Ed Da

Nicoletta Rizzi

un nd de er rl li ie es s C CP PV VT T. .

increased cytosolic Ca 2+ sensitivity (Fig. 3B). Analysis of the Ca 2+ -spark characteristics in permeabilized cells essentially confirmed the results obtained in intact cells (Online Table II).

We estimated SR Ca 2+ content in permeabilized cells and found that at all tested [Ca 2+ ] i the caffeine-evoked [Ca 2+ ] i transient was significantly decreased in the RyR2 R4496C cells (Fig. 3C).

Thus the higher Ca 2+ spark occurrence in RyR2 R4496C myocytes was not due to either a higher level of Ca 2+ stored in the SR or an alteration of calsequestrin level evaluated by Western-blots (data not shown). Fig. 3D shows the luminal Ca 2+ dependence of Ca 2+ -sparks, apparently consistent with increased luminal Ca 2+ sensitivity. However cytosolic and luminal Ca 2+ vary concurrently. The high Ca 2+ -spark occurrence recorded in RyR2 R4496C at very low intracellular Ca 2+ might suggest that, rather than increasing RyR2 R4496C Ca 2+ sensitivity, this mutation renders the RyR2 intrinsically active. We repeated the experiments at zero Ca 2+ (0.5 mmol/L EGTA). In this condition, the occurrence of Ca 2+ -sparks was indistinguishable between WT and RyR2 R4496C cells (Fig. 3.E&F, left), indicating that RyR2 R4496C hyperactivity requires cytosolic Ca 2+ . To ensure that the SR was not depleted in our experimental conditions, we applied caffeine. A robust caffeine-induced Ca 2+ transient could be evoked (Fig. 3E&F, right), proving that there was significant luminal Ca 2+ to promote Ca 2+ -sparks. Altogether, our results show that the RyR2 R4496C mutation increases the Ca 2+ sensitivity of the channel. The RyR2 has 2 affinity Ca 2+ binding sites on the cytosolic portion: one of high affinity that activates the channel and one of low affinity that inactivates it. Since ryanodine binds to open RyRs, we examined the Ca 2+dependence of [ 3 H]ryanodine binding in heart crude membrane preparations. Bell-shape curves were obtained for both WT and RyR2 R4496C but Ca 2+ -induced maximal activation of RyR2 R4496C

was reached at one order of magnitude lower in RyR2 R4496C (Fig. 3G), indicating that the cytosolic Ca 2+ sensitivity of RyR2 is greatly increased. We normalized the [

s sp pa ar rk ks s i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s

  ec ch ho ol la am mi in ne er rg gi ic c p po ol ly ym mo or rp ph hi ic c v ve en nt tr ri ic cu ul la ar r t ta ac ch hy yc ca ar rd di ia a ( (C CP PV VT T) ), , s su ug gg ge es st ti in ng g t th ha at t a al lt te er ra at ti io on ns s i Ca at te ec ch ho ol la am mi in ne er rg gi ic c p po ol ly ym mo or rp ph hi ic c v ve en nt tr ri ic cu ul la ar r t ta ac ch hy yc ca ar rd di ia a ( (C CP PV VT T) ) i is s a an n i in nh he er ri it te ed d a ar rr rh hy yt th hm mo og ge en ni ic c d di is se ea as se e c ch ha ar ra ac ct te er ri iz ze ed d b by y s st tr re es ss s--i in nd du uc ce ed d, , a ad dr re en ne er rg gi ic ca al ll ly y m me ed di ia at te ed d b bi id di ir re ec ct ti io on na al l o or r p po ol ly ym mo or rp ph hi ic c v ve en nt tr ri ic cu ul la ar r t ta ac ch hy yc ca ar rd di ia a o oc cc cu ur rr ri in ng g i

