The role of pulmonary vascular contractile protein

Journal of Molecular and Cellular Cardiology 65 (2013) 147–155

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Original article

The role of pulmonary vascular contractile protein expression in pulmonary arterial hypertension☆ Ewa A. Konik, Young Soo Han, Frank V. Brozovich ⁎ Mayo Clinic, Division of Cardiovascular Diseases, 200 First Street SW, Rochester, MN 55905, USA

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Article history: Received 29 July 2013 Received in revised form 30 September 2013 Accepted 15 October 2013 Available online 23 October 2013 Keywords: Myosin light chain phosphatase Myosin phosphatase targeting subunit 1 Smooth muscle myosin Nonmuscle myosin Vascular reactivity Nitric oxide

a b s t r a c t Pulmonary arterial hypertension (PAH) is associated with refractory vasoconstriction and impaired NO-mediated vasodilatation of the pulmonary vasculature. Vascular tone is regulated by light chain (LC) phosphorylation of both nonmuscle (NM) and smooth muscle (SM) myosins, which are determined by the activities of MLC kinase and MLC phosphatase. Further, NO mediated vasodilatation requires the expression of a leucine zipper positive (LZ+) isoform of the myosin targeting subunit (MYPT1) of MLC phosphatase. The objective of this study was to define contractile protein expression in the pulmonary arterial vasculature and vascular reactivity in PAH. In severe PAH, compared to controls, relative LZ+MYPT1 expression was decreased (100 ± 14% vs. 60 ± 6%, p b 0.05, n = 7–8), and NM myosin expression was increased (15 ± 4% vs. 53 ± 5% of total myosin, p b 0.05, n = 4–6). These changes in contractile protein expression should alter vascular reactivity; following activation with Ang II, force activation and relaxation were slowed, and sustained force was increased. Further, the sensitivity to ACh-mediated relaxation was reduced. These results demonstrate that changes in the pulmonary arterial SM contractile protein expression may participate in the molecular mechanism producing both the resting vasoconstriction and the decreased sensitivity to NO-mediated vasodilatation associated with PAH. © 2013 The Authors. Published by Elsevier Ltd. All rights reserved.

1. Introduction In PAH, the pulmonary vasculature is characterized by a resting vasoconstriction and impairment of NO-mediated vasodilatation, and only ~20% of PAH patients respond to conventional vasodilators [1,2]. For untreated patients with PAH, median survival is 2.8 years [3]. Currently, 5-year survival for WHO category I patients is 61%, and the 1-year mortality is ~15% [4]. In addition, pulmonary hypertension contributes to the morbidity and mortality of patients with both pulmonary and cardiovascular diseases [5]. An increase in the proliferation/apoptosis ratio has been suggested to produce PAH. It is hypothesized that widespread endothelial apoptosis results in selection of apoptosis-resistant endothelial precursor cells that proliferate and eventually form plexiform lesions [1,2,6]. In the media, pulmonary artery SM cell apoptosis is suppressed and proliferation is enhanced [3,7,8]. These changes result in the histological findings present in PAH, including intimal hyperplasia, adventitial proliferation and plexiform lesions, which produce an elevation of pulmonary arterial pressure (reviewed in [4,9]). Despite these known factors that contribute to the molecular mechanism that produces ☆ This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-ShareAlike License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited. ⁎ Corresponding author at: 200 First Street SW, Rochester, MN 55905, USA. Tel.: +1 507 774 3727; fax: +1 507 538 6418. E-mail address: [email protected] (F.V. Brozovich).

PAH, the contribution of dysfunction at the level of SM contractility to the pathogenesis of PAH has not been elucidated. Vascular tone is regulated by regulatory light chain (LC) phosphorylation of both nonmuscle (NM) and smooth muscle (SM) myosins, which are determined by the activities of MLC kinase and MLC phosphatase [5,10–12]. MLC kinase is regulated by Ca2+-calmodulin, whereas MLC phosphatase activity is regulated by several signaling pathways including those mediated by both NO and Rho kinase [11]. MLC phosphatase is a trimeric enzyme consisting of catalytic, 20 kDa and MYPT1 subunits [10]. Alternative splicing of a 3′ exon produces MYPT1 isoforms that differ by the presence/absence of a LZ (LZ+/LZ−, [13]), and a LZ+MYPT1 isoform is required for NO-mediated activation of MLC phosphatase [14]. We have demonstrated that, in isolation, a decrease in LZ+MYPT1 expression results in a decrease in the sensitivity of SM to NO-mediated relaxation [14]. Thus, changes in LZ+MYPT1 expression contribute to the mechanism that regulates the sensitivity of the vasculature to NO-mediated relaxation [14–16]. Further, LZ+MYPT1 isoform expression decreases in heart failure, pre-eclampsia, and portal hypertension [15–18], and thus, a decrease in LZ+MYPT1 expression contributes to the decrease in sensitivity to NO associated with these diseases. Recent evidence suggests that NM myosin also participates in the force maintenance phase of SM contraction and hence to the regulation of vascular tone. Compared with SM myosin, the kinetics of NM myosin are slow [19,20]. Consequently, NM myosin spends a majority of its kinetic cycle attached to actin to support force [19], and changes in NM myosin expression have been demonstrated to influence vascular tone [21].

