Ci a ion: Malague a-Viei a, L.;
Fe nández-Ruocco, J.;
Ho igón-Vinag e, M.P.; Zamo a, V.;
Zayas-A abal, J.; Echeaza a, L.;
Smi h, G.L.; Vila Pe o , M.; Medei,
E.; Casis, Ó.; e al. Me o min
Reduces Po assium Cu en s and
P olongs Repola iza ion in
Non-Diabe ic Hea . In . J. Mol. Sci.
2022,23, 6021. h ps://doi.o g/
10.3390/ijms23116021
Academic Edi o s: An onio
Fe e -Mon iel and An onio Felipe
Recei ed: 14 Ap il 2022
Accep ed: 24 May 2022
Published: 27 May 2022
Publishe ’s No e: MDPI s ays neu al
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published maps and ins i u ional a il-
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Copy igh : © 2022 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
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A ibu ion (CC BY) license (h ps://
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In e na ional Jou nal o
Molecula Sciences
A icle
Me o min Reduces Po assium Cu en s and P olongs
Repola iza ion in Non-Diabe ic Hea
Layse Malague a-Viei a 1, Julie a Fe nández-Ruocco 2, Ma ía P. Ho igón-Vinag e 3, Víc o Zamo a 3,
Julián Zayas-A abal 1, Ley e Echeaza a 1, God ey L. Smi h 3, Ma ín Vila Pe o 4, Emiliano Medei 2,
Ósca Casis 1,* and Mónica Gallego 1
1Depa amen o de Fisiología, Facul ad de Fa macia, Uni e sidad del País Vasco UPV/EHU,
01006 Vi o ia-Gas eiz, Spain; [email p o ec ed] (L.M.-V.); [email p o ec ed] (J.Z.-A.);
[email p o ec ed] (L.E.); [email p o ec ed] (M.G.)
2Ins i u e o Biophysics Ca los Chagas Filho, Uni e sidade Fede al do Rio de Janei o,
Rio de Janei o 21941-902, B azil; [email p o ec ed] (J.F.-R.); [email p o ec ed] (E.M.)
3Ins i u e o Ca dio ascula and Medical Sciences, College o Medical, Ve e ina y and Li e Science,
Uni e si y o Glasgow, 126 Uni e si y Place, Glasgow G12 8TA, UK; [email p o ec ed] (M.P.H.-V.);
[email p o ec ed] (V.Z.); god ey.smi [email protected] (G.L.S.)
4Cen o de In es igaciones Ca dio ascula es, Conice La Pla a, Facul ad de Ciencias Médicas,
Uni e sidad Nacional de La Pla a, La Pla a 1900, A gen ina; [email p o ec ed]g.a
*Co espondence: osca [email p o ec ed]; Tel.: +34-945013033
Abs ac :
Me o min is he i s choice d ug o he ea men o ype 2 diabe es due o posi i e esul s
in educing hype glycaemia and insulin esis ance. Howe e , diabe ic pa ien s ha e highe isk
o en icula a hy hmia and sudden ca diac dea h, and me o min ailed o educe en icula
a hy hmia in clinical ials. In o de o explo e he mechanisms esponsible o he lack o p o ec i e
e ec , we in es iga ed
in i o
he e ec o me o min on ca diac elec ical ac i i y in non-diabe ic a s;
and
in i o
in isola ed en icula myocy es, HEK293 cells exp essing he hERG channel and human
induced plu ipo en s em cells de i ed ca diomyocy es (hIPS-CMs). Su ace elec oca diog ams
showed ha long- e m me o min ea men (7 weeks) a he apeu ic doses p olonged ca diac epo-
la iza ion, e lec ed as QT and QTc in e al du a ion, and inc eased en icula a hy hmia du ing
he ca eine/dobu amine challenge. Pa ch-clamp eco dings in en icula myocy es isola ed om
ea ed animals showed ha he cellula mechanism is a educ ion in he ca diac ansien ou wa d
po assium cu en (I
o
).
In i o
, incuba ion wi h me o min o 24 h also educed I
o
, p olonged
ac ion po en ial du a ion, and inc eased spon aneous con ac ions in en icula myocy es isola ed
om con ol a s. Me o min incuba ion also educed I
hERG
in HEK293 cells. Finally, me o min
incuba ion p olonged ac ion po en ial du a ion a 30% and 90% o epola iza ion in hIPS-CMs, which
is compa ible wi h he educ ion o I
o
and I
hERG
. Ou esul s show ha me o min di ec ly modi ies
he elec ical beha io o he no mal hea . The mechanism consis s in he inhibi ion o epola izing
cu en s and he subsequen dec ease in epola iza ion capaci y, which p olongs AP and QTc du a ion.
