Preclinical research. Introduction. * Corresponding author. Tel: þ ; fax: þ address:

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1 European Heart Journal (2007) 28, do: /eurheartj/ehm241 Preclncal research Mult-parameter n vvo cardac magnetc resonance magng demonstrates normal perfuson reserve despte severely attenuated b-adrenergc functonal response n neuronal ntrc oxde synthase knockout mce Morel H. Vandsburger 1, Brent A. French 1,2,3,4, Patrck A. Helm 2,4, Rene Jack Roy 2, Chrstopher M. Kramer 2,3,4, Alstar A. Young 5, and Frederck H. Epsten 1,2,4 * 1 Department of Bomedcal Engneerng, Unversty of Vrgna, Charlottesvlle, VA 22908, USA; 2 Department of Radology, Unversty of Vrgna, Charlottesvlle, VA 22908, USA; 3 Department of Medcne, Unversty of Vrgna, Charlottesvlle, VA 22908, USA; 4 Cardovascular Research Center, Unversty of Vrgna Charlottesvlle, VA 22908, USA; and 5 Department of Anatomy wth Radology, The Unversty of Auckland, Auckland, New Zealand Receved 29 September 2006; revsed 14 May 2007; accepted 23 May 2007; onlne publsh-ahead-of-prnt 30 June 2007 KEYWORDS Neuronal ntrc oxde synthase; Cardac functon; Heart; MRI; Adrenergc stmulaton; Myocardal taggng Ams The role of neuronal ntrc oxde synthase (nnos) n regulatng contractle functon remans controversal, and n regulatng myocardal perfuson s unnvestgated. We used magnetc resonance magng (MRI) to phenotype nnos 2/2 and wld-type (WT) mce regardng left ventrcular (LV) structure, baselne functon, b-adrenergc responsveness, and perfuson reserve. Methods and results Cne MRI showed hgher LV mass to end-dastolc volume rato ( mg/ml nnos 2/2 vs mg/ml WT;P¼0.032) and LV ejecton fracton ( % nnos 2/2 vs % WT; P ¼ 0.003) n nnos 2/2. Myocardal taggng demonstrated smlar baselne systolc crcumferental stran (E cc ) n nnos 2/2 and WT. Wth dobutamne, the normal change n E cc was nearly absent n nnos 2/2 ( % nnos 2/2 vs % WT; P ¼ 0.001), and the systolc stran rate (de cc /dt) response to dobutamne seen n WT was reduced n nnos 2/2 ( %/s nnos 2/2 vs %/s WT; P ¼ 0.001). Dastolc stran rate ncreased sgnfcantly wth dobutamne only n WT. Arteral spn labellng showed that baselne perfuson and perfuson reserve wth ether dobutamne or an adenosne receptor agonst are normal n nnos 2/2. Concluson MRI provdes non-nvasve n vvo evdence that nnos does not play a role n basal contractle functon or myocardal perfuson, but s requred for ncreasng cardac notropy and lustropy upon b-adrenergc stmulaton. Introducton Ntrc oxde (NO) s generated n normal cardomyocytes by both neuronal ntrc oxde synthase (nnos) and endothelal NOS (enos) and, under dfferent crcumstances, can postvely or negatvely modulate cardac functon. 1 4 However, the specfc role of nnos n basal left ventrcular (LV) functon and also durng b-adrenergc stmulaton remans controversal. 1 Specfcally, one seres of studes found that, compared wth wld-type (WT) mce, solated myocytes from nnos 2/2 mce have a blunted cell shortenng response to b-adrenergc stmulaton wth soproterenol, 5 and smlarly, ntact hearts from nnos 2/2 mce have a blunted dp/dt response to soproterenol 5 or ncreased pacng frequency. 6 Conversely, other studes found greater shortenng under basal condtons 7,8 and n response to soproterenol n the solated myocytes of nnos 2/2 mce. 7 * Correspondng author. Tel: þ ; fax: þ E-mal address: fredepsten@vrgna.edu Interestngly, n vvo data from ths group showed decreased end-systolc elastance (E es ) and preload recrutable stroke work (PRSW) responses to dobutamne n nnos 2/2 mce. 9 The most recent n vtro data from ths seres of studes confrmed earler fndngs of greater cell shortenng at baselne and durng b-adrenergc stmulaton n nnos 2/2 myocytes when compared wth myocytes from WT mce, although both WT and nnos 2/2 showed smlar ncreases n shortenng over baselne. 