Characterization of muscle ankyrin repeat proteins in human skeletal muscle

Abstract

Muscle ankyrin repeat proteins (MARPs) are a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. In cardiac muscle, cardiac ankyrin repeat protein (CARP) and diabetes-related ankyrin repeat protein (DARP) reportedly redistribute from binding sites on titin to the nucleus following a prolonged stretch. However, it is unclear whether ankyrin repeat domain protein 2 (Ankrd 2) shows comparable stretch-induced redistribution to the nucleus. We measured the following in rested human skeletal muscle: 1) the absolute amount of MARPs and 2) the distribution of Ankrd 2 and DARP in both single fibers and whole muscle preparations. In absolute amounts, Ankrd 2 is the most abundant MARP in human skeletal muscle, there being ~3.1 µmol/kg, much greater than DARP and CARP (~0.11 and ~0.02 µmol/kg, respectively). All DARP was found to be tightly bound at cytoskeletal (or possibly nuclear) sites. In contrast, ~70% of the total Ankrd 2 is freely diffusible in the cytosol [including virtually all of the phosphorylated (p)Ankrd 2-Ser99 form], ~15% is bound to non-nuclear membranes, and ~15% is bound at cytoskeletal sites, likely at the N2A region of titin. These data are not consistent with the proposal that Ankrd 2, per se, or pAnkrd 2-Ser99 mediates stretch-induced signaling in skeletal muscle, dissociating from titin and translocating to the nucleus, because the majority of these forms of Ankrd 2 are already free in the cytosol. It will be necessary to show that the titin-associated Ankrd 2 is modified by stretch in some as-yet-unidentified way, distinct from the diffusible pool, if it is to act as a stretch-sensitive signaling molecule.

Keywords: Ankrd 1; Ankrd 2; DARP; MARP; protein quantification; single fibers; titin.

Fig. 5.

Distribution of diffusible and nondiffusible Ankrd 2 and DARP pools in human fibers. A: schematic of single fiber experiments where skinned fibers were either washed to separate cytosol (W) and remnant fiber pools (F; referred to as Wash only) or given the same wash step, then Triton treated (T), and fiber remnant collected (F; referred to as Wash and Triton). All washes and Triton treatments were for 10 min. Molecular mass of protein markers (kDa) indicated. B: Western blot showing protein markers for the three subcellular compartments collected from W (all GAPDH is present; cytosol), T (majority of SERCA2a is present; membrane), and F (all myosin is present; nuclear-cytoskeletal). Dotted line is for ease of visualizing separate lanes. C: Western blots of Ankrd 2 and DARP proteins in W, T, and F samples from two individuals (subjects 1 and 2) on the same gel. Fiber type of fiber segments used in Wash Only and Wash & Triton experiments identified using MHC isoform-specific antibodies shown in Fig. 4A. Note that DARP (bottom band, ~36 kDa, Mb425-575a antibody) was probed after first probing for Ankrd 2, which is still apparent as indicated (top band, ~42 kDa). Molecular mass markers are indicated on the left. D: distribution of Ankrd 2 in the various subcellular compartments, as indicated, shown as amount of Ankrd 2 in each compartment relative to total pool (e.g., W/W + F) and expressed as means ± SD, with the number of fibers for Wash Only or Wash and Triton experiments indicated from n = 8 subjects (parentheses). E: DARP protein from n = 6 subjects, shown as for Ankrd 2 in D.

Fig. 1.

Muscle ankyrin repeat protein (MARP) antibody validation. Total protein from purified recombinant MARPs, human (skeletal and heart extract) and rat cardiac muscle samples, separated on 4–15% Criterion Stain-Free Gels, transferred to nitrocellulose, and probed for MARPs, as described below. A: two Ankrd 2 antibodies targeted to different regions of Ankrd 2 detected the DDK-Ankrd 2 pure proteins at ~46 kDa (transcript variant 1, TV1) and ~43 kDa (transcript variant 1, TV2), which is ~1 kDa larger (due to tag size) than single band detected in human skeletal muscle at ~42 kDa (indicated with arrows). B: two DARP antibodies targeted to different regions of DARP detected the GST-DARP fusion protein (~48 kDa) and a triplet band in skeletal muscle at ~36 kDa (brackets), which could not be resolved individually. DARP fusion protein has a predicted size of ~46 kDa, which includes the GST tag (~26 kDa) and 20 kDa DARP protein (60% of DARP’s predicted full-length sequence of ~34 kDa). C: two CARP antibodies targeted to the NH₂-terminal region of CARP detected the His-CARP pure protein at ~41 kDa, which is ~1 kDa larger than the band in rat heart (positive control) at ~40 kDa (arrows indicate CARP), in accord with the His tag size (~1 kDa). The same band at ~40 kDa detected in skeletal muscle with Sc-30181 CARP antibody (right). The Mb423-424 CARP antibody detected a ~40-kDa band in skeletal muscle, which migrated ~1 kDa slower than in human heart extract and rat heart samples. D: 11427-1-AP CARP antibody does not detect the ~40-kDa band in skeletal muscle or rat heart sample (left); compare Sc-30181 CARP antibody (right). Company name and catalog number of antibodies shown at top of blots (see Table 1 for full details). Molecular mass of protein markers (kDa) indicated.

