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Background In the DAPA-HF trial, SGLT inhibition reduces cardiovascular mortality in heart failure. However, the mechanism and a potential positive effect in HfpEF remain elusive. Introduction LA remodeling is a hallmark feature of HFpEF and commonly associated with LA enlargement and dysfunction. Previous studies of SGLT-2 inhibitor Empagliflozin suggest a utilization of alternative metabolites for energy consumption (i.e. ketone bodies). Additionally, alterations of sodium and calcium ion hemostasis have been reported. We investigated the effect of SGLT inhibition on mitochondrial (dys)function during atrial remodeling in HFpEF. Methods Rats (WT: Wistar Kyoto, HFpEF: ZFS-1 Obese (metabolic syndrome)) were obtained at ∼10w and fed Purina 5008 diet. At 17w, animals were randomized to treatment with either vehicle or Sota (30mg/kg/d) for 5w until primary adult cardiomyocytes were isolated for final experiments. Structural information of mitochondria was obtained with Mitotracker Red in either a glucose starved (1h incubation with mannitol) or saturated state. ROS production was assessed with H2-DCF in a starved and saturated condition. Mitochondrial calcium buffer capacity was imaged with Rhod-2 following perforation of the cellular membrane with saponin. Glycolytic dependency of calcium cycling was assessed upon glycolytic inhibition with 2-deoxyglucose during imaging of cytosolic calcium transients with Fura-2. Results In a glucose saturated state, LA cardiomyocytes in HFpEF showed increased mitochondrial density, which was ameliorated with Sota. Sota increased mitochondrial calcium buffer capacity in HFpEF, indicating a decrease in mitochondrial resting calcium. Differences in mitochondrial fission could not be detected. However, during glucose starvation cardiomyocytes showed a decrease in mitochondrial fission and ROS production with Sota. A difference in ROS production was not visible when cells were abruptly challenged with high glucose concentrations, but Sota decreased mitochondrial fission, indicating long term protective properties towards ROS. Glycolytic inhibition led to an increase of cytosolic diastolic calcium and calcium transient peak height in HFpEF vs. WT, indicating an increased glucose dependency of cytosolic calcium cycling, which was mitigated with Sota. Additionally, Sota negated an increase in diastolic calcium, when cardiomyocytes where challenged with high concentrations of glucose after starvation. Conclusion SGLT1/2 inhibition alters mitochondrial calcium uptake in HFpEF and positively affects mitochondrial structure with subsequent decreases of ROS production and enhanced calcium homeostasis. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Else-Kröner-Fresenius-Stiftung, Deutsches Zentrum für Herz-Kreislaufforschung

Heart failure (HF) manifestation and progression are driven by systemic activation of neuroendocrine signaling cascades, such as the renin–angiotensin aldosterone system (RAAS). Fibroblast growth factor 23 (FGF23), an endocrine hormone, is linked to HF and cardiovascular mortality. It is also a mediator of left-ventricular hypertrophy (LVH). In vivo, high circulating levels of FGF23 are associated with an altered systemic RAAS response. FGF23 is proposed to trigger pathological signaling mediated by Ca2+-regulated transcriptional pathways. In the present study, we investigated Ca2+-dependent signaling of FGF23 in ventricular cardiomyocytes and its association with angiotensin II (ATII). In neonatal rat ventricular myocytes (NRVMs), both ATII and FGF23 induced hypertrophy as observed by an increase in cell area and hypertrophic gene expression. Furthermore, FGF23 activates nuclear Ca2+-regulated CaMKII–HDAC4 pathway, similar to ATII. In addition to a global increase in cytoplasmic Ca2+, FGF23, like ATII, induced inositol 1, 4, 5-triphosphate (IP3)-induced Ca2+ release from the nucleoplasmic Ca2+ store, associated with cellular hypertrophy. Interestingly, ATII receptor antagonist, losartan, significantly attenuated FGF23-induced changes in Ca2+ homeostasis and cellular hypertrophy suggesting an involvement of ATII receptor-mediated signaling. In addition, application of FGF23 increased intracellular expression of ATII peptide and its secretion in NRVMs, confirming the participation of ATII. In conclusion, FGF23 and ATII share a common mechanism of IP3-nuclear Ca2+-dependent cardiomyocyte hypertrophy. FGF23-mediated cellular hypertrophy is associated with increased production and secretion of ATII by cardiomyocytes. These findings indicate a pathophysiological role of the cellular angiotensin system in FGF23-induced hypertrophy in ventricular cardiomyocytes.

