High throughput cell mechanotyping of cell response to cytoskeletal modulations using a microfluidic cell deformation system.

Cellular mechanical properties influence cellular functions across pathological and physiological systems. The observation of these mechanical properties is limited in part by methods with a low throughput of acquisition or with low accessibility. To overcome these limitations, we have designed, developed, validated, and optimized a microfluidic cellular deformation system (MCDS) capable of mechanotyping suspended cells on a population level at a high throughput rate of ∼300 cells pers second. The MCDS provides researchers with a viable method for efficiently quantifying cellular mechanical properties towards defining prognostic implications of mechanical changes in pathology or screening drugs to modulate cytoskeletal integrity.

Detyrosinated microtubule arrays drive myofibrillar malformations in mdx muscle fibers.

Altered myofibrillar structure is a consequence of dystrophic pathology that impairs skeletal muscle contractile function and increases susceptibility to contraction injury. In murine Duchenne muscular dystrophy (mdx), myofibrillar alterations are abundant in advanced pathology (>4 months), an age where we formerly established densified microtubule (MT) arrays enriched in detyrosinated (deTyr) tubulin as negative disease modifiers impacting cell mechanics and mechanotransduction. Given the essential role of deTyr-enriched MT arrays in myofibrillar growth, maintenance, and repair, we examined the increased abundance of these arrays as a potential mechanism for these myofibrillar alterations. Here we find an increase in deTyr-tubulin as an early event in dystrophic pathology (4 weeks) with no evidence myofibrillar alterations. At 16 weeks, we show deTyr-enriched MT arrays significantly densified and co-localized to areas of myofibrillar malformation. Profiling the enzyme complexes responsible for deTyr-tubulin, we identify vasohibin 2 (VASH2) and small vasohibin binding protein (SVBP) significantly elevated in the mdx muscle at 4 weeks. Using the genetic increase in VASH2/SVBP expression in 4 weeks wild-type mice we find densified deTyr-enriched MT arrays that co-segregate with myofibrillar malformations similar to those in the 16 weeks mdx. Given that no changes in sarcomere organization were identified in fibers expressing sfGFP as a control, we conclude that disease-dependent densification of deTyr-enriched MT arrays underscores the altered myofibrillar structure in dystrophic skeletal muscle fibers.

Editing of the myosin phosphatase regulatory subunit suppresses angiotensin II induced hypertension via sensitization to nitric oxide mediated vasodilation.

Alternative splicing of exon 24 (E24) of the myosin phosphatase regulatory subunit (Mypt1) tunes smooth muscle sensitivity to NO/cGMP-mediated vasorelaxation and thereby controls blood pressure (BP) in otherwise normal mice. This occurs via the toggling in or out of a C-terminal leucine zipper (LZ) motif required for hetero-dimerization with and activation by cGMP-dependent protein kinase cGK1α. Here we tested the hypothesis that editing (deletion) of E24, by shifting to the LZ positive isoform of Mypt1, would suppress the hypertensive response to angiotensin II (AngII). To test this, mice underwent tamoxifen-inducible and smooth muscle-specific deletion of E24 (E24 cKO) at age 6 weeks followed by a chronic slow-pressor dose of AngII (400 ng/kg/min) plus additional stressors. E24 cKO suppressed the hypertensive response to AngII alone or with the addition of a high salt diet. This effect was not a function of altered salt balance as there were no differences in intake or renal excretion of sodium. This effect was NO dependent as L-NAME in the drinking water caused an exaggerated hypertensive response in the E24cKO mice. E24cKO mouse mesenteric arteries were more sensitive to DEA/NO-induced vasorelaxation and less responsive to AngII- and α-adrenergic-induced vasoconstriction at baseline. Only the latter two effects were still present after 2 weeks of chronic AngII treatment. We conclude that editing of Mypt1 E24, by shifting the expression of naturally occurring isoforms and sensitizing to NO-mediated vasodilation, could be a novel approach to the treatment of human hypertension.

Electroblotting from Polyacrylamide Gels.

Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N-terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose. In addition to the commonly used tank or wet transfer system, protocols are provided for electroblotting using semidry and dry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents for specialized applications.

