Effects of Na⁺/Ca²⁺ exchanger downregulation on contractility and [Ca²⁺]_i transients in adult rat myocytes

Abstract

Postmyocardial infarction (MI) rat myocytes demonstrated depressed Na(+)/Ca(2+) exchange (NCX1) activity, altered contractility, and intracellular Ca(2+) concentration ([Ca(2+)](i)) transients. We investigated whether NCX1 downregulation in normal myocytes resulted in contractility changes observed in MI myocytes. Myocytes infected with adenovirus expressing antisense (AS) oligonucleotides to NCX1 had 30% less NCX1 at 3 days and 66% less NCX1 at 6 days. The half-time of relaxation from caffeine-induced contracture was twice as long in ASNCX1 myocytes. Sarcoplasmic reticulum (SR) Ca(2+)-ATPase abundance, SR Ca(2+) uptake, resting membrane potential, action potential amplitude and duration, L-type Ca(2+) current density and cell size were not affected by ASNCX1 treatment. At extracellular Ca(2+) concentration ([Ca(2+)](o)) of 5 mM, ASNCX1 myocytes had significantly lower contraction and [Ca(2+)](i) transient amplitudes and SR Ca(2+) contents than control myocytes. At 0.6 mM [Ca(2+)](o), contraction and [Ca(2+)](i) transient amplitudes and SR Ca(2+) contents were significantly higher in ASNCX1 myocytes. At 1.8 mM [Ca(2+)](o), contraction and [Ca(2+)](i) transient amplitudes were not different between control and ASNCX1 myocytes. This pattern of contractile and [Ca(2+)](i) transient abnormalities in ASNCX1 myocytes mimics that observed in rat MI myocytes. We conclude that downregulation of NCX1 in adult rat myocytes resulted in decreases in both Ca(2+) influx and efflux during a twitch. We suggest that depressed NCX1 activity may partly account for the contractile abnormalities after MI.

Low-intensity exercise training delays onset of decompensated heart failure in spontaneously hypertensive heart failure rats

“…Analysis of sarco(endo)plasmic reticulum Ca 2ϩ -ATPase (SERCA2), phospholamban (PLB), and Na ϩ /Ca 2ϩ exchanger (NCX1) protein amounts was performed by using previously described methods (34,62). A portion of the LV was removed, and tissue was homogenized in an ice-cold buffer containing (in mmol/l) 50 Tris (pH 8.0), 150 NaCl, 1 Na 3VO4, 1 PMSF, 100 NaF, 1 EDTA, and 1 EGTA and 0.5% Nonidet P-40, 10 g/ml leupeptin, and 10 g/ml aprotinin.…”

Effects of sarcoplasmic reticulum Ca2+-ATPase overexpression in postinfarction rat myocytes

“…Cardiac myocytes were isolated from the septum and LV free wall of rat hearts by successive perfusion with collagenase and hyaluronidase (6). Isolated myocytes were seeded on laminin-coated coverslips and cultured in modified serum-free medium 199 [extracellular Ca 2ϩ concentration ([Ca 2ϩ ]o) ϭ 1.8 mM], as described previously (22,23,25,33,34). After 2 h, media were changed to remove nonadherent myocytes.…”

“…Cell contraction was measured in myocytes incubated in HEPES-buffered (20 mM, pH 7.4) medium 199 (37°C), containing either 0.6, 1.8, or 5.0 mM [Ca 2ϩ ]o, by using a charged-couple device video camera and edge-detection software (Ionoptix) as described previously (22,23,25,31,33,34). For calibration of pixels vs. micrometer, a high-resolution test target (model 22-8635, Ealing Electro-Optics; Natick, MA) was used.…”

“…Myocytes were exposed to 0.67 M of fura 2-AM for 15 min at 37°C. Fura 2 epifluorescence (excitation 360 and 380 Ϯ 10 nm bandpass; emission 510 Ϯ 18 nm bandpass) from paced myocytes (1 Hz, 37°C) was measured with our quantitative fluorescence microscopy system described previously (22,25,28,(32)(33)(34) using an Olympus DApo Ultraviolet ϫ40/1.30 numerical aperature oil objective. Background and cellular autofluorescence measured in sham-GFP and MI-GFP myocytes not loaded with fura 2 accounted for Ͻ10% of the fura 2 signal (34).…”

“…Intracellular fura 2 fluorescence was calibrated daily for each batch of myocytes, as previously described (7). [Ca 2ϩ ]i transient data derived from fura 2 signals were analyzed with custom-written software (Ionoptix) (22,25,28,29,(32)(33)(34).…”