Supplementary Figures Supplementary Figure 1. Development of the camp biosensor targeted to the SERCA2a microdomain.
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1 Supplementary Figures Supplementary Figure 1. Development of the camp biosensor targeted to the SERCA2a microdomain. A B C (A) Schematic representation of the new constructs designed for local camp imaging. Epac1-camps sequences (comprised of YFP, Epac1 camp binding domain and CFP) was fused to a full length or N-terminally truncated phospholamban (PLN, 1-18). Furthermore, a mutant Epac1-PLN (R279E) sensor insensitive to camp was developed. To study possible involvement of intramolecular FRET into Epac1-PLN responses, CFP or YFP sequences in the sensor construct were mutated to generate dark YFP (dyfp, contains the Y66G point mutation in the YFP sequence) and dark CFP (dcfp, contains the W66G point mutation in the CFP sequence) sensor constructs. (B) FRET responses of the sensors to camp stimulation with isoproterenol (ISO) in transiently transfected 293A cells show higher signal amplitude for the full-length PLN construct compared to PLN ( 1-18) and virtually abolished
2 signals for R279E and co-transfected dcfp/dyfp mutants. Quantification is in C, shown are means± SE, n= 8-12 cells each. * - significant difference, p=4x1-6 ; p=.57 by one-way ANOVA. D CFP channel YFP channel Overlay Epac1-PLN Epac1-PLN dyfp Epac1-PLN dcfp Epac1-PLN dcfp + Epac1-PLN dyfp (D) Representative (n>5) confocal images of 293A cells transfected with Epac1PLN, dyfp, dcfp or with the combination of the two mutant constructs. Fluorescence was measured in individual CFP (45 nm laser excitation) and YFP (514 nm laser excitation) channels. Scale bars, 5 µm. 2
3 Supplementary Figure 2. Characterization of Epac1-PLN transgenic mice. (A) Heart-weight-to-body-weight (HW/BW) and heart-weight-to-tibia-length (HW/TL) ratios at the age of 3 and 6 months (means ± SE, n= 1-11 each) do not show any abnormalities in the animals. (B) Representative (n>5) hematoxylin and eosin stainings of heart cross sections at the age of 3 and 6 months. Scale bars, 1 mm. (C) Diameter of transgenic () Epac1-PLN cardiomyocytes was not altered compared to wildtype () cells at the age of 3 and 6 months as investigated by WGA assay. Data are means ± SE from 3-5 and 3
4 hearts with 1 cells counted per each section. Scale bars, 5 µm. All p-values were >.3, by one-way ANOVA. Supplementary Figure 3. Epac1-PLN is localized in the membrane fraction of transgenic mouse hearts. kda Epac1-PLN Monomer (PLN antibody) 7 Epac1-PLN Monomer (GFP antibody) 7 SERCA2a 1 GAPDH 35 cytosol membrane Immunoblot analysis of cytosolic and membrane fractions of wildtype () and transgenic () hearts show that the Epac1-PLN sensor is localized in the membrane fraction of hearts together with SERCA2a. Representative immunoblots (n=4). 4
5 2+ (ms) Ca % Ratio change SERCA2a/GAPDH PLN/SERCA2a PLN/GAPDH (normalized to ) Supplementary Figure 4. PLN and SERCA2a expression and calcium cycling. A * B kda 2 SERCA2a 1 GAPDH 35 1 Sensor PLN 25 Endogenous PLN 25 C 1. * D E F G /2 38/2 51/2 28/2 NT ISO 1nM F 34 /F Caffeine, 1 mm Time (s) Peak Amplitude 16/2 19/2 Caffeine 1 mm (A) Quantitative real time PCR analysis showed a ~2-fold overexpression of PLN in Epac1- PLN hearts (means ± SE, n=4 hearts each, p=2x1-16 by one-way ANOVA). (B-D) Immunoblot analysis revealed an increase in SERCA2a protein expression (p=.2) but unchanged PLN/SERCA2a ratio (p=.93 by one-way ANOVA). Epac1-PLN and endogenous PLN were detected with the same total PLN antibody. (E) Quantification of Ca 2+ transients shows no dramatic changes in Ca 2+ -decay time constant (τ 2+ Ca ) in cardiomyocytes at basal (p=.28) and ISO (1 nm for 5 min, p=.1 by one-way ANOVA) stimulated states (means ± SE, n=28-56 cells from 2 hearts each). (F) Analysis of SR-calcium load by the addition of caffeine to beating cardiomyocytes demonstrates no changes in compared to cells (means ± SE, n=18-19 cells from 2 hearts each, p=.43 by one-way ANOVA). 5
