Yong-Xiao Wang

Oestrogen protects FKBP12.6 null mice from cardiac hypertrophy

FK506 binding proteins 12 and 12.6 (FKBP12 and FKBP12.6) are intracellular receptors for the immunosuppressant drug FK506 (ref. 1). The skeletal muscle ryanodine receptor (RyR1) is isolated as a hetero-oligomer with FKBP12 (ref. 2), whereas the cardiac ryanodine receptor (RyR2) more selectively associates with FKBP12.6 (refs 3, 4, 5). FKBP12 modulates Ca2+ release from the sarcoplasmic reticulum in skeletal muscle and developmental cardiac defects have been reported in FKBP12-deficient mice, but the role of FKBP12.6 in cardiac excitation–contraction coupling remains unclear. Here we show that disruption of the FKBP12.6 gene in mice results in cardiac hypertrophy in male mice, but not in females. Female hearts are normal, despite the fact that male and female knockout mice display similar dysregulation of Ca2+ release, seen as increases in the amplitude and duration of Ca2+ sparks and calcium-induced calcium release gain. Female FKBP12.6-null mice treated with tamoxifen, an oestrogen receptor antagonist, develop cardiac hypertrophy similar to that of male mice. We conclude that FKBP12.6 modulates cardiac excitation–contraction coupling and that oestrogen plays a protective role in the hypertrophic response of the heart to Ca2+ dysregulation.

Type-3 Ryanodine Receptors Mediate Hypoxia-, but Not Neurotransmitter-induced Calcium Release and Contraction in Pulmonary Artery Smooth Muscle Cells

In this study we examined the expression of RyR subtypes and the role of RyRs in neurotransmitter- and hypoxia-induced Ca2+ release and contraction in pulmonary artery smooth muscle cells (PASMCs). Under perforated patch clamp conditions, maximal activation of RyRs with caffeine or inositol triphosphate receptors (IP3Rs) with noradrenaline induced equivalent increases in [Ca2+]i and Ca2+-activated Cl− currents in freshly isolated rat PASMCs. Following maximal IP3-induced Ca2+ release, neither caffeine nor chloro-m-cresol induced a response, whereas prior application of caffeine or chloro-m-cresol blocked IP3-induced Ca2+ release. In cultured human PASMCs, which lack functional expression of RyRs, caffeine failed to affect ATP-induced increases in [Ca2+]i in the presence and absence of extracellular Ca2+. The RyR antagonists ruthenium red, ryanodine, tetracaine, and dantrolene greatly inhibited submaximal noradrenaline– and hypoxia-induced Ca2+ release and contraction in freshly isolated rat PASMCs, but did not affect ATP-induced Ca2+ release in cultured human PASMCs. Real-time quantitative RT-PCR and immunofluorescence staining indicated similar expression of all three RyR subtypes (RyR1, RyR2, and RyR3) in freshly isolated rat PASMCs. In freshly isolated PASMCs from RyR3 knockout (RyR3−/−) mice, hypoxia-induced, but not submaximal noradrenaline–induced, Ca2+ release and contraction were significantly reduced. Ruthenium red and tetracaine can further inhibit hypoxic increase in [Ca2+]i in RyR3−/− mouse PASMCs. Collectively, our data suggest that (a) RyRs play an important role in submaximal noradrenaline– and hypoxia-induced Ca2+ release and contraction; (b) all three subtype RyRs are expressed; and (c) RyR3 gene knockout significantly inhibits hypoxia-, but not submaximal noradrenaline–induced Ca2+ and contractile responses in PASMCs.

Effects of Doxorubicin on Excitation-Contraction Coupling in Guinea Pig Ventricular Myocardium

