Evaluation of myocardial glucose metabolism in hypertrophic cardiomyopathy using 18F-fluorodeoxyglucose positron emission tomography

Background The purposes of this study were to assess the usefulness of myocardial 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography (PET)/computed tomography (CT) for evaluating myocardial metabolic status in hypertrophic cardiomyopathy (HCM) and the therapeutic efficacy of alcohol septal ablation (ASA) in hypertrophic obstructive cardiomyopathy (HOCM). Methods Thirty HCM patients (64.4±10.5 years, 14 male, 12 hypertrophic non-obstructive cardiomyopathy [HNCM], 16 HOCM, and 2 dilated phase of HCM) underwent 18F-FDG-PET/CT. 18F-FDG uptake was semi-quantitatively evaluated using an uptake score in each 17 segment and the entire LV or regional standardized uptake value (SUV). Results 18F-FDG uptake was observed mostly in a hypertrophied myocardium in HNCM patients, whereas 18F-FDG was extensively accumulated beyond the hypertrophied myocardium in HOCM patients. There was a positive correlation between the summed uptake score of 18F-FDG and high-sensitive troponin T level in HNCM patients (r = 0.603, p = 0.049), whereas the score was positively correlated with brain natriuretic peptide level (r = 0.614, p = 0.011) in HOCM patients. In 10 patients who received ASA, the maximum SUV of the entire LV was significantly reduced from 5.6±2.6 to 3.2±2.1 (p = 0.040) after ASA. Reduction of that maximum SUV was particularly significant in the lateral region (from 5.5±2.6 to 2.9 ±2.2, p = 0.024) but not significant in the anteroseptal region (from 4.5±2.6 to 2.9±1.6, p = 0.12). Conclusion Extensive 18F-FDG uptake beyond the hypertrophied myocardium was observed in HOCM. ASA attenuates 18F-FDG uptake in a remote lateral myocardium.


Introduction
Hypertrophic cardiomyopathy (HCM) is characterized by asymmetric left ventricular (LV) hypertrophy in the absence of other cardiac or systemic diseases that may cause cardiac hypertrophy [1]. The symptom and clinical course of HCM vary, regardless of the type and severity of HCM [2]. Although many patients stay asymptomatic and experience no serious cardiac event during their life time, some develop sudden death, refractory heart failure, repetitive syncope, and/or severe angina [1]. These various clinical presentations are closely associated with narrowing LV capacity, intra-LV obstruction, severe tissue degradation, extensive myocardial fibrosis, and diastolic dysfunction [1,3]. Myocardial ischemia also plays an important role in those conditions [4,5]. The pathohistological degradation involves not only the myocytes but also small coronary arteries, which cause microcirculation disturbance in some HCM patients [6]. Demand ischemia caused by supply/demand mismatch can also occur, particularly when the myocardium contracts against pressure overload [7]. It is important to understand whether myocardial ischemia is responsible for these symptoms when treating symptomatic HCM patients.
The significance of radionuclide imaging with a metabolic tracer in HCM has been reported [8,9]. These methods are useful, particularly for evaluating myocardial ischemia in HCM, since fatty acid metabolism shifts to glucose metabolism in the setting of ischemia [10,11]. We therefore focused on regional myocardial energy metabolism in various types of HCM patients using myocardial 18 F-fluorodeoxyglucose ( 18 F-FDG) positron emission tomography (PET)/computed tomography (CT). We sought to determine the relationship between the 18 F-FDG uptake and type and severity of HCM or clinical parameters in HCM patients. Furthermore, we also assessed whether regional 18 F-FDG uptake indicating energy metabolic shift or inflammatory response, can be altered by septal reduction therapy with alcohol septal ablation (ASA) in hypertrophic obstructive cardiomyopathy (HOCM) patients suffering from heart failure or angina symptoms refractory to medical treatment.

