Effects of Fixed-Dose Isosorbide Dinitrate/Hydralazine onDiastolic Function and Exercise Capacity in

Hypertension-Induced Diastolic Heart FailureRichard M. Wilson, Deepa S. De Silva, Kaori Sato, Yasuhiro Izumiya, Flora Sam

Abstract—Hypertension-induced diastolic heart failure accounts for a large proportion of all heart failure presentations.Hypertension also induces left ventricular (LV) hypertrophy. Fixed-dose isosorbide dinitrate/hydralazine (HISDN)decreased mortality in human systolic heart failure but it is unknown whether it improves maladaptive myocardialremodeling. We sought to test the hypothesis that chronic HISDN modulates LV hypertrophy and myocardialremodeling in hypertension-induced diastolic heart failure. FVB mice underwent either saline (n�18) or aldosterone(n�28) infusion. All underwent uninephrectomy and drank 1% salt water for 4 weeks. Mice were randomized aftersurgery to regular chow or chow containing HISDN (isosorbide dinitrate: 26 mg/kg per day; hydralazine: 50 mg/kg perday) for 4 weeks. Aldosterone infusion increased tail-cuff blood pressure (161�3 mm Hg) versus saline-infused mice(129�2 mm Hg). Aldosterone induced LV hypertrophy versus saline-infused mice (LV:body weight ratio: 4.2�0.1versus 3.6�0.1 mg/g). HISDN attenuated the aldosterone-induced increased in systolic blood pressure (137�5 mm Hg)and also lowered blood pressure in saline-infused mice (114�2 mm Hg). However, HISDN did not cause LVhypertrophy regression in aldosterone-infused mice. Aldosterone increased LV end-diastolic dimensions that were notattenuated by HISDN. Similarly, neither aldosterone infusion nor HISDN affected LV end-systolic dimensions. LVejection fraction and wet:dry lung ratio were not different between aldosterone-untreated and aldosterone-HISDN mice.However, mitral Doppler E/A ratio (a measure of diastolic function), exercise capacity, and plasma soluble vascular celladhesion molecule 1 levels were improved in aldosterone-HISDN hearts. In conclusion, fixed-dose HISDN improvedhypertension, diastolic function, and exercise capacity and reduced soluble vascular cell adhesion molecule 1 levels.There were no reductions in LV hypertrophy, cardiac fibrosis, or pulmonary congestion. These functional improvementsare likely related to extracardiac effects, such as effects on the vasculature. (Hypertension. 2009;54:583-590.)

Key Words: diastolic heart failure � hydralazine � nitrates � hypertension � exercise capacity

Hypertension induces left ventricular (LV) hypertrophy(LVH) and is a major cause of both diastolic and

systolic heart failures (HF).1 Diastolic HF accounts for up to�50% of all HF presentations2 and is associated withincreasing morbidity and mortality.3 Diastolic HF refers tothe clinical syndrome of pulmonary congestion in the pres-ence of a normal LV ejection fraction, whereas diastolicdysfunction denotes an abnormality of mechanical propertiesthat exist during LV relaxation and filling.4,5 Diastolic dys-function may be an intermediary between hypertension andHF.6 In the setting of hypertension, diastolic HF represents adiverse clinical syndrome with various associated comorbidi-ties (eg, age and sex) and manifests a spectrum of symptomsranging from exercise intolerance to acute pulmonary edema.In addition to LVH, cardiac fibrosis, altered myocyte calciumhandling,7 and ventricular-vascular stiffening plays a signif-icant role in the pathophysiology of diastolic HF.7

In the African-American Heart Failure Trial (AHeFT),fixed-dose isosorbide dinitrate and hydralazine (HISDN)reduced mortality in black patients with advanced systolicHF.8,9 The predominant cause of HF in AHeFT was hyper-tension.9 Benefits of HISDN are partially derived from nitricoxide (NO),10 which results in vasorelaxation, inhibition ofcardiac hypertrophy,11 and improved cardiac remodeling.11–13

Unfortunately the use of NO donors is limited by thedevelopment of tolerance. With HISDN, the component ofhydralazine (a potent antioxidant) is thought to diminish NOconsumption by reactive oxygen species14,15 and reactivenitrogen species.16 The survival benefits seen in AHeFT werereported to be attributed to myocardial effects, because LVejection fraction improved, and LV mass was reduced.17

However, there are no experimental studies thus far that testthis hypothesis. We, therefore, sought to test the hypothesisthat chronic fixed-dose HISDN modulates LVH and adversemyocardial remodeling in hypertension-induced diastolic HF.

