Role of SIRT1 in Modulating Acetylation of the Sarco-Endoplasmic Reticulum Ca²⁺-ATPase in Heart Failure

Animal models.

All mice were housed and treated in accordance with NIH and Institutional Animal Care and Use Committee (IACUC) guidelines, and used protocols approved by the Icahn School of Medicine at Mount Sinai or the Gwangju Institute of Science and Technology Animal Care and Use Committees. Studies were conducted in male C57BL/6J mice aged 8–10 weeks (weight, 25 ~ 30 g) purchased from Jackson Laboratories. Cardiac-specific Sirt1 knockout (SIRT1^−/−) mice were generated by crossing Sirt1 ^flox/flox mice (Jackson Laboratory) with α-MHC-MerCreMer mice (αMHC-MerCreMer, Jackson Laboratory)¹⁹. Conditional cardiomyocyte-specific Serca2 knockout mouse model has been previously described¹⁴. All animal experiments were described in the Supplemental material.

Adult cardiomyocyte isolation and physiology.

Ventricular myocytes were isolated from mouse hearts using the method previously described¹¹. The isolation process, analysis of mechanical property, and molecular analysis were described in the Supplemental material.

Production, purification, and administration of the adenoviruses and adeno-associated viruses.

The production and purification, and gene transfer of adenoviruses and adeno-associated viruses were described in the Supplemental material.

In vitro acetylation and deacetylation of SERCA2a.

For in vitro analysis of acetylation and deacetylation of SERCA2a, we performed cell-based and purified protein-based assays. Methods were described in detail in the Supplemental material.

SERCA2a activity assays.

For analysis of in vitro activity of SERCA2a, we performed Ca²⁺ uptake assay and ATPase activity assay. Methods were described in detail in the Supplemental material.

Expression plasmids.

For expression in HEK293 cells, cDNAs encoding wild type SERCA2a, K492Q SERCA2a, K492R SERCA2a, SIRT1, SIRT2, and p300 were cloned into a pcDNA vector. For expression in primary cardiomyocytes and mice, cDNAs encoding wild type SERCA2a, K492Q SERCA2a, and SIRT1 shRNA were cloned into adenoviral and adeno-associated viral vectors. Methods were described in detail in the Supplemental material.

Generation of the anti-acetylated (Ac)-K492 of SERCA2a antibody.

A peptide encompassing K492 of SERCA2a, F⁴⁸⁸SRDKSMSVYC⁴⁹⁸, was synthesized, and the lysine residue was chemically acetylated (Anygen, Korea). Antibody for the acetylated peptide was generated in mice and purified by Abfrontier (Korea).

Statistical analysis.

Statistical analysis was described in the Supplemental material.

SERCA2a acetylation is elevated in HF and SERCA2a interacts with SIRT1.

A large-scale analysis of the acetylome in human cancer cell lines has revealed that SERCA2 undergoes acetylation²². Heart samples obtained from the left ventricles (LV) of patients with HF and normal human controls were analyzed by immunoblotting. In line with previous observations, SERCA2a levels were lower in failing hearts than in normal controls. Interestingly, the acetylation of SERCA2a was markedly higher in failing hearts than the normal controls (Fig. 1a). In addition, this increase in SERCA2a acetylation was also observed in a murine model of HF induced by pressure overload (Fig. 1b) and a porcine model of HF induced by myocardial infarction (MI) (Online Fig. I), implying that an increase in acetylation is a general mechanism underlying the impaired function of SERCA2a in failing hearts.

Through proteomic analysis of the SERCA2a interactome, we identified and confirmed that SIRT1 interacts with SERCA2a in porcine hearts (Online Fig. IIa-c). To verify this interaction, lysates of mouse adult cardiomyocytes (ACMs) were immunoprecipitated with an anti-SERCA2a antibody and probed with an anti-SIRT1 antibody. Inversely, the ACM lysates were also immunoprecipitated with an anti-SIRT1 antibody and probed with an anit-SERCA2a antibody. This reciprocal immunoprecipitaiton experiment showed that SERCA2a and SIRT1 are complexed in ACMs (Fig. 1c). SIRT2 was not co-immunoprecipitated with SERCA2a (Online Fig. IId) suggesting the specificity of the interaction between SERCA2a and SIRT1. Furthermore, HEK293 cells were transfected with expression plasmids for HA-SIRT1 and Flag-SERCA2a, and then cell lysates were subjected to reciprocal immunoprecipitation experiments. The results indicated that SERCA2a and SIRT1 constitute a complex in HEK293 cells (Fig. 1d). In a similar experiment, SIRT2 was not co-immunoprecipitated with SERCA2a in HEK293 cells (Online Fig. IIe). Confocal microscopy also revealed co-localization of SERCA2a and SIRT1 in ACMs (Fig. 1e).

