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• W2000624583 abstract "In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca2+/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo. In contrast to studies on skeletal and smooth muscles, the identity of kinases in the heart that are important physiologically for direct phosphorylation of myosin regulatory light chain (RLC) is not known. A Ca2+/calmodulin-activated myosin light chain kinase is expressed only in cardiac muscle (cMLCK), similar to the tissue-specific expression of skeletal muscle MLCK and in contrast to the ubiquitous expression of smooth muscle MLCK. We have ablated cMLCK expression in male mice to provide insights into its role in RLC phosphorylation in normally contracting myocardium. The extent of RLC phosphorylation was dependent on the extent of cMLCK expression in both ventricular and atrial muscles. Attenuation of RLC phosphorylation led to ventricular myocyte hypertrophy with histological evidence of necrosis and fibrosis. Echocardiography showed increases in left ventricular mass as well as end-diastolic and end-systolic dimensions. Cardiac performance measured as fractional shortening decreased proportionally with decreased cMLCK expression culminating in heart failure in the setting of no RLC phosphorylation. Hearts from female mice showed similar responses with loss of cMLCK associated with diminished RLC phosphorylation and cardiac hypertrophy. Isoproterenol infusion elicited hypertrophic cardiac responses in wild type mice. In mice lacking cMLCK, the hypertrophic hearts showed no additional increases in size with the isoproterenol treatment, suggesting a lack of RLC phosphorylation blunted the stress response. Thus, cMLCK appears to be the predominant protein kinase that maintains basal RLC phosphorylation that is required for normal physiological cardiac performance in vivo. Sarcomeric proteins in myocytes account for contraction of the heart that depends on the molecular motor myosin in the thick filaments binding to actin in thin filaments to initiate shortening and force development (1Gordon A.M. Homsher E. Regnier M. Physiol. Rev. 2000; 80: 853-924Crossref PubMed Scopus (1342) Google Scholar, 2Moss R.L. Razumova M. Fitzsimons D.P. Circ. Res. 2004; 94: 1290-1300Crossref PubMed Scopus (124) Google Scholar, 3Solaro R.J. J. Biol. Chem. 2008; 283: 26829-26833Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Myosin cross-bridges contain an actin-binding surface and ATP pocket in the motor domain that taper to an α-helical neck connecting to the myosin rod region responsible for the self-assembly into thick filaments. Two small protein subunits, the essential light chain and the phosphorylatable RLC, 2The abbreviations used are: RLCregulatory light chainMLC2vventricular RLCMLC2aatrial RLCcMLCKcardiac myosin light chain kinaseMLCKmyosin light chain kinaseZIPKzipper-interacting protein kinasecTnIinhibitory subunit of cardiac troponinMYPT2myosin protein targeting subunit 2 of myosin light chain phosphataseLVleft ventricularLVEDDleft ventricular end-diastole dimensionLVESDleft ventricular end-systole dimensionLVIDleft ventricular internal diameterLVPWleft ventricular posterior wall. wrap around each α-helical neck region providing mechanical stability (4Rayment I. J. Biol. Chem. 1996; 271: 15850-15853Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). RLC is necessary for assembly of thick filaments in cardiac myocytes, and mutations in RLC are linked to inherited hypertrophic cardiomyopathy (5Rottbauer W. Wessels G. Dahme T. Just S. Trano N. Hassel D. Burns C.G. Katus H.A. Fishman M.C. Circ. Res. 2006; 99: 323-331Crossref PubMed Scopus (110) Google Scholar, 6Poetter K. Jiang H. Hassanzadeh S. Master S.R. Chang A. Dalakas M.C. Rayment I. Sellers J.R. Fananapazir L. Epstein N.D. Nat. Genet. 1996; 13: 63-69Crossref PubMed Scopus (493) Google Scholar). There are two types of cardiac RLCs, a ventricular myosin light chain, MLC2v, and an atrium-specific form, MLC2a (7Collins J.H. J. Muscle Res. Cell Motil. 