Cardiac MyBP-C phosphorylation levels in normal and pathological states. A, One dimensional IEF (pH 5–7) of total myofilament proteins followed by western blot analysis using cMyBP-C antibody.18 Proteins were obtained from the following samples: (lane 1) hearts that had undergone sham operation for TAC, 24 hours. (lane 2) 24 hours post-TAC. (lane 3) 18 day sham. (lane 4) 18 day post-TAC. (lane 5) a NTG control. (lane 6) 8 weeks old calcineurin TG mouse. (lane 7) 7–8 months old MLP deficient mouse. (lane 8) a β-MHC TG mouse.24 (lane 9) Ischemic reperfusion (IR), 24 hours sham. (lane 10) 24 hours post-IR. (lane 11) 5 day post-IR sham. (lane 12) 5 day post-IR. The four forms of cMyBP-C based on its phosphorylation status are shown as P0, P1, P2 and P3, which correspond to the de-, mono-, bi- and tri-phosphorylated forms, respectively with isoelectric points of 6.1, 5.9, 5.7 and 5.5. B, MHC isoform shifts. Using the samples from panel A, the α- and β-MHC isoforms were separated in a 5% glycerol gel. The data show the expected isoform shifts characteristic of activation of the fetal gene program in the disease models (lanes 4, 6, 7 and 12). C, cMyBP-C phosphorylation in cardiac disease. Samples are as in panel A and average values are shown as a percentage of total cMyBPC (n = 3). The quantitative data are shown in tabular form in Table 1S, Online Supplement.

MyBP-C^AllP− transgene expression. A, Schematic diagram of cMyBP-C. The eight IgI-like domains are shown as ovals and the three F3 domains as octagons. Interaction sites are shown above the diagram. The cardiac-specific phosphorylation motif, which is highly conserved between species, is located between domains C1 and C2 and the sequence shown. The three known phosphorylation sites (Ser-273, -282 and -302) and two potential alternative phosphorylation sites (Thr-272 and -281) were each altered to a non-phosphorylatable alanine. B, Northern blot analyses of RNAs from left ventricles derived from 8–12 week old MyBP-C^WT, NTG and three lines of MyBP-C^AllP− mice. The RNAs were hybridized with cMyBP-C and GAPDH probes to confirm expression of the intact and correctly sized transcript. C, Dot-blot analyses using the same RNAs and probes as in panel B were used to quantitate the levels of TG expression. D, Western blot analyses of myofibrillar proteins from NTG, MyBP-C^WT (WT); expresses TG normal cMyBP-C such that approximately 40% of the endogenous protein is replaced with the transgenically encoded protein2 and three MyBP-C^AllP− (lines 45, 262 and 272) hearts. Note the conservation of normal levels of total cMyBP-C expression. E, Total cMyBP-C levels as well as the presence of myc-tagged cMyBP-C protein in the same mice was confirmed by western blot analysis. α-sarcomeric actin was used as a loading control. F, Phosphorylation state of NTG, MyBP-C^WT (WT) and MyBP-C^AllP− (AllP−) cMyBP-C in myofibrillar extracts assessed by one-dimensional IEF. Normal cMyBP-C in these extracts could be further phosphorylated by PKA but MyBP-C^AllP− was not a PKA substrate. Phosphatase treatment (Phosp) of myofibrillar extracts dephosphorylated normal cMyBP-C.

Phenotypic analyses of MyBP-C^AllP− (AllP⁻) mouse hearts. A, Longitudinal sections derived from 3 months old left ventricle stained with hematoxylin-eosin (top) or Masson trichrome (bottom) demonstrate the absence of obvious pathology (×10). B, Transmission electron micrographs of the sarcomeres. Areas of the MyBP-C^AllP− ventricles occasionally show altered sarcomeric organization, with high magnification (lower panels) showing altered M-line definition (M). Magnification: upper panels, ×10000; lower panels, ×30000. C, Transmission electron micrographs of sarcomeres from 12-week-old NTG and MyBP-C^AllP− TG hearts. The abnormal H-lines and I-zones (right bottom panel) and perturbation of the normal sarcomere-mitochondria architecture (left bottom panel) in MyBP-C^AllP− are indicated (◂). D, Immunofluorescent staining of cMyBP-C with either anti-cMyBP-C (top) or anti-myc antibodies (bottom) show normal incorporation. E, RNA dot blot analyses of hypertrophic markers. Four μg of total RNA was loaded in each dot. The quantitative data are summarized in F. *P<0.001, **P<0.01 MyBP-C^AllP− (AllP⁻) vs NTG or MyBP-C^WT (WT).

