Calsequestrin Accumulation in Rough Endoplasmic Reticulum Promotes Perinuclear Ca2+ Release*

Background: Calsequestrin is a high-capacity Ca2+-binding protein that stores Ca2+ within the sarcoplasmic reticulum. Results: Calsequestrin retention in the rough endoplasmic reticulum promotes perinuclear Ca2+ release and spontaneous Ca2+ wave initiation from perinuclear regions. Conclusion: Subcellular redistribution of calsequestrin affects spatial distribution of Ca2+ signals and myocyte function. Significance: Our data provide a new perspective of calsequestrin in perinuclear Ca2+ homeostasis. Molecular mechanisms underlying Ca2+ regulation by perinuclear endoplasmic/sarcoplasmic reticulum (ER/SR) cisternae in cardiomyocytes remain obscure. To investigate the mechanisms of changes in cardiac calsequestrin (CSQ2) trafficking on perinuclear Ca2+ signaling, we manipulated the subcellular distribution of CSQ2 by overexpression of CSQ2-DsRed, which specifically accumulates in the perinuclear rough ER. Adult ventricular myocytes were infected with adenoviruses expressing CSQ2-DsRed, CSQ2-WT, or empty vector. We found that perinuclear enriched CSQ2-DsRed, but not normally distributed CSQ2-WT, enhanced nuclear Ca2+ transients more potently than cytosolic Ca2+ transients. Overexpression of CSQ2-DsRed produced more actively propagating Ca2+ waves from perinuclear regions than did CSQ2-WT. Activities of the SR/ER Ca2+-ATPase and ryanodine receptor type 2, but not inositol 1,4,5-trisphosphate receptor type 2, were required for the generation of these perinuclear initiated Ca2+ waves. In addition, CSQ2-DsRed was more potent than CSQ2-WT in inducing cellular hypertrophy in cultured neonatal cardiomyocytes. Our data demonstrate for the first time that CSQ2 retention in the rough ER/perinuclear region promotes perinuclear Ca2+ signaling and predisposes to ryanodine receptor type 2-mediated Ca2+ waves from CSQ2-enriched perinuclear compartments and myocyte hypotrophy. These findings provide new insights into the mechanism of CSQ2 in Ca2+ homeostasis, suggesting that rough ER-localized Ca2+ stores can operate independently in raising levels of cytosolic/nucleoplasmic Ca2+ as a source of Ca2+ for Ca2+-dependent signaling in health and disease.

Ca 2ϩ is the most universal signal used by different cell systems to encode diverse information, which is decoded from the temporal and spatial dynamics of Ca 2ϩ signals by specific mechanisms. It has been recognized that the subcellular locations of Ca 2ϩ events determine the specific biological outcomes of Ca 2ϩ signaling (1). In cardiomyocytes, Wu et al. (2) showed that inositol 1,4,5-trisphosphate receptor type 2 (IP 3 R2) 3 -mediated nuclear envelope Ca 2ϩ signals have a primary effect on regulating gene transcription. More recently, it was demonstrated that nuclear Ca 2ϩ elevations, but not the global contraction-associated Ca 2ϩ elevations, induce cardiomyocyte hypertrophy (3). However, mechanisms regulating the nuclear or perinuclear Ca 2ϩ events still remain obscure.
During cardiac excitation-contraction coupling, sarcoplasmic reticulum (SR) Ca 2ϩ release occurs primarily through the type 2 ryanodine receptors (RyR2s), which are located at junctional SR sites (4 -6). RyR2 functionally associates with three additional SR proteins: the luminal SR protein cardiac calsequestrin (CSQ2) and two smaller SR transmembrane proteins, triadin and junctin (6 -9). CSQ2 is a low-affinity, high-capacity Ca 2ϩ -binding protein that can store Ca 2ϩ within the SR (10). Each molecule of CSQ2 can bind 18 -50 Ca 2ϩ ions. CSQ2 is also believed to regulate the activity of RyR2 Ca 2ϩ release channels by controlling the local luminal Ca 2ϩ concentration in the vicinity of the RyR2 channels (11)(12)(13).
