Myocardial oxidative stress is increased in early reperfusion, but systemic antioxidative therapy does not prevent ischemia-reperfusion arrhythmias in pigs

BackgroundArrhythmias in the early phase of reperfusion after myocardial infarction (MI) are common, and can lead to hemodynamic instability or even cardiac arrest. Reactive oxygen species (ROS) are thought to play a key role in the underlying mechanisms, but evidence from large animal models is scarce, and effects of systemic antioxidative treatment remain contentious.MethodsMI was induced in 7 male and 7 female pigs (Norwegian landrace, 35–40 kg) by clamping of the left anterior descending artery (LAD) during open thorax surgery. Ischemia was maintained for 90 min, before observation for 1 h after reperfusion. Pigs were randomized 1:1 in an operator-blinded fashion to receive either i.v. N-acetylcysteine (NAC) from 70 min of ischemia and onwards, or 0.9% NaCl as a control. Blood samples and tissue biopsies were collected at baseline, 60 min of ischemia, and 5 and 60 min of reperfusion. ECG and invasive blood pressure were monitored throughout.ResultsThe protocol was completed in 11 pigs. Oxidative stress, as indicated by immunoblotting for Malondialdehyde in myocardial biopsies, was increased at 5 min of reperfusion compared to baseline, but not at 60 min of reperfusion, and not reduced with NAC. We found no significant differences in circulating biomarkers of myocardial necrosis, nor in the incidence of idioventricular rhythm (IVR), non-sustained ventricular tachycardia (NSVT), ventricular tachycardia (VT) or ventricular fibrillation (VF) between NAC-treated and control pigs during reperfusion.ConclusionMyocardial oxidation was increased early after reperfusion in a porcine model of MI, but systemic antioxidative treatment did not protect against reperfusion arrhythmias

Secretoneurin Is an Endogenous Calcium/Calmodulin-Dependent Protein Kinase II Inhibitor That Attenuates Ca2+-Dependent Arrhythmia

BACKGROUND: Circulating SN (secretoneurin) concentrations are increased in patients with myocardial dysfunction and predict poor outcome. Because SN inhibits CaMKII delta (Ca2+/calmodulin-dependent protein kinase II delta) activity, we hypothesized that upregulation of SN in patients protects against cardiomyocyte mechanisms of arrhythmia. METHODS: Circulating levels of SN and other biomarkers were assessed in patients with catecholaminergic polymorphic ventricular tachycardia (CPVT; n=8) and in resuscitated patients after ventricular arrhythmia-induced cardiac arrest (n=155). In vivo effects of SN were investigated in CPVT mice (RyR2 [ryanodine receptor 2]-R2474S) using adeno-associated virus-9-induced overexpression. Interactions between SN and CaMKII delta were mapped using pull-down experiments, mutagenesis, ELISA, and structural homology modeling. Ex vivo actions were tested in Langendorff hearts and effects on Ca2+ homeostasis examined by fluorescence (fluo-4) and patchclamp recordings in isolated cardiomyocytes. RESULTS: SN levels were elevated in patients with CPVT and following ventricular arrhythmia-induced cardiac arrest. In contrast to NT-proBNP (N-terminal proB- type natriuretic peptide) and hs-TnT (high-sensitivity troponin T), circulating SN levels declined after resuscitation, as the risk of a new arrhythmia waned. Myocardial pro-SN expression was also increased in CPVT mice, and further adeno-associated virus-9-induced overexpression of SN attenuated arrhythmic induction during stress testing with isoproterenol. Mechanistic studies mapped SN binding to the substrate binding site in the catalytic region of CaMKII delta. Accordingly, SN attenuated isoproterenol induced autophosphorylation of Thr287-CaMKII delta in Langendorff hearts and inhibited CaMKII delta-dependent RyR phosphorylation. In line with CaMKII delta and RyR inhibition, SN treatment decreased Ca2+ spark frequency and dimensions in cardiomyocytes during isoproterenol challenge, and reduced the incidence of Ca2+ waves, delayed afterdepolarizations, and spontaneous action potentials. SN treatment also lowered the incidence of early afterdepolarizations during isoproterenol; an effect paralleled by reduced magnitude of L-type Ca2+ current. CONCLUSIONS: SN production is upregulated in conditions with cardiomyocyte Ca2+ dysregulation and offers compensatory protection against cardiomyocyte mechanisms of arrhythmia, which may underlie its putative use as a biomarker in at-risk patients.Peer reviewe

Reduksjon av adrenalininhalasjoner hos hospitaliserte barn med bronkiolitt. Et kvalitetsforbedringsprosjekt ved pediatrisk avdeling, Lillehammer sykehus

Innledning og problemstilling Til tross for at det foreligger god evidens for at inneliggende barn med bronkiolitt ikke har effekt av inhalasjonsbehandling med racemisk adrenalin, er dette likevel vanlig praksis mange steder. Vi søker å skissere tiltak for å redusere adrenalinbruken i behandlingen av barn med bronkiolitt ved pediatrisk avdeling ved Sykehuset Innlandet Lillehammer. Kunnskapsgrunnlaget Ved hjelp av et PICO-søk i McMaster Plus fant vi som øverste kunnskapsnivå en UpToDate- artikkel, «Bronchiolitis in infants and children: Treatment; outcome; and prevention». Vi har brukt metaanalysene, retningslinjene og artiklene som er referert i denne artikkelen for å kartlegge evidensgrunnlaget rundt vår problemstilling nærmere. En metaanalyse i Cochrane viste at behandling av inneliggende barn med bronkiolitt med adrenalininhalasjoner verken hadde effekt på ”antall liggedøgn” eller ”clinical score”. Både amerikanske og britiske retningslinjer fraråder bruk av adrenalin i denne sammenheng. Akuttveilederen i pediatri (1) er imidlertid uklar i sine anbefalinger og åpner for at adrenalinbehandling kan forsøkes hos barn med spesifikk komorbiditet. Nyere enkeltstudier indikerer imidlertid at selv ikke utsatte grupper har nytte av adrenalininhalasjoner. Dagens praksis, tiltak og indikatorer Årlig innlegges 50-200 barn med bronkiolitt på Lillehammer. Avdelingen forholder seg til Akuttveilederen i pediatri. De ønsker å redusere bruken av adrenalin ved bronkiolitt, men det er kjent at det fremdeles brukes i ukjent omfang. Som konkrete tiltak foreslår vi internundervisning og informasjon til personalet via flere kanaler, innføring av sjekkliste i pasientmappen med opptelling og begrunnelse for ordinering av adrenalin, opptelling av antall dager siden siste adrenalinadministrasjon på oppslagstavlen, samt at adrenalin skal føres som engangsdose og ikke som "ved behov"-medisin i pasientkurven. For å vurdere kvaliteten av våre tiltak har vi valgt å bruke én struktur- og én prosessindikator, samt at vi legger opp til muligheten for også å bruke en resultatindikator. Kort oppsummert betyr dette at vi ønsker å få bekreftet at informasjon er gitt som planlagt, at alle tiltakene er iverksatt på avdelingen ved oppstart av prosjektet, at vi aktivt måler bruk av adrenalin hos barn med bronkiolitt under prosjektets varighet. Prosess, ledelse og organisering For å systematisk kunne gjennomføre kvalitetsforbedringsprosjektet har vi valgt å ta utgangspunkt i Langley and Nolans totrinnsmodell for kvalitetsforbedring (2) og vi har brukt Kunnskapssenterets modell for kvalitetsforbedring (3) som arbeidsverktøy. Vi har forsøkt å forberede og planlegge prosjektet godt og detaljert slik at utførelse, evaluering og oppfølging kan foregå på best mulig måte. Diskusjon/konklusjon Det er god evidens for endring av praksis, tiltakene er enkle og krever verken stor omlegging av rutiner eller mye ressurser. Vi mener derfor kvalitetsforbedringsprosjektet er høyst gjennomførbart og bør implementeres

The oxidation-resistant CaMKII-MM281/282VV mutation does not prevent arrhythmias in CPVT1

Catecholaminergic polymorphic ventricular tachycardia type 1 (CPVT1) is an inherited arrhythmogenic disorder caused by missense mutations in the cardiac ryanodine receptors (RyR2), that result in increased β-adrenoceptor stimulation-induced diastolic Ca2+ leak. We have previously shown that exercise training prevents arrhythmias in CPVT1, potentially by reducing the oxidation of Ca2+/calmodulin-dependent protein kinase type II (CaMKII). Therefore, we tested whether an oxidation-resistant form of CaMKII protects mice carrying the CPVT1-causative mutation RyR2-R2474S (RyR2-RS) against arrhythmias. Antioxidant treatment (N-acetyl-L-cysteine) reduced the frequency of β-adrenoceptor stimulation-induced arrhythmogenic Ca2+ waves in isolated cardiomyocytes from RyR2-RS mice. To test whether the prevention of CaMKII oxidation exerts an antiarrhythmic effect, mice expressing the oxidation-resistant CaMKII-MM281/282VV variant (MMVV) were crossed with RyR2-RS mice to create a double transgenic model (RyR2-RS/MMVV). Wild-type mice served as controls. Telemetric ECG surveillance revealed an increased incidence of ventricular tachycardia and an increased arrhythmia score in both RyR2-RS and RyR2-RS/MMVV compared to wild-type mice, both following a β-adrenoceptor challenge (isoprenaline i.p.), and following treadmill exercise combined with a β-adrenoceptor challenge. There were no differences in the incidence of arrhythmias between RyR2-RS and RyR2-RS/MMVV mice. Furthermore, no differences were observed in β-adrenoceptor stimulation-induced Ca2+ waves in RyR2-RS/MMVV compared to RyR2-RS. In conclusion, antioxidant treatment reduces β-adrenoceptor stimulation-induced Ca2+ waves in RyR2-RS cardiomyocytes. However, oxidation-resistant CaMKII-MM281/282VV does not protect RyR2-RS mice from β-adrenoceptor stimulation-induced Ca2+ waves or arrhythmias. Hence, alternative oxidation-sensitive targets need to be considered to explain the beneficial effect of antioxidant treatment on Ca2+ waves in cardiomyocytes from RyR2-RS mice

Datasheet1_Myocardial oxidative stress is increased in early reperfusion, but systemic antioxidative therapy does not prevent ischemia-reperfusion arrhythmias in pigs.docx

BackgroundArrhythmias in the early phase of reperfusion after myocardial infarction (MI) are common, and can lead to hemodynamic instability or even cardiac arrest. Reactive oxygen species (ROS) are thought to play a key role in the underlying mechanisms, but evidence from large animal models is scarce, and effects of systemic antioxidative treatment remain contentious.MethodsMI was induced in 7 male and 7 female pigs (Norwegian landrace, 35–40 kg) by clamping of the left anterior descending artery (LAD) during open thorax surgery. Ischemia was maintained for 90 min, before observation for 1 h after reperfusion. Pigs were randomized 1:1 in an operator-blinded fashion to receive either i.v. N-acetylcysteine (NAC) from 70 min of ischemia and onwards, or 0.9% NaCl as a control. Blood samples and tissue biopsies were collected at baseline, 60 min of ischemia, and 5 and 60 min of reperfusion. ECG and invasive blood pressure were monitored throughout.ResultsThe protocol was completed in 11 pigs. Oxidative stress, as indicated by immunoblotting for Malondialdehyde in myocardial biopsies, was increased at 5 min of reperfusion compared to baseline, but not at 60 min of reperfusion, and not reduced with NAC. We found no significant differences in circulating biomarkers of myocardial necrosis, nor in the incidence of idioventricular rhythm (IVR), non-sustained ventricular tachycardia (NSVT), ventricular tachycardia (VT) or ventricular fibrillation (VF) between NAC-treated and control pigs during reperfusion.ConclusionMyocardial oxidation was increased early after reperfusion in a porcine model of MI, but systemic antioxidative treatment did not protect against reperfusion arrhythmias.</p

Beta-Adrenoceptor Stimulation Reveals Ca2+ Waves and Sarcoplasmic Reticulum Ca2+ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure.

Abnormal cellular Ca2+ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca2+ transient amplitude due to decreased sarcoplasmic reticulum Ca2+ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca2+ release events which occur when sarcoplasmic reticulum Ca2+ content exceeds a certain threshold level. Such Ca2+ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca2+ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca2+ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca2+ transients and Ca2+ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca2+ content, decreased Ca2+ transient amplitude, and increased frequency of Ca2+ waves. These results are in line with a decreased threshold for diastolic Ca2+ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca2+ release were increased THR286 phosphorylation of Ca2+/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca2+ content and increased propensity for diastolic Ca2+ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca2+ handling after a myocardial infarction

Immunoblotting.

<p>Immunoblot analysis of key Ca²⁺ handling proteins and phosphorylation was performed on tissue from left ventricles. SERCA2A abundance (A). PLB abundance (B) and phosphorylation on SER16 (C) and THR17 (D). NCX abundance (E). RyR abundance (F) with phosphorylation on SER2808 (G) and SER2814 (H). CaMKII abundance (I) and CaMKII phosphorylation on THR286 (J). PP1 (K) and PP2A (L) abundance. n_heart = 6 for all analysis. *p<0.05 (T-test).</p

Echocardiographic measurements.

<p>Representative echocardiographic parasternal long axis (A) and m-mode (B) images. LVDd, left ventricular diameter in diastole, LADd, left atrial diameter in diastole, PWd, posterior wall thickness in diastole.</p

Beta-Adrenoceptor Stimulation Reveals Ca²⁺ Waves and Sarcoplasmic Reticulum Ca²⁺ Depletion in Left Ventricular Cardiomyocytes from Post-Infarction Rats with and without Heart Failure

<div><p>Abnormal cellular Ca²⁺ handling contributes to both contractile dysfunction and arrhythmias in heart failure. Reduced Ca²⁺ transient amplitude due to decreased sarcoplasmic reticulum Ca²⁺ content is a common finding in heart failure models. However, heart failure models also show increased propensity for diastolic Ca²⁺ release events which occur when sarcoplasmic reticulum Ca²⁺ content exceeds a certain threshold level. Such Ca²⁺ release events can initiate arrhythmias. In this study we aimed to investigate if both of these aspects of altered Ca²⁺ homeostasis could be found in left ventricular cardiomyocytes from rats with different states of cardiac function six weeks after myocardial infarction when compared to sham-operated controls. Video edge-detection, whole-cell Ca²⁺ imaging and confocal line-scan imaging were used to investigate cardiomyocyte contractile properties, Ca²⁺ transients and Ca²⁺ waves. In baseline conditions, i.e. without beta-adrenoceptor stimulation, cardiomyocytes from rats with large myocardial infarction, but without heart failure, did not differ from sham-operated animals in any of these aspects of cellular function. However, when exposed to beta-adrenoceptor stimulation, cardiomyocytes from both non-failing and failing rat hearts showed decreased sarcoplasmic reticulum Ca²⁺ content, decreased Ca²⁺ transient amplitude, and increased frequency of Ca²⁺ waves. These results are in line with a decreased threshold for diastolic Ca²⁺ release established by other studies. In the present study, factors that might contribute to a lower threshold for diastolic Ca²⁺ release were increased THR286 phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II and increased protein phosphatase 1 abundance. In conclusion, this study demonstrates both decreased sarcoplasmic reticulum Ca²⁺ content and increased propensity for diastolic Ca²⁺ release events in ventricular cardiomyocytes from rats with heart failure after myocardial infarction, and that these phenomena are also found in rats with large myocardial infarctions without heart failure development. Importantly, beta-adrenoceptor stimulation is necessary to reveal these perturbations in Ca²⁺ handling after a myocardial infarction.</p></div