Matching ATP supply and demand in cardiac pacemaker cells

匹配心脏起搏细胞中的 ATP 供应和需求

• 批准号: 8931611
• 负责人: Edward Lakatta
• 金额: $ 11.59万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8931611
• 关键词: Action Potentials; Adenosine Triphosphate; Adenylate Cyclase; Calmodulin; Cells; Consumption; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Feedback; Flavoproteins; Fluorescence; Glycolysis Inhibition; Heart; Homeostasis; Intervention; Isoproterenol; Link; Measures; Mediating; Membrane; Mitochondria; Muscle Cells; Nature; Oryctolagus cuniculus; Phosphorylation; Physiological; Production; Protein phosphatase; Proteins; Pump; Receptor Activation; Saline; Sarcoplasmic Reticulum; Signal Transduction; Sinoatrial Node; Surface; Suspension substance; Suspensions; Tissues; Ventricular; beta-adrenergic receptor; calmodulin-dependent protein kinase II; density; falls; feeding; meetings; nodal myocyte; phosphoric diester hydrolase; response

In sinoatrial node cells (SANC), Ca2+ activates adenylate cyclase (AC) to generate a high basal level of cAMP-mediated/protein kinase A (PKA)-dependent phosphorylation and Ca2+/calmodulin-dependent protein kinase II (CaMKII) protein phosphorylation of Ca2+ cycling proteins. These result in spontaneous sarcoplasmic reticulum (SR)-generated rhythmic Ca2+ oscillations during diastolic depolarization that not only trigger the surface membrane to generate rhythmic action potentials (APs), but, in a feed-forward manner, also activate AC/PKA/CaMKII signaling. ATP is consumed to pump Ca2+ to the SR, to produce cAMP, to support contraction and to maintain cell ionic homeostasis. Since feedback mechanisms link ATP-demand to ATP production, we hypothesized that (1) both basal ATP supply and demand in SANC would be Ca2+-cAMP/PKA/CaMKII dependent. (2) Due to its feed-forward nature, a decrease in flux through the Ca2+-cAMP/PKA signaling axis will reduce the basal ATP production rate. (3) Basal state calmodulin-CaMKII signaling is also linked to basal ATP production rate. SANC possess a high mitochondrial density, similar to other heart tissues O2 consumption in spontaneous beating SANC was comparable to ventricular myocytes (VM) stimulated at 3 Hz. Graded reduction of basal Ca2+-cAMP/PKA/CaMKII signaling to reduce ATP demand in rabbit SANC produced graded ATP depletion, and reduced O2 consumption and flavoprotein fluorescence. Graded reductions in basal CaMKII activity also reduced the cAMP level. In contrast to SANC, reductions in ATP demand in VM do not appreciably change the steady ATP levels. Neither inhibition of glycolysis, selectively blocking contraction nor specific inhibition of mitochondrial Ca2+ flux reduced the ATP level. We can conclude that feed-forward basal Ca2+-cAMP/PKA/CaMKII signaling both consumes ATP to drive spontaneous APs in SANC and is tightly linked to mitochondrial ATP production. Interfering with Ca2+-cAMP/PKA /CaMKII signaling not only slows the firing rate and reduces ATP consumption, but also appears to reduce ATP production so that ATP levels fall. This distinctly differs from VM, which lack this basal feed-forward basal cAMP/PKA/CaMKII signaling, and in which the ATP level remains constant when the demand changes. In response to beta-adrenergic receptor activation, the spontaneous AP firing rate and the demand for ATP increase. To increase ATP demand, single isolated rabbit SANC were superfused by physiological saline at 350.5C with isoproterenol, or by phosphodiesterase or protein phosphatase inhibition. We measured cytosolic and mitochondrial Ca2+ and flavoprotein fluorescence in single SANC and we measured cAMP, ATP and O2 consumption in SANC suspensions. Although the increase in spontaneous AP firing rate was accompanied by an increase in O2 consumption, the ATP level and flavoprotein fluorescence remained constant, indicating that ATP production had increased. Both Ca2+m and cAMP increased concurrently with the increase in AP firing rate. When Ca2+m was reduced by Ru360, the increase in spontaneous AP firing rate in response to isoproterenol was reduced by 25%. Thus, both an increase in Ca2+m and an increase in Ca2+ activated cAMP-PKA-CaMKII signaling regulate the increase in ATP supply to meet ATP demand above the basal level.

在窦房结细胞(SANC)中，Ca~(2+)激活腺苷环化酶(AC)，产生基础水平较高的cAMP/蛋白激酶A(PKA)依赖的磷酸化和Ca~(2+)/钙调素依赖的蛋白激酶II(CaMKII)蛋白的磷酸化。这导致肌浆网(SR)在舒张期去极化过程中自发地产生节律性的钙振荡，不仅触发表膜产生节律性动作电位(AP)，而且以前馈的方式激活AC/PKA/CaMKII信号。ATP被消耗来泵出钙离子到SR，产生cAMP，支持收缩和维持细胞离子平衡。由于反馈机制将ATP需求与ATP生产联系在一起，我们假设(1)SANC的基础ATP供需都是依赖于钙-cAMP/PKA/CaMKII的。(2)由于其前馈性质，通过钙-cAMP/PKA信号轴的流量减少将降低基础的ATP产生速率。(3)基础态钙调素-CaMKII信号也与基础ATP生成率有关。SANC具有较高的线粒体密度，与其他心脏组织相似，自发搏动SANC的耗氧量与3赫兹刺激的心室肌细胞(Vm)相当。分级降低基础钙-cAMP/PKA/CaMKII信号以减少兔SARC对ATP的需求，造成分级的ATP耗竭，并减少氧消耗和黄素蛋白荧光。基础CaMKII活性的逐步降低也降低了cAMP水平。与SANC不同的是，VM对ATP需求的减少并没有明显改变稳定的ATP水平。抑制糖酵解、选择性阻断收缩或特异性抑制线粒体钙通量都不会降低ATP水平。我们可以得出结论，前馈基础钙-cAMP/PKA/CaMKII信号既消耗ATP来驱动SANC的自发AP，又与线粒体ATP的产生密切相关。干扰Ca~(2+)-cAMP/PKA/CaMKII信号系统不仅可以减慢细胞的放电速率，减少ATP的消耗，而且还可以减少ATP的产生，从而降低细胞内的ATP水平。这与VM明显不同，在VM中，缺乏基本的前馈基础cAMP/PKA/CaMKII信号，当需求变化时，ATP水平保持不变。 随着β-肾上腺素能受体的激活，自发性AP放电频率和对ATP的需求增加。为增加对三磷酸腺苷的需求，用350.5℃生理盐水和异丙肾上腺素、磷酸二酯酶或蛋白磷酸酶抑制法灌流分离的兔心。测定单个SAEC的胞浆和线粒体的钙、黄素蛋白荧光，以及SANC悬液中cAMP、ATP和O2的消耗。虽然自发性AP放电频率的增加伴随着O2消耗的增加，但ATP水平和黄素蛋白荧光保持不变，表明ATP的产生增加。Ca~(2+)~(2+)m和cAMP均随AP放电频率增加而增加。当钙离子m被Ru360降低时，异丙肾上腺素引起的自发性AP放电频率的增加减少了25%。因此，Ca~(2+)m的增加和Ca~(2+)激活的cAMP-PKA-CaMKII信号的增加都调节了ATP供应的增加，以满足高于基础水平的ATP需求。