Dual thick and thin filament linked regulation of stretch- and L-NAME-induced tone in young and senescent murine basilar artery

Abstract

Stretch-induced vascular tone is an important element of autoregulatory adaptation of cerebral vasculature to maintain cerebral flow constant despite changes in perfusion pressure. Little is known as to the regulation of tone in senescent basilar arteries. We tested the hypothesis, that thin filament mechanisms in addition to smooth muscle myosin-II regulatory-light-chain-(MLC20)-phosphorylation and non-muscle-myosin-II, contribute to regulation of stretch-induced tone. In young BAs (y-BAs) mechanical stretch does not lead to spontaneous tone generation. Stretch-induced tone in y-BAs appeared only after inhibition of NO-release by L-NAME and was fully prevented by treatment with 3 μmol/L RhoA-kinase (ROK) inhibitor Y27632. L-NAME-induced tone was reduced in y-BAs from heterozygous mice carrying a point mutation of the targeting-subunit of the myosin phosphatase, MYPT1 at threonine696 (MYPT1-T696A/+). In y-BAs, MYPT1-T696A-mutation also blunted the ability of L-NAME to increase MLC20-phosphorylation. In contrast, senescent BAs (s-BAs; >24 months) developed stable spontaneous stretch-induced tone and pharmacological inhibition of NO-release by L-NAME led to an additive effect. In s-BAs the MYPT1-T696A mutation also blunted MLC20-phosphorylation, but did not prevent development of stretch-induced tone. In s-BAs from both lines, Y27632 completely abolished stretch- and L-NAME-induced tone. In s-BAs phosphorylation of non-muscle-myosin-S1943 and PAK1-T423, shown to be down-stream effectors of ROK was also reduced by Y27632 treatment. Stretch- and L-NAME tone were inhibited by inhibition of non-muscle myosin (NM-myosin) by blebbistatin. We also tested whether the substrate of PAK1 the thin-filament associated protein, caldesmon is involved in the regulation of stretch-induced tone in advanced age. BAs obtained from heterozygotes Cald1+/− mice generated stretch-induced tone already at an age of 20–21 months old BAs (o-BA). The magnitude of stretch-induced tone in Cald1+/− o-BAs was similar to that in s-BA. In addition, truncation of caldesmon myosin binding Exon2 (CaD-▵Ex2−/−) did not accelerate stretch-induced tone. Our study indicates that in senescent cerebral vessels, mechanisms distinct from MLC20 phosphorylation contribute to regulation of tone in the absence of a contractile agonist. While in y-and o-BA the canonical pathways, i.e., inhibition of MLCP by ROK and increase in pMLC20, predominate, tone regulation in senescence involves ROK regulated mechanisms, involving non-muscle-myosin and thin filament linked mechanisms involving caldesmon.

Aging related decreases in NM myosin expression and contractility in a resistance vessel

“…The disparities between our results and those reported by others ( Barton et al, 1997 ; Lubomirov et al, 2016 ; 2018 ; 2021 ; Luttrell et al, 2020 ; Zhong et al, 2021 ) could be due to differences in species (rat vs. mouse), strain of animal and/or vessel studied. In addition, MYPT1 has multiple phosphorylation sites which regulate MLC phosphatase activity ( Somlyo and Somlyo, 1994 ; Hartshorne et al, 1998 ; Brozovich et al, 2016 ), and in murine basilar arteries, others have demonstrated that MYPT1 phosphorylation at T853 increases during aging ( Lubomirov et al, 2023 ). We did not measure MYPT1 phosphorylation, and changes in MYPT1 phosphorylation could contribute to the disparities among studies.…”

“…Similar to SM myosin, NM myosin is regulated by phosphorylation of its light chain ( Cremo et al, 2001 ). Further, NM myosin phosphorylation is regulated during contraction of smooth muscle ( Yuen et al, 2009 ; Zhang and Gunst, 2017 ) and NM myosin has been demonstrated to participate in force maintenance ( Morano et al, 2000 ; Löfgren et al, 2003 ; Rhee et al, 2006 ; Yuen et al, 2009 ; Zhang and Gunst, 2017 ; Lubomirov et al, 2023 ); both a change in NM myosin expression ( Morano et al, 2000 ; Löfgren et al, 2003 ; Yuen et al, 2009 ) and inhibition of the NM myosin AMATPase ( Rhee and Brozovich, 2003 ; Lubomirov et al, 2023 ) have been demonstrated to produce a reduction in force, which demonstrate that changes in NM myosin expression will alter force. In tertiary mesenteric vessels, during aging, our data show NM myosin expression is reduced by ∼10% ( Figure 2 ), while there is no change in NM myosin expression in the aorta, a large conduit vessel.…”

“…The data also demonstrate that SM MyHC and α-SM actin expression does not change with aging ( Figure 5 ), suggesting that the smooth muscle phenotype does not change from contractile to synthetic during aging ( Rensen et al, 2007 ). However, NM myosin has been shown to participate in smooth muscle contraction ( Morano et al, 2000 ; Löfgren et al, 2003 ; Yuen et al, 2009 ), and both decreasing NM myosin expression ( Yuen et al, 2009 ) and inhibiting of NM myosin ( Rhee et al, 2006 ; Lubomirov et al, 2023 ) reduce force. Our data demonstrating a decrease in NM myosin expression in mesenteric arteries ( Figure 2 ) would decrease contractile force, which is consistent with our results ( Figure 3 ).…”

“…The expression of a LZ+ MYPT1 isoform has been demonstrated to be necessary for NO/cGMP/PKG mediated activation of the MLC phosphatase ( Huang et al, 2004 ; Yuen et al, 2014 ), and the sensitivity to NO mediated vasodilatation has been demonstrated to be defined by the relative expression of LZ+/LZ- MYPT1 ( Khatri et al, 2001 ; Huang et al, 2004 ; Payne et al, 2006 ; Yuen et al, 2011 ; Reho et al, 2016 ). Further, in addition to smooth muscle (SM) myosin, nonmuscle (NM) myosin is expressed in smooth muscle and has been demonstrated to participate in smooth muscle contraction ( Morano et al, 2000 ; Löfgren et al, 2003 ; Rhee et al, 2006 ; Yuen et al, 2009 ; Zhang and Gunst, 2017 ; Lubomirov et al, 2023 ); changes in NM myosin expression and NM myosin activation have been demonstrated to influence vascular tone ( Morano et al, 2000 ; Löfgren et al, 2003 ; Rhee et al, 2006 ; Yuen et al, 2009 ; Lubomirov et al, 2023 ).…”