10 Dvergent results may be due to methodologcal dfferences nvolvng temperature and concentratons of soproterenol. 2,11 Clarfcaton of the role of nnos s mportant as recent evdence suggests a sgnfcant role for nnos n heart falure. 9,12 14 Although the role of nnos n cardac functon has been examned prevously, less s known about the role of nnos n vascular tone and perfuson. Assessments of the nvolvement of nnos n basal vascular tone based upon measurements of systolc blood pressure have shown varablty. 15 Wth respect to mantanng vascular & The European Socety of Cardology All rghts reserved. For Permssons, please e-mal: journals.permssons@oxfordjournals.org

2 Mult-parameter MRI phenotypng of nnos 2/2 mce 2793 homeostass, 16 one study showed preferental augmentaton of vascular nnos expresson durng cerebral hypoxa. 17 In a separate study, hyperoxa caused a temporary depresson of regonal cerebral blood flow n WT mce whch was prolonged n nnos 2/2 mce. 18 Also, nnos has been shown to cause vasodlataton of the afferent arterole n the kdney, 15 as well as to contrbute to mantenance of renal homeostass. 15 The role of nnos n myocardal perfuson has not been nvestgated prevously. Cardac magnetc resonance (CMR) has become a reference standard modalty for non-nvasve phenotypng of the mouse heart n vvo. 19 In addton to cne MRI 20 for quantfyng myocardal mass, 21 LV volume, wall thckness, and ejecton fracton (EF), myocardal taggng 20,22 can be used to accurately assess contractle functon by measurng myocardal stran and stran rate, and arteral spn labellng (ASL) can unquely measure myocardal perfuson. 23,24 The purpose of the present study was to use these CMR technques to non-nvasvely and quanttatvely phenotype nnos 2/2 mce n terms of cardac structure, functon, responsveness to b-adrenergc stmulaton, and perfuson reserve upon pharmacologcal vasodlaton. Methods Anmals Sxteen WT and 16 nnos 2/2 male mce on a 129S4/SvJae * C57BL/6 background (Jackson Laboratory, Bar Harbor, Mane, USA) were studed under protocols that conformed to the Declaraton of Helsnk as well as the Gude for the Care and Use of Laboratory Anmals (NIH publcaton no , revsed 1996) and were approved by the Anmal Care and Use Commttee at our nsttuton. The WT mce were F2 hybrds of a B6129SF1/JB6129SF1/J cross used by Jackson Laboratory to approxmate lttermate controls. Assessment of basal LV structure and functon as well as stran and stran rate was performed at weeks of age, 2 days after whch systolc blood pressure was measured usng a non-nvasve tal plethysmography system (Model BP 2000,Vstech Systems, Apex, NC, USA). One week later, ASL was used to assess myocardal perfuson. Wth peak myocardal crcumferental shortenng (E cc ) as the prmary endpont, sample szes were powered to detect a dfference n mean DE cc wth dobutamne of 15% over baselne, wth an SD of 7%, power of 0.95 and sgnfcance of Cardac magnetc resonance preparaton Imagng was performed on a 4.7 T MRI system (Varan, Inc., Palo Alto, CA, USA) usng a custom-bult radofrequency (RF) col (Doty Scentfc, Columba, SC, USA). The RF col, desgned for ASL, conssted of an outer 6 cm long cylndrcal Ltz col for whole-body RF transmsson (used for non-selectve nverson n ASL) and an