Functional culture and in vitro genetic and small-molecule manipulation of adult mouse cardiomyocytes.
ABSTRACT: Primary adult cardiomyocyte (aCM) represent the mature form of myocytes found in the adult heart. However, culture of aCMs in particular is challenged by poor survival and loss of phenotype, rendering extended in vitro experiments unfeasible. Here, we establish murine aCM culture methods that enhance survival and maintain sarcomeric structure and Ca2+ cycling to enable physiologically relevant contractile force measurements. We also demonstrate genetic and small-molecule manipulations that probe mechanisms underlying myocyte functional performance. Together, these refinements to aCM culture present a toolbox with which to advance our understanding of myocardial physiology.
Project description:BACKGROUND:Adult mammalian hearts have a limited ability to generate new cardiomyocytes. Proliferation of existing adult cardiomyocytes (ACMs) is a potential source of new cardiomyocytes. Understanding the fundamental biology of ACM proliferation could be of great clinical significance for treating myocardial infarction (MI). We aim to understand the process and regulation of ACM proliferation and its role in new cardiomyocyte formation of post-MI mouse hearts. METHODS:?-Actin-green fluorescent protein transgenic mice and fate-mapping Myh6-MerCreMer-tdTomato/lacZ mice were used to trace the fate of ACMs. In a coculture system with neonatal rat ventricular myocytes, ACM proliferation was documented with clear evidence of cytokinesis observed with time-lapse imaging. Cardiomyocyte proliferation in the adult mouse post-MI heart was detected by cell cycle markers and 5-ethynyl-2-deoxyuridine incorporation analysis. Echocardiography was used to measure cardiac function, and histology was performed to determine infarction size. RESULTS:In vitro, mononucleated and bi/multinucleated ACMs were able to proliferate at a similar rate (7.0%) in the coculture. Dedifferentiation proceeded ACM proliferation, which was followed by redifferentiation. Redifferentiation was essential to endow the daughter cells with cardiomyocyte contractile function. Intercellular propagation of Ca2+ from contracting neonatal rat ventricular myocytes into ACM daughter cells was required to activate the Ca2+-dependent calcineurin-nuclear factor of activated T-cell signaling pathway to induce ACM redifferentiation. The properties of neonatal rat ventricular myocyte Ca2+ transients influenced the rate of ACM redifferentiation. Hypoxia impaired the function of gap junctions by dephosphorylating its component protein connexin 43, the major mediator of intercellular Ca2+ propagation between cardiomyocytes, thereby impairing ACM redifferentiation. In vivo, ACM proliferation was found primarily in the MI border zone. An ischemia-resistant connexin 43 mutant enhanced the redifferentiation of ACM-derived new cardiomyocytes after MI and improved cardiac function. CONCLUSIONS:Mature ACMs can reenter the cell cycle and form new cardiomyocytes through a 3-step process: dedifferentiation, proliferation, and redifferentiation. Intercellular Ca2+ signal from neighboring functioning cardiomyocytes through gap junctions induces the redifferentiation process. This novel mechanism contributes to new cardiomyocyte formation in post-MI hearts in mammals.
Project description:Atypically-shaped cardiomyocytes (ACMs) constitute a novel subpopulation of beating heart cells found in the cultures of cardiac myocyte-removed crude fraction cells obtained from adult mouse cardiac ventricles. Although ~500 beating ACMs are observed under microscope in the cell cultures obtained from the hearts of either male or female mice, the origin of these cells in cardiac tissue has yet to be elucidated due to the lack of exclusive markers. In the present study, we demonstrate the efficacy of cellular prion protein (PrP) as a surface marker of ACMs. Cells expressing PrP at the plasma membrane in the culture of the crude fraction cells were found to develop into beating ACMs by themselves or fuse with each other to become larger multinuclear beating ACMs. Combining PrP with a cardiac-specific contractile protein cardiac troponin T (cTnT) allowed us to identify native ACMs in the mouse cardiac ventricles as either clustered or solitary cells. PrP- and cTnT-marked cells were also found in the adult, even aged, human cardiac ventricles. These findings suggest that interstitial cells marked by PrP and cTnT, native ACMs, exhibit life-long survival in the cardiac ventricles of both mice and humans.
Project description:It has been believed that mammalian adult cardiomyocytes (ACMs) are terminally-differentiated and are unable to proliferate. Recently, using a bi-transgenic ACM fate mapping mouse model and an in vitro culture system, we demonstrated that adult mouse cardiomyocytes were able to dedifferentiate into cardiac progenitor-like cells (CPCs). However, little is known about the molecular basis of their intrinsic cellular plasticity. Here we integrate single-cell transcriptome and whole-genome DNA methylation analyses to unravel the molecular mechanisms underlying the dedifferentiation and cell cycle reentry of mouse ACMs. Compared to parental cardiomyocytes, dedifferentiated mouse cardiomyocyte-derived CPCs (mCPCs) display epigenomic reprogramming with many differentially-methylated regions, both hypermethylated and hypomethylated, across the entire genome. Correlated well with the methylome, our transcriptomic data showed that the genes encoding cardiac structure and function proteins are remarkably down-regulated in mCPCs, while those for cell cycle, proliferation, and stemness are significantly up-regulated. In addition, implantation of mCPCs into infarcted mouse myocardium improves cardiac function with augmented left ventricular ejection fraction. Our study demonstrates that the cellular plasticity of mammalian cardiomyocytes is the result of a well-orchestrated epigenomic reprogramming and a subsequent global transcriptomic alteration.
