Interaction of Acidosis and Increased Extracellular Potassium on Action Potential Characteristics and Conduction in Guinea Pig Ventricular Muscle

Transcription

1 614 Interactin f Acidsis and Increased Extracellular Ptassium n Actin Ptential Characteristics and Cnductin in Guinea Pig Ventricular Muscle Yutaka Kagiyama, Jeffrey L. Hill, and Lenard S. Gettes Frm the Department f Medicine, University f Nrth Carlina, Chapel Hill, Nrth Carlina SUMMARY. We studied the individual and cmbined effects f extracellular acidsis and increases in extracellular ptassium n actin ptential characteristics and cnductin in rder t gain a better understanding f the effects f acute ischemia. At each level f ptassium between 2.7 and 17 ITIM, acidsis induced by increasing Pc2 (respiratry acidsis) and by decreasing HCC>3~ (metablic acidsis) decreased resting membrane ptential, the maximum rate f rise f the actin ptential upstrke (V max ), and slwed cnductin. Metablic acidsis cnsistently and significantly lengthened the steady state actin ptential duratin whereas respiratry acidsis did nt. Respiratry acidsis caused changes in resting membrane ptential, V ma x, and cnductin velcity; which ccurred mre rapidly and were f greater magnitude than the changes induced by metablic acidsis. The changes in Vrnax induced by bth types f acidsis were due t a change in the resting membrane ptential- V m ai relatinship as well as t the changes in resting membrane ptential. The cnductin slwing induced by acidsis was greater when ptassium was 9 and 13 HIM than when ptassium was 5.4 HIM. Our results suggest that acidsis causes imprtant changes in the electrphysilgical prperties f ventricular fibers and that many f the knwn electrphysilgical effects f acute ischemia can be mimicked by the cmbined effects f extracellular acidsis and an increase in extracellular ptassium. (Circ Res 51: , 1982) THE precise cause f the slwing f cnductin which ccurs within the first few minutes fllwing the acute reductin f crnary flw is nt well understd. Harris et al. (1954) cnsidered the increase in extracellular ptassium t be the majr factr respnsible fr these changes and, thus, t be a majr determinant f the ventricular arrhythmias, particularly ventricular fibrillatin, which ccur fllwing acute crnary Iigatin (Harris and Rjas, 1943; Kaplinsky et al., 1979). Mre recent studies have clearly shwn that the cnductin slwing induced by acute ischemia cannt be attributed slely t the changes in extracellular ptassium (Dwnar et al., 1976; Hill and Gettes, 1980; Mrena et al., 1980). There is increasing evidence t implicate a variety f ther factrs in the genesis f the cnductin slwing. These include hypxia and glucse lack, acidsis, and pssibly the accumulatin f lysphsphglycerides (Vaughan Williams and Whyte, 1967; Crr et al., 1970; Marrannes et al., 1979; Wjtczak, 1979; DeMell, 1980; Mrena et al., 1980). Until very recently, it has been difficult t mdel precisely the inic changes assciated with acute ischemia because the precise values fr ptassium and ph were nt knwn. The values derived using inselective electrdes (Gebert et al., 1971; Wiegand, 1979; Hill and Gettes, 1980; Cbbe and Ple-Wilsn, 1980; Hirche et al., 1980; Hill et al., 1981) nw permit the assessment f the individual and cmbined cntributin f the changes in ptassium and ph t the electrphysilgical abnrmalities induced by acute ischemia. The purpse f ur investigatin was t study the effects f the cmbined changes in ptassium and ph n cnductin velcity and actin ptential characteristics in the islated superfused guinea pig papillary muscle in rder t gain greater insight int the cause f the electrphysilgical changes induced by acute ischemia. Methds Guinea pigs weighing g were killed by cervical fracture. The hearts were rapidly remved and papillary muscles 3-5 mm lng and mm in diameter were munted in a single cmpartment bath (3-ml capacity) and superfused at a rate f 6 ml/min. The temperature was maintained at C. The cmpsitin f the cntrl superfusin slutin was (ITIM): NaCl, 125; KC1, 5.4; CaCl 2, 1.8; MgCl 2, 1.05; NaHCO 3, 24; NaH 2 PO 4, 0.42; and glucse, 5. The cntrl slutin was equilibrated fr at least 30 minutes with a humidified gas mixture cntaining 5% CO2 and 95% O2. ph was lwered in steps frm 7.4 t 6.0 by decreasing NaHCO3 (metablic acidsis), r by increasing the CO2 in the equilibrating gas t levels as high as 40% (respiratry acidsis). In all slutins, the ttal sdium cncentratin was kept cnstant (Table 1). The ptassium cncentratin in the bathing slutin was changed in steps frm 2.7 t 17 HIM by subtracting r adding KCI t the cntrl slutin. The values f ph and ptassium were chsen t lie within the range which has been reprted t ccur in the extracellular space in the early stage fllwing acute crnary cclusin (Gebert et al., 1971; Wiegand et al., 1979; Cbbe and Ple-Wilsn, 1980; Hill and Gettes, 1980; Hirche et al., 1980; Hill et al., 1981). Perfusate ph was measured using a micrcmbinatin ph prbe (Micrelectrdes, Inc.) cnnected t an Orin