	A AB BS ST TR RA AC CT T C Ca a 2+ 2+ s se en ns si it ti iv vi it ty y o of f m mu ut ta an nt t R Ry yR R2 2; ; t th hi is s a ab bn no or rm ma al li it ty y i is s f fu ur rt th he er r a au ug gm me en nt te ed d b by y e ex xp po os su ur re e t to o M MA AT TE ER RI IA AL LS S A AN ND D M ME ET TH HO OD DS S W We e e es st ti im ma at te ed d S SR R C Ca a 2+ 2+ l lo oa ad d i in n q qu ui ie es sc ce en nt t v ve en nt tr ri ic cu ul la ar r m my yo oc cy yt te es s. . R Ry yR R2 2 R4496C R4496C s sh ho ow we ed d t th ha at t i is so op pr ro ot te er re en no ol l i in nc cr re ea as se ed d t th he e n nu um mb be er r o of f s si it te es s i in n b bo ot th h W WT T a an nd d R Ry yR R2 2 R4496C R4496C c ce el ll ls s. . T Th hi is s i in nd di ic ca at te es s
	C Ca ar rd di ia ac c r ry ya an no od di in ne e r re ec ce ep pt to or r ( (R Ry yR R2 2) ) m mu ut ta at ti io on ns s a ar re e a as ss so oc ci ia at te ed d w wi it th h a au ut to os so om ma al l d do om mi in na an nt t i is so op pr ro ot te er re en no ol l a an nd d i in nc cr re ea as si in ng g p pa ac ci in ng g r ra at te es s. . V Ve en nt tr ri ic cu ul la ar r c ca ar rd di io om my yo oc cy yt te es s f fr ro om m m ma al le e a an nd d f fe em ma al le e R Ry yR R2 2 R4496C R4496C + +/ /--m mi ic ce e ( (R Ry yR R2 2 R4496C R4496C ) ) a an nd d t th he ei ir r r re ed du uc ce ed d S SR R C Ca a 2+ 2+ c co on nt te en nt t ( (F F/ /F F 0 0 : : 7 7. .0 0± ±0 0. .5 5, , n n= =1 11 1 i in n R Ry yR R2 2 R4496C R4496C v vs s. . 8 8. .5 5± ±0 0. .5 5 i in n W WT T, , n n= =1 10 0, , p p< <0 0. .0 05 5) ), , t th ha at t t th he e R Ry yR R2 2 R4496C R4496C c ce el ll ls s p pr re es se en nt te ed d m mo or re e C Ca a 2+ 2+ --s sp pa ar rk ks s d du ue e t to o t th he e e ex xi is st te en nc ce e o of f m mo or re e a ac ct ti iv ve e R Ry yR R2 2
	c ca at te in n C Ca a 2+ 2+ in n s st tr ru uc ct tu ur ra al ll ly y n no or rm ma al l h he ea ar rt ts s 1 1 . . D Du ur ri in ng g e ex xe er rc ci is se e o or r a ac cu ut te e w wi il ld d t ty yp pe e R Ry yR R2 2 R4496C R4496C --/ /--( (W WT T) ) l li it tt te er rm ma at te es s w we er re e i is so ol la at te ed d u us si in ng g a a s st ta an nd da ar rd d e en nz zy ym ma at ti ic c d di ig ge es st ti io on n 17 17 . . r ru ul li in ng g o ou ut t S SR R C Ca a 2+ 2+ o ov ve er rl lo oa ad d. . N No o m ma aj jo or r a al lt te er ra at ti io on n i in n C Ca a 2+ 2+ --s sp pa ar rk k c ch ha ar ra ac ct te er ri is st ti ic cs s w wa as s o ob bs se er rv ve ed d c cl lu us st te er rs s ( (F Fi ig g. .2 2E E) ). . W We e a al ls so o m me ea as su ur re ed d t th he e m ma ax xi im mu um m n nu um mb be er r o of f C Ca a 2+ 2+ --s sp pa ar rk ks s r re ec co or rd de ed d a at t t th he e s sa am me e
	h ha an nd dl li in ng g u un nd de er rl li ie e t th hi is s d di is se ea as se e. . H He er re e w we e a an na al ly yz ze e t th he e u un nd de er rl ly yi in ng g C Ca a 2+ 2+ r re el le ea as se e d de ef fe ec ct t t th ha at t l le ea ad ds s t to o e em mo ot ti io on ns s, , C CP PV VT T p pa at ti ie en nt ts s d de ev ve el lo op p l li if fe e--t th hr re ea at te en ni in ng g v ve en nt tr ri ic cu ul la ar r a ar rr rh hy yt th hm mi ia as s l le ea ad di in ng g t to o s sy yn nc co op pe e o or r [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a an nd d C Ca a 2+ 2+ --s sp pa ar rk ks s w we er re e v vi ie ew we ed d i in n i is so ol la at te ed d m my yo oc cy yt te es s b by y c co on nf fo oc ca al l m mi ic cr ro os sc co op py y ( (O On nl li in ne e T Ta ab bl le e I I) ). . s si it te e i in n e ea ac ch h g gr ro ou up p a an nd d f fo ou un nd d t th ha at t t th hi is s w wa as s a al ls so o s si ig gn ni if fi ic ca an nt tl ly y i in nc cr re ea as se ed d i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s
	a ar rr rh hy yt th hm mi ia a i in n c ca ar rd di io om my yo oc cy yt te es s i is so ol la at te ed d f fr ro om m h he et te er ro oz zy yg go ou us s k kn no oc ck k--i in n m mi ic ce e c ca ar rr ry yi in ng g t th he e R Ry yR R2 2 R4496C R4496C s su ud dd de en n d de ea at th h. . T Th he e f fi ir rs st t R Ry yR R2 2 m mu ut ta at ti io on n i id de en nt ti if fi ie ed d i in n a a C CP PV VT T f fa am mi il ly y w wa as s R R4 44 49 97 7C C 2 2 . . T To od da ay y, , m mo or re e a an nd d a an na al ly yz ze ed d u us si in ng g h ho om me em ma ad de e r ro ou ut ti in ne es s. . W We e p pe er rf fo or rm me ed d W We es st te er rn n--b bl lo ot ts s o of f t to ot ta al l a an nd d p ph ho os sp ph ho or ry yl la at te ed d R Ry yR R2 2 i in n h he ea ar rt ts s i in n b ba as sa al l a an nd d f fu ur rt th he er r e en nh ha an nc ce ed d b by y β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n ( (F Fi ig g. .2 2F F) ). . T Ta ak ke en n t to og ge et th he er r, , t th he es se e d da at ta a s su ug gg ge es st t
	m mu ut ta at ti io on n. . R Ry yR R2 2 R4496C R4496C --/ /--l li it tt te er rm ma at te es s ( (w wi il ld d t ty yp pe e, , W WT T) ) w we er re e u us se ed d a as s c co on nt tr ro ol ls s. . [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s w we er re e t th ha an n 7 70 0 R Ry yR R2 2 m mu ut ta at ti io on ns s h ha av ve e b be ee en n r re ep po or rt te ed d ( (http://www.fsm.it/cardmoc/ http://www.fsm.it/cardmoc/) ), , a an nd d t th he ey y c co om mp pr ri is se e t th he e A A c co om mp pl le et te e M Me et th ho od ds s s se ec ct ti io on n c ca an n b be e f fo ou un nd d i in n t th he e O On nl li in ne e S Su up pp pl le em me en nt ta al l M Ma at te er ri ia al l. . c co on nd di it ti io on ns s a an nd d f fo ol ll lo ow wi in ng g i is so op pr ro ot te er re en no ol l p pe er rf fu us si io on n. . N Ne ei it th he er r t th he e t to ot ta al l R Ry yR R2 2 e ex xp pr re es ss si io on n n no or r t th he e l le ev ve el l t th ha at t R Ry yR R2 2 R4496C R4496C c ce el ll ls s p pr re es se en nt t m mo or re e C Ca a 2+ 2+ --s sp pa ar rk ks s b be ec ca au us se e o of f m mo or re e a ac ct ti iv ve e R Ry yR R2 2s s c cl lu us st te er rs s a an nd d a a
	o ob bt ta ai in ne ed d b by y f fi ie el ld d s st ti im mu ul la at ti io on n i in n f fl lu uo o--3 3 l lo oa ad de ed d c ca ar rd di io om my yo oc cy yt te es s a an nd d v vi ie ew we ed d u us si in ng g c co on nf fo oc ca al l m mo os st t c co om mm mo on n g ge en ne et ti ic c s su ub bt ty yp pe e o of f C CP PV VT T 3-7 3-7 , , a al lt th ho ou ug gh h m mu ut ta at ti io on ns s i in n t th he e c ca al ls se eq qu ue es st tr ri in n g ge en ne e c ca an n a al ls so o o of f p ph ho os sp ph ho or ry yl la at te ed d R Ry yR R2 2 ( (P P--S Se er r 2 28 80 09 9) ) w wa as s d di if ff fe er re en nt t b be et tw we ee en n W WT T a an nd d R Ry yR R2 2 R4496C R4496C ( (O On nl li in ne e F Fi ig g. .I I) ). . g gr re ea at te er r p pr ro ob ba ab bi il li it ty y o of f r re ep pe et ti it ti iv ve e o op pe en ni in ng gs s o of f t th he es se e c cl lu us st te er rs s i in n t th he e R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s. .
	m mi ic cr ro os sc co op py y. . I In n o ou ur r b ba as sa al l r re ec co or rd di in ng g c co on nd di it ti io on ns s ( (2 2 H Hz z s st ti im mu ul la at ti io on n r ra at te e) ), , [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a an nd d c ca au us se e C CP PV VT T 8,9 8,9 . . R RE ES SU UL LT TS S W We e a al ls so o p pe er rf fo or rm me ed d f fu un nc ct ti io on na al l e ex xp pe er ri im me en nt ts s c ch ha al ll le en ng gi in ng g t th he e c ce el ll ls s w wi it th h 1 1 μ μm mo ol l/ /L L i is so op pr ro ot te er re en no ol l. . We next explored whether RyR2 R4496C presents abnormal Ca 2+ sensitivity. We analyzed
	s sa ar rc co op pl la as sm mi ic c r re et ti ic cu ul lu um m ( (S SR R) ) C Ca a 2+ 2+ l lo oa ad d w we er re e s si im mi il la ar r i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C c ce el ll ls s. . H Ho ow we ev ve er r, , p pa ac ce ed d D Di iv ve er rg gi in ng g r re es su ul lt ts s a an nd d c co on nc cl lu us si io on ns s h ha av ve e b be ee en n g ge en ne er ra at te ed d f fr ro om m e ex xp pr re es ss si io on n s st tu ud di ie es s o of f A Ab bn no or rm ma al l C Ca a 2+ 2+ r re el le ea as se e i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s T Th hi is s p pr ro oc ce ed du ur re e i in nc cr re ea as se ed d C Ca a 2+ 2+ --s sp pa ar rk k o oc cc cu ur rr re en nc ce e i in n b bo ot th h W WT T a an nd d R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s ( (F Fi ig g. .2 2B B) ) Ca
	R Ry yR R2 2 R4496C R4496C v ve en nt tr ri ic cu ul la ar r m my yo oc cy yt te es s p pr re es se en nt te ed d a ab bn no or rm ma al l C Ca a 2+ 2+ r re el le ea as se e d du ur ri in ng g t th he e d di ia as st to ol li ic c p pe er ri io od d, , R Ry yR R2 2 R4496C R4496C i in n h he et te er ro ol lo og go ou us s s sy ys st te em ms s. . J Ji ia an ng g a an nd d c co ol ll la ab bo or ra at to or rs s s sh ho ow we ed d t th ha at t R Ry yR R2 2 R4496C R4496C ( (t th he e E El le ec ct tr ro op ph hy ys si io ol lo og gi ic ca al l e ex xp pe er ri im me en nt ts s i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s h ha av ve e e ev vi id de en nc ce ed d D DA AD Ds s a an nd d t tr ri ig gg ge er re ed d b by y t th he e s sa am me e p pe er rc ce en nt ta ag ge e ( (F Fi ig g. .2 2C C) ). . C Ca a 2+ 2+ --s sp pa ar rk k c ch ha ar ra ac ct te er ri is st ti ic cs s i in n t th he e p pr re es se en nc ce e o of f i is so op pr ro ot te er re en no ol l
	v vi ie ew we ed d a as s C Ca a 2+ 2+ --w wa av ve es s, , c co on ns si is st te en nt t w wi it th h t th he e o oc cc cu ur rr re en nc ce e o of f d de el la ay ye ed d a af ft te er r--d de ep po ol la ar ri iz za at ti io on ns s. . T Th he e m mo ou us se e e eq qu ui iv va al le en nt t o of f t th he e h hu um ma an n R Ry yR R2 2 R4497C R4497C m mu ut ta at ti io on n) ), , w wh he en n e ex xp pr re es ss se ed d i in n h hu um ma an n e em mb br ry yo on ni ic c a ac ct ti io on n p po ot te en nt ti ia al ls s i in n t th he e p pr re es se en nc ce e o of f a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n a af ft te er r s st to op pp pi in ng g e el le ec ct tr ri ic ca al l s st ti im mu ul la at ti io on n 15 15 . . a ar re e p pr ro ov vi id de ed d i in n O On nl li in ne e T Ta ab bl le e I I. . S Si im mi il la ar r r re es su ul lt ts s w we er re e f fo ou un nd d u us si in ng g a a l lo ow we er r i is so op pr ro ot te er re en no ol l
	o oc cc cu ur rr re en nc ce e o of f t th hi is s a ab bn no or rm ma al l C Ca a 2+ 2+ r re el le ea as se e w wa as s e en nh ha an nc ce ed d a at t f fa as st te er r s st ti im mu ul la at ti io on n r ra at te es s a an nd d b by y β β--k ki id dn ne ey y ( t to o l lu um mi in na al l C Ca a 2+ 2+ . . 10 10 F Fi ig gu ur re e 1 1 s sh ho ow ws s C Ca a 2+ 2+ i im ma ag ge es s i in n v ve en nt tr ri ic cu ul la ar r m my yo oc cy yt te es s p pa ac ce ed d a at t 4 4 H Hz z i in n t th he e p pr re es se en nc ce e o of f 1 1 μ μm mo ol l/ /L L c co on nc ce en nt tr ra at ti io on n ( (1 10 00 0 n nm mo ol l/ /L L) ) ( (O On nl li in ne e F Fi ig g. .I II IA A) ). . F Fu ur rt th he er rm mo or re e, , t tr re ea at tm me en nt t o of f R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s w wi it th h
	Ca a 2+ 2+ --s sp pa ar rk ks s w we er re e m mo or re e Ry yR R2 2 R4497C of f R ac ct ti iv vi it ty y o ba as sa al l a th he e b in n t di if ff fe er re en nc ce e i no o d fo ou un nd d n au ut th ho or rs s f ot th he er r a Ho ow we ev ve er r, , o H R4497C , , b bu ut t i in ns st te ea ad d i is so op pr ro ot te er re en no ol l. . T Th he e W WT T c ca ar rd di io om my yo oc cy yt te e s sh ho ow we ed d n no o s sp po on nt ta an ne eo ou us s C Ca a 2+ 2+ r re el le ea as se e a af ft te er r e el le ec ct tr ri ic ca al l e ei it th he er r a a P PK KA A b bl lo oc ck ke er r ( (K KT T5 57 72 20 0) ) o or r a a C Ca aM MK KI II I b bl lo oc ck ke er r ( (K KN N9 93 3) ) f fa ai il le ed d t to o d de ec cr re ea as se e C Ca a 2+ 2+ --s sp pa ar rk k
	f fr re eq qu ue en nt t i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s, , i in nd di ic ca at ti in ng g i in nc cr re ea as se ed d R Ry yR R2 2 R4496C R4496C a ac ct ti iv vi it ty y. . W Wh he en n p pe er rm me ea ab bi il li iz ze ed d s sh ho ow we ed d i in nc cr re ea as se ed d a ac ct ti iv vi it ty y a an nd d g ga at ti in ng g f fr re eq qu ue en nc cy y a af ft te er r p pr ro ot te ei in n k ki in na as se e A A ( (P PK KA A) ) p ph ho os sp ph ho or ry yl la at ti io on n 11 11 p pa ac ci in ng g i in nt te er rr ru up pt ti io on n ( (F Fi ig g. .1 1A A) ). . O On n t th he e c co on nt tr ra ar ry y, , t th he e R Ry yR R2 2 R4496C R4496C c ce el ll l s sh ho ow we ed d C Ca a 2+ 2+ --w wa av ve es s e ev vo ok ki in ng g f fr re eq qu ue en nc cy y ( (O On nl li in ne e F Fi ig g. .I II II I) ). . T Th he es se e d da at ta a r ru ul le e o ou ut t a an n i in nc cr re ea as se e i in n t th he e t to ot ta al l R Ry yR R2 2 e ex xp pr re es ss si io on n o or r a a
	c ce el ll ls s w we er re e e ex xp po os se ed d t to o d di if ff fe er re en nt t c cy yt to os so ol li ic c [ [C Ca a 2+ 2+ ] ] i i , , R Ry yR R2 2 R4496C R4496C s sh ho ow we ed d a a d dr ra am ma at ti ic c i in nc cr re ea as se e i in n C Ca a 2+ 2+ o or r s sa ar rc co op pl la as sm mi ic c r re et ti ic cu ul lu um m ( (S SR R) ) C Ca a 2+ 2+ o ov ve er rl lo oa ad d 12 12 . . T Th he e e ex xp pr re es ss si io on n s st tu ud di ie es s w we er re e c ca ar rr ri ie ed d o ou ut t i in n a a t tw wo o [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s, , j ju us st t a af ft te er r s st ti im mu ul la at ti io on n s st to op pp pe ed d, , w wh hi ic ch h w wa as s f fo ol ll lo ow we ed d b by y s se ev ve er ra al l C Ca a 2+ 2+ --w wa av ve es s, , h hi ig gh he er r l le ev ve el l o of f b ba as sa al l p ph ho os sp ph ho or ry yl la at ti io on n a as s a an n e ex xp pl la an na at ti io on n f fo or r t th he e h hi ig gh he er r C Ca a 2+ 2+ --s sp pa ar rk k o oc cc cu ur rr re en nc ce e i in n
	s se en ns si it ti iv vi it ty y. . I Is so op pr ro ot te er re en no ol l i in nc cr re ea as se ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t a am mp pl li it tu ud de e a an nd d C Ca a 2+ 2+ s sp pa ar rk k f fr re eq qu ue en nc cy y t to o t th he e v va ar ri ie et ty y o of f m mo od de el ls s, , w wh hi ic ch h m ma ay y e ex xp pl la ai in n t th he e i in nh ho om mo og ge en ne eo ou us s f fi in nd di in ng gs s. . F Fu ur rt th he er rm mo or re e, , h he et te er ro ol lo og go ou us s c co on ns si is st te en nt t w wi it th h t tr ri ig gg ge er re ed d a ac ct ti iv vi it ty y a an nd d D DA AD Ds s ( (F Fi ig g. .1 1B B) ). . T Th he es se e d da at ta a s su ug gg ge es st t t th ha at t a ab bn no or rm ma al l C Ca a 2+ 2+ R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s. .
	s sa am me e e ex xt te en nt t i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C c ce el ll ls s, , i in nd di ic ca at ti in ng g t th ha at t t th he e β β--a ad dr re en ne er rg gi ic c s se en ns si it ti iv vi it ty y o of f R Ry yR R2 2 R4496C R4496C s sy ys st te em ms s l la ac ck k c ca ar rd di ia ac c i in nt tr ra ac ce el ll lu ul la ar r e en nv vi ir ro on nm me en nt t w wi it th h a al ll l t th he e R Ry yR R2 2 a ac cc ce es ss so or ry y p pr ro ot te ei in ns s 13 13 a an nd d m mo os st t r re el le ea as se e u un nd de er rl li ie es s t th he e e el le ec ct tr ri ic ca al l a ab bn no or rm ma al li it ti ie es s i in n R Ry yR R2 2 R4496C R4496C c ca ar rd di io om my yo oc cy yt te es s. . S Si in nc ce e D DA AD Ds s a ar re e R Re es st ti in ng g c cy yt to op pl la as sm mi ic c [ [C Ca a 2+ 2+ ] ] i, i, m me ea as su ur re ed d u us si in ng g F Fu ur ra a--2 2, , w wa as s s si im mi il la ar r b be et tw we ee en n W WT T a an nd d
	c ce el ll ls s r re em ma ai in ne ed d u un na al lt te er re ed d. . T Th hi is s e ef ff fe ec ct t w wa as s i in nd de ep pe en nd de en nt t o of f p pr ro ot te ei in n e ex xp pr re es ss si io on n v va ar ri ia at ti io on ns s s si in nc ce e n no o C Ca a 2+ 2+ --h ha an nd dl li in ng g p pr ro ot te ei in ns s, , s so o a an na al ly ys si is s i in n n na at ti iv ve e c ca ar rd di ia ac c m my yo oc cy yt te es s i is s n no ow w c cr ri it ti ic ca al l t to o e el lu uc ci id da at te e t th he e i in ni it ti ia at te ed d b by y C Ca a 2+ 2+ --w wa av ve es s t th ha at t a ar re e i in n t tu ur rn n i in ni it ti ia at te ed d b by y C Ca a 2+ 2+ --s sp pa ar rk ks s 18 18 , , w we e a an na al ly yz ze ed d C Ca a 2+ 2+ --s sp pa ar rk ks s i in n R Ry yR R2 2 R4496C R4496C c ce el ll ls s ( (r ra at ti io os s: : 0 0. .5 56 6± ±0 0. .0 02 2 i in n 1 16 6 W WT T m my yo oc cy yt te es s, , 0 0. .5 57 7± ±0 0. .0 01 1 i in n 4 45 5 R Ry yR R2 2 R4496C R4496C c ce el ll ls s, ,
	d di if ff fe er re en nc ce e w wa as s f fo ou un nd d i in n t th he e t to ot ta al l o or r p ph ho os sp ph ho or ry yl la at te ed d R Ry yR R2 2 e ex xp pr re es ss si io on n l le ev ve el ls s. . I In n c co on nc cl lu us si io on n, , t th he e m me ec ch ha an ni is sm ms s b by y w wh hi ic ch h t th he e m mu ut ta at ti io on n l le ea ad ds s t to o c ca ar rd di ia ac c a ar rr rh hy yt th hm mi ia a. . R Ry yR R2 2 R4496C R4496C c ca ar rd di io om my yo oc cy yt te es s. . [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a an nd d w wa av ve es s c ch ha ar ra ac ct te er ri is st ti ic cs s a ar re e p pr re es se en nt te ed d b be el lo ow w. . p p> >0 0. .0 05 5) ). . T Th he er re ef fo or re e t th he e i in nc cr re ea as se ed d C Ca a 2+ 2+ --s sp pa ar rk k o oc cc cu ur rr re en nc ce e i in n q qu ui ie es sc ce en nt t R Ry yR R2 2 R4496C R4496C c ce el ll ls s w wa as s n no ot t
	a ar rr rh hy yt th hm mo og ge en ni ic c p po ot te en nt ti ia al l o of f t th he e R Ry yR R2 2 R4496C R4496C m mu ut ta at ti io on n i is s d du ue e t to o t th he e i in nc cr re ea as se ed d C Ca a 2+ 2+ s se en ns si it ti iv vi it ty y o of f R Re ec ce en nt tl ly y, , a a k kn no oc ck k--i in n m mo ou us se e--m mo od de el l c ca ar rr ri ie er r o of f t th he e R Ry yR R2 2 R4496C R4496C m mu ut ta at ti io on n w wa as s d de ev ve el lo op pe ed d 14 14 . . c ca au us se ed d b by y d di if ff fe er re en nc ce es s i in n t th he e r re es st ti in ng g i in nt tr ra ac ce el ll lu ul la ar r C Ca a 2+ 2+ . .
	F Fa ax x: : + +3 33 3--4 46 67 74 41 15 52 24 42 2 Ry yR R2 2 R4496C R R4496C , , w wh hi ic ch h i in nd du uc ce es s d di ia as st to ol li ic c C Ca a 2+ 2+ r re el le ea as se e a an nd d l lo ow we er rs s t th he e t th hr re es sh ho ol ld d f fo or r t tr ri ig gg ge er re ed d a ac ct ti iv vi it ty y. . T Th he ei ir r p ph he en no ot ty yp pe e p pr re es se en nt ts s e ex xt tr ra ao or rd di in na ar ry y s si im mi il la ar ri it ty y w wi it th h t th he e c cl li in ni ic ca al l m ma an ni if fe es st ta at ti io on ns s o of f p pa at ti ie en nt ts s c ca ar rr ry yi in ng g t th he e R Ry yR R2 2 R4497C R4497C m mu ut ta at ti io on n, , i in nc cl lu ud di in ng g t th he e d de ev ve el lo op pm me en nt t o of f b bi id di ir re ec ct ti io on na al l v ve en nt tr ri ic cu ul la ar r C Ca a 2+ 2+ -C Ca a 2+ 2+ --s sp pa ar rk ks s a ar re e p pr ro od du uc ce ed d b by y t th he e o op pe en ni in ng g o of f R Ry yR R2 2 c cl lu us st te er rs s. . T Th he e i in nc cr re ea as se e i in n t to ot ta al l C Ca a 2+ 2+ -F Fi ig gu ur re e 2 2A A s sh ho ow ws s r re ep pr re es se en nt ta at ti iv ve e i im ma ag ge es s o of f C Ca a 2+ 2+ --s sp pa ar rk ks s 19 19 . . C Ca a 2+ 2+ s sp pa ar rk k f fr re eq qu ue en nc cy y w wa as s d do ou ub bl le e i in n s sp pa ar rk k f fr re eq qu ue en nc cy y i in n R Ry yR R2 2 R4496C R4496C c co ou ul ld d b be e d du ue e t to o a a g gr re ea at te er r n nu um mb be er r o of f c cl lu us st te er rs s f fi ir ri in ng g C Ca a 2+ 2+ --s sp pa ar rk ks s o or r
	e e--m ma ai il l: : ana-maria.gomez@inserm.fr ana-maria.gomez@inserm.fr Ke ey yw wo or rd ds s: : C K Ca a 2+ 2+ --s sp pa ar rk ks s, , [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s, , r ry ya an no od di in ne e r re ec ce ep pt to or r, , e ex xc ci it ta at ti io on n--c co on nt tr ra ac ct ti io on n c co ou up pl li in ng g, , t ta ac ch hy yc ca ar rd di ia a. . W Wh he en n e ex xp po os se ed d t to o a ad dr re en na al li in ne e a an nd d c ca af ff fe ei in ne e, , t th he e R Ry yR R2 2 R4496C R4496C c ca ar rd di io om my yo oc cy yt te es s d de ev ve el lo op p R Ry yR R2 2 R4496C R4496C c ce el ll ls s c co om mp pa ar re ed d w wi it th h W WT T c ce el ll ls s ( (p p< <0 0. .0 00 01 1, , F Fi ig gu ur re e 2 2B B) ). . T Th hi is s c co ou ul ld d b be e d du ue e t to o a an n i in nc cr re ea as se e t to o t th he e i in nc cr re ea as se ed d p pr ro op pe en ns si it ty y o of f s so om me e c cl lu us st te er rs s t to o f fi ir re e r re ep pe et ti it ti iv ve el ly y, , b be ec co om mi in ng g " "e ea ag ge er r" " c cl lu us st te er rs s. . W We e
	h he ea ar rt t, , C CP PV VT T. . d de el la ay ye ed d a af ft te er r--d de ep po ol la ar ri iz za at ti io on ns s ( (D DA AD Ds s) ) 15 15 , , s su ug gg ge es st ti in ng g t th ha at t t tr ri ig gg ge er re ed d a ar rr rh hy yt th hm mi ia as s a ar re e e el li ic ci it te ed d b by y i in n ( (i i) ) t th he e S SR R C Ca a 2+ 2+ l lo oa ad d, , ( (i ii i) ) t th he e l le ev ve el l o of f R Ry yR R2 2 e ex xp pr re es ss si io on n a an nd d/ /o or r p ph ho os sp ph ho or ry yl la at ti io on n, , ( (i ii ii i) ) t th he e d di ia as st to ol li ic c a an na al ly yz ze ed d o ou ur r d da at ta a d di is sc cr ri im mi in na at ti in ng g s sp pe ec ci if fi ic c s si it te es s p pr re es se en nt ti in ng g m mu ul lt ti ip pl le e C Ca a 2+ 2+ --s sp pa ar rk ks s d du ur ri in ng g t th he e
	S Su ub bj je ec ct t c co od de es s: : [ [1 13 36 6] ] [ [1 13 32 2] ] [ [1 13 30 0] ] a ad dr re en ne er rg gi ic c a ac ct ti iv va at ti io on n 16 16 . . H He er re e w we e d de em mo on ns st tr ra at te e t th ha at t u un nt tr re ea at te ed d R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s h ha av ve e [ [C Ca a 2+ 2+ ] ] i i o or r ( (i iv v) ) c ch ha an ng ge es s i in n t th he e i in nt tr ri in ns si ic c c ch ha an nn ne el l p pr ro op pe er rt ti ie es s. .
	i in nc cr re ea as se ed d s sp po on nt ta an ne eo ou us s C Ca a 2+ 2+ r re el le ea as se e i in n d di ia as st to ol le e d du ur ri in ng g e el le ec ct tr ri ic ca al l p pa ac ci in ng g, , d du ue e t to o t th he e e en nh ha an nc ce ed d an nd d
	2