Project description:BACKGROUND:Asbestos-containing materials (ACMs) can still be found in many homes in Australia and other countries. ACMs present a health risk when they are damaged or disturbed, such as during do-it-yourself home renovations. However, community members lack knowledge and awareness about asbestos identification and its safe management in residential settings. OBJECTIVE:The objective of our study was to describe the process of developing a mobile phone app, ACM Check, that incorporates a questionnaire designed to identify and assess ACMs located in residential settings. METHODS:A multidisciplinary team was involved in the formative development and creation of the mobile phone app. The formative development process comprised 6 steps: defining the scope of the app; conducting a comprehensive desktop review by searching online literature databases, as well as a wider online search for gray literature; drafting and revising the content, questionnaire, conditional branching rules, and scoring algorithms; obtaining expert input; manually pretesting the questionnaire; and formulating a final content document to be provided to the software development company. We then constructed ACM Check on the iOS platform for use in a validation study, and then updated the app, replicated it on Android, and released it to the public. RESULTS:The ACM Check app identifies potential ACMs, prioritizes the materials based on their condition and likelihood of disturbance, and generates a summary report for each house assessed. CONCLUSIONS:ACM Check is an initiative to raise community members' awareness of asbestos in the residential environment and also serves as a data collection tool for epidemiologic research. It can potentially be modified for implementation in other countries or used as the basis for the assessment of other occupational or environmental hazards.
Project description:Mammalian cardiac myocytes (CMs) stop proliferating soon after birth and subsequent heart growth comes from hypertrophy, limiting the adult heart's regenerative potential after injury. The molecular events that mediate CM cell cycle exit are poorly understood. To determine the epigenetic mechanisms limiting CM cycling in adult CMs (ACMs) and whether trimethylation of lysine 9 of histone H3 (H3K9me3), a histone modification associated with repressed chromatin, is required for the silencing of cell cycle genes, we developed a transgenic mouse model where H3K9me3 is specifically removed in CMs by overexpression of histone demethylase, KDM4D. Although H3K9me3 is found across the genome, its loss in CMs preferentially disrupts cell cycle gene silencing. KDM4D binds directly to cell cycle genes and reduces H3K9me3 levels at these promotors. Loss of H3K9me3 preferentially leads to increased cell cycle gene expression resulting in enhanced CM cycling. Heart mass was increased in KDM4D overexpressing mice by postnatal day 14 (P14) and continued to increase until 9-weeks of age. ACM number, but not size, was significantly increased in KDM4D expressing hearts, suggesting CM hyperplasia accounts for the increased heart mass. Inducing KDM4D after normal development specifically in ACMs resulted in increased cell cycle gene expression and cycling. We demonstrated that H3K9me3 is required for CM cell cycle exit and terminal differentiation in ACMs. Depletion of H3K9me3 in adult hearts prevents and reverses permanent cell cycle exit and allows hyperplastic growth in adult hearts in vivo.
Project description:Asbestos-containing materials (ACMs) were used extensively throughout much of the 20th century and can still be found in many Australian homes. Therefore, we developed a mobile application ("app"), called ACM Check, which guides users through a home inspection to identify and assess certain types of in situ ACM. A cross-sectional study was conducted using the app to collect data on the type and condition of in situ asbestos in Australian residential settings. Since being released in June 2017, we have received data for 702 home inspections. Of these, 578 (82.3%) houses contained a total of 1895 in situ materials categorised as positive for asbestos by the app. The most prevalent ACMs were used for the backing board to electrical meter boxes (50% of homes), eaves and soffit linings (44.2% of homes), and fencing (28.1% of homes). While the majority of ACMs were categorised as 'very low' or 'low' priority for removal or remediation, 6.6% of all ACMs were considered to be of 'high' priority. Mobile apps offer a platform to help increase people's awareness of possible health hazards found in the residential environment, such as asbestos, while also being used to collect data for public and environmental health research.
