Patent Application: US-201615281269-A

Abstract:
methods for treating cardiovascular disease , and in particular heart failure , are provided comprising administering a therapeutically effective amount of a modulator of serca2a post - translation modification such as sumoylation or acetylation . also provided are methods of treating cardiovascular disease by inhibiting serca2a degradation . further provided are methods of diagnosing a propensity to develop heart failure comprising determining if a serca2a mutant is present or determining the level of expression of sumo1 in cardiomyocytes . the disclosure also provides methods of screening for therapeutics that modulate the post - translational modification of serca2a , such as by modulating post - translational sumoylation and / or acetylation .

Description:
disclosed herein are data establishing that serca2a is sumoylated at lysine residues 480 and 585 and that this sumoylation preserves the atpase activity and stability of serca2a . the significance of sumoylation was further demonstrated by the observation that a serca2a variant ( k480r / k585r ) lacking the sumoylated residues possessed a significantly reduced atpase activity and stability . in isolated cardiomyocytes , adenovirus - mediated sumo1 overexpression augmented contractility and calcium transients with an accelerated calcium decay . transgene - mediated sumo1 overexpression rescued pressure overload - induced cardiac dysfunction concomitantly with increased serca2a function . in contrast , down - regulation of sumo1 level using shrna accelerated pressure overload - induced deterioration of cardiac function accompanied by a decreased serca2a function . taken together , the work disclosed herein shows that sumoylation is a critical post - translational modification regulating serca2a function , and provides a method for treating a cardiac dysfunction or disorder , e . g ., heart failure , by modifying intracellular calcium in the heart . human clinical trials with a recombinant adeno - associated virus encoding serca2a ( raav1 / serca2a ) have been initiated and the results indicate that targeting serca2a is a safe and effective modality for the treatment of human hf . disclosed herein for the first time is the sumoylation of serca2a at two lysine residues . interestingly , both serca2a levels and sumoylation of serca2a were significantly reduced in failing hearts . compelling evidence , disclosed in the detailed description below , established that the reduced sumoylation of serca2a is a direct result of the reduced sumo1 level in failing hearts . this reduced sumoylation was strictly correlated with reduced atpase activity of serca2a and with reduced serca2a stability . moreover , restoration of sumo1 reversed contractile dysfunctions in failing hearts . these results are summarized in fig7 . the conserved nature of the amino acid sequence of serca2a is illustrated in fig8 , which provides an aligned amino acid sequences for the serca2a of human ( seq id no : 2 ), pig ( seq id no : 4 ), rat ( seq id no : 6 ) and mouse ( seq id no : 8 ). in fig9 , aligned polynucleotide sequences encoding these serca2a amino acid sequences , with human ( seq id no : 1 ), pig ( seq id no : 3 ), rat ( seq id no : 5 ) and mouse ( seq id no : 7 ) polynucleotide sequences being presented . the sumoylation motif noted in the brief description of fig3 are found at positions 479 - 482 ( mkke , seq id nos : 10 , 12 , 14 , and 16 for human , pig , rat and mouse , respectively ) and at positions 584 - 587 ( ikye , seq id nos : 10 , 12 , 14 , and 16 ). it is expected that single amino acid changes in either or both of these four - amino - acid motifs will modify serca2a in a manner that modulates its effect on cardiac function . it is further expected that most of these variations will interfere or inhibit sumoylation of serca2a , leading to increased likelihood of cardiovascular disease such as hf . accordingly , one method for diagnosing a disposition towards cardiovascular disease comprises obtaining a biological sample from a patient and determining the amino acid sequence of serca2a at positions 479 - 482 and / or positions 584 - 587 and diagnosing a disposition towards cardiovascular disease if the amino acid sequence varies from the wild - type sequence disclosed herein . analogous diagnostic methods are contemplated for the encoding polynucleotide sequence ( s ). amino acid sequences of the sumo - 1 protein mediating the ptm of serca2a that affects cardiac function are also presented for human ( seq id no : 10 ), pig ( seq id no : 12 ), rat ( seq id no : 14 ) and mouse ( seq id no : 16 ). polynucleotide sequences encoding these amino acid sequences are set forth in seq id nos : 9 , 11 , 13 , and 15 for human , pig , rat , and mouse , respectively . without wishing to be bound by theory , it is possible that the effect of sumoylation on serca2a atpase activity results from an induced conformational change in serca2a ; alternatively , sumoylation may lead to an additional interface for atp binding , leading to an increase in atpase activity . it is also possible that sumoylation may affect other post - translational modifications ( ptms ) of serca2a , such as the acetylation of particular residues . previous studies indicated that a host of regulatory proteins were reciprocally and competitively regulated by sumoylation and acetylation . for example , the transcriptional activity of tumor repressor gene hic1 is promoted by sumoylation and inhibited by acetylation ( van rechem et al ., 2010 ). interestingly , acetylation of serca2a has been recently identified in a large - scale analysis of human acetylome in cancer cell lines ( choudhary et al ., 2009 ). also , it has been found that serca2a is acetylated and that this acetylation is more prominent in failing hearts and can be reversed by sirt1 deacetylase . the reciprocal regulation of sumoylation and ubiquitination is consistent with sumoylation stabilizing serca2a . this type of sumoylation - mediated inhibition of protein degradation has been shown for other proteins . for example , sumoylation of axin , a negative regulator of wnt signaling , prevented ubiquitination and thus conferred a prolonged half - life of axin ( kim et al ., 2008 ). similarly , sumoylation of p68 and p72 rna helicases increased the stabilities of these proteins by reducing ubiquitin - proteasome - mediated protein degradation ( mooney et al ., 2010 ). the data disclosed herein establish that the sumo1 level is significantly reduced in failing hearts , providing experimental basis for the position that cellular sumo1 level should be precisely maintained and controlled for proper functions of cardiomyocytes . the finding disclosed herein that replenishment of sumo1 reversed tac - induced failing phenotypes indicated that reduced sumo1 level is the direct cause of contractile dysfunction . the therapeutic effect of sumo1 gene transfer was profound . in contrast to the reduced sumo1 level , the protein level of the sumoylating and de - sumoylating enzymes , ubc9 and senp1 , were unaltered in failing hearts and when shsumo1 was administered . the level of ubc9 and senp1 was also unaltered when the sumo1 level was restored . therefore , the specificity and capacity of sumoylation is unlikely to be changed in failing hearts . what matters most appears to be the reduced supply of sumo1 . in this regard , it is intriguing to note that depletion of cellular ubiquitin level is sufficient to cause neuronal dysfunction and death ( ryu et al ., 2008 ). in the experiments disclosed hereinbelow , a novel regulatory mechanism is disclosed whereby sumoylation affects or modulates serca2a activity and overall contractile properties of the cardiac muscle cells . in addition , the impressive beneficial effects of sumo1 on cardiac contractility and survival indicates that targeting sumo1 will have a therapeutic value in the treatment of heart failure . the following examples illustrate embodiments of the disclosure . example 1 discloses the materials and methods used in the experiments described herein . example 2 discloses data establishing that sumo1 interacts with serca2a . example 3 shows that sumoylation of serca2a is reduced in failing hearts . example 4 reveals that serca2a is sumoylated at lysine residues k480 and k585 . example 5 shows that sumoylation of serca2a increases the atpase activity of serca2a . example 6 establishes that sumoylation enhances serca2a stability . example 7 shows that sumo1 overexpression enhances cardiomyocyte contractility and enhances ca 2 + transients in isolated cardiomyocytes . example 8 further shows that sumo1 overexpression improves cardiac function in tac - induced heart failure . example 9 shows that small hairpin rna mediates down - regulation of sumo1 , which accelerates cardiac dysfunction . this example provides a description of the materials and methods used in the experiments disclosed herein . to analyze sumoylation within cells , lipofectamine 2000 was used to transfect hek293 cells with plasmids encoding serca2a wild type ( wt ) or serca2a sumoylation site mutants , along with flag - tagged sumo1 and myc - tagged ubc9 . cells were lysed by sonication in ice - cold lysis buffer ( 50 mm tris - cl , ph 8 . 