Myocardial SERCA2 Protects Against Cardiac Damage and Dysfunction Caused by Inhaled Bromine

Abstract

Myocardial sarcoendoplasmic reticulum calcium ATPase 2 (SERCA2) activity is critical for heart function. We have demonstrated that inhaled halogen (chlorine or bromine) gases inactivate SERCA2, impair calcium homeostasis, increase proteolysis, and damage the myocardium ultimately leading to cardiac dysfunction. To further elucidate the mechanistic role of SERCA2 in halogen-induced myocardial damage, we used bromine-exposed cardiac-specific SERCA2 knockout (KO) mice [tamoxifen-administered SERCA2 (flox/flox) Tg (αMHC-MerCreMer) mice] and compared them to the oil-administered controls. We performed echocardiography and hemodynamic analysis to investigate cardiac function 24 hours after bromine (600 ppm for 30 minutes) exposure and measured cardiac injury markers in plasma and proteolytic activity in cardiac tissue and performed electron microscopy of the left ventricle (LV). Cardiac-specific SERCA2 knockout mice demonstrated enhanced toxicity to bromine. Bromine exposure increased ultrastructural damage, perturbed LV shape geometry, and demonstrated acutely increased phosphorylation of phospholamban in the KO mice. Bromine-exposed KO mice revealed significantly enhanced mean arterial pressure and sphericity index and decreased LV end diastolic diameter and LV end systolic pressure when compared with the bromine-exposed control FF mice. Strain analysis showed loss of synchronicity, evidenced by an irregular endocardial shape in systole and irregular vector orientation of contractile motion across different segments of the LV in KO mice, both at baseline and after bromine exposure. These studies underscore the critical role of myocardial SERCA2 in preserving cardiac ultrastructure and function during toxic halogen gas exposures.

SIGNIFICANCE STATEMENT

Due to their increased industrial production and transportation, halogens such as chlorine and bromine pose an enhanced risk of exposure to the public. Our studies have demonstrated that inhalation of these halogens leads to the inactivation of cardiopulmonary SERCA2 and results in calcium overload. Using cardiac-specific SERCA2 KO mice, these studies further validated the role of SERCA2 in bromine-induced myocardial injury. These studies highlight the increased susceptibility of individuals with pathological loss of cardiac SERCA2 to the effects of bromine.

Introduction

Halogens such as chlorine (Cl₂) and bromine (Br₂) are commonly produced highly reactive and very toxic gases that can be weaponized for use in warfare and terrorism. Because of their abundant industrial use and production, they pose extreme occupational and accidental hazards as well (Makarovsky et al., 2007). We and others have previously demonstrated that inhaled halogen exposure, both by Br₂ or Cl₂, causes cardiac injury by modifying and inactivating sarcoendoplasmic reticulum calcium ATPase 2 (SERCA2), leading to a cytosolic calcium ion (Ca²⁺) overload, ATP depletion, decrease in mitochondrial transmembrane gradient, and activation of proteases like calpains (Zaky et al., 2015b; Shintani-Ishida and Yoshida 2011). Calpains exert their proteolytic activity and degrade proteins essential for cardiac contractility such as titin (Barta et al., 2005; Carmignac et al., 2007). This proteolytic activity expands to even SERCA2 itself, leading to further impaired Ca²⁺ transport (French et al., 2006; Ahmad et al., 2019). Cardiac SERCA2 activity is impaired acutely and persists long term in the survivors in the Br₂ exposure model (Masjoan Juncos et al., 2020).

In the United States, heart disease is the most common cause of mortality and morbidity, while about half the adult population has hypertension, one of the main risk factors for developing heart disease (https://www.cdc.gov/nchs/fastats/heart-disease.htm). The most common feature of heart failure is a reduction in myocardial SERCA2 activity (Hasenfuss et al., 1994; Luo and Anderson 2013). Cardiac function is highly dependent on Ca²⁺ transport to and from the cytosol during contraction and relaxation, respectively (Bers 2002). Reduction in SERCA2 leads to impaired myocardial relaxation as showed by reduced end diastolic volumes (EDVs) and increased end diastolic pressures (EDPs) (Andersson et al., 2009; Land et al., 2013; Boardman et al., 2014). The impaired relaxation can progress to global heart failure with the appearance of systolic dysfunction, characterized by reduction of ejection fraction (EF) and cardiac output (CO) (Louch et al., 2010; Hillestad et al., 2013; Boardman et al., 2014).

Experimental models of cardiac SERCA2 knockout (KO) are available, where the phenotype is induced after birth as SERCA2 KO is not compatible with viable intrauterine development (Land et al., 2013). In this cardiac-specific SERCA2 KO model, SERCA2 is reduced to 5% of that of controls. This leads to a cytosolic accumulation of Ca²⁺ in the heart of the KO mice (Li et al., 2012). Changes in the transport of ions through the cell membrane were reflected in changes in myocardial contraction and relaxation capabilities and velocity (Stokke et al., 2010; Hillestad et al., 2013). These cardiac-specific SERCA2 KO animals also presented with systolic dysfunction as observed by a decrease in their ejection fraction, EF, stroke volume (SV), fraction shortening (FS) and myocardial contractility (Louch et al., 2010; Hillestad et al., 2013; Boardman et al., 2014). There was diastolic dysfunction as evidenced by decreased left ventricle (LV) volumes and mechanical efficiency, while isovolumetric relaxation time, EDP, and LV relaxation time constant were increased (Andersson et al., 2009; Land et al., 2013; Boardman et al., 2014). All this leads to heart failure when the heart is no longer capable of preserving cardiac pump function to meet the metabolic demands of the body. Presently, there are no studies on how changes in baseline SERCA2 expression could influence cardiac injury caused by chemicals or halogens including Br₂. Given the crucial role of SERCA2 in both normal cardiac function and cardiac pathophysiological changes, it is essential to further investigate the impact of SERCA2 expression on Br₂-induced cardiac injury. In this manuscript, we investigated the role of cardiac-specific SERCA2 in bromine-induced cardiopulmonary injury by utilizing cardiac-specific SERCA2 knockout mice and comparing them to their respective control mice.

Materials and Methods

Animals.

