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. Author manuscript; available in PMC: 2022 Jan 1.
Published in final edited form as: Arch Toxicol. 2020 Sep 26;95(1):179–193. doi: 10.1007/s00204-020-02919-8

Chronic cardiac structural damage, diastolic and systolic dysfunction following acute myocardial injury due to bromine exposure in rats

Juan Xavier Masjoan Juncos 1, Shazia Shakil 1, Wayne E Bradley 2, Chih-Chang Wei 2, Iram Zafar 1, Pamela Powell 2, Nithya Mariappan 1, William E Louch 3, David A Ford 4, Aftab Ahmad 1, Louis J Dell’Italia 2,$, Shama Ahmad 1,$,*
PMCID: PMC7855670  NIHMSID: NIHMS1632796  PMID: 32979061

Abstract

Accidental bromine spills are common and its large industrial stores risk potential terrorist attacks. The mechanisms of bromine toxicity and effective therapeutic strategies are unknown. Our studies demonstrate that inhaled bromine causes deleterious cardiac manifestations. In this manuscript we describe mechanisms of delayed cardiac effects in the survivors of a single bromine exposure. Rats were exposed to bromine (600 ppm for 45 minutes) and the survivors were sacrificed at 14 or 28 days. Echocardiography, hemodynamic analysis, histology, transmission electron microscopy (TEM) and biochemical analysis of cardiac tissue were performed to assess functional, structural and molecular effects. Increases in right ventricular (RV) and left ventricular (LV) end-diastolic pressure and LV end-diastolic wall stress with increased LV fibrosis were observed. TEM images demonstrated myofibrillar loss, cytoskeletal breakdown and mitochondrial damage at both time points. Increases in cardiac troponin I (cTnI) and N-terminal pro brain natriuretic peptide (NT-proBNP) reflected myofibrillar damage and increased LV wall stress. LV shortening decreased as a function of increasing LV end-systolic wall stress and was accompanied by increased sarcoendoplasmic reticulum calcium ATPase (SERCA) inactivation and a striking dephosphorylation of phospholamban. NADPH oxidase-2 and protein phosphatase 1 were also increased. Increased circulating eosinophils and myocardial 4-hydroxynonenal content suggested increased oxidative stress as a key contributing factor to these effects. Thus, a continuous oxidative stress-induced chronic myocardial damage along with phospholamban dephosphorylation are critical for bromine-induced chronic cardiac dysfunction. These findings in our preclinical model will educate clinicians and public health personnel and provide important endpoints to evaluate therapies.

Subject codes: Delayed, injury, Remodeling, Physiology, Echocardiography, Mechanisms, Animal models of human disease, Translational studies

Introduction

Exposure to toxic gases such as bromine (Br2) can result in significant morbidity and mortality (Mackie et al. 2014). Our limited understanding of the pathophysiology stems from few case reports of victims of accidental Br2 inhalation demonstrating respiratory and myocardial injury, cardiac arrest and circulatory collapse (Mackie et al. 2014; Makarovsky et al. 2007). To overcome this barrier, studies utilizing animal models for Br2 inhalation are emerging (Aggarwal et al. 2016; Ahmed et al. 2017; Duerr et al. 2018; Jilling et al. 2018; Lam et al. 2016; Lambert et al. 2017; Pavicevic et al. 1989; Summerhill et al. 2017; Zaky et al. 2015a; Zaky et al. 2015b; Zhou et al. 2018). We have established that inhalation of halogens (Cl2 or Br2) causes acute ischemia-reperfusion-type injury to the heart with biventricular dysfunction (Ahmad et al. 2019; Zaky et al. 2015b). These acute cardiac effects may persist in survivors and cause severe complications. However, there is a big gap in understanding of the long-term effects and progression of disease in such individuals.

Once inhaled, as with other halogens, Br2 reacts with the moist airway surface forming highly bioactive brominated intermediates (brominated fatty acids and brominated fatty aldehydes) (Ahmad et al. 2019; Duerr et al. 2018; Juncos et al. 2020). Halogenated reactants reach the heart and react with important cardiac proteins such as SERCA2, modifying and inactivating it, causing a cytosolic Ca2+ overload, ATP depletion, decrease in mitochondrial transmembrane gradient and calpain activation (Ahmad et al. 2015; Ahmad et al. 2019). Modification of SERCA2 and Ca2+ overload is a central pathophysiological mechanism of various cardiac diseases, including cardiac hypertrophy and heart failure (Ahmad et al. 2015; Ahmad et al. 2019; Zaky et al. 2015a; Zaky et al. 2015b). We have shown that acute Br2 inhalation inactivates SERCA2 resulting in increased Ca2+-sensitive LV calpain activity (Ahmad et al. 2019). Calpain activation causes degradation of myocardial contractile proteins such as titin and the major cytoskeletal protein desmin, resulting in cardiac contractile dysfunction (Ahmad et al. 2015; Ahmad et al. 2019).

Cardiac SERCA2 is regulated by a key molecule called phospholamban (PLN), phosphorylation of which increases calcium uptake into the sarcoplasmic reticulum by increasing SERCA activity (Kranias and Solaro 1982). PLN phosphorylation also activates SERCA2 by releasing itself from the SERCA2 molecule. Moreover, PLN forms a multimeric protein complex to interact with SERCA2 and endoplasmic reticulum stress signaling molecules (Kranias and Hajjar 2017). The fine tuning of PLN phosphorylation to regulate SERCA2 is carried out by binding of anchoring subunits of protein phosphatase 1 (PP1) and protein kinase A to PLN (Kranias and Hajjar 2017). Therefore, perturbation of these networks of signaling molecules may lead to altered calcium cycling and cell death and is emerging at the forefront of cardiac disease pathogenesis (Kranias et al. 2018; Shintani-Ishida and Yoshida 2011). In this study we demonstrate that a single Br2 exposure causes chronic cardiac hypertrophy, ultrastructural damage, myocardial remodeling and biventricular dysfunction in survivors. We also highlight critical mechanisms wherein oxidative stress, decreased PLN phosphorylation and increased Nox-2 and PP1 play a significant role.

Methods:

In vivo exposures to Br2:

All animal procedures were approved by the University of Alabama at Birmingham Institutional Animal Care and Use Committee. Un-anesthetized male Sprague Dawley rats (200–250 g, Envigo, Indianapolis, Indiana) were exposed (whole body) to 600 ppm Br2 for 45 min as described previously (Aggarwal et al. 2016; Ahmad et al. 2019; Leustik et al. 2008). At this concentration and time of exposure our studies have demonstrated about 50% mortality (animals either die or are euthanized based on predetermined euthanasia criterion (SpO2 <70%, clinical score>7 and weight loss>15%)) and increased clinical/respiratory distress (Ahmad et al. 2019; Manzoor et al. 2020; Mariappan et al. 2020; Rana et al. 2020). Rats were then returned to room air and monitored continuously up to 8 h, again at 24 h and at 48 h for clinical scores (Ahmad et al. 2019; Veress et al. 2013). Clinical scoring is a composite score of activity and respiratory quality as previously described (Ahmad et al. 2019; Veress et al. 2013). Oxygen saturation and heart rates were also monitored using the MouseOX small animal oximeter (Starr Life Sciences, Oakmont PA) as previously described in our laboratory (Ahmad et al. 2019; Zaky et al. 2015b). Animals were monitored until 14- or 28-day endpoint was reached. One month in adult rat age translates to approximately 3 years in human adult age duration (Sengupta 2013). Therefore, these durations would provide sufficient insight into the disease pathogenesis after acute injury.

