Abnormal cardiac function in the
streptozotocin-diabetic rat. Changes in active
and passive properties of the left ventricle.
S E Litwin, … , S Daugherty, S Goldman
J Clin Invest.
1990;
86(2)
:481-488.
https://doi.org/10.1172/JCI114734
.
To provide an integrated assessment of changes in systolic and diastolic function in
diabetic rats, we measured conscious hemodynamics and performed ex vivo analysis of left
ventricular passive-elastic properties. Rats given streptozotocin (STZ) 65 mg/kg i.v. (n = 14)
were compared with untreated age-matched controls (n = 15) and rats treated with insulin
after administration of STZ (n = 11). After 7 d, diabetic rats exhibited decreases in heart rate
and peak developed left ventricular (LV) pressure during aortic occlusion. After 26 d of
diabetes there were significant decreases in resting LV systolic pressure, developed
pressure, and maximal +dP/dt, whereas LV end-diastolic pressure increased and the time
constant of LV relaxation was prolonged. The passive LV pressure-volume relationship was
progressively shifted away from the pressure axis, and the overall chamber stiffness
constant was decreased. However, "operating chamber stiffness" calculated at end-diastolic
pressure was increased at 7 d, and unchanged at 26 d. LV cavity/wall volume and
end-diastolic volume were increased after 26 d of diabetes. Myocardial stiffness was unchanged
at both time intervals. All of the above abnormalities were reversed by the administration of
insulin. We conclude that the hemodynamic and passive-elastic changes that occur in
diabetic rats represent an early dilated cardiomyopathy which is reversible with insulin.
Research Article
Find the latest version:
Abnormal Cardiac Function in the Streptozotocin-Diabetic
Rat
Changes
in Activeand
Passive Properties of the Left Ventricle
SheldonE.Litwin,Thomas E.Raya, Peter G.Anderson, Sherry Daugherty, and StevenGoldman Department ofInternal Medicine, Veterans Administration Medical Center, Tucson, Arizona85 723; andUniversity Heart Center, University ofArizona College ofMedicine, Tucson,Arizona85724
Abstract
To
provide
an integrated assessment of changes in systolic anddiastolic function
indiabetic
rats, we measuredconscious
he-modynamics
andperformed
ex vivo analysis of left ventricularpassive-elastic properties.
Ratsgiven
streptozotocin (STZ)65
mg/kg i.v. (n = 14) were compared with untreated
age-matched controls (n
=15)
and rats treatedwith
insulin afteradministration
of STZ(n
=11).
After 7 d,diabetic
rats exhib-iteddecreases
in heart rate andpeak developed left ventricular
(LV) pressure during aortic occlusion. After 26 d of diabetes there were
significant
decreases in resting LV systolic pres-sure,developed
pressure, and maximal+dP/dt,
whereas LVend-diastolic
pressureincreased
and the time constantof
LVrelaxation
was prolonged. The passive LVpressure-volume
relationship
wasprogressively shifted
awayfrom
thepressureaxis,
andthe overall chamber stiffness
constantwas decreased. However,"operating
chamberstiffness"
calculated atend-dia-stolic
pressure wasincreased
at7d,
andunchanged
at26 d. LVcavity/wall
volume andend-diastolic
volume were increasedafter 26
dof diabetes.
Myocardial stiffness
wasunchanged
at both timeintervals.
Allof
the aboveabnormalities
were re-versed by theadministration of insulin.
Weconcludethat thehemodynamic and
passive-elastic
changes
that occur india-betic
ratsrepresent anearly
dilatedcardiomyopathy
which isreversible with insulin. (J. Clin.
Invest. 1990. 86:481-488.) Key words:diabetes
*diastole
*passive-elastic-
systolicfunc-tion
-ventricular relaxation
Introduction
Diabetes mellitus is associated with
thedevelopment
ofmyo-cardial
dysfunction
inthe absence
of coronary arterydisease,
systemic hypertension,
orvalvular heart disease(1, 2).
Experi-mental
modelsof
diabetes, especially
thestreptozotocin
(STZ)l
rat, have been usedtohelp
define thepathophysiology
of this disorder
(3, 4).
Thesestudies,
which utilized isolatedpapillary
muscle and
perfused
heartpreparations,
suggest thatThisstudywaspresentedinpartatthe Federation of American Societ-ies forExperimental Biology (FASEB) 1989 scientific session, New Orleans,LA.
Dr.Anderson is with theDepartment ofPathology, Universityof Alabama inBirmingham.
Addressreprintrequests to Dr. Litwin, Cardiology 11IC,Tucson VAMC, Tucson,AZ85723.
Received
for
publication 29August 1989 andinrevisedform21 February 1990.1.Abbreviations used in this paper: LV, leftventricular; STZ, strepto-zotocin.
TheJournal ofClinicalInvestigation,Inc. Volume86, August 1990, 481-488
the
cardiomyopathy of diabetes mellitus is associated
withab-normalities of
systolic performance andimpairment
ofleft ventricular (LV) relaxation.The
effect of diabetes
on LVdiastolic function
has not beenthoroughly examined.
