Download Failure to Precondition Pathological Human Myocardium

Medical Research Society
R2 SARCOMERE PROTEIN
DILATED CARDIOMYOPATW
MUTATIONS
ZIP
CAUSE
SD SHARMA, M KAMISACO, SR DEPALMA,S SOLOMON,P
SHARMA,
B MCDONOUGH,
J JARCHO,L SMOOT,MP MULLEN,
LR
SHA~IRO,
PK WOOLF, ED WIGLE,JG SEIDMAN
and CE SEIDMAN
Harvard Medial School, Brigham and Women's Hospital &
Massachusetts GeneralHospital, Boston USA
B a c k g r o u ~The incidence of dilated cardiomyopathy (DCM) i s
36/100.000, with one-third due to inherited genetic defects. Five
disease loci have been identified for non-syndromic cases of familial
DCM but only two disease causing mutations characterised, both in
the cardiac actin gene.
Methods: A large family with DCM was recruited. Pedigree analysis
suggested an autosomal dominant mode of inheritance. A systematic
genome-screen was undertaken using a standard set of fluorescently
labeled informative microsatellite markers,. PCR products were
pooled and resolved on an ABI377 sequencer. Data was analyzed
using Genotypex and Genescan dedicated software (Applied
Biosystems). Two-point LOD scores were calculated using MLINK
and multipoint LOD scores were calculated using LINKMAP.
Results: Genomewide linkage analysis identified a locus on
chromosome 14q11.2-13 with a maximum LOD score = 5.11. The
interval was d e h e d by 14S283-Dl4S597 and spanned 14cM.
The region contained the pcardiac myosin heavy chain gene which is
hewn to be involved in hypertrophic cardiomyopathy. As this gene
was thought to be a likely candidate gene, all 38 exons were
amplified using standard PCR protocols and sequenced using
fluorescent based sequencing chemistry. A single T1680C mutation
was identified in exon 16. A unique mismatch primer was
constructed which allowed a restriction recognition site to be used to
quickly confirm cosegregation of the mutation with affected
individuals.
Conclusion: We show that p-cardiac myosin heavy chain gene
mutations cause DCM. This result suggests mechanisms for distinct
pathways for cardiac remodeling.
R4
FAILURE TO PRECONDITION PATHOLOGICAL HUMAN
MYOCARDIUM
S GHOSH, N B STANDEN, M GALRhNFS
Dept of Cardiac Surgery, University of Lcicester, UK
Background There is conflicting evidence to suggest that ischemic
preconditioning is a healthy heart phenomenon.
Objectives: We investigated the effects of preconditioning on diabetic and
the failing human myocardium and the role of mitochondria1K A Tchannels
~
on the response in these discased tissues.
Methods: Right atrial appendages were obtained 60m 7 different groups of
patients: nondiabetics; diet contmlled diabetics (DCD); NIDD receiving
K A T ~ channel blockers, insulin dependant diabetics (IDD), with
LVEDSO%, LVEF between 30-50% and LVEFOO%. After stabilisation,
the muscle slices were mdomised into 5 experimental pooups (n=6/group):
(1) Aerobic Control- incubated in oxygenated buffer for 2lOmin. (2)
Ischemia Alone - %in ischemia followed by l2Omin reoxygenation, (3)
Preconditioning by Smin ischemd5min reoxygenation before 9Omin
ischemia/lZOmin reoxygenation, (4) Diazoxide (MltoKAw opener, 0.lmM)
- for lomin before the 9Omh ischemia/l2Omin reoxygenation and ( 5 )
Glibenclamide (low)- lOmin exposure prior to preconditioning (only in
the diabetic patient groups). Creatine kinase leakage into the medium (CK,
U/g wet wt) and MTT reduction (OD/mg wet wt), an index of cell viability,
were assessed at the end of the experiment.
Results: Ischemia caused similar d e p of injury in both normal and
diseased tissue. PC prevented the effects of ischemia in all groups except
NIDD, IDD and poor cardiac function (<300!). In the diazoxide keatcd
groups, protection was mimicked in all groups except the NIDD and IDD
groups. Interestingly, Glybenclamide abolished protection in nondiabetics
21?
