STIM1 enhances SR Ca2+ content through binding phospholamban in rat ventricular myocytes.

In ventricular myocytes, the physiological function of stromal interaction molecule 1 (STIM1), an endo/sarcoplasmic reticulum (ER/SR) Ca(2+) sensor, is unclear with respect to its cellular localization, its Ca(2+)-dependent mobilization, and its action on Ca(2+) signaling. Confocal microscopy was used to measure Ca(2+) signaling and to track the cellular movement of STIM1 with mCherry and immunofluorescence in freshly isolated adult rat ventricular myocytes and those in short-term primary culture. We found that endogenous STIM1 was expressed at low but measureable levels along the Z-disk, in a pattern of puncta and linear segments consistent with the STIM1 localizing to the junctional SR (jSR). Depleting SR Ca(2+) using thapsigargin (2-10 µM) changed neither the STIM1 distribution pattern nor its mobilization rate, evaluated by diffusion coefficient measurements using fluorescence recovery after photobleaching. Two-dimensional blue native polyacrylamide gel electrophoresis and coimmunoprecipitation showed that STIM1 in the heart exists mainly as a large protein complex, possibly a multimer, which is not altered by SR Ca(2+) depletion. Additionally, we found no store-operated Ca(2+) entry in control or STIM1 overexpressing ventricular myocytes. Nevertheless, STIM1 overexpressing cells show increased SR Ca(2+) content and increased SR Ca(2+) leak. These changes in Ca(2+) signaling in the SR appear to be due to STIM1 binding to phospholamban and thereby indirectly activating SERCA2a (Sarco/endoplasmic reticulum Ca(2+) ATPase). We conclude that STIM1 binding to phospholamban contributes to the regulation of SERCA2a activity in the steady state and rate of SR Ca(2+) leak and that these actions are independent of store-operated Ca(2+) entry, a process that is absent in normal heart cells.

Quarky calcium release in the heart.

RATIONALE: In cardiac myocytes, "Ca(2+) sparks" represent the stereotyped elemental unit of Ca(2+) release arising from activation of large arrays of ryanodine receptors (RyRs), whereas "Ca(2+) blinks" represent the reciprocal Ca(2+) depletion signal produced in the terminal cisterns of the junctional sarcoplasmic reticulum. Emerging evidence, however, suggests possible substructures in local Ca(2+) release events. OBJECTIVE: With improved detection ability and sensitivity provided by simultaneous spark-blink pair measurements, we investigated possible release events that are smaller than sparks and their interplay with regular sparks. METHODS AND RESULTS: We directly visualized small solitary release events amid noise: spontaneous Ca(2+) quark-like or "quarky" Ca(2+) release (QCR) events in rabbit ventricular myocytes. Because the frequency of QCR events in paced myocytes is much higher than the frequency of Ca(2+) sparks, the total Ca(2+) leak attributable to the small QCR events is approximately equal to that of the spontaneous Ca(2+) sparks. Furthermore, the Ca(2+) release underlying a spark consists of an initial high-flux stereotypical release component and a low-flux highly variable QCR component. The QCR part of the spark, but not the initial release, is sensitive to cytosolic Ca(2+) buffering by EGTA, suggesting that the QCR component is attributable to a Ca(2+)-induced Ca(2+) release mechanism. Experimental evidence, together with modeling, suggests that QCR events may depend on the opening of rogue RyR2s (or small cluster of RyR2s). CONCLUSIONS: QCR events play an important role in shaping elemental Ca(2+) release characteristics and the nonspark QCR events contribute to "invisible" Ca(2+) leak in health and disease.

Sarcoplasmic reticulum Ca2+ pumping kinetics regulates timing of local Ca2+ releases and spontaneous beating rate of rabbit sinoatrial node pacemaker cells.

RATIONALE: Sinoatrial node cells (SANCs) generate local, subsarcolemmal Ca(2+) releases (LCRs) from sarcoplasmic reticulum (SR) during late diastolic depolarization. LCRs activate an inward Na(+)-Ca(2+) exchange current (I(NCX)), which accelerates diastolic depolarization rate, prompting the next action potential (AP). The LCR period, ie, a delay between AP-induced Ca(2+) transient and LCR appearance, defines the time of late diastolic depolarization I(NCX) activation. Mechanisms that control the LCR period, however, are still unidentified. OBJECTIVE: To determine dependence of the LCR period on SR Ca(2+) refilling kinetics and establish links between regulation of SR Ca(2+) replenishment, LCR period, and spontaneous cycle length. METHODS AND RESULTS: Spontaneous APs and SR luminal or cytosolic Ca(2+) were recorded using perforated patch and confocal microscopy, respectively. Time to 90% replenishment of SR Ca(2+) following AP-induced Ca(2+) transient was highly correlated with the time to 90% decay of cytosolic Ca(2+) transient (T-90(C)). Local SR Ca(2+) depletions mirror their cytosolic counterparts, LCRs, and occur following SR Ca(2+) refilling. Inhibition of SR Ca(2+) pump by cyclopiazonic acid dose-dependently suppressed spontaneous SANCs firing up to ≈50%. Cyclopiazonic acid and graded changes in phospholamban phosphorylation produced by ß-adrenergic receptor stimulation, phosphodiesterase or protein kinase A inhibition shifted T-90(C) and proportionally shifted the LCR period and spontaneous cycle length (R(2)=0.98). CONCLUSIONS: The LCR period, a critical determinant of the spontaneous SANC cycle length, is defined by the rate of SR Ca(2+) replenishment, which is critically dependent on SR pumping rate, Ca(2+) available for pumping, supplied by L-type Ca(2+) channel, and ryanodine receptor Ca(2+) release flux, each of which is modulated by cAMP-mediated protein kinase A-dependent phosphorylation.

