Chronic in vivo nitric oxide deficiency impairs cardiac functional recovery after ischemia in female (but not male) mice

Journal of Molecular and Cellular Cardiology 112 (2017) 8–15

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Chronic in vivo nitric oxide deficiency impairs cardiac functional recovery after ischemia in female (but not male) mice

MARK

Laura A. Bienvenua,b, James Morgana, Melissa E. Reicheltc, Lea M.D. Delbridgeb,⁎,1, Morag J. Younga,1 a b c

Centre for Endocrinology and Metabolism, Hudson Institute of Medical Research, Melbourne, Australia School of Biomedical Sciences, University of Melbourne, Australia School of Biomedical Sciences, University of Queensland, Brisbane, Australia

A R T I C L E I N F O

A B S T R A C T

Keywords: Heart Nitric oxide Ischemia/reperfusion Sex Cardioprotection Mineralocorticoid receptor

Nitric oxide (NO) is an important regulator of cardiac function and plays a key role in ischemic cardioprotection. The role of chronic NO deficiency in coordinating ischemic vulnerability in female myocardium has not been established. The aim of this study was to determine the influence of chronic in vivo NO synthase inhibition in modulating ex vivo ischemia-reperfusion responses in female hearts (relative to males). Mice were subjected to LNAME (L-NG-Nitroarginine-methyl-ester) treatment in vivo for 8 weeks. Cardiac fibrotic, inflammatory and cardiomyocyte Ca2 + handling related gene expression changes were assessed. Hearts were Langendorff-perfused, subjected to 20 min global ischemia with 45 min reperfusion. In response to this moderate ex vivo ischemic insult, hearts derived from L-NAME treated female animals exhibited increased incidence of reperfusion arrhythmias, diastolic abnormality and reduced contractile recovery in reperfusion. This differential response was observed even though baseline performance of hearts from L-NAME treated animals was not different to vehicle controls, myocardial inflammatory and fibrotic indices were similar in males and females and the systolic blood pressure effect of L-NAME administration was equivalent in both sexes. Evaluation of a subgroup of mice with cardiomyocyte specific mineralocorticoid receptor deletion suggests involvement of this receptor in NO-deficiency mediated responses. To examine underlying pre-disposing mechanisms, expression of a panel of candidate genes encoding proteins involved in electromechanical homeostasis (particularly relevant to ischemic challenge) was evaluated in normoxic myocardial tissues from the L-NAME- and vehicle-treated animals. Analysis revealed that L-NAME treatment in females selectively regulated expression of genes related directly and indirectly to cardiomyocyte Ca2 + handling in a manner consistent with destabilization of Ca2 + homeostasis and arrhythmogenesis. Our investigation provides new insight into the role of sustained decrease in NO bioavailability in determining distinctive female cardiac vulnerability to ischemic challenge.

1. Introduction Ischemic heart disease is the leading cause of death and disability for men and women. The incidence of cardiovascular disease differs between the sexes with earlier onset in men and rising incidence in women post-menopause [1]. Ischemic heart disease due to microvascular dysfunction is more prevalent in women, and in both women and men the importance of ischemic disease which may not be the consequence of obstructive coronary artery pathology is increasingly recognized [2,3]. Local production of vascular and myocardial regulatory mediators (i.e. nitric oxide, NO) plays an important role in maintaining coronary flow and protecting against ischemic

⁎

1

myocardium pathology. Low levels of NO are implicated in heart failure [4] and failure is linked with generalized systemic impairment of vasodilatory reserve [5]. Various lines of evidence indicate sex differences in NO-dependent responses. In rodent models, sex differences in NO production capacity have been linked with differential cardiac functional responses to ischemia [6,7]. Preclinical studies have also shown that in females (but not males) endogenous estrogen-mediated NO production is permissive in modulating effectiveness of PDE5-inhibitor treatment in the prefailure setting [8,9]. Regulating NO bioavailability may therefore be an attractive target for sex-specific therapies in cardiac ischemia and failure.

Corresponding author at: School of Biomedical Sciences, University of Melbourne, Parkville 3031, Australia. E-mail address: [email protected] (L.M.D. Delbridge). These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.yjmcc.2017.08.012 Received 30 May 2017; Received in revised form 24 August 2017; Accepted 26 August 2017 0022-2828/ © 2017 Published by Elsevier Ltd.

