Rationale Increased activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) is thought to promote heart failure progression. However, the importance of CaMKII phosphorylation of ryanodine receptors (RyR2) in heart failure (HF) development and associated diastolic sarcoplasmic reticulum (SR) Ca2+ leak is unclear. Objective Determine the role of CaMKII phosphorylation of RyR2 in patients and mice with non-ischemic and ischemic forms of HF. Methods and Results Phosphorylation of the primary CaMKII site S2814 on RyR2 was increased in patients with non-ischemic but not with ischemic HF. Knock-in mice with an inactivated S2814 phosphorylation site were relatively protected from HF development following transverse aortic constriction (TAC) compared to wildtype (WT) littermates. After TAC, S2814A mice did not exhibit pulmonary congestion and had reduced levels of atrial natriuretic factor (ANF). Cardiomyocytes from S2814A mice exhibited significantly lower SR Ca2+ leak and improved SR Ca2+ loading compared to WT mice after TAC. Interestingly, these protective effects on cardiac contractility were not observed in S2814A mice following experimental myocardial infarction. Conclusions Our results suggest that increased CaMKII phosphorylation of RyR2 plays a role in the development of pathological SR Ca2+ leak and heart failure development in non-ischemic forms of HF such as transverse aortic constriction in mice.

Following the deadly anthrax attacks of 2001, the Centers for Disease Control and Prevention (CDC) determined that Bacillus anthracis and Yersinia pestis that cause anthrax and plague, respectively, are two Tier 1 select agents that pose the greatest threat to the national security of the United States. Both cause rapid death, in 3 to 6 days, of exposed individuals. We engineered a virus nanoparticle vaccine using bacteriophage T4 by incorporating key antigens of both B. anthracis and Y. pestis into one formulation. Two doses of this vaccine provided complete protection against both inhalational anthrax and pneumonic plague in animal models. This dual anthrax-plague vaccine is a strong candidate for stockpiling against a potential bioterror attack involving either one or both of these biothreat agents. Further, our results establish the T4 nanoparticle as a novel platform to develop multivalent vaccines against pathogens of high public health significance.

j Currently, no plague vaccine exists in the United States for human use. The capsular antigen (Caf1 or F1) and two type 3 secretion system (T3SS) components, the low-calcium-response V antigen (LcrV) and the needle protein YscF, represent protective antigens of Yersinia pestis. We used a replication-defective human type 5 adenovirus (Ad5) vector and constructed recombinant monovalent and trivalent vaccines (rAd5-LcrV and rAd5-YFV) that expressed either the codon-optimized lcrV or the fusion gene designated YFV (consisting of ycsF, caf1, and lcrV). Immunization of mice with the trivalent rAd5-YFV vaccine by either the intramuscular (i.m.) or the intranasal (i.n.) route provided protection superior to that with the monovalent rAd5-LcrV vaccine against bubonic and pneumonic plague when animals were challenged with Y. pestis CO92. Preexisting adenoviral immunity did not diminish the protective response, and the protection was always higher when mice were administered one i.n. dose of the trivalent vaccine (priming) followed by a single i.m. booster dose of the purified YFV antigen. Immunization of cynomolgus macaques with the trivalent rAd5-YFV vaccine by the prime-boost strategy provided 100% protection against a stringent aerosol challenge dose of CO92 to animals that had preexisting adenoviral immunity. The vaccinated and challenged macaques had no signs of disease, and the invading pathogen rapidly cleared with no histopathological lesions. This is the first report showing the efficacy of an adenovirus-vectored trivalent vaccine against pneumonic plague in mouse and nonhuman primate (NHP) models. Yersinia pestis is the causative agent of plague and can be transmitted to humans via an infected flea bite or by direct inhalation of the aerosolized bacilli from an infected person or an animal (1, 2). Plague manifests itself in three major forms in humans, namely, bubonic, septicemic, and pneumonic (2). Pneumonic plague is the most feared form due to its rapid onset and associated high mortality rate (1, 2). Y. pestis has been responsible for at least three pandemics in the past, which killed more than 200 million people (3). Current epidemiological records suggest that there are 4,000 human plague cases annually worldwide (2). The emergence of multi-antibiotic-resistant Y. pestis strains from plague patients and the potential of malicious dissemination of recombinantly engineered bacteria as an airborne bioweapon necessitate the development of an effective preexposure and/or postexposure prophylaxis treatment (1, 2).Currently, no Food and Drug Administration (FDA)-licensed plague vaccine exists in the United States, and recent efforts have focused on the development of recombinant subunit plague vaccines consisting of two well-characterized Y. pestis antigens, the F1 capsular antigen and the type 3 secretion system (T3SS) component and effector LcrV (4-8). F1, encoded by the caf1 gene, has a polymeric structure and confers bacterial resistance to phagocytosis (9). The F1-V-based vaccines are generally protective ag...