Project description:Nitrogen-doped porous activated carbon monoliths (NDP-ACMs) have long been the most desirable materials for supercapacitors. Unique to the conventional template based Lewis acid/base activation methods, herein, we report on a simple yet practicable novel approach to production of the three-dimensional NDP-ACMs (3D-NDP-ACMs). Polyacrylonitrile (PAN) contained carbon nanotubes (CNTs), being pre-dispersed into a tubular level of dispersions, were used as the starting material and the 3D-NDP-ACMs were obtained via a template-free process. First, a continuous mesoporous PAN/CNT based 3D monolith was established by using a template-free temperature-induced phase separation (TTPS). Second, a nitrogen-doped 3D-ACM with a surface area of 613.8?m2/g and a pore volume 0.366?cm3/g was obtained. A typical supercapacitor with our 3D-NDP-ACMs as the functioning electrodes gave a specific capacitance stabilized at 216?F/g even after 3000 cycles, demonstrating the advantageous performance of the PAN/CNT based 3D-NDP-ACMs.
Project description:The cAMP-dependent protein kinase (PKA) signaling pathway is the primary means by which the heart regulates moment-to-moment changes in contractility and metabolism. We have previously found that PKA signaling is dysfunctional in the diabetic heart, yet the underlying mechanisms are not fully understood. The objective of this study was to determine if decreased insulin signaling contributes to a dysfunctional PKA response. To do so, we isolated adult cardiomyocytes (ACMs) from wild type and Akita type 1 diabetic mice. ACMs were cultured in the presence or absence of insulin and PKA signaling was visualized by immunofluorescence microscopy using an antibody that recognizes proteins specifically phosphorylated by PKA. We found significant decreases in proteins phosphorylated by PKA in wild type ACMs cultured in the absence of insulin. PKA substrate phosphorylation was decreased in Akita ACMs, as compared to wild type, and unresponsive to the effects of insulin. The decrease in PKA signaling was observed regardless of whether the kinase was stimulated with a beta-agonist, a cell-permeable cAMP analog, or with phosphodiesterase inhibitors. PKA content was unaffected, suggesting that the decrease in PKA signaling may be occurring by the loss of specific PKA substrates. Phospho-specific antibodies were used to discern which potential substrates may be sensitive to the loss of insulin. Contractile proteins were phosphorylated similarly in wild type and Akita ACMs regardless of insulin. However, phosphorylation of the glycolytic regulator, PFK-2, was significantly decreased in an insulin-dependent manner in wild type ACMs and in an insulin-independent manner in Akita ACMs. These results demonstrate a defect in PKA activation in the diabetic heart, mediated in part by deficient insulin signaling, that results in an abnormal activation of a primary metabolic regulator.
Project description:NimbleGen Mouse DNA Methylation 3x720K CpG Islands Pus RefSeq Promoter Array was used to perform a DNA methylome analysis to unravel the molecular mechanism underlying the dedifferentiation and cell cycle reentry of mouse adult cardiomyocytes (ACMs). A total of six DNA samples (10 ng each) derived from the population cells from either control adult cardiomyocytes or mCPCs were subject to whole genome amplification and then 2.5 Âµg of amplified products were used for DNA methylome analysis. There were three biological replicates in each group (both ACM controls and mCPCs). The whole genome methylome data using NimbleGen Mouse 2.1M array and genomic DNA derived from population cells for both control adult cardiomyocytes and mCPC can be found in E-MTAB-3984. Single-cell whole-transcriptome microarray data using Affymetrix Mouse Genome 430 2.0 Array can be found in E-MTAB-3981.
Project description:Amyotrophic lateral sclerosis (ALS) is a fatal paralytic disorder caused by dysfunction and degeneration of motor neurons. Multiple disease-causing mutations, including in the genes for SOD1 and TDP-43, have been identified in ALS. Astrocytes expressing mutant SOD1 are strongly implicated in the pathogenesis of ALS: we have shown that media conditioned by astrocytes carrying mutant SOD1(G93A) contains toxic factor(s) that kill motoneurons by activating voltage-sensitive sodium (Na v ) channels. In contrast, a recent study suggests that astrocytes expressing mutated TDP43 contribute to ALS pathology, but do so via cell-autonomous processes and lack non-cell-autonomous toxicity. Here we investigate whether astrocytes that express diverse ALS-causing mutations release toxic factor(s) that induce motoneuron death, and if so, whether they do so via a common pathogenic pathway. We exposed primary cultures of wild-type spinal cord cells to conditioned medium derived from astrocytes (ACM) that express SOD1 (ACM-SOD1(G93A) and ACM-SOD1(G86R)) or TDP43 (ACM-TDP43(A315T)) mutants; we show that such exposure rapidly (within 30-60 min) increases dichlorofluorescein (DCF) fluorescence (indicative of nitroxidative stress) and leads to extensive motoneuron-specific death within a few days. Co-application of the diverse ACMs with anti-oxidants Trolox or esculetin (but not with resveratrol) strongly improves motoneuron survival. We also find that co-incubation of the cultures in the ACMs with Na v channel blockers (including mexiletine, spermidine, or riluzole) prevents both intracellular nitroxidative stress and motoneuron death. Together, our data document that two completely unrelated ALS models lead to the death of motoneuron via non-cell-autonomous processes, and show that astrocytes expressing mutations in SOD1 and TDP43 trigger such cell death through a common pathogenic pathway that involves nitroxidative stress, induced at least in part by Na v channel activity.