A TRICk to Improve the Effectiveness of RIC: Role of Limb Temperature in Enhancing the Effectiveness of Remote Ischemic Conditioning

SIMPLE SUMMARY: Remote ischemic conditioning is a simple cardioprotective practice consisting in brief intermittent ischemia applied to a limb. Remote ischemic conditioning has been repeatedly validated in animal models. However, translation from animal experiments to clinics for remote ischemic conditioning has been disappointing. We have demonstrated that keeping the animal’s limb warm while performing intermittent ischemia reduces infarct size more effectively than cold intermittent ischemia; thus, we propose that a more accurate temperature control of the limb undergoing remote ischemic conditioning can increase the efficacy of this cardioprotective maneuver. A simple thermal blanket around the ischemic limb while performing remote ischemic conditioning could be an easy approach to test in humans, as it is simple and safe. ABSTRACT: Background: Treatment of myocardial ischemia/reperfusion (IR) injury is still an unmet clinical need. A large variability of remote ischemic conditioning (RIC) protection has been reported; however, no studies have considered the temperature of the ischemic limb. We analyzed the effects of temperature on RIC protection. Methods: Left hind-limbs of anesthetized male mice were immersed in warm (40 °C, warm-RIC) or cold (20 °C, cold-RIC) water and subjected to a RIC protocol (4 × 5 min limb ischemia/reperfusion). In the control groups (warm-CTR or cold-CTR), the limbs underwent thermic conditions only. Isolated hearts underwent 30 min ischemia and 60 min reperfusion. A PI3K-inhibitor, LY294002 (5 µM), was infused in warm-RIC hearts before the IR protocol (warm-RIC LY). Infarct size was evaluated by nitro blue tetrazolium staining and expressed as the percent of risk area. Results: While cold-RIC did not reduce the infarct size compared to cold-CTR (51 ± 1.62% vs. 54 ± 1.07% of risk area, p = NS), warm-RIC (44 ± 1.13%) significantly reduced the infarct size with respect to either cold-RIC (p < 0.001) or warm-CTR (58 ± 1.41%, p < 0.0001). LY294002 infusion revealed the PI3K/Akt involvement in the warm-RIC protection. Infarct size reduction was abrogated by LY294002 pretreatment (warm-RIC: 44 ± 1.13% vs. warm-CTR 58 ± 1.41% p < 0.0001; vs. warm-RIC LY 54 ± 1.69% p = 0.0002). Conclusion: our study shows a remarkable difference between warm-RIC and cold-RIC in terms of infarct size reduction, supporting a pivotal role for limb temperature in RIC-induced cardioprotection

Cardioprotective Properties of Human Platelets Are Lost in Uncontrolled Diabetes Mellitus: A Study in Isolated Rat Hearts

Platelets affect myocardial damage from ischemia/reperfusion. Redox-dependent sphingosine-1-phosphate production and release are altered in diabetic platelets. Sphingosine-1-phosphate is a double-edged sword for ischemia/reperfusion injury. Therefore, we aimed to verify whether: (1) human healthy- or diabetic-platelets are cardioprotective, (2) sphingosine-1-phosphate receptors and downstream kinases play a role in platelet-induced cardioprotection, and (3) a correlation between platelet redox status and myocardial ischemia/reperfusion injury exists. Isolated rat hearts were subjected to 30-min ischemia and 1-h reperfusion. Infarct size was studied in hearts pretreated with healthy- or diabetic-platelets. Healthy-platelets were co-infused with sphingosine-1-phosphate receptor blocker, ERK-1/2 inhibitor, PI3K antagonist or PKC inhibitor to ascertain the cardioprotective mechanisms. In platelets we assessed (i) aggregation response to ADP, collagen, and arachidonic-acid, (ii) cyclooxygenase-1 levels, and (iii) AKT and ERK-phosphorylation. Platelet sphingosine-1-phosphate production and platelet levels of reactive oxygen species (ROS) were quantified and correlated to infarct size. Infarct size was reduced by about 22% in healthy-platelets pretreated hearts only. This cardioprotective effect was abrogated by either sphingosine-1-phosphate receptors or ERK/PI3K/PKC pathway blockade. Cyclooxygenase-1 levels and aggregation indices were higher in diabetic-platelets than healthy-platelets. Diabetic-platelets released less sphingosine-1-phosphate than healthy-platelets when mechanical or chemically stimulated in vitro. Yet, ROS levels were higher in diabetic-platelets and correlated with infarct size. Cardioprotective effects of healthy-platelet depend on the platelet’s capacity to activate cardiac sphingosine-1-phosphate receptors and ERK/PI3K/PKC pathways. However, diabetic-platelets release less S1P and lose cardioprotective effects. Platelet ROS levels correlate with infarct size. Whether these redox alterations are responsible for sphingosine-1-phosphate dysfunction in diabetic-platelets remains to be ascertained

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

Mitochondria in Cardiac Postconditioning

Mitochondria play a pivotal role in cardioprotection. Here we report some fundamental studies which considered the role of mitochondrial components (connexin 43, mitochondrial KATP channels and mitochondrial permeability transition pore) in postconditioning cardioprotection. We briefly discuss the role of mitochondria, reactive oxygen species and gaseous molecules in postconditioning. Also the effects of anesthetics—used as cardioprotective substances—is briefly considered in the context of postconditioning. The role of mitochondrial postconditioning signaling in determining the limitation of cell death is underpinned. Issues in clinical translation are briefly considered. The aim of the present mini-review is to discuss in a historical perspective the role of main mitochondria mechanisms in cardiac postconditioning

Redox Aspects of Chaperones in Cardiac Function

Molecular chaperones are stress proteins that allow the correct folding or unfolding as well as the assembly or disassembly of macromolecular cellular components. Changes in expression and post-translational modifications of chaperones have been linked to a number of age- and stress-related diseases including cancer, neurodegeneration, and cardiovascular diseases. Redox sensible post-translational modifications, such as S-nitrosylation, glutathionylation and phosphorylation of chaperone proteins have been reported. Redox-dependent regulation of chaperones is likely to be a phenomenon involved in metabolic processes and may represent an adaptive response to several stress conditions, especially within mitochondria, where it impacts cellular bioenergetics. These post-translational modifications might underlie the mechanisms leading to cardioprotection by conditioning maneuvers as well as to ischemia/reperfusion injury. In this review, we discuss this topic and focus on two important aspects of redox-regulated chaperones, namely redox regulation of mitochondrial chaperone function and cardiac protection against ischemia/reperfusion injury