FKBP12 Levels Research Articles

Dual effect of cardiac FKBP12.6 overexpression on excitation-contraction coupling and the incidence of ventricular arrhythmia depending on its expression level

FKBP12.6, a binding protein to the immunosuppressant FK506, which also binds the ryanodine receptor (RyR2) in the heart, has been proposed to regulate RyR2 function and to have antiarrhythmic properties. However, the level of FKBP12.6 expression in normal hearts remains elusive and some controversies still persist regarding its effects, both in basal conditions and during β-adrenergic stimulation. We quantified FKBP12.6 in the left ventricles (LV) of WT (wild-type) mice and in two novel transgenic models expressing distinct levels of FKBP12.6, using a custom-made specific anti-FKBP12.6 antibody and a recombinant protein. FKBP12.6 level in WT LV was very low (0.16 ± 0.02 nmol/g of LV), indicating that <15% RyR2 monomers are bound to the protein. Mice with 14.1 ± 0.2 nmol of FKBP12.6 per g of LV (TG1) had mild cardiac hypertrophy and normal function and were protected against epinephrine/caffeine-evoked arrhythmias. The ventricular myocytes showed higher [Ca2+]i transient amplitudes than WT myocytes and normal SR-Ca2+ load, while fewer myocytes showed Ca2+ sparks. TG1 cardiomyocytes responded to 50 nM Isoproterenol increasing these [Ca2+]i parameters and producing RyR2-Ser2808 phosphorylation. Mice with more than twice the TG1 FKBP12.6 value (TG2) showed marked cardiac hypertrophy with calcineurin activation and more arrhythmias than WT mice during β-adrenergic stimulation, challenging the protective potential of high FKBP12.6. RyR2R420Q CPVT mice overexpressing FKBP12.6 showed fewer proarrhythmic events and decreased incidence and duration of stress-induced bidirectional ventricular tachycardia. Our study, therefore, quantifies for the first time endogenous FKBP12.6 in the mouse heart, questioning its physiological relevance, at least at rest due its low level. By contrast, our work demonstrates that with caution FKBP12.6 remains an interesting target for the development of new antiarrhythmic therapies.

Rapalogs can promote cancer cell stemness in vitro in a Galectin-1 and H-ras-dependent manner.

Currently several combination treatments of mTor- and Ras-pathway inhibitors are being tested in cancer therapy. While multiple feedback loops render these central signaling pathways robust, they complicate drug targeting.Here, we describe a novel H-ras specific feedback, which leads to an inadvertent rapalog induced activation of tumorigenicity in Ras transformed cells. We find that rapalogs specifically increase nanoscale clustering (nanoclustering) of oncogenic H-ras but not K-ras on the plasma membrane. This increases H-ras signaling output, promotes mammosphere numbers in a H-ras-dependent manner and tumor growth in ovo. Surprisingly, also other FKBP12 binders, but not mTor-inhibitors, robustly decrease FKBP12 levels after prolonged (>2 days) exposure. This leads to an upregulation of the nanocluster scaffold galectin-1 (Gal-1), which is responsible for the rapamycin-induced increase in H-ras nanoclustering and signaling output. We provide evidence that Gal-1 promotes stemness features in tumorigenic cells. Therefore, it may be necessary to block inadvertent induction of stemness traits in H-ras transformed cells by specific Gal-1 inhibitors that abrogate its effect on H-ras nanocluster. On a more general level, our findings may add an important mechanistic explanation to the pleiotropic physiological effects that are observed with rapalogs.

