A piece of the human heart: variance of protein phosphorylation in left ventricular samples from end-stage primary cardiomyopathy patients
- 530 Downloads
Cardiomyocyte contraction is regulated by phosphorylation of sarcomeric proteins. Throughout the heart regional and transmural differences may exist in protein phosphorylation. In addition, phosphorylation of sarcomeric proteins is altered in cardiac disease. Heterogeneity in protein phosphorylation may be larger in hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) as it may be caused by multiple mutations in genes encoding different sarcomeric proteins. Moreover, HCM is characterized by asymmetric remodelling of the heart. In the present study we assessed if local differences in sarcomeric protein phosphorylation are more evident in primary HCM or DCM than in non-failing donors. Thereto, phosphorylation of the two main target proteins of the beta-adrenergic receptor pathway, troponin I (cTnI) and myosin binding protein C (cMyBP-C) was analysed in different parts in the free left ventricular wall of end–stage failing HCM and DCM patients and donors obtained during transplant surgery. Intra-patient variability in protein phosphorylation within tissue samples of approximately 2 g wet weight was comparable between donor, HCM and DCM samples and could partly be attributed to the precision of the technique. Thus, our data indicate that within the precision of the measurements small, biopsy-sized cardiac tissue samples are representative for the region of the free left ventricular wall from which they were obtained.
KeywordsCardiomyopathy Phosphorylation Physiology
Primary hypertrophic cardiomyopathy (HCM) is characterized by asymmetrical hypertrophy of the left ventricle and septum, without underlying cardiac or vascular abnormalities (Richardson et al. 1996) The prevalence of primary HCM is 0.2% in the general population and in about 60% of the cases a mutation is identified in one of the 13 genes encoding the sarcomeric proteins (Ramaraj 2008; Richard et al. 2003, 2009). The clinical presentation and development of HCM is very diverse, ranging from no symptoms at all to end–stage heart failure. Primary dilated cardiomyopathy (DCM) is characterized by eccentric remodelling of the heart (Richardson et al. 1996). Although 20–48% of DCM cases are familial, only a small number of mutations are known to date (Chang and Potter 2005). The asymmetric remodelling observed in HCM and the variable geno- and phenotypes in familial HCM and DCM raise the question how representative small cardiac tissue sample are for the region of the left ventricle (LV) they are taken from.
A characteristic feature of end-stage heart failure is down regulation and desensitization of the β-adrenergic receptor pathway, as a consequence of chronic stimulation of the sympathetic system (Bristow et al. 1993; Hamdani et al. 2008). Consequently, the downstream sarcomeric target proteins cardiac troponin I (cTnI) and myosin binding protein C (cMyBP-C) may become less phosphorylated by protein kinase A (PKA) (Bodor et al. 1997; El Armouche et al. 2007; van der Velden et al. 2003; van Dijk et al. 2008). Therefore, we compared the variability in phosphorylation of the β-adrenergic target proteins cTnI and cMyBP-C within the left ventricle from HCM and DCM tissue samples to cardiac tissue from donors.
Tissue from the free LV wall from end-stage heart failure patients clinically characterized with familial hypertrophic (n = 5) or dilated (n = 5) cardiomyopathy was obtained during cardiac transplantation surgery. Control cardiac LV tissue was obtained from donor hearts (n = 5) for which no suitable transplant recipient was found. The donors had no history of cardiac disease, a normal ECG and normal ventricular function on echocardiography performed within 24 h prior to heart explantation.
The tissue was collected in cold cardioplegic solution, divided into pieces of approximately 2 g wet weight and immediately frozen in liquid nitrogen. Samples were obtained after informed consent and with approval of the local ethical committee (St. Vincent’s Hospital Human research Ethics Committee: File number H03/118; Title: Molecular Analysis of Human heart Failure).
Data are presented as mean ± standard error of the mean (SEM). The variance within each sample was expressed as the coefficient of variation. A comparison was made between the mean coefficient of variation in HCM, DCM and donor with one-way ANOVA. P < 0.05 was considered significant.
