MA3-919

antibody from Invitrogen Antibodies

Targeting: ATP2A2 ATP2B, DAR, SERCA2

Provider product page for MA3-919

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Antibody data

Antibody Data
Antigen structure
References [248]
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Validations
Western blot [2]
Immunocytochemistry [4]
Other assay [65]

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Product number: MA3-919 - Provider product page
Provider: Invitrogen Antibodies
Product name: SERCA2 ATPase Monoclonal Antibody (2A7-A1)
Antibody type: Monoclonal
Antigen: Purifed from natural sources
Description: MA3-919 detects sarcoplasmic or endoplasmic reticulum calcium (SERCA) 2 ATPase from canine, porcine, human, mouse, rabbit and rat tissues. MA3-919 recognizes the SERCA2a and SERCA2b isoforms identically. However, the antibody does not recognize the skeletal muscle (SERCA1) or housekeeping isoforms (SERCA3). MA3-919 has been successfully used in Western blot, immunocytochemistry and immunoprecipitation procedures. By Western blot, this antibody detects an ~110 kDa protein representing SERCA2 ATPase in rat cardiac tissue. The MA3-919 antigen is purified canine cardiac sarcoplasmic reticulum vesicles.
Reactivity: Human, Mouse, Rat, Canine, Guinea Pig, Porcine, Rabbit
Host: Mouse
Isotype: IgG
Antibody clone number: 2A7-A1
Vial size: 100 µL
Concentration: Conc. Not Determined
Storage: -20° C, Avoid Freeze/Thaw Cycles

ATP2A2 protein structure - MA3-919 shown in red.

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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of DU-145 (Lane 1), L6 (Lane 2), U-87MG (Lane 3) and PANC 1 (Lane 4). The blots were probed with Anti- SERCA2ATPASE Mouse Monoclonal Antibody (Product # MA3-919, 1 in 1000 and detected by chemiluminescence Goat anti-Mouse IgG (H+L) Secondary Antibody, HRP conjugate (Product # 62-6520, 1:4000 dilution). A 110 kDa band corresponding to SERCA2ATPASE was observed across cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE®10 % Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane by iBlot® 2 Dry Blotting System (Product # IB21001).The membrane was probed with the relevant primary and secondary Antibody following blocking with 5 % skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western Blotting Substrate (Product # 32106).

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Experimental details: Knockdown of SERCA2 ATPase was achieved by transfecting A549 with SERCA2 ATPase specific siRNAs (Silencer® select Product # S1748). Western blot analysis (Fig. a) was performed using Whole cell extracts from the SERCA2 ATPase knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with SERCA2 ATPase Monoclonal Antibody (2A7-A1) (Product # MA3-919, 1:1000 dilution ) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to SERCA2 ATPase.

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Experimental details: Immunofluorescent analysis of SERCA2 ATPase using SERCA2 ATPase Monoclonal antibody (2A7-A1) (Product # MA3-919) shows staining in A549 cells. SERCA2 ATPase staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing SERCA2 ATPase (Product # MA3-919) at a dilution of 1:100-1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

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Experimental details: Immunofluorescent analysis of SERCA2 ATPase using SERCA2 ATPase Monoclonal antibody (2A7-A1) (Product # MA3-919) shows staining in HeLa cells. SERCA2 ATPase staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing SERCA2 ATPase (Product # MA3-919) at a dilution of 1:100-1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

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Experimental details: Immunofluorescent analysis of SERCA2 ATPase using SERCA2 ATPase Monoclonal antibody (2A7-A1) (Product # MA3-919) shows staining in U251 glioma cells. SERCA2 ATPase staining (green), F-Actin staining with Phalloidin (red) and nuclei with DAPI (blue) is shown. Cells were grown on chamber slides and fixed with formaldehyde prior to staining. Cells were probed without (control) or with or an antibody recognizing SERCA2 ATPase (Product # MA3-919) at a dilution of 1:100-1:200 over night at 4 °C, washed with PBS and incubated with a DyLight-488 conjugated secondary antibody (Product # 35552 for GAR, Product # 35503 for GAM). Images were taken at 60X magnification.

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Experimental details: Immunofluorescence analysis of SERCA2 ATPase was performed using 70 confluent log phase A549 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with SERCA2 ATPase Monoclonal Antibody (2A7-A1) (Product # MA3-919) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing endoplasmic reticulum localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

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Experimental details: Figure 3 Ca 2+ handling proteins. (A) Representative western blot images of Ca V 1.1, RyR1, SERCA1, SERCA2, calsequestrin (1 and 2), and sarcolipin. To allow the use of data in multiple western blots each band within a single western blot was normalized to GAPDH for that sample as a loading control and then normalized to the average WT values for that particular gel to give %WT. (B) Analysis of muscle levels of Ca V 1.1 normalized to GAPDH. (C) Analysis of muscle levels of RyR1 normalized to GAPDH. (D) Analysis of muscle levels of SERCA1 normalized to GAPDH. (E) Analysis of muscle levels of SERCA2 normalized to GAPDH. (F) Analysis of muscle levels of CSQ normalized to GAPDH. (G) Analysis of sarcolipin normalized to GAPDH. (H) SERCA activity as a function of Ca 2+ concentration. (I) Scheme of changes in Ca 2+ handling proteins. Values are shown as mean +- SEM. * P