Abstract 1877: A new H-ras specific feedback loop from the TOR-pathway impacts on tumorigenicity

Abstract Introduction: The lack of direct Ras inhibitors, has led to the development of inhibitors of downstream components of the Ras pathway albeit with mixed success. Unexpected mechanistic complexity, feedback loops and tumor specific synthetically lethal requirements continue to be major obstacles. In the past we investigated the mechanisms of the Ras membrane signaling system on the nanoscale (nanoclustering). Ras nanoclustering on the plasma membrane is critical for its signaling activity and we and others recently showed how it is exploited in a number of physiological and pathophysiological conditions, including cancer. Importantly, Ras isoform specificity seems to emerge at the level of laterally segregated nanocluster. This is supported by isoform specific nanocluster scaffolds, such as galectin-1 (Gal-1), which specifically augments GTP-H-ras nanocluster. Methods: The effect of compounds and siRNAs targeting the TOR pathway and associated proteins on H- vs. K-ras4B (hereafter K-ras)-nanoclustering was evaluated using FRET-measurements in BHK and HEK cells, or point pattern analysis of electron micrographs from BHK cell plasma membrane sheets. Ras specific effects of treatments were furthermore assessed in PC12 differentiation assays, driven by oncogenic H- or K-ras. Changes in downstream signaling were evaluated using Western Blotting of treated BHK and HEK cells. The effect of treatments (compounds or siRNAs) on mammosphere formation was assessed in MDA-MB-231 cells. Results: Here we describe a new feedback mechanism from the TOR-pathway specifically to H-ras, but not K-ras. Inhibition of FKBP12 inhibitors cycloheximide (CHX), Rapamycin (Rapa) and FK506, as well as knockdown of FKBP12 specifically increased H-ras nanoclustering. While all compounds could also specifically increase differentiation of H- but not K-rasG12V transfected PC12 cells, their effect on cellular signaling pathways differed in BHK cells. Both, CHX and Rapa increased Erk phosphorylation, but only Rapa blocked S6K and S6 phosphorylation, while CHX did the opposite. Surprisingly, siRNA-mediated ablation of FKBP12 upregulated Gal-1 protein levels, suggesting that the observed increase in H-ras nanoclustering is due to this response. This was however not observed for FKBP12 inhibitors. On the other hand overexpression of Gal-1 also increased FKBP12 levels. Intriguingly, only FKBP12 ablation (with concomitant Gal-1 upregulation) was sufficient to increase the mammosphere forming ability, but not Gal-1 overexpression alone. This effect could be mimicked by FKBP12 inhibition with CHX or Rapa, but not FK506. Conclusions: Our data indicate a H-ras specific feedback from inhibition of the TOR pathway, which can lead to increased mammosphere formation. These data suggest a tumor promoting effect by TOR pathway inhibitors via H-ras probably in a specific cellular and genetic context. Citation Format: Itziar M. D. Posada, Benoit Lectez, Christina Oetken-Lindholm, Daniel Abankwa. A new H-ras specific feedback loop from the TOR-pathway impacts on tumorigenicity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1877.

Rapamycin-mediated mTORC2 inhibition is determined by the relative expression of FK506-binding proteins.

The mechanism by which the drug rapamycin inhibits the mechanistic target of rapamycin (mTOR) is of intense interest because of its likely relevance in cancer biology, aging, and other age-related diseases. While rapamycin acutely and directly inhibits mTORC1, only chronic administration of rapamycin can inhibit mTORC2 in some, but not all, cell lines or tissues. The mechanism leading to cell specificity of mTORC2 inhibition by rapamycin is not understood and is especially important because many of the negative metabolic side effects of rapamycin, reported in mouse studies and human clinical trials, have been attributed recently to mTORC2 inhibition. Here, we identify the expression level of different FK506-binding proteins (FKBPs), primarily FKBP12 and FKBP51, as the key determinants for rapamycin-mediated inhibition of mTORC2. In support, enforced reduction of FKBP12 completely converts a cell line that is sensitive to mTORC2 inhibition to an insensitive cell line, and increased expression can enhance mTORC2 inhibition. Further reduction of FKBP12 in cell lines with already low FKBP12 levels completely blocks mTORC1 inhibition by rapamycin, indicating that relative FKBP12 levels are critical for both mTORC1 and mTORC2 inhibition, but at different levels. In contrast, reduction of FKBP51 renders cells more sensitive to mTORC2 inhibition. Our findings reveal that the expression of FKBP12 and FKBP51 is the rate limiting factor that determines the responsiveness of a cell line or tissue to rapamycin. These findings have implications for treating specific diseases, including neurodegeneration and cancer, as well as targeting aging in general.

