Abstract
The homozygous appearance of the intronic mutation (IVS6-1) in the MYL2 gene encoding for myosin ventricular/slow-twitch skeletal regulatory light chain (RLC) was recently linked to the development of slow skeletal muscle fiber type I hypotrophy and early cardiac death. The IVS6-1 (c403-1G>C) mutation resulted from a cryptic splice site in MYL2 causing a frameshift and replacement of the last 32 codons by 19 different amino acids in the RLC mutant protein. Infants who were IVS6-1+∕+-positive died between 4 and 6 months of age due to cardiomyopathy and heart failure. In this report we have investigated the molecular mechanism and functional consequences associated with the IVS6-1 mutation using recombinant human cardiac IVS6-1 and wild-type (WT) RLC proteins. Recombinant proteins were reconstituted into RLC-depleted porcine cardiac muscle preparations and subjected to enzymatic and functional assays. IVS6-1-RLC showed decreased binding to the myosin heavy chain (MHC) compared with WT, and IVS6-1-reconstituted myosin displayed reduced binding to actin in rigor. The IVS6-1 myosin demonstrated a significantly lower Vmax of the actin-activated myosin ATPase activity compared with WT. In stopped-flow experiments, IVS6-1 myosin showed slower kinetics of the ATP induced dissociation of the acto-myosin complex and a significantly reduced slope of the kobs-[MgATP] relationship compared to WT. In skinned porcine cardiac muscles, RLC-depleted and IVS6-1 reconstituted muscle strips displayed a significant decrease in maximal contractile force and a significantly increased Ca2+ sensitivity, both hallmarks of hypertrophic cardiomyopathy-associated mutations in MYL2. Our results showed that the amino-acid changes in IVS6-1 were sufficient to impose significant conformational alterations in the RLC protein and trigger a series of abnormal protein-protein interactions in the cardiac muscle sarcomere. Notably, the mutation disrupted the RLC-MHC interaction and the steady-state and kinetics of the acto-myosin interaction. Specifically, slower myosin cross-bridge turnover rates and slower second-order MgATP binding rates of acto-myosin interactions were observed in IVS6-1 vs. WT reconstituted cardiac preparations. Our in vitro results suggest that when placed in vivo, IVS6-1 may lead to cardiomyopathy and early death of homozygous infants by severely compromising the ability of myosin to develop contractile force and maintain normal systolic and diastolic cardiac function.
Highlights
A new skeletal muscle fiber type-I myopathy with progressive cardiomyopathy and the early death of infants due to cardiac failure was reported in three unrelated Dutch families by Barth et al (1998)
The splice site intronic mutation in gene encoding for human ventricular regulatory light chain (RLC) (MYL2) (IVS6-1) mutation originates from a frameshift within the MYL2 gene and results in a replacement of the last 32 amino acids by 19 different amino acids, severely altering the Cterminus of the human cardiac RLC protein resulting in a shorter protein sequence (153 aa for IVS6-1 vs. 166 aa for WT) (Figure 1A)
The purity of the recombinant RLC WT and IVS6-1 proteins was tested by Western blotting with antibodies against the C-terminus of RLC (CT-1) and its N-terminus (NT1) (Figure 1B), both produced in this laboratory (SzczesnaCordary et al, 2005; Wang et al, 2006)
Summary
A new skeletal muscle fiber type-I myopathy with progressive cardiomyopathy and the early death of infants due to cardiac failure was reported in three unrelated Dutch families by Barth et al (1998). In this report we aimed at elucidating the potential molecular mechanism by which the IVS6-1 mutation may exert its effects on cardiac muscle contraction. This process is highly dependent upon the integrity of myosin, including its two heavy chains (MHC) and the regulatory and essential (ELC) light chains (Holmes and Geeves, 2000), and such drastic amino acid changes in the sequence of RLC due to IVS6-1 are likely to affect the interaction of myosin with actin, force production and lead to cardiac dysfunction responsible for infantile death of IVS6-1 homozygous patients (Barth et al, 1998; Weterman et al, 2013)
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