Smooth muscle myosin heavy chain isoform levels and unloaded shortening in single smooth muscle cells
Abstract
The relative abundance of the smooth muscle myosin heavy chain (SM-MHC) isoforms, SM1 and SM2, is known to vary with species, age, tissue, and in various disease states. However, unique functions for these myosin isoforms have not been distinguished. One possible explanation for this is most measurements of smooth muscle mechanics have been performed on multicellular or in vitro isolated myosin preparations. Because it has been shown that the myosin isoform content of a vascular smooth muscle cell varies with its distance from the lumen of the vessel, the contractile properties of cells with different isoform content must be studied at the single cell level. In vitro studies isolate the myosin molecule from the myosin filament and may therefore remove any influence the myosin isoforms have on filament structure. This project has led to the development of an experimental protocol which allows the correlation of the contractile properties of individual smooth muscle cells (SMCs) with their SM2/SM1 expression levels. The polymerase chain reaction (PCR) was performed on cDNA produced via reverse transcription (RT) from single smooth muscle cells or smooth muscle tissue utilizing two oligonucleotide primers from the known sequence of rabbit uterus myosin. These primers flank the alternative splice site which generates the difference between the 3$\prime$ ends of the SM1 and SM2 SM-MHC isoforms. Densitometric analyses of the PCR products corresponding to the mRNA of SM1 and SM2 were compared to the relative protein levels of SM1 and SM2 in adjacent tissue samples generating a correlation of R = 0.92. Duplicate RT reactions performed on aliquots of the same RNA extracted from smooth muscle tissues and individual SMCs yielded PCR amplified SM2:SM1 ratios which were not significantly different (P $<$ 0.05, n = 6 for tissue; P $<$ 0.05, n = 11 for cells) indicating reproducibility of the methods. Also, the RT-PCR assay was able to reproduce known mixtures of SM2:SM1 after 35 and 60 cycles of amplification. RT-PCR performed on single vascular SMCs from the same tissue exhibit a large range of SM2:SM12 ratios (0.00-1.78, n = 59, mean = 0.44, S.D. = 0.29) with 90% falling between 0.1 and 0.8. These results suggest the relative content of SM1 and SM2 MHC is regulated at the single cell level and that the SM-MHC content of individual SMCs within a tissue is not uniform. This RT-PCR technique was applied to individual SMCs after measuring untethered shortening. Untethered permeabilized ($\alpha$-toxin, saponin) isolated SMCs were simultaneously activated with Ca$\sp{++}$ (pCa 6.0), phenylephrine (1 $\mu$M), and histamine (1 $\mu$M), to insure maximal activation. Before, during, and after unloaded shortening, cell length was constantly recorded at high magnification by a CCD camera. Digitized cell images were analyzed with respect to initial length, final length, total change in length, and maximum velocity of shortening (V$\sb{\rm max}$). These values were correlated with the relative expression level of SM1 and SM2 in individual SMCs. The correlation between SM-MHC expression and V$\sb{\rm max}$ was very low (n = 20, R = 0.03, $\alpha$-toxin; n = 8, R = 0.27, saponin). However, the correlation between SM2:SM1 ratio and the minimum length an individual SMC could achieve was significant (n = 20, R = 0.72, $\alpha$-toxin; n = 8, R = 0.78, saponin). These results suggest a structural difference in myosin filaments with increasing SM2 expression.
This paper has been withdrawn.