Mechanism of motoneuronal and pyramidal cell death in amyotrophic lateral sclerosis and its potential therapeutic modulation.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder clinically characterized by muscle atrophy and progressive paralysis. Loss of motoneurons and pyramidal cells is thought to be the center piece of the complex and multifaceted ALS pathology, however, the exact mechanisms laying behind motoneuronal cell death in the spinal cord and motor cortex are still unknown. It was originally proposed that apoptosis plays a fundamental role in motoneuronal demise, nonetheless, later it became clear that other forms of regulated cell death, including necroptosis, pyroptosis, ferroptosis, and autophagy-dependent cell death, may also contribute to motoneuron loss. Over the past years, multiple studies aimed to improve our understanding of the contributory role of these mechanisms as well as to offer novel targets for potential therapeutic interventions. The pharmacological inhibition of the ferroptotic pathway and the modulation of the autophagic machinery seem to have particularly promising effects, reducing motoneuron loss and slowing disease progression in transgenic models of ALS. Nevertheless, the potential beneficial effects of necroptosis-targeting interventions were mostly disproven in the latest studies. In this review we aim to summarize the current view on regulated cell death mechanisms that lead to motoneuronal and pyramidal cell degeneration in ALS and showcase their applicability as future drug targets.

The Proteoglycans Biglycan and Decorin Protect Cardiac Cells against Irradiation-Induced Cell Death by Inhibiting Apoptosis.

Radiation-induced heart disease (RIHD), a common side effect of chest irradiation, is a primary cause of mortality among patients surviving thoracic cancer. Thus, the development of novel, clinically applicable cardioprotective agents which can alleviate the harmful effects of irradiation on the heart is of great importance in the field of experimental oncocardiology. Biglycan and decorin are structurally related small leucine-rich proteoglycans which have been reported to exert cardioprotective properties in certain cardiovascular pathologies. Therefore, in the present study we aimed to examine if biglycan or decorin can reduce radiation-induced damage of cardiomyocytes. A single dose of 10 Gray irradiation was applied to induce radiation-induced cell damage in H9c2 cardiomyoblasts, followed by treatment with either biglycan or decorin at various concentrations. Measurement of cell viability revealed that both proteoglycans improved the survival of cardiac cells post-irradiation. The cardiocytoprotective effect of both biglycan and decorin involved the alleviation of radiation-induced proapoptotic mechanisms by retaining the progression of apoptotic membrane blebbing and lowering the number of apoptotic cell nuclei and DNA double-strand breaks. Our findings provide evidence that these natural proteoglycans may exert protection against radiation-induced damage of cardiac cells.

Migraine with aura and stroke - the role of warning signs in the context of secondary headaches: case report / Enxaqueca com aura e acidente vascular cerebral ­ o papel dos sinais de alerta no contexto das cefaleias secundárias: relato de caso

Introduction: Headache is a very common complaint in doctors' offices, with primary causes being the majority in relation to secondary ones. Despite this, the identification of secondary headaches is very relevant in clinical practice, since these can be a life-threatening condition, functionality or even a reversible cause. However, imaging screening for all individuals with headache is costly and unrewarding. Therefore, it is important to know the warning signs that, together with the clinical context, lead to a more precise indication of these exams and early and well-targeted therapeutic interventions. Clinical case: This is a 60-year-old man, previously dyslipidemic and smoker, with migraine with aura reported since childhood, who underwent treatment with sodium valproate, with headache attack suppression. About 4 months before admission, he presented with an alteration in the pain pattern, amaurosis fugax in the right eye, dizziness and mild paresis and hypoesthesia in the left side of the body, primarily treated by him as migraine crises, without improvement with the use of triptans. A new outpatient investigation was carried out, which showed multiple small infarcts in the right hemisphere secondary to atheromatous plaque in the right carotid bulb with an obstruction of approximately 85%. Diagnostic and therapeutic arteriography was performed, with stent implantation, uneventfully. Conclusion: The differential diagnosis between migraine with aura and a cerebrovascular event has already been widely reported in the literature and constitutes a pitfall in the routine of headaches, since a serious and potentially disabling condition can be overlooked. The joint evaluation of the alarm signs with the global context becomes an important tool in the propaedeutics of these patients, with knowledge of this casuistry being something relevant within clinical practice.

