Droplet Digital PCR Is a Novel Screening Method Identifying Potential Cardiac G-Protein-Coupled Receptors as Candidate Pharmacological Targets in a Rat Model of Pressure-Overload-Induced Cardiac Dysfunction.

The identification of novel drug targets is needed to improve the outcomes of heart failure (HF). G-protein-coupled receptors (GPCRs) represent the largest family of targets for already approved drugs, thus providing an opportunity for drug repurposing. Here, we aimed (i) to investigate the differential expressions of 288 cardiac GPCRs via droplet digital PCR (ddPCR) and bulk RNA sequencing (RNAseq) in a rat model of left ventricular pressure-overload; (ii) to compare RNAseq findings with those of ddPCR; and (iii) to screen and test for novel, translatable GPCR drug targets in HF. Male Wistar rats subjected to transverse aortic constriction (TAC, n = 5) showed significant systolic dysfunction vs. sham operated animals (SHAM, n = 5) via echocardiography. In TAC vs. SHAM hearts, RNAseq identified 69, and ddPCR identified 27 significantly differentially expressed GPCR mRNAs, 8 of which were identified using both methods, thus showing a correlation between the two methods. Of these, Prostaglandin-F2α-receptor (Ptgfr) was further investigated and localized on cardiomyocytes and fibroblasts in murine hearts via RNA-Scope. Antagonizing Ptgfr via AL-8810 reverted angiotensin-II-induced cardiomyocyte hypertrophy in vitro. In conclusion, using ddPCR as a novel screening method, we were able to identify GPCR targets in HF. We also show that the antagonism of Ptgfr could be a novel target in HF by alleviating cardiomyocyte hypertrophy.

Addition of epidermal growth factor receptor inhibitors to standard chemotherapy increases survival of advanced head and neck squamous cell carcinoma patients: A systematic review and meta-analysis.

Head and neck squamous cell carcinoma (HNSCC) is among the common tumors associated with high mortality. The aim of our meta-analysis was to determine how additional anti-epidermal growth factor receptor (EGFR) therapy to standard chemotherapy affects the progression-free (PFS) and overall survival (OS) of the patients, besides the most common side effects. We used CENTRAL, MEDLINE, and Embase databases until October 26, 2020, and included 13 eligible randomized controlled trials in our systematic research. The pooled hazard ratios (HR) for the main outcomes from the original data were estimated and for the other dichotomous outcomes, odds ratios (ORs) with their 95% confidence intervals (CI) were calculated. Addition of EGFR inhibitors to conventional chemotherapy significantly decreased the death and disease progression (for PFS HR: 0.68, 95% CI: 0.55-0.81, I2  = 65.5%, p = 0.005) and mortality (for OS HR: 0.83, 95% CI: 0.72-0.94, I2  = 42.3%, p = 0.076). In the EGFR inhibitor group, we revealed an increased chance of the over Grade 3 skin rashes (OR: 4.86; 95% CI: 1.52-15.49, I2  = 2.3%, p = 0.407), and all Grade skin rashes (OR: 18.32, 95% CI: 8.07-41.60, I2  = 56.6%, p = 0.032). Despite their unwanted dermatological side effects, the addition of EGFR inhibitors is recommended to be included in advanced HNSCC therapy.

Tenodesis yields better functional results than tenotomy in long head of the biceps tendon operations-a systematic review and meta-analysis.

BACKGROUND: Pathology of the long head of the biceps tendon (LHBT) is a common disorder affecting muscle function and causing considerable pain for the patient. The literature on the two surgical treatment methods (tenotomy and tenodesis) is controversial; therefore, our aim was to compare the results of these interventions. METHODS: We performed a meta-analysis using the following strategy: (P) patients with LHBT pathology, (I) tenodesis, (C) tenotomy, (O) elbow flexion and forearm supination strength, pain assessed on the ten-point Visual Analog Scale (VAS), bicipital cramping pain, Constant, ASES, and SST score, Popeye deformity, and operative time. We included only randomized clinical trials. We searched five databases. During statistical analysis, odds ratios (OR) and weighted mean differences (WMD) were calculated for dichotomous and continuous outcomes, respectively, using the Bayesian method with random effect model. RESULTS: We included 11 studies in the systematic review, nine of these were eligible for the meta-analysis, containing data about 572 patients (279 in the tenodesis, 293 in the tenotomy group). Our analysis concluded that tenodesis is more beneficial considering 12-month elbow flexion strength (WMD: 3.67 kg; p = 0.006), 12-month forearm supination strength (WMD: 0.36 kg; p = 0.012), and 24-month Popeye deformity (OR: 0.19; p < 0.001), whereas tenotomy was associated with decreased 3-month pain scores on VAS (WMD: 0.99; p < 0.001). We did not find significant difference among the other outcomes. CONCLUSION: Tenodesis yields better results in terms of biceps function and is non-inferior regarding long-term pain, while tenotomy is associated with earlier pain relief.

