Matching ATP supply and demand in cardiac pacemaker cells

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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.