Temporal Changes in Key Signal Transduction Pathways Mediating Muscle Protein Synthesis with Adaptive and Maladaptive Right Ventricular Hypertrophy in Pulmonary Arterial Hypertension

Pulmonary Arterial Hypertension (PAH) is a progressive cardiopulmonary disease and is characterized by occlusive remodeling of pulmonary arterioles and increased pulmonary vascular resistance. With the onset of PAH, the right ventricle (RV) of the heart adapts to the increased afterload pressure by undergoing adaptive hypertrophic remodeling to maintain adequate blood ow. However, for unknown reasons, maladaptive inuences ensue, resulting in impaired RV function with progressive decompensation and right heart failure. Using a rodent model of PAH, we evaluated key signaling pathways mediating cardiac muscle protein synthesis in the RV during the adaptive hypertrophy phase, with preserved right heart function, and the decompensated maladaptive phase, in which right heart failure (RHF) was clinically present. direct comparison of and non-phosphorylated states. Blots were a the and identied bands by densitometry using ImageJ 1.43u (NIH). Western blot data from the experimental groups were expressed relative to measured mean values from the CTL group. All results are expressed as the ratio of phosphorylated protein/total protein.

Pulmonary arterial hypertension (PAH) is a progressive condition for which there is no cure. Even with substantial pharmacologic advances in the modern treatment era, survival remains unacceptably poor [1][2][3]. PAH is characterized by occlusive remodeling of pulmonary arterioles and increased pulmonary vascular resistance. With the onset of PAH, the right ventricle (RV) adapts to the increased afterload presented to it by adaptive hypertrophy to maintain adequate blood ow. This is driven by signaling events that promote enhanced muscle protein synthesis and physiologic growth. However, for unknown reasons, maladaptive in uences ensue, resulting in impaired RV function with progressive decompensation and failure [4]. Indeed, RV function is the major factor determining survival of patients with PAH [5][6][7][8].
We postulate that these three signaling pathways would be key in mediating adaptive changes in the RV muscle exposed to a slow progressive rise in RV afterload facing the RV as PAH develops. We thus studied the temporal changes in signaling of these three pathways as PAH develops in an animal model of PAH, for which there are limited data. In addition, we serially evaluated insulin like growth factor-1 (IGF-1) as this growth factor has been shown to both impact upon these three pathways [21] and has been implicated in reports of physiologic cardiac hypertrophy [10,[22][23][24]. Further, we also evaluated the expression of proteins related to these three signaling pathways together with IGF-1 levels in the decompensated maladapted RV muscle of rats with established right ventricular dysfunction and failure. The latter was con rmed by echocardiographic parameters of impaired RV systolic function along with con rmatory changes at the cellular level, which included changes in RV wall brosis and capillarity.
In the monocrotaline (MCT) rat model of PAH, our observations in both the adapted and maladapted states describe several directions of change in the RV muscle that were unanticipated based on skeletal muscle data [15] and provide robust data and important questions for future research endeavors in fully understanding RV physiologic and pathobiologic changes that occur in the RV with progressive PAH. We also demonstrate in several instances, a continuum of changes as progression from an apparent adaptive state evolves into a full-blown maladaptive milieu.

Animals
Adult male Sprague-Dawley rats (initial body weight ~ 225 g; n=52) were divided into 2 major groups, namely control (CTL) and PAH. The CTL rats were injected subcutaneously with saline, while the PAH animals received a single subcutaneous injection of MCT (Sigma, St. Louis, MO) at a dose of 60 mg/kg body weight, dissolved in 0.5mL saline. All animals were provided with food (Purina rat chow) and water ad libitum. Animals were individually housed with a dark: light cycle of 12 hours each and with the ambient temperature maintained at 22º C. Terminal experiments were performed at days 7, 14, 24 and 28 (adapted cohort) and at 28 or more days (maladapted cohort, based on echocardiography).

Cardiac Muscle Weights and Measurement of RV pressures
The animals were anesthetized with either a combination of ketamine (100 mg/kg) and xylazine (10 mg/kg) injected intraperitoneally or with inhalational iso urane. The right external jugular vein was exposed via a minor surgical cut-down and the vein cannulated with a 1.4F Mikro-Tip catheter pressure transducer (SPR-671; Millar Instruments, Houston, TX). The catheter was advanced into the RV with measurements of RV systolic pressure via a dual channel pressure control unit (PCU-2000; Millar Instruments). Pressures were monitored and stored for later analysis using a PowerLab 8/30 (ML870) data acquisition system (AD Instruments, Colorado Springs, CO) connected to an IBM ThinkPad computer with LabChart 7 Pro data acquisition and analysis software (v7.2; AD Instruments). Subsequently the heart was excised, and the RV wall was separated from the left ventricle (LV) and the interventricular septum (S) using a dissecting microscope and their weights recorded. Both the RV mass and the ratio of RV/LV+S were used as indices of RV hypertrophy.