AIM2-driven inflammasome activation in heart failure.

AIMS: Interleukin-1ß (IL-1ß) is an important pathogenic factor in cardiovascular diseases including chronic heart failure (HF). The CANTOS trial highlighted that inflammasomes as primary sources of IL-1 ß are promising new therapeutic targets in cardiovascular diseases. Therefore, we aimed to assess inflammasome activation in failing hearts to identify activation patterns of inflammasome subtypes as sources of IL-1ß. METHODS AND RESULTS: Out of the four major inflammasome sensors tested, expression of the inflammasome protein absent in melanoma 2 (AIM2) and NLR family CARD domain-containing protein 4 (NLRC4) increased in human HF regardless of the aetiology (ischaemic or dilated cardiomyopathy), while the NLRP1/NALP1 and NLRP3 (NLR family, pyrin domain containing 1 and 3) inflammasome showed no change in HF samples. AIM2 expression was primarily detected in monocytes/macrophages of failing hearts. Translational animal models of HF (pressure or volume overload, and permanent coronary artery ligation in rat, as well as ischaemia/reperfusion-induced HF in pigs) demonstrated activation pattern of AIM2 similar to that of observed in end-stages of human HF. In vitro AIM2 inflammasome activation in human Tohoku Hospital Pediatrics-1 (THP-1) monocytic cells and human AC16 cells was significantly reduced by pharmacological blockade of pannexin-1 channels by the clinically used uricosuric drug probenecid. Probenecid was also able to reduce pressure overload-induced mortality and restore indices of disease severity in a rat chronic HF model in vivo. CONCLUSIONS: This is the first report showing that AIM2 and NLRC4 inflammasome activation contribute to chronic inflammation in HF and that probenecid alleviates chronic HF by reducing inflammasome activation. The present translational study suggests the possibility of repositioning probenecid for HF indications.

Telomerase/myocardin expressing mesenchymal cells induce survival and cardiovascular markers in cardiac stromal cells undergoing ischaemia/reperfusion.

Cardiac stromal cells (CSCs) contain a pool of cells with supportive and paracrine functions. Various types of mesenchymal stromal cells (MSCs) can influence CSCs in the cardiac niche through their paracrine activity. Ischaemia/reperfusion (I/R) leads to cell death and reduction of the paracrine activity of CSCs. The forced co-expression of telomerase reverse transcriptase (TERT) and myocardin (MYOCD), known to potentiate anti-apoptotic, pro-survival and pro-angiogenic activities of MSCs isolated from the adipose tissue (AT-MSCs), may increase CSC survival, favouring their paracrine activities. We aimed at investigating the hypothesis that CSCs feature improved resistance to simulated I/R (SI/R) and increased commitment towards the cardiovascular lineage when preconditioned with conditioned media (CM) or extracellular vesicles (EV) released from AT-MSCs overexpressing TERT and MYOCD (T/M AT-MSCs). Murine CSCs were isolated with the cardiosphere (CSps) isolation technique. T/M AT-MSCs and their secretome improved spontaneous intracellular calcium changes and ryanodine receptor expression in aged CSps. The cytoprotective effect of AT-MSCs was tested in CSCs subjected to SI/R. SI/R induced cell death as compared to normoxia (28 ± 4 vs 10 ± 3%, P = .02). Pre-treatment with CM (15 ± 2, P = .02) or with the EV-enriched fraction (10 ± 1%, P = .02) obtained from mock-transduced AT-MSCs in normoxia reduced cell death after SI/R. The effect was more pronounced with CM (7 ± 1%, P = .01) or the EV-enriched fraction (2 ± 1%, P = .01) obtained from T/M AT-MSCs subjected to SI/R. In parallel, we observed lower expression of the apoptosis marker cleaved caspase-3 and higher expression of cardiac and vascular markers eNOS, sarcomeric α-actinin and cardiac actin. The T/M AT-MSCs secretome exerts a cytoprotective effect and promotes development of CSCs undergoing SI/R towards a cardiovascular phenotype.

PACAP-38 in Acute ST-Segment Elevation Myocardial Infarction in Humans and Pigs: A Translational Study.

