Loss of enigma homolog protein results in dilated cardiomyopathy

Abstract

Rationale: The Z-line, alternatively termed the Z-band or Z-disc, is a highly ordered structure at the border between 2 sarcomeres. Enigma subfamily proteins (Enigma, Enigma homolog protein, and Cypher) of the PDZ-LIM domain protein family are Z-line proteins. Among the Enigma subfamily, Cypher has been demonstrated to play a pivotal role in the structure and function of striated muscle, whereas the role of Enigma homolog protein (ENH) in muscle remains largely unknown.

Objective: We studied the role of Enigma homolog protein in the heart using global and cardiac-specific ENH knockout mouse models.

Methods and results: We identified new exons and splice isoforms for ENH in the mouse heart. Impaired cardiac contraction and dilated cardiomyopathy were observed in ENH null mice. Mice with cardiac specific ENH deletion developed a similar dilated cardiomyopathy. Like Cypher, ENH interacted with Calsarcin-1, another Z-line protein. Moreover, biochemical studies showed that ENH, Cypher short isoform and Calsarcin-1 are within the same protein complex at the Z-line. Cypher short isoform and Calsarcin-1 proteins are specifically downregulated in ENH null hearts.

Conclusions: We have identified an ENH-CypherS-Calsarcin protein complex at the Z-line. Ablation of ENH leads to destabilization of this protein complex and dilated cardiomyopathy.

Fig.1

ENH genomic structure and splice isoforms. Colored boxes are used to represent the 20 exons which encode the murine ENH gene and blank boxes are non-coding regions. The translational start site is in the exon 2. Two stop codons for ENH gene are in exon 11 and exon 20 respectively. The N-terminal exons in red encode PDZ domain. The C-terminal exons in yellow encode three LIM domains. ENH1b is encoded using short exon 5′ instead of exon 5 in ENH1/1a, which was reported as ENH1 in NCBI database (NM_019808). Exons 12-14 are newly identified exons. ENH1c includes the short exon 5′ and exon 14. ENH1d includes exon 5′, exon 13 and exon 14. ENH1e includes exon 5′ and exons 12-14. ENH4 includes the small 17-bp exon 6. ENH3b was renamed from ENH3 (NM_022554) and ENH3a does not include the small 15-bp exon 7. (*) ENH isoforms (ENH2 and ENH4) are only expressed in skeletal muscle.

Fig. 2

Dilated cardiomyopathy assessed by echocardiography in ENH^−/− mice. ENH^−/− mice (n=8, blank) and WT mice (n=8, grey) were measured at 1-, 3-, 5-, 9- and 12-month-of-age. A, Reduced fractional shortening (FS) in ENH^−/− hearts (* P < 0.05). B, Enlarged left ventricles as shown by left ventricular internal dimension (LVIDd) at end diastole in ENH^−/− hearts (* P < 0.05). C, The ratios of heart weight to body weight (HW/BW) (mg/g) for ENH^−/− mice (male, n=5) and WT mice (male, n=5) at 3-months. (* P < 0.05)

Fig. 3

Characterization of the dilation and widened Z-lines in ENH^−/− mouse hearts. A, mRNA levels for cardiac fetal genes (ANF, α-MHC, β-MHC and skeletal α-actin) was shown by dot-blot analysis of 1-month-old ENH^−/− male mice and WT controls. GAPDH was used as a RNA loading control. Quantification of RNA levels normalized to WT levels of the dot densities was shown in the bottom panel (* P < 0.05). B, Representative morphology of an ENH^−/− heart and WT heart at 3-months following H&E stain. High-power field was shown in the right panel (20X). C, The ultrastructure of cardiac muscle from the left ventricles of 3-month-old WT and ENH^−/− mice was shown by electron microscopy. The quantitative width (distances between two arrows) of Z-lines is shown in the bottom panel (* P < 0.05).

