Publications by authors named "Simone Spinozzi"

10 Publications

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Cardiolipin Remodeling Defects Impair Mitochondrial Architecture and Function in a Murine Model of Barth Syndrome Cardiomyopathy.

Circ Heart Fail 2021 Jun 15;14(6):e008289. Epub 2021 Jun 15.

Department of Medicine (S.Z., Z.C., M.Z., S.S., C.T., Y.G., A.N., W.F., S.M.E., X.F.), University of California, San Diego, La Jolla.

Background: Cardiomyopathy is a major clinical feature in Barth syndrome (BTHS), an X-linked mitochondrial lipid disorder caused by mutations in (), encoding a mitochondrial acyltransferase required for cardiolipin remodeling. Despite recent description of a mouse model of BTHS cardiomyopathy, an in-depth analysis of specific lipid abnormalities and mitochondrial form and function in an in vivo BTHS cardiomyopathy model is lacking.

Methods: We performed in-depth assessment of cardiac function, cardiolipin species profiles, and mitochondrial structure and function in our newly generated cardiomyocyte-specific knockout mice and Cre-negative control mice (n≥3 per group).

Results: cardiomyocyte-specific knockout mice recapitulate typical features of BTHS and mitochondrial cardiomyopathy. Fewer than 5% of cardiomyocyte-specific knockout mice exhibited lethality before 2 months of age, with significantly enlarged hearts. More than 80% of cardiomyocyte-specific knockout displayed ventricular dilation at 16 weeks of age and survived until 50 weeks of age. Full parameter analysis of cardiac cardiolipin profiles demonstrated lower total cardiolipin concentration, abnormal cardiolipin fatty acyl composition, and elevated monolysocardiolipin to cardiolipin ratios in Taz cardiomyocyte-specific knockout, relative to controls. Mitochondrial contact site and cristae organizing system and F1F0-ATP synthase complexes, required for cristae morphogenesis, were abnormal, resulting in onion-shaped mitochondria. Organization of high molecular weight respiratory chain supercomplexes was also impaired. In keeping with observed mitochondrial abnormalities, seahorse experiments demonstrated impaired mitochondrial respiration capacity.

Conclusions: Our mouse model mirrors multiple physiological and biochemical aspects of BTHS cardiomyopathy. Our results give important insights into the underlying cause of BTHS cardiomyopathy and provide a framework for testing therapeutic approaches to BTHS cardiomyopathy, or other mitochondrial-related cardiomyopathies.
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http://dx.doi.org/10.1161/CIRCHEARTFAILURE.121.008289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8210459PMC
June 2021

Calpains for dummies: What you need to know about the calpain family.

Biochim Biophys Acta Proteins Proteom 2021 05 3;1869(5):140616. Epub 2021 Feb 3.

Genethon, 1 bis, Rue de l'Internationale - 91000 Evry, France; Université Paris-Saclay, Univ Evry, Inserm, Genethon, Integrare Research Unit UMR_S951, 91000, Evry, France. Electronic address:

This review was written in memory of our late friend, Dr. Hiroyuki Sorimachi, who, following the steps of his mentor Koichi Suzuki, a pioneer in calpain research, has made tremendous contributions to the field. During his career, Hiro also wrote several reviews on calpain, the last of which, published in 2016, was comprehensive. In this manuscript, we decided to put together a review with the basic information a novice may need to know about calpains. We also tried to avoid similarities with previous reviews and reported the most significant new findings, at the same time highlighting Hiro's contributions to the field. The review will cover a short history of calpain discovery, the presentation of the family, the life of calpain from transcription to activity, human diseases caused by calpain mutations and therapeutic perspectives.
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http://dx.doi.org/10.1016/j.bbapap.2021.140616DOI Listing
May 2021

Homozygous G650del nexilin variant causes cardiomyopathy in mice.

JCI Insight 2020 08 20;5(16). Epub 2020 Aug 20.

Department of Medicine, UCSD, La Jolla, California, USA.

