Cytosolic Dynamics of Annexin A6 Trigger Feedback Regulation of Hypertrophy via Atrial Natriuretic Peptide in Cardiomyocytes*

Background: Altered annexin A6 expression is associated with several cardiovascular disorders, including myocyte hypertrophy, but precise functions of the protein remain elusive. Results: Dynamic association of annexin A6 with intracellular atrial natriuretic peptide precursor protects against adrenergic stimulation-induced hypertrophy of H9c2 cardiomyocytes. Conclusion: Annexin A6 negatively regulates hypertrophic progression of cardiomyocytes. Significance: Annexin A6 exhibits potential for antihypertrophic therapeutics. Malfunctions in regulatory pathways that control cell size are prominent in pathological cardiac hypertrophy. Here, we show annexin A6 (Anxa6) to be a crucial regulator of atrial natriuretic peptide (ANP)-mediated counterhypertrophic responses in cardiomyocytes. Adrenergic stimulation of H9c2 cardiomyocytes by phenylephrine (PE) increased the cell size with enhanced expression of biochemical markers of hypertrophy, concomitant with elevated expression and subcellular redistribution of Anxa6. Stable cell lines with controlled increase in Anxa6 levels were protected against PE-induced adverse changes, whereas Anxa6 knockdown augmented the hypertrophic responses. Strikingly, Anxa6 knockdown also abrogated PE-induced juxtanuclear accumulation of secretory granules (SG) containing ANP propeptides (pro-ANP), a signature of maladaptive hypertrophy having counteractive functions. Mechanistically, PE treatment prompted a dynamic association of Anxa6 with pro-ANP-SG, parallel to their participation in anterograde traffic, in an isoform-specific fashion. Moreover, Anxa6 mutants that failed to associate with pro-ANP hindered ANP-mediated protection against hypertrophy, which was rescued, at least partially, by WT Anxa6. Additionally, elevated intracellular calcium (Ca2+) stimulated Anxa6-pro-ANP colocalization and membrane association. It also rescued pro-ANP translocation in cells expressing an Anxa6 mutant (Anxa6ΔC). Furthermore, stable overexpression of Anxa6T356D, a mutant with superior flexibility, provided enhanced protection against PE, compared with WT, presumably due to enhanced membrane-binding capacity. Together, the present study delivers a cooperative mechanism where Anxa6 potentiates ANP-dependent counterhypertrophic responses in cardiomyocytes by facilitating regulated traffic of pro-ANP.

Mechanisms regulating cell size in metazoans remain poorly understood. In actively dividing cells, a balance between increase in cell number (hyperplasia) and size (hypertrophy) is instrumental for systemic homeostasis (1). However, in terminally differentiated cells, like cardiomyocytes of postnatal myocardium, regulation of hypertrophic process becomes crucial (2). Hypertrophy in cardiomyocytes occurs as a physiological phenomenon, as well as an adaptive response to stress, which may turn maladaptive. Pathological hypertrophy is a major risk factor in several cardiovascular disorders that culminate in heart failure (3). Understanding the molecular mechanisms of hypertrophic signaling and development of strategies to check hypertrophic growth remains a challenging quest in the cardiovascular arena of cell biology (4).
Anxa6, 3 largest member of the annexin family of Ca 2ϩ -and phospholipid-binding proteins, is a major myocardial annexin (5). It is ubiquitously expressed and associated with a variety of physiological functions. Anxa6 predominantly serves as a scaffold protein for linking mediators of signaling pathways (6), preferably in a Ca 2ϩ -dependent and membrane-proximal manner (7), which includes sarcolemma (8) as well as intracellular membranous structures (9 -11). Functional redundancies arising from the highly conserved nature of annexins (12) remain a major reason for the lack of overt phenotypes in Anxa6 knockout animal models (13), where loss of Anxa6 function is believed to be mitigated by compensatory responses (14). However, it is known that end stage heart failure is associated with down-regulated Anxa6 (15), and the protein is unchanged to lowered in transition from hypertrophy to heart failure.
Onsets of cardiovascular pathological conditions are usually associated with an up-regulation of Anxa6 (16). We have characterized earlier a role of Anxa6 in the regulation of cardiomyocyte contractility (17). Moreover, Anxa6 is also considered to be a negative inotropic factor playing compensatory roles in chronic pathology (18). However, the mechanistic significance of altered Anxa6 expression in hypertrophied cardiomyocytes remains elusive.
In the present study, we investigated whether differential expression and spatiotemporal dynamics of Anxa6 are critically involved in the hypertrophic process of cardiomyocytes. For this purpose, a mechano-deficient cardiomyocyte cell line has been chosen. This line, H9c2(2-1), derived from rat heart, has been extensively characterized (19) and is an established animal origin-free model for studying signal transduction pathways in cardiomyocytes, including hypertrophy (20). Recently, it has also been shown that H9c2 and neonatal cardiomyocytes display identical hypertrophic responses (21), thus rendering the cells ideal for screening of antihypertrophic therapeutics.