Introdução: A cefaleia é uma queixa muito comum nos consultórios médicos, sendo as causas primárias majoritárias em relação às secundárias. Apesar disso, a identificação de cefaleias secundárias é muito relevante na prática clínica, uma vez que estas podem ser uma condição potencialmente fatal, funcional ou mesmo uma causa reversível. No entanto, o rastreio imagiológico para todos os indivíduos com cefaleias é dispendioso e pouco recompensador. Portanto, é importante conhecer os sinais de alerta que, juntamente com o contexto clínico, levam a uma indicação mais precisa destes exames e a intervenções terapêuticas precoces e bem direcionadas. Caso clínico: Trata-se de um homem de 60 anos, previamente dislipidémico e fumador, com queixa de enxaqueca com aura desde a infância, que realizou tratamento com valproato de sódio, com supressão das crises de cefaleia. Cerca de 4 meses antes da internação apresentou alteração do padrão álgico, amaurose fugaz em olho direito, tontura e leve paresia e hipoestesia no lado esquerdo do corpo, tratada por ele primariamente como crises de enxaqueca, sem melhora com o uso de triptanos. Foi realizada nova investigação ambulatorial que evidenciou múltiplos pequenos infartos no hemisfério direito secundários a placa de ateroma no bulbo carotídeo direito com obstrução de aproximadamente 85%. Foi realizada arteriografia diagnóstica e terapêutica, com implante de stent, sem intercorrências. Conclusão: O diagnóstico diferencial entre enxaqueca com aura e evento cerebrovascular já foi amplamente relatado na literatura e constitui uma armadilha na rotina das cefaleias, uma vez que uma condição grave e potencialmente incapacitante pode ser negligenciada. A avaliação conjunta dos sinais de alarme com o contexto global torna-se uma ferramenta importante na propedêutica destes pacientes, sendo o conhecimento desta casuística algo relevante dentro da prática clínica.

Ischemic preconditioning protects the heart against ischemia-reperfusion injury in chronic kidney disease in both males and females.

BACKGROUND: Uremic cardiomyopathy is a common cardiovascular complication of chronic kidney disease (CKD) characterized by left ventricular hypertrophy (LVH) and fibrosis enhancing the susceptibility of the heart to acute myocardial infarction. In the early stages of CKD, approximately 60% of patients are women. We aimed to investigate the influence of sex on the severity of uremic cardiomyopathy and the infarct size-limiting effect of ischemic preconditioning (IPRE) in experimental CKD. METHODS: CKD was induced by 5/6 nephrectomy in 9-week-old male and female Wistar rats. Two months later, serum and urine laboratory parameters were measured to verify the development of CKD. Transthoracic echocardiography was performed to assess cardiac function and morphology. Cardiomyocyte hypertrophy and fibrosis were measured by histology. Left ventricular expression of A- and B-type natriuretic peptides (ANP and BNP) were measured by qRT-PCR and circulating BNP level was measured by ELISA. In a subgroup of animals, hearts were perfused according to Langendorff and were subjected to 35 min global ischemia and 120 min reperfusion with or without IPRE (3 × 5 min I/R cycles applied before index ischemia). Then infarct size or phosphorylated and total forms of proteins related to the cardioprotective RISK (AKT, ERK1,2) and SAFE (STAT3) pathways were measured by Western blot. RESULTS: The severity of CKD was similar in males and females. However, CKD males developed more severe LVH compared to females as assessed by echocardiography. Histology revealed cardiac fibrosis only in males in CKD. LV ANP expression was significantly increased due to CKD in both sexes, however, LV BNP and circulating BNP levels failed to significantly increase in CKD. In both sexes, IPRE significantly decreased the infarct size in both the sham-operated and CKD groups. IPRE significantly increased the phospho-STAT3/STAT3 ratio in sham-operated but not in CKD animals in both sexes. There were no significant differences in phospho-AKT/AKT and phospho-ERK1,2/ERK1,2 ratios between the groups. CONCLUSION: The infarct size-limiting effect of IPRE was preserved in both sexes in CKD despite the more severe uremic cardiomyopathy in male CKD rats. Further research is needed to identify crucial molecular mechanisms in the cardioprotective effect of IPRE in CKD.

