Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.actin-thin filaments | cardiomyopathy | cytoskeletal dynamics S triated muscle cells contain arrays of protein filaments assembled into contractile units that are nearly crystalline in structure. Efficient contraction at the molecular level is predicated upon accurate overlap of actin-containing thin and myosin-containing thick filaments. Therefore, proper control of filament assembly is absolutely critical.In striated muscle it is currently thought that the thin-filament pointed end capping protein tropomodulin (Tmod) is the predominant regulator of thin filament length, with Tmod1 being the sole isoform expressed in cardiomyocytes (1). Extensive in vitro work has revealed that Tmod1 uses two actin-and two tropomyosin-binding sites to associate with the end of the thin filament and to prevent addition or loss of actin monomers, thereby controlling length of the thin filament (2-7). Tmod1 is essential for life; Tmod1-KO mice are embryonic lethal because of cardiac defects (8-11).Identification of additional but structurally different members of the Tmod family of proteins, the leiomodins (Lmods), raises the possibility that thin filament lengths are not regulated solely by Tmod at thin filament pointed ends (12). Although there are three Lmod genes (Lmod1-3), Lmod2 and 3 are expressed in striated muscle with Lmod2 being the predominant isoform in cardiac muscle and Lmod3 the predominant isoform in skeletal muscle (12-16). The Lmods share ∼40% sequence identity at the protein level with the Tmods but do not contain a recognizable second tropomyosin-binding domain and have an additional C-terminal extension that includes a proline-rich region and an actin-binding Wiskott-Aldrich syndrome protein homology 2 (WH2) domain (12, 17). Lmod2 has been proposed to be the long-sought muscle actin filament nucleator because it robustly nucleates actin filament formation in ...

Most current drug screening assays used to identify new drug candidates are 2D cell-based systems, even though such in vitro assays do not adequately recreate the in vivo complexity of 3D tissues. Inadequate representation of the human tissue environment during a preclinical test can result in inaccurate predictions of compound effects on overall tissue functionality. Screening for compound efficacy by focusing on a single pathway or protein target, coupled with difficulties in maintaining long-term 2D monolayers, can serve to exacerbate these issues when utilizing such simplistic model systems for physiological drug screening applications. Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens. In this review, we discuss the development of microengineered 3D tissue models which accurately mimic the physiological properties of native tissue samples, and highlight the advantages of using such 3D micro-tissue models over conventional cell-based assays for future drug screening applications. We also discuss biomimetic 3D environments, based-on engineered tissues as potential preclinical models for the development of more predictive drug screening assays for specific disease models.

Oxygen is a key modulator of many cellular pathways, but current devices permitting in vitro oxygen modulation fail to meet the needs of biomedical research. A microfabricated insert for multiwell plates has been developed to more effectively control the temporal and spatial oxygen concentration to better model physiological phenomena found in vivo. The platform consists of a polydimethylsiloxane insert that nests into a standard multiwell plate and serves as a passive microfluidic gas network with a gas-permeable membrane aimed to modulate oxygen delivery to adherent cells. Equilibration time is on the order of minutes and a wide variety of oxygen profiles can be attained based on the device design, such as the cyclic profile achieved in this study, and even oxygen gradients to mimic those found in vivo. The proper biological consequences of the device's oxygen delivery were confirmed in cellular models via a proliferation assay and western analysis of the upregulation of hypoxia inducible transcription factor-1α. These experiments serve as a demonstration for the platform as a viable tool to increase experimental throughput and permit novel experimental possibilities in any biomedical research lab.