Mechanism of enhanced cardiac function in mice with hypertrophy induced by overexpressed Akt

Transgenic mice with cardiac-specific overexpression of active Akt (TG) not only exhibit hypertrophy but also show enhanced left ventricular (LV) function. In 3-4-month-old TG, heart/body weight was increased by 60% and LV ejection fraction was elevated (84 +/- 2%, p < 0.01) compared with nontransgenic littermates (wild type (WT)) (73 +/- 1%). An increase in isolated ventricular myocyte contractile function (% contraction) in TG compared with WT (6.1 +/- 0.2 versus 3.5 +/- 0.2%, p < 0.01) was associated with increased Fura-2 Ca2+ transients (396 +/- 50 versus 250 +/- 24 nmol/liter, p < 0.05). The rate of relaxation (+dL/dt) was also enhanced in TG (214 +/- 15 versus 98 +/- 18 microm/s, p < 0.01). L-type Ca2+ current (ICa) density was increased in TG compared with WT (-9.0 +/- 0.3 versus 7.2 +/- 0.3 pA/pF, p < 0.01). Sarcoplasmic reticulum Ca2+ ATPase 2a (SERCA2a) protein levels were increased (p < 0.05) by 6.6-fold in TG, which could be recapitulated in vitro by adenovirus-mediated overexpression of Akt in cultured adult ventricular myocytes. Conversely, inhibiting SERCA with either ryanodine or thapsigargin affected myocyte contraction and relaxation and Ca2+ channel kinetics more in TG than in WT. Thus, myocytes from mice with overexpressed Akt demonstrated enhanced contractility and relaxation, Fura-2 Ca2+ transients, and Ca2+ channel currents. Furthermore, increased protein expression of SERCA2a plays an important role in mediating enhanced LV function by Akt. Up-regulation of SERCA2a expression and enhanced LV myocyte contraction and relaxation in Akt-induced hypertrophy is opposite to the down-regulation of SERCA2a and reduced contractile function observed in many other forms of LV hypertrophy.     

SERCA2a activity correlates with the force-frequency relationship in human myocardium

The present investigation addresses whether protein expression and function of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) and phospholamban (PLB) correlate in failing and nonfailing human myocardium. SERCA2a activity and protein expression, PLB phosphorylation, and the force-frequency relationship (FFR) have been determined in right atrium (RA) and left ventricle (LV) from nonfailing (NF, n = 12) and terminally failing [dilated cardiomyopathy (DCM), n = 12] human hearts. Only in LV of DCM hearts was SERCA2a activity significantly decreased [maximal turnover rate (V(max)) = 196 +/- 11 and 396 +/- 30 nmol. mg(-1). min(-1) in LV and RA, respectively], whereas protein expression of SERCA2a in the different chambers was unchanged in NF (3.9 +/- 0.3 and 3.2 +/- 0.4 densitometric units in LV and RA, respectively) and DCM hearts (4.8 +/- 0.8 and 3.4 +/- 0.1 densitometric units in LV and RA, respectively). Phosphorylation of PLB was higher in LV than in RA in NF (Ser(16): 180.5 +/- 19.0 vs. 56.8 +/- 6.0 densitometric units; Thr(17): 174.6 +/- 11.2 vs. 37.4 +/- 8.9 densitometric units) and DCM hearts (Ser(16): 132.0 +/- 5.4 vs. 22.4 +/- 3.5 densitometric units; Thr(17): 131.2 +/- 10.9 vs. 9.2 +/- 2.4 densitometric units). SERCA2a function, but not protein expression, correlated well with the functional parameters of the FFR in DCM and NF human hearts. Regulation of SERCA2a function depends on the phosphorylation of PLB at Ser(16) and Thr(17). However, direct SERCA2a regulation might also be affected by an unknown mechanism.     

