arrhythmia (mechanisms); calcium; ion channels or ion channel; structural heart disease; CaMKII
Understanding relationships between heart failure and arrhythmias, important causes of suffering and sudden death, remains an unmet goal for biomedical researchers and physicians. Evidence assembled over the last decade supports a view that activation of the multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) favors myocardial dysfunction and cell membrane electrical instability. CaMKII activation follows increases in intracellular Ca2+ or oxidation, upstream signals with the capacity to transition CaMKII into a Ca2+ and calmodulin-independeant, constitutively active enzyme. Constitutively active CaMKII appears poised to participate in disease pathways by catalyzing the phosphorylation of classes of protein targets important for excitation-contraction coupling and cell survival, including ion channels and Ca2+ homeostatic proteins, and transcription factors that drive hypertrophic and inflammatory gene expression. This rich diversity of downstream targets helps to explain the potential for CaMKII to simultaneously affect mechanical and electrical properties of heart muscle cells. Proof of concept studies from a growing number of investigators show that CaMKII inhibition is beneficial for improving myocardial performance and reducing arrhythmias. Here we review the molecular physiology of CaMKII, discuss CaMKII actions at key cellular targets and results of animal models of myocardial hypertrophy, dysfunction and arrhythmias that suggest CaMKII inhibition may benefit myocardial function while reducing arrhythmias.
CaMKII; Arrhythmias; Heart Failure; Ion channels; Remodeling
Neisseria gonorrhoeae is the causative agent of gonorrhea and an obligate pathogen of humans. The Opa proteins of these bacteria are known to mediate attachment and internalization by host cells, including neutrophils. The Opa protein repertoire of a typical N. gonorrhoeae isolate is encoded on ∼11 genes distributed throughout the chromosome and is subject to stochastic changes in expression through phase variation. Together, these characteristics make Opa proteins a critical yet unpredictable aspect of any experimental investigation into the interaction of N. gonorrhoeae with host cells. The goal of this study was to identify novel virulence factors of N. gonorrhoeae by assessing the contribution of a set of uncharacterized hydrogen peroxide-induced genes to bacterial survival against neutrophil-mediated killing. To this end, a strain harboring an engineered mutation in the NGO0322 gene was identified that exhibited increased sensitivity to neutrophil-mediated killing, enhanced internalization by neutrophils, and the ability to induce high levels of neutrophil-generated reactive oxygen species. Each of these phenotypes reverted to near wild-type levels following genetic complementation of the NGO0322 mutation. However, after immunoblot analysis of Opa proteins expressed by the isogenic parent, mutant, and genetically complemented strains, it was determined that phase variation had resulted in a disparity between the Opa profiles of these strains. To determine whether Opa phase variation, rather than NGO0322 mutation, was the cause of the observed neutrophil-related phenotypes, NGO0322 function was investigated in N. gonorrhoeae strains lacking all Opa proteins or constitutively expressing the OpaD variant. In both cases, mutation of NGO0322 did not alter survival of gonococci in the presence of neutrophils. These results demonstrate the importance of controlling for the frequent and random variation in Opa protein production by N. gonorrhoeae when investigating host cell interactions.
Sustained hemodynamic stress mediated by high blood flow promotes arteriogenesis, the outward remodeling of existing arteries. Here, we examined whether Ca2+/calmodulin-dependent kinase II (CaMKII) regulates arteriogenesis.
Methods and Results
Ligation of the left common carotid led to an increase in vessel diameter and perimeter of internal and external elastic lamina in the contralateral, right common carotid. Deletion of CaMKIIδ (CaMKIIδ−/−) abolished this outward remodeling. Carotid ligation increased CaMKII expression and was associated with oxidative activation of CaMKII in the adventitia and endothelium. Remodeling was abrogated in a knock-in model in which oxidative activation of CaMKII is abolished. Early after ligation, matrix metalloproteinase 9 (MMP9) was robustly expressed in the adventitia of right carotid arteries of WT but not CaMKIIδ−/− mice. MMP9 mainly colocalized with adventitial macrophages. In contrast, we did not observe an effect of CaMKIIδ deficiency on other proposed mediators of arteriogenesis such as expression of adhesion molecules or smooth muscle proliferation. Transplantation of WT bone marrow into CaMKIIδ−/− mice normalized flow-mediated remodeling.
CaMKIIδ is activated by oxidation under high blood flow conditions and is required for flow-mediated remodeling through a mechanism that includes increased MMP9 expression in bone marrow-derived cells invading the arterial wall.
The multifunctional Ca2+ and calmodulin-dependent protein kinase II (CaMKII) is now recognized to play a central role in pathological events in the cardiovascular system. CaMKII has diverse downstream targets that promote vascular disease, heart failure and arrhythmias, so improved understanding of CaMKII signaling has the potential to lead to new therapies for cardiovascular disease. CaMKII is a multimeric serine-threonine kinase that is initially activated by binding calcified calmodulin (Ca2+/CaM). Under conditions of sustained exposure to elevated Ca2+/CaM CaMKII transitions into a Ca2+/CaM-autonomous enzyme by two distinct but parallel processes. Autophosphorylation of threonine 287 in the CaMKII regulatory domain ‘traps’ CaMKII into an open configuration even after Ca2+/CaM unbinding. More recently, our group identified a pair of methionines (281/282) in the CaMKII regulatory domain that undergo a partially reversible oxidation which, like autophosphorylation, prevents CaMKII from inactivating after Ca2+/CaM unbinding. Here we review roles of CaMKII in cardiovascular disease with an eye to understanding how CaMKII may act as a transduction signal to connect pro-oxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of cardiovascular disease.
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is directly linked to mutations in proteins (e.g., RyR2R4496C) responsible for intracellular Ca2+ homeostasis in the heart. However, the mechanism of Ca2+ release dysfunction underlying CPVT has only been investigated in isolated cells but not in the in situ undisrupted myocardium.
Methods and Results
We investigated in situ myocyte Ca2+ dynamics in intact Langendorff perfused hearts (ex vivo) from wildtype (WT) and RyR2R4496C+/− mice using laser scanning confocal microscopy. We found that myocytes from both WT and RyR2R4496C+/− hearts displayed uniform, synchronized Ca2+ transients. Ca2+ transients from beat to beat were comparable in amplitude with identical activation and decay kinetics in WT and RyR2R4496C+/− hearts, suggesting that excitation-contraction (EC) coupling between the sarcolemmal Ca2+ channels and mutated RyR2R4496C+/− channels remains intact under baseline resting conditions. Upon adrenergic stimulation, RyR2R4496C+/− hearts exhibited a high degree of Ca2+ release variability (CRV). The varied pattern of Ca2+ release was absent in single isolated myocytes, independent of cell cycle length, synchronized among neighboring myocytes, and correlated with CPVT. A similar pattern of action potential variability, which was synchronized among neighboring myocytes, was also revealed under adrenergic stress in intact hearts but not in isolated myocytes.
Our studies using in situ confocal imaging approach suggest that mutated RyR2s are functionally normal at rest but display a high degree of CRV upon intense adrenergic stimulation. CRV is a Ca2+ release abnormality resulting from electrical defects rather than the failure of the Ca2+ release response to action potentials in mutated ventricular myocytes. Our data provide important insights into Ca2+ release and electrical dysfunction in an established model of CPVT.
arrhythmia (mechanisms); calcium; catecholaminergic polymorphic ventricular tachycardia; sarcoplasmic reticulum; ryanodine receptors
Bacterial pathogens have specific virulence factors (e.g., toxins) that contribute significantly to the virulence and infectivity of microorganisms within the human hosts. Virulence factors are molecules expressed by pathogens that enable colonization, immunoevasion, and immunosuppression, obtaining nutrients from the host or gaining entry into host cells. They can cause pathogenesis by inhibiting or stimulating certain host functions. For example, in systemic Staphylococcus aureus infections, virulence factors such as toxic shock syndrome toxin 1 (TSST-1), staphylococcal enterotoxin A (SEA), and staphylococcal enterotoxin B (SEB) cause sepsis or toxic shock by uncontrolled stimulation of T lymphocytes and by triggering a cytokine storm. In vitro, these superantigens stimulate the proliferation of human peripheral blood mononuclear cells (PBMC) and the release of many cytokines. NVC-422 (N,N-dichloro-2,2-dimethyltaurine) is a broad-spectrum, fast-acting topical anti-infective agent against microbial pathogens, including antibiotic-resistant microbes. Using mass spectrometry, we demonstrate here that NVC-422 oxidizes methionine residues of TSST-1, SEA, SEB, and exfoliative toxin A (ETA). Exposure of virulence factors to 0.1% NVC-422 for 1 h prevented TSST-1-, SEA-, SEB-, and ETA-induced cell proliferation and cytokine release. Moreover, NVC-422 also delayed and reduced the protein A- and clumping factor-associated agglutination of S. aureus cultures. These results show that, in addition to its well-described direct microbicidal activity, NVC-422 can inactivate S. aureus virulence factors through rapid oxidation of methionines.
Large-offset oceanic detachment faults are a characteristic of slow- and ultraslow-spreading ridges, leading to the formation of oceanic core complexes (OCCs) that expose upper mantle and lower crustal rocks on the seafloor. The lithospheric extension accommodated by these structures is now recognized as a fundamentally distinct “detachment-mode” of seafloor spreading compared to classical magmatic accretion. Here we demonstrate a paleomagnetic methodology that allows unequivocal recognition of detachment-mode seafloor spreading in ancient ophiolites and apply this to a potential Jurassic detachment fault system in the Mirdita ophiolite (Albania). We show that footwall and hanging wall blocks either side of an inferred detachment have significantly different magnetizations that can only be explained by relative rotation during seafloor spreading. The style of rotation is shown to be identical to rolling hinge footwall rotation documented recently in OCCs in the Atlantic, confirming that detachment-mode spreading operated at least as far back as the Jurassic.
Myocardial infarction is a major cause of morbidity and mortality in the developing and developed world. Although current interventions have been successful in prolonging life, they are inadequate because mortality is still high among MI patients. The multifunctional Ca2+/calmodulin dependent protein kinase (CaMKII) plays a key role in the structure and contractility of the myocardium. CaMKII activity is increased in MI hearts and CaMKII promotes cardiac hypertrophy and inflammation, processes consistently activated by myocardial injury. Hypertrophy and inflammation are also related to neurohumoral and redox signaling that uncouple CaMKII activation from Ca2+/calmodulin dependence. Thus, CaMKII may act as a nodal point for integrating hypertrophic and inflammatory signaling in myocardium.
CaMKII; Heart; Myocardium; Myocardial infarction; Heart attack; Inflammation; Hypertrophy; Toll-like receptors; Oxidative stress; ROS; AngII
The current clinical practice has established guidelines to assess influences of severity of autonomic injury on the control of heart and blood pressure following spinal cord injury (SCI). However, the influences of SCI-induced autonomic impairment on microvascular dysfunction have not yet been established. Heart rate variability (HRV) has been shown to be a potential tool for quantifying residual sympathovagal regulation of the cardiovascular system following SCI, and may be used to assess the effect of autonomic injury on skin microvascular dysfunction. A total of 26 people were recruited into the study, including 12 people with SCI and 14 healthy controls. R-R intervals and sacral skin perfusion were continually recorded during 10-min upright and 10-min prone postures. The sympathovagal balance was defined as the ratio of the power of the low frequency to the high frequency of HRV. The results showed that postural changes of healthy people produced significant changes in the sympathovagal balance; lower sympathovagal balance was associated with higher skin perfusion (p<0.05). People with SCI did not show a significant change of HRV and skin perfusion in response to postural changes. In this study, we have demonstrated that the sympathovagal balance assessed by HRV was associated with the skin vasoconstrictive response to postural changes.
autonomic nervous system; blood flow oscillations; laser Doppler; microvascular function; pressure ulcer; skin blood flow; spectral analysis
Cancer of the testes is currently the most frequent neoplasm and a leading cause of morbidity in men 15–35 years of age. Its incidence is increasing. Embryonal carcinoma is its most malignant form, which either may be resistant or may develop resistance to therapies, which results in relapses. Cancer stem cells are hypothesized to be drivers of these phenomena.
The specific aim of this work was identification and isolation of spectra of single, living cancer stem cells, which were acquired directly from the patients’ biopsies, followed by testing of their pluripotency.
Biopsies were obtained from the patients with the clinical and histological diagnoses of the primary, pure embryonal carcinomas of the testes. The magnetic and fluorescent antibodies were genetically engineered. The SSEA-4 and TRA-1–60 cell surface display was analyzed by multiphoton fluorescence spectroscopy (MPFS), flow cytometry (FCM), immunoblotting (IB), nuclear magnetic resonance spectroscopy (NMRS), energy dispersive x-ray spectroscopy (EDXS), and total reflection x-ray spectroscopy (TRXFS). The single, living cells were isolated by magnetic or fluorescent sorting followed by their clonal expansion. The OCT4A, SOX2, and NANOG genes’ transcripts were analyzed by qRTPCR and the products by IB and MPFS.
The clones of cells, with the strong surface display of TRA-1–60 and SSEA-4, were identified and isolated directly from the biopsies acquired from the patients diagnosed with the pure embryonal carcinomas of the testes. These cells demonstrated high levels of transcription and translation of the pluripotency genes: OCT4A, SOX2, and NANOG. They formed embryoid bodies, which differentiated into ectoderm, mesoderm, and endoderm.
In the pure embryonal carcinomas of the testes, acquired directly from the patients, we identified, isolated with high viability and selectivity, and profiled the clones of the pluripotent stem cells. These results may help in explaining therapy-resistance and relapses of these neoplasms, as well as, in designing targeted, personalized therapy.
Cancer of the testis; testicular carcinoma; germ cell tumor; embryonal carcinoma; pluripotency; stem cell; Fv; TRA-1–60; SSEA-4; OCT4A; SOX2; NANOG
Chronic Inflammatory Demyelinating Polyneuropathy is a debilitating autoimmune disease characterized by peripheral nerve demyelination and dysfunction. How the autoimmune response is initiated, identity of provoking antigens, and pathogenic effector mechanisms are not well-defined. The Autoimmune Regulator (Aire) plays a critical role in central tolerance by promoting thymic expression of self-antigens and deletion of self-reactive T cells. Here, we utilized mice with hypomorphicAire function and two patients with Aire mutations to define how Aire deficiency results in spontaneous autoimmune peripheral neuropathy. Autoimmunity against peripheral nerves in both mice and humans targets Myelin Protein Zero (P0), an antigen whose expression is Aire-regulated in the thymus. Consistent with a defect in thymic tolerance, CD4+ T cells are sufficient to transfer disease in mice and produce IFN-gamma in infiltrated peripheral nerves. Our findings suggest that defective Aire-mediated central tolerance to P0 initiates an autoimmune Th1 effector response toward peripheral nerves.
Electrical and structural remodeling during the progression of cardiovascular disease is associated with adverse outcomes subjecting affected patients to overt heart failure (HF) and/or sudden death. Dysfunction in integral membrane protein trafficking has long been linked with maladaptive electrical remodeling. However, little is known regarding the molecular identity or function of these intracellular targeting pathways in the heart. Eps15 homology domain-containing (EHD) gene products (EHD1-4) are polypeptides linked with endosomal trafficking, membrane protein recycling, and lipid homeostasis in a wide variety of cell types. EHD3 was recently established as a critical mediator of membrane protein trafficking in the heart. Here, we investigate the potential link between EHD3 function and heart disease. Using four different HF models including ischemic rat heart, pressure overloaded mouse heart, chronic pacing-induced canine heart, and non-ischemic failing human myocardium we provide the first evidence that EHD3 levels are consistently increased in HF. Notably, the expression of the Na/Ca exchanger (NCX1), targeted by EHD3 in heart is similarly elevated in HF. Finally, we identify a molecular pathway for EHD3 regulation in heart failure downstream of reactive oxygen species and angiotensin II signaling. Together, our new data identify EHD3 as a previously unrecognized component of the cardiac remodeling pathway.
EHD proteins; heart failure; cardiac remodeling; regulation; animal models
Significance: In heart failure (HF), contractile dysfunction and arrhythmias result from disturbed intracellular Ca handling. Activated stress kinases like cAMP-dependent protein kinase A (PKA), protein kinase C (PKC), and Ca/calmodulin-dependent protein kinase II (CaMKII), which are known to influence many Ca-regulatory proteins, are mechanistically involved. Recent Advances: Beside classical activation pathways, it is becoming increasingly evident that reactive oxygen species (ROS) can directly oxidize these kinases, leading to alternative activation. Since HF is associated with increased ROS generation, ROS-activated serine/threonine kinases may play a crucial role in the disturbance of cellular Ca homeostasis. Many of the previously described ROS effects on ion channels and transporters are possibly mediated by these stress kinases. For instance, ROS have been shown to oxidize and activate CaMKII, thereby increasing Na influx through voltage-gated Na channels, which can lead to intracellular Na accumulation and action potential prolongation. Consequently, Ca entry via activated NCX is favored, which together with ROS-induced dysfunction of the sarcoplasmic reticulum can lead to dramatic intracellular Ca accumulation, diminished contractility, and arrhythmias. Critical Issues: While low amounts of ROS may regulate kinase activity, excessive uncontrolled ROS production may lead to direct redox modification of Ca handling proteins. Therefore, depending on the source and amount of ROS generated, ROS could have very different effects on Ca-handling proteins. Future Directions: The discrimination between fine-tuned ROS signaling and unspecific ROS damage may be crucial for the understanding of heart failure development and important for the investigation of targeted treatment strategies. Antioxid. Redox Signal. 18, 1063–1077.
Diabetes increases oxidant stress and doubles the risk of dying after myocardial
infarction, but the mechanisms underlying increased mortality are unknown. Mice with
streptozotocin-induced diabetes developed profound heart rate slowing and doubled
mortality compared with controls after myocardial infarction. Oxidized
Ca2+/calmodulin-dependent protein kinase II (ox-CaMKII) was
significantly increased in pacemaker tissues from diabetic patients compared with
that in nondiabetic patients after myocardial infarction. Streptozotocin-treated mice
had increased pacemaker cell ox-CaMKII and apoptosis, which were further enhanced by
myocardial infarction. We developed a knockin mouse model of oxidation-resistant
CaMKIIδ (MM-VV), the isoform associated with cardiovascular disease.
Streptozotocin-treated MM-VV mice and WT mice infused with MitoTEMPO, a mitochondrial
targeted antioxidant, expressed significantly less ox-CaMKII, exhibited increased
pacemaker cell survival, maintained normal heart rates, and were resistant to
diabetes-attributable mortality after myocardial infarction. Our findings suggest
that activation of a mitochondrial/ox-CaMKII pathway contributes to increased sudden
death in diabetic patients after myocardial infarction.
Right ventricular failure (RVF) is the main cause of death in patients with pulmonary artery hypertension (PAH). Sildenafil, a phosphodiesterase type 5 (PDE5) inhibitor, was recently approved for treatment of PAH patients. However, the mechanisms underlying RV contractile malfunction and the benefits of sildenafil on RV function are not well understood. We aimed to investigate 1) the ultrastructural and excitation-contraction coupling alterations underlying PAH-induced RVF; 2) whether the ultrastructural changes are reversible; 3) the mechanisms underlying the therapeutic benefits of sildenafil in PAH-RVF. We used a single injection of monocrotaline (MCT) in Wistar rats to induce pulmonary vascular proliferation, which led to PAH and RVF. RV myocytes displayed severe T-tubule loss and disorganization as well as blunted and dys-synchronous SR Ca2+ release. Sildenafil prevented and reversed the MCT-induced PAH and LV filling impairment. Early intervention with sildenafil prevented RV hypertrophy and the development of RVF, T-tubule remodeling and Ca2+ handling dysfunction. While late treatment with sildenafil did not reverse RV hypertrophy in animals with established RVF, RV systolic function was improved. Furthermore, late intervention partially reversed both the impairment of myocyte T-tubule integrity and Ca2+ handling protein and SR Ca2+ release function in MCT-treated rats. In conclusion, PAH-induced increase in RV afterload causes severe T-tubule remodeling and Ca2+ handling dysfunction in RV myocytes, leading to RV contractile failure. Sildenafil prevents and partially reverses ultrastructural, molecular and functional remodeling of failing RV myocytes. Reversal of pathological T-tubule remodeling, although incomplete, is achievable without the regression of RV hypertrophy.
right ventricle failure; pulmonary artery hypertension; PDE5 inhibitor; calcium; t-tubule; excitation-contraction coupling
Nicotinamidases catalyze the hydrolysis of nicotinamide to nicotinic acid and ammonia. Nicotinamidases are absent in mammals but function in NAD+ salvage in many bacteria, yeast, plants, protozoa, and metazoans. We have performed structural and kinetic investigations of the nicotinamidase from S. cerevisiae (Pnc1). Steady-state product inhibitor analysis revealed an irreversible reaction where ammonia is the first product released, followed by nicotinic acid. A series of nicotinamide analogs acting as inhibitors or substrates were examined revealing that the nicotinamide carbonyl oxygen and ring nitrogen are critical for binding and reactivity. X-ray structural analysis revealed a covalent adduct between nicotinaldehyde and Cys167 of Pnc1 and coordination of the nicotinamide ring nitrogen to the active-site zinc ion. Using this structure as a guide, the function of several residues was probed via mutagenesis and primary 15N and 13C kinetic isotope effects (KIE) on V/K for amide bond hydrolysis. The KIE values of almost all variants were increased indicating that C-N bond cleavage is at least partially rate limiting; however, a decreased KIE for D51N was observed indicative of a higher commitment to catalysis. In addition, KIE values using slower alternate substrates indicated that C-N bond cleavage is at least partially rate limiting with nicotinamide to highly rate limiting with thionicotinamide. A detailed mechanism is discussed involving nucleophilic attack of Cys167, followed by elimination of ammonia and then hydrolysis to liberate nicotinic acid. These results will aid design of mechanism-based inhibitors to target pathogens that rely on nicotinamidase activity.
Prolonged seizures and status epilepticus are a common acute neurological presentation in pediatric practice. As a result, there is a need for effective and safe medications that can be delivered to convulsing children to effect rapid seizure termination both in hospital and community settings. The challenges of achieving intravenous access, particularly in young children, mandate alternative routes of administration for these drugs. Over the last ten years, midazolam delivered via the buccal mucosa has been demonstrated to be efficacious, safe, and acceptable to children and their caregivers, and a formulation has recently been licensed for use in Europe. The aim of this article is to review the clinical pharmacology with respect to these issues.
pediatrics; neurology; pharmacology; buccal midazolam
Chronic non-healing wounds, such as venous stasis ulcers, diabetic ulcers, and pressure ulcers are serious unmet medical needs that affect a patient’s morbidity and mortality. Common pathogens observed in chronic non-healing wounds are Staphylococcus including MRSA, Pseudomonas, Enterobacter, Stenotrophomonas, and Serratia spp. Topical and systemically administered antibiotics do not adequately decrease the level of bacteria or the associated biofilm in chronic granulating wounds and the use of sub-lethal concentrations of antibiotics can lead to resistant phenotypes. Furthermore, topical antiseptics may not be fully effective and can actually impede wound healing. We show 5 representative examples from our more than 30 clinical case studies using NeutroPhase® as an irrigation solution with chronic non-healing wounds with and without the technique of negative pressure wound therapy (NPWT). NeutroPhase® is pure 0.01% hypochlorous acid (i.e. >97% relative molar distribution of active chlorine species as HOCl) in a 0.9% saline solution at pH 4-5 and is stored in glass containers. NovaBay has three FDA cleared 510(k)s. Patients showed a profound improvement and marked accelerated rates of wound healing using NeutroPhase® with and without NPWT. NeutroPhase® was non-toxic to living tissues.
NeutroPhase®; hypochlorous acid; chronic non-healing wounds; 510(k); negative pressure wound therapy (NPWT)
The cardiovascular system operates under demands ranging from conditions of rest to extreme stress. One mechanism of cardiac stress tolerance is action potential duration shortening driven by ATP-sensitive potassium (KATP) channels. KATP channel expression has a significant physiologic impact on action potential duration shortening and myocardial energy consumption in response to physiologic heart rate acceleration. However, the effect of reduced channel expression on action potential duration shortening in response to severe metabolic stress is yet to be established. Here, transgenic mice with myocardium-specific expression of a dominant negative KATP channel subunit were compared with littermate controls. Evaluation of KATP channel whole cell current and channel number/patch was assessed by patch clamp in isolated ventricular cardiomyocytes. Monophasic action potentials were monitored in retrogradely perfused, isolated hearts during the transition to hypoxic perfusate. An 80-85% reduction in cardiac KATP channel current density results in a similar magnitude, but significantly slower rate, of shortening of the ventricular action potential duration in response to severe hypoxia, despite no significant difference in coronary flow. Therefore, the number of functional cardiac sarcolemmal KATP channels is a critical determinant of the rate of adaptation of myocardial membrane excitability, with implications for optimization of cardiac energy consumption and consequent cardioprotection under conditions of severe metabolic stress.
