301 results on '"Leinwand LA"'
Search Results
2. Plasma membrane calcium-ATPase inhibits cardiomyocyte hypertrophy by interaction with calcineurin and induces apoptosis by a calmodulin-dependent mechanism
- Author
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Maass, AH, Leinwand, LA, and Cardiovascular Centre (CVC)
- Published
- 2005
3. Mechanisms of sudden cardiac death after adrenergic stress in mouse models of hypertrophic cardiomyopathy
- Author
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Maier, SK, Scheuer, T, Feigl, EO, Leinwand, LA, Catterall, WA, Maass, AH, and Cardiovascular Centre (CVC)
- Published
- 2004
4. Corrigenda for vol. Cardiac and skeletal muscle adaptations to voluntary wheel running in the mouse (vol 90, pg 1900, 2001)
- Author
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Allan, DL, Harrison, BC, Maass, A, Bell, ML, Byrnes, WC, Leinwand, LA, and Cardiovascular Centre (CVC)
- Published
- 2001
5. Overexpression of the plasma membrane calcium ATPase isoform 4b dose-dependently affects growth and survival of neonatal rat cardiomyocytes
- Author
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Maass, A, Oberdorf-Maass, S, Langer, SJ, Leinwand, LA, and Cardiovascular Centre (CVC)
- Published
- 2001
6. Cardiac and skeletal muscle adaptations to voluntary wheel running in the mouse (vol 90, pg 1900, 2001)
- Author
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Allan, DL, Harrison, BC, Maass, A, Bell, ML, Byrnes, WC, Leinwand, LA, and Cardiovascular Centre (CVC)
- Published
- 2001
7. Voluntary wheel running leads to cardiac adaptation in troponin T mutant and wild type mice
- Author
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Maass, A, Oberdorf-Maass, S, Leinwand, LA, and Cardiovascular Centre (CVC)
- Published
- 2000
8. Right ventricular function and failure: report of a National Heart, Lung, and Blood Institute working group on cellular and molecular mechanisms of right heart failure.
- Author
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Voelkel NF, Quaife RA, Leinwand LA, Barst RJ, McGoon MD, Meldrum DR, Dupuis J, Long CS, Rubin LJ, Smart FW, Suzuki YJ, Gladwin M, Denholm EM, Gail DB, and US National Heart, Lung, and Blood Institute. Working Group on Cellular and Molecular Mechanisms of Right Heart Failure
- Published
- 2006
- Full Text
- View/download PDF
9. Hypertrophy, fibrosis, and sudden cardiac death in response to pathological stimuli in mice with mutations in cardiac troponin T.
- Author
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Maass AH, Ikeda K, Oberdorf-Maass S, Maier SKG, and Leinwand LA
- Published
- 2004
- Full Text
- View/download PDF
10. Molecular regulation of reversible cardiac remodeling: lessons from species with extreme physiological adaptations.
- Author
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Martin TG and Leinwand LA
- Subjects
- Animals, Ventricular Remodeling physiology, Heart physiology, Cardiomegaly physiopathology, Boidae physiology, Adaptation, Physiological
- Abstract
Some vertebrates evolved to have a remarkable capacity for anatomical and physiological plasticity in response to environmental challenges. One example of such plasticity can be found in the ambush-hunting snakes of the genus Python, which exhibit reversible cardiac growth with feeding. The predation strategy employed by pythons is associated with months-long fasts that are arrested by ingestion of large prey. Consequently, digestion compels a dramatic increase in metabolic rate and hypertrophy of multiple organs, including the heart. In this Review, we summarize the post-prandial cardiac adaptations in pythons at the whole-heart, cellular and molecular scales. We highlight circulating factors and cellular signaling pathways that are altered during digestion to affect cardiac form and function and propose possible mechanisms that may drive the post-digestion regression of cardiac mass. Adaptive physiological cardiac hypertrophy has also been observed in other vertebrates, including in fish acclimated to cold water, birds flying at high altitudes and exercising mammals. To reveal potential evolutionarily conserved features, we summarize the molecular signatures of reversible cardiac remodeling identified in these species and compare them with those of pythons. Finally, we offer a perspective on the potential of biomimetics targeting the natural biology of pythons as therapeutics for human heart disease., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)
- Published
- 2024
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- View/download PDF
11. Regression of postprandial cardiac hypertrophy in burmese pythons is mediated by FoxO1.
- Author
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Martin TG, Hunt DR, Langer SJ, Tan Y, Ebmeier CC, and Leinwand LA
- Subjects
- Animals, Autophagy, Myocytes, Cardiac metabolism, Postprandial Period, Signal Transduction, Transcriptome, Boidae, Forkhead Box Protein O1 metabolism, Forkhead Box Protein O1 genetics, Heart physiology
- Abstract
As ambush-hunting predators that consume large prey after long intervals of fasting, Burmese pythons evolved with unique adaptations for modulating organ structure and function. Among these is cardiac hypertrophy that develops within three days following a meal (Andersen et al., 2005, Secor, 2008), which we previously showed was initiated by circulating growth factors (Riquelme et al., 2011). Postprandial cardiac hypertrophy in pythons also rapidly regresses with subsequent fasting (Secor, 2008); however, the molecular mechanisms that regulate the dynamic cardiac remodeling in pythons during digestion are largely unknown. In this study, we employed a multiomics approach coupled with targeted molecular analyses to examine remodeling of the python ventricular transcriptome and proteome throughout digestion. We found that forkhead box protein O1 (FoxO1) signaling was suppressed prior to hypertrophy development and then activated during regression, which coincided with decreased and then increased expression, respectively, of FoxO1 transcriptional targets involved in proteolysis. To define the molecular mechanistic role of FoxO1 in hypertrophy regression, we used cultured mammalian cardiomyocytes treated with postfed python plasma. Hypertrophy regression both in pythons and in vitro coincided with activation of FoxO1-dependent autophagy; however, the introduction of a FoxO1-specific inhibitor prevented both regression of cell size and autophagy activation. Finally, to determine whether FoxO1 activation could induce regression, we generated an adenovirus expressing a constitutively active FoxO1. FoxO1 activation was sufficient to prevent and reverse postfed plasma-induced hypertrophy, which was partially prevented by autophagy inhibition. Our results indicate that modulation of FoxO1 activity contributes to the dynamic ventricular remodeling in postprandial Burmese pythons., Competing Interests: Competing interests statement:L.A.L. is a Co-Founder of MyoKardia, acquired by Bristol Myers Squibb. MyoKardia and Bristol Myers Squibb were not involved in this study. The other authors have no competing interests to disclose.
- Published
- 2024
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12. Postprandial cardiac hypertrophy is sustained by mechanics, epigenetic, and metabolic reprogramming in pythons.
- Author
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Crocini C, Woulfe KC, Ozeroff CD, Perni S, Cardiello J, Walker CJ, Wilson CE, Anseth K, Allen MA, and Leinwand LA
- Subjects
- Animals, Postprandial Period physiology, Energy Metabolism, Myofibrils metabolism, Calcium metabolism, Adaptation, Physiological, Myocardium metabolism, Metabolic Reprogramming, Cardiomegaly metabolism, Cardiomegaly genetics, Cardiomegaly physiopathology, Boidae physiology, Boidae genetics, Epigenesis, Genetic
- Abstract
Constricting pythons, known for their ability to consume infrequent, massive meals, exhibit rapid and reversible cardiac hypertrophy following feeding. Our primary goal was to investigate how python hearts achieve this adaptive response after feeding. Isolated myofibrils increased force after feeding without changes in sarcomere ultrastructure and without increasing energy cost. Ca
2+ transients were prolonged after feeding with no changes in myofibril Ca2+ sensitivity. Feeding reduced titin-based tension, resulting in decreased cardiac tissue stiffness. Feeding also reduced the activity of sirtuins, a metabolically linked class of histone deacetylases, and increased chromatin accessibility. Transcription factor enrichment analysis on transposase-accessible chromatin with sequencing revealed the prominent role of transcription factors Yin Yang1 and NRF1 in postfeeding cardiac adaptation. Gene expression also changed with the enrichment of translation and metabolism. Finally, metabolomics analysis and adenosine triphosphate production demonstrated that cardiac adaptation after feeding not only increased energy demand but also energy production. These findings have broad implications for our understanding of cardiac adaptation across species and hold promise for the development of innovative approaches to address cardiovascular diseases., Competing Interests: Competing interests statement:The authors declare no competing interest.- Published
- 2024
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13. Hearts apart: sex differences in cardiac remodeling in health and disease.
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Martin TG and Leinwand LA
- Subjects
- Humans, Female, Male, Animals, Heart Diseases pathology, Heart Diseases metabolism, Heart Diseases physiopathology, Heart Diseases genetics, Gonadal Steroid Hormones metabolism, Heart physiopathology, Heart physiology, Myocardium pathology, Myocardium metabolism, Sex Characteristics, Ventricular Remodeling
- Abstract
Biological sex is an important modifier of physiology and influences pathobiology in many diseases. While heart disease is the number one cause of death worldwide in both men and women, sex differences exist at the organ and cellular scales, affecting clinical presentation, diagnosis, and treatment. In this Review, we highlight baseline sex differences in cardiac structure, function, and cellular signaling and discuss the contribution of sex hormones and chromosomes to these characteristics. The heart is a remarkably plastic organ and rapidly responds to physiological and pathological cues by modifying form and function. The nature and extent of cardiac remodeling in response to these stimuli are often dependent on biological sex. We discuss organ- and molecular-level sex differences in adaptive physiological remodeling and pathological cardiac remodeling from pressure and volume overload, ischemia, and genetic heart disease. Finally, we offer a perspective on key future directions for research into cardiac sex differences.
- Published
- 2024
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14. Turn(over) the Page: Advancing Understanding of Proteome Dynamics After Heart Attack.
- Author
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Martin TG and Leinwand LA
- Abstract
Competing Interests: Dr Leinwand acknowledges support from the National Institutes of Health (R01GM029090). Dr Martin acknowledges support from the National Institutes of Health (F32HL170637). Dr Leinwand is a co-founder of MyoKardia, acquired by Bristol Myers Squibb. MyoKardia and Bristol Myers Squibb were not involved in this study. Dr Martin has reported that he has no relationships relevant to the contents of this paper to disclose.
- Published
- 2024
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15. A Laing distal myopathy-associated proline substitution in the β-myosin rod perturbs myosin cross-bridging activity.
- Author
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Buvoli M, Wilson GC, Buvoli A, Gugel JF, Hau A, Bönnemann CG, Paradas C, Ryba DM, Woulfe KC, Walker LA, Buvoli T, Ochala J, and Leinwand LA
- Subjects
- Animals, Mice, Humans, Mutation, Missense, Cardiac Myosins genetics, Cardiac Myosins metabolism, Myosin Heavy Chains genetics, Myosin Heavy Chains metabolism, Myosin Heavy Chains chemistry, Female, Male, Mice, Transgenic, Muscle Contraction genetics, Muscle, Skeletal metabolism, Muscle, Skeletal pathology, Proline genetics, Proline metabolism, Distal Myopathies genetics, Distal Myopathies metabolism, Distal Myopathies pathology, Amino Acid Substitution
- Abstract
Proline substitutions within the coiled-coil rod region of the β-myosin gene (MYH7) are the predominant mutations causing Laing distal myopathy (MPD1), an autosomal dominant disorder characterized by progressive weakness of distal/proximal muscles. We report that the MDP1 mutation R1500P, studied in what we believe to be the first mouse model for the disease, adversely affected myosin motor activity despite being in the structural rod domain that directs thick filament assembly. Contractility experiments carried out on isolated mutant muscles, myofibrils, and myofibers identified muscle fatigue and weakness phenotypes, an increased rate of actin-myosin detachment, and a conformational shift of the myosin heads toward the more reactive disordered relaxed (DRX) state, causing hypercontractility and greater ATP consumption. Similarly, molecular analysis of muscle biopsies from patients with MPD1 revealed a significant increase in sarcomeric DRX content, as observed in a subset of myosin motor domain mutations causing hypertrophic cardiomyopathy. Finally, oral administration of MYK-581, a small molecule that decreases the population of heads in the DRX configuration, significantly improved the limited running capacity of the R1500P-transgenic mice and corrected the increased DRX state of the myofibrils from patients. These studies provide evidence of the molecular pathogenesis of proline rod mutations and lay the groundwork for the therapeutic advancement of myosin modulators.
- Published
- 2024
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16. Homologous mutations in human β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation.
- Author
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Liu C, Karabina A, Meller A, Bhattacharjee A, Agostino CJ, Bowman GR, Ruppel KM, Spudich JA, and Leinwand LA
- Subjects
- Humans, Adenosine Triphosphatases metabolism, Mutation, Actins metabolism, Muscle, Skeletal metabolism, Ventricular Myosins genetics, Myosins metabolism
- Abstract
Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in β-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human β, embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins but minimal effects in β myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing overall enzymatic (ATPase) cycle rate. In contrast, the only measured effect of R671C in β myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not β, myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are a testament to myosin's highly allosteric nature., Competing Interests: Competing interests statement:J.A.S. is cofounder and on the Scientific Advisory Board of Cytokinetics, Inc., a company developing small molecule therapeutics for treatment of HCM. J.A.S. is cofounder and CEO, and K.M.R. is cofounder and Research and Clinical Advisor, of Kainomyx, Inc., a company developing small molecule therapeutics targeting myosins in parasites. G.R.B. is cofounder and equity holder in Decrypt Biomedicine.
- Published
- 2024
- Full Text
- View/download PDF
17. A Conserved Mechanism of Cardiac Hypertrophy Regression through FoxO1.
- Author
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Martin TG, Hunt DR, Langer SJ, Tan Y, Ebmeier CC, Crocini C, Chung E, and Leinwand LA
- Abstract
The heart is a highly plastic organ that responds to diverse stimuli to modify form and function. The molecular mechanisms of adaptive physiological cardiac hypertrophy are well-established; however, the regulation of hypertrophy regression is poorly understood. To identify molecular features of regression, we studied Burmese pythons which experience reversible cardiac hypertrophy following large, infrequent meals. Using multi-omics screens followed by targeted analyses, we found forkhead box protein O1 (FoxO1) transcription factor signaling, and downstream autophagy activity, were downregulated during hypertrophy, but re-activated with regression. To determine whether these events were mechanistically related to regression, we established an in vitro platform of cardiomyocyte hypertrophy and regression from treatment with fed python plasma. FoxO1 inhibition prevented regression in this system, while FoxO1 activation reversed fed python plasma-induced hypertrophy in an autophagy-dependent manner. We next examined whether FoxO1 was implicated in mammalian models of reversible hypertrophy from exercise and pregnancy and found that in both cases FoxO1 was activated during regression. In these models, as in pythons, activation of FoxO1 was associated with increased expression FoxO1 target genes involved in autophagy. Taken together, our findings suggest FoxO1-dependent autophagy is a conserved mechanism for regression of physiological cardiac hypertrophy across species., Competing Interests: COMPETING INTERESTS LAL is a Co-Founder of MyoKardia, acquired by Bristol Myers Squibb. MyoKardia and Bristol Myers Squibb were not involved in this study. The other authors have no competing interests to disclose.
- Published
- 2024
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18. Assessment of Autophagy Markers Suggests Increased Activity Following LVAD Therapy.
- Author
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Martin TG, Juarros MA, Cleveland JC Jr, Bristow MR, Ambardekar AV, Buttrick PM, and Leinwand LA
- Abstract
Left ventricular reverse remodeling in heart failure is associated with improved clinical outcomes. However, the molecular features that drive this process are poorly defined. Left ventricular assist devices (LVADs) are the therapy associated with the greatest reverse remodeling and lead to partial myocardial recovery in most patients. In this study, we examined whether autophagy may be implicated in post-LVAD reverse remodeling. We found expression of key autophagy factors increased post-LVAD, while autophagic substrates decreased. Autolysosome numbers increased post-LVAD, further indicating increased autophagy. These findings support the conclusion that mechanical unloading activates autophagy, which may underly the reverse remodeling observed., Competing Interests: The authors have received support from the National Institute of Health (R01HL117138 and R01GM029090 to Dr Leinwand, T32 HL007822 to Dr Martin, and T32 GM142607 to Dr Juarros). REDCap was provided by National Institutes of Health/NCATS Colorado CTSA Grant Number UL1 TR002535. The contents are the authors’ sole responsibility and do not necessarily represent the official views of the National Institutes of Health. Dr Leinwand is a cofounder of MyoKardia, acquired by Bristol Myers Squibb; and is a paid member of their Scientific Advisory Board. All other authors have reported that they have no relationships relevant to the contents of this paper to disclose., (© 2023 The Authors.)
- Published
- 2023
- Full Text
- View/download PDF
19. Homologous mutations in β , embryonic, and perinatal muscle myosins have divergent effects on molecular power generation.
- Author
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Liu C, Karabina A, Meller A, Bhattacharjee A, Agostino CJ, Bowman GR, Ruppel KM, Spudich JA, and Leinwand LA
- Abstract
Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in β -cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known if their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human β , embryonic, and perinatal myosin subfragment-1. We found large effects in the developmental myosins, with the most dramatic in perinatal, but minimal effects in β myosin, and magnitude of changes correlated partially with clinical severity. The mutations in the developmental myosins dramatically decreased the step size and load-sensitive actin-detachment rate of single molecules measured by optical tweezers, in addition to decreasing ATPase cycle rate. In contrast, the only measured effect of R671C in β myosin was a larger step size. Our measurements of step size and bound times predicted velocities consistent with those measured in an in vitro motility assay. Finally, molecular dynamics simulations predicted that the arginine to cysteine mutation in embryonic, but not β , myosin may reduce pre-powerstroke lever arm priming and ADP pocket opening, providing a possible structural mechanism consistent with the experimental observations. This paper presents the first direct comparisons of homologous mutations in several different myosin isoforms, whose divergent functional effects are yet another testament to myosin's highly allosteric nature., Competing Interests: Competing interest statement J.A.S. is cofounder and on the Scientific Advisory Board of Cytokinetics, Inc., a company developing small molecule therapeutics for treatment of hypertrophic cardiomyopathy. J.A.S. is cofounder and CEO, and K.M.R. is cofounder and Research and Clinical Advisor, of Kainomyx, Inc., a company developing small molecule therapeutics targeting myosins in parasites. G.R.B. is cofounder and equity holder in Decrypt Biomedicine.
- Published
- 2023
- Full Text
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20. Utility of the burmese Python as a model for studying plasticity of extreme physiological systems.
- Author
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Tan Y, Martin TG, Harrison BC, and Leinwand LA
- Subjects
- Animals, Mammals, Myanmar, Boidae genetics, Boidae metabolism, Boidae physiology, Postprandial Period physiology
- Abstract
Non-traditional animal models present an opportunity to discover novel biology that has evolved to allow such animals to survive in extreme environments. One striking example is the Burmese python (Python molurus bivittatus), which exhibits extreme physiological adaptation in various metabolic organs after consuming a large meal following long periods of fasting. The response to such a large meal in pythons involves a dramatic surge in metabolic rate, lipid overload in plasma, and massive but reversible organ growth through the course of digestion. Multiple studies have reported the physiological responses in post-prandial pythons, while the specific molecular control of these processes is less well-studied. Investigating the mechanisms that coordinate organ growth and adaptive responses offers the opportunity to gain novel insight that may be able to treat various pathologies in humans. Here, we summarize past research on the post-prandial physiological changes in the Burmese python with a focus on the gastrointestinal tract, heart, and liver. Specifically, we address our recent molecular discoveries in the post-prandial python liver which demonstrate transient adaptations that may reveal new therapeutic targets. Lastly, we explore new biology of the aquaporin 7 gene that is potently upregulated in mammalian cardiac myocytes by circulating factors in post-prandial python plasma., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)
- Published
- 2023
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21. Modulating fast skeletal muscle contraction protects skeletal muscle in animal models of Duchenne muscular dystrophy.
