The effects of over-expression of the FK506-binding protein FKBP12.6 on K+ currents in adult rabbit ventricular myocytes

This study examines the effects of the intracellular protein FKBP12.6 on action potential and associated K+ currents in isolated adult rabbit ventricular cardiomyocytes. FKBP12.6 was over-expressed by ~6 times using a recombinant adenovirus coding for human FKBP12.6. This over-expression caused prolongation of action potential duration (APD) by ~30%. The amplitude of the transient outward current (Ito) was unchanged, but rate of inactivation at potentials positive to +40 mV was increased. FKBP12.6 over-expression decreased the amplitude of the inward rectifier current (IK1) by ~25% in the voltage range −70 to −30 mV, an effect prevented by FK506 or lowering intracellular [Ca2+] below 1 nM. Over-expression of an FKBP12.6 mutant, which cannot bind calcineurin, prolonged APD and affected Ito and IK1 in a similar manner to wild-type protein. These data suggest that FKBP12.6 can modulate APD via changes in IK1 independently of calcineurin binding, suggesting that FKBP12.6 may affect APD by direct interaction with IK1

Sphingomyelin Synthases Regulate Protein Trafficking and Secretion

Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD), to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN), the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP) from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2) are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi

FKBP12 Activates the Cardiac Ryanodine Receptor Ca2+-Release Channel and Is Antagonised by FKBP12.6

Changes in FKBP12.6 binding to cardiac ryanodine receptors (RyR2) are implicated in mediating disturbances in Ca2+-homeostasis in heart failure but there is controversy over the functional effects of FKBP12.6 on RyR2 channel gating. We have therefore investigated the effects of FKBP12.6 and another structurally similar molecule, FKBP12, which is far more abundant in heart, on the gating of single sheep RyR2 channels incorporated into planar phospholipid bilayers and on spontaneous waves of Ca2+-induced Ca2+-release in rat isolated permeabilised cardiac cells. We demonstrate that FKBP12 is a high affinity activator of RyR2, sensitising the channel to cytosolic Ca2+, whereas FKBP12.6 has very low efficacy, but can antagonise the effects of FKBP12. Mathematical modelling of the data shows the importance of the relative concentrations of FKBP12 and FKBP12.6 in determining RyR2 activity. Consistent with the single-channel results, physiological concentrations of FKBP12 (3 µM) increased Ca2+-wave frequency and decreased the SR Ca2+-content in cardiac cells. FKBP12.6, itself, had no effect on wave frequency but antagonised the effects of FKBP12

Electrophysiological and Structural Remodeling in Heart Failure Modulate Arrhythmogenesis. 1D Simulation Study

Background: Heart failure is a final common pathway or descriptor for various cardiac pathologies. It is associated with sudden cardiac death, which is frequently caused by ventricular arrhythmias. Electrophysiological remodeling, intercellular uncoupling, fibrosis and autonomic imbalance have been identified as major arrhythmogenic factors in heart failure etiology and progression. Objective: In this study we investigate in silico the role of electrophysiological and structural heart failure remodeling on the modulation of key elements of the arrhythmogenic substrate, i.e., electrophysiological gradients and abnormal impulse propagation. Methods: Two different mathematical models of the human ventricular action potential were used to formulate models of the failing ventricular myocyte. This provided the basis for simulations of the electrical activity within a transmural ventricular strand. Our main goal was to elucidate the roles of electrophysiological and structural remodeling in setting the stage for malignant life-threatening arrhythmias. Results: Simulation results illustrate how the presence of M cells and heterogeneous electrophysiological remodeling in the human failing ventricle modulate the dispersion of action potential duration and repolarization time. Specifically, selective heterogeneous remodeling of expression levels for the Na+ /Ca2+ exchanger and SERCA pump decrease these heterogeneities. In contrast, fibroblast proliferation and cellular uncoupling both strongly increase repolarization heterogeneities. Conduction velocity and the safety factor for conduction are also reduced by the progressive structural remodeling during heart failure. Conclusion: An extensive literature now establishes that in human ventricle, as heart failure progresses, gradients for repolarization are changed significantly by protein specific electrophysiological remodeling (either homogeneous or heterogeneous). Our simulations illustrate and provide new insights into this. Furthermore, enhanced fibrosis in failing hearts, as well as reduced intercellular coupling, combine to increase electrophysiological gradients and reduce electrical propagation. In combination these changes set the stage for arrhythmias.This work was partially supported by (i) the "VI Plan Nacional de Investigacion Cientifica, Desarrollo e Innovacion Tecnologica" from the Ministerio de Economia y Competitividad of Spain (grant number TIN2012-37546-C03-01) and the European Commission (European Regional Development Funds - ERDF - FEDER), (ii) the Direccion General de Politica Cientifica de la Generalitat Valenciana (grant number GV/2013/119), and (iii) Programa Prometeo (PROMETEO/2012/030) de la Conselleria d'Educacio Formacio I Ocupacio, Generalitat Valenciana. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Gómez García, JF.; Cardona, K.; Romero Pérez, L.; Ferrero De Loma-Osorio, JM.; Trénor Gomis, BA. (2014). Electrophysiological and Structural Remodeling in Heart Failure Modulate Arrhythmogenesis. 1D Simulation Study. PLoS ONE. 9(9). https://doi.org/10.1371/journal.pone.0106602S9

Ca2+-handling proteins and heart failure: Novel molecular targets?

Calcium (Ca2+) ions are the currency of heart muscle activity. During excitation-contraction coupling Ca2+ is rapidly cycled between the cytosol (where it activates the myofilaments) and the sarcoplasmic reticulum (SR), the Ca2+ store. These fluxes occur by the transient activity of Ca2+-pumps and -channels. In the failing human heart, changes in activity and expression profile of Ca2+-handling proteins, in particular the SR Ca2+-ATPase (SERCA2a), are thought to cause an overall reduction in the amount of SR-Ca2+ available for contraction. In the steady state, the Ca2+-content of the SR is essentially a balance between Ca2+-uptake via SERCA2a pump and Ca2+-release via the cardiac SR Ca2+-release channel complex (Ryanodine receptor, RyR2). This review discusses current pharmacological options available to enhance cardiac SR Ca2+ content and the implications of this approach as an inotropic therapy in heart failure. Two options are considered: (i) activation of the SERCA2a pump to increase SR Ca2+-uptake, and (ii) reduction of SR Ca2+-leakage through RyR2. RyR2 forms a macromolecular complex with a number of regulatory proteins that either remain permanently bound or that interact in a time- and/or Ca2+-dependant manner. These regulatory proteins can dramatically affect RyR2 function, e.g. over-expression of the accessory protein FK 506-binding protein 12.6 (FKBP12.6) has recently been shown to reduce SR Ca2+-leak. Recent attempts to design positive inotropes for chronic administrations have focussed on the use of phosphodiesterase III inhibitors (PDE III inhibitors). These compounds, which increase intracellular cAMP-levels, have failed in clinical trials. Therefore medical researchers are seeking new drugs that act through alternative pathways. Novel cardiac inotropes targeting SR Ca2+-cycling proteins may have the potential to fill this gap

FK506-binding Protein FKBP12.6 Overexpression Alters the Characteristics of the Ca2+ Transient and Caffeine-induced Ca2+ Release in Adult Rabbit Cardiac Myocytes

No abstract available

FK506-binding Protein FKBP12.6 Overexpression Alters the Characteristics of the Ca2+ Transient and Caffeine-induced Ca2+ Release in Adult Rabbit Cardiac Myocytes

No abstract available

Overexpression of FK506-Binding protein FKBP12.6 in cardiomyocytes reduces ryanodine receptor-mediated Ca2+ leak from the sarcoplasmic reticulum and increases contractility

The FK506-binding protein FKBP12.6 is tightly associated with the cardiac sarcoplasmic reticulum (SR) Ca2+-release channel (ryanodine receptor type 2 [RyR2]), but the physiological function of FKBP12.6 is unclear. We used adenovirus (Ad)-mediated gene transfer to overexpress FKBP12.6 in adult rabbit cardiomyocytes. Western immunoblot and reverse transcriptase-polymerase chain reaction analysis revealed specific overexpression of FKBP12.6, with unchanged expression of endogenous FKBP12. FKBP12.6-transfected myocytes displayed a significantly higher (21%) fractional shortening (FS) at 48 hours after transfection compared with Ad-GFP-infected control cells (4.8+/-0.2% FS versus 4+/-0.2% FS, respectively; n=79 each; P=0.001). SR-Ca2+ uptake rates were monitored in beta -escin-permeabilized myocytes using Fura-2. Ad-FKBP12.6-infected cells showed a statistically significant higher rate of Ca2+ uptake of 0.8+/-0.09 nmol/s(-1)/10(6) cells (n = 8, P<0.05) compared with 0.52+/-0.1 nmol/s(-1)/10(6) cells in sham-infected cells (n = 8) at a [Ca2+] of 1 <mu>mol/L. In the presence of 5 mu mol/L ruthenium red to block Ca2+ efflux via RyR2, SR-Ca2+ uptake rates were not significantly different between groups. From these measurements, we calculate that SR-Ca2+ leak through RyR2 is reduced by 53% in FKBP12.6-overexpressing cells. Caffeine-induced contractures were significantly larger in Ad-FKBP12.6-infected myocytes compared with Ad-GFP-infected control cells, indicating a higher SR-Ca2+ load. Taken together, these data suggest that FKBP12.6 stabilizes the closed conformation state of RyR2. This may reduce diastolic SR-Ca2+ leak and consequently increase SR-Ca2+ release and myocyte shortening

Downregulation of the Na(+)-creatine cotransporter in failing human myocardium and in experimental heart failure.

BACKGROUND:The failing myocardium is characterized by depletion of phosphocreatine and of total creatine content. We hypothesized that this is due to loss of creatine transporter protein. METHODS AND RESULTS:Creatine transporter protein was quantified in nonfailing and failing human myocardium (explanted hearts with dilated cardiomyopathy [DCM; n=8] and healthy donor hearts [n=8]) as well as in experimental heart failure (residual intact left ventricular tissue, rats 2 months after left anterior descending coronary artery ligation [MI; n=8] or sham operation [sham; n=6]) by Western blotting. Total creatine content was determined by high-performance liquid chromatography. Donor and DCM hearts had total creatine contents of 136.4+/-6.1 and 68.7+/-4.6 nmol/mg protein, respectively (*P<0.05); creatine transporter protein was 25.4+/-2.2 optical density units in donor and 17.7+/-2.5 in DCM (*P<0.05). Total creatine was 87.5+/-4.2 nmol/mg protein in sham and 65.7+/-4.2 in MI rats (*P<0.05); creatine transporter protein was 139.0+/-8.7 optical density units in sham and 82.1+/-4.0 in MI (*P<0.05). CONCLUSIONS:Both in human and in experimental heart failure, creatine transporter protein content is reduced. This mechanism may contribute to the depletion of creatine compounds and thus to the reduced energy reserve in failing myocardium. This finding may have therapeutic implications, suggesting a search for treatment strategies targeted toward creatine transport