In e na ional Jou nal o
Molecula Sciences
A icle
Molecula and Elec ophysiological Role o
Diabe es-Associa ed Ci cula ing In lamma o y Fac o s in
Ca diac A hy hmia Remodeling in a Me abolic-Induced Model
o Type 2 Diabe ic Ra
Julian Zayas-A abal 1, Amaia Alquiza 1, E kan Tuncay 2, Belma Tu an 3, Monica Gallego 1and Osca Casis 1,*
Ci a ion: Zayas-A abal, J.; Alquiza,
A.; Tuncay, E.; Tu an, B.; Gallego, M.;
Casis, O. Molecula and
Elec ophysiological Role o
Diabe es-Associa ed Ci cula ing
In lamma o y Fac o s in Ca diac
A hy hmia Remodeling in a
Me abolic-Induced Model o Type 2
Diabe ic Ra . In . J. Mol. Sci. 2021,22,
6827. h ps://doi.o g/10.3390/
ijms22136827
Academic Edi o : Tzong-Shyuan Lee
and Hec o Ba ajas-Ma inez
Recei ed: 18 May 2021
Accep ed: 22 June 2021
Published: 25 June 2021
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A ibu ion (CC BY) license (h ps://
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1Depa amen o Physiology, 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] (J.Z.-A.); [email p o ec ed] (A.A.);
[email p o ec ed] (M.G.)
2Depa men o Biophysics, Facul y o Medicine, Anka a Uni e si y, 06100 Anka a, Tu key;
E kan.T[email p o ec ed]
3Depa men o Biophysics, Facul y o Medicine, Lokman Hekim Uni e si y, 06510 Anka a, Tu key;
Belma.T[email p o ec ed]
*Co espondence: osca [email p o ec ed]; Tel.: +34-945013033
Abs ac :
Backg ound: Diabe ic pa ien s ha e p olonged ca diac epola iza ion and highe isk
o a hy hmia. Besides, diabe es ac i a es he inna e immune sys em, esul ing in highe le els
o plasma ic cy okines, which a e desc ibed o p olong en icula epola iza ion. Me hods: We
cha ac e ize a me abolic model o ype 2 diabe es (T2D) wi h p olonged ca diac epola iza ion.
Sp ague-Dawley a s we e ed on a high- a die (45% Kcal om a ) o 6 weeks, and a low dose
o s ep ozo ozin in ape i oneally injec ed a week 2. Body weigh and as ing blood glucose we e
measu ed and elec oca diog ams o conscious animals we e eco ded weekly. Plasma ic lipid p o ile,
insulin, cy okines, and a hy hmia suscep ibili y we e de e mined a he end o he expe imen al
pe iod. Ou wa d K
+
cu en s and ac ion po en ials we e eco ded in isola ed en icula myocy es by
pa ch-clamp. Resul s: T2D animals showed insulin esis ance, hype glycemia, and ele a ed le els o
plasma choles e ol, iglyce ides, TNF
α
, and IL-1b. They also de eloped b adyca dia and p olonged
QTc-in e al du a ion ha esul ed in inc eased suscep ibili y o se e e en icula achyca dia
unde ca diac challenge. Ac ion po en ial du a ion (APD) was p olonged in con ol ca diomyocy es
incuba ed 24 h wi h plasma isola ed om diabe ic a s. Howe e , adding TNF
α
and IL-1b ecep o
blocke s o he se um o diabe ic animals p e en ed he inc eased APD. Conclusions: The ele a ion
o he ci cula ing le els o TNF
α
and IL-1b a e esponsible o impai ed en icula epola iza ion
and highe suscep ibili y o ca diac a hy hmia in ou me abolic model o T2D.
Keywo ds: cy okines; a hy hmia; o sade de poin es; po assium cu en ; insulin esis ance
1. In oduc ion
Diabe es a ec s he elec ical unc ion o he hea , making i mo e p one o de elop
a ial ib illa ion, abno mali ies in ac ion po en ial conduc ion o epola iza ion, and a -
hy hmic sudden dea h [
1
,
2
]. The main ea u e o he ca diac elec ical emodeling is a
p olonged en icula epola iza ion, measu ed as a p olonga ion o he QT in e al in he
elec oca diog am. This inc eases he isk o o sade de poin es, a ype o a hy hmia ha
can degene a e in o en icula ib illa ion and sudden dea h [
3
]. Leng hening o he QT
in e al du a ion associa es wi h sudden dea h in ype 1 and ype 2 diabe ic pa ien s [
4
–
6
].
A he cellula le el, p olonged epola iza ion is he consequence o educed exp ession and
ac i i y o ca diac ionic channels, mainly he epola izing K 4.3 po assium channels [
7
,
8
].
K 4.3 channels a e esponsible o he ansien ou wa d po assium cu en , I
o
, ac i e
du ing phase 1 o he ca diac ac ion po en ial [9].
