A natural loss-of-function deletion of the cytohesin 1 (Cyth1) gene in BALB/cByJ mice does not impact cardiomyocyte polyploidy.

Mammalian cardiomyocytes (CMs) mostly become polyploid shortly after birth. Because this feature may relate to several aspects of heart biology, including regeneration after injury, the mechanisms that cause polyploidy are of interest. BALB/cJ and BALB/cByJ mice are highly related sister strains that diverge substantially in CM ploidy. We identified a large deletion in the Cyth1 gene that arose uniquely in BALB/cByJ mice that creates a null allele. The deletion also results in ectopic transcription of the downstream gene Dnah17, although this transcript is unlikely to encode a protein. By evaluating the natural null allele from BALB/cByJ and an engineered knockout allele in the C57BL/6J background, we determined that absence of Cyth1 does not by itself influence CM ploidy. The ready availability of BALB/cByJ mice may be helpful to other investigations of Cyth1 in other biological processes.

A kinase-dead natural polymorphism in the canine Tnni3k gene.

Most mammalian cardiomyocytes become polyploid in the neonatal period, concurrent with their loss of proliferative capacity. In mice, natural or engineered mutation of the cardiomyocyte-specific kinase gene Tnni3k causes a higher level of diploid CMs and a higher capacity to support proliferation after adult injury. Here, we identified a polymorphism in the canine Tnni3k gene that is particularly common in the West Highland White Terrier breed, and show that this variant eliminates Tnni3k kinase activity. Thus, in several species, natural Tnni3k polymorphisms exist that are predicted to contribute to variation in diploid CM level and heart regenerative ability.

Purkinje Cardiomyocytes of the Adult Ventricular Conduction System Are Highly Diploid but Not Uniquely Regenerative.

Adult hearts are characterized by inefficient regeneration after injury, thus, the features that support or prevent cardiomyocyte (CM) proliferation are important to clarify. Diploid CMs are a candidate cell type that may have unique proliferative and regenerative competence, but no molecular markers are yet known that selectively identify all or subpopulations of diploid CMs. Here, using the conduction system expression marker Cntn2-GFP and the conduction system lineage marker Etv1CreERT2, we demonstrate that Purkinje CMs that comprise the adult ventricular conduction system are disproportionately diploid (33%, vs. 4% of bulk ventricular CMs). These, however, represent only a small proportion (3%) of the total diploid CM population. Using EdU incorporation during the first postnatal week, we demonstrate that bulk diploid CMs found in the later heart enter and complete the cell cycle during the neonatal period. In contrast, a significant fraction of conduction CMs persist as diploid cells from fetal life and avoid neonatal cell cycle activity. Despite their high degree of diploidy, the Purkinje lineage had no enhanced competence to support regeneration after adult heart infarction.

Mesowestern Blot: Simultaneous Analysis of Hundreds of Submicroliter Lysates.

Western blotting is a widely used technique for molecular-weight-resolved analysis of proteins and their posttranslational modifications, but high-throughput implementations of the standard slab gel arrangement are scarce. The previously developed Microwestern requires a piezoelectric pipetting instrument, which is not available for many labs. Here, we report the Mesowestern blot, which uses a 3D-printable gel casting mold to enable high-throughput Western blotting without piezoelectric pipetting and is compatible with the standard sample preparation and small (∼1 µL) sample sizes. The main tradeoffs are reduced molecular weight resolution and higher sample-to-sample CV, making it suitable for qualitative screening applications. The casted polyacrylamide gel contains 336, ∼0.5 µL micropipette-loadable sample wells arranged within a standard microplate footprint. Polyacrylamide % can be altered to change molecular weight resolution profiles. Proof-of-concept experiments using both infrared-fluorescent molecular weight protein ladder and cell lysate (RIPA buffer) demonstrate that the protein loaded in Mesowestern gels is amenable to the standard Western blotting steps. The main difference between Mesowestern and traditional Western is that semidry horizontal instead of immersed vertical gel electrophoresis is used. The linear range of detection is at least 32-fold, and at least ∼500 attomols of ß-actin can be detected (∼29 ng of total protein from mammalian cell lysates: ∼100-300 cells). Because the gel mold is 3D-printable, users with access to additive manufacturing cores have significant design freedom for custom layouts. We expect that the technique could be easily adopted by any typical cell and molecular biology laboratory already performing Western blots.