Leiomodin is a potent actin nucleator related to tropomodulin, a capping protein localized at the pointed end of the thin filaments. Mutations in leiomodin-3 are associated with lethal nemaline myopathy in humans, and leiomodin-2–knockout mice present with dilated cardiomyopathy. The arrangement of the N-terminal actin- and tropomyosin-binding sites in leiomodin is contradictory and functionally not well understood. Using one-dimensional nuclear magnetic resonance and the pointed-end actin polymerization assay, we find that leiomodin-2, a major cardiac isoform, has an N-terminal actin-binding site located within residues 43–90. Moreover, for the first time, we obtain evidence that there are additional interactions with actin within residues 124–201. Here we establish that leiomodin interacts with only one tropomyosin molecule, and this is the only site of interaction between leiomodin and tropomyosin. Introduction of mutations in both actin- and tropomyosin-binding sites of leiomodin affected its localization at the pointed ends of the thin filaments in cardiomyocytes. On the basis of our new findings, we propose a model in which leiomodin regulates actin polymerization dynamics in myocytes by acting as a leaky cap at thin filament pointed ends.
A novel cardiac-specific transgenic mouse model was generated to identify the physiological consequences of elongated thin filaments during post-natal development in the heart. Remarkably, increasing the expression levels in vivo of just one sarcomeric protein, Lmod2, results in ~10% longer thin filaments (up to 26% longer in some individual sarcomeres) that produce up to 50% less contractile force. Increasing the levels of Lmod2 in vivo (Lmod2-TG) also allows us to probe the contribution of Lmod2 in the progression of cardiac myopathy because Lmod2-TG mice present with a unique cardiomyopathy involving enlarged atrial and ventricular lumens, increased heart mass, disorganized myofibrils and eventually, heart failure. Turning off of Lmod2 transgene expression at postnatal day 3 successfully prevents thin filament elongation, as well as gross morphological and functional disease progression. We show here that Lmod2 has an essential role in regulating cardiac contractile force and function.
Increases in lung vascular permeability is a cardinal feature of inflammatory disease and represents an imbalance in vascular contractile forces and barrier-restorative forces, with both forces highly dependent upon the actin cytoskeleton. The current study investigates the role of Ena-VASP-like (EVL), a member of the Ena-VASP family known to regulate the actin cytoskeleton, in regulating vascular permeability responses and lung endothelial cell barrier integrity. Utilizing changes in transendothelial electricial resistance (TEER) to measure endothelial cell barrier responses, we demonstrate that EVL expression regulates endothelial cell responses to both sphingosine-1-phospate (S1P), a vascular barrier-enhancing agonist, and to thrombin, a barrier-disrupting stimulus. Total internal reflection fluorescence demonstrates that EVL is present in endothelial cell focal adhesions and impacts focal adhesion size, distribution, and the number of focal adhesions generated in response to S1P and thrombin challenge, with the focal adhesion kinase (FAK) a key contributor in S1P-stimulated EVL-transduced endothelial cell but a limited role in thrombin-induced focal adhesion rearrangements. In summary, these data indicate that EVL is a focal adhesion protein intimately involved in regulation of cytoskeletal responses to endothelial cell barrier-altering stimuli. Keywords: cytoskeleton, vascular barrier, sphingosine-1-phosphate, thrombin, focal adhesion kinase (FAK), Ena-VASP like protein (EVL), cytoskeletal regulatory protein.
Significance Modulation of actin filament architecture underlies a plethora of cellular processes including cell shape, division, adhesion, and motility. In heart muscle cells actin-containing thin filaments form highly organized structures with precisely regulated lengths. This precision is required for efficient interaction with myosin-containing filaments and provides the basis for contraction. The mechanism whereby heart muscle cells regulate thin filament assembly and its consequences for cardiac physiology are largely unknown. We discovered that Leiomodin 2 (Lmod2) elongates thin filaments to a proper length. Mice lacking Lmod2 have abnormally short thin filaments, experience severe contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy, and die at age ∼3 wk. Therefore, Lmod2 and proper thin filament lengths are essential for heart function.
The leiomodin (Lmod) family of actin-binding proteins play a critical role in muscle function, highlighted by the fact that mutations in all three family members (LMOD1-3) result in human myopathies. Mutations in the cardiac predominant isoform, LMOD2 lead to severe neonatal dilated cardiomyopathy. Most of the disease-causing mutations in the LMOD gene family are nonsense, or frameshift, mutations predicted to result in expression of truncated proteins. However, in nearly all cases of disease, little to no LMOD protein is expressed. We show here that nonsense-mediated mRNA decay, a cellular mechanism which eliminates mRNAs with premature termination codons, underlies loss of mutant protein from two independent LMOD2 disease-causing mutations. Furthermore, we generated steric-blocking oligonucleotides that obstruct deposition of the exon junction complex, preventing nonsense-mediated mRNA decay of mutant LMOD2 transcripts, thereby restoring mutant protein expression. Our investigation lays the initial groundwork for potential therapeutic intervention in LMOD-linked myopathies.
Muscle contraction is a regulated process driven by the sliding of actin-thin filaments over myosin-thick filaments. Lmod2 is an actin filament length regulator and essential for life since human mutations and complete loss of Lmod2 in mice lead to dilated cardiomyopathy and death. To study the little-known role of Lmod2 in skeletal muscle, we created a mouse model with Lmod2 expressed exclusively in the heart but absent in skeletal muscle. Loss of Lmod2 in skeletal muscle results in decreased force production in fast- and slow-twitch muscles. Soleus muscle from rescued Lmod2 knockout mice have shorter thin filaments, increased Lmod3 levels, and present with a myosin fiber type switch from fast myosin heavy chain (MHC) IIA to the slower MHC I isoform. Since Lmod2 regulates thin-filament length in slow-twitch but not fast-twitch skeletal muscle and force deficits were observed in both muscle types, this work demonstrates that Lmod2 regulates skeletal muscle contraction, independent of its role in thin-filament length regulation.
Prior to menopause, women are protected against cardiovascular disease (CVD) compared to age-matched men; this protection is gradually lost after menopause. Mechanisms responsible for loss of CVD protection are unknown. We previously demonstrated that menopause and CVD suppress the AMP-activated protein kinase (AMPK) signaling pathway in mice. We also validated the cellular mechanism by which estrogen (E2) potentiates AMPK activity through a direct interaction of estrogen receptors (ER) with members of the AMPK kinase complex. Because AMPK signaling is down in CVD and menopause, we hypothesized that activation of AMPK will prevent pathological cardiac remodeling in menopausal female mice. First, we demonstrated that E2 potentiates AMPK activity in neonatal rat cardiomyocytes (NRCMs) subjected to energy stress. NRCMs, cultured in estrogen-free media, were treated (10-30 minutes at 100nm) with the electron transport chain uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenyphydrazone (FCCP). As expected, AMPK activity determined by phosphorylation of threonine 172 (p-AMPK172) was increased over controls. Adding 1-100nm of E2 potentiated p-AMPK172 over control-treated NRCMs by 5-fold. Next, we used our novel model of menopause with 4-vinylcyclohexene diepoxide (VCD), which induces gradual ovarian failure, preserving the perimenopause transitional period and androgen secreting capacity of residual ovarian tissue. Starting at 2 months, females received daily (i.p.) injections of VCD (160mg/kg, 20 consecutive days) or sesame oil as vehicle. Peri/menopause were confirmed by vaginal cytology. Menopausal females receiving angiotensin II (Ang II, 800 ng/kg/min via alzet s.c. mini-pump, 14 days) demonstrated exacerbation of hypertension and pathological cardiac remodeling compared to pre- and peri-menopausal mice. Female mice treated with Ang II following surgical removal of ovaries (OVX) experienced a similar exacerbation of cardiac remodeling. Daily adminstration of the AMPK activator (A-769662, s.c. 30mg/kg) prevented pathological remodeling in menopausal and OVX female mice subjected to Ang II. We conclude that AMPK represents a non-canonical target for the mitigation of menopausal susceptibly to CVD.
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