Loss of alpha2B-adrenoceptors increases magnitude of hypertension following nitric oxide synthase inhibition

Vascular alpha(2B)-adrenoceptors (alpha(2B)-AR) may mediate vasoconstriction and contribute to the development of hypertension. Therefore, we hypothesized that blood pressure would not increase as much in mice with mutated alpha(2B)-AR as in wild-type (WT) mice following nitric oxide (NO) synthase (NOS) inhibition with N(omega)-nitro-l-arginine (l-NNA, 250 mg/l in drinking water). Mean arterial pressure (MAP) was recorded in heterozygous (HET) alpha(2B)-AR knockout mice and WT littermates using telemetry devices for 7 control and 14 l-NNA treatment days. MAP in HET mice was increased significantly on treatment days 1 and 4 to 14, whereas MAP did not change in WT mice (days 0 and 14 = 113 +/- 3 and 114 +/- 4 mmHg in WT, 108 +/- 0.3 and 135 +/- 13 mmHg in HET, P < 0.05). MAP was significantly higher in HET than in WT mice days 10 through 14 (P < 0.05). Thus blood pressure increased more rather than less in mice with decreased alpha(2B)-AR expression. We therefore examined constrictor responses to phenylephrine (PE, 10(-9) to 10(-4) M) with and without NOS inhibition to determine basal NO contributions to arterial tone. In small pressurized mesenteric arteries (inner diameter = 177 +/- 5 microm), PE constriction was decreased in untreated HET arteries compared with WT (P < 0.05). l-NNA (100 microM) augmented PE constriction more in HET arteries than in WT arteries, and responses were not different between groups in the presence of l-NNA. Acetylcholine dilated preconstricted arteries from HET mice more than arteries from WT mice. Endothelial NOS expression was increased in HET compared with WT mesenteric arteries by Western analysis. Griess assay showed increased NO(x) concentrations in HET plasma compared with those in WT plasma. These data demonstrate that diminished alpha(2B)-AR expression increases the dependence of arterial pressure and vascular tone on NO production and that vascular alpha(2B)-AR either directly or indirectly regulates vascular endothelial NOS function.     

Altered dose response to beta-agonists in SERCA1a-expressing hearts ex vivo and in vivo

In this study we evaluated the contractile characteristics of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)1a-expressing hearts ex vivo and in vivo and in particular their response to beta-adrenergic stimulation. Analysis of isolated, work-performing hearts revealed that transgenic (TG) hearts develop much higher maximal rates of contraction and relaxation than wild-type (WT) hearts. Addition of isoproterenol only moderately increased the maximal rate of relaxation (+20%) but did not increase contractility or decrease relaxation time in TG hearts. Perfusion with varied buffer Ca(2+) concentrations indicated an altered dose response to Ca(2+). In vivo TG hearts displayed fairly higher maximal rates of contraction (+ 25%) but unchanged relaxation parameters and a blunted but significant response to dobutamine. Our study also shows that the phospholamban (PLB) level was decreased (-40%) and its phosphorylation status modified in TG hearts. This study clearly demonstrates that increases in SERCA protein level alter the beta-adrenergic response and affect the phosphorylation of PLB. Interestingly, the overall cardiac function in the live animal is only slightly enhanced, suggesting that (neuro)hormonal regulations may play an important role in controlling in vivo heart function.     

Gender differences in myosin heavy chain-beta and phosphorylated phospholamban in diabetic rat hearts

The objective of this study was to determine whether a gender difference exists in myosin heavy chain (MHC) isoform or sarcoplasmic reticulum protein levels in diabetic rat hearts. As is the case with normal rodent hearts, all four chambers of the control rat hearts expressed almost 100% MHC-alpha. In 6-wk diabetic rats, MHC-beta expression in ventricles of males was significantly greater (78 +/- 7%) than in females (50 +/- 5%). The cardiac sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA2a) protein level was decreased and the phospholamban (PLB) protein level was increased in the left ventricle of diabetic rats, but there was no difference between male and female diabetic rats. The phosphorylated PLB level was decreased more in male than in female diabetic rats. Insulin treatment completely normalized blood glucose level, cardiac SERCA2a and PLB protein levels, and the decrease in MHC-beta levels in both male and female diabetic rats. Insulin treatment completely normalized serum insulin and almost completely normalized phosphorylation of PLB at serine 16 in male diabetic rats. Although insulin treatment completely normalized serum insulin levels in male diabetic rats, in females it only partially normalized serum insulin levels. Also, insulin treatment almost completely normalized phosphorylation of PLB at threonine 17 in female diabetic rats; however, the increase was significantly greater than that identified for insulin-treated male diabetic rats. We conclude that higher levels of MHC-beta and dephosphorylated PLB may contribute to more contractile dysfunction in male than in female diabetic rat hearts, and that phosphorylation of PLB at threonine 17 is more responsive to insulin in female diabetic rat hearts.     

