The muscle ankyrin repeat proteins CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to pressure overload

Abstract

Ankrd1/CARP, Ankrd2/Arpp, and Ankrd23/DARP belong to a family of stress inducible ankyrin repeat proteins expressed in striated muscle (MARPs). The MARPs are homologous in structure and localized in the nucleus where they negatively regulate gene expression as well as in the sarcomeric I-band, where they are thought to be involved in mechanosensing. Together with their strong induction during cardiac disease and the identification of causative Ankrd1 gene mutations in cardiomyopathy patients, this suggests their important roles in cardiac development, function, and disease. To determine the functional role of MARPs in vivo, we studied knockout (KO) mice of each of the three family members. Single KO mice were viable and had no apparent cardiac phenotype. We therefore hypothesized that the three highly homologous MARP proteins may have redundant functions in the heart and studied double and triple MARP KO mice. Unexpectedly, MARP triple KO mice were viable and had normal cardiac function both at basal levels and in response to mechanical pressure overload induced by transverse aortic constriction as assessed by echocardiography and hemodynamic studies. Thus, CARP, Ankrd2, and DARP are not essential for normal cardiac development and function at basal conditions and in response to mechanical pressure overload.

Figure 1. Generation of CARP and DARP KO mice.

(A, E) Targeting strategies. Restriction maps of relevant genomic regions of Ankrd1 (A) and Ankrd23 (E) are shown on top, targeting constructs are shown in the center, and mutated loci after homologous recombination are shown at the bottom. B, BamHI; E, EcoRI; H, HindIII; K, KpnI; P, PstI; S1, SalI, neo, neomycin resistance gene; TK, thymidine kinase, E, Exon, P, Primer. (B, F) Detection of WT and targeted alleles by Southern blot analysis. Ankrd1 (B) and Ankrd23 (F) DNAs from electroporated ES cells were digested with BamHI and HindIII, respectively and analyzed by Southern blot analysis with probes as shown in A. (C, G) Detection of Ankrd1 (C) or Ankrd23 (G) mRNA by Northern blot analysis. Aliquots of 10 µg of total RNA isolated from adult ventricles of WT and KO mice were analyzed by using a cDNA probe spanning the entire coding regions of Ankrd1 and Ankrd23, respectively. A GAPDH probe was used as a control for equal loading. (D, H) Detection of CARP (D) and DARP (H) protein by Western blot analysis. Protein prepared from adult ventricles of WT and KO mice were analyzed with polyclonal antibodies against CARP and DARP. Monoclonal antibodies against GAPDH were used as loading control.