At experimental endpoint surgery was performed after echocardiographic and hemodynamical analysis. Blood was collected from the descending aorta and arterial blood gases (ABG) were measured using the EPOC automated blood-gas analysis reader (Ottawa, ON, Canada) (Zaky et al. 2015b). To measure the complete blood count (CBC), an aliquot of whole blood was transferred to EDTA-coated vials and analyzed using hematology analyzer (HEMAVET 950 FS; Drew Scientific, Dallas, TX). Plasma was analyzed for cardiac injury markers, NT-proBNP and cTnI by ELISA (Zaky et al. 2015b). Bronchioalveolar lavage fluid was collected, and the right upper lobe of the lung was isolated and collected, weighed for a wet weight measurement and then dried at 98 °C for 48 h, then weighted for a dry weight measurement in order to do a wet weight to dry weight ratio (WW/DW)(Ahn et al. 1993). Heart was collected and weighed in order to obtain heart weight to body weight ratio (HW/BW) (Richer et al. 1980). Then parts of heart (LV or RV) were fixed or frozen for later analyses.

Transthoracic Two-dimensional Echocardiography/Doppler (ECHO/Doppler) and RV and LV hemodynamics:

All experiments were performed under 2% isoflurane anesthesia in compressed room air as previously performed in our laboratory(Lindsey et al. 2018). The body temperature was maintained at 37°C during measurements. Transthoracic two-dimensional ECHO/Doppler was performed using a Vevo2100 high-resolution ultrasound system (VisualSonics Inc., Toronto, ON, Canada) as previously described in our laboratory. Parasternal long- and short-axis two-chamber M-mode, and B-mode views were obtained at mid papillary level and averaged to determine LV dimensions at end-systole and end-diastole. LV volumes, cardiac output (CO), fractional shortening (Hansen et al.), and ejection fraction were calculated (Visualsonics software). Spectral Doppler was used to determine trans-mitral early (E) and atrial (A) wave peak velocities. Operators blinded to exposure performed image collection and analyses. A 1.4 F high-fidelity catheter (SPR-671, Millar Institute, Houston, TX, USA) was inserted into the LV via the right carotid artery and the RV via the left external jugular vein. LV and RV high-fidelity pressures were measured with a Biopac data acquisition system interfaced with a PC with AcqKnowledge III (ACQ 3.2) software (Ahmad et al. 2019; Zaky et al. 2015b).

Collagen Analysis:

Hydroxyproline assay was performed using 10 mg of LV and following instructions for Hydroxyproline Assay Kit (MAK008, Sigma-Aldrich) (Nehra et al. 2016), and normalized by wet tissue weight.

Histological Imaging and Transmission Electron Microscopy (TEMs):

Tissue was obtained at terminal surgery and processed for imaging following previously described methods and stained with Picro-Sirius Red (PSR) (Ahmad et al. 2019). Image acquisition was performed on a Leica DM6000 epifluorescence microscope with SimplePCI software (Compix, Inc., Cranberry Township, PA) (Chen et al. 2011). TEMs were performed as previously described (EMLabs, Birmingham Al) (Ahmed et al. 2016; Guichard et al. 2017).

Immunohistochemistry

Rat hearts were immersion-fixed in 10% neutral buffered formalin and paraffin-embedded. 5 μm sections were mounted on + slides, deparaffinized in xylene and rehydrated in a graded series of ethanol. HIER was performed with citrate buffer (Vector Laboratories, #H-3300, Burlingame, CA). Sections were blocked with 5% goat serum (in 1% bovine serum/PBS), followed by overnight incubation at 4°C with primary antibody (4-Hydroxynonenal, Abcam #ab46545, Cambridge, MA, 1:100; Myosin, DSHB #MF20, University of Iowa Hybridoma Bank, 1:10). Sections were incubated with Alexa Fluor 488- or 594-conjugated secondary antibody (Life Technologies/Molecular Probes, Eugene, OR, 1:700) to visualize lipid peroxidation products and cardiomyocyte structure, as indicated in figure legends. Nuclei were stained with DAPI (1.5 μg/ml, Vector Laboratories #H-1500). Image acquisition was performed on a Leica DM6000 epifluorescence microscope with SimplePCI software (Compix, Inc., Cranberry Township, PA). Images were adjusted appropriately to remove background fluorescence.

Immunoprecipitation and immunoblots

Rat hearts lysates (500 mg protein) were immunoprecipitated for SERCA2 using monoclonal mouse antibody (2 μg/ml, Thermo Scientific) with 20 μl protein G magnetic beads (Cell Signaling Technology). Immunoprecipitated protein captured on the beads was eluted in the sample buffer and resolved on polyacrylamide gels for subsequent Western blotting using SERCA and Br-Tyr antibody as described previously (Ahmad et al. 2019). Immunoblots on cardiac tissues were performed according to previously described methods using antibodies against SERCA2 (1:1000 abcam), Br-Tyrosine (1:1000; JaICA), phospho-phospholamban (1:1000, Cell Signaling Technology), phospholamban (1:1000, Cell Signaling Technology), PP1 (1:1000 R&D Systems), NOX2 & NOX4 (1:500 Novus Biologicals), GAPDH (1:5000, Cell Signaling Technology), and 4-Hydroxynonenal (1:1000 abcam).

LV SERCA activity assay:

We measured SERCA activity according to previously published methods and using a commercial phosphate (Pi) assay kit (Ahmad et al. 2015; Giroud et al. 2013). Briefly frozen LV tissue was weighed (25 mg) and homogenized in 500 μl of 100 mM Tris buffer pH 7.0 containing 250 mM sucrose, 600 mM KCl, 0.5 mM dithiothreitol and protease inhibitor cocktail using a glass homogenizer on ice. The homogenate was transferred to a centrifuge tube and centrifuged at 13,800 g for 15 min at 4 °C. The supernatant containing the sarcoplasmic reticulum was collected and 50 μl of glycerol was added before freezing at −80 °C. SERCA activity was determined by spectrophotometric assessment of rate of ATP hydrolysis with or without thapsigargin (100 nM), a specific SERCA inhibitor. The reaction was carried out in a buffer containing 100 mM HEPES pH 8.5 containing 65 mM NaCl and 5% glycerol (HNG) as described before(Rule et al. 2016). In a final volume of 250 μl HNG buffer, the SR containing SERCA protein and 100 mM MgCl2-ATP mixture (1:1) and 1mM CaCl2 was added. The reaction was set at 37 °C and aliquots were removed and snap frozen at 0, 30 and 60 min to evaluate the hydrolyzed ATP by measuring the Pi content using a phosphate (Pi) assay kit as described by the manufacturer (Cytoskeleton Inc, Denver, CO). Thapsigargin inhibitable LV SERCA activity assayed here was defined as nmols of Pi released per mg of SR protein per min.

Statistical Analysis:

Data are expressed as mean±Standard Error (SE) analyzed by one-way analysis of variance and unpaired Welch’s t test was performed. A p value <0.05 was considered significant. Analysis was conducted using GraphPad Prism version 7 software (LaJolla, CA). All echocardiography analysis and calculations were performed using SPSS version 19.0 (SPSS, Inc., Chicago, IL). Two observers with expertise in echocardiography assessed the studies for intra- and inter-observer reproducibility.

Results:

Increase in Cardiac Mass and Myofibrillar breakdown

Figure 1A represents the schematic of the experimental paradigm and shows survival after bromine (Br2) exposure, at 28 days after exposure survival which is about 50%. Most of the deaths after Br2 inhalation occur acutely. Figure 1B demonstrates an increase in circulating NT-proBNP at 14 and 28 days with values of 44.78±3.79 and 14.81±3.05 pg/ml in Br2 exposed rats, respectively, compared to 1.02±0.53 pg/ml in naïve animals (p<0.001). This increase in NT-proBNP was supported by a significant increase in heart weight by body weight ratio (HW/BW) at 14-day (0.0037 ± 0.00007, p<0.05) and 28 day (0.0035 ± 0.00005 p<0.05) compared to those of naïves (0.0031 ± 0.00005) (Figure 1C). This is also supported by LV volumes and LV mass determined by echocardiography (Table 1), demonstrating a statistically significant increase in the 28-day LV mass (1010 ± 29 mg, p<0.05) in Br2 exposed rats as compared to naïve rat LV mass (856.0 ± 21.3 mg).

