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Ca2+ sparks constitute the fundamental units of Ca2+ release in cardiomyocytes. Here we investigate how ryanodine receptors (RyRs) collectively generate these events by employing a transgenic mouse with a photoactivated label on RyR2. This allowed correlative imaging of RyR localization, by super-resolution photoactivated localization microscopy, and Ca2+ sparks, by high-speed imaging. Two populations of Ca2+ sparks were observed: stationary events and ‘traveling’ events that spread between neighboring RyR clusters. Traveling sparks exhibited up to eight distinct releases, sourced from local or distal junctional sarcoplasmic reticulum. Quantitative analyses showed that sparks may be triggered by any number of RyRs within a cluster, and that acute β-adrenergic stimulation augments intracluster RyR recruitment to generate larger events. In contrast, RyR ‘dispersion’ during heart failure facilitates the generation of traveling sparks. Thus, RyRs cooperatively generate Ca2+ sparks in a complex, malleable fashion, and channel organization regulates the propensity for local propagation of Ca2+ release.

Aims Guanylyl cyclase-B (GC-B; natriuretic peptide receptor-B, NPR-B) stimulation by C-type natriuretic peptide (CNP) increases cGMP and causes a lusitropic and negative inotropic response in adult myocardium. These effects are not mimicked by NPR-A (GC-A) stimulation by brain natriuretic peptide (BNP), despite similar cGMP increase. More refined methods are needed to better understand the mechanisms of the differential cGMP signaling and compartmentation. The aim of this work was to measure cGMP near proteins involved in regulating contractility to understand compartmentation of cGMP signaling in adult cardiomyocytes. Methods and results We constructed several fluorescence resonance energy transfer (FRET)-based biosensors for cGMP subcellularly targeted to phospholamban (PLB) and troponin I (TnI). CNP stimulation of adult rat cardiomyocytes increased cGMP near PLB and TnI, whereas BNP stimulation increased cGMP near PLB, but not TnI. The phosphodiesterases PDE2 and PDE3 constrained cGMP in both compartments. Local receptor stimulation aided by scanning ion conductance microscopy (SICM) combined with FRET revealed that CNP stimulation both in the t-tubules and on the cell crests increases cGMP similarly near both TnI and PLB. In ventricular strips, CNP stimulation, but not BNP, induced a lusitropic response, enhanced by inhibition of either PDE2 or PDE3, and a negative inotropic response. In cardiomyocytes from heart failure rats, CNP increased cGMP near PLB and TnI more pronounced than in cells from sham-operated animals. Conclusions These targeted biosensors demonstrate that CNP, but not BNP, increases cGMP near TnI in addition to PLB, explaining how CNP, but not BNP is able to induce lusitropic and negative inotropic responses. Translational perspective Although best known as heart failure biomarkers, natriuretic peptides (ANP, BNP and CNP) are important signaling molecules in the heart and other organs through increasing cyclic GMP (cGMP). Treatment preventing their degradation improves heart failure prognosis. To better understand their cardiac signaling, we employed fluorescent cGMP biosensors targeted to troponin I and phospholamban and found that BNP and CNP increase cGMP differently around these proteins in both normal and failing cardiomyocytes. This may explain the different effects of BNP and CNP on cardiac contractility and relaxation, with possible implications for understanding and treatment of heart failure.

