Time-restricted eating improves quality of life, heart rate, and mitochondrial function in patients with postural orthostatic tachycardia syndrome. An open-label pilot study

This clinical study was approved by the University of California, San Diego Institutional Review Board (IRB #802200) and registered on ClinicalTrials.gov (NCT05409651) on 16-05-2022. All methods were performed in accordance with the relevant guidelines and regulations set forth by the University of California, San Diego IRB. Informed written consent was obtained from all participants prior to their inclusion in the study.

Study design

Following recruitment by the study team, and a 2-week baseline period, eligible patients with an eating window of ≥ 12-hours were entered into a 12-week TRE intervention, during which they adhered to an 8-10-hour eating window (Fig. 1A).

Patient selection

Participants were screened and recruited from the cardiovascular and internal medicine clinics at the University of California, San Diego, and from www.clinicaltrials.gov. Patients ages 18–70 years with a self-reported dietary intake of ≥ 12-hour window, regular daytime schedule of activity (no shift workers) before study enrollment, and a formal diagnosis of POTS with chronic symptoms that have lasted for longer than six months, were enrolled. Pregnant and/or breastfeeding women and patients with other disorders, medications, or functional states that are known to predispose to orthostatic tachycardia, were excluded (Supplemental Material 1). All patients were taking medications for POTS, including beta blockers, midodrine, ivabradine, and fludrocortisone. On average, they were diagnosed with POTS 3.5 years before enrolling in the study, with 3 participants having a post-COVID POTS diagnosis.

MyCircadianClock (mCC) app for monitoring eating pattern

The smartphone app, myCircadianClock (mCC), serves as an electronic food, activity, and sleep diary^27,28 and has been used in several studies to monitor adherence to TRE^{29,30,31,32,33,34}. Participants who met preliminary eligibility entered a 2-week baseline phase of screening where they were instructed to utilize the mCC app to record all ingestion events. The study team tracked patient eating patterns to confirm ≥ 12 h eating windows and determine individual 8–10 h TRE intervention windows. The length of this TRE interval was at least 4 h shorter than their average eating window during their pre-screening, with a minimum eating window of 8 h (from a 12-hour eating window baseline) and a maximum of 10 h (from a baseline of 14-hours or greater). The interval was entered in the app, so participants could visualize their daily eating pattern and consume all meals within the designated interval. Participants continued to utilize the mCC app throughout the 12 weeks of intervention and received study-related nudges and reminders from the study team to encourage adherence to the intervention (Supplemental Table 1).

If a participant fails to log any food data for more than 1 day, the dashboard flags the participant and sends an alert to the research coordinator. The coordinators will login to the dashboard at least twice weekly to monitor food intake data, and follow up with flagged participants as necessary. If any participant faces difficulty logging data, or has questions about any of the features of the app, they can contact the study coordinator through the feedback feature of the app. Their questions are encrypted and delivered to a HIPAA compliant email server specifically set up for this study.

Intervention

Following a 2-week baseline period, participants whose initial eating windows met study criteria were invited to the clinic for their first visit. Patients were instructed to hold all POTS medications including those that impact heart rate (beta blockers, calcium channel blockers, ivabradine) for 48 h before both Visit 1 (baseline) and Visit 2 (after the 12-week intervention). During Visit 1, a 10-minute active standing test was conducted to confirm the diagnosis of POTS and establish baseline orthostatic values without medications³⁵. The stand test was conducted by first having patients rest in the supine position for 5 min prior to standing. Blood pressure and heart rate were recorded at both the 3-minute and 5-minute supine time points; however, only the 5-minute measurements—taken immediately prior to standing—were used for calculating orthostatic changes. Participants were then instructed to stand for 10 min. During the 10 min, blood pressure and heart rate were taken at the 3-, 5-, 7-, and 10-minute time points. All supine and standing blood pressure and heart rate measures were recorded as single measurements, not averaged. All orthostatic measurements were obtained using an automated blood pressure cuff and a pulsometer.Participants failing to meet POTS criteria (30-point increase in heart rate) during this test were excluded from further study procedures. After completion of the 10-minute active standing test, a fasted blood draw was performed, and participants completed general health and sleep questionnaires. Screening for depression utilized the Beck Depression Inventory-II (BDI-II), with scores ≥ 19, without stable medication or therapy, resulting in exclusion. QOL measurements were assessed using the Malmö POTS Symptom Score Survey (MAPS) and the General Health Questionnaire Short Form-36 (SF-36). All assessments took place at the University of California, San Diego.

Upon completion of Visit 1, data was reviewed, and eligible participants worked with a research coordinator to select an 8–10 h eating window tailored to fit their lifestyle, based on pre-screening eating patterns. Subsequently, participants entered a 12-week intervention period during which dietary intake was documented using the mCC app. Participants were instructed to hydrate well during the fasting period and not to make any other changes to their diet. Participants were told not to consume commercial electrolyte supplements during fasting periods, and instead to consume non-caloric beverages (carbonated water, water, decaffeinated tea etc.). Periodic phone contacts reinforced nutritional practices, supplemented by daily push notifications on their phones.

