Dataset Information

Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility.

ABSTRACT: Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter subcellular protein kinase activation, intracellular Ca2+ and contractility.Ventricular myocytes, isolated from adult Sprague Dawley rats, were treated with the cholesterol reducing agent methyl-?-cyclodextrin (M?CD, 5 mM, 1 hr, room temperature). Total cellular cholesterol levels, caveolin-3 localization, subcellular, ERK and p38 mitogen activated protein kinase (MAPK) signaling, contractility, and [Ca2+]i were assessed.Treatment with M?CD reduced cholesterol levels by ~45 and shifted caveolin-3 from cytoskeleton and triton-insoluble fractions to the triton-soluble fraction, and increased ERK isoform phosphorylation in cytoskeletal, cytosolic, triton-soluble and triton-insoluble membrane fractions without altering their subcellular distributions. In contrast the primary effect of M?CD was on p38 subcellular distribution of p38? with little effect on p38 phosphorylation. Cholesterol depletion increased cardiomyocyte twitch amplitude and the rates of shortening and relaxation in conjunction with increased diastolic and systolic [Ca2+]i.These results indicate that acute reductions in membrane cholesterol levels differentially modulate basal cardiomyocyte subcellular MAPK signaling, as well as increasing [Ca2+]i and contractility.

SUBMITTER: Haque MZ

PROVIDER: S-EPMC4956108 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility.

Haque Mohammed Z MZ McIntosh Victoria J VJ Abou Samra Abdul B AB Mohammad Ramzi M RM Lasley Robert D RD

PloS one 20160721 7

<h4>Unlabelled</h4>Membrane cholesterol levels play an important factor in regulating cell function. Sarcolemmal cholesterol is concentrated in lipid rafts and caveolae, which are flask-shaped invaginations of the plasma membrane. The scaffolding protein caveolin permits the enrichment of cholesterol in caveolae, and caveolin interactions with numerous proteins regulate their function. The purpose of this study was to determine whether acute reductions in cardiomyocyte cholesterol levels alter s ...[more]

PMID: 27441649

Similar Datasets

Project description:We have previously demonstrated that substitution of ATP with 2 deoxy-ATP (dATP) increased the magnitude and rate of force production at all levels of Ca(2+)-mediated activation in demembranated cardiac muscle. In the current study we hypothesized that cellular [dATP] could be increased by viral-mediated overexpression of the ribonucleotide reductase (Rrm1 and Rrm2) complex, which would increase contractility of adult rat cardiomyocytes. Cell length and ratiometric (Fura2) Ca(2+) fluorescence were monitored by video microscopy. At 0.5Hz stimulation, the extent of shortening was increased ~40% and maximal rate of shortening was increased ~80% in cardiomyocytes overexpressing Rrm1+Rrm2 as compared to non-transduced cardiomyocytes. The maximal rate of relaxation was also increased ~150% with Rrm1+Rrm2 overexpression, resulting in decreased time to 50% relaxation over non-transduced cardiomyocytes. These differences were even more dramatic when compared to cardiomyocytes expressing GFP-only. Interestingly, Rrm1+Rrm2 overexpression had no effect on minimal or maximal intracellular [Ca(2+)], indicating increased contractility is primarily due to increased myofilament activity without altering Ca(2+) release from the sarcoplasmic reticulum. Additionally, functional potentiation was maintained with Rrm1+Rrm2 overexpression as stimulation frequency was increased (1Hz and 2Hz). HPLC analysis indicated cellular [dATP] was increased by approximately 10-fold following transduction, becoming ~1.5% of the adenine nucleotide pool. Furthermore, 2% dATP was sufficient to significantly increase crossbridge binding and contractile force during sub-maximal Ca(2+) activation in demembranated cardiac muscle. These experiments demonstrate the feasibility of directly targeting the actin-myosin chemomechanical crossbridge cycle to enhance cardiac contractility and relaxation without affecting minimal or maximal Ca(2+). This article is part of a Special issue entitled "Possible Editorial".

Project description:Aims:Cardiomyocyte Ca2+ homeostasis is altered with aging via poorly-understood mechanisms. The Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is an osmotically-activated Ca2+ channel, and there is limited information on the role of TRPV4 in cardiomyocytes. Our data show that TRPV4 protein expression increases in cardiomyocytes of the aged heart. The objective of this study was to examine the role of TRPV4 in cardiomyocyte Ca2+ homeostasis following hypoosmotic stress and to assess the contribution of TRPV4 to cardiac contractility and tissue damage following ischaemia-reperfusion (I/R), a pathological condition associated with cardiomyocyte osmotic stress. Methods and results:TRPV4 protein expression increased in cardiomyocytes of Aged (24-27?months) mice compared with Young (3-6?months) mice. Immunohistochemistry revealed TRPV4 localization to microtubules and the t-tubule network of cardiomyocytes of Aged mice, as well as in left ventricular myocardium of elderly patients undergoing surgical aortic valve replacement for aortic stenosis. Following hypoosmotic stress, cardiomyocytes of Aged, but not Young exhibited an increase in action-potential induced Ca2+ transients. This effect was mediated via increased sarcoplasmic reticulum Ca2+ content and facilitation of Ryanodine Receptor Ca2+ release and was prevented by TRPV4 antagonism (1??mol/L HC067047). A similar hypoosmotic stress-induced facilitation of Ca2+ transients was observed in Young transgenic mice with inducible TRPV4 expression in cardiomyocytes. Following I/R, isolated hearts of Young mice with transgenic TRPV4 expression exhibited enhanced contractility vs. hearts of Young control mice. Similarly, hearts of Aged mice exhibited enhanced contractility vs. hearts of Aged TRPV4 knock-out (TRPV4-/-) mice. In Aged, pharmacological inhibition of TRPV4 (1??mol/L, HC067047) prevented hypoosmotic stress-induced cardiomyocyte death and I/R-induced cardiac damage. Conclusions:Our findings provide a new mechanism for hypoosmotic stress-induced cardiomyocyte Ca2+ entry and cell damage in the aged heart. These finding have potential implications in treatment of elderly populations at increased risk of myocardial infarction and I/R injury.

Project description:The measurement of the contractile behavior of single cardiomyocytes has made a significant contribution to our understanding of the physiology and pathophysiology of the myocardium. However, the isolation of cardiomyocytes introduces various technical and statistical issues. Traditional video and fluorescence microscopy techniques based around conventional microscopy systems result in low-throughput experimental studies, in which single cells are studied over the course of a pharmacological or physiological intervention. We describe a new approach to these experiments made possible with a new piece of instrumentation, the CytoCypher High-Throughput System (CC-HTS). We can assess the shortening of sarcomeres, cell length, Ca2+ handling, and cellular morphology of almost 4 cells per minute. This increase in productivity means that batch-to-batch variation can be identified as a major source of variability. The speed of acquisition means that sufficient numbers of cells in each preparation can be assessed for multiple conditions reducing these batch effects. We demonstrate the different temporal scales over which the CC-HTS can acquire data. We use statistical analysis methods that compensate for the hierarchical effects of clustering within heart preparations and demonstrate a significant false-positive rate, which is potentially present in conventional studies. We demonstrate a more stringent way to perform these tests. The baseline morphological and functional characteristics of rat, mouse, guinea pig, and human cells are explored. Finally, we show data from concentration response experiments revealing the usefulness of the CC-HTS in such studies. We specifically focus on the effects of agents that directly or indirectly affect the activity of the motor proteins involved in the production of cardiomyocyte contraction. A variety of myocardial preparations with differing levels of complexity are in use (e.g., isolated muscle bundles, thin slices, perfused dual innervated isolated heart, and perfused ventricular wedge). All suffer from low throughput but can be regarded as providing independent data points in contrast to the clustering problems associated with isolated cell studies. The greater productivity and sampling power provided by CC-HTS may help to reestablish the utility of isolated cell studies, while preserving the unique insights provided by studying the contribution of the fundamental, cellular unit of myocardial contractility.

