Muscle Ankyrin Repeat Proteins Research Articles

Rbm20ΔRRM Mice, Expressing a Titin Isoform with Lower Stiffness, Are Protected from Mechanical Ventilation-Induced Diaphragm Weakness.

Diaphragm weakness frequently develops in mechanically ventilated critically ill patients and is associated with increased morbidity, including ventilator weaning failure, mortality, and health care costs. The mechanisms underlying diaphragm weakness are incompletely understood but may include the elastic properties of titin, a giant protein whose layout in the muscle's sarcomeres makes it an ideal candidate to sense ventilation-induced diaphragm unloading, resulting in downstream signaling through titin-binding proteins. In the current study, we investigated whether modulating titin stiffness affects the development of diaphragm weakness during mechanical ventilation. To this end, we ventilated genetically engineered mice with reduced titin stiffness (Rbm20ΔRRM), and robust (TtnΔIAjxn) or severely (TtnΔ112-158) increased titin stiffness for 8 h, and assessed diaphragm contractility and protein expression of titin-binding proteins. Mechanical ventilation reduced the maximum active tension of the diaphragm in WT, TtnΔIAjxn and TtnΔ112-158 mice. However, in Rbm20ΔRRM mice maximum active tension was preserved after ventilation. Analyses of titin binding proteins suggest that muscle ankyrin repeat proteins (MARPs) 1 and 2 may play a role in the adaptation of the diaphragm to mechanical ventilation, and the preservation of diaphragm contractility in Rbm20ΔRRM mice. Thus, Rbm20ΔRRM mice, expressing titin isoforms with lower stiffness, are protected from mechanical ventilation-induced diaphragm weakness, suggesting that titin elasticity may modulate the diaphragm's response to unloading during mechanical ventilation.

Muscle ankyrin repeat protein 1 (MARP1) locks titin to the sarcomeric thin filament and is a passive force regulator.

Muscle ankyrin repeat protein 1 (MARP1) is frequently up-regulated in stressed muscle, but its effect on skeletal muscle function is poorly understood. Here, we focused on its interaction with the titin-N2A element, found in titin's molecular spring region. We show that MARP1 binds to F-actin, and that this interaction is stronger when MARP1 forms a complex with titin-N2A. Mechanics and super-resolution microscopy revealed that MARP1 "locks" titin-N2A to the sarcomeric thin filament, causing increased extension of titin's elastic PEVK element and, importantly, increased passive force. In support of this mechanism, removal of thin filaments abolished the effect of MARP1 on passive force. The clinical relevance of this mechanism was established in diaphragm myofibers of mechanically ventilated rats and of critically ill patients. Thus, MARP1 regulates passive force by locking titin to the thin filament. We propose that in stressed muscle, this mechanism protects the sarcomere from mechanical damage.

Expression of titin-linked putative mechanosensing proteins in skeletal muscle after power resistance exercise in resistance-trained men.

