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www.sciencemag.org/content/351/6270/271/suppl/DC1
Supplementary Materials for
A peptide encoded by a transcript annotated as long noncoding RNA enhances SERCA activity in muscle
Benjamin R. Nelson, Catherine A. Makarewich, Douglas M. Anderson, Benjamin R. Winders, Constantine D. Troupes, Fenfen Wu, Austin L. Reese, John R. McAnally,
Xiongwen Chen, Ege T. Kavalali, Stephen C. Cannon, Steven R. Houser, Rhonda Bassel-Duby, Eric N. Olson*
*Corresponding author. E-mail: [email protected]
Published 15 January 2016, Science 351, 271 (2016) DOI: 10.1126/science.aad4076
This PDF file includes
Materials and Methods Figs. S1 to S17 Tables S1 to S4 References
1
Materials and Methods
Identification of Conserved Small Open Reading Frames
Total RNA was extracted from adult mouse heart tissue using Trizol (Invitrogen).
An RNA sequencing library was prepared using the TruSeq RNA Library Prep Kit
(Illumina) according to the manufacturer’s protocol. Reads were mapped to the UCSC
mm9 reference genome (23) using TopHat (24). Final transcripts were assembled using
Cufflinks and consolidated with the mm9 reference annotations (25). Novel transcripts
(i.e. those not existing in the reference annotation) were extracted and combined with
transcripts annotated as long non-coding RNA in the UCSC database. Sequence
alignments from fourteen mammalian species (mouse, rabbit, rat, human, chimpanzee,
rhesus monkey, shrew, dog, cat, horse, cow, armadillo, elephant and tenrec) were
extracted using the “Stitch Gene blocks” tool in Galaxy (26-28). PhyloCSF was used to
calculate conservation scores for potential open reading frames (ORFs) in three frames on
both strands (11). Only the highest scoring ORF for each transcript was reported.
Quantitative mRNA Measurement
Total RNA was extracted from adult mouse tissues using Trizol and reverse
transcribed using the SuperScript III First-Strand Synthesis System (Invitrogen) with a
1:1 mix of random hexamer and oligo-dT primers. Quantitative Polymerase Chain
Reaction (qPCR) was performed using 5’ nuclease assays on the StepOne Real-Time
PCR System (Life Technologies). The following oligonucleotides were ordered from
Integrated DNA Technologies to measure endogenous Dworf abundance:
Forward – 5’-TTCTTCTCCTGGTTGGATGG-3’
Reverse – 5’-TCTTCTAAATGGTGTCAGATTGAAGT-3’
Probe – 5’-TTTACATTGTCTTCTTCTAGAAAAGGAAGAAG-3’
Probes were labeled with 5’ 6-FAM, an internal ZEN quencher, and a 3’ Iowa Black
quencher and used in a 2:1 ratio with primers. Human cDNAs were quantified using
SYBR Green with the following primers:
Forward 5’-CCACCCACCAACAGGAATA-3’
Reverse 5’-TTATGATGCAGCCCACAATC-3’
2
Each sample was normalized to a Eukaryotic 18S rRNA Endogenous Control (Life
Technologies) reaction.
Northern Blot Analysis
A Dworf cDNA fragment was PCR purified from heart RNA extract with the
following primers:
Forward – 5’-TTTCCAAAAGATAGGAAATACTACAGC-3’
Reverse – 5’-ACTCCTGGCCCTGACTAAGC-3’
The fragment was gel purified and cloned into the TOPO pCR2.1 plasmid (Life
Technologies). Following amplification in E. coli, the probe template was excised with
EcoRI and gel purified. Radiolabeled probe was prepared using the RadPrime DNA
Labeling System (Life Technologies) with -32P-dCTP. Radiolabeled probe was
hybridized overnight at 68C to a commercial northern blot (Zyagen, MN-MT-1)
containing 20 g of total RNA per sample. The hybridized blot was washed four times
and then exposed to autoradiography film for twenty-four hours. The blot was then
stripped using a published protocol and probed for the 18S rRNA loading control using
the same procedure described for Dworf.
