Bi-allelic LETM1 variants perturb mitochondrial ion homeostasis leading to a clinical spectrum with predominant nervous system involvement.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies.

The Link of the Prion Protein with Ca2+ Metabolism and ROS Production, and the Possible Implication in Aß Toxicity.

The cellular prion protein (PrPC) is an ubiquitous cell surface protein mostly expressed in neurons, where it localizes to both pre- and post-synaptic membranes. PrPC aberrant conformers are the major components of mammalian prions, the infectious agents responsible for incurable neurodegenerative disorders. PrPC was also proposed to bind aggregated misfolded proteins/peptides, and to mediate their neurotoxic signal. In spite of long-lasting research, a general consensus on the precise pathophysiologic mechanisms of PrPC has not yet been reached. Here we review our recent data, obtained by comparing primary neurons from PrP-expressing and PrP-knockout mice, indicating a central role of PrPC in synaptic transmission and Ca2+ homeostasis. Indeed, by controlling gene expression and signaling cascades, PrPC is able to optimize glutamate secretion and regulate Ca2+ entry via store-operated channels and ionotropic glutamate receptors, thereby protecting neurons from threatening Ca2+ overloads and excitotoxicity. We will also illustrate and discuss past and unpublished results demonstrating that Aß oligomers perturb Ca2+ homeostasis and cause abnormal mitochondrial accumulation of reactive oxygen species by possibly affecting the PrP-dependent downregulation of Fyn kinase activity.

Alisporivir rescues defective mitochondrial respiration in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a severe muscle disease of known etiology without effective, or generally applicable therapy. Mitochondria are affected by the disease in animal models but whether mitochondrial dysfunction is part of the pathogenesis in patients remains unclear. We show that primary cultures obtained from muscle biopsies of DMD patients display a decrease of the respiratory reserve, a consequence of inappropriate opening of the permeability transition pore (PTP). Treatment with the cyclophilin inhibitor alisporivir - a cyclosporin A derivative that desensitizes the PTP but does not inhibit calcineurin - largely restored the maximal respiratory capacity without affecting basal oxygen consumption in cells from patients, thus reinstating a normal respiratory reserve. Treatment with alisporivir, but not with cyclosporin A, led to a substantial recovery of respiratory function matching improved muscle ultrastructure and survival of sapje zebrafish, a severe model of DMD where muscle defects are close to those of DMD patients. Alisporivir was generally well tolerated in HCV patients and could be used for the treatment of DMD.

NIM811, a cyclophilin inhibitor without immunosuppressive activity, is beneficial in collagen VI congenital muscular dystrophy models.

Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are inherited muscle diseases due to mutations in the genes encoding the extracellular matrix protein collagen (Col) VI. Opening of the cyclosporin A-sensitive mitochondrial permeability transition pore (PTP) is a causative event in disease pathogenesis, and a potential target for therapy. Here, we have tested the effect of N-methyl-4-isoleucine-cyclosporin (NIM811), a non-immunosuppressive cyclophilin inhibitor, in a zebrafish model of ColVI myopathy obtained by deletion of the N-terminal region of the ColVI α1 triple helical domain, a common mutation of UCMD. Treatment with antisense morpholino sequences targeting col6a1 exon 9 at the 1-4 cell stage (within 1 h post fertilization, hpf) caused severe ultrastructural and motor abnormalities as assessed by electron and fluorescence microscopy, birefringence, spontaneous coiling events and touch-evoked responses measured at 24-48 hpf. Structural and functional abnormalities were largely prevented when NIM811--which proved significantly more effective than cyclosporin A--was administered at 21 hpf, while FK506 was ineffective. Beneficial effects of NIM811 were also detected (i) in primary muscle-derived cell cultures from UCMD and BM patients, where the typical mitochondrial alterations and depolarizing response to rotenone and oligomycin were significantly reduced; and (ii) in the Col6a1(-/-) myopathic mouse model, where apoptosis was prevented and muscle strength was increased. Since the PTP of zebrafish shares its key regulatory features with the mammalian pore, our results suggest that early treatment with NIM811 should be tested as a potential therapy for UCMD and BM.

Forty years later: Mitochondria as therapeutic targets in muscle diseases.

