The calcilytic drug Calhex-231 ameliorates vascular hyporesponsiveness in traumatic hemorrhagic shock by inhibiting oxidative stress and mitochondrial fission CURRENT STATUS: UNDER REVIEW

Background The calcium-sensing receptor (CaSR) plays a fundamental role in extracellular calcium homeostasis in humans. Surprisingly, CaSR is also expressed in non-homeostatic tissues and is involved in regulating diverse cellular functions. The objective of this study was to determine if Calhex-231 (Cal), a negative modulator of CaSR, may be beneficial in the treatment of traumatic hemorrhagic shock (THS) by improving cardiovascular function, and investigated its relationship to oxidative stress and the mitochondrial fusion-fission pathway. Methods Rats that had been subjected to traumatic hemorrhagic shock were used as models in this study. Hypoxia-treated vascular smooth muscle cells (VSMCs) were also used. The effects of Cal on cardiovascular function, animal survival, hemodynamic parameters, and vital organ function in THS rats were observed, and the relationship to oxidative stress and mitochondrial fusion-fission was investigated. Results Cal significantly improved hemodynamics, elevated blood pressure, increased vital organ blood perfusion and local oxygen supply, and markedly improved the survival outcomes of THS rats. Furthermore, Cal significantly improved vascular reactivity after THS, including the pressor response of THS rats to norepinephrine (NE), and also the contractile response of superior mesenteric arteries, mesenteric arterioles, and isolated VSMCs to NE. Cal also restored the THS-induced decrease in myosin light chain (MLC) phosphorylation, which is the principal mechanism responsible for VSMC contraction and vascular reactivity. Inhibition of MLC phosphorylation antagonized the Cal-induced restoration of vascular reactivity following THS. Cal decreased oxidative stress indexes and increased antioxidant enzyme levels in THS rats, and also reduced reactive oxygen species levels in hypoxic VSMCs. In addition, THS induced expression of mitochondrial fission proteins Drp1 and Fis1, and decreased expression of mitochondrial fusion protein

Mfn1 in vascular tissues. Cal reduced expression of Drp1 and Fis1, but did not affect Mfn1 expression. In hypoxic VSMCs, Cal inhibited hypoxia-induced mitochondrial fragmentation and preserved mitochondrial morphology.
Conclusions Calhex-231 exhibits outstanding potential for effective therapy of traumatic hemorrhagic shock, due to its ability to improve hemodynamics, increase vital organ blood perfusion, and markedly prolong animal survival. These beneficial effects result from its protection of vascular function via inhibition of oxidative stress and mitochondrial fission.