Ambroxol Treatment Suppresses the Proliferation of Chlamydia pneumoniae in Murine Lungs.

Ambroxol (Ax) is used as a mucolytics in the treatment of respiratory tract infections. Ax, at a general dose for humans, does not alter Chlamydia pneumoniae growth in mice. Therefore, we aimed to investigate the potential anti-chlamydial effect of Ax at a concentration four timed higher than that used in human medicine. Mice were infected with C. pneumoniae and 5-mg/kg Ax was administered orally. The number of recoverable C. pneumoniae inclusion-forming units (IFUs) in Ax-treated mice was significantly lower than that in untreated mice. mRNA expression levels of several cytokines, including interleukin 12 (IL-12), IL-23, IL-17F, interferon gamma (IFN-γ), and surfactant protein (SP)-A, increased in infected mice treated with Ax. The IFN-γ protein expression levels were also significantly higher in infected and Ax-treated mice. Furthermore, the in vitro results suggested that the ERK 1/2 activity was decreased, which is essential for the C. pneumoniae replication. SP-A and SP-D treatments significantly decreased the number of viable C. pneumoniae IFUs and significantly increased the attachment of C. pneumoniae to macrophage cells. Based on our results, a dose of 5 mg/kg of Ax exhibited an anti-chlamydial effect in mice, probably an immunomodulating effect, and may be used as supporting drug in respiratory infections caused by C. pneumoniae.

Pathomechanisms and therapeutic opportunities in radiation-induced heart disease: from bench to bedside.

Cancer management has undergone significant improvements, which led to increased long-term survival rates among cancer patients. Radiotherapy (RT) has an important role in the treatment of thoracic tumors, including breast, lung, and esophageal cancer, or Hodgkin's lymphoma. RT aims to kill tumor cells; however, it may have deleterious side effects on the surrounding normal tissues. The syndrome of unwanted cardiovascular adverse effects of thoracic RT is termed radiation-induced heart disease (RIHD), and the risk of developing RIHD is a critical concern in current oncology practice. Premature ischemic heart disease, cardiomyopathy, heart failure, valve abnormalities, and electrical conduct defects are common forms of RIHD. The underlying mechanisms of RIHD are still not entirely clear, and specific therapeutic interventions are missing. In this review, we focus on the molecular pathomechanisms of acute and chronic RIHD and propose preventive measures and possible pharmacological strategies to minimize the burden of RIHD.

Kynurenine Pathway Metabolites as Potential Clinical Biomarkers in Coronary Artery Disease.

Coronary artery disease (CAD) is one of the leading cause of mortality worldwide. Several risk factors including unhealthy lifestyle, genetic background, obesity, diabetes, hypercholesterolemia, hypertension, smoking, age, etc. contribute to the development of coronary atherosclerosis and subsequent coronary artery disease. Inflammation plays an important role in coronary artery disease development and progression. Pro-inflammatory signals promote the degradation of tryptophan via the kynurenine pathway resulting in the formation of several immunomodulatory metabolites. An unbalanced kynurenic pathway has been implicated in the pathomechanisms of various diseases including CAD. Significant improvements in detection methods in the last decades may allow simultaneous measurement of multiple metabolites of the kynurenine pathway and such a thorough analysis of the kynurenine pathway may be a valuable tool for risk stratification and determination of CAD prognosis. Nevertheless, imbalance in the activities of different branches of the kynurenine pathway may require careful interpretation. In this review, we aim to summarize clinical evidence supporting a possible use of kynurenine pathway metabolites as clinical biomarkers in various manifestations of CAD.

