Hypertension-related intermyocyte junction remodelling is associated with a higher incidence of...

It has been reported that long-term inhibition of nitricoxide (NO) production by the NO synthase inhibitor-NAME results in a decrease in vascular relaxation(Holecyova et al. 1996) accompanied by an increase insystemic blood pressure (Riberio et al. 1992) and vascularremodelling (Nugamuchi et al. 1995; Kristek & Gerova,1996). The development of NO-deficient hypertension isaccompanied by a significant decrease in the activity of NOsynthase activity (Pechanova et al. 1997; Tribulova et al.2000) and Na+,K+-ATPase (Vrbjar et al. 1999), and

histochemical techniques have shown heterogenouslyreduced activities of glycogen phosphorylase, succinicdehydrogenase, 5‚-nucleotidase, alkaline phosphatase andATPases (Tribulova et al. 2000).

These metabolic alterations were linked with morphologicalchanges manifested particularly by interstitial fibrosis,moderate myocardial hypertrophy and ischaemia-like injury(Moreno et al. 1996; Pechanova et al. 1997, 1999; Tribulovaet al. 2000) as well as by both angiopathy and angiogenesis(Okruhlicova et al. 2000). The angiotensin-converting

Hypertension-related intermyocyte junction remodelling isassociated with a higher incidence of low-K+-induced lethal

arrhythmias in isolated rat heart

N. Tribulova*, L. Okruhlicova, S. Novakova, D. Pancza, I. Bernatova†,

O. Pechanova†, P. Weismann‡, M. Manoach§, S. Seki|| and S. Mochizuki||

Institute for Heart Research, Slovak Academy of Sciences, Bratislava, † Institute of Normal

and Pathological Physiology, SAS, Bratislava, ‡ Department of Anatomy, Medical Faculty of

Comenius University, Bratislava, Slovak Republic, § Department of Physiology,

Tel Aviv University, Tel Aviv, Israel and || Department of Internal Medicine 4, Jikei University,

Tokyo, Japan

(Manuscript received 29 November 2001; accepted 14 January 2002)

The aim of this study was to characterise the arrhythmogenic mechanisms involved in hypokalaemia-inducedsustained ventricular fibrillation (SVF), in hypertensive rats. The hearts from rats with hypertension inducedby the nitric oxide synthase inhibitor -NAME, and age-matched normotensive controls, were perfused inLangendorff mode with oxygenated Krebs-Henseleit solution followed by a K+-deficient solution. In additionalexperiments, free intracellular Ca2+ concentration ([Ca2+]i) was measured using fura-2 in conjunction with anepicardial optical probe. The epicardial electrocardiogram was continuously monitored during allexperiments. The gap junction protein connexin-43 and the ultrastructure of the cardiomyocytes wereexamined, and selected enzyme activities were measured in situ. There was a higher incidence of low-K+-induced SVF in the hearts of hypertensive compared to normotensive rats (83 % vs. 33 %, P < 0.05). Perfusionwith a low-K+-containing solution lead to elevation of diastolic [Ca2+]i that was accompanied by prematurebeats, bigeminy, ventricular tachycardia and transient ventricular fibrillation. These events occurred earlierwith increased incidence and duration in the hearts of hypertensive rats (arrhythmia scores: hypertensive,4.9 ± 0.7; normotensive, 3.1 ± 0.1; P < 0.05), which exhibited apparent remodelling accompanied by asignificant decrease in the density of connexin-43-positive gap junctions. Moreover, low-K+-related myocardialchanges, including local impairment of intermyocyte junctions, ultrastructural alterations due to Ca2+ overloadand intercellular uncoupling, and decreased enzyme activities were more pronounced and more dispersed inhypertensive than normotensive rats. In conclusion, nitric oxide-deficient hypertension is associated withdecreased myocardial coupling at gap junctions. The further localised deterioration of junctional coupling, dueto low-K+-induced Ca2+ disturbances, as well as spatial heterogeneity of myocardial alterations includinginterstitial fibrosis, probably provide the mechanisms for re-entry and sustaining ventricular fibrillation.Experimental Physiology (2002) 87.2, 195–205.

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enzyme inhibitor captopril prevented hypertension andhypertrophy, and reduced the collagen concentration(Pechanova et al. 1997) although it did not affect nitricoxide synthase activity (Tribulova et al. 2000; Bernatova etal. 2000) and angiogenesis related to NO deficiency(Okruhlicova et al. 2000). This indicates that NO-deficienthypertension is accompanied by metabolic and structuralalterations of the heart that are probably not induced bythe renin–angiotensin system alone (Cunha et al. 1993;Moreno et al. 1996).

