CHAPTER 3 RESULTS
3.4 NRIP is a novel Z-disc protein and co-localized with ACTN2
After a series of protein-protein interaction assays, we demonstrated that NRIP interacts with α-actinin-2 in vitro and in vivo, defined their interacting domain.
Therefore, it raised a question that NRIP is co-localized with ACTN2 in cardiac tissue.
To answer the question, the hearts tissues excised from WT and NRIP-/- mice were fixed and co-stained with antibodies against NRIP, ACTN2, or myomesin to perform immunofluorescence assay (IF). IF with control antibodies such as α-actinin-2 and Myomesin labeling Z-disc and M-band, respectively indicated that both WT and NRIP deficient myocardium are well organized with regular cross-striations (Figure 8A).
Furthermore, NRIP was found to co-localize with ACTN2 and flanked by M-band labeled by myomesin. The subcellular distribution of NRIP was also confirmed within cardiomyocytes cultured from WT and NRIP-null mice at 12 weeks (Figure 8B).
According to the previous definition, a z-disc protein must conform to these
requirements. First, a suspected Z-disc protein must co-localize with a bona fide Z-disc protein, such as ACTN2, in immunofluorescence assay. Second, the Z-disc localization of the suspected protein should be detected by electron microscopy with immunogold labelling or biochemical evidences of direct protein–protein interaction with known
have demonstrated that NRIP interacts with ACTN2 in vitro and in vivo and these two proteins co-localize in Z-disc. Hence, we characterized that NRIP is a novel Z-disc protein.
3.5 Loss of NRIP reduces I-band width and widen the Z-disc of sarcomere.
Z-disc protein in the traditional concepts is a passive constituent scaffold of
sarcomeric structure. It cross-links with thin filament to stabilize the muscle contraction and transmit the force generated by the myofilaments. Many Z-disc proteins have proved that mutations or defects of these proteins disrupt cardiac cytoarchitectural
organization and lead to cardiomyopathy (Arber et al, 1997; Hassel et al, 2009; Knoll et al, 2011). Because NRIP has proved as a Z-disc protein and loss of NRIP leads to cardiac hypertrophy. Therefore, it raised a question whether lack of NRIP disrupts sarcommeric structure. To clarify the question, hearts were excised from adult mice, and fixed for transmission electron microscopy (TEM) analysis (Figure 9). As shown in Figure 9, deficiency of NRIP leads to narrower I-band and loose Z-disc. Because of the shortening of I-band, the boundary of H-zone is not clear in myocardium of NRIP -/-mice. To analyze the widths of sarcomeric structures (Figure 10 and Figure 11), the I-band width of NRIP-/- mice was reduced 57.6% (0.11 microns short) and the Z-disc width was 15.2% widen (10.17 nm wide), but the A-band width was similar with WT.
The results of TEM and statistical analyses from hearts of embryonic day 17.5 (E17.5) and postnatal day 2 (P2) (Figure 12 and Figure 13) are corresponded with the
consequence from adult mice. The I-band width was reduced in NRIP-/- mice at age E17.5 and P2. To further confirm the results of ultrastructural analysis, the frozen
sections of hearts from WT and NRIP-/- were co-stained with phalloidin (F-actin) and anti-actinin antibody to perform immunofluorescence assay (IF) (Figure 14). As shown in Figure 14, the expression pattern of F-actin was more concentrative in Z-disc than the pattern of WT. This might implies deficiency of NRIP affects the expression pattern of F-actin and leads to narrower I-band. Following with the results above, we concluded that loss of NRIP affects the sarcomeric structure, especially the width of I-band. The narrower width of I-band might affect the contractility of hearts, and finally leads to dilated cardiomyopathy progressively.
