Background
Hypertrophic cardiomyopathy (HCM) is the most prevalent cardiac genetic disease with an estimated population prevalence of HCM ranging from 1:200 to 1:500 individuals [
1,
2] and mainly caused by distinct genetic variants in sarcomeric proteins. The major clinical feature of HCM is left ventricular (LV) hypertrophy (LVH), which is often asymmetric and preferentially affects the interventricular septum, in the absence of any other conditions that could induce LVH, such as environmental factors (stress or physical activities), hypertension, or aortic stenosis [
9]. Sudden cardiac death can be the first manifestation of HCM, particularly in young [
3,
4] and asymptomatic patients [
5,
6]. The key sarcomere genes [
7] most frequently affected genes in HCM are the cardiac myosin-binding protein C (
MYBPC3) [
8] and β-myosin-heavy chain (
MYH7) [
9]. Aberrant proteins trigger myocardial tissue remodelling which is a complex process of transcriptional, signaling, structural, electrophysiological, and functional events occurring within the cardiac myocyte and myocardium [
10]. The myocardial tissue remodelling contributes to small vessel disease (namely vascular remodelling), cardiac hypertrophy, myocyte disarray, myocardial fibrosis and ultimately compromise the cardiac function [
11,
12]. While the early detection of myocardial remodelling is a key to effective disease management [
13], at present no in vivo imaging markers of dynamic changes of microvasculature have been found [
14]. CMR can detect ventricular wall hypertrophy and fibrosis progression, two main features that are useful in predicting HCM prognoses [
15], but monitoring those morphological changes on the macroscopic and mesoscopic scales cannot detect subclinical microvascular dysfunctions, which potentially might be more sensitive markers of disease progression [
16]. Here we focused on the remodelling of small vessels and microvascular dysfunction in the myocardium, which may lead to a reduction of myocardial blood supply and a subsequent progressive loss or deterioration of cardiac function. In response to varying physiological or pathological conditions and functional demands, blood vessels are continuously adapting their structural change [
12].
Microvascular dysfunction in HCM patients has been proposed as a strong predictor of clinical outcomes and mortality risk [
17], but the relationship between this and myocardial remodelling is poorly understood. The small vessels of the microvascular network supply the myocardium with oxygen to maintain functional cardiac tissue integrity [
18]. Myocardial wall thickening connected to pressure overload is thought to play a role in impairing coronary inflow and trigger a progressive ventricular dysfunction [
19]. Hypertrophy-induced deficits in myocardial perfusion and micro-circulation might also trigger the pathological deposition of collagen in the myocardium. This reduces myocardial blood flow and might be the key determinant of subsequent heart failure [
20]. This suggests that monitoring changes in micro-perfusion might make an effective early indicator to diagnose the disease and have the chance to provide early procedures that improve blood flow.
Local myocardial blood volume and blood flow are important indicators of changes in micro-perfusion [
21]. Recent work has investigated the relationship between hypertrophy and the myocardial microvascular network in HCM [
22]. In these studies, the fractional tissue blood volume per cardiac tissue volume (Bvf), defined as the volume in the microvascular network, was directly probed by Bvf-sensitive CMR using blood oxygenation-weighted imaging contrast [
22]. Myocardial regions with reduced Bvf were highly correlated with ventricular wall thickening [
22,
23]. Yet the factors responsible for the reduction of microvascular network volume remain unknown.
We hypothesize that vascular dysregulation caused by hypertrophy or excessive collagen deposits may contribute to the microstructural changes in HCM and cause the reduction. Using a mouse model that closely reflects human HCM, we quantified the amount of blood delivered to myocardial tissue per unit time using myocardial blood flow (MBF) CMR based on the labelling of arterial proton spins [
24,
25]. Unlike widely used methods based on contrast agent enhanced first pass perfusion CMR, arterial spin labelling (ASL)-CMR does not require an injection of exogenous contrast material, and thus can be repeated [
26]. Employing this approach, the goal was to find any sign of small vessel change indicated by myocardial perfusion and then correlate the changes detectable by CMR with microstructural histology in the murine heart – something that is difficult to do in human patients. For this purpose MBF, capillary density, perivascular fibrosis and interstitial fibrosis were determined.