The junctional SR appears to be contiguous with the nuclear envelope in cardiomyocytes (14). Meanwhile, the cardiac rough endoplasmic reticulum (ER) exists in adult cardiomyocytes as perinuclear cisternae, perhaps including the outer leaf of the nuclear envelope, from where CSQ2 is synthesized and traffics anterogradely along an as-yet unidentified pathway (15). Well defined changes in CSQ2 co-translocational processing during cardiac hypertrophy or heart failure suggest that retention of CSQ2 in the rough ER of the perinuclear cisternae is greatly augmented (16). We postulated that the accumulation of CSQ2 in the perinuclear rough ER could play a role in the regulation of local Ca 2ϩ events, which may lead to pathophysiological response.
To test this hypothesis, we used adenovirus-mediated expression of a CSQ2 fusion protein, CSQ2-DsRed, in cultured cardiomyocytes. When CSQ2 is overexpressed as a fusion protein with DsRed, it is retained mostly in the rough ER (15). This CSQ2 accumulation in the rough ER/perinuclear cisternae allows us to study the effects of rough ER retention of CSQ2 on myocyte perinuclear Ca 2ϩ handling and its implication for cardiac disease. We compared triggered and spontaneous Ca 2ϩ release in cultured adult cardiomyocytes expressing CSQ2 in either the junctional SR (CSQ2-WT) or the perinuclear region (CSQ2-DsRed). Our data show that enrichment of CSQ2 in the perinuclear cisternae was sufficient to enhance nuclear Ca 2ϩ transients, promoting Ca 2ϩ -dependent transcription and myocyte hypertrophy, and also led to the shift of spontaneous arrhythmogenic Ca 2ϩ waves emanating from the perinuclear region instead of the junctional SR.

EXPERIMENTAL PROCEDURES
Adult Ventricular Myocyte Culture and Adenoviral Infection of CSQ2 Constructs-Rat and mouse ventricular myocytes were isolated and cultured as described (17). Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1996) and were approved by the Institutional Animal Care and Use Committee at the University of Iowa. IP 3 R2 Ϫ/Ϫ and wild-type Black Swiss mice were kindly provided by Dr. Ju Chen (University of California, San Diego) (18). Two hours after plating cells, adenoviruses were applied at a multiplicity of infection of 100. Adenoviruses containing cDNAs for wild-type CSQ2 (Ad-CSQ2-WT) and CSQ2-DsRed fusion protein (Ad-CSQ2-DsRed) were described previously (15). Empty Ad5 vector (Ad-empty) was purchased from the Gene Transfer Vector Core of the University of Iowa. Experiments were performed 40ϳ48 h after infection.
Western Blot Analysis and Immunofluorescence of CSQ2 and RyR2 Proteins-Western blotting was performed as described previously (17). Mouse anti-CSQ2 antibody was used to detect endogenous and exogenous CSQ2. GAPDH (Cell Signaling Technology, Inc.) was used as a loading control. Immunostaining of CSQ2 and RyR2 proteins was performed as described previously (15,19). CSQ2-DsRed was detected as DsRed fluorescence. Nuclear staining was performed with TO-PRO-3 (Invitrogen).
Confocal Ca 2ϩ Imaging of Cultured Adult Ventricular Myocytes-Confocal Ca 2ϩ imaging was performed as described (19). Briefly, cells were loaded with Fluo-4 AM at 37°C for 30 min. The cells were then washed with Tyrode's solution at room temperature for 15 min before Ca 2ϩ imaging. Confocal images were acquired using a 63ϫ, 1.3 numerical aperture oil immersion objective mounted on a Zeiss LSM 510 confocal microscope (Carl Zeiss MicroImaging GmbH). Confocal line scan or two-dimensional frame scan was used to record Ca 2ϩ signals. Steady-state Ca 2ϩ transients were measured in Tyrode's solution containing 1.8 mM Ca 2ϩ under a field stimulation of 1 Hz. Ca 2ϩ waves were examined after field stimulation was halted, and each cell was imaged for 24 -40 s. In some experiments, ouabain (100 M), isoproterenol (100 nM), or 10 mM extracellular Ca 2ϩ was added to induce Ca 2ϩ waves. The fluorescence of CSQ2-DsRed was also recorded to identify the infected myocytes and perinuclear localized protein. For Ad-CSQ2-WTand Ad-empty-infected myocytes, the nuclear region was readily identified by the strong Ca 2ϩ indicator staining of the nuclear envelope and the immediate surrounding ER/SR and also by delayed Ca 2ϩ transients compared with adjacent cytosolic regions. SR Ca 2ϩ content was measured as the maximum Ca 2ϩ release induced by a local application of 10 mM caffeine.