- Author
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Russell AJ, DuVall M, Barthel B, Qian Y, Peter AK, Newell-Stamper BL, Hunt K, Lehman S, Madden M, Schlachter S, Robertson B, Van Deusen A, Rodriguez HM, Vera C, Su Y, Claflin DR, Brooks SV, Nghiem P, Rutledge A, Juehne TI, Yu J, Barton ER, Luo YE, Patsalos A, Nagy L, Sweeney HL, Leinwand LA, and Koch K
- Subjects
- Mice, Animals, Dogs, Mice, Inbred mdx, Muscle, Skeletal metabolism, Dystrophin genetics, Muscle Contraction physiology, Disease Models, Animal, Muscular Dystrophy, Duchenne metabolism, Muscular Dystrophy, Animal genetics, Muscular Dystrophy, Animal metabolism
- Abstract
Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by absence of the protein dystrophin, which acts as a structural link between the basal lamina and contractile machinery to stabilize muscle membranes in response to mechanical stress. In DMD, mechanical stress leads to exaggerated membrane injury and fiber breakdown, with fast fibers being the most susceptible to damage. A major contributor to this injury is muscle contraction, controlled by the motor protein myosin. However, how muscle contraction and fast muscle fiber damage contribute to the pathophysiology of DMD has not been well characterized. We explored the role of fast skeletal muscle contraction in DMD with a potentially novel, selective, orally active inhibitor of fast skeletal muscle myosin, EDG-5506. Surprisingly, even modest decreases of contraction (<15%) were sufficient to protect skeletal muscles in dystrophic mdx mice from stress injury. Longer-term treatment also decreased muscle fibrosis in key disease-implicated tissues. Importantly, therapeutic levels of myosin inhibition with EDG-5506 did not detrimentally affect strength or coordination. Finally, in dystrophic dogs, EDG-5506 reversibly reduced circulating muscle injury biomarkers and increased habitual activity. This unexpected biology may represent an important alternative treatment strategy for Duchenne and related myopathies.
- Published
- 2023
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22. Regression of cardiac hypertrophy in health and disease: mechanisms and therapeutic potential.
- Author
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Martin TG, Juarros MA, and Leinwand LA
- Subjects
- Animals, Female, Cardiomegaly metabolism, Exercise physiology, Risk Factors, Ventricular Remodeling, Quality of Life, MicroRNAs therapeutic use
- Abstract
Left ventricular hypertrophy is a leading risk factor for cardiovascular morbidity and mortality. Although reverse ventricular remodelling was long thought to be irreversible, evidence from the past three decades indicates that this process is possible with many existing heart disease therapies. The regression of pathological hypertrophy is associated with improved cardiac function, quality of life and long-term health outcomes. However, less than 50% of patients respond favourably to most therapies, and the reversibility of remodelling is influenced by many factors, including age, sex, BMI and disease aetiology. Cardiac hypertrophy also occurs in physiological settings, including pregnancy and exercise, although in these cases, hypertrophy is associated with normal or improved ventricular function and is completely reversible postpartum or with cessation of training. Studies over the past decade have identified the molecular features of hypertrophy regression in health and disease settings, which include modulation of protein synthesis, microRNAs, metabolism and protein degradation pathways. In this Review, we summarize the evidence for hypertrophy regression in patients with current first-line pharmacological and surgical interventions. We further discuss the molecular features of reverse remodelling identified in cell and animal models, highlighting remaining knowledge gaps and the essential questions for future investigation towards the goal of designing specific therapies to promote regression of pathological hypertrophy., (© 2023. Springer Nature Limited.)
- Published
- 2023
- Full Text
- View/download PDF
23. Distinct effects of two hearing loss-associated mutations in the sarcomeric myosin MYH7b.
- Author
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Lee LA, Barrick SK, Buvoli AE, Walklate J, Stump WT, Geeves M, Greenberg MJ, and Leinwand LA
- Subjects
- Animals, Humans, Mice, Actins metabolism, Cell Line, Chlorocebus aethiops, COS Cells, Kinetics, Mutation, Protein Aggregates genetics, Recombinant Proteins genetics, Recombinant Proteins metabolism, Hearing Loss genetics, Hearing Loss physiopathology, Myosin Heavy Chains genetics, Myosin Heavy Chains metabolism
- Abstract
For decades, sarcomeric myosin heavy chain proteins were assumed to be restricted to striated muscle where they function as molecular motors that contract muscle. However, MYH7b, an evolutionarily ancient member of this myosin family, has been detected in mammalian nonmuscle tissues, and mutations in MYH7b are linked to hereditary hearing loss in compound heterozygous patients. These mutations are the first associated with hearing loss rather than a muscle pathology, and because there are no homologous mutations in other myosin isoforms, their functional effects were unknown. We generated recombinant human MYH7b harboring the D515N or R1651Q hearing loss-associated mutation and studied their effects on motor activity and structural and assembly properties, respectively. The D515N mutation had no effect on steady-state actin-activated ATPase rate or load-dependent detachment kinetics but increased actin sliding velocity because of an increased displacement during the myosin working stroke. Furthermore, we found that the D515N mutation caused an increase in the proportion of myosin heads that occupy the disordered-relaxed state, meaning more myosin heads are available to interact with actin. Although we found no impact of the R1651Q mutation on myosin rod secondary structure or solubility, we observed a striking aggregation phenotype when this mutation was introduced into nonmuscle cells. Our results suggest that each mutation independently affects MYH7b function and structure. Together, these results provide the foundation for further study of a role for MYH7b outside the sarcomere., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2023
- Full Text
- View/download PDF
24. Breath analysis by ultra-sensitive broadband laser spectroscopy detects SARS-CoV-2 infection.
- Author
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Liang Q, Chan YC, Toscano J, Bjorkman KK, Leinwand LA, Parker R, Nozik ES, Nesbitt DJ, and Ye J
- Subjects
- Humans, SARS-CoV-2, Breath Tests, Spectrum Analysis, Lasers, Sensitivity and Specificity, COVID-19
- Abstract
Rapid testing is essential to fighting pandemics such as coronavirus disease 2019 (COVID-19), the disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Exhaled human breath contains multiple volatile molecules providing powerful potential for non-invasive diagnosis of diverse medical conditions. We investigated breath detection of SARS-CoV-2 infection using cavity-enhanced direct frequency comb spectroscopy (CE-DFCS), a state-of-the-art laser spectroscopic technique capable of a real-time massive collection of broadband molecular absorption features at ro-vibrational quantum state resolution and at parts-per-trillion volume detection sensitivity. Using a total of 170 individual breath samples (83 positive and 87 negative with SARS-CoV-2 based on reverse transcription polymerase chain reaction tests), we report excellent discrimination capability for SARS-CoV-2 infection with an area under the receiver-operating-characteristics curve of 0.849(4). Our results support the development of CE-DFCS as an alternative, rapid, non-invasive test for COVID-19 and highlight its remarkable potential for optical diagnoses of diverse biological conditions and disease states., (Creative Commons Attribution license.)
- Published
- 2023
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25. Drug specificity and affinity are encoded in the probability of cryptic pocket opening in myosin motor domains.
- Author
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Meller A, Lotthammer JM, Smith LG, Novak B, Lee LA, Kuhn CC, Greenberg L, Leinwand LA, Greenberg MJ, and Bowman GR
- Subjects
- Protein Isoforms, Probability, Heterocyclic Compounds, 4 or More Rings pharmacology, Heterocyclic Compounds, 4 or More Rings chemistry, Myosins metabolism, Myosin Type II metabolism
- Abstract
The design of compounds that can discriminate between closely related target proteins remains a central challenge in drug discovery. Specific therapeutics targeting the highly conserved myosin motor family are urgently needed as mutations in at least six of its members cause numerous diseases. Allosteric modulators, like the myosin-II inhibitor blebbistatin, are a promising means to achieve specificity. However, it remains unclear why blebbistatin inhibits myosin-II motors with different potencies given that it binds at a highly conserved pocket that is always closed in blebbistatin-free experimental structures. We hypothesized that the probability of pocket opening is an important determinant of the potency of compounds like blebbistatin. To test this hypothesis, we used Markov state models (MSMs) built from over 2 ms of aggregate molecular dynamics simulations with explicit solvent. We find that blebbistatin's binding pocket readily opens in simulations of blebbistatin-sensitive myosin isoforms. Comparing these conformational ensembles reveals that the probability of pocket opening correctly identifies which isoforms are most sensitive to blebbistatin inhibition and that docking against MSMs quantitatively predicts blebbistatin binding affinities (R
2 =0.82). In a blind prediction for an isoform (Myh7b) whose blebbistatin sensitivity was unknown, we find good agreement between predicted and measured IC50s (0.67 μM vs. 0.36 μM). Therefore, we expect this framework to be useful for the development of novel specific drugs across numerous protein targets., Competing Interests: AM, JL, LS, BN, LL, CK, LG, LL, MG, GB No competing interests declared, (© 2023, Meller et al.)- Published
- 2023
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26. Functional divergence of the sarcomeric myosin, MYH7b, supports species-specific biological roles.
- Author
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Lee LA, Barrick SK, Meller A, Walklate J, Lotthammer JM, Tay JW, Stump WT, Bowman G, Geeves MA, Greenberg MJ, and Leinwand LA
- Subjects
- Animals, Humans, Mammals metabolism, Protein Isoforms genetics, Protein Isoforms metabolism, Muscle, Skeletal metabolism, Myosin Heavy Chains genetics, Myosin Heavy Chains metabolism, Cardiac Myosins genetics, Cardiac Myosins metabolism
- Abstract
Myosin heavy chain 7b (MYH7b) is an evolutionarily ancient member of the sarcomeric myosin family, which typically supports striated muscle function. However, in mammals, alternative splicing prevents MYH7b protein production in cardiac and most skeletal muscles and limits expression to a subset of specialized muscles and certain nonmuscle environments. In contrast, MYH7b protein is abundant in python cardiac and skeletal muscles. Although the MYH7b expression pattern diverges in mammals versus reptiles, MYH7b shares high sequence identity across species. So, it remains unclear how mammalian MYH7b function may differ from that of other sarcomeric myosins and whether human and python MYH7b motor functions diverge as their expression patterns suggest. Thus, we generated recombinant human and python MYH7b protein and measured their motor properties to investigate any species-specific differences in activity. Our results reveal that despite having similar working strokes, the MYH7b isoforms have slower actin-activated ATPase cycles and actin sliding velocities than human cardiac β-MyHC. Furthermore, python MYH7b is tuned to have slower motor activity than human MYH7b because of slower kinetics of the chemomechanical cycle. We found that the MYH7b isoforms adopt a higher proportion of myosin heads in the ultraslow, super-relaxed state compared with human cardiac β-MyHC. These findings are supported by molecular dynamics simulations that predict MYH7b preferentially occupies myosin active site conformations similar to those observed in the structurally inactive state. Together, these results suggest that MYH7b is specialized for slow and energy-conserving motor activity and that differential tuning of MYH7b orthologs contributes to species-specific biological roles., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)
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- 2023
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27. Remembering Jeff Robbins: The Father of Cardiac Transgenesis.
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Molkentin JD and Leinwand LA
- Abstract
Competing Interests: The authors have reported that they have no relationships relevant to the contents of this paper to disclose.
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- 2022
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28. Cardiomyocyte-Specific Long Noncoding RNA Regulates Alternative Splicing of the Triadin Gene in the Heart.
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Zhao Y, Riching AS, Knight WE, Chi C, Broadwell LJ, Du Y, Abdel-Hafiz M, Ambardekar AV, Irwin DC, Proenza C, Xu H, Leinwand LA, Walker LA, Woulfe KC, Bristow MR, Buttrick PM, and Song K
- Subjects
- Animals, Arrhythmias, Cardiac, Catecholamines, Heart physiology, Humans, In Situ Hybridization, Fluorescence, Intracellular Signaling Peptides and Proteins metabolism, Mice, Mice, Knockout, Myocytes, Cardiac metabolism, Protein Isoforms genetics, Protein Isoforms metabolism, RNA Splicing Factors genetics, RNA Splicing Factors metabolism, Alternative Splicing, Carrier Proteins genetics, Heart Failure genetics, Heart Failure metabolism, Muscle Proteins genetics, Muscle Proteins metabolism, RNA, Long Noncoding genetics
- Abstract
Background: Abnormalities in Ca
2+ homeostasis are associated with cardiac arrhythmias and heart failure. Triadin plays an important role in Ca2+ homeostasis in cardiomyocytes. Alternative splicing of a single triadin gene produces multiple triadin isoforms. The cardiac-predominant isoform, mouse MT-1 or human Trisk32, is encoded by triadin exons 1 to 8. In humans, mutations in the triadin gene that lead to a reduction in Trisk32 levels in the heart can cause cardiac dysfunction and arrhythmias. Decreased levels of Trisk32 in the heart are also common in patients with heart failure. However, mechanisms that maintain triadin isoform composition in the heart remain elusive., Methods: We analyzed triadin expression in heart explants from patients with heart failure and cardiac arrhythmias and in hearts from mice carrying a knockout allele for Trdn-as , a cardiomyocyte-specific long noncoding RNA encoded by the antisense strand of the triadin gene, between exons 9 and 11. Catecholamine challenge with isoproterenol was performed on Trdn-as knockout mice to assess the role of Trdn-as in cardiac arrhythmogenesis, as assessed by ECG. Ca2+ transients in adult mouse cardiomyocytes were measured with the IonOptix platform or the GCaMP system. Biochemistry assays, single-molecule fluorescence in situ hybridization, subcellular localization imaging, RNA sequencing, and molecular rescue assays were used to investigate the mechanisms by which Trdn-as regulates cardiac function and triadin levels in the heart., Results: We report that Trdn-as maintains cardiac function, at least in part, by regulating alternative splicing of the triadin gene. Knockout of Trdn-as in mice downregulates cardiac triadin, impairs Ca2+ handling, and causes premature death. Trdn-as knockout mice are susceptible to cardiac arrhythmias in response to catecholamine challenge. Normalization of cardiac triadin levels in Trdn-as knockout cardiomyocytes is sufficient to restore Ca2+ handling. Last, Trdn-as colocalizes and interacts with serine/arginine splicing factors in cardiomyocyte nuclei and is essential for efficient recruitment of splicing factors to triadin precursor mRNA., Conclusions: These findings reveal regulation of alternative splicing as a novel mechanism by which a long noncoding RNA controls cardiac function. This study indicates potential therapeutics for heart disease by targeting the long noncoding RNA or pathways regulating alternative splicing.- Published
- 2022
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29. Extracellular matrix stiffness controls cardiac valve myofibroblast activation through epigenetic remodeling.
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Walker CJ, Batan D, Bishop CT, Ramirez D, Aguado BA, Schroeder ME, Crocini C, Schwisow J, Moulton K, Macdougall L, Weiss RM, Allen MA, Dowell R, Leinwand LA, and Anseth KS
- Abstract
Aortic valve stenosis (AVS) is a progressive fibrotic disease that is caused by thickening and stiffening of valve leaflets. At the cellular level, quiescent valve interstitial cells (qVICs) activate to myofibroblasts (aVICs) that persist within the valve tissue. Given the persistence of myofibroblasts in AVS, epigenetic mechanisms have been implicated. Here, we studied changes that occur in VICs during myofibroblast activation by using a hydrogel matrix to recapitulate different stiffnesses in the valve leaflet during fibrosis. We first compared the chromatin landscape of qVICs cultured on soft hydrogels and aVICs cultured on stiff hydrogels, representing the native and diseased phenotypes respectively. Using assay for transposase-accessible chromatin sequencing (ATAC-Seq), we found that open chromatin regions in aVICs were enriched for transcription factor binding motifs associated with mechanosensing pathways compared to qVICs. Next, we used RNA-Seq to show that the open chromatin regions in aVICs correlated with pro-fibrotic gene expression, as aVICs expressed higher levels of contractile fiber genes, including myofibroblast markers such as alpha smooth muscle actin (αSMA), compared to qVICs. In contrast, chromatin remodeling genes were downregulated in aVICs compared to qVICs, indicating qVICs may be protected from myofibroblast activation through epigenetic mechanisms. Small molecule inhibition of one of these remodelers, CREB Binding Protein (CREBBP), prevented qVICs from activating to aVICs. Notably, CREBBP is more abundant in valves from healthy patients compared to fibrotic valves. Our findings reveal the role of mechanical regulation in chromatin remodeling during VIC activation and quiescence and highlight one potential therapeutic target for treating AVS., Competing Interests: The authors declare no conflicts of interests., (© 2022 The Authors. Bioengineering & Translational Medicine published by Wiley Periodicals LLC on behalf of American Institute of Chemical Engineers.)
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- 2022
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30. Ablation of lysophosphatidic acid receptor 1 attenuates hypertrophic cardiomyopathy in a mouse model.
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Axelsson Raja A, Wakimoto H, DeLaughter DM, Reichart D, Gorham J, Conner DA, Lun M, Probst CK, Sakai N, Knipe RS, Montesi SB, Shea B, Adam LP, Leinwand LA, Wan W, Choi ES, Lindberg EL, Patone G, Noseda M, Hübner N, Seidman CE, Tager AM, Seidman JG, and Ho CY
- Subjects
- Animals, Carrier Proteins, Disease Models, Animal, Endothelial Cells pathology, Fibrosis, Hypertrophy pathology, Mice, Cardiomyopathy, Hypertrophic genetics, Cardiomyopathy, Hypertrophic pathology, Receptors, Lysophosphatidic Acid genetics
- Abstract
Myocardial fibrosis is a key pathologic feature of hypertrophic cardiomyopathy (HCM). However, the fibrotic pathways activated by HCM-causing sarcomere protein gene mutations are poorly defined. Because lysophosphatidic acid is a mediator of fibrosis in multiple organs and diseases, we tested the role of the lysophosphatidic acid pathway in HCM. Lysphosphatidic acid receptor 1 (LPAR1), a cell surface receptor, is required for lysophosphatidic acid mediation of fibrosis. We bred HCM mice carrying a pathogenic myosin heavy-chain variant (403
+/- ) with Lpar1 -ablated mice to create mice carrying both genetic changes (403+/- LPAR1-/- ) and assessed development of cardiac hypertrophy and fibrosis. Compared with 403+/- LPAR1WT , 403+/- LPAR1-/- mice developed significantly less hypertrophy and fibrosis. Single-nucleus RNA sequencing of left ventricular tissue demonstrated that Lpar1 was predominantly expressed by lymphatic endothelial cells (LECs) and cardiac fibroblasts. Lpar1 ablation reduced the population of LECs, confirmed by immunofluorescence staining of the LEC markers Lyve1 and Ccl21a and, by in situ hybridization, for Reln and Ccl21a . Lpar1 ablation also altered the distribution of fibroblast cell states. FB1 and FB2 fibroblasts decreased while FB0 and FB3 fibroblasts increased. Our findings indicate that Lpar1 is expressed predominantly by LECs and fibroblasts in the heart and is required for development of hypertrophy and fibrosis in an HCM mouse model. LPAR1 antagonism, including agents in clinical trials for other fibrotic diseases, may be beneficial for HCM.- Published
- 2022
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31. Targeting the sarcomere in inherited cardiomyopathies.
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Lehman SJ, Crocini C, and Leinwand LA
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- Humans, Mutation, Myocardium metabolism, Myosins genetics, Myosins metabolism, Sarcomeres genetics, Sarcomeres metabolism, Cardiomyopathies metabolism, Cardiomyopathy, Dilated genetics, Cardiomyopathy, Hypertrophic drug therapy, Cardiomyopathy, Hypertrophic genetics
- Abstract
Variants in >12 genes encoding sarcomeric proteins can cause various cardiomyopathies. The two most common are hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM). Current therapeutics do not target the root causes of these diseases, but attempt to prevent disease progression and/or to manage symptoms. Accordingly, novel approaches are being developed to treat the cardiac muscle dysfunction directly. Challenges to developing therapeutics for these diseases include the diverse mechanisms of pathogenesis, some of which are still being debated and defined. Four small molecules that modulate the myosin motor protein in the cardiac sarcomere have shown great promise in the settings of HCM and DCM, regardless of the underlying genetic pathogenesis, and similar approaches are being developed to target other components of the sarcomere. In the setting of HCM, mavacamten and aficamten bind to the myosin motor and decrease the ATPase activity of myosin. In the setting of DCM, omecamtiv mecarbil and danicamtiv increase myosin activity in cardiac muscle (but omecamtiv mecarbil decreases myosin activity in vitro). In this Review, we discuss the therapeutic strategies to alter sarcomere contractile activity and summarize the data indicating that targeting one protein in the sarcomere can be effective in treating patients with genetic variants in other sarcomeric proteins, as well as in patients with non-sarcomere-based disease., (© 2022. Springer Nature Limited.)
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- 2022
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32. Regression from pathological hypertrophy in mice is sexually dimorphic and stimulus specific.
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Muehleman DL, Crocini C, Swearingen AR, Ozeroff CD, and Leinwand LA
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- Angiotensin II pharmacology, Animals, Female, Isoproterenol pharmacology, Male, Mice, Myocytes, Cardiac metabolism, Sex Factors, Signal Transduction, Hypertrophy, Left Ventricular chemically induced, Hypertrophy, Left Ventricular metabolism, Sex Characteristics
- Abstract
Pathological cardiac hypertrophy is associated with increased morbidity and mortality. Understanding the mechanisms whereby pathological cardiac growth can be reversed could be of therapeutic value. Here, we show that pathways leading to regression of pathological cardiac hypertrophy are strongly dependent on the hypertrophic trigger and are significantly modified by sex. Two pathological stimuli causing hypertrophy via distinct pathways were administered to male and female mice: angiotensin II (ANG II) or isoproterenol (Iso). Stimuli were removed after 7 days of treatment, and left ventricles (LVs) were studied at 1, 4, and 7 days. ANG II-treated females did not show regression after stimulus removal. Iso-treated males showed rapid LV hypertrophy regression. Somewhat surprisingly, RNAseq analysis at day 1 after removal of triggers revealed only 45 differentially regulated genes in common among all the groups, demonstrating distinct responses. Ingenuity pathway analysis predicted strong downregulation of the TGFβ1 pathway in all groups except for ANG II-treated females. Consistently, we found significant downregulation of Smad signaling after stimulus removal including in ANG II-treated females. In addition, the ERK1/2 pathway was significantly reduced in the groups showing regression. Finally, protein degradation pathways were significantly activated only in Iso-treated males 1 day after stimulus removal. Our data indicate that TGFβ1 downregulation may play a role in the regression of pathological cardiac hypertrophy via downregulation of the ERK1/2 pathway and activation of autophagy and proteasome activity in Iso-treated males. This work highlights that the reversal of pathological hypertrophy does not use universal signaling pathways and that sex potently modifies this process. NEW & NOTEWORTHY Pathological cardiac hypertrophy is a major risk factor for mortality and is thought to be largely irreversible in many individuals. Although cardiac hypertrophy itself has been studied extensively, very little is understood about its regression. It is important that we have a better understanding of mechanisms leading to regression, why this process is not reversible in some individuals and that sex differences need to be considered when contemplating therapies.
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- 2022
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33. Burmese pythons exhibit a transient adaptation to nutrient overload that prevents liver damage.
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Magida JA, Tan Y, Wall CE, Harrison BC, Marr TG, Peter AK, Riquelme CA, and Leinwand LA
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- Adaptation, Physiological, Animals, Humans, Liver, Mammals, Nutrients, Postprandial Period physiology, Boidae metabolism
- Abstract
As an opportunistic predator, the Burmese python (Python molurus bivittatus) consumes large and infrequent meals, fasting for up to a year. Upon consuming a large meal, the Burmese python exhibits extreme metabolic responses. To define the pathways that regulate these postprandial metabolic responses, we performed a comprehensive profile of plasma metabolites throughout the digestive process. Following ingestion of a meal equivalent to 25% of its body mass, plasma lipoproteins and metabolites, such as chylomicra and bile acids, reach levels observed only in mammalian models of extreme dyslipidemia. Here, we provide evidence for an adaptive response to postprandial nutrient overload by the python liver, a critical site of metabolic homeostasis. The python liver undergoes a substantial increase in mass through proliferative processes, exhibits hepatic steatosis, hyperlipidemia-induced insulin resistance indicated by PEPCK activation and pAKT deactivation, and de novo fatty acid synthesis via FASN activation. This postprandial state is completely reversible. We posit that Burmese pythons evade the permanent hepatic damage associated with these metabolic states in mammals using evolved protective measures to inactivate these pathways. These include a transient activation of hepatic nuclear receptors induced by fatty acids and bile acids, including PPAR and FXR, respectively. The stress-induced p38 MAPK pathway is also transiently activated during the early stages of digestion. Taken together, these data identify a reversible metabolic response to hyperlipidemia by the python liver, only achieved in mammals by pharmacologic intervention. The factors involved in these processes may be relevant to or leveraged for remediating human hepatic pathology., (© 2022 Magida et al.)
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- 2022
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34. Genes That Escape X Chromosome Inactivation Modulate Sex Differences in Valve Myofibroblasts.
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Aguado BA, Walker CJ, Grim JC, Schroeder ME, Batan D, Vogt BJ, Rodriguez AG, Schwisow JA, Moulton KS, Weiss RM, Heistad DD, Leinwand LA, and Anseth KS
- Subjects
- Actins genetics, Actins metabolism, Animals, Aortic Valve Stenosis etiology, Aortic Valve Stenosis metabolism, Aortic Valve Stenosis pathology, Biomarkers, Cells, Cultured, Disease Models, Animal, Female, Gene Expression Regulation drug effects, Humans, Immunohistochemistry, Male, Myofibroblasts drug effects, Sex Factors, Signal Transduction, Swine, Transcriptome, Aortic Valve cytology, Gene Expression, Genes, X-Linked, Myofibroblasts metabolism, X Chromosome Inactivation
- Abstract
Background: Aortic valve stenosis is a sexually dimorphic disease, with women often presenting with sustained fibrosis and men with more extensive calcification. However, the intracellular molecular mechanisms that drive these clinically important sex differences remain underexplored., Methods: Hydrogel biomaterials were designed to recapitulate key aspects of the valve tissue microenvironment and to serve as a culture platform for sex-specific valvular interstitial cells (VICs; precursors to profibrotic myofibroblasts). The hydrogel culture system was used to interrogate intracellular pathways involved in sex-dependent VIC-to-myofibroblast activation and deactivation. RNA sequencing was used to define pathways involved in driving sex-dependent activation. Interventions with small molecule inhibitors and siRNA transfections were performed to provide mechanistic insight into sex-specific cellular responses to microenvironmental cues, including matrix stiffness and exogenously delivered biochemical factors., Results: In both healthy porcine and human aortic valves, female leaflets had higher baseline activation of the myofibroblast marker α-smooth muscle actin compared with male leaflets. When isolated and cultured, female porcine and human VICs had higher levels of basal α-smooth muscle actin stress fibers that further increased in response to the hydrogel matrix stiffness, both of which were higher than in male VICs. A transcriptomic analysis of male and female porcine VICs revealed Rho-associated protein kinase signaling as a potential driver of this sex-dependent myofibroblast activation. Furthermore, we found that genes that escape X-chromosome inactivation such as BMX and STS (encoding for Bmx nonreceptor tyrosine kinase and steroid sulfatase, respectively) partially regulate the elevated female myofibroblast activation through Rho-associated protein kinase signaling. This finding was confirmed by treating male and female VICs with endothelin-1 and plasminogen activator inhibitor-1, factors that are secreted by endothelial cells and known to drive myofibroblast activation through Rho-associated protein kinase signaling., Conclusions: Together, in vivo and in vitro results confirm sex dependencies in myofibroblast activation pathways and implicate genes that escape X-chromosome inactivation in regulating sex differences in myofibroblast activation and subsequent aortic valve stenosis progression. Our results underscore the importance of considering sex as a biological variable to understand the molecular mechanisms of aortic valve stenosis and to help guide sex-based precision therapies.
- Published
- 2022
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35. Nuclear mechanosensing drives chromatin remodelling in persistently activated fibroblasts.
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Walker CJ, Crocini C, Ramirez D, Killaars AR, Grim JC, Aguado BA, Clark K, Allen MA, Dowell RD, Leinwand LA, and Anseth KS
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- Animals, Cell Differentiation, Extracellular Matrix, Humans, Myofibroblasts, Swine, Chromatin Assembly and Disassembly, Fibroblasts
- Abstract
Fibrotic disease is caused by the continuous deposition of extracellular matrix by persistently activated fibroblasts (also known as myofibroblasts), even after the resolution of the injury. Using fibroblasts from porcine aortic valves cultured on hydrogels that can be softened via exposure to ultraviolet light, here we show that increased extracellular stiffness activates the fibroblasts, and that cumulative tension on the nuclear membrane and increases in the activity of histone deacetylases transform transiently activated fibroblasts into myofibroblasts displaying condensed chromatin with genome-wide alterations. The condensed structure of the myofibroblasts is associated with cytoskeletal stability, as indicated by the inhibition of chromatin condensation and myofibroblast persistence after detachment of the nucleus from the cytoskeleton via the displacement of endogenous nesprins from the nuclear envelope. We also show that the chromatin structure of myofibroblasts from patients with aortic valve stenosis is more condensed than that of myofibroblasts from healthy donors. Our findings suggest that nuclear mechanosensing drives distinct chromatin signatures in persistently activated fibroblasts., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)
- Published
- 2021
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36. Author Correction: Nuclear mechanosensing drives chromatin remodelling in persistently activated fibroblasts.
- Author
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Walker CJ, Crocini C, Ramirez D, Killaars AR, Grim JC, Aguado BA, Clark K, Allen MA, Dowell RD, Leinwand LA, and Anseth KS
- Published
- 2021
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37. Matters of the heart: Cellular sex differences.
- Author
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Walker CJ, Schroeder ME, Aguado BA, Anseth KS, and Leinwand LA
- Subjects
- Animals, Cardiovascular Diseases genetics, Chromosomes genetics, Extracellular Matrix metabolism, Female, Genotype, Gonadal Steroid Hormones metabolism, Humans, Male, Sex Factors, Transcriptome genetics, Cardiovascular Diseases metabolism, Cardiovascular Diseases physiopathology, Heart physiopathology, Myocardium cytology, Myocardium metabolism
- Abstract
Nearly all cardiovascular diseases show sexual dimorphisms in prevalence, presentation, and outcomes. Until recently, most clinical trials were carried out in males, and many animal studies either failed to identify the sex of the animals or combined data obtained from males and females. Cellular sex in the heart is relatively understudied and many studies fail to report the sex of the cells used for in vitro experiments. Moreover, in the small number of studies in which sex is reported, most of those studies use male cells. The observation that cells from males and females are inherently different is becoming increasingly clear - either due to acquired differences from hormones and other factors or due to intrinsic differences in genotype (XX or XY). Because of the likely contribution of cellular sex differences in cardiac health and disease, here, we explore differences in mammalian male and female cells in the heart, including the less-studied non-myocyte cell populations. We discuss how the heart's microenvironment impacts male and female cellular phenotypes and vice versa, including how secretory profiles are dependent on cellular sex, and how hormones contribute to sexually dimorphic phenotypes and cellular functions. Intracellular mechanisms that contribute to sex differences, including gene expression and epigenetic remodeling, are also described. Recent single-cell sequencing studies have revealed unexpected sex differences in the composition of cell types in the heart which we discuss. Finally, future recommendations for considering cellular sex differences in the design of bioengineered in vitro disease models of the heart are provided., (Copyright © 2021 Elsevier Ltd. All rights reserved.)
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- 2021
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38. Higher Viral Load Drives Infrequent Severe Acute Respiratory Syndrome Coronavirus 2 Transmission Between Asymptomatic Residence Hall Roommates.
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Bjorkman KK, Saldi TK, Lasda E, Bauer LC, Kovarik J, Gonzales PK, Fink MR, Tat KL, Hager CR, Davis JC, Ozeroff CD, Brisson GR, Larremore DB, Leinwand LA, McQueen MB, and Parker R
- Subjects
- COVID-19 diagnosis, COVID-19 epidemiology, COVID-19 virology, Humans, Pandemics prevention & control, Pandemics statistics & numerical data, SARS-CoV-2 isolation & purification, Students, Young Adult, Asymptomatic Infections epidemiology, COVID-19 transmission, SARS-CoV-2 pathogenicity, Viral Load
- Abstract
Background: The coronavirus disease 2019 pandemic spread to >200 countries in <6 months. To understand coronavirus spread, determining transmission rate and defining factors that increase transmission risk are essential. Most cases are asymptomatic, but people with asymptomatic infection have viral loads indistinguishable from those in symptomatic people, and they do transmit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, asymptomatic cases are often undetected., Methods: Given high residence hall student density, the University of Colorado Boulder established a mandatory weekly screening test program. We analyzed longitudinal data from 6408 students and identified 116 likely transmission events in which a second roommate tested positive within 14 days of the index roommate., Results: Although the infection rate was lower in single-occupancy rooms (10%) than in multiple-occupancy rooms (19%), interroommate transmission occurred only about 20% of the time. Cases were usually asymptomatic at the time of detection. Notably, individuals who likely transmitted had an average viral load approximately 6.5-fold higher than individuals who did not (mean quantification cycle [Cq], 26.2 vs 28.9). Although students with diagnosed SARS-CoV-2 infection moved to isolation rooms, there was no difference in time to isolation between cases with or without interroommate transmission., Conclusions: This analysis argues that interroommate transmission occurs infrequently in residence halls and provides strong correlative evidence that viral load is proportional to transmission probability., (© The Author(s) 2021. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)
- Published
- 2021
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39. High-resolution within-sewer SARS-CoV-2 surveillance facilitates informed intervention.
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Reeves K, Liebig J, Feula A, Saldi T, Lasda E, Johnson W, Lilienfeld J, Maggi J, Pulley K, Wilkerson PJ, Real B, Zak G, Davis J, Fink M, Gonzales P, Hager C, Ozeroff C, Tat K, Alkire M, Butler C, Coe E, Darby J, Freeman N, Heuer H, Jones JR, Karr M, Key S, Maxwell K, Nelson L, Saldana E, Shea R, Salveson L, Tomlinson K, Vargas-Barriga J, Vigil B, Brisson G, Parker R, Leinwand LA, Bjorkman K, and Mansfeldt C
- Subjects
- Humans, Pandemics, Universities, Wastewater, COVID-19, SARS-CoV-2
- Abstract
To assist in the COVID-19 public health guidance on a college campus, daily composite wastewater samples were withdrawn at 20 manhole locations across the University of Colorado Boulder campus. Low-cost autosamplers were fabricated in-house to enable an economical approach to this distributed study. These sample stations operated from August 25th until November 23rd during the fall 2020 semester, with 1512 samples collected. The concentration of SARS-CoV-2 in each sample was quantified through two comparative reverse transcription quantitative polymerase chain reactions (RT-qPCRs). These methods were distinct in the utilization of technical replicates and normalization to an endogenous control. (1) Higher temporal resolution compensates for supply chain or other constraints that prevent technical or biological replicates. (2) The data normalized by an endogenous control agreed with the raw concentration data, minimizing the utility of normalization. The raw wastewater concentration values reflected SARS-CoV-2 prevalence on campus as detected by clinical services. Overall, combining the low-cost composite sampler with a method that quantifies the SARS-CoV-2 signal within six hours enabled actionable and time-responsive data delivered to key stakeholders. With daily reporting of the findings, wastewater surveillance assisted in decision making during critical phases of the pandemic on campus, from detecting individual cases within populations ranging from 109 to 2048 individuals to monitoring the success of on-campus interventions., (Copyright © 2021. Published by Elsevier Ltd.)
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- 2021
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40. Nonproductive Splicing Prevents Expression of MYH7b Protein in the Mammalian Heart.
- Author
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Lee LA, Broadwell LJ, Buvoli M, and Leinwand LA
- Subjects
- Animals, Blotting, Western, Cadaver, Cardiomyopathies metabolism, Cardiomyopathies pathology, Disease Models, Animal, Heart Ventricles metabolism, Humans, Mammals, Mice, Myocardium pathology, Myocytes, Cardiac pathology, Myosin Heavy Chains biosynthesis, Myosin Type II biosynthesis, RNA genetics, RNA, Messenger genetics, Rats, Cardiomyopathies genetics, Gene Expression Regulation, Heart Ventricles pathology, Myocardial Contraction physiology, Myocardium metabolism, Myocytes, Cardiac metabolism, Myosin Heavy Chains genetics, Myosin Type II genetics
- Abstract
Background Although the roles of alpha-myosin heavy chain (α-MyHC) and beta-myosin heavy chain (β-MyHC) proteins in cardiac contractility have long been appreciated, the biological contribution of another closely related sarcomeric myosin family member, MYH7b (myosin heavy chain 7b), has become a matter of debate. In mammals, MYH7b mRNA is transcribed but undergoes non-productive alternative splicing that prevents protein expression in a tissue-specific manner, including in the heart. However, several studies have recently linked MYH7b variants to different cardiomyopathies or have reported MYH7b protein expression in mammalian hearts. Methods and Results By analyzing mammalian cardiac transcriptome and proteome data, we show that the vast majority of MYH7b RNA is subject to exon skipping and cannot be translated into a functional myosin molecule. Notably, we discovered a lag in the removal of introns flanking the alternatively spliced exon, which could retain the non-coding RNA in the nucleus. This process could play a significant role in controlling MYH7b expression as well as the activity of other cardiac genes. Consistent with the negligible level of full-length protein coding mRNA, no MYH7b protein expression was detected in adult mouse, rat, and human hearts by Western blot analysis. Furthermore, proteome surveys including quantitative mass spectrometry analyses revealed only traces of cardiac MYH7b protein and even then, only in a subset of individual samples. Conclusions The comprehensive analysis presented here suggests that previous studies showing cardiac MYH7b protein expression were likely attributable to antibody cross-reactivity. More importantly, our data predict that the MYH7b disease-associated variants may operate through the alternately spliced RNA itself.
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- 2021
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41. Identification of sequence changes in myosin II that adjust muscle contraction velocity.
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Johnson CA, McGreig JE, Jeanfavre ST, Walklate J, Vera CD, Farré M, Mulvihill DP, Baines AJ, Ridout M, Leinwand LA, Wass MN, and Geeves MA
- Subjects
- Adaptation, Physiological, Adenosine Diphosphate metabolism, Adenosine Triphosphate metabolism, Amino Acid Sequence, Animals, Body Weight, Cell Line, Conserved Sequence, Humans, Phylogeny, Protein Domains, Protein Isoforms chemistry, Protein Isoforms metabolism, Rats, Muscle Contraction physiology, Myosin Type II chemistry
- Abstract
The speed of muscle contraction is related to body size; muscles in larger species contract at slower rates. Since contraction speed is a property of the myosin isoform expressed in a muscle, we investigated how sequence changes in a range of muscle myosin II isoforms enable this slower rate of muscle contraction. We considered 798 sequences from 13 mammalian myosin II isoforms to identify any adaptation to increasing body mass. We identified a correlation between body mass and sequence divergence for the motor domain of the 4 major adult myosin II isoforms (β/Type I, IIa, IIb, and IIx), suggesting that these isoforms have adapted to increasing body mass. In contrast, the non-muscle and developmental isoforms show no correlation of sequence divergence with body mass. Analysis of the motor domain sequence of β-myosin (predominant myosin in Type I/slow and cardiac muscle) from 67 mammals from 2 distinct clades identifies 16 sites, out of 800, associated with body mass (padj < 0.05) but not with the clade (padj > 0.05). Both clades change the same small set of amino acids, in the same order from small to large mammals, suggesting a limited number of ways in which contraction velocity can be successfully manipulated. To test this relationship, the 9 sites that differ between human and rat were mutated in the human β-myosin to match the rat sequence. Biochemical analysis revealed that the rat-human β-myosin chimera functioned like the native rat myosin with a 2-fold increase in both motility and in the rate of ADP release from the actin-myosin crossbridge (the step that limits contraction velocity). Thus, these sequence changes indicate adaptation of β-myosin as species mass increased to enable a reduced contraction velocity and heart rate., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: LAL owns stock in MyoKardia, Inc. and has a Sponsored Research Agreement with MyoKardia, Inc.
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- 2021
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42. Cardiac Fibroblasts Mediate a Sexually Dimorphic Fibrotic Response to β-Adrenergic Stimulation.
- Author
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Peter AK, Walker CJ, Ceccato T, Trexler CL, Ozeroff CD, Lugo KR, Perry AR, Anseth KS, and Leinwand LA
- Subjects
- Adrenergic beta-Agonists pharmacology, Animals, Disease Models, Animal, Disease Progression, Female, Fibroblasts drug effects, Fibroblasts metabolism, Fibrosis metabolism, Fibrosis pathology, Heart Diseases metabolism, Male, Myocardium metabolism, Rats, Rats, Sprague-Dawley, Sex Factors, Fibroblasts pathology, Heart Diseases pathology, Isoproterenol pharmacology, Myocardium pathology, Receptors, Adrenergic, beta metabolism
- Abstract
Background Biological sex is an important modifier of cardiovascular disease and women generally have better outcomes compared with men. However, the contribution of cardiac fibroblasts (CFs) to this sexual dimorphism is relatively unexplored. Methods and Results Isoproterenol (ISO) was administered to rats as a model for chronic β-adrenergic receptor (β-AR)-mediated cardiovascular disease. ISO-treated males had higher mortality than females and also developed fibrosis whereas females did not. Gonadectomy did not abrogate this sex difference. To determine the cellular contribution to this phenotype, CFs were studied. CFs from both sexes had increased proliferation in vivo in response to ISO, but CFs from female hearts proliferated more than male cells. In addition, male CFs were significantly more activated to myofibroblasts by ISO. To investigate potential regulatory mechanisms for the sexually dimorphic fibrotic response, β-AR mRNA and PKA (protein kinase A) activity were measured. In response to ISO treatment, male CFs increased expression of β1- and β2-ARs, whereas expression of both receptors decreased in female CFs. Moreover, ISO-treated male CFs had higher PKA activity relative to vehicle controls, whereas ISO did not activate PKA in female CFs. Conclusions Chronic in vivo β-AR stimulation causes fibrosis in male but not female rat hearts. Male CFs are more activated than female CFs, consistent with elevated fibrosis in male rat hearts and may be caused by higher β-AR expression and PKA activation in male CFs. Taken together, our data suggest that CFs play a substantial role in mediating sex differences observed after cardiac injury.
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- 2021
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43. Saliva TwoStep for rapid detection of asymptomatic SARS-CoV-2 carriers.
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Yang Q, Meyerson NR, Clark SK, Paige CL, Fattor WT, Gilchrist AR, Barbachano-Guerrero A, Healy BG, Worden-Sapper ER, Wu SS, Muhlrad D, Decker CJ, Saldi TK, Lasda E, Gonzales P, Fink MR, Tat KL, Hager CR, Davis JC, Ozeroff CD, Brisson GR, McQueen MB, Leinwand LA, Parker R, and Sawyer SL
- Subjects
- COVID-19 metabolism, COVID-19 Testing, Humans, Molecular Diagnostic Techniques methods, Nucleic Acid Amplification Techniques methods, RNA, Viral genetics, SARS-CoV-2 genetics, Sensitivity and Specificity, Specimen Handling methods, COVID-19 diagnosis, COVID-19 virology, Carrier State diagnosis, Carrier State virology, SARS-CoV-2 isolation & purification, Saliva virology
- Abstract
Here, we develop a simple molecular test for SARS-CoV-2 in saliva based on reverse transcription loop-mediated isothermal amplification. The test has two steps: (1) heat saliva with a stabilization solution and (2) detect virus by incubating with a primer/enzyme mix. After incubation, saliva samples containing the SARS-CoV-2 genome turn bright yellow. Because this test is pH dependent, it can react falsely to some naturally acidic saliva samples. We report unique saliva stabilization protocols that rendered 295 healthy saliva samples compatible with the test, producing zero false positives. We also evaluated the test on 278 saliva samples from individuals who were infected with SARS-CoV-2 but had no symptoms at the time of saliva collection, and from 54 matched pairs of saliva and anterior nasal samples from infected individuals. The Saliva TwoStep test described herein identified infections with 94% sensitivity and >99% specificity in individuals with sub-clinical (asymptomatic or pre-symptomatic) infections., Competing Interests: QY, NM, CP Some of the authors of this study (NRM, QY, CLP, SLS) are founders of Darwin Biosciences, who licenses the Saliva TwoStep assay described herein. SC is an employee of Darwin Biosciences. WF, AG, AB, BH, EW, SW, DM, CD, TS, EL, PG, MF, KT, CH, JD, CO, GB, MM, LL, RP No competing interests declared, SS Senior editor, eLife, (© 2021, Yang et al.)
- Published
- 2021
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44. Associations Between Female Sex, Sarcomere Variants, and Clinical Outcomes in Hypertrophic Cardiomyopathy.
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Lakdawala NK, Olivotto I, Day SM, Han L, Ashley EA, Michels M, Ingles J, Semsarian C, Jacoby D, Jefferies JL, Colan SD, Pereira AC, Rossano JW, Wittekind S, Ware JS, Saberi S, Helms AS, Cirino AL, Leinwand LA, Seidman CE, and Ho CY
- Subjects
- Adult, Aged, Cardiac Myosins genetics, Cardiomyopathy, Hypertrophic complications, Cardiomyopathy, Hypertrophic genetics, Carrier Proteins genetics, Female, Genotype, Heart Failure etiology, Heart Failure mortality, Humans, Male, Middle Aged, Myosin Heavy Chains genetics, Polymorphism, Genetic, Proportional Hazards Models, Registries, Retrospective Studies, Sex Characteristics, Survival Rate, Ventricular Function, Left, Cardiomyopathy, Hypertrophic diagnosis, Sarcomeres genetics
- Abstract
Background: The impact of sex on phenotypic expression in hypertrophic cardiomyopathy (HCM) has not been well characterized in genotyped cohorts., Methods: Retrospective cohort study from an international registry of patients receiving care at experienced HCM centers. Sex-based differences in baseline characteristics and clinical outcomes were assessed., Results: Of 5873 patients (3788 genotyped), 2226 (37.9%) were women. At baseline, women were older (49.0±19.9 versus 42.9±18.4 years, P <0.001) and more likely to have pathogenic/likely pathogenic sarcomeric variants (HCM patients with a sarcomere mutation; 51% versus 43%, P <0.001) despite equivalent utilization of genetic testing. Age at diagnosis varied by sex and genotype despite similar distribution of causal genes. Women were 3.6 to 7.1 years older at diagnosis ( P <0.02) except for patients with MYH7 variants where age at diagnosis was comparable for women and men (n=492; 34.8±19.2 versus 33.3±16.8 years, P =0.39). Over 7.7 median years of follow-up, New York Heart Association III-IV heart failure was more common in women (hazard ratio, 1.87 [CI, 1.48-2.36], P <0.001), after controlling for their higher burden of symptoms and outflow tract obstruction at baseline, reduced ejection fraction, HCM patients with a sarcomere mutation, age, and hypertension. All-cause mortality was increased in women (hazard ratio, 1.50 [CI, 1.13-1.99], P <0.01) but neither implantable cardioverter-defibrillator utilization nor ventricular arrhythmia varied by sex., Conclusions: In HCM, women are older at diagnosis, partly modified by genetic substrate. Regardless of genotype, women were at higher risk of mortality and developing severe heart failure symptoms. This points to a sex-effect on long-term myocardial performance in HCM, which should be investigated further.
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- 2021
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45. Myosin 7b is a regulatory long noncoding RNA (lncMYH7b) in the human heart.
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Broadwell LJ, Smallegan MJ, Rigby KM, Navarro-Arriola JS, Montgomery RL, Rinn JL, and Leinwand LA
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- Cardiac Myosins chemistry, Humans, Induced Pluripotent Stem Cells, MicroRNAs genetics, Molecular Dynamics Simulation, Myocardium cytology, Myocytes, Cardiac metabolism, Myosin Heavy Chains chemistry, Protein Conformation, Cardiac Myosins metabolism, Myocardium metabolism, Myosin Heavy Chains metabolism, RNA, Long Noncoding genetics
- Abstract
Myosin heavy chain 7b (MYH7b) is an ancient member of the myosin heavy chain motor protein family that is expressed in striated muscles. In mammalian cardiac muscle, MYH7b RNA is expressed along with two other myosin heavy chains, β-myosin heavy chain (β-MyHC) and α-myosin heavy chain (α-MyHC). However, unlike β-MyHC and α-MyHC, which are maintained in a careful balance at the protein level, the MYH7b locus does not produce a full-length protein in the heart due to a posttranscriptional exon-skipping mechanism that occurs in a tissue-specific manner. Whether this locus has a role in the heart beyond producing its intronic microRNA, miR-499, was unclear. Using cardiomyocytes derived from human induced pluripotent stem cells as a model system, we found that the noncoding exon-skipped RNA (lncMYH7b) affects the transcriptional landscape of human cardiomyocytes, independent of miR-499. Specifically, lncMYH7b regulates the ratio of β-MyHC to α-MyHC, which is crucial for cardiac contractility. We also found that lncMYH7b regulates beat rate and sarcomere formation in cardiomyocytes. This regulation is likely achieved through control of a member of the TEA domain transcription factor family (TEAD3, which is known to regulate β-MyHC). Therefore, we conclude that this ancient gene has been repurposed by alternative splicing to produce a regulatory long-noncoding RNA in the human heart that affects cardiac myosin composition., Competing Interests: Conflict of interest No conflicts of interest were reported., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2021
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46. Defining the Cardiac Fibroblast Secretome in a Fibrotic Microenvironment.
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Ceccato TL, Starbuck RB, Hall JK, Walker CJ, Brown TE, Killgore JP, Anseth KS, and Leinwand LA
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- Animals, Cell Differentiation, Cells, Cultured, Mechanotransduction, Cellular, Rats, Signal Transduction, Cardiomegaly metabolism, Fibrosis metabolism, Membrane Proteins metabolism, Myocytes, Cardiac metabolism, Myofibroblasts metabolism, Paracrine Communication physiology, Transforming Growth Factor beta1 metabolism
- Abstract
Background Cardiac fibroblasts (CFs) have the ability to sense stiffness changes and respond to biochemical cues to modulate their states as either quiescent or activated myofibroblasts. Given the potential for secretion of bioactive molecules to modulate the cardiac microenvironment, we sought to determine how the CF secretome changes with matrix stiffness and biochemical cues and how this affects cardiac myocytes via paracrine signaling. Methods and Results Myofibroblast activation was modulated in vitro by combining stiffness cues with TGFβ1 (transforming growth factor β 1) treatment using engineered poly (ethylene glycol) hydrogels, and in vivo with isoproterenol treatment. Stiffness, TGFβ1, and isoproterenol treatment increased AKT (protein kinase B) phosphorylation, indicating that this pathway may be central to myofibroblast activation regardless of the treatment. Although activation of AKT was shared, different activating cues had distinct effects on downstream cytokine secretion, indicating that not all activated myofibroblasts share the same secretome. To test the effect of cytokines present in the CF secretome on paracrine signaling, neonatal rat ventricular cardiomyocytes were treated with CF conditioned media. Conditioned media from myofibroblasts cultured on stiff substrates and activated by TGFβ1 caused hypertrophy, and one of the cytokines in that media was insulin growth factor 1, which is a known mediator of cardiac myocyte hypertrophy. Conclusions Culturing CFs on stiff substrates, treating with TGFβ1, and in vivo treatment with isoproterenol all caused myofibroblast activation. Each cue had distinct effects on the secretome or genes encoding the secretome, but only the secretome of activated myofibroblasts on stiff substrates treated with TGFβ1 caused myocyte hypertrophy, most likely through insulin growth factor 1.
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- 2020
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47. miR-206 enforces a slow muscle phenotype.
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Bjorkman KK, Guess MG, Harrison BC, Polmear MM, Peter AK, and Leinwand LA
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- Animals, Female, Male, Mice, Muscle Contraction genetics, Muscle Fibers, Skeletal, Phenotype, MicroRNAs genetics, Muscle, Skeletal
- Abstract
Striated muscle is a highly specialized collection of tissues with contractile properties that vary according to functional needs. Although muscle fiber types are established postnatally, lifelong plasticity facilitates stimulus-dependent adaptation. Functional adaptation requires molecular adaptation, which is partially provided by miRNA-mediated post-transcriptional regulation. miR-206 is a muscle-specific miRNA enriched in slow muscles. We investigated whether miR-206 drives the slow muscle phenotype or is merely an outcome. We found that miR-206 expression increases in both physiological (including female sex and endurance exercise) and pathological conditions (muscular dystrophy and adrenergic agonism) that promote a slow phenotype. Consistent with that observation, the slow soleus muscle of male miR-206-knockout mice displays a faster phenotype than wild-type mice. Moreover, left ventricles of male miR-206 knockout mice have a faster myosin profile, accompanied by dilation and systolic dysfunction. Thus, miR-206 appears to be necessary to enforce a slow skeletal and cardiac muscle phenotype and to play a key role in muscle sexual dimorphisms., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)
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- 2020
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48. Three-dimensional encapsulation of adult mouse cardiomyocytes in hydrogels with tunable stiffness.
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Crocini C, Walker CJ, Anseth KS, and Leinwand LA
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- Animals, Capsules, Cell Proliferation drug effects, Cell Survival drug effects, Cellular Microenvironment drug effects, Intracellular Space drug effects, Intracellular Space metabolism, Mice, Polyethylene Glycols chemistry, Protein Transport drug effects, Biocompatible Materials chemistry, Biocompatible Materials pharmacology, Hydrogels chemistry, Hydrogels pharmacology, Mechanical Phenomena, Myocytes, Cardiac cytology, Myocytes, Cardiac drug effects
- Abstract
Numerous diseases, including those of the heart, are characterized by increased stiffness due to excessive deposition of extracellular matrix proteins. Cardiomyocytes continuously adapt their morphology and function to the mechanical changes of their microenvironment. Because traditional cell culture is conducted on substrates that are many orders of magnitude stiffer than any environment encountered by a cardiomyocyte in health or disease, alternate culture systems are necessary to model these processes in vitro. Here, we employ photo-clickable thiol-ene poly(ethylene glycol) (PEG) hydrogels for three-dimensional cell culture of adult mouse cardiomyocytes. PEG hydrogels serve as versatile biocompatible scaffolds, whose stiffness can be precisely tuned to mimic physiological and pathological microenvironments. Compared to traditional culture, adult cardiomyocytes encapsulated in PEG hydrogels exhibited longer survival and preserved sarcomeric and T-tubular architecture. Culture in PEG hydrogels of varying stiffnesses regulated the subcellular localization of the mechanosensitive transcription factor, YAP, in adult cardiomyocytes, indicating PEG hydrogels offer a versatile platform to study the role of mechanical cues in cardiomyocyte biology., (Copyright © 2019 Elsevier Ltd. All rights reserved.)
- Published
- 2020
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49. Estrogen receptor-α in female skeletal muscle is not required for regulation of muscle insulin sensitivity and mitochondrial regulation.
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Iñigo MR, Amorese AJ, Tarpey MD, Balestrieri NP, Jones KG, Patteson DJ, Jackson KC, Torres MJ, Lin CT, Smith CD, Heden TD, McMillin SL, Weyrauch LA, Stanley EC, Schmidt CA, Kilburg-Basnyat BB, Reece SW, Psaltis CE, Leinwand LA, Funai K, McClung JM, Gowdy KM, Witczak CA, Lowe DA, Neufer PD, and Spangenburg EE
- Subjects
- Animals, Estrogen Receptor alpha deficiency, Female, Humans, Mice, Mice, Knockout, Muscle, Skeletal cytology, Estrogen Receptor alpha metabolism, Insulin metabolism, Mitochondria metabolism, Muscle, Skeletal metabolism
- Abstract
Objective: Estrogen receptor-α (ERα) is a nuclear receptor family member thought to substantially contribute to the metabolic regulation of skeletal muscle. However, previous mouse models utilized to assess the necessity of ERα signaling in skeletal muscle were confounded by altered developmental programming and/or influenced by secondary effects, making it difficult to assign a causal role for ERα. The objective of this study was to determine the role of skeletal muscle ERα in regulating metabolism in the absence of confounding factors of development., Methods: A novel mouse model was developed allowing for induced deletion of ERα in adult female skeletal muscle (ERαKO
ism ). ERαshRNA was also used to knockdown ERα (ERαKD) in human myotubes cultured from primary human skeletal muscle cells isolated from muscle biopsies from healthy and obese insulin-resistant women., Results: Twelve weeks of HFD exposure had no differential effects on body composition, VO2 , VCO2 , RER, energy expenditure, and activity counts across genotypes. Although ERαKOism mice exhibited greater glucose intolerance than wild-type (WT) mice after chronic HFD, ex vivo skeletal muscle glucose uptake was not impaired in the ERαKOism mice. Expression of pro-inflammatory genes was altered in the skeletal muscle of the ERαKOism , but the concentrations of these inflammatory markers in the systemic circulation were either lower or remained similar to the WT mice. Finally, skeletal muscle mitochondrial respiratory capacity, oxidative phosphorylation efficiency, and H2 O2 emission potential was not affected in the ERαKOism mice. ERαKD in human skeletal muscle cells neither altered differentiation capacity nor caused severe deficits in mitochondrial respiratory capacity., Conclusions: Collectively, these results suggest that ERα function is superfluous in protecting against HFD-induced skeletal muscle metabolic derangements after postnatal development is complete., (Copyright © 2019 The Author(s). Published by Elsevier GmbH.. All rights reserved.)- Published
- 2020
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50. Differences in microRNA-29 and Pro-fibrotic Gene Expression in Mouse and Human Hypertrophic Cardiomyopathy.
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Liu Y, Afzal J, Vakrou S, Greenland GV, Talbot CC Jr, Hebl VB, Guan Y, Karmali R, Tardiff JC, Leinwand LA, Olgin JE, Das S, Fukunaga R, and Abraham MR
- Abstract
Background: Hypertrophic cardiomyopathy (HCM) is characterized by myocyte hypertrophy and fibrosis. Studies in two mouse models (R92W-TnT/R403Q-MyHC) at early HCM stage revealed upregulation of endothelin (ET1) signaling in both mutants, but TGFβ signaling only in TnT mutants. Dysregulation of miR-29 expression has been implicated in cardiac fibrosis. But it is unknown whether expression of miR-29a/b/c and profibrotic genes is commonly regulated in mouse and human HCM. Methods: In order to understand mechanisms underlying fibrosis in HCM, and examine similarities/differences in expression of miR-29a/b/c and several profibrotic genes in mouse and human HCM, we performed parallel studies in rat cardiac myocyte/fibroblast cultures, examined gene expression in two mouse models of ( non-obstructive ) HCM (R92W-TnT, R403Q-MyHC)/controls at early (5 weeks) and established (24 weeks) disease stage, and analyzed publicly available mRNA/miRNA expression data from obstructive -HCM patients undergoing septal myectomy/controls (unused donor hearts). Results: Myocyte cultures: ET1 increased superoxide/H
2 O2 , stimulated TGFβ expression/secretion, and suppressed miR-29a expression in myocytes. The effect of ET1 on miR-29 and TGFβ expression/secretion was antagonized by N-acetyl-cysteine, a reactive oxygen species scavenger. Fibroblast cultures: ET1 had no effect on pro-fibrotic gene expression in fibroblasts. TGFβ1/TGFβ2 suppressed miR-29a and increased collagen expression, which was abolished by miR-29a overexpression. Mouse and human HCM: Expression of miR-29a/b/c was lower, and TGFB1 /collagen gene expression was higher in TnT mutant-LV at 5 and 24 weeks; no difference was observed in expression of these genes in MyHC mutant-LV and in human myectomy tissue. TGFB2 expression was higher in LV of both mutant mice and human myectomy tissue. ACE2 , a negative regulator of the renin-angiotensin-aldosterone system, was the most upregulated transcript in human myectomy tissue. Pathway analysis predicted upregulation of the anti-hypertrophic/anti-fibrotic liver X receptor/retinoid X receptor (LXR/RXR) pathway only in human myectomy tissue. Conclusions: Our in vitro studies suggest that activation of ET1 signaling in cardiac myocytes increases reactive oxygen species and stimulates TGFβ secretion, which downregulates miR-29a and increases collagen in fibroblasts, thus contributing to fibrosis. Our gene expression studies in mouse and human HCM reveal allele-specific differences in miR-29 family/profibrotic gene expression in mouse HCM, and activation of anti-hypertrophic/anti-fibrotic genes and pathways in human HCM., (Copyright © 2019 Liu, Afzal, Vakrou, Greenland, Talbot, Hebl, Guan, Karmali, Tardiff, Leinwand, Olgin, Das, Fukunaga and Abraham.)- Published
- 2019
- Full Text
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