Thestudies which
have been done have producedconflicting
results. For example, in diabeticdogs the diastolic pressure-volume relation
was reported toshift
toward the
pressureaxis, which would indicate
decreased chambercompliance, increased myocardial
stiffness, or both(5). Conversely, in the STZ
rat, thepressure-volume relation
of
theisolated left ventricle
isshifted
awayfrom
the pressureaxis,
suggesting increased
chambercompliance
(4). In neitherstudy
was theentire
diastolic pressure-volume relation
exam-ined,
nor wasmyocardial stiffness
measured. Furthermore, theinfluence of
LVrelaxation abnormalities
on the shape andposition
of
thediastolic pressure-volume relation
were nottaken into
account. Thus, theprevious studies
have notade-quately
resolved thequestion of whether diabetes is associated
with abnormalities
of thepassive-elastic properties of
the heart,independent of
changesin relaxation.
Our
goal
was tocharacterize
the changesin
diastolic
func-tion (i.e.,
LVrelaxation
andpassive-elastic behavior) with
dia-betesand
their relation
toalterations
insystolic function.
We measuredconscious hemodynamics,
open-chest LV developed pressure,and
exvivo
LVpressure-volume
relations. We pos-tulated that thepreviously described
decrease in LVcompli-ance
might reflect abnormalities of
thecontraction-relaxation
sequence,
rather
thanchanges
inintrinsic
passive-elastic
prop-erties of the
myocardium.
Wealsotreated
agroupof diabetic
rats
with insulin, after
aperiod of untreated diabetes,
toestab-lish
whetherthechanges incardiac function
werereversible.
This
is the first
study tointegrate hemodynamic
datafrom
conscious
animals, with
aquantitative
analysis of the
me-chanical
properties
of
the heart indiabetes.
Methods
Diabeteswasinduced byasingle tail veininjectionof STZat adoseof 65 mg/kg (SigmaChemical Co., St. Louis, MO) in male
Sprague-Dawley rats (220-250 g). Diabetic animalsweredivided into three
groups.GroupI(n = 5)wasstudied after7dof untreated diabetes.
Group II (n=9)wasstudied after 21-28 d (mean =26)of untreated diabetes.GroupIII(n= I
1)
wasuntreatedfor21d,thenweretreated with subcutaneousNPH humaninsulin (8U/kg twice daily) for21 d. Insulin wasnotgiven onthemorningof thestudy. Untreated, age-matchedrats(n= 15) servedasthe controlgroup. Allanimalswerefed standard ratlaboratory diet andwere allowed freeaccessto water.Animalswerehousedtwotothreeper cageandweremaintainedon a
12-hlight-dark cycle.
LVperformanceinconsciousrats. Toobtainconscious
measure-ments ofLV performance, ratswereanesthetized with inhaled
me-thoxyflurane.A
1-mm
diammicromanometer-tipped
catheter(Millar
insertion. The left femoral vein was cannulated with Silastic tubing which was advanced to the thoracic inferior vena cava. Both catheters were then secured and exteriorized to the dorsal cervical region. Rats were allowed to recover for 4 h. Left ventricular dP/dt was obtained from a differentiating circuit in the physiologic recorder (model 2400,
Gould, Inc., Cleveland,OH)with the
high-frequency
filter cutoffsetat100 Hz. In conscious rats 100-150 consecutive beats of LV pressure were recorded directly onto a computer (model AT, IBM Corp., White Plains, NY) and digitized at 1,000 Hz (6, 7). An example of an LV pressure recording in a conscious rat is shown in Fig. 1. These rats were then used for determination of peak developed LV pressure and the LVpressure-volume relationship as described below.
LVrelaxation. From the recordings of LV pressures and dP/dt, LV relaxation was determined in untreated diabetic, treated diabetic, and control rats using previously established methods (6, 7). Briefly, the decay of LV pressure with time can be closely approximated by the
exponentialrelation:
P=PBe +PA, (1)
where PB + PA is LV pressure at maximum negative LV dP/dt, PA is LVasymptote pressure, and a is the constant of the exponential rela-tion. The value ofris calculated by plotting the derivative of pressure with time versus pressure. LV pressures from 100-150 consecutive beats wereanalyzed. For each cardiac cycle maximum positive and negativedP/dt, peak systolic and end-diastolic pressure points were identified. The value of end-diastolic pressure for each cycle was sub-tracted fromeach pressure point on the pressure decay curve of that cycle beginningatmaximum negative dP/dt and ending at a point equal to end-diastolic pressure. The resulting values were then fit to the relation:
dP/dt=-a(P-PA)- (2)
ais calculated by the least squares method, andT= -I/a.
Open-chest determinations ofpeak developed LV pressure. After
conscioushemodynamicswererecorded, rats were anesthetized with 100mg/kg thiobutarbital(Inactin,Buikgulden Pharmaceutical,
Kon-stanz, FRG) i.p., intubated,andventilated with a volume-cycled respi-rator(Harvard Apparatus Co., Inc., S. Natick, MA).Aleft anterior thoracotomywasperformed andasnarewasplaced around the
as-cendingaorta.WhilerecordingLVpressure with thepreviously
posi-tioned Millarcatheter, anabrupt aortic occlusion wasproducedby suddentighteningofthesnare.Peakdevelopedpressurewasdefinedas themeanof thesystolicpressure minus the end-diastolic pressure of the first five stable beats after aorticocclusion.
.. i...5 s
150----r-
-----LVP I
(mm Hg) .
0 .. ,. .
-1-r-- --l-- -l .-~~...-:...-.
8000[
!..1.1::: .1...-:..:1. ,:
dP/dt
(mm Hg/s)
IsolatedLVpressure-volume relationship. Pressure-volumedata were recorded using methods previously described (6, 7). After the measurement of peak developed pressure, potassium chloride (2meq/
ml) wasinjected through the femoral vein catheter to arrest the heart. The heart was rapidly removed and the right ventricle was incised. A doublelumen catheter, attached to a pressure transducer (Statham 23 Id, Gould, Inc.) and an infusion pump (Sage model 341, Sage Instru-mentsDiv., Cambridge, MA) was passed into the left ventricle. The atrioventricular groove was identified andaligaturewaspassed around the heart and tiedtoisolate theleft atrium from the left ventricle. After gentleaspirationof theLVcavitytoremoveany residual blood, and to reduce pressure to -5 mm Hg, normal saline was infused at 0.70
ml/mininto the suspended left ventricle while pressure was recorded. Saline was infused until the pressure increased to 40 mm Hg. Two or three curves were obtained from each ventricle within 10 min of car-diac arrest, and before onset of rigor mortis.Anexampleof a pres-sure-volume recording is shown in Fig. 2.
Fromthe pressure-volume data recorded ex vivo, the overall chamber stiffness constant
KI(
was determined. Atleast 15 pairs of simultaneous pressure-volume points from each curve were digitized and stored. The pressure-volume data were fitted to the monoexpo-nential equation:P=Poe-V+PB
(3)
(mean correlation coefficient r = 0.98±0.002), where Pand Vare pressure and volumerespectively,and P0 isamodelingconstant.PB is defined below. Differentiating thisequationwith respecttovolume yieldsdP/dV= KC(P-
PB).
(4)
dP/dV is calculatednumericallyfrom the continuous pressure record-ing and plotted vs. pressure.KCand PB, the pressure intercept, are then calculatedby the method of least squares. This derivation allows for thepossibility ofthe curvepassingthrough thezeropressure-volume
point.For purposes ofcomparison,ventricular volumeswereindexed tobodyweight(millilitersperkilogram)andto LVwallvolume, be-causebodyweight indexingmaynotbeappropriateinanimals that lose weight (8).Operatingchamberstiffness
(C,)
wasdefinedasC.
=KI(P-PB),
(5)
where P= end-diastolicpressure measured in the conscious animal. Thiscalculationwasalso doneusingboth forms ofindexing.
Myocardial stiffness.
The incremental modulus(ENc)
and themyocardialstiffness constant
(K.)
werecalculatedassumingaspheri-cal geometry(innerandouterradii=aand
b, respectively)
for theleftventricle, according topreviously described methods(7, 9). Briefly, midwallradialstress(a)ateach pressurepointwascalculated from the measured values for LVcavityvolume and wall volume(mass/
1.05)
usingtheexpression:
(6)
a=
(3/2)P(V/V.Xbl/R3),
40o-Pressure
(mm Hg)
0-4~~-t4-~~~~ q + t -t * *
+... ...
.. +. -hit-.... ... ....-_t--.l .,,4,... .... I-il!I.|.!_ *
~~~~~~~~~~~~~~~~~~..r-
in*
~~~~~~~~~~~~~~~~~~...
;:t::t...u ..|, ,
*1'.:'; ... : --:: . z-. i... ;
[
-t
~~~~~~~~~~..
...,.'. _t.-...5
5s
Figure 1. LV pressure measured inconsciousnormalratwith
solid-state
micromanometer-tipped
catheter. Peakpositive
andnegative
dP/dt,and Tarederivedfrom these pressuremeasurements.
Figure 2.Example ofan exvivoLV
pressure-time recording.
LV vol-umeiscalculated from the time andthe knownrateofvolume in-flow. Chamber andmyocardial
stiffness constants, and LV end-dia-stolic volumearederived from thesecurves.TableLBody and Heart Weights and Glucose Levelsin Ratswith STZ-induced Diabetes
Control Group I Group II Group III
(n=15) (n=5) (n=9) (n=II)
Bodyweight (kg) 0.28±0.02 0.25±0.005 0.23±0.01 0.35±0.01
Leftventricle weight(g) 0.60±0.03 0.55±0.02 0.51±0.03* 0.67±0.02
Leftventricleweight/bodyweight(g/kg) 2.13±0.05 2.19±0.06 2.23±0.04 2.11±0.04
Left ventricle/tibia(g/cm) 0.188±0.004 0.146±0.006* 0.196±0.011
Glucose(mmol/liter) 9.1±0.6 31.8±0.7* 36.2±2.0$ 21.1±7.1
GroupI, untreated diabetes for7d; group II, untreated diabetes for 26 d; group III, insulin treatment for 26 d after 26 d of untreated diabetes.
*P<0.05 compared withcontrol; $ P<
0.01
comparedwith control.where
R =(a+b)/2, (7)
V=4/3 ira3, (8) and
V,
=4/3 rb3 - 4/3Ira3. (9)Theincremental modulus is obtained from the formula:
EINC
=1/2
Ao,(AR/R).
(10)The relation between the incremental modulus and stress is linear and can be described by the equation:
EINC=
Kmo+c, (11)where
Ki,
the slopeof the line, is the myocardial stiffness constant, and cis a constant.Cavity/wall volume. Ventricular cavityvolume atadistending
pressure of 10mmHgwasdetermined from the passive pressure-vol-umerelation. Aftercompletionof the pressure-volume recordings, the heart was separatedinto right ventricle, and left ventricle plus septum, andweighed. LV wall volume(Vw)wasdetermined from the mass of the leftventricle, suchthatVw= LVmass/l.05(density of muscle).
Operatingend-diastolic volumewascalculatedfromthe
pressure-vol-ume curvesusingthe measured end-diastolic pressure from the
con-sciousanimal.
LVweight/tibia length.Becausebody weight maybe an unreliable reference fornormalizingheartweightinsituations where bodyweight changes, LV weight was normalized toboth bodyweight andtibia length todetermine whether hypertrophy was present. Tibiaswere preparedusingpreviouslydescribed methods (8).
Glucoselevels. Blood was drawnjust priorto KCI arrest at the conclusion of each study.It wasimmediatelyspun, and theserum was frozen at-20'C for lateranalysis. Glucose wasmeasuredin the thawed serumusingaglucoseoxidase method(modelCX3glucose
analyzer, Beckman Instruments, Inc.,Fullerton, CA).
Histology. Heartswereimmersion fixed in 10% neutral buffered formalin. The heartwasseriallyslicedinto 3-mm sections
perpendicu-larto theaxis of the heart from apexto base. Thesesectionswere embedded in parafin, sectioned at 5
Mm,
andserial sectionswerestained withhematoxylin-eosinandwithGomori's aldehydefuchsin trichrome staintohighlight collagen.
Statisticalanalysis. Differences between the three diabetic and control groupsweredetectedusing
analysis
of variance. Where differ-ences werefound, significancewasdetermined withDunnett'stestformultiple comparisons with a singlecontrol (10). Values forLV chamberstiffnessconstantsandrweredetermined byregression
anal-ysis using the methodof least squares. Results are presented as
mean±SEM. Statisticalsignificancewasdefinedatthe P<0.05 level.
Results
Diabetes
wasconfirmed
by thefinding of glycosuria 2 d after theinjection.
None of the rats showed more than traceketon-uria.
Severe
hyperglycemia
was seenin
the untreateddiabetic
rats
(Table
I).Diabetic
animals had lower body weights andleft
ventricle weights,
butleft ventricle/body weight
wasun-changed compared
to control (Table I). Left ventricle/tibialength
wasdecreased in group IIanimals.
Conscious hemodynamics and peak developed pressure. Heart rate was
progressively
slowed, and systolic function wasimpaired in
thediabetic
rats (Table II). LVsystolic
pressure, andmaximal
positive
dP/dt were unchanged at 7 d (groupI),
but
decreased
at 4wk (group
II) of untreated diabetes. Peak
developed
pressure inopen-chest
rats wasdecreased
in group I,and
further decreased in
groupII.
LVend-diastolic
pressure had a tendency toincrease in
groupI,
and wassignificantly
increased in
group II.Indices
ofdiastolicfunction.
Relaxation ofthe left ventricle
in
conscious
rats, as assessed by thetime
constantof
isovolu-mic
pressure decay (T),and maximal
negative dP/dt,
wasslowed
in group II(Table III). There
werealso
alterations inthe
passive-elastic properties of the ventricle. The overall
chamber
stiffness
constant(Ku)
wasdecreased
in group II based on LVwall volume
indexing, but
notbody
weight
in-dexing.
Theexvivopressure-volume
relations withLV vol-umeindexed
tobody
weight
areshown
inFig.
3.
In contrast to overall chamberstiffness, operating
chamber stiffness(CQ)
wasincreased in
groupI,
andunaltered in
group IIusing
both
forms of
indexing. Fig.
4shows thepressure-volume
relations ofthe control and untreated diabeticratswith the tangent lines at meanend-diastolic
pressure. Theslope
of the tangent=C,.
Operating
chamberstiffness is influenced by
theend-diastolic
pressure,
and
hence maybe
different than
theoverall
chamberstiffness
constant. Themyocardial
stiffness
constant(K.)
wasunchanged
indiabeticrats(Table III).
Thestiffness-stressrela-tions
arelinear
(Fig. 5).
Thereare nosignificant
differencesinthe
slope
orposition
ofthe
lines. Ventricular dilatationdevel-oped
in groups Iand II,
asdefined
by
increases in end-diastolic
volume. This
wasaccompanied by
anincrease in
cavity/wall
volume
(V/Vw) in
group II(Table III).
Effects of
insulin
therapy.
Administration
of insulinre-sulted in
weight gain
andlowering
ofserumglucose (Table I).
All
conscious
hemodynamic variables,
and
peak developed
LVpressure
returned
tobaseline
after3-4
wkoftreatmentwithinsulin
(Table II).
Treatment wasassociated
with theTableII. ConsciousHemodynamic Data and Peak Developed LV Pressure in Control and STZ-induced Diabetic Rats
Control Group I Group II Group III (n=15) (n=5) (n=9) (n=11)
Heart rate(beats/min) 372±5 329±12t 307±11t 407±6
LV systolic pressure (mm Hg) 150±2 146±13 124±5t 144±4
LV end-diastolic pressure (mm Hg) 8±2 15±2 15±3* 7±1
dP/dt (mmHg/s) 9,609±171 9,948±1,229 7,015+344$ 9,699±865
-dP/dt (mm Hg/s) 8,253±257 6,952±929
5,193+339t
7,324±390Peak developed LV pressure (mm Hg) 217±9 182±10* 133±3t 217±9
Group I, untreated diabetes for7d; groupII,untreated diabetes for 26 d; group III, insulintreatment for 26 d after 26 d of untreated diabetes.
*P <0.05 comparedwith control,tP<
0.01
compared with control.tion of the abnormalities ofdiastolic function, including LV relaxation, LV end-diastolic pressure and volume, and LV chamberstiffness (Table
III).
Histology. Qualitative
light microscopy revealed
nohisto-logic changes in myocardium from diabetic animals. Specifi-cally, there was no evidence of interstitialcollagen
accumula-tion
seen withstain
specific for collagen
(Fig. 6).
Discussion
The major findings
ofthis study
were that bothactive
andpassive properties
ofthe left ventricle
werealtered
inconscious
diabetic
rats.The
former
wasmanifest
asimpairment of
LVrelaxation and systolic function,
while the latterconsisted
of
decreased
LV chamberstiffness, unchanged myocardial
stiff-ness, and increased LV end-diastolic volume.
Despite
a de-crease in the overall chamber stiffness constant,operating
chamber stiffness was increased at 1 wk and
unchanged
at 4 wk.This seemingly
paradoxical change
inoperating
chamberstiffness resulted from
theopposing
effects of increased
end-di-TableIII.Indices
of
DiastolicFunction inControl andSTZ-induced
DiabeticRatsControl GroupI GroupII GroupIII (n=15) (n=5) (n=9) (n= 11) T(ms) 13.1±0.3 15.1±1.0 16.5±0.6t 12.5±1.0 Kc
(V.)
4.4±0.4 4.4±0.6 3.0±0.2* 4.9±0.3 Kc(BW) 2.0±0.2 2.1±0.3 1.5±0.1 2.5±0.2C.
(V.)
27.9±6.6 68.3±15.2* 46.0±9.6 42.4±6.3C6
(BW) 12.6±2.7 32.8±7.2* 22.3±4.7 21.0±8.75Km 7.3±0.1 7.3±0.4 7.3±0.3 8.1±0.3
EDV(BW)
(ml/kg) 0.72±0.11 1.54±0.28t 1.74±0.15t 0.83±0.1
V/V. 0.54±0.04 0.67±0.09 0.74±0.03* 0.51±0.05 Group I, untreated diabetes for7d;groupII,untreated diabetes for 26d; group III, insulintreatmentfor 26 dafter 26 d of untreated
dia-betes.Abbreviations: EDV,LVend-diastolic volume
(ml/kg); BW,
indexedtobodyweight;
C,,
operating
chamberstiffness;
Kc,chamber stiffness constant;Km,
myocardial
stiffness constant; T, timeconstantof left ventricularrelaxation; V/V,,,
LVcavity/wall
volumeat 10 mmHgdistendingpressure; V.,indexedtoLVwall volume.*P<0.05compared withcontrol;tP<0.01comparedwith control.
astolic pressure, and decreased chamber
stiffness.
All of the hemodynamic and mechanical abnormalities were correctedby
insulin therapy.
Nohistologic alterations
in themyocar-dium of diabetic
rats wereevident
onlight microscopy.
Thus,diabetic
ratshave characteristics of
anearlydilated
cardiomy-opathy which is
due tometabolic,
rather thanstructural,alter-ations of
themyocardium.
Cardiac
performance is
afunction of ventricular filling
andejection.
As such,it
is important
to understand thevariableswhich effect
both systole and diastole when studying a givendisease
state.Wehaveattempted
todissect systole
anddiastole
into
componentswhich
could bequantitatively
analyzed. Measurementsof active
processes,i.e., relaxation
anddevel-oped
pressure, were done inintact
rats. Thepassive-elastic
properties of
themyocardium, i.e.,
overall chamberstiffness
(Kr),
operating
chamberstiffness
(C,),
and
myocardial stiffness
(Km),
were evaluated exvivo. This
typeof analysis of
LVfunction
provides
aframework
tounderstandcardiovascular
function
indiabetes
and mayhelp
toexplain
conflicting
datafrom previous studies.
