Spatiotemporal characteristics of SR Ca(2+) uptake and release in detubulated rat ventricular myocytes

In cardiac ventricular myocytes, sarcoplasmic reticulum (SR) Ca(2+) load is a key determinant of SR Ca(2+) release. This release normally occurs predominantly from SR junctions at sarcolemmal invaginations (t-tubules), ensuring synchronous SR Ca(2+) release throughout the cell. However under conditions of Ca(2+) overload, spontaneous SR Ca(2+) release and propagating Ca(2+) waves can occur, which are pro-arrhythmic. We used detubulated rat ventricular myocytes to determine the dependence of Ca(2+) wave propagation on SR Ca(2+) load, and the role of t-tubules in SR Ca(2+) uptake and spontaneous release. After SR Ca(2+) depletion, recovery of Ca(2+) transient amplitude (and SR Ca(2+) load) was slower in detubulated than control myocytes (half-maximal recovery: 9.9+/-1.4 vs. 5.5+/-0.7 beats). In detubulated myocytes the extent and velocity of Ca(2+) propagation from the cell periphery increased with each beat and depended steeply on SR Ca(2+) load. Isoproterenol (ISO) accelerated recovery, increased maximal propagation velocity and reduced the threshold SR Ca(2+) load for propagation. Ca(2+) spark frequency was uniform across control cell width and was similar at the periphery of detubulated cells. However, internal Ca(2+) spark frequency in detubulated cells was 75% lower (despite comparable local SR Ca(2+) load); this transverse spark frequency profile was similar to that in atrial myocytes. We conclude that: (1) t-tubule Ca(2+) fluxes normally control SR Ca(2+) refilling; (2) Ca(2+) wave propagation depends steeply on SR Ca(2+) content (3) SR-t-tubule junctions are important in initiating SR Ca(2+) release and (4) ISO enhances propagation of SR Ca release, but not the initiation of SR Ca release events (for given SR Ca(2+) loads).     

Termination of cardiac Ca2+ sparks: role of intra-SR [Ca2+], release flux, and intra-SR Ca2+ diffusion

Ca(2+) release from cardiac sarcoplasmic reticulum (SR) via ryanodine receptors (RyRs) is regulated by dyadic cleft [Ca(2+)] and intra-SR free [Ca(2+)] ([Ca(2+)](SR)). Robust SR Ca(2+) release termination is important for stable excitation-contraction coupling, and partial [Ca(2+)](SR) depletion may contribute to release termination. Here, we investigated the regulation of SR Ca(2+) release termination of spontaneous local SR Ca(2+) release events (Ca(2+) sparks) by [Ca(2+)](SR), release flux, and intra-SR Ca(2+) diffusion. We simultaneously measured Ca(2+) sparks and Ca(2+) blinks (localized elementary [Ca(2+)](SR) depletions) in permeabilized ventricular cardiomyocytes over a wide range of SR Ca(2+) loads and release fluxes. Sparks terminated via a [Ca(2+)](SR)-dependent mechanism at a fixed [Ca(2+)](SR) depletion threshold independent of the initial [Ca(2+)](SR) and release flux. Ca(2+) blink recovery depended mainly on intra-SR Ca(2+) diffusion rather than SR Ca(2+) uptake. Therefore, the large variation in Ca(2+) blink recovery rates at different release sites occurred because of differences in the degree of release site interconnection within the SR network. When SR release flux was greatly reduced, long-lasting release events occurred from well-connected junctions. These junctions could sustain release because local SR Ca(2+) release and [Ca(2+)](SR) refilling reached a balance, preventing [Ca(2+)](SR) from depleting to the termination threshold. Prolonged release events eventually terminated at a steady [Ca(2+)](SR), indicative of a slower, [Ca(2+)](SR)-independent termination mechanism. These results demonstrate that there is high variability in local SR connectivity but that SR Ca(2+) release terminates at a fixed [Ca(2+)](SR) termination threshold. Thus, reliable SR Ca(2+) release termination depends on tight RyR regulation by [Ca(2+)](SR).     