Patient population
This study has been approved by Institutional Review Board in Nippon Medical School, and has been conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from the participants. Between April 2013 and May 2016, 36 new patients with HCM were referred to the HCM clinic from other institutions for ASA or further medical treatment. Two patients with a dilated phase of HCM (DHCM) who used to have a typical HCM apparatus and currently had dilated cardiomyopathy-like echocardiographic findings were also included. Among them, 30 patients who agreed to receive 18 F-FDG-PET/CT were included in this study. The inclusion criteria of this study were 1) the presence of a maximal LV wall thickness !15mm by transthoracic echocardiography (TTE) performed before initiation of medical treatment [12], and 2) the absence of other conditions that might explain left ventricular hypertrophy (LVH) during the clinical course. The diagnosis of HCM was based on electrocardiogram (ECG) and echocardiographic demonstrations. ECG and TTE showed a non-dilated, asymmetrical, hypertrophic LV in the absence of other cardiac or systemic diseases that could produce hypertrophy. All patients underwent either coronary angiography or coronary CT angiography and were confirmed to have no significant (>50%) coronary artery disease. Those who had significant valvular heart disease except mitral regurgitation, concomitant neoplasm, or poorly controlled diabetes mellitus (fasting blood sugar level ! 120 mg/dL) were not included in this study. HOCM was defined when intra-LV pressure gradient was > 30 mmHg at rest as seen on TTE [13]. A complete history was obtained and clinical examination was performed, along with assessments of New York Heart Association (NYHA) functional class, present drug therapy, and device therapy. We measured serum high-sensitive troponin T (HSTnT) and brain natriuretic peptide (BNP) levels with an AIA-2000ST analyzer (TOSOH, Tokyo, Japan) in accordance with the manufacturer's instructions. The imaging protocol consisted of TTE, 18 F-FDG-PET/CT, and cardiac magnetic resonance (CMR) imaging. They received those examinations within 3 weeks. The patients who received ASA underwent repeat imaging protocol approximately 6 months after ASA. Medication was kept constant during the study.

TTE study
We performed two-dimensional (2D), M-mode, and Doppler echocardiographic studies with PHILIPS IE 33 (Philips Healthcare, Best, The Netherlands) or GE Vivid E 9 ultrasound systems. We measured wall thickness (intraventricular septal thickness [IVST]; posterior wall thickness [PWT]), LV diastolic function (E/A [peak early transmitral filling velocity/peak late transmitral filling velocity], and E/e' [peak early transmitral filling velocity/peak early diastolic mitral annulus velocity] of the septal side or lateral side on tissue Doppler imaging) and maximum pressure gradient in the LV at rest. 18 F-FDG-PET/CT imaging 18 F-FDG-PET/CT imaging proceeded as described [14]. Briefly, patients underwent dietary preparation with 24 hours of carbohydrate restriction (glucose < 10g) to suppress the physiological uptake of 18 F-FDG. 60 min after intravenous administration of a 4MBq/kg dose of 18 F-FDG, we acquired PET and CT images using a PET/CT scanner with 16-slice CT (GEM-INI TF 16, Philips Healthcare, Best, The Netherlands). We checked the blood sugar level before the test to confirm that it is under 120mg/dl. Diabetic patients who received oral hypoglycemic agents or insulin were not included in the present study. 18

F-FDG-PET/CT imaging analysis
We analyzed all PET/CT data as described [14] with a computer workstation. For the visual analysis of PET, we defined the 18 F-FDG uptake as positive if it was greater than that for a physiologically normal liver. We used the AHA 17-segment model of the LV myocardium to visually localize 18 F-FDG accumulated myocardium [15] using a semi-quantitative uptake score. Scores were defined as the following; 0: no uptake, 1: slight uptake, 2: mild uptake 3: moderate uptake, and 4: dense uptake. Extent score was calculated as the number of segments with 18 F-FDG uptake. To compare 18 F-FDG uptake between pre-and post-ASA, The difference score was calculated by subtracting the post-value from the pre-value for each segment. We also performed another semi-quantitative analysis. 2D regions of interest (ROI) were drawn on the transaxial slices of the PET images to measure the standardized uptake value (SUV) of the entire or regional (anteroseptal, inferior, or lateral) LV myocardium; SUV = (peak kBq/mL in ROI)/(injected activity/g body weight). Three investigators (two radiologists and a cardiologist) separately interpreted 18 F-FDG-PET/CT findings. Consensus was reached in case of discrepancy.

CMR imaging
The electrocardiography-gated CMR protocol proceeded with breath-holding as described [16] using an Achieva 1.5 and 3.0 T (Philips Healthcare, Best, The Netherlands). Patients who had a pacemaker, claustrophobia, or renal dysfunction were excluded. Twenty-four patients received the CMR imaging with gadolinium enhancement and one received that without enhancement.

CMR imaging analysis
We measured the LV myocardial mass index (g/cm2), LV ejection fraction using Simpson's method, mitral regurgitation (MR) jet, left ventricular outflow tract (LVOT) jet, and left atrial diameter on cine steady state free precession using a workstation (View Forum, Philips, Best, The Netherlands). Additionally, experienced radiologists evaluated imaging of late gadolinium enhancement (LGE) to clarify its presence. We applied the American Heart Association (AHA) 17-segments method to directly compare imaging and 18 F-FDG-PET/CT for the visual evaluation of the extent of LGE.

ASA procedure
We performed ASA in 10 hypertrophic obstructive cardiomyopathy (HOCM) patients out of 20 candidates. Indication for HOCM was determined according to the following criteria: 1) symptoms were life limiting after optimization of medication, 2) resting or provoked gradient > 50 mmHg that was confirmed by at least one method during simultaneous pressure recordings, and 3) appropriate target branch(es) leading to the septal myocardium were responsible for intra-LV obstruction. ASA was performed as previously described [17].

Statistical analysis
Data were expressed as a mean ± the standard deviation. We performed all statistical analyses with IBM SPSS statistics version 21 and compared continuous variables between HNCM and LVOT obstruction (LVOTO) or segments with and without LGE using a Student's t-test or the Mann-Whitney's U test when appropriate. We compared categorical variables using the chisquare test and assessed the relationships between the summed uptake score and clinical parameters using Pearson's correlation. Pre-and post-ASA values were compared using a paired t-test or the Wilcoxon signed-rank test. Statistical significance was determined when a P-value was less than 0.05.

F-FDG and CMR analyses
The 18 F-FDG uptake in each type of HCM is shown in Fig 1A. Patients with HNCM had 18 F-FDG uptake mostly in the basal and mid-anteroseptal segments, corresponding to a hypertrophied myocardium. On the other hand, HOCM patients with LVOTO had strong 18 F-FDG uptake not only in the basal septal segments but also in the mid-lateral segments. HOCM patients with isolated MVO had 18 F-FDG uptake mainly in the segments close to the apical or mid-free wall. The detailed degree of 18 F-FDG uptake in each segment is shown in S2  The frequency of LGE in each 17 segment in the HNCM and LVOTO types of HOCM is shown in Fig 1B. In HNCM patients, LGE was frequently observed in the anteroseptal segments. On the other hand, LGE was randomly observed in HOCM with LVOTO. The segments with LGE had a higher 18 F-FDG uptake score compared to those without (1.4 ± 1.3 vs. 0.7 ± 1.2, P = 0.02) in HNCM patients, whereas the difference was not significant (2.0 ± 1.5 vs. 1.7 ± 1.5, P = 0.29) in HOCM patents. The frequency of LGE on each segment is shown in S3 Table. The relationships between the summed uptake score of 18 F-FDG and HSTnT and BNP are shown in Fig 2. In HNCM patients, there was a positive correlation between the uptake score and HSTnT level (r = 0.603, P = 0.049) but not between the uptake score and BNP level (r = 0.419, P = 0.154). On the other hand, in HOCM patients, there as a positive correlation between the uptake score and BNP level (r = 0.614, P = 0.011) but not between the uptake score and HSTnT level (r = 0.256, P = 0.357).

ASA procedure
Ten HOCM cases received ASA. A representative case of a 72-year-old woman with HOCM who received ASA is described in Fig 3. As well as the significant reduction of the intra-LV pressure gradient, 18 F-FDG uptake in the lateral LV wall was attenuated (Fig 3).  Table 2 shows the details of the procedures and the changes of clinical parameters after ASA. ASA reduced the intra-LV pressure gradient from 60.2±39.9 to 21.7±22.7 mmHg   P = 0.022). The semiquantitative analysis of 18 F-FDG uptake in the LV myocardium, expressed as maximum SUV of the entire LV, was significantly reduced (from 5.6±2.6 to 3.2±2.1, P = 0.040). The reduction was particularly significant in the lateral region (from 5.5±2.6 to 2.9±2.2, P = 0.024), not in the anteroseptal or inferior regions (from 4.5±2.6 to 2.9±1.6, P = 0.12, or from 4.4±2.9 to 2.5±1.5, P = 0.085, respectively). The semi-quantitative scoring method also revealed the significant reduction of 18 F-FDG uptake in the mid-anterolateral segment after ASA (Fig 4). The detailed 18 F-FDG uptake score before and after ASA and difference of uptake score at each segment are shown in S4

Discussion
In the present study, we addressed regional myocardial 18 F-FDG uptake which indicate energy metabolic shift or inflammatory response using 18 F-FDG-PET/CT in each type of HCM. Uptake of 18 F-FDG was limited in a hypertrophied myocardium in HNCM whereas extensive 18 F-FDG uptake beyond the hypertrophied myocardium was observed in HOCM. The extent and degree of 18 F-FDG uptake was closely related with the HSTnT level, as well as the parameters of diastolic LV function and BNP. Reduction of intra-LV obstruction using ASA can affect myocardial metabolic shift in the lateral myocardium of HOCM.