Echocardiography
Transthoracic two-dimensional, M-mode echocardiography and pulsed-wave Doppler imaging were performed on anesthetized (iso urane) rats (Vevo 770 Micro-Ultrasound imaging system; Visual Sonics: Toronto, Canada). RV systolic function was determined by tricuspid annular plane systolic excursion (TAPSE), recorded in M-mode. Doppler of pulmonary out ow measured pulmonary artery ow velocity time (PA VTI). To obtain stroke volume (SV), the cross-sectional area of the pulmonary artery was multiplied by PA VTI, and estimated cardiac output (CO) was derived by multiplying SV by heart rate.
Serial studies were performed before and post MCT administration.

Muscle Proteins
Protein extraction: RV muscle segments were rapidly frozen in liquid nitrogen and stored at -80°C until analysis. Sample protein was extracted in a 1:10 ratio of cold RIPA buffer (Cell Signaling Technologies, Beverly, MA), with protease and phosphatase inhibitors (Roche, South San Francisco, CA) added, according to manufacturer's protocol. For mTOR analysis samples, 0.4% CHAPS was also added to the lysis buffer. Homogenization was performed with a Polytron homogenizer (Kinematica, Bohemia, NY) and tissue lysates were incubated on ice for 4 hours and then were centrifuged at 13,200 RPM. The supernatant was aliquoted in microcentrifuge tubes. Protein concentration was determined using a commercial protein assay kit (Bio-Rad, Hercules, CA) and measured with a spectrophotometer (SmartSpecä 3000, Bio-Rad).
Immunoprecipitation: For mTOR analysis only, protein lysates were precleared with normal IgG (Santa Cruz Biotechnology, Dallas, TX) and protein A/G Plus-agarose beads (Santa Cruz Biotechnology). The lysates were incubated with the primary antibody (mTOR or phosphorylated mTOR (Ser 2448 ); Cell Signaling Technology, Beverly, MA) overnight at 4°C.
Protein A/G Plus-agarose beads were added to the immunocomplex, incubated for 4 hours at 4°C, and centrifuged. The immunocomplex pellet was washed, resuspended in sample buffer, heated at 70°C for 15 min and centrifuged. The supernatant was used to load gels for electrophoresis as described below.
SDS-PAGE and Western blotting: Electrophoresis of protein extracts was performed by SDS-PAGE. In brief, samples were heated at 70°C for 15 min and cooled prior to being used for electrophoresis. Protein extracts (40 mg per well) were loaded on NuPAGE 4-12% Bis-Tris gradient gels (Invitrogen, Grand Island, NY). For the analysis of 4E-BP1 (PHAS-I) samples, extracts were loaded on 12% Bis-Tris gels. Separated proteins were then electrophoretically transferred to nitrocellulose membranes (Bio-Rad). The membranes were treated with blocking buffer for 1 hour at room temperature. Blots were incubated with primary antibodies at 4°C overnight, washed and incubated with an appropriate peroxidase-conjugated secondary antibody at room temperature for 1 hour. The blots were visualized following development with enhanced chemiluminescence (ECL) Western blotting detection reagents (GE Healthcare Life Sciences, Pittsburgh, PA), according to manufacturer's protocol. Blots were re-used by exposing them to stripping buffer (Restoreä, Thermo Scienti c, Rockford, IL) and re-probed with a different primary antibody to enable direct comparison of phosphorylated and non-phosphorylated states. Blots were exposed to X-ray lm in a cassette, the lms scanned, and identi ed bands analyzed by densitometry using ImageJ 1.43u (NIH). Western blot data from the experimental groups were expressed relative to measured mean values from the CTL group. All results are expressed as the ratio of phosphorylated protein/total protein.
IGF-1 protein measurement by ELISA: Determination of IGF-1 in the adaptive hypertrophy experiments was performed as previously described [25]. Brie y, frozen RV muscle segments were pulverized in liquid nitrogen. Protein was extracted twice on cold acetic acid (1M; 1 mg/10 µl) and supernatant stored at -80ºC overnight. Aliquots (100 µl) were lyophilized and stored frozen overnight. Protein pellets were resuspended in 40 µl H 2 O and protein concentration determined as described above. Duplicate 10 µl protein samples were assayed using a commercial high sensitivity ELISA kit for rodent IGF-1 (AC-42F1) from Immunodiagnostic Systems, Inc. (IDS; Gaithersburg, MD) according to manufacturer's protocol.
Note, for the maladapted RV muscle IGF-1 assays a different kit was used, as the Immunodiagnostic Systems kit (AC-42F1) had been discontinued. Thus, for these experiments, we used a rat IGF-1 ELISA Kit (Catalog # 80573) from Crystal Chem (Elk Grove Village, IL). Results are expressed as ng/ mg protein. Real-time quantitative PCR: RT-qPCR was performed in two steps. One ug of total RNA per sample was reverse transcribed with Superscript III using Oligo (dT) 20 (Invitrogen) according to manufacturer's protocol. qPCR was performed in 10 ul reactions using 2X Sybr Green Master Mix (ABi P/N 4309155), 50 nM gene speci c primers, and 2 ul of the reverse transcribed samples. Samples were analyzed in triplicates across the genes on the ABi Viia7. In order to compare the relative mRNA expression between control and experimental groups, the comparative threshold cycle (C T ; the fractional cycle number at which the amount of ampli ed target reaches a xed threshold) method was used.

