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European Journal of Heart Failure 2003 5(3):229-239; doi:10.1016/S1388-9842(03)00010-2
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© 2003 European Society of Cardiology

Role of nitric oxide in posthypoxic contractile dysfunction of diabetic cardiomyopathy

Magdi M. El-Omara, Roger Lordb, Nick J. Drapera and Ajay M. Shahc,*

a Department of Cardiology, University of Wales College of Medicine Heath Park, Cardiff CF4 4XN, UK
b Discipline of Surgery, University of Tasmania Hobart, Australia
c Guy's, King's & St. Thomas's School of Medicine, King's College London Bessemer Road, London SE5 9PJ, UK

* Corresponding author. Tel: +44-207-346-3865; fax: +44-207-346-4771. E-mail address: ajay.shah{at}kcl.ac.uk


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
We investigated the role of nitric oxide synthase (NOS) in the contractile dysfunction of diabetic cardiomyopathy, comparing streptozotocin-treated (60 mg/kg) diabetic Wistar rats with matched non-diabetic controls. Isolated isovolumic heart function was studied during normoxia and in response to brief hypoxia-reoxygenation. Diabetic hearts had significantly lower left-ventricular pressure and slower isovolumic relaxation than controls (relaxation time constant, T 40.2±2.3 vs. 27.7±0.9 ms; P<0.05) and a blunted response to hypoxia. These abnormalities were unaffected by NOS inhibition. Upon reoxygenation after brief hypoxia, diabetic hearts exhibited substantial worsening of LV relaxation compared to normal hearts (T 69.1±3.3 vs. 56.6±7.9 ms; P<0.05). This post-hypoxic diastolic dysfunction was significantly attenuated either by the non-selective NOS inhibitor L-NAME, the iNOS inhibitor L-NIL, or the reactive-oxygen-species (ROS) scavenger thiourea. Only diabetic hearts expressed iNOS protein, whereas eNOS expression was similar in both groups. In conclusion, diabetic hearts exhibit markedly abnormal post-hypoxic relaxation, which is attributable to both ROS and NO derived from iNOS.

Key Words: Diastole • Nitric oxide synthase (NOS) • Hypoxia • Relaxation • Reactive oxygen species

Received August 16, 2002; Revised October 18, 2002; Accepted November 12, 2002


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 
Diabetes mellitus is associated with a specific cardiomyopathy, independent of hypertension, coronary artery disease or hyperlipidaemia, as evidenced by clinical studies and experimental animal models, such as streptozotocin-induced diabetes [1–3]. Prominent early features of diabetic cardiomyopathy include impaired myocardial relaxation and increased diastolic stiffness (i.e. diastolic dysfunction), abnormalities which are exacerbated following even brief ischaemia [1–3]. More advanced cardiomyopathy is characterised by systolic and autonomic dysfunction. The underlying mechanisms of diabetic cardiomyopathy remain incompletely understood. Intrinsic abnormalities of excitation–contraction coupling and the myofilaments, as well as metabolic dysfunction, are likely to contribute [1–3].

Previous work from our laboratory and others has established that nitric oxide (NO) is an important modulator of myocardial function [4,5]. Endothelium-derived NO modulates myocardial relaxation, diastolic tone and oxygen consumption in preparations ranging from single myocytes and isolated hearts to the intact heart in vivo [5,6]. NO may also be generated physiologically within cardiac myocytes by an endothelial-type nitric oxide synthase (eNOS), and can modulate β-adrenergic inotropic response. In certain pathological settings, the inducible form of nitric oxide synthase (iNOS) is expressed in cardiac myocytes and endothelial cells, as well as in other cell types. Expression of iNOS in dilated cardiomyopathy and endotoxic shock is suggested to contribute to myocardial dysfunction [4,5]. In the context of ischaemia-reperfusion or hypoxia-reoxygenation, the reaction of NO with superoxide radicals to form peroxynitrite may induce deleterious effects, depending upon the level of peroxynitrite generated [6,7].