Doxorubicin, an anticancer drug, was recently shown to release Ca2+ from cardiac sarcoplasmic reticulum (SR) by increasing the open probability of Ca2+ release channels. In the present study, we investigated the effects of doxorubicin on excitation-contraction coupling of guinea pig heart preparations. In papillary muscles contracting at 0.5 Hz, 100 mumol/L doxorubicin produced within 3 hours the following effects: it increased the force of contraction by 269.3 +/- 19.8% (n = 6) and prolonged the time to peak force by 75.1 +/- 8.7% (n = 6), relaxation time by 54.7 +/- 8.7% (n = 6), and action potential duration (APD) at 90% repolarization (APD90) by 38.6 +/- 2.9% (n = 3). Despite its positive inotropic effect, doxorubicin depressed the early contraction component by increasing the latency between stimulus and the onset of force development. In single myocytes, 100 mumol/L doxorubicin prolonged APD90 by 62.1% (n = 18) and blocked time-dependent delayed rectifier K+ current (IK) by 44% (n = 9). Ca2+ inward current and inward rectifier K+ current were not affected by doxorubicin. Ca2+ transients elicited in myocytes loaded with the fluorescent Ca2+ indicator fura 2 were strongly suppressed by doxorubicin in their initial rising phase. There-after, doxorubicin produced a delayed rise in intracellular Ca2+, which reached a late peak exceeding that of the control peak by 52 +/- 8% (n = 5). The results suggest that doxorubicin decreases Ca(2+)-induced Ca2+ release from cardiac SR, probably by increasing the SR Ca2+ leak. On the other hand, prolongation of APD due to inhibition of IK allows more Ca2+ to enter the cell. After being only temporarily buffered by the SR, Ca2+ may accumulate in the cytosol as long as depolarization is maintained and lead to a more complete activation of contractile proteins.

Membrane depolarization causes a direct activation of G protein-coupled receptors leading to local Ca2+ release in smooth muscle

Membrane depolarization activates voltage-dependent Ca2+ channels (VDCCs) inducing Ca2+ release via ryanodine receptors (RyRs), which is obligatory for skeletal and cardiac muscle contraction and other physiological responses. However, depolarization-induced Ca2+ release and its functional importance as well as underlying signaling mechanisms in smooth muscle cells (SMCs) are largely unknown. Here we report that membrane depolarization can induce RyR-mediated local Ca2+ release, leading to a significant increase in the activity of Ca2+ sparks and contraction in airway SMCs. The increased Ca2+ sparks are independent of VDCCs and the associated extracellular Ca2+ influx. This format of local Ca2+ release results from a direct activation of G protein-coupled, M3 muscarinic receptors in the absence of exogenous agonists, which causes activation of Gq proteins and phospholipase C, and generation of inositol 1,4,5-triphosphate (IP3), inducing initial Ca2+ release through IP3 receptors and then further Ca2+ release via RyR2 due to a local Ca2+-induced Ca2+ release process. These findings demonstrate an important mechanism for Ca2+ signaling and attendant physiological function in SMCs.

Functional and molecular evidence for impairment of calcium-activated potassium channels in type-1 diabetic cerebral artery smooth muscle cells

Cerebral vascular dysfunction and associated diseases often occur in type-1 diabetes, but the underlying mechanisms are largely unknown. In this study, we sought to determine whether big-conductance, Ca2+-activated K+ (BK) channels were impaired in vascular (cerebral artery) smooth muscle cells (CASMCs) from streptozotocin-induced type-1 diabetic mice using patch clamp, molecular biologic, and genetic approaches. Our data indicate that the frequency and amplitude of spontaneous transient outward currents (STOCs) are significantly decreased, whereas the activity of spontaneous Ca2+ sparks is increased, in diabetic CASMCs. The sensitivity of BK channels to voltage, Ca2+, and the specific inhibitor iberiotoxin are all reduced in diabetic myocytes. Diabetic mice show increased myogenic tone and decreased contraction in response to iberiotoxin in cerebral arteries and elevated blood pressure. The expression of the BK channel β1, but not α-subunit protein, is markedly decreased in diabetic cerebral arteries. Diabetic impairment of BK channel activity is lost in CASMCs from BK channel β1-subunit gene deletion mice. In conclusion, the BK channel β1-subunit is impaired in type-1 diabetic vascular SMCs, resulting in increased vasoconstriction and elevated blood pressure, thereby contributing to vascular diseases in type-1 diabetes.

