The phosphatase and actin regulator 1 (PHACTR1) locus is a very commonly identified hit in genome-wide association studies investigating coronary artery disease and myocardial infarction (MI). However, the function of PHACTR1 in the heart is still unknown. We characterized the mechanisms regulating Phactr1 expression in the heart, used adenoviral gene delivery to investigate the effects of Phactr1 on cardiac function, and analyzed the relationship between MI associated PHACTR1 allele and cardiac function in human subjects. Phactr1 mRNA and protein levels were markedly reduced (60%, P<0.01 and 90%, P<0.001, respectively) at 1 day after MI in rats. When the direct myocardial effects of Phactr1 were studied, the skeletal α-actin to cardiac α-actin isoform ratio was significantly higher (1.5-fold, P<0.05) at 3 days but 40% lower (P<0.05) at 2 weeks after adenovirus-mediated Phactr1 gene delivery into the anterior wall of the left ventricle. Similarly, the skeletal α-actin to cardiac α-actin ratio was lower at 2 weeks in infarcted hearts overexpressing Phactr1. In cultured neonatal cardiac myocytes, adenovirus-mediated Phactr1 overexpression for 48 hours markedly increased the skeletal α-actin to cardiac α-actin ratio, this being associated with an enhanced DNA binding activity of serum response factor. Phactr1 overexpression exerted no major effects on the expression of other cardiac genes or LV structure and function in normal and infarcted hearts during 2 weeks’ follow-up period. In human subjects, MI associated PHACTR1 allele was not associated significantly with cardiac function (n = 1550). Phactr1 seems to regulate the skeletal to cardiac α-actin isoform ratio.

The transforming growth factor (TGF)-β is one of the key mediators in cardiac remodelling occurring after myocardial infarction (MI) and in hypertensive heart disease. The TGF-β-stimulated clone 22 (TSC-22) is a leucine zipper protein expressed in many tissues and possessing various transcription-modulating activities. However, its function in the heart remains unknown.

Objectives: Transforming-growth-factor β-stimulated clone 22 (TSC-22) is a leucine zipper protein expressed in many tissues and possessing various transcription-modulating activities. However, its function in the heart remains largely unknown. The aim of the present study was to characterize the cardiac TSC-22 expression. Methods: Acute pressure overload was accomplished in conscious Sprague-Dawley (SD) rats by intravenous infusion of arginine 8 -vasopressin (AVP, 0.05 μg/kg/min) for 4 hours and subcutaneous infusion of angiotensin II (Ang II, 33 μg/kg/h) with and without Ang II receptor type 1 blocker losartan (400 μg/kg/h) by using osmotic minipumps for 2 weeks. Adenovirus-mediated intramyocardial gene transfer of TSC-22 was performed into left ventricle (LV) of SD rats. Experimental myocardial infarction (MI) was produced by ligation of the left anterior descending coronary artery. Cultured neonatal rat ventricular myocytes (NRVM) were treated with endothelin-1 (ET-1, 100 nM). Results: A significant 1.6-fold increase ( P <0.05) in LV TSC-22 mRNA levels was noted already after 1 hour AVP infusion. Moreover, Ang II infusion markedly upregulated TSC-22 expression, LV mRNA levels being highest at 6 hours (11-fold, P <0.001). Simultaneous infusion of losartan completely abolished Ang II-induced increase in TSC-22 mRNA levels. Adenovirus-mediated gene transfer of TSC-22 into LV resulted a 1.9-fold ( P <0.001) increase in TSC-22 mRNA levels, accompanied by upregulated BNP mRNA levels (1.4-fold, P <0.01). In response to experimental MI, TSC-22 mRNA levels were elevated 4.1-fold ( P <0.001) at 1 day and 1.9-fold ( P <0.05) at 4 weeks. In cultured NRVM, ET-1 treatment increased TSC-22 mRNA levels from 1 h to 24 h, the greatest increase being observed at 12 h (2.7-fold, P <0.001). TSC-22 protein levels were upregulated from 4 h to 24 h with the highest increase at 24 h (4.7-fold, P <0.01). Conclusion: These results indicate that TSC-22 expression is rapidly activated in response to pressure overload, MI and in ET-1 treated cultured NRVM. Moreover, adenovirus-mediated overexpression of TSC-22 mRNA was associated with elevated left ventricular BNP mRNA levels.

Background: The PDZ-LIM proteins are a family of signalling adaptors that interact with the actin cross-linking protein, alpha-actinin, via their PDZ domains or via internal regions between the PDZ and LIM domains. Three of the PDZ-LIM proteins have a conserved 26-residue ZM motif in the internal region, but the structure of the internal region is unknown.Results: In this study, using circular dichroism and nuclear magnetic resonance (NMR), we showed that the ALP internal region (residues 107-273) was largely unfolded in solution, but was able to interact with the alpha-actinin rod domain in vitro, and to co-localize with alpha-actinin on stress fibres in vivo. NMR analysis revealed that the titration of ALP with the alpha-actinin rod domain induces stabilization of ALP. A synthetic peptide (residues 175-196) that contained the N-terminal half of the ZM motif was found to interact directly with the alpha-actinin rod domain in surface plasmon resonance (SPR) measurements. Short deletions at or before the ZM motif abrogated the localization of ALP to actin stress fibres.Conclusion: The internal region of ALP appeared to be largely unstructured but functional. The ZM motif defined part of the interaction surface between ALP and the alpha-actinin rod domain.

The Z-line is a specialized structure connecting adjacent sarcomeres in muscle cells. alpha-Actinin cross-links actin filaments in the Z-line. Several PDZ-LIM domain proteins localize to the Z-line and interact with alpha-actinin. Actinin-associated LIM protein (ALP), C-terminal LIM domain protein (CLP36), and Z band alternatively spliced PDZ-containing protein (ZASP) have a conserved region named the ZASP-like motif (ZM) between PDZ and LIM domains. To study the interactions and function of ALP we used purified recombinant proteins in surface plasmon resonance measurements. We show that ALP and alpha-actinin 2 have two interaction sites. The ZM motif was required for the interaction of ALP internal region with the alpha-actinin rod and for targeting of ALP to the Z-line. The PDZ domain of ALP bound to the C terminus of alpha-actinin. This is the first indication that the ZM motif would have a direct role in a protein-protein interaction. These results suggest that the two interaction sites of ALP would stabilize certain conformations of alpha-actinin 2 that would strengthen the Z-line integrity.