Acute myocardial infarction (MI) is one of the most common causes of death worldwide. Pituitary adenylate cyclase activating polypeptide (PACAP) is a cardioprotective neuropeptide expressing its receptors in the cardiovascular system. The aim of our study was to examine tissue PACAP-38 in a translational porcine MI model and plasma PACAP-38 levels in patients with ST-segment elevation myocardial infarction (STEMI). Significantly lower PACAP-38 levels were detected in the non-ischemic region of the left ventricle (LV) in MI heart compared to the ischemic region of MI-LV and also to the Sham-operated LV in porcine MI model. In STEMI patients, plasma PACAP-38 level was significantly higher before percutaneous coronary intervention (PCI) compared to controls, and decreased after PCI. Significant negative correlation was found between plasma PACAP-38 and troponin levels. Furthermore, a significant effect was revealed between plasma PACAP-38, hypertension and HbA1c levels. This was the first study showing significant changes in cardiac tissue PACAP levels in a porcine MI model and plasma PACAP levels in STEMI patients. These results suggest that PACAP, due to its cardioprotective effects, may play a regulatory role in MI and could be a potential biomarker or drug target in MI.

Natural and synthetic antioxidants targeting cardiac oxidative stress and redox signaling in cardiometabolic diseases.

Cardiometabolic diseases (CMDs) are metabolic diseases (e.g., obesity, diabetes, atherosclerosis, rare genetic metabolic diseases, etc.) associated with cardiac pathologies. Pathophysiology of most CMDs involves increased production of reactive oxygen species and impaired antioxidant defense systems, resulting in cardiac oxidative stress (OxS). To alleviate OxS, various antioxidants have been investigated in several diseases with conflicting results. Here we review the effect of CMDs on cardiac redox homeostasis, the role of OxS in cardiac pathologies, as well as experimental and clinical data on the therapeutic potential of natural antioxidants (including resveratrol, quercetin, curcumin, vitamins A, C, and E, coenzyme Q10, etc.), synthetic antioxidants (including N-acetylcysteine, SOD mimetics, mitoTEMPO, SkQ1, etc.), and promoters of antioxidant enzymes in CMDs. As no antioxidant indicated for the prevention and/or treatment of CMDs has reached the market despite the large number of preclinical and clinical studies, a sizeable translational gap is evident in this field. Thus, we also highlight potential underlying factors that may contribute to the failure of translation of antioxidant therapies in CMDs.

Cardiac dysfunction in cancer patients: beyond direct cardiomyocyte damage of anticancer drugs: novel cardio-oncology insights from the joint 2019 meeting of the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart.

In western countries, cardiovascular (CV) disease and cancer are the leading causes of death in the ageing population. Recent epidemiological data suggest that cancer is more frequent in patients with prevalent or incident CV disease, in particular, heart failure (HF). Indeed, there is a tight link in terms of shared risk factors and mechanisms between HF and cancer. HF induced by anticancer therapies has been extensively studied, primarily focusing on the toxic effects that anti-tumour treatments exert on cardiomyocytes. In this Cardio-Oncology update, members of the ESC Working Groups of Myocardial Function and Cellular Biology of the Heart discuss novel evidence interconnecting cardiac dysfunction and cancer via pathways in which cardiomyocytes may be involved but are not central. In particular, the multiple roles of cardiac stromal cells (endothelial cells and fibroblasts) and inflammatory cells are highlighted. Also, the gut microbiota is depicted as a new player at the crossroads between HF and cancer. Finally, the role of non-coding RNAs in Cardio-Oncology is also addressed. All these insights are expected to fuel additional research efforts in the field of Cardio-Oncology.

Cardiac miRNA Expression and their mRNA Targets in a Rat Model of Prediabetes.

Little is known about the mechanism of prediabetes-induced cardiac dysfunction. Therefore, we aimed to explore key molecular changes with transcriptomic and bioinformatics approaches in a prediabetes model showing heart failure with preserved ejection fraction phenotype. To induce prediabetes, Long-Evans rats were fed a high-fat diet for 21 weeks and treated with a single low-dose streptozotocin at week 4. Small RNA-sequencing, in silico microRNA (miRNA)-mRNA target prediction, Gene Ontology analysis, and target validation with qRT-PCR were performed in left ventricle samples. From the miRBase-annotated 752 mature miRNA sequences expression of 356 miRNAs was detectable. We identified two upregulated and three downregulated miRNAs in the prediabetic group. We predicted 445 mRNA targets of the five differentially expressed miRNAs and selected 11 mRNAs targeted by three differentially expressed miRNAs, out of which five mRNAs were selected for validation. Out of these five targets, downregulation of three mRNAs i.e., Juxtaposed with another zinc finger protein 1 (Jazf1); RAP2C, member of RAS oncogene family (Rap2c); and Zinc finger with KRAB and SCAN domains 1 (Zkscan1) were validated. This is the first demonstration that prediabetes alters cardiac miRNA expression profile. Predicted targets of differentially expressed miRNAs include Jazf1, Zkscan1, and Rap2c mRNAs. These transcriptomic changes may contribute to the diastolic dysfunction and may serve as drug targets.

