Supplemental Methods - Circulation

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CirculationAHA/2005/583351/R1



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1

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Supplement
for Materials and Methods


Generation and Identification of T
ransgenic Mice Expressing Human
-
PLN


The Invitrogen mutant kit was used to substitute the PLN Asn
27

with PLN Lys
27

in mouse
PLN cDNA
.
1

The pIBI
31 vectors containing the fusion gene (

-
MHCp
-
PLN cDNA
-
SV40 poly
(A) signal), which was used previously
,
2,3

was chosen as template. The 5’ end PLN mutant
primer with the site
-
directed PLN A
sn
27

(AAT) to Lys (AAG) mutation
: GCA

AGC ACG TCA
GAA GCT CCA GAA CCT ATT TAT C and the 3’ end of complementary chain mutant primer:
GAT AAA TAG GTT CTG GAG CTT CTG ACG TGC TTG C were used to amplify a minor
product harboring the PLN mutation. A human grow
th hormone (HGH) polyA was used and was
ligated to PLN cDNA with the N27K mutation. The resultant amplification product (1.2
-
kb)
contained the PLN cDNA with the N27K mutation and HGH poly(A) signal. This fragment was
cleaved by SalI and HindIII, and ligate
d into the pIBI31 fusion gene vector, which had been
digested with SalI and HindIII to release a 0.9
-
kb fragment, containing the wild
-
type PLN cDNA
and SV40
poly(
A). Therefore, the mutant PL
N cDNA, which encodes human PLN

and HGH
poly(A), replaced the wild
-
type PLN cDNA and SV40 poly(A). Positive clones were identified
by Sal I and HindIII digestion, as well as DNA sequencing.

The complete 6.7
-
kb fragment of the

-
MHCp
-
PLN N27K mutant cDNA
-
HGH poly (A)
was released from the pIBI31 vector by Kpn I (or BamHI
) and Hind III digestion and was gel
purified. Pronuclear microinjection of fertilized eggs from FVB/N mice was performed by
standard methods
.
3

Three transgenic lines were identified by PCR methodology of genomic
DNA isolated from tail biopsies
.
2,3

The transgenic expression was 210

19%, 300

28% and
CirculationAHA/2005/583351/R1



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2

-

240

25% of the total

PLN levels, respectively, compared to wild type hearts (100

9.6%).
Subsequently, all three transgenic lines were crossed with the PLN deficient mouse and
backcrossed for 3 generations. PCR analysis indicated three transgenic expression lines in the
PLN nu
ll background (TGL1, TGL2 and TGL3).
Genomic DNA was isolated from mouse
-
tail
clips and mice with
human
N27K
-
PLN expression were identified using PCR.


Echocardiographic Measurements of Cardiac Function

2D guided M
-
mode echocardiography (9 MHz) and color
-
flow directed Doppler (5
-
7MHz) were performed using an Interspec Apogee CX
-
200 ultrasonograph (Interspec
-
ATL,
Ambler, PA), as described previously
.
4,5

Mice were lightly anesthetized with 2.
5% avertin
(
15
ml/
k
g), they were allowed to breathe spontaneously and studies were performed at baseline.
Left ventricular dimensions, fractional shortening, velocity of circumferential shortening and
mitral and aortic spectral Dopplar waveforms were analyz
ed
.
4,5


Langendorff Perfused Heart
s


Retrograde aortic Langendorff heart perfusions were performed as described
.
1

Briefly,
the hearts were perfused wi
th modified Krebs
-
Henseleit buffer at 37

C. The heart rate and left
ventricular pressure were monitored continuously by a pressure transducer, which was hooked to
a computerized heart performance analyzer (HPA
-

, MicroMed, Inc). The perfused hearts were
pa
ced to heart rates of 400 beats/min for 30 minutes, with 10
-
V square pulse via platinum
electrodes coupled to a Grass S4 stimulatory.
The electrode was attached to the right atria

and
there was one to one atri
o
-
ventricular conduction observed.