Histological Studies
Fibrosis: To assess brosis, cryosections of RV free wall from 5 adapted, 5 maladapted and 3 CTL rats were stained with Picrosirius Red as described [26,27]. Sections were imaged by bright eld microscopy and analysis was performed using ImageJ (Fiji), by measuring pixel density within the green channel throughout the entire RV free wall [28,29]. One section was analyzed for each animal and normalized to the total area of the RV free wall. Results are presented as percent brosis.

Statistical Analysis
The distribution of all data was tested for normality and then statistical analysis was performed using ANOVA (Instat v. 3.06, GraphPad, San Diego, CA) to compare differences between the independent groups in the adaptive state study. If a signi cant interaction was found, post hoc analysis (Student-Newman-Keuls test) was used to compare differences within independent groups. A Student t-test was used to compare differences between the groups in the maladaptive state study. An a level of 0.05 was used to compare differences in independent groups and to determine overall signi cance. Values are expressed as means ± SEM.

ADAPTIVE RV HYPERTROPHY Animal Weights and RV Pressures
During the RV adaptive phase of PAH, there was a progressive increment in CTL body weight, as expected. No difference in body weight was observed at day 7 between CTL and PAH rats. At days 14, 24 and 28 body weight gains were attenuated by 6, 8% and 21% respectively, for PAH animals compared to CTL (P < 0.01; Figure 1). An assessment of RV systolic pressures found similar pressures between CTL and PAH animals at day 7. Thereafter, a signi cant and progressive rise in RV pressures was observed in PAH rats at days 14, 24 and 28 (1.9, 2.6 and 2.8-fold-increment respectively; P < 0.001) compared with CTL animals (P < 0.001). RV systolic pressures of PAH rats at days 24 and 28 were not signi cantly different. [see Supplemental gure 1A in ref. 30].

Indices of RV Hypertrophy
RV mass and the ratio of RV/LV+S were similar between the CTL and PAH rats at days 7 and 14, indicating no RV hypertrophy at these times, despite signi cantly increased RV pressures at day 14. Signi cant RV hypertrophy however was evident at day 24, with RV mass 1.6-fold larger than CTL (P < 0.001) and the RV/LV+S index increased by 1.8-fold (P < 0.001). At day 28, RV mass was increased by by1.8-fold (P < 0.001) and RV/LV+S index by 2. Echocardiography TAPSE, a measure of RV systolic function, was measured in MCT rats and compared to CTL animals. No signi cant differences were observed between CTL rats and PAH animals 28 days post MCT administration. (CTL: 2.9 +/-0.7 mm; PAH: 2.8 +/-0.14 mm).
Estimated CO for PAH rats all time frames during the compensatory period were not signi cantly different from CTL values, and so the data was pooled for PAH animals (CTL: 124 +/-8 ml/min; PAH: 100 +/-4 ml/min). Values for CO for CTL rats were similar to those previously reported for male Sprague-Dawley rats of the same weight range [31].