Figure 1: Survivors of bromine exposure have persistent myocardial remodeling and cardiac hypertrophy.

Figure 1:

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A) Schematic representation of 14 d and 28 d study after bromine inhalation. The inset shows the Kaplan-Meier survival plot 28 d after bromine exposure (n=25). Rats were exposed to 600 ppm Br2 for 45 minutes and transferred to room air. Surviving rats were sacrificed at 14 or 28 days after exposure and blood was collected from the descending aorta. NT-proBNP (B) and Heart weight to body weight ratios (C). Data shown are mean±SE (n=8–12 for each group), *indicates p<0.05 compared to naïve controls.

Table 1:

Bromine exposure alters hemodynamic and echocardiographic parameters at 14d and 28d after exposure timepoints.

Control 14d after bromine exposure 28d post bromine exposure
Unit Mean SE n Mean SE n Mean SE n
Hemodynamics
BP max mmHg 107.58 2.96 8 120.0* 3.72 11 116.41* 1.68 14
BP min mmHg 69.02 3.54 8 80.55* 2.21 12 77.54* 1.61 14
MAP mmHg 88.25 3.30 8 101.4* 2.75 12 99.50* 1.85 14
Heart Rate** bpm 339.8 5.39 8 350.9 7.97 12 336.6 4.84 15
LVESP mmHg 62.85 2.75 8 78.53* 2.6 12 71.81* 1.47 14
LVEDP mmHg 5.02 0.52 8 7.50* 0.67 12 8.11* 0.60 14
LV wall stress d % 10.49 0.83 7 15.96* 1.43 12 17.95* 1.84 14
RV peak pressure mmHg 25.86 0.86 8 30.45* 0.71 11 31.93* 1.33 14
RVEDP mmHg 1.25 0.41 7 1.45 0.12 11 3.20* 0.36 12
Echocardiography
Sphere Index L/D axis 1.89 0.04 8 1.94 0.04 12 1.99 0.04 15
LVEDV ul 448.94 15.81 8 492.68 23.37 12 500.50* 13.87 14
LVESV ul 114.48 7.77 8 153.97 16.74 12 175.61* 10.94 14
LVEDD mm 7.64 0.10 8 7.56 0.13 12 7.76 0.11 13
LVESD mm 4.48 0.14 8 4.53 0.20 12 4.97* 0.15 13
LV EF % 67.61 1.93 8 69.12 3.02 12 63.98 2.08 13
LV FS ul 41.33 1.89 8 40.22 2.26 12 36.64* 1.2 14
LVWT: d mm 1.80 0.05 8 1.78 0.05 12 1.79 0.07 13
LVRWT: d ratio 0.47 0.02 8 0.47 0.02 12 0.47 0.02 13
VCFr % 8.21 0.43 8 8.31 0.71 11 7.14 0.34 14
MV E/A ratio 1.46 0.12 8 1.23 0.04 11 1.28 0.06 14
RVFWT: d mm 0.55 0.04 8 0.59 0.03 12 0.64 0.06 13
LV mass/LVEDV ratio 1.918 0.06 8 1.78 0.11 11 1.99 0.06 12
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Mean and SE ± (n= 7–14).

*

indicates statistical significance p<0.05.

**

Heart rate was measured under anesthesia. Systolic blood pressure (BP max), diastolic blood pressure (BP min), mean arterial blood pressure (MAP), heart rate, left ventricular end systolic pressure (LVESP), left ventricular end diastolic pressure (LVEDP), left ventricular diastolic wall stress (LV wall stress d), right ventricular peak pressure (RV peak pressure), Right ventricular end diastolic pressure (RVEDP), sphere index, left ventricular end diastolic volume (LVEDV), left ventricular end systolic volume (LVESV), left ventricular end diastolic diameter (LVEDD), left ventricular end systolic diameter (LVESD), left ventricle ejection fraction (LV EF), left ventricular fraction shortening (LV FS), left ventricular wall thickness in diastole (LVWT: d), ), left ventricular relative wall thickness in diastole (LVRWT: d), Velocity of circumferential fiber shortening (VCFr), mitral valve E wave/A wave (MV E/A), Right ventricle free wall thickness in diastole (RVFWT: d) and left ventricular mass / left ventricular end diastolic volume (LV mass/LVEDV).

We have previously reported severe myofibrillar loss and mitochondrial damage and an increase in cardiac injury markers at 24 h and 7 days after an exposure to 600 ppm of Br2 for 45 minutes. In this study at 14- and 28-day post Br2 exposure we observed a persistent marked myofibrillar breakdown along with mitochondrial damage and disorganization and disruption of the sarcomeric and mitochondrial structure (Figure 2). In addition, inset of Figure 2C demonstrates a near five-fold increase in circulating troponin levels at 14 and 28 days after Br2 exposure (4.82±1.21 ng/ml and 2.51±0.56 ng/ml), compared to naïve controls (1.21±0.06 ng/ml, p<0.05) further supporting the myofibrillar damage.

Figure 2: Br2 inhalation causes persistent disruption of cardiomyocyte cytoskeleton and loss of the normal highly organized linear mitochondrial-sarcomere integrity.

Figure 2:

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As described in legend to Figure 1 rats were exposed Br2, transferred to room air, and surviving animals were sacrificed and cardiac tissue collected at 14- and 28-day time points and fixed for transmission electron microscopy (TEM). Representative TEM of control (4000X A and 13000X D) and Br2 exposed rats (4000X B-C and 13000X E-F). Br2 exposed rats have extensive myofibrillar loss (yellow arrows) and disruption of z-discs (yellow arrowheads) in addition to mitochondrial swelling, cristae lysis and extensive mitochondrial vacuolization (red asterisks). Aortic blood troponin I, cTnI, as a further measure of myofibrillar damage was also evaluated (inset of C). Data shown are mean±SE (n=6 for each group), * indicates p<0.05 vs unexposed control (naïve).

Extracellular Matrix Pathology

Picrosirius red, PSR, stained cardiac histological images demonstrated an increase in spacing between perimysial myofiber bundles that disrupts the laminar structure throughout the endocardium and mesocardium at the 14-day timepoint (Figure 3B and 3E) and at the 28-day timepoint (Figure 3C and 3F) compared to the naïve group (Figure 3A and 3D). This breakdown of the larger laminar structure of the LV was previously observed at 2 and 7 days and as demonstrated here persists at 14 and 28 days, most likely represents myocardial edema (Ahmad et al. 2019). However, TEM images demonstrated marked increase in extracellular endomysial fibrillar collagen (Figure 4A–F). Since we could not quantify the collagen staining due to the loss of continuity as spacing between fibers generate noise and artificial lack of signal and therefore we measured collagen by quantifying hydroxyproline. Analysis of LV hydroxyproline content demonstrates an increase at 14 days (0.587 ± 0.058) that achieves statistical significance at 28 days (0.770 ± 0.032 p<0.05) compared to naïve rats (0.502 ± 0.062) (Figure 4C). This supports both persistent edema as well as fibrosis in the hearts at the 28-day timepoint after Br2 inhalation. We also investigated if there was pulmonary injury and edema present in these animals at these later timepoints. As opposed to the hearts, there was no significant difference in BALF protein content (an indicatory of lung injury) in 14-day (0.12 ± 0.01 mg/ml, p=0.129) or 28-day (0.15 ± 0.02 mg/ml, p= 0.143) after Br2 exposure as compared to naïve rats (0.18 ± 0.02 mg/ml). In addition, lung WW/DW ratio (an indicator of pulmonary edema) did not differ in 14-day (4.91 ± 0.14) or 28-day (4.77 ± 0.05) after Br2 exposure as compared to naïve rats (4.84 ± 0.02).