2 Kagiyama et al./cmbined Effects f Acidsis and K n Ventricular Muscle 615 Cntrl Slutin Metablic acidsis Respiratry acidsis TABLE 1 Cmpsitin f Varius Test Slutins PH NaCl (ITIM) NaHCO 3 (UM) c 2 (%) Pc 2 (mm Hg) mdel 601A digital inalyzer (Orin Research, Inc.). The effect f the changes in ph n calcium activity increasing Pc 2 r lwering HCC>3~ was assessed as described by Spitzer and Hgan (1979) using an Orin calcium and reference electrde (Orin 90-01) and the Orin digital ph meter. When ph was lwered t 6.5 by either increasing CO 2 t 30% r lwering HCO 3 ~ t 3 mm, the calcium activity was equivalent t a calcium cncentratin f 2.05 ± 0.05 min (/i=6), i.e., a 14% increase. When ph was lwered t 6.0 by lwering HCO3" t 1%, calcium activity was equivalent t a calcium cncentratin f 2.21 ± 0.1 mtu (n=6), a 23% increase. These changes are similar t thse reprted by Frye and Ple-Wilsn (1981), wh used a slutin cntaining 1.8 mm Ca ++, but less than thse described by Spitzer and Hgan (1979), wh used a slutin cntaining 2.7 mm Ca ++. The effect f increases in extracellular calcium n cnductin was assessed by increasing the calcium chlride cntent f the perfusin t achieve a calcium cncentratin f 3.6 mm (2 times calcium) and 7.2 mm (4 times calcium). This increase did nt alter the ph. Calcium activity increased in prprtin t the increase in cncentratin. The ptassium cncentratin f the perfusing slutin was mnitred by means f a miniature K + -sensitive electrde designed in ur labratry (Hill et al., 1978). Bth electrdes were psitined as clsely as pssible t the surface f the papillary muscle. Steady state values f ph and ptassium in the bath were achieved within 20 secnds f changing the perfusate. The muscles were stimulated at the cut end at a rate f 0.5/sec by pulses 2.5 msec in duratin and times diastlic threshld strength using biplar silver/silver chlride surface electrdes. This rate f stimulatin was chsen t limit the pssibility f use-dependent effects. The stimulus strength was adjusted t maintain a cnstant latency. Transmembrane ptentials were recrded with glass micrelectrdes filled with 3 M KCI cupled t a Transidyne mdel MPA 6 DC amplifier via Ag/AgCl wires. The maximum rate f rise f the actin ptential upstrke (V m a*) was determined by electrnic differentiatin as previusly described (Gettes and Reuter, 1974). Actin ptential duratin was determined at 90% replarizatin. Cnductin velcity was determined as the time between peaks f the differentiated upstrke signals recrded simultaneusly frm micrelectrdes placed mm apart in line with the stimulatin site alng the lng axis f the fiber divided int the interelectrde distance determined by a micrmeter eyepiece in a Nikn dissectin micrscpe. The cmmn extracellular reference electrde was psitined as clse as pssible t the midpint f the line between the intracellular electrdes. The actin ptentials and the differentiated upstrkes were displayed n a Tektrnix mdel 565 dual beam scillscpe and phtgraphed n Plarid film. The muscles were superfused with cntrl slutin fr at least 60 minutes befre changing t a test slutin. The steady state data were recrded 20 minutes after changing slutins because initial experiments indicated that apprximately 15 minutes were required fr all the actin ptential characteristics t reach a steady state. The recrds were accepted fr analysis when resting membrane ptentials were mre negative than 80 mv and V ma, was mre than 200 V/sec in cntrl slutin. In all reprted experiments, the micrelectrdes were maintained in the same cells thrughut the entire prtcl. The results are expressed as mean values ± SD. Statistical evaluatin was perfrmed using Student's t-test fr paired and unpaired bservatins. Results Steady State Effects The effects f respiratry and metablic acidsis n cnductin velcity and actin ptential characteristics were examined at ptassium cncentratins f 5.4, 9, and 13 mm. In eight experiments, ph was lwered frm 7.4 t 6.5 by bth methds. A typical experiment is shwn in Figure 1. Increasing ptassium frm 5.4 t 9 at ph 7.4 caused an 8% increase in cnductin velcity, a 12 mv decrease in resting membrane ptential, and a 6.5% decrease in V max. The further increase in ptassium t 13 mm caused a further 10-mV decrease in resting membrane ptential, a greater decrease in V max, and a decrease in cnductin velcity. At each level f ptassium, bth types f acidsis decreased resting ptential, decreased V m ax, and slwed cnductin. In the experiment shwn in Figure 1, bth types f acidsis als prlnged actin ptential duratin. Hwever, the prlngatin induced by metablic acidsis was greater than that induced by respiratry acidsis. The cmpiled results f the eight experiments are shwn in Table 2. The effects f respiratry acidsis n resting membrane ptential, V ma x, and cnductin velcity were slightly but significantly greater than thse induced by metablic acidsis except fr the effect n V ma x at K + = 5.4. Actin ptential duratin was significantly prlnged by metablic acidsis. Hwever, respiratry acidsis did nt cause cnsistent r significant steady state changes in the actin ptential duratin. The magnitude f decrease in cnductin velcity induced by each type f acidsis at ptassium levels f 5.4, 9, and 13 mm is pltted in Figure 2. The cnductin slwing induced by respiratry acidsis at each level f ptassium was significantly greater than that induced by metablic acidsis. The figure als shws that the magnitude f the decrease in cnductin velcity induced by bth types f acidsis at ptassium levels f 9 and 13 mm was significantly greater than that induced at a ptassium level f 5.4 mm. N statistically significant difference was bserved between the decrease in cnductin induced by acidsis at K + = 9 and K + = 13 mm. T determine whether the maximum cnductin speeding effect f increasing ptassium ccurred at the same ptassium cncentratin befre and after acidsis and t determine the effect f acidsis n the relatinship between cnductin velcity and Vmax, we perfrmed three experiments in which ptassium

