Abstract 14694: Ventricular Fibroblasts Expression of Orai1 and Ca2+ -influx Through ICRAC is Increased in Human Heart Failure
Introduction: Increased ventricular fibrosis contributes to the progression of heart failure (HF); interventions that limit fibrosis are proposed to improve cardiovascular outcomes. Intracellular Ca2+-dependent processes like excessive fibroblast (FB) proliferation, activation and secretion of extracellular matrix and cytokines are critical for progression of fibrosis. Defining pathways that increase Ca2+ mobilization in FB from HF patients could identify a novel target to prevent fibrosis and HF progression.
Hypothesis: We hypothesized that human ventricular FB (hVF) from a failing heart will have enhanced intracellular Ca2+ release and ICRAC (Ca2+-release activated channel)-mediated Ca2+ influx.
Methods: Primary cultures of hVF obtained from HF patients undergoing left ventricular assist device implantation (n = 9) were compared to non-diseased hVF from trauma victims (n = 3). Cytosolic free Ca2+ imaging was performed in FB preloaded with fluo-3 AM (confocal microscopy) and thapsigargin (2.5 μM) was used to release and deplete intracellular Ca2+ stores in Ca2+-free KRH solution followed by addition of CaCl2 (2 mM) to assess Ca2+ influx (ICRAC). Ionomycin (2 μM) was used for normalization. Protein expression of STIM1, Orai1 and GAPDH was performed by immunoblotting. Data were analyzed by unpaired Student’s t-test.
Results: Ca2+ release from intracellular stores by thapsigargin was not different (p = 0.7) between control (16 ± 1%) and HF groups (15 ± 1%), but store-operated Ca2+-influx (ICRAC) was significantly (p = 0.05) elevated in HF (43 ± 2%) compared to control group (32 ± 5%). The Ca2+-influx was sensitive to YM-58483 (10 μM), an ICRAC inhibitor (9 ± 2%, n = 4, p <0.01). Immunoblots of the components of ICRAC channel, STIM1 and Orai1, showed significant (p = 0.01) increase in the Orai1 expression in HF group (0.09 ± 0.01 AU, n = 8) compared to control (0.04 ± 0.01 AU, n = 3) but no difference (p = 0.7) in STIM1 expression (control [0.19 ± 0.02 AU, n = 3] vs HF [0.2 ± 0.02 AU, n = 8]).
Conclusions: Store-operated Ca2+ influx is significantly increased in hVF from HF patients and may play an important role in the progression of cardiac fibrosis. Selective fibroblast Orai1 inhibition may be a potential therapeutic target to prevent progressive worsening of HF.
Author Disclosures: G. Ross: None. T. Bajwa: None. K. Kraft: None. M. Cosic: None. L. Emelyanova: None. F. Rizvi: None. E. Holmuhamedov: None. P. Werner: None. A. Tajik: None. A. Jahangir: None.
- © 2015 by American Heart Association, Inc.