A Model Multi-systems Approach for Understanding the Role of the PIX Pathway in Cardiac Muscle and Cardiomyopathy

Myofibrils located at the periphery of the muscle cell are attached to the sarcolemma via costameres, muscle-
specific integrin adhesion complexes (IACs), that transmit the force of muscle contraction to the outside of the
cell. IACs consist of the transmembrane protein integrin and hundreds of proteins in a complex both in the
ECM and especially intracellularly. Although much is known about the composition of IACs and mechanisms
that initiate their assembly, we do not know how the composition of an IAC is determined, and what determines
where an IAC will form. We exploit the conservation of muscle structure/function and the power of genetics in
C. elegans. From a genetic screen, we identified a gene, pix-1, which is required for the formation of IACs at
muscle cell boundaries (MCBs), but not the other IACs (M-lines and dense bodies). PIX-1 is the nematode
ortholog of human b-PIX, which contains a RhoGEF domain known to be an activator of Rac1 and Cdc42. pix-
1 mutants show decreased whole animal locomotion. As compared to wild type, a pix-1 null mutant, shows
50% reduction in the level of activated (GTP bound) Rac in muscle. In addition to deficiency of PIX-1,
overexpression of wild type PIX-1 protein results in decreased locomotion and disrupted MCBs. PIX-1 localizes
to all 3 IACs in muscle—M-lines, dense bodies and MCBs. Loss of function mutants in each of the known PIX
pathway proteins (PIX, Rac, GIT) result in disrupted MCBs. The protein kinase activity of PAK-1 is essential for
its MCB function: a kinase-dead PAK-1 mutant has disrupted MCBs. Although PIX proteins are known to be
important in several cell types in mammals and nematodes, our results are the first to demonstrate that a PIX
protein is required for assembly of an IAC, and is required in striated muscle. A GAP protein (inactivates Rac)
for this pathway has not been reported for any cell type or organism, but we now have two candidate GAPs.
Remarkably, we found that heart specific knockout of the mouse ortholog b-PIX results in dilated
cardiomyopathy at 8 months of age. Similarly, heart-specific knockdown of the Drosophila ortholog, dPix, also
results in cardiomyopathy. We hypothesize that PIX proteins, through their RacGEF activity, direct assembly
of IACs by activating PAK kinases to phosphorylate key substrate(s) in muscle. We will leverage the unique
advantages of 3 model systems to learn new conserved functions of the PIX pathway in striated and cardiac
muscle. Aim 1: Exploiting the power of studying muscle in C. elegans we will: (a) identify the GAP protein(s);
(b) determine the localization, function and substrates of PAK kinases, and (c) test the hypothesis that PIX-1 is
only required at MCBs because there are additional RacGEF proteins at the other IAC locations. Aim 2: We
will determine how b-PIX functions in the mouse heart and how its absence leads to cardiomyopathy. Aim 3:
We will take advantage of the power of genetic modifier screens in Drosophila to identify new members of the
PIX pathway in muscle, and more specifically, cardiac muscle. There is a need to identify additional
cardiomyopathy genes in humans and genes encoding members of the PIX pathway may be new candidates.

Grant Number: 5R01HL160693-04
NIH Institute/Center: NIH
Principal Investigator: GUY BENIAN