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.

The effects of pentoxifylline on equine platelet aggregation.

BACKGROUND: Pentoxifylline (PTX) possesses a number of vasomotor, immunomodulatory, and hemorheologic properties. Based upon the hypothesis that equine laminitis and navicular disease result from microthrombosis, the inhibitory effects of PTX on inflammatory cytokines, and its inhibitory effects on human platelet aggregation, PTX has been widely used to treat equine endotoxemia, navicular disease, and laminitis. Despite this, the effects of PTX on equine platelet aggregation have not been investigated previously. HYPOTHESIS: PTX decreases platelet aggregation in equine whole blood at concentrations approximating those achieved in horses given clinically relevant doses of PTX. ANIMALS: Seven healthy adult horses from a research herd. METHODS: Whole blood impedance aggregometry using whole equine blood incubated with varying concentrations of PTX. Adenosine diphosphate (ADP) and collagen were used to initiate aggregation. RESULTS: The onset time of collagen-induced equine platelet aggregation was significantly shortened by PTX. The maximum slope of resistance change (dR/dt) and total resistance change of collagen-induced platelet aggregation were unaffected by PTX. No effects of PTX on ADP-induced onset time of aggregation, dR/dt, or total resistance change were observed. CONCLUSIONS AND CLINICAL IMPORTANCE: Our hypothesis is not supported by the results. PTX hastens the onset of collagen-induced platelet aggregation in equine whole blood, but has no effect on the rate of collagen-induced aggregation. PTX does not affect ADP-dependent equine platelet aggregation. Given these findings, PTX may not be a reasonable therapeutic option to decrease platelet aggregation in horses.

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.

The calcimimetic R-467 potentiates insulin secretion in pancreatic beta cells by activation of a nonspecific cation channel.

The extracellular, G protein-linked Ca(2+)-sensing receptor (CaSR), first identified in the parathyroid gland, is expressed in several tissues and cells and can be activated by Ca(2+) and some other inorganic cations and organic polycations. Calcimimetics such as NPS (R)-N-(3-phenylpropyl)-alpha-methyl-3-methoxybenzylamine hydrochloride (R-467), a phenylalkylamine, are thought to activate CaSR by allosterically increasing the affinity of the receptor for Ca(2+). When tested for its effect on insulin release in C57BL/6 mice, R-467 had no effect under basal conditions but enhanced both phases of glucose-stimulated release. The betaHC9 cell also responded to R-467 and to the enantiomer S-467 with a stimulation of insulin release. In subsequent studies with the betaHC9 cell, it was found that the stimulatory effect was due to activation of a nonspecific cation channel, depolarization of the beta-cell, and increased Ca(2+) entry. No other stimulatory mechanism was uncovered. The depolarization of the cell induced by the calcimimetic could be due to a direct action on the channel or via the CaSR. However, it appeared not to be mediated by G(i), G(o), G(q/11), or G(s). The novel mode of action of the calcimimetic, combined with the glucose-dependence of the stimulation on islets, raises the possibility of a totally new class of drugs that will stimulate insulin secretion during hyperglycemia but which will not cause hypoglycemia.

A syndrome resembling idiopathic noncirrhotic portal hypertension in 4 young Doberman pinschers.

We describe 4 young male Doberman Pinschers (3 littermates and 1 unrelated dog) with a syndrome resembling idiopathic or noncirrhotic portal hypertension of humans. Each dog was evaluated for a hepatopathy resulting in portal hypertension, development of portosystemic collateral vessels, and hepatic encephalopathy. These dogs differ from previous reports of young dogs with hepatic insufficiency associated with portal hypertension and acquired portal systemic shunting by their lack of intrahepatic arteriovenous fistulae, portal vein atresia, or intrahepatic fibrosis. Clinicopathologic features included erythrocyte microcytosis, normal to mildly increased liver enzyme activities, increased concentrations of serum bile acids, reduced plasma indocyanine green clearance, and normal total bilirubin concentration. Abdominal ultrasonography disclosed a small liver and portosystemic collateral vessels. Radiographic imaging studies confirmed hepatofugal portal circulation and discounted hepatic arteriovenous fistulae. Histopathologic features in liver tissue from each dog were similar and consistent in all sections examined. Common findings included increased cross-sectional views of hepatic arterioles; hepatic lobular atrophy; scanty increase in connective tissue around some large portal triads; and absence of inflammation, disturbed lobular architecture, bile duct proliferation, or intrahepatic cholestasis.

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.

Age dependence of the development of ventricular arrhythmias in a canine model of sudden cardiac death.

OBJECTIVES: The age-dependence of the development of ventricular arrhythmias was studied in German shepherd dogs with inherited ventricular arrhythmias and sudden death. BACKGROUND: A colony of German shepherd dogs has been established that exhibit inherited ventricular arrhythmias and sudden death. The incidence of arrhythmias increases with age. Because ventricular tachycardia is associated with bradycardia, it was hypothesized that the increased incidence of arrhythmias was related to age-dependent slowing of heart rate. METHODS: Arrhythmia counts and RR intervals were measured from serial ambulatory ECG recordings obtained in 71 dogs (1-48 weeks). In addition, 19 dogs were challenged with phenylephrine (10 micrograms/kg i.v.) at 15, 28, and 45 weeks of age, 10 dogs were challenged with epinephrine (1 microgram/kg i.v.) at 3, 5, 7, 9, 11, 18, and 28 weeks of age, and 10 dogs were challenged at 28 weeks with epinephrine (2.5 micrograms/kg i.v.), before and after propranolol (0.5 mg/kg i.v.). RESULTS: The incidence and severity of ventricular arrhythmias increased between 7 and 28 weeks of age and decreased between 28 and 44 weeks of age. The age-dependent increase in the incidence of ventricular tachycardia was associated with age-dependent reductions in sinus rate. Baroreflex-mediated slowing of the heart rate unmasked arrhythmias in young animals that did not spontaneously display arrthythmias and exacerbated existing arrhythmias in older animals. However, the magnitude of baroreflex-induced bradycardia was similar from 7-18 weeks of age, yet the incidence of arrhythmias increased progressively. Moreover, the waning of ventricular arrhythmias in older animals was not associated with more rapid sinus rates. CONCLUSION: The risk for sudden death in dogs with inherited ventricular arrhythmias increases with age in part because of age-dependent slowing of heart rate and in part because of other heart-rate-independent factors. The correspondence between the development of ventricular tachycardia and sinus pauses is consistent with the hypothesis that ventricular arrhythmias are initiated by early afterdepolarization-induced triggered activity.

Inherited ventricular arrhythmias and sudden death in German shepherd dogs.

OBJECTIVES: This report describes a unique group of German shepherd dogs with inherited ventricular arrhythmias and sudden death. Before death, these dogs have no evidence of cardiovascular failure. BACKGROUND: There are few spontaneous animal models of sudden death that permit intensive investigation. METHODS: To determine the temporal evolution of ventricular arrhythmias and to characterize the syndrome of sudden cardiac death in these dogs, 24-h ambulatory electrocardiographic (ECG) monitoring, echocardiograms, electrophysiologic testing and breeding studies were conducted. RESULTS: The 24-h ambulatory ECGs from dogs that died showed frequent ventricular arrhythmias with rapid polymorphic ventricular tachycardia (rates > 480 beats/min). Affected dogs had a window of vulnerability for arrhythmias, with the highest incidence and severity of arrhythmias between 20 to 30 and 40 to 50 weeks of age. Affected dogs that died did not have prolongation of the QT interval over a spectrum of heart rates compared with unaffected dogs. The clinical arrhythmia was not induced in dogs during programmed electrical stimulation. Severely affected dogs monitored > 5 years did not develop any evidence of heart failure or cardiomyopathy, and no histopathologic abnormalities existed. Seventeen dogs died suddenly (age 4 to 30 months) and were either 1) found dead at first observation in the morning (n = 8), 2) observed to die during sleep (n = 4), 3) observed to die while resting after exercise (n = 3), or 4) observed to die during exercise (n = 2). All sudden deaths occurred between the end of September and April, with most (n = 11) during January and February. CONCLUSIONS: The cause of the inherited severe ventricular arrhythmias and sudden death in these young German shepherd dogs is still undetermined. A purely arrhythmic disorder is supported by the lack of cardiac pathology. Moreover, the window of vulnerability to ventricular arrhythmias and the age and circumstances of death invite speculation about the role of the autonomic nervous system.

Cloning and characterization of the gene coding for the human acrosomal protein SP-10.

SP-10 is a human intra-acrosomal protein that is first detected in the developing acrosome of round spermatids and is observed associated with the acrosomal membranes and matrix of mature sperm. Recombinant SP-10 is currently being tested as a contraceptive vaccine immunogen on the basis of its tissue specificity as well as functional assays indicating that anti-SP-10 antisera inhibit sperm-egg interactions. In the present study, structural characterization of the human SP-10 gene was undertaken to investigate the expression of the gene and to identify possible functions of the SP-10 protein within the acrosome. Genomic blots indicate that SP-10 is encoded by a single-copy gene. Mapping and sequencing of the 8-kb SP-10 gene show that the SP-10 mRNA consists of exons of 119, 487, 113, and 390 bp with each exon coding for a distinct structural domain within the SP-10 protein. An in-frame alternatively spliced form of the SP-10 mRNA, identified during SP-10 cDNA characterization, employs the same 3' splice site as the 487-bp exon and a unique 5' splice site within the 487-bp exon. Previous comparisons of human, baboon, and macaque SP-10 cDNA sequences indicated that the baboon and macaque cDNAs contained 60 bp (20 aa) not present in the human SP-10 cDNA. Analysis of the human SP-10 genomic sequence suggests that a 60-bp deletion may have occurred during the evolution of the human SP-10 gene by homologous recombination.(ABSTRACT TRUNCATED AT 250 WORDS)