Background
Trauma is the leading cause of death for people under 44 years of age, claiming more than 5 million victims per year worldwide [1]. About 40% of trauma-related mortality is attributed to hemorrhage and its sequelae [2]. Despite the development of new technologies and therapeutic methods in recent years, the management of trauma patients with severe hemorrhage and hemorrhagic shock remains a challenge.
Cardiovascular dysfunction, such as vascular hyporesponsiveness, is a well-documented phenomenon and a major cause of death in trauma patients with severe hemorrhage or hemorrhagic shock. Vascular hyporesponsiveness is characterized by an impaired response of blood vessels to vasoactive agents, which leads to refractory hypotension and multiple organ failure, even when the patient is supported by traditional fluid resuscitation and vasopressors [3][4][5][6]. In order to develop more effective treatments, it is necessary to investigate the underlying mechanisms of vascular hyporesponsiveness in trauma and hemorrhagic shock.
The calcium-sensing receptor (CaSR), a member of the C family of G protein-coupled receptors (GPCR), plays a fundamental role in extracellular calcium homeostasis in humans by regulating parathyroid hormone (PTH) secretion and renal calcium reabsorption [7,8]. Surprisingly, CaSR is also expressed outside of the parathyroid gland and kidney in non-homeostatic neural and cardiovascular tissues. In the cardiovascular system, functional CaSR is present in cardiomyocytes, perivascular nerves, vascular endothelial cells (VECs), and vascular smooth muscle cells (VSMCs) [9]. Several studies have shown that CaSR plays an important role in the regulation of vascular tone and blood pressure [8][9][10][11]. However, the precise mechanism by which CaSR regulates blood pressure has not been determined.
CaSR can be activated by many kinds of ligands in addition to extracellular calcium (Ca o 2+ ), the prototypical activator of CaSR. Type I agonists include cations such as Mg 2+ and Gd 3+ , polyamines, and some antibiotics. In addition to these biological ligands, a series of type II allosteric modulators of CaSR have been developed. Positive allosteric modulators of CaSR, such as Cinacalcet and Calindol, are named calcimimetics, while negative modulators of CaSR, such as NPS2143 and Calhex-231, are named calcilytics [12][13][14]. Cinacalcet is the only allosteric CaSR modulator that is currently approved for use in humans, and is used to treat uraemic secondary hypercalcaemia and secondary hyperparathyroidism caused by chronic kidney disease [13]. Calcilytics inhibit CaSR and stimulate PTH secretion, and are thus potential treatments for osteoporosis [14]. Recently, some studies on the effect of calcimimetics and calcilytics in the vasculature suggest that CaSR modulators may have therapeutic potential in the treatment of cardiovascular disease [15,16]. However, it is unknown whether CaSR is involved in trauma/shockinduced cardiovascular dysfunction, or if CaSR modulators might exert protective effects on cardiovascular function during traumatic shock.
Oxidative stress and mitochondrial dysfunction play critical roles in the pathogenesis of many cardiovascular diseases, such as atherosclerosis, ischemic heart disease, and hypertension. An increased understanding of the tight link between oxidative stress and 5 mitochondria offers tantalyzing prospects for the treatment and prevention of these diseases [17]. Oxidative stress is an imbalance in the production of oxidants and antioxidants. Reactive oxygen species (ROS) are produced during oxidative stress and are involved in multiple organ damage in patients experiencing severe trauma and hemorrhage. While mitochondria are the primary source of ROS, ROS also cause mitochondrial damage and dysfunction. Mitochondrial function is closely linked to the balance between the opposing processes of mitochondrial fusion and fission. In mammalian cells, the primary regulators of mitochondrial fission are dynamin-related protein 1 (Drp1) and fission 1 (Fis1), whereas mitochondrial fusion is regulated by mitofusin 1 (Mfn1) and mitofusin 2 (Mfn 2). Interactions between ROS and mitochondrial dynamic fusion-fission have been implicated in aging, cancer, neuropathies, and cardiovascular disorders [18,19]. We previously showed that oxidative stress and mitochondrial dysfunction are both involved in the pathogenesis of vascular hyporesponsiveness following shock. The damage caused by ROS may be due to an impairment of mitochondrial permeability [6], which is closely related to the status of mitochondrial fusion-fission. Based on the literature and our previous findings, we hypothesized that CaSR and its modulators play important roles in cardiovascular function, possibly via a mechanism that is related to oxidative stress and mitochondrial dynamic processes.
In this study, we tested whether the calcilytic (negative modulator of CaSR) Calhex-231 improves cardiovascular function when used to treat traumatic hemorrhagic shock (THS).
We also investigated the relationship between oxidative stress and the mitochondrial fusion-fission pathway, using traumatic hemorrhagic shock-induced rats and hypoxiatreated vascular smooth muscle cells (VSMCs). rats (190-220 g) were housed in a containment facility at the animal center at an ambient temperature of 25 °C and a light/dark cycle of 12 hr each. Animals were provided with food and water ad libitum. Food was withdrawn 12 hr prior to the experiment, but access to water was maintained.

Traumatic hemorrhagic shock models and resuscitation
Rats were anesthetized with sodium pentobarbital (initial dosage, 30 mg/kg ip) and Jingsongling (xylidinothiazole; initial dosage, 0.1 mg/kg im). The heparinized catheters were placed in the right femoral artery and vein. Blood pressure and bleeding were monitored from the femoral artery and drugs were administered through the femoral vein.
The right carotid artery and vein were catheterized for monitoring hemodynamic values or cardiac output. Traumatic hemorrhagic shock was induced as previously described [6].
Briefly, a fracture of the left femur was made, and hemorrhage was induced via the right In these groups the volume of LR was twice the volume of blood lost. The shock control group did not receive any treatment after shock. The shamoperated rats experienced the same operation but no shock was induced and they received no LR infusion. LR was administered with an infusion pump (Model AS 50, B. Braun Melsungen AG, Germany).