Proteomic analysis of the secretome of adipose tissue-derived murine mesenchymal cells overexpressing telomerase and myocardin.

RATIONALE: Understanding mechanisms of the therapeutic effects of stem/progenitor cells, among which adipose tissue-derived mesenchymal stromal cells (AT-MSCs), has important implications for clinical use. Since the majority of such cells die within days or weeks after transplantation and do not persist in the transplanted organ or tissue, their effects appear to be largely mediated by paracrine signaling pathways, and are enhanced by overexpression of the antisenescent protein telomerase reverse transcriptase (TERT), and the anti-apoptotic transcription factor myocardin (MYOCD). AIM: By a proteomic approach combining two-dimensional gel electrophoresis (2DE) with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF/TOF) mass spectrometry, we aimed at analyzing how soluble and vesicular secretomes of aged murine AT-MSCs and their angiogenic function are modulated by the overexpression of TERT and MYOCD. METHODS: We cultured murine mock-transduced AT-MSCs and "rejuvenated" AT-MSCs overexpressing TERT and MYOCD (rTMAT-MSCs) harvested from 1-year-old male C57BL/6 mice. We established proteomes from 3 mock-transduced AT-MSCs and rTMAT-MSCs cultures in serum-free conditions, as well as their corresponding conditioned medium (CM) and exosome-enriched fractions (Exo+). RESULTS AND CONCLUSIONS: Proteomic analysis revealed a 2-fold increase of matrix metalloproteinase-2 (MMP-2) and its inhibitor metalloproteinase inhibitor 2 (TIMP2) in the CM - but not in the Exo + - of rTMAT-MSCs as compared to mock-transduced AT-MSCs. At the functional level, rTMAT-MSCs-CM, and - to a lesser extent - its Exo + fraction, increased tube formation of human vein endothelial cells (HUVECs), which could be blocked by anti-MMP2 and enhanced by anti-TIMP2 antibodies, respectively. Altogether, our results identify MMP2 and its inhibitor TIMP2 as novel candidates by which rTMAT-MSCs can support angiogenesis. Our strategy also illustrates the usefulness of comparative targeted proteomic approach to decipher molecular pathways underlying rTMAT-MSCs properties.

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.

Targeting Mitochondrial Dysfunction with L-Alpha Glycerylphosphorylcholine.

BACKGROUND: We hypothesized that L-alpha-glycerylphosphorylcholine (GPC), a deacylatedphosphatidylcholine derivative, can influence the mitochondrial respiratory activity and in this way, may exert tissue protective effects. METHODS: Rat liver mitochondria were examined with high-resolution respirometry to analyze the effects of GPC on the electron transport chain in normoxic and anoxic conditions. Besides, Sprague-Dawley rats were subjected to sham operation or standardized liver ischemia-reperfusion (IR), with or without GPC administration. The reduced glutathione (GSH) and oxidized glutathione disulfide (GSSG), the tissue myeloperoxidase, xanthine oxidoreductase and NADPH oxidases activities were measured. Tissue malondialdehyde and nitrite/nitrate formation, together with blood superoxide and hydrogen-peroxide production were assessed. RESULTS: GPC increased the efficacy of complex I-linked mitochondrial oxygen consumption, with significantly lower in vitro leak respiration. Mechanistically, liver IR injury was accompanied by deteriorated mitochondrial respiration and enhanced ROS production and, as a consequence, by significantly increased inflammatory enzyme activities. GPC administration decreased the inflammatory activation in line with the reduced oxidative and nitrosative stress markers. CONCLUSION: GPC, by preserving the mitochondrial complex I function respiration, reduced the biochemical signs of oxidative stress after an IR episode. This suggests that GPC is a mitochondria-targeted compound that indirectly suppresses the activity of major intracellular superoxide-generating enzymes.

