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Low K+-induced subcellular, histochemical and
connexin-43 alterations initiate ventricular fibrillation

Tribulova N; Manoach M1; Varon D1; Sheinberg A2; Okruhlicova L and Stetka R

Inst. for Heart Research, Slovak Academy of Sciences Dubravska, Bratislava, Slovakia
1Dept. Physiol. Sackler Sch. Med. Tel Aviv
2Bar Ilan University, Bar Ilan, Israel

Abstract
Introduction
Material and Methods
Results
Discussion
References

Abstract
Introduction: we hypothesize that hypokalemia-related electrolyte disbalance linked with abnormal elevation of intracellular free Ca [Ca]i can cause metabolic disturbances, subcellular alterations and intercellular uncoupling which favour occurence of malignant arrhythmias.
Objective: the aim of the study was to examin young, adult and old (n=30) guinea pig hearts perfused by Langendorff mode with standard oxygenated Tyrode solution followed by K+ defficient (1.4mM) perfusion, before and after occurence of sustained ventricular fibrillation.
Material and Methods: ventricular tissue was analyzed for ultrastructure; for succinic dehydrogenase, glycogen phosphorylase and 5-nucleotidase activities using in situ catalytic histochemistry; for immunodetection of Cx43 by using mouse monoclonal Ab and FITC conjugated gout antimouse Ab; [Ca]i was measured by indo1 in neonatal cell culture exposed to K+ free medium.
Results: age-independent sustained VF appeared within 15-30 min of low K+ perfusion. Hypokalemia induced ischemia-like reversible and irreversible heterogenously distributed subcellular alterations and patchy areas with decreased enzyme activities. Impairment of intercellular junctions was locally found which correlated with abolished immunoreactivity of Cx43. Ni+, verapamil, d-sotalol or tedisamil efficiently defibrillate the heart.
Discussion: results indicate that increased [Ca]i, dispersion of altered cardiomyocytes and local intercellular uncoupling can create reentry and favour fibrillation in the setting of K+ defficiency
Conclusion: we suggest that impairment of cell-to-cell coupling can be crucial arrhythmogenic factor involved in the occurence of ventricular fibrillation.

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Introduction

Potassium is known as one of the possible chemical mediator of potentially life threatening arrhythmias, whereby both increase and decrease of external K+ might be arrhythmogenic. Thus any disturbances in extracellular basal concentration of K+ might be associated with a risk of triggering various arrhythmias including ventricular fibrillation (VF), whereby it seems that key factor involved in this process is acummulation of cytoplasmic free calcium [1].
Whereas hyperkalemia in experimental as well as clinical studies is linked particularly with ischemic conditions and ischemia-induced VF, hypokalemia is associated particularly with incidence of Torsade de point during diuretic and other therapies [2]. Moreover, hypokalemia is frequently associated not only with cardiovascular but also gastrointestinal and urogenital diseases [3]. Both, transient hyperkalemia or hypokalemia can be induced by increase of catecholamines and by strain or exercise [4,5 ].
Despite of the data pointing out that hypokalemia decreases myocardial electrical stability, by alterations in excitability, by increase of membrane potential, duration of action potential (APD) and effective refractory period (ERP) and by decreasing conduction velocity [6], the all spectrum of low K+ induced alterations involved in the initiation of VF is still not known.
Our previous studies elucidating mechanisms involved in the appearance of transient versus sustained VF [7] as well as our recent examination of the electrically [8] and/or ischemia-induced [9] cardiac fibrillation indicate that viability of the cardiomyocytes and disturbances in intercellular coupling and communication at gap junctions can be critical in the process of initiation and perpetuance of asynchronous activity, which can degenerate to fibrillation. Moreover, we have found impairment of intermyocyte coupling most likely induced by excess of [Ca2+]i as a results of hypoxia [10] or elevated external Ca2+concentration in ventricular muscle strips [10,11]. In cultured ventricular myocytes increase of [Ca2+]i was accompanied by asynchronous contraction or even by lost of spontaneous beating, and synchronization was recovered by drugs normalizing intracellular Ca2+concentration [12]. It is generally accepted that gap junctional uncoupling lead to discontinous propagation and nonunifrom anisotropy in small circuits which can initiate reentry [13].
Since the understanding how hypokalemia affects cardiac metabolism, structure and function, which result in life threatening arrhythmias is still incomplete, the aim of this study was to examin some of these parameters, focusing particularly on alterations in intermyocyte junctions and coupling.

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Material and Methods

The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health, Publication No 85-23, revised 1996.
The experiments were conducted on the hearts of young (n=10), adult (n=22) and old (n=12) guinea pigs of both sexes. The animals were sacrificed by stunning followed by carotid exsanguination and the aorta of excised heart was immediately cannulated for perfusion with crystaloid 370C worm Tyrode solution oxygenated by 95% 02 and 5% CO2 at the pressure of 65 mmHg. After15 min stabilization with standard solution the heart was perfused with K+ deficient (1.4mM) ones. Bipolar epicardial electrocardiograms from the left atria and ventricle were continously monitored and the incidence of arrhythmias was evaluated.
The efficacy of Ca2+ channel blockers, verapamil (2.5-4 mM) and diltiazem (4-6 mM), Na+/Ca2+ inhibitor Ni+ (1mM) and class III drug d-sotalol (1-10 mM), tedisamil (1-10 mM) in the prevention of sustained ventricular fibrillation were examined.
Cytosolic [Ca2+]i was estimated by indo-1 in 3-6 days old cultured rat ventricular cardiomyocytes bathed in Tyrode solution with or without K+. The cardiac cells grown on cover glass coated with gelatin/collagen were transfered to a chamber on the stage of Zeiss inverted microscope (Axiovert 135 TV), filtered with UV epifluorescence illumination. Indo-1 was excited at 355 nm. The fluorescence ratio 405/490, which is proportional to [Ca2+]i was monitored.
Left ventricular tissue was collected during stabilization, low K+ perfusion and at the onset of ventricular fibrillation. For in situ demonstration of succinic dehydrogenase (1.3.99.1), glycogen phosphorylase (2.4.1.18) and 5-nucleotidase (3.1.3.5) enzyme activities and for immunodetection of gap junction protein connexin-43, the left ventricle was immediately frozen in liquid nitrogen and cutted in 10 um thick cryostat sections. The immunolabelling of connexin43 was performed using monoclonal mouse anticonnexin-43 Ab and FITC goat antimouse IgG (Zymed laboratories Inc.). Histochemical and immunostaining reactions were examined under light and/or fluorescence microscopes (Carl Zeiss Jena).
For transmision electron microscopic examination small tissue blocks from subepicardial and subendocardial layer of the left ventricle were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate for 6 hours. After postfixation in 1% osmium tetroxide the tissue was subsequently washed in cacodylate buffer, dehydrated in ethanol, infiltrated by propylene oxide and embedded in Epon 812. The ultrathin sections were stained with uranyl aceteate and lead phopshate and examined in electron microscope Tesla 500.

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Results

Perfusion of isolated guinea pig heart with K+ deficient Tyrode solution lasting 15-30 min induced 100% incidence of ventricular fibrillation, independent on the age and sex, however earlier onset of fibrillation was usually occured in female and old animals. The continual recordings of ventricular bipolar electrocardiogram during hypokalemia showed changes in R and T configuration, bigeminy, various ectopic activity followed by clear changes in R vector and sudden ventricular tachycardia which preceeded sustained ventricular fibrillation (Fig 1).

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Figure 1: The originals of the ECG records from two isolated guinea pig hearts during stabilization perfusion with standard Tyrode solution (C) and during 20-30 min of K+ defficient one. Salves of bigeminy and tachyarrhythmias or changes in the R vector preceeded occurence of ventricular fibrillation Time in seconds. A - left atrium; V - left ventricle.

Administration of either verapamil and diltiazem or Ni+ (Fig 2) decreased incidence of transient arrhythmias and prevented occurence of sustained ventricular fibrillation, whereas tedisamil (Fig 3) and d-sotalol defibrillated the heart.

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Figure 2: 30 min perfusion of guinea pig heart with K+ deficient Tyrode solution containg 1mM Ni+ prevented ventricular fibrillation. A - left atrium, V- left ventricle, time in seconds.


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Figure 3: Administration of 10mM Tedisamil during low K+ induced ventricular fibrillation caused clear ventricular defibrillation . A - left atrium, V - left ventricle, time in seconds.

In 3-6 days old cultured ventricular cardiomyocytes subjected to K+ free Tyrode solution was Ca2+ concentration significantly increased from 100 nM to 340 nM (Fig 4).

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Figure 4: Indo1 measurement of intracellular free Ca2+ in the cultured ventricular myocytes subjected to K+ free Tyrode solution showed significant increase of basal concentration.


The myocardium of the left ventricle taken during stabilization perfusion did not exibit any alterations in the histochemically determined enzyme activities of succinic dehydrogenase, glycogen phosphorylase and 5-nucleotidase. However, 15-30 min of low K+ perfusion caused decrease of enzyme activities detected by heterogenously diminished intensity of histochemical reactions. Accordingly, some cardiomyocytes were more and some less affected. Moreover, at the interface with the severely affected cardiomyocytes showing unregularly contracted myofibers poit out the disturbances in intermyocyte coupling and desynchronisation of contraction-relaxation processess. All these changes were more pronounced in the fibrillating myocardium.
In correlation with histochemical changes the connexin-43 labelling revealed patchy microareas with lost of immunoreaction indicating impairment of the integrity of gap junction proteins. Diffrent from control myocardial tissue showing numerous gap junctions regularly distributed at the site of intercalated discs. Patterns of irregularly labeled gap junctions as well as microareas with abolished immunoreactivity were more pronounced during fibrillation. These changes strongly indicate disruption and disturbances in intermyocyte junctions and communication.
Perfusion of the isolated heart with standard Tyrode solution did not affect normal subcellular architecture of cardiomyocyte, including intermyocyte junctions. The myocardial tissue from the heart subjected to K+ deficient perfusion was characterized by presence of slightly or markedly but still reversibly altered cardiomyocytes as well as by irreversibly injured cardiomyocytes, wich were in minority. Nonuniformly affected cardiomyocytes were heterogenously distributed in the subepicardial and subendocardial regions. Severely damaged cardiomyocytes showed oedema, apparently injured mitochondria and impaired integrity of intermyocyte connections at the fascia adherens and gap junctions. Less affected cardiomyocytes exibited mild oedema and moderate mitochondria alterations as well as mild dissociation of adhesive, fascia adherens, junctions and no visible changes in gap junctions. However, these alterations were frequently accompanied by nonuniform pattern of sarcomers in adjacent cardiomyocyte, indicating electrical and metabolic uncoupling and desynchronisation of contraction. Moreover, occurence of hypercontracted myofibres and even contraction bands especially in irreversibly altered cardiomyocytes indicated Ca overload injury. The subcellular changes were again much more pronounced in fibrillating myocardium.
Morphological alterations of the isolated guinea pig heart caused by K+ deficiency are summarized in the Table.

Table
tabla.gif (8385 bytes)

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Discussion

Our results showed that isolated guinea pig heart subjected to low K+ perfusion for relatively short period, exibited ectopic activity, episodes of premature beats, bigeminy and tachycardia with consequent sustained ventricular fibrillation. In human and other experimental models, hypokalemia was associated particularly with polymorphic tachycardia, Torsade de pointes [14], which often degenerated into fibrillation [15].
Hypokalemia is known to increase the resting membrane potential, APD and ERP [15]. Nevertheles, despite of antiarrhythmic nature of prolongation of refractory period the reentry arrhythmias occur, thus other electrophysiological alterations can determine appearance of VF. In this respect, it should be note, that prolongation of APD is accompanied by increase of Ca2+ influx followed by increase of cytoplasmic free calcium, which can be even enhanced by hypokalemia-induced dysfunction of Na pump [16] and consequent reverse mode of Na+/Ca2+ exchange. This assumption was supported also by our findings of elevated [Ca]i in cultured ventricular cells subjected to K+ free solution and by subcellular alterations of the cardiomyocytes indicating Ca2+ overload. Excess of [Ca2+]i may favour abnormal automaticity, triggered activity and induce aftercontractions [17] as well as it can induce intermyocyte uncoupling and block of conduction favouring reentry.
Besides supression of Na,K-ATPase activity [16] we found that hypokalemia decreases activity of sarcolemmal 5-nucleodidase, which indicate disturbances in purine nucleotide metabolism. Further metabolic disturbances were manifested by decreased mitochondrial succinic dehydrogenase activity and by cytoplasmic glycogen phosphorylase activity pointing out the break-down of energy production. These results coincide with hypokalemia induced decrease of ATP concentrations and impaired respiratory activity of the mitochondria [18]. Observed metabolic disturbances can contribute to further deterioration of low K+ induced changes including accumulation of [Ca]i and its effect associated with increase of internal and gap junctional resistance and decreased conductivity [19,20].
It was established that abnormal [Ca2+]i inhibits gap junctional channels and downregulates of intermyocyte communication, thus it can affect conduction velocity and myocardial symchronisation [1,21]. Cell-to-cell uncoupling at the gap junction channels can even aggravate pathophysiological inhomogeneity in APD [22] and contribute to the dispersion of repolarization. All these higly arrhythmogenic events may determine electrical instability of the heart prior occurence of fibrillation. In addition, dispersed and dynamic character of gap junctional alterations detected in our experiments might account for perpetuance of fibrillating proces [13,23,24].
Taking into consideration that similar like chronic also acute hypokalemia [18] increase cytoplasmic [Ca]i, we suggest, that the main factor involved in hypokalemia-induced arrhythmias and fibrillation is abnormal elevation of Ca2+ and/or Ca2+ overload with its deleterious effects particularly on intermyocyte coupling and myocardial synchronisation.
This suggestion was supported by our findings that inhibition of sarcolemmal Ca2+ transport systems, Na+/Ca2+ exchanger and Ca2+ channels, prevented occurence of fibrillation. Moreover, class III drugs, which were shown to attenuate Ca overload [12,25] most likely by enhancement of Ca2+ uptake by sarcoplasmic reticulum [26], facilitated conversion of fibrillation to sinus rythm.
Hypokalemia-induced electrolyte disturbances resulting in accumulation of [Ca2+]i and metabolic disturbances were associated furtermore with the changes in immunodetection of major gap junction protein connexin-43. It should be note that not only enzyme histochemical and subcellular changes but also connexin-43 alterations were heterogenpusly distributed throughout myocardium. The cardiomyocytes with diminished immunoreactivity suggests the local disruption of gap junctional function. Similar feature of alterations we have found in the myocardial tissue during burst pacing-induced sustained atrial or ventricular fibrillation [8].
Connexin-43 related alterations were detected for the first time in our acute models of cardiac fibrillation and they coincide by some way with alterations in connexin-43 or 40 [27], which were found in chronic experiments or in the human disseased heart prone to arrhytmias and fibrillation [23,28]. Both, chronic and acute alterations indicate nonuniform disruption of gap junctional distribution. Lost or decreased intensity of immunofluorescence can be attributed to degradation of gap junction proteins as well as to their conformational changes and masking of connexin43 epitops [29]. Accordingly, degradation and permanent lost of immunoreactivity can be associated particularly with irreversibly injured cardiomyocytes. These were surrounded or in the vicinity of less affected cardiomyocytes most likely with reversible alterations of gap junction channels. We suggest that local and dispersed character of these myocardial changes might provide substrat for multiply circuits of reentry similar as it was suggested for a border zone of the infarcts (28).
Examination of the ultrastructure revealed besides Ca overload-related injury also impairment of intercellular connections, characterized by widenning of adhesive junctions and lost of integrity of gap junctions. Moreover, nonuniform patterns of sarcomers in neighboring cardiomyocytes strongly indicate desynchronisation of contraction, most likely as a results of disturbances in mechanical and gap junctional coupling.
Similar feature of the subcellular alterations was found in the heart prone to ischemia and/ or reperfusion-related arrhythmias [9,30] or in the heart during period of burst pacing resulting in sustained atrial or ventricular fibrillations [8]. They point out a close relationship between heterogenously injured, but still viable cardiomyocytes, and occurence of fibrillations. Thus, despite of species-related differences as well as multiplicity of potencialy contributing factors involved in the arrhythmogenesis, disturbances in Ca2+ homeostasis and accumulation of [Ca]i resulting in intermyocyte uncoupling, seems to be crucial and common pathway in the occurence of fibrillation. Conversely, prevention or attenuation of Ca2+ overload with consequent protection of gap junction channels as well as direct upregulation of cell-to-cell coupling and synchronisation by endogenous or by exogenous compounds may be very promising target for antifibrillating/defibrillating drug therapy.
Summarizing our results showed that perfusion of isolated guinea pig heart with K+ defficient solution results in significant qualitative changes heterogenously distributed within myocardium and characterized by decreased enzyme activities, abolished immunodetection of connexin-43 and subcellular injury with impairment of intermyocyte coupling. These changes correlate each other and they reflect of low K+ induced electrolyte disturbances accompanied by Ca overload. We suggest, that high [Ca]i -related alterations may account for both initiation of arrhythmias as well as for their degeneration to sustained ventricular fibrillation, whereby dispersion of intermyocyte uncoupling might be fundamental in this process.

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References

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Nov/17/1999