Primary Culture of Neonatal Cardiomyocytes and Immunostaining Assay-Neonatal myocytes were prepared as described previously (20) and infected with viral constructs at a multiplicity of infection of 50. Cells were used 48 h after adenoviral infection. ␣-Actinin (sigma) immunofluorescence was used to detect cardiomyocytes. The cell surface area was measured using NIH ImageJ 1.43.
Quantitative RT-PCR-Total RNA extraction from cell samples was performed using TRIzol (Invitrogen). 0.6 g of DNasetreated total RNA samples for each group was reverse-transcribed using SuperScript II (Invitrogen). Quantitative PCR using iQ SYBR Green Supermix (Bio-Rad) was performed on a Bio-Rad iQ5 real-time PCR cycler. Primers were specific for mouse sequences. The sequences were as follows: atrial natriuretic factor (ANF), GTCTTGGCCTTTTGGCTTC (forward) and TTCCTCAGTCTGCTCACTC (reverse); and GAPDH, CA-TTTCCTGGTATGACAATGAATACG (forward) and TCC-AGGGTTTCTTACTCCTTGGA (reverse). Relative transcript quantities of target (ANF) and endogenous (GAPDH) genes were determined using the comparative C T method. The amount of ANF was normalized to the amount of GAPDH for each group.
Statistics-Data are expressed as means Ϯ S.E. Analysis of variance, Student's t test, and the 2 test were applied when appropriate. A p value of Ͻ0.05 was considered statistically significant.
We next investigated how expression of perinuclear enriched CSQ2-DsRed and CSQ2-WT affected Ca 2ϩ transients in rat ventricular myocytes. Steady-state, field-stimulated Ca 2ϩ transients (1 Hz) were acquired using line scan confocal imaging, with the scanning line across the center of the nucleus (nuclei) longitudinally (Fig. 3A). Cytosolic Ca 2ϩ ([Ca 2ϩ ] cyt ) and nuclear Ca 2ϩ ([Ca 2ϩ ] nu ) transients were obtained, normalized, and plotted against time (Fig. 3A). The [Ca 2ϩ ] nu transients were distinguished from corresponding [Ca 2ϩ ] cyt transients by a smaller magnitude and prolonged time-to-peak and decay kinetics. Overexpression of CSQ2-WT significantly increased the amplitudes of [Ca 2ϩ ] cyt and [Ca 2ϩ ] nu transients proportionally ( Fig. 3, B-D). Overexpression of CSQ2-DsRed also increased the amplitudes of [Ca 2ϩ ] cyt transients to a similar level compared with CSQ2-WT even though CSQ2-DsRed was localized predominantly around the nucleus. These data imply that the rough ER can serve as a Ca 2ϩ store and contributes to bulk [Ca 2ϩ ] cyt signals, as suggested previously (14). Compared with the effects of CSQ2-WT on [Ca 2ϩ ] nu , CSQ2-DsRed expression induced a much greater effect on [Ca 2ϩ ] nu when normalized to the amplitude of [Ca 2ϩ ] cyt transients (Fig. 3D). Assessment of SR Ca 2ϩ stores demonstrated that overexpression of CSQ2-WT and CSQ2-DsRed similarly increased the bulk Ca 2ϩ loading of caffeine-sensitive Ca 2ϩ stores (Fig. 3E). These data indicate that overexpression of CSQ2-WT enhances [Ca 2ϩ ] cyt and [Ca 2ϩ ] nu equally, whereas perinuclear enriched CSQ2 increases the [Ca 2ϩ ] nu signal more potently than it does the [Ca 2ϩ ] cyt signal, suggesting that CSQ2 plays a critical role in mobilizing Ca 2ϩ release from ER/SR lumens.
