This is the first study evaluating atrial remodelling in aortic stenosis patients with chronic AF, both fibrosis quantification and target extracellular matrix protein gene expression analysis. AF patients were older, had increased cardiomyocyte area and atrial fibrosis on histologic quantification, increased collagen type III gene expression, as well as decreased TIMP1 and TIMP2 gene expression. Moreover, MMP16/TIMP4 ratio was decreased in AF patients. Serum TIMP1 and TIMP2 proteins were both increased in the AF patient subgroup.
Aortic stenosis patients with AF were older than their SR counterparts. Age is the most common and consistently described risk factor for AF, regardless of the cardiovascular disease background.[4],[14] Patients with AF had increased fibrosis, as demonstrated by histology sections, as well as increased cardiomyocyte size, suggested by the higher cell area. There is extensive evidence on the role of atrial fibrosis in sustaining the arrhythmia [15], although studies on aortic stenosis are scarce. Studies on animal models with increased afterload but absent hypertension suggest atrial remodeling increases AF inducibility through atrial myocardium fibrosis [16]. On the other hand, fibrosis evaluated by cardiac magnetic resonance imaging suggests a correlation with atrial fibrillation in hypertrophic cardiomyopathy. [17] Aortic stenosis causes pressure overload in both the ventricle and atrium, thus stimulating fibroblast proliferation which leads to cardiac fibrosis. [18] Fibroblast-cardiomyocyte coupling might slow conduction, therefore promoting re-entrance mechanisms, as well as enhance phase 4 depolarization, inducing ectopic impulse generation.[19] This cell-to-cell interaction depends on the degree of coupling, the number of coupled fibroblasts to each cardiomyocyte, and the relative size of cardiomyocytes versus fibroblasts.[20] Furthermore, the increased cardiomyocyte cell area in AF patients might correspond to a glycogen accumulation, with depletion of contractile material, as demonstrated in a study of sustained AF in goats with a substantial proportion of atrial myocytes with marked ultrastructure changes, including loss of myofibrils and accumulation of glycogen. [21] Studies on dogs with mitral valve stenosis have reported atrial cardiomyocyte hypertrophy with decreased myofibrils.[21],[22] Despite the absence of studies on aortic stenosis patients, it has been reported similar degenerative cardiomyocyte changes in patients submitted to cardiac surgery, which correlate with atrial size and pressure, along with diastolic dysfunction.[23]
In addition to the increase in atrial fibrosis in histology sections, mRNA expression of collagen type III was higher in AF patients, although collagen I and collagen I/III ratio did not present differences between groups. Zhang et al reported an increase in atrial mRNA expression of collagen I and III in AF patients with rheumatic heart disease [24]. Likewise, Cao et al reported a similar increase in patients with permanent AF undergoing valvular replacement.[25] Conversely, Yoshihara et al found a decrease in mRNA expression of both collagen type I and III in patients with AF submitted to Kosakai's modified maze procedure.[26] The present study focused on AF patients with aortic stenosis submitted to valve replacement, which could explain the isolated increase in type III collagen gene expression between patient groups.
Regarding atrial extracellular matrix gene expression, TGF-β1 was similar between groups. TGFβ1 is an established positive regulator of cardiac fibrosis [27], is upregulated in AF patients with mitral valve disease submitted to valve replacement, and [28] postoperative AF in patients submitted to myocardial revascularization [29]. Nevertheless, studies on aortic stenosis are scarce. MMP2, MMP9, and MMP16 showed no statistically significant differences between AF and SR patient subgroups. However, TIMP1 and TIMP2 were decreased in AF (p = 0.052 and p = 0.026, respectively), with no differences in gene expression for TIMP4. Regarding MMP/TIMP ratios, MMP2/TIMP1, MMP2/TIMP2, and MMP9/TIMP1 were similar in AF participants when compared to their SR counterparts. Conversely, MMP2/TIMP4 demonstrated a tendency towards significance, being decreased in AF patients, while MMP16/TIMP4 was significantly lower in this patient subgroup. Extracellular matrix remodeling is induced by collagen digestion through MMPs with tightly opposing inhibitors (TIMPs).[30] Findings vary according to experimental conditions, target extracellular matrix proteins, type of cardiovascular disease, and AF progression. [1] Polyakova et al found increased atrial expression of MMP2, MMP9, and TIMP1, although no differences in TIMP4, in AF patients submitted to a mini-Maze procedure.[31] Conversely, there were no differences in MMP9 atrial expression in AF patients with heart failure with reduced ejection fraction.[32] Despite a similar expression of TIMP4 between groups, MMP2/TIMP4 and MMP16/TIMP4 ratios demonstrated a decrease in AF patients. MMP2/TIMP4 ratio has been reported to be increased in heart failure induced by occlusion of the left coronary artery in spontaneously hypertensive rats. [33] Additionally, Wetzl et al. found a correlation between MMP2/TIMP4 ratio and mean pulmonary arterial pressure, pulmonary vascular resistance, estimated glomerular filtration rate, and tricuspid annular plane systolic excursion. Remarkably, MMP16/TIMP4 was overwhelmingly decreased in AF patients. This is the first study that suggests MMP16/TIMP4 as a marker of disease, specifically, a marker of chronic AF in aortic stenosis patients. Furthermore, although a significantly decreased atrial gene expression (TIMP1 and TIMP2) was observed, AF patients’ serum TIMP1 and TIMP2 proteins were significantly increased. This might represent a downregulation of expression when considering increased protein levels. The increase in matrix inhibitors when comparing with proteolytic enzymes (decreased MMP/TIMP ratios) may explain the collagen accumulation seen by histology, in addition to the increased mRNA collagen type III expression.
Concerning aortic stenosis severity, Aortic Valve Maximum and Mean Gradients were negatively correlated with collagen type I gene expression, collagen type I/III ratio, and serum TIMP1 protein levels. Additionally, MMP2/TIMP1 ratio was inversely correlated with aortic valve mean gradient. Aortic valve area was positively correlated with collagen type I gene expression and collagen type I/III ratio. Moreover, aortic valve area was negatively associated with cardiomyocyte cell area. Although the classical aortic valve stenosis severity definition remains dependent on transthoracic echocardiography and its parameters, such as maximal aortic velocity (severe, > 4 m/s), mean pressure gradient (severe, > 40 mmHg) and aortic valve area (severe, > 1 cm2), the interplay between comorbidities and aortic stenosis risk of progression remains incompletely understood.[34],[35] AF in aortic stenosis is associated with lower maximum and mean pressure gradients, which could lead to a misclassification of disease severity, since AF represents an independent risk factor for adverse outcomes, including all-cause mortality.[36] The obstruction caused by aortic stenosis leads to pressure overload and, ultimately, to ventricular hypertrophy and cardiac fibrosis.[37] Azevedo et al. found the amount of myocardial fibrosis is associated with all-cause mortality after aortic valve replacement in patients with severe aortic stenosis, indicating tissue fibrosis could be a marker of disease severity.[38] Overall, our results indicate an adverse remodelling in patients with lower maximum and mean pressure gradients, which can be explained by the presence of AF in this cohort. AF patients have worse remodelling and lower gradients, thus patients with more collagen deposition will be those with, apparently, less severe stenosis. However, a higher degree of fibrosis is associated with worse outcomes. Our results suggest several matrix proteins which could be implicated in disease severity besides histological fibrosis. Serum TIMP1 could be a marker of adverse outcomes in aortic stenosis, in addition to conventional echocardiography.
This study indicates an atrial matrix remodeling in aortic stenosis patients with chronic AF submitted to valve replacement while suggesting TIMP1 and TIMP2 as biomarkers of disease, readily available with peripheral blood sampling. MMP16/TIMP4 ratio is increased in AF and potentially a novel marker of this arrhythmia. Although the number of patients analysed is relatively low and thus further studies on chronic AF are needed, this is the first study on atrial matrix remodelling in aortic stenosis patients with atrial fibrillation.