The possible role of eNOS or iNOS in the development of diabetic cardiac dysfunction is unclear. However, many recent studies have reported alterations in the expression of NOS isoforms and/or NO bioactivity in several diabetic tissues. For example, iNOS is reported to be expressed in the diabetic kidney [8], vascular smooth muscle [9] and retina [10]. Vascular endothelial dysfunction, in part due to reduced bioavailability of NO, is known to be a feature of diabetes [7,11]. The aims of this study were to examine: (a) the potential roles of NOS in baseline cardiac dysfunction in the streptozotocin-treated rat, an experimental model of insulin-dependent diabetic cardiomyopathy independent of coronary atherosclerosis [2,3]; and (b) the response of the diabetic heart to brief hypoxia-reoxygenation, an insult that exacerbates diastolic dysfunction.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 5. Conclusions
 References
 
2.1. Experimental animals
Experiments were performed in accordance with the UK Animals (Scientific Procedures) Act 1986 and institutional guidelines. Diabetes was induced in male Wistar rats (200–300 g) by intraperitoneal streptozotocin injection (60 mg/kg in 0.1 M citrate buffer). Diabetes was confirmed on the third day post-streptozotocin by hyperglycaemia (>20 mmol/l, Glucometer). Approximately 15% of rats had glucose levels <20 mmol/l and were excluded from the study. Weekly urinalysis was performed for ketonuria. At killing 4–6 weeks later, blood/serum were collected for estimation of glycosylated haemoglobin A1 (HbA1C) and random glucose. A total of 64 diabetic animals were studied.

The control group comprised 55 age- and sex-matched non-diabetic litter mates, which were studied contemporaneously. All studies were comparisons of diabetic animals against matched non-diabetic (control) animals.

2.2. Isolated perfused hearts
Animals were terminally anaesthetised with intraperitoneal sodium pentobarbitone (60 mg/kg). Hearts were rapidly excised and mounted on a non-recirculating Langendorff apparatus. The perfusion solution comprised (in mM): NaCl 117; KCl 5.7; NaHCO3 4.4; NaH2PO4 1.2; CaCl2 1.25; MgCl2 1.7; hydroxyethylpiperazine ethanesulfonic acid (HEPES) 20; and glucose 10; pH 7.4, 37 °C. The pH was adjusted to 7.4 with NaOH. This was gassed with 100% O2 during normoxic perfusion (pO2{approx}700 mmHg) and 100% N2 during hypoxic periods (pO2{approx}40 mmHg). Coronary flow rate was adjusted to give a mean coronary perfusion pressure (CPP) of 80 mmHg, and was then maintained constant. Hearts were paced at 10% above intrinsic rate by a right atrial electrode at 10% above threshold voltage. Left ventricular pressure (LVP) was monitored using an intraventricular balloon connected to a Statham (P23XL) pressure transducer. LV end-diastolic pressure (LVEDP) was set to 10 mmHg. Pressure data were sampled via a MacLab data module (AD Instruments, UK). LV dP/dtmax was obtained from the first derivative of the LVP signal. The exponential time-constant of isovolumic LV relaxation, T, was calculated as previously described [12].

2.3. Experimental protocol
LV function of diabetic and control hearts was compared during normoxia (after at least 20 min of equilibration) and during exposure to brief hypoxia (10 min, pO2{approx}40 mmHg) and reoxygenation. We studied the effects of the following interventions, each in a separate group of diabetic or non-diabetic hearts (n≥6, except n=4 for control +L-NIL group): (a) no additional agents added; (b) a non-selective NOS inhibitor, L-NAME (50 µM); (c) a selective iNOS inhibitor, L-NIL (10 µM); (d) a cyclooxygenase inhibitor, indomethacin (10 µM); (e) a lipooxygenase inhibitor, diethylcarbamazine (0.25 mM); (f) a scavenger of reactive oxygen species (ROS), thiourea (10 mM); and (g) insulin 0.01 IU/ml. In hypoxia-reoxygenation experiments, test compounds were added at least 5 min prior to hypoxia and remained present subsequently. At the end of experiments, hearts were blotted dry and weighed.

2.4. Western analyses for eNOS and iNOS
Cardiac myocytes were isolated from perfused hearts by collagenase digestion [13]. After passage through a 2% BSA cushion, they comprised >95% rod-shaped striated cells with no significant endothelial cell contamination. Myocytes were lysed in 1 ml of boiling 1% SDS with 10 mM Tris (pH 7.4). Protein concentration was measured using a micro BCA kit (Pierce). Equal amounts of protein (100 µg/lane) were run on 8.5% SDS-polyacrylamide gels and electroblotted onto nitrocellulose membrane. Membranes were incubated either with a rabbit anti-iNOS polyclonal antibody (Calbiochem, La Jolla, CA) or a rabbit anti-eNOS polyclonal antibody (Alpha Diagnostics, TX). The secondary antibody was a horseradish peroxidase-linked donkey anti-rabbit antibody (Amersham, UK). Blots were assessed by densitometry (Bio Rad GS-700 densitometer, Hercules, CA).

