Diaphragm Atrophy and Contractile Dysfunction in a Murine Model of Pulmonary Hypertension

Pulmonary hypertension (PH) causes loss of body weight and inspiratory (diaphragm) muscle dysfunction. A model of PH induced by drug (monocrotaline, MCT) has been extensively used in mice to examine the etiology of PH. However, it is unclear if PH induced by MCT in mice reproduces the loss of body weight and diaphragm muscle dysfunction seen in patients. This is a pre-requisite for widespread use of mice to examine mechanisms of cachexia and diaphragm abnormalities in PH. Thus, we measured body and soleus muscle weight, food intake, and diaphragm contractile properties in mice after 6–8 weeks of saline (control) or MCT (600 mg/kg) injections. Body weight progressively decreased in PH mice, while food intake was similar in both groups. PH decreased (P<0.05) diaphragm maximal isometric specific force, maximal shortening velocity, and peak power. Protein carbonyls in whole-diaphragm lysates and the abundance of select myofibrillar proteins were unchanged by PH. Our findings show diaphragm isometric and isotonic contractile abnormalities in a murine model of PH induced by MCT. Overall, the murine model of PH elicited by MCT mimics loss of body weight and diaphragm muscle weakness reported in PH patients.


Introduction
Pulmonary hypertension (PH) is a chronic, progressive disease characterized by inflammation and pulmonary vascular remodeling leading to high blood pressure in the pulmonary circulation [1]. Symptoms of PH include breathlessness, weakness, and fatigue. These symptoms have been traditionally attributed to pulmonary gas exchange abnormalities [2]. However, important factors in PH symptomatology that have emerged in recent years are cachexia and skeletal muscle weakness [3,4]. Inspiratory muscle weakness has been documented in PH patients [5]. The diaphragm is the primary muscle used during inspiratory breathing efforts; hence, depression of diaphragm force is of particular relevance for symptoms of breathlessness and fatigue experienced by patients with PH. Depression of muscle force can result from atrophy (decrease in fiber size or cross-sectional area) and contractile dysfunction (defined as force normalized for muscle cross-sectional area). Patients with PH also experience loss of body weight [4], which is associated with poor prognosis in chronic diseases, e.g., heart failure [6]. However, diaphragm weakness and loss of body weight remain poorly understood in PH.
Diaphragm weakness in PH results from loss of muscle fiber size (atrophy) and contractile dysfunction [3,7]. Contractile dysfunction has been shown by lower maximal diaphragm isometric tension normalized for cross sectional area in rats and humans with PH [7][8][9], whereas the effects of PH on diaphragm isometric force in a mouse model of PH remains unknown. In addition, impaired ventilatory function and diminished inspiratory pressure during dynamic maneuvers suggest diaphragm isotonic contractile impairments in patients with PH, but could also be due to abnormalities extrinsic to the diaphragm muscle (e.g., neurological or airway narrowing). However, it is unclear whether PH impairs diaphragm isotonic contractile properties.
Monocrotaline (MCT) has been widely used to induce PH and right ventricular (RV) failure in rodents [10][11][12][13][14][15]. To our knowledge, studies focusing on skeletal muscle abnormalities caused by PH and RV failure induced via MCT have been done solely in rats [8,14,16,17]. Rats are hypersensitive to MCT -10 times more sensitive than mice [12] -and become severely anorexic upon exposure to MCT [14]. While PH causes loss of body weight [4], it appears that PH per se does not necessarily cause anorexia in humans [18,19]. Mice receiving MCT develop PH and show a progressive loss of body weight like rats [15,20], but it is unknown if mice receiving MCT are anorexic. This aspect is relevant for further understanding diaphragm abnormalities in rodent models of PH.
The primary purpose of the present study was to investigate the effects of PH on diaphragm fiber atrophy and contractile properties in the mouse model of the disease induced by MCT. We found that PH caused diaphragm fiber atrophy and impaired isometric and isotonic contractile function in mice. As potential mechanisms for contractile dysfunction in chronic diseases, we tested for protein oxidation and selective degradation of myofibrillar proteins and found those variables were unaffected by the disease. A secondary purpose of our study was to examine changes in body weight and food intake in mice treated with MCT. We observed that progressive loss of body weight in PH mice occurred in the absence of anorexia.