Keywo ds:
ca diac elec ophysiology; epola iza ion; ca diomyocy e; ion channels; en icula
a hy hmia; ca diac ac ion po en ial; diabe es
1. In oduc ion
In he las decades, diabe es p e alence in adul s has inc eased in almos e e y coun y
a ec ing 422 million adul s in 2014 [
1
,
2
]. Type 2 diabe es (T2D) accoun s o mo e han
90% o diagnosed cases o diabe es. Since he posi i e esul s wi h o e weigh diabe ic
pa ien s epo ed in he Uni ed Kingdom Diabe es P ospec i e S udy in 1998 [
3
], me o min
became he i s he apeu ic choice o he ea men o T2D. The bene icial e ec s o
me o min include imp o ing glycemic con ol; educing insulin esis ance; educing
as ing hype insulinemia and p og ession o p ediabe es; educing ca dio ascula isk;
In . J. Mol. Sci. 2022,23, 6021. h ps://doi.o g/10.3390/ijms23116021 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2022,23, 6021 2 o 14
and may also imp o e blood lipid p o ile and educe p oin lamma o y s a e. Howe e ,
me o min mechanisms o ac ion in he ea men o T2D a e complex and no ye ully
unde s ood [4].
The incidence o en icula a hy hmia and sudden ca diac dea h is highe in dia-
be ic pa ien s [
5
,
6
], as diabe es-induced elec ical emodeling can con ibu e o he isk o
en icula a hy hmia [
7
]. The mos common elec ical dis u bance in diabe ic pa ien s
is p olonged en icula epola iza ion ha can be seen on he elec oca diog am as an
excessi e leng hening o hea a e co ec ed QT in e al (QTc). P olonged QTc is associa ed
wi h po en ially le hal en icula a hy hmias and p edic s ca dio ascula mo ali y e en
in heal hy indi iduals [8].
Since many pa ien s wi h T2D ecei e medica ion immedia ely a e diagnosis, da a
ega ding he incidence o QT in e al p olonga ion usually come om s udies o pa ien s
on glucose-lowe ing he apy. In addi ion, la ge s udies do no usually di e en ia e pa ien s
on me o min mono he apy om hose on combined he apy o achie e glycemic con ol.
A c oss-sec ional s udy o 501 ype 2 diabe ic pa ien s showed a high p e alence o p o-
longed QTc (44%). In e es ingly, he pe cen age o pa ien s on me o min was simila in
he no mal and in he p olonged QTc g oup [
9
]. In his sense, we ha e ecen ly published
ha me o min ea men con ols glycaemia in T2D a s, bu does no no malize QTc in e -
al du a ion and does no educe he suscep ibili y o a hy hmia [
10
]. Among he main
amilies o hypoglycemic d ugs, sul onylu eas cause a highe isk o ca diac a hy hmias.
Rega ding he o he amilies, compa ed wi h dipep idyl pep idase 4 inhibi o s, hiazolidine-
diones o Glucagon-like pep ide-1 ecep o agonis s, me o min was no associa ed wi h
a di e ence in he isk o de eloping en icula a hy hmia [
11
]. Fu he mo e, despi e
he ca dio ascula bene i s, me o min ailed o educe en icula a hy hmia in clinical
ials [12–14].
The aim o his wo k was o s udy a po en ial mechanism o explain his lack o
p o ec i e e ec o me o min agains a hy hmia. We in es iga ed he e ec o me o min
ea men on ca diac elec ical beha io in heal hy non-diabe ic a s, HEK293 cells and
human induced plu ipo en s em cells de i ed ca diomyocy es (hIPSC-CMs). Ca diac
ansien ou wa d po assium cu en (I
o
) and hERG cu en (I
hERG
), ca diac ac ion po en ial
(AP) and elec oca diog ams (ECG) we e eco ded.
2. Resul s
2.1. Me o min T ea men P olongs QTc
To in es iga e he e ec s o ch onic me o min ea men , we ea ed heal hy Sp ague-
Dawley a s o 7–8 weeks wi h me o min 35 mg/kg daily o ehicle (d inking
wa e
).
Al hough
in ou p e ious wo k highe me o min doses we e equi ed o keep glycemic
con ol in T2D Sp ague-Dawley a s [
10
], we used 35 mg/kg daily because is he maximal
app o ed o al daily dose o he ea men o ype 2 diabe es [
15
]. Th oughou he ea men ,
me o min did no cause hypoglycemia o a ec body weigh gain (
Supplemen a y Figu e S1
).
The ECG in conscious animals was moni o ed weekly (Figu e 1). We ound no di -
e ences in he du a ions o RR, PR, and QRS in e als be ween animals ea ed wi h
me o min and con ol g oup (no shown). Howe e , me o min leng hened QT and QTc
in e al du a ions. A he end o he expe imen al pe iod, QTc was signi ican ly longe in
he me o min g oup (131 ±1.9 ms) compa ed o con ol (120 ±2.2 ms; p< 0.01).
Bea - o-bea a iabili y o epola iza ion du a ion is an al e na i e ma ke used o
p edic a hy hmia and sudden ca diac dea h [
16
,
17
]. Howe e , no di e ence was ound
be ween con ol and me o min- ea ed animals (2.40 ±0.17 ms s. 2.45 ±0.19 ms).
2.2. Me o min T ea men Inc eases In Vi o A hy hmia Suscep ibili y
To assess he unc ional ele ance o me o min-induced QTc p olonga ion, we sea ched
o a hy hmic e en s. ECG in conscious a s ea ed wi h me o min o 7 o 8 weeks,
he pe iod when all changes in he elec oca diog aphic pa ame e s we e well es ablished
was analyzed. The incidence and ype o spon aneous a hy hmia (Figu e 2a,b) we e
In . J. Mol. Sci. 2022,23, 6021 3 o 14
di e en in he wo expe imen al g oups. O e all, spon aneous a hy hmic e en s we e
in equen and mild, mainly en icula p ema u e bea s and pe sis en bigeminy/sal o.
Howe e , he pe cen age o animals who showed no e en s was smalle in he me o min
g oup (
35% s. 67%
in con ol) and one me o min- ea ed animal de eloped non-sus ained
polymo phic en icula achyca dia.
Figu e 1
QT in e al
W0
W7
40
50
60
70
80
90
*
7
4
1
QT (ms)
T ea men (weeks)
Con ol
Me o min
0
0
200
400
600
Me o min
W7
Con ol
W7
HR (bea s/min)
Con ol
W0
75
100
125
150
175
Me o min
W7
Con ol
W7
QTc (ms)
Con ol
W0
**
ab
cd
Figu e 1.
Me o min ea men p olongs QTc in e al du a ion. (
a
) Rep esen a i e ECGs o a conscious
animal be o e s a ing (W0) and 7 weeks a e (W7) me o min ea men (35 mg/kg
×
day), g ey lines
show he QT in e al. (
b
) Mean QT in e al du a ion h oughou me o min o ehicle ea men .
Hea a e (
c
) and QTc in e al (
d
), whe e QT du a ion is adjus ed o hea a e using F ide icia’s
o mula. Da a ep esen indi idual da a poin s wi h mean
±
SEM ( ed ma ks). * p< 0.05 and
** p< 0.01
compa ed o con ol a he same week. (Con ol W0 n = 22; Con ol W7 n = 15; Me o min
n = 15).
In i o
a hy hmia suscep ibili y assays unde ca diac challenge using he ca eine
and dobu amine es (Figu e 2c,d) we e also ca ied ou . Anaes he ized animals did no
de elop a hy hmia a basal condi ions be o e he challenge. Dobu amine plus ca eine
injec ion di e en ially induced a hy hmia in con ol and me o min- ea ed g oups. Again,
con ol animals su e ed mino e en s, mos ly pe sis en bigeminy o sal o (PB/S), whe eas
me o min- ea ed a s esponded mo e se e ely o he challenge and de eloped en icula
achyca dia and o sades de poin es.
An adap ed sco ing sys em was used o calcula e a e age a hy hmia sco e. The
mean a hy hmia sco e was signi ican ly highe in he me o min g oup (
3.5 ±1.05 me
,
1.33 ±0.44 con
,
p< 0.05
). Taken oge he , hese esul s suppo he hypo hesis ha ch onic
me o min ea men induces ca diac emodeling esul ing in p olonged epola iza ion and
enhanced suscep ibili y o a hy hmias a e ca diac challenge.