Fig. 2.

Quantification of MARP absolute amounts in human skeletal muscle. A: Western blot (bottom) of Ankrd 2, using 11821-1-AP Ankrd 2 antibody and total protein (top; myosin in the Stain-Free Gel) for whole muscle homogenate samples loaded in different amounts (5–20 µg muscle wet weight) from two subjects (S1 and S2, left), together with 125–1,000 pg purified recombinant DDK-Ankrd 2 TV2 (~43 kDa, right). B: quantification of Ankrd 2 shown for two subjects. Standard curve for pure protein constructed by plotting density of Ankrd 2 band (y-axis) for each amount of Ankrd 2 pure protein (125–1,000 pg; x-axis). Ankrd 2 absolute amount in each individual muscle sample determined from this pure protein standard curve (S2: ~207 pg in ~3.5 and ~553 pg in ~10 µg muscle corresponding to 1.64 and 1.59 µmol/kg wet weight, respectively). C: Western blot (bottom) of DARP, using Ab122320 DARP antibody and total protein (top; Stain-Free Gel) for homogenate samples (5–20 µg muscle wet weight) from two subjects (S1 and S2; left), together with 31–124 pg purified recombinant GST-DARP fusion protein (~48 kDa; right). D: quantification of DARP in two subjects as in B. Sum of all GST-DARP pure protein bands and DARP protein bands (~36 kDa) used in quantifying DARP amounts. DARP absolute amount in each individual muscle sample determined from this standard curve (S2: ~71 pg in ~10 µg and ~122 pg in ~27 µg of muscle corresponding to 0.14 and 0.13 µmol/kg wet weight, respectively). E: Western blot (bottom) of CARP using Mb423-424 CARP antibody and total protein (top; Stain-Free Gel) for homogenate samples (5–20 µg muscle wet weight) from two subjects (S2 and S3; left) and 10–80 pg purified recombinant HIS-CARP pure protein (~41 kDa; right). F: quantification of CARP in two subjects as in B and D. Absolute amount of CARP (~40 kDa band) in each individual muscle sample determined from this standard curve (S2: ~15 pg in ~55 µg and ~6 pg in ~27 µg muscle, indicated with × symbol, corresponding to 0.007 and 0.006 µmol/kg wet weight, respectively). The line of best fit was extrapolated to the y-intercept (dotted line) so that the amount of CARP in samples that fell below the lowest point (10 pg) could be determined. A.U., arbitrary units. Molecular mass of protein markers (kDa) indicated.

Fig. 3.

Relative amounts of Ankrd 2 in different human single fiber types. A: representative blot of myosin heavy chain (MHC) I, MHCII, and Ankrd 2 proteins detected in the same fiber segment, showing qualitatively more Ankrd 2 in type I compared with type II fibers. Myosin in Stain-Free Gel (top), indicative of total protein in each fiber. Type I fibers identified by the presence of MHCI protein and absence of MHCIIa protein and type II fibers identified by opposite MHC protein expression. Western blots first probed with MHCIIa and then MHCI. B: amount of Ankrd 2 in type II (n = 32, n = 8) relative to type I fibers (n = 19, n = 7) and expressed as means ± SD. The number of subjects contributing type I and type II fibers is shown in parentheses. For each fiber segment, the densities of Ankrd 2 and total protein expressed relative to their respective calibration curves and then Ankrd 2 relative amount normalized to total protein present in that lane and re-expressed relative to mean amount in type I fibers on a given gel. 11821-1-AP Ankrd 2 antibody used for all single fiber analyses. *Significant difference between type I and type II fibers (P ≤ 0.05, unpaired t-test, n = 10 individuals). Molecular mass of protein markers (kDa) indicated.

Fig. 4.

Properties of diffusible Ankrd 2 in human single fibers at rest. A: Western blot showing relative amount of Ankrd 2 diffusing out of skinned fiber in 10 min wash (W) and amount remaining in skinned fiber (F) of a type I and II fiber. B: mean percentage (±SD) of Ankrd 2 diffusing out of skinned type I (n = 6, n = 4) and II (n = 8, n = 5) rested muscle fibers in 10 min, showing no significant fiber-type difference (P = 0.26, unpaired t-test). Number of fibers examined shown with the number of subjects shown in parentheses. C: Western blots of calpain-3 (full-length and autolysed products as indicated) and Ankrd 2 in W and F following 15 s, 30 s, 1 min, and 10 min washes; W represents cytosolic compartment, and F represents total of membrane, nuclear, and cytoskeletal compartments in skinned fiber. Myosin present exclusively in the F lane, showing W not contaminated with myofibrils. D: time course of Ankrd 2 diffusing out of resting human fibers. Data expressed as mean amount of Ankrd 2 present in W as a percentage of the total Ankrd 2 protein pool (i.e., W/W + F) in F/W pairs from n = 5 subjects. Each data point is fitted with a 1-phase exponential function, y = M/100(1 − e^−t/τ), where M is maximum % of Ankrd 2 diffusing out (determined as 66%), t is time, and τ is the time constant (0.68 min). Molecular mass of protein markers (kDa) indicated.