Medical Research Society
and diet controlled NIDD groups and did not affect NIDD receiving K A T ~
channel blockers and IDD groups.
Coneladons: These d t s haw shown that the failure to p d i t i o n the
diabetic heart is due to dysfunction of the mitochondrialKAR channels and
the mechanism of hilure in the failing heart lies in other elements of the
signal transduEtion pathway different from the mitochondrial K Achannels.
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R5
EFFECFSOFFUlWONTJ5JCPERMEABILFFYOFTHE
RABBIT AORTIC WALL NEAR BRANCHES
T. J. mAUGETON, M J. LEVER* and P.D.WEINBERG
School of Animal and Mlu0bl.l Sdencea, UntVerrity of Reading,
RG6 6AJ. *Dcprrbnentof B I o ~ aand
l Medial Systems,Imperial
College, London, SW7 2BX
Intmductiou Spontanwus lipid deposition occurs downskeam of
branch ostia in immaturehuman aortas but upstream of branch Ostia in
mature -1s.
Spontaneousdiand uptake of macromoleculesby
the aortic wall of normal rabbits show similar patterns (Sebkhi and
Weinberg 1994; 1996, Barnes and Weinberg, 1998). We examined the
effect of flow on the pattern of uptake.
Methods. Elmn male New zealand White rabbits were anaesthetized
(3mgkg i.m. Hyplorm, 2mg/kg i.v. midazolam). Intemxtal arteries in
each either tied, sham operated or left alone. Twenty minutes later,
modamin-lobelledalbumin was introduced mto thecirculation. Animals
wac sauificcd& a fdmxfifteen minutes and the thoracic aorta was
fixed in situ. Tracer concentrationswere mcasurcd in sections through
intercostal branch Ostia using digital imaging fluomacence. microscopy
(Sebkhi and Weinberg, 1994).
Rea& and Diacnsslon. There was no significant difference in the
pattern of transport betweencontrol, sham operated and tied branches in
the immature animals (ANOVA F4.008, P 0.05) but the tied branches
showed the rcvwc pattern of transport (T-test v. control e3.811, P 0.01;
v, sham e2.383, P 0.05). I~NIS
mature but not immature animals showed
a changcin hp116poTt with a change in side branch flow. In combination
with previous aperimnts showing that NOS inhibitors afTect the mature
but not the immaturepattern of transport (Forster and Weinberg, 1997),
thescrcsultsarc coosistnawith the mature pattembeing determined by
a flow-dependentrelease of NO.
References. Sebkhi, A and Weinberg PD. (1994). A f h c l e r o s i s . 106,
1-8. Sebkhi. A. and Weinberg,PD. (1996) Artm'ascler. Thromb. VUSC.
Bid. 16,317-327;ForstaB.A. and Weinberg, P.D. (1997) Artm'ascler.
Thromb. VUSC.Bid. 17.1361-1368. Barnes, S.E. and Weinberg, P.D.
(1998). A~~W~OSCILT.l7~0mb.VUSC.BWl. 18,300-308.
