Complete Atrial-Specific Knockout of Sodium-Calcium Exchange Eliminates Sinoatrial Node Pacemaker Activity

The origin of sinoatrial node (SAN) pacemaker activity in the heart is controversial. The leading candidates are diastolic depolarization by “funny” current (I_f) through HCN4 channels (the “Membrane Clock“ hypothesis), depolarization by cardiac Na-Ca exchange (NCX1) in response to intracellular Ca cycling (the "Calcium Clock" hypothesis), and a combination of the two (“Coupled Clock”). To address this controversy, we used Cre/loxP technology to generate atrial-specific NCX1 KO mice. NCX1 protein was undetectable in KO atrial tissue, including the SAN. Surface ECG and intracardiac electrograms showed no atrial depolarization and a slow junctional escape rhythm in KO that responded appropriately to β-adrenergic and muscarinic stimulation. Although KO atria were quiescent they could be stimulated by external pacing suggesting that electrical coupling between cells remained intact. Despite normal electrophysiological properties of I_f in isolated patch clamped KO SAN cells, pacemaker activity was absent. Recurring Ca sparks were present in all KO SAN cells, suggesting that Ca cycling persists but is uncoupled from the sarcolemma. We conclude that NCX1 is required for normal pacemaker activity in murine SAN.     

Mechanistic distinction between activation and inhibition of (Na(+)+K(+))-ATPase-mediated Ca2+ influx in cardiomyocytes

(Na(+)+K(+))-ATPase (NKA) mediates positive inotropy in the heart. Extensive studies have demonstrated that the reverse-mode Na(+)/Ca(2+)-exchanger (NCX) plays a critical role in increasing intracellular Ca(2+) concentration through the inhibition of NKA-induced positive inotropy by cardiac glycosides. Little is known about the nature of the NCX functional mode in the activation of NKA-induced positive inotropy. Here, we examined the effect of an NKA activator SSA412 antibody on (45)Ca influx in isolated rat myocytes and found that KB-R7943, a NCX reverse-mode inhibitor, fails to inhibit the activation of NKA-induced (45)Ca influx, suggesting that the Ca(2+) influx via the reverse-mode NCX does not mediate this process. Nifedipine, an L-type Ca(2+) channel (LTCC) inhibitor, completely blocks the activation of NKA-induced (45)Ca influx, suggesting that the LTCC is responsible for the moderate increase in intracellular Ca(2+). In contrast, the inhibition of NKA by ouabain induces 4.7-fold (45)Ca influx compared with the condition of activation of NKA. Moreover, approximately 70% of ouabain-induced (45)Ca influx was obstructed by KB-R7943 and only 30% was impeded by nifedipine, indicating that both the LTCC and the NCX contribute to the rise in intracellular Ca(2+) and that the NCX reverse-mode is the major source for the (45)Ca influx induced by the inhibition of NKA. This study provides direct evidence to demonstrate that the activation of NKA-induced Ca(2+) increase is independent of the reverse-mode NCX and pinpoints a mechanistic distinction between the activation and inhibition of the NKA-mediated Ca(2+) influx path ways in cardiomyocytes.     

Three-dimensional distribution of cardiac Na+-Ca2+ exchanger and ryanodine receptor during development

Mechanisms of cardiac excitation-contraction coupling in neonates are still not clearly defined. Previous work in neonates shows reverse-mode Na(+)-Ca(2+) exchange to be the primary route of Ca(2+) entry during systole and the neonatal sarcoplasmic reticulum to have similar capability as that of adult in storing and releasing Ca(2+). We investigated Na(+)-Ca(2+) exchanger (NCX) and ryanodine receptor (RyR) distribution in developing ventricular myocytes using immunofluorescence, confocal microscopy, and digital image analysis. In neonates, both NCX and RyR clusters on the surface of the cell displayed a short longitudinal periodicity of approximately 0.7 microm. However, by adulthood, both proteins were also found in the interior. In the adult, clusters of NCX on the surface of the cell retained the approximately 0.7-microm periodicity whereas clusters of RyR adopted a longer longitudinal periodicity of approximately 2.0 microm. This suggests that neonatal myocytes also have a peri-M-line RyR distribution that is absent in adult myocytes. NCX and RyR colocalized voxel density was maximal in neonates and declined significantly with ontogeny. We conclude in newborns, Ca(2+) influx via NCX could potentially activate the dense network of peripheral Ca(2+) stores via peripheral couplings, evoking Ca(2+)-induced Ca(2+) release.     

