Myocardial susceptibility to ischaemia/reperfusion in obesity: a re-evaluation of the effects of age

The effect of ischaemia/reperfusion (I/R) on the myocardium as well as its response to cardioprotective interventions are influenced by several variables, amongst others, age and obesity. The respective contributions of the latter two factors have recently been extensively reviewed [1‐4].

It is of significance that the vast majority of studies on the response of the heart to I/R injury, the processes involved and the consequences of interventions, have been conducted on healthy or mature populations of experimental animals. As cardiovascular diseases are the leading cause of death, particularly in the elderly, cognizance should be taken of the role of age-induced changes in the response of the heart to pathophysiological conditions. The effects of increasing age on the cardiovascular system are well-established and entail, amongst others, an increase in left ventricular mass, myocyte hypertrophy and a reduction in myocyte number [5‐7]. Apart from the significant changes in β-adrenergic signalling [8, 9], mitochondrial ROS production also increases with age, which in turn, may cause mitochondrial damage [6]. The amounts of ROS generated are critical for the fate of the cell, since low amounts can function as signalling molecules and are important in cardioprotection, whereas high amounts are detrimental by opening the mitochondrial permeability transition pore, causing cell death [10].

Aging decreases the intrinsic tolerance of the heart to ischaemic injury. For example, it has been shown that loss of myocardial tolerance to ischaemia in the mouse begins during the middle-age (~12 months) and becomes outspoken at 18–24 months of age [11]. This increased susceptibility of the heart to I/R damage is likely to be the consequence of enhanced oxidative stress [11‐13].

In addition to the effects of old age on the response of the heart to I/R, obesity is also considered to be a serious risk factor in the development of cardiovascular disorders and is regarded to impact negatively on the outcome of myocardial ischaemia. Since obesity associated with insulin resistance has been demonstrated to increase oxidative stress, and is associated with coronary artery disease [14‐16], I/R occurring in this condition could provoke a further increase in oxidative stress. However experimental studies reported a variety of outcomes. A decreased myocardial tolerance to I/R damage was observed in vivo [17] and ex vivo studies using hyperphagia-induced obese insulin resistant male Wistar rats [18‐21]. These studies have shown that an increased infarct size is associated with concomitant poor functional recovery in hearts from obese rats compared with controls. In contrast to the above, smaller infarcts and an improved functional recovery during reperfusion were reported by Donner and coworkers [22] also using a hyperphagia-induced obesity rat model. A recent study from our own laboratory also showed that hearts from rats fed either a sucrose-supplemented diet or a high fat diet for 16 weeks presented with a significant reduction in infarct size when subjected to 35 min regional ischaemia/60 min reperfusion, when compared to age-matched controls [23]. Furthermore, the degree of left ventricular (LV) dysfunction in Zucker obese rats during I/R was reported to be similar to that of lean rats [24]. At the moment the reason(s) for these discrepancies are not clear and could be another manifestation of the phenomenon of the “obesity-paradox” which has been observed in humans: Observational studies have shown that obese heart failure patients tend to have higher survival rates compared to patients with heart failure and normal BMI values [25, 26].

In agreement with the marked effects of age on the susceptibility of the heart to I/R damage, a number of experimental studies showed that the cardioprotective power of ischaemic pre- and postconditioning waned with ageing (for reviews see [1‐3]), despite variations in age and preconditioning protocols. The majority of these studies used haemodynamic recovery as endpoint [27‐30], with only a few employing the more robust endpoint of infarct size [31, 32]. . It should be borne in mind that in these ex vivo studies, using the isolated perfused heart, the period of ischaemia was followed by immediate reperfusion, usually for 60 min, during which time functional recovery was determined. This protocol therefore does not allow evaluation of chronic recovery. Clearly the effects of age and obesity should also be evaluated in an in vivo model which allows study of the consequences of I/R on chronic recovery.

In view of earlier observations made in our laboratory, namely that hearts from rats with hyperphagia-induced insulin-resistance, aged 22 weeks, are characterized by an unaltered functional performance and an improvement in resistance to I/R injury [23], the aim of the present study was to re-evaluate the effects of age and obesity-induced insulin resistance on myocardial susceptibility to I/R damage and functional performance during reperfusion as well as the capacity to be protected by prior ischaemic preconditioning. For this purpose two age groups were used, namely 22 weeks and 17 months. The precise correlation between the age of laboratory rats and the human is still a matter of debate and complicated by the different phases of life. It has been estimated that in general, considering their entire life span, a human month resembles one every-day life of a laboratory rat [33]. Based on these assumptions, it was calculated that the 22 weeks old and 17 months old rats represent 13 and 43 human years respectively and were referred to as “young” and “middle-aged” throughout the article.

The hyperphagia-induced model of obesity, as originally described by Pickavance and coworkers [34], was used in this study. This diet resulted in an initial significant rise in body weight and intraperitoneal fat seen after 16 weeks on the diet. This difference was still present after 17 months, although aging per se also caused a significant increase in intraperitoneal fat in the age-matched controls. We have previously demonstrated that the diet induced insulin resistance associated with a significant rise in serum insulin but not glucose levels [38].