Successful treatment of inhalation anthrax, pneumonic plague and tularemia can be achieved with fluoroquinolone antibiotics, such as ciprofloxacin and levofloxacin, and initiation of treatment is most effective when administered as soon as possible following exposure. Bacillus anthracis Ames, Yersinia pestis CO92, and Francisella tularensis SCHU S4 have equivalent susceptibility in vitro to ciprofloxacin and levofloxacin (minimal inhibitory concentration is 0.03 μg/ml); however, limited information is available regarding in vivo susceptibility of these infectious agents to the fluoroquinolone antibiotics in small animal models. Mice, guinea pig, and rabbit models have been developed to evaluate the protective efficacy of antibiotic therapy against these life-threatening infections. Our results indicated that doses of ciprofloxacin and levofloxacin required to protect mice against inhalation anthrax were approximately 18-fold higher than the doses of levofloxacin required to protect against pneumonic plague and tularemia. Further, the critical period following aerosol exposure of mice to either B. anthracis spores or Y. pestis was 24 h, while mice challenged with F. tularensis could be effectively protected when treatment was delayed for as long as 72 h postchallenge. In addition, it was apparent that prolonged antibiotic treatment was important in the effective treatment of inhalation anthrax in mice, but short-term treatment of mice with pneumonic plague or tularemia infections were usually successful. These results provide effective antibiotic dosages in mice, guinea pigs, and rabbits and lay the foundation for the development and evaluation of combinational treatment modalities.

TWIK-2 (KCNK6) is a member of the two-pore domain (K2P) family of potassium channels which are highly expressed in the vascular system. We tested the hypothesis that TWIK-2 deficiency leads to pulmonary hypertension. TWIK-2 knockout mice and their wildtype littermates at 8 weeks of age had similar mean right ventricular systolic pressures (mRVSP: 24 ± 3 mm Hg& 21 ± 3 mm Hg, respectively.) Significantly, by 20 weeks of age, the mRVSP in TWIK-2 knockout mice increased to 35 ± 3 mm Hg (p ≤ 0.036) while mRVSP in wildtype littermates remained at 22 ± 3 mm Hg. Elevated mRVSP in the TWIK-2 knockout mice was accompanied pulmonary vascular remodeling as determined by a 25% increase in the cross-sectional area of the vessels occupied by the vessel wall. Additionally, secondary branches of the pulmonary artery from 20 week old TWIK-2 knockout mice showed an enhanced contractile response to U46619 (10−6 moles/liter), a thromboxane A2 mimetic, which was completely abolished with the Rho-kinase inhibitor, Y27632 (10−6 and 10−5moles/liter). Treatment of TWIK-2 knockout mice with the Rho-kinase inhibitor, fasudil, in the drinking water for 12 weeks, abolished the development of pulmonary hypertension and attenuated the vessel remodeling.. We conclude that mice deficient the TWIK-2 channel develop pulmonary hypertension between 8 and 20 weeks of age through a mechanism involving Rho-kinase. Our results suggest that down-regulation of TWIK-2 in the pulmonary vasculature may be an underlying mechanism in the development of pulmonary hypertension.

