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A icleTi le P e en ion o adenosine A2A ecep o ac i a ion diminishes bea - o-bea al e na ion in human a ial
myocy es
A icle Sub-Ti le
A icle CopyRigh Sp inge -Ve lag Be lin Heidelbe g
(This will be he copy igh line in he inal PDF)
Jou nal Name Basic Resea ch in Ca diology
Co esponding Au ho Family Name Ho e-Madsen
Pa icle
Gi en Name Lei
Su ix
Di ision Ca diac Rhy hm and Con ac ion, Ca dio ascula Resea ch Cen e CSIC-
ICCC and IIB San Pau
O ganiza ion Hospi al de la San a C eu i San Pau
Add ess S An oni Mª Cla e 167, Ba celona, 08025, Spain
Email [email p o ec ed]
Au ho Family Name Molina
Pa icle
Gi en Name C is ina E.
Su ix
Di ision Ca diac Rhy hm and Con ac ion, Ca dio ascula Resea ch Cen e CSIC-
ICCC and IIB San Pau
O ganiza ion Hospi al de la San a C eu i San Pau
Add ess S An oni Mª Cla e 167, Ba celona, 08025, Spain
Di ision Ins i u e o Pha macology, Facul y o Medicine
O ganiza ion Uni e si y Duisbu g-Essen
Add ess Essen, 45122, Ge many
Email
Au ho Family Name Llach
Pa icle
Gi en Name Anna
Su ix
Di ision Depa men o Ca diology and IIB San Pau
O ganiza ion Hospi al de San Pau
Add ess Ba celona, Spain
Email
Au ho Family Name He aiz-Ma ínez
Pa icle
Gi en Name Adela
Su ix
Di ision Ca diac Rhy hm and Con ac ion, Ca dio ascula Resea ch Cen e CSIC-
ICCC and IIB San Pau
O ganiza ion Hospi al de la San a C eu i San Pau
Add ess S An oni Mª Cla e 167, Ba celona, 08025, Spain
Email
Au ho Family Name Ta i a
Pa icle
Gi en Name Ca men
Su ix
Di ision Ca diac Rhy hm and Con ac ion, Ca dio ascula Resea ch Cen e CSIC-
ICCC and IIB San Pau
O ganiza ion Hospi al de la San a C eu i San Pau
Add ess S An oni Mª Cla e 167, Ba celona, 08025, Spain
Email
Au ho Family Name Ba iga
Pa icle
Gi en Name Mon se a
Su ix
Di ision Ca diac Rhy hm and Con ac ion, Ca dio ascula Resea ch Cen e CSIC-
ICCC and IIB San Pau
O ganiza ion Hospi al de la San a C eu i San Pau
Add ess S An oni Mª Cla e 167, Ba celona, 08025, Spain
Email
Au ho Family Name Wiege inck
Pa icle
Gi en Name Rob F.
Su ix
Di ision Depa men o Ca diology and IIB San Pau
O ganiza ion Hospi al de San Pau
Add ess Ba celona, Spain
Email
Au ho Family Name Fe nandes
Pa icle
Gi en Name Jacqueline
Su ix
Di ision Depa men o Ca diology and IIB San Pau
O ganiza ion Hospi al de San Pau
Add ess Ba celona, Spain
Email
Au ho Family Name Cabello
Pa icle
Gi en Name Nu ia
Su ix
Di ision Ca diac Rhy hm and Con ac ion, Ca dio ascula Resea ch Cen e CSIC-
ICCC and IIB San Pau
O ganiza ion Hospi al de la San a C eu i San Pau
Add ess S An oni Mª Cla e 167, Ba celona, 08025, Spain
Email
Au ho Family Name Vallmi jana
Pa icle
Gi en Name Alex
Su ix
Di ision Depa men o Au oma ic Con ol
O ganiza ion Uni e si a Poli ècnica de Ca alunya
Add ess Ba celona, Spain
Email
Au ho Family Name Beni éz
Pa icle
Gi en Name Raúl
Su ix
Di ision Depa men o Au oma ic Con ol
O ganiza ion Uni e si a Poli ècnica de Ca alunya
Add ess Ba celona, Spain
Email
Au ho Family Name Mon iel
Pa icle
Gi en Name José
Su ix
Di ision Depa men o Ca diac Su ge y and IIB San Pau
O ganiza ion Hospi al de San Pau
Add ess Ba celona, Spain
Email
Au ho Family Name Cinca
Pa icle
Gi en Name Juan
Su ix
Di ision Depa men o Ca diology and IIB San Pau
O ganiza ion Hospi al de San Pau
Add ess Ba celona, Spain
Email
Schedule
Recei ed 8 July 2015
Re ised
Accep ed 20 No embe 2015
Abs ac A ial ib illa ion (AF) has been associa ed wi h inc eased spon aneous calcium elease om he
sa coplasmic e iculum and linked o inc eased adenosine A2A ecep o (A2AR) exp ession and ac i a ion.
He e we es ed whe he his may a o a ial a hy hmogenesis by p omo ing bea - o-bea al e na ion and
i egula i y. Pa ch-clamp and con ocal calcium imaging was used o measu e he bea - o-bea esponse o
he calcium cu en and ansien in human a ial myocy es. Responses we e classi ied as uni o m,
al e na ing o i egula and s imula ion o Gs-p o ein coupled ecep o s dec eased he equency whe e a
uni o m esponse could be main ained om 1.0 ± 0.1 o 0.3 ± 0.1 Hz; p < 0.001 o be a-ad ene gic
ecep o s and om 1.4 ± 0.1 o 0.5 ± 0.1 Hz; p < 0.05 o A2ARs. The la e was linked o inc eased
spon aneous calcium elease and a e depola iza ions. Mo eo e , A2AR ac i a ion inc eased he ac ion o
non-uni o mly esponding cells in HL-1 myocy e cul u es (19 ± 3–51 ± 9 %; p < 0.02), and elec ical
mapping in pe used po cine a ia e ealed ha adenosine induced elec ical al e nans a longe cycle
leng hs, doubled he ac ion o elec odes showing al e na ion, and inc eased he ampli ude o al e na ions.
