Severe Hypoglycemia–Induced Lethal Cardiac Arrhythmias Are

12 Pages·2013·1.54 MB·English

ORIGINALARTICLE
–
Severe Hypoglycemia Induced Lethal Cardiac
Arrhythmias Are Mediated by Sympathoadrenal Activation
Candace M. Reno,1 Dorit Daphna-Iken,1 Y. Stefanie Chen,1 Jennifer VanderWeele,1 Krishan Jethi,1
and Simon J. Fisher1,2
Duringhypoglycemia,lackofglucosesupplytoneurons
For people with insulin-treated diabetes, severe hypoglycemia
can be lethal, though potential mechanisms involved are poorly canleadtoconfusion,braindamage(12),seizures(13,14),
understood. To investigate how severe hypoglycemia can be and even death. Indeed, 6–10% of deaths in young people
fatal, hyperinsulinemic, severe hypoglycemic (10–15 mg/dL) with type 1 diabetes are directly attributable to hypogly-
clamps were performed in Sprague-Dawley rats with simulta- cemia (15–17). It has been hypothesized that abrupt
neous electrocardiogram monitoring. With goals of reducing hy- cardiac arrhythmias contribute to severe hypoglycemia–
poglycemia-induced mortality, the hypotheses tested were that: induced sudden death. Clinical studies using electro-
1) antecedent glycemic control impacts mortality associated
cardiograms(ECGs)havefoundthatcardiacarrhythmias
with severe hypoglycemia; 2) with limitation of hypokalemia,
associated with corrected QT (QTc) prolongation occur
potassium supplementation could limit hypoglycemia-associated
deaths;3)withpreventionofcentralneuroglycopenia,brainglu- during moderate hypoglycemia (18–21). QTc prolongation
represents dispersion of ventricle depolarization and can
cose infusion could prevent hypoglycemia-associated arrhyth-
mias and deaths; and 4) with limitation of sympathoadrenal lead to increased risk of fatal cardiac arrhythmias (22–24).
activation, adrenergic blockers could prevent hypoglycemia- In the setting of insulin-induced severe hypoglycemia, it
inducedarrhythmicdeaths.Severehypoglycemia–inducedmortality remains unknown whether fatal cardiac arrhythmias are
wasnotedtobeworsenedbydiabetes,butrecurrentantecedent mediated by brain neuroglycopenia per se or systemic
hypoglycemia markedly improved the ability to survive an epi- factors acting directly on the heart. Systemically, both the
sodeofseverehypoglycemia.Potassiumsupplementationtended insulin-induced decrement in potassium levels and the
to reduce mortality. Severe hypoglycemia caused numerous car-
counterregulatory-inducedincreaseincatecholaminelevels
diacarrhythmiasincludingprematureventricularcontractions, havebeenspeculatedtocontributetoarrhythmias(25–27).
tachycardia,andhigh-degreeheartblock.Intracerebroventricular
glucose infusion reduced severe hypoglycemia–induced arrhyth- It has been shown previously that the administration of
mias and overall mortality. b-Adrenergic blockade markedly re- potassium or b-blockers can reduce mild cardiac arrhyth-
duced cardiac arrhythmias and completely abrogated deaths due mias during moderate hypoglycemia (;45 mg/dL) (26).
to severe hypoglycemia. Under conditions studied, sudden deaths However, it remains to be determined whether the pre-
caused by insulin-induced severe hypoglycemia were mediated by vention of hypokalemia or blocking the actions of the
lethal cardiac arrhythmias triggered by brain neuroglycopenia and catecholamines protects against lethal arrhythmias and
themarkedsympathoadrenalresponse. cardiorespiratory arrest during severe hypoglycemia.
In order to determine preventable causes of death dur-
ing hypoglycemia, severe hypoglycemic clamps were per-
T
formed in Sprague-Dawley rats with simultaneous arterial
he rate of sudden death in young people with
blood sampling and ECG monitoring. It was hypothesized
type 1 diabetes is 10-fold higher than in age-
that brain neuroglycopenia and the marked sympatho-
matched control subjects (1). Sudden nocturnal
adrenal response during severe hypoglycemia trigger fatal
deaths account for up to 27% of all unexplained
cardiac arrhythmias.
deaths in people with type 1 diabetes (2,3). The aptly
named “dead in bed syndrome” identifies otherwise
healthy young individuals with type 1 diabetes who were RESEARCHDESIGNANDMETHODS
founddeadinbedwithnoclearcauseofdeath(1–9).Ithas Nine-week-old male Sprague-Dawley rats (CharlesRiver Laboratories) were
been speculated that their deaths were due to excess in- housedindividuallyinatemperature-andlight-controlledenvironmentandfed
adlibitum withstandard ratchow dietandwater.Allstudieswere donein
sulin administration that resulted in severe hypoglycemia
accordancewithandapprovedbytheAnimalStudiesCommitteeatWashington
(3,5–11). However, the mechanisms by which severe hy-
UniversitySchoolofMedicine.
poglycemia can be lethal is unclear. Implantation of vascular catheters and ECG leads. Vascular catheters
wereimplantedaspreviouslydescribed(28).OneECGwireleadwasplacedin
therightsupraclavicularfossaandanotherwasplacedexteriortothelower
left rib cage and sutured to underlying muscle. Reference lead was placed
From the 1Division of Endocrinology, Metabolism, & Lipid Research, De- subcutaneously.
partment of Medicine, Washington University, St. Louis, Missouri; and Hyperinsulinemic-severehypoglycemicclamp.Fourtotendayspostsurgery,
the2DepartmentofCellBiologyandPhysiology,WashingtonUniversity, overnight fasted, precannulated, awake, unrestrained rats were subjected to
St.Louis,Missouri. hyperinsulinemic(0.2units$kg21$min21)severehypoglycemic(10–15mg/dL)
Correspondingauthor:SimonFisher,sfi[email protected]. clamps as previously described (28–30). Hypoglycemia was maintained for a
Received11February2013andaccepted15May2013.
predeterminedduration(60minforstudies1and2,90minforstudy3,and180
DOI:10.2337/db13-0216
This article contains Supplementary Data online at http://diabetes minforstudies4and5).
