Donor hearts and the K280N HCM sample were supplied by the Sydney Heart Bank (Li et al., 2013; Messer et al., 2016). Donor sample (NH) had no history of cardiac disease and normal ECG and ventricular function and were obtained when no suitable transplant recipient was found. HOCM sample (MV) was obtained from a patient undergoing septal myectomy operation at The Heart Hospital (UCL), London (Jacques et al., 2008). Clinical data for NH and MV has been previously reported in Messer et al. (2007), Bayliss et al. (2012) and in Supplementary Table 1. Measurements of TnI and MyBP-C phosphorylation were described in Messer et al. (2009) and Copeland et al. (2010).

Biopsies were taken from septum and free wall of patients undergoing valve replacement surgery to relieve aortic stenosis at the Royal Brompton Hospital, London and Careggi University Hospital, Florence. The ACTC E99K patient sample was kindly supplied by Dr. Lorenzo Monserrat, La Coruña, Spain (Monserrat et al., 2007; Song et al., 2011). Available clinical data on these samples is given in the Supplementary Material.

Patients gave written consent with PIS approved by the relevant ethical committee. All samples are anonymised. The investigations conform to the principles of the Declaration of Helsinki.

Ethical approval for collection and distribution of the human heart samples was granted by the Research Integrity, Human Research Ethics Committee, University of Sydney (Protocol No. 15401); the Joint UCL/UCLH Ethics Committee Rec No: 04/0035; Outer North East London Research Ethics Committee REC ref: 10/H0701/8; Careggi University Hospital Ref: 2006/0024713 and Comite Ético de Investigatio ń Clìnica de Galicia. Permission for study of the samples was granted by the NHS National Research Ethics Service, South West London REC3 (10/H0803/147).

Twenty milligrams of heart muscle sample, stored in liquid nitrogen were crushed in a liquid nitrogen cooled percussion mortar and then homogenized in 200 μl buffer (5 mmol/l NaH₂PO₄, 5 mmol/l Na₂HPO₄ pH 7.0, 0.1 mol/l NaCl, 5 mmol/l MgCl₂, 0.5 mmol/l EGTA, 0.1% Triton X-100, 20 mmol/l NaF and 5 mmol/l DTT with 2 μg/ml each of the protease inhibitors E64, chymostatin, leupeptin and pepstatin A). The homogenate was then centrifuged at 16,500 × g for 5 min and the supernatant discarded. The wash process was repeated and then the pellet was dissolved in sample buffer containing 8 M urea, 2 M thiourea, 0.05 M Tris-HCl, pH 6.8, 75 mM DTT, 3% SDS and 0.05% bromophenol blue as decribed (Layland et al., 2005a; Messer et al., 2007).

TnI phosphorylation levels in heart muscle myofirils was measured by Phosphate affinity SDS-PAGE as described by Messer et al. (2009). Discontinuous SDS-PAGE gels were hand-cast and run using the Mini-PROTEAN system (Bio-Rad). Gel compositions are as follows: stacking gel: 4% acrylamide (29:1 acrylamide:bis-acrylamide), resolving gel: 10% acrylamide (29:1 acrylamide:bis-acrylamide, 100 μM MnCl₂ (from 10 mM stock) and 50 μM Phos-TagTM acrylamide [from 5 mM stock solution of Phos-TagTM acylamide AAL-107 (NARD Institute, Hyogo, Japan)] prepared according to suppliers instructions (Kinoshita et al., 2006).

Gels were probed with the phosphorylation-independent anti-human-cardiac troponin I (hcTnI) clone 14G5 mouse mAb (Abcam plc antibodies), 1/2,000 dilution on Western blots. Secondary antibody was HRP- conjugated anti-rabbit IgG (1:1,000) and the blots were visualized using ECL (GE Biosciences). Chemiluminescence was detected by a cooled CCD camera-based gel imager (G:BOX Chemi HR16, Syngene).

To resolve MyBP-C phosphospecies the myofibril samples were run on the gels for 165 min. The current was initially 25 mA, raised to 35 mA once the samples had entered the resolving gel. The gels were Western blotted and probed with a rabbit polyclonal antibody against cMyBP-C residues 2–14 which recognizes total cMyBP-C or with phosphorylation site-specific antibodies (Bardswell et al., 2009; Sadayappan et al., 2009; Copeland et al., 2010).