0022-2828/$ – see front matter © 2013 The Authors. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.yjmcc.2013.10.009

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E.A. Konik et al. / Journal of Molecular and Cellular Cardiology 65 (2013) 147–155

This study was designed to determine the expression of contractile proteins in pulmonary arterial smooth muscle and pulmonary vascular reactivity in PAH. Our data suggest that MYPT1 and NM myosin expression is altered in PAH, and that these changes contribute to the molecular mechanism that produces PAH. 2. Material and methods 2.1. MCT rat model of PAH and pulmonary artery preparation The Institutional Animal Care and Use Committee of the Mayo Clinic approved all experimental protocols and animal care, and the study conformed to the guidelines of the National Institutes of Health. We used the rat model of monocrotaline (MCT)-induced PAH, in which a single MCT injection leads to severe PAH within 4 wks [22]. The pathological changes occurring in the lung vasculature as well PAH progression in this model have been well characterized [23,24]. Briefly, PAH was induced in male Sprague–Dawley rats weighing 250 g by a single i.p. injection of MCT (60 mg/kg). Control rats were injected with normal saline. The rats were sacrificed at 2 and 4 wks after injection. Prior to sacrifice, RV pressure was assessed using a 22G catheter connected to a pressure transducer and inserted into RV from abdominal cavity via the diaphragm. Subsequently, the heart was exposed by a midline thoracotomy and the heart and lungs were harvested. Tissue was then placed in Ca2+-free Tyrode's solution (in mM: 135 NaCl, 4 KCl, 1 MgCl2, 0.33 Na2HPO4, 0.03 EDTA, 10 glucose, 10 HEPES; pH 7.4) on ice. The main, right and left pulmonary arteries, and their lobar branches were isolated (with an intact endothelium) and cleaned of connective tissue. PA tissue was processed immediately as appropriate for studies described below. The RV was separated from the LV and the interventicular septum (IVS). The RV wt and RV to (LV + IVS) ratio (Fulton index) was used to assess RV hypertrophy. The left lung was placed in the neutral buffered 10% formalin and submitted to the Mayo Clinic Pathology Department for further processing. 2.2. Echocardiography Echocardiography was performed at baseline, 2-wk, and 4-wk time points using the Vevo 770 ultrasound system (VisualSonics), as previously described [23]. Briefly, transthoracic 2D, M-mode, and pulsed wave (PW) Doppler were obtained with a 17 MHz broadband scan head. M-mode and 2D modalities were applied to measure RV free-wall thickness during end diastole using the right parasternal short-axis view. Main pulmonary artery diameter was measured at the level of pulmonary outflow tract during midsystole using superior angulation of the parasternal short-axis view. PW Doppler was used to obtain pulmonary artery flow velocity time integral (VTI) and to measure the pulmonary artery acceleration time (PAAT). For these measurements, the sample volume was centrally positioned within the main pulmonary artery, just distal to the pulmonary valve with orientation of the beam parallel to flow. 2.3. Pathology The lung tissue preserved in the neutral buffered 10% formalin was processed by the Mayo Clinic Pathology Department. The tissue was embedded in paraffin, cut in 5 μm slices (thickness), and stained with either haematoxylin and eosin (H&E) or Van Gieson. Morphometric analysis of pulmonary vasculature was performed using ImageJ. 2.4. Mechanical studies Mechanical studies were conducted following protocols established in our laboratory as described previously [25]. Briefly, isolated main pulmonary artery and the right and left pulmonary arteries were cut

