Mei Chi

Ablation of smooth muscle myosin heavy chain SM2 increases smooth muscle contraction and results in postnatal death in mice

The physiological relevance of smooth muscle myosin isoforms SM1 and SM2 has not been understood. In this study we generated a mouse model specifically deficient in SM2 myosin isoform but expressing SM1, using an exon-specific gene targeting strategy. The SM2 homozygous knockout (SM2−/−) mice died within 30 days after birth, showing pathologies including segmental distention of alimentary tract, retention of urine in renal pelvis, distension of bladder, and the development of end-stage hydronephrosis. In contrast, the heterozygous (SM2+/−) mice appeared normal and reproduced well. In SM2−/− bladder smooth muscle the loss of SM2 myosin was accompanied by a concomitant down-regulation of SM1 and a reduced number of thick filaments. However, muscle strips from SM2−/− bladder showed increased contraction to K+ depolarization or in response to M3 receptor agonist Carbachol. An increase of contraction was also observed in SM2−/− aorta. However, the SM2−/− bladder was associated with unaltered regulatory myosin light chain (MLC20) phosphorylation. Moreover, other contractile proteins, such as α-actin and tropomyosin, were not altered in SM2−/− bladder. Therefore, the loss of SM2 myosin alone could have induced hypercontractility in smooth muscle, suggesting that distinctly from SM1, SM2 may negatively modulate force development during smooth muscle contraction. Also, because SM2−/− mice develop lethal multiorgan dysfunctions, we propose this regulatory property of SM2 is essential for normal contractile activity in postnatal smooth muscle physiology.

Loss of the Serum Response Factor Cofactor, Cysteine-Rich Protein 1, Attenuates Neointima Formation in the Mouse

Objective—Cysteine-rich protein (CRP) 1 and 2 are cytoskeletal lin-11 isl-1 mec-3 (LIM)-domain proteins thought to be critical for smooth muscle differentiation. Loss of murine CRP2 does not overtly affect smooth muscle differentiation or vascular function but does exacerbate neointima formation in response to vascular injury. Because CRPs 1 and 2 are coexpressed in the vasculature, we hypothesize that CRPs 1 and 2 act redundantly in smooth muscle differentiation. Methods and Results—We generated Csrp1 (gene name for CRP1) null mice by genetic ablation of the Csrp1 gene and found that mice lacking CRP1 are viable and fertile. Smooth muscle–containing tissues from Csrp1-null mice are morphologically indistinguishable from wild-type mice and have normal contractile properties. Mice lacking CRPs 1 and 2 are viable and fertile, ruling out functional redundancy between these 2 highly related proteins as a cause for the lack of an overt phenotype in the Csrp1-null mice. Csrp1-null mice challenged by wire-induced arterial injury display reduced neointima formation, opposite to that seen in Csrp2-null mice, whereas Csrp1/Csrp2 double-null mice produce a wild-type response. Conclusion—Smooth muscle CRPs are not essential for normal smooth muscle differentiation during development, but may act antagonistically to modulate the smooth muscle response to pathophysiological stress.

The C-terminal calcium-sensitive disordered motifs regulate isoform-specific polymerization characteristics of calsequestrin

Calsequestrin (CASQ) exists as two distinct isoforms CASQ1 and CASQ2 in all vertebrates. Although the isoforms exhibit unique functional characteristic, the structural basis for the same is yet to be fully defined. Interestingly, the C‐terminal region of the two isoforms exhibit significant differences both in length and amino acid composition; forming Dn‐motif and DEXn‐motif in CASQ1 and CASQ2, respectively. Here, we investigated if the unique C‐terminal motifs possess Ca2+‐sensitivity and affect protein function. Sequence analysis shows that both the Dn‐ and DEXn‐motifs are intrinsically disordered regions (IDRs) of the protein, a feature that is conserved from fish to man. Using purified synthetic peptides, we show that these motifs undergo distinctive Ca2+‐mediated folding suggesting that these disordered motifs are Ca2+‐sensitivity. We generated chimeric proteins by swapping the C‐terminal portions between CASQ1 and CASQ2. Our studies show that the C‐terminal portions do not play significant role in protein folding. An interesting finding of the current study is that the switching of the C‐terminal portion completely reverses the polymerization kinetics. Collectively, these data suggest that these Ca2+‐sensitivity IDRs located at the back‐to‐back dimer interface influence isoform‐specific Ca2+‐dependent polymerization properties of CASQ.