According to early models of GPCR signaling, G proteins only interact with activated receptors. However, some GPCRs were shown to assemble with G proteins before receptor activation, in accordance with more recent models. Previously, we found that the 5-HT7 receptor, as opposed to the 5-HT4 receptor, was preassociated with Gs, but the molecular determinants for this interaction are still elusive. In a series of chimeric 5-HT7 receptors with intracellular segments from 5-HT4, we determined the receptor-G protein interaction by performing antibody-immobilized fluorescence recovery after photobleaching and fluorescence resonance energy transfer. We identified the intracellular loop 3 and C-tail of the 5-HT7 receptor to be responsible for the preassociation with Gs, and we further delineated the TM5 extension in the intracellular loop 3 and helix 8 in the C-tail as the molecular determinants. These chimeric exchanges converted the 5-HT7 receptor into a collision-coupled receptor that recruited G proteins only upon agonist activation, whereas reciprocal exchanges converted 5-HT4 to a preassociated receptor. The 5-HT7 receptor displayed 2-component agonist-induced Gs signaling with high and low potency. In addition, the same segments were involved in low-potency signaling and preassociation. The correspondence between Gs preassociation and low-potency Gs signaling is a novel aspect of GPCR pharmacology.-Ulsund, A. H., Dahl, M., Frimurer, T. M., Manfra, O., Schwartz, T. W., Levy, F. O., Andressen, K. W. Preassociation between the 5-HT7 serotonin receptor and G protein Gs: molecular determinants and association with low potency activation of adenylyl cyclase.

Background Whereas heart failure with reduced ejection fraction (HFrEF) is associated with ventricular dilation and markedly reduced systolic function, heart failure with preserved ejection fraction (HFpEF) patients exhibit concentric hypertrophy and diastolic dysfunction. Impaired cardiomyocyte Ca2+ homeostasis in HFrEF has been linked to disruption of membrane invaginations called t-tubules, but it is unknown if such changes occur in HFpEF. Objectives This study examined whether distinct cardiomyocyte phenotypes underlie the heart failure entities of HFrEF and HFpEF. Methods T-tubule structure was investigated in left ventricular biopsies obtained from HFrEF and HFpEF patients, whereas cardiomyocyte Ca2+ homeostasis was studied in rat models of these conditions. Results HFpEF patients exhibited increased t-tubule density in comparison with control subjects. Super-resolution imaging revealed that higher t-tubule density resulted from both tubule dilation and proliferation. In contrast, t-tubule density was reduced in patients with HFrEF. Augmented collagen deposition within t-tubules was observed in HFrEF but not HFpEF hearts. A causative link between mechanical stress and t-tubule disruption was supported by markedly elevated ventricular wall stress in HFrEF patients. In HFrEF rats, t-tubule loss was linked to impaired systolic Ca2+ homeostasis, although diastolic Ca2+ removal was also reduced. In contrast, Ca2+ transient magnitude and release kinetics were largely maintained in HFpEF rats. However, diastolic Ca2+ impairments, including reduced sarco/endoplasmic reticulum Ca2+-ATPase activity, were specifically observed in diabetic HFpEF but not in ischemic or hypertensive models. Conclusions Although t-tubule disruption and impaired cardiomyocyte Ca2+ release are hallmarks of HFrEF, such changes are not prominent in HFpEF. Impaired diastolic Ca2+ homeostasis occurs in both conditions, but in HFpEF, this mechanism for diastolic dysfunction is etiology-dependent.

Supplemental Digital Content is available in the text., Rationale: Hypokalemia occurs in up to 20% of hospitalized patients and is associated with increased incidence of ventricular and atrial fibrillation. It is unclear whether these differing types of arrhythmia result from direct and perhaps distinct effects of hypokalemia on cardiomyocytes. Objective: To investigate proarrhythmic mechanisms of hypokalemia in ventricular and atrial myocytes. Methods and Results: Experiments were performed in isolated rat myocytes exposed to simulated hypokalemia conditions (reduction of extracellular [K+] from 5.0 to 2.7 mmol/L) and supported by mathematical modeling studies. Ventricular cells subjected to hypokalemia exhibited Ca2+ overload and increased generation of both spontaneous Ca2+ waves and delayed afterdepolarizations. However, similar Ca2+-dependent spontaneous activity during hypokalemia was only observed in a minority of atrial cells that were observed to contain t-tubules. This effect was attributed to close functional pairing of the Na+-K+ ATPase and Na+-Ca2+ exchanger proteins within these structures, as reduction in Na+ pump activity locally inhibited Ca2+ extrusion. Ventricular myocytes and tubulated atrial myocytes additionally exhibited early afterdepolarizations during hypokalemia, associated with Ca2+ overload. However, early afterdepolarizations also occurred in untubulated atrial cells, despite Ca2+ quiescence. These phase-3 early afterdepolarizations were rather linked to reactivation of nonequilibrium Na+ current, as they were rapidly blocked by tetrodotoxin. Na+ current-driven early afterdepolarizations in untubulated atrial cells were enabled by membrane hyperpolarization during hypokalemia and short action potential configurations. Brief action potentials were in turn maintained by ultra-rapid K+ current (IKur); a current which was found to be absent in tubulated atrial myocytes and ventricular myocytes. Conclusions: Distinct mechanisms underlie hypokalemia-induced arrhythmia in the ventricle and atrium but also vary between atrial myocytes depending on subcellular structure and electrophysiology.

