Akt/PKB信号转导通路与脑缺血

Akt被生长因子介导的受体酪氨酸激酶磷酸化激活后可激活一系列底物分子,包括Forkhead转录因子等,对细胞生存和死亡进行调控.随着脑缺血后Akt磷酸化水平(Ser473)的改变,其上游、下游蛋白磷酸化水平也发生变化.预处理可能通过改变Akt蛋白磷酸化水平而产生缺血耐受.Akt/PKB信号转导通路功能障碍可能介导了脑缺血后神经元死亡.     

MCI-186减轻Aβ1-40诱导PC12细胞tau蛋白磷酸化的研究

目的 探讨依达拉奉(MCI-186)对β样淀粉蛋白(Aβ1-40)引起的PC12细胞tau蛋白磷酸化的保护作用及其机制.方法 采用 Western 印迹等检测Ser396,Ser199/202,Tau-5及GSK-3β,GSK-3βSer9磷酸化水平,观察MCI-186对Aβ25-35致PC12细胞的损伤保护作用.结果 模型组 tau蛋白在Ser396、Ser199/202位点的磷酸化水平及总tau蛋白水平在Aβ1-40作用3 h后开始升高,同时GSK-3β的表达增多,磷酸化GSK-3βSer9的表达减少,MCI-186保护组tau蛋白在Ser396,Ser199/202位点的磷酸化水平和总tau蛋白水平均明显低于Aβ模型组(P＜0.05),GSK-3β的表达减少,磷酸化GSK-3βSer9的表达增多(P＜0.05).结论 在Aβ1-40诱导PC12细胞损伤过程中,出现了tau蛋白的过度磷酸化,可以使Ser396,Ser199/202位点及Tau-5蛋白升高.激活GSK-3β是产生tau蛋白过度磷酸化的主要途径.MCI-186可通过抑制GSK-3β的活性,从而减轻Aβ1-40诱导的tau蛋白过度磷酸化,而达到保护神经细胞的目的 .     

连接蛋白43磷酸化状态与心脏缝隙连接通道功能的关系

缝隙连接是介导相邻细胞间直接通讯的特殊膜结构.心室肌细胞间的缝隙连接通道主要由连接蛋白43(Cx43)构成.Cx43的磷酸化状态除了快速调节通道的开放/闭合状态(单通道传导性和通道开放概率),还可通过影响Cx43的合成、转运、聚集/解聚和降解等不同环节,改变活化通道数量,最终实现对缝隙连接功能的调控.迄今,已发现多种激酶和蛋白磷酸酶可直接和(或)间接调控Cx43羧基末端的丝氨酸残基和酪氨酸残基的磷酸化状态,从而影响缝隙连接通道的功能.磷酸化/去磷酸化影响Cx43和缝隙连接通道功能的确切作用和具体机制未明.本文就Cx43磷酸化状态与心脏缝隙连接通道功能的关系作一综述.     

游离脂肪酸诱导3T3-L1脂肪细胞胰岛素抵抗的分子机制

目的研究游离脂肪酸（FFA）对3T3-L1脂肪细胞IKKβ及胰岛素信号转导蛋白的影响,探讨FFA诱导胰岛素抵抗（IR）的分子机制。方法诱导成熟的3T3-L1脂肪细胞与0.3-1.0mmol/L的软脂酸（PA）培养6-24h,以2-脱氧-〔^3H〕-D-葡萄糖摄入法观察葡萄糖的转运率,用Western blot检测IKKβ蛋白、IKKβ Ser181磷酸化、IRS-1蛋白、IRS-1 Ser307磷酸化、PI3Kp85蛋白及GluT4蛋白的表达。结果0.3-1.0mmol/LPA作用6-24h后,3T3-L1脂肪细胞的葡萄糖消耗明显减少,同时,Western blot显示,PA对IKKβ及GluT4蛋白的表达无明显影响,却能明显增加IKKβ Ser181及IRS-1 Ser307磷酸化,同时减少IRS-1蛋白和PI3Kp85蛋白的表达。结论FFA可以诱导IR,其分子机制可能与FFA激活IKKβ,使IRS-1丝氨酸残基磷酸化增加而酪氨酸残基磷酸化减少,进而使其下游的PI-3Kp85蛋白表达减少抑制葡萄糖转运有关。     

短时重复游泳调节SAM8鼠AMPA受体GluR1亚单位的磷酸化

目的观察短时重复游泳训练对SAM鼠AMPA受体GluR1亚单位磷酸化的影响,探讨运动改善脑功能的可能机制。方法选取3月龄SAMP8(prone/8)亚系为研究对象,运动模型采用2 w游泳方案:2次/d,每次6 min的游泳,结束后给予浴巾擦干放回鼠笼;对照组则在相同时间每天给予两次相同的浴巾安抚刺激。采用Western印迹方法,检测SAM8鼠海马和皮层AMPA受体GluR1亚单位Ser831和Ser 845位点的磷酸化水平的变化。结果 SAMP8海马、皮层中AMPA受体GluR1亚单位Ser831和Ser845磷酸化水平与对照组相比均增加(P<0.05)。结论 2 w的短时间重复游泳运动作为一种应激诱导剂促进了AMPA受体的活化,这可能是运动改善脑功能的机制之一。     

运动对胰岛素抵抗大鼠丝氨酸/酪氨酸磷酸化通路干预效应的研究

目的 观察运动对高脂膳食诱导胰岛素抵抗(IR)大鼠骨骼肌胰岛素受体底物1(IRS-1)丝氨酸307(Ser307)磷酸化和酪氨酸(Tyr)磷酸化通路的影响.方法 Wistar大鼠75只,分为正常对照(NC)组、高脂膳食(HF)组.10周后再将HF组分为高脂膳食运动(HFE)组和高脂膳食非运动(HFNE)组.以钳夹技术观察各组大鼠胰岛素敏感性(IS)变化,Western Blot法检测大鼠骨骼肌IRS-1 Ser307磷酸化和Tyr磷酸化水平.结果 (1) 10周后HF组葡萄糖输注率(GIR60～120)明显低于NC组;与NC组比较,HF组IRS-1 Tyr磷酸化水平降低,IRS-1 Ser307磷酸化水平升高(P均＜0.01).(2) 14周后HFE组GIR60～120明显高于HFNE组;与HFNE组比较,HFE组IRS-1 Tyr磷酸化水平升高,IRS-1 Ser307磷酸化水平降低(P均＜0.01).结论 运动干预改善IR大鼠IS,其机制可能与改善IRS-1 Ser307/Tyr异常磷酸化有关.     

缺氧/复氧引起H9C2细胞eNOS Ser633磷酸化水平降低的调控机制

目的:初步探讨缺氧/复氧(H/R)损伤过程心肌细胞株H9C2中内皮型一氧化氮合酶(eNOS)Ser633磷酸化水平的变化及其可能的调控机制。方法:采用缺氧4h培养后恢复常氧条件培养12、16或24h造成H9C2细胞H/R损伤。选取缺氧4h/复氧12h(H4/R12)进行后续实验。(1)H/R后用钙离子泵抑制剂毒胡萝卜素(thapsigargin,TG),1.0×10~(-6) mol/L处理细胞1h;用PI3K/Akt抑制剂LY294002(5.0×10~(-5) mol/L)预处理1h,再进行H/R及TG处理1h。(2)用PP2A/PP1抑制剂冈田酸Okadaic acid(OA)低剂量(5×10~(-8) mol/L)、高剂量(1×10~(-6) mol/L)预处理细胞30 min,再进行H/R。用Western blot检测eNOS总蛋白及eNOS Ser633磷酸化水平,化学比色法检测培养基中一氧化氮(NO)含量。结果:(1)H/R损伤后心肌细胞eNOS Ser633磷酸化水平降低(P0.05);TG明显上调eNOS Ser633磷酸化水平(P0.05),LY294002预处理可抑制TG的上调作用(P0.05);低剂量OA、高剂量OA预处理均可上调H/R损伤后eNOS Ser633磷酸化水平(P0.05),且高、低剂量组间无明显差异。(2)H/R损伤后培养基中NO含量减少(P0.05);TG、低剂量OA、高剂量OA处理均可增加H/R损伤后培养基中NO含量(P0.05)。结论:H/R损伤可明显降低H9C2细胞中eNOS Ser633水平,这可能是由于H/R过程中既有PI3K/Akt通路被抑制,又有PP2A活性增强所致。     