Little is known about the molecular responses to power resistance exercise that lead to skeletal muscle remodeling and enhanced athletic performance. We assessed the expression of titin-linked putative mechanosensing proteins implicated in muscle remodeling: muscle ankyrin repeat proteins (Ankrd 1, Ankrd 2, and Ankrd 23), muscle-LIM proteins (MLPs), muscle RING-finger protein-1 (MuRF-1), and associated myogenic proteins (MyoD1, myogenin, and myostatin) in skeletal muscle in response to power resistance exercise with or without a postexercise meal, in fed, resistance-trained men. A muscle sample was obtained from the vastus lateralis of seven healthy men on separate days, 3 h after 90 min of rest (Rest) or power resistance exercise with (Ex + Meal) or without (Ex) a postexercise meal to quantify mRNA and protein levels. The levels of phosphorylated HSP27 (pHSP27-Ser15) and cytoskeletal proteins in muscle and creatine kinase activity in serum were also assessed. The exercise increased (P ≤ 0.05) pHSP27-Ser15 (∼6-fold) and creatine kinase (∼50%), whereas cytoskeletal protein levels were unchanged (P > 0.05). Ankrd 1 (∼15-fold) and MLP (∼2-fold) mRNA increased, whereas Ankrd 2, Ankrd 23, MuRF-1, MyoD1, and myostatin mRNA were unchanged. Ankrd 1 (∼3-fold, Ex) and MLPb (∼20-fold, Ex + Meal) protein increased, but MLPa, Ankrd 2, Ankrd 23, and the myogenic proteins were unchanged. The postexercise meal did not affect the responses observed. Power resistance exercise, as performed in practice, induced subtle early responses in the expression of MLP and Ankrd 1 yet had little effect on the other proteins investigated. These findings suggest possible roles for MLP and Ankrd 1 in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise.NEW & NOTEWORTHY This is the first study to assess the early changes in the expression of titin-linked putative mechanosensing proteins and associated myogenic regulatory factors in skeletal muscle after power resistance exercise in fed, resistance-trained men. We report that power resistance exercise induces subtle early responses in the expression of Ankrd 1 and MLP, suggesting these proteins play a role in the remodeling of skeletal muscle in individuals who regularly perform this type of exercise.

Effect of a 12-week endurance training program on force transfer and membrane integrity proteins in lean, obese, and type 2 diabetic subjects.

The mechanisms accounting for the loss of muscle function with obesity and type 2 diabetes are likely the result of a combination of neural and muscular factors. One muscular factor that is important, yet has received little attention, is the protein machinery involved in longitudinal and lateral force transmission. The purpose of this study was to compare the levels of force transfer and membrane integrity proteins before and after a 12‐week endurance training program in lean, obese, and obese type 2 diabetic adults. Nineteen sedentary subjects (male = 8 and female = 11) were divided into three groups: Lean (n = 7; 50.3 ± 4.1 y; 69.1 ± 7.2 kg); Obese (n = 6; 49.8 ± 4.1 y; 92.9 ± 19.5 kg); and Obese with type 2 diabetes (n = 6; 51.5 ± 7.9 years; 88.9 ± 15.1 kg). Participants trained 150 min/week between 55% and 75% of VO2max for 12 weeks. Skeletal muscle biopsies were taken before and after the training intervention. Baseline dystrophin and muscle LIM protein levels were higher (~50% p < .01) in lean compared to obese and type 2 diabetic adults, while the protein levels of the remaining force transfer and membrane integrity proteins were similar between groups. After training, obese individuals decreased (−53%; p < .01) the levels of the muscle ankyrin repeat protein and lean individuals decreased dystrophin levels (−45%; p = .01), while the levels of the remaining force transfer and membrane integrity proteins were not affected by training. These results suggest that there are modest changes to force transfer and membrane integrity proteins in middle‐aged individuals as a result of 12 weeks of lifestyle and training interventions.

Ankrd2 in Mechanotransduction and Oxidative Stress Response in Skeletal Muscle: New Cues for the Pathogenesis of Muscular Laminopathies.

Ankrd2 (ankyrin repeats containing domain 2) or Arpp (ankyrin repeat, PEST sequence, and proline-rich region) is a member of the muscle ankyrin repeat protein family. Ankrd2 is mostly expressed in skeletal muscle, where it plays an intriguing role in the transcriptional response to stress induced by mechanical stimulation as well as by cellular reactive oxygen species. Our studies in myoblasts from Emery-Dreifuss muscular dystrophy 2, a LMNA-linked disease affecting skeletal and cardiac muscles, demonstrated that Ankrd2 is a lamin A-binding protein and that mutated lamins found in Emery-Dreifuss muscular dystrophy change the dynamics of Ankrd2 nuclear import, thus affecting oxidative stress response. In this review, besides describing the latest advances related to Ankrd2 studies, including novel discoveries on Ankrd2 isoform-specific functions, we report the main findings on the relationship of Ankrd2 with A-type lamins and discuss known and potential mechanisms involving defective Ankrd2-lamin A interplay in the pathogenesis of muscular laminopathies.