Impo an ly, p o ein kinase A inhibi ion inc eased he highes equency whe e uni o m esponses could be
main ained (0.84 ± 0.12–1.86 ± 0.11 Hz; p < 0.001) and p e en ion o A2AR-ac i a ion wi h exogenous
adenosine deaminase selec i ely inc eased he h eshold om 0.8 ± 0.1 o 1.2 ± 0.1 Hz; p = 0.001 in
myocy es om pa ien s wi h AF. A2AR-ac i a ion p omo es bea - o-bea i egula i ies in he calcium
ansien in human a ial myocy es, and p e en ion o A2AR ac i a ion may be a no el means o main ain
uni o m bea - o-bea esponses a highe bea ing equencies in pa ien s wi h a ial ib illa ion.
Keywo ds (sepa a ed by '-') Adenosine ecep o - A ial myocy e - Elec ophysiology - L-Type calcium cu en - Sa coplasmic
e iculum
Foo no e In o ma ion Elec onic supplemen a y ma e ial The online e sion o his a icle (doi:10.1007/s00395-015-0525-2)
con ains supplemen a y ma e ial, which is a ailable o au ho ized use s.
UNCORRECTED PROOF
ORIGINAL CONTRIBUTION
1
2P e en ion o adenosine A
2A
ecep o ac i a ion diminishes
3bea - o-bea al e na ion in human a ial myocy es
4C is ina E. Molina
1,5
•Anna Llach
2
•Adela He aiz-Ma ı
´nez
1
•Ca men Ta i a
1
•
5Mon se a Ba iga
1
•Rob F. Wiege inck
2
•Jacqueline Fe nandes
2
•Nu ia Cabello
1
•
6Alex Vallmi jana
4
•Rau
´l Beni e
´z
4
•Jose
´Mon iel
3
•Juan Cinca
2
•Lei Ho e-Madsen
1
7Recei ed: 8 July 2015 / Accep ed: 20 No embe 2015
8Sp inge -Ve lag Be lin Heidelbe g 2015
9Abs ac A ial fib illa ion (AF) has been associa ed wi h
10 inc eased spon aneous calcium elease om he sa coplas-
11 mic e iculum and linked o inc eased adenosine A
2A
12 ecep o (A
2A
R) exp ession and ac i a ion. He e we es ed
13 whe he his may a o a ial a hy hmogenesis by p omo -
14 ing bea - o-bea al e na ion and i egula i y. Pa ch-clamp
15 and con ocal calcium imaging was used o measu e he bea -
16 o-bea esponse o he calcium cu en and ansien in
17 human a ial myocy es. Responses we e classified as uni-
18 o m, al e na ing o i egula and s imula ion o Gs-p o ein
19 coupled ecep o s dec eased he equency whe e a uni o m
20 esponse could be main ained om 1.0 ±0.1 o
21 0.3 ±0.1 Hz; p 0.001 o be a-ad ene gic ecep o s and
22 om 1.4 ±0.1 o 0.5 ±0.1 Hz; p 0.05 o A
2A
Rs. The
23 la e was linked o inc eased spon aneous calcium elease
24 and a e depola iza ions. Mo eo e , A
2A
R ac i a ion
25
inc eased he ac ion o non-uni o mly esponding cells in
26
HL-1 myocy e cul u es (19 ±3–51 ±9%;p 0.02), and
27
elec ical mapping in pe used po cine a ia e ealed ha
28
adenosine induced elec ical al e nans a longe cycle
29
leng hs, doubled he ac ion o elec odes showing al e na-
30
ion, and inc eased he ampli ude o al e na ions. Impo -
31
an ly, p o ein kinase A inhibi ion inc eased he highes
32
equency whe e uni o m esponses could be main ained
33
(0.84 ±0.12–1.86 ±0.11 Hz; p 0.001) and p e en ion
34
o A
2A
R-ac i a ion wi h exogenous adenosine deaminase
35
selec i ely inc eased he h eshold om 0.8 ±0.1 o
36
1.2 ±0.1 Hz; p=0.001 in myocy es om pa ien s wi h
37
AF. A
2A
R-ac i a ion p omo es bea - o-bea i egula i ies in
38
he calcium ansien in human a ial myocy es, and p e-
39
en ion o A
2A
R ac i a ion may be a no el means o main ain
40
uni o m bea - o-bea esponses a highe bea ing equencies
41
in pa ien s wi h a ial fib illa ion. 42
43
Keywo ds Adenosine ecep o A ial myocy e
44
Elec ophysiology L-Type calcium cu en Sa coplasmic
45
e iculum
46
In oduc ion
47
Elec omechanical al e nans has been obse ed in di e en
48
pa hological se ings [16,35], p eceding he occu ence o
49
a ial fib illa ion (AF) [15,16] and he iden ifica ion o
50
molecula mechanisms ha egula e he s abili y o he
51
bea - o-bea esponse could help p e en ing he induc ion
52
o ecu ence o AF.
