Cardiomyocyte-specific PCSK9 deficiency compromises mitochondrial bioenergetics and heart function.

AIMS: Pro-protein convertase subtilisin-kexin type 9 (PCSK9), which is expressed mainly in the liver and at low levels in the heart, regulates cholesterol levels by directing low-density lipoprotein receptors to degradation. Studies to determine the role of PCSK9 in the heart are complicated by the close link between cardiac function and systemic lipid metabolism. Here, we sought to elucidate the function of PCSK9 specifically in the heart by generating and analysing mice with cardiomyocyte-specific Pcsk9 deficiency (CM-Pcsk9-/- mice) and by silencing Pcsk9 acutely in a cell culture model of adult cardiomyocyte-like cells. METHODS AND RESULTS: Mice with cardiomyocyte-specific deletion of Pcsk9 had reduced contractile capacity, impaired cardiac function, and left ventricular dilatation at 28 weeks of age and died prematurely. Transcriptomic analyses revealed alterations of signalling pathways linked to cardiomyopathy and energy metabolism in hearts from CM-Pcsk9-/- mice vs. wild-type littermates. In agreement, levels of genes and proteins involved in mitochondrial metabolism were reduced in CM-Pcsk9-/- hearts. By using a Seahorse flux analyser, we showed that mitochondrial but not glycolytic function was impaired in cardiomyocytes from CM-Pcsk9-/- mice. We further showed that assembly and activity of electron transport chain (ETC) complexes were altered in isolated mitochondria from CM-Pcsk9-/- mice. Circulating lipid levels were unchanged in CM-Pcsk9-/- mice, but the lipid composition of mitochondrial membranes was altered. In addition, cardiomyocytes from CM-Pcsk9-/- mice had an increased number of mitochondria-endoplasmic reticulum contacts and alterations in the morphology of cristae, the physical location of the ETC complexes. We also showed that acute Pcsk9 silencing in adult cardiomyocyte-like cells reduced the activity of ETC complexes and impaired mitochondrial metabolism. CONCLUSION: PCSK9, despite its low expression in cardiomyocytes, contributes to cardiac metabolic function, and PCSK9 deficiency in cardiomyocytes is linked to cardiomyopathy, impaired heart function, and compromised energy production.

Glucosylceramide synthase deficiency in the heart compromises ß1-adrenergic receptor trafficking.

AIMS: Cardiac injury and remodelling are associated with the rearrangement of cardiac lipids. Glycosphingolipids are membrane lipids that are important for cellular structure and function, and cardiac dysfunction is a characteristic of rare monogenic diseases with defects in glycosphingolipid synthesis and turnover. However, it is not known how cardiac glycosphingolipids regulate cellular processes in the heart. The aim of this study is to determine the role of cardiac glycosphingolipids in heart function. METHODS AND RESULTS: Using human myocardial biopsies, we showed that the glycosphingolipids glucosylceramide and lactosylceramide are present at very low levels in non-ischaemic human heart with normal function and are elevated during remodelling. Similar results were observed in mouse models of cardiac remodelling. We also generated mice with cardiomyocyte-specific deficiency in Ugcg, the gene encoding glucosylceramide synthase (hUgcg-/- mice). In 9- to 10-week-old hUgcg-/- mice, contractile capacity in response to dobutamine stress was reduced. Older hUgcg-/- mice developed severe heart failure and left ventricular dilatation even under baseline conditions and died prematurely. Using RNA-seq and cell culture models, we showed defective endolysosomal retrograde trafficking and autophagy in Ugcg-deficient cardiomyocytes. We also showed that responsiveness to ß-adrenergic stimulation was reduced in cardiomyocytes from hUgcg-/- mice and that Ugcg knockdown suppressed the internalization and trafficking of ß1-adrenergic receptors. CONCLUSIONS: Our findings suggest that cardiac glycosphingolipids are required to maintain ß-adrenergic signalling and contractile capacity in cardiomyocytes and to preserve normal heart function.