Effects of
diabetes
onsystolic
function
and
ventricular
re-laxation.
Inconscious
rats,resting systolic
function,
assessedby
LVsystolic
pressure anddP/dt,
was normalafter
7 dof
30T
251+
-11J
E
E
w
(f)
w
0-
201-
15t-10
5
0.0
*-* CONTROL
A-A STZ 7 DAYS i-c F- -
0-0-0 STZ 26 DAYS
c-c STZ +INSUUN
/-F 0--4-A
0.5 1.0 1.5 2.0 2.5
VOLUME
(ml/kg)
Figure 3.Exvivo left ventricularpressure-volume relations.Note that in diabeticratsthecurves are
progressively
shifted away from thepressure axis. Insulintreatment restoresthe normalpressure-vol-umerelation.Errorbarsshow SEM forvolumeateachpressure. *P
30
20
10
CONTROL STZ 7 STZ 26
/ ,
1.0 1.5
VOLUME
(ml/kg)
Figure4.Operatingchamber stiffness in control and untreated dia-beticrats. Thecurvesshownarethebestmonoexponentialfittedto
the datapoints.Theslopeof thepressure-volumecurves(tangent line)atmeanend-diastolicpressurefor eachgroup=operating
chamber stiffness. This illustrates howoperatingchamberstiffness
canbe increased(group I),andunchanged (group II)whenthe
over-all chamber stiffnessconstantsareunchanged (group I),and de-creased(group II).
untreated diabetes.
However, systolic
reserve wascompro-mised,
asevidencedby
decreasedpeakdeveloped
LVpressureinopen-chest rats. After 3-4wk ofdiabetes,bothrestingand
stressed
systolic
functionwereabnormal. These observationsin conscious andanesthetizedratssupportthe
findings
instud-ies of papillary muscle from diabetic rats, in which time to
peak
tension wasincreased andpeak
rate of tensionrisewasdecreased
(3). Likewise,
inprevious
studiesusing
isolatedper-fused heartsfrom diabetic rats,
peak
LV pressure response toprogressive increases
inpreload
wasattenuated(4).
One of the best measuresof relaxationin vivo is the time constantofventricularpressuredecay (r).We found thatTwas
progressively prolonged
inconscious diabeticrats. Ventricularrelaxation is altered
by changes
intemperature(1 1),
catechol-amines(12), inotropic state(13), heart rate(14), andloading conditions (15). Diabetes is associated with several of these
factors, including impaired contractility, bradycardia,
in-creased bloodvolume
(16),
and increased catecholamines(17).
Sincethe combined effect ofthese factors is difficult topredict,
~~CONTROL
U)_ 300
STZ DAY 7 A
CIO
Z g - - ~~STZ DAY26 , /
LLs ---
~STZ
+INSULINat,
LL_
(n ° 200
n
100
2
010 20 30 40
WALL STRESS aY (g/cm2)
Figure5.Diastolic stiffness-stressrelations of the left ventriclein di-abeticand control hearts.Thelinesrepresentthe best linearfit for the stiffness-stresspointsfor therats in eachgroup(meancorrelation
coefficientr=0.98).Thecurvesarenotsignificantlydifferent.
it is
important
todocument
therateof ventricular relaxation
in
conscious
rats.The
prolongation
of
rthat
weobserved
ex-pands
onthe
findings
of
previous
in vitrostudies,
which
showed
prolongation
of time
to one-half relaxation andde-pressed
peak
rateof tension fall in
papillary muscles,
and
de-creased
-dP/dt
inisolated
hearts(3, 4).
The
mechanisms of
impaired
systolic
function
andslowed
relaxation
are unclear. Thesechanges
mayshare
a commonmechanism since both
arerelated
tothe
binding
orrelease
of
calcium from
troponin
C.
Itis
likely
that altered
sarcoplasmic
reticular calcium
transportplays
arole
inthese
changes (18,
19). Large
increases
in heartrate(100%)
have been shown
todecrease
Tby
-20% in
conscious
dogs (14). Thus,
partof the
slowed
rateof relaxation
seenin the
diabetic
ratsmaybe
attrib-utableto
bradycardia; however,
it
is
unlikely
that
bradycardia
alone was
responsible
for slowed relaxation since heart
rate wasonly
decreasedby
17% in thediabetic
rats,while
Twasincreased
by
26%.
Bradycardia
and loss
of normal baroreflexes
have been
previously
observed in diabetic
rats(16, 20).
These
findings
mayreflect
decreased
sensitivity
toadrenergic
stimu-lation,
orincreased
sensitivity
tomuscarinic
stimulation
(21,
22).
Alterations in
adrenergic
tone may havecontributed
toslowed
relaxation in the diabetic
rats.Effects of
diabetes
onpassive-elastic properties of
the
ven-tricle.
