Expression patterns of cardiac myofilament proteins: genomic and protein analysis of surgical myectomy tissue from patients with obstructive hypertrophic cardiomyopathy

Abstract

Background: Mutations in myofilament proteins, most commonly MYBPC3-encoded myosin-binding protein C and MYH7-encoded beta-myosin heavy chain, can cause hypertrophic cardiomyopathy (HCM). Despite significant advances in structure-function relationships pertaining to the cardiac sarcomere, there is limited knowledge of how a mutation leads to clinical HCM. We, therefore, set out to study expression and localization of myofilament proteins in left ventricular tissue of patients with HCM.

Methods and results: Frozen surgical myectomy specimens from 47 patients with HCM were examined and genotyped for mutations involving 8 myofilament-encoding genes. Myofilament protein levels were quantified by Western blotting with localization graded from immunohistochemical staining of tissue sections. Overall, 25 of 47 (53%) patients had myofilament-HCM, including 12 with MYBPC3-HCM and 9 with MYH7-HCM. As compared with healthy heart tissue, levels of myofilament proteins were increased in patients manifesting a mutation in either gene. Patients with a frameshift mutation predicted to truncate MYBPC3 exhibited marked disturbances in protein localization as compared with missense mutations in either MYBPC3 or MYH7.

Conclusions: In this first expression study in human HCM tissue, increased myofilament protein levels in patients with either MYBPC3- or MYH7-mediated HCM suggest a poison peptide mechanism. Specifically, the mechanism of dysfunction may vary according to the genetic subgroup suggested by a distinctly abnormal distribution of myofilament proteins in patients manifesting a truncation mutation in MYBPC3.

Figure 1. SDS polyacrylamide gels and ECL blots of myofilament proteins from a healthy individual or patients manifesting a mutation

Each lane is labeled either as normal or with the corresponding case (See Table 1) in which a mutation was identified in (A) MYBPC3 or (B) MYH7. Coomassie staining of the gel illustrated overall protein profiles and GAPDH served as a quantitative loading control to which other proteins were normalized. Myosin binding protein C and myosin heavy chain blots are shown for each individual.

Figure 2. Protein levels in cardiac tissue of patients with MYBPC3-HCM or MYH7-HCM

Levels of myosin heavy chain (A) and myosin binding protein C (B) are shown. Samples were run in triplicate with 3 to 4 normal samples run on each gel for comparison to diseased tissue. Statistically significant differences from normal were determined by generalized estimating equations, using a gamma distribution and a log link (*, p<0.001). Values are represented as adjusted least square means ± S.E., after adjusting for the sample and gel effects.

Figure 3. Immunohistochemistry of myosin heavy chain and myosin binding protein C in patients with myofilament HCM

(A) Homogeneous pattern of expression myosin heavy chain and myosin binding protein C in normal heart tissue. (B) Even distribution noted in Case 20 having a missense mutation in MYH7 and (C) Case 4 having a missense mutation in MYBPC3. Splotchy pattern of distribution in patients with a truncation mutation in MYBPC3 is demonstrated in (D) case 9 and (E) case 7 with the polyclonal antibody (middle panel) and the new monoclonal antibody (right panel). Score is represented in upper right hand corner for staining with myosin heavy chain and the polyclonal MYBPC3 antibody. Scores ranged from 0 (100% homogeneity of protein throughout the tissue) to 16 (complete disruption of protein staining). All images shown are at 20X magnification and scale bars indicate a 50 µM distance.

Figure 3. Immunohistochemistry of myosin heavy chain and myosin binding protein C in patients with myofilament HCM

(A) Homogeneous pattern of expression myosin heavy chain and myosin binding protein C in normal heart tissue. (B) Even distribution noted in Case 20 having a missense mutation in MYH7 and (C) Case 4 having a missense mutation in MYBPC3. Splotchy pattern of distribution in patients with a truncation mutation in MYBPC3 is demonstrated in (D) case 9 and (E) case 7 with the polyclonal antibody (middle panel) and the new monoclonal antibody (right panel). Score is represented in upper right hand corner for staining with myosin heavy chain and the polyclonal MYBPC3 antibody. Scores ranged from 0 (100% homogeneity of protein throughout the tissue) to 16 (complete disruption of protein staining). All images shown are at 20X magnification and scale bars indicate a 50 µM distance.

Figure 4. High magnification illustrating intracellular disruption of protein expression

(A) 200X magnification confirms homogeneous expression of myosin binding protein C in normal heart tissue. (B) 200X magnification illustrates heterogeneous expression of MYBPC3 in Case 7 manifesting a splice mutation. (C) 400X magnification of Case 7 showing striations are still present with lighter staining (asterisk) and distinct from fibrosis (arrow).

Figure 5. Quantification of Localization Patterns

Scores were averaged for both the distribution of myosin binding protein C (A) and myosin heavy chain (B) and converted to a percentage representing the total disruption of the protein in the tissue section. This takes into account both intracellular as well as intercellular distribution pattern of the protein. Statistically significant differences were determined by unpaired Student’s t-test and represented as the mean ± S.D.