Mitochondrial Kinase Signaling for Cardioprotection.

Myocardial ischemia/reperfusion injury is reduced by cardioprotective adaptations such as local or remote ischemic conditioning. The cardioprotective stimuli activate signaling cascades, which converge on mitochondria and maintain the function of the organelles, which is critical for cell survival. The signaling cascades include not only extracellular molecules that activate sarcolemmal receptor-dependent or -independent protein kinases that signal at the plasma membrane or in the cytosol, but also involve kinases, which are located to or within mitochondria, phosphorylate mitochondrial target proteins, and thereby modify, e.g., respiration, the generation of reactive oxygen species, calcium handling, mitochondrial dynamics, mitophagy, or apoptosis. In the present review, we give a personal and opinionated overview of selected protein kinases, localized to/within myocardial mitochondria, and summarize the available data on their role in myocardial ischemia/reperfusion injury and protection from it. We highlight the regulation of mitochondrial function by these mitochondrial protein kinases.

Mitochondrial pannexin1 controls cardiac sensitivity to ischaemia/reperfusion injury.

AIMS: No effective therapy is available in clinics to protect the heart from ischaemia/reperfusion (I/R) injury. Endothelial cells are activated after I/R, which may drive the inflammatory response by releasing ATP through pannexin1 (Panx1) channels. Here, we investigated the role of Panx1 in cardiac I/R. METHODS AND RESULTS: Panx1 was found in cardiac endothelial cells, neutrophils, and cardiomyocytes. After in vivo I/R, serum Troponin-I, and infarct size were less pronounced in Panx1-/- mice, but leukocyte infiltration in the infarct area was similar between Panx1-/- and wild-type mice. Serum Troponin-I and infarct size were not different between mice with neutrophil-specific deletion of Panx1 and Panx1fl/fl mice, suggesting that cardioprotection by Panx1 deletion rather involved cardiomyocytes than the inflammatory response. Physiological cardiac function in wild-type and Panx1-/- hearts was similar. The time to onset of contracture and time to maximal contracture were delayed in Panx1-/- hearts, suggesting reduced sensitivity of these hearts to ischaemic injury. Moreover, Panx1-/- hearts showed better recovery of left ventricle developed pressure, cardiac contractility, and relaxation after I/R. Ischaemic preconditioning failed to confer further protection in Panx1-/- hearts. Panx1 was found in subsarcolemmal mitochondria (SSM). SSM in WT or Panx1-/- hearts showed no differences in morphology. The function of the mitochondrial permeability transition pore and production of reactive oxygen species in SSM was not affected, but mitochondrial respiration was reduced in Panx1-/- SSM. Finally, Panx1-/- cardiomyocytes had a decreased mitochondrial membrane potential and an increased mitochondrial ATP content. CONCLUSION: Panx1-/- mice display decreased sensitivity to cardiac I/R injury, resulting in smaller infarcts and improved recovery of left ventricular function. This cardioprotective effect of Panx1 deletion seems to involve cardiac mitochondria rather than a reduced inflammatory response. Thus, Panx1 may represent a new target for controlling cardiac reperfusion damage.

Connexin 43 in Mitochondria: What Do We Really Know About Its Function?

Connexins are known for their ability to mediate cell-cell communication via gap junctions and also form hemichannels that pass ions and molecules over the plasma membrane when open. Connexins have also been detected within mitochondria, with mitochondrial connexin 43 (Cx43) being the best studied to date. In this review, we discuss evidence for Cx43 presence in mitochondria of cell lines, primary cells and organs and summarize data on its localization, import and phosphorylation status. We further highlight the influence of Cx43 on mitochondrial function in terms of respiration, opening of the mitochondrial permeability transition pore and formation of reactive oxygen species, and also address the presence of a truncated form of Cx43 termed Gja1-20k. Finally, the role of mitochondrial Cx43 in pathological conditions, particularly in the heart, is discussed.

The Mitochondrial Disruptor Devimistat (CPI-613) Synergizes with Genotoxic Anticancer Drugs in Colorectal Cancer Therapy in a Bim-Dependent Manner.