Animals and procedures
We strictly followed all recommendations for the care and use of laboratory animals established by the National Institute of Health [21]. The study was also approved by the Institutional Animal Care and Use Committee at the University of Florida (protocol # 201105762), and all efforts were made to minimize animal suffering. We used C57BL6 male mice aged 8-12 wks old at the beginning of the experiments (Harlan, Indianapolis, IN).
Mice were individually caged, exposed to a 12:12 hr dark:light cycle, and had access to standard water and chow ad libitum. We administered weekly injections of 600 mg/kg MCT (20 mL/g body weight, subcutaneous) or equivalent volume of sterile saline for 6-8 weeks to induce PH as shown by Yamazato et al. [15]. We measured body weight and food consumption 2-36 per week. On the day of the experiment, we anesthetized the mice with subcutaneous injection of ketamine (100 mg/kg) and xylazine (15 mg/kg). When the animals reached the surgical plane of anesthesia, we performed a laparotomy to excise diaphragm, heart, and lungs. One hemidiaphragm was quickly frozen in liquid nitrogen for biochemical analyses. The other hemidiaphragm was used to for histology and to measure contractile properties in vitro (see below). The heart was dissected to remove atria, major vessels, and separate the right ventricle (RV) from left ventricle plus septum (LV+S). Lungs, RV, and LV+S were blotted dry and weighed. We also removed soleus and blotted dry prior to measuring weight.

Histology
We used standard procedures for tissue preparation as outlined elsewhere [22,23]. Briefly, diaphragm bundles were were embedded in Tissue-Tek freezing medium, frozen in liquid-nitrogencooled isopentane, and stored at 280uC. We sliced diaphragm bundles into 10 mm cross-sections using a Microm HM 550 cryostat (Microm International, Walldorf, Germany). Sections were incubated in 0.5% Triton X-100 solution for 5 minutes at room temperature, then washed in PBS for 5 minutes. Sections were subsequently incubated in anti-Laminin primary antibody (1:200; Sigma-Aldrich) for 1 hour at room temperature in a humid chamber. After primary antibody incubation, sections were washed in PBS 36 for 5 minutes and incubated in anti-Rabbit rhodamine red secondary antibody (1:100; Sigma-Aldrich) in blocking solution for 1 hour at room temperature in a humid chamber. Sections were then washed 36 for 5 min in PBS. We used a Zeiss Axio Observer-A1 microscope (Carl Zeiss Microscopy, Jena, Germany) and AxioCam MRm3 camera to capture images and NIH ImageJ software for image analysis. We traced the cell membrane envelope delineated by laminin to measure the area of all visible fibers from each diaphragm muscle section.

Isolated diaphragm contractile properties
We dissected a diaphragm strip along with rib and central tendon in bicarbonate-buffered solution (in mM: 137 NaCl, 5 KCl, 1 MgSO 4 , 1 NaH 2 PO 4 , 24 NaHCO 3 , 2 CaCl 2 ) gassed with a mixture of 95% O 2 and 5% CO 2 at room temperature. We used 4.0 silk suture to tie the rib to a glass rod and to attach the central tendon to a Dual-Mode Muscle Lever System (300C-LR, Aurora Scientific Inc., Aurora, Canada). The diaphragm strip was placed in an organ bath containing bicarbonate-buffered solution. We adjusted bundle length to attain maximal twitch tension (optimal length, Lo), increased the temperature of the organ bath to 37uC, added D-tubocurarine (25 mM) to the solution, and after thermoequilibration (20 min) started our force-frequency protocol. The isometric force-frequency protocol consisted of pulse frequencies of 1-300 Hz interspersed by 1 min intervals. The stimulation protocol consisted of supramaximal electrical current (600 mA) delivered through platinum electrodes using a biphasic high-power stimulator (701C, Aurora Scientific Inc.) with pulse duration of 0.25 ms and 300 ms train duration.
To determine the force-velocity relationship, we used afterloaded contractions employing a protocol similar to previous studies [24][25][26]. In this protocol, the bundle was maximally stimulated (300 Hz) to shorten against an external load corresponding to 2-80% P o above the resting tension. Each stimulus lasted for 200 ms and was delivered with 2 min intervals. We measured velocity at least 10 ms after the initial change in length in the linear portion of the tracing. We normalized shortening velocity per optimal length (L o ) and force per cross-sectional area (CSA, kN/m 2 ). To estimate the bundle CSA, we divided diaphragm bundle weight (g) by bundle length (cm) multiplied by muscle specific density (1.056) [27]. The force-velocity curve was plotted and fitted to the Hill equation [28]. We determined maximal shortening velocity (V max ) as the velocity at zero force in the force-velocity relationship. Power output for each isotonic contraction was also calculated as Force 6Velocity and is given in kN/m 2 6 Lo/s.

