Transcriptional coactivation of NRF2 signaling in cardiac fibroblasts promotes resistance to oxidative stress.

We recently discovered that steroid receptor coactivators (SRCs) SRCs-1, 2 and 3, are abundantly expressed in cardiac fibroblasts (CFs) and their activation with the SRC small molecule stimulator MCB-613 improves cardiac function and dramatically lowers pro-fibrotic signaling in CFs post-myocardial infarction. These findings suggest that CF-derived SRC activation could be beneficial in the mitigation of chronic heart failure after ischemic insult. However, the cardioprotective mechanisms by which CFs contribute to cardiac pathological remodeling are unclear. Here we present studies designed to identify the molecular and cellular circuitry that governs the anti-fibrotic effects of an MCB-613 derivative, MCB-613-10-1, in CFs. We performed cytokine profiling and whole transcriptome and proteome analyses of CF-derived signals in response to MCB-613-10-1. We identified the NRF2 pathway as a direct MCB-613-10-1 therapeutic target for promoting resistance to oxidative stress in CFs. We show that MCB-613-10-1 promotes cell survival of anti-fibrotic CFs exposed to oxidative stress by suppressing apoptosis. We demonstrate that an increase in HMOX1 expression contributes to CF resistance to oxidative stress-mediated apoptosis via a mechanism involving SRC co-activation of NRF2, hence reducing inflammation and fibrosis. We provide evidence that MCB-613-10-1 acts as a protectant against oxidative stress-induced mitochondrial damage. Our data reveal that SRC stimulation of the NRF2 transcriptional network promotes resistance to oxidative stress and highlights a mechanistic approach toward addressing pathologic cardiac remodeling.

A simple drying solution that minimizes cracking during air-drying of polyacrylamide gels.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis is a routine technique used in biochemistry. Air-drying is an economical method of gel preservation that does not require expensive equipment. Our laboratory uses drying frames from RPI, which recommends a drying solution of 20% ethanol and 10% glycerol. The solution performs well for gels up to 10% acrylamide and 0.75 mm thickness; however, crack formation may occur if nicks or bubbles are present. The literature shows various drying methods and combinations of alcohol (30-100%) and glycerol (5-35%), but still reports cracking problems. Tests were conducted to independently evaluate the effects of ethanol and glycerol concentration on gel cracking. Here we introduce a simple solution that does not require glycerol or modified frames to generate preserved, crack-free sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.