Sex Difference in Cardioprotection against Acute Myocardial Infarction in MAO-B Knockout Mice In Vivo.

The cardiomyocyte-specific knockout (KO) of monoamine oxidase (MAO)-B, an enzyme involved in the formation of reactive oxygen species (ROS), reduced myocardial ischemia/reperfusion (I/R) injury in vitro. Because sex hormones have a strong impact on MAO metabolic pathways, we analyzed the myocardial infarct size (IS) following I/R in female and male MAO-B KO mice in vivo. METHOD AND RESULTS: To induce the deletion of MAO-B, MAO-B KO mice (Myh6 Cre+/MAO-Bfl/fl) and wild-type (WT, Cre-negative MAO-Bfl/fl littermates) were fed with tamoxifen for 2 weeks followed by 10 weeks of normal mice chow. Myocardial infarction (assessed by TTC staining and expressed as a percentage of the area at risk as determined by Evans blue staining)) was induced by 45 min coronary occlusion followed by 120 min of reperfusion. RESULTS: The mortality following I/R was higher in male compared to female mice, with the lowest mortality found in MAO-B KO female mice. IS was significantly higher in male WT mice compared to female WT mice. MAO-B KO reduced IS in male mice but had no further impact on IS in female MAO-B KO mice. Interestingly, there was no difference in the plasma estradiol levels among the groups. CONCLUSION: The cardiomyocyte-specific knockout of MAO-B protects male mice against acute myocardial infarction but had no effect on the infarct size in female mice.

Cardioprotective efficacy of limb remote ischaemic preconditioning in rats: discrepancy between a meta-analysis and a three-centre in vivo study.

AIMS: Remote ischaemic preconditioning (RIPC) is a robust cardioprotective intervention in preclinical studies. To establish a working and efficacious RIPC protocol in our laboratories, we performed randomized, blinded in vivo studies in three study centres in rats, with various RIPC protocols. To verify that our experimental settings are in good alignment with in vivo rat studies showing cardioprotection by limb RIPC, we performed a systematic review and meta-analysis. In addition, we investigated the importance of different study parameters. METHODS AND RESULTS: Male Wistar rats were subjected to 20-45 min cardiac ischaemia followed by 120 min reperfusion with or without preceding RIPC by 3 or 4 × 5-5 min occlusion/reperfusion of one or two femoral vessels by clamping, tourniquet, or pressure cuff. RIPC did not reduce infarct size (IS), microvascular obstruction, or arrhythmias at any study centres. Systematic review and meta-analysis focusing on in vivo rat models of myocardial ischaemia/reperfusion injury with limb RIPC showed that RIPC reduces IS by 21.28% on average. In addition, the systematic review showed methodological heterogeneity and insufficient reporting of study parameters in a high proportion of studies. CONCLUSION: We report for the first time the lack of cardioprotection by RIPC in rats, assessed in individually randomized, blinded in vivo studies, involving three study centres, using different RIPC protocols. These results are in discrepancy with the meta-analysis of similar in vivo rat studies; however, no specific methodological reason could be identified by the systematic review, probably due to the overall insufficient reporting of several study parameters that did not improve over the past two decades. These results urge for publication of more well-designed and well-reported studies, irrespective of the outcome, which are required for preclinical reproducibility, and the development of clinically translatable cardioprotective interventions.

Altered Cellular Protein Quality Control System Modulates Cardiomyocyte Function in Volume Overload-Induced Hypertrophy.