Design and application of a class of sensors to monitor Ca2+ dynamics in high Ca2+ concentration cellular compartments

Quantitative analysis of Ca(2+) fluctuations in the endoplasmic/sarcoplasmic reticulum (ER/SR) is essential to defining the mechanisms of Ca(2+)-dependent signaling under physiological and pathological conditions. Here, we developed a unique class of genetically encoded indicators by designing a Ca(2+) binding site in the EGFP. One of them, calcium sensor for detecting high concentration in the ER, exhibits unprecedented Ca(2+) release kinetics with an off-rate estimated at around 700 s(-1) and appropriate Ca(2+) binding affinity, likely attributable to local Ca(2+)-induced conformational changes around the designed Ca(2+) binding site and reduced chemical exchange between two chromophore states. Calcium sensor for detecting high concentration in the ER reported considerable differences in ER Ca(2+) dynamics and concentration among human epithelial carcinoma cells (HeLa), human embryonic kidney 293 cells (HEK-293), and mouse myoblast cells (C2C12), enabling us to monitor SR luminal Ca(2+) in flexor digitorum brevis muscle fibers to determine the mechanism of diminished SR Ca(2+) release in aging mice. This sensor will be invaluable in examining pathogenesis characterized by alterations in Ca(2+) homeostasis.     

Local Ca(2+) signaling and EC coupling in heart: Ca(2+) sparks and the regulation of the [Ca(2+)](i) transient

The elementary event of Ca(2+) release in heart is the Ca(2+) spark. It occurs at a low rate during diastole, activated only by the low cytosolic [Ca(2+)](i). Synchronized activation of many sparks is due to the high local [Ca(2+)](i) in the region surrounding the sarcoplasmic reticulum (SR) Ca(2+) release channels and is responsible for the systolic [Ca(2+)](i) transient. The biophysical basis of this calcium signaling is discussed. Attention is placed on the local organization of the ryanodine receptors (SR Ca(2+) release channels, RyRs) and the other proteins that underlie and modulate excitation-contraction (EC) coupling. A brief review of specific elements that regulate SR Ca(2+) release (including SR lumenal Ca(2+) and coupled gating of RyRs) is presented. Finally integrative calcium signaling in heart is presented in the context of normal heart function and heart failure.     

Role of Ca2+, membrane excitability, and Ca2+ stores in failing muscle contraction with aging

Excitation-contraction (EC) coupling in a population of skeletal muscle fibers of aged mice becomes dependent on the presence of external Ca(2+) ions (Payne, A.M., Zheng, Z., Gonzalez, E., Wang, Z.M., Messi, M.L., Delbono, O., 2004b. External Ca(2+)-dependent excitation - contraction coupling in a population of aging mouse skeletal muscle fibers. J. Physiol. 560, 137-155.). However, the mechanism(s) underlying this process remain unknown. In this work, we examined the role of (1) extracellular Ca(2+); (2) voltage-induced influx of external Ca(2+) ions; (3) sarcoplasmic reticulum (SR) Ca(2+) depletion during repeated contractions; (4) store-operated Ca(2+) entry (SOCE); (5) SR ultrastructure; (6) SR subdomain localization of the ryanodine receptor; and (7) sarcolemmal excitability in muscle force decline with aging. These experiments show that external Ca(2+), but not Ca(2+) influx, is needed to maintain force upon repetitive fiber electrical stimulation. Decline in fiber force is associated with depressed SR Ca(2+) release. SR Ca(2+) depletion, SOCE, and the putative segregated Ca(2+) release store do not play a significant role in external Ca(2+)-dependent contraction. More importantly, a significant number of action potentials fail in senescent mouse muscle fibers subjected to a stimulation frequency. These results indicate that failure to generate action potentials accounts for decreased intracellular Ca(2+) mobilization and tetanic force in aging muscle exposed to a Ca(2+)-free medium.     

Junctate is a Ca2+-sensing structural component of Orai1 and stromal interaction molecule 1 (STIM1)

Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca(2+) sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca(2+)-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca(2+) binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca(2+)-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.     

Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum

The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca(2+) storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca(2+) that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca(2+) store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca(2+) uptake. Direct measurements with ER-targeted Ca(2+)-sensitive fluorescent "cameleon" proteins revealed that Bcl-2 decreased the free Ca(2+) concentration within the lumen of the ER, [Ca(2+)](ER). Analysis of the kinetics of Ca(2+) store depletion in response to the Ca(2+)-ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca(2+) concentration within the ER lumen by increasing the Ca(2+) permeability of the ER membrane. The increased ER Ca(2+) permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane.     