Chlorine inhalation induces acute chest syndrome in humanized sickle cell mouse model and ameliorated by postexposure hemopexin.

Triggering factors of Acute Chest Syndrome (ACS) is a leading cause of death in patients with Sickle Cell Disease (SCD) and targeted therapies are limited. Chlorine (Cl2) inhalation happens frequently, but its role as a potential trigger of ACS has not been determined. In this study, we hypothesized that Cl2 exposure resembling that in the vicinity of industrial accidents induces acute hemolysis with acute lung injury, reminiscent of ACS in humanized SCD mice. When exposed to Cl2 (500 ppm for 30 min), 64% of SCD mice succumbed within 6 h while none of the control mice expressing normal human hemoglobin died (p<0.01). Surviving SCD mice had evidence of acute hemolysis, respiratory acidosis, acute lung injury, and high concentrations of chlorinated palmitic and stearic acids (p<0.05) in their plasmas and RBCs compared to controls. Treatment with a single intraperitoneal dose of human hemopexin 30 min after Cl2 inhalation reduced mortality to around 15% (p<0.01) with reduced hemolysis (decreased RBCs fragility (p<0.001) and returned plasma heme to normal levels (p<0.0001)), improved oxygenation (p<0.0001) and reduced acute lung injury scores (p<0.0001). RBCs from SCD mice had significant levels of carbonylation (which predisposes RBCs to hemolysis) 6 h post-Cl2 exposure which were absent in RBCs of mice treated with hemopexin. To understand the mechanisms leading to carbonylation, we incubated RBCs from SCD mice with chlorinated lipids and identified sickling and increased hemolysis compared to RBCs obtained from control mice and treated similarly. Our study indicates that Cl2 inhalation induces ACS in SCD mice via induction of acute hemolysis, and that post exposure administration of hemopexin reduces mortality and lung injury. Our data suggest that SCD patients are vulnerable in Cl2 exposure incidents and that hemopexin is a potential therapeutic agent.

Integrative transcriptomic analysis of tissue-specific metabolic crosstalk after myocardial infarction.

Myocardial infarction (MI) promotes a range of systemic effects, many of which are unknown. Here, we investigated the alterations associated with MI progression in heart and other metabolically active tissues (liver, skeletal muscle, and adipose) in a mouse model of MI (induced by ligating the left ascending coronary artery) and sham-operated mice. We performed a genome-wide transcriptomic analysis on tissue samples obtained 6- and 24 hr post MI or sham operation. By generating tissue-specific biological networks, we observed: (1) dysregulation in multiple biological processes (including immune system, mitochondrial dysfunction, fatty-acid beta-oxidation, and RNA and protein processing) across multiple tissues post MI and (2) tissue-specific dysregulation in biological processes in liver and heart post MI. Finally, we validated our findings in two independent MI cohorts. Overall, our integrative analysis highlighted both common and specific biological responses to MI across a range of metabolically active tissues.

The human body is like a state-of-the-art car, where each part must work together with all the others. When a car breaks down, most of the time the problem is not isolated to only one part, as it is an interconnected system. Diseases in the human body can also have systemic effects, so it is important to study their implications throughout the body. Most studies of heart attacks focus on the direct impact on the heart and the cardiovascular system. Learning more about how heart attacks affect rest of the body may help scientists identify heart attacks early or create improved treatments. Arif and Klevstig et al. show that heart attacks affect the metabolism throughout the body. In the experiments, mice underwent a procedure that mimics either a heart attack or a fake procedure. Then, Arif and Klevstig et al. compared the activity of genes in the heart, muscle, liver and fat tissue of the two groups of mice 6- and 24-hours after the operations. This revealed disruptions in the immune system, metabolism and the production of proteins. The experiments also showed that changes in the activity of four important genes are key to these changes. This suggests that this pattern of changes could be used as a way to identify heart attacks. The experiments show that heart attacks have important effects throughout the body, especially on metabolism. These discoveries may help scientists learn more about the underlying biological processes and develop new treatments that prevent the harmful systemic effects of heart attacks and boost recovery.