R6 NF-KBIS ESSENTIAL, FOR METALLOPROTEINASE
SECRETION BY SMOOTH MUSCLE CEUS AND FOAM CELLS
ALEX JCHASE AND ANDREW CNEWBY
Bristol Heart Instie, University of Bristol, Bristol, UK
Id&i@iUgthebiocbRmcal
. pathwaysthatlmdertit~*
plaquinstabilityisnecdedtodefmenewthcarepies.We~b~
adeaoviral( rAd) gene delivery of the inhibitory subrmit I-& to
investigate the importance of tmsmiphn factor NF-KB m reghthg
MMP 1,3 and 9 sccrction in human smooth muscle cells (VSMC),
macrophBBes(Maand
) rabbit foam cells. Interleukin-laand platelet
derived growth &or S p e r g i s t i i increase m i o n ofeachMMP
by VSMCs. O p t i d adenoviralgene delivery into human VSMCs
(detamined using a ~galactosidanemporter g m ) with rAd: I-*
but not rAd:ft-galsctosidaseor r A d d led to dPny reducrd
M M P induction;MMP-1by 97i4%, MMP-3 by 94*7% and MMP-9
by 77i12% (d~ . o o O l ~5-8).culturcd
,
~UD
IUI
MO C0-b
expresd MMP-9 but not MMP-I or -3. Rat I-& idcctcd lnmm
)
m
M c o S ~ ~ I - & b u t t h c r ~c 1 y-difFerma
MMP-9 secretioncompared with minktedor rAd:null inf&tion,
MMP(-difference
11i16%,~ 1 0 )This
. show that
9 SeCrctiDnis regulsted by anNF-KB independentmcbsman
*
We
next investigated the role of NF-KB m the pathobgical ovaqms io n
ofMMP-land3mmacropbagc-denvbd
. foamctllsisolatedfrom1%
cholestero1fedrabbits.usiog0ptimaladerroviralgenedelivery
conditbm, ovemcpressionof I-& m foam cells with rAd1-m
resuhedmmdiffereaa m MMP-9 semtionbut an 8B12% and
69i11% reduction in MMP-1and MMP-3, reqectkly, as coupad
with uninfected controls. No significant decrease was seenfolbwing
rAd$-galactosidase or rAd:rmll inkction. Thtsc data dummhak an
essential role f
i
xNF-KBm r e g u h i q ofboth- "
ryinducedMMP expresion by VSMCs and the pnthobgical
overexpmsion of MMP-1 and 3 charact&
of foam cells.
I n h i i n of NF-KB is thmfore an amsctive caddate
.
promote plaque stability.
+
R7
IS THERE A SECOND VOLTAGE DEPENDENT
MECHANISM FOR EC COUPLING IN HEART?
K C R PATEL, J V JONES and AJ LEV1
University of Bristol, Bristol, U.K.
Excitationantraction coupling (ECC) in heart is thought to be
exclusively triggered by Ca entry triggering Ca release from the
sarcoplasmic reticulum (SR), i.e. Ca induced Ca release.
IntracellularCa (Ca,) decline is due to Ca, extrusion (by NdCa
exchange) and termination of SR Ca release ((SRCR) enabling SR
uptake by SR CaATPase). How ECC, Ca entry and termination of
SRCR axe affected by adrenergic stimulation axe unknown. Whole
cell patch clamp experiments were performed on isolated guinea-pig
ventricular cells at 37°C. Cells were dialysed with CAMPor CAMPfree, Na-free pipette solution. Ca, was measured using FURA 2.
8mM Ni (blocks ,I
and NdCa exchange) was used to abolish Ca
entry and extrusion. To assess SRCR, cells held at -6OmV were
depolarised from -60 to +8OmV for 500 ms. For termination of
SRCR, cells were repolarisedto varying potentials. Under control
conditions, voltage dependence (VD)of the Ca,transient peaked
between 0 - +2OmV and stayed elevated at higher potentials. With
Ca entry blocked depolarizationstill released 7of7%, (meanhem;
n=8 cells) of SR Ca, with a VD peaking at +2OmV. A lOmM
caffeine prepulse abolished any Ca, transient elicited in the presence
of Ni, suggesting it was SRCR elicited. This SRCR mechanism was
absent in CAMP-& dialysed cells. Repolarisation in the presence of
Na-freet8mM Ni, elicited Ca, decline suggesting that repolarisation
per se terminated SRCR, allowing unopposed SR Ca ATPase
mediated SR Ca reuptake. Ca entry contribution to the Ca transient
peaked at +2OmV, contributing 67 f 12% (n=29) of the control Ca,
transient in CAMPdialysed cells. In CAMP-& cells, the
contributionpeaked at 38.W 6.7% (n=8). These data suggest that
there is a second voltage dependent mechanism able, under
conditions of adrenergic stimulation, to not only trigger but also
terminate SRCR. Furthermore, Ca entry, under these conditions,
makes a significantcontribution to the source of Ca for myocyte
contraction, in addition to providing trigger Ca for SRCR