An Update to Calcium Binding Proteins.

Ca2+ binding proteins (CBP) are of key importance for calcium to play its role as a pivotal second messenger. CBP bind Ca2+ in specific domains, contributing to the regulation of its concentration at the cytosol and intracellular stores. They also participate in numerous cellular functions by acting as Ca2+ transporters across cell membranes or as Ca2+-modulated sensors, i.e. decoding Ca2+ signals. Since CBP are integral to normal physiological processes, possible roles for them in a variety of diseases has attracted growing interest in recent years. In addition, research on CBP has been reinforced with advances in the structural characterization of new CBP family members. In this chapter we have updated a previous review on CBP, covering in more depth potential participation in physiopathological processes and candidacy for pharmacological targets in many diseases. We review intracellular CBP that contain the structural EF-hand domain: parvalbumin, calmodulin, S100 proteins, calcineurin and neuronal Ca2+ sensor proteins (NCS). We also address intracellular CBP lacking the EF-hand domain: annexins, CBP within intracellular Ca2+ stores (paying special attention to calreticulin and calsequestrin), proteins that contain a C2 domain (such as protein kinase C (PKC) or synaptotagmin) and other proteins of interest, such as regucalcin or proprotein convertase subtisilin kexins (PCSK). Finally, we summarise the latest findings on extracellular CBP, classified according to their Ca2+ binding structures: (i) EF-hand domains; (ii) EGF-like domains; (iii) ɣ-carboxyl glutamic acid (GLA)-rich domains; (iv) cadherin domains; (v) Ca2+-dependent (C)-type lectin-like domains; (vi) Ca2+-binding pockets of family C G-protein-coupled receptors.

Simvastatin Promotes Cardiac Myocyte Relaxation in Association with Phosphorylation of Troponin I.

The number of people taking statins is set to increase across the globe due to recent changes in prescription guidelines. For example, half the US population over 40 is now eligible for these drugs, whether they have high serum cholesterol or not. With such development in policy comes a stronger need for understanding statins' myriad of effects. Surprisingly little is known about possible direct actions of statins on cardiac myocytes, although claims of a direct myocardial toxicity have been made. Here, we determine the impact of simvastatin administration (40 mg/kg/day) for 2 weeks in normocholesterolemic rats on cardiac myocyte contractile function and identify an underlying mechanism. Under basal conditions, statin treatment increased the time to half (t0.5) relaxation without any effect on the magnitude of shortening, or the magnitude/kinetics of the [Ca2+]i transient. Enhanced myocyte lusitropy could be explained by a corresponding increase in phosphorylation of troponin I (TnI) at Ser23,24. Statin treatment increased expression of eNOS and Ser1177 phosphorylated eNOS, decreased expression of the NOS-inhibitory proteins caveolins 1 and 3, and increased (P = 0.06) NO metabolites, consistent with enhanced NO production. It is well-established that NO stimulates protein kinase G, one of the effectors of TnI phosphorylation at Ser23,24. Trends for parallel changes in phospho-TnI, phospho-eNOS and caveolin 1 expression were seen in atrial muscle from patients taking statins. Our data are consistent with a mechanism whereby chronic statin treatment enhances TnI phosphorylation and myocyte lusitropy through increased NO bioavailability. We see no evidence of impaired function with statin treatment; the changes we document at the level of the cardiac myocyte should facilitate diastolic filling and cardiac performance.

The Golgi apparatus is a functionally distinct Ca2+ store regulated by the PKA and Epac branches of the ß1-adrenergic signaling pathway.