Trends in strong opioid prescribing in Ireland: A repeated cross-sectional analysis of a national pharmacy claims database between 2010 and 2019.

PURPOSE: Significant increases in opioid utilisation have been reported in many countries in recent decades. This study investigated strong opioid prescribing in Irish General Medical Services (GMS) patients over a 10-year period. METHODS: A retrospective repeated cross-sectional analysis of a national pharmacy claims database between January 2010 and December 2019 was conducted. Strong opioid prescribing in GMS patients was evaluated, including by route of administration, age (16-64 years and ≥65 years) and gender. Measures of consumption included prescribing prevalence and defined daily dose (DDD)/1000 population/day. Prevalence ratios (PRs) with 95% confidence intervals (CIs), and percentage and absolute changes were determined. RESULTS: Strong opioid prescribing prevalence increased from 14.43% in 2010 to 16.28% in 2019, with the greatest increase in the ≥65 years age group. Tramadol was the most frequently prescribed product, constituting 63.9% of total strong opioid prescribing. The prescribing prevalence of oxycodone increased from 0.95% in 2010 to 2.68% in 2019 (PR 2.81, 95% CI 2.76, 2.87), with steep increases in oxycodone-naloxone since it became available (PR 5.23, 95% CI 4.98, 5.50). The prescribing prevalence of tapentadol increased from 0.18% to 1.58% between 2012 (first complete year available for reimbursement) and 2019 (PR 8.79, 95% CI 8.43, 9.16). Strong opioid prescribing was highest in females aged ≥65 years. CONCLUSIONS: This study found an overall increase in strong opioid prescribing in Ireland between 2010 and 2019, particularly in older adults. Tramadol was the most frequently prescribed product, with oxycodone and tapentadol prescribing increasing markedly over the study period.

Multi-omics approaches for revealing the complexity of cardiovascular disease.

The development and progression of cardiovascular disease (CVD) can mainly be attributed to the narrowing of blood vessels caused by atherosclerosis and thrombosis, which induces organ damage that will result in end-organ dysfunction characterized by events such as myocardial infarction or stroke. It is also essential to consider other contributory factors to CVD, including cardiac remodelling caused by cardiomyopathies and co-morbidities with other diseases such as chronic kidney disease. Besides, there is a growing amount of evidence linking the gut microbiota to CVD through several metabolic pathways. Hence, it is of utmost importance to decipher the underlying molecular mechanisms associated with these disease states to elucidate the development and progression of CVD. A wide array of systems biology approaches incorporating multi-omics data have emerged as an invaluable tool in establishing alterations in specific cell types and identifying modifications in signalling events that promote disease development. Here, we review recent studies that apply multi-omics approaches to further understand the underlying causes of CVD and provide possible treatment strategies by identifying novel drug targets and biomarkers. We also discuss very recent advances in gut microbiota research with an emphasis on how diet and microbial composition can impact the development of CVD. Finally, we present various biological network analyses and other independent studies that have been employed for providing mechanistic explanation and developing treatment strategies for end-stage CVD, namely myocardial infarction and stroke.

Effect of an Online Reimbursement Application System on Prescribing of Lidocaine 5% Medicated Plaster in the Republic of Ireland.

BACKGROUND: The lidocaine 5% medicated plaster, Versatis®, has one therapeutic indication listed on the Summary of Product Characteristics-symptomatic relief of post-herpetic neuralgia (PHN) in adults. Increased expenditure on Versatis® suggests that there is considerable off-label use. To support the appropriate use of Versatis®, the Health Service Executive's Primary Care Reimbursement Service (PCRS) introduced a reimbursement application system for Versatis® from 1 September 2017. OBJECTIVE: The aim of this study was to investigate the effect of introducing a reimbursement application system on Versatis® prescribing under the General Medical Services (GMS) scheme. METHODS: This study was carried out using prescription dispensing data from the PCRS pharmacy claims database. We carried out segmented linear regression to assess changes in the Versatis® prescribing rate per 1000 GMS eligible population, before and after the introduction of the online reimbursement application system. RESULTS: The results of the segmented regression analysis show that there was a statistically significant level (- 4.91, p < 0.001) and trend change (- 0.69, p < 0.001) in the rate of Versatis® prescribing post-introduction of the reimbursement application system. In the year prior to the introduction of the system, 2016, the annual GMS expenditure on Versatis® lidocaine 5% patches was over €27 million, whereas the GMS expenditure in 2018 was reduced to just over €2 million. CONCLUSION: In our study, a substantial decrease in the dispensing of Versatis® was seen after the implementation of a reimbursement application system. Prescribing of Versatis® should be restricted to patients with a diagnosis of PHN not only to reduce costs, but to ensure evidence-based use of this medication.