Species- and chamber-specific responses of 12 kDa FK506-binding protein to temperature in fish heart

The sarcoplasmic reticulum (SR) Ca(2+) release channel or ryanodine receptor (RyR) of the vertebrate heart is regulated by the FK506-binding proteins, FKBP12 and FKBP12.6. This study examines whether temperature-related changes in the SR function of fish hearts are associated with changes in FKBP12 expression. For this purpose, a polyclonal antibody against trout FKBP12 was used to compare FKPB12 expression in cold-acclimated (4°C, CA) and warm-acclimated (18°C, WA) rainbow trout (Oncorhynchus mykiss), burbot (Lota lota) and crucian carp (Carassius carassius) hearts. FKBP12 expression was modulated in a species- and tissue-specific manner. Temperature acclimation affected FKBP12 expression only in atrial tissue. Changes in the ventricular FKBP12 expression were not detected in any of the fish species. In the atria of rainbow trout and crucian carp, temperature acclimation produced opposite thermal responses: FKBP12 increased in the trout atrium and decreased in the crucian carp atrium under cold acclimation. In the burbot heart, chronic temperature changes did not affect cardiac FKBP12 levels. Expression of FKBP12 mRNA in rainbow trout and crucian carp hearts suggests that the transcript levels are higher in the ventricle than in the atrium and are elevated by cold acclimation in trout, but not in crucian carp. Since FKBP12 is known to increase the Ca(2+) sensitivity of cardiac RyRs and thereby the opening frequency of the Ca(2+) release channels, temperature-related changes in FKBP12 expression may modify the SR function in excitation-contraction coupling. The cold-induced increase in FKBP12 in the trout atrium and decrease in the crucian carp atrium are consistent with the previously noted increase and decrease, respectively, of SR Ca(2+) stores in cardiac contraction in these species.

FKBP12.6 overexpression does not protect against remodelling after myocardial infarction

Reducing the open probability of the ryanodine receptor (RyR) has been proposed to have beneficial effects in heart failure. We investigated whether conditional FKBP12.6 overexpression at the time of myocardial infarction (MI) could improve cardiac remodelling and cell Ca(2+) handling. Wild-type (WT) mice and mice overexpressing FKBP12.6 (Tg) were studied on average 7.5 ± 0.2 weeks after MI and compared with sham-operated mice for in vivo, myocyte function and remodelling. At baseline, unloaded cell shortening in Tg was not different from WT. The [Ca(2+)](i) transient amplitude was similar, but sarcoplasmic reticulum (SR) Ca(2+) content was larger in Tg, suggesting reduced fractional release. Spontaneous spark frequency was similar despite the increased SR Ca(2+) content, consistent with a reduced RyR channel open probability in Tg. After MI, left ventricular dilatation and myocyte hypertrophy were present in both groups, but more pronounced in Tg. Cell shortening amplitude was unchanged with MI in WT, but increased with MI in Tg. The amplitude of the [Ca(2+)](i) transient was not affected by MI in either genotype, but time to peak was increased; this was most pronounced in Tg. The SR Ca(2+) content and Na(+)- Ca(2+) exchanger function were not affected by MI. Spontaneous spark frequency was increased significantly after MI in Tg, and larger than in WT (at 4 Hz, 2.6 ± 0.4 sparks (100 μm)(-1) s(-1) in Tg MI versus 1.6 ± 0.2 sparks (100 μm)(-1) s(-1) in WT MI; P < 0.05). We conclude that FKPB12.6 overexpression can effectively reduce RyR open probability with maintained cardiomyocyte contraction. However, this approach appears insufficient to prevent and reduce post-MI remodelling, indicating that additional pathways may need to be targeted.

