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）以产生高基础水平的camp介导/蛋白激酶a （PKA）依赖性磷酸化和Ca2+/钙调素依赖性蛋白激酶II （CaMKII）蛋白磷酸化的Ca2+循环蛋白。这些导致自发性肌浆网（SR）在舒张去极化期间产生节律性Ca2+振荡，不仅触发表面膜产生节律性动作电位（APs），而且以前馈方式激活AC/PKA/CaMKII信号传导。ATP被消耗以泵送Ca2+到SR，产生cAMP，支持收缩和维持细胞离子稳态。由于反馈机制将ATP需求与ATP生产联系起来，我们假设(1)SANC中基础ATP供应和需求都依赖于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生成速率。(3)基础状态钙调素- camkii信号也与基础ATP生成速率有关。SANC具有高线粒体密度，与其他心脏组织相似，自发跳动时SANC的氧气消耗与3hz刺激的心室肌细胞（VM）相当。逐渐减少基础Ca2+-cAMP/PKA/CaMKII信号以减少兔SANC的ATP需求，产生逐渐的ATP消耗，降低O2消耗和黄蛋白荧光。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、选择性阻断收缩和特异性抑制线粒体Ca2+通量都不能降低ATP水平。我们可以得出结论，前馈基底Ca2+-cAMP/PKA/CaMKII信号既消耗ATP来驱动SANC中自发的APs，又与线粒体ATP产生密切相关。干扰Ca2+-cAMP/PKA /CaMKII信号不仅会减慢燃烧速度和减少ATP消耗，而且还会减少ATP的产生，从而使ATP水平下降。这与VM明显不同，VM缺乏这种基础前馈cAMP/PKA/CaMKII信号，并且当需求变化时ATP水平保持不变。