Tauri ne depl eti on, a novel mechani sm for

cardi oprotecti on from regi onal i schemi a

SI MON N. ALLO,
1
LI LLI AN BAGBY,
2
AND STEPHEN W. SCHAFFER
2
Departments of
1
I nternal Medicine and
2
Pharmacology, School of Medicine,
University of South Alabama, Mobile, Alabama 36688
Allo, Simon N., Lillian Bagby, and Stephen W. Schaf-
fer. Tauri ne depl eti on, a novel mechani sm for cardi oprotec-
ti on from regi onal i schemi a. Am. J . Physiol. 273 (Heart Circ.
Physiol. 42): H1956H1961, 1997.Three processes that
have been i mpl i cated i n i schemi c i njury are i mpai red Ca
2
movement, al tered osmoregul ati on, and membrane remodel -
i ng. Because the ami no aci d, tauri ne, affects al l three pro-
cesses, i t seemed l ogi cal that changes i n the myocardi al
content of tauri ne mi ght affect i schemi c i njury. To test thi s
hypothesi s, i nfarct si ze and areas at ri sk were compared i n
i sol ated hearts from control and tauri ne-depl eted rats after a
45-mi n l i gati on of the l eft anteri or descendi ng coronary
artery and 2 h of reperfusi on. Hearts of rats treated for 4 wk
wi th the tauri ne i nhi bi tor, -al ani ne, exhi bi ted a 57% reduc-
ti on i n the i nfarct si ze-to-ri sk area rati o. The degree of
cardi oprotecti on was found to correl ate (r 0.85) wi th the
extent of tauri ne depl eti on, the l atter dependent on the
l ength of -al ani ne feedi ng. When the tauri ne-depl eted rats
were fed tauri ne, myocardi al tauri ne l evel s were restored and
the cardi oprotecti on was l ost. However, addi ti on of nei ther
-al ani ne (3%) nor tauri ne (20 mM) to the perfusi on medi um
al tered i nfarct si ze. We concl ude that tauri ne depl eti on
renders the heart resi stant to i njury caused by regi onal
i schemi a.
-al ani ne; i nfarct si ze; osmoregul ati on
THE UBI QUI TOUS sul fur-contai ni ng ami no aci d, tauri ne,
i s found i n hi gh concentrati ons i n the heart, where i t
accounts for 50% of the free ami no aci d pool (12). The
exact rol e of tauri ne i n cardi ac functi on i s not ful l y
understood, al though i t has been purported to medi ate
a pl ethora of effects at the physi ol ogi cal , bi ochemi cal
and mol ecul ar l evel (2, 12). Among i ts most i mportant
physi ol ogi cal acti ons i n the heart are the modul ati on
of Na

and Ca
2
homeostasi s (2, 9), the al terati on i n
membrane structure and functi on (8), and the regul a-
ti on of i ntracel l ul ar osmol al i ty (24). The i mportance of
these acti ons i s borne out by nutri ti onal studi es show-
i ng that cats fed a tauri ne-deci ent di et devel op a
cardi omyopathy (21).
Pharmacol ogi cal doses of tauri ne al so medi ate sev-
eral effects. One i mportant acti on i s i ts abi l i ty to exert a
posi ti ve i notropi c effect, whi ch i s i ndependent of adeno-
si ne 3,5-cycl i c monophosphate or Na

-K

-adenosi ne-
tri phosphatase (2). Of equal i mportance i s the ndi ng
that tauri ne treatment protects agai nst i njury i n sev-
eral model s of heart fai l ure, i ncl udi ng the Ca
2
para-
dox, cardi omyopathi c hamster, i soproterenol cardi otox-
i ci ty, and doxorubi ci n-i nduced cardi ac damage (2, 16,
23). However, the most dramati c effect of tauri ne i s
observed i n experi mental heart fai l ure, i n whi ch tau-
ri ne treatment i s reported to si gni cantl y reduce mor-
tal i ty (29). These ani mal studi es have encouraged the
use of tauri ne as a therapeuti c agent. The most cl i ni -
cal l y rel evant rol e of tauri ne to date has been i ts use for
congesti ve heart fai l ure i n Japan, where cl i ni cal tri al s
have reveal ed an i mprovement i n New York Heart
Associ ati on cl assi cati on of pati ents, who have been
treated wi th or wi thout di goxi n (1).
Al though most studi es to date have supported the
noti on that mai ntenance of hi gh i ntracel l ul ar tauri ne
l evel s i n the heart i s beneci al to normal myocardi al
functi on, recent studi es by Chapman et al . (5) suggest
that tauri ne effl ux may hel p el i mi nate excess Na