A number of interesting observations were made using the well-characterized isolated working perfused rat heart for evaluation of necrosis and function: (i) exposure of hearts of young rats to 35 min of regional ischaemia resulted in a significant reduction in infarct size in DIO hearts compared to their AMC (Fig. 1), confirming previous observations made in the same model [22, 23]. With increasing the age to 17 months, however, the difference between AMC and DIO hearts was no longer observed (ii) baseline functional performance of the working heart model, was similar in hearts from AMC and DIO rats and not affected by age (iii) contrary to expectations, after regional ischaemia hearts from middle-aged AMC rats recovered functionally during reperfusion as well as their younger (22 weeks) counterparts (iv) the reduction in infarct size of DIO hearts after 22 weeks, was not associated with an improvement in functional recovery, probably due to concomitant stunning (v) infarct size as well as functional recovery during reperfusion of hearts from DIO rats were not affected by age and no differences were observed in these parameters after 17 months when compared with those after 22 weeks (vi) although hearts from middle-aged AMC and DIO rats can be preconditioned, functional recovery was depressed in both middle-aged groups despite the very significant reduction in infarct size (Fig. 3).

Effect of age: It is clear from the above summary, that the results obtained in the present study differ from the generally accepted viewpoint, namely that aging has detrimental effects on the mechanical performance of the heart as well as on its response to I/R injury. The aging cardiomyocyte has been described to develop a reduction in tolerance to stress, mitochondrial function and contractile function associated with increased susceptibility to apoptosis and necrosis (for review see [39]) with ROS playing a pivotal role. Interestingly, many of the studies on the aging myocardium have been done on mouse hearts, with mice aged 12 months being regarded as “middle-aged” and those aged 24–28 months as “aged” [11]. Since rats aged 24 months have been regarded as old [1, 2], the rats used in the present study (aged 17 months), were regarded as “middle-aged” rather than aged and could account for the fact that no marked differences in baseline mechanical performance and in the response to regional ischaemia were seen in the present study, regardless of age. However, no significant increase in infarct size between young and aging cohorts has also been reported by two other groups namely Schulman [31] and Fenton and coworkers [40]. Interestingly, in both the latter studies rats aged 18–20 months [31] and 22 months [40] were regarded as “aged”.

It should be kept in mind that the mechanical performance of the working heart model was evaluated under carefully controlled baseline conditions, such as fixed pre-and afterloads, without pacing. Their response to increased workloads, which may disclose defects in performance, was not investigated in the present study and is a topic for further experiments. It should be noted that exposure to more severe ischaemic stress, namely15 min global ischaemia, indicated that hearts from middle-aged DIO rats are more susceptible to ischaemic damage than their age-matched controls (Table 3).

Effect of obesity: Since obesity-induced insulin resistance as well I/R are known to be associated with increased oxidative stress [14‐16], it was expected that the combination of age and obesity should exacerbate myocardial I/R injury. However, indications are that the model of hyperphagia-induced obesity used in the present study, is not characterized by significant oxidative stress: normalized values for serum TBARS, conjugated dienes and lipid hydroperoxide were similar to those of age-matched controls [20], suggesting that this phenomenon may not play a role in the results obtained.

Thus in the working heart model subjected to regional ischaemia, functional recovery during reperfusion of DIO hearts did not differ significantly between the young and middle-aged groups, although there are indications of deterioration in the hearts of the latter groups when subjected to global ischaemia (Table 3). This possibility is supported by the echocardiographic studies done in the middle- aged group which indicated a significant increase in LVIDd and EDV (Table 6). Dilated left ventricles with preserved systolic function are indicative of early stage dilated cardiomyopathy [41]. Whether this was the case in the DIO hearts remains to be determined. Interestingly, a similar increase in LVIDd was seen in young (16–20 week) DIO rats [42]. Obesity however had no significant effect on fractional shortening. Using a similar model of obesity, Wensley and coworkers could not find any echocardiographic changes after 32 weeks on the diet [18].

The reduction in infarct size observed in young DIO rats as well as the fact that all these hearts subjected to I/R were able to produce aortic output during reperfusion suggest that the diet protect against I/R damage, confirming previous data from our laboratory [23]. These observations are also in agreement with the findings in a similar model [22] and in fructose-fed rats [43]. However, our results are in contrast with those obtained in genetic deletion models of obesity [44‐46] and in diet models [24, 47‐49] where infarct size was either increased or unchanged respectively. The reasons for these controversies are not clear yet but may be due to, amongst other factors, differences in experimental protocols, diet, age and species of the animals etc. In our own hands, preference was given to use of infarct size as the more robust endpoint instead of left ventricular function which can be influenced by other factors such as concomitant stunning during reperfusion.

Explanations for the beneficial effects observed of obesity and insulin resistance on the outcome of I/R remain to be established. A previous study from our laboratory on 22 week old DIO rats did not show an association between the reduction in infarct size and activation of the RISK pathway during reperfusion [23], a signalling pathway which has been shown to be activated during cardioprotection in many experimental interventions [50, 51]. As far as we know, the role of the JAK-STAT pathway has not been investigated in this regard.