All animals were obtained, housed, and cared for and animal experiments performed according to approved protocols (APN10282) and University of Alabama at Birmingham Institutional Animal Care and Use Committee guidelines. Healthy adult (8–12 weeks, both sexes) cardiac-specific SERCA2 knockout [Serca2^flox/flox Tg (αMHC-MerCreMer)] were obtained by crossing Serca2^flox/flox (provided by Dr. William E Louch, University of Norway) and αMHC-MerCreMer mice, Jackson Laboratory (Bar Harbor, ME, strain no 005650) (Sohal et al., 2001). The newly acquired mice underwent a 4-week quarantine period before being released to the regular housing after screening for pathogens. Genotyping was performed in the litter upon weaning by utilizing the services of Transnetyx (Cordova, TN) and published protocols (Andersson et al., 2009). Six to seven mice per cage were housed under a 12-hour dim light/12-hour dark cycle in standard polycarbonate cages with wood chip bedding and nesting enrichment (EnviroPak). Standard diet (Labdiet NIH 31, PMI Nutrition, Arden Hills, MN) and tap water were provided ad libitum; the room temperature was maintained at 70°F and humidity was maintained between 40% and 60%. A priori power analysis was performed using ANOVA F-test to compute sample size. Assuming the group sizes with a power of 95% and α-error probability of 5% or less, expected mortality, and an effect size of 0.5 to 0.7, the number of animals required for proposed studies was calculated. An equal number of male and female mice were used for exposures (two mice per exposure chamber), which were done separately as described later. Survivors obtained from various exposures at a given time point were evaluated. Mice were euthanized at the end of the experiment in accordance with the 2013 American Veterinary Medical Association Panel on Euthanasia Guidelines to allow for specimen collection. For blood and tissue collection, general anesthesia was induced by 5% isoflurane (Piramal Critical Care, Telangana, India) and maintained by 2% isoflurane. After ensuring depth of anesthesia, incisions were made to open the abdominal cavity, bowels were gently displaced, and abdominal aorta was exposed. Blood was collected using a heparinized syringe with a 25G needle. Exsanguination was then performed, and cardiac and pulmonary tissues were collected.

Phenotype Induction.

αMHC-MerCreMer mice were injected intraperitoneally with peanut oil (P2144, Sigma-Aldrich, St. Louis, MO) (FF) or tamoxifen (1 mg/100 μl peanut oil, T5648, Sigma-Aldrich) (KO) 2 weeks after weaning to obtain cardiomyocyte-specific disruption of the SERCA2 (KO) gene (Andersson et al., 2009; Land et al., 2013). We used the mice at 6 to 8 weeks after injection for exposure to bromine as this duration is reported to have maximum functional effects of SERCA2 knockout (Andersson et al., 2009; Louch et al., 2010; Li et al., 2012; Hillestad et al., 2013; Land et al., 2013; Boardman et al., 2014).

In Vivo Br₂ Exposure, Echocardiography, and Hemodynamics.

Unanesthetized mice (FF or KO) were exposed (whole body) to 600 ppm Br₂ (Airgas, Birmingham AL; gas concentrations in balance air was certified within 2% by the manufacturer) for 30 minutes as previously described (Leustik et al., 2008; Aggarwal et al., 2016; Ahmad et al., 2019). Mice were then returned to room air (RA) and monitored continuously for 6 hours and then at 24 hours and every 24 hours after that until 4 days after exposure. Animals for echocardiographic and hemodynamic studies were used at 24 hours after exposure. All hemodynamics and echocardiography measurements were performed under 2% isoflurane in compressed room air and body temperature was maintained at 37°C as previously described (Lindsey et al., 2018). Intraventricular pressures were measured using 1.4-Fr high-fidelity catheter (SPR 671, Millar Institute, Huston, TX) and analyzed using AcqKnowledge III software (ACQ 3.2; Biopac Systems, Galeto, CA) as previously described (Ahmad et al., 2019). Echo/Doppler data were acquired using Vevo 2100 high-resolution ultrasound system with the 21-MHz MS250 MicroScanTransducer (VisualSonics, Toronto, ON, Canada) as previously described (Zaky et al., 2015b). Data obtained from echo/Doppler were assessed as described in our previous publication (Ahmad et al., 2019). Operators were blinded to exposure performed during image collection and analysis. Strain analysis was done by performing echo with ultrasound probe (MX400, axial resolution: 50μm) applied to the chest at an 11° angle to the sternum as described in previous publications (Halade et al. 2018). Electrocardiogram data were obtained by placing paws on electrode pads while performing echo as previously published (Halade et al. 2018; Ahmad et al., 2019).

Tissue Analysis.

Myocardial tissue was obtained and processed for transmission electron microscopy (TEM) as previously described in our laboratory (EMLabs, Birmingham, AL) (Ahmad et al., 2019). Calpain activity was also measured in cardiac tissue lysates prepared as previously published using commercial calpain activity assay kits (G8501, Promega, Madison, WI) (Ahmad et al., 2019). Western blots were performed in cardiac tissue lysates using anti-mouse phospholamban (PLN) (Abcam, Waltham, MA), phospho-phospholamban (Cell Signaling Technology, Beverly MA), and SERCA2 (Abcam), glyceraldehyde-3-phosphate dehydrogenase, and sarcoplasmic actin antibodies (Abcam).

Cardiac Injury Marker Measurement.

Blood was collected from descending aorta and plasma was stored at –80°C. Samples were processed using muscle injury panel 3 (Mesoscale Discovery, Rockville MD) to measure levels of heart fatty acid binding protein (h-FABP), cardiac troponin I (cTnI), skeletal troponin (sTNI), and myosin light chain 3 (Myl3) following instructions provided in the manufacturer’s datasheet.

Statistical Analysis.

Data are expressed as mean ± S.E. Groups were analyzed by one-way ANOVA with a Student’s paired t test. Statistical significances were considered for P values of < 0.05. GraphPad Prism version 8 software (GraphPad, La Jolla, CA) was used to perform the analysis. Survival was assessed using the Kaplan–Meier method and compared using a log-rank test (Mantel–Cox test).

Results

Cardiac-Specific SERCA2 Knockout Mice.

SERCA2 regulates cytosolic Ca²⁺ by packing it back in the sarcoendoplasmic reticulum lumen for subsequent release and initiation of cardiomyocyte contraction. Since more than 70% of cytosolic calcium is removed by SERCA2 activity, it is a major regulator of cardiac relaxation and normal cardiac function. Our studies have shown that highly reactive species produced in the pulmonary bed upon halogen inhalation reach the heart and modify and inactivate cardiac SERCA2 and cause biventricular cardiac dysfunction (Ahmad et al., 2014, 2015, 2019; Zakyet al., 2015a,b; Juncos et al., 2020; Masjoan Juncos et al., 2021). To further understand the role of SERCA2 in inhaled bromine-induced cardiotoxicity, we used inducible cardiomyocyte-specific excision of SERCA2 (Supplemental Fig. 1). As shown by the original investigators (Andersson et al., 2009), these αMHC-MerCreMer (male and female KO) mice develop diastolic dysfunction by 7 weeks after tamoxifen administration (Andersson et al., 2009; Louch et al., 2010). We used these mice (male and female) close to this time point (6–8 weeks) when they had reduced (by ∼80%) SERCA2 [LV lower panel Supplemental Fig. 1 and both LV and right ventricle (RV) shown in Supplemental Fig. 1] as compared with the control mice (FF).

