Noriko Saegusa

Coupled Ca2+/H+ transport by cytoplasmic buffers regulates local Ca2+ and H+ ion signaling

Significance The concentration of Ca2+ ions is kept low in cells by specialized ion-pumping proteins at the membrane. We show that in cardiac cells, cytoplasm also has an intrinsic ability to pump Ca2+. Histidyl dipeptides and ATP are diffusible cytoplasmic buffer molecules. By exchanging Ca2+ for H+, they act like local “pumps,” producing uphill Ca2+ movement within cytoplasm in response to H+ ion gradients. Intracellular H+ ions are generated locally by metabolism and competitively inhibit many Ca2+-activated biochemical processes. Recruiting Ca2+ to acidic zones facilitates these processes. Cytoplasmic histidyl dipeptides and ATP thus act like a biological pump without a membrane. Ca2+ signaling regulates cell function. This is subject to modulation by H+ ions that are universal end-products of metabolism. Due to slow diffusion and common buffers, changes in cytoplasmic [Ca2+] ([Ca2+]i) or [H+] ([H+]i) can become compartmentalized, leading potentially to complex spatial Ca2+/H+ coupling. This was studied by fluorescence imaging of cardiac myocytes. An increase in [H+]i, produced by superfusion of acetate (salt of membrane-permeant weak acid), evoked a [Ca2+]i rise, independent of sarcolemmal Ca2+ influx or release from mitochondria, sarcoplasmic reticulum, or acidic stores. Photolytic H+ uncaging from 2-nitrobenzaldehyde also raised [Ca2+]i, and the yield was reduced following inhibition of glycolysis or mitochondrial respiration. H+ uncaging into buffer mixtures in vitro demonstrated that Ca2+ unloading from proteins, histidyl dipeptides (HDPs; e.g., carnosine), and ATP can underlie the H+-evoked [Ca2+]i rise. Raising [H+]i tonically at one end of a myocyte evoked a local [Ca2+]i rise in the acidic microdomain, which did not dissipate. The result is consistent with uphill Ca2+ transport into the acidic zone via Ca2+/H+ exchange on diffusible HDPs and ATP molecules, energized by the [H+]i gradient. Ca2+ recruitment to a localized acid microdomain was greatly reduced during intracellular Mg2+ overload or by ATP depletion, maneuvers that reduce the Ca2+-carrying capacity of HDPs. Cytoplasmic HDPs and ATP underlie spatial Ca2+/H+ coupling in the cardiac myocyte by providing ion exchange and transport on common buffer sites. Given the abundance of cellular HDPs and ATP, spatial Ca2+/H+ coupling is likely to be of general importance in cell signaling.

Influence of pH on Ca2+ current and its control of electrical and Ca2+ signaling in ventricular myocytes

Modulation of L-type Ca2+ current (ICa,L) by H+ ions in cardiac myocytes is controversial, with widely discrepant responses reported. The pH sensitivity of ICa,L was investigated (whole cell voltage clamp) while measuring intracellular Ca2+ (Ca2+i) or pHi (epifluorescence microscopy) in rabbit and guinea pig ventricular myocytes. Selectively reducing extracellular or intracellular pH (pHo 6.5 and pHi 6.7) had opposite effects on ICa,L gating, shifting the steady-state activation and inactivation curves to the right and left, respectively, along the voltage axis. At low pHo, this decreased ICa,L, whereas at low pHi, it increased ICa,L at clamp potentials negative to 0 mV, although the current decreased at more positive potentials. When Ca2+i was buffered with BAPTA, the stimulatory effect of low pHi was even more marked, with essentially no inhibition. We conclude that extracellular H+ ions inhibit whereas intracellular H+ ions can stimulate ICa,L. Low pHi and pHo effects on ICa,L were additive, tending to cancel when appropriately combined. They persisted after inhibition of calmodulin kinase II (with KN-93). Effects are consistent with H+ ion screening of fixed negative charge at the sarcolemma, with additional channel block by H+o and Ca2+i. Action potential duration (APD) was also strongly H+ sensitive, being shortened by low pHo, but lengthened by low pHi, caused mainly by H+-induced changes in late Ca2+ entry through the L-type Ca2+ channel. Kinetic analyses of pH-sensitive channel gating, when combined with whole cell modeling, successfully predicted the APD changes, plus many of the accompanying changes in Ca2+ signaling. We conclude that the pHi-versus-pHo control of ICa,L will exert a major influence on electrical and Ca2+-dependent signaling during acid–base disturbances in the heart.

Cardiomyocyte-specific p65 NF-κB deletion protects the injured heart by preservation of calcium handling.

NF-κB is a well-known transcription factor that is intimately involved with inflammation and immunity. We have previously shown that NF-κB promotes inflammatory events and mediates adverse cardiac remodeling following ischemia reperfusion (I/R). Conversely, others have pointed to the beneficial influence of NF-κB in I/R injury related to its anti-apoptotic effects. Understanding the seemingly disparate influence of manipulating NF-κB is hindered, in part, by current approaches that only indirectly interfere with the function of its most transcriptionally active unit, p65 NF-κB. Mice were generated with cardiomyocyte-specific deletion of p65 NF-κB. Phenotypically, these mice and their hearts appeared normal. Basal and stimulated p65 expression were significantly reduced in whole hearts and completely ablated in isolated cardiomyocytes. When compared with wild-type mice, transgenic animals were protected from both global I/R by Langendorff as well as regional I/R by coronary ligation and release. The protected, transgenic hearts had less cytokine activity and decreased apoptosis. Furthermore, p65 ablation was associated with enhanced calcium reuptake by the sarcoplasmic reticulum. This influence on calcium handling was related to increased expression of phosphorylated phospholamban in conditional p65 null mice. In conclusion, cardiomyocyte-specific deletion of the most active, canonical NF-κB subunit affords cardioprotection to both global and regional I/R injury. The beneficial effects of NF-κB inhibition are related, in part, to modulation of intracellular calcium homeostasis.

