Polycystin-1 loss of function increases susceptibility to atrial fibrillation through impaired DNA damage response.

BACKGROUND: The increasing prevalence of atrial fibrillation (AF) and chronic kidney diseases highlights the need for a deeper comprehension of the molecular mechanisms linking them. Mutations in PKD1, the gene encoding Polycystin-1 (PKD1 or PC1), account for 85% of autosomal dominant polycystic kidney disease (ADPKD) cases. This disease often includes cardiac complications such as AF. In cardiomyocytes, PC1 deletion reduces hypertrophic response to pressure overload but promotes baseline ventricular dysfunction, while deletion in fibroblasts ameliorates post-myocardial infarction fibrosis. Despite its known cardiac impact, the role of PC1 in atrial cardiomyocytes and arrhythmias is less understood. Here, we sought to investigate the role of PC1 in AF. METHODS: We used intracardiac programmed stimulation and optical mapping to evaluate AF inducibility in two mouse models, Pkd1 R3277C, which recapitulates human ADPKD progression, and cardiomyocyte-specific Pkd1 deletion, and their respective controls. Isolated adult mouse atrial cardiomyocytes, human iPSC-derived atrial cardiomyocytes (hiPSC-aCM), and HL-1 cells served as in vitro cellular models. Molecular mechanisms were evaluated using optical mapping and molecular and biochemical approaches. RESULTS: Loss-of-function PC1 mutations significantly increased AF susceptibility in vivo and facilitated local reentry in ex vivo left atrial appendages. Comprehensive in vitro experiments supported a direct effect of PC1 in atrial cardiomyocytes. PC1-deficient monolayers exhibited increased arrhythmic events, escalating into reentrant spiral waves post-tachypacing. Transcriptomics analysis revealed PC1-dependent regulation of DNA repair, with PC1 deficiency leading to increased DNA damage under stress. PARP1 inhibitors or nicotinamide riboside, which counteract DNA damage-related metabolic consequences, reduced in vitro arrhythmias PC1-deficient monolayers. Overexpression of the C-terminus of PC1 had the opposite effects in DNA repair genes, suggesting its regulatory effects in atrial cardiomyocytes through retinoblastoma/E2F. Analyses of human atrial tissue from non-ADPKD patients showed reduced levels of mature PC1, suggesting a broader relevance of impaired PC1 in AF. CONCLUSIONS: Impaired PC1 increases in vivo AF inducibility under programmed electrical stimulation and promotes in vitro arrhythmias in hiPSC-aCM and HL-1 cells. Our findings indicate that PC1 protects against DNA damage to reduce AF susceptibility.

Folate as a potential treatment for lethal ventricular arrhythmias in TANGO2-deficiency disorder.

TANGO2-deficiency disorder (TDD) is an autosomal-recessive genetic disease caused by biallelic loss-of-function variants in the TANGO2 gene. TDD-associated cardiac arrhythmias are recalcitrant to standard antiarrhythmic medications and constitute the leading cause of death. Disease modeling for TDD has been primarily carried out using human dermal fibroblast and, more recently, in Drosophila by multiple research groups. No human cardiomyocyte system has been reported, which greatly hinders the investigation and understanding of TDD-associated arrhythmias. Here, we established potentially novel patient-derived induced pluripotent stem cell differentiated cardiomyocyte (iPSC-CM) models that recapitulate key electrophysiological abnormalities in TDD. These electrophysiological abnormalities were rescued in iPSC-CMs with either adenoviral expression of WT-TANGO2 or correction of the pathogenic variant using CRISPR editing. Our natural history study in patients with TDD suggests that the intake of multivitamin/B complex greatly diminished the risk of cardiac crises in patients with TDD. In agreement with the clinical findings, we demonstrated that high-dose folate (vitamin B9) virtually abolishes arrhythmias in TDD iPSC-CMs and that folate's effect was blocked by the dihydrofolate reductase inhibitor methotrexate, supporting the need for intracellular folate to mediate antiarrhythmic effects. In summary, data from TDD iPSC-CM models together with clinical observations support the use of B vitamins to mitigate cardiac crises in patients with TDD, providing potentially life-saving treatment strategies during life-threatening events.

Histone demethylase KDM5 regulates cardiomyocyte maturation by promoting fatty acid oxidation, oxidative phosphorylation, and myofibrillar organization.