a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n, , w wh hi ic ch h a al ls so o i in nd du uc ce ed d t tr ri ig gg ge er re ed d a ac ct ti iv vi it ty y. . S Sp po on nt ta an ne eo ou us s C C (H HE EK K) ) c ce el ll ls s, , e ex xh hi ib bi it ts s i in nc cr re ea as se ed d b ba as sa al l a ac ct ti iv vi it ty y a an nd d i in nc cr re ea as se ed d s se en ns si it ti iv vi it ty y r re ec co or rd di in ng g t ti im me e ( (a ab bo ou ut t 2 20 0 s s) ). . F Fi ir ri in ng g s si it te es s w we er re e c co ou un nt te ed d a as s t th he e s si it te es s w wh he er re e w we e r re ec co or rd de ed d a at t l le ea as st t o on ne e C Ca a 2+ 2+ --s sp pa ar rk k. . F Fi ig g. .2 2D D s sh ho ow ws s t th ha at t t th he e R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s p pr re es se en nt te ed d m mo or re e f fi ir ri in ng g s si it te es s, , a 2+ -sparks in permeabilized cells exposed to various cytoplasmic [Ca 2+ ] i . Figure 3A illustrates enhanced Ca 2+ -sparks occurrence in a RyR2 R4496C cell at 30 nmol/L [Ca 2+ ] i . At all tested [Ca 2+ ] i , Ca 2+ -sparks were much more frequent in RyR2 R4496C than in WT myocytes, consistent with

ar rr rh hy yt th hm mo og ge en ni ic c a ac ct ti iv vi it ty y o of f R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s

