Arrhythmia and cardiac defects are a feature of spinal muscular

13 Pages·2010·0.56 MB·English

Human Molecular Genetics, 2010, Vol. 19, No. 20 3906–3918
doi:10.1093/hmg/ddq330
Advance Access published on August 6, 2010
Arrhythmia and cardiac defects are a feature
of spinal muscular atrophy model mice
Christopher R.Heier1,2, RosalbaSatta1,2, CathleenLutz3 and Christine J.DiDonato1,2,∗
1Human Molecular Genetics Program, Children’s Memorial Research Center, 2300 Children’s Plaza, PO Box 211,
Chicago,IL60614,USA,2DepartmentofPediatrics,FeinbergSchoolofMedicine,NorthwesternUniversity,Chicago,
IL 60611, USA and 3The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609, USA
D
o
w
n
ReceivedJune3,2010;RevisedJuly13,2010;AcceptedJuly30,2010 lo
a
d
e
d
Proximal spinal muscular atrophy (SMA) is the leading genetic cause of infant mortality. Traditionally, SMA fro
m
hasbeendescribedasamotorneurondisease;however,thereisagrowingbodyofevidencethatarrhythmia h
and/or cardiomyopathy may present in SMA patients at an increased frequency. Here, we ask whether SMA ttp
s
model micepossesssuchphenotypes. WefindSMA micesuffer from severebradyarrhythmia characterized ://a
c
byprogressiveheartblockandimpairedventriculardepolarization.Echocardiographyfurtherconfirmsfunc- a
d
e
tionalcardiacdeficitsinSMAmice.Additionalinvestigationsshowevidenceofbothsympatheticinnervation m
defectsanddilatedcardiomyopathyatlatestagesofdisease.Baseduponthesedata,weproposeamodelin ic.o
u
whichdecreasedsympatheticinnervationcausesautonomicimbalance.Suchimbalancewouldbecharacter- p
.c
ized by a relative increase in the level of vagal tone controlling heart rate, which is consistent with bradyar- o
m
rhythmiaandprogressiveheartblock.Finally,treatmentwiththehistonedeacetylaseinhibitortrichostatinA, /h
m
a drug known to benefit phenotypes of SMA model mice, produces prolonged maturation of the SMA heart- g
/a
beatandanincreaseincardiacsize.Treatedmicemaintainmeasuresofmotorfunctionthroughoutextended rtic
survivalthoughtheyultimatelyreachdeathendpointsinassociationwithaprogressionofbradyarrhythmia. le
-a
These data represent the novel identification of cardiac arrhythmia as an early and progressive feature of bs
murine SMA while providing several new, quantitative indices of mouse health. Together with clinical tra
c
casesthatreportsimilarsymptoms,thisrevealsanewareaofinvestigationthatwillbeimportanttoaddress t/1
9
as we move SMA therapeutics towards clinical success. /2
0
/3
9
0
6
/6
4
7
INTRODUCTION with severe symptomatic bradycardia, with four of these 61
8
patients dying as bradycardia progressed to cardiac standstill b
y
Spinal muscular atrophy (SMA) is the leading monogenic (5). In addition to these severe patients, instances of heart g
u
cause of infant mortality (1). It is caused by insufficient block progressing to complete atrioventricular (AV) block e
s
dosage of the survival motor neuron (SMN) genes. Patients have been observed in adults with mild SMA (Type III) (6– t o
n
are homozygous for SMN1 deletion or loss of function 8). Arrhythmias such as these can develop from either auto- 0
4
mutations and possess varying copy numbers of the nearly nomicnervoussystem(ANS)defectsorfromcardiomyopathies. A
liodwentliecvaellsbuotf SalMteNrnaptrivoetelyinspprloicdeudcedSMfrNom2 gSeMnNe2(2re–s4u)l.t iTnhea AloNngSsbyemenptcoamsusaslulychoabssheyrvpeedrhiidnroSsMisAandpaptoieonrtcsi.rcCualasetiosntuhdaivees pril 2
0
disease pathology that is characterized by motor neuron loss confirm that such effects can manifest in tachyarrhythmia, 19
and skeletal muscle atrophy. However, a growing number altered RR interval variation, decreased sympathetic skin
of SMA case studies suggest cardiac and autonomic response and affected vasodilation in severe to intermediate
complications may either be associated with SMA or be SMA (9–11). Cardiomyopathies have also been observed to
uncovered as patient survival is extended by improved co-occur with SMA, in both Type I and Type III patients
medical intervention. (6,8,10,12–17).Inaddressingthisissue,arecentstudyfocusing
Recently,acomprehensivesurveyof63severeSMA(TypeI) onsevereSMAbySchonebornetal.(18)foundthatthreeoutof
patientsattendingonepediatricclinicfoundthat24%presented four random patients possessing a single copy of SMN2 had
∗Towhomcorrespondenceshouldbeaddressed.Tel: [phone];Fax: [phone];Email:[email protected]
# The Author 2010. Published by Oxford University Press. All rights reserved.
For Permissions, please email: [email protected]
Human Molecular Genetics, 2010, Vol. 19, No. 20 3907
congenitalseptaldefects,acomplicationthatstatisticallyshould
onlyco-occurwithSMAin1outof50millionindividuals.