CSQ2-DsRed Promotes Ca 2ϩ Waves Originating from Perinuclear Region-On the basis of our finding that perinuclear CSQ2 favorably increases nuclear Ca 2ϩ , we next investigated whether overexpression of CSQ2-DsRed promotes spontane-ous Ca 2ϩ release events in a similar spatial pattern. We therefore compared spontaneous Ca 2ϩ waves originating from cells expressing either wild-type or perinuclear CSQ2. Perinuclear originated Ca 2ϩ waves were defined as those for which the firing spot fell within the region of the nucleus and the 4-m flanking regions (Fig. 4A, right panels; for more details, see supplemental Figs. S2 and S3). Both perinuclear and non-perinuclear originated Ca 2ϩ waves were detected in the presence and absence of CSQ2 overexpression (Fig. 4A). In empty virus-infected myocytes, the perinuclear originated Ca 2ϩ waves accounted for 30% of the total recorded waves (Fig. 4B). Overexpression of CSQ2-WT slightly increased the percentage to 39%. By sharp contrast, in CSQ2-DsRed-overexpressing myocytes, the perinuclear originated Ca 2ϩ waves were significantly increased (to 86%). To promote the generation of spontaneous Ca 2ϩ waves, the myocytes were also exposed to high extracellular Ca 2ϩ (10 mM), 100 M ouabain, or 100 nM isoproterenol. Under each of these conditions, overexpression of CSQ2-DsRed, but not CSQ2-WT, maintained this increased percentage of perinuclear originated Ca 2ϩ waves of Ͼ80% (Fig. 4

, C-E).
Overexpression of CSQ2-WT only slightly increased the percentage of perinuclear originated Ca 2ϩ waves in the presence of ouabain or excess extracellular Ca 2ϩ (Fig. 4, C and D), but not isoproterenol (Fig. 4E). Taken together, these results indicate that the localization of CSQ2 is the critical factor for spatial distribution of spontaneous Ca 2ϩ waves such that increased expression of CSQ2 in the perinuclear region promotes Ca 2ϩ wave generation from this area.
Ryanodine Receptors, but Not IP 3 Receptors, Are Necessary for Generation of Ca 2ϩ Waves Originating from Perinuclear Region-IP 3 Rs, especially IP 3 R2s, are localized to the nuclear envelope (21), where they contribute to nuclear Ca 2ϩ signaling (22)(23)(24)(25). To examine whether the CSQ2-DsRed-induced spatial redistribution of Ca 2ϩ wave firing requires IP 3 R activity, we first tested whether inhibition of IP 3 Rs with xestospongin C (XeC) and 2-aminoethoxydiphenyl borate (2-APB) affects the generation of Ca 2ϩ waves from the perinuclear region. Fig. 5A and supplemental Fig. S4 show a sequence of two-dimensional images displaying representative non-perinuclear and perinuclear originated Ca 2ϩ waves from myocytes infected with either Ad-CSQ2-WT (left panels) or Ad-CSQ2-DsRed (right panels). Unexpectedly, the generation of neither non-perinuclear nor perinuclear originated Ca 2ϩ waves was blocked by XeC and 2-APB. Although the initiation of Ca 2ϩ waves was not affected by XeC or 2-APB, application of a high concentration of 2-APB reduced the size and amplitude of the Ca 2ϩ waves (supplemental Fig. S4B), possibly due to the nonspecific effects of 2-APB, e.g. its partial inhibition of the SERCA pump (26).
To further test the contribution of the perinuclear localized IP 3 R2 to the generation of perinuclear originated Ca 2ϩ waves, ventricular myocytes from wild-type or IP 3 R2 knock-out (IP 3 R2 Ϫ/Ϫ ) mice were used. Similar to the results obtained with rat myocytes (Fig. 4), overexpression in wild-type myocytes of CSQ2-DsRed, but not CSQ2-WT, resulted in mostly (95%) perinuclear originated Ca 2ϩ waves (Fig. 5, B and D). The percentage of Ca 2ϩ waves generated in the perinuclear region remained unchanged (90%) in IP 3 R2 Ϫ/Ϫ myocytes expressing CSQ2-DsRed (Fig. 5, C and E). These data indicate that the enhanced release of perinuclear Ca 2ϩ that occurs with CSQ2-DsRed in the rough ER/perinuclear cisternae occurs through a mechanism not involving IP 3 R2.
