The childhood heart disease of Friedreich’s Ataxia (FRDA) is characterized by hypertrophy and failure. It is caused by loss of frataxin (FXN), a mitochondrial protein involved in energy homeostasis. FRDA model hearts have increased mitochondrial protein acetylation and impaired sirtuin 3 (SIRT3) deacetylase activity. Protein acetylation is an important regulator of cardiac metabolism and loss of SIRT3 increases susceptibility of the heart to stress-induced cardiac hypertrophy and ischemic injury. The underlying pathophysiology of heart failure in FRDA is unclear. The purpose of this study was to examine in detail the physiologic and acetylation changes of the heart that occur over time in a model of FRDA heart failure. We predicted that increased mitochondrial protein acetylation would be associated with a decrease in heart function in a model of FRDA.A conditional mouse model of FRDA cardiomyopathy with ablation of FXN (FXN KO) in the heart was compared to healthy controls at postnatal days 30, 45 and 65. We evaluated hearts using echocardiography, cardiac catheterization, histology, protein acetylation and expression.Acetylation was temporally progressive and paralleled evolution of heart failure in the FXN KO model. Increased acetylation preceded detectable abnormalities in cardiac function and progressed rapidly with age in the FXN KO mouse. Acetylation was also associated with cardiac fibrosis, mitochondrial damage, impaired fat metabolism, and diastolic and systolic dysfunction leading to heart failure. There was a strong inverse correlation between level of protein acetylation and heart function.These results demonstrate a close relationship between mitochondrial protein acetylation, physiologic dysfunction and metabolic disruption in FRDA hypertrophic cardiomyopathy and suggest that abnormal acetylation contributes to the pathophysiology of heart disease in FRDA. Mitochondrial protein acetylation may represent a therapeutic target for early intervention. Heart disease and heart failure exert a significant morbidity and mortality burden worldwide. Abnormal metabolism and metabolic remodeling are common pathologic features in heart disease, such as in obesity and diabetes-related cardiomyopathy , 2, isch+-dependent deacetylase, sirtuin 3 (SIRT3). SIRT3 responds to cellular energy status and utilizes an “acetylation switch” to modify protein function [Mitochondrial protein acetylation is an important post-translational regulatory mechanism of heart metabolism that has emerged in recent years. Acetylation in mitochondria is controlled by the NADfunction  in orderfunction . SIRT3 tfunction , 11, pyrfunction , acetyl function , and elefunction –16. Addifunction . It is wfunction .Beneficial effects of SIRT3 activity in the heart are well documented. For example, SIRT3 has been shown to protect against oxidative stress, attenuate fatty acid accumulation in the heart , and pre+ precursors can increase NAD+ levels in mitochondria, activate sirtuins to reduce protein acetylation, and improve cardiac outcome [Acetylation is most exciting in the context of heart disease in that it has potential as a therapeutically modifiable target. For example, recent work has shown that exogenous treatment with NAD outcome , 24.+/NADH ratio, and increased oxidative stress [The heart disease of Friedreich’s Ataxia (FRDA) results from inherited deficiency of frataxin (FXN), a mitochondrial protein important in energy homeostasis. FXN is a highly conserved mitochondrial matrix protein that functions in iron-sulfur cluster assembly, which is integral to mitochondrial metabolic machinery . Reducede stress –28. In ae stress . There iMice with conditional loss of FXN in the heart develop cardiac hypertrophy as early as 5 weeks of age, followed by transition to dilated cardiomyopathy and heart failure by approximately 8 weeks of age . Certain+ bioavailability as a result of dysfunctional energy metabolism, and possibly by direct oxidative modification of the native protein. The impact of abnormal acetylation in the FRDA heart has not been investigated. Because of the important role that acetylation plays in cardiac function, we believe that loss of SIRT3 and resultant mitochondrial protein hyper-acetylation contributes to the heart disease of FRDA.We previously reported that mitochondrial proteins in FRDA mouse model hearts have increased acetylation and decreased SIRT3 activity, resulting in increased acetylation of several important metabolic enzymes, including LCAD, MCAD, AceCS2, and the ETC , 31. TheThe purpose of the present study was to document the changes in physiology and function that evolve over time in a model of FRDA heart failure using both non-invasive (ECHO) and invasive left heart catheterization, and match the trajectory of cardiac pathophysiology to the protein acetylation profile along the course of disease in order to determine the link between acetylation of mitochondrial proteins and FRDA heart disease.fl/fl) [We used an established conditional mouse model of FRDA heart failure with absence of FXN in the heart (FXN KO) compared to healthy littermates (FXNfl/fl) . We examfl/fl) .There were no functional or anatomic differences between hearts of FXN KO and control mice at day 30 .fl/fl = 0.64 ±0.08 mm; p<0.001), increased relative wall thickness (RWT) . FXN KO at day 45 also show findings consistent with both cardiomegaly and hypertrophy with increased ECHO-derived LV:body  and gross heart (mg):body weight (g) ratios .Cardiac hypertrophy is manifest by day 45 in FXN KO animals . CompareEarly-to-Atrial waves (E/A) on Doppler imaging is increased at day 45  and 65 , as well as isovolumic relaxation time (IVRT) at day 45  and day 65 . Left ventricle relaxation rate (-dP/dt) is decreased in FXN KO compared to controls at days 45  and 65 . Finally, Tau, the LV relaxation time constant, is increased in FXN KO compared to controls at both day 45  and 65 , demonstrating prolonged relaxation phase in diastole.Diastolic dysfunction occurs early, is concomitant with presentation of LV hypertrophy, and is persistent in FXN KO hearts. FXN KO mice demonstrate multiple relaxation abnormalities at day 45 , Table 1fl/fl = 143.3 ±32.1 mWatt; p<0.001) as well as rate of contraction (+dP/dt) . Again, at day 65, FXN KO mice demonstrate significantly depressed contractility (+dP/dt)  compared to control mice  (p<0.001) in addition to decreased maxPower .Contractility abnormalities occur in FXN KO mice as early as postnatal day 45 on invasive measurements, with progressive decline in systolic indices on day 65 measurements , Table 1fl/fl = 47.7 ±8.2%, p<0.001) and fractional shortening (FS) . FXN KO mice at day 65 also demonstrate reductions in both cardiac output (CO)  and cardiac index (CI)  compared to controls.FXN KO mice progress to overt systolic heart failure on ECHO by postnatal day 65. Compared to controls, FXN KO animals have significantly reduced EF (FXN KO = 23.9 ±12.5% vs. FXNfl/fl (3.91 ±0.14 mm)(p = 0.006), providing evidence of a dilated LV. Further, the end-cycle blood volumes of the chamber in diastole are markedly increased in FXN KO at 65 days. Left ventricle end-diastolic volume (EDV) data collected with catheter-based direct volume measurements show that day 65 FXN KO mice have an increased EDV that is almost twice that of FXNfl/fl , causing a dramatic rightward-shift in their pressure-volume curves  (p<0.001). FXN KO mice at day 65 also demonstrated close to a 50% increase in gross heart:body weight ratio (8.81 ±1.5) than that measured at day 45 (5.90 ±1.7), and significantly higher than age-matched controls (4.70 ±0.26) (p<0.001).FXN KO mice at 65 days of age maintain earlier findings of cardiomegaly on ECHO-derived LV:body weight ratios (5.71 ±1.19) compared to FXNWe previously demonstrated that acetylation in the conditional FXN KO heart is dramatically increased in late stages of heart disease and that the majority of protein acetylation was localized to mitochondria . Here, wWe show that acetylation is modestly increased prior to onset of detectable cardiac dysfunction in the FXN KO heart at day 30. This is followed by a dramatically rapid and progressive increase in protein acetylation compared to controls . We measr = -0.923) and FS (r = -0.927)  .To demonstrate that mitochondrial deacetylase activity is decreased or absent in the FXN KO hearts, we probed for acetylation of known SIRT3 mitochondrial protein targets, SOD2 and LCAD. SOD2 acts to destroy superoxide radicals and is inhibited when acetylated at lysine 68. SIRT3 reverses acetylation , 34. LCAConsistent with previous reports , 32, we p = 0.011), and the average percentage of mitochondrial area containing abnormal mitochondria in FXN KO (78.1±26.7%) was significantly higher than in control micrographs in which no abnormalities were detected (p = 0.002)  .p<0.001) when measured by Clark Oxygen electrodes using complex I substrates glutamate and malate  in FXN KO cardiac mitochondria (1.67 ±0.12) were significantly decreased compared to controls (4.86 ±0.37)   . We did = 0.002) . DegenerVentricular tissue of FXN KO at day 65 demonstrated a combined pattern of macro- and microsteatosis in cardiomyocytes .Because mice with loss of MCAD, or VLCAD in the heart quickly become hypothermic when exposed to cold environment and die , 39, andThis study is the first to document the progression of lysine-acetylated proteins and demonstrate a correlation between acetylation and cardiac function in the heart of the conditional FXN KO mouse model of FRDA heart disease. We have characterized the initial stages of cardiac dysfunction and evolution to heart failure in this animal model, which mirrors that of the FRDA human heart disease. We conclude that protein lysine acetylation increases in a temporally progressive pattern and that there is a strong negative correlation between level of acetylation in the heart and global heart function: as acetylation increases, ejection fraction and fractional shortening decrease.We found that abnormal post-translational modification of key metabolic enzymes has occurred prior to detectable impairment in heart function in the FXN KO. Acetylation is increased at day 30 and continues to progress over time. The first notable abnormal traits on cardiac pathophysiology occur at 45 days of age, with thickening of the left ventricular wall and diastolic dysfunction. As acetylation progresses, heart function continues to decline and demonstrates features of decompensated left ventricular dilation, decreased ejection fraction and fractional shortening, consistent with systolic heart failure. These results suggest that targeting acetylation early would provide the best chance to halt or slow progression of FRDA heart disease. Modifying acetylation only after detectable cardiac dysfunction occurs may miss the opportunity to prevent irreversible hemodynamic changes in the FXN KO heart.