AIMS: Human pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) provide a platform to identify and characterize factors that regulate the maturation of CMs. The transition from an immature foetal to an adult CM state entails coordinated regulation of the expression of genes involved in myofibril formation and oxidative phosphorylation (OXPHOS) among others. Lysine demethylase 5 (KDM5) specifically demethylates H3K4me1/2/3 and has emerged as potential regulators of expression of genes involved in cardiac development and mitochondrial function. The purpose of this study is to determine the role of KDM5 in iPSC-CM maturation. METHODS AND RESULTS: KDM5A, B, and C proteins were mainly expressed in the early post-natal stages, and their expressions were progressively downregulated in the post-natal CMs and were absent in adult hearts and CMs. In contrast, KDM5 proteins were persistently expressed in the iPSC-CMs up to 60 days after the induction of myogenic differentiation, consistent with the immaturity of these cells. Inhibition of KDM5 by KDM5-C70 -a pan-KDM5 inhibitor, induced differential expression of 2372 genes, including upregulation of genes involved in fatty acid oxidation (FAO), OXPHOS, and myogenesis in the iPSC-CMs. Likewise, genome-wide profiling of H3K4me3 binding sites by the cleavage under targets and release using nuclease assay showed enriched of the H3K4me3 peaks at the promoter regions of genes encoding FAO, OXPHOS, and sarcomere proteins. Consistent with the chromatin and gene expression data, KDM5 inhibition increased the expression of multiple sarcomere proteins and enhanced myofibrillar organization. Furthermore, inhibition of KDM5 increased H3K4me3 deposits at the promoter region of the ESRRA gene and increased its RNA and protein levels. Knockdown of ESRRA in KDM5-C70-treated iPSC-CM suppressed expression of a subset of the KDM5 targets. In conjunction with changes in gene expression, KDM5 inhibition increased oxygen consumption rate and contractility in iPSC-CMs. CONCLUSION: KDM5 inhibition enhances maturation of iPSC-CMs by epigenetically upregulating the expressions of OXPHOS, FAO, and sarcomere genes and enhancing myofibril organization and mitochondrial function.

Polycystin-1 regulates cardiomyocyte mitophagy.

Polycystin-1 (PC1) is a transmembrane protein found in different cell types, including cardiomyocytes. Alterations in PC1 expression have been linked to mitochondrial damage in renal tubule cells and in patients with autosomal dominant polycystic kidney disease. However, to date, the regulatory role of PC1 in cardiomyocyte mitochondria is not well understood. The analysis of mitochondrial morphology from cardiomyocytes of heterozygous PC1 mice (PDK1+/- ) using transmission electron microscopy showed that cardiomyocyte mitochondria were smaller with increased mitochondria density and circularity. These parameters were consistent with mitochondrial fission. We knocked-down PC1 in cultured rat cardiomyocytes and human-induced pluripotent stem cells (iPSC)-derived cardiomyocytes to evaluate mitochondrial function and morphology. The results showed that downregulation of PC1 expression results in reduced protein levels of sub-units of the OXPHOS complexes and less functional mitochondria (reduction of mitochondrial membrane potential, mitochondrial respiration, and ATP production). This mitochondrial dysfunction activates the elimination of defective mitochondria by mitophagy, assessed by an increase of autophagosome adapter protein LC3B and the recruitment of the Parkin protein to the mitochondria. siRNA-mediated PC1 knockdown leads to a loss of the connectivity of the mitochondrial network and a greater number of mitochondria per cell, but of smaller sizes, which characterizes mitochondrial fission. PC1 silencing also deregulates the AKT-FoxO1 signaling pathway, which is involved in the regulation of mitochondrial metabolism, mitochondrial morphology, and processes that are part of cell quality control, such as mitophagy. Together, these data provide new insights about the controls that PC1 exerts on mitochondrial morphology and function in cultured cardiomyocytes dependent on the AKT-FoxO1 signaling pathway.

Xbp1s-FoxO1 axis governs lipid accumulation and contractile performance in heart failure with preserved ejection fraction.

Heart failure with preserved ejection fraction (HFpEF) is now the dominant form of heart failure and one for which no efficacious therapies exist. Obesity and lipid mishandling greatly contribute to HFpEF. However, molecular mechanism(s) governing metabolic alterations and perturbations in lipid homeostasis in HFpEF are largely unknown. Here, we report that cardiomyocyte steatosis in HFpEF is coupled with increases in the activity of the transcription factor FoxO1 (Forkhead box protein O1). FoxO1 depletion, as well as over-expression of the Xbp1s (spliced form of the X-box-binding protein 1) arm of the UPR (unfolded protein response) in cardiomyocytes each ameliorates the HFpEF phenotype in mice and reduces myocardial lipid accumulation. Mechanistically, forced expression of Xbp1s in cardiomyocytes triggers ubiquitination and proteasomal degradation of FoxO1 which occurs, in large part, through activation of the E3 ubiquitin ligase STUB1 (STIP1 homology and U-box-containing protein 1) a novel and direct transcriptional target of Xbp1s. Our findings uncover the Xbp1s-FoxO1 axis as a pivotal mechanism in the pathogenesis of cardiometabolic HFpEF and unveil previously unrecognized mechanisms whereby the UPR governs metabolic alterations in cardiomyocytes.