Bromine Exposure Causes Enhanced Mortality and Cardiac Injury in Cardiac-Specific SERCA2 KO Mice.

Approximately 6 to 8 weeks after tamoxifen injection, mice (male and female FF or KO) were exposed to Br₂ and followed for 96 hours for survival analysis and 24 hours for all other endpoint measurements (Fig. 1A). Exposure to 600 ppm Br₂ for 30 minutes was fatal and caused about 25% mortality within the first 12 hours after exposure in the KO mice, whereas no fatality was observed at this time point in the FF control mice. By 24 hours, increased mortality was observed in both groups, and by 48 hours more than 50% of KO and more than 30% of FF mice died (Fig. 1A). Four days after exposure, only ∼30% of the KO mice and approximately 60% of FF mice survived. We therefore used the 24-hour time point after Br₂ exposure for our subsequent studies. The baseline heart weight of SERCA2 knockout mice was enhanced but not statistically significant from the FF controls (P = 0.08, Fig. 1B). Br₂ exposure caused significantly increased cardiac weights in the FF control mice, 24 hours after exposure. Br₂-induced cardiac hypertrophy was significant in the cardiac-specific SERCA2 KO animals when compared with the FF controls. However, this did not achieve significance when compared with the room air SERCA2 KO animals. The mean body weights of these mice were not significantly different at the endpoint measurements (24 hours) (Table 1). Knockdown of cardiac SERCA2 caused significantly increased baseline cTnI in the plasma (Fig. 1C). However, levels of sTNI did not increase at baseline following cardiac-specific SERCA2 knockdown (Fig. 1D). Br₂ exposure caused significantly increased cTnI and sTNI in the plasma of FF mice. Since the KO animals already had increased baseline cTnI, Br₂ exposure-induced cTnI was not statistically significant in this group. The sTNI increased to similar levels in the KO animals as compared with the FF upon Br₂ exposure. In the plasma of unexposed SERCA2 KO animals, the acute cardiac injury marker, h-FABP, was present at concentrations of 52.99 ± 8.71 pg/ml, which was similar to the h-FABP content in the plasma of unexposed FF animals with concentrations of 52.38 ± 9.80 pg/ml. Notably, h-FABP was elevated only in the plasma of Br₂-exposed KO animals with concentrations of 85.87 ± 10.59 pg/ml when compared with the Br₂-exposed FF animals with concentrations of 54.57 ± 7.76 pg/ml (P = 0.04) (Fig. 1E). Additionally, plasma levels of Myl3, an indicator of contractile fibers breakdown, was also assessed. Both unexposed groups (FF and KO) had almost undetectable Myl3 contents (FF with 0.204 ± 0.08 pg/ml and SERCA2 KO with 0.158 ± 0.07 pg/ml), whereas Br₂ exposure increased Myl3 concentrations to 1.709 ± 0.31 pg/ml and 2.83±1.11 pg/ml, respectively, in the plasma of FF and KO mice (Fig. 1F).

TABLE 1.

Effect of bromine exposure on LV pump functions in control (FF) and cardiac-specific SERCA2 KO mice

Measurements		0 ppm		600 ppm
	Unit	FF Mean ± S.E. (n = 8: 3 M, 5 F)	KO Mean ± S.E. (n = 6: 3 M, 3 F)	FF Mean ± S.E. (n = 9: 6 M, 3 F)	KO Mean ± S.E. (n = 10: 6 M, 4 F)
Weights
Body weight	g	26 ± 1.6	24 ± 1.6	24 ± 1.2	23.90 ± 1.56
Left ventricle
Hemodynamics
Diastolic BP	mmHg	63 ± 2.2	62 ± 1.5	67 ± 4.7	66 ± 2.7
HR	bpm	490 ± 18.8	501 ± 20	484.00 ± 21	498.00 ± 23
LV +dp/dt	mmHg/s	8094 ± 453	8043 ± 795	9473.83 ± 613	8867.00 ± 673
LV-dp/dt	mmHg/s	−6886 ± 433	−6730 ± 622	−8770 ± 464*b	−7383 ± 438*d
Echo
Avg HR	bpm	480 ± 15	512 ± 28	496 ± 12.76	501 ± 18
IVSd	mm	0.93 ± 0.05	0.97 ± 0.04	0.94 ± 0.04	0.98 ± 0.05
IVSs	mm	1.34 ± 0.05	1.44 ± 0.08	1.32 ± 0.05	1.44 ± 0.07
LVPWd	mm	0.74 ± 0.03	0.86 ± 0.06*a	0.93 ± 0.04***b	0.88 ± 0.04
LVPWs	mm	1.03 ± 0.04	1.11 ± 0.01	1.25 ± 0.04**b	1.21 ± 0.05
LV Vol; s	μl	25.30 ± 2.56	23.83 ± 3.13	21.56 ± 1.99	17.30 ± 2.56
LV Vol; d	μl	56.14 ± 2.30	53.33 ± 1.62	48.11 ± 1.40*b	42.80 ± 1.78*#d
LV SV	μl	28.88 ± 2.16	30.50 ± 1.27	26.78 ± 1.28	25.50 ± 1.54*c
LV EF	%	53.25 ± 2.76	56.17 ± 4.28	56.00 ± 3.13	60.70 ± 4.02
LV CO	ml/min	13.88 ± 1.06	15.00 ± 0.55	13.56 ± 0.90	12.13 ± 0.79*c
LV mass	mg	96.88 ± 5.19	103.67 ± 7.05	104.33 ± 5.04	114.50 ± 6.04
LV FS	%	32.38 ± 0.91	28.80 ± 2.08	32.67 ± 2.52	37.67 ± 1.35**c
RWT; d (2 × pw/id)	mm	0.40 ± 0.02	0.50 ± 0.04*a	0.53 ± 0.03**b	0.54 ± 0.02
RWT; s	mm	0.82 ± 0.04	0.85 ± 0.09	1.08 ± 0.08*b	1.19 ± 0.06*c
IVS/LVPW; d		1.30 ± 0.1	1.15 ± 0.1	1.03 ± 0.06*b	1.13 ± 0.05
IVS/LVPW; s		1.30 ± 0.06	1.34 ± 0.13	1.06 ± 0.03**b	1.19 ± 0.05*d
PWTH (PW thickening)	%	29.04 ± 2.31	21.72 ± 3.80	25.18 ± 3.30	27.62 ± 2.72
VCFr	%	10.16 ± 0.61	11.13 ± 2.24	12.11 ± 0.99	12.53 ± 1.29
Wall stress; d	%	6.91 ± 0.97	6.42 ± 1.11	6.23 ± 1.36	6.24 ± 0.73
Wall stress; s	%	63.80 ± 4.69	58.63 ± 8.16	46.63 ± 3.17*b	47.50 ± 2.46
Vent-art coupling		0.23 ± 0.03	0.20 ± 0.04	0.17 ± 0.02	0.17 ± 0.05
VCFr / ESWS		0.16 ± 0.03	0.25 ± 0.08	0.30 ± 0.03**b	0.31 ± 0.03
ESWS / ESV		2.64 ± 0.27	2.60 ± 0.43	2.54 ± 0.13	3.01 ± 0.26
Aorta
MV E/A		1.29 ± 0.05	1.30 ± 0.06	1.22 ± 0.06	1.17 ± 0.13c

