Tnni3k alleles influence ventricular mononuclear diploid cardiomyocyte frequency

Figures

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  Fig 1. Tnni3k deletion mutants cause nonsense-mediated mRNA decay. A. Western blot of protein extracted 293 cells transfected with constructs to express wild-type full-length mouse Tnni3k (WT), the 4bp or 8bp deletion mutant variants (Δ4 and Δ8), and a truncation mutation at position 492 corresponding to the premature stop codon observed in H. glaber. All constructs were based on the mouse protein, and all carried a Flag epitope at the N-terminus. Transfected protein was detected by anti-Flag antibody. MG132 was added at the concentrations indicated to test for proteasomal degradation. Gapdh was used as a loading control. B. Semi-quantitative RT-PCR analysis of Tnni3k transcript abundance in ventricular tissue from adult mice (2 of each genotype) of the indicated genotypes. C. Western blot to detect Tnni3k protein in ventricular lysate from adult mice of the indicated genotypes. The filled arrow indicates full-length Tnni3k (93kDa), the open arrow indicates the expected size of the Δ4 and Δ8 truncated proteins (62kDa) if they were stably expressed. D. Ventricular mononuclear CM% in adult hearts of the indicated genotypes; in both evaluations, littermate +/- control mice were used as a…

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  Fig 2. The Tnni3k gene in naked mole-rats (H. glaber). A. Genomic DNA sequence at the end of intron 15 and beginning of exon 16 and deduced protein coding sequence (in 1 letter code above) in mouse (Mmu) and in H. glaber (Hgla). The in-frame premature stop codon is indicated in red type, and lies at position 22288559–561 (reverse complement) of NW_004624742.1 and at 1144837–839 (reverse complement) of NW_004629930.1. The sequence trace confirms the accuracy of the H. glaber genomic sequence. B. Western blot to detect Tnni3k; the blot contains ventricular protein from one wild-type (WT) C57BL/6 mouse, one homozygous null (-/-) mouse, and two individual H. glaber animals. C. Ventricular mononuclear CM% in adult hearts of two inbred mouse strains (SJL/J and A/J), which represent the lowest and highest known values of mononuclear CM% among common inbred strains, and in adult H. glaber. Data for SJL/J and A/J mice are from our previous evaluation [20] and are shown here for reference. D. Evaluation of nuclear ploidy in single cell preparations from one C57BL/6J mouse (at left) and four naked mole-rats, as measured by DAPI fluorescence. Values shown represent fluorescence intensity per…

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  Fig 3. Consequences of a kinase-dead Tnni3k allele in mice. A. Western blot of Tnni3k protein from mice of the indicated genotypes, demonstrating that the K489R protein is stable in vivo. B. Ventricular mononuclear CM% in mice of the indicated Tnni3k genotypes; heterozygous (+/-) and null (-/-) mice were littermates of the K489R/- mice, but not of each other. C. Evaluation of nuclear ploidy specifically in the mononuclear CM subpopulation of mice of the indicated Tnni3k genotypes. See also S4 Fig.

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  Fig 4. Consequences on in vitro kinase activity of common human Tnni3k kinase domain variants. A. Representative Western blot showing autophosphorylation on Ser and Tyr residues of wild-type and variant Tnni3k extracted from transfected 293 cells prior to (-) or following (+) incubation in an in vitro kinase reaction. Proteins were expressed with an N-terminal Flag epitope, which was detected separately as a loading control. B. Quantitation of kinase activity for the four indicated variants based on data shown in S5 Fig.

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  Fig 5. Tnni3k converges with oxidative stress. A. Western blot of ventricular protein from two wild-type and two Tnni3k null P3 mice showing phosphorylated (activated) and total protein for the three MAPK members p38, Jnk, and Erk. The very low level of phospho-p38 in P3 heart is compared against the normal level in adult heart in S6 Fig. B-C. Ventricular mononuclear CM% in adult hearts, comparing absence vs. presence of the mCAT transgene in a background that is wild-type for Tnni3k and heterozygous for Nnt (B), and comparing absence vs. presence of the mCAT transgene in a Tnni3k null and Nnt null background (C).