SMB myosin heavy chain knockout enhances tonic contraction and reduces the rate of force generation in ileum and stomach antrum

Abstract

The role of SMA and SMB smooth muscle myosin heavy chain (MHC) isoforms in tonic and phasic contractions was studied in phasic (longitudinal ileum and stomach circular antrum) and tonic (stomach circular fundus) smooth muscle tissues of SMB knockout mice. Knocking out the SMB MHC gene eliminated SMB MHC protein expression and resulted in upregulation of the SMA MHC protein without altering the total MHC protein level. Switching from SMB to SMA MHC protein expression decreased the rate of the force transient and increased the sustained tonic force in SMB((-/-)) ileum and antrum with high potassium (KPSS) but not with carbachol (CCh) stimulation. The increased tonic contraction under the depolarized condition was not through changes in second messenger signaling pathways (PKC/CPI-17 or Rho/ROCK signaling pathway) or LC(20) phosphorylation. Biochemical analyses showed that the expression of contractile regulatory proteins (MLCK, MLCP, PKCδ, and CPI-17) did not change significantly in tissues tested except for PKCα protein expression being significantly decreased in the SMB((-/-)) antrum. However, specifically activating PKCα with phorbol dibutyrate (PDBu) was not significantly different in knockout and wild-type tissues, with total force being a fraction of the force generation with KPSS or CCh stimulation in SMB((-/-)) ileum and antrum. Taken together, these data show removing the SMB MHC protein expression with a compensatory increase in the SMA MHC protein results in enhanced sustained KPSS-induced tonic contraction with a reduced rate of force generation in these phasic tissues.

Jun kinase-induced overexpression of leukemia-associated Rho GEF (LARG) mediates sustained hypercontraction of longitudinal smooth muscle in inflammation

“…Consistent with the inhibition of sustained contraction by Y27632, treatment of cultured muscle cells for 10 min with ACh induced phosphorylation of MYPT1 at Thr 696 and MLC 20 at Ser 19 ; phosphorylation of both MYPT1 and MLC 20 was blocked by LARG siRNA (Fig. 5C).…”

“…Functional differences are evident between muscle layers (e.g., circular vs. longitudinal muscle of the intestine) or within the same layer (proximal tonic vs. distal rhythmic circular muscle of the stomach) (10,19,31,35,38,58,59). In all types of smooth muscle, an essential requirement for contraction is phosphorylation of the 20-kDa regulatory light chain (MLC) of myosin II (MLC 20 ), which in turn is regulated by the balance between Ca 2ϩ -dependent or -independent MLC kinase (MLCK) activity and MLC phosphatase (MLCP) activity (8,13,25,37,55).…”

Differential expression of multidrug resistance protein 5 and phosphodiesterase 5 and regulation of cGMP levels in phasic and tonic smooth muscle

“…The phasic and tonic behavior of smooth muscles may be related to differences in content and isoform composition of contractile proteins and intracellular signaling molecules that regulate the activities of MLCK and MLC phosphatase (4,6,7,13,15,17,(21)(22)(23)(24)36). For example, previous studies have shown that phasic muscle differs from the tonic muscle in the relative abundance of the ratio of acidic to basic isoforms of the 17-kDa essential light chain, in the ratio of seven-amino acid inserted to noninserted myosin heavy chain isoforms (SMA/ SMB isoforms) (2,17,21), in thin-filament-associated proteins such as caldesmon and calponin, and in the signaling pathways that regulate MLC 20 phosphorylation (45,46).…”

“…For example, previous studies have shown that phasic muscle differs from the tonic muscle in the relative abundance of the ratio of acidic to basic isoforms of the 17-kDa essential light chain, in the ratio of seven-amino acid inserted to noninserted myosin heavy chain isoforms (SMA/ SMB isoforms) (2,17,21), in thin-filament-associated proteins such as caldesmon and calponin, and in the signaling pathways that regulate MLC 20 phosphorylation (45,46). These differences in the expression of contractile proteins and biochemical pathways highly correlate with the functional phasic phenotype of antrum and tonic phenotype of fundus.…”

“…The phasic and tonic behavior of smooth muscles may also be related to differences in content and isoform composition of contractile proteins and intracellular signaling molecules that regulate the activities of MLCK and MLCP (4,6,7,13,15,17,[21][22][23]36). However, differences in the mechanisms involved in the termination of cGMP signaling in phasic and tonic muscle is not known.…”