Mena/VASP and αII-Spectrin complexes regulate cytoplasmic actin networks in cardiomyocytes and protect from conduction abnormalities and dilated cardiomyopathy.

BACKGROUND: In the heart, cytoplasmic actin networks are thought to have important roles in mechanical support, myofibrillogenesis, and ion channel function. However, subcellular localization of cytoplasmic actin isoforms and proteins involved in the modulation of the cytoplasmic actin networks are elusive. Mena and VASP are important regulators of actin dynamics. Due to the lethal phenotype of mice with combined deficiency in Mena and VASP, however, distinct cardiac roles of the proteins remain speculative. In the present study, we analyzed the physiological functions of Mena and VASP in the heart and also investigated the role of the proteins in the organization of cytoplasmic actin networks. RESULTS: We generated a mouse model, which simultaneously lacks Mena and VASP in the heart. Mena/VASP double-deficiency induced dilated cardiomyopathy and conduction abnormalities. In wild-type mice, Mena and VASP specifically interacted with a distinct αII-Spectrin splice variant (SH3i), which is in cardiomyocytes exclusively localized at Z- and intercalated discs. At Z- and intercalated discs, Mena and ß-actin localized to the edges of the sarcomeres, where the thin filaments are anchored. In Mena/VASP double-deficient mice, ß-actin networks were disrupted and the integrity of Z- and intercalated discs was markedly impaired. CONCLUSIONS: Together, our data suggest that Mena, VASP, and αII-Spectrin assemble cardiac multi-protein complexes, which regulate cytoplasmic actin networks. Conversely, Mena/VASP deficiency results in disrupted ß-actin assembly, Z- and intercalated disc malformation, and induces dilated cardiomyopathy and conduction abnormalities.

Characterization and expression of a heart-selective alternatively spliced variant of alpha II-spectrin, cardi+, during development in the rat.

Spectrin is a large, flexible protein that stabilizes membranes and organizes proteins and lipids into microdomains in intracellular organelles and at the plasma membrane. Alternative splicing occurs in spectrins, but it is not yet clear if these small variations in structure alter spectrin's functions. Three alternative splice sites have been identified previously for alpha II-spectrin. Here we describe a new alternative splice site, a 21-amino acid sequence in the 21st spectrin repeat that is only expressed in significant amounts in cardiac muscle (GenBank GQ502182). The insert, which we term alpha II-cardi+, results in an insertion within the high affinity nucleation site for binding of alpha-spectrins to beta-spectrins. To assess the developmental regulation of the alpha II-cardi+ isoform, we used qRT-PCR and quantitative immunoblotting methods to measure the levels of this form and the alpha II-cardi- form in the cardiac muscles of rats, from embryonic day 16 (E16) through adulthood. The alpha II-cardi+ isoform constituted approximately 26% of the total alpha II-spectrin in E16 hearts but decreased to approximately 6% of the total after 3 weeks of age. We used long-range RT-PCR and Southern blot hybridization to examine possible linkage of the alpha II-cardi+ alternatively spliced sequence with alternatively spliced sequences of alpha II-spectrin that had been previously reported. We identified two new isoforms of alpha II-spectrin containing the cardi+ insert. These were named alpha II Sigma 9 and alpha II Sigma 10 in accordance with the spectrin naming conventions. In vitro studies of recombinant alpha II-spectrin polypeptides representing the two splice variants of alpha II-spectrin, alpha II-cardi+ and alpha II-cardi-, revealed that the alpha II-cardi+ subunit has lower affinity for the complementary site in repeats 1-4 of betaII-spectrin, with a K(D) value of approximately 1 nM, as measured by surface plasmon resonance (SPR). In addition, the alpha II-cardi+ form showed 1.8-fold lower levels of binding to its site on beta II-spectrin than the alpha II-cardi- form, both by SPR and blot overlay. This suggests that the 21-amino acid insert prevented some of the alpha II-cardi+ form from interacting with beta II-spectrin. Fusion proteins expressing the alpha II-cardi+ sequence within the two terminal spectrin repeats of alpha II-spectrin were insoluble in solution and aggregated in neonatal myocytes, consistent with the possibility that this insert removes a significant portion of the protein from the population that can bind beta subunits. Neonatal rat cardiomyocytes infected with adenovirus encoding GFP-fusion proteins of repeats 18-21 of alpha II-spectrin with the cardi+ insert formed many new processes. These processes were only rarely seen in myocytes expressing the fusion protein lacking the insert or in controls expressing only GFP. Our results suggest that the embryonic mammalian heart expresses a significant amount of alpha II-spectrin with a reduced avidity for beta-spectrin and the ability to promote myocyte growth.

