A Central Role for TRPM4 in Ca2+-Signal Amplification and Vasoconstriction.

Transient receptor potential melastatin-4 (TRPM4) is activated by an increase in intracellular Ca2+ concentration and is expressed on smooth muscle cells (SMCs). It is implicated in the myogenic constriction of cerebral arteries. We hypothesized that TRPM4 has a general role in intracellular Ca2+ signal amplification in a wide range of blood vessels. TRPM4 function was tested with the TRPM4 antagonist 9-phenanthrol and the TRPM4 activator A23187 on the cardiovascular responses of the rat, in vivo and in isolated basilar, mesenteric, and skeletal muscle arteries. TRPM4 inhibition by 9-phenanthrol resulted in hypotension and a decreased heart rate in the rat. TRPM4 inhibition completely antagonized myogenic tone development and norepinephrine-evoked vasoconstriction, and depolarization (high extracellular KCl concentration) evoked vasoconstriction in a wide range of peripheral arteries. Vasorelaxation caused by TRPM4 inhibition was accompanied by a significant decrease in intracellular Ca2+ concentration, suggesting an inhibition of Ca2+ signal amplification. Immunohistochemistry confirmed TRPM4 expression in the smooth muscle cells of the peripheral arteries. Finally, TRPM4 activation by the Ca2+ ionophore A23187 was competitively inhibited by 9-phenanthrol. In summary, TRPM4 was identified as an essential Ca2+-amplifying channel in peripheral arteries, contributing to both myogenic tone and agonist responses. These results suggest an important role for TRPM4 in the circulation. The modulation of TRPM4 activity may be a therapeutic target for hypertension. Furthermore, the Ca2+ ionophore A23187 was identified as the first high-affinity (nanomolar) direct activator of TRPM4, acting on the 9-phenanthrol binding site.

Titin isoforms are increasingly protected against oxidative modifications in developing rat cardiomyocytes.

During the perinatal adaptation process N2BA titin isoforms are switched for N2B titin isoforms leading to an increase in cardiomyocyte passive tension (Fpassive). Here we attempted to reveal how titin isoform composition and oxidative insults (i.e. sulfhydryl (SH)-group oxidation or carbonylation) influence Fpassive of left ventricular (LV) cardiomyocytes during rat heart development. Moreover, we also examined a hypothetical protective role for titin associated small heat shock proteins (sHSPs), Hsp27 and αB-crystallin in the above processes. Single, permeabilized LV cardiomyocytes of the rat (at various ages following birth) were exposed either to 2,2'-dithiodipyridine (DTDP) to provoke SH-oxidation or Fenton reaction reagents (iron(II), hydrogen peroxide (H2O2), ascorbic acid) to induce protein carbonylation of cardiomyocytes in vitro. Thereafter, cardiomyocyte force measurements for Fpassive determinations and Western immunoblot assays were carried out for the semiquantitative determination of oxidized SH-groups or carbonyl-groups of titin isoforms and to monitor sHSPs' expressions. DTDP or Fenton reagents increased Fpassive in 0- and 7-day-old rats to relatively higher extents than in 21-day-old and adult animals. The degrees of SH-group oxidation or carbonylation declined with cardiomyocyte age to similar extents for both titin isoforms. Moreover, the above characteristics were mirrored by increasing levels of HSP27 and αB-crystallin expressions during cardiomyocyte development. Our data implicate a gradual build-up of a protective mechanism against titin oxidation through the upregulation of HSP27 and αB-crystallin expressions during postnatal cardiomyocyte development.