The superposition of work on a pathologic microenvironmental stiffness may regulate actin assembly via phosphorylation and sarcomeric redistribution of PKCɛ

Abstract

Moderate exercise benefits the heart, but chronic demand leads to failure. Thus, acute and chronic loading may drive cell contractility and cell hypertrophy differentially. We test the hypothesis that, in response to changes in demand, the actin assembly is regulated through PKCɛ signaling to alter cell hypertrophy. Cultured neonatal rat ventricular myocytes (NRVMs) were chronically stressed by increased stiffness of the microenvironment, resembling tissue fibrosis in diseased hearts. Physiologic or pathologic work load was mimicked by one‐hour or 24‐hour treatment with 2.5 μM isoproterenol, respectively. NRVMs were plated on polyacrylamide (PAA) or polydimethylsiloxane (PDMS) substrata mimicking the stiffness of normal (10 kPa) or diseased (100 kPa) hearts, in addition to plating directly on glass (>1 GPa). Cell area increased with increasing stiffness following three‐day culture but not with 24‐hour isoproterenol treatment. Contractility was assessed by beat frequency and time to peak tension by line‐scan kymographs, increasing with substrata stiffness and also with one or 24‐hour isoproterenol treatment. Actin dynamics, as assessed by fluorescence recovery after photobleaching (FRAP) using actin‐GFP, were altered by changes in work load. Both chronic stiffness and one‐hour isoproterenol treatment increased kFRAP to much higher values of ≈0.00075 s−1 on glass or on 100 kPa with isoproterenol treatment compared to untreated cells on 100 kPa, ≈0.0004 s−1 (P<0.05, n≥10 cells). To seek a mechanistic basis for regulation of actin assembly and cell hypertrophy, GFP‐PKCɛ positive NRVMs were observed using time lapse imaging over 1 hour. PKCɛ translocated to the sarcomere within minutes, was sustained for approximately 30 minutes, and declined by one hour. Immunostaining confirmed that translocated PKCɛ is localized with alpha‐actinin at the Z‐band. In order to study whether PKCɛ phosphorylation was necessary, we used a phospho‐specific PKCɛ antibody on fixed cells, which showed an increased Z‐disk localization between alpha‐actinin and phospho‐PKCɛ at 15 min as quantified by Pearson's coefficient (P<0.05), but this difference dissipated by 1 hour. Since the PKCɛ translocation was transient and actin assembly was sustained, hypertrophy may be triggered by the arrival of phospho‐PKCɛ at the Z‐band. The superposition of work on a pathologic microenvironmental stiffness may regulate actin assembly and maladaptive hypertrophy by localization of signaling molecules. This basic research may help understanding of hypertrophy found in heart failure in many cardiac disease states.Support or Funding InformationHL 62426.