PKC epsilon signaling effect on actin assembly is diminished in cardiomyocytes when challenged to additional work in a stiff microenvironment

Abstract

The stiffness of the microenvironment surrounding a cell can result in cytoskeletal remodeling, leading to altered cell function and tissue macrostructure. In this study, we tuned the stiffness of the underlying substratum on which neonatal rat cardiomyocytes were grown in culture to mimic normal (10 kPa), pathological stiffness of fibrotic myocardium (100 kPa), and a nonphysiological extreme (glass). Cardiomyocytes were then challenged by beta adrenergic stimulation through isoproterenol treatment to investigate the response to acute work demand for cells grown on surfaces of varying stiffness. In particular, the PKCɛ signaling pathway and its role in actin assembly dynamics were examined. Significant changes in contractile metrics were seen on cardiomyocytes grown on different surfaces, but all cells responded to isoproterenol treatment, eventually reaching similar time to peak tension. In contrast, the assembly rate of actin was significantly higher on stiff surfaces, so that only cells grown on soft surfaces were able to respond to acute isoproterenol treatment. Förster Resonance Energy Transfer of immunofluorescence on the cytoskeletal fraction of cardiomyocytes confirmed that the molecular interaction of PKCɛ with the actin capping protein, CapZ, was very low on soft substrata but significantly increased with isoproterenol treatment, or on stiff substrata. Therefore, the stiffness of the culture surface chosen for in vitro experiments might mask the normal signaling and affect the ability to translate basic science more effectively into human therapy.

CapZ integrates several signaling pathways in response to mechanical stiffness

“…7) suggests that the actin binding face (site 1) and the β-tentacle (site 2) cooperate to regulate CapZ docking to the actin filament. This model is based on the new results, other investigations (Kim et al, 2007; Kuhn and Pollard, 2007; Smith et al, 2006), and our prior work (Hartman et al, 2009; Li and Russell, 2013; Lin et al, 2013, 2015, 2016; Li et al, 2014, 2016; Mkrtschjan et al, 2018). We propose the tight binding is loosened in growth states of hypertrophy triggered by stimuli such as mechanical flexing, loading, stiffer substrate, angiotensin II, and phenylephrine.…”

“…Substrate stiffness varies in health and disease (Bhana et al, 2010; Fomovsky and Holmes, 2010), and stiffness has been shown to regulate actin assembly and cell size (Li et al, 2016; Mkrtschjan et al, 2018). FRAP data (Fig.…”

“…This improved the quality of FRET analysis and the preservation of fluorescent probe localization in the samples. FRET measurements were conducted in a confocal light scanning microscope (LSM 880) at room temperature by the acceptor photobleaching method (König et al, 2006; Mkrtschjan et al, 2018). Briefly, images of the donor (CapZ-GFP) and the acceptor (PIP2 secondary antibody) were captured with alternating, partial photobleaching of the acceptor dye in an ROI of the cell.…”

Cardiac CapZ regulation during acute exercise

“…Cardiac myofilament phosphorylation is mediated by a number of different kinases. Work by us (7)(8)(9)(10)14) and others (18)(19)(20)(21) have found that CapZ influences myofilament regulation by protein kinase C (PKC) and is a substrate for this family of kinases. To determine how exercise alters myofilament-associated PKC and if a decrease in cardiac CapZ levels alters exercisedependent changes in sarcomeric PKC we probed isolated cardiac myofilaments for PKC- (calciumdependent isoform) and PKC- (calcium independent isoform).…”

CapZ actúa como integrador de señalamiento intracelular en respuesta a estímulos mecánicos en el miofilamento cardiaco

“…Los virus adeno-asociados que contienen los ADN codificantes de CapZβ1-GFP y la forma truncada de CapZ que carece de los 12 últimos amino ácidos en la C-terminal (CapZβ1ΔC-GFP) fueron preparados como se ha descrito previamente (Lin et al 2013) Ensayos de Fluorescence Recovery After Photobleaching (FRAP) fueron realizados en cardiomiocitos infectados con CapZβ1-GFP o CapZβ1ΔC-GFP, mientras que los ensayos de Förster Resonance Energy Transfer (FRET) fueron hechos mediante la incorporación de un anticuerpo específico para PIP2 (Abcam #2335) como se ha descrito previamente (Mkrtschjan et al 2018). Neomicina (500 μM, 30 min) fue utilizada para inhibir la disponibilidad intracelular de PIP2.…”