Dantrolene prevents arrhythmogenic Ca2+ release in heart failure

In heart failure (HF), arrhythmogenic Ca(2+) release and chronic Ca(2+) depletion of the sarcoplasmic reticulum (SR) arise due to altered function of the ryanodine receptor (RyR) SR Ca(2+)-release channel. Dantrolene, a therapeutic agent used to treat malignant hyperthermia associated with mutations of the skeletal muscle type 1 RyR (RyR1), has recently been suggested to have effects on the cardiac type 2 RyR (RyR2). In this investigation, we tested the hypothesis that dantrolene exerts antiarrhythmic and inotropic effects on HF ventricular myocytes by examining multiple aspects of intracellular Ca(2+) handling. In normal rabbit myocytes, dantrolene (1 μM) had no effect on SR Ca(2+) load, postrest decay of SR Ca(2+) content, the threshold for spontaneous Ca(2+) wave initiation (i.e., the SR Ca(2+) content at which spontaneous waves initiate) and Ca(2+) spark frequency. In cardiomyocytes from failing rabbit hearts, SR Ca(2+) load and the wave initiation threshold were decreased compared with normal myocytes, Ca(2+) spark frequency was increased, and the postrest decay was potentiated. Using a novel approach of measuring cytosolic and intra-SR Ca(2+) concentration (using the low-affinity Ca(2+) indicator fluo-5N entrapped within the SR), we showed that treatment of HF cardiomyocytes with dantrolene rescued postrest decay and increased the wave initiation threshold. Additionally, dantrolene decreased Ca(2+) spark frequency while increasing the SR Ca(2+) content in HF myocytes. These data suggest that dantrolene exerts antiarrhythmic effects and preserves inotropy in HF cardiomyocytes by decreasing the incidence of diastolic Ca(2+) sparks, increasing the intra-SR Ca(2+) threshold at which spontaneous Ca(2+) waves occur, and decreasing the loss of Ca(2+) from the SR. Furthermore, the observation that dantrolene reduces arrhythmogenicity while at the same time preserves inotropy suggests that dantrolene is a potentially useful drug in the treatment of arrhythmia associated with HF.     

Increases in diastolic [Ca2+] can contribute to positive inotropy in guinea pig ventricular myocytes in the absence of changes in amplitudes of Ca2+ transients

Increases in contraction amplitude following rest or in elevated extracellular Ca(2+) concentration ([Ca(2+)]) have been attributed to increased sarcoplasmic reticulum (SR) Ca(2+) stores and/or increased trigger Ca(2+). However, either manipulation also may elevate diastolic [Ca(2+)]. The objective of this study was to determine whether elevation of diastolic [Ca(2+)] could contribute to positive inotropy in isolated ventricular myocytes. Voltage-clamp experiments were conducted with high-resistance microelectrodes in isolated myocytes at 37 degrees C. Intracellular free [Ca(2+)] was measured with fura-2, and cell shortening was measured with an edge detector. SR Ca(2+) stores were assessed with 10 mM caffeine (0 mM Na(+), 0 mM Ca(2+)). Following a period of rest, cells were activated with trains of pulses, which generated contractions of increasing amplitude, called positive staircases. Positive staircases were accompanied by increasing diastolic [Ca(2+)] but no change in Ca(2+) transient amplitudes. When extracellular [Ca(2+)] was elevated from 2.0 to 5.0 mM, resting intracellular [Ca(2+)] increased and resting cell length decreased. Amplitudes of contractions and L-type Ca(2+) current increased in elevated extracellular [Ca(2+)], although SR Ca(2+) stores, assessed by rapid application of caffeine, did not increase. Although Ca(2+) transient amplitude did not increase in 5.0 mM extracellular [Ca(2+)], diastolic [Ca(2+)] continued to increase with increasing extracellular [Ca(2+)]. These data suggest that increased diastolic [Ca(2+)] contributes to positive inotropy following rest or with increasing extracellular [Ca(2+)] in guinea pig ventricular myocytes.     

