Non-responsiveness to cardioprotection by ischaemic preconditioning in Ossabaw minipigs with genetic predisposition to, but without the phenotype of the metabolic syndrome

There is still a need for cardioprotection beyond that by early reperfusion. Interventional reperfusion is increasingly used in patients with acute myocardial infarction, but 1-year mortality in a large contemporary registry was still around 14% in 2017 [86], and the morbidity from post-infarct heart failure is even increasing [29]. Injury to both, cardiomyocytes, i.e., infarct size [82], and the coronary microcirculation, i.e., microvascular obstruction [11], contribute to patients’ prognosis. Whereas encouraging preclinical and clinical proof-of-concept studies demonstrated reduced infarct size by mechanical and pharmacological cardioprotection strategies [27], their translation to better clinical outcome in phase III trials in patients with acute myocardial infarction has been largely disappointing [1, 4, 25]. Such failure of translation has primarily been attributed to co-morbidities and co-medications of patients with acute myocardial infarction which may impair cardioprotective signalling [26]. In contrast, animals in most preclinical studies have no such co-morbidities and co-medications [4, 13, 39]. The metabolic syndrome is a typical co-morbidity which predisposes patients to acute myocardial infarction and comprises several features which impair cardioprotective signalling, including glucose intolerance and hyperinsulinaemia that progress to type 2 diabetes, obesity, hypertension, and dyslipidaemia. We originally aimed to better understand the mechanisms through which a metabolic syndrome interferes with cardioprotection and possibly overcome this obstacle. We, therefore, used our established clinically relevant model of coronary occlusion/reperfusion [32, 37, 40, 49, 78] in minipigs of a particular strain (Ossabaw) which develop a metabolic syndrome upon a hypercaloric, atherogenic diet and consequently coronary atherosclerosis and occasional myocardial infarction [83, 88, 94]. To induce cardioprotection we used ischaemic preconditioning (IPC) [56] which is unequivocally the strongest and most robust stimulus for cardioprotection, but failed to reduce infarct size when using our established protocol of IPC [14] in preliminary experiments.

We then hypothesised that there may also be a primordial [7], genetically determined non-responsiveness to cardioprotection and designed a prospective study to look at infarct size reduction by IPC. The Ossabaw minipigs used for these experiments had the major genotype associated with the thrifty phenotype and develop a robust metabolic syndrome [83]. These Ossabaw minipigs have a single nucleotide polymorphism encoding for isoleucine (I) rather than valine (V) in the 199^th position of the γ-subunit of adenosine monophosphate-activated protein kinase (AMPK) [50, 83]. AMPK is an important sensor of the metabolic and energetic state in myocardial ischaemia [51, 92] and causally involved in cardioprotection by IPC and by atorvastatin in mice [53, 57]. We, therefore, studied Ossabaw minipigs homozygous for both γ-subunit AMPK genotypes (V/V and I/I). For IPC, we now used the consensus protocol of the Consortium for preclinicAl assESsment of cARdioprotective therapies (CAESAR) [35].

Again, both genotypes had no infarct size reduction with IPC. We performed DNA sequencing in the Ossabaw minipigs and a bioinformatic comparison to the genomes of Sus scrofa and Göttingen minipigs [67] to identify genes that might explain the lack of cardioprotection in Ossabaw minipigs. Major differences in genes encoding janus kinase (JAK)—signal transducer and activator of transcription (STAT) signalling were identified and were consistent with a lack of increased STAT3 phosphorylation at early reperfusion in the Ossabaw minipigs which we have demonstrated before as causal for cardioprotection by ischaemic conditioning in Göttingen minipigs [14, 31, 78]. For comparison, we used contemporary and published data [40] from our Göttingen minipigs.

The authors declare that all supporting data of the present study are available in the article and its Online supplement. Experiments were performed between September 2017 and June 2021. Unless otherwise specified, materials were obtained from Sigma-Aldrich (Deisenhofen, Germany).