Colorectal cancer is one of the most frequent tumor entities, with an increasing incidence and mortality in younger adults in Europe and the United States. Five-year survival rates for advanced colorectal cancer are still low, highlighting the need for novel targets in colorectal cancer therapy. Here, we investigated the therapeutic potential of the compound devimistat (CPI-613) that targets altered mitochondrial cancer cell metabolism and its synergism with the antineoplastic drugs 5-fluorouracil (5-FU) and irinotecan (IT) in colorectal cancer. Devimistat exerted a comparable cytotoxicity in a panel of established colorectal cancer cell lines and patient-derived short-term cultures independent of their genetic and epigenetic status, whereas human colonic epithelial cells were more resistant, indicating tumor selectivity. These findings were corroborated in intestinal organoid and tumoroid models. Mechanistically, devimistat disrupted mitochondrial membrane potential and severely impaired mitochondrial respiration, resulting in colorectal cancer cell death induction independent of p53. Combination treatment of devimistat with 5-FU or IT demonstrated synergistic cell killing in colorectal cancer cells as shown by Combenefit modeling and Chou-Talalay analysis. Increased cell death induction was revealed as a major mechanism involving downregulation of antiapoptotic genes and accumulation of proapoptotic Bim, which was confirmed by its genetic knockdown. In human colorectal cancer xenograft mouse models, devimistat showed antitumor activity and synergized with IT, resulting in prolonged survival and enhanced therapeutic efficacy. In human tumor xenografts, devimistat prevented IT-triggered p53 stabilization and caused synergistic Bim induction. Taken together, our study revealed devimistat as a promising candidate in colorectal cancer therapy by synergizing with established antineoplastic drugs in vitro and in vivo.

Regulation of STAT3 and its role in cardioprotection by conditioning: focus on non-genomic roles targeting mitochondrial function.

Ischemia-reperfusion injury (IRI) is one of the biggest challenges for cardiovascular researchers given the huge death toll caused by myocardial ischemic disease. Cardioprotective conditioning strategies, namely pre- and post-conditioning maneuvers, represent the most important strategies for stimulating pro-survival pathways essential to preserve cardiac health. Conditioning maneuvers have proved to be fundamental for the knowledge of the molecular basis of both IRI and cardioprotection. Among this evidence, the importance of signal transducer and activator of transcription 3 (STAT3) emerged. STAT3 is not only a transcription factor but also exhibits non-genomic pro-survival functions preserving mitochondrial function from IRI. Indeed, STAT3 is emerging as an influencer of mitochondrial function to explain the cardioprotection phenomena. Studying cardioprotection, STAT3 proved to be crucial as an element of the survivor activating factor enhancement (SAFE) pathway, which converges on mitochondria and influences their function by cross-talking with other cardioprotective pathways. Clearly there are still some functional properties of STAT3 to be discovered. Therefore, in this review, we highlight the evidence that places STAT3 as a promoter of the metabolic network. In particular, we focus on the possible interactions of STAT3 with processes aimed at maintaining mitochondrial functions, including the regulation of the electron transport chain, the production of reactive oxygen species, the homeostasis of Ca2+ and the inhibition of opening of mitochondrial permeability transition pore. Then we consider the role of STAT3 and the parallels between STA3/STAT5 in cardioprotection by conditioning, giving emphasis to the human heart and confounders.

Connexin 43 phosphorylation by casein kinase 1 is essential for the cardioprotection by ischemic preconditioning.

