Exogenous Ca2+-ATPase isoform effects on Ca2+ transients of embryonic chicken and neonatal rat cardiac myocytes

Abstract

1. Sarco-endoplasmic reticulum Ca2+-ATPase from fast skeletal (SERCA1) or cardiac muscle (SERCA2a) was expressed in embryonic chicken and neonatal rat cardiac myocytes by adenovirus vectors, with c-myc tags on both constructs to compare expression and distinguish exogenous from endogenous SERCA2a in myocytes. 2. Expression of the two isoforms was similar (approximately 3-fold higher than endogenous SERCA). However, SERCA1 activity was 2-fold greater than SERCA2a activity, due to intrinsic differences in turnover rates. Activation of both exogenous SERCA isoforms by Ca2+ was displaced to slightly lower [Ca2+], suggesting that the overexpressed isoforms were independent of phospholamban. In fact, phospholamban and calsequestrin expression were unchanged. 3. Decay time constants of cytosolic Ca2+ transients from cells overexpressing SERCA1 were reduced by 30-40 % and half-widths by 10-15 % compared to controls. SERCA2a overexpression produced much less acceleration of transients in chick than in rat, and less acceleration than SERCA1 overexpression in either species. There was no significant change in resting [Ca2+], peak amplitudes, or in the amount of Ca2+ releasable by caffeine from overexpression of either SERCA isoform. However, the amplitudes of the transients increased with SERCA1 overexpression when pacing frequency limited refilling of the sarcoplasmic reticulum. 4. It is concluded that total SERCA transport velocity has a primary effect on the decay phase of transients. Transport velocity is affected by SERCA isoform turnover rate, temperature, and/or SERCA copy number.

Figure 1

Immunostaining of SERCA in embryonic chicken cardiac myocytes A and B, cultured embryonic chicken cardiac myocytes stained with monoclonal antibodies specific for endogenous SERCA2. A, phase-contrast image. B, fluorescence image of the same field. C and D, fluorescence images of myocytes stained with antibodies specific for exogenous SERCA1. C, uninfected myocytes. D, cells infected with Ad.Sr1. E and F, higher-magnification images showing targeting of exogenous SERCA to the SR. E, uninfected myocytes stained with a monoclonal antibody specific for endogenous SERCA2 and Texas red-conjugated secondary antibody. F, cells infected with Ad.Sr1 and stained with a monoclonal antibody specific for exogenous SERCA1 and fluorescein-conjugated secondary antibody. Scale bar in F corresponds to 60 μm in A-D and to 20 μm in E and F.

Figure 2

Protein expression in embryonic chicken cardiac myocytes Embryonic chicken cardiac myocytes were infected with Ad.Sr1 (expressing SERCA1) or Ad.Sr2a (expressing SERCA2a), and cell lysates were probed with various monoclonal antibodies. A, right, antibody to the c-myc tag found only in exogenous SERCA1 and SERCA2a; middle, antibody to SERCA1; left, antibody to endogenous and exogenous SERCA2a. Lane headings: C, control; Sr1, Ad.Sr1 infected; Sr2a, Ad.Sr2a infected. B, antibody to phospholamban. C, antibody to calsequestrin. For B and C, each lane was run in duplicate.

Figure 3

SERCA activity in embryonic chicken cardiac myocytes Embryonic chicken cardiac myocytes were infected with Ad.Sr1 or Ad.Sr2a. A, rates of ⁴⁵Ca²⁺ uptake by SERCA (nmol Ca²⁺ per mg protein) in cell lysates. Inset, Western blot shows similar expression levels of SERCA1 and SERCA2a detected by the same anti-c-myc antibody. B, Ca²⁺-dependent ATPase activity (%) measured at varying pCa. •, endogenous activity in control cells; ▪, activity in cells overexpressing SERCA1; □, activity in cells overexpressing SERCA2a.

Figure 4

Cytosolic Ca²⁺ transients in embryonic chicken and neonatal rat cardiac myocytes Embryonic chicken and neonatal rat cardiac myocytes were infected with Ad.Sr1 or Ad.Sr2. Cytosolic Ca²⁺ transients were measured in single cells using fluo-4 (A-D) or fura-2 (E and F). Each trace represents the average of transients from 15–30 cells over 3–5 preparations. Note the different time scales of the displayed transients from embyronic chicken (left) and neonatal rat (right). Ca²⁺ concentrations in E and F were calculated from the fura-2 fluorescence at 380 and 358 nm excitation (see Methods).

Figure 5

SR Ca²⁺ content in embryonic chicken cardiac myocytes Embryonic chicken cardiac myocytes loaded with fura-2 were exposed to a modified Ringer solution containing 10 mm caffeine and 1 μm thapsigargin while fluorescence was sampled at 358 and 380 nm excitation every 500 ms. A, control cell. B, cell overexpressing SERCA1.

Figure 6

Time course of refilling of Ca²⁺ stores in embryonic chicken cardiac myocytes A, chicken cardiac myocytes loaded with fluo-4 were stimulated with a two-pulse protocol after pacing at 1·0 Hz. The last paced transient and the extrasystolic transient were recorded while varying the extrasystolic interval. Each record was normalized to its conditioning transient and superimposed as a series. Upper series, control myocyte. Lower series, myocyte overexpressing SERCA1. Time intervals are indicated above the traces (in ms). B, relative amplitudes of the Ca²⁺ transients as a function of the extrasystolic interval. Amplitude was calculated as the difference between the peak of the transient and the baseline fluorescence just prior to the stimulus pulse. Points are means ± s.e.m. •, control cells (n = 20); ▪, cells overexpressing SERCA1 (n = 16).

Figure 7

Effects of temperature on Ca²⁺ uptake and Ca²⁺ transients A, cytosolic Ca²⁺ transients recorded at 22 and 30 °C in control rat cardiac myocytes. B, rates of ⁴⁵Ca²⁺ uptake by endogenous SERCA2a (nmol Ca²⁺ per mg protein) in rat cardiac myocyte lysates. ▪, uptake at 22 °C; •, uptake at 30 °C.