Histone methyltransferase Smyd1 regulates mitochondrial energetics in the heart.

Smyd1, a muscle-specific histone methyltransferase, has established roles in skeletal and cardiac muscle development, but its role in the adult heart remains poorly understood. Our prior work demonstrated that cardiac-specific deletion of Smyd1 in adult mice (Smyd1-KO) leads to hypertrophy and heart failure. Here we show that down-regulation of mitochondrial energetics is an early event in these Smyd1-KO mice preceding the onset of structural abnormalities. This early impairment of mitochondrial energetics in Smyd1-KO mice is associated with a significant reduction in gene and protein expression of PGC-1α, PPARα, and RXRα, the master regulators of cardiac energetics. The effect of Smyd1 on PGC-1α was recapitulated in primary cultured rat ventricular myocytes, in which acute siRNA-mediated silencing of Smyd1 resulted in a greater than twofold decrease in PGC-1α expression without affecting that of PPARα or RXRα. In addition, enrichment of histone H3 lysine 4 trimethylation (a mark of gene activation) at the PGC-1α locus was markedly reduced in Smyd1-KO mice, and Smyd1-induced transcriptional activation of PGC-1α was confirmed by luciferase reporter assays. Functional confirmation of Smyd1's involvement showed an increase in mitochondrial respiration capacity induced by overexpression of Smyd1, which was abolished by siRNA-mediated PGC-1α knockdown. Conversely, overexpression of PGC-1α rescued transcript expression and mitochondrial respiration caused by silencing Smyd1 in cardiomyocytes. These findings provide functional evidence for a role of Smyd1, or any member of the Smyd family, in regulating cardiac energetics in the adult heart, which is mediated, at least in part, via modulating PGC-1α.

Conduction in the right and left ventricle is differentially regulated by protein kinases and phosphatases: implications for arrhythmogenesis.

The "stress" kinases cAMP-dependent protein kinase (PKA) and calcium/calmodulin-dependent protein kinase II (CaMKII), phosphorylate the Na+ channel Nav1.5 subunit to regulate its function. However, how the channel regulation translates to ventricular conduction is poorly understood. We hypothesized that the stress kinases positively and differentially regulate conduction in the right (RV) and the left (LV) ventricles. We applied the CaMKII blocker KN93 (2.75 µM), PKA blocker H89 (10 µM), and broad-acting phosphatase blocker calyculin (30 nM) in rabbit hearts paced at a cycle length (CL) of 150-8,000 ms. We used optical mapping to determine the distribution of local conduction delays (inverse of conduction velocity). Control hearts exhibited constant and uniform conduction at all tested CLs. Calyculin (15-min perfusion) accelerated conduction, with greater effect in the RV (by 15.3%) than in the LV (by 4.1%; P < 0.05). In contrast, both KN93 and H89 slowed down conduction in a chamber-, time-, and CL-dependent manner, with the strongest effect in the RV outflow tract (RVOT). Combined KN93 and H89 synergistically promoted conduction slowing in the RV (KN93: 24.7%; H89: 29.9%; and KN93 + H89: 114.2%; P = 0.0016) but not the LV. The progressive depression of RV conduction led to conduction block and reentrant arrhythmias. Protein expression levels of both the CaMKII-δ isoform and the PKA catalytic subunit were higher in the RVOT than in the apical LV (P < 0.05). Thus normal RV conduction requires a proper balance between kinase and phosphatase activity. Dysregulation of this balance due to pharmacological interventions or disease is potentially proarrhythmic. NEW & NOTEWORTHY We show that uniform ventricular conduction requires a precise physiological balance of the activities of calcium/calmodulin-dependent protein kinase II (CaMKII), PKA, and phosphatases, which involves region-specific expression of CaMKII and PKA. Inhibiting CaMKII and/or PKA activity elicits nonuniform conduction depression, with the right ventricle becoming vulnerable to the development of conduction disturbances and ventricular fibrillation/ventricular tachycardia.

Mesenchymal stromal cells and TGF-ß1 in tracheal aspirate of premature infants: early predictors for bronchopulmonary dysplasia?