Baseline body weight, left ventricular pressure and laboratory data are presented in Tables 1 and 2. There were only minor differences between genotypes (Table 1) and only minor differences to data from the contemporary cohort of female and castrated Göttingen minipigs of the same age (14–15 months) [40] (Table 2). Obviously, the Ossabaw minipigs in the absence of a high-fat diet had no increased body weight or signs of a metabolic syndrome by comparison to Göttingen minipigs of equal age (Table 2). We lost 8 Ossabaw minipigs due to surgical problems (n = 5) or intractable ventricular fibrillation (n = 3).

For the present study, we have used a prospective design including a power analysis, as typically used for a randomised clinical trial. The main result of our study is: Ossabaw minipigs, in contrast to Göttingen minipigs, are not protected from infarction by IPC. This raises issues with respect to the mechanisms of cardioprotection and with respect to the clinical translation of cardioprotection.

There was no significant reduction in infarct size and area of no-reflow no matter what AMPK γ-subunit genotype the Ossabaw minipigs had. In the pre-specified secondary analysis, it did also not matter whether they were females or castrated males. IPC is the strongest cardioprotective stimulus known [20, 27], and we have used the IPC protocol which reduced infarct size robustly in the multi-centre studies of the CAESAR [35] and CIBER–CLAP consortia [69]. Apparently, the lack of cardioprotection by IPC was not related to the single nucleotide polymorphism resulting in a change from valine 199 to isoleucine in the γ-subunit of AMPK. The bioinformatic analysis identified more variants in the AMPK coding exons of Göttingen minipigs than of Ossabaw minipigs. In addition, there was apparently no difference between the AMPK γ-subunit genotypes in their protein phosphorylation/activity. Thus, it is not surprising that the AMPK γ-subunit genotype also had no impact on cardioprotection or the lack of it. AMPK has been demonstrated to be activated by IPC in mice [34, 57] and a dominant negative mutation of the α₂-subunit abrogated enzyme activity and infarct size reduction by IPC in mice [84]. However, the AMPK γ-subunit genotype made no difference for the lack of cardioprotection, as defined by infarct size reduction, by IPC whether the Ossabaw minipigs were females or castrated males. In our contemporary study in Göttingen minipigs [40], IPC was also not associated with an increase in AMPK phosphorylation (data not shown). Apparently, AMPK is not important for cardioprotection by IPC in pigs.

A protective role for female sex against myocardial I/R injury has been proposed [62, 70], but in our contemporary study in Göttingen minipigs infarct size did not display sex-related differences [40]. In addition, infarct size was larger in the female Yorkshire pigs of the CAESAR consortium with 60 min LAD occlusion and thus even larger than in our Göttingen minipigs but still reduced significantly by IPC [35]. Thus, the lack of cardioprotection by IPC in our female and castrated male Ossabaw minipigs cannot be related to their sex being not male. Interestingly, the area of no-reflow was less in castrated males than in females - there were no apparent differences in systemic haemodynamics and regional blood flow during ischaemia and reperfusion (Table 4) -, but we could not identify pertinent data in the literature and have no explanation for this finding.

Whereas isoflurane anaesthesia may have attenuated the effects of IPC, since isoflurane as compared to, e.g., barbiturates is cardioprotective per se [36], such effect is unlikely to account for the failure of IPC to reduce infarct size in the Ossabaw pigs in our current study. In fact, infarct size without IPC was even larger than that in our contemporary Göttingen minipigs (female: 56 ± 9 vs. 45 ± 8% area at risk; castrated male: 53 ± 12 vs. 45 ± 13% area at risk), arguing against a more pronounced genuine cardioprotection in the Ossabaw than the Göttingen minipigs, and on the other hand IPC still decreased infarct size markedly in the contemporary Göttingen minipigs [40] but not in the Ossabaw minipigs. Isoflurane, in contrast to propofol, also permits cardioprotection by remote ischemic conditioning in humans [43, 93].