3.6 Deficiency of NRIP decreases the amplitude of calcium transient.
According to the previous study, we know that NRIP is a calcium-dependent
calmodulin binding protein (Chang et al, 2011). Calmodulin (CaM) has been shown as a regulator of many ion channels, such as L-type Ca2+ channel, ryanodine receptor and IP3 receptor or as the Ca2+ sensor for signaling pathways in cardiac myocytes (Saucerman & Bers,
contractility of heart muscles (Marks, 2003). Hence, we interested to know whether loss of NRIP affects calcium transient of cardiomyocytes. Adult cardiomyocytes of WT and NRIP-/- mice were isolated at 12 weeks and measured the calcium transient of
contractions (Figure 15). The preliminary result of calcium transient measurement shows that the calcium transient amplitude of NRIP-/- mice is lower comparing with WT (Figure 15A). Further analyzed the ratiomeric calcium concentrations of base, peak and the calcium variation of an action, we found that the peak of calcium transient and the total calcium variation of an action are lower than WT (Figure 15B-D). Besides, the time to relaxation is longer in NRIP-/- than in WT (Figure 15F). According the
investigation of calcium transient, we speculated NRIP might involve in regulating the calcium concentration in cardiomyocytes. Deficiency of NRIP decreases the variation of calcium concentration and impairs cardiac function.
Chapter 4
DISCUSSION
In summary, loss of NRIP defects cardiac function and leads to cardiac hypertrophy and fibrosis progressively. NRIP affects cardiac function might through interacting with a cardiac Z-disc protein, ACTN2. The IQ motif of NRIP associates with the CaM-like domain of ACTN2. Besides associates with ACTN2 biochemically, NRIP also co-localizes with ACTN2 in Z-disc histologically. Being a novel Z-disc protein, NRIP affects organizations of sarcomere, which narrows I-band and widens Z-disc.
Moreover, as a calcium-dependent calmodulin binding protein, deficiency of NRIP decreases the amplitude of calcium transient. According to these results, we speculated that the effects of sarcomeric structure and calcium concentration impair the
contractility of myocardium. To compensate the cardiac function, the hearts of NRIP -/-mice trend to hypertrophic cardiomyopathy progressively.
4.1 Deficiency of NRIP leads to cardiac hypertrophy.
According to our study, we know that deficiency of NRIP causes cardiac hypertrophy.
In most forms of cardiac hypertrophy, the expression of embryonic genes is increased, including the genes for natriuretic peptides and fetal contractile proteins (Hunter &
Chien, 1999). In our study, we have found the pathological morphology of hypertrophic hearts in NRIP-/- mice, including the thickened ventricular walls and the enlarged cardiac myocytes. To further confirm the cardiac hypertrophy, we might investigate the gene expression of hypertrophic markers, such as atrial natriuretic factor (ANF) and
brain natriuretic peptide (BNP) (Kim et al, 2008; Vikstrom et al, 1998). Because we have found fibrosis in NRIP-/- mice at elder stages, maybe the gene expression of some fibrosis markers, such as connective tissue growth factor (Ctgf), procollagen, type I, α2 (Col1a2), and procollagen, type III, α1 (Col3a1), could also be investigated.
4.2 NRIP reduces I-band width through affecting proteins involving in actin filament assembly.
Being a Z-disc protein, the defects of NRIP deletion are not similar with other Z-disc proteins, such as MLP or Nexilin, which deletion of MLP or Nexilin leads to Z-disc disarrangement(Arber et al, 1997; Hassel et al, 2009). Lack of NRIP mainly affects the length of actin filament, no matter in embryonic or in adult stages. Actin filament (F-actin) is organized from the polymerization of actin monomer (Ono, 2010; Taylor et al, 2011). Proteins involved in the process of actin polymerization such as CapZ, tropomodulin-1 (Tmod1) and nebulin have been reported that defects of these proteins
affects the length of actin filaments (Cooper & Schafer, 2000; Gokhin & Fowler, 2011;
Hart & Cooper, 1999; Littlefield et al, 2001; Littlefield & Fowler, 1998; Witt et al, 2006). From the study of Witt C. et al, deletion of nebulin lead to reduction of I-band length and ~15% extension of Z-disc, these phenotype are similar with our
observations. In the study of Witt C et al, they speculated that the loss of nebulin might affect actin filament stability by decelerating actin nucleation, affecting actin
termination. Therefore, lengths of actin filaments are reduced. According to the study of Witt c et al, we speculated that NRIP might affect the expression or localization of proteins involving in actin filament formations. For clarify the speculation, we can investigate the protein expression or expression pattern of these proteins in
cardiomyocytes of NRIP-/- mice foremost.