0 , 150 mm nacl , 0 . 1 % triton x - 100 , 10 mm edta , complete protease inhibitor [ one tablet per 10 ml ; roche ], and protein phosphatase inhibitor cocktail ( sigma )) containing 20 mm n - ethylmaleimide . lysates were cleared by centrifugation at 30 , 000 g for 20 minutes . cell lysates were then subjected to immune - precipitation by incubation with a flag - specific affinity matrix gel ( sigma ) overnight at 4 ° c ., after which the immunoprecipitates were washed in cold lysis buffer immunocomplexes were resolved by sds - page , and subjected to western blotting with serca2a - specific antibody , i . e ., anti - serca2a antibody . fresh tissue extracts were prepared in lysis buffer for in vivo sumoylation assays . hearts from each experimental and control group were frozen in liquid nitrogen . frozen tissues were crushed and homogenized in lysis buffer , as described above , using the mp homogenate system ( fastprep homogenizer ). the insoluble portion was removed by centrifugation at 30 , 000 g for 20 minutes . extracts were incubated with anti - sumo1 agarose resin with agitation overnight . the sumo conjugated forms were detected by western blotting with specific primary antibodies . crude microsome was prepared as previously described ( clarke et al ., 1989 ). serca2a activity assays were performed using pyruvate / nadh - coupled reactions , as previously described ( hajjar et al ., 1997 ). the activity of the ca 2 + - atpase was calculated as follows : δabsorbance / 6 . 22 × protein × time ( in nmol atp / mg protein × min ) all assays were done in triplicate . the αmhc - flox - mouse sumo1 transgene was subcloned into the pml2g vector , which has an egfp cdna between two loxp sites . the dna construct was microinjected into fertilized eggs from b6c3 mice and transgenic integration was confirmed by pcr . the analysis was performed using the student &# 39 ; s t test , with significant differences demarcated by a single asterisk (*), indicating p & lt ; 0 . 05 , or by a double asterisk (**), indicating p & lt ; 0 . 001 . data in figures represent mean ± sd . as an approach to identify novel modifiers of serca2a , the serca2a - associated protein complex was isolated from porcine heart lysates by immunoprecipitation with anti - serca2a antibody and then analyzed by two - dimensional electrophoresis ( fig1 a ). ms / ms analysis of the stained protein spots revealed that sumo1 is co - precipitated with serca2a . a representative ms / ms peptide fingerprint for sumo1 is shown in fig1 b . hek293 cells were co - transfected with plasmids encoding serca2a and flag - tagged sumo1 immunoprecipitation with anti - flag antibody followed by western blotting with anti - serca2a antibody showed that serca2a indeed binds to sumo1 ( fig1 c ). a similar experiment performed with hek293 cells co - transfected with plasmids encoding serca2a and myc - tagged ubc9 , a sumo conjugating enzyme , also confirmed that serca2a binds to ubc9 ( fig1 d ). these data establish that serca2a is a target of sumoylation . we then examined whether serca2a is indeed sumoylated in hearts . human heart lysates were immunoprecipitated with anti - sumo1 antibody and probed with anti - serca2a antibody . in addition to a normal serca2a band (˜ 110 kda ), slowly migrating serca2a bands ( 150 ˜ 250 kda ) were detected ( fig2 a , top ) which represent the sumoylated serca2a . the level of sumoylated serca2a was significantly reduced in failing hearts ( hf ) compared to normal hearts ( nf ). in addition , serca2a levels were significantly reduced in the failing hearts , consistent with previous reports , and supporting the validity of the heart sample preparations . along with this reduced serca2a level , sumo1 level was also significantly reduced in the failing hearts . in contrast , the levels of ubc9 and senp1 , the critical sumoylating and de - sumoylating enzymes , respectively , were unaltered in the failing hearts ( fig2 a , bottom ). these data indicated that the reduced sumoylation of serca2a might be primarily due to the reduced level of sumo1 but not to the reduced sumoylating or elevated de - sumoylating activities . we have also observed that sumo1 level as well as serca2a level were significantly reduced in a murine model of hf induced by pressure - overload ( fig2 b ) and in a porcine model of hf induced by volume - overload ( fig2 