Calcium influx through the voltage-gated L-type calcium channels (LTCC) mediates a wide range of physiological processes from contraction to secretion. Despite extensive research on regulation of LTCC conductance by PKA phosphorylation in response to β-adrenergic stimulation, the science remains incomplete. Here, we show that Wnt11, a non-canonical Wnt ligand, through its G protein-coupled receptor (GPCR) Fzd7 attenuates the LTCC conductance by preventing the proteolytic processing of its C terminus. This is mediated across species by protein kinase A (PKA), which is compartmentalized by A-kinase anchoring proteins (AKAP). Systematic analysis of all AKAP family members revealed AKAP2 anchoring of PKA is central to the Wnt11-dependent regulation of the channel. The identified Wnt11/AKAP2/PKA signalosome is required for heart development, controlling the intercellular electrical coupling in the developing zebrafish heart. Altogether, our data revealed Wnt11/Fzd7 signaling via AKAP2/PKA as a conserved alternative GPCR system regulating Ca2+homeostasis.

Heart failure (HF) manifestation and progression are driven by systemic activation of neuroendocrine signaling cascades, such as the renin-angiotensin aldosterone system (RAAS). Fibroblast growth factor 23 (FGF23), an endocrine hormone, is linked to HF and cardiovascular mortality. It is also a mediator of left-ventricular hypertrophy (LVH). In vivo, high circulating levels of FGF23 are associated with an altered systemic RAAS response. FGF23 is proposed to trigger pathological signaling mediated by Ca

Glucagon-like peptide-1 receptor (GLP-1R) agonists are a rapidly growing class of drugs developed for treating type-2 diabetes mellitus. Patients with diabetes carry an up to 5-fold greater mortality risk compared to non-diabetic patients, mainly as a result of cardiovascular diseases. Although beneficial cardiovascular effects have been reported, exact mechanisms of GLP-1R-agonist action in the heart, especially in human myocardium, are poorly understood. The effects of GLP-1R-agonists (exenatide, GLP-1(7-36)NH2, PF-06446009, PF-06446667) on cardiac contractility were tested in non-failing atrial and ventricular trabeculae from 72 patients. The GLP-1(7-36)NH2 metabolite, GLP-1(9-36)NH2, was also examined. In electrically stimulated trabeculae, the effects of compounds on isometric force were measured in the absence and presence of pharmacological inhibitors of signal transduction pathways. The role of β-arrestin signaling was examined using a β-arrestin partial agonist, PF-06446667. Expression levels were tested by immunoblots. Translocation of GLP-1R downstream molecular targets, Epac2, GLUT-1 and GLUT-4, were assessed by fluorescence microscopy. All tested GLP-1R-agonists significantly increased developed force in human atrial trabeculae, whereas GLP-1(9-36)NH2 had no effect. Exendin(9-39)NH2, a GLP-1R-antagonist, and H-89 blunted the inotropic effect of exenatide. In addition, exenatide increased PKA-dependent phosphorylation of phospholamban (PLB), GLUT-1 and Epac2 translocation, but not GLUT-4 translocation. Exenatide failed to enhance contractility in ventricular myocardium. Quantitative real-time PCR (qRT-PCR) revealed a significant higher GLP-1R expression in the atrium compared to ventricle. Exenatide increased contractility in a dose-dependent manner via GLP-1R/cAMP/PKA pathway and induced GLUT-1 and Epac2 translocation in human atrial myocardium, but had no effect in ventricular myocardium. Therapeutic use of GLP-1R-agonists may therefore impart beneficial effects on myocardial function and remodelling.

Heart failure (HF) with preserved ejection fraction (HFpEF) is present in about 50% of HF patients. Atrial remodeling is common in HFpEF and associated with increased mortality. We postulate that atrial remodeling is associated with atrial dysfunction in vivo related to alterations in cardiomyocyte Calcium (Ca) signaling and remodeling. We examined atrial function in vivo and Ca transients (CaT) (Fluo4-AM, field stim) in atrial cardiomyocytes of ZSF-1 rats without (Ln; lean hypertensive) and with metabolic syndrome (Ob; obese, hypertensive, diabetic) and HFpEF. Results At 21 weeks Ln showed an increased left ventricular (LV) mass and left ventricular end-diastolic pressure (LVEDP), but unchanged left atrial (LA) size and preserved atrial ejection fraction vs. wild-type (WT). CaT amplitude in atrial cardiomyocytes was increased in Ln (2.9 ± 0.2 vs. 2.3 ± 0.2 F/F0 in WT; n = 22 cells/group; p Summary In hypertensive ZSF-1 rats, a possibly compensatory increase of cytosolic CaT amplitude and increased SR Ca leak precede atrial remodeling and HFpEF. Atrial remodeling in ZSF-1 HFpEF is associated with an altered tubular network in-vitro and atrial contractile dysfunction in vivo, indicating insufficient compensation. Atrial cardiomyocyte dysfunction in vitro is induced by the addition of angiotensin II.