Figure 3: PSR staining for collagen in rat hearts after Br2 exposure.

Figure 3:

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As described in legend to Figures 1 and 2, 14- and 28-days post Br2 exposure cardiac tissue was fixed and embedded in paraffin and stained with picric acid sirius red (PSR). Images at 1X magnification for naïve group and Br2 groups demonstrate, ventricular cavity (1), endocardium (2) and meso/myocardium (3). Control LV demonstrates a compact myocardium while the images from 14 d group or the 28 d groups show loss of continuity in the endocardium and increase in spacing between myocardial muscle fibers (arrows). 20X images (bottom panels) of mid myocardium demonstrates diffused fracturing and increased interstitial space in the 14 d and 28 d group (arrows). Arrow heads indicate interstitial collagen fiber deposition in the 14 and 28 d groups.

Figure 4: Br2 inhalation causes myocardial remodeling and fibrosis.

Figure 4:

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Representative TEM (4,000X (A and B) and 13,000X (D-F)) images demonstrate marked collagen fiber deposition in the interstitium (red arrows, the dots are fibers that are trimmed in a cross-section) of the Br2 exposed rat hearts at 28 days (28 d). LV hydroxyproline content (C) at 28 days was significantly increased compared to controls. Data shown are mean±SE (n=5–6 for each group), * indicates p<0.05 vs unexposed control.

Hemodynamic and LV functional Analysis

Mean arterial blood pressure was increased at both 14- and 28- days post Br2 exposure (101 ± 3 mmHg, p<0.05 and 100 ± 2 mmHg, p<0.05) compared to naïve rats (88 ± 3 mmHg) (Table 1). RVEDP and RV peak pressure increased only at the 28-day time point compared to naïve (1.25 ± 0.40 mmHg to 3.20 ± 0.36 mmHg (p<0.05) (Table 1). LVED pressure and LVED wall stress increased significantly at 14- and 28- day time points that culminates into a statistically significant increase in LVEDV at 28 days post Br2 exposure (Table 1). LV sphericity index, LV relative wall thickness measured from LVED dimension and LVED wall thickness and LV Mass/LVEDV ratio were unchanged compared to naïve rat values (Table 1).

There was also a significant increase in both LV end-systolic volume (LVESV) and end-systolic wall stress (ESWS) at 28 days coupled with a decrease in LV velocity of circumferential fiber shortening (VCFr) with a trend toward a decrease in VCFr at 28 days (Figure 5A and 5B). To further analyze the relationship between LVES wall stress and LV rate of shortening other parameters were evaluated. A significant decrease was found in the VCFr/ESWS ratio at both 14-day (0.11 ± 0.01, p< 0.05) and the 28-day (0.12 ± 0.01, p<0.05) time points after Br2 exposure as compared to naïve naive rats (0.164 ± 0.016). A change in the distribution of LV end-systolic wall stress to LVESV, LVESD, VCFr and FS, individual animals are plotted in the graph along with lines marking the mean values for naïve group, we observe how there is a shift of the distribution of these animals to quadrants showing more stress and less effective work (Figure 5C–F). Taken together, the adverse systolic remodeling of the heart with underlying myofibrillar loss is associated with progressive decrease in both rate and extent of LV shortening.

Figure 5: Br2 inhalation increases left ventricular wall stress.

Figure 5:

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At 14 and 28 days after exposure LV high-fidelity pressure measurements and echocardiography were obtained under isoflurane anesthesia. LV end-systolic wall stress (A, B) was increased at 28 days, while VCFr/ESWS (C) was decreased at both time points. Linear regression with quadrant representations demonstrate the relation between LV end-systolic wall stress to ESV (D), FS (E) and LVESD (F) with 95% confidence intervals. Mean±SE values for the naïve group were used to make quadrants (dotted lines). Data shown are mean±SE (n=8–14 for each group), * indicates p<0.05 vs. naïve controls.

Calcium Handling Proteins and oxidative stress

In conjunction with the decrease in contractile function, we also observed significant increase in cardiac SERCA modification (bromination of tyrosine residues) 28 days after Br2 inhalation (Figure 6A–B). Quantitation of the Westerns revealed increased brominated SERCA2/SERCA2 ratio in the hearts of animals 28 days after Br2 exposure as compared to naïve animals (p<0.01), however at 14 days after exposure it was not significantly elevated (Figure 6A–B). We also evaluated thapsigargin inhibitable SERCA ATPase activity in the LV of naïve and bromine exposed animals. SERCA activity was significantly reduced at both 14-and 28-day timepoints (Figure 6C). Our previous study demonstrated an early increase in the protein content of phospholamban, an important SERCA regulator (Ahmad et al. 2019). Here we evaluated both total phospholamban and phosphorylated phospholamban (Ser16/Thr17) in the hearts of the Br2 exposed animals. A dramatic loss of phospholamban phosphorylation in the LV of both the 14 day and in the 28-day group compared to naïve animals was observed (Figure 6D and 6E) without a change in total phospholamban expression (Figure 6D and 6F). To further establish the time course of PLN phosphorylation we evaluated the LVs of rats from 24h, 3 and 7-day post Br2 exposure group. PLN phosphorylation was decreased at 24h and 7-day post Br2e exposure (Supplementary Figure 1B). There seemed to be a transient compensatory increase of phosphorylation at 3-day time point which could not be sustained and perhaps became permanently altered beginning at 7 day (Supplementary Figure 1). Protein phosphatase 1, PP1, the major phosphatase of phospholamban trended towards an increase at 14 days and achieved a near threefold increase at 28 days (Figure 6G and 6H). As NOX plays a significant role in the activation of PP1, we evaluated the expression of both NOX-2 and NOX-4. and found increased NOX-2 content but no change in the NOX-4 content in the 14-day or 28-day group compared to the naïve group (Figure 6IJ and 6K–L). Another factor that induces PP1 is oxidative stress (Shintani-Ishida and Yoshida 2011). We demonstrated increased myocardial neutrophils in our acute studies. Therefore, we looked at the circulating blood cell counts. Circulating neutrophils were not significantly different than our unexposed controls in the 14 or 28d post-bromine exposed group. However, we found a significant and persistent (at both 14 and 28d) increase in blood eosinophils (Figure 7A–B). In contrast to neutrophils, eosinophils are known to persist in tissues for extended (up to two weeks) durations and produce reactive brominated oxidants that can lead to pathogenesis of different cardiopulmonary diseases (Heinecke 2000; Strassheim et al. 2019; Wu et al. 2000). Oxidation also produces hydroxynonenal species and myocardial hydroxynonenal (HNE) accumulation and subsequent protein modifications are known to contribute largely to cardiac and vascular disease pathology (Csala et al. 2015). Therefore, to further understand the basis of these underlying effects of a single Br2 exposure we evaluated the 4-HNE content in the hearts of animals 14- and 28-day after Br2 exposure. A significant increase in the 4-HNE content was observed in the Br2 exposed hearts (at 28- day timepoint post Br2 exposure) as compared to naïve animals (Figure 7C and 7D). Immunohistochemistry, IHC, was performed for 4-HNE and myosin. A significant increase in HNE was observed and the staining was primarily located intracellularly in exposed animals compared to extracellularly in naïve animals (Figure 7E). Furthermore, normal myofibrillar structure was shown to be present in naïve animals, while in animals at 14 days after exposure, cardiomyocytes also presented loss of normal myofibrillar structure, and at 28 days cardiomyocytes that have lost their nuclei and myofibrillar structure indicating necrosis were observed (Figure 7E). Thus, our results demonstrate that the survivors of a single Br2 inhalation event may have significant cardiac dysfunction due to increased cardiac hypertrophy and stress caused by oxidative stress and perturbations in important cardiac function regulators in vasculature, intracellular and extracellular compartments in the heart (Figure 8).