Withagrowingbodyofevidencethatarrhythmiaand/orcardi-
omyopathymaybepresentinSMApatientswithincreasedfre-
quency, we ask here whether or not SMA model mice show
suchphenotypes.WefindthatSMAmicesufferfromseverebra-
dyarrhythmia. A natural history of SMA heart rhythms reveals
that SMA mice present with bradycardia as early as 2 days of
age, before quantifiable onset of neuromuscular symptoms.
Their bradyarrhythmia is characterized by progressive heart
blockandreducedventriculardepolarizationefficiency.Further
Figure1.ConsciousSMAmiceexhibitbradycardia.Initialsurveyofseveral
investigations show evidence of both ANS defects and cardio-
SMAmicerevealedareductioninheartrateincomparisontocontrollitter- D
myopathy at the late stages of disease. Drug-based rescue of mates. Tracingshere come from representative ECGsobtainedin conscious ow
SMAsurvivaldemonstratestheutilityoftheelectrocardiogram SMA(top)andunaffectedheterozygous(bottom)mice.Sometremorofthe nlo
(ECG) as a quantitative, non-invasive biomarker, withthe pro- isoelectric line is present in all readings due to the mice being conscious a
d
and unrestrained. Individual waveforms are denoted by P, Q, R or S. Scale e
gression of bradycardia further predictive of death events in d
micethatexhibitextendedsurvivalwithoutprogressionofneuro- bar ¼100ms. fro
m
muscular phenotypes. Together, these data represent the novel h
identificationofcardiacarrhythmiaasanearlyandprogressive ttp
s
featureofmurineSMA.Withagrowingnumberofcasestudies ://a
andfindingsincomparablediseases,itisessentialthatwedeter- c
a
d
minethefullextentofcardiacinvolvementinpatientsaswemove e
m
forwardindevelopingtherapeuticscapableofextendingsurvival. ic
.o
u
p
.c
o
RESULTS m
/h
m
We utilized the SMND7 SMA mouse model (JAX line 5025) g
d(2e0v)eloanpdedibnyaLgereeetmale.n(t19w)iftohrpthreesveiosutusdsietus.diIensou(1r9la,2b1o)r,attohriys /article
model exhibited a phenotype apparent by postnatal day -a
b
(PND) 5 with an average survival of approximately 2 weeks. stra
Debilitating neuromuscular symptoms were overtly visible c
by PND8, at which point ambulation was severely affected t/19
and mice were grossly undersized. Around PND10, SMA /20
mice began to lose weight and exhibit a decreased level of /39
0
consciousness (LOC). All ECG values were obtained from 6
/6
resting heart rates recorded using a non-invasive method 4
7
described previously (22), with a sample size of ten mice Figure2.TransgenicbackgroundhasnoeffectonECG.ECGswererecorded 61
every2daysfromunaffected5025micethatwereeitherhomozygouswild- 8
unless otherwise noted. type (Smn+/+) or heterozygous carriers of a knockout allele (Smn+/2) at by
the Smn locus. All data were obtained from conscious, unrestrained mice, g
u
withheartratepresentedhereasmean+SEM.Nodifferenceswerepresent e
s
SMA mice exhibit bradyarrhythmia between genotypes at any time point in this or other ECG parameters (n¼ t o
10miceperdatapoint). n
To determine whether SMA mice show functional cardiac 04
abnormalities, we examined ECGs recorded from affected A
mutants and unaffected carrier mice. Readings at PND8, this model(SMN2+/+;SMND7+/+).Nodifferenceinheartrate pril 2
when mutant mice show overt SMA symptoms, revealed that between genotypes was detected at any time point assayed 0
1
bradyarrhythmia was present in SMA mice relative to unaf- (Fig.2),norwasanydifferenceobservedinPRintervalduration, 9
fected heterozygous littermates (Fig. 1). To further examine QRSintervaldurationorinmeasuresofheartratevariability(data
the electrophysiological nature and developmental time not shown). Comparison with the previous data obtained from
course of cardiac arrhythmia in SMA mice, we characterized wild-type FVB/N mice showed that all values and time points
the natural history of ECG waveform parameters throughout were consistent with established ECG values (22). Together,
thedevelopmentofSMAmiceandtheircontrollittermates. this demonstrated that there is no effect of transgene insertion
ECGwaveformsestablishedinpreviousworksareequivalent orSmnheterozygosityonheartrateorotherECGwaveforms.