53
In physiological condi ions, al e na ion in ac ion
54
po en ial shape can be induced by a ificially inc easing he
55
hea a e [8,16]. Fu he mo e, mechanical al e nans is
56
modula ed by he plasma ic calcium le el [8], and episodes
A1 Elec onic supplemen a y ma e ial The online e sion o his
A2 a icle (doi:10.1007/s00395-015-0525-2) con ains supplemen a y
A3 ma e ial, which is a ailable o au ho ized use s.
A4 &Lei Ho e-Madsen
A5 [email p o ec ed]
A6
1
Ca diac Rhy hm and Con ac ion, Ca dio ascula Resea ch
A7 Cen e CSIC-ICCC and IIB San Pau, Hospi al de la San a
A8 C eu i San Pau, S An oni M
a
Cla e 167, 08025 Ba celona,
A9 Spain
A10
2
Depa men o Ca diology and IIB San Pau, Hospi al de San
A11 Pau, Ba celona, Spain
A12
3
Depa men o Ca diac Su ge y and IIB San Pau, Hospi al de
A13 San Pau, Ba celona, Spain
A14
4
Depa men o Au oma ic Con ol, Uni e si a Poli e
`cnica de
A15 Ca alunya, Ba celona, Spain
A16
5
P esen Add ess: Ins i u e o Pha macology, Facul y o
A17 Medicine, Uni e si y Duisbu g-Essen, 45122 Essen,
Ge many
AQ1
123
Jou nal : La ge 395 Dispa ch : 23-11-2015 Pages : 15
A icle No. : 525 hLE hTYPESET
MS Code : BRIC-D-15-00242 hCP hDISK
44
Basic Res Ca diol _#####################_
DOI 10.1007/s00395-015-0525-2
Au ho P oo
UNCORRECTED PROOF
57 can be e e sed by calcium adminis a ion in humans [35].
58 In isola ed mammalian myocy es, al e na ions in he cal-
59 cium ansien (calcium al e nans) can be induced by
60 lowe ing calcium en y h ough L- ype calcium channels
61 [10,22,29,36], by me abolic inhibi ion [19]o by
62 inc easing s imula ion equencies [1,36], and has been
63 asc ibed o in e - and/o in a-cellula inhomogenei y in
64 calcium handling [1,10,22,29,36]. In human a ial
65 myocy es, calcium al e nans can also be induced by ele-
66 a ing he s imula ion equency [26]. Mo eo e , human
67 a ial myocy es wi h la ge L- ype calcium cu en (I
Ca
) and
68 equen sa coplasmic e iculum (SR) calcium elease a
69 es we e ound mo e p one o p esen calcium al e nans
70 upon ele a ion o he s imula ion equency while myo-
71 cy es wi h less equen SR calcium elease and smalle I
Ca
72 could main ain a uni o m bea - o-bea esponse a highe
73 s imula ion equencies [26]. In e es ingly, a ial myocy es
74 om pa ien s wi h AF ha e a highe equency o spon a-
75 neous calcium elease [17] bu smalle I
Ca
densi y [11,27,
76 39], which would ha e opposi e e ec s on he bea - o-bea
77 esponse.
78 The highe equency o spon aneous calcium elease in
79 myocy es om pa ien s wi h AF has been linked o phos-
80 pho yla ion o he SR calcium elease channel/ yanodine
81 ecep o (RyR2) media ed by p o ein kinase A (PKA) o
82 calmodulin kinase II (CaMKII) [27,32,41]. Mo eo e ,
83 ac i a ion o he Gs-p o ein coupled adenosine A
2A
84 ecep o (A
2A
R) induce a PKA-media ed s imula ion o
85 spon aneous calcium elease in human a ial myocy es [18]
86 ha is mo e p onounced in myocy es om pa ien s wi h AF
87 and linked o a concu en inc ease in A
2A
R exp ession.
88 The abo e-men ioned findings may no only p omo e
89 a hy hmogenic calcium elease and a e depola iza ions in
90 pa ien s wi h AF [27] bu could also p omo e a ial
91 a hy hmia by a o ing al e na ing and i egula bea - o-
92 bea esponses. Howe e , his hypo hesis has ne e been
93 es ed and he aim o he p esen wo k was o es whe he
94 A
2A
R-ac i a ion educes he abili y o human a ial myo-
95 cy es o main ain a uni o m bea - o-bea esponse.
96 Me hods
97
98 A o al o 275 a ial myocy es we e isola ed om he igh
99 a ial appendix om 191 pa ien s as p e iously desc ibed
100 [17]. Pa ien s ea ed wi h Ca
2?
an agonis s we e excluded
101 om he s udy. Table 1 in he supplemen a y ma e ial
102 summa izes he clinical pa ame e s a baseline and pha -
103 macological ea men s o he pa ien s included in his
104 s udy. Pa ien s wi h AF included hose ha had a p e ious
105 his o y o AF, i.e. pa oxysmal o ch onic AF (see Table 1 in
106 he supplemen a y ma e ial). Pe mission o use he issue
107
samples was ob ained om each pa ien , and he s udy was
108
app o ed by he E hical Commi ee o ou ins i u ion and
109
conduc ed in acco dance wi h he Decla a ion o Helsinki
110
p inciples. The s udy also con o ms o he guidelines o he
111
Ca e and Use o Labo a o y Animals, and was app o ed by
112
he Ins i u ional Animal Ca e and Use Commi ee a ou
113
ins i u ion. Specific expe imen al p o ocols and condi ions
114
used in he s udy a e desc ibed in he supplemen a y ma e-
115
ial. Values a e exp essed as mean ±SEM. Fo human a ial
116
myocy es, he numbe o cells and pa ien s a e indica ed as
117
n=(cells/pa ien s). Da a se s we e es ed o no mali y.
