Disruption of Z-Disc Function Promotes Mechanical Dysfunction in Human Myocardium: Evidence for a Dual Myofilament Modulatory Role by Alpha-Actinin 2

Figures

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  Figure A1. Sequence of α-actinin 2 EF 3–4 region across 25 different species. Multi-species sequence alignment of the region where the A868T variant is located. Highlighted by the red box is the amino acid that is mutated in the human patient.

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  Figure A2. Sequence of α-actinin 2 EF 3–4 in four small mammals that have threonine (T) at the equivalent residue as to human A868. Multi-species sequence alignment of the region where the A868T variant is located. Highlighted by the red box is the amino acid that is mutated in the human patient, aligned with four small mammal species that, surprisingly, normally have a threonine (T) residue at that location.

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  Figure 1. ACTN2 (α-actinin 2) variant as a strong candidate for diverse cardiac signs and symptoms. De-identified human free left ventricular wall was obtained from an A868T variant patient after heart transplant. (A) Family pedigree of A868T variant patient, red arrow marks proband patient. (MI: myocardial infarction, MVA: motor vehicle accident, CHD: congenital heart disease). (B) Representative echocardiographic longitudinal strain of proband patient. (C) Presenting electrocardiogram of proband patient. (D) Location of the A868T variant in the sarcomere (top left), primary sequence (bottom left), the red arrow shows where the variant is located and the amino acid substitution, schematic (top right), and crystal structure (bottom right) of α-actinin 2. (E) Representative H&E stained sections from explanted left ventricle free wall from the proband patient at 10× (left) and 40× (right) magnification.

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  Figure 2. Ultrastructural analysis of the A868T variant patient heart. (A,B) Representative electron microscopy of (A) normal and (B) A868T variant myocardium. Scale bars are 800 nm. (C–F) Representative immunofluorescence images with α-actinin 2 (green) and titin (red) antibodies. Scale bars are 10 µm. Additional immunofluorescence images are shown in Supplementary Figures S2 and S3. (G) Sarcomere length measured from EM images (5 EM images of the same patient’s heart and 5 EM images of the same donor heart were analyzed). (H) Myofibrillar width measured from EM images (5 EM images of the same patient’s heart and 5 EM images of the same donor heart were analyzed). (I) Z-disc width measured from EM images (5 EM images of the same patient’s heart and 5 EM images of the same donor heart were analyzed). (J,K) Antibody fluorescence intensity quantification. (L) Sarcomere length measured from immunofluorescence images. (M) Z-bodies quantification measured from immunofluorescence images. (N) Titin spots measured from immunofluorescence images. Data are shown as mean ± S.E; the data points are technical replicates. * p < 0.05. ** p < 0.01.

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  Figure 3. Impacts of the A868T variant on the mechanics of cardiac muscle contraction. (A,B) Isometric force and Ca2+ sensitivity: A868T CMPs presented a reduction in maximum steady-state and higher myofilament Ca2+ sensitivity. (C,D) Sinusoidal stiffness: A868T CMPs showed lower cross-bridge populations at all levels of activation. (E,F) Kinetics of tension redevelopment (kTR): A868T CMPs displayed faster kTR at all levels of calcium activation. Data are shown as mean ± S.D.; n = 4–5 per group (technical replicates).

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  Figure 4. Small-angle equatorial X-ray diffraction patterns from A868T cardiac tissue. (A,B) Representative images of small angle equatorial X-ray diffraction patterns from control and A868T cardiac tissue. Histogram plots demonstrate the intensity of the equatorial reflections in both tissues. Overall, a reduction i intensity of the 1,0 and 1,1 reflections was observed in A868T variant cardiac tissue. (C) I1,1/I1,0 is increased in ACNT2 A868T variant cardiac tissue as compared to WT. (D) Interfilament lattice spacing, d10, is elevated in A868T variant cardiac tissue. Data are shown as mean ± S.E; n = 6 per group (technical replicates), ** p < 0.01.

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  Figure 5. Principal component analysis. (A) Free energy landscapes along PC1 and PC2 of WT (left column) and A868T (right column) of the three systems. Free energies were converted from the probability density distributions using G = −kBTP, where kB is Boltzmann’s constant, T is temperature (310 K), and P is the probability density. Low energy areas are color-coded and indicated by asterisks. (B) PC1 (cyan arrows) and PC2 (green arrows) projected onto the EF 3–4–actinin complex. The bound peptide is colored red. (C) Superposition of structures of EF 3–4 WT–actinin from region 1 (white), EF 3–4 WT–titin from region 2 (orange), and EF 3–4 A868T–titin from region 3 (cyan).

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  Figure 6. Summary of proposed pathophysiological pathways. (A) Communication pathways. Communication Pathway 1 (CP1) proposes a relationship between α-actinin 2 and actin that regulates thin filament activation. Communication Pathway 2 (CP2) proposes an interaction between α-actinin 2/titin/myosin that controls LS, thick filament activation, and cross-bridges kinetics. (B) Model for α-actinin 2 variant A868T influences cardiac function, leading to pathology. Summary of the ultrastructural and mechanical changes caused by the A868T variant on α-actinin 2 and the predicted role in the Z-disc of the sarcomere.