Rationale : Synchronized release of Ca 2+ into the cytosol during each cardiac cycle determines cardiomyocyte contraction. Objective: We investigated synchrony of cytosolic [Ca 2+ ] decay during diastole and the impact of cardiac remodeling. Methods and Results: Local cytosolic [Ca 2+ ] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca 2+ ] decay based on the local time constants of decay (TAU local ). The SD of TAU local as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca 2+ release. Stimulation of sarcoplasmic reticulum Ca 2+ ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAU local significantly more in slowCaR, thus altering the relationship between SD of TAU local and global [Ca 2+ ] decay (TAU global ). Na + /Ca 2+ exchanger inhibitor SEA0400 prolonged TAU local similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca 2+ uniporter blocker Ru360. Variation in TAU local was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAU local correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAU local was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density. Conclusions: In cardiomyocytes, cytosolic [Ca 2+ ] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca 2+ ] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

Objectives Heart failure with preserved ejection fraction (HFpEF) is increasingly common but the underlying cellular mechanisms are not well understood. We investigated cardiomyocyte function and the role of SEA0400, a Na+/Ca2+ exchanger (NCX) inhibitor in a rat model of chronic kidney disease (CKD) with HFpEF. Methods and results Male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (Sham). After 8 weeks and 24 weeks, in vivo (haemodynamics, echocardiography) and in vitro function [left ventricular (LV) cardiomyocyte cell shortening (CS) and Ca2+ transients (CaT)] were determined without and with SEA0400. In a subgroup of rats SEA0400 or vehicle was given p.o. (1 mg/kg body weight) between weeks 8 and 24. NXT resulted in stable compensated CKD and HFpEF [hypertrophied LV, prolonged LV isovolumetric relaxation constant TAU (IVRc TAU), left and upward shift of end-diastolic pressure (EDP) volume relationship, increased lung weight (pulmonary congestion) and preserved LV systolic function (EF, dP/dt)]. In NXT cardiomyocytes amplitude of CS and CaT were unchanged but relaxation and CaT decay were progressively prolonged at 8 weeks and 24 weeks vs. Sham, individually correlating with diastolic dysfunction in vivo. NCX forward mode activity (caffeine response) was progressively reduced, while NCX protein expression was upregulated, suggesting increased NCX reverse mode activity in NXT. SEA0400 acutely improved relaxation in NXT in vivo and in cardiomyocytes, and improved cardiac remodelling and diastolic function when given chronically. Conclusions This model of renal HFpEF is associated with slowed relaxation of LV cardiomyocytes. Treatment with SEA0400 improved cardiomyocyte function, remodelling, and HFpEF.

BACKGROUND AND PURPOSE Ca2+ leak from the sarcoplasmic reticulum (SR) via ryanodine receptors (RyR2s) contributes to cardiomyocyte dysfunction. RyR2 Ca2+ leak has been related to RyR2 phosphorylation. In these conditions, JTV519 (K201), a 1,4-benzothiazepine derivative and multi-channel blocker, stabilizes RyR2s and decrease SR Ca2+ leak. We investigated whether JTV519 stabilizes RyR2s without increasing RyR2 phosphorylation in mice and in non-failing human myocardium and explored underlying mechanisms. EXPERIMENTAL APPROACH SR Ca2+ leak was induced by ouabain in murine cardiomyocytes. [Ca2+]-transients, SR Ca2+ load and RyR2-mediated Ca2+ leak (sparks/waves) were quantified, with or without JTV519 (1 µmol·L−1). Contribution of Ca2+-/calmodulin-dependent kinase II (CaMKII) was assessed by KN-93 and Western blot (RyR2-Ser2814 phosphorylation). Effects of JTV519 on contractile force were investigated in non-failing human ventricular trabeculae. KEY RESULTS Ouabain increased systolic and diastolic cytosolic [Ca2+]i, SR [Ca2+], and SR Ca2+ leak (Ca2+ spark (SparkF) and Ca2+ wave frequency), independently of CaMKII and RyR-Ser2814 phosphorylation. JTV519 decreased SparkF but also SR Ca2+ load. At matched SR [Ca2+], Ca2+ leak was significantly reduced by JTV519, but it had no effect on fractional Ca2+ release or Ca2+ wave propagation velocity. In human muscle, JTV519 was negatively inotropic at baseline but significantly enhanced ouabain-induced force and reduced its deleterious effects on diastolic function. CONCLUSIONS AND IMPLICATIONS JTV519 was effective in reducing SR Ca2+ leak by specifically regulating RyR2 opening at diastolic [Ca2+]i in the absence of increased RyR2 phosphorylation at Ser2814, extending the potential use of JTV519 to conditions of acute cellular Ca2+ overload.

Insulin-like growth factor 1 (IGF1) promotes a physiological type of cardiac hypertrophy and has therapeutic effects in heart disease. Here, we report the relationship of IGF1 to GATA4, an essential transcription factor in cardiac hypertrophy and cell survival. In cultured neonatal rat ventricular myocytes, we compared the responses to IGF1 (10 nmol/liter) and phenylephrine (PE, 20 μmol/liter), a known GATA4 activator, in concentrations promoting a similar extent of hypertrophy. IGF1 and PE both increased nuclear accumulation of GATA4 and phosphorylation at Ser(105) (PE, 2.4-fold; IGF1, 1.8-fold; both, p0.05) and increased GATA4 DNA binding activity as indicated by ELISA and by chromatin IP of selected promoters. Although IGF1 and PE each activated GATA4 to the same degree, GATA4 knockdown by RNA interference only blocked hypertrophy by PE but not by IGF1. PE induction of a panel of GATA4 target genes (Nppa, Nppb, Tnni3, Myl1, and Acta1) was inhibited by GATA4 knockdown. In contrast, IGF1 regulated only Acta1 in a GATA4-dependent fashion. Consistent with the in vitro findings, Gata4 haploinsufficiency in mice did not alter cardiac structure, hyperdynamic function, or antifibrotic effects induced by myocardial overexpression of the IGF1 receptor. Our data indicate that GATA4 is activated by the IGF1 pathway, but although it is required for responses to pathological stimuli, it is not necessary for the effects of IGF1 on cardiac structure and function.

Introduction Fundamental differences in Ca2+ homeostasis between mice and larger mammals require the validation of the mechanisms of arrhythmogenesis before translation into human pathophysiology. The purpose of this study was to create transgenic rabbits that express defective human cardiac ryanodine receptor (hRyR2) with a mutation (R4497C) causing a clinically relevant arrhythmogenic syndrome. Methods The construct pcDNA3-EGFP-hRyR2-R4497C with the CMV promoter was used to generate transgenic rabbits. The founder animals were created by microinjection and identified by PCR with specific primers for the EGFP sequence. The copy number of the transgene was quantified by real-time PCR using genomic DNA from blood cells. mRNA expression of EGFP-hRyR2-R4497C was quantified using RT-PCR with specific primers for the RyR2 and EGFP sequence. Protein expression of the transgene in heart and non-cardiac tissues was determined using immunoblots with antibodies directed against EGFP and RyR2. Results Real-time PCR in peripheral blood cells identified several rabbit lines with the construct integrated into their genome. Transcription levels of the transgene were low (Ct > 30). On the protein level, neither EGFP nor hRyR2 R4497C was detected in either cardiac or non-cardiac tissue. A truncated gene product (3′ end and central part of hRyR2 R4497C, but not EGFP) could be detected at the mRNA level in the heart. Discussion Lack of significant protein expression of the EGFP-RyR2 R4497C gene construct despite successful incorporation into the genomic DNA is due to combination of at least two factors: low mRNA expression, and truncation of the transgene on the mRNA level. Our results suggest that the CMV promoter may not be well suited for creating transgenic rabbits.

Mutations in the cardiac ryanodine receptor Ca(2+) release channel, RyR2, underlie catecholaminergic polymorphic ventricular tachycardia (CPVT), an inherited life-threatening arrhythmia. CPVT is triggered by spontaneous RyR2-mediated sarcoplasmic reticulum (SR) Ca(2+) release in response to SR Ca(2+) overload during beta-adrenergic stimulation. However, whether elevated SR Ca(2+) content--in the absence of protein kinase A activation--affects RyR2 function and arrhythmogenesis in CPVT remains elusive.Isolated murine ventricular myocytes harbouring a human RyR2 mutation (RyR2(R4496C+/-)) associated with CPVT were investigated in the absence and presence of 1 micromol/L JTV-519 (RyR2 stabilizer) followed by 100 micromol/L ouabain intervention to increase cytosolic [Na(+)] and SR Ca(2+) load. Changes in membrane potential and intracellular [Ca(2+)] were monitored with whole-cell patch-clamping and confocal Ca(2+) imaging, respectively. At baseline, action potentials (APs), Ca(2+) transients, fractional SR Ca(2+) release, and SR Ca(2+) load were comparable in wild-type (WT) and RyR2(R4496C+/-) myocytes. Ouabain evoked significant increases in diastolic [Ca(2+)], peak systolic [Ca(2+)], fractional SR Ca(2+) release, and SR Ca(2+) content that were quantitatively similar in WT and RyR2(R4496C+/-) myocytes. Ouabain also induced arrhythmogenic events, i.e. spontaneous Ca(2+) waves, delayed afterdepolarizations and spontaneous APs, in both groups. However, the ouabain-induced increase in the frequency of arrhythmogenic events was dramatically larger in RyR2(R4496C+/-) when compared with WT myocytes. JTV-519 greatly reduced the frequency of ouabain-induced arrhythmogenic events.The elevation of SR Ca(2+) load--in the absence of beta-adrenergic stimulation--is sufficient to increase the propensity for triggered arrhythmias in RyR2(R4496C+/-) cardiomyocytes. Stabilization of RyR2 by JTV-519 effectively reduces these triggered arrhythmias.