.diabetesjournals.org/lookup/suppl/doi:10.2337/db13-0216/-/DC1. InsulinwasmeasuredbyELISA(CrystalChem,DownersGrove,IL).Glu-
(cid:1)2013bytheAmericanDiabetesAssociation.Readersmayusethisarticleas cagonwasmeasuredbyradioimmunoassay(Millipore,Billerica,MA).Epinephrine
longastheworkisproperlycited,theuseiseducationalandnotforprofit, and norepinephrine levels were determined by a single isotope derivative
and the work is not altered. See http://creativecommons.org/licenses/by (radioenzymatic)methodaspreviouslydescribed(28).Bloodpressure(BP)
-nc-nd/3.0/fordetails. (noninvasive BP monitor; Columbus Instruments, Columbus, OH) was mea-
See accompanying commentary, p. XXX. suredbytailcuffinselectstudies.
diabetes.diabetesjournals.org DIABETES 1
Diabetes Publish Ahead of Print, published online July 24, 2013
SEVEREHYPOGLYCEMIA–INDUCEDSUDDENDEATH
Arterialbloodgases.Bloodsamplesweretakenthroughouttheclampfor had markedly less mortality (4%) during severe hypo-
analysisofelectrolytesandbloodgases(pHOxPlusCarterialbloodgasma- glycemia (P , 0.05) (Fig. 1A). Differences in death rates
chine;NovaBiomedical,Waltham,MA).Respirationwascalculatedbyvisibly
were hypothesized to be due to hypokalemia, neuro-
countingbreaths.
glycopenia, or the sympathoadrenal response, each of
Seizures.Seizure-likebehaviorwasnotedduringthehypoglycemicclampsas
previouslydescribed(28,29).Asubgroupoffourratshadelectroencephalo- which may have increased susceptibility to cardiac
gram(EEG)recording(XLTEKVideoEEGSystem,Oakville,Ontario)toan- arrhythmias.
alyzebrainwaveactivityduringhypoglycemia. Study 2: potassium supplementation. In order to de-
Study1:pooledmortalityanalysisincontrol,diabetic,andrecurrently termine if the cause of death due to severe hypoglycemia
hypoglycemic rat experiments. Control (n = 123), diabetic (n = 95), and
resulted from hypokalemia, potassium was supplemented
recurrenthypoglycemic(n=27)ratsthatunderwent1hofseverehypogly-
cemia(28–30)wereanalyzedformortality. in nondiabetic and diabetic rats during a 1 h severe hypo-
Diabetes.Diabeteswasinducedbyinjectionofstreptozotocin(STZ)(65mg/kg glycemic clamp. Control rats had basal glucose levels of
i.p.;Sigma,St.Louis,MO)2weekspriortotheclampaspreviouslydescribed 127 6 7 mg/dL and 116 6 12 mg/dL with or without po-
(29). Control rats received injections of 0.1 mmol/L sodium citrate (Fisher tassiumsupplementation,respectively.Basalglucoselevels
Scientific)buffer. fordiabeticratswere479677mg/dLand394654mg/dL,
Recurrent hypoglycemia. Moderate hypoglycemia (25–40 mg/dL) was in-
respectively. Mean glucose levels during severe hypogly-
ducedfor3h/3daysinnondiabeticratsaspreviouslydescribed(28).Control
ratsreceivedsalineinjections.Severehypoglycemicclampswereperformed cemia were not different among the nondiabetic rats that
onthefourthday. did (12.6 6 1.18 mg/dL) or did not (12.5 6 0.76 mg/dL)
Study 2: potassium supplementation. Potassium (KCl, 0.375 mEq/mL; receivepotassiumorthediabeticratsthatdid(12.160.84
Hospira,LakeForest,IL)wasinfusedwithsalineat4mL/min.Nondiabeticrats mg/dL) or did not (13.4 6 0.39 mg/dL) receive potassium.
received potassium (n = 12) or saline (n = 15), and diabetic rats received
potassium(n=28)orsaline(n=45)throughouta1-hhyperinsulinemic/severe Potassiumlevelsdecreasedduringseverehypoglycemiain
hypoglycemicclampasdescribedabove. bothnondiabeticrats(4.260.15to2.960.06mmol/L)and
Study3:cardiacarrhythmias.Nondiabeticrats(n=6)underwentsevere diabeticrats(4.060.11to2.960.23mmol/L)thatdidnot
hypoglycemic clamps for 90 min as described above, with continuous ECG receive potassium (Fig. 1B). From basal to severe hypo-
recording(ADI Powerlab 26T; ADInstruments, Colorado Springs, CO). ECG glycemia, potassium supplementation maintained plasma
analyses included heart rate, QTc length, and other arrhythmias (ADI Lab- potassium levels in nondiabetic (4.4 6 0.26 to 3.9 6 0.38
ChartPro7).QTcwascalculatedusingBazettformula(31).
Study4:intracerebroventricularglucoseinfusion.Intracerebroventricular mmol/L) and diabetic (4.5 6 0.25 to 3.8 6 0.47 mmol/L)
(ICV)cannulas(internalcannula31g,outerguidecannula24g,9.5mmdepth; rats (Fig. 1B). Mortality associated with severe hypogly-
PlasticsOne,Roanoke, VA)wereplacedontheskull(22.8mmposteriorto cemia trended to be reduced with potassium supplemen-
bregma)usingstereotaxicequipmentunderisoflourane.Ratsrecovered1week tationcomparedwithratsthatdidnotreceivepotassiumin
before vascular and lead implantations. D-glucose (2 mmol/L i.c.v.) (n = 9) both nondiabetic (from 33 to 8%) and diabetic (from 44 to
(Sigma)orD-mannitol(2mmol/Li.c.v.)(n=9)(Sigma)wasinfused(0.1mL/min) 25%) rats (P , 0.08) (Fig. 1C).