Adrenergic stimulation of cardiomyocytes is a major contributor for triggering prohypertrophic cascades in vivo (22). Phenotypes in pathological hypertrophy are usually associated with changes in the gene expression or cytosolic distribution pattern of certain biochemical markers, predominantly the natriuretic peptides atrial natriuretic peptide (ANP) and brain natriuretic peptide and cytoskeletal proteins like ␣-SkA (␣-skeletal actin). These constitute part of what is known as hypertrophy-associated "fetal gene reprogramming," a signature of the maladaptive pathology (4). ANP has also been shown to possess vital autocrine and paracrine functions locally, including antihypertrophic activities (23). Here we show that spatiotemporal alterations in Anxa6 expression are associated with PE-induced hypertrophic changes of H9c2 cardiomyocytes. Using stable cell lines of H9c2 cardiomyocytes, we found that Anxa6 confers substantial protection against hypertrophy through its association with pro-ANP, which is crucial for ANP-dependent protection against hypertrophy, acting ensemble as a negative feedback loop. Thus, the present study identifies a novel mechanistic spectrum of Anxa6 facilitating ANP-dependent counterhypertrophic cascades.
Molecular Cloning and Mutagenesis-Oligonucleotides used for cloning and mutagenesis are listed in Table 1. GFP-or YFPtagged plasmid vectors expressing rat Anxa6 or pro-ANP were generated by standard molecular biology procedures. Dendra2tagged plasmid was constructed by PCR-amplifying Dendra2 from Addgene (Cambridge, MA) plasmid 29574:tol2-mpx-Dendra2 (24) and swapping with EGFP in pEGFP-N1. The Anxa6 NLS mutant was generated by ligating indicated nuclear localization signal (NLS) as a C-terminal fusion of Anxa6 in the AcGFP1-Anxa6 construct, without intervening stop codons. Site-directed mutagenesis was performed using the QuikChange Lightning kit (Stratagene, La Jolla, CA), following the manufacturer's instructions. Schematic maps of Anxa6 constructs are depicted in Figs. 3A and 6A. The pro-ANP-EGFP construct has been described elsewhere (25). Constructs were verified by automated sequencing at Eurofins Genomics (Bangalore, Karnataka, India).
Cell Culture, Transfection, Stable Cell Line Generation, and Treatments-The rat cardiomyocyte cell line H9c2(2-1), acquired from the cell repository of the National Centre for Cell Science (Ganeshkhind, Pune, India), was maintained in DMEM supplemented with 4.5 g/liter glucose, 1.5 g/liter sodium bicarbonate, 10% FBS, and antibiotics at 37°C in a humidified atmosphere containing 5% CO 2 . Before experimentation, cells were cultured in serum-and antibiotic-free growth medium for 24 h. For transient expression, 1-2 ϫ 10 5 cells, seeded in 35-mm dishes, were transfected with 2-6 g of plasmid DNA and 2-8 l of FuGENE HD (Roche Applied Science) reagent, as per the manufacturer's instructions. Downstream experiments were performed after 72-96 h (knockdown experiments) or 48 h (all others) post-transfection. Adding selection antibiotics to transfected cells, according to predetermined kill curves, generated  Table 2. Relative quantification of transcript profiles was performed as described earlier (28). Briefly, cDNA synthesized from 500 ng of total RNA extracted from cells, were subjected to quantitative PCR using SYBR Green chemistry. Computation of -fold changes in mRNA levels from C T values using 2 Ϫ⌬⌬CT methods have been described elsewhere (29). 18 S rRNA levels were used as an internal reference standard.
Subcellular Fractionation, Co-IP, and Western Immunoblotting-Whole cell lysates (WCL) were prepared in CellLytic MT lysis buffer (Sigma) supplemented with Halt protease and phosphatase inhibitor mixture (Pierce). Subcellular fractionation, membrane protein extraction using a Mem-PER kit, and co-IP were carried out as per the manufacturer's instructions. For co-IP, about 1 mg of lysate was subjected to IP, and 10% of the starting material was taken as input. Lysates were incubated with antibody-coupled or control resins overnight at 4°C with end-on rotation. 25-100 g of elutes were used for subsequent Western blot analysis. SDS-PAGE, immunoblotting, detection, and densitometric quantification were performed as described elsewhere (28).
Enzyme-linked Immunosorbent Assay (ELISA)-The levels of ANP in the cell culture medium were measured by a doubleantibody sandwich ELISA, using a commercially available kit from Qayee-Bio (Shanghai, China) according to the manufacturer's instructions. Briefly, culture media from control and treated cells were collected and centrifuged at 3000 rpm for 20 min, and the supernatant was incubated with HRP conjugates in antibody-coated wells of microwell plates. Signal detection was carried out in a Spectramax Paradigm multimode detection platform (Molecular Devices, Sunnyvale, CA).