Glucagon-like peptide-1 receptor (GLP-1R) agonists are a rapidly growing class of drugs developed for treating type-2 diabetes mellitus. Patients with diabetes carry an up to 5-fold greater mortality risk compared to non-diabetic patients, mainly as a result of cardiovascular diseases. Although beneficial cardiovascular effects have been reported, exact mechanisms of GLP-1R-agonist action in the heart, especially in human myocardium, are poorly understood. The effects of GLP-1R-agonists (exenatide, GLP-1(7-36)NH2, PF-06446009, PF-06446667) on cardiac contractility were tested in non-failing atrial and ventricular trabeculae from 72 patients. The GLP-1(7-36)NH2 metabolite, GLP-1(9-36)NH2, was also examined. In electrically stimulated trabeculae, the effects of compounds on isometric force were measured in the absence and presence of pharmacological inhibitors of signal transduction pathways. The role of β-arrestin signaling was examined using a β-arrestin partial agonist, PF-06446667. Expression levels were tested by immunoblots. Translocation of GLP-1R downstream molecular targets, Epac2, GLUT-1 and GLUT-4, were assessed by fluorescence microscopy. All tested GLP-1R-agonists significantly increased developed force in human atrial trabeculae, whereas GLP-1(9-36)NH2 had no effect. Exendin(9-39)NH2, a GLP-1R-antagonist, and H-89 blunted the inotropic effect of exenatide. In addition, exenatide increased PKA-dependent phosphorylation of phospholamban (PLB), GLUT-1 and Epac2 translocation, but not GLUT-4 translocation. Exenatide failed to enhance contractility in ventricular myocardium. Quantitative real-time PCR (qRT-PCR) revealed a significant higher GLP-1R expression in the atrium compared to ventricle. Exenatide increased contractility in a dose-dependent manner via GLP-1R/cAMP/PKA pathway and induced GLUT-1 and Epac2 translocation in human atrial myocardium, but had no effect in ventricular myocardium. Therapeutic use of GLP-1R-agonists may therefore impart beneficial effects on myocardial function and remodelling.

Cardiac hypertrophy is commonly observed in conditions of increased hemodynamic or metabolic stress. This hypertrophy is not compensatory but rather reflects activation of maladaptive cellular processes that promote disease progression. Myocardial hypertrophy serves as a diagnostic and prognostic marker of cardiac remodeling, and underlying regulatory processes have provided effective therapeutic targets to slow disease progression and improve outcome. We review hypertrophic signaling pathways in cardiomyocytes and discuss established and novel targets for pharmacological intervention. New drugs in the pipeline include the third generation aldosterone antagonists (PF-03882845 and BAY94-8862) and biased angiotensin II receptor agonists. Furthermore, different approaches to stimulate cGMP-dependent protective signaling are currently evaluated in clinical trials, including the combination of the vasopeptidase neprilysin inhibitor and an angiotensin receptor blocker (ARNi). In an overview on cardiomyocyte hypertrophic signaling, we also highlight emerging experimental treatment concepts such as inhibition of Ca-mediated transcriptional regulation, adeno-associated viruses for sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA2a), PI3 kinase gene transfer and microRNA-based therapy. We conclude that antihypertrophic therapy extends beyond blocking the classical β-adrenergic and renin-angiotensin-aldosterone system-dependent signaling cascades, although new therapies require clinical validation regarding outcome.

Urocortin 2 (Ucn2) is a cardioactive peptide exhibiting beneficial effects in normal and failing heart. In cardiomyocytes, it elicits cAMP- and Ca2+-dependent positive inotropic and lusitropic effects. We tested the hypothesis that, in addition, Ucn2 activates cardiac nitric oxide (NO) signaling and elucidated the underlying signaling pathways and mechanisms. In isolated rabbit ventricular myocytes, Ucn2 caused concentration- and time-dependent increases in phosphorylation of Akt (Ser473, Thr308), endothelial NO synthase (eNOS) (Ser1177), and ERK1/2 (Thr202/Tyr204). ERK1/2 phosphorylation, but not Akt and eNOS phosphorylation, was suppressed by inhibition of MEK1/2. Increased Akt phosphorylation resulted in increased Akt kinase activity and was mediated by corticotropin-releasing factor 2 (CRF2) receptors (astressin-2B sensitive). Inhibition of phosphatidylinositol 3-kinase (PI3K) diminished both Akt as well as eNOS phosphorylation mediated by Ucn2. Inhibition of protein kinase A (PKA) reduced Ucn2-induced phosphorylation of eNOS but did not affect the increase in phosphorylation of Akt. Conversely, direct receptor-independent elevation of cAMP via forskolin increased phosphorylation of eNOS but not of Akt. Ucn2 increased intracellular NO concentration ([NO]i), [cGMP], [cAMP], and cell shortening. Inhibition of eNOS suppressed the increases in [NO]iand cell shortening. When both PI3K-Akt and cAMP-PKA signaling were inhibited, the Ucn2-induced increases in [NO]iand cell shortening were attenuated. Thus, in rabbit ventricular myocytes, Ucn2 causes activation of cAMP-PKA, PI3K-Akt, and MEK1/2-ERK1/2 signaling. The MEK1/2-ERK1/2 pathway is not required for stimulation of NO signaling in these cells. The other two pathways, cAMP-PKA and PI3K-Akt, converge on eNOS phosphorylation at Ser1177 and result in pronounced and sustained cellular NO production with subsequent stimulation of cGMP signaling.