To further understand the mechanism of CSQ2-mediated perinuclear Ca 2ϩ wave generation, we then evaluated the effects of RyR2 inhibitors on perinuclear originated Ca 2ϩ waves. Treatment with the RyR2 inhibitor tetracaine (100 M) (Fig. 6A) or the more specific RyR2 blocker ryanodine (5 M) (Fig. 6B) of rat ventricular myocytes expressing either CSQ2-WT or CSQ2-DsRed completely blocked both the perinuclear and non-perinuclear originated Ca 2ϩ waves. Removal of the inhibitor tetracaine restored Ca 2ϩ wave generation from both regions in myocytes expressing either CSQ2-WT or CSQ2-DsRed. In addition, the irreversible SERCA inhibitor thapsigargin completely abolished all Ca 2ϩ waves that initiated from any regions in both Ad-CSQ2-WT-and Ad-CSQ2-DsRed-infected myocytes (Fig. 6C). Taken together, these results indicate that IP 3 Rs contribute minimally to CSQ2-DsRed-induced redistribution of Ca 2ϩ waves, whereas RyR2-mediated Ca 2ϩ release is necessary for the generation of both perinuclear and non-perinuclear originated Ca 2ϩ waves, consistent with the colocalized pattern of RyR2 with CSQ2-DsRed in a confined perinuclear region. Our data also suggest that SERCA-regulated Ca 2ϩ storage is necessary for the generation of either type of Ca 2ϩ wave, independent of CSQ2 localization.
Overexpression of CSQ2-DsRed and CSQ2-WT Enhances Hypertrophy of Neonatal Cardiomyocytes-Nuclear Ca 2ϩ plays an important role in the regulation of gene expression (2, 27-30). Enhanced nuclear Ca 2ϩ signals induced by CSQ2 redis-tribution could affect hypertrophic processes in cardiomyocytes. We therefore studied the consequence of overexpressing CSQ2-WT and CSQ2-DsRed in mouse neonatal cardiomyocytes, which display autonomous hypertrophy in culture (20). Using an antibody specific for ␣-actinin to define myocyte dimension, we found that overexpression of CSQ2-DsRed increased the surface area of neonatal cardiomyocytes by 27 Ϯ 2%, whereas equal overexpression of CSQ2-WT ( Fig. 1) produced a modest increase in surface area (14 Ϯ 3%) (Fig. 7, A and  B). These data were consistent with the results from quantitative analysis of the hypertrophic marker ANF in neonatal myocytes upon these treatments (Fig. 7C). Taken together, our results demonstrate that overexpression of CSQ2 enhances hypertrophy of neonatal cardiomyocytes, and the perinuclear enriched form of CSQ2 produces a more pronounced effect compared with CSQ2-WT, consistent with their differential contributions to nuclear Ca 2ϩ levels.

DISCUSSION
We recently developed a method to direct CSQ2 localization to the perinuclear rough ER by appending the fluorescent protein DsRed to CSQ2 (15). CSQ2-DsRed overexpression thus provides an interesting model for studying the biological consequences of the perinuclear enriched form of CSQ2. In this study, by overexpressing CSQ2-WT or CSQ2-DsRed in rat and mouse ventricular myocytes, we found that 1) CSQ2-DsRed increases the bulk Ca 2ϩ storage in the ER/SR, similar to CSQ2-WT; 2) CSQ2-DsRed, but not CSQ2-WT, preferentially enhances the nuclear Ca 2ϩ tran-   sients and promotes active propagated Ca 2ϩ waves initiating from the perinuclear region; 3) perinuclear originated Ca 2ϩ waves are mediated by RyR2 instead of IP 3 R2; and 4) CSQ2-DsRed induces a greater degree of cellular hypertrophy in cultured neonatal cardiomyocytes compared with CSQ2-WT.
