Amy L. Tucker

Phospholemman-Phosphorylation Mediates the β-Adrenergic Effects on Na/K Pump Function in Cardiac Myocytes

Cardiac sympathetic stimulation activates β-adrenergic (β-AR) receptors and protein kinase A (PKA) phosphorylation of proteins involved in myocyte Ca regulation. The Na/K-ATPase (NKA) is essential in regulating intracellular [Na] ([Na]i), which in turn affects [Ca]i via Na/Ca exchange. However, how PKA modifies NKA function is unknown. Phospholemman (PLM), a member of the FXYD family of proteins that interact with NKA in various tissues, is a major PKA substrate in heart. Here we tested the hypothesis that PLM phosphorylation is responsible for the PKA effects on cardiac NKA function using wild-type (WT) and PLM knockout (PLM-KO) mice. We measured NKA-mediated [Na]i decline and current (IPump) to assess β-AR effects on NKA function in isolated myocytes. In WT myocytes, 1 &mgr;mol/L isoproterenol (ISO) increased PLM phosphorylation and stimulated NKA activity mainly by increasing its affinity for internal Na (Km decreased from 18.8±1.4 to 13.6±1.5 mmol/L), with no significant effect on the maximum pump rate. This led to a significant decrease in resting [Na]i (from 12.5±1.8 to 10.5±1.4 mmol/L). In PLM-KO mice under control conditions Km (14.2±1.5 mmol/L) was lower than in WT, but comparable to that for WT in the presence of ISO. Furthermore, ISO had no significant effect on NKA function in PLM-KO mice. ATPase activity in sarcolemmal vesicles also showed a lower Km(Na) in PLM-KO versus WT (12.9±0.9 versus 16.2±1.5). Thus, PLM inhibits NKA activity by decreasing its [Na]i affinity, and this inhibitory effect is relieved by PKA activation. We conclude that PLM modulates the NKA function in a manner similar to the way phospholamban affects the related SR Ca-ATPase (inhibition of transport substrate affinity, that is relieved by phosphorylation).

Phospholemman Inhibition of the Cardiac Na+/Ca2+ Exchanger ROLE OF PHOSPHORYLATION

We have demonstrated previously that phospholemman (PLM), a 15-kDa integral sarcolemmal phosphoprotein, inhibits the cardiac Na+/Ca2+ exchanger (NCX1). In addition, protein kinase A phosphorylates serine 68, whereas protein kinase C phosphorylates both serine 63 and serine 68 of PLM. Using human embryonic kidney 293 cells that are devoid of both endogenous PLM and NCX1, we first demonstrated that the exogenous NCX1 current (INaCa) was increased by phorbol 12-myristate 13-acetate (PMA) but not by forskolin. When co-expressed with NCX1, PLM resulted in: (i) decreases in INaCa, (ii) attenuation of the increase in INaCa by PMA, and (iii) additional reduction in INaCa in cells treated with forskolin. Mutating serine 63 to alanine (S63A) preserved the sensitivity of PLM to forskolin in terms of suppression of INaCa, whereas mutating serine 68 to alanine (S68A) abolished the inhibitory effect of PLM on INaCa. Mutating serine 68 to glutamic acid (phosphomimetic) resulted in additional suppression of INaCa as compared with wild-type PLM. These results suggest that PLM phosphorylated at serine 68 inhibited INaCa. The physiological significance of inhibition of NCX1 by phosphorylated PLM was evaluated in PLM-knock-out (KO) mice. When compared with wild-type myocytes, INaCa was significant larger in PLM-KO myocytes. In addition, the PMA-induced increase in INaCa was significantly higher in PLM-KO myocytes. By contrast, forskolin had no effect on INaCa in wild-type myocytes. We conclude that PLM, when phosphorylated at serine 68, inhibits Na+/Ca2+ exchange in the heart.

A1 Adenosine Receptor Activation Promotes Angiogenesis and Release of VEGF From Monocytes

Adenosine is a proangiogenic purine nucleoside released from ischemic and hypoxic tissues. Of the 4 adenosine receptor (AR) subtypes (A1, A2A, A2B, and A3), the A2 and A3 have been previously linked to the modulation of angiogenesis. We used the chicken chorioallantoic membrane (CAM) model to determine whether A1 AR activation affects angiogenesis. We cloned and pharmacologically characterized chicken AR subtypes to evaluate the selectivity of various agonists and antagonists. Application of the A1 AR-selective agonist N6-cyclopentyladenosine (CPA; 100 nmol/L) to the CAM resulted in a 40% increase in blood vessel number (P<0.01), which was blocked by the A1 AR-selective antagonist C8-(N-methylisopropyl)-amino-N6-(5′-endohydroxy)-endonorbornan-2-yl-9-methyladenine (WRC-0571; 1 &mgr;mol/L). Selective A2A AR agonists did not stimulate angiogenesis in the CAM. In an ex vivo rat aortic ring model of angiogenesis that includes cocultured endothelial cells, fibroblasts, and smooth muscle cells, 50 nmol/L CPA did not directly stimulate capillary formation; however, medium from human mononuclear cells pretreated with CPA, but not vehicle, increased capillary formation by 48% (P<0.05). This effect was blocked by WRC-0571 (1.5 &mgr;mol/L) or anti-VEGF antibody (1 &mgr;g/mL). CPA (5 nmol/L) stimulated a 1.7-fold increase in VEGF release from the mononuclear cells. This is the first study to show that A1 AR activation induces angiogenesis. Stimulation of A2 ARs on endothelial cells results in proliferation and tube formation, and A2 and A3 ARs on inflammatory cells modulate release of angiogenic factors. We conclude that adenosine promotes a coordinated angiogenic response through its interactions with multiple receptors on multiple cell types.