Cross-linking of C-terminal residues of phospholamban to the Ca2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain

Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+-phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all cross-linked strongly to V89C-SERCA2a, coupling to 70-100% of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca2+ for cross-linking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2 x ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2 x TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca2+ pump is complete, not partial, when the pump binds Ca2+ (E1 x Ca2) or adopts the E2P or E2 x TG conformations. V49C of PLB cross-linked to 100% of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2a is presented, showing that Val49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA.     

Role of conformational sampling of Ser16 and Thr17-phosphorylated phospholamban in interactions with SERCA

Phosphorylation of phospholamban (PLB) at Ser16 and/ or Thr17 is believed to release its inhibitory effect on sarcoplasmic reticulum calcium ATPase. Ser16 phosphorylation of PLB has been suggested to cause a conformational change that alters the interaction between the enzyme and protein. Using computer simulations, the conformational sampling of Ser16 phosphorylated PLB in implicit membrane environment is compared here with the unphosphorylated PLB system to investigate these conformational changes. The results suggest that conformational changes in the cytoplasmic domain of PLB upon phosphorylation at Ser16 increase the likelihood of unfavorable interactions with SERCA in the E2 state prompting a conformational switch of SERCA from E2 to E1. Phosphorylation of PLB at Thr17 on the other hand does not appear to affect interactions with SERCA significantly suggesting that the mechanism of releasing the inhibitory effect is different between Thr17 phosphorylated and Ser16 phosphorylated PLB.     

Defective Ca(2+) handling proteins regulation during heart failure

Abnormal release of Ca(2+) from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction in heart failure (HF). We previously demonstrated that RyR2 macromolecular complexes from HF rat were significantly more depleted of FK506 binding protein (FKBP12.6). Here we assessed expression of key Ca(2+) handling proteins and measured SR Ca(2+) content in control and HF rat myocytes. Direct measurements of SR Ca(2+) content in permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF (HF: DeltaF/F(0) = 26.4+/-1.8, n=12; control: DeltaF/F(0) = 49.2+/-2.9, n=10; P<0.01). Furthermore, we demonstrated that the expression of RyR2 associated proteins (including calmodulin, sorcin, calsequestrin, protein phosphatase 1, protein phosphatase 2A), Ca(2+) ATPase (SERCA2a), PLB phosphorylation at Ser16 (PLB-S16), PLB phosphorylation at Thr17 (PLB-T17), L-type Ca(2+) channel (Cav1.2) and Na(+)- Ca(2+) exchanger (NCX) were significantly reduced in rat HF. Our results suggest that systolic SR reduced Ca(2+) release and diastolic SR Ca(2+) leak (due to defective protein-protein interaction between RyR2 and its associated proteins) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a, PLB-S16 and PLB-T17), abnormal Ca(2+) extrusion (due to down-regulation of NCX) and defective Ca(2+) -induced Ca(2+) release (due to down-regulation of Cav1.2) could contribute to HF.     

Frequency-dependent contractile strength in mice over- and underexpressing the sarco(endo)plasmic reticulum calcium-ATPase

One of the prominent markers of end-stage heart failure at the molecular level is a decrease in function and/or expression of the sarcoplasmic reticulum ATPase protein [sarco(endo)plasmic reticulum calcium-ATPase, SERCA]. It has been often postulated that a decrease in SERCA pump activity can contribute in a major way to decreased cardiac function. To establish a functional relationship, we assessed how alterations in SERCA activity level affect basic contractile function in healthy myocardium devoid of other significant molecular changes. We investigated baseline contractile function, frequency-dependent activation, and beta-adrenergic response in ultrathin trabeculae isolated from hearts of mice overexpressing SERCA (transgenic, TG), underexpressing SERCA2a (heterozygous knockout, Het), and their respective wild-type (WT) littermates. At physiological temperature and frequency, compared with their respective WT littermates, SERCA1a mice displayed increased developed force at frequencies of 4-8 Hz ( approximately 90% increase at 4 Hz) and force equal to WT mice at 10-14 Hz. Force development at 4 Hz in presence of 1 muM isoproterenol was similar in TG and WT mice. In Het mice, developed force was nearly identical at the lower end of the frequency range (4-8 Hz) but slightly depressed at higher frequency (P < 0.05 at 14 Hz). In presence of 1 muM isoproterenol, developed force at 4 Hz was equal to that in WT mice. Compared with normal levels, increased SERCA activity enhanced force development only at subphysiological frequencies. A reduction in SERCA activity only showed a depression of force at the higher frequency range. Thus generalizations regarding the correlation between SERCA activity and contractility can be highly ambiguous, because this relationship is critically dependent on other factors including stimulation frequency.     