Myocardial connexin 43 (Cx43) forms gap junctions and hemichannels, and is also present within subsarcolemmal mitochondria. The protein is phosphorylated by several kinases including mitogen-activated protein kinase (MAPK), protein kinase C (PKC), and casein kinase 1 (CK1). A reduction in Cx43 content abrogates myocardial infarct size reduction by ischemic preconditioning (IPC). The present study characterizes the contribution of Cx43 phosphorylation towards mitochondrial function, hemichannel activity, and the cardioprotection by IPC in wild-type (WT) mice and in mice in which Cx43-phosphorylation sites targeted by above kinases are mutated to non-phosphorylatable residues (Cx43MAPKmut, Cx43PKCmut, and Cx43CK1mut mice). The amount of Cx43 in the left ventricle and in mitochondria was reduced in all mutant strains compared to WT mice and Cx43 phosphorylation was altered at residues not directly targeted by the mutations. Whereas complex 1 respiration was reduced in all strains, complex 2 respiration was decreased in Cx43CK1mut mice only. In Cx43 epitope-mutated mice, formation of reactive oxygen species and opening of the mitochondrial permeability transition pore were not affected. The hemichannel open probability was reduced in Cx43PKCmut and Cx43CK1mut but not in Cx43MAPKmut cardiomyocytes. Infarct size in isolated saline-perfused hearts after ischemia/reperfusion (45 min/120 min) was comparable between genotypes and was significantly reduced by IPC (3 × 3 min ischemia/5 min reperfusion) in WT, Cx43MAPKmut, and Cx43PKCmut, but not in Cx43CK1mut mice, an effect independent from the amount of Cx43 and the probability of hemichannel opening. Taken together, our study shows that alterations of Cx43 phosphorylation affect specific cellular functions and highlights the importance of Cx43 phosphorylation by CK1 for IPC's cardioprotection.

Cardioprotection with esmolol-based cardioplegia for non-infarcted and infarcted rat hearts.

OBJECTIVES: Esmolol-based cardioplegic arrest offers better cardioprotection than crystalloid cardioplegia but has been compared experimentally with blood cardioplegia only once. We investigated the influence of esmolol crystalloid cardioplegia (ECCP), esmolol blood cardioplegia (EBCP) and Calafiore blood cardioplegia (Cala) on cardiac function, metabolism and infarct size in non-infarcted and infarcted isolated rat hearts. METHODS: Two studies were performed: (i) the hearts were subjected to a 90-min cardioplegic arrest with ECCP, EBCP or Cala and (ii) a regional myocardial infarction was created 30 min before a 90-min cardioplegic arrest. Left ventricular peak developed pressure (LVpdP), velocity of contractility (dLVP/dtmax), velocity of relaxation over time (dLVP/dtmin), heart rate and coronary flow were recorded. In addition, the metabolic parameters were analysed. The infarct size was determined by planimetry, and the myocardial damage was determined by electron microscopy. RESULTS: In non-infarcted hearts, cardiac function was better preserved with ECCP than with EBCP or Cala relative to baseline values (LVpdP: 100 ± 28% vs 86 ± 11% vs 57 ± 7%; P = 0.002). Infarcted hearts showed similar haemodynamic recovery for ECCP, EBCP and Cala (LVpdP: 85 ± 46% vs 89 ± 55% vs 56 ± 26%; P = 0.30). The lactate production with EBCP was lower than with ECCP (0.6 ± 0.7 vs 1.4 ± 0.5 µmol/min; P = 0.017). The myocardial infarct size and (ECCP vs EBCP vs Cala: 16 ± 7% vs 15 ± 9% vs 24 ± 13%; P = 0.21) the ultrastructural preservation was similar in all groups. CONCLUSIONS: In non-infarcted rat hearts, esmolol-based cardioplegia, particularly ECCP, offers better myocardial protection than Calafiore. After an acute myocardial infarction, cardioprotection with esmolol-based cardioplegia is similar to that with Calafiore.

Importance of Cx43 for Right Ventricular Function.

In the heart, connexins form gap junctions, hemichannels, and are also present within mitochondria, with connexin 43 (Cx43) being the most prominent connexin in the ventricles. Whereas the role of Cx43 is well established for the healthy and diseased left ventricle, less is known about the importance of Cx43 for the development of right ventricular (RV) dysfunction. The present article focusses on the importance of Cx43 for the developing heart. Furthermore, we discuss the expression and localization of Cx43 in the diseased RV, i.e., in the tetralogy of Fallot and in pulmonary hypertension, in which the RV is affected, and RV hypertrophy and failure occur. We will also introduce other Cx molecules that are expressed in RV and surrounding tissues and have been reported to be involved in RV pathophysiology. Finally, we highlight therapeutic strategies aiming to improve RV function in pulmonary hypertension that are associated with alterations of Cx43 expression and function.

Lack of Contribution of p66shc to Pressure Overload-Induced Right Heart Hypertrophy.

