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: eric.olson@utsouthwestern.edu

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

REFERENCES AND NOTES 1. D. M. Bers, Cardiac excitation-contraction coupling. Nature 415, 198–205 (2002). Medline

doi:10.1038/415198a

2. D. H. MacLennan, M. Asahi, A. R. Tupling, The regulation of SERCA-type pumps by phospholamban and sarcolipin. Ann. N. Y. Acad. Sci. 986, 472–480 (2003). Medline doi:10.1111/j.1749-6632.2003.tb07231.x

3. E. G. Kranias, R. J. Hajjar, Modulation of cardiac contractility by the phospholamban/SERCA2a regulatome. Circ. Res. 110, 1646–1660 (2012). Medline doi:10.1161/CIRCRESAHA.111.259754

4. D. M. Anderson, K. M. Anderson, C. L. Chang, C. A. Makarewich, B. R. Nelson, J. R. McAnally, P. Kasaragod, J. M. Shelton, J. Liou, R. Bassel-Duby, E. N. Olson, A micropeptide encoded by a putative long noncoding RNA regulates muscle performance. Cell 160, 595–606 (2015). Medline

5. N. C. Bal, S. K. Maurya, D. H. Sopariwala, S. K. Sahoo, S. C. Gupta, S. A. Shaikh, M. Pant, L. A. Rowland, E. Bombardier, S. A. Goonasekera, A. R. Tupling, J. D. Molkentin, M. Periasamy, Sarcolipin is a newly identified regulator of muscle-based thermogenesis in mammals. Nat. Med. 18, 1575–1579 (2012). Medline doi:10.1038/nm.2897

6. E. G. Magny, J. I. Pueyo, F. M. Pearl, M. A. Cespedes, J. E. Niven, S. A. Bishop, J. P. Couso, Conserved regulation of cardiac calcium uptake by peptides encoded in small open reading frames. Science 341, 1116–1120 (2013). Medline

7. G. W. Dorn 2nd, J. D. Molkentin, Manipulating cardiac contractility in heart failure: Data from mice and men. Circulation 109, 150–158 (2004). Medline doi:10.1161/01.CIR.0000111581.15521.F5

8. S. A. Slavoff, A. J. Mitchell, A. G. Schwaid, M. N. Cabili, J. Ma, J. Z. Levin, A. D. Karger, B. A. Budnik, J. L. Rinn, A. Saghatelian, Peptidomic discovery of short open reading frame-encoded peptides in human cells. Nat. Chem. Biol. 9, 59–64 (2013). Medline doi:10.1038/nchembio.1120

9. M. C. Frith, A. R. Forrest, E. Nourbakhsh, K. C. Pang, C. Kai, J. Kawai, P. Carninci, Y. Hayashizaki, T. L. Bailey, S. M. Grimmond, The abundance of short proteins in the mammalian proteome. PLOS Genet. 2, e52 (2006). Medline

10. B. R. Nelson, D. M. Anderson, E. N. Olson, Small open reading frames pack a big punch in cardiac calcium regulation. Circ. Res. 114, 18–20 (2014). Medline doi:10.1161/CIRCRESAHA.113.302716

11. M. F. Lin, I. Jungreis, M. Kellis, PhyloCSF: A comparative genomics method to distinguish protein coding and non-coding regions. Bioinformatics 27, i275–i282 (2011). Medline doi:10.1093/bioinformatics/btr209

12. C. Xie, J. Yuan, H. Li, M. Li, G. Zhao, D. Bu, W. Zhu, W. Wu, R. Chen, Y. Zhao, NONCODEv4: Exploring the world of long non-coding RNA genes. Nucleic Acids Res. 42 (D1), D98–D103 (2014). Medline doi:10.1093/nar/gkt1222

30

13. E. L. Sonnhammer, G. von Heijne, A. Krogh, A hidden Markov model for predicting transmembrane helices in protein sequences. Proc. Int. Conf. Intell. Syst. Mol. Biol. 6, 175–182 (1998). Medline