118
S uden ’s es was used o assess significan di e ences
119
when es ing a specific e ec . Di e ences we e conside ed
120
significan a p 0.05. Two-way ANOVA and Holm-Sidak
121
pos - es was used o compa ison o mul iple e ec s in
122
pe used po cine a ial p epa a ions. Fo mul iple compa -
123
isons o bea - o-bea esponses in human a ial myocy es, a
124
Mixed-e ec s logis ic eg ession analysis was pe o med
125
using he S a a 12 p og am (S a aCo p, USA). The au ho s
126
had ull access o he da a and ake esponsibili y o i s
127
in eg i y. All au ho s ha e ead and ag ee o he manusc ip
128
as w i en. Expe imen s we e pe o med wi hou knowledge
129
abou clinical da a.
130
Resul s
131
P o ein kinase A inhibi ion inc eases he s abili y
132
o he bea - o-bea esponse in human a ial
133
myocy es
134
To de e mine i baseline ac i a ion o Gs-p o ein coupled
135
memb ane ecep o s, modula e he a e-dependen bea - o-
136
bea esponse in human a ial myocy es h ough PKA
137
ac i a ion, we fi s examined he e ec s o he PKA-in-
138
hibi o H-89. As shown in Fig. 1a, PKA inhibi ion
139
inc eased he equency whe e a s able I
Ca
ampli ude (blue
140
squa es) and he ime in eg al o he ail cu en (o ange
141
squa es) could be main ained, esul ing in a s ong inc ease
142
in he ac ion o uni o m esponses a all s imula ion e-
143
quencies examined (Fig. 1b). S a is ical analysis e ealed
144
ha H-89 p o ec ed agains non-uni o m bea - o-bea
145
esponses by inc easing he ac ion o uni o m esponses
146
(p 0.001) and dec easing al e na ing (p 0.001) and
147
i egula esponses (p 0.001). Consequen ly, he maxi-
148
mal equency whe e a uni o m bea - o-bea esponse could
149
be main ained was doubled by H-89 (Fig. 1c).
150
Be a-ad ene gic s imula ion p omo es bea - o-bea
151
al e na ion in human a ial myocy es
152
To es i ac i a ion o Gs-p o ein coupled be a-ad ene gic
153
ecep o s had he opposi e e ec o H-89, myocy es we e
_####_ Page 2 o 15 Basic Res Ca diol _#####################_
123
Jou nal : La ge 395 Dispa ch : 23-11-2015 Pages : 15
A icle No. : 525 hLE hTYPESET
MS Code : BRIC-D-15-00242 hCP hDISK
44
Au ho P oo
UNCORRECTED PROOF
154 s imula ed wi h he agonis isop o e enol (ISO). As shown
155 in Fig. 2a, his induced a p onounced al e na ion in he
156 ime in eg al o he ail cu en , causing a s ong inc ease in
157 al e na ing (p 0.001) and i egula (p 0.05) esponses
158 and consequen ly a educ ion in he ac ion o uni o m
159 esponses (p 0.001; Fig. 2b). Acco dingly, ISO s ongly
160 educed he h eshold o he induc ion o non-uni o m
161 esponses (Fig. 2c). Subsequen ly, we de e mined whe he
162 myocy es om pa ien s ea ed wi h be a-blocke s had a
163 di e en esponse han myocy es om pa ien s ecei ing
164 no ea men . As shown in Fig. 2d, e he e we e nei he
165 di e ences in he esponse o myocy es om he wo
166 pa ien g oups no any di e ence in he maximal equency
167 whe e a uni o m esponse could be main ained.
168 Adenosine A
2A
ecep o s egula e bea - o-bea
169 changes in he calcium ansien
170 Since A
2A
R ac i a ion induces PKA-dependen s imula ion
171 o spon aneous SR calcium elease wi hou a ec ing I
Ca
,
172 we used con ocal calcium imaging (see supplemen a y
173 ma e ial figu e S1) o in es iga e how ac i a ion o his
174 ecep o wi h i s na u al ligand adenosine (ADO) a ec ed
175 bea - o-bea changes in he calcium ansien and calcium
176 fluxes ac oss he sa colemma. Inclusion o 30 lM ADO in
177 he pa ch pipe e p omo ed bea - o-bea changes in he
178
calcium ansien in a ime-dependen manne (Fig. 3a, b).
179
Thus, he highes equency whe e uni o m calcium an-
180
sien s could be main ained was 1.40 ±0.12 Hz a he
181
beginning o ADO in usion (2–4 min a e pa ch b eak)
182
and only 0.45 ±0.08 Hz a e 18–24 min o ADO in usion
183
(p=0.002, n=9/6). Analysis o local calcium ansien s
184
e ealed ha ADO in usion g adually changed he bea - o-
185
bea esponse om uni o m and synch onized calcium
186
ansien s o synch onized local non-uni o m esponses
187
(panel 3c) ha e en ually deg aded in o non-uni o m
188
esponses wi h un-synch onized spon aneous calcium
189
wa es. Figu e 3d illus a es how he ac ion o synch o-
190
nized non-uni o m esponses and calcium wa es inc eases
191
wi h he ime ADO is in used in o myocy es s imula ed a
192
1 Hz.
193
Simul aneous measu emen s o in acellula calcium
194
ansien s and ionic cu en s we e used o in es iga e he
195
mechanisms unde lying his adenosine-media ed e ec ,
196
and e ealed ha ele a ion o he s imula ion equency
197
induced concu en al e na ion (see supplemen a y ma e ial
198
figu e S4) o non-uni o m esponses in he calcium an-
199
sien , he I
Ca
ampli ude and he ail cu en elici ed upon
200
epola iza ion (Fig. 4a).