According to early models of GPCR signaling, G proteins only interact with activated receptors. However, some GPCRs were shown to assemble with G proteins before receptor activation, in accordance with more recent models. Previously, we found that the 5-HT

Aims Regional heterogeneities in contraction contribute to heart failure with reduced ejection fraction (HFrEF). We aimed to determine whether regional changes in myocardial relaxation similarly contribute to diastolic dysfunction in post-infarction HFrEF, and to elucidate the underlying mechanisms. Methods and results Using the magnetic resonance imaging phase-contrast technique, we examined local diastolic function in a rat model of post-infarction HFrEF. In comparison with sham-operated animals, post-infarction HFrEF rats exhibited reduced diastolic strain rate adjacent to the scar, but not in remote regions of the myocardium. Removal of Ca2+ within cardiomyocytes governs relaxation, and we indeed found that Ca2+ transients declined more slowly in cells isolated from the adjacent region. Resting Ca2+ levels in adjacent zone myocytes were also markedly elevated at high pacing rates. Impaired Ca2+ removal was attributed to a reduced rate of Ca2+ sequestration into the sarcoplasmic reticulum (SR), due to decreased local expression of the SR Ca2+ ATPase (SERCA). Wall stress was elevated in the adjacent region. Using ex vivo experiments with loaded papillary muscles, we demonstrated that high mechanical stress is directly linked to SERCA down-regulation and slowing of relaxation. Finally, we confirmed that regional diastolic dysfunction is also present in human HFrEF patients. Using echocardiographic speckle-tracking of patients enrolled in the LEAF trial, we found that in comparison with controls, post-infarction HFrEF subjects exhibited reduced diastolic train rate adjacent to the scar, but not in remote regions of the myocardium. Conclusion Our data indicate that relaxation varies across the heart in post-infarction HFrEF. Regional diastolic dysfunction in this condition is linked to elevated wall stress adjacent to the infarction, resulting in down-regulation of SERCA, disrupted diastolic Ca2+ handling, and local slowing of relaxation.

Background and Purpose Classically, ligands of GPCRs have been classified primarily upon their affinity and efficacy to activate a signal transduction pathway. Recent reports indicate that the efficacy of a particular ligand can vary depending on the receptor-mediated response measured (e.g. activating G proteins, other downstream responses, internalization). Previously, we reported that inverse agonists induce both homo- and heterologous desensitization, similar to agonist stimulation, at the Gs-coupled 5-HT7 receptor. The primary objective of this study was to determine whether different inverse agonists at the 5-HT7 receptor also induce internalization and/or degradation of 5-HT7 receptors. Experimental Approach HEK293 cells expressing 5-HT7(a, b or d) receptors were pre-incubated with 5-HT, clozapine, olanzapine, mesulergine or SB269970 and their effects upon receptor density, AC activity, internalization, recruitment of β-arrestins and lysosomal trafficking were measured. Key Results The agonist 5-HT and three out of four inverse agonists tested increased internalization independently of β-arrestin recruitment. Among these, only the atypical antipsychotics clozapine and olanzapine promoted lysosomal sorting and reduced 5-HT7 receptor density (∼60% reduction within 24 h). Inhibition of lysosomal degradation with chloroquine blocked the clozapine- and olanzapine-induced down-regulation of 5-HT7 receptors. Incubation with SB269970 decreased both 5-HT7(b) constitutive internalization and receptor density but increased 5-HT7(d) receptor density, indicating differential ligand regulation among the 5-HT7 splice variants. Conclusions and Implications Taken together, we found that various ligands differentially activate regulatory processes governing receptor internalization and degradation in addition to signal transduction. Thus, these data extend our understanding of functional selectivity at the 5-HT7 receptor.