nner 2 cm long cylndrcal Ltz col for localzed sgnal recepton. Body temperature was mantaned at C by crculatng thermostated water, and anaesthesa was mantaned usng 1.25% soflurane n O 2 nhaled through a nose cone. The entre LV functon magng study took 2 h, and the ASL study took 2.5 h. Durng both experments the mouse lay prone wthn the scanner. Heart rate (HR), respraton, and rectal temperature were montored durng magng usng a fbre optc, MR compatble system (Small Anmal Imagng Inc., Stony Brook, NY, USA). Baselne left ventrcular structure and functon Baselne LV structure and functon were assessed usng a black blood cne technque as descrbed prevously. 20 Sx short-axs slces were acqured from base to apex, wth slce thckness equal to 1 mm, n-plane spatal resoluton of mm 2, and temporal resoluton of 8 12 ms. Baselne EF, end-dastolc volume (EDV), endsystolc volume (ESV), myocardal mass, wall thckness, and wall thckenng were measured from the cne mages usng the ARGUS analyss program (Semens Medcal Solutons, Prnceton, NJ, USA). EDV and ESV were then ndexed to body mass (EDVI and ESVI, respectvely). Mass to volume rato (MVR) was calculated as the rato of myocardal mass to EDV. One long-axs slce was also acqured at baselne wth dentcal magng parameters n seven WT and eght nnos 2/2 studes. Myocardal stran and stran rate Myocardal taggng was performed on two md-ventrcular short-axs slces as descrbed prevously 20,22 at baselne and durng the constant nfuson of dobutamne nto the ntrapertoneal (IP) cavty at doses of 20 and 40mg/kgmn durng the same magng sesson. To measure two-dmensonal myocardal stran wth hgh spatal resoluton, two orthogonal sets of lne-tagged mages were acqured at each slce locaton. Stran analyss of the tagged mages was performed usng the Fndtags method 25,26 generatng crcumferental shortenng data for three layers of myocardum: subendocardum, md-wall, and subepcardum. Peak md-wall crcumferental systolc stran (E cc ) and systolc stran rate (de cc /dt) were used to assess regonal contractle functon. Dastolc stran rate (de cc / dt) dastolc was used to assess regonal dastolc functon. Systolc and dastolc stran rates were calculated as the slopes of the contracton and relaxaton portons of the E cc -tme curve, respectvely. Longtudnal shortenng The global baselne shortenng n the longtudnal drecton was estmated by measurng the length of a lne drawn from the center of the mtral valve to the epcardal apex of the LV at end-dastole (ED) and end-systole (ES). Systolc blood pressure Systolc blood pressure was measured n WT and nnos /2 mce at baselne (n ¼ 10) and after constant IP nfuson of dobutamne at a dose of 20 mg/kgmn (n ¼ 6) usng dentcal anaesthesa and nfuson delvery as durng the MR procedure. A mnmum of fve baselne waveforms were obtaned for each mouse at both stmulaton levels. Systolc blood pressure measurements were obtaned from each waveform, and then averaged together for each ndvdual mouse. Myocardal perfuson ASL was used to quantfy myocardal perfuson at rest and ether durng IP nfuson of dobutamne (20 mg/kgmn) or after bolus IP njecton of the hghly selectve A 2A adenosne receptor agonst ATL313 (12.5 mg/kg body weght) (Adenosne Therapeutcs, Charlottesvlle, VA, USA). 23,24 The advantage of selectve A 2A adenosne receptor stmulaton s that t acheves maxmum vasodlataton wthout the deleterous sde-effects of adenosne. 