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Experimental details: Fig 2 PLN phosphorylation and expression levels of other Ca 2+ regulatory proteins. (A) Western blot analyses demonstrating that monomeric p-PLN/PLN ratio is significantly lower in both Pln OE and Pln OE / Sln KO soleus muscles compared with WT (n = 5-6 per genotype). Western blotting for other major Ca 2+ regulatory proteins SERCA1a (B, 110 kDa), SERCA2a (C, 110 kDa), RyR (D, 565 kDa), DHPR (E, 170 kDa), and CSQ (F, 63 kDa) did not reveal any significant differences across genotypes (n = 5-8 per genotype, with a 6 mug load for all proteins). *Significantly different from WT using a a one-way ANOVA with a Tukey's post-hoc, p

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Experimental details: Figure 2--figure supplement 2. Analysis of RNA and protein expression levels of the major cardiac Ca 2+ -handling proteins in PLN/DWORF Tg mice. ( A ) RNA levels of the indicated genes as quantified by qRT-PCR in 16-week-old heart tissue. Atp2a2 , SERCA2a; Ryr2 , ryanodine receptor 2; Cacna1c, alpha1C-subunit of the L-type Ca 2+ channel; Casq2 , calsequestrin 2. Data are normalized to 18S and presented as expression level relative to WT, mean +-SD for n = 10 mice per genotype. ( B ) Representative immunoblots of cardiac homogenates from mice with the indicated genotypes. PS16, phospho-serine 16 on PLN; PT17, phospho-threonine 17 on PLN; tPLN, total phospholamban; RyR2, ryanodine receptor 2; LTCC, L-type Ca 2+ channel (alpha1C-subunit); Casq2, calsequestrin 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ( C ) Western blots for n = 9-10 mice of each genotype were quantified using ImageJ and data are normalized to GAPDH and expressed as mean +-SD relative to WT. ( D ) Quantification of total phospholamban and its phosphorylation status as assessed by western blot ( B ) and expressed as relative to WT. Western blots were quantified with ImageJ software. Phosphorylation blots (PS16 and PT17) were normalized to total PLN (tPLN). Total PLN was normalized to GAPDH. Data are expressed as mean +-SD for n = 9-10 mice per genotype. Statistical comparisons between groups were evaluated by Student's t-test. p-value *p

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Experimental details: Figure 2--figure supplement 3. DWORF binding to SERCA2a displaces PLN in a dose-dependent manner and enhances SERCA activity. ( A ) Immunoprecipitations of Myc-SERCA2a were performed on lysates of HEK293 cells co-transfected with equal amounts of HA-PLN and Myc-SERCA2a (1 ug) and increasing amounts of HA-DWORF. Western blots on input samples (bottom) and bound immunoprecipitated fractions (top) reveal that DWORF binding to SERCA2a competitively displaces PLN. ( B, C ) Ca 2+ -dependent Ca 2+ -uptake assays were performed using homogenates from HEK293 cells co-transfected with equal amounts of SERCA2a and PLN (1 ug) and increasing amounts of DWORF. Co-transfection of cells with PLN (dark grey line) results in a rightward shift of the Ca 2+ -affinity curve as compared to control cells (SERCA2a alone, black) ( B ). Co-transfection with increasing levels of DWORF relieves the inhibitory effect of PLN on SERCA2a in a dose-dependent manner as evidenced by a leftward shift of the affinity curve back toward control values. Cells expressing SERCA2a and DWORF in the absence of PLN do not exhibit enhanced SERCA activity (purple), indicating that DWORF exerts its' stimulatory effect on SERCA through the displacement of PLN. ( C ) Mean K Ca values from n = 4 separate experiments are represented as bar graphs (+-SD). p-value *p

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Experimental details: Figure 6--figure supplement 1. Expression and post-translational modifications of Ca 2+ -handling proteins. ( A ) Immunoblots of cardiac homogenates from mice with the indicated genotypes. ( B ) Immunoblots were quantified using ImageJ and normalized to GAPDH. LTCC, L-type Ca 2+ channel (alpha1C-subunit); Casq2, calsequestrin 2; RyR2, ryanodine receptor 2; GAPDH, glyceraldehyde 3-phosphate dehydrogenase. ( C ) Quantification of the phosphorylation status and oligomerization of phospholamban as assessed by western blot (panel A ) and expressed as relative to WT. Western blots were quantified with ImageJ software. Phosphorylation blots (PS16 and PT17) were normalized to total PLN (tPLN). Total PLN was normalized to GAPDH. tPLN, total phospholamban; PS16, phospho-serine 16 on PLN; PT17, phospho-threonine 17 on PLN. Data are expressed as mean +-SD for n = 4-6 mice per genotype. ( D ) RNA levels of the indicated genes as quantified by qRT-PCR in 8-week-old heart tissue. Atp2a2 , SERCA2a; Cacna1c, alpha1C-subunit of the L-type Ca 2+ channel; Casq2 , calsequestrin 2; Pln , phosholamban; Ryr2 , ryanodine receptor 2. Data are normalized to 18S values and presented as expression level relative to WT, mean +-SD for n = 4-5 mice per genotype.