Volume-induced hypertrophy is one of the risk factors for cardiac morbidity and mortality. In addition, mechanical and metabolic dysfunction, aging, and cellular redox balance are also contributing factors to the disease progression. In this study, we used volume overload (VO), which was induced by an aortocaval fistula in 2-month-old male Wistar rats, and sham-operated animals served as control. Functional parameters were measured by transthoracic echocardiography at termination 4- or 8-months after VO. The animals showed hypertrophic remodeling that was accompanied by mechanical dysfunction and increased cardiomyocyte stiffness. These alterations were reversible upon treatment with glutathione. Cardiomyocyte dysfunction was associated with elevated oxidative stress markers with unchanged inflammatory signaling pathways. In addition, we observed altered phosphorylation status of small heat shock proteins 27 and 70 and diminished protease expression caspases 3 compared to the matched control group, indicating an impaired protein quality control system. Such alterations might be attributed to the increased oxidative stress as anticipated from the enhanced titin oxidation, ubiquitination, and the elevation in oxidative stress markers. Our study showed an early pathological response to VO, which manifests in cardiomyocyte mechanical dysfunction and dysregulated signaling pathways associated with enhanced oxidative stress and an impaired protein quality control system.

Proprotein Convertase Subtilisin Kexin Type 9 (PCSK9) Deletion but Not Inhibition of Extracellular PCSK9 Reduces Infarct Sizes Ex Vivo but Not In Vivo.

Hypoxia upregulates PCSK9 expression in the heart, and PCSK9 affects the function of myocytes. This study aimed to investigate the impact of PCSK9 on reperfusion injury in rats and mice fed normal or high-fat diets. Either the genetic knockout of PCSK9 (mice) or the antagonism of circulating PCSK9 via Pep2-8 (mice and rats) was used. Isolated perfused hearts were exposed to 45 min of ischemia followed by 120 min of reperfusion. In vivo, mice were fed normal or high-fat diets (2% cholesterol) for eight weeks prior to coronary artery occlusion (45 min of ischemia) and reperfusion (120 min). Ischemia/reperfusion upregulates PCSK9 expression (rats and mice) and releases it into the perfusate. The inhibition of extracellular PCSK9 does not affect infarct sizes or functional recovery. However, genetic deletion largely reduces infarct size and improves post-ischemic recovery in mice ex vivo but not in vivo. A high-fat diet reduced the survival rate during ischemia and reperfusion, but in a PCSK9-independent manner that was associated with increased plasma matrix metalloproteinase (MMP)9 activity. PCSK9 deletion, but not the inhibition of extracellular PCSK9, reduces infarct sizes in ex vivo hearts, but this effect is overridden in vivo by factors such as MMP9.

Ca2+/calmodulin-dependent protein kinase II and protein kinase G oxidation contributes to impaired sarcomeric proteins in hypertrophy model.

AIMS: Volume overload (VO) induced hypertrophy is one of the hallmarks to the development of heart diseases. Understanding the compensatory mechanisms involved in this process might help preventing the disease progression. METHODS AND RESULTS: Therefore, the present study used 2 months old Wistar rats, which underwent an aortocaval fistula to develop VO-induced hypertrophy. The animals were subdivided into four different groups, two sham operated animals served as age-matched controls and two groups with aortocaval fistula. Echocardiography was performed prior termination after 4- and 8-month. Functional and molecular changes of several sarcomeric proteins and their signalling pathways involved in the regulation and modulation of cardiomyocyte function were investigated. RESULTS: The model was characterized with preserved ejection fraction in all groups and with elevated heart/body weight ratio, left/right ventricular and atrial weight at 4- and 8-month, which indicates VO-induced hypertrophy. In addition, 8-months groups showed increased left ventricular internal diameter during diastole, RV internal diameter, stroke volume and velocity-time index compared with their age-matched controls. These changes were accompanied by increased Ca2+ sensitivity and titin-based cardiomyocyte stiffness in 8-month VO rats compared with other groups. The altered cardiomyocyte mechanics was associated with phosphorylation deficit of sarcomeric proteins cardiac troponin I, myosin binding protein C and titin, also accompanied with impaired signalling pathways involved in phosphorylation of these sarcomeric proteins in 8-month VO rats compared with age-matched control group. Impaired protein phosphorylation status and dysregulated signalling pathways were associated with significant alterations in the oxidative status of both kinases CaMKII and PKG explaining by this the elevated Ca2+ sensitivity and titin-based cardiomyocyte stiffness and perhaps the development of hypertrophy. CONCLUSIONS: Our findings showed VO-induced cardiomyocyte dysfunction via deranged phosphorylation of myofilament proteins and signalling pathways due to increased oxidative state of CaMKII and PKG and this might contribute to the development of hypertrophy.