The leading cause of death in pulmonary arterial hypertension (PAH) is right ventricular (RV) failure (RVF). Reactive oxygen species (ROS) have been suggested to play a role in the development of RV hypertrophy (RVH) and the transition to RVF. The hydrogen peroxide-generating protein p66shc has been associated with left ventricular (LV) hypertrophy but its role in RVH is unclear. The purpose of this study was to determine whether genetic deletion of p66shc affects the development and/or progression of RVH and RVF in the pulmonary artery banding (PAB) model of RV pressure overload. The impact of p66shc on mitochondrial ROS formation, RV cardiomyocyte function, as well as on RV morphology and function were studied three weeks after PAB or sham operation. PAB in wild type mice did not affect mitochondrial ROS production or RV cardiomyocyte function, but induced RVH and impaired cardiac function. Genetic deletion of p66shc did also not alter basal mitochondrial ROS production or RV cardiomyocyte function, but impaired RV cardiomyocyte shortening was observed following PAB. The development of RVH and RVF following PAB was not affected by p66shc deletion. Thus, our data suggest that p66shc-derived ROS are not involved in the development and progression of RVH or RVF in PAH.

Exacerbation of adverse cardiovascular effects of aircraft noise in an animal model of arterial hypertension.

Arterial hypertension is the most important risk factor for the development of cardiovascular disease. Recently, aircraft noise has been shown to be associated with elevated blood pressure, endothelial dysfunction, and oxidative stress. Here, we investigated the potential exacerbated cardiovascular effects of aircraft noise in combination with experimental arterial hypertension. C57BL/6J mice were infused with 0.5 mg/kg/d of angiotensin II for 7 days, exposed to aircraft noise for 7 days at a maximum sound pressure level of 85 dB(A) and a mean sound pressure level of 72 dB(A), or subjected to both stressors. Noise and angiotensin II increased blood pressure, endothelial dysfunction, oxidative stress and inflammation in aortic, cardiac and/or cerebral tissues in single exposure models. In mice subjected to both stressors, most of these risk factors showed potentiated adverse changes. We also found that mice exposed to both noise and ATII had increased phagocytic NADPH oxidase (NOX-2)-mediated superoxide formation, immune cell infiltration (monocytes, neutrophils and T cells) in the aortic wall, astrocyte activation in the brain, enhanced cytokine signaling, and subsequent vascular and cerebral oxidative stress. Exaggerated renal stress response was also observed. In summary, our results show an enhanced adverse cardiovascular effect between environmental noise exposure and arterial hypertension, which is mainly triggered by vascular inflammation and oxidative stress. Mechanistically, noise potentiates neuroinflammation and cerebral oxidative stress, which may be a potential link between both risk factors. The results indicate that a combination of classical (arterial hypertension) and novel (noise exposure) risk factors may be deleterious for cardiovascular health.

Cardioprotection in right heart failure.

Ischaemic and pharmacological conditioning of the left ventricle is mediated by the activation of signalling cascades, which finally converge at the mitochondria and reduce ischaemia/reperfusion (I/R) injury. Whereas the molecular mechanisms of conditioning in the left ventricle are well characterized, cardioprotection of the right ventricle is principally feasible but less established. Similar to what is known for the left ventricle, a dysregulation in signalling pathways seems to play a role in I/R injury of the healthy and failing right ventricle and in the ability/inability of the right ventricle to respond to a conditioning stimulus. The maintenance of mitochondrial function seems to be crucial in both ventricles to reduce I/R injury. As far as currently known, similar molecular mechanisms mediate ischaemic and pharmacological preconditioning in the left and right ventricles. However, the two ventricles seem to respond differently towards exercise-induced preconditioning. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.23/issuetoc.

Cardiac surgery in acute myocardial infarction: crystalloid versus blood cardioplegia - an experimental study.