27 Specfcally, the ASL method descrbed by Kober et al. 23 was used to measure myocardal T1 recovery curves after applcaton of both nonselectve and (2 mm thck) slce-selectve nverson pulses. A hyperbolc secant RF pulse was used for magnetzaton nverson. Estmaton of T1 followng the non-selectve (T1 ns ) and slce-selectve (T1 sel ) nverson pulses was performed usng a least-squares ft of the measured data to the theoretcal T1 recovery curve. ASL was performed n one mdventrcular short-axs slce wth an magng slce thckness of 1 mm and n-plane spatal resoluton of mm 2. After estmatng T1 ns and T1 sel, perfuson was calculated usng the equaton P ¼ (1/T1 sel 21/T1 ns )T1 ns /T1 blood l, 24 where T1 blood was measured as 1.5 s and the partton coeffcent (l) was

3 2794 M.H. Vandsburger et al. Statstcal analyses Statstcal analyses of myocardal structure, global functon, systolc blood pressure, and perfuson were performed usng SgmaStat (Systat Software Inc., Pont Rchmond, CA, USA). All these comparsons were made usng t-tests. Dfferences n E cc,de cc /dt, and (de cc /dt) dastolc at baselne, 20 mg/kgmn, and 40 mg/ kgmn dobutamne, and between WT and nnos 2/2 mce were assessed usng repeated measures analyses, assumng compound symmetry. Analyses repeated wth other assumed covarance structures, ncludng an unstructured covarance matrx, produced nearly dentcal results. Comparson of dose effects between WT and nnos 2/2 mce was performed usng F-tests. Comparsons between WT and nnos 2/2 mce for the dfference between baselne and 20 mg/kgmn dobutamne, or baselne and 40 mg/kgmn dobutamne were examned usng contrasts. The analyses were carred out n SAS PROC MIXED (SAS Inc., Cary, NC, USA). All values n the text, tables, and graphs are presented as mean + SEM. Results Left ventrcular structure Examples of mdventrcular ED and ES mages of WT and nnos 2/2 mce are shown n Fgure 1. As ndcated by lower EDV and ESV, as well as EDVI and ESVI (Table 1), nnos 2/2 mce had sgnfcantly smaller LV cavty sze and lower LV mass when compared wth WT mce at 8 weeks. However, MVR was sgnfcantly hgher (Table 1). No dfferences were observed n LV wall thckness, body mass, or LV mass ndexed to body mass (LVMI) between WT and nnos 2/2 mce (Table 1). Baselne left ventrcular functon and haemodynamcs Cne CMR revealed that nnos 2/2 mce have a sgnfcantly lower stroke volume (SV) than WT mce (Table 2). Also, as Table 1 Baselne left ventrcular structure for wld-type and nnos 2/2 mce WT (n ¼ 12) nnos 2/2 (n ¼ 10) P End-dastolc volume (ml) End-systolc volume (ml) End-dastolc volume ndex (ml/g) End-systolc volume ndex (ml/g) LV mass (mg) MVR (mg/ml) Wall thckness (mm) Body mass (g) LV mass/body mass (mg/g) Table 2 Baselne left ventrcular functon for wld-type and nnos 2/2 mce WT (n ¼ 12) nnos 2/2 (n ¼ 10) P Stroke volume (ml) Heart rate (b.p.m.) Cardac output ndex (ml/gmn) Ejecton fracton Wall thckenng (%) Table 3 Baselne and dobutamne (20 mg/kgmn) systolc blood pressure for wld-type and nnos 2/2 mce WT nnos 2/2 P Baselne systolc blood pressure (mmhg) (n ¼ 10) Dobutamne systolc blood pressure (mmhg) (n ¼ 6) Fgure 1 Typcal black-blood mdventrcular short-axs end-dastolc and end-systolc mages from cne MRI sets of wld-type and nnos 2/2 mce. Sx contguous short-axs cne mage slces spannng the heart from base to apex are used to assess three-dmensonal parameters such as chamber volumes and ejecton fracton. Cne MRI detected reduced end-dastolc volume ndex and end-systolc volume ndex as well as ncreased ejecton fracton and mass to volume rato n nnos 2/2 mce when compared wth WT mce. HR s hgher n nnos 2/2 mce (Table 2), cardac output ndexed to body mass was smlar between WT and nnos 2/2 