In a prospective multicenter study conducted at University of Campania “Luigi Vanvitelli”, Naples, at “S. Maria della Pietà Hospital”, Naples, at “Vecchio Pellegrini Hospital”, Naples, at “Catholic University of the Sacred Heart”, Rome, Italy, at Catholic University of Sacred Heart, Campobasso, and at Gemelli Molise, Campobasso, Italy, we investigated from June 2018 to March 2021 a population of 4794 consecutive patients who had reported at least two syncopal episodes of unknown origin during the previous 6 months, and who had a recurrence of syncope during Head-Up Tilt Test (HUT). We defined the VVS as a transient loss of consciousness whose cause was not determined by the following series of examinations performed in affected patients: clinical history, clinical examination (auscultation, carotid sinus massage, blood pressure measurement in supine, and upright position), electrocardiogram (ECG), chest X-ray, echocardiography, 24 h Holter ambulatory monitoring, late potentials, and a complete neurological examination (head imaging and electroencephalograms), according to last international guidelines (10). However, all enrolled patients performed a HUT. These patients were in stable sinus rate before performing the HUT, and they performed a 24 hours ECG Holter to assess sinus rhythm, HR, HRV, and the MIBG myocardial scintigraphy before receiving a HUT. Thus, we included only T2DM patients (n 607) from the study populations, according to diagnostic criteria for T2DM (10). Figure 1. The study complies with the Declaration of Helsinki, and the locally appointed Ethics Committee approved the research protocol (n 06062.18), and informed consent was obtained from all the subjects. The study population respected the following inclusion/exclusion criteria.
Inclusion criteria: eligible adults’ patients with T2DM (≥18 years of age); previous VVS event; indication to receive a HUT; indication to practice cardiac MIBG scintigraphy; patients without previous cardiovascular disease; patients with an estimated glomerular filtration rate (eGFR) of at least 30 ml per minute per 1.73 m2 of body-surface area.
Exclusion criteria: patients with diagnosis of type 1 diabetes, neuropathy, heart failure and coronary heart disease or depression of left ventricle ejection fraction (LVEF < 55%); patients with severe anemia, thyroid dysfunction, concomitant treatment with anticholinergic agents, adrenergic antagonists, and vaso-constrictive agents that can affect the results of autonomic function test; patients with uncontrolled blood pressure (blood pressure > 140/90 mmHg on two occasions 2 weeks apart).
The enrolled patients were regularly followed by clinical visits 10 days, and at 3th, 6th and 12th months after clinical discharge by the treating physician. These patients had a diagnosis of VVS in accordance to the HUT result (1). All the patients gave their written informed to participate in the study. Clinical evaluations included physical examination, vital signs, and review of adverse events. We performed fasting blood (at least 12 hours from last meal) for glycemia, lipid profile (total cholesterol (TC), triglycerides, HDL-C, and LDL-C) at every visit. Syncope recurrence and other clinical events were collected during patients’ interviews, visits, and hospital discharge schedules. The diagnosis of T2DM was made according to international recommendations, by plasma glucose values as fasting plasma glucose level ≥ 7.0 mmol/L (126 mg/dL), plasma glucose ≥ 11.1 mmol/L (200 mg/dL) either while fasting or not fasting, glycated hemoglobin ≥ 48 mmol/mol, and by the known clinical history of diabetes and by the current use of anti-diabetic medications (11). In the T2DM patients we defined SGLT2-I users as those receiving a SGLT2-I therapy for at least 6 months without discontinuation. We defined the rest of the T2DM patients as Non-SGLT2-I users. All the T2DM patients enrolled in the study had a glycated hemoglobin level of at least 7.0% and no more than 9.0%. The SGLT2-I users received either 10 mg or 25 mg of empagliflozin once daily, and/or canaglifozin 100 mg daily (mean duration of SGLT2-I therapy 16±4.8 months).
Cardiovascular autonomic neuropathy evaluation
We evaluated the autonomic function of study population by classical Ewing cardiovascular autonomic function tests, heart and pulse rate variability (3). The test was performed in the morning (08:00–10:30) in a room with quiet ambiance and temperature of 19–22 °C, in fasting condition from midnight and refraining from smoking and caffeine-containing beverages for almost 12 hours before the evaluations. Two experienced physicians blinded to study protocol evaluated the cardiovascular autonomic reflex function tests as parasympathetic and sympathetic tests (3, 4, 5). The parasympathetic tests evaluated the HR response during deep breathing, the Valsalva maneuver, and quick standing (3, 4, 5). The sympathetic tests evaluated the blood pressure response during the sustained handgrip test and quick standing (3, 4, 5). We evaluated the deep breathing test to determine the maximum and minimum R–R intervals during each breathing cycle during six deep breaths in 1 min (3). However, we expressed the R–R intervals during inspiration and expiration as the R–R inspiration/R–R expiration ratio (3). Again, we evaluated the Valsalva maneuver as a forced expiration in a manometer against 40 mmHg for 15 s, and the Valsalva ratio as division of longest R–R interval by shortest R–R interval (3). Moreover, during the quick standing test, we measured the HR response after standing from the R–R intervals calculation at 15 and 30 beats after standing and reported as the ratio of the longest vs. the shortest R–R interval (3). Thus, we calculated the sympathetic component of the standing test by the values of the systolic blood pressure response 2 min after standing (3). Finally, we evaluated the diastolic blood pressure response during the sustained handgrip by a dynamometer to establish a maximum developed force, followed by a handgrip squeeze of 30% of the maximum force for 5 min (3). Furthermore, we defined Cardiovascular autonomic neuropathy (CAN) as the composite CAN index including the HR ratio <1.30 plus Valsalva ratio <1.5 or a decrease of >10 mm Hg in diastolic blood pressure (DBP) upon standing (CAN+) (3).