Sacubitril/valsartan (Entresto) utilisation and prescribing patterns in the context of a reimbursement application system.

AIMS: Entresto (sacubitril/valsartan) is used to treat symptomatic chronic heart failure with reduced ejection fraction. Given its high potential budget impact, the Health Services Executive introduced a reimbursement application system (RAS) to ensure its appropriate use. The aim of this study was to evaluate the utilisation of Entresto in Ireland and compare patient characteristics to those of the pivotal PARADIGM-HF trial. METHODS: We used dispensed claims data from the Primary Care Reimbursement Services, clinical data obtained from the RAS, and data from published studies of Entresto utilisation. Differences in the baseline characteristics in the study populations vs the Entresto arm of the PARADIGM-HF trial were analysed. We also investigated cardiovascular medication use in the 6 months pre- and post-Entresto initiation. RESULTS: In 2018, there were 1043 individuals receiving Entresto, corresponding to an expenditure of €1.2 million. Patients prescribed Entresto in Ireland were older, had lower left ventricular ejection fraction and were more symptomatic than those in the PARADIGM-HF trial. Irish patient characteristics were reflective of Entresto-treated populations in other real-world studies. More than 63% of patients were commenced on the lowest Entresto dose. Entresto initiation was associated with a reduction in the use of other medications for heart failure. CONCLUSION: The utilisation of Entresto has been steadily increasing in Ireland since its reimbursement approval. The expenditure in the first year was substantially lower than predicted, and the RAS is an example of how health technology management can facilitate appropriate and cost-effective use of medicines.

Addressing the heterogeneity in liver diseases using biological networks.

The abnormalities in human metabolism have been implicated in the progression of several complex human diseases, including certain cancers. Hence, deciphering the underlying molecular mechanisms associated with metabolic reprogramming in a disease state can greatly assist in elucidating the disease aetiology. An invaluable tool for establishing connections between global metabolic reprogramming and disease development is the genome-scale metabolic model (GEM). Here, we review recent work on the reconstruction of cell/tissue-type and cancer-specific GEMs and their use in identifying metabolic changes occurring in response to liver disease development, stratification of the heterogeneous disease population and discovery of novel drug targets and biomarkers. We also discuss how GEMs can be integrated with other biological networks for generating more comprehensive cell/tissue models. In addition, we review the various biological network analyses that have been employed for the development of efficient treatment strategies. Finally, we present three case studies in which independent studies converged on conclusions underlying liver disease.

Vascular permeability disruption explored in the proteomes of mouse lungs and human microvascular cells following acute bromine exposure.