mce (Table 2). In addton, nnos 2/2 mce have a hgher baselne EF than WT mce (Table 2), whch s explaned by ther hgher MVR (Appendx). Indeed, the rato of EF between WT and nnos 2/2 mce (0.86) was essentally equal to the rato of MVR between WT and nnos 2/2 mce (0.87). There were no dfferences between WT and nnos 2/2 mce n wall thckenng (Table 2). Baselne systolc blood pressure was lower n nnos 2/2 mce (Table 3). Baselne stran and stran rate Examples of ED and ES tagged mages of WT mce at baselne and durng dobutamne nfuson at 20 mg/kgmn are shown

4 Mult-parameter MRI phenotypng of nnos 2/2 mce 2795 smlar for WT and nnos 2/2 mce, ndcatng smlar baselne contractle functon. Baselne de cc /dt ( %/s WT, n ¼ 10 vs %/s nnos 2/2, n ¼ 10, P ¼ 0.047) was hgher n nnos 2/2 mce, whch, gven the baselne E cc results, reflects the ncreased heart rate of nnos 2/2 mce. Also, both groups had smlar baselne dastolc functon as assessed by (de cc /dt) dastolc ( %/s WT, n ¼ 10 vs %/s nnos 2/2, n ¼ 10, P ¼ 0.111). Baselne longtudnal shortenng as measured from the long-axs cne mages was also smlar between the two groups ( % WT, n ¼ 7, vs % nnos 2/2, n ¼ 8, P ¼ 0.857). Fgure 2 Examples of end-dastolc and end-systolc tagged mages from cne data sets of a wld-type mouse acqured at baselne and durng dobutamne nfuson (20 mg/kgmn). For each slce, two sets of mages were acqured wth tags appled n orthogonal drectons (the second drecton s not shown). Orthogonal lne-tagged mages are combned durng mage analyss for the computaton of two-dmensonal mdwall crcumferental shortenng (E cc ). Dobutamne response At 20 mg/kgmn dobutamne, there was a four-fold dfference n the change n E cc from baselne between WT and nnos 2/2 mce ( % WT, n ¼ 10 vs % nnos 2/2, n ¼ 10, P ¼ 0.001), whch was mantaned at 40 mg/kgmn dobutamne ( % WT, n ¼ 10 vs % nnos 2/2, n ¼ 10, P ¼ 0.001) (Fgure 4). In addton, sgnfcant ncreases n de cc /dt over baselne at 20 ( %/s WT, n ¼ 10 vs %/s nnos 2/2, n ¼ 10, P ¼ 0.001) and 40 mg/kgmn dobutamne ( %/s WT, n ¼ 10 vs %/s nnos 2/2, n ¼ 10, P ¼ 0.001) seen n WT mce were absent n nnos 2/2 mce. Smlarly, sgnfcant ncreases n (de cc /dt) dastolc n WT mce at both 20 (55 + 9%/s WT, n ¼ 10 vs %/s nnos 2/2, n ¼ 10, P ¼ 0.001) and 40 mg/kgmn dobutamne ( %/s WT, n ¼ 10 vs %/s nnos 2/2, n ¼ 10, P ¼ 0.001) were essentally absent n nnos 2/2 mce (Fgure 5). Whle dfferences n E cc and de cc /dt were apparent, WT and nnos 2/2 mce dsplayed smlar systolc blood pressure n response to 20 mg/ kgmn dobutamne (Table 3). Fnally, smlar HR responses to dobutamne were observed n WT and nnos 2/2 mce at 20 mg/kgmn dobutamne ( b.p.m. WT, n ¼ 10 vs b.p.m. nnos 2/2, n ¼ 10, P ¼ 0.435) and 40 mg/ kgmn dobutamne ( b.p.m. WT, n ¼ 10 vs b.p.m. nnos 2/2, n ¼ 10, P ¼ 0.791). Myocardal perfuson Myocardal perfuson was not sgnfcantly dfferent n nnos 2/2 mce at baselne ( ml/gmn WT, Fgure 3 E cc vs. tme curves showng mdwall crcumferental shortenng (E cc ) at baselne and durng dobutamne nfuson for wld-type and nnos 2/2 mce. Sgnfcant ncreases n E cc and stran rate (de cc /dt) are observed n wld-type mce upon dobutamne nfuson, however, ths ncrease s essentally absent n nnos 2/2 mce. n Fgure 2. Also, examples of E cc tme curves are shown for WT and nnos 2/2 mce at baselne and wth 20 mg/kgmn dobutamne n Fgure 3. Baselne E cc ( % WT, n ¼ 10 vs % nnos 2/2, n ¼ 10, P ¼ 0.640) was Fgure 4 Effect of b-adrenergc stmulaton on E cc. The change n E cc vs. baselne was greater n wld-type (n ¼ 10) vs. nnos 2/2 (n ¼ 10) mce at 20 and 40 mg/kgmn dobutamne.