in 4 rings of ~2 mm (length). Rings isolated from the same animal were used for both mechanical and molecular studies. For the mechanical studies, rings were transferred to a vessel chamber containing continuously oxygenated physiological saline solution (PSS, in mM: 140 NaCl, 3.7 KCl, 2.5 CaCl2, 0.81 MgSO4, 1.19 KH2PO4, 0.03 EDTA, 5.5 glucose, 25 HEPES; pH 7.4), mounted on a DMT (Mulvany) 4-channel myograph system and stretched to Lo (the length for maximal force, which was determined in control preparations). Following the stretch to Lo, the rings were allowed to equilibrate for 30 min, and then the passive force was recorded. The rings were stimulated to contract with 80 mM KCl depolarization (in mM: 64.5 NaCl, 80 KCl, 2.5 CaCl2, 0.81 MgSO4, 1.19 KH2PO4, 0.03 EDTA, 5.5 glucose, 25 HEPES; pH 7.4) or 1 μM Ang II. Active force is defined as the increase in force above the resting passive force, and the force traces are the active component of force. To determine the sensitivity to ACh-mediated relaxation, the SM was depolarized (80 mM KCl) and after force reached a steady state, the dose response relationship for ACh-mediated relaxation was determined by sequential addition of a higher [ACh]. 2.5. Determination of NM and SM expression NM and SM myosin expression was determined using 2D SDS-PAGE as described [21,25]. We have previously demonstrated that this technique resolves the nonphosphorylated and phosphorylated SM myosin light chain (SM LC) and NM myosin light chain (NM LC) as four distinct spots [21]. Briefly, rings from the main PA, the right and left PAs and their lobar branches were manually homogenized in 2D gel extraction buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1% 3–5.6 immobilized pH gradient (IPG) buffer and EDTA-free Protease Inhibitor and PhosStop Phosphatase Inhibitor (Roche, Indianapolis, Ind., USA)). The homogenates were cleared of lipids and extraneous salts using the 2D gel clean up kit (GE Healthcare). The acidic halves of 13-cm IPG DryStrip gels (pH 3–5.6 NL) were rehydrated in the presence of suitable amounts of sample in rehydration buffer solution (7 M urea, 2 M thiourea, 2% CHAPS, 0.5% pH 3.5–5 IPG buffer, 0.002% bromophenol blue and 12 μM/ml Destreak Reagent) for at least 10 h in the ‘facedown’ mode on the Ettan IPG rehydration tray and then resolved by isoelectric focusing in the ‘face-up’ mode on an Ettan IPGphor III (GE Healthcare). Following isoelectric focusing, the gel strips were equilibrated in 6 M urea, 50 mM Tris–HCl, pH 6.4, 30% glycerol, 2% (w/v) SDS and 0.002% bromophenol blue, first containing 130 mM DTT for 15 min and then containing 135 mM iodoacetamide for 15 min before undergoing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) for protein separation by molecular weight using the BisTris buffering system with 12% gels (29:1). Subsequently, resolved 2D SDS-PAGE gels were silver stained. Gels were scanned using a Personal Densitometer SI, and the spots were quantified using ImageQuant TL software. The two spots closest to the anode (spots 1 & 2) represent the phosphorylated and nonphosphorylated NM LC and the two spots nearest the cathode (spots 3 & 4) represent the phosphorylated and nonphosphorylated SM LC. The expression of NM myosin is calculated as [(1 + 2) / (1 + 2 + 3 + 4)] × 100%], while LC phosphorylation for SM myosin is (3/(3+4))×100% and LC phosphorylation for NM myosin is (1 / (1 + 2)) × 100% [21]. 2.6. Immunoblotting Western blots were used to determine protein expression in pulmonary arteries of normal versus rats with PAH as previously described [21,25]. Briefly, pulmonary arterial rings were homogenized in an SDS sample buffer. The total extracted protein was resolved by SDS-PAGE using the Bis-Tris buffering system with 8% gels (29:1). The actin band on Coomassie stained gels was used to normalize protein loading among samples. After SDS-PAGE separation, proteins were transferred onto a Hybond™ (GE Healthcare) membrane. MYPT1, LZ+MYPT1, SM myosin, NMIIA, NMIIB, and actin were visualized


using appropriate Abs; a rabbit polyclonal anti-MYPT1 (#2634 Cell Signaling), a mouse mAb anti-LZ+MYPT1 isoform [15,26], a mouse mAb anti-SM myosin (MYH11 sc-6956 from Santa Cruz), a rabbit polyclonal anti-NMIIA (M8064 Sigma), a rabbit polyclonal anti-NMIIB (M7939 Sigma), and a rabbit polyclonal anti-actin (A2066, Sigma) Ab. Following washing, the blots were incubated with Cy3-labeled antimouse IgG (Jackson Immunoresearch) or Cy5-labeled anti-rabbit IgG (GE Life Sciences). Blots were scanned on a Typhoon 9410 imager, and analyzed using ImageQuant TL software. All blots were normalized for actin, and to normalize expression across blots, one of the sample preparations was chosen as a standard sample and loaded on all Western blots. The level in the controls was set as 1.

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2.7. Statistical analysis All data are presented as mean ± SEM, and n represents the number of animals in each group. Student's t-test was performed to evaluate significant differences between the two groups. Differences were considered to be significant at p b 0.05, and when multiple comparisons between groups were necessary, a Bonferroni correction was performed.

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3. Results 3.1. Physiology At 2 wks, there were no significant hemodynamic differences between control and MCT treated rats. However as reported by many others [23,27], 4 wks following MCT injection, the rats had findings consistent with the development of RV hypertrophy and severe pulmonary hypertension (Supplemental Table 1). Although the wet wt of pulmonary arteries was no different at 2 wks, at 4 wks, the pulmonary artery wet wt was higher in MCT treated rats vs. controls (0.040 ± 0.005 g vs. 0.090 ± 0.010 g, p b 0.05, n = 4). Histological lung examination was performed on a subset of animals at weeks 2 and 4; at week 4, multiple small pulmonary vessels with increased wall thickness appeared throughout the lung parenchyma, which were not apparent at 2 wks (data not shown). These findings are consistent with the development of severe PAH at 4 wks following MCT administration [23]. 3.2. Mechanical Studies In PAH, the pulmonary vasculature is characterized by a resting vasoconstriction and impairment of NO-mediated vasodilatation. Thus, we determined the reactivity of pulmonary arterial SM in control and MCT treated rats. Total force is the sum of active and passive forces. Passive force was similar in control and MCT treated animals at 2 wks, but at 4 wks, passive force was significantly higher in the MCT treated animals (2.6 ± 0.2 mN vs. 3.0 ± 0.1 mN, p b 0.05, n = 4), which is consistent with a significant change in the architecture of the smooth muscle strip. Following KCl depolarization, force rose to a plateau in control and MCT treated rats, and force declined with return of the preparation to PSS. At 2 wks, there was no significant difference in the rate of force activation between control and MCT rats (t1/2; 0.30 ± 0.05 min vs. 0.19 ± 0.03 min, p N 0.05, n = 4 per group) but, relaxation was slower (t1/2; 0.95 ± 0.11 min vs. 1.74 ± 0.31 min, p b 0.05) and force maintenance was lower (1.98 ± 0.21 mN vs. 1.48 ± 0.28 mN, p b 0.05) in the MCT treated rats. At wk 4 (Fig. 1A), the MCT rats had a lower sustained active force (2.45 ± 0.24 mN vs. 1.28 ± 0.31 mN, p b 0.05, n = 4 per group) as well as slower rates of force activation (t1/2; 0.29 ± 0.02 min vs. 0.83 ± 0.26 min, p b 0.05) and force relaxation (t1/2; 1.23 ± 0.09 min vs. 2.16 ± 0.17 min, p b 0.05). Stimulation with Ang II resulted in a phasic contractile response in pulmonary arterial rings from control animals; force rapidly rose to a