Absence of keratin 19 in mice causes skeletal myopathy with mitochondrial and sarcolemmal reorganization.

Intermediate filaments, composed of desmin and of keratins, play important roles in linking contractile elements to each other and to the sarcolemma in striated muscle. We examined the contractile properties and morphology of fast-twitch skeletal muscle from mice lacking keratin 19. Tibialis anterior muscles of keratin-19-null mice showed a small but significant decrease in mean fiber diameter and in the specific force of tetanic contraction, as well as increased plasma creatine kinase levels. Costameres at the sarcolemma of keratin-19-null muscle, visualized with antibodies against spectrin or dystrophin, were disrupted and the sarcolemma was separated from adjacent myofibrils by a large gap in which mitochondria accumulated. The costameric dystrophin-dystroglycan complex, which co-purified with gamma-actin, keratin 8 and keratin 19 from striated muscles of wild-type mice, co-purified with gamma-actin but not keratin 8 in the mutant. Our results suggest that keratin 19 in fast-twitch skeletal muscle helps organize costameres and links them to the contractile apparatus, and that the absence of keratin 19 disrupts these structures, resulting in loss of contractile force, altered distribution of mitochondria and mild myopathy. This is the first demonstration of a mammalian phenotype associated with a genetic perturbation of keratin 19.

Role of an alternatively spliced form of alphaII-spectrin in localization of connexin 43 in cardiomyocytes and regulation by stress-activated protein kinase.

Decreases in the expression of connexin 43 and the integrity of gap junctions in cardiac muscle, induced by the constitutive activation of the c-Jun N-terminal kinase (JNK) signaling pathway, have been linked to conduction defects and sudden cardiac failure in mice [Petrich BG, Gong X , Lerner DL , Wang X , Brown JH , Saffitz JE , Wang Y. c-Jun N-terminal kinase activation mediates downregulation of connexin 43 in cardiomyocytes. Circ Res. 91 (2002) 640-647; B.G. Petrich, B.C. Eloff, D.L. Lerner, A. Kovacs, J.E. Saffitz, D.S. Rosenbaum, Y. Wang, Targeted activation of c-Jun N-terminal kinase in vivo induces restrictive cardiomyopathy and conduction defects. J. Biol. Chem. 2004;279: 15330-15338]. We examined the membrane cytoskeletal protein, alphaII-spectrin, which associates with connexin 43, to learn if changes in its association with connexin 43 are linked to the instability of gap junctions. Several forms of alphaII-spectrin are expressed in the heart, including one, termed alphaII-SH3i, which contains a 20-amino-acid sequence next to the SH3 domain of repeat 10. In adult mouse heart, antibodies to all forms of alphaII-spectrin labeled the sarcolemma, transverse ("t-") tubules and intercalated disks of cardiomyocytes. In contrast, antibodies specific for alphaII-SH3i labeled only gap junctions and transverse tubules. In transgenic hearts, in which the JNK pathway was constitutively activated, alphaII-SH3i was lost specifically from gap junctions but not from t-tubules while other isoforms of alphaII-spectrin were retained at intercalated disks. Immunoprecipitations confirmed the decreased association of alphaII-SH3i with connexin 43 in transgenic hearts compared to controls. Furthermore, activation of JNK in neonatal myocytes blocked the formation of gap junctions by exogenously expressed Cx43-GFP fusion protein. Similarly, overexpression of the SH3i fragment in the context of repeats 9-11 of alphaII-spectrin specifically caused the accumulation of Cx43-GFP in the perinuclear region and inhibited its accumulation at gap junctions. These results support a critical role for the alphaII-SH3i isoform of spectrin in intracellular targeting of Cx43 to gap junctions and implicates alphaII-SH3i as a potential target for stress signaling pathways that modulate intercellular communication.