Figure 6: Mechanisms of Br2 exposure-induced myocardial SERCA2 modification.

Figure 6:

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(A-B) Role of chemical modification: Lysates were prepared from the LV of naïve or bromine exposed rats and immunoprecipitations were performed using 1 μg/ml anti-rat SERCA2 antibody and 500 μg protein lysate. Western blots were performed using immunoprecipitated proteins separated by magnetic beads as described in the Methods. (A) Antibodies against Br-Tyrosine (Br-Tyr) and SERCA2 (representative blots shown in the top and middle panel) were used to determine SERCA2 modification and SERCA2 expression. IgG released from the beads was used as a loading control. Blots were quantified and values are expressed as Br-SERCA/SERCA ratio, and corrected by IgG AU are shown in (B). SERCA activity was determined in the LV of controls and bromine exposed groups as described in the Methods (C). Data shown are mean±SE (n=4–7 for each group), * indicates p<0.05 vs. naïve controls. Role of Br2-induced loss of phospholamban phosphorylation in the myocardium. Left ventricle of naïve rats or of the rats exposed to bromine 14 or 28 days before were collected and lysates were prepared for Western blots as described in the Methods. Antibodies against anti rat phospho-phospholamban (P-PLN), phospholamban (PLN) were used at a dilution of 1:1000. GAPDH expression was used as a loading control. Representative blots of at least two reproducible experiments are shown (D). Data are mean±SE (n=8 for each group), * indicates p<0.05 as compared to controls. (E & F) Br2 inhalation causes increased protein phosphatase (PP1) and NOX-2 expression in the myocardium. Left ventricle of naïve rats or of the rats exposed to bromine 14 or 28 days before were collected and lysates were prepared for Western blots as described in the Methods. Antibodies against protein phosphatase 1 (PP1) and NADPH oxidase 2 (NOX-2) and NADPH oxidase 4 (NOX-4) were used at a dilution of 1:1000. GAPDH expression was used as loading control. Representative blots of at least two-three reproducible experiments are shown (G, I & J). Data of quantified blots shown in H, K & L are mean±SE (n=4–7 for each group), * indicates p<0.05 as compared to naïve control.

Figure 7: Bromine exposure causes increased circulating eosinophils and induces increased cardiac oxidative stress in survivors.

Figure 7:

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Rats were exposed to bromine and blood was collected and complete blood cell counts (expressed as % (A) or k/μl (B)) were measured in naïve or bromine exposed rats as described in the methods. Data shown are mean±SE (n=8 for naïve, n=15 for 14d group and n=13 for 28d group), (C-D) Left ventricle of naïve rats or of the rats exposed to bromine 14 or 28 days before were collected and lysates were prepared for Western blots as described in the Methods. Antibodies against 4-hydroxynonenal (HNE) were used at a dilution of 1:1000. GAPDH expression was used as a loading control (A). Values are expressed in arbitrary units (AU), and corrected by GAPDH (B). Data shown are mean±SE (n=5–6 for each group), * indicates p<0.05 vs. naïve control. E) HNE localization and myosin structural changes in the hearts of Br2-exposed rats. Immunohistochemistry was performed for hydroxynonenal (HNE) (red), myosin (green) and DAPI as a nuclear stain (blue). HNE staining as shown in panels for 14 and 28 d groups demonstrates increase in oxidative stress (more obvious in the sample collected 28 days after exposure). Myosin + DAPI staining shows conserved myofibrillar structure in naïve group (red arrows) fibers that are in degradation process (white arrows) and cardiomyocytes that have gone through necrosis cells (white arrowheads) in 14 or 28 d post Br2-exposed groups. In the overlay one can observe the location of HNE as compared to myocardial structures.

Figure 8: Schematic representation of mechanisms of delayed Br2 induced cardiac stress, dysfunction and remodeling leading to heart failure in survivors.

Figure 8:

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High concentrations of bromine inhalation cause deaths in the victims due to cardiopulmonary damage. The survivors have continuous release of cardiac damage markers and circulating eosinophils in the blood and increased edema and fibrosis in the heart. These could be results of increased oxidative stress in the myocardium causing a vicious cycle of enhanced protein phosphatase 1 (PP1) and loss of phospholamban (PLN) phosphorylation. SERCA is modified and inhibited by PLN and hence inactivated causing calcium overload and subsequent heart failure.

Discussion:

Halogens such as bromine (Br2) are produced, transported and stored in large quantities to be used in various industrial applications (including disinfectants and chemical syntheses) all over the United States, which is one of the top global halogen gas producer. Besides being an occupational hazard for the factory employees, the increased production and abundance of halogens such as Br2, further enhances the risk of accidental or intentional exposure to mass populations. Therefore, there is a growing need to understand the mechanisms and nature of injuries caused by exposures to these toxic gases to invent potential therapeutic strategies and educate clinicians and public health personnel. We have previously reported acute myocardial injury with significant ultrastructural changes leading to biventricular cardiac dysfunction after Br2 inhalation. Here we demonstrate the persistence of myocardial pathology with hemodynamic and functional evidence of heart failure in survivors of a single Br2 inhalation incident. These results for the first time demonstrate the long-term effect on cardiac function in survivors of a single Br2 inhalation. This mechanistic study was designed to understand the progression of such a pathogenic process.

There is also very sparse clinical information regarding the heart in humans exposed to halogens. Cardiomegaly has been reported on autopsy in 8 of 9 victims of acute halogen (Cl2) poisoning in the 2005 South Carolina train derailment (Achanta and Jordt 2019; Hoyle and Svendsen 2016; Van Sickle et al. 2009). Other reports have also described cardiomegaly in association with pulmonary edema and vascular congestion of the lungs, liver, and other organs. Lung congestion and pulmonary edema may explain subsequent RV hypertrophy and dilation; however, these case reports provide no insight into whether cardiomegaly results from primary RV and LV injury or a combined cardiopulmonary process. Our studies demonstrate that halogens induce acute and chronic cardiac injury that is independent and separate from lung injury (Ahmad et al. 2019; Juncos et al. 2020). We demonstrated increased contractility that subsequently reversed to basal levels after 7 days. Diastolic dysfunction was also observed acutely and persisted. Increases in cTnI and NT-proBNP were observed and these injury markers remain elevated at the timepoints presented in this study. SERCA bromination was increased acutely and returned to basal values by 7 days, leading to a decrease in SERCA activity that persists at 14 and 28 days after exposure timepoints. SERCA inactivation causes cytosolic calcium overload in acute timepoints triggering an increase in calpain activity and degeneration of myocardial fibers. PLN was increased acutely, while we observed no changes at later timepoints, its phosphorylation was dramatically affected in the delayed time points. TEM Imaging revealed myofibrillar degeneration and mitochondrial injury that remains present at 14 and 28 days after exposure (Ahmad et al. 2019).