Functional Role of Canonical Transient Receptor Potential 1 and Canonical Transient Receptor Potential 3 in Normal and Asthmatic Airway Smooth Muscle Cells

Canonical transient receptor potential (TRPC)-encoded nonselective cation channels (NSCCs) are crucial for many cellular responses in a variety of cells; however, their molecular expression and functional roles in airway smooth muscle cells (ASMCs) remain obscure. The objective of this study was to determine whether TRPC1 and TRPC3 molecules could be important molecular constituents of native NSCCs controlling the resting membrane potential (Vm) and [Ca(2+)](i) in freshly isolated normal and ovalbumin (OVA)-sensitized/-challenged mouse ASMCs. Western blotting, RT-PCR, single-channel recording, whole-cell current-clamp recording, and a fluorescence imaging system were used to determine TRPC expression, NSCC activity, resting Vm, and resting [Ca(2+)](i). Specific individual TRPC antibodies and siRNAs were applied to test their functional roles. TRPC1 and TRPC3 proteins and mRNAs were expressed in freshly isolated ASM tissues. TRPC3 antibodies blocked the activity of NSCCs and hyperpolarized the resting Vm in ASMCs, whereas TRPC1 antibodies had no effect. TRPC3, but not TRPC1 gene silencing, largely diminished NSCC activity, hyperpolarized the resting Vm, lowered the resting [Ca(2+)](i), and inhibited methacholine-induced increase in [Ca(2+)](i). In OVA-sensitized/-challenged ASMCs, NSCC activity was greatly augmented, resting Vm was depolarized, and TRPC3 protein expression was increased. TRPC1 and TRPC3 antibodies blocked the increased activity of NSCCs and membrane depolarization in OVA-sensitized/-challenged cells. TRPC3 is an important molecular component of native NSCCs contributing to the resting Vm and [Ca(2+)](i) in normal ASMCs, as well as membrane depolarization and hyperresponsiveness in OVA-sensitized/-challenged cells, whereas TRPC1-encoded NSCCs are only activated in OVA-sensitized/-challenged airway myocytes.

Heterogeneous Gene Expression and Functional Activity of Ryanodine Receptors in Resistance and Conduit Pulmonary as well as Mesenteric Artery Smooth Muscle Cells

Background: Hypoxia causes heterogeneous contractile responses in resistance and conduit pulmonary as well as systemic (mesenteric) artery smooth muscle cells (RPASMCs, CPASMCs and MASMCs), but the underlying mechanisms are largely unknown. In this study, we aimed to investigate whether the gene expression and functional activity of ryanodine receptors (RyRs) would be different in these 3 cell types. Methods: RyR mRNA expression, Ca2+ sparks and [Ca2+]i were measured by real-time quantitative RT-PCR, laser scanning confocal microscopy and wide-field fluorescence microscopy, respectively. Results: All 3 RyR subtype (RyR1, RyR2 and RyR3) mRNAs are expressed in RPASMCs, CPASMCs and MASMCs, but their expression levels are different. Spontaneous Ca2+ sparks (functional events of RyRs) show distinct frequency, amplitude, duration, size and kinetics in these 3 cell types. Similarly, activation of RyRs by caffeine, 4-chloro-m-cresol or high K+ induces differential Ca2+ release. Moreover, hypoxia-induced increase in [Ca2+]i is largest in MASMCs relative to CPSAMCs and smallest in RPASMCs. Conclusion: This study provides comprehensive evidence that RyRs are heterogeneous in gene expression and functional activity in RPASMCs, CPASMCs and MASMCs, which may contribute to the diversity of excitation-contraction coupling and hypoxic Ca2+ responses in different vascular smooth muscle cells.

Effects of doxorubicinol on excitation–contraction coupling in guinea pig ventricular myocytes

The cardiotoxicity of the anticancer drug doxorubicin may be related to its main metabolite doxorubicinol. In this study, the acute effects of doxorubicinol on excitation-contraction coupling in isolated guinea pig ventricular myocytes were investigated and compared with doxorubicin using the whole-cell patch-clamp-, fura-2 fluorescence- and cell-edge tracking techniques. Both drugs were applied intracellularly by diffusion from the patch electrode for 15--20 min. Doxorubicin (100 microM) prolonged the action potential duration (APD) by 31% and enhanced cell shortening by 26%. Contrary to doxorubicin, doxorubicinol (10 microM) shortened APD by 25% and decreased cell shortening by 31%. APD shortening by doxorubicinol was due to an increase of the delayed rectifier K(+) current. Neither the inward rectifier K(+) current nor the L-type Ca(2+) current was influenced by doxorubicinol. The decline in cell shortening induced by doxorubicinol was not exclusively due to APD shortening because doxorubicinol reduced the peak Ca(2+) transient by 23% in cells clamped with an action potential of constant duration. Despite opposite effects on APD and contractility, both doxorubicin and doxorubicinol produced a considerable delay in the activation and inactivation of contraction and Ca(2+) transient, compatible with an impaired function of the sarcoplasmic reticulum. It is suggested that doxorubicinol-induced APD shortening may amplify the detrimental effects of both doxorubicin and doxorubicinol on sarcoplasmic reticulum Ca(2+) load and hence on contractile function. The accumulation of doxorubicinol in the cardiac myocytes may play an important role in the time-dependent development of doxorubicin-induced ventricular dysfunction.