Altered Ca2+ sparks and gating properties of ryanodine receptors in aging cardiomyocytes

To investigate the cellular mechanisms for altered cardiac function in senescence, we measured Ca(2+) transients and Ca(2+) sparks in ventricular cardiomyocytes from 6- to 24-month-old Fisher 344 (F344) rat hearts. The single channel properties of ryanodine receptors from adult and senescent hearts were also studied. In senescent myocytes, we observed a decreased peak [Ca(2+)](i) amplitude and an increased time constant for decay (tau), both of which correlated with a reduced Ca(2+) content of the sarcoplasmic reticulum (SR). Our studies also revealed that senescent cardiomyocytes had an increased frequency of Ca(2+) sparks and a slight but statistically significant decrease in average amplitude, full-width-at-half-maximum (FWHM) and full-duration-at-half-maximum (FDHM). Single channel recordings of ryanodine receptors (RyR2) demonstrated that in aging hearts, the open probability (P(o)) of RyR2 was increased but the mean open time was shorter, providing a molecular correlate for the increased frequency of Ca(2+) sparks and decreased size of sparks, respectively. Thus, modifications of normal RyR2 gating properties may play a role in the altered Ca(2+) homeostasis observed in senescent myocytes.     

Mechanistic distinction between activation and inhibition of (Na+K)-ATPase-mediated Ca influx in cardiomyocytes

(Na⁺+K⁺)-ATPase (NKA) mediates positive inotropy in the heart. Extensive studies have demonstrated that the reverse-mode Na⁺/Ca²⁺-exchanger (NCX) plays a critical role in increasing intracellular Ca²⁺ concentration through the inhibition of NKA-induced positive inotropy by cardiac glycosides. Little is known about the nature of the NCX functional mode in the activation of NKA-induced positive inotropy. Here, we examined the effect of an NKA activator SSA412 antibody on ⁴⁵Ca influx in isolated rat myocytes and found that KB-R7943, a NCX reverse-mode inhibitor, fails to inhibit the activation of NKA-induced ⁴⁵Ca influx, suggesting that the Ca²⁺ influx via the reverse-mode NCX does not mediate this process. Nifedipine, an L-type Ca²⁺ channel (LTCC) inhibitor, completely blocks the activation of NKA-induced ⁴⁵Ca influx, suggesting that the LTCC is responsible for the moderate increase in intracellular Ca²⁺. In contrast, the inhibition of NKA by ouabain induces 4.7-fold ⁴⁵Ca influx compared with the condition of activation of NKA. Moreover, approximately 70% of ouabain-induced ⁴⁵Ca influx was obstructed by KB-R7943 and only 30% was impeded by nifedipine, indicating that both the LTCC and the NCX contribute to the rise in intracellular Ca²⁺ and that the NCX reverse-mode is the major source for the ⁴⁵Ca influx induced by the inhibition of NKA. This study provides direct evidence to demonstrate that the activation of NKA-induced Ca²⁺ increase is independent of the reverse-mode NCX and pinpoints a mechanistic distinction between the activation and inhibition of the NKA-mediated Ca²⁺ influx path ways in cardiomyocytes.     