Preparation of diaphragm tissue lysates
We homogenized diaphragm samples on ice using Kontes Duall Tissue Grinders in muscle extraction buffer (20 mM Hepes; pH 7.4, 2 mM EGTA, 1% Triton-X100, 50% Glycerol, 50 mM b-Glycerophosphate, 16 Protease Inhibitor Cocktail, 16 Phosphatase Inhibitor Cocktail (Thermoscientific)) [29]. Homogenates were rotated end over end for 1 hr at 4uC and then sonicated. The samples were then centrifuged at 15,000 g for 2 min at room temperature to pellet insoluble debris. We kept the supernatant and determined its protein content using the DC protein assay (Bio-Rad Laboratories).

Immunoblotting
We loaded 10-15 mg of protein into 4-15% polyacrylamide gradient gels (Bio-Rad Laboratories) and performed electrophoresis at 200 V for 50 mins at room temperature, and then transferred proteins to low fluorescence PVDF membranes (Immobilon-FL) at 100 mA overnight at 4uC. We blocked the membranes in either Odyssey Blocking Buffer (LI-COR) for 1 hr at room temperature, and probed with a sarcomeric actin specific antibody (JLA 20; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) and a anti-troponin T1-T2-T3 antibody (TT-98; AbCam) diluted 1:1000 and incubated overnight at 4uC, followed by secondary antibodies (IRDye, LI-COR) diluted 1:10,000 and incubated for 45 minutes at room temperature. We quantified the bands using Odyssey Infrared Imaging system (LI-COR, Lincoln, NE). We stripped the membranes using 16 stripping buffer (LI-COR) for 20 minutes, re-probed with atubulin antibody (Developmental Studies Hybridoma Bank), and determined the abundance of a-tubulin, which was used for normalization purposes.

Protein Carbonyls Assay
We measured protein carbonyls in whole diaphragm homogenates using the Oxyblot kit according to manufacturer's instructions (Millipore Corporation). Briefly, we reacted 20 mg of protein with 2,4 dinitrophenylhydrazine (DNPH) and immuno-blotted (see above) using anti-DNP primary antibody and antirabbit secondary antibody (IRDye 800CW). We scanned the membranes and quantified integrated intensity in each lane using the Odyssey Infrared Imaging system (LI-COR). We stripped the membranes using 16 stripping buffer (LI-COR) for 20 minutes, re-probed with a-tubulin antibody (Developmental Studies Hybridoma Bank), and determined the abundance of a-tubulin. Protein carbonyls were normalized for a-tubulin.