Matrix Metalloproteinases Repress Hypertrophic Growth in Cardiac Myocytes.

PURPOSE: Matrix metalloproteinases (MMPs) are identified as modulators of the extracellular matrix in heart failure progression. However, evidence for intracellular effects of MMPs is emerging. Pro- and anti-hypertrophic cardiac effects are described. This may be due to the various sources of different MMPs in the heart tissue. Therefore, the aim of the present study was to determine the role of MMPs in hypertrophic growth of isolated rat ventricular cardiac myocytes. METHODS: Cardiomyocytes were isolated form ventricular tissues of the rat hearts by collagenase perfusion. RT-qPCR, western blots, and zymography were used for expression and MMP activity analysis. Cross-sectional area and the rate of protein synthesis were determined as parameters for hypertrophic growth. RESULTS: MMP-1, MMP-2, MMP-3, MMP-9 and MMP-14 mRNAs were detected in cardiomyocytes, and protein expression of MMP-2, MMP-9, and MMP-14 was identified. Hypertrophic stimulation of cardiomyocytes did not enhance, but interestingly decreased expression of MMPs, indicating that downregulation of MMPs may promote hypertrophic growth. Indeed, the nonselective MMP inhibitors TAPI-0 or TIMP2 and the MMP-2-selective ARP-100 enhanced hypertrophic growth. Furthermore, TAPI-0 increased phosphorylation and thus activation of extracellular signaling kinase (ERK) and Akt (protein kinase B), as well as inhibition of glycogen synthase 3ß (GSK3ß). Abrogation of MEK/ERK- or phosphatidylinositol-3-kinase(PI3K)/Akt/GSK3ß-signaling with PD98059 or LY290042, respectively, inhibited hypertrophic growth under TAPI-0. CONCLUSION: MMPs' inhibition promotes hypertrophic growth in cardiomyocytes in vitro. Therefore, MMPs in the healthy heart may be important players to repress cardiac hypertrophy.

Cardioprotective Effect of Novel Matrix Metalloproteinase Inhibitors.

BACKGROUND: We recently developed novel matrix metalloproteinase-2 (MMP-2) inhibitor small molecules for cardioprotection against ischemia/reperfusion injury and validated their efficacy in ischemia/reperfusion injury in cardiac myocytes. The aim of the present study was to test our lead compounds for cardioprotection in vivo in a rat model of acute myocardial infarction (AMI) in the presence or absence of hypercholesterolemia, one of the major comorbidities affecting cardioprotection. METHODS: Normocholesterolemic adult male Wistar rats were subjected to 30 min of coronary occlusion followed by 120 min of reperfusion to induce AMI. MMP inhibitors (MMPI)-1154 and -1260 at 0.3, 1, and 3 µmol/kg, MMPI-1248 at 1, 3, and 10 µmol/kg were administered at the 25th min of ischemia intravenously. In separate groups, hypercholesterolemia was induced by a 12-week diet (2% cholesterol, 0.25% cholic acid), then the rats were subjected to the same AMI protocol and single doses of the MMPIs that showed the most efficacy in normocholesterolemic animals were tested in the hypercholesterolemic animals. Infarct size/area at risk was assessed at the end of reperfusion in all groups by standard Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC) staining, and myocardial microvascular obstruction (MVO) was determined by thioflavine-S staining. RESULTS: MMPI-1154 at 1 µmol/kg, MMPI-1260 at 3 µmol/kg and ischemic preconditioning (IPC) as the positive control reduced infarct size significantly; however, this effect was not seen in hypercholesterolemic animals. MVO in hypercholesterolemic animals decreased by IPC only. CONCLUSIONS: This is the first demonstration that MMPI-1154 and MMPI-1260 showed a dose-dependent infarct size reduction in an in vivo rat AMI model; however, single doses that showed the most efficacy in normocholesterolemic animals were abolished by hypercholesterolemia. The further development of these promising cardioprotective MMPIs should be continued with different dose ranges in the study of hypercholesterolemia and other comorbidities.

Capsaicin-Sensitive Sensory Nerves and the TRPV1 Ion Channel in Cardiac Physiology and Pathologies.

Cardiovascular diseases, including coronary artery disease, ischemic heart diseases such as acute myocardial infarction and postischemic heart failure, heart failure of other etiologies, and cardiac arrhythmias, belong to the leading causes of death. Activation of capsaicin-sensitive sensory nerves by the transient receptor potential vanilloid 1 (TRPV1) capsaicin receptor and other receptors, as well as neuropeptide mediators released from them upon stimulation, play important physiological regulatory roles. Capsaicin-sensitive sensory nerves also contribute to the development and progression of some cardiac diseases, as well as to mechanisms of endogenous stress adaptation leading to cardioprotection. In this review, we summarize the role of capsaicin-sensitive afferents and the TRPV1 ion channel in physiological and pathophysiological functions of the heart based mainly on experimental results and show their diagnostic or therapeutic potentials. Although the actions of several other channels or receptors expressed on cardiac sensory afferents and the effects of TRPV1 channel activation on different non-neural cell types in the heart are not precisely known, most data suggest that stimulation of the TRPV1-expressing sensory nerves or stimulation/overexpression of TRPV1 channels have beneficial effects in cardiac diseases.

Chronic treatment with rofecoxib but not ischemic preconditioning of the myocardium ameliorates early intestinal damage following cardiac ischemia/reperfusion injury in rats.

There is some recent evidence that cardiac ischemia/reperfusion (I/R) injury induces intestinal damage within days, which contributes to adverse cardiovascular outcomes after myocardial infarction. However, it is not clear whether remote gut injury has any detectable early signs, and whether different interventions aiming to reduce cardiac damage are also effective at protecting the intestine. Previously, we found that chronic treatment with rofecoxib, a selective inhibitor of cyclooxygenase-2 (COX-2), limited myocardial infarct size to a comparable extent as cardiac ischemic preconditioning (IPC) in rats subjected to 30-min coronary artery occlusion and 120-min reperfusion. In the present study, we aimed to analyse the early intestinal alterations caused by cardiac I/R injury, with or without the above-mentioned infart size-limiting interventions. We found that cardiac I/R injury induced histological changes in the small intestine within 2 h, which were accompanied by elevated tissue level of COX-2 and showed positive correlation with the activity of matrix metalloproteinase-2 (MMP-2), but not of MMP-9 in the plasma. All these changes were prevented by rofecoxib treatment. By contrast, cardiac IPC failed to reduce intestinal injury and plasma MMP-2 activity, although it prevented the transient reduction in jejunal blood flow in response to cardiac I/R. Our results demonstrate for the first time that rapid development of intestinal damage follows cardiac I/R, and that two similarly effective infarct size-limiting interventions, rofecoxib treatment and cardiac IPC, have different impacts on cardiac I/R-induced gut injury. Furthermore, intestinal damage correlates with plasma MMP-2 activity, which may be a biomarker for its early diagnosis.

Myocardial ischaemia reperfusion injury and cardioprotection in the presence of sensory neuropathy: Therapeutic options.

During the last decades, mortality from acute myocardial infarction has been dramatically reduced. However, the incidence of post-infarction heart failure is still increasing. Cardioprotection by ischaemic conditioning had been discovered more than three decades ago. Its clinical translation, however, is still an unmet need. This is mainly due to the disrupted cardioprotective signalling pathways in the presence of different cardiovascular risk factors, co-morbidities and the medication being taken. Sensory neuropathy is one of the co-morbidities that has been shown to interfere with cardioprotection. In the present review, we summarize the diverse aetiology of sensory neuropathies and the mechanisms by which these neuropathies may interfere with ischaemic heart disease and cardioprotective signalling. Finally, we suggest future therapeutic options targeting both ischaemic heart and sensory neuropathy simultaneously. 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.