Taken together, these data showed that SIRT1 directly interacts with SERCA2a, and raised a possibility that SIRT1 regulates the acetylation/deacetylation of SERCA2a.

SIRT1 deacetylates SERCA2a.

We next sought to determine the role of SIRT1 in acetylation/deacetylation of SERCA2a. In ACMs isolated from normal mice, acetylation of SERCA2a was significantly enhanced when treated simultaneously with EX-527 (SIRT1 inhibitor) and Trichostatin A (TSA, class I and II HDAC inhibitor)(data not shown), and the elevated acetylation of SERCA2a was significantly reduced by adenovirus-mediated overexpression of SIRT1 but not a control β-galactosidase (β-gal) (Fig. 2a). ACMs isolated from mice harboring cardiomyocyte-specific deletion of Sirt1 (SIRT1^−/−) exhibited significantly elevated acetylation of SERCA2a compared to normal ACMs (Online Fig. III), and the elevated acetylation of SERCA2a was normalized by re-introduction of SIRT1 but not by overexpression of β-gal (Fig. 2b).

Flag-SERCA2a expressed in HEK293 cells was acetylated as determined by immnoprecipitation with an anti-Flag antibody followed by probing with anti-acetyl-lysine antibody. Acetylation of Flag-SERCA2a was significantly reduced when HA-SIRT1 was co-expressed (Fig. 2c), whereas it was significantly increased in cells where SIRT1 expression was knocked-down by transfection of a short hairpin RNA (shRNA) directed against SIRT1 (sh-SIRT1) (Fig. 2d). The level of SERCA2a was unaltered. The reciprocal immunoprecipitation experiments consistently showed that acetylation of SERCA2a was regulated by SIRT1.

To further verify the role of SIRT1, acetylated Flag-SERCA2a was partially purified from the transfected HEK293 cells treated with nicotinamide (NAM, SIRT1 inhibitor) and TSA. Purified acetylated Flag-SERCA2a was incubated with recombinant human SIRT1 (rhSIRT1) in the presence or absence of nicotinamide adenine dinucleotide (NAD⁺, SIRT1 cofactor) and/or NAM. Immunoblotting with an anti-acetyl-lysine antibody revealed that rhSIRT1 deacetylated Flag-SERCA2a in the presence of NAD⁺, and that this modification was inhibited completely by NAM (Online Fig. IV).

Taken together, these data demonstrated that SIRT1 directly deacetylates SERCA2a.

Acetylation suppresses SERCA2a activity.

SIRT1 supports and promotes a wide variety of cellular processes such as apoptosis/cell survival, endocrine signaling, and gene transcription. In addition, SIRT1 regulates the function of many transcription factors and cofactors through the mechanism of deacetylation¹⁷. In failing hearts, SIRT1 levels are significantly reduced, and the reduction appears to correlate with the increased SERCA2a acetylation. Thus, the increase in SERCA2a acetylation caused by the reduced SIRT1 level may contribute to SERCA2a dysfunction in failing hearts.

To test this hypothesis, we measured Ca²⁺ transient profiles and mechanical properties of ACMs isolated from SIRT1^−/− mice using video-based edge-detection system. The SIRT1^−/− ACMs exhibited significantly reduced Ca²⁺ handling as demonstrated by decreased Ca²⁺ amplitude and increased tau, and reduced contractility as demonstrated by decreased cell shortening and maximal rates of contraction and relaxation in comparison to wild type (WT) ACMs (Fig. 2e). However, the dysregulated Ca²⁺ handling and contractility in SIRT1^−/− ACMs were significantly normalized by re-introduction of SIRT1 (Fig. 2e). These data suggest that increased acetylation of SERCA2a caused by knockdown of SIRT1 may impair its functions.

