Pressure Overload Is Associated With Low Levels of Troponin I and Myosin Binding Protein C Phosphorylation in the Hearts of Patients With Aortic Stenosis

In previous studies of septal heart muscle from HCM patients with hypertrophic obstructive cardiomyopathy (HOCM, LVOT gradient 50–120 mmHg) we found that the level of phosphorylation of troponin I (TnI) and myosin binding protein C (MyBP-C) was extremely low yet samples from hearts with HCM or DCM mutations that did not have pressure overload were similar to donor heart controls. We therefore investigated heart muscle samples taken from patients undergoing valve replacement for aortic stenosis, since they have pressure overload that is unrelated to inherited cardiomyopathy. Thirteen muscle samples from septum and from free wall were analyzed (LVOT gradients 30–100 mmHg) 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 antibodies. TnI was predominantly monophosphorylated and total phosphorylation was 0.85 ± 0.03 molsPi/mol TnI. This phosphorylation level was significantly different (p < 0.0001) from both donor heart TnI (1.6 ± 0.06 molsPi/mol TnI) and HOCM heart TnI (0.19 ± 0.04 molsPi/mol TnI). MyBP-C is phosphorylated at up to four sites. In donor heart the 4P and 3P species predominate but in the pressure overload samples the 4P species was much reduced and 3P and 1P species predominated. Total phosphorylation was 2.0 ± 0.2 molsPi/mol MyBP-C (n = 8) compared with 3.4 ± 0.07 (n = 21) in donor heart and 1.1 ± 0.1 (n = 10) in HOCM heart. We conclude that pressure overload may be associated with substantial dephosphorylation of troponin I and MyBP-C.

In previous studies of septal heart muscle from HCM patients with hypertrophic obstructive cardiomyopathy (HOCM, LVOT gradient 50-120 mmHg) we found that the level of phosphorylation of troponin I (TnI) and myosin binding protein C (MyBP-C) was extremely low yet samples from hearts with HCM or DCM mutations that did not have pressure overload were similar to donor heart controls. We therefore investigated heart muscle samples taken from patients undergoing valve replacement for aortic stenosis, since they have pressure overload that is unrelated to inherited cardiomyopathy. Thirteen muscle samples from septum and from free wall were analyzed (LVOT gradients 30-100 mmHg) 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 antibodies. TnI was predominantly monophosphorylated and total phosphorylation was 0.85 ± 0.03 molsPi/mol TnI. This phosphorylation level was significantly different (p < 0.0001) from both donor heart TnI (1.6 ± 0.06 molsPi/mol TnI) and HOCM heart TnI (0.19 ± 0.04 molsPi/mol TnI). MyBP-C is phosphorylated at up to four sites. In donor heart the 4P and 3P species predominate but in the pressure overload samples the 4P species was much reduced and 3P and 1P species predominated. Total phosphorylation was 2.0 ± 0.2 molsPi/mol MyBP-C (n = 8) compared with 3.4 ± 0.07 (n = 21) in donor heart and 1.1 ± 0.1 (n = 10) in HOCM heart. We conclude that pressure overload may be associated with substantial dephosphorylation of troponin I and MyBP-C.

INTRODUCTION
Cardiac muscle contractility is modulated by the β-adrenergic system that primarily acts via activation of protein kinase A (PKA). In the cardiac muscle sarcomere the main targets of PKA are myosin binding protein C (MyBP-C) and Troponin I (TnI) and phosphorylation of these proteins plays a vital role in the enhanced contraction and relaxation kinetics induced by adrenergic stimulation (Layland et al., 2005b;Barefield and Sadayappan, 2010;Messer and Marston, 2014). In non-diseased heart muscle from donor human hearts or from mice, the level of phosphorylation of TnI and MyBP-C are both relatively high (Messer et al., 2009;Copeland et al., 2010). TnI has a total level of phosphorylation of 1.6 molsPi/mol in human and 1.2 in mouse with the majority of TnI being bis-phosphorylated. MyBP-C has a total level of phosphorylation of 3.4 molsPi/mol MyBP-C with 4P and 3P species predominating.
In heart disease the phosphorylation level is often low. For instance in idiopathic (non-ischaemic) end stage heart failure explants, phosphorylation levels are 0.26 molsPi/mol TnI and 0.62 molsPi/mol MyBP-C (van der Velden et al., 2003;Messer et al., 2007;Zaremba et al., 2007;Messer et al., 2009). In genetic heart disease the situation appears to be more complex. Explanted heart samples with inherited DCM can be associated with high or low levels of phosphorylation in the range 0.3-1.5 molsPi/mol TnI (Memo et al., 2013). However, myectomy samples taken from patients with inherited hypertrophic obstructive cardiomyopathy (HOCM) always have a low level of phosphorylation (Messer et al., 2009;Copeland et al., 2010;Bayliss et al., 2012). Thus, there appears to be no direct relationship between mutations causing heart disease and the TnI and MyBP-C phosphorylation level.
The HCM-associated mutations often cause a hypertrophied interventricular septum that can lead to left ventricular outflow tract obstruction (LVOTO). The septal myectomy operation for patients with HCM is usually indicated to reduce the obstruction when there is a high Aorta/LV pressure difference, typically 100 mmHg (Firoozi et al., 2002;Elliott and McKenna, 2004). We hypothesized that the pressure gradient may be a major factor in inducing the secondary phenotype of HOCM heart. To test this we have studied TnI and MyBP-C phosphorylation levels in heart muscle from patients with HCM but without pressure overload and heart muscle from patients without HCM but with pressure overload due to aortic stenosis (Bates, 2011).

Tissue Sources
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.
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 sitespecific antibodies (Bardswell et al., 2009;Sadayappan et al., 2009;Copeland et al., 2010).

Reduced Level of Phosphorylation in Pressure Overloaded Heart
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 bisphosphorylated 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.

Normal Level of Phosphorylation in HCM Samples Without Pressure Overload
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.

CONCLUSION
In these limited studies on human heart muscle samples we have found that a reduced level of phosphorylation of TnI and MyBP-C, the sarcomeric targets of PKA, is associated with pressure overload, i.e., a mean transvalvular pressure gradient >40 mmHg, but is not associated with hypertrophic cardiomyopathy in the absence of pressure overload.
Since we are using human samples obtained during surgery our results could be confounded by a number of uncontrolled variables such as the time between excision of the sample and freezing and the medication, particularly beta-blockers, taken by the patients. On the other hand we have more than 12 years experience making measurements with human heart samples and have not yet been able to detect a correlation between these variables and any measurements we made (Messer et al., 2007(Messer et al., , 2009Jacques et al., 2008;Copeland et al., 2010;Song et al., 2011;Bayliss et al., 2012;Memo et al., 2013).
We do not know the underlying cause of the reduced phosphorylation levels but in HCM and heart failure there is evidence for both the reduction in PKA activity and the increase in phosphatase activity in pathological heart muscle that could account for this observation. In the diseased myocardium β adrenoceptors are often downregulated via receptor phosphorylation and β-arrestin binding (Mangmool et al., 2018) whilst protein phosphatase activity is enhanced via inactivation of phosphatase inhibitor-1 (El-Armouche et al., 2004;Champion, 2005).

DATA AVAILABILITY STATEMENT
All datasets generated for this study are included in the article/Supplementary Material.

ETHICS STATEMENT
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