Effects of K-channel blockers, calcium, and verapamil suggest different pacemaker mechanisms in cultured neonatal rat and embryonic chick ventricle cells

We compared the determinants of spontaneous activity in explanted neonatal (2-day-old) rat ventricle cells and in reaggregates derived from 15-day-old chick embryos. We studied the beating rate with an optical recording method and the underlying electrical activity with glass microelectrodes using the K current blockers cesium (Cs) and tetraethylammonium, varied Ca concentrations, and the Ca antagonist verapamil. In the rat (i) Cs increased the beating rate that was mediated by an increase in the slope of the diastolic potential. (ii) Ca increased the beating rate dramatically at low and medium concentrations to decrease it again at 8 mM Cao. This increase in the beating rate was mediated by an increase of the slope of the diastolic depolarization. (iii) The beating rate decreased with verapamil at concentrations between 0.5 and 2.0 microM. The effects of Cs and Ca suggest that an increase in net inward current (block of IK1) underlies the positive chronotropic effect of Cs and that the pacemaker mechanism is determined by a Ca inward current or an IT1 type current modulated by variations of Cai. In the chick reaggregates (i) Cs and tetraethylammonium decreased the beating rate that was mainly brought about by a decrease in the slope of diastolic depolarization. (ii) Ca increased the beating rate but to a lesser degree than in the rat and there was no decrease of the beating rate at higher concentrations. (iii) The increase in the beating rate was not mediated by an increase in the slope of the diastolic potential but mainly by a depolarization of the maximum diastolic potential. (iv) Verapamil inhibited electrogenesis before any change in the diastolic potential was evident. The negative chronotropic effect of Cs and tetraethylammonium is compatible with the notion that a voltage- and time-dependent K current was inhibited and that this current determines the pacemaker. Moreover, the Ca component of the pacemaker mechanism in explanted rat ventricle cells resembles either that of the sinoatrial node or represents triggered activity.     

Defective Ca(2+) handling proteins regulation during heart failure

Abnormal release of Ca(2+) from sarcoplasmic reticulum (SR) via the cardiac ryanodine receptor (RyR2) may contribute to contractile dysfunction in heart failure (HF). We previously demonstrated that RyR2 macromolecular complexes from HF rat were significantly more depleted of FK506 binding protein (FKBP12.6). Here we assessed expression of key Ca(2+) handling proteins and measured SR Ca(2+) content in control and HF rat myocytes. Direct measurements of SR Ca(2+) content in permeabilized cardiac myocytes demonstrated that SR luminal [Ca(2+)] is markedly lowered in HF (HF: DeltaF/F(0) = 26.4+/-1.8, n=12; control: DeltaF/F(0) = 49.2+/-2.9, n=10; P<0.01). Furthermore, we demonstrated that the expression of RyR2 associated proteins (including calmodulin, sorcin, calsequestrin, protein phosphatase 1, protein phosphatase 2A), Ca(2+) ATPase (SERCA2a), PLB phosphorylation at Ser16 (PLB-S16), PLB phosphorylation at Thr17 (PLB-T17), L-type Ca(2+) channel (Cav1.2) and Na(+)- Ca(2+) exchanger (NCX) were significantly reduced in rat HF. Our results suggest that systolic SR reduced Ca(2+) release and diastolic SR Ca(2+) leak (due to defective protein-protein interaction between RyR2 and its associated proteins) along with reduced SR Ca(2+) uptake (due to down-regulation of SERCA2a, PLB-S16 and PLB-T17), abnormal Ca(2+) extrusion (due to down-regulation of NCX) and defective Ca(2+) -induced Ca(2+) release (due to down-regulation of Cav1.2) could contribute to HF.     

ATP promotes NCX-reversal in aortic smooth muscle cells by DAG-activated Na+ entry

Reversal of the plasma membrane Na(+)/Ca(2+) exchanger (NCX) has been shown to mediate Ca(2+) influx in response to activation of G-protein linked receptors. Functional coupling of reverse-mode NCX with canonical transient receptor potential channels (TRPC), specifically TRPC6, has recently been demonstrated by our laboratory to mediate Ca(2+) influx in rat aortic smooth muscle cells (RASMCs) following ATP stimulation. In this communication, we provide further detail of this functional coupling by indirectly measuring NCX reversal. We found that NCX reversal, induced by the removal of extracellular Na(+), was increased following stimulation with ATP and the diacylglycerol analog 1-Oleoyl-2-acetyl-sn-glycerol. This increased NCX reversal was attenuated by SKF-96365, an inhibitor of non-selective cation channels, and by activation of protein kinase C with phorbol ester 12-tetradecanoylphorbol-13 acetate. These data are consistent with the known properties of TRPC6 and further support that functional coupling of TRPC6 and NCX occurs via a receptor-operated, rather than store-operated, cascade in RASMCs.     