PL - 041 Effects of power resistance exercise and feeding on the expression of putative mechanosensing proteins in skeletal muscle of resistance-trained men

Objective Power resistance exercise involves high intensity (load and velocity) dynamic muscular contractions and is frequently performed by athletes to enhance performance via improved muscle function. To investigate the remodelling processes that contribute to improved muscle function, we investigated the expression of putative mechanosensing genes implicated in this process (Kojic et al., 2011): titin-linked Muscle Ankyrin Repeat Protein (MARPs) family CARP, Ankrd 2 and DARP, and the Z-disc associated muscle-LIM protein (MLP) in healthy, resistance-trained men (n = 7) following 90 min of rest (Rest) or power resistance exercise, with (Ex + Meal) or without (Ex only) feeding during recovery.&#x0D; Methods Percutaneous needle biopsy samples were obtained from the vastus lateralis of resistance-trained males using local anesthetic (2% Xylocaine), 3 h after performing each of the three experimental trials on separate days.&#x0D; Previously, we presented results from this study showing that the mRNA levels of CARP (~15-fold) and MLP (~2.5-fold) were upregulated in human skeletal muscle 3 h post power resistance exercise (Wette et al., 2012). Based on these results, we performed protein analyses on the same muscle samples to determine the protein levels of all MARPs and MLP in whole muscle homogenates after Rest, Ex only and Ex + Meal. To assess whether the exercise elicited a stress response in these resistance-trained individuals, the level of phosphorylated heat shock protein 27 at serine 15 (pHSP27-Ser15) was measured at Rest and 3 h after Ex only and Ex + Meal. The levels of pHSP27-Ser15 are typically upregulated 3 h after eccentric exercise in human skeletal muscle (Frankenberg et al., 2014).&#x0D; Results The 90 min exercise session consisted of 180 intermittent muscular contractions at high intensity (70-96% maximal strength). Compared to Rest, there were ~5.8- and 12.6-fold increases in pHSP27-Ser15 levels at 3 h post Ex only and Ex + Meal (both P = 0.049, one-way ANOVA) respectively. CARP protein levels were elevated ~2.7-fold after Ex only (P = 0.049, one-way ANOVA) and ~7.6-fold after Ex + Meal (P = 0.326), due to markedly higher levels (6-40-fold) in three of the seven participants. Pearson correlation analysis revealed a significant positive correlation between the levels of pHSP27-Ser-15 and CARP protein (r = 0.56, P = 0.008). Ankrd 2, DARP and MLP protein levels were unchanged (all P &gt; 0.05) following Ex only and Ex + Meal.&#x0D; Conclusions These findings indicate that CARP is highly responsive to increased mechanical loading because the protein levels in skeletal muscle can be substantially increased as early as 3 h after stressful resistance exercise. This suggests a specialised role for CARP protein during the early phases of muscle remodelling that occur as a consequence of performing high intensity resistance exercise.

Characterization of zebrafish (Danio rerio) muscle ankyrin repeat proteins reveals their conserved response to endurance exercise.

Muscle proteins with ankyrin repeats (MARPs) ANKRD1 and ANKRD2 are titin-associated proteins with a putative role as transcriptional co-regulators in striated muscle, involved in the cellular response to mechanical, oxidative and metabolic stress. Since many aspects of the biology of MARPs, particularly exact mechanisms of their action, in striated muscle are still elusive, research in this field will benefit from novel animal model system. Here we investigated the MARPs found in zebrafish for protein structure, evolutionary conservation, spatiotemporal expression profiles and response to increased muscle activity. Ankrd1 and Ankrd2 show overall moderate conservation at the protein level, more pronounced in the region of ankyrin repeats, motifs indispensable for their function. The two zebrafish genes, ankrd1a and ankrd1b, counterparts of mammalian ANKRD1/Ankrd1, have different expression profiles during first seven days of development. Mild increase of ankrd1a transcript levels was detected at 72 hpf (1.74±0.24 fold increase relative to 24 hpf time point), while ankrd1b expression was markedly upregulated from 24 hpf onward and peaked at 72 hpf (92.18±36.95 fold increase relative to 24 hpf time point). Spatially, they exhibited non-overlapping expression patterns during skeletal muscle development in trunk (ankrd1a) and tail (ankrd1b) somites. Expression of ankrd2 was barely detectable. Zebrafish MARPs, expressed at a relatively low level in adult striated muscle, were found to be responsive to endurance exercise training consisting of two bouts of 3 hours of forced swimming daily, for five consecutive days. Three hours after the last exercise bout, ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change), while ankrd1b and ankrd2 were upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively). This study provides the foundation to establish zebrafish as a novel in vivo model for further investigation of MARPs function in striated muscle.

Reply to “Letter to the editor: Comments on Wette et al. (2017): ‘Characterization of muscle ankyrin repeat proteins in human skeletal muscle’”

we appreciate the interest in our recently published study ([6][1]) shown by the Letter to the editor by Dr. Cenni ([2][2]), but we are compelled to point out that the main criticisms made are entirely misplaced and that the Letter includes some major misstatements about findings in the literature

Ankyrin Repeat Domain 1 Protein: A Functionally Pleiotropic Protein with Cardiac Biomarker Potential.

The ankyrin repeat domain 1 (ANKRD1) protein is a cardiac-specific stress-response protein that is part of the muscle ankyrin repeat protein family. ANKRD1 is functionally pleiotropic, playing pivotal roles in transcriptional regulation, sarcomere assembly and mechano-sensing in the heart. Importantly, cardiac ANKRD1 has been shown to be highly induced in various cardiomyopathies and in heart failure, although it is still unclear what impact this may have on the pathophysiology of heart failure. This review aims at highlighting the known properties, functions and regulation of ANKRD1, with focus on the underlying mechanisms that may be involved. The current views on the actions of ANKRD1 in cardiovascular disease and its utility as a candidate cardiac biomarker with diagnostic and/or prognostic potential are also discussed. More studies of ANKRD1 are warranted to obtain deeper functional insights into this molecule to allow assessment of its potential clinical applications as a diagnostic or prognostic marker and/or as a possible therapeutic target.

Characterization of muscle ankyrin repeat proteins in human skeletal muscle.

Muscle ankyrin repeat proteins (MARPs) are a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. In cardiac muscle, cardiac ankyrin repeat protein (CARP) and diabetes-related ankyrin repeat protein (DARP) reportedly redistribute from binding sites on titin to the nucleus following a prolonged stretch. However, it is unclear whether ankyrin repeat domain protein 2 (Ankrd 2) shows comparable stretch-induced redistribution to the nucleus. We measured the following in rested human skeletal muscle: 1) the absolute amount of MARPs and 2) the distribution of Ankrd 2 and DARP in both single fibers and whole muscle preparations. In absolute amounts, Ankrd 2 is the most abundant MARP in human skeletal muscle, there being ~3.1 µmol/kg, much greater than DARP and CARP (~0.11 and ~0.02 µmol/kg, respectively). All DARP was found to be tightly bound at cytoskeletal (or possibly nuclear) sites. In contrast, ~70% of the total Ankrd 2 is freely diffusible in the cytosol [including virtually all of the phosphorylated (p)Ankrd 2-Ser99 form], ~15% is bound to non-nuclear membranes, and ~15% is bound at cytoskeletal sites, likely at the N2A region of titin. These data are not consistent with the proposal that Ankrd 2, per se, or pAnkrd 2-Ser99 mediates stretch-induced signaling in skeletal muscle, dissociating from titin and translocating to the nucleus, because the majority of these forms of Ankrd 2 are already free in the cytosol. It will be necessary to show that the titin-associated Ankrd 2 is modified by stretch in some as-yet-unidentified way, distinct from the diffusible pool, if it is to act as a stretch-sensitive signaling molecule.