Antibody Production
A custom polyclonal antibody was derived against the N-terminal region of the
predicted DWORF protein by New England Peptide. Rabbits were immunized with a
synthetic peptide with the following sequence MAEKESTSPHLIC. Sera were collected
and affinity purified against the peptide immunogen.
Human Heart Failure Tissue
Samples were acquired from the Temple University human heart tissue bank (IRB
#21319). All ischemic heart failure samples were core biopsies of post-MI heart failure
patients at the time of transplant. An n of 8 was used for both failing and non-failing
hearts.
3
Western Blot Analysis
Lysates were prepared by pulverizing snap frozen tissues in liquid nitrogen and then
homogenizing in RIPA buffer (150 mM NaCl; 1% v/v Igepal CA-630; 50 mM Tris-Cl,
pH 8.3; 0.5% w/v sodium deoxycholate; 0.1% w/v sodium dodecyl sulfate) with added
protease inhibitors (cOmplete ULTRA mini tablet, Roche) on ice using a ‘tight’ glass
dounce homogenizer. Protein concentrations were determined using a BCA Protein
Assay Kit (Pierce). For DWORF and PLN, samples were separated on a 16.5% tricine
buffered polyacrylamide gel (BioRad) or 20% bis/acrylamide gels made by standard gel
preparation. Samples for other experiments were separated on Any kD tris-glycine
buffered polyacrylamide gels (BioRad). DWORF was electroblotted onto Immobilon PSQ
membranes (Millipore) using a semi-dry apparatus for 30 min at 20 V. Other experiments
were electroblotted onto Immobilon P membranes using a semi-dry apparatus for 50 min
at 20 V. Membranes were blocked overnight at 4C in blotto (5% w/v non-fat dry milk in
TBST). Primary antibody hybridization was carried out overnight at 4C with the
following antibodies: DWORF, 1:5,000; total PLN (2D12, Pierce), 1:5,000; pSer16-PLN
(Badrilla), 1:5,000; pThr17-PLN (Badrilla), 1:5,000; SERCA2 (2A7-A1, Pierce), 1:2000;
NCX1 (Abcam), 1:500; RyR2 (Pierce, C3-33), 1:1000; LTCC (Millipore, α1C), 1:1000;
PMCA (Pierce, 5F10), 1:250; GAPDH (Millipore); 1:10,000. Blots were washed five
times for five minutes each in TBST and then incubated with HRP-conjugated secondary
antibodies (BioRad) at 1:20,000. Blots were then developed with chemiluminescent
substrate and exposed to either autoradiograph film or a digital ChemiDoc system
(G:BOX, GeneSys).
Site Directed Mutagenesis
PCR-based site directed mutagenesis of SERCA1 was performed using a
QuikChange Lightening Site Directed Mutagenesis kit (Agilent Technologies). Point
mutations were used to convert amino acids valine 795, leucine 802, and phenylalanine
809 to alanine. Mutations were confirmed by DNA sequencing.
4
Generation of Mouse Lines
All experiments involving animals were approved by the Institutional Animal Care
and Use Committee at the University of Texas Southwestern Medical Center. Knockout
mice were generated using the CRISPR/Cas9 system by pronuclear and cytoplasmic
injection of mouse embryos with guide RNA (gRNA) and Cas9 mRNA as described
previously (29). Briefly, a gRNA was cloned that targeted the predicted coding sequence
of Dworf. Cleavage efficiency was tested in cell culture, and then the gRNA and Cas9
mRNA were transcribed in vitro and spin-column purified. Mouse embryos were injected
with an equal ratio (w/w) of gRNA and Cas9 mRNA into the pronucleus and cytoplasm
and then transferred to a surrogate dam for gestation. Because F0 founders are expected to
be mosaic, allelic disruption was confirmed in the F0 generation pups using the T7E1
endonuclease assay on tail biopsies, and positive founders were bred to wild-type
C57/BL6 mice to isolate potential knockout alleles. Mutants in the F1 generation were
identified by T7E1 assay and then the alleles were cloned and sequenced. One mouse line
with a two-bp insertion was chosen for further study.
