Investigation of the cardiac effects of exercise testing on apparently healthy Boxer dogs.

BACKGROUND: Holter electrocardiographic monitoring is a cornerstone of diagnostic testing for arrhythmogenic cardiomyopathy (ACM) in Boxer dogs, but physical activity during monitoring is not controlled. In humans, exercise testing (ExT) can identify latent tachyarrhythmias associated with cardiomyopathy, and exercise increases serum cardiac troponin-I concentrations ([hs-cTnI]). These effects have not yet been investigated in Boxer dogs. HYPOTHESIS/OBJECTIVES: Subjecting Boxer dogs to brief, moderate-intensity ExT can identify changes in Holter recordings and [hs-cTnI] compared to baseline results. ANIMALS: Thirty overtly healthy, client-owned Boxer dogs. METHODS: Prospective interventional study. Dogs underwent baseline diagnostic testing including 24-hour Holter monitoring and [hs-cTnI], followed by brief ExT (accompanied, brisk stair-climbing and -descending for <5 minutes). RESULTS: Eleven dogs (37%) had >100 premature ventricular complexes (PVCs)/24 hours at baseline (3), ExT (3), or both (5). After ExT, these dogs had more PVCs/24 hours and greater increases in [hs-cTnI] compared to those with ≤100 PVCs/24 hours. Dogs with the striatin mutation had more PVCs/24 hours and a greater increase in [hs-cTnI] after ExT than did dogs without the striatin mutation. CONCLUSIONS AND CLINICAL IMPORTANCE: Exercise testing may improve the binary classification of Boxer dogs with or without ACM by increasing the number of PVCs and [hs-cTnI] in affected dogs to a greater degree than in unaffected dogs. This effect also is associated with presence or absence of the striatin mutation. Exercise should be a controlled variable when screening Boxer dogs for ACM.

Effects of handling and storage on potassium concentration in plasma and serum samples obtained from cats.

OBJECTIVE: To compare potassium concentrations in feline plasma and serum samples analyzed promptly after collection or after 20 to 28 hours of refrigerated storage. ANIMALS: 41 cats. PROCEDURES: A venous blood sample was obtained from each cat. Aliquots were placed in 2 tubes without anticoagulant (blood was allowed to clot to derive serum) and 2 tubes with heparin (to derive plasma). One serum and 1 plasma sample were kept at room temperature and analyzed within 60 minutes after collection (baseline); the other serum and plasma samples were analyzed after 20 to 28 hours of refrigerated storage. At both time points, serum and plasma potassium concentrations were measured. RESULTS: Median baseline serum potassium concentration (4.3 mmol/L) was significantly higher than median baseline plasma potassium concentration (4.1 mmol/L). The median difference between those values was 0.4 mmol/L (95% CI, 0.2 to 0.5 mmol/L). Compared with their respective baseline measurements, the median serum plasma concentration (4.8 mmol/L) and median plasma potassium concentration (4.6 mmol/L) were higher after 20 to 28 hours of refrigeration. CLINICAL RELEVANCE: Results indicated that with regard to potassium concentration in feline blood samples, clotting or refrigerated storage for 20 to 28 hours results in a significant artifactual increase. Detection of an unexpectedly high potassium concentration in a cat may represent pseudohyperkalemia, especially if the blood sample was placed in a no-additive tube, was stored for 20 to 28 hours prior to analysis, or both.

Comparison of major complication and survival rates between surgical ligation and use of a canine ductal occluder device for treatment of dogs with left-to-right shunting patent ductus arteriosus.

OBJECTIVE To compare rates of major intraoperative complications and survival to hospital discharge between surgical ligation (SL) and canine ductal occluder (CDO) implantation for treatment of dogs with left-to-right shunting patent ductus arteriosus (PDA). DESIGN Retrospective cohort study. ANIMALS 120 client-owned dogs with left-to-right shunting PDA (62 treated by SL and 58 treated by CDO implantation). PROCEDURES Data were retrieved from medical records of included dogs regarding signalment, medical history, vertebral heart scale, preoperative echocardiographic findings, complications encountered during surgery, and durations of anesthesia and surgery (SL or CDO implantation). Data were compared between dogs treated by SL and those treated by CDO implantation. RESULTS Dogs treated by CDO implantation were significantly older and heavier than dogs treated by SL and had more pathological cardiac remodeling (as indicated by mitral regurgitation scores, left atrial-to-aortic root diameter ratios, and fractional shortening values). Durations of anesthesia and surgery were also significantly longer for CDO implantation versus SL. The major complication rate for dogs treated by SL (6/62 [10%]) was significantly greater than that for dogs treated by CDO implantation (0/58 [0%]). One dog in the SL group died during surgery. Overall rate of survival to hospital discharge was 99% (119/120). CONCLUSIONS AND CLINICAL RELEVANCE Both SL and CDO implantation were viable methods for PDA attenuation in the evaluated dogs. Although a greater proportion of dogs had major complications during the SL procedure, the 2 procedures had comparable rates of survival to hospital discharge.