Data have not yet been obtained to determine whetherthese myocardial changes are associated with electro-physiological alterations that may affect the electricalstability of the heart. Nevertheless, hypertension is knownto increase significantly the risk of morbidity and mortalityas a result of cardiovascular alterations (Weber & Brilla,1991; Griendling et al. 1993) and fatal arrhythmias. Itmight be expected that NO-deficient hypertension wouldbe accompanied not only by myocardial remodelling butalso by alterations in Ca2+ handling, in line with othermodels of hypertension, and consequently increase thesensitivity of the heart to Ca2+ overload. Both Ca2+

overload and structural remodelling may contribute to thedevelopment of arrhythmogenic substrates and susceptibilityto arrhythmias. Fibrosis, infarct scars or necrotic lesionswere shown to reduce cardiomyocyte interconnections anddecrease myocardial coupling at the gap junctions (Spach& Heidlage, 1995; Saffitz et al. 1999; Tribulova et al. 1998).

This can create alternative pathways and conduction blockor slowed conduction, facilitating re-entry arrhythmias inboth ischaemic (Peters et al. 1997) and senescent hearts(Spach & Heidlage, 1995; Tribulova et al. 1999).

Arrhythmias are known to be induced during variouspathophysiological conditions such as ischaemia and/orreperfusion. Likewise, electrolyte imbalance due topotassium deficiency can enhance the risk of arrhythmiasnot only in an experimental setting (Tribulova et al.2001a,b), but also in clinical conditions, particularlyduring therapy with antihypertensive or other medications(Janko et al. 1992). It was reported that hypokalaemiadecreased myocardial electrical stability by alterations inexcitability, action potential duration and conductionvelocity (Helfant, 1998). Moreover, our previous resultsshowed that lack of potassium can cause disturbances ofmetabolism and Ca2+ handling, resulting in subcellularinjury and impairment of intermyocyte coupling (Tribulovaet al. 2001a,b). An increase in diastolic free [Ca2+] inhibitsgap junction channels, increases gap junctional resistanceand decreases conductance (for review see Dhein, 1998),thus it may be involved in uncoupling, and conductiondisturbances that facilitate microre-entry and the occurrenceof re-entry arrhythmias.

The aim of this study was to examine the susceptibility ofrats with NO-deficient hypertension, and age-matchedcontrols, to low-K+-induced arrhythmias (i.e. ventricularfibrillation), and to investigate the involvement ofintermyocyte junctions in the development of arrhythmiastructural substrate.

METHODSThe investigation conformed with the Guide for the Care andUse of Laboratory Animals published by the United StatesNational Institutes of Health, 1985. Male, 12-week-old Wistarrats (n = 20) were treated with -NAME at a daily dose of40 mg kg_1 in the drinking water for 4 weeks. Age-matchedcontrol (n = 25) were used. Systolic blood pressure was measuredweekly by tail-cuff plethysmography.

Isolated Langendorff heart preparation

Rats were killed by cervical dislocation and the heart was quicklyexcised and placed into ice-cold Krebs-Henseleit solution. Theaorta was cannulated and the heart was perfused at constantpressure equivalent to 70 mmHg with oxygenated (95 % O2–5 %CO2), warm Krebs-Henseleit solution (37 °C), containing(mmol l_1) NaCl 118, NaHCO3 25, KCl 2.9, MgSO4 1.2, CaCl2

1.8, KH2PO4 1.3 and glucose 11.5. (pH 7.4). Epicardial electro-cardiogram (ECG) was continuously recorded (MingographELEMA, Sweden) via two stainless steel electrodes attached to theapex and to the aortic cannula for monitoring electrical activity,heart rate and the incidence of arrhythmias. Coronary flow wasmeasured by collection of perfusion effluent.

After a 20 min equilibration period in standard Krebs-Henseleitsolution, the hearts of normotensive and hypertensive rats wereperfused with a low-K+ (1.0 mmol l_1) solution for a period of60 min, unless sustained ventricular fibrillation occurred earlier.The hearts of both groups were taken at the end of theequilibration period (n = 10), and after 15 and 60 min of low-K+

perfusion (n = 22), and 2 min after ventricular fibrillation(n = 14), for histochemical and ultrastructural measurements,and for immunodetection of connexin-43. Arrhythmias weredefined in accordance with Lambeth Conventions using thefollowing score: 1, ventricular premature beats; 2, bigeminy; 3,ventricular tachycardia; 4, transient ventricular fibrillation; 5,sustained (> 2 min) ventricular fibrillation.

Histochemistry

For in situ demonstration of selected enzyme activities, the heartwas immediately frozen in liquid nitrogen. Transverse cryostatsections, 10 mm thick, from each heart containing both ventriclesand septum were used for histochemistry and for Van Giesonstaining of fibrosis. Myocardial oxidative and glycolytic metabolismwas monitored by histochemically determined activities ofsuccinic dehydrogenase, b-hydroxybutyrate dehydrogenase andglycogen phosphorylase, sarcolemmal function by detection ofATPases and 5‚-nucleotidase. Alkaline phosphatase and dipeptidylpeptidase IV were used as capillary network markers (Lojda et al.1976). Interstitial fibrosis was analysed by planimetry andfibrotic tissue was expressed as a percentage of the total measuredarea of the heart slice (Pechanova et al. 1999).