In . J. Mol. Sci. 2021,22, 6827. h ps://doi.o g/10.3390/ijms22136827 h ps://www.mdpi.com/jou nal/ijms
In . J. Mol. Sci. 2021,22, 6827 2 o 14
Al hough he ela ion be ween in lamma ion and hea ailu e is known [
10
,
11
], he
media o s ha link he in lamma o y sys em wi h ype 2 diabe es-induced elec ical ab-
no mali ies a e unknown. In s e ile condi ions, low-g ade in lamma ion can be s a ed
by he inna e immune mechanisms and be p olonged by bo h inna e and adap i e im-
muni y. In his sense, ac i a ion o he inna e immune sys em has been p oposed as he
main pa hophysiological mechanism o de eloping ca diac elec ical emodeling in ype 1
diabe es [12]. Howe e , mo e han 90% o diabe ic pa ien s ha e ype 2 diabe es.
On he o he hand, in heal hy a en icula ca diomyocy es TLR ac i a ion inhibi ed
I o, leading o ac ion po en ial p olonga ion and igge ed ac i i y [
13
]. Simila ly, cy okines
such as TNF
α
and IL-1b also inhibi ed he ansien ou wa d K
+
cu en and p olonged
en icula epola iza ion [
12
,
14
]. Type 1 and ype 2 diabe ic pa ien s had inc eased TLR
ac i a ion and p oin lamma o y cy okine p oduc ion [
15
–
17
] and, when ype 1 diabe es was
induced o Tl 2
−/−
and IL-1
−/−
mice, animals we e mo e esis an o de elop a hy hmias
a e ca diac challenge han wild- ype diabe ic mice [12].
Mos o he knowledge on diabe ic ca diac elec ical emodeling, om ECGs o be-
ha io o ionic cu en s, de i es om ype 1 diabe ic animals [
7
,
18
–
20
]. Rega ding ype
2 diabe es, me abolic-induced non-gene ic animal models eplica e he biochemical cha -
ac e is ics o ype 2 diabe es in humans: insulin esis ance, mode a e hype glycemia, and
hype lipidemia. These models combine high calo ic ood ha p og essi ely leads o glu-
cose in ole ance and insulin esis ance, wi h low-dose STZ o p o ide he loss o unc ional
be a-cell mass equi ed o es ablish diabe es [21–24].
In his wo k, we aimed o c ea e and cha ac e ize a me abolic-induced ype 2 diabe ic
model ha de eloped ca diac elec ical emodeling and o iden i y he key p oin lamma o y
ac o s in ol ed in he suscep ibili y o a hy hmia.
2. Resul s
2.1. Me abolic Cha ac e iza ion o he Type 2 Diabe ic Model
The induc ion o ype 2 diabe es begins by eeding o a s wi h a high- a die ha
p og essi ely led o insulin esis ance. Two weeks la e , we injec ed a low-dose STZ
(Figu e 1A) in o de o p o ide some be a cell loss.
Since he se e i y o diabe es and associa ed complica ions depend on he composi ion
o he die and he dose o STZ [
23
], we i s con i med ha ou weeks we e su icien
o he animals o de elop he me abolic cha ac e is ic o ype 2 diabe es. We moni o ed
he as ing plasma glucose (FPG) le els weekly. As can be obse ed in Figu e 1B, a e
STZ injec ion FPG le els apidly ose om 95 mg/dL o app oxima ely 170 mg/dL in
he diabe ic g oup (p< 0.01). Th oughou he expe imen al pe iod, plasma glucose le els
emained ele a ed and signi ican ly highe han hose o he con ol, chow- ed g oup.
In Chow + STZ and HFD g oups, FPG le els we e signi ican ly lowe han hose o he
diabe ic g oup. They we e also highe han hose o he con ol g oup, bu emained close
o he expec ed physiological limi s (Supplemen a y Table S1).
A e ou weeks o diabe es, insulin le els we e no signi ican ly di e en be ween he
expe imen al g oups (Figu e 1C; Supplemen a y Table S1). To con i m ha he high FPG
obse ed in diabe ic animals was due o insulin esis ance, we pe o med an in ape i oneal
glucose/insulin ole ance es a he end o he expe imen al pe iod. A e he injec ion
o he bolus o glucose and he insulin, plasma glucose le els we e signi ican ly highe in
diabe ics han in con ol a s a all he ime poin s es ed (Figu e 1D,E). In con ol animals,
insulin no malized blood glucose in 30 min. Howe e , diabe ic animals could no es o e
glycemia e en a e 60 min, indica ing he p esence o signi ican insulin esis ance. Thus,
ou model displayed he high plasma glucose and insulin esis ance cha ac e is ic o ype
2 diabe es.
In . J. Mol. Sci. 2021,22, 6827 3 o 14
In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 3 o 14
Figu e 1. Type 2 diabe ic a s show ele a ed plasma glucose and insulin esis ance. (A) Desc ip ion
o he expe imen al p o ocol o induc ion o ype 2 diabe es. (B) Weekly as ing plasma glucose
h oughou he expe imen al pe iod. The do ed line indica es he injec ion o STZ o ehicle. (C)
Plasma insulin le els a e 4 weeks o diabe es. (D) IPIGTT pe o med a he end o he expe imen al
pe iod (week 6). (E) The co esponding a ea unde he cu e (AUC). In pa en hesis (numbe o an-
imals); ** p < 0.01.