To explore the effects of SIRT1-mediated deacetylation on the enzyme activity of SERCA2a, HL-1 cardiac muscle cells were transfected with a siRNA directed against SIRT1 (si-SIRT1). Immunoblotting showed that SIRT1 levels were markedly reduced in si-SIRT1-transfected cells, but those of SERCA2a and its endogenous regulator phospholamban (PLN) were not affected. Acetylation of p53, a well-studied substrate of SIRT1¹⁷, was significantly increased in si-SIRT1-transfected cells compared to si-Scramble-transfected cells (si-Scr), indicating successful knockdown of SIRT1 in si-SIRT1-transfected cells. As expected, SIRT1 knockdown significantly elevated levels of acetylated SERCA2a, indicating that SIRT1 deacetylates SERCA2a in HL-1 cells (Online Fig. V). Microsomal fractions were isolated and their Ca²⁺ uptake and ATPase activities, which are contributed mainly by SERCA2a, were determined. Knockdown of SIRT1 significantly reduced Ca²⁺ uptake activity (Vmax= 89.53±3.81 and Km=6.92±0.14 of si-Scr vs Vmax= 46.41±4.09 and Km=6.63±0.24 of si-Sirt1, Fig. 2f) and ATPase activity (Vmax= 51.33±3.16 and Km=0.69±0.16 of si-Scr vs Vmax= 15.89±1.34 and Km=0.46±0.16 of si-Sirt1, Fig. 2g). These data showed that the enzymatic activity of SERCA2a is regulated by its acetylation.

To further evaluate the role of SIRT1-mediated deacetylation of SERCA2a in vivo, mice were injected with a recombinant adeno-associated virus serotype 9 designed to express either a SIRT1-specific shRNA (rAAV9-sh-SIRT1) or a scrambled sequence (rAAV9-Scr) (Fig. 3a). A rAAV9-sh-SIRT1-mediated reduction in SIRT1 level was evident as early as 4 weeks after viral injection, and persisted for up to several months (data not shown). Immunoblotting and immunoprecipitation analyses were performed 6 weeks after the injection of the viruses. Knockdown of SIRT1 significantly increased acetylation of SERCA2a and slightly decreased the SERCA2a level (Fig. 3b). The increased acetylation of SERCA2a was associated reduced Ca²⁺ uptake (Vmax= 73.67±2.86 and Km=6.88±0.13 of sh-Scr vs Vmax= 48.95±5.51 and Km=6.42±0.25 of sh-Sirt1, Fig. 3c) and ATPase activities (Vmax= 68.68±2.78 and Km=0.52±0.09 of sh-Scr vs Vmax= 50.35±2.78 and Km=0.73±0.15 of sh-Sirt1, Fig. 3d) in the microsomal fractions. In addition, systolic function was significantly decreased in rAAV9-sh-SIRT1-injected mice as demonstrated by decreased left ventricular (LV) fractional shortening (FS) and ejection fraction (EF). Adverse LV remodeling was also evident in mice that received rAAV9-sh-SIRT1 compared to mice that received rAAV9-Scr, as demonstrated by reduced interventricular septal thickness at systole (IVSs) and increased LV internal dimensions at systole (LVIDs) (Fig. 3e and Online Table I). These data showed that SIRT1-mediated deacetylation plays an important role on SERCA2a activity in vivo.

SIRT1 activation reduces SERCA2a acetylation and restores its activity in HF.

We have previously shown that β-lapachone (β-lap), a natural quinine compound, indirectly activates SIRT1 by elevating intracellular levels of NAD⁺, a critical regulator of SIRT1 activity^{23, 24}. In mice, transverse aortic constriction (TAC) caused a significant reduction in the intracellular NAD⁺/NADH ratio and SIRT1 protein expression levels (Online Fig. VIa and Fig. 4b), which was consistent with an increase in SERCA2a acetylation (Fig. 1b and 4b). In parallel, increased acetylation of known SIRT1 substrates (p53, PARP-1, and Hif-2α) in TAC mouse hearts were observed²⁵, further indicating a reduction in the levels of SIRT1 expression and activity (Online Fig. VIb).