We studied 13 heart muscle biopsies from intraventricular septum and free wall taken from patients undergoing valve surgery to relieve pressure overload and compared them with previously studied donor heart samples and myectomy samples from patients with HOCM (Messer et al., 2009; Copeland et al., 2010). LVOT gradients ranged from 30 to 100 mmHg in the pressure overload patients compared with 90–120 mmHg in the HOCM patients’ hearts and close to zero in the donor hearts (see Supplementary Tables 1 and 2).

The levels of TnI and MyBP-C phosphorylation were determined in muscle myofibrils by separating phosphospecies using phosphate affinity SDS-PAGE and detecting with TnI and MyBP-C specific, but phosphorylation-independent antibodies previously characterized. This technique measures the proportions of bis-phosphorylated, monophosphorylated and unphosphorylated species of TnI. We previously showed that in donor hearts, 70% of the troponin I is bis-phosphorylated and 21% is monophosphorylated with a calculated total phosphorylation of 1.6 ± 0.06 molsPi/mol TnI. The HOCM samples were just 5% bis-phosphorylated and 30% monophosphorylated with a calculated total phosphorylation level of 0.18 ± 0.02 molsPi/mol TnI (Messer et al., 2009; Figure 1).

In the pressure overload samples, we found that TnI was predominantly monophosphorylated (0P = 24 ± 2%, 1P = 67 ± 2%, 2P = 9 ± 1%, n = 21) and total phosphorylation was 0.85 ± 0.03 molsPi/mol TnI and was not significantly different in septum and free wall samples (see Supplementary Figure 1). Thus in pressure overload samples, the phosphorylation level was significantly less than donor heart and significantly more than in HOCM heart Troponin I (p ≤ 0.0001). Western blotting using antibodies to bis-phosphorylated troponin I confirmed that phosphorylation at serines 22 and 23 was low relative to donor heart samples. Mass spectrometry measurements showed that only Ser22 or Ser23 were phosphorylated with no evidence for phosphorylation at other sites in Troponin I.

MyBP-C is phosphorylated at up to four sites. In donor heart samples we showed that the 4P and 3P species predominate (4P = 43%, 3P = 57%, 2P = 0%, 1P = 0%, 0P = 0%) (Copeland et al., 2010). In the pressure overload samples the 4P species was much reduced and 3P and 1P species predominated (4P = 9 ± 3%, 3P = 41 ± 5%, 2P = 1 ± 1%, 1P = 33 ± 2%, 0P 15 ± 4%, n = 15) (Figure 2). Total phosphorylation was 2.0 ± 0.2 molsPi/mol MyBP-C (n = 15) compared with 3.4 ± 0.07 (n = 21) in donor heart and 1.1 ± 0.1 (n = 10) in HOCM heart (Copeland et al., 2010). Comparison of free wall and septal samples indicated higher phosphorylation levels in the free wall that was barely statistically significant (see Supplementary Figure 2). We used antibodies specific to the main targets of PKA in MYBP-C: phosphorylation sites at Ser 302, 273 and 283 but found no consistent pattern of phosphorylation in the pressure overload samples that was different from the pan-MyBP-C antibody, 2–14.

To test whether the low level of phosphorylation in HOCM myectomy samples is due to the mutation or the pressure overload, we investigated samples from HCM patients that did not have pressure overload. The ACTC E99K mutation is associated with HCM, with the hypertrophy often confined to the apex of the heart and does not develop HOCM (Monserrat et al., 2007). We have previously studied a biopsy from a 33 year old patient with the ACTC E99K mutation who showed no signs of LVOTO and was not on any medication (Song et al., 2011). The tissue sample was obtained from an operation to repair an atrial septal defect. Troponin I, troponin T, and MyBP-C phosphorylation levels in myofibrils were the same as donor heart samples (see Figure 1D). Likewise, The ACTC E99K mouse model of HCM develops apical hypertrophy without LVOTO or symptoms of heart failure at 21 weeks (Song et al., 2011). The HCM mouse myofibrils have the same level of troponin I, troponin T, and MyBP-C phosphorylation as NTG littermates (Figure 1D).

We have also studied a heart muscle sample from a patient with an HCM-causing mutation [homozygous TNNT2 K280N (Sequeira et al., 2013; Messer et al., 2016; Piroddi et al., 2019)]. The patient had a myectomy operation that relieved LVOTO permanently but later developed heart failure requiring a heart transplant. This sample is from the explanted heart. The troponin I phosphorylation level was comparable to donor heart (Figure 1D). These two examples suggest that reduced levels of phosphorylation are related primarily to the pressure overload.