The hypothesis that mitochondrial dysfunction can be a general mechanism for cell death in muscle diseases is 40 years old. The key elements of the proposed pathogenetic sequence (cytosolic Ca2+ overload followed by excess mitochondrial Ca2+ uptake, functional and then structural damage of mitochondria, energy shortage, worsened elevation of cytosolic Ca2+ levels, hypercontracture of muscle fibers, cell necrosis) have been confirmed in amazing detail by subsequent work in a variety of models. The explicit implication of the hypothesis was that it "may provide the basis for a more rational treatment for some conditions even before their primary causes are known" (Wrogemann and Pena, 1976, Lancet, 1, 672-674). This prediction is being fulfilled, and the potential of mitochondria as pharmacological targets in muscle diseases may soon become a reality, particularly through inhibition of the mitochondrial permeability transition pore and its regulator cyclophilin D.

Activation of c-SRC underlies the differential effects of ouabain and digoxin on Ca(2+) signaling in arterial smooth muscle cells.

Cardiotonic steroids (CTS) of the strophanthus and digitalis families have opposing effects on long-term blood pressure (BP). This implies hitherto unrecognized divergent signaling pathways for these CTS. Prolonged ouabain treatment upregulates Ca(2+) entry via Na(+)/Ca(2+) exchanger-1 (NCX1) and TRPC6 gene-encoded receptor-operated channels in mesenteric artery smooth muscle cells (ASMCs) in vivo and in vitro. Here, we test the effects of digoxin on Ca(2+) entry and signaling in ASMC. In contrast to ouabain treatment, the in vivo administration of digoxin (30 µg·kg(-1)·day(-1) for 3 wk) did not raise BP and had no effect on resting cytolic free Ca(2+) concentration ([Ca(2+)](cyt)) or phenylephrine-induced Ca(2+) signals in isolated ASMCs. Expression of transporters in the α2 Na(+) pump-NCX1-TRPC6 Ca(2+) signaling pathway was not altered in arteries from digoxin-treated rats. Upregulated α2 Na(+) pumps and a phosphorylated form of the c-SRC protein kinase (pY419-Src, ~4.5-fold) were observed in ASMCs from rats treated with ouabain but not digoxin. Moreover, in primary cultured ASMCs from normal rats, treatment with digoxin (100 nM, 72 h) did not upregulate NCX1 and TRPC6 but blocked the ouabain-induced upregulation of these transporters. Pretreatment of ASMCs with the c-Src inhibitor PP2 (1 µM; 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) but not its inactive analog eliminated the effect of ouabain on NCX1 and TRPC6 expression and ATP-induced Ca(2+) entry. Thus, in contrast to ouabain, the interaction of digoxin with α2 Na(+) pumps is unable to activate c-Src phosphorylation and upregulate the downstream NCX1-TRPC6 Ca(2+) signaling pathway in ASMCs. The inability of digoxin to upregulate c-Src may underlie its inability to raise long-term BP.

Melanocytes--a novel tool to study mitochondrial dysfunction in Duchenne muscular dystrophy.

Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal-epidermal junction. In addition the full-length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments.

Visceral adipocytes: old actors in obesity and new protagonists in Crohn's disease?

OBJECTIVE: Crohn's disease (CD) is a chronic inflammatory bowel disease characterised by a peculiar accumulation of mesenteric adipose tissue covering the inflamed intestinal wall. METHODS: The authors characterised different adipose tissue compartments of patients with CD using morphological and molecular techniques and compared them to those of subjects with obesity (OB) and healthy subjects with normal weight (N). Adipose tissue samples were taken from subcutaneous adipose tissue, omental visceral adipose tissue (VAT) and healthy mesenteric depot (hMES), as well as from fat wrapping the affected (unhealthy) intestinal tracts (uhMES). Microarray analyses, validated by real-time quantitative PCR technique, were performed in whole adipose tissue and in isolated adipocytes. RESULTS: The morphology of subcutaneous adipose tissue was similar in subjects with CD and those with N. In patients with CD, VAT adipocytes were smaller than those derived from uhMES and hMES and were smaller than VAT adipocytes of subjects with N. The molecular profiles of CD, VAT and uhMES were characterised by upregulation of genes related to inflammation and downregulation of those involved in lipid metabolism. Adipocytes isolated from VAT of subjects with CD and those with OB exhibited similar upregulation of genes involved in inflammation and immunity. VAT adipocytes of patients with CD compared to those of patients with OB also showed a greater upregulation of several anti-inflammatory genes. CONCLUSION: In patients with CD, VAT distant from uhMES is affected by inflammation and displays features similar to those of VAT of patients with severe OB. The small diameter of VAT adipocytes of CD, together with their high expression of anti-inflammatory genes, suggests a potentially protective role for this tissue. VAT adipocytes may play an important role in the pathophysiology and/or activity of CD.

Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor).

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.

The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide.

We have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig. We conclude that selective NRB transport into rat mitochondria occurs via TSPO in the OMM, which allows its translocation to PT-regulating sites in the inner membrane. Thus, species-specificity of NRB toward the rat PT depends on subtle differences in the structure of TSPO or of TSPO-associated proteins affecting its substrate specificity.

Increased arterial smooth muscle Ca2+ signaling, vasoconstriction, and myogenic reactivity in Milan hypertensive rats.

The Milan hypertensive strain (MHS) rats are a genetic model of hypertension with adducin gene polymorphisms linked to enhanced renal tubular Na(+) reabsorption. Recently we demonstrated that Ca(2+) signaling is augmented in freshly isolated mesenteric artery myocytes from MHS rats. This is associated with greatly enhanced expression of Na(+)/Ca(2+) exchanger-1 (NCX1), C-type transient receptor potential (TRPC6) protein, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2) compared with arteries from Milan normotensive strain (MNS) rats. Here, we test the hypothesis that the enhanced Ca(2+) signaling in MHS arterial smooth muscle is directly reflected in augmented vasoconstriction [myogenic and phenylephrine (PE)-evoked responses] in isolated mesenteric small arteries. Systolic blood pressure was higher in MHS (145 ± 1 mmHg) than in MNS (112 ± 1 mmHg; P < 0.001; n = 16 each) rats. Pressurized mesenteric resistance arteries from MHS rats had significantly augmented myogenic tone and reactivity and enhanced constriction to low-dose (1-100 nM) PE. Isolated MHS arterial myocytes exhibited approximately twofold increased peak Ca(2+) signals in response to 5 µM PE or ATP in the absence and presence of extracellular Ca(2+). These augmented responses are consistent with increased vasoconstrictor-evoked sarcoplasmic reticulum (SR) Ca(2+) release and increased Ca(2+) entry, respectively. The increased SR Ca(2+) release correlates with a doubling of inositol 1,4,5-trisphosphate receptor type 1 and tripling of SERCA2 expression. Pressurized MHS arteries also exhibited a ∼70% increase in 100 nM ouabain-induced vasoconstriction compared with MNS arteries. These functional alterations reveal that, in a genetic model of hypertension linked to renal dysfunction, multiple mechanisms within the arterial myocytes contribute to enhanced Ca(2+) signaling and myogenic and vasoconstrictor-induced arterial constriction. MHS rats have elevated plasma levels of endogenous ouabain, which may initiate the protein upregulation and enhanced Ca(2+) signaling. These molecular and functional changes provide a mechanism for the increased peripheral vascular resistance (whole body autoregulation) that underlies the sustained hypertension.

Variant Selection and Interpretation: An Example of Modified VarSome Classifier of ACMG Guidelines in the Diagnostic Setting.

Variant interpretation is challenging as it involves combining different levels of evidence in order to evaluate the role of a specific variant in the context of a patient's disease. Many in-depth refinements followed the original 2015 American College of Medical Genetics (ACMG) guidelines to overcome subjective interpretation of criteria and classification inconsistencies. Here, we developed an ACMG-based classifier that retrieves information for variant interpretation from the VarSome Stable-API environment and allows molecular geneticists involved in clinical reporting to introduce the necessary changes to criterion strength and to add or exclude criteria assigned automatically, ultimately leading to the final variant classification. We also developed a modified ACMG checklist to assist molecular geneticists in adjusting criterion strength and in adding literature-retrieved or patient-specific information, when available. The proposed classifier is an example of integration of automation and human expertise in variant curation, while maintaining the laboratory analytical workflow and the established bioinformatics pipeline.