Toll-like receptor 4 inhibition within the paraventricular nucleus attenuates blood pressure and inflammatory response in a genetic model of hypertension.

BACKGROUND: Despite the availability of several antihypertensive medications, the morbidity and mortality caused by hypertension is on the rise, suggesting the need for investigation of novel signaling pathways involved in its pathogenesis. Recent evidence suggests the role of toll-like receptor (TLR) 4 in various inflammatory diseases, including hypertension. The role of the brain in the initiation and progression of all forms of hypertension is well established, but the role of brain TLR4 in progression of hypertension has never been explored. Therefore, we investigated the role of TLR4 within the paraventricular nucleus (PVN; an important cardioregulatory center in the brain) in an animal model of human essential hypertension. We hypothesized that a TLR4 blockade within the PVN causes a reduction in mean arterial blood pressure (MAP), inflammatory cytokines and sympathetic drive in hypertensive animals. METHODS: Spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats were administered either a specific TLR4 blocker, viral inhibitory peptide (VIPER), or control peptide in their PVN for 14 days. MAP was recorded continuously by radiotelemetry. PVN and blood were collected for the measurement of pro-inflammatory cytokines (Tumor Necrosis Factor (TNF)-α, interleukin (IL)-1ß), anti-inflammatory cytokine IL-10, inducible nitric oxide synthase (iNOS), TLR4, nuclear factor (NF) κB activity and plasma norepinephrine (NE) and high mobility group box (HMGB)1 expression, respectively. RESULTS: Hypertensive rats exhibited significantly higher levels of TLR4 in the PVN. TLR4 inhibition within the PVN attenuated MAP, improved cardiac hypertrophy, reduced TNF-α, IL-1ß, iNOS levels, and NFκB activity in SHR but not in WKY rats. These results were associated with a reduction in plasma NE and HMGB1 levels and an increase in IL-10 levels in SHR. CONCLUSIONS: This study demonstrates that TLR4 upregulation in PVN plays an important role in hypertensive response. Our results provide mechanistic evidence that hypertensive response in SHR are mediated, at least in part, by TLR4 in the PVN and that inhibition of TLR4 within the PVN attenuates blood pressure and improves inflammation, possibly via reduction in sympathetic activity.

Central blockade of TLR4 improves cardiac function and attenuates myocardial inflammation in angiotensin II-induced hypertension.

AIMS: Understanding the novel signalling pathways involved in the pathogenesis of hypertension is vital for the development of effective therapeutic strategies. Recent evidence suggests a role for Toll-like receptor (TLR) 4 in the development of cardiovascular diseases. Although brain has been implicated in the pathogenesis of hypertension, the role of brain TLR4 in hypertension is largely unexplored. Therefore, we investigated the role of brain TLR4 in angiotensin (Ang) II-induced hypertension and whether central TLR4 blockade has cardioprotective effects in hypertension. METHODS AND RESULTS: Hypertension was induced in male Sprague-Dawley rats by delivering AngII for 14 days. The rats were administered either specific TLR4 blocker, viral inhibitory peptide (VIPER), or control peptide, intracerebroventricularly. Blood pressure, and cardiac hypertrophy and function, was evaluated by radiotelemetry and echocardiography, respectively. Blood and paraventricular nucleus were collected for measurement of plasma norepinephrine (NE), tumour necrosis factor-alpha (TNF-α), interleukin (IL)-1ß, and TLR4 expression, respectively. Heart was analysed for TNF-α, IL-1ß, inducible nitric oxide synthase (iNOS), nuclear factor-kappa B (NFκB), and renin-angiotensin system (RAS) components. Hypertensive rats had dramatically increased TLR4 expression compared with normotensive rats. Central blockade of TLR4 delayed progression of hypertension and improved cardiac hypertrophy and function in hypertensive rats. TLR4 blockade significantly reduced myocardial TNF-α, IL-1ß, iNOS levels, NFκB activity, and altered RAS components in hypertensive rats. These results were associated with reduced circulating NE levels in VIPER-treated hypertensive rats. CONCLUSION: These results provide mechanistic evidence that AngII-induced hypertensive effects are mediated, at least in part, by brain TLR4, and that brain TLR4 blockade attenuates AngII-induced hypertensive response, possibly via down-regulation of myocardial inflammatory molecules and sympathetic activity.