Epigenetic Reader BRD4 (Bromodomain-Containing Protein 4) Governs Nucleus-Encoded Mitochondrial Transcriptome to Regulate Cardiac Function.

BACKGROUND: BET (bromodomain and extraterminal) epigenetic reader proteins, in particular BRD4 (bromodomain-containing protein 4), have emerged as potential therapeutic targets in a number of pathological conditions, including cancer and cardiovascular disease. Small-molecule BET protein inhibitors such as JQ1 have demonstrated efficacy in reversing cardiac hypertrophy and heart failure in preclinical models. Yet, genetic studies elucidating the biology of BET proteins in the heart have not been conducted to validate pharmacological findings and to unveil potential pharmacological side effects. METHODS: By engineering a cardiomyocyte-specific BRD4 knockout mouse, we investigated the role of BRD4 in cardiac pathophysiology. We performed functional, transcriptomic, and mitochondrial analyses to evaluate BRD4 function in developing and mature hearts. RESULTS: Unlike pharmacological inhibition, loss of BRD4 protein triggered progressive declines in myocardial function, culminating in dilated cardiomyopathy. Transcriptome analysis of BRD4 knockout mouse heart tissue identified early and specific disruption of genes essential to mitochondrial energy production and homeostasis. Functional analysis of isolated mitochondria from these hearts confirmed that BRD4 ablation triggered significant changes in mitochondrial electron transport chain protein expression and activity. Computational analysis identified candidate transcription factors participating in the BRD4-regulated transcriptome. In particular, estrogen-related receptor α, a key nuclear receptor in metabolic gene regulation, was enriched in promoters of BRD4-regulated mitochondrial genes. CONCLUSIONS: In aggregate, we describe a previously unrecognized role for BRD4 in regulating cardiomyocyte mitochondrial homeostasis, observing that its function is indispensable to the maintenance of normal cardiac function.

FoxO1-Dio2 signaling axis governs cardiomyocyte thyroid hormone metabolism and hypertrophic growth.

Forkhead box O (FoxO) proteins and thyroid hormone (TH) have well established roles in cardiovascular morphogenesis and remodeling. However, specific role(s) of individual FoxO family members in stress-induced growth and remodeling of cardiomyocytes remains unknown. Here, we report that FoxO1, but not FoxO3, activity is essential for reciprocal regulation of types II and III iodothyronine deiodinases (Dio2 and Dio3, respectively), key enzymes involved in intracellular TH metabolism. We further show that Dio2 is a direct transcriptional target of FoxO1, and the FoxO1-Dio2 axis governs TH-induced hypertrophic growth of neonatal cardiomyocytes in vitro and in vivo. Utilizing transverse aortic constriction as a model of hemodynamic stress in wild-type and cardiomyocyte-restricted FoxO1 knockout mice, we unveil an essential role for the FoxO1-Dio2 axis in afterload-induced pathological cardiac remodeling and activation of TRα1. These findings demonstrate a previously unrecognized FoxO1-Dio2 signaling axis in stress-induced cardiomyocyte growth and remodeling and intracellular TH homeostasis.

Poliposis vesicular y riesgo de malignidad: una revisión sistemática y metaanálisis / Vesicular polyposis and risk of malignancy: a systematic review and meta-analysis

La prevalencia de poliposis vesicular en la población general oscila entre el 1.5% y 5.5%. Estudios han demostrado que el Ecuador hasta el 5,3% de las personas presentan esta característica. La progresión a cáncer de vesícula biliar es temida por una baja tasa de sobrevida de hasta menos del 4% a los 5 años; la incidencia de cáncer vesicular en Ecuador bordea 12,9/100.000 habitantes. El objetivo de este estudio fue determinar si existe una asociación entre el tamaño de los pólipos y el riesgo de malignidad. Metodología se realizó una búsqueda en PubMed, ScienceDirect, Google Scholar, ResearchGate y Virtual Health Library (VHL), para estudios de cohorte retrospectivos o prospectivos, que reportaran factores de riesgo de malignidad en pólipos vesiculares. Se realizó un metaanálisis para el tamaño de pólipo >10 mm vs <10mm y el riesgo de malignidad. Resultados 15 publicaciones se incluyeron en esta revisión. El tamaño medio de pólipos fue 11,6mm (DS: ±3,1mm), entre 10 de 15 estudios. El tamaño de los pólipos vesiculares fue el factor de riesgo más evaluado para malignidad, reportándose entre >10mm y ≥15mm. Una edad > 50 años se asoció con riesgo de malignidad en varios estudios. El metaanálisis para pólipos >10mm vs <10mm y riesgo de malignidad reportó un OR global de 13,4 (IC 95%: 11,456 a 26,431; p<0,001) (I2: 45,6%; IC 95%: 0,00 a 72,21; p=0,0424). Conclusiones el tamaño de pólipo >10mm se considera un factor de riesgo significativo para malignidad. El diagnóstico y tratamiento oportuno de esta patología contribuirá a la reducción de la mortalidad por cáncer de vesícula biliar.