Background: Heart failure with preserved ejection fraction (HFPEF) and renal impairment are often associated but the underlying pathomechanisms are largely unknown. We investigated tissue fibrosis and cardiomyocyte contractile function in a rat model with HFPEF and compensated renal failure.Methods: Seventy young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP). Blood/urine samples, echocardiography, pressure-volume loops were performed after 8 and 24 weeks. Left ventricular (LV) hypertrophy, fibrosis (picosirius-red-staining) and NCX protein expression (Western blot) were determined after sacrification. Cardiomyocyte function (Ca2+ transients, cell shortening) was quantified in isolated cardiomyocytes without and with the NCX inhibitor SEA0400 (300nM).Results: NXT rats showed stable renal impairment with preserved urine excretion and elevated arterial pressure (148±8 and 154±7 vs. 105±3 and 109±2 mmHg, p

Background— A hallmark of heart failure is impaired cytoplasmic Ca 2+ handling of cardiomyocytes. It remains unknown whether specific alterations in nuclear Ca 2+ handling via altered excitation-transcription coupling contribute to the development and progression of heart failure. Methods and Results— Using tissue and isolated cardiomyocytes from nonfailing and failing human hearts, as well as mouse and rabbit models of hypertrophy and heart failure, we provide compelling evidence for structural and functional changes of the nuclear envelope and nuclear Ca 2+ handling in cardiomyocytes as remodeling progresses. Increased nuclear size and less frequent intrusions of the nuclear envelope into the nuclear lumen indicated altered nuclear structure that could have functional consequences. In the (peri)nuclear compartment, there was also reduced expression of Ca 2+ pumps and ryanodine receptors, increased expression of inositol-1,4,5-trisphosphate receptors, and differential orientation among these Ca 2+ transporters. These changes were associated with altered nucleoplasmic Ca 2+ handling in cardiomyocytes from hypertrophied and failing hearts, reflected as increased diastolic Ca 2+ levels with diminished and prolonged nuclear Ca 2+ transients and slowed intranuclear Ca 2+ diffusion. Altered nucleoplasmic Ca 2+ levels were translated to higher activation of nuclear Ca 2+ /calmodulin-dependent protein kinase II and nuclear export of histone deacetylases. Importantly, the nuclear Ca 2+ alterations occurred early during hypertrophy and preceded the cytoplasmic Ca 2+ changes that are typical of heart failure. Conclusions— During cardiac remodeling, early changes of cardiomyocyte nuclei cause altered nuclear Ca 2+ signaling implicated in hypertrophic gene program activation. Normalization of nuclear Ca 2+ regulation may therefore be a novel therapeutic approach to prevent adverse cardiac remodeling.

Synchronized release of Ca²⁺ into the cytosol during each cardiac cycle determines cardiomyocyte contraction.We investigated synchrony of cytosolic [Ca²⁺] decay during diastole and the impact of cardiac remodeling.Local cytosolic [Ca²⁺] transients (1-µm intervals) were recorded in murine, porcine, and human ventricular single cardiomyocytes. We identified intracellular regions of slow (slowCaR) and fast (fastCaR) [Ca²⁺] decay based on the local time constants of decay (TAUlocal). The SD of TAUlocal as a measure of dyssynchrony was not related to the amplitude or the timing of local Ca²⁺ release. Stimulation of sarcoplasmic reticulum Ca²⁺ ATPase with forskolin or istaroxime accelerated and its inhibition with cyclopiazonic acid slowed TAUlocal significantly more in slowCaR, thus altering the relationship between SD of TAUlocal and global [Ca²⁺] decay (TAUglobal). Na⁺/Ca²⁺ exchanger inhibitor SEA0400 prolonged TAUlocal similarly in slowCaR and fastCaR. FastCaR were associated with increased mitochondrial density and were more sensitive to the mitochondrial Ca²⁺ uniporter blocker Ru360. Variation in TAUlocal was higher in pig and human cardiomyocytes and higher with increased stimulation frequency (2 Hz). TAUlocal correlated with local sarcomere relengthening. In mice with myocardial hypertrophy after transverse aortic constriction, in pigs with chronic myocardial ischemia, and in end-stage human heart failure, variation in TAUlocal was increased and related to cardiomyocyte hypertrophy and increased mitochondrial density.In cardiomyocytes, cytosolic [Ca²⁺] decay is regulated locally and related to local sarcomere relengthening. Dyssynchronous intracellular [Ca²⁺] decay in cardiac remodeling and end-stage heart failure suggests a novel mechanism of cellular contractile dysfunction.