Suppression of EGFR Autophosphorylation by FKBP12

FK506 binding proteins (FKBPs) represent a subfamily of peptidyl prolyl cis/trans isomerases that can control receptor-mediated intracellular signaling. The prototypic PPIase FKBP12 functionally interacts with EGFR. FKBP12 was shown to inhibit EGF-induced EGFR autophosphorylation with all internal phosphorylation sites equally affected. The inhibition of EGFR catalytic activity is conducted by targeting the EGFR kinase domain. The change of intracellular FKBP12 levels resulted in a change of EGFR autophosphorylation level. Collectively, our results demonstrate that FKBP12 forms an endogenous inhibitor of EGFR phosphorylation directly involved in the control of cellular EGFR activity.

Targeted immunosuppression: No longer naïve

Targeted immunosuppression: No longer naïve

AQP4 knockout mice manifest abnormal expressions of calcium handling proteins possibly due to exacerbating pro-inflammatory factors in the heart

We tested the hypothesis that aquaporin-4 (AQP4) knockout (KO) mice might exhibit abnormal Ca 2+ modulating proteins resulting from the exacerbation of pro-inflammatory factors in the heart. Downregulation of FKBP12.6, SERCA2a, and CASQ2 and calcium leak in diastole have been recognized as endpoints for assessing cardiac failure and arrhythmias. The AQP4 KO mice and wild-type (WT) mice were randomly divided into 3 groups, such as control, isoproterenol (ISO, β-receptor agonist) injected (1 mg/kg, sc, 5 d), and treated with aminoguanidine (AMG, 100 mg/kg, po, a selective inhibitor of the iNOS) during the last 3 d. RT-PCR, western blot and calcium transient measurements were conducted. The results demonstrated that the cardiac weight index was increased in AQP4 KO mice and further increased following treatment with ISO. The expression levels of FKBP12.6, SERCA2a, and CASQ2 were downregulated and diastolic calcium concentrations were elevated in the AQP4 KO mice, indicative of a calcium leak. In the myocardium, expressions of pro-inflammatory biomarkers, including ET A, pPKCɛ, NADPH oxidase p67 phox were upregulated and associated with downregulation of Cx43. The aforementioned changes were exacerbated in response to ISO medication and were attenuated by AMG; however, its treatment effectiveness was less in the AQP4 KO mice. We concluded AQP4 KO caused abnormalities of calcium modulating proteins leading to an exacerbation of risk for cardiac arrhythmias and failure. These changes are likely due to an increase in pro-inflammatory factors which are exacerbated by stress. Therefore, AQP4 KO mice are prone to cardiac failure and arrhythmias through exacerbating pro-inflammatory factors in the myocardium.

FKBP12.6 ‘Stabilises’ Cardiac SR Ca 2+-Release by Antagonising High-Affinity Reversible Activation of RyR2 by FKBP12

FKBP12.6 is thought to play an important cardioprotective role, however, the underlying mechanism is not understood. Since FKBP12 is structurally similar to FKBP12.6 but is found at much higher levels (1-3 µM), we investigated the effects of both FKBP12 and FKBP12.6 on RyR2 single-channel function and on SR Ca2+-release in rat isolated permeabilised cardiomyocytes. FKBP12 increased RyR2 open probability (Po) in a concentration-dependent, reversible manner (EC50 51 nM). Physiological levels of FKBP12 (3 µM) increased Po from 0.187±0.051 to 0.657±0.111 (SEM; n=14; P<0.001). FKBP12.6 (200 nM), itself, did not significantly alter RyR2 Po, but was a very effective antagonist of FKBP12, shifting the FKBP12 EC50 to 4 μM. In permeabilised myocytes perfused with Fluo-5F, spontaneous waves of Ca2+-induced Ca2+-release were induced by 234 nM Ca2+ in the mock cytosolic solution. Perfusion with FKBP12 (3 µM) increased wave frequency from 0.34±0.04 Hz to 0.52±0.07 Hz (SEM; n=14; p<0.03). 10 mM caffeine produced a larger Ca2+-transient in control (2.21±0.11; F/Fo) than in FKBP12 (1.47±0.16; n=6; p<0.003) indicating lower SR Ca2+-content. Perfusion with FKBP12.6 (200 nM) alone, had no significant effect yet it reduced the ability of FKBP12 to increase wave frequency (49.9±5.8% increase over control in the absence of FKBP12.6 vs. 16.2±2.1% in the presence). Our single-channel experiments demonstrate that FKBP12 is a high affinity, potent activator of RyR2. FKBP12.6 acts as an antagonist of FKBP12 at RyR2 but itself possesses minimal efficacy. Our cellular experiments suggest that this is the underlying mechanism by which FKBP12.6 acts to ‘stabilise’ or reduce SR Ca2+-release in cardiac cells. Thus, the balance between the opposing actions of FKBP12 and FKBP12.6 on RyR2 gating may be crucial for normal EC-coupling in cardiac cells. British Heart Foundation Funding

