Dyssynchronous calcium removal in heart failure-induced atrial remodeling.

We tested the hypothesis that in atrial myocytes from a rabbit left ventricular heart failure (HF) model, spatial inhomogeneity and temporal dyssynchrony of Ca removal during excitation-contraction coupling together with increased Na/Ca exchange (NCX) activity generate a substrate for proarrhythmic Ca release. Ca removal occurs via Ca reuptake into the sarcoplasmic reticulum and extrusion via NCX exclusively in the cell periphery since rabbit atrial myocytes lack transverse tubules. Ca removal kinetics were assessed by the time constant τ of decay of local peripheral subsarcolemmal (SS) and central (CT) action potential (AP)-induced Ca transients (CaTs) recorded in confocal line scan mode (using Fluo-4). Spatial and temporal dyssynchrony of Ca removal was quantified by CV TAU, defined as the standard deviation of local τ along the transverse cell axis divided by mean τ. In normal cells CT CaT decline was slower compared with the SS domain, while in HF cells decline was accelerated, became equal in SS and CT regions, and a significant increase of CV TAU indicated an increased Ca removal dyssynchrony. In HF atrial cells NCX upregulation was accompanied by an overall higher incidence of spontaneous Ca waves and a higher propensity of arrhythmogenic Ca waves, defined as waves that triggered APs due to NCX-mediated membrane depolarization. NCX inhibition normalized CV TAU in HF atrial cells and decreased the propensity of Ca waves. In summary, HF atrial myocytes show accelerated but dyssynchronous diastolic Ca removal and altered sarcoplasmic reticulum Ca-ATPase (SERCA) and NCX activity that result in increased susceptibility to arrhythmia.

Excitation-contraction coupling in ventricular myocytes is enhanced by paracrine signaling from mesenchymal stem cells.

In clinical trials mesenchymal stem cells (MSCs) are transplanted into cardiac ischemic regions to decrease infarct size and improve contractility. However, the mechanism and time course of MSC-mediated cardioprotection are incompletely understood. We tested the hypothesis that paracrine signaling by MSCs promotes changes in cardiac excitation-contraction (EC) coupling that protects myocytes from cell death and enhances contractility. Isolated mouse ventricular myocytes (VMs) were treated with control tyrode, MSC conditioned-tyrode (ConT) or co-cultured with MSCs. The Ca handling properties of VMs were monitored by laser scanning confocal microscopy and whole cell voltage clamp. ConT superfusion of VMs resulted in a time dependent increase of the Ca transient amplitude (ConT(15min): ΔF/F(0)=3.52±0.38, n=14; Ctrl(15min): ΔF/F(0)=2.41±0.35, n=14) and acceleration of the Ca transient decay (τ: ConT: 269±18ms n=14; vs. Ctrl: 315±57ms, n=14). Voltage clamp recordings confirmed a ConT induced increase in I(Ca,L) (ConT: -5.9±0.5 pA/pF n=11; vs. Ctrl: -4.04±0.3 pA/pF, n=12). The change of τ resulted from increased SERCA activity. Changes in the Ca transient amplitude and τ were prevented by the PI3K inhibitors Wortmannin (100nmol/L) and LY294002 (10µmol/L) and the Akt inhibitor V (20µmol/L) indicating regulation through PI3K signal transduction and Akt activation which was confirmed by western blotting. A change in τ was also prevented in eNOS(-/-) myocytes or by inhibition of eNOS suggesting an NO mediated regulation of SERCA activity. Since paracrine signaling further resulted in increased survival of VMs we propose that the Akt induced change in Ca signaling is also a mechanism by which MSCs mediate an anti-apoptotic effect.

Ca2+ /H+ exchange via the plasma membrane Ca2+ ATPase in skeletal muscle.