Elementary calcium release events from the sarcoplasmic reticulum in the heart.

Ca(2+) release events underlie global Ca(2+) signaling yet they are regulated by local, subcellular signaling features. Here we review the latest developments of different elementary Ca(2+) release features that include Ca(2+) sparks, Ca(2+) blinks (the corresponding depletion of Ca(2+) in the sarcoplasmic reticulum (SR) during a spark) and the recently identified small Ca(2+) release events called quarky SR Ca(2+) release (QCR). QCR events arise from the opening of only a few type 2 ryanodine receptors (RyR2s) - possibly only one. Recent reports suggest that QCR events can be commingled with Ca(2+) sparks and may thus explain some variations observed in Ca(2+) sparks. The Ca(2+) spark termination mechanism and the number of RyR2 channels activated during a Ca(2+) spark will be discussed with respect to both Ca(2+) sparks and QCR events.

Ca2+/calmodulin kinase II-dependent phosphorylation of ryanodine receptors suppresses Ca2+ sparks and Ca2+ waves in cardiac myocytes.

The multifunctional Ca(2+)/calmodulin-dependent protein kinase II delta(C) (CaMKIIdelta(C)) is found in the macromolecular complex of type 2 ryanodine receptor (RyR2) Ca(2+) release channels in the heart. However, the functional role of CaMKII-dependent phosphorylation of RyR2 is highly controversial. To address this issue, we expressed wild-type, constitutively active, or dominant-negative CaMKIIdelta(C) via adenoviral gene transfer in cultured adult rat ventricular myocytes. CaMKII-mediated phosphorylation of RyR2 was reduced, enhanced, or unaltered by dominant-negative, constitutively active, or wild-type CaMKIIdelta(C) expression, whereas phosphorylation of phospholamban at Thr17, an endogenous indicator of CaMKII activity, was at 73%, 161%, or 115% of the control group expressing beta-galactosidase (beta-gal), respectively. In parallel with the phospholamban phosphorylation, the decay kinetics of global Ca(2+) transients was slowed, accelerated, or unchanged, whereas spontaneous Ca(2+) spark activity was hyperactive, depressed, or unchanged in dominant-negative, constitutively active, or wild-type CaMKIIdelta(C) groups, respectively. When challenged by high extracellular Ca(2+), both wild-type and constitutively active CaMKIIdelta(C) protected the cells from store overload-induced Ca(2+) release, manifested by a approximately 60% suppression of Ca(2+) waves (at 2 to 20 mmol/L extracellular Ca(2+)) in spite of an elevated sarcoplasmic reticulum Ca(2+) content, whereas dominant-negative CaMKIIdelta(C) promoted Ca(2+) wave production (at 20 mmol/L Ca(2+)) with significantly depleted sarcoplasmic reticulum Ca(2+). Taken together, our data support the notion that CaMKIIdelta(C) negatively regulates RyR2 activity and spontaneous sarcoplasmic reticulum Ca(2+) release, thereby affording a negative feedback that stabilizes local and global Ca(2+)-induced Ca(2+) release in the heart.

Mitsugumin 29 regulates t-tubule architecture in the failing heart.

Transverse tubules (t-tubules) are uniquely-adapted membrane invaginations in cardiac myocytes that facilitate the synchronous release of Ca2+ from internal stores and subsequent myofilament contraction, although these structures become disorganized and rarefied in heart failure. We previously observed that mitsugumin 29 (Mg29), an important t-tubule organizing protein in skeletal muscle, was induced in the mouse heart for the first time during dilated cardiomyopathy with heart failure. Here we generated cardiac-specific transgenic mice expressing Mg29 to model this observed induction in the failing heart. Interestingly, expression of Mg29 in the hearts of Csrp3 null mice (encoding muscle LIM protein, MLP) partially restored t-tubule structure and preserved cardiac function as measured by invasive hemodynamics, without altering Ca2+ spark frequency. Conversely, gene-deleted mice lacking both Mg29 and MLP protein showed a further reduction in t-tubule organization and accelerated heart failure. Thus, induction of Mg29 in the failing heart is a compensatory response that directly counteracts the well-characterized loss of t-tubule complexity and reduced expression of anchoring proteins such as junctophilin-2 (Jph2) that normally occur in this disease. Moreover, preservation of t-tubule structure by Mg29 induction significantly increases the function of the failing heart.