Background The pathogenesis of bronchopulmonary dysplasia (BPD) includes arrest of alveolar septation and enhanced fibrosis. We hypothesized that mesenchymal stromal cells (MSC) and transforming growth factor-ß1 (TGF-ß1) in tracheal aspirates of mechanically ventilated premature infants differ in BPD and non-BPD infants. Methods Tracheal aspirates were collected during the first week of life. Mononuclear cells were separated, cultured and immunophenotyped by flow cytometry. MSCs colony/cluster ratio was calculated as an index for dysplastic potentials. TGF-ß1 was assessed by enzyme-linked immunosorbent assay (ELISA). Setting: Neonatal intensive care unit. Patients Premature infants at risk for BPD. Results A total of 121 preterm infants were enrolled; 27 of them died and among the 94 survivors 23 infants had BPD. MSCs were identified in younger [gestational age (GA): 30.9±1.7 vs. 31.8±1.8, P=0.025] and smaller [birth weight (BW): 1.3±0.28 vs. 1.44±0.37 kg, P=0.04] infants with lower Apgar scores. The recovery rate of MSCs in BPD and non-BPD groups did not differ. BPD group had significantly smaller colony/cluster ratio compared to non-BPD (0.97 vs. 4.25, P=0.002). TGF-ß1 was significantly greater in BPD infants (4173.9±864.3 vs. 3705.8±540.5 pg/mL, P=0.021). Conclusion Infants with BPD had different MSCs morphology and greater TGF-ß1 expression. The pathogenesis for these morphological changes of resident lung MSCs needs further studying.

A De Novo Optimized Cell-Free System for the Expression of Soluble and Active Human Tumor Necrosis Factor-Alpha.

Cell-free (in vitro) expression is a robust alternative platform to the cell-based (in vivo) system for recombinant protein production. Tumor necrosis factor-alpha (TNF-α) is an effective pro-inflammatory cytokine with pleiotropic effects. The aim of the current study was de novo optimized expression of soluble and active human TNF-α by an in vitro method in an E. coli-based cell-free protein synthesis (CFPS) system and its biological activity evaluation. The codon-optimized synthetic human TNF-α gene was constructed by a two-step PCR, cloned into pET101/D-TOPO vector and then expressed by the E. coli CFPS system. Cell-free expression of the soluble protein was optimized using a response surface methodology (RSM). The anticancer activity of purified human TNF-α was assessed against three human cancer cell lines: Caco-2, HepG-2 and MCF-7. Data from RSM revealed that the lowest value (7.2 µg/mL) of cell-free production of recombinant human TNF-α (rhTNF-α) was obtained at a certain incubation time (6 h) and incubation temperature (20 °C), while the highest value (350 µg/mL) was recorded at 4 h and 35 °C. This rhTNF-α showed a significant anticancer potency. Our findings suggest a cell-free expression system as an alternative platform for producing soluble and functionally active recombinant TNF-α for further research and clinical trials.

PERM1 regulates energy metabolism in the heart via ERRα/PGC-1α axis.

