Post-extra systouc potentiation: Influence of calcium and verapamil in rat and rabbit he arts

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1 urpean Heart Jurnal (1985) bl Pst-extra systuc ptentiatin: Influence f calcium and verapamil in rat and rabbit he arts J. P. KRKR T. J. C. RUIGROK J. H. M. NIUWNHUIJS* C. BORST AND F. L. MIJLR Department fcardilgy University Hspital Utrecht The Netherlands *and the Department f Physilgy State University Utrecht Utrecht The Netherlands KY WORDS: PSP calcium calcium antagnist cntractility. The interactins fthe intrpic effects fverapamil ( 5-2' Jlml Z-1 ) calcium ('33-5'2 mml Z-1 ) and pst extra systlic ptentiatin (PSP) as induced by paired stimulatin were studied in islated rabbit and rat hearts under isvlumic and istnic cnditins. At lw dses f verapamil cntractins were depressed but thse elicited by paired stimulatin shwed less depressin than cntractins f the same rate during single stimulatin and even exceeded the unptentiated cntractins withut verapamil. At high dses f verapamil cnlractility culd nt be restred by paired stimulatin. Althugh cntractins we re restred t cntrllevel by an increase in extra-cellular calcium they were still abnrmal in the sense that PSP culd nt be elicited. The excitatin-cntractin (e-c) uncupling due t lw calcium perfusin culd be cunteraeted by paired stimulatin but e- c uncupling due t high dse verapamil culd nt be reversed by paired stimulatin. Dur results supprt the view that PSP des nt nly depend n augmented slw channel calcium influx but als n an enhanced calcium shift within the sarcplasmic retieulum. We are dubtful abut the idea that P SP can be used clinically t cunteract the negative intrpic effect f high dses f verapamil. Intrductin The mechanism by which an extrasystlic beat elicits ptentiatin f the next beat (pst extra systlic ptentiatin PSP) is nt fully understd[l - 1. During paired stimulatin every regular cardiac cntractin is fllwed by an extra systle. Therefre paired stimulatin is a frm f sustained PSP. The premature deplarizatin has t be induced early enugh t be mechanically ineffective and thus there will be tw deplarizatins fr ne mechanical cntractin[1 71. Recently the clinical relevance fpsp has been re-emphasized[81. xcitatin--cntractin cupling and thus the relatinship between cycle length and strength f cntractin may be interpreted in terms f calcium in mvements[ The effect f calcium antagnists has been attributed t inhibitin f these calcium in mvements[121. In the present study the interactins f the intrpic effects f Address Jr reprints: F. L. Meijler Department f Cardilgy University Hspital Catharijnesingel GV Utrecht The Netherlands. Received fr publicatin n 2 Nvember 1984 and in revised frm 4 March X $2. verapamil calcium and paired stimulatin were investigated t explre further the mechanisms respnsible fr PSP and fr the negative intrpic effect fverapamil. Materials and methds Perfusin experiments we re carried ut with islated hearts f New Zealand white rabbits (25-3 g) and Wistar rats (2-25 g). The rabbits were anesthetized with sdium pentbarbital and heparinized. After islatin f the heart the arta and mitral rifice were cannulated[ An e1ectric valve in the artic cannula allwed fr crnary perfusin (37 C; mmhg) during a cntrlled perid f diastle but was therwise clsed. An electric valve in the mitral cannula was pened during every diastlic pause. nd-diastlic pressure was maintained at 4 mmhg. Intraventri cu lar pressure during isvlumic cntractins was measured with a Millar Mikr-Tip pressure transducer inserted via the mitral cannula. The rats were anesthetized with diethyl ether and heparinized. The hearts we re quickly remved and subsequently perfused at 37 C by the Langendrff 1985 The urpean Sciety f Cardilgy