Fig. 6.

Subcellular distribution of Ankrd 2 and DARP in human skeletal muscle homogenate. A: Western blots showing Ankrd 2, DARP, and calpain-3 (full-length ~94 kDa, and autolysed products ~60–56 kDa as indicated) in 6 μl of cytosolic (Cyt; majority of GAPDH present), membrane (Mem; majority of SERCA2a present), and nuclear-cytoskeletal (Nuc-Csk) fractions (all topoisomerase IIB and myosin present) and whole homogenate samples (Wh; all proteins present) from 1 rested individual. Note, DARP (bottom band, ~36 kDa, Mb425-575a antibody), probed after first probing for Ankrd 2 (11821-1-AP antibody), and previous Ankrd 2 band (top band, ~42 kDa) are still apparent as indicated. Equal volumes (6 μl) of each fraction and whole muscle samples loaded, so that for each given protein band, the sum of the densities across the three fraction lanes should approximately match that in the Wh sample (i.e., Cyt + Mem + Nuc-Csk = Wh). Molecular mass of protein markers (kDa) indicated. Distribution of Ankrd 2 (B) and DARP (C) in muscle fractions, shown as amount of Ankrd 2/DARP in cytosol, membrane, and nuclear-cytoskeletal fractions relative to total Ankrd 2/DARP protein pool (i.e., Cyt/Cyt + Mem + Nuc-Csk), expressed as means ± SD, from n = 4 individuals. Dotted line is for ease of visualizing separate lanes.

Fig. 7.

Intracellular Ankrd 2 localization in human single fibers. Single muscle fiber segments were treated with 1% Triton X-100 to examine localization Ankrd 2 in the nuclear-cytoskeletal compartment. A: Western blot validation of the 11821-1-AP Ankrd 2 antibody for IHC analyses, showing a single Ankrd 2 band (~42 kDa) across ~200–26-kDa molecular mass range. Pinned fiber segments were fixed, then labeled with DAPI (blue; B), Ankrd 2 (green; C), and desmin (red; D). Digital images were captured at the same pinhole setting of 1 Airy unit using a Zeiss 780 confocal laser-scanning microscope. E: merged image at ×63 (oil) objective. F, inset: merged image at higher magnification (×2 zoom) scan of E showing detail of Ankrd 2 double bands separated by the Z-disk (red/desmin). Scale bars: 10 μM (B–E) and 5 μM (F).

Fig. 8.

Subcellular distribution of phosphorylated Ankrd 2-Serine 99 (pAnkrd 2-Ser99) in human skeletal muscle homogenate. A: Western blots showing pAnkrd 2-Ser99 protein in 6 μl of cytosolic (Cyt), membrane (Mem), and nuclear-cytoskeletal (Nuc-Csk) fractions and whole homogenate (Wh) samples from one rested individual, as in Fig. 6A. Molecular mass of protein markers (kDa) indicated. B: distribution of pAnkrd 2-Ser99 in whole muscle fractions, as described in Fig. 6B, shown as amount of pAnkrd 2-Ser99 in cytosol, membrane, and nuclear-cytoskeletal fractions relative to total pAnkrd 2-Ser99 protein pool, expressed as means ± SD, from n = 4 individuals. Dotted line is for ease of visualizing separate lanes. The same individual samples used in Fig. 6 were used here.

Fig. 9.

Subcellular distribution of CARP in whole homogenate of rat heart muscle. A: Western blot showing CARP protein (~40 kDa; indicated with arrow; Mb423-424 antibody) present in 8 μl of cytosolic (Cyt), membrane (Mem), and nuclear-cytoskeletal (Nuc-Csk) fractions and whole homogenate (Wh) samples from two animals with protein markers, as in Fig. 6A. Molecular mass of protein markers (kDa) indicated. Equal volumes of each fraction and whole homogenate samples loaded so that sum of protein densities in fractions should approximately match that in Wh sample (i.e., Cyt + Mem + Nuc-Csk = Wh). B: CARP protein distribution shown as the amount of CARP in the cytosol, membrane, nuclear-cytoskeletal fractions relative to total CARP protein pool (i.e., Cyt/Cyt + Mem + Nuc-Csk), expressed as means ± SD from n = 3 animals. Dotted lines are for ease of visualizing separate lanes.