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perfusion fluid and the aorta cannulated. Hearts were retrogradely perfused and instrumented for pressure recording as previously [17]. Hearts were perfused for 30 min prior to 20 min of global “no flow” ischemia and 45 min of reperfusion. Arrhythmia incidence was assessed from left ventricle pressure records as previously validated from electrocardiogram measurements [22].

The particular contexts in which NO intervention may achieve beneficial outcomes have not yet been resolved. Findings in relation to NO augmentation using pharmacological donor treatments in the setting of cardiac ischemia/reperfusion are equivocal. Experimentally, NO treatments and increased coronary NO levels after myocardial infarction induction have been linked with improved cardiac outcomes (in male animals) but clinical studies have not demonstrated that NO donor treatment administered at the time of an acute ischemic event can confer infarct sparing benefit [10–13]. Experimental investigation of the impact of NO deficiency have utilized L-NAME (L-arginine, NG-nitro-L-arginine methyl-ester), a nonspecific NO synthase (NOS) inhibitor [14]. In rodent studies L-NAME has been shown to abrogate the beneficial effects of β-adrenoceptor modulation when administered at the time of an ischemic insult (in vivo and ex vivo), and this effect is observed to be more pronounced in females [15,16]. Preclinical studies investigating the sex-specific effects of chronic in vivo NO deficiency on cardiac responses to ischemic insult are lacking. Given the clinically demonstrated link between NO deficiency and cardiac functional decline, this knowledge gap is important. Thus, the aim of this study was to determine the influence of sustained in vivo NOS inhibition in modulating acute cardiac responses to ischemia-reperfusion in females and males. Mice were subjected to chronic L-NAME treatment in vivo. Cardiac structural remodelling was evaluated and sex-specific shifts in expression of a panel of genes relevant to electromechanical function were investigated. The influence of NO inhibition on female and male arrhythmogenic and contractile responses to an ex vivo cardiac global ischemia-reperfusion insult were compared. Given that we have recently demonstrated involvement of the cardiomyocyte mineralocorticoid receptor (MR) in mediating cardiac response to ischemia-reperfusion [17], and that an MR-NO signalling link in vascular tissues has been previously reported [18,19], we also sought evidence of sex-specific MR involvement in modulating LNAME functional cardiopathology. Our findings provide new insight into the role of established NO deficiency in determining distinctive female cardiac vulnerability to ischemic challenge.

2.3. Histological and immunohistochemical analyses Cardiac tissues were harvested for immunohistochemical analysis from a cohort of animals treated in parallel with those evaluated for ex vivo function. Histological analysis for collagen was performed on tissues stained with 0.1% Sirius Red (Sigma-Aldrich, Australia) using an unbiased systematic sampling approach as previously described [17]. For immunostaining to evaluate macrophage infiltration, paraffin-embedded sections were incubated with MAC2 (eBioscience, CA), followed by the appropriate biotinylated secondary antibody and then incubated with ABC complex (Vectastain, Vector Laboratories, CA). Positive staining was visualized by incubation with 3,3-diaminodenzidine (DAKO Corp., CA), and the tissues were counterstained with hemotoxylin. 2.4. Quantitative PCR Cardiac tissues were harvested for gene expression analysis from a cohort of animals treated in parallel with those evaluated for ex vivo function. Total RNA was isolated from snap frozen left ventricle, cDNA synthesis from total RNA was performed. Quantitative digital PCR was carried out. Gene Expression assays using the Integrated Fluidic Circuits on the Biomark HD platform (Fluidigm, CA, USA). Data were analyzed by Fluidigm Real-Time PCR Analysis software (version 4.12). Relative quantification of change in gene expression was calculated with the formula 2 (−ΔΔCt) and normalized to housekeeping gene (GAPDH) and expressed as fold-change versus female VEH group. 2.5. Statistical analyses

2. Materials and methods Data sets were assessed for normality and analyzed by two way ANOVA and Bonferroni's post hoc test (GraphPad Prism version 6.0a and 7.0, GraphPad Software, CA). Differences between mean values were considered significant at p < 0.05. All data are reported as mean ± SEM and with n value to examine the effects of sex, treatment or genetic intervention achieving 95% confidence in detecting changes of ≥20% in groups with a standard deviation of ≤ 15%. In Figure legends, significant ANOVA factor effects are identified, followed by posthoc significance testing outcomes. To summarise figure panel annotations utilized: # = ANOVA factor sex effect. § = ANOVA factor treatment effect. † = ANOVA factor interaction effect. * = Bonferroni post hoc test.

The detailed Materials and Methods are available in the online Supplementary File. 2.1. Experimental animals All procedures involving animals were approved by the relevant Institutional Animal Ethics and Biosafety Committees, and conducted in compliance with the NHMRC/CSIRO/ACC Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2013). The nitric oxide synthase inhibitor L-NG-Nitroarginine methyl ester (LNAME, 150 mg/kg/day) or vehicle (VEH) was administered to male and female mice (age 8 weeks, 20–30 g) in the drinking solution for an 8 week treatment period. L-NAME dose was adjusted based on animal fluid consumption to ensure standardized treatment as previously reported [20]. To accelerate progression of cardiopathology over the 8 week treatment period and to provide co-morbidity context, all mice were uninephectomized as previously described and drinking water supplemented with 0.9% NaCl, 0.4% KCl [17]. A subset of experiments was performed in age-matched male and female cardiomyocyte-specific mineralocorticoid receptor null mice (MR-KO) of C57Bl6 lineage identical to receptor intact mice [17,21]. After 8 weeks treatment, mice were allocated either for histological assessment and gene expression studies or ex vivo functional evaluation.

3. Results 3.1. NO deficiency modulates cardiac structure in both sexes Systolic blood pressure (SBP) was increased similarly in male and female mice after 8 weeks of L-NAME treatment when compared with vehicle (VEH) treated animals (Fig. 1A). The timecourse of development of hypertension was similar in L-NAME treated groups. These data confirmed our previous report that female and male systemic hemodynamic endpoint responses were well matched [20], with modest blood pressure increase as observed in prior studies. For each sex, heart weight/tibia length indices were not different between L-NAME and VEH treatment groups. Higher male heart weight indices were observed for both treatment groups, reflecting established intrinsic sex difference

2.2. Assessment of cardiac function ex vivo Mice were anesthetised with sodium pentobarbitone (70 mg/kg, i.p). A thoracotomy was performed, hearts excised into ice-cold 9

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Systolic Blood Pressure

A

20

*

*

150 100

10

Fig. 1. L-NAME increases systolic blood pressure, fibrosis and macrophage infiltration similarly in female and male mice. A. Systolic blood pressure (plethysmography), B. Heart weight to tibia length ratio, C. Picro-Sirius red measurement of interstitial myocardial fibrosis, D. Myocardial Mac2 + macrophage infiltration, E. PicroSirius red representative images, 20 × magnification. All data analyzed by two-way ANOVA, #p < 0.05 sex effect, §p < 0.05 treatment effect, *p < 0.05 by Bonferroni's post hoc test. Mean ± SEM, n = 7–8.

0

0 F

M

F

VEH

F

M

M

*

Mac2+ cell/field

5

2

1

M

L-NAME

Macrophage Infiltration

D

3

§

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VEH

L-NAME

Interstitial Fibrosis

C Percent area

*

15

5

50

*

4 3 2 1

0

0

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M VEH

E

#

* mg/mm

mmHg

Heart weight:Tibia length ratio

B

200

F

F

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M

F

VEH

L-NAME

M

L-NAME

Sirius red - fibrosis F VEH

M VEH

F L-NAME

M L-NAME

treatment. Interestingly, expression of collagen type 1a (COL1), but not collagen type 3a (COL3), was significantly greater in female compared to male hearts in response to L-NAME treatment. In summary, chronic NO deficiency produced a similar (modest) elevation in blood pressure and extent of myocardial fibrosis and inflammation in males and females.

Table 1 Nitric oxide deficiency induced fibrotic and inflammatory gene expression responses. VEH

Collagen 1a1 (COL1) Collagen 3a1 (COL3) Transforming growth factor beta1 (TGFβ-1) Vascular endothelial growth factor a (VEGFa)

L-NAME

Female

Male

Female

Male

1.00 ± 0.12

0.97 ± 0.10#

1.53 ± 0.12⁎

0.88 ± 0.09#

1.00 ± 0.13

0.83 ± 0.05

1.14 ± 0.11

0.98 ± 0.12

§

3.2. In vivo NO modulation of ex vivo cardiac function §

1.00 ± 0.08

1.05 ± 0.06

1.22 ± 0.08

1.18 ± 0.09

1.00 ± 0.07

1.28 ± 0.14

1.67 ± 0.19§

1.48 ± 0.19§

At baseline, ex vivo cardiac contractile function and coronary flow indices measured under control perfusion conditions were similar for male and female hearts and were not modulated by in vivoL-NAME treatment in either sex. Left ventricular developed pressure and the rates of contraction and relaxation were equivalent, and intrinsic heart rates were not different (Fig. 2A–D). Thus basal function was preserved in female and male hearts exposed to chronic NO deficiency. Performance characteristics of ex vivo hearts were tracked from baseline, through the 20 min ischemic period (global no-flow), and in both early and later reperfusion to 45 min (Fig. 3A). As has been shown previously, a period of 20 min ischemia would be expected to incorporate elements of stunning as well as necrotic cell death [23]. During ischemia, contracture development (indexed by time to rise in end-diastolic pressure of > 15 mmHg) was more rapid in male hearts compared to female in both L-NAME and VEH treatment groups (Fig. 3B). Peak contracture was also greater in male compared to female hearts in both treatment groups (Fig. 3C). These data are consistent with other reports describing a more pronounced ischemic functional response in male hearts compared to female. Interestingly, the post-hoc analysis indicates much of this difference is driven by loss of female

Data analyzed by two-way ANOVA. n = 6–8. Mean ± SEM. § p < 0.05 treatment effect. # p < 0.05 sex effect. ⁎ p < 0.05 Female VEH vs all other groups by Bonferroni's post hoc test.

in this parameter (Fig. 1B). L-NAME treatment significantly increased cardiac collagen content and macrophage infiltration equivalently in male and female hearts (Fig. 1C–E). As expected, expression of transforming growth factor beta (TGF-β1) was increased with L-NAME in males [20], and in this study a similar female response was observed (Table 1). Expression of vascular endothelial growth factor (VEGFa) was increased equivalently in both sexes in response to L-NAME 10

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Left Ventricular Developed Pressure 100

4000

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D 500

3000

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Fig. 2. L-NAME does not regulate basal cardiac function in female or male hearts. A. Left ventricular (LV) developed pressure, B. LV cardiac contraction (dP/dtmax), C. LV cardiac relaxation (dP/dtmin), D. Intrinsic heart rate (ex vivo) non paced. All data analyzed by two-way ANOVA, p = ns by Bonferroni post hoc test. Mean ± SEM, n = 7–8.

dP/dtmax

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L-NAME

Ca2 +, sodium (Na+) and proton (H+) flux. Expression levels of a panel of these potentially participant genes was compared in myocardial tissues harvested from L-NAME and VEH treated mice (from hearts not exposed to the ex vivo ischemia protocol). L-NAME treatment produced higher expression levels of genes encoding key proteins involved in Ca2 +/Na+/H+ handling in female hearts only. Expression upregulation was detected for a constellation of genes involved directly and indirectly in Ca2 + homeostasis: sodium hydrogen exchanger (NHE-1), sodium calcium exchanger (NCX), sodium channel subunit 1.5 (Nav1.5), L-type subunit Cav1.2 (Cav1.2), L-type subunit 1D (Cav1.3), T-type subunit 1G (Cav3.1d), T-type subunit 1H (Cav3.2), and ryanodine receptor (Ryr2) (Fig. 5A, Table S2). Down-regulation of the RyR2 associated protein FKBP12.6 was also detected only in female hearts (Fig. 5A, Table S2). Protein phosphorylation is critical in functional outcomes post-ischemia, determining the extent to which phosphorylation-mediated activation of proteins may be curtailed. Two protein phosphatase 2A (PP2A) subunits, PP2A Aβ and PP2A Cβ, were significantly up-regulated in female hearts (Fig. 5A, Table S2). These data demonstrated a selective responsiveness of female hearts to chronic in vivo NO deficiency in the regulation of expression of transporters involved directly or indirectly in the Ca2 + homeostatic response.

protection in L-NAME compared to VEH. Arrhythmic activity was not detected in female or male hearts under basal conditions, suggesting that modification of NO levels does not affect baseline cardiac rhythm in this setting. Early reperfusion arrhythmias were increased in L-NAME treated female hearts indicating that electrical instability during early reperfusion may be promoted by NO deficiency (Fig. 3D). This effect was not observed in male hearts, indicating a selective ectopic vulnerability in female hearts subjected to chronic NO deficiency in vivo. After 45 min reperfusion, recovery of left ventricular developed pressure (LVDevP) and rate of cardiac contraction (dP/dtmax) were significantly reduced in the L-NAME treatment group in female hearts only (Fig. 4A–B). Rate of cardiac relaxation (dP/dTmin) was not significantly lower in female hearts (Fig. 4C). End diastolic pressure was higher at the end of reperfusion in hearts from females treated with LNAME, confirming impaired relaxation (Fig. 4D). In contrast, functional recovery in reperfusion in male hearts was similar in L-NAME and VEHtreated groups. Together these data revealed that females were more susceptible to acute ischemic stress after exposure to chronic NO deficiency in vivo. We have previously shown that the cardiomyocyte mineralocorticoid receptor (MR) may modulate cardiac functional response to ex vivo ischemic insult [17]. In a parallel subset of experiments with MR-KO mice, the effects of the L-NAME treatment protocol was examined. In MR-KO mice, evidence of cardiac inflammation and fibrosis was completely suppressed in both males and females. In addition, in the hearts of these mice no significant differences in functional responses to ischemia and during reperfusion were detected, and the female selective post-ischemia arrhythmogenic response associated with L-NAME treatment was absent (Table S1).

4. Discussion This study provides novel demonstration that chronic in vivo NO deficiency adversely modulates acute post-ischemic functional responses in female, but not male, murine hearts. In response to a moderate ex vivo ischemic insult, hearts derived from L-NAME treated female animals exhibited increased incidence of reperfusion arrhythmias, diastolic abnormality and reduced contractile recovery in reperfusion. This differential response was observed even though baseline performance of hearts from L-NAME treated animals was not different to vehicle controls, myocardial inflammatory and fibrotic indices were similar in males and females and the systolic blood pressure effect of LNAME administration was equivalent in both sexes. To examine underlying pre-disposing mechanisms, expression of a panel of candidate

3.3. Marked modulation of gene expression in NO deficiency in females Cardiac recovery post-ischemia is reliant on homeostatic mechanisms which suppress cardiomyocyte calcium (Ca2 +) overload, and involves participation a number of transporters/channels which regulate 11

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A LV Pressure (mmHg)

Female L-NAME 120 100 80 60 40 20 0

LV Pressure (mmHg)

Male L-NAME 120 100 80 60 40 20 0

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% Ectopic beats

(2 min reperfusion) 50

#†

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30 20 10 0 F

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L-NAME

Fig. 3. L-NAME increases reperfusion arrhythmia in female hearts. A. Time-compressed representative left ventricular pressure traces showing basal, ischemic, early reperfusion and end reperfusion function in hearts from female and male L-NAME treated mice, B. Time to contracture, C. Peak contracture, D. Ectopic beats in early reperfusion. All data analyzed by two-way ANOVA, #p < 0.05 sex effect, *p < 0.05 by Bonferroni's post hoc test, †p < 0.05 interaction effect. Mean ± SEM, n = 7–8.

has female-specific negative impact on intrinsic cardiac ischemic resilience. An implication of this finding is that greater jeopardy for females exists in response to a transient ischemic event where there is an underlying NO-deficient condition, such as would be likely associated with microvascular pathology (even in the absence of macrovascular disease). Furthermore, these findings indicate that this differential female responsiveness is observed even when markers of fibrosis and inflammation are similar. Our findings provide new evidence supporting the contention that strategies seeking to achieve beneficial outcomes in relation to cardiac NO interventions need to consider sex-specific response nuances. It has been demonstrated experimentally that NOS expression levels are increased in females and that female hearts are more responsive to disruption of NO signalling [25,26]. In this investigation L-NAME was administered at relatively low dose, and interestingly no basal effect on cardiac performance was apparent. In models of murine L-NAME administration, blood pressure effects are modest (as observed here and reported previously) and this suggests that the L-NAME generated phenotype reflects a cardiac-specific impact which is not secondary to a systemic hemodynamic effect in this setting. In other studies, using male animals only, chronic L-NAME treatment at higher dose level has been linked with contractility impairment and severe structural remodelling [27–29]. No previous studies have investigated female responses. Our observations suggest that with low