2006; 27: 69-74Crossref PubMed Scopus (7) Google Scholar). regulatory light chain ventricular RLC atrial RLC cardiac myosin light chain kinase myosin light chain kinase zipper-interacting protein kinase inhibitory subunit of cardiac troponin myosin protein targeting subunit 2 of myosin light chain phosphatase left ventricular left ventricular end-diastole dimension left ventricular end-systole dimension left ventricular internal diameter left ventricular posterior wall. In heart and skeletal muscle Ca2+ binds to troponin in the actin thin filament, thereby allowing myosin heads to attach to actin for sarcomeric force development and shortening (8Kobayashi T. Solaro R.J. Annu. Rev. Physiol. 2005; 67: 39-67Crossref PubMed Scopus (277) Google Scholar). Additionally, phosphorylation of RLC in fast-twitch skeletal muscle fibers by a skeletal muscle-specific Ca2+/calmodulin-dependent MLCK modulates the contractile response by potentiating frequency-dependent force development (9Sweeney H.L. Bowman B.F. Stull J.T. Am. J. Physiol. 1993; 264: C1085-C1095Crossref PubMed Google Scholar, 10Zhi G. Ryder J.W. Huang J. Ding P. Chen Y. Zhao Y. Kamm K.E. Stull J.T. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 17519-17524Crossref PubMed Scopus (159) Google Scholar, 11Ryder J.W. Lau K.S. Kamm K.E. Stull J.T. J. Biol. Chem. 2007; 282: 20447-20454Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In the heart, phosphorylation of multiple sarcomeric proteins adjusts myofilament protein interactions and thus fine-tunes the troponin-dependent contraction (3Solaro R.J. J. Biol. Chem. 2008; 283: 26829-26833Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 12Olsson M.C. Patel J.R. Fitzsimons D.P. Walker J.W. Moss R.L. Am. J. Physiol. Heart Circ. Physiol. 2004; 287: H2712-H2718Crossref PubMed Scopus (103) Google Scholar, 13Sweeney H.L. Stull J.T. Am. J. Physiol. 1986; 250: C657-C660Crossref PubMed Google Scholar). The basal phosphorylation of RLC (40–50%) in beating hearts is maintained by slow rates of phosphorylation and dephosphorylation (14High C.W. Stull J.T. Am. J. Physiol. 1980; 239: H756-H764PubMed Google Scholar, 15Herring B.P. England P.J. Biochem. J. 1986; 240: 205-214Crossref PubMed Scopus (25) Google Scholar, 16Silver P.J. Buja L.M. Stull J.T. J. Mol. Cell. Cardiol. 1986; 18: 31-37Abstract Full Text PDF PubMed Scopus (36) Google Scholar, 17Scruggs S.B. Hinken A.C. Thawornkaiwong A. Robbins J. Walker L.A. de Tombe P.P. Geenen D.L. Buttrick P.M. Solaro R.J. J. Biol. Chem. 2009; 284: 5097-5106Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). RLC phosphorylation in skinned fibers increases the extent and rate of force development while decreasing sarcomeric interfilament spacing (9Sweeney H.L. Bowman B.F. Stull J.T. Am. J. Physiol. 1993; 264: C1085-C1095Crossref PubMed Google Scholar, 12Olsson M.C. Patel J.R. Fitzsimons D.P. Walker J.W. Moss R.L. Am. J. Physiol. Heart Circ. Physiol. 2004; 287: H2712-H2718Crossref PubMed Scopus (103) Google Scholar, 18Colson B.A. Locher M.R. Bekyarova T. Patel J.R. Fitzsimons D.P. Irving T.C. Moss R.L. J. Physiol. 2010; 588: 981-993Crossref PubMed Scopus (124) Google Scholar). It is also proposed that altered RLC phosphorylation may contribute to compensatory responses and contractile dysfunction in human diseases (19van der Velden J. Papp Z. Boontje N.M. Zaremba R. de Jong J.W. Janssen P.M. Hasenfuss G. Stienen G.J. Cardiovasc. Res. 2003; 57: 505-514Crossref PubMed Scopus (118) Google Scholar). Kinases that phosphorylate RLC in the heart have not been clearly identified, although there are two primary candidates, cMLCK and ZIPK. Skeletal muscle MLCK was reported to be present in heart (20Davis J.S. Hassanzadeh S. Winitsky S. Lin H. Satorius C. Vemuri R. Aletras A.H. Wen H. Epstein N.D. Cell. 2001; 107: 631-641Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar), but its abundance is too low to maintain RLC phosphorylation (10Zhi G. Ryder J.W. Huang J. Ding P. Chen Y. Zhao Y. Kamm K.E. Stull J.T. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 17519-17524Crossref PubMed Scopus (159) Google Scholar). Cardiac RLC is not a good substrate for the ubiquitous smooth muscle MLCK, which is present in cardiac myocytes and probably phosphorylates nonmuscle cytoplasmic myosin II-B (10Zhi G. Ryder J.W. Huang J. Ding P. Chen Y. Zhao Y. Kamm K.E. Stull J.T. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 17519-17524Crossref PubMed Scopus (159) Google Scholar, 21Stull J.T. Nunnally M.H. Michnoff C.H. Krebs E.G. Boyer P.D. The Enzymes. Academic Press, Orlando1986: 113-166Google Scholar, 22Dudnakova T.V. Stepanova O.V. Dergilev K.V. Chadin A.V. Shekhonin B.V. Watterson D.M. Shirinsky V.P. Cell. Motil. Cytoskeleton. 2006; 63: 375-383Crossref PubMed Scopus (7) Google Scholar, 23Ma X. Takeda K. Singh A. Yu Z.X. Zerfas P. Blount A. Liu C. Towbin J.A. Schneider M.D. Adelstein R.S. Wei Q. Circ. Res. 2009; 105: 1102-1109Crossref PubMed Scopus (45) Google Scholar). Recently, a novel cMLCK was identified in human heart failure and found to be regulated by the cardiac homeobox protein Nkx2-5 during development (24Seguchi O. Takashima S. Yamazaki S. Asakura M. Asano Y. Shintani Y. Wakeno M. Minamino T. Kondo H. Furukawa H. Nakamaru K. Naito A. Takahashi T. Ohtsuka T. Kawakami K. Isomura T. Kitamura S. Tomoike H. Mochizuki N. Kitakaze M. J. Clin. Invest. 2007; 117: 2812-2824Crossref PubMed Scopus (123) Google Scholar, 25Chan J.Y. Takeda M. Briggs L.E. Graham M.L. Lu J.T. Horikoshi N. Weinberg E.O. Aoki H. Sato N. Chien K.R. Kasahara H. Circ. Res. 2008; 102: 571-580Crossref PubMed Scopus (122) Google Scholar). Suppression of cMLCK expression in zebra fish embryos led to ventricular dilation with incomplete sarcomere formation, whereas overexpression in neonatal myocytes promoted sarcomere organization and increased cell contractility. Cardiac RLC is also a good biochemical substrate for ZIPK, and knockdown of the kinase in neonatal myocytes by siRNA inhibited RLC phosphorylation (26Chang A.N. Chen G. Gerard R.D. Kamm K.E. Stull J.T. J. Biol. Chem. 2010; 285: 5122-5126Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Thus, ZIPK has emerged as another candidate kinase that may phosphorylate RLC. We have ablated cMLCK expression in mice to determine its role in RLC phosphorylation. Because it appears to be the primary kinase for basal RLC phosphorylation that may modulate cardiac function, we also evaluated cardiac adaptations resulting from RLC dephosphorylation to obtain insights into the physiological role for cMLCK. The strategy to study the physiological role of cMLCK in cardiac myocytes was to modify the cMLCK gene to ablate expression at different developmental times from embryonic to adult stages (27Maillet M. Davis J. Auger-Messier M. York A. Osinska H. Piquereau J. Lorenz J.N. Robbins J. Ventura-Clapier R. Molkentin J.D. J. Biol. Chem. 2010; 285: 6716-6724Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Our approach involved development of a LoxP-targeted allele in mice that will allow different cardiac expressing Cre transgenes to affect cMLCK expression at different developmental times. However, because others suggested that cMLCK was a cardiac myocyte-specific kinase (24Seguchi O. Takashima S. Yamazaki S. Asakura M. Asano Y. Shintani Y. Wakeno M. Minamino T. Kondo H. Furukawa H. Nakamaru K. Naito A. Takahashi T. Ohtsuka T. Kawakami K. Isomura T. Kitamura S. Tomoike H. Mochizuki N. Kitakaze M. J. Clin. Invest. 2007; 117: 2812-2824Crossref PubMed Scopus (123) Google Scholar, 25Chan J.Y. Takeda M. Briggs L.E. Graham M.L. Lu J.T. Horikoshi N. Weinberg E.O. Aoki H. Sato N. Chien K.R. Kasahara H. Circ. Res. 2008; 102: 571-580Crossref PubMed Scopus (122) Google Scholar), we decided initially to affect kinase expression by retaining the marker cassette containing the neomycin resistance gene (neo) as part of the initial floxed allele to generate a hypomorphic allele (28Lewandoski M. Nat. Rev. Genet. 