Androstano-arylpyrimidines: Novel small molecule inhibitors of MDR1 for sensitizing multidrug-resistant breast cancer cells.

Apart from the numerous physiological functions of MDR1, it is widely known for its role in granting multidrug resistance to cancer cells. This ATP-driven transmembrane protein exports a wide range of chemotherapeutic agents from cancer cells, thereby deterring drugs to reach effective intracellular concentrations. Thus, inhibition of MDR1 expression or function would be a viable option to enhance the accumulation of cytotoxic agents in cancer cells which in turn could improve significantly the success rate of chemotherapy. Although, several pharmacological inhibitors have been designed and tested in the past, due to their unsuccessful translation to clinical application, there is still ongoing research to find suitable compounds to manipulate MDR1 function and potentially overturn multidrug resistance. In the present study, we demonstrate that novel DHT-derived A-ring-fused arylpyrimidinone derivatives, based on their acetylation status, can inhibit MDR1 efflux activity in MDR1 overexpressing multidrug-resistant breast adenocarcinoma cells. Strikingly, all derivatives carrying an acetoxy group on the sterane d-ring were highly potent in hindering Rhodamine 123 export via MDR1, however deacetylated molecules were not capable to exert a similar effect on multidrug resistant cancer cells. The possible molecular and cellular mechanisms underlying the efflux pump inhibiting function of acetylated derivatives were dissected using the most potent MDR1 inhibitor, compound 10g and its deacetylated counterpart (11g). Importantly, molecule 10g was able to sensitize drug resistant cells to doxorubicin-induced apoptosis, further verifying the highly advantageous nature of efflux pump inhibition upon chemotherapy. Our experiments also revealed that neither mitochondrial damage, nor MDR1 gene regulation could lay behind the MDR1 inhibitory function of compound 10g. Molecular docking studies were carried out to analyze the interactions of 10g and 11g with MDR1, however no significant differences in their binding properties were observed. Nevertheless, our results indicate that the ER stress inducing potential of molecule 10g might be the fundamental mechanism behind its inhibitory action on MDR1. With additional studies, our work can yield a structural platform for a new generation of small molecule MDR1 inhibitors to sensitize drug resistant cancer cells and at the same time it elucidates the exemplary involvement of endoplasmic reticulum stress in the molecular events to defeat multidrug resistance.

Human papillomavirus and Its Association with Other Sexually Transmitted Coinfection among Sexually Active Women from the Northeast of Brazil.

OBJECTIVE: To verify the association between HPV infection and the presence of coinfections (Chlamydia trachomatis, Trichomonas vaginalis, and Neisseria gonorrhoeae) in women in the state of Maranhão. METHODS: HPV-DNA detection was performed by the nested PCR, using the primers PGMY09/11 and GP + 5/GP + 6. For the identification of sexually transmitted agents, conventional PCR was performed using the following primers: KL1/KL2 (Chlamydia trachomatis), TVA5/TVA6 (Trichomonas vaginalis), and HO1/HO3 (Neisseria gonorrhoeae). DNA-HPV positive samples were subjected to automated sequencing for genotyping. RESULTS: Among the 353 women evaluated, 204 (57.8%) had HPV-DNA, of which 140 (68.6%) exhibited HPV/STIs, while 64 (31.4%) had the only HPV. T. vaginalis infection showed a positive association with HPV (p=0.003). Women without cervical lesions were predominant (327/92.6%); however, the largest number of lesions was reported in women who had HPV/coinfections (18/8.8%). Multiple regression analysis showed that both HPV only and the concomitant presence of HPV/STI were able to indicate the occurrence of epithelial lesions (R = 0.164; R2 = 0.027). CONCLUSION: The findings suggest that the presence of T. vaginalis can contribute to HPV infection, and HPV/IST association may influence the development of cervical intraepithelial lesions that are precursors of cervical cancer.

