Loss of type 9 adenylyl cyclase triggers reduced ... - Semantic Scholar

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Deletion of AC9 does not alter PKA phosphorylation of Troponin I and ... or AC9-/- mice with rabbit IgG (control) or anti-Yotiao were subjected to WB analysis for ...
Lossoftype9adenylylcyclasetriggersreducedphosphorylationofHsp20and diastolicdysfunction  YongLi1,TanyaA.Baldwin1,YanWang,JananiSubramaniam1,AnibalGarzaCarbajal1,CameronS Brand2,ShaneRCunha1,andCarmenWDessauer1,* 1 Dept.IntegrativeBiologyandPharmacology,McGovernMedicalSchool,UniversityofTexasHealth ScienceCenter,Houston,TX77030   SupplementaryTable1.CardiacparametersforWTandAC9Ͳ/Ͳmice. WT AC9Ͳ/Ͳ Pvalue Parameter# MͲmodeEchocardiography  LVanteriorwall,diastole(mm) 0.70+/Ͳ0.06 0.72+/Ͳ0.05 n.s. LVanteriorwall,systole(mm) 0.90+/Ͳ0.08 1.01+/Ͳ0.08 n.s. LVinternaldiastolicdiameter 3.6+/Ͳ0.1 3.7+/Ͳ0.1 n.s. (mm) LVinternalsystolicdiameter 2.6+/Ͳ0.1 2.7+/Ͳ0.1 n.s. (mm) LVposteriordiastolicwall 0.76+/Ͳ0.08 0.69+/Ͳ0.04 n.s. (mm) LVposteriorsystolicwall 0.94+/Ͳ0.06 1.14+/Ͳ0.07 n.s. (mm) EF(%) 53+/Ͳ3 51+/Ͳ4 n.s. FS(%) 27+/Ͳ2 26+/Ͳ3 n.s. LVMass,corrected(mg) 73+/Ͳ12 73+/Ͳ7 n.s. Enddiastolicvolume(μL) 55+/Ͳ4 58+/Ͳ4 n.s. Endsystolicvolume(μL) 26+/Ͳ3 28+/Ͳ3 n.s.   



SupplementaryFigureLegends. FigS1.SANshowsconnexin45(Cx45)butnotCx43expression. FigS2.InhibitionofPKAabolishesisoproterenolͲstimulatedphosphorylationofHsp20.Ratneonatal cardiomyocyteswerepretreatedintheabsenceorpresenceof10ʅMofthePKAinhibitorH89for10 minfollowedbyvehicle(AT)orisoproterenol(1ʅM)for5min.CellswerelysedandsubjectedtoWB analysisforphosphorylationofHsp20(n=3).  FigS3.DeletionofAC9doesnotalterPKAphosphorylationofTroponinIandphospholamban.WTand AC9Ͳ/Ͳmicewereinjectedwithsalineorisoproterenol(2μg/gbodyweight,IP).Animalsweresacrificed4 minlaterandhearttissuewasharvested.Cardiacextractswerepreparedinthepresenceof phosphataseinhibitors.EqualproteinsupernatantsweresubjectedtoWBanalysiswithA)antiͲpͲ Troponin1andB)antiͲpͲPLN.ThecorrespondingtotalproteinwasquantitatedbyWB(n=5Ͳ7)andthe ratioofphosphoproteintototalisshownforeachreplicate. FigS4.ImmunoprecipitationofYotiaofailstopullͲdownHsp20inheart.IPofheartextractsfromWT orAC9Ͳ/ͲmicewithrabbitIgG(control)orantiͲYotiaoweresubjectedtoWBanalysisforHsp20and Yotiao(n=3).  FigS5.MutationofD399AinAC9hasdramaticallyreducedcatalyticactivity.Membraneswere preparedfromSf9cellsexpressingɴͲgal,AC9,orAC9ͲD399A.ACactivitywasmeasuredupon stimulationwith300nMGTPɶSͲGɲs(n=3).WBofmembraneproteinsisshown.  FigS6.FullͲlengthwesternblotsforfigures1Ͳ5inthemainpaper. 

Supplementary Figures. Fig S1

SAN

LV

SAN

LV

SAN

93 70

93 70

93 70

53

53

Cx43

LV

Cx45

53

41

41

41 30

30

GAPDH 30

22

22

18

22 18

18

Fig S2 AT

Iso

Iso +H89

1.0

pHsp20/ total

22

pHsp20

RNCMs

1.2

30

18 14

30

0.8 0.6 0.4 0.2

22

Hsp20

0.0

18 14

AT

Iso

Iso+H89

ISO

-

Fig S3 KO ISO

-

+

B

WT

- +

48 35 25 20 17 48 35

Trpn I

WT p-Trpn I / total

p-Trpn I

KO

+

WT

- +

53 41

KO

1.2

30

p-PLN

0.8

22 18

0.4

14 0.0

ISO

KO

1.2 0.8 0.4 0.0

ISO

- + - +

41

25 20 17

WT 1.6

p-PLN / total

A

30

PLN 22 18 14

- + - +

Fig S4 Lysates WT

KO

IP: IgG Yotiao IgG Yotiao WT KO M

IgG

22

Hsp20 18

235

Yotiao

170 135 93

IgG

70

Fig S5 AC activity (pmol/min/mg)



E-gal AC9 AC9d

1600

anti-AC9 1200

170 117 71

800 55 400

0

E-gal AC9 AC9d

41

Supplemental Fig 6. Full length western blots.

1C

WT IP: PI Yot AC9-

KO PI

Yot

E-galAC9 (Sf9)(Sf9) -245 -180 -135 -100 -75 -63 -48

Yotiao-

-245 -180 -135 -100 -75 -63 -48

Supplemental Fig 6. Full length western blots, cont.

2C

2D

AC9-/-

WT

IgG AK5 IgG AK5

PI Yot AK5 PI Yot AK5

-235 -170 -130 -93

Yotiao-

AC9-/-

WT

-245 -180 -135

AKAP5IgG

-100

-70

-75 -63

IgG

2F

2E

WT -235 -170 -130 -93 -70 -53

Yotiao-

IgG

-41

-30

-22 IgG

Yot

SAN

IgG

LV

Yot

IgG

Yot

RA

PI

Yotiao-

Yot

AC9-/PI

Yot -235 -170 -130

Supplemental Fig 6. Full length western blots, cont. 3A

3B

3C

-235 -170

-235 -170 -130

-235 -170

-93

-93

-70

-70

-130

-130 -93 -70

-53

-53

pi-PKA

pCREB-

-53

-41

-41

-41

-30 -30

-22

-22

-18 -14

-30 -22

pHsp20-

-18

-18

-9

ISO

- + WT

-9

-14

-14

ISO

- +

- + WT

- +

ISO

-9

KO

-235

KO

-130

-130

-170

-93

-93

-130

WT

- + -235 -170

-235 -170

KO

- +

-70

-93

-70

-70

-53

-53

-41

-41

-30

-22

-22

-18 -14

-18 -14

-9

-53

E-actin

CREB-

-30

-30

Hsp20-

-22 -18

-9

-14 -9

3E 3D

WT

SAN

LV

RA -235 -170 -130

KO -235 -170 -130 -93

-93 -70 -53

-70 -53

-41

-41 -30 -30

pHsp20-

-22

pHsp20-

-22 -18

-18 -14 -235 -170 -130

-235 -170 -130 -93

-93 -70

-70

-53 -53 -41

-41

Hsp20-

-41

-30

-30

-22

-22

-18

Hsp20-

-18 -14

KO

H sp 20 Ig G

Ig G

WT

WT KO

H sp 20

4A

Lysates -235 -170 -130 -93 -70

IgG -53

-41 -30 -22

Hsp20-

-18 -14

4B

IPs Flag-AC9

Total

4C

Flag AC9

-235 -170 -130

AC9-

-235 -170 -130

-93

-93

-70

-70

-53

-53

-41

-41

-30

-30

-22

-22

-18

-18 -14

-14

Yotiao-

Total Flag-AC9

t t Yo Yo 0 20+ 0 0 20+ 0 2 2 2 2 p p p p p p Hs -- Hs Hs Hs -- Hs Hs

t ot Yo +Y 0 20+ 0 0 0 0 2 2 2 2 2 p p p p p p Hs -- Hs Hs Hs -- Hs Hs

Hsp20-

IPs Flag-AC9

-235 -170

Yotiao-

-235 -170

-130

-130

-93

-93

-70 -70 -53 -53 -41 -41 -30 -30

-22

-22

-18 -14

-18 -14

AC9-

-235 -170 -130

-235 -170

-93

-130

-70

-93

-53

-70

-41

-53

-30

-41

-22 -30

-18 -14

Hsp20-

-22 -18 -14

Supplemental Fig 6. Full length western blots, cont.

5

--

ISO

GFP AC9d

- + - + - +

-235 -170 -130 -93 -70 -53 -41 -30 -22

p-Hsp20

-18 -14 -235 -170 -130 -93 -70 -53 -41 -30 -22

Hsp20

-18 -14 -235 -170 -130 -93 -70 -53 -41 -30

GFP -18 -14 -235 -170 -130

AC9

-93 -70 -53 -41 -30

Sf9 AC9