In Vitro Assessment of Cardiac Fibroblast Activation at Physiologic Stiffness.

Cardiac fibroblasts (CF) are an essential cell type in cardiac physiology, playing diverse roles in maintaining structural integrity, extracellular matrix (ECM) synthesis, and tissue repair. Under normal conditions, these cells reside in the interstitium in a quiescent state poised to sense and respond to injury by synthesizing and secreting collagen, vimentin, hyaluronan, and other ECM components. In response to mechanical and chemical stimuli, these "resident" fibroblasts can undergo a transformation through a continuum of activation states into what is commonly known as a "myofibroblast," in a process critical for injury response. Despite progress in understanding the contribution of fibroblasts to cardiac health and disease, much remains unknown about the signaling mediating this activation, in part owing to technical challenges in evaluating CF function and activation status in vitro. Given their role in monitoring the ECM, CFs are acutely sensitive to stiffness and pressure. High basal activation of isolated CFs is common due to the super-physiologic stiffness of traditional cell culture substrates, making assays dependent on quiescent cells challenging. To overcome this problem, cell culture parameters must be tightly controlled, and the use of dishes coated with biocompatible reduced-stiffness substrates, such as 8-kPa polydimethylsiloxane (PDMS), has shown promise in reducing basal activation of fibroblasts. Here, we describe cell culture protocol for maintaining CF quiescence in vitro to enable a dynamic range for the assessment of activation status in response to fibrogenic stimuli using PDMS-coated coverslips. Our protocol provides a cost-effective tool to study fibroblast signaling and activity, allowing researchers to better understand the underlying mechanisms involved in cardiac fibrosis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of 8-kPa polydimethylsiloxane (PDMS)/gelatin-coated coverslips for cardiac fibroblast cell culture Basic Protocol 2: Isolation of adult cardiac fibroblasts and plating onto PDMS coverslips Basic Protocol 3: Assessment of cardiac fibroblast activation by α smooth muscle actin (αSMA) immunocytochemistry.

Altered reward sensitivity to sucrose outcomes prior to drug exposure in alcohol preferring rats.

Addiction involves key impairments in reward sensitivity (RS). The current study explored impaired RS to natural reward as a predisposing factor to addictive-like behavior. Alcohol preferring (P) rats are selectively bred based on significantly greater ethanol consumption and preference and offer the ability to inspect differences in subjects with a positive family history of addictive-like behavior. P rat's RS was compared to RS in the well-used Sprague-Dawley (SD) strain. To assess RS in a novel manner, instrumental incentive contrast, discrimination and consumption of sucrose solution were examined. Animals performed in a free operant situation for different sucrose concentration solutions using a block of 'mixed' trials with alternating outcome concentrations (e.g., 5 and 10 % sucrose) to change outcome value in a predictable manner. Animals also performed for reward in blocks of single outcome trials (5 or 10 or 20 or 40 % sucrose daily exposure) surrounding the mixed block. RS (e.g., reward discrimination and contrast effects between and within-sessions) was measured by changes in trials completed, instrumental response latency and consumption. P rats expressed an altered profile of RS with a greater tendency toward equivalent responding to different outcomes within the same session and an absence of incentive contrast from diverse reward comparisons. In contrast, SD animals expressed within-session reward discrimination and a subset of incentive contrast effects. These effects were moderated by food deprivation more consistently in SD compared to P rats. P rat alterations in processing natural rewards could predispose them to addictive-like behaviors including greater alcohol consumption and preference.

Sirtuin 5 levels are limiting in preserving cardiac function and suppressing fibrosis in response to pressure overload.

Heart failure (HF) is the inability of the heart to pump blood sufficiently to meet the metabolic demands of the body. HF with reduced systolic function is characterized by cardiac hypertrophy, ventricular fibrosis and remodeling, and decreased cardiac contractility, leading to cardiac functional impairment and death. Transverse aortic constriction (TAC) is a well-established model for inducing hypertrophy and HF in rodents. Mice globally deficient in sirtuin 5 (SIRT5), a NAD+-dependent deacylase, are hypersensitive to cardiac stress and display increased mortality after TAC. Prior studies assessing SIRT5 functions in the heart have all employed loss-of-function approaches. In this study, we generated SIRT5 overexpressing (SIRT5OE) mice, and evaluated their response to chronic pressure overload using TAC. Compared to littermate controls, SIRT5OE mice were protected against adverse functional consequences of TAC, left ventricular dilation and impaired ejection fraction. Transcriptomic analysis revealed that SIRT5 suppresses key HF sequelae, including the metabolic switch from fatty acid oxidation to glycolysis, immune activation, and fibrotic signaling pathways. We conclude that SIRT5 is a limiting factor in the preservation of cardiac function in response to experimental pressure overload.

Effort-reward balance and work motivation in rats: Effects of context and order of experience.

Being motivated means exerting effort toward a goal. The 'law of least work' emphasizes a preference for exerting relatively less effort. The law crosses boundaries among species and between physical and mental work. Organisms should be highly sensitive to shifts in effort-reward balance (ERB) in order to make optimal choices. We used a free operant-foraging task to investigate changes in ERB on choice between options requiring more or less effort. Results showed a consistent preference for the option with less effort and insensitivity to shifts in ERB. A second aim explored the influence of order of experience on effort choice. Choice for the more effortful option significantly increased after experiencing an equal effort-reward relationship during the initial free operant-foraging session. This relative increase in choice for the effortful option persisted even after effort-reward imbalance. The findings highlight the importance of contextual factors such as order of experience when examining the impact of shifting effort-reward associations. Instead of ignoring or reducing order effects, the sequence of experience (e.g. for shifts in ERB) could be manipulated to enhance or reduce value of outcomes or effort itself.

Medical marijuana in the workplace: challenges and management options for occupational physicians.

Although possession and use of marijuana is prohibited by federal law, legalization in four states (Alaska, Colorado, Oregon, and Washington) and allowance for palliation and therapy in 19 others may reposition the drug away from the fringes of society. This evolving legal environment, and growing scientific evidence of its effectiveness for select health conditions, requires assessment of the safety and appropriateness of marijuana within the American workforce. Although studies have suggested that marijuana may be used with reasonable safety in some controlled environments, there are potential consequences to its use that necessitate employer scrutiny and concern. Several drug characteristics must be considered, including Δ-tetrahydrocannabinol (Δ-THC, or THC) concentration, route of administration, dose and frequency, and pharmacokinetics, as well as the risks inherent to particular workplace environments.