High-resolution images were acquired sequentially with a slow unidirectional scanning speed (200 -400 Hz; beam expander: 6), and smaller frame sizes were used for higher magnification. For time lapse imaging, faster scanning speeds (800 -1000 Hz) coupled with low line averaging were used to preserve temporal resolution. Pixel dimensions and Z-steps were estimated to satisfy Nyquist sampling criteria. The disc spinning speed of the CSU unit was set to 1800 rpm. All comparable sets of images were acquired under identical stack parameters, laser power, detector gain, amplifier offset, and pinhole aperture windows.
Atomic force microscopy of live cells was carried out in CO 2independent live cell imaging media (Molecular Probes) by Magnetic AC mode using an Agilent (La Jolla, CA) 5500-ILM AFM with a piezo-scanner (maximum range, 100 m). Microfabricated Type-II Magnetic AC silicon cantilevers (Agilent) of 235-m length and with a nominal spring force constant of 0.5-9.5 newtons/m were used. Cantilever oscillation frequency was tuned into resonance frequency of about 45-115 kHz. Images were captured at frame sizes of 512 ϫ 512 pixels with a scanning speed of 0.5 lines/s. Immunocytochemistry and Live Cell Imaging-Cells cultured in LabTek-II chambered coverglass (Nunc, Roskilde, Denmark) were fixed with 3.7% paraformaldehyde for 10 min, quenched in 50 mM NH 4 Cl for 15 min, and permeabilized using 0.2% Triton X-100 for 5 min, followed by blocking with Image-iT FX signal enhancer (Molecular Probes) for 30 min and 1% BSA for 1 h at room temperature. F-actin staining was performed with Alexa Fluor 532 phalloidin as per the manufacturer's instructions. For immunofluorescence, primary antibody incubation was performed in 1% BSA for 2 h at room temperature or overnight at 4°C, followed by incubation with Alexa Fluor-conjugated secondary antibodies (1:500) in 1% BSA for 1 h (room temperature). Nuclear counterstaining was performed with DAPI or TO-PRO-3. Cells were washed 3 times after each step with DPBS, which was also used as solvent in previous steps. Stained cells were kept under DPBS and imaged within 24 h.
For live cell imaging, cells cultured in 35-mm glass bottom dishes (MatTek, Ashland, MA) were imaged in CO 2 -independent live cell imaging medium within a climate box mounted onstage. Dendra2 photoswitching was performed with little modification of previously described methods (30). Briefly, unconverted Dendra2 (green) was irreversibly photoswitched to its red form (supplemental Movie 2) by UV laser, for 500 ms, which enabled simultaneous detection of EGFP signal in cotransfected cells without bleaching EGFP (data not shown). Unconverted and photoconverted forms were simultaneously tracked at 500 -540 and 575-625 nm with 488-and 543-nm laser lines, respectively. Image Processing-All images were processed with ImageJ (National Institutes of Health, Bethesda, MD) using identical linear adjustment parameters across comparable groups. Images were iteratively deconvolved for improving the signal/ noise ratio. For quantification purposes where binary images were instrumental, an adaptive threshold algorithm was applied for dynamic segmentation. All time stacks were bleachcorrected using a histogram-matching macro. For measurement of hypertrophy, individual cell areas of thresholded 8-bit grayscale images were outlined with a tracing tool macro. Output area measurements were acquired from the masked outlines and expressed as -fold change in treated groups over control. Only cells whose areas lie completely within the field of view were considered. Colocalization analysis was performed with JACoP, as described elsewhere (31). Briefly, the percentage of colocalization was calculated from Manders' coefficients as the fraction of green channel overlapping red. In-built particletracking algorithms in ImageJ were used to measure vesicular structures in cytosol. Tracking of clusters in time lapse series were performed using the MTrackJ plugin, and data were plotted as described (32). Atomic force microscopy images were flattened using PicoView version 1.12 (Agilent) and assigned the indicated LUT using Pico Image advanced (Agilent).
Statistical Analysis-All experiments were repeated at least three times, and representative data are displayed. Statistics were performed using Prism 5 (GraphPad Software, La Jolla, CA). Unless stated otherwise, values represent mean Ϯ S.E. Unpaired, two-tailed Student's t test was used to compare means, and p Ͻ 0.05 was considered statistically significant.

PE Induces Reversible Hypertrophic Transformation of H9c2
Cardiomyocytes-Treatment with 100 M PE for 24 h resulted in increased volume over control as revealed by atomic force microscopy of live cells (Fig. 1A). Measurement of cell surface area by actin staining with fluorescent phalloidin (Fig. 1B) displayed a 2.2-fold increase in the area of PE-treated cells (Fig. 1C) and transformed relaxed actin filaments into a complex web of stress fibers ( Fig. 1D) with prominent actin incorporation into the fibers (Fig. 1E). Analysis of relative transcript levels of hypertrophic markers Nppa and ␣-SkA by qRT-PCR (Fig. 1F) showed a 6-fold up-regulation for Nppa and 5-fold for ␣-SkA. Immunoblotting of WCL for ANP precursor, pro-ANP, and ␣-SkA (Fig. 1G) displayed a ϳ2.5-fold increase of either candidate by PE (Fig. 1H). It also increased perinuclear accumulation of secretory granules containing pro-ANP (Fig. 1I). Furthermore, PE-induced increases in cell surface area of H9c2 cells were found to be reversible (Fig. 1J), as shown by significant restoration of cell area after PE withdrawal (Fig. 1K).
