Figure 4.

Nontruncating MyBP-C (myosin binding protein C) mutant protein localizes to the myofilament for C3 and C6 domain mutants but does not incorporate into myofilaments for C10 domain mutants. To determine whether mutant MyBP-C proteins integrate normally into the myofilaments, both FLAG-tagged control and mutant constructs were cloned into an adenoviral vector that was then used to transduce neonatal rat ventricular myocytes (NRVMs). Forty-eight hours following transduction, NRVMs were immunofluorescently labeled with an anti-MyBP-C antibody to detect both endogenous and exogenously expressed MyBP-C (left column) and an anti-FLAG antibody to detect only the transduced MyBP-C (middle column). This system achieved stable integration of FLAG-control MyBP-C into myofilaments (top row) with no FLAG signal detected without viral transduction (second row). Nontruncating mutant MyBP-C for C3 and C6 domain pathogenic variants exhibited normal myofilament integration while C10 mutant MyBP-C exhibited poor or no myofilament localization.