from
the myocyte. Moreover, under pathol ogi cal condi ti ons
i n whi ch the i ntracel l ul ar osmoti c pressure ri ses, i t i s
l i kel y that the heart shoul d benet from a l oss of the
osmol yte, tauri ne. Thus, because both water and Na

accumul ate duri ng i schemi a and are thought to contri b-

ute to the severi ty of i njury, i t i s l ogi cal to assume that
changes i n the si ze of the i ntracel l ul ar tauri ne pool
shoul d affect the outcome of an i schemi c i nsul t. To test
thi s hypothesi s, we exami ned the effect of drug-i nduced
tauri ne depl eti on on cel l ul ar necrosi s i n a regi onal
model of i schemi a.
METHODS
Hearts from mal e Wi star rats wei ghi ng 250300 g were
tauri ne depl eted by mai ntai ni ng the rats on tap water
contai ni ng 3% -al ani ne for 428 days (9, 25). Some rats,
referred to as the tauri ne-repl ete group, were mai ntai ned for
8 days on tap water contai ni ng 1.5% tauri ne after a 28-day
treatment wi th 3%-al ani ne. Control rats were age matched
and mai ntai ned on normal tap water for the durati on of the
experi ment. Nei ther tauri ne depl eti on nor repl eti on si gni -
cantl y al tered rat body wei ght rel ati ve to the control s; body
wei ght was 382 28, 350 21, and 365 31 g for the control ,
tauri ne-depl eted, and tauri ne-repl ete groups, respecti vel y.
Moreover, the dry wei ght of the heart was i denti cal (0.29
0.01 g) i n al l three groups. However, the protocol s l ed to an
al terati on i n the wet-to-dry wei ght rati o of the heart, whi ch
was 4.76 0.06, 5.05 0.10, and 4.73 0.06 for the control ,
tauri ne-depl eted, and tauri ne-repl ete groups, respecti vel y.
After the appropri ate peri od of tauri ne depl eti on and
repl eti on, most hearts were perfused on a Langendoff appara-
tus wi th Krebs-Hensel ei t buffer contai ni ng 118 mM NaCl ,
27.1 mM NaHCO
3
, 2.8 mM KCl , 1 mM KH
2
PO
4
, 1.2 mM
MgSO
4
, 2.5 mM CaCl
2
, and 11 mM gl ucose, whi ch was
saturated wi th 95% O
2
-5% CO
2
and mai ntai ned at 37C.
However, when the effect of exogenous tauri ne and -al ani ne
on i schemi c i njury of untreated, control hearts was exami ned,
the Krebs-Hensel ei t buffer was suppl emented wi th ei ther 20
mM tauri ne or 3% -al ani ne. For al l experi ments, the coro-
nary perfusi on pressure was xed at 100 cmH
2
O.
To i ni ti ate the experi ment, a 2-O si l k suture was l oosel y
pl aced around the l eft mai n coronary artery and passed
through a smal l vi nyl tubi ng to form a snare. The hearts were
then perfused under normal condi ti ons for 20 mi n. After the
stabi l i zati on peri od, coronary occl usi on was effected i n some
0363-6135/97 $5.00 Copyri ght