ATP-dependent effects of halothane on SR Ca2+ regulation in permeabilized atrial myocytes

OBJECTIVE: Previous work suggests that modification of sarcoplasmic reticulum (SR) function may contribute to the cardioprotective effect of halothane during ischaemia and reperfusion. The aim of this study was to investigate the effects of halothane on spontaneous Ca(2+) release from the sarcoplasmic reticulum (Ca(2+) sparks and waves). METHODS: Rat atrial myocytes were permeabilized with saponin and perfused with solutions approximating to the intracellular milieu and containing fluo-3. SR Ca(2+) release was detected using confocal microscopy. RESULTS: In the presence of 5 mM ATP, halothane (0.25-2 mM) had no significant effect on the amplitude or frequency of spontaneous Ca(2+) waves. However, in the presence of 0.05 mM ATP, halothane (0.25-2 mM) induced a concentration-dependent decrease in the amplitude and an increase in the frequency of spontaneous Ca(2+) waves, e.g., 1 mM halothane decreased the amplitude by 34.7+/-3.5% (n=9) and increased the frequency by 67+/-19.9% (n=7). In the presence of 5 mM ATP, 1 mM halothane had no significant effect on the amplitude or frequency of Ca(2+) sparks. When [ATP] was reduced to 0.05 mM, Ca(2+) spark frequency decreased by 67.9+/-14% and the amplitude increased by 27.5+/-4.9% (n=13). Subsequent introduction of halothane (0.5-1 mM) induced a transient burst of Ca(2+) sparks, consistent with ryanodine receptor (RyR) activation. Further experiments showed that the decrease in Ca(2+) spark frequency following ATP depletion was associated with a progressive increase in the SR Ca(2+) content over 1-2 min. This rise in SR Ca(2+) content did not occur when 1 mM halothane was present during ATP depletion. CONCLUSIONS: These data suggest that the sensitivity of the RyR to activation by halothane increases at low [ATP]. In metabolically impaired cells, halothane would be expected to lessen any rise in SR Ca(2+) content and to reduce the amplitude of spontaneous Ca(2+) release. These effects of halothane are considered in relation to the events that occur during ischaemia and reperfusion.     

Store-operated Ca2+ entry and TRPC expression; possible roles in cardiac pacemaker tissue

Store-operated Ca(2+) channels (SOCCs) were first identified in non-excitable cells by the observation that depletion of Ca(2+) stores caused increased influx of extracellular Ca(2+). Recent studies have suggested that SOCCs might be related to the transient receptor potential (TRPC) gene family. The mechanism of cardiac pacemaking involves voltage-dependent pacemaker current; in addition there is growing evidence that intracellular sarcoplasmic reticulum (SR) Ca(2+) release plays an important role. In the present short review we assess preliminary evidence for Ca(2+) entry related to SR store depletion and expression of TRPCs in pacemaker tissue. These newer findings suggest that Ca(2+) entry and inward current triggered by store depletion might also contribute to the pacemaker current. Many hormones, drugs and interventions such as ischaemia and stretch, which alter Ca(2+) handling, will also modulate pacemaker firing thought their effect on SOCCs.     