BP, blood pressure; d, diastolic; EF, ejection fraction; ESWS, end systolic wall stress; ESV, end systolic volume; HR, heart rate; IVS, intraventricular septum; LVPW, left ventricular posterior wall; RWT, relative wall thickness; s, systolic; SV, stroke volume; VCFr, velocity of circumferential fractional shortening; MV E/A, early to late transmitral diastolic velocity.

* P < 0.05, ** P < 0.01, *** P < 0.001, # indicates significant difference from both FF (control) and KO (SERCA2 KO) unexposed and significant difference are shown as FF Unexposed versus KO Unexposed^a; FF Unexposed versus FF Exposed^b; KO Unexposed versus KO Exposed^c; FF Exposed versus KO Exposed^d.

Fig. 1.

Cardiac-specific SERCA2 KO mice have increased cardiac injury and decreased survival after Br₂ exposure. The top panel demonstrates the experimental design for obtaining heart-specific SERCA2 KO mice and subsequent Br₂ exposure. Serca2^flox/flox Tg (αMHC-MerCreMer) mice were injected with peanut oil (FF) or tamoxifen (KO) 2 weeks after weaning. Six to 8 weeks after injections mice were exposed to room air (0 ppm) or Br₂ (600 ppm for 30 minutes) and returned to room air and various endpoints were evaluated 24 to 96 hours later. Kaplan–Meier curves were generated for survival data of animals that were followed for four days after exposure (A) (….) lines are for KO and (––-) lines demonstrate FF after Br₂ exposure (600 ppm for 30 minutes) and the room air (0 ppm) exposed animal are represented by the combined bold line. Data are mean ± S.E. (n = 12: 6 males and 6 females) for each group in survival analysis. *Statistical significance from 0 ppm P < 0.05. Other animals (survivors of separate Br₂ exposures) were euthanized 24 hours after exposure, and tissues and plasma (from arterial blood collected from descending aorta) were evaluated. (B) Demonstrates heart weights of the animals after Br₂ exposure where (●) indicates RA-exposed FF, (▲) indicates RA-exposed KOs, (○) indicates Br₂ exposed FF and (△) indicates Br₂ exposed KOs. Similarly, cardiac injury markers such as cardiac troponin I, cTnI (C), sTNI (D), h-FABP3 (E), and Myl3 (F) were measured in plasma of the groups as indicated previously using commercial ELISA kits as described in the methods. Data are mean ± S.E. (n = 5–8 for each group).

Bromine Inhalation Causes Increased Cardiac Ultrastructure Damage and Cytosolic Protease Activation in Cardiac-Specific SERCA2 KO Mice.

We evaluated the cardiac ultrastructure by electron microscopy to further examine the effect of Br₂ in FF and KOs. Corresponding with the increased plasma biomarker cTnI, we observed increased ultrastructural damage at baseline in the naïve cardiac-specific SERCA2 KOs, which corroborates the source of plasma cTnI (Fig. 2A, top right panel). This damage included marked mitochondrial disarray and swelling, disruption of z-disks, and loss of I bands. Ultrastructural damage was evident in both FF and KOs upon Br₂ exposure as depicted in the lower panel of Fig. 2A. Mitochondrial swelling, cristae lysis, increased vacuolization, and lipid droplet formation were also observed. Disruption of z-disks and loss of I bands were more prominent in the hearts of the cardiac-specific SERCA2 KOs (Fig. 2A, lower panel and Supplemental Fig. 2, A and B).

Fig. 2.

Increased Br₂-induced ultrastructural damage and protease activation in the LV of cardiac-specific SERCA2 KO mice. Control (FF) and cardiac-specific SERCA2 KO mice were exposed to bromine and transferred to RA. Twenty-four hours later, the animals were sacrificed, and pieces of hearts (LV) were fixed for TEM or frozen to assay for calpain activity (A, B). (A) Images were obtained at 13,000 × magnification. Images demonstrated mitochondrial injury with swelling and loss of cristae (arrows), myosin fiber degeneration (asterisks) and Z line degeneration (triangle). Calpain activity was measured in cardiac tissue lysates (B) where (●) indicates RA-exposed FF, (▲) indicates RA-exposed KOs, (○) indicates Br₂-exposed FF, and (△) indicates Br₂-exposed KOs (n = 5–8 for each group). (C–F) Demonstrate SERCA2 and phospholamban, PLN and phosphorylated phospholamban in FF and KO mice hearts after Br₂ exposure. Data are mean ± S.E. (n = 5–8 for each group).

We have previously described an increase in calpain activity owing to the loss of SERCA2 and accumulation of cytosolic Ca²⁺, which are major drivers in Br₂-induced cardiac injury. Calpain activity was increased in the RA KO mice hearts indicating an increased baseline effect as compared with the RA FF controls and further confirming our findings (Fig. 2B). Both FF and KOs mice exposed to Br₂ had significantly increased calpain activity in their hearts (Fig. 2B). Br₂ exposure decreased cardiac SERCA2 content in the FF mice, but in the survivors where the protein content was measured, statistical significance was not achieved (P = 0.06) (Fig. 3, C and D). The cardiac SERCA2 content of KO mice remained lower after Br₂ inhalation and was significantly reduced compared with the FF mice, both in RA and after Br₂ exposure.

Fig. 3.