Transgenic mice were derived by pronuclear injection of mouse embryos. Briefly,
the Dworf coding sequence was cloned into an MHC promoter-driven plasmid with a
polyadenylation sequence from the human growth hormone (hGH) gene. The plasmid
was injected into the pronucleus of mouse embryos and then implanted in a surrogate
dam for gestation. F0 generation pups were selected by presence of transgene by PCR
from a tail biopsy and bred to wild-type. F1 generation pups were bred to C57/BL6 mice
and expression of the transgene was verified in the F2 progeny by qPCR.
Genotyping of Mouse Lines
Knockout mice were genotyped using a custom TaqMan genotyping assay (Life
Technologies). Briefly, tail biopsies were digested in lysis buffer (50 mM KCl; 10 mM
Tris-Cl, pH 8.3; 2.5 mM MgCl2; 0.1 mg/mL porcine gelatin; 0.45% v/v Igepal CA-630;
0.45% v/v Tween 20) with proteinase K (6 U/mL) overnight at 55 C. Particulates were
removed by high-speed centrifugation, and the supernatant was diluted 1:10 in water. The
tail samples were then analyzed by qPCR with a mixture of the following
oligonucleotides:
5
Forward Primer 5’-TCATTGCTTCTAAGCAGAGTCAACA-3’
Reverse Primer 5’-ATGCAGCCTACAATCCATCCAA-3’
WT Probe 5’-CCAGGAGAAGAATG-3’
KO Probe 5’-CAGGAGACAAGAATG-3’
Transgenic mice were genotyped based on presence or absence of the hGH
sequence. Tail biopsies were processed as described for the Dworf KO mice and used for
duplex PCR with a 1:1:1:1 mixture of the following primers (myogenin primers are used
as a positive control):
hGH Forward Primer 5’-GTCTGACTAGGTGTCCTTCT-3’
hGH Reverse Primer 5’-CGTCCTCCTGCTGGTATAG-3’
Myogenin Forward Primer 5’-TTACGTCCATCGTGGACAGC-3’
Myogenin Reverse Primer 5’-TGGGCTGGGTGTTAGCCTTA-3’
PCR products were analyzed by agarose gel electrophoresis.
Co-immunoprecipitations (CoIPs)
CoIPs were performed as previously described in detail (30). HEK 293 cells or
COS7 cells were transfected with GFP-DWORF, GFP-PLN, HA-DWORF, HA-PLN,
HA-SLN, HA-MLN and Myc-tagged SERCA. Immunoprecipitations were carried out
using mouse anti-GFP (Life Technologes) or mouse anti-Myc antibody (Invitrogen) and
collected with Dynabeads (Life Technologies). Standard western blot procedures were
performed on IP fractions using HRP-conjugated GFP (Pierce, GF28R), Myc (Pierce,
9E10) or HA (Pierce, 2-2.2.14) antibodies.
Mouse Cardiomyocyte Isolation
Adult mouse myocytes were isolated as described previously (31, 32). Anesthesia
was induced using 3% isoflurane and maintained using 1% isoflurane. Mouse hearts were
rapidly excised and the aorta was cannulated on a constant-flow Langendorff apparatus.
The heart was digested by perfusion of Tyrode’s solution containing 0.2 mg/mL Liberase
DH (Roche), 0.14 mg/mL Trypsin (Gibco/Invitrogen) and (mM): CaCl2 0.02, glucose 10,
HEPES 5, KCl 5.4, MgCl2 1.2, NaCl 150, sodium pyruvate 2, pH 7.4. When the tissue
softened, the left ventricle was isolated and gently minced, filtered, and equilibrated in
6
Tyrode’s solution with 200 μM CaCl2 and 1% bovine serum albumin (BSA) at room
temperature.
Ca2+ Transients and SR Load
Myocytes were loaded with 5 μM fluo-4 AM (Molecular Probes) and placed in a
heated chamber (35°C) on the stage of an inverted microscope and perfused with
Tyrode’s solution containing in mM: CaCl2 1, glucose 10, HEPES 5, KCl 5.4, MgCl2 1.2,
NaCl 150, sodium pyruvate 2, pH 7.4. Myocytes were paced at 0.5 Hz and fractional
shortening data was collected using edge detection. For intracellular Ca2+ fluorescence
measurements, the F0 (or F unstimulated) was measured as the average fluorescence of
the cell 50 ms prior to stimulation. The maximal fluo-4 fluorescence (F) was measured at
peak amplitude. Background fluorescence was subtracted from each parameter before
representing the peak Ca2+ transient as F/F0 (31, 32). Tau (τ) was measured as the decay
rate of the average Ca2+ transient trace. Isoproterenol (Iso, Sigma) was used at 10 nM.
