Cardiac myosin-binding protein C is a novel marker of myocardial injury and fibrosis in aortic stenosis

Objective Cardiac myosin-binding protein C (cMyC) is an abundant sarcomeric protein and novel highly specific marker of myocardial injury. Myocyte death characterises the transition from hypertrophy to replacement myocardial fibrosis in advanced aortic stenosis. We hypothesised that serum cMyC concentrations would be associated with cardiac structure and outcomes in patients with aortic stenosis. Methods cMyC was measured in two cohorts in which serum had previously been prospectively collected: a mechanism cohort of patients with aortic stenosis (n=161) and healthy controls (n=46) who underwent cardiac MRI, and an outcome cohort with aortic stenosis (n=104) followed for a median of 11.3 years. Results In the mechanism cohort, cMyC concentration correlated with left ventricular mass (adjusted Î²=11.0 g/m2 per log unit increase in cMyC, P<0.001), fibrosis volume (adjusted Î²=8.0 g, P<0.001) and extracellular volume (adjusted Î²=1.3%, P=0.01) in patients with aortic stenosis but not in controls. In those with late gadolinium enhancement (LGE) indicative of myocardial fibrosis, cMyC concentrations were higher (32 (21–56) ng/L vs 17 (12–24) ng/L without LGE, P<0.001). cMyC was unrelated to coronary calcium scores. Unadjusted Cox proportional hazards analysis in the outcome cohort showed greater all-cause mortality (HR 1.49 per unit increase in log cMyC, 95% CI 1.11 to 2.01, P=0.009). Conclusions Serum cMyC concentration is associated with myocardial hypertrophy, fibrosis and an increased risk of mortality in aortic stenosis. The quantification of serum sarcomeric protein concentrations provides objective measures of disease severity and their clinical utility to monitor the progression of aortic stenosis merits further study. Clinical trial registration NCT1755936; Post-results.


Tissue Sampling
At the time of open heart surgery myocardial biopsies were obtained from the basal segment of the septum using a Tru-Cut needle biopsy gun. In order to minimise the chance of missed biopsies at least two samples per patient were collected. Immediately after collection, tissue was placed in buffered 10% formalin and subsequently embedded in paraffin.

Tissue processing
For apoptosis 7µm thick sections were deparaffinised in xylene, rehydrated through a graded series of alcohols and subsequently the DeadEndTM Fluorometric TUNEL System (Promega Co, US) was applied according to the manufacturer's guidelines. The system labels fragmented DNA which is a hallmark of apoptosis. After cell membrane permeabilisation the 3' OH ends of the cleaved DNA multimers were "tailed" with labelled fluorescin-12-dUTP by

Histological image analysis
The TUNEL stained tissue samples were analysed using confocal microscopy with FITC and UV filter cubes. All measurements have been performed using 40x objectives. Apoptotic cells (co-positive for both DAPI and TUNEL) were counted manually on entire tissue sections.
The total cell number present on each slide was derived from two sets of data: the total sample area and the number of cells positively stained with DAPI which was evaluated manually in three random areas of interest. Eventually the number of apoptotic cells was expressed as a percentage of the total cell number.
All C9 and ubiquitin stained slides images were acquired on the AxioScan Z1 (Carl Zeiss, Oberkochen, Germany) and analysed using Image-Pro Premiere 9.1 (MediaCybernetics, Rockville, MD, USA). In the first step the number of oncotic/autophagic cells was calculated using the counting toll after manual protocol adjustment. Cut off values of signal intensity, object size (area) and a roundness criterion was used to distinguish myocytes positively stained with DAB from artefacts. For Oncosis a pixel intensity of 0-163 on the Mono scale together with an object area range of 400-3000 square pixels and roundness criterion of 1-1.7 was applied. For autophagy the settings were as follows: a pixel intensity of 0-60 and an object area range of 200-7000 square pixels. In the second step the average cell area and the 20 total tissue area was measured using a threshold of 0-238 on the Mono pixel intensity scale.
Finally the number of oncotic or autophagic cells was expressed as a percentage of the total cell count.

Cell counts
The median number of cells examined per patient was 115,161 [IQR 78,427  193,585]

Example apoptotic cell
As demonstrated by confocal microscopy and immunofluorescence. A: DAPI; B: TUNEL; C: fused image demonstrating co-staining.