2.5. Measurement of total nitrite/nitrate (NOx)
Plasma samples from control and streptozotocin-treated rats were stored at –20 °C. Total plasma NOx was measured using a 2,3-diamino-napthalene fluorometric assay, adapted for use in a 96-well plate, after treating with nitrate reductase to convert nitrate to nitrite [14]. Total NOx concentration was assessed from the 450-nm fluorescence emission after excitation at 375 nm, using sodium nitrite and sodium nitrate to generate standard curves.

2.6. Materials
Unless stated otherwise, all agents were obtained from Sigma Chemical Co (UK) and were prepared fresh in physiological buffer, apart from indomethacin, which was initially dissolved in dimethylsulfoxide (DMSO). The final DMSO concentration (<0.01%) had no significant effects.

2.7. Data analysis
Results are presented as mean±standard error. Responses to hypoxia-reoxygenation, as well as baseline effects of compounds, were compared by two-way repeated-measures ANOVA, followed by a Student–Neumann–Keul test to isolate differences. Comparison of single effects among groups was by one-way ANOVA, followed by a Student–Neumann–Keul test to isolate differences. P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 5. Conclusions
 References
 
3.1. Baseline characteristics
Streptozotocin-treated rats had severe hyperglycaemia, elevated HbA1C, and reduced body and heart weights compared to controls (Table 1). The relative loss of heart weight was less than that of body weight, as reflected in higher heart/body weight ratios (Table 1). None of the animals had any clinical evidence of infection either before sacrifice or at post-mortem. In a subset of eight diabetic animals, rectal temperatures measured in the week prior to killing were normal (36.4±0.1 °C).


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  		Table 1 Baseline characteristics of control and diabetic groups

 
3.2. Expression of eNOS and iNOS
Fig. 1a shows the expression of eNOS protein in control and diabetic cardiac myocytes. The level of eNOS protein was similar in the two groups (11.5±0.79 and 14.83±2.21 densitometric units, respectively; P=NS; n=6 hearts per group). In contrast, iNOS expression was significantly different between groups. iNOS protein was expressed in all diabetic samples, but was undetectable in any of the control samples (Fig. 1b). Similar results were also obtained with the use of whole-heart protein samples in the two groups (n=5 hearts per group; data not shown).


Figure 1
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  		Fig. 1 eNOS and iNOS protein expression in diabetic and control myocardium. (a) Western blot showing isolated cardiac myocyte protein probed for eNOS: lanes a–c, control myocytes, lanes d–f, diabetic myocytes, lane g, eNOS positive control (Alpha Diagnostics, TX). Similar results were obtained in two independent experiments. (b) Isolated cardiac myocyte protein probed for iNOS: lanes a–e, diabetic myocytes, lanes f–j, control myocytes, lane k, iNOS positive control (Calbiochem). Similar results were obtained in two experiments.

 
Plasma NOx levels were significantly higher in diabetic animals compared to controls (24.42±6.74 vs. 6.97±0.84 µM; n≥7 per group; P<0.002)

3.3. Isolated heart function during normoxia
Diabetic hearts had significantly lower heart rates, LV developed pressures (LVDP) and LV dP/dtmax, and a higher T (slower isovolumic relaxation) than controls (Table 1). CPP and coronary flow per gram of heart tissue were similar in the two groups (Table 1). Parameters of LV function remained stable for >50 min under normoxic conditions in both diabetic and non-diabetic hearts (data not shown).

3.4. Effect of NOS inhibitors and other agents during normoxia
Neither L-NAME nor L-NIL affected LVDP, LV dP/dtmax, or T in diabetic hearts at baseline (Table 2; n≥6 per group). Likewise, there was no significant effect of indomethacin, diethylcarbamazine or thiourea (Table 2). CPP was significantly increased by L-NAME (+6.1±1.7%; P<0.05) and indomethacin (+14.3±4.3%; P<0.05), whereas thiourea significantly decreased CPP (–14.6±3.1%; P<0.05). Acute treatment of diabetic hearts with insulin significantly increased baseline LVDP and LV dP/dtmax, and decreased T (Table 2). However, insulin had no effect on baseline CPP in diabetic hearts (81±1.3 mmHg before and 78±2.4 mmHg after insulin; P=NS).


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  		Table 2 Effect of interventions on baseline contractile function in diabetic hearts

 
In control hearts, L-NAME increased CPP to a greater extent (+33.3±4.5% compared to baseline; P<0.05) than in diabetics, whereas indomethacin did not significantly alter CPP. L-NAME, L-NIL, indomethacin or thiourea had no effect on other LV contractile parameters in non-diabetic hearts (Table 3).


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  		Table 3 Effect of interventions on baseline contractile function in non-diabetic (control) hearts

 
3.5. Isolated heart function during brief hypoxia-reoxygenation
Fig. 2 shows the effects of 10-min hypoxia-reoxygenation in control and diabetic hearts. In controls, hypoxia caused marked reduction in LVDP, LV dP/dtmax and CPP, and prolonged T within 1–2 min. In marked contrast, in diabetic hearts there was a much slower decline in LVDP and LV dP/dtmax, and a more gradual prolongation of T. Thus, end-hypoxic levels of LVDP and LV dP/dtmax were higher in diabetic than control hearts, despite significantly lower pre-hypoxic values. However, the reduction in CPP in diabetic hearts was similar to controls, reaching a nadir slightly lower than the controls.


Figure 2
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  		Fig. 2 Effect of 10-min hypoxia-reoxygenation in control (C) and diabetic (STZ) hearts. H denotes the period of hypoxia. CPP, coronary perfusion pressure; LVDP, LV developed pressure; T, time constant for isovolumic relaxation. *P<0.05.

 
Upon reoxygenation of control hearts, all indices recovered fairly rapidly. There was a minor deterioration in T soon after reoxygenation (average 3.2±1.0 ms) (Fig. 2). By 15 min after reoxygenation, LVDP, CPP and T were not significantly different from pre-hypoxic levels, but LV dP/dtmax was still slightly lower than the pre-hypoxic level.

In diabetic hearts, however, upon reoxygenation there was a striking further prolongation in T (average 18.6±3.0 ms or 50.2±9.3% of the baseline value), which gradually recovered over the next 10–15 min (Fig. 2). In contrast, LVDP, LV dP/dtmax and CPP in diabetic hearts recovered rapidly following reoxygenation. Thus, reoxygenation was accompanied by a transient, but profound, further slowing of LV relaxation in the diabetic group.

3.6. Effects of NOS inhibitors and other agents on the response to hypoxia-reoxygenation
In the presence of L-NAME, L-NIL or thiourea, the marked prolongation of T upon reoxygenation of diabetic hearts was significantly attenuated (Figs. 3 and 4). The improvement in LV relaxation with these agents occurred in the absence of any significant effects on LVDP or LV dP/dtmax (Figs. 5 and 6). None of the other agents tested significantly affected the post-hypoxic relaxation abnormality of diabetic hearts (Figs. 3 and 4). Of note, post-hypoxic prolongation of T was unaltered in the insulin-treated diabetic group (Fig. 3), and remained significantly greater than non-diabetic (control) hearts despite the improvement in pre-hypoxic LV relaxation. Interestingly, in the presence of indomethacin, hypoxic coronary vasodilatation was significantly blunted in diabetic hearts, and this was associated with impaired post-hypoxic recovery of LVDP and LV dP/dtmax (Fig. 6).


Figure 3
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  		Fig. 3 Effect of interventions on the isovolumic relaxation constant, T, in diabetic hearts subjected to hypoxia-reoxygenation. *P<0.05 compared with untreated group.

 


Figure 4
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  		Fig. 4 Effect of interventions on the increase in isovolumic relaxation constant, T, induced by reoxygenation in (a) diabetic and (b) non-diabetic hearts. The maximal change in T compared to the corresponding end-hypoxic value for each group is shown. *P<0.05 for each column compared with the untreated group. Diethylcarb, diethylcarbamazine.

 


Figure 5
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  		Fig. 5 Effect of L-NAME (left panels) or L-NIL (right panels) on the response of diabetic hearts to hypoxia-reoxygenation. Abbreviations as in Fig. 2.

 


Figure 6
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  		Fig. 6 Effect of thiourea (left panels) or indomethacin (Indom, right panels) on the response of diabetic hearts to hypoxia-reoxygenation. Abbreviations as in Fig. 2.

