Key Words: FKBP12.6 Ⅲ FKBP12 Ⅲ Ca sparks Ⅲ binding properties Ⅲ rapamycin Ⅲ RyR2 C ardiac ryanodine receptors (RyR2) are sarcoplasmic reticulum (SR) Ca release channels, crucial in excitation-contraction coupling. 1 Dysfunctional RyR2, exhibiting enhanced Ca leak has been implicated in arrhythmogenesis and heart failure (HF). 1 Homotetrameric RyR2s have a transmembrane channel domain, and regulatory and scaffolding cytoplasmic domain.In heart, FK506 binding protein (FKBP) isoforms FKBP12 and FKBP12.6 are coexpressed and can bind RyR2 at a stoichiometry of 4 FKBP per RyR tetramer. 2,3 FKBP12 binds RyR2 with much lower affinity, but is much higher in concentration in heart than FKBP12.6. 4 Nevertheless, FKBP12 and 12.6 share 85% sequence homology and similar 3D structures. 5 Human and rat FKBP12 differ in only 3 residues, and human and rat FKBP12.6 are identical. This makes study of human FKBP function in rat myocytes reasonable.Effects of FKBPs on RyR2 activity in myocytes are controversial. Some groups reported that dissociation of FKBP12.6 from RyR2 by immunosuppressants (rapamycin or FK506) activated RyR2 channels and induced subconductance states. 6 -8 RyR2 point mutations associated with cardiac sudden death may also exhibit altered FKBP12.6 interaction, 9 and FK506 can alter resting Ca 2ϩ spark frequency (CaSpF) and SR Ca 2ϩ content. 7,8,10 FKBP12.6 overexpression also increased SR load and enhanced contraction. 11 However, others reported that FKBP12.6 removal had no effect on RyR2 activity, 3,12 and failed to observe RyR2 subconductance states in channels from FKBP12.6 knockout mice. 13,14 Some groups suggest that FKBP12 affects RyR2 differently from FKBP12.6. [15][16][17]20 Therefore, FKBP12/12.6 binding and RyR2 effects remain unclear, especially in the cardiomyocyte environment (as studied here).Altered FKBP-RyR2 interaction is a prominent hypothesis explaining increased SR Ca leak in HF via RyR2 hyperphos- Here we characterize in the myocyte environment the interaction between FKBP12/12.6 and RyR2, its functional consequences, and modulation by PKA-dependent phosphorylation. Using fluorescent FKBP12/12.6 (F-FKBP), we simultaneously assessed physical association-dissociation of FKBP12/12.6 with RyR2, and RyR2 activity (via Ca sparks) in permeabilized ventricular myocytes. We found that: (1) both FKBP12.6 and FKBP12 bound to RyR2 (K d Ϸ1 nmol/L and 200 nmol/L, respectively); (2) FKBP12.6 but not FKBP12 inhibited CaSpF; (3) the binding properties of FKBP12.6/12-RyR2 were not changed by PKA-dependent phosphorylation of RyR2; and (4) endogenous FKBP12 was Ϸ1 mol/L cytosol and FKBP12.6 Ϸ100 nmol/L cytosol. MethodsRat and mouse ventricular myocytes were isolated and permeabilized as previously described (see the Online Data Supplement, available at http://circres.ahajournals.org). 23 F-FKBP12.6 and F-FKBP12 were characterized using circular dichroism spectroscopy and ligandbinding studies (Online Figure I). 24 Consistent with previous reports, 25 F-FKBP constructs have the same secondary structure, RyR ...

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beta3-Adrenoceptor is involved in altered positive inotropic response to beta-adrenoceptor stimulation in diabetic cardiomyopathy. This effect is mediated by NOS1-derived nitric oxide in diabetic cardiomyocyte.

Ryanodine receptors (RyRs) and inositol triphosphate receptors (InsP3Rs) are structurally related intracellular calcium release channels that participate in multiple primary or secondary amplified Ca2+ signals, triggering muscle contraction and oscillatory Ca2+ waves, or activating transcription factors. In the heart, RyRs play an indisputable role in the process of excitation–contraction coupling as the main pathway for Ca2+ release from sarcoplasmic reticulum (SR), and a less prominent role in the process of excitation–transcription coupling. Conversely, InsP3Rs are believed to contribute in subtle ways, only, to contraction of the heart, and in more important ways to regulation of transcription factors. Because uncontrolled activity of either RyRs or InsP3Rs may elicit life-threatening arrhythmogenic and/or remodeling Ca2+ signals, regulation of their activity is of paramount importance for normal cardiac function. Due to their structural similarity, many regulatory factors, accessory proteins, and post-translational processes are equivalent for RyRs and InsP3Rs. Here we discuss regulation of RyRs and InsP3Rs by CaMKII phosphorylation, but touch on other kinases whenever appropriate. CaMKII is emerging as a powerful modulator of RyR and InsP3R activity but interestingly, some of the complexities and controversies surrounding phosphorylation of RyRs also apply to InsP3Rs, and a clear-cut effect of CaMKII on either channel eludes investigators for now. Nevertheless, some effects of CaMKII on global cellular activity, such as SR Ca2+ leak or force-frequency potentiation, appear clear now, and this constrains the limits of the controversies and permits a more tractable approach to elucidate the effects of phosphorylation at the single channel level.

