Cardiothoracic surgeries are complex procedures during which the patient cardiovascular physiology is constantly changing due to various factors. Physiological changes begin with the induction of anesthesia, whose effects remain active into the postoperative period. Depending on the surgery, patients may require the use of cardiopulmonary bypass and cardioplegia, both of which affect postoperative physiology such as cardiac index and vascular resistance. Complications may arise due to adverse reactions to the surgery, causing hemodynamic instability. In response, fluid resuscitation and/or vasoactive agents with varying effects may be used in the intraoperative or postoperative periods to improve patient hemodynamics. These factors have important implications for lumped-parameter computational models which aim to assist surgical planning and medical device evaluation. Patient-specific models are typically tuned based on patient clinical data which may be asynchronously acquired through invasive techniques such as catheterization, during which the patient may be under the effects of drugs such as anesthesia. Due to the limited clinical data available and the inability to foresee short-term physiological regulation, models often retain preoperative parameters for postoperative predictions; however, without accounting for the physiologic changes that may occur during surgical procedures, the accuracy of these predictive models remains limited. Understanding and incorporating the effects of these factors in cardiovascular models will improve the model fidelity and predictive capabilities. There are several factors that can cause significant changes in the cardiovascular physiology during cardiothoracic surgeries and patient management. For instance, prior to surgery, patients undergo the induction of anesthesia and remain under its influence in the intraoperative and early postoperative periods. Surgeries may also require cardiopulmonary bypass and cardioplegia to isolate and depress the heart. Complications may arise due to unforeseen circumstances, and patients may be treated with fluids and vasoactive agents to improve their hemodynamic state.The implications of these factors on the fidelity of computational cardiovascular models are far-reaching. Current cardiovascular models, in particular lumped-parameter models, seek to simulate patient physiology to aid in clinical decision making; these models are constructed using primarily pre-operative clinical data and do not consider the events which may affect patient physiology during surgery , 2. TherSeveral anesthetic combinations are used in surgery, each with significant and unique effects on the cardiovascular system. Currently, there is no universally accepted combination of anesthetic agents for cardiac surgeries. The choice of drugs is determined by the pathophysiologic state of the patient and the knowledge of the anesthesiologist. For initial hypnosis, inhalational agents or intravenous agents are typically used, while narcotics and muscle relaxants are used during periods of high stimulation such as intubation. The management of anesthesia is important in maintaining stable hemodynamics and an adequate blood supply to body tissues.Volatile inhalational agents typically cause dose-dependent myocardial depression. Nitrous oxide is an inhalational anesthetic sometimes used in the pre-bypass period due to its rapid onset and elimination. It can be used to increase pulmonary pressures, and, to a lesser extent, depress contractility  . HoweverOther types of anesthetic agents include intravenous agents such as Barbiturates, Ketamine, and Propofol. Barbiturates are occasionally used in cardiac surgery, acting to depress the metabolic demands of the brain and providing protection against ischemia. They are usually safe only in hemodynamically stable patients, making their use rather limited. Their use is associated with a decrease in blood pressure and an increase in heart rate (HR) due to vasodilation and decreased contractility . KetaminOpioids and Muscle relaxants are other types of anesthetic drugs, which may be administered to control pain and muscle movement, respectively. Muscle relaxants such as succinylcholine may result in abnormal heart beats, however both opioids and muscle relaxants cause very minimal changes to CO and blood pressure when compared to volatile or intravenous agents .Although the current line of anesthetic drugs has been used for some time, there is an active search for agents with fewer adverse effects. One candidate is the inhalational use of Xenon. Other inert gases such as Argon have been found to have anesthetic qualities, but Xenon possesses the most potent anesthetic capabilities of the inert gasses. In an animal trial using Xenon on guinea pigs, Stowe et al. demonstrated that it had no mechanical, electrical, or metabolic effects on isolated guinea pig hearts . In a stSome operations such as valve replacement, congenital heart defect surgery, and coronary artery bypass graft (CABG) surgery require the use of a CPB machine to take over the function of the patient's heart and lungs, enabling the stopping of the heart. The heart is most often stopped by a cardioplegic (CP) solution decreasing the resting potential of the heart. This stops the heart, reduces its metabolism and oxygen demand, and allows the cardiac surgeon to work on a bloodless and motionless heart during the procedure.The CPB machine operation and the CP solution directly impact postoperative states. Davidson et al. found that increased doses of CP were associated with greater degrees of increased left anterior descending coronary artery flow after congenital cardiac surgery . A meta-Typically, there is an increased oxygen demand as patients are weaned off CPB and anesthesia. There are similar increases in oxygen metabolism in both CABG and off-pump CABG patients, likely due to inflammatory responses to surgical trauma . The sur2) and tissue hypoxia. It is characterized by a CI <2.0 L/min/m2 and a systolic blood pressure <90 mmhg (A common complication following cardiac surgery is low cardiac output syndrome (LCOS). This is typically due to diminished heart pump function, leading to reduced oxygen delivery . LV diastolic dysfunction is the inability of the left ventricle to accept an adequate volume of blood, despite a normal preload. This may be caused by severe tachycardia, decreased myocardial compliance, or impaired ventricular relaxation. Diastolic dysfunction is a common occurrence in cardiac patients in the postoperative period after CABG due to increased chamber stiffness (LV systolic dysfunction is commonly a result of myocyte necrosis due to impaired coronary circulation and ischemia/reperfusion injury. The ventricle's impaired response to preload (reduced ejection fraction) causes a decrease in CO and DOtiffness . Right vtiffness .Another common postoperative condition occurring in 5–15% of patients following cardiac surgery involving CPB is vasoplegia, characterized by low systemic vascular resistance and high or normal CO . A lack 2 due to inadequate left ventricular preload. However, there is little change in VO2 due to a redistribution of blood flow to tissues with greater metabolic needs caused by the lowering of regional vascular resistance. As the volume of blood loss increases, blood pressure continues to drop, and heart rate increases in an compensatory effort to increase CO  symptoms with suspected or proven infection. SIRS is a stress response which occurs as a result of injury such as that caused by surgical trauma or cardiopulmonary bypass, whose symptoms include abnormalities in body temperature, heart rate, respiratory rate, and white cell count . The iniFluid resuscitation is often the first step in treating patients with low levels of tissue perfusion. The primary reason for treating patients with intravenous fluids is to increase stroke volume through an increase in preload. Following the Frank-Starling principle, increasing LV preload will increase LV stroke volume until an optimal preload is reached, after which the stroke volume will remain relatively constant. However, just 50% of hemodynamically unstable patients respond positively to fluids (volume responsive) . For thoIf fluid resuscitation fails to bring the patient's postoperative hemodynamics into a stable range, vasoactive agents are often employed. The most common vasoactive agents are catecholamines. These agents induce sympathetic responses of the autonomic nervous system, triggering the “fight or flight” response. Catecholamines induce cardiovascular effects primarily through α1, β1, β2, and dopaminergic receptors. The activation of α1 adrenergic receptors results in vascular smooth muscle contraction and subsequent increase in SVR. β1 adrenergic receptor activation causes enhanced myocardial contractility through calcium ion channel activation and increased HR. β2 adrenergic stimulation results in an increased calcium ion uptake and vasodilation in skeletal muscle vessels. Lastly, dopaminergic receptor stimulation causes renal and mesenteric vasodilation .Epinephrine is a naturally occurring catecholamine and adrenergic agonist of both α and β receptors. It is a potent vasoconstrictor with inotropic and chronotropic effects. High and prolonged doses can cause cardiac toxicity by damaging arterial walls . AlthougAnother catecholamine is norepinephrine, which is primarily an α adrenergic agent, with a modest effect on β adrenergic receptors. It can generate significant vasoconstriction, exhibiting inotropic and chronotropic effects to a much lesser degree. Norepinephrine is generally used for treatment of hypotensive and normovolemic patients experiencing shock . ProlongDopamine is a naturally occurring catecholamine that acts on dopaminergic and adrenergic receptors to cause a range of hemodynamic effects. At low doses, β receptor responses dominate, resulting in increased contractility and HR with a modest increase in SVR. However, with increasing doses, α1 adrenergic vasoconstriction effects dominate . Side efDobutamine is a synthetic catecholamine that is primarily β1 adrenergic. It enhances ventricular contraction with only slight vasodilation effects. It is a potent inotrope that causes mild increases in chronotropic effects, and consequently causes a dose dependent increase in CO. However, it increases myocardial oxygen demand, limiting its clinical use due to risk of ischemia, and may cause ventricular arrhythmias , 32.2 or HR, and is commonly used as a secondary agent when hemodynamic improvement cannot be accomplished with catecholamines (Another class of vasoactive agents are phosphodiesterase (PDE) inhibitors. PDE inhibitors increase intracellular cyclic adenosine monophosphate which produces inotropic effects as well as systemic and pulmonary vasodilatory effects. Milrinone is the most commonly used PDE inhibitor. Unlike catecholamines, it does not cause increases in myocardial VOolamines .Vasopressin, also known as “antidiuretic hormone,” is stored in the pituitary gland and released after hypotension or pain stimulus, and leads to vasoconstriction, consequently increasing SVR . It is n2. It also causes the opening of potassium channels on vascular smooth muscle, resulting in vasodilation, sometimes leading to undesired hypotension  and a rapid decrease in calcium during diastole (increasing lusitropy). These combined effects produce increased systolic and diastolic performance without increasing myocardial oxygen consumption, offering a major advantage over existing inotropes .Another group of vasoactive agents with promising capabilities are cardiac myosin activators. In a trial of patients with stable heart failure and left systolic function, the agent omecamtiv mecarbil demonstrated an ability to increase the duration of left ventricular systole, increasing the ejection fraction . This inAssessing the efficacy of cardiothoracic surgeries in practice is difficult, relying on past surgeries for guidance, but advances in the field of computational cardiovascular modeling have opened the door to the evaluation of surgeries and medical devices in a virtual setting. One approach to simulate cardiovascular