Bromine exposure induces LV dysfunction in control FF and cardiac-specific SERCA2 KO mice. Control (FF) and cardiac-specific SERCA2 KO mice were exposed to Br₂ and transferred to RA. Echocardiography and hemodynamics were performed in anesthetized animals 24 hours later and mean arterial pressure (A), systolic blood pressure (B), LV end diastolic pressure (C), LV end systolic pressure (D), LV end diastolic diameter (E), and LV end systolic diameter (F) were determined. Systolic and diastolic sphericity indices (L/D axis) (G, H) were also determined to assess shape changes. (●) indicates RA-exposed FF, (▲) indicates RA-exposed KOs, (○) indicates Br₂-exposed FF, and (△) indicates Br₂-exposed KOs. Data are mean ± S.E. (n = 5–8 for each group).

Phospholamban, an allosteric regulator of SERCA2 activity in the sarcoendoplasmic reticulum, is activated by phosphorylation, which removes it from the SERCA2 molecule. Br₂ inhalation in the KO mice significantly increased phosphorylation of phospholamban compared with both the RA FF and KO, and bromine-exposed FF mice (Fig. 2, C, E, and F and Supplemental Fig. 2C). The total cardiac phospholamban levels remained unaffected in all the groups, suggesting a possible bromine-induced adaptive or regulatory response aimed at activating the remaining cardiac SERCA2 in KO mice.

Bromine Inhalation-Induced Exacerbation of Biventricular Cardiac Dysfunction in SERCA2 KO Mice.

Under our conditions of housing and the tamoxifen administration schedule, the cardiac-specific SERCA2 KOs in room air (0 ppm) did not exhibit any obvious pathologic phenotype. The mean body weight of all the animal groups, including the ones that were exposed to Br₂, were not significantly different when measured at the 24-hour time point (Table 1). We assessed both LV (Table 1 and Fig. 4) and RV function (Table 2) by performing both hemodynamics and echocardiography in Br₂-exposed FF and KO mice 24 hours after exposure. Br₂ exposure significantly increased the mean arterial pressure in cardiac-specific SERCA2 KO mice (Fig. 3A). The systolic blood pressure was significantly increased in both FF and KOs upon bromine exposure (Fig. 3B). The diastolic blood pressure (blood pressure minimum) and heart rate were similar in all groups both in RA and 24 hours after Br₂ exposure (Table 1). The LV end diastolic pressure of the KO animals was significantly increased after Br₂ exposure as compared with the RA FF but not when compared with RA KO mice (Fig. 3C). The LV end systolic pressure was significantly increased upon Br₂ inhalation in the KO mice as compared the RA FF and KO (Fig. 3D). The LV contractility as measured by rate of rise of systolic LV pressure, positive pressure change rate (dp/dt) was not significantly different between the RA FF and KO mice (Table 1). Br₂ exposure seemed to enhance the LV +dp/dt but was not statistically significant in both FF and KO. The rate of decline of diastolic LV pressure was significantly reduced by Br₂ inhalation in both FF and KO mice, and it was significantly different between Br₂-exposed FF and Br₂-exposed KO mice (Table 1). Echocardiography of the mice revealed that LV end diastolic dimension (diameter) was significantly reduced in the KO animals as compared with the FF mice (Fig. 3E). A trend toward reduced LV end systolic dimension (diameter) was also demonstrated in the KO mice (Fig. 3F). Br₂ exposure significantly reduced both LV end diastolic diameter and LV end systolic diameter in the KO animals (Fig. 3, E and F). The LV posterior wall thickness at end diastole was significantly increased in the RA KO as compared with the RA FF mice (Table 1). Br₂ inhalation significantly increased both the end systolic and end diastolic left ventricular posterior wall thickness in the FF mice, but the KO animals did not have a significant increase. The LV relative wall thickness was significantly increased in the RA KO mice when compared with the RA FF mice (Table 1). Br₂ exposure significantly increased both relative wall thickness at end diastole and at end systole in the FF mice, whereas only relative wall thickness was significantly increased upon Br₂ exposure in the KOs (Table 1). The left ventricular septal to posterior wall ratio was significantly increased in the Br₂-exposed KOs as compared with Br₂-exposed FF mice (Table 1). The LV end diastolic volume was significantly reduced upon Br₂ inhalation between Br₂-exposed FF and KO (Table 1). Although not significantly different in FF versus KO, Br₂ exposure caused significantly reduced stroke volume and increased LV FS in the KO mice as compared with the RA KO (Table 1). Ratio of velocity of fractional shortening by end systolic wall stress was significantly increased in the Br₂-exposed FF mice but was not statistically significant in the KO mice exposed to Br₂. The mitral valve ratio of early E wave to late A wave ventricular filling velocities (E/A) was significantly decreased upon Br₂ exposure in the KO mice, further suggesting impaired relaxation of the LV (Table 1). Br₂ exposure significantly increased the systemic vascular resistance in the KO mice (378.37 ± 23.90 mmHg min/ml in RA KO vs 524 ± 43.27 mmHg min/ml in Br₂-exposed KO) but not in the FF controls.

TABLE 2.

Effect of bromine inhalation on RV function in control FF and cardiac-specific SERCA2 KO mice

Measurements		0 ppm		600 ppm
	Unit	FF Mean ± S.E. (n = 8: 3 M, 5 F)	KO Mean ± S.E. (n = 6: 3 M, 3 F)	FF Mean ± S.E. (n = 9: 6 M, 3 F)	KO Mean ± S.E. (n = 10: 6 M, 4 F)
Hemodynamics
RV peak press	mmHg	28.17 ± 5.00	30.20 ± 3.02	29.00 ± 1.52	30.22 ± 1.36
RV mean press	mmHg	10.83 ± 0.98	11.00 ± 1.82	8.80 ± 1.16	9.89 ± 0.68
RV EDP	mmHg	2.67 ± 0.95	2.20 ± 0.73	1.20 ± 0.20	3.44 ± 1.61
RV +dP/dT	mmHg/s	2024 ± 160	2241 ± 76	2416 ± 300	2482 ± 185
RV-dP/dT	mmHg/s	−1784 ± 182	−1695 ± 102	−2197 ± 56	−1852 ± 204
HR (RV)	bpm	459 ± 30	421 ± 23	464 ± 29	468 ± 24
Echo
RV FW; d	mm	0.32 ± 0.03	0.34 ± 0.03	0.33 ± 0.02	0.35 ± 0.03
RV FW; s	mm	0.61 ± 0.07	0.66 ± 0.08	0.64 ± 0.06	0.68 ± 0.08
PA Hemodynamics
PAT	ms	14.75 ± 0.78	16.78 ± 0.87	17.35 ± 0.80*b	16.17 ± 0.76
PET	ms	43.96 ± 1.93	42.54 ± 1.44	43.69 ± 1.31	39.14 ± 1.52*d
PV diam	mm	1.34 ± 0.03	1.48 ± 0.06*a	1.46 ± 0.05	1.44 ± 0.05
PV VTI	mm	21 ± 1.4	23.31 ± 0.56	21.11 ± 0.85	19.99 ± 1.05*c
PV VTI	mm/s	−320 ± 10	−357 ± 15	−314 ± 10	−327 ± 16
PV VTI	mmHg	0.41 ± 0.03	0.48 ± 0.03	0.40 ± 0.03	0.44 ± 0.04
PV VTI	mm/s	−629 ± 14	−663 ± 29	−610 ± 17	−658 ± 26
PV VTI	mmHg	1.59 ± 0.07	1.78 ± 0.16	1.50 ± 0.09	1.76 ± 0.15
MPAP	mmHg	80.86 ± 0.48	78.92 ± 0.81	78.53 ± 1.00	79.97 ± 0.47
PAT/PET		0.33 ± 0.01	0.42 ± 0.03*a	0.42 ± 0.03*b	0.42 ± 0.03
PV CO	mL/min	16.19 ± 1.19	23.29 ± 2.88*a	18.00 ± 1.59	16.92 ± 0.83*c
PV SV	μl	31.27 ± 2.70	42.70 ± 4.16*a	35.34 ± 2.16	31.96 ± 1.30*c
TV E/A		0.56 ± 0.06	0.63 ± 0.03	0.47 ± 0.04	0.47 ± 0.04*c
TAPSE	mm	0.67 ± 0.06	0.42 ± 0.06**a	0.48 ± 0.05*b	0.42 ± 0.04
IVC diameter		0.31 ± 0.04	0.35 ± 0.05	0.27 ± 0.04	0.33 ± 0.04
HV Hemodynamics
HV velocity S/A		1.09 ± 0.20	1.28 ± 0.24	0.99 ± 0.26	1.09 ± 0.18
HV velocity S/D		−1.09 ± 0.20	−1.28 ± 0.24	−0.99 ± 0.26	−1.09 ± 0.18
HV VTI S/A		15.26 ± 6.28	9.12 ± 3.33	2.40 ± 0.72	3.99 ± 1.45
HV VTI S/D		1.57 ± 0.48	1.90 ± 0.43	1.66 ± 0.65	1.75 ± 0.48
Vascular
Systemic Vasc Resist.	mmHg*min/mL	450 ± 41	378 ± 23	446 ± 27	524 ± 43*c

FW, free wall; MPAP, mean pulmonary artery pressure; IVC, inferior vena cava; TV E/A, tricuspid valve ratio of early to late diastolic filling.

* P < 0.05, ** P < 0.01, and significant difference are shown as FF Unexposed versus KO Unexposed^a; FF Unexposed versus FF Exposed^b; KO Unexposed versus KO Exposed^c; FF Exposed versus KO Exposed^d.

Fig. 4.

LV circumferential strain measurement using two-dimensional speckle tracking echocardiography in control (FF) and cardiac-specific SERCA2 KO mice before and after Br₂ inhalation. Shown are echocardiographic representation of speckle tracking analysis in short axis B-mode wall trace and circumferential three-dimensional strain and representative echocardiographic traces of short axis circumferential segmental synchronicity in the mice measured in RA- or bromine-exposed FF and KO mice. The LV images are representative of n = 4 male or female mice/group.

Right ventricle hemodynamics and echocardiographic measurements did not reflect any significant difference between the RA FF and KO mice. Our hemodynamic studies did demonstrate an increased pulmonary artery acceleration time (PAT) in the FF mice after Br₂ exposure and pulmonary ejection time (PET) was significantly decreased in Br₂-exposed KOs compared with the Br₂-exposed FF (Table 2). The pulmonary vein (PV) velocity time integral (VTI) was significantly decreased in the KO mice upon Br₂ exposure. The ratio of pulmonary artery acceleration time to right ventricular ejection time (PAT/PET) was significantly increased in the RA KO mice compared with the RA FF mice. The PAT/PET was increased in FF mice upon Br₂ exposure while it remained at baseline values in the KOs (Table 2). Right ventricular stroke volume and tricuspid valve ratio of early to late diastolic filling were significantly decreased in the KO mice upon Br₂ exposure. Tricuspid annular plane systemic excursion (TAPSE) was significantly reduced in the RA KOs as compared with the RA FF (Table 2). Br₂ exposure significantly decreased TAPSE in the FF mice. We also measured the hepatic vein hemodynamics and vascular function, yet no significant differences between or within groups were noticed. Reduction in TAPSE, PV VTI, RV stroke volume, and CO are all indicators of severe intrinsic RV systolic dysfunction. A significant reduction in the tricuspid valve ratio of early to late diastolic filling denotes an impaired relaxation pattern of the RV.

The sphericity index is an index used to evaluate cardiac geometry in response to loading conditions (DeVore et al., 2018). We measured the sphericity index in the RA FF and KO mice and observed that the sphericity index was significantly altered in the RA KOs (Fig. 3, G and H). LV of Br₂ exposed KO mice had elevated diastolic and systolic sphericity index in the KO mice as compared with the Br₂-exposed FF mice principally due to a reduction in LV longitudinal diameter during both systole and diastole denoting a more spherical shape of LV during the cardiac cycle. LV strain measures myocardial deformation and is used in the assessment of subclinical cardiac dysfunction in a variety of cardiac diseases as recommended by international echocardiography guidelines (Smiseth et al., 2016). To further evaluate the LV function, we used two-dimensional speckle tracking echocardiography to measure global longitudinal and circumferential strains in RA FF and KO mice, as well as Br₂-exposed FF and KO animals. There was a reduction in peak global circumferential strain (value less negative) in KO mice both at RA and after Br₂ exposure denoting a reduction in circumferential systolic shortening of LV myocardium across the short axis. Time to peak deformation varied between different segments of the LV upon Br₂ exposure denoting intraventricular dys-synchrony (Fig. 4 and Supplemental Fig. 3, A and B). LV cardiac electrical abnormalities were apparent by electrocardiogram findings as demonstrated in Fig. 5 in both FF and KO after Br₂ inhalation. The QT interval was increased in the SERCA2 KOs at baseline (Fig. 5, A and B).

Fig. 5.

Electrophysiological dysfunction in cardiac-specific SERCA2 KO mice and effect of Br₂ inhalation. Control (FF) and cardiac-specific SERCA2 KO mice were exposed to Br₂ and transferred to RA. Mice were anesthetized 24 hours later, and electrocardiogram was collected by a two-limb (upper right leg–lower left leg) lead connection. For each animal, the QT interval for 10 subsequent complexes was measured and averaged.