in males and females during neonatal time points (22,23). To
provide further baseline data in our control genotypes of mice,
Bradyarrhythmia with progressive heart block
we recorded ECGs from ages PND2 to PND14 in unaffected
and impaired conduction
mice that were either wild-type at the endogenous Smn locus
(Smn+/+) or heterozygous at the endogenous locus (Smn+/2). Heart rates were reduced in SMA mice at all time points
All mice were homozygous for the two transgenes present in examined (Fig. 3A). At PND2, the earliest age we could
3908 Human Molecular Genetics, 2010, Vol. 19, No. 20
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/h
m
g
/a
rtic
le
-a
b
s
tra
c
t/1
9
/2
0
/3
9
0
6
/6
4
7
6
1
8
b
y
g
u
e
s
t o
n
0
4
A
p
ril 2
0
1
Figure3.LifehistoryofarrhythmiainSMAmice.RestingECGsrecordedforSMAandunaffectedlittermatesevery2daysrevealsignificantphysiological 9
differencesinwaveforms.Allmicewereconsciousandunrestrained.(A)Heartrate,(B)PRinterval,(C)QRSintervaland(D)pNN6msvaluesareexpressed
asmean+SEM.(∗P,0.05,∗∗P,0.005,∗∗∗P,5E26,n¼10miceperdatapoint).(E)RepresentativeECGfromaPND14mousethatdisplayedprogressive
heartblockmanifestinginskippedbeats.
reliably assay, SMA mice had an average heart rate of rates that were approximately 100b.p.m. lower than
378+50b.p.m., whereas unaffected littermates had a unaffected mice. At PND10, the heart rate of SMA mice
heart rate of 451+22b.p.m. (n¼10 per genotype, P≤ became further reduced compared with control mice
0.0005). From PND4 to PND8, SMA heart rates increased (505+51 versus 642+24b.p.m., respectively). This
at a steady rate of approximately 25b.p.m. each day, con- deficit progressed at PND12 and PND14 where there was
sistent with littermate controls as well as previous studies a large drop from previous time points in the heart rate of
characterizing the development of neonatal heart rhythms SMA mice. This resulted in heart rates in SMA mice that
(22). Throughout this time, SMA mice exhibited heart were approximately half those in unaffected mice as they
Human Molecular Genetics, 2010, Vol. 19, No. 20 3909
D
o
w
n
Figure4.EchocardiographydeficitsinSMAmice.EchocardiographywasperformedonsedatedPND6mice.SMAmicehaddepressedvaluesin(A)PVpeak lo
a
velocity(P,0.005)and(B)PVpeakgradient(P,0.05).(C)Heartratewasdecreasedinsedatedmicewhencomparedwithconsciousmice.Consistentwith d
e
datafromconsciousmice,SMAmiceshowedreducedheartratescomparedwithcontrollittermates(P,0.005;n¼6–7micepergenotype). d
fro
m
h
approached death endpoints (368+119 versus 657+ Functional deficits detected by echocardiography ttp
s
83b.p.m.; P,5E211). Together, these results elucidate Echocardiography was performed on PND6 mice to gain ://a
two points. First, a significant bradyarrhythmia phenotype c
further insight into the level of cardiac function in SMA ad
is present in SMA mice as early as PND2. Second, the e
mice. Deficiencies in blood flow out of the right ventricle m
ebnraddystaargrheyothfmliifaepirnogSrMesAsinmgitcoeciasrdcihaacrasctatenrdiszteidll.by severe woceirteydweatescstiegdnaifitcthanetplyuldmeocrneaarsyedvailnvealilnSSMMAAmmiiccee.rePleaatikvveetlo- ic.oup
Themostcommoncauseofbradyarrhythmiaisheartblock, all littermate controls (489.7+162.2 versus 1617.09+ .co
detected through the elongation of PR interval duration. In m
SMA mice, PR interval was significantly elongated at every 6v3er0s.u2sm1m1/.s8;+P1,0.50.m00m5H),g;asPw,as0.0p5e)ak(Fgigra.d4ieAntan(1d.1B+).0I.5n /hm
time point ECGs were recorded (Fig. 3B). As with heart both groups, heart rates measured while performing echocar- g/a
rate, PR interval showed two phases during the growth of diography were lower than previous measurements due to rtic
SMA mice. At PND4, SMA mice possessed a first-degree le
the mice being sedated. Consistent with earlier findings, -a
heart block characterized by elongated PR interval durations b
however, heart rates were significantly lower in SMA mice s
c3o6m.1p+are5d.7mwsit,hP≤un0a.f0f0ec1t)e;dthilsitwtearsmmataeisnta(i4n8e.d6+asP8.R8invteerrsvuasl t0h.0an05)in(Fcigo.ntr4oCls). (T2h4u3s+, t5h8e SveMrsAushe3a5r1t+sh2o4wbs.ps.imgn.;ifiPca,nt tract/1
development paralleled that of unaffected mice until PND10. reductions in both pumping efficiency and blood flow from 9/2
After this age, there was a sharp increase in the PR interval the right ventricle to the lungs. Together, these data establish 0/3
and breakdown of the cardiac rhythm as mice exhibited pro- that gross deficits in cardiac function are present in neonate 906
gressive heart block as they neared the end stage of survival. SMA mice. /64
Wedetectedasignificantelongationofthetimeofventricu- 7
6
lar depolarization for SMA mice through an increase in QRS 18
interval duration, beginning by PND4 (Fig. 3C). At this b
y
point, SMA mice possessed a QRS interval lasting 15.9+ Examination of SMA heart morphology and sympathetic gu
innervation e
4.9ms, compared with 11.9+3.0ms for unaffected litter- s
mates (P≤0.05). Ventricular depolarization times were pre- We examined heart to body size ratios to determine whether t on
cisely maintained in unaffected mice. SMA mice exhibited gross defects in heart morphology were present. Defects 04
larger variations in this parameter as they matured until such as cardiac hypertrophy or hypotrophy could affect A
p
around PND10, at which point there was a sharp increase as cardiac function and result in hemodynamic insufficiency ril 2
mice began to approach death endpoints. during periods of normally rapid development in growing 0
1
9
We found low values of heart rate variability in unaffected SMAneonates. After peaking atPND10,SMAmice typically
neonate mice, as previously observed in neonatal stages of lost weight each day before dying around 13 days of age.