Discussion
In this study, we demonstrate that cine ASL-CMR is capable of detecting changes in myocardial perfusion that are correlated to a reduction in small vessel density in the myocardium. The reduced myocardial perfusion provides an indication for microvascular dysfunction in HCM. The myocardial perfusion deficiency is correlated with LVH and associated with myocardial fibrosis and loss of small vessel densities, features which are presumably mediated by the genetic variants commonly found in HCM. We analysed the global as well as the regional differences of MBF based on the cardiac segmentation model. However, motion artefacts have been recognized as a limiting factor for the precise detection of myocardial regional differences. These artefacts can be induced by myocardial contraction/relaxation, by respiratory motion causing a shift a in the chest wall and the diaphragm, by susceptibility changes due to motion of the heart–lung or heart–liver interface or by non-uniform regionally motion patterns (different twist and strain) of the heart [
35]. Another confounding factor could be the different rotational behaviour during early systole in mice [
31]. The assessment of regional differences in myocardial perfusion pattern has to be further validated with respect to their functional component.
Subclinical myocardial remodelling dictates the progression of HCM, but currently diagnosis and treatment of the disease are restricted to measurements of myocardial hypertrophy. Diagnosing microvascular dysfunction has been a challenge. Standard tests used to diagnose coronary artery disease are not designed to detect small vessel disease, so a better understanding of the factors associated with microvascular dysfunction and its relation to the development of HCM would represent a major step toward earlier diagnosis and better management of the disease. Our results can partially explain previous findings showing that HCM patients have lower myocardial blood volume than healthy counterparts [
22]. In the presence of LVH, effective perfusion is critical to compensate for hypertrophy-induced micro-vessel restriction in the HCM heart. This hypothesis will need to be refined, tested and extended to obtain a fuller picture of the development of the pathological features of the disease and their consequences. In addition, ASL-CMR is not a standard clinical technique, while quantitative first-pass contrast agent based techniques are clinically established for myocardial perfusion imaging. Therefore, despite the necessity of bolus injections, robust quantitative first-pass post-processing methods are now becoming more widely available and currently represent the method of choice when similar studies are conducted in humans.
En route to non-contrast myocardial perfusion imaging such as ASL-CMR, more studies are needed to prove that both methods are equally relevant for clinical applications [
36].
Although a number of post-mortem studies have demonstrated marked impairment of the coronary microcirculation in the absence of significant coronary lesions in HCM patients [
37,
38], the influence of genetic variants on microvascular function in HCM myocardium has remained unexplored. Measuring myocardial perfusion using CMR or other imaging modalities may reveal deficiencies in microvascular function even in mild cases or asymptomatic HCM patients, giving it potential as an independent predictor of clinical outcome [
17,
39]. For instance, many patients with HCM have symptoms of myocardial ischemia and cardiac dysfunction. Abnormal intramural coronary arteries with markedly thickened walls and narrowed lumens are observed in HCM patients and may represent a genetic component of the underlying myocardial remodelling process [
40]. Although the clinical relevance of microvascular dysfunction in HCM remains unclear, the fact that intramural coronary arteries exhibit structural alterations in areas of substantial myocardial fibrosis suggests a causal role for these arteries in producing ischemia [
41]. In line with previous findings, we show in this study that the abnormal microvasculature was substantially pronounced in the inferior septum. Although it has become evident that perivascular fibrosis, but not interstitial fibrosis is associated with the impairment of coronary blood flow [
42], we observed both perivascular and interstitial fibrosis are more pronounced in our HCM mouse model. Fibrosis is a dynamic process, thus the characterization of temporal pattern of both perivascular fibrosis and interstitial fibrosis requires additional attention [
43].
Classically, HCM is characterized by varying degrees of LVH with a preserved or sometimes even increased LVEF [
44]. Our data suggest that the quantification of cyclic changes of myocardial perfusion under resting conditions is sensitive to detect differences in HCM when using cineASL-based perfusion CMR. The incidence of heart failure (HF) in HCM patients is about ∼50%, with symptoms varying from mild to severe [
45,
46]. Due to a substantial heterogeneity, ascertaining the incidence of which HF in HCM is challenging. In HCM, HF has two distinct clinical features: HFpEF or HFrEF. In the majority of HCM patients, HF is manifested as HFpEF phenotype, known as “diastolic heart failure” while only a minority develops HFrEF at a later stage. A recent large cohort study confirmed that systolic dysfunction (manifest by low LVEF) is highly associated with prognosis [
47]. On the other hand, in HFpEF, microvascular dysfunction was evident as the major determinant of the pathological cascade that justifies clinical manifestations [
48,
49]. Therefore it is extremely relevant to identify microvascular dysfunction, including the cause and its mechanisms. Recent reports have suggested that patients with HFpEF exhibit an increased incidence of small vessel disease as shown by abnormal blood flow and increased microvascular resistance [
49,
50]. Our results support the idea that myocardial perfusion changes are connected to hypertrophy and the extent of fibrosis and thus can be an additional remodelling marker of the HCM phenotype regardless of cardiac dysfunction.
Blood flow through small vessels in the myocardium is influenced by changes in myocardial tissue pressure during heartbeats. Most myocardial perfusion occurs during the diastolic heart phase, when the myocardial pressure is low [
51]. It may also be affected by total capillary density. Vascular endothelial dysfunction obviously impairs myocardial perfusion [
52,
53]; our study shows that capillary density is a potential key confounding factor for myocardial perfusion.
The accuracy of this non-invasive MBF measurement using cineASL has already been validated against the more common FAIR Look-Locker Gradient Echo technique, which was in turn validated against fluorescent microspheres [
54]. The variations of blood flow throughout the cardiac cycle under different conditions were previously assessed using cineASL [
30]. Notably, cyclic variation of MBF across the cardiac cycle has been thoroughly assessed using this technique in rats [
24]. Since the MBF value in mice has been shown to be strongly dependent on the anesthetic conditions, the relatively high values found here could be due to differences in anesthesia. The anesthesia is also affected by the individual experimental conditions: actual isoflurane concentration inhaled, which is dependent on the face mask, space around the animal and gas recovery and therefore difficult to match across different lab setups. Other influencing factors are temperature and breathe rate. Therefore, one has to be cautious that the results on MBF may not reflect perfect resting conditions due to the vasodilating effect of isoflurane.
Sex differences are known in several facets of cardiac physiology [
55]. Sex differences in myocardial perfusion have previously been demonstrated in healthy subjects and in patients with nonischemic heart failure using positron emission tomography (PET) or CMR [
56,
57]. These reports suggested that females have higher resting myocardial perfusion compared to males. Our data showed the tendency of higher resting MBF in both diastolic and systolic cardiac phases. The sex difference of myocardial perfusion will therefore need to be takin into account in future studies.
Limitations
While we observed the sex differences in end-diastolic MBF in D2 mice but not in other experimental groups, this observation might be attributed to the small sample size used, which is a recognized limitation of our study. We are therefore unable to comment on the exact sex influence that might exist with perfusion abnormalities. To address this scientific question future work will need to incorporate a larger sample size. The cardiac function and MBF measurements were not derived from the same group of mice. Notably, the physiological effects of anaesthesia during CMR have to be minimized. Consequently, we have designed the CMR measurement protocol such that the total duration of an individual in vivo experiment does not exceed a maximum of 60 min. Therefore all measurements were performed in the same time window.
We noted that MBF values in mice found in this study are higher when compared with published findings [
30,
58,
59]. Notwithstanding our findings remain comparable and within the error ranges given in the literature [
60]. Multiple reasons can contribute to modifications of the resting perfusion in rodents such as the level of anesthesia, gas mixture or temperature. The comparatively high control values have also been obtained by Abdesselam et al. [
58], who attributed this to a moderate cardiac stress condition due to isoflurane-dependent vasodilation to the elevated baseline values. In previous studies in healthy mice using ASL-CMR reported myocardial perfusion ranging from 5.0 ± 0.8 to 6.9 ± 1.7 ml/g/min [
30,
59,
61] at 4.7 T and 7.0 ± 0.5 ml/g/min at 7 T [
60]. Our experiments were performed at 9.4 T and revealed an average MBF of 8.2 ± 2.6 ml/g/min, which is in accordance with the literature values. No specific validation against gold-standard techniques has been done for perfusion measured in this study. However, the employed techniques were earlier validated in rats but not mice.
Our study did not tackle the influence of single genetic mutations. Therefore, the specific effects of Mybpc3 or Myh7 point mutations need to be pursued, as does the relation between oxygen consumption and myocardial perfusion in HCM.
To tackle the diastolic dysfunction, we did not include myocardial strain measurements in our study because we would like to first emphasize the early microstructural change such as vascular deficiency in HCM. MR tagging can be useful for detecting changes in myocardial strain in the mouse heart. We anticipate including this approach into our future studies. Our preliminary results provide the basis for future investigations of the entire disease course, including the early disease phase when the pathological changes are subtle. Then by performing a comprehensive protocol of CMR measurements including parametric mapping (T1, T2 and T2*) and ASL, we can observe how CMR biomarkers change over time, and how these changes can predict later disease outcomes.
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