4.3 NRIP disrupts myofibrilar arrangements through decreasing gene expressions of genes involving in actin filament formation.
Many Z-disc proteins have been reported as a mechanical or biochemical sensors, such MLP, zyxin and myopodin (Frank et al, 2006). MLP is a sensor of mechanical stimuli, cyclic stretch triggers MLP shuttles from Z-disc to nucleus (Boateng et al, 2007), and then induces downstream gene expression, such as MyoD (Frank et al, 2006).
stress of heat shock (Weins et al, 2001). According to our previous studies, we knew that NRIP is a ligand-dependent transcriptional co-activator of androgen receptor in prostate cancer cell lines. (Chen et al, 2008; Tsai et al, 2005). In addition, except seven WD-40 repeats and one IQ motif, NRIP also contains a nuclear localization sequence (NLS) (Tsai et al, 2005). Consistent with the study of Tsai et al, we found that besides localizing in Z-disc, NRIP is also expressed in nuclei of cardiomyocytes at P2 (data not shown) or adult stages. Furthermore, treated neonatal cardiomyocytes with A23187 (a divalent cation ionophore) (Reed & Lardy, 1972) enhanced the nuclear expression of NRIP (Data not shown). As described previously, NRIP interacts with calmodulin in calcium-dependent manner. In myocytes, calmodulin plays as a calcium sensor to regulate calcium-dependent signaling or activities the ion channels (Frank et al, 2006;
Ohrtman et al, 2008). In the study of Wyszynski et al, elevation of the cellular calcium concentration triggers calmodulin to compete the interactions between NMDA receptor and α-actinin, and then to release α-actinin from NMDA receptor(Wyszynski et al, 1997). Therefore, we speculated that in cardiomyocytes calmodulin senses the calcium signals and then associated with NRIP. The interactions of each other release NRIP to translocate into nucleus and to regulate the expressions of genes associated with actin filament formations and stabilizations. To clarify our speculations, the microarray data is essential to help us find out the downstream targets of NRIP. In addition, the
translocation of NRIP and the competition between calmodulin and ACTN2 must further confirm.
4.4 NRIP plays a role in regulating calcium homeostasis.
According to the calcium transient measurement of WT and NRIP-/- mice, we found that loss of NRIP decreases the amplitude of calcium transient. But the
mechanism of NRIP regulating calcium homeostasis is still unknown. Many ion channel inhibitors or drugs are applied to investigate the calcium storage or the ability of
calcium removal of cells. For examples, caffeine is applied to measure the Ca2+ content of sarcoplasmic reticulum (SR) and thapsigargin (TG) or cyclopiazonic acid (CPA) are applied as SR/ER Ca2+ pump inhibitors (Campbell et al, 1991; Smith & Steele, 1998).
Therefore, ion channel inhibitors or drugs could be applied to further investigate which NRIP participate in which state of calcium regulation, calcium influx or removal.
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53
FIGURES
Table 1. Echocardiographic analysis of NRIP mutant (KO) mice.
The cardiac function of wild-type and NRIP-/- mice were analyzed by
echocardiography from ages of 8-week to 39-week. Multiple indexes including LVEDD, IVSd, IVSs, IVSs and LVPWd reveal that deficiency of NRIP progressively lead to cardiac hypertrophy. Besides, the measurements of EF and FS imply the cardiac functions of NRIP-/- mice are defected. B6-129 NRIP+/+ (n=4), NRIP-/- (n=2), LV, left ventricle; FS, fractional shortening of LV; EF, ejection fraction of LV; EDD,
Table.1 Echocardiographic analysis of NRIP+/+and NRIP-/-mice
FS,% 39.89±4.09 32.41±18.34 33.06±1.78 22.96±10.37 33.53±5.09 27.37±6.07
EF,% 71.15±4.73 58.47±26.19 62.42±2.81 45.35±17.36 63.04±6.85 53.3±9.66
LVEDD,mm 3.73±0.28 4.52±0.12 3.67±0.58 4.64±0.1 3.52±0.24 4.26±0.24*
LVEDS,mm 2.24±0.19 3.06±0.92 2.51±0.43 3.57±0.4* 2.34±0.28 3.09±0.43 IVSd,mm 0.97±0.34 1.15±0.27 0.59±0.11 0.85±0.049* 0.57±0.04 0.78±0.09*
IVSs,mm 0.69±0.24 0.69±0.11 0.43±0.04 0.66±0.028** 0.47±0.05 0.66±0.12*
LVPWd,mm 1.04±0.17 1.015±0.31 0.64±0.04 0.78±0.049* 0.63±0.04 0.97±0.02**
LVPWs,mm 0.72±0.12 0.63±0.03 0.49±0.08 0.645±0.049 0.51±0.06 0.745±0.049*
HR 381.5±77.25 284.5±0.26 417±47.14 365±65.05 511±37.64 399±8.48*
LVM,mg 146.7±44.88 74.04±0.19 73.25±20.91 155.36±6.32** 65.79±13.78 147.65±28.11**
30-40 wk
-/-dimension. *P ＜0.05; **P ＜0.01 represent significant differences between the measurements in NRIP-/- mice compared with NRIP+/+ at the same age.
Figure 1. Progressively hypertrophic response of the heart in NRIP-deficient mice.
Hearts from NRIP+/+ and NRIP-/- mice at age 12 (A) and 39 (B) weeks were excised, respectively following perfused with 0.5% lidocaine and fixed with 4%
paraformaldehyde (PFA) at 4℃ overnight. The hearts of NRIP-/- mice are enlarged and the enlargements are more significant with aging. Scale bar, 5 mm.
Figure 2. NRIP deficiency leads to pathological hypertrophy.
Hearts from WT and NRIP-null mice were excised and stained with hematoxylin and eosin (H&E) at age 12 weeks (A). Compared with NRIP+/+, the left ventricle walls of NRIP-/- are thicken, including left ventricle posterior wall (LVPW) and interventricular septum (IVS), and the left ventricle dimension is decreased. (B) The myocytes
diameters of NRIP-/- mice are widened. (C) The cell width of NRIP-/- cardiomyocyte is enlarged. The short axis was measured as the width of cell by using Image J software.
NRIP+/+ ( n=1, 178 cells), NRIP-/- ( n=1, 135 cells). (D) The number of cells per field is reduced in NRIP-/-. NRIP+/+ ( n=1, 4 fields), NRIP-/- ( n=1, 9 fields). Results are mean
±SD. **P ＜0.01 versus NRIP+/+ .Scale bars, 2.0 mm (A), 20 μm (B).
Figure 3. Lack of NRIP leads to dilated cardiomyopathy progressively.
Hearts from WT and NRIP-/- mice were excised and stained with hematoxylin and eosin (H&E) at age 39 weeks (A). The LVPW and IVS are thickened and the right ventricle wall is thinned in NRIP-/- mice. The chambers of both left and right ventricles are dilated in hearts of NRIP-/- mice. (B) The cell width of NRIP-/- cardiomyocyte is increased. The quantitative results are shown in panel (C). NRIP+/+ (n=2, 182 cells), NRIP-/- (n=2, 200 cells) (D) The number of cells per field is reduced in NRIP-/-. NRIP+/+ ( n=1, 5 fields), NRIP-/- ( n=1, 10 fields) Results are mean ±SD. **P ＜0.01 versus NRIP+/+ .Scale bars, 2.0 mm (A), 20 μm (B).
Figure 4. Cardiac fibrosis in LV was observed in NRIP-/- mice at elder stage.
Paraffin-embedded sections from the hearts of WT and NRIP-/- mice were analyzed with Massion’s trichrome staining at 12 weeks (A) and 39 weeks (B), respectively. The collagen deposits are shown in blue color. Massive collagen deposits in the left
ventricle of NRIP-/- mice at elder stage (B), which are indicated by arroews. Scale bars,
ventricle of NRIP-/- mice at elder stage (B), which are indicated by arroews. Scale bars,