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Enhanced TRPV4‐Mediated Ca 2+ Influx in Ventricular Fibroblasts from Human Hypertrophic Cardiomyopathy
Author(s) -
Ross Gracious R.,
Chahal Bikrum,
Sra Amar,
Ryan Sean D.,
Rizvi Farhan,
Werner Paul,
Tajik A. Jamil,
Jahangir Arshad
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.lb538
Subject(s) - thapsigargin , ionomycin , trpc6 , intracellular , extracellular , medicine , hypertrophic cardiomyopathy , chemistry , endocrinology , transient receptor potential channel , calcium , biochemistry , receptor
Background Excessive cardiac fibrosis (CF) is a major factor contributing to mechanical and electrical dysfunction of the heart in patients with hypertrophic cardiomyopathy (HCM). However, the molecular mechanisms of increased CF are not clearly known. Pro‐fibrotic processes like excessive fibroblast (FB) proliferation, activation, and secretion of extracellular matrix and cytokines are dependent on intracellular Ca 2+ . Therefore, defining mechanisms that increase Ca 2+ mobilization in FB from HCM patients could identify novel therapeutic targets to prevent excessive CF and contractile dysfunction. Hence, we tested the hypothesis that human ventricular FB (hVF) from HCM will have enhanced intracellular Ca 2+ release and ion channel‐mediated Ca 2+ influx. Methods Primary hVF cultures obtained from ventricular or septal tissue resection from HCM patients undergoing surgical intervention (n=7) and non‐diseased hVF from trauma victims (n=4) were used at passage 3 or 4. Cytosolic free Ca 2+ imaging was performed in FB preloaded with fluo‐3 AM (confocal microscopy), and thapsigargin (2.5 μM) was used to release and deplete intracellular Ca 2+ stores in Ca 2+ ‐free KRH solution followed by the addition of CaCl 2 (2 mM) to assess store‐operated Ca 2+ ‐entry (SOCE). Transient receptor potential (TRP) channels including TRPC6‐ and TRPV4‐mediated Ca 2+ influx were elicited by activators: Hyp9 (1 μM) and GSK‐1016790A (1μM), respectively, in the presence of thapsigargin and CaCl 2 (2 mM). Ionomycin (2 μM) was used for maxima normalization. Gene expression of TRPV4 and GAPDH were quantified by real time‐polymerase chain reaction (rt‐PCR). Respective area under the curves (as % of ionomycin response*min) were calculated for Ca 2+ imaging data and analyzed by unpaired Student t‐test. Results Intracellular Ca 2+ release from stores following inhibition of endoplasmic reticulum Ca 2+ ‐ATPase by thapsigargin was not different ( p =0.7) between the control (56±22 %·min) and HCM groups (48±13 %·min). Store‐operated Ca 2+ influx also was not different ( p =0.14) between the HCM group (168±19 %·min) and the control group (238±48 %·min). However, TRPV4‐mediated (GSK‐1016790A) Ca 2+ influx was significantly ( p =0.03) greater in the HCM group (144±24 %·min) than the control group (51±8 %·min), although TRPC6‐mediated (Hyp9) Ca 2+ influx was not different (Control: 149±23 %·min, HCM: 111±19 %·min; p =0.34). Quantitative rt‐PCR data, normalized to GAPDH, showed no difference in the gene expression of TRPV4 between the two groups. Conclusion Unlike intracellular Ca 2+ , SOCE and TRPC6, TRPV4‐mediated Ca 2+ influx is significantly increased in hVF from HCM patients and may play an important role in the development of cardiac fibrosis by facilitating the intracellular Ca 2+ ‐dependent pro‐fibrotic processes. Selective fibroblast TRPV4 inhibition may be a potential therapeutic target in the management of electrical and contractile dysfunction in HCM. Support or Funding Information Aurora Health Care‐Cardiovascular Surgery Research Award (CVSRA 2016) to Gracious R. Ross.