c ). these results established that reduced sumoylation of serca2a caused by a reduced sumo1 level is a prevailing characteristic associated with the development of hf in diverse mammalian species . sumoylation of target proteins is known to occur on lysine residues in the context of a highly conserved recognition motif , γψkχe / d ( where ψ stands for a large hydrophobic amino acid and χ for any amino acid ) ( sampson et al ., 2001 ). three independent sumoylation prediction programs ( http :// bioinformatics . lcd - ustc . org , http :// www . abgent . com . cn / doc / sumoplot , http :// sumosp . biocuckoo . org / prediction . php ) identified two putative sumo conjugating sites in serca2a , lysines 480 ( k480 ) and 585 ( k585 ). these lysine residues are located in the cytosolic nucleotide - binding domain where atp binds and are perfectly conserved in mouse , rat , pig , and human serca2a ( fig3 a ). to investigate the role of serca2a k480 and k585 during sumoylation , we generated three serca2a variants in which k480 or k585 was replaced by arginine ( k480r and k585r , respectively ) or both k480 and k585 were replaced by arginine ( k480r / k585r ). hek293 cells were transfected with plasmids encoding wild type ( wt ) and these serca2a variants , and then the cell lysates were immunoprecipitated with anti - sumo1 antibody and probed with anti - serca2a antibody . while k480r and k585r were sumoylated indistinguishably from wt serca2a , k480r / k585r was completely unsumoylated ( fig3 b ). the sumoylation of wt serca2a was enhanced when increasing amounts of the sumo1 plasmid were co - transfected in a dose - dependent manner , whereas sumoylation of k480r / k585r was completely abolished ( fig4 c ). taken together , these results indicate that serca2a is sumoylated at the k480 and k585 residues . since the sumoylated lysine residues , k480 and k585 , reside in the nucleotide - binding domains of serca2a , sumoylation may affect the serca2a atpase activity . wt and sumoylation - defective k480r / k585r serca2a were immune - precipitated from the lysates of hek293 cells transfected with the corresponding plasmids , along with the empty or sumo1 - expressing plasmids , and atpase activities were determined . k480r / k585r possessed a significantly decreased vmax compared to wt serca2a ( wt ; 94 . 60 ± 1 . 63 , k480r / k585r ; 37 . 95 ± 5 . 40 nmol / min / mg ) and a significantly increased ec50 value compared to wt serca2a ( wt ; 0 . 24 ± 0 . 09 , k480r / k585r ; 0 . 76 ± 0 . 17 nmol ca 2 + / l ). co - expression of sumo1 significantly increased vmax ( 98 . 58 ± 1 . 83 nmol / min / mg ) and decreased ec50 ( 0 . 11 ± 0 . 09 nmol ca 2 + / l ) in wt serca2a , whereas it does not affect the atpase activity of k480r / k585r ( fig3 d ). further tests addressed whether sumoylation affected the atp - binding affinity of serca2a . hek293 cells were transfected with wt or k480r / k585r serca2a - expressing plasmids , along with the empty or sumo1 - expressing plasmids . cell lysates were incubated with atp - sepharose and the resulting precipitates were probed with anti - serca2a antibody . the results indicated that co - expression of sumo significantly increased the atp - binding affinity of serca2a . in contrast , k480r / k585r possessed a significantly reduced atp - binding affinity , which was not affected by the co - expression of sumo1 ( fig3 e ). these data establish that sumoylation increased the atpase activity of serca2a , at least partly by enhancing atp - binding affinity . the data disclosed herein establish that the serca2a level was reduced in failing hearts concomitantly with a reduced sumo1 level ( fig2 ). as a consequence , the possibility that sumoylation affects the stability of serca2a was investigated . hek293 cells were transfected with wt or k480r / k585r serca2a expressing plasmids , along with the empty or sumo1 - expressing plasmids . at forty - eight hours after transfection , the cells were treated with cycloheximide , an inhibitor of protein synthesis , to block further de novo synthesis of serca2a . after incubation for an additional three and five days , the serca2a levels were determined by western blotting . the estimated half - life of wt serca2a was 4 . 90 ± 0 . 70 days , whereas it increased to 5 . 90 ± 0 . 20 days when sumo1 was co - expressed . the estimated half - life of k480r / k585r was significantly reduced ( 2 . 42 ± 0 . 50 and 2 . 35 ± 0 . 80 days with and without co - expression of sumo1 , respectively ) compared to wt serca2a ( fig3 f ). these data indicate that sumoylation increased the stability of serca2a . sumo1 overexpression enhances cardiomyocyte contractility and ca 2 + transients in isolated cardiomyocytes to examine the physiological function of sumo1 , mouse adult cardiomyocytes were isolated from normal ( sham ) or tac - induced failing hearts ( hf ), and then infected with either adenovirus expressing β - gal ( ad - β - gal ) or sumo1 ( ad - sumo1 ). contractile properties were determined using a dual - excitation spectrofluorometer equipped with a video - edge detection system . when infected with ad - sumo1 , normal cardiomyocytes showed enhanced contractility with an 11 % increase in cell shortening , a 17 % increase in maximal rate of contraction , and a 9 % increase in the maximal relaxation in comparison with the ad - β - gal - infected cardiomyocytes . more prominent enhancement in contractility was observed when the failing cardiomyocytes were infected with ad - sumo1 with a 27 % increase in cell shortening , a 30 % increase in maximal rate of contraction , and a 27 % increase in maximal relaxation . ad - sumo1 - infected cardiomyocytes showed increased calcium amplitude and ca 2 + decay in comparison with ad - β - gal - infected cardiomyocytes . the overall inotropic effect of sumo1 overexpression was comparable to the effect when serca2a is overexpressed ( fig4 ). it is expected that sumo1 overexpression enhanced cardiomyocyte contractility , at least partly through increasing the enzymatic activity and stability of serca2a . we proceeded to define the physiological consequences of sumo1 overexpression in vivo . for this purpose , we utilized a cre / loxp conditional expression system in which administration of tamoxifen induced heart - specific sumo1 overexpression in exchange of egfp expression ( fig5 a ). no apparent cardiac dysfunctions were seen in this transgenic mouse ( table s 1 ). western blotting revealed that tamoxifen - induced sumo1 expression level in transgenic mice ( tg ) was approximately 5 - fold higher than that of wild type littermates ( wt ) ( fig5 b ). wt and tg mice were subjected to tac operation . hf with an approximately 40 - 50 % decrease in fractional shortening ( fs ) was developed in two months . tamoxifen was then administered for four days to induce sumo1 overexpression . along with the increased sumo1 level , sumoylation and the protein level of serca2a were significantly induced by the administration of tamoxifen ( figure s2 ). one month later ( three months post - tac in total ), cardiac functions were examined by histology and echocardiography . representative heart sections and m - mode echocardiographic data are shown in fig5 c . wt and tg mice were indistinguishable at baseline . at three months post - tac , wt mice exhibited severe failing phenotypes with significant left ventricular ( lv ) dilation and reduced fs and ejection fraction ( ef ). tamoxifen - induced sumo1 overexpression , however , dramatically reversed these failing phenotypes with less lv dilation ( lv internal diastolic dimension ( lvidd ), 3 . 24 ± 0 . 33 mm in tg vs . 4 . 40 ± 0 . 57 mm in wt , p & lt ; 0 . 001 ; lv internal systolic dimension ( lvids ), 1 . 44 ± 0 . 30 mm in tg vs . 2 . 93 ± 0 . 54 mm in wt , p & lt ; 0 . 001 ) and improved fs ( 55 . 91 ± 6 . 70 % in tg vs . 34 . 30 ± 5 . 56 % in wt , p & lt ; 0 . 001 ) and ef ( 90 . 26 ± 4 . 27 % in tg vs . 69 . 57 ± 7 . 28 % in wt , p & lt ; 0 . 001 ) ( fig5 d ). hemodynamic analyses also showed improved lv function in tg . the end - systolic pressure - volume relationship ( espvr ) in lv was slightly steeper in tg animals than wt , suggesting an increased cardiac contractility ( figure s 3 ). in contrast , the slope of lv end - diastolic pressure - volume relationship ( edpvr ) was decreased in tg mice , indicating a decreased end - diastolic lv chamber stiffness . parameters of lv dilation including stroke volume , end - diastolic volume , and end - systolic volume were likewise restored in tg . in addition , an increase in heart weight to body weight ratio was significantly inhibited in tg ( table s 2 ). the recovery of cardiac dysfunction by sumo1 was also manifested by increased survival of tg under prolonged pressure - overload ( fig5 e ). the survived mice were 14 out of 14 ( 100 %) in tg , whereas they were only 7 out of 15 ( 47 %) in wt at 100 days after administration of tamoxifen . we performed western blotting to monitor expression levels of key regulatory proteins involved in ca 2 + homeostasis . notable changes under tac were a