Angiotensin II causes imbalance between pro- and anti-inflammatory cytokines by modulating GSK-3ß in neuronal culture.

BACKGROUND AND PURPOSE: Emerging evidence indicates that the balance between pro-inflammatory cytokines (PICs) and anti-inflammatory cytokines (AICs) within the brain is an important determinant in the outcome of hypertension. However, the mechanism by which this dysregulation occurs is not known. We aimed to investigate whether AngII induces imbalance between PIC and AIC by modulating downstream transcription factors, NFκB and cyclic AMP response element-binding protein (CREB), and whether AngII-induced effects are mediated by glycogen synthase kinase-3ß (GSK-3ß). EXPERIMENTAL APPROACH: CATH.a neurons were exposed to AngII (10 nM-1 µM) over a preset time course. In another set of experiments, GSK-3ß was knock down by using lentivirus containing short hairpin RNA targeting GSK-3ß (L-sh-GSK3ß) before AngII exposure. Cell extracts were subjected to RT-PCR, immunoblot and immunoprecipitation. KEY RESULTS: AngII caused time-dependent increase in PICs (TNF-α and IL-1ß) and reduction in AIC (IL-10). AngII exposure caused reduced phosphorylated CREB(Ser-133) and increased p-NFκB(Ser-276) levels, leading to reduced CREB-CBP and increased NFκB-CBP binding. These results were accompanied by increased activation of GSK-3ß, as indicated by increased p-GSK3(Tyr-216) to p-GSK3(Ser-9) ratio. In a subsequent study, pretreatment with L-sh-GSK3ß attenuated AngII-induced alterations in PICs and IL-10 by augmenting CREB-CBP and attenuating NFκB-CBP binding. CONCLUSIONS AND IMPLICATIONS: Collectively, these findings are the first to provide direct evidence that AngII-induced dysregulation in cytokines is mediated by GSK-3ß-mediated alterations in downstream transcription factors in neuronal cells. Our data also reveal that AngII-induced effects could be alleviated by GSK-3ß inhibition, suggesting GSK-3ß as an important therapeutic target for hypertension that is characterized by increased PICs and NFκB activation.

Diminazene attenuates pulmonary hypertension and improves angiogenic progenitor cell functions in experimental models.

RATIONALE: Studies have demonstrated that angiotensin-converting enzyme 2 (ACE2) plays a protective role against lung diseases, including pulmonary hypertension (PH). Recently, an antitrypanosomal drug, diminazene aceturate (DIZE), was shown to exert an "off-target" effect of enhancing the enzymatic activity of ACE2 in vitro. OBJECTIVES: To evaluate the pharmacological actions of DIZE in experimental models of PH. METHODS: PH was induced in male Sprague Dawley rats by monocrotaline, hypoxia, or bleomycin challenge. Subsets of animals were simultaneously treated with DIZE. In a separate set of experiments, DIZE was administered after 3 weeks of PH induction to determine whether the drug could reverse PH. MEASUREMENTS AND MAIN RESULTS: DIZE treatment significantly prevented the development of PH in all of the animal models studied. The protective effects were associated with an increase in the vasoprotective axis of the lung renin-angiotensin system, decreased inflammatory cytokines, improved pulmonary vasoreactivity, and enhanced cardiac function. These beneficial effects were abolished by C-16, an ACE2 inhibitor. Initiation of DIZE treatment after the induction of PH arrested disease progression. Endothelial dysfunction represents a hallmark of PH pathophysiology, and growing evidence suggests that bone marrow-derived angiogenic progenitor cells contribute to endothelial homeostasis. We observed that angiogenic progenitor cells derived from the bone marrow of monocrotaline-challenged rats were dysfunctional and were repaired by DIZE treatment. Likewise, angiogenic progenitor cells isolated from patients with PH exhibited diminished migratory capacity toward the key chemoattractant stromal-derived factor 1α, which was corrected by in vitro DIZE treatment. CONCLUSIONS: Our results identify a therapeutic potential of DIZE in PH therapy.