Animal survival
Ninety-six rats were randomly divided into the six groups (n=16/group) and subjected to the procedures described above. After resuscitation and group-specific treatments, all catheters were removed and the incisions were closed in two layers with nonabsorbable suture. Rats were returned to their cages and allowed free access to food and water.

Blood pressure and hemodynamics
Forty-eight rats were randomly divided into the six groups (n=8/group) and subjected to

Cardiac output and myocardial contractility
Forty-eight rats were used for this experiment, randomly divided into six groups and subjected to the procedures described above. Using a Cardiomax-III Thermodilution  Papillary muscle contractility, normalized for muscle cross-sectional area (g/mm 2 ), was calculated using the formula: G/π(D/2) 2 (G: tension; D: diameter).

Vital organ blood perfusion, local oxygen supply, and vascular reactivity of isolated mesenteric vessels
Forty-eight rats were divided into six groups (n=8/group) and subjected to the treatments described above. Two hours after resuscitation, blood perfusion of the liver and kidney was assessed by laser speckle contrast analysis (LASCA) using a PeriCam PSI system (PSI-9 ZR; Perimed, Jarfalla, Sweden). Briefly, the rats were placed in the supine position, sodium pentobarbital and xylidinothiazole anesthesia was maintained, and a laparotomy was performed exposing the liver and kidney. Dynamic blood perfusion was recorded in realtime with a PSI camera, and the data were expressed as perfusion units (PU) calculated using PIMSoft version 1.5 (Perimed). Subsequently, oxygen saturation of the liver and kidney was assessed by tissue spectrophotometry using an "oxygen to see" (O2C) system (LEA Medizintechnik, Giessen, Germany). The O2C probe was placed on the surface of the liver and kidney, and the capillary venous oxygen saturation was recorded.
After these procedures, rats were euthanized and the superior mesenteric arteries (SMAs) were rapidly excised and placed in ice-cold Krebs-Henseleit (K-H) solution. SMAs were sectioned into rings 2-3 mm long, and the endothelium was denuded by gently rubbing the intimal surface as previously described [3]. The vascular contraction function to NE was measured using an isolated organ perfusion system (Scientific Instruments, Barcelona, Spain). Maximal contraction (Emax) and NE concentration-response curves were used to compare vascular reactivity.
A cohort of twenty-four rats was divided into three groups (n=8/group): shock+LR+Cal 1 mg/kg, shock+LR+Cal+ML-9 10 -6 mol/L (a selective MLCK inhibitor, Sigma, St. Louis, MO, USA), and shock+LR+Cal+ML-9 5×10 -6 mol/L. The procedures, including production of the shock model, Cal treatment, and isolation of SMA rings were identical to those described above. Some SMA rings were treated with ML-9 10 -6 mol/L or 5×10 -6 mol/L for 30 min before reactivity to NE was determined.

Microvascular reactivity of mesenteric microvessels in vivo
Thirty-two rats were randomly divided into four groups (n=8/group): normal control, shock control, shock+LR, and shock+LR+Cal 1 mg/kg. Two hours after resuscitation, the rats Changes in diameter were calculated using the expression (D 1 -D 2 )/D 1 ×100% (D 1 and D 2 : diameter before and after the addition of NE, respectively).

Isolation of cardiomyocytes and VSMCs, and contraction in isolated myocytes
Thirty-two rats were randomly divided into four groups (n=8/group): normal control, shock control, shock+LR, and shock+LR+Cal 1 mg/kg. Two hours after resuscitation, hearts and mesenteric arteries were rapidly removed.

Oxidative stress assay, preparation of tissue lysates, and western blot analysis
Thirty-two rats were divided into four groups (n=8/group), and treated as described above. Two hours after resuscitation, blood samples and SMA tissues were collected.
Serum levels for four oxidative stress biomarkers, including malondialdehyde (MDA), superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH), were detected with commercial assay kits (Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer's instructions. SMA tissue protein extracts were prepared and western blot analysis was performed as described previously [3]. Antibodies were as follows: MLC

Primary culture of VSMCs and hypoxic treatment
Rat VSMCs were obtained from the SMAs of twenty adult SD rats using an explant technique as previously described [3].