201
Mo eo e , his p omo ion o non-uni o m bea - o-bea
202
esponses was linked o a concu en inc ease in spon a-
203
neous calcium wa es du ing ADO in usion ( om
Fig. 1 PKA inhibi ion a o s
uni o m bea - o-bea esponses.
aConsecu i e cu en aces
eco ded in a human a ial
myocy e paced a 2 Hz be o e
( op panel) and a e exposu e o
1lM H-89 (lowe panel). Blue
squa es indica e I
Ca
and o ange
squa es he ail cu en elici ed
upon epola iza ion. The fi s
inwa d peak o each cu en
ace is he Na
?
-cu en elici ed
by a p epulse o -50 mV.
bF equency-dependen
dis ibu ion o uni o m,
al e na ing, and i egula bea -
o-bea esponses among 15
myocy es om 15 pa ien s
be o e (con ol,le panel) and
a e PKA inhibi ion wi h 1 lM
H-89 ( igh panel). cMaximal
equency whe e a uni o m
esponse could be main ained.
***p 0.001
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204 1.1 ±0.2 e en s/min a he onse o 13.7 ±3.2 e en s/min
205 a e 18–24 min). T ansien inwa d cu en s (I
TI
) elici ed
206 by hese calcium wa es occu ed bo h a es and du ing
207 elec ical s imula ion and had simila kine ics (Fig. 4b).
208 Mo eo e , he I
TI
equency du ing s imula ion was p o-
209 po ional o he I
TI
equency a es (Fig. 4c). Conse-
210 quen ly, he e was an in e se ela ionship be ween he I
TI
211 equency and he highes equency whe e a uni o m
212 esponse could be main ained (Fig. 4d). By con as , he I
Ca
213 densi y was no changed by ADO in usion (1.5 ±0.2 pA/
214 pF a he onse s. 1.6 ±0.2 pA/pF a e ADO in usion) o
215
deple ion (1.5 ±0.2 pA/pF a he onse s. 1.6 ±0.3 pA/
216
pF a e in usion o ADO ee solu ion), and he e was no
217
co ela ion be ween he I
Ca
ampli ude and he highes e-
218
quency whe e a uni o m esponse could be main ained
219
(Fig. 4e).
220
Figu e 5analyzes how p olonged in usion (18–24 min)
221
o ADO-con aining and ADO- ee solu ion a ec ed he
222
bea - o-bea esponse, and e ealed ha myocy es in used
223
wi h ADO- ee solu ion we e able o main ain uni o m
224
esponses a he highe s imula ion equencies. By con as
225
ADO in usion elici ed i egula esponses wi h nume ous
Fig. 2 Be a-ad ene gic s imula ion a o s he induc ion o bea - o-
bea al e na ion. aRep esen a i e eco dings showing he e ec s o
30 nM ISO on he ime in eg al o he ail cu en (uppe panel) and
I
Ca
(lowe panel) in a human a ial myocy e paced a 0.5 Hz. The
ime-dependen changes in he ime in eg al and I
Ca
a e shown on he
igh .Le e s deno e he ime poin whe e cu en s shown on he le
we e eco ded. bF equency-dependen dis ibu ion o uni o m
(black), al e na ing (g ey), and i egula (whi e) bea - o-bea
esponses among 29 myocy es om 27 pa ien s be o e (con ol) and
a e be a-ad ene gic s imula ion wi h 30 nM ISO. The s imula ion
equency is indica ed below each ba .cMaximal equency o
main enance o a uni o m esponse wi h and wi hou ISO. dF e-
quency-dependen dis ibu ion o uni o m (black), al e na ing (g ey),
and i egula (whi e) bea - o-bea esponses among 65 myocy es om
43 pa ien s ea ed wi h be a blocke s (BB) and 137 myocy es om 95
pa ien s wi hou bea -blocke ea men (no BB). eMaximal equency
o main enance o a uni o m esponse wi h and wi hou BB ea men
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404 s imula ion pulse, uling ou ha hese wa es a e igge ed
405 e en s. Mo eo e , he A
2A
R agonis CGS21680 caused a
406 pa allel inc ease in calcium elease-induced I
TI
s and
407 spon aneous memb ane depola iza ions (Fig. 7 , g).
408 CGS21680 also s ongly inc eased he ampli ude hese
409 memb ane depola iza ions (Fig. 7h) and hey pe sis ed
410 when myocy es we e s imula ed, demons a ing ha A
2A
R
411 ac i a ion s imula es spon aneous calcium elease ha
412 a o s elec ical ins abili y. L-Type calcium channels did
413 no appea o be a majo a ge o A
2A
R-media ed egu-
414 la ion since nei he adenosine no he selec i e A
2A
R
415 agonis CGS21680 had any significan e ec on I
Ca
den-
416 si y. Acco dingly, he e was no co ela ion be ween I
Ca
417 densi y and he h eshold equency o loss o a uni o m
418 esponse (see Fig. 4).
419
The abili y o A
2A
R-ac i a ion o p omo e spon aneous
420
calcium elease has p e iously been linked o PKA-de-
421
penden phospho yla ion o he RyR2 a se 2808 [27]. In
422
line wi h his, he PKA inhibi o H-89 d ama ically educed
423
he incidence o non-uni o m esponses a all s imula ion
424
equencies s udied, suppo ing he no ion ha PKA-de-
425
penden signaling in e ene in he egula ion o he bea - o-
426
bea s abili y a baseline.