恶性疟原虫和寄生原虫的PP2磷酸酶家族

可逆性蛋白磷酸化在大多数生物的细胞功能调节中具有重要作用,疟原虫也属此列。目前仅报道恶性疟原虫(P.f.)几个蛋白磷酸酶(PP)基因,提示其催化亚基(C)对磷酸化底物的专一性不甚严格,Ser/Thr PP功能的多样性主要取决于C与多种调节亚基(A)相作用的能力。     

肌钙蛋白I对不稳定型心绞痛患者预后评价

目的观察不稳定型心绞痛（UAP）患者入院后血清肌钙蛋白I（cTnI）的变化,探讨其与ECG改变、心绞痛分级、冠状动脉病变的关系及对心脏事件的短期预报价值。方法34例UAP患者分别在入院时,入院后12h、24h、48h、10d各采血一次。用免疫发光法测定cTnI浓度,并分析其与临床状况、冠状动脉病变以及心脏事件的关系。结果在30d内,UAP患者中cTnI≥0.4μg/L者46.67%发生心脏事件;cTnI＜0.4μg/L者10.53%发生心脏事件（P＜0.05）。心绞痛Ⅲ级cTnI阳性率高,cTnI有否升高与心电图改变及冠状动脉造影、冠状动脉病变支数、狭窄部位无关（P>0.05）。cTnI对预测发生心脏事件的敏感性77.8%、特异性68%、阳性预测值46.7%、阴性预测值89.5%、准确性70.6%。结论cTnI定量测定对判断UAP患者短期内发生心脏事件有重要的预报价值。     

金雀异黄素对人胃腺癌细胞中Akt及其磷酸化蛋白表达的影响

目的:研究金雀异黄素(Genistein)对人胃腺癌细胞SGC-7901中Akt及其磷酸化蛋白p-Akt(Ser473)和p-Akt(Thr308)表达的影响.方法:用浓度为0,5,10,20,40和80μmo1/L的Genistein处理SGC-7901细胞24 h后,采用四甲基偶氮唑盐(MTT)方法检测Genistein对SGC-7901增殖的抑制作用,Hoechst 33342染色观察Genistein对SGC-7901凋亡的诱导作用,Westem blot方法检测不同浓度Genistein处理后的SGC-7901细胞中Akt,p-Akt(Ser473)和p-Akt(Thr308)的表达情况.结果:5,10,20,40和80 μmol/L的Genistein对SGC-7901细胞增殖的抑制率分别为6.85%±3.71%,13.19%±1.90%,20.94%±1.83%,29.58%±1.19%和41.75%±1.92%.20-80μmo1/L的Genistein处理组细胞呈现明显的凋亡形态学改变.Genistein对细胞中Akt蛋白的表达没有明显影响.不同浓度Genistein处理SGC-7901细胞24 h后,均有两种磷酸化Akt蛋白的表达,两种磷酸化蛋白表达均随Genistein浓度的增加而逐渐减弱,80 μmo1/L Genistein处理组表达最弱.结论:Genistein的抗癌活性与其抑制磷酸化Akt蛋白表达有关.     

Protein kinase D is a novel mediator of cardiac troponin I phosphorylation and regulates myofilament function