BACKGROUND: Because hearts in acute myocardial infarction are often prone to ischemia-reperfusion damage during cardiac surgery, we investigated the influence of intracellular crystalloid cardioplegia solution (CCP) and extracellular blood cardioplegia solution (BCP) on cardiac function, metabolism, and infarct size in a rat heart model of myocardial infarction. METHODS: Following euthanasia, the hearts of 50 rats were quickly excised, cannulated, and inserted into a blood-perfused isolated heart apparatus. A regional myocardial infarction was created in the infarction group (18 hearts) for 120 min; the control group (32 hearts) was not subjected to infarction. In each group, either Buckberg BCP or Bretschneider CCP was administered for an aortic clamping time of 90 min. Functional parameters were recorded during reperfusion: coronary blood flow, left ventricular developed pressure (LVDP) and contractility (dp/dt max). Infarct size was determined by planimetry. The results were compared between the groups using analysis of variance or parametric tests, as appropriate. RESULTS: Cardiac function after acute myocardial infarction, 90 min of cardioplegic arrest, and 90 min of reperfusion was better preserved with Buckberg BCP than with Bretschneider CCP relative to baseline (BL) values (LVDP 54 ± 11% vs. 9 ± 2.9% [p = 0.0062]; dp/dt max. 73 ± 11% vs. 23 ± 2.7% [p = 0.0001]), whereas coronary flow was similarly impaired (BCP 55 ± 15%, CCP 63 ± 17% [p = 0.99]). The infarct in BCP-treated hearts was smaller (25% of myocardium) and limited to the area of coronary artery ligation, whereas in CCP hearts the infarct was larger (48% of myocardium; p = 0.029) and myocardial necrosis was distributed unevenly to the left ventricular wall. CONCLUSIONS: In a rat model of acute myocardial infarction followed by cardioplegic arrest, application of BCP leads to better myocardial recovery than CCP.

Ageing, sex, and cardioprotection.

Translation of cardioprotective interventions aimed at reducing myocardial injury during ischaemia-reperfusion from experimental studies to clinical practice is an important yet unmet need in cardiovascular medicine. One particular challenge facing translation is the existence of demographic and clinical factors that influence the pathophysiology of ischaemia-reperfusion injury of the heart and the effects of treatments aimed at preventing it. Among these factors, age and sex are prominent and have a recognised role in the susceptibility and outcome of ischaemic heart disease. Remarkably, some of the most powerful cardioprotective strategies proven to be effective in young animals become ineffective during ageing. This article reviews the mechanisms and implications of the modulatory effects of ageing and sex on myocardial ischaemia-reperfusion injury and their potential effects on cardioprotective interventions. LINKED ARTICLES: This article is part of a themed issue on Risk factors, comorbidities, and comedications in cardioprotection. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.23/issuetoc.

P66shc and its role in ischemic cardiovascular diseases.

Oxidative stress caused by an imbalance in the formation and removal of reactive oxygen species (ROS) plays an important role in the development of several cardiovascular diseases. ROS originate from various cellular origins; however, the highest amount of ROS is produced by mitochondria. One of the proteins contributing to mitochondrial ROS formation is the adaptor protein p66shc, which upon cellular stresses translocates from the cytosol to the mitochondria. In the present review, we focus on the role of p66shc in longevity, in the development of cardiovascular diseases including diabetes, atherosclerosis and its risk factors, myocardial ischemia/reperfusion injury and the protection from it by ischemic preconditioning. Also, the contribution of p66shc towards cerebral pathologies and the potential of the protein as a therapeutic target for the treatment of the aforementioned diseases are discussed.

Investigating and re-evaluating the role of glycogen synthase kinase 3 beta kinase as a molecular target for cardioprotection by using novel pharmacological inhibitors.

