Purpose. The objective of this study was to determine the efficacy of dietary supplementation with the metal chelators, trientine or citric acid, in preventing the development of cardiomyopathy in the Zucker diabetic rat. Hypothesis. We hypothesized that dietary chelators would attenuate metal-catalyzed oxidative stress and damage in tissues and protect against pathological changes in ventricular structure and function in type II diabetes. Methods. Animals (10 weeks old) included lean control , untreated Zucker diabetic fatty , and ZDF rats treated with either trientine (triethylenetetramine) or citrate at 20 mg/d in drinking water, starting when rats were frankly diabetic. Cardiac functional assessment was determined using a Millar pressure/volume catheter placed in the left ventricle at 32 weeks of age. Results. End diastolic volume for the ZDF animals increased by 36% indicating LV dilatation (P < .05) and was accompanied by a 30% increase in the end diastolic pressure (P ≤ .05). Both trientine and citric acid prevented the increases in EDV and EDP (P < .05). Ejection fraction and myocardial relaxation were also significantly improved with chelator treatment. Conclusion. Dietary supplementation with trientine and citric acid significantly prevented structural and functional changes in the diabetic heart, supporting the merits of mild chelators for prevention of cardiovascular disease in diabetes. Cardiovascular disease is the primary cause of increased mortality in diabetes. Clinically, over 60% of diabetics succumb to heart failure. Moreover, the 5-year survival rate following the first documented heart injury is less than 50% . These s Trientine and AGE breakers and inhibitors are potent chelators of copper, and to a lesser extent iron, and are thought to act by inhibition of metal-catalyzed oxidative stress. Oxidative stress is implicated in the development of the full range of diabetic complications, including nephropathy, neuropathy, retinopathy, and micro- and macrovascular disease –13. AlthAccordingly, we designed this study to determine if the relatively inexpensive and well-tolerated chelators, trientine and citric acid, would alter the onset of cardiomyopathy in the Zucker diabetic rat, a model of type II diabetes, which accounts for more than 90% of the diabetic population. We show here that both of these mild chelators provide protection against the development of diabetes induced cardiomyopathy, supporting the merits of further research on chelation therapy for prevention and treatment of this and other diabetic complications. Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health . All chemicals and reagents were purchased from Sigma-Aldrich , unless otherwise indicated. All animals were housed two to a cage in a 12:12 hours dark/light cycle controlled room. Male Zucker diabetic fatty rats (ZDF fa/fa) and male Zucker lean control heterozygous litter mates (LC fa/+) were obtained at 6 weeks of age (~200 g) from Charles River Laboratories . The ZDF is an inbred rat model of adult onset type II diabetes, characterized by gradual development of insulin resistance, hyperinsulinemia and hyperglycemia. All Animals were maintained on Purina 5008 rat chow with water ad libitum. At the age of 10 weeks, after confirming hyperglycemia by tail vein blood samples , the animals were divided into four groups: (1) lean control rats , (2) untreated ZDF rats (n = 10), (3) ZDF rats treated with trientine  administered in drinking water (1 g/L) as previously reported [n = 4) administered at the same dose in drinking water (1 g/L). Left ventricular systolic and diastolic structural and functional parameters were assessed at 32 weeks of age, just prior to sacrifice. Plasma samples were collected, the heart and kidneys were removed, rinsed in ice cold PBS and weighed, and then portions either snap frozen in liquid nitrogen for biochemical analysis or placed in 4% paraformaldehyde fixative for histological analysis.All protocols were approved by the Institutional Animal Care and Use Committee of the University of South Carolina and conform to the As previously described, LV systolic and diastolic pressure and volume functional analysis were performed in anesthetized rats using a high-fidelity Millar pressure/volume  conductance catheter inserted into the LV via the right carotid artery . CharactP ≤ .05.Statistical analyses were performed with Graphpad 5.0 software . All grouped data are expressed as means ± SEM unless otherwise noted. Grouped data comparisons were made by one-way analysis of variance with intergroup comparisons analyzed using Bonferroni's posthoc testing. Non parametric Kruskal-Wallis test with Gaussian approximation was utilized to analyze the rank/scoring system of the hydronephrosis ratings. Statistical significance was taken to be Compared to lean control animals, there was a significant reduction in whole body and LV weight in the ZDF and ZDF + CA groups