physiology and hemodynamics is by implementing a zero-dimensional lumped-parameter network (LPN). The LPN models the circulatory system as a network of electrical circuit elements, which accounts for the effects of friction, inertia, and vessel compliance with electrical analogs of resistance, inductance, and capacitance, respectively. This gives rise to a system of ordinary differential equations whose solution provides values of hemodynamic variables such as blood pressure and flow rate in different locations of the circulation.in vivo studies are impractical (LPN has been used to predict hemodynamic responses under varying circumstances such as surgical intervention, disease conditions, and ventricular assist device support . For exaractical . LPN canractical . Researcractical .When constructing a patient-specific LPN physiology model, it is necessary to tune the model to accurately reflect the patient's cardiovascular system. This is accomplished using available clinical data acquired through methods such as cardiac catheterization and magnetic resonance imaging. Standard-of-care patient data acquired prior to surgery are often obtained at different times and when the physiological state of the patient varies due to the influences of factors such as anesthesia. Furthermore, once they have been tuned, LPN parameters are often assumed unchanged in simulations predicting postoperative hemodynamics , 2. HoweAs discussed, patient cardiovascular physiology in cardiothoracic surgery changes due to many factors: induction of anesthesia, use of cardiopulmonary bypass and cardioplegia, responses to surgical trauma, fluid resuscitation, and administration of vasoactive agents. Without accounting for these factors in lumped-parameter cardiovascular models, the ability of models to predict postoperative hemodynamics will remain limited. The goal of this paper is to provide a summary describing the primary sources of physiologic changes during surgery that affect hemodynamics as the first step in guiding future model improvement; only after recognizing these sources can we know where to direct focus to incorporate their effects quantitatively into models. For example, transient dissipation of anesthesia may be accounted for by integrating pharmacodynamic models to reflect a period of postoperative recovery. As fluid resuscitation and vasoactive agents are available for intentional hemodynamic management, implementing their effects in models may encourage their use in developing surgical strategies, accounting for post-operative management flexibility. With an increasing volume of available data and advances in modeling algorithms, incorporating the complex nature of these factors in cardiovascular models is an area ripe for exploration and pursuit.JK performed literature search and drafted the manuscript, with EK providing the conception and structural design of the paper. EK critically revised the manuscript. JK and EK both performed analysis and interpretation of the literature search results and reviewed the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, significantly improves cardiovascular outcomes in diabetic patients; however, the mechanism is unclear. We hypothesized that empagliflozin might have beneficial effects on cardiac function, structure, adiposity, and myocardial diffuse fibrosis. This prospective study enrolled 35 patients  with type 2 diabetes mellitus (T2DM) from June 1, 2017, to November 31, 2018. The patients received an SGLT2 inhibitor (empagliflozin 25 or 12.5 mg/d) for 6 months in addition to stable oral hypoglycaemic treatment. All patients underwent cardiac magnetic resonance imaging (CMRI) before and after empagliflozin treatment. Left ventricular (LV) function and structure were quantified using cine CMRI. Cardiac adiposity was defined based on pericardial fat and intracardiac triglyceride contents, whereas myocardial diffuse fibrosis was indicated by extracellular volume (ECV). The statistical significance of parameter changes was assessed using paired t-test and stepwise multiple linear regression. There were no significant differences in LV function and structure changes. Cardiac adiposity and diffuse fibrosis indices were also not different before and after empagliflozin treatment. Concerning clinical parameters, only a significant decrease in systolic blood pressure (by 6.4 mmHg) was observed (p = 0.013). Stepwise multiple linear regression revealed that worse baseline MRI parameters were associated with better improvements. Intracardiac triglyceride content decrease was inversely associated with baseline intracardiac triglyceride content (p < 0.001). Pericardial fat changes were negatively correlated with baseline pericardial fat (p < 0.001) and ECV changes (p = 0.028). ECV changes were inversely associated with baseline ECV (p < 0.001), baseline LV ejection fraction (p < 0.001), and LV mass index changes (p = 0.020). This study demonstrated that 6 months of empagliflozin treatment did not significantly improve the LV function, structure, adiposity, and diffuse fibrosis in patients with T2DM. Further, the beneficial effects of empagliflozin treatment might be more evident in patients with worse baseline LV substrate and structure. However, the underlying mechanisms leading to these beneficial effects remain not well elucidated. As the human myocardium does not express SGLT2 receptors, many researchers have been attempting to find the link between pharmacological mechanisms and favourable outcomes.Empagliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, represents a new milestone in the treatment of type 2 diabetes mellitus (T2DM). The EMPA-REG OUTCOME trial showed that empagliflozin significantly reduced the heart failure hospitalization rate and lowered the cardiac mortality rate in patients with T2DM with a high cardiovascular (CV) risk2. In addition, the benefits of empagliflozin on heart failure hospitalization and CV death were evident despite the magnitude of HbA1c reduction or the baseline HbA1c3. These findings suggest that the effect of empagliflozin is independent of glycaemia control. Empagliflozin significantly lowers the systolic and diastolic blood pressure, which contributes to the reduction of heart loading. However, the fact that the incidence of stroke did not decrease in the EMPA-REG OUTCOME trial casts doubt on the osmotic diuretic effect as the only explanation4. Empagliflozin treatment also changes the fuel and enhances the energy status of the heart by increasing ATP production5. An experimental model demonstrated that empagliflozin could increase the rate of glucose and fatty acid oxidation, rather than ketone oxidation, in diabetic mice, leading to an increase in the circulating ketone levels and thus providing the failing heart with additional source of energy6. The study has set a new translational horizon; however, it focused more on systolic rather than diastolic function. Further, as glucagon increases the heart rate, the theory of shifting fuel does not fully support the results of a relative stable heart rate in the EMPA-REG OUTCOME trial7. Recently, one study showed that treatment with empagliflozin for 3 months significantly reduced the left ventricular (LV) mass index (LVMi) and improved diastolic function8. This study indicated that the beneficial effects of empagliflozin might be attributed to LV reverse remodelling.A potential explanation for the beneficial effects is better glycaemic control. However, a systematic review showed no difference in haemoglobin A1c (HbA1c) levels among SGLT2 inhibitors and other oral pharmacological treatment of T2DM11. CMRI could also quantify the cardiac adiposity by measuring the pericardial fat volume and the intramyocardial triglyceride (TG) content (cardiac steatosis). It is well known that pericardial fat and cardiac steatosis are associated with LV diastolic dysfunction and might contribute to the generation and progression of diffuse myocardium fibrosis14.Cardiac magnetic resonance imaging (CMRI) is a promising tool for evaluating the structure, function, and myocardial contents of the left ventricle. With T1 mapping, CMRI could measure the extracellular volume (ECV) of the myocardium, which is an indicator of diffuse myocardial fibrosis. Diffuse myocardial fibrosis is the core of LV remodelling and plays a unique role in the pathogenesis of heart failure with preserved ejection fractionThe current study was designed to investigate whether empagliflozin treatment for 6 months could have beneficial effects on the cardiac function, structure, and myocardial contents of the left ventricle. The aim of this study was to analyse the changes of CMRI parameters before and after empagliflozin treatment, and to identify the predictors leading to favourable LV remodelling.A total of 35 patients were included. The average age was 63.5 ± 9.7 years, and 17 (48%) patients were men. The mean systolic/diastolic blood pressure was 132.3 ± 17.4/75.5 ± 12.6 mmHg. Most patients had hypertension (82.9%) and dyslipidaemia (74.3%), and about half of the patients had a history of coronary artery disease . All patients were enrolled from the outpatient department and had no signs of acute decompensated heart failure. The mean serum N-terminal-pro-brain natriuretic peptide (NT-proBNP) level was 64.32 ± 66.58 pg/mL. The mean HbA1c was 7.0 ± 1.1%. Beta-blocker, angiotensin-converting enzyme inhibitor, and angiotensin II receptor blocker were administered in 45.7%, 22.9%, and 48.6% of the patients, respectively. With respect to T2DM medications, 74.3% received metformin, 14.3% received sulfonylurea, 2.9% received glinide, 31.1% received pioglitazone, and 5.7% of the patients received insulin treatment. As the national health insurance does not allow the combination use of dipeptidyl-peptidase-4 inhibitor and SGLT2, no patient received this combination treatment during the study. There was only one patient with impaired systolic function, with a mean LV ejection fraction (LVEF) of 70.1 ± 8.0%. The amount of intracardiac TG content, pericardial fat, ECV, and other CMRI parameters are listed in Table The CMRI parameters of the patients are summarized in Table In this study, we showed that, overall, 6 months of empagliflozin treatment did not significantly improve the LV function, structure, adiposity, and diffuse fibrosis in patients with T2DM. We also demonstrated that the beneficial effects of empagliflozin treatment might be more evident in patients with worse baseline LV substrate and structure.15. The development of diabetic cardiomyopathy is slow and silent when there are no other confounding factors such as CAD, hypertension, and valvular heart disease. Diastolic dysfunction is considered the most frequent and an early sign of myocardial dysfunction that could emerge even in the early stage of T2DM18. The pathogenesis of diabetic cardiomyopathy is complex. In normal individuals, myocardial energy is mainly derived from the metabolism of glucose and free fatty acids through the lipolysis of adipose tissue; however, in diabetic patients, the energy sources are shifted to the beta-oxidation of fatty acids because of the depletion of glucose transporters 1 and 4, in which the oxygen demand is higher and energy imbalance occurs. Free fatty acids inhibit pyruvate dehydrogenase, accumulate glycolytic intermediates and ceramide, and further promote apoptosis19. In a previous study using 1 H magnetic resonance spectroscopy (MRS), myocardial TG content was increased in diabetic patients and associated with impaired LV diastolic function20. Although the proportion of patients with systolic versus diastolic dysfunction in the EMPA-REG OUTCOME trial is unknown, diastolic dysfunction was reported to be present in >60% of subjects with diabetes21.T2DM is an independent risk factor of heart failure with adverse effects on the function and structure of the heart23. However, the effects of therapeutic interventions on the myocardial substrate of patients with T2DM have been seldom studied. One report showed that 16 weeks of caloric restriction in patients with T2DM seemed to decrease the myocardial TG content along with improvements in LV diastolic function, LV mass, and E/A