Depar t ment of Cel l Phy s i ol ogy
Satoshi Kurihara,Professor Iwao Ohtsuki,Visiting Professor Masato Konishi,Visiting Professor Norio Suda,Assistant Professor Yoichiro Kusakari,Assistant Professor Norio Fukuda,Assistant Professor
Gener al Summar y
The ma i n r e s e a r c h i nt e r e s t s of our de pa r t me nt a r e t he phy s i ol og y of mus c l e c ont r a c t i on a nd r e l a t e d s ubj e c t s .
Res ear ch Act i v i t i es
I nt r ac e l l ul ar r e g ul at i o n me c hani s ms o f t he c hang e s i n L- t y p e Ca c ur r e nt i nduc e d b y α- adr e no c e p t o r s t i mul at i o n i n t he p r e s e nc e o f aβ - adr e no c e p t o r ag o ni s t
We ha v e pr e v i ous l y s hown t ha t α- a dr e noc e pt or( AR)s t i mul a t i on a l one pot e nt i a t e s L- t y pe Ca c ur r e nt( I )t hr oug h α - AR- Gq- phos phol i pa s e C( PLC) ― pr ot e i n ki na s e C( PKC) ― c a l c i um / c a l modul i n de pe nde nt pr ot e i n ki na s e( Ca MK)I I pa t hwa y . How- e v e r , t he i nt e r a c t i on of α-a ndβ - AR s i g na l i ng on I ha s not be e n f ul l y c l a r i f i e d. Thi s y e a r ,we e x a mi ne d t he e f f e c t of α- AR s t i mul a t i on on L- t y pe Ca c ur r e nt( I )whe n β - AR i s s t i mul a t e d i n r a t v e nt r i c ul a r my oc y t e s us i ng a pe r f or a t e d pa t c h- c l a mp t e c hni que . We f ound t ha t α - AR s t i mul a t i on i nhi bi t s I i n t he pr e s e nc e of aβ - AR a g oni s t , whi c h i s oppos i t e t o t he e f f e c t obs e r v e d i n t he a bs e nc e of aβ - AR a g oni s t . We a l s o c onf i r me d t ha t t y r os i ne ki na s e ,whi c h mi g ht be a c t i v a t e d by α - AR- Gq s i g na l i ng ,i nhi bi t s β - AR s i g na l i ng a t t he r e c e pt or s i t e( or Gs pr ot e i n) . Thus ,I i s i nhi bi t e d. Thi s nov e l s i g na l i ng pa t hwa y by α - AR s t i mul a t i on c oul d s e r v e a s a r e g ul a t or y f e e dba c k me c ha - ni s m whe n t he c a t e c hol a mi ne c onc e nt r a t i on i nc r e a s e s unde r pa t hophy s i ol og i c a l c ondi - t i ons f or pr ot e c t i ng c a r di omy oc y t e s f r om Ca ov e r l oa d.
I nt r ac e l l ul ar r e g ul at i o n me c hani s ms o f t he c hang e s i n L- t y p e Ca c ur r e nt i nduc e d b y e ndo t he l i n- 1 s t i mul at i o n
Endot he l i n- 1( ET- 1 )i s a pot e nt v a s oc ons t r i c t i v e pe pt i de . Thi s pe pt i de ha s a di r e c t e f f e c t on c a r di omy oc y t e s whi c h pr oduc e s a pos i t i v e i not r opi c e f f e c t t hr oug h a n i nc r e a s e i n t he i nt r a c e l l ul a r Ca t r a ns i e nt . Howe v e r ,t he i nt r a c e l l ul a r me c ha ni s m of t he pos i t i v e i not r opi c e f f e c t r e ma i ns unc l e a r . Us i ng a pe r f or a t e d pa t c h- c l a mp a nd bi oc he m- i c a l me t hod,we f ound t ha t ET- 1 a c t i v a t e d I t hr oug h t he ET - r e c e pt or - Gq- PKC- Ca MKI I pa t hwa y ,a s i n t he c a s e of α - AR s i g na l i ng . We a s s ume t ha t ET- 1 s t i mul a - t i on ha s a pos i t i v e i not r opi c e f f e c t pa r t l y by i nc r e a s i ng Ca e nt r y t hr oug h L- t y pe Ca c ha nne l s . The de t a i l e d mol e c ul a r me c ha ni s m of t he c oupl i ng of ET- 1 s t i mul a t i on a nd Ca s i g na l i ng wi l l pr ov i de ne w i ns i g ht s i nt o t he f unc t i ona l r ol e s of ET- 1 s i g na l i ng unde r phy s i ol og i c a l a nd pa t hophy s i ol og i c a l c ondi t i ons i n c a r di a c mus c l e .
