Abstract
It is unclear why mutations in the filament-forming tail of myosin heavy chain (MHC) cause hypertrophic or dilated cardiomyopathy as these mutations should not directly affect contraction. To investigate this, we first investigated the impact of five hypertrophic cardiomyopathy-causing (N1327K, E1356K, R1382W, E1555K, and R1768K) and one dilated cardiomyopathy-causing (R1500W) tail mutations on their ability to incorporate into muscle sarcomeres in vivo. We used adenoviral delivery to express full-length wild type or mutant enhanced GFP-MHC in isolated adult cardiomyocytes. Three mutations (N1327K, E1356K, and E1555K) reduced enhanced GFP-MHC incorporation into muscle sarcomeres, whereas the remainder had no effect. No mutations significantly affected contraction. Fluorescence recovery after photobleaching showed that fluorescence recovery for the mutation that incorporated least well (N1327K) was significantly faster than that of WT with half-times of 25.1 ± 1.8 and 32.2 ± 2.5 min (mean ± S.E.), respectively. Next, we determined the effects of each mutation on the helical properties of wild type and seven mutant peptides (7, 11, or 15 heptads long) from the myosin tail by circular dichroism. R1382W and E1768K slightly increased the α-helical nature of peptides. The remaining mutations reduced α-helical content, with N1327K showing the greatest reduction. Only peptides containing residues 1301-1329 were highly α-helical suggesting that this region helps in initiation of coiled coil. These results suggest that small effects of mutations on helicity translate into a reduced ability to incorporate into sarcomeres, which may elicit compensatory hypertrophy.
Highlights
It is unclear how mutations in the coiled-coil tail of -cardiac myosin cause heart disease
We first investigated the impact of five hypertrophic cardiomyopathy-causing (N1327K, E1356K, R1382W, E1555K, and R1768K) and one dilated cardiomyopathy-causing (R1500W) tail mutations on their ability to incorporate into muscle sarcomeres in vivo
Expression of Full-length myosin heavy chain (MHC) in Cardiomyocytes—wild type (WT) eGFP-MHC was expressed successfully in isolated adult cardiomyocytes, and the eGFP-tagged MHC (in which eGFP is fused to the MHC N terminus) incorporated into the thick filaments in muscle sarcomeres. eGFP-MHC began to be expressed and to incorporate into sarcomeres 6 –7 h after viral infection
Summary
It is unclear how mutations in the coiled-coil tail of -cardiac myosin cause heart disease. Significance: A change in myosin tail structure can lead to heart disease It is unclear why mutations in the filament-forming tail of myosin heavy chain (MHC) cause hypertrophic or dilated cardiomyopathy as these mutations should not directly affect contraction. Effects on coiled-coil structure and/or packing of the coiled coil into the thick filament might affect myosin incorporation into thick filaments in sarcomeres, and indirectly affect force output over the longer term To test this idea, we investigated five different mutations that result in HCM [15,16,17,18,19,20,21] and one (R1500W) that causes DCM [17]. We used peptides that were 7, 11, or 15 heptads long from the myosin coiled-coil tail to determine whether any or all of the five different mutations affect molecular structure and to determine whether the effects observed depended on peptide length
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