Ker_197-202.qxd INTRODUCTION In 1925 Wiggers observed that, if ventricular acti- vation proceeds from the ventricular epicardium instead of from the atrium, the consequence will be a lower peak left ventricular pressure as well as a lower dP/dt. He also concluded that the normal sequence of ventricular activation is essential for optimum ventricular function (Varna & Camm 2001). In the mammalian heart, cardiac activation is initi- ated in the atria and proceeds to the ventricles via the specialized conduction system (SCS). This SCS consists of the His bundle, the left and right bundle branches with their major fascicles and the peripheral Purkinje network (Van Dam 1989). The terminal ramifications of this Purkinje network merge into the ventricular myocardium at sites of Purkinje-myocardial coupling. Proximal to these sites the SCS has no functional connections with the myocardium because of a thin, collagenous sheath (Van Dam 1989). Ventricular dyssynchrony can be defined as an ab- normality of the normal, organized electromechani- cal coupling of the ventricles and this disturbance is the consequence of an interventricular conduction delay (Aranda & Schofield 2002). Ventricular dys- synchrony is not an uncommon clinical problem and has numerous causes, such as left bundle branch 197 Onderstepoort Journal of Veterinary Research, 71:197–202 (2004) Ventricular dyssynchrony as a cause of structural disease in the heart of Dorper sheep J. KER1, E.C. WEBB2 and C.F. VAN DER MERWE3 ABSTRACT KER, J., WEBB, E.C. & VAN DER MERWE, C.F. 2004. Ventricular dyssynchrony as a cause of structural disease in the heart of Dorper sheep. Onderstepoort Journal of Veterinary Research, 71: 197–202 Ventricular dyssynchrony is a disturbance of the normal, organized electromechanical coupling of the two ventricles. This condition has many causes, such as left bundle branch block, ventricular pre- excitation, right ventricular pacing and right ventricular premature ventricular complexes (PVCs). Ventricular dyssynchrony has many adverse haemodynamic effects on the left ventricle and we wanted to know whether these adverse haemodynamic effects might have any structural conse- quences on the left ventricles of such hearts. Six healthy Dorper wethers were subjected to numerous right ventricular PVCs to induce ventricular dyssynchrony in order to determine whether any structural consequences will occur in the left ven- tricles of these hearts. Myocarditis in the musculature of the left ventricles of all six these hearts was seen. Keywords: Dorper sheep, premature ventricular complexes, ventricular dyssynchrony 1 Department of Physiology, Faculty of Medicine, University of Pretoria, P.O. Box 24318, Gezina, Pretoria, 0031 South Africa. E-mail: james.ker@med.up.ac.za 2 Department of Animal and Wildlife Sciences, Faculty of Natural and Agricultural Sciences, University of Pretoria, Pre- toria, 0002 South Africa 3 Department of Microscopy and Microanalysis, University of Pretoria, Pretoria, 0002 South Africa Accepted for publication 28 January 2004—Editor block (Littmann & Symanski 2000), right ventricular pacing (Badke & Boinay 1980; Grover & Glantz 1983; DiCarlo & Morady 1987; Rosenqvist & Isaaz 1991), ventricular pre-excitation (Sutherland & Ku- kulski 2000) and premature ventricular complexes (Sutherland & Kukulski 2000). During periods of ventricular dyssynchrony, right ventricular activation begins and is completed be- fore the initiation of left ventricular activation (Dunn 1987). Right ventricular depolarization is then fol- lowed by a reversed activation sequence of the interventricular septum, proceeding from right to left, which is then followed by a parallel, rather than radial, spread of depolarization towards the left ven- tricular free wall (Dunn 1987). This delayed activa- tion of the left ventricle, more specifically the lateral wall of the left ventricle, has several important haemodynamic consequences (Aranda & Schofield 2002): • There is delayed contraction of the posteromedi- al papillary muscle, which leads to systolic mitral regurgitation. • The delayed activation of the left ventricle leads to a delay in the onset of early diastolic ventric- ular filling, but atrial activation proceeds normal- ly. The result is that early, passive diastolic filling of the left ventricle and atrial contraction occur simultaneously. This has been demonstrated by the merging of the E and A waves on mitral Doppler inflow patterns in these hearts. • Atrial activation during the early, passive dias- tolic filling phase of the left ventricle decreases the total transmitral flow which results in a dimin- ished preload of the left ventricle. • The delayed activation of the posteromedial papillary muscle also leads to diastolic mitral regurgitation. • The interventricular septum is a crucial structure for the maintenance of interventricular depen- dance. However, with delayed activation of the left ventricular lateral wall, left ventricular sys- tolic pressure will be increased well after depo- larization of the interventricular septum is com- pleted. The consequence is septal dyskinesis, with the septum moving away from the left ven- tricular wall during left ventricular contraction. The result is a decreased septal contribution to left ventricular stroke volume. Ventricular dyssynchrony is a common clinical problem and has been shown to occur in approxi- mately 30 % of humans with chronic heart failure, both of ischaemic and idiopathic origin (Barold 2001; Abraham & Fisher 2002). The objective of this investigation was to determine if ventricular dyssynchrony per se can cause struc- tural heart disease, instead of it always being con- sidered a consequence of such structural disease of the heart. MATERIALS AND METHODS Six clinically normal Dorper wethers, all between the ages of 9 and 12 months and weighing between 35 and 40 kg, were used in this study. They were fed lucerne hay ad libitum, received 300 g per day of pelleted concentrate (10 MJ ME/kg DM with 14 % crude protein) and had free access to water at all times. Right ventricular premature ventricular complexes (PVCs) were used to induce ventricular dyssyn- chrony. Right ventricular PVCs were induced in each wether as previously described by Ker & Webb (2003): A spring-wire guide, diameter 0.81 mm and length 600 mm, was advanced into the right ventri- cle via the left internal jugular vein, using the Sel- dinger technique. The position of the wire was con- firmed in every case by an X-ray. Right ventricular PVCs were induced by mechanical movement of the spring-wire guide inside the right ventricle. These right ventricular PVCs were induced on a daily basis in every wether for variable periods. Thus PVCs were induced in sheep 1 for 15 days, in sheep 2 for 36 days, in sheep 3 for 28 days, in sheep 4 for 16 days, in sheep 5 for 53 days and in sheep 6 for 34 days. On completion of each series of PVCs, each wether was subsequently slaughtered and their hearts sub- jected to histological examination. Only the mus- culature of the left ventricles were examined histo- logically as the right ventricles were subjected to mechanical trauma by the spring-wire guides used to induce the right ventricular PVCs and therefore some histological changes in the musculature of the right ventricles are to be expected. However, because the left ventricle is isolated from the right by the interventricular septum the wire will have no direct histological effects on the left ventricle. Left ventricular dissection • The musculature of each left ventricle (LV) was dissected into three regions: Two transverse in- 198 Ventricular dyssynhrony in heart of Dorper sheep cisions were made, one at the level of the base and the other at the level of the apex of the pos- teromedial papillary muscle. This divides the LV into three regions: base, mid-region and apex. • Each of these three regions were then dissected into four parts: anterior, posterior, septal and lat- eral. • In this way every LV was dissected into 12 pieces, representing the musculature of the entire LV, which were subsequently all subjected to histological examination. • These 12 segments were numbered as follows: A = Anterior part of base B = Anterior part of mid region C = Anterior part of apex D = Septal part of base E = Septal part of mid region F = Septal part of apex G = Lateral part of base H = Lateral part of mid region I = Lateral part of apex J = Posterior part of base K = Posterior part of mid region L = Posterior part of apex Histological evaluation Tissue blocks from the 12 sites were fixed in 10 % buffered formalin and paraffin-embedded sections for light microscopy were prepared using routine his- tological procedures. They were stained with hema- toxylin and eosin (HE). All the sections were evalu- ated for the following: 1. Myocardial cellular abnormalities and/or 2. Myocardial interstitial abnormalities. Histologic categories On the basis of the morphological findings each of the 12 specimens was assigned to one of four cat- egories: 1. Normal 2. Myocardial cellular abnormalities 3. Myocardial interstitial abnormalities 4. Both myocardial cellular and interstitial abnor- malities. The heart of a normal Dorper wether, of similar age and mass, served as a histological control (see Fig. 1). RESULTS No sheep in this study showed any signs of infec- tion as a result of the spring-wire guide during the entire study period. The induction of PVCs was possible throughout the study period in all the ani- mals. Histologic abnormalities As compared to the histological control (Fig. 1) we observed histological changes in all six of the exper- imental animals. These changes consisted of both myocardial cellular and interstitial abnormalities in the musculature of the left ventricle (Table 1). According to the Dallas criteria (Hare & Baughman 1994; Kühl, Noutsias, Seeberg & Schultheiss 1996; Pisani, Taylor & Mason 1997; Feldman & McNamara 2000) these observed myocardial cellular and inter- stitial changes are indicative of myocarditis. Relation between the PVC load and number of abnormal left ventricular segments We found no relation between the PVC load, as well as the number of days subjected to PVCs, and the number of abnormal left ventricular segments (Table 2). Location of left ventricular histologic abnormalities The location of these histologic abnormalities in the left ventricles of the experimental animals was evenly distributed between the anterior, posterior, septal and lateral wall (Table 3). 199 J. KER, E.C. WEBB & C.F. VAN DER MERWE FIG. 1 Longitudinal section through the musculature of the left ventricle of a normal Dorper wether heart 200 Ventricular dyssynhrony in heart of Dorper sheep TABLE 1 Histologic outcomes Sheep Histologic abnormalities in left ventricle 1 Myocardial interstitial abnormalities* in segments C, D, E, F, G, H, I, J, K 2 Myocardial interstitial abnormalities in segments B, C, D, E, I, J, K, L Myocardial cellular abnormalities** in segments B, E, K Fibrosis in segments B, K 3 Myocardial interstitial abnormalities in segments A, B, D, G, K Myocardial cellular abnormalities in segments A, D, G 4 Myocardial cellular abnormalities in segments A, C 5 Myocardial interstitial abnormalities in segments A, B, D, E, H, I, K Myocardial cellular abnormalities in segment A Fibrosis in segments D, F, I 6 Myocardial interstitial abnormalities in segments D, I * Myocardial interstitial abnormalities consist of an infiltration of white blood cells (> 5 WBCs/high power field) ** Myocardial cellular abnormalities consist of myocytolysis TABLE 2 Days subjected to PVCs, PVC load and number of abnormal left ventricular segments Days subjected PVC load* Number of abnormal to PVCs left ventricular segments 53 902 8 36 575 8 34 908 2 28 371 5 16 1 887 2 15 221 9 * The spring-wire guides were left in situ in the right ventricles and electrocardiography was done once daily. Therefore, the number of actual PVCs may be much higher than the num- ber of PVCs counted FIG. 2 Infiltration of the left ventricular interstitium by a mixed inflammatory cell infiltrate, a feature of myocarditis FIG. 3 Myocytolysis in the left ventricle, another feature of myocarditis TABLE 3 Location of left ventricular histologic abnormalities Location of abnormal Number of abnormal left left ventricular segments ventricular segments (total of 6) Anterior 5 Posterior 4 Septal 5 Lateral 5 DISCUSSION Any alteration in the normal sequence of left ventri- cular activation, thereby leading to interventricular dyssynchrony, has several important consequences. These include haemodynamic, myocardial and metabolic disturbances (Aranda & Schofield 2002; Van Oosterhout, Prinzen, Arts, Schreuder, Vanagt, Cleutjens & Reneman 1998; Zanco, Desideri, Mo- bilia, Carguel, Milan, Celegon, Buchberger & Ferlin 2000). Firstly, haemodynamic consequences are mainly due to delayed activation of the lateral wall of the left ventricle. These include systolic and diastolic mitral regurgitation, a diminished preload of the left ventricle and septal dyskinesis causing a decreased septal contribution to left ventricular stroke volume (Aranda & Schofield 2002). Secondly, myocardial consequences have been described (Van Oosterhout, Prinzen, Arts, Schreu- der, Vanagt, Cleutjens & Reneman 1998). Asynchro- nous activation of the left ventricle induces asym- metrical left ventricular hypertrophy. When there is a period of asynchronous activation of the left ven- tricle, as induced by right ventricular pacing or PVCs, this causes regional differences in the work- load of the left ventricle. The workload is lower in early than in late-activated regions and as a result the early-activated regions become thinner and the late-activated regions become thicker. Thirdly, metabolic consequences have been de- scribed as well (Zanco, Desideri, Mobilia, Cargnel, Milan, Celegon, Buchberger & Ferlin 2000). There is a reduction of septal glucose uptake and metab- olism in the left ventricle during periods of ventricu- lar dyssynchrony. According to the Dallas criteria (Hare & Baughman 1994; Kühl, Noutsias, Seeberg & Schultheiss 1996; Pisani, Taylor & Mason 1997; Feldman & McNamara 2000) all six of the experimental animals developed myocarditis. Myocarditis has many causes (Feldman & McNamara 2000). Some of these include infec- tious agents, physical agents, drugs, heavy metals, systemic diseases and various miscellaneous causes, such as snake- and spider bites (Feldman & McNamara 2000). Furthermore, myocarditis consists of various histo- pathologic subclasses and these include a lympho- cytic, eosinophilic, neutrophilic, giant cell and a granulomatous type (Pisani, Taylor & Mason 1997). Might it be possible that there is also an immuno- logical consequence of ventricular dyssynchrony, that presents with myocardial inflammation, as seen in our study? A search of the Medline database from 1966 to the present revealed only two studies where myocarditis occurred in situations of ventric- ular dyssynchrony. Basso, Corrado, Rossi & Thiene (2001) examined the hearts of eight human patients who died from Wolff-Parkinson-White (WPW) syndrome and found isolated atrial myocarditis in four (50 %) of these hearts. This syndrome is a condition in which strands of atrial-like muscle form atrioventricular bypass tracts which activate the right ventricle causing right ventricular activation to precede left ventricular acti- vation. It is therefore a cause of ventricular dyssyn- chrony. The syndrome is a congenital disorder and therefore its relationship to myocarditis is either co- incidental or possibly causal. Because PVCs do not constitute a congenital disor- der, but rather a complication of various cardiac and non-cardiac disorders, its possible relation to myo- carditis is more complex. Biase, Chiddo, Caruso, Tritto, Marchese & Rizzon (1992) examined ventric- ular endomyocardial biopsies from 26 human patients with PVCs and found acute myocarditis in 7 % and borderline myocarditis in 3.5 %. However, it is not possible to determine from this data whether PVCs are a consequence or a cause of myocardi- tis. We found in this study that the induction of right ventricular PVCs consistently led to the develop- ment of myocarditis of the left ventricle in all of the experimental animals. An interesting finding is that there was no relation between the PVC load and the number of abnormal left ventricular segments (see Table 2). As stated before, the spring-wire guides were left in situ in the right ventricles and electrocardiography was done once daily. It is therefore possible that the actual number of PVCs may be higher in sheep with less days exposed to PVCs. Alternatively, it is possible that the critical time period for the development of myocarditis is 2 weeks or less and/or that the critical number of PVCs needed to induce myocarditis is 221 or less. Another interesting finding is the distribution of myocardial inflammation (Table 2). The histological manifestations of myocarditis are evenly distributed among the anterior, posterior, septal and lateral walls of the left ventricle of all the experimental ani- mals. In conclusion, myocarditis was detected in the left ventricles in all of the experimental animals. In this study this was a diffuse process, which involved all 201 J. KER, E.C. WEBB & C.F. VAN DER MERWE four walls of the left ventricle. 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