Anxa6 Is Spatiotemporally Reorganized in the Course of Hypertrophic Progression-To assess the Anxa6 expression pattern in hypertrophied cells, relative mRNA levels of Anxa6 ( Fig.  2A) were analyzed by qRT-PCR, which showed 3.5-fold increase. Immunoblot analysis on WCL (Fig. 2B) showed a progressive increase in Anxa6 protein levels by PE at the indicated time points (Fig. 2C), with significant increases at 12 and 24 h. Assessment of changes in intracellular distribution of Anxa6 by time lapse immunostaining (Fig. 2D) displayed an increase in the signal of Anxa6 across the time points (Fig. 2D, insets) that spread out from the nuclear periphery toward the membrane after 24 hours of PE treatment. Interestingly, Anxa6 displayed a punctate staining pattern in the juxtanuclear cytosol, the number and size of which, increased with duration of PE treatment (Fig. 2, E and F). A punctate appearance of Anxa6 has been reported earlier in some cell types (33,34), where Anxa6 participated in membrane trafficking. To inquire whether such a punctate staining pattern of Anxa6 indicates its association with membranous structures, a subcellular fractionation followed by immunoblot analysis was performed. It showed that Anxa6 was associated mostly with membrane fractions, including the limiting membrane as well as the intracellular membranous entities (Fig. 2, G and H).
Controlled Up-regulation of Anxa6 Protects against PE-induced Hypertrophy-To gain functional insight into the differential expression and localization of Anxa6 between control and hypertrophied states, stable cell lines expressing Anxa6-YFP (H9c2 Anxa6-YFP ) or empty vector (H9c2 YFP ) were generated ( Fig. 3B), with transfection and clonal selection titered to match Anxa6 protein levels observed in PE-treated cells (Figs. 2A and 3C). Such calibrations were instrumental, given that a 10-fold overexpression of Anxa6 targeted to the heart negatively affected cardiomyocyte function in transgenic mice (35). H9c2 Anxa6-YFP cells displayed reduced nuclear signal of YFP and speckled signals from granular structures in juxtanuclear cytosol ( Fig. 3B) that transformed into profuse clusters extending from the nuclear to cellular periphery upon PE treatment (supplemental Movies 1 and 5), similar to the ones observed in Fig. 2D. Surprisingly, AFM screening displayed grossly unaltered cellular volume following PE treatment of H9c2 Anxa6-YFP cells (Fig. 3D), unlike that observed in Fig. 1A. To address whether the controlled up-regulation of Anxa6 prevented PEinduced hypertrophy, cellular morphometric analysis was carried out by phalloidin staining of H9c2 YFP or H9c2 Anxa6-YFP cells (Fig. 3E) at the indicated time points after PE addition. The H9c2 YFP cells showed an increase in area similar to WT cells (Fig. 1J). In contrast, quantification of cell surface area (Fig. 3F) revealed that not only were the H9c2 Anxa6-YFP cells significantly protected from the PE-induced increase in cell surface area, but also their rates of recovery during the withdrawal phase were significantly faster than those of H9c2 YFP cells. Furthermore, transcript levels of Nppa and ␣-SkA were analyzed by qRT-PCR (Fig. 3G). In contrast to WT cells, which displayed ϳ6-fold higher transcript levels of the two markers (Fig. 1F) by PE, H9c2 Anxa6-YFP cells showed 2.5-fold rise, indicating a truncated hypertrophic response. Thus, it is likely that Anxa6 confers a sustained (both under treatment and postwithdrawal) protection against PE-induced hypertrophy of cardiomyocytes.
Down-regulation of Anxa6 Augments Hypertrophy, albeit It Abrogates the Perinuclear Vesicular Architecture of Pro-ANP in Hypertrophied Cells-To assess whether Anxa6 is necessary for the endogenous regulatory mechanism to resist hypertrophy-associated changes, stable cell lines (H9c2 shR ) expressing Anxa6 (H9c2 Anxa6shR ) or scrambled (H9c2 ScrambshR ) shRNA were generated (Fig. 4A). qRT-PCR and immunoblot analysis showed an 80% reduction in Anxa6 expression levels (Fig. 4, B-D). AFM analysis of live H9c2 Anxa6shR cells showed a substantial increase in cell volume by PE, compared with control (Fig. 4E). Phalloidin staining (Fig. 4F) revealed a 15% additional increase in the area of H9c2 Anxa6shR cells compared with H9c2 ScrambshR (Fig. 4G). Surprisingly, immunocytochemistry showed unchanged to an insignificant accumulation of pro-ANP-SG (Fig. 4H) in H9c2 Anxa6shR cells under PE treatment.