Nexilin (NEXN) was recently identified as a component of the junctional membrane complex required for development and maintenance of cardiac T-tubules. Loss of Nexn in mice leads to a rapidly progressive dilated cardiomyopathy (DCM) and premature death. A 3 bp deletion (1948-1950del) leading to loss of the glycine in position 650 (G650del) is classified as a variant of uncertain significance in humans and may function as an intermediate risk allele. To determine the effect of the G650del variant on cardiac structure and function, we generated a G645del-knockin (G645del is equivalent to human G650del) mouse model. Homozygous G645del mice express about 30% of the Nexn expressed by WT controls and exhibited a progressive DCM characterized by reduced T-tubule formation, with disorganization of the transverse-axial tubular system. On the other hand, heterozygous Nexn global KO mice and genetically engineered mice encoding a truncated Nexn missing the first N-terminal actin-binding domain exhibited normal cardiac function, despite expressing only 50% and 20% of the Nexn, respectively, expressed by WT controls, suggesting that not only quantity but also quality of Nexn is necessary for a proper function. These findings demonstrated that Nexn G645 is crucial for Nexn's function in tubular system organization and normal cardiac function.
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http://dx.doi.org/10.1172/jci.insight.138780DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7455123PMC
August 2020

Nexilin Is Necessary for Maintaining the Transverse-Axial Tubular System in Adult Cardiomyocytes.

Circ Heart Fail 2020 07 8;13(7):e006935. Epub 2020 Jul 8.

Department of Medicine (S.S., C.L., Z.C., W.F., L.Z., S.M.E., J.C.), University of California San Diego, La Jolla.

Background: NEXN (nexilin) is a protein of the junctional membrane complex required for development of cardiac T-tubules. Global and cardiomyocyte-specific loss of in mice leads to a rapidly progressive dilated cardiomyopathy and premature death. Therefore, little is known as to the role of NEXN in adult cardiomyocytes. Transverse-axial tubular system remodeling are well-known features in heart failure. Although NEXN is required during development for T-tubule formation, its role, if any, in mature T-tubules remains to be addressed.

Methods: inducible adult cardiomyocyte-specific KO mice were generated. Comprehensive morphological and functional analyses were performed. Heart samples (n>3) were analyzed by molecular, biochemical, and electron microscopy analyses. Isolated single adult cardiomyocytes were analyzed by confocal microscopy, and myocyte shortening/re-lengthening and Ca transient studies were conducted.

Results: Inducible cardiomyocyte-specific loss of in adult mice resulted in a dilated cardiomyopathy with reduced cardiac function (13% reduction in percentage fractional shortening; <0.05). In vivo and in vitro analyses of adult mouse heart samples revealed that NEXN was essential for optimal contraction and calcium handling and was required for maintenance of T-tubule network organization (transverse tubular component in inducible adult cardiomyocyte-specific KO mice reduced by 40% with respect to controls, <0.05).

Conclusions: Results here reported reveal NEXN to be a pivotal component of adult junctional membrane complexes required for maintenance of transverse-axial tubular architecture. These results demonstrate that NEXN plays an essential role in the adult cardiomyocyte and give further understanding of pathological mechanisms responsible for cardiomyopathy in patients carrying mutations in the NEXN gene.
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http://dx.doi.org/10.1161/CIRCHEARTFAILURE.120.006935DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7583668PMC
July 2020

Atypical ALPK2 kinase is not essential for cardiac development and function.

Am J Physiol Heart Circ Physiol 2020 06 8;318(6):H1509-H1515. Epub 2020 May 8.

Department of Medicine, University of California, San Diego, La Jolla, California.

Protein kinases play an integral role in cardiac development, function, and disease. Recent experimental and clinical data have implied that protein kinases belonging to a family of atypical α-protein kinases, including α-protein kinase 2 (ALPK2), are important for regulating cardiac development and maintaining function via regulation of WNT signaling. A recent study in zebrafish reported that loss of ALPK2 leads to severe cardiac defects; however, the relevance of ALPK2 has not been studied in a mammalian animal model. To assess the role of ALPK2 in the mammalian heart, we generated two independent global Alpk2-knockout (Alpk2-gKO) mouse lines, using CRISPR/Cas9 technology. We performed physiological and biochemical analyses of Alpk2-gKO mice to determine the functional, morphological, and molecular consequences of Alpk2 deletion at the organismal level. We found that Alpk2-gKO mice exhibited normal cardiac function and morphology up to one year of age. Moreover, we did not observe altered WNT signaling in neonatal Alpk2-gKO mouse hearts. In conclusion, Alpk2 is dispensable for cardiac development and function in the murine model. Our results suggest that Alpk2 is a rapidly evolving gene that lost its essential cardiac functions in mammals. Several studies indicated the importance of ALPK2 for cardiac function and development. A recent study in zebrafish report that loss of ALPK2 leads to severe cardiac defects. In contrast, murine Alpk2-gKO models developed in this work display no overt cardiac phenotype. Our results suggest ALPK2, as a rapidly evolving gene, lost its essential cardiac functions in mammals.
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http://dx.doi.org/10.1152/ajpheart.00249.2020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7311700PMC
June 2020

Identifying the Cardiac Dyad Proteome In Vivo by a BioID2 Knock-In Strategy.

Circulation 2020 03 16;141(11):940-942. Epub 2020 Mar 16.

Department of Medicine (W.F., C.L., S.S., S.M.E.), University of California, San Diego, La Jolla.

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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.043434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7100982PMC
March 2020

Parallel Lineage-Tracing Studies Establish Fibroblasts as the Prevailing In Vivo Adipocyte Progenitor.

Cell Rep 2020 01;30(2):571-582.e2

Institute of Cardiovascular Regeneration, Goethe University, Frankfurt 60590, Germany; German Center for Cardiovascular Research, Berlin (partner site Frankfurt Rhine-Main), Germany. Electronic address:

Despite decades of studies suggesting that the in vivo adipocyte progenitor resides within the vascular niche, the exact nature of this progenitor remains controversial because distinct studies have attributed adipogenic properties to multiple vascular cell types. Using Cre recombinases labeling distinct vascular lineages, we conduct parallel lineage tracing experiments to assess their degree of contribution to de novo adipogenesis. Although we detect occasional adipocytes that were lineage traced by endothelial or mural recombinases, these are rare events. On the other hand, platelet-derived growth factor receptor alpha (PDGFRα)-expressing adventitial or capsular fibroblasts make a significant contribution to adipocytes in all depots and experimental settings tested. Our data also suggest that fibroblasts transition to an intermediate beige adipocyte phenotype prior to differentiating to a mature white adipocyte. These observations, together with histological analyses revealing that adipose tissue fibroblasts express the mural cell marker PDGFRβ, harmonize a highly controversial field with implications for multiple human diseases, including the pandemic of obesity.
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http://dx.doi.org/10.1016/j.celrep.2019.12.046DOI Listing
January 2020

Nexilin Is a New Component of Junctional Membrane Complexes Required for Cardiac T-Tubule Formation.

Circulation 2019 07 15;140(1):55-66. Epub 2019 Apr 15.

Department of Medicine (C.L., S.S., X.F., W.F., N.D.D., K.L.P., T.W., S.M.E., J.C.), University of California, San Diego, La Jolla.

Background: Membrane contact sites are fundamental for transmission and translation of signals in multicellular organisms. The junctional membrane complexes in the cardiac dyads, where transverse (T) tubules are juxtaposed to the sarcoplasmic reticulum, are a prime example. T-tubule uncoupling and remodeling are well-known features of cardiac disease and heart failure. Even subtle alterations in the association between T-tubules and the junctional sarcoplasmic reticulum can cause serious cardiac disorders. NEXN (nexilin) has been identified as an actin-binding protein, and multiple mutations in the NEXN gene are associated with cardiac diseases, but the precise role of NEXN in heart function and disease is still unknown.

Methods: Nexn global and cardiomyocyte-specific knockout mice were generated. Comprehensive phenotypic and RNA sequencing and mass spectrometry analyses were performed. Heart tissue samples and isolated single cardiomyocytes were analyzed by electron and confocal microscopy.