throughout a 3-h hyperinsulinemic/severe hypoglycemic clamp as described
above.Correctcannulaplacementwasconfirmedbypostmortemstainingwith Study 3: cardiac arrhythmias. Severe hypoglycemia–
Evansbluedye. induced death seemed to be related to the proarrhythmic
Study5:adrenergicblockade.a-Blockers(prazosin[a1],1mg/mL,1mL/min; condition of hypokalemia; thus, cardiac arrhythmias were
Sigma)andb-blockers(propranolol[b1andb2],1mg/mL,2.5mL/min;Sigma)
hypothesized to mediate severe hypoglycemia-induced
wweitrhesianlfiunsee(dcoinnttrraovle,nnou=s1ly2)costnacrutirnrgen3t0lym(nin=pr1i3o)rotoriinnsduivliinduianlfluysi(onn=an5d–6c)oonr- mortality. Nondiabetic rats (n = 6) were subjected to se-
tinuedthroughouta3-hhypoglycemicclampasdescribedabove. vere hypoglycemic clamps with continuous ECG re-
Statisticalanalyses.Alldataarerepresentedasmeans6SEM.ANOVA(one- cording. Glucose was clamped between 10–15 mg/dL for
and two-way), repeated-measures ANOVA, and Student t test were used to 90 min during which all rats died (Fig. 2A and B).
determine significance. Holm-Sidak was used for post hoc analyses. Fisher Duringinsulininfusion,heartrateremainednormaluntil
exacttestwiththeFreeman-Haltonextensionwasusedformortalityandar- itincreasedattheonsetofseverehypoglycemia(Fig.2C).
rhythmiaincidence.SignificancewasdeterminedatP,0.05.
Severe hypoglycemia increased the QTc interval (172 6
8ms)comparedwithbasal(12262ms),whichrepresents
RESULTS a 40% increased ventricular depolarization and repolar-
Study1:pooledmortalityanalysisincontrol,diabetic, ization phase (P , 0.001) (Fig. 2D).
and recurrently hypoglycemic rat experiments. For Epinephrine and norepinephrine increased during se-
an understanding of how diabetes and recurrent hypogly- vere hypoglycemia compared with the basal period (P ,
cemia might impact survival during severe hypoglycemia, 0.001)(Fig.2EandF).Plasmaepinephrinepeakedshortly
pooled data were analyzed from 1) nondiabetic, 2) un- aftertheonsetofseverehypoglycemiaandthendecreased
controlled STZ diabetic, and 3) recurrent moderate hypo- as severe hypoglycemia continued, possibly indicating
glycemic prior to severe hypoglycemic rats. Basal glucose adrenal exhaustion. Plasma norepinephrine continued to
levels for nondiabetic, diabetic, and recurrently hypogly- increase throughout severe hypoglycemia.
cemic rats were 88 6 5, 348 6 37, and 74 6 2 mg/dL, re- Cardiac arrhythmias did not occur during moderate
spectively. During the 1 h of hypoglycemia, glucose levels hypoglycemia. After the onset of severe hypoglycemia,
were similar for control (12 6 0.2 mg/dL), diabetic (12 6 premature ventricular contractions (PVCs) and narrow
0.2 mg/dL), and recurrent hypoglycemia (12 6 0.4 mg/dL) complexseconddegreeheartblock(MobitzII)werenoted
rats. As expected, the recurrently hypoglycemic rats had asinitial arrhythmias (Fig.3).Premature atrial contractions
a blunted epinephrine response to hypoglycemia (2,001 6 (PACs) and first-degree heart block were also observed as
241 vs. 3,487 6 474 pg/mL in controls) (28). During se- early arrhythmias (Fig. 3). Occasionally, brief runs of non-
verehypoglycemia,allratslosttheirrightingreflex,went sustained ventricular tachycardia occurred, but these
into a coma, and experienced brief seizure-like activity. rhythms did not precede death. With longer duration of se-
Mortality due to severe hypoglycemia was 21% in non- vere hypoglycemia, a sequential pattern and progression of
diabetic rats and increased to 36% in diabetic rats (P , arrhythmias that preceded death were noted. Frequent and
0.05) (Fig. 1A). Interestingly, rats that had been pre- higher-grade second-degree heart block (4:1) was noted fol-
viously treated with recurrent moderate hypoglycemia lowedbythird-degreeheartblock(completeatrioventricular
2 DIABETES diabetes.diabetesjournals.org
C.M.RENOANDASSOCIATES
FIG.1.Severehypoglycemia–inducedmortalityindiabetic,nondiabetic,andrecurrentlyhypoglycemicratsandtheeffectsofpotassiumsupple-
mentation(studies1and2).A:Analysisofmoralityassociatedwithseverehypoglycemiarevealed21%mortalityinnondiabeticcontrolrats(n=
123).Mortalitywasincreasedindiabeticratsto36%(n=95)anddecreasedto4%(n=27)inratsthatunderwentrecurrenthypoglycemiapriorto
severehypoglycemia(*P<0.05,Fisherexacttest,Freeman-Haltonextension).B:Potassiumlevelsinratsthathadnopotassiumsupplementation
versusratswithpotassiumsupplementationbothinthebasalstateandduringseverehypoglycemia.Ratswithoutsupplementationhadpotassium
levelssignificantlydecreaseduringseverehypoglycemia,whileratswithsupplementationmaintainednormalpotassiumduringseverehypogly-
cemia.Dataexpressedasmeans6SEM.C:Potassiumsupplementationinnondiabeticanddiabeticratstrendedtoreduceseverehypoglycemia–
inducedmortalityfrom33to8%(n=12–15)and44to25%(n=28–45),respectively,butthisdidnotreachsignificance.
dissociation), characterized by a terminal bradycardic required a higher peripheral glucose infusion rate (5.4 6
ventricular rhythm (Fig. 3). 0.6 mg/kg/min; P , 0.005) during severe hypoglycemia
Oxygen and carbon dioxide levels remained normal compared with MAN (4.1 6 0.41 mg/kg/min) (Fig. 4C).