Altered beta-adrenergic signal transduction in nonfailing hypertrophied myocytes from Dahl salt-sensitive rats

Desensitization of the beta-adrenergic receptor (beta-AR) response is well documented in hypertrophied hearts. We investigated whether beta-AR desensitization is also present at the cellular level in hypertrophied myocardium, as well as the physiological role of inhibitory G (G(i)) proteins and the L-type Ca(2+) channel in mediating beta-AR desensitization. Left ventricular (LV) myocytes were isolated from hypertrophied hearts of hypertensive Dahl salt-sensitive (DS) rats and nonhypertrophied hearts of normotensive salt-resistant (DR) rats. Cells were paced at a rate of 300 beats/min at 37 degrees C, and myocyte contractility and intracellular Ca(2+) concentration ([Ca(2+)](i)) were simultaneously measured. In response to increasing concentrations of isoproterenol, DR myocytes displayed a dose-dependent augmentation of cell shortening and the [Ca(2+)](i) transient amplitude, whereas hypertrophied DS myocytes had a blunted response of both cell shortening and the [Ca(2+)](i) transient amplitude. Interestingly, inhibition of G(i) proteins did not restore beta-AR desensitization in DS myocytes. The responses to increases in extracellular Ca(2+) and an L-type Ca(2+) channel agonist were also similar in both DS and DR myocytes. Isoproterenol-stimulated adenylyl cyclase activity, however, was blunted in hypertrophied myocytes. We concluded that compensated ventricular hypertrophy results in a blunted contractile response to beta-AR stimulation, which is present at the cellular level and independent of alterations in inhibitory G proteins and the L-type Ca(2+) channel.     

Cellular and molecular determinants of altered Ca2+ handling in the failing rabbit heart: primary defects in SR Ca2+ uptake and release mechanisms

Myocytes from the failing myocardium exhibit depressed and prolonged intracellular Ca(2+) concentration ([Ca(2+)](i)) transients that are, in part, responsible for contractile dysfunction and unstable repolarization. To better understand the molecular basis of the aberrant Ca(2+) handling in heart failure (HF), we studied the rabbit pacing tachycardia HF model. Induction of HF was associated with action potential (AP) duration prolongation that was especially pronounced at low stimulation frequencies. L-type calcium channel current (I(Ca,L)) density (-0.964 +/- 0.172 vs. -0.745 +/- 0.128 pA/pF at +10 mV) and Na(+)/Ca(2+) exchanger (NCX) currents (2.1 +/- 0.8 vs. 2.3 +/- 0.8 pA/pF at +30 mV) were not different in myocytes from control and failing hearts. The amplitude of peak [Ca(2+)](i) was depressed (at +10 mV, 0.72 +/- 0.07 and 0.56 +/- 0.04 microM in normal and failing hearts, respectively; P < 0.05), with slowed rates of decay and reduced Ca(2+) spark amplitudes (P < 0.0001) in myocytes isolated from failing vs. control hearts. Inhibition of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a revealed a greater reliance on NCX to remove cytosolic Ca(2+) in myocytes isolated from failing vs. control hearts (P < 0.05). mRNA levels of the alpha(1C)-subunit, ryanodine receptor (RyR), and NCX were unchanged from controls, while SERCA2a and phospholamban (PLB) were significantly downregulated in failing vs. control hearts (P < 0.05). alpha(1C) protein levels were unchanged, RyR, SERCA2a, and PLB were significantly downregulated (P < 0.05), while NCX protein was significantly upregulated (P < 0.05). These results support a prominent role for the sarcoplasmic reticulum (SR) in the pathogenesis of HF, in which abnormal SR Ca(2+) uptake and release synergistically contribute to the depressed [Ca(2+)](i) and the altered AP profile phenotype.     

Rescue of contractile abnormalities by Na+/Ca2+ exchanger overexpression in postinfarction rat myocytes.

