Crosstalk between Mitochondrial and Sarcoplasmic Reticulum Ca2+ Cycling Modulates Cardiac Pacemaker Cell Automaticity

Mitochondria dynamically buffer cytosolic Ca(2+) in cardiac ventricular cells and this affects the Ca(2+) load of the sarcoplasmic reticulum (SR). In sinoatrial-node cells (SANC) the SR generates periodic local, subsarcolemmal Ca(2+) releases (LCRs) that depend upon the SR load and are involved in SANC automaticity: LCRs activate an inward Na(+)-Ca(2+) exchange current to accelerate the diastolic depolarization, prompting the ensemble of surface membrane ion channels to generate the next action potential (AP).To determine if mitochondrial Ca(2+) (Ca(2+) (m)), cytosolic Ca(2+) (Ca(2+) (c))-SR-Ca(2+) crosstalk occurs in single rabbit SANC, and how this may relate to SANC normal automaticity.Inhibition of mitochondrial Ca(2+) influx into (Ru360) or Ca(2+) efflux from (CGP-37157) decreased [Ca(2+)](m) to 80 ± 8% control or increased [Ca(2+)](m) to 119 ± 7% control, respectively. Concurrent with inhibition of mitochondrial Ca(2+) influx or efflux, the SR Ca(2+) load, and LCR size, duration, amplitude and period (imaged via confocal linescan) significantly increased or decreased, respectively. Changes in total ensemble LCR Ca(2+) signal were highly correlated with the change in the SR Ca(2+) load (r(2) = 0.97). Changes in the spontaneous AP cycle length (Ru360, 111 ± 1% control; CGP-37157, 89 ± 2% control) in response to changes in [Ca(2+)](m) were predicted by concurrent changes in LCR period (r(2) = 0.84).A change in SANC Ca(2+) (m) flux translates into a change in the AP firing rate by effecting changes in Ca(2+) (c) and SR Ca(2+) loading, which affects the characteristics of spontaneous SR Ca(2+) release

Ca²+/calmodulin-dependent protein kinase II (CaMKII) activity and sinoatrial nodal pacemaker cell energetics.

: Ca(2+)-activated basal adenylate cyclase (AC) in rabbit sinoatrial node cells (SANC) guarantees, via basal cAMP/PKA-calmodulin/CaMKII-dependent protein phosphorylation, the occurrence of rhythmic, sarcoplasmic-reticulum generated, sub-membrane Ca(2+) releases that prompt rhythmic, spontaneous action potentials (APs). This high-throughput signaling consumes ATP.We have previously demonstrated that basal AC-cAMP/PKA signaling directly, and Ca(2+) indirectly, regulate mitochondrial ATP production. While, clearly, Ca(2+)-calmodulin-CaMKII activity regulates ATP consumption, whether it has a role in the control of ATP production is unknown.We superfused single, isolated rabbit SANC at 37°C with physiological saline containing CaMKII inhibitors, (KN-93 or autocamtide-2 Related Inhibitory Peptide (AIP)), or a calmodulin inhibitor (W-7) and measured cytosolic Ca(2+), flavoprotein fluorescence and spontaneous AP firing rate. We measured cAMP, ATP and O2 consumption in cell suspensions. Graded reductions in basal CaMKII activity by KN-93 (0.5-3 µmol/L) or AIP (2-10 µmol/L) markedly slow the kinetics of intracellular Ca(2+) cycling, decrease the spontaneous AP firing rate, decrease cAMP, and reduce O2 consumption and flavoprotein fluorescence. In this context of graded reductions in ATP demand, however, ATP also becomes depleted, indicating reduced ATP production.CaMKII signaling, a crucial element of normal automaticity in rabbit SANC, is also involved in SANC bioenergetics

The “Funny” Current (I<sub>f</sub>) Inhibition by Ivabradine at Membrane Potentials Encompassing Spontaneous Depolarization in Pacemaker Cells

Recent clinical trials have shown that ivabradine (IVA), a drug that inhibits the funny current (<em>I<sub>f</sub></em>) in isolated sinoatrial nodal cells (SANC), decreases heart rate and reduces morbidity and mortality in patients with cardiovascular diseases. While IVA inhibits<em> I<sub>f</sub></em>, this effect has been reported at essentially unphysiological voltages,<em> i.e.</em>, those more negative than the spontaneous diastolic depolarization (DD) between action potentials (APs). We tested the relative potency of IVA to block <em>I<sub>f</sub></em> over a wide range of membrane potentials, including those that encompass DD governing to the SANC spontaneous firing rate. A clinically relevant IVA concentration of 3 μM to single, isolated rabbit SANC slowed the spontaneous AP firing rate by 15%. During voltage clamp the maximal <em>I<sub>f</sub> </em>was 18 ± 3 pA/pF (at −120 mV) and the maximal <em>I<sub>f</sub> </em>reduction by IVA was 60 ± 8% observed at −92 ± 4 mV. At the maximal diastolic depolarization (~−60 mV) <em>I<sub>f</sub> </em>amplitude was only −2.9 ± 0.4 pA/pF, and was reduced by only 41 ± 6% by IVA. Thus, <em>I<sub>f</sub></em> amplitude and its inhibition by IVA at physiologically relevant membrane potentials are substantially less than that at unphysiological (hyperpolarized) membrane potentials. This novel finding more accurately describes how IVA affects SANC function and is of direct relevance to numerical modeling of SANC automaticity

Cardiac synthesis, processing, and coronary release of enkephalin-related peptides Downloaded from