Signaling Data
For all protein studies in the RV muscle, phosphorylated proteins are expressed as the ratio of phosphorylated (P) protein/total protein.
PI3K/Akt/mTOR pathway: Phosphorylated Akt, mTOR and down steam effectors, 4E-BP1 and p70 S6K were analyzed. As noted in Figure 2A, P-Akt and P-mTOR were not signi cantly different between CTL and PAH rats at days 7 and 14. However, signi cant increments for both signaling proteins were observed at day 24 in PAH animals compared to CTL (1.3 and 1.4-fold increment respectively; P < 0.05 and P < 0.01; Figure 2A). At day 28, levels of P-Akt and P-mTOR had returned to control levels.
The downstream effector P-p70 S6K was unchanged at days 7 and 14 in PAH rats, but signi cantly elevated at days 24 and 28 compared to CTL (1.6 and 2.4-fold increments respectively; P < 0.01 and P < 0.001; Figure 2B). The hyperphosphorylated γ form of 4E-BP1 was signi cantly elevated only at days 24 and 28 in PAH rats compared to CTL by 1.7 and 1.2-fold respectively (P < 0.001 and P < 0.05; Figure 2B). The ratio of γ/α+β+γ was increased only at day 24 (P < 0.05; not shown).

IGF-1 Protein Assays
IGF-1 protein expression in the RV muscle of PAH rats were similar to that in CTL animals between days 7and 24 (Figure 4). At day 28 a signi cant 45% reduction (P < 0.001) in IGF-1 expression in the RV of PAH rats was noted compared to CTL (Figure 4).

MALADAPTIVE RV and Right Heart Failure
Animal Weights and RV Pressures PAH rats were also studied 28 or more days post MCT administration until they develop RHF. During this period there was signi cant weight loss from peak weight achieved over time to weight at the time of terminal experiments (Peak weight: 362 +/-3 g; Terminal weight: 299 +/-11 g; P < 0.01). In addition, other observational signs suggestive of right heart failure were evident. This included a progressive listless state with obvious reduced mobility, poor grooming and ru ed fur and tachypnea with a pattern of increased work of breathing. Abdominal distension suggested the presence of ascites. Animals were sacri ced at this point, which was a joint decision by the investigators and veterinary staff. Post sacri ce, the presence of ascites +/-pleural effusions were con rmed.

Echocardiography
Mean value of TAPSE for this maladapted PAH cohort was signi cantly reduced compared to CTL and other adapted PAH animals (1.73 +/-0.144 mm; P < 0.01; Figure 5A).

Vascularity:
Changes in the RV free wall vascularity to PAH were assessed at both capillaries and arterioles levels. During adaptive RV hypertrophy the number of capillaries and arterioles in the RV free wall signi cantly decreased by 32 % (P < 0.05) and 21% (P < 0.05), respectively, compared to CTL ( Figure 6B). By contrast, in the maladaptive RHF/PAH cohort we observed a further signi cant decline in RV free wall capillaries and arterioles by 54% (P < 0.01) and 65% (P < 0.001), respectively, compared to the adaptive RV cohort ( Figure 6B).

Signaling Data
For all protein studies in the RV muscle, phosphorylated proteins are expressed as the ratio of phosphorylated (P) protein/total protein.

GSK-3 pathway:
P-GSK-3β was assessed in the hearts of maladapted PAH and CTL rats. There was no signi cant difference in phosphorylation of GSK-3β between maladapted/failed RV of PAH and CTL hearts ( Figure  8A).
Values for both P-p44 and P-p42 were similar between maladapted PAH and CTL rats ( Figure 8B & C). mTOR: In the maladapted RV muscle, phosphorylated mTOR was signi cantly increased (P < 0.01; Figure 9) suggesting activation by pathways separate from the PI3K/Akt signaling axis as P-Akt was markedly diminished ( Figure 7B). To further explain this, we examined another possible signaling pathway that has recently been described to activate mTOR with subsequent autophagy inhibition [32].
Autophagy pathway: The LC3BII/I ratio was analyzed as a surrogate marker of autophagosome formation. There was a signi cant decrease in the LC3BII/I ratio in maladapted/failed RV of PAH rats compared to CTL rats (P < 0.05; Figure 9). Given the well-known role mTOR plays in regulating autophagy, we analyzed phosphorylated mTOR as stated above and its downstream effector p70 S6K . P-mTOR and P-p70 S6K were signi cantly elevated in these PAH rats (P < 0.05; Figure 9). A recent publication identi ed a p27/CDK2 axis in the regulation of mTOR-dependent inhibition of autophagy during heart failure [32]. p27 was signi cantly decreased in maladapted/failed RV of PAH rats relative to CTL (P < 0.05; Figure 9). However, p-CDK2 was not statistically different (P = 0.22) in PAH rats which likely re ects variances and power in uences (Figure 9).