throughout the experiment until just before death, when Insulin levels were comparable between MAN (7.5 6
oxygen levels decreased and carbon dioxide levels in- 0.78 ng/mL) and glucose (8.8 6 0.49 ng/mL) groups dur-
creased (Supplementary Fig. 1A). As previously noted, ing severe hypoglycemia.
potassium levels decreased during severe hypoglycemia During severe hypoglycemia, heart rate was lower in the
compared with basal (P , 0.05) (Supplementary Fig. 1B). glucosecomparedwithMANgroup(P,0.05)(Fig.4D).Mean
BP increased from 105 6 14/60 6 2 mmHg during the heart rate increased in the MAN group during severe hypo-
basal period to 177 6 16/93 6 9 mmHg during severe hy- glycemiacomparedwithbasal(41469vs.37364bpm;P,
poglycemiaandremainedelevateduntilimmediatelypriorto 0.01),whileICVglucoseinfusionpreventedthisriseinheart
death,whenitsuddenlydecreased(SupplementaryFig.1C). rate(basal:35764bpm;hypoglycemia:35768bpm).QTc
Seizure-like activity was noted in all rats. A subset of increased during severe hypoglycemia compared with basal
rats had simultaneous EEG recordings during the clamp. in both MAN and glucose groups (P = NS) (Fig. 4E).
During severe hypoglycemia, EEG tracings showed char- Cardiac arrhythmias were absent in 25% of the ICV
acteristic high-amplitude slow waves. EEG isoelectricity glucose-infused rats. Frequencies of PVCs and second-
coincided with a nonresponsive comatose state (Supple- degree heart block were nonsignificantly reduced in the
mentary Fig. 1D). Witnessed seizurescoincided with high- glucose compared with the MAN group (P , 0.07) (Sup-
frequency, high-amplitude spikes of increased electrical plementary Fig. 2A and B). Incidence of third-degree
activity on the EEG. There was no correlation found be- heart block was decreased in the glucose group com-
tween seizures and mortality. pared with the MAN group (P , 0.05) (Supplementary
Study 4: ICV glucose infusion. For an understanding of Fig. 2C) and preceded death in both groups, suggesting
the extent to which neuroglycopenia mediates sudden that third-degree heart block was the terminal fatal ar-
death, glucose was infused into the third ventricle of the rhythmia associated with severe hypoglycemia. Other
brainduringseverehypoglycemia.Comparedwithcontrols, life-threateningarrhythmiassuchasventriculartachycardia
ICV glucose infusion reduced mortality due to severe hy- were witnessed in 33% of the MAN and 0% of the glucose
poglycemia from 86 to 33% (P , 0.05) (Fig. 4A). Systemic group (Supplementary Fig. 2D).
blood glucose was similar during severe hypoglycemia in Thenorepinephrineresponsewasbluntedduringsevere
ICVmannitol(MAN)(n=9)-infusedandICVglucose(n=9)- hypoglycemia in the glucose (464 6 48 pg/mL) compared
infusedrats(1260.1vs.1260.2mg/dL,respectively)(Fig. with the MAN group (1,441 6 646 pg/mL; P , 0.05) (Fig.
4B).Despitematchedglucoselevels,theICVglucosegroup 4F).Duringseverehypoglycemia,epinephrineincreasedto
diabetes.diabetesjournals.org DIABETES 3
SEVEREHYPOGLYCEMIA–INDUCEDSUDDENDEATH
FIG.2.Severehypoglycemicclampincontrolexperiments(study3).A:Glucoselevelsofcontrol,nondiabeticratsduringaseverehypoglycemic
clamp.Withinsulininfusionandcarefulglucoseinfusion,plasmaglucoselevelsaredecreasedovera2.5-hperiodto15mg/dL(time0).Severe
hypoglycemia(10–15mg/dL)wasmaintainedfor1.5h.B:Mortalityassociatedwithseverehypoglycemiastartedasearlyas25minaftertheonset
ofseverehypoglycemia.Allratshaddiedby90minofseverehypoglycemia.C:Heartrateduringtheclampremainednormaluntiljustbefore
severehypoglycemiawhenglucoselevelsreach;35mg/dL,atwhichtimeheartrateincreases.Attheonsetofseverehypoglycemia,heartrate
decreases.Heartrateincreasesagainimmediatelypriortodeath.Theslowerheartratesthatoccurtowardtheendoftheclampcanbeattributed
tomultiplecardiacarrhythmiasassociatedwithbradycardia.D:QTcprolongationstartstooccurduringinsulininfusionwhenglucoselevelsare
moderately hypoglycemic (;35 mg/dL) and persists throughout the period of severe hypoglycemia. Mean QTc during severe hypoglycemia in-
creasedto15865mscomparedwith12262msinthebasalperiod(P<0.01).E:Epinephrinelevelsincreasedfrom495660pg/mLinthebasal
periodandpeakat3,0346383pg/mLattheonsetofseverehypoglycemiabutdeclinedasseverehypoglycemiapersisted(P<0.001).F:Nor-
epinephrinelevelsincreasedfrom197620pg/mLinthebasalperiod,startedtoincreaseattheonsetofseverehypoglycemia,andcontinuedtorise
throughoutseverehypoglycemia,peakingat7486116pg/mL(P<0.001).n=6;dataexpressedasmeans6SEM.
asimilarextentinboththeMAN(1,9266320pg/mL)and completely prevented death (P , 0.029) (Fig. 5A).
glucose(2,1556448pg/mL)groups.Frombasaltosevere a-Blocker infusion alone had no effect on mortality
hypoglycemia,plasmapotassium levelsfellinMAN(4.76 (50%; n = 6).
0.3 to 3.6 6 0.4 mmol/L) and glucose (4.4 6 0.3 to 3.1 6 Glucoselevelsduringseverehypoglycemiawereequally
0.5 mmol/L) rats. matched in control, a/b-, a-, and b-blocker-infused rats
Study 5: adrenergic blockade. For testing of the hy- (12 6 0.2, 12 6 0.3, 12 6 0.5, and 12 6 0.5 mg/dL, re-
pothesis that the sympathoadrenal response increases spectively) (Fig. 5B). Consistent with a blunted counter-
risk of fatal cardiac arrhythmias during severe hypo- regulatory response, glucose infusion rate was increased
glycemia, rats had adrenergic receptor blockers infused during severe hypoglycemia in a/b-blocker-infused rats
during a hyperinsulinemic/severe hypoglycemic clamp. (P , 0.01) (Fig. 5C) compared with controls, with no
Severe hypoglycemia–induced mortality was 33% in differenceina-andb-blockeralone–infusedrats.Insulin
control rats (n = 12), while combined a/b-blocker in- levels were not different between the groups (Supple-
fusion (n = 13) and b-blocker infusion alone (n = 5) mentary Fig. 3A).
4 DIABETES diabetes.diabetesjournals.org
C.M.RENOANDASSOCIATES
FIG.3.ECGchangesinresponsetoseverehypoglycemia(study3).ECGrecordingsfromonerepresentativecontrolratfrombasal(euglycemia)
throughseverehypoglycemia–induceddeath.Theorderofthegraphsisatypicalrepresentationofwhatoccursinallratsduringseverehypo-
glycemiaandjustpriortodeath.Atthetopoftheleftcolumn,theECGshowsaratinnormalsinusrhythmduringeuglycemicconditions.During
moderatehypoglycemia,cardiacarrhythmiasarerarelywitnessed.Atthestartofseverehypoglycemia,heartratetendstoreturntobasallevels
followedbyQTcprolongationandtheinitiationofseveraltypesofcardiacarrhythmias.Cardiacarrhythmiasthatoccurduringseverehypogly-
cemia are PACs (noted by arrow), PVCs (noted by arrow), nonsustained ventricular tachycardia (V-Tach), first-degree heart block (PR pro-
longationnotedbyarrow),andsecond-degreeheartblock(absentQRScomplexnotedbyarrow).Withseverehypoglycemiaofincreasedfrequency
high-grade,second-degreeheartblockwasnotedfollowedbythird-degreeheartblock.Atthetimeofcardiorespiratoryarrest,alower-voltageECG
withamarkedlydecreasedheartratewasnoted.
During severe hypoglycemia, heart rate was lower in Arrhythmiaincidencewaslowerwitha/b-andb-blocker
a/b-blocker-infused rats (225 6 5 bpm) and b-blocker- infusioncomparedwithcontrols.PVCsandsecond-degree
infusedrats(19864bpm)thanincontrols(33165bpm) heart block occurred in only 25 and 8% of the a/b-blocker
and a-blocker-infused (339 6 9 bpm) rats (P , 0.05) (Fig. rats, respectively (P , 0.001). Frequency of PVCs was
5D). Sinus tachycardia was experienced in all rats that reduced in a/b- and b-blocker-infused rats compared with
died. More than 30 min of tachycardia resulted in 100% controls (P , 0.001) (Fig. 6A). a-blocker-infused rats had
mortality (Supplementary Fig. 3B). b-Blocker and com- nonsignificantly lower frequency of PVCs. Frequency of
bined a/b-blocker infusion completely abrogated hypo- second-degree heart block was significantly reduced in
glycemia-induced tachycardia and mortality. Sinus combined a/b-blocker-infused rats (P , 0.001) and trended
tachycardia is therefore a predictor of mortality in these to be reduced in a- and b-blocker-infused rats compared
experiments, and prevention of tachycardia was associ- with controls (Fig. 6B). Third-degree heart block was
ated with a mortality benefit. prevented in a/b-blocker-infused rats (P , 0.024) and
QTcprolongationincreasedduringseverehypoglycemia b-blocker-infused rats (P = NS), with no difference in
in control (163 6 2 ms), a-blocker-infused (210 6 5 ms), a-blocker-infused rats compared with controls (Fig. 6C).
and b-blocker-infused (164 6 2 ms) rats (P , 0.001), but Epinephrine and norepinephrine significantly increased
QTcprolongationwasbluntedwitha/b-blocker(13463ms; during severe hypoglycemia compared with basal in all
P , 0.001) infusion (Fig. 5E). QTc prolongation was asso- groups (Fig. 6D and E); however, the actions of epineph-
ciated with increased risk of mortality in control and rineandnorepinephrinewereblockedwiththeadrenergic
a-blockerrats(Supplementary Fig. 3C). Interestingly, the blockers. Consistent with decreased epinephrine clear-
b-blocker-infused rats had QTc prolongation similar to ance by b-blockers, epinephrine levels were significantly
that in control rats (Fig. 5E), yet all survived, indicating higher in b-blocker-infused rats compared with controls
that QTc prolongation may not be a direct mediator of (P , 0.004) and a/b-blocker rats (P , 0.011) (Fig. 6D).
severe hypoglycemia–induced lethality. Norepinephrinewassignificantlyincreasedinthea-blocker
diabetes.diabetesjournals.org DIABETES 5
SEVEREHYPOGLYCEMIA–INDUCEDSUDDENDEATH
FIG.4.SeverehypoglycemicclampwithICVglucoseinfusion(study4).A:MortalityduetoseverehypoglycemiawassignificantlyreducedinICV
glucose-infusedcomparedwithICVMAN-infused(control)(33vs.89%;*P<0.05,Fisherexacttest,Freeman-Haltonextension)rats.B:Blood
glucoselevelswereevenlymatched betweentheICVMAN-infusedandICVglucose-infusedrats.Oncebloodglucoselevelsreached15 mg/dL,
severehypoglycemia(startingattime0)wasmaintainedforadurationof3h.C:Meanglucoseinfusion(Ginf)ratesduringseverehypoglycemia
weresignificantlyelevatedinICVglucose-infused(5.460.6mg/kg/min)comparedwithICVMAN-infused(4.260.41mg/kg/min)rats.*P<0.01,
ttest.D:Heartrateremainednormalinbothgroupsuntil45minintoseverehypoglycemia,whenICVMAN-infusedratshadelevatedheartrate.
ICVglucose-infusedratshadlowerheartratesthroughoutseverehypoglycemiacomparedwithcontrols.Meanheartduringseverehypoglycemia
was41469bpmand35768bpminICVMAN-infusedandglucose-infusedrats,respectively(*P<0.05,ttest).E:QTclengthincreasedafterthe
startofinsulininfusionduringmoderatehypoglycemia.Duringseverehypoglycemia,QTclengthwasnotdifferentbetweenICVMAN-infusedand
glucose-infusedrats,withmeanQTcvaluesof18062and18763ms,respectively.F:Norepinephrinelevelswereincreasedduringseverehy-
poglycemiainICVMAN-infusedratscomparedwiththebasalstate,butthisresponsewasbluntedinICVglucose-infusedrats.*P<0.05,ttest.n=
9/group.Dataexpressedasmeans6SEM.
group (P , 0.001) compared with the control, a/b-, and Based on the composite observations in the presented
b-blocker groups (Fig. 6E). Glucagon was significantly experiments, mechanisms of sudden cardiac death due to
higher in a-blocker-infused rats compared with other insulin-inducedseverehypoglycemiaare proposed (Fig.7).
groups (P , 0.001 for AUC) (Fig. 6F), consistent with the
elevated norepinephrine-mediated, unopposed, b-adrenergic
receptor stimulation of pancreatic a-cells. DISCUSSION
Potassium levels decreased in all groups during severe Mortality owing to iatrogenic hypoglycemia represents
hypoglycemia (P , 0.01) (Table 1). Oxygen and carbon a major concern for insulin-treated diabetic patients and
dioxide levels remained normal in all groups until just their families. Determining the mechanisms by which hy-
before death (Table 1). Compared with controls, respi- poglycemia causes sudden death is critically important in
ration rates were lower in the a/b-, a-, and b-blocker- ordertofindtreatmentstrategiesthatcouldprotectat-risk
infused rats during severe hypoglycemia (P , 0.001) patients. Based on ECG anomalies reported during mod-
(Table1)anddroppedprecipitouslyimmediatelypriorto erate hypoglycemia (26), it has been speculated that the
death. “deadinbedsyndrome”maybemediatedbyhypoglycemia-
MeansystolicBPduringseverehypoglycemiaforcontrol, inducedfatalarrhythmias.Inthecurrentstudy,itisshown
a/b-,a-,andb-blocker-infusedratswas133614,140610, for the first time that fatal cardiac arrhythmias occur
129 6 8, and 112 6 4 mmHg, respectively (Supplementary during severe hypoglycemia and can be reduced by ICV
Fig. 3D). Mean diastolic BP during severe hypoglycemia glucose infusion and prevented by b-adrenergic blockade,
was91616,9069,9268,and7764mmHg,respectively indicating that brain neuroglycopenia and the striking
(Supplementary Fig. 3E). Systolic and diastolic BP was sympathoadrenal response mediate fatal cardiac arrhyth-
significantly lower in b-blocker-infused rats (P , 0.001). mias during severe hypoglycemia (Fig. 7A).
6 DIABETES diabetes.diabetesjournals.org
C.M.RENOANDASSOCIATES
FIG.5.Severehypoglycemicclampwithadrenergicblockade(study5).A:Controlratshadamortalityrateof33%(n=12)inresponsetosevere
hypoglycemia.a-Blockadehadnoeffectonmortality(50%;n=6).Deathwaspreventedinbotha/b-blocker-infused(n=13)andb-blocker-infused
(n=5)rats(*P<0.029,Fisherexacttest,Freeman-Haltonextension).B:Glucoselevelswereevenlymatchedthroughouttheclamp.Glucosewas
clampedbetween10and15mg/dLfor3h.C:Meanglucoseinfusion(Ginf)rateduringseverehypoglycemia.a/b-Blockadesignificantlyincreased
glucoseinfusionrate.a-Blockadeandb-blockadealonedidnotdiffercomparedwithcontrols,buta-blockerratshadalowerglucoseinfusionrate
compared with a/b-blocker-infused and b-blocker-infused rats (P < 0.05, ANOVA). D: Heart rate was consistently lower in a/b-blockade and
b-blockaderatsthroughouttheclampcomparedwithcontrolanda-blockaderats(*P<0.001forindividualtimepoints,ANOVA).E:QTclength
wassignificantlyincreasedinallgroupsduringseverehypoglycemia(P<0.001,ANOVA).Combineda/b-blockadesignificantlybluntedtheQTc
prolongationcomparedwithcontrols(*P<0.001formeanQTcduringseverehypoglycemia,ANOVA).Dataexpressedasmeans6SEM.CON,
control.
Intheseexperiments,diabetespersenearlydoubledthe maladaptations associated with insulin deficiency, it may
mortality risk associated with severe hypoglycemia. Un- be that the greater absolute decrement in blood glucose
controlleddiabetesishypothesizedtoincreaseriskoffatal levelsindiabetic(348to12mg/dL)versuscontrol(88to12
cardiac arrhythmias via altering the myocardium compo- mg/dL) rats may have contributed to increased mortality.
sition(32)andpotentialcardiacmechanicaldefects(33).It Finally,although STZdiabeticrats havebeen showntobe
is unclear whether these previously reported chronic at higher risk for arrhythmias (34), in the absence of ECG
maladaptivechangesinducedbydiabetesweremanifestin data from these preliminary experiments the cause of in-
the currents studies because the duration of diabetes in creased mortality rates in diabetic rats remains unknown.