Ignition of calcium sparks in arterial and cardiac muscle through caveolae

Ca(2+) sparks are localized intracellular Ca(2+) events released through ryanodine receptors (RyRs) that control excitation-contraction coupling in heart and smooth muscle. Ca(2+) spark triggering depends on precise delivery of Ca(2+) ions through dihydropyridine (DHP)-sensitive Ca(2+) channels to RyRs of the sarcoplasmic reticulum (SR), a process requiring a very precise alignment of surface and SR membranes containing Ca(2+) influx channels and RyRs. Because caveolae contain DHP-sensitive Ca(2+) channels and may colocalize with SR, we tested the hypothesis that caveolae are the structural element necessary for the generation of Ca(2+) sparks. Using methyl-ss-cyclodextrin (dextrin) to deplete caveolae, we found that dextrin dose-dependently decreased the frequency, amplitude, and spatial size of Ca(2+) sparks in arterial smooth muscle cells and neonatal cardiomyocytes. However, temporal characteristics of Ca(2+) sparks were not significantly affected. We ruled out the possibility that the decreases in Ca(2+) spark frequency and size are caused by changes in DHP-sensitive L-type channels, SR Ca(2+) load, or changes in membrane potential. Our results suggest a novel signaling model that explains the formation of Ca(2+) sparks in a caveolae microdomain. The transient elevation in [Ca(2+)](i) at the inner mouth of a single caveolemmal Ca(2+) channel induces simultaneous activation and thus opens several RyRs to generate a local Ca(2+) release event, a Ca(2+) spark. Alterations in the molecular assembly and ultrastructure of caveolae may lead to pathophysiological changes in Ca(2+) signaling. Thus, caveolae may be intimately involved in cardiovascular cell dysfunction and disease.     

Live-cell imaging reveals sequential oligomerization and local plasma membrane targeting of stromal interaction molecule 1 after Ca2+ store depletion

Stromal interaction molecule 1 (STIM1) has recently been identified by our group and others as an endoplasmic reticulum (ER) Ca(2+) sensor that responds to ER Ca(2+) store depletion and activates Ca(2+) channels in the plasma membrane (PM). The molecular mechanism by which STIM1 transduces signals from the ER lumen to the PM is not yet understood. Here we developed a live-cell FRET approach and show that STIM1 forms oligomers within 5 s after Ca(2+) store depletion. These oligomers rapidly dissociated when ER Ca(2+) stores were refilled. We further show that STIM1 formed oligomers before its translocation within the ER network to ER-PM junctions. A mutant STIM1 lacking the C-terminal polybasic PM-targeting motif oligomerized after Ca(2+) store depletion but failed to form puncta at ER-PM junctions. Using fluorescence recovery after photobleaching measurements to monitor STIM1 mobility, we show that STIM1 oligomers translocate on average only 2 mum to reach ER-PM junctions, arguing that STIM1 ER-to-PM signaling is a local process that is suitable for generating cytosolic Ca(2+) gradients. Together, our live-cell measurements dissect the STIM1 ER-to-PM signaling relay into four sequential steps: (i) dissociation of Ca(2+), (ii) rapid oligomerization, (iii) spatially restricted translocation to nearby ER-PM junctions, and (iv) activation of PM Ca(2+) channels.     

Effect of atorvastatin on intracellular calcium uptake in coronary smooth muscle cells from diabetic pigs fed an atherogenic diet.

Intracellular Ca(2+) store loading has been shown to alter proliferation and apoptosis of several cell types. In addition, HMG-CoA reductase inhibitors (i.e. atorvastatin) are effective in treating diabetic dyslipidemic patients. Thus, we hypothesized that chronic atorvastatin treatment would prevent increased Ca(2+) uptake into intracellular Ca(2+) stores in vascular smooth muscle cells from diabetic dyslipidemic pigs. Male Yucatan pigs were divided into four groups for 20 weeks-- (1) low fat fed (control); (2) hyperlipidemic (F); (3) alloxan-induced diabetic dyslipidemic (DF); and (4) diabetic dyslipidemic pigs treated with atorvastatin (DFA). The F, DF, and DFA groups were fed a high fat/cholesterol diet. Cells were isolated from the coronary artery and the myoplasmic Ca(2+) (Ca(m)) response measured using single cell fura-2 imaging. The Ca(m) response to caffeine (5 mM to release Ca(2+) from the sarcoplasmic reticulum, SR) and ionomycin (10 microM; to release the total Ca(2+) store) was determined in either the presence of low Na (19Na; inhibits Na(+)-Ca(2+) exchange), thapsigargin (TSG; inhibits the SR Ca(2+) pump), and a 19Na+TSG solution. Low Na induced the uptake of Ca(2+) into both SR and non-SR Ca(2+) stores in the DF group, but not the DFA group. Furthermore, after depletion of the SR Ca(2+) store with TSG, 19Na evoked Ca(2+) uptake into non-SR Ca(2+) stores in all three groups except in the DFA group. In summary, this study demonstrates that atorvastatin prevents the enhanced uptake of Ca(2+) by SR and non-SR Ca(2+) stores in diabetic dyslipidemic pigs.     