at 32 weeks of age . Body weAlthough heart rate was significantly reduced in the ZDF + CA treated group compared to LC, no differences in cardiac output were evident between any of the groups . Stroke μL; resp. P ≤ .05 versus ZDF). Likewise the optimal volume for minimum dP/dt (Vol@dP/dt min) was also significantly increased in the untreated ZDF group compared to LC . Further, ejection fraction which was 34% in the untreated ZDF group was significantly improved, but not completely corrected by treatment with trientine (45%) or citric acid (43%). Lastly, in a effort to assess alterations in passive tissue property reflective of changes in the extracellular matrix, we analyzed the time constant of LV pressure decay (Tau) according to Weiss et al. [P ≤ .05). Tau values for ZDF + TN and ZDF + CA were not significantly different from LC, indicating attenuation in the overall stiffening or loss of compliance seen in the ventricle of the ZDF animals.Neither maximum nor minimum first derivative of LV pressure (dP/dt)  was statistically different among groups . Howevered to LC . Changess et al. . RelativChelation Therapy: Unproven Claims and Unsound Theories. However, chelation has proven effective in numerous studies in attenuating microvascular  as well as cardiovascular complications in animal models of diabetes [Chelation therapy has a bad reputation in modern medicine. Indeed, the second citation in a Google search for “chelation therapy” yields the website “Quackwatch,” and an article titled: diabetes , 19, 23.diabetes , 24. Mordiabetes . Alteratdiabetes , 26, anddiabetes , 28. Prediabetes . These pIn our studies of cardiac function, the data indicate that significant changes in LV chamber morphology occurred in the untreated ZDF animals as a diabetes-induced adaptation in an attempt to maintain overall function. The two chelators inhibited adverse changes in LV chamber morphology and relaxation. Specifically, type II diabetic rats treated with trientine or citric acid had significantly better in vivo left ventricular end systolic and end diastolic pressure/volume parameters than untreated animals, without a significant effect on blood glucose concentration. Additionally, both volumes at maximum and minimum dP/dt were significantly improved by trientine indicating that LV chamber underwent dilatation in order to maintain contractility. These results indicate that defective transition metal(s) homeostasis makes a significant contribution to pathogenic LV chamber remodeling in type II diabetes and that this cardiovascular pathology is prevented by chelation therapy. Although the experiments with citric acid were designed as exploratory and were limited in scope, they are reported here because, even in a small number of rats, citric acid had significant beneficial effects on both cardiac structure and function. Further studies on the effects of dietary citrate on iron and copper uptake and excretion in type II diabetes may provide some insight into the beneficial effects of citrus fruits in the human diet.The difference in LV mass between the age-matched lean control and the untreated ZDF is an interesting observation. Indeed, changes in LV mass and or geometry greatly affect the overall function of the heart. Hypertrophy can be defined as either concentric  or eccentric . In either case, this is a progressive response to chronic physiologic stress such as alterations in cardiac output due to altered hemodynamic load on the heart . In the β and Smad, which subsequently suppressed collagen production, thereby limiting ECM accumulation common to the streptozotocin diabetic model. Further, trientine may not only act to sequester Cu for urinary excretion but also act to balance the activity or increase the expression of a number of oxidases  [Work by Cooper et al. suggests the beneficial effects of chelators may be at the extracellular level , 3. In toxidase) , 17. Cheoxidase) , 34, 35.oxidase) –38. Advaoxidase) –8.It should be noted that diabetic cardiomyopathy is often accompanied by the development of cardiovascular autonomic dysfunction. Although we were unable to assess that in this study, Schreihofer et al.  investigIn summary, chelation of transition metals by trientine or citric acid significantly attenuated structural and functional changes in the heart in the Zucker type 2 diabetic rat. These observations support a previous work in the streptozotocin-induced type 1 diabetic rat and in clinical studies, suggesting that transition metal chelation can attenuate maladaptive cardiovascular remodeling leading to cardiac dysfunction in diabetes. Thus, a pharmacologic regimen incorporating Cu (and Fe) chelation is a potentially beneficial treatment strategy to prevent or attenuate the progression of cardiomyopathy in diabetes, even in the presence of chronic hyperglycemia. However, the question of whether this treatment improves remodeling or inhibits damage remains uncertain.


