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- Material and Methods
- LITERATURE CITED
Chronic volume overload leads to cardiac hypertrophy and later to heart failure (HF), which are both associated with increased risk of cardiac arrhythmias. The goal of this study was to describe changes in myocardial morphology and to characterize arrhythmogenic substrate in rat model of developing HF due to volume overload. An arteriovenous fistula (AVF) was created in male Wistar rats between the inferior vena cava and abdominal aorta using needle technique. Myocardial morphology, tissue fibrosis, and connexin43 distribution, localization and phosphorylation were examined using confocal microscopy and Western blotting in the stage of compensated hypertrophy (11 weeks), and decompensated HF (21 weeks). Heart to body weight (BW) ratio was 89% and 133% higher in AVF rats at 11 and 21 weeks, respectively. At 21 weeks but not 11 weeks, AVF rats had pulmonary congestion (increased lung to BW ratio) indicating presence of decompensated HF. The myocytes in left ventricular midmyocardium were significantly thicker (+8% and +45%) and longer (+88% and +97%). Despite extensive hypertrophy, there was no excessive fibrosis in the AVF ventricles. Distribution and localization of connexin43 were similar between groups, but its phosphorylation was significantly lower in AVF hearts at 21st week, but not 11th week, suggesting that HF, rather than hypertrophy contributes to the connexin43 hypophosphorylation. In conclusion, volume overload leads to extensive eccentric hypertrophy, but not to myocardial fibrosis. Increased vulnerability to arrhythmia in this HF model is possibly related to gap junction remodeling with hypophosphorylation of connexin43. Anat Rec, 2010. © 2010 Wiley-Liss, Inc.
The increase in volume loading of the heart due to valve insufficiency or arteriovenous fistula (AVF) causes dilation of cardiac chambers and cardiac hypertrophy (Ford,1976). It is believed that this process is due to cardiomyocyte elongation and hypertrophy that compensate volume overload and normalize wall stress (Grossman et al.,1975). Despite cardiac output being often increased (such as in the case of chronic arteriovenous fistula–AVF), substantial part of stroke volume is shunted or recirculated and is not contributing to systemic perfusion. Diminished systemic perfusion leads to redistribution of cardiac output and neurohumoral activation. When compensatory mechanisms become inadequate, overt heart failure (HF) develops (Hood et al.,1968) in a way similar to other models of LV hypertrophy-HF transition (Hatt et al.,1979; Legault et al.,1990; Ruzicka et al.,1993; Ryan et al.,2007).
Regardless its etiology, cardiac hypertrophy is associated with increased incidence of potentially life-threatening ventricular arrhythmias (Artham et al.,2009) and is one of the strongest risk factors for sudden cardiac death (Haider et al.,1998). The mechanisms of arrhythmias in eccentric hypertrophy due to volume overload are known less than in cardiac hypertrophy due to pressure overload or chronic myocardial infarction. Arrhythmogenesis is often linked to increased electrical heterogeneity of myocardial tissue and slowed impulse conduction (Shah et al.,2005). The involved mechanisms consist of myocardial fibrosis, changes in cell and tissue architecture, membrane excitability, and alterations of gap-junctional coupling (Libby et al.,2008). Gap junctions are required for electrical impulse propagation and synchronous contraction in the healthy heart and their alterations might contribute to abnormal conduction and thus be a substrate for arrhythmia (von Olshausen et al.,1983; Kligfield et al.,1987; Kostin et al.,2003; Wiegerinck et al.,2008). The main protein forming gap junctions in rat ventricular myocardium is connexin43 (Sohl and Willecke,2004). Changes in amount and in localization of connexin43 have been reported in the diseased myocardium (Severs et al.,2004). Some of the previous studies demonstrated a reduction in connexin43 levels in left ventricles of transplant patients with end-stage HF (Dupont et al.,2001). Lateralization of connexin43 from intercalated discs to lateral membrane of myocytes occurs in experimentally induced hypertrophy of right and left ventricle of the rat (Uzzaman et al.,2000; Emdad et al.,2001). Apart from alterations in connexin43 levels, the dephosphorylation of connexin43 was described during several pathological states, including myocardial ischemia (Beardslee et al.,2000; Burstein et al.,2009). Taking all this into an account, new antiarrhythmic drugs targeting function of gap junctions are developed, with rotigaptide, a selective gap junction modifier, as an example (Haugan et al.,2006).
In our study, HF in rats was induced by AVF. Similar volume overload HF models were created also by other groups in dogs (Legault et al.,1990) and rats (Hatt et al.,1979; Ruzicka et al.,1993; Ryan et al.,2007). However, no previous study characterized cardiac morphology at two distinct phases of HF development. The main purpose of this study was thus to provide morphological characteristics of the AVF experimental rat HF model induced by volume overload with a focus on abnormalities in myocardial tissue potentially contributing to arrhythmogenesis, such as fibrosis and connexin43 distribution.
- Top of page
- Material and Methods
- LITERATURE CITED
Aortocaval fistula results in volume overload, which induces cardiac dilation and hypertrophy. Over time, this leads to HF and increased mortality. In our study, rats at 21 weeks demonstrated not only hypertrophy, but also decompensated HF phenotype with increased normalized weight of lungs and HF symptoms. In the failing rats, there was an increased heart weight, heart to BW ratio, and thickness of ventricular walls. The hypertrophy was present already after 11 weeks and the heart weight did not change with the time of volume overload, which shows that hypertrophy as a compensatory mechanism evolving early after creation of AVF. However, this compensated state became decompensated over time, resulting in development of congestive HF, as was demonstrated by increasing lung weight. The dynamic nature of changes during development of HF was best exemplified by increased heart to BW ratio, which was increased by almost 90% at 11 weeks but by more than 130% at 21 weeks in comparison with aged-matched sham controls (Table 1). Since the total weight of the animals did not change significantly, other organs have to shrink; this indicates increased catabolic state characteristic of HF and documented in a separate functional and metabolic study (Benes et al., submitted) by decreased fat reserves. At present, we are evaluating chronic changes in animals that have been in HF for 1 year to extend the longitudinal aspect of this study and to see the combined effects of HF and ageing. Cardiac dilation and hypertrophy were confirmed by increased size of cardiomyocytes, which were enlarged in both longitudinal and transverse diameters. Cardiomyocytes in midmyocardium were more affected than those in papillary muscles. This is in contrast with asymmetric cellular hypertrophy in pressure overload model (Campbell et al.,1989), where most pronounced hypertrophy was found in the subendocardial layers. The interesting discrepancy between continued increase in ventricular wall thickness that could not be explained by increased myocyte dimensions, especially in the right ventricle (Table 2) suggests, in the notable absence of fibrosis, that there could be activation of myocyte (or resident stem cells) proliferation, that was recognized previously in decompensated HF in humans (Kajstura et al.,1998).
AVF model of volume overload induced HF had been described before. However, no previous study described these changes in decompensated HF stage. The principal new findings of our study include distinction between compensated (11 weeks) and decompensated (21 weeks) stage of HF, complete morphological evaluation of possible arrhythmogenic substrates (cell size and shape, connexin expression and distribution, fibrosis) and providing links between these findings and ventricular arrhythmias (Fig. 5). Our data thus provide solid morphological grounds for ongoing functional, metabolic and pharmacological studies in this model. Ruzicka et al. used this model for describing renin-angiotensin system and effects of angiotensin converting enzyme inhibitors in situation of volume overload (Ruzicka et al.,1993). Ryan et al. described remodeling process induced by bradykinin (Ryan et al.,2007). Study of Hatt et al. (1979) is closest to our morphological approach. Hatt et al. measured cells in failing hearts and our study generally corresponds with their findings. Their sampling intervals were 1 and 6 months, so it is not possible to compare exactly our findings with theirs. In their study, the hearts of rats with volume overload increased their weight by 81% after 6 month (compare to our increase by 140% after 21 weeks). They also measured cell width in midwall and in subendocardium and described greater increase in cell width in subendocardium than in midwall, which is in contrast to our data. Generally, the changes they described in the group of rats 6 month after performing the fistula were much milder than our findings. The possible reason for this is that they used female rats and there is known strong gender difference in cardiac response to volume overload—females are far less prone to eccentric remodeling and to the development of HF symptoms (Gardner et al.,2009). Since the methodology of fistula creation was not described in detail in that study, it is also possible that their method of fistula creation may have differed (e.g., different diameter of the needle, no exclusion of animals where AVF closed spontaneously). The reason for different findings in subendocardial myocyte width changes can be explained by the fact that we measured width in papillary muscle from transversal sections through the cells while Hatt et al. measured cells in myocardial wall in transversal sections.
Figure 5. ECG tracing (2 leads, bottom) and the left ventricular pressure tracing (2F Millar catheter) from a rat with AVF. Several ectopic beats degenerate into fast polymorphic ventricular tachycardia and later to ventricular fibrillation with immediate collapse of the circulation, visible in the pressure channel.
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Hypertrophic or failing rat or human hearts have bigger predisposition to develop severe ventricular arrhythmias (von Olshausen et al.,1983; Kligfield et al.,1987). Increased risk of sudden, presumably arrhythmic death was found even in asymptomatic subjects with left ventricular volume overload due to mitral regurgitation (Grigioni et al., 1999). Similarly, we observed increased excitability of hypertrophied AVF hearts, characterized by high frequency of ventricular ectopic beats and bursts of ventricular tachycardia, particularly during intraventricular measurements of pressure using Millar catheter (our unpublished observations). In several AVF animals, this ectopy even degenerated into lethal ventricular fibrillation, which is exceedingly rare in normal rat hearts (Fig. 5). In previous study that examined long-term survival of rats with AVF, 27% of all animals died without preceding HF symptoms (Brower and Janicki2001), suggesting that arrhythmic sudden cardiac death occurs in substantial proportion of animals with AVF-induced chronic volume overload. Since no excessive collagen accumulation was found in the ventricular myocardium, we suggest that other arrhythmogenic mechanisms than fibrosis might be involved. In our study, we focused on connexin43 changes. Changes in connexin43 can lead to slowing of conduction velocity in ventricular wall, which may create a substrate for re-entry phenomenon (Libby et al.,2008). The main changes described in previous studies were changes in expression, localization, and phosphorylation (Severs et al.,2004). This is not, however, the only possible mechanism. Other contributing factors can be changes in ion channel expression, which can also substantially contribute to arrhythmogenesis (Shah et al.,2005).
In a partial contrast with previous studies of connexin43 changes in HF (Dupont et al.,2001; Emdad et al.,2001; Uzzaman et al.,2000), we found only mild difference in total connexin43 levels by immunohistochemistry (but there was over 60% reduction by Western blot), and the changes in localization were also insignificant. Possible reason for this discrepancy can be differences in HF models or methods used. Dupont et al. described the changes in humans and used Northern blot for quantification, detecting thus mRNA levels (Dupont et al.,2001). Emdad et al. described HF in rats induced by pressure overload from aortic banding and they used enzymatic separation of myocytes to measure connexin43 levels by immunohistochemistry (Emdad et al.,2001). Recent study of Burstein et al. described changes in dog HF atria and they found also no difference in the amount of connexin43, which corresponds to our immunohistochemistry data (Burstein et al.,2009). Study of Goldfine et al. describes connexin43 changes in volume overload HF model. They found decrease in connexin43 levels in acute state of volume overload HF but when compensatory hypertrophy developed, the amount of connexin43 seemed to normalize (Goldfine et al.,1999). In any case, the absolute levels of connexin43 have to be decreased over 50% to induce significant physiological phenotype per se, as indicated by apparent normality of connexin43 heterozygous mice as well as ventricular arrhythmias leading to sudden cardiac death observed in myocardium-restricted null animals (Gutstein et al.,2001). In this respect, the 60% decreased in total amount of connexin43 found in our Western blot seems biologically sufficiently significant to form (together with changes in cell shape) a proarrhythmogenic substrate, as ventricular arrhythmias were recorded in ∼10% of our HF animals.
Phosphorylation of connexin43 can influence conductance, assembly and degradation of gap junctions (Lampe and Lau,2004; Laird,2005; Solan and Lampe,2005). Recent work suggests an important role of connexin43 phosphorylation in HF (Akar et al.,2004; Ai and Pogwizd,2005). We found greatly decreased phosphorylation of connexin43 in the stage of decompensated HF (21 weeks after operation). The phosphorylation of connexin43 at the earlier stage, when congestive HF was not yet developed (11 weeks after operation), was also slightly decreased but without statistical significance. Thus, our data suggest that the HF and not the hypertrophy itself contribute to the hypophosphorylation. Targeting the phosphorylation status of connexin43 using specific drugs in patients with HF can be a method to prevent development of fatal ventricular arrhythmias.