2 lnfluence f calcium and verapamil n P SP 533 technique at a cnstant pressure f 75 mmhg. The istnic cntractins f the hearts were recrded as described earlier[141. The perfusin fluid reservirs were cnnected t the artic cannula by means f a specially designed stpcck allwing successive use f the reservirs. The standard perfusate had the fllwing cmpsitin (mmll- 1 ): NaCl 124: KCl 4'7; CaC1 2 13; MgClz 1; NaHC 3 24; Na z HP 4 5; glucse 11. The perfusin fluids were equilibrated with 95 % O2-5% COz t prvide a p2 f > mmhg and had a ph f7ao± 5. When required 5-2 Ilmll - l verapamil was added t the perfusate. In a number f experiments the CaClz cncentratin was increased t 2 r 5 2 mmll - 1 r decreased t 5 r O 33 mmll - 1. The left ventricular electrgram was recrded by means f biplar epicardial electrdes cnnected t a Guld amplifier and a Guld Brush 28 ink recrder. T prevent interference with prpagated sinus beats the atria were partially remved and atriventricular cnductin was blcked by means f an irn clip in the interventricular septum f the rat heart and by cauterizing the His bundie in the rabbit heart. Right ventricular stimulatin weil abve threshld with impulses f I ma with a duratin f I ms we re applied. In the rabbit he arts the fllwing prtcl was applied: single stimulatin fr 1-15 min at a frequency f 1 Hz paired stimulatin fr 1 s using stimulus pairs f equal strength and duratin with intervals f 2 and 8 ms alternatively fllwed by single stimulatin until steady state was reached. Subsequently the perfusin was changed t a medium cntaining 2 r 1 Ilmll- 1 verapamil and after 2 min f equilibratin the same prtcl was repeated. In the rat hearts the fllwing prtcl was applied: single stimulatin fr 1-15 min at a frequency f 2 Hz paired stimulatin fr 3 s with intervals f 7 and 43 ms alternatively single stimulatin at 2 Hz until steady state was reached fllwed by single stimulatin fr 3 min at 4 Hz. Subsequently the perfusin was changed t a medium cntaining '5-2'Ilmll - 1 verapamil '2 Ilmll - 1 verapamil and 2 mmll - 1 calcium r 1'IlillOll- 1 verapamil and 5 2 mmll - 1 calcium and after 2 min f equilibratin the same prtcl was repeated. STATISTICAL ANALYSIS Oa ta are presen ted as means ± standard errr f the mean (SM). Statistical significance was determined by the Wilcxn signed rank test. A P < 5 was cnsidered t indicate a significant difference between means. Results INFLUNC OF VRAPAMIL IN TH RABBIT HART The peak isvlumic intraventricular pressure during single stimulatin in the rabbit hearts amunted t 7 ± 8 mmhg (mean ± SM; n = 8). Ouring paired stimulatin it increased t 11 ± 12 mmhg. Figures land 2 shw the effect f 2 and 1 Ilmll - 1 verapamil n the unptentiated and ptentiated cntractins. Ouring perfusin with '2Ilmll- l verapamil paired stimulatin still increased the pressure abve the level f single stimulatin befre additin f verapamil 1 '" I L-...J 1 ms eg Figure 1 The intraventricular pressure f the rabbit heart during single stimulatin (lef! panel) and paired stimulatin (right panel) withut verapamil (a and a') with 2 mll -l verapamil (b and b') and with 1 mll -l verapamil (c and c'). Original tracings f a representative experiment are phtgraphically superimpsed.