+ and to oxidative damage of SIRT3 [Loss of SIRT3 results in maladaptive ventricular remodeling in response to stress , abnormaof SIRT3 . The finof SIRT3 , 19. Forof SIRT3 , mirrori+/NADH ratio in the FXN KO heart would stimulate SIRT3 function, recover activity of targeted metabolic enzymes, and improve measured cardiac outcome. Recent work has shown that exogenous treatment with NAD+ precursors can increase NAD+ levels in mitochondria, reduce protein acetylation, and activate sirtuins [More studies are needed in order to determine a causative role of abnormal acetylation in leading to impairment of hearts with FXN loss. For instance, it would be of great interest to determine whether normalizing the NADsirtuins , 24. TogOne limitation of this study may be that the MCK promoter used to drive Cre expression in this model is expressed in all sarcomeric tissues, including skeletal muscle. It is not expected that loss of FXN in skeletal muscle would significantly alter cardiac function in this study. Indeed, in patients with FRDA, and in the original report of this mouse model , there iIn conclusion, we demonstrate a close relationship between mitochondrial protein acetylation, cardiac dysfunction and metabolic disruption in a model of FRDA hypertrophic cardiomyopathy. Our results suggest that abnormal acetylation contributes to the pathophysiology of heart disease in FRDA and may represent a therapeutic target for early intervention.fl/fl mice to create FXN KO mice that were homozygous for heart and skeletal muscle deletion of FXN (MCK-Cre:FXN KO), as previously published [fl/fl).This study was approved by the Institutional Animal Care and Use Committee of Indiana University. We used site-specific Cre-lox recombination to carry out gene deletions of interest. We used FXNublished . Data wa® 2100 ultrasound machine for small animal imaging and MS400 transducer . Functional parameters of the left ventricle and outflow tracts were measured using standard assessment techniques. Relative wall thickness (RWT) was calculated by (2*LVPWd)/LVIDd).Mice were anesthetized with isoflurane and placed on a warming mat with continuous monitoring. Transthoracic ECHO images were obtained using a VisualSonicsMice were anesthetized with isoflurane and placed on a warming mat with continuous monitoring. Pressure-volume loops were obtained directly using a 1.2 F microconductance catheter , which was inserted into the right carotid artery through a small neck incision and advanced retrograde into the left ventricle. The ADVantage pressure volume system  was used to acquire pressure, admittance, phase shift, and amplitude, and real time pressure-volume data was displayed and analyzed offline using specialized software  .fl/fl (n = 6) were selected for cold exposure. Mice were deprived of food for 6 hours prior to onset of study and for the duration of experiment. A baseline rectal temperature was taken prior to placement in 4°C cold room and then every 30 minutes thereafter for a total of 3 hours. A study was terminated for humane reasons if core body temperature reached 19°C or less.This experiment was modeled after similar studies , 39. 65 Ventricular tissue selected for histology was fixed in 10% formalin and paraffin embedded. Histological analysis included hematoxylin and eosin (H&E) and Masson’s Trichrome to detect collagen as a measure of fibrosis. Collagen quantification was performed using ImageJ (IJ1.46) to measure percent area of tissue positively stained for collagen. Three to five 20x digital micrographs were obtained from each tissue section that underwent quantification studies.Ventricular tissue selected for electron microscopy was fixed in 2.5% glutaraldehyde and underwent sectioning and uranyl acetate staining by the Electron Microscopy Center of Indiana University School of Medicine. At least 3 separate sections from each animal strain were selected for quantification analysis. Quantification methods were similar to methods used in our previous work . AbnormaMitochondrial isolation followed the method described in our previous work . FreshlyMitochondrial isolates designated for use in respiration assays were resuspended in mitochondrial respiration buffer. A total of 2–3 mouse heart mitochondria were combined for each run, with n = 4–6 pooled heart mitochondria assayed for controls and n = 8–12 pooled heart mitochondria assayed for FXN KO. A Clark-oxygen electrode was used for respiration assays. Glutamate and malate were used as a substrate for all assays.Primary antibodies included anti-acetyl-lysine , anti-SIRT3 , anti-SOD2 (acetyl K68) (Abcam ab137037), anti-SOD2 , anti-VDAC , anti-GAPDH , anti-FXN , anti-LCAD (Abcam ab196655) and to the electron transport chain complexes: anti-CI-NDUFA9 (Abcam ab14713), anti-CII-SDHB (Abcam ab14714) and anti-CIII-UQCRFS1 (Abcam ab14746). Band intensities were measured using ImageJ (IJ1.46). To determine correlation between acetylation and heart function, relative density of acetylation for n = 3 western blot images was normalized to day 30 controls and averages for each group were used for correlation calculation.p-value of <0.05 was considered statistically significant.All calculations, analyses and graphs were performed using SigmaPlot . Two groups were compared using a two-tailed student’s t test for samples with equal variance. Final data are presented as mean (±SD). Alpha was set at 0.05 and power at 0.800. A S1 Figfl/fl controls. (h) FXN KO mice demonstrate significantly depressed global contractility function compared to controls for ejection fraction  and (i) fractional shortening . E/A = ratio of the early (E) to late (A) ventricular filling velocity; -dP/dt = -Δintra-ventricular pressure/Δtime; +dP/dt = +Δintra-ventricular pressure/Δtime. * = p<0.05.FXN KO mice demonstrate cardiac hypertrophy at ages 45 and 65 days on (a) ECHO-derived left ventricle:body mass ratio (LV:body) and (b) left ventricular posterior wall thickness in diastole (LVPWd). Diastolic indices in the FXN KO mice are abnormal; with increases in (c) mitral valve Doppler flow ratio (E/A) and (d) isovolumic relaxation time (IVRT), and (e) decreased rate of left ventricle relaxation (-dP/dt). (f) FXN KO (days 45 and 65) had significantly slower rates of contraction (+dP/dt) and (g) maximum left ventricular power (maxPower) compared to FXN(TIF)Click here for additional data file.S1 File(XLSX)Click here for additional data file.


Cardiac autonomic neuropathy (CAN) is a frequent but underdiagnosed complication of diabetes mellitus. It has a strong influence on various cardiac disorders including myocardial ischemia and infarction, hypertension, orthostatic hypotonia, heart failure, and arrhythmias. CAN can lead to severe morbidity and mortality and increase the risk of sudden cardiac death.  This review article summarizes the latest evidence regarding the epidemiology, pathogenesis, influence on the cardiovascular system, and diagnostic methods for CAN. The methodology of this review involved analyzing available data from recent papers relevant to the topic of diabetic autonomic neuropathy and cardiac disorders.  The early diagnosis of CAN can improve the prognosis and reduce adverse cardiac events. Methods based on heart rate variability enable the diagnosis of CAN even at a preclinical stage. These methods are simple and widely available for use in everyday clinical practice. According to the recently published Toronto Consensus Panel on Diabetic Neuropathy, all diabetic patients should be screened for CAN. Because diabetes mellitus often coexists with heart diseases and the most common methods used for diagnosis of CAN are based on ECG, not only diabetologists but also cardiologists should be responsible for diagnosis of CAN. Diabetes mellitus (DM) affects at least 8.5% of the global population, that is, about 422 million people worldwide . Diabetep < 0.01) [There is huge variation in CAN prevalence depending on the diagnostic methods used, population studied, and disease stage. As reported in major studies, the number of patients with CAN varies from 17% to 90% in patients with DM type 1 and 27.5% to 73% in patients with DM type 2 . The dur < 0.01) .CAN is caused by complex interactions and involves several mechanisms and pathways that lead to neuronal ischemia and finally neuronal death , 9. The The early stages of CAN damage the vagus nerve, thus leading to sympathetic predominance. This increase in sympathetic tone continues until advanced CAN, when sympathetic denervation also ensues .Recent papers have also reported an association between hypoglycemia and the autonomic nervous system. Cichosz et al. found that the HRV parameter low frequency (LF) was significantly reduced during hypoglycemic episodes in patients with and without CAN . Jaiswalp < 0.001) [Coronary artery disease (CAD) is a major complication of DM. The most typical clinical evidence of CAN in patients with concomitant CAD is silent myocardial ischemia (SMI) . A meta-< 0.001) . Several< 0.001) . The Fra< 0.001) . A study< 0.001) . Feature< 0.001) . AlthougAutonomic dysfunction, assessed as reduced heart rate variability, was also associated with coronary artery calcification , 23. WheHypertension (HT) is well-known to place patients at risk for heart disease and often coexists with diabetes mellitus. Persistent HT increases the morbidity and mortality risk. Both the parasympathetic and sympathetic nervous systems innervate the heart and control heart rate (HR) (chronotropic activity) and strength of compression (inotropic activity). Only the SNS innervates the vasculature, thereby controlling peripheral resistance, and mediates the baroreceptor reflex (BRR), which in turn mediates blood pressure (BP). The angiotensin-renin system controls fluid levels in the body, including blood volume. Angiotensin directly affects the SNS, and the SNS indirectly affects angiotensin . ConditiA frequent clinical manifestation of CAN is orthostatic hypotension. This phenomenon is defined as a decrease in systolic blood pressure > 20 mmHg (or >30 mmHg in hypertensive patients) or diastolic blood pressure > 10 mmHg when changing body position from supine to standing . A changAlso, there are clinical data indicating that CAN is associated with arterial stiffness –30. ArteCAN can lead to abnormalities in the left ventricular systolic and predominantly diastolic function. Echocardiographic studies showed that CAN is significantly associated with reduction in the peak diastolic filling and an increase in the atrial component of diastole . Also, MThe ANS has an important influence on heart rhythm. The sinus node is innervated by the PNS and SNS, and the balance between these systems is important for control of HR.Inappropriate sinus tachycardia is a common manifestation of CAN that occurs at a relatively early stage of the disease. An HR > 90 bpm can be observed as a result of parasympathetic withdrawal. A fixed HR without changes during sleep, exercise, or stress is a sign of complete cardiac denervation. An impaired HR response to exercise leads to exercise intolerance .p < 0.01). This study also revealed that the presence of CAN caused a significant increase in P-wave duration and dispersion. That inhomogeneous atrial depolarization is the potential trigger of paroxysmal AF in patients with CAN [The arrhythmia typically associated with changes of the ANS is atrial fibrillation (AF). In the 1990s, Phillipe Coumel stated that AF is not a homogeneous entity, and many factors are responsible for a number of different behaviors. He observed that the electrophysiological characteristics of atrial cells  are modulated differently by parasympathetic and sympathetic influences. Parasympathetic effects tend