Insights into sinus arrhythmia of the dog: Acetylcholine perfusion of canine right atrium results in beat-to-beat patterns that mimic sinus arrhythmia supporting exit block in the sinoatrial conduction pathways.

Sinus arrhythmia of the dog is unique because of the pronounced alternating beat-to-beat intervals. The clustering of these short (faster rates) and long (slower rates) intervals is not just influenced by autonomic input from breathing; sinus arrhythmia can persist in the panting or apneic dog. The multiplicity of central and peripheral influences on the sinus node complicates the unraveling of the mechanisms of sinus arrhythmia. Studies of the sinus node suggest that acetylcholine can slow cellular depolarization and block sinoatrial conduction. Electrocardiographic monitoring of the dog supports this notion in that abrupt bifurcation into short and long intervals develop at lower heart rates. We sought to determine whether this phenomenon could be recapitulated in canine atrial preparations perfused with acetylcholine and whether selective pharmacologic blockade of the voltage and calcium clocks could provide insight into its mechanism. Spontaneous beat to beat (A-A) intervals were obtained from monophasic action potential recordings of perfused canine right atrial preparations before and during perfusion with acetylcholine (2-5 µM). The calcium clock was blocked with ryanodine (2-3 µM). The membrane clock was blocked with diltiazem hydrochloride (ICa,L blocker; 0.25 µM) and ZD7288 (If blocker; 3 µM). Hyperpolarization was hindered by blockade of IK,Ado/IK,Ach with tertiapin Q (100 nM) before and during acetylcholine perfusion. Acetylcholine resulted in beat clusters similar to those seen in sinus arrhythmia of the dog. Beat clusters were consistent with intermittent 2:1 and 3:1 sinoatrial conduction block. Tertiapin Q abolished this patterning suggesting a role of IK,Ado/IK,ACh in the mechanism of these acetylcholine-induced beat-to-beat patterns.

Change in ß-catenin localization suggests involvement of the canonical Wnt pathway in Boxer dogs with arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited myocardial disease with high prevalence in the Boxer dog population. It is characterized by replacement of the myocardium with fatty or fibro-fatty tissue. Several mechanisms for the development of ARVC have been suggested, including dysfunction of the canonical Wnt pathway, which is linked to many cellular functions, including growth and differentiation of adipocytes. HYPOTHESIS: Wnt pathway dysfunction is involved in the development of ARVC in the Boxer as evidenced by mislocalization of ß-catenin, an integral Wnt pathway modulator, and striatin, a known Wnt pathway component. ANIMALS: Five dogs without ARVC and 15 Boxers with ARVC were identified by 24-hour Holter monitoring and histopathologic examination of the heart. METHODS: Right ventricular samples were collected and examined using confocal microscopy, Western blots, and quantitative (q) PCR. RESULTS: Confocal microscopy indicated that ß-catenin localized at sites of cell-to-cell apposition, and striatin localized in a diffuse intracellular pattern in hearts without ARVC. In hearts affected with ARVC, both ß-catenin and striatin were colocalized with the endoplasmic reticulum (ER) marker calreticulin. Western blots indentified a 50% increase in the amount of ß-catenin in ARVC samples. No change in ß catenin mRNA was detected using qPCR. CONCLUSIONS: Our data suggest that trafficking of Wnt pathway proteins from the ER to their proper location within the cell is inhibited in Boxers with ARVC. These results suggest that disturbances in the Wnt pathway may play a role in the development of ARVC in the Boxer.

Fluorescence in-situ hybridization for the identification of bacterial species in archival heart valve sections of canine bacterial endocarditis.

Bacterial endocarditis (BE) is defined as inflammation of cardiac valve structures and/or the endocardium secondary to bacterial infection. Canine valvular BE is associated with significant morbidity and mortality and ante-mortem diagnosis and post-mortem identification of causative organisms is problematic. Identification of bacteria in canine BE has traditionally relied on visualization of organisms on histological sections stained with haematoxylin and eosin (HE), Gram and modified Steiner's stains. Each of these staining techniques has limitations with respect to identification of bacterial species in cases of BE. Fluorescence in-situ hybridization (FISH) has been introduced recently as a technique to identify bacteria in biological specimens. To our knowledge, FISH has not been used previously to identify bacteria in archival samples of heart valves from dogs with naturally occurring BE. We sought to determine whether FISH could detect the presence and species of bacteria in archival heart valve sections from dogs with BE, and to compare FISH to histochemical stains in the identification of bacteria. FISH detected bacteria in seven of 17 cases of canine BE and showed near perfect agreement with modified Steiner's stain for the detection of bacteria. FISH identified Streptococcus spp. and/or Staphylococcus spp. in all of these cases, but Bartonella spp. were not identified.

Identification of C-terminal domain residues involved in protein kinase A-mediated potentiation of kainate receptor subtype 6.

Glutamate receptors are the major excitatory receptors in the vertebrate CNS and have been implicated in a number of physiological and pathological processes. Previous work has shown that glutamate receptor function may be modulated by protein kinase A (PKA)-mediated phosphorylation, although the molecular mechanism of this potentiation has remained unclear. We have investigated the phosphorylation of specific amino acid residues in the C-terminal cytoplasmic domain of the rat kainate receptor subtype 6 (GluR6) as a possible mechanism for regulation of receptor function. The C-terminal tail of rat GluR6 can be phosphorylated by PKA on serine residues as demonstrated using [gamma-32P]ATP kinase assays. Whole cell recordings of transiently transfected human embryonic kidney (HEK) 293 cells showed that phosphorylation by PKA potentiates whole cell currents in wildtype GluR6 and that removal of the cytoplasmic C-terminal domain abolishes this potentiation. This suggested that the C-terminal domain may contain residue(s) involved in the PKA-mediated potentiation. Single mutations of each serine residue in the C-terminal domain (S815A, S825A, S828A, and S837A) and a truncation after position 855, which removes all threonines (T856, T864, and T875) from the domain, do not abolish PKA potentiation. However, the S825A/S837A mutation, but no other double mutation, abolishes potentiation. These results demonstrate that phosphorylation of the C-terminal tail of GluR6 by PKA leads to potentiation of whole cell response, and the combination of S825 and S837 in the C-terminal domain is a vital component of the mechanism of GluR6 potentiation by PKA.

Right atrioventricular valve malformation in dogs and cats: an electrocardiographic survey with emphasis on splintered QRS complexes.

The purposes of this study were 2-fold: (1) to determine the prevalence of splintered QRS complexes (Rr', RR', rR', rr') and other electrocardiographic abnormalities in dogs and cats with congenital right atrioventricular valve malformation (RAVM) and (2) to determine if the Labrador Retriever was at greater risk for RAVM and splintered QRS complexes. EKGs from 39 dogs and 6 cats with echocardiographically diagnosed RAVM were studied retrospectively. Splintered QRS complexes were commonly found in affected Labrador Retrievers (9 of 19, 47%), non-Labrador Retrievers (12 of 20, 60%), and cats (4 of 6, 67%). Right ventricular enlargement was most commonly detected by precordial leads (CV6LL[V2], CV6LU[V4]) in the dogs and by the standard limb leads in the cats. Arrhythmias were uncommon. The Labrador Retriever was significantly overrepresented (P < .001) In the RAVM group when compared to the general hospital population (50% versus 8%). Males were also significantly overrepresented (P < .01). It was concluded that splintered QRS complexes are a distinctive and common electrocardiographic finding in dogs and cats with RAVM. Moreover, this congenital cardiac defect is most common in the Labrador Retriever, although this breed does not have proportionately more or less splintering of the QRS complexes than other breeds.