3 534 J. P. Kerker et al. '" Cl> - 2- :; 1- ().: e Cl> > -Cl>...J 1 e c:> a :; 5 V> " D x.. Verapamil (flmll- l ) Figure 2 The relative effect f verapamil ( 2 and 1 J.lmll- ') n the peak systlic intraventricular pressure f the rabbit heart during single and paired stimulatin. Intraventricular pressure was cmpared t the initial unptentiated cntractins befre verapamil was added (1% level). The data indicate mean±sm f 8 hearts. The carsely dtted clumn indicates single stimulatin. The empty clumn indicates paired stimulatin. whereas paired stimulatin during perfusin with 1 Ilmll-l verapamil althugh still causing cnsiderable ptentiatin was unable t equal this level. In Fig. 3 the depressin by verapamil during single stimulatin was cmpared with that during paired stimulatin. Fr bth verapamil cncentratins the depressin f the ptentiated cntractins was significantly less than that f the unptentiated cntractins (P < '5). INFLUNC OF VRAPAMIL IN TH RAT HART The effect f six cncentratins f verapamil at a calcium cncentratin f I 3 mmll- 1 in the perfusin fluid n the unptentiated and ptentiated cntractins f rat hearts is shwn in Fig. 4. Befre expsure t verapamil the height f the ptentiated cntractins amunted t 15 ± 5% f the unptentiated cntractins. Af ter 3 s f paired stimulatin resumptin f single stimulatin resulted in a transient pst-stimulatin depressin. Recvery was always cmplete befre verapamil was added. Increasing the stimulatin frequency 5 Single slimulalin (%) 1 Figure 3 The relative effect f verapamil n the peak systlic intraventricular pressure f isvlumically cntracting rabbit hearts (N = 8) during single stimulatin at 1 Hz and paired stimulatin. Intraventricular pressure befre verapamil was 1%. The brken line is the line fidentity. VerapamilcncentratininJ.lmll- 1 : x ='2 =1. frm 2 t 4 Hz resulted in a transient increase in cntractin height. After 3 min a new steady state was reached which did nt differ significantly frm the cntractin height at 2 Hz. The cardidepressive effect f verapamil was cncentratin dependent and mre prnunced at higher stimulatin rates. At verapamil cncentratins f 2Ilmll- l and higher paired stimulatin caused cnsiderable ptentiatin but failed t bring the cntractin height back t the level f the unptentiated cntractins in the absence f verapamil. At verapamil cncentratins f 5 1 and '2Ilmll-l the ptentiated cntractins were significantly less depressed than the unptentiated cntractins (P < '5) (Fig. 5). INTRACTION OF VRAPAMIL AND CALCIUM IN TH RATHART In Fig. the relatin between a test interval (see insert: x) and the crrespnding apex t base displacement is demnstrated. This relatinship is called the restitutin curve[15j. As has been shwn previusly[1j the restitutin kinetics under cn trl cnditins first fllw a nearly expnential curve with a time cnstant f abut 3 ms reaching a plateau at 1 t 2 s smetimes via a slight versht. Paired stimulatin resulted in an upward

4 Injluence f calcium and verapamil n PSP J I : c: g 1 Q. I) " I) C.!l >< Q. « ' Verpmil (flmll- l ) Figure 4 The relative effect f verapamil (5-2!lmll- l ) n the apex t base displacement f rat hearts at different stimulatin patterns. Displacement was cmpared t the initial unptentiated displacement (1% level). The data indicate mean ± SM f 8 hearts in each grup. The carsely dtted clumn indicates single stimulatin at 2 Hz the empty clumn indicates paired stimulatin the finely dtted clumn indicates single stimulatin at 4 Hz ' c.2 Od). rt 5 1 Single slimulalin (%l Figure 5 The relative effect f verapamil (5-2!lmll- l) n the apex t base displacement f rat hearts (n = 8) during single stimulatin at 2 Hz and paired stimulatin. Displacement befre verapamil was 1%. The brken line is the line f identity. Verapamil cncentratin in!lmll- l : À ='5 =1 ='2 8 ='4 = 1' =2. shift f the restitutin curve[1] [Fig. (a)] whereas verapamil and lw calcium resulted in a dwnward shift f the restitutin curve[17.18] [Fig. (c) and (d)]. When the verapamil induced depressin f cntractins during single stimulatin was nrmalized by an increased calcium cncentratin paired stimulatin was unable t prduce a further upward shift f the restitutin curve [Fig. (b)]. Verapamil ( 2 Ilml 1-1) and a lw calcium cncentratin ('5 mmll- 1 ) caused an almst identical dwnward shift f the restitutin curve [Fig. (c) and (d)]. Paired stimulatin ptentiated cntractins mre in lw-calcium depressed than in verapamil depressed rat hearts [Fig. (c) and (d) Fig. 7]. When depressed cntractins due t high cncentratins f verapamil were nrmalized by an increased calcium cncentratin a further effect f paired stimulatin culd nt be btained. Discussin In ur experiments paired stimulatin f rat hearts culd vercme the negative intrpic effect