To measure SR Ca2+ content, myocytes were paced at 0.5 Hz for 10 consecutive
contractions, and 10 mM caffeine (Sigma) was then rapidly applied via a glass pipette
close to the myocyte with a Pico spritzer (33). The decay of caffeine-induced Ca2+
transients was fit with a single-exponential function, and time constant (τ) values indicate
Na+/Ca2+-exchanger (NCX) activity.
All data analysis, including cardiomyocyte contractility parameters, were assessed
using pCLAMP software.
Oxalate-Supported Ca2+ Uptake Measurements
Oxalate-supported Ca2+ uptake in cardiac homogenates was measured by a modified
Millipore filtration technique (4, 34-35). Heart, soleus and quadriceps tissues were
isolated from WT, Tg and KO mice and rapidly frozen in liquid nitrogen and stored at
80°C until processed. Frozen tissue samples were homogenized in 50 mM phosphate
buffer, pH 7.0 containing 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, 0.3 mM PMSF and
0.5 mM DTT. Ca2+ uptake was measured in reaction solution containing 40 mM
imidazole pH 7.0, 95 mM KCl, 5 mM NaN3, 5 mM MgCl2, 0.5 mM EGTA, 5 mM K+
oxalate, 1 μM ruthenium red and various concentrations of CaCl2 to yield 0.02 to 5 μM
7
free Ca2+. Homogenates were incubated at 37°C for 2 minutes in the above reaction
buffer and the reaction was initiated by the addition of ATP (final concentration 5 mM).
The data were analyzed by nonlinear regression with computer software (GraphPad
Software), and the KCa values were calculated using an equation for a general cooperative
model for substrate activation. The values for maximal SERCA activity were taken
directly from the experimental data and normalized for total protein concentration
(μmol/mg protein/min). Statistical analyses were performed using an unpaired t-test and
data are presented as mean ± SEM.
Oxalate-supported Ca2+-dependent Ca2+-ATPase activity in homogenates from
COS7 cells was measured as described previously (4). COS7 cells were co-transfected
with equal amounts of an expression plasmid encoding SERCA2a and untagged
DWORF, PLN, MLN or SLN. Approximately 36 hr after transfection, COS7 cells were
homogenized in the buffer detailed above and assayed the same way as tissue samples.
Measurement of Contractility in Soleus Muscle
The soleus muscle was dissected free and suspended in an organ bath maintained at
37°C. One end was tied to a rigid post and the other was fastened to a force transducer
(Myobath, WPI Inc.). Isometric contractions were elicited by application of brief current
pulses of 0.2 msec duration at 80 mA (A385 Stimulator; WPI Inc.). Low frequency pulse
trains (< 10 Hz) elicited unfused twitch responses and progressively higher stimulation
frequencies produced a smooth tetanic contraction. Contraction tests were separated by a
2-minute rest interval to prevent fatigue. Muscle contraction was quantified as the peak
force and the time constant for relaxation that was obtained from a single exponential fit
to the decay in force from 50% of the maximum back to baseline. The bath contained 118
mM NaCl, 4.7 mM KCl, 1.18 mM MgSO4, 2.5 mM CaCl2, 1.18 mM NaH2PO4, 10 mM
glucose, 24.8 mM NaHCO3 and was continuously bubbled with 95% O2 and 5% CO2. d-
turbocurarine (0.25 M) was added to prevent muscle activation from nerve fiber
endings.
8
Skeletal Muscle Electroporation
Flexor digitorum brevis muscles were electroporated with GFP-tagged constructs
and visualized fresh in physiologic buffer using two-photon laser scanning microscopy as
described previously (19, 36).
9
Fig. S1. Dworf cDNA sequence from mouse.
Nucleotide sequence of a cloned fragment of Dworf from mouse heart cDNA. The open
reading frame is highlighted in red with the amino acid sequence below.