ratio24. Another study showed that although pioglitazone, in comparison with metformin, could improve LV function and alter the myocardial substrate metabolism in patients with T2DM, no therapeutic effect on myocardial TG content was noted25. Empagliflozin is the first oral anti-hyperglycaemic agent proven to have benefits on the CV outcome. Therefore, it is of clinical interest to determine if empagliflozin would have beneficial effects on the heart function/structure and myocardial substrate. In one human study, 3 months of empagliflozin treatment was associated with a significant reduction in LVMi and an improvement in diastolic function8. Recently, Verma et al. conducted the EMPA-HEART CardioLink-6 trial and enrolled 97 patients with T2DM and established CAD. This study showed that 6 months of empagliflozin treatment could lower the LVMi and that, when LVMi was >60 g/m2, the regression of LV mass was associated with the baseline LV mass26.Cardiac MRI has provided a unique view of subclinical lipid overload-related myocardial remodelling. Studies using cardiac MRI have established an association between myocardial steatosis and LV dysfunction independent of age, diastolic blood pressure, and BMI in T2DM30. Recent studies further demonstrated that SGLT2 inhibitors could reset the tubuloglomerular feedback, leading to volume contraction without the activation of the sympathetic nervous system and harmful glomerular hyperfiltration31. Moreover, SGLT2 inhibitors might improve the efficiency of myocardial energy utilization by offering β-hydroxybutyrate as an attractive fuel for oxidation6. These multiple non-glycaemic effects support SGLT2 inhibitors as the preferred glucose-lowering drug for treating diabetic patients with heart failure.Despite the impressive results, the potential mechanisms remain largely unexplained. The unique pharmacological profile of SGLT2 inhibitors places them at the crossroads of important haemodynamic, neurohumoral, metabolic, and vascular endothelial pathways influencing cardiac and renal diseases. There are several potential direct and indirect pathways leading to the improvement of heart structure, function, and myocardial substrate induced by SGLT2. Treatment with SGLT2 inhibitors in patients with diabetes could provide better glycaemic control and insulin sensitivity and reduce arterial blood pressure, arterial stiffness, body weight, and visceral fat26 might be attributed to the different study populations and the lower severity of T2DM, fewer comorbidities, normal or milder heart function impairment, and myocardial substrate change in our study. Nevertheless, our results are in accordance with the current observations that patients with worse baseline characteristics benefit more from the treatment.Although the current study failed to demonstrate improvements in heart function/structure and myocardial substrate, we found that the greater the accumulation of intracardiac TG content, pericardial fat, and diffuse fibrosis at baseline, the better the improvements of these components after 6 months of empagliflozin treatment. The discrepancy between our findings and those of previous studies35. The impact of SGLT2 inhibitors on the adiposity and fibrosis content of the liver has been addressed by several studies. For example, tofogliflozin and ipragliflozin have been shown to prevent hepatic TG accumulation and fibrosis in obese rats36. A study pooling randomized trials including EMPA-REG demonstrated a highly consistent result that empagliflozin reduces aminotransferases in patients with T2DM, which potentially indicates a reduction in liver fat30. Thus far, there is no report on the effects of SGLT2 on the adiposity  and fibrosis content of the heart. As the first study enrolling patients with worse baseline disease severity and cardiac characteristics, our current study might be important in clarifying these issues.The adiposity of the heart has been proven to be significantly associated with heart function. For example, pericardial fat is associated with metabolic risks, severity of CAD, adverse LV remodelling, and sudden cardiac deathThis study has some limitations. First, the study population was small and no control (non-diabetic) group was enrolled in this study. Second, instead of echocardiographic measurements, we used only LVPFR to represent LV diastolic function, which might be an insensitive indicator. Third, we used non-invasive MRS to quantify the intracardiac TG content and myocardial diffuse fibrosis without performing histological examination to validate the results. Fourth, whether the results of this study on empagliflozin can be extrapolated to all SGLT2 inhibitors is questionable. Finally, the lack of a significant reduction of HbA1c may raise the possibility that some patients might have poor compliance with the medication despite our careful efforts to confirm the compliance of patients. Nevertheless, the significant increase in haematocrit, reduction in blood pressure, and persistent glucosuria during the study imply that most of the patients had fair compliance.In conclusion, we demonstrated that, overall, 6 months of empagliflozin treatment did not significantly improve the LV function, structure, adiposity, and diffuse fibrosis in patients with T2DM. However, we found that the beneficial effects of empagliflozin on the intracardiac TG content, pericardial fat, and ECV might be more evident in patients with worse baseline LV substrate and structure.The study was approved by the institutional review board of the National Taiwan University Hospital Ethics Committee. All participants provided written informed consent. We prospectively enrolled a total of 41 patients with T2DM aged >20 years from June 1, 2017, to November 31, 2018. The patients received an SGLT2 inhibitor (empagliflozin 25 or 12.5 mg/d) for 6 months in addition to a stable regimen of oral hypoglycaemic agents. The diagnosis of T2DM was based on the 2018 American Diabetes Association criteria, as follows: fasting plasma glucose ≥126 mg/dL (7.0 mmol/L), or 2-h plasma glucose ≥200 mg/dL (11.1 mmol/L) during an oral glucose tolerance test, or A1C ≥6.5% (48 mmol/mol), or random plasma glucose ≥200 mg/dL (11.1 mmol/L) in a patient with classic symptoms of hyperglycaemia or hyperglycaemic crisis. Detailed medical history was obtained including comorbid diseases and current medications. The exclusion criteria were chronic kidney disease stage V or end-stage renal disease that precluded the treatment of empagliflozin or injection of gadolinium. As LV function might be influenced by CAD during the study, patients with significant CAD with active ischaemia or those who had undergone a coronary intervention procedure within 6 months before enrolment were also excluded. The clinical data, body weight, systolic blood pressure, diastolic blood pressure, and laboratory information including total cholesterol, TG, low-density lipoprotein, high-density lipoprotein, HbA1c, and NT-proBNP were all recorded before and after empagliflozin treatment. All enrolled patients underwent CMRI before and after empagliflozin therapy. Among them, four patients were excluded because of poor CMRI quality or incomplete data and two patients withdrew from the study because of intolerance to empagliflozin.37.A 1.5-T MRI system  with an eight-channel CV phased array torso coil was used. The Modified Look Locker Inversion recovery (MOLLI) sequence for myocardial T1 mapping was performed in a single breath hold before and 10 min after the intravenous administration of 0.15 mmol/kg gadolinium-based contrast medium . During three to five heartbeats after the inversion pulse, the electrocardiography (ECG)-triggered MOLLI protocol obtained images intermittently in the diastole phases, generating each inversion recovery curves for the region of interest (ROI) and each pixel location. The information of multiple inversion recovery of each pixel location can be applied to generate T1 mappingCine MRI was performed with an ECG-triggered segmented, balanced, steady-state gradient echo pulse sequence. Short-axis slices were separated from the mitral orifice to the LV apex with a slice thickness of 8 mm and a gap of 2 mm. The T1 maps were reconstructed using the scanner console. We adjusted the centre frequency to avoid off-resonance artefacts and excluded image with poor quality due to severe motion artefacts secondary to arrhythmia or difficulty in breath holding.3. The myocardial TG signal was acquired at 1.4 ppm from the spectra with water suppression, and the water signal was acquired at 4.7 ppm from the spectra without water suppression38.Cardiac MRS was performed by using a standard flex coil for signal reception. Four-chamber and short-axis cine images were used to prescribe the locations of MRS voxels. The H1-magnetic resonance spectra of water and TG were acquired using cardiac-gated point-resolved spectroscopy during a single breath hold. The acquisition parameters were as follows: nominal repetition time/echo time, 550 ms/30 ms; 10–12 average; and voxel size, 4*23*20 mmThe main MRI parameters in this study were ECV, LVEDV, LVESV, LVEF, LVPER, LVPFR, and LV mass at end-diastolic volume, LV intracardiac fat (TG) content, and pericardial fat.ECV: The quantitative analysis of myocardial ECV was based on T1 maps. For each slice, the ROIs in blood and in the LV myocardium were drawn in the central area of the LV cavity and the septal myocardium. The T1 values of the segmented ROIs were averaged. The difference between the pre-contrast value and the post-contrast value represented the changes of the relaxation rate (1/T1). The ratio of the change in relaxation rate (1/T1) in the myocardium to that in blood, adjusted by multiplying by (1-haematocrit), is defined as ECV.3. All indices of LV volumes and mass were normalized by dividing with the body surface area.LV function and structure: The parameters were obtained from cine MRI. For each slice level, the endocardial and epicardial contours of the left ventricle were determined. Using Simpson’s rule for the volume-time curve of the left ventricle, the LV volumes for each heart cycle were calculated. LV end-systolic diameter and LVESV were defined as the maximal and minimal LV values in the volume-time curve. LV mass was defined as the difference between LV epicardial volume at end-diastole and LVEDV, multiplied by the myocardial density, which is 1.05 g/cmPericardial fat: Consecutive short-axis images during the end-diastolic phase were examined and multiplied by the slice thickness. Pericardial fat was defined as regions of high-signal intensity between the epicardium and the endocardium.Intracardiac fat: In the magnetic resonance spectrum, signal suppression with hydrogen was manipulated, and there were two peaks representing the water and TG proportions, respectively. The myocardial TG content was expressed as the integral ratio of TG to water (%).The definitions were as follows:All parameters were analysed using a custom-developed MATLAB 7.9 program .Continuous variables are presented as means ± standard deviations, and categorical variables are presented as numbers and percentages. The differences of clinical, laboratory, and MRI parameters before and after the treatment with empagliflozin were analysed using paired Student’s t-test. To investigate the determinants of the changes of MRI parameters before and after empagliflozin treatment, stepwise multiple linear regression analysis was performed using changes of the cardiac TG content, pericardial fat, or ECV as dependent variables and the baseline values or changes of other MRI parameters including normalized LV end-diastolic diameter, normalized LV end-systolic diameter, LVMi, LVEF, LVPER, and LVPFR as independent variables. Two-tailed p < 0.05 was considered statistically significant. All analyses were conducted using Statistical Package for the Social Sciences software .The study was approved by the institutional review board of the National Taiwan University Hospital Ethics Committee.Written consent was collected from all patients. The study complied with the second Declaration of Helsinki. The design of the study was approved by a regional scientific ethics committee.


LMNA c.1824C > T mutation, which provokes the expression of a dominant-negative mutant protein called progerin. Therapies proven effective in HGPS-like mouse models have yielded only modest benefit in HGPS clinical trials. To overcome the gap between HGPS mouse models and patients, we have generated by CRISPR-Cas9 gene editing the first large animal model for HGPS, a knockin heterozygous LMNA c.1824C > T Yucatan minipig. Like HGPS patients, HGPS minipigs endogenously co-express progerin and normal lamin A/C, and exhibit severe growth retardation, lipodystrophy, skin and bone alterations, cardiovascular disease, and die around puberty. Remarkably, the HGPS minipigs recapitulate critical cardiovascular alterations seen in patients, such as left ventricular diastolic dysfunction, altered cardiac electrical activity, and loss of vascular smooth muscle cells. Our analysis also revealed reduced myocardial perfusion due to microvascular damage and myocardial interstitial fibrosis, previously undescribed readouts potentially useful for monitoring disease progression in patients. The HGPS minipigs provide an appropriate preclinical model in which to test human-size interventional devices and optimize candidate therapies before advancing to clinical trials, thus accelerating the development of effective applications for HGPS patients.Hutchinson-Gilford progeria syndrome (HGPS) is an extremely rare genetic disorder for which no cure exists. The disease is characterized by premature aging and inevitable death in adolescence due to cardiovascular complications. Most HGPS patients carry a heterozygous de novo  This mutation activates usage of an alternative 5′ splice donor site in exon 11 that results in deletion of 150 nucleotides from LMNA mRNA and the synthesis of a truncated protein called progerin. This aberrant protein accumulates in the nuclear envelope due to irreversible farnesylation and causes severe alterations in multiple cellular functions2  is an extremely rare disorder , featuring atherosclerosis, vascular stiffening and calcification, electrocardiographic (ECG) alterations, and left ventricular (LV) diastolic dysfunction11. Despite their limitations, HGPS-like mice have been the gold-standard preclinical model and have led to clinical trials testing the ability of repurposed drugs to reduce progerin farnesylation12. Targeting progerin farnesylation resulted in a mild benefit in body weight, bone, and vascular alterations in a subset of HGPS patients and was associated with lower mortality rate after 2.2 years of follow-up; however, the estimated increase in life expectancy is only 1.6 years15, highlighting the limitations in translating results of preclinical mouse studies to HGPS patients.The extreme rarity of HGPS makes the organization of any clinical trial a huge challenge where the inevitable limitation of a small patient cohort adds to the difficulty of deciding which therapies effective in HGPS-like mice should be tested in patients. Available HGPS mouse models either ectopically express progerin, lack or overexpress A-type lamin isoforms, or accumulate farnesylated prelamin A -Cas9 (CRISPR-associated protein 9) gene editing26 in male Yucatan minipig skin fibroblasts, followed by somatic cell nuclear transfer (SCNT) by handmade cloning to enucleated oocytes from Large White sows27  were constructed to target exon 11 in the porcine LMNA gene. sgRNA1 was selected for subsequent gene editing experiments due to its consistency when tested in an in vitro validation assay28. Information on the different molecular tools is available in Supplementary Tables LMNA c.1824C > T mutation without unwanted insertions, deletions, or base changes in the region involved in homologous recombination , of which 10 survived the postnatal period  Fig. . One HGPLMNA c.1824C > T Yucatan minipigs, like HGPS patients, endogenously co-express progerin mRNA and protein in all tissues tested.Primers for PCR-based detection of progerin mRNA in porcine tissues were based on the human progerin mRNA sequence (NCBI accession number NM_001282626.1) Fig. . Sequenc30 was significantly lower in HGPS minipigs  in 4.3- to 5.4-month-old HGPS and WT Yucatan minipigs. T1 and T2 mapping and overall LV extracellular volume fraction showed no significant differences between WT and HGPS minipigs. However, qualitative analysis of late gadolinium contrast enhancement images revealed patchy gadolinium deposition in the anterior LV wall and septal regions in three out of eight HGPS minipigs Fig. . Cardiacigs Fig. , indicatWe next performed hematoxilyn & eosin (H&E) and Masson trichrome (MT) staining in transverse heart sections (between the atrium and apex) obtained after necropsy. Compared with controls, the LV of the HGPS minipigs had double amount of medium and large coronary arteries . Moreover, in five out of eight HGPS minipigs, ~21% of these vessels showed signs of atherosclerotic disease including medial degeneration and intimal hypertrophy   Fig. . Togethech) Fig. .E′ wave, and higher E/E′ ratios  or LV mass  and N-Cadherin (N-Cadh); these proteins are normally expressed in the intercalated disk between adjacent cardiomyocytes and are essential for proper mechanical and electrical coupling and propagation of the electrical impulseion Fig. .Cardiac electrical activity was monitored continuously in vivo by placing implanted loop recorders (ILR) subcutaneously in five WT and four HGPS minipigs Fig. . Single-MRI analysis of brain showed no cerebrovascular disease in the HGPS minipigs. None of the images indicated any localization with hyper-intense regions on FLAIR images or hypo-intense regions on T1-weighted images, which would be indicative of localized regions of stroke. Vascular microbleeds were not detected, as indicated by the lack of localized signal decay in T2*-weighted images (data not shown).Gross histological examination of kidney, testicle, spleen, liver, and lung revealed pathological features only in the kidneys and testicles of HGPS minipigs. In the case of kidney, 88% of the HGPS minipigs showed dilation of cortical tubules and different degrees of glomerulosclerosis  and none of the pigs survived to a post-pubertal age. As an alternative strategy, we collected and froze epididymal sperm from three necropsied HGPS minipig testicles and attempted surgical intrauterine insemination of two gilts treated with hormones to induce ovulation. One gilt received 300 × 10LMNA c.1824C > T mutation, the HGPS minipigs co-express endogenous progerin, lamin A and C, thus circumventing the limitation of HGPS-like mouse models lacking or overexpressing A-type lamin isoforms11. Progerin expression in the HGPS minipigs induced all major progeroid symptoms that, like in patients, are variable in severity along the time-course of disease, increase with age and culminate in early death. We focused our studies on CVD, which is considered the main cause of death in HGPS.The generation of a large animal model for HGPS addresses the need for a more translational preclinical model of this devastating disease. Like patients carrying the heterozygous 7. LV systolic dysfunction and valve disease were infrequently seen in the first decade of life but became more prevalent in the second decade7. HGPS minipigs develop both systolic and diastolic dysfunction , which are common underlying causes of heart failure. Also consistent with reports in HGPS patients40, we found signs of mild mitral valve degeneration accompanied by mild mitral regurgitation in two of eight HGPS minipigs.LV diastolic dysfunction was the most frequent echocardiographic abnormality in a cohort of 27 HGPS patients, with prevalence higher in the oldest patients7. However, ECG alterations were more prevalent (47%) in our previous study of 15 HGPS patients and 13 sex- and aged-matched healthy volunteers8. This apparent discrepancy may reflect differences in the median age at evaluation in the two cohorts (median age of 5.6 [2–17] years7 versus 11.5 [2–19] years8), with the older cohort predicted to have a greater burden of ECG alterations9. Notably, the HGPS minipigs exhibited significant ECG abnormalities at advanced disease stages, in particular longer QRS complex duration and significant bradycardia, in agreement with ECG alterations reported in progeroid mice at advanced disease stages8. The HGPS minipigs also showed Cnx43 mislocalization in the heart as an underlying cellular alteration related to ECG abnormalities, consistent with previous findings in HGPS patients and progeroid mice8. We further investigated cardiac electrical activity through continuous in vivo recordings, which revealed pre-mortem advanced atrio-ventricular conduction block in three out of four HGPS minipigs. Moreover, the short-duration polymorphic ventricular tachycardia observed in one HGPS minipig before complete atrio-ventricular block (likely related to severe bradycardia) is in agreement with our previous findings demonstrating premature ventricular complexes in progeroid mice during sequential ECG examination8. Another HGPS minipig had overt ST elevation at the moment of death, suggesting that myocardial ischemia may have contributed to the atrio-ventricular block. Consistent with this, our in vivo cardiac MRI studies identified reduced myocardial perfusion and interstitial fibrosis, both contributing to the diastolic LV dysfunction phenotype in the HGPS minipigs. These MRI findings are confirmed by the histopathological studies revealing a significant proportion of degenerated medium-to-large coronaries and above-normal collagen deposition in cardiac arterioles and myocardial tissue. The defective myocardial perfusion and interstitial fibrosis are previously unidentified cardiovascular alterations in genetically diagnosed HGPS patients that deserve attention when monitoring disease progression in patients and can be used as in vivo readouts in clinical trials. Our HGPS minipig model can guide future research into the natural history of CVD in HGPS and aid in the important task of elucidating primary molecular and cellular mechanisms of progerin-induced damage. These knockin minipigs should also help to identify early biomarkers of disease progression and responses to therapies.Prakash et al. reported normal ECG parameters in 25 of 27 HGPS patients12 before moving them forward to clinical trials. The expectation is that any preclinical tests in HGPS minipigs will yield highly translatable results regarding drug regimes, administration routes, bioavailability, pharmacokinetics, efficacy, and acute toxicity. Likewise, these knockin minipigs should help in the development and optimization of cell-based therapies and systemic or tissue-specific gene therapy. Notably, the c.1824C > T mutation in the pig LMNA gene provokes progerin expression, suggesting that HGPS minipigs may be useful for testing therapies targeting aberrant LMNA exon 11–12 splicing, such as morpholino-oligonucleotides, which showed efficacy in progeroid mice33 but await testing in a large animal model.The course of disease in the HGPS minipigs correlates well with the timing of disease onset, progression, and death in patients, with both species manifesting symptoms a certain period after birth and dying around puberty. With a mean life span of just ∼6 months, our HGPS minipig model is ideal for preclinical studies, because it offers a wide window for therapeutic interventions to prevent the appearance of symptoms or revert the phenotype, while also permitting longevity studies over a relatively short period. This favorable timing will enormously facilitate the execution of future preclinical tests. Indeed, this HGPS minipig model can provide an appropriate filter for drug-based strategies with proven efficiency in HGPS-like miceThe human-like size of the HGPS minipig model is another key advantage that allowed the use of the same MRI, echography, ECG, and telemetry devices used in hospitals for human diagnosis. Due to dentition and mandibular problems, anesthesia is considered a risky procedure in HGPS patients. The HGPS minipigs recapitulate these dental and facial bone alterations, and due to the narrow airway in these animals, standard intubation for anesthesia was substituted in some cases by ventilation with canine laryngeal masks. Any interventional trial in HGPS minipigs involving anesthesia will therefore face similar challenges to those encountered with HGPS patients, and will thus produce invaluable translational results. For all these reasons, we expect this progeroid minipig model to have a strong and positive impact on HGPS by expediting the development of effective therapies for HGPS patients.LMNA 1824C > T Yucatan minipig fibroblast clone or fibroblasts isolated from HGPS minipigs. While disadvantageous from the perspective of colony generation, the lack of reproductive fitness is a further example of the recapitulation of disease symptoms seen in patients, who experience delayed or absent pubertal development. For example, a 12-year-old HGPS female patient and a 17-year-old HGPS male patient were classified as Tanner developmental stage II 6. Additionally, a group of female HGPS patients aged over 12 years and who declared having experienced menarche remained between the Tanner I and II pubertal stages even 2 years after their menarche41. To our knowledge, there are no reports of the potential viability of gametes from HGPS patients. Although the dose of A-type lamins can affect spermatogenesis in rodents42, A-type lamins were not detected in human spermatids or mature spermetazoa43. Consequently, no progerin-induced damage would be expected in mature HGPS spermatozoids. Recently, it was reported that mouse LmnaG609G/G609G oocytes are meiotically competent and can be fertilized by WT spermatozoids and that LmnaG609G/G609G sperm, although not abundant, is motile and able to fertilize WT oocytes44. The same authors propose that the sub-fertility in LmnaG609G/G609G mice may simply be a consequence of the lack of physical fitness. This idea is supported by the physical condition of the HGPS minipigs and the low numbers of interstitial Leydig cells and mature spermatids, indicating delayed pubertal development. These findings may reflect a failure of the minipigs to achieve the necessary weight or fat threshold to trigger sexual maturation.The difficulty encountered in establishing a HGPS minipig colony either by mating HGPS boars with WT sows or by surgery-assisted fertilization of sows with sperm obtained from HGPS minipigs was unsurprising given the lack of fitness and delayed pubertal development among the male HGPS minipigs. Although we were able to cryopreserve and freeze small amounts of sperm from HGPS necropsied testicles, attempts to surgically inseminate two hormone-treated gilts were unsuccessful. Therefore, the generation of new HGPS minipigs for future preclinical trials will require either in vitro fertilization or recloning using our heterozygous A potential limitation of our study is that we only included male HGPS minipigs because all the cloned animals were derived from genetically modified fibroblasts obtained from a male Yucatan minipig. However, no gender-specific alterations have been reported in HGPS patients, and we expect future preclinical results generated in our HGPS minipig model to be equally translatable to male and female patients.LMNA c.1824C > T Yucatan minipig model is a bona fide and necessary bridge between HPGS mouse models and patients. These knockin minipigs provide an appropriate large animal model in which to test human-size interventional devices (like stents or pacemakers), and standardize surgical models or medical protocols using the same methodologies utilized with patients. Importantly, HGPS minipigs will facilitate the optimization of drug regimens escalated from small- to large-body-size mammals, and will accelerate the unraveling of potential counteracting toxicities in combined new therapies, or side effects in challenging future cures. This model will therefore facilitate decision-making about which therapeutic strategies should advance to clinical trials and potentially expedite the development of effective therapeutic applications for HGPS patients. Moreover, as progerin is expressed at low levels in tissues from aged non-HGPS individuals (reviewed in ref. 2), we believe that research in this HGPS minipig model may also shed light into the complex mechanisms underlying physiological aging.We believe that our heterozygous The data, analytic methods, and study materials will be made available to other researchers for purposes of reproducing the results or replicating the procedure (available at the authors’ laboratories). Supplementary Fig. All animal procedures at Aarhus University (AU) were approved by the Danish Animal Experiments Inspectorate (licence no. 2014-15-0201-00434). Experimental procedures at the Centro Nacional de Investigaciones Cardiovasculares (CNIC), Universidad de Murcia, and the Spanish National Institute for Agricultural and Food Research and Technology conform to EU Directive 2010/63EU and Recommendation 2007/526/EC, enforced in Spanish law under Real Decreto 53/2013. Animal protocols were approved by the respective local ethics committees. Animal work at the CNIC was approved by the Comunidad Autónoma de Madrid (PROEX #313/16). The cloned HGPS animals were generated at AU and housed in a specific pathogen-free facility under standard conditions. At ~3.5 months of age, animals were transported to Agropardal S.L. , a farm dedicated exclusively to the breeding of experimental pigs. Phenotypic characterization of minipigs was conducted at the CNIC’s animal facility. Surgical intrauterine insemination of gilts was carried out at the Universidad de Murcia. All minipigs were uncastrated Yucatan males except for two WT minipigs that were castrated and excluded in weight and survival studies. We used non-littermate WT controls because it was not possible to cross the male HGPS minipigs with WT gilts.Anesthesia was administered to all Yucatan minipigs before proceeding with non-invasive procedures and implantation of a telemetry monitor. Anesthesia was induced by intramuscular injection of ketamine (20 mg) followed by continuous intravenous infusion of ketamine (2 mg/kg/h), xylazine (0.2 mg/kg/h), and midazolam (0.2 mg/kg/h) throughout the protocol.46. Likewise, valve function was analyzed according to European Society of Cardiology guidelines45. Ultrasound images were analyzed with Qlab R 9.1  by two expert observers blinded to genotype. Pulsed-wave Doppler imaging was used to determine mitral valve inflow velocity during early (E) and late (A) diastole, E/A velocity ratio, mitral E velocity DT, septal early (E′) diastolic myocardial velocity, and E/E′ ratio. In addition, left atrium antero-posterior dimensions were obtained and normalized to body surface area using the modified Brody’s formula48. Images were analyzed by two researchers blinded to genotype.Diastolic function patterns were studied with a Commercial scanner  and the standard techniques49. T1 mapping images were captured using a 5(3)3 MOLLI scheme with a single-shot balanced acquisition  and a spatial resolution of 1.5 × 1.8 × 8.0 mm3. Tissue characterization parameters were acquired from a single mid-ventricular slice in short-axis orientation. Microvascular status was studied with a previously reported methodology to assess absolute quantitative perfusion using a dual saturation recovery sequence30. Macro areas of increased extracellular space (a fibrosis surrogate) were assessed by delayed gadolinium enhancement (10–15 min after intravenous administration of 0.20 mmol gadobutrol contrast agent per kg body weight) using a T1-weighted three-dimensional inversion recovery sequence with an adjusted inversion time to null the healthy myocardium and an isotropic resolution of 1.5 × 1.5 × 1.5 mm3. CMR images were analyzed using dedicated software  by two observers experienced in CMR analysis who were blinded to genotype. LVEF, LV volumes, and LV mass were calculated by tracing endocardial/epicardial borders during end-systole and end-diastole in short-axis slices. Mapping analyses were performed with a region of interest in a mid-apical LV short-axis slice. All volumes and masses were normalized to body surface area using the modified Brody’s formula48. Images were analyzed by two researchers blinded to genotype.CMRI studies were performed in a 3 Tesla Philips Achieva Tx whole-body scanner  equipped with a 32-element phased-array cardiac coil. The imaging protocol included anatomical and functional assessment using a segmented cine steady-state free-precession (SSFP) sequence with spatial resolution of 1.8 × 1.8 mm, end-diastolic acquisition, thickness 6 mm with no gap, and 30 fully acquired cardiac phases. Segmented cine SSFP was performed to acquire 11–13 contiguous short-axis slices covering the heart from the base to the apex in order to evaluate global and regional LV motion. Multiparametric tissue characterization was performed using T2 mapping and pre- (native) and post-contrast T1 mapping techniques and absolute quantitative perfusion. T2 mapping data were acquired using a previously validated T2-GraSE sequence with eight equally spaced echo-times ranging from 6.7 to 53.7 ms50. QT intervals were corrected with the Bazzet formula. A set of 10 beats per animal was analyzed and the mean value of each ECG parameter was assigned to the animal. Two researchers blinded to the genotype performed the analysis.The 12-lead ECG recordings were obtained before imaging studies using the same anesthesia period. ECG recordings were acquired at 1 kHz for at least 5 min . PR, QRS, and QT intervals were measured using manual calipers at a 200 mm/s sweep speed. QRS complex duration was measured as the time from the lead showing the earliest deflection (positive or negative) to the lead with the latest offset of the QRS complexILR  were placed subcutaneously in four HGPS and five WT Yucatan minipigs under local anesthesia with lidocaine 1% . The device was placed subcutaneously on the left side of the sternum to ensure accurate QRS complex recordings.Blood samples were analyzed in an ADVIA 2120i hematology system (Siemens). Serum samples were analyzed in a Dimension RxL Max analyzer (Siemens).t-test using GraphPad Prism software, except for the % fibrosis in LV and septal myocardium in Fig. t-test was used. A log-rank (Mantel-Cox) test was used for the Kaplan-Meier survival curve. Differences were considered significant at p < 0.05.Statistical significance was assessed by unpaired two-tailed Student’s Supplementary InformationSupplementary Movie S1Supplementary Movie S2Supplementary Movie S3Supplementary Movie S4Supplementary Movie S5


Transthoracic 3D cardiac analysis is enticing in its potential simplicity and wealth of data available. It has been suggested to be accurate vs magnetic resonance imaging in relatively stable patients, but feasibility and agreement with conventional echocardiographic assessment of stroke volume (SV) have not been thoroughly assessed in critically ill patients, who are traditionally harder to image. The objectives of this study were to compare 3D transthoracic volumetric analysis vs Doppler assessment of SV  and Simpson’s biplane assessment in a cohort typical of the intensive care unit (ICU), where accurate assessment is important: mechanically ventilated patients with a significant ventilation/perfusion (V/Q) mismatch. We hypothesised that it would be feasible but might lack agreement.Patients were imaged within 24 hours of admission. Inclusion criteria were adult patients, V/Q mismatch present , and mechanically ventilated with Doppler SV assessment possible. Biventricular echocardiographic volumetric analysis was performed using Siemens SC2000 along with standard Simpson’s biplane and Doppler SV assessment. 3D images were unacceptable if two segments or more were unable to be seen in two volumetric planes. 3D left ventricular (3DLV) and 3D right ventricular (3DRV) analyses were performed with the Tomtec Imaging and Siemens Acuson platforms, respectively.Ninety-two patients were included . 3DLV and 3DRV analyses were feasible in 72% and 55% of patients, respectively; however, they underestimated SV compared with Doppler by 2.6 ml (± 10.4) and 4.1 ml (± 15.4), respectively. Limits of agreement for 2D, 3DLV and 3DRV volumetric analysis techniques were large.3DLV and 3DRV volumetric analyses appear feasible (obtainable) in the majority of mechanically ventilated ICU patients. Compared with the Doppler method, 3DLV and 3DRV volumetric analyses underestimate SV. The large limits of agreement between the methods also cast doubt on their comparability. Given the scenarios in which SV analysis is required , our study cautions against the use of 3D SV clinically. Assessing ventricular size and function is the foundation for the diagnosis of cardiac dysfunction in the critically ill. Stroke volume (SV), end-diastolic volume (EDV), end-systolic volume (ESV) and ejection fraction (EF) are important when considering management in many circumstances, such as heart failure, fluid administration and effect of treatment. SV and cardiac output estimation using Doppler echocardiography (echo) has been suggested to have sufficient precision to be able to estimate cardiac output in the critically ill , 2, and see Fig. 3D transthoracic echo has been a feature of most major ultrasound systems since 2008, being used for valvular analysis and volumetric and left ventricular (LV) mass estimation [3D volumetric measurements by echo were originally made by acquiring images over multiple heartbeats, obtaining the full-volume image through stitching together the data. More recently real-time 3D echo has been developed, which allows for the full volume to be recorded in one beat and prevents stitching artefacts, which can occur with respiratory movement or with arrhythmias such as atrial fibrillation. This can be particularly attractive in imaging the critically ill because breath holds can be challenging and arrhythmias are common. With real-time 3D transthoracic echo, there is reduced temporal and spatial resolution , and theWe sought to assess if SV obtained by different methods, namely pulsed-wave Doppler, Simpson’s biplane and 3D echocardiography, is comparable and to assess if 3DLV and 3D right ventricular (3DRV) echo are feasible in an ICU population who were mechanically ventilated. We assumed that the SV of the LV would equal the SV of the right ventricle (RV). We chose a cohort of patients who would be considered typical ICU patients in whom ventricular volumetric analysis was important: mechanically ventilated critically ill patients with a significant ventilation/perfusion (V/Q) mismatch .see Fig. Adult patients admitted to the ICU of Nepean Hospital, Sydney, Australia, over an 18-month period were considered in this study. The project was approved by the Nepean Blue Mountains Local Health District (LNR/13/NEPEAN/154), and written consent was provided prospectively by the authorised representatives (next of kin) or retrospectively by the patient, given the non-invasive nature of the imaging. Patients were included if they were over the age of 18 years, were mechanically ventilated with a P/F ratio < 300 and were able to have SV assessed by Doppler echo. Patients were excluded if they had intracardiac shunts, previous cardiac surgery or congenital heart disease or were pregnant. We did not include consecutive patients, because SO was the sole investigator performing the 3D analysis and the majority of 2D studies  using either a Vivid 7 machine  with an M45 probe or a Siemens SC2000 with a 4V1c transducer . Accurate SV was ensured by estimating the left ventricular outflow tract (LVOT) diameter in a zoomed view of the LVOT and averaging LVOT velocity time integral measures with pulsed-wave Doppler , with a closing click present and ensuring optimal Doppler angle and Doppler trace [Real-time 3DLV and 3DRV assessment was performed using the 4Z1c full-volume 1.5–3.5-MHz matrix array transducer on the SC2000 echo machine by an experienced 3D operator (SO). The apical view was used, with the ventricle being analysed placed in the middle of the sector and the depth, sector size and angle adjusted to ensure maximal volumes per second . Three cardiac cycles were recorded for sinus rhythm and five for atrial fibrillation. Images were analysed at stand-alone stations: LV images were transferred to a Tomtec system , and RV images were transferred to the SC2000 workstation using the RV analysis application; both systems use similar voxel analysis techniques and hence were felt to be comparable. Analysis was performed by clinicians with experience in 3D echo (SO and MS) using the automated analysis packages. SO completed all the offline 3D RV analyses, and MS completed all the offline 3D LV analyses. A 10% random population was assessed by both for inter- and intra-rater variability. Image quality was assessed in a manner similar to that in recent studies by reviewing the three planes that are provided. If two consecutive segments or more in any two views were not visualised, then the image was considered poor and unsuitable .see Fig. see Fig. The automated analysis packages were used to estimate volumes for both the LV and RV. LV volumes were estimated in end-diastole (ED) and end-systole (ES) by the software tracing the endocardium in three planes: apical four-, two- and three-chamber cut planes see Fig. . The opeP values < 0.05 were considered statistically significant. Bias (mean of difference), precision (SD of difference) and limits of agreement (95% CI of the bias) statistics were performed using methods described by Bland and Altman [Statistical analysis was performed with JMP Pro version 13 . Continuous variables are expressed as mean ± SD if normally distributed and as median with IQR if not normally distributed. Normality was assessed using the Shapiro-Wilk test. d Altman . To corrd Altman . Thirty d Altman , 11. FeaNinety-nine patients were imaged, but seven patients were excluded because Doppler SV estimation could not be obtained reliably (7%). The characteristics of the 92 patients included in the study, along with ventilation data, are shown in Table The echo data are shown in Table Using Doppler as the reference method, 2D Simpson’s biplane, 3DLV and 3DRV analysis all underestimated SV  with relatively wide ranges of limits of agreement . The corrected percentage error was greatest for LV end-systolic volumes (75%) and was the smallest when comparing SV and EF (40%); however, it was still considered to lack clinical comparison.A random ten patients were selected for blinded variability analysis of the offline 3DLV and 3DRV SV assessments . Inter-rater variability was reasonable for (1) 3DLV with mean absolute difference (±SD) of 3.6 ml (± 8.6) and expressed as percentage of the mean 8% (± 19) and (2) 3DRV with mean absolute difference (±SD) of − 2.1 (± 7.3) and expressed as percentage of the mean 10% (± 22). Intra-rater variability also showed reasonable repeatability for (1) 3DLV with mean absolute difference (±SD) of − 3.6 ml (± 8.8) and expressed as percentage of mean 7% (± 19) and (2) 3DRV with mean absolute difference (SD) of − 1.6 (10) and expressed as percentage of the mean difference of 4% (± 27).We found real-time 3DLV and 3DRV transthoracic echo analysis of SV to be possible in a majority of critically ill patients, defined as patients on mechanical ventilation with significant V/Q mismatch; however, it did not have sufficient agreement with Doppler echo assessment to be considered clinically or statistically acceptable. In the intensive care clinical setting, cardiac volume analysis needs to be feasible, but low variability and high precision are key. We found that 3D transthoracic echo did not have sufficient agreement to be either statistically or clinically satisfactory for SV estimation in this study. 2D Simpson’s biplane analysis of SV was also assessed (because use of Doppler is considered to require higher levels of training ), and thThe ability to accurately measure SV in the critically ill plays an important role in analysis of cardiac function and haemodynamics, which are often abnormal in the ICU setting. Doppler echocardiography has been shown to be an accurate and precise method for estimating cardiac output and SV in the critically ill patient  and is tThere are several limitations to our study. It is a single-centre study performed by echocardiography enthusiasts. The 3D volumetric data were acquired on a single platform by a single operator, and data were analysed by the same operator, and therefore we cannot exclude bias. We attempted to limit bias by ignoring Doppler data prior to analysis of 3D volumes; however, a more structured blinding of data, or random assessment, would have meant greater scientific rigour. Data were analysed by a second experienced user and inter-rater variability was small and not statistically significant. The lack of consecutive assessment of patients indicates selection bias, but pragmatically, performing the study meant only one operator was regularly available for 3D imaging. The use of Doppler echo as the reference standard for SV estimation is controversial . Basic e. RV volumes in particular are not easily assessed by 2D echo, and given the complex shape of this ventricle and the extent of RV dysfunction in the critically ill, 3D RV analysis is enticing. 3D transoesophageal echo for volumetric analysis in the critically ill has been studied, particularly in the peri-operative setting, and has been suggested to be both feasible and as accurate as other forms of echocardiography [Further studies are warranted in this area for analysis of precision (vs robust reference standards such as thermodilution of MRI), as well as in assessing the change in SV. In addition, comparison among groups of physicians with different levels of experience may be useful to confirm these resultsiography . Cardiac3DLV and 3DRV echo imaging in the critically ill is feasible and reproducible, but SV estimation by real-time 3D echo analysis did not have sufficient statistical or clinical agreement with Doppler evaluation of SV in this study.


LV systolic dysfunction presents an elevated risk of thromboembolism. Previous studies demonstrated low left ventricular ejection fraction (LVEF), ischemic cardiomyopathy and increased myocardial scarring as independent risk factors for LV thrombus formation. Structural changes that alter the size and shape of LV apex may have a significant role in predicting LV thrombus, but there is no definite evidence exists in this entity.A case-control cardiac magnetic resonance (CMR) study of 150 patients with LV systolic dysfunction  was performed. Factors associated with thrombus including sphericity index and ‘new’ apical area index  were evaluated.p <  0.001) while sphericity index in both groups was not different . Univariate analysis revealed that male gender, prior myocardial infarction, presence of apical aneurysm, ischemic-typed scar, apical scar and apical area index were associated with thrombus. Further, multivariate analysis showed only apical area index and apical scar as independent predictors for thrombus formation.Average age was 63.48 ± 12.82 years and mean LVEF was 29.22 ± 8.53%. Patients with LV thrombus had significantly higher apical area index than those without thrombus (46.5 ± 3.27 vs. 42.71 ± 3.02, Apical area index from CMR is a new index to predict LV thrombus in patients with LV systolic dysfunction and may have a future role in early anticoagulant therapy. Patients with left ventricular (LV) systolic dysfunction are at increased risk of thromboembolic events that may result in major adverse cardiac sequelae. According to previous echocardiographic studies, the prevalence of LV thrombus in patients with LV systolic dysfunction was 8–19% . Thus, aCardiac magnetic resonance (CMR) provides high-resolution images, good reproducibility and tissue characterization for detection of thrombus . VariousA recent comparative study demonstrated that CMR yielded more than 3-fold higher diagnostic accuracy for detection of LV thrombus compared with transthoracic and transesophageal echocardiography which detected thrombus based on anatomical appearance rather than tissue characteristics .Two CMR studies demonstrated independent predictors of LV thrombus such as worsening left ventricular ejection fraction (LVEF), ischemic etiology, prior anterior wall myocardial infarction, and increased myocardial scarring , 6. AparPrevious echocardiographic studies reported that some parameters including sphericity index (long-to-short axis ratio in a four-chamber view) and apical conicity index  affected LV remodeling after anterior wall myocardial infarction . NeverthFrom our hypothesis, apical remodeling may associate with thrombus formation. Previous indices involved particular length measurements without a comprehensive evaluation of LV apex, thus we would like to introduce ‘apical area index’  as a new predictor of LV thrombus. The primary objective of this study was to compare apical area index of patients with LV thrombus with no thrombus group. The secondary objective was to determine the predictors of LV thrombus in patients with systolic dysfunction.This case-control, single center study was approved by the institutional ethics committee. A total of 150 patients with systolic dysfunction (30 consecutive cases with thrombus and 120 consecutive controls without thrombus) who underwent cine, adenosine stress, and LGE-CMR during the same period at Siriraj Hospital  between August 2014 and January 2016 were enrolled. Systolic dysfunction was defined as LVEF by cine-CMR below 40%. Patients were most commonly referred for CMR examination to assess myocardial ischemia, myocardial viability, or causes of cardiomyopathy. Patients exhibiting the following criteria were excluded: 1) inability to perform CMR due to a permanent pacemaker or implantable cardioverter defibrillator implantation, 2) incomplete CMR examination, 3) history of allergy to gadolinium, 4) pregnancy, and (5) history of claustrophobia.On the day of the CMR procedure, a complete medical history including cardiac risk factors, current medications including antithrombotic and heart failure drugs, information regarding coronary angiography results, prior coronary revascularization, previous myocardial infarction, and thromboembolic events were collected to assess potential predictors of thrombus.Additionally, regarding the excellent ability of LGE-CMR for the diagnosis of infarcted myocardium, patients were considered to have ischemic cardiomyopathy if there was transmural or subendocardial scar pattern from LGE-CMR. All other patients were classified as having nonischemic cardiomyopathy.Cardiac structure, function and thrombus were assessed from two CMR techniques, cardiac function by a SSFP technique and LGE using a 1.5 Tesla Gyroscan NT Philips scanner . Functional study was performed by the acquisition of images by a SSFP technique in Short-axis images were acquired every 8 mm throughout the entire LV. Long-axis images were obtained in standard 2-, 3-, and four-chamber orientations. Parameters for cardiac function were as follows: repetition time/echo time/number of excitations = 3.7 ms/1.8 ms/ 2, 390 × 312 mm field of view, 256 × 240 matrix, 1.52 × 1.21 reconstruction pixel, 8 mm slice thickness, and 70 flip angles.For LGE imaging, images were acquired 15 to 20 min after intravenous injection of 0.2 mmol/kg gadolinium contrast agent  with the following scanning parameters; echo time 1.25 ms, repetition time 4.1 ms, 15-degree flip angle, 303 × 384 mm field of view, 240 × 256 matrix, in-plane resolution 1.26 × 1.5 mm, slice thickness 8 mm and 1.5 Sensitivity-Encoding (SENSE) factor.Additionally, long inversion time sequence was applied for increasing the accuracy for detection of LV thrombus, as mentioned in previous study .CMR indices of LV function, geometry, and scarring were measured to determine whether these parameters were related to the presence of thrombus. CMR images in short-axis view were classified as the basal, mid, or apical part of the left ventricle. Segmentation of each slice was performed according to the recommendation of American Heart Association . Left veLeft ventricular thrombi detected by CMR on cine sequences were defined as filling defects within the LV cavity, typically adherent to regions of abnormal wall motion  then confirmed by LGE technique in Fig. Here, two indices were evaluated as sphericity index and ‘new’ apical area index. The sphericity index was described as the LV long-axis length divided by the mid LV diameter at end diastole for each in Fig. To represent the importance of apical remodeling, a ‘new’ apical area index was introduced to evaluate the predictor of thrombus. The apical area index was simply calculated as a percentage of an apical-half LV area (mid to apex) divided by an entire LV area from a cine four-chamber view in end diastole. Similar to the sphericity index, LV long-axis length was measured from the endocardial border of the mitral valve annulus plane to tip of apex, and the mid LV diameter was measured at the half-length from the LV long axis to tip of apex Fig. .Fig. 3CaTo assess intra- and inter-observer reliability of apical area index measurements by CMR, 20 patients were randomly selected to measure variability by the same observer 4 weeks after the initial analysis, and by the second independent observer blinded to the initial results.Descriptive statistics, including frequency and percentage, were used for categorical variables. Continuous variables were reported as mean ± standard deviation for normally distributed variables and median (percentile 25 and percentile 75) for non-normally distributed variables. Normality of distribution of variables was examined by Kolmogorov-Smirnov test. Comparisons of categorical variables between patients with and without thrombus were performed using chi-square test or Fisher’s exact test. Continuous variables were compared using Student’s t-test or Mann-Whiney U test. Univariate and Multivariate analyses of thrombus predictions were evaluated using binary logistic regression analysis (Backward method) and presented as Odds ratio (OR) . Intra- and inter-observer reliability were analyzed by the intraclass correlation coefficient.p-value < 0.05 was statistically significant. PASW Statistic (SPSS) 18.0  was used to perform all statistical analyses.For all tests performed, a two-tailed Clinical characteristics of the overall population are shown in Table Imaging parameters are demonstrated in Table Thirty patients with LV thrombus from cine and LGE-CMR were mostly male and exhibited prior myocardial infarction without any differences in other coronary artery disease risk factors, atrial fibrillation, prior thromboembolic events, or baseline medication regimen including anticoagulation therapy.p-value < 0.0001). Significantly extensive apical scarring was found in patients with LV thrombus compared with no thrombus .Patients with LV thrombus did not differ from those without thrombus in aspects of LV volume and LVEF. Apical aneurysm was presented in significantly more patients with LV thrombus than without , while no difference of sphericity index was detected between patients with and without thrombus .For LV geometrical indexes, patients with LV thrombus had significantly greater apical area index than those without thrombus . Only the presence of apical scar and apical area index were determined as independent markers for thrombus.Six clinical and CMR variables including male gender, history of myocardial infarction, presence of apical aneurysm, ischemic cardiomyopathy scar pattern, apical scar, and apical area index were analyzed using multivariate analysis Table . Only thr = 0.73, p = 0.001) for the first observer in the initial analysis and 4 weeks later, respectively, and 42.52 ± 3.51  for the second observer in the initial analysis.Moderate intra- and inter-observer reliability were shown for apical area index measurements by cine-CMR. In 20 patients, mean apical area index values were 44.05 ± 4.79 and 43.18 ± 4.13 , Srichai et al. reported that CMR yielded more than 3-fold higher diagnostic accuracy for detection of thrombus then transthoracic echocardiography (87% vs. 27%) . ImproveScreening algorithms have been proposed that use the extent of wall-motion abnormalities on transthoracic echocardiography to trigger consideration of subsequent imaging with CMR . High apPrevious evidence indicated CMR as a non-invasive modality of choice to diagnose LV thrombus. Moreover, our study emphasized the importance of apical remodeling to provide apical area index assessment.From our study, patients with LV thrombus did not differ from those without thrombus in aspects of LV volume and systolic function, while male gender, history of myocardial infarction, apical aneurysm, apical scar, and ischemic scar were higher in the thrombus group. Most factors corresponded with prior studies such as history of myocardial infarction, ischemic scar and apical aneurysm , 14–17. p-value < 0.0001) as additional evidence demonstrating the significance value of apical change for thrombus formation.We determined apical scarring as a new predictor of LV thrombus. Extensive apical scarring was found significantly more in patients with thrombus than without thrombus .Scar volume has previously been established as a risk factor for thrombus formation . Our stuSphericity index and apical area index were the two geometrical parameters assessed for LV thrombus prediction in our study. The sphericity index was described according to substantial echocardiographic studies which evaluated LV systolic and diastolic dysfunction , 12. OneFrom our results, sphericity index failed to demonstrate any difference between the two groups, while apical area index in the LV thrombus group was significantly higher than the group without thrombus. Apical area index measurement is simple with acceptable reproducibility and add-on information to predict LV thrombus in patients with systolic dysfunction.Moreover, from multivariate analysis, only apical scar and apical area index were determined as independent predictors of LV thrombus. Combinations of these two factors will provide additional value in this aspect. This new index in other cardiomyopathies involving apex warrants further study.There were some study limitations, First, our study design cannot demonstrate prevalence of LV thrombus in the population; however, this was evident in previous study . Second,Apical area index is a new simple predictor of LV thrombus formation from cine-CMR in patients with systolic dysfunction.Results will be of value for two future research areas as 1) to identify the cut-off level of apical area index to accurately predicted thrombus in larger populations, and 2) to evaluate the association between apical area index and hard clinical outcomes such as stroke. If an association is present, patients may get benefits from early coagulation therapy. This entity warrants further study.