AIMS: Glycogen synthase kinase 3 beta (GSK3ß) link with the mitochondrial Permeability Transition Pore (mPTP) in cardioprotection is debated. We investigated the role of GSK3ß in ischaemia (I)/reperfusion (R) injury using pharmacological tools. METHODS AND RESULTS: Infarct size using the GSK3ß inhibitor BIO (6-bromoindirubin-3'-oxime) and several novel analogues (MLS2776-MLS2779) was determined in anaesthetized rabbits and mice. In myocardial tissue GSK3ß inhibition and the specificity of the compounds was tested. The mechanism of protection focused on autophagy-related proteins. GSK3ß localization was determined in subsarcolemmal (SSM) and interfibrillar mitochondria (IFM) isolated from Langendorff-perfused murine hearts (30'I/10'R or normoxic conditions). Calcium retention capacity (CRC) was determined in mitochondria after administration of the inhibitors in mice and in vitro. The effects of the inhibitors on mitochondrial respiration, reactive oxygen species (ROS) formation, ATP production, or hydrolysis were measured in SSM at baseline. Cyclosporine A (CsA) was co-administered with the inhibitors to address putative additive cardioprotective effects. Rabbits and mice treated with MLS compounds had smaller infarct size compared with control. In rabbits, MLS2776 and MLS2778 possessed greater infarct-sparing effects than BIO. GSK3ß inhibition was confirmed at the 10th min and 2 h of reperfusion, while up-regulation of autophagy-related proteins was evident at late reperfusion. The mitochondrial amount of GSK3ß was similar in normoxic SSM and IFM and was not altered by I/R. The inhibitors did not affect CRC or respiration, ROS and ATP production/hydrolysis at baseline. The co-administration of CsA ensured that cardioprotection was CypD-independent. CONCLUSION: Pharmacological inhibition of GSK3ß attenuates infarct size beyond mPTP inhibition.

Mitochondria "THE" target of myocardial conditioning.

Several interventions, such as ischemic preconditioning, remote pre/perconditioning, or postconditioning, are known to decrease lethal myocardial ischemia-reperfusion injury. While several signal transduction pathways become activated by such maneuvers, they all have a common end point, namely, the mitochondria. These organelles represent an essential target of the cardioprotective strategies, and the preservation of mitochondrial function is central for the reduction of ischemia-reperfusion injury. In the present review, we address the role of mitochondria in the different conditioning strategies; in particular, we focus on alterations of mitochondrial function in terms of energy production, formation of reactive oxygen species, opening of the mitochondrial permeability transition pore, and mitochondrial dynamics induced by ischemia-reperfusion.

Blood cardioplegia for cardiac surgery in acute myocardial infarction: rat experiments with two widely used solutions.

OBJECTIVES: Blood cardioplegia (BCP) can be used in different ways to protect the heart from ischaemia-reperfusion injury during cardiac surgery. Because there could be differences between warm and cold intermittent cardioplegia with or without warm reperfusion, we investigated the influence of 2 blood cardioplegia solutions on cardiac function, metabolism and infarct size in stable and infarcted rat hearts. METHODS: The hearts of 32 male Wistar rats were excised and inserted into a blood-perfused isolated heart apparatus. In 16 hearts, an acute myocardial infarction was induced by ligation of the left anterior descending coronary artery at least 30 min before aortic clamping. After aortic clamping, either Calafiore or Buckberg BCP was administered. During reperfusion, coronary blood flow, left ventricular developed pressure and dp/dt max were recorded, and oxygen consumption and lactate production were determined. The infarct size after 90 min of reperfusion was measured by triphenyl tetrazolium chloride staining. The hearts of rats without infarction were investigated using transmission electron microscopy. RESULTS: In hearts without infarction, haemodynamic recovery was similar for Calafiore and Buckberg solutions: left ventricular developed pressure [Cala 62% of baseline (BL), Buck 58% BL] and dp/dt max (Cala 83% BL, Buck 89% BL). Coronary flow, which was slightly less in infarcted hearts, also recovered similarly after the administration of the 2 BCP solutions (Cala 65% BL, Buck 68% BL). During reperfusion, lactate production was similar (Cala 0.85 ml/min, Buck 1.0 ml/min), and the cellular oedema index and mitochondrial swelling were comparable between the 2 groups. In hearts with infarction, left ventricular developed pressure (Cala 58% BL, Buck 56% BL) and dp/dt max (Cala 79% BL, Buck 72% BL) showed similar recovery for reperfusion with Calafiore or Buckberg BCP. In addition, coronary flow recovered similarly (Cala 54% BL, Buck 57% BL). During reperfusion, myocardial oxygen consumption was lower in the Cala (67% BL) than in the Buck (82% BL) group, but lactate production was similar between the Cala (1.1 ml/min) and the Buck (1.1 ml/min) groups. Myocardial infarct size was also similar in the Cala group (24%) and in the Buck group (26%). CONCLUSIONS: In stable perfused rat hearts and in an in vitro model of acute myocardial infarction, the 2 BCP solutions offer equally good myocardial protection.