15 Research Activities 2008 The Jikei University School of Medicine
I nt r ac e l l ul ar me c hani s ms o f t he i nc r e as e i n Ca l e ak f r o m r y ano di ne r e c e p t o r b y β - AR s t i mul at i o n i n mo us e c ar di ac mus c l e
I n he a r t f a i l ur e ,c hr oni c c a t e c hol a mi ne r g i c s t i mul a t i on i nc r e a s e s di a s t ol i c Ca l e a ka g e f r om r y a nodi ne r e c e pt or s( Ry Rs )of t he s a r c opl a s mi c r e t i c ul um( SR) ,l e a di ng t o a r r hy t h- mi a a nd a de c r e a s e i n c ont r a c t i l i t y . The i nc r e a s e d Ca l e a ka g e f r om t he SR byβ - AR s t i mul a t i on mi g ht be due t o t he phos phor y l a t i on of Ry Rs t hr oug h t he a c t i v a t i on of PKA or Ca MKI I or bot h. I n t he pr e s e nt s t udy ,we i nt e nde d t o i de nt i f y whi c h ki na s e a c t i v a t i on i s r e s pons i bl e f or t he e nha nc e d Ca l e a ka g e f r om t he SR i nduc e d byβ - AR s t i mul a t i on us i ng a s a poni n- s ki nne d mul t i c e l l ul a r pr e pa r a t i on. We e x a mi ne d t he phos phor y l a t i on l e v e l s of Ry R a f t e r β - AR s t i mul a t i on by us i ng c omme r c i a l l y a v a i l a bl e a nt i bodi e s a g a i ns t t he PKA-a nd Ca MKI I - s pe c i f i c phos phor y l a t i on s i t e of Ry R. We f ound t ha t t he i nc r e a s e i n Ca l e a ka g e f r om t he SR a f t e r β - AR s t i mul a t i on i s r e s pon- s i bl e a t l e a s t f or t he i nc r e a s e i n PKA- de pe nde nt Ry R phos phor y l a t i on.
Si ng l e s ar c o me r e i mag i ng i n c ar di ac mus c l e
Ski nne d c a r di a c f i be r s e x hi bi t s pont a ne ous os c i l l a t or y c ont r a c t i ons( SPOCs )ov e r a br oa d r a ng e of i nt e r me di a t e a c t i v a t i ng c ondi t i ons ,na me l y ,a t a pCa of 6 . 0 t o 5 . 0 ( Ca - SPOC) ,or i n t he pr e s e nc e of Mg ADP a nd Pi unde r r e l a x i ng c ondi t i ons( ADP- SPOC) . We ha v e r e por t e d t ha t t he pe r i od of s a r c ome r i c os c i l l a t i ons i n f i be r s c or r e l a t e s wi t h t ha t of t he r e s t i ng he a r t be a t i n v a r i ous a ni ma l s pe c i e s . The pr e s e nt s t udy wa s pe r f or me d t o a na l y z e SPOCs i n s i ng l e c a r di omy oc y t e s of t he r a t . To e nha nc e t he qua l i t y of s a r c ome r e l e ng t h me a s ur e me nt ,we us e d qua nt um dot s c onj ug a t e d wi t h a n a nt i body a g a i ns t α - a c t i ni n t o v i s ua l i z e t he Z- l i ne pos i t i on dur i ng SPOC i n a s i ng l e s a r c ome r e . We me a s ur e d t he pe r i od a nd a mpl i t ude of ADP-SPOC a nd Ca - SPOC a t v a r i ous s a r c ome r e l e ng t hs a nd f ound t ha t t he pe r i od of s a r c ome r i c os c i l l a t i ons i s s i mi l a r t o t ha t obs e r v e d a t t he f i be r l e v e l . We a l s o me a s ur e d s a r c ome r e l e ng t hs i n i nt a c t c a r di omy oc y t e s wi t h qua nt um dot s a t v a r i ous s t i mul a t i on f r e que nc i e s . At l ow f r e - que nc i e s( e . g . ,1 Hz ) ,t he s hor t e ni ng a nd r e l e ng t he ni ng of t he s a r c ome r e dur i ng c ont r a c - t i on s i mpl y r e f l e c t e d t he c ha ng e s i n ［Ca ］ . Howe v e r ,a n i nc r e a s e i n s t i mul a t i on f r e que nc y t o t he phy s i ol og i c a l l e v e l( 3 ‑ 5 Hz )c a us e d a pha s e s hi f t of s hor t e ni ng a nd r e l e ng t he ni ng due t o e nha nc e me nt of t he r e l e ng t he ni ng s pe e d,r e s ul t i ng i n t he wa v e f or m be i ng s i mi l a r t o t ha t obs e r v e d dur i ng SPOC i n s ki nne d my oc y t e s . The s e f i ndi ng s s ug g e s t t ha t t he i nt r i ns i c a ut o- os c i l l a t or y pr ope r t y of s a r c ome r e s ma y c ont r i but e t o my oc a r di a l be a t i ng i n v i v o .
Pat ho p hy s i o l o g y o f c ar di ac mus c l e i n di l at e d c ar di o my o p at hy
We we r e s uppl i e d wi t h a di l a t e d c a r di omy opa t hy mode l mous e by Ky us hu Uni v e r s i t y ( knoc k- i n of di l a t e d c a r di omy opa t hy t r oponi n i n mous e he a r t ) . The my oc a r di a l c ont r a c t i l e pr ot e i ns of t he mode l mous e s howe d a de c r e a s e i n Ca s e ns i t i v i t y ,whi c h wa s pr ov e n by me a s ur i ng t he pCa - t e ns i on r e l a t i on of t he s ki nne d pr e pa r a t i ons . Pat ho p hy s i o l o g y o f s ke l e t al mus c l e
1 . Di s us e - i nduc e d c ha ng e s i n f a t i g a bi l i t y i n s ke l e t a l mus c l e
We ha v e r e por t e d t ha t l ong - t e r m hi ndl i mb i mmobi l i z a t i on( 6 we e ks )l owe r s t he e x pr e s -
16Research Activities 2008 The Jikei University School of Medicine
s i on of t he g i a nt pr ot e i n t i t i n i n t he s ol e us mus c l e of t he r a t ,r e s ul t i ng i n a de c r e a s e i n a c t i v e f or c e pr oduc t i on v i a a bnor ma l s a r c ome r i c or g a ni z a t i on. I n t he pr e s e nt s t udy ,we i nv e s t i g a t e d how i mmobi l i z a t i on a f f e c t s f a t i g a bi l i t y by us i ng Tr i t on X- 1 0 0 - t r e a t e d s i ng l e f i be r s t a ke n f r om t he s a me a ni ma l mode l . The i nt r a c e l l ul a r c onc e nt r a t i ons of i nor g a ni c phos pha t e( P)a nd H i nc r e a s e i n s ke l e t a l mus c l e dur i ng i nt e ns e e x e r c i s e ,r e s ul t i ng i n a f a l l i n a c t i v e f or c e . The r e f or e ,we e x a mi ne d t he e f f e c t s of c ha ng e s i n pH a nd t he i nor g a ni c phos pha t e( P)c onc e nt r a t i on on ma x i ma l Ca - a c t i v a t e d f or c e i n c ont r ol f i be r s a nd i mmobi l i z e d f i be r s . We f ound t ha t l owe r i ng t he pH f r om 7 . 0 t o 6 . 2 de c r e a s e d t he ma x i ma l f or c e i n bot h mus c l e s ,wi t h t he ma g ni t ude s i g ni f i c a nt l y g r e a t e r i n i mmobi l - i z e d f i be r s . Li ke wi s e ,t he i nhi bi t or y e f f e c t of P up t o 2 0 mM wa s mor e pr onounc e d i n i mmobi l i z e d f i be r s . The s e r e s ul t s s ug g e s t t ha t f a t i g a bi l i t y i s e nha nc e d i n i mmobi l i z e d mus c l e a nd t ha t t he me c ha ni s m i nc l ude s a de c r e a s e i n t he f r a c t i on of f or c e - g e ne r a t i ng c r os s - br i dg e s c oupl e d a nd t he a bnor ma l s a r c ome r i c or g a ni z a t i on.
Publications
O-Uchi J,Morimoto S,Kusakari Y,Shinji H, Obata T,Hongo K,Komukai K,Kurihara S.
Interaction ofα1-adrenoceptor subtypes with different G proteins induces opposite effects on cardiac L-type Ca channel.Circ Res 2008;
102:1378‑88.
Tanaka H,Suzuki H,Oht suki I,Ojima T (Lab Marine Biotechnol Microbiol,Grad Sch Fisheries Sci,Hokkaido Univ).Structure-function relation- ships of molluscan troponin T revealed by limited proteolysis.BBA 2008;1784:1037‑42.
Reviews and Books
Mizuno J,Arita H,Hanaoka K,Kusakari Y, Kurihara S(Dept Anesthesiol,Teikyo Univ,Dept Anesthesiol Pain Relief Center,JR Tokyo Gen Hosp).Novel assessment of intracellular cal- cium transient decay in cardiac muscle by curve- fitting with half-logistic function (in Japanese). Masui 2008;57:408‑9.
Fukuda N.Mechanism of the heart beat(in Japanese).Pariti 2008;23:44.
Mizuno J,Morita S,Araki J,Otsuji M ,Hanao- ka K,Kurihara S (Dept Anes thesiol,Teikyo Univ,Dept Cardiovasc Physiol,Okayama Univ Grad Sch Med,Dent Pharm Sci,Dept Anesth- esiol Fac Med,Univ Tokyo, Dept Anesthesiol Pain Relief Cent,JR Tokyo Gen Hosp).Curve-fit with hybrid logistic function for isovolumetric left ventricular pressure curve and isometric tension of cardiac muscles(in Japanese).Masui 2008;
57:1472‑84.
Mizuno J,Morita S,Araki J,Otsuji M ,Hanao- ka K,Kurihara S (Dept Anesthesiol,Teikyo Univ,Dept Cardiovasc Phys iol,Okayama Univ Grad Sch Med,Dent Pharm Sci,Dept Anesth- esiol Fac Med,Univ Tokyo, Dept Anesthesiol Pain Relief Cent,JR Tokyo Gen Hosp).Curve-fit with hybrid logistic function for intracellular cal- cium transient(in Japanese).Masui 2009;58:
67‑76.
17 Research Activities 2008 The Jikei University School of Medicine