Titin, a Central Mediator for Hypertrophic Signaling, Exercise-Induced Mechanosignaling and Skeletal Muscle Remodeling.

Titin is a giant scaffold protein with multiple functions in striated muscle physiology. Due to the elastic I-band domains and the filament-like integration in the half-sarcomere titin is an important factor for sarcomere assembly and serves as an adaptable molecular spring that determines myofilament distensibility. Protein-interactions e.g., with muscle ankyrin repeat proteins or muscle LIM-protein link titin to hypertrophic signaling and via p62 and Muscle Ring Finger proteins to mechanisms that control protein quality control. This review summarizes our current knowledge on titin as a central node for exercise-induced mechanosignaling and remodeling and further highlights the pathophysiological implications.

Focused Proteomics Revealed a Novel Rho-kinase Signaling Pathway in the Heart.

Protein phosphorylation plays an important role in the physiological regulation of cardiac function. Myocardial contraction and pathogenesis of cardiac diseases have been reported to be associated with adaptive or maladaptive protein phosphorylation; however, phosphorylation signaling in the heart is not fully elucidated. We recently developed a novel kinase-interacting substrate screening (KISS) method for exhaustive screening of protein kinase substrates, using mass spectrometry and affinity chromatography. First, we examined protein phosphorylation by extracellular signal-regulated kinase (ERK) and protein kinase A (PKA), which has been relatively well studied in cardiomyocytes. The KISS method showed that ERK and PKA mediated the phosphorylation of known cardiac-substrates of each kinase such as Rps6ka1 and cTnI, respectively. Using this method, we found about 330 proteins as Rho-kinase-mediated substrates, whose substrate in cardiomyocytes is unknown. Among them, CARP/Ankrd1, a muscle ankyrin repeat protein, was confirmed as a novel Rho-kinase-mediated substrate. We also found that non-phosphorylatable form of CARP repressed cardiac hypertrophy-related gene Myosin light chain-2v (MLC-2v) promoter activity, and decreased cell size of heart derived H9c2 myoblasts more efficiently than wild type-CARP. Thus, focused proteomics enable us to reveal a novel signaling pathway in the heart.

Diabetes-Related Ankyrin Repeat Protein (DARP/Ankrd23) Modifies Glucose Homeostasis by Modulating AMPK Activity in Skeletal Muscle.

Skeletal muscle is the major site for glucose disposal, the impairment of which closely associates with the glucose intolerance in diabetic patients. Diabetes-related ankyrin repeat protein (DARP/Ankrd23) is a member of muscle ankyrin repeat proteins, whose expression is enhanced in the skeletal muscle under diabetic conditions; however, its role in energy metabolism remains poorly understood. Here we report a novel role of DARP in the regulation of glucose homeostasis through modulating AMP-activated protein kinase (AMPK) activity. DARP is highly preferentially expressed in skeletal muscle, and its expression was substantially upregulated during myotube differentiation of C2C12 myoblasts. Interestingly, DARP-/- mice demonstrated better glucose tolerance despite similar body weight, while their insulin sensitivity did not differ from that in wildtype mice. We found that phosphorylation of AMPK, which mediates insulin-independent glucose uptake, in skeletal muscle was significantly enhanced in DARP-/- mice compared to that in wildtype mice. Gene silencing of DARP in C2C12 myotubes enhanced AMPK phosphorylation, whereas overexpression of DARP in C2C12 myoblasts reduced it. Moreover, DARP-silencing increased glucose uptake and oxidation in myotubes, which was abrogated by the treatment with AICAR, an AMPK activator. Of note, improved glucose tolerance in DARP-/- mice was abolished when mice were treated with AICAR. Mechanistically, gene silencing of DARP enhanced protein expression of LKB1 that is a major upstream kinase for AMPK in myotubes in vitro and the skeletal muscle in vivo. Together with the altered expression under diabetic conditions, our data strongly suggest that DARP plays an important role in the regulation of glucose homeostasis under physiological and pathological conditions, and thus DARP is a new therapeutic target for the treatment of diabetes mellitus.