Annexins are a family of 13 proteins known to bind phospholipids (PL) in a Ca(2+)-dependent way. They are ubiquitous proteins and share a similar structure characterized by a conserved C-terminal domain with Ca(2+) binding sites and a variable N-terminal domain. Depending on Ca(2+) concentration, they have been reported to participate in a variety of membrane-related events such as exocytosis, endocytosis, apoptosis and binding to cytoskeletal proteins. They have also been reported to regulate protein activities. This review will focus on annexins in the heart, and particularly on annexins A2, A5, A6 and A7. Annexin A2 has been found in endothelial cells and reported to play a central role in control of plasmin-mediated processes. Annexin A5 is mainly localized in cardiomyocytes. However, it could be relocated to interstitial tissue in ischemic and failing hearts or it could be externalized and exhibit a proapoptotic effect in cardiomyocytes. Annexin A6 is the most abundant annexin in the heart, and has been localized in various cell types including myocytes. Overexpression of annexin A6 has underlined physiological alterations in contractile mechanics leading to dilated cardiomyopathy, whereas knockout has been found to induce faster changes in Ca(2+) transient and increased contractility, suggesting a negative inotropic role for annexin A6. Annexin A7 is expressed in heart and skeletal muscle. In annexin A7 null mutant mice decreases in the force-frequency relationship were observed in adult cardiomyocytes, consistent with regulation of Ca(2+) handling. In conclusion, while annexin A2 was involved in regulation of fibrin homeostasis, alterations in expression and activity of annexins A5, A6 and A7 have been associated with regulation of Ca(2+) handling in the heart, but the target of each annexin has not yet been identified.

Ankyrin-B (AnkB) loss-of-function may cause ventricular arrhythmias and sudden cardiac death in humans. Cardiac myocytes from AnkB heterozygous mice (AnkB+/−) show reduced expression and altered localization of Na/Ca exchanger (NCX) and Na/K-ATPase (NKA), key players in regulating [Na]i and [Ca]i. Here we investigate how AnkB reduction affects cardiac [Na]i, [Ca]i and SR Ca release. We found reduced NCX and NKA transport function but unaltered [Na]i and diastolic [Ca]i in myocytes from AnkB+/− vs. wild-type (WT) mice. Ca transients, SR Ca content and fractional SR Ca release were larger in AnkB+/− myocytes. The frequency of spontaneous, diastolic Ca sparks (CaSpF) was significantly higher in intact myocytes from AnkB+/− vs. WT myocytes (with and without isoproterenol), even when normalized for SR Ca load. However, total ryanodine receptor (RyR)-mediated SR Ca leak (tetracaine-sensitive) was not different between groups. Thus, in AnkB+/− mice SR Ca leak is biased towards more Ca sparks (vs. smaller release events), suggesting more coordinated openings of RyRs in a cluster. This is due to local cytosolic RyR regulation, rather than intrinsic RyR differences, since CaSpF was similar in saponin-permeabilized myocytes from WT and AnkB+/− mice. The more coordinated RyRs openings resulted in an increased propensity of pro-arrhythmic Ca waves in AnkB+/− myocytes. In conclusion, AnkB reduction alters cardiac Na and Ca transport and enhances the coupled RyR openings, resulting in more frequent Ca sparks and waves although the total SR Ca leak is unaffected. This could enhance the propensity for triggered arrhythmias in AnkB+/− mice.

During physical exercise or stress, the sympathetic system stimulates cardiac contractility via β-adrenergic receptor (β-AR) activation, resulting in protein kinase A (PKA)–mediated phosphorylation of the cardiac ryanodine receptor RyR2. PKA-dependent “hyperphosphorylation” of the RyR2 channel has been proposed as a major impairment that contributes to progression of heart failure. However, the sites of PKA phosphorylation and their phosphorylation status in cardiac diseases are not well defined. Among the known RyR2 phosphorylation sites, serine 2030 (S2030) remains highly controversial as a site of functional impact. We examined the contribution of RyR2-S2030 to Ca2+ signaling and excitation–contraction coupling (ECC) in a transgenic mouse with an ablated RyR2-S2030 phosphorylation site (RyR2-S2030A+/+). We assessed ECC gain by using whole-cell patch–clamp recordings and confocal Ca2+ imaging during β-ARs stimulation with isoproterenol (Iso) and consistent SR Ca2+ loading and L-type Ca2+ current (ICa) triggering. Under these conditions, ECC gain is diminished in mutant compared with WT cardiomyocytes. Resting Ca2+ spark frequency (CaSpF) with Iso is also reduced by mutation of S2030. In permeabilized cells, when SR Ca2+ pump activity is kept constant (using 2D12 antibody against phospholamban), cAMP does not change CaSpF in S2030A+/+ myocytes. Using Ca2+ spark recovery analysis, we found that mutant RyR Ca2+ sensitivity is not enhanced by Iso application, contrary to WT RyRs. Furthermore, ablation of RyR2-S2030 prevents acceleration of Ca2+ waves and increases latency to the first spontaneous Ca2+ release after a train of stimulations during Iso treatment. Together, these results suggest that phosphorylation at S2030 may represent an important step in the modulation of RyR2 activity during β-adrenergic stimulation and a potential target for the development of new antiarrhythmic drugs.

This study indicated for the first time that annexin A5 was externalized at a very early stage of apoptosis and could have a proapoptotic effect in cardiomyocytes.