Determination of expression profile and localization of ANKRD2 protein in human cardiac and skeletal muscle

Protein Ankrd2 pripada familiji mišićnih proteina sa ankirinskim ponovcima (eng. Muscle Ankyrin Repeat Proteins – MARP). U sarkomeri ima ulogu strukturnog elementa i signalnog molekula u procesu mehanotransdukcije. Pod dejstvom fiziĉkih i hemijskih stresora migrira u jedro, gde ostvaruje ulogu regulatora transkripcije. Ekspresija Ankrd2 je indukovana mehaniĉkim stresom i zavisna od stepena diferencijacije mišićne ćelije i faze razvića organizma. Osim u skeletnim mišićima, protein ANKRD2 je u manjoj meri eksprimiran i u srcu ĉoveka. U cilju otkrivanja novih funkcija koje ANKRD2 ima u ovom organu odreĊeni su njegov profil ekspresije i lokalizacija u srcu ĉoveka i uporeĊeni sa karakteristikama homolognog ANKRD1, koji je eksprimiran samo u srcu i detaljno okarakterisan. Utvrdili smo da je nivo ekspresije ANKRD2 dosta niţi u poreĊenju sa ANKRD1 i da je preteţno lokalizovan u sarkomeri kardiomiocita. Interkalirajući diskovi su identifikovani kao novo mesto lokalizacije oba proteina. S obzirom da su ĉesto i uspešno korišćen model sistem za prouĉavanje funkcije srĉano-specifiĉnih gena, kardiomiociti neonatalnih pacova (KMNP) su okarakterisani po pitanju ekspresije i lokalizacije endogenog Ankrd2 i egzogenog ANKRD2. Ankrd2 je lokalizovan i u jedru i u citoplazmi KMNP, a sliĉnu dualnu lokalizaciju je pokazivao i egzogeno eksprimirani ANKRD2. Za razliku od inhibitornog dejstva na ekspresiju Ankrd1, kardiotoksiĉni agens doksorubicin nije doveo do promene nivoa ekspresije proteina Ankrd2 u KMNP. U ovoj tezi su okarakterisane tri izoforme ANKRD2 od 446 (L-ANKRD2), 360 (M-ANKRD2) i 333 (S-ANKRD2) aminokiselina. Diskriminacija izoformi u popreĉno prugastim mišićima ĉoveka je postignuta korišćenjem specifiĉno dizajniranih antitela i graniĉnika. Utvrdili smo brojne sliĉnosti izmeĊu izoformi S- i M-ANKRD2, kao i znaĉajne razlike koje mogu da se odraze na njihove specifiĉne funkcije. Izoforma LANKRD2 nije detektovana ni u srcu, ni u skeletnim mišićima...Ankrd2 is a member MARP (Muscle Ankyrin Repeat Proteins) protein family. In sarcomere, it has a role of a structural element and signaling molecule in mechanotransduction. Ankrd2 is highly responsive to physical and chemical stress, which results in its translocation to the nucleus where it acts as a transcriptional regulator. Expression of Ankrd2 is upregulated in response to mechanical stresses such as exercise and stretch, as well as during muscle development and myoblast differentiation. Apart from skeletal muscles, ANKRD2 has been also detected in adult human heart, but at lower levels. In order to explore the cardiac-specific function of ANKRD2, we determined its expression and localization in human heart and compared them with the profiles of homologous cardiac protein ANKRD1. It was found that ANKRD2 is preferentially localized in sarcomere of cardiomyocytes and that level of its expression was lower when compared to ANKRD1. Intercalated disks were identified as a novel site of localization of both proteins. Neonatal rat cardiomyocytes (NRCM) are commonly used as a model system for functional analysis of cardiac-specific genes. One of the goals of this thesis was to characterize this model regarding expression and intracellular localization of endogenous Ankrd2 and exogenous ANKRD2. Nuclear and cytoplasmic localization of Ankrd2 was observed in NRCM, while similar dual localization was established for exogenous ANKRD2 as well. In the contrast to inhibitory effect on Ankrd1 expression, cardio-toxic drug doxorubicin did not change the protein level of Ankrd2 in NRCM. In this thesis we also characterized three ANKRD2 isoforms, containing 446 (LANKRD2), 360 (M-ANKRD2) i 333 (S-ANKRD2) amino acids. Their discrimination in human striated muscles was achieved by specifically designed antibodies and PCR primers. Various similarities between S- and M-ANKRD2 isoforms were discovered, as well as several significant differences, which could reflect on their specific functions..

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

Expression profiling of ANKRD1 in rhabdomyosarcoma cell lines

Introduction: Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in children and adolescents. Respecting the age of the patients and the tumor aggressiveness, investigation of the molecular mechanisms of RMS tumorigenesis is essential, most notably due to the possible identification of novel therapeutic targets. To contribute to a better understanding of the molecular pathology of RMS, we investigated ANKRD1 (ankyrin repeat domain 1) gene, considered a potential RMS diagnostic marker. The changes in its expression are related to carcinogenesis and resistance to chemotherapy in several types of tumors.EACR 2023: Innovative Cancer Science, 12-15 June 2023, Torino, Ital

Spatial profile of ankrd1a activation during regeneration of zebrafish heart

Introduction: In contrast to humans, zebrafish have a remarkable ability to regenerate injured heart through a complex and highly orchestrated processinvolving all cardiac structures. The majorsource of new myocardial cells are resident cardiomyocytes, which dedifferentiate and reinitiate proliferation, invading the area of injury to replace the lost myocardium. The response of the myocardium and coronary vasculature is preceded by activation of epi- and endocardium, which form active scaffolds to guide regeneration. The aim of thisstudy wasto identify cardiac structuresin which ankrd1a gene is activated during zebrafish heart regeneration. Methods: We crossed several zebrafish reporter lines: TgBAC(ankrd1a:EGFP) (to identify cells expressing ankrd1a), Tg(myl7:nls-dsRedExpress) (for labeling cardiomyocyte nuclei) and Tg(kdrl:RAS-mCherry) (for labeling endocardial/endothelial cells). Zebrafish hearts were cryoinjured and left to regenerate for 3 and 7 days. Dedifferentiating cardiomyocytes and epicardial cells were immunostained with anti-MYH7 and anti-caveolin1 antibody, respectively. Cells labeled with transgenes and immunostaining were visualized on tissue cryosections by fluorescent microscopy. Results: Zebrafish ankrd1a was activated in the injury border zone cardiomyocytes, located between the injured and remote myocardium. Its expression preceded that of a dedifferentiation marker, MYH7. The TgBAC(ankrd1a:EGFP) transgene was not detected in epicardial or endocardial cells of regenerating zebrafish heart. Conclusion: Activation of ankrd1a during regeneration of zebrafish heart is restricted to borderzone cardiomyocytes, implicating this gene in dedifferentiation and proliferation of cardiomyocytes. The absence of ankrd1a expression in epicardium and endocardium indicatesthat this gene does not contribute to the regeneration process occuring in these layers of the heart

Expression profile of ankrd1a during repair of injured zebrafish skeletal muscle

In our previous work, using transgenic zebrafish line TgBAC(ankrd1a:EGFP), we showed activation of the zebrafish ankrd1a gene in border zone cardiomyocytes of cryoinjured heart and in close proximity of needle-stab wounds in skeletal muscle, indicating its involvement in muscle regeneration. Our results implicated ankrd1a in zebrafish skeletal muscle tissue repair and remodeling, as a sensor of stressed muscle. Here we take a closer look at the spatio-temporal expression profile of the ankrd1a gene in injured zebrafish skeletal muscle by analyzing cryosections prepared from wounded tissue of TgBAC(ankrd1a:EGFP) adults at 1, 3, 5, 7 and 10 days post-injury (dpi). The expression of the fluorescent reporter was observed from 3 dpi and remained until 10 dpi. At 3dpi, new GFP-positive muscle cells emerged inside the injury zone, at the site of needle entry, while in the later days (5, 7 and 10 dpi), newly formed GFP-positive myofibers were visible in the deeper tissue layers within the injury, indicating active repair of the injured tissue. To identify cells in which ankrd1a is activated after injury, we stained the sections for markers of satellite-like cells, undifferentiated and differentiated muscle cells, and mature myofibers. Since the reporter was detected both in the newly formed myofibers that invade the wound and in the apparently uninjured tissue surrounding the injury, we hypothesize that ankrd1a is not only involved in satellite celldependent tissue repair, but its expression might be a hallmark of adaptive process in undamaged myofibers surrounding the physical injury.EZM2023 Abstract Book: 12th European Zebrafish Meeting July 9-13, 2023 Krakow, Polan

Violacein enhances the cytotoxic effect of commonly used chemotherapeutics on rhabdomyosarcoma cells

Investigati on of natural compounds showing specific toxicity to tumor cells aims to improve the efficacy of available therapies. Our previous research demonstrated the cytotoxic acti vity of the bacterial pigment violacein against rhabdomyosarcoma (RMS) cell lines. RMS is the most common soft tissue malignancy in children. In this study, we evaluated the cytotoxicity of violacein on RMS cells in combinati on with conventi onal chemotherapeutics doxorubicin, irinotecan, and vinflunine