Decorin Protects Cardiac Myocytes against Simulated Ischemia/Reperfusion Injury.

Search for new cardioprotective therapies is of great importance since no cardioprotective drugs are available on the market. In line with this need, several natural biomolecules have been extensively tested for their potential cardioprotective effects. Previously, we have shown that biglycan, a member of a diverse group of small leucine-rich proteoglycans, enhanced the expression of cardioprotective genes and decreased ischemia/reperfusion-induced cardiomyocyte death via a TLR-4 dependent mechanism. Therefore, in the present study we aimed to test whether decorin, a small leucine-rich proteoglycan closely related to biglycan, could exert cardiocytoprotection and to reveal possible downstream signaling pathways. Methods: Primary cardiomyocytes isolated from neonatal and adult rat hearts were treated with 0 (Vehicle), 1, 3, 10, 30 and 100 nM decorin as 20 h pretreatment and maintained throughout simulated ischemia and reperfusion (SI/R). In separate experiments, to test the mechanism of decorin-induced cardio protection, 3 nM decorin was applied in combination with inhibitors of known survival pathways, that is, the NOS inhibitor L-NAME, the PKG inhibitor KT-5823 and the TLR-4 inhibitor TAK-242, respectively. mRNA expression changes were measured after SI/R injury. Results: Cell viability of both neonatal and adult cardiomyocytes was significantly decreased due to SI/R injury. Decorin at 1, 3 and 10 nM concentrations significantly increased the survival of both neonatal and adult myocytes after SI/R. At 3nM (the most pronounced protective concentration), it had no effect on apoptotic rate of neonatal cardiac myocytes. No one of the inhibitors of survival pathways (L-NAME, KT-5823, TAK-242) influenced the cardiocytoprotective effect of decorin. MYND-type containing 19 (Zmynd19) and eukaryotic translation initiation factor 4E nuclear import factor 1 (Eif4enif1) were significantly upregulated due to the decorin treatment. In conclusion, this is the first demonstration that decorin exerts a direct cardiocytoprotective effect possibly independent of NO-cGMP-PKG and TLR-4 dependent survival signaling.

Hypercholesterolemia Interferes with Induction of miR-125b-1-3p in Preconditioned Hearts.

Ischemic preconditioning (IPre) reduces ischemia/reperfusion (I/R) injury in the heart. The non-coding microRNA miR-125b-1-3p has been demonstrated to play a role in the mechanism of IPre. Hypercholesterolemia is known to attenuate the cardioprotective effect of preconditioning; nevertheless, the exact underlying mechanisms are not clear. Here we investigated, whether hypercholesterolemia influences the induction of miR-125b-1-3p by IPre. Male Wistar rats were fed with a rodent chow supplemented with 2% cholesterol and 0.25% sodium-cholate hydrate for 8 weeks to induce high blood cholesterol levels. The hearts of normo- and hypercholesterolemic animals were then isolated and perfused according to Langendorff, and were subjected to 35 min global ischemia and 120 min reperfusion with or without IPre (3 × 5 min I/R cycles applied before index ischemia). IPre significantly reduced infarct size in the hearts of normocholesterolemic rats; however, IPre was ineffective in the hearts of hypercholesterolemic animals. Similarly, miR-125b-1-3p was upregulated by IPre in hearts of normocholesterolemic rats, while in the hearts of hypercholesterolemic animals IPre failed to increase miR-125b-1-3p significantly. Phosphorylation of cardiac Akt, ERK, and STAT3 was not significantly different in any of the groups at the end of reperfusion. Based on these results we propose here that hypercholesterolemia attenuates the upregulation of miR-125b-1-3p by IPre, which seems to be associated with the loss of cardioprotection.