Buckberg's blood cardioplegia for protection of adult and senile myocardium in a rat in vitro model of acute myocardial infarction.

BACKGROUND: In patients undergoing surgical myocardial revascularization for acute myocardial infarction, excellent myocardial protection can be achieved by blood cardioplegia. We investigated the influence of age on cardiac function, metabolism, and infarct size using Buckberg's blood cardioplegia (BCP). METHODS: The hearts of male Wistar rats ("adult", age 3 months, n = 8; "senile", age 24 months, n = 8) were excised and mounted on a blood-perfused isolated heart apparatus. An acute myocardial infarction was induced by coronary artery ligation for 30 min before aortic clamping and infusion of Buckberg's BCP. Throughout the experiment, functional parameters were recorded: coronary blood flow (normalized by heart weight), left ventricular peak developed pressure (LVpdP), and positive and negative derived left ventricular pressure over time (dLVPdtmax and dLVPdtmin). Oxygen consumption (MVO2) and lactate production of the hearts were calculated. The infarct size after 90 min of reperfusion (in % of the area at risk) was measured with triphenyl tetrazolium chloride staining of the myocardium. RESULTS: The baseline coronary flow normalized by heart weight was significantly lower in the senile hearts (1.6 ±â€¯0.4 ml/(min ∗ g)) compared with the adult hearts (2.0 ±â€¯0.3 ml/(min ∗ g); p = 0.04). After 90 min of aortic clamping, hemodynamic function of senile hearts recovered better than that of adult hearts: LVpdP (adult 57% of baseline [BL]; senile 88% BL; p = 0.044) and dLVPdtmax (adult 74% BL, senile 102% BL; p = 0.12). In contrast, myocardial infarct size was similar between the adult (26%) and senile (21%; p = 0.45) hearts, and coronary flow recovered to a similar extent (55% BL and 58% BL, respectively). During reperfusion, MVO2 (80% BL and 81% BL) and lactate production (1.2 and 1.3 µmol/min) were similar in the two groups. CONCLUSION: After acute myocardial infarction in a rat model, hearts recovered function after reperfusion with Buckberg's BCP solution. Hearts from aged animals recovered better than those from younger animals.

Nagarse treatment of cardiac subsarcolemmal and interfibrillar mitochondria leads to artefacts in mitochondrial protein quantification.

INTRODUCTION: In the heart, subsarcolemmal (SSM), interfibrillar (IFM) and perinuclear mitochondria represent three subtypes of mitochondria. The most commonly used protease during IFM isolation is the nagarse, however, its effect on the detection of mitochondrial proteins is still unclear. Therefore, we investigated whether nagarse treatment influences the quantification of mitochondrial proteins. METHODS: SSM and IFM were isolated from hearts of mice and rats. During IFM isolation, nagarse activity was either stopped by centrifugation (common protocol, IFM+N) or inhibited by phenylmethylsulfonyl fluoride (PMSF, IFM+N+I). The amounts of proteins located in different mitochondrial compartments (outer membrane: mitofusin 1 (MFN1) and 2 (MFN2); intermembrane space: p66shc; inner membrane (connexin 43 (Cx43)), and of protein deglycase DJ-1 were determined by Western blot. RESULTS: MFN2 and Cx43 were found predominantly in SSM isolated from mouse and rat hearts. MFN1 and p66shc were present in similar amounts in SSM and IFM+N, whereas the level of DJ-1 was higher in IFM+N compared to SSM. In IFM+N+I samples from mice, the amount of MFN2, but not that of Cx43 increased. Nagarse or nagarse inhibition by PMSF had no effect on oxygen consumption of SSM or IFM. DISCUSSION: Whereas the use of the common protocol indicates the localization of MFN2 predominantly in SSM, the inhibition of nagarse by PMSF increases the signal of MFN2 in IFM to that of in SSM, indicating an underestimation of MFN2 in IFM. Therefore, protease sensitivity should be considered when assessing distribution of mitochondrial proteins using nagarse-based isolation.