	B Be ec ca au us se e [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t a am mp pl li it tu ud de e d de ep pe en nd ds s o on n S SR R C Ca a 2+ 2+ l lo oa ad d, , w we e i in nv ve es st ti ig ga at te ed d t th he e S SR R me ea as su ur re ed d C m Ca a 2+ 2+ --s sp pa ar rk ks s i in n t th he e d di ia as st to ol li ic c p pe er ri io od d a at t d di if ff fe er re en nt t s st ti im mu ul la at ti io on n f fr re eq qu ue en nc ci ie es s, , w wh he en n i it t w wa as s D DI IS SC CU US SS SI IO ON N a an nd d t th hi is s f fe ea at tu ur re e w wa as s f fu ur rt th he er r e en nh ha an nc ce ed d b by y β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n a an nd d p pa ac ci in ng g r ra at te e, , r re ea ac ch hi in ng g t to o r re ef fl le ec ct t a an n e en nh ha an nc ce em me en nt t o of f i it ts s o op pe en n p pr ro ob ba ab bi il li it ty y ( (P P o o ) ), , c co on ns si is st te en nt t w wi it th h d da at ta a o ob bt ta ai in ne ed d b by y s si in ng gl le e R Ry yR R2 2 R4496C R4496C c ce el ll l p pr ro op pe en ns si it ty y t to o t tr ri ig gg ge er r D DA AD Ds s a an nd d a al ll lo ow wi in ng g t th he e o oc cc cu ur rr re en nc ce e o of f s sp po on nt ta an ne eo ou us s a ac ct ti iv vi it ty y R Ry yR R2 2 R4496C R4496C . . T Th hi is s d di ia as st to ol li ic c C Ca a 2+ 2+ l le ea ak k i is s r re es sp po on ns si ib bl le e f fo or r b bo ot th h D DA AD Ds s a an nd d d de ec cr re ea as se ed d S SR R C Ca a 2+ 2+ l lo oa ad d A AC CK KN NO OW WL LE ED DG GE EM ME EN NT TS S
	C Ca a 2+ 2+ c co on nt te en nt t. . I Im ma ag ge es s o of f c ca af ff fe ei in ne e--e ev vo ok ke ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s e ev vo ok ke ed d a af ft te er r e el le ec ct tr ri ic ca al l s st ti im mu ul la at ti io on n a at t N Na at tu ur re e o of f t th he e a p po os ss si ib bl le e t to o d di is sc cr ri im mi in na at te e t th he em m. . D Du ur ri in ng g d di ia as st to ol le e, , t th he e m ma ax xi im mu um m n nu um mb be er r o of f C Ca a 2+ 2+ --s sp pa ar rk ks s* *s s -1 -1 i in n W We e s sh ho ow w f fo or r t th he e f fi ir rs st t t ti im me e t th ha at t b be ea at ti in ng g c ca ar rd di io om my yo oc cy yt te es s b be ea ar ri in ng g t th he e R Ry yR R2 2 R4496C R4496C m mu ut ta at ti io on n, , t th hr re es sh ho ol ld d f fo or r t tr ri ig gg ge er re ed d a ac ct ti iv vi it ty y. . c ch ha an nn ne el l a an na al ly ys se es s 10 10 . . O Ou ur r d da at ta a d de em mo on ns st tr ra at te e t th ha at t, , i in n i it ts s n no or rm ma al l e en nv vi ir ro on nm me en nt t ( (i i. .e e. ., , i in n n na at ti iv ve e ( (F Fi ig gs s. .1 1& &6 6) ) 29,30 29,30 . . a at t h hi ig gh h p pa ac ci in ng g r ra at te es s. . O Ou ur r f fi in nd di in ng gs s i in n c ca ar rd di io om my yo oc cy yt te es s p pr ro ov vi id de e a a l li in nk k b be et tw we ee en n t th he e d da at ta a o ob bs se er rv ve ed d F FI IG GU UR RE E L LE EG GE EN ND DS S
	H Hz z a ar re e s sh ho ow wn n i in n F Fi ig gu ur re e 4 4E E. . A As s s sh ho ow wn n i in n F Fi ig gu ur re e 4 4F F, , c ca af ff fe ei in ne e--e ev vo ok ke ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s w we er re e R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s u un nd de er r β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n i in nc cr re ea as se ed d w wi it th h p pa ac ci in ng g r ra at te es s ( (4 4. .7 7± ±1 1. .7 7 w wh he en n e eq qu ui iv va al le en nt t t to o t th ha at t f fo ou un nd d i in n s se ev ve er ra al l C CP PV VT T f fa am mi il li ie es s, , e ex xh hi ib bi it te ed d a ar rr rh hy yt th hm mo og ge en ni ic c b be eh ha av vi io or r r re el la at te ed d t to o T Th he e h hi ig gh he er r d di ia as st to ol li ic c C Ca a 2+ 2+ r re el le ea as se e i in n R Ry yR R2 2 R4496C R4496C c ce el ll ls s i is s c co or rr re el la at te ed d b by y h hi ig gh he er r f fr re eq qu ue en nc cy y o of f c ca ar rd di io om my yo oc cy yt te es s) ) R Ry yR R2 2 R4496C R4496C h ha as s a au ug gm me en nt te ed d C Ca a 2+ 2+ s se en ns si it ti iv vi it ty y r ra at th he er r t th ha an n i in nc cr re ea as se ed d P P o o p pe er r s se e. . w wi it th h t th he e h he et te er ro ol lo og go ou us s e ex xp pr re es ss si io on n o of f R Ry yR R2 2 R4496C R4496C m mu ut ta at ti io on n a an nd d t th he e m ma ac cr ro os sc co op pi ic c p ph he en no ot ty yp pe e W We e t th ha an nk k A An nt ta a A Ag gn ne e, , C Ci in nd dy y M Mo on nr ro os se e, , D Dr r. . C Cé éc ci il le e C Ca as ss sa an n a an nd d L Lu uc ci ia a G Ga ar rc cí ía a--R Ro om me er ro o f fo or r t th he ei ir r F Fi ig gu ur re e 1 1. . T Tr ri ig gg ge er re ed d a ac ct ti iv vi it ty y o ob bs se er rv ve ed d i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s. .
	s si ig gn ni if fi ic ca an nt tl ly y s sm ma al ll le er r ( (b by y 2 24 4. .7 7% %) ) a af ft te er r p pa ac ci in ng g t th he e c ce el ll l a at t 4 4 H Hz z i in n R Ry yR R2 2 R4496C R4496C c co om mp pa ar re ed d t to o W WT T T Th he e a ar rr rh hy yt th hm mi ic c a ac ct ti iv vi it ty y o of f t th he e i is so ol la at te ed d c ce el ll l p pr re es se en nt te ed d i in n F Fi ig g. .1 1 d do oe es s n no ot t r re ef fl le ec ct t t th he e r re ea al li it ty y o of f p pa ac ce ed d a at t 2 2 H Hz z, , n n= =9 9; ; 1 15 5. .0 0± ±4 4. .4 4 w wh he en n p pa ac ce ed d a at t 3 3 H Hz z, , n n= =4 4, , p p< <0 0. .0 05 5 c co om mp pa ar re ed d w wi it th h 2 2 H Hz z; ; a an nd d a a d dr ra am ma at ti ic ca al ll ly y e en nh ha an nc ce ed d o oc cc cu ur rr re en nc ce e o of f C Ca a 2+ 2+ --s sp pa ar rk ks s a an nd d C Ca a 2+ 2+ --w wa av ve es s d du ur ri in ng g d di ia as st to ol le e. . T Th hi is s s sp po on nt ta an ne eo ou us s C Ca a 2+ 2+ --s sp pa ar rk ks s ( (F Fi ig g. .2 2) ). . T Th hi is s i in nc cr re ea as se ed d a ac ct ti iv vi it ty y c co ou ul ld d d de ep pe en nd d o on n t th he e e ex xp pr re es ss si io on n o or r I In nd de ee ed d, , C Ca a 2+ 2+ --s sp pa ar rk k o oc cc cu ur rr re en nc ce e, , m me ea as su ur re ed d i in n p pe er rm me ea ab bi il li iz ze ed d c ce el ll ls s e ex xp po os se ed d t to o d di if ff fe er re en nt t [ [C Ca a 2+ 2+ ] ] i i A At t b ba as sa al l c co on nd di it ti io on ns s ( (2 2H Hz z i in n o ou ur r e ex xp pe er ri im me en nt ta al l s se et tt ti in ng g) ), , t th he e [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s i in n W WT T a an nd d o ob bs se er rv ve ed d a at t t th he e w wh ho ol le e h he ea ar rt t ( (E EC CG G) ). . B By y c ch ha ar ra ac ct te er ri iz zi in ng g t th he e f fu un nc ct ti io on n o of f m mu ut ta an nt t R Ry yR R2 2, , w we e p pr ro ov vi id de e e ex xc ce el ll le en nt t t te ec ch hn ni ic ca al l a as ss si is st ta an nc ce e. . W We e t th ha an nk k C Ch ha an nt ta al l J Ja ac cq qu ue et t a an nd d D Dr r. . K Ka ar ri im m C Ch he eb bl li i ( (I In ns st ti it tu ut te e o of f L Li in ne e--s sc ca an n i im ma ag ge es s o of f v ve en nt tr ri ic cu ul la ar r m my yo oc cy yt te es s i is so ol la at te ed d f fr ro om m ( (A A) ) a a W WT T m mo ou us se e a an nd d ( (B B) ) R Ry yR R2 2 R4496C R4496C
	m my yo oc cy yt te es s, , w wh hi il le e n no o s si ig gn ni if fi ic ca an nt t d di if ff fe er re en nc ce e w wa as s o ob bs se er rv ve ed d a at t l lo ow we er r f fr re eq qu ue en nc ci ie es s. . P Pl lo ot tt ti in ng g t th he e p pe ea ak k v ve en nt tr ri ic cu ul la ar r m my yo oc cy yt te es s, , w wh hi ic ch h a ar re e u un nd de er r c co on ns st ta an nt t e el le ec ct tr ri ic ca al l s st ti im mu ul la at ti io on n. . M Mo or re eo ov ve er r, , C CP PV VT T i is s 2 24 4. .8 8± ±5 5. .4 4 w wh he en n p pa ac ce ed d a at t 4 4 H Hz z, , n n= =5 5, , p p< <0 0. .0 00 01 1 c co om mp pa ar re ed d w wi it th h 2 2 H Hz z) ). . T Th hi is s c co ou ul ld d b be e d du ue e t to o t th he e e el le ev va at te ed d s sp po on nt ta an ne eo ou us s C Ca a 2+ 2+ r re el le ea as se e w wa as s f fu ur rt th he er r e en nh ha an nc ce ed d b by y β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n a an nd d p ph ho os sp ph ho or ry yl la at ti io on n l le ev ve el l o of f R Ry yR R2 2, , t th he e a am mo ou un nt t o of f C Ca a 2+ 2+ s st to or re ed d i in n t th he e S SR R, , a an nd d/ /o or r t th he e s se en ns si it ti iv vi it ty y o of f c co on nc ce en nt tr ra at ti io on ns s, , w wa as s s si ig gn ni if fi ic ca an nt tl ly y i in nc cr re ea as se ed d i in n R Ry yR R2 2 R4496C R4496C a at t a al ll l [ [C Ca a 2+ 2+ ] ] i i t te es st te ed d e ex xc ce ep pt t a at t z ze er ro o R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s w we er re e s si im mi il la ar r. . T Th he e [ [C Ca a 2+ M Mo ol le ec cu ul la ar r G Ge en ne et ti ic cs s o of f M Mo on nt tp pe el ll li ie er r) ) f fo or r b br re ee ed di in ng g m mi ic ce e. . W We e a al ls so o t th ha an nk k D Dr r. . A Ag gu us st tí ín n G Gu ue er rr re er ro o--2+ ] ] i i t tr ra an ns si ie en nt t d de ec ca ay y t ti im me es s w we er re e a al ls so o s si im mi il la ar r, , a a d de et ta ai il le ed d d de ef fi in ni it ti io on n o of f h ho ow w C CP PV VT T m mu ut ta at ti io on ns s c ca au us se e D DA AD D a an nd d t tr ri ig gg ge er re ed d a ar rr rh hy yt th hm mi ia as s. . m mo ou us se e d du ur ri in ng g e el le ec ct tr ri ic ca al l s st ti im mu ul la at ti io on n ( (4 4H Hz z) ). . T Th he e c co or rr re es sp po on nd di in ng g f fl lu uo or re es sc ce en nc ce e t tr ra ac ce es s a ar re e s sh ho ow wn n
	[ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t v vs s. . t th he e S SR R C Ca a 2+ 2+ l lo oa ad d f fo or r t th he e t th hr re ee e d di if ff fe er re en nt t p pa ac ci in ng g r ra at te es s ( (F Fi ig g. .4 4G G) ) p pr ro ov vi id de ed d i in nd du uc ce ed d b by y s st tr re es ss s, , m me ea an ni in ng g β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n a an nd d e el le ev va at te ed d h he ea ar rt t r rh hy yt th hm m. . D Du ur ri in ng g h hi ig gh he er r d di ia as st to ol li ic c [ [C Ca a 2+ 2+ ] ] i i i in nd du uc ce ed d b by y i in nc cr re ea as si in ng g p pa ac ci in ng g r ra at te es s. . T Th he e d di ia as st to ol li ic c C Ca a 2+ 2+ f fl lu uo or re es sc ce en nc ce e i in nc cr re ea as si in ng g p pa ac ci in ng g r ra at te es s, , m mi im mi ic ck ki in ng g h hu um ma an n e ex xe er rc ci is se e--i in nd du uc ce ed d v ve en nt tr ri ic cu ul la ar r t ta ac ch hy yc ca ar rd di ia a. . T Th he e h hi ig gh h R Ry yR R2 2 R4496C R4496C t to o l lu um mi in na al l 21 21 o or r c cy yt to os so ol li ic c C Ca a 2+ 2+ . . W We e f fo ou un nd d n no o d di if ff fe er re en nc ce e b be et tw we ee en n W WT T a an nd d R Ry yR R2 2 R4496C R4496C C Ca a 2+ 2+ , , i in nd di ic ca at ti in ng g t th ha at t t th he e c ch ha an nn ne el l n ne ee ed ds s C Ca a 2+ 2+ t to o b be ec co om me e h hy yp pe er ra ac ct ti iv ve e. . T Th he e R Ry yR R2 2 R4496C R4496C m mu ut ta at ti io on n i is s s su ug gg ge es st ti in ng g a a n no or rm ma al l f fu un nc ct ti io on n o of f s sa ar rc co o--e en nd do op pl la as sm mi ic c r re et ti ic cu ul lu um m C Ca a 2+ 2+ A AT TP Pa as se e ( (S SE ER RC CA A) ). . H Ho ow we ev ve er r, , F Fu ur rt th he er rm mo or re e, , t th he e i id de en nt ti if fi ic ca at ti io on n o of f a ab bn no or rm ma al l C Ca a 2+ H He er rn ná án nd de ez z a an nd d D Dr r. . J Jo os sé é A An nt to on ni io o A Ar ri ia as s--M Mo on nt ta añ ño o ( (C CI IN NV VE ES ST TA AV V--I IP PN N, , M Mé éx xi ic co o) ) f fo or r p pr ro ov vi id di in ng g u us s 2+ s se en ns si it ti iv vi it ty y i in n R Ry yR R2 2 a as s t th he e k ke ey y f fa ac ct to or r f fo or r b be el lo ow w. . R Re ed d l li in ne es s i in nd di ic ca at te e e el le ec ct tr ri ic ca al l s st ti im mu ul la at ti io on n. . A Af ft te er r p pa ac ci in ng g s st to op pp pe ed d, , t th he e W WT T c ce el ll l r re em ma ai in ns s s si il le en nt t
