intracellular nitric oxide ( noi ) is a free radical that is synthesized by a family of nitric oxide synthases ( noss ) , consisting of endothelial nos ( enos or nos1 ) , inducible nos ( inos or nos2 ) , and neuronal nos ( nnos or nos3 ) isoforms . enos and nnos are constitutively expressed , whereas inos is up - regulated under pathological conditions such as sepsis , hypertension , hypertrophy and heart failure [ 1 , 2 ] . thus , noi production plays an important role in regulating cardiomyocyte function during excitation - contraction coupling , mitochondrial respiration , hypertrophic remodeling , apoptosis , and myocardial regeneration . the local effects of endogenous noi production on cardiomyocyte structure and function are dependent upon the localization of the different nos isoforms . for instance , enos is associated with caveolin-3 within caveoli , where it locally regulates  - adrenergic receptor stimulation . nnos is associated with sarcoplasmic reticulum ( sr ) membranes , where it modulates sr ca  uptake and release [ 24 ] . during different pathological conditions , noi production shifts from spatially and temporally regulated noi production to dysregulated , excessive release . the relative abundance of nos expression and activity in diseased hearts also changes , with nnos and inos being upregulated , whereas enos is downregulated . during these pathological states , inos is localized throughout the cytoplasm and increased inos expression and noi production then contribute to myocardial dysfunction and reduced myocardial responsiveness to  - adrenergic stimulation [ 68 ] . previous studies have demonstrated that inos expression is significantly increased in the intact , volume - overloaded heart , which is mediated in part by systemic production of tnf- , il-1 , angiotensin ii , and other cytokines [ 1 , 9 ] . however , inos expression and noi production can also be induced ex vivo by static stretch of cultured neonatal rat ventricular myocytes ( nrvm ) , suggesting the involvement of a mechanosensitive signaling pathway in cardiomyocytes that operates independently of circulating growth factors and cytokines . indeed , cardiomyocytes can directly transduce physical forces into biochemical signals and generate appropriate responses leading to alterations in cellular structure and function . costameres ( and focal adhesions , their functional equivalent in cultured cells ) are important subcellular structures responsible for mechanotransduction in cardiomyocytes ( for review , see ) . a complex signaling web connects mechanosensory and growth factor - dependent signal transduction pathways in cardiomyocytes , and many different downstream effectors are activated in response to mechanical loading . mechanotransduction via integrins and their accessory nonreceptor protein tyrosine kinases ( focal adhesion kinase ( fak ) and proline - rich tyrosine kinase 2 ( pyk2 ) ) resembles the downstream signals generated following activation of receptor tyrosine kinases , and their interconnecting signaling pathways share critical components with signaling pathways activated in response to peptide growth factors and cytokines . these responses include acute alterations in contractile function , as well as long - term , structural changes in cardiomyocyte size , shape , and gene expression .   in a previous study , we demonstrated that electrical pacing of quiescent , freshly isolated adult cat ventricular myocytes acutely increased noi  production by a mechanochemical signaling pathway that required ca - calmodulin ( cam ) , phosphatidylinositol-3-kinase ( pi(3)k ) , and akt . based upon relatively specific small - molecule inhibitors of the various nos isoforms , acute stimulation of noi production by electrical pacing and mechanical activity involved both enos and nnos . in this paper , we examined whether cardiomyocyte contractile activity also regulated noi production over a longer time period , via the contraction - dependent upregulation of inos expression in spontaneously contracting nrvm . we also investigated the roles of fak , pyk2 , pi(3)k , and akt in this process . pc-1 tissue culture medium was obtained from biowhittaker ( walkersville , md , usa ) . dulbecco 's modified eagle medium ( dmem ) and medium 199 were obtained from gibco brl ( grand island , ny , usa ) . fak , inos , enos and c - terminal pyk2/crnk monoclonal antibodies ( mab ) were purchased from bd transduction laboratories ( san jose , ca , usa ) . phospho - specific fak - y397 and fak - y577 polyclonal ( pab ) antibodies were purchased from biosource international , camarillo , ca . phospho - specific pyk2-y402 , akt - t308 , akt - s473 , and total akt pab were purchased from cell signaling technology ( danvers , ma , usa ) . n - terminal pyk2 pab was obtained from biolegend ( san diego , ca , usa ) . horseradish - peroxidase - conjugated goat anti - rabbit and goat anti - mouse iggs were obtained from biorad ( hercules , ca , usa ) . pf573228 and blebbistatin were obtained from tocris bioscience ( minneapolis , mn , usa ) . all other reagents were of the highest grade commercially available and were obtained from sigma ( st . louis , mo , usa ) and baxter s / p ( mcgaw park , il ) . animals used in these experiments were handled in accordance with the guiding principles in the care and use of animals , approved by the council of the american physiological society . nrvms were isolated from the hearts of 2-day old sprague - dawley rats by collagenase digestion , as previously described . cells were preplated for 1  h in serum - free pc-1 medium to reduce nonmyocyte contamination . the nonadherent nrvms were then plated at a density of 1600 cells per mm onto collagen - coated 60  mm dishes and left undisturbed in a 5% co2 incubator at 37c for 36  h. unattached cells were removed by aspiration , washed twice in hbss , and the attached cells were maintained in a solution of dmem / medium 199 ( 4  :  1 ) containing antibiotic / antimycotic solution . at this density , spontaneous contractile activity ( ~100150 beats per min ) was visible within 24  h of plating . nrvms were then infected ( 24  h ) with replication - defective adenoviruses ( adv ) diluted in dmem / medium 199 . medium was replaced with virus - free dmem / medium 199 , treated with inhibitors or cultured for an additional 672  h.   advs expressing shrnas specific for rat fak ( shfak ) , rat pyk2 ( shpyk2 ) , and firefly luciferase ( shluc ) were generated as previously described . to inhibit fak - dependent signal transduction , nrvms were infected ( 5  moi , 24  h ) with replication - defective adenoviruses ( adv ) expressing either gfp - frnk   or y397f - fak , the latter of which was kindly provided by dr . to inhibit pyk2-dependent signaling , a replication - defective adv - expressing flag - tagged , human cell adhesion kinase- related non - kinase ( crnk ) was kindly provided by dr . fak - dependent signaling was increased using replication - defective adv expressing either wildtype ( wt ) fak   or a  constitutively active  fak fusion protein ( cd2-fak )   which was constructed as previously described . akt - dependent signaling was increased using a replication - defective adv - expressing either wt - akt , or constitutively active akt ( myr - akt ) , which were kindly provided by dr . k. walsh , tufts university school of medicine , boston , ma , usa . in each experiment , a replication - defective adv expressing either gfp   or nuclear - encoded -galactosidase ( negal )   was used to control for nonspecific effects of adv infection . the multiplicity of viral infection ( moi ) was determined by viral dilution assay in hek293 cells grown in 96 well clusters . at moi of 510 , > 95% of the cells were infected , as determined by adv - negal infection and x - gal staining , and there were no cytotoxic effects of adv infection during the 2472  h following adv infection . nrvms were homogenized in lysis buffer , and equal amounts of extracted proteins ( 50100  g ) were separated by sds - page and western blotting on 10% polyacrylamide gels . primary antibody binding was detected with horseradish peroxidase - conjugated goat anti - mouse or goat anti - rabbit secondary antibodies and visualized by enhanced chemiluminescence ( pierce biotechnology , rockford , il , usa ) . developed films were then scanned on a hp deskjet 4890 scanner , and band intensity was quantified using un - scan - it gel software , ver . band intensity obtained with the phosphospecific antibody was divided by the band intensity for total protein ( phosphorylated + nonphosphorylated ) and then normalized to the intensity of the control sample . measurements of noi production were obtained using the fluorescent no - sensitive dye 4,5-diaminofluorescein diacetate ( daf-2 da ) [ 25 , 26 ] as previously described . cells were incubated with membrane - permeant daf-2 da ( 2  m ; 40  min ) at room temperature in 3  ml standard tyrode solution containing 100  m l - arginine . daf-2 fluorescence was excited at 480  nm and emitted cellular fluorescence was recorded at 540  nm . cellular daf-2 fluorescence intensity ( f ) was quantified with image j software . control measurements of noi obtained in different experiments were grouped together and compared to drug treatment or adenoviral infection groups . data were compared using one - way anova followed by student - newman - keuls test , one - way anova on ranks followed by dunn 's test , student - newman - keuls test , or paired t - test , where appropriate . data were analyzed using sigmaplot for windows , ver . 9.0 ( systat software , san jose , ca , usa ) . spontaneously contracting nrvms were treated with the l - type calcium channel blockers nifedipine and verapamil ( which inhibit both spontaneous [ ca]i transients and contractile activity ) or the myosin ii atpase inhibitors butanedione monoxime ( bdm ) and blebbistatin ( which block contractile activity but have relatively little effects on spontaneous [ ca]i transients ) [ 14 , 2830 ] . inos and enos expression was then examined by sds - page and western blotting under basal conditions and in response to contractile arrest . as seen in figure 1(a ) , nrvm expressed substantial quantities of both enos and inos under basal conditions . treatment with the various inhibitors of spontaneous contractile activity all significantly reduced inos expression in a dose - dependent fashion ( figures 1(b ) and 1(c ) ) , with little or no effect on enos expression levels ( figure 1(a ) ) . as both types of contractile inhibitors substantially reduced inos expression , these results suggested that mechanical activity per se , rather than a ca - dependent , contraction - independent signaling pathway was required . as shown in figure 1(d ) , nifedipine , bdm and blebbistatin all reduced noi production as compared to spontaneously contracting , control nrvm . interestingly , nifedipine reduced noi production to a greater extent than either bdm or blebbistatin , suggesting that at the time of noi measurement , both [ ca]i and mechanical activity contributed to enhanced noi production . the partial [ ca]i dependence of noi production is consistent with the known role of ca - cam in regulating enos activity . 
 to investigate the role of fak and pi(3)k in regulating contraction - dependent inos expression , nrvms were first treated with pf573228 or ly294002 , which are potent and highly selective inhibitors of fak   and pi(3)k   activities , respectively . as seen in figures 2(a ) and 2(b ) , both agents substantially reduced inos levels in nrvm . of note , at the concentrations used here , neither agent appeared to affect spontaneous contractile activity in these high - density cultures . to confirm the role of fak in regulating inos expression , nrvms were also infected with adv - expressing shrnas specific for fak ( shfak ) . an adv - expressing a shrna specific for firefly luciferase ( shluc ) was used to control for nonspecific effects of adv infection and shrna expression . as seen in figure 2(c ) , inos expression was also reduced by using 2 other inhibitors of fak - dependent signaling ( i.e. , gfp - frnk and y397f - fak ; data not shown ) . previous studies have demonstrated that fak autophosphorylation at y397 provides a docking site for the binding and tyrosine phosphorylation of the p85 subunit of pi(3)k , which is required for its activation in nrvm [ 34 , 35 ] and other cell types [ 3641 ] . as both fak and pi(3)k activities appeared necessary to regulate contraction - dependent inos expression , we next examined whether fak was also involved in regulating akt . as seen in figures 3(a ) and 3(b ) , spontaneously contracting nrvm demonstrated high levels of basal akt activation , as detected by western blotting with antibodies specific for akt phosphorylated at t308 and s473 . in contrast , nrvms arrested with l - type ca channel blockers or inhibitors of myosin atpase demonstrated reduced fak and akt phosphorylation . shrna - induced fak knockdown also reduced akt phosphorylation at both sites ( figure 3(c ) ) . in contrast , shrna - mediated knockdown of pyk2 , the other member of the fak family of protein tyrosine kinases , had much less of an effect on contraction - dependent akt phosphorylation . similar negative results were observed by overexpressing crnk , the c - terminal inhibitor of pyk2 ( data not shown ) . finally , adv - mediated overexpression of a form of fak ( cd2-fak )   was sufficient to further increase akt phosphorylation in spontaneously contracting nrvms ( figures 4(a ) and 4(b ) ) .   to further examine the role of fak , pi(3)k , and akt in regulating contraction - dependent inos expression bdm inhibition of inos expression by overexpressing wildtype ( wt ) fak ,  constitutively active fak ( i.e. , cd2-fak ) , wt - akt , or a constitutively active form of akt ( myr - akt ) . as seen in figures 5(a ) and 5(b ) , overexpression of wt - fak had no effect on the bdm - mediated inhibition of inos expression . overexpression of cd2-fak in bdm - arrested nrvm caused a relative ~8-fold increase in inos expression , thus restoring its expression to levels above that observed in contracting cells ( figures 5(a ) and 5(b ) ) . similarly , myr - akt overexpression also reversed the inhibitory effect of bdm ( figures 5(a ) and 5(c ) ) . to confirm these results , we performed the rescue experiment on bdm - treated cells but measured noi production using daf-2-da . both wt - akt and myr - akt were sufficient to restore noi production under bdm inhibition ( myr - akt > wt - akt ) ( figure 5(d ) ) . these results confirmed that fak and akt are both necessary and sufficient for contraction - mediated inos expression and noi production in nrvm . as demonstrated in this report and our previous study , cardiomyocyte contractile activity is an important factor that regulates noss and noi production via ca - dependent and ca - independent signaling pathways . as demonstrated in figure 1 , nifedipine reduced noi production to a greater extent than either bdm or blebbistatin . previous studies have also shown that acute mechanical stimulation , such as , by sheer stress [ 43 , 44 ] and static stretch   stimulate enos - dependent noi production in endothelial cells . a similar effect of acute mechanical loading on noi production was observed in cardiomyocytes [ 14 , 31 , 46 , 47 ] . however , in these studies , the acute load - dependent phosphorylation of enos at s1177 was likely the responsible mechanism . furthermore , enos activity can be regulated via activation of pi(3)k - akt or ca - cam signaling in different cell types [ 14 , 44 , 48 ] . in our study , the additive effect of inhibition of ca influx and contractile arrest led to a further reduction in noi production as compared to the inhibition of mechanical activity alone . these results are consistent with other observations that ca - dependent and ca - independent noss are both involved in mechanical stimulation - induced noi production . in our previous study , we showed that either decreased ( bdm or blebbistatin ) or increased ( emd 57033 or cgp 48506 ) contractile strength affected changes in field stimulation - induced noi production without changing ca influx ( 12 ) . here we demonstrate that contractile activity can also induce inos gene expression and noi production through a fak - pi(3)k - akt signaling pathway without interfering with ca handling . a schematic diagram outlining this signaling pathway is depicted in figure 5(e ) . previous studies have shown that mechanical stretch can induce both inos and enos expression in nrvm [ 10 , 49 ] . however in the present study , we could only detect a significant reduction in inos ( but not enos ) expression when cells were treated with l - type ca channel blockers or myosin ii atpase inhibitors . these results suggest that load - induced enos upregulation may be mediated by a different signaling mechanism . .   also indicated that the stretch - induced ca signal was very likely the trigger for subsequent noi signaling in cardiomyocytes , and the noi signal further amplified itself by increasing inos expression . however , both types of inhibitors could not completely eliminate inos expression or noi production , suggesting that contraction - induced inos expression may also be subjected to noi autoregulation . our experiments utilized nrvm plated at high density ( ~1  10  cells / cm ) in order to enhance spontaneous contractile activity and increase inos expression . under these conditions , fak and akt were also highly phosphorylated , but their phosphorylation could be reduced by inhibitors of contractile activity . have also shown that dense myocyte cultures displayed higher metabolic activity and contraction rates as compared to cells plated at low density . for instance , obese type 2 diabetic ( db / db ) mice have significantly reduced cardiac contractility and depressed pi(3)k - akt signaling . conversely , acute pressure overload increased fak , pi(3)k , and akt activation in the intact heart . these data suggest that cardiomyocyte contractility can activate several downstream signaling cascades in order to maintain cardiac function . although fak appears both necessary and sufficient to induce akt activation in nrvm , the intermediary steps linking contractile activity to fak and akt activation remain unclear . as indicated in our previous paper , contraction - induced noi production required pi(3)k and akt , as the highly specific pi(3)k inhibitor ly294002 , or overexpression of a dominant - negative mutant of akt , partially blocked contraction - dependent noi production . similar results were obtained by del re et al . , who showed that static stretch - induced akt activation required fak and pi(3)k . furthermore , we recently showed that adhesion - dependent akt activation in adult atrial myocytes was also dependent on fak and pi(3)k . in this regard , there are several potential mechanisms whereby pi(3)k may be involved . first , previous studies in nonmuscle cells have indicated that the p85 regulatory subunit of pi(3)k is tyrosine phosphorylated by fak and can directly bind to the fak - y397 autophosphorylation site , thereby activating the lipid kinase [ 3641 ] . wei and vander heide   showed that fak and p85 formed a complex in unstimulated nrvms , and their association increased in response to heat stress . the heat stress - induced complexation of fak and p85 was associated with an increase in akt phosphorylation , which was prevented by frnk overexpression . del re et al .   