The prevalence of vesicular polyposis in the general population ranges from 1.5% to 5.5%. Studies have shown that Ecuador up to 5.3% of people have this characteristic. Progression to gallbladder cancer is feared by a low survival rate of up to less than 4% at age 5; the incidence of vesicular cancer in Ecuador borders 12.9/100,000 inhabitants. The objective of this study was to determine whether there is an association between the size of the polyps and the risk of malignancy. Methodology A search was conducted in PubMed, ScienceDirect, Google Scholar, ResearchGate and Virtual Health Library (VHL), for retrospective or prospective cohort studies, which reported risk factors for malignancy in vesicular polyps. A meta-analysis was performed for the size of polyp >10 mm vs <10mm and the risk of malignancy. Results 15 publications were included in this review. The average polyp size was 11.6mm (DS: ±3.1mm), among 10 out of 15 studies. The size of the vesicular polyps was the most evaluated risk factor for malignancy, reporting between >10mm and ≥15mm. An age > 50 years was associated with risk of malignancy in several studies. Meta-analysis for polyps >10mm vs <10mm and risk of malignancy reported a global OR of 13.4 (95% CI: 11,456 to 26,431; p<0.001) (I2: 45.6%; 95% CI: 0.00 to 72.21; 0.0424). Conclusions the size of polyp >10mm is considered a significant risk factor for malignancy. Timely diagnosis and treatment of this pathology will contribute to the reduction of gallbladder cancer mortality.

Polycystin-2-dependent control of cardiomyocyte autophagy.

AIMS: Considerable evidence points to critical roles of intracellular Ca2+ homeostasis in the modulation and control of autophagic activity. Yet, underlying molecular mechanisms remain unknown. Mutations in the gene (pkd2) encoding polycystin-2 (PC2) are associated with autosomal dominant polycystic kidney disease (ADPKD), the most common inherited nephropathy. PC2 has been associated with impaired Ca2+ handling in cardiomyocytes and indirect evidence suggests that this protein may be involved in autophagic control. Here, we investigated the role for PC2 as an essential regulator of Ca2+ homeostasis and autophagy. METHODS AND RESULTS: Activation of autophagic flux triggered by mTOR inhibition either pharmacologically (rapamycin) or by means of nutrient depletion was suppressed in cells depleted of PC2. Moreover, cardiomyocyte-specific PC2 knockout mice (αMhc-cre;Pkd2F/F mice) manifested impaired autophagic flux in the setting of nutrient deprivation. Stress-induced autophagy was blunted by intracellular Ca2+ chelation using BAPTA-AM, whereas removal of extracellular Ca2+ had no effect, pointing to a role of intracellular Ca2+ homeostasis in stress-induced cardiomyocyte autophagy. To determine the link between stress-induced autophagy and PC2-induced Ca2+ mobilization, we over-expressed either wild-type PC2 (WT) or a Ca2+-channel deficient PC2 mutant (PC2-D509V). PC2 over-expression increased autophagic flux, whereas PC2-D509V expression did not. Importantly, autophagy induction triggered by PC2 over-expression was attenuated by BAPTA-AM, supporting a model of PC2-dependent control of autophagy through intracellular Ca2+. Furthermore, PC2 ablation was associated with impaired Ca2+ handling in cardiomyocytes marked by partial depletion of sarcoplasmic reticulum Ca2+ stores. Finally, we provide evidence that Ca2+-mediated autophagy elicited by PC2 is a mechanism conserved across multiple cell types. CONCLUSION: Together, this study unveils PC2 as a novel regulator of autophagy acting through control of intracellular Ca2+ homeostasis.

Down Syndrome Critical Region 1 Gene, Rcan1, Helps Maintain a More Fused Mitochondrial Network.