Paroxysmal atrial fibrillation (PAF) is often asymptomatic but nonetheless harmful. We evaluated the performance of disease-related blood biomarkers and CHA(2)DS(2)-VASc score to discriminate for PAF in patients with continuous rhythm monitoring. Clinical data and blood samples were obtained from patients with dual-chamber pacemakers selected according to the absence (no_AHRE) or presence of Atrial High-Rate Episodes (AHRE) > 6 min in recent device history (case-control approach). We included 93 patients (n = 49 AHRE, n = 44 no_AHRE). In a subgroup with high AHRE burden and confirmed PAF 15 biomarkers were evaluated (n = 19 AHRE-AF vs. n = 20 no_AHRE). Significantly regulated biomarkers were then tested in all patients to distinguish no_AHRE from AHRE (receiver operating characteristics analysis). Hsp27, TGF beta 1, cystatin C, matrix metalloproteinases MMP-2,-3,-9, albumin, and serum uric acid were not altered in the subgroup. Tissue inhibitors of metalloproteinases (TIMP) -1,-2,-4;NT-proANP, NT-proBNP, IL-6 and serum amyloid protein A were significantly different in AHRE vs. no_AHRE (subgroup and whole cohort), with best discriminatory performance for TIMP-4. Biomarkers performed better than CHADS(2)-VASc for AHRE discrimination. Intracardial electrograms and medical history from seven AHRE patients suggested atrial tachycardia and not AF (AHRE-AT). Four of the most relevant regulated biomarkers (TIMP-4, TIMP-2, SAA, NT-proBNP) behaved similarly in AHRE-AT and AHRE-AF. NT-proBNP > 150 pg/mL indicated an odds ratio of 12.9 for AHRE. Combining two biomarkers significantly improved discrimination of AHRE. TIMP-4, NT-proANP, NT-proBNP were strongest associated with PAF and AHRE. The discriminatory performance of CHADS(2)-VASc for PAF was increased by addition of selected biomarkers.

It is reasonably well understood how the initiation of translation is controlled by reversible phosphorylation of the eukaryotic translation initiation factors eIF2alpha, eIF2Bepsilon and eIF4E. Other initiation factors, including eIF2beta, are also established phosphoproteins but the physiological impact of their phosphorylation is not known. Using a sequence homology search we found that the central region of eIF2beta contains a putative PP1-(protein phosphatase-1) binding RVxF-motif. The predicted eIF2beta-PP1 interaction was confirmed by PP1 binding and co-immunoprecipitation assays on cell lysates as well as with the purified components. Site-directed mutagenesis showed that eIF2beta contains, in addition to an RVxF-motif, at least one other PP1-binding site in its C-terminal half. eIF2beta functioned as an inhibitor for the dephosphorylation of glycogen phosphorylase and Ser51 of eIF2alpha by PP1, but did not affect the dephosphorylation of Ser464 of eIF2Bepsilon by this phosphatase. Strikingly, eIF2beta emerged as an activator of its own dephosphorylation (Ser2, Ser67, Ser218) by associated PP1, since the substrate quality of eIF2beta was decreased by the mere mutation of its RVxF-motif. These results make eIF2beta an attractive candidate substrate for associated PP1 in vivo. The overexpression of wild-type eIF2beta or eIF2beta with a mutated RVxF-motif did not differentially affect the rate of translation, indicating that the binding of PP1 is not rate-limiting for translation under basal conditions.

Most interactors of protein phosphatase-1 (PP1) contain a variant of a so-called "RVXF" sequence that binds to a hydrophobic groove of the catalytic subunit. A combination of sequence alignments and site-directed mutagenesis has enabled us to further define the consensus sequence for this degenerate motif as [RK]-X(0-1)-[VI]-[P]-[FW], where X denotes any residue and [P] any residue except Pro. Naturally occurring RVXF sequences differ in their affinity for PP1, and we show by swapping experiments that this binding affinity is an important determinant of the inhibitory potency of the regulators NIPP1 and inhibitor-1. Also, inhibition by NIPP1-(143-224) was retained when the RVXF motif (plus the preceding Ser) was swapped for either of two unrelated PP1-binding sequences from human inhibitor-2, i.e. KGILK or RKLHY. Conversely, the KGILK motif of inhibitor-2 could be functionally replaced by the RVXF motif of NIPP1. Our data provide additional evidence for the view that the RVXF and KGILK motifs function as anchors for PP1 and thereby promote the interaction of secondary binding sites that determine the activity and substrate specificity of the enzyme.