genes encoding proteins involved in electromechanical homeostasis (particularly relevant to ischemic challenge) was evaluated in normoxic myocardial tissues derived from L-NAME and VEH treated animals. Analysis revealed that L-NAME treatment in females selectively regulated the expression of genes related directly and indirectly to cardiomyocyte Ca2 + handling in a manner consistent with destabilization of Ca2 + homeostasis and arrhythmogenesis. Our investigation provides new insight into the role of sustained decrease in NO bioavailability in determining distinctive female cardiac vulnerability to ischemic challenge. These findings may assist in identifying contexts in which NO signalling could be targeted in a sex-selective manner to achieve therapeutic outcome. As a clinically relevant established model, chronic L-NAME treatment (combined with uninephrectomy/high salt) provides a pre-failure cardiac setting of maintained NO deficiency combined with modest hypertension and renal impairment [20,24]. For women in particular, these co-morbidites coincide in the increasingly prevalent condition of heart failure characterized by diastolic dysfunction with maintained systolic function. In failure, underlying loss of vasodilatory reserve (including NO-dependent vasodilation) is apparent and has been linked with impaired stress responses (notably in a study cohort comprising 75% female participants) [5]. In the present experimental investigation, where a defined ischemia stress was applied to ex vivo hearts (i.e. innervation independent), the findings show that chronic NO deficiency 12

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mmHg/s (%Basal)

120

#†

*

100 80 60 40 20 0

M

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40 20 F

80 60

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20 0

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L-NAME

120

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F

Fig. 4. L-NAME reduces recovery in female hearts at end reperfusion (45 min). A. Recovery of left ventricular developed pressure, B. Recovery of LV cardiac contraction (dP/dtmax), C. Recovery of LV cardiac relaxation (dP/dtmin), D. LV end diastolic pressure. All data analyzed by two-way ANOVA, #p < 0.05 sex effect, *p < 0.05 by Bonferroni's post hoc test, †p < 0.05 interaction effect. Mean ± SEM, n = 7–8.

dP/dtmax

B mmHg/s (%Basal)

LV Developed Pressure

A

F

M VEH

F

0

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M

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VEH

L-NAME

M

L-NAME

rodents in modifying serum NO and nitrate/nitrite levels [29,31] and downstream cGMP generation [32]. Detailed studies of nitrite/nitrate and ROS levels will assist in determining the metabolic and signalling basis for a sex difference in L-NAME cardiac performance modulation. Given the rapid nature of NO production and action, further experiments need to be specifically designed to directly confirm the extent of NO signalling suppression in male and female hearts. The efficacy of NO-targeted interventions in the acute reperfusion setting may be dependent on pre-existing NO bioavailability status – and also on sex-specific NO signalling characteristics. Intervention to

dose L-NAME, the NO deficiency produced may more closely mimic endogenous disease state, with maintained basal cardiac performance, and providing the context in which increased susceptibility of females to ischemic stress can be identified. Further studies are necessary to establish whether equivalent L-NAME dosage in males and females has differential effect on NO metabolism. Beyond inducing NO deficiency, we and others have shown that chronic L-NAME increases myocardial tissue production of reactive oxygen species [20,30]. In this study we did not measure the levels of NO or other related molecules. Other investigators have reported on the efficacy of L-NAME treatment in

A 0.5

1.0

1.5

SL C 9 SL A 1 C 8A SC 1 N 5A R YR FK 2 B P FK 1B B C P1 A C A N A C 1 A C C N A C 1 A C D N A C 1 A C G N A A 1H TP 2A 2 PL C A N M K PP 2D P3 PP CA P PP 3C P2 B PP R 1 P2 A R PP 1B P2 PP CA P2 C B

