was not significantly different for ABKO hearts (n = 22) vs. WT (n = 38) hearts: time to peak pressure was 61 ± 0.7 vs. 62 ± 0.6 ms, respectively; time to half relaxation was 43 ± 0.8 vs. 43 ± 0.7 ms, respectively (P > 0.05). The maximum rate of pressure rise (+dP/dt) was greater (P < 0.01) for ABKO than WT hearts (4093 ± 150 mmHg/s,
n = 22 vs. 3198 ± 105 mmHg/s, n = 38). Likewise, the maximum rate of pressure fall (–dP/dt) was greater (P < 0.01) for ABKO than WT hearts (–2956 ± 86 mmHg/s, n = 22 vs. –2246 ± 156 mmHg/s, n = 38). These differences largely reflected the higher pressures developed by ABKO hearts. To investigate if increased pressure of ABKO hearts was due to acute regulatory effects on contraction we treated WT hearts with the a1–AR-antagonist prazosin (5 µM). Relative to before prazosin, the developed pressure of WT hearts was not significantly changed by prazosin treatment (103 ± 3% initial, P = 0.84, n = 4), consistent with previous studies [13,14]. 3.2. Knockout of ␣1–ARs increased submaximal force of RV trabeculae To investigate the basis for increased contractility in ABKO hearts we measured force, Ca2+ transients, and intracellular pH in RV trabeculae set to the same diastolic sarcomere length (2.1 µm). Fig. 2A shows superimposed records of submaximal contractions of ABKO and WT trabeculae at 2 mM extracellular [Ca2+]. Similar to results from Langendorff-perfused hearts, there was increased contraction force of ABKO trabeculae compared to WT. Fig. 2C shows that for all experiments, increased submaximal force in ABKO myocardium was statistically significant (P < 0.05, n = 8 per group). Similar to findings in the perfused heart, the timing of contraction and relaxation was not significantly different for ABKO (n = 9) vs. WT trabeculae (n = 8): time-to-peak force was 184 ± 10 vs. 178 ± 7 ms, respectively; time to half relaxation was 134 ± 7 vs. 130 ± 5 ms, respectively (P > 0.05). In contrast to the higher forces of ABKO trabeculae, Ca2+ transients for ABKO and WT trabeculae were almost superimposable (Fig. 2B). Fig. 2D shows that for all experiments, there was no statistical difference between ABKO and WT trabeculae in peak cytosolic [Ca2+] ([Ca2+]c) (P > 0.05). Thus, increased submaximal force in ABKO trabeculae was not due to increased Ca2+ transients, suggesting increased myofilament Ca2+–sensitivity in ABKO hearts.
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Fig. 2. Knockout of a1–ARs caused increased submaximal force but unchanged systolic [Ca2+]c in trabeculae. Superimposed records of force and Ca2+ transients for ABKO and WT trabeculae (left panels). Pooled data from all experiments (right panels). The peak force at 2 mM extracellular Ca2+ was increased in the ABKO trabeculae (*P < 0.05) (top panels) but the systolic Ca2+ was not statistically different (lower panels). Data are mean ± S.E., n = 7–8 per group.
There was no statistically significant difference in the timing of the Ca2+ transient for ABKO (n = 7) vs. WT trabeculae (n = 8): time to peak was 47 ± 3 vs. 62 ± 6 ms, respectively; time to half relaxation was 243 ± 16 vs. 252 ± 35 ms, respectively (P > 0.05 for both). Similarly, there was no difference in the diastolic [Ca2+]c in the ABKO (103 ± 26 nM, n = 12) vs. the WT (120 ± 24 nM, n = 10, P > 0.05). 3.3. Knockout of ␣1–ARs decreased maximal force of RV trabeculae To determine if increased submaximal force in ABKO trabeculae was due to increased myofilament function we assessed myofilament function by raising the extracellular [Ca2+] up to 6 mM. Fig. 3A shows superimposed maximal twitch contractions of ABKO and WT trabeculae. In contrast to increased submaximal force in ABKO trabeculae, we found that maximum twitch force for ABKO trabeculae was 21% less than that for WT (P < 0.01) (Fig. 3C). However, Ca2+ transients at high extracellular Ca2+ were almost superimposable (Fig. 3B), and there was no statistical difference between ABKO and WT trabeculae in peak systolic [Ca2+]c (P > 0.05) (Fig. 3D). Thus, decreased maximal force in ABKO trabeculae was not due to decreased Ca2+ transients suggesting there was impaired myofilament function in ABKO hearts. Furthermore, this result also suggests that increased submaximal force in ABKO trabeculae was not caused by increased myofilament function. Similar to submaximal activation, with maximal activation there were no differences between ABKO and WT for
diastolic [Ca2+]c or in the timings of contraction, relaxation, and Ca2+ transients (not shown).
3.4. Effect of knockout of ␣1–ARs on the Ca2+–force relationship To more directly determine if ABKO hearts had increased myofilament Ca2+–sensitivity, we measured the steady-state Ca2+–force relationship by tetanizing trabeculae to produce steady-state levels of force and [Ca2+]c. Fig. 4A shows tetanic forces recorded from a single trabecula at increasing concentrations of extracellular [Ca2+] and the simultaneously recorded [Ca2+]c (Fig. 4B). By relating the maximum plateau levels of force and [Ca2+]c, the steady-state Ca2+–force relation was obtained (Fig. 4C). Fig. 4C shows that for contractions of ABKO trabeculae the Ca2+–force relation lay to the left of that for WT trabeculae. Data for WT trabeculae were fit to the Hill equation (solid line); however, data for ABKO trabeculae appeared more irregular and could not be fit. For each experiment the [Ca2+]c that resulted in half maximal activation (Ca50) was determined and was lower for ABKO trabeculae (0.8 ± 0.05 µM, n = 5) than for WT (1.1 ± 0.06 µM, n = 5, P < 0.01) consistent with increased myofilament Ca2+–sensitivity for ABKO trabeculae. The Ca50 for WT trabeculae in this study was similar to that previously reported for mouse myocardium [15]. Fig. 4D shows that the maximum tetanic force of ABKO trabeculae was lower than that of the WT.
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Fig. 3. Knockout of a1–ARs caused decreased maximal force but unchanged systolic [Ca2+]c in trabeculae. The peak force at up to 6 mM extracellular [Ca2+] was reduced in the ABKO trabeculae (**P < 0.01) (top panels) but the systolic [Ca2+]c was not statistically different (lower panels). Data are mean ± S.E., n = 7–8 per group.