MyBP-C^AllP− fails to rescue the MyBP-C^(t/t) cardiac phenotype. A, Representative Northern (upper two panels) and dot-blot (lower panel) analyses of cMyBP-C mRNA from the left ventricles of 12-week old NTG (lane 1), homozygous MyBP-C^(t/t) (lane 2), MyBP-C^WT:(t/t) (lane 3) and MyBP-C^{AllP−:(t/t)} (lane 4) mice hybridized with a cMyBP-C probe. GAPDH was used as a loading control. B, SDS-PAGE analysis shows the absence of cMyBP-C in the homozygous MyBP-C^(t/t) hearts, replacement of endogenous cMyBP-C at normal levels in the MyBP-C^WT:(t/t) and MyBP-C^{AllP−:(t/t)} crosses and conservation of the other sarcomeric protein levels in these animals. Lanes are numbered as in panel A. C, Western blot analysis shows the absence of endogenous cMyBP-C in the homozygous MyBP-C^(t/t) hearts and the presence of myc-tagged TG cMyBP-C in MyBP-C^WT:(t/t) and MyBP-C^{AllP−:(t/t)} mice. D, One dimensional IEF showing the complete replacement of MyBP-C^AllP− in the cMyBP-C null background. Samples from NTG (lane 1), MyBP-C^WT:(t/t) (lane 3) and MyBP-C^{AllP−:(t/t)} (lane 4) were either untreated (Control), or treated with PKA or phosphatase (Phosp). PKA treated myofilaments show levels of MyBP-C^AllP− in the MyBP-C^(t/t) background that are essentially identical to NTG levels. Lane 2, which corresponds to the protein derived from MyBP-C^(t/t) hearts, showed no detectable signal and was omitted. E, Myosin isoform shifts in the samples shown in panel D. MyBP-C^WT expression completely rescued the cardiac phenotype observed in MyBP-C^(t/t) hearts in terms of the shift to the fetal hypertrophic pattern as evidenced by β-MHC expression. In contrast, MyBP-C^AllP− failed to prevent reversion to the fetal MHC isoform program. M, size markers.

Gross cardiac morphology and histological comparisons. A, Rescue and non-rescue of overt hypertrophy by MyBP-C^WT and MyBP-C^AllP−, respectively. Gross morphology of 12–14 week old hearts. B, Longitudinal sections of the entire heart stained with hematoxylin-eosin (×4). C, Hematoxylin-eosin; D, Masson trichrome stained myocardial sections to assess fibrosis (×20). Shown are representative paraffin-embedded sections prepared from 10% buffered formalin perfusion-fixed hearts from 12-week-old mice. Interstitial fibrosis, calcification and myocardial disarray are seen in the MyBP-C^(t/t) and MyBP-C^{AllP−:(t/t)} sections. E, Heart weight/body weight ratios. All data are presented as means±SE (n=3). #Significant difference vs NTG (P<0.05), *Significant difference vs MyBP-C^WT:(t/t) (P<0.05).

Ultrastructural and immunohistochemical analyses. A, Transmission electron micrographs showing ultrastructure of sarcomeres in MyBP-C^(t/t), MyBP-C^WT:(t/t) and MyBP-C^{AllP−:(t/t)} at 12-weeks. Note the loss of M-lines in the MyBP-C^(t/t). Although the MyBP-C^{AllP−:(t/t)} hearts displayed regular A- and I-bands, and M-lines, the overall architecture was disrupted, with sarcomeres slightly misaligned. Magnification: upper panels, ×10000; lower panels, ×30000. B, Incorporation of MyBP-C^AllP− into the MyBP-C^(t/t) background. Ventricular myocardial sections were immunolabeled for cMyBP-C using either anti-cMyBP-C (top) or anti-myc antibodies (bottom). There is no staining of cMyBP-C in the MyBP-C^(t/t) background (×60).

In vivo cardiac function and analysis of molecular responses. A, M-mode echocardiographic tracings show left ventricular dilation in the MyBP-C^t/t and MyBP-C^{AllP−:(t/t)} hearts. The MyBP-C^WT:(t/t) hearts show relatively preserved cardiac function. B, Dot-blot analyses of hypertrophic markers. Two μg of total mRNA from NTG (lanes 1–3), homozygous MyBP-C^(t/t) (lanes 4–6), MyBP-C^WT:(t/t) (lanes 7–9) and MyBP-C^{AllP−:(t/t)} (lanes 10–12) were dotted and hybridized with GAPDH, cMyBP-C, α- and β-MHC, atrial natriuretic factor (ANF), brain natriuretic peptide (BNP), α-skeletal-actin, phospholamban (PLN) and sarcoplasmic reticulum Ca²⁺-ATPase 2a (SERCA) γ-³²P labeled probes. Hypertrophic molecular markers such as ANF, BNP, skeletal actin and β-MHC were significantly up regulated in the MyBP-C^t/t and MyBP-C^{AllP−:(t/t)} groups, while the NTG pattern of expression is conserved in the MyBP-C^WT:(t/t) hearts. The quantitative data are summarized in C.