Subantimicrobial Dose Doxycycline Worsens Chronic Arthritis-Induced Bone Microarchitectural Alterations in a Mouse Model: Role of Matrix Metalloproteinases?

Background: Rheumatoid arthritis (RA) is a chronic inflammatory joint disease hallmarked by irreversible damage of cartilage and bone. Matrix metalloproteinases (MMPs) involved in connective tissue remodeling play an important role in this process. Numerous MMPs have been examined in humans and animals, but their functions are still not fully understood. Therefore, we investigated the role of MMPs in the K/BxN serum-transfer model of RA with the broad-spectrum MMP inhibitor subantimicrobial dose doxycycline (SDD) using complex in vivo and in vitro methodolgy. Methods: Chronic arthritis was induced by repetitive i.p. injections of K/BxN serum in C57BL/6J mice. SDD was administered daily in acidified drinking water (0.5 mg/mL, 80 mg/kg) during the 30 days experimental period. Mechanonociceptive threshold of the paw was evaluated by aesthesiometry, grasping ability by grid test, arthritis severity by scoring, neutrophil myeloperoxidase activity by luminescence, vascular hyperpermeability and MMP activity by fluorescence in vivo imaging and the latter also by gelatin zymography, bone structure by micro-computed tomography (micro-CT). Plasma concentrations of doxycycline were determined by liquid chromatography-mass spectrometry analysis. Results: K/BxN serum induced significant inflammatory signs, mechanical hyperalgesia, joint function impairment, increased myeloperoxidase activity and vascular hyperpermeability. Significant increase of MMP activity was also observed both in vivo and ex vivo with elevation of the 57-60, 75, and 92 kDa gelatinolytic isoforms in the arthritic ankle joints, but neither MMP activity nor any above described functional parameters were influenced by SDD. Most importantly, SDD significantly reduced bone mineral density in the distal tibia and enhanced the Euler number in the ankle. Arthritis-induced microarchitectural alterations demonstrating increased irregularity and cancellous bone remodeling, such as increased Euler number was significantly elevated by SDD in both regions. Conclusion: We showed increase of various MMP activities in the joints by in vivo fluorescence imaging together with ex vivo zymography, and investigated their functional significance using the broad-spectrum MMP inhibitor SDD in the translational RA model. This is the first demonstration that SDD worsens arthritis-induced bone microarchitectural alterations, but it appears to be independent of MMP inhibition.

The impact of age on cardiac function and extracellular matrix component expression in adverse post-infarction remodeling in mice.

The aim of this study was to describe the potential associations of the expression of matricellular components in adverse post-infarction remodeling of the geriatric heart. In male geriatric (OM, age: 18 months) and young (YM, age: 11 weeks) OF1 mice myocardial infarction (MI) was induced by permanent ligation of the left anterior descending coronary artery. Cardiac function was evaluated by MRI. Plasma and myocardial tissue samples were collected 3d, 7d, and 32d post-MI. Age and MI were associated with impaired cardiac function accompanied by left-ventricular (LV) dilatation. mRNA expression of MMP-2 (7d: p < 0.05), TIMP-1 (7d: p < 0.05), TIMP-2 (7d: p < 0.05), Collagen-1 (3d and 7d: p < 0.05) and Collagen-3 (7d: p < 0.05) in LV non-infarcted myocardium was significantly higher in YM than in OM after MI. MMP-9 activity in plasma was increased in OM after MI (3d: p < 0.01). Tenascin-C protein levels assessed by ELISA were decreased in OM as compared to YM after MI in plasma (3d: p < 0.001, 7d: p < 0.05) and LV non-infarcted myocardium (7d: p < 0.01). Dysregulation in ECM components in non-infarcted LV might be associated and contribute to adverse LV remodeling and impaired cardiac function. Thus, targeting ECM might be a potential therapeutic approach to enhance cardiac function in geriatric patients following MI.