m-Calpain is required for preimplantation embryonic development in mice

Abstract
Background
μ-calpain and m-calpain are ubiquitously expressed proteases implicated in cellular migration, cell cycle progression, degenerative processes and cell death. These heterodimeric enzymes are composed of distinct catalytic subunits, encoded by Capn1 (μ-calpain) or Capn2 (m-calpain), and a common regulatory subunit encoded by Capn4. Disruption of the mouse Capn4 gene abolished both μ-calpain and m-calpain activity, and resulted in embryonic lethality, thereby suggesting essential roles for one or both of these enzymes during mammalian embryogenesis. Disruption of the Capn1 gene produced viable, fertile mice implying that either m-calpain could compensate for the loss of μ-calpain, or that the loss of m-calpain was responsible for death of Capn4-/- mice.

Results
To distinguish between the alternatives described above, we deleted an essential coding region in the mouse Capn2 gene in embryonic stems cells and transmitted this mutant allele through the mouse germline. Breeding of heterozygous animals failed to produce homozygous mutant live offspring or implanted embryos. A nested PCR genotyping protocol was established, and homozygous preimplantation mutant embryos were detected at the morula but not at the blastocyts stage.

Conclusion
We conclude that homozygous disruption of the Capn2 gene results in pre-implantation embryonic lethality between the morula and blastocyst stage. This establishes that μ-calpain and m-calpain have distinct functions, and that m-calpain is vital for development of the preimplantation murine embryo.

Background
The two ubiquitous Ca2+-dependent, cysteine proteases known as μ-calpain (calpain-1) and m-calpain (capain-2), are the founding members of a gene family comprising 13 genes in mammals [1-3]. Both are heterodimeric enzymes consisting of distinct 80 kDa catalytic subunits, encoded by the Capn1 (μ-80 k) and Capn2 (m-80 k) genes, respectively, that associate with a common 28 kDa regulatory subunit encoded by the Capn4 gene. The μ-80 k and m-80 k subunits share 62% amino acid sequence identity, and are very similar in terms of structure, protein chemistry, and in vitro substrate specificity. Despite these similarities, the differential expression patterns of μ- and m-calpain in mammalian tissues suggest they have some isoform specific and distinct functions. The μ and m designations derive from the levels of Ca2+ required in vitro for optimal activation; 10–50 μM Ca2+ for μ-calpain and 0.3–0.35 mM Ca2+ for m-calpain. It is generally assumed that μ- and m-calpain maintain their differential sensitivities to calcium in vivo, although this has not yet been strictly demonstrated. Furthermore, since the cytoplasmic free Ca2+ concentration is typically less than 1 μM, it is also assumed that other in vi