The aims of this work were to determine: 1) whether Ca2+ exit via the plasmalemmal Ca2+ ATPase (PMCA) is coupled to H+ entry via a Ca2+/H+ exchange; 2) whether operation of PMCA has an absolute requirement on external H+ (Ho); and 3) the stoichiometry and voltage-dependence of the Ca2+/H+ exchange. Barnacle muscle cells were used because of the ease with which they can be internally-perfused (e.g., with 45Ca), voltage-clamped and impaled with a pH electrode. Thus, the simultaneous measurement of plasmalemmal Ca2+ and H+ fluxes can be measured. The effects of Ho, intracellular ATP, PMCA blockers, and membrane potential (VM) were studied on PMCA-mediated Ca2+/H+ exchange. The results indicate that: i) Ca2+ efflux is promoted by external acidification, is accompanied by a membrane depolarization, and by an intracellular acidification greater than the one resulting from Ho "leak" and PMCA-mediated ATP hydrolysis; ii) Ho-dependent Ca2+ efflux is inhibited by PMCA blockers and by ATP depletion and is accelerated by membrane depolarization (~3 fold by 20 mV depolarization); iii) the coupling ratio of the Ca2+/H+ exchange depends on Ho: at an extracellular pH (pHo)=6.5, the ratio is 1Ca2+:~3H+; at pHo=8.2, Ca2+ efflux rate is 3 times slower and the ratio is 1Ca2+: <1H+.

Phospholamban is required for CaMKII-dependent recovery of Ca transients and SR Ca reuptake during acidosis in cardiac myocytes.

Initially during acidosis, Ca transient amplitude (Delta[Ca]i) and the rate constant of [Ca]i decline (k(Ca)) are decreased, but later during acidosis Delta[Ca]i and k(Ca) partially recover. This recovery in rat myocytes could be inhibited by KN-93 suggesting that CaMKII-dependent protein phosphorylation (and enhanced SR Ca uptake) may be responsible. To test whether phospholamban (PLB) is required for the Delta[Ca]i and k(Ca) recovery during acidosis, we used isolated myocytes from PLB knockout (PLB-KO) vs. wild-type (WT) mice. [Ca]i was measured using fluo-3. During the initial phase of acidosis (1-4 min), Delta[Ca]i decreased in WT myocytes (n = 8) from 1.75 +/- 0.19 to 1.10 +/- 0.13 DeltaF/F0 (P < 0.05) and k(Ca) decreased from 3.20 +/- 0.22 to 2.38 +/- 0.18 s(-1) (P < 0.05). Later during acidosis (6-12 min), Delta[Ca]i partially recovered to 1.41 +/- 0.18 DeltaF/F0 and k(Ca) to 2.78 +/- 0.22 s(-1) (i.e. both recovered by approximately 50%). CaMKII inhibition using KN-93 completely prevented this recovery of Delta[Ca]i and k(Ca) during late acidosis in WT myocytes. In PLB-KO myocytes (n = 11) Delta[Ca]i decreased during early acidosis from 2.92 +/- 0.31 to 1.33 +/- 0.17 DeltaF/F0 (P < 0.05) and k(Ca) decreased from 10.45 +/- 0.56 to 7.58 +/- 0.68 s(-1) (P < 0.05). However, Delta[Ca]i did not recover during late acidosis and k(Ca) decreased even more (6.59 +/- 0.65 s(-1)). Parallel results were seen for contractile parameters. We conclude that PLB is crucial to the recovery of Delta[Ca]i and k(Ca) during acidosis. Moreover, PLB phosphorylation by CaMKII plays an important role in limiting the decline in Ca transients (and contraction) during acidosis.

Phosphorylation of phospholamban and troponin I in beta-adrenergic-induced acceleration of cardiac relaxation.

Activation of cAMP-dependent protein kinase A (PKA) in ventricular myocytes by isoproterenol (Iso) causes phosphorylation of both phospholamban (PLB) and troponin I (TnI) and accelerates relaxation by up to twofold. Because PLB phosphorylation increases sarcoplasmic reticulum (SR) Ca pumping and TnI phosphorylation increases the rate of Ca dissociation from the myofilaments, both factors could contribute to the acceleration of relaxation seen with PKA activation. To compare quantitatively the role of TnI versus PLB phosphorylation, we measured relaxation rates before and after maximal Iso treatment for twitches of matched amplitudes in ventricular myocytes and muscle from wild-type (WT) mice and from mice in which the PLB gene was knocked out (PLB-KO). Because Iso increases contractions, even in the PLB-KO mouse, extracellular [Ca] or sarcomere length was adjusted to obtain matching twitch amplitudes (in the presence and absence of Iso). In PLB-KO myocytes and muscles (which were allowed to shorten), Iso did not alter the time constant (tau) of relaxation ( approximately 29 ms). However, with increasing isometric force development in the PLB-KO muscles, Iso progressively but modestly accelerated relaxation (by 17%). These results contrast with WT myocytes and muscles where Iso greatly reduced tau of cell relaxation and intracellular Ca concentration decline (by 30-50%), independent of mechanical load. The Iso treatment used produced comparable increases in phosphorylation of TnI and PLB in WT. We conclude that the effect of beta-adrenergic activation on relaxation is mediated entirely by PLB phosphorylation in the absence of external load. However, TnI phosphorylation could contribute up to 14-18% of this lusitropic effect in the WT mouse during maximal isometric contractions.