Fig. 4. Steady-state Ca2+–force relationship in trabeculae. Force traces (A) and corresponding Ca2+ records (B) during tetanization at various extracellular [Ca2+] (indicated in mM next to each trace). (C) Ca2+–force relationships for ABKO (closed symbols) lay to the left of the WT. (open symbols; solid line shows fit to Hill equation). (D) Summary of maximum tetanic forces, ** P < 0.01. Data are mean ± S.E., n = 4–5 per group.
3.5. Knockout of ␣1–ARs did not affect intracellular pH For other species, inotropic responses to a1–AR stimulation involve changes of intracellular pH (pHi) [16]. Intracellular alkalinization due to stimulation of Na+/H+ exchange causes enhanced Ca2+ binding to the myofilaments [16].
Therefore, we measured pHi using the fluorescent pH indicator BCECF to determine if increased myofilament Ca2+– sensitivity of ABKO trabeculae involved intracellular alkalinization. We found that there was no difference in pHi between the ABKO (7.41 ± 0.01) and WT trabeculae (7.39 ± 0.02) at 2 mM extracellular [Ca2+] (P > 0.05, n = 4–6).
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Thus, increased myofilament Ca2+–sensitivity in ABKO myocardium must involve mechanisms other than increased pHi. 3.6. Knockout of ␣1–ARs decreased TnI phosphorylation Phosphorylation of TnI results in decreased myofilament Ca2+–sensitivity [17–19]. Thus, increased myofilament Ca2+–sensitivity in ABKO myocardium could result from decreased TnI phosphorylation. We assessed TnI phosphorylation for WT and ABKO myocytes using a phosphorylationspecific antibody. Western blot analysis showed that for quiescent myocytes TnI phosphorylation levels were undetectable for both WT and ABKO myocytes (not shown). However, with isoproterenol stimulation, the TnI phosphorylation level was significantly less for ABKO compared to WT myocytes (Fig. 5A). There was no difference in total TnI levels between ABKO and WT. The pooled data (Fig. 5B) show that after isoproterenol stimulation, the maximum TnI phosphorylation level for ABKO was 73 ± 1% of that found for WT (n = 3, P < 0.01). Moreover, Fig. 5 shows that TnI phosphorylation levels were also lower for ABKO myocytes after stimulation using the cAMP agonists dibutyryl cAMP
(72 ± 7% of WT response, n = 2) or forskolin (47% of WT response). These data suggest that ABKO hearts had impaired TnI phosphorylation when stimulated by b-ARs or by cAMP agonists. Impaired TnI phosphorylation in ABKO myocardium could contribute to increased myofilament Ca2+–sensitivity. 3.7. Effect of acute ␣1 –AR stimulation Previous studies by us and others have demonstrated that for in vitro preparations of mouse myocardium, nonsubtype-specific a1–AR stimulation causes a sustained negative inotropic response [6,20,21]. The contractile response of a WT mouse RV trabecula to phenylephrine (10 µM) in the presence of timolol (10 µM) is shown in Fig. 6. As we previously reported, approximately 10 min after adding phenylephrine, force fell markedly to 52 ± 4.6% initial (P < 0.001 compared to control, n = 8); force subsequently recovered slightly to 67 ± 4.4% initial (P < 0.001 compared to control, n = 8) [6]. Fig. 6 also shows that ABKO trabeculae, lacking the two predominant a1–AR subtypes, had no response to phenylephrine [7]. 3.8. Effect of b-adrenergic stimulation To investigate if deletion of a1–ARs altered b-AR function we monitored the responses of ABKO trabeculae that were stimulated with isoproterenol at 0.3 mM extracellular [Ca2+]. Fig. 7 shows that following isoproterenol stimulation the rise of force for ABKO trabeculae was blunted compared to WT (P < 0.01, two-way repeated measures ANOVA). Thus, deletion of a1–ARs caused decreased b-AR responsiveness. To investigate receptor-independent effects, we stimulated adenylyl cyclase directly using 20 µM forskolin and 0.3 mM extracellular [Ca2+]. For both WT and ABKO trabeculae, force was appreciably higher with forskolin compared to with isoproterenol (P < 0.01). However, in contrast to the
Fig. 5. Knockout of a1–ARs caused decreased TnI phosphorylation: (A) Western blots of total– and phospho-TnI (p–TnI) for WT and ABKO myocytes after stimulation with isoproterenol and dibutyryl cAMP (db cAMP) (see Section 2); (B) summary of TnI phosphorylation in response to isoproterenol and the cAMP agonists db cAMP (circles) and forskolin (triangle) (intensity of TnI-P bands were normalized to TnI intensity and scaled relative to the WT response).
Fig. 6. Effect of a1–AR stimulation of ABKO and WT trabeculae. Slow time-base recording of peak twitch force of ABKO and WT mouse RV trabeculae. Arrow indicates addition of the a1–AR-agonist phenylephrine in the presence of the b-AR-antagonist timolol.
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Fig. 7. Summary of the effect of b-AR stimulation of trabeculae on contraction force (mean ± S.E., n = 6 WT and n = 5 ABKO). The response to isoproterenol was significantly lower for ABKO myocardium (P < 0.01, two-way repeated measures ANOVA). The response to forskolin is shown to the right of the x–axis (and where the y–axis has a break and change of scale).
blunted response of ABKO trabeculae to isoproterenol, in the presence of forskolin there was no difference in force developed by ABKO and WT trabeculae (30.6 ± 3.2 vs. 34.3 ± 2.3 mN/mm2, n = 5–6, P > 0.05). Thus, decreased b-AR responsiveness of ABKO myocardium was not caused by impaired adenylyl cyclase function. This suggests decreased b-AR responsiveness involved impaired receptormediated cyclase stimulation. 3.9. Effect of pacing frequency Some studies suggest that in models of disease, contractility may appear normal at low pacing rates but that impaired function may become apparent at higher pacing rates [22]. Therefore, for WT and ABKO trabeculae we determined the effect of increasing pacing frequency on force and systolic [Ca2+]c over the frequency range 0.25–2 Hz at submaximal extracellular [Ca2+] (2 mM). Similar to results from previous studies [15,23], Fig. 8 shows that for mouse myocardium the relationship between force and frequency was negative below 0.5 Hz pacing but positive above 0.5 Hz. Furthermore, Fig. 8 shows increased pacing rate was not associated with impaired function of ABKO trabeculae compared to WT. In contrast, ABKO trabeculae developed higher forces at all pacing rates such that there was a significant difference between ABKO and WT myocardium in response to pacing (P < 0.05, two-way repeated measures ANOVA). Although the peak force of ABKO trabeculae tended to be higher than for WT trabeculae, peak systolic [Ca2+]c was not statistically different. Relating systolic force to systolic [Ca2+]c over all pacing frequencies (Fig. 8C) shows that the force–Ca2+ relationship for ABKO trabeculae lay above that for WT, consistent with increased myofilament Ca2+–sensitivity in ABKO trabeculae as was suggested by the data in Figs. 2 and 4.