Methodological considerations
In order to more precisely evaluate myocardial metabolism, we contrived a new carbohydrate restriction diet protocol in addition to conventional 18 F-FDG-PET. A major obstacle in diagnosing myocardial metabolism by using 18 F-FDG-PET is the high physiological accumulation of 18 F-FDG in the myocardium, which interferes with the recognition of abnormal 18 F-FDG uptake [14]. Suppression of this unfavorable uptake is important to identify myocardium metabolism status. It is reported that carbohydrate restriction over 24 hours significantly suppresses physiological accumulation of 18 F-FDG in the myocardium [14]. Therefore, all patients underwent over 24 hours carbohydrate restriction (glucose, <10g) before 18 F-FDG-PET/CT study.
In addition to the semi-quantitative scoring method, we also measure SUV which is considered to be more objective evaluation. The summed uptake score was positively correlated with mean SUV of entire LV (R = 0.673, P<0.001, S3 Fig). This indicates the appropriateness of our uptake scoring evaluation and sufficient restriction of physiological 18 F-FDG uptake to other organ.

The significance of 18 F-FDG uptake in HCM
The energy source of a normal myocardium is mainly fatty acids (over 90%) [10]. In some pathologic conditions such as ischemia or inflammation, the energy source of the myocardium shifts to glucose metabolism from fatty acids [8]. 18 F-FDG is an analogue of glucose, and 18 F-FDG-PET is used to visualize glucose metabolism in vivo [18]. In HCM, we postulate that the following four mechanisms are involved, in which glucose metabolism is necessary: 1) increased energy demand due to myocardial hypertrophy [19], 2) inflammatory response caused by inflammatory cell infiltration [20], 3) myocardial ischemia due to microangiopathy [21,22], and 4) demand myocardial ischemia due to supply/demand mismatch of blood flow [23]. We speculate that the Change of 18F-FDG uptake score after ASA. Mean uptake score of 18 F-FDG before and after ASA and difference score at each LV 17-segment. The mean uptake score was calculated with a semi-quantitative scoring method for each segment. Light gray dot indicates the individual with slight uptake, middle gray dot mild uptake, dark gray dot moderate uptake, and black dot dense uptake. The difference score was calculated by subtracting the post-value from the pre-value for each segment. The values were expressed by the density of gray color. * P<0.05 between pre-and post-ASA uptake score.
https://doi.org/10.1371/journal.pone.0188479.g004 accumulation of 18 F-FDG that we observed at non-hypertrophied lesions in LVOTO patients mainly involves the demand myocardial ischemia, because blood supply decreases due to increased extravascular compressive forces and oxygen demand increases in order to contract against wall stress under the increased pressure overload condition.
The usefulness of 18 F-FDG-PET in HCM has been reported in previous studies [24][25][26]. Uehara reported that 18 F-FDG uptake is increased not only in hypertrophied but also in the non-hypertrophied myocardium in HCM with asymmetrical septal hypertrophy or a dilated phase of HCM, whereas it was limited in a hypertrophied myocardium in HNCM [26]. The present study, in agreement with Uehara's report, identified extensive pathophysiological metabolism beyond the hypertrophied myocardium in HOCM using 18 F-FDG-PET/CT although the PET protocol was different from that in the previous study. As novel findings, we demonstrated that abnormal metabolism at a non-hypertrophied myocardium can be reversed by the attenuation of intra-LV obstruction.
We found that 18 F-FDG uptake score was positively correlated with HSTnT level in HNCM patients. Because HSTnT is generally considered as the cardiac marker of ongoing myocardial injury, 18 F-FDG uptake observed in hypertrophic myocardium may be reflecting inflammatory response or ischemia caused by microcirculation disturbance. On the other hand, we found the positive correlation between 18 F-FDG uptake score and BNP level in HOCM patients, indicating that 8 F-FDG uptake was more extensively observed in failing heart in this condition. Flow disturbance at LVOT due to obstruction causes metabolic abnormality in not only hypertrophied but also remote myocardium possibly due to demand ischemia, as well as increased secretion of BNP.
LGE and 18 F-FDG uptake Since LGE seen on CMR is known to reflect myocardial fibrosis, the existence of LGE in HCM indicates an advanced stage of this disease [27]. The pathohistological changes of HCM are characterized by myocyte disarray, enlarged cardiomyocytes, and deposition of interstitial fibrosis [27]. To develop dense myocardial fibrosis, detected as LGE on CMR, sustained pathological stress is considered to be necessary. In this process, microvascular ischemia or inflammatory response plays an important role. A previous study has reported a significant relationship between microvascular ischemia and myocardial fibrosis [28]. In the present study, by comparing CMR and 18 F-FDG-PET/CT, we clarified the spatial relationship between LGE and 18 F-FDG uptake in each type of HCM. In patients with HNCM, we found LGE as well as 18 F-FDG uptake mostly in hypertrophied segments. This may indicate that the myocardium requiring glucose metabolism also has fibrotic changes. Therefore, it is possible that the 18 F-FDG uptake may indicate the risk of the development of fibrosis in HNCM patients. On the other hand, in patients with LVOTO, 18 F-FDG was widely distributed in both hypertrophied septal and remote lateral segments whereas LGE was rarely observed in those segments. The possible explanation of less frequent LGE segments compared with 18 F-FDG uptake segments in LVOTO is that LGE reflects progressed myocardial cell damage, whereas 18 F-FDG uptake reflects metabolic disorders which emerge from the early stage of myocardial cell damage. Since the pathological condition responsible for their symptom in patients with LVOTO who were referred for ASA was mainly due to mechanical intra LV obstruction, LVOTO patients enrolled in the present study might be still on the way to the development of myocardial fibrosis.
Serial assessment of 18 F-FDG-PET/CT before and after ASA ASA is an optional treatment for symptomatic HOCM patients who are refractory to medical therapy [29]. Increasing evidence has supported the beneficial effect of ASA in improving symptoms associated with HOCM [30,31]. Timmer et al. have reported that ASA has favorable effects on myocardial metabolism. 15 O-H 2 O PET and 11 C-acetate PET showed the changes of microvascular function and myocardial metabolism by relief of LVOTO [32]. In the present study, we found altered metabolism or myocardial inflammation detected as 18 F-FDG uptake which observed in a non-hypertrophied myocardium can be reversed by ASA. We attributed this improvement to attenuation of demand myocardial ischemia, which is associated with wall stress from pressure overload. A previous study has reported the beneficial effect of ASA on the systolic movement of lateral (remote) wall using CMR. In the present study, we demonstrated the disappearance of 18 F-FDG uptake in the remote myocardium as well as diastolic function in the lateral side. The metabolic improvement may explain, at least in part, the mechanism of the improvement of systolic function observed in the previous study [33].
Currently, indication for ASA is fundamentally determined by the degree of pressure gradient and assessment of life-limiting symptoms, assessed by NYHA grade [30,31]. However, a discrepancy between these two factors is frequently observed [17]. We therefore postulate that the severity and extent of 18 F-FDG uptake may be helpful for determining the indication for ASA, although further study is necessary to confirm the generalizability of our findings. 18 F-FDG-PET/CT can be also useful to evaluate the therapeutic efficacy of ASA.

Limitations
The number of patients was small due to the limited capacity of PET imaging in our institution. Particularly, we only included two isolated MVO patients and two dilated HCM patients in this study. Therefore, we did not include their data in our analysis and avoided commenting on isolated MVO or dilated HCM, although data are shown in the figures and tables as references. Secondly, the study was basically cross-sectional and no long-term follow-up data were addressed. Thus, prognostic implication of 18 F-FDG-PET/CT could not be assessed in this study. Finally, although we observed significant 18 F-FDG uptake in hypertrophied myocardium in HNCM patients, it remains unclear whether the uptake is indeed pathophysiological or mostly due to the greater thickness of the hypertrophied septum. The increase of 18 F-FDG uptake might be related to a relative, partial-volume dependent overestimation of the true 18 F-FDG tissue concentration.

Conclusion
We addressed regional myocardial energy metabolic shift using 18 F-FDG-PET/CT in HNCM and HOCM patients. Uptake of 18 F-FDG is limited in the hypertrophied myocardium in HNCM whereas extensive 18 F-FDG uptake beyond the hypertrophied myocardium was observed in HOCM. The fact that ASA can contribute to the improvement of myocardial metabolism or inflammation in the remote myocardium suggests the novel, beneficial effect of ASA besides the symptomatic improvement.
Supporting information S1  Table. The uptake score of 8