A molecular complex of Ca _v 1.2/CaMKK2/CaMK1a in caveolae is responsible for vascular remodeling via excitation–transcription coupling

Abstract

Significance Excitation–transcription (E-T) coupling can initiate and modulate essential physiological or pathological responses in cells, such as neurons and cardiac myocytes. Although vascular myocytes also exhibit E-T coupling in response to membrane depolarization, the underlying molecular mechanisms are unknown. Our study reveals that E-T coupling in vascular myocytes converts intracellular Ca 2+ signals into selective gene transcription related to chemotaxis, leukocyte adhesion, and inflammation. Our discovery identifies a mechanism for vascular remodeling as an adaptation to increased circumferential stretch.

Local Ca²⁺ Signals within Caveolae Cause Nuclear Translocation of CaMK1α in Mouse Vascular Smooth Muscle Cells

“…Animals Wild type (WT) and caveolin1-knockout (cav1-KO) mice (C57BL/6 background, 8-15 weeks, male or female) were obtained from Japan SLC (Hamamatsu, Japan) and Jackson Labs (Bar Harbor, ME, U.S.A.), respectively. 20) All experiments were approved by the Ethics Committee of Nagoya City University (H30-P-1) and conducted in accordance with the Guide for the Care and Use of Laboratory Animals of the Japanese Pharmacological Society.…”

“…Single Cell Isolation and Unpassaged SMC Culture To obtain unpassaged primary ASMCs, 20) SMCs were isolated from mouse aorta. These isolated myocytes were then seeded on glass bottom dishes (Matsunami, Osaka, Japan) coated with 20 µg/mL laminin (MilliporeSigma, St. Louis, MO, U.S.A.) for 2 h and kept in an incubator at 37 °C with 5% CO 2 for 5-7 d to allow expansion to a semi-confluent state.…”

“…These isolated myocytes were then seeded on glass bottom dishes (Matsunami, Osaka, Japan) coated with 20 µg/mL laminin (MilliporeSigma, St. Louis, MO, U.S.A.) for 2 h and kept in an incubator at 37 °C with 5% CO 2 for 5-7 d to allow expansion to a semi-confluent state. Then, the medium was switched to Dulbecco's modified Eagle's medium (DMEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) with 0.1% fetal bovine serum (FBS) (MilliporeSigma), 1 × ITS (insulin, transferrin and selenium, FUJIFILM Wako Pure Chemical Corporation) supplement and 200 µM L-ascorbic acid sodium (FUJIFILM Wako Pure Chemical Corporation) for an additional 2-3 d. Adenovirus vectors (ViraPower™Adenoviral Expression System, Thermo Fisher Scientific, Waltham, MA, U.S.A.) 20) were applied to the myocytes when the medium was switched. Single MASMCs were freshly isolated from second-and third-order mesenteric arteries as reported previously.…”

“…Single MASMCs were freshly isolated from second-and third-order mesenteric arteries as reported previously. 20) These isolated cells were incubated with standard (5 mM KCl) N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES)-buffered solution: (mM) 137 NaCl, 5.9 KCl, 2. Transfection Using Adenovirus Vectors CaMK1α and cav1 were labeled with green fluorescent protein (GFP) and mCherry, respectively at the N termini.…”

“…This results in macrophage accumulation and medial hypertrophy in vessels after sustained pressure loading. 20) In this previous study, CaMK1α was suggested to be the prime candidate to deliver Ca 2+ signals generated at the cell surface to the nucleus; however, direct evidence was not presented. In addition, it was unclear if CaMK1α actually translocates from the cell surface/caveolae to the nucleus in freshly-isolated vascular SMCs.…”