On he o he hand, wo weeks on a high- a die signi ican ly inc eased he body
weigh o he diabe ic animals, bu he STZ injec ion unca ed he endency (Figu e 2A,
see weeks 2 o 3). Thus, a he end o he expe imen al pe iod, body weigh was simila in
con ol and diabe ic animals. Consis en wi h ha , HFD alone inc eased he o al body
weigh whe eas STZ injec ion in chow- ed animals educed he o al weigh (Supplemen-
a y Table S1).
Diabe ic animals ha e simila weigh han con ols, bu ha e mo e pe iepididimal,
abdominal a (Figu e 2B). Mo eo e , hey show signi ican highe le els o plasma i-
glyce ides and choles e ol han hose o con ol animals (Figu e 2C,D), indica ing a diabe-
es-induced wo sening in plasma lipid p o ile.
Figu e 1.
Type 2 diabe ic a s show ele a ed plasma glucose and insulin esis ance. (
A
) Desc ip ion o he expe imen al
p o ocol o induc ion o ype 2 diabe es. (
B
) Weekly as ing plasma glucose h oughou he expe imen al pe iod. The do ed
line indica es he injec ion o STZ o ehicle. (
C
) Plasma insulin le els a e 4 weeks o diabe es. (
D
) IPIGTT pe o med a
he end o he expe imen al pe iod (week 6). (
E
) The co esponding a ea unde he cu e (AUC). In pa en hesis (numbe o
animals); ** p< 0.01.
On he o he hand, wo weeks on a high- a die signi ican ly inc eased he body
weigh o he diabe ic animals, bu he STZ injec ion unca ed he endency (
Figu e 2A
,
see weeks 2 o 3). Thus, a he end o he expe imen al pe iod, body weigh was simila
in con ol and diabe ic animals. Consis en wi h ha , HFD alone inc eased he o al body
weigh whe eas STZ injec ion in chow- ed animals educed he o al weigh (Supplemen a y
Table S1).
Diabe ic animals ha e simila weigh han con ols, bu ha e mo e pe iepididimal,
abdominal a (Figu e 2B). Mo eo e , hey show signi ican highe le els o plasma iglyc-
e ides and choles e ol han hose o con ol animals (Figu e 2C,D), indica ing a diabe es-
induced wo sening in plasma lipid p o ile.
In . J. Mol. Sci. 2021,22, 6827 4 o 14
In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 o 14
Figu e 2. Me abolic dis u bances o he ype 2 diabe ic model. (A) Two weeks o HFD inc eased he
body weigh , bu his e ec was e e sed by he STZ injec ion (do ed line). (B) Al hough o al body
weigh is no di e en be ween g oups a week 6, abdominal a is signi ican ly g ea e in diabe ic
animals. Plasma lipid p o ile wo sened in diabe ic animals as can be seen in he inc eased ci cula ing
le els o (C) choles e ol and (D) iglyce ides. In pa en hesis (numbe o animals); * p > 0.05; ** p < 0.01.
2.2. Ca diac Elec ical Remodeling in he Type 2 Diabe ic Model
As expec ed o a sho - e m diabe es, no ca diac ib osis was obse ed (supple-
men al Figu e 1). Th oughou he expe imen al pe iod, we eco ded ECGs (Figu e 3A)
and analyzed he ca diac elec ical cha ac e is ics o he diabe ic animals. The ECGs o he
diabe ic animals and age-ma ched con ols showed clea di e ences. F om he i s week
a e STZ injec ion, RR-in e al du a ion in diabe ic a s was signi ican ly longe han ha
o con ols. Thus, as expec ed, diabe ic animals had a lowe hea a e compa ed o ha o
con ols (Figu e 3B). The PR-in e al du a ion showed ha ca diac impulse conduc ion a
he a io en icula node was no a ec ed by diabe es (Figu e 3C; Supplemen a y Table
S2). Diabe es did no modi y ei he a ial o en icula depola iza ion, measu ed as P
wa e and QRS complex du a ion espec i ely (Figu e 3D; Supplemen a y Table S2). How-
e e , du a ions o QT-in e al, QTc (QT in e al co ec ed o he hea a e) and Tpeak-Tend
we e longe in diabe ic animals han in con ols (Figu e 3E–G), con i ming ha ca diac
epola iza ion is se e ely a ec ed by diabe es. Las , al hough he RR-du a ion in he
Chow + STZ g oup was longe han ha in he con ol g oup, no signi ican e ec on QTc
du a ion was obse ed (Supplemen a y Table S2).
P olonged QTc-in e al and Tpeak-Tend du a ions a e a hy hmia su oga es, so we
nex es ed he a hy hmia suscep ibili y o con ol and diabe ic animals unde ca -
eine/dobu amine challenge (Figu e 4A). Ven icula achya hy hmias we e mo e e-
quen in ype 2 diabe es han in con ol animals (55% s 23%). Mo eo e , a hy hmia in
he diabe ic g oup we e mo e se e e han in he con ol g oup, since 33% o diabe ic ani-
mals and 0% o con ols de eloped he li e- h ea ening a hy hmia, o sade de poin es
(Figu e 4B).
Figu e 2.
Me abolic dis u bances o he ype 2 diabe ic model. (
A
) Two weeks o HFD inc eased he body weigh , bu his
e ec was e e sed by he STZ injec ion (do ed line). (
B
) Al hough o al body weigh is no di e en be ween g oups a
week 6, abdominal a is signi ican ly g ea e in diabe ic animals. Plasma lipid p o ile wo sened in diabe ic animals as
can be seen in he inc eased ci cula ing le els o (
C
) choles e ol and (
D
) iglyce ides. In pa en hesis (numbe o animals);
*p> 0.05; ** p< 0.01.