Determination of muscle fiber types expressing ANKRD2

Introduction: Ankyrin Repeat Domain 2 (ANKRD2) is expressed in skeletal muscle, where plays a role inmuscle development, differentiation and adaptation to stress. Human skeletal muscle consists of threemajor fiber types: type 1 (slow-twitch, oxidative), type 2A (fast-twitch, oxidative) and type 2X (fast-twitch,glycolytic). ANKRD2 is reported to be primarily expressed in type 1 myofibers. However, recent findingson human single myofibers and our study of chicken muscles have shown that this protein may also beexpressed in type 2A fibers. Hence, our objective was to examine whether ANKRD2 is present in humanfast, type 2A muscle fibers using immunohistochemistry.Methods: Samples of large leg musclessoleus, gastrocnemius, vastusintermedius and vastuslateralis wereobtained from human cadaveric tissue. Serial cryosections were independently stained with anti-ANKRD2and antibodies for different myosin heavy chain isoforms (6H1 for type 2X, BF35 for type 1 and 2A, antiMHCs for type 1 and anti-MHCf for type 2A and 2X fibers). Immunostained tissues were analyzed by fluorescent microscopy.Results: In addition to slow, type 1, ANKRD2 wasfound expressed in fast, type 2A myofibers, which bothhave oxidative metabolism. Further, we did not observe ANDRD2 expression in glycolytic, type 2Xmyiofibers. This pattern of ANKRD2 expression was consistent across all examined muscles.Conclusion: Our resultsimplicate that the regulatory mechanism of ANKRD2 expression in human skeletal muscle is associated with oxidative metabolism, rather than muscle contraction speed

Determination of muscle fiber types expressing ANKRD2

Introduction: Ankyrin Repeat Domain 2 (ANKRD2) is expressed in skeletal muscle, where plays a role in muscle development, differentiation and adaptation to stress. Human skeletal muscle consists of three major fiber types: type 1 (slow-twitch, oxidative), type 2A (fast-twitch, oxidative) and type 2X (fast-twitch, glycolytic). ANKRD2 is reported to be primarily expressed in type 1 myofibers. However, recent findings on human single myofibers and our study of chicken muscles have shown that this protein may also be expressed in type 2A fibers. Hence, our objective was to examine whether ANKRD2 is present in human fast, type 2A muscle fibers using immunohistochemistry. Methods: Samples of large leg musclessoleus, gastrocnemius, vastusintermedius and vastuslateralis were obtained from human cadaveric tissue. Serial cryosections were independently stained with anti-ANKRD2 and antibodies for different myosin heavy chain isoforms (6H1 for type 2X, BF35 for type 1 and 2A, antiMHCs for type 1 and anti-MHCf for type 2A and 2X fibers). Immunostained tissues were analyzed by fluorescent microscopy. Results: In addition to slow, type 1, ANKRD2 wasfound expressed in fast, type 2A myofibers, which both have oxidative metabolism. Further, we did not observe ANDRD2 expression in glycolytic, type 2X myiofibers. This pattern of ANKRD2 expression was consistent across all examined muscles. Conclusion: Our resultsimplicate that the regulatory mechanism of ANKRD2 expression in human skeletal muscle is associated with oxidative metabolism, rather than muscle contraction speed

Zebrafish ankrd1a as a common player in heart regeneration and skeletal muscle repair

In contrast to humans, zebrafish have a remarkable ability to regenerate their hearts after injury, while both humans and zebrafish efficiently repair the wounded skeletal muscle. Common players in these two processes might represent potential targets for the development of efficient therapies to stimulate human heart to regenerate after injury. We identified ankrd1a expression to be upregulated in both regenerating zebrafish hearts and in repairing skeletal muscle. Its mammalian homolog ANKRD1/CARP encodes a stress responsive cardiac ankyrin repeat protein involved in transcriptional regulation, sarcomere assembly and mechanosensing. Using a TgBAC(ankrd1a:EGFP) line, we showed that activation of ankrd1a in cryoinjured heart is restricted to border zone cardiomyocytes, implicating this gene in dedifferentiation and proliferation of regenerating cardiomyocytes. After stab wound injury of skeletal muscle expression of the fluorescent reporter was observed from 3 dpi, when new EGFP-positive muscle cells emerged inside the injury zone. At later time points, EGFP-positive myofibers were visible in the deeper tissue layers, concomitant with active repair of the injured tissue. In cryoinjured skeletal muscle, strong activation of ankrd1a was also observed in myofibers adjacent to the injury, and in those on uninjured side. Detection of the transgene in both newly formed myofibers that invade the wound and in the apparently uninjured tissue surrounding the injury suggests the role of ankrd1a in skeletal muscle tissue repair and adaptive processes in uninjured myofibers surrounding the injury site. Our results implicate ankrd1a in zebrafish muscle regeneration, repair and remodeling, promoting it as an attractive target for translational studies, as a player in muscle healing and as a sensor of stressed muscle.10th Strategic Conference of Zebrafish Investigators, January 6-9, 2024 at the Asilomar Conference Grounds in Pacific Grove, Californi