H9c2 ScrambshR showed juxtanuclear pro-ANP accumulation, similar to WT (Fig. 1I). Immunoblotting also displayed H9c2 ScrambshR cells having significantly higher levels of pro-ANP and ␣-SkA upon PE treatment. However, pro-ANP levels were found to be unaltered in H9c2 Anxa6shR cells treated with PE (Fig. 4, I and J). In contrast, the Nppa transcript level was 6-fold higher in both H9c2 ScrambshR and H9c2 Anxa6shR cells compared with control (Fig. 4K). Thus, it seems that a lack of Anxa6 in H9c2 cardiomyocytes aggravates hypertrophy but abrogates hypertrophy-associated accumulation of pro-ANP vesicles in cytosol, without affecting ANP gene expression. Anxa6 Progressively Associates with Pro-ANP in the Course of PE Treatment-Negative modulation of pro-ANP in hypertrophied H9c2 Anxa6shR cells raised the possibility of interaction of the two proteins or formation of a shared complex that can accommodate the load of cytosolic pro-ANP accumulation in hypertrophied cells. To address this question, colocalization analysis was performed on confocal images of WT cells, double-immunostained for Anxa6 and pro-ANP at the indicated time points after PE addition (Fig. 5A). As shown, Anxa6 increasingly colocalized with pro-ANP in course of PE treatment (Fig. 5B), where high magnification sections revealed an increase in colocalized cluster sizes in the course of PE treatment. To assess whether Anxa6 physically associates with pro-ANP or pro-ANP-SG, a co-IP experiment was performed with WT cells, without PE (Ϫ) or treated with PE (ϩ) (Fig. 5C). pro-ANP was present in Anxa6 immunoprecipitate in both control and treated cells. Similarly, reverse IP showed the presence of Anxa6 in pro-ANP immunoprecipitate of either group, and the association was seemingly higher under PE-treated conditions, indicating a progressive order of association between the two candidates.
Anxa6 Dynamically Associates with Pro-ANP in Hypertrophied Cardiomyocytes and Participates in Anterograde Traffic-PE-induced changes in Anxa6 gene expression and the punctate nature of cytosolic distribution, together with its pattern of colocalization with pro-ANP, indicated a dynamic nature of Anxa6 in cytosol of the cells undergoing hypertrophic transformation. To examine the dynamic nature of Anxa6-pro-ANP association and its involvement in anterograde traffic, cells transiently cotransfected with plasmids encoding pro-ANP-EGFP and Anxa6-Dendra2 were treated with PE for 6 h (time point of appearance of Anxa6 puncta). Photoconversion of Dendra2 tagged with Anxa6 showed dynamic association of Anxa6 with pro-ANP vesicles (supplemental Movie 3), resulting in its punctate appearance. Tracking overlapped paths of pro-ANP-EGFP and Anxa6-Dendra2 raised the possibility of Anxa6 participating in anterograde vesicular transport of pro-ANP (data not shown). To visualize whether Anxa6 participates in such forward traffic, movement of a single Anxa6 cluster was tracked in H9c2 Anxa6-YFP cells, 6 h post-PE treatment (supplemental Movie 4), and of multiple such clusters in H9c2 Anxa6-EGFP cells, 24 h post-PE treatment (supplemental Movie 5). As shown, the cluster of Anxa6 scans the perinuclear region before adapting a linear path toward the plasma membrane (Fig. 5D).
Anxa6 Provides Protection against PE via ANP-dependent Counterhypertrophic Mechanisms-To evaluate whether antihypertrophic functions of Anxa6 depend upon pro-ANP-dependent mechanisms, we adopted a site-directed mutagenesis approach (Fig. 6A). As shown in Fig. 6, B and C, mutations disrupting the N-terminal (Anxa6 ⌬N ) tail (residues 1-89) or first annexin (Anxa6 ⌬N1 ) repeat (residues 29 -89) altered the cytosolic localization pattern of Anxa6 and completely abol- ished association between Anxa6 and pro-ANP. Deletion of the C-terminal (Anxa6 ⌬C ) tail (residues 600 -673) did not have any gross effect on localization patterns but partially abrogated the association. Obligatory compartmentalization by nuclear retention of Anxa6 (Anxa6 NLS ) abolished its association with pro-ANP, whereas deletion of a putative nuclear export sequence (NES; predicted by the NetNES server) partially abrogated the association (Anxa6 ⌬NES ). The T356D phosphomimic of Anxa6 (Anxa6 T356D ) was more similar to Anxa6 FL . To test the necessity of Anxa6-pro-ANP association for Anxa6-mediated protection, we checked the protective nature of overexpressed ANP and questioned whether the association-deficient mutants of Anxa6 alter ANP-mediated protection against PE (Fig. 6D). Strikingly, ANP overexpression severely restricted hypertrophic transformation of H9c2 cells. Coexpression of Anxa6 ⌬N1 or Anxa6 NLS with ANP-EGFP significantly abrogated such protective functions of ANP, which were partially rescued by transiently transfecting the cells with Anxa6 FL (Fig.  6E). To further investigate the mechanism behind such rescue operations, we measured the release of ANP by H9c2 ScrambshR and H9c2 Anxa6shR into the culture medium in response to PE and Iso, which are agonists of the hormonal signals known to regulate the process. For the scrambled group, in comparison with the control cells, PE-and Iso-treated cells displayed significantly higher levels of ANP release into the media. However, for the Anxa6 shRNA group, there was no significant difference in ANP release between the control and agonist-treated cells (Fig. 6F).
