Michelle Batthish

Selective Inhibition of Inward Rectifier K+ Channels (Kir2.1 or Kir2.2) Abolishes Protection by Ischemic Preconditioning in Rabbit Ventricular Cardiomyocytes

Volume regulatory Cl− channels are key regulators of ischemic preconditioning (IPC). Because Cl− efflux must be balanced by an efflux of cations to maintain cell membrane electroneutrality during volume regulation, we hypothesize that IK1 channels may play a role in IPC. We subjected cultured cardiomyocytes to 60-minute simulated ischemia (SI) followed by 60-minute of simulated reperfusion (SR) and assessed percent cell death using trypan blue staining. Ischemic preconditioning (10-minute SI/10-minute SR) significantly (P<0.0001) reduced the percent cell death in nontransfected cardiomyocytes [IPCCM 18.0±2.1% versus control (CCM) 48.3±1.0%]. IPC protection was not altered by overexpression of the reporter gene (enhanced green fluorescent protein, EGFP). However, overexpression of dominant-negative Kir2.1 or Kir2.2 genes using adenoviruses (AdEGFPKir2.1DN or AdEGFPKir2.2DN) encoding the reporter gene EGFP prevented IPC protection [both IPCCM+AdEGFPKir2.1DN 45.8±2.3% (mean±SEM) and IPCCM+AdEGFPKir2.2DN 47.9±1.4% versus IPCCM; P<0.0001] in cultured cardiomyocytes (n=8 hearts). Transfection of cardiomyocytes with AdEGFPKir2.1DN or AdEGFPKir2.2DN did not affect cell death in control (nonpreconditioned) cardiomyocytes (both CCM+ AdEGFPKir2.1DN 45.8±0.7% and CCM+AdEGFPKir2.2DN 46.2±1.3% versus CCM; not statistically significant). Similar effects were observed in both cultured (n=5 hearts) and freshly isolated (n=4 hearts) ventricular cardiomyocytes after IK1 blockade with 20 &mgr;mol/L BaCl2 plus 1 &mgr;mol/L nifedipine (to prevent Ba2+ uptake). Nifedipine alone neither protected against ischemic injury nor blocked IPC protection. Our findings establish that IK1 channels play an important role in IPC protection.

Enhanced cell volume regulation: a key protective mechanism of ischemic preconditioning in rabbit ventricular myocytes

Accumulation of osmotically active metabolites, which create an osmotic gradient estimated at ~60 mOsM, and cell swelling are prominent features of ischemic myocardial cell death. This study tests the hypothesis that reduction of ischemic swelling by enhanced cell volume regulation is a key mechanism in the delay of ischemic myocardial cell death by ischemic preconditioning (IPC). Experimental protocols address whether: (i) IPC triggers a cell volume regulation mechanism that reduces cardiomyocyte swelling during subsequent index ischemia; (ii) this reduction in ischemic cell swelling is sufficient in magnitude to account for the IPC protection; (iii) the molecular mechanism that mediates IPC also mediates cell volume regulation. Two experimental models with rabbit ventricular myocytes were studied: freshly isolated pelleted myocytes and 48-h cultured myocytes. Myocytes were preconditioned either by distinct short simulated ischemia (SI)/simulated reperfusion protocols (IPC), or by subjecting myocytes to a pharmacological preconditioning (PPC) protocol (1 microM calyculin A, or 1 microM N(6)-2-(4-aminophenyl)ethyladenosine (APNEA), prior to subjecting them to either different durations of long SI or 30 min hypo-osmotic stress. Cell death (percent blue square myocytes) was monitored by trypan blue staining. Cell swelling was determined by either the bromododecane cell flotation assay (qualitative) or video/confocal microscopy (quantitative). Simulated ischemia induced myocyte swelling in both the models. In pelleted myocytes, IPC or PPC with either calyculin A or APNEA produced a marked reduction of ischemic cell swelling as determined by the cell floatation assay. In cultured myocytes, IPC substantially reduced ischemic cell swelling (P < 0.001). This IPC effect on ischemic cell swelling was related to an IPC and PPC (with APNEA) mediated triggering of cell volume regulatory decrease (RVD). IPC and APNEA also significantly (P < 0.001) reduced hypo-osmotic cell swelling. This IPC and APNEA effect was blocked by either adenosine receptor, PKC or Cl(-) channel inhibition. The osmolar equivalent for IPC protection approximated 50-60 mOsM, an osmotic gradient similar to the estimated ischemic osmotic load for preconditioned and non-preconditioned myocytes. The results suggest that cell volume regulation is a key mechanism that accounts for most of the IPC protection in cardiomyocytes.