Diabetes-related defects in sarcoplasmic Ca2+ release are prevented by inactivation of Gα11 and Gαq in murine cardiomyocytes

Neurohumoral stimulation of Gq-coupled receptors has been proposed as a central mechanism in the pathogenesis of diabetic heart disease. The resulting contractile dysfunction is closely related to abnormal intracellular Ca(2+) handling with functional defects of the sarcoplasmic reticulum (SR). The present study was therefore designed to determine the role of G(q)-protein signaling via G(alpha)(11) and G(alpha)(q) in diabetes for the induction of functional and structural changes in the Ca(2+) release complex of the SR. An experimental type 1-diabetes was induced in wild type, G(alpha)(11) knockout, and G(alpha)(11/q)-knockout mice by injection of streptozotocin. Cardiac morphology and function was assessed in vivo by echocardiography. SR Ca(2+) leak was tested in vitro based on a (45)Ca(2+) assay and protein densities as well as gene expression of ryanodine receptor (RyR2), FKBP12.6, sorcin, and annexin A7 were analyzed by immunoblot and RT-PCR. In wild type animals 8 weeks of diabetes resulted in cardiac hypertrophy and SR Ca(2+) leak was increased. In addition, diabetic wild type animals showed reduced protein levels of FKBP12.6 and annexin A7. In G(alpha)(11)- and G(alpha)(11/q)-knockout animals, however, SR Ca(2+) release and cardiac phenotype remained unchanged upon induction of diabetes. Densities of the proteins that we presently analyzed were also unaltered in G(alpha)(11)-knockout mice. G(alpha)(11/q)-knockout animals even showed increased expression of sorcin and annexin A7. Thus, based on the present study we suggest a signaling pathway via the G(q)-proteins, G(alpha)(11) and G(alpha)(q), that could link increased neurohumoral stimulation in diabetes with defective RyR2 channel function by regulating protein expression of FKBP12.6, annexin A7, and sorcin.

FKBP12 is a High Affinity, Reversible Activator of RyR2, and FKBP12.6 Antagonises Its Actions

FKBP12.6 binds tightly to RyR2 and evidence suggests that it plays a vital physiological role in regulating channel activity. Moreover, changes in FKBP12.6/RyR2 interactions have been implicated in heart failure. Controversy exists, however, as to how FKBP12.6 affects the single-channel behaviour of RyR2. Furthermore, although higher levels of FKBP12 than FKBP12.6 are present in cardiac cells, the effects of FKBP12 on RyR2 are virtually unresearched. We have therefore compared the effects of FKBP12 and FKBP12.6 on the single-channel function of sheep RyR2 incorporated into bilayers under voltage-clamp conditions. We find that FKBP12 increases RyR2 open probability (Po) in a dose-dependant, reversible manner with an EC50 of 51 nM. In the presence of 10 μM cytosolic Ca2+, physiological levels of FKBP12 (3 μM) increased Po from 0.187±0.051 in controls to 0.657±0.111 (SEM; n=14; P<0.001). In contrast, under identical experimental conditions, FKBP12.6 did not significantly increase or decrease RyR2 Po, however, it was able to antagonise the actions of FKBP12, shifting the EC50 value for FKBP12 to 4 μM. Our experiments demonstrate that FKBP12 has high affinity for RyR2 and that at physiological concentrations (1-3 μM) is an effective activator of the channel thereby suggesting that FKBP12 may have a more important role in cardiac excitation-contraction coupling than previously thought. We hypothesise that FKBP12.6 is a very low efficacy (but high affinity) partial agonist of RyR2 and that the balance between the effects of FKBP12 and FKBP12.6 is crucial for normal EC-coupling in cardiac cells. Supported by the British Heart Foundation