The synthesis and pharmacological data of some new and potent hydrophilic 5-HT4 receptor antagonists are described. Propanediol derivative 25 was identified as a potent antagonist with low affinity for the hERG potassium channel and promising pharmacokinetics.

The sodium (Na

The sodium (Na+ )-calcium (Ca2+ ) exchanger 1 (NCX1) is an antiporter membrane protein encoded by the SLC8A1 gene. In the heart, it maintains cytosolic Ca2+ homeostasis, serving as the primary mechanism for Ca2+ extrusion during relaxation. Dysregulation of NCX1 is observed in end-stage human heart failure. In this study, we used affinity purification coupled with MS in rat left ventricle lysates to identify novel NCX1 interacting proteins in the heart. Two screens were conducted using: (1) anti-NCX1 against endogenous NCX1 and (2) anti-His (where His is histidine) with His-trigger factor-NCX1cyt recombinant protein as bait. The respective methods identified 112 and 350 protein partners, of which several were known NCX1 partners from the literature, and 29 occurred in both screens. Ten novel protein partners (DYRK1A, PPP2R2A, SNTB1, DMD, RABGGTA, DNAJB4, BAG3, PDE3A, POPDC2, STK39) were validated for binding to NCX1, and two partners (DYRK1A, SNTB1) increased NCX1 activity when expressed in HEK293 cells. A cardiac NCX1 protein-protein interaction map was constructed. The map was highly connected, containing distinct clusters of proteins with different biological functions, where "cell communication" and "signal transduction" formed the largest clusters. The NCX1 interactome was also significantly enriched with proteins/genes involved in "cardiovascular disease" which can be explored as novel drug targets in future research.

Purpose: To determine how inotropic responses are regulated in heart failure (HF) through signalling pathways increasing cAMP and cGMP is crucial for understanding how new drugs can be of benefit to patients. Myocardial levels of C-type natriuretic peptide (CNP) are increased in HF. CNP activates the guanylyl cyclase NPR-B receptor and increases cGMP. Our group has previously shown that CNP induces a negative inotropic and positive lusitropic response in failing hearts through cGMP-mediated phosphorylation of phospholamban and troponin I. In addition, we have shown that activating these receptors enhances the positive inotropic response to β1-adrenergic and 5-HT4 receptors via a mechanism where increased cGMP levels inhibit phosphodiesterase 3 (PDE3) and thereby the degradation of cAMP from these receptors. Neither of these effects were observed when activating other NPR receptors with ANP and BNP, indicating a tightly compartmented response of both the NPR-B, β1- and 5-HT4 receptors. We therefore hypothesize that cGMP is increased in distinct subcellular compartments in cardiac cells. In the current work, we want to clarify the organization of functional cGMP compartments, importance of PDEs and how cGMP regulates inotropic responses. Methods: We have constructed several intracellular FRET-based sensors for cGMP targeted to different subcellular compartments that allow us to measure the local levels of cGMP in single cardiac myocytes. Results and conclusions: In the current work, we have constructed new FRET-based cGMP sensors, located to specific anchoring and structural proteins regulating inotropic responses, thus enabling measurement of local changes in cGMP concentrations after stimulation of the different natriuretic peptide receptors. Understanding compartmented cGMP signaling would help explain how natriuretic peptides can regulate calcium handling (phospholamban) and calcium sensitivity (troponin I) of cardiac myocytes. FRET = Fluorescence resonance energy transfer