Two distinct signaling pathways for regulation of spontaneous local Ca2+ release by phospholipase C in airway smooth muscle cells

Spontaneous local Ca2+ release events have been observed in airway smooth muscle cells (SMCs), but the underlying mechanisms are largely unknown. Considering that each type of SMCs may use its own mechanisms to regulate local Ca2+ release events, we sought to investigate the signaling pathway for spontaneous local Ca2+ release events in freshly isolated mouse airway SMCs using a laser scanning confocal microscope. Application of ryanodine to block ryanodine receptors (RyRs) abolished spontaneous local Ca2+ release events, indicating that these events are RyR-mediated Ca2+ sparks. Inhibition of inositol 1,4,5-triphosphate receptors (IP3Rs) by 2-aminoethoxydiphenyl-borate (2-APB) or xestospongin-C significantly blocked the activity of Ca2+ sparks. Under patch clamp conditions, dialysis of IP3 to activate IP3Rs increased the activity of local Ca2+ events in control cells but had no effect in ryanodine-pretreated cells. The RyR agonist caffeine augmented the frequency of Ca2+ sparks in cells pretreated with and without 2-APB or xestospongin-C. The specific phospholipase C (PLC) blocker U73122 decreased the activity of Ca2+ sparks and prevented xestospongin-C from producing the inhibitory effect. The protein kinase C (PKC) activator 1-oleoyl-2-acetyl-glycerol or phorbol-12-myristate-13-acetate inhibited Ca2+ sparks, whereas the PKC inhibitor chelerythrine, PKCɛ inhibitory peptide, or PKCɛ gene knockout produced an opposite effect. Collectively, our data suggest that the basal activation of PLC regulates the activity of RyR-mediated, spontaneous Ca2+ sparks in airway SMCs through two distinct signaling pathways: a positive IP3-IP3R pathway and a negative diacylglycerol–PKCɛ pathway.

Hypoxia Induces Intracellular Ca2+ Release by Causing Reactive Oxygen Species-Mediated Dissociation of FK506-Binding Protein 12.6 from Ryanodine Receptor 2 in Pulmonary Artery Myocytes

Here we attempted to test a novel hypothesis that hypoxia may induce Ca(2+) release through reactive oxygen species (ROS)-mediated dissociation of FK506-binding protein 12.6 (FKBP12.6) from ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) in pulmonary artery smooth muscle cells (PASMCs). The results reveal that hypoxic exposure significantly decreased the amount of FKBP12.6 on the SR of PAs and increased FKBP12.6 in the cytosol. The colocalization of FKBP12.6 with RyRs was decreased in intact PASMCs. Pharmacological and genetic inhibition of intracellular ROS generation prevented hypoxia from decreasing FKBP12.6 on the SR and increasing FKBP12.6 in the cytosol. Exogenous ROS (H(2)O(2)) reduced FKBP12.6 on the SR and augmented FKBP12.6 in the cytosol. Oxidized FKBP12.6 was absent on the SR from PAs pretreated with and without hypoxia, but it was present with a higher amount in the cytosol from PAs pretreated with than without hypoxia. Hypoxia and H(2)O(2) diminished the association of FKBP12.6 from type 2 RyRs (RyR2). The activity of RyRs was increased in PAs pretreated with hypoxia or H(2)O(2). FKBP12.6 removal enhanced, whereas RyR2 gene deletion blocked the hypoxic increase in [Ca(2+)](i) in PASMCs. Collectively, we conclude that hypoxia may induce Ca(2+) release by causing ROS-mediated dissociation of FKBP12.6 from RyR2 in PASMCs.