Nonanticoagulant heparin reduces myocyte Na+ and Ca2+ loading during simulated ischemia and decreases reperfusion injury

Heparin desulfated at the 2-O and 3-O positions (ODSH) decreases canine myocardial reperfusion injury. We hypothesized that this occurs from effects on ion channels rather than solely from anti-inflammatory activities, as previously proposed. We studied closed-chest pigs with balloon left anterior descending coronary artery occlusion (75-min) and reperfusion (3-h). ODSH effects on [Na(+)](i) (Na Green) and [Ca(2+)](i) (Fluo-3) were measured by flow cytometry in rabbit ventricular myocytes after 45-min of simulated ischemia [metabolic inhibition with 2 mM cyanide, 0 glucose, 37 degrees C, pacing at 0.5 Hz; i.e., pacing-metabolic inhibition (PMI)]. Na(+)/Ca(2+) exchange (NCX) activity and Na(+) channel function were assessed by voltage clamping. ODSH (15 mg/kg) 5 min before reperfusion significantly decreased myocardial necrosis, but neutrophil influx into reperfused myocardium was not consistently reduced. ODSH (100 microg/ml) reduced [Na(+)](i) and [Ca(2+)](i) during PMI. The NCX inhibitor KB-R7943 (10 microM) or the late Na(+) current (I(Na-L)) inhibitor ranolazine (10 microM) reduced [Ca(2+)](i) during PMI and prevented effects of ODSH on Ca(2+) loading. ODSH also reduced the increase in Na(+) loading in paced myocytes caused by 10 nM sea anemone toxin II, a selective activator of I(Na-L). ODSH directly stimulated NCX and reduced I(Na-L). These results suggest that in the intact heart ODSH reduces Na(+) influx during early reperfusion, when I(Na-L) is activated by a burst of reactive oxygen production. This reduces Na(+) overload and thus Ca(2+) influx via NCX. Stimulation of Ca(2+) extrusion via NCX later after reperfusion may also reduce myocyte Ca(2+) loading and decrease infarct size.

Platelet-activating factor stimulates sodium-hydrogen exchange in ventricular myocytes

Sodium-hydrogen exchanger (NHE), the principal sarcolemmal acid extruder in ventricular myocytes, is stimulated by a variety of autocrine/paracrine factors and contributes to myocardial injury and arrhythmias during ischemia-reperfusion. Platelet-activating factor (PAF; 1-o-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent proinflammatory phospholipid that is released in the heart in response to oxidative stress and promotes myocardial ischemia-reperfusion injury. PAF stimulates NHE in neutrophils and platelets, but its effect on cardiac NHE (NHE1) is unresolved. We utilized quiescent guinea pig ventricular myocytes bathed in bicarbonate-free solutions and epifluorescence to measure intracellular pH (pH(i)). Methylcarbamyl-PAF (C-PAF; 200 nM), a metabolically stable analog of PAF, significantly increased steady-state pH(i). The alkalosis was completely blocked by the NHE inhibitor, cariporide, and by sodium-free bathing solutions, indicating it was mediated by NHE activation. C-PAF also significantly increased the rate of acid extrusion induced by intracellular acidosis. The ability of C-PAF to increase steady-state pH(i) was completely blocked by the PAF receptor inhibitor WEB 2086 (10 μM), indicating the PAF receptor is required. A MEK inhibitor (PD98059; 25 μM) also completely blocked the rise in pH(i) induced by C-PAF, suggesting participation of the MAP kinase signaling cascade downstream of the PAF receptor. Inhibition of PKC with GF109203X (1 μM) and chelerythrine (2 μM) did not significantly affect the alkalosis induced by C-PAF. In summary, these results provide evidence that PAF stimulates cardiac NHE1, the effect occurs via the PAF receptor, and signal relay requires participation of the MAP kinase cascade.

Modulation of ventricular transient outward K+ current by acidosis and its effects on excitation-contraction coupling

The contribution of transient outward current (Ito) to changes in ventricular action potential (AP) repolarization induced by acidosis is unresolved, as is the indirect effect of these changes on calcium handling. To address this issue we measured intracellular pH (pHi), Ito, L-type calcium current (ICa,L), and calcium transients (CaTs) in rabbit ventricular myocytes. Intracellular acidosis [pHi 6.75 with extracellular pH (pHo) 7.4] reduced Ito by ~50% in myocytes with both high (epicardial) and low (papillary muscle) Ito densities, with little effect on steady-state inactivation and activation. Of the two candidate α-subunits underlying Ito, human (h)Kv4.3 and hKv1.4, only hKv4.3 current was reduced by intracellular acidosis. Extracellular acidosis (pHo 6.5) shifted Ito inactivation toward less negative potentials but had negligible effect on peak current at +60 mV when initiated from -80 mV. The effects of low pHi-induced inhibition of Ito on AP repolarization were much greater in epicardial than papillary muscle myocytes and included slowing of phase 1, attenuation of the notch, and elevation of the plateau. Low pHi increased AP duration in both cell types, with the greatest lengthening occurring in epicardial myocytes. The changes in epicardial AP repolarization induced by intracellular acidosis reduced peak ICa,L, increased net calcium influx via ICa,L, and increased CaT amplitude. In summary, in contrast to low pHo, intracellular acidosis has a marked inhibitory effect on ventricular Ito, perhaps mediated by Kv4.3. By altering the trajectory of the AP repolarization, low pHi has a significant indirect effect on calcium handling, especially evident in epicardial cells.