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

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 min) 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.

Keywords: Animal models of human disease; Delayed; Echocardiography; Injury; Mechanisms; Physiology; Remodeling; Translational studies.

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

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.

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

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).

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

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.

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.

Figure 5:. Br₂ inhalation increases left ventricular wall stress.

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.

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

(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.

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.

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.