humans and multiple mammalian species (24–27). However, Accordingly, we sacrificed mice at PND10 and PND13 to
as SMA mice neared the end stage of disease, they displayed examine the heart at these two points. General observation
a sharp spike in measures of heart rate variability (Fig. 3D). of heart morphology and histological sections (data not
This manifested in ECGs as heart rhythms that were erratic shown)wereentirelyconsistentwithvehiclegroupspresented
and/or contained skipped beats (Fig. 3E). Ultimately, SMA later, with SMA mouse hearts appearing smaller and flaccid
mice displayed adjacent RR intervals that were highly incon- while lacking defined shape with gross attenuation of walls.
sistent, resulting in average pNN6 values of (cid:4)50–60% for Here, SMA mice sacrificed at PND13 lost an average of
PND12 and PND14 mice. Thus, roughly half of the consecu- 21+5% of their body weight, whereas unaffected genotypes
tiveheartbeatsinendstageSMAmicedifferedindurationby gainedanaverageof26+5%(Fig.5A).WefoundatPND10
an amount significantly exceeding the threshold set for a thatSMAmicedisplayedahearttobodyweightratiocompar-
regular rhythm. abletothatofunaffectedlittermates.AtPND13,thatratiowas
3910 Human Molecular Genetics, 2010, Vol. 19, No. 20
D
o
w
n
lo
a
Figure5.Lossofcardiacmassinendstageoflife.(A)Bodymassdecreased21+5%inSMAmicefromPND10toPND13,whereasthatofunaffectedlit- d
termatesincreasedby26+5%(∗P,0.05,n¼7micepergenotype).(B)Heart:bodymassratioswerepreciselymaintainedinbothunaffectedandSMAmice, ed
evenasSMAmicelostonefifthoftheirbodyweight.(C)Heart:bodylengthratiosweremaintainedbetweengenotypesandtimepoints. fro
m
h
maintained, with values comparable between both groups and of ECG as a non-invasive biomarker. Additionally, we ttp
s
ages (Fig. 5B). Equivalent ratios of heart to body length were wanted to gain insight into the effects that a drug established ://a
also observed between both groups and time points (Fig. 5C). to display positive-acting effects in SMA mice has on novel c
a
These data suggest first that heart to body size is precisely cardiac phenotypes we observed, alongside the effects on de
m
maintained in both SMA and heterozygous mice, so the motor function and survival. For these studies, we chose to ic
decline in SMA body mass does not appear to result from usetrichostatinA(TSA).TSA,ahistonedeacetylaseinhibitor, .ou
p
the developing SMA mouse ‘outgrowing’ its cardiac size nor haspreviouslybeen showntoincrease SMNtranscriptionand .c
o
from relative cardiac hypertrophy. Second, the hearts of protein both in vitro and in vivo and to provide benefits to m
SMA mice appear to be losing mass at the end stages of motor function and survival of SMA model mice (21,28). /hm
disease, since their mass ratio is precisely maintained even Here, SMAandunaffectedheterozygouslittermatemicewere g
/a
as the mice lose one-fifth of their body mass. injectedoncedailywitheitherTSAat10mg/kgoranequivalent rtic
Heart rate in healthy mice is controlled by a balance of the volumeofvehicle(DMSO)beginningatPND2.ECGwasper- le
-a
sympathetic and vagal components of the ANS. Sympathetic formedonevennumbereddaysbyareaderblindedtogenotype, b
s
nerve activity accelerates the heart rate, whereas the vagal drugidentityandphenotypingstudies,withasamplesizeof10 tra
c
systemdeceleratesit.Arrhythmiacharacterizedbybradycardia micepergroup.Motorfunctionwasassayedbymeasuringthe t/1
andprogressiveheartblockasdetectedherecanresultfroman time to right after the placement of each mouse on its back, 9/2
autonomic imbalance resulting from decreased sympathetic which was done on each odd numbered day to prevent the 0/3
tone relative to vagal tone. To investigate whether such a elevationofheartratebyaphysicalactivity. 90
6
decrease in sympathetic innervation may be present, we har- Asexpected,injectionwithTSAsuccessfullyimprovedsur- /6
4
vested hearts from PND12 mice and performed whole-mount vival, body weight and motor function of SMA mice (Fig. 7). 7
6
immunostaining with an antibody to tyrosine hydroxylase InductionofSMNlevelsandhistoneacetylationwasalsocon- 1
8
(TH),amarkerofsympatheticnerves(Fig.6).Wefounddistinct firmed through molecular analyses (Supplementary Material, b
y
differencesintheTHstainingpatternofSMAmiceversuscon- Fig. S1). Survival of SMA mice was increased to a median g
u
e
trols in both ventral and dorsal views. Ventral views of unaf- of 22 days when compared with 15.5 days for those injected s
fected hearts contained prominent neurons not observed in with vehicle (P,0.0005) (Fig. 7B). Body weights of SMA t on
SMA hearts (Fig. 6A and B). In dorsal views, we observed and unaffected groups were comparable at PND2 and PND4, 04
that TH-positive neurons from SMA mice seemed to have with vehicle-injected SMA mice weighing 2.5+0.5g versus A
p
fewer branches while also appearing thinner and less distinct unaffected mice weighing 2.7+0.4g at PND4 (Fig. 7C). ril 2
(Fig. 6C and D), though fine structure was still present upon Afterthis,SMAmicewerelowerinweightthanunaffectedlit- 0
1
highermagnification.Quantificationofthetwomostprominent termates(P,0.005).TSAproducedaweightbenefitinSMA 9
sympatheticneuronsvisibleinthedorsalviewoftheheartcon- mice beginning at PND10, at which point vehicle-injected
firmedthattherewerefewermajorneuronalbranchesdetected SMA mice began to lose weight, whereas TSA-treated SMA
in SMA hearts (Fig. 6E; n¼7 mice per group, P≤0.05). mice continued to gain/maintain weight until approximately
These results, in conjunction with the types of arrhythmia we PND20. Motor function benefits were observed in SMA
observed, suggest that there is a decrease in sympathetic tone mice from PND6 onwards upon TSA treatment (Fig. 7D).
