Characterization of muscle ankyrin repeat proteins in human skeletal muscle

Muscle ankyrin repeat proteins (MARPs) are a family of titin-associated, stress-response molecules and putative transducers of stretch-induced signaling in skeletal muscle. In cardiac muscle, cardiac ankyrin repeat protein (CARP) and diabetes-related ankyrin repeat protein (DARP) reportedly redistribute from binding sites on titin to the nucleus following a prolonged stretch. However, it is unclear whether ankyrin repeat domain protein 2 (Ankrd 2) shows comparable stretch-induced redistribution to the nucleus. We measured the following in rested human skeletal muscle: 1) the absolute amount of MARPs and 2) the distribution of Ankrd 2 and DARP in both single fibers and whole muscle preparations. In absolute amounts, Ankrd 2 is the most abundant MARP in human skeletal muscle, there being ~3.1 µmol/kg, much greater than DARP and CARP (~0.11 and ~0.02 µmol/kg, respectively). All DARP was found to be tightly bound at cytoskeletal (or possibly nuclear) sites. In contrast, ~70% of the total Ankrd 2 is freely diffusible in the cytosol [including virtually all of the phosphorylated (p)Ankrd 2-Ser99 form], ~15% is bound to non-nuclear membranes, and ~15% is bound at cytoskeletal sites, likely at the N2A region of titin. These data are not consistent with the proposal that Ankrd 2, per se, or pAnkrd 2-Ser99 mediates stretch-induced signaling in skeletal muscle, dissociating from titin and translocating to the nucleus, because the majority of these forms of Ankrd 2 are already free in the cytosol. It will be necessary to show that the titin-associated Ankrd 2 is modified by stretch in some as-yet-unidentified way, distinct from the diffusible pool, if it is to act as a stretch-sensitive signaling molecule.

Abstract 308: Targeted Inhibition of Cardiac Ankyrin Repeat Protein Disrupts Sarcomeric ERK-GATA4 Signaling at the Titin I-band and Abrogates Agonist-Induced Cardiac Hypertrophy

Hypertrophic cardiomyocyte growth occurs in response to various stress stimuli including biomechanical stress and neurohormonal factors. Accumulating evidence suggest that sarcomere signaling complexes play a pivotal role in the cardiomyocyte hypertrophic response by transmitting signals to the nucleus to induce gene expression. Cardiac ankyrin repeat protein (CARP, Ankrd1) is a transcriptional regulatory protein that also associates with the titin I-band spring domain, however the exact role of CARP in the heart remains to be elucidated. We report that CARP directly interacts with mitogen activated protein kinase ERK1/2 and cardiac transcription factor GATA4. Phenylephrine (PE) stimulation in cardiomyocytes induced ERK1/2 and GATA4 to transiently co-localize with sarcomeric CARP, followed by translocation of CARP and GATA4 to the nucleus. Four-and-a-half-LIM (FHL) domains proteins are part of a sarcomeric ERK2 sensory complex and knockdown of CARP by small interfering RNA (siRNA) resulted in disruption of FHL1 and FHL2. Moreover, loss of CARP attenuated PE-induced phosphorylation of ERK1/2 and GATA4, decreased GATA4 DNA binding, and prevented PE-induced cardiomyocyte growth. Mice lacking CARP have decreased FHL1 levels, and PE stimulation in wild-type mice resulted in elevated GATA4 phosphorylation and a hypertrophic response, which were completely abrogated in CARP-KO mice. We demonstrate that CARP plays an important role in PE-induced hypertrophic signaling by recruiting ERK2 and GATA4 into a titin I-band macro-molecular complex to induce GATA4 activation, followed by translocation of CARP and GATA4 to the nucleus to enhance GATA4 DNA binding and hypertrophic gene expression. Loss of CARP destabilizes FHL1 and FHL2, resulting in disruption of the PE-induced sarcomeric complex and abrogation of the cardiomyocyte hypertrophic response. These data reveal a novel role for sarcomeric titin I-band as a transcription factor activation hub that induces downstream nuclear signaling in response to agonist-induced hypertrophic stimuli.