Identification of New, Translatable ProtectomiRs against Myocardial Ischemia/Reperfusion Injury and Oxidative Stress: The Role of MMP/Biglycan Signaling Pathways.

INTRODUCTION: Ischemic conditionings (ICon) were intensively investigated and several protective signaling pathways were identified. Previously, we have shown the role of matrix metalloproteinases (MMP) in myocardial ischemia/reperfusion injury (MIRI) and the cardioprotective role of biglycan (BGN), a small leucine-rich proteoglycan in vitro. Here, we hypothesized that cardiac MMP and BGN signaling are involved in the protective effects of ICon. METHODS: A reverse target-microRNA prediction was performed by using the miRNAtarget™ 2.0 software to identify human microRNAs with a possible regulatory effect on MMP and BGN, such as on related genes. To validate the identified 1289 miRNAs in the predicted network, we compared them to two cardioprotective miRNA omics datasets derived from pig and rat models of MIRI in the presence of ICons. RESULTS: Among the experimentally measured miRNAs, we found 100% sequence identity to human predicted regulatory miRNAs in the case of 37 porcine and 24 rat miRNAs. Upon further analysis, 42 miRNAs were identified as MIRI-associated miRNAs, from which 24 miRNAs were counter-regulated due to ICons. CONCLUSIONS: Our findings highlight 24 miRNAs that potentially regulate cardioprotective therapeutic targets associated with MMPs and BGN in a highly translatable porcine model of acute myocardial infarction.

An Observational Study on the Pharmacokinetics of Oseltamivir in Lactating Influenza Patients.

Influenza infection may lead to serious complications in the postpartum period, therefore, oseltamivir treatment in these patients and their breastfed infants is of great importance. However, the pharmacokinetics of oseltamivir in postpartum lactating women with acute influenza infection, and the consequent infant exposure to oseltamivir are still unknown, and these data would help in assessing risk and the need for dose adjustment in breastfed infants. Six lactating women with influenza-like symptoms, at a standard dose of 75 mg oral oseltamivir twice daily for 5 days, were recruited in this phase IV clinical study during the 2011/2012 H1N1 pandemic seasons. Breast milk/colostrum and venous blood samples were taken at multiple timepoints, maternal urine samples were obtained from total output within the 12-hour observational period following the seventh dose of oseltamivir. Oseltamivir phosphate (OP) reached a maximum 69.5 ± 29.4 ng/mL concentration in breast milk, higher than that found in the plasma, and showed elimination within ~ 8 hours. Oseltamivir carboxylate (active metabolite of OP) showed a lower, nearly steady-state concentration in breast milk during the observational period (maximum plasma concentration (Cmax ) = 38.4 ± 12.9 ng/mL). Based on estimated daily milk consumption of exclusively breastfed infants, their calculated daily exposure is < 0.1% of the infant dose of oseltamivir for treatment of influenza as per marketing authorization. Here, we provide the first maternal breast milk pharmacokinetic data for oral multiple-dose oseltamivir in lactating patients with influenza and showed that its concentration in the breast milk is not sufficient to reach a therapeutic dose for breastfed infants.

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.

Distally Tilted Implants According to the All-on-Four® Treatment Concept for the Rehabilitation of Complete Edentulism: A 3.5-Year Retrospective Radiographic Study of Clinical Outcomes and Marginal Bone Level Changes.

Bone grafting procedures during the use of dental implants may be avoided by the use of tilted implants in the maxilla and the mandible; advantages of angled implants are associated with the extension of the distal cantilever, leading to better implant survival rates. However, the bending effect on the single tilting implants may increase the marginal bone stress. The purpose of the present study was to retrospectively assess the clinical success and proximal bone loss rate following the implantation of distally tilted implants according to the All-on-Four™ prosthetic concept­based on radiographic findings­in a single-center experience, in addition to the assessment of the outcomes in the context of various clinico-epidemiological correlates. During the study period, n = 36 patients (24 males and 12 females) with complete records of periapical radiographs, received a full-arch fixed bridge supported by two axial and two distal tilted implants; overall n = 144 and n = 144 implants (Nobel B) were place in the maxilla and mandibles of patients, respectively. Mean age of patients at the time of fixture installation was 58.75 ± 13.71 years; n =11 patients presented with relevant underlying conditions/habits. To assess peri-implant bone-level changes, matched and calibrated orthopantomogram (OPT) images were taken at follow-ups after 1.5 years, 2.5 years, and 3.5 years post-restoration, and marginal bone levels were assessed on the mesio- (MA) and disto-approximal (DA) aspects. All implants were successful, resulting in a 100% overall survival rate. The radiographic mean bone loss levels at baseline (mean ± SEM) were 0.181 ± 0.011 mm and 0.178 ± 0.017 mm in the maxilla and mandible, respectively, while by the 3.5-year mark, bone loss was 0.770 ± 0.029 mm and 0.713 ± 0.026 mm in the maxilla and mandible (p > 0.05), respectively; bone-level changes were significant over time (p = 0.035 and p = 0.033). Peri-implant bone loss was more aggressive around titled distal implants versus mesial actual position implants. The effect of smoking and other underlying conditions showed significantly higher (p < 0.05) bone resorption levels when assessed on an individual implant-level, while during patient-level analysis, only a tendency was shown for higher bone loss rates for both MA and DA implants (p > 0.05). Within its limitations, our study has concluded that the use of All-on-Four™ prosthetic concept for total arch rehabilitation yields higher bone loss in association with tilted implants and, in some cases, on the MA surfaces at vertically positioned implants after >40 months in function.

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.

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.

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.

Swiprosin-1/EFhD-2 Expression in Cardiac Remodeling and Post-Infarct Repair: Effect of Ischemic Conditioning.

Swiprosin-1 (EFhD2) is a molecule that triggers structural adaptation of isolated adult rat cardiomyocytes to cell culture conditions by initiating a process known as cell spreading. This process mimics central aspects of cardiac remodeling, as it occurs subsequent to myocardial infarction. However, expression of swiprosin-1 in cardiac tissue and its regulation in vivo has not yet been addressed. The expression of swiprosin-1 was analyzed in mice, rat, and pig hearts undergoing myocardial infarction or ischemia/reperfusion with or without cardiac protection by ischemic pre- and postconditioning. In mouse hearts, swiprosin-1 protein expression was increased after 4 and 7 days in myocardial infarct areas specifically in cardiomyocytes as verified by immunoblotting and histology. In rat hearts, swiprosin-1 mRNA expression was induced within 7 days after ischemia/reperfusion but this induction was abrogated by conditioning. As in cultured cardiomyocytes, the expression of swiprosin-1 was associated with a coinduction of arrestin-2, suggesting a common mechanism of regulation. Rno-miR-32-3p and rno-miR-34c-3p were associated with the regulation pattern of both molecules. Moreover, induction of swiprosin-1 and ssc-miR-34c was also confirmed in the infarct zone of pigs. In summary, our data show that up-regulation of swiprosin-1 appears in the postischemic heart during cardiac remodeling and repair in different species.

Ischaemic post-conditioning in rats: Responder and non-responder differ in transcriptome of mitochondrial proteins.

Ischaemic post-conditioning (IPoC) is a clinical applicable procedure to reduce reperfusion injury. Non-responsiveness to IPoC possibly caused by co-morbidities limits its clinical attractiveness. We analysed differences in the expression of mitochondrial proteins between IPoC responder (IPoC-R) and non-responder (IPoC-NR). Eighty rats were randomly grouped to sham, ischaemia/reperfusion (I/R), IPoC or ischaemic pre-conditioning (IPC, as positive cardioprotective intervention) in vivo. Infarct sizes were quantified by plasma troponin I levels 60 minutes after reperfusion. After 7 days, rats were sacrificed and left ventricular tissue was taken for post hoc analysis. The transcriptome was analysed by qRT-PCR and small RNA sequencing. Key findings were verified by immunoblots. I/R increased plasma troponin I levels compared to Sham. IPC reduced troponin I compared to I/R, whereas IPoC produced either excellent protection (IPoC-R) or no protection (IPoC-NR). Twenty-one miRs were up-regulated by I/R and modified by IPoC. qRT-PCR analysis revealed that IPoC-R differed from other groups by reduced expression of arginase-2 and bax, whereas the mitochondrial uncoupling protein (UCP)-2 was induced in IPC and IPoC-R. IPoC-R and IPoC-NR synergistically increased the expression of non-mitochondrial proteins like VEGF and SERCA2a independent of the infarct size. Cardiac function was more closely linked to differences in mitochondrial proteins than on regulation of calcium-handling proteins. In conclusion, healthy rats could not always be protected by IPoC. IPoC-NR displayed an incomplete responsiveness which is reflected by different changes in the mitochondrial transcriptome compared to IPoC-R. This study underlines the importance of mitochondrial proteins for successful long-term outcome.

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.

Sensory Neuropathy Affects Cardiac miRNA Expression Network Targeting IGF-1, SLC2a-12, EIF-4e, and ULK-2 mRNAs.

BACKGROUND: Here we examined myocardial microRNA (miRNA) expression profile in a sensory neuropathy model with cardiac diastolic dysfunction and aimed to identify key mRNA molecular targets of the differentially expressed miRNAs that may contribute to cardiac dysfunction. METHODS: Male Wistar rats were treated with vehicle or capsaicin for 3 days to induce systemic sensory neuropathy. Seven days later, diastolic dysfunction was detected by echocardiography, and miRNAs were isolated from the whole ventricles. RESULTS: Out of 711 known miRNAs measured by miRNA microarray, the expression of 257 miRNAs was detected in the heart. As compared to vehicle-treated hearts, miR-344b, miR-466b, miR-98, let-7a, miR-1, miR-206, and miR-34b were downregulated, while miR-181a was upregulated as validated also by quantitative real time polymerase chain reaction (qRT-PCR). By an in silico network analysis, we identified common mRNA targets (insulin-like growth factor 1 (IGF-1), solute carrier family 2 facilitated glucose transporter member 12 (SLC2a-12), eukaryotic translation initiation factor 4e (EIF-4e), and Unc-51 like autophagy activating kinase 2 (ULK-2)) targeted by at least three altered miRNAs. Predicted upregulation of these mRNA targets were validated by qRT-PCR. CONCLUSION: This is the first demonstration that sensory neuropathy affects cardiac miRNA expression network targeting IGF-1, SLC2a-12, EIF-4e, and ULK-2, which may contribute to cardiac diastolic dysfunction. These results further support the need for unbiased omics approach followed by in silico prediction and validation of molecular targets to reveal novel pathomechanisms.

Chronic kidney disease induces left ventricular overexpression of the pro-hypertrophic microRNA-212.

Chronic kidney disease (CKD) is a public health problem that increases the risk of cardiovascular morbidity and mortality. Heart failure with preserved ejection fraction (HFpEF) characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction is a common cardiovascular complication of CKD. MicroRNA-212 (miR-212) has been demonstrated previously to be a crucial regulator of pathologic LVH in pressure-overload-induced heart failure via regulating the forkhead box O3 (FOXO3)/calcineurin/nuclear factor of activated T-cells (NFAT) pathway. Here we aimed to investigate whether miR-212 and its hypertrophy-associated targets including FOXO3, extracellular signal-regulated kinase 2 (ERK2), and AMP-activated protein kinase (AMPK) play a role in the development of HFpEF in CKD. CKD was induced by 5/6 nephrectomy in male Wistar rats. Echocardiography and histology revealed LVH, fibrosis, preserved systolic function, and diastolic dysfunction in the CKD group as compared to sham-operated animals eight and/or nine weeks later. Left ventricular miR-212 was significantly overexpressed in CKD. However, expressions of FOXO3, AMPK, and ERK2 failed to change significantly at the mRNA or protein level. The protein kinase B (AKT)/FOXO3 and AKT/mammalian target of rapamycin (mTOR) pathways are also proposed regulators of LVH induced by pressure-overload. Interestingly, phospho-AKT/total-AKT ratio was increased in CKD without significantly affecting phosphorylation of FOXO3 or mTOR. In summary, cardiac overexpression of miR-212 in CKD failed to affect its previously implicated hypertrophy-associated downstream targets. Thus, the molecular mechanism of the development of LVH in CKD seems to be independent of the FOXO3, ERK1/2, AMPK, and AKT/mTOR-mediated pathways indicating unique features in this form of LVH.

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.

Definition of hidden drug cardiotoxicity: paradigm change in cardiac safety testing and its clinical implications.

Unexpected cardiac adverse effects are the leading causes of discontinuation of clinical trials and withdrawal of drugs from the market. Since the original observations in the mid-90s, it has been well established that cardiovascular risk factors and comorbidities (such as ageing, hyperlipidaemia, and diabetes) and their medications (e.g. nitrate tolerance, adenosine triphosphate-dependent potassium inhibitor antidiabetic drugs, statins, etc.) may interfere with cardiac ischaemic tolerance and endogenous cardioprotective signalling pathways. Indeed drugs may exert unwanted effects on the diseased and treated heart that is hidden in the healthy myocardium. Hidden cardiotoxic effects may be due to (i) drug-induced enhancement of deleterious signalling due to ischaemia/reperfusion injury and/or the presence of risk factors and/or (ii) inhibition of cardioprotective survival signalling pathways, both of which may lead to ischaemia-related cell death and/or pro-arrhythmic effects. This led to a novel concept of 'hidden cardiotoxicity', defined as cardiotoxity of a drug that manifests only in the diseased heart with e.g. ischaemia/reperfusion injury and/or in the presence of its major comorbidities. Little is known on the mechanism of hidden cardiotoxocity, moreover, hidden cardiotoxicity cannot be revealed by the routinely used non-clinical cardiac safety testing methods on healthy animals or tissues. Therefore, here, we emphasize the need for development of novel cardiac safety testing platform involving combined experimental models of cardiac diseases (especially myocardial ischaemia/reperfusion and ischaemic conditioning) in the presence and absence of major cardiovascular comorbidities and/or cotreatments.