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，Ca2+ 激活腺苷酸环化酶 (AC)，产生高基础水平的 cAMP 介导/蛋白激酶 A (PKA) 依赖性磷酸化和 Ca2+/钙调蛋白依赖性蛋白激酶 II (CaMKII) 蛋白磷酸化Ca2+ 循环蛋白。这些导致舒张期去极化期间自发的肌浆网 (SR) 产生节律性 Ca2+ 振荡，不仅触发表面膜产生节律性动作电位 (AP)，而且以前馈方式激活 AC/PKA/CaMKII 信号传导。消耗 ATP 将 Ca2+ 泵送到 SR、产生 cAMP、支持收缩并维持细胞离子稳态。由于反馈机制将 ATP 需求与 ATP 产生联系起来，我们假设 (1) SANC 中的基础 ATP 供应和需求均依赖于 Ca2+-cAMP/PKA/CaMKII。 (2) 由于其前馈性质，通过 Ca2+-cAMP/PKA 信号轴的通量减少将降低基础 ATP 生产率。 (3) 基础状态钙调蛋白-CaMKII 信号传导也与基础 ATP 生成率相关。 SANC 具有高线粒体密度，与其他心脏组织类似，自发跳动中的 O2 消耗量与 3 Hz 刺激的心室肌细胞 (VM) 相当。基础 Ca2+-cAMP/PKA/CaMKII 信号传导的分级减少可减少兔 SANC 中 ATP 的需求，从而产生分级的 ATP 耗竭，并减少 O2 消耗和黄素蛋白荧光。基础 CaMKII 活性的逐渐降低也降低了 cAMP 水平。与 SANC 相比，VM 中 ATP 需求的减少不会明显改变稳定的 ATP 水平。抑制糖酵解、选择性阻断收缩或特异性抑制线粒体 Ca2+ 流量均不会降低 ATP 水平。我们可以得出结论，前馈基础 Ca2+-cAMP/PKA/CaMKII 信号传导既消耗 ATP 来驱动 SANC 中的自发 AP，又与线粒体 ATP 产生紧密相关。干扰 Ca2+-cAMP/PKA/CaMKII 信号传导不仅会减慢放电速率并减少 ATP 消耗，而且还会减少 ATP 产生，从而导致 ATP 水平下降。这与 VM 明显不同，VM 缺乏这种基础前馈基础 cAMP/PKA/CaMKII 信号传导，并且当需求变化时 ATP 水平保持恒定。 为了响应 β-肾上腺素能受体激活，自发的 AP 放电率和对 ATP 的需求增加。为了增加 ATP 需求，用 350.5℃ 的生理盐水和异丙肾上腺素灌注单个分离的兔 SANC，或者通过磷酸二酯酶或蛋白磷酸酶抑制。我们测量了单一 SANC 中的胞质和线粒体 Ca2+ 和黄素蛋白荧光，并测量了 SANC 悬浮液中的 cAMP、ATP 和 O2 消耗。尽管自发 AP 放电率的增加伴随着 O2 消耗的增加，但 ATP 水平和黄素蛋白荧光保持恒定，表明 ATP 产量增加。随着AP放电率的增加，Ca2+m和cAMP同时增加。当 Ru360 减少 Ca2+m 时，响应异丙肾上腺素的自发 AP 放电率增加减少了 25%。因此，Ca2+m 的增加和 Ca2+ 激活的 cAMP-PKA-CaMKII 信号传导的增加都可以调节 ATP 供应的增加，以满足高于基础水平的 ATP 需求。