Receptor-interacting protein kinase 3 (RIP3) is a convergence point of multiple signalling pathways, including necroptosis, inflammation and oxidative stress; however, it is completely unknown whether it underlies acute myocardial ischemia/reperfusion (I/R) injury. Langendorff-perfused rat hearts subjected to 30 min ischemia followed by 10 min reperfusion exhibited compromised cardiac function which was not abrogated by pharmacological intervention of RIP3 inhibition. An immunoblotting analysis revealed that the detrimental effects of I/R were unlikely mediated by necroptotic cell death, since neither the canonical RIP3–MLKL pathway (mixed lineage kinase-like pseudokinase) nor the proposed non-canonical molecular axes involving CaMKIIδ–mPTP (calcium/calmodulin-dependent protein kinase IIδ–mitochondrial permeability transition pore), PGAM5–Drp1 (phosphoglycerate mutase 5–dynamin-related protein 1) and JNK–BNIP3 (c-Jun N-terminal kinase–BCL2-interacting protein 3) were activated. Similarly, we found no evidence of the involvement of NLRP3 inflammasome signalling (NOD-, LRR- and pyrin domain-containing protein 3) in such injury. RIP3 inhibition prevented the plasma membrane rupture and delayed mPTP opening which was associated with the modulation of xanthin oxidase (XO) and manganese superoxide dismutase (MnSOD). Taken together, this is the first study indicating that RIP3 regulates early reperfusion injury via oxidative stress- and mitochondrial activity-related effects, rather than cell loss due to necroptosis.

In this study, a role of cell loss due to necroptosis and its linkage with pyroptosis in organ damage under the conditions of pulmonary arterial hypertension (PAH) was examined. Monocrotaline (MCT) was used to induce PAH in Wistar rats, and depending on the severity of the disease progression, they were further divided into two subgroups: MCT group—sacrificed 4 weeks after MCT administration and ptMCT group—prematurely sacrificed due to rapid deterioration in vital functions (on Day 24,11 ± 0,7). The elevation of respiratory rate and right ventricular (RV) hypertrophy were more evident in ptMCT group, while the heart rate and cardiac haemodynamic stress markers were comparably higher in both diseased groups. Detailed immunoblotting analysis revealed that the upregulation of pThr231/Ser232‐RIP3 proceeded into necroptosis execution in the RVs, unlike in the lungs of both PAH stages. The elevated pulmonary pThr231/Ser232‐RIP3 levels in both PAH subgroups were associated rather with GSDMD‐mediated pyroptosis. On the contrary, other inflammasome forms, such as AIM2 and NLRC4, were higher in the RV, unlike in the lungs, of diseased groups. The PAH‐induced increase in the plasma RIP3 levels was more pronounced in ptMCT group, and positively correlated with RV hypertrophy, but not with haemodynamic stress. Taken together, we indicated for the first time that pThr231/Ser232‐RIP3 upregulation resulting in two different necrosis‐like cell death modes might underlie the pathomechanisms of PAH and that the plasma RIP3 might serve as an additional diagnostic and prognostic marker of cardiac injury under these conditions.

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Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Slovak Research and Development Agency Medical Research Council Grant Necroptosis, a necrosis-like programmed cell death modality dependent on the activity of receptor-interacting protein kinase 3 (RIP3) and mixed lineage domain kinase domain-like pseudokinase (MLKL), plays a prominent role in mediating myocardial ischemia/reperfusion injury. However, the extent to which necroptosis contributes to such damage under short and long reperfusion has not been evaluated in detail. In Wistar rat hearts, subjected to global 30-min ischemia followed by an acute 10-min reperfusion period, with compromised cardiac function, no changes in the protein expression of the main necroptotic markers (pThr231/Ser232-RIP3, MLKL) were found. Likewise, the non-canonical pathways of necroptosis involving Ca2+/calmodulin dependent protein kinase II–mitochondrial permeability transition pore (CaMKII–mPTP) or phosphoglycerate mutase 5–dynamin-related protein 1 (PGAM5–Drp1) axes were unlikely affected by such short reperfusion. In contrast, hearts subjected to global 30-min ischemia followed by a prolonged 40-min reperfusion period exhibited worsened hemodynamic parameters what was accompanied by the increased levels of RIP3, pSer229-RIP3 and MLKL. Moreover, this reperfusion period induced MLKL translocation to the plasma membrane, indicating necroptosis execution with resultant very likely cell disruption. Similarly, activated necroptosis, evidenced by the higher levels of proteins of the canonical pathway, has been suggested to contribute to the pathogenesis of post-infarction heart failure (30-min ischemia, 42-day reperfusion). Collectively, these findings suggest that short reperfusion seems to be insufficient to induce necroptosis in the heart and the molecular mechanisms being activated during the longer reperfusion phase are needed to promote necroptotic cell dying. Therefore, inhibition of necroptosis might represent a cardioprotective strategy in the settings of chronic, but not acute myocardial ischemia/reperfusion injury.

Funding Acknowledgements Type of funding sources: Public Institution(s). Main funding source(s): Scientific Grant Agency of the Ministry of Education, Science, Research and Sport of the Slovak Republic The Slovak Research and Development Agency Introduction Necroptosis has been found to underlie myocardial damage in various pathological states, however its role as well as its potential interconnection to another cell damaging processes under conditions of pulmonary arterial hypertension (PAH) remains insufficiently understood. Purpose In view of these facts, we aimed to investigate the signalling of both necroptosis and pyroptosis in the lung and right ventricular (RV) tissue affected by PAH and we also examined the circulating levels of receptor-interacting protein kinase 3 (RIP3). Methods Male Wistar rats were administrated either vehicle (Control group) or monocrotaline (MCT) (60 mg/kg, s. c.) to induce PAH. The MCT-treated animals were sacrificed 4 weeks after the treatment (MCT group) or prematurely, based on rapid health deterioration, thereby indicating the advanced stage of PAH (ptMCT group). Pulse oximetry, gravimetry and evaluation of myocardial damage markers were used to assess the development of PAH. The molecular analyses (ELISA, RT-qPCR and immunoblotting) were used to examine the potential underlying mechanisms of tissue damage due to cell death. Results In the RVs, the expression of both pThr231/Ser232-RIP3 and pSer345-mixed linkage kinase domain-like protein was elevated equally in both PAH stages, strongly indicating promotion of necroptosis. Contrary in the lungs affected by PAH, the upregulation of pThr231/Ser232-RIP3 likely proceeded to pyroptotic rather than necroptotic cell death activation, as evidenced by caspase-1 and N-terminal gasdermin D increase. Moreover, we found that the plasma RIP3 levels increased with the severity of PAH stage and positively correlated with RV hypertrophy, but not with cardiac hemodynamic stress markers. Conclusions In summary, we showed for the first time that PAH-induced damage of RV and lung tissue might be mediated by different necrosis-like cell death forms with a crucial role pThr231/Ser232-RIP3. Furthermore, we suggested that the plasma RIP3 might serve as an additional diagnostic and prognostic marker of cardiac damage due to PAH.