Right Ventricular Myocardial Oxygen Partial Pressure Is Reduced in Monocrotaline-induced Pulmonary Hypertension in the Rat and Restored by Myo-inositol Trispyrophosphate

Pulmonary arterial hypertension (PAH) initially results in compensatory RV hypertrophy, but eventually in RV failure. This transition is poorly understood, but may be triggered by hypoxia. Measurements of RV oxygen tension (pO 2 ) in PAH are lacking. We hypothesized that RV hypoxia occurs in monocrotaline-induced PAH in rats and that myo-inositol trispyrophosphate (ITPP), facilitating oxygen dissociation from hemoglobin, can relieve it. Rats received monocrotaline (PAH) or saline (control) and 24 days later echocardiograms, pressure-volume loops were obtained and myocardial pO 2 was measured using a uorescent probe. In PAH mean pulmonary artery pressure more than doubled (35±5 vs. 15±2 in control), RV was hypertrophied, though its contractility was augmented. RV and LV pO 2 was 32±5 and 15±8 mmHg, respectively, in control rats. In PAH RV pO 2 was reduced to 18±9 mmHg, while LV pO 2 was unchanged. RV pO 2 correlated with RV diastolic wall stress (negatively) and LV systolic pressure (positively). Acute ITPP administration did not affect RV or LV pO 2 in control animals, but increased RV pO 2 to 26±5 mmHg without affecting LV pO 2 in PAH. RV oxygen balance is impaired in PAH and as such can be an important target for PAH therapy. ITPP may be one of such potential therapies.


Introduction
Pulmonary arterial hypertension (PAH) is a condition involving increased pulmonary vascular resistance (PVR) that results in increased mean pulmonary artery pressure (mPAP) and afterload of the cardiac right ventricle (RV). This initially results in compensatory RV hypertrophy, but when RV is no longer able to compensate for the increased afterload, eventually RV failure occurs resulting in patient's death [1].
The cause of this transition from compensatory RV hypertrophy to decompensated RV failure is unclear [2]. Several lines of evidence indicate that impaired RV metabolism [3] and in particular RV hypoxia, i.e. reduced myocardial oxygen tension, may underlie this phenomenon [4,5]. First, in PAH oxygen delivery is impaired due to increased extravascular compression of RV coronary vessels (due to both RV hypertrophy and increased RV pressures) and reduced coronary perfusion pressure (due to reduced aortic pressure as a consequence of reduced LV cardiac output). Second, increased RV afterload results in proportionally increased energy demand. Third, capillary rarefaction was found in various animal PAH models as well as in humans [6]. Fourth, these changes are accompanied by adverse metabolic reprogramming [7]. Fifth, increased activation of right ventricular hypoxia-inducible factor-1 (HIF-1) pathway, a marker of tissue hypoxia, was found in various models of PAH [8].
However, direct measurements of RV myocardial oxygen content are lacking due to technical di culties. Indirect methods, utilizing e.g. 1 H NMR techniques which estimate the ratio of deoxygenated to oxygenated myoglobin or magnetic resonance imaging based on differences in magnetic properties between oxygenated and deoxygenated hemoglobin [9] exhibit poor speci city and spatial resolution, but indeed suggest that RV oxygen partial pressure (pO 2 ) may be reduced in humans with PAH. However, some studies suggest normal RV oxygen availability [10], while other studies indicate the presence of so called pseudohypoxia, i.e. activation of hypoxia-dependent molecular pathways without true tissue hypoxia [11]. delivery to hypoxic tumors [14], and reduces HIF-1 expression by vascular endothelial cells [15]. This is accompanied by a potent anti-cancer effect in various tumor models in the mouse [14,16]. Moreover, we have recently shown that chronic ITPP treatment partially prevents post-myocardial infarction heart failure in the rat model [17]. However effects of ITPP on myocardial pO 2 have never been studied.
Thus the aim of our study was to verify the hypothesis that RV is hypoxic in the rat model of monocrotaline (MCT)induced PAH and that acute ITPP administration is able to relieve it. For this purpose we used a speci c uorescencebased method to directly measure myocardial pO 2 in situ in PAH and assess the effects of ITPP treatment.

Methods
Forty four male Wistar rats, weighing 180-220 g, were used. All study animals were used in compliance with local and institutional regulations. The study conformed to the Guide for the Care and Use of Laboratory Animals, US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and was approved by the local ethics committee (Second Warsaw Local Ethics Committee for Animal Experimentation). The manuscript follows the recommendations in the ARRIVE guidelines. ITPP was a kind gift from professor Jean-Marie Lehn, UNISTRA, University of Strasbourg to CK.