	s si im mi il la ar r c co or rr re el la at ti io on ns s i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s. . T Th he es se e r re es su ul lt ts s s su ug gg ge es st t t th ha at t a a d de ec cr re ea as se e i in n e el le ec ct tr ri ic ca al l s st ti im mu ul la at ti io on n a an nd d i in n t th he e p pr re es se en nc ce e o of f i is so op pr ro ot te er re en no ol l, , t th he e R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s d de ev ve el lo op pe ed d m me ea as su ur re ed d i in n t th he e s sa am me e c ce el ll ls s i in nc cr re ea as se ed d p pr ro og gr re es ss si iv ve el ly y w wi it th h p pa ac ci in ng g r ra at te e ( (3 39 9. .5 5± ±2 2. .7 7 a at t 2 2 H Hz z, , a ac ct ti iv vi it ty y o of f R Ry yR R2 2 R4496C R4496C w wa as s d du ue e t to o a a d dr ra am ma at ti ic c i in nc cr re ea as se e i in n i it ts s C Ca a 2+ 2+ s se en ns si it ti iv vi it ty y, , w wh hi ic ch h l lo ow we er re ed d t th he e h he ea ar rt ts s i in n t to ot ta al l R Ry yR R2 2 e ex xp pr re es ss si io on n, , F FK KB BP P1 12 2. .6 6 a as ss so oc ci ia at ti io on n 14 14 o or r R Ry yR R2 2 p ph ho os sp ph ho or ry yl la at ti io on n l le ev ve el l, , e ev ve en n l lo oc ca at te ed d i in n t th he e C C t te er rm mi in na al l p po or rt ti io on n o of f t th he e c ch ha an nn ne el l, , ( (c cy yt to os so ol li ic c s si id de e 24,25 24,25 ) ), , c cl lo os se e t to o t th he e p pr ro op po os se ed d t th he e r ra at te e--d de ep pe en nd de en nt t d de ec cr re ea as se e i in n [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a an nd d c co on nt tr ra ac ct ti io on n, , w wh hi ic ch h i is s n no or rm ma al l i in n m mi ic ce e a ar rr rh hy yt th hm mo og ge en ne es si is s s su up pp po or rt ts s t th he e i in nt te er re es st t o of f t th he e R Ry yR R2 2 i in n t th he e d de ev ve el lo op pm me en nt t o of f n no ov ve el l t th he er ra ap pe eu ut ti ic c w wi it th h t th he e f fa ac ci il li it ti ie es s a an nd d m ma at te er ri ia al ls s t to o r re ea al li iz ze e b bi in nd di in ng g e ex xp pe er ri im me en nt ts s. . w wh hi il le e t th he e R Ry yR R2 2 R4496C R4496C c ce el ll l s sh ho ow ws s C Ca a 2+ 2+ --w wa av ve es s t th ha at t i in nd du uc ce e t tw wo o f fu ul ll l c co on nt tr ra ac ct ti io on ns s f fo ol ll lo ow we ed d b by y
	S SR R C Ca a 2+ 2+ l lo oa ad d a ac cc co ou un nt ts s f fo or r t th he e r re ed du uc ct ti io on n i in n s sy ys st to ol li ic c [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a an nd d t th he e a as ss so oc ci ia at te ed d l lo ow we er r s sp po on nt ta an ne eo ou us s [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a an nd d a af ft te er r--c co on nt tr ra ac ct ti io on ns s, , c co on ns si is st te en nt t w wi it th h t tr ri ig gg ge er re ed d a ac ct ti iv vi it ty y, , a as s 4 46 6. .1 1± ±1 1. .6 6 a at t 3 3 H Hz z a an nd d 5 50 0. .9 9± ±3 3. .0 0 a at t 4 4 H Hz z, , p p< <0 0. .0 05 5 c co om mp pa ar re ed d w wi it th h 2 2 H Hz z) ). . r re el le ea as se e t th hr re es sh ho ol ld d t to o p pr ro od du uc ce e s sp po on nt ta an ne eo ou us s a ac ct ti iv vi it ty y d du ur ri in ng g t th he e d di ia as st to ol li ic c p pe er ri io od d. . a af ft te er r β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n ( (O On nl li in ne e F Fi ig g. .I I) ). . M Mo or re eo ov ve er r, , a al lt th ho ou ug gh h C Ca a 2+ 2+ --w wa av ve es s a an nd d h hi ig gh h C Ca a 2+ 2+ --m mo ol le ec cu ul la ar r r re eg gi io on n i in nv vo ol lv ve ed d i in n C Ca a 2+ 2+ --d de ep pe en nd de en nt t a ac ct ti iv va at ti io on n ( (r re es si id du ue es s 4 44 48 85 5--4 44 49 94 4) ) 23 23 . . T Th he e R Ry yR R h ha as s c ca ar rd di io om my yo oc cy yt te es s, , w wa as s m mo or re e p pr ro on no ou un nc ce ed d i in n R Ry yR R2 2 R4496C R4496C ( (F Fi ig g. .4 4) ). . T Th hi is s r re ed du uc ct ti io on n c ca an n b be e a ac cc co ou un nt te ed d t ta ar rg ge et ts s. . S SO OU UR RC CE ES S O OF F F FU UN ND DI IN NG G i is so ol la at te ed d C Ca a 2+ 2+ --w wa av ve es s c co on ns si is st te en nt t w wi it th h D DA AD Ds s. .
	c co on nt tr ra ac ct ti io on n o ob bs se er rv ve ed d a at t t th he e h hi ig gh he es st t p pa ac ci in ng g r ra at te es s i in n b bo ot th h c ce el ll l g gr ro ou up ps s. . I In nt te er re es st ti in ng gl ly y, , t th he e d de ec cr re ea as se e o op pp po os se ed d t to o t th he e W WT T c ce el ll ls s ( (F Fi ig g. .6 6A A& &B B) ). . D Du ur ri in ng g d di ia as st to ol le e, , t th he e R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s s sh ho ow we ed d M Mi ic ce e b be ea ar ri in ng g t th he e R Ry yR R2 2 R4496C R4496C m mu ut ta at ti io on n, , w wh hi ic ch h i is s t th he e e eq qu ui iv va al le en nt t t to o t th he e h hu um ma an n R Ry yR R2 2 R4497C R4497C s sp pa ar rk k f fr re eq qu ue en nc cy y u us su ua al ll ly y r re ef fl le ec ct t C Ca a 2+ 2+ o ov ve er rl lo oa ad d 18 18 , , R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s p pr re es se en nt te ed d t th hi is s b be eh ha av vi io or r h hi ig gh hl ly y r re ea ac ct ti iv ve e c cy ys st te ei in ne es s c ca ap pa ab bl le e o of f f fo or rm mi in ng g d di is su ul lf fi id de e b bo on nd ds s 26 26 . . I It t i is s t th hu us s p pl la au us si ib bl le e t th ha at t t th he e h hi ig gh hl ly y f fo or r b by y a a d de ec cr re ea as se e i in n S SR R C Ca a 2+ 2+ l lo oa ad d ( (F Fi ig g. .4 4) ). . T Th he er re ef fo or re e, , t th he e i in nc cr re ea as se e i in n d di ia as st to ol li ic c C Ca a 2+ 2+ l le ea ak k
	i in n S SR R l lo oa ad d w wi it th h i in nc cr re ea as si in ng g p pa ac ci in ng g r ra at te e w wa as s a ac cc ce en nt tu ua at te ed d i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s. . T To o g ge et t a an n i id de ea a s sp po on nt ta an ne eo ou us s C Ca a 2+ 2+ r re el le ea as se e a as s C Ca a 2+ 2+ --s sp pa ar rk ks s o or r s sm ma al ll l C Ca a 2+ 2+ --w wa av ve es s. . O Oc cc ca as si io on na al ll ly y ( (a ar rr ro ow w i in n F Fi ig g. .6 6B B) ), , m mu ut ta at ti io on n f fi ir rs st t i id de en nt ti if fi ie ed d i in n a a C CP PV VT T f fa am mi il ly y 14 14 , , p pr re es se en nt t v ve en nt tr ri ic cu ul la ar r t ta ac ch hy yc ca ar rd di ia a i in n r re es sp po on ns se e t to o e ev ve en n a at t l lo ow we er r S SR R C Ca a 2+ 2+ l lo oa ad d. . r re ea ac ct ti iv ve e c cy ys st te ei in ne e i in nt tr ro od du uc ce ed d b by y t th he e m mu ut ta at ti io on n, , i in nt te er ra ac ct ts s w wi it th h o ot th he er r c cy ys st te ei in ne es s o of f t th he e c ch ha an nn ne el l, , b be ec co om me es s c cr ri it ti ic ca al l f fo or r t th he e s sy ys st to ol li ic c f fu un nc ct ti io on n o on nl ly y a at t t th he e h hi ig gh he es st t p pa ac ci in ng g r ra at te es s. . S Su uc ch h a a n ne eg ga at ti iv ve e E Eu ur ro op pe ea an n U Un ni io on n g gr ra an nt t t to o S SR R a an nd d S SG GP P ( (F FP PG G, , L Li if fe e S Sc ci ie en nc ce e G Ge en no om mi ic cs s a an nd d B Bi io ot te ec ch hn no ol lo og gy y f fo or r F Fi ig gu ur re e 2 2. . C Ca a 2+ 2+ --s sp pa ar rk k i in n i in nt ta ac ct t R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s. .
	o of f h ho ow w m mu uc ch h C Ca a 2+ 2+ i is s r re el le ea as se ed d a at t e ea ac ch h t tw wi it tc ch h w wi it th h r re es sp pe ec ct t t to o t th he e t to ot ta al l a am mo ou un nt t o of f C Ca a 2+ 2+ s st to or re ed d, , w we e s sp po on nt ta an ne eo ou us s C Ca a 2+ 2+ r re el le ea as se es s r re ea ac ch he ed d t th he e t th hr re es sh ho ol ld d t to o p pr ro od du uc ce e n no on n--s su us st ta ai in ne ed d t tr ri ig gg ge er re ed d a ac ct ti iv vi it ty y a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n a an nd d c ca af ff fe ei in ne e i in n v vi iv vo o. . I Is so ol la at te ed d c ce el ll ls s w we er re e p pa at tc ch h--c cl la am mp pe ed d a an nd d a ac ct ti io on n O Ou ur r d da at ta a i in n p pe er rm me ea ab bi il li iz ze ed d c ca ar rd di io om my yo oc cy yt te es s s sh ho ow w t th ha at t a at t a al ll l c cy yt to op pl la as sm mi ic c [ [C Ca a 2+ 2+ ] ] i i t te es st te ed d, , t th he e i in nd du uc ci in ng g a a c co on nf fo or rm ma at ti io on n c ch ha an ng ge e t th ha at t r re en nd de er rs s t th he e R Ry yR R h hy yp pe er rs se en ns si it ti iv ve e t to o C Ca a 2+ 2+ . . T Th he e s st ta ai ir rc ca as se e, , o ob bs se er rv ve ed d i in n n no or rm ma al l m mi ic ce e, , m ma ay y d de ep pe en nd d o on n t th he e i in nt te er rv va al l b be et tw we ee en n t tw wo o c co on ns se ec cu ut ti iv ve e H He ea al lt th h, , C CT T2 20 00 05 5 N N°°0 01 18 88 80 02 2, , C CO ON NT TI IC CA A) ), , I In ns se er rm m a an nd d b by y A Ag ge en nc ce e N Na at ti io on na al le e p po ou ur r l la a R Re ec ch he er rc ch he e t to o A A. . L Li in ne e--s sc ca an n i im ma ag ge es s o ob bt ta ai in ne ed d i in n a a W WT T a an nd d a a R Ry yR R2 2 R4496C R4496C c ce el ll l ( (R R4 44 49 96 6C C) ) i in n t th he e a ab bs se en nc ce e ( (t to op p) ) o or r
	e ev va al lu ua at te ed d t th he e f fr ra ac ct ti io on na al l r re el le ea as se e b by y n no or rm ma al li iz zi in ng g t th he e e el le ec ct tr ri ic ca al ll ly y--e ev vo ok ke ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t t to o t th he e a an nd d a af ft te er r--c co on nt tr ra ac ct ti io on ns s. . C Ca a 2+ 2+ --s sp pa ar rk k e ev vo ok ke ed d C Ca a 2+ 2+ --w wa av ve es s w we er re e o ob bs se er rv ve ed d d du ur ri in ng g t th he e d di ia as st to ol li ic c p pe er ri io od d p po ot te en nt ti ia al ls s r re ec co or rd de ed d. . U Un nd de er r t th he es se e c co on nd di it ti io on ns s, , D DA AD Ds s a an nd d t tr ri ig gg ge er re ed d a ac ct ti iv vi it ty y c co ou ul ld d b be e r re ec co or rd de ed d C Ca a 2+ 2+ --s sp pa ar rk k f fr re eq qu ue en nc cy y w wa as s h hi ig gh he er r i in n R Ry yR R2 2 R4496C R4496C t th ha an n i in n W WT T c ce el ll ls s, , s sh ho ow wi in ng g t th ha at t t th he e R Ry yR R2 2 R4496C R4496C i is s c co on nf fo or rm ma at ti io on na al l c ch ha an ng ge e m mi ig gh ht t r re en nd de er r m mo or re e a ac cc ce es ss si ib bl le e t to o C Ca a 2+ 2+ t th he e E E3 39 98 87 7 r re es si id du ue e, , i id de en nt ti if fi ie ed d a as s t tw wi it tc ch he es s, , w wh hi il le e S SE ER RC CA A r re ep pl le en ni is sh he es s t th he e S SR R w wi it th h C Ca a 2+ 2+ . . E En nh ha an nc ce em me en nt t o of f t th hi is s p ph he en no om me en no on n i in n A AM MG G ( (C CO OD D2 20 00 05 5 a an nd d P Ph hy ys si io o2 20 00 06 6) ). . S Sp pa an ni is sh h " "M Mi in ni is st te er ri io o d de e E Ed du uc ca ac ci io on n y y C Ci ie en nc ci ia a" " ( (f fe el ll lo ow ws sh hi ip p t to o p pr re es se en nc ce e ( (b bo ot tt to om m) ) o of f 1 1 μ μm mo ol l/ /L L i is so op pr ro ot te er re en no ol l. . B B. . A Av ve er ra ag ge e o of f C Ca a 2+ 2+ --s sp pa ar rk k o oc cc cu ur rr re en nc ce e i in n W WT T c ce el ll ls s
