Overexpression of Na⁺/Ca²⁺exchanger alters contractility and SR Ca²⁺content in adult rat myocytes

Abstract

The functional consequences of overexpression of rat heart Na+/Ca2+ exchanger (NCX1) were investigated in adult rat myocytes in primary culture. When maintained under continued electrical field stimulation conditions, cultured adult rat myocytes retained normal contractile function compared with freshly isolated myocytes for at least 48 h. Infection of myocytes by adenovirus expressing green fluorescent protein (GFP) resulted in >95% infection as ascertained by GFP fluorescence, but contraction amplitude at 6-, 24-, and 48-h postinfection was not affected. When they were examined 48 h after infection, myocytes infected by adenovirus expressing both GFP and NCX1 had similar cell sizes but exhibited significantly altered contraction amplitudes and intracellular Ca2+ concentration ([Ca2+]i) transients, and lower resting and diastolic [Ca2+]i when compared with myocytes infected by the adenovirus expressing GFP alone. The effects of NCX1 overexpression on sarcoplasmic reticulum (SR) Ca2+ content depended on extracellular Ca2+ concentration ([Ca2+]o), with a decrease at low [Ca2+]o and an increase at high [Ca2+]o. The half-times for [Ca2+]i transient decline were similar, suggesting little to no changes in SR Ca2+-ATPase activity. Western blots demonstrated a significant (P < or = 0.02) threefold increase in NCX1 but no changes in SR Ca2+-ATPase and calsequestrin abundance in myocytes 48 h after infection by adenovirus expressing both GFP and NCX1 compared with those infected by adenovirus expressing GFP alone. We conclude that overexpression of NCX1 in adult rat myocytes incubated at high [Ca2+]o resulted in enhanced Ca2+ influx via reverse NCX1 function, as evidenced by greater SR Ca2+ content, larger twitch, and [Ca2+]i transient amplitudes. Forward NCX1 function was also increased, as indicated by lower resting and diastolic [Ca2+]i.

Sarcoplasmic reticulum Ca2+ release channel ryanodine receptor (RyR2) plays a crucial role in aconitine-induced arrhythmias

“…An increased propensity for spontaneous Ca 2+ oscillations and decreased propagating Ca 2+ waves under the condition of intracellular Ca 2+ overload triggered by excess enhancement of the SR Ca 2+ release may underlie a mechanism of aconitine-induced cardiac arrhythmia. It has now become clear that alterations in RyR 2 channel function as a result of genetic defects can cause ventricular arrhythmias [30]. Our findings also provide evidence that acquired dysfunction of RyR 2 could lead to arrhythmia susceptibility.…”

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.…”

“…After 2 h, media were changed to remove nonadherent myocytes. The myocytes were incubated for an additional 3-4 h before initiation of pacing (1 Hz, 5-ms pulses of alternating polarity, field strength of 4 V/cm) using a custom-designed amplifier described previously (22,34). In later experiments, the C-Pacer system (Ionoptix, Milton, MA) was used to pace myocytes in culture (1 Hz, 4-ms pulses of alternating polarity, field strength of 5.3 V/cm).…”

“…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).…”