Familial Hypertrophic Cardiomyopathy Mutations in the Regulatory Light Chains of Myosin Affect Their Structure, Ca2+Binding, and Phosphorylation

Abstract

The effect of the familial hypertrophic cardiomyopathy mutations, A13T, F18L, E22K, R58Q, and P95A, found in the regulatory light chains of human cardiac myosin has been investigated. The results demonstrate that E22K and R58Q, located in the immediate extension of the helices flanking the regulatory light chain Ca 2؉ binding site, had dramatically altered Ca 2؉ binding properties. The K Ca value for E22K was decreased by ϳ17-fold compared with the wild-type light chain, and the R58Q mutant did not bind Ca 2؉ . Interestingly, Ca 2؉binding to the R58Q mutant was restored upon phosphorylation, whereas the E22K mutant could not be phosphorylated. In addition, the ␣-helical content of phosphorylated R58Q greatly increased with Ca 2؉ binding. The A13T mutation, located near the phosphorylation site (Ser-15) of the human cardiac regulatory light chain, had 3-fold lower K Ca than wild-type light chain, whereas phosphorylation of this mutant increased the Ca 2؉ affinity 6-fold. Whereas phosphorylation of wildtype light chain decreased its Ca 2؉ affinity, the opposite was true for A13T. The ␣-helical content of the A13T mutant returned to the level of wild-type light chain upon phosphorylation. The phosphorylation and Ca 2؉ binding properties of the regulatory light chain of human cardiac myosin are important for physiological function, and alteration any of these could contribute to the development of hypertrophic cardiomyopathy.There is substantial evidence that myosin regulatory light chains (RLC) 1 play a primary regulatory role in scallop and smooth muscle contraction, but their functional role in mammalian striated (skeletal and cardiac) muscle contraction is unclear. RLC, together with the essential light chain, stabilizes the 8.5-nm-long ␣-helical neck of the myosin head, with the N terminus of RLC wrapped around the heavy chain (1). Smooth muscle contraction is initiated by RLC phosphorylation with a Ca 2ϩ -calmodulin-activated myosin light chain kinase (MLCK) (2, 3). However, in skeletal and cardiac muscle, RLC phosphorylation does not activate contraction but appears to play a modulatory role (4). It was shown that RLC phosphorylation increased the Ca 2ϩ sensitivity of force in skinned skeletal (5-7) and cardiac (8) muscle fibers. In the human heart, several RLC isoforms are expressed (9, 10) preferentially in the atrium and in the ventricle. Recent studies have revealed that the ventricular RLC is one of the sarcomeric proteins associated with familial hypertrophic cardiomyopathy (FHC) (11,12). FHC is an autosomal dominant disease, characterized by left ventricular hypertrophy, myofibrillar disarray, and sudden death. It is caused by missense mutations in various genes that encode for ␤-myosin heavy chain (13), myosin-binding protein C (14), ventricular RLC and essential light chain (11,12,15), troponin T (16), troponin I (17), ␣-tropomyosin (18), actin (19), and titin (20). Depending on the affected gene, and the site of the mutation, FHC has variable presentation with regard to its degree and severity and t...

Familial Hypertrophic Cardiomyopathy-linked Alterations in Ca2+ Binding of Human Cardiac Myosin Regulatory Light Chain Affect Cardiac Muscle Contraction

“…All proteins were equilibrated in this buffer prior to the measurements. The flow dialysis experiments were performed according to Colowick and Womack (19) with slight modifications described in detail previously (18). Data were calculated using Scatchard analysis (20) as described (18).…”

“…The CDNAs of the proteins were transformed into BL21 expression host cells, and proteins were expressed in large (16 liters) cultures. Expressed proteins were purified as described previously (18).…”

Systematic analysis of the regulatory and essential myosin light chain genes: genetic variants and mutations in hypertrophic cardiomyopathy

“…Szsesna et al 23 investigated the effects of the Glu22Lys and Arg58Gln mutations in the RLC on Ca 2+ binding, phosphorylation and secondary structural properties. They demonstrated that the Glu22Lys mutant could not be phosphorylated and showed decreased Ca 2+ affinity.…”

Pseudophosphorylation of cardiac myosin regulatory light chain: a promising new tool for treatment of cardiomyopathy

“…Szczesna et al (2001) reported that A13T (in close proximity to Ser-15) led to decreased phosphorylation levels, impaired secondary structure and reduced calcium binding properties of RLC, which were normalized following phosphorylation. DCMlinked D94A-RLC mutation was shown to significantly alter the N-terminal α-helical region of the RLC, and trigger intramolecular rearrangements in the RLC molecule that ultimately resulted in alterations of RLC function .…”

“…In red, HCM phenotypes of decreased RLC phosphorylation in transgenic mouse myocardium. Adapted from Szczesna et al (2001) Myosin regulatory light chain phosphorylation in health and disease…”