Results: Global and cardiomyocyte-specific loss of Nexn in mice resulted in a rapidly progressive dilated cardiomyopathy. In vivo and in vitro analyses revealed that NEXN interacted with junctional sarcoplasmic reticulum proteins, was essential for optimal calcium transients, and was required for initiation of T-tubule invagination and formation.

Conclusions: These results demonstrated that NEXN is a pivotal component of the junctional membrane complex and is required for initiation and formation of T-tubules, thus providing insight into mechanisms underlying cardiomyopathy in patients with mutations in NEXN.
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http://dx.doi.org/10.1161/CIRCULATIONAHA.119.039751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6889818PMC
July 2019

Identification and characterization of three novel mutations in the CASQ1 gene in four patients with tubular aggregate myopathy.

Hum Mutat 2017 12 26;38(12):1761-1773. Epub 2017 Sep 26.

Department of Molecular and Developmental Medicine, Molecular Medicine Section, University of Siena, Siena, Italy.

Here, we report the identification of three novel missense mutations in the calsequestrin-1 (CASQ1) gene in four patients with tubular aggregate myopathy. These CASQ1 mutations affect conserved amino acids in position 44 (p.(Asp44Asn)), 103 (p.(Gly103Asp)), and 385 (p.(Ile385Thr)). Functional studies, based on turbidity and dynamic light scattering measurements at increasing Ca concentrations, showed a reduced Ca -dependent aggregation for the CASQ1 protein containing p.Asp44Asn and p.Gly103Asp mutations and a slight increase in Ca -dependent aggregation for the p.Ile385Thr. Accordingly, limited trypsin proteolysis assay showed that p.Asp44Asn and p.Gly103Asp were more susceptible to trypsin cleavage in the presence of Ca in comparison with WT and p.Ile385Thr. Analysis of single muscle fibers of a patient carrying the p.Gly103Asp mutation showed a significant reduction in response to caffeine stimulation, compared with normal control fibers. Expression of CASQ1 mutations in eukaryotic cells revealed a reduced ability of all these CASQ1 mutants to store Ca and a reduced inhibitory effect of p.Ile385Thr and p.Asp44Asn on store operated Ca entry. These results widen the spectrum of skeletal muscle diseases associated with CASQ1 and indicate that these mutations affect properties critical for correct Ca handling in skeletal muscle fibers.
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http://dx.doi.org/10.1002/humu.23338DOI Listing
December 2017

A mutation in the CASQ1 gene causes a vacuolar myopathy with accumulation of sarcoplasmic reticulum protein aggregates.

Hum Mutat 2014 Oct 10;35(10):1163-70. Epub 2014 Sep 10.

Molecular Medicine Section, Department of Molecular and Developmental Medicine, University of Siena and Azienda Ospedaliera Universitaria Senese, Siena, 53100, Italy; IIM, Interuniversity Institute of Myology.

A missense mutation in the calsequestrin-1 gene (CASQ1) was found in a group of patients with a myopathy characterized by weakness, fatigue, and the presence of large vacuoles containing characteristic inclusions resulting from the aggregation of sarcoplasmic reticulum (SR) proteins. The mutation affects a conserved aspartic acid in position 244 (p.Asp244Gly) located in one of the high-affinity Ca(2+) -binding sites of CASQ1 and alters the kinetics of Ca(2+) release in muscle fibers. Expression of the mutated CASQ1 protein in COS-7 cells showed a markedly reduced ability in forming elongated polymers, whereas both in cultured myotubes and in in vivo mouse fibers induced the formation of electron-dense SR vacuoles containing aggregates of the mutant CASQ1 protein that resemble those observed in muscle biopsies of patients. Altogether, these results support the view that a single missense mutation in the CASQ1 gene causes the formation of abnormal SR vacuoles containing aggregates of CASQ1, and other SR proteins, results in altered Ca(2+) release in skeletal muscle fibers, and, hence, is responsible for the clinical phenotype observed in these patients.
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http://dx.doi.org/10.1002/humu.22631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4177304PMC
October 2014
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