427
This is also compa ible wi h p e ious wo k sugges ing
428
he p esence o a cyclic adenosine monophospha e- onus a
429
baseline in human a ial myocy es [43]. Mechanis ically,
430
A
2A
Rs a e coupled o G
s
p o eins [33] and linked o cAMP
431
p oduc ion and PKA-ac i a ion [34] and could s imula e
432
phospho yla ion o phospholamban a he esidue se ine16
433
(s16) o he RyR2 a he esidues s2808 o s2030. This
Fig. 8 P e en ion o A
2A
R
ac i a ion a o s uni o m bea -
o-bea esponses in myocy es
om pa ien s wi h AF.
aRep esen a i e cu en
eco dings om a human a ial
myocy e be o e (con ol) and
a e exposu e o ADA o
ADA ? he non-deg adable
ADO analog CGS21680
(ADA ?CGS). bF equency-
dependen dis ibu ion o
uni o m (black), al e na ing
(g ey), and i egula (whi e)
bea - o-bea esponses among
14 myocy es om 12 pa ien s
wi h AF eco ded in con ol and
wi h ADA. cF equency-
dependen dis ibu ion o
uni o m, al e na ing, and
i egula bea - o-bea esponses
among 20 myocy es om 18
pa ien s wi hou AF eco ded in
con ol and wi h ADA.
dTh eshold equency o
main enance o a uni o m
esponse ***p 0.001.
eF equency o calcium elease
induced I
TI
s eco ded in pa ien s
wi hou (no AF) o wi h AF.
Measu emen s we e done be o e
(CON; black ba s) and a e
exposu e o ADA (g ey ba s).
Rela ionship be ween he I
TI
equency and h eshold o he
main enance o a uni o m bea -
o-bea esponse. **p 0.01
***p 0.001
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UNCORRECTED PROOF
434 would be in acco dance wi h p e ious s udies linking
435 dys unc ional calcium handling o inc eased phospholam-
436 ban phospho yla ion a s16 in ch onic and pa oxysmal AF
437 [12,42] and RyR2 phospho yla ion a s2808 in ch onic AF
438 [27,43]. In line wi h his, he PKA-inhibi o H-89 has been
439 shown o e e se A
2A
R-media ed s imula ion o spon a-
440 neous calcium elease [18]. Howe e , A
2A
R-dependen
441 cAMP p oduc ion could also ac i a e calmodulin kinase II
442 (CamKII)-dependen phospho yla ion o he RyR a s2814,
443 which would be in ag eemen wi h o he s udies linking
444 spon aneous calcium elease in AF o s2814 phospho yla-
445 ion [32,42,43]. Mo eo e , i egula hy hm has been
446 shown o inc ease dias olic [Ca
2?
] and ac i a ion o
447 CaMKII and AMP-ac i a ed p o ein kinase [25] ha could
448 c ea e a icious cycle a o ing i egula bea ing.
449 Be a-ad ene gic s imula ion wi h ISO also educed he
450 h eshold o he induc ion o non-uni o m esponses and
451 p e e en ially induced al e na ing esponses. This is oppo-
452 si e o he epo ed abili y o ISO o escue calcium al e -
453 nans in ca a ial myocy es [13], bu in acco dance wi h he
454 no ion ha concu en s imula ion o I
Ca
and SR calcium
455 elease, as epo ed o ISO he e and in o he s udies [20,
456 45], p omo es al e nans in human a ial myocy es [26].
457 Simila ly, s imula ion o SR calcium elease bu no I
Ca
by
458 adenosine, as shown in Fig. 4and epo ed by Llach e al.
459 [27] is mo e likely o induce i egula bea - o-bea
460 esponses [26]. In e es ingly, i has been epo ed ha ISO
461 p omo es and p op anolol dec eases he ampli ude and
462 incidence o T-wa e al e nans in human a ia om pa ien s
463 su e ing om sup a en icula achya hy hmia when he
464 a ia we e paced a 110 bpm (1.8 Hz) [24], unde sco ing
465 he physiological ele ance o ou findings. Indeed, ou
466 esul s would sugges ha he mechanism unde lying he
467 abili y o ISO o induce T-wa e al e nans in human a ia is
468 a PKA- o CaMKII-dependen s imula ion o SR calcium
469 elease ha educes he h eshold equency o induc ion
470 o bea - o-bea al e na ion in he calcium ansien . The
471 opposi e e ec s o ISO in ca and human a ial myocy es
472 a e possibly due o di e en expe imen al condi ions o
473 species-dependen di e ences in he cellula calcium
474 homeos asis. In a o o he la e possibili y, ma hema ical
475 models ha e shown ha small changes in SR calcium
476 up ake o RyR2 ga ing p ope ies can p o oundly a ec he
477 a ial bea - o-bea esponse [7,28].
478 Regula ion o he bea - o-bea esponse in a ial
479 ib illa ion
480 AF has p e iously been linked o PKA-media ed phos-
481 pho yla ion o he RyR2 a s2808 in pa ien s wi h pe ma-
482 nen AF [41] and phospholamban a s16 [12,42], which
485485485
would bo h a o ele a ion o spon aneous SR calcium
486
elease e en s a baseline [17]. Mo eo e , inc eased A
2A
R
487
exp ession in pa ien s wi h AF has been shown o inc ease
488
RyR2 phospho yla ion a s2808 and p oposed o accoun
489
o he highe a e o spon aneous calcium elease [27].