1997 the Ameri can Physi ol ogi cal Soci ety H1956
of the hearts by pul l i ng the suture through the snare and
cl ampi ng the snare wi th a hemostat. The desi red peri od of
coronary artery occl usi on (45 mi n) was fol l owed by reperfu-
si on for 2 h. The experi ment was termi nated by reti ghteni ng
the snare and i nfusi ng a 0.2% sol uti on contai ni ng 110 m
zi nc-cadmi um uorescent parti cl es (Duke Sci enti c, Pal o
Al to, CA) i nto the aorta. The uorescent parti cl es were abl e to
del i neate the ri sk zone, whi ch was dened as the regi on
l acki ng uorescence when observed wi th a 366-nm uores-
cent l amp. After admi ni strati on of the uorescent parti cl es,
the hearts were removed from the perfusi on apparatus and
frozen for at l east 2 h before bei ng cut i nto 2-mm-thi ck sl i ces.
The sl i ces were i ncubated for a peri od of 20 mi n at 37C i n a
1% sol uti on of tri phenyl tetrazol i um chl ori de di ssol ved i n
phosphate buffer (pH 7.4). The sl i ces were then pl aced
between gl ass pl ates, and the areas of the ri sk zones (del i n-
eated as nonuorescent zones) and i nfarcted areas (l acki ng
stai ni ng wi th tetrazol i um) were determi ned by pl ani metry.
The vol umes of the ri sk and i nfarcted zones were determi ned
by mul ti pl yi ng the area by the sl i ce thi ckness. The i nfarct si ze
was expressed as a percentage of the ri sk zone that was
i nfarcted.
Determination of taurine levels. Cardi ac tauri ne content
was measured by the method of Shaffer and Kocsi s (25).
Hearts were perfused wi th Krebs-Hensel ei t buffer vi a aorti c
cannul ati on to remove the bl ood and then bl otted, wei ghed,
and frozen. After freeze dryi ng, the sampl es were rewei ghed
and homogeni zed wi th 2% perchl ori c aci d. After neutral i za-
ti on wi th K
2
CO
3
, the supernatant was used for tauri ne
determi nati on.
An al i quot of the supernatant (20 l ) was di l uted to 400 l
and then reacted wi th 0.1 ml of 2,4-di ni trouoro-1-benzene
(DNFB) i n the presence of 0.1 ml of 1 M NaOH and 0.5 ml of
di methyl sul foxi de. The reacti on was termi nated after 30 s by
the addi ti on of 0.1 ml of 3 M HCl , whi ch l owered the pH to
1.52.0. Dei oni zed water was added to a nal vol ume of 5 ml .
Sampl es were then extracted wi th ethyl acetate (20 ml ) for 10
mi n to remove the deri vati zed carboxyl i c ami no aci ds and
unreacted DNFB. The opti cal densi ty at 355 nm of the
aqueous fracti on contai ni ng 2,4-di ni trophenyl tauri ne was
determi ned. A tauri ne standard curve usi ng 240 g tauri ne
was obtai ned for each assay.
Hemodynamicmeasurements. Hearts from control , tauri ne-
depl eted, and tauri ne-repl ete rats were perfused on a stan-
dard worki ng heart apparatus wi th Krebs-Hensel ei t buffer
suppl emented wi th 11 mM gl ucose. After a 20-mi n stabi l i za-
ti on peri od, several hemodynami c parameters were mea-
sured. Peak ventri cul ar systol i c pressure and heart rate were
measured wi th a Statham P23 Gb pressure transducer by
i nserti ng a 22-gauge needl e through the ventri cul ar wal l .
Coronary ow was determi ned by col l ecti ng the coronary
effl uent.
Statistical analysis. Al l data i nvol vi ng mul ti pl e groups
(control , tauri ne depl eted, and tauri ne repl ete) were anal yzed
by anal ysi s of vari ance, wi th the Newman-Keul s test used to
determi ne the source of the si gni cant di fference. When onl y
the tauri ne-depl eted and control groups were compared, the
Students t-test was used to determi ne si gni cant di fferences.
RESULTS
Taurinedepletion. Previ ous studi es have shown that
cardi ac tauri ne pool s can be si gni cantl y reduced by
treati ng rats wi th the tauri ne transport i nhi bi tor,
-al ani ne (9, 25). I n accordance wi th those studi es, we
found that cardi ac tauri ne l evel s were reduced from
105.0 2.2 to 63.2 5.3 mol /g dry wt after 4 wk of
-al ani ne treatment (Tabl e 1). Thi s process i s readi l y
reversi bl e, si nce the addi ti on of tauri ne to the dri nki ng
water rapi dl y restored the myocardi al tauri ne pool .
The degree of tauri ne depl eti on was dependent on
the l ength of ti me the ani mal s recei ved -al ani ne i n
thei r dri nki ng water. Wi thi n the rst few days after
treatment wi th -al ani ne, myocardi al tauri ne l evel s
fel l abruptl y. Thereafter, there was a sl ow decl i ne i n the
si ze of the myocardi al tauri ne pool , whi ch reached a
new steady state by 2 wk of treatment (Fi g. 1).
Tabl e 2 demonstrates that the basel i ne hemody-
nami c parameters were unaffected by ei ther tauri ne
depl eti on or repl eti on.
Effect of taurine depletion and repletion on myocar-