At two weeks post TAC surgery, β-lap administration was initiated to induce SIRT1 activation (Fig. 4a). As expected, daily oral administration of β-lap for 4 consecutive weeks corrected these metabolic abnormalities and inhibited acetylation of known SIRT1 substrates in failing mouse hearts (Online Fig. VI). TAC-induced acetylation of SERCA2a was also reduced by β-lap treatment (Fig. 4b). As expected, the TAC-induced acetylation of SERCA2a caused a significant decrease in SERCA2a activity compared to Sham mice. The maximum rate of Ca²⁺ uptake (Vmax= 96.95±2.38 and Km=6.82±0.07 of Sham+Veh vs Vmax= 58.59±2.03 and Km=6.77±0.10 of TAC+Veh) was reduced by approximately 40% in TAC heart tissues, as compared to Sham hearts. However, this SERCA2a dysfunction was almost completely restored in β-lap-treated TAC-operated mice (Vmax= 104.30±3.65 and Km=6.23±0.07 of Sham+ βL and Vmax= 91.14±3.42 and Km=6.48±0.08 of TAC+ βL) (Fig. 4c). The ATPase activity of SERCA2a was also reduced in TAC-operated mice (Vmax= 74.75±2.61 and Km=0.52±0.08 of Sham+Veh vs Vmax= 44.26±2.13 and Km=0.50±0.10 of TAC+Veh), and this defect was significantly normalized by β-lap (Vmax= 81.66±3.29 and Km=0.48±0.08 of Sham+ βL and Vmax= 56.76±2.51 and Km=0.55±0.10 of TAC+ βL) (Fig. 4d). The levels of PLN phosphorylation and expression of SIRT1 were not altered by β-lap (Online Fig. VII), suggesting that the observed changes in the microsomal Ca²⁺ uptake and ATPase activities were the result of decreased SERCA2a acetylation due to activation of SIRT1. β-lap also restored TAC-induced contractile dysfunction and adverse cardiac remodeling, as determined by echocardiography (Fig. 4e and Online Table II).

Taken together, these results indicate that pharmacological activation of SIRT1 protects cardiac function by reducing SERCA2a acetylation during pressure overload.

The effects of β-lap on SERCA2a are SIRT1-dependent.

To confirm that the observed beneficial effect of β-lap on SERCA2a function is SIRT1-dependent, we utilized two independent SIRT1 deficient mouse models. First, WT mice were intravenously injected with rAAV9-sh-SIRT1 or rAAV9-Scr for 6 weeks and then exposed to β-lap for 4 weeks (Fig. 5a). In rAAV9-sh-SIRT1-injected mice, SERCA2a acetylation was significantly elevated, which was not restored by β-lap (Fig. 5b). In these mice, Ca²⁺ uptake and ATPase activities of SERCA2a were also significantly impaired, and these defects were not normalized by β-lap (Fig. 5c, d, and Online Table VII). The contractile dysfunction and adverse cardiac remodeling in rAAV9-sh-SIRT1-treated mice were not rescued by β-lap (Fig. 5e and Online Table III).

Second, SIRT1^−/− mice¹⁹ were fed with β-lap for four weeks (Fig. 5f). While SIRT1 level was prominently reduced, the acetylation of p53, a well-known substrate of SIRT1¹⁷, was significantly elevated (Online Fig. VIIIa). SIRT1 deficiency did not alter the expression levels of SIRT2 and SIRT3 nor the acetylation of tubulin, a well-known target of SIRT2. However, overall lysine acetylation levels were significantly elevated in these mice (Online Fig. VIIIb). In SIRT1^−/− mice, elevated SERCA2a acetylation was not restored by β-lap (Fig. 5g). The contractile dysfunction and adverse cardiac remodeling in SIRT1^−/− mice were not rescued by β-lap (Fig. 5h and Online Table IV). It is of note that both Serca2a mRNA and protein levels were not significantly altered by β-lap in SIRT1^−/− mice (Fig. 5g and Online Fig. IX).

In order to further address the critical link between SIRT1 activity and SERCA2a function, we set out to determine the effects of β-lap on cardiac function of mice harboring cardiac-specific deletion of Serca2 (SERCA2^−/−). Two weeks after induction of Serca2 gene excision, the SERCA2^−/− mice were subjected to β-lap administration for 4 consecutive weeks (Online Fig. Xa). These mice displayed significantly decreased cardiac function in terms of EF, FS, IVSs, and LVIDs. β-lap-mediated activation of SIRT1 did not rescue the cardiac dysfunction in SERCA2^−/− mice (Online Fig. Xb). The endogenous SERCA2a level was barely detectable and the SERCA2a acetylation was not significantly affected by β-lap in SERCA2^−/− mice (Online Fig. Xc). ACMs isolated from SERCA2^−/− mice exhibited defective Ca²⁺ handling and contractility. The cardioprotective effects of β-lap were not observed in these ACMs (Online Fig. Xd-h). These data suggest that β-lap exerts its beneficial effects through SIRT1-mediated deacetyaltion of SERCA2a.