The PIROUETTE (PIRfenidOne in patients with heart failUre and preserved lEfT venTricular Ejection fraction) trial is designed to evaluate the efficacy and safety of the anti-fibrotic pirfenidone in patients with chronic heart failure and preserved ejection fraction (HFpEF) and myocardial fibrosis. HFpEF is a diverse syndrome associated with substantial morbidity and mortality. Myocardial fibrosis is a key pathophysiological mechanism of HFpEF and myocardial fibrotic burden is strongly and independently associated with adverse outcome. Pirfenidone is an oral anti-fibrotic agent, without haemodynamic effect, that leads to regression of myocardial fibrosis in preclinical models. It has proven clinical effectiveness in pulmonary fibrosis.31phosphorus magnetic resonance spectroscopy.The PIROUETTE trial is a randomised, double-blind, placebo-controlled phase II trial evaluating the efficacy and safety of 52 weeks of treatment with pirfenidone in patients with chronic HFpEF  and myocardial fibrosis (extracellular matrix (ECM) volume ≥ 27% measured using cardiovascular magnetic resonance). The primary outcome measure is change in myocardial ECM volume. A sub-study will investigate the relationship between myocardial fibrosis and myocardial energetics, and the impact of pirfenidone, using PIROUETTE will determine whether pirfenidone is superior to placebo in relation to regression of myocardial fibrosis and improvement in myocardial energetics in patients with HFpEF and myocardial fibrosis (NCT02932566).clinicaltrials.gov (NCT02932566) https://clinicaltrials.gov/ct2/show/NCT02932566The online version of this article (10.1007/s10557-019-06876-y) contains supplementary material, which is available to authorized users. As highlighted in recent commentaries, there is a disconnect between phase II and phase III drug trials in heart failure (HF); despite often promising phase II results, most phase III trials prove neutral or negative –3. The rHeart failure (HF) affects approximately 1–2% of the adult population in developed countries , 5. PoteExtracellular matrix (ECM) expansion secondary to excess collagen accumulation  is consistently demonstrated on a group level in myocardial tissue from patients with HFpEF, and there are considerable data demonstrating both the potential for myocardial ECM to have a primary aetiological role in HFpEF, and the adverse impact that ECM expansion has on myocardial mechanical, electrical and microvascular function –21. Notan = 410) with HFpEF or at risk for HFpEF (B-type natriuretic peptide (BNP) > 100 pg/ml but no clinical HF), with a clear “dose-response” relationship between ECM volume and outcome [Importantly, following previous smaller studies, Schelbert et al. showed myocardial ECM volume, measured using cardiovascular magnetic resonance (CMR) imaging (see below), was strongly associated with adverse outcome on multivariable analysis in a large cohort of patients , and human in vitro work –40. In kPirfenidone has proven to be safe and well tolerated in patients with pulmonary fibrosis in randomised controlled trials (RCT) and post-marketing surveillance –37, 51. PIROUETTE is a randomised, double-blind, placebo-controlled phase II trial designed to evaluate the efficacy and safety of pirfenidone in patients with HFpEF and myocardial fibrosis. The hypothesis is that pirfenidone will target a fundamental, prognostically important underlying pathophysiological mechanism of HFpEF, i.e. myocardial fibrosis, in individual HFpEF patients with evidence of myocardial fibrosis, leading to regression of myocardial fibrosis. If true, it is hypothesised that this will lead onto improvements in cardiac structure and function, fluid status and quality of life, and thus, ultimately, translate into improved outcome. The trial was designed by the research team. The trial has been registered (NCT02932566).The primary objective of this study is to evaluate whether pirfenidone compared to placebo leads to regression of myocardial fibrosis in patients with HFpEF and myocardial fibrosis. The secondary objectives are to determine the efficacy of pirfenidone compared to placebo with regard to improving ventricular structure and function, left atrial volume and function, aortic function, myocardial energetics, circulating markers of fluid status and myocardial injury, exercise tolerance and quality of life in patients with HFpEF and myocardial fibrosis. The study will also evaluate the safety of pirfenidone in patients with HFpEF and compare it to that of placebo, and record screening and recruitment data in order to inform a subsequent phase III study. Outcome measures are listed in full in Table The eligibility criteria are summarised in Table Recruitment to PIROUETTE began on March 7, 2017, after approval by a NHS Research Ethics Committee, the UK Medicines and Healthcare Products Regulatory Agency (MHRA) and the UK Health Research Authority (HRA). The study is being conducted in accordance with Good Clinical Practice and the Declaration of Helsinki.The study design is summarised in Fig. Potential participants are identified at four NHS hospital trusts in the North West of England, UK see , and areAfter confirmation of eligibility, participants are randomised in a 1:1 ratio to double-blind treatment with either pirfenidone or placebo. Randomisation is done using web randomisation software accessed using a secure website provided via the Clinical Trials Unit. Block randomisation, stratified by sex , is implemented, with computer generated randomisation allocations.The active treatment is pirfenidone (Esbriet) 2403 mg daily, taken orally as three 267 mg capsules three times per day. The comparator is placebo , taken as three capsules three times per day. After treatment with IMP is started, it is titrated, as tolerated, to the full dose of three capsules three times a day over a 14-day period, as follows: Days 1 to 7: one capsule, three times a day; days 8 to 14: two capsules, three times a day; day 15 onward: three capsules, three times a day. In participants who experience side effects, the IMP dose may be reduced, and subsequently re-escalated as appropriate. Every effort is made to maintain patients on the optimal dose (i.e. 9 capsules per day). The treatment period is 52 weeks.A target dose of pirfenidone of 2403 mg daily was chosen because it proved clinically effective and safe in pulmonary fibrosis –37. A trFollow-up visits are conducted at week 1 (telephone interview) and weeks 2, 4, 8, 13, 17, 21, 26, 39 and 52 . Unscheduled visits can occur at the investigators discretion . At follow-up visits, patients undergo a review of symptoms and concomitant medications, assessment of vital signs, physical examination, biochemistry and haematological laboratory investigations and an ECG. At the final visit (week 52), baseline procedures are repeated in order to assess the primary and secondary outcome measures. Participants who request to withdraw from the trial early undergo ‘exit’ data collection, equivalent to the final visit, provided they have received at least 6 months of IMP, for use in a sensitivity analysis. With specific consent, an additional blood sample is taken at baseline, 13, 26 and 52 weeks and stored in a central biorepository for future analysis.Whilst myocardial fibrosis is an important pathophysiological mechanism in HFpEF, the mechanisms by which it exerts a deleterious effect are not clear. A widely held hypothesis is that myocardial fibrosis impairs myocyte capillary blood supply and causes arteriolar vasomotor dysfunction, which leads to energy starvation of cells and impaired energetics. Indeed, there is evidence of impaired myocardial energetics in HFpEF, but the relationship between ECM expansion and myocardial energy metabolism has not been investigated . The hyp31phosphorus magnetic resonance spectroscopy (31P MRS) in a subgroup of patients who are due to be randomised  and a subgroup of patients without ECM expansion (i.e. ECM volume < 27%) but who otherwise meet eligibility criteria, in order to assess the relationship between myocardial ECM volume and energetic status. 31P MRS will then be repeated in randomised patients after 52 weeks of treatment in order to compare the change in energetic status in the pirfenidone group with the change in energetic status in the placebo group. The relationships between myocardial ECM volume, energetic status and mechanical properties will be investigated. Details of the 31P MRS procedure are given in the PCr:ATP ratio will be measured at baseline using Modifications to the PIROUETTE protocol are summarised in Table PIROUETTE is conducted by the research team, under the guidance of C.A.M. (Chief Investigator), in conjunction with Liverpool Clinical Trials Research Centre (CTRC), which is a UK Clinical Research Collaboration fully registered Clinical Trials Unit. The sponsor is Manchester University NHS Foundation Trust. The trial is funded by the UK National Institute for Health Research (NIHR). Roche Products Limited has gifted the IMP. NIHR and Roche Products Limited have had no role in the study design other than through their external peer review processes. A trial steering committee (TSC) provides overall supervision for the trial and provides advice through its independent Chairman. An independent data and safety monitoring committee (IDSMC) is responsible for reviewing and assessing recruitment, interim monitoring of safety and effectiveness, trial conduct and external data and submits periodic reports to the TSC. Further details are in the Thirty-seven participants per group are required to detect an absolute minimum difference, between pirfenidone and placebo groups, of 2% in terms of change in CMR ECM volume from baseline following 52 weeks of treatment, with 80% power at a 5% significance level (2-sided), assuming a standard deviation of the within-patient differences from baseline equal to 3%, as per Garg et al. . This efThirty-three participants per group are required to detect an absolute minimum difference in PCr/ATP ratio of 0.37 between ECM expansion and no ECM expansion groups at baseline , assuming a standard deviation of the between group differences of 0.52, as per Phan . Twenty-The trial will be analysed and reported using the ‘Consolidated Standard of Reporting Trials’ (CONSORT) and the International Conference on Harmonisation E9 guidelines. All primary analyses will be on an intention to treat basis including all randomised participants retained in their randomised treatment groups. Secondary causal analyses (according to dose and duration of intervention) will also be undertaken to assess the causal impact of treatment received. Analyses of covariance will be used to compare myocardial ECM volume (and other measures) between pirfenidone and placebo groups, adjusting for baseline ECM volume. The conventional 5% significance level will be used.t test assuming the measurements are normally distributed, with transformation as necessary. Correlation analysis will be used to assess the relationships between PCr/ATP ratio, mechanics and ECM volume. PCr/ATP ratio will be compared between pirfenidone and placebo groups using analyses of covariance, adjusting for baseline PCr/ATP ratio.For the sub-study, PCr/ATP ratio and mechanical variables will be compared between patients with and without ECM expansion at baseline using an independent ‘The path forward to improve HF trials needs the connecting of biological pathways, drug mechanisms of action, and underlying pathophysiology’ .HFpEF is a diverse syndrome that involves multiple pathophysiological mechanisms . Indeed,The PIROUETTE trial specifically targets HFpEF patients with myocardial fibrosis. Myocardial fibrosis is a key pathophysiological mechanism of HFpEF and myocardial fibrotic burden is strongly and independently associated with adverse outcome in HFpEF . RecruitThe intervention, pirfenidone, is an anti-fibrotic agent that leads to substantial regression of myocardial fibrosis in preclinical models, and has proven clinical effectiveness in pulmonary fibrosis. Importantly from a mechanistic point of view for this study and for the wider cardiovascular field, pirfenidone does not have a haemodynamic effect. Myocardial fibrosis regression has been observed in humans following interventions with haemodynamic effects, both drug and mechanical, but, to our knowledge, myocardial fibrosis regression has not been observed in humans with a ‘dedicated’ anti-fibrotic, agent i.e. without haemodynamic effect –27, 60. Choice of primary outcome variable in phase II HF trials has proven challenging . For exaElevated circulating natriuretic peptide levels are required for study entry in order to increase the diagnostic confidence of HF . A LVEF The sub-study is designed to provide additional mechanistic insight into the pathophysiology of HFpEF, the action of pirfenidone and, more broadly, the myocardial fibrosis paradigm. The data generated by the sub-study will provide additional support for a subsequent phase III study, if the primary outcome is reached.The PIROUETTE trial will evaluate the efficacy and safety of pirfenidone in patients with HFpEF and myocardial fibrosis. By targeting a known prognostically important mechanism of HFpEF, i.e. myocardial fibrosis, with an intervention designed to modulate this mechanism, using a primary outcome measure specific to this mechanism and sensitive to its biological modulation, and patient recruitment personalised to individual expression of this mechanism, PIROUETTE has been designed to overcome the disconnect between phase II and III HF trials and maximise the chances of both successfully modulating an important mechanism of HFpEF, and translating the findings, if positive, into phase III success. As such, PIROUETTE could serve as a blueprint for future phase II HF trials.ESM 1(DOCX 53 kb)