3 Circulatin Research/Vy. 51, N. 5, Nvember KmM-5.4 Cntrl Or (ph=74) -80 mv M.Acid. (ph=6.5) 0 (m/feec) 0.87 Vmax(V/sec) RMP(-mV) 86 APD(msec) 186 R.Acid. (ph=6.5) 100 V/sec 6 (m/sec) 0.85 Vmax(V/sec) 198 RMP(- mv) 84 APD(msec) msec 4msec FIGURE 1. Effects f extracellular metablic (M) and respiratry (R) acidsis (Acid) n cnductin velcity and actin ptential characteristics at three levels f extracellular ptassium cncentratins. 0 = cnductin velcity; V m n i = maximum rate f rise f the actin ptential upstrke; RMP = resting membrane ptential; and APD = actin ptential duratin. In each panel, the actin ptential clsest t the stimulating electrde is recrded at the slwer sweep speed. The mre distal actin ptential and the differentiated upstrkes are recrded at the mre rapid sweep speed. Thus, nly the upstrke f this actin ptential is displayed. The arrws indicate Vmajt. At each ptassium level, bth types f acidsis decrease 0, Vmax, and RMP, and lengthen APD. Nte that respiratry acidsis causes mre marked changes in 0, Vmax, and RMP and less marked changes in APD than des metablic acidsis. was increased in prgressive 20-minute steps frm 2.7 t 5.4, 7, 9,11, 13,15, and/r 17 min at ph 7.4 and 6.5. In these experiments, acidsis was induced by increasing Pc2. Figure 3 illustrates the results f ne f these experiments. In all ptassium cncentratins at any level f Vmax, cnductin velcity was slwer at ph 6.5 than at ph 7.4. The figure als shws that the relatinship between Vmax and cnductin velcity was similar at bth ph levels. That is: (1) maximal speeding ccurred at K+ = 9 ITIM, (2) cnductin velcity decreased t a value less than that bserved at K+ = 5.4 when K+ was between 11 and 13 mm, and (3) decreasing K+ t 2.7 mm caused a decrease in cnductin velcity with n change in Vma!I. Similar results were btained in the ther tw experiments. T characterize further the interactin between changes in ptassium and ph n Vmax and cnductin velcity, we perfrmed three experiments in which ph was lwered in steps frm 7.4 t 6.9, 6.5, and 6.0 by decreasing HCC>3~ at ptassium levels f 5.4, 9, and 13 mm. The results f ne experiment are shwn in Figure 4. At each level f ptassium, Vmax and cnductin velcity decreased prgressively as ph was lwered. As a result, the speeding in cnductin

4 Kagiyama et al. /Cmbined Effects f Acidsis and K n Ventricular Muscle 617 TABLE 2 Cmpiled Mean Results ± 1 SD frm the Eight Experiments in which Bth Types f Acidsis (ph = 6.5) were Induced at the Three Levels f Extracellular Ptassium RMP (mv) APD (ms) V, (V/sec) 6 (m/sec) C 86.4 ± ± ± ±0.206 K = 5.4 MA 83.8* ± * ±37 216* ± * ±0.184 RA 81.5 ±1.9t 167 ±37t 209* ± * ±0.184$ C 75.1 ± ± ± ±0.227 K = 9 MA 72.9* ± * ±27 200* ± * ±0.211 RA 71.6* ±0.8f 136 ±36$ 194* ±14$ 0.780* ±0.203$ c 65.0 ± ± ± ±0.216 K = 13 MA 63.3* ± * ±20 141* ± * ±0.194 C = cntrl, MA = metablic acidsis, RA = respiratry acidsis. Definitins f RMP, APD, V max, and 8 are in Figure 1. *P < 0.001: cntrl vs. metablic and respiratry acidsis fp < 0.001: metablic vs. respiratry acidsis $P < 0.05: metablic vs. respiratry acidsis RA 61.7* ±1.2f 107 ±28f 133* ±17$ 0.668* ±0.187$ which ccurred when the ptassium cncentratin was changed frm 5.4 t 9 mm at ph 7.4 was eliminated when the ptassium cncentratin was changed frm 5.4 m\i at ph 7.4 t 9 mm at ph 6.5 (see Fig. 1 and Table 2). As indicated abve, acidsis caused a cnsistent decrease in resting ptential and V ma x- Hwever, the results suggested that the changes in V ma x were nt due slely t the change in resting ptential, since the effect f acidsis n Vmax at K + = 5.4 mm was greater than that induced by increasing the ptassium cncentratin t 9 mm, althugh the effect f acidsis n resting ptential was less than that induced by increasing the ptassium cncentratin. This bservatin suggests an acidsis-induced effect n Vmax which was independent f changes in membrane ptential. Figure 5 illustrates the results f an experiment perfrmed t assess the effects f acidsis n the relatinship between resting ptential and V ma x at ph r n=8-1*001- -p<005- I p<0.0l - I (S3 M.Acid ph=65 EZ1 R.Acid 65 -(KQ05- and 6.5 as the ptassium cncentratin was changed in 20-minute steps frm 2.7 t 17.0 mm when ph was 7.4 and frm 2.7 t 15 mm when ph was 6.5. In this experiment, acidsis was induced by increasing Pc2. At resting ptentials between 100 and 70 mv (Fig. 5A), V m ax was less at ph 6.5 than at ph 7.4. Hwever, at resting ptentials less negative than 70 mv, V m ax was greater at ph 6.5 than at ph 7.4. This results in a shift f the nrmalized curve (Fig. 5B) alng the a) 150 x E K mm KmM FIGURE 2. Cmparisn f the percentage decrease in cnductin velcity induced by metablic (M) and respiratry (R) acidsis (Acid) at ptassium levels f 5.4, 9 and 13 mm. The change in cnductin velcity induced by respiratry acidsis was significantly greater than that induced by metablic acidsis at each ptassium level. The changes induced by bth types f acidsis were significantly greater at K* = 9 and K + = 13 than at K* = 5.4, but the differences between K* = 9 and K* = 13 were nt statistically significant. I I I J v ll Q CONDUCTION VELOCITY (m/sec) FIGURE 3. Effects f respiratry acidsis n the relatinship between Vmi and cnductin velcity during stepwise elevatin f extracellular ptassium frm 2.7 t 17 mm. At all levels f ptassium cnductin velcity was faster at ph 7.4 than at ph 6.5. The cnductin speeding effects f increasing ptassium were similar at bth levels f ph, and maximum speeding ccurred at a ptassium cncentratin f 9 mm.