Bromine (Br2) is an organohalide found in nature and is integral to many manufacturing processes. Br2 is toxic to living organisms, and high concentrations can prove fatal. To meet industrial demand, large amounts of purified Br2 are produced, transported, and stored worldwide, providing a multitude of interfaces for potential human exposure through either accidents or terrorism. To identify the key mechanisms associated with acute Br2 exposure, we have surveyed the lung proteomes of C57BL/6 male mice and human lung-derived microvascular endothelial cells (HMECs) at 24 h following exposure to Br2 in concentrations likely to be encountered in the vicinity of industrial accidents. Global discovery proteomics applications combined with systems biology analysis identified robust and highly significant changes in proteins associated with three biological processes: 1) exosome secretion, 2) inflammation, and 3) vascular permeability. We focused on the latter, conducting physiological studies on isolated perfused lungs harvested from mice 24 h after Br2 exposure. These experiments revealed significant increases in the filtration coefficient (Kf) indicating increased permeability of the pulmonary vasculature. Similarly, confluent monolayers of Br2 and Br-lipid-treated HMECs exhibited differential levels of zona occludens-1 that were found to be dissociated from cell wall localization, an increase in phosphorylation and internalization of E-cadherin, as well as increased actin stress fiber formation, all of which are consistent with increased permeability. Taken as a whole, our discovery proteomics and systems analysis workflow, combined with physiological measurements of permeability, revealed both profound and novel biological changes that contribute to our current understanding of Br2 toxicity.

Vascular Endothelial Growth Factor-121 Administration Mitigates Halogen Inhalation-Induced Pulmonary Injury and Fetal Growth Restriction in Pregnant Mice.

Background Circulating levels of sFLT-1 (soluble fms-like tyrosine kinase 1), the extracellular domain of vascular endothelial growth factor (VEGF) receptor 1, and its ratio to levels of placental growth factor are markers of the occurrence and severity of preeclampsia. Methods and Results C57BL/6 pregnant mice on embryonic day 14.5 (E14.5), male, and non-pregnant female mice were exposed to air or to Br2 at 600 ppm for 30 minutes and were treated with vehicle or with VEGF-121 (100 µg/kg, subcutaneously) daily, starting 48 hours post-exposure. Plasma, bronchoalveolar lavage fluid, lungs, fetuses, and placentas were collected 120 hours post-exposure. In Br2-exposed pregnant mice, there was a time-dependent and significant increase in plasma levels of sFLT-1 which correlated with increases in mouse lung wet/dry weights and bronchoalveolar lavage fluid protein content. Supplementation of exogenous VEGF-121 improved survival and weight gain, reduced lung wet/dry weights, decreased bronchoalveolar lavage fluid protein levels, enhanced placental development, and improved fetal growth in pregnant mice exposed to Br2. Exogenous VEGF-121 administration had no effect in non-pregnant mice. Conclusions These results implicate inhibition of VEGF signaling driven by sFLT-1 overexpression as a mechanism of pregnancy-specific injury leading to lung edema, maternal mortality, and fetal growth restriction after bromine gas exposure.

Upregulation of airway smooth muscle calcium-sensing receptor by low-molecular-weight hyaluronan.

We investigated the mechanisms involved in the development of airway hyperresponsiveness (AHR) following exposure of mice to halogens. Male mice (C57BL/6; 20-25 g) exposed to either bromine (Br2) or Cl2 (600 or 400 ppm, respectively, for 30 min) developed AHR 24 h after exposure. Nifedipine (5 mg/kg body wt; an L-type calcium channel blocker), administered subcutaneously after Br2 or Cl2 exposure, produced higher AHR compared with Br2 or Cl2 alone. In contrast, diltiazem (5 mg/kg body wt; a nondihydropyridine L-type calcium channel blocker) decreased AHR to control (air) values. Exposure of immortalized human airway smooth muscle cells (hASMC) to Br2 resulted in membrane potential depolarization (Vm Air: 62 ± 3 mV; 3 h post Br2:-45 ± 5 mV; means ± 1 SE; P < 0.001), increased intracellular [Ca2+]i, and increased expression of the calcium-sensing receptor (Ca-SR) protein. Treatment of hASMC with a siRNA against Ca-SR significantly inhibited the Br2 and nifedipine-induced Vm depolarization and [Ca2+]i increase. Intranasal administration of an antagonist to Ca-SR in mice postexposure to Br2 reversed the effects of Br2 and nifedipine on AHR. Incubation of hASMC with low-molecular-weight hyaluronan (LMW-HA), generated by exposing high-molecular-weight hyaluronan (HMW-HA) to Br2, caused Vm depolarization, [Ca2+]i increase, and Ca-SR expression to a similar extent as exposure to Br2 and Cl2. The addition of HMW-HA to cells or mice exposed to Br2, Cl2, or LMW-HA reversed these effects in vitro and improved AHR in vivo. We conclude that detrimental effects of halogen exposure on AHR are mediated via activation of the Ca-SR by LMW-HA.