Pharmacological preconditioning in rabbit myocardium is blocked by chloride channel inhibition

OBJECTIVES We have recently proposed that chloride (Cl(-)) channels contribute to ischemic preconditioning (IPC) in the myocardium. To further evaluate this hypothesis, we investigated the role of Cl(-) channels in pharmacological preconditioning. METHODS Isolated rabbit cardiomyocytes and isolated buffer-perfused rabbit hearts were initially preconditioned with a 10 min exposure to either an adenosine receptor agonist [2-chloro-N(6)-cyclopentyladenosine (CCPA, 200 nM) and/or N(6)-2-(4-aminophenyl)ethyladenosine (APNEA, 1 microM)] or the PKC activator phorbol 12-myristate 13-acetate (PMA, 1 microM) followed by a 10 or 20 min washout or not preconditioned (control). Cardiomyocytes or whole hearts were then subjected to prolonged ischemic period (45 min simulated ischemia or 40 min of regional myocardial ischemia, respectively) followed by 60 min reperfusion (resuspension in oxygenated medium or release of the transient coronary occlusion, respectively). RESULTS Indanyloxyacetic acid 94, a selective Cl(-) channel inhibitor that produced substantial inhibition of the regulatory volume decrease (RVD) when given at 10 microM concentration in cultured cardiomyocytes, was administered before ischemia to block RVD through Cl(-) channel inhibition. CCPA, APNEA and PMA significantly (P<0.01) reduced the % of dead cardiomyocytes (by trypan blue staining) after 45 min SI/60 min SR, as compared to controls, while IAA-94 abolished this protection but did not affect PKCepsilon translocation by IPC. We confirmed that IAA-94 blocked IPC-, APNEA- and PMA-induced protection against infarction in the isolated heart model. CONCLUSIONS These findings support our contention that Cl(-) channels are downstream effectors of IPC.

Development of System-level Performance Measures for Evaluation of Models of Care for Inflammatory Arthritis in Canada

Objective. To develop system-level performance measures for evaluating the care of patients with inflammatory arthritis (IA), including rheumatoid arthritis (RA), psoriatic arthritis, ankylosing spondylitis, and juvenile idiopathic arthritis. Methods. This study involved several methodological phases. Over multiple rounds, various participants were asked to help define a set of candidate measurement themes. A systematic search was conducted of existing guidelines and measures. A set of 6 performance measures was defined and presented to 50 people, including patients with IA, rheumatologists, allied health professionals, and researchers using a 3-round, online, modified Delphi process. Participants rated the validity, feasibility, relevance, and likelihood of use of the measures. Measures with median ratings ≥ 7 for validity and relevance were included in the final set. Results. Six performance measures were developed evaluating the following aspects of care, with each measure being applied separately for each type of IA except where specified: waiting times for rheumatology consultation for patients with new onset IA, percentage of patients with IA seen by a rheumatologist, percentage of patients with IA seen in yearly followup by a rheumatologist, percentage of patients with RA treated with a disease-modifying antirheumatic drug (DMARD), time to DMARD therapy in RA, and number of rheumatologists per capita. Conclusion. The first set of system-level performance measures for IA care in Canada has been developed with broad input. The measures focus on timely access to care and initiation of appropriate treatment for patients with IA, and are likely to be of interest to other arthritis care systems internationally.

Refractory Primary Central Nervous System Vasculitis of Childhood: Successful Treatment with Infliximab

To the Editor: Childhood primary angiitis of the central nervous system (cPACNS) is an increasingly recognized inflammatory brain disease causing devastating brain injury in previously healthy children1. Early recognition and initiation of treatment may reverse the severe deficits caused by inflammation and lead to complete neurological recovery. cPACNS has a broad clinical spectrum including acute ischemic stroke, intractable seizures, and severe cognitive decline2. cPACNS is classified into angiography-positive vasculitis and angiography-negative small-vessel vasculitis (SV-cPACNS)2. We reported the efficacy and safety of a treatment protocol for SV-cPACNS3. This immunosuppressive regimen led to complete neurological recovery in the majority of children3. There is limited knowledge of the approach to children who fail standard therapy. Tumor necrosis factor (TNF) inhibition has been considered in the treatment of refractory systemic vasculitis4. In addition, histological studies of SV-cPACNS brain biopsies demonstrated primary lymphocytic vessel wall infiltrates5. We describe 2 children with refractory SV-cPACNS who failed standard treatment with cyclophosphamide and high-dose corticosteroids. Treatment with anti-TNF therapy with infliximab (IFX) controlled disease activity and resolved neurologic symptoms. In Case 1, a 7-year-old previously healthy girl presented with fever and headaches. She was admitted to hospital and developed a generalized tonic-clonic seizure. … Address correspondence to Dr. S. Benseler, Division of Rheumatology, Department of Pediatrics, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. E-mail: susanne.benseler{at}sickkids.ca

Predictors of hip disease in the systemic arthritis subtype of juvenile idiopathic arthritis.

