Striated preferentially expressed gene deficiency leads to mitochondrial dysfunction in developing cardiomyocytes.

A deficiency of striated preferentially expressed gene (Speg), a member of the myosin light chain kinase family, results in abnormal myofibril structure and function of immature cardiomyocytes (CMs), corresponding with a dilated cardiomyopathy, heart failure and perinatal death. Mitochondrial development plays a role in cardiomyocyte maturation. Therefore, this study investigated whether Speg deficiency ( - / - ) in CMs would result in mitochondrial abnormalities. Speg wild-type and Speg-/- C57BL/6 littermate mice were utilized for assessment of mitochondrial structure by transmission electron and confocal microscopies. Speg was expressed in the first and second heart fields at embryonic (E) day 7.5, prior to the expression of mitochondrial Na+/Ca2+/Li+ exchanger (NCLX) at E8.5. Decreases in NCLX expression (E11.5) and the mitochondrial-to-nuclear DNA ratio (E13.5) were observed in Speg-/- hearts. Imaging of E18.5 Speg-/- hearts revealed abnormal mitochondrial cristae, corresponding with decreased ATP production in cells fed glucose or palmitate, increased levels of mitochondrial superoxide and depolarization of mitochondrial membrane potential. Interestingly, phosphorylated (p) PGC-1α, a key mediator of mitochondrial development, was significantly reduced in Speg-/- hearts during screening for targeted genes. Besides Z-line expression, Speg partially co-localized with PGC-1α in the sarcomeric region and was found in the same complex by co-immunoprecipitation. Overexpression of a Speg internal serine/threonine kinase domain in Speg-/- CMs promoted translocation of pPGC-1α into the nucleus, and restored ATP production that was abolished by siRNA-mediated silencing of PGC-1α. Our results demonstrate a critical role of Speg in mitochondrial development and energy metabolism in CMs, mediated in part by phosphorylation of PGC-1α.

Dilated-Left Ventricular Non-Compaction Cardiomyopathy in a Pediatric Case with SPEG Compound Heterozygous Variants.

Left Ventricular Non-Compaction (LVNC) is defined by the triad prominent myocardial trabecular meshwork, thin compacted layer, and deep intertrabecular recesses. LVNC associated with dilation is characterized by the coexistence of left ventricular dilation and systolic dysfunction. Pediatric cases with dilated-LVNC have worse outcomes than those with isolated dilated cardiomyopathy and adult patients. Herein, we report a clinical and genetic investigation using trio-based whole-exome sequencing of a pediatric case with early-onset dilated-LVNC. Compound heterozygous mutations were identified in the Striated Muscle Enriched Protein Kinase (SPEG) gene, a key regulator of cardiac calcium homeostasis. A paternally inherited mutation: SPEG; p.(Arg2470Ser) and the second variant, SPEG; p.(Pro2687Thr), is common and occurred de novo. Subsequently, Sanger sequencing was performed for the family in order to segregate the variants. Thus, the index case, his father, and both sisters carried the SPEG: p.(Arg2470Ser) variant. Only the index patient carried both SPEG variants. Both sisters, as well as the patient's father, showed LVNC without cardiac dysfunction. The unaffected mother did not harbor any of the variants. The in silico analysis of the identified variants (rare and common) showed a decrease in protein stability with alterations of the physical properties as well as high conservation scores for the mutated residues. Interestingly, using the Project HOPE tool, the SPEG; p.(Pro2687Thr) variant is predicted to disturb the second fibronectin type III domain of the protein and may abolish its function. To our knowledge, the present case is the first description of compound heterozygous SPEG mutations involving a de novo variant and causing dilated-LVNC without neuropathy or centronuclear myopathy.

Loss of SPEG Inhibitory Phosphorylation of Ryanodine Receptor Type-2 Promotes Atrial Fibrillation.

BACKGROUND: Enhanced diastolic calcium (Ca2+) release through ryanodine receptor type-2 (RyR2) has been implicated in atrial fibrillation (AF) promotion. Diastolic sarcoplasmic reticulum Ca2+ leak is caused by increased RyR2 phosphorylation by PKA (protein kinase A) or CaMKII (Ca2+/calmodulin-dependent kinase-II) phosphorylation, or less dephosphorylation by protein phosphatases. However, considerable controversy remains regarding the molecular mechanisms underlying altered RyR2 function in AF. We thus aimed to determine the role of SPEG (striated muscle preferentially expressed protein kinase), a novel regulator of RyR2 phosphorylation, in AF pathogenesis. METHODS: Western blotting was performed with right atrial biopsies from patients with paroxysmal AF. SPEG atrial knockout mice were generated using adeno-associated virus 9. In mice, AF inducibility was determined using intracardiac programmed electric stimulation, and diastolic Ca2+ leak in atrial cardiomyocytes was assessed using confocal Ca2+ imaging. Phosphoproteomics studies and Western blotting were used to measure RyR2 phosphorylation. To test the effects of RyR2-S2367 phosphorylation, knockin mice with an inactivated S2367 phosphorylation site (S2367A) and a constitutively activated S2367 residue (S2367D) were generated by using CRISPR-Cas9. RESULTS: Western blotting revealed decreased SPEG protein levels in atrial biopsies from patients with paroxysmal AF in comparison with patients in sinus rhythm. SPEG atrial-specific knockout mice exhibited increased susceptibility to pacing-induced AF by programmed electric stimulation and enhanced Ca2+ spark frequency in atrial cardiomyocytes with Ca2+ imaging, establishing a causal role for decreased SPEG in AF pathogenesis. Phosphoproteomics in hearts from SPEG cardiomyocyte knockout mice identified RyR2-S2367 as a novel kinase substrate of SPEG. Western blotting demonstrated that RyR2-S2367 phosphorylation was also decreased in patients with paroxysmal AF. RyR2-S2367A mice exhibited an increased susceptibility to pacing-induced AF, and aberrant atrial sarcoplasmic reticulum Ca2+ leak, as well. In contrast, RyR2-S2367D mice were resistant to pacing-induced AF. CONCLUSIONS: Unlike other kinases (PKA, CaMKII) that increase RyR2 activity, SPEG phosphorylation reduces RyR2-mediated sarcoplasmic reticulum Ca2+ release. Reduced SPEG levels and RyR2-S2367 phosphorylation typified patients with paroxysmal AF. Studies in S2367 knockin mouse models showed a causal relationship between reduced S2367 phosphorylation and AF susceptibility. Thus, modulating SPEG activity and phosphorylation levels of the novel S2367 site on RyR2 may represent a novel target for AF treatment.

Mechanisms underlying pathological Ca2+ handling in diseases of the heart.

Cardiomyocyte contraction relies on precisely regulated intracellular Ca2+ signaling through various Ca2+ channels and transporters. In this article, we will review the physiological regulation of Ca2+ handling and its role in maintaining normal cardiac rhythm and contractility. We discuss how inherited variants or acquired defects in Ca2+ channel subunits contribute to the development or progression of diseases of the heart. Moreover, we highlight recent insights into the role of protein phosphatase subunits and striated muscle preferentially expressed protein kinase (SPEG) in atrial fibrillation, heart failure, and cardiomyopathies. Finally, this review summarizes current drug therapies and new advances in genome editing as therapeutic strategies for the cardiac diseases caused by aberrant intracellular Ca2+ signaling.

Clinical and genetic analysis of a case with centronuclear myopathy caused by SPEG gene mutation: a case report and literature review.

BACKGROUND: Centronuclear myopathy (CNM), a subtype of congenital myopathy (CM), is a group of clinical and genetically heterogeneous muscle disorders. Since the discovery of the SPEG gene and disease-causing variants, only a few additional patients have been reported. CASE PRESENTATION: The child, a 13-year-old female, had delayed motor development since childhood, weakness of both lower extremities for 10 years, gait swinging, and a positive Gower sign. Her distal muscle strength of both lower extremities was grade IV. The electromyography showed myogenic damage and electromyographic changes. Her 11-year-old sister had a similar muscle weakness phenotype. Gene sequencing revealed that both sisters had SPEG compound heterozygous mutations, and the mutation sites were c.3715 + 4C > T and c.3588delC, which were derived from their parents. These variant sites have not been reported before. The muscle biopsy showed the nucleic (> 20% of fibers) were located in the center of the cell, the average diameter of type I myofibers was slightly smaller than that of type II myofibers, and the pathology of type I myofibers was dominant, which agreed with the pathological changes of centronuclear myopathy. CONCLUSIONS: The clinical phenotypes of CNM patients caused by mutations at different sites of the SPEG gene are also different. In this case, there was no cardiomyopathy. This study expanded the number of CNM cases and the mutation spectrum of the SPEG gene to provide references for prenatal diagnosis and genetic counseling.

Mass Spectrometry-Based Glycoproteomics and Prostate Cancer.

Aberrant glycosylation has long been known to be associated with cancer, since it is involved in key mechanisms such as tumour onset, development and progression. This review will focus on protein glycosylation studies in cells, tissue, urine and serum in the context of prostate cancer. A dedicated section will cover the glycoforms of prostate specific antigen, the molecule that, despite some important limitations, is routinely tested for helping prostate cancer diagnosis. Our aim is to provide readers with an overview of mass spectrometry-based glycoproteomics of prostate cancer. From this perspective, the first part of this review will illustrate the main strategies for glycopeptide enrichment and mass spectrometric analysis. The molecular information obtained by glycoproteomic analysis performed by mass spectrometry has led to new insights into the mechanism linking aberrant glycosylation to cancer cell proliferation, migration and immunoescape.

SpeG polyamine acetyltransferase enzyme from Bacillus thuringiensis forms a dodecameric structure and exhibits high catalytic efficiency.