Fig. 8. Summary of the effect of stimulation frequency on: (A) force, (B) [Ca2+]c, and (C) relationship between force and [Ca2+]c at all stimulation frequencies (mean ± S.E., n = 7–8 per group).
4. Discussion The major finding of this study was that knockout of both a1–AR subtypes in the mouse (ABKO) caused marked myocardial contraction abnormalities involving increased Ca2+– sensitivity, decreased maximal contraction, and decreased
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responsiveness to b-AR stimulation. Inhibition of a1–ARs using prazosin in WT hearts did not mimic the ABKO phenotype suggesting that contraction abnormalities of ABKO hearts were not due to acute regulatory effects of altered a1–AR signaling. Taken together our results suggest that a1–ARs are required for development of normal myocardial function and that a1–ARs mediate long-term effects on contractile function. Consistent with our finding that loss of a1–AR function is detrimental in the long term, a massive clinical trial (ALLHAT) of an a1–AR-antagonist was stopped prematurely because of an increase in heart failure in the a1–AR-antagonisttreated group [24]. Furthermore, our recent study found 50% mortality due to heart failure in ABKO mice after the stress of transverse aortic constriction (TAC) vs. 100% survival for WT mice after TAC [5]. Thus, impaired a1–AR function may be a contributory factor in the development of heart failure. Interestingly, similar to the contraction abnormalities we observed in the present study, previous studies of human and animal failing hearts also found increased myofilament Ca2+–sensitivity [25–27], decreased maximal Ca2+–activated force [28–30], and decreased b-AR responsiveness [31–33]. Thus, loss of a1–ARs is detrimental to the heart and may produce some (but not all) of the pathophysiological changes that are involved in the development of heart failure. A striking result in this study was that contractile function was actually higher for ABKO myocardium compared to WT at a physiological level of external [Ca2+]. This was so for both isovolumic Langendorff-perfused hearts, and for in vitro RV trabeculae. Previous studies found that the heart size of ABKO male mice was smaller than WT males; however, heart size for ABKO females was normal [5]. In the present study, we found that ABKO myocardium for both male and female mice had increased submaximal contractions. Therefore, hypercontractility of ABKO myocardium was not related to smaller heart size found only in males. To investigate the mechanism for increased myocardial contraction in ABKO hearts we studied RV trabeculae. Several results suggest that increased submaximal contractions of ABKO myocardium were caused by increased myofilament Ca2+–sensitivity relative to WT. First, increased submaximal contractions of ABKO trabeculae were not associated with increased Ca2+ transients. Second, the steady-state Ca2+–force relation for ABKO trabeculae lay to the left of that for the WT. Finally, for ABKO trabeculae the relationship between peak [Ca2+]c and peak force obtained using a range of pacing rates lay above that for the WT. One mechanism by which a1–AR signaling is known to influence Ca2+– sensitivity is via effects on intracellular pH. However, we found that increased Ca2+–sensitivity of ABKO myocardium was not associated with increased pHi. Interestingly, we found that stimulation of the Gs-signaling pathway using isoproterenol caused less TnI phosphorylation in ABKO myocytes compared to WT. Thus, for contracting ABKO myocardium, which will likely have some basal Gs tone,
impaired TnI phosphorylation could contribute to increased myofilament Ca2+–sensitivity. We recently reported that increased acute signaling by a1–ARs resulted in decreased Ca2+–sensitivity for mouse myocardium [6]. By extension, increased Ca2+–sensitivity of ABKO myocardium could arise from elimination of basal a1–AR signaling. However, the failure of prazosin to mimic the ABKO phenotype in WT myocardium suggests that the ABKO phenotype is not simply due to acute effects of inhibiting a1–AR signaling. Our results suggest that knockout of a1–ARs in the heart has long-term effects on cardiac function. In contrast to increased submaximal contraction of ABKO myocardium, the maximal contraction force of ABKO myocardium was reduced at high extracellular [Ca2+]. Reduced maximal twitch force was not due to a decreased Ca2+ transient, which suggested decreased myofilament function. Decreased maximal force of ABKO myocardium was also evident by decreased maximal tetanic force. The heart normally operates under conditions of submaximal activation, therefore, the functional significance of decreased maximal force for ABKO myocardium is unclear. Possibly ABKO hearts may respond worse than WT to conditions that require use of their contractile reserve. Indeed, the response of ABKO myocardium to b-AR stimulation was decreased. Furthermore, exercise capacity is decreased and mortality in response to TAC is increased in ABKO mice [5]. Despite differences in contraction between ABKO and WT myocardium with both submaximal and maximal contractions, Ca2+ transients of ABKO myocardium were not different from WT. This suggests that a1–ARs are not required for normal Ca2+ handling. The interaction between a1– and b-ARs in myocardium has been unclear. There has been a suggestion of an antagonistic interplay between the a1– and b-ARs, where norepinephrine exerts an inhibitory effect through a1–ARs on b1– AR-stimulated Ca2+ current in rat ventricular myocytes [34,35]. The ABKO model has provided an opportunity to assess if b1–AR function is altered by knockout of a1–AR function. We found that the response of ABKO myocardium to b-AR stimulation was blunted in ABKO myocardium compared to WT. However, adenylyl cyclase stimulation by forskolin was not impaired; therefore, decreased b-AR responsiveness in ABKO myocardium involved impaired receptor-mediated adenylyl cyclase stimulation (for example due to decreased agonist binding or impaired receptor coupling to the cyclase). Thus, loss of both a1–AR subtypes in the heart did not cause upregulation of the function of b-ARs, instead there was a reduction in the b-AR response. Decreased b-AR responsiveness could also limit the ability of ABKO myocardium to respond to conditions of increased stress, such as exercise or aortic banding (see above). Previous studies suggested that growth and contraction were normal for myocardium lacking only one a1–AR subtype. This suggests that a1–AR subtypes may mediate redundant functions. Consistent with this, we recently demon-
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strated that for mouse myocardium when a1–AR subtypes were stimulated separately, the functional response to a1A–AR stimulation was very similar to the a1B–AR response [6]. However, in the present study, contraction was abnormal when both a1–AR subtypes were knocked out. This suggests that at least one a1–AR subtype has to be present for normal cardiac growth and function. From this study we conclude that: (a) a1–ARs are required for normal contractile function; (b) loss of a1–AR function induces long-term trophic effects on contraction; and (c) loss of a1–AR function results in a pathophysiological phenotype with some (but not all) features of the failing heart.
Acknowledgements This work was supported by NIH grants HL 56257 and P01 HL68738 – project 3 (A.J.B.), HL 54890 and HL 31113 (P.C.S.); and a postdoctoral fellowship HL10422 (D.T.M.); and by a Grant-in-Aid from the American Heart Association, Western States Affiliate (A.J.B.). A.J. Baker is an Established Investigator of the American Heart Association. Some of these data were published in abstract form: Biophys J 2002;82:190a.
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in ischemic versus idiopathic dilated cardiomyopathy. Circulation 1991;84:1024–39. [33] Feldman MD, Copelas L, Gwathmey JK, Phillips P, Warren SE, Schoen FJ, et al. Deficient production of cyclic AMP: pharmacologic evidence of an important cause of contractile dysfunction in patients with end-stage heart failure. Circulation 1987;75:331–9. [34] Boutjdir M, Restivo M, Wei Y, el-Sherif N. a1– and b-adrenergic interactions on L–type calcium current in cardiac myocytes. Pflug Arch Eur J Physiol 1992;421:397–9. [35] Chen L, Elsherif N, Boutjdir M. a1–adrenergic activation inhibits b-adrenergic-stimulated unitary Ca2+ currents in cardiac ventricular myocytes. Circ Res 1996;79:184–93.
Original Article
Abnormal Myocardial Contraction in a1A– and a1B–adrenoceptor double-knockout mice Diana T. McCloskey a,b, Lynne Turnbull a,b, Philip Swigart a,b, Timothy D. O’Connell a,b, Paul C. Simpson a,b, Anthony J. Baker a,b,* b
a Departments of Medicine and Radiology, Cardiovascular Research Institute, University of California, San Francisco, CA, USA Cardiology Division (111C), Veterans Affairs Medical Center, University of California, 4150 Clement Street, San Francisco, CA 94121, USA
Received 9 May 2003; accepted 16 June 2003
Abstract We used double-knockout mice (ABKO) lacking both predominant myocardial a1–adrenergic receptor (AR) subtypes (a1A and a1B) to determine if a1–ARs are required for normal myocardial contraction. Langendorff-perfused ABKO hearts had higher developed pressure than wild type (WT) hearts (123 ± 3 mmHg n = 22 vs. 103 ± 3 mmHg, n = 38, P < 0.001). Acutely inhibiting a1–ARs in WT hearts with prazosin did not increase pressure, suggesting that the increased pressure of ABKO hearts was mediated by long-term trophic effects on contraction rather than direct regulatory effects of a1–AR removal. Similar to perfused hearts, ABKO ventricular trabeculae had higher submaximal force at 2 mM extracellular [Ca2+] than WT (11.4 ± 1.7 vs. 6.9 ± 0.6 mN/mm2, n = 8, P < 0.05); however, the peaks of fura-2 Ca2+ transients were not different (0.79 ± 0.11 vs. 0.75 ± 0.16 µM, n = 10–12, P > 0.05), suggesting ABKO myocardium had increased myofilament Ca2+–sensitivity. This conclusion was supported by measuring the Ca2+–force relationship using tetanization. Increased myofilament Ca2+–sensitivity was not explained by intracellular pH, which did not differ between ABKO and WT (7.41 ± 0.01 vs. 7.39 ± 0.02, n = 4–6, P > 0.05; from BCECF fluorescence). However, ABKO displayed impaired troponin I phosphorylation, which may have played a role. In contrast to increased submaximal force, ABKO trabeculae had lower maximal force than WT at high extracellular [Ca2+] (29.6 ± 1.9 vs. 37.6 ± 1.4 mN/mm2, n = 7, P < 0.01). However, peak cytosolic [Ca2+] was not different (1.13 ± 0.15 vs. 1.19 ± 0.04 µM, n = 6–7, P > 0.05), suggesting ABKO myocardium had impaired myofilament function. Finally, ABKO myocardium had decreased responsiveness to b-AR stimulation. We conclude: a1–ARs are required for normal myocardial contraction; a1–ARs mediate long-term trophic effects on contraction; loss of a1–AR function causes some of the functional abnormalities that are also found in heart failure. © 2003 Elsevier Ltd. All rights reserved. Keywords: a1–adrenergic receptor; Langendorff; Trabeculae; Ca2+; pH; ABKO; Myofilament Ca2+–sensitivity; b-adrenergic receptor
1. Introduction Several studies suggest that a1–adrenergic receptors (ARs) play a trophic role in regulating both myocardial growth during development and the growth response to hypertropic stimuli [1,2]. In the myocardium, a1–ARs exist in two predominant subtypes, a1A and a1B. To investigate the trophic role of a1–AR subtypes, a1–AR subtype-knockout mice were generated [3,4]. Heart size was normal in singleknockout mice lacking the gene for the a1A–AR or the a1B–AR subtype [3,4]. Therefore, a double-knockout mouse
* Corresponding author. Tel.: +1-415-221-4810x4790; fax: +1-415-750-6950. E-mail address: [email protected] (A.J. Baker). © 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0022-2828(03)00227-X
lacking both a1A–AR and a1B–AR subtypes (ABKO) was developed to investigate if a1–ARs are required for normal myocardial growth. Recent studies have shown that ABKO mice had smaller hearts suggesting a1–ARs are required for normal cardiac growth [5]. In addition to a trophic role, a1–ARs also have a functional role in regulating myocardial contraction. The goal of this study was to determine if a1–ARs are required for normal myocardial function. Previously, the baseline function of right ventricular (RV) trabeculae from a1A–AR-knockout mice was not different from wild type (WT) [6]; moreover, baseline blood pressure and heart rate of a1B–AR knockout were similar to WT [4]. Therefore, here we determined the effect of knockout of both a1–AR subtypes on myocardial function using ABKO mice.
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We found that ABKO myocardium displayed significant functional abnormalities that included increased Ca2+–sensitivity, decreased maximal force generation, and decreased responsiveness to b-AR stimulation. We also found that the ABKO phenotype could not be mimicked by inhibition of a1–ARs in WT using prazosin. These findings suggest that a1–ARs are required for normal myocardial contraction; secondly, the contraction abnormalities of ABKO myocardium are not solely due to acute regulatory signaling effects from loss of a1–ARs but also involve long-term effects on cardiac function.