Phospholemman Phosphorylation Mediates the Protein Kinase C–Dependent Effects on Na+/K+ Pump Function in Cardiac Myocytes

Because phospholemman (PLM) regulates the Na+/K+ pump (NKA) and is a major cardiac phosphorylation target for both protein kinase A (at Ser68) and protein kinase C (PKC) (at both Ser63 and Ser68), we evaluated whether PLM mediates the PKC-dependent regulation of NKA function and protein kinase A/PKC crosstalk in ventricular myocytes. PKC was activated by PDBu (300 nmol/L), and we measured NKA-mediated [Na+]i decline (fluorescence measurements) and current (Ipump) (voltage clamp). In wild-type mouse myocytes, PDBu increased PLM phosphorylation at Ser63 and Ser68, Ipump (both at 10 and 100 mmol/L Na+ in the pipette solution) and maximal NKA-mediated Na+ extrusion rate (Vmax) from 7.9±1.1 to 12.7±1.9 mmol·L−1 per minute without altering NKA affinity for internal Na+ (K0.5). In PLM knockout mice, PDBu had no effect on either Vmax or K0.5. After pretreatment with isoproterenol (ISO) (1 &mgr;mol/L), PDBu still increased the NKA Vmax and PLM phosphorylation at Ser63 and Ser68. Conversely, after pretreatment with PDBu, ISO further increased the Na+ affinity of NKA and phosphorylation at Ser68, as it did alone without PDBu. The final NKA activity was independent of the application sequence. The NKA activity in PLM knockout myocytes, after normalizing the protein level, was similar to that after PDBu and ISO treatment. We conclude that (1) PLM mediates the PKC-dependent activation of NKA function in cardiac myocytes, (2) PDBu and ISO effects are additive in the mouse (affecting mainly Vmax and K0.5, respectively), and (3) PDBu and ISO combine to activate NKA in wild-type to the level found in the PLM knockout mouse.

Phospholemman-Mediated Activation of Na/K-ATPase Limits [Na]i and Inotropic State During β-Adrenergic Stimulation in Mouse Ventricular Myocytes

Background— Cardiac Na/K-ATPase (NKA) regulates intracellular Na ([Na]i), which in turn affects intracellular Ca and thus contractility via Na/Ca exchange. Recent evidence shows that phosphorylation of the NKA-associated small transmembrane protein phospholemman (PLM) mediates &bgr;-adrenergic–induced NKA stimulation. Methods and Results— Here, we tested whether PLM phosphorylation during &bgr;-adrenergic activation limits the rise in [Na]i, Ca transient amplitude, and triggered arrhythmias in mouse ventricular myocytes. In myocytes from wild-type (WT) mice, [Na]i increased on field stimulation at 2 Hz from 11.1±1.8 mmol/L to a plateau of 15.2±1.5 mmol/L. Isoproterenol induced a decrease in [Na]i to 12.0±1.2 mmol/L. In PLM knockout (PLM-KO) mice in which &bgr;-adrenergic stimulation does not activate NKA, [Na]i also increased at 2 Hz (from 10.4±1.2 to 17.0±1.5 mmol/L) but was unaltered by isoproterenol. The PLM-mediated decrease in [Na]i in WT mice could limit the isoproterenol-induced inotropic state. Indeed, the isoproterenol-induced increase in the amplitude of Ca transients was significantly smaller in the WT mice (5.2±0.4- versus 7.1±0.5-fold in PLM-KO mice). This also was the case for the sarcoplasmic reticulum Ca content, which increased by 1.27±0.09-fold in WT mice versus 1.53±0.09-fold in PLM-KO mice. The higher sarcoplasmic reticulum Ca content in PLM-KO versus WT mice was associated with an increased propensity for spontaneous Ca transients and contractions in PLM-KO mice. Conclusions— These data suggest that PLM phosphorylation and NKA stimulation are an integral part of the sympathetic fight-or-flight response, tempering the rise in [Na]i and cellular Ca loading and perhaps limiting Ca overload–induced arrhythmias.

RNA toxicity in myotonic muscular dystrophy induces NKX2-5 expression

Myotonic muscular dystrophy (DM1) is the most common inherited neuromuscular disorder in adults and is considered the first example of a disease caused by RNA toxicity. Using a reversible transgenic mouse model of RNA toxicity in DM1, we provide evidence that DM1 is associated with induced NKX2-5 expression. Transgene expression resulted in cardiac conduction defects, increased expression of the cardiac-specific transcription factor NKX2-5 and profound disturbances in connexin 40 and connexin 43. Notably, overexpression of the DMPK 3′ UTR mRNA in mouse skeletal muscle also induced transcriptional activation of Nkx2-5 and its targets. In human muscles, these changes were specific to DM1 and were not present in other muscular dystrophies. The effects on NKX2-5 and its downstream targets were reversed by silencing toxic RNA expression. Furthermore, using Nkx2-5+/− mice, we show that NKX2-5 is the first genetic modifier of DM1-associated RNA toxicity in the heart.

Lysophosphatidic acid (LPA) and angiogenesis

Lysophosphatidic acid (LPA) is a simple lipid with many important biological functions such as the regulation of cellular proliferation, cellular migration, differentiation, and suppression of apoptosis. Although a direct angiogenic effect of LPA has not been reported to date, there are indications that LPA promotes angiogenesis. In addition, LPA is a chemoattractant for cultured endothelial cells and promotes barrier function in such cultures [1]. To test the hypothesis that LPA is angiogenic, we used the chicken chorio-allantoic membrane (CAM) assay. Sequence analysis of the cloned, full-length chicken LPA receptor cDNAs revealed three receptor types that are orthologous to the mammalian LPA1, LPA2, and LPA3 receptors. We document herein that LPA is angiogenic in the CAM system and further that synthetic LPA receptor agonists and antagonists mimic or block this response, respectively. Our results predict that LPA receptor antagonists are a possible therapeutic route to interdicting angiogenesis

Cloning and expression of a bovine adenosine A1 receptor cDNA

A bovine brain adenosine A1 receptor cDNA encoding a 326 amino acid protein has been identified. This cDNA, which encodes a protein >90% identical to analogous rat and dog receptors, was transiently expressed in COS‐1 cells. Recombinant receptors exhibited the features of bovine A1 receptors that distinguish it from rat and canine receptors, including subnanomolar K i for 1,3‐dipropyl‐8‐cyclopentylxanthine, R‐phenylisopropyl‐adenosine (R‐PIA) and xanthine amino conjugate, and the distinct potency order: R‐PIA > S‐PIA > 5′‐N‐ethylcarboxamidoadenosine > 2′‐chloroadenosine. The results indicate that the pharmacological differences between A1 adenosine receptors among species result from only minor differences in receptor structures.

Automated quantitative analysis of angiogenesis in the rat aorta model using Image-Pro Plus 4.1

This paper explains the automated image-processing steps for the quantification of microvascular growth formation in the rat thoracic aortic ring model, an ex vivo model using excised rings of rat aorta embedded in a collagen matrix which produce outgrowths of microvessels. This model of angiogenesis is useful to study the mechanism by which external agents inhibit or stimulate endothelial cell proliferation and tube formation. The manual quantification of blood vessel growth in this model is normally a time-consuming, error prone process. Former automated image analysis methods of the ring model are outdated and cannot be used with current software technology. A macro was created using Image-Pro Plus 4.1 software which was chosen for image analysis because it allows a high degree of control and replication of image-processing steps. The accuracy of this macro was determined by comparing automated counts to manual counts in 161 aortic rings. The square root of the manual count versus the square root of the automated count resulted in a root mean square value of 0.8305.

Regulation of Cardiac Na+/Ca2+ Exchanger by Phospholemman

Abstract:  Phospholemman (PLM) is the first sequenced member of the FXYD family of regulators of ion transport. The mature protein has 72 amino acids and consists of an extracellular N terminus containing the signature FXYD motif, a single transmembrane (TM) domain, and a cytoplasmic C‐terminal domain containing four potential sites for phosphorylation. PLM and other members of the FXYD family are known to regulate Na+‐K+‐ATPase. Using adenovirus‐mediated gene transfer into adult rat cardiac myocytes, we showed that changes in contractility and intracellular Ca2+ homeostasis associated with PLM overexpression or downregulation are not consistent with the effects expected from inhibition of Na+‐K+‐ATPase by PLM. Additional studies with heterologous expression of PLM and cardiac Na+/Ca2+ exchanger 1 (NCX1) in HEK293 cells and cardiac myocytes isolated from PLM‐deficient mice demonstrated by co‐localization, co‐immunoprecipitation, and electrophysiological and radioactive tracer uptake techniques that PLM associates with NCX1 in the sarcolemma and transverse tubules and that PLM inhibits NCX1, independent of its effects on Na+‐K+‐ATPase. Mutational analysis indicates that the cytoplasmic domain of PLM is required for its regulation of NCX1. In addition, experiments using phosphomimetic and phospho‐deficient PLM mutants, as well as activators of protein kinases A and C, indicate that PLM phosphorylated at serine68 is the active form that inhibits NCX1. This is in sharp contrast to the finding that the unphosphorylated PLM form inhibits Na+‐K+‐ATPase. We conclude that PLM regulates cardiac contractility by modulating the activities of NCX and Na+‐K+‐ATPase.