In . J. Mol. Sci. 2022,23, 6021 4 o 14
Figu e 2
Con ol Me o min
a b
TdP
(32%)
NE
(66%)
NE
(33%)
VPB
(22%)
PB/S
(11%)
PB/S
(33%)
VPB
(33%)
c d
PVT
(10%)
+ Ø + Ca /Dob
Con ol
Me o min
No e en Spon e en
Con ol
Me o min
Con ol Me o min
0
1
2
3
4
5
6
Me o min
W7
Con ol
W7
A hy hmia Sco e
*
0
1
2
3
4
5
6
Me o min
W7
Con ol
W7
A hy hmia Sco e
*
NE
(35%)
VPB
(50%)
PB/S
(5%)
NE
(16%)
VPB
(16%)
PB/S
(16%)
PVT
(16%)
Figu e 2.
Me o min ea men inc eases he isk o se e e a hy hmia unde condi ions o ca diac
challenge. (
a
) ECGs o conscious animals du ing weekly moni o ing. (Le ), ep esen a i e eco dings
om a con ol and a me o min- ea ed animal (35 mg/kg
×
day) ha did no de elop a hy hmic
e en s. (Righ ), ECGs o he animals ha showed he se e es spon aneous e en : pe sis en bigeminy
in he con ol and non-sus ained polymo phic en icula achyca dia in he me o min- ea ed
animal. (
b
) Pie cha s and sca e plo s showing he p opo ion and se e i y o each spon aneous
e en . F om mildes o mos se e e: NE, no e en s; VPB, en icula p ema u e bea ; PB/S, pe sis en
bigeminy/sal os; PVT, polymo phic en icula achyca dia; TdP, o sades de poin es. n = 9 con ol
and 19 me o min- ea ed animals. (
c
) Rep esen a i e ECGs o a con ol and a me o min- ea ed
animal be o e (le ) and a e ( igh ) he
in i o
a hy hmia suscep ibili y assay pe o med a he
end o he expe imen al pe iod. Ca eine/dobu amine challenge igge s pe sis en bigeminy in he
con ol, bu o sades de poin es in he me o min- ea ed animal. (
d
) Pie cha s and sca e plo s
showing he p opo ion and se e i y he p opo ion o each e en a e ca diac challenge. Da a
ep esen indi idual da a poin s wi h mean
±
SEM ( ed ma ks). * p< 0.05 compa ed o con ol.
n = 9 con ol and 6 me o min- ea ed animals.
2.3. Me o min T ea men Reduces T ansien Ou wa d Po assium bu No L-Type Calcium
Cu en (ICa-L)
P olonged epola iza ion can esul om a educ ion in epola izing cu en s o an
inc ease in depola izing cu en s. The ansien ou wa d po assium cu en (I
o
) is he main
epola izing cu en in he a hea and he esponsible o he phase 1 o he ac ion po en-
ial in humans. As shown in Figu e 3a–c, in isola ed ca diomyocy es ch onic me o min
ea men caused a 26.7% educ ion in he peak I
o
densi y wi hou a ec ing he biophysical
p ope ies o he cu en (Supplemen a y Figu e S2). Rega ding he depola izing I
Ca-L
,
(Figu e 3d– ), we ound simila cu en densi y in bo h g oups a all ol ages es ed. These
esul s indica e ha I
o
bu no I
Ca-L
con ibu es o he me o min-induced leng hening in
QTc du a ion.
In . J. Mol. Sci. 2022,23, 6021 5 o 14
0
5
10
15
20
25
30
Me o min
W7
Con ol
W7
I
o
Densi y a +50 mV (pA/pF)
**
-15
-10
-5
0
Me o min
W7
Con ol
W7
I
Ca-L
Densi y a +10 mV (pA/pF)
-40 -20 0 20 40 60
5
10
15
20
25
I
o
Densi y (pA/pF)
Memb ane Po en ial (mV)
Con ol
Me o min
**
-40 -20 20 40 60
-10
-8
-6
-4
-2
I
Ca-L
Densi y (pA/pF)
Memb ane Po en ial (pA/pF)
Con ol
Me o min
abc
de
Figu e 3
100 ms
10 pA/pF
100 ms
2,5 pA/pF
Con ol
Me o min
Con ol
Me o min
Figu e 3.
Ch onic me o min ea men (35 mg/kg
×
day) educes I
o
and does no a ec I
Ca-L
.