Taken together, these results demonstrate that SIRT1 play a critical role in modulating SERCA2a activity through deacetylation in the heart.

Identification and characterization of acetylated lysine residues in SERCA2a.

To identify critical acetylation sites on SERCA2a, a series of one-dimensional SDS-PAGE and LC-MS/MS analyses were performed on SERCA2a acetylated in vitro and in vivo. Using this approach, a total of 12 acetylation sites on SERCA2a were identified (data not shown). Among these sites, we recognized a previously identified acetylation site on SERCA, Lys464²². To identify SIRT1-specific deacetylation sites in SERCA2a, semi-quantitative LC-MS/MS analyses were performed on SERCA2a isolated from mouse hearts in which SIRT1 was either inhibited or activated. Among the 12 putative lysine residues, K492 was highly acetylated in TAC-operated mice, which was normalized by β-lap (Fig. 6a, b). In addition, a peptide containing acetylated K492 was identified in hearts of SIRT1^−/− mice (Online Fig. XI).

To confirm these results, an antibody specific for acetyl-K492 of SERCA2a (Ac-K492) was generated. To test the specificity of Ac-K492 antibody against acetyl-K492 of SERCA2a, Flag-tagged WT SERCA2a (Flag-SERCA2a) or K492R SERCA2a (non-acetylated K492) was expressed in HEK293 cells. To induce SERCA2a acetylation, the transfected cells were treated simultaneously with Ex-527 and TSA. Immunoblotting with Ac-K492 antibody detected a strong SERCA2a band in WT SERCA2a-transfected cells, but not in cells transfected with the K492R mutant of SERCA2a (Online Fig. XII). Next, immunoblotting with this antibody revealed that the level of acetylation at K492 was signifcantly elevated in the TAC-induced HF mice and was normalized upon SIRT1 activation by β-lap (Fig. 6c). Up-regulation of K492 acetylation was also found in SIRT1^−/− mice (Fig. 6d) and failing human hearts (Fig. 6e). K492 is highly conserved among the family of human P-type ATPases supporting its essential role in the regulation of ATPase activity (Fig. 6f).

Taken together, these results suggest that acetylation at K492 may be associated with the reduced SERCA2a activity in failing hearts and its deacetyaltion may be mediated by SIRT1.

Effects of acetylated K492 on SERCA2a activity.

To explore the functional significance of acetylation of SERCA2a at K492, we generated Flag-tagged acetylation mimicking (K492Q) and non-acetylated (K492R) mutants of SERCA2a. HEK293 cells were transfected with plasmids expressing these recombinant proteins. The protein levels of both K492Q and K492R SERCA2a were comparable to WT SERCA2a level. Total lysine acetylation levels in these mutants were slightly lower than WT (Online Fig. XIIIa). Ca²⁺ uptake, ATPase activity, and ATP-binding affinity were measured and the values were normalized by the protein levels of SERCA2a. K492Q SERCA2a exhibited significantly decreased Ca²⁺ uptake and ATPase activities compared to WT SERCA2a, which is consistent with our view that acetylation at 492 significantly reduces the SERCA2a activity (Fig. 7a, b). However, K492R SERCA2a also exhibited significantly reduced Ca²⁺ uptake and ATPase activities, although the reduction was lesser than K492Q SERCA2a (Fig. 7a, b). ATP binding affinity was completely abolished in K492Q and significantly reduced in K492R SERCA2a, which suggest that K492 might play a key role in ATP-binding activity of SERCA2a (Fig. 7c). We carried out molecular modeling of SERCA2a based on the crystal structure of the rabbit SERCA1a complexed with ATP²⁶. The deduced model suggested that acetylation at K492 might alter the ATP-binding pocket to render the ATP-binding of SERCA2a less favorable (Fig. 7d). Notably, previous mutagenesis studies of SERCA1a, the major SERCA isoform in skeletal muscles, showed that K492 plays a critical role in ATP binding^{26, 27}. In addition, our modeling revealed that the K492R mutant might exert steric hindrance to the ATP-binding pocket that is composed of R677, V678, and E679 in the phosphorylation domain of the SERCA2a. Thus, K492R, like K492Q, appears to interfere with the binding of ATP to SERCA2a. Therefore, it is likely that K492 is highly vulnerable to modification (i.e. acetylation) or substitution so that any alteration at K492 can negatively influence the ATP-binding properties of ultimately leading to decreased activity of SERCA2a.