dial infarct size. The si ze of the ri sk zone after l i gati on
of the l eft anteri or descendi ng coronary artery was
0.4 cm
3
i n al l hearts exami ned (Tabl e 3). After 45 mi n
of i schemi a and 2 h of reperfusi on, control hearts
exhi bi ted an i nfarct si ze-to-ri sk area rati o of 55.68
2.04%, whi ch i s si mi l ar to the val ue reported by other
i nvesti gators for the same peri od of i schemi a (4, 11, 17).
Si gni cantl y, a 40%reducti on i n the si ze of the myocar-
Tabl e 1. Effect of taurinedepletion and repletion
on myocardial taurinecontent
Tauri ne Content, mol /g dry wt
Control 105.02.2
Tauri ne depl eted 63.25.3*
Tauri ne repl ete 103.63.5
Val ues are means SE of 35 hearts. Hearts were i sol ated from
control , tauri ne-depl eted, and tauri ne-repl ete rats and perfused on a
Langendoff apparatus for 5 mi n to wash out the bl ood. Hearts were
then frozen, freeze dri ed, and extracted wi th 2%perchl ori c aci d. After
neutral i zati on wi th K
2
CO
3
, tauri ne l evel s were determi ned as de-
scri bed i n METHODS. *Si gni cant di fference between tauri ne depl eted
and other 2 groups (P 0.05).
Fi g. 1. Ti me course for parti al depl eti on of myocardi al tauri ne pool
by -al ani ne treatment. After i ndi cated peri od of -al ani ne treat-
ment, hearts were i sol ated, perfused to remove bl ood, and then freeze
dri ed. After extracti on wi th 2% perchl ori c aci d and neutral i zati on
wi th K
2
CO
3
, tauri ne l evel s were determi ned. Val ues shown represent
means SE of 45 hearts. Si gni cant decreases i n tauri ne l evel s
were noted by 4 days of -al ani ne feedi ng (P 0.05).
H1957 TAURI NE AND MYOCARDI AL I SCHEMI C I NJURY
di al tauri ne pool resul ted i n a 57% decrease i n the
i nfarct si ze-to-ri sk area rati o (Tabl e 3).
To further del i neate the effect of tauri ne depl eti on on
i nfarct si ze, we eval uated hearts whose tauri ne content
was vari ed by treati ng rats wi th -al ani ne for di fferent
i nterval s. I nfarct si ze was maxi mal l y reduced after 2
wk of -al ani ne feedi ng, coi nci di ng wi th maxi mal depl e-
ti on of the myocardi al tauri ne pool (Fi gs. 1 and 2).
Conti nuous feedi ng wi th -al ani ne beyond 2 wk nei ther
al tered myocardi al tauri ne l evel s nor affected the ex-
tent of cardi oprotecti on from regi onal i schemi a. How-
ever, exposure to -al ani ne for shorter peri ods of ti me
resul ted i n l ess tauri ne depl eti on and a correspondi ng
smal l er decl i ne i n i nfarct si ze.
Fi gure 3 reveal s that a si gni cant correl ati on (r
0.85) exi sts between cardi ac tauri ne l evel s and the
i nfarct si ze-to-ri sk area rati o. Because -al ani ne does
not accumul ate i n the heart (data not shown), the
observed reducti on i n the i nfarct si ze-to-ri sk area rati o
appears to be caused by tauri ne depl eti on. Thi s i s
supported by the observati on that acute exposure to
-al ani ne (3%) i n vi tro had no effect on i nfarct si ze
(Tabl e 4). Si mi l arl y, addi ti on of 20 mM tauri ne to the
perfusi on buffer throughout the experi mental protocol
di d not al ter i nfarct si ze. However, repl eti on of the
cardi ac tauri ne pool by mai ntai ni ng tauri ne-depl eted
rats for 8 days on water contai ni ng 1.5% tauri ne
compl etel y el i mi nated the cardi oprotecti ve effects of
tauri ne depl eti on (Tabl e 3).
DISCUSSION
The present study i s the rst i nvesti gati on exami n-
i ng the effect of tauri ne i n a myocardi al regi onal
i schemi a model . The rel evance of thi s study i s that i t
uncovers a new and novel means of cardi oprotecti on. I t
al so i ntroduces a new model to i nvesti gate mechani sms
that contri bute to cardi oprotecti on agai nst i schemi c-
i nduced cel l necrosi s.
Tabl e 2. Effect of taurinedepletion on
hemodynamicproperties
Hemodynami c Property Control Tauri ne Depl eted Tauri ne Repl ete
Heart rate, beats/mi n 25022 25025 24927
Coronary ow, ml /mi n 21.90.9 23.11.6 26.31.2
Peak systol i c pressure,
cmH
2
O 18511 17912 1746
Rate-pressure product
1,000 46.13 45.810 43.23
Val ues are means SE of 46 hearts. Hearts from control ,
tauri ne-depl eted, and tauri ne-repl ete rats were perfused on a work-
i ng heart apparatus wi th buffer contai ni ng 11 mM gl ucose. After 20
mi n of stabi l i zati on, heart rate, coronary ow, and peak systol i c
pressure were measured. No si gni cant di fference was noted be-
tween groups.
Tabl e 3. Effect of taurinedepletion and repletion
on infarct size
Parameter Control Tauri ne Depl eted Tauri ne Repl ete
Ri sk area, cm
3
0.400.03 0.390.02 0.470.04
I nfarct si ze, cm
3
0.220.02 0.100.02* 0.240.03
I nfarct si ze/ri sk area, % 55.72.04 24.15.2* 51.65.1