also demonstrated a similar static stretch - induced complexation of fak and p85 in nrvm . thus , a direct interaction between fak and pi(3)k appears to be operative in cardiomyocytes . have shown that angiotensin ii stimulated the tyrosine phosphorylation and activation of pdk1 in vascular smooth muscle cells via a pyk2- and c - src - dependent pathway . have shown that in nrvm , a similar pathway is activated by h2o2 , leading to downstream phosphorylation of akt at both t308 and s473 . although we demonstrate that pyk2 is not necessary for contraction - induced akt activation , it is conceivable that fak , or the fak / src complex may function in a similar manner , thereby causing the tyrosine phosphorylation of pdk1 and subsequent akt activation . thus , the requirement for pi(3)k to mediate contraction - induced akt activation would be conferred through the generation of phosphoinositides necessary for pdk1 and akt recruitment to the cell membrane and not directly via formation of a fak / pi(3)k complex . of note , xia et al .   demonstrated that ligation of 1 integrins ( accomplished with an anti-1 integrin antibody ) activated both fak and akt in a src- and pi(3)k - dependent manner , although fak - p85 complexation could not be demonstrated . several studies have used genetically modified animal models to address the function of inos in the heart . three different research groups have shown that inos deletion improved ventricular function , reduced myocardial nitrotyrosine content , decreased apoptosis , and improved survival after experimental myocardial infarction [ 5658 ] . zhang et al .   also demonstrated that inos deletion protected against pressure overload - induced ventricular hypertrophy and heart failure , in association with decreased myocardial nitrotyrosine and 4-hydroxy-2-nonenal . have shown that cardiac - specific inos overexpression caused increased myocardial peroxynitrite production , myocardial fibrosis , ventricular hypertrophy , heart failure , and sudden cardiac death . however , other studies have shown contradictory results , in that inos deficiency did not reduce myocardial infarct - induced ventricular dysfunction or mortality . in fact , other studies demonstrated that inos plays a cardioprotective role during myocardial ischemia / reperfusion [ 62 , 63 ] . thus , the function of inos in the cardiomyocyte remains unclear , and additional studies are needed to evaluate the role of contractile activity - induced inos expression in regulating cardiomyocyte function and cell survival .
intracellular nitric oxide ( noi ) is a physiological regulator of excitation - contraction coupling , but is also involved in the development of cardiac dysfunction during hypertrophy and heart failure . to determine whether contractile activity regulates nitric oxide synthase ( nos ) expression , spontaneously contracting , neonatal rat ventricular myocytes ( nrvm ) 
 were treat with l - type calcium channel blockers ( nifedipine and verapamil ) or myosin ii atpase inhibitors ( butanedione monoxime ( bdm ) and blebbistatin ) to produce contractile arrest . 
 both types of inhibitors significantly reduced inos but not enos expression , and also reduced noi production . inhibiting 
 contractile activity also reduced focal adhesion kinase ( fak ) and akt phosphorylation . 
 contraction - induced inos expression required fak and phosphatidylinositol 3-kinase ( pi(3)k ) , as both pf573228 and ly294002 ( 10  m , 24  h ) eliminated contraction - induced inos expression . 
 similarly , shrnas specific for fak ( shfak ) caused fak knockdown , reduced akt phosphorylation at t308 and s473 , and reduced inos expression . in contrast , shrna - mediated knockdown of pyk2 , the other member of the fak - family of protein tyrosine kinases , had much less of an effect . 
 conversely , overexpression of a constitutively active form of fak ( cd2-fak ) or akt ( myr - akt ) reversed the inhibitory effect of bdm on inos expression and noi production . 
 thus , contraction - induced inos expression and noi production in nrvm are mediated via a fak - pi(3)k - akt signaling pathway .