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Experimental details: Figure 5 Effects of vehicle and neuregulin treatment on calcium handling proteins not associated with the myofilament . A-F) Quantitation of proteins compared with respective total protein or GAPDH for each treatment group is shown. Values for hearts from non-treated (white bars), vehicle-treated (gray bars), and neuregulin-treated (black bars) animals are shown. Sample number is indicated within each bar. Significance was determined by 1-way ANOVA and Newman-Keuls multiple comparison test using GraphPad software. *P

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Experimental details: Figure 1 Expression of NCX1 and other Ca handling proteins in atrial-specific NCX1 knockout mice. A . Immunoblot (top) of mouse atrial (A) and ventricular (V) homogenate using an antibody to NCX1 in wildtype control (C) and atrial-specific NCX1 KO mice. There is complete absence of NCX1 protein in the immunoblot. The new band in the KO lanes at ~110 kDa represents nonfunctional NCX in KO atria after excision of exon 11 by Cre recombinase [13] . Ventricular expression of NCX1 is unaffected in atrial-specific KO mice. The bottom panel shows immunofluorescence of isolated SAN node myocytes from control (C) and KO hearts. Myocytes were co-immunolabled with antibodies against HCN4 and NCX1. Both control and KO SAN cells stained positive for HCN4, but only control SAN cells showed staining of NCX at the membrane. B . Immunoblots of sarcoendoplasmic reticulum Ca ATPase (SERCA), plasma membrane Ca pump (PMCA; the lower band at 72 kDa represents an active proteolytic fragment), and dihydropyridine receptor (DHPR) in control (C) and NCX1 KO atria. Note the reduction in SERCA and the increase in PMCA and DHPR in KO.

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Experimental details: Figure 5 Comparison of protein expression in LV sample from control and cardiomyopathic patients. A: Top; representative SERCA2 and respective GAPDH stainings obtained in normal and cardiomyopathic LV samples. Bottom; Mean+-SEM, *** P

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Experimental details: Figure 7 Expression of Ca 2+ handling protein by isolated cardiomyocytes and electron micrographs of myofibrils in TRPV2-deficient hearts. ( a ) Time-dependent changes in the localization of the Ca 2+ regulatory proteins LTCC, RyR and NCX1 in tamoxifen-treated TRPV2 flox/flox ;Cre +/ - mice. Double staining with anti-LTCC antibody (green) and DAPI (blue) in cardiac muscle (left panels). Double staining with anti-RyR antibody (green) and DAPI (blue) in cardiac muscle (middle panels). Double staining with anti-NCX1 antibody (green) and DAPI (blue) in cardiomyocytes (right panels). Scale bar, 100 mum. ( b ) Expression of NCX1, LTCC, RyR and SERCA proteins in TRPV2-deficient heart extracts (10 mug per lane), using GAPDH as the internal control in cardiac muscle, (see full blots in Supplementary Fig. 6 ). ( c ) Electron micrographs of myofibrils in cardiomyocytes from TRPV2 flox/flox ;Cre +/ - mice, untreated or treated with tamoxifen. Scale bar, 5 mum.

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Experimental details: Fig 6 Immunoblotting. Immunoblot analysis of key Ca 2+ handling proteins and phosphorylation was performed on tissue from left ventricles. SERCA2A abundance (A). PLB abundance (B) and phosphorylation on SER16 (C) and THR17 (D). NCX abundance (E). RyR abundance (F) with phosphorylation on SER2808 (G) and SER2814 (H). CaMKII abundance (I) and CaMKII phosphorylation on THR286 (J). PP1 (K) and PP2A (L) abundance. n heart = 6 for all analysis. *p

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Experimental details: Figure 3. Increased KAI1 levels in AICD transgenic mice. (A) The AICD transgenic mice, but not the control or Fe.27 mice, show expression of KAI1 gene. Membrane fractions from indicated mice were probed with anti-KAI1 antibody. FeCgamma.25 mice showed higher expression of KAI1 protein compared with FeCgamma.12 mice. (B) Membrane fractions from indicated mice were probed with anti-SERCA 2b antibody. No significant changes in SERCA 2b levels were reproducibly observed in FeCgamma transgenic mice.

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Experimental details: FIGURE 4. Effect of UCP3 knockdown on ER Ca 2+ refilling in intact and permeabilized cells. A , SERCA2 immunoblot of HeLa cells transfected with Ctrl or UCP3 siRNA for 48 h. 50 mug/lane of protein from cell extracts was analyzed, using actin as loading control. B , left , averaged [Ca 2+ ] ER recordings of intact HeLa cells during ER Ca 2+ refilling. After 15 mu m BHQ induced Ca 2+ release in Ca 2+ -free medium, cells were washed, and Ca 2+ was then added to assess the kinetics of store refilling. Data were fitted with the sigmoidal equation to extract the EC 50 ( right ), and they are mean +- S.E. of 40 ( n = 5) and 30 cells ( n = 4) for Ctrl and UCP3 siRNA, respectively. **, p < 0.01. C , left , averaged [Ca 2+ ] ER recordings during ER Ca 2+ release and refilling in permeabilized cells. The K + -rich intracellular buffer was supplemented with 1 m m Mg-ATP and 1 m m MgCl 2 to allow ER Ca 2+ refilling. Where indicated, 100 mu m digitonin ( dig ), 15 mu m BHQ, and 100 n m free Ca 2+ were added. Right , statistical evaluation of the ER Ca 2+ refilling kinetics. EC 50 was determined as described for B . Data are mean +- S.E. of 47 ( n = 3) and 33 cells ( n = 3) for Ctrl and UCP3 siRNA, respectively. NS , not stimulated.

