Yevgeniya E. Koshman

Mechanical stress-induced sarcomere assembly for cardiac muscle growth in length and width

A ventricular myocyte experiences changes in length and load during every beat of the heart and has the ability to remodel cell shape to maintain cardiac performance. Specifically, myocytes elongate in response to increased diastolic strain by adding sarcomeres in series, and they thicken in response to continued systolic stress by adding filaments in parallel. Myocytes do this while still keeping the resting sarcomere length close to its optimal value at the peak of the length-tension curve. This review focuses on the little understood mechanisms by which direction of growth is matched in a physiologically appropriate direction. We propose that the direction of strain is detected by differential phosphorylation of proteins in the costamere, which then transmit signaling to the Z-disc for parallel or series addition of thin filaments regulated via the actin capping processes. In this review, we link mechanotransduction to the molecular mechanisms for regulation of myocyte length and width.

Stimulus interval, rate and direction differentially regulate phosphorylation for mechanotransduction in neonatal cardiac myocytes

The effect of interval, direction and rate of strain on mechanotransduction in neonatal rat cardiomyocytes is determined for focal adhesion kinase (Y397pFAK), extracellular signal‐regulated kinase ERK1/2 (Thr183/Tyr185) and paxillin (pY31) and phosphorylation time courses to 10% strain assessed. Cells are non‐responsive at 5 min but recover at 15 min (P < 0.03) with FAK nuclear translocation by 30 min. Cyclic biaxial strain increased phosphorylation from slower to faster rates (P < 0.05). Uniaxial strain to groove‐aligned myocytes increased FAK and ERK1/2 phosphorylation transversely more than longitudinally (P < 0.05). Mechanotransduction may have a refractory period of 5 min and differentiate directions and rates of strain.

Delivery and visualization of proteins conjugated to quantum dots in cardiac myocytes.

The design of a novel transduction complex has permitted the introduction of protein-quantum dot conjugates into the cytoplasm of living cells. Appropriate subcellular localization of quantum dot-conjugated cardiac troponin C to the myofibrils and a nuclear peptide to the nucleus was attained in living cardiac myocytes using this approach. This new methodology opens the possibility for live tracking of exogenous proteins and study of protein dynamics.

Talin1 has unique expression versus talin 2 in the heart and modifies the hypertrophic response to pressure overload.

Background: Talin is an integrin-actin linker essential for integrin activation. Results: Talin1 has distinct developmental and postnatal expression in heart versus Talin2. Cardiac-myocyte specific Talin1 deletion alters physiological and molecular responses of the myocardium to stress. Conclusion: Talin1 has a unique mechanotransductive role in the cardiomyocyte. Significance: Reduction of talin1 in cardiomyocytes may have beneficial effects in the stressed myocardium. Integrins are adhesive, signaling, and mechanotransduction proteins. Talin (Tln) activates integrins and links it to the actin cytoskeleton. Vertebrates contain two talin genes, tln1 and tln2. How Tln1 and Tln2 function in cardiac myocytes (CMs) is unknown. Tln1 and Tln2 expression were evaluated in the normal embryonic and adult mouse heart as well as in control and failing human adult myocardium. Tln1 function was then tested in the basal and mechanically stressed myocardium after cardiomyocyte-specific excision of the Tln1 gene. During embryogenesis, both Tln forms are highly expressed in CMs, but in the mature heart Tln2 becomes the main Tln isoform, localizing to the costameres. Tln1 expression is minimal in the adult CM. With pharmacological and mechanical stress causing hypertrophy, Tln1 is up-regulated in CMs and is specifically detected at costameres, suggesting its importance in the compensatory response to CM stress. In human failing heart, CM Tln1 also increases compared with control samples from normal functioning myocardium. To directly test Tln1 function in CMs, we generated CM-specific Tln1 knock-out mice (Tln1cKO). Tln1cKO mice showed normal basal cardiac structure and function but when subjected to pressure overload showed blunted hypertrophy, less fibrosis, and improved cardiac function versus controls. Acute responses of ERK1/2, p38, Akt, and glycogen synthase kinase 3 after mechanical stress were strongly blunted in Tln1cKO mice. Given these results, we conclude that Tln1 and Tln2 have distinct functions in the myocardium. Our data show that reduction of CM Tln1 expression can lead to improved cardiac remodeling following pressure overload.

Serine 910 Phosphorylation of Focal Adhesion Kinase is Critical for Sarcomere Reorganization In Cardiomyocyte Hypertrophy