Tight interplay between the Ca2+ affinity of the cardiac SERCA2 Ca2+ pump and the SERCA2 expression level

A reduced activity of the sarcoplasmic reticulum Ca2+ pump SERCA2a is a hallmark of cardiac dysfunction in heart failure. In SERCA2b/b mice, the normal SERCA2a isoform is replaced by SERCA2b, displaying a higher Ca2+ affinity. This elicited decreased cardiac SERCA2 expression and cardiac hypertrophy. Here, the interplay was studied between the increased Ca2+ affinity and a reduced expression of the pump and its role in the cardiac remodeling was investigated. First, SERCA2b/b mice were crossed with SERCA2b transgenes to boost cardiac SERCA2b expression. However, the enforced expression of SERCA2b was spontaneously countered by an increased inhibition by phospholamban (PLB), reducing the pump's Ca2+ affinity. Moreover, the higher SERCA2 content did not prevent hypertrophy. Second, we studied heterozygous SERCA2b/WT mice, which also express lower SERCA2 levels compared to wild-type. Hypertrophy was not observed. In heterozygotes, SERCA2b expression was specifically suppressed, explaining the reduced SERCA2 content. The SERCA2b/WT model strikingly differs from the homozygote models because SERCA2a (not SERCA2b) is the major isoform and because the inhibition of the pump by PLB is decreased instead of being increased. Thus, a tight correlation exists between the SERCA2 levels and Ca2+ affinity (controlled by PLB). This compensatory response may be important to prevent cardiac remodeling.     

Synthetic Phosphopeptides Enable Quantitation of the Content and Function of the Four Phosphorylation States of Phospholamban in Cardiac Muscle

We have studied the differential effects of phospholamban (PLB) phosphorylation states on the activity of the sarcoplasmic reticulum Ca-ATPase (SERCA). It has been shown that unphosphorylated PLB (U-PLB) inhibits SERCA and that phosphorylation of PLB at Ser-16 or Thr-17 relieves this inhibition in cardiac sarcoplasmic reticulum. However, the levels of the four phosphorylation states of PLB (U-PLB, P16-PLB, P17-PLB, and doubly phosphorylated 2P-PLB) have not been measured quantitatively in cardiac tissue, and their functional effects on SERCA have not been determined directly. We have solved both problems through the chemical synthesis of all four PLB species. We first used the synthetic PLB as standards for a quantitative immunoblot assay, to determine the concentrations of all four PLB phosphorylation states in pig cardiac tissue, with and without left ventricular hypertrophy (LVH) induced by aortic banding. In both LVH and sham hearts, all phosphorylation states were significantly populated, but LVH hearts showed a significant decrease in U-PLB, with a corresponding increase in the ratio of total phosphorylated PLB to U-PLB. To determine directly the functional effects of each PLB species, we co-reconstituted each of the synthetic peptides in phospholipid membranes with SERCA and measured calcium-dependent ATPase activity. SERCA inhibition was maximally relieved by P16-PLB (the most highly populated PLB state in cardiac tissue homogenates), followed by 2P-PLB, then P17-PLB. These results show that each PLB phosphorylation state uniquely alters Ca²⁺ homeostasis, with important implications for cardiac health, disease, and therapy.     

Hydroxyl radical-induced apoptosis in human tumor cells is associated with telomere shortening but not telomerase inhibition and caspase activation

Reactive oxygen species (ROS) have been found to trigger apoptosis in tumor cells. At the same time, telomerase is found to be associated with malignancy and reduced apoptosis. However little is known about the linkage between ROS such as *OH and telomerase/telomere. To address the interrelations between *OH and telomerase/telomere in tumor cell killing, HeLa, 293 and MW451 cells were induced to undergo apoptosis with *OH radicals generated via Fe(2+)-mediated Fenton reactions (0.1 mM FeSO(4) plus 0.3-0.9 mM H2O2) and telomerase activity, telomere length were measured during apoptosis. We found that during *OH-induced apoptosis, telomere shortening took place while no changes in telomerase activity were observed. Our results suggest that *OH-induced telomere shortening is not through telomerase inhibition but possibly a direct effect of *OH on telomeres themselves indicating that telomere shortening but not telomerase inhibition is the primary event during *OH-induced apoptosis. Strikingly, we also found that *OH-induced apoptosis in HeLa cells is caspase-3-independent but is associated with reduction of mitochondrial transmembrane potential. Our results indicate that *OH triggers apoptotic tumor cell death through a telomere-related, caspase-independent pathway.     