Discussion
In this study, we evaluated over time, signaling events related to 3 key pathways known to be involved the early adaptive and physiologic response of the RV to the increased afterload presented to it in terms of in muscle protein synthesis and muscle hypertrophy in the adaptive phase of RV hypertrophy in the MCT model of PAH. We further examined in the phase of RV systolic decompensation /failure (often described as the maladaptive phase) what changes in these signaling pathways ensue and their consequences, particularly with prolonged mTOR activation [33]. What became evident is that even in the adaptive hypertrophy phase, there was the start of increased RV muscle brosis and a decline in RV muscle capillarity both of which markedly progressed further in the hemodynamically de ned state of maladaptive hypertrophy. With regard to early increased

Adaptive RV Hypertrophy
The time course studies revealed that a signi cant increase in RV systolic pressure predated the development of RV hypertrophy in PAH animals, like that described in previous studies [30,34]. This suggests that pressure overload was an important early stimulus to RV hypertrophy in an adaptive fashion. Several phosphorylated proteins in the RV of PAH rats were upregulated from all 3 of the major signaling pathways evaluated, peaking for the most part at day 24 in which established RV hypertrophy was evident. During this phase TAPSE and CO were preserved indicative of preserved RV systolic function.

RV Fibrosis and Capillarity
An interesting nding in our studies was that even in the hemodynamically de ned state of adaptive RV hypertrophy in which TAPSE and CO were preserved and in which clinically, the rats exhibited no clinical features of RV failure (see below), there was evident a 2-fold increase in RV brosis compared with healthy controls and a 34% decrease in capillarity as depicted in Figure 6. This in part may not be that unexpected, as there are robust data that RV diastolic dysfunction is evident in animal models of PAH and which precedes RV systolic dysfunction. RV diastolic dysfunction, with associated RV wall stiffness, impaired lling and prolonged relaxation is felt to be associated with RV myocardial brosis [35][36][37]. The reduced capillarity in the adaptive phase is intriguing and suggests multiple impacts on angiogenesis even at this early stage. While not examined in this study, the reduced levels of IGF-1 in the RV myocardium may have in part contributed as IGF-1 is a potent angiogenesis factor [38]. Thus, a continuum of effects likely ensues in this progressive condition. Exactly what promotes the change is unknown. We speculate that when the burden of pathobiologic factors reaches a critical threshold, pathophysiologic features of RV systolic dysfunction and decompensation ensues.

Signaling Pathways
While there is an abundance of literature on the signaling pathways associated with cardiac hypertrophy, this re ects predominantly events in the LV. Data for the RV is limited, and especially so for serial measures over time.
PI3K/Akt/ mTOR pathway: In the present study, P-Akt, and P-mTOR were signi cantly upregulated at day 24 while downstream effectors, P-4E-BP1 and P-p70 S6K were signi cantly upregulated at days 24 and 28, suggesting signaling events likely to impact on cardiac muscle protein synthesis. Akt activation phosphorylates a key regulatory domain of mTOR, which acts on downstream targets 4E-BP1 and p70 S6K . Under conditions with low demand for mRNA translation and cardiac muscle protein synthesis, the translation repressor protein, 4E-BP1 sequesters eukaryotic initiation factor 4E (eIF4E) preventing its interaction with other initiation factors (e.g., eIF4G). Phosphorylation of 4E-BP1 leads to the dissociation of the 4E-BP1.eIF4e complex and the start of translation initiation [15]. This was indeed borne out in cardiac muscle (total heart homogenates) in which Vary and Lang [13] administered IGF-1 to rats showing increased phosphorylation of 4E-BP1and reduced association with eIF4E. IGF-1 administration also markedly increased P-Akt, P-mTOR and P-p70 S6K [13]). Of note, p70 S6K phosphorylates a 40S ribosomal protein S6, resulting in enhanced ribosomal binding capacity and protein translation for essential components of the muscle protein synthesis apparatus. The PI3K/Akt/mTOR pathway has also been shown to be upregulated in several models of hypertrophy of the LV, including in response to pressure overload [e.g., 10-12].
GSK-3 pathway: GSK-3 is known to play an important role in regulating glycogen metabolism in cardiac muscle. Further, its role in the integration of hypertrophic signaling in the heart has been increasingly emphasized [17]. While much importance has been placed on the β isoform of GSK-3, there is increasing appreciation that the α isoform can also mediate cardiac hypertrophy [16]. Phosphorylation of GSK-3α (Ser 21 ) and β (Ser 9 ) inhibits their protein kinase activities, which diminishes GSK-3's inhibitory effects on eIF2 to promote protein translation and hypertrophy. While inhibition of GSK-3 in cardiac myocytes can be mediated by several other kinases (see below), its inhibition is likely largely mediated via PI3K/Akt signaling, with IGF-1 (and insulin) the most potent activators of Akt [39].
In the present study, there was increased phosphorylation of both GSK-3α and GSK-3β to similar extents, which coincided in time with the onset of RV hypertrophy at day 24. While P-GSK-3α remained at control levels at day 28, surprisingly the levels of P-GSK-3β in the RV of PAH animals, was signi cantly reduced.
It is unclear what signal/s mediated this effect.
MAPK/ERK pathway: The MAPK comprises several family subtypes and is ubiquitously expressed amongst the body tissues, including in cardiac muscle [39]. The ERK1/2 subfamily is a prototypical pathway mainly responsive to growth factors (e.g., IGF-1 and insulin), while the p38 and JNK subfamilies are more "stress" responsive [40]. Activation of ERK1/2 by IGF-1 can occur via its tyrosine kinase receptor or independently, possibly via G proteins [41]. The Ras/Raf/Mek/ERK1/2 signaling pathways are regarded as having signi cant in uences in promoting cardiomyocyte hypertrophy, in both in vitro as well as in vivo cardiac speci c genetic models [18,20]. While ERK1/2 activation has been described with leftsided pressure overload [42], there is a paucity of data on ERK pathways in the context of right-sided pressure overload and RV hypertrophy. Increased levels of activated ERK were however reported in the RV in pulmonary artery banded fetal sheep [19]. It is thus of interest to report signi cantly increased expression of P-ERK1 and P-ERK2 at day 24 in PAH animals compared to controls, a time point at which signi cant RV hypertrophy emerged. In and of itself, it is unclear whether the ERK pathways are a dominant component of RV hypertrophy per se, or whether they only exert positive in uences in conjunction with the effects of other signaling pathways [39].