Angiotensin II-induced hypertension is modulated by nuclear factor-κBin the paraventricular nucleus.

Hypertension is considered a low-grade inflammatory condition, and understanding the role of transcription factors in guiding this response is pertinent. A prominent transcription factor that governs inflammatory responses and has become a focal point in hypertensive research is nuclear factor-κB (NFκB). Within the hypothalamic paraventricular nucleus (PVN), a known brain cardioregulatory center, NFκB becomes potentially even more important in ultimately coordinating the systemic hypertensive response. To definitively demonstrate the role of NFκB in the neurogenic hypertensive response, we hypothesized that PVN NFκB blockade would attenuate angiotensin II-induced hypertension. Twelve-week-old male Sprague-Dawley rats were implanted with radiotelemetry probes for blood pressure measurement and allowed a 7-day recovery. After baseline blood pressure recordings, rats were administered either continuous NFκB decoy oligodeoxynucleotide infusion or microinjection of a serine mutated adenoviral inhibitory-κB vector, or their respective controls, bilaterally into the PVN to inhibit NFκB at two levels of its activation pathway. Simultaneously, rats were implanted subcutaneously with an angiotensin II or saline-filled 14-day osmotic minipump. After the 2-week treatments, rats were euthanized and brain tissues collected for PVN analysis. Bilaterally inhibited NFκB rats had a decrease in blood pressure, NFκB p65 subunit activity, proinflammatory cytokines, and reactive oxygen species, including the angiotensin II type 1 receptor, angiotensin-converting enzyme, tumor necrosis factor, and superoxide in angiotensin II-treated rats. Moreover, after NFκB blockade, key protective antihypertensive renin-angiotensin system components were upregulated. This demonstrates the important role that transcription factor NFκB plays within the PVN in modulating and perpetuating the hypertensive response via renin-angiotensin system modulation.

Chronic exercise preserves renal structure and hemodynamics in spontaneously hypertensive rats.

AIMS: Exercise training (ExT) is a recommended adjunct to many pharmaceutical antihypertensive therapies. The effects of chronic ExT on the development of hypertension-induced renal injury remain unknown. We examined whether ExT would preserve renal hemodynamics and structure in the spontaneously hypertensive rat (SHR), and whether these effects were mediated by improved redox status and decreased inflammation. Normotensive WKY rats and SHR underwent moderate-intensity ExT for 16 weeks. One group of SHR animals was treated with hydralazine to investigate the pressure-dependent/independent effects of ExT. Acute renal clearance experiments were performed prior to sacrifice. Tissue free radical production rates were measured by electron paramagnetic resonance; gene and protein expression were measured by real time RT-PCR and Western blot or immunofluorescence, respectively. Plasma angiotensin II levels and kidney antioxidants were assessed. Training efficacy was assessed by citrate synthase activity assay in hind-limb muscle. RESULTS: ExT delayed hypertension, prevented oxidative stress and inflammation, preserved antioxidant status, prevented an increase in circulating AngII levels, and preserved renal hemodynamics and structure in SHR. In addition, exercise-induced effects, at least, in part, were found to be pressure-independent. INNOVATION: This study is the first to provide mechanistic evidence for the renoprotective benefits of ExT in a model of hypertension. Our results demonstrate that initiation of ExT in susceptible patients can delay the development of hypertension and provide renoprotection at the functional and ultrastructural level. CONCLUSION: Chronic ExT preserves renal hemodynamics and structure in SHR; these effects are partially mediated by improved redox status and decreased inflammation.

Detraining differentially preserved beneficial effects of exercise on hypertension: effects on blood pressure, cardiac function, brain inflammatory cytokines and oxidative stress.

AIMS: This study sought to investigate the effects of physical detraining on blood pressure (BP) and cardiac morphology and function in hypertension, and on pro- and anti-inflammatory cytokines (PICs and AIC) and oxidative stress within the brain of hypertensive rats. METHODS AND RESULTS: Hypertension was induced in male Sprague-Dawley rats by delivering AngiotensinII for 42 days using implanted osmotic minipumps. Rats were randomized into sedentary, trained, and detrained groups. Trained rats underwent moderate-intensity exercise (ExT) for 42 days, whereas, detrained groups underwent 28 days of exercise followed by 14 days of detraining. BP and cardiac function were evaluated by radio-telemetry and echocardiography, respectively. At the end, the paraventricular nucleus (PVN) was analyzed by Real-time RT-PCR and Western blot. ExT in AngII-infused rats caused delayed progression of hypertension, reduced cardiac hypertrophy, and improved diastolic function. These results were associated with significantly reduced PICs, increased AIC (interleukin (IL)-10), and attenuated oxidative stress in the PVN. Detraining did not abolish the exercise-induced attenuation in MAP in hypertensive rats; however, detraining failed to completely preserve exercise-mediated improvement in cardiac hypertrophy and function. Additionally, detraining did not reverse exercise-induced improvement in PICs in the PVN of hypertensive rats; however, the improvements in IL-10 were abolished. CONCLUSION: These results indicate that although 2 weeks of detraining is not long enough to completely abolish the beneficial effects of regular exercise, continuing cessation of exercise may lead to detrimental effects.

Chronic exercise modulates RAS components and improves balance between pro- and anti-inflammatory cytokines in the brain of SHR.

Recently, exercise has been recommended as a part of lifestyle modification for all hypertensive patients; however, the precise mechanisms of its effects on hypertension are largely unknown. Therefore, this study aimed to investigate the mechanisms within the brain that can influence exercise-induced effects in an animal model of human essential hypertension. Young normotensive WKY rats and SHR were given moderate-intensity exercise for 16 weeks. Blood pressure was measured bi-weekly by tail-cuff method. Animals were then euthanized; paraventricular nucleus (PVN) and rostral ventrolateral medulla (RVLM), important cardiovascular regulatory centers in the brain, were collected and analyzed by real-time RT-PCR, Western blot, EIA, and fluorescent microscopy. Exercise of 16-week duration attenuated systolic, diastolic, and mean arterial pressure in SHR. Sedentary SHR exhibited increased pro-inflammatory cytokines (PICs) and decreased anti-inflammatory IL-10 levels in the PVN and RVLM. Furthermore, SHR(sed) rats exhibited elevated levels of ACE, AT1R, and decreased levels of ACE2 and receptor Mas in the PVN and RVLM. Chronic exercise not only prevented the increase in PICs (TNF-α, IL-1ß), ACE, and AT1R protein expression in the brain of SHR, but also dramatically upregulated IL-10, ACE2, and Mas receptor expression in SHR. In addition, these changes were associated with reduced plasma AngII levels, reduced neuronal activity, reduced NADPH-oxidase subunit gp91(phox) and inducible NO synthase in trained SHRs indicating reduced oxidative stress. These results suggest that chronic exercise not only attenuates PICs and the vasoconstrictor axis of the RAS but also improves the anti-inflammatory defense mechanisms and vasoprotective axis of the RAS in the brain, which, at least in part, explains the blood pressure-lowering effects of exercise in hypertension.

Role of proinflammatory cytokines and redox homeostasis in exercise-induced delayed progression of hypertension in spontaneously hypertensive rats.

Hypertension is a well-known risk factor for various cardiovascular diseases. Recently, exercise has been recommended as a part of lifestyle modification for all hypertensive patients. However, the precise mechanisms of exercise training (ExT)-induced effects on the development of hypertension are poorly understood. Therefore, we hypothesized that chronic ExT would delay the progression of hypertension in young spontaneously hypertensive rats (SHRs). In addition, we explored whether the beneficial effects of chronic ExT were mediated by reduced proinflammatory cytokines and improved redox status. We also investigated the involvement of nuclear factor-kappaB in exercise-induced effects. To test our hypotheses, young normotensive (Wistar-Kyoto) and SHRs were given moderate-intensity ExT for 16 weeks. Blood pressure was determined by the tail-cuff method, and cardiac function was assessed by echocardiography. Myocardial total reactive oxygen species and superoxide production were measured by electron paramagnetic resonance spectroscopy; tumor necrosis factor-alpha, interleukin-1beta, gp91(phox), and inducible NO synthase by real-time PCR; and nuclear factor kappaB activity by electrophoretic mobility shift assay. Chronic ExT in hypertensive rats resulted in significantly reduced blood pressure, reduced concentric hypertrophy, and improved diastolic function. ExT significantly reduced proinflammatory cytokines and inducible NO synthase, attenuated total reactive oxygen species and superoxide production, and increased antioxidants in SHRs. ExT also resulted in increased NO production and decreased nuclear factor kappaB activity in SHRs. In summary, chronic ExT delays the progression of hypertension and improves cardiac function in young SHRs; these ExT-induced beneficial effects are mediated by reduced proinflammatory cytokines and improved redox homeostasis via downregulation of nuclear factor-kappaB.