Biology subjects

Leinwand, L. A.

Publications and source records attributed to Leinwand, L. A..

6 recordsLinked to original sources

bioRxiv2019

miR-1/206 down-regulates splicing factor Srsf9 to promote myogenesis

BackgroundMyogenesis is driven by specific changes in the transcriptome that occur during the different stages of muscle differentiation. In addition to controlled transcriptional transitions, several other post-transcriptional mechanisms direct muscle differentiation. Both alternative splicing and miRNA activity regulate gene expression and production of specialized protein isoforms. Importantly, disruption of either process often results in severe phenotypes as reported for several muscle diseases. Thus, broadening our understanding of the post-transcriptional pathways that operate in muscles will lay the foundation for future therapeutic interventions.\n\nMethodsWe employed bioinformatics analysis in concert with the well-established C2C12 cell system for predicting and validating novel miR-1 and miR-206 targets engaged in muscle differentiation. We used reporter gene assays to test direct miRNA targeting and studied C2C12 cells stably expressing one of the cDNA candidates fused to a heterologous, miRNA-resistant 3 UTR. We monitored effects on differentiation by measuring fusion index, myotube area, and myogenic gene expression during time course differentiation experiments.\n\nResultsGene ontology analysis revealed a strongly enriched set of putative miR-1 and miR-206 targets associated with RNA metabolism. Notably, the expression levels of several candidates decreased during C2C12 differentiation. We discovered that the splicing factor Srsf9 is a direct target of both miRNAs during myogenesis. Persistent Srsf9 expression during differentiation impaired myotube formation and blunted induction of the early pro-differentiation factor myogenin as well as the late differentiation marker sarcomeric myosin, Myh8.\n\nConclusionsOur data uncover novel miR-1 and miR-206 cellular targets and establish a functional link between the splicing factor Srsf9 and myoblast differentiation. The finding that miRNA-mediated clearance of Srsf9 is a key myogenic event illustrates the coordinated and sophisticated interplay between the diverse components of the gene regulatory network.

cell biology↗

bioRxiv2019

Cardiac myocytes respond differentially and synergistically to matrix stiffness and topography

During cardiac disease progression, myocytes undergo molecular, functional and structural changes, including increases in cell size and shape, decreased myocyte alignment and contractility. The heart often increases extracellular matrix production and stiffness, which affect myocytes. The order and hierarchy of these events remain unclear as available in vitro cell culture systems do not adequately model both physiologic and pathologic environments. Traditional cell culture substrates are 5-6 orders of magnitude stiffer than even diseased native cardiac tissue. Studies that do account for substrate stiffness often do not consider intercellular alignment and vice versa. We developed a cardiac myocyte culture platform that better recapitulates native tissue stiffness while simultaneously introducing topographical cues that promote cellular alignment. We show that stiffness and topography impact myocyte molecular and functional properties. We used a spatiotemporally-tunable, photolabile hydrogel platform to generate a range of stiffness and micron-scale topographical patterns to guide neonatal rat ventricular myocyte morphology. Importantly, these substrate patterns were of subcellular dimensions to test whether cells would spontaneously respond to topographical cues rather than an imposed geometry. Cellular contractility was highest and the gene expression profile was most physiologic on gels with healthy cardiac tissue stiffness. Surprisingly, while elongated patterns in stiff gels yielded the greatest cellular alignment, the cells actually had more pathologic functional and molecular profiles. These results highlight that morphological measurements alone are not a surrogate for overall cellular health as many studies assume. In general, substrate stiffness and micropatterning synergistically affect cardiac myocyte phenotype to recreate physiologic and pathologic microenvironments.\n\nSignificance StatementHeart disease is accompanied by organ- and cellular-level remodeling, and deconvoluting their interplay is complex. Cellular-level change is best studied in vitro due to greater control and uniformity of cell types compared to animals. One common metric is degree of cellular alignment as misalignment of myocytes is a hallmark of disease. However, most studies utilize featureless culture surfaces that are orders of magnitude stiffer than, and do not mimic the scaffolding of, the heart. We developed a hydrogel platform with tunable stiffness and patterns providing topographical alignment cues. We cultured heart cells on and characterized multifactorial responses to these dynamic surfaces. Interestingly, conditions that yielded greatest alignment did not yield the healthiest functional and molecular state. Thus, morphology alone is not an indicator of overall cellular health.

cell biology↗

bioRxiv2019

Regression from pathological hypertrophy is sexually dimorphic and stimulus-specific

AimsPathological cardiac hypertrophy is the result of increased cardiomyocyte size, leading to thickening of the left ventricular walls and a decrease in the left ventricular chamber. With early treatment of the underlying cause, cardiac hypertrophy can be reversed in some individuals, while it persists in others. Here, we investigate mechanisms leading to regression of pathological cardiac hypertrophy in two mouse models, in addition to defining the sex differences associated with hypertrophy and regression.\n\nMethods and ResultsTwo pathological hypertrophic stimuli were used in male and female mice (Isoproterenol or Angiotensin II). The stimulus was removed after 7 days of treatment, then the left ventricle was studied at intervals up to 7 days following the removal of the stimulus. Following Isoproterenol removal, male hearts returned to baseline sizes in 4 days while it took 7 days for female hearts to regress. After Angiotensin II removal, the left ventricular masses of males and females did not regress. ERK1/2 was activated in response to both Isoproterenol and Angiotensin II in males, then decreased back to baseline one day after stimulus removal. Expression of ECM genes was greater in response to Angiotensin II and remained elevated longer after Angiotensin II removal, compared to Isoproterenol. Further, collagen content may be playing a role in the irreversible state of Angiotensin II induced hypertrophy as hydroxyproline content was increased following the removal of Angiotensin II in both males and females.\n\nConclusionsRegression of pathological cardiac hypertrophy is possible in some people and in some mouse models; however, the ability for the heart to regress is dependent on the stimulus and biological sex. Further, molecular changes including cellular signaling, protein degradation pathways and the formation of a fibrotic network may contribute to the ability to reverse pathological cardiac hypertrophy and are stimulus- and sex-dependent.\n\nTranslational PerspectivePathological cardiac hypertrophy is a major risk factor for mortality. If cardiac hypertrophy persists for an extended time, there can be many maladaptive changes to the myocardium. With early treatment of the underlying cause, cardiac hypertrophy can be reversed in some individuals, but not in others. While cardiac hypertrophy has been studied extensively, very little is understood about regression of cardiac hypertrophy. It is important that we have a better understanding of mechanisms leading to regression and why this process might not be reversible in some individuals.

molecular biology↗

bioRxiv2019

Cardiac contraction velocity has evolved to match heart rate with body size through variation in β-cardiac myosin sequence.

Heart rate and the maximum velocity of contraction of striated muscle are inversely related to species size. As mammals evolve to different sizes, adaptations are required such as slower contracting heart and skeletal muscles. Analysis of the motor domain of {beta}-myosin from 67 mammals from two clades identifies 14 sites, out of 800, strongly associated with body mass (p<0.01) but not with the clade (p>0.05). Nine of these sites were mutated in the human {beta}-myosin to make it resemble the rat sequence. Biochemical analysis revealed that the rat-human {beta}-myosin chimera functioned like the native rat myosin with a two fold increase in both motility and in the rate of ADP release from the actin.myosin cross-bridge (the step that limits contraction velocity). Both clades use the same small set of amino acids to adjust contraction velocity, suggesting a limited number of ways in which velocity can be manipulated.

biochemistry↗

bioRxiv2019

The ATPase cycle of Human Muscle Myosin II Isoforms: Adaptation of a single mechanochemical cycle for different physiological roles.

Striated muscle myosins are encoded by a large gene family in all mammals, including human. These isoforms define several of the key characteristics of the different striated muscle fiber types including maximum shortening velocity. We have previously used recombinant isoforms of the motor domains of eight different human myosin isoforms to define the actin.myosin cross-bridge cycle in solution. Here, we use a recently developed modeling approach MUSICO to explore how well the experimentally defined cross-bridge cycles for each isoform in solution can predict the characteristics of muscle fiber contraction including duty ratio, shortening velocity, ATP economy and the load dependence of these parameters. The work shows that the parameters of the cross-bridge cycle predict many of the major characteristics of each muscle fiber type and raises the question of what sequence changes are responsible for these characteristics.

biochemistry↗

bioRxiv2019

Pediatric and adult-onset HCM mutations in the myosin motor domain have similar properties

Hypertrophic Cardiomyopathy (HCM) is a common genetic disorder that typically involves left ventricular hypertrophy and cardiac hypercontractility. Mutations in {beta} cardiac myosin heavy chain ({beta}-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to the disease remain incompletely understood. Predicting the severity of any single {beta}-MyHC mutation is hindered by a lack of detailed evaluation at the molecular level. In addition, since the cardiomyopathy can take 20 or more years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations which result in early onset disease may show more severe molecular changes in function compared to later onset mutations. In this work, we performed steady-state and transient kinetic analyses of myosins carrying 1 of 7 missense mutations in the motor domain. Of these 7, 4 have been identified in early onset cardiomyopathy screens. The derived parameters were used to model the ATP driven cross-bridge cycle. Contrary to our hypothesis, the results show no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin.myosin.ADP complex at [Actin] = 3 Kapp along with the closely related Duty Ratio (DR; fraction of myosin in strongly attached force-holding states) and the measured ATPases all change in parallel (in both sign and degree of change) compared to wild type (WT) values. Six of the 7 HCM mutations are clearly distinct from a set of DCM mutations previously characterized.

biophysics↗