IGF-1
The IGF-1 data we report in this time series study of adaptive RV hypertrophy are intriguing. While the marked increases in mRNA abundance in the RV at days 24 and 28 appeared to support IGF-1 as the key driver of the signaling events and RV muscle hypertrophy, this was not borne out by the protein assays for IGF-1. Indeed, no signi cant increment in IGF-1 expression was noted at any time point and at day 28 a signi cant 45% decrement was noted. How do we explain this discordance and the signi cant increments in activated signaling proteins observed? We speculate that there was a signi cant stimulus to produce IGF-1 induced by the increasing loads imposed on the RV over time, as noted by the 6.5 and 7.4 -fold increment in RV IGF-1 mRNA abundance at days 24 and 28. However, this stimulus did not result in concomitant increments in protein expression of IGF-1.
We speculate that several possible factors may have acted to impair translation of IGF-1 mRNA in our model. Firstly, in ammation has recently been highlighted in the pathobiology of PAH and most certainly plays a role in the MCT model [43 -45]. Indeed, in ammatory cytokines such as tumor necrosis factor (TNF)-α can inhibit the action of IGF-1 in muscle [46]. Of interest, MCT -induced PAH in rats was attenuated by TNF-α antagonists via the suppression of TNF-α expression and the NF-κB pathway [47].
Further, MCT has been reported to activate NF-κB expression in the RV of the MCT-induced PAH model [48]. In addition, several in ammatory cytokines, including TNF-α, IL-1β and IL-6, are expressed in heart muscle and upregulated in heart failure and in the MCT model [49,50]. Thus IGF-1 expression may have been suppressed by the action of in ammatory cytokines. Signi cant body weight loss was observed in the PAH animals at days 24 and 28 compared with controls and malnutrition may also suppress IGF-1 expression in muscle tissue [25].
Secondly, it is possible that epigenetic factors such as in uences of microRNAs may have repressed mRNA translation of IGF-1. MicroRNAs are small conserved non-coding RNA molecules that ne tune gene expression by either enhancing the degradation of mRNA or by inhibiting its translation [51]. Indeed, microRNA-1(miR-1) was recently described to exhibit a feedback loop between it and the IGF-1 signaling cascade in cardiac muscle and that IGF-1 and its receptor are targets of miR-1 [52]. Further, there is an inverse relation between muscle IGF-1 and miR-1 [52], and several other miRs have been reported to in uence IGF-1 in muscle [53]. We thus speculate that several possible in uences in our model may have suppressed IGF-1 expression in the RV of PAH rats, despite a high stimulus to increase IGF-1protein expression.
While we focused our studies on 3 major signaling pathways, we cannot rule out in uences due to crosstalk with other pathways known to be implicated in cardiac muscle hypertrophy.