reduction of the serca2a level ( 65 % decrease vs . sham ), which is consistent with the numerous previous reports , and an increase in ncx1 level ( 56 % increase vs . sham ) ncx1 is responsible for cytosolic ca 2 + elimination during diastole . it was previously shown that a decrease in serca function is coupled with an increase in ncx function in failing hearts ( schillinger et al ., 2003 ; studer et al ., 1994 ) and in isolated cardiomyocytes after delivery of sirna against serca2a ( seth et al ., 2004 ). these tac - induced changes in the levels of serca2a and ncx1 were normalized in tg ( fig5 f ). tac resulted in a significant reduction in the atpase activity of serca2a in wt with a 50 % decrease in vmax ( tac ; 40 . 39 ± 5 . 08 , sham ; 81 . 03 ± 7 . 11 nmol / min / mg ) and a 120 % increase in ec50 ( tac ; 0 . 31 ± 0 . 021 , sham ; 0 . 14 ± 0 . 08 μnmol ca 2 + / l ). this tac - induced reduction in the atpase activity was significantly ameliorated in tg with a 15 % decrease in vmax ( tac ; 70 . 32 ± 5 . 54 , sham ; 82 . 29 ± 5 . 39 nmol / min / mg ) and a 59 % increase in ec50 ( tac ; 0 . 27 ± 0 . 12 , sham ; 0 . 166 ± 0 . 09 μnmol ca 2 + / l ), however . taken together , these data indicate that sumo1 overexpression restores cardiac dysfunction induced by pressure - overload . to evaluate the effects of down - regulation of sumo1 in hearts , we generated recombinant adeno - associated viruses serotype 9 ( raav9 ) that express sumo1 - directed short hairpin rna , or shrna , ( raav9 / shsumo1 ), or a scrambled sequence ( raav9 / sc ) under the control of the u6 promoter ( fig6 a ). these raav constructs were injected into b6c3 / f1 male mice through the tail vein at a dose of 5 × 10 10 viral genomes ( vg )/ mouse . at three weeks after injection , western blotting with heart extracts revealed that sumo1 levels were reduced by approximately 70 % in the raav9 / shsumo1 - injected hearts but not in the raav9 / sc - injected hearts . expression levels of sumo2 , sumo3 , ubc9 , and senp1 were not affected by raav9 / shsumo1 ( fig6 b ). at six weeks after injection , cardiac functions were evaluated . gross morphology of the hearts and representative m - mode images of echocardiographic analyses are shown in fig6 c . hearts from raav9 / shsumo1 - injected mice showed prominent left ventricle ( lv ) dilation and functional deterioration compared with the hearts from raav9 / sc - injected mice with an increased left ventricular internal dimension - diastole , or lvidd , ( shsumo1 ; 3 . 85 ± 0 . 17 mm , sc ; 3 . 42 ± 0 . 19 mm , p & lt ; 0 . 001 ), and left ventricular internal dimension - systole , or lvids , ( shsumo1 ; 2 . 02 ± 0 . 17 mm , sc ; 1 . 30 ± 0 . 12 mm , p & lt ; 0 . 001 ), and decreased fs ( shsumo1 ; 47 . 63 ± 3 . 07 %, sc ; 61 . 77 ± 4 . 77 %, p & lt ; 0 . 001 ) and ef ( shsumo1 ; 84 . 51 ± 2 . 63 %, sc ; 93 . 85 ± 1 . 23 %, p & lt ; 0 . 001 ) ( fig6 d ). hemodynamic analyses showed that injection of raav9 / shsumo1 resulted in a rightward shift of the lv pressure - volume loops and a decreased end - systolic pressure - volume relationship , or espvr , indicating negative inotropic effects of sumo1 down - regulation . injection of an increased dose of raav9 / shsumo1 resulted in a more severe cardiac dysfunction ( figures s 4 a and s 4 b ). an increased heart weight to body weight ratio was also observed in raav9 / shsumo1 - injected hearts ( table s 3 ). the raav9 / shsumo1 - induced cardiac dysfunction was manifested by sudden deaths of the raav9 / shsumo1 - injected mice . all the mice received 1 × 10 11 vg of raav9 / shsumo1 died within three weeks . death rates of mice received 3 × 10 10 vg and 5 × 10 10 vg of raav9 / shsumo1 were slightly higher than , but not statistically significant from , that of control mice received raav9 / sc . during the period of experiments , none of the control mice died ( fig6 e ). western blotting revealed that serca2a protein level was decreased by approximately 40 % in raav9 / shsumo1 - injected hearts . related to this , the sodium / calcium exchanger ncx1 protein level was slightly elevated , although the elevation was not statistically significant . pln ( phospholamban ) and ryr2 ( ryanodine receptor 2 ) protein levels , however , were not altered . as expected , sumoylation of serca2a was also significantly blunted ( fig6 f ). sumoylation of pln and ryr2 were not altered ( figure s4c ). these results establish that sumo1 increases serca2a sumoylation and serca2a stability . sumo1 down - 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