F Veh M Veh F LNAME M LNAME

Na+ flux

SR load

Ca2+ L+T

SR CaMKII

Protein phosphatases

B

13

Fig. 5. Nitric oxide deficiency and cardiomyocyte expressed gene levels – a female specific response with electromechanical resilience implications. A. Gene expression heat map, red indicates 1.5 expression fold change (i.e. upregulation), green indicates 0.5 expression fold change (i.e. downregulation). Refer Table S2 for full gene descriptions and statistical annotation. B. Mechanism hypothesis: summary of significant gene expression shifts induced in female myocardium with chronic NO deficiency and possible implications for mechanisms underlying acute ischemia-reperfusion vulnerability. All data analyzed by two-way ANOVA with Bonferroni's post hoc test, fold change indicated by colour gradient, n = 7–8. Full abbreviations, refer Table S2. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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5. Conclusions

suppress NO synthesis during reperfusion may be protective if ROS production is reduced but may also be associated with increased inflammation and infarct exacerbation [13,33–35]. Similarly, NO donor intervention can potentially be of benefit or of detriment in contexts where additional stressors are present [10,36–38]. The challenge in translating experimental findings to the clinical context reflects these complexities. Our current findings indicate that evaluation of acute NO interventions using female and male animal disease models of compromised NO bioavailability may be most informative. A relationship between NO signalling and MR activation has been identified in several cell types [18,20,39]. In particular it has been demonstrated that NOdependent endothelial cell dysfunction can be abrogated by genetic deletion of the MR in females [18,19]. We have previously shown that cardiomyocyte MR deletion improves ex vivo cardiac functional responses to ischemia/reperfusion [17]. In this study, our findings show that in animals with cardiomyocyte-specific MR deletion, L-NAME associated differences in functional responses to ischemia/reperfusion were absent. This may suggest a protective role for MR blockade in suppressing NO-deficiency mediated responses, but this proposition requires further direct testing. Male and female MR-KO also showed suppressed and similar macrophage recruitment and fibrosis induction with L-NAME treatment – and this argues against an inflammatory driven mechanism for the difference in functional response. The present study identified a distinctive gene expression signature in myocardium of females exposed to chronic L-NAME treatment. The panel of genes evaluated was constructed to focus on cardiomyocyte expressed genes involved in Na+, Ca2 + and H+ flux impacting electromechanical stability, on important Ca2 + cycling operational substrates regulated by phosphorylation and on the genes encoding proteins responsible for dephosphorylation. It is notable that L-NAME treatment did not induce any significant gene expression responses in male animals. In overview (summarized Fig. 5B), in females the expression shifts suggest a cardiomyocyte transition to higher levels of Na+ & Ca2 + influx from external sources, reliance on increased exchanger activity to control ion homeostasis, an unstable internal Ca2 + store, and regulatory mechanisms with increased dependence on the phosphatase ‘off’ switch. Further studies are required to evaluate protein expression levels and protein post-translational modification status. With the caveat that the gene expression responses require protein verification, they are strongly suggestive of a specific female NO-induced cardiomyocyte electromechanical phenotype vulnerable to ischemia/reperfusion ionic derangement and arrhythmogenesis (as indicated in Fig. 5B) [40]. Importantly, even though these significant expression changes can be detected, the basal function of L-NAME treated hearts is maintained. This indicates an underlying remodelling of operational mode to preserve basal function, with latent potential for stress instability. These analyses represent a collective assessment of expression shifts derived from multiple cell types present in the tissue – including endothelial cells and the most volumetrically significant cardiac myocyte population. Cardiomyocyte impacts of NO deficiency both directly, and indirectly via endothelial mediation seem likely. We and others have shown that female and male cardiomyocytes, subjected to simulated ischemia/reperfusion insults in vitro, perform differently and exhibit specific Ca2 + management responses [41,42]. Accumulating experimental evidence is available to indicate a role for NO modulation of cardiomyocyte Ca2 +[27,43] and Na+[42,44] handling. There is also evidence that L-NAME treatment modifies myocyte Ca2 + levels [27]. To date, these studies have involved male animals, and new information is required to systematically assess male and female cardiomyocyte Ca2 + handling responses. Our findings provide impetus for further work investigating sex difference in cardiomyocyte functional Ca2 + and Na+ responses in settings of disease associated with a deficit in NO bioavailability.