Myofibrillar protein isolation [30]
We homogenized diaphragm samples with Kontes Duall Tissue Grinders in Standard Relax Buffer with Triton X-100 (SRB-X100; 75 mM KCl, 10 mM imidazole, 2 mM MgCl 2 , 2 mM EDTA, 1 mM NaN 3 , 1% v/v Triton X-100), and then centrifuged for 1 minute at 20,0006g at 4uC. We washed the pellet once with SRB-X100 and once with SRB (75 mM KCl, 10 mM imidazole, 2 mM MgCl 2 , 2 mM EDTA, 1 mM NaN 3 ) centrifuging at 20,0006 g for 1 min at 4uC and discarding the supernatant each time. The pellet was then resuspended in 1:20 (w/v) SDS-Page industrial buffer (8M urea, 2M thiourea, 0.05M Tris pH 6.8, 75 mM DTT, 3% SDS), vortexed for 1 h and centrifuged at 20,000 g for 10 min at room temperature. The supernatant containing the clarified soluble sample is the myofibrillar protein enriched fraction and was stored at 280uC. We determined protein concentrations of the myofibrillar protein enriched fraction using the RC DC Assay (Bio-Rad, Hercules, CA). We loaded 15 mg of protein into a 4-15% polyacrylamide gradient gel (Criterion precast gels; Bio-Rad, Hercules, CA), protein molecular weight standards (Precision Plus, Bio-Rad), and subjected to gel electrophoresis for 55 min at 200 V at room temperature. The gel was stained with Coomassie blue overnight (Thermo Fisher Scientific, Waltham, MA) and washed with dH 2 O three times. We quantified the optical density of individual bands and the whole lane (total protein) using the Odyssey Infrared Imaging system (LI-COR, Lincoln, NE) to determine the abundance of specific and total myofibrillar proteins. Specific proteins of interest were identified based on their known molecular weights, the band was quantified and data were normalized to total protein.

Statistical analysis
Data are shown as mean 6 SE. Statistical analyses were conducted using either Student's t-tests or two-way repeatedmeasures ANOVA followed by Bonferroni post hoc test (Prism 5.0b, GraphPad Software Inc., La Jolla, CA). We declared statistical significance when P,0.05.

Results
We administered MCT to a total of 14 mice. Of those, 2 animals died spontaneously and 2 were euthanized due to poor health conditions as recommended by institutional veterinarians. Thus, data from a total of 10 mice that survived the entire duration of the study (6-8 weeks) are reported here. In the cases of spontaneous death or euthanasia, the profile of changes in body weight resembled that of animals surviving the 6-8 weeks of treatment.
Several laboratories have used MCT to induce PH in mice [12,13,20]. The protocol we used has been shown to promote a 2.5 fold increase in right ventricular systolic pressure (RVSP) in C57BL6 mice over 8 weeks [13,15,20]. In this setting, PH causes RV hypertrophy defined by an increase in the ratio of RV weight to LV+S weight [RV/(LV+S)] of ,20-25%. Due to technical problems, we were not able to measure RVSP, but MCT increased RV/(LV+S) by ,1862% in our study. We also observed other typical signs of PH after 6-8 weeks of MCT injections, such as increased RV and lung weight (Table 1). Hence, we consider that mice in the MCT group developed PH consistent with previous studies [13,15,20] and, heretofore, refer to this group as PH for clarity.
PH caused a progressive decrease in body weight after the first week of treatment ( Figure 1). The decrease in body weight was accompanied by ,1561% lower soleus weight (Table 1). Importantly, food intake normalized for body weight was similar between groups and unchanged over the course of 8 weeks (Fig. 1C). The outcome was the same when absolute food intake was analyzed as average food intake during the 8-week period was 4.260.2 g/day for control and 4.360.4 g/day for PH.
We conducted morphological analysis in cross-sections of diaphragm bundles and measured diaphragm function in isolated bundles in vitro. Fiber cross-sectional area was decreased by 25% in PH mice ( Fig. 2A-B). Due to problems in trials of RVSP measurements (see above), we were unable to determine diaphragm contractile properties in 4 saline-injected mice. Saline injections had no effect on diaphragm contractile properties when compared to data from age-matched mice. Thus, we included in our control group data of diaphragm function from age-matched mice that did not receive injections. PH decreased submaximal and maximal specific isometric tetanic force by 25-30% (Fig. 2C). Twitch force was unchanged (6.360.4 N/cm 2 for control; 5.560.45 for PH) and, as a consequence, the twitch-to-tetanus ratio increased in PH. Time to peak tension (1460.5 ms for control; 1561 ms for PH) and one half relaxation time (1761.1 ms for control; 1761.4 ms for PH) did not differ between groups.
An important and novel observation was that PH impaired isotonic contractile properties in the diaphragm. Specifically, maximal shortening velocity (V max ) was 40% slower and peak power was 63% lower in PH than control (P,0.05; Fig. 3), despite a 138% increase in curvature of the force velocity relationship. The differences in the force-velocity characteristics remained after normalizing for P o (Fig. 3A).
Based on our findings of impaired isometric and isotonic diaphragm contractile function, we examined post-translational modifications of myofibrillar proteins as potential mechanisms of dysfunction. We measured total protein carbonyls as marker of oxidation and found no change in the diaphragm of PH mice ( Fig. 4). Nor were there changes in the abundance of carbonyls in individual bands (data not shown). Protein content measured by the RC-DC assay in myofibrillar protein enriched fractions was similar in control (1.2060.17 mg/ ml and PH (1.1060.38 mg/ml). Based on the existing literature, we focused our analysis of myofibrillar protein abundance on MHC, actin, and troponin T. We found that the optical density of the band corresponding to myosin heavy chain (MHC, ,220 kDa) in myofibrillar protein enriched fractions was similar in PH and controls (Fig. 5A). We also found that sarcomeric actin (Fig. 5B) or troponin T (Fig. 5C) were unchanged in whole muscle homogenates (Western blots).

Discussion
The main findings of our study were that PH caused diaphragm atrophy and impaired both isometric and isotonic contractile properties in mice. Moreover, food consumption of PH mice was unchanged despite a progressive decrease in body weight over 6-8 weeks. These alterations in contractile function occurred in the absence of increases in protein oxidation or decreases in myofibrillar protein abundance.
Patients with PH experience loss of body weight, although the exact prevalence is unknown [4]. Patients with PH who experience loss of body weight have increased mortality rate after lung transplantation [31]. Loss of muscle mass is an important component of loss of body weight in chronic diseases [32][33][34]. Accordingly, soleus muscle mass of PH mice was 15% lower than control. The decreases in body and muscle weight were not caused by differences in food intake. In contrast, rats show ,78% decrease in food intake 20 days after a single MCT injection compared to control [14]. This is an important observation when considering the experimental design of studies using MCT in rodents. To our knowledge, there are no published data showing that PH patients become anorexic, and it appears that PH per se does not cause anorexia in patients [18]. Our study shows that loss of body weight in PH mice is accompanied by decreases in muscle mass, and these changes are not caused by anorexia. Hence, our findings suggest that mice can be used to investigate the mechanisms of diaphragm contractile dysfunction in PH without confounding effects of anorexia induced by MCT.
Patients with PH have a 25-35% lower maximal inspiratory pressure than healthy peers [6,35], which suggests diaphragm weakness. This weakness can arise from fiber atrophy and contractile dysfunction. Previous studies found diaphragm fiber atrophy in rats and patients [3]. Moreover, there was diminished specific force (force normalized for cross sectional area) in diaphragm bundles of rats with PH induced by MCT [3]. The decrease in specific force was evident in permeabilized single fibers of rats and patients with PH [3,7], showing that PH causes dysfunction of the contractile apparatus.
In agreement with previous studies [3,7], we found diaphragm fiber atrophy and decreased submaximal and maximal specific isometric tension in PH mice. The effects of PH on the diaphragm force-frequency relationship were slightly different in mice and rats. In mice, force was depressed predominantly at high stimulus frequencies (.50 Hz). In rats, the depression of force occurred at all frequencies [3]. These discrepancies might reflect the hypersensitivity of rats to MCT. The important point, however, is that  mice injected with MCT to cause PH show diminished isometric specific tetanic force as seen in patients with PH. Specific force (reported here in kN/m 2 ) represents force normalized for bundle cross sectional area, thus a decrease in specific force suggests contractile dysfunction independent of changes in muscle or fiber size. Thus, the ability of the diaphragm to generate force (or pressure) in PH is dimished due to combined effects of decreased fiber size (atrophy) and contractile dysfunction.
The force-frequency characteristics provide useful information regarding the force generating capacity of diaphragm, but existing data are limited to isometric contractions. The diaphragm rarely performs isometric contractions in vivo [25]. Rather, it shortens against submaximal loads. Therefore, shortening velocity and power output are more physiologically relevant properties of diaphragm function. Patients with PH show a 20% decrease in maximal sniff nasal pressure [6] -a volitional dynamic maneuver that involves diaphragm shortening. Similarly, maximal voluntary ventilation is 30% lower in patients with primary PH than in healthy controls [2]. The decrease in sniff nasal pressure and maximal voluntary ventilation in PH patients suggest, among other factors, impairment in isotonic contractile properties