Cardiac insults such as ischemia, infarction, hypertrophy and dilatation are often accompanied by altered abundance and/or localization of the connexin43 gap junction protein, which may predispose towards arrhythmic complications. Models of chronic dyssynchronous cardiac activation have also been shown to result in redistribution of connexin43 in cardiomyocytes. We hypothesized that alterations in connexin43 expression and localization in the mouse heart might be induced by ventricular pacing over a short period of time.The subdiaphragmatic approach was used to pace a series of wild type mice for six hours before the hearts were removed for analysis. Mice were paced at 10–15% above their average anesthetized sinus rate and monitored to ensure 1:1 capture. Short-term pacing resulted in a significant reduction in connexin43 mRNA abundance, a partial redistribution of connexin43 from the sarcolemma to a non-sarcolemmal fraction, and accumulation of ubiquitinated connexin43 without a significant change in overall connexin43 protein levels. These early pacing-induced changes in connexin43 expression were not accompanied by decreased cardiac function, prolonged refractoriness or increased inducibility into sustained arrhythmias.Our data suggest that short-term pacing is associated with incipient changes in the expression of the connexin43 gap junction, possibly including decreased production and a slowed rate of degradation. This murine model may facilitate the study of early molecular changes induced by pacing and may ultimately assist in the development of strategies to prevent gap junction remodeling and the associated arrhythmic complications of cardiac disease. Sudden cardiac death is a common and tragic complication of heart disease that affects more than 400,000 Americans annually . The patChronically altered ventricular activation, such as that induced in canine models using pacing or radiofrequency ablation of the left bundle branch, has been associated with focal structural gap junction remodeling ,11. AreaTo test our hypothesis, we adapted the subdiaphragmatic approach for programmed electrical stimulation in the mouse heart  to allowThese data suggest that alterations in the expression and distribution of Cx43 occur after limited exposure to pacing. Changes in Cx43 expression patterns may contribute to increased arrhythmogenicity in the presence of additional insults to the heart. Since gap junction alterations may be preventable or reversible, elucidating the pathways involved may ultimately allow for the pharmacologic targeting of intermediaries in those pathways and the prevention of arrhythmic complications.Mice aged 3 – 4 months were used for these experiments. All studies were performed in accordance with the regulations of the Institutional Animal Care and Use Committee of the New York University School of Medicine . The C57BL/6J strain was used for all experiments except for immunoblotting of ventricular lysates and quantification of mRNA expression, for which CD-1 mice were used. Mice were anesthetized with inhaled isoflurane  and immobilized on a heating pad set to 37°C. Electrocardiographic signals from limb leads were monitored during the experiment and recordings were obtained prior to the initiation of pacing, during pacing (to document 1:1 capture) and following cessation of pacing.Following recording of the baseline electrocardiogram, a 1 cm midline incision was made in the epigastric region. A custom-designed UE-GM1 cardiac stimulating electrode with a 200 μm monopolar platinum tip  mounted on a micromanipulator  was inserted through the diaphragm directly into contact with the surface of the beating right ventricle. Electrode contact was ensured by monitoring the electrocardiographic activity. Pacing was performed with a Model 2352 Programmable Stimulator . Output was set at twice the stimulating threshold in all animals, with a pulse width of 1.0 ms. Pacing cycle lengths were set and maintained at 10–15% above the underlying heart rate, to ensure 100% capture for 6 hours. To serve as controls, age-, sex- and strain-matched mice were prepared and anesthetized with the pacing electrode placed into contact with the heart identically to the paced cohort, but the programmable stimulator was not switched on ("sham-paced").Electrocardiographic signals were recorded as previously described. Electrocardiographic intervals were calculated from leads I, II and III recorded at the beginning of each study and after the completion of the