IP3 constricts cerebral arteries via IP3 receptor-mediated TRPC3 channel activation and independently of sarcoplasmic reticulum Ca2+ release

Vasoconstrictors that bind to phospholipase C-coupled receptors elevate inositol-1,4,5-trisphosphate (IP(3)). IP(3) is generally considered to elevate intracellular Ca(2+) concentration ([Ca(2+)](i)) in arterial myocytes and induce vasoconstriction via a single mechanism: by activating sarcoplasmic reticulum (SR)-localized IP(3) receptors, leading to intracellular Ca(2+) release. We show that IP(3) also stimulates vasoconstriction via a SR Ca(2+) release-independent mechanism. In isolated cerebral artery myocytes and arteries in which SR Ca(2+) was depleted to abolish Ca(2+) release (measured using D1ER, a fluorescence resonance energy transfer-based SR Ca(2+) indicator), IP(3) activated 15 pS sarcolemmal cation channels, generated a whole-cell cation current (I(Cat)) caused by Na(+) influx, induced membrane depolarization, elevated [Ca(2+)](i), and stimulated vasoconstriction. The IP(3)-induced I(Cat) and [Ca(2+)](i) elevation were attenuated by cation channel (Gd(3+), 2-APB) and IP(3) receptor (xestospongin C, heparin, 2-APB) blockers. TRPC3 (canonical transient receptor potential 3) channel knockdown with short hairpin RNA and diltiazem and nimodipine, voltage-dependent Ca(2+) channel blockers, reduced the SR Ca(2+) release-independent, IP(3)-induced [Ca(2+)](i) elevation and vasoconstriction. In pressurized arteries, SR Ca(2+) depletion did not alter IP(3)-induced constriction at 20 mm Hg but reduced IP(3)-induced constriction by approximately 39% at 60 mm Hg. [Ca(2+)](i) elevations and constrictions induced by endothelin-1, a phospholipase C-coupled receptor agonist, were both attenuated by TRPC3 knockdown and xestospongin C in SR Ca(2+)-depleted arteries. In summary, we describe a novel mechanism of IP(3)-induced vasoconstriction that does not occur as a result of SR Ca(2+) release but because of IP(3) receptor-dependent I(Cat) activation that requires TRPC3 channels. The resulting membrane depolarization activates voltage-dependent Ca(2+) channels, leading to a myocyte [Ca(2+)](i) elevation, and vasoconstriction.     

No role for a voltage sensitive release mechanism in cardiac muscle

We have investigated the possibility that some component of calcium release from the cardiac sarcoplasmic reticulum (SR) may occur directly in response to the surface membrane action potential rather than by calcium induced calcium release (CICR). Experiments were performed on rat ventricular myocytes and intracellular calcium concentration ([Ca(2+)](i)) measured with fluo-3. In order to mimic physiological conditions, experiments were performed at 37 degrees C, using the perforated patch technique (to avoid intracellular dialysis) with pulses from -80 to 0 mV. The addition of 500 microM Cd(2+) to inhibit the L-type Ca current reduced the rate of increase of the Ca transient to 2.8 +/- 1% of control. When experiments were performed with Na-free solutions in the pipette, Cd(2+) abolished the transient completely suggesting that the residual Ca entry was on Na-Ca exchange. The addition of Ni(2+) produced a concentration dependent inhibition of the Ca transient with 5 mM being sufficient to completely inhibit the transient. The inhibitory effects of Ni(2+) were unaffected by prior exposure to isoprenaline. These results provide no evidence for a voltage activated calcium release mechanism in cardiac muscle and are consistent with SR Ca(2+) release being triggered by a process of Ca(2+) induced Ca(2+) release.     