5 618 Circulatin Research/V/. 51, N. 5, Nvember r > K=5.4mM /«''K=I3 X ph = CONDUCTION VELOCITY (rrv%ec) FIGURE 4. Effect f three steady state levels f ptassium at varying ph n the maximum rate f rise f actin ptential upstrke (V m,,x) and cnductin velcity. At all studied ph levels, increasing ptassium frm 5.4 t 9.0 HIM speeds cnductin, and the further increase t 13 mm slws cnductin. Lwering ph in steps frm 7.4 t 6.0 causes a prgressive decrease in bth V mtlx and cnductin velcity at each ptassium level. Nte that the decrease in ph als lessens the speeding effect induced by increasing ptassium frm 5.4 t 9.0 mm. vltage axis in the deplarizing directin. The level f resting ptential assciated with a 50% reductin in V max was -63 mv at ph 7.4 and -60 mv at ph 6.5, a shift f 3 mv. In tw ther experiments f this type, the membrane ptential assciated with a 50% reductin in V m ax was shifted by 4 and 5 mv, respectively. In tw experiments in which acidsis was induced by lwering bicarbnate, the shift was 2.5 mv. Thus, the average shift in the five experiments was 3.4 ± 1.0 mv Because f the similarity f the acidsis-induced shift in the membrane ptential-v ma x relatinship t that induced by increasing extracellular calcium cncentratin (Weidmann, 1955; Gettes and Reuter, 1974; Windisch and Tritthart, 1981) and the knwn increase in calcium activity which ccurs when ph is lwered by decreasing bicarbnate and by increasing PcO2 (Spitzer and Hgan, 1979; Frye and Ple-Wilsn, 1981), we analyzed the effect f increasing extracellular calcium n the Vma X cnductin velcity relatinship. Figure 6 illustrates the results f an experiment in which calcium was varied between 1.8 mm and 7.6 mm at ph f 7.4, nrmalized t the results achieved when calcium equals 1.8 mm. The figure als displays the results illustrated in Figure 4 when ph was lwered frm 7.4 t 6.0 at K + =5.4 mm. These results are als nrmalized t the cntrl value at K + =5.4 mm, Ca ++ =1.8 mm, and ph=7.4. The figure shws that a 2-fld increase in extracellular calcium cncentratin t 3.6 mm causes a 6% decrease in cnductin velcity. This decrease is nly slightly less than that assciated with a change in ph frm 7.4 t 6.5 induced by lwering bicarbnate. As mentined in Methds, this degree f bicarbnate-induced ph change resulted in a change in calcium activity equivalent t a cncentratin f 2.05 mm. The change in cnductin assciated with a 4-fld increase in extracellular calcium t 7.6 mm is similar t the change in cnductin which ccurs when ph is lwered t 6.0 by decreasing bicarbnate. As indicated in Methds, this degree f bicarbnate induced ph change resulted in a change in calcium activity equivalent t a cncentratin f Thus, the results f this experiment indicate that the changes in cnductin induced by acidsis cannt be attributed slely t the increase in extracellular calcium activity. Dynamic Effects The dynamic nature f the effects f bth types f acidsis was investigated at cntrl and elevated ptassium levels. Ptassium was altered either separately r simultaneusly with the change in ph. As indicated in the methds, the new steady state values f ptas > IIO TO 60 RESTING POTENTIAL (-mv) RESTING POTENTIAL (-mv) 50 FIGURE 5. Panel A: Effects f respiratry acidsis n the relatinship between resting membrane ptential and V mi, Extracellular ptassium was increased in steps frm 2.7 t 17 mm. Panel B. Nrmalized curves in which 100% equals the maximum V ma * btained at bth levels f PH.

6 Kagiyama et al. /Cmbined Effects f Acidsis and K n Ventricular Muscle 619 a e Cnductin 1.8 (mm Ca> A 0 0 B c 0 D K = 5.4 Velcity A = ph 7.4 B = ph 6.9 C = ph 6.5 D = ph 6.0 FIGURE 6. Cmparisn f effects f increasing extracellular calcium cncentratin (clsed circles) and metablic acidsis (pen circles) n V m ax and cnductin velcity. The ph data are the same as illustrated in Figure 4 fr K + = 5.4 mi*. The values are nrmalized t thse btained at K + = 5.4 mm, Ca** = 1.8. mm, and ph = 7.4. Nte that a decrease in ptit between 6.9and 6.5 causes cnductin slwing similar t that assciated with a 2-fld increase in calcium, and that a decrease in ph t 6.0 causes cnductin slwing which is nly slightly less than that assciated with a 4-fld increase in extracellular calcium cncentratin. sium and ph were achieved within 20 secnds f changing the perfusate. Figure 7 illustrates the time curse f change in cnductin velcity and actin ptential duratin induced by bth types f acidsis. At ptassium levels f bth 5.4 and 9 mm, cnductin velcity reached a steady state within 4 minutes f inducing respiratry acidsis (clsed symbls), whereas apprximately 15 minutes were required fr the steady state values t be achieved after the inductin f metablic acidsis (pen symbls). The dynamic changes in the actin ptential duratin induced by the tw types f acidsis als differed. At bth levels f ptassium, respiratry acidsis caused either a di- r triphasic effect n actin ptential duratin. Actin ptential duratin first increased with a maximum value ccurring within 2 minutes f the change in ph. Thereafter, actin ptential duratin decreased t a level which, in the experiment illustrated, was nly slightly greater than the cntrl value. As shwn in Table 2, the average change in the steady state value was nt statistically significant. Metablic acidsis caused a prgressive and, as shwn in Table 2, a statistically significant lengthening in actin ptential duratin at bth ptassium levels. Fllwing the return f ph t 7.4 at bth ptassium levels, cnductin velcity and actin ptential duratin returned t the steady state values within 4 minutes, regardless f the type f acidsis. Figure 8 shws the results f an experiment in which ptassium and ph were changed separately and then simultaneusly t levels which are knwn t ccur in the ischemic mycardium fllwing crnary cclusin. ph was lwered by increasing Pc2. The effect f the cmbined changes in ptassium and ph were generally the sum f the effects f the a> «? E LU i I.I Q9 240r 220 E g K-9mM Acidsis -H- 1 -!!