Study protocol
At the age of 4-5 weeks the rats received a subcutaneous injection of MCT (60 mg/kg, Sigma) to induce pulmonary hypertension (n=26) or saline (n=18) as a control group. Twenty four days later the animals from both control (n=13) and MCT (n=14) groups were anesthetized using inhaled 2% iso urane, underwent echocardiographic examination, followed by hemodynamic evaluation and pO 2 measurements. Thereafter they were euthanized and their lungs were processed for histological examination.
The remaining animals from the control (n=5) and MCT (n=5) groups underwent echocardiographic examination, received a single intraperitoneal dose of ITPP (1.5 g/kg b.wt.) and 1 hour later were re-anesthetized, underwent echocardiographic examination, followed by hemodynamic evaluation and pO 2 measurements. Thereafter they were euthanized and their lungs were processed for histological examination. A total of 7 rats died in the MCT group, while there were no deaths in the control group.

Echocardiographic evaluation
Transthoracic echocardiography was performed using E-cube 15 Platinum (Alpinion Medical Systems) with 17 MHz linear transducer under a light iso urane sedation. The rats were placed on a heating pad to sustain proper body temperature.
Images of the parasternal short-axis view (SAX) at the papillary muscle level, parasternal long axis view (PLAX) and the apical 4-chamber view (4CH) were. LV and RV fractional shortening as well as LV and RV diastolic and systolic wall thickness were assessed using the M-mode in PLAX view. All measurements were obtained by one observer blinded to the study groups.

Hemodynamic evaluation
As reported previously [18], rats were put on a heating pad, anesthetized with 2% iso urane, intubated and put on an animal ventilator. The upper abdominal cavity was opened and the heart was exposed through cutting of the diaphragm. The left ventricular and subsequently right ventricular apex was punctured with a 25G needle and a microtip pressurevolume (PV) catheter (SPR-838, Millar Instruments; Houston, TX) was inserted into the LV. Its position was established based on pressure and volume signals. After stabilization for 5 minutes, the signals were continuously recorded at sampling rate of 1000/s using an ARIA P-V conductance system (Millar Instruments) coupled to a PowerLab/4SP A/D converter (AD Instruments; Mountain View, CA) and a personal computer. To characterize cardiac function, rst the inferior vena cava was compressed for 10 seconds and then released to achieve reduction and augmentation of venous return and cardiac preload, respectively. Heart rate, maximal LV and RV systolic pressure (ESP), end-diastolic pressure (EDP), maximal slope of systolic pressure increment (+dP/dt max) and diastolic pressure decrement (−dP/dt max), ejection fraction (EF), end-diastolic volume (EDV), end-systolic volume (ESV), stroke volume (SV), and cardiac output (CO) were computed using a cardiac P-V analysis program (PVAN3.2, Millar Instruments). Indexes of contractility and stiffness [slope of end-systolic and end-diastolic P-V relations (ESPVR and EDPVR)] were also calculated using PVAN3.2.

pO 2 measurements
Myocardial tissue pO 2 was measured using a beroptic oxygen-sensing device, the OxyLite Pro pO 2 monitor (Oxford Optronics Ltd., Oxford, UK). This device measures pO 2 by determining the oxygen-dependent uorescent lifetime of ruthenium chloride. Probes are supplied precalibrated by the manufacturer. The tip of an optical ber probe is covered with ruthenium chloride, which uorescence lifetime is O 2 -dependent and is inversely proportional to the pO 2 in the tissue.
Following excitation by a ash of green light, the measured half-life of the phosphorescence signal can be quantitatively related to the oxygen tension.
The rats were anesthetized using inhaled iso urane (2%) and connected to a ventilator. The heart was exposed by leftsided thoracotomy, and a 20-gauge needle was used to pierce the epicardium and guide the probe into the myocardium immediately beneath the epicardium. A 100-µm diameter sensing tip was inserted into three locations in the cardiac RV and LV as well as in the liver and spleen. The pO2 signal was recorded until the signal was stable. Subsequently data were averaged for each location.
Under baseline conditions the animals were mechanically ventilated with 30-40% inspiration oxygen, which is above the ambient air content of oxygen (21%) with spontaneously breathing. The higher inspiration oxygen levels are necessary to compensate for the known ventilation-perfusion defects caused by anesthesia and mechanical ventilation, especially in diseased animals. Mechanical ventilation with a FiO 2 of 21% resulted in an arterial tension of approximately 80 mmHg, below the 100 mmHg normally obtained with spontaneous breathing, indicating that the use of ambient air oxygen levels with mechanical ventilation and anesthesia was indeed associated with insu cient blood oxygenation [10]. Finally 100% oxygen was used to ventilate the rat lungs and the pO 2 measurements were repeated.