Project description:Cellular structure and function are interdependent. To understand this relationship in beating heart cells, individual neonatal rat ventricular myocytes (NRVMs) were analyzed one and 3 days after plating when cultured on different stiffness (100, 400 kPa) and surface structures (flat or [Formula: see text] high, [Formula: see text] diameter, microposts spaced [Formula: see text] apart) manufactured from polydimethylsiloxane. Myofibril structure seen by immunohistochemistry was organized in three dimensions when NRVMs were attached to microposts. On day three, paxillin distribution near the post serving as cellular anchorage was quantified on both soft posts (12.04 % of total voxel count) and stiff posts (8.16 %). Living NRVMs were analyzed using line scans for sarcomeric shortening and shortening velocity, and traction force microscopy for surface stress and surface tension. One day after plating, NRVMs shortened more on soft posts ([Formula: see text] at [Formula: see text]) compared to either soft flat ([Formula: see text] at [Formula: see text]), stiff posts ([Formula: see text] at [Formula: see text]) or stiff flat ([Formula: see text] at [Formula: see text]). NRVMs have decreased shortening and shortening velocity on soft posts ([Formula: see text] at [Formula: see text]) compared to soft flat ([Formula: see text] at [Formula: see text]) substrates. The surface stress and surface tension increased over time for both soft post ([Formula: see text] and [Formula: see text] to [Formula: see text] and [Formula: see text]) and flat ([Formula: see text] and [Formula: see text] to [Formula: see text] and [Formula: see text]) substrates. Paxillin displacement during contraction on day three was significantly greater in NRVMs attached to soft posts [Formula: see text] compared to flat [Formula: see text] substrates. The volume and time creating four-dimensional data, interpreted by structural engineering theory, demonstrate subdomain structure is maintained by the counterbalance between the external load acting upon and the internal forces generated by the cardiomyocyte. These findings provide further insight into localized regulation of cellular mechanical function.

Project description:This study examines the relationship between cellular sphingomyelin content and the distribution of unesterified cholesterol between the plasma-membrane pool and the putative intracellular regulatory pool. The sphingomyelin content of cultured human skin fibroblasts was reduced by treatment of intact cells with extracellularly added neutral sphingomyelinase, and subsequent changes in the activities of cholesterol-metabolizing enzymes were determined. Exposure of fibroblasts to 0.1 unit of sphingomyelinase/ml for 60 min led to the depletion of more than 90% of the cellular sphingomyelin, as determined from total lipid extracts. In a time-course study, it was found that within 10 min of the addition of sphingomyelinase to cells, a dramatic increase in acyl-CoA:cholesterol acyltransferase activity could be observed, whether measured from the appearance of plasma membrane-derived [3H]cholesterol or exogenously added [14C]oleic acid, in cellular cholesteryl esters. In addition, the cholesteryl ester mass was significantly higher in sphingomyelin-depleted fibroblasts at 3 h after exposure to sphingomyelinase compared with that in untreated fibroblasts [7.1 +/- 0.4 nmol of cholesterol/mg equivalents of esterified cholesterol compared with 4.2 +/- 0.1 nmol of cholesterol/mg equivalents of cholesteryl ester in control cells (P less than 0.05)]. The sphingomyelin-depleted cells also showed a reduction in the rate of endogenous synthesis of cholesterol, as measured by incorporation of sodium [14C]acetate into [14C]cholesterol. These results are consistent with a rapid movement of cholesterol from sphingomyelin-depleted plasma membranes to the putative intracellular regulatory pool of cholesterol. This mass movement of cholesterol away from the plasma membranes presumably resulted from a decreased capacity of the plasma membranes to solubilize cholesterol, since sphingomyelin-depleted cells also had a decreased capacity to incorporate nanomolar amounts of [3H]cholesterol from the extracellular medium, as compared with control cells. These findings confirm previous assumptions that the membrane sphingomyelin content is an important determinant of the overall distribution of cholesterol within intact cells.

Dataset Information

Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility.

Publications

Cholesterol Depletion Alters Cardiomyocyte Subcellular Signaling and Increases Contractility.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure