Die Bedeutung der Proteinkinase A und der Ca2+/Calmodulin abhängigen Proteinkinase II für das Sarkoplasmatische Retikulum Kalziumleck in der humanen Hypertrophie und Herzinsuffizienz

Sarcoplasmic reticulum (SR) Ca2+ leak through ryanodine receptor type 2 (RyR2) hyperphosphorylation is a key pathomechanism in human heart failure (HF). The resulting increased spontaneous diastolic calcium release promotes arrhythmic events and compromises cardiac contractility. Protein kinase A (PKA) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) have been identified as potential mediators of RyR2 dysregulation, but their respective contribution to the development of HF has been controversially discussed. Sossalla et al. (Circulation 2010) demonstrated that inhibition of CaMKII in human HF reduces SR Ca2+ leak and improves contractility. The present work is the first to comprehensively investigate the role of both kinases in the transition from human afterload-induced hypertrophy to HF. Analyses of RyR2 expression and phosphorylation demonstrated increased RyR2 expression in compensated afterload-induced hypertrophy compared to healthy donor hearts. Relative RyR2 phosphorylation was not significantly altered at the specific phosphorylation sites of either PKA or CaMKII. In HF, however, there was a pronounced CaMKII-dependent hyperphosphorylation of RyR2 at an unchanged RyR2 expression, whereas PKA-dependent regulation was not detectable. This finding was independent from previous β-blocker treatment. In addition, Ca2+ cycling was investigated by epifluorescence and confocal laser scanning microscopy in isolated cardiomyocytes from both pathologies. In HF, the SR Ca2+ leak was nearly doubled in comparison to compensated cardiac hypertrophy. This was associated with a significant reduction of systolic Ca2+ release and SR Ca2+ load. Accordingly, in cardiac hypertrophy inhibitors of both kinases reduced the SR Ca2+ leak. In HF, CaMKII inhibition but not inhibition of PKA yielded a reduction of the SR Ca2+ leak. Moreover, PKA inhibition further reduced SR Ca2+ load and systolic Ca2+ transients. In conclusion, these results show that in compensated hypertrophy both CaMKII and PKA functionally regulate RyR2 and may induce SR Ca2+ leak. In the transition from hypertrophy to HF, the diastolic Ca2+ leak increases and disturbed Ca2+ cycling occurs. This is associated with an increase in CaMKII- but not PKA-dependent RyR2 phosphorylation. Inhibition of CaMKII may thus represent a promising therapeutic approach for the treatment of arrhythmias and contractile dysfunction in human HF.2022-09-2

Utvärdering och optimering av filtreringstekniker i reningsverk för att separera skadliga nanopartiklar

Silvernanopartiklar används i en ökande utsträckning, främst på grund av deras anti-bakteriella egenskaper, vilket utnyttjas i produkter som kläder och beläggningar för att till exempel motverka svettlukt. Framtida scenarier för emissioner av silvernanopartiklar har pekat på risker för vattenlevande organismer efter det att silverpartiklarna släppts ut i avloppsvatten och passerat genom vattenreningsverk och därefter spridits till vattenrecipienter. Därför är det viktigt att förstå hur partiklarnas egenskaper förändras i en kommunal vattenreningsprocess och hur dessa partiklar binds till organiskt material och andra partiklar i avloppsvattnet. Detta ger information huruvida silvernanopartiklarna hamnar i slammet eller i det utgående vattnet samt i vilken omfattning. Denna kunskap kan sedan användas för att kunna uppskatta risker med eventuella ökade halter silvernanopartiklar i dessa strömmar. En annan viktig aspekt är design av filtreringstekniker för nanopartiklar. I detta projekt behandlas dessa frågeställningar genom experiment i ett pilotvatten-reningsverk som är en kopia av ett standardiserat kommunalt avloppsvattenreningsverk (ARV). Det nya i denna studie är att undersökningarna genomförts under helt realistiska förhållanden i en komplett vattenreningsprocess, vilken inkluderar samtliga reningssteg. Dessutom följs växelverkan av silverpartiklar/silverkomplex genom alla steg i reningen, även i den efterföljande rötningen. Resultaten i denna rapport visar att silver i form av avsiktligt tillsatta nanopartiklar i det inkommande spillvattnet till reningsverket (Stockholm innerstads avloppsvatten) till en betydande del (80-100 procent) hamnar i slammet vid en vattenreningsprocess. Silvernanopartiklarnas beteende är likartat med silverjoner i detta avseende. Mikroskopimätningar visar att det är rimligt att anta att silvernanopartiklarna till viss del fastnar på material i slammet. Liknande resultat erhölls för både tillsatta silvernanopartiklar och tillsatta silverjoner, med den lilla skillnaden att silverjonerna sedimenterade något snabbare än silvernanopartiklarna. Vid rötning av slam innehållande silvernanopartiklar/silverjoner, följt av centrifugering för avvattning, visar det sig att majoriteten av silvret (>95 procent) fortfarande är bundet till slammet. Efter att silvernanopartiklar passerat vattenreningsverket uppför de sig alltså inte som singulära nanopartiklar dispergerade i lösning. De adsorberas till slammet där de troligtvis löses upp till stor del och transformeras till silversulfid. Resultaten visar att fokus bör ligga på spridningen av silver i slam och de kemiska förekomstformerna för vidare studier av de risker som en ökad användning av silver i konsumentprodukter, medför

Interactions of Calcium Fluctuations during Cardiomyocyte Contraction with Real-Time cAMP Dynamics Detected by FRET.

Calcium (Ca2+) and 3',5'-cyclic adenosine monophosphate (cAMP) play a critical role for cardiac excitation-contraction-coupling. Both second messengers are known to interact with each other, for example via Ca2+-dependent modulation of phosphodiesterase 1 (PDE1) and adenylyl cyclase 5/6 (AC 5/6) activities, which is supposed to occur especially at the local level in distinct subcellular microdomains. Currently, many studies analyze global and local cAMP signaling and its regulation in resting cardiomyocytes devoid of electrical stimulation. For example, Förster resonance energy transfer (FRET) microscopy is a popular approach for visualization of real time cAMP dynamics performed in resting cardiomyocytes to avoid potential contractility-related movement artifacts. However, it is unknown whether such data are comparable with the cell behavior under more physiologically relevant conditions during contraction. Here, we directly compare the cAMP-FRET responses to AC stimulation and PDE inhibition in resting vs. paced adult mouse ventricular cardiomyocytes for both cytosolic and subsarcolemmal microdomains. Interestingly, no significant differences in cAMP dynamics could be detected after β-adrenergic (isoproterenol) stimulation, suggesting low impact of rapidly changing contractile Ca2+ concentrations on cytosolic cAMP levels associated with AC activation. However, the contribution of the calcium-dependent PDE1, but not of the Ca2+-insensitive PDE4, to the regulation of cAMP levels after forskolin stimulation was significantly increased. This increase could be mimicked by pretreatment of resting cells with Ca2+ elevating agents. Ca2+ imaging demonstrated significantly higher amplitudes of Ca2+ transients in forskolin than in isoproterenol stimulated cells, suggesting that forskolin stimulation might lead to stronger activation of PDE1. In conclusion, changes in intracellular Ca2+ during cardiomyocyte contraction dynamically interact with cAMP levels, especially after strong AC stimulation. The use of resting cells for FRET-based measurements of cAMP can be justified under β-adrenergic stimulation, while the reliable analysis of PDE1 effects may require electric field stimulation

Sacubitrilat reduces pro‐arrhythmogenic sarcoplasmic reticulum Ca2+^{2+} leak in human ventricular cardiomyocytes of patients with end‐stage heart failure

Aims Inhibition of neprilysin and angiotensin II receptor by sacubitril/valsartan (Val) (LCZ696) reduces mortality in heart failure (HF) patients compared with sole inhibition of renin–angiotensin system. Beneficial effects of increased natriuretic peptide levels upon neprilysin inhibition have been proposed, whereas direct effects of sacubitrilat (Sac) (LBQ657) on myocardial Ca$^{2+}$ cycling remain elusive. Methods and results Confocal microscopy (Fluo‐4 AM) was used to investigate pro‐arrhythmogenic sarcoplasmic reticulum (SR) Ca$^{2+}$ leak in freshly isolated murine and human ventricular cardiomyocytes (CMs) upon Sac (40 μmol/L)/Val (13 μmol/L) treatment. The concentrations of Sac and Val equalled plasma concentrations of LCZ696 treatment used in PARADIGM‐HF trial. Epifluorescence microscopy measurements (Fura‐2 AM) were performed to investigate effects on systolic Ca$^{2+}$ release, SR Ca$^{2+}$ load, and Ca$^{2+}$‐transient kinetics in freshly isolated murine ventricular CMs. The impact of Sac on myocardial contractility was evaluated using in toto‐isolated, isometrically twitching ventricular trabeculae from human hearts with end‐stage HF. Under basal conditions, the combination of Sac/Val did not influence diastolic Ca$^{2+}$‐spark frequency (CaSpF) nor pro‐arrhythmogenic SR Ca$^{2}$ leak in isolated murine ventricular CMs (n CMs/hearts = 80/7 vs. 100/7, P = 0.91/0.99). In contrast, Sac/Val treatment reduced CaSpF by 35 ± 9% and SR Ca$^{2+}$ leak by 45 ± 9% in CMs put under catecholaminergic stress (isoproterenol 30 nmol/L, n = 81/7 vs. 62/7, P < 0.001 each). This could be attributed to Sac, as sole Sac treatment also reduced both parameters by similar degrees (reduction of CaSpF by 57 ± 7% and SR Ca2+ leak by 76 ± 5%; n = 101/4 vs. 108/4, P < 0.01 each), whereas sole Val treatment did not. Systolic Ca2+ release, SR Ca$^{2+}$ load, and Ca$^{2+}$‐transient kinetics including SERCA activity (k$_{SERCA}$) were not compromised by Sac in isolated murine CMs (n = 41/6 vs. 39/6). Importantly, the combination of Sac/Val and Sac alone also reduced diastolic CaSpF and SR Ca$^{2+}$ leak (reduction by 74 ± 7%) in human left ventricular CMs from patients with end‐stage HF (n = 71/8 vs. 78/8, P < 0.05 each). Myocardial contractility of human ventricular trabeculae was not acutely affected by Sac treatment as the developed force remained unchanged over a time course of 30 min (n trabeculae/hearts = 3/3 vs. 4/3). Conclusion This study demonstrates that neprilysin inhibitor Sac directly improves Ca$^{2+}$ homeostasis in human end‐stage HF by reducing pro‐arrhythmogenic SR Ca$^{2+}$ leak without acutely affecting systolic Ca$^{2+}$ release and inotropy. These effects might contribute to the mortality benefits observed in the PARADIGM‐HF trial

cAMP dynamics in adult mouse ventricular Epac1-camps expressing cardiomyocytes upon treatment with cAMP elevating agents and PDE4 inhibition.

<p><b>(A)</b> Representative calcium traces in Fura2-AM loaded Epac1-camps transgenic cardiomyocytes under resting conditions (left) and upon electric field stimulation at 1 Hz (right). <b>(B)</b> Non-normalized FRET ratios do not differ between resting and paced Epac1-camps cardiomyocytes under basal and stimulated conditions (isoproterenol—ISO, 100 nM—plus 3-isobutyl-1-methylxathin—IBMX, 100 μM). <b>(C)</b> Representative FRET traces from Epac1-camps cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) or <b>(E)</b> with the direct AC activator forskolin (10 μM) leading to an increase of cAMP visualized as a decrease in the FRET ratio. Inhibition of PDE4 by rolipram (Roli, 10 μM) strongly enhances this effect, whereas the unselective PDE inhibitor IBMX (100 μM) has only little additional effect. <b>(D and F)</b> Quantification of the FRET results reveal no significant differences in FRET ratio changes between resting and paced cardiomyocytes. Cells were paced at 1 Hz. Values are means ± SEM; from n = 6 cells isolated from 3 hearts per condition; n.s.—not significant by one-way ANOVA.</p

cAMP dynamics in adult mouse cardiomyocytes upon treatment with cAMP elevating agents and PDE1 inhibition.

<p><b>(A)</b> Representative FRET traces of Epac1-camps cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) or with the direct AC activator forskolin (10 μM) <b>(C)</b>. Subsequent application of the PDE1 inhibitor 8-methoxymethyl-3-isobutyl-1-methylxanthine (8-MMX, 30 μM) enhances the cAMP stimulatory effect of ISO and forskolin. Stimulation with the unselective PDE inhibitor 3-isobutyl-1-methylxanthin (IBMX, 100 μM) leads to a further increase of cAMP. <b>(B and D)</b> Quantification of experiments shows no significant difference in FRET responses between control and paced cardiomyocytes stimulated with ISO. Forskolin stimulated cardiomyocytes show significant differences in PDE1 contribution to total PDE inhibition which is significantly higher in paced cardiomyocytes as compared to resting cells. Pretreatment of resting cardiomyocytes with calcium elevating reagents such as thapsigargin (100 nM) and calcium ionophore A23187 (10 μM) mimics the effect of field stimulation. Cells were paced at 1 Hz. Values are means ± SEM; n = 6–10 cells isolated from 3 hearts per condition; *—significant difference at p<0.05 by one-way ANOVA; n.s.- not significant.</p

cAMP dynamics in adult mouse cardiomyocytes upon preincubation with IBMX and after low concentration of ISO.

<p><b>(A)</b> Representative FRET traces of Epac1-camps cardiomyocytes preincubated with 3-isobutyl-1-methylxanthin (IBMX, 100 μM) and then stimulated with non-saturating concentrations of the β-AR agonist isoproterenol (ISO, 1nM). The AC activator forskolin (10 μM) was used to reach the maximal FRET response. <b>(B)</b> Representative control traces (n = 4 and 8 for unpaced and paced, respectively) showing FRET response to IBMX response over the whole time-course involved in these experiments. Representative FRET responses (n = 7 each) to 1 nM ISO applied along (without IBMX prestimulation), followed by forskolin plus IBMX <b>(D)</b> Quantification of experiments from A and C shows no significant difference in FRET responses between control and paced cardiomyocytes stimulated with IBMX and ISO. Cells were paced at 1 Hz. Values are means ± SEM, n = 8 cells for each A graph and n = 7 cells for each C graph isolated from 3 hearts per condition; n.s.—not significant by one-way ANOVA.</p

Subsarcolemmal cAMP dynamics in adult mouse ventricular cardiomyocytes transgenically expressing pmEpac1 biosensor.

<p><b>(A)</b> Representative FRET traces from pmEpac1 cardiomyocytes stimulated with the β-AR agonist isoproterenol (ISO, 100 nM) and subsequently by the PDE4 inhibitor rolipram (Roli, 10 μM) followed by the unselective PDE inhibitor IBMX (100 μM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167974#pone.0167974.g001" target="_blank">Fig 1C and 1D</a>. <b>(C)</b> Representative FRET traces from pmEpac1 cardiomyocytes stimulated with ISO (100 nM) and subsequently by the PDE1 inhibitor 8-MMX (30 μM) followed by the unselective PDE inhibitor IBMX (100 μM) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0167974#pone.0167974.g003" target="_blank">Fig 3A and 3B</a>. <b>(B and D)</b> Quantification of the FRET results reveal no significant differences in FRET ratio changes between resting and paced cardiomyocytes treated with ISO, rolipram or 8-MMX. Cells were paced at 1 Hz. Values are means ± SEM; from n = 12 and n = 11 cells (unpaced and paced, respectively) isolated from 3 hearts per condition in B and n = 9 cell from 2 hearts each in D; n.s.—not significant by one-way ANOVA.</p

Ca²⁺ transient amplitudes in ISO and forskolin treated cardiomyocytes.

<p>Cells were loaded with Fura2-AM, paced at 1 Hz and treated with 100 nM ISO or 10 μM forskolin with subsequent applications of PDE inhibitors rolipram 10 μM, 8-MMX 30 μM and IBMX 100 μM. Shown are baseline Ca²⁺ amplitudes (black bars) and systolic Ca²⁺ transient amplitudes (grey bars) measured in paced <b>(A)</b> and resting <b>(B)</b> cells. Means ± SEM, *—p<0.05; **—p<0.01; ***—p<0.001 with ANOVA (compared to control, second value after / as compared to previous stimulation) followed by the Gasser-Greenhouse correction. n = 7 for ISO and n = 9 for forskolin cells in A, and n = 4 and 5 for ISO and forskolin cells in B, respectively (all isolated from at least 3 mice for each condition). Effects of ISO and forskolin in A are significantly different (p = 0.03 by one-way ANOVA).</p