The Z-line is a specialized structure connecting adjacent sarcomeres in muscle cells. ␣-Actinin cross-links actin filaments in the Z-line. Several PDZ-LIM domain proteins localize to the Z-line and interact with ␣-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 ␣-actinin 2 have two interaction sites. The ZM motif was required for the interaction of ALP internal region with the ␣-actinin rod and for targeting of ALP to the Z-line. The PDZ domain of ALP bound to the C terminus of ␣-actinin. This is the first indication that the ZM motif would have a direct role in a proteinprotein interaction. These results suggest that the two interaction sites of ALP would stabilize certain conformations of ␣-actinin 2 that would strengthen the Z-line integrity.The muscle Z-line is a highly specialized structure between adjacent sarcomeres in muscle fibers that maintain the organization of the contractile machinery (for review, see Ref. 1). ␣-Actinin is one of the major components at the Z-line. The ␣-actinin polypeptide is composed of an N-terminal actin binding domain, four spectrin repeats that form the central rod region (2, 3), and two pairs of C-terminal EF-hands (4). ␣-Actinin forms an antiparallel homodimer. ␣-Actinin cross-links actin filaments from opposite sarcomeres to the Z-line and, therefore, has a major mechanical function in keeping the sarcomeres together.Several PDZ-LIM proteins have been detected in the muscle Z-line and shown to interact with ␣-actinin (5-12). PDZ domain is a widely expressed protein-protein interaction domain (for review, see Ref. 13). PDZ-LIM proteins form one subgroup of PDZ proteins (13) and are regarded as mediators between cytoskeletal structures and signaling cascades.PDZ-LIM proteins can be divided in two subclasses; they either contain one or three LIM domains. Actinin-associated LIM protein (ALP) 1 (5, 10), C-terminal LIM domain protein (CLP36) (14) (also called hCLIM1 (15) or Elfin (16)), Reversion-induced LIM protein (RIL) (17), and Mystique (Uniprot accession number Q7Z584) belong to the first class, which has one N-terminal PDZ domain and one C-terminal LIM domain. ALP is expressed in muscle (5, 10), whereas CLP36 and RIL are mainly expressed in nonmuscle tissues (11,18,19). CLP36 shows also high expression levels in muscle (7,15,16). Enigma, Enigma homology protein, and ZASP/Cypher/Oracle form the second class, with one N-terminal PDZ domain and three C-terminal LIM domains. They all are expressed mainly in muscle (6, 9, 20 -22). An interesting feature of these seven PDZ-LIM proteins is that ALP, CLP36, and ZASP contain a conserved region, named ZASP-like motif (ZM) (SMART prediction (23) accession number SM 00735, Interpro 006643), in the internal r...

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.

Background: The PDZ-LIM proteins are a family of signalling adaptors that interact with the actin cross-linking protein, α-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.

BackgroundThe 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.MethodsThe aim of the present study was to characterize cardiac TSC-22 expression in vivo in cardiac remodelling and in myocytes in vitro. In addition, we used TSC-22 gene transfer in order to examine the effects of TSC-22 on cardiac gene expression and function.ResultsWe found that TSC-22 is rapidly up-regulated by multiple hypertrophic stimuli, and in post-MI remodelling both TSC-22 mRNA and protein levels were up-regulated (4.1-fold, P <0.001 and 3.0-fold, P <0.05, respectively) already on day 1. We observed that both losartan and metoprolol treatments reduced left ventricular TSC-22 gene expression. Finally, TSC-22 overexpression by local intramyocardial adenovirus-mediated gene delivery showed that TSC-22 appears to have a role in regulating collagen type IIIα1 gene expression in the heart.ConclusionsThese results demonstrate that TSC-22 expression is induced in response to cardiac overload. Moreover, our data suggests that, by regulating collagen expression in the heart in vivo, TSC-22 could be a potential target for fibrosis-preventing therapies.

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.