(
a
) Rep esen a i e aces o I
o
eco ded om ca diomyocy es isola ed om a con ol and a me o min-
ea ed a . (
b
) A e age cu en densi y– ol age ela ionships o I
o
and (
c
) peak alues a +50 mV.
(
n = 17
con ol and 12 me o min) (
d
) I
Ca-L
aces eco ded om ca diomyocy es isola ed om a
con ol and a me o min- ea ed a , (
e
) cu en densi y– ol age ela ion and (
) peak cu en densi y
alues a +10 mV (n = 7 con ol and 8 me o min). Diag ams o I
o
and I
Ca-L
eco ding p o ocols
depic ed in he inse s (
a
,
d
). Poin s ep esen indi idual da a wi h mean
±
SEM ( ed ma ks), excep
o he ol age-cu en cu es, whe e mean
±
SEM is shown. ** p< 0.01 compa ed o con ol g oup.
2.4. In Vi o Me o min Rep oduces he E ec s Obse ed In Vi o
To assess whe he he e ec o me o min on he hea was di ec ,
in i o
expe imen s
in en icula myocy es isola ed om con ol a s and hen incuba ed wi h 10 mM me -
o min o 24 h we e pe o med. Fi s , we es ed whe he cells incuba ed wi h me o min
could de elop a hy hmic beha io unde challenge condi ions. Pacing in e up ion p o-
ocol was used as a igge and he numbe o spon aneous con ac ions we e analyzed.
Ca diomyocy es incuba ed wi h me o min had a signi ican ly highe numbe o spon a-
neous con ac ions a e in e up ion o he s imula ion ains a 1 and 2 Hz (Figu e 4a–c).
Mo eo e , 100% o me o min- ea ed cells de eloped spon aneous con ac ions a he
wo equencies es ed, whe eas in he con ol g oup only 50% and 40% o cells p esen ed
spon aneous con ac ions a 1 Hz and 2 Hz, espec i ely. These esul s in isola ed cells a e
consis en wi h he highe suscep ibili y o de elop a hy hmias obse ed in he whole
animal. We hen explo ed he mechanism o a hy hmia by measu ing calcium ansien s
in ca diomyocy es isola ed om con ol hea s and incuba ed o 24 h wi h o wi hou
me o min. The ampli udes o calcium ansien s eco ded a 0.5 and 1 Hz we e simila
in he wo g oups (Supplemen a y Figu e S3), indica ing ha me o min does no cause
in acellula calcium o e load.
In . J. Mol. Sci. 2022,23, 6021 6 o 14
90
100
110
Cell Leng h (mm)
0
10
20
30
Me o min
24h
Spon aneous Con ac ions (2Hz)
Con ol 24h
**
0
10
20
30
Me o min
24h
Spon aneous Con ac ions (1Hz)
Con ol 24h
**
bc
Con ol 24h
Me o min 24h
a
Figu e 4
0
10
20
30
40 Con ol
Me o min
*
800
800
APD
30
(ms)
300 300
Basic Cycle Leng h (ms)
*
0
50
100
150
200 Con ol
Me o min
*
800
800
APD
90
(ms)
300 300
Basic Cycle Leng h (ms)
*
de
25 s
90
100
110
Cell Leng h (mm)
25 s
100 ms
50 mV
Con ol 24h
Me o min 24h
Figu e 4.
Me o min inc eases spon aneous con ac ions and p olongs AP du a ion
in i o
. Ca -
diomyocy es isola ed om con ol animals we e incuba ed wi h 10 mM me o min o ehicle o
24 h. (
a
) Rep esen a i e eco dings o spon aneous con ac ions de eloped a e in e up ion o a
s imula ion ain a 2 Hz. Mean numbe o spon aneous con ac ions du ing he i s 100 s a e
pacing in e up ion a s imula ion ains o 2 Hz (n = 10 con ol and 10 me o min) (
b
) and 1 Hz
(
n = 15
con ol and 8 me o min) (
c
). Rep esen a i e aces o AP (
d
) and AP du a ion a 30% (APD
30
)
and 90% o epola iza ion (APD
90
) (
e
,
) o epola iza ion a s imula ion equencies o 300 and 800 ms
(
n = 11
con ol and 11 me o min). Poin s ep esen indi idual da a wi h mean
±
SEM ( ed ma ks),
** p< 0.01 and * p< 0.05 compa ed o con ol a he same equency.
Nex , he e ec o me o min on epola iza ion
in i o
was analyzed. Ven icula s ips
we e incuba ed wi h o wi hou 10 mM me o min and AP we e eco ded (
Figu e 4d–
).