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Experimental details: Fig 5 Densitometric analysis of Western blots. (A) SERCA2a; (B) phospholamban (PLB), (C) PLB to SERCA2a ratio, in hearts from male and female sham and gonadectomized rats. (SM: sham male; SF: sham female; GM: gonadectomized male; GF: gonadectomized female). The values are expressed as the means +-S.E.M (n = 6 per group). *P < 0.05 vs. females of the same group; + P < 0.05 vs. sham animals of the same sex (two-way ANOVA and Fischer test).

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Experimental details: Fig 2 Left atrial expression of Ca 2+ handling proteins. (A) Original Western blots of Ca 2+ handling proteins from Sham and AoB LA. Note the GAPDH blots for normalization. (B) Mean band intensity expressed relative to Sham as 100%. Data represent mean +- SEM from 6 Sham and 6 AoB hearts. *, P

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Experimental details: Fig. 2 Evaluation of the expression levels, phosphorylation status, and subcellular distribution of Ca 2+ -cycling proteins in 1-year-old homozygous female knock-in hearts. ( A ) Representative immunoblots and relative quantification of the expression levels of major Ca 2+ -handling proteins showed that the amounts of SERCA2 are significantly increased (~10%), whereas the amounts of pentameric (but not monomeric) PLN are significantly reduced (~15%) in knock-in compared to wild-type hearts; t test, * P < 0.05; error bars represent SEM; n = 3 animals per group, two replica blots per heart. HSP90alpha/beta served as loading and normalization control. ( B to G' ) Immunolabeling of freshly isolated cardiomyocytes from wild-type and knock-in hearts with antibodies to Ca 2+ -cycling proteins did not show any apparent changes in their localization; scale bar, 5 mum; note that all images from wild-type and knock-in animals were obtained using the same laser power and microscope settings. ( H ) Representative immunoblots and relative quantification of the phosphorylation levels of select Ca 2+ -cycling proteins using antibodies to phospho-Ser 2808 and phospho-Ser 2814 of RyR2, phospho-Ser 38 of SERCA2, and phospho-Ser 16 of PLN did not reveal any differences between wild-type and knock-in hearts. Phosphorylation levels were normalized to total levels for each protein; t test, P > 0.05; error bars represent SEM; n = 3 animals per group, two replica blots per heart. As in (A), HSP9

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Experimental details: Figure 3 Notch Inhibition Cooperates with AKT1 to Enhance Cardiac Reprogramming alphaMHC-GFP MEFs (or GCaMP MEFs in C) were infected with GHMT or AGHMT, and treated with DMSO (vehicle) or DAPT. (A) Representative immunostaining images of GFP, alpha-actinin, and cTnT at day 15 of reprogramming. Scale bar, 200 mum. (B) Quantification of cells positive for alpha-actinin and cTnT as determined by immunostaining; n = 3 biological replicates. (C) Quantification of Ca 2+ flux-positive cells in GCaMP MEFs at day 15; n = 3 biological replicates. (D) Percentage of beating cells, relative to the number of input cells; n = 3 biological replicates. (E) Immunoblot against the Ca 2+ handling proteins ryanodine receptor (RyR) and SERCA2 at day 15 of reprogramming. Densitometric quantification is shown as the average of every replicate, relative to GAPDH. Data are presented as mean +- SD. * p < 0.05, ** p < 0.01, *** p < 0.001.

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Experimental details: Fig 2 Neither contractile proteins nor excitation-contraction coupling proteins are altered in PH diaphragm muscles. A : Stain free images of myosin heavy chain (MyHC) and western blots of troponin (Tn) T. B : the expression levels of MyHC or TnT normalized to actin content. C : electrophoretically separated MyHC isoforms. D : percentage distribution of MyHC isoforms: I, slow myosin isoform; IIa, IId/x, and IIb, fast myosin isoforms. E : representative western blots of ryanodine receptor (RyR1), dihydropyridine receptor alpha2 subunit (DHPR), sarcoplasmic reticulum Ca 2+ -ATPase (SERCA) 1, and SERCA2. F : the expression levels of RyR1, DHPR, SERCA1, or SERCA2 normalized to actin content. Control (CNT), white bars; AIA, black bars. Data represent mean +- SD for 4-8 rats in each group.

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Experimental details: Figure 6 Expression of Ca 2+ handling proteins in hearts from sham and OVX mice. A . Detection of ~240 kDa bands corresponding to Ca V 1.2 in ventricular tissue from sham and OVX mice. Na/K ATPase (110 kDa band) was used as a loading control (15 ug of protein were loaded in each lane). Average Ca V 1.2 band intensity was lower in OVX ventricular tissue compared to sham controls (n=3 hearts/group). B . Representative immunoblot illustrating ~116 kDa bands corresponding to NCX in sham and OVX ventricle. The loading control was Na-K ATPase as in A. Mean normalized NCX band intensity was similar in ventricular tissue from sham and OVX mice (n=3 hearts in each group). C . Detection of ~110 kDa bands corresponding to SERCA2 in the ventricles of sham and OVX mice. Amido black was used as a loading control (60 ug of protein were loaded in each lane). Average intensity of the SERCA2 bands was similar in sham and OVX ventricles (n=3 hearts in each group). In experiments where Na-K ATPase was used as a loading control, there was no significant difference in Na-K ATPase protein levels between sham and OVX (t-test, p=0.164) (*denotes p