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AngII Fig. 1. Pulmonary vascular reactivity is altered in severe PAH. (A) Representative tracings of active force for a pulmonary artery ring from control (black) and rats with severe PAH (red, MCT at 4 wks) activated with 80 mM KCl depolarization. Compared to controls, both contraction and relaxation phases were prolonged in pulmonic SM from rats with severe PAH. (B) Representative tracings of active force for pulmonary arterial rings from control (black) and severe PAH (red) stimulated with Ang II. Compared to controls, the contractile properties of pulmonary arterial SM from rats with severe PAH were more tonic displaying slower rates of force activation and relaxation, and an increase in the level of force maintenance.

peak before falling to a lower steady state. At 2 wks, there was no difference in the phasic response; t1/2 for contraction (0.40 ± 0.04 min vs. 0.45 ± 0.07 min, p N 0.05, n = 4 per group) and the t1/2 for relaxation (3.9 ± 0.2 min vs. 4.0 ± 0.2, p N 0.05) were not different. However at wk 4, the force profile following Ang II stimulation was significantly different when compared to control (Fig. 1B); the rate of force activation and relaxation was significantly slower for MCT treated animals (t1/2 contraction: 0.30 ± 0.01 min vs. 0.40 ± 0.03 min, p b 0.05 & t1/2 relaxation: 2.3 ± 0.2 min vs. 8.7 ± 0.7 min, p b 0.05, n = 4 per group). Further, although peak active force was lower (2.87 ± 0.33 mN vs 0.65 ± 0.21 mN, p b 0.05), at 15 min following Ang II stimulation, force maintenance for pulmonary arterial SM from the rats with severe PAH was significantly higher (0.16 ± 0.05 mN vs. 0.41 ± 0.18 mN, p b 0.05, n = 4–5 per group). Thus for agonist activation, force maintenance (both the active and the total force (2.76 ± 0.05 mN vs

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3.41±0.20mN, pb0.05)) was higher in rats with severe PAH, compared to controls. We investigated NO-mediated signaling by determining the dose– response relationship of ACh-induced relaxation of pulmonary arterial rings. At 2 wks, ACh produced a dose dependent SM relaxation for both control and MCT treated rats (data not shown). However at 4 wks, ACh did not produce a significant relaxation of pulmonary SM from MCT treated animals (Fig. 2). This contrasted with control preparations, in which ACh produced a dose-dependent relaxation; relaxation averaged 9.8 ± 2.3% at 0.1 μM ACh and 23 ± 3% at 1 μM ACh. 3.3. Molecular studies NM myosin has been shown to contribute to force maintenance in SM and hence to the regulation of vascular tone, whereas the expression of the LZ+MYPT1 isoform is required for NO-mediated SM relaxation. Thus to explain the changes in vascular reactivity present in severe PAH (Figs. 1, 2), we examined whether PAH was associated with a change in the expression of NM myosin and MYPT1. The absolute expression of NM vs. SM myosin was determined by 2D SDS-PAGE, which separates the non and phosphorylated SM and NM myosins as four distinct spots (Fig. 3). At 2 wks, NM expression represented ~15–20% of total myosin expression and there was no significant difference in the expression of NM myosin in control vs. MCT-treated rats (16 ± 2% vs. 23 ± 3%, p N 0.05, n = 4). However at wk 4, there was a significant increase in NM myosin expression in MCT-treated rats (15 ± 4% vs. 53 ± 5%, p b 0.05, n = 5–6); with severe PAH, NM myosin expression was equal to that of the SM MHC. Western blots were consistent with the 2D SDS-PAGE findings. At 4 wks (Fig. 4), there was a significant decrease in SM MHC expression (1.00 ± 0.07 vs. 0.70 ± 0.06, p b 0.05, n = 5–6), whereas there was a significant increase in both NMIIA (1.00 ± 0.06 vs. 2.71 ± 0.55, p b 0.05, n = 4–6) and NMIIB (1.00 ± 0.06 vs. 1.25 ± 0.05, p b 0.05, n = 4–6) expression. In addition, there was no difference in the resting phosphorylation of the regulatory LCs of either SM (15 ± 4% vs 7 ± 3%, p N 0.05) or NM (3 ± 3% vs 3 ± 3%, p N 0.05) myosin. Both total MYPT1 and LZ+MYPT1 isoform expression was assessed with Western blots using a polyclonal MYPT1 Ab and a mAb antiLZ+MYPT1 Ab [15,26]. At 2 wks, there was no difference in the relative expression of the LZ+MYPT1 isoform, defined as LZ+MYPT1/total MYPT1, between control and MCT treated rats. However in pulmonary arterial SM from MCT rats with severe PAH (wk 4), relative LZ+MYPT1 expression was significantly reduced (1.00±0.14 vs. 0.60±0.06, n=7– 8, Fig. 5), and the decrease in relative LZ+MYPT1 expression was predominantly due to an increase in total MYPT1 expression (total MYPT1/actin; 1.00 ± 0.10 vs 1.60 ± 0.10). 4. Discussion PAH is a syndrome that is characterized by increased pulmonary vascular resistance. The histological findings in PAH include vascular

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Fig. 2. The sensitivity to ACh-mediated relaxation is reduced in severe PAH. Representative tracings for active force for pulmonary arterial rings during ACh-mediated relaxation from control (black) and rats with severe PAH (red) are overlaid to more easily compare the response to ACh. In controls, ACh produced a dose dependent relaxation, while there was no significant response to ACh in the preparations from animals with severe PAH.

remodeling and angioproliferative plexiform lesions [9]. However in PAH, the contribution of changes in SM contractile protein expression to the increase in pulmonary vascular resistance has not been defined. In SM, others have demonstrated that NM myosin is responsible for the sustained force response [28,29] and a decrease in NM myosin expression, in isolation, decreases vascular tone [21]. Additionally, changes in the expression of LZ+/LZ− MYPT1, in isolation, have been demonstrated to produce changes in the sensitivity to NO-mediated vasodilatation [14]. Thus, our data suggests that changes in the expression of pulmonary arterial SM contractile proteins, namely the increase in NM myosin and decrease in relative LZ+MYPT1 expression, will produce abnormalities of vascular reactivity and these changes may contribute to the pathogenesis of PAH. Within the media of the pulmonary arteries, PAH could be associated with an increase in fibroblasts and/or myofibroblasts. However for both MCT and hypoxia induced PAH, there is a proportional increase in both SM and connective tissues [30,31]. Additionally in PAH, the staining of the cells within the media is consistent with an increase in smooth muscle cells [30,31]. Thus in our study, we would argue that the increase in the ratio of NM/total myosin observed in severe PAH is due to an increase in the expression of NM myosin in the SM cells. This increase in NM myosin could mark a transition from a contractile to a proliferative SM phenotype. However, the increase in NM myosin expression would also alter SM contractility [19–21,25,32,33]. In severe PAH, the significant increase in passive force suggests that there is a change in tissue architecture, possibly including the orientation of the smooth muscle cells within the tissue. These changes in smooth muscle orientation and tissue architecture would alter the force length relationship of the pulmonary smooth muscle strips [34], which could have affected our results; active force would be reduced if the MCT treated preparations were not at Lo. We performed our experiments using larger pulmonary vessels isolated from the main pulmonary artery up to the lobar arteries. It was technically not feasible to dissect the resistance vessels due to their small diameter (b 100 μm), but dissection of the larger arteries allowed us to use the same vessels for both molecular and contractility studies. It is unclear if the changes we demonstrated in the pulmonary arterial vessels reflect changes that occur in the pulmonary resistance vessels. However, we have demonstrated that cardiac ischemia alters both NM myosin and LZ+MYPT1 expression in the resistance vessels isolated from the mesenteric circulation [25], suggesting that the changes in NM myosin and MYPT1 expression documented in the pulmonary arteries (Figs. 3–5) could reflect changes in protein expression in the resistance vessels of the pulmonary vasculature. 4.1. NM myosin expression With the development of severe PAH, 4 wks after MCT injection, there is a 3.5 fold increase of NM myosin expression (15 ± 4% vs. 53 ± 5%, Figs. 3, 4), which is accompanied by markedly abnormal vascular reactivity; both force activation and relaxation are prolonged, with a markedly increased and prolonged phase of force maintenance for Ang II stimulation (Fig. 1). Others have suggested that the expression of NM myosin increases in hypoxia induced PAH [33]. However in this study [33], NM and SM MHCs were identified only based on molecular weight. The SM1 and SM2 smooth muscle MHC isoforms are readily resolved and separated from NM myosin [35]. NMIIA has a predicted MW of 228 kDa and NMIIB has three isoforms, with predicted MWs of 229, 230 & 232 kDa. But, NMIIA and NMIIB are not separated by SDS PAGE [36], and thus, it is unclear if the two bands (MW 196 kDa & 190 kDa) denoted as NM myosin in Packer's study [33] represent NMIIA and NMIIB. Nonetheless, our study is the first to document that NM myosin expression significantly increases in severe PAH. The kinetics of NM myosin are slower than SM myosin [19,20,32]. In the presence of actin, NM myosin has a high ADP affinity and it spends majority of kinetic cycle attached to actin [19,20]. For NMIIB, the rate of


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Fig. 3. NM myosin expression increases with the development of PAH. 2D SDS-PAGE resolves the NM myosin and SM MHC as 4 distinct spots. Spots 1 and 2 represent phosphorylated and nonphosphorylated NM LC, respectively, while spots 3 and 4 represent phosphorylated and nonphosphorylated SM LC. The expression of NM myosin is calculated as [(1 + 2) / (1 + 2 + 3 + 4)] × 100% [21]. Bar graphs summarize NM myosin expression in the pulmonary artery in MCT-treated rats at wk 2 and 4 (*, p b 0.05). (A) 2 weeks following MCT treatment. (B) 4 weeks following MCT treatment.

