Carol C Gregorio
Co-Director, Sarver Heart Center
Department Head, Cellular and Molecular Medicine
Director, Molecular Cardiovascular Research Program
Professor, BIO5 Institute
Professor, Cellular and Molecular Medicine
Professor, Molecular and Cellular Biology
Primary Department
(520) 626-8113
Work Summary
The research in my laboratory is focused on identifying the components and molecular mechanisms regulating actin architecture in cardiac and skeletal muscle during normal development and disease. Control of actin filament lengths and dynamics is important for cell motility and architecture and is regulated in part by capping proteins that block elongation and depolymerization at both the fast-growing (barbed) and slow-growing (pointed) ends of the filaments.
Research Interest
Carol Gregorio, PhD, performs research in her lab that is focused on identifying the components and molecular mechanisms regulating actin architecture in cardiac and skeletal muscle during normal development and disease. Control of actin filament lengths and dynamics is important for cell motility and architecture and is regulated in part by capping proteins that block elongation and depolymerization at both the fast-growing (barbed) and slow-growing (pointed) ends of the filaments. Striated muscle is an ideal model system to test for the functional properties of various actin regulatory proteins due to the precise organization and polarity of cytoskeletal components within repeating sarcomeric units (for example, the ~1 mm long actin filaments are easily resolved by light microscopy). Using this system, she can combine advanced cell biological and biochemical approaches with direct tests of physiological function in live beating muscle cells.The research objectives of the laboratory can be broadly summarized as follows: 1) understanding the cellular mechanisms involved in the assembly, regulation and maintenance of contractile proteins in cardiac muscle in health and disease; 2) deciphering the mechanisms critical for precisely specifying and maintaining the lengths of actin filaments; 3) discovery of novel models of de novo cardiac muscle assembly, with special emphasis on differentiating murine embryonic stem (ES) cells to study all stages of heart muscle development. Actin is an indispensable structural element of cells and is the major component of heart muscle. Changes in actin, caused by genetic mutations, which have been identified in humans, are a frequent cause of several forms of cardiomyopathy. Her lab is determining how genetic defects in this protein affect muscle force generation and muscle contraction, leading to sudden cardiac death.


Kazmierski, S. T., Antin, P. B., Witt, C. C., Huebner, N., McElhinny, A. S., Labeit, S., & Gregorio, C. C. (2003). The complete mouse nebulin gene sequence and the identification of cardiac nebulin. Journal of Molecular Biology, 328(4), 835-846.
BIO5 Collaborators
Parker B Antin, Carol C Gregorio

PMID: 12729758;Abstract:

Nebulin is a giant (Mr 750-850kDa), modular sarcomeric protein proposed to regulate the assembly, and to specify the precise lengths of actin (thin) filaments in vertebrate skeletal muscles. Nebulin's potential role as a molecular template is based on its structural and biochemical properties. Its central ∼700kDa portion associates with actin along the entire length of the thin filament, its N-terminal region extends to thin filament pointed ends, and ∼80kDa of its C-terminal region integrates within the Z-line lattice. Here, we determined the exon/intron organization of the entire mouse nebulin gene, which contains 165 exons in a 202kb segment. We identified 16 novel exons, 15 of which encode nebulin-repeat motifs (12 from its central region and 3 from its Z-line region). One novel exon shares high sequence homology to the 20 residue repeats of the tight-junction protein, ZO-1. RT-PCR analyses revealed that all 16 novel exons are expressed in mouse skeletal muscle. Surprisingly, we also amplified mRNA transcripts from mouse and human heart cDNA using primers designed along the entire length of nebulin. The expression of cardiac-specific nebulin transcripts was confirmed by in situ hybridization in fetal rat cardiomyocytes and in embryonic Xenopus laevis (frog) heart. On the protein level, antibodies specific for skeletal muscle nebulin's N and C-terminal regions stained isolated rat cardiac myofibrils at the pointed and barbed ends of thin filaments, respectively. These data indicate a conserved molecular layout of the nebulin filament systems in both cardiac and skeletal myofibrils. We propose that thin filament length regulation in cardiac and skeletal muscles may share conserved nebulin-based mechanisms, and that nebulin isoform diversity may contribute to thin filament length differences in cardiac and skeletal muscle. © 2003 Elsevier Science Ltd. All rights reserved.

Ono, Y., Schwach, C., Antin, P. B., & Gregorio, C. C. (2005). Disruption in the tropomodulin1 (Tmod1) gene compromises cardiomyocyte development in murine embryonic stem cells by arresting myofibril maturation. Developmental Biology, 282(2), 336-348.
BIO5 Collaborators
Parker B Antin, Carol C Gregorio

PMID: 15950601;Abstract:

Tropomodulins (Tmods) comprise a family of capping proteins for actin filament pointed ends. To decipher the significance of Tmod1 functions during de novo myofibrillogenesis, we generated Tmod1 null embryonic stem (ES) cells and studied their differentiation into cardiomyocytes. Strikingly, in vitro cardiomyocyte differentiation of wild type (WT) ES cells faithfully recapitulates in vivo cardiomyocyte differentiation, allowing us to evaluate the phenotypes of Tmod1 knockout (KO) myofibrils irrespective of embryonic lethality of Tmod1 KO mice. Immunofluorescence and electron microscopy studies revealed that Tmod1 null cardiac myocytes were round, morphologically immature, and contained underdeveloped myofibrils that were shorter, narrower, and had fewer thin filaments than those in WT cells. Unexpectedly, clear gaps in the staining pattern for F-actin at the H-zone were detected in most KO cells, indicating the presence of filaments at uniform lengths. This indicates that additional mechanisms other than capping proteins are responsible for thin filament length maintenance in cardiac myocytes. Also unexpectedly, ∼40% of the KO cardiac myocytes exhibited contractile activity. Our data indicate that differentiating ES cells are a powerful system to investigate the functional properties of contractile proteins and that Tmod1 functions are critical for late stages of myofibrillogenesis, and for the maturation of myofibrils. © 2005 Elsevier Inc. All rights reserved.