14. A. Krogh, B. Larsson, G. von Heijne, E. L. Sonnhammer, Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J. Mol. Biol. 305, 567–580 (2001). Medline doi:10.1006/jmbi.2000.4315

15. M. Goujon, H. McWilliam, W. Li, F. Valentin, S. Squizzato, J. Paern, R. Lopez, A new bioinformatics analysis tools framework at EMBL-EBI. Nucleic Acids Res. 38 (Web Server), W695–W699 (2010). Medline doi:10.1093/nar/gkq313

16. A. N. Guido, G. E. Campos, H. S. Neto, M. J. Marques, E. Minatel, Fiber type composition of the sternomastoid and diaphragm muscles of dystrophin-deficient mdx mice. Anat. Rec. (Hoboken) 293, 1722–1728 (2010). Medline doi:10.1002/ar.21224

17. S. Schiaffino, C. Reggiani, Fiber types in mammalian skeletal muscles. Physiol. Rev. 91, 1447–1531 (2011). Medline doi:10.1152/physrev.00031.2010

18. J. D. Molkentin, J. R. Lu, C. L. Antos, B. Markham, J. Richardson, J. Robbins, S. R. Grant, E. N. Olson, A calcineurin-dependent transcriptional pathway for cardiac hypertrophy. Cell 93, 215–228 (1998). Medline doi:10.1016/S0092-8674(00)81573-1

19. B. R. Nelson, F. Wu, Y. Liu, D. M. Anderson, J. McAnally, W. Lin, S. C. Cannon, R. Bassel-Duby, E. N. Olson, Skeletal muscle-specific T-tubule protein STAC3 mediates voltage-induced Ca2+ release and contractility. Proc. Natl. Acad. Sci. U.S.A. 110, 11881–11886 (2013). Medline doi:10.1073/pnas.1310571110

20. M. Asahi, Y. Kimura, K. Kurzydlowski, M. Tada, D. H. MacLennan, Transmembrane helix M6 in sarco(endo)plasmic reticulum Ca(2+)-ATPase forms a functional interaction site with phospholamban. Evidence for physical interactions at other sites. J. Biol. Chem. 274, 32855–32862 (1999). Medline doi:10.1074/jbc.274.46.32855

21. A. R. Tupling, E. Bombardier, S. C. Gupta, D. Hussain, C. Vigna, D. Bloemberg, J. Quadrilatero, M. G. Trivieri, G. J. Babu, P. H. Backx, M. Periasamy, D. H. MacLennan, A. O. Gramolini, Enhanced Ca2+ transport and muscle relaxation in skeletal muscle from sarcolipin-null mice. Am. J. Physiol. Cell Physiol. 301, C841–C849 (2011). Medline doi:10.1152/ajpcell.00409.2010

22. B. A. Davis, A. Schwartz, F. J. Samaha, E. G. Kranias, Regulation of cardiac sarcoplasmic reticulum calcium transport by calcium-calmodulin-dependent phosphorylation. J. Biol. Chem. 258, 13587–13591 (1983). Medline

23. W. J. Kent, C. W. Sugnet, T. S. Furey, K. M. Roskin, T. H. Pringle, A. M. Zahler, D. Haussler, The human genome browser at UCSC. Genome Res. 12, 996–1006 (2002). Medline doi:10.1101/gr.229102

24. C. Trapnell, L. Pachter, S. L. Salzberg, TopHat: Discovering splice junctions with RNA-Seq. Bioinformatics 25, 1105–1111 (2009). Medline doi:10.1093/bioinformatics/btp120

25. C. Trapnell, A. Roberts, L. Goff, G. Pertea, D. Kim, D. R. Kelley, H. Pimentel, S. L. Salzberg, J. L. Rinn, L. Pachter, Differential gene and transcript expression analysis of

