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

Abstract

Introduction: Vasodilatation in response to NO is a fundamental response of the vasculature, and during aging, the vasculature is characterized by an increase in stiffness and decrease in sensitivity to NO mediated vasodilatation. Vascular tone is regulated by the activation of smooth muscle and nonmuscle (NM) myosin, which are regulated by the activities of myosin light chain kinase (MLCK) and MLC phosphatase. MLC phosphatase is a trimeric enzyme with a catalytic subunit, myosin targeting subunit (MYPT1) and 20 kDa subunit of unknown function. Alternative mRNA splicing produces LZ+/LZ- MYPT1 isoforms and the relative expression of LZ+/LZ- MYPT1 determines the sensitivity to NO mediated vasodilatation. This study tested the hypothesis that aging is associated with changes in LZ+ MYPT1 and NM myosin expression, which alter vascular reactivity. Methods: We determined MYPT1 and NM myosin expression, force and the sensitivity of both endothelial dependent and endothelial independent relaxation in tertiary mesenteric arteries of young (6mo) and elderly (24mo) Fischer344 rats. Results: The data demonstrate that aging is associated with a decrease in both the expression of NM myosin and force, but LZ+ MYPT expression and the sensitivity to both endothelial dependent and independent vasodilatation did not change. Further, smooth muscle cell hypertrophy increases the thickness of the medial layer of smooth muscle with aging. Discussion: The reduction of NM myosin may represent an aging associated compensatory mechanism to normalize the stiffness of resistance vessels in response to the increase in media thickness observed during aging.

Keywords: MYPT1; NM myosin; NO; endothelial independent and dependent vasodilatation; vascular reactivity.

FIGURE 1

During aging, MYPT1 and LZ + MYPT1 expression does not change. Western blots of MYPT1 and LZ + MYPT1 expression in the aorta and tertiary mesenteric artery. MYPT1 and LZ + MYPT1 expression were normalized to total protein (TP) as previously described (Schaible et al., 2016; Yap et al., 2020; Han et al., 2021). Box blots summarize the data; neither MYPT1/TP nor LZ + MYPT1/TP expression is altered during aging, p > 0.05.

FIGURE 2

During aging, NM myosin expression decreases in a resistance, but not a conduit vessel. Two-dimensional SDS-PAGE was used to separate the NM myosin light chain (1) and SM light chain (2). The expression of NM myosin as a percentage of total myosin is the density of (1/(1 + 2))x100% (Yuen et al., 2009). Box plots summarize NM myosin expression; during aging, NM myosin does not change in the aorta (31% ± 2% vs. 26% ± 4%, p > 0.05), but declines significantly in the tertiary mesenteric artery (22% ± 3% vs. 12% ± 2%, p = 0.025); *, p < 0.05.

FIGURE 3

During aging, neither endothelial independent nor endothelial dependent relaxation are altered in a resistance vessel, but force was reduced. (A) Force trace demonstrating relaxation to ACh of tertiary mesenteric artery. Sensitivity of ACh mediated relaxation is similar (p > 0.05) in mesenteric vessel from young and old animals. (B) Relaxation stimulated by 100 μM 8Br-cGMP is no different in tertiary mesenteric arteries from young and old rats (46% ± 7% vs. 43% ± 5%, p > 0.05). However, the contraction to 80 mM KCl is significantly depressed in mesenteric vessels from old rats (35 ± 13 mN/mm² vs. 8 ± 4 mN/mm², p = 0.016). Box plots summarize the data for both maximal force in response to 80 mM KCl and relaxation to 100 μM 8Br-cGMP (*, p < 0.05).

FIGURE 4

Aging is associated with an increase in media thickness in a resistance vessel. HE stained tertiary mesenteric artery (6mo & 24mo); scale bar, 50 μm (for both images). Box plot summarize the data of the thickness of the media at 6mo (36 ± 3 μm, n = 4) and 24 mo (51 ± 5 μm, n = 4). Aging is associated with a significant (p = 0.022) increase in the thickness of the media layer of smooth muscle; *, p < 0.05.

FIGURE 5

During aging, smooth muscle myosin and α-smooth muscle actin expression did not change. Western blots of smooth muscle myosin heavy chain (SM MyHC) and α-smooth muscle actin (α-SM actin) expression in the aorta and tertiary mesenteric artery. SM MyHC and α-SM actin were normalized to total protein (TP) as previously described (Schaible et al., 2016; Yap et al., 2020; Han et al., 2021). Box blots summarize the data; neither SM MyHC/TP nor α-SM actin/TP expression are altered during aging, p > 0.05.