Previous studies on myocytes isolated from rat hearts 3 wk after myocardial infarction (MI) demonstrated increased cell length, reduced Na(+)/Ca(2+) exchange (NCX1) activity, altered contractility, and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients. In the present study, we investigated whether NCX1 overexpression in MI myocytes would restore contraction and [Ca(2+)](i) transients to normal. When myocytes were placed in culture under continued electrical-field stimulation conditions, differences in contraction amplitudes and cell lengths between sham and MI myocytes were preserved for at least 48 h. Infection of both sham and MI myocytes by adenovirus expressing green fluorescent protein resulted in >95% infection, as evidenced by green fluorescent protein fluorescence, but contraction amplitudes at 6-, 24-, and 48-h postinfection were not affected. NCX1 overexpression in MI myocytes resulted in lower diastolic [Ca(2+)](i) levels at all extracellular Ca(2+) concentrations ([Ca(2+)](o)) examined, suggesting enhanced forward NCX1 activity. At 5 mM [Ca(2+)](o), subnormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were restored toward normal levels by overexpressing NCX1. At 0.6 mM [Ca(2+)](o), supranormal contraction and [Ca(2+)](i) transient amplitudes in MI myocytes (compared with sham myocytes) were lowered by NCX1 overexpression. We conclude that overexpression of NCX1 in MI myocytes was effective in improving contractile dysfunction, most likely because of enhancement of both Ca(2+) efflux and influx during a cardiac cycle. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in postinfarction myocytes.     

Functional consequence of protein kinase A-dependent phosphorylation of the cardiac ryanodine receptor: sensitization of store overload-induced Ca2+ release

The phosphorylation of the cardiac Ca(2+)-release channel (ryanodine receptor, RyR2) by protein kinase A (PKA) has been extensively characterized, but its functional consequence remains poorly defined and controversial. We have previously shown that RyR2 is phosphorylated by PKA at two major sites, serine 2,030 and serine 2,808, of which Ser-2,030 is the major PKA site responding to beta-adrenergic stimulation. Here we investigated the effect of the phosphorylation of RyR2 by PKA on the properties of single channels and on spontaneous Ca(2+) release during sarcoplasmic reticulum Ca(2+) overload, a process we have referred to as store overload-induced Ca(2+) release (SOICR). We found that PKA activated single RyR2 channels in the presence, but not in the absence, of luminal Ca(2+). On the other hand, PKA had no marked effect on the sensitivity of the RyR2 channel to activation by cytosolic Ca(2+). Importantly, the S2030A mutation, but not mutations of Ser-2,808, diminished the effect of PKA on RyR2. Furthermore, a phosphomimetic mutation, S2030D, potentiated the response of RyR2 to luminal Ca(2+) and enhanced the propensity for SOICR in HEK293 cells. In intact rat ventricular myocytes, the activation of PKA by isoproterenol reduced the amplitude and increased the frequency of SOICR. Confocal line-scanning fluorescence microscopy further revealed that the activation of PKA by isoproterenol increased the rate of Ca(2+) release and the propagation velocity of spontaneous Ca(2+) waves, despite reduced wave amplitude and resting cytosolic Ca(2+). Collectively, our data indicate that PKA-dependent phosphorylation enhances the response of RyR2 to luminal Ca(2+) and reduces the threshold for SOICR and that this effect of PKA is largely mediated by phosphorylation at Ser-2,030.     

Two populations of pancreatic islet alpha-cells displaying distinct Ca2+ channel properties