2.2. Ca diac Elec ical Remodeling in he Type 2 Diabe ic Model
As expec ed o a sho - e m diabe es, no ca diac ib osis was obse ed (supplemen al
Figu e S1). Th oughou he expe imen al pe iod, we eco ded ECGs (Figu e 3A) and
analyzed he ca diac elec ical cha ac e is ics o he diabe ic animals. The ECGs o he
diabe ic animals and age-ma ched con ols showed clea di e ences. F om he i s week
a e STZ injec ion, RR-in e al du a ion in diabe ic a s was signi ican ly longe han ha
o con ols. Thus, as expec ed, diabe ic animals had a lowe hea a e compa ed o ha o
con ols (Figu e 3B). The PR-in e al du a ion showed ha ca diac impulse conduc ion a
he a io en icula node was no a ec ed by diabe es (Figu e 3C; Supplemen a y Table S2).
Diabe es did no modi y ei he a ial o en icula depola iza ion, measu ed as P wa e
and QRS complex du a ion espec i ely (Figu e 3D; Supplemen a y Table S2). Howe e ,
du a ions o QT-in e al, QTc (QT in e al co ec ed o he hea a e) and T
peak-
T
end
we e longe in diabe ic animals han in con ols (Figu e 3E–G), con i ming ha ca diac
epola iza ion is se e ely a ec ed by diabe es. Las , al hough he RR-du a ion in he Chow
+ STZ g oup was longe han ha in he con ol g oup, no signi ican e ec on QTc du a ion
was obse ed (Supplemen a y Table S2).
P olonged QTc-in e al and T
peak-
T
end
du a ions a e a hy hmia su oga es, so we nex
es ed he a hy hmia suscep ibili y o con ol and diabe ic animals unde ca eine/dobu amine
challenge (Figu e 4A). Ven icula achya hy hmias we e mo e equen in ype 2 diabe es
han in con ol animals (55% s 23%). Mo eo e , a hy hmia in he diabe ic g oup we e mo e
se e e han in he con ol g oup, since 33% o diabe ic animals and 0% o con ols de eloped
he li e- h ea ening a hy hmia, o sade de poin es (Figu e 4B).
In . J. Mol. Sci. 2021,22, 6827 5 o 14
In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 5 o 14
Figu e 3. Ca diac elec ical emodeling in diabe ic hea . (A) Elec oca diog aphic eco dings in a
con ol and in a diabe ic animal be o e and a e he 6-weeks expe imen al pe iod. The do ed line
shows he end o he T wa e. (B–D) Ca diac impulse gene a ion (RR in e al) and conduc ion (PR
in e al and QRS complex) h oughou he 6 weeks. (E,F) The main epola iza ion pa ame e s (QT,
QTc, and Tpeak-Tend) a e longe in diabe ic han in con ol animals. In pa en hesis (numbe o animals);
* p < 0.05; ** p < 0.01. The do ed line in (B–G) shows he momen o STZ o ehicle injec ion.
Figu e 3.
Ca diac elec ical emodeling in diabe ic hea . (
A
) Elec oca diog aphic eco dings in a con ol and in a diabe ic
animal be o e and a e he 6-weeks expe imen al pe iod. The do ed line shows he end o he T wa e. (
B
–
D
) Ca diac
impulse gene a ion (RR in e al) and conduc ion (PR in e al and QRS complex) h oughou he 6 weeks. (
E
,
F
) The main
epola iza ion pa ame e s (QT, QTc, and T
peak-Tend
) a e longe in diabe ic han in con ol animals. In pa en hesis (numbe o
animals); * p< 0.05; ** p< 0.01. The do ed line in (B–G) shows he momen o STZ o ehicle injec ion.
In . J. Mol. Sci. 2021,22, 6827 6 o 14
In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 6 o 14
Figu e 4. Inc eased a hy hmia suscep ibili y in diabe ic hea . (A) Examples o non-a hy hmic and
a hy hmic elec oca diog aphic eco dings in con ol and diabe ic animals a e ca eine/dobu a-
mine challenge (Ca /Dob). The ECG o he con ol animal displays only en icula p ema u e bea s
and sal o, whe eas he diabe ic animal shows episodes o o sade de poin es (TdP). (B) Incidence
and se e i y o en icula achyca dias and TdP in con ol and diabe ic animals. Each a hy hmia
was sco ed as ollows: No e en s (NE) = 0; en icula achyca dia (VT) = 1; and TdP = 2. The inci-
dence and se e i y o en icula a hy hmias a e ca diac challenge a e highe in diabe ic animals
han in con ols. * p < 0.05.