Protein kinase D (PKD) is a serine kinase whose myocardial substrates are unknown. Yeast 2-hybrid screening of a human cardiac library, using the PKD catalytic domain as bait, identified cardiac troponin I (cTnI), myosin-binding protein C (cMyBP-C), and telethonin as PKD-interacting proteins. In vitro phosphorylation assays revealed PKD-mediated phosphorylation of cTnI, cMyBP-C, and telethonin, as well as myomesin. Peptide mass fingerprint analysis of cTnI by liquid chromatography-coupled mass spectrometry indicated PKD-mediated phosphorylation of a peptide containing Ser22 and Ser23, the protein kinase A (PKA) targets. Ser22 and Ser23 were replaced by Ala, either singly (Ser22Ala or Ser23Ala) or jointly (Ser22/23Ala), and the troponin complex reconstituted in vitro, using wild-type or mutated cTnI together with wild-type cardiac troponin C and troponin T. PKD-mediated cTnI phosphorylation was reduced in complexes containing Ser22Ala or Ser23Ala cTnI and completely abolished in the complex containing Ser22/23Ala cTnI, indicating that Ser22 and Ser23 are both targeted by PKD. Furthermore, troponin complex containing wild-type cTnI was phosphorylated with similar kinetics and stoichiometry (approximately 2 mol phosphate/mol cTnI) by both PKD and PKA. To determine the functional impact of PKD-mediated phosphorylation, Ca2+ sensitivity of tension development was studied in a rat skinned ventricular myocyte preparation. PKD-mediated phosphorylation did not affect maximal tension but produced a significant rightward shift of the tension-pCa relationship, indicating reduced myofilament Ca2+ sensitivity. At submaximal Ca2+ activation, PKD-mediated phosphorylation also accelerated isometric crossbridge cycling kinetics. Our data suggest that PKD is a novel mediator of cTnI phosphorylation at the PKA sites and may contribute to the regulation of myofilament function.     

Why does troponin I have so many phosphorylation sites? Fact and fancy

We discuss a current controversy regarding the relative role of phosphorylation sites on cardiac troponin I (cTnI) (Fig. 1) in physiological and patho-physiological cardiac function. Studies with mouse models and in vitro studies indicate that multi-site phosphorylations are involved in both control of maximum tension and sarcomeric responsiveness to Ca²⁺. Thus one hypothesis is that cardiac function reflects a balance of cTnI phosphorylations and a tilt in this balance may be maladaptive in acquired and genetic disorders of the heart. Studies on human heart samples taken mainly at end-stage heart failure, and in depth proteomic analysis of human and rat heart samples demonstrate that Ser23/Ser24 are the major and perhaps the only sites likely to be relevant to control cardiac function. Thus functional significance of Ser23/Ser24 phosphorylation is taken as fact, whereas the function of some other sites is treated as fancy. Maybe the extremes will meet: in any case we both agree that further work needs to be carried out with relatively large mammals and with determination of the time course of changes in phosphorylation to identify transient modifications that may be relevant at a beat-to-beat basis. Moreover, we agree that the changes and effects of cTnI phosphorylation need to be fully integrated into the effects of other phosphorylations in the cardiac myocyte.     

Partial replacement of cardiac troponin I with a non-phosphorylatable mutant at serines 43/45 attenuates the contractile dysfunction associated with PKCepsilon phosphorylation

We have previously reported a transgenic mouse that over-expresses constitutively active PKCepsilon in the myocardium and exhibits a steady progression to heart failure. Associated with the decline in function was an increased phosphorylation of sarcomeric proteins including cardiac troponin I (cTnI). To determine whether PKCepsilon phosphorylation of cTnI is sufficient to induce cardiac maladaptation, we have generated a double transgenic mouse (DbTG) that expresses constitutively active PKCepsilon and cTnI harboring non-phosphorylatable mutations in the putative PKC phosphorylation sites (S43A, S45A). We compared the hemodynamic and biochemical properties of the hearts from the DbTG mice to the non-transgenic and single transgenic lines at both 3 and 12 months of age. While no significant differences in LV function were noted in 3-month groups, the depression of function in the PKCepsilon mice was attenuated in the double transgenic mice at 12 months. The improvement in cardiac function was correlated with decreased beta-myosin heavy chain and ANF mRNA expression in the 12m DbTG mice. The extent of cTnI phosphorylation was determined using a novel one-dimensional, non-equilibrium isoelectric focusing technique. At 3 months the migration of cTnI phospho-species was different in the PKCepsilon mice and to a lesser degree in the DbTG compared to all other groups. At 12 months additional phospho-species were observed in both the PKCepsilon and DbTG samples, along with an overall shift in the distribution of phospho-species in all groups due to age. These results suggest that phosphorylation of cTnI by PKCepsilon is associated with contractile dysfunction and partial replacement of serines 43/45 improves cardiac performance. Therefore, we conclude that phosphorylation of cTnI at Ser 43 and 45 may contribute to the progression of failure.     