Diastolic
filling
is determined
by
acomplex
interaction
of ventricular
relaxation, passive-elastic properties
of the
ven-tricle,
myocardial
blood
flow,
atrial
pressureand
contractility,
and
ventricular interaction
(23). Early
diastolic
filling
is
in-fluenced
predominantly by
the
rateof
ventricular
relaxation,
mid-diastolic
filling
is determined
moreby
themechanical and
geometric
characteristics of
theventricle
(chamber
stiffness),
and
late
diastolic
filling depends
onthe force of atrial
contrac-tion.
Itis
difficult
toseparatethe
varying
effects of these forces.
Inour
study,
thepassive-elastic properties
of the left ventricle
were
studied
in the arrested heartprior
torigor mortis; thus,
the
aboveconfounding
variables
werealleliminated
andwewere
able
tostudy
passive filling
overthefull
rangeof
physio-logic
pressures.The
passive pressure-volume
relationship
is curvilinear
and
is
uniquely
determined for
agiven
ventricle
by
myocardial
stiffness,
andthe ratio of ventricular
cavity
and
wall volume(VIV,)
(9).
Anincrease in end-diastolic
pressure willincrease
"operating
chamberstiffness"
by
causing
ashift
to asteeper
portion
of the
curve. Purevolume
expansion,
or adecline
insystolic
function
alonecan causethis
type
of
change (23).
This
movement
along
the curveshould
bedistinguished
from
anactual
change
in
position,
orshape
of
theentire
curve,which
representsa
primary change
indiastolic
properties
(23).
Inthe presentstudy,
myocardial
stiffness
wasunchanged,
and bothVand
VI
VF,
wereincreased.
Thus,
passive
chamber stiffness
(Kr)
was
decreased. The
changes
in
passive
stiffness
shouldnotbeviewed
inisolation. There
are concurrentchanges
insystolic
and
diastolic function
inthis
model. Thesumof
thesechanges
results ina
pressure-volume
curvethatis
lesssteep
andshifted
away
from
the pressureaxis.
However,
LVend-diastolic
pres-sure rises
before
LVdilatation
occurs, and theresult is
anincrease in
operating
chamberstiffness
at7d. After 26
doper-ating
chamber stiffness
returns tobaseline
duetotheopposing
effects of the increased end-diastolic
pressure,which
causes ashift
to asteeperportion
of
the curve, and the decrease in the slopeofthepressure-volume curve.Ventricular Function in DiabeticRats 485
e-N
I
E
E
D
Un
w
Figure 6. Lightmicroscopy from representative control and diabetic hearts. Transmural low power (X30) photomicrographs of control
(JA)
and diabetic (2A) free wall show no significant lesions in diabetic heart. Higher power magnification (X450) of control(IB)and (X425)diabetic (2B) hearttissue demonstrates normal myocyte morphology in the subendomyocardial region of the left ventricle.The
forces which
governventricular dilatation
are notwell-understood.
Inthe diabetic
ratchronic
systolic
dysfunction
almost
certainly contributes
to the LVdilatation. This may
not be the same
in
humans orother animal
modelsofdiabetes.
Prolonged
increases in end-systolic
volume arebelieved
to causeventricular
remodelling
due
toslippage of
myocytes (24).Diabetes is also thought
to beassociated with
ahyperad-renergic
state(17).
Itis
possible that this sympathetic
stimula-tion
results inarterial
andvenoconstriction which could
alterloading
conditions, and thus play
arole in the
ventricular
dilatation
seen inthis model.
Blood volumehas been
reported toincrease in
experimental
diabetes, which also could
predis-pose to
ventricular dilatation
(16).
Afinal
factor which
couldcontribute
to LVdilatation is increased
ventricular
filling time
due to
bradycardia.
Effects
of insulin treatment.
Allof the hemodynamic
ab-normalities
werereversedwith insulin therapy. This confirms
similar
findings from previous studies in
rats, butdiffers from
observations in diabetic dogs
(16, 25, 26).
Itcannotbeascer-tained from currently available
datawhetherit is
thehypergly-cemia
itself,
the lackof
insulin,
or somecombination of
the twowhich
causesthe
cardiomyopathy
of diabetes. The
finding
that
insulin markedly increases calcium
uptakein
response tocatecholamine stimulation of
thediabetic
heartsupports thecontention
thatinsulin is
animportant determinant of
cardiacfunction (27).
Furthermore, treatment withinsulin
causesnor-malization of myosin isoforms, which
shift to afetal pattern in thediabetic
state(28).
Alternatively,
thereis
reason tobelieve thathyperglycemia
maydirectly
contribute
to the develop-mentof
thecardiomyopathy.
The glycation of intracellularproteins
may alterenzyme
orreceptor
function in a mannerwhich
depressescardiac
function (29). In supportof
such amechanism
are thefindings of
increased
proteinkinase
Cac-tivity
and decreased Na-K ATPaseactivity
inretinal capillary
endothelial
cellscultured in
high-glucose medium (30).
Simi-larly,increased protein kinase
Cactivity
and decreasedpoly-phosphoinositol
turnover have been observedin glomeruli
from
diabetic rats (31).Relationship of the present study to previous work.