Polyamines are important for regulating biofilms and the exopolysaccharide of the biofilm matrix of Bacillus subtilis. Understanding how enzymes can regulate polyamine concentrations is critical for learning more about how these processes occur in diverse bacteria. Here, we describe the structure and function of another member of the spermidine/spermine acetyltransferases (SSAT) found in Bacilli. The SpeG enzyme from B. thuringiensis (BtSpeG) binds polyamines in its allosteric site and adopts a dodecameric oligomeric state similar to other SpeG enzymes from Gram-negative bacteria. Our kinetic results show the catalytic efficiency of BtSpeG was greater than any previously characterized SpeG to date, and in contrast to other SpeG proteins it exhibited very similar kinetic properties toward both spermine and spermidine. Similar to the SpeG enzyme from E. coli, BtSpeG was able to acetylate spermidine on the N1 and N8 positions. The turnover of BtSpeG toward spermine and spermidine was also two to three orders of magnitude greater than any other Bacilli SSAT enzyme that has been previously characterized. SpeG proteins from Bacilli, including B. cereus, B. thuringiensis and B. anthracis share nearly identical sequences and therefore our results likely provide insight into the structure/function relationship across multiple Bacillus species.

Novel SPEG variant cause centronuclear myopathy in China.

BACKGROUND: Centronuclear myopathy (CNM), a subtype of congenital myopathy (CM), is a group of clinical and genetically heterogeneous muscle disorders. Centronuclear myopathy is a kind of disease difficult to diagnose due to its genetic diversity. Since the discovery of the SPEG gene and disease-causing variants, only a few additional patients have been reported. METHODS: A radiograph test, ultrasonic test, and biochemical tests were applied to clinical diagnosis of CNM. We performed trio medical exome sequencing of the family and conservation analysis to identify variants. RESULTS: We report a pair of severe CNM twins with the same novel homozygous SPEG variant c. 8710A>G (p.Thr2904Ala) identified by clinical trio medical exome sequencing of the family and conservation analysis. The twins showed clinical symptoms of facial weakness, hypotonia, arthrogryposis, strephenopodia, patent ductus arteriosus, and pulmonary arterial hypertension. CONCLUSIONS: Our report expands the clinical and molecular repertoire of CNM and enriches the variant spectrum of the SPEG gene in the Chinese population and helps us further understand the pathogenesis of CNM.

Novel SPEG mutations in congenital myopathies: Genotype-phenotype correlations.

INTRODUCTION: Centronuclear myopathies (CNMs) are a subtype of congenital myopathies (CMs) characterized by muscle weakness, predominant type 1 fibers, and increased central nuclei. SPEG (striated preferentially expressed protein kinase) mutations have recently been identified in 7 CM patients (6 with CNMs). We report 2 additional patients with SPEG mutations expanding the phenotype and evaluate genotype-phenotype correlations associated with SPEG mutations. METHODS: Using whole exome/genome sequencing in CM families, we identified novel recessive SPEG mutations in 2 patients. RESULTS: Patient 1, with severe muscle weakness requiring respiratory support, dilated cardiomyopathy, ophthalmoplegia, and findings of nonspecific CM on muscle biopsy carried a homozygous SPEG mutation (p.Val3062del). Patient 2, with milder muscle weakness, ophthalmoplegia, and CNM carried compound heterozygous mutations (p.Leu728Argfs*82) and (p.Val2997Glyfs*52). CONCLUSIONS: The 2 patients add insight into genotype-phenotype correlations of SPEG-associated CMs. Clinicians should consider evaluating a CM patient for SPEG mutations even in the absence of CNM features. Muscle Nerve 59:357-362, 2019.

Mycosphaerellaceae - Chaos or clarity?

The Mycosphaerellaceae represent thousands of fungal species that are associated with diseases on a wide range of plant hosts. Understanding and stabilising the taxonomy of genera and species of Mycosphaerellaceae is therefore of the utmost importance given their impact on agriculture, horticulture and forestry. Based on previous molecular studies, several phylogenetic and morphologically distinct genera within the Mycosphaerellaceae have been delimited. In this study a multigene phylogenetic analysis (LSU, ITS and rpb2) was performed based on 415 isolates representing 297 taxa and incorporating ex-type strains where available. The main aim of this study was to resolve the phylogenetic relationships among the genera currently recognised within the family, and to clarify the position of the cercosporoid fungi among them. Based on these results many well-known genera are shown to be paraphyletic, with several synapomorphic characters that have evolved more than once within the family. As a consequence, several old generic names including Cercosporidium, Fulvia, Mycovellosiella, Phaeoramularia and Raghnildiana are resurrected, and 32 additional genera are described as new. Based on phylogenetic data 120 genera are now accepted within the family, but many currently accepted cercosporoid genera still remain unresolved pending fresh collections and DNA data. The present study provides a phylogenetic framework for future taxonomic work within the Mycosphaerellaceae.