The neutral results of our prospectively designed study resemble the neutral results of several larger randomised controlled trials in patients undergoing interventional reperfusion of myocardial infarction [21] or cardiovascular surgery [19, 54] which used an all-comer approach and did not confirm prior smaller proof-of-concept studies on remote ischaemic conditioning [5, 18, 87]. These larger trials were neutral with respect to clinical outcome as the primary endpoint but also with respect to infarct size. In the discussion of these trials several points of critique were pointed out. The neutral cardiosurgical trials were conducted under propofol anaesthesia which is known to interfere with cardioprotection [28, 93]. In the neutral myocardial infarction trial, infarct size was determined from somewhat incomplete troponin data which do not consider for the ischaemic area at risk, and more importantly the 1-year mortality was so low in the placebo group (< 3%) that it was not possibly further reduced by any intervention [17, 30]. In fact, cardioprotection by remote ischaemic conditioning is clinically only apparent in patients with acute myocardial infarction who most need it, i.e., those with cardiac arrest or cardiogenic shock prior to interventional reperfusion [9]. In our present study, infarct size in both female and castrated male Ossabaw minipigs was somewhat larger than in female and castrated male Göttingen minipigs with the same duration of coronary occlusion and reperfusion [40]. Our primary endpoint infarct size in the power analysis-based prospective study design was not significantly changed; therefore, secondary sub-group analyses are, therefore, in a strict sense not permitted and, if anything, hypothesis generating. Nevertheless, when overstepping the do-not-test rule of the two-way ANOVA, a significant reduction of infarct size by IPC in the I/I AMPK γ-subunit genotype Ossabaw minipigs became apparent, which was, however, much less pronounced than in the Göttingen minipigs (50 ± 11 vs. 25 ± 11% area at risk in females and 51 ± 13 vs. 30 ± 8% area at risk in castrated males) [40]. When looking closer at the data, the protection in the I/I Ossabaw minipigs was mostly confined to those of female sex, i.e., a very specific subgroup had protection which was, however, hidden in the all-comer approach. Somewhat different from the above clinical studies, the I/I female Ossabaw minipigs undergoing I/R were not in greater need for cardioprotection, since infarct size in those not undergoing IPC was not different from the other subgroups. The reduction in the I/I females Ossabaw minipigs may have entirely been by chance, and it would require another prospective study to confirm it as true. Of note, however, females of both genotypes had greater coronary microvascular injury than castrated males, and we have no explanation for this difference.