AIMS Tyrosine-phosphorylated focal adhesion kinase (FAK) is required for the hypertrophic response of cardiomyocytes to growth factors and mechanical load, but the role of FAK serine phosphorylation in this process is unknown. The aims of the present study were to characterize FAK serine phosphorylation in cultured neonatal rat ventricular myocytes (NRVM), analyse its functional significance during hypertrophic signalling, and examine its potential role in the pathogenesis of human dilated cardiomyopathy (DCM). METHODS AND RESULTS Endothelin-1 (ET-1) and other hypertrophic factors induced a time- and dose-dependent increase in FAK-S910 phosphorylation. ET-1-induced FAK-S910 phosphorylation required ET(A)R-dependent activation of PKCδ and Src via parallel Raf-1 → MEK1/2 → ERK1/2 and MEK5 → ERK5 signalling pathways. Replication-deficient adenoviruses expressing wild-type (WT) FAK and a non-phosphorylatable, S910A-FAK mutant were then used to examine the functional significance of FAK-S910 phosphorylation. Unlike WT-FAK, S910A-FAK increased the half-life of GFP-tagged paxillin within costameres (as determined by total internal reflection fluorescence microscopy and fluorescence recovery after photobleaching) and increased the steady-state FAK-paxillin interaction (as determined by co-immunoprecipitation and western blotting). These alterations resulted in reduced NRVM sarcomere reorganization and cell spreading. Finally, we found that FAK was serine-phosphorylated at multiple sites in non-failing, human left ventricular tissue. FAK-S910 phosphorylation and ERK5 expression were dramatically reduced in patients undergoing heart transplantation for end-stage DCM. CONCLUSION FAK undergoes S910 phosphorylation via PKCδ and Src-dependent pathways that are important for cell spreading and sarcomere reorganization. Reduced FAK-S910 phosphorylation may contribute to sarcomere disorganization in DCM.

Role of FRNK tyrosine phosphorylation in vascular smooth muscle spreading and migration

AIMS Focal adhesion kinase (FAK) and its autonomously expressed, C-terminal inhibitor FAK-related non-kinase (FRNK), are important regulators of vascular smooth muscle cell (VSMC) spreading and migration. However, the mechanisms of FRNK-mediated inhibition of FAK-dependent signalling are not fully defined. The aim of this study was to determine the potential role of FRNK tyrosine phosphorylation in regulating these processes. METHODS AND RESULTS Rat carotid arteries were balloon-injured and FAK and FRNK expression and phosphorylation were examined by immunocytochemistry, immunoprecipitation, and western blotting with total and phosphospecific antibodies. FAK and FRNK expression increased four- and nine-fold, respectively, in alpha-smooth muscle actin-positive VSMCs of injured arteries when compared with contralateral control arteries, and the upregulated FRNK was phosphorylated at residues Y168 and Y232. In A7r5 cells (an embryonic rat VSMC line), endogenously expressed FRNK was also phosphorylated at Y168 and Y232 under basal conditions, and Y168/Y232 phosphorylation increased in response to angiotensin II treatment. When overexpressed in A7r5 cells and adult rat aortic smooth muscle cells (RASM), wild-type (wt) GFP-tagged FRNK was also phosphorylated at residues Y168 and Y232, and GFP-wtFRNK inhibited cell spreading and migration. Mutation of GFP-FRNK at Y168 (GFP-Y168F-FRNK) abrogated FRNK-mediated inhibition of cell spreading and migration, but did not affect its localization in VSMC focal adhesions or its ability to inhibit FAK tyrosine phosphorylation. CONCLUSION Phosphorylation of Y168 on FRNK may represent a novel mechanism by which FRNK inhibits cell spreading and migration in VSMCs.

Connective tissue growth factor regulates cardiac function and tissue remodeling in a mouse model of dilated cardiomyopathy.

Cardiac structural changes associated with dilated cardiomyopathy (DCM) include cardiomyocyte hypertrophy and myocardial fibrosis. Connective tissue growth factor (CTGF) has been associated with tissue remodeling and is highly expressed in failing hearts. Our aim was to test if inhibition of CTGF would alter the course of cardiac remodeling and preserve cardiac function in the protein kinase Cε (PKCε) mouse model of DCM. Transgenic mice expressing constitutively active PKCε in cardiomyocytes develop cardiac dysfunction that was evident by 3 months of age, and that progressed to cardiac fibrosis, heart failure, and increased mortality. Beginning at 3 months of age, PKCε mice were treated with a neutralizing monoclonal antibody to CTGF (FG-3149) for an additional 3 months. CTGF inhibition significantly improved left ventricular (LV) systolic and diastolic functions in PKCε mice, and slowed the progression of LV dilatation. Using gene arrays and quantitative PCR, the expression of many genes associated with tissue remodeling was elevated in PKCε mice, but significantly decreased by CTGF inhibition. However total collagen deposition was not attenuated. The observation of significantly improved LV function by CTGF inhibition in PKCε mice suggests that CTGF inhibition may benefit patients with DCM. Additional studies to explore this potential are warranted.