Immunodetection of connexin-43

Immunoreactivity was used for the in situ detection of the majorgap junction protein, connexin-43, on 10 mm cryostat sections.Exposure of the tissue slices to monoclonal mouse anticonnexin-43 antibody was followed by incubation with secondary goatantimouse IgG antibody conjugated to fluorescein isothiocyanate(FITC). Specificity of the immunoreaction was verified byincubation of the slices without primary antibody. The FITCsignal was examined using a fluorescence microscope (Carl ZeissJena, Germany), evaluated using the image analySIS program(GmBh Soft Imaging System, Germany) and expressed as aproportion of the total tissue area. Ten cryostat sections wereanalysed for each heart.

N. Tribulova and others196 Exp. Physiol. 87.2



Electron microscopy

For examination of the ultrastructure, the heart was fixed byperfusion with buffered glutaraldehyde (2.5 %). Small blocksrandomly taken from the ventricular tissue were excised, post-fixed in osmium tetroxide, dehydrated and embedded in Epon812. Semithick sections were examined under light microscopyand selected areas were cut to produce ultrathin sections, whichwere then stained in uranyl acetate and lead phosphate andexamined using electron microscopy (Tesla BS 500, CzechRepublic). Semi-quantitative evaluation of subcellular alterationswas performed using 10 ultrathin slices from each heart.

Measurement of intracellular free Ca2+ concentration

Another group of rat hearts (n = 6) was examined to determinewhether K+-deficient perfusion, and arrhythmias, induce changesin [Ca2+]i. The hearts were Langendorff perfused with Hepes-buffered and oxygenated Tyrode solution at a flow rate of13–14 ml min_1. [Ca2+]i was measured using the acetoxymethylester of fura-2, with an optical fibre and analysing system(CAF-110, CA-200 DP, Japan Spectroscopic, Tokyo, Japan), withthe probe located on the epicardial surface of the left ventricle.The ratio of the fluorescence intensity at 340 nm to that at380 nm (F340/380) was monitored as a qualitative index of [Ca2+]i

(Seki et al. 1998). ECG and left ventricular pressure (LVP) werealso simultaneously monitored.

All reagents used in the study were purchased from Sigma andantibodies for connexin-43 labelling were from ZymedLaboratories Inc. (USA).

Statistical procedures

The data were expressed as means ± ... Student’s unpaired ttest was used with P < 0.05 considered as significant fordifferences between groups.

RESULTSThe -NAME-treated rats showed a significant increase insystolic blood pressure by 38 ± 2 % (P < 0.05). Duringstabilisation, there were no significant differences in heartrate (280 ± 10 vs. 295 ± 5 beats min_1) and coronary flow(12 ± 1 vs. 13 ± 1.5 ml min_1) between normotensive andhypertensive rats. Perfusion with low-K+ resulted in avariable heart rate and a small decrease in coronary flow(to 10 ± 1.2 and 11 ± 0.8 ml min_1, respectively) in bothgroups.

Within 10–20 min of the beginning of perfusion with alow-K+ solution, the incidence of premature beats,bigeminy and ventricular tachycardia preceded theoccurrence of transient and/or sustained ventricularfibrillation. Transient arrhythmias occurred earlier andwere more frequent in hypertensive than in normotensiverat hearts (Fig. 1); moreover, the duration of ventriculartachycardia was significantly longer in the hypertensiverats (Fig. 2). The incidence of sustained ventricularfibrillation that occurred during 15–40 min of low-K+

Intermyocyte junctions and arrhythmiasExp. Physiol. 87.2 197

Figure 1

Susceptibility of normotensive (NT, n = 12) andhypertensive (HT, n = 12) rat hearts to low-K+-inducedsevere arrhythmias. Open bars, ventricular tachycardia;grey bars, transient ventricular fibrillation; filled bars,sustained ventricular fibrillation. * P < 0.05, HT vs. NT.

Figure 2

Duration of low-K+-inducedsevere arrhythmias innormotensive (NT, n = 10) andhypertensive (HT, n = 10) rathearts. Open bars, ventriculartachycardia; grey bars, transientventricular fibrillation. P < 0.05HT vs. NT.



perfusion was increased in hypertensive animals (83 % vs.33 %, respectively, P < 0.05; Fig. 1). Arrhythmia scoreanalysis revealed differences between groups withsignificantly higher scores in hypertensive compared tonormotensive rats (4.9 ± 0.7 vs. 3.1 ± 0.1, respectively,P < 0.05).