Phosgene inhalation causes hemolysis and acute lung injury.

Phosgene (Carbonyl Chloride, COCl2) remains an important chemical intermediate in many industrial processes such as combustion of chlorinated hydrocarbons and synthesis of solvents (degreasers, cleaners). It is a sweet smelling gas, and therefore does not prompt escape by the victim upon exposure. Supplemental oxygen and ventilation are the only available management strategies. This study was aimed to delineate the pathogenesis and identify novel biomarkers of acute lung injury post exposure to COCl2 gas. Adult male and female C57BL/6 mice (20-25 g), exposed to COCl2 gas (10 or 20 ppm) for 10 min in environmental chambers, had a dose dependent reduction in PaO2 and an increase in PaCO2, 1 day post exposure. However, mortality increased only in mice exposed to 20 ppm of COCl2 for 10 min. Correspondingly, these mice (20 ppm) also had severe acute lung injury as indicated by an increase in lung wet to dry weight ratio, extravasation of plasma proteins and neutrophils into the bronchoalveolar lavage fluid, and an increase in total lung resistance. The increase in acute lung injury parameters in COCl2 (20 ppm, 10 min) exposed mice correlated with simultaneous increase in oxidation of red blood cells (RBC) membrane, RBC fragility, and plasma levels of cell-free heme. In addition, these mice had decreased plasmalogen levels (plasmenylethanolamine) and elevated levels of their breakdown product, polyunsaturated lysophosphatidylethanolamine, in the circulation suggesting damage to cellular plasma membranes. This study highlights the importance of free heme in the pathogenesis of COCl2 lung injury and identifies plasma membrane breakdown product as potential biomarkers of COCl2 toxicity.

Organ Donation and Declaration of Death: Combined Neurologic and Cardiopulmonary Standards.

Prolonged survival after the declaration of death by neurologic criteria creates ambiguity regarding the validity of this methodology. This ambiguity has perpetuated the debate among secular and nondissenting Catholic authors who question whether the neurologic standards are sufficient for the declaration of death of organ donors. Cardiopulmonary criteria are being increasingly used for organ donors who do not meet brain death standards. However, cardiopulmonary criteria are plagued by conflict of interest issues, arbitrary standards for candidacy, and the lack of standardized protocols for organ procurement. Combining the neurological and cardiopulmonary standards into a single protocol would mitigate the weaknesses of both and provide greater biologic and moral certainty that a donor of unpaired vital organs is indeed dead. SUMMARY: Before a person's organs can be used for transplantation, he or she must be declared "brain-dead." However, sometimes when someone is declared brain-dead, that person can be maintained on life-support for days or even weeks. This creates some confusion about whether the person has truly died. For patients who have a severe neurologic injury but are not brain-dead, organ donation can also occur after his or her heart stops beating. However, this protocol is more ambiguous and lacks standardized protocols. We propose that before a person can donate organs, he or she must first be declared brain-dead, and then his or her heart must irreversibly stop beating before organs are taken.

Organ Donation and the Ars Moriendi.

Organ donation is rightly understood as a gift that is a genuine act of love. Organ donation as an act of love requires it to be an act of freedom that honors the integrity of the human person who is in the process of dying. However, the process of organ donation, by necessity, inserts a third party of interest whose primary aim is to assist someone other than the dying person. Caregivers can become "organ focused" instead of "patient focused." The procurement of organs potentially results in the commodification of the potential organ donor. Furthermore, death is not a momentary event but rather an ontological change in the person where the union of body and soul becomes divided. This Catholic understanding of death is important to assess the impact of organ donation on the process of dying. Family members of organ donors often have traumatic memories associated with the organ donation process, potentially overshadowing the ars moriendi-the art of dying. SUMMARY: While organ donation is an act of love, the donation process can be distraction from the care of the dying patient, who may be treated differently than other dying patients who are not organ donors. A Catholic understanding of death is helpful in assessing the impact of the organ donation process.