to favor macroreentry phenomena, whereas sympathetic ones favor abnormal automaticity and triggered activity . In normwith CAN .The influence of CAN on nonsustained ventricular arrhythmias, according to our knowledge, is not well-documented. However, life-threatening ventricular arrhythmias and sudden cardiac deaths are clearly associated with CAN , 7, 8.p < 0.0001) for CAN patients. The magnitude of the association was stronger for those studies for which two or more measures were used to define CAN. The pooled relative risk for studies that defined CAN with the presence of two or more abnormalities was 3.45  compared with 1.20  for studies that used one measure. CAN also had the strongest association with mortality among other risk factors in the EURODIAB IDMM Complications Study [CAN is associated with an increased total and cardiovascular mortality. In a meta-analysis of 15 studies, Maser et al.  found thns Study . In the ns Study .CAN is also associated with a higher risk of malignant ventricular arrhythmias and sudden death . On the Other mechanisms depending on autonomic imbalance, such as impaired response to hypoxic state, reduced hypoglycemia awareness, and prolonged hypoglycemia episodes, can also be responsible for malignant ventricular arrhythmias and finally lead to sudden death , 44.Traditionally, the diagnostic methods of cardiac autonomic neuropathy are based on heart rate variability (HRV) and changes in BP during certain physiological maneuvers. In the 1970s, Ewing et al.  proposedThe first two tests measure parasympathetic function—mainly the ability of the vagal nerve to slow the HR during procedures which increase HR. The Valsalva maneuver primarily represents also parasympathetic activity, but autonomic changes also include a sympathetic component. The last two tests show changes in sympathetic function and involve baroreflex-mediated blood presser fluctuations. The American Diabetes Association recommends the use of these Ewing's tests in the diagnosis of CAN .According to the Toronto Diabetic Neuropathy Expert Group, the most widely used tests assessing cardiac parasympathetic function are based on the time-domain HR response to deep breathing, a Valsalva maneuver, and postural change. Of these tests, HR to deep breathing has the greatest specificity (∼80%). Cardiovascular sympathetic function is assessed by measuring the BP response to orthostatic change and a Valsalva maneuver . The expThe short-term ECG recordings can be analyzed by dedicated software in the frequency domain. This method usually uses the Fourier method, which transforms R-R intervals into waves with three basic components: very low frequency ≤ 0.04 Hz (VLF), low frequency 0.04–0.15 Hz (LF), and high frequency 0.15–0.4 Hz (HF). HF represents vagal activity, whereas LF combines the effect of sympathetic and parasympathetic influence. A decrease in HF is a sign of parasympathetic dysfunction, in the early stages of autonomic dysfunction in diabetes, when sympathetic predominance is observed it leads to an increase in LF/HF .It is not clear if classical Ewing's tests or time-domain methods are better for diagnosis of CAN. Studies comparing the two methods showed high—over 80%—correlations between their results . HoweverAnother diagnostic method of CAN can be based on HRV assessed during classic 24 h Holter ECG monitoring and use of statistical indexes in the time and frequency domain. It is obvious that reduction in HRV is associated with CAN, but this method has no standard values for the diagnosis of CAN . Also, dThe baroreceptor reflex (BRR) is another method that can be used for detecting CAN. In the physiological BRR, an increase of BP induces an increase in the vagal cardiac efferent and reduction in the sympathetic activity, resulting in bradycardia, hypotension, and peripheral vasodilatation . A reducAnother Holter-based technique for evaluating CAN is heart rate turbulence (HRT). HRT is an indirect measurement of baroreflex sensitivity and refers to sinus rhythm fluctuations following premature ventricular beat. Physiologically, after ventricular beat, sinus rate acceleration and next deceleration are observed. There are two components of HRT—turbulence onset (TO) and turbulence slope (TS). The initial heart rhythm acceleration after ventricular premature beat is defined as TO and the ensuring deceleration as TS . HRT par123I-metaiodobenzylguanidine (123I-MIBG), 11C-metahydroxyephedrine (11C-HED), and 11C-epinephrine [Other methods currently used in the detection of CAN are single photon emission computed tomography (SPECT) and positron emission tomography (PET) with sympathetic neurotransmitter analogs such as nephrine .The lack of standardization, the high cost, and the requirement of specific equipment and skilled staff restrict the role of scintigraphy as a research tool, and it is not a part of everyday clinical practice.According to the latest Standards of Medical Care in Diabetes published by the American Diabetes Association, symptoms and signs of autonomic neuropathy should be assessed in patients with microvascular and neuropathic complications. CAN may be completely asymptomatic and detected only by decreased heart rate variability with deep breathing. Major clinical manifestations of CAN include resting tachycardia (>100 bpm) and orthostatic hypotension  .Ewing's tests  are the For bedside testing, a dedicated software can be used. The authors have personal experience with ProSciCard III , which offers the possibility of an online ECG measurement and a subsequent offline detailed analysis with comparison to normal values. The tests included in the software are RR variability at rest , RR variability during deep breathing, Valsalva test (10 or 15 s), and orthostasis test. The results are not only typical Ewing's parameters but also time and frequency domain HRV during each test. Examples of normal tests are presented in Figures CAN is a frequent chronic complication of DM with potentially life-threatening outcomes. Although there are available simple bedside tests for diagnosis of CAN, it is often overlooked. The statements of the Toronto Diabetic Neuropathy Experts Group recommended screening for all diabetic patients . Because


Both papillary muscle infarction (PMI) and chronic ischemic mitral regurgitation (CIMR) are associated with reduced survival after myocardial infarction. The influence of PMI on CIMR and factors influencing both entities are incompletely understood.We sought to determine the influence of PMI on CIMR after primary percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI) and to define independent predictors of PMI and CIMR.Between January 2011 and May 2013, 263 patients (mean age 57.8 ± 11.5 years) underwent late gadolinium-enhanced cardiac magnetic resonance imaging and transthoracic echocardiography 4 months after PCI for STEMI. Infarct size, PMI, and mitral valve and left ventricular geometric and functional parameters were assessed. Univariate and multivariate analyses were performed to identify predictors of PMI and CIMR (≥grade 2+).P < 0.001). In multivariate analyses, infarct size , inferior MI , and circumflex infarct-related artery  were independent predictors of PMI. Age , infarct size , tethering height , and interpapillary muscle distance  were independent predictors of CIMR.PMI was present in 61 patients (23 %) and CIMR was present in 86 patients (33 %). In patients with PMI, 52 % had CIMR, and in patients without PMI, 27 % had CIMR (The risk of PMI is mainly associated with inferior infarction and infarction in the circumflex coronary artery. Although the prevalence of CIMR is almost doubled in the presence of PMI, PMI is not an independent predictor of CIMR. Tethering height and interpapillary muscle distance are the strongest independent predictors of CIMR. Both papillary muscle infarction (PMI) and chronic ischemic mitral regurgitation (CIMR) are associated with reduced survival after myocardial infarction (MI) –3.Ischemic mitral regurgitation (IMR) is a common complication of MI with an estimated incidence of 20–50 % –8. IMR iPMI has a strong (negative) prognostic value after MI . This maIn this study, we sought to determine the influence of PMI detected by LGE cardiac MRI on CIMR after primary percutaneous coronary intervention (PCI) for ST-segment elevation myocardial infarction (STEMI) and to determine independent predictors of PMI and CIMR.This study was performed as a substudy of the glycometabolic intervention as adjunct to primary percutaneous intervention in ST elevation myocardial infarction (GIPS)-III trial  . The GIPBetween January 2011 and May 2013, 380 patients were enrolled in the GIPS-III trial. A total of 275 patients underwent cardiac MRI and transthoracic echocardiography (TTE) 4 months after PCI. 263 patients had an evaluable cardiac MRI, and were eligible for the current substudy. None of these patients had a history of (organic) mitral valve disease.Standard laboratory assessment including serum concentrations of creatinine phosphokinase (CK) was performed.Coronary angiography and coronary intervention were performed using standard techniques. The choice and order of coronary intervention  was left to the discretion of the operator. Perfusion was evaluated according to the TIMI criteria . MyocardCardiac MRI was performed 4 months post-PCI with a 3.0 Tesla clinical scanner  using a phased array cardiac receiver coil. Electrocardiogram-gated cine steady-state, free precision magnetic resonance images were acquired during repeated breath holds in the standard long-axis views  and contiguous short-axis slices of 1 cm covering the entire LV were used to assess global and regional ventricular function and to calculate LVEF. Using identical slice locations, late contrast-enhanced images were acquired 10 min after intravenous administration of a gadolinium-based contrast agent  with an inversion recovery, gradient echo pulse sequence to identify the location and extent of MI and PMI. The inversion time was set to null the signal of viable myocardium for every individual patient.Images were stored and sent to an independent cardiac MRI core laboratory  for assessment by fully blinded operators. Additional assessment of PMI and mitral valve geometry was performed using an open-source software package (OsiriX Imaging Software).Summation of the volumes per slice of areas of hyperenhancement was outlined, allowing calculation of total infarct size (% LV myocardium). PMI was evaluated by LGE cardiac MRI images. Cine images of the same location were used as a side-by-side reference for localizing the PM within the blood pool during interpretation of contrast-enhanced images. PMI was considered present if any papillary hyperenhancement was present on LGE images. PMI was further categorized by location  and extent  or complete (>50 % hyperenhanced papillary myocardium) on LGE short-axis images Fig.  11].Fig.Fig11].FLeft atrial volume was calculated using the summation of slices method multiplied by slice thickness. Left ventricular end-diastolic diameter (LVEDD) and left ventricular end-systolic diameter (LVESD) were measured in the short-axis view at mid-LV level. Additionally, the systolic sphericity index (SSI) (ratio of LV width to length) was measured in the four-chamber view at