10
Fig. S2. Overview of the Dworf locus.
(A) In mice, Dworf is transcribed from a 2.8 kb locus on chromosome 3 to produce two
transcript isoforms of approximately 300 bp that differ by inclusion of three additional
base pairs, producing a polyadenylated RNA. The predicted open reading frame
(highlighted in red) begins in exon one and ends near the 3’ end of exon two. In humans
the transcript is annotated as a lncRNA named LOC100507537 and appears to only
produce a single isoform. (B) Amino acid sequence alignment of vertebrate DWORF
proteins. The symbols below the alignment represent the biochemical similarity of
aligned amino acids. Asterisks indicate identical conservation, colons represent high
similarity, periods are somewhat similar. (C) PhyloCSF score plot for the Dworf locus as
seen in the UCSC genome browser using the PhyloCSF track hub.
11
Fig. S3. Expression of Dworf in adult tissue and heart development.
(A) Detection of Dworf RNA by qPCR in adult mouse tissues. (B) Detection of Dworf
RNA by qPCR in hearts of mice at the indicated ages. Mean ± SD; n=4.
12
Fig. S4. Protein-coding capacity of the Dworf 5’ UTR.
The 5’ UTR and the first thirteen codons of Dworf were cloned into a HaloTag
expression vector that contains a protein tag but lacks a Kozak sequence and start codon.
The empty HaloTag vector lacks an initiation codon and is not translated. The Dworf 5’
UTR is capable of initiating translation and can be detected by immunoblotting with an
antibody for the HaloTag protein or an antibody against DWORF.
α-HaloTag
α-Tubulin
α-DWORF
Empty 5' UTR
HaloTagEmpty
HaloTag5' UTR
No start codon
Dworf 5' UTR +13 codons (69 bp)
13
Fig. S5. DWORF binding to SERCA displaces PLN in a dose-dependent manner.
Immunoprecipitation of Myc-SERCA from lysates of COS7 cells co-transfected with
equal amounts of HA-PLN and Myc-SERCA and increasing amounts of either GFP-PLN
or GFP-DWORF. Western blots on input samples and bound immunoprecipitated
fractions reveal that DWORF binding to SERCA competitively displaces PLN from
SERCA. GFP-PLN is used as a positive control for HA-PLN displacement.
GFP-DWORF:
MYC
HA
GFPInput
GFP-PLN:- 5x1x-- -- - -
25x-
SERCA
PLN
PLN/DWORF
MYC
HA
GFP
MYC(SERCA)
IP
SERCA
PLN
PLN/DWORF
1x 5x 25x
14
Fig. S6. Mutations in the M6 helical transmembrane domain of SERCA reduce association of SERCA with PLN and DWORF.
COS7 cells were co-transfected with GFP-PLN or GFP-DWORF and Myc-tagged WT-
SERCA or constructs with mutations in the M6 helical transmembrane domain of
SERCA that are known to reduce PLN binding. Homogenates were immunoprecipitated
with an antibody for GFP and western blot analysis was used to assess the relative
amount of SERCA pulled down in association with PLN or DWORF. Mutation of each
of the indicated residues to alanine reduced SERCA pull-down in association with both
PLN and DWORF and mutation of all three residues (V795A/L802A/F809A) had the
most dramatic reduction in binding (V795A, valine 795 to alanine; L802A, leucine 802 to
alanine; F809A, phenylalanine 809 to alanine).
Input
GFP IP(PLN/DWORF)
V795A
WT
WT
GFPGFP-PLN GFP-DWORF
Myc
GFP
Myc
GFP
L802
A
F809A
V795A
WT
V795A
/L80
2A/F
809A
L802
A
F809A
V795A
/L80
2A/F
809A
Myc-SERCA
Myc-SERCA
GFP-PLNGFP-DWORF GFP
GFP-PLNGFP-DWORF GFP
15
Fig. S7. Competitive binding of DWORF and PLN to SERCA.
Immunoprecipitation of Myc-SERCA in COS7 cells transfected with varying ratios of
GFP-DWORF and GFP-PLN and detection with an antibody for GFP indicates that
DWORF and PLN have similar binding affinities for SERCA.