The increases in cytosolic Ca 2ϩ were initially thought to be transmitted passively to the nucleus via Ca 2ϩ diffusion through nuclear pore complexes (31)(32)(33). More recent studies showed that cardiomyocytes contain a separate membrane source around and within the nucleus that is sensitive to IP 3 (2, 14, 22, 24, 25, 29). IP 3 R2s localized to the inner FIGURE 5. IP 3 Rs are not necessary for initiation of perinuclear originated Ca 2؉ waves. A, the potent IP 3 R inhibitor XeC (IC 50 ϭ 358 nM) did not abolish either the perinuclear originated (Peri-Nu) or non-perinuclear originated (non-Peri-Nu) Ca 2ϩ waves. The Ca 2ϩ waves were recorded by two-dimensional confocal imaging before and after treatment with 3 M XeC for 10 min. Note that the Ca 2ϩ wave firing spots were not affected by XeC. B and C, wild-type or IP 3 R2 Ϫ/Ϫ mouse ventricular myocytes, respectively, were infected with Ad-CSQ2-WT or Ad-CSQ2-DsRed. Ca 2ϩ waves were recorded in line scan mode, with the scanning line run across the nuclear center longitudinally. D and E, summary of the percentage of perinuclear originated and non-perinuclear originated Ca 2ϩ waves. Note that deletion of IP 3 R2 did not alter the effect of CSQ2-DsRed on the spatial redistribution of Ca 2ϩ waves. The number of waves detected is denoted as numbers within the bars. n ϭ 8, 23, and 20 for Ad-empty, Ad-CSQ2-WT, and Ad-CSQ2-DsRed in WT myocytes and n ϭ 15, 17, and 28 for Ad-empty, Ad-CSQ2-WT, and Ad-CSQ2-DsRed in IP 3 R2 Ϫ/Ϫ myocytes. **, p Ͻ 0.01 between the indicated groups by 2 test. membrane surface of the nuclear envelope can function as a source of Ca 2ϩ that leads to hypertrophic growth (2,24). Other studies have described a nucleoplasmic reticulum that contains IP 3 Rs and RyR2s (29), which is likely due to nuclear envelope membrane extension or in-foldings deep into the nucleoplasm, as revealed earlier by Vaux and co-workers (34) and more recently by Franzini-Armstrong and co-workers (21) with high magnification of electron micrographs. Escobar et al. (21) described perinuclear dyads in close proximity to the nuclear envelope, aligned along a perinuclear basket of longitudinal T-tubules. These authors suggested that Ca 2ϩ microdomains emanating from these RyR-sensitive sites could support or augment IP 3 -dependent activation. In this study, we found that CSQ2 localized to the rough ER preferentially enhanced [Ca 2ϩ ] nu transients as opposed to [Ca 2ϩ ] cyt transients, whereas the more widely distributed (junctional SR-localized) CSQ2 proportionally enhanced [Ca 2ϩ ] nu and [Ca 2ϩ ] cyt transients. This differential effect was observed despite similar increases in the general SR Ca 2ϩ stores by the expression of the two Ca 2ϩ -binding proteins. These findings suggest that both passive Ca 2ϩ diffusion and active Ca 2ϩ release may contribute to [Ca 2ϩ ] nu homeostasis, and the contribution of these two modes of [Ca 2ϩ ] nu regulation may depend on the compartmentalization of Ca 2ϩ storage and Ca 2ϩbinding proteins. Increasing the Ca 2ϩ storage in the junctional SR may therefore affect the [Ca 2ϩ ] nu by enhancing the passive flooding of cytosolic Ca 2ϩ into the nucleoplasm, whereas recruiting Ca 2ϩ to perinuclear ER/SR Ca 2ϩ stores may directly and more effectively increase the Ca 2ϩ released into nucleoplasm.
Recent studies reported that CSQ2 regulates the activity of Ca 2ϩ release by controlling the local luminal [Ca 2ϩ ] in the vicinity of the RyR channels (11,13,35). The data showed that overexpression of CSQ2-WT attenuates the generation of Ca 2ϩ waves (11,35), indicating its important role for the luminal Ca 2ϩ sensing of RyR2. Thus, we initially predicted that myocytes overexpressing perinuclear CSQ2 would have fewer Ca 2ϩ waves originating around nucleus compared FIGURE 7. Overexpression of CSQ2-DsRed and CSQ2-WT enhances neonatal cardiomyocyte hypertrophy. A, confocal micrographs of neonatal myocytes following infection with Ad-empty, Ad-CSQ2-WT, or Ad-CSQ2-DsRed and staining with ␣-actinin (green). B, summary data of A in which the cell surface area was measured in three independent experiments. The results are presented as means Ϯ S.E. relative to the Ad-empty control, which was assigned a value of 1. Statistical significance was first tested by one-way analysis of variance, and then the differences between groups were determined by Student's t test. n ϭ 257, 284, and 418 for Ad-empty, Ad-CSQ2-WT, and Ad-CSQ2-DsRed, respectively. **, p Ͻ 0.01 between the indicated groups by Student's t test. C, quantitative PCR analysis of the hypertrophic marker ANF in neonatal myocytes. n ϭ three batches of cells per group. *, p Ͻ 0.05. with control myocytes. Surprisingly, we observed that the fraction of perinuclear originated Ca 2ϩ waves in CSQ2-DsRed-expressing myocytes is dramatically higher than in control myocytes. Two possible mechanisms may be responsible for these apparently contradictory results. First, the RyR2 luminal Ca 2ϩ -sensing mechanism in the rough ER may be different from that in the junctional SR. The junctional SR is a specialized subcellular compartment where CSQ2 co-localizes with and acts in concert with triadin-1 and junctin to regulate the luminal Ca 2ϩ sensing of RyR2 (6,7,13,36,37). However, this complex has not been shown to exist in the rough ER. The second possible mechanism is that the ER form of CSQ2 in the heart is more fully phosphorylated, whereas junctional SR forms of CSQ2 are relatively dephosphorylated (38). The result of increased level of CSQ2 phosphorylation is thought to be increased rough ER retention (15,39). This mechanism is predicted to underlie increased levels of perinuclear CSQ2 in tissue from tachycardia-induced canine heart failure (16).