Altered sinus nodal and atrioventricular nodal function in freely moving mice overexpressing the A1 adenosine receptor

To investigate whether altered function of adenosine receptors could contribute to sinus node or atrioventricular (AV) nodal dysfunction in conscious mammals, we studied transgenic (TG) mice with cardiac-specific overexpression of the A1 adenosine receptor (A1AR). A Holter ECG was recorded in seven freely moving littermate pairs of mice during normal activity, exercise (5 min of swimming), and 1 h after exercise. TG mice had lower maximal heart rates (HR) than wild-type (WT) mice (normal activity: 437 +/- 18 vs. 522 +/- 24 beats/min, P < 0.05; exercise: 650 +/- 13 vs. 765 +/- 28 beats/min, P < 0.05; 1 h after exercise: 588 +/- 18 vs. 720 +/- 12 beats/min, P < 0.05; all values are means +/- SE). Mean HR was lower during exercise (589 +/- 16 vs. 698 +/- 34 beats/min, P < 0.05) and after exercise (495 +/- 16 vs. 592 +/- 27 beats/min, P < 0.05). Minimal HR was not different between genotypes. HR variability (SD of RR intervals) was reduced by 30% (P < 0.05) in TG compared with WT mice. Pertussis toxin (n = 4 pairs, 150 microg/kg ip) reversed bradycardia after 48 h. TG mice showed first-degree AV nodal block (PQ interval: 42 +/- 2 vs. 37 +/- 2 ms, P < 0.05), which was diminished but not abolished by pertussis toxin. Isolated Langendorff-perfused TG hearts developed spontaneous atrial arrhythmias (3 of 6 TG mice vs. 0 of 9 WT mice, P < 0.05). In conclusion, A1AR regulate sinus nodal and AV nodal function in the mammalian heart in vivo. Enhanced expression of A1AR causes sinus nodal and AV nodal dysfunction and supraventricular arrhythmias.     

Phospholamban oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase binding measured by fluorescence resonance energy transfer in living cells

Phospholamban (PLB) oligomerization, quaternary structure, and sarco(endo)plasmic reticulum calcium ATPase (SERCA) binding were quantified by fluorescence resonance energy transfer (FRET) in an intact cellular environment. FRET between cyan fluorescent protein-PLB and yellow fluorescent protein-PLB in AAV-293 cells showed hyperbolic dependence on protein concentration, with a maximum efficiency of 45.1 +/- 1.3%. The observed FRET corresponds to a probe separation distance of 58.7 +/- 0.5A(,) according to a computational model of intrapentameric FRET. This is consistent with models of the PLB pentamer in which cytoplasmic domains fan out from the central bundle of transmembrane helices. An I40A mutation of PLB did not alter pentamer conformation but increased the concentration of half-maximal FRET (K(D)) by >4-fold. This is consistent with the previous observation that this putatively monomeric mutant still oligomerizes in intact membranes but forms more dynamic pentamers than wild type PLB. PLB association with SERCA, measured by FRET between cyan fluorescent protein-SERCA and yellow fluorescent protein-PLB, was increased by the I40A mutation without any detectable change in probe separation distance. The data indicate that the regulatory complex conformation is not altered by the I40A mutation. A naturally occurring human mutation (L39Stop) greatly reduced PLB oligomerization and SERCA binding and caused mislocalization of PLB to the cytoplasm and nucleus. Overall, the data suggest that the PLB pentamer adopts a "pinwheel" shape in cell membranes, as opposed to a more compact "bellflower" conformation. I40A mutation decreases oligomerization and increases PLB binding to SERCA. Truncation of the transmembrane domain by L39Stop mutation prevents anchoring of the protein in the membrane, greatly reducing PLB binding to itself or its regulatory target, SERCA.     