end-systole. Interpapillary muscle distance (IPMD) was measured in the short-axis view at end-systole. On the stack of short-axis cines, the endocardial and epicardial borders were outlined in end-systolic and end-diastolic images. Left ventricular end-diastolic volume (LVEDV) and left ventricular end-systolic volume (LVESV) were calculated using the summation of slice method multiplied by slice thickness. LVEF was calculated as LVEF = 100 % × (LVEDV-LVESV)/LVEDV. Regional LV contractile function was graded with the wall motion score index (WMSI) using a 17-segment, 5-point scoring system .Mitral annular diameter, tethering height , tethering area  posterior tethering angle, and anterior tethering angle were measured in the 3-chamber view (mid-systolic) Fig.  9].Fig..Fig.9].FTTE was performed with commercially available equipment  with a phased array transducer. CIMR was defined as MR caused by MI in the absence of structural mitral valve abnormalities and present 4 months after PCI. Based on the echocardiography guidelines –19, the t test or Mann–Whitney U test as appropriate for continuous variables. Univariate variables with P <0.10 were included in the multivariate analysis. Age and gender were forced in all multivariate models. Multivariate logistic regression analyses by means of a forward stepwise algorithm  were performed to identify independent predictors of PMI and CIMR. Odds ratios were reported with 95 % confidence intervals (CI). Goodness-of-fit of the final logistic regression models was assessed with the Hosmer–Lemeshow statistic.Continuous variables were expressed as mean ± SD. Categorical variables were expressed as percentages. Comparisons between groups were performed using Pearson’s Chi-square test or Fisher`s exact test as appropriate for categorical variables and the independent samples P < 0.05.All calculations were performed using commercially available statistical packages . Statistically significant differences were defined as P < 0.001).A flowchart for this substudy is shown in Fig. PMI was found in 61 patients (23 %). Posteromedian PMI was found in 42 patients (69 %), Anterolateral PMI was found in nine patients (15 %), and combined PMI was found in ten patients (16 %). An overview of the IRA according to type of PMI is provided in Table χ2 = 13.85, df = 8, P = 0.086).Univariate and multivariate logistic regression analyses of PMI are shown in Tables χ2 = 4.87, df = 8, P = 0.772).Univariate and multivariate logistic regression analyses of CIMR are shown in Tables LGE cardiac MRI is the technique of choice for detecting scar tissue and fibrosis formation after MI and the high resolution of this technique permits careful delineation of partial or complete involvement of the PM in the infarcted area , 11, 20.In patients with PMI, the posteromedian PM was involved in 85 % and the anterolateral PM was involved in 31 %. The posteromedian PM is known to be more prone to ischemia/infarction (and rupture) due to its dependence on single blood supply from the posterior descending coronary artery (which is either derived from the LCx or from the RCA) , 29. TheInfarct size, inferior MI, and the LCx as IRA were independent predictors of PMI in this study. Two other studies also showed that in patients with PMI infarct size is generally larger on MRI, that myocardial scar most often involves the lateral and inferior walls, and that the IRA is most often the RCA or LCx , 11.PMI also has been shown to have a strong (negative) prognostic value , which cCIMR was defined as MR 4 months after PCI for STEMI present on TTE with a jet area/LA area of ≥10 % (grade 2+) in the mid-systolic apical four-chamber view. Different studies use different time intervals to characterize IMR as chronic. The minimum interval is usually 6 weeks. We chose a 4 month period to make sure the majority of myocardial remodeling has occurred  and thatCIMR increases the risk of heart failure and mortality in a graded fashion according to MR severity , 2. BecaThe exact mechanism for the development of CIMR after MI remains a subject of debate . Both anIn a quest to unravel these mechanisms and their underlying pathological abnormalities, several studies have tried to shed light on the precise role of PM involvement in the development of CIMR , 35, 36.Undocumented pre-existing mitral valve disease may have been present at the time of MI in some patients although none of the patients had a history of (organic) mitral valve disease or evidence of structural mitral valve disease on cardiac MRI or TTE. Baseline post-MI MRI data were not available in this study. Other limitations are related to methods of CIMR quantification. Alternative validated methods for CIMR severity assessment, including regurgitant volume and effective regurgitant orifice area were not available in this study. Inherent limitations of two-dimensional imaging, such as viewing plane selection and regional asymmetry or localized annular distortions, may have biased results. Future studies with three-dimensional imaging may have the potential to overcome some of these limitations.In conclusion, our findings indicate that 4 months after primary PCI for STEMI CIMR rates are higher in patients with PMI, but PMI is not an independent predictor of CIMR. The geometric parameters tethering height and interpapillary muscle distance are the strongest independent predictors of CIMR. Inferior infarction and infarction in the circumflex coronary artery are independent predictors of PMI.


The left bundle-branch block (LBBB) is related to worsening cardiac performance. We describe a case of a patient with an intermittent left bundle-branch block who underwent biventricular pacemaker implantation. We noted a detrimental effect on the performance of her left ventricle with the presence of the LBBB as compared to the performance after she underwent biventricular pacing or after reversal of LBBB .Her LBBB was accompanied by increased isovolumetric contraction time, longer pre-ejection period, and increased myocardial performance index  compared to her sinus rhythm with a narrow QRS complex with or under biventricular pacing. Hemodynamic studies in heart failure patients with left bundle-branch block (LBBB) show that LBBB leads to worsening cardiac performance ,2. We deA 55-year-old Caucasian woman presented to our hospital with a new onset dyspnea on exertion, lower extremity swelling, and orthopnea. Her electrocardiogram (ECG) showed sinus rhythm and a complete LBBB. She underwent a transthoracic echocardiogram (TTE) that revealed left ventricular (LV) enlargement and severe LV systolic dysfunction. She had no history of hypertension or alcohol abuse, and the results of her serology evaluation were negative for Chagas. A multislice computed tomography scan showed no lesions in the coronary arteries. She was diagnosed with idiopathic dilated cardiomyopathy.Pharmaceutical treatment was initiated and optimized to reach the target doses of carvedilol, enalapril, spironolactone, digoxin, and furosemide. While the patient showed clinical improvement, she remained symptomatic after several months of optimized doses of these treatments. She subsequently experienced clinical worsening, remaining in New York Heart Association (NYHA) functional class III to IV. She was then admitted for implantation of a biventricular pacemaker.During hospitalization, she showed rapid clinical improvement after reversal of the LBBB. An ECG showed sinus rhythm and a QRS complex interval of 110 ms. The medical team opted for not implanting the pacemaker at that time.However, the patient returned 10 days later, presenting with clinical deterioration in NYHA functional class III. The ECG showed LBBB once more. She also brought the TTE performed on the day of discharge revealing LV enlargement with LV end-diastolic dimension of 67 mm, LV end-systolic dimension of 61 mm, LV end-systolic volume of 155 ml, an ejection fraction (EF) of 19% (by Simpson’s method), and Grade III diastolic dysfunction. We also noted a wide QRS complex in the ECG signal obtained during the TTE. Implantation of a biventricular pacemaker was scheduled.The implantation of the biventricular pacing was performed uneventfully, with the LV lead positioned through the coronary sinus in the posterolateral wall. During the procedure, we observed alternating narrow QRS complex intervals and LBBB. This allowed the electrode positioned in the posterolateral wall of the LV to capture the ECG of a narrow QRS sinus rhythm of 110 ms and LBBB . We documented an increase in the LV electrical delay. The interval time was defined as the time from the first deflection on a surface ECG to the local intrinsic activation at the LV stimulation site (Q-LV) after the installation of LBBB , sinus rhythm with a narrow QRS complex, and LBBB pattern Table .The sinus rhythm with a narrow QRS is the underlying rhythm, which we obtained after inhibiting the pacemaker. The LBBB pattern was obtained after atrial pacing at a rate of 75 beats/minute (the LBBB was rate-dependent).The LVEF remained unchanged for each condition measured. There was a clear increase of the pre-ejection period (PEP), isovolumetric contraction time (ICT), and myocardial performance index in the presence of LBBB when compared to biventricular pacing and narrow QRS. We also observed abnormal interventricular septum motion during LBBB, which reversed at biventricular pacing or sinus rhythm with narrow QRS Figure .The patient presented good clinical function and echocardiographic response after the intervention. An echocardiogram done eight months later showed an increase in LVEF to 34% compared to the value of 19% before pacemaker implantation and an end-systolic volume reduction to 141 mL (before pacemaker implantation=155 ml). These parameters have been measured by Simpson’s method.The induction of LBBB is accompanied by changes in LV systolic and diastolic intervals, with a delay of the onset and termination of systolic ejection and therefore, a reduction of the diastolic time, stroke volume, and arterial blood pressure -3.In this case, we observed an increase of the LV electrical delay  and a detrimental effect at the LV during LBBB. These changes were reversed by biventricular stimulation or sinus rhythm with narrow QRS complex. LBBB increased the ICT, PEP, and myocardial performance index. LBBB increased the duration of systole and shortened the total time of diastole compared to the sinus rhythm with narrow QRS or biventricular pacing. Cardiac intervals and stroke volume (and cardiac output) presented similar values at sinus rhythm with narrow QRS and under biventricular pacing.One study shows that the LV myocardial performance index, which is the ratio of the sum of the LV isovolumetric contraction time + LV isovolumetric relaxation time divided by the LV ejection time exhibited an acute and sustained improvement after cardiac resynchronization therapy (CRT) .Bourassa et al.  reportedThe deleterious effects of LBBB on cardiac intervals are attenuated after their reversion to a narrow QRS rhythm or through biventricular pacing. The comparison of the parameters  measured in native rhythm and LBBB with a wide QRS complex and after implantation of biventricular pacing could be studied as a means of predicting the response to resynchronization therapy.