Val ues are means SE of 46 hearts. Hearts from control ,
tauri ne-depl eted, and tauri ne-repl ete rats were perfused for a stabi -
l i zi ng peri od of 20 mi n. Hearts were then subjected to 45 mi n of
regi onal i schemi a and 2 h of reperfusi on. I nfarct si ze and ri sk area
were determi ned as descri bed i n METHODS. Percent of i nfarct si ze/ri sk
area was cal cul ated by di vi di ng i nfarct zone vol ume by ri sk zone
vol ume for each experi ment. *Stati sti cal si gni cance from control
and tauri ne-repl ete groups (P 0.05).
Fi g. 2. Effect of -al ani ne treatment on i nfarct si ze reducti on. Hearts
were i sol ated from rats treated for vari ous peri ods of ti me wi th
-al ani ne. After a 20-mi n stabi l i zati on perfusi on peri od, control and
tauri ne-depl eted hearts were subjected to 45 mi n of regi onal i sch-
emi a by occl usi on of l eft mai n coronary artery. Hearts were then
reperfused for 2 h before determi nati on of area of ri sk and i nfarcted
vol ume. Data are expressed as percent i nfarct si ze-to-ri sk area.
Val ues shown represent means SE of 46 hearts. Si gni cant
decreases i n percent i nfarct si ze-to-ri sk area were found by 4 days of
-al ani ne treatment (P 0.05).
Fi g. 3. Correl ati on between myocardi al tauri ne l evel s and i nfarct
si ze-to-ri sk area rati o for control and -al ani ne-treated hearts. Rats
were subjected to varyi ng peri ods of -al ani ne feedi ng, resul ti ng i n
di fferi ng degrees of tauri ne depl eti on. These hearts al so showed
varyi ng i nfarct si zes after an i schemi c-reperfusi on i nsul t. Shown i s
correl ati on curve for i nfarct si ze and tauri ne content data obtai ned
after vari ous peri ods of tauri ne depl eti on (r 0.85).
H1958 TAURI NE AND MYOCARDI AL I SCHEMI C I NJURY
The effects of tauri ne on the i schemi c heart has been
an area of controversy. I n the rst study exami ni ng thi s
questi on, Kramer et al . (16) reported that addi ti on of 10
mM tauri ne to the perfusi on medi um di d not i mprove
the recovery of cardi ac work i n i sol ated rat hearts
subjected to severe gl obal i schemi a. The data i n Tabl e 4
showi ng that addi ti on of 20 mM tauri ne to the perfu-
si on medi um di d not attenuate the degree of i schemi c
i njury i s consi stent wi th that ndi ng. However, both
studi es are i n apparent coni ct wi th the work of
Franconi et al . (6), who reported an i mprovement i n the
recovery of contracti l e functi on after a 30-mi n hypoxi c
i nsul t i n gui nea pi g hearts perfused wi th gl ucose-free
buffer. Recentl y, Raschke et al . (23) al so observed no
i mprovement i n recovery of cardi ac work after 15 mi n
of gl obal i schemi a when 15 mM tauri ne was i ncl uded i n
the reperfusi on buffer. However, they found that tau-
ri ne prevented the decl i ne i n mechani cal functi on
i nduced by addi ti on of neutrophi l s to the reperfusi on
medi um. Thi s protecti ve effect of tauri ne was attri b-
uted to i ts abi l i ty to scavenge hypochl orous aci d, thereby
reduci ng the degree of oxi dant damage medi ated by the
neutrophi l s. Si mi l arl y, the anti oxi dant effect of tauri ne
has been i mpl i cated i n tauri ne-medi ated reducti ons i n
cel l damage that occur duri ng coronary artery bypass
grafti ng (19). I nteresti ngl y, i n the l atter two studi es,
cardi oprotecti on was onl y observed when extracel l ul ar
tauri ne l evel s were si gni cantl y el evated, suggesti ng
that the neutrophi l neutral i zi ng effect of tauri ne repre-
sents a pharmacol ogi cal , rather than a physi ol ogi cal
effect.
The present study contrasts wi th al l previ ous i sch-
emi a studi es because the experi mental protocol focuses
on changes i n the i ntracel l ul ar tauri ne pool . A standard
procedure was used to sl owl y l ower the i ntracel l ul ar
tauri ne pool . The val ue of thi s procedure was twofol d. I t
permi tted the regul ati on of myocardi al tauri ne l evel s
over a fai rl y wi de concentrati on range and al so al l owed
the reversal of the tauri ne defect merel y by addi ng
tauri ne to the ani mal s water suppl y.
The most si gni cant ndi ng of thi s study i s that
tauri ne depl eti on resul ts i n cardi oprotecti on from re-
gi onal i schemi a. The maxi mal effect, a 57%reducti on i n
ri sk zone i nfarcted, i s comparabl e i n scope to other
cardi oprotecti ve procedures. One of the most wi del y
studi ed cardi oprotecti ve mechani sms, precondi ti oni ng,
has been reported to reduce i nfarct si ze-to-ri sk area
between 63 and 73% after 45 mi n of regi onal i schemi a
(4). Equal l y effecti ve i n reduci ng i nfarct si ze-to-ri sk
area have been the Na