16
Fig. S8. Western blot analysis of heart tissue homogenates from αMHC-DWORF transgenic mice analyzing expression of Ca2+ handling proteins.
(A) Immunoblots of heart homogenates from αMHC-DWORF transgenic mice reveals no
significant changes in total expression of relevant Ca2+ handling proteins as compared to
wild-type mice. (B) Immunoblots were quantified using ImageJ. Protein samples were
normalized to GAPDH. RyR (ryanodine receptor 2), LTCC (α1C-subunit of the voltage
regulated L-Type Ca2+ channel), PMCA (plasma membrane Ca2+-ATPase), SERCA2 (SR
Ca2+-ATPase 2), NCX (Na+/Ca2+-exchanger), GAPDH (glyceraldehyde 3-phosphate
dehydrogenase). Mean ± SEM; WT n=10, Tg n=12.
RyR
LTCC
WT DWORF Tg
150
250
kDa
PMCA 150
SERCA2150
NCX 100
DWORF 10
GAPDH 37
0.0
0.5
1.0
1.5
Rel
ativ
e E
xpre
ssio
n
RyR LTCC PMCA SERCA NCX
WTTg
B
A
17
Fig. S9. Western blot analysis of heart tissue homogenates from αMHC-DWORF transgenic mice analyzing PLN expression, phosphorylation state and oligomerization.
(A) Immunoblot of heart homogenates from αMHC-DWORF transgenic mice reveals no
significant changes in total PLN expression, phosphorylation status, or oligomerization
state as compared to wild-type mice. (B) Immunoblots were quantified using ImageJ.
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), GAPDH (glyceraldehyde 3-phosphate
dehydrogenase, M/P (monomer/pentamer ratio of PLN). Mean ± SEM; WT n=10, TG
n=12.
PS16
tPLN
WT Tg10
10
10
10
kDa
PT17
tPLN
tPLN 20
GAPDH
10
37
37
WTTg
B
0.0
0.5
1.0
1.5
Re
lativ
e E
xpre
ssio
n
PS16 PT17 tPLN M/P
A
monomer
pentamer
18
Fig. S10. RT-PCR and western blot analysis of heart and skeletal muscle from Dworf knockout mice.
(A) Dworf mRNA is increased approximately four fold in the hearts of adult knockout
mice, but other notable genes are not altered. Mean ± SD; WT n=3, KO n=3; *P-value =
0.006 (B-C) Immunoblot of Ca2+ regulatory proteins in heart (B) and soleus muscle (C)
homogenates from Dworf KO mice reveals no significant change in total protein
expression levels of relevant Ca2+ handling proteins as compared to WT mice.
Immunoblots were quantified using ImageJ. Protein samples were normalized to
GAPDH. RyR (ryanodine receptor 2), LTCC (α1C-subunit of the voltage regulated L-
Type Ca2+ channel), PMCA (plasma membrane Ca2+-ATPase), SERCA2 (SR Ca2+-
ATPase 2), NCX (Na+/Ca2+-exchanger), GAPDH (glyceraldehyde 3-phosphate
dehydrogenase). Mean ± SEM; WT n=9, KO n=9.
B
RyR
LTCC
WT DWORF KO
150
250
kDa
PMCA 150
SERCA2150
NCX100
DWORF 10
GAPDH 37
0.0
0.5
1.0
1.5
Re
lativ
e E
xpre
ssio
n
WTKO
RyR LTCC PMCA SERCA NCX
0
1
2
4
3
5
Rel
ativ
e to
WT
mR
NA
Dworf Serca2 Myh7 Nppa Pln
A
WTKO
*
C
RyR
DWORF
WT DWORF KO
150
250
kDa
PMCA
150SERCA2
10
37GAPDH
0.0
0.5
1.0
1.5
2.0
Re
lativ
e E
xpre
ssio
n
WTKO
RyR PMCA SERCA
19
Fig. S11. Western blot analysis of heart tissue homogenates from αMHC-DWORF transgenic mice analyzing PLN expression, phosphorylation state and oligomerization.