presentinSMAmiceresultinginautonomicimbalance. Interestingly, TSA improved the motor function of SMA
micethroughrightingtimetowardslevelscomparabletounaf-
fected controls through PND21, though many reached death
DrugtreatmentimprovesSMAmotorfunctionthroughout
endpoints during this time or shortly after. Maintenance of
extended survival
motor function at late time points was further demonstrated
With SMA mouse models becoming a common system for using ambulation and negative geotaxis assays (Supplemental
preclinical drug studies, we next sought to explore the utility Material, Videos S1–S4). Thus, TSA-treated SMA mice with
Human Molecular Genetics, 2010, Vol. 19, No. 20 3911
extendedsurvivalwerefrequentlyfoundtodiewithoutexperi-
encing a visible decline in motor function.
TSA prolongs heart beat maturation until death
of SMA mice
Heart rates of SMA mice were significantly elevated at
PND10–14 upon TSA treatment, along with concurrent
decreases in PR and QRS interval durations (Fig. 8). These
benefits appeared to be maintained until approximately
PND20,afterwhichweobservedtheprogressionofbradycardia
associated with mice reaching death endpoints. In regard to D
heart rate, we observed no difference between treated and ow
n
untreated SMA mice before the age of PND10 (Fig. 8A). lo
a
Before this point, both groups displayed slopes of heart rate d
e
growth that were parallel to those of unaffected mice. At d
PND10 andonward, however, thevehicle groupwasfoundto fro
m
declinesignificantly,whereasdrug-injectedmicecontinuedto h
exhibit a rate of increase comparable to unaffected mice for ttp
s
several more time points. In unaffected littermates, ://a
TSA-injected mice showed a trend of lower heart rates; ca
d
however, this difference was not significant and was present e
m
betweenthesegroupsatPND2,beforethefirstinjection. ic
.o
First-degreeheartblockwasstillpresentinSMAmiceupon u
p
TSA treatment, though we did not observe the same pro- .c
o
gression seen in vehicle or untreated SMA mice (Fig. 8B). m
/h
We found a sharp increase in PR interval duration at m
g
PND22, but did not observe highly erratic and/or skipped /a
beats as were detected in untreated SMA mice. Consistent rtic
withalackofbreakdowninheartrateregularity,weobserved le-a
noincreaseinmeasuresofheartratevariabilityinTSA-treated bs
SMA mice over that found in unaffected littermates of either tra
c
treatment group at any time point (Fig. 8C). t/1
9
Timeofventriculardepolarizationimprovedupondrugtreat- /2
0
ment. Values for QRS interval duration in treated SMA mice /3
9
were equivalent to those in unaffected littermates from 0
6
PND10 to PND14 (Fig. 8D). However, at the end stage of /6
4
life,thiswasnotmaintained,asSMAmiceshowedanincreased 76
1
QRSintervalwhennearingadeathendpointoverunaffectedlit- 8
b
termates(15.1+5.4versus9.38+0.6;P,0.05). y
g
u
e
s
TSA treatment increases the size of SMA hearts t o
n
0
We next investigated the cardiac morphology in SMA mice 4 A
andourtreatmentgroupsatPND12,whenclearelectrocardio- p
graphic differences are present. Observations of heart mor- ril 2
0
phology in vehicle-injected mice were consistent with those 1
9
in mice that received no injections. Upon dissection, hearts
Figure6.SMAheartsshowadecreaseinsympatheticstaining.Whole-mount from vehicle-injected SMA mice appeared grossly undersized
immunostaining with an antibody to TH was used to visualize sympathetic andflaccid(Fig.9A).Incontrast,TSA-treatedSMAmicepos-
innervation.HeartswereharvestedfromPND12SMAmiceandunaffectedlit-
sessedlargerheartsthatwerecloserinsizetounaffectedmice
termates.(A)Reducedsympatheticpatterningwasvisibleinventralviewsof
injected with TSA. In addition, both groups treated with TSA
representativehearts,furthervisiblebylackofprominentneuronsinenlarged
views of the right ventricle (B). Dorsal views of representative hearts dis- were smaller than unaffected mice injected with vehicle (P,
played a decrease in neuronal branching (C) and neurons that appeared to 0.005). Hematoxylin and eosin staining of hearts from
bethinnerwithareductioninstainingintensity(D).Finestructurewasstill vehicle-injected SMA mice revealed gross attenuation of
visible in high magnifications. (E) Quantification confirmed a reduction of
walls and dilated cardiomyopathy (Fig. 9B and C). Often,
neuronalbranching.Valuesrepresentmean+SEMoftotalobservedbranches
counted from the two largest neurons present in dorsal views of the heart this resulted in hearts that appeared misshapen or ‘deflated’
(∗P,0.05, n¼7 hearts per genotype). Scale bars are equivalent to and again was consistent with histological observations from
2000mm(A)or1000mm(B–D). SMA mice receiving no injections (data not shown). Upon
3912 Human Molecular Genetics, 2010, Vol. 19, No. 20
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/h
m
g
/a
rtic
le
-a
b
s
tra
c
t/1
9
/2
Figure7.TSAimprovesmotorfunctionthroughoutextendedsurvival.TSAwasadministeredtoSMAmiceandunaffectedheterozygouslittermatesat10mg/kg 0/3
byIPinjectiononcedailybeginningatPND2.Controlgroupsreceivedanequivalentvolumeofvehicle(DMSO).(A)Micethatareunaffectedinjectedwith 9
0
vehicle(top),unaffectedinjectedwithTSA(middle)andSMAtreatedwithTSA(bottom)arepictured.NoSMAmiceinjectedwithvehiclelivedbeyond16 6
/6
days. (B) Kaplan–Meier survival curve of treatment groups. TSA treatment extended SMA survival to a median of 22 days, compared with 15.5 days for 4
vehicle-injectedmice(log-ranktest,P,0.0005,n¼10micepergroup).(C)BodymassofmicewascomparablebetweenSMAandunaffectedlittermate 76
groups at PND2 and PND4. Significant reduction in weight of SMA mice compared with unaffected controls was observed beginning on PND6 (∗P, 18
0.005,n¼10micepergroup).(D)TimetorightforSMAmiceimprovedtovaluescomparablewithunaffectedlittermatesuponTSAtreatment.Thisimprove- b
y
mentwasmaintainedasmicereachedtimepointscorrespondingtodeathendpoints. g
u
e
s
TSA treatment, SMA hearts more closely resembled those of clinic, case studies are mounting, which suggest that this t o
n
unaffected mice injected with TSA. may be true in SMA patients as well. We find that SMA 0
4
TreatmentwithTSAresultedinheartsthatwerelargerinpro- mice suffer from congenital bradyarrhythmia and heart A
p
portiontobodysizethaninmiceinjectedwithvehicle(Fig.9D). block. The end stage of their life is characterized by the pro- ril 2
This was true for the heart:body mass ratio of both SMA and gression of these two features to cardiac standstill. Patients 0
1
unaffected mice (P,0.05, n≥5 hearts per group). Drug- in recent studies have shown similar cardiac arrhythmias, 9
treated SMA mice also showed an increase in the ratio of the autonomic defects and/or cardiomyopathies at what appears
lengthoftheirhearttotheirbody(P,0.05)(Fig.9E).Atthis to be developing into a higher than chance co-occurrence
time, SMA mice treated with drug were continuing to gain (Table 1), with severe symptomatic bradycardia being
body weight. Thus, SMA mice appear to have hearts that are observed in severe forms of SMA and progressive heart
larger both in weight and in volume when treated with TSA block also found in mild or adult onset cases of SMA.
and do not appear to lose cardiac mass at this stage of their
lifeaswasfoundforuntreatedmiceatPND13.
Natural history of SMA ECGs and clinical relevance
ECG studies of SMA mice throughout development reveal
DISCUSSION
severe bradycardia characterized by progressive heart block
Here, we provide the first evidence that cardiac arrhythmias and impaired ventricular conduction. At early time points,
and abnormalities are a feature of SMA model mice. In the heart rate parameters are elongated but appear to mature at
Human Molecular Genetics, 2010, Vol. 19, No. 20 3913
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/h
m
g
/a
rtic
le
-a
b
s
tra
c
t/1
9
/2
0
/3
Figure8.ECGofTSAtreatmentgroups.ExtendedmaturationofECGwaveformswasobservedinSMAmicetreatedwithTSA.ECGswereobtainedevery2 90
dmaeyasnf+romSEPMND(∗2Pto,P0N.0D11,4∗,∗wPi,th0a.l0l0a1n,im∗∗a∗lPsc,on1sEci2o5u,sna¼nd1u0nmreisctreaipneerdd.a(tAa)pHoienatr)t.rate,(B)PRinterval,(C)pNN6msand(D)QRSintervalallexpressedas 6/64
7
6
1
8
b
y
rates that closely parallel those of unaffected littermates, metabolic stress. At the end stages of the disease in SMA g
u
despite the onset of large differences in both the rate of mice, we do see an exacerbation of ECG deficits consistent e
s
growth displayed by mice (setting in around PND3 and with such an event. Therefore, the ECG natural history we t o
n
PND4) and motor function. At the end stage of disease, provide may be accurately describing the SMA death 0
4
approximately 4–6 days preceding death, we see a sharppro- process of these mice. A
p
gbrreasdsyicoanrdoifaEaCndGhdiegfihceirtsdeagsrmeeicheeadretvbelloocpks,euvletirmeasytemlyptloemadaitnigc ril 20
Impaired cardiac function present by PND2 1
tocardiacstandstill.AlthoughthoroughECGnaturalhistories 9
ofSMApatientsarecurrentlylackingintheliterature,several Quantitativeconsistencyingagingtheonsetofdiseasepheno-
groups have noted precisely these two features of severe types in fragile groups of mice such as those studied here can
symptomatic bradycardia and progressive heart block in both bedifficult.Thiscanbefurthercomplicatedbysubjectivecalls
random pools and isolated cases of SMA patients (5–8). in applying motor scales and by inter-rater variability. Pre-
We find evidence of both myocardial and ANS deficits vious investigations describe the onset of neuromuscular
present in SMA mice. Cases of both autonomic and myocar- symptoms in the SMND7 mouse model as becoming apparent
dial defects are also found in clinical studies (9–11,16). aroundPND5(19–20,28,38–40).Othershavepredictedident-
Instances of each are also found in other neuromuscular and ifiablephenotypicdifferencesatearliertimepointswhenusing
motorneurondiseases(29–37).Respiratorydistress,typically largenumbers(greaterthan60)ofmice (41).Here wereadily
the ultimate cause of death in SMA and other neuromuscular detect impaired cardiac function using a system that is auto-
diseases, can further exacerbate arrhythmia and myocardial mated, non-invasive and requires no restraint or anesthesia.
defects by putting the body into states of oxidative and/or We find clear differences present in relatively small groups
3914 Human Molecular Genetics, 2010, Vol. 19, No. 20
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/h
m
g
/a
rtic
le
-a
b
s
tra
c
t/1
9
/2
0
/3
9
0
6
/6
4
7
6
1
8
b
y
g
u
e
s
t o
n
Figure9.CardiacmorphologyofSMAtreatmentgrouphearts.HeartsharvestedfromSMAmiceinjectedwithvehicleweresmallerthanthoseofunaffected 0
4
littermatesandtypicallyappearedflaccidorunabletoholdtheirshape.TSAtreatmentcorrespondedwithanincreaseinheart:bodyratioandanincreaseinthe A
absolutemassofSMAhearts.(A)Imagesofheartsrepresentativeforeachtreatmentgroup,fromatleastfiveheartspergroup.(BandC)Hematoxylinandeosin p
stainingofrepresentativeheartsectionsrevealedcardiomyopathyinvehicleSMAmicecharacterizedbygrossattenuationofwallsanddilationofventricles. ril 2
Sections in (B) are cut in a longitudinal orientation to display the four chambers of the heart. A black line represents the level at which additional hearts 0
1
werecutinacross-sectionaldirectionforimagesin(C),wherethepapillarymuscleusedasapointofreferenceisdenotedintheTSA-treatedSMAheart 9
byablackarrowhead.(D)Heart:bodymassratioswereincreasedinTSA-treatedgroupsofmice.(E)Heart:bodylengthratioswereincreasedinTSA-treated
SMAmiceonly(∗P,0.05,n¼5–6heartspertreatmentgroup).
ofmiceasearlyasPND2,withheartratesthatarereducedby Such a decrease represents a functional reduction in the
greater than 20% of littermate controls and PR intervals that hearts’abilitytomovedeoxygenatedbloodtothelungs.Inan
areelevatedby(cid:4)15%.Atthispoint,miceareweight-matched organism that is modeling a human disease where respiratory
anddonotdisplayconsistent,quantifiablemotorfunctiondef- distressisaprimaryhealthconcern,thiscouldrepresentasig-
icits. nificanthealthdeficitforSMAmice.Together,ourdatapresent
FunctionaldeficitofSMAheartsisfurthervalidatedbyecho- thefirstevidenceofimpairedcardiacfunctioninSMAmiceand
cardiographyatPND6,atwhichpointweobserveadecreasein establish new, quantitative phenotypes that are readily detect-
hemodynamics at the pulmonary valve of the right ventricle. ableasearlyasPND2.