Received April 20, 2009; first decision May 12, 2009; revision accepted June 17, 2009.From the Whitaker Cardiovascular Institute (R.M.W., D.S.D.S., K.S., Y.I., F.S.), Cardiovascular Section (F.S.), and Evans Department of Medicine

(F.S.), Boston University School of Medicine, Boston, Mass.Correspondence to Flora Sam, Whitaker Cardiovascular Institute, Boston University School of Medicine, Evans Department of Medicine and

Cardiovascular Section, 715 Albany St, Room W507, Boston, MA 02118. E-mail flora.sam@bmc.org© 2009 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.109.134932

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Methods and MaterialsAn expanded Materials and Methods section is available in theonline Data Supplement at http://hyper.ahajournals.org. Ten-week–old male FVB mice (Charles River) were maintained on a 12-hourlight/dark cycle in a temperature-controlled (19°C to 21°C) room.Mice were fed standard rodent chow ad libitum. The BostonUniversity School of Medicine Institutional Animal Care and UseCommittee approved all of the study procedures related to handlingand surgery of the mice.

ResultsHISDN Improved Hypertension But Had NoEffect on Cardiac HypertrophyAll of the mice survived (Table 1). HISDN prevented thealdosterone-induced increase in systolic blood pressure (SBP;137�5 versus 161�3 mm Hg; P�0.001). Heart rate tendedto be lower with aldosterone infusion but was unaffected byHISDN. The body weight (BW) of all of the HISDN-treatedmice was increased; therefore, morphological measurementswere normalized to BW. This was attributed to the HISDN-chow being made with lactose. Aldosterone caused LVH, asshown by an increase in the LV weight:BW ratio (4.2�0.1).However, despite the reduction in SBP in aldosterone-HISDNmice, there was no decrease in the LV weight:BW ratio.Cardiomyocyte cross-sectional area was increased in re-sponse to aldosterone infusion. Similar to the LV weight:BWratio, cardiomyocyte hypertrophy was no different betweenaldosterone-untreated and aldosterone-HISDN hearts. Aldo-sterone infusion induced pulmonary congestion that wasunaffected by HISDN.

HISDN Had No Effect on Cardiac Structure andSystolic FunctionAldosterone infusion increased LV end-diastolic dimensionsbut not end-systolic dimensions. LV end-diastolic dimensions

and LV end-systolic dimensions were unaltered by HISDN(Figure 1A and 1B). LV fractional shortening was unchangedand not different between aldosterone-untreated and aldoste-rone-HISDN hearts (Figure 1C). Consistent with LVH andcardiomyocyte hypertrophy, total wall thickness was in-creased with aldosterone and unaffected by HISDN (Figure1D and Table 1).

HISDN Improved Diastolic DysfunctionAldosterone infusion induced diastolic HF (normal LV sys-tolic function and pulmonary congestion; Table 2).18 Mitralvalve inflow velocity was determined by Doppler echocardi-ography. Measurements were standardized at a lower andcomparable heart rate in all of the groups to control forloading.19,20 The normal physiological heart rate in mice israpid (�600 bpm) and results in the loss of the A velocity.19,20

Aldosterone increased the mitral E velocity (early diastolicfilling velocity) with minimal change in late diastolic filling(mitral A wave). The resultant E/A ratio (diastolic dysfunc-tion) was, therefore, increased, indicating reduced LV com-pliance (increased stiffness). Consistent with the elevated E/Aratio, the isovolumetric contraction time was increased,whereas deceleration time tended to shorten with aldosteroneinfusion. HISDN treatment significantly decreased mitral Evelocity and increased the mitral A wave (Table 2). As aresult, the E/A ratio was decreased, indicating a morecompliant LV. HISDN shortened both deceleration time andisovolumetric contraction time, indicating improved LV re-laxation and compliance (Figure 1E). HISDN improveddiastolic function, although “clinical” evidence of pulmonarycongestion was unaltered (Table 1).

HISDN Had No Effect on Cardiac Fibrosis andCytokine ExpressionTo determine whether the improvement in diastolic dysfunc-tion was associated with a reduction in cardiac fibrosis, bothinterstitial and perivascular fibrosis were measured. Totalfibrosis (interstitial and perivascular fibrosis) was increasedin both groups of aldosterone-infused mice but was notdifferent between aldosterone-untreated and aldosterone-HISDN hearts by quantitative analysis (Figure 2A and 2B).

Aldosterone also induces a proinflammatory response.21

Macrophage infiltration by ED-1 staining was measured andshown to be present in the interstitial areas of the LV. HISDNdid not alter ED-1 staining (Figure 3A). Myocardial cytokinemRNA levels and quantitative RT-PCR were performed withspecific primer sets with normalization to GAPDH. Aldoste-rone infusion increased LV interferon-� expression 2.6-fold(P�0.05), interleukin (IL) 6 expression 2.8-fold (P�0.05),and tumor necrosis factor (TNF)-� expression 1.8-fold(P�0.01) compared with saline infusion (Figure 3B through3E). HISDN decreased interferon-� and IL-6 transcripts(P�0.05 versus aldosterone untreated) with no effect onTNF-� expression. LV IL-1� expression was negligiblyaltered by aldosterone infusion or therapy with HISDN(Figure 3E).

HISDN and Fetal Gene Re-ExpressionAldosterone infusion increased myocardial atrial natriureticpeptide (ANP) mRNA expression by �2.0-fold (P�0.05

Table 1. Characteristics of Untreated and HISDN-Treated Mice4 Weeks After Saline or Aldosterone Infusion

Groups

Untreated HISDN

Saline Aldosterone Saline Aldosterone

No. 5 10 5 10

Body weight, g 28.1�0.9 28.4�0.7 30.8�0.7§ 31.1�0.5¶

SBP, mm Hg 129�2 161�3† 114�2†† 137�5‡�

HR, bpm 729�5 690�9 745�22 683�17

LV/BW, mg/g 3.6�0.1 4.2�0.1†† 3.8�0.1 4.3�0.1**

Lung, wet:dry ratio

4.3�0.2 5.4�0.5§ 4.3�0.3 5.4�0.3**

Myocytecross-sectionalarea, �m2

180�24 380�38* 169�21 366�42#

Data are represented as mean�SEM.*P�0.01 vs saline-untreated.†P�0.0001 vs saline-untreated.‡P�0.01 vs saline-HISDN.§P�0.05 vs saline-untreated.�P�0.001 vs aldosterone-untreated.¶P�0.05 vs aldosterone-untreated.#P�0.0001 vs saline-HISDN.**P�0.05 vs saline-HISDN.††P�0.001 vs saline-untreated.

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versus saline infusion) and had negligible effects on SERCA2mRNA expression. Both ANP and sarcoplasmic reticulumCa2�-ATPase (SERCA2) transcripts were decreased significantlyin aldosterone-HISDN hearts (P�0.05 and P�0.001, respec-tively, versus aldosterone untreated; Figure 4A through 4B).

HISDN Improved Exercise CapacityExercise limitation occurs in human diastolic HF,22 and“quality-of-life” scores were improved in AHeFT withHISDN therapy.23 We sought to determine whether diastolicdysfunction improvement was associated with improvedfunctional outcome by measuring exercise capacity in thesemice. Exercise capacity (determined by running distance) was

measured in a blinded manner. There were no differencesbetween the saline-untreated and saline-HISDN groups (Fig-ure 5A). Exercise capacity was impaired in aldosterone-untreated mice (P�0.05 versus saline infusion). HISDNimproved exercise capacity by �40% in aldosterone-infusedmice (P�0.05 versus aldosterone untreated). Running time wasalso increased in aldosterone-HISDN (999�68 seconds) versusaldosterone-untreated (776�47 seconds; P�0.05) mice.

HISDN Improved Vascular InflammationPlasma soluble vascular cell adhesion molecule (sVCAM-1; amarker of vascular inflammation) was increased in hyperten-sive, untreated mice with diastolic HF (P�0.01 versus saline

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Figure 1. In vivo transthoracic echo-cardiography measurements with orwithout fixed-dose HISDN. Two- and4-week measurements. A, LV end-dia-stolic diameter (LVEDD); (B) LV end-systolic diameter (LVESD); (C) frac-tional shortening (FS); and (D) totalwall thickness (TWT). Saline-untreated(f), aldosterone-untreated ( ), saline-HISDN (�), and aldosterone-HISDN(o) mice. (*P�0.05 vs wild-type saline;‡P�0.01 vs wild-type saline). Data aremean�SEM; n�5 to 9 per group.*P�0.05 vs saline-HIDN at 4 weeks;‡P�0.001 vs saline-untreated at 2weeks; ¶P�0.001 vs saline-untreatedat 4 weeks; *P�0.05 vs saline-HISDNat 2 weeks; †P�0.001 vs saline-HISDN at 4 weeks. E, Representativemitral Doppler inflow pattern: an aldo-sterone-infused mouse on no HISDNand an aldosterone-infused mouse onHISDN. E indicates early ventricularfilling; A, late filling caused by atrialcontraction; IVRT, isovolumetric con-traction time; DT, deceleration time.