/H

exchange i nhi bi tors (4, 13).

Two procedures that are sl i ghtl y l ess potent i n medi at-
i ng cardi oprotecti on are hyperthermi a, a form of heat-
shock protei n i nducti on, and streptozotoci n-i nduced
di abetes, whose mechani sm of cardi oprotecti on i s un-
known; both condi ti ons di mi ni sh the i nfarct si ze-to-ri sk
area rati o 33%(11, 17).
Al though tauri ne depl eti on si gni cantl y reduces i n-
farct si ze i n the reperfused heart, i t does not si gni -
cantl y i mprove recovery of contracti l e functi on (data
not shown). Two expl anati ons can be provi ded to ac-
count for thi s paradoxi cal observati on. Fi rst, the area
at ri sk i s not xed i n the regi onal i schemi a model ,
causi ng consi derabl e vari abi l i ty i n the recovery of
mechani cal functi on even among the control group.
Second, tauri ne affects myocardi al contracti l e functi on
through al terati ons i n Ca
2
movement and i ncreased
sensi ti vi ty of the myobri l s to Ca
2
(2, 9, 26). Al though
the degree of tauri ne depl eti on achi eved i n the -
al ani ne-treated rats does not i nduce a change i n me-
chani cal functi on (Tabl e 2), more severe decreases i n
the i ntracel l ul ar tauri ne pool are associ ated wi th the
devel opment of a cardi omyopathy (21). Thi s i s rel evant
because massi ve amounts of tauri ne effl ux the heart
duri ng an i schemi c-reperfusi on i nsul t (16, 18). Thus,
al though the protected regi ons of the heart do not di e,
they exi st i n an unusual l y severe state of stunni ng.
Consequentl y, the favorabl e effect of reduced i nfarct
si ze may be bal anced by the unfavorabl e effect of
tauri ne depl eti on on contracti l e functi on.
Several factors support the concl usi on that the -
al ani ne-medi ated reducti on i n i nfarct si ze i s di rectl y
rel ated to tauri ne depl eti on. Fi rst, a negati ve correl a-
ti on exi sts between tauri ne l evel s and the extent of
cardi oprotecti on (Fi g. 3). Second, the cardi oprotecti on
i s compl etel y reversed by repl eti ng the cardi ac tauri ne
pool . Thi rd, the onl y known cardi ovascul ar effects of
-al ani ne rel ate to the i nhi bi ti on of tauri ne transport
and the promoti on of tauri ne effl ux from the myocyte (9,
25). Fourth, the effects of -al ani ne feedi ng cannot be
dupl i cated by acute exposure of the i sol ated heart to
ei ther 3%-al ani ne or 20 mM tauri ne (Tabl e 4).
The mechani sm by whi ch tauri ne depl eti on i s cardi o-
protecti ve remai ns to be compl etel y exami ned. One
attracti ve hypothesi s i s that tauri ne, an effecti ve osmo-
l yte, pl ays a cri ti cal rol e i n osmoregul ati on duri ng
i schemi a-reperfusi on (5). I t has been establ i shed that
tauri ne i s rapi dl y l ost from the heart after l i gati on of
the ci rcumex branch of the l eft mai n artery (18) or
after gl obal i schemi a fol l owed by reperfusi on (16). Thi s
i schemi a-i nduced tauri ne l oss may merel y reect a
response of the i schemi c heart to the accumul ati on of
osmoti cal l y acti ve agents, such as Na