Head-Up Tilt Test (HUT)
In the overall study population, and selectively in SGLT2-I-users vs. Non-SGLT2-I users, we performed the HUT in the morning in a quiet room with lights slightly dimmed after overnight fasting (1-3). Using a motorized tilt table with foot support, we had a first 5-minute supine control phase, and then we moved the patients to the 60°upright position for a maximum duration of 45minutes or until syncope developed (1-3). However, at 20 minutes, we administered to the patients 400 mg of nitroglycerin spray sublingually (1-3). To date, at the time of syncope, we immediately tilted back the patients to the horizontal position (1-3). We defined the syncope as an abrupt, transient loss of consciousness and a loss of postural tone associated with bradycardia, hypotension, or both (1-3). Therefore, we classified the VVS into three groups according to HUT response during the onset of syncope: i) vasodepressor VVS by evidence of a decrease in systolic blood pressure to <60 mmHg without decrease in HR during symptoms; ii) cardioinhibitory by evidence of an abrupt decrease in HR by ≥20% without any antecedent decrease in systolic blood pressure; iii) mixed response by evidence of a concurrent decrease in systolic blood pressure to <60 mmHg and a decrease in HR by ≥20% compared with averages 4 min before the onset of symptoms (1-3).
At hospital admission for the VVS clinical event, and before performing the HUT, the authors practiced the 123I-MIBG myocardial scintigraphy. We used a standardized protocol at the time of hospitalization and at 1 year of follow-up (6). The 123I-MIBG myocardial scintigraphy was performed in VVS patients to assess the cardiac sympathetic nerve activity (7). Moreover, we used the 123I-MIBG, a norepinephrine analogous, to calculate the late heart-to-mediastinum ratio (H/Mlate) and washout rate (WR). Thus, we evaluated the H/Mlate as index of global neuronal function due to norepinephrine uptake and the WR as index of sympathetic tone. We withheld the drugs interfering with 123I-MIBG uptake, and thyroid uptake of unbound 123I was blocked with 500 mg of potassium perchlorate given orally 30 min before 123I-MIBG injection (6, 7). We injected intravenously at rest, the dose ranging from 148 MBq to 370 MBq of 123I-MIBG. Then, we acquired both planar and SPECT images at 15 min after injection (early) and 4 h after injection (delayed, by the use of a dual-head gamma camera - ECAM Siemens, Erlangen, Germany) equipped with a low-energy, high-resolution collimator. A 20% window was usually centered over the 159-keV photopeak of 123I for imaging. Anterior planar images of the chest for global assessment of cardiac innervation were acquired using a 256x256 matrix. Thereafter, we acquired the SPECT images for the regional evaluation, using a 64x64 matrix over 180˚, from the right anterior oblique position to the left posterior diagonal position. Thus, we performed the quantitative evaluations with a standard protocol from this imaging (6). Moreover, we evaluated at baseline and at 1 year of follow-up the H/Mlate and WR in the study cohorts.
The formula to calculate WR was: WR BKG corrected =
The BKG is background; H is heart mean counts per pixel; M is mediastinum mean counts per pixel; "e" is early; "l" is late; and 1.21 is the correction factor for 123I decay at 3 h and 45 min (6, 7).
The study endpoints were: 1) the evaluation of the HRV parameters in SGLT2-I-users vs. Non-SGLT2-I users at baseline and at 1 year of follow-up; 2) the evaluation of cardiac 123I-MIBG indexes in SGLT2-I-users vs. Non-SGLT2-I users at baseline and at 1 year of follow-up; 3) the rate of VVS recurrence after HUT diagnosis in SGLT2-I-users vs. Non-SGLT2-I users at 1 year of follow-up.
We made the descriptive statistical analysis by the correct measurements on the collected data. However, before the HUT we divided the VVS patients in SGLT2-I-users vs. Non-SGLT2-I users. Then, at follow-up after HUT, and during follow-up visits and controls, we divided patients with VVS recurrence vs. patients without VVS recurrence. Thus, as appropriate, we tested the study variables by parametric and non-parametric tests. The normality of the distribution was tested with the Kolmogorov-Smirnov test. We compared the categorical variables by chi-square or Fisher exact test where appropriate. The statistical significance was set at p <0.05 (two-sided tests), and for multiple testing, we used a statistical significance of p <0.01. The Kaplan curves were made at 1 year of follow-up to show the cumulative risk to have VVS recurrence (all causes, mixed, cardio-inhibitory, and vasodepressor VVS recurrence). The Log Rank test evaluated the comparison between SGLT2-I users and Non-SGLT2-I users regarding the cumulative risk of VVS recurrence (all causes, mixed, cardio-inhibitory, and vasodepressor) VVS recurrence) at 1 year of follow-up. We made the multivariate Cox multivariate regression analysis to predict all causes of VVS recurrence at 1 year of follow-up in the study population. Among all risk factors and all clinical and angiographic parameters evaluated (age, smoking, resting HR, systolic and diastolic blood pressure, etc.), only the variables presenting a p ≤0.25 at univariate analysis were included in the model, and we used Hazard Ratios (HR) with 95% confidence intervals was calculated. We performed the statistical analysis using the SPSS software package for Windows 17.0 (SPSS Inc., Chicago, Illinois).