Left atrium (LA) size and function are powerful biomarkers of cardiovascular outcomes in many diseases. We sought to determine if the expected age-associated increase in arterial stiffness (AS) and left ventricular (LV)-LA afterload leads to corresponding effects on LA function and this can be measured with cardiovascular magnetic resonance (CMR). Additionally, we investigated the significance of these markers in asymptomatic individuals with cardiovascular risk factors (CRF).Female subjects from the Twins UK cohort with no overt cardiac disease were prospectively recruited for a CMR study on a 1.5 Tesla scanner  with tissue characterization (T1 mapping and late gadolinium enhancement). Patients with atrial fibrillation, valvular disease, regional wall motion abnormalities at rest or areas of myocardial enhancement were excluded from the analysis. LA reservoir, conduit and contractile functions were quantified by both fractional volume changes and CMR feature tracking derived strain and strain rate. Additionally, CMR feature tracking derived myocardial deformation indices and pulse wave velocity (PWV) (foot-to-foot methodology), were calculated.40 female twins were enrolled. Baseline characteristics are shown in table LA mechanics correlates with LV deformation parameters and PWV and differs significantly in elderly subjects with CRF compared to their healthy age-matched peers. LA structural remodeling is predicted by AS (as expressed by PWV/LV parameters) independently of conventional CRF, thus supporting the hypothesis of arterial-ventricular-atrial coupling (AVAC). These novel markers of LA performance can potentially uncover abnormal AVAC in patients with CRF but no overt cardiac disease and give valuable insights into ventricular dysfunction beyond standard volumetrics.


Perioperative myocardial ischemia and infarction are the leading causes of morbidity and mortality following anesthesia and surgery. The discovery of endogenous cardioprotective mechanisms has led to testing of new methods to protect the human heart. These approaches have included ischemic pre-conditioning, per-conditioning, post-conditioning, and remote conditioning of the myocardium. Pre-conditioning and per-conditioning include brief and repetitive periods of sub-lethal ischemia before and during prolonged ischemia, respectively; and post-conditioning is applied at the onset of reperfusion. Remote ischemic conditioning involves transient, repetitive, non-lethal ischemia and reperfusion in one organ or tissue (remote from the heart) that renders myocardium more resistant to lethal ischemia/reperfusion injury. In healthy, young hearts, many conditioning maneuvers can significantly increase the resistance of the heart against ischemia/reperfusion injury. The large multicenter clinical trials with ischemic remote conditioning have not been proven successful in cardiac surgery thus far. The lack of clinical success is due to underlying risk factors that interfere with remote ischemic conditioning and the use of cardioprotective agents that have activated the endogenous cardioprotective mechanisms prior to remote ischemic conditioning. Future preclinical research using remote ischemic conditioning will need to be conducted using comorbid models. Although medical advances in cardiac treatment have been significant, there is very little that can be done pharmacologically or mechanically to prevent the injury of the reperfused myocardium, which can lead to heart failure and death. The development of novel cardioprotective strategies that would mitigate further myocardial injury, secondary to ischemia/reperfusion (I/R) injury, is required and is the focus of many preclinical and clinical investigations3. The endogenous cardioprotection—mediated via ischemic pre-conditioning, ischemic per-conditioning, and ischemic post-conditioning—has some clear drawbacks because it has to be administered to the compromised myocardium. On the other hand, the maneuvers implemented with remote ischemic conditioning (RIC)  have the advantage of applying the protective ischemia to an organ distant from the heart4. Therefore, in this short review article, we focus mostly on RIC because of its popularity, non-invasive nature, and relative safety.When the coronary arterial blood flow is blocked, it is critical to re-establish the blood flow to the ischemic area of the heart as soon as possible. During the return of blood supply to the ischemic myocardium, there is paradoxical myocardial damage; the excess oxygen may trigger further myocardial cell death and greater cardiac injury, termed myocardial reperfusion injury4. It represents a strategy for harnessing the body’s endogenous, protective capabilities against the myocardial injury incurred by I/R. In experimental animals, brief episodes of ischemia and reperfusion in an arm or a leg dramatically reduce myocardial infarct size when applied prior to lethal myocardial ischemia (remote ischemic pre-conditioning), during lethal myocardial ischemia (remote ischemic per-conditioning), or at the onset of reperfusion (remote ischemic post-conditioning)10. Remote ischemic pre-conditioning and post-conditioning produce similar efficacy of cardioprotection against I/R injury11.RIC is the phenomenon in which transient, repetitive, non-lethal ischemia and reperfusion in one organ or tissue (remote from the heart) render myocardium resistant to lethal I/R injury13. In experimental animals, if the sensory nerve to the ischemic limb, spinal cord, or the vagus nerve is transected or silenced, the cardioprotective effects of RIC are lost17. These studies reveal the importance of neural pathways in transmitting the signal for cardioprotection in RIC. Over the past few years, many humoral factors have also been implicated, including nitric oxide, adenosine, bradykinin, opioid peptides, prostaglandins, natriuretic peptides, endocannabinoids, angiotensin I and calcitonin gene-related peptide, hypoxia-inducible factor 1α, erythropoietin, stromal-derived factor 1α, hypoxia-inducible factor prolyl hydroxylase 2 (encoded byEGLN1 gene), and microRNAs24. Currently, it is believed that the stimulus of remote ischemic pre-conditioning activates afferent C fiber sensory nerves by locally released autacoids to transmit cardioprotective signal25. In the meantime, the aforementioned humoral factors released by the tissue undergoing transient I/R are relayed in the blood to the heart, where they trigger cardioprotection. Humoral pathways may be more prominent in remote ischemic post-conditioning relative to remote ischemic pre-conditioning14.The mechanisms of cardioprotection by RIC are complex and have not been fully elucidated. However, it has been established that the signaling pathways from the tissue/organ subject to transient, repetitive I/R to the heart consist of three entities: remote stimulus to generate protective signal, the transfer of the signal to the heart, and myocardial responses to the transferred signal resulting in cardioprotection26. Autacoids, neurohormones, and humoral factors generated by remote stimulus bind to G protein-coupled receptors in myocardium or activate intracellular signal pathways in a receptor-independent manner or do both25. Three main intracellular signaling pathways may be critical in RIC-elicited cardioprotection against I/R injury: the endothelial nitric oxide synthase/protein kinase G pathway, the reperfusion injury salvage kinase pathway, and the survivor activating factor enhancement pathway27. Ultimately, these signaling pathways converge on the mitochondria and the cytoskeleton, resulting in inhibition of opening of the mitochondria permeability transition pore (mPTP), preservation of mitochondrial integrity and function, and reduction of cytoskeleton damage29.The intracellular signaling pathways of RIC in myocardium are thought to have much in common with local ischemic pre- and post-conditioning30. Moreover, it was shown that individuals with cardiovascular disease who participate in vigorous exercise prior to their cardiac event have improved ejection fraction and may obtain a protective benefit similar to that of RIC31.It is worth noting that there is considerable similarity between remote conditioning and exercise. Exercise appears to act as a physiological stress leading to similar accumulation of metabolic mediators such as adenosine, bradykinin, and calcitonin gene-related peptide along with baroreflex responses that induce beneficial myocardial adaptive responses at a cellular level36. Notwithstanding more recent clinical outcome studies , most of the clinical studies were conducted on small cohorts of selected patients and in controlled conditions. These smaller trials on the effectiveness of RIC during elective interventional revascularization, other forms of cardiac surgeries, non-cardiac vascular surgeries, and others have shown various degrees of beneficial effects of RIC38.RIC was shown to reduce cardiac injury in patients undergoing revascularization and other cardiac surgeries, as seen by reduction of cardiac biomarker release39. Some additional negative studies on RIC in humans were attributed mostly to underlying risk factors or medications that interfere with different cardioprotective interventions. One recent review covered the cardioprotection by ischemic pre-conditioning, ischemic post-conditioning, and remote conditioning in various clinical settings40. In addition to comorbidities and medications, the negative results may be due to the anesthetics used during the surgeries, which have been proven to prevent the protection by RIC. For instance, propofol has been shown to eliminate the benefits of RIC. It is of interest that most of the negative studies on the protection of RIC so far have used propofol as anesthetic41.One of the larger trials, which involved 1,280 patients and used both remote ischemic pre- and post-conditioning, did not show a reduction in the number of major adverse outcomes42. In the ERICCA study, with 1,612 patients undergoing elective on-pump coronary artery bypass grafting with or without valve surgery and without standardization of the anesthetic regimen, remote ischemic pre-conditioning using transient-arm I/R did not improve clinical outcomes43. Similarly, the RIPHeart study, in which the upper-limb remote ischemic pre-conditioning was performed while 1,385 patients were under propofol-induced anesthesia, did not show a relevant benefit among patients undergoing elective cardiac surgery44. The trials also failed to confirm the presence of initial cardioprotection by RIC-induced reduction of cardiac troponin release. Both of these studies are a clear disappointment in the cardioprotection efforts45. These large trials (ERICCA and RIPHeart) during cardiac surgery contradict previous smaller trials on the role of RIC36. Taken together, the recent large multicenter trials using RIC have not proven successful in cardiac surgery.Owing to both positive and negative results from various clinical trials, the results from large multicenter randomized controlled trials (such as ERICCA and RIPHeart) were meant to close that gap48. Another cause is the utility of cardioprotective agents that have activated the endogenous cardioprotective mechanisms prior to