Selective Heart Irradiation Induces Cardiac Overexpression of the Pro-hypertrophic miR-212.

Background: A deleterious, late-onset side effect of thoracic radiotherapy is the development of radiation-induced heart disease (RIHD). It covers a spectrum of cardiac pathology including also heart failure with preserved ejection fraction (HFpEF) characterized by left ventricular hypertrophy (LVH) and diastolic dysfunction. MicroRNA-212 (miR-212) is a crucial regulator of pathologic LVH via FOXO3-mediated pathways in pressure-overload-induced heart failure. We aimed to investigate whether miR-212 and its selected hypertrophy-associated targets play a role in the development of RIHD. Methods: RIHD was induced by selective heart irradiation (50 Gy) in a clinically relevant rat model. One, three, and nineteen weeks after selective heart irradiation, transthoracic echocardiography was performed to monitor cardiac morphology and function. Cardiomyocyte hypertrophy and fibrosis were assessed by histology at week 19. qRT-PCR was performed to measure the gene expression changes of miR-212 and forkhead box O3 (FOXO3) in all follow-up time points. The cardiac transcript level of other selected hypertrophy-associated targets of miR-212 including extracellular signal-regulated kinase 2 (ERK2), myocyte enhancer factor 2a (MEF2a), AMP-activated protein kinase, (AMPK), heat shock protein 40 (HSP40), sirtuin 1, (SIRT1), calcineurin A-alpha and phosphatase and tensin homolog (PTEN) were also measured at week 19. Cardiac expression of FOXO3 and phospho-FOXO3 were investigated at the protein level by Western blot at week 19. Results: In RIHD, diastolic dysfunction was present at every time point. Septal hypertrophy developed at week 3 and a marked LVH with interstitial fibrosis developed at week 19 in the irradiated hearts. In RIHD, cardiac miR-212 was overexpressed at week 3 and 19, and FOXO3 was repressed at the mRNA level only at week 19. In contrast, the total FOXO3 protein level failed to decrease in response to heart irradiation at week 19. Other selected hypertrophy-associated target genes failed to change at the mRNA level in RIHD at week 19. Conclusions: LVH in RIHD was associated with cardiac overexpression of miR-212. However, miR-212 seems to play a role in the development of LVH via FOXO3-independent mechanisms in RIHD. As a central regulator of pathologic remodeling, miR-212 might become a novel target for RIHD-induced LVH and heart failure.

L-Alpha-glycerylphosphorylcholine can be cytoprotective or cytotoxic in neonatal rat cardiac myocytes: a double-edged sword phenomenon.

L-Alpha-glycerylphosphorylcholine (GPC) is a widely used food supplement. GPC has been shown to exert beneficial effects in several organs; however, the cardiac effects of GPC have yet to be investigated. The aim of the present study was therefore to map out the effects of GPC on cardiac myocytes, with or without ischemia-reperfusion insult. Neonatal rat cardiac myocytes were treated with GPC at 1, 10, 80, and 100 µM concentrations for 15 min, 3 h, or 24 h, respectively. Cell viability by calcein assay and the degree of oxidative stress by DHE (superoxide level) and H2DCF (total ROS accumulation) staining were measured. In separate experiments, cardiomyocytes were pre-treated with the optimal concentration of GPC for 3 h and then cells were exposed to 4 h of simulated ischemia followed by 2 h of reperfusion (SI/R). Cell viability was measured at the end of the SI/R protocol. In normoxic conditions, the 15-min and the 3-h GPC treatment did not affect cell viability, total ROS, and superoxide levels. Under SI/R conditions, the 3-h GPC treatment protected the cardiac myocytes from SI/R-induced cell death and did not alter the level of oxidative stress. The 24-h GPC treatment in normoxic conditions resulted in significant cell death and increased oxidative stress at each concentration. Here we provide the first evidence for the cytoprotective effect of short-term GPC treatment. However, long-term administration of GPC may exert cytotoxicity in a wide concentration range in cardiac myocytes. These results may draw attention to a comprehensive cardiac safety protocol for the testing of GPC.