Background: Heart failure with preserved ejection fraction (HFPEF) is increasingly common but the established heart failure (HF) drugs are not effective. The underlying cellular mechanisms are incompletely understood. Therefore we investigated cardiomyocyte function and intracellular Ca2+ homeostasis in a model of HFPEF. Methods: Young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP). Serial blood/urine samples, echocardiography and pressure-volume loops at 8 and 24 weeks were performed. After sacrifice, left ventricular (LV) hypertrophy and NCX function (Caffeine induced Ca2+ transient, TAU) and protein expression (Western blot) were determined. Cardiomyocyte function (Ca2+ transients, sarcoplasmic reticulum (SR) diastolic Ca2+ leak (Ca2+ sparks) and SR Ca2+ content; Fluo4-AM) were quantified in isolated LV cardiomyocytes without and with the NCX inhibitor SEA0400 (300nM). Results: NXT rats showed stable compensated renal impairment and significantly hypertrophied LV at 8 weeks with a further increase after 24 weeks. LV systolic function (EF, dP/dt) was preserved. End diastolic pressure (EDP) volume relationship was markedly shifted left- and upwards and lung weight were significantly increased, indicating HFPEF with pulmonary congestion. LV cardiomyocytes from NXT showed no significant differences in amplitudes of Ca2+ transients. However, time for early (50%) relaxation of the Ca2+ transients at 8 weeks were significantly prolonged with a further increase after 24 weeks (RT50 17.2±2.9 and 30.8±2.7 vs. 27.6±1.8 and 41.8±2.6 ms; n≥20; p< 0.05). TAU was significantly prolonged at 8 and 24 weeks indicating reduced NCX forward mode activity, while NCX protein expression was upregulated. At 8 weeks, Ca2+ spark frequency tended to be increased (p=0.07) while SR Ca2+ content was unchanged. SEA0400 accelerated Ca2+ transient decay but did not affect Ca2+ spark frequency. At 24 weeks, Ca2+ spark frequency was increased (4.3±0.7 vs. 11.5±1.8 sparks/s/μm3; n≥20; p

Background: Renal impairment and heart failure with preserved ejection fraction (HFPEF) are often associated but the underlying mechanisms are largely unresolved. Therefore we investigated cardiomyocyte function in a rat model with compensated renal failure and diastolic dysfunction. Methods: Fifty-six young male Wistar rats were subjected to subtotal nephrectomy (NXT) or sham operation (SOP) and observed up to 24 weeks. Urine and blood samples for calculating the glomerular filtration rate (GFR) and echocardiography were performed during disease progression. Invasive hemodynamics (P/V-loops), morphometry, protein expression analysis and Ca2+ transients (confocal and ratiometric) as well as cell shortening (CS) were studied in isolated left ventricle (LV) cardiomyocytes in final experiments at 8 and 24 weeks. Results: Following NXT rats develop compensated renal failure (GFR 22±2 and 16±2 vs. 32±2 and 28±3 ml/min/m2 at 8 and 24 weeks, resp. p

Chronic beta-adrenergic stimulation leads to heart failure (HF). In mice overexpressing beta1-adrenoceptors (TG), increased diastolic Ca load in cardiomyocytes at early age is pivotal for the development of HF. The mechanisms underlying intracellular Ca dysregulation are unclear. We examined cytosolic Ca transients (Fluo4-AM, field stimulation), Na-Ca-exchanger (NCX) function and protein expression, cytosolic Na (SBFI) and T-tubular structures (Di8-ANEPPS) in cardiomyocytes from young (8-16 wks) TG mice and wildtype (WT) littermates.Results: Systolic [Ca] amplitude was unchanged, time to peak [Ca] (140±5 vs. 127±3 ms) and [Ca] decay (time constant, tau, 223±16 vs. 182±9 ms) were significantly prolonged in TG vs. WT. Diastolic Ca leak from the SR (quantified as tetracaine-sensitive change in diastolic [Ca] or diastolic Ca spark frequency) was unchanged. However, cytosolic Ca removal by NCX during coffein application was significantly slower (tau, 3683±337 in TG vs. 2304±272 ms in WT), indicating reduced forward mode NCX activity. NCX protein expression was unchanged. Preliminary results indicate increased cytosolic [Na] in young TG. Furthermore, confocal line scans revealed delayed (> 15 ms until half-maximal) systolic Ca release in 24.7±2.6 (TG) vs. 4.6±1.4% (WT) of the intracellular regions (n=32 and 31 cells, resp., p

The Wnt/frizzled signalling pathways play a key role in cardiogenesis and in adult hearts during cardiac remodelling. We investigated whether the expression of Wnt-11 and its anticipated downstream signalling components is altered in human failing hearts and whether Wnt-11 acutely affects Ca2+ transients in adult murine cardiomyocytes.Left ventricles (LV) homogenates of human non-failing hearts (NF, n=4) and end-stage failing hearts with ischemic (ICM, n=5) and dilated (DCM, n=5) cardiomyopathy were used for western blotting with antibodies against Wnt-11, Fzd-4 and Fzd-7 (Wnt receptors) and Dvl-1 (downstream cytoplasmic regulator). Expression of proteins was normalized to GAPDH and is given as a fraction of average protein expression in NF. Ca2+ transients were measured during confocal line scan imaging with Fluo-4 in isolated murine adult cardiomyocytes after 5 (ncells=20) and 25 minutes (ncells=18) incubation with recombinant Wnt-11 (2 µg/ml). Untreated cells served as controls (ncells=27).Wnt-11 expression in LV in ICM (0.85 ± 0.05) and DCM (0.91 ± 0.34) was not significantly changed when compared to NF. However, the expression of Wnt-11 receptors was highly increased: Fzd-4 in ICM (6.25 ± 1.23; p