Acetyl Writers and Erasers in Muscle Signal Transduction and Contractility
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Authors
Habibian, Justine Shabnam
Issue Date
2023
Type
Dissertation
Language
Keywords
Alternative Title
Abstract
Striated muscle includes skeletal and cardiac muscle as a result of repeating sections of sarcomeres within their respective myofibrils. The coordination of various signal transduction pathways and their post-translational modifications function to regulate the health and activity of striated muscle tissue in the generation of force, contractions, and movement. Recent evidence highlights histone deacetylase (HDAC) and histone acetyltransferase (HAT) inhibitors as potential therapeutics in pre-clinical animal models of skeletal muscle atrophy and congestive heart failure. To date, most of these studies have focused on the epigenetic actions of HDACs and HATs in the control of histone acetylation and gene expression. However, emerging evidence demonstrates that many lysine residues (>14,000) can be acetylated on many different proteins (>4,500). Our group and others have shown that protein acetylation occurs on proteins critical for protein synthesis, autophagy, energy metabolism, muscle contraction, and intracellular signaling. Not surprisingly, we have published that HDAC inhibitors attenuate pathological cardiac signaling via epigenetic and non-epigenetic mechanisms. However, a role for HDACs in the regulation of muscle and cardiac signal transduction remains a 'black box.' In this dissertation, we have demonstrated that HDAC8 inhibition attenuated phorbol-12-myristate-13-acetate (PMA), angiotensin II (Ang II), and dexamethasone (Dex)-induced increases in protein kinase D (PKD) phosphorylation at S744/S748 in response to cellular stress in C2C12 myoblasts. Moreover, we demonstrated that romidepsin selectively targets HDAC1, 2, and 8 for inhibition in vitro and in cell culture. We also report that protein kinase C delta (PKC) phosphorylation is decreased in skeletal muscle at threonine 505 (T505) and serine 643 (S643) in C2C12 myotubes in response to muscle atrophy. Interestingly, PKC phosphorylation was restored in atrophied myotubes treated with a pan-HDAC inhibitor or a class I selective HDAC inhibitor targeting HDAC1, -2, or -8. Phosphorylation of PKCδ at S643 is necessary and sufficient for its activity, suggesting that PKCδ activity is inhibited with muscle atrophy and re-activated with HDAC inhibitors while total PKCδ expression remained unchanged. HDAC inhibition restored myotube size under atrophy conditions that was not restored when myotubes were treated with a PKCδ inhibitor, Rotterlin, or infected with an adenovirus overexpressing a dominant negative PKCδ. Additionally, the overexpression of a constitutively active PKCδ prevented Dex-induced myotube atrophy. Not only does acetylation appear to change signal transduction, but emerging evidence suggests that myofilament protein acetylation is also important for the regulation of cardiac contractility and relaxation. Our lab previously demonstrated that sarcomeric proteins, such as skeletal muscle alpha-actin (ACTA1), can be acetylated. Lysine acetyltransferase (HAT) enzymes serve as writer proteins to acetylate histone and non-histone proteins. We show that garcinol, a p300/PCAF inhibitor, attenuated isoproterenol-induced left ventricular hypertrophy (LVH), cardiac output (CO), and stroke volume (SV) in C57BL6J mice. Of interest, isoproterenol increased skeletal muscle alpha-actin (ACTA1) protein acetylation in the left ventricle (LV) of mice and this was attenuated by garcinol treatment. Moreover, pseudo-acetylation of ACTA1 increased LVH, and reduced fractional shortening (FS) and ejection fraction (EF) in mice. Lastly, we show that PCAF acetylated ACTA1 but not cardiac myosin; p300 failed to acetylate ACTA1 in vitro. Combined, these data suggest a non-canonical role for HDACs and HATs in the regulation of striated muscle physiology on signal transduction and cardiac contractility outside of its role in chromatin condensation and gene regulation.