Molecular Epidemiology in 591 Italian Probands With Nonsyndromic Retinitis Pigmentosa and Usher Syndrome.

Purpose: To describe the molecular epidemiology of nonsyndromic retinitis pigmentosa (RP) and Usher syndrome (US) in Italian patients. Methods: A total of 591 probands (315 with family history and 276 sporadics) were analyzed. For 155 of them, we performed a family segregation study, considering a total of 382 relatives. Probands were analyzed by a customized multigene panel approach. Sanger sequencing was used to validate all genetic variants and to perform family segregation studies. Copy number variants of selected genes were analyzed by multiplex ligation-dependent probe amplification. Four patients who tested negative to targeted next-generation sequencing analysis underwent clinical exome sequencing. Results: The mean diagnostic yield of molecular testing among patients with a family history of retinal disorders was 55.2% while the diagnostic yield including sporadic cases was 37.4%. We found 468 potentially pathogenic variants, 147 of which were unpublished, in 308 probands and 66 relatives. Mean ages of onset of the different classes of RP were autosomal dominant RP, 19.3 ± 12.6 years; autosomal recessive RP, 23.2 ± 16.6 years; X-linked RP, 13.9 ± 9.9 years; and Usher syndrome, 18.9 ± 9.5 years. We reported potential new genotype-phenotype correlations in three probands, two revealed by TruSight One testing. All three probands showed isolated RP caused by biallelic variants in genes usually associated with syndromes such as PERCHING and Senior-Loken or with retinal dystrophy, iris coloboma, and comedogenic acne syndrome. Conclusions: This is the largest molecular study of Italian patients with RP in the literature, thus reflecting the epidemiology of the disease in Italy with reasonable accuracy.

Ablation of collagen VI leads to the release of platelets with altered function.

Hemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by collagen VI-related disorders and investigated the defects in platelet functionality, whose mechanisms are unknown. We demonstrated that megakaryocytes express collagen VI that is involved in the regulation of functional platelet production. By exploiting a collagen VI-null mouse model (Col6a1-/-), we found that collagen VI-null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1-/- megakaryocytes and platelets showed increased expression of stromal interaction molecule 1 (STIM1) and ORAI1, the components of store-operated calcium entry (SOCE), and activation of the mammalian target of rapamycin (mTOR) signaling pathway. In vivo mTOR inhibition by rapamycin reduced STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell autonomous, because transplantation of lineage-negative bone marrow cells from Col6a1-/- mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation seen in Col6a1-/- mice. Peripheral blood platelets of patients affected by collagen VI-related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of collagen VI in the production of functional platelets by megakaryocytes in mouse models and in collagen VI-related diseases.

Switch from inhibition to activation of the mitochondrial permeability transition during hematoporphyrin-mediated photooxidative stress. Unmasking pore-regulating external thiols.

We have studied the mitochondrial permeability transition pore (PTP) under oxidizing conditions with mitochondria-bound hematoporphyrin, which generates reactive oxygen species (mainly singlet oxygen, (1)O(2)) upon UV/visible light-irradiation and promotes the photooxidative modification of vicinal targets. We have characterized the PTP-modulating properties of two major critical sites endowed with different degrees of photosensitivity: (i) the most photovulnerable site comprises critical histidines, whose photomodification by vicinal hematoporphyrin causes a drop in reactivity of matrix-exposed (internal), PTP-regulating cysteines thus stabilizing the pore in a closed conformation; (ii) the most photoresistant site coincides with the binding domains of (external) cysteines sensitive to membrane-impermeant reagents, which are easily unmasked when oxidation of internal cysteines is prevented. Photooxidation of external cysteines promoted by vicinal hematoporphyrin reactivates the PTP after the block caused by histidine photodegradation. Thus, hematoporphyrin-mediated photooxidative stress can either inhibit or activate the mitochondrial permeability transition depending on the site of hematoporphyrin localization and on the nature of the substrate; and selective photomodification of different hematoporphyrin-containing pore domains can be achieved by fine regulation of the sensitizer/light doses. These findings shed new light on PTP modulation by oxidative stress.