Effect of isosmotic removal of extracellular Na+ on cell volume and membrane potential in muscle cells.

Isosmotic removal of extracellular Na+ (Nao) is a frequently performed manipulation. With the use of isolated voltage-clamped barnacle muscle cells, the effect of this manipulation on isosmotic cell volume was studied. Replacement of Nao by tris(hydroxymethyl)aminomethane produced membrane depolarization (approximately 20 mV) and cell volume loss (approximately 14%). The membrane depolarization was verapamil insensitive but depended on extracellular Ca2+ (Cao) and was probably due to activation of intracellular Ca2+ (Cai)-dependent nonselective cation channels. The cell volume loss did not require membrane depolarization but depended on Cao. This was probably due to an increase in Cai, mediated by activation of Ca2+ influx via Na+/Ca2+ exchange. Nao replacement by Li+ also promoted membrane depolarization (approximately 20 mV) and cell volume loss (20%). Both effects were reduced (approximately 73%) but were not abolished by Cao removal. Under this condition, the remaining membrane depolarization was probably due to a higher membrane permeability of Li+ over Na+. The remaining cell volume loss was due to membrane depolarization, which probably induced Ca2+ release from intracellular stores.

Extracellular Mg(2+)-dependent Na+, K+, and Cl- efflux in squid giant axons.

An extracellular Na+ (Nao)-dependent Mg2+ efflux process that requires intracellular ATP has been proposed as the sole mechanism responsible for Mg2+ extrusion in internally dialyzed squid axons (12). We have shown that this exchanger can also "reverse" and mediate an extracellular Mg2+ (Mgo)-dependent Na+ efflux (16). We have extended these studies and found that, in the presence of ouabain, bumetanide, tetrodotoxin, and K+ channel blockers and in the absence of extracellular Na+, K+, and bicarbonate, intracellular K+ and Cl- are also involved in the Mgo-dependent Na+ efflux process. Two main observations support this view: 1) operation of the Mgo-dependent Na+ efflux requires the presence of intracellular K+ and Cl-, and 2) Mgo removal produces a reversible and nearly identical reduction in the magnitude of the simultaneous efflux of the ionic pairs K(+)-Na+ and Cl(-)-Na+. These results suggest that the putative bumetanide-insensitive Na-Mg exchanger also transports K+ and Cl-.

Alpha-chymotrypsin deregulation of the sodium-calcium exchanger in barnacle muscle cells.

To gain insight into the mechanism by which the protease alpha-chymotrypsin (alpha-chym) activates the Na-Ca exchanger in muscle cells we studied 1) the ability of this enzyme to remove the intracellular "catalytic" Ca2+ requirement for activation of all the modes of exchange mediated by the Na-Ca exchanger (i.e., Nao-Cai, Nai-Cao, Nao-Nai, and Cao-Cai, where the subscripts o and i represent extracellular and intracellular, respectively), and 2) the ability of certain monovalent cations to stimulate Cao-Cai exchange after activation of the exchanger by alpha-chym. Barnacle muscle cells were used as models because these cells are so large that they can be internally perfused and voltage clamped. The results show that alpha-chym produces a highly activated Na-Ca exchanger able to operate in all its modes of exchange independently of catalytic Cai. The concentration-dependent effect of alpha-chym was biphasic; maximal activation occurred at 0.5 mg alpha-chym/ml perfusate for 20 min of perfusion at a perfusion rate of 2.5 microliters/min. The results are discussed in terms of the possible effects of alpha-chym on the kinetic modulation of the exchanger.