5 53 J. P. Kerker et al. ' M"'t'f ' " r- X ---. f l llll I----.J r-x---j.!i!i!i!i!i I! ; <Il 2 c: ::l >. % "..c b ' 1 t:j u Q. <Il "t:i <Il..c 2 )( Q. «1 '1' M M ' Ö M I û u () (l? b "u I Test interval (ms) Figure The influence f verapamil and calcium n apex t base displacement- test interval relatinships f a single istnic cntractin fllwing single (intervals 5 ms) r paired (intervals 7 and 43 ms) stimulatin f an islated rat heart. (a) cntrl (b) verapamill'o ilmll- t and calcium 5 2 mmll - t (c) calcium 5 mmll- t (d) verapamil 2ilmll- t. f 5 and 1 mll - 1 verapamil bringing the cntractin height abve the level f the unptentiated cntractin in the absence f verapamil. At the higher verapamil cncentratins hwever paired stimulatin was unable t restre cntractin height t cntrl levels. In the rabbit hearts paired stimulatin at 2 mll-l verapamil restred the intraventricular pressure till abve the level f single stimulatin withut verapamil but at 1 mll- I verapamil paired stimulatin was unable t restre intraventricular pressure t cn trl levels. In a previus study[18] it was demnstrated that paired stimulatin can vercme in part the e-c uncupling induced by Iw calcium perfusin ( 3 mmll- 1). The present study shws that paired stimulatin cannt d s if e-c uncupling has been caused by high dses f verapamil. Our

6 Influence f calcium and verapamil n P SP c '" 1 - " '" x '" - ct Calcium 2 Verapamil Figure 7 The effect fsimultaneus additin fverapamil and increase fthe calcium cncentratin (Ieft) a decreased calcium cncentratin (middle) and verapamil (right) n the apex t base displacement during single and paired stimulatin. Apex t base displacement was cmpared t the initial unptentiated displacement during standard perfusin (1% level). The data indicate mean ± SM f 8 rat hearts in each grup. data slightly differ frm the results f Wiggins et al.[4 J btained frm papillary muscies and trabeculae carneae f dg cat and pig-tail mnkey hearts. These authrs demnstrated that paired stimulatin in the presence f high dses f verapamil (lllmll- 1 ) brught the frce f cntractin t a level exceeding that f an unptentiated cntractin withut verapamil. Given ur results (Figs 1-5) with islated perfused he arts we are dubtful abut the idea that paired stimulatin culd be used ciinically t cunteract the negative intrpic effect f high dses f verapamil. When cntractin height was equally depressed by either lw calcium r verapamil the restring effect f paired stimulatin was less prnunced in the latter [Fig. (c) and (d) Fig. 7]. At the higher verapamil cncentratin paired stimulatin culd nt restre cntractility while an increased calcium cncentratin culd [Figs (b) and 7]. In these restred cntraetins hwever n further ptentiatin culd be elicited by PSP unveiling an abnrmal cntractiie behaviur. It has been suggested that ptentiatin induced by paired stimulatin is mediated nt nly by an increased transfer f calcium acrss the cell membrane but als by an enhanced shift f calcium inside the sarcplasmie reticulum frm uptake t release sites[5.19j. This culd explain the upward shift f the restitutin curve[ j. Verapamil is nt nly a slw channel blcker but mayals slw dwn the kinetics f calcium reavailability frm these internal stres[17j. Our results shw that lw dses f verapamil ( '5-2 Ilml 1-1 ) had less effect n the cntractins during paired stimulatin than n thse during single stimulatin. Since the inhibitin f the slw calcium channels is mre prnunced at a higher stimulatin rate[2j a mre prnunced effect fverapamil wuld be expected during paired stimulatin if an increased transfer f calcium acrss the cell mem bra ne were the nly mechanism f PSP. Hwever if PSP is als mediated by an enhanced shift f calcium inside the