2001; 2: 743-755Crossref PubMed Scopus (630) Google Scholar). Genomic regions of the mouse cMLCK (MYLK3) locus were isolated from 129SvEv genomic DNA by LA Taq™ polymerase (Takara Bio) and cloned into targeting vector OS.DUP/DEL with a TK cassette for negative selection and a neo cassette for positive selection. A 2.6-kb genomic sequence (5′-targeting arm, short arm) upstream of cMLCK exon 4, bounded by an upstream XhoI site and a downstream ClaI site, was cloned upstream of the 5′ loxP sequence in the targeting vector. A 4.4-kb genomic sequence (3′-targeting arm, long arm) downstream of cMLCK exon 4, bounded by an upstream SalI site and a downstream SalI site (including BamHI BglII recognition sequence at the 3′ end for subsequent screening), was cloned downstream of the 3′ loxP sequence. A 1.2-kb region (conditional knock-out region, knock-out arm), including exon 4, bounded by an upstream NdeI site and a downstream AflII site, was cloned between the 5′ loxP sequence and the 5′ FRT sequence. The resulting vector was verified by DNA sequencing and restriction mapping. The vector was linearized at the PvuI site downstream of the 3′-targeting arm and electroporated into 129SvEv-derived embryonic stem cells. Cells were then treated with G418, and negative selection was accomplished by gancyclovir. Southern blot analysis was performed using probes located 5′ of the 5′-targeting arm and 3′ of the 3′-targeting arm (Fig. 1). Accurate recombination was verified by sequencing genomic PCR products derived from primers located 5′ of the 5′-targeting arm and within the neomycin resistance cassette and 3′ of the 3′-targeting arm and within the neomycin resistance cassette. Three cMLCK-targeted embryonic stem clones were identified. Two clones were expanded and injected into C57BL/6 blastocysts that were transferred to the uterus of pseudopregnant females. High percentage chimeric male mice (cMLCK+/neo) were bred into a C57BL/6 background to obtain germ line transmission. We generated mice with a cMLCK hypomorphic allele (cMLCKneo/neo) by intercrossing cMLCK+/neo to each other. All experiments on mice were conducted in a 129SvEv/C57Bl/6 mixed background. Genotyping was performed by Southern blotting with 5′ and 3′ probes. Animals were housed under standard conditions and maintained on commercial mouse chow and water ad libitum. The environment was maintained at 22 °C with a 12-h light/12-h dark cycle. All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Texas Southwestern Medical Center. Southern blot probes were generated by PCR using the following primer sets: 5′ probe forward, 5′-CTGGGACTGGGATTATAGACAATTGTG-3′, reverse, 5′-GGTCTAATTAACAGCATGGCCAATGG-3′; and 3′ probe forward, 5′-GGGTCATAGCCATCATTGCACAG-3′, reverse, 5′-GTTAAAGACCATACTTGAGACTCGAGCC-3′. In brief, tail genomic DNA was digested with BglII (5′ screening) or BamHI (3′ screening) and analyzed using a standard Southern blot protocol. Total RNA was purified from isolated heart ventricles with TRIzol reagent (Invitrogen) according to the manufacturer's instructions. Two micrograms of RNA were used as template to synthesize cDNA using random hexamers. Quantitative PCR was performed using the following TaqMan® probes purchased from Applied Biosystems: ANP, Mm01255748_g1; BNP, Mm00435304_g1; Col1a2, Mm00483888_m1; Myh6, Mm00440354_m1; Myh7, Mm00600555_m1; and rodent GAPDH, 4308313. Analyses were performed by the comparative CT method. Initial data were normalized to GAPDH; relative values were obtained by normalizing to the mean for cMLCK+/+ ventricles. For Western blot analysis, hearts were isolated for dissection in less than 2 min, frozen in liquid nitrogen, and stored at −80 °C until homogenization. Changes in RLC phosphorylation occur on the order of 30–45 min in heart, so immediate fixation in situ is not essential to measure the extent of phosphorylation that reflects in vivo values (15Herring B.P. England P.J. Biochem. J. 1986; 240: 205-214Crossref PubMed Scopus (25) Google Scholar, 16Silver P.J. Buja L.M. Stull J.T. J. Mol. Cell. Cardiol. 