Ourfindings of
decreasedoverall chamber
stiffness with
normalmyocardial
stiffness
are atvariance with previous
investiga-tions
inwhich
anexaggerated
increase in LV end-diastolic pressureduring intraventricular saline infusion,
wasinter-preted as
increased
myocardial
stiffness (5).
Thelatter study
wasdone in achronic (1 yr) canine model of mild
alloxan-in-duced
diabetes. Thereare
severalpotential explanations
for the difference between the studies. First, it is possible that theeffects
ofdiabetes
onthe heart arespecies-specific.
Histologiccharacteristics
of themyocardium
appear to be different in various experimental models of diabetes. Chronically diabetic dogs have been reported to haveincreased periodic
acid-Schiffstaining of interstitial glycoproteinlike
materialin the ventric-ular wall (5) andincreased
collagen content (26), whereas ourfindings
strengthen previous observations that diabetic rats do nothavehistologic
evidence
offibrosis
(32) orincreased colla-gencontent(33).
Lackof
significant
alterations in myocardialhistology support
theconcept
thatthe functional
changes weobserved have
ametabolic, rather than
a structuralbasis.
Secondly,
theeffects ofdiabetes
may betime-dependent.
If so, chamber andmyocardial stiffness
in the ratmight
increase
after a
longer duration
ofhyperglycemia.
Thedeposition of
interstitial advanced glycation products occurs
later in the course ofdiabetes and
potentially
couldcontribute
toalter-ations
inpassive
myocardial stiffness (29).
Itis
notpresently known whetherthis
process occurs in the heart. Furthermore,in
other modelsof cardiomyopathy,
such as thespontaneously
hypertensive
rat,increases in myocardial stiffness
occuronlyafter
aprolongedperiod of hypertension (34).
Finally, methodologic differences
may beimportant.
Forexample,
in thepreviously cited study: (a) pressure-volume
data were
gathered
over arelatively
small
pressure range;(b)
only
twopressure-volume
points
wererecordedfor
eachani-mal;
(c)
noattempt was made tofit
the data to anexponential
curve, nor to
quantify
chamberstiffness
constants;(d)
possible
contributions of baroreflexes
were nottakeninto
account; and(e)
measurements were madeat end-diastole
and thus mayreflect viscous effects
related toatrial contraction,
rather than truepassive
behavior. The
severity
of
thediabetes
may alsoaffect
thefindings
of
agiven
study.
We
believe
that someof the changes
insystolic
anddia-stolic
function
thatwehavedescribed
in theratmay becom-mon to other
animal
species,
and topatients with diabetes
as well. The exaggeratedend-diastolic
pressure response tointra-ventricular saline infusion
inprevious
experiments (5) could
be
explained
by
impaired
relaxation,
which is known
toshiftthe
pressure-volume
curveupwards. Large-volume
loads,
such as those used in that
study,
have beenassociated
withprolongation of relaxation (15).
To separatethe
effects of
re-laxation abnormalities
from changes in
myocardial
stiffness
inintact
animals,
we measured thepassive properties of
themyocardium
inpotassium arrested,
exvivo hearts.
Inthis
preparation,
the heartis arrested withpotassium
chloride,
andpersistent
cross-bridge
activation
should notcomplicate
theanalysis.
This
technique
allowedus todetermine
thatchanges
in
passive
musclestiffness
were notresponsible
for
the in-creasedend-diastolic
pressure and volume.Clinical
significance.
In mostcardiac diseases diastolic
ab-normalities
arethought
to be a consequenceof systolic
dys-function, although
symptomsreferable
toisolated diastolic
dysfunction
maypredominate
incertain
patients.
Recentdata suggest thatdiastolic
dysfunction
maycontribute
to the in-creasedmortality
rate seen indiabetic patients after
myocar-dial infarction (35).
Furtherevidence of diastolic
dysfunction
in
patients
with diabetes
comesfrom Doppler
echocardio-graphic evaluation of trans-mitral
diastolic flow
indiabetic
patients
without ischemic heart disease(36). Diabetic patients
wereobserved to have a decrease in the early/late (E/A) flow velocity, which is suggestive of impaired relaxation. At present, there is still considerable debate about the use of noninvasive indices of diastolic function and the meaning of isolated ab-normalitiesin a given index (37). This lack ofconsensus makes study of diastolic function in humans difficult. Although there aresignificant differences between STZ-rats and humans with diabetes, our data are consistent with the speculation that im-paired relaxation and contractility may contribute to the car-diac dysfunction seen in diabetic patients. Moreover, our find-ings emphasize that the early cardiovascular dysfunction of diabetes is reversible with insulin replacement.
Acknowledgments
Wegratefully acknowledge the technical assistance of Chuck Houg-land forwriting the computer programsused inthis study,andthe laboratory workprovidedbyTeresaWesthoffandMichelle Timmons. This studywassupportedinpartbygrantsfromthe Arizona
Affili-ateof the American HeartAssociation,ArizonaDisease Control Re-searchCommission,the GlaxoCorporation,theNationalInstitutesof
Health (HL-20984), and the Veterans Administration.
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