RIC49. Potential cardioprotective agents that would mitigate/interfere with cardioprotective interventions may include volatile anesthetics, propofol, P2Y12 blocking agents, beta blockers, morphine, nicorandil, sulfonylureas, statins, angiotensin-converting enzyme inhibitors, and nitrates53. In addition, the beneficial effect of ischemic conditioning is not detectable in patients with the small extent of myocardial injury54.The lack of success in cardiac surgery using RIC is multifactorial. One cause is that cardiovascular risk factors can interfere with RIC. Most of the risk factors responsible for human ischemic heart disease in the first place include hypertension, chronic obstructive pulmonary disease, heart failure, atherosclerosis, diabetes and other metabolic diseases, age, and routine drug therapies55,56, and infarct size is the main determinant of prognosis. Reduction of infarct size is a main goal of treatment and can be achieved efficiently with primary angioplasty. Successful and timely reperfusion with percutaneous coronary intervention (PCI) or primary angioplasty attenuates infarct size and improves cardiac function and clinical outcomes58. However, sudden reperfusion can cause fatal myocardial injury59, which may limit therapeutic benefits. Thus, supplementary cardioprotection such as RIC may be considered in elective and emergent PCI60. Clinical trials using small cohorts of selected patients generally suggest that RIC can provide cardioprotection by lowering peak troponin I or reducing infarct size (or both) in patients undergoing elective PCI64.ST segment elevation myocardial infarction is one of the leading causes of mortalities and morbidities worldwide65, and the use of propofol, rather than volatile anesthesia, appears to be a common denominator of studies that failed to protect with RIC67. It is likely that the use of volatile anesthetics would have made the ERICCA and RIPHeart trials more complete. Indeed, the successful cardioprotection by RIC was also documented in acute myocardial infarction, where the type of anesthesia was not an issue69.One could speculate that the lack of protection in these two phase III clinical trials is because of the use of propofol anesthesia in most of the patients in the ERICCA trial and all patients in the RIPHeart trial. Attenuation of RIC in the presence of propofol anesthesia has been reported70, pharmacologic cardioprotection with volatile anesthetics emerged as a considerably less risk-bearing and equally effective intervention71. The American College of Cardiology Foundation and the American Heart Association Task Force on Practice Guidelines adopted a recommendation for the use of volatile anesthetics in surgical patients at risk for myocardial ischemia72. Since anesthetic cardioprotection was discovered, experimental and clinical research has focused on elucidating the mechanisms of anesthetic cardioprotection with the anticipation of finding an anesthetic agent or approach that would be the most beneficial for patients with coronary artery disease. Clinical studies with sufficient power to detect differences between process variables and outcome among anesthetic agents or techniques have confirmed the relevance of anesthetic cardioprotection for patients79. The loss of cardioprotection is strongly associated with the risk of death/non-fatal myocardial infarction within the year after determining the absence of pre-conditioning with the PCI model of coronary occlusion80. At one year, the risks of death were reduced by 85% in patients who manifested pre-conditioning as compared with patients who did not. The ability for cardioprotection to remain significantly, and inversely, associated with the risk of death/non-fatal myocardial infarction in one year affirms the clinical significance of this phenomenon.Parallel to the most powerful endogenous cardioprotective mechanism of ischemic pre-conditioning51. In contrast, other small-scale clinical trials indicate that RIC can provide additional protective effects in isoflurane-anesthetized patients undergoing cardiac surgery67. However, recent studies from two large-scale, multicenter, clinical trials of RIC show negative results in cardiac surgery44. The reasons for the differences among these studies are complex and have not been fully understood. To use better RIC and lessen myocardial I/R injury in human cardiac surgery, a greater understanding of the interaction between RIC and volatile anesthetic conditioning is necessary.Every year, volatile anesthetics are used in millions of patients undergoing cardiac surgery. Whether RIC provides more cardioprotection to the myocardium of the volatile anesthetic-anesthetized patients undergoing cardiac surgery has been examined, yet the clinical outcomes remain uncertain. Some clinical studies show that the cardioprotective effect of RIC is unable to be detected in isoflurane-anesthetized patients undergoing coronary artery bypass grafting81; and the prevalence of this disease is expected to increase by over 200% in the next several decades. Impaired glucose tolerance currently affects 20% to 35% of all middle-aged and elderly Americans, and hyperglycemia alone is a significant independent predictor of cardiovascular morbidity and mortality in patients undergoing cardiac surgery85. The mechanisms that contribute to increased risk in diabetes and hyperglycemia are poorly understood but likely are related to insufficient activation of pro-survival signaling pathways, elevated nitrosative stress, activation of the PI3K/Akt/mTOR  pathway, and autophagy46. Aggressive control of blood glucose concentration using insulin is one approach, but it is unlikely that insulin alone can substantially improve cardiovascular outcomes in patients with diabetes.Diabetes is a significant predictor of increased perioperative risk due to a greater susceptibility to I/R injury. Both preclinical and clinical results indicate that the cardioprotective effect of ischemic and pharmacologic conditioning is impaired in the presence of diabetes. Type 2 diabetes occurs in 9.3% of the US population, affecting 29.1 million individuals86. At present, 15% to 30% of the patients who undergo coronary artery surgery are diabetic89. During cardiac surgery, the heart is inevitably subjected to I/R injury due to pre-existing coronary occlusion and heart arrest subsequent to aortic artery cross-clamp. After cardiac surgery, patients with diabetes have an increased mortality and poorer clinical recovery than non-obese, non-diabetic patients90. Therefore, diabetic populations may obtain a greater benefit from therapies shown to be effective in treating ischemic heart disease.Patients with diabetes have a significantly higher incidence of coronary heart disease compared with non-diabetic individuals93. The mechanisms underlying the impaired cardioprotective effect of RIC by diabetes are not fully elucidated. About 60% to 70% of people with diabetes mellitus eventually develop diabetic peripheral neuropathy94. In many of these patients, peripheral neuropathy, including sensory C fibers which are required by the cardioprotective effect of RIC, is damaged95. This damage may be an important contributor to the reduction of RIC cardioprotection during diabetes97. In future clinical trials, selection of patients with functional sensory C fibers may help to improve the clinical outcome of RIC. In addition, diabetes impairs the PI3K/Akt/glycogen synthase kinase 3 beta (PI3K/Akt/GSK3-β) signaling pathway and phosphorylation of ERK1/2 , decreases generation and release of nitric oxide, inactivates ATP-sensitive potassium channels, and elevates oxidative stress100. These intracellular signaling pathways are crucial for endogenous cardioprotection. Diabetes-elicited impairments in these signaling pathways may critically contribute to the attenuation of RIC cardioprotection92.The cardioprotective effect of single-dose RIC in diabetes is often disappointing102. Compared with clinical single-dose RIC and local ischemic conditioning, repeated RIC appeared to have more consistently yielded significantly beneficial results against remodeling in both preclinical and clinical studies of RIC103. It has been demonstrated that repeated RIC reduces adverse cardiac remodeling after myocardial infarction, elevates survival of animals in a dose-dependent fashion, improves endothelial function and skin microcirculation, and modulates the systemic inflammatory responses105. Intriguingly, repeated RIC is beneficial for healing in lower-extremity diabetic ulcers106. Whether repeated RIC is effective in I/R protection of diabetic hearts remains elusive. The efficacy, potential, and safety of repeated RIC in protection of diabetic hearts need to be tested in future experimental studies and clinical trials.Repeated RIC, where short periods of limb ischemia are repeatedly applied over days or weeks, is the extension of RIC. Recent studies have identified repeated RIC as a newer strategy for cardioprotection108. The studies to restore myocardial sensitivity to local ischemic conditioning in diabetes have focused on GSK3-β and the mPTP. Pharmacological inhibition of either GSK3-β or the mPTP restores the protective potential of local ischemic conditioning in the diabetic heart110. Whether pharmacological interventions also are effective in restoring the cardioprotective potential of RIC in diabetes has not been investigated. Given that intact neural pathway is required for the cardioprotective effect of RIC13, it is likely that a combination of approaches that target both neural integrity and the final common signaling molecules and effectors would be the best strategy for restoring the myocardial response to RIC in diabetes.Restoring myocardial sensitivity to RIC in the setting of diabetes is of primary importance. Since diabetes impairs multiple signal transduction pathways of RIC, it is reasonable to believe that a therapy that targets one pathway may not completely restore the sensitivity of the myocardium to RIC. In the various signal transduction pathways, GSK3-β is the intervention point of convergence, and the mPTP is thought to be the final effector of cardioprotectionIt is fair to say that owing to underlying risk factors that interfere with different cardioprotective interventions and the use of cardioprotective agents, most of the clinical trials with cardioprotective drugs have not been very successful. The results of various ischemic conditioning in humans appear to follow the same unsuccessful path, although RIC is a potent form of endogenous cardioprotection in healthy animals. In future research endeavors, the validation of drug targets and various cardiac conditioning needs to be conducted in comorbid animal models to have a successful clinical translation.ERICCA, Effect of Remote Ischemic Preconditioning on Clinical Outcomes in Patients Undergoing Coronary Artery Bypass Surgery; GSK3-β, glycogen synthase kinase 3 beta; I/R, ischemia/reperfusion; mPTP, mitochondria permeability transition pore; PCI, percutaneous coronary intervention; PI3K/Akt, phosphatidylinositol 3-kinase, serine/threonine kinase also known as protein kinase B; RIC, remote ischemic conditioning; RIPHeart, Remote Ischemic Preconditioning for Heart Surgery.


Cine derived myocardial WT explained 16–36% of the histological myocardial fibrosis. The peak circumferential and radial strain measured by feature tracking could explain 15 and 10% of the variance of myocardial fibrosis, respectively. The used method to systematically compare CMR image data with digital histological images is novel and feasible. Myocardial WT and strain were only modestly related with the amount of fibrosis. The fully automatic FWHM analysis technique is the preferred method to detect myocardial fibrosis.The noninvasive reference standard for myocardial fibrosis detection on cardiovascular magnetic resonance imaging (CMR) is late gadolinium enhancement (LGE). Currently there is no consensus on the preferred method for LGE quantification. Moreover myocardial wall thickening (WT) and strain are measures of regional deformation and function. The aim of this research was to systematically compare in vivo CMR parameters, such as LGE, WT and strain, with histological fibrosis quantification. Eight weeks after 90 min ischemia/reperfusion of the LAD artery, 16 pigs underwent in vivo Cine and LGE CMR. Histological sections from transverse heart slices were digitally analysed for fibrosis quantification. Mean fibrosis percentage of analysed sections was related to the different CMR techniques (using segmentation or feature tracking software) for each slice using a linear mixed model analysis. The full width at half maximum (FWHM) technique for quantification of LGE yielded the highest RThe online version of this article (doi:10.1007/s10554-017-1187-y) contains supplementary material, which is available to authorized users. Myocardial fibrosis has been associated with heart failure and can act as a substrate for cardiac arrhythmias . FollowiFunctional assessment of the local myocardium is typically performed visually on cine CMR images. Quantitative assessment of local myocardial function  can also be performed with (semi-)automatic segmentation software packages , 12. MorWe have recently developed a method for high resolution systematic digital histological quantification of  cardiac fibrosis in a whole heart slice , which cAll in vivo experiments were conducted in accordance with the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources. Experiments were approved (protocol no.: 2012.II.09.145) by the local Animal Experiments Committee (DEC) .Our protocol regarding a porcine chronic MI model has been described in detail before . Eight w2, bandwidth = 1200 Hz and flip angle = 45°.Pigs were positioned supine with a dedicated 32-channel phased-array receiver coil over the chest and scanned using a standardized protocol. For image planning scout images were obtained in short-axis and two-chamber long-axis views. ECG-gated steady-state free precession (SSFP) short-axis (from apex to base of LV) and two chamber long-axis cine images were acquired. Thirty frames were acquired per RR cycle. Cine parameters: echo time (TE)/repetition time (TR) 1.6/3.2 ms, 13 slices, slice thickness 8 mm, resolution = 2 × 2 mm, field of voxel (FOV) = 320 × 320 mm2, FOV = 300 × 300 mm2, flip angle = 25°, 63 TFE shots, bandwidth = 300 Hz, number of signals averaged = 2, SENSE acceleration = 2.Late gadolinium enhancement CMR was performed using an inversion recovery 3D-turbo-gradient-echo-technique 15 min after an intravenous bolus injection of 0.2 ml/kg gadobutrol . First, a look-locker scout was performed for the optimal inversion time. Acquisition parameters for the LGE scan: inversion time (TI) = 200–270 ms, TE/TR = 1.5/4.7 ms, slice thickness = 6 mm, spatial resolution = 1.5 × 1.5 mmhttp://segment.heiberg.se, Medviso AB, Lund, Sweden) [Offline image analysis to derive WT and LGE was performed using Segment software version v1.9 R3590 ( Sweden) . In all cc), radial strain (εrr) and WT (endo to epi distance), was exported and used for further analysis . This technology tracks gray value image feature in the myocardium in the CINE images in which the end diastolic frame serves as the reference phase. The software quantifies the strain in 48 segments equally spaced over the LV myocardium. The end-diastolic endo- and epicardial contours of the selected slice were manually traced  Fig. c, standaFollowing CMR, the animals were sacrificed by exsanguination under general anaesthesia and the hearts were excised and cut into transverse (short-axis) 1 cm thick slices from apex to base. Each third transverse slice was fixed in formalin, cut into smaller sections and an overview of the heart slice was drawn to annotate the origin of each tissue specimen Fig. a. These http://sourceforge.net/projects/fibroquant) [Digital histological analysis was performed systematically as previously described, using the in-house developed open source software package Fibroquant (roquant) . The epiA landmark in the mid-lateral wall , was calculated as 100% minus the ratio of the full and null models . We compared different CMR techniques with percentages of fibrosis per section using a linear mixed model analysis. For fibrosis, the amount of residual variance  were successfully stained, segmented Fig. c and anaMyocardial WT curves were successfully extracted for 15 animals in 60 segments. Mean end systolic absolute WT was 2.4 ± 2.4 mm (range −2.1 to 7.2 mm) , 26.9 ± 34.7% (range −26.0 to 171.9%) and −1.6 ± 2.5 mm (range −4.6 to 7.7 mm), respectively  decreased (0.94 to 0.65) with more stringent automatic segmentation methods (2–5 SD), while it remained similar in the manually corrected SD subgroup. The MSI of the LGE images, in which no additional interaction is required, explained the myocardial fibrosis for 52%.The mean LGE scar transmurality and MSI 15 animals) is shown in Fig. 5 animalscc and εrr, ranged from −15.0 to −7.7% and 40.0 to −1.0% respectively between the infarct transmurality from 0 to 100%.In the boxplots Fig. , the medThis study demonstrates a novel systematic method to compare different in vivo CMR techniques with high spatial resolution histological analyses in a chronic porcine infarct model. To the best of our knowledge, this is the first time such a systematic comparison has been performed and this method can be readily adapted for use with other imaging modalities such as SPECT, CT, PET and echocardiography. For LGE imaging, we found that the FWHM and the 2, 3 or 5SD from remote methods with manual correction were the best methods to quantify the amount of myocardial fibrosis. Although it is expected that measures of myocardial deformation would be affected in infarcted myocardium, myocardial WT and strain were only modestly related with the amount of myocardial fibrosis.2 = 36 vs. 16%). In Segment, both the endo- and epicardial contours of the selected slice were semi-automatically traced in every frame. In Image-Arena the segmentation was performed in only one frame and the tracing in subsequent frames was automatically propagated by the software algorithm. A limitation in the FT implementation in the TomTec software is caused by the inability to perform manual adjustments in multiple frames, limiting accuracy and causing variations in the tracings. Furthermore, the slightly lower spatial resolution of the FT export compared to the Segment export  may have influenced the results. Additionally, through plane motion and filtering algorithms resulting in loss of detail are likely to introduce erroneously tracked features, thereby affecting the FT results.The reference standard for the noninvasive assessment of cardiac anatomy and function is CMR . While c2 = 10 and 15% respectively). As previously described by Cowan et al., differences in strain could occur between two regions indicated as healthy by LGE [In our analysis of the LV slices, both radial and circumferential strain based on FT were poorly associated with myocardial fibrosis . The between animal variance of LGE MSI compared to FWHM was higher microtome could be considered to preserve the gross cardiac anatomy. The resulting high resolution images of the complete transversal heart slice can than be analyzed and subdivided to the resolution of the imaging datasets with the lowest spatial resolution to allow for a more detailed comparison with high detail imaging modalities. Hereby obviating the requirement to reduce the details of any of the datasets.The selected mid-lateral point was used as a landmark for registration of the different modalities due to a standardized workflow for histological processing as it was used as a starting point for cutting the heart slice into smaller sections . Selecticc and εrr), next to the known poor intra- and interobserver variability associated with FT analysis on a segmental level, it is likely that a higher fibrosis percentage would have led to a stronger relation with worsening of functional parameters. The RV was excluded from all comparisons because the conventional analyses applied on the cine and LGE images in this study did not allow an accurate analysis of the right ventricular parameters.In the assessment of local cardiac deformation  and can also be translated for use with a 3D model. While the proximal LAD ischemia/reperfusion model used in this study produced consequent isolated anteroseptal infarctions, it would be in the interest of external validity to study other infarct sizes and locations using this method. Improved fibrosis detection with CMR will be applicable to a broad clinical spectrum ranging from diagnostic to therapeutic outcomes, including ischemic heart disease, ablation therapy, valvular diseases, cardiomyopathies and cell therapy. For example, precise identification of the fibrotic region allows for accurate therapy guidance to the target area with the aim to ultimately improve clinical outcome for patients.In conclusion, the novel systematic method to compare high resolution in vivo CMR imaging with detailed histological fibrosis data was feasible and can readily be applied to other imaging data. Locally measured functional parameters such as WT, and measures of myocardial deformation derived from FT: radial and circumferential strain related modestly with local myocardial fibrosis, yet can be used to gain insight into local cardiac mechanics. The fully automatic FWHM algorithm applied on the reference standard LGE CMR showed to be preferred to detect myocardial fibrosis in a chronic in vivo infarct model.Below is the link to the electronic supplementary material.Supplementary material 1 (DOCX 1062 KB)


P < 0.05), left ventricular end diastolic volume(LVEDV) and area(LVEDA) decreased significantly (P < 0.05), while E/A ratio of mitral valve, tricuspid valve systolic velocity E and A peaks and E/A ratio remained unchanged (P > 0.05). PFO hemodynamic changes mainly occurred in the left ventricle when the Valsalva maneuver was performed. The Valsalva maneuver increased pressure in the chest, then pulmonary venous return was impeded, which resulted in left ventricular limited filling, and E and A peaks decreased. The pressure of the left ventricle and atrium was lower than that of the right side, which resulted in right-to-left shunt (RLS) through PFO.The purpose of this study was to investigate the influence of the Valsalva maneuver (VM) on cardiac hemodynamics in patients with patent foramen ovale (PFO). Sixty-five patients who were highly suspected to have PFO were included. The changes in E, A, E/A ratio of mitral valve blood flow, E, A, E/A ratio of tricuspid valve blood flow, left ventricular end-diastolic volume, area and right atrial area during the resting state and the strain phase of the Valsalva maneuver were observed by transthoracic echocardiography (TTE). Statistical analyses were performed using SPSS Version18.0. Compared to the resting state, mitral valve diastolic velocity E and A peaks at the strain phase of the Valsalva maneuver significantly decreased ( Patent foramen ovale (PFO) is believed to be a common finding in adults according to a study of 965 normal adult hearts in postmortem studies891012The study population consisted of 65 patients with high clinical suspicion of patent foramen ovale who suffered from migraine, vertigo, transient ischemic attack (TIA), limb weakness, blurred vision and unknown causes of cerebral ischemia. All patients were enrolled from April 2015 to November 2015 in Xi’an, Shaanxi Province, China. All patients gave written consent prior to the examination. The study protocol was performed with approval by the ethics committee of the Second Affiliated Hospital of Xi’an Jiaotong University and was performed in accordance with the CONSORT 2010 guidelines. Written informed consent was obtained from all subjects. Before TTE with the micro-bubbles test, all patients were instructed to perform the standard Valsalva maneuver. Patients who were unable to perform the maneuver during the test because of coordination impairment were excluded from our study. Routine ultrasound, Computed tomography (CT), and magnetic resonance imaging (MRI) were used to rule out cardiac, intracranial, or extracranial arterial disease and pulmonary arteriovenous malformation.The simple pressure measurement device used in the study is shown in P value of <0.05 was considered statistically significant.Categorical and continuous variables were expressed as percentages (%) and mean ± standard deviation, respectively. Statistical analyses were performed using SPSS Version 18.0 . We used paired t-tests to compare the parameters between the resting and Valsalva maneuver states. A P > 0.05). Results of TTE with the microbubbles test at rest and during the Valsalva maneuver were demonstrated in Parameters of left ventricular and right ventricular at rest and during the Valsalva maneuver was shown in P < 0.05), left ventricular end diastolic volume (LVEDV) and area (LVEDA) decreased significantly (P < 0.05), while E/A ratio of mitral valve, tricuspid valve systolic velocity, E and A peak and E/A ratio remained unchanged (P > 0.05). The results also indicated that the Valsalva maneuver significantly increased the number of micro-bubbles shunting.Our results indicated that compared to the resting state, mitral valve diastolic velocity E peak and A peak at the strain phase of the Valsalva maneuver significantly decreased  at rest. When the Valsalva maneuver was performed, the test result was positive in 55 patients (84.6%). The results were consistent with previous studies9However, our work also had limitations. First, the major problem was the small number of sample size with only 55 cases of patent foramen ovale confirmed by TTE with the micro-bubbles test. Second, there is no apparent power calculation in our work and given the drop-out of non-PFO patients present, it is likely that type I statistical errors could be present. Besides, our patients did not undergo transesophageal echocardiography (TEE) to confirm the presence of patent foramen ovale.In conclusion, our results indicated that patients with patent foramen ovale hemodynamic changes mainly occurred in the left ventricle when the Valsalva maneuver was performed, mitral valve diastolic velocity E peak and A peak at the strain phase of Valsalva maneuver significantly decreased, left ventricular end diastolic volume (LVEDV) and area (LVEDA) decreased significantly, while E/A ratio of mitral valve, tricuspid valve systolic velocity E peak, A peak and E/A ratio remained unchanged. The results also indicated that the Valsalva maneuver significantly increased the number of micro-bubbles shunting. The Valsalva maneuver increased the pressure of the chest, and pulmonary venous return was impeded, which resulted in left ventricular filling, and E and A peaks decreased. The pressure of left ventricle and atrium was lower than that of the right side, which resulted in right-to- left shunt (RLS) through PFO.How to cite this article: Zhao, E. et al. Influence of the Valsalva maneuver on the cardiac hemodynamics and right to left shunt in patients with patent foramen ovale. Sci. Rep.7, 44280; doi: 10.1038/srep44280 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


The prevalence of intrauterine growth restriction (IUGR) is about 3–10% of live-born newborns and can be as high as 20% in developing countries. It may result in the occurrence of cardiovascular diseases later in life.The aim of this study was echocardiographic evaluation, with the use of conventional and tissue Doppler parameters, of cardiac function in children born with IUGR, and comparison with healthy peers born as normally grown foetuses.In the IUGR group, E wave and E/A ratio were significantly lower compared to the control group. A wave, isovolumetric relaxation time, deceleration time, myocardial performance index as well as E/E′ septal and E/E′ lateral indices were significantly higher compared to healthy peers.Children with IUGR presented with subclinical myocardial dysfunction. Intrauterine growth restriction (IUGR) is a major problem in present-day medicine. It is defined as a birth weight below the 10th percentile for gestational age and involves about 5–10% of neonates.It is well known that such pathology may be associated with the later occurrence of cardiovascular diseases. Barker’s hypothesis, published in 1989,4Due to the sparsity of reports, the increase in incidence of myocardial dysfunction in children born with IUGR remains unclear. The aim of our prospective study was echocardiographic evaluation of cardiac function in children born with IUGR, compared to children born at normal gestational age and birth weight.The analysis included 77 children , aged from 5–11 years, who were randomly selected from the obstetrics and gynecology out-patient clinic. We included those born at term as small-for-gestational-age (SGA) babies  with IUGR features, detected prenatally by foetal size measurements on obstetric ultrasonography. All the children were single births. The control group included 30 healthy subjects , born with normal birth weight, gender and age matched to the study group. Gestational age was calculated from the mother’s last menstrual period.2]  = weight (kg)/height (m)2] . All subThe exclusion criteria were: evidence of chromosomal or infectious aetiology for IUGR, gestational diabetes, hypothyroidism, systemic or acute disease, and the mother smoking cigarettes or using medication. This study was approved by the medical ethics committee of the Health Sciences Faculty of Lodz University (No: RNN/150/09/KB).All patients underwent a full echocardiographic study, using the Aloka Prosound α 10 device, evaluating anatomy and cardiac function. In standard projections, systolic and diastolic function of the left ventricle was estimated.Left ventricular diameter, ejection fraction (EF) and shortening fraction (SF) were calculated in the parasternal, long-axis view, in M-mode presentation according to the Teichholz formula.In the apical, four-chamber view, with the use of pulsed Doppler, evaluation of mitral inflow velocities was done. The sample volume was placed at the mitral valve annulus and the peak of early mitral inflow velocity (E wave in early diastole), the peak of atrial mitral inflow velocity  and the E/A ratio were assessed.In the apical, five-chamber view, with the use of pulsed Doppler, left ventricular inflow and outflow were recorded with the sample volume placed between the aortic and mitral valves to evaluate isovolumetric relaxation time (IRT) and deceleration time (DecT). On the basis of these measurements, myocardial performance index, defined as quotient of the sum of the isovolumetric contraction time (ICT) and IRT-to-left ventricular ejection time (ET), were calculated.In the apical, five-chamber view, with the use of pulsed Doppler, aortic flow was recorded and heart rate calculated.Each measurement was obtained for three cardiac cycles. For statistical analysis the mean value was used.Pulsed and colour tissue Doppler imaging were performed in the apical four-chamber view. The sample volume was positioned as parallel as possible with the lateral mitral annular motion. The maximal myocardial velocities during systole (S), and early (E′) and late (A′) diastole were measured at the interventricular septum  and lateral annulus . The ratio of the early to late diastolic velocities was calculated for IVS  and for the posterior wall . The ratio of peak transmitral E velocity to early diastolic mitral annular velocity (E/E′) was calculated for both the interventricular septum and posterior wall .Descriptive statistics were executed by computing the mean and standard deviation (SD) for scale variables, or frequencies for nominal variables. The significance level was computed for the differences between variables in the IUGR and control groups. To evaluate the differences between the two groups, a parametric t-test and a non-parametric Mann–Whitney test were performed. Distributions were checked for normality with the Kolmogorow–Smirnov test. Statistical significance was defined as a p-value < 0.05. Pearson and Spearman correlation coefficients were computed to evaluate the degree of association between variables for either the control or study group.Analysis of the medical records confirmed statistically significant differences in birth weight , while there was no significant difference for gestational age between the groups. Physical examinations did not reveal statistically significant differences between the mean values for weight and body mass index, whereas in the IUGR group, the children were significantly smaller compared to healthy subjects .Analysis of echocardiographic left ventricular diameters did not reveal any significant differences in diastolic wall dimensions  or left ventricular lumen diameters between the two groups. Left atrial diameter also did not differ significantly.The mean values of ejection fraction as well as shortening fraction were within normal limits and similar between the groups. The mean values were as follows: IUGR group: EF 69.32 ± 2.99%, SF 38.33 ± 2.55%; control group: EF 69.20 ± 3.14%, SF 38.30 ± 2.73%.According to parameters evaluating left ventricular diastolic function, there were statistically significant differences between mean values of E wave, A wave and E/A ratio. The mean values of E wave and E/A ratio were significantly lower in children with intrauterine growth retardation compared to the control group. The mean values of the A wave were significantly higher in that group of patientsThe mean values of isovolumetric relaxation time and deceleration time were significantly higher in children with IUGR compared to the healthy peers . The mean values of myocardial performance index obtained in the IUGR group were significantly higher than those of the control group. The heart rate was similar in both groups of patients (p = 0.095) .Estimation of left ventricular function using tissue Doppler imaging did not reveal significant differences between the mean values of systolic myocardial velocity, either at the level of IVS, or at the posterior wall. The mean values of septal and posterior wall E′/A′ ratio were also similar. The mean values of E/E′ for the interventricular septum and E/E′ for the posterior wall were significantly higher in patients with IUGR .In recent years, more attention has been paid to changes in cardiac function in children with features of intrauterine growth retardation. Many articles describe the disorders appearing as early as in the foetus, revealing subclinical changes in the myocardium detected on echocardiographic examination.9In our study we evaluated the cardiac function in small-forgestational- age children with features of IUGR with the use of conventional as well as tissue Doppler echocardiographic parameters. We analysed left ventricular diameters  but did not find significant differences between the groups. This is similar to the findings of Bjarnedard et al. and Crispi et al., who studied, respectively, young adults and children the same age as our study patients.10On the other hand, Turkish authors demonstrated that left ventricular diastolic dimension in neonates and infants were significantly higher in children with IUGR.Other conventional echocardiographic parameters assessed in our study were shortening fraction (SF) and ejection fraction (EF) of the left ventricle. As in other reports, there were no differences between the groups.12Altin et al. describes a significantly higher heart rate in children with growth restriction, which was not observed in our study.7E/A ratio is a conventional index evaluating ventricular filling during diastole. In adults, reduced E/A ratios indicate diastolic dysfunction. Cohen et al. measured a mean E/A index of 1.9 in healthy people over 21 years of age and Eto et al. of 1.77 ± 0.53 in children. We had similar findings in the control group, but in children with IUGR, the mean E/A ratio was lower.14Other articles on children with IUGR describe different values for this index. In most recent reports in foetuses, the E/A ratio was significantly higher compared with the control group. In older children authors describe a normalisation of the E/A ratio, with no differences between a group of healthy children and those born with IUGR features.16The lower E/A index in children with IUGR seems to be dependent on the increase in A wave and decrease in E wave in those patients. This could be explained by a lower free inflow (E wave) than active left ventricular filling (A wave), which is associated with susceptibility of the left ventricle to abnormalities, and may be one of the symptoms of diastolic dysfunction. Such abnormalities are described in children with connective tissue diseases, due to the higher content of connective tissue in cardiac muscle, which diminishes its susceptibility to stretching.The results of research on deceleration time in adults, which is an indicator of left ventricular ‘stiffness’, support this theory.With regard to the IRT, results in the literature are not consistent. Sehgal et al., who studied a slightly younger group of children with growth restrictions, described a similar tendency to our results.7In our study, MPI was significantly higher in the IUGR group.There are few reports on diastolic cardiac function in children with IUGR. Altin et al. evaluated diastolic function in neonates and infants and revealed that indices that were abnormal in the foetal period  tended to decrease with age.Other researchers evaluated a similar age group to those in our study and, as in our analysis, some diastolic function parameters were significantly higher in IUGR children. Perhaps the observed anomalies due to intrauterine chronic hypoxia leading to cardiac volume overload and cardiac remodelling resulted in impaired cardiac function.7From our results, we found that diastolic function may be impaired in IUGR patients, but further studies with larger sample sizes are needed. This group of patient should be monitored long term and evaluated for cardiovascular status, due to the high risk of cardiovascular disease in adulthood.