The cardiac-specific N-terminal region of troponin I positions the regulatory domain of troponin C

The cardiac isoform of troponin I (cTnI) has a unique 31-residue N-terminal region that binds cardiac troponin C (cTnC) to increase the calcium sensitivity of the sarcomere. The interaction can be abolished by cTnI phosphorylation at Ser22 and Ser23, an important mechanism for regulating cardiac contractility. cTnC contains two EF–hand domains (the N and C domain of cTnC, cNTnC and cCTnC) connected by a flexible linker. Calcium binding to either domain favors an “open” conformation, exposing a large hydrophobic surface that is stabilized by target binding, cTnI[148–158] for cNTnC and cTnI[39–60] for cCTnC. We used multinuclear multidimensional solution NMR spectroscopy to study cTnI[1–73] in complex with cTnC. cTnI[39–60] binds to the hydrophobic face of cCTnC, stabilizing an alpha helix in cTnI[41–67] and a type VIII turn in cTnI[38–41]. In contrast, cTnI[1–37] remains disordered, although cTnI[19–37] is electrostatically tethered to the negatively charged surface of cNTnC (opposite its hydrophobic surface). The interaction does not directly affect the calcium binding affinity of cNTnC. However, it does fix the positioning of cNTnC relative to the rest of the troponin complex, similar to what was previously observed in an X-ray structure [Takeda S, et al. (2003) Nature 424(6944):35–41]. Domain positioning impacts the effective concentration of cTnI[148–158] presented to cNTnC, and this is how cTnI[19–37] indirectly modulates the calcium affinity of cNTnC within the context of the cardiac thin filament. Phosphorylation of cTnI at Ser22/23 disrupts domain positioning, explaining how it impacts many other cardiac regulatory mechanisms, like the Frank–Starling law of the heart.The balance between contraction and relaxation must be carefully regulated in the heart. Impaired relaxation can lead to diastolic heart failure, whereas systolic failure is characterized by insufficient contractility. Despite having different etiologies, both forms of heart failure are similar in terms of prevalence, symptoms, and mortality (1). Of all of the signaling pathways that regulate contractile function, the best studied is sympathetic β₁-adrenergic stimulation (2), which leads to cardiomyocyte cAMP production and activation of protein kinase A (PKA). Downstream phosphorylation of L-type calcium channels and phospholamban increases calcium fluxes, whereas phosphorylation of sarcomeric proteins, cardiac troponin I (cTnI), cardiac myosin binding protein-C, and titin (3) regulates the calcium-induced mechanical response.In human cTnI, Ser22 and Ser23 are the residues most consistently phosphorylated (4, 5). (There are some numbering inconsistencies in the literature, and we will refer to Ser22/23 instead of Ser23/24 to account for physiologic removal of the N-terminal methionine residue.) Originally identified as PKA targets, Ser22/23 are now known to be phosphorylated by other kinases, including PKG, PKCβ, PKCδ, and PKD1 (6), showing it to be an important locus at which multiple signaling pathways converge. Phosphorylation at cTnI Ser22/23 decreases the calcium sensitivity of the cardiac sarcomere (7). High levels of phosphorylation are seen in healthy individuals, but decreased phosphorylation levels occur in a number of pathologic states, including heart failure with reduced ejection fraction, heart failure with preserved ejection fraction, dilated cardiomyopathy, and hypertrophic cardiomyopathy (5, 8). Although dephosphorylation is likely a compensatory mechanism in many cases, it may be a disease-driving dysregulation in others.Other regulatory mechanisms are strongly influenced by the phosphorylation state of Ser22/23. The Frank–Starling law of the heart, also known as length-dependent activation or stretch activation, is more pronounced when Ser22/23 are phosphorylated (9, 10). In contrast, Ser5 (11) or Ser41/43 (12, 13) phosphorylation has more of an impact when Ser22/23 are unphosphorylated. Finally, some mutations that cause familial dilated cardiomyopathy have been shown to mitigate the effect of Ser22/23 phosphorylation (14). Despite the physiologic importance of Ser22/23 phosphorylation in regulating cardiac calcium sensitivity, the extent of its modulatory capacity has remained elusive.Ser22/23 lie within the cardiac-specific N-terminal region, cTnI[1–31], not present in the skeletal muscle isoforms. cTnI[1–209] forms long stretches of helical structure along a winding course that binds to troponin C, troponin T, and actin–tropomyosin. The X-ray structure of the cardiac troponin complex (15) did not include cTnI[1–34], so the structure of this region has not been determined, although there have been some preliminary investigations (16, 17). It is known that cTnI[1–31] interacts with cTnC in its unphosphorylated state (18), but phosphorylation abolishes this interaction, having an effect similar to truncation or removal of cTnI[1–31] (19). Our present study provides a detailed analysis of the structure and dynamics of cTnI[1–73] in complex with cTnC using solution NMR spectroscopy, highlighting its unique mechanism of action and physiologic implications.     

The troponin C G159D mutation blunts myofilament desensitization induced by troponin I Ser23/24 phosphorylation

Striated muscle contraction is regulated by the binding of Ca(2+) to the N-terminal regulatory lobe of the cardiac troponin C (cTnC) subunit in the troponin complex. In the heart, beta-adrenergic stimulation induces protein kinase A phosphorylation of cardiac troponin I (cTnI) at Ser23/24 to alter the interaction of cTnI with cTnC in the troponin complex and is critical to the regulation of cardiac contractility. We investigated the effect of the dilated cardiomyopathy linked cTnC Gly159 to Asp (cTnC-G159D) mutation on the development of Ca(2+)-dependent tension and ATPase rate in whole troponin-exchanged skinned rat trabeculae. Even though this mutation is located in the C-terminal lobe of cTnC, the G159D mutation was demonstrated to depress ATPase activation and filament sliding in vitro. The effects of this mutation within the cardiac myofilament are unknown. Our results demonstrate that the cTnC-G159D mutation by itself does not alter the myofilament response to Ca(2+) in the cardiac muscle lattice. However, in the presence of cTnI phosphorylated at Ser23/24, which reduced Ca(2+) sensitivity and enhanced cross-bridge cycling in controls, cTnC-G159D specifically blunted the phosphorylation induced decrease in Ca(2+)-sensitive tension development without altering cross-bridge cycling. Measurements in purified troponin confirmed that this cTnC-G159D blunting of myofilament desensitization results from altered Ca(2+)-binding to cTnC. Our results provide novel evidence that modification of the cTnC-cTnI interaction has distinct effects on troponin Ca(2+)-binding and cross-bridge kinetics to suggest a novel role for thin filament mutations in the modulation of myofilament function through beta-adrenergic signaling as well as the development of cardiomyopathy.     

Differential roles of cardiac myosin-binding protein C and cardiac troponin I in the myofibrillar force responses to protein kinase A phosphorylation

The heart is remarkably adaptable in its ability to vary its function to meet the changing demands of the circulatory system. During times of physiological stress, cardiac output increases in response to increased sympathetic activity, which results in protein kinase A (PKA)-mediated phosphorylations of the myofilament proteins cardiac troponin (cTn)I and cardiac myosin-binding protein (cMyBP)-C. Despite the importance of this mechanism, little is known about the relative contributions of cTnI and cMyBP-C phosphorylation to increased cardiac contractility. Using engineered mouse lines either lacking cMyBP-C (cMyBP-C(-/-)) or expressing a non-PKA phosphorylatable cTnI (cTnI(ala2)), or both (cMyBP-C(-/-)/cTnI(ala2)), we investigated the roles of cTnI and cMyBP-C phosphorylation in the regulation of the stretch-activation response. PKA treatment of wild-type and cTnI(ala2) skinned ventricular myocardium accelerated stretch activation such that the response was indistinguishable from stretch activation of cMyBP-C(-/-) or cMyBP-C(-/-)/cTnI(ala2) myocardium; however, PKA had no effect on stretch activation in cMyBP-C(-/-) or cMyBP-C(-/-)/cTnI(ala2) myocardium. These results indicate that the acceleration of stretch activation in wild-type and cTnI(ala2) myocardium is caused by phosphorylation of cMyBP-C and not cTnI. We conclude that the primary effect of PKA phosphorylation of cTnI is reduced Ca(2+) sensitivity of force, whereas phosphorylation of cMyBP-C accelerates the kinetics of force development. These results predict that PKA phosphorylation of myofibrillar proteins in living myocardium contributes to accelerated relaxation in diastole and increased rates of force development in systole.     

Regulation by phosphodiesterase isoforms of protein kinase A-mediated attenuation of myocardial protein kinase D activation

Protein kinase D (PKD) targets several proteins in the heart, including cardiac troponin I (cTnI) and class II histone deacetylases, and regulates cardiac contraction and hypertrophy. In adult rat ventricular myocytes (ARVM), PKD activation by endothelin-1 (ET1) occurs via protein kinase Cε and is attenuated by cAMP-dependent protein kinase (PKA). Intracellular compartmentalisation of cAMP, arising from localised activity of distinct cyclic nucleotide phosphodiesterase (PDE) isoforms, may result in spatially constrained regulation of the PKA activity that inhibits PKD activation. We have investigated the roles of the predominant cardiac PDE isoforms, PDE2, PDE3 and PDE4, in PKA-mediated inhibition of PKD activation. Pretreatment of ARVM with the non-selective PDE inhibitor isobutylmethylxanthine (IBMX) attenuated subsequent PKD activation by ET1. However, selective inhibition of PDE2 [by erythro-9-(2-hydroxy-3-nonyl) adenine, EHNA], PDE3 (by cilostamide) or PDE4 (by rolipram) individually had no effect on ET1-induced PKD activation. Selective inhibition of individual PDE isoforms also had no effect on the phosphorylation status of the established cardiac PKA substrates phospholamban (PLB; at Ser16) and cTnI (at Ser22/23), which increased markedly with IBMX. Combined administration of cilostamide and rolipram, like IBMX alone, attenuated ET1-induced PKD activation and increased PLB and cTnI phosphorylation, while combined administration of EHNA and cilostamide or EHNA and rolipram was ineffective. Thus, cAMP pools controlled by PDE3 and PDE4, but not PDE2, regulate the PKA activity that inhibits ET1-induced PKD activation. Furthermore, PDE3 and PDE4 play redundant roles in this process, such that inhibition of both isoforms is required to achieve PKA-mediated attenuation of PKD activation.     

Phosphorylation of troponin I controls cardiac twitch dynamics: evidence from phosphorylation site mutants expressed on a troponin I-null background in mice

The cardiac myofilament protein troponin I (cTnI) is phosphorylated by protein kinase C (PKC), a family of serine/threonine kinases activated within heart muscle by a variety of agonists. cTnI is also a substrate for cAMP-dependent protein kinase (PKA) activated during beta-adrenergic signaling. To investigate the role of cTnI phosphorylation in contractile regulation by these pathways, we generated transgenic mice harboring a mutated cTnI protein lacking phosphorylation sites for PKC (serine(43/45) and threonine(144) mutated to alanine) and for PKA (serine(23/24) mutated to alanine). Transgenic mice were interbred with cTnI-knockout mice to ensure the absence of endogenous phosphorylatable cTnI. Here, we report that regulation of myocyte twitch kinetics by beta-stimulation and by endothelin-1 was altered in myocytes containing mutant cTnI. In wild-type myocytes, the beta-agonist isoproterenol decreased twitch duration and relaxation time constant (tau) by 37% to 44%. These lusitropic effects of isoproterenol were reduced by about half in nonphosphorylatable cTnI mutant myocytes and were absent in cTnI mutants also lacking phospholamban (generated by crossing cTnI mutants with phospholamban-knockout mice). These observations are consistent with important roles for both cTnI and phospholamban phosphorylation in accelerating relaxation after beta-adrenergic stimulation. In contrast, endothelin-1 increased twitch duration by 32% and increased tau by 58%. These endothelin-1 effects were substantially blunted in nonphosphorylatable cTnI myocytes, indicating that PKC phosphorylation of cTnI slows cardiac relaxation and increases twitch duration. We propose that beta-agonists and endothelin-1 regulate cardiac twitch dynamics in opposite directions in part through phosphorylation of the myofilament protein cTnI on distinct sites.     

Intracellular localization and functional effects of P21-activated kinase-1 (Pak1) in cardiac myocytes

We investigated intracellular localization and substrate specificity of P21-activated kinase-1 (Pak1) in rat cardiac myocytes. Pak1 is a serine/threonine protein kinase that is activated by Rac1/Cdc42 and important in signaling of stress responses. Yet the localization and in vivo function of Pak1 in heart cells is poorly understood. Studies reported here indicate that Pak1 physically interacts with protein phosphatase 2a and localizes to the Z-disk, cell membrane, intercalated disc, and nuclear membrane of adult rat heart myocytes. We compared levels of phosphorylation of cardiac troponin I (cTnI) in control myocytes with phosphorylation of cTnI and myosin binding protein C (C-protein) in myocytes with increased Pak1 activity. The increase in activity was induced by infection of myocytes with a recombinant adenovirus (AdPak1) containing cDNA for a constitutively active Pak1. Control cells were infected with a virus (AdLacZ) containing LacZ. Basal levels of phosphorylation of cTnI and C-protein were relatively high in the myocytes infected with AdLacZ. However, phosphorylation of cTnI and C-protein in cells expressing constitutively active Pak1 was significantly reduced compared with those expressing LacZ. Measurement of Ca2+ tension relations in single myocytes demonstrated that this reduction in phosphorylation of cTnI and C-protein was associated with the predicted increase in sensitivity to Ca2+. Our data provide evidence for a novel pathway of phosphatase regulation in cardiac myocytes.     

Effect of troponin I Ser23/24 phosphorylation on Ca-sensitivity in human myocardium depends on the phosphorylation background

Protein kinase A (PKA)-mediated phosphorylation of Ser23/24 of cardiac troponin I (cTnI) causes a reduction in Ca²⁺-sensitivity of force development. This study aimed to determine whether the PKA-induced modulation of the Ca²⁺-sensitivity is solely due to cTnI phosphorylation or depends on the phosphorylation status of other sarcomeric proteins. Endogenous troponin (cTn) complex in donor cardiomyocytes was partially exchanged (up to 66 ± 1%) with recombinant unphosphorylated human cTn and in failing cells similar exchange was achieved using PKA-(bis)phosphorylated cTn complex. Cardiomyocytes immersed in exchange solution without complex added served as controls. Partial exchange of unphosphorylated cTn complex in donor tissue significantly increased Ca²⁺-sensitivity (pCa₅₀) to 5.50 ± 0.02 relative to the donor control value (pCa₅₀ = 5.43 ± 0.04). Exchange in failing tissue with PKA-phosphorylated cTn complex did not change Ca²⁺-sensitivity relative to the failing control (pCa₅₀ = 5.60 ± 0.02). Subsequent treatment of the cardiomyocytes with the catalytic subunit of PKA significantly decreased Ca²⁺-sensitivity in donor and failing tissue. Analysis of phosphorylated cTnI species revealed the same distribution of un-, mono- and bis-phosphorylated cTnI in donor control and in failing tissue exchanged with PKA-phosphorylated cTn complex. Phosphorylation of myosin-binding protein-C in failing tissue was significantly lower compared to donor tissue. These differences in Ca²⁺-sensitivity in donor and failing cells, despite similar distribution of cTnI species, could be abolished by subsequent PKA-treatment and indicate that other targets of PKA are involved the reduction of Ca²⁺-sensitivity. Our findings suggest that the sarcomeric phosphorylation background, which is altered in cardiac disease, influences the impact of cTnI Ser23/24 phosphorylation by PKA on Ca²⁺-sensitivity.