the current study was only of 2–4 weeks’ duration. It was Recurrent hypoglycemia significantly reduced severe
speculatedthatrelativewhole-bodypotassiumdepletionin hypoglycemia–induced mortality. These findings are con-
diabetic rats might predispose to arrhythmic deaths. Al- sistent with a preconditioning effect of recurrent hypo-
thoughsystemicpotassiumlevelsindiabeticratswerenot glycemia to limit severe hypoglycemia-induced seizures,
different from those in controls, a potential role for an braindamage,andcognitivedysfunction(28).Theblunted
alteredcardiacmyo-cellularpotassiumgradientindiabetic catecholamine response in the recurrently hypoglycemic
rats could not be ruled out. Aside from potential chronic ratswouldrenderthemmorepronetoseverehypoglycemia
diabetes.diabetesjournals.org DIABETES 7
SEVEREHYPOGLYCEMIA–INDUCEDSUDDENDEATH
FIG. 6. Severe hypoglycemia–induced arrhythmias and catecholamine levels during adrenergic blockade (study 5). A: Compared with that in
controlrats,PVCfrequencyduringseverehypoglycemiawassignificantlydecreasedina/b-andb-blockaderats(*P<0.001,ANOVA),withatrend
fordecreasedfrequencyina-blockaderats.B:Second-degreeheartblockfrequencywassignificantlyreducedina/b-blockaderats,withonlyone
ratexperiencingsecond-degreeheartblock.a-andb-Blockadealonetrendedtoreducefrequencyofsecond-degreeheartblock,butthiswasnot
significantlydifferentcomparedwithcontrols.C:Incidenceofthird-degreeheartblockwas40%incontrolratsand33%ina-blockaderats.No
third-degreeheartblockoccurredina/b-orb-blockaderats.(*P<0.02,Fisherexacttest,Freeman-Haltonextension).D:Epinephrinelevels
increasedsignificantlyinallgroupsduringseverehypoglycemia.Theb-blockadegrouphadsignificantlyhigherepinephrinelevelscomparedwith
allothergroups(*P<0.01,ANOVA).E:Norepinephrinelevelsincreasedsignificantlyinallgroupsduringseverehypoglycemia.Norepinephrine
levelsinthea-blockadegroupweresignificantlyhighercomparedwithallthreeothergroups.(*P<0.001,ANOVA).F:Glucagonareaunderthe
curve(AUC)wassignificantlyhigherwitha-blockadeandsimilarwitha/b-andb-blockadecomparedwithcontrols(*P<0.005,two-wayANOVA).
n=5–13.Dataexpressedasmeans6SEM.CON,control.
but, paradoxically, lessvulnerableto severe hypoglycemia- heart rate, absent Q waves, and absent isoelectric ST-
induceddeath.Thisputativebluntedadrenergicmechanism segments (35). Supporting our assertion that the reported
leading to improved survival is supported by both the re- rat ECG changes truthfully reflect human ECG responses
duced norepinephrine response in the ICV glucose study, during hypoglycemia are clinical ECG studies that note
as well as the reduced cardiac stimulation noted in the similarhypoglycemia-inducedQTcprolongation(20,21,26,36),
b-blockade study, with both studies noting a reduction of sinus bradycardia (18,36), and multifocal ventricular
arrhythmiasanddeath.IntheabsenceofECGrecordings, ectopies(18).Furthermore,thereductioninheartrateand
though, the extent to which cardiac arrhythmias were BP with b-blockade in rats during hypoglycemia corrobo-
reduced by antecedent recurrent hypoglycemia remains rates with responses seen in humans (26). Therefore, the
unknown. rat ECG changes presented here, that suggest b-blockers
Compared with human ECG recordings, rat ECGs are have the potential to prevent severe hypoglycemia–
characterized by shorter interval durations, more rapid induced sudden death, may have clinical applicability.
8 DIABETES diabetes.diabetesjournals.org
C.M.RENOANDASSOCIATES
TABLE1
Arterial blood gases, electrolytes, and respiratory rates during basal, severe hypoglycemia, and predeath conditions for adrenergic
blockadestudy(study5)
Control a/b-Blockers a-Blocker b-Blocker
Potassium(mmol/L)
Basal 4.460.14 4.060.11 4.760.1 5.060.14
Severehypoglycemia 3.1260.18* 2.8460.08* 3.0160.21* 3.7460.05*†
Predeath 3.660.48 N/A 4.260.21 N/A
Oxygensaturation,%
Basal 9661.9 9760.28 9760.3 9760.32
Severehypoglycemia 9760.34 9860.15 9860.36 9860.14
Predeath 26612* N/A 3560.28* N/A
Carbondioxide(mmHg)
Basal 3261.7 3360.69 3260.87 3460.91
Severehypoglycemia 3261.4 3860.97*† 3662.2 4262.2*†
Predeath 4862.5* N/A 63611A N/A
Respiration (breaths/min)
Basal 8664 8864 9668 8664
Severehypoglycemia 7663 5162*† 6668* 5462*†
Predeath 2868* N/A 8 N/A
Dataaremeans6SEM.Potassiumlevelsdecreasedinallgroupsduringseverehypoglycemia.b-Blockaderesultedinhigherpotassiumlevels
duringseverehypoglycemiacomparedwithcontrols.Oxygensaturationremainednormalinallgroupsthroughoutseverehypoglycemia.In
controlanda-blocker-infusedrats,oxygendecreasedandcarbondioxidelevelsincreasedjustpriortodeath.Respirationwassignificantly
decreasedduringseverehypoglycemiaina/b-,a-,andb-blocker-infusedratscomparedwithbasal.Respirationdecreasedjustpriortodeathin
controlanda-blocker-infusedrats.Ofthea-blocker-infusedratsthatdied,predeathrespirationwasonlynotedfor1rat;therefore,thereare
no SEM values and the data are not significant. Note that since a/b- and b-blocker-infused rats did not die, predeath values were not
applicable.*P,0.05vs.basal,†P,0.05vs.control.n=5–13/group.
A key concept highlighted in the findings is the impor- per se does not lead to cardiac arrhythmias and death.
tance of the duration of severe hypoglycemia, and not Takentogether,thesestudiesindicatethatQTcprolongation
hypoglycemic nadir alone, as a critically important com- is a predictive marker of ensuing potentially lethal cardiac
ponent in determining arrhythmias and sudden death. arrhythmias but alone is not sufficient to cause cardiac
Indeed a consistent, time-dependent sequence of events arrhythmias.
leading to severe hypoglycemia–induced sudden death A role for potassium in potentiating ECG anomalies
was observed (Fig. 7B). The ability to survive an episode during moderate hypoglycemia has been noted (25,26).
of severe hypoglycemia depended on the ability to escape Potassium supplementation in the current studies tended
fromsinustachycardia.Inratsthatdied,sinustachycardia to reduce mortality in nondiabetic and diabetic rats, in-
wasfollowedbyincreasedfrequencyofPVCsandsecond- dicating that hypokalemia increases risk of death due to
degree heart block, which culminated into third-degree severehypoglycemia.However,intheECGstudies,similar
heart block associated with bradycardia followed by the degrees of hypokalemia were noted in rats that lived and
rapidsequentialonsetofreducedcardiacoutput(notedby died, suggesting that in the setting of insulin-induced hy-
hypotension), respiratory depression, hypoxemia, hyper- poglycemia, the level of hypokalemia achieved was not
carbia,andacidosis.Thesensitivityandspecificityofsinus sufficient to cause lethal cardiac arrhythmias.
tachycardia (heart rate .400 bpm) as a predictor of mor- It was questioned whether cardiac arrhythmias could
tality was 88 and 74%, respectively, while third-degree have been secondary to respiratory depression. In these
heart block was 89 and 95%, respectively. The sensitivity studies,theoverallrespiratoryrateandoxygenandcarbon
and specificity of QTc prolongation (.200 ms) as a pre- dioxidelevelsdidnotchange.Thus,changesinrespiratory
dictor of mortality was 81 and 60%, respectively, while drivewerenotprimarycontributorstoseverehypoglycemia–
second-degree heart block was 94 and 53%, respectively. induced sudden death. In these studies, respiratory arrest
Hypoglycemic seizures were also noted to occur in most (noted by apnea, hypoxemia, and hypercarbia) only devel-
rats but were not associated with severe hypoglycemia– oped after fatal cardiac arrhythmias.
induced mortality, consistent with other studies demon- The hypothalamus also plays a critical role in regulat-
strating survival after hypoglycemia-induced seizures ingthehypoglycemiccounterregulatoryresponse(37)and
(28,29). heart contractility (38). The reduced mortality rate with
Moderate hypoglycemia has been shown to lead to ICV glucose infusion demonstrated that the brain is im-
QTc prolongation (ventricular dispersion) and be portant in reducing cardiac arrhythmias during severe
proarrhythmic (18–21,26). In the present studies, QTc hypoglycemia. Of note, the intracerebroventricular glu-
lengtheningwasassociatedwithincreasedriskofmortality. cosedoseinfusedwasnotenoughtoblunttheepinephrine
With adrenergic blockade during hypoglycemia, blunted response,consistentwithotherstudies(37).Thus,evenin
QTc prolongation may have contributed to reduced in- the presence of elevated epinephrine, prevention of brain
cidenceofarrhythmiasandpreventionofdeath.However, neuroglycopenia reduces severe hypoglycemia–induced
hypoglycemia-induced QTc prolongation in both the cardiac arrhythmias and mortality.
b-blocker and the ICV glucose-infused rats was similar to Although adrenergic receptors located within the brain
theirrespectivecontrols,indicatingthat QTcprolongation have been shown to be involved in mediating the
diabetes.diabetesjournals.org DIABETES 9
SEVEREHYPOGLYCEMIA–INDUCEDSUDDENDEATH
FIG.7.Proposedmechanismofsuddencardiacdeathduetoinsulin-inducedseverehypoglycemia.A:Inthesettingofinsulin-inducedhypoglycemia,
alteredlevelsofcirculatingnutrients(glucose),electrolytes(potassium),andhormones(insulin,epinephrine,andnorepinephrine)mayhaveadi-
rectarrhythmogeniceffectontheheart.Additionally,sincehypoglycemiaisdetectedatthelevelofthebrain,indirect(i.e.,centralnervoussystem)
effectsofhypoglycemiaalsocontributetofatalcardiacarrhythmiasviatheefferentnervoussystemandlocalreleaseofnorepinephrineatnerve
terminalswithintheheart.Autonomicinnervationoftheadrenalglandresultsinepinephrinerelease.Bothepinephrineandnorepinephrineactat
thelevelofthehearttoincreaseriskoffatalcardiacarrhythmias.Inthesettingofinsulinadministration,hypokalemiaisalsothoughttocontribute
tocardiacarrhythmias.DirectandindirectactionsontheheartleadtoQTcprolongation.Hypoglycemia-inducedsuddencardiacdeathresultsfrom
enhancedadrenergicsignalingattheleveloftheheartthatleadstosinustachycardia.Tachycardiaisfollowedbythird-degreeheartblockthat
culminatesintoafatalbradycardicrhythmcausingcardiorespiratoryfailureandsuddendeath.Red,circulatingglucose,electrolytes,andhormones;
blue,autonomicinnervations.B:Inresponsetohyperinsulinemicconditions,yetbeforehypoglycemiastarts,hypokalemiaandQTcprolongationwere
notedtooccur.Duringmoderatelevelsofhypoglycemia(;40mg/dL),PACsoccuroccasionally.Atthestartofseverehypoglycemia(15mg/dL),
epinephrinepeaks.Asseverehypoglycemiaprolongs,hypokalemiaworsens,QTcprolongationpeaks,andcardiacarrhythmiasdevelop(PVCsand
first-andsecond-degreeheartblock).Norepinephrinepeaks;1hintoseverehypoglycemiaandisfollowedbysinustachycardia.Iftachycardiais
followedbyareturntonormalsinusrhythm,theratsurvives(blueboxes).IftachycardiaisfollowedbyanincreaseinthefrequencyofPVCsand
second-degreeheartblock,thenthird-degreeheartblockassociatedwithbradycardiawillmanifestandleadtosuddencardiacdeath(redboxes).
10 DIABETES diabetes.diabetesjournals.org