Cloning and characterization of cytokeratins 8 and 19 in adult rat striated muscle. Interaction with the dystrophin glycoprotein complex.

We used degenerate primers for the amino- and carboxyl-terminal ends of the rod domains of intermediate filament proteins in reverse transcriptase-PCR experiments to identify and clone cytokeratins 8 and 19 (K8 and K19) from cardiac muscle of the adult rat. Northern blots showed that K8 has a 2.2-kb transcript and K19 has a 1.9-kb transcript in both adult cardiac and skeletal muscles. Immunolocalization of the cytokeratins in adult cardiac muscle with isoform-specific antibodies for K8 and K19 showed labeling at Z-lines within the muscle fibers and at Z-line and M-line domains at costameres at the sarcolemmal membrane. Dystrophin and K19 could be co-immunoprecipitated and co-purified from extracts of cardiac muscle, suggesting a link between the cytokeratins and the dystrophin-based cytoskeleton at the sarcolemma. Furthermore, transfection experiments indicate that K8 and K19 may associate with dystrophin through a specific interaction with its actin-binding domain. Consistent with this observation, the cytokeratins are disrupted at the sarcolemmal membrane of skeletal muscle of the mdx mouse that lacks dystrophin. Together these results indicate that at least two cytokeratins are expressed in adult striated muscle, where they may contribute to the organization of both the myoplasm and sarcolemma.

Sarcoplasmic reticulum calcium defect in Ras-induced hypertrophic cardiomyopathy heart.

The small G protein Ras-mediated signaling pathway has been implicated in the development of hypertrophy and diastolic dysfunction in the heart. Earlier cellular studies have suggested that the Ras pathway is responsible for reduced L-type calcium channel current and sarcoplasmic reticulum (SR) calcium uptake associated with sarcomere disorganization in neonatal cardiomyocytes. In the present study, we investigated the in vivo effects of Ras activation on cellular calcium handling and sarcomere organization in adult ventricular myocytes using a newly established transgenic mouse model with targeted expression of the H-Ras-v12 mutant. The transgenic hearts expressing activated Ras developed significant hypertrophy and postnatal lethal heart failure. In adult ventricular myocytes isolated from the transgenic hearts, the calcium transient was significantly depressed but membrane L-type calcium current was unchanged compared with control littermates. The expressions of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2a and phospholamban (PLB) were significantly reduced at mRNA levels. The amount of SERCA2a protein was also modestly reduced. However, the expression of PLB protein and gross sarcomere organization remained unchanged in the hypertrophic Ras hearts, whereas Ser(16) phosphorylation of PLB was dramatically inhibited in the Ras transgenic hearts compared with controls. Hypophosphorylation of PLB was also associated with a significant induction of protein phosphatase 1 expression. Therefore, our results from this in vivo model system suggest that Ras-induced contractile defects do not involve decreased L-type calcium channel activities or disruption of sarcomere structure. Rather, suppressed SR calcium uptake due to reduced SERCA2a expression and hypophosphorylation of PLB due to changes in protein phosphatase expression may play important roles in the diastolic dysfunction of Ras-mediated hypertrophic cardiomyopathy.

Costameres: repeating structures at the sarcolemma of skeletal muscle.

Costameres, structures at the plasma membrane of skeletal muscle, are present in a rectilinear array that parallels the organization of the underlying contractile apparatus. Costameres have three major functions: to keep the plasma membrane, or sarcolemma, aligned and in register with nearby contractile structures; to protect the sarcolemma against contraction-induced damage; and to transmit some of the forces of contraction laterally, to the extracellular matrix. These functions require that costameres link the contractile apparatus through the membrane to the extracellular matrix. Mutations to key components of costameres cause these structures to lose their rectilinear organization and can result in muscle weakness or death. This article summarizes the evidence that costameres are composed of large complexes of integral and peripheral membrane proteins that are linked to the contractile apparatus by intermediate filaments and to the extracellular matrix by laminin. They also present evidence that costameres are altered when key costameric components are missing, as in a murine form of muscular dystrophy.