2. Methods 2.1. ␣1–AR-knockout mouse Double-knockout mice lacking both a1A–ARs and a1B– ARs (ABKO) were generated by crossing single knockouts as recently described [5]. Control mice consisted of WT littermates. Male and female mice (25–30 g) were used. 2.2. Langendorff-perfused heart preparations As previously described [7], hearts were mounted on a Langendorff perfusion apparatus (Radnoti constant pressure non-recirculating system, Radnoti Glass Technology Inc, Monrovia, CA). Briefly, hearts were retrogradely perfused (at a perfusion pressure of 70 mmHg) with Krebs–Henseleit solution ([Ca2+] 2.5 mM, 37 °C, pH 7.4). Hearts were electrically paced at a rate of 6 Hz and left ventricular (LV) pressure was monitored by placing into the LV a fluid-filled balloon that was coupled to a pressure transducer. The balloon volume was adjusted to achieve a diastolic pressure of 10 mmHg. Potentially, differences in diastolic stiffness between ABKO and WT myocardium could result in setting of different diastolic sarcomere lengths. Therefore, we also used RV trabeculae where diastolic sarcomere length was set. 2.3. RV trabeculae As previously described [6] mouse trabeculae were isolated from the right ventricle, mounted in a muscle chamber, set to a diastolic sarcomere length of 2.1 µm, superfused with Krebs–Henseleit solution at 22 °C, electrically stimulated (0.5 Hz) and measurements were made of force, cytosolic [Ca2+] ([Ca2+]c), and intracellular pH (pHi). The width and thickness of trabeculae from WT mice (192 ± 23 and 101 ± 15 µm, respectively, n = 17) were not statistically different (P > 0.05) from those of ABKO mice (221 ± 32 and 105 ± 18 µm, respectively, n = 13). 2.4. Steady-state tetanization Ryanodine and cyclopiazonic acid (CPA) were used to enable steady-state tetanization as previously described [8,9]. After 30 min of exposure to ryanodine (1 µM) and CPA
(100 µM), tetanization was induced briefly (~4–8 s) by stimulating trabeculae at 10 Hz. The extracellular [Ca2+] was varied (1–20 mM) to obtain different steady-state levels of force and [Ca2+]c. 2.5. ␣1– and b-AR stimulation a1–ARs were stimulated with the non-subtype selective agonist phenylephrine [6,10] (10 µM) added to the Krebs– Henseleit solution containing 2 mM extracellular [Ca2+]. This agonist dose was sufficient to produce a maximal response. The b-antagonist timolol (10 µM) was present >15 min before and throughout phenylephrine stimulation. The b-AR-agonist isoproterenol was added in a dose-dependent manner to the superfusate. For these experiments low extracellular [Ca2+] was used (0.3 mM) to avoid saturation of the force response at the higher doses of isoproterenol. Forskolin (20 µM) was used to stimulate adenylyl cyclase directly. 2.6. Troponin I phosphorylation assay Troponin I (TnI) phosphorylation was detected using a phospho-specific antibody (cardiac phospho-TnI: Ser23/24) and a total TnI antibody (both were a generous gift from Dr. Qingyuan Ge, Cell Signaling Technology Inc). The phosphoantibody detects endogenous levels of cardiac TnI only when phosphorylated on both Ser23 and Ser24. Thus, the degree of phosphorylation measured will differ from estimates using incorporation of 32P, which detects total phosphorylation at either or both sites. Adult mouse myocytes were isolated, plated at 50,000 cells per 35–mm tissue culture dish, and cultured overnight [11]. Myocytes were treated in duplicate at 18 h, with isoproterenol (1 µM for 10 min) or forskolin (100 µM for 10 min) or dibutyryl cAMP (5 mM for 20 min). After treatment, each dish was harvested by scraping in 75 µl of 1.5 times Laemmli sample buffer [12] supplemented with protease inhibitor cocktail (#1836153 Roche), and phosphatase inhibitor cocktails (P–2850 and P–5726, Sigma). Samples placed in 1.5–ml Eppendorf tubes were boiled for 3 min and 20 µl per lane was run on an 18% SDS-PAGE gel (Bio Rad 345–0023). Gels were transferred to nitrocellulose membranes (Millipore HAHY00010) using wet transfer (25 mM Tris–HCl, 192 mM glycine, 0.1% SDS, and 20% methanol) at 250 mA for 1 h. Membranes were blocked for 1 h at room temperature with Tris-buffered saline (TBS) containing Tween-20 (0.1%) and 5% non-fat dried milk, then washed 3 times for 5 min with TBS/Tween-20 solution. Membranes were incubated overnight at 4 °C in TBS/Tween-20 solution containing BSA (5%) and primary antibody diluted 1:2000. Membranes were washed 3 times for 5 min with TBS/Tween-20 and incubated with anti-rabbit horseradish peroxidase diluted 1:3000 for 1 h at room temperature in TBS/Tween-20 with 5% non-fat dried milk. Membranes were washed 3 times for 5 min in TBS/Tween-20 and 2 times for 5 min in TBS. Proteins were visualized using ECL reagents (Amersham RPN2106) and exposed to X–ray
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Fig. 1. Knockout of a1A– and a1B–ARs caused increased pressure in Langendorff-perfused hearts: (A) representative examples of pressure transients for ABKO (bold) and WT hearts; (B) pooled LV developed pressure (LVDP: systolic–diastolic pressure) (mean ± S.E., n = 22 (ABKO) and 38 (WT); ** P < 0.01).
film and quantified using Molecular Dynamics ImageQuant Version 1.2. 2.7. Materials Timolol maleate, CPA, L–phenylephrine hydrochloride, and Forskolin were from Sigma-Aldrich (St. Louis, MO). Ryanodine was from Calbiochem-Novabiochem Corp. (San Diego, CA). Isoproterenol HCL (Isuprel) was from Abbott Labs (Chicago, IL). Fura-2 and BCECF AM were from Molecular Probes Inc., (Eugene, Oregon). 2.8. Statistical analysis Data are presented as mean ± S.E. Comparisons between WT and ABKO mice were made using Students t–test and one– and two-way repeated measures ANOVA, where values of P < 0.05 were considered statistically significant.
