Variation in stiffness regulates cardiac myocyte hypertrophy via signaling pathways

Abstract

Much diseased human myocardial tissue is fibrotic and stiff, which increases the work that the ventricular myocytes must perform to maintain cardiac output. The hypothesis tested is that the increased load due to greater stiffness of the substrata drives sarcomere assembly of cells, thus strengthening them. Neonatal rat ventricular myocytes (NRVM) were cultured on polyacrylamide or polydimethylsiloxane substrates with stiffness of 10 kPa, 100 kPa, or 400 kPa, or glass with stiffness of 61.9 GPa. Cell size increased with stiffness. Two signaling pathways were explored, phosphorylation of focal adhesion kinase (p-FAK) and lipids by phosphatidylinositol 4,5-bisphosphate (PIP2). Subcellular distributions of both were determined in the sarcomeric fraction by antibody localization, and total amounts were measured by Western or dot blotting, respectively. More p-FAK and PIP2 distributed to the sarcomeres of NRVM grown on stiffer substrates. Actin assembly involves the actin capping protein Z (CapZ). Both actin and CapZ dynamic exchange were significantly increased on stiffer substrates when assessed by fluorescence recovery after photobleaching (FRAP) of green fluorescent protein tags. Blunting of actin FRAP by FAK inhibition implicates linkage from mechano-signalling pathways to cell growth. Thus, increased stiffness of cardiac disease can be modeled with polymeric materials to understand how the microenvironment regulates cardiac hypertrophy.

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

“…PKCɛ translocation has also been shown in vitro following CM stretch (Vincent et al, ). Our laboratory has demonstrated that mechanical stimulation through increased substrate stiffness or cyclic mechanical stretch leads to greater actin dynamics, which is mediated through a variety of signaling molecules, including PKCɛ, PIP2, and FAK (Li, Mkrtschjan, Lin, & Russell, ; Lin, Swanson, Li, Mkrtschjan, & Russell, ). It has been shown that isoproterenol treatment leads to rapid translocation of PKCɛ to the cell particulate fraction (Li, Cai, Liu, & Guo, ), making it a strong candidate here for implication in increased actin assembly rate.…”

“…PKCɛ translocation has also been shown in vitro following CM stretch [Vincent et al, 2006]. Our lab has demonstrated that mechanical stimulation through increased substrate stiffness or cyclic mechanical stretch leads to greater actin dynamics, which is mediated through a variety of signaling molecules, including PKCɛ, PIP2, and FAK [J. Li et al, 2015; Lin et al, 2015].…”

“…Primary heart cultures were obtained from 1–2 day old neonatal rats according to the Institutional Animal Care and Use Committee at the University of Illinois at Chicago. Hearts were removed, and CMs were isolated from 1–2 day old Sprague–Dawley rats with collagenase type II (Worthington, Lakewood, NJ) as previously described (Li et al, ). Neonatal rat ventricular CMs were re‐suspended, filtered through a 70 μm nylon sieve to remove large material, and plated in PC–1 medium (Lonza Group, Basel, Switzerland).…”

“…Primary heart cultures were obtained from 1–2 day old neonatal rats according to the Institutional Animal Care and Use Committee at the University of Illinois at Chicago. Hearts were removed and CMs isolated from 1–2 day old Sprague-Dawley rats with collagenase type II (Worthington, Lakewood, NJ) as previously described [J. Li et al, 2015].…”

“…Dishes were coated with fibronectin for at least 2 hours prior to plating. Polyacrylamide (10 kPa and 100 kPa) substrata were functionalized according to previous studies prior to incubation with fibronectin [J. Li et al, 2015].…”