Determination of expression profile and localization of ANKRD2 protein in human cardiac and skeletal muscle

Protein Ankrd2 pripada familiji mišićnih proteina sa ankirinskim ponovcima (eng. Muscle Ankyrin Repeat Proteins – MARP). U sarkomeri ima ulogu strukturnog elementa i signalnog molekula u procesu mehanotransdukcije. Pod dejstvom fiziĉkih i hemijskih stresora migrira u jedro, gde ostvaruje ulogu regulatora transkripcije. Ekspresija Ankrd2 je indukovana mehaniĉkim stresom i zavisna od stepena diferencijacije mišićne ćelije i faze razvića organizma. Osim u skeletnim mišićima, protein ANKRD2 je u manjoj meri eksprimiran i u srcu ĉoveka. U cilju otkrivanja novih funkcija koje ANKRD2 ima u ovom organu odreĊeni su njegov profil ekspresije i lokalizacija u srcu ĉoveka i uporeĊeni sa karakteristikama homolognog ANKRD1, koji je eksprimiran samo u srcu i detaljno okarakterisan. Utvrdili smo da je nivo ekspresije ANKRD2 dosta niţi u poreĊenju sa ANKRD1 i da je preteţno lokalizovan u sarkomeri kardiomiocita. Interkalirajući diskovi su identifikovani kao novo mesto lokalizacije oba proteina. S obzirom da su ĉesto i uspešno korišćen model sistem za prouĉavanje funkcije srĉano-specifiĉnih gena, kardiomiociti neonatalnih pacova (KMNP) su okarakterisani po pitanju ekspresije i lokalizacije endogenog Ankrd2 i egzogenog ANKRD2. Ankrd2 je lokalizovan i u jedru i u citoplazmi KMNP, a sliĉnu dualnu lokalizaciju je pokazivao i egzogeno eksprimirani ANKRD2. Za razliku od inhibitornog dejstva na ekspresiju Ankrd1, kardiotoksiĉni agens doksorubicin nije doveo do promene nivoa ekspresije proteina Ankrd2 u KMNP. U ovoj tezi su okarakterisane tri izoforme ANKRD2 od 446 (L-ANKRD2), 360 (M-ANKRD2) i 333 (S-ANKRD2) aminokiselina. Diskriminacija izoformi u popreĉno prugastim mišićima ĉoveka je postignuta korišćenjem specifiĉno dizajniranih antitela i graniĉnika. Utvrdili smo brojne sliĉnosti izmeĊu izoformi S- i M-ANKRD2, kao i znaĉajne razlike koje mogu da se odraze na njihove specifiĉne funkcije. Izoforma LANKRD2 nije detektovana ni u srcu, ni u skeletnim mišićima...Ankrd2 is a member MARP (Muscle Ankyrin Repeat Proteins) protein family. In sarcomere, it has a role of a structural element and signaling molecule in mechanotransduction. Ankrd2 is highly responsive to physical and chemical stress, which results in its translocation to the nucleus where it acts as a transcriptional regulator. Expression of Ankrd2 is upregulated in response to mechanical stresses such as exercise and stretch, as well as during muscle development and myoblast differentiation. Apart from skeletal muscles, ANKRD2 has been also detected in adult human heart, but at lower levels. In order to explore the cardiac-specific function of ANKRD2, we determined its expression and localization in human heart and compared them with the profiles of homologous cardiac protein ANKRD1. It was found that ANKRD2 is preferentially localized in sarcomere of cardiomyocytes and that level of its expression was lower when compared to ANKRD1. Intercalated disks were identified as a novel site of localization of both proteins. Neonatal rat cardiomyocytes (NRCM) are commonly used as a model system for functional analysis of cardiac-specific genes. One of the goals of this thesis was to characterize this model regarding expression and intracellular localization of endogenous Ankrd2 and exogenous ANKRD2. Nuclear and cytoplasmic localization of Ankrd2 was observed in NRCM, while similar dual localization was established for exogenous ANKRD2 as well. In the contrast to inhibitory effect on Ankrd1 expression, cardio-toxic drug doxorubicin did not change the protein level of Ankrd2 in NRCM. In this thesis we also characterized three ANKRD2 isoforms, containing 446 (LANKRD2), 360 (M-ANKRD2) i 333 (S-ANKRD2) amino acids. Their discrimination in human striated muscles was achieved by specifically designed antibodies and PCR primers. Various similarities between S- and M-ANKRD2 isoforms were discovered, as well as several significant differences, which could reflect on their specific functions..