Inter- and intra-patient variability can partly be attributable to imprecision of the technique used to determine protein phosphorylation. The precision of the technique was estimated by analyzing the difference between duplicate measurements. The coefficient of variation of the duplicate measurements of cMyBP-C phosphorylation was higher in HCM and DCM samples compared to donor (38 ± 10 and 39 ± 9 vs. 11 ± 2%, respectively). Lower variability was seen in cTnI phosphorylation, though again the coefficient of variation was higher in HCM and DCM than in donor (17 ± 3 and 24 ± 6 vs 10 ± 2%, respectively). The variabilities between duplicate measurements are inversely related with the absolute phosphorylation levels (i.e. higher variability on low phosphorylation levels), indicating that differences in variability are largely accountable to noise in the measurement.
The average phosphorylation of cMyBP-C was lower in HCM and DCM than in donor (HCM 0.2 ± 0.05, DCM 0.1 ± 0.03 and donor 0.4 ± 0.01). Post hoc analysis indicated that cMyBP-C phosphorylation was significantly lower in DCM compared to donor. The average cTnI phosphorylation was significantly lower in both HCM and DCM compared to donor (0.4 ± 0.01 and 0.6 ± 0.4 vs. 1.5 ± 0.1, respectively).
In this study the variability in phosphorylation of β-adrenergic target proteins within patients with HCM and DCM was determined. A comparison was made between protein phosphorylation status of cMyBP-C and cTnI in HCM and DCM patients and in donor.
Although some variation was found between different pieces from one heart sample, this variation was not significantly different in HCM and DCM samples compared to donor samples. Furthermore, the coefficient of variation of the cMyBP-C and cTnI phosphorylation in hearts was in the same range as the variability of the duplicate measurements on the ProQ stained gels, indicating that part of the variability within hearts can be ascribed to imprecision of the technique. Since the samples weighted approximately 2 g, our results do not rule out the possibility of transmural differences in protein phosphorylation (Cazorla et al. 2005; Davis et al. 2001).
Figure 2 demonstrates inter-patient variability in protein phosphorylation. Furthermore, it can be appreciated that in general the phosphorylation of cMyBP-C and cTnI is lower in HCM and DCM compared to donor, in accordance with previous data obtained in ischemic heart disease (van der Velden et al. 2003).
In conclusion, we demonstrate comparable variability of cMyBP-C and cTnI phosphorylation within hearts from HCM and DCM patients and donors, indicating that, within the precision of our measurements, small left ventricular tissue samples of end stage-failing hearts from primary cardiomyopathy patients are representative for the region they were taken from.
This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.
- Hamdani N, de Waard M, Messer AE, Boontje NM, Kooij V, van Dijk S, Versteilen A, Lamberts R, Merkus D, dos Remedios C, Duncker DJ, Borbely A, Papp Z, Paulus W, Stienen GJ, Marston SB, van der Velden J (2008) Myofilament dysfunction in cardiac disease from mice to men. J Muscle Res Cell Motil 29:189–201CrossRefPubMedGoogle Scholar
- Richard P, Charron P, Carrier L, Ledeuil C, Cheav T, Pichereau C, Benaiche A, Isnard R, Dubourg O, Burban M, Gueffet JP, Millaire A, Desnos M, Schwartz K, Hainque B, Komajda M (2003) Hypertrophic cardiomyopathy: distribution of disease genes, spectrum of mutations, and implications for a molecular diagnosis strategy. Circulation 107:2227–2232CrossRefPubMedGoogle Scholar
- Richardson P, McKenna W, Bristow M, Maisch B, Mautner B, O’Connell J, Olsen E, Thiene G, Goodwin J, Gyarfas I, Martin I, Nordet P (1996) Report of the 1995 world health organization/international society and federation of cardiology task force on the definition and classification of cardiomyopathies. Circulation 93:841–842PubMedGoogle Scholar
- van der Velden J, Papp Z, Zaremba R, Boontje NM, de Jong JW, Owen VJ, Burton PB, Goldmann P, Jaquet K, Stienen GJ (2003) Increased Ca2+-sensitivity of the contractile apparatus in end–stage human heart failure results from altered phosphorylation of contractile proteins. Cardiovasc Res 57:37–47CrossRefPubMedGoogle Scholar