Anxa6 Is Isoform-specific and "Necessary" but Not "Sufficient" for Evoking ANP-mediated Protection against Hypertrophy-Isoform specificity has been questioned as a determinant of Anxa6 involvement in vesicular trafficking (36,37). We found that the shorter isoform (lacking the VAAEIL residues between 525-530) of Anxa6 (Anxa6.2), although unnoticeable in control cells, expressed to a detectable limit upon PE treatment (Fig.  7A). To address whether association of Anxa6 with pro-ANP is an isoform-specific activity, a VAAEIL deletion mutant of the longer isoform Anxa6.1 (Figs. 6A and 7A) was generated (Anxa6 ⌬VAAEIL ). Localization of Anxa6 ⌬VAAEIL resembled that of Anxa6 FL , with more prominent puncta (Fig. 6B). However, mobility of such puncta (supplemental Movie 6) resembled more of retrograde traffic than anterograde, as shown for Anxa6.1 (supplemental Movies 4 and 5). Anxa6 ⌬VAAEIL did not colocalize with pro-ANP (Fig. 7C) and showed partial association with pro-ANP (Fig. 6C) as compared with Anxa6 FL or T356D constructs.
When H9c2 cells cotransfected with ANP-EGFP and Anxa6 NLS were treated with PE, cellular hypertrophy was induced, accompanied by inconspicuous punctate structures in juxtanuclear cytosol that extended to the cellular periphery (Fig. 7D). Such structures were negligible to undetectable in corresponding control group or vector-transfected cells (Fig. 7D, inset). In the absence of Anxa6-pro-ANP association due to restricted localization of Anxa6, the appearance of such vesicular structures might be indicative of subtle pro-ANP traffic. To address whether such an occurrence represents a "leaky" phenotype of basal pro-ANP traffic, we examined involvement of factor(s), like Ca 2ϩ or cholesterol, which are known to alter localization, membrane binding, and subsequently the activity of Anxa6 (38) and, in general, vesicular traffic. Pretreatment of the H9c2 cells with the Ca 2ϩ ionophore ionomycin triggered enhanced membrane association of both Anxa6 and pro-ANP even without PE treatment, whereas pretreatment with the Ca 2ϩ chelator BAPTA did not affect basal membrane association of Anxa6 but reduced the membrane association of Anxa6 under PE treatment (Fig. 7, E and F). In either condition, BAPTA pretreatment caused total loss of pro-ANP in the membrane fraction, suggesting that association of these two proteins is Ca 2ϩ -dependent. Furthermore, pretreatment of cells with the cholesterol chelator M␤CD abolished colocalization between Anxa6 and pro-ANP, even after 24 h of PE treatment (Fig. 7G).
To evaluate the necessity of Ca 2ϩ in directing pro-ANP-SG trafficking, pro-ANP-EGFP was overexpressed in the H9c2 Anxa6(⌬C) cell line and treated with PE for 9 h (time point having a high anisotropy value of Anxa6). As shown (Fig. 7H and supplemental Movie 7), the lack of the Anxa6 C terminus affected anterograde movement of pro-ANP-SG, which was partially restored (within 2-5 min) by the addition of ionomycin. Thus, it is possible that a cholesterol-mediated recruitment of Anxa6 to pro-ANP-SG might be necessary to deliver Ca 2ϩ for regulated exocytosis, which in turn, is essential for ANPmediated regulation of hypertrophy. In other words, Anxa6 seems to be "necessary" but not "sufficient" for driving ANPdependent counterhypertrophic signaling, which may account for the partial restoration of cell size by Anxa6 FL , as observed in Fig. 6E.
Dynamics of Anxa6 Facilitate Counterhypertrophic Responses in Cardiomyocytes-To ascertain further that the dynamics of Anxa6 (Fig. 5 and supplemental  are involved in its counterhypertrophic function, we used the T356D phosphomimic of Anxa6, which has a more open and flexible struc- ture that can simultaneously bind multiple membranes (39,40). We found that membrane association of Anxa6 T356D under PE treatment was more prominent than that of Anxa6 FL (Fig. 8, A and B), and H9c2 Anxa6(T356D) displayed superior protection over H9c2 Anxa6(FL) in regulating cell surface area (Fig. 8,  C and D), thus indicating a necessity of conformation-dependent dynamics for promoting counterhypertrophic activity of Anxa6.

DISCUSSION
Protein dynamics play a vital role in the regulation of fundamental biochemical processes in living cells, deregulations of which are known to be associated with a host of clinical conditions (41). Participation of Anxa6 in vesicular trafficking has been well characterized, and the protein is known to associate with endosomes and secretory vesicles (37,42,43). Here we show that the cytosolic 4dynamics of Anxa6 confer sustained protection against cardiomyocyte hypertrophy by associating with and regulating pro-ANP traffic, an endogenous counterhypertrophic factor (44). These conclusions are based on gainand loss-of-function approaches involving Anxa6 in H9c2 cardiomyocytes.