Histamine-induced Ca(2+) signaling in human valvular myofibroblasts

In this study, we examined histamine-induced calcium signaling in cultured human valvular myofibroblasts (hVMFs), which are the most prominent interstitial cells in cardiac valves mediating valvular contraction, extracellular matrix secretion, and wound repair. Despite the functional importance of VMFs in cardiac valves, the cellular-signaling pathways, especially those mediated by Ca(2+), are still poorly understood. Using fluorescence imaging microscopy, we measured intracellular Ca(2+) ([Ca(2+)](i)) levels in fura-2-loaded hVMFs. Activation of H(1) receptors released Ca(2+) from one compartment of the endoplasmic reticulum (ER) of hVMFs, but did not induce Ca(2+) entry. This histamine-induced Ca(2+) release was oscillatory and dependent on Ca(2+) re-uptake into the ER by sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA). Application of the reversible SERCA blocker, cyclopiazonic acid (CPA), after depletion of the histamine-sensitive Ca(2+) store revealed the presence of a second, smaller histamine-insensitive Ca(2+) store in the ER. The Ca(2+) content ratio of the histamine-sensitive and histamine-insensitive Ca(2+) stores in the ER was found to be approximately 1.15:1. Another effect of CPA in hVMFs was the activation of store-operated Ca(2+) channels, as demonstrated by maintained [Ca(2+)](i) elevation as well as accelerated Mn(2+) entry. In conclusion, this study establishes for the first time an agonist-induced Ca(2+)-signaling pathway in hVMFs.     

CaMKII inhibition targeted to the sarcoplasmic reticulum inhibits frequency-dependent acceleration of relaxation and Ca2+ current facilitation

Cardiac Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in heart has been implicated in Ca(2+) current (I(Ca)) facilitation, enhanced sarcoplasmic reticulum (SR) Ca(2+) release and frequency-dependent acceleration of relaxation (FDAR) via enhanced SR Ca(2+) uptake. However, questions remain about how CaMKII may work in these three processes. Here we tested the role of CaMKII in these processes using transgenic mice (SR-AIP) that express four concatenated repeats of the CaMKII inhibitory peptide AIP selectively in the SR membrane. Wild type mice (WT) and mice expressing AIP exclusively in the nucleus (NLS-AIP) served as controls. Increasing stimulation frequency produced typical FDAR in WT and NLS-AIP, but FDAR was markedly inhibited in SR-AIP. Quantitative analysis of cytosolic Ca(2+) removal during [Ca(2+)](i) decline revealed that FDAR is due to an increased apparent V(max) of SERCA. CaMKII-dependent RyR phosphorylation at Ser2815 and SR Ca(2+) leak was both decreased in SR-AIP vs. WT. This decrease in SR Ca(2+) leak may partly balance the reduced SERCA activity leading to relatively unaltered SR-Ca(2+) load in SR-AIP vs. WT myocytes. Surprisingly, CaMKII regulation of the L-type Ca(2+) channel (I(Ca) facilitation and recovery from inactivation) was abolished by the SR-targeted CaMKII inhibition in SR-AIP mice. Inhibition of CaMKII effects on I(Ca) and RyR function by the SR-localized AIP places physical constraints on the localization of these proteins at the junctional microdomain. Thus SR-targeted CaMKII inhibition can directly inhibit the activation of SR Ca(2+) uptake, SR Ca(2+) release and I(Ca) by CaMKII, effects which have all been implicated in triggered arrhythmias.     

Calreticulin and calsequestrin are differentially distributed in canine heart

Cardiac sarcoplasmic reticulum (SR) consists of three continuous yet distinct regions: longitudinal, corbular, and junctional. Ca(2+)uptake, catalyzed by the Ca(2+)-dependent ATPase, is thought to occur throughout the SR whereas Ca(2+)release occurs in the region of the junctional SR. In the SR, Ca(2+)is stored in a complex with Ca(2+)-binding proteins such as calsequestrin. In the present study, the distribution of the high-affinity calcium-binding protein, calreticulin, in canine cardiac SR was determined and compared with the distribution of other SR proteins. Three types of distribution were observed. Many proteins, including the Ca(2+)-ATPase and two mannose-containing glycoproteins (52 and 131 kDa), were present in all subfractions. These proteins were absent or depleted from the sarcolemma-enriched fraction. The ryanodine receptor/Ca(2+)release channel and calsequestrin were localized to the junctional SR. Calreticulin immunoreactivity was detected in fractions containing longitudinal SR but not in fractions enriched in sarcolemma or junctional SR. Hence calreticulin is present in a unique vesicular fraction and the Ca(2+)-binding proteins calreticulin and calsequestrin display different patterns of distribution in canine heart.     