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infusion). Therapy with HISDN decreased sVCAM-1 levelsin aldosterone-infused mice (P�0.05 versus aldosterone un-treated; Figure 5B).

DiscussionIn this study, aldosterone-induced hypertension resulted inLVH, diastolic dysfunction, and diastolic HF. Fixed-doseHISDN lowered SBP and improved diastolic dysfunction andexercise capacity. It also decreased plasma sVCAM-1 (amarker of vascular inflammation) and myocardial interferon-�and IL-6 expression. HISDN did not ameliorate LVH or cardio-myocyte size. Similarly pulmonary congestion and cardiacfibrosis remained unaffected.

HISDN and ExerciseExercise intolerance is seen in diastolic HF,22; therefore,exercise capacity was determined in the mice. The improved

exercise capacity seen in our study may be because of thefollowing: (1) a reduction in SBP despite the presence ofLVH; (2) an improvement in diastolic dysfunction24; and (3)a decrease in vascular inflammation/stiffness. Although notdirectly addressed in our study, the effect of HISDN was notlimited to the myocardium. Vascular-ventricular stiffeningduring exercise (or other stressors) may lead to an exagger-ated hypertensive response and further load-dependent dia-stolic dysfunction.25 Therefore, it is possible that HISDNenhanced vascular and ventricular interactions (that affect LVfilling and ejection) and, thus, improved diastolic dysfunctionand exercise capacity.

HISDN and Diastolic DysfunctionDiastolic dysfunction is an independent risk factor for HF andcardiovascular death.26,27 If diastolic dysfunction is consid-ered a “preclinical” diagnosis, its early recognition mayrepresent an important way to reduce the incidence ofcongestive HF.28 Conversely, diastolic dysfunction may alsobe an early marker of cardiac end-organ damage in hyperten-sion that precedes the development of LVH.6

The presence of hypertension also increases afterload anddecreases early diastolic filling and myocardial lengthening.29,30

Increased preload and afterload affect the LV relaxation rate, andthese become more pronounced during neurohormonal stimula-tion (eg, aldosterone infusion or during exercise), which thenfurther exacerbates diastolic dysfunction. In our study, SBPreduction improved diastolic dysfunction and exercise capacity.In humans, diastolic dysfunction need not be present to meet thedefinition of diastolic HF.31 Similarly, diastolic dysfunction mayherald the development of diastolic HF26,27 or systolic HF.28 Wecannot exclude the possibility that, if mice were followed for alonger period, improvement in diastolic dysfunction may pre-cede potential decreases in pulmonary congestion (ie, diastolicHF) with HISDN therapy. Thus, recognition of the beneficialeffects of HISDN on Doppler diastolic indices and exercisecapacity may potentially translate into relief of pulmonarycongestion because of vasodilatory effects of HISDN on thevasculature.14,15

Table 2. Measurements of Diastolic Function by MitralDoppler Imaging in Untreated and HISDN-Treated Mice After 4Weeks of Saline- or Aldosterone Infusion

Groups

Untreated HISDN

Saline Aldosterone Saline Aldosterone

No. 5 7 5 7

Mitral Evelocity, mm/s

623�29 1026�44** 545�25 768�77†*

Mitral Avelocity, mm/s

400�20 456�20 374�12 553�35¶*

E/A ratio 1.6�0.1 2.3�0.1** 1.5�0.1 1.4�0.1‡

IVRT, ms 20�0.9 28�1.9** 20�1.1 13.5�0.8§‡

DT, ms 23�1 21�0.5 22�1 15.5�1.1#¶

Data are represented as mean�SEM.*P�0.01 vs aldosterone-untreated.†P�0.05 vs saline-HISDN.‡P�0.0001 vs aldosterone-untreated.§P�0.001 vs saline-HISDN.¶P�0.01 vs saline-HISDN.#P�0.001 vs aldosterone-untreated.**P�0.001 vs saline-untreated.

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Figure 2. Representative Masson’s trichrome-stained cross-sections. A, Interstitial and perivascular areas in LV sections from the myo-cardium from saline- and aldosterone-infused mice with or without HISDN. Scale bar�20 �m. B, Quantitatively, there is more cardiacfibrosis in aldosterone-untreated (*P�0.05 vs saline-untreated) and aldosterone-HISDN hearts (†P�0.01 vs saline-untreated hearts).Data reflect 10 measurements from the 3 sections on the slide from each animal; n�2 to 3 per group.

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Characterizing LV diastolic properties in humans is usuallydone by measuring pressure and volume with high-fidelitymicromanometers.31 In our study, the Vevo 770 High-Resolution In Vivo Imaging System provided the measures ofmurine diastolic function.19,32 Mitral Doppler measurements aregood measures of diastolic function in mice.32 In a subset ofmice, increased LVEDP correlated with an increased E/A ratio(data not shown). SBP control and LVH regression improve LVdiastolic filling33; however, LVH regression was not seen in ourstudy. Once again, it is possible that, if mice were followed for anextended period, SBP control with HISDN and improved diastolicdysfunction may precede potential reductions in cardiac mass.6

Equally plausible is that a delay in measurable change in structuremay allow functional changes to precede structural changes.

HISDN and Cardiac RemodelingControversy surrounds the roles of LVH and HF. LVHregression, an intermediary between hypertension and HF,6

may prevent both systolic and diastolic HFs. Conversely,although LVH increases the risk of HF, hypertensive subjectswithout LVH may also develop HF,1 suggesting that targetingLVH may be unnecessary to prevent HF. The effect of LVHregression on diastolic dysfunction, one of the earliestchanges in hypertension, is unclear. Even in the absence ofLVH, SBP control improves diastolic filling irrespective ofthe regimen used.6 It is unclear why LVH regression was notseen in the present study, but this indicates that LVH andhypertension may not have a causal relationship in thismodel. Furthermore, study duration may have been insuffi-

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Figure 3. Myocardial inflammation. A, Representative ED-1 staining for macrophages in cross-sections of the LV at 4 weeks. Photomi-crographs of LV sections from the 4 groups: saline-untreated, saline HISDN, aldosterone-untreated, and aldosterone-HISDN. Blackarrows indicate the presence of immunostaining specific for the ED-1 antibody in both groups of aldosterone-infused hearts (magnifica-tion: �200). Increased myocardial cytokine expression in aldosterone-untreated hearts: (B) interferon-�, (C) IL-6, and (D) TNF-� expres-sion. E, IL-1� expression was not significantly increased. HISDN decreased interferon-� and IL-6 expression but not TNF-� expressionin aldosterone-infused mice. *P�0.05 vs saline-untreated; **P�0.01 vs saline-untreated; †P�0.05 vs saline-HISDN; n�4 to 5 per group.

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Figure 4. Molecular markers of cardiac remodeling.A, ANP gene expression by RT-PCR is significantlyincreased in untreated aldosterone-infused hearts.Chronic therapy with HISDN reduced myocardialANP expression; *P�0.05 vs saline-untreated. B,SERCA2 expression in the hearts was not signifi-cantly altered in untreated aldosterone-infusedhearts. SERCA2 expression was decreased withHISDN; P�0.001. Data are mean�SEM; n�4 to 5per group.

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cient; however, other experimental models with other thera-pies have shown LVH regression within 4 weeks.34,35

AHeFT suggested that chronic HISDN mediated positiveeffects on the myocardium.17,23 NO exerts beneficial effectson the vasculature,15,36 lowers SBP,37 and also has directeffects on cardiac remodeling,11 partly by increasing endo-thelial NO synthase and inhibiting LVH.11–13 In our study,aldosterone induced LVH and diastolic HF,18 and NO givenin the form of ISDN was ineffective in reducing LVH orcardiomyocyte size despite SBP reduction. The form in whichNO is given is important38,39: uncoupled NO synthase (seen inhypertension, ischemia-reperfusion injury, and LVH withchamber remodeling38) generates oxygen free radicals andless NO, shifts the nitroso-redox balance, and all maycontribute to the development of diastolic dysfunction. Hy-dralazine also lowers SBP40 and suppresses reactive oxygenspecies production15 (which mediates cardiac hypertrophy41).The addition of hydralazine to NO should theoretically shiftthe nitroso-redox balance (although not tested in our study)and result in LVH regression.

The persistence of LVH with HISDN in our study may bean adverse finding. Cardiac fetal gene re-expression, that is,upregulation of ANP and downregulation of SERCA2, is amarker of pathological remodeling. Because LVH persisted,we expected no change in myocardial ANP expression.However, both ANP and SERCA2 transcripts were de-creased. It has been suggested that the development of LVHcan, in part, be dissociated from activating the fetal geneprogram.42,43 Others have shown that an abnormal structuralphenotype may exits without fetal gene expression but cancoexist with molecular signaling pathways that ultimatelylead to an abnormal phenotype.44 Therefore, although specula-tive, HISDN may modulate gene expression and control signaltransduction pathways involved in cardiac remodeling anddiastolic dysfunction but may not modulate the cardiac pheno-type. Our study does not exclude the possibility that reducedANP expression may precede the eventual reduction in LVH ifthe animals had been followed for a longer time period.