Crosstalk between signaling pathways
The pathways leading to RV hypertrophy in our model of PAH are clearly complex and likely involve signals from multiple key pathways as well as interaction with or crosstalk between the varieties of signals elaborated in this model. The calcium-dependent phosphatase, calcineurin, for example, has been implicated in pathological models of LV hypertrophy. Calcineurin dephosphorylates the transcription factor, NFAT-3 (nuclear factor of activated Tcells-3), which in combination with another transcription factor, GATA4, synergistically promotes cardiac gene transcription and hypertrophy [54,55]. Of note, IGF-1 increases P-GATA4 and its nuclear accumulation in cardiac myocytes [56], while GSK-3β and ERK1/2 have also been shown to activate GATA4 in vitro [57,58]. However, GATA4 has robustly been shown not to be required for IGF-1 induced cardiac hypertrophy, which can proceed in its absence [56]. Further, activation of ERK1/2 by IGF-1 in cardiomyocytes may in part be mediated by protein kinase C alpha (PKCα) [59]. In our model, P-p70 S6K was upregulated in the RV of PAH animals at days 24 and 28 likely mediated in large part by Akt/mTOR pathways. Interestingly, crosstalk between ERK and p70 S6K has been reported in cardiomyocytes [60].
Several pathways can signal through ERK to facilitate cardiomyocyte hypertrophy. In addition to growth factor signaling via tyrosine kinase receptors, signaling by adrenergic agonists, angiotensin and ET-1 via GqPCRs can also occur via MEK/ERK pathway signaling to mediate cardiac muscle protein synthesis and hypertrophy [39,62].

Maladapted RV Hypertrophy and Right Heart Failure
Following the period of adaptive and physiologic RV hypertrophy the RV becomes progressively decompensated with eventual development of overt RHF. Further, echocardiography data support the notion that these animals had failing RVs as evinced by very low TAPSE and CO values together with a marked increase in RV muscle brosis and marked decrement in RV muscle capillarity, compared to CTL and MCT-treated animals in the adaptive phase. To gain insight into possible alterations in those signaling pathway patterns we described during the adaptive phase, we re-evaluated the GSK-3, MAPK/ERK, and mTOR pathways in the RV muscle of rats in whom clear RV dysfunction and failure had ensued.

IGF-1
Reduced expression of RV muscle IGF-1 has been well described in animal models of PH complicated by RV failure and was reduced by almost 50% in our RHF cohort compared to control values. Indeed, reduced IGF-1 has been described as part of the RV muscle failure molecular signature complicating PAH [63]. This suggests that many of the protean actions of IGF-1 on cardiac muscle would be severely impaired, such as its anabolic in uences on RV muscle protein synthesis, as well as its anti-apoptotic, pro-angiogenic and positive inotropic actions, all of which could impair RV muscle function and contribute to the "sick" RV muscle milieu with RV decompensation and failure [64 -67].

Signaling Pathways
As highlighted above, much of the literature that relates to the signaling pathways we evaluated relates to the left ventricle. There are however limited data on the RV in the context of PAH which we refer to in support of our own data.

Phosphorylated Akt:
Data in our PH model complicated by a decompensated/failed RV show marked reduction in phosphorylated Akt. Similar ndings have been reported in several animal models of the pressure overloaded LV accompanied by LV hypertrophy and heart failure due to transverse aortic constriction [68,69] or with doxorubicin-induced cardiomyopathy [70]. The PI3K/Akt pathway has been highlighted as important in physiologic adaptive cardiac hypertrophy and to exert a protective role particularly under conditions of stress. Thus, the markedly diminished expression of p-Akt in the decompensated RV muscle would be expected to negatively impact on several signaling pathways known to promote a healthy RV milieu. Our ndings are in contrast to those reported for the Sugen/Hypoxia model of PAH and RV decompensation in which mTORC2-Akt levels were unaffected, while mTORC1 signaling in the RV muscle was upregulated [14].
GSK3 pathway: We were surprised that the expression of GSK-3β in the decompensated RV muscle was preserved. In states of nutritional stress, we reported that GSK-3 and ERK expression was suppressed along with reduced p-Akt [15]. However, it is of interest that GSK-3β was previously reported as increased in the RV of MCT treated rats [71] as well as in the LV of LV pressure overload and heart failure [69] with a late reduction reported. We speculate that the preservation of GSK-3 expression may re ect an attempt to preserve muscle protein synthesis in an otherwise stress-induced catabolic and apoptotic environment.

MAPK/ERK pathway:
In the present study, the RV expression of p42 and p44 in the maladapted state was preserved in our cohort. Similar to the data for GSK-3β, ERK1/2, levels in the pressure overloaded maladapted LV were initially increased [69]. The implications are likely similar as speculated for GSK-3β and there are data suggesting an anti-apoptotic function of cardiac ERK signaling [72]. mTOR: Another unexpected nding was a signi cant and marked increase in p-mTOR expression in the maladapted RV muscle, given the markedly reduced p-Akt. Thus, it appears that mTOR is not activated via the PI3K/Akt pathway in maladaptive RV hypertrophy, unlike signaling in adaptive RV hypertrophy.
Similarly, Deng and coworkers [73] reported increased p-mTOR in the decompensated/failed RV muscle 42 days post MCT administration in rats. Further, Pena and coworkers reported upregulation of mTORC1 in the maladapted RV of rats with severe PAH induced by Sugen/Hypoxia [14]. We thus evaluated other possible sources of mTOR activation independent of Akt and ERK signaling, as described below.

