The muscle ankyrin repeat proteins: CARP, ankrd2/Arpp and DARP as a family of titin filament-based stress response molecules

CARP, ankrd-2/Arpp, and DARP, are three members of a conserved gene family, referred to here as MARPs (muscle ankyrin repeat proteins). The expression of MARPs is induced upon injury and hypertrophy (CARP), stretch or denervation (ankrd2/Arpp), and during recovery following starvation (DARP), suggesting that they are involved in muscle stress response pathways. Here, we show that MARP family members contain within their ankyrin repeat region a binding site for the myofibrillar elastic protein titin. Within the myofibril, MARPs, myopalladin, and the calpain protease p94 appear to be components of a titin N2A-based signaling complex. Ultrastructural studies demonstrated that all three endogenous MARP proteins co-localize with I-band titin N2A epitopes in adult heart muscle tissues. In cultured fetal rat cardiac myocytes, passive stretch induced differential distribution patterns of CARP and DARP: staining for both proteins was increased in the nucleus and at the I-band region of myofibrils, while DARP staining also increased at intercalated discs. We speculate that the myofibrillar MARPs are regulated by stretch, and that this links titin-N2A-based myofibrillar stress/strain signals to a MARP-based regulation of muscle gene expression.     

Cytosolic CARP Promotes Angiotensin II- or Pressure Overload-Induced Cardiomyocyte Hypertrophy through Calcineurin Accumulation

The gene ankyrin repeat domain 1 (Ankrd1) is an enigmatic gene and may exert pleiotropic function dependent on its expression level, subcellular localization and even types of pathological stress, but it remains unclear how these factors influence the fate of cardiomyocytes. Here we attempted to investigate the role of CARP on cardiomyocyte hypertrophy. In neonatal rat ventricular cardiomyocytes (NRVCs), angiotensin II (Ang II) increased the expression of both calpain 1 and CARP, and also induced cytosolic translocation of CARP, which was abrogated by a calpain inhibitor. In the presence of Ang-II in NRVCs, infection with a recombinant adenovirus containing rat Ankrd1 cDNA (Ad-Ankrd1) enhanced myocyte hypertrophy, the upregulation of atrial natriuretic peptide and β-myosin heavy chain genes and calcineurin proteins as well as nuclear translocation of nuclear factor of activated T cells. Cyclosporin A attenuated Ad-Ankrd1-enhanced cardiomyocyte hypertrophy. Intra-myocardial injection of Ad-Ankrd1 in mice with transverse aortic constriction (TAC) markedly increased the cytosolic CARP level, the heart weight/body weight ratio, while short hairpin RNA targeting Ankrd1 inhibited TAC-induced hypertrophy. The expression of calcineurin was also significantly increased in Ad-Ankrd1-infected TAC mice. Olmesartan (an Ang II receptor antagonist) prevented the upregulation of CARP in both Ang II-stimulated NRVCs and hearts with pressure overload. These findings indicate that overexpression of Ankrd1 exacerbates pathological cardiac remodeling through the enhancement of cytosolic translocation of CARP and upregulation of calcineurin.     

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

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.     

心肌锚定重复序列蛋白CARP的研究进展

CARP是新发现的具有锚定重复序列,并在哺乳类动物中呈心肌特异性表达的蛋白,它在肌肉发育过程中对转录调控、肌纤维组装和拉伸信号传递等方面发挥重要的作用。本文综述了CARP基因与蛋白质结构、CARP的表达模式及其表达调控、参与调节CARP的细胞内信号转导通路、CARP在肌肉发育中的作用,以及MARP家族其他成员。     

Early depression of Ankrd2 and Csrp3 mRNAs in the polyribosomal and whole tissue fractions in skeletal muscle with decreased voluntary running

The wheel-lock (WL) model for depressed ambulatory activity in rats has shown metabolic maladies ensuing within 53-173 h after WL begins. We sought to determine if WL beginning after 21-23 days of voluntary running in growing female Wistar rats affected the mRNA profile in the polyribosomal fraction from plantaris muscle shortly following WL. In experiment 1, WL occurred at 0200 and muscles were harvested at 0700 daily at 5 h (WL5h, n = 4), 29 h (WL29h, n = 4), or 53 h (WL53h, n = 4) after WL. Affymetrix Rat Gene 1.0 ST Arrays were used to test the initial question as to whether WL affects mRNA occupancy on skeletal muscle polyribosomes. Using a false discovery rate of 15%, no changes in mRNAs in the polyribosomal fraction were observed at WL29h and eight mRNAs (of over 8,200 identified targets) were altered at WL53h compared with WL5h. Interestingly, two of the six downregulated genes included ankyrin repeat domain 2 (Ankrd2) and cysteine-rich protein 3/muscle LIM protein (Csrp3), both of which encode mechanical stretch sensors and RT-PCR verified their WL-induced decline. In experiment 2, whole muscle mRNA and protein levels were analyzed for Ankrd2 and Csrp3 from the muscles of WL5h (4 original samples + 2 new), WL29h (4 original), WL53h (4 original + 2 new), as well as WL173 h (n = 6 new) and animals that never ran (SED, 4-5 new). Relative to WL5h controls, whole tissue Ankrd2 and Csrp3 mRNAs were lower (P < 0.05) at WL53h, WL173h, and SED; Ankrd2 protein tended to decrease at WL53h (P = 0.054) and Csrp3 protein was less in WL173h and SED rats (P < 0.05). In summary, unique early declines in Ankrd2 and Csrp3 mRNAs were identified with removal of voluntary running, which was subsequently followed by declines in Csrp3 protein levels during longer periods of wheel lock.     

Ankrd2 is a modulator of NF-κB-mediated inflammatory responses during muscle differentiation

Adaptive responses of skeletal muscle regulate the nuclear shuttling of the sarcomeric protein Ankrd2 that can transduce different stimuli into specific adaptations by interacting with both structural and regulatory proteins. In a genome-wide expression study on Ankrd2-knockout or -overexpressing primary proliferating or differentiating myoblasts, we found an inverse correlation between Ankrd2 levels and the expression of proinflammatory genes and identified Ankrd2 as a potent repressor of inflammatory responses through direct interaction with the NF-κB repressor subunit p50. In particular, we identified Gsk3β as a novel direct target of the p50/Ankrd2 repressosome dimer and found that the recruitment of p50 by Ankrd2 is dependent on Akt2-mediated phosphorylation of Ankrd2 upon oxidative stress during myogenic differentiation. Surprisingly, the absence of Ankrd2 in slow muscle negatively affected the expression of cytokines and key calcineurin-dependent genes associated with the slow-twitch muscle program. Thus, our findings support a model in which alterations in Ankrd2 protein and phosphorylation levels modulate the balance between physiological and pathological inflammatory responses in muscle.     

14-3-3 Family Members Act Coordinately to Regulate Mitotic Progression

CARP1 and CARP2 proteins (CARPs) are E3 ligases that target p53 as well as phospho-p53 for degradation. Because MDM2 is a critical regulator of p53 turnover, we investigated and found that CARPs associate with MDM2. We provide evidence that CARPs stabilize MDM2 by inhibiting MDM2 self-ubiquitination. CARPs together with MDM2 enhance p53 degradation, thereby inhibiting p53-mediated cell death. CARP protein levels correlate with MDM2 levels including under hypoxia where both are reduced. CARP2 was found to target 14-3-3σ for degradation, leading to MDM2 stabilization. MDMX, a homolog of MDM2, is not absolutely required for MDM2 stabilization by CARPs, although overexpression of CARP2 enhances MDM2/MDMX interaction. Taken together, our study identifies novel mechanisms by which CARP proteins regulate the p53 signaling pathway.