Morphological examination showed that NO-deficienthypertension was accompanied by apparent myocardialremodelling. There was activation of fibroblasts andsignificantly increased localised interstitial and perivascularfibrosis (12.24 ± 0.30 vs. 1.55 ± 0.1 % of total area,P < 0.05). Myocardial tissue consisted of a heterogeneouspopulation of cardiomyocytes, with those exhibitingconventional architecture in the majority (Fig. 3A).Additionally, hypertrophic cardiomyocytes with abundantpolyribosomes, neoformation of mitochondria, myo-filaments and intermyocyte junctions were observed(Fig. 3B). Moreover, cardiomyocytes exhibiting markers of

ischaemia-related changes (i.e. oedematous mitochondriaand nuclei; Fig. 3C) and impaired intermyocyte junctionswere occasionally found (Fig. 3D).

Immunolabelling of connexin-43 revealed a loss ofimmunopositivity in the areas of fibrosis and non-uniformdistribution of gap junctions throughout the tissue(Fig. 4C) compared to control rat hearts (Fig. 4A). Theoverall density of gap junctions was significantly decreasedin the ventricular tissue of hypertensive compared tonormotensive rats (Fig. 5).

In addition to both ultrastructural and immunofluorescencechanges induced by NO-deficient hypertension, histo-chemical reactions corresponding to enzyme activitiesrevealed a decrease in succinic (Fig. 6B) and b-hydroxy-butyrate dehydrogenases as well as glycogen phosphorylase(Fig. 6A) activities in the cardiomyocytes adjacent to areasof fibrosis. Similarly, activities of ATPases, 5‚-nucleotidase,


Figure 3

Electron micrographs of the myocardium from hypertensive rat heart showing a heterogeneouspopulation of cardiomyocytes exhibiting structural remodelling. Besides cardiomyocytes withconventional architecture (A), there are cardiomyocytes exhibiting hypertrophy, shown by increasednumber of polyribosomes, neoformation of myofilaments and lateral junctions (B). NO-deficienthypertension is also accompanied by myocardial injury, which is demonstrated by cardiomyocytesexhibiting ischaemia-like alterations: cellular oedema, electrolucent nucleus and mitochondria (C),marked dehiscence of cell-to-cell adhesive junctions and less frequent gap junctions (D).i, mitochondria; e, extracellular matrix; b, fibroblast; n, nucleus; arrows show adhesive junctions;arrowhead shows gap junction. Scale bars, 1 mm.



alkaline phosphatase and dipeptidyl peptidase IV wereheterogeneously diminished, while the latter two enzymeswere increased in the area of fibrosis (Fig. 6C and D).

Lowering the external potassium concentration caused asmall increase in the amplitude of the Ca2+ transients andan apparent increase of diastolic [Ca2+]i. A further increasein [Ca2+]i was more pronounced during the incidence of

transient arrhythmias (Fig. 7), which were observed on theECG and LVP recordings.

Perfusion with low-K+ accompanied by transient arrhythmiasinduced changes in subcellular, connexin-43 immuno-reactivity and enzyme histochemistry in both normotensiveand hypertensive rat hearts. Myocardial alterations precedingsustained ventricular fibrillation were heterogenously


Figure 4

Immunolabelling of connexin-43 shows uniform distribution and higher density of gap junctions innormotensive (A) than hypertensive (C) rat hearts. The localised reduction of immunopositivity after15 min of low-K+ perfusion in both normotensive (B) and hypertensive (D) groups is more pronouncedin hypertensive rat hearts. Magnification, w 60.

Figure 5

Immunofluorescence analysis ofconnexin-43 in the hearts ofnormotensive (NT, n = 18) andhypertensive (HT, n = 16) rats afterperfusion with standard and low-K+

solution (a.u., arbitrary units). There is asignificant decrease in immunopositivegap junctions in hypertensive vs.normotensive rat heart as well as amarked decrease during low-K+

perfusion, with transient arrhythmias inboth groups (* P < 0.01), which aremore pronounced in hypertensive ratheart (+ P < 0.01).




Figure 6

Enzyme histochemistry of the hypertensive rat myocardium shows a decrease (arrows) of glycogenphosphorylase (A), succinic dehydrogenase (B) activities and their absence in the fibrotic tissue.Decreases in alkaline phosphatase (C) and dipeptidyl peptidase IV (D) in the capillary network, withincreases (arrowheads) in the areas of extracellular matrix alterations are also demonstrated.Magnification, w 100.

Table 1. Semi-quantitative evaluation of histochemical and subcellular alterations of normotensiveand hypertensive rat heart before and after perfusion with low-K+ solution

Normotensive rat hearts Hypertensive rat hearts

Control Low-K+ Control Low-K+

Histochemistry

SDH, b-HBDH, GPh 3 3, 2, 1 3, 2, 1 3, 2, 1, 0ATPases, 5‚-NC 3 3, 2, 1 3, 2 3, 2, 1 AlP, DAP IV 3 3, 1 3, 2, 1 3, 2, 1, 0

Electron microscopy

Mitochondria N N, M, S N, M N, M, S Myofibrils N N, M, S N, M N, M, SIntermyocyte junctions N N, M, S N, M N, M, S

Scores indicate intensity of histochemical reactions corresponding to enzyme activities: 0, absent; 1,weak; 2, moderate; 3, high. Higher numbers indicate spatial heterogeneity of staining. Succinicdehydrogenase (SDH), b-hydroxybutyrate dehydrogenase (b-HBDH), glycogen phosphorylase (GPh),ATPases, 5‚-nucleotidase (5‚-NC), alkaline phosphatase (AlP) and dipeptidyl peptidase IV (DAP IV)enzyme activities were measured. Scores indicate the variations of subcellular alterations: N, none; M,moderate; S, severe.



distributed. These changes included impairment ofintercellular junctions (Fig. 8), local abolition of connexin-43 immunoreactivity (Fig. 4B and D, and Fig. 5) anddecreased enzyme activity (Fig. 9).

Dysfunction of cell-to-cell coupling and communicationwere observed as a non-uniform pattern of sarcomeres andasynchronous contraction between neighbouring cardio-myocytes (Fig. 8B and C). Ca2+ overload was not onlyobserved by direct measurement of [Ca2+]i, but was alsosuggested by the presence of myofibrillar hypercontraction,contraction bands and ruptured myofilaments (Fig. 8Band D).

As with the incidence and onset of low-K+-inducedarrhythmias, myocardial changes including subcellularalterations of the intermyocyte junctions and connexin-43immunoreactivity were observed earlier and were moredispersed in the hearts of hypertensive compared to

normotensive rats. The histochemical and ultrastructuralchanges are summarised in Table 1.

DISCUSSIONThe main finding of this study is that structuralremodelling, as a consequence of -NAME-inducedhypertension, is associated with abnormalities in myocardialcell-to-cell communications at the gap junctions. Indeed,immunodetection of connexin-43 showed a decreasedfluorescence signal in the ventricle of hypertensive rats andalso an irregular distribution of gap junctions throughoutthe myocardium. Immunoreactivity was abolished in theareas of extensive fibrosis and diminished within themyocardium due to alterations of the extracellular matrix.Accordingly, overall density of the fluorescence signal wassignificantly decreased, as confirmed by quantitative imageanalysis.


Figure 7

A, original traces showing simultaneous recordings of fura-2 fluorescence excited at 340 nm and 380 nm,the ratio of the fluorescence intensity at 340 nm and 380 nm (ratio F340/380) and left ventricular pressure(LVP) during perfusion with standard solution. B, original records of fura-2 fluorescence ratio and LVPduring control and low-K+ perfusion. A small increase in the amplitude of the Ca2+ transients and anapparent increase in diastolic [Ca2+]i is accompanied by an increase in both the level and variability ofLVP due to transient arrhythmias.



Cell-to-cell junctions are responsible for electrical andmechanical couplings, as well as for intercellular signalling,resulting in myocardial synchronisation. Since gapjunctions play a central role in conduction, alterations inconnexin-43 expression and gap junction distributionhave been implicated in the pathogenesis of re-entrantarrhythmias (Peters et al. 1997). Reduced gap junctionalcoupling in areas of fibrosis could disrupt wave-frontpropagation and this might interfere with the uniform andsynchronised function of the cardiomyocyte. Localiseduncoupling clearly contributes to the pathogenesis ofconduction abnormalities and unidirectional conductionblock (Saffitz et al. 1999). Moreover, altered and/orabnormal intermyocyte coupling may enhance theheterogeneity of hypertension-related myocardial alterations(Ahmmed et al. 2000) that probably contribute to earlierand faster degeneration of transient arrhythmias intosustained ventricular fibrillation during hypokalaemia.

Accordingly, areas with sparse coupling between cardio-myocytes, either due to chronic remodelling or acutedown-regulation of gap junction proteins, are highlyarrhythmogenic (Manoach et al. 1986; Spach & Heidlage,1995; Peters et al. 1997; Saffitz et al. 1999; Tribulova et al.1999, 2001a,b). Our present findings support previousinvestigations. Electromechanical desynchronisation maybe enhanced, in addition, by abnormal adhesive junctionsmediating mechanical coupling (Dhein, 1998, Tribulova etal. 2001a,b) observed also in this study. Moreover,hypertension-related coronary artery remodelling (Kristek& Gerova, 1996), and endothelial and capillary dysfunction(Okruhlicova et al. 2000; Tribulova et al. 2000) associatedwith ischaemia-like injury of the cardiomyocytes, and thecorresponding histochemical changes, may contribute tothe pathophysiological heterogeneity of the myocardiumthat decreases its electrical stability. Indeed, spatialheterogeneity of the myocardial alterations revealed byelectron microscopy and in situ histochemistry was


Figure 8

Low-K+-related subcellular alterations of the cardiomyocytes, preceding the occurrence of SVF aresimilar in normotensive (A and B) and hypertensive (C and D) rat hearts. In addition to cardiomyocyteswith only small subcellular alterations (A) there are moderately (C) and severely (B and D) affectedmyocytes. They exhibit detached adhesive junctions of various degrees (arrows), loss of integrity of gapjunctions (arrowheads), desynchronisation of contraction between neighbouring cardiomyocytes (†),hypercontraction and contraction bands, and injured mitochondria (i). It should be noted that thesechanges are dispersed throughout the myocardium and are much more pronounced in the hypertensivegroup. Scale bars, 1 mm.



accompanied by heterogeneity of connexin-43 immuno-staining, which indicates the widespread nature of theimpairment of myocardial coupling.

Not only experimental but also clinical studies indicatethat the progression of maladaptive myocardial structuralchanges in heart failure correlates with an increasedincidence of lethal arrhythmias (Eckardt et al. 2000). Thismaladaptive consequence of remodelling is thought tocreate an anatomic substrate for arrhythmogenesis (Peterset al. 1997; Tribulova et al. 1998, 1999; Saffitz et al. 1999).Slowing and/or fractionated conduction (Spach &Heidlage, 1995) facilitate re-entry as well as the inductionof sustained tachycardia and/or fibrillation (Peters et al.1997). Furthermore, it was reported that intercellularuncoupling enhances the dispersion of repolarisation,which has long been considered a major electrophysiologicalsubstrate for re-entrant arrhythmias.

Several studies support the possibility that down-regulationof connexin-43 causes conduction disturbances (Guerreroet al. 1997; Dhein, 1998) and enhances arrhythmogenesisin the diseased heart (Peters et al. 1997; Dhein, 1998; Saffitzet al. 1999). A direct relationship between reducedconnexin-43 expression and the incidence of ischaemia-

induced tachyarrhythmias was documented for the firsttime in the study of Lerner et al. (2000). Accordingly,myocardial architecture is intimately involved in theelectrical and mechanical function of the heart, andstructural disarrangement or remodelling may affect bothelectrical and mechanical properties of the cardiac muscle.Today, functional intermyocyte coupling is thought to becrucial not only for coordinated electrical function, but alsofor the maintenance of intermyocyte signal transduction,and thus overall myocardial tissue synchronisation (Delmar,2000). Nevertheless, the crucial role of myocardial couplingin the development of life-threatening arrhythmias andparticularly in spontaneous defibrillation was suggestedseveral years ago by Manoach et al. (1986).

The increased susceptibility of the hypertensive rat heart tolow-K+-induced lethal arrhythmias, preceded by a muchhigher incidence of premature contractions and transientarrhythmias, indicates an apparent electrical instability.Since hypokalaemia is accompanied by an electrolyteimbalance and a prolongation of the action potentialduration (Akita et al. 1998), we can assume their morepronounced appearance in hypertensive rat heart thatexhibits metabolic, enzymatic, subcellular and perhaps


Figure 9

Histochemical reactions for the demonstration of succinic dehydrogenase (A and C), glycogenphosphorylase (B) and 5‚-nucleotidase activities in both normotensive (A and B) and hypertensive (Cand D) rat hearts show patchy areas (arrows) with markedly reduced activity. Some cardiomyocytesexhibit contraction bands (arrowheads). Magnification, w 85.



electrophysiological and Ca2+ handling alterations. It wasreported that hypokalaemia-induced Ca2+ oscillations(Akita et al. 1998) probably account for the occurrence ofpremature beats due to early or delayed after-depolarisation. It seems that both prolongation of theaction potential duration (Helfant, 1998) and suppressionof Na+,K+-ATPase activity (Knochel, 1987) during hypo-kalaemia may contribute to the increased influx of Ca2+, itsabnormal level and even the Ca2+ overload with furtherdeterioration due to transient arrhythmias.

Indeed, loss of Ca2+ homeostasis and its elevation due to K+

deficiency was previously detected using the fluorescentdye indo-1 in cultured cardiomyocytes (Tribulova et al.2001a) and with fura-2 in a whole heart preparation inthis study. Ca2+-related alterations of the cardiomyocyteswere observed as non-uniform contraction of thesarcomeres and/or hypercontraction of myofilaments,while desynchronisation of the contraction betweenneighbouring cardiomyocytes indicated the impairment ofintercellular coupling. It is noteworthy that these changeswere not homogeneously distributed throughout themyocardium but spatially dispersed. As with hypo-kalaemia, post-ischaemia/reperfusion-induced increase of[Ca2+]i was found to be linked with the incidence oftransient arrhythmias and ventricular fibrillation (Seki et al.2001). Transient arrhythmias enhanced Ca2+ disturbancesand myocardial injury that cause a marked deterioration incell-to-cell coupling. This can contribute to the perpetuationof re-entry and the degeneration of these arrhythmias tofibrillation, and to sustaining the fibrillation process(Tribulova et al. 2001a,b). Available data indicate thatpotassium depletion results not only in electrolyte andelectrophysiological abnormalities but also leads tometabolic changes (Shapiro et al. 1998) and alterations inenzyme activity (Knochel, 1987). In agreement with thesestudies, we found that acute severe hypokalaemiadecreased the activity of succinic and b-hydroxybutyratedehydrogenase and particularly glycogen phosphorylase,indicating disturbances in energy supply. Furthermore, thedecrease in membrane-bound 5‚-nucleotidase and ATPaseactivities point to an alteration of sarcolemmal function.These changes coincide with subcellular changes of thecardiomyocytes exhibiting ischaemia-like and Ca2+-overload injury (Ravingerova et al. 1995), as well as withimpairment of intermyocyte coupling and electro-mechanical synchronisation (Tribulova et al. 2001a,b). Itshould be noted that the incidence of transientarrhythmias, including ventricular fibrillation, aggravatedmyocardial alterations and their dispersion.

In conclusion, these results suggest that NO-deficienthypertension is accompanied by structural remodelling ofthe heart and decreased myocardial coupling. Thesehearts are more vulnerable to hypokalaemia-induced Ca2+

disturbances and the consequent transient arrhythmiasthat lead to further impairment of cell-to-cellcommunication. This may result in a higher incidence ofsustained ventricular fibrillation in hypertensive rats.

A, G. U., D, P. H., S, G., B, N. A., X, Y., W,R. A. & C, N. (2000). Changes in Ca2+ cyclingproteins underlie cardiac action potential prolongation in apressure-overload guinea pig model with cardiac hypertrophy andfailure. Circulation Research 86, 558–570.

A, M., K, M., T, H. & S, S. (1998). ECGchanges during furosemide-induced hypokalemia in the rat.Journal of Electrocardiology 31, 45–49.

B, P., P, O., B, I. & S, S. (1997).Chronic inhibition of NO synthesis produces myocardial fibrosisand arterial media hyperplasia. Histology and Histopathology 12,623–629.

B, I., P, O., P, V. & S, F. (2000).Regression of chronic L-NAME-treatment-induced left ventricularhypertrophy: Effect of captopril. Physiological Research 32,177–185.

C, R. S., C, A. M. & V, E. C. (1993). Evidencethat the autonomic nervous system plays a major role in the L-NAME-induced hypertension in conscious rats. American Journalof Hypertension 6, 806–809.

D, M. (2000). Gap junctions as active signaling molecules forsynchronous cardiac function. Journal of CardiovascularElectrophysiology 11, 118–120.

D, S. (1998). Gap junction channels in the cardiovascularsystem: pharmacological and physiological modulation. Trends inPharmacological Sciences 19, 229–241.

E, L., H, W., J, R., B, D., D, M. C.,B, G. & B, M. (2000). Arrhythmias in heartfailure: current concept of mechanisms and therapy. Journal ofCardiovascular Electrophysiology 11, 106–117.

G, K. K., M, T. J. & A, R. W. (1993).Molecular biology of the renin-angiotensin system. Circulation 87,1824–1828.

G, P. A., S, R. B., D, L. M., B, E. C.,J, C. M., Y, K. A. & S, J. E. (1997). Slowventricular conduction in mice heterozygous for a connexin-43null mutation. Journal of Clinical Investigation 99, 1991–1998.

H, R. H. (1998). Hypokalemia and arrhythmias. AmericanJournal of Medicine 80, 13–22.

H, A., T, J., B, I. & P, O. (1996).Restriction of nitric oxide rather than elevated blood pressure isresponsible for vascular responses in nitric oxide-deficienthypertension. Physiological Research 45, 317–321.

J, O., S, J. & Z, J. (1992). Hypokalemia incidenceand severity in a general hospital. Wien medizinische Wochenschrift142, 78–81.

K, J. P. (1987). Hypokalemia. Advances in Internal Medicine30, 315–335.

K, F. & G, M. (1996). Long-term NO synthaseinhibition affects heart weight and geometry of coronary andcarotid arteries. Physiological Research 45, 361–367.

L, D. L., Y, K. A., S, R. B. & S, J. F.(2000). Accelerated onset and increased incidence of ventriculararrhythmias induced by ischaemia in Cx43-deficient mice.Circulation 101, 547–552.

L, Z., G, R. & S, T. H. (1976). Enzym-histochemische Methoden. Springer-Verlag, Berlin, Heidelberg,New York.




M, M., N, H., V, D., A, G., W, M.,K, A. & A, M. (1986). Factors influencing spontaneousinitiation and termination of ventricular fibrillation. JapaneseHeart Journal 27, 365–375.

M, H., M, K., B, A. C., A, E., Z, R. & DN, G. (1996). Chronic nitric oxide inhibition as a model ofhypertensive heart muscle disease. Basic Research in Cardiology 91,248–255.

N, K., E, K., T, M., K, T.,S, H., S, K. & T, A. (1995). Chronicinhibition of nitric oxide synthesis causes coronary microvascularremodeling in rats. Hypertension 26, 957–962.

O, L., T, N., B, I. & P, O.(2000). Induction of angiogenesis in NO-deficient heart.Physiological Research 49, 71–76.

P, O., B, I., P,V. & B, P. (1999).L-NAME-induced protein remodeling and fibrosis in the rat heart.Physiological Research 48, 353–362.

P, O., B, I., P, V. & S, F. (1997).Protein remodeling of the heart in NO-deficient hypertension: theeffect of captopril. Journal of Molecular and Cellular Cardiology 29,3365–3374.

P, N. S., C, J., S, N. J. & W, A. L. (1997).Disturbed connexin-43 gap junction distribution correlates withlocation of re-entrant circuits in the epicardial border zone ofhealing infarcts that cause ventricular tachycardia. Circulation 95,988–996.

R, T., T, N., S, J. & C, M. J. (1995).Brief, intermediate and prolonged ischaemia in the isolatedcrystalloid perfused rat heart: Relationship between susceptibilityto arrhythmias and degree of ultrastructural injury. Journal ofMolecular and Cellular Cardiology 27, 1937–1951.

R, M. O., A, E., D N, G., L, S. M. &Z, R. (1992). Chronic inhibition of nitric oxide synthesis. Anew model of arterial hypertension. Hypertension 20, 298–303.

S, J., S, R. B. & Y, K. A. (1999). Mechanismsof remodeling of gap junction distribution and the development ofanatomic substrates of arrhythmias. Cardiovascular Research 42,309–317.

S, S., H, K., T, H., I, T., N, A.,O, H., T, M. & M, S. (2001). Effect ofsustained low-flow ischaemia and reperfudion on Ca2+ transientsand contractility in perfused rat hearts. Molecular and CellularBiochemistry 216,111–119.

S, S. & M, S. (1998). Role of the Na+/Ca2+ exchanger inintracellular Ca2+ overload during ischaemia and reperfusion. InProtection against Ischaemia/Reperfusion Damage of the Heart,ed. A, Y. & K, M., pp. 39–48. Springer-Verlag,Tokyo.

S, J. I., B, A., R, O. K. & E, N. (1998). Acuteand chronic hypokalemia sensitize the isolated heart to hypoxicinjury. American Journal of Physiology 274, H598–1604.

S, M. S. & H, J. F. (1995). The stochastic nature ofcardiac propagation at a microscopic level. An electricaldescription of myocardial architecture and its application toconduction. Circulation Research 76, 366–380.

T, N., M, M., V, D., O, L.,Z, T. & S, A. (2001a). Dispersion of cell-to-celluncoupling precedes of low K+-induced ventricular fibrillation.Physiological Research 50, 247–259.

T, N., O, L., B, I. & P, O.(2000). Chronic disturbances in NO production results inhistochemical and subcellular alterations of the rat heart.Physiological Research 49, 77–88.

T, N., S, S., M, M., T, H., O,L. & M, S. (2001b). Restorations of basal cytoplasmicCa2+ and recovery of intermyocyte coupling precede stobadine-induced ventricular defibrillation in whole heart preparation.European Heart Journal 22, 547.

T, N., V, D. & M, M. (1998). Structuraldeterminants underlying conduction disturbances resulting incardiac fibrillations. Circulation 98, 683–684.

T, N., V, D., P-C, S., B, P.,S, J. & M, M. (1999). Aged heart as a model forprolonged atrial fibrillo-flutter. Experimental and ClinicalCardiolology 4, 64–72.

V, N., B, I. & P, O. (1999). Changes ofsodium and ATP affinities of the cardiac Na,K-ATPase during andafter nitric oxide deficient hypertension. Molecular and CellularBiochemistry 202, 141–147.

W, K. T. & B, C. G. (1991). Pathological hypertrophy andcardiac intersticium. Fibrosis and renin-angiotensin-aldosteronesystem. Circulation 83, 1849–1865.

Acknowledgements

This work was supported by the Slovak Grant Agency, VEGAgrant nos 2/7155/20 and 2/7123/20. Part of the study wassupported by the Japanese Society for Promotion of Science andby the Department of Internal Medicine 4, Jikei University,Tokyo. We express our thanks to Mrs Anna Brichtova andAdelka Maczaliova for skilful technical assistance.




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