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Background: Loss of brain-derived neurotrophic factor (BDNF)/TrkB (tropomyosin kinase receptor B) signaling accounts for brain and cardiac disorders. In neurons, β-adrenergic receptor stimulation enhances local BDNF expression. It is unclear if this occurs in a pathophysiological relevant manner in the heart, especially in the β-adrenergic receptor-desensitized postischemic myocardium. Nor is it fully understood whether and how TrkB agonists counter chronic postischemic left ventricle (LV) decompensation, a significant unmet clinical milestone. Methods: We conducted in vitro studies using neonatal rat and adult murine cardiomyocytes, SH-SY5Y neuronal cells, and umbilical vein endothelial cells. We assessed myocardial ischemia (MI) impact in wild type, β3AR knockout, or myocyte-selective BDNF knockout (myoBDNF KO) mice in vivo (via coronary ligation [MI]) or in isolated hearts with global ischemia-reperfusion (I/R). Results: In wild type hearts, BDNF levels rose early after MI ( Conclusions: BDNF loss underscores chronic postischemic heart failure. TrkB agonists can improve ischemic LV dysfunction via replenished myocardial BDNF content. Direct cardiac β3AR stimulation, or β-blockers (via upregulated β3AR), is another BDNF-based means to fend off chronic postischemic heart failure.

BackgroundThough acute mortality by myocardial infarction (MI) has declined in past decades, MI still represents one of the leading causes of heart failure (HF) development. We recently demonstrated the accumulation of toxic desmin aggregates in patients with HF of ischemic origin. Since desmin aggregates are toxic for the heart we aimed to test whether their formation can be induced by oxidative stress as a proxy for reperfusion injury, as well as addressing the effects of therapeutic strategies aimed at reducing desmin aggregation with cardiac oxidative stress.Methods and ResultsWe demonstrate here that oxidative stress is able to induce desmin aggregation, acutely, in a cell-specific and dose-dependent fashion. We also show that elevation ofO-linked β-N-acetylglucosamine (O-GlcNAc) prior to or after oxidative stress reduces the formation of toxic desmin aggregates and its pro-aggregating desmin post-translational modifications (PTM). In addition, we show for the first time a role for the transmembrane protease serine 13 (TMPRSS13) with desmin cleavage in response to oxidative stress while desmin’s single cysteine plays a protective role from I/R injury, which is independent of gain or loss of desmin function.ConclusionsThe proliferation of desmin PTM-forms (i.e., proteoforms) and its aggregation hallmark acute and chronic cardiac stress and result in both loss of and gain of desmin function. We report here two novel mechanisms that could be targeted for therapy to preserve desmin homeostasis and cardiac function in the acute settings of oxidative stress and reperfusion injury.

Introduction: Post-translational modifications of cardiac troponin I (cTnI), an essential sarcomeric protein, have been known to play a vital role in regulating heart function. Site-specific phosphorylation on cTnI Ser199 is upregulated in human ischemic heart failure, negatively affects diastolic function but preserves ejection fraction in mice in vivo , and promotes contractility recovery after ischemia/reperfusion (I/R) in male isolated mouse hearts. However, its cardioprotective mechanism is largely unknown. Hypothesis: We assessed the hypothesis that cTnI Ser199 hyperphosphorylation protects hearts during I/R via down-regulating reactive oxygen species (ROS). Methods: Due to the current methodology limitation, there is no way to phosphorylate one specific site on cTnI in vivo . We, therefore, generated a transgenic mouse model named TgD that carried Serine (S) to Aspartic Acid (D) mutation at cTnI Ser200 (equivalent to Ser199 in human sequence) to mimic the site-specific hyperphosphorylation, which was a widely accepted method for in vivo studies on sarcomeric protein phosphorylation. The cardiac function was examined using Langendorff isolated hearts with pacing at baseline and after 30-minute global ischemia followed by 2-hour reperfusion. Results: At baseline, TgD hearts (both genders, 7-8 mice/gender, age 3-5 months) showed comparable rate pressure product (RPP), left ventricular developed pressure (LVDevP), and d P /d t max but decreased d P /d t min relative to wildtype (WT) littermates. After I/R, TgD presented significantly better RPP, LVDevP, d P /d t max , and d P /d t min with remarkable contractile function recovery rate compared to WT (i.e., 47% ±2% vs. 24%±2% in RPP, P Conclusions: In conclusion, human heart-failure-related cTnI Ser199 hyperphosphorylation protects heart function during global I/R in isolated mouse hearts in both genders . The protective mechanism, at least in part, involves the downregulation of ROS.

Arsenic exposure though drinking water is widespread and well associated with adverse cardiovascular outcomes, yet the pathophysiological mechanisms by which iAS induces these effects are largely unknown. Recently, an epidemiological study in an American population with a low burden of cardiovascular risk factors found that iAS exposure was associated with altered left ventricular geometry. Considering the possibility that iAS directly induces cardiac remodeling independently of hypertension, we investigated the impact of an environmentally relevant iAS exposure on the structure and function of male and female hearts. Adult male and female C56BL/6J mice were exposed to 615 μg/L iAS for 8 wk. Males exhibited increased systolic blood pressure via tail cuff photoplethysmography, left ventricular wall thickening via transthoracic echocardiography, and increased plasma atrial natriuretic peptide via enzyme immunoassay. RT-qPCR revealed increased myocardial RNA transcripts of Acta1, Myh7, and Nppa and decreased Myh6, providing evidence of pathological hypertrophy in the male heart. Similar changes were not detected in females, and nitric oxide-dependent mechanisms of cardioprotection in the heart appeared to remain intact. Further investigation found that Rcan1 was upregulated in male hearts and that iAS activated NFAT in HEK-293 cells via luciferase assay. Interestingly, iAS induced similar hypertrophic gene expression changes in neonatal rat ventricular myocytes, which were blocked by calcineurin inhibition, suggesting that iAS may induce pathological cardiac hypertrophy in part by targeting the calcineurin-NFAT pathway. As such, these results highlight iAS exposure as an independent cardiovascular risk factor and provide biological impetus for its removal from human consumption. NEW & NOTEWORTHY This investigation provides the first mechanistic link between an environmentally relevant dose of inorganic arsenic (iAS) and pathological hypertrophy in the heart. By demonstrating that iAS exposure may cause pathological cardiac hypertrophy not only by increasing systolic blood pressure but also by potentially activating calcineurin-nuclear factor of activated T cells and inducing fetal gene expression, these results provide novel mechanistic insight into the theat of iAS exposure to the heart, which is necessary to identify targets for medical and public health intervention. Listen to this article’s corresponding podcast at https://ajpheart.podbean.com/e/inorganic-arsenic-induces-cardiac-hypertrophy/.

In order to extract sulforaphane (SFN) from broccoli via green and efficient ways, a novel method based on salting-out assisted deep eutectic solvent (DES) has been developed. Compared to known organic solvent- (such as dichloromethane, ethyl acetate, n-hexane, etc.) based liquid-liquid extraction, this new N

Adult mice with cardiac-specific overexpression of adenylyl cyclase (AC) type VIII (TGAC8) adapt to an incessantly increased cAMP-induced cardiac workload (∼30% increases in heart rate, ejection fraction and cardiac output) for up to a year without signs of heart failure or excessive mortality. Here we show that despite markedly increased cardiac work, classical cardiac hypertrophy markers were absent in TGAC8, total left ventricular (LV) mass was not increased: a reduced LV cavity volume in TGAC8 was encased by thicker LV walls harboring an increased number of small cardiac myocytes and a network of small interstitial non-cardiac myocytes, manifesting increased proliferation markers and compared to WT. Protein synthesis, proteosome activity, autophagy, and Nrf-2, Hsp90α, ACC2 protein levels were increased in TGAC8, but LV ATP and phosphocreatine levels in vivo did not differ by genotype. 2,323 transcripts and 2,184 proteins identified in unbiased omics analyses, spanning a wide array of biological processes and molecular functions in numerous cellular compartments differed in TGAC8 vs WT; and over 250 canonical signaling pathways characteristic of adaptive survival circuitry of cancers, including PI3K and growth factor signaling, cytokine and T cell receptor signaling, immune responses, ROS scavenging, proliferation, protection from apoptosis, and nutrient sensing, were activated in TGAC8; and compared to WT there was a shift from fatty acid oxidation to increased aerobic glycolysis in the context of increased utilization of the pentose phosphate shunt and nucleotide synthesis. Thus, the adaptive paradigm, that becomes activated in the LV of TGAC8 in response to severe chronic, intense AC/PKA/Ca2+ signaling embodies many hallmarks of cancer.

HIV-associated neurocognitive disorders (HAND) remain prevalent despite implementation of antiretroviral therapy (ART). Development of HAND is linked to mitochondrial dysfunction and oxidative stress in the brain; therefore, upregulation of antioxidant defenses is critical to curtail neuronal damage. Superoxide dismutase 2 (SOD2) is a mitochondrial antioxidant enzyme essential for maintaining cellular viability. We hypothesized that SOD2 was upregulated during retroviral infection. Using a simian immunodeficiency virus (SIV)-infected macaque model of HIV, quantitative PCR showed elevated SOD2 mRNA in cortical gray (GM, 7.6-fold for SIV vs. uninfected) and white matter (WM, 77-fold for SIV vs. uninfected) during SIV infection. Further, SOD2 immunostaining was enhanced in GM and WM from SIV-infected animals. Double immunofluorescence labeling illustrated that SOD2 primarily co-localized with astrocyte marker glial fibrillary acidic protein (GFAP) in SIV-infected animals. Interestingly, in ART-treated SIV-infected animals, brain SOD2 RNA levels were similar to uninfected animals. Additionally, using principal component analysis in a transcriptomic approach, SOD2 and GFAP expression separated SIV-infected from uninfected brain tissue. Projection of these data into a HIV dataset revealed similar expression changes, thereby validating the clinical relevance. Together, our findings suggest that novel SOD2-enhancing therapies may delay the onset or reduce severity of HAND seen in ART-treated HIV-infected patients.

BACKGROUND: Abnormalities in cardiac energy metabolism occur in heart failure (HF) and contribute to contractile dysfunction, but their role, if any, in HF-related pathologic remodeling is much less established. CK (creatine kinase), the primary muscle energy reserve reaction which rapidly provides ATP at the myofibrils and regenerates mitochondrial ADP, is down-regulated in experimental and human HF. We tested the hypotheses that pathologic remodeling in human HF is related to impaired cardiac CK energy metabolism and that rescuing CK attenuates maladaptive hypertrophy in experimental HF., METHODS: First, in 27 HF patients and 14 healthy subjects, we measured cardiac energetics and left ventricular remodeling using noninvasive magnetic resonance P-31 spectroscopy and magnetic resonance imaging, respectively. Second, we tested the impact of metabolic rescue with cardiac-specific overexpression of either Ckmyofib (myofibrillar CK) or Ckmito (mitochondrial CK) on HF-related maladaptive hypertrophy in mice., RESULTS: In people, pathologic left ventricular hypertrophy and dilatation correlate closely with reduced myocardial ATP levels and rates of ATP synthesis through CK. In mice, transverse aortic constriction-induced left ventricular hypertrophy and dilatation are attenuated by overexpression of CKmito, but not by overexpression of CKmyofib. CKmito overexpression also attenuates hypertrophy after chronic isoproterenol stimulation. CKmito lowers mitochondrial reactive oxygen species, tissue reactive oxygen species levels, and upregulates antioxidants and their promoters. When the CK capacity of CKmito-overexpressing mice is limited by creatine substrate depletion, the protection against pathologic remodeling is lost, suggesting the ADP regenerating capacity of the CKmito reaction rather than CK protein per se is critical in limiting adverse HF remodeling., CONCLUSIONS: In the failing human heart, pathologic hypertrophy and adverse remodeling are closely related to deficits in ATP levels and in the CK energy reserve reaction. CKmito, sitting at the intersection of cardiac energetics and redox balance, plays a crucial role in attenuating pathologic remodeling in HF.

Myocyte death occurs in many inherited and acquired cardiomyopathies, including arrhythmogenic cardiomyopathy (ACM), a genetic heart disease plagued by the prevalence of sudden cardiac death. Individuals with ACM and harboring pathogenic desmosomal variants, such as desmoglein-2 (DSG2), often show myocyte necrosis with progression to exercise-associated heart failure. Here, we showed that homozygous Dsg2 mutant mice (Dsg2(mut/mut)), a model of ACM, die prematurely during swimming and display myocardial dysfunction and necrosis. We detected calcium (Ca(2+)) overload in Dsg2(mut/mut) hearts, which induced calpain-1 (CAPN1) activation, association of CAPN1 with mitochondria, and CAPN1-induced cleavage of mitochondrial-bound apoptosis-inducing factor (AIF). Cleaved AIF translocated to the myocyte nucleus triggering large-scale DNA fragmentation and cell death, an effect potentiated by mitochondrial-driven AIF oxidation. Posttranslational oxidation of AIF cysteine residues was due, in part, to a depleted mitochondrial thioredoxin-2 redox system. Hearts from exercised Dsg2(mut/mut) mice were depleted of calpastatin (CAST), an endogenous CAPN1 inhibitor, and overexpressing CAST in myocytes protected against Ca(2+) overload–induced necrosis. When cardiomyocytes differentiated from Dsg2(mut/mut) embryonic stem cells (ES-CMs) were challenged with β-adrenergic stimulation, CAPN1 inhibition attenuated CAPN1-induced AIF truncation. In addition, pretreatment of Dsg2(mut/mut) ES-CMs with an AIF-mimetic peptide, mirroring the cyclophilin-A (PPIA) binding site of AIF, blocked PPIA-mediated AIF-nuclear translocation, and reduced both apoptosis and necrosis. Thus, preventing CAPN1-induced AIF-truncation or barring binding of AIF to the nuclear chaperone, PPIA, may avert myocyte death and, ultimately, disease progression to heart failure in ACM and likely other forms of cardiomyopathies.

Exposure to inorganic arsenic (iAS) through drinking water is well-associated with adverse cardiovascular outcomes, yet the mechanisms through which it induces these effects are not fully understood. Recent epidemiological findings highlight an association between iAS exposure and altered left ventricular geometry in both the presence and absence of hypertension. We therefore tested the hypothesis that iAS exposure has a bimodal impact on cardiac-intrinsic and hemodynamic mechanisms that together induce pathological remodeling of the myocardium. Adult male and female mice were exposed to an environmentally relevant dose of 615 μg/L NaAsO 2 for eight weeks. Males (n=9-10 mice/group) exhibited increased systolic blood pressure (115.1±3.0 vs. 106.0±2.3 mmHg, p=0.0350) via tail cuff photoplethysmography, left ventricular wall thickening (0.98±0.01 vs. 0.88±0.01 mm, pActa1 (1.36±0.18 vs. 0.73±0.11, p=0.0037), Myh7 (1.53±0.15 vs. 1.04±0.10, p=0.0138), and Nppa (2.40±0.29 vs. 1.02±0.07, p=0.0001) were increased, and Myh6 (0.92±0.17 vs. 1.14±0.23, p=0.0001) was decreased, evidencing pathological hypertrophy in the male heart. Female hearts, however, were largely protected at this eight-week timepoint as similar changes were not detected. Further investigation found that Rcan1 was upregulated (1.47±0.19 vs. 0.97±0.04, p=0.0161; n=10 hearts/group) in male hearts, suggesting that calcineurin-NFAT was activated. Interestingly, iAS was sufficient to activate NFAT (0.82±0.11 vs. 0.46±0.05, p=0.0214; n=8 wells/group) independent of blood pressure via luciferase assay. In conclusion, these results demonstrate for the first time that iAS may cause pathological cardiac hypertrophy not only by increasing hemodynamic load, but also by activating calcineurin-NFAT and inducing fetal gene expression in the male heart, thus providing novel mechanistic insight into the threat of iAS exposure to the cardiovascular system.

Many forms of cardiac disease, including heart failure, present with inadequate protein quality control (PQC). Pathological conditions often involve impaired removal of terminally misfolded proteins. This results in the formation of large protein aggregates, which further reduce cellular viability and cardiac function. Cardiomyocytes have an intricately collaborative PQC system to minimize cellular proteotoxicity. Increased expression of chaperones or enhanced clearance of misfolded proteins either by the proteasome or lysosome has been demonstrated to attenuate disease pathogenesis, whereas reduced PQC exacerbates pathogenesis. Recent studies have revealed that phosphorylation of key proteins has a potent regulatory role, both promoting and hindering the PQC machinery. This review highlights the recent advances in phosphorylations regulating PQC, the impact in cardiac pathology, and the therapeutic opportunities presented by harnessing these modifications.

Physiological stressors, such as exercise, can precipitate sudden cardiac death or heart failure progression in patients with arrhythmogenic cardiomyopathy (ACM). Yet, whether and to what extent a highly prevalent and more elusive environmental factor, such as psychosocial stress (PSS), can also increase ACM disease progression is unexplored. Here, we first quantified perceived stress levels in patients with ACM and found these levels correlated with the extent of arrhythmias and cardiac dysfunction. To determine whether the observed correlation is due to causation, we inflicted PSS-via the resident-intruder (RI) paradigm&mdash, upon Desmoglein-2 mutant mice, a vigorously used mammalian model of ACM. We found that ACM mice succumbed to abnormally high in-trial, PSS mortality. Conversely, no sudden deaths occurred in wildtype (WT) counterparts. Desmoglein-2 mice that survived RI challenge manifested markedly worse cardiac dysfunction and remodeling, namely apoptosis and fibrosis. Furthermore, WT and ACM mice displayed similar behavior at baseline, but Desmoglein-2 mice exhibited heightened anxiety following RI-induced PSS. This outcome correlated with the worsening of cardiac phenotypes. Our mouse model demonstrates that in ACM-like subjects, PSS is incisive enough to deteriorate cardiac structure and function per se, i.e., in the absence of any pre-existing anxious behavior. Hence, PSS may represent a previously underappreciated risk factor in ACM disease penetrance.

Treatment with nitroxyl (HNO) improves cardiac function in failing hearts by inducing release of SR Ca2+-ATPase from phospholamban (PLN), thereby enhancing Ca2+ reuptake. Keceli et al. use 15N-edited NMR spectroscopy to show that HNO achieves this by reversibly modifying PLN cysteines 41 and 46., Nitroxyl (HNO) positively modulates myocardial function by accelerating Ca2+ reuptake into the sarcoplasmic reticulum (SR). HNO-induced enhancement of myocardial Ca2+ cycling and function is due to the modification of cysteines in the transmembrane domain of phospholamban (PLN), which results in activation of SR Ca2+-ATPase (SERCA2a) by functionally uncoupling PLN from SERCA2a. However, which cysteines are modified by HNO, and whether HNO induces reversible disulfides or single cysteine sulfinamides (RS(O)NH2) that are less easily reversed by reductants, remain to be determined. Using an 15N-edited NMR method for sulfinamide detection, we first demonstrate that Cys46 and Cys41 are the main targets of HNO reactivity with PLN. Supporting this conclusion, mutation of PLN cysteines 46 and 41 to alanine reduces the HNO-induced enhancement of SERCA2a activity. Treatment of WT-PLN with HNO leads to sulfinamide formation when the HNO donor is in excess, whereas disulfide formation is expected to dominate when the HNO/thiol stoichiometry approaches a 1:1 ratio that is more similar to that anticipated in vivo under normal, physiological conditions. Thus, 15N-edited NMR spectroscopy detects redox changes on thiols that are unique to HNO, greatly advancing the ability to detect HNO footprints in biological systems, while further differentiating HNO-induced post-translational modifications from those imparted by other reactive nitrogen or oxygen species. The present study confirms the potential of HNO as a signaling molecule in the cardiovascular system.

Introduction Obesity and psychosocial stress (PS) co-exist in individuals of Western society. Nevertheless, how PS impacts cardiac and hippocampal phenotype in obese subjects is still unknown. Nor is it clear whether changes in local brain-derived neurotrophic factor (BDNF) account, at least in part, for myocardial and behavioral abnormalities in obese experiencing PS. Methods In adult male WT mice, obesity was induced via a high-fat diet (HFD). The resident-intruder paradigm was superimposed to trigger PS. In vivo left ventricular (LV) performance was evaluated by echocardiography and pressure-volume loops. Behaviour was indagated by elevated plus maze (EPM) and Y-maze. LV myocardium was assayed for apoptosis, fibrosis, vessel density and oxidative stress. Hippocampus was analyzed for volume, neurogenesis, GABAergic markers and astrogliosis. Cardiac and hippocampal BDNF and TrkB levels were measured by ELISA and WB. We investigated the pathogenetic role played by BDNF signaling in additional cardiac-selective TrkB (cTrkB) KO mice. Findings When combined, obesity and PS jeopardized LV performance, causing prominent apoptosis, fibrosis, oxidative stress and remodeling of the larger coronary branches, along with lower BDNF and TrkB levels. HFD/PS weakened LV function similarly in WT and cTrkB KO mice. The latter exhibited elevated LV ROS emission already at baseline. Obesity/PS augmented anxiety-like behaviour and impaired spatial memory. These changes were coupled to reduced hippocampal volume, neurogenesis, local BDNF and TrkB content and augmented astrogliosis. Interpretation PS and obesity synergistically deteriorate myocardial structure and function by depleting cardiac BDNF/TrkB content, leading to augmented oxidative stress. This comorbidity triggers behavioral deficits and induces hippocampal remodeling, potentially via lower BDNF and TrkB levels. Fund J.A. was in part supported by Rotary Foundation Global Study Scholarship. G.K. was supported by T32 National Institute of Health (NIH) training grant under award number 1T32AG058527. S.C. was funded by American Heart Association Career Development Award (19CDA34760185). G.A.R.C. was funded by NIH ( K01HL133368-01 ). APB was funded by a Grant from the Friuli Venezia Giulia Region entitled: “Heart failure as the Alzheimer disease of the heart; therapeutic and diagnostic opportunities”. M.C. was supported by PRONAT project ( CNR ). N.P. was funded by NIH ( R01 HL136918 ) and by the Magic-That-Matters fund (JHU) . V.L. was in part supported by institutional funds from Scuola Superiore Sant'Anna (Pisa, Italy) , by the TIM-Telecom Italia (WHITE Lab, Pisa, Italy) , by a research grant from Pastificio Attilio Mastromauro Granoro s.r.l. (Corato, Italy) and in part by ETHERNA project (Prog. n. 161/16, Fondazione Pisa, Italy). Funding source had no such involvement in study design, in the collection, analysis, interpretation of data, in the writing of the report; and in the decision to submit the paper for publication.

Objectives: This review summarizes the spectroscopic results, which will provide useful suggestions for future research. In addition, the fields that urgently need more information are also advised. Background: Nitrite-NO-cGMP has been considered as an important signaling pathway of NO in human cells. To date, all the four known human molybdenum-containing enzymes, xanthine oxidase, aldehyde oxidase, sulfite oxidase, and mitochondrial amidoxime-reducing component, have been shown to function as nitrite reductases under hypoxia by biochemical, cellular, or animal studies. Various spectroscopic techniques have been applied to investigate the structure and catalytic mechanism of these enzymes for more than 20 years. Methods: We summarize the published data on the applications of UV-vis and EPR spectroscopies, and X-ray crystallography in studying nitrite reductase activity of the four human molybdenum-containing enzymes. Results: UV-vis has provided useful information on the redox active centers of these enzymes. The utilization of EPR spectroscopy has been critical in determining the coordination and redox status of the Mo center during catalysis. Despite the lack of substrate-bound crystal structures of these nitrite reductases, valuable structural information has been obtained by X-ray crystallography. Conclusions: To fully understand the catalytic mechanisms of these physiologically/pathologically important nitrite reductases, structural studies on substrate-redox center interaction are needed.

The prevalence of heart failure with a preserved ejection fraction (HFpEF) is increasing, but therapeutic options are limited. Oxidative stress is suggested to play an important role in the pathophysiology of HFpEF. However, whether oxidative stress is a bystander due to comorbidities or causative in itself remains unknown. Recent results have shown that depletion of 5-oxoprolinase (OPLAH) leads to 5-oxoproline accumulation, which is an important mediator of oxidative stress in the heart. We hypothesize that oxidative stress induced by elevated levels of 5-oxoproline leads to the onset of a murine HFpEF-like phenotype.Oplah full body knock-out (KO) mice had higher 5-oxoproline levels coupled to increased oxidative stress. Compared with wild-type (WT) littermates, KO mice had increased cardiac and renal fibrosis with concurrent elevated left ventricular (LV) filling pressures, impaired LV relaxation, yet a normal LV ejection fraction. Following the induction of cardiac ischaemia/reperfusion (IR) injury, 52.4% of the KO mice died compared with only 15.4% of the WT mice (P 0.03). Furthermore, KO mice showed a significantly increased atrial, ventricular, kidney, and liver weights compared with WT mice (P 0.05 for all). Cardiac and renal fibrosis were more pronounced following cardiac IR injury in the KO mice and these mice developed proteinuria post-IR injury. To further address the link between 5-oxoproline and HFpEF, 5-oxoproline was measured in the plasma of HFpEF patients. Compared with healthy controls (3.8 ± 0.6 µM), 5-oxoproline levels were significantly elevated in HFpEF patients (6.8 ± 1.9 µM, P 0.0001). Furthermore, levels of 5-oxoproline were independently associated with more concentric remodelling on echocardiography.Oxidative stress induced by 5-oxoproline results in a murine phenotype reminiscent of the clinical manifestation of HFpEF without the need for surgical or pharmacological interference. Better understanding of the role of oxidative stress in HFpEF may potentially lead to novel therapeutic options.

Exercise increases disease penetrance in arrhythmogenic cardiomyopathy (ACM). Yet, how exercise contributes to disease pathogenesis is unclear. Mitochondria potentiate reactive oxygen species (ROS) generation during exercise that are scavenged by antioxidants, such as thioredoxin-2 (Trx2). Here we tested if deficits in Trx2-based ROS buffering act as substrates for exercise-induced cardiac apoptosis in ACM. Homozygote Desmoglein-2 mutant mice ( Dsg2 mut/mut ) model ACM features, thus WT and Dsg2 mut/mut mice were enrolled in a 10 week swimming protocol to evaluate survival and post-effort cardiac function, ROS production by EPR, and mitochondrial ROS-gating protein levels. Survival rate after swimming was 91% (20/22) in WT, but only 60% (15/25) in Dsg2 mut/mut mice (p=0.008). Of the survivors, Dsg2 mut/mut mice displayed cardiac dysfunction (Ejection Fraction 57±4 vs 84±0.4% in WT; n≥14/cohort, PDsg2 mut/mut right ventricles (RV) than in WT (25±3 vs 15±1 Gauss/total protein [G/tp]; PDsg2 mut/mut left ventricles (LV) (39±5 G/tp vs mutant RV; Pvs WT in RV&LV). When mitochondria bound, Apoptosis-Inducing Factor (AIF) acts as a NADH oxidoreductase, yet upon oxidation, AIF translocates to the nucleus and initiates apoptosis. After exercise, Dsg2 mut/mut hearts displayed marked AIF nuclear and chromatin-bound protein levels and increased AIF immunostained nuclei vs WT. Additionally, direct H 2 O 2 exposure or Trx2 inhibition (by auranofin) in Dsg2 mut/mut embryonic stem-cell derived myocytes elevated AIF-nuclear translocation and apoptosis (via AnnexinV/PI FaCS analysis). Our study reveals a novel causal link between exercise-evoked cardiac redox imbalance and aberrant AIF-Trx2 signaling in ACM, which was associated with increased apoptosis, propensity of arrhythmias and sudden cardiac death. These findings offer a new targetable mechanism for preventing one of the most cited, yet poorly understood, pathological phenotypes (apoptosis) in ACM.

Objective— KLF15 (Kruppel-like factor 15) has recently been shown to suppress activation of proinflammatory processes that contribute to atherogenesis in vascular smooth muscle, however, the role of KLF15 in vascular endothelial function is unknown. Arginase mediates inflammatory vasculopathy and vascular injury in pulmonary hypertension. Here, we tested the hypothesis that KLF15 is a critical regulator of hypoxia-induced Arg2 (arginase 2) transcription in human pulmonary microvascular endothelial cells (HPMEC). Approach and Results— Quiescent HPMEC express ample amounts of full-length KLF15. HPMECs exposed to 24 hours of hypoxia exhibited a marked decrease in KLF15 protein levels and a reciprocal increase in Arg2 protein and mRNA. Chromatin immunoprecipitation indicated direct binding of KLF15 to the Arg2 promoter, which was relieved with HPMEC exposure to hypoxia. Furthermore, overexpression of KLF15 in HPMEC reversed hypoxia-induced augmentation of Arg2 abundance and arginase activity and rescued nitric oxide (NO) production. Ectopic KLF15 also reversed hypoxia-induced endothelium-mediated vasodilatation in isolated rat pulmonary artery rings. Mechanisms by which hypoxia regulates KLF15 abundance, stability, and compartmentalization to the nucleus in HPMEC were then investigated. Hypoxia triggered deSUMOylation of KLF15 by SENP1 (sentrin-specific protease 1), and translocation of KLF15 from nucleus to cytoplasm. Conclusions— KLF15 is a critical regulator of pulmonary endothelial homeostasis via repression of endothelial Arg2 expression. KLF15 abundance and nuclear compartmentalization are regulated by SUMOylation/deSUMOylation—a hypoxia-sensitive process that is controlled by SENP1. Strategies including overexpression of KLF15 or inhibition of SENP1 may represent novel therapeutic targets for pulmonary hypertension.

The selective reaction of chemical reagents with reduced protein thiols is critical to biological research. This reaction is utilized to prevent cross-linking of cysteine-containing peptides in common proteomics workflows and is applied widely in discovery and targeted redox investigations of the mechanisms underlying physiological and pathological processes. However, known and commonly used thiol blocking reagents like iodoacetamide, N-ethylmaleimide, and others were found to cross-react with oxidized protein sulfenic acids (-SOH) introducing significant errors in studies employing these reagents. We have investigated and are reporting here a new heteroaromatic alkylsulfone, 4-(5-methanesulfonyl-[1,2,3,4]tetrazol-1-yl)-phenol (MSTP), as a selective and highly reactive -SH blocking reagent compatible with biological applications.

Background: In human heart failure, Ser199 (equivalent to Ser200 in mouse) of cTnI (cardiac troponin I) is significantly hyperphosphorylated, and in vitro studies suggest that it enhances myofilament calcium sensitivity and alters calpain-mediated cTnI proteolysis. However, how its hyperphosphorylation affects cardiac function in vivo remains unknown. Methods and Results: To address the question, 2 transgenic mouse models were generated: a phospho-mimetic cTnIS200D and a phospho-silenced cTnIS200A, each driven by the cardiomyocyte-specific α-myosin heavy chain promoter. Cardiac structure assessed by echocardiography and histology was normal in both transgenic models compared with littermate controls (n=5). Baseline in vivo hemodynamics and isolated muscle studies showed that cTnIS200D significantly prolonged relaxation and lowered left ventricular peak filling rate, whereas ejection fraction and force development were normal (n=5). However, with increased heart rate or β-adrenergic stimulation, cTnIS200D mice had less enhanced ejection fraction or force development versus controls, whereas relaxation improved similarly to controls (n=5). By contrast, cTnIS200A was functionally normal both at baseline and under the physiological stresses. To test whether either mutation impacted cardiac response to ischemic stress, isolated hearts were subjected to ischemia/reperfusion. cTnIS200D were protected, recovering 88±8% of contractile function versus 35±15% in littermate controls and 28±8% in cTnIS200A (n=5). This was associated with less cTnI proteolysis in cTnIS200D hearts. Conclusions: Hyperphosphorylation of this serine in cTnI C terminus impacts heart function by depressing diastolic function at baseline and limiting systolic reserve under physiological stresses. However, paradoxically, it preserves heart function after ischemia/reperfusion injury, potentially by decreasing proteolysis of cTnI.

Reactions based on the transfer of one or more electrons from a donor (reductant) to an acceptor (oxidant) are at the basis of several physiologically relevant cellular processes. Both reactive oxygen and nitrogen species (ROS and RNS, respectively) can signal through these reduction/oxidation (redox) reactions, particularly via reversible interaction with highly reactive thiols. Nitroxyl (HNO), the one-electron reduction product of nitric oxide (NO•), is an RNS that in the last few lusters has garnered a lot of attention owing to its pharmacological properties that are quite dissimilar from those exhibited by its sibling NO• or other RNS such as nitrite/nitrate. HNO uniqueness becomes particularly evident in the cardiovascular system. One aim of this chapter is to put the following three fundamental chemical properties of HNO in a cardiovascular physiology and therapeutic perspective: (1) HNO elective, and likely selective thiophilic nature; (2) inertness toward ROS; and (3) modest reactivity with molecular O 2 . With this conceptual framework in mind, here we will first review routes accounting for possible HNO endogenous formation as they may be relevant to govern basal and stress-stimulated cardiac and vascular function. Then, we will provide an updated account of the pharmacological properties of HNO donors in the heart and in vessels, both in vivo and in vitro, under normal and disease conditions, flanking this evidence with additional HNO pharmacological properties that may nicely complement its main cardiovascular actions. We will conclude discussing the perspective of HNO donors as a treatment for heart failure, more specifically for acute decompensated heart failure (ADHF), comparing HNO therapeutic portfolio to current mainstay ADHF therapies.

Recent discoveries of important pharmacological properties have drawn attention to the reactivity of HNO (azanone, nitroxyl) with biologically relevant substrates. Apart from its role in thiol oxidation, HNO has been reported to have nitrosative properties, for example, with tryptophan resulting in N-nitrosotryptophan formation. We have investigated the reactivity of HNO with tryptophan and small peptides containing either tryptophan or both a tryptophan and a cysteine residue. Our results point to the more reactive nature of cysteine towards HNO compared with tryptophan.

Congestive heart failure frequently involves reduced cardiac contractility and slowed muscle relaxation. The reactive nitrogen species HNO (nitroxyl, azanone) has been shown to augment contractility and accelerate relaxation in both normal and failing hearts. In humans, greater than 70% of cytosolic Ca2+ is pumped into the sarco(endo)plasmic reticulum (SR) by SR Ca2+-ATPase (SERCA2a). Phospholamban (PLN) is a small transmembrane protein, which is associated with SERCA2a, and depending on its phosphorylation state, regulates the activity of the Ca2+ pump. Our previous work has shown that HNO modifies PLN and affects SERCA2a activity independent of the β-adrenergic pathway. We have applied our 15N-edited NMR method for sulfinamide detection and biochemical approaches to investigate HNO-derived modifications on PLN. These studies demonstrate that PLN cysteines 46 and 41 are the major sites of HNO reactivity. Furthermore, our results indicate that sulfinamide formation is the major HNO-derived modification in the presence of excess HNO donor, whereas disulfide formation is expected to dominate under physiologically relevant conditions.

Sulfinamide [RS(O)NH(2)] formation is known to occur upon exposure of cysteine residues to nitroxyl (HNO), which has received recent attention as a potential heart failure therapeutic. Because this modification can alter protein structure and function, we have examined the reactivity of sulfinamides in several systems, including a small organic molecule, peptides, and a protein. Although it has generally been assumed that this thiol to sulfinamide modification is irreversible, we show that sulfinamides can be reduced back to the free thiol in the presence of excess thiol at physiological pH and temperature. We have examined this sulfinamide reduction both in peptides, where a cyclic intermediate analogous to that proposed for asparagine deamidation reactions potentially can contribute, and in a small organic molecule, where the mechanism is restricted to a direct thiolysis. These studies suggest that the contribution from the cyclic intermediate becomes more important in environments with lower dielectric constants. In addition, although sulfinic acid [RS(O)OH] formation is observed upon prolonged incubations in water, reduction of sulfinamides is found to dominate in the presence of thiols. Finally, studies with the cysteine protease, papain, suggest that the reduction of sulfinamide to the free thiol is viable in a protein environment.

Exercise increases arrhythmic risk and sudden cardiac death (SCD) in arrhythmogenic cardiomyopathy (ACM) subjects via unknown mechanisms. Exercise increases reactive oxygen species (ROS) generation. Yet antioxidant defenses, such as the thioredoxin (Trx) system effectively buffer ROS, preventing cardiac oxidative damage. Desmoglein-2 (DSG2) mutations are commonly associated to ACM, accordingly we generated a knock-in ACM mouse model bearing a mutation in murine Dsg2 (Dsg2mut/mut). Then, we subjected WT and Dsg2mut/mut mice to a 10 week swimming protocol, monitoring cardiac function, and terminally assessing cardiac tissue Trx1/2 protein levels and ROS production by EPR. Of note, only 60% (15/25) of Dsg2mut/mutmice survived to exercise end-point, compared to WT mice (91%, 20/22, p=0.008). No SCD occurred in age-matched sedentary mice. While sedentary and exercised Dsg2mut/mut mice (EF: 57±4 vs 84±0.4% in WT; n≥14/cohort, P

Heart failure (HF) is characterized by perturbed myocyte Ca2+ handling. Altered SR Ca2+-ATPase (SERCA2a) activity sizably contributes to impair Ca2+ cycling in the heart, affecting its contraction/relaxation. Strategies to rescue or improve SERCA2a expression/activity are being clinically tested in HF patients. Donors of nitroxyl (HNO) augment cardiac muscle performance in failing hearts, by modulating phospholamban (PLN) and SERCA2a interaction. They do so independently from β-adrenergic activation. In fact, HNO increases SERCA2a activity by promoting redox-dependent PLN oligomerization, thus reducing the amount of monomeric/inhibitory PLN. Yet, the precise sites (cysteines that are modified by HNO in PLN) and the chemical nature of these redox modifications (disulfide bond vs. sulfinamide formation) remain elusive. To fill this gap, we have applied our 15N-edited NMR method for sulfinamide detection and biochemical approaches to investigate HNO-derived modifications on PLN. We found that cysteines 46 and 41 are the sites of HNO action in PLN, and that sulfinamide formation is the major HNO-derived modification when HNO is in excess with respect to thiols. However, when HNO/thiol approach the 1:1 ratio (thus reflecting more physiological conditions), intramolecular disulfide bonds are expected to be the dominant HNO-induced species. To investigate the intramolecular disulfide bond formation further, we have started to employ fluorescence labeling methodologies. Present findings provide more mechanistic insight into the salutary actions of HNO donors that are currently tested for safety and efficacy in patients with acute decompensated HF.

HNO, a potential heart failure therapeutic, is known to post-translationally modify cysteine residues. Among reactive nitrogen oxide species, the modification of cysteine residues to sulfinamides [RS(O)NH2] is unique to HNO. Because this modification can alter protein structure and function, we have examined the reactivity of sulfinamides in several systems, including small organic molecules, peptides, and a protein. At physiological pH and temperature, relevant reactions of sulfinamides involve reduction to free thiols in the presence of excess thiol and hydrolysis to form sulfinic acids [RS(O)OH]. In addition to utilizing ESI-MS and other spectroscopic methods to study sulfinamide reduction, we have applied 15N-edited 1H-NMR techniques to sulfinamide detection and used this method to explore sulfinamide hydrolysis. We have also investigated the effect of local environment on the reactivity of HNO with C-terminal cysteines. Our findings indicate that the nature of HNO-derived modifications of C-terminal ...

The failing heart is energy-starved and redox imbalanced. Understanding how, and where cardiac energetic and redox alterations intersect in the failing heart are paramount to understanding and treating heart failure (HF). Creatine kinase (CK) is the major myocardial energy reserve reaction and ATP buffer with isozymes in the cytosol (CK-M) and mitochondria (Mt-CK). CK activity is reduced in human HF and following transverse aortic constriction (TAC). Mt-CK is an octamer particularly prone to oxidative modifications. It is not known whether HF changes in Mt-CK contribute to alter myocardial redox state in HF. We found that, in failing TAC WT hearts, a marked rise in reactive oxygen species (ROS) emission occurs (+93%, n=13, p

Nitroxyl (HNO), a potential heart failure therapeutic, is known to target cysteine residues to form sulfinamides and/or disulfides. Because HNO-derived modifications may depend on their local environment, we have investigated the reactivity of HNO with cysteine derivatives and C-terminal cysteine-containing peptides at physiological pH and temperature. Our findings indicate that the nature of HNO-derived modifications of C-terminal cysteines is affected by the C-terminal carboxylate. Apart from the lack of sulfinamide formation, these studies have revealed the presence of new products, a sulfohydroxamic acid derivative (RS(O)2NHOH) and a thiosulfonate (RS(O)2SR), presumably produced under our experimental conditions via the intermediacy of a cyclic structure that is hydrolyzed to give a sulfenic acid (RSOH). Moreover, these modifications are formed independent of oxygen.

It is currently not known whether caffeine has an enhancing effect on long-term memory in humans. We used post-study caffeine administration to test its effect on memory consolidation using a behavioral discrimination task. Caffeine enhanced performance 24 h after administration according to an inverted U-shaped dose-response curve; this effect was specific to consolidation and not retrieval. We conclude that caffeine enhanced consolidation of long-term memories in humans.

Aims: Nitroxyl (HNO) interacts with thiols to act as a redox-sensitive modulator of protein function. It enhances sarcoplasmic reticular Ca2+ uptake and myofilament Ca2+ sensitivity, improving cardiac contractility. This activity has led to clinical testing of HNO donors for heart failure. Here we tested whether HNO alters the inhibitory interaction between phospholamban (PLN) and the sarcoplasmic reticulum Ca2+-ATPase (SERCA2a) in a redox-dependent manner, improving Ca2+ handling in isolated myocytes/hearts. Results: Ventriculocytes, sarcoplasmic reticulum (SR) vesicles, and whole hearts were isolated from control (wildtype [WT]) or PLN knockout (pln−/−) mice. Compared to WT, pln−/− myocytes displayed enhanced resting sarcomere shortening, peak Ca2+ transient, and blunted β-adrenergic responsiveness. HNO stimulated shortening, relaxation, and Ca2+ transient in WT cardiomyocytes, and evoked positive inotropy/lusitropy in intact hearts. These changes were markedly blunted in pln−/− cells/hearts. HNO enhanced SR Ca2+ uptake in WT but not pln−/− SR-vesicles. Spectroscopic studies in insect cell microsomes expressing SERCA2a±PLN showed that HNO increased Ca2+-dependent SERCA2a conformational flexibility but only when PLN was present. In cardiomyocytes, HNO achieved this effect by stabilizing PLN in an oligomeric disulfide bond-dependent configuration, decreasing the amount of free inhibitory monomeric PLN available. Innovation: HNO-dependent redox changes in myocyte PLN oligomerization relieve PLN inhibition of SERCA2a. Conclusions: PLN plays a central role in HNO-induced enhancement of SERCA2a activity, leading to increased inotropy/lusitropy in intact myocytes and hearts. PLN remains physically associated with SERCA2a; however, less monomeric PLN is available resulting in decreased inhibition of the enzyme. These findings offer new avenues to improve Ca2+ handling in failing hearts. Antioxid. Redox Signal. 19, 1185–1197.

Although misuse and abuse of Cannabis is well known, the health benefits have been proved by various biomedical studies. Tetrahydrocannabinol (THC) is the major active substance in leaves of Cannabis, which is the common target for drug testing. In field drug testing, oral fluid (OF) has its unique advantages over other specimens such as blood, urine, and hair. Thus the study of THC in OF is gaining popularity in Cannabis research. In this review, extraction methods are introduced in three categories, which are Liquid-Liquid Extraction (LLE), Solid Phase Extraction (SPE), and Supercritical Fluid Extraction (SFE). Examples of application with each method will be covered. Advantages and disadvantages of these methods will be compared. In addition, methods in analysis following extraction will be briefly discussed.

Our enjoyable collaboration with Dr. Jeffrey P. Froehlich focused on the role of phospholamban (PLN) in HNO-induced enhancement of SR Ca2+-ATPase (SERCA2a) activity. We hypothesized that given HNO thiophilic nature it modifies cysteine residues in PLN transmembrane domain, altering its interaction with SERCA2a, thus enhancing pump activity. When HNO, donated by Angeli's salt (AS), was administered to control isolated myocytes, it enhanced both sarcomere shortening and Ca2+ transient. However, when AS/HNO was applied to PLN-/- ventriculocytes, HNO inotropy was reduced by ≅ 50% (the remaining likely stemming from enhanced myofilament sensitivity to Ca2+). PLN centrality to HNO cardiotropic action was confirmed incubating SR vesicles from WT and PLN-/- mice with AS/HNO to measure ATP-dependent Ca2+ uptake by stopped-flow mixing. In WT, HNO increased Ca2+ uptake rate, but it failed to do so in PLN-/- vesicles. The role of cysteines in PLN emerged from studies using ER microsomes from Sf21 insect cells expressing SERCA2a±PLN (WT or Cys 36-41-46→Ala mutant) where we assessed SERCA2a dephosphorylation, a measure of E2P hydrolysis, i.e. a rate-limiting step of SERCA2a activity. AS/HNO augmented SERCA2a dephosphorylation in ER microsomes co-expressing SERCA2a and WT PLN, but this stimulation was absent in microsomes expressing SERCA2a and Cys 36-41-46→Ala mutant PLN. Thus, Jeff's elegant, creative and passionate approach helped us to show that PLN is essential for HNO-induced faster Ca2+ uptake by SERCA2a, suggesting that HNO action occurs, at least partly, via modifications of critical cysteines in PLN transmembrane domain. In Jeff's view, “forbidden” disulfide bonds in PLN are involved, and one of his legacies for us is unearthing the cysteine pairs that are involved in HNO-induced formation of an intramolecular bond that could distort the conformation of PLN, thus perturbing its interaction with SERCA2a and relieving the inhibition.

Nitroxyl (HNO) donated by Angeli's salt activates uptake of Ca 2+ by the cardiac SR Ca 2+ pump (SERCA2a). To determine whether HNO achieves this by a direct interaction with SERCA2a or its regulatory protein, phospholamban (PLN), we measured its effects on SERCA2a activation (as reflected in dephosphoryla- tion) using insect cell microsomes expressing SERCA2a with or without PLN (wild-type and Cys f Ala mutant). The results show that activation of SERCA2a dephos- phorylation by HNO is PLN-dependent and that PLN thiols are targets for HNO. We conclude that HNO produces a disulfide bond that alters the conformation of PLN, relieving inhibition of the Ca 2+ pump. Stimulation of the cardiac SR Ca 2+ pump (SERCA2a) 1 in isolated SR vesicles by cAMP/protein kinase A-dependent phosphorylation of phospholamban (PLN) was originally demonstrated by Tada et al. (1). Subsequent studies showed that phosphorylation of PLN at Ser 16 increases the apparent Ca 2+ affinity of SERCA2a (2), the rates of phosphorylation and dephosphorylation (2, 3), and the Vmax of Ca 2+ transport (3, 4). Phosphorylation of Ser 16 induces a change in the conformation of PLN (5, 6), which relieves its inhibition and activates the pump. Recent work with ex- pressed Ca 2+ pump proteins suggests that activation of the cardiac SR Ca 2+ pump following the relief of PLN inhibition involves changes in the kinetic behavior of the Ca 2+ -ATPase consistent with SERCA2a oligomerization (7). These changes include stabilization of the ADP-sensitive phosphoenzyme, E1P, and allosteric activation of dephosphorylation by ATP, resulting in an increased rate of turnover of the ADP- insensitive phosphoenzyme, E2P. We have recently shown that nitroxyl (HNO) donated by Angeli's salt (AS, Na2N2O3) activates the SR Ca 2+ pump in isolated murine cardiac myocytes (8). This effect is inde- pendent of � -adrenergic activation (9) and results from a direct effect of HNO on the SR Ca 2+ pump as evidenced by stimulation of Ca 2+ uptake by HNO in isolated murine heart SR vesicles (8). We hypothesize that HNO stimulates Ca 2+ uptake by covalently modifying critical thiol residues in the SERCA2a pump and/or its regulatory protein, PLN, altering the conformation of these proteins, and relieving the inhibi- tion of the pump. Chemical modification of protein thiols by HNO can proceed by two pathways leading to either (a) the formation of a sulfinamide (RS(O)NH2) when one thiol group is involved or (b) the formation of a disulfide (RSSR) plus hydroxylamine (H2NOH) when two thiols are in the proximity of each other (10, 11). The fact that HNO-induced activation of RyR2 receptors reconstituted in lipid planar bilayers can be reversed by DTT (8) suggests that disulfide bond formation may be important in activating Ca 2+ release. In this study, we investigated the mechanism of activation of the SR Ca 2+ pump by HNO using SERCA2a expressed in the absence or presence of PLN in High Five (HF) insect cell microsomes. To study Ca 2+ pump activation by HNO, we measured the kinetics of dephosphorylation of SERCA2a, which is accelerated by HNO. To test whether thiol residues in PLN are critical for activation, we carried out these experiments in microsomes expressing SERCA2a and null-Cys PLN (PLN(-C)) in which the three transmembrane (TM) domain cysteine residues were replaced with alanine. Immunoblotting with an anti-PLN antibody was used to test whether HNO-induced PLN oligomer formation contributes to the relief of inhibition of SERCA2a by PLN. The results show that PLN is necessary for activation of SERCA2a by HNO and that cysteine residues in the TM domain of PLN play a critical role in PLN-dependent HNO activation of the Ca 2+ pump.

Nat. Neurosci. 17, 201–203; published online 12 January 2014; corrected online 17 January 2014; corrected after print 30 June 2014 In the version of this article initially published, there were errors in the reporting of statistics. In the Figure 1b legend, the asterisked P value was given in the HTML version as *P = 0.