CirculationAHA/2005/583351/R1



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3

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Quantitativ
e Immunoblotting

Hearts from
mouse
-
PLN

and
human
-
PLN mice were homogenized and subjected to
quantitative immunoblotting
with
a
specif
ic primary monoclonal antibody

to phospholamban,

which recognizes amino acids 3
-
25, that are identical between human and mo
use PLN.

Quantitative immunoblotting with specific primary monoclonal antibodies

to d
ihydropyridine
receptor

2

(DHPR),
sodium/calcium exchanger (NCX)
,
ryanodine receptor

(Affinity
Bioreagents Inc., Golden, CO.) and

-
myosin heavy chain (

-
MHC, Alexis Bioc
hemicals)
, and
polyclonal antibodies to SERCA2a
,
6

calsequestrin, FKBP12 (Affinity Bioreagents Inc., Golden,
CO
.),
protein phosph
a
tase 1, 2A, calcineurin (protein phosph
a
tase 2B) A


or A


and calcium
-
calmodulin
-
dependent kinase II,
phospho
-

and total: Akt, p
-
38, and Erk1/2(Cell Signaling). An
alkaline phosphatase
-
conjugated anti
-
mouse (PLN
, DHPR, NCX,


-
MHC

and protein
phosph
a
tase 1
)
, or anti
-
rabbit (SERCA2a and calsequestrin, ryanodine receptor, FKBP12,
phospho
-

and total: Akt, p
-
38 and Erk1/2
, protein phosph
a
tase 2A,
calcineurin (protein
phosph
a
tase 2B) A


or A


and calcium
-
calmodulin
-
dependent kinase II
) secondary

antibodies
(Cappe
l Division of Organon Teknika) were then used according to standard procedures.
6

All
antibodies used in the current study have be
en confirmed
for
the
ir

absolute specificity for the
protein of interest
1,6
-
14
. Mouse wild type pool
ed

heart
s were used
as

a standard control
on each
gel
,

to assure that

the immunoblot signal for each protein target
was

within the dynamic range

of
detection

and
that
the signal saturation ha
d

not
occurred.


Isolation of Mouse Left Ventricular Myocytes and Measure
ments of Cell Shortening and
Ca
2+

Transients

CirculationAHA/2005/583351/R1



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4

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Isolation of mouse left ventricular myocytes was carried out as described previously
.
6,12

To obtain intracellular Ca
2+

signals,
the cells were loaded with the acetoxymethyl ester form of
fura
-
2 (Fura
-
2/AM; 2

M). Myocytes were field
-
stimulated by a Grass S5 stimulator (0.5 Hz,
square waves). Ca
2+

transients were recorded as the 340/380 nm ratio of the resulting 510 nm
emissions. Al
l data were analyzed using software from FeliX and Ionwizard.


L
-
type Calcium Current


Isolated ventricular myocytes were maintained at room temperature (24°C) and perfused
with Tyrode’s solution containing (in mM) NaCl 140, KCl 5.4, MgCl
2

1, CaCl
2

1.8,

HEPES 5,
and glucose 10 (pH = 7.4). Ca
2+

currents were recorded using the whole
-
cell patch clamp
technique with an Axonpatch
-
200B amplifier
(Axon Instruments, Foster City, CA)
.
15

All drugs
were purchased from Sigma

(St. Louis, MO)
.
Data collection and analysis were performed using
pCLAMP software (Axo
n Instruments, Foster City, CA).


Structural NMR
-
Analysis

A shorter peptide, which contains amino acids (AA) 1 to 36 of phospholamban (PL
N 1
-
36), was used as a structural model. PL
N1
-
36 contains only 6 AA of the transmembrane domain
but is soluble in wate
r. Human
-

and mouse
-
PL
N1
-
36 were synthesized and purified as described
previously
.
16

Two
-
dimensional proton correlation NMR spectra were acqui
red at 17

C using an
800 MHz NMR spectrometer. Spectra were assigned in the conventio
nal way
. Structures were
generated by torsion angle
simulated annealing (DYANA 1.5)
.
17

Families of structures were
analyzed for restraint violation, backbone dihedral, and energy.