It is possible that the frequent perinuclear Ca 2ϩ waves in myocytes expressing CSQ2-DsRed may be simply due to the overloading of Ca 2ϩ in the rough ER as a consequence of overexpression of a Ca 2ϩ buffer in this region. New approaches that can spatially resolve the differences in Ca 2ϩ levels between the rough ER and junctional SR are warranted and will provide a further understanding of the findings observed in this study. Our findings regarding the significance of perinuclear CSQ2 have three implications. 1) Our data suggest that the regulation of RyR2 by luminal Ca 2ϩ is environment-dependent and determined differently by accessory proteins such as CSQ2.
2) The functional role of CSQ2 in cardiomyocytes is dependent on its spatial distribution. 3) If CSQ2 is redistributed through a regulated retention to the rough ER, as is observed in failing hearts, Ca 2ϩ homeostasis is likely to undergo a redistribution as well, from a normal contractile to an arrhythmogenic or transcriptional phenotype. These conclusions are supported by our observations that CSQ2-DsRed preferentially enhances perinuclear Ca 2ϩ release, promotes arrhythmogenic Ca 2ϩ waves from the perinuclear region, and stimulates myocyte hypertrophy to a greater extent than CSQ2-WT. It is worthwhile to note that our findings of CSQ2-DsRed-mediated transcriptional and hypertrophic changes in cultured neonatal cardiomyocytes are interesting but limited. Future studies using alternative experimental systems, including a transgenic mouse approach, may be necessary to further examine this postulation. IP 3 R2 was shown to be localized to the nuclear envelope of ventricular myocytes (21), supporting its potential role as a regulator of perinuclear Ca 2ϩ release. Several groups have demonstrated in neonatal, atrial, and ventricular cardiomyocytes that IP 3 -dependent Ca 2ϩ release contributes to nuclear Ca 2ϩ signals (2,3,(22)(23)(24)(25). However, in this study, using pharmacological tools and genetic mouse models, we found that IP 3 R2 does not contribute to the generation of CSQ2enhanced, perinuclear originated Ca 2ϩ waves. Instead, RyR2 is necessary for the generation of Ca 2ϩ waves by perinuclear CSQ2. These data do not exclude the possibility that IP 3 -dependent Ca 2ϩ release may contribute to delayed responses such as Ca 2ϩ -dependent transcriptional regulation (2).
In summary, by manipulating the subcellular distribution of CSQ2, we have revealed several new aspects of its biology. Specifically, our data demonstrate that perinuclear CSQ2 accumulation enhances [Ca 2ϩ ] nu transients disproportionate to the increases in [Ca 2ϩ ] cyt transients. CSQ2-DsRed promotes the generation of Ca 2ϩ waves predominantly from the perinuclear region. The spatial redistribution of Ca 2ϩ waves mediated by perinuclear CSQ2 requires RyR2s but not IP 3 R2s. Thus, two Ca 2ϩ microdomains for regulation of Ca 2ϩ -dependent transcription may exist in the adult cardiomyocytes: the IP 3 -sensitive nuclear envelope and the perinuclear rough ER (Fig. 8). Finally, our data provide a new perspective of CSQ2 in perinuclear Ca 2ϩ homeostasis and Ca 2ϩ -dependent transcription in health and heart disease.