Human Molecular Genetics, 2010, Vol. 19, No. 20 3915
Table1. Clinicalco-occurrenceofarrhythmia,cardiomyopathyandANSdefectswithSMA
Reference SMAtype Inclusioncriteriaand Typeofabnormality Finding #Observed(%oftotal)
samplesize
Bach(5) I All63patients Arrhythmia LOCw/severebradycardia,four 15(24%)
attending1clinic diedfromprogressiontostandstill
II Observed,not Arrhythmia LOCw/severebradycardia
reported
Roosetal.(7) Adultonset 2relatedindividuals Arrhyhtmia AVblock Both
Elkohenetal.(6) III 8randomcases Cardiomyopathy Fataldilatedcardiomyopathy, 1(13%)
reducedSFdetectedviaecho
Arrhythmia Symptomaticconductiondisease, 1(13%)
progressiveAVblock
D
Tanakaetal.(8) III 1isolatedcase Arrhythmia CompleteAVblock,atrialflutter o
w
Cardiomyopathy Fibrosisofrightventricle n
Schonebornetal.(18) I(1copy) 4randomcases CHD ASD,VSD,PDA,dilatedventricle 3(75%) lo
a
Arryhthmia Fetal,tachycardia,bradycardia d
e
I(2copies) 56randomcases Minorcardio PFO,PDA,VSD 6(11%) d
AraujoAdeetal.(10) I 2unrelatedcases Vascular Poorperfusion,digitalnecrosis 2(100%) fro
ANS Hyperhidrosis,absentreflexes 1(50%) m
Araietal.(9) IandII 9randomcases ACaNrSdiomyopathy AHSypDerahnyddraossyims,mdeectrriecaVseHdSSRsand 17((5707%%)) https
vasodilationresponse ://a
Arrhythmia Restingtachycardia 7(77%) ca
Arrhythmia AbnormalRRvariation 3(33%) de
Hachiyaetal.(11) I 2caseswith Arrhythmia AbnormalRRvariation 2(67%) m
extendedsurvival ANS Affectedvasodilationresponse 2(67%) ic.o
Arrhythmia Paroxysmaltachycardia u
p
Tanakaetal.(16) III 1isolatedcase Cardiomyopathy EM:lossofmyosinfilaments,Z-band .c
o
defects,leptomericfibrils m
/h
m
CHD,congenitalheartdisease;ASD,atrialseptaldefect;SF,shorteningfraction;VSD,ventricularseptaldefect;PDA,patentductusarteriosus;PFO,patent g/a
aforeradmeennotoevdailne;bVoHld,.ventricularhypertrophy;SSRs,sympatheticskinresponsetosound;EM,electronmicroscopy.ConditionsalsoobservedinSMAmodelmice rticle
-a
b
s
ECG of drug treated mice yet we were able to detect a progression of bradyarrhythmia tra
c
and weight loss. This suggests that progression of arrhythmia t/1
Maturation of neonatal heart rate in healthy mice is linear, maybebothpredictiveofadeatheventandseparatefromthat 9/2
with a rate of approximately 25b.p.m. each day (22). ofneuromuscularfunctioninSMAmice.Together,thesefind- 0/3
Untreated SMA mice deviate from this maturation pattern ings establish non-invasive ECG as a sensitive and quantitat- 90
6
beginning at PND10, with a decreased rate of growth and/or ive index of SMA mouse health. /6
4
crash in heart rate. Treatment of SMA mice with TSA cor- 7
6
rected this, resulting in SMA mice with extended periods of 18
normal ECG maturation rates. Mass and body temperature by
do not appear to cause the reduction in heart rate in SMA Conclusions and working model gu
e
mice, because at periods when the slope of SMA heart rate Based on the compilation of our data and SMA patient case s
maturation is comparable to healthy mice, these mouse reports,weproposethatlowlevelsofSMN,besidescontribut- t on
groups have very different growth rates and are known to ing to motor neuron loss, also result in a primary autonomic 04
possess opposite trends of body temperature (39). Specific imbalance. In the SMND7 mouse model of SMA, this mani- A
p
time points when groups here have comparable body mass festsasadecreaseinsympatheticinnervation.Suchanimbal- ril 2
but very different ECGs (PND2, 4 and 10) support this. ance would be characterized by a relative increase in vagal 0
1
9
Thus, these data show that discrepancies in ECG are not tone,whichisconsistentwiththemanifestationofbradycardia
simplyaconsequenceofdifferencesinsize,bodytemperature and progressive heart block observed here. Resulting func-
or growth rate of SMA mice. It instead suggests that, since tional deficits at critical developmental periods could prevent
cardiac function is impaired at a very early age, a decrease propermyocardialdevelopmentand/orinducesecondarymyo-
in the ability of the SMA heart to supply blood to rapidly cardial defects. Cardiac insufficiency could also slow the
developing neonates may actually cause or contribute to the growth rate of neonate mice and be compounded as mice,
stunted growth and body temperature development of SMA unable to maintain rapid development, enter states of cardior-
mice. espiratory/oxidative/metabolic distress. Such events are
UntreatedSMAmiceshowadecrease intheirLOCincon- observed in SMA mice around the ages of PND4 through a
junctionwiththeprogressionofbradyarrhythmia,heartblock, reduction in growth rate and of PND10 when there is a
weight loss and motor function decline beginning around decrease in LOC, onset of weight loss and exacerbation of
PND10.TSA-treatedSMAmicewithextendedsurvivaleven- arrhythmias. During these ages, maturation of autonomic
tually died without exhibiting a clear loss of motor function, neurotransmission and control of heart rate is still occurring