RATIONALE: The regulator of calcineurin 1 (RCAN1) inhibits CN (calcineurin), a Ca2+-activated protein phosphatase important in cardiac remodeling. In humans, RCAN1 is located on chromosome 21 in proximity to the Down syndrome critical region. The hearts and brains of Rcan1 KO mice are more susceptible to damage from ischemia/reperfusion (I/R); however, the underlying cause is not known. OBJECTIVE: Mitochondria are key mediators of I/R damage. The goal of these studies was to determine the impact of RCAN1 on mitochondrial dynamics and function. METHODS AND RESULTS: Using both neonatal and isolated adult cardiomyocytes, we show that, when RCAN1 is depleted, the mitochondrial network is more fragmented because of increased CN-dependent activation of the fission protein, DRP1 (dynamin-1-like). Mitochondria in RCAN1-depleted cardiomyocytes have reduced membrane potential, O2 consumption, and generation of reactive oxygen species, as well as a reduced capacity for mitochondrial Ca2+ uptake. RCAN1-depleted cardiomyocytes were more sensitive to I/R; however, pharmacological inhibition of CN, DRP1, or CAPN (calpains; Ca2+-activated proteases) restored protection, suggesting that in the absence of RCAN1, CAPN-mediated damage after I/R is greater because of a decrease in the capacity of mitochondria to buffer cytoplasmic Ca2+. Increasing RCAN1 levels by adenoviral infection was sufficient to enhance fusion and confer protection from I/R. To examine the impact of more modest, and biologically relevant, increases in RCAN1, we compared the mitochondrial network in induced pluripotent stem cells derived from individuals with Down syndrome to that of isogenic, disomic controls. Mitochondria were more fused, and O2 consumption was greater in the trisomic induced pluripotent stem cells; however, coupling efficiency and metabolic flexibility were compromised compared with disomic induced pluripotent stem cells. Depletion of RCAN1 from trisomic induced pluripotent stem cells was sufficient to normalize mitochondrial dynamics and function. CONCLUSIONS: RCAN1 helps maintain a more interconnected mitochondrial network, and maintaining appropriate RCAN1 levels is important to human health and disease.

Whole Body Periodic Acceleration Improves Muscle Recovery after Eccentric Exercise.

INTRODUCTION: The aim of this study was to determine whether whole body periodic acceleration (pGz) could improve muscle recovery after unaccustomed eccentric exercise (EE). METHODS: Downhill treadmill running was used to elicit EE-induced muscle damage in mice, and pGz treatment (480 cycles per minute, 1 h·d) was applied daily for 10 d after the initial EE bout (day 0). Every 2 d during the pGz treatment course starting at day 0, we 1) assessed intracellular Ca and Na concentrations and membrane potential (as indicators of intracellular ion dysfunction) in vivo in gastrocnemius muscle from anesthetized animals and 2) quantified creatine kinase (CK), tumor necrosis factor α (TNF-α), monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6), and interleukin-10 (IL-10) concentrations in plasma or muscle lysates (as indicators of muscle damage and inflammation). RESULTS: EE significantly increased intracellular Ca and Na, CK, TNF-α, MCP-1, IL-6, and IL-10, all of which peaked on day 2 with the exception of IL-10 and declined slowly over 10 d of recovery. pGz treatment after the EE bout mitigated ion dyshomeostasis and expedited recuperation to control values after 6 d of treatment. pGz treatment also accelerated the normalization of CK, TNF-α, MCP-1, and IL-6 while further increasing IL-10 concentrations. The nitric oxide synthase inhibitor L-N-nitroarginine methyl ester, administered in drinking water before and maintained throughout the treatment course, was sufficient to abrogate the salutary effects of pGz after EE. CONCLUSIONS: The present study demonstrates whole body periodic acceleration as an effective therapeutic strategy to accelerate muscle recovery after EE-induced skeletal muscle injury, as indicated by a faster normalization of all the studied parameters.

Altered ROS production, NF-κB activation and interleukin-6 gene expression induced by electrical stimulation in dystrophic mdx skeletal muscle cells.

Duchenne muscular dystrophy is a fatal X-linked genetic disease, caused by mutations in the dystrophin gene, which cause functional loss of this protein. This pathology is associated with an increased production of reactive oxygen (ROS) and nitrogen species. The aim of this work was to study the alterations in NF-κB activation and interleukin-6 (IL-6) expression induced by membrane depolarization in dystrophic mdx myotubes. Membrane depolarization elicited by electrical stimulation increased p65 phosphorylation, NF-κB transcriptional activity and NF-κB-dependent IL-6 expression in wt myotubes, whereas in mdx myotubes it had the opposite effect. We have previously shown that depolarization-induced intracellular Ca2+ increases and ROS production are necessary for NF-κB activation and stimulation of gene expression in wt myotubes. Dystrophic myotubes showed a reduced amplitude and area under the curve of the Ca2+ transient elicited by electrical stimulation. On the other hand, electrical stimuli induced higher ROS production in mdx than wt myotubes, which were blocked by NOX2 inhibitors. Moreover, mRNA expression and protein levels of the NADPH oxidase subunits: p47phox and gp91phox were increased in mdx myotubes. Looking at ROS-dependence of NF-κB activation we found that in wt myotubes external administration of 50 µM H2O2 increased NF-κB activity; after administration of 100 and 200 µM H2O2 there was no effect. In mdx myotubes there was a dose-dependent reduction in NF-κB activity in response to external administration of H2O2, with a significant effect of 100 µM and 200 µM, suggesting that ROS levels are critical for NF-κB activity. Prior blockage with NOX2 inhibitors blunted the effects of electrical stimuli in both NF-κB activation and IL-6 expression. Finally, to ascertain whether stimulation of NF-κB and IL-6 gene expression by the inflammatory pathway is also impaired in mdx myotubes, we studied the effect of lipopolysaccharide on both NF-κB activation and IL-6 expression. Exposure to lipopolysaccharide induced a dramatic increase in both NF-κB activation and IL-6 expression in both wt and mdx myotubes, suggesting that the altered IL-6 gene expression after electrical stimulation in mdx muscle cells is due to dysregulation of Ca2+ release and ROS production, both of which impinge on NF-κB signaling. IL-6 is a key metabolic modulator that is released by the skeletal muscle to coordinate a multi-systemic response (liver, muscle, and adipocytes) during physical exercise; the alteration of this response in dystrophic muscles may contribute to an abnormal response to contraction and exercise.