Histological lung analysis
The lungs were in ated with OCT (Optimal Cutting Temperature) compound and then placed in plastic molds lled with OCT and snap-frozen in liquid nitrogen. All tissues were maintained at −80°C until further analysis.
The representative lung parts were cut into 5 µm-thick sections using Cryostat Microm HM550 (Thermo Fisher Scienti c, Massachusetts, USA) and stained with the hematoxylin and eosin (H&E) stain for microscopic analysis. Arterioles with an external diameter of 30-90 µm were assessed and % wall thickness was expressed as (2×wall thickness / external vessel diameter).

Statistical analysis
Shapiro-Wilk test was used to test normality of data distribution. Homogeneity of variances was tested by Bartlett's. Normally distributed data were expressed as means ± SEM. One-way ANOVA was used to test differences between groups. Tukey post hoc test was used to compare data pairs. Non-normally distributed data were presented as median ± inter-quartile ranges + outliers. Subsequently statistical analysis of differences was tested using non-parametric methods (Mann-Whitney test to compare two groups or Kruskal-Wallis ANOVA to compare three or more groups followed by Dunn's post hoc test). Pearson correlation analysis was used to analyze the correlations. Differences were considered signi cant when P < 0.05. Statistical analyses were performed using SigmaPlot version 14.
RV was dilated, which was re ected by increased RV end-diastolic volume (RVEDV, Fig. 1E-H Fig. 2A-B), while LVEDV was reduced ( Fig. 1E-H, Fig. 2A-B). Normalized right ventricular (RV) mass (Table 1) and RV diastolic wall thickness (Fig. 2C) were increased by 50%, while the corresponding parameters for LV were unchanged in PAH rats. Ventricular function was characterized using echocardiography and pressure-volume (PV) loops. RV fractional shortening (FS, Fig. 2D) and ejection fraction (EF, Table 1) were impaired and RV PV loops were shifted to the right (Fig. 1E-H).
However, maximum rate of RV pressure increase (RV dP/dt max) was slightly increased, while the most precise and loadindependent index of contractility, slope of end-systolic pressure-volume relation (ESPVR) was increased by almost 3-fold (Table 1), indicating that RV contractility was actually augmented in our model and impairment of EF and FS was due to increased afterload. RV hypertrophy was not su cient to prevent increase of RV wall stress: the diastolic wall stress was more than doubled, while the systolic wall stress -more than tripled (Table 1).
On the other hand, LV was unloaded and a result of reduced RV function, resulting in leftward and downward shift of LV PV loops (Fig. 1H) and a low grade reduction of LV diastolic and systolic wall stress (Table 1).

Myocardial oxygen content
Mean myocardial pO 2 was 32±5 mmHg and 15±8 mmHg in RV and LV (Fig. 3A-D), respectively, in the control animals, while that in the liver and spleen was 19±11 and 15±11 mmHg, respectively. Breathing with 100% oxygen did not affect these values (Fig. 3A-D).
Myocardial oxygen delivery depends on 2 crucial factors, perfusion pressure and vascular resistance. Therefore, to gain insight into potential causes of this reduction of RV pO 2 we correlated myocardial pO 2 with LV systolic pressure (LVSP, as a measure of aortic pressure, supplying perfusion pressure to coronary arteries) and with RV wall stress (as a marker of external vascular compression). As Fig. 3F and 3H show, RV pO 2 was highly negatively correlated with RV diastolic wall stress and positively correlated with LVSP. No such correlation was found for the LV pO 2 values ( Fig. 3G and 3I).

Effect of ITPP
In additional experiments (n = 5) a single dose of ITPP was given intraperitoneally and 1 hour later pO 2 measurements were performed.
ITPP had no effect on pO 2 values in the control animals: both LV and RV (Fig. 3D) as well as hepatic and splenic (not shown) oxygen partial pressures were not affected. 100% oxygen breathing had no effect in this setting, either. However, in PAH animals, RV pO 2 was signi cantly increased by acute ITPP administration by 44% to 26±5 mmHg. ITPP did not affect pO 2 values in the LV (Fig. 3E) or spleen or liver (not shown). 100% oxygen did not affect RV pO 2 values in MCT-treated rats that received ITPP.

Discussion
In this paper we reveal two new crucial facts regarding myocardial oxygen partial pressure in a rat model of monocrotaline-induced PH: (1) we demonstrate that PAH is associated with reduced RV pO 2 and that this reduction correlates with both reduced LV systolic pressure and increased RV diastolic wall stress and that (2) a new agent, ITPP, that increases oxygen dissociation from hemoglobin, improves RV pO 2 in PAH without affecting it in the healthy RV or LV.
RV function and structure in our pulmonary arterial hypertension model In our model of MCT-induced PAH, RV exhibited augmented contractile function as manifested by more than 3-fold increase of ESPVR and marked hypertrophy and dilation corresponding to a compensated stage of RV hypertrophy required to overcome more than 4-fold increase of RV afterload. However, this was accompanied by reduction of cardiac output that in turn reduced LV preload and its output, resulting in decreased LV pressures and afterload. Thus, paradoxically RV wall stress increased, LV wall stress decreased and RV wall stresses were higher than those in LV, unlike in normal hearts.