Aims: PERM1 is a striated muscle-specific regulator of mitochondrial bioenergetics. We previously demonstrated that PERM1 is downregulated in the failing heart and that PERM1 positively regulates metabolic genes known as targets of the transcription factor ERRα and its coactivator PGC-1α in cultured cardiomyocytes. The aims of this study were to determine the effect of loss of PERM1 on cardiac function and energetics using newly generated Perm1-knockout (Perm1 -/-) mice and to investigate the molecular mechanisms of its transcriptional control. Methods and results: Echocardiography showed that ejection fraction and fractional shortening were lower in Perm1 -/- mice than in wild-type mice (both p < 0.05), and the phosphocreatine-to-ATP ratio was decreased in Perm1 -/- hearts (p < 0.05), indicating reduced contractile function and energy reserves of the heart. Integrated proteomic and metabolomic analyses revealed downregulation of oxidative phosphorylation and upregulation of glycolysis and polyol pathways in Perm1 -/- hearts. To examine whether PERM1 regulates energy metabolism through ERRα, we performed co-immunoprecipitation assays, which showed that PERM1 bound to ERRα in cardiomyocytes and the mouse heart. DNA binding and reporter gene assays showed that PERM1 was localized to and activated the ERR target promoters partially through ERRα. Mass spectrometry-based screening in cardiomyocytes identified BAG6 and KANK2 as potential PERM1's binding partners in transcriptional regulation. Mammalian one-hybrid assay, in which PERM1 was fused to Gal4 DNA binding domain, showed that the recruitment of PERM1 to a gene promoter was sufficient to activate transcription, which was blunted by silencing of either PGC-1α, BAG6, or KANK2. Conclusion: This study demonstrates that PERM1 is an essential regulator of cardiac energetics and function and that PERM1 is a novel transcriptional coactivator in the ERRα/PGC-1α axis that functionally interacts with BAG6 and KANK2.

Efficacy of single phototherapy with low-cost reflective sheets versus single phototherapy alone in mild-to-moderate unconjugated hyperbilirubinaemia in full-term neonates.

BACKGROUND: An economical alternative method of increasing the light intensity of phototherapy for neonatal jaundice is the use of reflective sheets placed on the sides of the incubator. AIM: To determine whether reflective sheets in addition to phototherapy increase the reduction of bilirubin levels and the duration of hospital stay. METHODS: The study was undertaken in the neonatal intensive care unit of Cairo University Children's Hospital. There were two groups: a study group of 90 full-term neonates with neonatal jaundice who received single phototherapy in incubators covered with white plastic reflective sheets and a control group of 90 full-term neonates with neonatal jaundice who received single phototherapy without the reflective sheets. RESULTS: The mean (SD) rate of bilirubin decline in the first 24 hours of phototherapy was greater in the study group [3.7 (0.86) µmol/L/hr] than in the control group [2.2 (0.14) µmol/L/hr] (p<0.001). CONCLUSION: Use of reflective sheets decreases the total duration of phototherapy and the cost and duration of hospitalisation without any added complications.Abbreviations: AAP: American Academy of Pediatrics; HIDS: high-intensity double-surface; LMIC: low- and middle-income countries; MCTP: mirror-covered tunnel phototherapy; NICU: neonatal intensive care unit; TSB: total serum bilirubin.

Multiple Levels of PGC-1α Dysregulation in Heart Failure.

Metabolic adaption is crucial for the heart to sustain its contractile activity under various physiological and pathological conditions. At the molecular level, the changes in energy demand impinge on the expression of genes encoding for metabolic enzymes. Among the major components of an intricate transcriptional circuitry, peroxisome proliferator-activated receptor γ coactivator 1 alpha (PGC-1α) plays a critical role as a metabolic sensor, which is responsible for the fine-tuning of transcriptional responses to a plethora of stimuli. Cumulative evidence suggests that energetic impairment in heart failure is largely attributed to the dysregulation of PGC-1α. In this review, we summarize recent studies revealing how PGC-1α is regulated by a multitude of mechanisms, operating at different regulatory levels, which include epigenetic regulation, the expression of variants, post-transcriptional inhibition, and post-translational modifications. We further discuss how the PGC-1α regulatory cascade can be impaired in the failing heart.

Perm1 regulates cardiac energetics as a downstream target of the histone methyltransferase Smyd1.