20-HETE对小鼠葡萄糖刺激胰岛素分泌反应的影响

为了考察20-羟基二十碳四烯酸(20-hydroxyeicosatetraenoic acids, 20-HETE)对葡萄糖刺激胰岛素分泌反应的影响,本研究选择CYP4F2转基因小鼠和小鼠胰岛素瘤INS-1E细胞作为研究材料,通过LCMS/MS检测WT和TG小鼠的胰腺20-HETE水平。通过IPGTT测定小鼠葡萄糖耐量,通过ELISA测定小鼠血浆C肽水平来检测胰岛素分泌。通过Western blotting、Real time PCR、免疫组化和免疫荧光来检测小鼠胰腺或INS-1E细胞中Glut2、GSK-3β(Ser9点)和AKT (Ser473点)的磷酸化水平。TG小鼠的20-HETE水平((7.26±2.03) ng/mg蛋白)显著高于WT小鼠((2.14±0.76) ng/mg蛋白)。在用20-HETE合成的选择性抑制剂HET0016处理后,TG小鼠((0.33±0.07) ng/mg蛋白)和WT小鼠((0.27±0.06) ng/mg蛋白)胰腺组织中的20-HETE水平均急剧降低。给予葡萄糖处理30 min后,TG小鼠的血糖水平均显著高于WT小鼠,而血浆C肽水平显著低于WT小鼠(p<0.05)。与WT小鼠相比,TG小鼠的胰腺组织中Glut2 m RNA和蛋白水平显著降低。与WT小鼠相比,CYP4F2转基因小鼠的GSK-3β和AKT磷酸化均显著降低。20-HETE处理可导致INS-1E细胞中AKT/GSK-3β磷酸化水平和Glut2表达水平显著降低(p<0.05)。此外,用17 mmol/L葡萄糖处理INS-1E细胞1 h,20-HETE处理组的胰岛素分泌显著降低。应用GSK-3β选择性抑制剂TWS119预处理INS-1E细胞3 h后,TWS119 (一种GSK-3β选择性抑制剂)预处理显著逆转了Glut2表达水平的降低以及胰岛素分泌的减少。20-HETE主要通过AKT/GSK-3β信号通路来下调Glut2的表达,进而减弱胰岛素分泌,导致胰岛素分泌功能障碍。     

Decreased cardiac SERCA2 expression, SR Ca uptake, and contractile function in hypothyroidism are attenuated in SERCA2 overexpressing transgenic rats

The sarco/endoplasmic reticulum (SR) Ca(2+)-ATPase SERCA2a has a key role in controlling cardiac contraction and relaxation. In hypothyroidism, decreased expression of the thyroid hormone (TH)-responsive SERCA2 gene contributes to slowed SR Ca(2+) reuptake and relaxation. We investigated whether cardiac expression of a TH-insensitive SERCA2a cDNA minigene can rescue SR Ca(2+) handling and contractile function in female SERCA2a-transgenic rats (TG) with experimental hypothyroidism. Wild-type rats (WT) and TG were rendered hypothyroid by 6-N-propyl-2-thiouracil treatment for 6 wk; control rats received no treatment. In vivo measured left ventricular (LV) hemodynamic parameters were compared with SERCA2a expression and function in LV tissue. Hypothyroidism decreased LV peak systolic pressure, dP/dt(max), and dP/dt(min) in both WT and TG. However, loss of function was less in TG. Thus slowed relaxation in hypothyroidism was found to be 1.5-fold faster in TG compared with WT (P < 0.05). In parallel, a 1.4-fold higher V(max) value of homogenate SR Ca(2+) uptake was observed in hypothyroid TG (P < 0.05 vs. hypothyroid WT), and the hypothyroidism-caused decline of LV SERCA2a mRNA expression in TG by -24% was markedly less than the decrease of -49% in WT (P < 0.05). A linear relationship was observed between the SERCA2a/PLB mRNA ratio values and the V(max) values of SR Ca(2+) uptake when the respective data of all experimental groups were plotted together (r = 0.90). The data show that expression of the TH-insensitive SERCA2a minigene compensates for loss of expressional activity of the TH-responsive native SERCA2a gene in the female hypothyroid rat heart. However, SR Ca(2+) uptake and in vivo heart function were only partially rescued.     