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.

Mechanisms and Modulation of Oxidative/Nitrative Stress in Type 4 Cardio-Renal Syndrome and Renal Sarcopenia.

Chronic kidney disease (CKD) is a public health problem and a recognized risk factor for cardiovascular diseases (CVD). CKD could amplify the progression of chronic heart failure leading to the development of type 4 cardio-renal syndrome (T4CRS). The severity and persistence of heart failure are strongly associated with mortality risk in T4CRS. CKD is also a catabolic state leading to renal sarcopenia which is characterized by the loss of skeletal muscle strength and physical function. Renal sarcopenia also promotes the development of CVD and increases the mortality in CKD patients. In turn, heart failure developed in T4CRS could result in chronic muscle hypoperfusion and metabolic disturbances leading to or aggravating the renal sarcopenia. The interplay of multiple factors (e.g., comorbidities, over-activated renin-angiotensin-aldosterone system [RAAS], sympathetic nervous system [SNS], oxidative/nitrative stress, inflammation, etc.) may result in the progression of T4CRS and renal sarcopenia. Among these factors, oxidative/nitrative stress plays a crucial role in the complex pathomechanism and interrelationship between T4CRS and renal sarcopenia. In the heart and skeletal muscle, mitochondria, nicotinamide adenine dinucleotide phosphate (NADPH) oxidases, uncoupled nitric oxide synthase (NOS) and xanthine oxidase are major ROS sources producing superoxide anion (O2·-) and/or hydrogen peroxide (H2O2). O2·- reacts with nitric oxide (NO) forming peroxynitrite (ONOO-) which is a highly reactive nitrogen species (RNS). High levels of ROS/RNS cause lipid peroxidation, DNA damage, interacts with both DNA repair enzymes and transcription factors, leads to the oxidation/nitration of key proteins involved in contractility, calcium handling, metabolism, antioxidant defense mechanisms, etc. It also activates the inflammatory response, stress signals inducing cardiac hypertrophy, fibrosis, or cell death via different mechanisms (e.g., apoptosis, necrosis) and dysregulates autophagy. Therefore, the thorough understanding of the mechanisms which lead to perturbations in oxidative/nitrative metabolism and its relationship with pro-inflammatory, hypertrophic, fibrotic, cell death and other pathways would help to develop strategies to counteract systemic and tissue oxidative/nitrative stress in T4CRS and renal sarcopenia. In this review, we also focus on the effects of some well-known and novel pharmaceuticals, nutraceuticals, and physical exercise on cardiac and skeletal muscle oxidative/nitrative stress in T4CRS and renal sarcopenia.

JDP2 overexpression provokes cardiac dysfunction in mice.

The transcriptional regulator JDP2 (Jun dimerization protein 2) has been identified as a prognostic marker for patients to develop heart failure after myocardial infarction. We now performed in vivo studies on JDP2-overexpressing mice, to clarify the impact of JDP2 on heart failure progression. Therefore, during birth up to the age of 4 weeks cardiac-specific JDP2 overexpression was prevented by doxycycline feeding in transgenic mice. Then, JDP2 overexpression was started. Already after 1 week, cardiac function, determined by echocardiography, decreased which was also resembled on the cardiomyocyte level. After 5 weeks blood pressure declined, ejection fraction and cardiac output was reduced and left ventricular dilatation developed. Heart weight/body weight, and mRNA expression of ANP, inflammatory marker genes, collagen and fibronectin increased. Collagen 1 protein expression increased, and fibrosis developed. As an additional sign of elevated extracellular matrix remodeling, matrix metalloproteinase 2 activity increased in JDP2 mice. Thus, JDP2 overexpression is deleterious to heart function in vivo. It can be concluded that JDP2 overexpression provokes cardiac dysfunction in adult mice that is accompanied by hypertrophy and fibrosis. Thus, induction of JDP2 is a maladaptive response contributing to heart failure development.