RV myocardial oxygen content
Here we used direct myocardial pO 2 measurements utilizing a novel uoroscopic method. Brie y the system uses a ruthenium chloride uorescent compound immobilized in a polymer at the tip of a ber-optic probe, which is 100 µm in diameter. Green light produced by diodes within the unit is propagated down the optical ber to the tip, where it excites the ruthenium chloride. The lifetime of the resultant uorescence is inversely proportional to the amount of oxygen at the circular area with a diameter of 230 µm around the tip. Thus this method offers great spatial resolution.
Here we showed that RV pO 2 was more than 2-fold higher than LV pO 2 (32±5 mmHg and 15±8 mmHg, respectively) in control rats. These values for LV are within the ranges reported by other authors and obtained using different methods: 45 mmHg in the LV of humans undergoing bypass surgery using voltammetric microelectrode technique [19], 35 mmHg of mitochondrial O 2 in the rat LV [20], 10 mmHg in the rat LV [21] and 8.6 mmHg in the swine LV using electron paramagnetic resonance oximetry [22]. These results indicate that LV pO 2 values in the literature vary considerably and are probably method-dependent.
However, we are the rst to report that RV pO 2 is markedly higher than LV pO 2 in control animals. It has long been known that the pO 2 in the veins draining RV is higher than in those draining LV [23] and that O 2 extraction is higher in the LV than in the RV [23]. As RV oxygen consumption increases from rest to exercise, the initial effect is an increasing oxygen extraction (indicated by the falling venous oxygen tension). This increased oxygen extraction in the RV occurs with little change in ow when venous tension is above 10-12 mmHg. Conversely, in the LV increases in myocardial oxygen consumption result in negligible decreases in venous oxygen tension and large increases in coronary ow [23]. Our data nicely support these observations, indicating that indeed resting myocardial pO 2 is much higher in RV than in LV. Higher extravascular compression in LV than in RV may be responsible for this phenomenon [24].
Furthermore we showed here that RV pO 2 was reduced by more than 40% in PAH, while that in the LV was unchanged.
This reduction correlated with both indices of reduced perfusion pressure (LVESP) as well as with indices of extravascular compression and oxygen utilization (RV wall stress). Thus it is di cult to determine, which of these factors was the main culprit here. Of note, no such correlation was found for LV pO2.

Effects of ITPP on myocardial oxygen
A nity of hemoglobin for molecular oxygen is regulated in human red blood cells by 2,3-diphospho-D-glycerate II (DPG), an allosteric effector of hemoglobin. ITPP binds to the same allosteric pocket, resulting in rightward shift of hemoglobin-O 2 dissociation curve, especially under low O 2 conditions [12], which could explain its speci c effects found in hypoxic tumor tissues [14]. Moreover, ITPP enters red blood cells through the Band3 transporter, which distribution is mainly restricted to erythrocytes, which is responsible for its red blood cell speci c a nity [13,15].
Here we showed that ITTP increased RV pO 2 in the PAH rats. What is interesting, RV pO 2 in PAH rats (18 mmHg) was still higher than LV pO 2 both in control rats (15 mmHg) and PAH rats (14 mmHg). However, acute administration of a single dose of ITPP improved only RV pO 2 in PAH rats (to 26 mmHg), without affecting LV pO 2 . This again stresses the fact that oxygen supply and utilization is differently regulated in RV and LV and what is acceptable for LV, is hypoxic for RV. This may be due to different density of mitochondria and capillaries between these two ventricles [10], but factors responsible for these differences remain unknown.

Conclusions
We show that RV pO 2 is markedly reduced in the rat model of MCT-induced PAH and that a new agent, ITPP, that facilitates oxygen release from hemoglobin peci cally under hypoxic conditions, is able to restore it upon acute administration. This indicates that RV is indeed hypoxic in PAH and oxygen supply and more broadly RV energetics can be an important target for PAH therapy; ITPP may be one of such potential therapies. Future studies are needed to verify this hypothesis.   ventricle on panels (D) and (E), respectively. Correlations between RV and LV pO2 and wall stress as well as LV systolic pressure are shown on panels (F) through (I). Horizontal lines represent mean (red) and median (black), the bottom and the top of the boxes represent the upper and the lower quartile, he whiskers represent 10th and 90th percentile, while the solid circles represent the individual data points. * p<0.05.