of the diaphragm. Indeed, we found 40% slower V max and 60% lower peak power in the diaphragm of mice with PH. There was also a downward shift in the force-velocity relationship when submaximal loads were normalized for maximal tetanic force, e.g., Fig. 3A. Because we used afterloaded isotonic contractions, the downward shift reflects slower cross-bridge kinetics due to factors intrinsic to cross-bridge cycling or the level of calcium activation [24]. Importantly, afterloaded isotonic tests simulate the contractile profile in vivo, and our data supports the notion that PH impairs the ability of the diaphragm to sustain ventilatory tasks. Impairment of isotonic function could, then, contribute to dyspnea in PH. However, the mechanisms underlying contractile dysfunction are unclear.
Oxidants decrease calcium release and calcium sensitivity of the contractile apparatus [36,37], and impair sarcomeric protein function [38]. However, PH did not change protein oxidation status measured by abundance of carbonyls in whole-diaphragm homogenates. Our results corroborate the data from a recent study showing unchanged protein carbonyls in PH rats [9]. Administration of MCT in rats increases protein carbonyls in limb muscles [16]. Thus, protein oxidation may be specific of limb muscles or become apparent after the development of right ventricular failure. Additionally, we cannot discard the possibility that PH increases other oxidant-mediated modifications (e.g., thiol oxidation). Based on the current and recent data [16], we consider that PH-induced diaphragm isometric and isotonic contractile dysfunctions are not caused by increased protein oxidation.
A mechanism that has been proposed to explain diaphragm weakness in chronic diseases is selective degradation of myofibrillar proteins [39][40][41]. A recent study has shown that MHC abundance was unchanged in rats with PH induced by MCT [16], which is consistent with our data in mice. Loss of thin-filament proteins could also contribute to contractile dysfunction. PH elevates systemic levels of pro-inflammatory cytokines [17]. The pro-inflammatory cytokine interleukin 1 promotes degradation of sarcomeric actin [42], which would cause contractile dysfunction. Tumor necrosis factor-a stimulates selective degradation of troponin T that results in diminished isometric force [43]. We measured the abundance of actin and troponin T and found that both were unchanged in MCT mice. Thus, our findings substantiate the concept that selective degradation of myofibrillar proteins does not appear cause contractile dysfunction in PH [7]. Rather, post-translational modifications are the likely cause of isometric and isotonic contractile impairments in PH. Identification of these post-translational modifications was beyond the scope of our investigation, but PH may affect the phosphorylation or thiol oxidation status of myofibrillar proteins [44][45][46].

Conclusion
Our conclusion is that MCT-induced PH causes diaphragm atrophy and impairs diaphragm isometric and isotonic contractile properties in mice. These perturbations in isotonic diaphragm contractile properties likely play a role in impaired ventilatory responses of PH patients [47]. Contractile dysfunction in PH mice occurs in the absence of selective degradation of myofibrillar proteins or accumulation of protein carbonyls, which suggests post-translational modifications of myofibrillar proteins such as phosphorylation and thiol oxidation as alternative mechanisms for contractile impairments. Importantly, MCT causes loss of body weight but not anorexia.
Hence, our findings suggest that mice can be used to investigate the mechanisms of diaphragm muscle weakness and atrophy in PH without confounding effects of anorexia induced by MCT.

Author Contributions
Conceived and designed the experiments: BA LFF. Performed the experiments: BA HME MA LFF. Analyzed the data: BA HME MA ARJ LFF. Contributed reagents/materials/analysis tools: ARJ LFF. Wrote the paper: BA HME ARJ LFF.