ADP release is near the steady state ATPase rate, and the addition of actin does not accelerate ADP release, but instead enhances ADP binding [19,37]. For NMIIA, the rate of ADP release is an order of magnitude faster than the steady state ATPase [20], which implies that compared to NMIIB, NMIIA spends a smaller fraction of its ATPase cycle bound to actin [19,35]. However when compared to SM myosin, these kinetic properties (Vmax; NMIIB 0.1 s−1 [19], NMIIA 1.2 s−1 [20], SM MHC 6 s−1 [32]) allow NM myosin to remain attached to actin for a greater fraction of AMATPase cycle, which would increase the number of NM myosin cross-bridges attached to actin and predict that an increase in NM myosin expression would result in sustained force at low energetic cost. Thus, NM myosin should contribute to force maintenance in SM. Mechanical studies support these biochemical predictions. Morano and colleagues [28] demonstrated that bladder SM from transgenic mice lacking SM myosin contract, albeit very slowly and without the transient peak force; i.e., the contraction was phasic in WT mice and tonic in SM KO mice. The KO animals did not express SM myosin, and the expression of NM myosin was similar in SM KO and WT mice. These data suggest that SM myosin is responsible for the rapid initial rise in force while NM myosin participates in the sustained phase of force maintenance. Similarly, force maintenance in the aorta of heterozygous NMIIB KO mice was lower than that in WT mice [21], and in WT mice, inhibition of NM myosin decreases force maintenance [38]. Our force tracings in severe PAH are similar to those in transgenic mice lacking SM myosin [28,29]. For Ang II stimulation, force had a slower rate of activation and a markedly prolonged relaxation phase, which when compared to control, resulted in a significant increase in sustained force (Fig. 1B). Similarly for KCl depolarization of preparations from rats with severe PAH, the rates of both force activation and

relaxation were slow (Fig. 1A). In bladder smooth muscle from WT mice, Lofgren et al. [29] demonstrated that force displayed a phasic response with a rapid peak before falling to a lower level of force maintenance, while in SM KO mice, force slowly rose to a plateau, which was 70% lower than that in WT mice. These results may suggest that SM myosin is required for a rapid increase in force, and NM myosin is primarily responsible for the maintenance of force. Thus, there could be differences for tonic and phasic contractions, which would be consistent with our results. For the tonic contractile response to KCl, the lack of a rapid increase in force could lead to a lower level of force maintenance. Further in normal rats and rats with PAH, the pulmonary vasculature is not innervated [39,40], which could question the physiologic significance of depolarization. For the phasic response to Ang II stimulation, the decrease in the expression of SM myosin would be expected to blunt the peak force, while the increase in NM myosin would be expected to increase force maintenance, which is consistent with our results (Fig. 1B). These results agree with recent evidence that suggests that NM myosin regulates vascular tone [20,21,37,38]. In aortic SM from heterozygous NM IIB KO mouse, we have demonstrated that a decrease in NM myosin expression (13% vs. 6% of total myosin expression) resulted in a 25% reduction in maintenance force [41]. Similarly, we have also demonstrated that inhibition of NM myosin with blebbistatin decreased force maintenance by 8% in the mouse bladder and 24% in the mouse aorta [38]. Further, Morano and Arner's groups [29] demonstrated that the bladder from the SM MHC KO mouse produced ~11% of the force compared to WT. Others have also reported a decrease in peak force in pulmonary smooth muscle isolated from animals with PAH [30,42–44]. However for agonist activation, it is unclear why these other studies only reported peak force rather than sustained force, since force maintenance is more

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Fig. 4. SM MHC and NM myosin expression change with the development of severe PAH. Immunoblotting demonstrates the changes in SM MHC, NMIIA and NMIIB expression in pulmonary artery in MCT rats with severe PAH (*, p b 0.05).

important for the regulation vascular tone, and thus, vascular resistance. Nonetheless for agonist activation of pulmonary arterial SM isolated from animals with severe PAH, the passive, active and total components of sustained force are significantly increased, which would produce an increase in both pulmonary vascular tone and resistance.