Dysregulated Autophagy and Implications for Right Heart Failure
Recently a p27/cyclin dependent kinase inhibitor (CDK2)/mTOR signaling pathway has been described in cardiac muscle regulated by miR-221 [32]. Activation of this pathway has been shown to increase the expression and activity of mTOR. In cardiomyocytes, p27 has been shown to be anti-hypertrophic and is down regulated in heart failure in response to pressure overload [74,75]. Moreover, mice de cient of p27 develop cardiac hypertrophy and heart failure [76]. What has also been observed in transgenic mice in which miR-221 (or miR-222) are overexpressed, is that there is the induction of heart failure with associated mTOR activation and autophagy inhibition [32,77].
Autophagy is a conserved intracellular process geared to remove misfolded proteins and damaged intracellular organelles which are digested in autophagolysosomes [78]. Autophagy inhibition mediated by activated mTOR, transcription factor EB, miR-365 and Eva-1 homologue A knockout, are all associated with the development of heart failure [79 -82]. Autophagy inhibition may also promote apoptosis of cardiomyocytes [77].
In the present study, we demonstrated that p27 is signi cantly downregulated in the maladapted RV muscle and that mTOR is increased with evidence to suggest autophagy inhibition as evinced by signi cant decrease in the LC3BII/I ratio, suggesting this signaling axis is active in our model. Further it has been proposed that pronged mTORC1 activation in the RV muscle in the Sugen/Hypoxia model of PAH results in decreased autophagy [33].

Conclusions
We began this investigation examining those signaling pathways thought to be key in cardiac muscle protein synthesis (PI3K/Akt/mTOR; GSK-3; ERK) in a temporal fashion during the development of adaptive hypertrophy in the MCT model of PAH. While the direction of change seen in the 3 major signaling pathways appear to support enhanced cardiac muscle protein synthesis during this evolving physiologic adaptation, the discordance between high IGF-1 mRNA and protein suggests that in the MCT model IGF-1 is not a major driver or that its attempts to drive anabolism are inhibited by multiple factors, as alluded to above. Examination of the 3 pathways in the maladapted/failed RV yield unexpected data and new data for RHF that autophagy inhibition may contribute to adverse RV pathobiology and poor RV function. Lastly we speculate taking all data into account that a continuum of changes characterizes the adaptive and maladaptive phases in the RV muscle and that a threshold effect in certain parameters likely determines whether overt pathophysiological changes (such as RV systolic dysfunction/failure) ensue as the disease progresses.  Ratio of "The p42/p44 MAP Kinase Pathway" (A) Ratio of phosphorylated p-GSK-3α (Ser21) to total GSK-3α and (B) p-GSK-3β (Ser9) to total GSK-3β in the right ventricle of control (CTL) and pulmonary arterial hypertension (PAH) animals at days 0, 7, 14, 24, and 28. (C) Ratio of phosphorylated p-p44 (Thr202) to total p44 and (D) p-p42 (Tyr204) to total p42 in the right ventricle of control (CTL) and pulmonary arterial hypertension (PAH) animals at days 0, 7, 14, 24, and 28. Values are means ± SEM; signi cantly different from CTL; signi cantly different from day 7 PAH; signi cantly different from day 14 PAH; # signi cantly different from day 24 PAH.

Figure 4
IGF-1 mRNA and proteins levels in the RV free wall during the adaptive phase of PAH (A) Abundance of IGF-1 mRNA and (B) IGF-1 protein in the right ventricle of control (CTL) and pulmonary arterial hypertension (PAH) animals at days 0, 7, 14, 24, and 28. Values are means ± SEM; signi cantly different from CTL; signi cantly different from day 7 PAH; signi cantly different from day 14 PAH; # signi cantly different from day 24 PAH.

Figure 5
Echocardiographic measurements of the RV in healthy and PAH animals (A) Values of TAPSE in normal controls (CTL) compared to that in pulmonary arterial hypertension (PAH) rats complicated by right heart failure (RHF). (B) Values of cardiac output (CO) in CTL animals compared to that in pulmonary arterial hypertension (PAH) rats complicated by RHF. signi cantly different from CTL. Values are means ± SEM.