490
These obse a ions, combined wi h p esen finding o an
491
in e se ela ionship be ween spon aneous calcium elease
492
e en s and he abili y o main ain a uni o m bea - o-bea
493
esponse (see Figs. 4,7,8), sugges ha pha macological
494
con ol o A
2A
R ac i a ion could be a means o egula e he
495
bea - o-bea esponse a high s imula ion equencies in
496
myocy es om AF pa ien s. Indeed, we he e show ha
497
in usion o adenosine- ee solu ion h ough he pa ch-pip-
498
e e o applica ion o exogenous ADA inc eases he
499
h eshold equency o induc ion o non-uni o m espon-
500
ses. The la e was mo e p onounced in myocy es om AF
501
pa ien s, demons a ing ha inhibi ion o A
2A
R ac i a ion
502
s abilizes calcium handling in pa ien s wi h AF. This s a-
503
bilizing ac ion is e en mo e p onounced i he e ec o
504
empe a u e on bea ing a e is aken in o accoun . Thus, a
505
bea ing a e nea 0.5 Hz a 22 C would co espond o
506
1.25 Hz a 37 C[
5], and be ep esen a i e o myocy es in
507
pa ien s wi h a no mal bea ing a e. A his equency mo e
508
han 90 % o he myocy es ha e a uni o m esponse and
509
inhibi ion o A
2A
R ac i a ion wi h ADA has li le e ec .
510
By con as , bea ing a es o 1 and 1.3 Hz a 22 C would
511
co espond o a es a ound 2.5 and 3.3 Hz a 37 C, co -
512
esponding o a ial a hy hmia. A hese equencies only
513
15 and 25 % o he myocy es espond uni o mly, and ADA
514
inc eases he numbe o uni o mly esponding myocy es o
515
45 and 70 % a 1.3 and 1 Hz, espec i ely.
516
As illus a ed in Fig. 6a–c, such a s ong educ ion in he
517
ac ion o myocy es wi h i egula esponses could
518
po en ially e e he o e all esponse o an i egula ly
519
bea ing mul icellula myocy e p epa a ion o a uni o m
520
esponse.
521
I is he e o e concei able ha physiologically ele an
522
fluc ua ions in he cy osolic adenosine le el such as hose
523
induced by s ess, deficien ci cula ion o he a ial
524
appendices [4] o ischemia [3] ha e he po en ial o p o-
525
mo e non-uni o m bea - o-bea esponses by educing he
526
h eshold equency o hei induc ion. In suppo o his
527
no ion, elec ical mapping in pe used po cine a ia
528
e ealed ha adenosine in usion induces T-wa e al e nans
529
a lowe bea ing a es and inc eases he al e nans ampli ude
530
(see Fig. 6).
531
Impo an ly, selec i e A
2A
R inhibi ion was able o
532
e e se he e ec s o massi e inc eases in he cy osolic
533
adenosine le el (see Fig. 5) and exogenous adenosine
534
deaminase inc eased he h eshold equency o induc ion
535
o non-uni o m esponses in myocy es om pa ien s wi h
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UNCORRECTED PROOF
538538538 AF by educing spon aneous calcium elease e en s in bo h
539 es ing and bea ing myocy es, demons a ing ha pha ma-
540 cological con ol o A
2A
R ac i a ion can be used o
541 coun e ac a hy hmogenic e ec s o pa hological inc ea-
542 ses in he cellula adenosine le el.
543 S udy limi a ions
544 A challenge encoun e ed wi h human ca diomyocy e
545 models is ha clinical o he apeu ical he e ogenei y
546 among pa ien s wi h and wi hou AF can po en ially bias
547 he esul s. To minimize his issue, pa ien s ea ed wi h
548 calcium an agonis s we e excluded om he s udy. We also
549 uled ou ha he e we e po en ially con ounding e ec s o
550 educed le en icula ejec ion ac ion ( 40 %), gende ,
551 and be a-blocke ea men (see Fig. 2d, e). Mo eo e , he
552 obse ed changes in I
Ca
and spon aneous calcium elease
553 a e consis en wi h p e ious epo s on human AF con-
554 fi ming ha he model can ai h ully ep oduce obse a-
555 ions om isola ed human a ial myocy es [17,27,39].
556 The use o fluo escen dyes, such as fluo-4, o moni o
557 in acellula calcium ansien s has p e iously been epo -
558 ed o a o i egula esponses a he expense o al e na -
559 ing esponses in human a ial myocy es [11], and we
560 he e o e only used calcium imaging in expe imen s
561 specifically add essing e ec s o A
2A
R-media ed e ec on
562 spa io- empo al changes in he calcium ansien and he
563 bea - o-bea esponse.
564 While pha macological ools used o manipula e A
2A
R
565 ac i a ion a e highly selec i e, he selec i i y o H-89 o
566 PKA inhibi ion is con o e sial and depends on he p es-
567 ence o o he kinases [9]. Ne e heless, unpublished da a
568 om ou labo a o y show ha H-89 and KT5720, consid-
569 e ed a mo e selec i e PKA inhibi o , ha e simila abili ies
570 o p e en spon aneous and igge ed calcium elease in
571 mouse ca diomyocy es whe e con ounding e ec s o con-
572 cu en ca dio ascula disease and pha macological ea -
573 men s a e a oided.
574 Func ionally, adenosine also ac i a es adenosine A
1
575 ecep o s and is associa ed o sho ening o he a ial
576 e ac o y pe iod [44] and elec ical e-en y [2], which
577 likely a ec s he a ial elec ical signals in he pe used
578 po cine a ia. Howe e , i is no clea ha his would
579 p omo e he obse ed -wa e al e nans. Ins ead, ou esul s
580 a e consis en wi h findings in humans [24,30] and in
581 animal models [1,21] whe e a ial ac ion po en ial al e -
582 nans and spon aneous depola iza ions associa ed o
583 abno mal calcium handling ha e been p oposed o unde lie
584 a hy hmic ulne abili y and inc eased suscep ibili y o AF.