Me o min signi ican ly p olonged APD
30
and APD
90
, consis en wi h he p olonga ion
o QTc in e al du a ion obse ed in he animals. In cells incuba ed wi h me o min
o 24 h I
o
peak densi y was lowe (37%) in he me o min g oup compa ed o con ol
(Figu e 5a–c). The possibili y ha me o min could ac as an I
o
channel blocke was
uled ou , since acu e exposu e o he d ug (by 30-min pe usion) did no al e cu en
ampli ude (Supplemen a y Figu e S4). The e o e, his inhibi o y e ec is p obably due o
ansc ip ional and/o ansla ional causes. Rega ding he I
Ca-L
, no e ec was obse ed
a e exposu e o me o min o 24 h (Figu e 5d– ). Taken oge he , hese
in i o
esul s
o e AP and ionic cu en s a e in ag eemen wi h he esul s obse ed ex i o, and con i m
he e idence ha me o min di ec ly egula es ca diac epola iza ion.
On he o he hand, he apid delayed ec i ie po assium cu en , I
K
, is he main
epola izing cu en ha d i es he human ca diac cells o he es ing po en ial. Since I
K
,
which is ca ied h ough hERG channels in human ca diac myocy es, is no signi ican ly
exp essed in he a hea , we used HEK293 cells ha cons i u i ely exp ess he hERG
channel (HEK-hERG cells) o s udy he e ec o me o min on his cu en . As shown in
Figu e 6a–c, in HEK-hERG cells incuba ed wi h me o min o 24 h he I
hERG
densi y was
55.7% lowe compa ed wi h con ol.
In . J. Mol. Sci. 2022,23, 6021 7 o 14
0
10
20
30
Me o min
24h
Con ol 24h
I
o
Densi y a +50 mV (pA/pF)
**
-15
-10
-5
0
Me o min
24h
Con ol 24h
I
Ca-L
Densi y a +10 mV (pA/pF)
-40 -20 0 20 40 60
5
10
15
20
25
I
o
Densi y (pA/pF)
Memb ane Po en ial (mV)
Con ol 24h
Me o min 24h
**
-40 -20 20 40 60
-10
-8
-6
-4
-2
I
Ca-L
Densi y (pA/pF)
Memb ane Po en ial (pA/pF)
Con ol 24h
Me o min 24h
b ca
d e
Figu e 5
100 ms
10 pA/pF
100 ms
2,5 pA/pF
Con ol 24h
Me o min 24h
Con ol 24h
Me o min 24h
Figu e 5.
Incuba ion wi h me o min educes I
o in i o
. Ca diomyocy es isola ed om con ol
animals we e incuba ed wi h 10 mM me o min o ehicle o 24 h. (
a
) Rep esen a i e I
o
aces,
(
b
) cu en densi y– ol age cu e and (
c
) peak densi y a +50 mV (n = 8 con ol and 14 me o min).
(
d
) Rep esen a i e I
Ca-L
eco dings, (
e
) cu en – ol age cu e and (
) I
Ca-L
-densi y a +10 mV (
n = 11
con ol and 4 me o min). Same p o ocols as in Figu e 3. Poin s ep esen indi idual da a wi h
mean ±SEM
( ed ma ks), excep o cu en – ol age cu es, whe e mean
±
SEM is shown.
** p< 0.01
compa ed o con ol.
Figu e 6
-40 -20 0 20 40
10
20
30
40
50
I
hERG
Densi y (pA/pF)
Memb ane Po en ial (mV)
Con ol 24 h
Me o min 24 h
Con ol 24 h
Me o min 24 h
0
25
50
75
Me o min
24h
Con ol 24h
I
hERG
Densi y a +10 mV (pA/pF)
**
b ca 50 mV
-40 mV
-80 mV
Figu e 6.
Me o min educes I
hERG
. (
a
) Rep esen a i e I
hERG
aces eco ded in HEK-hERG cells
incuba ed wi h ehicle o 10 mM me o min o 24 h. (
b
) Mean cu en densi y– ol age ela ion and
(
c
) peak-densi y alues a +10 mV (n = 8 con ol and 9 me o min). Poin s ep esen indi idual da a
wi h mean
±
SEM excep , o he cu en - ol age cu e, whe e mean
±
SEM ( ed ma ks) is shown.