With increasing needs to reduce the number of animals in laboratory research, a cardiomyocyte cell line like H9c2 provides a valuable animal origin-free alternative (21). In this regard, our data on single cell-based analysis of PE-treated H9c2 cells (Fig. 1) are comparable with changes brought about in primary cardiomyocytes exposed to hypertrophic insults in vivo (45). Consistent with earlier reports that Anxa6 is increased in hypertrophied and failing heart (16), it is up-regulated in hypertrophied H9c2 cardiomyocytes with a distinct punctate appearance (Fig. 2). Involvement of Anxa6 in the hypertrophic process is apparent because gain-of-function restricts the changes brought about by PE, at least partially (Fig.  3). Such findings are further confirmed by Anxa6 knockdown, which augments hypertrophic transformation of H9c2 cells, both phenotypically and biochemically (Fig. 4).
Increased expression of ANP is a well established marker of pathological cardiac hypertrophy (4). Despite the appearance of augmented hypertrophied phenotypes, the level and characteristic spatial distribution of pro-ANP were unchanged in PE-treated H9c2 Anxa6shR cells (Fig. 4, H-J). It is not due to an upstream signaling failure to elevate ANP gene expression because qRT-PCR reveals an up-regulated Nppa expression profile in H9c2 Anxa6shR cells, similar to PE-treated H9c2 ScrambshR cells (Fig. 4K). This brought into question whether Anxa6 is necessary for maintaining stability or turnover rates of pro-ANP. The absence of Anxa6 might have affected PE-induced accumulation of juxtanuclear pro-ANP-SG, a prerequisite for vesicle aggregation and regulated exocytosis of ANP (46,47). Interestingly, several reports demonstrated that ANP exhibits local counterhypertrophic activities (23,48,49), and Anxa6 is an atrial SG-binding protein, a major component of which is pro-ANP (50). Therefore, we inquired whether Anxa6 associates with pro-ANP to confer its protective functions via ANP-dependent mechanisms.
A progressive association of Anxa6 with pro-ANP is evidenced, where they seem to aggregate in the course of PE treat-ment (Fig. 5). Intriguingly, such clustering of puncta also represents a signature pattern of regulated exocytosis (47). Localization of pro-ANP in cardiomyocytes is restricted to punctate vesicular structures (51) that follow a regulated secretory pathway rather than a constitutive one under pathological hypertrophy (44). Anxa6-pro-ANP association also appears to be dynamic, as visualized in cells co-expressing these proteins, where a real-time overlap of Anxa6 and pro-ANP is evident (supplemental Movie 3). Moreover, movement of Anxa6 clusters occurs parallel to anterograde (41) vesicular traffic (supplemental Movies 4 and 5) in the course of PE treatment. It appears that adrenergic stimulation of H9c2 cells provokes spatiotemporal dynamics of Anxa6 involving pro-ANP vesicular traffic. The functional significance of such association between Anxa6 and pro-ANP is apparent from the fact that overexpression of pro-ANP offers substantial protection against PE, which is markedly abrogated when Anxa6 fails to interact with ANP due to domain deletion or forced compartmentalization. Such loss of protection is significantly rescued by Anxa6 FL (Fig. 6, B-E), suggesting that the counterhypertrophic activity of Anxa6 depends on its association with pro-ANP. Moreover, reduced levels of Anxa6 in H9c2 Anxa6shR cells severely abrogated PE-or Iso-induced release of ANP, showing that putative changes in pro-ANP modulated by Anxa6 translate into functionally significant differences in ANP release (Fig. 6F). Such activity also FIGURE 7. A "necessary" but not "sufficient" mechanism of isoform-dependent Anxa6 activity drives pro-ANP traffic. A, total RNA extracted from control, PE-treated, or Anxa6 ⌬VAAEIL cells was subjected to RT-PCR using the primers indicated in Table 2, and the PCR products were analyzed in 2. seems to be specific for the larger isoform of Anxa6 rather than the smaller one (Anxa6 ⌬VAAEIL ), which displays a punctate distribution pattern but does not associate with pro-ANP or participate in anterograde trafficking (Fig. 7, A-C, and supplemental Movie 6). This is consistent with the finding that the structural differences between the two isoforms due to the lack of the VAAEIL residues alters their functional behavior and interaction with membranes (45). It is likely that, as also noted by others, Anxa6 isoforms interact with different targets and behave differentially in vesicular and endocytic trafficking pathways (36,37).