In low or absence of glucose, alpha-cells generate rhythmic action potentials and secrete glucagon. alpha-Cell T-type Ca(2+) channels are believed to be pacemaker channels, which are expected to open near the resting membrane potential (around -60 mV) to initiate a small depolarization. A previous publication, however, showed that alpha-cell T-type Ca(2+) channels have an activation threshold of -40 mV, which does not appear to fulfill their role as pacemakers. In this work, we investigated the Ca(2+) channel characteristics in alpha-cells of mouse-insulin-promoter green-fluorescent-protein (MIP-GFP) mouse. The beta-cells of MIP-GFP were conveniently distinguished as green cells, while immunostaining indicated that the majority of non-green cells were alpha-cells. We found that majority of alpha-cells possessed T-type Ca(2+) channels having an activation threshold of -40 mV; these cells also had high-voltage-activated (HVA) Ca(2+) channels (activation threshold of -20 mV). A novel finding here is that a minority of alpha-cells had T-type Ca(2+) channels with an activation threshold of -60 mV. This minor population of alpha-cells was, surprisingly, devoid of HVA Ca(2+) channels. We suggest that this alpha-cell subpopulation may act as pacemaker cells in low or absence of glucose.     

Sex differences in expression of calcium-handling proteins and beta-adrenergic receptors in rat heart ventricle

Human studies reveal sex differences in myocardial function as well as in the incidence and manifestation of heart disease. Myocellular Ca(2+) cycling regulates normal contractile function; whereas cardiac dysfunction in heart failure has been associated with alterations in Ca(2+)-handling proteins. Beta-adrenergic receptor (beta-AR) signaling regulates activity of several Ca(2+)-handling proteins and alterations in beta-AR signaling are associated with heart disease. This study examines sex differences in expression of beta(1)-AR, beta(2)-AR, and Ca(2+)-handling proteins including: L-type calcium channel (Ca(v)1.2) , ryanodine calcium-release channels (RyR), sarcoplasmic reticular Ca(2+) ATPase (SERCA2), phospholamban (PLB) and Na(+)-Ca(2+) exchange protein (NCX) in healthy hearts from male and female Sprague-Dawley rats. Protein levels were examined using Western blot analysis. Abundance of mRNA was determined by real time RT-PCR normalized to abundance of GAPDH mRNA. Contraction parameters were measured in right ventricular papillary muscle in the presence and absence of isoproterenol. Results demonstrate that female ventricle has significantly higher levels of Ca(v)1.2, RyR, and NCX protein compared to males. Messenger RNA abundance for RyR, and NCX protein was significantly higher in females whereas Ca(v)1.2 mRNA was higher in males. No differences were detected in beta-ARs, SERCA2 or PLB. Female right papillary muscle had a faster maximal rate of force development and decline (+/- dF/dt). There were no sex differences in response to isoproterenol. Results show significant sex differences in expression of key ventricular Ca(2+)-handling proteins that are associated with small functional differences in +/- dF/dt. Further studies will determine whether differences in the abundance of these key proteins play a role in sex disparities in the incidence and manifestation of heart disease.     

Vectorial Ca2+ release via ryanodine receptors contributes to Ca2+ extrusion from freshly isolated rabbit aortic endothelial cells

In this study, we identified ryanodine receptors (RyRs) as a component of a cytosolic Ca(2+) removal pathway in freshly isolated rabbit aortic endothelial cells. In an earlier article, we reported that the sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) and Na(+)/Ca(2+) exchanger (NCX) function in series to extrude cytosolic Ca(2+) to the extracellular space. Here we employed caffeine and ryanodine as modulators of RyR and showed that they act as the linkage between SERCA and NCX in removing Ca(2+) from the cytoplasm. Our data indicate that both 15 mM caffeine and 1 microM ryanodine facilitated Ca(2+) extrusion by activating RyRs while 100 microM ryanodine had the opposite effect by blocking RyRs. A further attempt to investigate RyR pharmacology revealed that in the absence of extracellular Ca(2+), ryanodine at 1 microM, but not 100 microM, stimulated Ca(2+) loss from the endoplasmic reticulum (ER). Blockade of RyR had no effect on the Ca(2+) removal rate when NCX had been previously blocked. In addition, the localization of RyR was determined using confocal microscopy of BODIPY TR-X fluorescent staining. Taken together, our findings suggest that in freshly isolated endothelial cells Ca(2+) is removed in part by transport through SERCA, RyR, and eventually NCX, and that RyR and NCX are in close functional proximity near the plasma membrane. After blockade of this component, Ca(2+) extrusion could be further inhibited by carboxyeosin, indicating a parallel contribution by the plasmalemmal Ca(2+)-ATPase (PMCA).     

