The major findings of our systematic review and meta-analysis are that H 2 S has a consistent and robust infarct-limiting effect against MIRI in pre-clinical studies. This robust effect was comparable when H 2 S boosters were given before the onset of ischemia (preconditioning) or at the time of reperfusion (postconditioning) based on in vivo data from almost 900 animals. This cardioprotection also was independent from the animal size or the class of H 2 S booster.

The mechanism of H 2 S-induced conditioning-like phenomena is not fully understood yet, despite several signalling molecules and pathways have been suggested to play a role. However, we here discussed potential conditioning mechanism(s) of H 2 S based on the in vivo evidence included in this study. We took into consideration the causal and temporal consequences of conditioning events and used a structuring scheme previously proposed by Heusch [24]. This scheme is based on the general consensus that conditioning maneuver triggers a “stimulus” which in turn activates a “mediator” to transfer the cardioprotective signal to its “target”. In fact, H 2 S itself has been demonstrated to be a crucial “chemical stimulus” of ischemic pre- [67] and postconditioning [28] to elicit their infarct-limiting effect. Augmented level of H 2 S activates similar signalling molecules and pathways to act as mediators to transmit its cardioprotective signal to its target(s). These signalling pathways mainly involve activating the RISK pathway components in the first minutes of reperfusion [3, 9, 10, 13, 33, 38, 45, 49, 65]. Notably, the activity of some micro-RNAs, namely micro-RNA-21 [58] and mirco-RNA-1 [30], were also reported to serve as mediators of H 2 S-induced cardioprotection. The key target of H 2 S’s protection is the mitochondria, where the majority of salvage signalling pathways converges. Enhanced H 2 S level protects against myocardial infarction via preserving mitochondrial function [17], maintaining membrane integrity [17, 65], limiting mitochondrial ROS generation [32] and inhibiting the opening of mitochondrial permeability transition pore (PTP) [10, 32, 65]. Moreover, mitochondrial K ATP channel is another target of H 2 S protection [54, 55, 57]. However, the question yet to be answered is how H 2 S triggers these signalling pathways to exert its infarct-limiting effect? It is highly unlikely that H 2 S activates the RISK pathway through a ligand/receptor-based mechanism as H 2 S is a gaseous molecule and not a ligand. The most plausible mechanism could be through inducing post-translation modifications (PTMs). Similar to nitrosylation, sulfhydration (or persulfidation) is a PTM induced by H 2 S which could modify the structure and eventually the function of several proteins and channels. Recently, it has been demonstrated that H 2 S activates PI3K/Akt signalling pathways through sulfhydrating phosphatase and tensin homolog (PTEN) abrogating its inhibitory effect [62]. Furthermore, sulfhydration is demonstrated to modify the activity of mitochondrial K ATP channel, another target of H 2 S [54, 55, 57], and ATP synthase (F 1 F 0 ATP synthase/complex V) [39], the current proposed main component of PTP, which either is known to protect the mitochondria and eventually limit infarct size. Taken together, the role of sulfhydration in conditioning with H 2 S needs further investigation.

There are a number of important aspects which we observed in our review. Despite highly consistent overall effect size, we noticed a high degree of heterogeneity between the included studies. We conducted subgroup analyses to investigate whether some of the experimental variables which we predefined could influence the observed effect size and/or heterogeneity using meta-regression. Others have previously shown, applying the same approach, that experimental model size could have a significant impact on effect size and heterogeneity observed with meta-analysis. For example, Lim et al. [37] reported that cyclosporine-induced infarct limitation in rodent models was absent in a large model (swine) of MIRI in vivo. Noteworthily, this could potentially explain the neutral clinical data of cyclosporine treatment in STEMI patients [25]. However, Bromage et al. [6] recently showed that the infarct limitation by remote ischemic conditioning manoeuvre was consistent across in vivo studies, independently of the model size. Similarly, we previously demonstrated that enhanced level of nitric oxide (NO) in vivo, using different NO treatments, exerted infarct limitation independently of the model size across (22) pre-clinical studies [4]. Our subgroup analyses showed that model size (rodent vs. non-rodent model) did not have a significant effect on either effect size or heterogeneity of H 2 S treatments in both pre-H 2 S and post-H 2 S groups.

We also assessed whether using different H 2 S boosters as a pharmacological approach to enhance H 2 S level could behave differently in terms of infarct limitation and heterogeneity. There have been number of approaches employed to enhance H 2 S level in vivo to investigate its effect on myocardial infarction. Inorganic sulfide salts, namely NaHS and Na 2 S, were the first class of H 2 S boosters initially utilised to investigate the significance of enhancement H 2 S on myocardial infarction. However, they are impure salts that cause a sharp and short-lasting increase in H 2 S level in vivo which make them unreliable H 2 S boosters. Furthermore, off-target or even toxic effects are highly likely with the burst of H 2 S achieved using sulfide salts due to the fact that H 2 S has a narrow therapeutic window. More stable and controllable organic H 2 S donors have been designed to overcome this limitation and have demonstrated infarct-limiting effect in vivo [10, 33, 57, 68]. Utilising triphenylphosphonium scaffold approach to target the mitochondria, we and others have recently reported infarct limitation in vivo using AP39, a mitochondrial-targeting H 2 S donor [10, 32], which have a significant implication considering the central role of mitochondria in MIRI. In a similar context, we have recently reported that the limit of infarct reduction by different NO donors at reperfusion was consistently comparable [4]. Although there was a pattern of increased efficacy of postconditioning with H 2 S enhancers, there was significant difference in the efficacy of any of the H 2 S booster groups in terms of infarct limitation at the two times of intervention. To note, the number of studies that employed large animal models was less than those that used small animals. Furthermore, we also noticed that the cardioprotective dose of some H 2 S boosters could vary between different animal models. For instance, cardioprotective dose of GYY4137, a slow-releasing H 2 S donor, was (26.6 µmol/kg) in the mouse model [10], while it was 10 times more in rat [33]. There is no obvious reason why the cardioprotection dose of these boosters might vary. However, it has been shown that there is a certain degree of dependency of H 2 S on NO signalling to induce its cardioprotection. Arguably, this dependency on NO seems to be high in mouse [3] and partially fading as the animal size increase, such as in rat [33] until it becomes insignificant in large animals, such as rabbit [3]. Whether this hypothesis explains the variation in the cardioprotective dose of some H 2 S boosters requires further investigation.