sarcplasmie reticulum this effect may be cunteracted in a dse-related way by verapamil. Pssibly this intracellular effect f verapamil cannt be vercme by an increased extracellular calcium cncentratin. This wuld imply th at neither the term 'slw channel blcker' nr 'calcium antagnist' is fully apprpriate t describe the mechanisms f actin f verapamil[12 1. It might als explain the reprted failure f calcium infusin in the treatment f severe verapamil pisning[21j. In cnciusin ur results are cmpatible with the view that PSP depends nt nly n an augmented calcium influx thrugh the slw channels but als n an enhanced calcium shift within the sarcplasmie reticulum. Verapamil in high dses

7 538 J. P. Kerker et al. ablishes the psitive intrpic effect f paired stimulatin. References [I] Cranefield PF. The frce f cntractin f extrasystles and the ptentiatin f frce f the pstextrasystlic cntractin: a histrical review. Bull NY Acad Med 195; 41: [2] Cranefield PF. The present status f paired pulse stimulatin and pstextrasystlic ptentiatin in the heart. Bull NY Acad Med 195; 41: [3] Suk J Ueba Y Chidsey CA. 1. lntracellul<lr calcium and mycardial cntractility. Il. ffects f pstextrasystlic ptentiatin in the islated rabbit heart. Circ Res 197; 27: [4] Wiggins JR Leary JM Cranefield PF. The effects f verapamil and paired pulse stimulatin n mammalian ventricle. ur J Cardil1975; 3: [5] De Mell WC. Pst-extrasystlic ptentiatin; effect f Ca histamine caffeine and epinephrine. Arch Int Pharmacdyn Ther 1977; 23: [] Iinuma H Kat K. The effects f extracellular ptassium and several drugs n the premature actin ptential and pstextrasystlic ptentiatin. ur J Cardil 1978; 7: [7] Hffman BF Bartelstne HJ Scherlag BJ Cranefield PF. ffects f pstextrasystlic ptentiatin n nrmal and failing hearts. Bull NY Acad Med 195; 41 : [8] Meijler FL Kuijer PJP Van Dam-Kpman I Heethaar RM Van der Werf T. Clinical relevance f pstextrasystlic ptentiatin. May Clin Prc 1982; 57 (Suppl): [9] Wd H Heppner RL Weidmann S. lntrpic effects f electric currents 1. Psitive and negative effects f cnstant electric currents r current pulses applied during cardiac actin ptentials Il. Hyptheses: calcium mvements excitatin-cntractin cupling and intrpic effects. Circ Res 199; 24: [lol Mrad M Gldman Y. xcitatin-cntractin cupling in heart muscie: membrane cntrl f develpment f tensin. Prg Biphys Ml Bil 1973; 27: [11] Kaufmann R Bayer R Fürniss T Krause H Tritthart H. Calcium mvement cntrlling cardiac cntractility Il. Analg cmputatin f cardiac excitatincntractin cupling n the basis f calcium kinetics in a multicmpartment mdel. J Ml Cell Cardil 1974; : [12] Nayler WG Ple-Wilsn P. Calcium antagnists: definitin and mde f actin. Basic Res Cardil 1981; 7: [13] Bm HBK Denier Van Der Gn JJ Nieuwenhuijs JHM Schiereck P. Cardiac cntractility: Actin- mysin interactin as measured [rm the left ventricular pressure curve. ur J Cardil1973; I: [14] Meijler FL Van Den Bgaard F Van Der Tweel H Durrer D. Pstextrasystlic ptentiatin in the islated rat heart. Am J Physil192: 22: [15] Kruta V Braveny P. Perfrmance f heart muscie in rhythmic activity. Restitutin f cntractility and ptentiatin phenmena. Prc Int Cng Physil 192; 22: [1] Meijler FL Nieuwendijk S Durrer D. Physilgical basis f paired stimulatin ptentiatin. In: Cranefield PF Hffman BF eds. Paired pulse stimulatin f the heart. New Yrk: The Rckefeller University Press 198: [17] Bayer R Hennekes R Kaufmann R Mannhld R. Intrpic and electrphysilgical actins fverapamil and D in mammalian mycardium I. Pattern f intrpic effects f the racemic cmpunds. Naunyn Schmiedebergs Arch Pharmacll975; 29: [18] Nieuwendijk S Meijler FL Durrer D. Influence f paired stimulatin n the electrmechanical dissciatin during lw calcium perfusin f islated rat hearts. Cardivasc Res 197; I: [19] Pidgen J Lab M Seed A lzinga G Papadyannis D Nble MIM. The cntractile state f cat and dg heart in relatin t the interval between beats. Circ Res 198; 47: [2] hara T Kaufmann R. The vltage- and timedependent effects f ( - ) verapamil n the slw inward current in islated cat ventricular mycardium. J Pharmacl xp Ther 1978; 27: [21] Crump BJ Hlt DW Vale JA. Lack f respnse t intravenus calcium in severe verapamil pisning. Lancet 1982; ii: