Quantification of calsequestrin 2 (CSQ2) in sheep cardiac muscle and Ca2+-binding protein changes in CSQ2 knockout mice

Abstract

Calsequestrin 2 (CSQ2) is generally regarded as the primary Ca2+-buffering molecule present inside the sarcoplasmic reticulum (SR) in cardiac cells, but findings from CSQ2 knockout experiments raise major questions about its role and necessity. This study determined the absolute amount of CSQ2 present in cardiac ventricular muscle to gauge its likely influence on SR free Ca2+ concentration ([Ca2+]) and maximal Ca2+ capacity. Ventricular tissue from hearts of freshly killed sheep was examined by SDS-PAGE without any fractionation, and CSQ2 was detected by Western blotting; this method avoided the >90% loss of CSQ2 occurring with usual fractionation procedures. Band intensities were compared against those for purified CSQ2 run on the same blots. Fidelity of quantification was verified by demonstrating that CSQ2 added to homogenates was detected with equal efficacy as purified CSQ2 alone. Ventricular tissue from sheep (n=8) contained 24±2 μmol CSQ2/kg wet wt. Total Ca2+ content of the ventricular tissue, measured by atomic absorption spectroscopy, was 430±20 μmol/kg (with SR Ca2+ likely<250 μmol/kg) and displayed a linear correlation with CSQ2 content, with gradient of ∼10 Ca2+ per CSQ2. The large amount of CSQ2 bestows the SR with a high theoretical maximal Ca2+-binding capacity (∼1 mmol Ca2+/kg ventricular tissue, assuming a maximum of ∼40 Ca2+ per CSQ2) and would keep free [Ca2+] within the SR relatively low, energetically favoring Ca2+ uptake and reducing SR leak. In mice with CSQ2 ablated, histidine-rich Ca2+-binding protein was upregulated ∼35% in ventricular tissue, possibly in compensation.

Fig. 1.

Loss of calsequestrin 2 (CSQ2) during fractionation when preparing crude sarcoplasmic reticulum (SR). A: Western blot of whole homogenate samples of sheep cardiac ventricular tissue (lanes 1 and 6-9, loaded with 120, 60, 40, 20, and 10 μg of tissue, respectively) and fractions (lanes 2-5) collected at each step during production of crude SR vesicles from another 120-μg sample of the same whole homogenate. Each fraction was made up to the same final volume as for the 120-μg whole homogenate in lane 1, enabling direct comparison of band intensity between lane 1 and lanes 2-5. More than 60% of the total CSQ2 was lost in the first pellet (lane 2), and only 5% of the total CSQ2 was recovered in the final crude SR preparation (lane 5). Homogenization was performed in buffer 1 (Na-EGTA solution). Myosin heavy chain (MHC) in the posttransferred gel was detected with Coomassie blue staining and used to verify the relative amount of muscle tissue loaded in each lane. Of the total MHC, 78% was found in the first pellet (lane 2) and <2% was found in the crude SR preparation (lane 5). Molecular mass markers are shown in position on left (see materials and methods). B: calibration curve derived by plotting CSQ2 band intensity (in arbitrary units) vs. the amount of whole homogenate loaded for lanes 1 and 6-9 in A, with the linear regression (y = mx + c) and the r² value for the line of best fit indicated. Such calibration on the same gel is required to accurately relate the band intensity for a given fraction sample to the proportion of CSQ2 present. P, pellet; S, supernatant.

Fig. 2.

CSQ2 in sheep cardiac ventricular muscle. A: Western blot of CSQ2 in homogenate samples of sheep cardiac ventricular tissue (5–60 μg tissue, lanes 1–5) compared with purified sheep cardiac CSQ2 (60–5 ng, lanes 6–10) and with homogenate samples (10 μg of tissue) with added purified CSQ2 (5–40 ng, lanes 11–14). B: MHC and actin were identified in gels before transfer (see materials and methods) to verify relative loading in each lane. Band intensities for CSQ2 (Western blot, C) and actin (pretransfer, D) in homogenate samples (lanes 1–5 in A and B, respectively) are shown with linear regressions (y = mx + c), and r² values for lines of best fit are indicated. E: silver gels showing a single band for recombinant human CSQ2 and CSQ2 purified from sheep heart. Gels also compare the same amounts (5 and 20 ng) of CSQ2 purified from sheep hearts and from cells recombinantly expressing human CSQ2.

Fig. 3.

Validation of directly comparing signals for purified CSQ2 and homogenate samples. Western blot CSQ2 band intensities for various amounts of purified CSQ2 (lanes 6–10 in Fig. 2A, closed triangles) and for 10 μg ventricular homogenate samples with added CSQ2 (lanes 11–14 in Fig. 2A, open triangles). CSQ2 present in the homogenate samples was detected with similar efficacy as purified CSQ2 by itself. Linear regressions for lines of best fit are y = 0.73x − 4.0, r² = 0.995 (pure CSQ2) and y = 0.66x − 15.5, r² = 0.999 (pure CSQ2 + homogenate). Similar results were obtained on 4 separate Western blots.

Fig. 4.

Correlation of total Ca²⁺ content and CSQ2 content in ventricular samples from hearts of 8 sheep. Ca²⁺ content was measured by atomic absorption spectroscopy, and CSQ2 content was measured by Western blotting as in Fig. 2; values/kg wet wt of muscle. Linear regression was performed, and r², P value, and the line of best fit are indicated. The fit values were 10 ± 2.5 (slope) and 198 ± 60 (y-intercept).

Fig. 5.

Histidine-rich Ca²⁺-binding protein (HRC) is upregulated in ventricular muscle from CSQ2 knockout (KO) mice. A: 10-μg samples of ventricular muscle from CSQ2 KO mice (n = 7) and wild-type (WT) mice (n = 4) were separated on 10% Stainfree gel (see materials and methods). Calibration curve was generated using 2.5, 5, 10, and 30 μg muscle (S1-S4) of a mixture made from equal portions of all homogenates. Blot was cut at ∼72 kDa, and separate parts were probed for HRC (top) and CSQ2 (middle). The relative amount of protein loaded in each lane was apparent on the pretransfer Stainfree image (bottom). B: band intensities for HRC and CSQ2 (Western blot) and MHC (Stainfree gel) for homogenate standards (lanes S1, S2, S3, and S4), normalized to respective amount in S3. Range of HRC densities in homogenate samples for CSQ2 HRC KO and WT mice is indicated by vertical lines (WT range: 0.59–1.02, solid line; KO range: 0.90–1.48, broken line). C: mean (+SE) of HRC density in WT and KO homogenate samples; each HRC band was normalized to density of corresponding MHC band, and the value is expressed relative to the average of that for all WT samples on the same gel. Ventricular tissue from KO mice had ∼35% more HRC protein than WT mice (*P < 0.05, 2-tailed unpaired t-test, average of triplicate runs of same samples) (see text).

Fig. 6.

Comparison of CSQ2 amounts in sheep, rat, and mouse ventricular muscle. Homogenized ventricular samples (without any fractionation) separated on 10% (A) or 8% (C) SDS-PAGE. A: pretransfer Stainfree image of gel (top) and corresponding Western blot of CSQ2 (bottom) for indicated amounts (μg wet wt muscle) of ventricular tissue from sheep (lanes 1–3), rat (lanes 4–6), and mouse (lanes 7 and 8). B: relative amount of CSQ2 in ventricular tissue of different species found by Western blotting. CSQ2 band density for a given sample was first normalized to amount of tissue loaded (gauged by density of corresponding actin band on Stainfree image) and then expressed relative to the average for all mouse samples run on the same gel. Mean data were derived from 3 gels. *P < 0.05 different from rat, 1-way ANOVA, Newman-Keuls post hoc analyses. C: gel of separated proteins stained with Stains-all (see materials and methods). Purified CSQ2 was seen as a single dark band at ∼55 kDa (arrowhead in lane 5); a band was apparent at the same molecular mass in sheep and rat ventricular tissue and also in rat soleus muscle tissue. The blue band at ∼120–130 kDa (long arrow), most apparent in rat ventricular tissue (lane 3), is likely to be HRC. CSQ1 is seen as a dark band in soleus muscle at ∼63 kDa (thick arrow in lane 2). Molecular mass markers are on left (lane 1). An equal amount of unfractionated muscle tissue (1.5 mg wet wt) was loaded in lanes 2, 3, and 4, and 3 μg pure CSQ2 prepared from sheep heart were loaded in lane 5.