Fig. 4

Impaired contractility in ENH^−/− hearts. 5-month-old male ENH^−/− (n=8) and WT mice (n=8) were subjected to hemodymic measurements. A and B, ENH^−/− and WT mice had the same heart rates and maximum left ventricular pressure. C, ENH^−/− mice showed lower dP/dt maximum (dP/dt max) when stimulated with 4-8U (U=μg/kg/min) of dobutamine (* p < 0.05). D, ENH^−/− mice had lower dP/dt minimum (dP/dt min) under basal conditions and with various doses of dobutamine (* p < 0.05). E, Tau, a load independent measure of relaxation, was increased in EHN^−/− mice under basal conditions and following various doses of dobutamine ranging from 2-8U (* p < 0.05).

Fig. 5

Increased Systolic Dysfuntion in ENH-null Mice Following Biomechanical Stress. 2-month-old WT or ENH^−/− mice underwent either sham operation (n=3) or TAC surgery (n=8). A, Systolic heart function, as shown by percent fractional shortening ((FS (%)), was assessed by echocardiography. B, Left ventricular posterior wall thickness (LVPWd) at end of diastole is shown for sham, 1-, and 4-week post-TAC. C, Ratios of heart weight to body weight (HW/BW) (mg/g) are shown for sham and 4-week post-TAC surgery. D, Left ventricular internal dimension (LVIDd) at end of diastole is shown for sham, 1-, and 4-week post-TAC. *, p < 0.05 between WT and ENH^−/− groups. @, p < 0.05 between sham and 1-week post-TAC. #, p < 0.05 between 1-week and 4-week post- TAC. &, p < 0.05 between sham and 4-week post-TAC.

Fig. 6

Loss of Cypher short isoform and Calsarcin-1 proteins in ENH^−/− hearts. A, CypherS and Calsarcin-1 were downregulated in 3-month-old ENH^−/− mice (left panel) and almost depleted in 12-month-old mice (right panel). Myotilin was upregualted in both 3-month-old and 12-month-old ENH^−/− mice. α B-crystallin, α-actinin 2 and Desmin were not different between WT and ENH^−/− mice at either age analyzed. GAPDH was shown as loading control. B, CypherS and Calsarcin-1 were slightly downregulated in 1-month ENH^−/− mice compared with age-matched WT controls. C, The downregulation of CypherS and Calsarcin-1 in ENH^−/− hearts is age dependent. D, The mRNA of Myotilin, Calsarcin-1 and CypherS were assessed by real-time RT-PCR. 18S RNA was used as internal RNA standard. The ratio to 18S RNA was further normalized to WT. *, P < 0.05.

Fig. 7

ENH-Cypher-Calsarcin protein complex. A, Calsarcin-1, Myotilin, CypherS and ENH long isoforms were dominantly localized in the filament fraction as shown by biochemical isolation and western blot analysis. CypherL and ENH short isoforms are in both filament and non-filament fractions. GAPDH (cytosolic protein), Integrinβ1D (membrane protein) and Troponin I (TnI, filament protein) were used as fractionation controls. B, ENH, CypherS, and Calsarcin are localized in the same fractions as shown by sucrose gradient sediment analysis. A total of 14 fractions were collected (from low to high of sucrose concentrations) and resolved by SDS-PAGE before western blot analysis. C, The ENH PDZ domain shares high similarity in amino acid residues with the Cypher PDZ domain. Identical amino acid residues are shown in red star underneath and similar amino acid residues are shown in green for both the Cypher (2-83) and the ENH PDZ domains (3-84). D, ENH interacted with Calsarcin-1 and Myotilin in vitro. FLAG-tagged Calsarcin-1 (lanes 2 and 5) or Myotilin (lanes 3 and 6) were coexpressed with HA-tagged ENH1 (ENH long isoform, lanes 1-3) or ENH3 (ENH short isoform, lanes 4-6) in HEK 293 cells. Exogenous protein expression was verified by immunobolting with FLAG or HA antibodies. ANTI-FLAG M2 Affinity Gel was used to purify FLAG-tagged proteins and interacting proteins were visualized by immunoblotting with a HA antibody. FLAG-tagged control proteins (protein kinase A RI subunit) (lanes 1 and 4) were used as a control and did not bind to HA-tagged ENH proteins.