ANKRD1 modulates inflammatory responses in C2C12 myoblasts through feedback inhibition of NF-κB signaling activity

Transcription factors of the nuclear factor-kappa B (NF-κB) family play a pivotal role in inflammation, immunity and cell survival responses. Recent studies revealed that NF-κB also regulates the processes of muscle atrophy. NF-κB activity is regulated by various factors, including ankyrin repeat domain 2 (AnkrD2), which belongs to the muscle ankyrin repeat protein family. Another member of this family, AnkrD1 is also a transcriptional effector. The expression levels of AnkrD1 are highly upregulated in denervated skeletal muscle, suggesting an involvement of AnkrD1 in NF-κB mediated cellular responses to paralysis. However, the molecular mechanism underlying the interactive role of AnkrD1 in NF-κB mediated cellular responses is not well understood. In the current study, we examined the effect of AnkrD1 on NF-κB activity and determined the interactions between AnkrD1 expression and NF-κB signaling induced by TNFα in differentiating C2C12 myoblasts. TNFα upregulated AnkrD1 mRNA and protein levels. AnkrD1-siRNA significantly increased TNFα-induced transcriptional activation of NF-κB, whereas overexpression of AnkrD1 inhibited TNFα-induced NF-κB activity. Co-immunoprecipitation studies demonstrated that AnkrD1 was able to bind p50 subunit of NF-κB and vice versa. Finally, CHIP assays revealed that AnkrD1 bound chromatin at a NF-κB binding site in the AnrkD2 promoter and required NF-κB to do so. These results provide evidence of signaling integration between AnkrD1 and NF-κB pathways, and suggest a novel anti-inflammatory role of AnkrD1 through feedback inhibition of NF-κB transcriptional activity by which AnkrD1 modulates the balance between physiological and pathological inflammatory responses in skeletal muscle.

Probing muscle ankyrin-repeat protein (MARP) structure and function.

Muscle ankyrin-repeat proteins (MARPs) have been shown to serve diverse functions within cardiac and skeletal muscle cells. Apart from their interactions with sarcomeric proteins like titin or myopalladin that locate them along myofilaments, MARPs are able to shuttle to the nucleus where they act as modulators for a variety of transcription factors. The deregulation of MARPs in many cardiac and skeletal myopathies contributes to their use as biomarkers for these diseases. Many of their functions are attributed to their domain composition. MARPs consist of an N-terminal coiled-coil domain responsible for their dimerization. The C-terminus contains a series of ankyrin repeats, whose best-characterized function is to bind to the N2A region of the giant sarcomeric protein titin. Here we investigate the nature of their dimerization and their interaction with titin more closely. We demonstrate that the coiled-coil domain in all MARPs enables their homo- and hetero-dimerization in antiparallel fashion. Protein complementation experiments indicate further antiparallel binding of the ankyrin repeats to titin's N2A region. Binding of MARP to titin also affects its PKA mediated phosphorylation. We demonstrate further that MARPs themselves are phosphorylated by PKA and PKC, potentially altering their structure or function. These studies elucidate structural relationships within the stretch-responsive MARP/titin complex in cross-striated muscle cells, and may relate to disease relevant posttranslational modifications of MARPs and titin that alter muscle compliance.

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.