MicroRNA-1 and -133 increase arrhythmogenesis in heart failure by dissociating phosphatase activity from RyR2 complex

In heart failure (HF), arrhythmogenic spontaneous sarcoplasmic reticulum (SR) Ca(2+) release and afterdepolarizations in cardiac myocytes have been linked to abnormally high activity of ryanodine receptors (RyR2s) associated with enhanced phosphorylation of the channel. However, the specific molecular mechanisms underlying RyR2 hyperphosphorylation in HF remain poorly understood. The objective of the current study was to test the hypothesis that the enhanced expression of muscle-specific microRNAs (miRNAs) underlies the HF-related alterations in RyR2 phosphorylation in ventricular myocytes by targeting phosphatase activity localized to the RyR2. We studied hearts isolated from canines with chronic HF exhibiting increased left ventricular (LV) dimensions and decreased LV contractility. qRT-PCR revealed that the levels of miR-1 and miR-133, the most abundant muscle-specific miRNAs, were significantly increased in HF myocytes compared with controls (2- and 1.6-fold, respectively). Western blot analyses demonstrated that expression levels of the protein phosphatase 2A (PP2A) catalytic and regulatory subunits, which are putative targets of miR-133 and miR-1, were decreased in HF cells. PP2A catalytic subunit mRNAs were validated as targets of miR-133 by using luciferase reporter assays. Pharmacological inhibition of phosphatase activity increased the frequency of diastolic Ca(2+) waves and afterdepolarizations in control myocytes. The decreased PP2A activity observed in HF was accompanied by enhanced Ca(2+)/calmodulin-dependent protein kinase (CaMKII)-mediated phosphorylation of RyR2 at sites Ser-2814 and Ser-2030 and increased frequency of diastolic Ca(2+) waves and afterdepolarizations in HF myocytes compared with controls. In HF myocytes, CaMKII inhibitory peptide normalized the frequency of pro-arrhythmic spontaneous diastolic Ca(2+) waves. These findings suggest that altered levels of major muscle-specific miRNAs contribute to abnormal RyR2 function in HF by depressing phosphatase activity localized to the channel, which in turn, leads to the excessive phosphorylation of RyR2s, abnormal Ca(2+) cycling, and increased propensity to arrhythmogenesis.     

Sprint training improves contractility in postinfarction rat myocytes: role of Na+/Ca2+ exchange.

Previous studies in adult myocytes isolated from rat hearts 3-9 wk after myocardial infarction (MI) demonstrated abnormal contractility and decreased Na(+)/Ca(2+) exchanger (NCX1) activity. In addition, a program of high-intensity sprint training (HIST) instituted shortly after MI restored both contractility and NCX1 activity toward normal. The present study examined the hypotheses that reduced NCX1 activity caused abnormal contractility in myocytes isolated from sedentary (Sed) rat hearts 9-11 wk after coronary artery ligation and that HIST ameliorated contractile dysfunction in post-MI myocytes by increasing NCX1 activity. The approach was to upregulate NCX1 in MI-sedentary (MISed) myocytes and downregulate NCX1 in MI-exercised (MIHIST) myocytes by adenovirus-mediated gene transfer. Overexpression of NCX1 in MISed myocytes did not affect sarco(endo)plasmic reticulum Ca(2+)-ATPase and calsequestrin levels but rescued contractile abnormalities observed in MISed myocytes. That is, at 5 mM extracellular Ca(2+) concentration, the subnormal contraction amplitude in MISed myocytes (compared with Sham myocytes) was increased toward normal by NCX1 overexpression, whereas at 0.6 mM extracellular Ca(2+) concentration the supernormal contraction amplitude in MISed myocytes was lowered. Conversely, NCX1 downregulation by antisense in MIHIST myocytes abolished the beneficial effects of HIST on contraction amplitudes in MI myocytes. We suggest that decreased NCX1 activity may play an important role in contractile abnormalities in post-MI myocytes and that HIST ameliorated contractile dysfunction in post-MI myocytes partly by enhancing NCX1 activity.