3. Results 3.1. Knockout of ␣1–ARs increased LV pressure We measured the LV pressure in the Langendorffperfused mouse heart at 2.5 mM extracellular [Ca2+]. In Fig. 1A representative superimposed LV pressure transients recorded from WT and ABKO hearts show greater pressure development in ABKO hearts. The pooled data for all experiments (Fig. 1B) show that the increased LV pressure developed in ABKO hearts was statistically significant (P< 0.01). There was no difference (P > 0.05) in LV pressure developed by males vs. females for ABKO (125 ± 4 mmHg, n = 11 vs. 122 ± 4 mmHg, n = 11) or WT (104 ± 4 mmHg, n = 22 vs. 102 ± 4 mmHg, n = 16). The time course of contraction and relaxation for all experiments was not significantly different for ABKO hearts (n = 22) vs. WT (n = 38) hearts: time to peak pressure was 61 ± 0.7 vs. 62 ± 0.6 ms, respectively; time to half relaxation was 43 ± 0.8 vs. 43 ± 0.7 ms, respectively (P > 0.05). The maximum rate of pressure rise (+dP/dt) was greater (P < 0.01) for ABKO than WT hearts (4093 ± 150 mmHg/s,
n = 22 vs. 3198 ± 105 mmHg/s, n = 38). Likewise, the maximum rate of pressure fall (–dP/dt) was greater (P < 0.01) for ABKO than WT hearts (–2956 ± 86 mmHg/s, n = 22 vs. –2246 ± 156 mmHg/s, n = 38). These differences largely reflected the higher pressures developed by ABKO hearts. To investigate if increased pressure of ABKO hearts was due to acute regulatory effects on contraction we treated WT hearts with the a1–AR-antagonist prazosin (5 µM). Relative to before prazosin, the developed pressure of WT hearts was not significantly changed by prazosin treatment (103 ± 3% initial, P = 0.84, n = 4), consistent with previous studies [13,14]. 3.2. Knockout of ␣1–ARs increased submaximal force of RV trabeculae To investigate the basis for increased contractility in ABKO hearts we measured force, Ca2+ transients, and intracellular pH in RV trabeculae set to the same diastolic sarcomere length (2.1 µm). Fig. 2A shows superimposed records of submaximal contractions of ABKO and WT trabeculae at 2 mM extracellular [Ca2+]. Similar to results from Langendorff-perfused hearts, there was increased contraction force of ABKO trabeculae compared to WT. Fig. 2C shows that for all experiments, increased submaximal force in ABKO myocardium was statistically significant (P < 0.05, n = 8 per group). Similar to findings in the perfused heart, the timing of contraction and relaxation was not significantly different for ABKO (n = 9) vs. WT trabeculae (n = 8): time-to-peak force was 184 ± 10 vs. 178 ± 7 ms, respectively; time to half relaxation was 134 ± 7 vs. 130 ± 5 ms, respectively (P > 0.05). In contrast to the higher forces of ABKO trabeculae, Ca2+ transients for ABKO and WT trabeculae were almost superimposable (Fig. 2B). Fig. 2D shows that for all experiments, there was no statistical difference between ABKO and WT trabeculae in peak cytosolic [Ca2+] ([Ca2+]c) (P > 0.05). Thus, increased submaximal force in ABKO trabeculae was not due to increased Ca2+ transients, suggesting increased myofilament Ca2+–sensitivity in ABKO hearts.
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Fig. 2. Knockout of a1–ARs caused increased submaximal force but unchanged systolic [Ca2+]c in trabeculae. Superimposed records of force and Ca2+ transients for ABKO and WT trabeculae (left panels). Pooled data from all experiments (right panels). The peak force at 2 mM extracellular Ca2+ was increased in the ABKO trabeculae (*P < 0.05) (top panels) but the systolic Ca2+ was not statistically different (lower panels). Data are mean ± S.E., n = 7–8 per group.
There was no statistically significant difference in the timing of the Ca2+ transient for ABKO (n = 7) vs. WT trabeculae (n = 8): time to peak was 47 ± 3 vs. 62 ± 6 ms, respectively; time to half relaxation was 243 ± 16 vs. 252 ± 35 ms, respectively (P > 0.05 for both). Similarly, there was no difference in the diastolic [Ca2+]c in the ABKO (103 ± 26 nM, n = 12) vs. the WT (120 ± 24 nM, n = 10, P > 0.05). 3.3. Knockout of ␣1–ARs decreased maximal force of RV trabeculae To determine if increased submaximal force in ABKO trabeculae was due to increased myofilament function we assessed myofilament function by raising the extracellular [Ca2+] up to 6 mM. Fig. 3A shows superimposed maximal twitch contractions of ABKO and WT trabeculae. In contrast to increased submaximal force in ABKO trabeculae, we found that maximum twitch force for ABKO trabeculae was 21% less than that for WT (P < 0.01) (Fig. 3C). However, Ca2+ transients at high extracellular Ca2+ were almost superimposable (Fig. 3B), and there was no statistical difference between ABKO and WT trabeculae in peak systolic [Ca2+]c (P > 0.05) (Fig. 3D). Thus, decreased maximal force in ABKO trabeculae was not due to decreased Ca2+ transients suggesting there was impaired myofilament function in ABKO hearts. Furthermore, this result also suggests that increased submaximal force in ABKO trabeculae was not caused by increased myofilament function. Similar to submaximal activation, with maximal activation there were no differences between ABKO and WT for
diastolic [Ca2+]c or in the timings of contraction, relaxation, and Ca2+ transients (not shown).
3.4. Effect of knockout of ␣1–ARs on the Ca2+–force relationship To more directly determine if ABKO hearts had increased myofilament Ca2+–sensitivity, we measured the steady-state Ca2+–force relationship by tetanizing trabeculae to produce steady-state levels of force and [Ca2+]c. Fig. 4A shows tetanic forces recorded from a single trabecula at increasing concentrations of extracellular [Ca2+] and the simultaneously recorded [Ca2+]c (Fig. 4B). By relating the maximum plateau levels of force and [Ca2+]c, the steady-state Ca2+–force relation was obtained (Fig. 4C). Fig. 4C shows that for contractions of ABKO trabeculae the Ca2+–force relation lay to the left of that for WT trabeculae. Data for WT trabeculae were fit to the Hill equation (solid line); however, data for ABKO trabeculae appeared more irregular and could not be fit. For each experiment the [Ca2+]c that resulted in half maximal activation (Ca50) was determined and was lower for ABKO trabeculae (0.8 ± 0.05 µM, n = 5) than for WT (1.1 ± 0.06 µM, n = 5, P < 0.01) consistent with increased myofilament Ca2+–sensitivity for ABKO trabeculae. The Ca50 for WT trabeculae in this study was similar to that previously reported for mouse myocardium [15]. Fig. 4D shows that the maximum tetanic force of ABKO trabeculae was lower than that of the WT.
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Fig. 3. Knockout of a1–ARs caused decreased maximal force but unchanged systolic [Ca2+]c in trabeculae. The peak force at up to 6 mM extracellular [Ca2+] was reduced in the ABKO trabeculae (**P < 0.01) (top panels) but the systolic [Ca2+]c was not statistically different (lower panels). Data are mean ± S.E., n = 7–8 per group.
Fig. 4. Steady-state Ca2+–force relationship in trabeculae. Force traces (A) and corresponding Ca2+ records (B) during tetanization at various extracellular [Ca2+] (indicated in mM next to each trace). (C) Ca2+–force relationships for ABKO (closed symbols) lay to the left of the WT. (open symbols; solid line shows fit to Hill equation). (D) Summary of maximum tetanic forces, ** P < 0.01. Data are mean ± S.E., n = 4–5 per group.