Objective. Hip involvement occurs in 20%–40% of all cases of juvenile idiopathic arthritis (JIA). Patients with systemic JIA (sJIA) are affected most frequently. The aim of our study was to investigate the predictors of clinical hip disease and radiographic hip damage in sJIA. Methods. The medical records (1997–2007) of all children (n = 98) with sJIA were reviewed. Potential clinical and laboratory predictors were examined at presentation and at 3 and 6 months. To account for censored observations, we used survival analysis. Results. During the study period, 59 children met our inclusion criteria. The mean age at diagnosis was 7.8 years. Thirty patients (51%) developed clinical hip disease, with 12 (20%) developing radiographic evidence of hip damage. The median time to develop clinical hip disease was 24 months. Using Kaplan-Meier estimates, 25% of patients develop radiographically evident hip damage within 43 months. At presentation, patients in whom clinical hip disease later developed had polyarthritis (hazard ratio 2.51, p = 0.01), elevated IgG (HR 1.12, p = 0.01) and IgM (HR 2.71, p = 0.02), and higher CHAQ scores (HR 1.65, p = 0.02). At 3 months after disease onset, patients in whom radiographic hip damage later developed had fever (HR 4.78, p = 0.02), polyarthritis (HR 4.63, p = 0.02), and higher CHAQ scores (HR 3.20, p = 0.005). At 6 months, polyarthritis was the strongest predictor of both clinical hip disease and radiographic hip damage. Conclusion. Half of patients with sJIA develop clinical hip disease a median time of 24 months from diagnosis. Early identification of predictors of hip disease and damage in patients with sJIA may suggest earlier, more aggressive interventions to prevent joint destruction.

Intra-rater reliability of the bath ankylosing spondylitis disease activity index (BASDAI) and the bath ankylosing spondylitis functional index (BASFI) in children with spondyloarthritis

Purpose Juvenile spondyloarthritis (JSpA), referred to as enthesitisrelated arthritis (ERA) sub-type under the International League of Associations for Rheumatology (ILAR) classification of juvenile idiopathic arthritis (JIA), is characterized by inflammation in the joints and entheses. Axial involvement is uncommon at presentation but may develop in the second decade of life. Several instruments assessing disease activity in ankylosing spondylitis have been validated including the BASDAI and BASFI. At this time, there are no disease activity scores for JSpA or ERA. The objective of this study is to measure the intra-rater reliability of the BASDAI and the BASFI in JSpA/ERA. Methods

Raynaud’s Phenomenon as a Presenting Feature of Hypothyroidism in an 11-year-old Girl

patients with RP, 65% of patients with RP had a secondary condition, with over 50% having a connective tissue disease; only 2 patients (0.3%) were found to have associated hypothyroidism 1 . A causal relationship between thyroid deficiency and RP has been reported in a number of adult patients 2-6 . We describe an 11-year-old patient with severe undiagnosed primary hypothyroidism presenting with RP. An 11-year-old girl was referred with a history of increasingly cold hands and feet for about 2 years. She described no pain but significant color changes affecting all fingers. Her parents also reported that she had had low energy levels, and had become notably inactive and lethargic over the pre vious 12 months. She had no history of fever or rash. She had been previ ously investigated for nasal speech and had attended speech and language therapy. Her parents had noted that her growth was delayed in comparison to her peers. On examination, her height was less than third centile and weight was on the tenth centile for her age. There was evidence of mild dysmorphic features including hypertelorism and a thin, long, flat philtrim with a thin upper lip. She had a flattened nasal bridge with a prominent nose. She had evidence of hirsutism. Her peripheries were cool on examination. She had dry scaling and a purplish discoloration of the tips of her toes. She had prominent dilated loops of her nailfold capillaries on capillaroscopy, although the vessels were not found to be tortuous. Mucosal and conjunc tival examinations were normal. Assessment of her musculoskeletal system showed stiff end range of movement of most joints, in particular both sub talars and elbows. She had early breast bud development but no evidence of pubarche. She had a prominent distended abdomen with a palpable liver approximately 2 cm below the costal margin. The rest of her physical