- -HH 200- Hh-MI TIME (min.) -Hl- 6 8 K) 15 FIGURE 7. Time curse f change in cnductin velcity (abve) and actin ptential duratin (belw) induced by metablic and respiratry acidsis at tw steady state ptassium levels. Arrws indicate nset and terminatin f acidtic perfusate. The ph f the bathing slutin reached a steady state within 20 secnds f slutin change; hwever, the dynamic changes in cnductin velcity and actin ptential duratin fllwed a slwer time curse. These changes ccurred mre rapidly with respiratry than with metablic acidsis. In additin, the changes in APD induced by respiratry acidsis were diphasicindividual changes. Of particular interest was the simultaneus change in ptassium and ph which caused a transient increase in cnductin velcity. This was fllwed by a decrease in cnductin velcity. The steady state value was less than bserved at a ptassium cncentratin f 5.4 mm and a ph f 7.4. All three parameters returned t the steady state within 5 minutes after the return t the cntrl slutin. Discussin Our experiments were perfrmed t determine the interactin between acidsis and increases in extracellular ptassium cncentratin n cnductin velcity and actin ptential characteristics. We hped that by s ding we wuld gain greater insight int the causes f the changes in cnductin and ther electrphysilgical parameters assciated with ischemia than was available frm previus studies (Dwnar et al., 1976; Marrannes et al., 1979; Senges et al., 1979; Gilmur and Zipes, 1980; Mrena et al., 1980). The effects f increasing ptassium n cnductin and actin ptential characteristics have been studied previusly (Han et al., 1967; Dmingues and Fzzard, 1970; Hlland and Brks, 1976; Arnsdrf et

7 620 Circulatin Research/Vi. 51, N. 5, Nvember 1982 > r r 4- > I 220 Test Slutin 1 ' 0 O O OO -J HhH H»e ee «5.4 9 :-. * = J Q g O ^ ^200- CL 180 < i ", 0»»» v * 8 6 HI-MM O O O O MI-HM TIME (min.) TIME (min.) FIGURE 8. Dynamic effects f separate and simultaneus changes in perfusate ptassium and ph n actin ptential characteristics and cnductin velcity. Arrws indicate pint f change frm cntrl perfusate (K = 5.4, ph = 7.4) t test slutin (cmpsitin shwn in insert). The intrductin f the varius test slutins was separated by at least 30 minutes f superfusin with cntrl slutin. The electrdes were maintained in the same cells thrughut the entire prtcl. The effect f the cmbined changes in ptassium and ph was generally the sum f the individual changes. al., 1977; Sait et al., 1978). These studies have shwn that mderate elevatins f ptassium speed cnductin, whereas higher cncentratins slw cnductin. Our results are cnsistent with these bservatins. The effects f acidsis induced by either an increase in Pc2 r by replacing bicarbnate with chlride r weak acids have als been studied in a variety f mycardial tissues (Vaughan Williams and Whyte, 1967; Jhannsn and Nilssn, 1975; Frye and Ple- Wilsn, 1979; Marrannes et al., 1979; Spitzer and Hgan, 1979; Mrena et al., 1980; Gilmre and Zipes, 1980). Mst f these studies have shwn that acidsis causes a decrease in cnductin velcity. Hwever, there is cnsiderable divergence f results when cnsidering the effects f acidsis n resting ptential, Vmax, and actin ptential duratin. Mrever, the dynamic effects f simultaneus changes in ptassium and ph have rit been previusly detailed. Our results may be summarized as fllws: (1) Extracellular acidsis caused by bth an increase in Pc2 and a decrease in HCO3"" reduced cnductin velcity, resting ptential, and Vmax at all studied ptassium cncentratins between 2.7 and 17.0 ITIM. (2) Decreasing ph t 6.5 ablished the steady state cnductin speeding assciated with an increase in K + frm 5.4 t 9 ITIM. (3) The effect f respiratry acidsis n cnductin velcity, V max, and resting ptential were f greater magnitude and ccurred mre rapidly than the effects f metablic acidsis. (4) Acidsis altered the relatinship between resting membrane ptential and Vmax. (5) The decrease in cnductin velcity induced by acidsis was mre marked at the higher K + levels. (6) Metablic acidsis lengthened steady state actin ptential duratin at all studied ptassium levels, whereas respiratry acidsis did nt cause cnsistent r significant changes. (7) The dynamic effects f simultaneus changes in ptassium and ph culd nt be predicted frm the steady state effects. The mechanism underlying the acidsis-induced decrease in cnductin velcity can be related t changes in bth active and passive membrane prperties (Dminguez and Fzzard, 1970). These include the magnitude f the rapid inward current as reflected by Vmax, threshld ptential, ther parameters f excitability, and cell-t-cell cupling (Fzzard, 1979). The decrease in Vmax is similar t that reprted by van Bgaert and Carmeleit (1972), Vaughan Williams and Whyte (1967), Marrannes et al. (1979), and Nattel et al. (1981). Hwever, neither Jhanssen and Neilsn (1975) nr Grant et al. (1980) were able t dcument a significant decrease in V ma x after the inductin f acidsis. The reasns fr this difference is unclear althugh differences in ph levels r fiber types may be partially respnsible. Tw mechanisms can be pstulated t explain the decrease in V max which we bserved: (1) a decrease in resting ptential with a secndary effect n V m ax; and (2) a direct effect f acidsis n the resting ptential-vmax relatinship. Our results indicate that decreasing ph t 6.5 was assciated with a 2- t 5-mV decrease in resting ptential at all levels f ptassium tested in this study, and that the effect f respiratry acidsis was mre marked than that f metablic acidsis. Vaughan Williams and Whyte (1967), Brwn and Nble (1978a), Spitzer and Hgan (1979), Marrannes et al. (1979), and Nattel et al. (1981) als reprted a decrease in resting ptential assciated with acidsis. Hwever, Jhannsn and Nilssn (1975), Frye and Ple- Wilsn (1979, 1981), and Grant et al. (1980) did nt detect a significant change in resting ptential. Again, the reasns fr this difference are nt bvius. The change in resting ptential may be due t intracellular