After the 12-week intervention, participants returned to the clinic for Visit 2, replicating procedures from Visit 1, including the standing test, fasted blood draw, and health questionnaires.

Outcomes

The primary outcome was a change in self-reported QOL, evaluated using two instruments: the 36-Item Short Form Health Survey questionnaire (SF-36) and the Malmö POTS Symptom Score (MAPS) survey. The SF-36 is widely used to assess health-related QOL, providing insights into patients’ physical and mental well-being³⁶.

The MAPS survey was specifically developed to assess the symptom burden experienced by POTS patients. It utilizes a semiquantitative system to evaluate the severity of 12 commonly reported symptoms: five cardiac symptoms (palpitations, dizziness, presyncope, dyspnea, and chest pain) and seven non-cardiac symptoms (gastrointestinal symptoms, insomnia, concentration difficulties, headache, myalgia, nausea, and fatigue)³⁷. Unlike the SF-36, which offers a broader evaluation of health-related QOL, the MAPS survey focuses specifically on symptoms relevant to POTS, providing detailed and condition-specific insights into patient experiences. Each of the 12 symptoms included in the survey is rated on a scale from 0 to 10, with 0 indicating no symptoms and 10 indicating the most severe symptoms. The total MAPS score ranges from 0 to 120, with higher scores reflecting greater symptom burden. The optimal cut-point value to differentiate POTS and healthy individuals is ≥ 42³⁷.

A secondary outcome of this study was the change in orthostatic heart rate (HR) when patients transitioned from a supine to an upright position.

To analyze the exploratory outcome of mitochondrial function, glycolytic and mitochondrial metabolism profiling was performed using established techniques³⁸. To analyze the metabolic effects of plasma samples, C2C12 cells, mouse myoblasts (ATCC, CRL-1772™) were used. C2C12 cells were maintained in culture with DMEM (ATCC, 30-2002™) supplemented with 10% fetal bovine serum (Gibco, A31604-01), 37 °C, 5% CO2, and controlled humidity. To perform the metabolic profiling assays, cells were resuspended with trypsin 0.25% (Gibco, 25200-056), centrifuged at 1,000RPM for 5 min, and washed with PBS1X (Gibco, 10010-023). The cells were seeded in Agilent Seahorse 96-well plates (Agilent, 103774-100) at a density of 10,000 cells per well and maintained in incubation for 24 h before the assay. All cells used for the metabolic profiling assays were at or before passage 5, meaning they were subcultured five times or fewer since initial isolation to minimize phenotypic drift and preserve metabolic integrity.

A glycolytic and mitochondrial metabolism profile was performed to analyze the plasma samples’ potential metabolic effects. C2C12 cells were treated with plasma samples (plasma 1%) prepared in Agilent Seahorse Assay Media (XF DMEM 103575-100, HEPES 5mM) supplemented with Agilent glucose (103577-100, final concentration 10mM), glutamine (103579-100, final concentration 2mM, and pyruvate (103578-100, final concentration 1mM). The cells were washed twice in supplemented Seahorse Assay Media, treated with plasma solutions (100mL final volume per well), and incubated for 1 h in a CO₂-free incubator as part of the standard Agilent Seahorse degassing procedure. After 1 h, the media was removed and replaced with fresh Agilent Seahorse Assay Media (180mL final volume per well) and a Mitochondrial Stress assay (Agilent Cell Mito Stress Test Kit, 103015-100) and a Real-time ATP Rate Assay (Agilent Real-Time ATP Rate Assay Kit, 103592-100) were performed in an Agilent Seahorse XF Pro. Both assays were run in parallel in two Seahorse XF Pro systems for data reliability. The final well concentrations of the Mitochondrial Stress assay used were oligomycin at 1.5mM, FCCP at 2.0mM, and rotenone/antimycin A at 0.5mM. For the Real-time ATP Rate Assay, the final well concentrations of the compounds used were oligomycin at 1.5mM and rotenone/antimycin A at 0.5mM. In both assays, during the Rotenone/Antimycin A injection Hoechst (ThermoScientific, 33342) was added. All data was normalized by cell count per well and presented as Oxygen Consumption Rate (OCR) (pmol O2/min/1,000 cells). ATP data is presented as ATP production rate (pmol/min/1,000 cells).

Statistical methods

Descriptive statistics and exploratory graphing techniques, including frequencies, means, standard deviations (SDs), box and whisker plots, stem and leaf diagrams, and scatter plots, were employed to assess the data for normality, skewness, and outliers. Additionally, z-scores for skewness and kurtosis were calculated for all outcomes. Final analyses for heart rate and QOL were conducted using both the original data (non-log-transformed) and nonparametric methods, yielding similar results. All reported analyses utilized the original non-log-transformed data format.

The comparability of baseline and post-intervention values was tested using analyses of variance (ANOVAs) for continuous variables and chi-square analyses for dichotomous variables. Treatment effect estimates, along with corresponding p-values, were derived from within-patient comparisons. All statistical tests were two-tailed. All available data was included in the analyses, with no patients excluded due to missing data. The SF-36 was scored according to RAND instructions³⁹. Differences were considered statistically significant if a p-value of 0.05 or less was obtained using R version 4.1.1.