Phosphorylated Myosin Light Chain 2 (p-MLC2) as a Molecular Marker of Antemortem Coronary Artery Spasm

Abstract

BACKGROUND It is not uncommon that only mild coronary artery stenosis is grossly revealed after a system autopsy. While coronary artery spasm (CAS) is the suspected mechanism of these deaths, no specific biomarker has been identified to suggest antemortem CAS. MATERIAL AND METHODS To evaluate the potential of using phosphorylated myosin light chain 2 (p-MLC2) as a diagnostic marker of antemortem CAS, human vascular smooth muscle cells (VSMCs) were cultured and treated with common vasoconstrictors, including prostaglandins F2α (PGF2α), acetylcholine (ACh), and 5-hydroxy tryptamine (5-HT). The p-MLC2 level was examined in the cultured cells using Western blot analysis and in a rat model of spasm provocation tests using immunohistochemistry (IHC). Effects of increased p-MLC2 level on VSMCs contractile activities were assessed in vitro using confocal immunofluorescence assay. Four fatal cases with known antemortem CAS were collected and subject to p-MLC2 detection. RESULTS The p-MLC2 was significantly increased in VSMCs after treatments with vasoconstrictors and in the spasm provocation tests. Myofilament was well-organized and densely stained in VSMCs with high p-MLC2 level, but disarrayed in VSMCs with low p-MLC2 level. Three of the 4 autopsied cases showed strongly positive staining of p-MLC2 at the stenosed coronary segment and the adjacent interstitial small arteries. The fourth case was autopsied at the 6th day after death and showed negative-to-mild positive staining of p-MLC2. CONCLUSIONS p-MLC2 might be a useful marker for diagnosis of antemortem CAS. Autopsy should be performed as soon as possible to collect coronary arteries for detection of p-MLC2.

Figure 1

Treatment of human VSMCs with PGF2α, ACh, and 5-HT increases the phosphorylated level of MLC2. Human VSMCs were treated with serial doses of PGF2α2α (A, B), ACh (C, D), and 5-HT (E, F). The p-MLC2 level was detected by Western blotting. Under constant doses of vasoconstrictors, VSMCs were treated with PGF2α (B, dose=30 μM/ml), ACh (D, dose=0.5 μM/ml), and 5-HT (F, dose=3 μM/ml) in incremental time periods, as indicated. The lower bars in each panel are relative p-MLC2 levels after normalization to GAPDH. Data were obtained from 3 independent experiments with each in triplicate. * p<0.05 vs. control (without any reagent treatment in A, C and E, or at the beginning of treatment in B, D, and F) by t-test.

Figure 2

MLC2 is hyper-phosphorylated in spasm provocation tests. Provocation of coronary artery spasm was performed with an intracoronary injection of 5-HT in rats. (A) IHC analysis of the p-MLC2 in the coronary artery at the PBS-injected segment. (B) IHC staining of p-MLC2 in the spastic coronary segment in the 5-HT-injected group of rats. (C) In the vehicle (PBS)-treated heart, the interstitial small artery adjacent to the injection site was stained with a specific antibody against p-MLC2 using IHC analysis. (D) In the 5-HT-treated coronary arteries, the interstitial small artery which was adjacent to the injection site was stained with p-MLC2 antibody using IHC. Red arrows highlight the positive staining of p-MLC2. Magnification: 400×.

Figure 3

Increased p-MLC2 level promotes VSMCs contractile activities in vitro. Staining of p-MLC2 using immunofluorescence assay was utilized to reveal myofilaments within the cytoplasm of VSMCs. VSMCs were exposed to PGF2α (A), ACh (B), and 5-HT (C) for indicated times. The best-organized myofilament was observed by exposure to PGF2α for 15 min, to ACh for 3 min, and to 5-HT for 2 min. Extension of exposure time led to dephosphorylation and, hence, less organized myofilament and dimmed fluorescence.

Figure 4

MLC2 is highly phosphorylated in sudden cardiac deaths with mild coronary artery stenosis. In Case 1, the p-MLC2 was positively stained around LADA wall (A) with a focally magnified view in (B). Adjacent to the stenosed LADA segment, the interstitial small artery was also positively stained of p-MLC2 (C). In Case 2, the p-MLC2 was strong-positively stained around the LADA wall (D) with a focally magnified view in (E). Adjacent to the stenosed LADA segment, interstitial small artery was also positively stained p-MLC2 (F). In Case 3, the p-MLC2 was strong-positively stained around the LADA wall (G) with a focally magnified view in (H). Adjacent to the stenosed LADA segment, the interstitial small artery also had positively stained p-MLC2 (I). Magnification: 100× for upper images and 400× for bottom images. Red arrows indicate strongly positive staining.

Figure 5

MLC2 is barely phosphorylated in Case 4. In the LADA, although the lumen was mildly stenosed, p-MLC2 was barely detected under low (A) and high (B) magnification views. Adjacent to the LADA with lesions, the interstitial small artery was also barely stained with p-MLC2 (C). In the RCA, p-MLC2 was barely detected under low (D) and high (E) magnification views. Adjacent to the LADA with lesions, the interstitial small artery was also barely stained with p-MLC2 (F). Magnification: 100× for upper images and 400× for bottom images. Blue arrows indicate mildly positive staining. Magnified insets at right bottoms of (B) and (E) highlight the almost negative staining of p-MLC2 within coronary artery walls.