Bromine inhalation mimics ischemia-reperfusion cardiomyocyte injury and calpain activation in rats.

Halogens are widely used, highly toxic chemicals that pose a potential threat to humans because of their abundance. Halogens such as bromine (Br2) cause severe pulmonary and systemic injuries; however, the mechanisms of their toxicity are largely unknown. Here, we demonstrated that Br2 and reactive brominated species produced in the lung and released in blood reach the heart and cause acute cardiac ultrastructural damage and dysfunction in rats. Br2-induced cardiac damage was demonstrated by acute (3-24 h) increases in circulating troponin I, heart-type fatty acid-binding protein, and NH2-terminal pro-brain natriuretic peptide. Transmission electron microscopy demonstrated acute (3-24 h) cardiac contraction band necrosis, disruption of z-disks, and mitochondrial swelling and disorganization. Echocardiography and hemodynamic analysis revealed left ventricular (LV) systolic and diastolic dysfunction at 7 days. Plasma and LV tissue had increased levels of brominated fatty acids. 2-Bromohexadecanal (Br-HDA) injected into the LV cavity of a normal rat caused acute LV enlargement with extensive disruption of the sarcomeric architecture and mitochondrial damage. There was extensive infiltration of neutrophils and increased myeloperoxidase levels in the hearts of Br2- or Br2 reactant-exposed rats. Increased bromination of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) and increased phosphalamban after Br2 inhalation decreased cardiac SERCA activity by 70%. SERCA inactivation was accompanied by increased Ca2+-sensitive LV calpain activity. The calpain-specific inhibitor MDL28170 administered within 1 h after exposure significantly decreased calpain activity and acute mortality. Bromine inhalation and formation of reactive brominated species caused acute cardiac injury and myocardial damage that can lead to heart failure. NEW & NOTEWORTHY The present study defines left ventricular systolic and diastolic dysfunction due to cardiac injury after bromine (Br2) inhalation. A calpain-dependent mechanism was identified as a potential mediator of cardiac ultrastructure damage. This study not only highlights the importance of monitoring acute cardiac symptoms in victims of Br2 exposure but also defines calpains as a potential target to treat Br2-induced toxicity.

Mechanisms and Treatment of Halogen Inhalation-Induced Pulmonary and Systemic Injuries in Pregnant Mice.

Inhalation of oxidant gases has been implicated in adverse outcomes in pregnancy, but animal models to address mechanisms and studies to identify potential pregnancy-specific therapies are lacking. Herein, we show that inhalation of bromine at 600 parts per million for 30 minutes by pregnant mice on the 15th day of embryonic development results in significantly lower survival after 96 hours than an identical level of exposure in nonpregnant mice. On the 19th embryonic day, bromine-exposed pregnant mice have increased systemic blood pressure, abnormal placental development, severe fetal growth restriction, systemic inflammation, increased levels of circulating antiangiogenic short fms-like tyrosine kinase-1, and evidence of pulmonary and cardiac injury. Treatment with tadalafil, an inhibitor of type 5 phosphodiesterase, by oral gavage 1 hour post-exposure and then once daily thereafter, attenuated systemic blood pressures, decreased inflammation, ameliorated pulmonary and cardiac injury, and improved maternal survival (from 36% to 80%) and fetal growth. These pathological changes resemble those seen in preeclampsia. Nonpregnant mice did not exhibit any of these pathological changes and were not affected by tadalafil. These findings suggest that pregnant women exposed to bromine may require particular attention and monitoring for signs of preeclampsia-like symptoms.

Nitrite therapy prevents chlorine gas toxicity in rabbits.