Endothelin receptor antagonist CPU0213 and vitamin E reverse downregulation of FKBP12.6 and SERCA2a: A role of hyperphosphorylation of PKCε

Downregulation of FKBP12.6 and sarcoplasmic reticulum Ca 2+ ATPase (SERCA2a) contributes to sudden cardiac death and heart failure. We aimed to test the hypothesis that (i) downregulation of FKBP12.6 and SERCA2a can be taken as molecular markers for drug interventions and (ii) such downregulation is produced by crosstalk between endothelin-reactive oxygen species and β-adrenoceptors stimulation, mediated by hyperphosphorylation of protein kinase Cɛ (PKCɛ). Rat cardiomyocytes were incubated with isoproterenol (1 μM), endothelin-1 (0.1 μM) or hydrogen peroxide (10 μM) for 18 h, resulting in downregulation of mRNA and protein of FKBP12.6 and SERCA2a, as well as upregulation of PKCɛ mRNA and phosphorylated PKCɛ protein. These changes were reversed by an application of either propranolol (1 μM), endothelin receptor antagonist CPU0213 (1 μM) or vitamin E (1 μM). As indicated by the fluorescent dye Fluo3, diastolic [Ca 2+] i in rat ventricular myocytes was increased after incubation with isoproterenol (0.1 μM). The increased [Ca 2+] i in diastole was dramatically decreased by CPU0213. Thus, the downregulation of FKBP12.6 and SERCA2a, and hyperphosphorylation of PKCɛ, appear to be related to crosstalk between over-activated endothelin-reactive oxygen species and a β-adrenoceptor pathway. CPU0213 is beneficial in treating cardiac insufficiency and preventing cardiac arrhythmias possibly by normalizing hyperphosphorylation of PKCɛ and abnormal FKBP12.6 and SERCA2a. The antioxidant activity of vitamin E was sufficient to normalize the levels of FKBP12.6 and SERCA2a and phosphorylation of PKCɛ. Thus by testing with biomarkers FKBP12.6 and SERCA2a, we have shown that the endothelin receptor antagonist CPU0213 and the antioxidant vitamin E may relieve risk of lethal arrhythmias and heart failure by suppressing PKCɛ.

Increased Striatal mRNA and Protein Levels of the Immunophilin FKBP-12 in Experimental Parkinson's Disease and Identification of FKBP-12-Binding Proteins

FKBP-12, a 12 kDa FK506-binding protein (neuroimmunophilin), acts as a receptor for the immunosuppressant drug FK506. Neuroimmunophilins, including FKBP-12, are abundant in the brain and have been shown to be involved in reversing neuronal degeneration and preventing cell death. In this report, we have utilized several analytical techniques, such as in situ hybridization, Western blotting, two-dimensional gel electrophoresis, and liquid chromatography electrospray tandem mass spectrometry to study the transcriptional expression as well as protein levels of FKBP-12 in the unilateral 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. The FKBP-12 protein was also detected directly on brain tissue sections using mass spectrometry profiling. We found increased levels of FKBP-12 mRNA and protein in the dorsal and middle part of the 6-OHDA lesioned striatum. Thus, these studies clearly demonstrate that FKBP-12 is increased in the brain of a common animal model of Parkinson's disease (PD). Additionally, we have identified potential binding partners to FKBP-12 that may be implicated in the pathophysiology of Parkinson's disease, such as alpha-enolase, 14-3-3 zeta/delta, pyruvate kinase isozymes, and heat shock protein 70, using surface plasmon resonance sensor technology in combination with mass spectrometry. In conclusion, these data strongly suggests that FKBP-12 is altered in an experimental model of PD.