To further verify the functional consequences of acetylation of SERCA2a at K492 on contractile function in vitro, adenoviruses expressing WT or K492Q SERCA2a were infected to ACMs isolated from SERCA2^+/− mice. In these mice, the SERCA2a level was reduced to ~ 30% of the normal level, and infection with the viruses restored the expression level of SERCA2a to ~70% (Online Fig. XIIIb). WT SERCA2a significantly restored the defective Ca²⁺ handling and contractility of SERCA2^+/− ACMs. However, K492Q SERCA2a had no effects on these properties, supporting the importance of K492 for the function of SERCA2a (Fig. 7e-i). We further extended these findings to in vivo studies. WT or K492Q SERCA2a was re-introduced via AAV-mediated gene delivery to SERCA2^+/− mice (Online Fig. XIVa). SERCA2^+/− mice showed significantly decreased cardiac function compared to WT mice (Online Fig. XIVb and Online Table VI). Re-introduction of WT SERCA2a restored the contractile function, whereas re-introduction of K492Q SERCA2a did not rescue the impaired cardiac function (Online Fig. XIVb and Online Table VI). AAV-mediated re-introduction of both WT and K492Q SERCA2a in SERCA2^+/− mice restored the total SERCA2a protein level to the range of WT mice (Online Fig. XIVc). ACMs were isolated from SERCA2^+/− mice injected with rAAV9-WT or K492Q SERCA2a. The defective Ca²⁺ handling and contractility was completely restored by WT but not by K492Q SERCA2a (Online Fig. XIVd-h).

Taken together, these results suggest that acetylation of SERCA2a at K492 profoundly impairs the SERCA2a activities.

p300 acetyltransferase acetylates SERCA2a.

We finally pursued to identify the acetyltransferase that is involved in the SERCA2a acetylation in failing hearts. Expression levels of acetyltransferases, p300, GCN5, and PCAF, were elevated in human failing hearts compared to normal donors (Online Fig. XV).

Flag-tagged SERCA2a was co-expressed either with or without Myc-tagged p300. Cell lysates were immunoprecipitated with an anti-Flag antibody and the resulting precipitates were probed with anti-acetyl-lysine antibody (Fig. 8a). Reverse immunoprecipitation experiment was also performed where cell lysates were immunoprecipitated with an anti-acetyl-lysine antibody and probed with anti-Flag antibody. Results from both experiments showed that p300 directly interacts with SERCA2a and that p300 acetylates SERCA2a.

To further confirm acetylation of SERCA2a by p300 in vitro, SERCA2a purified from normal porcine hearts was incubated in the presence or absence of recombinant human p300 (hp300). Co-immunoblotting with an anti-acetyl-lysine and anti-SERCA2a antibodies revealed that SERCA2a is acetylated when p300 is co-incubated (Fig. 8b upper). This SERCA2a acetylation was inhibited by C646 (selective p300 inhibitor) (Fig. 8b bottom). MS-based analysis and co-immunoblotting experiment revealed that the acetylation at K492 was prominently elevated by p300 treatment, which is prevented by co-incubation with rhSIRT1 (Fig. 8c, d). Acetylation at several other lysine residues were also increased by p300, but not prevented by co-incubation with rhSIRT1 (data not shown). Acetylation at K502 was shown as such an example (Fig. 8c).

It was previously shown that the transgenic (TG) mice harboring cardiac-specific overexpression of p300 displayed severe contractile dysfunction^{51, 52}. Total levels of SERCA2a acetylation and acetylation at K492 were significantly increased in the hearts of p300 TG mice compared to WT mice (Fig. 8e).

Taken together, our data suggest that p300 directly acetylates SERCA2a and that acetylation/deacetylation of SERCA2a at K492 is determined by SIRT1 and p300.