 
In control non-diabetic hearts, L-NAME, L-NIL or thiourea had no significant effect on T (Fig. 4) or on LV contractile parameters upon reoxygenation (data not shown).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
5. Conclusions
 References
 
The main new findings of this study were that: (1) streptozotocin-induced diabetic hearts exhibited a profound (although transient) post-hypoxic relaxation abnormality, which was attributable to both NO derived from iNOS and to ROS; and (2) baseline dysfunction of the diabetic heart (systolic impairment, reduced heart rate and slow LV relaxation) was not influenced by NOS inhibitors or a ROS scavenger. In addition, LV contractile depression during hypoxia was blunted in diabetic hearts, a response again unaffected by NOS inhibition.

4.1. Baseline dysfunction in diabetes and role of NO
The baseline dysfunction observed in this study is similar to that previously reported in many other studies of streptozotocin-induced diabetes [2,3]. Abnormal myocardial relaxation is an early feature of diabetic cardiomyopathy, and several different underlying mechanisms have been postulated including: abnormalities of cardiac repolarisation [15], Ca2+ handling [16], Na+/H+ exchange [17], myofilament Ca2+ responsiveness [3] and metabolism [18].

In the present study, we showed that, despite increased expression of iNOS in the diabetic heart, the overproduction of neither NO nor ROS significantly contributed to baseline contractile dysfunction, at least acutely. Similarly, neither the cyclooxygenase nor lipooxygenase pathways were involved, since indomethacin and diethylcarbamazine also had no effects on LV function. The lack of effect of NOS inhibitors on baseline dysfunction, despite documented iNOS expression, might appear surprising, but is consistent with several studies in vitro and in humans in vivo, where NO derived from iNOS was found to have little or no effect on basal function [4,5,19–21]. In some of these studies, an influence of NO on β-adrenergic inotropic responsiveness was nevertheless demonstrated [5,19,20]. The possibility that L-arginine availability may be rate-limiting is not consistent with the finding that NOS inhibitors altered CPP and that they had demonstrable effects during reoxygenation.

It is notable that heart rates in the diabetic group were slightly but significantly lower than the control group. The possibility that this might have contributed to reduced systolic function in diabetic hearts should therefore be considered. We believe this is unlikely for several reasons. First, although diabetic and control hearts had different rates as a group, there was a considerable overlap between them and there was no significant correlation between heart rate and LV dP/dtmax. Secondly, baseline function was acutely improved by insulin independent of heart rate, suggesting that a major component of contractile dysfunction during normoxia may be primarily metabolic. Thirdly, it has previously been shown that a substantial proportion of contractile dysfunction in the diabetic heart is intrinsic to the cardiac myocyte, independent of heart rate [2,3]. The abnormal myocardial relaxation observed at baseline in diabetic hearts is also unlikely to be related to lower heart rates, since relaxation in fact worsened at higher heart rates (not shown).

4.2. Response to brief hypoxia
Hypoxia-induced LV contractile depression was found to be significantly blunted in diabetic hearts compared to controls, implying increased tolerance to hypoxia. Similar findings have previously been reported, although some studies have also reported increased sensitivity of the diabetic heart to ischaemia (for review, see [22]). The precise definition of the mechanisms underlying this effect was not the focus of the present study. However, we were able to demonstrate that the blunted response to hypoxia of diabetic hearts was unaltered in the presence of either L-NAME or L-NIL. This finding is of interest, given the evidence of iNOS expression in the diabetic heart and recent data that iNOS expression can increase the tolerance to ischaemic insults (e.g. in ischaemic myocardial preconditioning) [23].

The other notable observation during hypoxia was that hypoxic coronary vasodilatation in diabetic hearts appeared to depend more on cyclooxygenase products than on NO (as opposed to the situation in control hearts), consistent with previous data that endothelial NO bioactivity is diminished and prostanoid production increased in diabetes [7,11,24]. Furthermore, cyclooxygenase inhibition caused substantial impairment of post-hypoxic contractile recovery in diabetic hearts, suggesting that cyclooxygenase products contribute significantly to the maintenance of diabetic cardiac function in this setting.

4.3. Reoxygenation-induced dysfunction
The most striking and novel finding in the present study was that of the dramatically abnormal post-hypoxic relaxation in diabetic hearts. Although contractile dysfunction (in particular diastolic dysfunction) upon reoxygenation/reperfusion following hypoxia/ischaemia is a recognised phenomenon in normal hearts, it is usually quite mild after brief hypoxic (or ischaemic) episodes at physiological temperatures (37 °C) [25]. The underlying mechanisms include ROS production at reoxygenation, cytosolic Ca2+ overload and changes in myofilament Ca2+ responsiveness [26]. The significant improvement in post-hypoxic relaxation dysfunction in diabetic hearts with the iNOS-selective inhibitor, L-NIL, and the presence of iNOS protein in these hearts strongly suggested that iNOS-derived NO was a major culprit. As might be expected, a non-selective NOS inhibitor (L-NAME), which inhibits all NOS isoforms, was also effective. Consistent with an increase in NO production in the diabetic heart, we found that plasma NOx levels were significantly elevated in this group.