Possible signalling mechanisms involved in the effects of obese-insulin resistance have been reviewed by Apaijai and coworkers [4]. Unfortunately, as was the case in the outcome of the effects of I/R, no clear-cut pattern of changes emerged. Another factor which could contribute to the many discrepancies observed, is the fact that the time of sampling for Western blotting varied from 30 min [52] to 10 min of reperfusion [23] or before induction of ischaemia [18, 22]. Clearly further studies are required.

Preconditioning: Cardioprotection in old age and obesity has become a very relevant clinical problem in view of the fact that the developed countries are faced with an increasing elderly population. With regard to cardioprotection in the aging heart, a number of comprehensive reviews recently appeared [1‐4] which indicated a loss in the ability to be preconditioned in the aging heart [27, 40, 53, 54]. For example, using infarct size as endpoint, Schulman and coworkers [31] showed that hearts from rats aged 18–20 months, could not be preconditioned by either multiple episodes of I/R or pharmacologically while the blunted response in middle-aged rat hearts could be improved by an increased preconditioning stimulus. Effective preconditioning in other aging species such as the rabbit [55] has been reported, although this was attributed to the possibility that the animals used in these two studies were probably not old enough to qualify as “old”, taking their lifespan into account [47]. Another explanation for these discrepancies is the fact that many of the previous studies used isolated hearts relying on functional recovery as endpoint [27, 28], which, in contrast to infarct size reduction, is regarded as less reliable.

Although type 2 diabetes has been associated with impaired PI3kinase/Akt signalling, GLUT4 expression as well as defects in AMPK and other kinases involved in preconditioning [56], our study showed that hearts from young DIO (insulin- resistant, but not diabetic) rats appear to be preconditioned already, while hearts from middle-aged DIO rats showed a significant reduction in infarct size when preconditioned (Figs. 2 and 3). Clearly the possibility that obesity may have beneficial effects on the myocardial response to I/R injury in young animals needs to be further investigated.

The very significant reduction in infarct size induced by ischaemic preconditioning in hearts from both middle-aged control and DIO rats (Fig. 3), was associated with a marked lowering in the ability to recover functionally during reperfusion as indicated by the overall reduction in % recovery in Total Work performed (see Table 5), when compared to their NPC counterparts. A lack of association between infarct size reduction and improved functional recovery has been observed by several groups [57‐59] and attributed to concomitant stunning. It is generally accepted that myocardial stunning is due to the formation of reactive oxygen species and cytosolic calcium overloading during reperfusion (for review see [60]). Using cardiomyocytes isolated from hearts of young and aged rats, O’Brien and coworkers [53] demonstrated that although the degree of contractile depression was comparable in both age groups, the aged cardiomyocytes exhibited a much greater and more prolonged accumulation of diastolic calcium in ischaemia. Increased cytosolic calcium as well as increased oxidative stress during early reperfusion, particularly in hearts from DIO rats, may cause severe stunning in the aged hearts. In fact, it has been shown that postischaemic stunning may be exacerbated in the senescent rat heart [61]. However, despite these possibilities, functional recovery by hearts from AMC and DIO rats albeit depressed, did not differ significantly and it is only when preconditioned, that hearts from middle-aged rats showed such a marked inability to recover function. A possible explanation could be that the additional generation of ROS during the preconditioning protocol could cause the severe mechanical dysfunction in the older animals. However, in view of the many complexities of ischaemic preconditioning this phenomenon should be further investigated. In a study of potential molecular mechanisms, Griecsova and coworkers [54] demonstrated that loss of preconditioning protection was associated with age-dependent reduced Akt/PKB phosphorylation and eNOS and PKCepsilon levels in hearts of mature rats compared with the younger ones, as well as a failure of preconditioning to upregulate these proteins, confirming data obtained in an earlier study by Iemitsu et al. [62]. The involvement of the RISK pathway, particularly in the middle-aged AMC and DIO rats, in the myocardial response to I/R injury, should be further investigated, with a specific focus on the time of sampling.

The data obtained in this study showed that, contrary to expectations, middle-age as well as obesity per se did not affect myocardial function at baseline level and did not increase the susceptibility of the heart to I/R damage, as indicated by infarct size and functional recovery during reperfusion after regional ischaemia. In fact, obesity appears to be beneficial in young animals as indicated by the reduction in infarct size when compared to their age-matched controls. These results are in contrast with the many well-established age-related modifications in the cardiovascular system [1‐3], but it should be kept in mind that our observations were made in healthy animals, without the comorbidities which may affect the outcome. There is also continued interest in the ability of cardioprotective interventions such as pre-or postconditioning to protect the ageing myocardium [1‐4]. Our data showed that although aging hearts exhibited normal function under baseline conditions, with an unchanged response to I/R and retention of the capacity to reduce infarct size when preconditioned, this was associated with a reduction in the ability to recover functionality. This is an important observation in view of the increasingly aging population in some countries where a loss of endogenous protection could have serious consequences for old patients. It is clear that further work is required to elucidate the effects of age and obesity on the outcome of cardioprotective interventions, with particular attention to the molecular mechanisms involved.