Ankrd1-mediated signaling is supported by its interaction with zonula occludens-1

The muscle ankyrin repeat protein Ankrd1 is localized in a mechanosensory complex of the sarcomeric I-band. It is involved in signaling pathways activated in response to mechanical stretch. It also acts as a transcriptional cofactor in the nucleus, playing an important role in cardiogenesis and skeletal muscle differentiation. To investigate its regulatory function in signaling we employed protein array methodology and identified 10 novel Ankrd1 binding partners among PDZ domain proteins known to act as platforms for multiprotein complex assembly. The zonula occludens protein-1 (ZO-1) was chosen for further analysis since its interaction with Ankrd2 had already been demonstrated. Both Ankrd2 and Ankrd1 have similar functions and localize in the same regions. We confirmed the interaction of Ankrd1 with ZO-1 protein and determined their subcellular distribution in HeLa cells, showing their colocalization in the cytoplasm. Our findings corroborate the role of Ankrd1 in intracellular signaling.

Molecular Characterization and Different Expression Patterns of the Muscle Ankyrin Repeat Protein (MARP) Family During Porcine Skeletal Muscle Development in vitro and in vivo

CARP, ANKRD2, and DARP belong to the ankyrin repeat protein (MARP) family and play a critical role in the integration of cytoskeletal architecture, stress response, and transcriptional regulation. In this study, we cloned the cDNA and promoter sequences of porcine ankyrin repeat protein (MARP) gene family. RT-PCR analysis revealed that porcine CARP gene was predominantly expressed in heart. ANKRD2 was widely expressed in many tissues, a high expression level was observed in the skeletal muscle and heart. DARP gene was expressed specifically in skeletal muscle and heart. Moreover, the expression of CARP and ANKRD2 was significantly different in porcine skeletal muscle among different developmental stages and between the two breeds. Expression analysis in porcine satellite cells showed that CARP and ANKRD2 were induced in differentiated porcine satellite cells, suggesting a role of them in myogenic differentiation. This result suggests that the MARP gene family may be important genes for skeletal muscle growth and provides useful information for further studies on their roles in porcine skeletal muscle.

The expression of Muscle ankyrin repeat proteins in brown adipose tissue

MARP family members CARP, Ankrd2 and DARP are expressed in the striated muscle, while DARP protein is also detected in brown adipose tissue (BAT). Taking into account recent findings concerning the common origin of muscle and brown fat, expression of CARP and Ankrd2 in mouse BAT was investigated. We demonstrated Ankrd2 expression in both inactive and thermogenically active BAT, while CARP expression was not detected. Our findings suggest that the expression of Ankrd2 in BAT could be a part of the ?myogenic transcriptional signature?, further supporting the evidence that muscle and brown adipose cells arise from the same myoblastic precursor.

A novel role for cardiac ankyrin repeat protein Ankrd1/CARP as a co-activator of the p53 tumor suppressor protein

The muscle ankyrin repeat protein (MARP) family member Ankrd1/CARP is a part of the titin-mechanosensory signaling complex in the sarcomere and in response to stretch it translocates to the nucleus where it participates in the regulation of cardiac genes as a transcriptional co-repressor. Several studies have focused on its structural role in muscle, but its regulatory role is still poorly understood. To gain more insight into the regulatory function of Ankrd1/CARP we searched for transcription factors that could interact and modulate its activity. Using protein array methodology we identified the tumor suppressor protein p53 as an Ankrd1/CARP interacting partner and confirmed their interaction both in vivo and in vitro. We demonstrate a novel role for Ankrd1/CARP as a transcriptional co-activator, moderately up regulating p53 activity. Furthermore, we show that p53 operates as an upstream effector of Ankrd1/CARP, by up regulating the proximal ANKRD1 promoter. Our findings suggest that, besides acting as a transcriptional co-repressor, Ankrd1/CARP could have a stimulatory effect on gene expression in cultured skeletal muscle cells. It is probable that Ankrd1/CARP has a role in the propagation of signals initiated by myogenic regulatory factors (MRFs) during myogenesis.