Discussion

Halogen gases interact with the pulmonary bed plasmalogens to produce highly reactive amines and fatty aldehydes that continue to cause detrimental organ damage long after the exposure (White and Martin 2010; Zaky et al., 2015b; Ford et al., 2016; Zhou et al., 2018; Ahmad et al., 2019; Juncos et al., 2020; Masjoan Juncos et al., 2021; Addis et al., 2021b). Acute halogen exposures can cause fatalities owing to both pulmonary and cardiac damage (Ball and Dworak 2005; White and Martin 2010; Howell et al., 2019). Our studies show that chlorine and bromine produce highly reactive amines and fatty aldehydes that modify important cardiac proteins like SERCA2 and alter downstream signaling pathways leading to myocardial failure (Ahmad et al., 2015; Zaky et al., 2015b; Juncos et al., 2020). Here we demonstrate that cardiac-specific SERCA2 knockdown can manifest baseline myocardial damage that can be partly attributed to ultrastructural damage caused by increased proteolysis due to calpain activation, subsequent hypertrophy, and alteration in ventricular strain and sphericity. Cardiac-specific SERCA2 KO mice are susceptible to halogen exposure, showing significant myocardial damage, increased h-FABP3 release in plasma, increased PLN phosphorylation, and significant changes in LV geometry, which lead to subclinical systolic dysfunction. This dysfunction was further exacerbated by Br₂ inhalation, as evidenced by LV strain measurements obtained through 2D speckle tracking.

Bromine inhalation has been demonstrated to be lethal in mice as evidenced by this study and a previous report (Lambert et al., 2017). However, susceptibility of cardiac-specific SERCA2 KO mice to Br₂ is unknown. Br₂ damages the airway and alveolar epithelium resulting in bronchitis and acute respiratory distress syndrome (Addis et al., 2021a). Additionally, systemic effects, vascular damage, and cardiac manifestations also occur (Zaky et al., 2015b; Ahmad et al., 2019; Addis et al., 2020, 2021a). While evidence of Br₂-induced skeletal muscle and contractile fiber damage has not been shown before, inhaled H₂S is known to cause skeletal muscle inflammation (Jing et al., 2019). Increased cytosolic calcium-induced skeletal muscle calpain increase and release of sTNI have been shown previously (Onuoha et al., 2001). Although the source of these proteins (sTNI and Myl3) is skeletal muscle, no differences in the FF and KO were observed in this regard. However, these findings underscore the importance of cardiac injury and cardiac SERCA2 in survival following bromine exposure.

Contractile abnormalities and cardiac pathogenesis have been associated with decreased SERCA2 protein levels (Takahashi et al., 1992; MacLennan and Kranias 2003; Kho 2023; Subramanian and Nikolaev 2023). There is a critical correlation between SERCA2 content in the myocardium and cardiac function, with diminished SERCA2 associated with increased arrhythmogenic potential due to elevated diastolic calcium levels. Reduced SERCA2 has also been observed in the hearts of patients with dilated cardiomyopathy and ischemic cardiomyopathy (Kho 2023). Inhibition of SERCA2 activity due to mutated PLN causes fatal arrhythmic/dilated cardiomyopathy and premature death (Haghighi et al., 2006). Furthermore, numerous studies have demonstrated that genetic or pharmacologic restoration of SERCA2 activity can reverse heart failure progression, confirming the significance of SERCA2 in maintaining normal cardiac function and its role in disease pathogenesis (del Monte et al., 2001; Bidwell et al., 2022). Therefore, heart failure patients with decreased SERCA2 activity and decreased cardiac function can be vulnerable during an event of a bromine spill.

Short-term (up to 4 weeks) cardiac-specific SERCA2 knockdown in mice caused a moderate reduction in cardiac function owing to compensatory increase in sympathetic tone, increased transmembrane calcium fluxes, and increased myofilament responsiveness (Antoons et al., 2003; Andersson et al., 2009). Prolonged cardiac-specific SERCA2 gene excision resulted in severe cardiac dysfunction, shown by impaired LV relaxation and a five fold increase in isovolumetric pressure decay and increased LV end diastolic pressure (Andersson et al., 2009). The cardiac specific KOs have been shown to have cardiac enlargement and significantly increased RV weights at baseline, which was not measured separately in this study (Andersson et al., 2009). The overall hemodynamic and echocardiographic picture in our study reveals a differential pattern of biventricular and vascular dysfunction between the right and left sides of the heart after Br₂ exposure in FF and KO mice. In the left heart, there is a severe diastolic dysfunction (reduction in left ventricular end diastolic diameter (LV EDD), LV EDV, and LV dp/dt increase in LV end diastolic pressure, increased LV mass and wall thickness, and reduced transmitral E/A) resulting from an increase in systemic vascular resistance and LV afterload with the ventricle acquiring a more spherical shape during the cardiac cycle, with preservation of LV EF. LV EF was preserved despite a reduction in SV mainly because of a severe reduction in EDV. The reduction in LV cardiac output and SV are primarily explained by a significant increase in afterload. This stage of Br₂ exposure resulted in enhancement of LV contractility manifested by increased LV dp/dt, LV FS, and velocity of circumferential fiber shortening (VCFr). Data on LV strain, though suggestive of a subclinical intrinsic reduction in LV contractility, remain exploratory at the present stage. In the right heart, there is severe RV systolic (reduction in PV VTI, TAPSE, CO, and SV) and diastolic (reduced tricuspid valve E/A) dysfunction without an increase in RV afterload or pulmonary vascular resistance, denoting an intrinsic RV dysfunction.

Ultrastructural damage shown by TEM and an increase in plasma cTnI, seen in unexposed cardiac-specific KO animals, is used to assess such cardiac remodeling (Hillestad et al., 2013). An increase in cTnI is due to myofibrillar degeneration in cardiac tissue, explaining the loss of definition in contractile bands in TEM images (Hanft et al., 2016; Masjoan Juncos et al., 2021). An increase in calpain has been reported as a central component of acute and chronic cardiac remodeling (Daniel 1975; Luo and Anderson 2013; Masjoan Juncos et al., 2021). An increase in h-FABP3 upon Br₂ exposure in the KOs indicates an altered energy demand and available energy pool and points to a critical mechanism of toxicant susceptibility (Binas et al., 1999; Iqubal et al., 2019). Serial measurements of h-FABP content are an accurate indicator of the extent of injury and long-term prognosis after acute injury (Ye et al., 2018). Cardiomyocytes of the cardiac-specific SERCA KO mice have a significantly altered phenotype, but ultrastructural damage has not been reported (Andersson et al., 2009). SERCA2 inactivation is central to cardiac cell death and injury caused by ischemia reperfusion and myocardial infarction (Gonnot et al., 2023). The ensuing mitochondrial fission fusion changes and subsequent damage are also critical in the cytosolic overload induced by Br₂ inhalation and/or SERCA2 inactivation (Ahmad et al., 2019; Hernandez-Resendiz et al., 2023; Murphy and Liu 2023). SERCA2 inactivation followed by calpain activation occurs in ischemia reperfusion injury and could be prevented by SERCA2 activation (French et al., 2006; Wang et al., 2021). Phospholamban phosphorylation upon Br₂ inhalation in the KO mice signifies a critical compensatory response to activate SERCA2. In contrast to this study, RA cardiac-specific SERCA2 KO mice at the 7-week timepoint demonstrated significantly increased PLN phosphorylation indicating possible differences in experimental materials and study protocols (Andersson et al., 2009).