Increased phospholamban phosphorylation limits the force-frequency response in the MLP-/- mouse with heart failure

Reduced Ca(2+) release from the sarcoplasmic reticulum (SR) and a negative force-frequency relation characterize end-stage human heart failure. The MLP(-/-) mouse with dilated cardiomyopathy is used as a model to explore novel therapeutic interventions but the alterations in Ca(2+) handling in MLP(-/-) remain incompletely understood. We studied [Ca(2+)](i) in left ventricular myocytes from MLP(-/-) and WT mice (3-4 months old; whole-cell voltage clamp, 30 degrees C). At 1 Hz stimulation, the amplitude of [Ca(2+)](i) transients was similar. However, in contrast to WT, at higher frequencies the [Ca(2+)](i) transient amplitude declined in MLP(-/-) and there was no increase in SR Ca(2+) content. Unexpectedly, the decline of [Ca(2+)](i) was faster in MLP(-/-) than in WT (at 1 Hz, tau of 80 +/- 9 vs. 174 +/- 29 ms, P < 0.001) and the frequency-dependent acceleration of the decline was abolished suggesting an enhanced basal SERCA activity. Indeed, the Ca(2+) affinity of SR Ca(2+) uptake in homogenates was higher in MLP(-/-), with the maximal uptake rate similar to WT. Phosphorylation of phospholamban in MLP(-/-) was increased (2.3-fold at Ser(16) and 2.9-fold at the Thr(17) site, P < 0.001) with similar SERCA and total phospholamban protein levels. On increasing stimulation frequency to 4 Hz, WT, but not MLP(-/-), myocytes had a net gain of Ca(2+), suggesting inadequate Ca(2+) sequestration in MLP(-/-). In conclusion, increased baseline phosphorylation of phospholamban in MLP(-/-) leads to a reduced reserve for frequency-dependent increase of Ca(2+) release. This represents a novel paradigm for altered Ca(2+) handling in heart failure, underscoring the importance of phosphorylation pathways.     

K201 modulates excitation-contraction coupling and spontaneous Ca2+ release in normal adult rabbit ventricular cardiomyocytes

OBJECTIVES: The drug K201 (JTV-519) increases inotropy and suppresses arrhythmias in failing hearts, but the effects of K201 on normal hearts is unknown. METHODS: The effect of K201 on excitation-contraction (E-C) coupling in normal myocardium was studied by using voltage-clamp and intracellular Ca(2+) measurements in intact cells. Sarcoplasmic reticulum (SR) function was assessed using permeabilised cardiomyocytes. RESULTS: Acute application of <1 micromol/L K201 had no significant effect on E-C coupling. K201 at 1 micromol/L decreased Ca(2+) transient amplitude (to 83+/-7%) without affecting I(Ca,L) or the SR Ca(2+) content. At 3 micromol/L K201 caused a larger reduction of Ca(2+) transient amplitude (to 60+/-7%) with accompanying reductions in I(Ca,L) amplitude (to 66+/-8%) and SR Ca(2+) content (74+/-9%). Spontaneous SR Ca(2+) release during diastole was induced by increasing intracellular [Ca(2+)]. At 1 micromol/L K201 reduced the frequency of spontaneous Ca(2+) release. The effect of K201 on SR-mediated Ca(2+) waves and Ca(2+) sparks was examined in beta-escin-permeabilised cardiomyocytes by confocal microscopy. K201 (1 micromol/L) reduced the frequency and velocity of SR Ca(2+) waves despite no change in SR Ca(2+) content. At 3 micromol/L K201 completely abolished Ca(2+) waves and reduced the SR Ca(2+) content (to approximately 73%). K201 at 1 micromol/L reduced Ca(2+) spark amplitude and frequency. Assays specific to SR Ca(2+)-ATPase and RyR2 activity indicated that K201 inhibited both SR Ca(2+) uptake and release. CONCLUSIONS: K201 modifies E-C coupling in normal cardiomyocytes. A dual inhibitory action on SERCA and RyR2 explains the ability of K201 to suppress spontaneous diastolic Ca(2+) release during Ca(2+) overload without significantly affecting Ca(2+) transient amplitude.