5 2796 M.H. Vandsburger et al. Fgure 5 Effect of dobutamne on (de cc /dt) dastolc. Wld-type (n ¼ 10) mce show sgnfcant ncreases n (de cc /dt) dastolc over baselne at 20 and 40 mg/ kg mn dobutamne. No sgnfcant ncrease was measured n nnos 2/2 mce (n ¼ 10). Fgure 6 Myocardal perfuson as measured by arteral spn labellng s smlar n wld-type and nnos 2/2 mce at baselne (n ¼ 16), upon nfuson of dobutamne (20 mg/kgmn) (n ¼ 6), and upon applcaton of the vasodlator ATL313 (12.5 mg/kg body weght) (n ¼ 10). n ¼ 16 vs ml/gmn nnos 2/2, n ¼ 16, P ¼ 0.081), durng admnstraton of 20 mg/kgmn dobutamne ( ml/gmn WT, n ¼ 6 vs ml/gmn nnos 2/2, n ¼ 6, P ¼ 0.946), or after admnstraton of ATL313 ( ml/gmn WT, n ¼ 10 vs ml/gmn nnos 2/2, n ¼ 10, P ¼ 0.187) (Fgure 6). However, at baselne there was a trend towards hgher perfuson n nnos 2/2 mce. Perfuson reserve was smlar n the two groups for dobutamne ( WT, n ¼ 6 vs nnos 2/2, n ¼ 6, P ¼ 0.976) as well as ATL313 ( WT, n ¼ 10 vs nnos 2/2, n ¼ 10, P ¼ 0.151). Dscusson The major fndngs of ths study are that: () LV cavty sze s sgnfcantly lower n nnos 2/2 when compared wth WT mce; () baselne contractle functon s smlar n WT and nnos 2/2 mce despte hgher EF n nnos 2/2 mce whch s attrbuted to ncreased MVR (Appendx); () nnos 2/2 mce have severely blunted n vvo notropc and lustropc responses to dobutamne despte a normal HR response; and (v) nnos 2/2 mce have normal baselne perfuson and perfuson reserve n response to both dobutamne and an adenosne receptor agonst. In contrast to other technques, such as studyng solated myocytes or usng nvasve pressure volume catheters, CMR enables the non-nvasve quanttatve assessment of n vvo structure, functon, and perfuson. Our fndng of decreased LV cavty sze n nnos 2/2 mce agrees wth a trend towards lower ESV and EDV n nnos 2/2 mce shown by Khan et al. 6 Lkewse, our fndng of smlar LVMI n nnos 2/2 and WT mce agrees wth prevous fndngs 8 ncludng those n mce aged 2 months, 5 and 2 3 months, 12 but not wth ncreased LVMI found n nnos 2/2 mce at 4 months. 9 The hgher LVMI at older ages reflects progresson towards LV hypertrophy n nnos 2/2 mce after 8 weeks of age. 5,9 Results of prevous studes regardng the role of nnos n basal contractle functon have shown consderable varablty. Fndngs n nnos 2/2 mce of hgher EF, 8 ventrcular elastance (E es ), 9 [(dp/dt)/p d ) 5 as well as PRSW n sham-operated nnos 2/2 mce 9 have prevously led to conclusons of elevated basal contractle functon. 1,3,8 However, n agreement wth fndngs of smlar couplng of ventrcular to arteral elastance (E es /E a ) 5,6 and whole heart relaxaton values, 6 our E cc and (de cc /dt) dastolc data suggest that basal contractle functon and relaxaton, respectvely, are smlar n nnos 2/2 and WT mce. Gven smlar E cc n WT and nnos 2/2 mce, the heghtened de cc / dt n nnos 2/2 mce can be explaned by ncreased basal HR. Although our EF results agree wth echocardography, 8,9 EF s senstve to LV geometry. 28 Usng a cylndrcal model of the LV, we demonstrate (Appendx) that the ncreased MVR of nnos 2/2 mce, wth mantaned crcumferental and longtudnal shortenng, s suffcent to explan the ncreased EF n these mce. The dvergence of these fndngs from those prevously mentoned may be attrbuted to the fact that ths study was conducted n younger mce (8 weeks). In addton, decreased HR compared wth prevous studes 5,6,8,9,29 or lower baselne systolc blood pressure n nnos 2/2 compared wth WT mce may have masked subtle dfferences n baselne contractle functon seen n other studes. 