Ca(2+) release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. We found that the Golgi apparatus was the source of prolonged Ca(2+) release events that originated near the nuclei of primary cardiomyocytes. Golgi Ca(2+) release was unaffected by depletion of sarcoplasmic reticulum Ca(2+), and disruption of the Golgi apparatus abolished Golgi Ca(2+) release without affecting sarcoplasmic reticulum function, suggesting functional and spatial independence of Golgi and sarcoplasmic reticulum Ca(2+) stores. ß1-Adrenoceptor stimulation triggers the production of the second messenger cAMP, which activates the Epac family of Rap guanine nucleotide exchange factors and the kinase PKA (protein kinase A). Phosphodiesterases (PDEs), including those in the PDE3 and PDE4 families, degrade cAMP. Activation of ß1-adrenoceptors stimulated Golgi Ca(2+) release, an effect that required activation of Epac, PKA, and the kinase CaMKII. Inhibition of PDE3s or PDE4s potentiated ß1-adrenergic-induced Golgi Ca(2+) release, which is consistent with compartmentalization of cAMP signaling near the Golgi apparatus. Interventions that stimulated Golgi Ca(2+) release appeared to increase the trafficking of vascular endothelial growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane of cardiomyocytes. In cardiomyocytes from rats with heart failure, decreases in the abundance of PDE3s and PDE4s were associated with increased Golgi Ca(2+) release events. These data suggest that the Golgi apparatus is a focal point for ß1-adrenergic-stimulated Ca(2+) signaling and that the Golgi Ca(2+) store functions independently from the sarcoplasmic reticulum and the global Ca(2+) transients that trigger contraction in cardiomyocytes.

Caveolin contributes to the modulation of basal and ß-adrenoceptor stimulated function of the adult rat ventricular myocyte by simvastatin: a novel pleiotropic effect.

The number of people taking statins is increasing across the globe, highlighting the importance of fully understanding statins' effects on the cardiovascular system. The beneficial impact of statins extends well beyond regression of atherosclerosis to include direct effects on tissues of the cardiovascular system ('pleiotropic effects'). Pleiotropic effects on the cardiac myocyte are often overlooked. Here we consider the contribution of the caveolin protein, whose expression and cellular distribution is dependent on cholesterol, to statin effects on the cardiac myocyte. Caveolin is a structural and regulatory component of caveolae, and is a key regulator of cardiac contractile function and adrenergic responsiveness. We employed an experimental model in which inhibition of myocyte HMG CoA reductase could be studied in the absence of paracrine influences from non-myocyte cells. Adult rat ventricular myocytes were treated with 10 µM simvastatin for 2 days. Simvastatin treatment reduced myocyte cholesterol, caveolin 3 and caveolar density. Negative inotropic and positive lusitropic effects (with corresponding changes in [Ca2+]i) were seen in statin-treated cells. Simvastatin significantly potentiated the inotropic response to ß2-, but not ß1-, adrenoceptor stimulation. Under conditions of ß2-adrenoceptor stimulation, phosphorylation of phospholamban at Ser16 and troponin I at Ser23/24 was enhanced with statin treatment. Simvastatin increased NO production without significant effects on eNOS expression or phosphorylation (Ser1177), consistent with the reduced expression of caveolin 3, its constitutive inhibitor. In conclusion, statin treatment can reduce caveolin 3 expression, with functional consequences consistent with the known role of caveolae in the cardiac cell. These data are likely to be of significance, particularly during the early phases of statin treatment, and in patients with heart failure who have altered ß-adrenoceptor signalling. In addition, as caveolin is ubiquitously expressed and has myriad tissue-specific functions, the impact of statin-dependent changes in caveolin is likely to have many other functional sequelae.

A novel approach to the Langendorff technique: preparation of isolated cardiomyocytes and myocardial samples from the same rat heart.

Researchers in biomedical sciences must continually re-evaluate their investment in experiments using laboratory animals. Our group is interested in various signalling pathways that underlie physiological and pathophysiological functioning of the mammalian heart. Two important resources for this type of work are isolated cardiomyocytes and homogenized or preserved sections of whole myocardium. In order to improve our experimental approach ethically, we devised an adaptation of the Langendorff whole-heart retrograde perfusion technique that allows the isolation of adult rat ventricular cardiomyocytes and processing/storage of myocardium from the same heart. This could result in a 50% reduction in the number of animals required for certain experiments. We believe that a generalized adoption of this method would represent a meaningful reduction of animal use in our field of research and, furthermore, strengthen data sets by permitting correlation between myocyte function and various parameters of myocardial biochemistry/structure in individual hearts. This approach is of particular relevance for studies of cardiac pathology, given the cost and time involved in generating experimental disease models.

Caveolae compartmentalise ß2-adrenoceptor signals by curtailing cAMP production and maintaining phosphatase activity in the sarcoplasmic reticulum of the adult ventricular myocyte.

Inotropy and lusitropy in the ventricular myocyte can be efficiently induced by activation of ß1-, but not ß2-, adrenoceptors (ARs). Compartmentation of ß2-AR-derived cAMP-dependent signalling underlies this functional discrepancy. Here we investigate the mechanism by which caveolae (specialised sarcolemmal invaginations rich in cholesterol and caveolin-3) contribute to compartmentation in the adult rat ventricular myocyte. Selective activation of ß2-ARs (with zinterol/CGP20712A) produced little contractile response in control cells but pronounced inotropic and lusitropic responses in cells treated with the cholesterol-depleting agent methyl-ß-cyclodextrin (MBCD). This was not linked to modulation of L-type Ca(2+) current, but instead to a discrete PKA-mediated phosphorylation of phospholamban at Ser(16). Application of a cell-permeable inhibitor of caveolin-3 scaffolding interactions mimicked the effect of MBCD on phosphorylated phospholamban (pPLB) during ß2-AR stimulation, consistent with MBCD acting via caveolae. Biosensor experiments revealed ß2-AR mobilisation of cAMP in PKA II signalling domains of intact cells only after MBCD treatment, providing a real-time demonstration of cAMP freed from caveolar constraint. Other proteins have roles in compartmentation, so the effects of phosphodiesterase (PDE), protein phosphatase (PP) and phosphoinositide-3-kinase (PI3K) inhibitors on pPLB and contraction were compared in control and MBCD treated cells. PP inhibition alone was conspicuous in showing robust de-compartmentation of ß2-AR-derived signalling in control cells and a comparatively diminutive effect after cholesterol depletion. Collating all evidence, we promote the novel concept that caveolae limit ß2-AR-cAMP signalling by providing a platform that not only attenuates production of cAMP but also prevents inhibitory modulation of PPs at the sarcoplasmic reticulum. This article is part of a Special Issue entitled "Local Signaling in Myocytes".

Effects of cholesterol depletion on compartmentalized cAMP responses in adult cardiac myocytes.

ß(1)-Adrenergic receptors (ß(1)ARs) and E-type prostaglandin receptors (EPRs) both produce compartmentalized cAMP responses in cardiac myocytes. The role of cholesterol-dependent lipid rafts in producing these compartmentalized responses was investigated in adult rat ventricular myocytes. ß(1)ARs were found in lipid raft and non-lipid raft containing membrane fractions, while EPRs were only found in non-lipid raft fractions. Furthermore, ß(1)AR activation enhanced the L-type Ca(2+) current, intracellular Ca(2+) transient, and myocyte shortening, while EPR activation had no effect, consistent with the idea that these functional responses are regulated by cAMP produced by receptors found in lipid raft domains. Using methyl-ß-cyclodextrin to disrupt lipid rafts by depleting membrane cholesterol did not eliminate compartmentalized behavior, but it did selectively alter specific receptor-mediated responses. Cholesterol depletion enhanced the sensitivity of functional responses produced by ß(1)ARs without having any effect on EPR activation. Changes in cAMP activity were also measured in intact cells using two different FRET-based biosensors: a type II PKA-based probe to monitor cAMP in subcellular compartments that include microdomains associated with caveolar lipid rafts and a freely diffusible Epac2-based probe to monitor total cytosolic cAMP. ß(1)AR and EPR activation elicited responses detected by both FRET probes. However, cholesterol depletion only affected ß(1)AR responses detected by the PKA probe. These results indicate that lipid rafts alone are not sufficient to explain the difference between ß(1)AR and EPR responses. They also suggest that ß(1)AR regulation of myocyte contraction involves the local production of cAMP by a subpopulation of receptors associated with caveolar lipid rafts.

Separate elements within a single IQ-like motif in adenylyl cyclase type 8 impart ca2+/calmodulin binding and autoinhibition.

The ubiquitous Ca(2+)-sensing protein calmodulin (CaM) fulfills its numerous signaling functions through a wide range of modular binding and activation mechanisms. By activating adenylyl cyclases (ACs) 1 and 8, Ca(2+) acting via calmodulin impacts on the signaling of the other major cellular second messenger cAMP. In possessing two CaM-binding domains, a 1-5-8-14 motif at the N terminus and an IQ-like motif (IQlm) at the C terminus, AC8 offers particularly sophisticated regulatory possibilities. The IQlm has remained unexplored beyond the suggestion that it bound CaM, and the larger C2b region of which it is part was involved in the relief of autoinhibition of AC8. Here we attempt to distinguish the function of individual residues of the IQlm. From a complementary approach of in vitro and cell population AC activity assays, as well as CaM binding, we propose that the IQlm alone, and not the majority of the C2b, imparts CaM binding and autoinhibitory functions. Moreover, this duality of function is spatially separated and depends on amino acid side-chain character. Accordingly, residues critical for CaM binding are positively charged and clustered toward the C terminus, and those essential for the maintenance of autoinhibition are hydrophobic and more N-terminal. Secondary structure prediction of the IQlm supports this separation, with an ideally placed break in the alpha-helical nature of the sequence. We additionally find that the N and C termini of AC8 interact, which is an association specifically abrogated by fully Ca(2+)-bound, but not Ca(2+)-free, CaM. These data support a sophisticated activation mechanism of AC8 by CaM, in which the duality of the IQlm function is critical.

Distinct mechanisms of regulation by Ca2+/calmodulin of type 1 and 8 adenylyl cyclases support their different physiological roles.

Nine membrane-bound mammalian adenylyl cyclases (ACs) have been identified. Type 1 and 8 ACs (AC1 and AC8), which are both expressed in the brain and are stimulated by Ca(2+)/calmodulin (CaM), have discrete neuronal functions. Although the Ca(2+) sensitivity of AC1 is higher than that of AC8, precisely how these two ACs are regulated by Ca(2+)/CaM remains elusive, and the basis for their diverse physiological roles is quite unknown. Distinct localization of the CaM binding domains within the two enzymes may be essential to differential regulation of the ACs by Ca(2+)/CaM. In this study we compare in detail the regulation of AC1 and AC8 by Ca(2+)/CaM both in vivo and in vitro and explore the different role of each Ca(2+)-binding lobe of CaM in regulating the two enzymes. We also assess the relative dependence of AC1 and AC8 on capacitative Ca(2+) entry. Finally, in real-time fluorescence resonance energy transfer-based imaging experiments, we examine the effects of dynamic Ca(2+) events on the production of cAMP in cells expressing AC1 and AC8. Our data demonstrate distinct patterns of regulation and Ca(2+) dependence of AC1 and AC8, which seems to emanate from their mode of regulation by CaM. Such distinctive properties may contribute significantly to the divergent physiological roles in which these ACs have been implicated.

Economic burden of deep-vein thrombosis, pulmonary embolism, and post-thrombotic syndrome.

PURPOSE: Deep-vein thrombosis (DVT) and pulmonary embolism (PE) are associated with major morbidity and mortality, with their burden often extending to longer-term complications such as event recurrence and post-thrombotic syndrome (PTS). Few data exist on the overall economic burden of DVT and PE and their sequelae. A retrospective observational cohort study was conducted to determine the direct medical costs of a DVT or PE patient across the entire continuum of care. SUMMARY: Administrative claims data for patients with a DVT or PE diagnosis (ICD-9-CM code) and patients with possible evidence of PTS between January 1, 1997, and March 31, 2004, were extracted from the PharMetrics Patient-Centric Database, which comprises fully adjudicated medical and pharmaceutical claims for U.S. health care-plan enrollees. Resource utilization and annualized direct medical costs of care for patients with DVT and/or PE were calculated and compared with matched controls. A total of 26,958 patients met the study inclusion criteria. Of the 17,634 patients evaluable for the PTS cohort, 663 (3.8%) patients experienced PTS. Patients with DVT, PE, or DVT and PE had higher annualized direct medical costs before the index (initial) DVT and/or PE event (median: $7227, $6381, and $6771, respectively) than controls (median: $1045). During and after the DVT/PE event, annualized median costs rose to $17,512, $18,901, and $25,554, respectively, compared with $680 in the control group. Annualized median total costs for the PTS group were $20,569 compared with $15,843 in matched controls with DVT and/or PE and no PTS. CONCLUSION: These data suggest that the initial acute DVT or PE event is associated with high total health care costs and that these costs are further increased by subsequent events such as recurrent DVT or PE and PTS. Early detection and appropriate treatment of this high-risk population have the potential for both clinical and economic benefits.