4.2. MYPT1 expression Our data also demonstrate that severe PAH is associated with a significant decrease in relative LZ+MYPT1 expression (Fig. 5) and a decrease in the sensitivity of pulmonary artery SM to NO mediated relaxation (Fig. 2). MLC phosphatase controls SM relaxation via dephosphorylation of the regulatory myosin light chains. Its regulatory subunit, MYPT1, is a key target of the NO/cGMP/PKG vasodilatorsignaling pathway. Alternative splicing generates LZ+and LZ− MYPT1 isoforms, and only the LZ+MYPT1 isoform undergoes site-specific phosphorylation by cGMP-dependent protein kinase (PKG), which is required for NO/cGMP-mediated activation of MLC phosphatase [41]. Further, we have demonstrated that a decrease in LZ+MYPT1 expression, in isolation, results in a decrease in the sensitivity of SM to NO [14]. Therefore, the decrease in relative LZ+MYPT1 expression that occurs with severe PAH would decrease PKG mediated activation

of MLC phosphatase and result in a decrease in the sensitivity of the pulmonary vasculature to NO-mediated relaxation. The requirement of an intact endothelium for ACh mediated vasodilatation has been established [45]. MCT is thought to damage the endothelial cells, and a decrease in endothelial function would produce a decrease in sensitivity to ACh mediated relaxation [45]. We would expect that MCT induced endothelial dysfunction should occur relatively early after MCT injection, which is not consistent with our results. Two wks after MCT injection, before the development of PAH, there was no change in either LZ+MYPT1 expression or the sensitivity to ACh-mediated relaxation. Four wks after MCT injection, concurrent with the development of severe PAH, there was a significant decrease in relative LZ+MYPT1 expression (Fig. 5) that was accompanied by severely blunted relaxation in response to ACh (Fig. 2). This decrease in sensitivity to NO would decrease flow-mediated vasodilatation, which in addition to the increase in NM myosin, would also produce an increase in vascular tone. Others have demonstrated a decrease in LZ+MYPT1 expression in cultured pulmonary SMC exposed to hypoxia [46]. Additionally, a decrease in the ratio of LZ+MYPT1/MYPT1 expression has been suggested to mark the transition from a phasic to tonic SM contractile phenotype [47]; SM with tonic contractile properties have increased vascular tone, similar to that observed in patients with PAH. The


2 Weeks Control

MCT LZ+ MYPT1

Actin

MYPT1

LZ+ MYPT1/MYPT1 (%)

A

153

0.8 0.6 0.4 0.2 0.0

Actin

B

1.0

Control

MCT

4 Weeks MCT LZ+ MYPT1

Actin

MYPT1

LZ+ MYPT1/MYPT1 (%)

Control

1.0 0.8

* 0.6 0.4 0.2 0.0

Actin

Control

MCT

Fig. 5. Relative LZ+MYPT1 expression decreases in severe PAH. Immunoblots reveal that the relative expression of LZ+MYPT1 isoform in pulmonary artery SM was significantly reduced in rats with severe PAH (*, p b 0.05). For the MYPT1 blots, the upper and lower bands represent the MYPT1 isoforms differing by an alternatively spliced central insert [10].

stimulus for the decrease in relative LZ+MYTP1 expression is unclear. This decrease may represent a pathological change that produces PAH. Alternatively, the increase in total MYPT1 expression demonstrated in this study could represent an attempt to increase flow mediated vasodilatation and decrease pulmonary vascular tone as an adaptive response to the resting vasoconstriction produced by both the increase in NM myosin expression and proliferative changes that occur in the pulmonary vasculature. In either scenario, we have demonstrated that although total MYPT1 increases, relative LZ+MYPT isoform expression falls. This is a result of an increase in LZ-MYPT1 expression, which would bind PKG, and decrease PKG interaction with and subsequent phosphorylation of the LZ+MYPT1 isoform, leading to a decrease in the sensitivity to NO, and thus, contribute to the vascular abnormalities that produce PAH. In cultured SMCs, we have previously demonstrated that adenoviral infection with LZ+/LZ− MYPT1 isoforms produces a change in sensitivity to cGMP mediated smooth muscle relaxation [14], and thus in isolation, changes in LZ+/LZ− MYPT1 regulate the sensitivity to cGMP mediated smooth muscle relaxation. Further, changes in relative LZ+MYPT1 isoform expression (LZ+MYPT1/total MYPT1) are a predominant mechanism for altered NO-sensitivity in vascular diseases such as heart failure [15,16], portal hypertension [17], preeclampsia [18], as well as nitrate tolerance [48]. Importantly, pathological LZ+MYPT1 isoform expression changes can be prevented. In the rat model of heart failure, captopril treatment maintained the normal expression of LZ+MYPT1 [16] by decreasing the activation of p42/44 MAPK [49], which results in preservation of the normal sensitivity to cGMP-mediated SM relaxation [16]. Thus for the treatment of heart failure, the beneficial effects of blocking Ang II signaling, compared to other vasodilatators, could be due to altering the vascular phenotype by increasing relative LZ+MYPT1 expression and normalizing vascular reactivity.