6 P Ser16 -PLN/PLN Supplementary Figure 5. Transgenic Epac1-PLN construct can form oligomers and be phosphorylated by the PKA. A C 35 B Sensor oligomers kda P Ser16 -PLN PLN C * Endogenous PLN P Ser16 -PLN PLN.4 * 1 nm ISO nm ISO (A) Immunoblot analysis of cardiomyocytes shows the ability of Epac1-PLN (monomers at 72 kda) to form oligomers, similar to endogenous PLN. (B) Immunoblot analysis of PLN Ser16 phosphorylation confirms PKA-dependent phosphorylation of Epac1-PLN upon stimulation with 1 nm of ISO, similar to endogenous PLN (n=3 mice each, means ± SE, * - significant differences, p=.2 and.1, respectively; not significant, p=.13 by one-way ANOVA). 6
7 Copy number (normalized) Fold change in expression (/) Copy number (normalized) Fold change in expression (/) Copy number (normalized) Fold change in expression (/) Supplementary Figure 6. Expression levels of the main components of the camp signaling pathway in cardiomyocytes isolated from and hearts. A Adrb1 Adrb2 Gnas Gnai2 Gnai3 Gnai1 Adrb1 Adrb2 Gnas Gnai2 Gnai3 Gnai1 B Adcy3 Adcy4 Adcy5 Adcy6 Adcy7 Adcy9 Adcy3 Adcy4 Adcy5 Adcy6 Adcy7 Adcy9 C Prkar1a Prkar2a Prkab2 Prkaca Prkacb Prkar1a Prkar2a Prkab2 Prkaca Prkacb A-E, Gene array data for -ARs, G-proteins (both in A) adenylyl cyclases (B), PKAs (C), its anchoring proteins (D), and PDEs (E). Data for further PDEs and Epac2 mrna (Rapgef4) are presented in the Supplementary Table 3. We could not measure the expression of Epac1 mrna since the Epac1-PLN sensor transcript led to a significant increase in Epac1 copy numbers. F, Heatmaps of all endogenous transcripts with >2-fold changed expression 7
8 Copy number (normalized) Fold change in expression (/) Copy number (normalized) Fold change in expression (/) between the genotypes (only the total of 9 genes). n=5 and 5 hearts, numbered 1-5 at the bottom. p>.5 by one-way ANOVA, except for the genes shown in F. PDE11A has previously not been characterized as a functionally-relevant cardiac PDE and was barely expressed compared to other classical camp-pdes (E). Copy numbers for all 9 transcripts are presented in the Supplementary Table 4. D Akap1 Akap5 Akap6 Akap7 Akap9 Akap13 Akap1 Akap5 Akap6 Akap7 Akap9Akap13 E Pde2a Pde3a Pde4a Pde4b Pde4d Pde11a Pde2a Pde3a Pde4a Pde4b Pde4dPde11a F Log2 Fold Tmem Cntn3 Cdh Kcnq M8Rik 1.9 Dusp Pde11a Plekhh Emilin
9 PDE2A/GAPDH PDE4D8/GAPDH PKA RII /GAPDH PKA RI/GAPDH Supplementary Figure 7. Protein levels of some important camp signaling pathway components in cardiomyocytes isolated from and hearts. A kda 1.6 PKA RI GAPDH B 1.2 PKA RII α kda GAPDH C PDE2A kda 1 D PDE4D8 kda 1 GAPDH 35 GAPDH Immunoblot analysis using available antibodies could further confirm unchanged protein levels of PKA type I and II regulatory subunits (A and B), PDE2A (C) and PDE4D8 (D). The latter PDE has been shown to interact with the β 1 -AR and locally confine the signaling of this receptor to camp 1., not significant (p-values were.43,.95,.56,.31 for A, B, C and D, respectively, by one-way ANOVA), n=4 and 4 hearts. 9
10 FRET response (% max) ISO cytosol ISO SERCA2a FRET response (% max) Ratio CFP/YFP (norm.) Ratio CFP/YFP (norm.) Supplemental Figure 8. Sensitivity of Epac1-camps and Epac1-PLN to camp and increasing isoproterenol (ISO) concentrations. A Epac1-camps 1 µm B Epac1-PLN 1 µm µm 1 µm Time (s) 1 µm Epac µm.1 µm 1 µm Time (s) C Epac1-camps Epac1-PLN basal cytosol basal SERCA2a D Epac1-camps Epac1-PLN Br-2'-O-Me-cAMP-AM (µm) ISO (nm) A-C, Representative titration experiments (A, B) and concentration-response dependencies (C) for the membrane-permeable camp analogue 8-Br-2 -O-Me-cAMP-AM (having the same affinity for Epac1 as camp) measured with Epac1-camps (EC 5 = 1.2 ±.5 μm, Hill coefficient 1. ±.3) and Epac1-PLN (EC 5 = 4.4 ± 2.1 μm, Hill coefficient.78 ±.8) transgenic cardiomyocytes (n=6-8 cells from 2-3 mice each). Dotted lines indicate conversion of the averaged FRET response values into absolute camp concentrations measured with both sensors in living cells. The y-values were calculated from the % max FRET response shown in Figure 2E added to the basal FRET levels (54. % max for Epac1-camps and 38. % max for Epac1-PLN) derived from the responses to the adenylyl cyclase inhibitor MDL12,33A. D, Stimulation of Epac1-camps or Epac1-PLN cells with increasing ISO concentrations reveals enhanced sensitivity of Epac1-PLN to ISO (n=6 cells from 2-3 mice each). 1
11 Change in FRET (% max) Ratio CFP/YFP (norm.) Ratio CFP/YFP (norm.) Supplementary Figure 9. The difference between Epac1-camps and Epac1-PLN in the magnitude of β-ar-camp signals receptor is abolished after combined PDE preinhibition. A Epac1-camps B Epac1-PLN IBMX ISO Forskolin 1.1 IBMX Forskolin ISO Time (s) Time (s) C Epac1-camps Epac1-PLN * 14/ * 16/ /3 13/ / /3 4 2 ISO after Cilo ISO after Roli ISO after Cilo+Roli Representative FRET traces from Epac1-camps (A) and Epac1-PLN (B) expressing cardiomyocytes stimulated with the unselective PDE inhibitor IBMX (1 μm), and subsequently with a submaximal dose of isoproterenol for β-ar stimulation (ISO, 1 nm). Forskolin (1 μm) was used to achieve a complete adenylyl cyclase activation at the end of each experiment. See Figure 2F for quantification. C, Combined inhibition of PDE3 and PDE4 by 1 µm cilostamide (Cilo) and 1 µm rolipram (Roli) but not of any PDE alone abolished the difference between Epac1-camps and Epac1-PLN FRET response to 1 nm 11
12 Intensity (a.u.) ISO (n=12-16 cells from 3-4 mice each; p-values were 2x1-6,.4 and.28 for the graphs shown in C. Supplementary Figure 1. Epac1-PLN localization is not altered in TAC mice. A B 12 Epac1- PLN SERCA2a Distance (µm) A, Confocal image of a representative (n>5) immunostained transgenic Epac1-PLN heart cross section 8 weeks after TAC surgery. Epac1-PLN and Serca2a show proper colocalization as demonstrated by the fluorescence intensity overlay (B). 12
13 Supplementary Figure 11. Uncropped immunoblots shown in Fig. 4F. Cardiac PLN runs and can be detected by Badrilla antibody at this correct size. kda P-PLN kda 15 PLN 1 13
14 PDE4D/CSQ PDE2/CSQ camp-pde activity (pmol/min/mg) Supplementary Figure 12. Whole-cell PDE activity and PDE protein levels are not significantly altered in TAC vs. sham mice. A B 5 4 Sham TAC PDE4D kda CSQ 55 1 PDE2 PDE3 PDE4 total PDE2A CSQ Sham TAC. Sham TAC A, camp-pde activities measured in cardiomyocyte lysates using in vitro assays were not significantly different between the TAC and sham cells (n=3 mice per group, p>.6 by oneway ANOVA). B, Immunoblot analysis of PDE4D and PDE2A protein levels in cardiomyocytes from sham vs. TAC mice shows no significant alterations of PDE4D and PDE2A protein levels (n=3 hearts per group, not significant, p=.8 and.4, respectively, by one-way ANOVA). 14
15 F 34 /F 38 SERCA2a/CSQ Supplementary Figure 13. No significant difference in SERCA2a expression and calcium transient decay in TAC cells. A 1.6 Sham TAC p=.2 SERCA2a CSQ kda B Sham TAC Ca 2+ (ms) /5 Sham TAC p=.7 58/ Time (s) 18 A, Immunoblot analysis of SERCA2a protein levels in cardiomyocytes shows a tendency (not significant) towards lower SERCA2a protein amounts in TAC cells (n=3 mice per group). B, Likewise, calcium transient measurements revealed only a tendency for a lowered diastolic calcium reuptake (means ± SE). Numbers of cells/hearts as well as p-values (by one-way ANOVA) are indicated above the bars. 15
16 YFP/CFP ratio YFP/CFP ratio Supplementary Figure 14. Non-normalized FRET ratios for Epac1-camps and Epac1- PLN sensors. A.6.5 Cardiomyocytes Epac1-camps n=28/9 Epac1-PLN n=32/11 p= basal fully stimulated B A cells Epac1-camps n=34 Epac1-PLN n= basal fully stimulated A, Data from Epac1-camps and Epac1-PLN transgenic cardiomyocytes either at basal or after full stimulation with forskolin (1 µm) plus IBMX (1 µm). B, Ratios measured in 293A cells transfected with either Epac1-camps or Epac1-PLN under the same conditions. Shown are means ± SE. Numbers of cardiomyocytes/hearts is indicated in the figure, for 293A cells n=34 for Epac1-camps and n=31 for Epac1-PLN, respectively., not significant (p>.1) by one-way ANOVA. 16
17 Supplementary Table 1. Echocardiographic phenotyping of the wildtype vs. Epac1- Genotype PLN transgenic mice at 3 months of age Parameter Wildtype Transgenic LV-EDD (mm) LV-ESD (mm) Septum (mm) FS (%) FAS (%) EF (%) HR (bpm) N * * * * * Shown are means ± SE, * - significant differences at p<.5 by one-way ANOVA. LV-EDD, left ventricular end-diastolic dimension; LV-ESD, left ventricular end-systolic dimension; FS, fractional shortening; FAS, fractional area shortening; EF, ejection fraction; HR, heart rate; bpm, beats per minute; n, number of mice analyzed per group. Heart rates were not significantly different between the two groups at p=.5 by one-way ANOVA.
18 Supplementary Table 2. Echocardiographic phenotyping of wildtype, Epac1-PLN and Epac1-camps transgenic mice 8 weeks after TAC vs Sham surgery Wildtype Epac1-PLN transgenic Epac1-camps transgenic Parameter Sham TAC Sham TAC Sham TAC Gradient (mm Hg) * * * FS (%) * * * FAS (%) * * EF (%) * * HW/BW (mg/g) * * * Ahd (mm) * * * LV-EDD (mm) * * HR (bpm) ** n Ahd, anterior wall thickness in diastole. HW/BW, calculated heat-to-body-weight ratio. All other parameters were as described in the legend to Supplementary Table 1. * - significant differences at p<.5, as comparted to the respective sham group by one-way ANOVA. ** - heart rates were significantly different compared to the sham or Epac1-camps sham groups by one-way ANOVA. No significant difference in contractile parameters or heart dimensions between the 3 genotypes at TAC could be found. 18
19 Copy number (normalized) Fold change Supplementary Table 3. Gene array data of transcript levels for Epac2 and additional PDEs Transcript Wildtype Transgenic (Transgenic/Wildtype) Rapgef4 66 ± ± ±. Pde1a 34 ± 4 3 ± 2.9 ±.1 Pde1b 14 ± 1 11 ± 2.8 ±.1 Pde1c 1186 ± ± 88.7 ±.1 Pde3b 9 ± ± ±.3 Pde4c 67 ± 8 65 ± 9 1. ±.1 Pde7a 631 ± ± ±.2 Pde7b 65 ± ± 67.9 ±.1 Pde8a 314 ± ± ±.1 Pde8b 5 ± 6 42 ± 3.8 ±.1 Pde1a 3 ± 2 33 ± ±. Pde ± ± ±. Shown are copy number and fold change (/) data of the transcripts for Epac2 (Rapgef4 gene) and additional PDEs not 19
20 included in Supplementary Figure 6E. Epac1 (Rapgef3) expression could not be analyzed since the Epac1-PLN sensor contains a sequence from Epac1, resulting in a significantly higher copy numbers in vs mrna. Shown are means ± SE. None of the transcripts showed significant differences between the genotypes. Supplementary Table 4. Gene array data of transcript levels for 9 endogenous transcripts with >2-fold changed expression between the genotypes Copy number (normalized) Transcript Wildtype Transgenic Tmem 56 Cntn3 Cdh22 Kcnq M8Rik Dusp26 Pde11a Plekhh1 Emilin2 31 ± 1.5 ±.3 26 ± 4 2 ± 2 16 ± 3 45 ± 5 63 ± ± ± ± 3 72 ± 5 74 ± 3 49 ± 6 42 ± 5 17 ± ± 4 1 ± ± 15 2
21 Shown are copy number (/) data for the transcripts displayed in the heatmap of Supplementary Figure 6F. Shown are means ± SE. 21
22 Supplementary Reference 1. Richter W, Day P, Agrawal R, Bruss MD, Granier S, Wang YL, et al. Signaling from beta1- and beta2-adrenergic receptors is defined by differential interactions with PDE4. Embo J 28, 27(2):
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