Imaging microdomain Ca2+ in muscle cells.

Ca2+ ions passing through a single or a cluster of Ca2+-permeable channels create microscopic, short-lived Ca2+ gradients that constitute the building blocks of cellular Ca2+ signaling. Over the last decade, imaging microdomain Ca2+ in muscle cells has unveiled the exquisite spatial and temporal architecture of intracellular Ca2+ dynamics and has reshaped our understanding of Ca2+ signaling mechanisms. Major advances include the visualization of "Ca2+ sparks" as the elementary events of Ca2+ release from the sarcoplasmic reticulum (SR), "Ca2+ sparklets" produced by openings of single Ca2+-permeable channels, miniature Ca2+ transients in single mitochondria ("marks"), and SR luminal Ca2+ depletion transients ("scraps"). As a model system, a cardiac myocyte contains a 3-dimensional grid of 104 spark ignition sites, stochastic activation of which summates into global Ca2+ transients. Tracking intermolecular coupling between single L-type Ca2+ channels and Ca2+ sparks has provided direct evidence validating the local control theory of Ca2+-induced Ca2+ release in the heart. In vascular smooth muscle myocytes, Ca2+ can paradoxically signal both vessel constriction (by global Ca2+ transients) and relaxation (by subsurface Ca2+ sparks). These findings shed new light on the origin of Ca2+ signaling efficiency, specificity, and versatility. In addition, microdomain Ca2+ imaging offers a novel modality that complements electrophysiological approaches in characterizing Ca2+ channels in intact cells.

Dual effect of initial [K] on vascular tone in rat mesenteric arteries.

A slight increase in extracellular concentration of potassium ([K(+)](o)) can act as a vasodilator in rat mesenteric vascular bed. However, in recent years, several groups have failed to consistently observe relaxation of rat mesenteric arteries in these conditions. The aim of the present study was to provide a mechanistic understanding of this discrepancy. In rat small mesenteric arteries, 37 of 40 arteries mounted for measurement of isometric force and pre-contracted with phenylephrine (PE) did not relax when ([K(+)](o) was raised from 5.9 mM (control ([K(+)](o) to 11.2 or 21.2 mM. However, when ([K(+)](o) was briefly lowered to 1.2 mM, increasing ([K(+)](o) to between 5.9 and 41.2 mM evoked relaxation. This relaxation was not reduced by barium or by removal of the endothelium, but was abolished by 0.1 mM ouabain. Raising ([K(+)](o) from concentrations between 0 and 5.9 mM to 13.8 mM elicited a relaxation of PE-induced tone that was inversely proportional to initial ([K(+)](o). Relaxation was associated with a ouabain-sensitive hyperpolarization of smooth muscle cells. In arteries exposed to dihydroouabain (DHO), raising ([K(+)](o) from 5.9 to 13.8 mM and simultaneously washing out DHO resulted in relaxation of PE-induced force. These results suggest that only when the initial ([K(+)](o) is less than approximately 5 mM do small elevations in ([K(+)](o) evoke smooth muscle hyperpolarization and relaxation via activation of Na,K-ATPase, and not inwardly rectifying K(+) channels. Therefore, small differences in the initial ([K(+)](o) (4.6 vs 5.9 mM) can strongly influence the variations of vascular tone to increases in ([K(+)](o).

Ca2+ blinks: rapid nanoscopic store calcium signaling.

Luminal Ca(2+) in the endoplasmic and sarcoplasmic reticulum (ER/SR) plays an important role in regulating vital biological processes, including store-operated capacitative Ca(2+) entry, Ca(2+)-induced Ca(2+) release, and ER/SR stress-mediated cell death. We report rapid and substantial decreases in luminal [Ca(2+)], called "Ca(2+) blinks," within nanometer-sized stores (the junctional cisternae of the SR) during elementary Ca(2+) release events in heart cells. Blinks mirror small local increases in cytoplasmic Ca(2+),orCa(2+) sparks, but changes of [Ca(2+)] in the connected free SR network were below detection. Store microanatomy suggests that diffusional strictures may account for this paradox. Surprisingly, the nadir of the store depletion trails the peak of the spark by about 10 ms, and the refilling of local store occurs with a rate constant of 35 s(-1), which is approximately 6-fold faster than the recovery of local Ca(2+) release after a spark. These data suggest that both local store depletion and some time-dependent inhibitory mechanism contribute to spark termination and refractoriness. Visualization of local store Ca(2+) signaling thus broadens our understanding of cardiac store Ca(2+) regulation and function and opens the possibility for local regulation of diverse store-dependent functions.