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.

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). SERCA2null 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.