Non-invasive technology that improves cardiac function after experimental myocardial infarction: Whole Body Periodic Acceleration (pGz).

Myocardial infarction (MI) may produce significant inflammatory changes and adverse ventricular remodeling leading to heart failure and premature death. Pharmacologic, stem cell transplantation, and exercise have not halted the inexorable rise in the prevalence and great economic costs of heart failure despite extensive investigations of such treatments. New therapeutic modalities are needed. Whole Body Periodic Acceleration (pGz) is a non-invasive technology that increases pulsatile shear stress to the endothelium thereby producing several beneficial cardiovascular effects as demonstrated in animal models, normal humans and patients with heart disease. pGz upregulates endothelial derived nitric oxide synthase (eNOS) and its phosphorylation (p-eNOS) to improve myocardial function in models of myocardial stunning and preconditioning. Here we test whether pGz applied chronically after focal myocardial infarction in rats improves functional outcomes from MI. Focal MI was produced by left coronary artery ligation. One day after ligation animals were randomized to receive daily treatments of pGz for four weeks (MI-pGz) or serve as controls (MI-CONT), with an additional group as non-infarction controls (Sham). Echocardiograms and invasive pressure volume loop analysis were carried out. Infarct transmurality, myocardial fibrosis, and markers of inflammatory and anti-inflammatory cytokines were determined along with protein analysis of eNOS, p-eNOS and inducible nitric oxide synthase (iNOS).At four weeks, survival was 80% in MI-pGz vs 50% in MI-CONT (p< 0.01). Ejection fraction and fractional shortening and invasive pressure volume relation indices of afterload and contractility were significantly better in MI-pGz. The latter where associated with decreased infarct transmurality and decreased fibrosis along with increased eNOS, p-eNOS. Additionally, MI-pGz had significantly lower levels of iNOS, inflammatory cytokines (IL-6, TNF-α), and higher level of anti-inflammatory cytokine (IL-10). pGz improved survival and contractile performance, associated with improved myocardial remodeling. pGz may serve as a simple, safe, non-invasive therapeutic modality to improve myocardial function after MI.

Ca2+ influx via the Na+/Ca2+ exchanger is enhanced in malignant hyperthermia skeletal muscle.

Malignant hyperthermia (MH) is potentially fatal pharmacogenetic disorder of skeletal muscle caused by intracellular Ca(2+) dysregulation. NCX is a bidirectional transporter that effluxes (forward mode) or influxes (reverse mode) Ca(2+) depending on cellular activity. Resting intracellular calcium ([Ca(2+)]r) and sodium ([Na(+)]r) concentrations are elevated in MH susceptible (MHS) swine and murine muscles compared with their normal (MHN) counterparts, although the contribution of NCX is unclear. Lowering [Na(+)]e elevates [Ca(2+)]r in both MHN and MHS swine muscle fibers and it is prevented by removal of extracellular Ca(2+) or reduced by t-tubule disruption, in both genotypes. KB-R7943, a nonselective NCX3 blocker, reduced [Ca(2+)]r in both swine and murine MHN and MHS muscle fibers at rest and decreased the magnitude of the elevation of [Ca(2+)]r observed in MHS fibers after exposure to halothane. YM-244769, a high affinity reverse mode NCX3 blocker, reduces [Ca(2+)]r in MHS muscle fibers and decreases the amplitude of [Ca(2+)]r rise triggered by halothane, but had no effect on [Ca(2+)]r in MHN muscle. In addition, YM-244769 reduced the peak and area under the curve of the Ca(2+) transient elicited by high [K(+)]e and increased its rate of decay in MHS muscle fibers. siRNA knockdown of NCX3 in MHS myotubes reduced [Ca(2+)]r and the Ca(2+) transient area induced by high [K(+)]e. These results demonstrate a functional NCX3 in skeletal muscle whose activity is enhanced in MHS. Moreover reverse mode NCX3 contributes to the Ca(2+) transients associated with K(+)-induced depolarization and the halothane-triggered MH episode in MHS muscle fibers.

Na+ dysregulation coupled with Ca2+ entry through NCX1 promotes muscular dystrophy in mice.