ROS level in VSMCs
Reactive oxygen species (ROS) levels in VSMCs were measured using the 2′,7′dichlorofluorescin diacetate (DCF-DA) method [6]. The cultured VSMCs were divided into three groups: normal control, hypoxia 2-h, and hypoxia+Cal (10 μmol/L, 30 min). After treatment with hypoxia and/or Cal, VSMCs were incubated with 10 μmol/L DCF-DA (Sigma) for 20 min at 37°C. DCF fluorescence was detected at 488 nm excitation and 525 nm emission using a Leica TCS SP5 confocal microscope. Images were collected and the mean fluorescence intensities of DCF were measured using the Image J application.

Mitochondrial morphology
To visualize mitochondria in living cells, a mitochondria-specific probe was used. The VSMCs were divided into 3 groups: normal control, hypoxia 2-h, and hypoxia+Cal. After 13 being subjected to hypoxia and/or treated with Cal, the VMSCs were washed with PBS and loaded with 100 nmol/L of MitoTracker deep red (Sigma) for 30 minutes. Fluorescence was detected with an excitation of 633 nm using a Leica confocal microscope.

Statistical analysis
Data are presented as means ± standard error (SEM). Animal survival was analyzed using the Kaplan-Meier survival curve, and the differences among groups were analyzed using a chi-square test. For the other experiments, differences between experimental groups were assessed using one or two-factor analysis of variance analyses, followed by post-hoc  to rats in the LR only group, but the difference was not statistically significant (Fig. 1A, B). were restored to 65.0%, 85.9%, 90.6%, and 80.4%, respectively, of normal control levels.
In the shock control rats, MAP and hemodynamic values remained depressed or decreased slightly 1 and 2 hr after shock ( Fig. 2A-D). 3.

Effect of Calhex-231 on blood perfusion and oxygen saturation of liver and kidney in rats with THS
A significant decrease in blood perfusion in liver and kidney was observed after shock.
Administration of 5 or 1 mg/kg Cal resulted in significantly increased perfusion in both liver and kidney, compared to rats in the LR group (P<0.01). There was no statistical difference between the 5 and 1 mg/kg Cal groups, nor was there between the 0.1 mg/kg and LR groups ( Fig. 3A-C). Not unexpectedly, the oxygen saturation results mirrored the blood perfusion results (Fig. 3D, E).

Effect of Calhex-231 on cardiac function in rats with THS
Cardiac function was evaluated in vivo by measuring cardiac output and in vitro by measuring the contractility of isolated ventricular papillary muscle and single cardiomyocytes. Cardiac output decreased significantly after shock. In all treatment groups, CO returned to near baseline levels 1 hr after treatment commenced and remained steady thereafter. Two hr after the beginning of treatment, CO in the LR group was about 92.9% of normal control levels. Cal did not further increase the CO, and there was no statistical difference between the Cal and the LR groups (Fig. 4A). Similar results were observed in vitro. Contractility of isolated papillary muscle and single cardiomyocytes from THS rats also decreased significantly, while contractility significantly 15 increased in the LR group. No significant differences were observed between the Cal and the LR groups ( Fig. 4B-D).

Effects of Calhex-231 on vascular function in rats with THS
To determine the effects of Cal on vascular function (vascular reactivity) following THS, we investigated pressor effect of NE, and the contractile response of superior mesenteric arteries (SMA), mesenteric arterioles, and isolated VSMCs to NE in vivo and in vitro. The pressor effect of NE (reflecting vascular reactivity in vivo) in THS rats decreased significantly, consistent with our previous report [6]. LR infusion slightly improved the pressor response to NE. Cal treatments at 1 and 5 mg/kg significantly increased the pressor effect of NE as compared with the LR group (P<0.01) (Fig. 5A). Similarly, the constriction reactivity of isolated SMAs from THS rats was significantly reduced, and LR improved slightly. Treatment with Cal at 1 and 5 mg/kg markedly increased the constriction of SMAs from THS rats (Fig. 5B). Similar results were obtained in the mesenteric arterioles and isolated VSMCs. Cal at 1 mg/kg also significantly restored the decreased constriction reactivity of mesenteric arterioles and VSMCs to NE (Fig. 5C-F).  7.