8 Kagiyama et al. /Cmbined Effects f Acidsis and K n Ventricular Muscle 621 acidsis inducing a lss f intracellular ptassium (Skinner and Kunze, 1976). We wuld attribute the greater effect f respiratry acidsis than metablic acidsis t a greater degree f intracellular acidsis (Ple-Wilsn and Camern, 1975). The decrease in resting ptential induced by acidsis wuld accunt, at least in part, fr the decrease in V max. Hwever, as shwn in Figure 5, the decrease in Vmax culd nt be accunted fr entirely by this mechanism. Rather, the change in V max represents a direct effect f acidsis n the relatinship between membrane ptential and Vmax- We fund that acidsis depressed V max at membrane ptentials mre negative than 70 mv but increased V max at less negative values, resulting in a shift f the curve describing the membrane ptential- Vmax relatinship alng the vltage axis in the directin f mre psitive ptentials. Nattel et al. (1981) shwed n change in the membrane ptential- Vmax relatinship t canine Purkinje fiber using metablic acidsis t a ph f 6.9. It is pssible that this result reflects the lesser degree f acidsis. Hwever, ur results are supprted by the bservatins f van Bgaert and Carmeleit (1972), wh studied the effect f acidsis in cw Purkinje fibers. This shift is analgus t that induced by a 4- fld increase in extracellular calcium (Weidmann, 1955; Beeler and Reuter, 1970; Gettes and Reuter, 1974; Windisch and Tritthart, 1981). Similar shifts in the steady state vltage dependence f the sdium channel cnductance variables by acidsis and by an increase in extracellular calcium have been reprted in the nde f Ranvier by Hille (1970). Brwn and Nble (1978b) attribute these changes in cnductance variables t the effects f hydrgen and calcium ins n fixed surface charges. It is likely that a decrease in rapid inward current, as reflected by the decrease in V max, is ne f the factrs respnsible fr the acidsis-induced decrease in cnductin velcity. Hwever, in sme situatins, decreases in V max have been shwn t be assciated with speeding f cnductin (Dminguez and Fzzard, 1970; Spear and Mre, 1974; Pen et al., 1978; Sait et al., 1978). An example f this is the increase in cnductin velcity which ccurs when the ptassium cncentratin is raised frm 5.4 t 9 mm. Mrever, the decrease in V ma x des nt explain the greater effect f acidsis n cnductin at ptassium levels f 9 and 13 mm than at 5.4 mm. Of ther factrs which may have influenced cnductin are thse related t excitability (Dminguez and Fzzard, 1970; Spear and Mre, 1974; Pen et al., 1978). This wuld include threshld ptential and the difference between resting and threshld ptentials. Brwn and Nble (1978b) reprted that the threshld ptential fr the sdium current was shifted in a psitive directin by extracellular acidsis. This effect may als be attributed, at least in part, t the increase in calcium activity, and might have cntributed t the slwing f cnductin. It is pssible that the mre marked slwing f cnductin induced by acidsis at ptassium levels f 9 and 13 mm than at a ptassium f 5.4 mm reflects a greater shift in the threshld ptentials at the higher ptassium levels. An increase in internal lngitudinal resistance wuld als cntribute t slwing f cnductin. DeMell (1980) has recently reprted that the intracellular injectin f HC1 caused a marked increase in intracellular lngitudinal resistance in canine Purkinje fibers. His reprt als cites unpublished bservatins f Weingart and Reber shwing similar results in Purkinje fibers expsed t high Pc2. The greater effect f respiratry than metablic acidsis n cnductin velcity which we have bserved may reflect a greater increase in lngitudinal resistance by respiratry acidsis. This culd be related t the fact that CO2 crsses the cell membrane and induces intracellular acidsis mre rapidly and t a greater degree than des a lwering f extracellular bicarbnate (Ple-Wilsn and Camern, 1975; Steenbergen et al., 1977). This effect might als be respnsible fr the shrter time required fr the effects f respiratry acidsis t reach a steady state. Intracellular acidsis might have caused changes in intracellular calcium activity r changes in calcium binding that wuld als tend t uncuple cells (Williamsn et al., 1975; Fabiat and Fabiat, 1978; DeMell, 1975). The intracellular acidsis might als be respnsible fr the shrter time required fr the effects f respiratry acidsis t reach a steady state. We wuld therefre attribute the effects f acidsis n cnductin t bth extracellularly and intracellularly mediated events. The shift in the curve relating resting ptential t V max and the shift in threshld ptential t mre psitive ptentials (Brwn and Nble, 1978b) may be attributed t the effects f extracellular acidsis. The intracellular mediated effects include the leakage f ptassium frm the intracellular t interstitial space (Skinner and Kunze, 1976) which wuld explain the reductin in resting ptential and an increase in lngitudinal resistance leading t cellular uncupling (DeMell, 1980). Bth the extracellularly and intracellularly mediated effects f acidsis may invlve changes in calcium activity. Our studies d nt define the precise cntributin f the changes in calcium activity t the changes induced by extracellular acidsis. A 4-fld increase in extracellular calcium cncentratin has been shwn t cause a 4-mV shift in the curve relating Vmax t membrane ptential (Weidmann, 1955; Beeler and Reuter, 1970; Gettes and Reuter, 1974; Windish and Tritthart, 1981). Thus, it is dubtful that the shift induced by the change in ph frm 7.4 t 6.5 which averaged 3.4 mv can be attributed entirely t this mechanism, since the increase in extracellular calcium activity assciated with this ph change is less than that assciated with a 4-fld increase in extracellular calcium cncentratin. Likewise, we cannt attribute the acidsis-induced changes in cnductin t the assciated change in calcium activity, since acidsis caused a smaller change in calcium activity than that required t cause a similar degree f cnductin slwing. We cannt rule ut the pssibility that intra-