(A) Immunoblot of Ca2+ regulatory proteins in hearts of Dworf KO mice reveals no
significant changes in total PLN expression, phosphorylation state or monomer/pentamer
ratio as compared to WT hearts. (B) Immunoblots were quantified using ImageJ. Total
PLN (tPLN) protein was normalized to GAPDH and phosphorylation blots (PS16 and
PT17) were normalized to tPLN. tPLN (total phospholamban), PS16 (phospho-serine 16
on PLN), PT17 (phospho-threonine on PLN), M/P (PLN monomer/pentamer ratio),
GAPDH (glyceraldehyde 3-phosphate dehydrogenase). Mean ± SEM; WT n=9, KO n=9.
PS16
tPLN
WT KO
10
10
10
10
kDa
PT17
tPLN
tPLN20
GAPDH
10
37
37
WTKO
B
0.0
0.5
2.0
1.5
1.0
2.5
Rel
ativ
e E
xpre
ssio
n
PS16 PT17 tPLN M/P
A
monomer
pentamer
20
Fig. S12. Characterization of αMHC-DWORF Tg Line 2.
(A) A second αMHC-DWORF transgenic line (Tg Line 2) was generated with a more
modest level of DWORF overexpression as assessed by DWORF immunoblotting of
heart lysates. Peak cardiomyocyte fractional shortening amplitude (B), peak systolic Ca2+
transient amplitude (C), and systolic Ca2+ transient decay rates (Tau) (D) were analyzed
in fluo-4 loaded cardiomyocytes from WT and Tg Line 2 mice. The lower Tau value seen
in αMHC-DWORF Tg cardiomyocytes indicates higher SERCA activity and faster Ca2+
reuptake. *P-value < 0.05, n=6.
DWORF
GAPDH
WT DWORF Tg Line 2
37
10kDa
A
B
WT Tg
*
Fra
ctio
nal
sh
orte
nin
g (%
)
0
2
4
6
8C
WT Tg
*
Pea
k a
mp
litud
e (F
/F0)
0
1
2
3
4D
WT Tg
*
SE
RC
A a
ctiv
ity(T
au
, ms)
0
50
100
150
21
Fig. S13. Contractility and Ca2+ transient parameters in αMHC-DWORF Tg and Dworf KO cardiomyocytes and response to isoproterenol.
(A) Mean values of decay time constants (Tau) of caffeine-induced Ca2+ transients as a
measure of Na+/Ca2+ exchanger (NCX) activity. n=6 for each group. (B) Representative
pacing-induced fractional shortening traces in isolated cardiomyocytes stimulated at 0.5
Hz as measured by edge detection. Peak fractional shortening amplitude (C), peak
systolic Ca2+ transient amplitude (D), and systolic Ca2+ transient decay rates (Tau) (E) of
WT, Tg and KO mice measured at baseline and in response to 10 nM isoproterenol (Iso).
Mean ± SEM; n=8 for all Iso response parameters tested. * P-value < 0.05; ** P-value <
0.01; *** P-value < 0.001.
22
Fig. S14. Ca2+-dependent Ca2+-uptake assay in hearts and quadriceps muscles from αMHC-DWORF Tg and Dworf KO mice.
(A) A second αMHC-DWORF transgenic line (Tg Line 2) was generated with a more
modest level of DWORF overexpression (fig. S12A). SERCA activity was measured
using the Ca2+-dependent Ca2+-uptake assay. This second transgenic line exhibited a
leftward shift of the Ca2+ affinity curve confirming that overexpression of DWORF
results in elevated SERCA activity and a higher affinity for Ca2+. Similar to our findings
in the first transgenic line, modulating DWORF expression has no apparent effect on
Vmax. (B) Ca2+-dependent Ca2+-uptake assays measuring SERCA affinity for Ca2+ was
unchanged in quadriceps muscle of Dworf KO.
23
Fig. S15. DWORF regulates the relative affinity of SERCA for Ca2+ by relieving the inhibition of PLN in a dose-dependent manner.