1986; 18: 31-37Abstract Full Text PDF PubMed Scopus (36) Google Scholar). Tissues were homogenized in 10% trichloroacetic acid and 10 mm dithiothreitol at 0 °C, and total proteins were collected by centrifugation at 2,000 rpm for 1 min in a tabletop centrifuge. Protein pellets were solubilized into 8 m urea as described previously (10Zhi G. Ryder J.W. Huang J. Ding P. Chen Y. Zhao Y. Kamm K.E. Stull J.T. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 17519-17524Crossref PubMed Scopus (159) Google Scholar, 29Huang G. Yao J. Zeng W. Mizuno Y. Kamm K.E. Stull J.T. Harding H.P. Ron D. Muallem S. J. Cell Sci. 2006; 119: 153-161Crossref PubMed Scopus (54) Google Scholar, 30Ding H.L. Ryder J.W. Stull J.T. Kamm K.E. J. Biol. Chem. 2009; 284: 15541-15548Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The protein pellets were readily solubilized following the low centrifugation force for a short time. Muscle samples were subjected to urea/glycerol-PAGE to separate phosphorylated and nonphosphorylated RLC as described previously (31Huang J. Shelton J.M. Richardson J.A. Kamm K.E. Stull J.T. J. Biol. Chem. 2008; 283: 19748-19756Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Because the urea/glycerol-PAGE system separates nonphosphorylated from the phosphorylated RLC, we have a direct quantitative measure of RLC phosphorylation in terms of percent phosphorylation. Because the separation results from a single phosphate, data may also be calculated as mol of phosphate/mol of RLC. Diphosphorylation results in additional migration of RLC in the urea-PAGE system, but cardiac muscle has very little diphosphorylated RLC (26Chang A.N. Chen G. Gerard R.D. Kamm K.E. Stull J.T. J. Biol. Chem. 2010; 285: 5122-5126Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 31Huang J. Shelton J.M. Richardson J.A. Kamm K.E. Stull J.T. J. Biol. Chem. 2008; 283: 19748-19756Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Quantitative measurements were processed on a Storm PhosphorImager and analyzed by ImageQuant software. Additional Western blotting was performed by SDS-PAGE on other proteins solubilized in the 8 m urea buffer. For preparation of soluble proteins, tissues were homogenized in buffer at pH 7.6 containing (in mm) Tris-HCl 50, EGTA 2, EDTA 2, NaCl 150, dithiothreitol, 1% Nonidet P-40, and 10 μl/ml protease inhibitor mixture (Sigma). Contractile proteins were pelleted by centrifugation at 7,000 × g for 10 min. Equal volumes of total and supernatant fractions were subjected to SDS-PAGE. Antibodies to cMLCK, MYPT2, and MLC2a were raised to bacterially expressed mouse protein (Proteintech Group, Inc.). Antibodies to MLC2v from bovine heart were previously described (31Huang J. Shelton J.M. Richardson J.A. Kamm K.E. Stull J.T. J. Biol. Chem. 2008; 283: 19748-19756Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Antibody to smooth muscle MLCK (K36) was obtained from Sigma. Antibody for GAPDH was obtained from Santa Cruz Biotechnology. Measurements of total cTnI and phosphorylation at Ser-23 and Ser-24 were performed by Western blots with antibodies from Research Diagnostics, Inc. (17Scruggs S.B. Hinken A.C. Thawornkaiwong A. Robbins J. Walker L.A. de Tombe P.P. Geenen D.L. Buttrick P.M. Solaro R.J. J. Biol. Chem. 2009; 284: 5097-5106Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 32Gomes A.V. Harada K. Potter J.D. J. Mol. Cell. Cardiol. 2005; 39: 754-765Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Before histological evaluation, hearts were dissected from anesthetized mice, fixed, and then processed into paraffin according to routine procedures (31Huang J. Shelton J.M. Richardson J.A. Kamm K.E. Stull J.T. J. Biol. Chem. 2008; 283: 19748-19756Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Four-chamber longitudinal views were sectioned at the level of the aortic and pulmonary valves (31Huang J. Shelton J.M. Richardson J.A. Kamm K.E. Stull J.T. J. Biol. Chem. 2008; 283: 19748-19756Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). The size of cardiac myocytes was measured following wheat germ agglutinin staining as described previously (23Ma X. Takeda K. Singh A. Yu Z.X. Zerfas P. Blount A. Liu C. Towbin J.A. Schneider M.D. Adelstein