Targeted mutation of MCU using TALENs. (A) Schematic representation of partial MCU genomic sequence demonstrating the TALEN binding sites. Open and solid boxes represent UTRs and coding exons, respectively. (B) Representative genotyping results. Amplification of the genomic region surrounding the deletion using a primer pair FP/RP shown in (A). The wildtype (152 bp) and mutant (141 bp) fragments were separated using polyacrylamide gel electrophoresis. (C) Sequencing analysis of cDNA identified a frameshift mutation caused by an 11-nucleotide deletion in exon 1, resulting in a premature termination of translation. (D)In situ hybridization detects MCU transcripts in the heart (arrow) in WT but not mcu mutant embryos at 3 days post fertilization (dpf). (E) Quantitative PCR analysis of MCU transcripts in 3 dpf embryos. (F) Quantification of Rhod 2 Signals in WT cardiomyocytes with and without MCU inhibitor Ru360 and MCU mutant cardiomyocytes. ****p < 0.0001; ***p < 0.001; and NS: p > 0.05.

Cardiac morphological analysis of adult MCU mutants. (A) Genotypic distribution of F1 generation from MCU heterozygous parents matches closely with Mendelian ratio and show no signs of embryonic lethality. (B) Comparable exterior phenotypic traits between wildtype and MCU mutant zebrafish. (C) Defects in cardiac structure observed in dissected adult MCU mutant hearts. Mutant hearts were characterized by enlarged atrial volume and limited ventricular blood flow. (D) Reduction in tissue density and disorganization of myocardium observed in histological sections (Hematoxylin and Eosin stains) of MCU mutant hearts. Nuclei rich regions stained blue and cytoplasmic regions and ECM stained pink. (E) Selected image frames during systolic and diastolic phases were analyzed to compare ventricular-to-atrial surface area ratio. Ventricular:atrial SA ratio were significantly reduced in MCU mutant hearts (n = 14 for each group, p < 0.0001). (F) α-actinin staining marks the Z-lines of intact sarcomeres in ventricular cardiomyocytes of wild type adult fish (yellow bracket). Adult MCU ventricles contain regions with intact myofibrils/sarcomeres (yellow bracket), but also exhibit areas of patchy α-actinin staining indicative of damaged myofibrils and broken-down sarcomeres (magenta asterisks). Scale bars indicate 10 μm.

Transmission electron microscopy analysis of MCU mutant ventricles. Representative TEM images of WT (A–C) and MCU(D–F) ventricular cardiomyocytes. (A,D) WT sarcomeres display clear Z-lines (arrows in A) separated by parallel bands of myosin thick filaments. MCU Z-lines are less well defined and patchy (arrows in D) and thick filaments are sometimes disorganized. (B,C) WT myofibrils are closely associated with oval-shaped mitochondria containing tightly packed cristae (arrowheads in B mark examples, C shows more examples). (E,F)MCU myofibrils are also closely associated with mitochondria, but they are less electron-dense and display a round and swollen morphology with more loosely packed cristae [arrowheads in (E) mark examples, (F) shows more examples]. Scale bars indicate 500 nm.

Electrocardiogram analysis of wildtype and MCU mutant fish. (A) ECG tracings of wildtype fish (top), Class I MCU mutant (clearly distinguishable QRS complex, middle) and Class II MCU mutant (highly abnormal QRS complex, bottom) highlight episodes of long beat-to-beat pauses. Arrows point to beat intervals that are greater than mean + 2σ. (B) Analysis and comparison of ECG indices between wildtype and MCU mutants (Class I and II combined). No significant differences were identified in heart rate (115.4 bpm v 115.0 bpm, p > 0.05), P-amplitude (0.045 mV v 0.055 mV, p > 0.05), and P-P interval (0.524 s v 0.539 s, p > 0.05) between wildtype and mutant, respectively. (C) Statistical differences were present in R-wave amplitudes (reduced in MCU Class I mutant, 0.248 mV v 0.062 mV, p < 0.0001), R-wave amplitude-to-P-wave amplitude ratio (increased in MCU Class I mutant, 0.182 v 0.884, p < 0.001), and duration of the QRS complex (prolonged in MCU Class I mutant, 0.0512 s v 0.0721 s, p < 0.05). *p < 0.05; ***p < 0.0001.

Analysis on cardiac arrhythmia in MCU mutant. (A) Increased heartbeat variability in MCU mutants displayed via Poincaré plots of consecutively occurring P-P intervals in adult wildtype and MCU mutant fish. (B) Summary of ECG data from 14 wildtype fish and 14 MCU mutant fish. Different cut-offs of P-P intervals were used to classify severity and definitive of occurring sinoatrial block episodes. Increased sample percentage with SA block episodes, per minute frequency, and consecutively occurring episodes were identified in MCU mutant (compared to wildtype), and further emphasized in MCU Class II mutants.

RNA-seq analysis of the MCU mutant adult heart. (A) Volcano plot of differential gene expression of MCU mutant hearts vs. wildtype. Each point represents the average value of one transcript in duplicate experiments. The expression difference is considered significant for an adjusted p-value (FDR) of less than 0.05. Red points represent genes significantly upregulated in MCU mutants and blue points represent significantly down regulated genes. FDR, false-discovery rate. (B–D) Selected lists of significantly differentially expressed (FDR < 0.05) ion transporter-encoding (B), structural protein-encoding (C), and mitochondrial protein-encoding (D) genes. The percent increase (+, red) or decrease (-, blue) in expression in MCU compared to WT, based on the fold-changes determined by Cufflinks, is displayed in the right column.

Upregulated genes in MCU hearts reflect altered mitochondrial biology. (A, B) Dot plots of over-represented Biological Process (A) and Molecular Function (B) gene ontology terms in the set of genes that is significantly upregulated in MCU mutant hearts. The dot size indicates the number of genes associated with each term and the dot color indicates the significance of the enrichment (adjusted p-values). (C) Gene concept networks of the top 10 over-represented Biological Process gene ontology terms in the set of genes that is significantly upregulated in MCU mutants. The dot size indicates the number of genes associated with each term and dots representing genes are colored based on their fold-change [log2(fold-change)].

Downregulated genes in MCU hearts suggest defects in cardiac structure and function. (A,B) Dot plots of over-represented Biological Process (A) and Molecular Function (B) gene ontology terms in the set of genes that is significantly downregulated in MCU mutant hearts. The dot size indicates the number of genes associated with each term and the dot color indicates the significance of the enrichment (adjusted p-values). (C) Gene concept networks of the top 10 over-represented Biological Process gene ontology terms in the set of genes that is significantly downregulated in MCU mutants. The dot size indicates the number of genes associated with each term and dots representing genes are colored based on their fold-change [log2(fold-change)].