CirculationAHA/2005/583351/R1



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5

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SR Calcium Uptake


Oxalate
-
supported Ca
2+

uptake

in cardiac homogenates was measured by a modified
Millipore filtration technique
.
18

The data were analyzed by nonlinear regression using Origin
(version 6.1) software.


In Vitro

Phosphorylation


Mouse
-
PLN

and
human
-
PLN

hearts were perfused in a Langendorff mode for 15 minutes
to dephosphorylate any prote
ins, which were phosphorylated
in vivo
. Cardiac homogenates
(60

g) were then incubated with c
yclic AMP
-
dependent protein kinase (PKA) and endogenous
Ca
2+
/
calmodulin
-
dependent protein kinase

(Ca
2+
/CaM)
,

as describe previously
.
9

Reactions were
terminated with 30

l of SDS sample buffer 2 min (PKA) or 5 min (Ca
2+
/CaM) incubation,
which was associated with optimal phosphate incorporation in PLN. The samples were then
boiled at 100

C for 10 min to dissociate PLN pentamer to monome
r. 15

g of protein for each
reaction was subjected to 13% SDS
-
PAGE, and site
-
specific antibodies to PLN Ser
16

(Upstate)
or Thr
17

(Badrilla) were used for quantitative
immunoblotting
.
An alkaline phosphatase
-
conjugated anti
-
rabbit secondary antibodies (Cap
pel Division of Organon Teknika) were then
used according to standard procedures
.
6


Radioligand Binding Experiments for β
-
Adrene
rgic Receptor Density


Radioligand binding studies were performed as described previously.
19

Briefly, mouse
hearts were homogenized in buffer containing 5 mM Tris, 2mM EDTA pH 7.4, benzamidine

(5

g/ml) and s
oybean typsin inhibitor (5

g/ml). The
homogenate was centrifuged at 40,000 x g
for 10 minutes at 4

C. The resulting pellets were resuspended in 10 volumes of homogenization
CirculationAHA/2005/583351/R1



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6

-

buffer and centrifuged again. The pellet was resuspended in assay buffer (75 mM T
ris, 12.5 mM
MgCl
2
, 2 mM EDTA, pH 7.4) and aliquots were then incubated in a total volume of 250 µl at
room temperature, for 2 hours with ~ 400 pM
125
I labeled iodocyanopindolol

in the presence of
various isoproterenol concentrations
.
Maximal stimulation o
ccurred at
higher

isoproterenol
concentrations
than
those
observed
in intact cells, due to the use of isolated membrane
preparations.
Non
-
specific binding, was determined in the presence of 1 µM propranolol. To
stop the reaction, cold wash buffer (10 mM T
ris, pH 7.4) was added and vacuum filtration was
performed through Whatman GF/C glass fiber filters.


Histopathology

Histopathological studies with H&E,
Mas
s
on
-
trichrome, and
fluorescein
-
conjugated
wheat
-
germ agglutinin (FITC
-
WGA, Vector Laboratories, Bur
lingame, CA)
for cardiomyocyte
cross
-
sectional area were performed as previously described.
20

Specifically, for
FITC
-
WGA

labeling of the cell wall, 40 or more cell cross
-
sectional areas (from multiple sections) were
determined for eac
h heart (n=
6

hearts per group).


Statistics

Results are expressed as mean


S.E.
.

The number (n) of mice used is indicated.
Comparisons were made between
mouse
-
PLN and human
-
PLN mice
,
using
One
-
Way ANOVA

by
Systat 9.0

software

for multiple groups

(Table I)
,

and student’s
t

test for two groups.

Values
of P<0.05 are considered statistically significant.


References:



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