3.5. Knockout of ␣1–ARs did not affect intracellular pH For other species, inotropic responses to a1–AR stimulation involve changes of intracellular pH (pHi) [16]. Intracellular alkalinization due to stimulation of Na+/H+ exchange causes enhanced Ca2+ binding to the myofilaments [16].
Therefore, we measured pHi using the fluorescent pH indicator BCECF to determine if increased myofilament Ca2+– sensitivity of ABKO trabeculae involved intracellular alkalinization. We found that there was no difference in pHi between the ABKO (7.41 ± 0.01) and WT trabeculae (7.39 ± 0.02) at 2 mM extracellular [Ca2+] (P > 0.05, n = 4–6).
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Thus, increased myofilament Ca2+–sensitivity in ABKO myocardium must involve mechanisms other than increased pHi. 3.6. Knockout of ␣1–ARs decreased TnI phosphorylation Phosphorylation of TnI results in decreased myofilament Ca2+–sensitivity [17–19]. Thus, increased myofilament Ca2+–sensitivity in ABKO myocardium could result from decreased TnI phosphorylation. We assessed TnI phosphorylation for WT and ABKO myocytes using a phosphorylationspecific antibody. Western blot analysis showed that for quiescent myocytes TnI phosphorylation levels were undetectable for both WT and ABKO myocytes (not shown). However, with isoproterenol stimulation, the TnI phosphorylation level was significantly less for ABKO compared to WT myocytes (Fig. 5A). There was no difference in total TnI levels between ABKO and WT. The pooled data (Fig. 5B) show that after isoproterenol stimulation, the maximum TnI phosphorylation level for ABKO was 73 ± 1% of that found for WT (n = 3, P < 0.01). Moreover, Fig. 5 shows that TnI phosphorylation levels were also lower for ABKO myocytes after stimulation using the cAMP agonists dibutyryl cAMP
(72 ± 7% of WT response, n = 2) or forskolin (47% of WT response). These data suggest that ABKO hearts had impaired TnI phosphorylation when stimulated by b-ARs or by cAMP agonists. Impaired TnI phosphorylation in ABKO myocardium could contribute to increased myofilament Ca2+–sensitivity. 3.7. Effect of acute ␣1 –AR stimulation Previous studies by us and others have demonstrated that for in vitro preparations of mouse myocardium, nonsubtype-specific a1–AR stimulation causes a sustained negative inotropic response [6,20,21]. The contractile response of a WT mouse RV trabecula to phenylephrine (10 µM) in the presence of timolol (10 µM) is shown in Fig. 6. As we previously reported, approximately 10 min after adding phenylephrine, force fell markedly to 52 ± 4.6% initial (P < 0.001 compared to control, n = 8); force subsequently recovered slightly to 67 ± 4.4% initial (P < 0.001 compared to control, n = 8) [6]. Fig. 6 also shows that ABKO trabeculae, lacking the two predominant a1–AR subtypes, had no response to phenylephrine [7]. 3.8. Effect of b-adrenergic stimulation To investigate if deletion of a1–ARs altered b-AR function we monitored the responses of ABKO trabeculae that were stimulated with isoproterenol at 0.3 mM extracellular [Ca2+]. Fig. 7 shows that following isoproterenol stimulation the rise of force for ABKO trabeculae was blunted compared to WT (P < 0.01, two-way repeated measures ANOVA). Thus, deletion of a1–ARs caused decreased b-AR responsiveness. To investigate receptor-independent effects, we stimulated adenylyl cyclase directly using 20 µM forskolin and 0.3 mM extracellular [Ca2+]. For both WT and ABKO trabeculae, force was appreciably higher with forskolin compared to with isoproterenol (P < 0.01). However, in contrast to the
Fig. 5. Knockout of a1–ARs caused decreased TnI phosphorylation: (A) Western blots of total– and phospho-TnI (p–TnI) for WT and ABKO myocytes after stimulation with isoproterenol and dibutyryl cAMP (db cAMP) (see Section 2); (B) summary of TnI phosphorylation in response to isoproterenol and the cAMP agonists db cAMP (circles) and forskolin (triangle) (intensity of TnI-P bands were normalized to TnI intensity and scaled relative to the WT response).
Fig. 6. Effect of a1–AR stimulation of ABKO and WT trabeculae. Slow time-base recording of peak twitch force of ABKO and WT mouse RV trabeculae. Arrow indicates addition of the a1–AR-agonist phenylephrine in the presence of the b-AR-antagonist timolol.
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Fig. 7. Summary of the effect of b-AR stimulation of trabeculae on contraction force (mean ± S.E., n = 6 WT and n = 5 ABKO). The response to isoproterenol was significantly lower for ABKO myocardium (P < 0.01, two-way repeated measures ANOVA). The response to forskolin is shown to the right of the x–axis (and where the y–axis has a break and change of scale).
blunted response of ABKO trabeculae to isoproterenol, in the presence of forskolin there was no difference in force developed by ABKO and WT trabeculae (30.6 ± 3.2 vs. 34.3 ± 2.3 mN/mm2, n = 5–6, P > 0.05). Thus, decreased b-AR responsiveness of ABKO myocardium was not caused by impaired adenylyl cyclase function. This suggests decreased b-AR responsiveness involved impaired receptormediated cyclase stimulation. 3.9. Effect of pacing frequency Some studies suggest that in models of disease, contractility may appear normal at low pacing rates but that impaired function may become apparent at higher pacing rates [22]. Therefore, for WT and ABKO trabeculae we determined the effect of increasing pacing frequency on force and systolic [Ca2+]c over the frequency range 0.25–2 Hz at submaximal extracellular [Ca2+] (2 mM). Similar to results from previous studies [15,23], Fig. 8 shows that for mouse myocardium the relationship between force and frequency was negative below 0.5 Hz pacing but positive above 0.5 Hz. Furthermore, Fig. 8 shows increased pacing rate was not associated with impaired function of ABKO trabeculae compared to WT. In contrast, ABKO trabeculae developed higher forces at all pacing rates such that there was a significant difference between ABKO and WT myocardium in response to pacing (P < 0.05, two-way repeated measures ANOVA). Although the peak force of ABKO trabeculae tended to be higher than for WT trabeculae, peak systolic [Ca2+]c was not statistically different. Relating systolic force to systolic [Ca2+]c over all pacing frequencies (Fig. 8C) shows that the force–Ca2+ relationship for ABKO trabeculae lay above that for WT, consistent with increased myofilament Ca2+–sensitivity in ABKO trabeculae as was suggested by the data in Figs. 2 and 4.
Fig. 8. Summary of the effect of stimulation frequency on: (A) force, (B) [Ca2+]c, and (C) relationship between force and [Ca2+]c at all stimulation frequencies (mean ± S.E., n = 7–8 per group).