Upregulation of Na+/Ca2+ exchanger and TRPC6 contributes to abnormal Ca2+ homeostasis in arterial smooth muscle cells from Milan hypertensive rats.

The Milan hypertensive strain (MHS) of rats is a model for hypertension in humans. Inherited defects in renal function have been well studied in MHS rats, but the mechanisms that underlie the elevated vascular resistance are unclear. Altered Ca(2+) signaling plays a key role in the vascular dysfunction associated with arterial hypertension. Here we compared Ca(2+) signaling in mesenteric artery smooth muscle cells from MHS rats and its normotensive counterpart (MNS). Systolic blood pressure was higher in MHS than in MNS rats (144 +/- 2 vs. 113 +/- 1 mmHg, P < 0.05). Resting cytosolic free Ca(2+) concentration (measured with fura-2) and ATP-induced Ca(2+) transients were augmented in freshly dissociated arterial myocytes from MHS rats. Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol (a measure of receptor-operated channel activity) was much greater in MHS than MNS arterial myocytes. This correlated with a threefold upregulation of transient receptor potential canonical 6 (TRPC6) protein. TRPC3, the other component of receptor-operated channels, was marginally, but not significantly, upregulated. The expression of TRPC1/5, components of store-operated channels, was not altered in MHS mesenteric artery smooth muscle. Immunoblots also revealed that the Na(+)/Ca(2+) exchanger-1 (NCX1) was greatly upregulated in MHS mesenteric artery (by approximately 13-fold), whereas the expression of plasma membrane Ca(2+)-ATPase was not altered. Ca(2+) entry via the reverse mode of NCX1 evoked by the removal of extracellular Na(+) induced a rapid increase in cytosolic free Ca(2+) concentration that was significantly larger in MHS arterial myocytes. The expression of alpha(1)/alpha(2) Na(+) pumps in MHS mesenteric arteries was not changed. Immunocytochemical observations showed that NCX1 and TRPC6 are clustered in plasma membrane microdomains adjacent to the underlying sarcoplasmic reticulum. In summary, MHS arteries exhibit upregulated TRPC6 and NCX1 and augmented Ca(2+) signaling. We suggest that the increased Ca(2+) signaling contributes to the enhanced vasoconstriction and elevated blood pressure in MHS rats.

Upregulation of Na+ and Ca2+ transporters in arterial smooth muscle from ouabain-induced hypertensive rats.

Prolonged ouabain administration (25 microg kg(-1) day(-1) for 5 wk) induces "ouabain hypertension" (OH) in rats, but the molecular mechanisms by which ouabain elevates blood pressure are unknown. Here, we compared Ca(2+) signaling in mesenteric artery smooth muscle cells (ASMCs) from normotensive (NT) and OH rats. Resting cytosolic free Ca(2+) concentration ([Ca(2+)](cyt); measured with fura-2) and phenylephrine-induced Ca(2+) transients were augmented in freshly dissociated OH ASMCs. Immunoblots revealed that the expression of the ouabain-sensitive alpha(2)-subunit of Na(+) pumps, but not the predominant, ouabain-resistant alpha(1)-subunit, was increased (2.5-fold vs. NT ASMCs) as was Na(+)/Ca(2+) exchanger-1 (NCX1; 6-fold vs. NT) in OH arteries. Ca(2+) entry, activated by sarcoplasmic reticulum (SR) Ca(2+) store depletion with cyclopiazonic acid (SR Ca(2+)-ATPase inhibitor) or caffeine, was augmented in OH ASMCs. This reflected an augmented expression of 2.5-fold in OH ASMCs of C-type transient receptor potential TRPC1, an essential component of store-operated channels (SOCs); two other components of some SOCs were not expressed (TRPC4) or were not upregulated (TRPC5). Ba(2+) entry activated by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol [a measure of receptor-operated channel (ROC) activity] was much greater in OH than NT ASMCs. This correlated with a sixfold upregulation of TRPC6 protein, a ROC family member. Importantly, in primary cultured mesenteric ASMCs from normal rats, 72-h treatment with 100 nM ouabain significantly augmented NCX1 and TRPC6 protein expression and increased resting [Ca(2+)](cyt) and ROC activity. SOC activity was also increased. Silencer RNA knockdown of NCX1 markedly downregulated TRPC6 and eliminated the ouabain-induced augmentation; silencer RNA knockdown of TRPC6 did not affect NCX1 expression but greatly attenuated its upregulation by ouabain. Clearly, NCX1 and TRPC6 expression are interrelated. Thus, prolonged ouabain treatment upregulates the Na(+) pump alpha(2)-subunit-NCX1-TRPC6 (ROC) Ca(2+) signaling pathway in arterial myocytes in vitro as well as in vivo. This may explain the augmented myogenic responses and enhanced phenylephrine-induced vasoconstriction in OH arteries (83) as well as the high blood pressure in OH rats.