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Experimental details: Figure 4 Effect of cecal ligation and puncture and treatment with Pep2.5 on SERCA2 expression in murine heart tissue. After CLP or sham 2-month-old male NMRI mice were treated with Pep2.5 (2.0 mug/h in saline 0.9%) or vehicle (100 mul/h saline 0.9%). ( a ) Representative immunofluorescence images of heart with antibodies reactive to sarcoplasmic reticulum Ca 2+ -ATP-ase (SERCA2) (green). ( b ) Quantification of SERCA2 expression in heart tissue was assessed 24 subsequent to sham or CLP by immunofluorescence and shown as relative optical density (OD); ( c ) Representative immunoblot of SERCA2 (bottom) and vinculin (top). OD of SERCA2 bands corrected for the corresponding vinculin bands and normalized using the related sham band; ( d ) Relative mRNA expression of SERCA2 corrected for S7 expression and normalized to sham. The following groups were studied: sham + vehicle (n = 8); CLP + vehicle (n = 8); CLP + Pep2.5 (n = 8). Data are expressed as means +- SD for n number of observations. * P < 0.05 vs. CLP + vehicle (Kruskall-Wallis test with Dunn's multiple comparisons test).

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Experimental details: Figure 8 Effect of treatment with Pep2.5 on SERCA2 expression in cardiomyocytes exposed to serum from septic shock patients. Cardiomyocytes (HL-1 cells) were exposed to medium (unstimulated) or serum from septic shock patients (SsP; 5%) in medium and treated with Pep2.5 (20 mug/ml in medium) or vehicle (medium) for 24 h. ( a ) Representative immunofluorescence images of cardiomyocytes stained against sarcoplasmic reticulum Ca2+-ATP-ase (SERCA2) (green), nucleus (blue) and cytoskeletal F-actin (red). ( b ) Quantification of SERCA2 expression in cardiomyocytes was assessed by immunofluorescence and shown as relative optical density (OD); ( c ) Representative immunoblot of SERCA2 (bottom) and vinculin (top). OD of SERCA2 bands corrected for the corresponding vinculin bands and normalized using the related sham band; ( d ) Relative mRNA expression of SERCA2 corrected for S7 expression and normalized to sham. The following groups were studied: unstimulated cells; SsP + vehicle; SsP + Pep2.5. All experiments have been performed in triplicates. Data are expressed as means +- SD for five independent experiments performed in triplicates. *P < 0.0001 vs. SsP + vehicle (Kruskall-Wallis test with Dunn's multiple comparisons test).

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Experimental details: Fig 10 Expression of Ca 2+ handling proteins. Immunoblots and densitometry analysis of total cardiac protein extracts from wildtype (WT) and Tnnt2 R141W/+ hearts at ages 2 and 8 weeks. CASQ2, calsequestrin 2; NCX1, sodium/calcium exchanger; P-PLN, Ser 16 phosphorylated phospholamban; SERCA2, sarcoplasmic reticulum cardiac Ca 2+ ATPase 2; T-PLN, total phospholamban. *, P

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Experimental details: Fig. 1 Isolated adult murine cardiomyocytes (aCMs) cultured on Geltrex and treated with blebbistatin show sustained viability and retention of functional protein expression patterns up to 7 days post-isolation. a Confocal analysis of aCMs of alpha-actinin and RyR2, DHPRalpha, SERCA2a, and STIM1 (green) up to one week in culture. Nuclei were visualized with Hoechst 33342 (blue). Scale bars equal 20 um. b Immunoblots for Akt, pAkt, and alpha-tubulin of aCM lysates after 24 h culture on Geltrex-coated surfaces ( N = 3, p = 0.023). c Cell counting revealed the survival of viable rod-shaped (red) relative to rounded aCMs (blue) ( N = 3). All data expressed as mean +- SEM; N denotes biological replicates; significance indicated by *( p < 0.05).

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Experimental details: Fig. 5 Super-resolution (dSTORM) microscopy reveals that aCMs are sensitive to chemical perturbations in vitro. a dSTORM imaging of aCM to visualize alpha-actinin, RyR2, DHPR, SERCA2a, and PLN in the absence or presence of tunicamycin-induced protein folding stress (5 umol L -1 for 24 h vs. a DMSO sham). Scale bars represent 10 um, magnified inset images are 4 x 4 um. b Voronoi tessellation cluster area and density analysis of dSTORM-imaged aCMs ( N = 3) for alpha-actinin ( b ), RyR2 ( c ), DHPR ( d ), SERCA2A ( e ), and PLN ( f ).

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Experimental details: Figure 4 Protein phosphorylation levels in heart failure (HF) left ventricles. Exercise did not decrease phosphorylation at sites that are known to be involved in destabilization of RyR2. Phosphoprotein levels were normalized to total protein levels. RyR2 phosphorylated at (A) serine 2814 (p-RyR2 ser 2814) and (B) serine 2808 (p-RyR2 ser 2808) were normalized to (C) total RyR2. Plb phosphorylated at (D) threonine 17 (p-Plb thr 17) and (E) serine 16 (p-Plb ser 16) were normalized to (F) total Plb and CaMKII phosphorylated at (G) threonine 286 (p-CaMKII thr 286) was normalized to (H) total CaMKII. (I) SERCA2a level was unaltered by exercise training. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as loading control, except for Plb, and the membrane was reprobed with the specified antibodies with stripping of the membrane between each antibody, shown at 37 kDa. (C,F,H,I) Total protein levels were normalized to GAPDH levels. Number of rats (SED/ET): 6/5.

Supportive validation

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Experimental details: 1 FIGURE FRET biosensor expression and localization in adult rat ventricular myocytes (ARVMs). Schematic representation of the (a) Epac2-alphaKAP and (c) Epac2-camps biosensors and representative confocal images of myocytes expressing each probe. Representative super-resolution images of fixed ARVMs expressing (b) Epac2-alphaKAP and (d) Epac2-camps ( n / N = 12/3 for each probe). Left hand panels are of each probe labelled with a GFP antibody. Centre panels are the same cells co-labelled with SERCA2 antibody. Right hand panels represent the merged images, with the ROI magnified 4x (insets). White scale bar: 3.0 mum. See Section 2.4 for details

Supportive validation

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Experimental details: Figure 4 Western blot analysis showing expression Ca 2+ handling proteins ( A ) SERCA -2A, ( B ) p- PLN ; ( C ) NCX . Representative Western blot shown above and quantification below. Data were obtained from 3-month-old mouse hearts (n=6 mice/group) that were born to preconception filtered air or particulate matter (

Supportive validation

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Experimental details: Figure 1 HiPSC line derivation and validation A Pedigree of the patient family. Arrow: Index patient; index patient and mother are PLN p.Arg14del mutation carriers and were diagnosed with DCM. B Sequencing of PLN patient-derived hiPSC (PLN p.Arg14del) and isogenic controls (PLNic) to confirm CRISPR/Cas9-mediated gene correction. Red box indicates codon for Arg14, and blue box indicates silent PAM (protospacer adjacent motif) mutation. C, D Relative PLN transcript level (normalized to ACTN2 and PLNic wt+mt) with PCR primers amplifying PLN wild type (wt) and p.Arg14del (mt), only wt or only p.Arg14del (mt). (C) Analysis of PLNic ( n = 9 EHTs from 5 batches, each replicate consists of a pool of 1-2 EHTs from separate batches) and PLN p.Arg14del ( n = 9 EHTs from 3 batches, each replicate consists of a pool of 2-4 EHTs from separate batches) EHTs, mean +- SEM. (D) Analysis of PLN p.Arg14del EHTs ( n = 9 EHTs from 3 batches, each replicate consists of a pool of 2-4 EHTs from separate batches) and two independent failing heart patient samples from heterozygous PLN p.Arg14del mutation carriers (each n = 1 RNA sample, 2 separate PCR analysis), mean +- SEM. E Immunofluorescence of 2D hiPSC-CM from each genotype with antibodies against PLN (red) and SERCA2 (green) and DAPI staining of nuclei (blue); scale bar 20 um.

Supportive validation

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Experimental details: Figure 7 SERCA2a calcium pump gene therapy reverses hiPSC-CM heart failure phenotype. ( A ) Viral transduction was verified by mCherry live-cell expression in hiPSC-CM monolayers. Further, SERCA2a protein expression was determined by Western Blotting. Quantification indicates that SERCA2a protein expression was reduced in metabolic stress-treated hiPSC-CMs at baseline (MACS = 2.11 +- 0.22, n = 3 vs. metabolic stress = 1.27 +- 0.06, n = 3). AdSERCA2a treatment restored SERCA2a levels to control levels (metabolic stress plus Ad SERCA2a = 2.15 +- 0.17, n = 3; ANOVA, * P < 0.05). ( B ) Calcium transient measurements indicate that metabolic stress selection media prolongs the cardiac action potential and spontaneous arrhythmias were observed in the metabolic stress selection purification approach. hiPSC-CM monolayers treated with AdSERCA2a did not develop arrhythmias. ( C ) CaTD50 was prolonged in metabolic stress media-treated cells at baseline and was corrected to control values with AdSERCA2a gene therapy (CaTD50s: MACS baseline = 475.5 +- 18.1 ms; metabolic stress baseline = 577.37 +- 17.7 ms; MACS plus AdSERCA2a = 396.6 +- 11.2 ms; metabolic stress plus Ad SERCA2a = 493.67 +- 19.9 ms; ANOVA, ** P = 0.002, ns). CaTD, calcium transient duration; hiPSC-CMs, human induced pluripotent stem cell-derived cardiomyocytes; MACS, magnetic-activated cell sorting.

Supportive validation

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Experimental details: Fig. 6 Western blot of EC-coupling proteins in human-induced pluripotent stem cell cardiomyocytes (iPSC-CM). ( a ) Representative original Western blots after treatment with empagliflozin (EMPA) or vehicle control (control) for 2 or 8 weeks from 4 differentiation experiments (Diff.) from 2 healthy donors. GAPDH was used as loading control. ( b ) Mean protein expression levels (normalized to the respective control group at 2 or 8 weeks) in iPSC-CM ( n = 4 differentiations, matched groups are displayed with matched individual symbols) and effects of 2 and 8 weeks treatment with empagliflozin (EMPA) on ryanodine-receptor type 2 (RyR2), ( c ) sodium-calcium exchanger (NCX), ( d ) sarcoplasmic reticulum Ca 2+ ATPase (SERCA), ( e ) phosphorylated Ca 2+ -/calmodulin-dependent protein kinase II (pCaMKII), ( f ) Ca 2+ -/calmodulin-dependent protein kinase II (CaMKII), and ( g ) phospholamban (PLB). Groups were statistically analyzed using two-way ANOVA with Sidak's test for multiple comparisons

Supportive validation

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Experimental details: Figure 4 Cardiac myocyte Ca 2+ -handling proteins in mouse mammary tumor virus-polyomavirus middle T antigen (MMTV-PyMT)- and MMTV-PyMT+ mice--phospholamban ( A ); SERCA2a (sarcoplasmic or endoplasmic reticulum calcium 2 ATPase) ( B ); phosphorilated phospholamban (p-phospholamban) ( C ); sodium/calcium exchanger (NCX) ( D ); and SERCA2a/phospholambam ratio ( E ). Western blot bands are shown for each protein. In the MMTV-PyMT+ mice, phospholamban protein expression was increased ( P =0.011) and SERCA2a protein expression was not different between the MMTV-PyMT+ and MMTV-PyMT- groups ( P =0.165). SERCA2a/phospholamban ratio was reduced in the MMTV-PyMT+ mice ( P =0.007). p-Phospholamban and NCX protein expression were decreased ( P =0.038 and P =0.017, respectively) compared with MMTV-PyMT- mice. Mann-Whitney test. Values are expressed as medians and interquartile ranges (IQRs). Animals per group: 7 MMTV-PyMT- and 7 MMTV-PyMT+.

Supportive validation

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Experimental details: Fig. 4 Repeated subcutaneous injection of PLN ASO in rats with a myocardial infarction improves improves contractility and reduces cardiac dimensions. a Experimental design of the rat post myocardial infarction intervention study. Six weeks after myocardial infarction, induced by permanent left anterior descending (LAD) artery ligation, an MRI was performed, and rats were randomized into groups. Treatment of ASO#136 was initiated at a 2x weekly dosing for the first 2 weeks followed by 1x weekly dosing (sham n = 5, PBS n = 7, Control-ASO n = 8 (50 mg/kg), PLN-ASO low dose n = 8 (25 mg/kg), and PLN-ASO high-dose n = 7 (50 mg/kg)). MRI, invasive hemodynamics and sacrifice was performed 5 weeks after treatment initiation, 11 weeks after myocardial infarction. b Western blot results of LV protein lysates stained for PLN and SERCA2 protein and semi-quantified relative to PBS treated control samples, intensities were normalized to GAPDH ( n = 5 for sham, n = 7 for PBS and PLN-ASO 50 mg/kg, and n = 8 for control ASO and PLN-ASO 25 mg/kg). Representative image of 1 out of 4 membrane stains (Supplementary Fig. 20 ). Ratio of SERCA2 to PLN is shown as fold change relative to PBS. c Individual MRI assessment and quantification of the change in left ventricular ejection fraction (LVEF), left ventricular end-systolic volume (LVESV), and left ventricular end-diastolic volume (LVEDV) between treatment initiation (6 weeks post MI) and 5 weeks after start of treatment showing ( n = 5 for sham,

Supportive validation

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Experimental details: Fig. 4 Immunoblot with pre- and post-test expression of SERCA1 (~ 110 kDa), SERCA2 (~ 110 kDa) and GAPDH (~ 37 kDa) in muscle biopsies from participants 1-8 in A maximal strength training group and B control group

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Experimental details: FIGURE 5 Endogenous L-type Ca 2+ currents and Ca 2+ transients measured at different pacing frequencies in ARCs expressing Ca v beta 2b -SH3. (A) Western blot analysis of the expression of Ca v beta 2 , Ca v 1.2, RyR2, SERCA2a, and GAPDH in ARCs after Ca v beta 2b -SH3 transduction (i) and bar plot of mean values of densitometry analyses from western blots (ii) . The expression of construct Ca v beta 2b -SH3, which contains a V5-tag at the C-terminus, was detected using an anti-V5 antibody. The protein expressions in the right panel were normalized vs GAPDH. (B) Voltage step protocol and representative whole-cell endogenous L-type Ca 2+ current traces of control (black) and Ca v beta 2b -SH3-expressing (red) cardiomyocytes. (C) Current-voltage curves of L-type Ca 2+ currents of control (black) and Ca v beta 2b -SH3-expressing (red) cardiomyocytes. (D) Scatter plot showing peak L-type Ca 2+ currents at 0 mV in control (black) and Ca v beta 2b -SH3-expressing (red) cardiomyocytes. (E) Representative fluorescence traces of Ca 2+ transients in control (black) and Ca v beta 2b -SH3-expressing (red) cardiomyocytes stimulated at pacing frequencies of 0.5, 1.0, 2.0, 3.0, and 4.0 Hz. (F) Diastolic (baseline) Ca 2+ concentration at the different pacing frequencies. (G) Ca 2+ transient amplitudes after baseline subtraction at the different pacing frequencies. (H) Scatter plot showing the Ca 2+ transient amplitudes for each cell after baseline subtraction at the different pacing frequenc

Supportive validation

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Experimental details: 1 Fig. NNAT promotes uncoupling of SERCA2a in the presence of a Ca 2+ gradient. (A) Representative Western blot images of NNAT and SERCA2a in HEK co-transfected cells. Ponceau staining was used as a loading control. (B) NNAT and (C) SERCA2a protein content in 1 : 0, 1 : 1, and 1 : 5 SERCA : NNAT co-transfected cells. (D) Change in [Ca 2+ ] free over time after Ca 2+ uptake was initiated with ATP without oxalate in the buffer in 1 : 0, 1 : 1, and 1 : 5 SERCA : NNAT co-transfected cells. (E) Rates of Ca 2+ uptake and (F) SERCA ATP hydrolysis (without ionophore) analyzed at a [Ca 2+ ] free of 1500 n m . (G) The calculated apparent coupling ratio (Ca 2+ uptake/SERCA activity) in 1 : 0, 1 : 1, and 1 : 5 SERCA : NNAT co-transfected cells. Data are means +- SEM; unpaired Student's t -test (B); one-way ANOVA (C) followed by a Tukey's post-hoc (E-G). * P < 0.05; ** P < 0.01; *** P < 0.005 ( n = 4-6 per group, technical replicates from passages 8-12). Values above bars indicate P value.

Supportive validation

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Experimental details: 2 FIGURE Sarco(endo)plasmic reticulum Ca 2+ ATPase (SERCA) function is impaired and SERCA2a T-nitration is increased in soleus muscles of SOD2 +/- mice. (a) SERCA activity- p Ca curves in wild-type (WT) and SOD2 +/- mice over Ca 2+ concentrations ranging from p Ca 6.83-6.10, presented as % of V max . p Ca 50 values are embedded in the graph with 95% confidence intervals (CIs). (b) No differences in maximal SERCA activity ( mu mol/g of protein/min) were observed between genotypes. (c) Densitometric analysis and representative images of Western blots for SERCA2a, SERCA1a, and sarcolipin protein content as well as analyses and representative images of SERCA-specific T-nitration (d). All values are mean +- SEM and are presented relative to WT. * p < 0.05 ** p < 0.01, values above bars indicate p values using Student's t -test ( n = 5-8 per group).

Supportive validation

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Experimental details: Figure 2. Nuclear envelope (NE) invaginations and localization of Ca 2&plus -regulating proteins in nonfailing and failing cardiomyocytes. A , Transmission electron micrograph of a nucleus from human left ventricular trabeculae ( left ), zoomed in on the NE invaginations ( middle and right ) and nuclear pore complexes (NPC; red arrows and inset). B , Original 2-dimensional images of cardiomyocytes isolated from sham-operated mice and mice subjected to transverse aortic constriction (TAC) 7 weeks after the intervention ( left ) and nonfailing and failing human cardiomyocytes ( right ) after immunostaining for NPC ( top ), inositol 1,4,5-trisphosphate receptor (IP 3 R; middle ), ryanodine receptor (RyR; middle ) and sarcoplasmic reticulum Ca 2&plus -ATPase 2a (SERCA2a; bottom ). Scale bars, 5 &mgrm. Transmission electron micrograph of control mouse myocardium stained for ( C ) RyR, NE and its surroundings ( top and middle ), and T-tubules (TT) and their surroundings ( bottom ); ( D ) IP 3 R, zoomed in on NE ( top ) and perinuclear region ( bottom ); and ( E ) SERCA2a, zoomed in on NE ( top and middle ), distribution between inner and outer nuclear membrane (n=11; bottom , left ), and expression level on NE in sham- vs TAC-operated mouse myocardium (n=6; bottom , right ). &ast P

Supportive validation

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Experimental details: Phospholamban is reduced in the soleus muscles of Ng +/- mice. Representative Western blots and their corresponding analyses for SERCA2a, SERCA1a, RYR, SLN and NNAT in the soleus of WT and Ng +/- mice (A) . Only reductions in SERCA1a content were observed, though this did not reach statistical significance. Characterizing PLN and its activation status showed reductions in monomeric PLN resulting in reductions in the monomer:pentamer ratio and statistically insignificant increases in the p-PLN : PLN ratio (B) . Molecular weight markers are included in the representative blots. * p < 0.05, values above bars indicate p values (n=4-5 per group).

Supportive validation

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Experimental details: Figure 5 SERCA content is reduced in iWAT and BAT from Ng +/- mice. Representative Western blots and corresponding analyses of SERCA2a, SERCA1a, and UCP1 from iWAT (A) and BAT (B) . In both depots, reductions in both SERCA isoforms were observed with no changes in UCP1. * p < 0.05, values above bars indicate p values (n=9-10 per group).

Supportive validation

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Experimental details: Figure 3 SERCA2a expression and tyrosine nitration in young (3-5 month) and old (10-12 month) TazKD mice compared with WT. (A) SERCA2a total protein levels determined via Western blotting was unaltered between genotypes. (B) SERCA2a tyrosine nitration was significantly elevated in LV obtained from TazKD mice, determined via Western blotting using co-IP eluent. (C) Correlational analyses between absolute SERCA2a tyrosine nitration levels and maximal SERCA activity. For (B), a significant main effect of genotype was detected using a two-way ANOVA ( n = 4-6 per group) and is depicted with an asterisk across the bar legends; and for (C), a significant negative correlation was detected using Pearson's correlational analyses, * P < 0.05

Supportive validation

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Experimental details: 8 Figure Immunoblots of major Ca 2+ -handling proteins in HFpEF A and B , representative western blots from ventricular cardiomyocytes (left) and pooled data (right) showing mean density relative to GAPDH for major Ca 2+ -handling proteins (Ca v 1.2, SERCA2, NCX and phospholamban (PLN) and assessment of phosphorylation at known sites (pSer1928 Ca v 1.2, pThr17-PLN and pSer16-PLN). C , immunoprecipitation studies of RyR2 and pSer2808 RyR2 showing significant increase in phosphorylation of RyR2 relative to expression of RyR2. Data were normalized to RyR2 in the immunoprecipitated sample. Data expressed as means + SD. Statistics are calculated as Mann-Whitney rank test. [Color figure can be viewed at wileyonlinelibrary.com ]