** p< 0.01 compa ed o con ol.
In . J. Mol. Sci. 2022,23, 6021 8 o 14
2.5. Me o min P olongs Repola iza ion in hIPSC-CMs
In a las se o expe imen s, hIPSC-CMs we e used. Cells we e incuba ed wi h 100
and 300
µ
M me o min o 24 h and eco ded AP using ol age-sensi i e dyes (Figu e 7).
Compa ed o he con ol g oup, me o min slowed down he epola iza ion o he ac-
ion po en ial a phases 1 and 3. Thus, cells incuba ed wi h 300
µ
M me o min showed
p olonged APD
30
, as expec ed om I
o
inhibi ion. Besides, APD
90
was longe in he
wo me o min- ea ed g oups, co obo a ing he inhibi ion o I
K
obse ed in p e ious
expe imen s. Fu he mo e, we obse ed ha e y high concen a ions o me o min
leng hened he AP du a ion o he poin o gene a ing ea ly a e depola iza ions (EADs)
(Supplemen a y Figu e S5).
300
400
500
600
700
**
300
APD
90
(ms)
0100
[Me o min] (mM)
**
150
200
250
300
350
300
APD
30
(ms)
0100
[Me o min] (mM)
**
200 ms
Con ol
300 mM Me
100 mM Me
b ca
Figu e 7
Figu e 7.
Me o min p olongs ac ion po en ial du a ion in hIPS-CMs. (
a
) Rep esen a i e AP aces
ob ained om hiPSC-CMs incuba ed wi h ehicle, o 100 and 300
µ
M me o min o 24 h. (
b
,
c
) Dose-
dependen e ec o me o min on APD a 30 and 90% o epola iza ion (n = 5). Poin s ep esen
indi idual da a wi h mean ±SEM ( ed ma ks). ** p< 0.01 compa ed o con ol.
3. Discussion
He e we ha e s udied he e ec s o me o min exposu e on ca diac elec ical p ope ies.
The majo indings a e ha me o min di ec ly modi ies he elec ical beha io o he no mal
hea making i mo e p one o de elop a hy hmia unde ca diac challenge. The mechanism
in ol es inhibi ion o epola izing cu en s, which dec eases epola izing capaci y and
p olongs AP and QTc in e al du a ion.
Compa ed o he con ol g oup we did no obse e hypoglycemia o changes in body
weigh h oughou he ea men , sugges ing ha ca diac al e a ions we e no caused by
me abolic de ec s seconda y o me o min- ea men . Nex ,
in i o
eco dings o AP and
ion cu en s in ca diomyocy es isola ed om con ol a s and incuba ed wi h me o min a e
consis en wi h he esul s obse ed ex i o, and con i m ha me o min di ec ly egula es
ca diac epola iza ion. These esul s a e in ag eemen wi h o he s ha epo ed di ec
e ec s o me o min in neona al a ca diomyocy es and HL-1 cells [18].
Me o min-induced educ ion o I
o
o I
hERG
mus in ol e in acellula signaling
pa hways, since me o min does no ac as a di ec channel blocke . Rega ding he I
Ca-L
,
Wang e al. epo ed ha me o min inhibi ed his cu en and hus sho ened APD and
QT in e al in ype 1 diabe ic mice [
19
]. Howe e , we obse ed no e ec on I
Ca-L
in
ca diomyocy es om con ol animals. Two possible explana ions o he disc epancy may
be ha hei ype 1 diabe ic animals ha e an inc eased I
Ca-L
and ha hey use doses o
me o min om wo o en imes highe han ou s.
Despi e hiPSC-CMs ha e an imma u e pheno ype cha ac e ized by a mo e depola ized
dias olic po en ial and he absence o no ch, due o small I
K1
and I
o
densi ies [
20
,
21
],
hese cells ec ea e ac ion po en ials simila o hose epo ed o human ca diomyocy es.
The e o e, hey ha e become a ele an model o d ug sa e y assays. Me o min leng hened
APD
30
and APD
90
in hese ca diomyocy es, which is compa ible wi h he educ ion in he
epola izing cu en s I o and IK ha we obse ed.
In . J. Mol. Sci. 2022,23, 6021 9 o 14
Me o min is known o ac i a e AMP-ac i a ed p o ein kinase o AMPK [
22
]. He e
ou esul s show ha me o min signi ican ly educes I
o
and I
hERG
densi y, in ag eemen
wi h s udies using he classical AMPK ac i a o AICAR ha also epo ed a dec ease in
hese cu en s in mice, xenopus oocy es and habdomyosa coma cells [
23
,
24
]. Despi e
his e idence, gi en he high concen a ions used in ou
in i o
expe imen s, AMPK-
independen mechanisms canno be exclude [15,25].