5,8,9 Also, t s mportant to note that WT mce used n ths study were not lttermate controls of the nnos 2/2 mce, but separate mce of smlar genetc background. Our fndng of a nearly completely absent notropc response to b-adrenergc stmulaton n nnos 2/2 mce agrees wth the n vvo normalzed dp/dt and E es results of Barouch et al. 5 and PRSW and E es results of Dawson et al. 9 Because mdwall myofbres are crcumferentally orented, mdwall E cc and de cc /dt are approxmately n vvo measurements of myocyte shortenng and shortenng velocty, respectvely. The ncrease n mdwall E cc of 17% n WT mce at 20 mg/kgmn dobutamne mrrors the normal dobutamne response n humans. 30 In addton, myocardal taggng detected a related change n (de cc /dt) dastolc wth ncreasng dobutamne dose n WT but not n nnos 2/2 mce, mrrorng the fndng of an attenuated lustropc response to b-adrenergc stmulaton by Dawson et al. 9 Whle the E cc,de cc /dt, and (de cc /dt) dastolc responses were dfferent between WT and nnos 2/2 mce, the HR responses of both groups were smlar, ncreasng by approxmately 10 and 20% at dobutamne doses of 20 and 40 mg/kgmn, respectvely. In addton, the HR ncrease from 20 to 40 mg/kgmn dobutamne demonstrates that the dose of 20 mg/kgmn s submaxmal. Although we cannot exclude the possblty that dfferences n changes n LV relaxaton

6 Mult-parameter MRI phenotypng of nnos 2/2 mce 2797 wth dobutamne could result from ncreased MVR n nnos 2/2 mce, the presence of a HR response but absence of E cc,de cc /dt, and (de cc /dt) dastolc responses n nnos 2/2 mce supports the hypothess that NO from nnos s crtcal for enhanced contractle functon under b-adrenergc stmulaton. The relatonshp between myocardal perfuson and contractle reserve n nnos 2/2 mce has not been nvestgated prevously. Perfuson reserve measurements for WT mce were smlar to those reported by other groups. 23 Our fndng that perfuson reserve s smlar n WT and nnos 2/2 mce despte an attenuated contractle response to b-adrenergc stmulaton n nnos 2/2 mce helps to elucdate the role of nnos n contractle functon. Dobutamne serves as a strong b 1 and b 2 agonst, and a weak a 1 agonst. 31 The net effect of strong vascular b 2 (vasodlataton) and weak vascular a 1 (vasoconstrcton) stmulaton s vasodlataton 31 and s manfested n both WTand nnos 2/2 mce as ncreased myocardal perfuson. The mpact of a strong cardac b 1 agonst s ncreased cardac notropy, whch was seen only n WT mce as ncreased E cc and de cc /dt. Vasodlataton wthout ncreased notropy n nnos 2/2 mce ndcates that the absence of a contractle reserve s not due to nadequate perfuson reserve. Further, smlar ncreases n myocardal perfuson wth ATL313 n WT and nnos 2/2 mce show that nnos 2/2 mce are not perfuson-lmted even under condtons of greater vasodlataton. When examned alongsde smlar ncreases n HR and systolc blood pressure n both WT and nnos 2/2 mce n response to dobutamne, the attenuated contractle response of nnos 2/2 mce ndcates a cardomyocyte-specfc role for nnos wth respect to modulaton of contractle functon. A lmtaton of ths study s that LV pressure was not measured durng magng, thus any potental dfferences n LV preload and afterload and consequent mpacts on LV contractle functon were unaccounted for. A second lmtaton s that our measurements were made under the nfluence of soflurane, whch at hgh concentratons s a vasodlator. 23 However, we mnmzed the dose of soflurane, and mantaned physologcal body temperature durng MR scannng. Furthermore, both groups of mce were maged under the same condtons and demonstrated smlar perfuson reserve. In addton, the nnos 2/2 mce used n ths study had systemc, and not cardac-specfc nnos ablaton. Through ts partcpaton n autonomc regulaton of perpheral targets, 15 nnos may affect n vvo LV functon n a manner not seen n n vtro experments. We were unable to solate ths effect n our experments, whch could be partally responsble for dfferences seen between n vvo and n vtro