R.S. Wei Q. Circ. Res. 2009; 105: 1102-1109Crossref PubMed Scopus (45) Google Scholar), except an optical fractionator probe of Stereo Investigator software (MBF Bioscience) was used to obtain an unbiased estimate of myocyte areas. Echocardiograms were performed on conscious, gently restrained mice using either a Sonos 5500 system with a 15-MHz linear probe or Vevo 2100 system with a MS400C scanhead. Left ventricular internal diameter at end-diastole (LVEDD) and end-systole (LVESD) were measured from M-mode recordings. Fractional shortening was calculated as (LVEDD − LVESD)/LVEDD (%). Measurements of interventricular septum thickness, left ventricular internal diameter, and left ventricular posterior wall thickness were made from two-dimensional parasternal short axis views in diastole. Left ventricular mass was calculated by the cubed method as 1.05 × ((IVS + LVID + LVPW)3 − LVID3) (mg), where IVS is interventricular septum thickness; LVID is left ventricular internal diameter; LVPW is left ventricular posterior wall thickness (33Collins K.A. Korcarz C.E. Shroff S.G. Bednarz J.E. Fentzke R.C. Lin H. Leiden J.M. Lang R.M. Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H1954-H1962Crossref PubMed Google Scholar). All measurements were made at the level of papillary muscles. Mice were treated with isoproterenol for 7 days to induce cardiac hypertrophy (31Huang J. Shelton J.M. Richardson J.A. Kamm K.E. Stull J.T. J. Biol. Chem. 2008; 283: 19748-19756Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Isoproterenol at 40 mg/ml/g of mouse in saline or saline itself was injected into an Alzet® mini-osmotic pump (model 2001, Durect Corp.), which releases at 1.0 μl/h. Pumps were surgically implanted on the back during anesthesia. Echocardiographic measurements were performed before and after the isoproterenol infusion. At the end of the treatment, mice were anesthetized (250 mg/kg Avertin, intraperitoneal) and weighed. Whole hearts were removed, weighed, and quick-frozen in liquid nitrogen. Tibial length was also measured. Data are expressed as mean ± S.E. Statistical evaluation was carried out by using an unpaired Student's t test for two comparisons or analysis of variance (plus the Newman-Keuls method) for multiple comparisons of data with variance homoscedasticity assessed by the Bartlett method. Kruskal-Wallis rank-sum and Nemenyi tests were used in multiple comparisons for data not meeting the homoscedastic variance test. Significance was accepted at a value of p < 0.05. The mRNA for cMLCK was previously shown to be expressed in ventricular and atrial muscle of the heart with no significant expression in other tissues, including skeletal or smooth muscles as well as nonmuscle tissues (24Seguchi O. Takashima S. Yamazaki S. Asakura M. Asano Y. Shintani Y. Wakeno M. Minamino T. Kondo H. Furukawa H. Nakamaru K. Naito A. Takahashi T. Ohtsuka T. Kawakami K. Isomura T. Kitamura S. Tomoike H. Mochizuki N. Kitakaze M. J. Clin. Invest. 2007; 117: 2812-2824Crossref PubMed Scopus (123) Google Scholar, 25Chan J.Y. Takeda M. Briggs L.E. Graham M.L. Lu J.T. Horikoshi N. Weinberg E.O. Aoki H. Sato N. Chien K.R. Kasahara H. Circ. Res. 2008; 102: 571-580Crossref PubMed Scopus (122) Google Scholar). We have obtained similar results with Northern and Western blotting of cMLCK in diverse tissues, which emphasizes the tissue-specific expression of cMLCK (data not shown). We also immunostained for cMLCK in adult mouse hearts showing specific expression in both ventricular and atrial cardiac myocytes (Fig. 2). The kinase appeared localized in the cytoplasm so we determined biochemically if it was associated with myofilaments (Fig. 2). Comparison of cMLCK in total tissue homogenates with that in supernatant fractions after removal of myofilaments by centrifugation showed that cMLCK was soluble in both ventricular and atrial myocytes, similar to the solubility of skeletal muscle MLCK (34Nunnally M.H. Stull J.T. J. Biol. Chem. 1984; 259: 1776-1780Abstract Full Text PDF PubMed Google Scholar), and in contrast to myofilament