9 622 Circulatin Research/Vi. 51, N. 5, Nvember 1982 cellularly mediated events invlving calcium may have participated in bth phenmena. The changes in ph induced by ischemia, which this study attempts t mdel, invlve changes in calcium activity, since ttal extracellular calcium cncentratin des nt change. Fr this reasn, ur results are applicable t an understanding f the effects f ischemia n cnductin, even thugh they d nt define precisely the cntributin f the calcium activity increase t these changes. We have als nt taken int cnsideratin the pssible changes in smlality and chlride assciated with the varius perfusing slutins. Hwever, based n the results f thers (Spitzer and Hgan, 1979; Kishida and Surawicz, 1979), it is unlikely that these changes cntributed significantly t the changes in resting ptential, V max, cnductin velcity r actin ptential duratin which we have btained. The steady state and dynamic changes in actin ptential duratin which we bserved with bth types f acidsis are similar t thse reprted by thers (Jhannsn and Nilssn, 1975; Spitzer and Hgan, 1979). Spitzer and Hgan (1979) shwed that the prlngatin f actin ptential duratin induced by lwered bicarbnate acidsis was due t the lwering f bicarbnate rather than t the acidsis. The nature f the acidsis induced by ischemia is cmplex (Williamsn et al., 1976; Gevers, 1977; Ple- Wilsn, 1978) and difficult t mdel. In acute ischemia, intracellular acidsis mst likely precedes extracellular acidsis and bth an increase in extracellular Pc 2 (Khuri et al., 1975; Case et al., 1979) and lactate are knwn t accmpany the fall in extracellular ph. We studied the effects f bth respiratry and metablic extracellular acidsis. In this way, ur study differs smewhat frm previus studies which attempt t mdel ischemia using metablic acidsis (Mrena et al., 1980; Gilmre and Zipes, 1980). Increasing Pc2 may be a mre apprpriate methd fr inducing acidsis than decreasing bicarbnate when seeking an explanatin fr the rapidly ccurring changes in cnductin velcity and actin ptential duratin assciated with ischemia. The dynamic changes which we recrded when ptassium was increased t 9, and ph was simultaneusly lwered t 6.5 by increasing Pc2 may accunt fr the cnductin speeding which has been bserved t precede the cnductin slwing effects f acute ischemia (Hlland and Brks, 1976). As shwn in Figure 8, this can be attributed t the fact that the cnductin speeding effects f the rising extracellular ptassium cncentratin were mre prnunced and nt immediately balanced by the cnductin slwing effects f increasing tissue Pc2. Clearly, ischemia als results in accumulatin f lactate in the extracellular space (Williamsn et al., 1976). This effect may be mst imprtant in cnsidering the mre slwly develping changes in actin ptential duratin (Hiraka et al., 1981). Our study als differs frm thers in that it prvides an analysis f the varius cmbinatins f ptassium and ph likely t be encuntered during acute ischemia. In this way, ur study permits an appreciatin f the effects f inhmgeneities in extracellular ptassium (Hill and Gettes, 1980) and ph (Hill et al., 1981) which have been reprted t exist, nt nly between the center and lateral margins f the ischemic zne, but als within the center f the ischemic zne (Hill and Gettes, 1980). Our study suggests that cnductin velcity may be simultaneusly increased, decreased r unchanged at varius lcatins within the ischemic zne, depending n the effect f the varius cmbinatins f ptassium and ph and ther factrs, as well as the varying sensitivity f the fiber types t these factrs (Han et al., 1967; Gilmre and Zipes, 1980). The inhmgeneities in cnductin may be imprtant in the creatin f reentry circuits and the develpment f reentrant ventricular arrhythmia, particularly ventricular fibrillatin knwn t be assciated with acute ischemia. This research was supprted by Grant NIH-HL Address fr reprints: Lenard 5. Gettes, M.D., Divisin f Cardilgy, 353 Burnett-Wmack Building 229H, University f Nrth Carlina, Chapel Hill, Nrth Carlina Received February 22, 1982; accepted fr publicatin August 6, References Arnsdrf MF, Schreiner E, Gambetta M, Friedlander I, Childers RW (1977) Electrphysilgical changes in the canine atrium and ventricle during prgressive hyperkalemia: Electrcardigraphical crrelates and the in viv validatin f in vitr predictins. Cardivasc Res 11: Beeler GW, Reuter H (1970) Membrane calcium current in ventricular mycardium fibers. J Physil (Lnd) 207: Bing RJ (1965) Cardiac metablism. Physil Review 45: Brwn RH, Nble D (1978a) The interactins f prtns, calcium and ptassium ins n cardiac Purkinje fibers. J Physil (Lnd) 282: Brwn RH, Nble D (1978b) Displacement f activatin threshlds in cardiac muscle by prtns and calcium ins. J Physil (Lnd) 282: Case RB, Felix A, Castellana FS (1979) Rate f rise f mycardial pcc>2 during early mycardial ischemia in the dg. Circ Res 45: Cbbe SM, Ple-Wilsn PA (1980) The time f nset and severity f acidsis in mycardial ischemia. J Ml Cell Cardil 12: Chn LH, Fujiwara Y, Cllins JJ Jr (1974) Mapping f ischemic mycardium by surface ph determinatins. J Surg Res 16: Crr PB, Cain ME, Witkwski FX, Price DA, Sbel BE (1979) Ptential arrhythmgenic electrphysilgical derangements in canine Purkinje fibers induced by lysphsphglycerides. Circ Res 44: Cranefield PF, Wit AL, Hffman BF (1973) Genesis f cardiac arrhythmias. Circulatin 47: DeMell WC (1975) Effect f intracellular injectin f calcium and strntium n cell cmmunicatin in heart. J Physil (Lnd) 250: DeMell WC (1980) Influence f intracellular injectin f H + n the electrical cupling in cardiac Purkinje fibres. Cell Bil Int Rep 4: Dminguez G, Fzzard H (1970) Influence f extracellular K + cncentratin n cable prperties and excitability f sheep cardiac Purkinje fibers. Circ. Res 26: Dwnar E, Janse MJ, Durrer D (1976) The effect f "ischemic" bld n transmembrane ptentials f nrmal prcine ventricu-

