The SMYD1 p.Asn101Ser is a partial loss-of-function variant that impairs mitochondrial function and leads to early-onset cardiomyopathy.

Figures

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  Figure 1.. Pedigree of a family carrying a novel SMYD1 variant (c.302A>G; p.Asn101Ser). Females are represented by circles and males by squares. Pregnancy losses are indicated as follows: miscarriages by triangles, stillbirth (SB) by a square with a diagonal line, termination of pregnancy (TOP) by a triangle with a diagonal line. A double line indicates consanguinity. Genotyped and negative for SMYD1 variant are shown as grey symbols. Unaffected and ungenotyped individuals are shown as open symbols. The parents are unaffected and heterozygous for the variant, as indicated by half-filled symbols. The proband P1 (arrow) is homozygous and affected, indicated by a black circle.

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  Figure 2.. Structural domain organization of SMYD1 and identification of a novel SMYD1 (c.302A>G; p.Asn101Ser) variant. A. Linear representation of the predicted structural domains of the SMYD1 protein in humans based on published mouse SMYD1 domains. A single amino acid substitution at position 101 (Asn to Ser) was identified in a pediatric patient (P1) with biventricular heart failure as indicated with a green arrow. B. SMYD1 protein sequence alignment from position 91 through position 110 spanning the MYND-SET-I domain boundary, showing the conserved Asn101 residue (highlighted in red) across 14 vertebrate species. Residues in bold capital letters are invariant across all 14 species, residues in capital letters (not bold) are highly conserved, and residues in lower case letters appear to be dispensable. C. Crystal structure of mouse SMYD1 (PDB ID: 3N71) showing the location of residue Asn101 (red) near the substrate binding site (black arrow).

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  Figure 3.. Structural and thermodynamic impact of the N101S substitution in SMYD1. A. Electrostatic surface representation of the wild-type SMYD1 structure (PDB ID: 3N71), with Asn101 highlighted (left), and the N101S variant with Ser101 indicated (right). The N101S substitution is predicted to modestly destabilize the protein structure, as indicated by a change in Gibbs free energy (ΔΔG = −0.55 kcal/mol), as calculated using DynaMut2. B. Enlarged view of the wild-type structure (left) showing the local interaction network of Asn101, including a hydrogen bond with Asp45 (dashed blue line, 3.1Å), and the N101S variant (right) demonstrating loss of this hydrogen bond with Asp45, resulting in perturbation of the local interaction network near the cofactor binding pocket. All structural models were generated based on the mouse SMYD1 crystal structure (PDB ID: 3N71) and visualized using PyMOL and UCSF Chimera.

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  Figure 4.. Validation of SMYD1-N101S variant expression in cultured myocytes. Undifferentiated C2C12 cells were transfected with wild-type SMYD1 or N101S constructs or an empty vector (EV) as a control. A. Sanger sequencing confirms the construct containing a single nucleotide substitution (A>G) in the mouse Smyd1 gene at position 302, resulting in a protein carrying the N101S variant. B. RT-qPCR analysis confirms increased Smyd1 transcript expression in both Smyd1- and N101S-transfected cells. n=2–3. Asterisk * indicates p<0.05. C. Immunoblotting showing expression of both wild-type SMYD1 protein and the MYC-tagged N101S variant, compared to untreated (Ctrl) and empty vector (EV) controls. D. Densitometric quantification of immunoblot band intensity for MYC and SMYD1, confirmed in three independent experiments. Data are represented as mean ± SEM.

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  Figure 5.. SMYD1-N101S impairs mitochondrial respiratory function in cultured myocytes. C2C12 cells were transfected with wild-type SMYD1 or N101S constructs or an empty vector (EV) as a control. A-C. Representative traces (A), quantification (B), and percent change (C) of mitochondrial oxygen consumption rate (OCR) using a Seahorse Cell Mito Stress Test demonstrating that the N101S variant leads to decreased mitochondrial respiration when compared to wild-type SMYD1 (green). n=6. Asterisks: * indicates p<0.05, ** indicates p<0.01. D. RT-qPCR analysis demonstrates no significant change in Ppargc1α transcript levels in N101S-expressing cells. n=3. E-F. Representative immunoblots (E) and quantification (F) demonstrating no significant change in PGC-1α protein abundance in both wild-type and N101S-expressing cells relative to empty vector control. n=3. G-H. Representative immunoblots of histone H3K4me3 (G) and densitometric quantification (H) showing similarly increased H3K4me3 levels in both wild-type and N101S-expressing cells compared to empty vector control, with no significant difference between wild-type SMYD1 and N101S. I. RT-qPCR analysis of hypertrophic markers demonstrates no…

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  Figure 6.. Increased SMYD1 protein abundance in cardiac but not skeletal muscle tissue from a patient with SMYD1 variant-associated biventricular heart failure. A. Representative immunoblots showing SMYD1 protein levels in cardiac and skeletal muscle tissues obtained from patient P1 at the time of heart transplantation, compared with an age- and sex-matched healthy control. GAPDH was used as a loading control. B. Densitometric quantification of SMYD1 protein abundance normalized to GAPDH, shown as relative protein expression. SMYD1 protein abundance was increased in cardiac tissue from P1 relative to the healthy control, whereas no significant difference was observed in skeletal muscle. C: healthy donor control; P: patient P1; sk.musc.: skeletal muscle. Asterisk * indicates p<0.05. Data are presented as mean ± SEM.

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  Figure 7.. Proposed mechanism of SMYD1 N101S variant dysfunction in the heart. Upper panel: Wild-type SMYD1 supports mitochondrial function and maintains metabolic homeostasis in the heart, contributing to normal cardiac function. Lower panel: the N101S variant impairs mitochondrial respiratory capacity, leads to compromised metabolic homeostasis and heart failure. The red circle on the SMYD1 N101S protein indicates the location of the N101S substitution within the SET-I domain.