Development of Matrix Metalloproteinase-2 Inhibitors for Cardioprotection.

The objective of our present study is to develop novel inhibitors for MMP-2 for acute cardioprotection. In a series of pilot studies, novel substituted carboxylic acid derivatives were synthesized based on imidazole and thiazole scaffolds and then tested in a screeening cascade for MMP inhibition. We found that the MMP-inhibiting effects of imidazole and thiazole carboxylic acid-based compounds are superior in efficacy in comparison to the conventional hydroxamic acid derivatives of the same molecules. Based on these results, a 568-membered focused library of imidazole and thiazole compounds was generated in silico and then the library members were docked to the 3D model of MMP-2 followed by an in vitro medium throughput screening (MTS) based on a fluorescent assay employing MMP-2 catalytic domain. Altogether 45 compounds showed a docking score of >70, from which 30 compounds were successfully synthesized. Based on the MMP-2 inhibitory tests using gelatin zymography, 7 compounds were then selected and tested in neonatal rat cardiac myocytes subjected to simulated I/R injury. Six compounds showed significant cardio-cytoprotecion and the most effective compound (MMPI-1154) significantly decreased infarct size when applied at 1 µM in an ex vivo model for acute myocardial infarction. This is the first demonstration that imidazole and thiazole carboxylic acid-based compounds are more efficacious MMP-2 inhibitor than their hydroxamic acid derivatives. MMPI-1154 is a promising novel cardio-cytoprotective imidazole-carboxylic acid MMP-2 inhibitor lead candidate for the treatment of acute myocardial infarction.

Transcriptomic alterations in the heart of non-obese type 2 diabetic Goto-Kakizaki rats.

BACKGROUND: There is a spectacular rise in the global prevalence of type 2 diabetes mellitus (T2DM) due to the worldwide obesity epidemic. However, a significant proportion of T2DM patients are non-obese and they also have an increased risk of cardiovascular diseases. As the Goto-Kakizaki (GK) rat is a well-known model of non-obese T2DM, the goal of this study was to investigate the effect of non-obese T2DM on cardiac alterations of the transcriptome in GK rats. METHODS: Fasting blood glucose, serum insulin and cholesterol levels were measured at 7, 11, and 15 weeks of age in male GK and control rats. Oral glucose tolerance test and pancreatic insulin level measurements were performed at 11 weeks of age. At week 15, total RNA was isolated from the myocardium and assayed by rat oligonucleotide microarray for 41,012 genes, and then expression of selected genes was confirmed by qRT-PCR. Gene ontology and protein-protein network analyses were performed to demonstrate potentially characteristic gene alterations and key genes in non-obese T2DM. RESULTS: Fasting blood glucose, serum insulin and cholesterol levels were significantly increased, glucose tolerance and insulin sensitivity were significantly impaired in GK rats as compared to controls. In hearts of GK rats, 204 genes showed significant up-regulation and 303 genes showed down-regulation as compared to controls according to microarray analysis. Genes with significantly altered expression in the heart due to non-obese T2DM includes functional clusters of metabolism (e.g. Cyp2e1, Akr1b10), signal transduction (e.g. Dpp4, Stat3), receptors and ion channels (e.g. Sln, Chrng), membrane and structural proteins (e.g. Tnni1, Mylk2, Col8a1, Adam33), cell growth and differentiation (e.g. Gpc3, Jund), immune response (e.g. C3, C4a), and others (e.g. Lrp8, Msln, Klkc1, Epn3). Gene ontology analysis revealed several significantly enriched functional inter-relationships between genes influenced by non-obese T2DM. Protein-protein interaction analysis demonstrated that Stat is a potential key gene influenced by non-obese T2DM. CONCLUSIONS: Non-obese T2DM alters cardiac gene expression profile. The altered genes may be involved in the development of cardiac pathologies and could be potential therapeutic targets in non-obese T2DM.

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.