The effectiveness of an ROS scavenger, thiourea, in reducing post-hypoxic relaxation dysfunction supported a role for ROS, as well as NO. This is consistent with previous data that ROS production is increased in diabetes [7]. An involvement of ROS in the post-hypoxic relaxation abnormality may explain the apparent paradox that NOS inhibitors did not influence baseline dysfunction, but attenuated post-hypoxic abnormalities. It may be that both NO and ROS are necessary for the occurrence of the post-hypoxic abnormality in diabetic hearts (possibly through peroxynitrite formation), and that this occurs to a significant extent only during reoxygenation. It is well recognised that ROS generation is particularly increased at reoxygenation, while NO production is also augmented during reoxygenation [6,7]. Peroxynitrite formation can contribute to contractile dysfunction via protein nitration, membrane damage, antioxidant depletion and inhibition of respiration [7], and it is possible that increased generation of both ROS and NO at reoxygenation could lead to peroxynitrite-induced effects on contractile function. Although potential sources of ROS production were not defined in the present study, it is of interest that NOS itself can generate superoxide, especially during co-factor (tetrahydrobiopterin) deficiency [7].

4.4. Induction of iNOS in diabetes
Several studies have reported the expression of iNOS in diabetic tissues, such as the kidney [8], vessels [9] and retina [10]. A potential mechanism for iNOS induction in diabetes may be the elevated systemic levels of cytokines (e.g. TNF-{alpha}) known to occur in the condition [27]. More recently, iNOS expression was also detected in the hearts of streptozotocin-diabetic rats [28,29]. However, neither of these studies investigated the response to hypoxia-reoxygenation in these hearts.

In the present study, we found no significant changes in eNOS expression in the diabetic heart. Previous studies have reported variable changes in eNOS expression in diabetic tissues, including increased eNOS expression in vessels and kidney or decreased eNOS [7,11,30]. In the diabetic heart too, eNOS expression has been reported to be increased [28,29], decreased [31] or unaltered [32]. Again, the latter studies did not investigate hypoxia-reoxygenation.

4.5. Study limitations
Extrapolation of the present data obtained in isolated hearts to the situation in vivo must be made with caution. In particular, no blood cells were present in the perfusing buffer, so that the potential contribution of these cells was not assessed. The absence of blood may also significantly alter the absolute levels and kinetics of ROS and NO generation. Secondly, diabetic hearts in this study had lower heart rates as a group than the non-diabetic hearts. However, as discussed earlier, it is unlikely that the present results are accounted for by this difference in heart rates. Finally, with respect to LV relaxation in vivo, factors such as cardiac load and prevailing neurohumoral status would also be important determinants of the overall response.


   5. Conclusions
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
References
 
The present study shows that streptozotocin-induced diabetes in the rat is associated with a distinctive response to brief hypoxia-reoxygenation, in particular an exaggerated post-hypoxic relaxation abnormality. Abnormal post-hypoxic relaxation is attributable to the formation of both iNOS-derived NO and ROS, whereas baseline systolic and diastolic dysfunctions appear to be independent of NO. In humans, abnormally delayed ventricular relaxation and/or an increase in ventricular diastolic stiffness contribute to elevated filling pressures and impairment of ventricular filling, and may compromise subendocardial perfusion, particularly in the diseased heart—so-called ‘diastolic dysfunction’. Diastolic LV dysfunction adversely affects prognosis in diabetic patients, e.g. after acute myocardial infarction [33]. The possible contribution of myocardial iNOS expression to ischaemic and post-ischaemic contractile dysfunction in diabetic patients merits investigation.


   Acknowledgements
 
Supported by the British Heart Foundation and the UK Medical Research Council. ME was supported by a British Heart Foundation (BHF) Junior Research Fellowship and NJD by a UK Medical Research Council PhD studentship. AMS holds the BHF Chair of Cardiology at King's College London. We are very grateful to Dr Phil James for undertaking the plasma NOx assays.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 5. Conclusions
 References
 

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