Unregulated Ca(2+) entry is thought to underlie muscular dystrophy. Here, we generated skeletal-muscle-specific transgenic (TG) mice expressing the Na(+)-Ca(2+) exchanger 1 (NCX1) to model its identified augmentation during muscular dystrophy. The NCX1 transgene induced dystrophy-like disease in all hind-limb musculature, as well as exacerbated the muscle disease phenotypes in δ-sarcoglycan (Sgcd(-/-)), Dysf(-/-), and mdx mouse models of muscular dystrophy. Antithetically, muscle-specific deletion of the Slc8a1 (NCX1) gene diminished hind-limb pathology in Sgcd(-/-) mice. Measured increases in baseline Na(+) and Ca(2+) in dystrophic muscle fibers of the hind-limb musculature predicts a net Ca(2+) influx state due to reverse-mode operation of NCX1, which mediates disease. However, the opposite effect is observed in the diaphragm, where NCX1 overexpression mildly protects from dystrophic disease through a predicted enhancement in forward-mode NCX1 operation that reduces Ca(2+) levels. Indeed, Atp1a2(+/-) (encoding Na(+)-K(+) ATPase α2) mice, which have reduced Na(+) clearance rates that would favor NCX1 reverse-mode operation, showed exacerbated disease in the hind limbs of NCX1 TG mice, similar to treatment with the Na(+)-K(+) ATPase inhibitor digoxin. Treatment of Sgcd(-/-) mice with ranolazine, a broadly acting Na(+) channel inhibitor that should increase NCX1 forward-mode operation, reduced muscular pathology.

Testosterone signals through mTOR and androgen receptor to induce muscle hypertrophy.

PURPOSE: The anabolic hormone testosterone induces muscle hypertrophy, but the intracellular mechanisms involved are poorly known. We addressed the question whether signal transduction pathways other than the androgen receptor (AR) are necessary to elicit hypertrophy in skeletal muscle myotubes. METHODS: Cultured rat skeletal muscle myotubes were preincubated with inhibitors for ERK1/2 (PD98059), PI3K/Akt (LY294002 and Akt inhibitor VIII) or mTOR/S6K1 (rapamycin), and then stimulated with 100 nM testosterone. The expression of α-actin and the phosphorylation levels of ERK1/2, Akt and S6K1 (a downstream target for mTOR) were measured by Western blot. mRNA levels were evaluated by real time RT-PCR. Myotube size and sarcomerization were determined by confocal microscopy. Inhibition of AR was assessed by bicalutamide. RESULTS: Testosterone-induced myotube hypertrophy was assessed as increased myotube cross-sectional area (CSA) and increased α-actin mRNA and α-actin protein levels, with no changes in mRNA expression of atrogenes (MAFbx and MuRF-1). Morphological development of myotube sarcomeres was evident in testosterone-stimulated myotubes. Known hypertrophy signaling pathways were studied at short times: ERK1/2 and Akt showed an increase in phosphorylation status after testosterone stimulus at 5 and 15 min, respectively. S6K1 was phosphorylated at 60 min. This response was abolished by PI3K/Akt and mTOR inhibition but not by ERK1/2 inhibition. Similarly, the CSA increase at 12 h was abolished by inhibitors of the PI3K/Akt pathway as well as by AR inhibition. CONCLUSIONS: These results suggest a crosstalk between pathways involving fast intracellular signaling and the AR to explain testosterone-induced skeletal muscle hypertrophy.

Cav1.1 controls frequency-dependent events regulating adult skeletal muscle plasticity.

An important pending question in neuromuscular biology is how skeletal muscle cells decipher the stimulation pattern coming from motoneurons to define their phenotype as slow or fast twitch muscle fibers. We have previously shown that voltage-gated L-type calcium channel (Cav1.1) acts as a voltage sensor for activation of inositol (1,4,5)-trisphosphate [Ins(1,4,5)P3]-dependent Ca(2+) signals that regulates gene expression. ATP released by muscle cells after electrical stimulation through pannexin-1 channels plays a key role in this process. We show now that stimulation frequency determines both ATP release and Ins(1,4,5)P3 production in adult skeletal muscle and that Cav1.1 and pannexin-1 colocalize in the transverse tubules. Both ATP release and increased Ins(1,4,5)P3 was seen in flexor digitorum brevis fibers stimulated with 270 pulses at 20 Hz, but not at 90 Hz. 20 Hz stimulation induced transcriptional changes related to fast-to-slow muscle fiber phenotype transition that required ATP release. Addition of 30 µM ATP to fibers induced the same transcriptional changes observed after 20 Hz stimulation. Myotubes lacking the Cav1.1-α1 subunit released almost no ATP after electrical stimulation, showing that Cav1.1 has a central role in this process. In adult muscle fibers, ATP release and the transcriptional changes produced by 20 Hz stimulation were blocked by both the Cav1.1 antagonist nifedipine (25 µM) and by the Cav1.1 agonist (-)S-BayK 8644 (10 µM). We propose a new role for Cav1.1, independent of its calcium channel activity, in the activation of signaling pathways allowing muscle fibers to decipher the frequency of electrical stimulation and to activate specific transcriptional programs that define their phenotype.