Cardiac hypertrophy and increased wall stress has been linked to progressive systolic dysfunction and other cardiac morbidities (Artham et al. 2009; Devereux et al. 2001; Sadler et al. 1997; Verdecchia et al. 1995). Toxic inhaled gases such as halogens and carbon monoxide may cause cardiac hypertrophy on one hand and inhalation of other gases such as nitric oxide and hydrogen may help reverse hypertrophy caused by other agents/mechanisms (Matsuoka et al. 2019; Roberts et al. 1995). Persistent increase in troponin and NT-proBNP is a great prognostic value for predicting serious cardiac outcomes in patients (Clark et al. 2019; du Fay de Lavallaz et al. 2019). These key circulating heart-specific biomarkers (brain natriuretic protein, BNP and troponin) were elevated at both 14 and 28 days. This is consistent with the increased in LV diastolic and systolic wall stress and heart weight/body weight in addition to the extensive myofibrillar breakdown by TEM, respectively. These same changes were present acutely in rats exposed to Br2 and persist at 28 days after exposure (Ahmad et al. 2019). There also is a persistence of a fractured laminar structure of the heart manifested by the spaces between myocyte bundles as shown before for other models (Pope et al. 2008). Studies in the rat and dog report a very organized laminar orientation of myofibers changing in direction from −70 to +70 form endo-to mesocardium (LeGrice et al. 1995). The orderly connection of this laminar structure is a necessary feature of LV contractile function and electrical propagation. At the same time, there is extensive collagen accumulation between individual cardiomyocytes by TEM that is verified by a significant increase in hydroxyproline. These early and continuous global damage can account for the diastolic dysfunction manifested by elevation of RV and LV filling pressures and LV diastolic wall stress. In addition to significant myofibrillar breakdown, there is complete disorganization of the sarcomere with breakdown of the z disc and disarray and proliferation of mitochondria. This pattern is also prominent in the acute state of Br2 exposure in the rat and persists in the chronic state (Ahmad et al. 2019). In combination with this severe ultrastructural damage is a marked dephosphorylation phospholamaban (PLN) that starts around day 7 and persists at 14 and 28 days coupled with a significant increase in PP1 leading to SERCA2 inactivation. Reduced SERCA2 expression and hypophosphorylated PLN are important contributors in impaired Ca2+ handling and reduced contractibility of failing cardiomyocytes (Cho et al. 2016). Further inactivation of SERCA derives from molecular modifications, such as bromination, generating Br-SERCA. Although, we did not find measurable amounts of brominated fatty acids in cardiac tissues in samples, the halogenation of SERCA at this later time point could be caused by a later accumulation of other brominated oxidants potentially produced by increased circulating eosinophils in these animals (Gamon et al. 2020; Heinecke 2000; Wildsmith et al. 2006; Wu et al. 2000). The HNE staining of the cardiac tissue further confirms a persistence of oxidative stress. Increased eosinophils themselves lead to heart failure (Strassheim et al. 2019). Therefore, we provide two mechanisms by which SERCA is affected in the heart of survivors of Br2 exposure: one by modulation of activity by PLN dephosphorylation and other by its chemical modification by brominated oxidants produced by increased eosinophils in blood.

Changes in SERCA activity through changes in expression, protein modification or PLN dephosphorylation are pathognomonic of the failing human heart; however, the stimulus for this critical regulation of contractile function resulting from acute Br2 exposure is an open question. LV NOX2 was increased while NOX4, and important regulator of PP1 regulation, protein expression at 14 and 28 days post Br2 exposure is unchanged compared to naïve rats. In contrast to this study increased NOX2 was also shown to increase SERCA activity by increasing phosphorylation of PLN and decreasing PP1 (Zhang et al. 2015). PP1 is also activated by increases of oxidative stress, in our model we can observe increased oxidative stress by the increase in HNE expression. Oxidative stress plays a critical role in pathological cardiac remodeling (Schiattarella and Hill 2017). PP1 is a predominant PLN phosphatase in the heart and acts at both Ser-16 and Thr-17 residues. Other phosphatases such as PP2Ce that act only on the Thr-17 of PLN were found to be increased in patients with cardiomyopathy and its expression caused decreased contractility and oxidative injury and susceptibility to ischemia-reperfusion injury in transgenic mice(Akaike et al. 2017).

The persistent cardiomyocyte ultrastructural and biochemical changes coupled with progressive systolic and diastolic dysfunction support the contention that acute Br2 exposure causes severe irreversible damage to the myocardium. There is a significant increase in both LV end-systolic volume and end-systolic wall stress at 28 days coupled with a decrease in LV fractional shortening with a trend toward a decrease in VCFr. In an attempt to connect LV remodeling with LV systolic function, Figure 5 demonstrates the correlation of LVES volume, LV fractional shortening, and VCFr with increasing LV end-systolic wall stress, consistent with the striking dephosphorylation of phospholamban and significant increase in protein phosphatase. As of now, there is no knowledge of how the survivors of acute bromine inhalation progress with their underlying cardiac injury. When one takes into consideration the hypertrophy, edema, fibrosis, increased cardiac injury markers, ventricular dilation, decreased contractility, diastolic and systolic dysfunction, we conclude that there is an ongoing deterioration and early stages of heart failure. Moreover, we demonstrate a marked cardiac PP1 dependent PLN hypophosphorylation post bromine exposure which is a molecular hallmark of heart failure (Hof et al. 2019; Kranias et al. 2018).

Supplementary Material

204_2020_2919_MOESM1_ESM

Supplementary Figure 1: Role of Br2-induced loss of phospholamban phosphorylation in the myocardium. Left ventricle of naïve rats or of the rats exposed to bromine (600 ppm for 45 min) 1, 3 or 7 days before were collected and lysates were prepared for Western blots as described in the Methods. Antibodies against anti rat phospho-phospholamban (P-PLN), phospholamban (PLN) were used at a dilution of 1:1000. GAPDH expression was used as a loading control. Representative blots of at least two reproducible experiments are shown. Data are mean±SE (n=4 for each group), * indicates p<0.05 as compared to controls.

Acknowledgements:

This work is supported by the CounterACT Program, National Institute of Health Office of the Director (NIH OD), the National Institute of Environmental Health Sciences (NIEHS) Grant Number U01ES028182 (SA & LJD), and U01ES025069 (AA) and the National Heart Lung and Blood Institute (NHLBI) R01HL114933 (AA).

Footnotes

Publisher's Disclaimer: This Author Accepted Manuscript is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication but has not been copyedited or corrected. The official version of record that is published in the journal is kept up to date and so may therefore differ from this version.

Conflict of Interest:

All authors declare that they have no conflicts of interest.

References:

  1. Achanta S, Jordt SE (2019) Toxic Effects of Chlorine Gas and Potential Treatments: A Literature Review. Toxicol Mech Methods:1–34 doi: 10.1080/15376516.2019.1669244 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Aggarwal S, Lam A, Bolisetty S, et al. (2016) Heme Attenuation Ameliorates Irritant Gas Inhalation-Induced Acute Lung Injury. Antioxidants & redox signaling 24(2):99–112 doi: 10.1089/ars.2015.6347 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ahmad S, Ahmad A, Hendry-Hofer TB, et al. (2015) Sarcoendoplasmic reticulum ca(2+) ATPase. A critical target in chlorine inhalation-induced cardiotoxicity. Am J Respir Cell Mol Biol 52(4):492–502 doi: 10.1165/rcmb.2014-0005OC [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ahmad S, Masjoan Juncos JX, Ahmad A, et al. (2019) Bromine inhalation mimics ischemia-reperfusion cardiomyocyte injury and calpain activation in rats. American journal of physiology Heart and circulatory physiology 316(1):H212–H223 doi: 10.1152/ajpheart.00652.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ahmed KA, Nichols AL, Honavar J, Dransfield MT, Matalon S, Patel RP (2017) Measuring nitrate reductase activity from human and rodent tongues. Nitric Oxide 66:62–70 doi: 10.1016/j.niox.2017.04.001 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Ahmed MI, Guichard JL, Soorappan RN, et al. (2016) Disruption of desmin-mitochondrial architecture in patients with regurgitant mitral valves and preserved ventricular function. J Thorac Cardiovasc Surg 152(4):1059–1070 e2 doi: 10.1016/j.jtcvs.2016.06.017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ahn CM, Sandler H, Glass M, Saldeen T (1993) Effect of a synthetic leukocyte elastase inhibitor on thrombin-induced pulmonary edema in the rat. Exp Lung Res 19(2):125–35 [DOI] [PubMed] [Google Scholar]
  8. Akaike T, Du N, Lu G, Minamisawa S, Wang Y, Ruan H (2017) A Sarcoplasmic Reticulum Localized Protein Phosphatase Regulates Phospholamban Phosphorylation and Promotes Ischemia Reperfusion Injury in the Heart. JACC Basic Transl Sci 2(2):160–180 doi: 10.1016/j.jacbts.2016.12.002 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Artham SM, Lavie CJ, Milani RV, Patel DA, Verma A, Ventura HO (2009) Clinical impact of left ventricular hypertrophy and implications for regression. Prog Cardiovasc Dis 52(2):153–67 doi: 10.1016/j.pcad.2009.05.002 [DOI] [PubMed] [Google Scholar]
  10. Chen YW, Pat B, Gladden JD, et al. (2011) Dynamic molecular and histopathological changes in the extracellular matrix and inflammation in the transition to heart failure in isolated volume overload. American journal of physiology Heart and circulatory physiology 300(6):H2251–60 doi: 10.1152/ajpheart.01104.2010 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Cho GW, Altamirano F, Hill JA (2016) Chronic heart failure: Ca(2+), catabolism, and catastrophic cell death. Biochim Biophys Acta 1862(4):763–777 doi: 10.1016/j.bbadis.2016.01.011 [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Clark CL, Gibson TA, Weiss RE, et al. (2019) Do High-sensitivity Troponin and Natriuretic Peptide Predict Death or Serious Cardiac Outcomes After Syncope? Acad Emerg Med 26(5):528–538 doi: 10.1111/acem.13709 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Csala M, Kardon T, Legeza B, et al. (2015) On the role of 4-hydroxynonenal in health and disease. Biochim Biophys Acta 1852(5):826–38 doi: 10.1016/j.bbadis.2015.01.015 [DOI] [PubMed] [Google Scholar]
  14. Devereux RB, Bella JN, Palmieri V, et al. (2001) Left ventricular systolic dysfunction in a biracial sample of hypertensive adults: The Hypertension Genetic Epidemiology Network (HyperGEN) Study. Hypertension 38(3):417–23 doi: 10.1161/01.hyp.38.3.417 [DOI] [PubMed] [Google Scholar]
  15. du Fay de Lavallaz J, Badertscher P, Nestelberger T, et al. (2019) B-Type Natriuretic Peptides and Cardiac Troponins for Diagnosis and Risk-Stratification of Syncope. Circulation 139:2403–2418 doi: 10.1161/CIRCULATIONAHA.118.038358 [DOI] [PubMed] [Google Scholar]
  16. Duerr MA, Palladino END, Hartman CL, et al. (2018) Bromofatty aldehyde derived from bromine exposure and myeloperoxidase and eosinophil peroxidase modify GSH and protein. J Lipid Res 59(4):696–705 doi: 10.1194/jlr.M083279 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gamon LF, Dieterich S, Ignasiak MT, Schrameyer V, Davies MJ (2020) Iodide modulates protein damage induced by the inflammation-associated heme enzyme myeloperoxidase. Redox Biol 28:101331 doi: 10.1016/j.redox.2019.101331 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Giroud S, Frare C, Strijkstra A, Boerema A, Arnold W, Ruf T (2013) Membrane phospholipid fatty acid composition regulates cardiac SERCA activity in a hibernator, the Syrian hamster (Mesocricetus auratus). PloS one 8(5):e63111 doi: 10.1371/journal.pone.0063111 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Guichard JL, Rogowski M, Agnetti G, et al. (2017) Desmin loss and mitochondrial damage precede left ventricular systolic failure in volume overload heart failure. American journal of physiology Heart and circulatory physiology 313(1):H32–H45 doi: 10.1152/ajpheart.00027.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hansen RR, Wufsus AR, Barton ST, Onasoga AA, Johnson-Paben RM, Neeves KB (2013) High content evaluation of shear dependent platelet function in a microfluidic flow assay. Annals of biomedical engineering 41(2):250–62 doi: 10.1007/s10439-012-0658-5 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Heinecke JW (2000) Eosinophil-dependent bromination in the pathogenesis of asthma. J Clin Invest 105(10):1331–2 doi: 10.1172/JCI10072 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hof IE, van der Heijden JF, Kranias EG, et al. (2019) Prevalence and cardiac phenotype of patients with a phospholamban mutation. Neth Heart J 27(2):64–69 doi: 10.1007/s12471-018-1211-4 [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hoyle GW, Svendsen ER (2016) Persistent effects of chlorine inhalation on respiratory health. Annals of the New York Academy of Sciences 1378(1):33–40 doi: 10.1111/nyas.13139 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Jilling T, Ren C, Yee A, et al. (2018) Exposure of neonatal mice to bromine impairs their alveolar development and lung function. American journal of physiology Lung cellular and molecular physiology 314(1):L137–L143 doi: 10.1152/ajplung.00315.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Juncos JXM, Shakil S, Ahmad A, et al. (2020) Circulating and tissue biomarkers as predictors of bromine gas inhalation. Annals of the New York Academy of Sciences doi: 10.1111/nyas.14422 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kranias EG, Doevendans PA, Glijnis PC, Hajjar RJ (2018) PLN Foundation. Circ Res 123(12):1276–1278 doi: 10.1161/CIRCRESAHA.118.314014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kranias EG, Hajjar RJ (2017) The Phospholamban Journey 4 Decades After Setting Out for Ithaka. Circ Res 120(5):781–783 doi: 10.1161/CIRCRESAHA.116.310007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Kranias EG, Solaro RJ (1982) Phosphorylation of troponin I and phospholamban during catecholamine stimulation of rabbit heart. Nature 298(5870):182–4 doi: 10.1038/298182a0 [DOI] [PubMed] [Google Scholar]
  29. Lam A, Vetal N, Matalon S, Aggarwal S (2016) Role of heme in bromine-induced lung injury. Annals of the New York Academy of Sciences 1374(1):105–10 doi: 10.1111/nyas.13086 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lambert JA, Carlisle MA, Lam A, et al. (2017) Mechanisms and Treatment of Halogen Inhalation-Induced Pulmonary and Systemic Injuries in Pregnant Mice. Hypertension 70(2):390–400 doi: 10.1161/HYPERTENSIONAHA.117.09466 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. LeGrice IJ, Smaill BH, Chai LZ, Edgar SG, Gavin JB, Hunter PJ (1995) Laminar structure of the heart: ventricular myocyte arrangement and connective tissue architecture in the dog. Am J Physiol 269(2 Pt 2):H571–82 doi: 10.1152/ajpheart.1995.269.2.H571 [DOI] [PubMed] [Google Scholar]