174: Immunophilin Ligand, GPI-1046, Attenuates Neuronal Cell Death Following Radiation

Nonimmunosuppressive immunophilin ligands act by binding to immunophilin receptor proteins such as FKBP-12 resulting in a decrease in the morbidity associated with neuronal crush injury. It is not known whether radiation-induced neuronal injury also results in alteration of FKBP-12 levels or whether the resulting injury can be modulated by immunophilin ligand binding to FKBP-12. To study this, we investigated the relationship between FKBP-12 expression patterns and radiation exposure as well as performed clonogenic survival assays to determine the protective effects of GPI-1046, an immunophilin ligand, in both an in vitro mammalian neuronal model as well as in human prostate cancer cell lines.

Dyssynchronous (non-uniform) Ca2+ release in myocytes from streptozotocin-induced diabetic rats

Using biochemical/pharmacological approaches, we previously showed that type 2 ryanodine receptors (RyR2) become dysfunctional in hearts of streptozotocin-induced type 1 diabetic rats. However, the functional consequence of this observation remains incompletely understood. Here we use laser confocal microscopy to investigate whether RyR2 dysfunction during diabetes alters evoked and spontaneous Ca2+ release from the sarcoplasmic reticulum (SR). After 7–8 weeks of diabetes, steady-state levels of RyR2 remain unchanged in hearts of male Sprague-Dawley rats, but the number of functional receptors decreased by >37%. Interestingly, residual functional RyR2 from diabetic rat hearts exhibited increased sensitivity to Ca2+ activation (EC50activation decreased from 80 μM to 40 μM, peak Ca2+ activation decreased from 425 μM to 160 μM). When field stimulated, intracellular Ca2+ release in diabetic ventricular myocytes was dyssynchronous (non-uniform) and this was independent of L-type Ca2+ currents. Time to peak Ca2+ increased 3.7-fold. Diabetic myocytes also exhibited diastolic Ca2+ release and 2-fold higher frequency of spontaneous Ca2+ sparks, albeit at a lower amplitude. The amplitude of caffeine-releasable Ca2+ was also lower in diabetic myocytes. RyR2 from diabetic rat hearts exhibited increased phosphorylation at Ser2809 and contained reduced levels of FKBP12.6 (calstablin2). Collectively, these data suggest that RyR2 becomes leaky during diabetes and this defect may be responsible to the reduced SR Ca2+ load. Diastolic Ca2+ release could also serve as a substrate for delayed after-depolarizations, contributing to the increased incidence of arrhythmias and sudden cardiac death in type 1 diabetes.

Ryanodine receptor mutations and ventricular arrhythmias: channel instability at the heart of Ca2+ release dysfunction

Cardiac ryanodine receptors (RyR2) are tetrameric Ca2+ release channels of enormous structural and functional complexity. Inherited or sporadic RyR2 mutations are associated with malignant ventricular arrhythmias induced by exercise or stress, and frequently lead to sudden cardiac death (SCD) in young adolescents. Since 2001, sixty-four mutations have been identified that cluster in three discrete regions of the protein that probably coalesce into close physical proximity in the folded channel architecture. These mutational ‘hot-spots’ in the amino-, central, and carboxyl- terminus are proposed to be critical determinants of intrinsic channel regulation via interdomain interaction. Acquired or mutation-linked weakening of these regional interactions (‘domain unzipping’) is proposed to underlie arrhythmogenesis in heart failure and stress-induced VT, respectively. We used a FRET-based strategy to monitor the dynamic intra-RyR2 interactions that occurred in wild-type and mutant channels in living cells. We showed that clinically severe mutations profoundly disrupted interdomain interactions and caused conformational instability in activated RyR2 channels. Mutation-linked channel instability occurred in diverse cell backgrounds (non-myocytic versus cardiomyocytes), and was independent of the cellular levels of FKBP12.6, a regulatory co-protein whose role in arrhythmia is highly controversial. These new insights provided good evidence for a structural basis of Ca2+ release dysfunction in RyR2-dependent arrhythmia. The implications of these data for the development of novel anti-arrhythmic therapies based on RyR2 stabilisation will be discussed.

Immunophilin expression in the HIV-infected brain

Immunophilins (IP) are receptors for immunosuppressive drugs and also aid protein folding, assembly, and trafficking. This autopsy study analyses the levels of FKBP12 in the brain of patients with HIV encephalitis. We show that the expression of FKBP12 is increased in the deep gray matter and frontal cortex where it colocalizes with markers of pathology. Changes in FKBP12 levels may be linked to the abnormal protein folding and axonal transport that characterize the neurodegenerative process and could also explain the selective vulnerability of the basal ganglia to HIV infection.

Immunophilins in nervous system degeneration and regeneration.

Immunophilins are receptors for immunosuppressive drugs like cyclosporin A, FK506, rapamycin and their non- immunosuppressive analogs, which are collectively referred to as "immunophilin ligands" (IPL). Cyclosporin A binds to a class of IP called cyclophilins, whereas the receptors for FK506 and rapamycin belong to the family of FK506- binding proteins (FKBP). The latter are designated according to their molecular weight: FKBP12, 25, 52 etc. FKBP levels in the rat brain are up to 50 times higher than in the immune system. FKBP12 is associated with IP3 and ryanodine receptors present on the endoplasmic reticulum and plays a role in stabilizing calcium release. It has also been proposed to be a modulator of the TGFbeta receptor activity. Crush injury of facial or sciatic nerves in rat leads to markedly increased FKBP12 levels in the respective nerve nuclei and this increase is related to nerve regeneration. Cyclophilin A protects cells from death following expression of mutant Cu/ Zn superoxide dismutase, which is associated with familial amyotrophic lateral sclerosis. Our recent studies show that FKBP12 and FKBP52 are expressed in the human nervous system, especially in the substantia nigra- deep gray matter axis. In neurodegenerative diseases, FKBP12 levels increase in neurons situated in areas of pathology. This IP colocalizes with synaptophysin and alpha- synuclein, suggesting that it may become a novel marker of pathology. Immunophilins participate in axonal transport, synaptic vesicle assembly and may play a role in neuroprotection against abnormal protein aggregation, suggesting a potential avenue of therapeutic interventions.

Changes in peptidyl-prolyl cis/trans isomerase activity and fk506 binding protein expression following neuroprotection by fk506 in the ischemic rat brain

FK506 is an immunosuppressant also showing neuroprotection following cerebral ischemia. FK506 binds to intracellular proteins (FKBP) which have a wide range of functions but have in common the peptidyl-prolyl cis/trans isomerase activity. Following transient focal ischemia, we have analyzed the expression of FKBP12, 52 and 65 and the total FKBP enzyme activity. Furthermore, we have investigated the effect of FK506 on signal transduction in neurons and perfusion changes in the infarct area.After 90 min of transient middle cerebral artery occlusion in male rats the expression of FKBP12, 52 and 65 was analyzed by Western blot in FK506-treated and control animals and the peptidyl-prolyl cis/trans isomerase activity was determined. Magnetic resonance imaging was used to measure tissue perfusion, development of vasogenic edema and infarct size. To investigate the neuronal stress signal cascade, activating transcription factor 2 (ATF-2), Fas-ligand (Fas-L) and c-Jun expression and phosphorylation were analyzed by immunohistochemistry.FK506 decreased the cerebral infarct volume by 53% and reduced the cytotoxic edema. The total FKBP enzymatic activity in the infarct area was increased and blocked dose dependently by FK506. FKBP expression was selectively up-regulated by cerebral ischemia. FK506 treatment does not influence the expression patterns. c-Jun phosphorylation in neurons of the peri-infarct area and Fas-L expression was reduced by FK506 treatment whereas ATF-2 expression was preserved.Cerebral ischemic damage to the brain was reduced by FK506. It was shown for the first time that neuroprotection by FK506 also included the suppression of the cerebral peptidyl-prolyl cis/trans isomerase activity of FKBP in vivo whereas the expression levels of FKBP12, 52 and 65 following ischemia changed slightly and FK506 treatment does not suppress the expression patterns. However, changes of FKBP enzymatic activity result in suppression of the stress cell body response in the peri-infarct area as observed by suppression of c-Jun phosphorylation and Fas-L expression.