Role of MLC phosphorylation in Cal-mediated effects on vascular reactivity after THS
To determine whether MLC phosphorylation is involved in the regulation of Cal on vascular reactivity after shock, we investigated MLC protein expression and phosphorylation in SMA tissues from THS rats after Cal treatment. Immunoblot analysis revealed that the phosphorylation of MLC in blood vessels decreased significantly after shock, and LR infusion had no significant influence on MLC phosphorylation. Cal treatment significantly increased MLC phosphorylation, while MLC protein expression did not change ( Fig. 7A-C).
We also measured the effects of ML9 (a selective MLCK inhibitor that inhibits phosphorylation of MLC) on the Cal-induced increase in vascular reactivity in THS rats.
Two doses of ML9 antagonized the Cal-induced restoration of vascular reactivity of SMA after shock (P<0.01). Relative to the group receiving Cal alone, the maximal contractile responses of SMA to NE in groups treated with Cal+ML9 10 -6 mol/L and Cal+ML9 5×10 -6 mol/L were 68.9% and 57.4%, respectively (Fig. 7D). 8.

Effects of Calhex-231 on mitochondrial fission/fusion and mitochondrial morphology following THS
Immunoblot analysis showed that expression of Drp1 and Fis1, two important mitochondrial fission proteins, increased significantly in blood vessels from THS rats. LR infusion further increased Drp1 and Fis1 levels. Cal treatment significantly reduced Drp1 and Fis1 expression (Fig. 8A-C). Expression of the fusion protein Mfn1 decreased significantly after shock, while LR infusion and Cal had no significant influence on Mfn1 levels under shock conditions (Fig. 8A, D). Expression of Mfn2, another fusion protein, did not change significantly after shock and/or Cal treatment (Fig. 8A, E).
To examine the effects of Cal treatment in vitro, VSMC mitochondria were stained with MitoTracker deep red. Confocal microscopy showed that hypoxia caused significant changes in mitochondrial morphology in rat VSMCs. Most mitochondria in normal cells were tubular, branched, and displayed a typical networked morphology. However, exposure to hypoxia disrupted the elongated networked structure, and mitochondria became shorter and fragmented. Treatment with Cal significantly reduced the hypoxiainduced mitochondrial fragmentation in VSMCs, as measured by the incidence of fragmented mitochondria and the formation of elongated networks (Fig. 8F). CaSR is known to control systemic calcium homeostasis in humans, and several synthetic CaSR modulators have been developed for the medical management of disorders of calcium metabolism [21]. Recently, CaSR expression has been conclusively demonstrated in various components of the cardiovascular system, including cardiomyocytes, vascular cells, and perivascular nerves, and has been shown to have a physiological role in the modulation of blood pressure [8][9][10][11]. The functions of CaSR and its modulators in cardiovascular physiology and pathophysiology have received much attention. Several studies show that the calcimimetic drug R-568 induces a sustained reduction in blood pressure in uremic and spontaneously hypertensive rats but not in normal rats [22,23].
However, Fryer et al. [25] reported that Cinacalcet (also termed AMG073, the only calcimimetic approved for clinical use) produces an acute increase in blood pressure in both uremic and normal rats. In in vitro experiments, NPS 2143 inhibits vascular contraction induced by vasoconstrictors in rat mesenteric arteries exposed to hypoxia/reoxygenation [26]. These conflicting results may be related to the fact that CaSR is expressed in a diverse range of tissues, or that the experiments were conducted in different pathophysiologic states or used different methods for drug administration.
In this study, we first evaluated the therapeutic effects of intravenous infusion of the To explore the mechanisms by which Calhex-231 improves vascular hyporesponsiveness following THS, we studied the relationships between the vascular action of Cal, oxidative stress, and mitochondrial dysfunction. Oxidative stress and mitochondrial dysfunction are correlated with pathogenesis in numerous cardiovascular diseases [17]. Oxidative stress results from an imbalance caused by excess production of oxidants (ROS) and damage to the ROS scavenging capacity provided by antioxidants (such as SOD and GSH).
Mitochondria are not only the most important source of ROS, but they are also damaged 20 by ROS excess [18,19]. Our previous studies showed that oxidative stress plays an important role in the development of vascular hyporesponsiveness following hemorrhagic shock. We found that antioxidant therapy can regulate mitochondrial membrane permeability of VSMCs and improve mitochondrial function after shock [6]. In the present study, levels of oxidative stress biomarkers, including MDA, SOD, CAT, and GSH, changed significantly after THS. Intracellular ROS levels in VSMCs also increased after hypoxia, confirming our previous observations [6].

Consent for publication
Not applicable.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.