4.3. Implications for human disease In PAH, aberrant signaling, altered protein expression levels, and histological findings have been documented [9,50]. However, the molecular mechanism that produces PAH has yet to be elucidated. Our results demonstrate that changes in both NM myosin and relative LZ+MYPT1 expression could contribute to the molecular mechanism that produces PAH in the MCT treated rat. Only 20% of PAH patients respond to conventional vasodilators [1]. Further, whether a patient with PAH will have significant pulmonary vasodilatation to NO cannot be predicted, and patients that lack a response to NO have a poorer prognosis [51]. Our results could suggest that patients that maintain normal NM myosin and LZ+MYPT1 expression would respond to NO based vasodilators, while those patients with an increase in NM myosin expression and a decrease in LZ+MYPT1 expression may have a poor prognosis. In heart failure, treatment with ACE inhibitors [16] or ARBs [49] maintains the normal expression of LZ+MYPT1 and sensitivity to NO. There is evidence in both animal and human clinical studies supporting a role of the renin angiotensin system in the development and progression of PAH, but the use of ACE inhibitors or ARBs in PAH is controversial [52–56]. Recent evidence has demonstrated that activation of guanylate cyclase improves hemodynamics in PAH [57,58], which suggests that there is a defect at the level of the SM, possibly an alteration in the expression of NM myosin and LZ+MYPT1 in the pulmonary vasculature, which contributes to the pathogenesis of PAH. Thus for PAH, therapies aimed at preventing and/or reversing the changes in NM myosin and MYPT1 in pulmonary arterial smooth muscle could restore NO-mediated vasodilatation and decrease pulmonary vascular tone, which would improve both symptoms and prognosis.

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5. Conclusion Our results suggest a novel paradigm that contributes to the mechanism that produces PAH. We propose that abnormalities at the level of the SM contribute to the mechanism for refractory vasoconstriction and impaired NO-mediated vasodilatation in PAH: (1) an increase in NM myosin expression results in a prolonged, sustained contraction that produces an increase in pulmonary vascular resistance; and (2) a decrease in relative LZ+MYPT1 expression results in a decrease in the sensitivity to NO-mediated relaxation. Vascular remodeling, angioproliferative plexiform lesions and excessive vasoconstriction are hallmarks of human PAH. Whereas proliferation is difficult to reverse, the changes in protein expression that contribute to both pulmonary arterial vasoconstriction and an impaired response to NO-mediated relaxation could be prevented and/or reversed with therapy aimed at preserving normal contractile protein expression in pulmonary arterial smooth muscle. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yjmcc.2013.10.009. Conflict of interest statement Ewa A. Konik none. Young Soo Han none. Frank V. Brozovich none. Acknowledgments We thank Dr Ozgur Ogut for his invaluable discussions, advice and comments on the manuscript. This study was supported by the Mayo Clinic. References [1] Sitbon O, Humbert M, Jaïs X, Ioos V, Hamid AM, Provencher S, et al. Long-term response to calcium channel blockers in idiopathic pulmonary arterial hypertension. Circulation 2005;111:3105–11. [2] Rich S, Kaufmann E, Levy PS. The effect of high doses of calcium-channel blockers on survival in primary pulmonary hypertension. N Engl J Med 1992;327:76–81. [3] Rich S, Dantzker DR, Ayres SM, Bergofsky EH, Brundage BH, Detre KM, et al. Primary pulmonary hypertension. A national prospective study. Ann Intern Med 1987;107: 216–23. [4] Thenappan T, Shah SJ, Rich S, Tian L, Archer SL, Gomberg-Maitland M. Survival in pulmonary arterial hypertension: a reappraisal of the NIH risk stratification equation. Eur Respir J 2010;35:1079–87. [5] Simonneau G, Robbins IM, Beghetti M, Channick RN, Delcroix M, Denton CP, et al. Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol 2009;54:S43–54. [6] Sakao S, Taraseviciene-Stewart L, Lee JD, Wood K, Cool CD, Voelkel NF. Initial apoptosis is followed by increased proliferation of apoptosis-resistant endothelial cells. FASEB J 2005;19:1178–80. [7] Morrell NW, Yang X, Upton PD, Jourdan KB, Morgan N, Sheares KK, et al. Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension to transforming growth factor-beta(1) and bone morphogenetic proteins. Circulation 2001;104:790–5. [8] McMurtry MS, Archer SL, Altieri DC, Bonnet S, Haromy A, Harry G, et al. Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension. J Clin Invest 2005;115:1479–91. [9] Archer SL, Weir EK, Wilkins MR. Basic science of pulmonary arterial hypertension for clinicians: new concepts and experimental therapies. Circulation 2010;121:2045–66. [10] Hartshorne DJ. Myosin phosphatase: subunits and interactions. Acta Physiol Scand 1998;164:483–93. [11] Somlyo AP, Somlyo AV. Ca2+ sensitivity of smooth muscle and nonmuscle myosin II: modulated by G proteins, kinases, and myosin phosphatase. Physiol Rev 2003;83: 1325–58. [12] Gong MC, Cohen P, Kitazawa T, Ikebe M, Masuo M, Somlyo AP, et al. Myosin light chain phosphatase activities and the effects of phosphatase inhibitors in tonic and phasic smooth muscle. J Biol Chem 1992;267:14662–8. [13] Khatri JJ, Joyce KM, Brozovich FV, Fisher SA. Role of myosin phosphatase isoforms in cGMP-mediated smooth muscle relaxation. J Biol Chem 2001;276:37250–7. [14] Huang QQ, Fisher SA, Brozovich FV. Unzipping the role of myosin light chain phosphatase in smooth muscle cell relaxation. J Biol Chem 2004;279:597–603. [15] Karim SM, Rhee AY, Given AM, Faulx MD, Hoit BD, Brozovich FV. Vascular reactivity in heart failure: role of myosin light chain phosphatase. Circ Res 2004;95:612–8.

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