Protection of neonatal rat cardiac myocytes against radiation-induced damage with agonists of growth hormone-releasing hormone.

Despite the great clinical significance of radiation-induced cardiac damage, experimental investigation of its mechanisms is an unmet need in medicine. Beneficial effects of growth hormone-releasing hormone (GHRH) agonists in regeneration of the heart have been demonstrated. The aim of this study was the evaluation of the potential of modern GHRH agonistic analogs in prevention of radiation damage in an in vitro cardiac myocyte-based model. Cultures of cardiac myocytes isolated from newborn rats (NRVM) were exposed to a radiation dose of 10Gy. The effects of the agonistic analogs, JI-34 and MR-356, of human GHRH on cell viability, proliferation, their mechanism of action and the protein expression of the GHRH/SV1 receptors were studied. JI-34 and MR-356, had no effect on cell viability or proliferation in unirradiated cultures. However, in irradiated cells JI-34 showed protective effects on cell viability at concentrations of 10 and 100nM, and MR-356 at 500nM; but no such protective effect was detected on cell proliferation. Both agonistic analogs decreased radiation-induced ROS level and JI-34 interfered with the activation of SAFE/RISK pathways. Using Western blot analysis, a 52kDa protein isoform of GHRHR was detected in the samples in both irradiated and unirradiated cells. Since GHRH agonistic analogs, JI-34 and MR-356 alleviated radiation-induced damage of cardiac myocytes, they should be tested in vivo as potential protective agents against radiogenic heart damage.

The cytoprotective effect of biglycan core protein involves Toll-like receptor 4 signaling in cardiomyocytes.

AIMS: Exogenously administered biglycan (core protein with high-molecular weight glycosaminoglycan chains) has been shown to protect neonatal cardiomyocytes against simulated ischemia/reperfusion injury (SI/R), however, the mechanism of action is not clear. In this study we aimed to investigate, which structural component of biglycan is responsible for its cardiocytoprotective effect and to further explore the molecular mechanisms involved in the cytoprotection. METHODS AND RESULTS: A pilot study was conducted to demonstrate that both native (glycanated) and deglycanated biglycan can attenuate cell death induced by SI/R in a dose-dependent manner in primary neonatal cardiomyocytes isolated from Wistar rats. In separate experiments, we have shown that similarly to glycanated biglycan, recombinant human biglycan core protein (rhBGNc) protects cardiomyocytes against SI/R injury. In contrast, the glycosaminoglycan component dermatan sulfate had no significant effect on cell viability, while chondroitin sulfate further enhanced cell death induced by SI/R. Treatment of cardiomyocytes with rhBGNc reverses the effect of SI/R upon markers of necrosis, apoptosis, mitochondrial membrane potential, and autophagy. We have also shown that pharmacological blockade of Toll-like receptor 4 (TLR4) signaling or its downstream mediators (IRAK1/4, ERK, JNK and p38 MAP kinases) abolished the cytoprotective effect of rhBGNc against SI/R injury. Pretreatment of cardiomyocytes with rhBGNc for 20h resulted in increased Akt phosphorylation and NO production without having significant effect on phosphorylation of ERK1/2, STAT3, and on the production of superoxide. Treatment over 10min and 1h with rhBGNc increased ERK1 phosphorylation, while the SI/R-induced increase in superoxide production was attenuated by rhBGNc. Blockade of NO synthesis also prevented the cardiocytoprotective effect of rhBGNc. CONCLUSIONS: The core protein of exogenous biglycan protects myocardial cells from SI/R injury via TLR4-mediated mechanisms involving activation of ERK, JNK and p38 MAP kinases and increased NO production. The cytoprotective effect of rhBGNc is due to modulation of SI/R-induced changes in necrosis, apoptosis and autophagy.

Stem Cell Aging and Age-Related Cardiovascular Disease: Perspectives of Treatment by Ex-vivo Stem Cell Rejuvenation.

Aging affects endogenous stem cells in terms of functionality and numbers. In particular, during aging, the stemness property can decrease because of enhanced apoptotic cell death and senescence. In addition, aging and aging-related co-morbidities affect the paracrine activity of stem cells and the efficiency of their transplantation. Collectively, this leads to a reduction of the capacity of organs to repair themselves, possibly due to a reduced functional capability of stem cells. Therefore, major efforts have been invested to improve the repair capability of stem cells in aged individuals by overexpressing antisenescence and antiapoptotic genes. In this review, we describe critical genes and signaling pathways in stem cell aging and discuss ex vivo genetic modification approaches aimed at stem cell rejuvenation that are of interest for the cardiovascular system.

Therapeutic potential of midkine in cardiovascular disease.

UNLABELLED: Ischaemic heart disease, stroke and their pathological consequences are life-threatening conditions that account for about half of deaths in developed countries. Pathology of these diseases includes cell death due to ischaemia/reperfusion injury, vascular stenosis and cardiac remodelling. The growth factor midkine plays a pivotal role in these events. Midkine shows an acute cytoprotective effect in ischaemia/reperfusion injury at least in part via its anti-apoptotic effect. Moreover, while midkine promotes endothelial cell proliferation, it also recruits inflammatory cells to lesions. These activities eventually enhance angiogenesis, thereby preventing cardiac tissue remodelling. However, midkine's activity in recruiting inflammatory cells into the vascular wall also triggers neointima formation, and consequently, vascular stenosis. Moreover, midkine is induced in cancer tissues where it enhances angiogenesis. Therefore, midkine may promote tumour formation through its angiogenic and anti-apoptotic activity. This review focuses on the roles of midkine in ischaemic cardiovascular disease and their pathological consequences, that is angiogenesis, vascular stenosis, and cardiac remodelling, and discusses the possible therapeutic potential of modulation of midkine in these diseases. LINKED ARTICLES: This article is part of a themed section on Midkine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-4.

Cholesterol-enriched diet inhibits cardioprotection by ATP-sensitive K+ channel activators cromakalim and diazoxide.

It has been previously shown that hyperlipidemia interferes with cardioprotective mechanisms. Here, we investigated the interaction of hyperlipidemia with cardioprotection induced by pharmacological activators of ATP-sensitive K(+) (KATP) channels. Hearts isolated from rats fed a 2% cholesterol-enriched diet or normal diet for 8 wk were subjected to 30 min of global ischemia and 120 min of reperfusion in the presence or absence of KATP modulators. In normal diet-fed rats, either the nonselective KATP activator cromakalim at 10(-5) M or the selective mitochondrial (mito)KATP opener diazoxide at 3 × 10(-5) M significantly decreased infarct size compared with vehicle-treated control rats. Their cardioprotective effect was abolished by coadministration of the nonselective KATP blocker glibenclamide or the selective mitoKATP blocker 5-hydroxydecanoate, respectively. However, in cholesterol-fed rats, the cardioprotective effect of cromakalim or diazoxide was not observed. Therefore, we further investigated how cholesterol-enriched diet influences cardiac KATP channels. Cardiac expression of a KATP subunit gene (Kir6.1) was significantly downregulated in cholesterol-fed rats; however, protein levels of Kir6.1 and Kir6.2 were not changed. The cholesterol diet significantly decreased cardiac ATP, increased lactate content, and enhanced myocardial oxidative stress, as shown by increased cardiac superoxide and dityrosine formation. This is the first demonstration that cardioprotection by KATP channel activators is impaired in cholesterol-enriched diet-induced hyperlipidemia. The background mechanism may include hyperlipidemia-induced attenuation of mitoKATP function by altered energy metabolism and increased oxidative stress in the heart.

Transient upregulation of connexin43 gap junctions and synchronized cell cycle control precede myoblast fusion in regenerating skeletal muscle in vivo.

The spatio-temporal expression of gap junction connexins (Cx) was investigated and correlated with the progression of cell cycle control in regenerating soleus muscle of Wistar rats. Notexin caused a selective myonecrosis followed by the complete recapitulation of muscle differentiation in vivo, including the activation, commitment, proliferation, differentiation and fusion of myogenic cells. In regenerating skeletal muscle, only Cx43 protein, out of Cx-s 26, -32, -37, -40, -43 and -45, was detected in desmin positive cells. Early expression of Cx43 in the proliferating single myogenic progenitors was followed by a progressive upregulation in interacting myoblasts until syncytial fusion, and then by a rapid decline in multinucleate myotubes. The significant upregulation of Cx43 gap junctions in aligned myoblasts preceding fusion was accompanied by the widespread nuclear expression of cyclin-dependent kinase inhibitors p21(waf1/Cip1) and p27(kip1) and the complete loss of Ki67 protein. The synchronized exit of myoblasts from the cell cycle following extensive gap junction formation suggests a role for Cx43 channels in the regulation of cell cycle control. The potential of Cx43 channels to stimulate p21(waf1/Cip1) and p27(kip1) is known. In the muscle, proving the involvement of Cx43 in either a direct or a bystander cell cycle regulation requires functional investigations.