The transcriptional regulatory machinery in mitochondrial bioenergetics is complex and is still not completely understood. We previously demonstrated that the histone methyltransferase Smyd1 regulates mitochondrial energetics. Here, we identified Perm1 (PPARGC-1 and ESRR-induced regulator, muscle specific 1) as a downstream target of Smyd1 through RNA-seq. Chromatin immunoprecipitation assay showed that Smyd1 directly interacts with the promoter of Perm1 in the mouse heart, and this interaction was significantly reduced in mouse hearts failing due to pressure overload for 4 weeks, where Perm1 was downregulated (24.4 ± 5.9% of sham, p<0.05). Similarly, the Perm1 protein level was significantly decreased in patients with advanced heart failure (55.2 ± 13.1% of donors, p<0.05). Phenylephrine (PE)-induced hypertrophic stress in cardiomyocytes also led to downregulation of Perm1 (55.7 ± 5.7% of control, p<0.05), and adenovirus-mediated overexpression of Perm1 rescued PE-induced downregulation of estrogen-related receptor alpha (ERRα), a key transcriptional regulator of mitochondrial energetics, and its target gene, Ndufv1 (Complex I). Pathway enrichment analysis of cardiomyocytes in which Perm1 was knocked-down by siRNA (siPerm1), revealed that the most downregulated pathway was metabolism. Cell stress tests using the Seahorse XF analyzer showed that basal respiration and ATP production were significantly reduced in siPerm1 cardiomyocytes (40.7% and 23.6% of scrambled-siRNA, respectively, both p<0.05). Luciferase reporter gene assay further revealed that Perm1 dose-dependently increased the promoter activity of the ERRα gene and known target of ERRα, Ndufv1 (Complex I). Overall, our study demonstrates that Perm1 is an essential regulator of cardiac energetics through ERRα, as part of the Smyd1 regulatory network.

Diagnostic Role of CD64 on Different Immune Cells in Early Diagnosis of Neonatal Sepsis.

Neonatal sepsis is an important cause of morbidity and mortality among neonates. Early diagnosis leads to better prognosis. CD64 can be used as an early marker to detect neonatal sepsis with promising results. Advances in flow cytometry have made it possible to assess its level on different white blood cells rapidly, precisely and with minimal blood. The aim of the present work was to assess the role of CD64 expressed on neutrophils, monocytes and lymphocytes in establishing diagnosis of neonatal sepsis as well as to compare its diagnostic value with CRP in diagnosis of neonatal sepsis. This study was performed on 80 neonates divided into 60 cases of neonatal sepsis (48 case of clinical sepsis & 12 cases of lab confirmed sepsis) and 20 healthy control neonates. Cases and controls were subjected to history taking, clinical examination and lab investigations in the form of CBC, CRP, CD64 expression on neutrophils, monocytes & lymphocytes and Blood culture (for cases only). Our study showed that CD64 expression on WBCs increases significantly in neonates with neonatal sepsis (P < 0.05) in comparison to controls. Results also showed that neutrophils CD 64 is the most sensitive indicator for detection of sepsis (sensitivity=95%, NPV=78.57%) at a cut off value of 0.18%, whereas CRP has shown the best specificity at a cut off value 3mg/Ml (specificity= 85%). In conclusion, neutrophil CD64 is superior to monocyte and lymphocyte CD64 and serum CRP in diagnosis of neonatal sepsis.

Functionality of intrinsic disorder in tumor necrosis factor-α and its receptors.

Tumor necrosis factor-α (TNF-α) is a pleiotropic inflammatory cytokine that exerts potent cytotoxic effects on solid tumor cells, while not affecting their normal counterparts. It is also known that TNF-α exerts many of its biological functions via interaction with specific receptors. To understand the potential roles of intrinsic disorder in the functioning of this important cytokine, we explored the peculiarities of intrinsic disorder distribution in human TNF-α and its homologs from various species, ranging from zebrafish to chimpanzee. We also studied the peculiarities of intrinsic disorder distribution in human TNF-α receptors, TNFR1 and TNFR2. Analysis revealed that cytoplasmic domains of TNF-α and its receptors are expected to be highly disordered. Furthermore, although the sequence identities of analyzed TNF-α homologs range from 99.57% (between human and chimpanzee proteins) to 22.33% (between frog and fish proteins), their intrinsic disorder profiles are characterized by a remarkable similarity. These observations indicate that the peculiarities of distribution of the intrinsic disorder propensity within the amino acid sequences are evolutionary conserved, and therefore could be of functional importance for this family of proteins. We also show that disordered and flexible regions of human TNF-α and its TNFR1 and TNFR2 receptors are crucial for some of their biological activities.