These findings indicate that Ca(2+) entry via the α(1A)-AR-Snapin-TRPC6-pathway plays an important role in physiological regulation of cardiac contractility and may be an important target for augmenting cardiac performance.

According to cDNA sequence homologies, Gef10 is related to the Rho-specific guanine nucleotide exchange factors GrinchGEF and p164-RhoGEF. Like these GEFs, Gef10 exhibits only weak homology to known pleckstrin homology domains, but contains a putative WD40-like domain. As detected by RT-PCR, Gef10 is transcribed in at least two splice variants in different human tissues. Although the Gef10 sequence contains two putative transmembrane segments, recombinantly expressed Gef10 displays a cytosolic localisation. As detected by guanine nucleotide exchange activity assay, precipitation assay of GTP-bound Rho proteins and serum response element dependent gene transcription Gef10 activates RhoA-C, but not Rac1 or Cdc42. In the reporter gene assay, Gef10 preferentially activated RhoB. When expressed in NIH3T3 cells, Gef10 induced actin stress fibre, but not lamellipodia or filopodia formation. We conclude that Gef10 is the third member of a Rho-specific GEF family with unusual protein architecture.

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AimsTo determine the mechanisms by which the α1A-adrenergic receptor (AR) regulates cardiac contractility.BackgroundWe reported previously that transgenic mice with cardiac-restricted α1A-AR overexpression (α1A-TG) exhibit enhanced contractility but not hypertrophy, despite evidence implicating this Gαq/11-coupled receptor in hypertrophy.MethodsContractility, calcium (Ca2+) kinetics and sensitivity, and contractile proteins were examined in cardiomyocytes, isolated hearts and skinned fibers from α1A-TG mice (170-fold overexpression) and their non-TG littermates (NTL) before and after α1A-AR agonist stimulation and blockade, angiotensin II (AngII), and Rho kinase (ROCK) inhibition.ResultsHypercontractility without hypertrophy with α1A-AR overexpression is shown to result from increased intracellular Ca2+ release in response to agonist, augmenting the systolic amplitude of the intracellular Ca2+ concentration [Ca2+]i transient without changing resting [Ca2+]i. In the absence of agonist, however, α1A-AR overexpression reduced contractility despite unchanged [Ca2+]i. This hypocontractility is not due to heterologous desensitization: the contractile response to AngII, acting via its Gαq/11-coupled receptor, was unaltered. Rather, the hypocontractility is a pleiotropic signaling effect of the α1A-AR in the absence of agonist, inhibiting RhoA/ROCK activity, resulting in hypophosphorylation of both myosin phosphatase targeting subunit 1 (MYPT1) and cardiac myosin light chain 2 (cMLC2), reducing the Ca2+ sensitivity of the contractile machinery: all these effects were rapidly reversed by selective α1A-AR blockade. Critically, ROCK inhibition in normal hearts of NTLs without α1A-AR overexpression caused hypophosphorylation of both MYPT1 and cMLC2, and rapidly reduced basal contractility.ConclusionsWe report for the first time pleiotropic α1A-AR signaling and the physiological role of RhoA/ROCK signaling in maintaining contractility in the normal heart.

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