(A) The Ca2+-dependence of the relative rate of Ca2+ uptake by SERCA is shown for
homogenates from COS7 cells co-transfected with SERCA2a and the indicated
constructs. Co-transfection of cells with PLN results in a rightward shift of the Ca2+
affinity curve as compared to control cells (GFP transfected). Co-transfection with
increasing amounts of DWORF relieves the inhibitory effect of PLN in a dose-dependent
manner. A ratio of 1:3 (PLN:DWORF) is sufficient to return SERCA activity to baseline
levels and this is maintained at a 1:10 ratio. Cells expressing SERCA2a and DWORF
alone do not exhibit enhanced SERCA activity indicating that DWORF itself does not
biochemically activate SERCA. (B) Mean KCa values from n=4 separate experiments are
represented as bar graphs. P-value ### p<0.001 vs GFP, ## p<0.01 vs GFP. ***p<0.001
vs. PLN, **p<0.01 vs. PLN.
A
B
24
Fig. S16. Co-expression of DWORF with the SERCA inhibitors PLN, SLN and MLN results in relief of their inhibitory effects.
(A) Ca2+-dependent Ca2+-uptake assays were performed using homogenates from COS7
cells co-transfected with SERCA2a and the indicated constructs. Co-transfection of cells
with PLN, SLN or MLN results in a characteristic rightward shift of the Ca2+ affinity
curve as compared to control cells (GFP transfected). Co-expressing DWORF in these
conditions at three-fold excess that of PLN, SLN or MLN was sufficient to completely
relieve the inhibitory effects of these peptides. (B) Mean KCa values from n=2
experiments are represented as bar graphs. P-value ### p<0.001 vs GFP, ## p<0.01 vs
GFP. ***p<0.001 vs. PLN, **p<0.01 vs. PLN.
A
B
25
Fig. S17. Alignment of SERCA inhibitory peptides compared to DWORF.
The SERCA inhibitory peptides MLN (myoregulin), PLN (phospholamban), and SLN
(sarcolipin) share the LFXXF motif (denoted by asterisks), which is absent in the
DWORF sequence.
26
Table S1.
Cardiomyocyte contractility properties in fluo-4 loaded cells from WT, αMHC-DWORF
Tg and Dworf KO mice.
Genotype Time to Peak
(ms) p-value
Rate of shortening (µm/msec)
p-value Rate of
re-lengthening (µm/msec)
p-value
WT 92.3 ± 5.9 NA -0.15 ± 0.03 NA 0.103 ± 0.031 NA
Tg Line 1 89.3 ± 5.8 0.720 -0.26 ± 0.08 0.220 0.259 ± 0.081 0.043
Tg Line 2 90.4 ± 6.4 0.831 -0.14 ± 0.03 0.887 0.169 ± 0.040 0.215
KO 102.8 ± 7.7 0.295 -0.19 ± 0.04 0.448 0.070 ± 0.026 0.444
!
27
Table S2.
Ca2+-dependent Ca2+-uptake assay KCa and Vmax values in total heart homogenates from
WT, αMHC-DWORF Tg and Dworf KO mice.
Genotype KCa (µM) p-value V
max (nmol/mg/min) p-value
WT 0.2038 ± 0.0067 NA 62.81 ± 10.26 NA
Tg Line 1 0.1670 ± 0.0072 0.0013 64.59 ± 5.81 0.824
Tg Line 2 0.1810 ± 0.0078 0.0279 63.25 ± 9.34 0.887
KO 0.2328 ± 0.0110 0.0478 66.15 ± 5.77 0.687
28
Table S3.
Ca2+-dependent Ca2+-uptake assay KCa and Vmax values in total soleus muscle
homogenates from WT and Dworf KO mice.
Genotype KCa (µM) p-value V
max (nmol/mg/min) p-value
WT 0.1663 ± 0.0058 NA 16.07 ± 1.33 NA
KO 0.1923 ± 0.0086 0.0478 14.72 ± 1.84 0.688
29
Table S4.
Ca2+-dependent Ca2+-uptake assay KCa and Vmax values in total quadriceps homogenates
from WT and Dworf KO mice.
Genotype KCa (µM) p-value V
max (nmol/mg/min) p-value
WT 0.1782 ± 0.0119 NA 44.64 ± 4.83 NA
KO 0.1809 ± 0.0047 0.8446 48.98 ± 3.91 0.511
!
29
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