2.3. Mechanisms o he Diabe es-Induced Elec ical Remodeling
In se e al animal models o diabe es, educ ion o he ansien ou wa d K+-cu en
(I o) has been p oposed as a main cause o p olonged epola iza ion [18,19]. We eco ded
I o in le en icula myocy es isola ed om con ol and diabe ic animals and con i med
ha ou me abolic-induced diabe ic model inhibi ed he cu en ampli ude. Figu e 5A
shows I o eco dings in a con ol and a diabe ic cell o simila size. The cu en aces we e
elici ed by depola izing pulses anging om −30 o + 50 mV, om a holding po en ial o
−60 mV. In diabe ic ca diomyocy es, I o was signi ican ly smalle han in con ol cells a
posi i e ol ages. A +50 mV, I o densi y was app oxima ely 50% smalle in diabe ic han
in con ol cells (Figu e 5B). The e o e, he al e a ion o he epola izing cu en is con-
sis en wi h he p olonged QTc obse ed in he ECG.
In ype 1 diabe ic animals, I o inhibi ion has been a ibu ed o he diabe ic in lamma-
o y s a us [12]. In ha ega d, TNFα and IL-1b a e ci cula ing p o-in lamma o y cy o-
kines known o educe he unc ional exp ession o I o. Nex , we compa ed he plasma ic
le els o hese cy okines in ou ype 2 diabe ic animals and age-ma ched con ols. A he
end o he expe imen al pe iod, diabe ic animals had signi ican ly highe le els o TNFα
and IL-1b han con ol animals (Figu e 5C).
Figu e 4.
Inc eased a hy hmia suscep ibili y in diabe ic hea . (
A
) Examples o non-a hy hmic and
a hy hmic elec oca diog aphic eco dings in con ol and diabe ic animals a e ca eine/dobu amine
challenge (Ca /Dob). The ECG o he con ol animal displays only en icula p ema u e bea s and
sal o, whe eas he diabe ic animal shows episodes o o sade de poin es (TdP). (
B
) Incidence and
se e i y o en icula achyca dias and TdP in con ol and diabe ic animals. Each a hy hmia was
sco ed as ollows: No e en s (NE) = 0; en icula achyca dia (VT) = 1; and TdP = 2. The incidence
and se e i y o en icula a hy hmias a e ca diac challenge a e highe in diabe ic animals han in
con ols. * p< 0.05.
2.3. Mechanisms o he Diabe es-Induced Elec ical Remodeling
In se e al animal models o diabe es, educ ion o he ansien ou wa d K
+
-cu en
(I
o
) has been p oposed as a main cause o p olonged epola iza ion [
18
,
19
]. We eco ded
I
o
in le en icula myocy es isola ed om con ol and diabe ic animals and con i med
ha ou me abolic-induced diabe ic model inhibi ed he cu en ampli ude. Figu e 5A
shows I
o
eco dings in a con ol and a diabe ic cell o simila size. The cu en aces we e
elici ed by depola izing pulses anging om
−
30 o + 50 mV, om a holding po en ial o
−
60 mV. In diabe ic ca diomyocy es, I
o
was signi ican ly smalle han in con ol cells a
posi i e ol ages. A +50 mV, I
o
densi y was app oxima ely 50% smalle in diabe ic han
in con ol cells (Figu e 5B). The e o e, he al e a ion o he epola izing cu en is consis en
wi h he p olonged QTc obse ed in he ECG.
In . J. Mol. Sci. 2021,22, 6827 7 o 14
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Figu e 5. Repola iza ion capaci y and in lamma ion. (A) Cu en aces o a cu en - ol age p o ocol
o he ansien ou wa d cu en elici ed in a con ol cell and a diabe ic en icula cell. (B) I o den-
si y/ ol age ela ionships and I o densi y a +50 mV in con ol and diabe ic myocy es. In pa en hesis
(numbe o cells/numbe o animals). (C) TNFα and IL-1b can educe he densi y o I o. Plasma le els
o TNFα and IL-1b a e highe in ype 2 diabe ic animals han in age-ma ched con ols. Da a a e
mean ± SEM; in pa en hesis (numbe o animals); * p < 0.05; ** p < 0.01.
2.4. Role o Ci cula ing Fac o s on Ca diac Elec ical Remodeling
These p e ious esul s sugges ha in his ype 2 diabe ic model, ele a ion o plas-
ma ic TNFα, and IL-1b le els could cause I o educ ion. This would lead o p olonged e-
pola iza ion and highe suscep ibili y o a hy hmia unde ca diac challenge. To con i m
whe he diabe es-associa ed ci cula ing ac o s could induce ca diac elec ical emodel-
ing, we exposed ca diomyocy es isola ed om con ol a s o plasma ex ac ed om dia-
be ic animals and eco ded ac ion po en ials.
Ven icula myocy es isola ed om con ol a s we e incuba ed 24 h in DMEM sup-
plemen ed wi h ei he plasma ob ained om non-diabe ic animals; o plasma ex ac ed
om ype 2 diabe ic animals, wi h o wi hou added TNFα (50 μM) and IL-1b (50 μg/mL)
ecep o blocke s (Figu e 6 and Table 1).
Res ing memb ane po en ial and AP ampli ude we e simila in he h ee expe i-
men al g oups. Howe e , incuba ion o ca diomyocy es wi h plasma ex ac ed om dia-
be ic animals signi ican ly p olonged he ac ion po en ial du a ion a he 30, 50, and 90%
o epola iza ion (Figu e 6 and Table 1). Blockade o he TNFα and IL-1b cy okine ecep-
o s p e en ed he diabe ic plasma-induced e ec . The e o e, we ound ha diabe ic
plasma componen s al e ed he elec ical beha io o no mal ca diomyocy es ega ding
majo p o-a hy hmic pa ame e s.
Figu e 5.
Repola iza ion capaci y and in lamma ion. (
A
) Cu en aces o a cu en - ol age p o ocol o he ansien
ou wa d cu en elici ed in a con ol cell and a diabe ic en icula cell. (
B
) I
o
densi y/ ol age ela ionships and I
o
densi y
a +50 mV in con ol and diabe ic myocy es. In pa en hesis (numbe o cells/numbe o animals). (
C
) TNF
α
and IL-1b
can educe he densi y o I
o
. Plasma le els o TNF
α
and IL-1b a e highe in ype 2 diabe ic animals han in age-ma ched
con ols. Da a a e mean ±SEM; in pa en hesis (numbe o animals); * p< 0.05; ** p< 0.01.
In ype 1 diabe ic animals, I
o
inhibi ion has been a ibu ed o he diabe ic in lamma-
o y s a us [
12
]. In ha ega d, TNF
α
and IL-1b a e ci cula ing p o-in lamma o y cy okines
known o educe he unc ional exp ession o I
o
. Nex , we compa ed he plasma ic le els
o hese cy okines in ou ype 2 diabe ic animals and age-ma ched con ols. A he end
o he expe imen al pe iod, diabe ic animals had signi ican ly highe le els o TNF
α
and
IL-1b han con ol animals (Figu e 5C).
2.4. Role o Ci cula ing Fac o s on Ca diac Elec ical Remodeling
These p e ious esul s sugges ha in his ype 2 diabe ic model, ele a ion o plas-
ma ic TNF
α
, and IL-1b le els could cause I
o
educ ion. This would lead o p olonged
epola iza ion and highe suscep ibili y o a hy hmia unde ca diac challenge. To con i m
whe he diabe es-associa ed ci cula ing ac o s could induce ca diac elec ical emodeling,
we exposed ca diomyocy es isola ed om con ol a s o plasma ex ac ed om diabe ic
animals and eco ded ac ion po en ials.
Ven icula myocy es isola ed om con ol a s we e incuba ed 24 h in DMEM sup-
plemen ed wi h ei he plasma ob ained om non-diabe ic animals; o plasma ex ac ed
om ype 2 diabe ic animals, wi h o wi hou added TNF
α
(50
µ
M) and IL-1b (50
µ
g/mL)
ecep o blocke s (Figu e 6and Table 1).
In . J. Mol. Sci. 2021,22, 6827 8 o 14
In . J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 8 o 14
Figu e 6. Role o ci cula ing media o s on ca diac elec ical emodeling. (A) Typical ac ion po en ials eco ded in myocy es
isola ed om he igh en icle o heal hy animals incuba ed o 24 h in DMEM supplemen ed wi h: plasma om heal hy
animals (C l Plsm); wi h plasma om diabe ic animals (Db Plsm); o plasma ex ac ed om ype 2 diabe ic animals plus
TNFα and IL-1b ecep o blocke s (Db Plsm Blck). (B) Incuba ion wi h diabe ic plasma p olongs he ac ion po en ial du-
a ion a 30% o epola iza ion (APD30), and his e ec is p e en ed by TNFα plus IL-1b ecep o blocke s. Da a a e mean
± SEM; in pa en hesis (numbe o cells/numbe o animals); * p < 0.05 wi h espec o con ol; # p < 0.05 wi h espec o Db
Plsm.
Table 1. Diabe ic ci cula ing ac o s p olongs ac ion po en ial du a ion in en icula myocy es. E -
ec s o con ol plasma, diabe ic plasma, and diabe ic plasma wi h blocke s o TNFα and IL1β e-
cep o s on en icula ac ion po en ial.
RMP (mV) APA (mV) APD90 (ms) APD50 (ms) APD30 (ms)
Con ol (17/10) −64.0 ± 6.7 72.4 ± 7.8 24.7 ± 4.5 9.0 ± 2.1 6.3 ± 1.2
Diabe ic Plasma (8/4) −63.0 ± 8.2 74.7 ± 8.0 57.4 ± 14.5 * 20.7 ± 5.6 * 13.0 ± 3.9 *
Diabe ic Plasma
Blocked (13/4) −64.4 ± 2.0 68.4 ± 4.4 33.3 ± 3.2 8.3 ± 1.9 # 5.2 ± 1.5 #
Res ing memb ane po en ial (RMP), ac ion po en ial ampli ude (APA), and ac ion po en ial du a-
ion (APD) a 30, 50, and 90% o epola iza ion. In pa en hesis (numbe o cells/numbe o ani-
mals); * p < 0.05 wi h espec o con ol; # p < 0.05 wi h espec o Db Plsm.
3. Discussion
In his wo k, we cha ac e ized a ype 2 diabe ic a model wi h ca diac elec ical e-
modeling. Diabe ic animals had p olonged QTc, educed I o and high suscep ibili y o de-
elop a hy hmia unde ca diac challenge. In diabe ic animals, we ound inc eased plas-
ma ic le els o TNFα and IL-1b. These ci cula ing p oin lamma o y ac o s we e espon-
sible o impai ed en icula epola iza ion ha unde lies he highe suscep ibili y o a -
hy hmia in ou me abolic model o ype 2 diabe es.
Di e en combina ions o high-calo ic die s ( uc ose ich, a ich) and low STZ
doses ha e been used o induce ype 2 diabe es in mammals [21–23]. In his wo k, we used
a die wi h 45% Kcal om lipids o cause p og essi e insulin esis ance. A e wo weeks,
we injec ed a low dose o STZ in ape i oneal o acili a e some be a cell des uc ion.
Abou 48 h a e STZ adminis a ion, diabe es was con i med by ele a ed FPG and ani-
mals con inued consuming a high- a die . We weekly measu ed he me abolic and elec-
oca diog aphic pa ame e s and ound ha he expec ed diabe es-associa ed changes
we e well es ablished o one week and main ained ou weeks a e STZ injec ion. Thus,
in his wo k, we cha ac e ized an animal model wi h he main me abolic and elec oca -
diog aphic cha ac e is ics o ype 2 diabe ic pa ien s in a easonable pe iod o 6 weeks.
Recen mo phological s udies show ha in he me abolically induced ype 2 diabe es
model in a s, he panc eas is cha ac e ized by a no mal exoc ine issue, oge he wi h
isle s wi h dis up ed bounda ies, dec eased size, and educed cell numbe . Isle cells ha e
Figu e 6.
Role o ci cula ing media o s on ca diac elec ical emodeling. (
A
) Typical ac ion po en ials eco ded in myocy es
isola ed om he igh en icle o heal hy animals incuba ed o 24 h in DMEM supplemen ed wi h: plasma om heal hy
animals (C l Plsm); wi h plasma om diabe ic animals (Db Plsm); o plasma ex ac ed om ype 2 diabe ic animals plus
TNF
α
and IL-1b ecep o blocke s (Db Plsm Blck). (
B
) Incuba ion wi h diabe ic plasma p olongs he ac ion po en ial
du a ion a 30% o epola iza ion (APD
30
), and his e ec is p e en ed by TNF
α
plus IL-1b ecep o blocke s. Da a a e
mean
±
SEM; in pa en hesis (numbe o cells/numbe o animals); * p< 0.05 wi h espec o con ol; # p< 0.05 wi h espec
o Db Plsm.
Table 1.
Diabe ic ci cula ing ac o s p olongs ac ion po en ial du a ion in en icula myocy es. E ec s o con ol plasma,
diabe ic plasma, and diabe ic plasma wi h blocke s o TNFαand IL1β ecep o s on en icula ac ion po en ial.
RMP (mV) APA (mV) APD90 (ms) APD50 (ms) APD30 (ms)
Con ol (17/10) −64.0 ±6.7 72.4 ±7.8 24.7 ±4.5 9.0 ±2.1 6.3 ±1.2
Diabe ic Plasma (8/4) −63.0 ±8.2 74.7 ±8.0 57.4 ±14.5 * 20.7 ±5.6 * 13.0 ±3.9 *
Diabe ic Plasma Blocked (13/4) −64.4 ±2.0 68.4 ±4.4 33.3 ±3.2 8.3 ±1.9 #5.2 ±1.5 #
Res ing memb ane po en ial (RMP), ac ion po en ial ampli ude (APA), and ac ion po en ial du a ion (APD) a 30, 50, and 90% o
epola iza ion. In pa en hesis (numbe o cells/numbe o animals); * p< 0.05 wi h espec o con ol; #p< 0.05 wi h espec o Db Plsm.
Res ing memb ane po en ial and AP ampli ude we e simila in he h ee expe imen al
g oups. Howe e , incuba ion o ca diomyocy es wi h plasma ex ac ed om diabe ic
animals signi ican ly p olonged he ac ion po en ial du a ion a he 30, 50, and 90% o
epola iza ion (Figu e 6and Table 1). Blockade o he TNF
α
and IL-1b cy okine ecep o s
p e en ed he diabe ic plasma-induced e ec . The e o e, we ound ha diabe ic plasma
componen s al e ed he elec ical beha io o no mal ca diomyocy es ega ding majo
p o-a hy hmic pa ame e s.
3. Discussion
In his wo k, we cha ac e ized a ype 2 diabe ic a model wi h ca diac elec ical
emodeling. Diabe ic animals had p olonged QTc, educed I
o
and high suscep ibili y o
de elop a hy hmia unde ca diac challenge. In diabe ic animals, we ound inc eased
plasma ic le els o TNF
α
and IL-1b. These ci cula ing p oin lamma o y ac o s we e
esponsible o impai ed en icula epola iza ion ha unde lies he highe suscep ibili y
o a hy hmia in ou me abolic model o ype 2 diabe es.
Di e en combina ions o high-calo ic die s ( uc ose ich, a ich) and low STZ doses
ha e been used o induce ype 2 diabe es in mammals [
21
–
23
]. In his wo k, we used a die
wi h 45% Kcal om lipids o cause p og essi e insulin esis ance. A e wo weeks, we in-
jec ed a low dose o STZ in ape i oneal o acili a e some be a cell des uc ion. Abou 48 h
a e STZ adminis a ion, diabe es was con i med by ele a ed FPG and animals con inued
consuming a high- a die . We weekly measu ed he me abolic and elec oca diog aphic
pa ame e s and ound ha he expec ed diabe es-associa ed changes we e well es ablished
In . J. Mol. Sci. 2021,22, 6827 9 o 14
o one week and main ained ou weeks a e STZ injec ion. Thus, in his wo k, we cha ac-
e ized an animal model wi h he main me abolic and elec oca diog aphic cha ac e is ics
o ype 2 diabe ic pa ien s in a easonable pe iod o 6 weeks.
Recen mo phological s udies show ha in he me abolically induced ype 2 diabe es
model in a s, he panc eas is cha ac e ized by a no mal exoc ine issue, oge he wi h
isle s wi h dis up ed bounda ies, dec eased size, and educed cell numbe . Isle cells ha e
acuola ed cy oplasm and small-sized de o med nucleus [
25
,
26
]. In addi ion, immuno-
his ochemical s aining show ha he educ ion in cell numbe is due o a selec i e loss o
β-cells in he damaged panc ea ic isle s [26,27].
Ou expe imen al model was de eloped o mimic he clinical ea u es o human ype
2 diabe es. Thus, we gene a ed a mild diabe ic condi ion a oiding massi e be a cells loss
and he consequen de elopmen o ype 1 diabe es. In his wo k, he le els o ci cula ing
insulin oge he wi h pe iphe al insulin esis ance ensu e ha ou expe imen al model
co esponds o ype 2 diabe es.
Diabe ic animals eplica ed he classical high- as ing plasma glucose and insulin esis-
ance cha ac e is ics o ype 2 diabe ic pa ien s. Since as ing plasma insulin was simila
in diabe ics han in con ol animals, he high blood glucose was due o insulin esis ance
and he inabili y o he panc eas o compensa e by inc easing insulin p oduc ion. Mo e-
o e , as desc ibed o ype 2 diabe ic pa ien s [
28
], ou ype 2 diabe ic animals had mo e
abdominal a han he con ol animals, hype choles e olemia, and hype iglyce idemia.
Ou expe imen al da a a e in line wi h he clinical indings in humans wi h ype 2 diabe es.
Co espondingly, he ECGs o diabe ic pa ien s show slowe hea a e and p olonged
du a ion o he QT-in e al, indica ing epola iza ion abno mali ies associa ed wi h in-
c eased isk o en icula a hy hmias and mo ali y [
2
,
29
]. Indeed, in ou p esen s udy,
diabe ic animals, as well as he animals in he Chow + STZ g oup, had a lowe hea
a e han con ol animals measu ed as longe RR-in e als. Howe e , only animals in he
diabe ic g oup (high- a die plus STZ) had epola iza ion abno mali ies. Diabe ic a s
showed he expec ed p olonga ion o he epola iza ion ime, seen as he p olonged QT
and QTc in e als and he p olonged T
peak-
T
end
, an elec oca diog aphic ma ke o he
ansmu al dispe sion o epola iza ion. P olonged QTc and T
peak-
T
end
a e used in clinical
se ings o p edic he isk o en icula a hy hmia and sudden ca diac dea h [
30
–
32
]. In
his sense, when subjec ed o p oa hy hmic condi ions diabe ic animals de eloped mo e
a hy hmic episodes and mo e se e e han con ol animals. I is impo an o no e ha
ca diac challenge induced he po en ially le hal a hy hmia o sade de poin es in one ou
o h ee ype 2 diabe ic animals. By con as , none o he con ol animals de eloped o sade
de poin es unde he same challenge.
A he cellula le el, diabe es can induce leng hening in he ca diac epola iza ion
ime by a ec ing he exp ession and ac i i y o se e al ca diac ionic channels and hei
co esponding ionic cu en s. In models o ype 1 diabe es, p olonged epola iza ion is
caused by a educ ion o he ansien ou wa d K
+
cu en , I
o
[
7
,
8
,
18
,
19
]. Ou esul s
in he ype 2 diabe ic model a e in ag eemen wi h ha . We ound ha I
o
densi y in
ype 2 diabe ic animals is smalle han in con ol animals. The s ong educ ion on I
o
densi y caused by diabe es educed he epola iza ion capaci y o he en icula cells.
This can explain he p olonged epola iza ion ime o he whole hea obse ed in he
elec oca diog am.
The accumula ion o abdominal a is ecognized as he o igin o a gene alized low-
g ade in lamma o y s a us o he whole body, wi h inc eased ci cula ing cy okines such
as IL-6, TNF
α
, IFN-
γ
, TGF-
β
, MCP-1, o IL-1b [
33
]. Among hem, TNF
α
and IL-1b
a e wo cy okines known o be up egula ed in ype 2 diabe ic pa ien s [
16
], which ha e
demons a ed
in i o
and
in i o
animal models he abili y o educe he unc ional
exp ession o he ca diac epola izing cu en I
o
. TNF
α
modi ies I
o
inac i a ion and
educes he channel exp ession h ough iNOS and oxidan species [
14
], whe eas IL-1b
ecep o ac i a ion educes I
o
exp ession h ough NLRP3 ac i a ion [
12
]. Mo eo e , i
has been ecen ly demons a ed he ole o bo h TNF
α
and IL-1b in he ini ia ion and