Electrical stimuli release ATP to increase GLUT4 translocation and glucose uptake via PI3Kγ-Akt-AS160 in skeletal muscle cells.

Skeletal muscle glucose uptake in response to exercise is preserved in insulin-resistant conditions, but the signals involved are debated. ATP is released from skeletal muscle by contractile activity and can autocrinely signal through purinergic receptors, and we hypothesized it may influence glucose uptake. Electrical stimulation, ATP, and insulin each increased fluorescent 2-NBD-Glucose (2-NBDG) uptake in primary myotubes, but only electrical stimulation and ATP-dependent 2-NBDG uptake were inhibited by adenosine-phosphate phosphatase and by purinergic receptor blockade (suramin). Electrical stimulation transiently elevated extracellular ATP and caused Akt phosphorylation that was additive to insulin and inhibited by suramin. Exogenous ATP transiently activated Akt and, inhibiting phosphatidylinositol 3-kinase (PI3K) or Akt as well as dominant-negative Akt mutant, reduced ATP-dependent 2-NBDG uptake and Akt phosphorylation. ATP-dependent 2-NBDG uptake was also inhibited by the G protein ßγ subunit-interacting peptide ßark-ct and by the phosphatidylinositol 3-kinase-γ (PI3Kγ) inhibitor AS605240. ATP caused translocation of GLUT4myc-eGFP to the cell surface, mechanistically mediated by increased exocytosis involving AS160/Rab8A reduced by dominant-negative Akt or PI3Kγ kinase-dead mutants, and potentiated by myristoylated PI3Kγ. ATP stimulated 2-NBDG uptake in normal and insulin-resistant adult muscle fibers, resembling the reported effect of exercise. Hence, the ATP-induced pathway may be tapped to bypass insulin resistance.

[Robotics in gynecology. Background, feasibility and applicability]. / La robótica en Ginecología. Antecedentes, viabilidad y conveniencia.

BACKGROUND: Robotic surgery is a technology that emerged from the fusion and improvement of laparoscopy, robotics and telepresence. All these three technologies underwent a long experimentation process in which several applications and innovations were tested until the only system approved for use in humans was developed: the Da Vinci system by Intuitive Surgical Inc. Gynecology, being one of the pioneer branches of Medicine involved in the development of laparoscopy, is one of the fields with the greatest possibilities for robotics, which offers great diversity of applications in hysterectomies, myomectomies, endometriosis, and in the fields of urogynecology and, most importantly, oncology. There are no publications in Mexico with a proper description of the clinical experience with gynecologic robotic surgery, though a great amount of clinical experience has been accumulated in institutions that already have such equipment. A serious evaluation of the cost-benefit ratio is required because of the high cost of this technology. OBJECTIVE: Evaluate and analyze the accumulated experience on this technology of foreign institutions in order to assess the benefits, cost and effectiveness of robotic surgery. CONCLUSION: The key to the optimal use of robotic technology is to diminish costs and speed the learning curve, and this implies the entry of other systems into the market as well as institutions with a high volume of patients and determined to invest in a highly trained and skilled surgical team. In order to recommend its implementation in our country an assessment of the efficiency and advantages of robotic technology considering institutional needs is mandatory.

[Family planning methods based on fertility awareness]. / Métodos de planificación familiar basados en el conocimiento de la fertilidad.

The desire to limit fertility is recognized both by individuals and by nations. The concept of family planning is based on the right of individuals and couples to regulate their fertility and is based in the area of health, human rights and population. Despite the changes in policies and family planning programs worldwide, there are large geographic areas that have not yet met the minimum requirements in this regard, the reasons are multiple, including economic reasons but also ideological or religious. Knowledge on the physiology of the menstrual cycle, specifically ovulation process has been further enhanced due to the advances in reproductive medicine research. The series of events around ovulation are used to detect the "fertile window", this way women will look for the possibility of postponing their pregnancy or actually start looking for it. The aim of this article is to review the current methods of family planning based on fertility awareness, from the historical methods like the core temperature determination and rhythm, to the most popular ones like the Billings ovulation method, the Sympto-thermal method and current methods like the two days, and the standard days method. There are also mentioned methods that require electronic devices or specifically computer designed ones to detect this "window of fertility". The spread and popularity of these methods is low and their knowledge among physicians, including gynecologists, is also quite scarce. The effectiveness of these methods has been difficult to quantify due to the lack of well designed, randomized studies which are affected by small populations of patients using these methods. The publications mention high effectiveness with their proper use, but not with typical use, what indicates the need for increased awareness among medical practitioners and trainers, obtaining a better use and understanding of methods and reducing these discrepancies.