Our experimental investigation provides new insight into the role of sustained in vivo decrease in NO bioavailability in determining distinctive female cardiac vulnerability to ischemic challenge. In female hearts, chronic L-NAME treatment was associated with increased ischemia/reperfusion induced arrhythmic activity, diastolic abnormality and reduced contractile recovery. Gene expression analyses provide direction for further studies to characterize the signalling pathways involved and to examine cardiomyocyte Ca2 + functional dysregulation. These findings may assist in identifying contexts in which NO signalling could be targeted in a sex-selective manner to achieve therapeutic outcome. Disclosures None. Acknowledgements This work was supported by a National Health and Medical Research Council (Aust.) (1010034) Project Grant (MJY), and by the Victorian Government's Operational Infrastructure Support Program. LAB was supported by an Australian Postgraduate Award. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.yjmcc.2017.08.012. References [1] M.J. Leening, B.S. Ferket, E.W. Steyerberg, M. Kavousi, J.W. Deckers, D. Nieboer, et al., Sex differences in lifetime risk and first manifestation of cardiovascular disease: prospective population based cohort study, BMJ 349 (2014) g5992. [2] C.N. Bairey Merz, C.J. Pepine, M.N. Walsh, J.L. Fleg, Ischemia and no obstructive coronary artery disease (INOCA): developing evidence-based therapies and research agenda for the next decade, Circulation 135 (2017) 1075–1092. [3] D.J. Campbell, J.B. Somaratne, A.J. Jenkins, D.L. Prior, M. Yii, J.F. Kenny, et al., Differences in myocardial structure and coronary microvasculature between men and women with coronary artery disease, Hypertension 57 (2011) 186–192. [4] S. Bhushan, K. Kondo, D.J. Polhemus, H. Otsuka, C.K. Nicholson, Y.X. Tao, et al., Nitrite therapy improves left ventricular function during heart failure via restoration of nitric oxide-mediated cytoprotective signaling, Circ. Res. 114 (2014) 1281–1291. [5] B.A. Borlaug, T.P. Olson, C.S. Lam, K.S. Flood, A. Lerman, B.D. Johnson, et al., Global cardiovascular reserve dysfunction in heart failure with preserved ejection fraction, J. Am. Coll. Cardiol. 56 (2010) 845–854. [6] C.C. Lim, N.S. Bryan, M. Jain, M.F. Garcia-Saura, B.O. Fernandez, D.B. Sawyer, et al., Glutathione peroxidase deficiency exacerbates ischemia-reperfusion injury in male but not female myocardium: insights into antioxidant compensatory mechanisms, Am. J. Physiol. Heart Circ. Physiol. 297 (2009) H2144–53. [7] J. Sun, E. Picht, K.S. Ginsburg, D.M. Bers, C. Steenbergen, E. Murphy, Hypercontractile female hearts exhibit increased S-nitrosylation of the L-type Ca2 + channel alpha1 subunit and reduced ischemia/reperfusion injury, Circ. Res. 98 (2006) 403–411. [8] H. Sasaki, T. Nagayama, R.M. Blanton, K. Seo, M. Zhang, G. Zhu, et al., PDE5 inhibitor efficacy is estrogen dependent in female heart disease, J. Clin. Invest. 124 (2014) 2464–2471. [9] E. Murphy, C. Steenbergen, Sex, drugs, and trial design: sex influences the heart and drug responses, J. Clin. Invest. 124 (2014) 2375–2377. [10] J.S. Bice, B.R. Jones, G.R. Chamberlain, G.F. Baxter, Nitric oxide treatments as adjuncts to reperfusion in acute myocardial infarction: a systematic review of experimental and clinical studies, Basic Res. Cardiol. 111 (2016) 23. [11] N. Siddiqi, C. Neil, M. Bruce, G. MacLennan, S. Cotton, S. Papadopoulou, et al., Intravenous sodium nitrite in acute ST-elevation myocardial infarction: a randomized controlled trial (NIAMI), Eur. Heart J. 35 (2014) 1255–1262. [12] R.H. Ritchie, G.R. Drummond, C.G. Sobey, T.M. De Silva, B.K. Kemp-Harper, The opposing roles of NO and oxidative stress in cardiovascular disease, Pharmacol. Res. 116 (2017) 57–69. [13] G.K. Couto, L.R. Britto, J.G. Mill, L.V. Rossoni, Enhanced nitric oxide bioavailability in coronary arteries prevents the onset of heart failure in rats with myocardial infarction, J. Mol. Cell. Cardiol. 86 (2015) 110–120. [14] S. Moncada, R.M. Palmer, E.A. Higgs, Nitric oxide: physiology, pathophysiology, and pharmacology, Pharmacol. Rev. 43 (1991) 109–142. [15] H.R. Cross, E. Murphy, W.J. Koch, C. Steenbergen, Male and female mice

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