Genetic test for the prescription of diets in support of physical activity.

Owing to the fields of nutrigenetics and nutrigenomics today we can think of devising approaches to optimize health, delay onset of diseases and reduce its severity according to our genetic blue print. However this requires a deep understanding of nutritional impact on expression of genes that may result in a specific phenotype. The extensive research and observational studies during last two decades reporting interactions between genes, diet and physical activity suggest a cross talk between various genetic and environmental factors and lifestyle interventions. Although considerable efforts have been made in unraveling the mechanisms of gene-diet interactions the scientific evidences behind developing commercial genetic tests for providing personalized nutrition recommendations are still scarce. In this scenario the current mini-review aims to provide useful insights into salient feature of nutrition based genetic research and its commercial application and the ethical issue and concerns related to its outcome.

Male Infertility Diagnosis: Improvement of Genetic Analysis Performance by the Introduction of Pre-Diagnostic Genes in a Next-Generation Sequencing Custom-Made Panel.

Background: Infertility affects about 7% of the general male population. The underlying cause of male infertility is undefined in about 50% of cases (idiopathic infertility). The number of genes involved in human spermatogenesis is over two thousand. Therefore, it is essential to analyze a large number of genes that may be involved in male infertility. This study aimed to test idiopathic male infertile patients negative for a validated panel of "diagnostic" genes, for a wide panel of genes that we have defined as "pre-diagnostic." Methods: We developed a next-generation sequencing (NGS) gene panel including 65 pre-diagnostic genes that were used in 12 patients who were negative to a diagnostic genetic test for male infertility disorders, including primary spermatogenic failure and central hypogonadism, consisting of 110 genes. Results: After NGS sequencing, variants in pre-diagnostic genes were identified in 10/12 patients who were negative to a diagnostic test for primary spermatogenic failure (n = 9) or central hypogonadism (n = 1) due to mutations of single genes. Two pathogenic variants of DNAH5 and CFTR genes and three uncertain significance variants of DNAI1, DNAH11, and CCDC40 genes were found. Moreover, three variants with high impact were found in AMELY, CATSPER 2, and ADCY10 genes. Conclusion: This study suggests that searching for pre-diagnostic genes may be of relevance to find the cause of infertility in patients with apparently idiopathic primary spermatogenic failure due to mutations of single genes and central hypogonadism.

Somatic Variant Analysis Identifies Targets for Tailored Therapies in Patients with Vascular Malformations.

Vascular malformations include various disorders characterized by morphological, structural and/or functional alterations of blood and lymph vessels. Most are sporadic, due to somatic mutations. Here, we report a cohort of patients with sporadic and/or unifocal vascular malformations, in whom we carried out next generation sequencing analysis of a panel of genes associated with vascular malformations. The 115 patients analyzed were from different clinical centres. In 37 patients (32%), we found pathogenic mutations: most of these were gain-of-function mutations in PIK3CA (18%, 21/115) and TEK (13/115, 11%). We also found mutations in GNAQ, CCM2 and PTEN. Identifying pathogenic variants in patients with vascular malformations can help improve management, particularly in cases with activating mutations that cause an increase in cell proliferation. Personalized pharmacological treatment, if possible, is now considered preferable to surgery and can help prevent recurrences, i.e., long-term complications of residual malformation or regrowth of tumors. For instance, rapamycin is currently being investigated for the treatment of various vascular malformations associated with hyperactivation of the phosphoinositide 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) pathway.