We were also interested in assessing the impact of other experimental variables which are also important on the external validity of our findings. As our main aim in this review was to characterise the effect of H 2 S on infarct size across the preclinical studies, we, accordingly, excluded all studies which utilised animals with co-morbidities, co-medications and risk factors such as diabetes, heart failure, hypertension or hypercholesterolemia. Therefore, insufficient number of studies in this review rendered these analyses not applicable. Nevertheless, in vivo preclinical studies utilised animals with co-morbidities which were identified in our literature search are summarised in (Table 3). This table is very helpful and has a considerable value for the field of cardioprotection with H 2 S as a starting point for future investigations characterising the impact of co-morbidities on H 2 S protection. Co-morbidities and risk factors associated with cardiovascular disease are important determinants of the efficacy of any cardioprotective therapy and this has recently been discussed in some position papers by others [8, 20, 21, 23]. There is a significant contrast in the biological milieu between the experimental animals and the patients. The majority of the cardioprotective interventions that have been tested in a “reductionist model” employing young and healthy animals, arguably to effectively control the experimental conditions [50]. However, the vast majority of patients recruited in the randomised clinical trials have co-morbidities and/or risk factors including diabetes, aging, hyperlipidemia and hypertension. These co-morbidities and risk factors are shown to modify the efficacy of several cardioprotective interventions [20, 22]. In addition, the potential impact of background medications on the examined efficacy of cardioprotective therapies is often neglected in the pre-clinical studies, despite the fact that most of the recruited patients are on standard medications. Similarly, current standard care could substantially alter the potency of cardioprotective therapies via either blocking the signalling pathway or elevating the threshold which is needed to produce the cardioprotection [22, 47]. Therefore, clinical translation could be considerably enhanced through conducting future preclinical studies on animals with co-morbidities and from a background of standard medications.

Table 3 Summary of pre-clinical studies investigated infarct-limiting effect of H 2 S using animals with co-morbidities Full size table

Another important experimental variable is gender, taking into consideration the cardioprotection of oestrogen which is mainly mediated by triggering the reperfusion injury salvage kinase (RISK) pathway [42], a common signalling pathway with H 2 S [33]. However, only 9% of included studies employed mixed gender. Another dimension to the reductionist model often employed in the pre-clinical studies is the use of a single therapy which is too simplistic and underestimates the clinical complexity. In the view of the current failure in clinical translation, the use of two or more drugs in what is often called “combination therapy” has been suggested as an alternative approach [22]. Especially, some combination treatments have shown promising benefits in vivo [64] and in human [16]. With the current advanced feasibility in designing H 2 S boosters which target different cellular compartments, it is tempting to suggest that combination therapy of different H 2 S boosters could potentially enhance the efficacy of H 2 S-induced cardioprotection. Especially, different H 2 S boosters signal through different protective mechanisms and could potentially have additive infarct-limiting effect to each other which maximise the beneficial effect [1, 32]. Despite this very tempting idea along with very encouraging experimental data, this concept has not been investigated yet and needs to be conducted in well-designed studies. We have listed H 2 S boosters which we think have potential clinical translatability along with proposed mechanism(s) of cardioprotection (Table 4). This table would have a great value for the field of cardioprotection and very helpful to test the concept of combination therapy in future investigations.

Table 4 List of H 2 S boosters with potential clinical translatability and proposed mechanism of infarct limitation Full size table

We also evaluated the internal validity of included studies including the quality of study reporting and publication bias and how these factors could have an impact on the observed results. The lack of full and comprehensive description of the methodological approach and study design could result in an overestimated effect size. By subjecting the included reports to our reporting quality assessment, included studies generally scored highly which is strengthening the validity of our study and it is due to our stringent inclusion criteria. Nevertheless, there was particularly poor reporting in a number of aspects including reporting any adverse effects (28%), a main determinant in any drug development. Reporting of sample size calculation was also poor (43%) which raises some important question regarding whether the study was sufficiently powered before commencing the experiments or allowed to continue until certain number of animals per group was achieved. Insufficient adherence to good quality research indicators could inevitably lead to false-positive results and overestimation of the effect size. As a consequence, this might subsequently lead to further testing of a particular treatment in clinical trial, as a logical consequence, which would be unethical and unnecessary. Furthermore, low standard study reporting makes it difficult to ascertain whether the study was conducted according to high-quality research standards which eventually assuring that the data are valid. Noteworthily, failing to report a good quality research could possibility account for the observed heterogeneity in this meta-analysis. Nevertheless, the effect size by H 2 S was consistent and robust despite the observed high heterogeneity which is reassuring.

We also investigated the publication bias within the included studies using funnel plot. The visual examination and the distribution of the effect size along with the precision of the measurement suggested that there might be an underrepresentation of studies with neutral or negative effect as well as studies with moderate precision in our analysis. However, it needs to be stressed here that studies with neutral or negative data are often not given priority, if at all, to be submitted for publication by the majority of the research groups especially that it is highly likely that they will be rejected at the peer review stage.