Severe protein aggregate myopathy in a knockout mouse model points to an essential role of cofilin2 in sarcomeric actin exchange and muscle maintenance.

Mutations in the human actin depolymerizing factor cofilin2 result in an autosomal dominant form of nemaline myopathy. Here, we report on the targeted ablation of murine cofilin2, which leads to a severe skeletal muscle specific phenotype within the first two weeks after birth. Apart from skeletal muscle, cofilin2 is also expressed in heart and CNS, however the pathology was restricted to skeletal muscle. The two close family members of cofilin2 - ADF and cofilin1 - were co-expressed in muscle, but unable to compensate for the loss of cofilin2. While primary myofibril assembly and muscle development were unaffected in cofilin2 mutant mice, progressive muscle degeneration was observed between postnatal days 3 and 7. Muscle pathology was characterized by sarcoplasmic protein aggregates, fiber size disproportion, mitochondrial abnormalities and internal nuclei. The observed muscle pathology differed from nemaline myopathy, but showed combined features of actin-associated myopathy and myofibrillar myopathy. In cofilin2 mutant mice, the postnatal expression pattern and turnover of sarcomeric α-actin isoforms were altered. Levels of smooth muscle α-actin were increased and remained high in developing muscles, suggesting that cofilin2 plays a crucial role during the exchange of α-actin isoforms during the early postnatal remodeling of the sarcomere.

Connexin39 deficient mice display accelerated myogenesis and regeneration of skeletal muscle.

During muscle development and regeneration of skeletal muscle in mice connexin43 (Cx43) and connexin39 (Cx39) are specifically expressed: Cx43 in satellite cells and myoblasts, whereas Cx39 is exclusively expressed in myogenin-positive cells. We generated Cx39 deficient mice by replacing the coding region of the Gjd4 gene by DNA coding for the enhanced green fluorescent protein eGFP. Adult Cx39 deficient mice exhibit no obvious phenotypic alterations of skeletal muscle compared to wild type mice in the resting state. However, myogenesis in Cx39 deficient embryos is accelerated as indicated by increased myogenin expression on ED13.5 and ED16.5 and increased expression of Cx43 in developing skeletal muscle. In addition, the regeneration process of skeletal muscle in Cx39 deficient mice is accelerated as shown by a 2day earlier onset of MyoD and myogenin expression, relative to wild type littermates. Interestingly, Cx43 expression was also upregulated in Cx39 deficient mice during regeneration of skeletal muscle. We hypothesize that Cx43 may compensate for the loss of Cx39 during myogenesis and regeneration.

C-terminal tagging with eGFP yields new insights into expression of connexin45 but prevents rescue of embryonic lethal connexin45-deficient mice.

Connexin45 (Cx45) is a member of the connexin family which can form gap junction channels and is known to be expressed in several cell types in the embryonic as well as adult mouse including working cardiomyocytes and certain types of neurons. Until now its subcellular localization could not be unequivocally determined in certain tissues due to the lack of sensitive and specific antibodies. In order to investigate the localization of Cx45, we have generated a transgenic mouse expressing a fusion protein composed of Cx45 and eGFP under control of the endogenous Cx45 promoter using a bacterial artificial chromosome (BAC). In previous studies it had been shown that a C-terminal tag of connexin proteins only slightly altered the properties of gap junction channels in cultured cells and allowed direct visualization of the fusion protein. In the adult brain the expression of the Cx45eGFP protein was found in the subventricular zone in transient amplifying cells as well as in neuroblasts and ependymal cells. In addition Cx45eGFP is expressed in the atrial and ventricular working myocardium, i.e. regions of the heart where divergent results regarding Cx45 expression had previously been published. In the lung we identified Cx45eGFP in the smooth muscle cell layer of bronchioles. The Cx45eGFP transgene could not rescue embryonic lethality of Cx45-deficient mice, i.e. Cx45eGFP//Cx45(-/-) mice die around ED10.5 presumably due to altered properties of gap junction channels as a result of C-terminal tagging of Cx45.