When searching for a mechanism for the observed lack of cardioprotection with IPC in Ossabaw minipigs, a number of differences between Ossabaw minipigs and Göttingen minipigs became apparent in the bioinformatic DNA sequencing analysis which notably identified variant protein coding sequences and subsequently differences of mitochondrial and inflammatory (including the JAK–STAT pathway) protein–protein associations. For both mitochondria and for STAT signalling, a causal role in cardioprotection is well-established [27]. Consistent with the bioinformatic analysis, the lack of infarct size reduction in the Ossabaw minipigs was associated with the lack of increased STAT3 phosphorylation at tyrosine 705 in the Western blot analysis of established cardioprotective proteins [24] which we performed in parallel. For STAT3 activation we have previously established causality by use of blocker experiments in Göttingen minipigs experiencing cardioprotection not only by IPC [14] but also by ischaemic postconditioning [31] and remote ischaemic preconditioning [78]. Such increased STAT3 phosphorylation at 10 min reperfusion with IPC was also seen in female, castrated male and male Göttingen minipigs in our contemporary study [40]. In principle, the unchanged STAT3 phosphorylation in the present study in Ossabaw minipigs could have resulted from a lack of increased kinase activity upstream of STAT3 or a simultaneous increase of phosphatase activity downstream of STAT3. PTEN is a phosphatase which decreases STAT3 phosphorylation [8, 91], but PTEN expression and phosphorylation were not increased in the present study. Of note, the STRING data bank reference is to human proteins, and STAT3 [90] and STAT5 [33, 90] activation is associated with cardioprotection by remote ischemic conditioning in cardiosurgical patients. We realise that STAT3 is a transcription factor that is causally involved in the protection of late IPC [3, 10], but its acute activation by phosphorylation during IPC within the immediate time frame of an ischaemia–reperfusion protocol cannot induce protection by initiating transcription and increased expression of proteins as in late IPC, but must occur through other mechanisms, such as improved mitochondrial respiration [31, 89]. In the present study, ERK phosphorylation at early reperfusion which in some studies including our contemporary study in Göttingen minipigs [40] was also associated with cardioprotection, was even decreased with IPC and thus consistent with the observed lack of cardioprotection. The phosphorylation of STAT5 in the present study was also decreased during ischaemia and returned back toward baseline during early reperfusion, but was not improved by IPC. Incidentally, the established genetic predisposition of the Ossabaw minipigs to develop a metabolic syndrome found its counterpart in the appearance of clusters of protein–protein associations of mitochondria and fat metabolism, where the genomes of Ossabaw minipigs differed from that of Göttingen minipigs and Sus scrofa, thus serving as a positive control. We realise that we did not identify specific genes which are responsible for lack of cardioprotection by IPC but just provided a transparently structured screening of genetic differences between Ossabaw minipigs and other pig strains which might be involved in the observed lack of cardioprotection. We also realise that we just identified variants in base pair sequences in the exons of protein-encoding genes between Ossabaw minipigs on one hand and Göttingen minipigs and Sus scrofa on the other hand, but we do not know whether the respective proteins are expressed at all and, if so, in which cell type and which subcellular compartment exactly. The lack of cardioprotection by IPC in the Ossabaw minipigs was apparent before the true risk factors for coronary atherosclerosis and its consequences had developed under the influence of a hypercaloric and hyperlipidic diet. At the time of the study, the Ossabaw minipigs still had the non-diseased phenotype in body weight, left ventricular pressure and blood glucose and lipid levels. We did not perform functional testing with a glucose tolerance test, but it has been reported to be not impaired in lean Ossabaw minipigs of this age [45]. Thus, this lack of cardioprotection by IPC cannot be related to established co-morbidities [39]. We realise that a different IPC algorithm than the one which we used might have induced cardioprotection, but we consider this possibility were unlikely. We have used the consensus IPC protocol advocated by the CAESRAR [35] and CIBER–CLAP [69] consortia, and all published studies using IPC in pigs of various strains have reported protection, no matter which IPC algorithm was used [12, 16, 35, 40, 42, 46, 52, 58, 64, 71, 73, 75‐77].

The lack of benefit from cardioprotection by IPC in the Ossabaw minipig strain is probably genetically determined as is the predisposition to develop a metabolic syndrome upon exposure to a hypercaloric and hyperlipidic diet, thus forming a double threat. Apparently, there are genuine responders and non-responders to a given cardioprotective intervention. When translating to humans, this double threat would make a strong case for primordial rather than primary prevention [15]. In patients with coronary artery disease, a genetically determined inability to develop a cardioprotective response to ischaemic conditioning as may occur during spontaneous episodes of myocardial ischaemia, i.e., pre-infarction angina [23, 68], would be considered a novel risk factor. The Ossabaw minipig is then a suitable model to further study such genetic lack of amenability to cardioprotection and to develop therapeutic strategies to circumvent this block in cardioprotective signal transduction. The signal transduction block could possibly be circumvented by agents and interventions that act distal to STAT3 in the cardioprotective signal transduction cascade, e.g., diazoxide [22, 24, 59] or malonate [65, 74] which target mitochondria as potential end-effectors of cardioprotection.