Signaling responses after exposure to 5α-dihydrotestosterone or 17β-estradiol in norepinephrine-induced hypertrophy of neonatal rat ventricular myocytes

Androgens appear to enhance, whereas estrogens mitigate, cardiac hypertrophy. However, signaling pathways in cells for short (3 min) and longer term (48 h) treatment with 17beta-estradiol (E2) or 5 alpha-dihydrotestosterone (DHT) are understudied. We compared the effect of adrenergic stimulation by norepinephrine (NE; 1 microM) alone or in combination with DHT (10 nM) or E2 (10 nM) treatment in neonatal rat ventricular myocytes (NRVMs) by cell area, protein synthesis, sarcomeric structure, gene expression, phosphorylation of extracellular signal-regulated (ERK), and focal adhesion kinases (FAK), and phospho-FAK nuclear localization. NE alone elicited the expected hypertrophy and strong sarcomeric organization, and DHT alone gave a similar but more modest response, whereas E2 did not alter cell size. Effects of NE dominated when used with either E2 or DHT with all combinations. Both sex hormones alone rapidly activated FAK but not ERK. Long-term or brief exposure to E2 attenuated NE-induced FAK phosphorylation, whereas DHT had no effect. Neither hormone altered NE-elicited ERK activation. Longer term exposure to E2 alone reduced FAK phosphorylation and reduced nuclear phospho-FAK, whereas its elevation was seen in the presence of NE with both sex hormones. The mitigating effects of E2 on the NE-elicited increase in cell size and the hypertrophic effect of DHT in NRVMs are in accordance with results observed in whole animal models. This is the first report of rapid, nongenomic sex hormone signaling via FAK activation and altered FAK trafficking to the nucleus in heart cells.

Focal Adhesion Kinase–Related Nonkinase Inhibits Vascular Smooth Muscle Cell Invasion by Focal Adhesion Targeting, Tyrosine 168 Phosphorylation, and Competition for p130 Cas Binding

Objective—Focal adhesion kinase–related nonkinase (FRNK), the C-terminal domain of focal adhesion kinase (FAK), is a tyrosine-phosphorylated, vascular smooth muscle cell (VSMC)–specific inhibitor of cell migration. FRNK inhibits both FAK and proline-rich tyrosine kinase 2 (PYK2) in cultured VSMCs, and both kinases may be involved in VSMC invasion during vascular remodeling. Methods and Results—Adenovirally mediated gene transfer of green fluorescent protein–tagged, wild-type (wt) FRNK into balloon-injured rat carotid arteries confirmed that FRNK overexpression inhibited both FAK and PYK2 phosphorylation and downstream signaling in vivo. To identify which kinase was involved in regulating VSMC invasion, adenovirally mediated expression of specific short hairpin RNAs was used to knock down FAK versus PYK2 in cultured VSMCs, but only FAK short hairpin RNA was effective in reducing VSMC invasion. The role of FRNK tyrosine phosphorylation was then examined using adenoviruses expressing nonphosphorylatable (Tyr168Phe-, Tyr232Phe-, and Tyr168,232Phe-) green fluorescent protein–FRNK mutants. wtFRNK and all FRNK mutants localized to FAs, but only Tyr168 phosphorylation was required for FRNK to inhibit invasion. Preventing Tyr168 phosphorylation also increased FRNK-paxillin interaction, as determined by coimmunoprecipitation, total internal reflection fluorescence microscopy, and fluorescence recovery after photobleaching. Furthermore, wtFRNK competed with FAK for binding to p130Cas (a critically important regulator of cell migration) and prevented its phosphorylation. However, Tyr168Phe-FRNK was unable to bind p130Cas. Conclusion—We propose a 3-stage mechanism for FRNK inhibition: focal adhesion targeting, Tyr168 phosphorylation, and competition with FAK for p130Cas binding and phosphorylation, which are all required for FRNK to inhibit VSMC invasion.

FRNK Inhibition of Focal Adhesion Kinase–Dependent Signaling and Migration in Vascular Smooth Muscle Cells

Objective—To examine whether interference with FRNK targeting to focal adhesions (FAs) affects its inhibitory activity and tyrosine phosphorylation. Methods and Results—Focal adhesion kinase and its autonomously expressed C-terminal inhibitor, focal adhesion kinase-related nonkinase (FRNK), regulate vascular smooth muscle cell (VSMC) signaling and migration. FRNK-paxillin binding was reduced by a point mutation in its FA targeting domain (L341S-FRNK). Green fluorescent protein-tagged wild type and L341S-FRNK were then adenovirally expressed in VSMCs. L341S-FRNK targeted to VSMC FAs, despite previous studies in other cell types. L341S-FRNK affected FA binding kinetics (assessed by total internal reflection fluorescnece [TIRF] microscopy and fluorescence recovery after photobleaching [FRAP]) and reduced its steady-state paxillin interaction (determined by coimmunoprecipitation). Both wt-FRNK and L341S-FRNK lowered basal and angiotensin II-stimulated focal adhesion kinase, paxillin, and extracellular signal-regulated kinase 1/2 phosphorylation. However, the degree of inhibition was significantly reduced by L341S-FRNK. L341S-FRNK also demonstrated significantly greater migratory activity compared with wt-FRNK-expressing VSMCs. Angiotensin II-induced Y168 phosphorylation was Src dependent, as evident by a significant reduction in Y168 phosphorylation by the Src family kinase inhibitor PP2 is 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Surprisingly, Y168 phosphorylation was unaffected by its targeting. Furthermore, Y232 phosphorylation increased approximately 3-fold in L341S-FRNK, which was less sensitive to PP2. Conclusion—FRNK inhibition of VSMC migration requires both FA targeting and Y168 phosphorylation by Src family kinases. FRNK-Y232 phosphorylation occurs outside of FAs, probably by a PP2-insensitive kinase.