binding of smooth muscle MLCK (35Smith L. Parizi-Robinson M. Zhu M.S. Zhi G. Fukui R. Kamm K.E. Stull J.T. J. Biol. Chem. 2002; 277: 35597-35604Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Additionally, the amount of cMLCK expression appeared greater in atria than in ventricles. cMLCK was distributed evenly throughout all portions of the ventricles. Because cMLCK is a cardiac myocyte-specific kinase, we decided initially to perturb kinase expression by retaining the marker cassette containing the neomycin resistance gene (neo) as part of the initial floxed allele to generate a hypomorphic allele (28Lewandoski M. Nat. Rev. Genet. 2001; 2: 743-755Crossref PubMed Scopus (630) Google Scholar). The long range strategy was to have animals in which the neo cassette could be removed and then ablate the gene in adult mice with conditional Cre expression. A targeting vector containing a neo cassette included frt sites as well as loxP sites flanking exon 4 (Fig. 1). Southern analysis of both the 5′ and 3′ arms of the targeted cMLCK gene demonstrated successful recombination and germ line transmission (Fig. 1). Additionally, insertion of the neo cassette disrupted cMLCK expression in both atrial and ventricular myocytes (Fig. 3). Expression of cMLCK protein in ventricular and atrial tissues from cMLCKneo/neo mice was undetectable, whereas the amount in cMLCK+/neo mice was about 50% that found in wild type mice. Interestingly, the partial reduction of cMLCK protein in cMLCK+/neo mice led to a partial reduction of RLC phosphorylation in both ventricular (MLC2v) and atrial (MLC2a) muscles (Fig. 3). The extent of RLC phosphorylation in ventricular and atrial tissues from cMLCKneo/neo mice was less than 5% that obtained for hearts from wild type animals. The extent of basal RLC phosphorylation appears to be dependent on the amount of cMLCK expressed, thus indicating the kinase activity is a limiting factor for RLC phosphorylation. We removed the neo cassette by crossing Flp-deleter mouse strain to mice containing the neo cassette flanked by frt sites and confirmed crossing results by Southern analysis. The resulting mice containing single (cMLCK+/f) or double (cMLCKf/f) floxed alleles without the neo cassette had similar amounts of cMLCK protein as wild type mice. The relative amounts were 100 ± 1, 104 ± 6, and 99 ± 10% (mean ± S.E., n = 5) for wild type, cMLCK+/f, and cMLCKf/f mice, respectively. The extent of MLC2v phosphorylation was also not different with 0.42 ± 0.2, 0.42 ± 0.01, and 0.41 ± 0.02 for wild type, cMLCK+/f, and cMLCKf/f mice, respectively. The morphological properties of hearts in the three different groups appeared normal. Thus, the insertion of the neo cassette appears to be selective for disrupting cMLCK expression. We also measured the protein contents of other related proteins, including MLC2v, MLC2a, MYPT2, cTnI, and smooth muscle MLCK (Table 1). There were no differences in the amounts of these proteins in cMLCK+/+, cMLCK+/neo, and cMLCKneo/neo mice. Thus, the loss of cMLCK protein or insertion of the neo cassette did not affect expression of these related proteins.TABLE 1Relative expression of proteins related to cardiac myosinMeasurementsap < 0.05 for comparisons to cMLCK+/+ mice.cMLCK+/+cMLCKneo/neo%%MLC2v100 ± 2099 ± 9MLC2a100 ± 7102 ± 4MYPT2100 ± 1188 ± 12cTnI100 ± 796 ± 10Smooth muscle MLCK100 ± 1687 ± 12a p < 0.05 for comparisons to cMLCK+/+ mice. Open table in a new tab The thin filament protein cTnI plays an important role in Ca2+ sensitivity of myofilaments, and it was recently reported that overexpression of a nonphosphorylatable MLC2v resulted in a marked compensatory decrease in cTnI phosphorylation (17" @default.
• W2000624583 created "2016-06-24" @default.
• W2000624583 creator A5004332219 @default.
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• W2000624583 title "Cardiac Myosin Light Chain Kinase Is Necessary for Myosin Regulatory Light Chain Phosphorylation and Cardiac Performance in Vivo" @default.
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