A limited vesicular network of pro-ANP extending from perinuclear cytosol to the cell periphery was visualized (Fig. 7D) in cells coexpressing ANP-EGFP and Anxa6 NLS . Such inconspicuous pro-ANP-SG is insufficient to protect the cells against hypertrophic insults and may be representative of a leaky basal pro-ANP traffic. This called into question the involvement of factors other than Anxa6 in regulation of pro-ANP traffic. Ca 2ϩ has long been known to be a critical packaging and driving factor for sorting of pro-ANP vesicles (47), and Anxa6 is reported to be a cytosolic Ca 2ϩ -sequestering agent (18). Consistent with these, ionomycin alone is sufficient to colocalize Anxa6 with pro-ANP, with increased deposition in membrane fractions, whereas removal of Ca 2ϩ by BAPTA completely abrogates the colocalization and membrane deposition, even under PE treatment (Fig. 7, E and F). Thus, a failure by Anxa6 NLS to bind cytosolic Ca 2ϩ may result in defective vesicular traffic of pro-ANP that can account for the loss of protection in these cells. Also, cholesterol has been described as a factor governing localization and membrane binding of Anxa6 in some cell types (34,57,58). Pretreatment of H9c2 cells with the cholesterol chelator M␤CD abolished colocalization of Anxa6 and pro-ANP (Fig. 7G), suggesting that the dynamic interaction between Anxa6 and pro-ANP also depends on membrane cholesterol content. pro-ANP is known to be a major membrane-associated protein of rat atrial SG (52). Because pro-ANP-EGFP clusters are relatively immobile in H9c2 Anxa6(⌬C) cells, which can be partially restored by ionomycin ( Fig. 7H and supplemental Movie 7), it is possible that Anxa6 is critical for delivering Ca 2ϩ necessary for pro-ANP trafficking. It has long been known that the C terminus of Anxa6 is essential for Ca 2ϩ and phospholipid binding (39). Given that the Anxa6 ⌬C mutant exhibits partial association with pro-ANP, it is likely that Ca 2ϩ binding at the C terminus induces the conformational change in the N terminus (53) necessary to associate with pro-ANP-SG, thereby driving regulated vesicular traffic of pro-ANP. Furthermore, the discovery of Ca 2ϩ -binding domains in pro-ANP (25) also strengthens the idea that Anxa6 delivers Ca 2ϩ to pro-ANP that may be necessary for its packaging or processing.
A functional role of Anxa6 dynamics receives further support from the stable expression of the Anxa6 T356D mutant, which displayed a considerably higher degree of membrane binding in hypertrophied cells (Fig. 8, A and B). This phosphomimic of Anxa6 reportedly exhibits a conformation resembling the Ca 2ϩ -bound state of WT and is assumed to possess a more flexible, open structure, enabling it to interact with multiple membranes simultaneously (39,40,54). Furthermore, cells stably expressing T356D were found to be superior to WT Anxa6 in defending PE-induced hypertrophy (Fig. 8, C and D). This may have multiple implications; a conformation reflecting Ca 2ϩ -bound Anxa6 would allow T356D to bypass the necessity of Ca 2ϩ to bind membranous structures, at least partially.
Moreover, the open architecture of T356D would allow Ca 2ϩ to induce vesicular aggregation more efficiently than WT, thereby accelerating regulated vesicular exocytosis of pro-ANP, a prerequisite for eliciting counterhypertrophic responses by natriuretic peptides (44,55,56). Therefore, our findings indicate that Anxa6 mediates counterhypertrophic signaling in cardiomyocytes by participating in the trafficking of pro-ANP (Fig. 8E). Because basal pro-ANP levels were unaltered in H9c2 Anxa6shR cells, it is possible that Anxa6 participates in the regulated rather than constitutive exocytosis of pro-ANP and participates in the formation of an ANP secretagogue under hypertrophic challenge. In this regard, earlier studies that described Anxa6 to be an inhibitor of secretion rather than a modulator, as described herein, relied on characterization of purified proteins and vesicles in competitive binding or analyzed tissue-specific distribution of the protein (9,59). Such studies did not take into account the dynamic changes that may occur in protein function under stress at the cellular level (60). But clearly, the mechanism of Anxa6 mobilization in hypertrophied cardiomyocytes and its subsequent integration into pro-ANP transport warrant further investiga-tion. The status of ANP signaling has not been studied in animal models of Anxa6, but knock-out mice lacking Anxa6 displayed faster calcium removal from cytosol (18). This, together with lowered Anxa6 levels in transition from hypertrophy to heart failure (16), leaves the question open about possible involvement of Anxa6 in regulation of ANP biology. However, as documented earlier (13,14), functional redundancies within the annexin family may well compensate for the lack of altered ANP signaling at the systemic level in Anxa6 knock-out mice and should be studied at the cellular level to rule out possible interference in determining changes in ANP signaling.
In conclusion, the present study depicts a hitherto uncharacterized function of Anxa6 in maneuvering regulated traffic of pro-ANP in hypertrophied H9c2 cardiomyocytes and thereby potentiating ANP-mediated counterhypertrophic responses. In this regard, it would be interesting to determine whether Anxa6 can afford similar protective functions in genetic animal models in vivo or whether the counterhypertrophic potential of Anxa6 could be harnessed therapeutically. Further studies would contribute to a better understanding of cardioprotective roles played by Anxa6 in the hypertrophied arena.