Myocardial Fibrosis and ExtracellularMatrix RemodelingMyocardial fibrosis is closely related to diastolic stiffness.45

Aldosterone-induced fibrosis was unaltered by HISDN, but

collagen and matrix metalloproteinases were not measured. Ithas been suggested that total collagen is unaltered in diastolicdysfunction.46 Posttranslational modification of myocardialcollagen occurs, and local formation of advanced glycationend products-collagen links correlate with LV stiffness.46

Myocardial fibrosis may contribute equally to LVH in dia-stolic HF. In the present study, HISDN had negligible effectson the extracellular “milieu” of the aldosterone-infused myo-cardium despite improvements in the E/A ratio. Thus, neithera reduction in myocardial fibrosis nor a decrease in LVHcontributed to the improved diastolic dysfunction seen inaldosterone-induced diastolic HF, suggesting possible “ex-tramyocardial” effects, such as a reduction in vascularinflammation.

HISDN and CytokinesMyocardial interferon-�, IL-6, and TNF-� expressions wereincreased in hypertension-induced diastolic HF. HISDN onlydecreased interferon-� and IL-6 expression. Chronic NO(with HISDN) may directly reduce interferon-� and IL-6expression, or these decreased cytokines may simply reflectimproved diastolic function. IL-6 has been shown to beprohypertrophic.47 It is interesting that TNF-� expressionremains unaffected by HISDN. It may be that concurrentTNF-� expression and the presence of myocardial NO mayfoster self-sustaining positive autocrine/paracrine feedbackinflammatory circuits in diastolic HF.48

HISDN and Vascular InflammationVascular inflammation, as reflected by sVCAM-1 levels, waselevated in hypertensive, aldosterone-infused mice. sVCAMlevels were decreased with HISDN. In humans, sVCAM-1 issignificantly associated with asymmetrical dimethylarginine,49

an important endogenous NO synthase inhibitor. Similarly, NOsynthesis inhibition is associated with an increase in endothelialadhesion molecule expression.50 Thus, HISDN likely exertsbeneficial effects on the vasculature.

LimitationsFirst, previous experimental studies using combination NOand hydralazine measured only acute hemodynamic ef-fects.14,15 There are no human or animal studies that address

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Figure 5. Exercise capacity. Aldosterone infu-sion for 4 weeks decreased maximal exercisecapacity as determined by running distance.Mice in the aldosterone-HISDN group had animprovement in running distance almost back tobaseline (P�0.05 vs untreated aldosterone-infused mice). B, Soluble VCAM-1 levels.Plasma sVCAM-1 levels were increased in aldo-sterone-infused mice and decreased withHISDN. *P�0.05 vs saline-untreated; **P�0.01vs saline-untreated. Data are mean�SEM; n�4per group.

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the effect of combined HISDN (which is used clinically) ondiastolic dysfunction and diastolic HF either acutely orchronically. Second, few animal models can successfullyreplicate human diastolic disease, and a single-cell study islimited in its inability to mimic diastolic disease given theimportance of the vasculature (in preload and afterload), heartrate, chamber geometry, and extracellular matrix in diastol-ogy. Therefore, this model of hypertension-induced diastolicHF is a clinically relevant model. It encompasses the myo-cardium, potential effects of the vasculature, and exerciseimpairment. Third, although Millar catheterization was notperformed in all of the mice, LVEDP correlated with the E/Aratio. Echocardiography allowed us to follow the micelongitudinally. Fourth, this study was performed exclusivelyin male mice. The prevalence of diastolic HF is greatest inelderly female mice.32 A future study proposes to study aged,female mice. Finally, although not directly addressed in thepresent study, our findings suggests that HISDN effects werenot limited exclusively to the myocardium, and the improve-ments seen were likely attributed to effects on the vasculatureand interactions of vascular and ventricular stiffness. Ourfindings represent not so much a change in diastolic functionas a change in myocardial load induced by alterations invascular inflammation and/or stiffness because of HISDN.

In conclusion, HISDN did not prevent the progression toLVH or diastolic HF. LVH regression is unnecessary for theimprovement in exercise capacity and diastolic dysfunction inhypertension. It is possible that improvement in ventricularinflammation/stiffness results in enhanced diastolic functionand will require further study.

PerspectivesDiastolic dysfunction may precede the development of LVHin hypertension and is the connection between hypertensionand diastolic HF. Although a lack of therapies continues toplague diastolic HF patients, understanding mechanisms pri-marily responsible for this clinical syndrome and its relation-ship to hypertension and diastolic dysfunction is important.Unlike human systolic HF studies,17 our findings indicate thatHISDN does not exert direct myocardial effects but ratherinfluences ventricular-vascular interactions in diastolic HF.Additional studies are warranted to determine whether LVHregression is beneficial in hypertension-mediated diastolicdysfunction and to determine causality in diastolic dysfunc-tion and cardiac remodeling.

Sources of FundingThis work was supported by funding from the National Institutes ofHealth grant HL079099 (to F.S.).

DisclosuresNone.

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590 Hypertension September 2009

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Richard M. Wilson, Deepa S. De Silva, Kaori Sato, Yasuhiro Izumiya and Flora SamCapacity in Hypertension-Induced Diastolic Heart Failure

Effects of Fixed-Dose Isosorbide Dinitrate/Hydralazine on Diastolic Function and Exercise

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2009 American Heart Association, Inc. All rights reserved.

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ONLINE SUPPLEMENT

Effects of Fixed-Dose Isosorbide Dinitrate/Hydralazine on Diastolic Function and Exercise Capacity in Hypertension-Induced Diastolic Heart Failure

Richard M. Wilson, BA*; Deepa S. De Silva, PhD*; Kaori Sato, MD, PhD*; Yasuhiro Izumiya MD PhD*, PhD; Flora Sam, MD *‡§

*Whitaker Cardiovascular Institute, ‡Cardiovascular Section and §Evans Department of Medicine;

Boston University School of Medicine, Boston, MA. Correspondence to: Flora Sam, MD Whitaker Cardiovascular Institute Boston University School of Medicine

Evans Department of Medicine and Cardiovascular Section 715 Albany Street, Room W507

Boston, MA 02118 Phone: (617) 638-8072

Fax: (617) 638-4066 E-mail: flora.sam@bmc.org

Expanded Materials And Methods Aldosterone infusion. Uninephrectomized mice (25-27g) received osmotic minipumps (Alzet, Durect Corporation) that delivered a continuous infusion of saline or d-aldosterone (0.15 μg/hour) (Sigma-Aldrich Co.) for 4 weeks1. All mice were maintained on 1% NaCl drinking water. Treatment. Forty-six mice were randomly assigned on the same day as the surgery to either regular chow or chow containing hydralazine: 50mg/kg/day and isosorbide dinitrate (ISDN): 26mg/kg/day, the combination known as HISDN, for 4 weeks. The dose of hydralazine and ISDN has been used by others2-4 and shown to be non-toxic. This combination maintains the same dose ratio that was used in A-HeFT5 i.e., the ISDN:hydralazine HCl ratio was 20mg:37.5mg. The 4 groups studied were: a) saline-untreated, n=9; b) saline-HISDN, n=9; c) aldosterone-untreated, n=14 and d) aldosterone-HISDN, n=14. Physiological measurements. Heart rate (HR) and tail cuff blood pressure (SBP) were determined non-invasively (BP-2000, VisiTech) as previously described1;6. Transthoracic echocardiography was performed in conscious mice biweekly after surgery using the Acuson Sequoia C-256 echocardiograph machine with a 15-MHz probe, as previously described1. Total wall thickness (TWT) was derived from an average of the interventricular septum and posterior wall thickness. Doppler echocardiography. Mitral Doppler flow study was performed using the Vevo 770 High-Resolution In Vivo Imaging System (VisualSonics,Toronto). Images were acquired using a high-resolution (30 MHz) transducer. Mice were anesthetized using isoflurane (0.5-1.5%) and titrated to achieve a HR around 350 beats/min since diastolic function measurement are sensitive to HR and loading conditions. The maximum 1.5% of isoflurane has minimal effects on diastolic function7. Images were recorded for 30–40 cardiac cycles and measurements were made from 3–5 representative cycles. The apical four-chamber view was used to record the mitral Doppler flow spectrum. Peak early (E) and late (A) mitral inflow velocities, deceleration time of early filling (DT) and isovolumetric relaxation time (IVRT) were measured as previously described8. In a subset of mice (n=5), hemodynamic measurements were performed 4 weeks after aldosterone infusion using a 1.4F catheter tip micromanometer (ARIA, Millar Instruments). Aldosterone infusion resulted in an increased LV end-diastolic pressure (EDP). Increased LVEDP correlated with the increased E/A ratio, R=0.95 (P<0.05; data not shown). Exercise Treadmill analysis. Exercise capacity was measured on a rodent motor-driven treadmill (Columbus Instruments) as previously described9. Mice were acclimated for at least 3 days (15% incline, speed of 15 m/min for 10 minutes and subsequently increased 2 m/min each 2 minutes, ending after 20 minutes). Total exercise time was recorded as the elapsed time to exhaustion and then converted to distance. Exhaustion was determined by an observer blinded to treatment groups and was defined as the point at which the animals could not keep pace with the treadmill and no longer avoided the electrical stimulus. Organ weight, tissue and blood analysis. After 4-weeks mice were sacrificed, at which time blood was obtained to determine plasma soluble vascular cell adhesion molecule (VCAM-1) levels (R&D Systems). Body weight (BW), heart weight (HW) and LV weights were also determined. Hearts were