Phosphomimetic mutations increase phospholamban oligomerization and alter the structure of its regulatory complex

To investigate the effect of phosphorylation on the interactions of phospholamban (PLB) with itself and its regulatory target, SERCA, we measured FRET from CFP-SERCA or CFP-PLB to YFP-PLB in live AAV-293 cells. Phosphorylation of PLB was mimicked by mutations S16E (PKA site) or S16E/T17E (PKA+CaMKII sites). FRET increased with protein concentration up to a maximum (FRET(max)) that was taken to represent the intrinsic FRET of the bound complex. The concentration dependence of FRET yielded dissociation constants (K(D)) for the PLB-PLB and PLB-SERCA interactions. PLB-PLB FRET data suggest pseudo-phosphorylation of PLB increased oligomerization of PLB but did not alter PLB pentamer quaternary structure. PLB-SERCA FRET experiments showed an apparent decrease in binding of PLB to SERCA and an increase in the apparent PLB-SERCA binding cooperativity. It is likely that these changes are secondary effects of increased oligomerization of PLB; a change in the inherent affinity of monomeric PLB for SERCA was not detected. In addition, PLB-SERCA complex FRET(max) was reduced by phosphomimetic mutations, suggesting the conformation of the regulatory complex is significantly altered by PLB phosphorylation.     

Overexpression of Na+/Ca2+ exchanger gene attenuates postinfarction myocardial dysfunction

We monitored myocardial function in postinfarcted wild-type (WT) and transgenic (TG) mouse hearts with overexpression of the cardiac Na(+)/Ca(2+) exchanger. Five weeks after infarction, cardiac function was better maintained in TG than WT mice [left ventricular (LV) systolic pressure: WT, 41 +/- 2; TG, 58 +/- 3 mmHg; P < 0.05; maximum rising rate of LV pressure (+dP/dt(max)): WT, 3,750 +/- 346; TG, 5,075 +/- 334 mmHg/s; P < 0.05]. The isometric contractile response to beta-adrenergic stimulation was greater in papillary muscles from TG than WT mice (WT, 13.2 +/- 0.9; TG, 16.3 +/- 1.0 mN/mm(2) at 10(-4) M isoproterenol). The sarcoplasmic reticulum (SR) Ca(2+) content investigated by rapid cooling contractures in papillary muscles was greater in TG than WT mouse hearts. We conclude that myocardial function is better preserved in TG mice 5 wk after infarction, which results from enhanced SR Ca(2+) content via overexpression of the Na(+)/Ca(2+) exchanger.     

Mechanism of reversal of phospholamban inhibition of the cardiac Ca2+-ATPase by protein kinase A and by anti-phospholamban monoclonal antibody 2D12

Our model of phospholamban (PLB) regulation of the cardiac Ca(2+)-ATPase in sarcoplasmic reticulum (SERCA2a) states that PLB binds to the Ca(2+)-free, E2 conformation of SERCA2a and blocks it from transitioning from E2 to E1, the Ca(2+)-bound state. PLB and Ca(2+) binding to SERCA2a are mutually exclusive, and PLB inhibition of SERCA2a is manifested as a decreased apparent affinity of SERCA2a for Ca(2+). Here we extend this model to explain the reversal of SERCA2a inhibition that occurs after phosphorylation of PLB at Ser(16) by protein kinase A (PKA) and after binding of the anti-PLB monoclonal antibody 2D12, which recognizes residues 7-13 of PLB. Site-specific cysteine variants of PLB were co-expressed with SERCA2a, and the effects of PKA phosphorylation and 2D12 on Ca(2+)-ATPase activity and cross-linking to SERCA2a were monitored. In Ca(2+)-ATPase assays, PKA phosphorylation and 2D12 partially and completely reversed SERCA2a inhibition by decreasing K(Ca) values for enzyme activation, respectively. In cross-linking assays, cross-linking of PKA-phosphorylated PLB to SERCA2a was inhibited at only two of eight sites when conducted in the absence of Ca(2+) favoring E2. However, at a subsaturating Ca(2+) concentration supporting some E1, cross-linking of phosphorylated PLB to SERCA2a was attenuated at all eight sites. K(Ca) values for cross-linking inhibition were decreased nearly 2-fold at all sites by PLB phosphorylation, demonstrating that phosphorylated PLB binds more weakly to SERCA2a than dephosphorylated PLB. In parallel assays, 2D12 blocked PLB cross-linking to SERCA2a at all eight sites regardless of Ca(2+) concentration. Our results demonstrate that 2D12 restores maximal Ca(2+)-ATPase activity by physically disrupting the binding interaction between PLB and SERCA2a. Phosphorylation of PLB by PKA weakens the binding interaction between PLB and SERCA2a (yielding more PLB-free SERCA2a molecules at intermediate Ca(2+) concentrations), only partially restoring Ca(2+) affinity and Ca(2+)-ATPase activity.