, P
i
, and l actate.
Because tauri ne i s an i mportant osmol yte, i ts effl ux
from the cel l effecti vel y reduces the i ntracel l ul ar os-
moti c l oad, thereby di mi ni shi ng the osmoti c gradi ent
across the cel l membrane. Accordi ng to Jenni ngs and
co-workers (14, 27), the i ntracel l ul ar osmoti c l oad can
l ead to excessi ve cel l swel l i ng, whi ch i s thought to pl ay
a cri ti cal rol e i n i rreversi bl e cel l damage.
Tabl e 4. Effect of acutetaurineand -alanineexposure
on infarct size
Parameter Control Tauri ne (20 mM) -Al ani ne (3%)
Ri sk area, cm
3
0.400.03 0.370.04 0.510.06
I nfarct si ze, cm
3
0.220.02 0.180.01 0.250.03
I nfarct si ze/ri sk area, % 55.72.04 48.85.0 48.86.4
Val ues shown are means SE; n 4 hearts. Hearts were perfused
on a Langendorff apparatus wi th Krebs-Hensel ei t buffer contai ni ng
no addi ti ons (control ), 20 mM tauri ne, or 3% -al ani ne. Perfusi on
protocol was i denti cal to that descri bed i n Tabl e 3.
H1959 TAURI NE AND MYOCARDI AL I SCHEMI C I NJURY
Several i nvesti gators have attempted to reduce i n-
farct si ze by decreasi ng the osmoti c gradi ent across the
cel l membrane by rai si ng the osmol al i ty of the extracel -
l ul ar medi um. Thi s strategy has l ed to mi xed resul ts.
Kl oner et al . (15) and Garci a-Dorado et al . (7) have
reported a decl i ne i n i nfarct si ze i n the hyperosmoti -
cal l y treated heart, whereas Harada et al . (10) found no
i nuence of hyperosmol ar manni tol on i nfarct si ze i n
the baboon heart. The present approach i s seemi ngl y
rel ated to that strategy; however, i nstead of rai si ng the
extracel l ul ar osmoti c l oad, tauri ne depl eti on reduces
the i ntracel l ul ar osmoti c l oad. Nonethel ess, two nd-
i ngs suggest that the cardi oprotecti on noted i n the
tauri ne-depl eted heart may not be rel ated sol el y to the
change i n the osmoti c pressure gradi ent. Fi rst, addi ti on
of hyperosmol ar concentrati ons of -al ani ne to the
perfusate fai l ed to reduce i nfarct si ze. Second, addi ti on
of 20 mM tauri ne to the perfusi on medi um wi th the ai m
of el i mi nati ng the tauri ne gradi ent across the myocyte
membrane, di d not i nuence i nfarct si ze.
Because the process of tauri ne depl eti on i n the
-al ani ne-fed rat i s compl ex, i t i s not surpri si ng that
mul ti pl e factors coul d contri bute to the observed cardi o-
protecti on. An i mportant consi derati on i s that the
tauri ne-depl eted heart, to mai ntai n an osmoti c bal -
ance, presumabl y undergoes an adjustment i nvol vi ng
modi cati ons i n the content of i ntracel l ul ar organi c
osmol ytes as wel l as the acti vi ty of transporters i n-
vol ved i n osmoregul ati on.
A transporter whose acti vi ty i s al tered after an
osmoti c pressure i nsul t i s the Na