10 Kagiyama et al./cmbined Effects f Acidsis and K n Ventricular Muscle 623 lar mycardium. Circulatin 55: Fabiat A, Fabiat F (T978) Effects f ph n the myfilaments and the sarcplasmic reticulum f skinned cells frm cardiac and skeletal muscles. J Physil (Lnd) 276: Fzzard HA (1979) Cnductin f the actin ptential. In Handbk f Physilgy, sec 2, The Cardivascular System, vl 1, edited by RM Berne, N Sperelakis. Bethesda, Maryland, American Physilgical Sciety, pp Frye CH, Ple-Wilsn PA (1979) Electrchemical effects f ph in guinea-pig cardiac ventricular muscle. J Physil (Lnd) 293: Frye CH, Ple-Wilsn PA (1981) Effects f acid-base changes n excitatin-cntractin cupling in guinea-pig and rabbit cardiac ventricular muscle. J Physil (Lnd) 313: Gebert G, Benzing H, Strhm M (1971) Changes in the interstitial ph f dg mycardium in respnse t lcal ischemia, hypxia, hyper- and hypercapnia, measured cntinuusly by means f glass micrelectrdes. Pfluegers Arch 329: Gettes LS, Reuter H (1974) Slw recvery frm inactivatin f inward currents in mammalian mycardial fibers. J Physil (Lnd) 240: Gevers W (1977) Generatin f prtns by metablic prcesses in heart cells. J Ml Cell Cardil 9: Gilmur RF, Zipes D (1980) Different electrphysilgical respnses f canine endcardium and epicardium t cmbined hyperkalemia, hypxia and acidsis. Circ Res 46: Grant AO, Strauss LJ, Strauss HC (1980) The influence f ph n the electrphysilgical effects f lidcaine in guinea pig ventricular mycardium. Circ. Res 47: Han J, Malzzi AM, Me GK (1967) Transient ventricular cnductin disturbances prduced by intra-atrial injectin f single dses f KC1. Circ Res 21: 3-8 Harris AS, Rjas AG (1943) Initiatin f ventricular fibrillatin due t crnary cclusin. Exp Med Surg 1: Harris AS, Bisteni A, Firestne JE (1954) Excitatry factrs in ventricular tachycardia resulting frm mycardial ischemia. Ptassium a majr excitant. Science 119: Hill JL, Gettes LS (1980) Effect f acute crnary artery cclusin n lcal mycardial extracellular K + activity in swine. Circulatin 61: Hill JL, Gettes LS, Lynch MR (1978) Flexible valinmycin electrdes fr n-line determinatin f intravascular and mycardial K +. Am J Physil 235: H455-H459 Hill JL, McCwn PM, Gettes LS (1981) Relatinship between changes in extracellular K + and ph in the ischemic swine heart (abstr). Circulatin 64: IV-152 Hille B (1970) Inic channels in nerve membranes. In Prgress in Biphysics, vl 21, edited by JAV Butler, D Nble. Oxfrd, Pergamn Press, pp 1-32 Hirche HJ, Franz CHR, Schramm M (1980) Mycardial extracellular K + and H + increase and nradrenaline release as pssible cause f early arrhythmias fllwing acute crnary artery cclusin in pigs. J Ml Cell Cardil 12: Hiraka M, Okamt Y, San T (1981) Electrphysilgic effects f Iactates in mammalian ventricular tissues, j Electrcardil 14: Hlland R, Brks H (1976) The QRS cmplex during mycardial ischemia: An experimental analysis in the prcine heart. J Clin Invest 57: Jhannsn M, Nilssn E (1975) Acid-base changes and excitatincntractin cupling in rabbit mycardium. II. Effects n resting membrane ptential, actin ptential characteristics and prpagatin velcity. Acta Physil Scand 93: Kaplinsky E, Ogawa S, Balke CW, Dreifus LS (1979) Tw perids f early ventricular arrhythmias in the canine acute mycardial infarctin mdel. Circulatin 60: Khun SF, Flaherty JT, O'Rirdn JB, Pitt B, Brawley RK, Dnahu JS, Gtt VL (1975) Changes in intramycardial ST segment vltage and tensins with reginal mycardial ischemia in the dg. Circ Res 37: Kishida H, Surawicz B, Fu LT (1979) Effects f K+ and K+-induced plarizatin n (dv/dt)max, threshld ptential, and membrane input resistance in guinea pig and cate ventricular mycardium. Circ Res 44: Marrannes R, dehemptinne, Leusen I (1979) Influence f lactate and ther rganic ins n cnductin velcity in mammalian heart fibers depressed by "metablic" acidsis. J Ml Cell Cardil 11: Mrena H, Janse ML, Durrer D (1980) Cmparisn f the effects f reginal ischemia, hypxia, hyperkalemia and acidsis n intracellular and extracellular ptentials and metablism in the islated prcine heart. Circ Res 46: Nattel S, Elharrar V, Zipes DP (1981) ph-dependent electrphysilgical effects f quinidine and lidcaine n canine cardiac Purkinje fibers. Circ Res 48: Pen J, Ferrier GR, Me GK (1978) The relatinship f excitability t cnductin velcity in canine Purkinje tissue. Circ Res 43: Ple-Wilsn PA (1978) Measurement f mycardial intracellular ph in pathlgical states. J Ml Cell Cardil 10(6): Ple-Wilsn PA, Camern IR (1975) Intracellular ph and K + f cardiac and skeletal muscle in acidsis and alkalsis. Am J Physil 229: Sait T, Hill JL, Gettes LS (1978) Determinants f cnductin velcity in guinea pig papillary muscle at varying K cncentratins (abstr). Circulatin 58(suppl II): 45 Senges J, Brachmann J, Pelzer D, Mizutani T and Kubler W (1979) Effects f sme cmpnents f ischemia n electrical activity and re-entry in the canine ventricular cnducting system. Circ Res 44: Skinner RB Jr, Kunze DL (1976) Changes in extracellular ptassium activity in respnse t decreased ph in rabbit atrial muscle. Circ Res 39: Spear JF, Mre EN (1974) Supernrmal excitability and cnductin in the His-Purkinje system f the dg. Circ Res 35: Spitzer KW, Hgan PM (1979) The effects f acidsis and bicarbnate n actin ptential replarizatin in canine cardiac Purkinje fibers. J Gen Physil 73: Steenbergen C, Deletuw G, Williamsn JR (1977) Effects f acidsis and ischemia n cntractility and intracellular ph f rat heart. Circ Res 41: van Bgaert PP, Carmeliet E (1972) Sdium inactivatin and ph in cardiac Purkinje-fibers (abstr). Arch Int Physil Bichem 80: Vaughan Williams EM, Whyte JM (1967) Chemsensitivity f cardiac muscle. J Physil (Lnd) 189: Weidmann S (1955) Effects f calcium ins and lcal anaesthetics n electrical prperties f Purkinje fibers. J Physil (Lnd) 129: Wiegand V, Guggi M, Messmann W, Kessler M, Greitschus F (1979) Extracellular ptassium activity changes in the canine mycardium after acute crnary cclusin and the influence f beta blckade. Cardivasc Res 13: Williamsn JR, Safer B, Rich T, Schaffer S, Kbayashi K (1975) Effects f acidsis n mycardial cntractility and metablism. Acta Med Scand Suppl 587: Williamsn JR, Schaffer SW, Frd C, Safer B (1976) Cntributin f tissue acidsis t ischemic injury in the perfused rat heart. Circulatin 53(suppl I):3-114 Windisch H, Tritthart HA (1981) Calcium in effects n the rising phases f actin ptentials btained frm guinea-pig papillary muscles at different ptassium cncentratins. J Ml Cell Cardil 13: Wjtczak J (1979) Cntractures and increase in internal lngitudinal resistance f cw ventricular muscle induced by hypxia. Circ Res 44: INDEX TERMS: Mdeled ischemia Impulse prpagatin Slw cnductin Cell-t-cell cupling