Chlorine (Cl2) gas exposure and toxicity remains a concern in military and industrial sectors. While post-Cl2 exposure damage to the lungs and other tissues has been documented and major underlying mechanisms elucidated, no targeted therapeutics that are effective when administered post-exposure, and which are amenable to mass-casualty scenarios have been developed. Our recent studies show nitrite administered by intramuscular (IM) injection post-Cl2 exposure is effective in preventing acute lung injury and improving survival in rodent models. Our goal in this study was to develop a rabbit model of Cl2 toxicity and test whether nitrite affords protection in a non-rodent model. Exposure of New Zealand White rabbits to Cl2 gas (600ppm, 45min) caused significant increases in protein and neutrophil accumulation in the airways and ∼35% mortality over 18h. Nitrite administered 30min post Cl2 exposure by a single IM injection, at 1mg/kg or 10mg/kg, prevented indices of acute lung injury at 6h by up to 50%. Moreover, all rabbits that received nitrite survived over the study period. These data provide further rationale for developing nitrite as post-exposure therapeutic to mitigate against Cl2 gas exposure injury.

Formation of chlorinated lipids post-chlorine gas exposure.

Exposure to chlorine (Cl2) gas can occur during accidents and intentional release scenarios. However, biomarkers that specifically indicate Cl2 exposure and Cl2-derived products that mediate postexposure toxicity remain unclear. We hypothesized that chlorinated lipids (Cl-lipids) formed by direct reactions between Cl2 gas and plasmalogens serve as both biomarkers and mediators of post-Cl2 gas exposure toxicities. The 2-chloropalmitaldehyde (2-Cl-Pald), 2-chlorostearaldehyde (2-Cl-Sald), and their oxidized products, free- and esterified 2-chloropalmitic acid (2-Cl-PA) and 2-chlorostearic acid were detected in the lungs and plasma of mouse and rat models of Cl2 gas exposure. Levels of Cl-lipids were highest immediately post-Cl2 gas exposure, and then declined over 72 h with levels remaining 20- to 30-fold higher at 24 h compared with baseline. Glutathione adducts of 2-Cl-Pald and 2-Cl-Sald also increased with levels peaking at 4 h in plasma. Notably, 3-chlorotyrosine also increased after Cl2 gas exposure, but returned to baseline within 24 h. Intranasal administration of 2-Cl-PA or 2-Cl-Pald at doses similar to those formed in the lung after Cl2 gas exposure led to increased distal lung permeability and inflammation and systemic endothelial dysfunction characterized by loss of eNOS-dependent vasodilation. These data suggest that Cl-lipids could serve as biomarkers and mediators for Cl2 gas exposure and toxicity.

Chlorine inhalation-induced myocardial depression and failure.

Victims of chlorine (Cl2) inhalation that die demonstrate significant cardiac pathology. However, a gap exists in the understanding of Cl2-induced cardiac dysfunction. This study was performed to characterize cardiac dysfunction occurring after Cl2 exposure in rats at concentrations mimicking accidental human exposures (in the range of 500 or 600 ppm for 30 min). Inhalation of 500 ppm Cl2 for 30 min resulted in increased lactate in the coronary sinus of the rats suggesting an increase in anaerobic metabolism by the heart. There was also an attenuation of myocardial contractile force in an ex vivo (Langendorff technique) retrograde perfused heart preparation. After 20 h of return to room air, Cl2 exposure at 500 ppm was associated with a reduction in systolic and diastolic blood pressure as well echocardiographic/Doppler evidence of significant left ventricular systolic and diastolic dysfunction. Cl2 exposure at 600 ppm (30 min) was associated with biventricular failure (observed at 2 h after exposure) and death. Cardiac mechanical dysfunction persisted despite increasing the inspired oxygen fraction concentration in Cl2-exposed rats (500 ppm) to ameliorate hypoxia that occurs after Cl2 inhalation. Similarly ex vivo cardiac mechanical dysfunction was reproduced by sole exposure to chloramine (a potential circulating Cl2 reactant product). These results suggest an independent and distinctive role of Cl2 (and its reactants) in inducing cardiac toxicity and potentially contributing to mortality.