4. Discussion The major finding of this study was that knockout of both a1–AR subtypes in the mouse (ABKO) caused marked myocardial contraction abnormalities involving increased Ca2+– sensitivity, decreased maximal contraction, and decreased
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responsiveness to b-AR stimulation. Inhibition of a1–ARs using prazosin in WT hearts did not mimic the ABKO phenotype suggesting that contraction abnormalities of ABKO hearts were not due to acute regulatory effects of altered a1–AR signaling. Taken together our results suggest that a1–ARs are required for development of normal myocardial function and that a1–ARs mediate long-term effects on contractile function. Consistent with our finding that loss of a1–AR function is detrimental in the long term, a massive clinical trial (ALLHAT) of an a1–AR-antagonist was stopped prematurely because of an increase in heart failure in the a1–AR-antagonisttreated group [24]. Furthermore, our recent study found 50% mortality due to heart failure in ABKO mice after the stress of transverse aortic constriction (TAC) vs. 100% survival for WT mice after TAC [5]. Thus, impaired a1–AR function may be a contributory factor in the development of heart failure. Interestingly, similar to the contraction abnormalities we observed in the present study, previous studies of human and animal failing hearts also found increased myofilament Ca2+–sensitivity [25–27], decreased maximal Ca2+–activated force [28–30], and decreased b-AR responsiveness [31–33]. Thus, loss of a1–ARs is detrimental to the heart and may produce some (but not all) of the pathophysiological changes that are involved in the development of heart failure. A striking result in this study was that contractile function was actually higher for ABKO myocardium compared to WT at a physiological level of external [Ca2+]. This was so for both isovolumic Langendorff-perfused hearts, and for in vitro RV trabeculae. Previous studies found that the heart size of ABKO male mice was smaller than WT males; however, heart size for ABKO females was normal [5]. In the present study, we found that ABKO myocardium for both male and female mice had increased submaximal contractions. Therefore, hypercontractility of ABKO myocardium was not related to smaller heart size found only in males. To investigate the mechanism for increased myocardial contraction in ABKO hearts we studied RV trabeculae. Several results suggest that increased submaximal contractions of ABKO myocardium were caused by increased myofilament Ca2+–sensitivity relative to WT. First, increased submaximal contractions of ABKO trabeculae were not associated with increased Ca2+ transients. Second, the steady-state Ca2+–force relation for ABKO trabeculae lay to the left of that for the WT. Finally, for ABKO trabeculae the relationship between peak [Ca2+]c and peak force obtained using a range of pacing rates lay above that for the WT. One mechanism by which a1–AR signaling is known to influence Ca2+– sensitivity is via effects on intracellular pH. However, we found that increased Ca2+–sensitivity of ABKO myocardium was not associated with increased pHi. Interestingly, we found that stimulation of the Gs-signaling pathway using isoproterenol caused less TnI phosphorylation in ABKO myocytes compared to WT. Thus, for contracting ABKO myocardium, which will likely have some basal Gs tone,
impaired TnI phosphorylation could contribute to increased myofilament Ca2+–sensitivity. We recently reported that increased acute signaling by a1–ARs resulted in decreased Ca2+–sensitivity for mouse myocardium [6]. By extension, increased Ca2+–sensitivity of ABKO myocardium could arise from elimination of basal a1–AR signaling. However, the failure of prazosin to mimic the ABKO phenotype in WT myocardium suggests that the ABKO phenotype is not simply due to acute effects of inhibiting a1–AR signaling. Our results suggest that knockout of a1–ARs in the heart has long-term effects on cardiac function. In contrast to increased submaximal contraction of ABKO myocardium, the maximal contraction force of ABKO myocardium was reduced at high extracellular [Ca2+]. Reduced maximal twitch force was not due to a decreased Ca2+ transient, which suggested decreased myofilament function. Decreased maximal force of ABKO myocardium was also evident by decreased maximal tetanic force. The heart normally operates under conditions of submaximal activation, therefore, the functional significance of decreased maximal force for ABKO myocardium is unclear. Possibly ABKO hearts may respond worse than WT to conditions that require use of their contractile reserve. Indeed, the response of ABKO myocardium to b-AR stimulation was decreased. Furthermore, exercise capacity is decreased and mortality in response to TAC is increased in ABKO mice [5]. Despite differences in contraction between ABKO and WT myocardium with both submaximal and maximal contractions, Ca2+ transients of ABKO myocardium were not different from WT. This suggests that a1–ARs are not required for normal Ca2+ handling. The interaction between a1– and b-ARs in myocardium has been unclear. There has been a suggestion of an antagonistic interplay between the a1– and b-ARs, where norepinephrine exerts an inhibitory effect through a1–ARs on b1– AR-stimulated Ca2+ current in rat ventricular myocytes [34,35]. The ABKO model has provided an opportunity to assess if b1–AR function is altered by knockout of a1–AR function. We found that the response of ABKO myocardium to b-AR stimulation was blunted in ABKO myocardium compared to WT. However, adenylyl cyclase stimulation by forskolin was not impaired; therefore, decreased b-AR responsiveness in ABKO myocardium involved impaired receptor-mediated adenylyl cyclase stimulation (for example due to decreased agonist binding or impaired receptor coupling to the cyclase). Thus, loss of both a1–AR subtypes in the heart did not cause upregulation of the function of b-ARs, instead there was a reduction in the b-AR response. Decreased b-AR responsiveness could also limit the ability of ABKO myocardium to respond to conditions of increased stress, such as exercise or aortic banding (see above). Previous studies suggested that growth and contraction were normal for myocardium lacking only one a1–AR subtype. This suggests that a1–AR subtypes may mediate redundant functions. Consistent with this, we recently demon-
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strated that for mouse myocardium when a1–AR subtypes were stimulated separately, the functional response to a1A–AR stimulation was very similar to the a1B–AR response [6]. However, in the present study, contraction was abnormal when both a1–AR subtypes were knocked out. This suggests that at least one a1–AR subtype has to be present for normal cardiac growth and function. From this study we conclude that: (a) a1–ARs are required for normal contractile function; (b) loss of a1–AR function induces long-term trophic effects on contraction; and (c) loss of a1–AR function results in a pathophysiological phenotype with some (but not all) features of the failing heart.
Acknowledgements This work was supported by NIH grants HL 56257 and P01 HL68738 – project 3 (A.J.B.), HL 54890 and HL 31113 (P.C.S.); and a postdoctoral fellowship HL10422 (D.T.M.); and by a Grant-in-Aid from the American Heart Association, Western States Affiliate (A.J.B.). A.J. Baker is an Established Investigator of the American Heart Association. Some of these data were published in abstract form: Biophys J 2002;82:190a.
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