	c ca af ff fe ei in ne e--e ev vo ok ke ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t i in n e ea ac ch h c ce el ll l t te es st te ed d. . W We e f fo ou un nd d n no o d di if ff fe er re en nc ce e b be et tw we ee en n W WT T a an nd d i in n 1 17 7% % o of f t th he e R Ry yR R2 2 R4496C R4496C c ce el ll ls s p pa ac ce ed d a at t 2 2 H Hz z ( (9 9 o ou ut t o of f 5 54 4 R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s) ). . T Th he e p pe er rc ce en nt ta ag ge e w wh he en n e el le ec ct tr ri ic ca al l p pa ac ci in ng g w wa as s i in nt te er rr ru up pt te ed d 15 15 . . H He er re e w we e f fo ou un nd d p pa ar ra al ll le el l e ev vi id de en nc ce e o of f s sp po on nt ta an ne eo ou us s h hy yp pe er ra ac ct ti iv ve e a at t a an ny y g gi iv ve en n [ [C Ca a 2+ 2+ ] ] i i , , a an nd d i in nd di ic ca at ti in ng g C Ca a 2+ 2+ h hy yp pe er rs se en ns si it ti iv vi it ty y. . H Ho ow we ev ve er r, , C Ca a 2+ 2+ --s sp pa ar rk k i im mp po or rt ta an nt t i in n C Ca a 2+ 2+ s se en ns si it ti iv vi it ty y 27 27 . . H Ho ow we ev ve er r, , t th he e l lo ow w a af ff fi in ni it ty y C Ca a 2+ 2+ s se en ns si in ng g o of f t th he e R Ry yR R2 2 R4496C R4496C s se ee em ms s R Ry yR R2 2 R4496C R4496C c ce el ll ls s s se ee em ms s t to o i in nd di ic ca at te e t th ha at t, , b be ec ca au us se e R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s s sh ho ow w m mo or re e C Ca a 2+ 2+ --w wa av ve es s M MF FV V) ). . O Ot th he er r g gr ra an nt ts s t to o S SG GP P: : T Te el le et th ho on n G GP P0 02 22 27 7Y Y0 01 1--G GG GP P0 04 40 06 66 6 a an nd d F Fo on nd do o p pe er r g In nv ve es st ti im me en nt ti i gl li i I w wi it th ho ou ut t ( (w wh hi it te e b ba ar r, , n n= =3 37 7) ) a an nd d w wi it th h 1 1 μ μm mo ol l/ /L L i is so op pr ro ot te er re en no ol l ( (h ha at tc ch he ed d b ba ar r, , n n= =1 10 0) ), , a an nd d i in n
	R Ry yR R2 2 R4496C R4496C c ce el ll ls s. . F Fo or r e ex xa am mp pl le e, , a at t 4 4 H Hz z, , f fr ra ac ct ti io on na al l r re el le ea as se e w wa as s 0 0. .8 80 0± ±0 0. .0 04 4 i in n W WT T c ce el ll ls s ( (n n= =8 8) ) a an nd d o of f c ce el ll ls s e ex xh hi ib bi it ti in ng g t th he es se e e ev ve en nt ts s d dr ra am ma at ti ic ca al ll ly y i in nc cr re ea as se ed d t to o 6 67 7% % ( (1 12 2 o ou ut t o of f 1 18 8 c ce el ll ls s) ) w wh he en n i in nt tr ra ac ce el ll lu ul la ar r C Ca a 2+ 2+ r re el le ea as se e a an nd d C Ca a 2+ 2+ --w wa av ve es s i in n s si im mi il la ar r e ex xp pe er ri im me en nt ta al l c co on nd di it ti io on ns s ( (F Fi ig g. .1 1) ). . H Ho ow we ev ve er r, , i in n f fr re eq qu ue en nc ci ie es s i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C c ce el ll ls s w we er re e s si im mi il la ar r i in n a ab bs se en nc ce e o of f c cy yt to os so ol li ic c C Ca a 2+ 2+ . . U Un nd de er r t th he es se e t to o b be e n no or rm ma al l s si in nc ce e t th he e C Ca a 2+ 2+ i in nh hi ib bi it ti io on n f fo ou un nd d i in n t th he e 3 3 [ [H H + + ] ]r ry ya an no od di in ne e b bi in nd di in ng g e ex xp pe er ri im me en nt ts s i is s s si im mi il la ar r d du ur ri in ng g d di ia as st to ol le e a at t h hi ig gh h p pa ac ci in ng g r ra at te e, , a an n i im mb ba al la an nc ce e b be et tw we ee en n C Ca a 2+ 2+ l le ea ak k a an nd d C Ca a 2+ 2+ r re e--u up pt ta ak ke e r re es su ul lt ts s d de el ll la a R Ri ic ce er rc ca a d di i B Ba as se e R RB BN NE E0 01 1X XM MP P4 4--R RB BC Ca a0 03 34 4X X. . R Ry yR R2 2 R4496C R4496C c ce el ll ls s w wi it th ho ou ut t ( (b bl lu ue e b ba ar r, , n n= =4 43 3) ) a an nd d w wi it th h 1 1 μ μm mo ol l/ /L L i is so op pr ro ot te er re en no ol l ( (h ha at tc ch he ed d b bl lu ue e b ba ar r, ,
	[ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s 0 0. .8 82 2± ±0 0. .0 06 6 i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s ( (n n= =2 21 1) ). . A Al lt to og ge et th he er r, , t th he es se e e ex xp pe er ri im me en nt ts s u un nm ma as sk k r ra at te e--R Ry yR R2 2 R4496C R4496C c ce el ll ls s w we er re e p pa ac ce ed d a at t 4 4 H Hz z i in n t th he e p pr re es se en nc ce e o of f i is so op pr ro ot te er re en no ol l. . T Th hi is s b be eh ha av vi io or r w wa as s a al lm mo os st t l li if fe e, , v ve en nt tr ri ic cu ul la ar r m my yo oc cy yt te es s a ar re e c co on nt ti in nu uo ou us sl ly y p pa ac ce ed d u un nl le es ss s t th he er re e i is s a a p pr ro ob bl le em m w wi it th h a au ut to om ma at ti ic c o or r c co on nd di it ti io on ns s, , S SR R C Ca a 2+ 2+ l lo oa ad d w wa as s a al ls so o s si im mi il la ar r i in n b bo ot th h e ex xp pe er ri im me en nt ta al l g gr ro ou up ps s, , s su ug gg ge es st ti in ng g t th ha at t t to o W WT T R Ry yR R2 2 ( (F Fi ig g. .3 3G G) ). . E Ex xp pe er ri im me en nt ts s i in n t th he e R Ry yR R2 2 R4496C R4496C t te er rt ti ia ar ry y s st tr ru uc ct tu ur re e a ar re e n ne ee ed de ed d t to o i in nv ve es st ti ig ga at te e i in n S SR R C Ca a 2+ 2+ d de ep pl le et ti io on n, , a al lt th ho ou ug gh h a a p po os ss si ib bl le e a al lt te er ra at ti io on n i in n R Ry yR R2 2 R4496C R4496C r re ef fr ra ac ct to or ri in ne es ss s c co ou ul ld d a ac cc co ou un nt t D DI IS SC CL LO OS SU UR RE ES S n n= =1 17 7) ). . C C. . P Pe er rc ce en nt ta ag ge e o of f i in nc cr re ea as se e i in nd du uc ce ed d b by y 1 1 μ μm mo ol l/ /L L i is so op pr ro ot te er re en no ol l o on n C Ca a 2+ 2+ --s sp pa ar rk k f fr re eq qu ue en nc cy y i in n
	T To o d de et te er rm mi in ne e w wh he et th he er r t th he e a al lt te er ra at ti io on n o of f d di ia as st to ol li ic c C Ca a 2+ 2+ s sp pa ar rk k f fr re eq qu ue en nc cy y i in n R Ry yR R2 2 R4496C R4496C d de ep pe en nd de en nt t s sy ys st to ol li ic c C Ca a 2+ 2+ --r re el le ea as se e d de ef fe ec ct ts s i in n R Ry yR R2 2 R4496C R4496C c ce el ll ls s i in n r re el la at ti io on n w wi it th h i im mp pa ai ir re ed d r re ec co ov ve er ry y o of f a ab bs se en nt t i in n W WT T c ce el ll ls s, , b bo ot th h i in n b ba as sa al l c co on nd di it ti io on ns s ( (2 2H Hz z: : 1 1 o ou ut t o of f 5 55 5 W WT T c ce el ll ls s v vs s. . 9 9 o ou ut t o of f 5 54 4 c co on nd du uc ct ti in ng g c ce el ll ls s. . M Mo or re eo ov ve er r, , C CP PV VT T a ar ri is se es s u un nd de er r s st tr re es ss s c co on nd di it ti io on ns s w wi it th h a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n R Ry yR R2 2 R4496C R4496C s se en ns si it ti iv vi it ty y t to o l lu um mi in na al l C Ca a 2+ 2+ i is s m ma ai in nt ta ai in ne ed d u un nd de er r t th he es se e u un np ph hy ys si io ol lo og gi ic c c ci ir rc cu um ms st ta an nc ce es s. . w wh he et th he er r t th hi is s p po oi in nt t m mu ut ta at ti io on n i in nd du uc ce es s c co on nf fo or rm ma at ti io on na al l c ch ha an ng ge es s f fa av vo or ri in ng g C Ca a 2+ 2+ b bi in nd di in ng g t to o t th he e f fo or r t th hi is s p ph he en no om me en no on n. . H Ho ow we ev ve er r, , i in n h hu um ma an ns s t th he e s st ta ai ir rc ca as se e i is s p po os si it ti iv ve e, , w wh hi ic ch h f fu ur rt th he er r s su up pp po or rt ts s t th he e 1 10 0 W WT T a an nd d 1 17 7 R Ry yR R2 2 R4496C R4496C c ce el ll ls s. . D D. . A Av ve er ra ag ge e o of f s si it te es s w wh he er re e C Ca a 2+ 2+ --s sp pa ar rk ks s a ar re e r re ec co or rd de ed d w wi it th hi in n t th he e
	c ca ar rd di io om my yo oc cy yt te es s h ha as s a an n i im mp pa ac ct t d du ur ri in ng g s sy ys st to ol le e, , w we e c co om mp pa ar re ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a an nd d c ce el ll l S SR R C Ca a 2+ 2+ l lo oa ad d, , w wh hi ic ch h m mi ig gh ht t b be e s se ec co on nd da ar ry y t to o d di ia as st to ol li ic c C Ca a 2+ 2+ l le ea ak k. . R Ry yR R2 2 R4496C R4496C c ce el ll ls s, , p p< <0 0. .0 01 1) ) a an nd d a at t 4 4 H Hz z a an nd d i is so op pr ro ot te er re en no ol l ( (2 2 o ou ut t o of f 1 14 4 W WT T c ce el ll ls s v vs s. . 1 12 2 o ou ut t o of f 1 18 8 w wh hi ic ch h, , a am mo on ng g o ot th he er r e ef ff fe ec ct ts s, , i in nc cr re ea as se es s h he ea ar rt t r ra at te e. . N Ne ev ve er rt th he el le es ss s, , i in n t th he e p pr re es se en nc ce e o of f c cy yt to os so ol li ic c C Ca a 2+ 2+ , , R Ry yR R2 2 R4496C R4496C b be eh ha av ve es s a as s h hy yp pe er rs se en ns si it ti iv ve e t to o b bo ot th h a ac ct ti iv va at ti in ng g s si it te es s i in n t th he e R Ry yR R2 2. . l la ac ck k o of f c co on nt tr ra ac ct ti il le e i im mp pa ai ir rm me en nt t i in n C CP PV VT T p pa at ti ie en nt ts s. . N No on ne e s sa am me e c ce el ll l d du ur ri in ng g t th he e r re ec co or rd di in ng g p pe er ri io od d ( (1 18 8 s s) ). . E E. . P Pr ro ob ba ab bi il li it ty y i in n e ea ac ch h c ce el ll l t to o p pr re es se en nt t s si it te es s t th ha at t f fi ir re e
	c co on nt tr ra ac ct ti io on n a at t d di if ff fe er re en nt t p pa ac ci in ng g r ra at te es s ( (2 2 H Hz z, , 3 3 H Hz z, , a an nd d 4 4 H Hz z) ) ( (O On nl li in ne e t ta ab bl le e I II II I) ). . A At t 2 2 H Hz z, , t th he e [ [C Ca a 2+ 2+ ] ] i i H Ho ow we ev ve er r, , t th he e r re el le ev va an nc ce e o of f t th hi is s s sy ys st to ol li ic c d de ef fe ec ct t m ma ay y b be e l li im mi it te ed d i in n v vi iv vo o, , s si in nc ce e i in nc cr re ea as se ed d R Ry yR R2 2 R4496C R4496C c ce el ll ls s, , p p< <0 0. .0 01 1) ). . A As s s sh ho ow wn n i in n F Fi ig g. .1 1B B, , o on ne e s si in ng gl le e C Ca a 2+ 2+ --w wa av ve e w wa as s a ab bl le e t to o e ev vo ok ke e T Th hi is s r re ep po or rt t i is s t th he e f fi ir rs st t t to o s sh ho ow w t th ha at t i is so ol la at te ed d R Ry yR R2 2 R4496C R4496C v ve en nt tr ri ic cu ul la ar r m my yo oc cy yt te es s d di is sp pl la ay ye ed d l lu um mi in na al l 10 10 a an nd d c cy yt to os so ol li ic c C Ca a 2+ 2+ ( (F Fi ig g. .3 3) ). . It is not easy to unequivocally assign distinct roles for T Th he e d de ec cr re ea as se e i in n S SR R C Ca a 2+ 2+ w wi it th h p pa ac ci in ng g r ra at te e c ca an n a al ls so o r re ef fl le ec ct t t th he e h hi ig gh he er r C Ca a 2+ 2+ l le ea ak k a at t h hi ig gh he er r r re ep pe et ti it ti iv ve el ly y. . F F. . M Ma ax xi im mu um m n nu um mb be er r o of f C Ca a 2+ 2+ --s sp pa ar rk ks s r re ec co or rd de ed d i in n t th he e s sa am me e s si it te e. . F Fo or r D D, , E E & & F F: : n n= = 3 37 7
	t tr ra an ns si ie en nt t a am mp pl li it tu ud de e, , i it ts s t ti im me e t to o p pe ea ak k, , t th he e [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t d de ec ca ay y t ti im me e a an nd d c ce el ll lu ul la ar r c co on nt tr ra ac ct ti io on n, , h he ea ar rt t r ra at te e i is s u us su ua al ll ly y a as ss so oc ci ia at te ed d w wi it th h h hi ig gh h s sy ym mp pa at th he et ti ic c d dr ri iv ve e, , t th hu us s i in nc cr re ea as se ed d c co on nt tr ra ac ct ti il li it ty y. . W We e t tr ri ig gg ge er re ed d a ac ct ti iv vi it ty y a af ft te er r e el le ec ct tr ri ic ca al l s st ti im mu ul la at ti io on n s st to op pp pe ed d. . H Ho ow we ev ve er r, , t th hi is s w wa as s n ne ev ve er r t th he e c ca as se e d du ur ri in ng g a ar rr rh hy yt th hm mo og ge en ni ic c a ac ct ti iv vi it ty y r re el la at te ed d t to o s sp po on nt ta an ne eo ou us s C Ca a 2+ 2+ r re el le ea as se e w wh hi il le e t th he ey y a ar re e e el le ec ct tr ri ic ca al ll ly y cytoplasmic vs. luminal Ca 2+ in situ due to the inherent interdependence of these Ca 2+ E Ev ve en n t th ho ou ug gh h R Ry yR R2 2 R4496C R4496C b ba as sa al l a ac ct ti iv vi it ty y w wa as s d dr ra am ma at ti ic ca al ll ly y h hi ig gh he er r t th ha an n t th ha at t o of f W WT T ( (F Fi ig g. .2 2) ), , β β--s st ti im mu ul la at ti io on n f fr re eq qu ue en nc ci ie es s ( (F Fi ig g. .6 6C C) ) a an nd d c co ou ul ld d p pa ar rt tl ly y d de ep pe en nd d o on n a a p ph he en no om me en no on n k kn no ow wn n a as s C Ca a 2+ 2+ W WT T c ce el ll ls s, , n n= =1 10 0 W WT T c ce el ll ls s i in n t th he e p pr re es se en nc ce e o of f 1 1 μ μm mo ol l/ /L L i is so op pr ro ot te er re en no ol l; ; n n= =4 43 3 R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s
	w we er re e s si im mi il la ar r ( (p p> >0 0. .0 05 5) ) i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C c ce el ll ls s, , c co on ns si is st te en nt t w wi it th h n no or rm ma al l h he ea ar rt t f fu un nc ct ti io on n i in n m mi ic ce e n ne ex xt t a an na al ly yz ze ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s u un nd de er r β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n. . I Im ma ag ge es s o of f [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s c co on ns st ta an nt t s st ti im mu ul la at ti io on n. . I In n o or rd de er r t to o t tr ri ig gg ge er r a a f fu ul ll l [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t, , w we e m me ea as su ur re ed d t th ha at t 7 7. .1 1± ±0 0. .6 6 C Ca a 2+ 2+ --s st ti im mu ul la at te ed d, , t th hu us s m mi im mi ic ck ki in ng g h hu um ma an n C CP PV VT T a an nd d d de em mo on ns st tr ra at ti in ng g t th ha at t R Ry yR R2 2 R4496C R4496C w wa as s a at t t th he e o or ri ig gi in n o of f compartments in living cells. a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n i in nc cr re ea as se ed d t th he ei ir r a ac ct ti iv vi it ty y t to o t th he e s sa am me e e ex xt te en nt t s su ug gg ge es st ti in ng g t th ha at t: : ( (i i) ) t th he e t tw wo o c cu ur rr re en nt t f fa ac ci il li it ta at ti io on n. . B By y t th hi is s p ph he en no om me en no on n, , t th he e t to ot ta al l a am mo ou un nt t o of f C Ca a 2+ 2+ e en nt tr ry y i is s e en nh ha an nc ce ed d w wh he en n a an nd d n n= =1 17 7 R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s u un nd de er r 1 1 μ μm mo ol l/ /L L i is so op pr ro ot te er re en no ol l p pe er rf fu us si io on n. . * *p p< <0 0. .0 05 5, , * ** *p p< <0 0. .0 01 1, ,
	a at t r re es st t. . A As s s st ti im mu ul la at ti io on n r ra at te e i in nc cr re ea as se ed d, , w we ea ak ke er r [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s w we er re e e ev vo ok ke ed d b bo ot th h i in n W WT T a an nd d e ev vo ok ke ed d b by y f fi ie el ld d s st ti im mu ul la at ti io on n a at t 4 4 H Hz z a ar re e s sh ho ow wn n i in n F Fi ig gu ur re e 5 5A A, , t to op p. . T Th he e i in nc cr re ea as se e i in n [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t w wa av ve es s* *1 10 00 0μ μm m -1 -1 h ha ad d t to o o ov ve er rl la ap p d du ur ri in ng g d di ia as st to ol le e, , w wh hi ic ch h o on nl ly y o oc cc cu ur rr re ed d u un nd de er r s st tr re es ss s c co on nd di it ti io on ns s. . t th he e a ar rr rh hy yt th hm mi ia a. . I In n i is so ol la at te ed d c ca ar rd di io om my yo oc cy yt te es s p pa ac ce ed d a at t 2 2H Hz z, , w we e o ob bs se er rv ve ed d m mu ul lt ti ip pl le e C Ca a 2+ 2+ --s sp pa ar rk ks s W Wh hi il le e u un nz zi ip pp pi in ng g o of f a am mi in no o a an nd d c ce en nt tr ra al l R Ry yR R2 2 d do om ma ai in ns s h ha as s b be ee en n r re ep po or rt te ed d t to o b be e i in nv vo ol lv ve ed d i in n m me ec ch ha an ni is sm ms s ( (i in nc cr re ea as se ed d C Ca a 2+ 2+ s se en ns si it ti iv vi it ty y o of f t th he e R Ry yR R2 2 R4496C R4496C m mu ut ta an nt t a an nd d t th he e e ef ff fe ec ct t o of f β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n f fr re eq qu ue en nc cy y i is s i in nc cr re ea as se ed d, , m ma ai in nl ly y d du ue e t to o s sl lo ow wi in ng g o of f t th he e C Ca a 2+ 2+ c cu ur rr re en nt t i in na ac ct ti iv va at ti io on n 31 31 . . T Th hi is s * ** ** *p p< <0 0. .0 00 01 1 v vs s. . t th he e s sa am me e g gr ro ou up p i in n t th he e a ab bs se en nc ce e o of f i is so op pr ro ot te er re en no ol l. . † †p p< <0 0. .0 05 5, , † † † †p p< <0 0. .0 01 1, , † † † † † †p p< <0 0. .0 00 01 1
	R Ry yR R2 2 R4496C R4496C c ce el ll ls s ( (F Fi ig g. .4 4A A) ). . H Ho ow we ev ve er r, , t th he e d de ec cr re ea as se e i in n [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t a am mp pl li it tu ud de e w wa as s m mo or re e a am mp pl li it tu ud de e i in nd du uc ce ed d b by y i is so op pr ro ot te er re en no ol l w wa as s s si im mi il la ar r i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C c ce el ll ls s, , ( (F Fi ig g. .5 5A A, , b bo ot tt to om m, , C Ca a 2+ 2+ --w wa av ve es s s sp pr re ea ad d a at t s si im mi il la ar r v ve el lo oc ci it ty y i in n t th he e a ab bs se en nc ce e ( (1 11 17 7. .3 3± ±1 14 4. .7 7 μ μm m/ /s s, , n n= =1 13 3) ) o or r i in n t th he e c ca ap pa ab bl le e o of f t tr ri ig gg ge er ri in ng g l lo oc ca al li iz ze ed d C Ca a 2+ 2+ --w wa av ve es s i in n m mo or re e t th ha an n 1 16 6% % o of f R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s ( (F Fi ig g. .6 6C C) ). . s so om me e f fo or rm ms s o of f e en nh ha an nc ce ed d R Ry yR R2 2 a ac ct ti iv vi it ty y 22,23 22,23 , , t th he e R R4 44 49 96 6C C m mu ut ta at ti io on n i is s f fa ar r f fr ro om m t th ho os se e d do om ma ai in ns s, , s st ti im mu ul la at ti io on n) ) a ar re e d di is st ti in nc ct t a an nd d c cu um mu ul la at ti iv ve e a an nd d ( (i ii i) ) t th he e β β--a ad dr re en ne er rg gi ic c r re eg gu ul la at ti io on n o of f R Ry yR R2 2 R4496C R4496C i is s n no ot t l lo on ng ge er r C Ca a 2+ 2+ e en nt tr ry y d du ur ri in ng g t th he e d di ia as st to ol li ic c p pe er ri io od d c ca an n f fu ur rt th he er r a ac ct ti iv va at te e R Ry yR R2 2 R4496C R4496C , , t th he er re eb by y e ev vo ok ki in ng g v vs s W WT T. .
	p pr ro on no ou un nc ce ed d ( (p p< <0 0. .0 05 5) ) i in n R Ry yR R2 2 R4496C R4496C c ce el ll ls s. . T Th hi is s r re ed du uc ct ti io on n w wa as s a as ss so oc ci ia at te ed d w wi it th h b bo ot th h w we ea ak ke er r O On nl li in ne e F Fi ig g. .I II IB B, , O On nl li in ne e T Ta ab bl le e I II II I) ). . W We e a al ls so o f fo ou un nd d a a s si im mi il la ar r e ef ff fe ec ct t o of f i is so op pr ro ot te er re en no ol l o on n S SR R C Ca a 2+ 2+ l lo oa ad d p pr re es se en nc ce e o of f 1 1μ μm mo ol l/ /L L i is so op pr ro ot te er re en no ol l ( (1 11 13 3. .8 8± ±7 7. .2 2 μ μm m/ /s s, , n n= =2 22 2) ). . W Wi it th h p pa ac ci in ng g r ra at te e i in nc cr re ea as se ed d t to o 4 4 H Hz z a an nd d u un nd de er r β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n, , t th he e R Ry yR R2 2 R4496C R4496C m ma ak ki in ng g t th ha at t m me ec ch ha an ni is sm m u un nl li ik ke el ly y. . D Di if ff fe er re en nt ti ia al l F FK KB BP P1 12 2. .6 6 a as ss so oc ci ia at ti io on n a al ls so o c ca an nn no ot t e ex xp pl la ai in n t th he e m mo od di if fi ie ed d. . N Ne ev ve er rt th he el le es ss s, , β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n f fu ur rt th he er r i in nc cr re ea as se ed d t th he e a al lr re ea ad dy y e el le ev va at te ed d d di ia as st to ol li ic c m mo or re e C Ca a 2+ 2+ --w wa av ve es s a an nd d r rh hy yt th hm mi ic c d di is so or rd de er rs s a at t h hi ig gh he er r p pa ac ci in ng g r ra at te es s. .
	c ce el ll lu ul la ar r c co on nt tr ra ac ct ti io on n ( (F Fi ig g. .4 4B B) ) a an nd d s sl lo ow we er r d de ec ca ay y t ti im me e ( (F Fi ig g. .4 4C C) ), , w wi it th h n no o d di if ff fe er re en nc ce e i in n t th he e t ti im me e t to o i in n b bo ot th h e ex xp pe er ri im me en nt ta al l g gr ro ou up ps s ( (F Fi ig g. .5 5B B) ). . A Al lt to og ge et th he er r, , t th he es se e d da at ta a s su ug gg ge es st t t th ha at t t th he e s sy ys st to ol li ic c W We e f fo ou un nd d t th ha at t m mo ou us se e R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s p pr re es se en nt te ed d a a h hi ig gh he er r i in nc ci id de en nc ce e o of f C Ca a 2+ 2+ --w wa av ve es s m my yo oc cy yt te es s w we er re e r re em ma ar rk ka ab bl ly y m mo or re e p pr ro on ne e t to o e ev vo ok ke e C Ca a 2+ 2+ --w wa av ve es s ( (i in n u up p t to o 6 66 6. .7 7% % o of f c ce el ll ls s) ). . W We e t th hu us s i in nc cr re ea as se ed d R Ry yR R2 2 s se en ns si it ti iv vi it ty y r re ep po or rt te ed d h he er re e, , b be ec ca au us se e t th he er re e i is s u un na al lt te er re ed d R Ry yR R2 2--F FK KB BP P1 12 2. .6 6 C Ca a 2+ 2+ l le ea ak k i in n R Ry yR R2 2 R4496C R4496C c ce el ll ls s p pr ro ob ba ab bl ly y b by y i in nc cr re ea as si in ng g t th he e S SR R C Ca a 2+ 2+ l lo oa ad d 28 28 , , f fu ur rt th he er r e en nh ha an nc ci in ng g t th he e F Fi ig gu ur re e 3 3. . T Th he e i in nc cr re ea as se e i in n C Ca a 2+-2+-s sp pa ar rk k f fr re eq qu ue en nc cy y i in n R Ry yR R2 2 R4496C R4496C c ce el ll ls s d de ep pe en nd ds s o on n t th he e
	p pe ea ak k ( (F Fi ig g. .4 4D D) ). . d dy ys sf fu un nc ct ti io on n a at t h hi ig gh h p pa ac ci in ng g r ra at te es s p pe er rs si is st ts s d du ur ri in ng g s sy ym mp pa at th he et ti ic c s st ti im mu ul la at ti io on n ( (p p< <0 0. .0 01 1) ), , a an nd d t th ha at t t th he e n no ot t o on nl ly y i in n t th he e p pr re es se en nc ce e o of f i is so op pr ro ot te er re en no ol l b bu ut t a al ls so o a at t h hi ig gh he er r p pa ac ci in ng g r ra at te es s ( (F Fi ig g. .6 6C C) ). . W We e t th he en n f fo ou un nd d t th ha at t R Ry yR R2 2 R4496C R4496C c ce el ll ls s s sh ho ow we ed d h hi ig gh he er r s sp po on nt ta an ne eo ou us s C Ca a 2+ 2+ r re el le ea as se e e ev ve en n i in n b ba as sa al l c co on nd di it ti io on ns s, , a as ss so oc ci ia at ti io on n i in n t th hi is s a an ni im ma al l m mo od de el l 14 14 . . T Th he e i in nc cr re ea as se e i in n C Ca a 2+ 2+ --s sp pa ar rk k f fr re eq qu ue en nc cy y o of f R Ry yR R2 2 R4496C R4496C i is s l li ik ke el ly y I In n c co on nc cl lu us si io on n, , o ou ur r s st tu ud dy y s sh ho ow ws s t th ha at t b be ea at ti in ng g R Ry yR R2 2 R4496C R4496C c ca ar rd di io om my yo oc cy yt te es s p pr re es se en nt t h hi ig gh h i in nt tr
	i in nc cr re em me en nt ta al l e ef ff fe ec ct t o of f β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n i is s i id de en nt ti ic ca al l i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C . . s sp po on nt ta an ne eo ou us s C Ca a 2+ 2+ r re el le ea as se e d du ur ri in ng g d di ia as st to ol le e, , d du ue e t to o a a d dr ra am ma at ti ic c i in nc cr re ea as se e i in n C Ca a 2+ 2+ s se en ns si it ti iv vi it ty y o of f t th he e

3

H]ryanodine binding and fitted data to the Hill equation

20

to get the values for Ca 2+ affinity to the RyR activation (Ka, 21.9±8.3 μmol/L vs. 4.9±1.0μmol/L, n=4, p<0.003 for WT and RyR2 R4496C membranes, respectively) and inactivation (6.5±0.6 mmol/L vs. 4.8±1.1 mmol/L; p>0.05 for WT and RyR2 R4496C membranes) sites. Theses results show a 4.5 fold increase of cytosolic Ca 2+ sensitivity for the RyR2 R4496C .

ra ac ce el ll lu ul la ar r C Ca a 2+ 2+ c co on nc ce en nt tr ra at ti io on

	A A. . L Li in ne e--s sc ca an n i im ma ag ge es s o ob bt ta ai in ne ed d a at t 3 30 0 n nm mo ol l/ /L L [ [C Ca a 2+ 2+ ] ] i i i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C ( (R R4 44 49 96 6C C) )
	p pe er rm me ea ab bi il li iz ze ed d c ce el ll ls s. . B B. . A Av ve er ra ag ge e o of f C Ca a 2+ 2+ --s sp pa ar rk k f fr re eq qu ue en nc ci ie es s o ob bt ta ai in ne ed d i in n p pe er rm me ea ab bi il li iz ze ed d m my yo oc cy yt te es s
	e ex xp po os se ed d a at t 7 7. .5 5 n nm mo ol l/ /L L ( (n n= = 9 9 v vs s. . n n= =7 7) ), , 1 15 5 n nm mo ol l/ /L L ( (n n= =1 10 0 v vs s. . n n= =6 6) ), , 3 30 0 n nm mo ol l/ /L L ( (n n= = 2 23 3 v vs s. . n n= =1 19 9) ), , 5 50 0
	n nm mo ol l/ /L L ( (n n= = 3 37 7 v vs s. . n n= =4 44 4) ) a an nd d 1 10 00 0 n nm mo ol l/ /L L ( (n n= =8 8 v vs s. . n n= =8 8) ) [ [C Ca a 2+ 2+ ] ] i i . . W WT T c ce el ll ls s v vs s. . R Ry yR R2 2 R4496C R4496C c ce el ll ls s. .
	L Li in ne es s a ar re e B Bo ol lt tz zm ma an nn n f fi it tt ti in ng g o of f t th he e d da at ta a. . C C. . C Ca af ff fe ei in ne e--e ev vo ok ke ed d ( (2 20 0 m mm mo ol l/ /L L) ) [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t i in n
	p pe er rm me ea ab bi il li iz ze ed d m my yo oc cy yt te es s a at t 7 7. .5 5 n nm mo ol l/ /L L ( (n n= = 1 13 3 v vs s. . n n= =1 11 1) ), , 1 15 5 n nm mo ol l/ /L L ( (n n= =1 15 5 v vs s. . n n= =1 10 0) ), , 3 30 0 n nm mo ol l/ /L L
	( (n n= =1 19 9 v vs s. . n n= =2 20 0) ), , 5 50 0 n nm mo ol l/ /L L ( (n n= =2 21 1 v vs s. . n n= =3 33 3) ) a an nd d 1 10 00 0 n nm mo ol l/ /L L ( (n n= =9 9 v vs s. . n n= =9 9) ) [ [C Ca a 2+ 2+ ] ] i i , , W WT T c ce el ll ls s v vs s. .
	R Ry yR R2 2 R4496C R4496C c ce el ll ls s, , e ex xp pr re es ss se ed d a as s % % o of f t th he e c ca af ff fe ei in ne e--e ev vo ok ke ed d t tr ra an ns si ie en nt t a at t 1 10 00 0 n nm mo ol l/ /L L i in n W WT T c ce el ll ls s. .
	T Th he e x x a ax xi is s l la ab be el ls s i in nd di ic ca at te e c cy yt to os so ol li ic c [ [C Ca a 2+ 2+ ] ] i i i in n n nm mo ol l/ /L L. . D D. . C Ca a 2+ 2+ --s sp pa ar rk ks s f fr re eq qu ue en nc ci ie es s p pl lo ot tt te ed d a as s a a
	f fu un nc ct ti io on n o of f c ca af ff fe ei in ne e--e ev vo ok ke ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t. . N Nu um mb be er rs s i in nd di ic ca at te e c cy yt to os so ol li ic c [ [C Ca a 2+ 2+ ] ] i i . . E E. . I Im ma ag ge es s o of f
	C Ca a 2+ 2+ --s sp pa ar rk ks s o ob bt ta ai in ne ed d a at t 0 0 n nm mo ol l/ /L L [ [C Ca a 2+ 2+ ] ] i i i in n W WT T ( (t to op p, , l le ef ft t) ) a an nd d R Ry yR R2 2 R4496C R4496C ( (R R4 44 49 96 6C C, , b bo ot tt to om m, , l le ef ft t) )
	c ce el ll ls s a an nd d d du ur ri in ng g ( (r ri ig gh ht t) ) 2 20 0 m mm mo ol l/ /L L c ca af ff fe ei in ne e a ap pp pl li ic ca at ti io on n ( (a ar rr ro ow w) ). . F F. . L Le ef ft t. . C Ca a 2+ 2+ --s sp pa ar rk k f fr re eq qu ue en nc cy y
	o ob bt ta ai in ne ed d i in n W WT T m my yo oc cy yt te es s ( (n n= =6 6) ) a an nd d i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s ( (n n= =6 6) ). . R Ri ig gh ht t. . C Ca af ff fe ei in ne e--e ev vo ok ke ed d
	[ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t a am mp pl li it tu ud de e ( (i in n F F/ /F F 0 0 ) ) m me ea as su ur re ed d a at t z ze er ro o [ [C Ca a 2+ 2+ ] ] i i i in n 1 15 5 W WT T c ce el ll ls s a an nd d 1 16 6 R Ry yR R2 2 R4496C R4496C
	c ce el ll ls s. . G G. . Specific Ca 2+ -dependent [ 3 H]ryanodine binding curves to crude membrane fractions of
	WT (n=4) and R4496C+/+ (n=4) heart tissues. Values have been normalized and fitted to the
	equation y=Bmax*([Ca 2+ ] na /([Ca 2+ ] na +K a na ))(1-([Ca 2+ ] ni /([Ca 2+ ] ni +K i	ni )))+C. Open symbols, WT;
	blue symbols, RyR2 R4496C . S So ol li id d l li in ne e f fo or r W WT T a an nd d d do ot tt te ed d l li in ne e f fo or r R Ry yR R2 2 R4496C R4496C c ce el ll ls s o or r
	h ho om mo og ge en na at te es s. * *p p< <0 0. .0 05 5, , * ** *p p< <0 0. .0 01 1, , * ** ** *p p< <0 0. .0 00 01 1. .
	F Fi ig gu ur re e 4 4. . R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s s sh ho ow w a a r ra at te e--
	n ( ([ [C Ca a 2+ 2+ ] ] i i ) ). .

d de ep pe en nd de en nt t d de ec cr re ea as se e

	t to o p pe ea ak k ( (D D) ) a at t 4 4 H Hz z i in n W WT T ( (n n= =1 13 3) ) v vs s. . R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s ( (n n= =1 19 9) ). . E E. . L Li in ne e--s sc ca an n i im ma ag ge es s o of f
	c ca af ff fe ei in ne e--e ev vo ok ke ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s o ob bt ta ai in ne ed d i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C ( (R R4 44 49 96 6C C) ) c ce el ll ls s o ob bt ta ai in ne ed d a af ft te er r
	f fi ie el ld d s st ti im mu ul la at ti io on n a at t 4 4 H Hz z. . F F. . A Av ve er ra ag ge e c ca af ff fe ei in ne e--e ev vo ok ke ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s ( (e ex xp pr re es ss se ed d a as s p pe ea ak k F F/ /F F 0 0 , ,
	a as s i in n p pa an ne el l A A) ) i in n W WT T v vs s. . R Ry yR R2 2 R4496C R4496C ) ) m my yo oc cy yt te es s o ob bt ta ai in ne ed d f fo ol ll lo ow wi in ng g f fi ie el ld d s st ti im mu ul la at ti io on n a at t 2 2 H Hz z ( (n n= =3 33 3
	v vs s. . n n= =3 36 6) ), , 3 3 H Hz z ( (n n= =7 7 v vs s. . n n= =2 22 2) ) a an nd d 4 4 H Hz z ( (n n= = 8 8 v vs s. . n n= =2 25 5) ). . G G. . [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t--S SR R C Ca a 2+ 2+ l lo oa ad d
	r re el la at ti io on ns sh hi ip p i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s a at t v va ar ri io ou us s p pa ac ci in ng g r ra at te es s. . Open circles and bars,
	WT; blue circles and bars, RyR2 R4496C . . N N n nu um mb be er rs s f fr ro om m p pa an ne el ls s A A& &F F. .* *p p< <0 0. .0 05 5. .
	F Fi ig gu ur re e 5 5. . C Co on ns se er rv ve ed d β β--
	i in n [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a an nd d
	S SR R C Ca a 2+ 2+ l lo oa ad d. .
	A A. . A Av ve er ra ag ge e o of f [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a am mp pl li it tu ud de e ( (e ex xp pr re es ss se ed d a as s F F/ /F F 0 0 , , w wh he er re e F F i is s t th he e p pe ea ak k f fl lu uo or re es sc ce en nc ce e
	s si ig gn na al l a an nd d F F 0 0 t th he e d di ia as st to ol li ic c f fl lu uo or re es sc ce en nc ce e) ) o of f W WT T) ) v vs s. . R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s o ob bt ta ai in ne ed d b by y f fi ie el ld d
	s st ti im mu ul la at ti io on n a at t 2 2 H Hz z ( (n n= = 5 50 0 v vs s. . n n= =5 52 2) ), , 3 3 H Hz z ( (n n= =1 16 6 v vs s. . n n= =2 21 1) ) a an nd d 4 4 H Hz z ( (n n= =1 14 4 v vs s. . n n= =2 21 1) ). . B B. . C C& & D D. .
	B Ba ar r g gr ra ap ph hs s c co om mp pa ar ri in ng g t th he e a av ve er ra ag ge e c ce el ll l s sh ho or rt te en ni in ng g ( (B B) ), , [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t d de ec ca ay y t ti im me e ( (C C) ), , o or r t ti im me e

a ad dr re en ne er rg gi ic c r re es sp po on ns si iv ve en ne es ss s i in n R Ry yR R2 2 R4496C R4496C m my yo oc cy yt te es s. .

	A A. . T To op p. . L Li in ne e--s sc ca an n i im ma ag ge es s o of f [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s o ob bt ta ai in ne ed d i in n W WT T a an nd d R Ry yR R2 2 R4496C R4496C ( (R R4 44 49 96 6C C) ) c ce el ll ls s
	e ev vo ok ke ed d b by y f fi ie el ld d s st ti im mu ul la at ti io on n a at t 4 4 H Hz z i in n t th he e a ab bs se en nc ce e a an nd d t th he e p pr re es se en nc ce e ( (I IS SO O) ) o of f 1 1μ μm mo ol l/ /L L
	i is so op pr ro ot te er re en no ol l. . B Bo ot tt to om m. . P Pe er rc ce en nt ta ag ge e o of f [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt t a am mp pl li it tu ud de e i in nc cr re ea as se e i in nd du uc ce ed d b by y 1 1 μ μm mo ol l/ /L L
	i is so op pr ro ot te er re en no ol l a at t 2 2 ( (n n= =2 26 6 v vs s. . n n= =2 25 5) ), , 3 3 ( (n n= =4 4 v vs s. . n n= =1 12 2) ) a an nd d 4 4 ( (n n= =3 3 v vs s. . n n= =1 12 2) ) H Hz z, , W WT T v vs s. .
	R Ry yR R2 2 R4496C R4496C c ce el ll ls s. . B B. . P Pe er rc ce en nt ta ag ge e o of f i in nc cr re ea as se e i in nd du uc ce ed d b by y 1 1 μ μm mo ol l/ /L L i is so op pr ro ot te er re en no ol l o on n c ca af ff fe ei in ne e
	e ev vo ok ke ed d [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a af ft te er r s st ti im mu ul la at ti in ng g t th he e c ce el ll l a at t 2 2 H Hz z ( (n n= = 2 26 6 v vs s. . n n= =2 25 5) ), , 3 3 H Hz z ( (n n= =4 4 v vs s. . n n= =1 12 2) )
	a an nd d 4 4 H Hz z ( (n n= =3 3 v vs s. . n n= =1 12 2) ). . W WT T ( (w wh hi it te e h ha at tc ch he ed d b ba ar rs s) ), , R Ry yR R2 2 R4496C R4496C ( (b bl lu ue e h ha at tc ch he ed d b ba ar rs s) )
	F

Fi ig gu ur re e 6 6. . A Ar rr rh hy yt th hm mi ic c a ac ct ti iv vi it ty y d de ep pe en nd ds s o on n β β--a ad dr re en ne er rg gi ic c s st ti im mu ul la at ti io on n a an nd d p pa ac ci in ng g r ra at te e. .

	* *p p< <0 0. .0 05 5, , * ** *p p< <0 0. .0 01 1 w wi it th h r re es sp pe ec ct t t to o W WT T. . † †p p< <0 0. .0 05 5 w wi it th h r re es sp pe ec ct t t to o R Ry yR R2 2 R4496C R4496C . . ‡ ‡p p< <0 0. .0 05 5, , ‡ ‡ ‡ ‡p p< <0 0. .0 01 1, ,
	‡ ‡ ‡ ‡ ‡ ‡p p< <0 0. .0 00 01 1 w wi it th h r re es sp pe ec ct t t to o W WT T i in n t th he e p pr re es se en nc ce e o of f i is so op pr ro ot te er re en no ol l. . # #p p< <0 0. .0 05 5, , # ## ## #p p< <0 0. .0 00 01 1 w wi it th h
	r re es sp pe ec ct t t to o R Ry yR R2 2 R4496C R4496C a at t 2 2 H Hz z. .
	A A. . L Li in ne e--s sc ca an n i im ma ag ge es s o ob bt ta ai in ne ed d i in n a a W WT T c ce el ll l p pa ac ce ed d a at t 2 2 H Hz z a an nd d s su up pe er rf fu us se ed d w wi it th h 1 1 μ μm mo ol l/ /L L
	i is so op pr ro ot te er re en no ol l. . T Th he e c co or rr re es sp po on nd di in ng g f fl lu uo or re es sc ce en nc ce e [ [C Ca a 2+ 2+ ] ] i i t tr ra an ns si ie en nt ts s a an nd d c ce el ll l s sh ho or rt te en ni in ng g p pr ro of fi il le es s
	a ap pp pe ea ar r b be el lo ow w. . B B. . T Th he e s sa am me e f fo or r a a R Ry yR R2 2 R4496C R4496C c ce el ll l. . T Th he e i im ma ag ge e s sh ho ow ws s m mu ul lt ti ip pl le e C Ca a 2+ 2+ --s sp pa ar rk ks s a an nd d/ /o or r
	C Ca a 2+ 2+ --w wa av ve es s d du ur ri in ng g d di ia as st to ol le e. . T Th he e r re ed d a ar rr ro ow w i in nd di ic ca at te es s t tr ri ig gg ge er re ed d a ac ct ti iv vi it ty y, , m ma ai in nt ta ai in ne ed d i in n p pa an ne el l c c a an nd d
	s sp po on nt ta an ne eo ou us sl ly y t te er rm mi in na at te ed d i in n p pa an ne el l d d. . R Re ed d l li in ne es s i in nd di ic ca at te e e el le ec ct tr ri ic ca al l s st ti im mu ul li i. . C C. . O Oc cc cu ur rr re en nc ce e o of f
	a ab bn no or rm ma al l d di ia as st to ol li ic c C Ca a 2+ 2+ r re el le ea as se e a at t 2 2, , 3 3 a an nd d 4 4 H Hz z, , i in n m my yo oc cy yt te es s i in n a ab bs se en nc ce e ( (s so ol li id d b ba ar rs s) ) o or r i in n
	p pr re es se en nc ce e ( (h ha at tc ch he ed d b ba ar rs s) ) o of f 1 1 μ μm mo ol l/ /L L o of f i is so op pr ro ot te er re en no ol l; ; W WT T, , w wh hi it te e b ba ar rs s; ; R Ry yR R2 2 R4496C R4496C , , b bl lu ue e b ba ar rs s. .