Rudy, D. E., Yatskievych, T. A., Antin, P. B., & Gregorio, C. C. (2001). Assembly of thick, thin, and titin filaments in chick precardiac explants. Developmental Dynamics, 221(1), 61-71.
BIO5 Collaborators
Parker B Antin, Carol C Gregorio

PMID: 11357194;Abstract:

De novo cardiac myofibril assembly has been difficult to study due to the lack of available cell culture models that clearly and accurately reflect heart muscle development in vivo. However, within precardiac chick embryo ex-plants, premyocardial cells differentiate and commence beating in a temporal pattern that corresponds closely with myocyte differentiation in the embryo. Immunofluorescence staining of explants followed by confocal microscopy revealed that distinct stages of cardiac myofibril assembly, ranging from the earliest detection of sarcomeric proteins to the late appearance of mature myofibrils, were consistently recognized in precardiac cultures. Assembly events involved in the early formation of sarcomeres were clearly visualized and accurately reflected observations described by others during chick heart muscle development. Specifically, the early colocalization of α-actinin and titin dots was observed near the cell periphery representing I-Z -I-like complex formation. Myosin-containing thick filaments assembled independently of actin-containing thin filaments and appeared centered within sarcomeres when titin was also linearly aligned at or near cell borders. An N-terminal epitope of titin was detected earlier than a C-terminal epitope; however, both epitopes were observed to alternate near the cell periphery concomitant with the earliest formation of myofibrils. Although vascular actin was detected within cells during early assembly stages, cardiac actin predominated as the major actin isoform in mature thin filaments. Well-aligned thin filaments were also observed in the absence of organized staining for tropomodulin at thin filament pointed ends, suggesting that tropomodulin is not required to define thin filament lengths. Based on these findings, we conclude that the use of the avian precardiac explant system accurately allows for direct investigation of the mechanisms regulating de novo cardiac myofibrillogenesis. © 2001 Wiley-Liss, Inc.

Tsukada, T., Pappas, C. T., Moroz, N., Antin, P. B., Kostyukova, A. S., & Gregorio, C. C. (2010). Leiomodin-2 is an antagonist of tropomodulin-1 at the pointed end of the thin filaments in cardiac muscle. Journal of cell science, 123(Pt 18), 3136-45.
BIO5 Collaborators
Parker B Antin, Carol C Gregorio

Regulation of actin filament assembly is essential for efficient contractile activity in striated muscle. Leiomodin is an actin-binding protein and homolog of the pointed-end capping protein, tropomodulin. These proteins are structurally similar, sharing a common domain organization that includes two actin-binding sites. Leiomodin also contains a unique C-terminal extension that has a third actin-binding WH2 domain. Recently, the striated-muscle-specific isoform of leiomodin (Lmod2) was reported to be an actin nucleator in cardiomyocytes. Here, we have identified a function of Lmod2 in the regulation of thin filament lengths. We show that Lmod2 localizes to the pointed ends of thin filaments, where it competes for binding with tropomodulin-1 (Tmod1). Overexpression of Lmod2 results in loss of Tmod1 assembly and elongation of the thin filaments from their pointed ends. The Lmod2 WH2 domain is required for lengthening because its removal results in a molecule that caps the pointed ends similarly to Tmod1. Furthermore, Lmod2 transcripts are first detected in the heart after it has begun to beat, suggesting that the primary function of Lmod2 is to maintain thin filament lengths in the mature heart. Thus, Lmod2 antagonizes the function of Tmod1, and together, these molecules might fine-tune thin filament lengths.

Gregorio, C., Conover, G. M., & Gregorio, C. C. (2011). The desmin coil 1B mutation K190A impairs nebulin Z-disc assembly and destabilizes actin thin filaments. Journal of cell science, 124(Pt 20).

Desmin intermediate filaments intimately surround myofibrils in vertebrate muscle forming a mesh-like filament network. Desmin attaches to sarcomeres through its high-affinity association with nebulin, a giant F-actin binding protein that co-extends along the length of actin thin filaments. Here, we further investigated the functional significance of the association of desmin and nebulin in cultured primary myocytes to address the hypothesis that this association is key in integrating myofibrils to the intermediate filament network. Surprisingly, we identified eight peptides along the length of desmin that are capable of binding to C-terminal modules 160-170 in nebulin. In this study, we identified a targeted mutation (K190A) in the desmin coil 1B region that results in its reduced binding with the nebulin C-terminal modules. Using immunofluorescence microscopy and quantitative analysis, we demonstrate that expression of the mutant desmin K190A in primary myocytes results in a significant reduction in assembled endogenous nebulin and desmin at the Z-disc. Non-uniform actin filaments were markedly prevalent in myocytes expressing GFP-tagged desmin K190A, suggesting that the near-crystalline organization of actin filaments in striated muscle depends on a stable interaction between desmin and nebulin. All together, these data are consistent with a model in which Z-disc-associated nebulin interacts with desmin through multiple sites to provide efficient stability to satisfy the dynamic contractile activity of myocytes.