either: (a) arrested in diastole by KCl (30mmol/l), weighed, perfused with 10% buffered formalin and sliced horizontally for histology or (b) snap-frozen in liquid nitrogen. Trichrome-stained sections (5μm) were visualized by light microscopy to measure fibrosis and the entire section was quantified using Bioquant Image analysis software. The wet-to-dry lung weight ratios were determined as an index of pulmonary congestion1. Determination of mRNA. Total RNA from 4 groups of mice hearts were extracted with PureLink™ Micro-to-Midi Total RNA purification system (Invitrogen). cDNA synthesis from total RNA was performed using a Thermoscript™ RT-PCR system (Invitrogen) according to the manufacturer’s instructions. Transcript expression levels of ANP, SERCA-2, IL-1β, IL6, INF-γ, TNF-α and GAPDH were quantified by iCycler iQ Real-Time PCR Detection Systems (BIO-RAD) using FastStart Universal SYBR Green Master Mix (Roche, IN, USA). Transcript levels were adjusted relative to the expression of GAPDH. Sequences of PCR primers are as listed. Primer sequence for mouse are as follows: ANP (F): 5'-GAG AGA CGG CAG TGC TTC TAG GC-3', ANP (R): 5'-CGT GAC ACA CCA CAA GGC CTT AGG-3'; SERCA2 (F): 5'-TAC TGA CCC TGT CCC TGA CC-3'; (R ): 5'-CAC CAC CAC TCC CAT AGC TT-3'; IL-1β (F): 5'-AGA CAC AGA TTC CAT GGT GAA GT-3', IL-1β (R): 5'-TCT CAG CTT CAA TGA AAG ACC TC-3'; IL-6 (F): CCC AAT TTC CAA TGC TCT CCT-3, IL-6 (R): TAA CGC ACT AGG TTT GCC GAG; INFγ (F): 5'-CAT GGC TGT TTC TGG CTG TTA C- 3', IFNγ (R): 5'-CCA GTT CCT CCA GAT ATC CAA GA-3'; TNF-α (F): 5'-CAT CTT CTC AAA ATT CGA GTG ACA A-3', (R): 5'-TGG GAG TAG ACA AGG TAC AAC CC-3', GAPDH (F): 5'-TCA CCA CCA TGG AGA AGG-3', GAPDH (R): 5'-GCT AAG CAG TTG GTG GTG CA-3'.

Immunohistochemistry. To visualize macrophages, ED-1 staining was used as previously described6. Sections were deparaffinized, rehydrated, and treated with 20μg proteinase K per mL of Tris-HCl (pH 8.5) for 25min at room temperature to recover antigenicity. Sections were then stained with a 1:25 dilution of rat anti-mouse cd68 primary antibody (Serotec, Raleigh, NC) in PBS with 1% BSA overnight at 4°C. Nonspecific binding was blocked by incubation with 10% horse serum in PBS (pH 7.4) for 30min before incubation with the antibody. A biotinylated anti-rat antibody (Vector) was used as secondary antibody and Vector Red alkaline phosphatase substrate (Vector) was applied. Sections were visualized under bright-field microscopy and images were recorded using an Optronics camera with Bioquant hardware and software.

Statistical analysis. Results are presented as mean±SEM. Statistical analyses of the data were carried out using the Student’s t test (2-sided). When necessary, 1- or 2-way ANOVA (followed by Student-Newman-Keuls post-hoc tests when appropriate) was applied. A value of P<0.05 was considered statistically significant.

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