ATP-sensitive potassium channel; K-ATP; heart; glyburide; monophasic action potential
N-chlorotaurine (NCT), the main representative of long-lived oxidants produced by granulocytes and monocytes, is known to exert broad-spectrum microbicidal activity. Here we show that NCT directly inactivates Shiga toxin 2 (Stx2), used as a model toxin secreted by enterohemorrhagic Escherichia coli (EHEC). Bacterial growth and Stx2 production were both inhibited by 2 mM NCT. The cytotoxic effect of Stx2 on Vero cells was removed by ≥5.5 mM NCT. Confocal microscopy and FACS analyses showed that the binding of Stx2 to human kidney glomerular endothelial cells was inhibited, and no NCT-treated Stx2 entered the cytosol. Mass spectrometry displayed oxidation of thio groups and aromatic amino acids of Stx2 by NCT. Therefore, long-lived oxidants may act as powerful tools of innate immunity against soluble virulence factors of pathogens. Moreover, inactivation of virulence factors may contribute to therapeutic success of NCT and novel analogs, which are in development as topical antiinfectives.
Doxorubicin (DOX) is one of the most effective chemotherapeutic agents, but cardiotoxicity limits DOX therapy. Although the mechanisms are not entirely understood, reactive oxygen species (ROS) appear to be involved in DOX cardiotoxicity. Ca/calmodulin dependent protein kinase II (CaMKII) can be activated by ROS through oxidation and is known to contribute to myocardial dysfunction through Ca leakage from the sarcoplasmic reticulum (SR).
We hypothesized that CaMKII contributes to DOX-induced defects in intracellular Ca ([Ca]i) handling.
Cardiac myocytes were isolated from wild-type (WT) adult rat hearts and from mouse hearts lacking the predominant myocardial CaMKII isoform (CaMKIIδ−/−, KO) vs. WT. Isolated cardiomyocytes were investigated 30 min after DOX (10 µmol/L) superfusion, using epifluorescence and confocal microscopy. Intracellular ROS-generation ([ROS]i) and [Ca]i handling properties were assessed. In a subset of experiments, KN-93 or AIP (each 1 µmol/L) were used to inhibit CaMKII. Melatonin (Mel, 100 µmol/L) served as ROS-scavenger. Western blots were performed to determine the amount of CaMKII phosphorylation and oxidation.
DOX increased [ROS]i and led to significant diastolic [Ca]i overload in rat myocytes. This was associated with reduced [Ca]i transients, a 5.8-fold increased diastolic SR Ca leak and diminished SR Ca content. ROS-scavenging partially rescued Ca handling. Western blots revealed increased CaMKII phosphorylation, but not CaMKII oxidation after DOX. Pharmacological CaMKII inhibition attenuated diastolic [Ca]i overload after DOX superfusion and led to partially restored [Ca]i transients and SR Ca content, presumably due to reduced Ca spark frequency. In line with this concept, isoform-specific CaMKIIδ-KO attenuated diastolic [Ca]i overload and Ca spark frequency.
DOX exposure induces CaMKII-dependent SR Ca leakage, which partially contributes to impaired cellular [Ca]i homeostasis. Pharmacological and genetic CaMKII inhibition attenuated but did not completely abolish the effects of DOX on [Ca]i. In light of the clinical relevance of DOX, further investigations seem appropriate to determine if CaMKII inhibition could reduce DOX-induced cardiotoxicity.
Twenty-five serial passages of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus and 50 passages of methicillin-resistant Staphylococcus aureus resulted in no significant increase in NVC-422 MICs, while ciprofloxacin MICs increased 256-fold for E. coli and 32-fold for P. aeruginosa and S. aureus. Mupirocin, fusidic acid, and retapamulin MICs for MRSA increased 64-, 256-, and 16-fold, respectively. No cross-resistance to NVC-422 was observed with mupirocin-, fusidic acid-, and retapamulin-resistant strains.