585 Finally, egional di e ences a e known o exis in
586 human a ial physiology, and we canno exclude ha he
587 p esen esul s om igh a ial appendages will di e om
588 he esponse o le a ial p epa a ions.
589
Clinical implica ions
590
The induc ion o pe pe ua ion o AF has been asc ibed o
591
emodeling o se e al ionic cu en s and calcium handling
592
mechanisms [6,14,17,39,40], including inc eased spon-
593
aneous calcium elease om he SR [17], hype phospho-
594
yla ion o he RyR2 a s2808 [27,41] and s2814 [32,42],
595
as well as inc eased A
2A
R exp ession and ac i a ion [27].
596
The p esen s udy shows ha emodeling o A
2A
R-medi-
597
a ed signaling in AF can also acili a e al e na ing o
598
i egula bea - o-bea esponses, a phenomenon ha has
599
been epo ed o p ecede and p omo e he onse o AF [23,
600
31,37,38]. Impo an ly, p e en ion o A
2A
R ac i a ion
601
significan ly imp o ed he bea - o-bea esponse in myo-
602
cy es om AF pa ien s. This, could p e en a massi e
603
induc ion o i egula bea - o-bea esponses upon ele a-
604
ion o he bea ing equency, and po en ially p e en e-
605
ini ia ion o he a hy hmia in pa ien s wi h pa oxysmal AF
606
exposed o achyca dic s ess o a e ca dio e sion in
607
pa ien s wi h AF. On he o he hand, he e ec o p e-
608
en ion o A
2A
R ac i a ion is smalle a he highes s im-
609
ula ion equency examined, sugges ing ha i s e ficacy
610
may be smalle in pa ien s wi h pe manen AF. Inhibi ion
611
o PKA-dependen signaling also p omo es uni o m bea -
612
o-bea esponses a high s imula ion equencies, bu he
613
ubiqui ous na u e and mul iple unc ions o PKA make i
614
di ficul o en isage PKA inhibi o s as a means o s abilize
615
calcium handling in AF. Ins ead, pha macological con ol
616
o A
2A
R ac i a ion may be a key o selec i ely educe
617
spon aneous calcium elease and p omo e uni o m bea - o-
618
bea esponses in a ial myocy es om pa ien s wi h AF
619
wi hou comp omising he L- ype calcium cu en , which is
620
c i ical o he ac i a ion o con ac ion.
621
In summa y, we show o he fi s ime ha A
2A
R ac i-
622
a ion educes he equency h eshold o induc ion o non-
623
uni o m bea - o-bea changes in he calcium ansien in
624
human a ial myocy es and T-wa e al e nans in pe used
625
po cine a ial p epa a ions. Impo an ly, he A
2A
R-media ed
626
e ec was mo e p onounced in pa ien s wi h AF, and p e-
627
en ion o A
2A
R ac i a ion a o ed uni o m esponses and
628
significan ly inc eased he h eshold o he induc ion o non-
629
uni o m esponses in myocy es om hese pa ien s. This
630
p oposes pha macological inhibi ion o A
2A
R ac i a ion as a
631
no el he apeu ical app oach o p e en bea - o-bea al e -
632
na ion o i egula esponses in a ial myocy es om pa ien s
633
wi h AF du ing s ess-induced ele a ion o he bea ing a e o
634
pa hological condi ions p omo ing ele a ion o cellula
635
adenosine le els such as hypoxia o ischemia.
636
Acknowledgmen s We would like o hank M . And eu Fe e o-
637
G ego i o in aluable help wi h he s a is ical analysis and g ea ly
638
app ecia e he collabo a ion o he su geons a he Ca diac Su ge y
639
Depa men a Hospi al de la San a C eu i San Pau.
AQ2
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640 Compliance wi h e hical s anda ds
641 Con lic o in e es None.
642 Funding This wo k has ecei ed unding om Spanish Minis y o
643 Educa ion and Science (SAF2011-30312), (SAF2014-58286-C2-1R)
644 and (CNIC2009-08), Ins i u o de Salud Ca los III (ne wo k RD12/
645 0042/0002), Gene ali a de Ca alunya (2014SGR1465) and Ma ie
646 Cu ie IEF G an (PIEF-GA-2012-331241).
647
648 Re e ences
649 1. Ais up GL, Kelly JE, Kapu S, Kowalczyk M, Sysman-Wolpin I,
650 Kadish AH, Wasse s om JA (2006) Pacing-induced he e o-
651 genei ies in in acellula Ca
2?
signaling, ca diac al e nans, and
652 en icula a hy hmias in in ac a hea . Ci c Res 99:e65–e73.
653 doi:10.1161/01.RES.0000244087.36230.b
654 2. A ienza F, Almend al J, Mo eno J, Vaidyana han R, Talkachou
655 A, Kali a J, A enal A, Villacas in JP, To ecilla EG, Sanchez A,
656 Plou z-Snyde R, Jali e J, Be en eld O (2006) Ac i a ion o
657 inwa d ec ifie po assium channels accele a es a ial fib illa ion
658 in humans: e idence o a een an mechanism. Ci cula ion
659 114:2434–2442. doi:10.1161/CIRCULATIONAHA.106.633735
660 3. Be ole BD, Hill JA, Ke ensky RA, Bela dinelli L (1997)
661 Myoca dial in a c ion ela ed a ial fib illa ion: ole o endoge-
662 nous adenosine. Hea 78:88–90
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