/H

exchanger (3).
Thi s transporter i s of parti cul ar i nterest because i nhi bi -
tors of the Na

/H

exchanger protect the heart agai nst

i schemi c i njury (13). Moreover, reducti on i n ux through
the exchanger, ei ther by mani pul ati on of the cati on
composi ti on of the myocyte or the i ntri nsti c acti vi ty of
the transporter, i nvari abl y l eads to l ess cel l damage
duri ng an i schemi c-reperfusi on or hypoxi c-reoxygen-
ati on i nsul t (13). Because tauri ne depl eti on appears to
i nduce an osmoti c stress, one woul d predi ct that the
acti vi ty of the Na

/H

exchanger shoul d be affected by

-al ani ne feedi ng.
Another i mportant osmoti c-sensi ti ve transporter i s
the Na

/Ca
2
exchanger (30). Previ ousl y, we demon-
strated that the acti vi ty of the Na

/Ca
2
exchanger i s
depressed i n the tauri ne-depl eted myocardi um (9).
Because thi s transporter i s thought to pl ay a pi vi tal
rol e i n Ca
2
overl oad-i nduced myocardi al i njury, i t i s a
l ogi cal candi date for the cardi oprotecti on of tauri ne
depl eti on.
Tauri ne has another potenti al l i nk to the regul ati on
of i ntracel l ul ar cati on homeostasi s. The process of
tauri ne uptake by the heart i nvol ves cotransport wi th
Na

. Accordi ng to Chapman et al . (5), tauri ne effl ux

uti l i zes thi s same Na

-tauri ne cotransporter. Thi s sce-

nari o woul d dramati cal l y affect the i schemi c heart
because tauri ne effl ux woul d be accompani ed by a
si gni cant decrease i n i ntracel l ul ar Na

concentrati on.
Thus damage to the heart woul d be mi ni mi zed because
both the osmoti c and Na

l oads woul d be reduced.

Al though thi s scenari o i s attracti ve, i n most noncardi ac
cel l s tauri ne effl ux occurs vi a a Na

-i ndependent vol -
ume sensi ti ve organi c osmol yte ani on channel rather
than the tauri ne-Na

cotransporter (28). Nonethel ess,

because the mode of tauri ne effl ux duri ng i schemi a
remai ns to be establ i shed, thi s i nteresti ng concept
deserves further consi derati on.
The nal possi bi l i ty i s that tauri ne depl eti on coul d
i nuence the stabi l i ty of the sarcol emmal membrane.
Hamaguchi et al . (8) have reported that tauri ne serves
as a potent i nhi bi tor of phosphol i pi d N-methyl transfer-
ase, the enzyme catal yzi ng the conversi on of phosphati -
dyl ethanol ami ne to phosphati dyl chol i ne. Because
phosphati dyl chol i ne i s a bi l ayer former, whereas phos-
phati dyl ethanol ami ne i s a nonbi l ayer former, tauri ne
can cause l ocal changes i n the bi l ayer-to-nonbi l ayer
content of the membrane. Recentl y, Post et al . (22) have
argued that an el evati on i n the membrane content of
bi l ayer formers protects the i schemi c myocardi um by
stabi l i zi ng the membrane. Thus l ocal changes i n phos-
phol i pi d content coul d occur i n the tauri ne-depl eted
heart, whi ch coul d affect the acti vi ty of a key enzyme or
transporter and modul ate the response to an i schemi c-
reperfusi on i nsul t.
Thi s study i s supported i n part by grants from the Southern
Medi cal Associ ati on (S. N. Al l o) and the Ameri can Heart Associ ati on
(S. W. Schaffer).
Address for repri nt requests: S. W. Schaffer, Dept. of Pharmacol -
ogy, School of Medi ci ne, Uni versi ty of South Al abama, Mobi l e, AL
36688.
Recei ved 15 September 1996; accepted i n nal form 11 June 1997.
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