results. In lght of the controversy regardng nnos, 1 3 our fndngs ndcate that systemc nnos gene deleton does not affect LV systolc functon under basal condtons but leads to a severe attenuaton of the n vvo contractle response to b-adrenergc stmulaton wthout affectng myocardal perfuson. Our study also ndcates that adequate ncreases n perfuson are avalable to fuel any ncreased oxygen demand requred to enhance contractle functon upon b-adrenergc stmulaton. As our study utlzed non-nvasve myocardal taggng and ASL CMR methods, these data add to our understandng of the n vvo role of nnos. In agreement wth prevous n vvo studes our fndngs ndcate that NO from nnos serves as a crtcal sgnallng agent for ncreasng the notropc 5 and lustropc 9 functonal responses to b-adrenergc stmulaton. Increased knowledge of the role of nnos may be clncally mportant gven recent fndngs of ncreased nnos mrna and proten expresson and translocaton of nnos to the sarcolemma n human heart falure. 14,32 Acknowledgements Ths work was supported by NIH grant RO1 EB and by an Amercan Heart Assocaton Establshed Investgator Award to F.H.E. Conflct of nterest: none declared. Appendx We propose that ncreased EF found n nnos 2/2 mce n ths study can be attrbuted to the ncreased MVR n ths group. It s well known that EF s senstve to LV geometry, and n partcular to the MVR. Followng 33 we use an ncompressble cylndrcal model of the LV, and calculate EF as a functon of geometry and shortenng. Let the nner and outer rad of the cylnder be R and R o, respectvely, at ED and r and r o at ES, respectvely. The EF s EF ¼ 1 LR2 lr 2 LR 2 ¼ 1 l L l 2 C where L s the LV length at ED, l s the length at ES. l L s the extenson rato n the longtudnal drecton. l L ¼ l L ¼ 1 %S L 100% ; l C ¼ r R ¼ 1 %S C 100% where %S L s the percent longtudnal shortenng and %S C s the percent crcumferental shortenng at the nner surface. l C s the extenson rato n the crcumferental drecton at the nner surface. l L ¼ l L ¼ %S L 100% ; l C ¼ r R ¼ 1 %S C 100% We defne a mdwall surface whch dvdes the mass of the heart nto equal parts,.e. a radus R m such that LR 2 o 2LR 2 m ¼ LR 2 m 2LR 2,or R 2 m ¼ (R 2 o þr 2 )/2. At ES, myocardum at R m has moved to r m. Owng to ncompressblty, the volume of myocardum between R m and R at ED s preserved at ES. lðrm 2 r2 Þ¼LðR2 m R2 Þ or LR 2 lr 2 ¼ LR 2 m lr2 m The EF s therefore related to the mdwall extenson rato l Cm ¼ r m /R m. EF ¼ LR2 lr 2 LR 2 ¼ LR2 m lr2 m LR 2 ¼ LR2 m lr2 m R 2 m LR 2 ¼ LR2 m lr2 m R 2 m R m LR 2 m R 2 ¼ð1 l L l 2 Cm Þ R m R Concentrc hypertrophy can be quantfed by the MVR: MVR ¼ LR2 o LR2 LR 2 ¼ R 2 o 1; R whch can be wrtten as MVR ¼ ðr2 o R2 Þ R 2 ¼ 2R2 m R 2 2:

7 2798 M.H. Vandsburger et al. Substtutng ths equaton nto the above relaton for EF yelds EF ¼ð1 l L l 2 Cm Þ 1 2 MVR þ 1 : The EF can therefore be seen to depend on the longtudnal shortenng (l L ), the mdwall crcumferental shortenng (l Cm ) and the degree of concentrc hypertrophy (MVR). In the current study, the contracton n the longtudnal and crcumferental drectons was found to be smlar between WT and nnos 2/2 mce. The rato of the mean WT EF to the mean nnos 2/2 EF was The rato of mean (0.5MVRþ1) n each group was These results agree wth the above equaton, and support the hypothess that the ncreased MVR s suffcent to explan the ncreased EF. References 1. Ballgand JL. La Donna e Moble. : s cardac neuronal ntrc oxde synthase such a dsconcertng enzyme? Crculaton 2005;112: Zolo MT, Bers DM. The real estate of NOS sgnalng: locaton, locaton, locaton. Crc Res 2003;92: Masson PB, Feron O, Dessy C, Ballgand JL. Ntrc oxde and cardac functon: ten years after, and contnung. 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