Electromagnetic fields at millimeter wave lengths are being developed for commercial and military use at power levels that can cause temperature increases in the skin. Previous work suggests that sustained exposure to millimeter waves causes greater heating of skin, leading to faster induction of circulatory failure than exposure to environmental heat (EH). We tested this hypothesis in three separate experiments by comparing temperature changes in skin, subcutis, and colon, and the time to reach circulatory collapse (mean arterial blood pressure, 20 mmHg) in male Sprague-Dawley rats exposed to the following conditions that produced similar rates of body core heating within each experiment: (1) EH at 42 degrees C, 35 GHz at 75 mW/cm, or 94 GHz at 75 mW/cm under ketamine and xylazine anesthesia; (2) EH at 43 degrees C, 35 GHz at 90 mW/cm, or 94 GHz at 90 mW/cm under ketamine and xylazine anesthesia; and (3) EH at 42 degrees C, 35 GHz at 90 mW/cm, or 94 GHz at 75 mW/cm under isoflurane anesthesia. In all three experiments, the rate and amount of temperature increase at the subcutis and skin surface differed significantly in the rank order of 94 GHz more than 35 GHz more than EH. The time to reach circulatory collapse was significantly less only for rats exposed to 94 GHz at 90 mW/cm, the group with the greatest rate of skin and subcutis heating of all groups in this study, compared with both the 35 GHz at 90 mW/cm and the EH at 43 degrees C groups. These data indicate that body core heating is the major determinant of induction of hemodynamic collapse, and the influence of heating of the skin and subcutis becomes significant only when a certain threshold rate of heating of these tissues is exceeded.

Background Chikungunya virus (CHIKV) infection can result in chikungunya fever (CHIKF), a self-limited acute febrile illness that can progress to chronic arthralgic sequelae in a large percentage of patients. A new measles virus-vectored vaccine was developed to prevent CHIKF, and we tested it for immunogenicity and efficacy in a nonhuman primate model. Methods Nine cynomolgus macaques were immunized and boosted with the measles virus-vectored chikungunya vaccine or sham-vaccinated. Sera were taken at multiple times during the vaccination phase to assess antibody responses against CHIKV. Macaques were challenged with a dose of CHIKV previously shown to cause fever and viremia, and core body temperature, viremia, and blood cell and chemistry panels were monitored. Results The vaccine was well tolerated in all macaques, and all seroconverted (high neutralizing antibody [PRNT80 titers, 40–640] and enzyme-linked immunosorbent assay titers) after the boost. Furthermore, the vaccinated primates were protected against viremia, fever, elevated white blood cell counts, and CHIKF-associated cytokine changes after challenge with the virulent La Reunión CHIKV strain. Conclusions These results further document the immunogenicity and efficacy of a measles-vectored chikungunya vaccine that shows promise in Phase I–II clinical trials. These findings are critical to human health because no vaccine to combat CHIKF is yet licensed.

SummaryObjectiveSympathetic predominance and ventricular repolarization abnormalities represent epilepsy‐associated cardiac alterations and may underlie seizure‐induced ventricular arrhythmias. Myocardial ion channel and electrical remodeling have been described early in epilepsy development and may contribute to ventricular repolarization abnormalities and excitability. Using the pilocarpine‐induced acquired epilepsy model we sought to examine whether altered myocardial ion channel levels and electrophysiological changes also occur in animals with long‐standing epilepsy.MethodsWe examined myocardial adrenergic receptor and ion channel protein levels of epileptic and age‐matched sham rats (9–20 months old) using western blotting. Cardiac electrical properties were examined using optical mapping ex vivo and electrophysiology in vivo. We investigated the propensity for ventricular tachycardia (VT) and the effects of β‐adrenergic blockade on ventricular electrical properties and excitability in vivo.ResultsIn animals with long‐standing epilepsy, we observed decreased myocardial voltage‐gated K+ channels Kv4.2 and Kv4.3, which are known to underlie early ventricular repolarization in rodents. Decreased β1 and increased α1A adrenergic receptor protein levels occurred in the myocardium of chronically epileptic animals consistent with elevated sympathetic tone. These animals exhibited many cardiac electrophysiological abnormalities, represented by longer QRS and corrected QT (QTc) intervals in vivo, slower conduction velocity ex vivo, and stimulation‐induced VT. Administration of a β‐adrenergic antagonist late in epilepsy was beneficial, as the therapy shortened the QTc interval and decreased stimulation‐induced VT.SignificanceOur findings demonstrate that myocardial ion channel remodeling and sympathetic predominance, risk factors for increased ventricular excitability and arrhythmias, persist in chronic epilepsy. The beneficial effects of β‐adrenergic antagonist treatment late in the course of epilepsy suggest that attenuating elevated sympathetic tone may represent a therapeutic target for ameliorating epilepsy‐associated cardiac morbidity.