Monitoring cardiac fibrosis: a technical challenge.

The heart contains a collagen network that contributes to the contractility of the heart and provides cardiac strength. In cardiac diseases, an increase in collagen deposition is often observed. This fibrosis formation causes systolic and diastolic dysfunction, and plays a major role in the arrythmogenic substrate. Therefore, accurate detection of cardiac fibrosis and its progression is of clinical importance with regard to diagnostics and therapy for patients with cardiac disease. To evaluate cardiac collagen deposition, both invasive and non-invasive techniques are used. In this review the different techniques that are currently used in clinical and experimental setting are summarised, and the advantages and disadvantages of these techniques are discussed.

Longitudinal arrhythmogenic remodelling in a mouse model of longstanding pressure overload.

INTRODUCTION: Sudden arrhythmogenic cardiac death is a major cause of mortality in patients with congestive heart failure due to adverse electrical remodelling. To establish whether abnormal conduction is responsible for arrhythmogenic remodelling in progressed stages of heart failure, we have monitored functional, structural and electrical remodelling in a murine model of heart failure, induced by longstanding pressure overload. METHODS: Mice were subjected to transverse aortic constriction (TAC; n=18) or sham operated (n=19) and monitored biweekly by echocardiography and electrocardiography. At the 16-week endpoint, electrical mapping was performed to measure epicardial conduction velocity and susceptibility to arrhythmias. Finally, tissue sections were stained for Cx43 and fibrosis. RESULTS: In TAC mice, fractional shortening decreased gradually and was significantly lower compared with sham at 16 weeks. Left ventricular hypertrophy was significant after six weeks. TAC mice developed PQ prolongation after 12 weeks, QT prolongation after 16 weeks and QRS prolongation after two weeks. Right ventricular conduction velocity was slowed parallel to fibre orientation. In 8/18 TAC hearts, polymorphic ventricular tachyarrhythmias were provoked and none in sham hearts. TAC mice had more interstitial fibrosis than sham. Immunohistology showed that Cx43 levels were similar but highly heterogeneous in TAC mice. All parameters were comparable in TAC mice with and without arrhythmias, except for Cx43 heterogeneity, which was significantly higher in arrhythmogenic TAC mice. CONCLUSION.: Chronic pressure overload resulted in rapid structural and electrical remodelling. Arrhythmias were related to heterogeneous expression of Cx43. This may lead to functional block and unstable reentry, giving rise to polymorphic ventricular tachyarrhythmias. (Neth Heart J 2010;18:509-15.).

Differences in distribution of fibrosis in the ventricles underlie dominant arrhythmia vulnerability of the right ventricle in senescent mice.

Mutations that are supposed to affect right (RV) and left ventricular (LV) electrophysiology equally, often reveal dominant conduction slowing and arrhythmia vulnerability in RV. In this study we investigated the mechanism of dominant arrhythmia vulnerability of RV in senescent mice. We performed epicardial ventricular activation mapping on adult and senescent Langendorff perfused hearts. Longitudinal and transversal conduction velocity, as well as arrhythmia inducibility were determined. Subsequently, hearts were processed for immunohisto-chemistry and Picro Sirius Red staining. Senescent mice revealed decreased conduction velocity, increased aniso-tropic ratio and reduced excitation wavelength in RV, but not in LV. Arrhythmias were mainly induced in RV of senescent hearts. No arrhythmias were induced in adult hearts. Immunohistochemistry revealed that the amount of Connexin 43 and cardiac sodium channel Nav1 .5 were equally decreased, and that collagen content was equally increased in senescent RV and LV. However, patches of replacement fibrosis were found throughout the RV wall, but only in the sub-endocardium and mid-myocardium of LV. The study shows that the dominant arrhythmia vulnerability in RV of senescent mice is caused by the distribution of replacement fibrosis which involves the entire RV but only part of the LV. (Neth Heart J 2008;16:356-8.).

Adrenergic regulation of conduction velocity in cultures of immature cardiomyocytes.

During cardiac maturation, increased exposure of the heart to circulating catecholamines correlates with increased conduction velocity and growth of the heart. We used an in vitro approach to study the underlying mechanisms of adrenergic stimulation induced changes in conduction velocity. By combining functional measurements and molecular techniques, we were able to demonstrate that the increased conduction velocity after beta-adrenergic stimulation is probably not caused by changes in intercellular coupling. Instead, RT-PCR experiments and action potential measurements have shown an increased excitability that may well explain the observed increase in conduction velocity. Apart from being relevant to cardiac maturation, our findings are relevant in the context of stem cells and cardiac repair. Preconditioning of stem cell derived cardiomyocytes may help to enhance electrical maturation of de novo generated cardiomyocytes and consequently reduce their proarrhythmogenic potential. (Neth Heart J 2008;16:106-9.).

Normal embryonic development and cardiac morphogenesis in mice with Wnt1-Cre-mediated deletion of connexin43.

Mice harboring a null mutation in the gap junction protein connexin43 (Cx43) die shortly after birth due to an obstruction of the right ventricular outflow tract of the heart. These hearts exhibit prominent pouches at the base of the pulmonary outlet, i.e., morphological abnormalities that were ascribed to Cx43-deficiency in neural crest cells. In order to examine the Cx43 expression pattern in neural crest cells and derived tissues and to test whether neural crest-specific deletion of Cx43 leads to the conotruncal defects seen in Cx43null mice, we ablated Cx43 using a Wnt1-Cre transgene. Deletion of Cx43 was complete and occurred in neural crest cells as well as in neural crest-derived tissues. Nevertheless, hearts of mice lacking Cx43 specifically in neural crest cells were indistinguishable from controls. Thus, the morphological heart abnormalities of Cx43 null mice are most likely not caused by lack of Cx43 in neural crest cells.

Functional role of connexin43 gap junction channels in adult mouse heart assessed by inducible gene deletion.

The gap junction protein Connexin43 (Cx43) is expressed in various cell types during embryonic development and in adult mice. Cx43 null mice (Cx43-/-) die perinatally due to cardiac malformation. In order to define the major functional role of Cx43 gap junction channels in adult mice and to circumvent perinatal death as well as direct or indirect compensation of Cx43 deficiency during development, we established a novel conditional Cx43 mouse mutant. To ablate Cx43 in adult mice in all cells that express Cx43 at a certain time, we targeted the 4-hydroxytamoxifen inducible Cre recombinase, Cre-ER(T), into the endogenous Cx43 locus. This approach left only one Cx43 coding region to be deleted upon induction of Cre-ER(T) activity. Highly efficient inducible ablation of Cx43 was shown in an embryonic stem cell test system and in adult mice. Although Cx43 protein was decreased in different tissues after induction of Cre-ER(T)-mediated recombination, cardiac abnormalities most likely account for death of those mice. Surface and telemetric ECG recordings revealed significant delay of ventricular activation and death during periods of bradyarrhythmia preceded by tachycardias. This novel approach of inducible ablation of Cx43 highlights the functional importance of normal activation of ventricular cardiomyocytes mediated by Cx43 gap junction channels in adult mouse heart to prevent initiation of fatal arrhythmias. The new mouse model should be useful for further analyses of molecular changes initiated by acute loss of Cx43 expression in various cell types.

Cardiac gap junction channels: modulation of expression and channel properties.

In the heart, intercellular gap junction channels constructed from connexin molecules are crucial for conduction of the electric impulse. Cardiomyocytes can be interconnected by channels composed of three types of connexin proteins: Cx40, Cx43 or Cx45. In mammalian hearts, these three isoforms are regionally differently expressed and even between the species differences exist. Each of these channel-types possesses specific properties and are susceptible to modulation by various mechanisms. In this paper we compare the differences in properties of these channels as deduced from studies on transfected cells and isolated cardiomyocytes and discuss the factors involved in modulation of channel properties. Next, we evaluate the consequences of alterations in expression and modulation of channel properties for cardiac function. Therefore, we have compared reports on genetically engineered animals and discuss this information in relation to various pathophysiological disorders.

Impaired conduction in the bundle branches of mouse hearts lacking the gap junction protein connexin40.

BACKGROUND: Connexin (Cx)40 and Cx45 are the major protein subunits of gap junction channels in the conduction system of mammals. To determine the role of Cx40, we correlated cardiac activation with Connexin distribution in normal and Cx40-deficient mice hearts. METHODS AND RESULTS: Epicardial and septal activation was recorded in Langendorff-perfused adult mice hearts with a 247-point compound electrode (interelectrode distance, 0.3 mm). After electrophysiological measurements, hearts were prepared for immunohistochemistry and histology to determine Connexin distribution and fibrosis. In both wild-type and Cx40-deficient animals, epicardial activation patterns were similar. The right and left ventricular septum was invariably activated from base to apex. Histology revealed a continuity of myocytes from the common bundle to the septal myocardium. Within this continuity, colocalization was found of Cx43 and Cx45 but not of Cx40 and Cx43. Both animals showed similar His-bundle activation. In Cx40-deficient mice, the proximal bundle branches expressed Cx45 only. The absence of Cx40 in the proximal bundles correlated with right bundle-branch block. Conduction in the left bundle branch was impaired as compared with wild-type animals. CONCLUSIONS: Our data show that (1) in mice, a continuity exists between the common bundle and the septum, and (2) Cx40 deficiency results in right bundle-branch block and impaired left bundle-branch conduction.

Electrical conductance of mouse connexin45 gap junction channels is modulated by phosphorylation.

In this study we report about the modulation of connexin45 (Cx45) gap junction channel properties by phosphorylation of the connexin molecules through different protein kinases. Phosphorylation of Cx45 was studied in HeLa cells transfected with mouse Cx45 (mCx45). Using Western blotting (WB) and immunocytochemistry, these cells were found exclusively positive for Cx45 and the protein was separated as a doublet of bands with a calculated mass of 46 and 48 kD. After dephosphorylation using calf intestine phosphatase (CIP), the 48 kD band disappeared almost completely leaving a single band at 46 kD. This effect can be prevented by including phosphatase inhibitors during CIP treatment. These results indicate that the 48 kD signal represents a phosphorylated form of Cx45. To investigate the effects of (de)phosphorylation of Cx45 on the conductive properties of gap junction channels built of this connexin, cell pairs were subjected to dual voltage clamp experiments and coupling was determined before and after addition of PMA, 4alpha-PDD, cAMP, cGMP, and pervanadate to the superfusate. 100 nM of the PKC activating phorbol ester PMA increased normalized junctional conductance by 50.9+/-28%. 100 nM of the inactive phorbol ester 4alpha-PDD had no significant effect. Activation of PKA with 1 mM 8-Br-cAMP decreased coupling by 20.9+/-5.7% while 1 mM 8-Br-cGMP (PKG-activation) was ineffective. 100 microM pervanadate, a tyrosine phosphatase inhibitor, reduced coupling by 43.7+/-11.1%. Single channel measurements, under identical phosphorylating conditions, were not significantly different from each other and all frequency histograms exhibited two conductance peaks at approximately 20 and 40 pS. WB analysis revealed, as compared to control conditions, a relative increase of the 48 kD signal upon stimulation with pervanadate (142+/-42%) and 8-Br-cAMP (50+/-23%) whereas neither stimulation with PMA nor 8-Br-cGMP had a significant effect. These experiments show that electrical intercellular conductance via Cx45 gap junction channels is differentially regulated by phosphorylation. However, regulation does not act by changing single channel conductance, but most likely by modulation of the open probability of Cx45 gap junction channels.

Human connexin40 gap junction channels are modulated by cAMP.

OBJECTIVE: Gap junction channels provide for direct electrical coupling between cells, and play an important role in homeostasis and electrical coupling. One of the proteins that form gap junctions, Connexin40 (Cx40), shows restricted expression in the body, and is found in blood vessels and in the atrium and conduction system of the heart. We have investigated whether gap junction channels formed of Cx40 are modulated by protein-kinase-A-mediated phosphorylation. METHODS: A communication-deficient human hepatoma cell line (SKHep1) was stably transfected with human Cx40 cDNA and the properties of Cx40 gap junctions channels and their modulation by cAMP were analyzed using immunocytochemistry, Western blotting, dual patch clamp, and dye coupling. RESULTS: Administration of 1 mM 8-Br-cAMP resulted in a mobility shift of Cx40 protein on western blot and increased macroscopic gap junctional conductance between cell pairs by 46.2 +/- 12.0% (mean +/- S.E.M., n = 8). Under control conditions, single channel experiments revealed three single channel conductances around 30, 80 and 120 pS. When cAMP was added, channel conductances of 46 and 120 pS were observed. In monolayers, cAMP also increased the permeability of Cx40 gap junction channels for Lucifer Yellow by 58%. CONCLUSIONS: Macroscopic conductance and permeability of Cx40 gap junctions is strongly increased by cAMP and may play a role in the regulation of intercellular communication in the heart and vasculature.

Quantitative analysis of dual whole-cell voltage-clamp determination of gap junctional conductance.

The dual whole-cell voltage-clamp technique is used widely for determination of kinetics and conductance of gap junctions. The use of this technique may, however, occasion to considerable errors. We have analysed the errors in steady state junctional conductance measurements under different experimental conditions. The errors in measured junctional conductance induced by series resistance alone, and by series resistance in combination with membrane resistance, were quantified both theoretically and experimentally, on equivalent resistive circuits with known resistance values in a dual voltage-clamp setup. We present and analyse a method that accounts for series resistance and membrane resistance in the determination of true junctional conductance. This method requires that series resistance is determined during the experiment, and involves some calculations to determine membrane resistance. We demonstrate that correction for both membrane and series resistance reduces the error in measured junctional conductance to near zero, even when membrane resistances on both sides of the gap junction are as low as 20 MOmega and the (true) junctional conductance is as high as 100 nS.

Tumour necrosis factor alpha alters the expression of connexin43, connexin40, and connexin37 in human umbilical vein endothelial cells.

Tumour necrosis factor alpha (TNF-alpha) plays an important role in orchestrating inflammatory responses with the vascular endothelium as main target cell type, and was found to promote migration of endothelial cells, as occurs in wound healing processes. Substantial evidence exists that endothelial cell migration in wound healing is related to changes in cell coupling by means of gap junctions. Gap junctions are agglomerates of cell-to-cell channels that allow direct electrical and metabolic communication between cells. The authors have investigated whether TNF-alpha alters the expression of gap junction proteins (connexins, Cx) between human umbilical vein endothelial cells (HUVEC), thereby changing the extent of intercellular communication, as measured by dye coupling. Under control conditions, Cx43, Cx40, and Cx37 protein and mRNA were present in HUVEC. After exposure to 0.5 nM TNF-alpha for 48 h, however, the authors were no longer able to detect Cx37 and Cx40 protein, whereas Cx43 levels seemed unaltered but showed more perinuclear staining. After 24 and 48 h exposure to TNF-alpha, levels of Cx37 and Cx40 mRNA, were reduced, while the level of Cx43 mRNA remained unaltered, suggesting transcriptional regulation. If TNF-alpha was removed from the medium, Cx37 and Cx40 expression was restored within 24 h. The modulation of connexin expression by TNF-alpha resulted in a decrease in dye coupling of 40%.