. Cardiac synthesis, processing, and coronary release of enkephalin-related peptides. Am J Physiol Heart Circ Physiol 279: H1989-H1998, 2000.-Although preproenkephalin mRNA is abundant in the heart, the myocardial synthesis and processing of proenkephalin is largely undefined. Isolated working rat hearts were perfused to determine the rate of myocardial proenkephalin synthesis, its processing into enkephalin-containing peptides, their subsequent release into the coronary arteries, and the influence of prior sympathectomy. Enkephalin-containing peptides were separated by gel filtration and quantified with antisera for specific COOH-terminal sequences. Proenkephalin, peptide B, and [Met 5 ]enkephalin-Arg 6 -Phe 7 (MEAP) comprised 95% of the extracted myocardial enkephalins (35 pmol/g). Newly synthesized enkephalins, estimated during a 1-h perfusion with [ 14 C]phenylalanine (4 pmol ⅐ h Ϫ1 ⅐ g wet wt Ϫ1 ), were rapidly cleared from the heart during a second isotope-free hour. Despite a steady release of enkephalins into the coronary effluent (4 pmol ⅐ h Ϫ1 ⅐ g wet wt Ϫ1 ), enkephalin replacement apparently exceeded its release, and tissue enkephalins actually accumulated during hour 2. In contrast to the tissue, methionine-enkephalin accounted for more than half of the released enkephalin. Chemical sympathectomy produced an increase in total enkephalin content similar to that observed after 2-h control perfusion. This observation suggested that the normal turnover of myocardial enkephalin may depend in part on continued sympathetic influences. enkephalin-containing peptides; opioids; rat heart ENDOGENOUS ENKEPHALIN PEPTIDES are involved in the normal regulation of cardiovascular function (14). These include actions at both central and peripheral locations. Enkephalin receptors are concentrated in the brain stem and hypothalamus in close proximity to the cardiovascular centers (2), and centrally administered enkephalins produce a variety of site-specific cardiovascular responses (12, 13). Some effects of peripherally released enkephalins may result from direct interaction with opiate receptors localized on cardiomyocytes (10, 18, 22-24, 26, 35, 39-41). There is growing support for the rationale that endogenous cardiac opioids are potent modulators of cardiovascular function with significant physiological and pathological influences. The effects of opioids on heart rate, contractile strength, arterial tonus, and arterial pressure were initially summarized by Holaday (14). Although many opioid effects were attributed to the prejunctional inhibition of neurotransmitter release, newer studies indicate that they also modify myocardial function through postjunctional interactions. The stimulation of ␦-and -opiate receptors in the cardiac sarcolemma modified both calcium homeostasis and associated intracellular signaling pathways (10, 18, 22-24, 26, 35, 39-41). We have recently shown that ␦-receptor stimulation with the agonist leucine-enkephalin (LE) reversed the positive inotropic effect induced by norepinephrine in isolated ventricular cardiac myocytes and isolated, Langendorff-mode perfused hearts from adult rat

Beat-to-Beat Variation in Periodicity of Local Calcium Releases Contributes to Intrinsic Variations of Spontaneous Cycle Length in Isolated Single Sinoatrial Node Cells

Spontaneous, submembrane local Ca(2+) releases (LCRs) generated by the sarcoplasmic reticulum in sinoatrial nodal cells, the cells of the primary cardiac pacemaker, activate inward Na(+)/Ca(2+)-exchange current to accelerate the diastolic depolarization rate, and therefore to impact on cycle length. Since LCRs are generated by Ca(2+) release channel (i.e. ryanodine receptor) openings, they exhibit a degree of stochastic behavior, manifested as notable cycle-to-cycle variations in the time of their occurrence. AIM: The present study tested whether variation in LCR periodicity contributes to intrinsic (beat-to-beat) cycle length variability in single sinoatrial nodal cells. METHODS: We imaged single rabbit sinoatrial nodal cells using a 2D-camera to capture LCRs over the entire cell, and, in selected cells, simultaneously measured action potentials by perforated patch clamp. RESULTS: LCRs begin to occur on the descending part of the action potential-induced whole-cell Ca(2+) transient, at about the time of the maximum diastolic potential. Shortly after the maximum diastolic potential (mean 54±7.7 ms, n = 14), the ensemble of waxing LCR activity converts the decay of the global Ca(2+) transient into a rise, resulting in a late, whole-cell diastolic Ca(2+) elevation, accompanied by a notable acceleration in diastolic depolarization rate. On average, cells (n = 9) generate 13.2±3.7 LCRs per cycle (mean±SEM), varying in size (7.1±4.2 µm) and duration (44.2±27.1 ms), with both size and duration being greater for later-occurring LCRs. While the timing of each LCR occurrence also varies, the LCR period (i.e. the time from the preceding Ca(2+) transient peak to an LCR’s subsequent occurrence) averaged for all LCRs in a given cycle closely predicts the time of occurrence of the next action potential, i.e. the cycle length. CONCLUSION: Intrinsic cycle length variability in single sinoatrial nodal cells is linked to beat-to-beat variations in the average period of individual LCRs each cycle

(A) CaMKII inhibition by KN-93 or AIP, or calmodulin inhibition by W-7 decreases O₂ consumption in SANC suspension (n = 5 for each drug), (B) AIP decreases mitochondrial flavoprotein fluorescence in single SANC (representative example), indicating a net reduction of flavoprotein pool.

<p>Washout of AIP reverses this effect. *p<0.05 vs. drug control.</p

Average cells AP induced Ca²⁺ parameters in control (CON) and following introduction of AIP (n = 6; 2 µmol/L), KN-93 (n = 6; 0.5 µmol/L) or KN-92(n = 5; 3 µmol/L), *p<0.05 vs. drug control.

<p>Average cells AP induced Ca²⁺ parameters in control (CON) and following introduction of AIP (n = 6; 2 µmol/L), KN-93 (n = 6; 0.5 µmol/L) or KN-92(n = 5; 3 µmol/L), *p<0.05 vs. drug control.</p