  32. Leustik M, Doran S, Bracher A, et al. (2008) Mitigation of chlorine-induced lung injury by low-molecular-weight antioxidants. Am J Physiol Lung Cell Mol Physiol 295(5):L733–43 doi: 10.1152/ajplung.90240.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Lindsey ML, Kassiri Z, Virag JAI, de Castro Bras LE, Scherrer-Crosbie M (2018) Guidelines for measuring cardiac physiology in mice. American journal of physiology Heart and circulatory physiology 314(4):H733–H752 doi: 10.1152/ajpheart.00339.2017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Mackie E, Svendsen E, Grant S, Michels JE, Richardson WH (2014) Management of chlorine gas-related injuries from the Graniteville, South Carolina, train derailment. Disaster medicine and public health preparedness 8(5):411–6 doi: 10.1017/dmp.2014.81 [DOI] [PubMed] [Google Scholar]
  35. Makarovsky I, Markel G, Hoffman A, et al. (2007) Bromine--the red cloud approaching. Isr Med Assoc J 9(9):677–9 [PubMed] [Google Scholar]
  36. Manzoor S, Mariappan N, Zafar I, et al. (2020) Cutaneous lewisite exposure causes acute lung injury. Annals of the New York Academy of Sciences doi: 10.1111/nyas.14346 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Mariappan N, Husain M, Zafar I, et al. (2020) Extracellular nucleic acid scavenging rescues rats from sulfur mustard analog-induced lung injury and mortality. Arch Toxicol 94(4):1321–1334 doi: 10.1007/s00204-020-02699-1 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Matsuoka H, Miyata S, Okumura N, et al. (2019) Hydrogen gas improves left ventricular hypertrophy in Dahl rat of salt-sensitive hypertension. Clin Exp Hypertens 41(4):307–311 doi: 10.1080/10641963.2018.1481419 [DOI] [PubMed] [Google Scholar]
  39. Nehra S, Bhardwaj V, Kar S, Saraswat D (2016) Chronic Hypobaric Hypoxia Induces Right Ventricular Hypertrophy and Apoptosis in Rats: Therapeutic Potential of Nanocurcumin in Improving Adaptation. High Alt Med Biol 17(4):342–352 doi: 10.1089/ham.2016.0032 [DOI] [PubMed] [Google Scholar]
  40. Pavicevic L, Frkovic A, Vukelic M (1989) [Changes in the anterior segment of the eye in workers in the coke manufacturing industry]. Arh Hig Rada Toksikol 40(4):405–8 [PubMed] [Google Scholar]
  41. Pope AJ, Sands GB, Smaill BH, LeGrice IJ (2008) Three-dimensional transmural organization of perimysial collagen in the heart. American journal of physiology Heart and circulatory physiology 295(3):H1243–H1252 doi: 10.1152/ajpheart.00484.2008 [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Rana T, Ahmad A, Zafar I, et al. (2020) MicroRNA-mediated inflammation and coagulation effects in rats exposed to an inhaled analog of sulfur mustard. Annals of the New York Academy of Sciences doi: 10.1111/nyas.14416 [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Richer C, Venturini-Souto N, Boissier JR, Giudicelli JF (1980) beta-Adrenoreceptor blockage and genetic hypertension development in rats. Clin Exp Hypertens 2(1):99–122 [DOI] [PubMed] [Google Scholar]
  44. Roberts JD Jr., Roberts CT, Jones RC, Zapol WM, Bloch KD (1995) Continuous nitric oxide inhalation reduces pulmonary arterial structural changes, right ventricular hypertrophy, and growth retardation in the hypoxic newborn rat. Circulation research 76(2):215–22 doi: 10.1161/01.res.76.2.215 [DOI] [PubMed] [Google Scholar]
  45. Rule CS, Patrick M, Sandkvist M (2016) Measuring In Vitro ATPase Activity for Enzymatic Characterization. Journal of visualized experiments : JoVE(114) doi: 10.3791/54305 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Sadler DB, Aurigemma GP, Williams DW, Reda DJ, Materson BJ, Gottdiener JS (1997) Systolic function in hypertensive men with concentric remodeling. Hypertension 30(4):777–81 doi: 10.1161/01.hyp.30.4.777 [DOI] [PubMed] [Google Scholar]
  47. Schiattarella GG, Hill JA (2017) Metabolic control and oxidative stress in pathological cardiac remodelling. Eur Heart J 38(18):1399–1401 doi: 10.1093/eurheartj/ehw199 [DOI] [PubMed] [Google Scholar]
  48. Sengupta P (2013) The Laboratory Rat: Relating Its Age With Human’s. Int J Prev Med 4(6):624–30 [PMC free article] [PubMed] [Google Scholar]
  49. Shintani-Ishida K, Yoshida K (2011) Ischemia induces phospholamban dephosphorylation via activation of calcineurin, PKC-alpha, and protein phosphatase 1, thereby inducing calcium overload in reperfusion. Biochimica et biophysica acta 1812(7):743–51 doi: 10.1016/j.bbadis.2011.03.014 [DOI] [PubMed] [Google Scholar]
  50. Strassheim D, Dempsey EC, Gerasimovskaya E, Stenmark K, Karoor V (2019) Role of Inflammatory Cell Subtypes in Heart Failure. J Immunol Res 2019:2164017 doi: 10.1155/2019/2164017 [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Summerhill EM, Hoyle GW, Jordt SE, et al. (2017) An Official American Thoracic Society Workshop Report: Chemical Inhalational Disasters. Biology of Lung Injury, Development of Novel Therapeutics, and Medical Preparedness. Ann Am Thorac Soc 14(6):1060–1072 doi: 10.1513/AnnalsATS.201704-297WS [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Van Sickle D, Wenck MA, Belflower A, et al. (2009) Acute health effects after exposure to chlorine gas released after a train derailment. The American journal of emergency medicine 27(1):1–7 doi: 10.1016/j.ajem.2007.12.006 [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Verdecchia P, Schillaci G, Borgioni C, et al. (1995) Adverse prognostic significance of concentric remodeling of the left ventricle in hypertensive patients with normal left ventricular mass. J Am Coll Cardiol 25(4):871–8 doi: 10.1016/0735-1097(94)00424-O [DOI] [PubMed] [Google Scholar]
  54. Veress LA, Hendry-Hofer TB, Loader JE, Rioux JS, Garlick RB, White CW (2013) Tissue plasminogen activator prevents mortality from sulfur mustard analog-induced airway obstruction. Am J Respir Cell Mol Biol 48(4):439–47 doi: 10.1165/rcmb.2012-0177OC [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wildsmith KR, Albert CJ, Anbukumar DS, Ford DA (2006) Metabolism of myeloperoxidase-derived 2-chlorohexadecanal. The Journal of biological chemistry 281(25):16849–60 doi: 10.1074/jbc.M602505200 [DOI] [PubMed] [Google Scholar]
  56. Wu W, Samoszuk MK, Comhair SA, et al. (2000) Eosinophils generate brominating oxidants in allergen-induced asthma. J Clin Invest 105(10):1455–63 doi: 10.1172/JCI9702 [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zaky A, Ahmad A, Dell’Italia LJ, et al. (2015a) Inhaled matters of the heart. Cardiovasc Regen Med 2 doi: 10.14800/crm.997 [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Zaky A, Bradley WE, Lazrak A, et al. (2015b) Chlorine inhalation-induced myocardial depression and failure. Physiological reports 3(6) doi: 10.14814/phy2.12439 [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zhang M, Prosser BL, Bamboye MA, et al. (2015) Contractile Function During Angiotensin-II Activation: Increased Nox2 Activity Modulates Cardiac Calcium Handling via Phospholamban Phosphorylation. J Am Coll Cardiol 66(3):261–272 doi: 10.1016/j.jacc.2015.05.020 [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhou T, Song WF, Shang Y, Yao SL, Matalon S (2018) Halogen Inhalation-Induced Lung Injury and Acute Respiratory Distress Syndrome. Chin Med J (Engl) 131(10):1214–1219 doi: 10.4103/0366-6999.231515 [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

204_2020_2919_MOESM1_ESM

Supplementary Figure 1: Role of Br2-induced loss of phospholamban phosphorylation in the myocardium. Left ventricle of naïve rats or of the rats exposed to bromine (600 ppm for 45 min) 1, 3 or 7 days before were collected and lysates were prepared for Western blots as described in the Methods. Antibodies against anti rat phospho-phospholamban (P-PLN), phospholamban (PLN) were used at a dilution of 1:1000. GAPDH expression was used as a loading control. Representative blots of at least two reproducible experiments are shown. Data are mean±SE (n=4 for each group), * indicates p<0.05 as compared to controls.

NIHMS1632796-supplement-204_2020_2919_MOESM1_ESM.tiff (154KB, tiff)

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