Cardiomyocyte Ca²⁺ and its homeostasis are largely controlled by SERCA2 and play a key role in cardiac dysfunction (Periasamy et al., 1999; Roe et al., 2015). SERCA2^null mutant mice die in utero, and deletion of one SERCA2 allele or its isoform SERCA2a results in mild concentric hypertrophy and impaired contractility (Ver Heyen et al., 2001; Antoons et al., 2003). Mice with systemic one allele deletion quickly proceed to heart failure when challenged with pressure overload and are sensitive to ischemia reperfusion injury (Schultz et al., 2004; Talukder et al., 2008). Although complete cardiomyocyte-specific deletion of the SERCA2 gene caused a modest cardiac dysfunction in the early few weeks, the mice eventually progressed to heart failure regardless of the presence of any other stress or insult (Andersson et al., 2009). An increase in Na⁺-Ca²⁺ exchanger (NCX) expression and activity compensate for the decrease in Ca²⁺ transport capabilities (Bers and Despa 2006; Louch et al., 2010; Li et al., 2012; Luo and Anderson 2013). Active transport by the NCX pump increases anaerobic metabolism that in turn increases cytosolic acidification, increasing activity by the Na⁺-H⁺ exchanger, which further increases anaerobic metabolic demand (Røe et al., 2019; Aksentijević and Shattock 2021). Anabolic metabolic demand leads to a cytosolic accumulation of both Ca²⁺ and Na⁺ (Røe et al., 2019; Aksentijević and Shattock 2021; Varró et al., 2021). These animals present with systolic dysfunction as observed by a decrease in their EF, SV, FS, and +dp/dt (Andersson et al., 2009). There is also diastolic dysfunction present as evidenced by decreased LV volumes, –dp/dt, and mechanical efficiency, while isovolumetric relaxation time, EDP, and left ventricular relaxation time constant are increased (Andersson et al., 2009; Li et al., 2012). All this leads to heart failure when the heart is no longer capable of maintaining a normal CO.

Under adverse pathologic conditions however, reduction or loss of SERCA2 activity can be detrimental resulting in deaths and long-term morbidity (Heitner and Hollenberg 2009; Guerrero-Beltrán et al., 2017; Ahmad et al., 2019; Goodman et al., 2020; Masjoan Juncos et al., 2021). Calpains degrade proteins essential for cardiac contractility, such as titin (Lim et al., 2004; H.E. Cizauskas et al., preprint, DOI: https://pubmed.ncbi.nlm.nih.gov/37961455/). This proteolytic activity expands to even SERCA2 itself, leading to further impaired Ca²⁺ transport (French et al., 2006; Ahmad et al., 2019). Altered ion transport across membranes is a well-established risk factor for cardiac injury, leading to arrhythmias, cardiac failure, cardiac arrest, and sudden death (Varró et al., 2021). SERCA2 KO increases NCX activity to compensate for reduced Ca²⁺ transport, and the inflow of Ca²⁺ to the sarcoendoplasmic reticulum slows the Ca²⁺wave velocity and rate decrease, resulting in a reduced heart rate. Further evidence is present as QT interval elongation, which is of significant prognostic value (Němec et al., 2016).

In summary, the disruption of Ca²⁺ transport due to impaired SERCA2 activity leads to structural remodeling through the induction of calpain activity. The normal hemodynamic and electrophysiological function is also impaired in the absence of regular SERCA2 activity. Therefore, a reduction in SERCA2 expression and the resulting decrease in SERCA2 activity before halogen exposure further accentuates the same injury mechanism and exacerbates cardiac injury caused by exposure to Br₂. Compared with our previous studies in rats, there are two major considerations that might affect our findings in this study: 1) mice may have different susceptibility to Br₂ concentrations used here, and 2) SERCA2 is acutely affected after Br₂ exposure, whereas, in this study, we investigate 6 to 8 weeks after SERCA2 knockout, when all other compensatory mechanisms have already been activated. Despite these considerations, we noted a significant difference in survival rates after bromine exposure in FF and KO mice, highlighting the crucial role of cardiac SERCA2. A notable decrease in cardiac SERCA2 content between FF Br₂-exposed and KO Br₂-exposed mice, along with a significant cardiac structural perturbation in SERCA2 KO, as demonstrated by LV sphericity index and strain measurements, is critical. Significant baseline cardiac injury attributed to decreased SERCA2 and increased proteolytic/calpain activity further contributed to the outcomes alongside other unmeasured factors. These studies not only identify SERCA2 as a critical therapeutic target but also highlight the susceptibility of individuals with pathologic SERCA2 loss to halogen/Br₂ exposure.

Abbreviations

Br₂

bromine

Cl₂

chlorine

Ca²⁺

calcium ion

cTnI

cardiac troponin I

CO

cardiac output

dp/dt

pressure change rate

EDP

end diastolic pressure

EDV

end diastolic volume

EF

ejection fraction

FF

peanut oil administered

FS

fraction shortening

h-FABP

heart fatty acid binding protein

KO

knockout

LV

left ventricle

MHC

myosin heavy chain

NCX

Na⁺-Ca²⁺ exchanger

PAT

pulmonary acceleration time

PET

pulmonary ejection time

PLN

phospholamban

PV

pulmonary vein

RA

room air

RV

right ventricle

SERCA2

sarcoendoplasmic reticulum calcium ATPase 2

sTNI

skeletal troponin I

SV

stroke volume

TAPSE

tricuspid annular plane systolic excursion

TEM

transmission electron microscopy

VTI

velocity time integral

Authorship Contributions

Participated in research design: Masjoan Juncos, Louch, A. Ahmad, S. Ahmad.

Conducted experiments: Masjoan Juncos, Shakil, El-Husari, Zafar.

Performed data analysis: Masjoan Juncos, Nadeem, Halade, Zaky, A. Ahmad.

Wrote or contributed to the writing of the manuscript: Masjoan Juncos, Louch, Zaky, A. Ahmad, S. Ahmad.