31

RNA-seq experiments with TopHat and Cufflinks. Nat. Protoc. 7, 562–578 (2012). Medline doi:10.1038/nprot.2012.016

26. D. Blankenberg, J. Taylor, A. Nekrutenko; Galaxy Team, Making whole genome multiple alignments usable for biologists. Bioinformatics 27, 2426–2428 (2011). Medline doi:10.1093/bioinformatics/btr398

27. B. Giardine, C. Riemer, R. C. Hardison, R. Burhans, L. Elnitski, P. Shah, Y. Zhang, D. Blankenberg, I. Albert, J. Taylor, W. Miller, W. J. Kent, A. Nekrutenko, Galaxy: A platform for interactive large-scale genome analysis. Genome Res. 15, 1451–1455 (2005). Medline doi:10.1101/gr.4086505

28. D. Blankenberg, G. V. Kuster, N. Coraor, G. Ananda, R. Lazarus, M. Mangan, A. Nekrutenko, J. Taylor, Galaxy: A Web-based genome analysis tool for experimentalists. Curr. Protoc. Mol. Biol. 89 (19.10), 19.10.1–19.10.21 (2010). DOI: 10.1002/0471142727.mb1910s89

29. C. Long, J. R. McAnally, J. M. Shelton, A. A. Mireault, R. Bassel-Duby, E. N. Olson, Prevention of muscular dystrophy in mice by CRISPR/Cas9-mediated editing of germline DNA. Science 345, 1184–1188 (2014). Medline doi:10.1126/science.1254445

30. D. M. Anderson, B. J. Beres, J. Wilson-Rawls, A. Rawls, The homeobox gene Mohawk represses transcription by recruiting the sin3A/HDAC co-repressor complex. Dev. Dyn. 238, 572–580 (2009). Medline doi:10.1002/dvdy.21873

31. C. A. Makarewich, H. Zhang, J. Davis, R. N. Correll, D. M. Trappanese, N. E. Hoffman, C. D. Troupes, R. M. Berretta, H. Kubo, M. Madesh, X. Chen, E. Gao, J. D. Molkentin, S. R. Houser, Transient receptor potential channels contribute to pathological structural and functional remodeling after myocardial infarction. Circ. Res. 115, 567–580 (2014). Medline

32. N. Jaleel, H. Nakayama, X. Chen, H. Kubo, S. MacDonnell, H. Zhang, R. Berretta, J. Robbins, L. Cribbs, J. D. Molkentin, S. R. Houser, Ca2+ influx through T- and L-type Ca2+ channels have different effects on myocyte contractility and induce unique cardiac phenotypes. Circ. Res. 103, 1109–1119 (2008). Medline doi:10.1161/CIRCRESAHA.108.185611

33. V. Piacentino 3rd, C. R. Weber, X. Chen, J. Weisser-Thomas, K. B. Margulies, D. M. Bers, S. R. Houser, Cellular basis of abnormal calcium transients of failing human ventricular myocytes. Circ. Res. 92, 651–658 (2003). Medline doi:10.1161/01.RES.0000062469.83985.9B

34. W. Luo, I. L. Grupp, J. Harrer, S. Ponniah, G. Grupp, J. J. Duffy, T. Doetschman, E. G. Kranias, Targeted ablation of the phospholamban gene is associated with markedly enhanced myocardial contractility and loss of beta-agonist stimulation. Circ. Res. 75, 401–409 (1994). Medline doi:10.1161/01.RES.75.3.401

35. T. Holemans, I. Vandecaetsbeek, F. Wuytack, P. Vangheluwe, Measuring Ca2+-dependent Ca2+-uptake activity in the mouse heart. Cold Spring Harb. Protoc. 2014, 876–886 (2014). Medline doi:10.1101/pdb.prot076893

36. M. DiFranco, M. Quinonez, J. Capote, J. Vergara, DNA transfection of mammalian skeletal muscles using in vivo electroporation. J. Vis. Exp. 32, 1520 (2009). Medline