Elec oca diog ams show ha me o min inc eases he p obabili y o de elop a -
hy hmia in ea ed animals
in i o
. Simila ly, 100% o he cells incuba ed
in i o
wi h
me o min had spon aneous con ac ili y a e pacing in e up ion. A classical igge o a -
hy hmias is he depola izing cu en ca ied by he sodium/calcium exchange , seconda y
o cellula Ca
2+
o e load ha can depola ize he memb ane om he es ing po en ial
and gene a e delayed a e depola iza ions [
26
]. Howe e , we ound no di e ences in
cy oplasmic calcium con en o he ampli ude o Ca
2+
ansien s in cells incuba ed wi h
me o min compa ed o con ols. The e o e, calcium o e load does no seem o be he
mechanism esponsible o me o min-induced a hy hmia.
P olonga ion o AP epola iza ion allows calcium and sodium channels o eco e om
inac i a ion. The eac i a ed channels can hen eopen, u he depola ize he memb ane,
and gene a e ea ly a e depola iza ions. Al hough o he mechanisms canno be uled ou ,
ou esul s suppo he hypo hesis ha he de eloping o EADs is he igge o me o min
dependen a hy hmias.
I is impo an o no e ha me o min use is ex ending beyond diabe es and diabe es
p e en ion [
27
]. Because o he po en ial e icacy o p omo e weigh loss, me o min is
used o educe obesi y in non-diabe ic pa ien s [
28
] and in he ea men o an ipsycho ic-
induced weigh gain [
29
]. I has also been used o ea he ep oduc i e and me abolic
abno mali ies associa ed wi h polycys ic o a y synd ome [
30
]. A limi a ion o he s udy is
ha he concen a ions used in he
in i o
expe imen s a e high and he e o e he obse ed
esul s may be maximal e ec s. Howe e , his could be ele an o acili a e he de el-
opmen o ec opic ac i i y in especially suscep ible indi iduals o unde a hy hmogenic
ci cums ances. Besides ion channel unc ion, o he me abolic and s uc u al ac o s can
con ibu e o diabe ic myoca dial dys unc ion and inc ease he isk o a hy hmia [5].
Expe imen s in animal models wi h ype 1 and ype 2 diabe es ha e shown ha se e al
ca diac ion cu en s, mainly I
o
and I
Ks
, a e signi ican ly educed. Ou esul s show ha
me o min (no diabe es) also educes I
o
and I
hERG
. Thus, al hough me o min co ec s
hype glycemia and migh imp o e o he me abolic aspec s, i canno e e he diabe es-
associa ed epola iza ion de ec s. In his sense, we ha e ecen ly published ha al hough
me o min ea men imp o es he me abolic and in lamma o y p o ile, i does no educe
he incidence and se e i y o ca diac a hy hmias in diabe ic animals [
10
]. Ou esul s help
o unde s and why me o min- he apy does no p o ec agains en icula a hy hmia in
diabe ic pa ien s [12–14].
4. Ma e ials and Me hods
4.1. E hics S a emen and In Vi o T ea men
The in es iga ion ul ills he Spanish (RD 53/2013) and Eu opean (D2003/65/CE and
R2007/526/CE) ules egula ions o he Ca e o Animals used o expe imen al and o he
esea ch pu poses, and has been app o ed by he E hics Commi ee o Animal Ca e o
he Uni e si y o he Basque Coun y unde e e ence CEBA/273M/2012. Female young
Sp ague-Dawley a s (200–220 g) om he Uni e sidad del País Vasco UPV/EHU animal
acili y we e housed in a empe a u e-con olled oom (24
◦
C) unde a 12:12 h ligh :da k
cycle and- wi h ee access o wa e and s anda d a chow. Me o min- ea ed animals
ecei ed me o min hyd ochlo ide 35 mg/kg daily (Sigma-Ald ich Co., Sain Louis, MO,
USA) in d inking wa e o 7 weeks. Animals we e pe iodically weigh ed and kep in
sepa a ed cages. Fo each animal, me o min was dissol ed in 30 mL o wa e , he a e age
daily d inking olume (n = 35). I needed, addi ional egula d inking wa e was also
p o ided. Con ol animals (n = 20) ecei ed egula ap wa e .