pacing protocol.Echocardiography was performed according to a previously described protocol  on pacedProgrammed electrical stimulation (PES) was carried out in paced and sham-paced mice at baseline and after the pacing protocol as described previously . The PESFollowing the pacing procedure, hearts destined for immunostaining were rapidly excised and frozen in Tissue Tek OCT compound . Five micron-thick sections were cut in an HM 560 cryostat  at -20°C, placed onto Superfrost/Plus microscope slides  and fixed in acetone. We selected frozen sections to evaluate the left ventricle from basal, mid-ventricular, and apical regions. We defined the basal region as those sections proximal to the papillary muscles; mid-ventricular regions were those sections of LV with clearly identified papillary muscles; and apical regions were distal to the papillary muscles. We also evaluated apical and basal sections of the right ventricle.Sections were blocked and then double-stained with a custom-made rabbit polyclonal anti-Cx43 antibody  at 1:100Images of immuno-stained sections from paced and sham-paced hearts were obtained from blinded slides and digitally archived for offline analysis. Blinded image files were uniformly thresholded by eliminating signal-free areas above and below the distribution of intensity values using the histogram function on Adobe Photoshop. Digital image processing was then performed according to previously established techniques , again iFor the evaluation of total protein levels by immunoblotting, endocardial ventricular tissue from paced and sham-paced hearts was prepared by Dounce homogenization in lysis buffer supplemented with Complete protease inhibitor cocktail . Endocardial samples were prepared by mounting the excised LV free wall in an OCT block, separating the endocardial, mid-myocardial and epicardial regions by sectioning on an HM 560 cryostat and homogenizing as above. The inner third of the LV free wall was considered the endocardial region, middle third mid-myocardial and outer third epicardial. Protein concentrations were determined by Bradford assay performed in triplicate and equal loading was confirmed with coomassie staining. Proteins were electrophoresed on 10% SDS-PAGE gels and transferred onto nitrocellulose blots . Immunoblots were blocked followed by incubation with appropriate primary antibodies directed against Cx43, cadherin (see above), Cx45 , Cx40  to the samples, the protein A suspension was centrifuged at 5000 × g and the supernatant was removed. The protein A beads were washed in IP buffer and resuspended in loading buffer prior to incubation at 100°C and analysis by SDS-PAGE. The resulting blots were incubated with a monoclonal antibody directed against ubiquitin  and processed as above.To isolate total RNA, the LV free walls of paced and sham-paced hearts were excised, mounted in OCT and snap frozen in liquid nitrogen. Endocardial and epicardial thirds were collected by sectioning through the LV free wall as described above. RNA was isolated using Trizol  according to the manufacturer's recommendations. RNA quality was verified with the Agilent 2100 Bioanalyzer . The concentration of RNA was determined by the Quant-iT RiboGreen RNA Assay Kit . Real time quantitative PCR was performed using the ABI Prism 7700 Sequence Detection System . Cx43 primer sequences, located in the 3' untranslated region, were as follows: forward primer sequence was 5'-GTGCCGGCTTCACTTTCATTAAG-3'; reverse primer sequence was 5'-ACTGACCTCGCGGAACC-3'; probe sequence was 5'-TTTCTCTCCACGGGTCT-3'. All data were normalized to Cyclophilin A (primer sequences as per ) and expData are expressed as mean ± SEM. Quantitative immunofluorescence data were compared between groups with ANOVA using StatView . Data from electrocardiography, echocardiography, PES, immunoblot densitometry and qRT-PCR were compared between groups with unpaired two-tailed t-tests (Microsoft Excel). Electrocardiographic indices, echocardiographic measurements and ERP values obtained during or after the pacing protocol were compared to baseline measurements with paired two-tailed t-tests (Microsoft Excel). P < 0.05 was considered statistically significant.For ease of pacing and reliability, we used the subdiaphragmatic approach, which allows for placement of the stimulating electrode on the heart without necessitating sternotomy, thoracotomy, mechanical ventilation or manipulation of the vasculature . For theSince our objective was to model altered activation rather than induce tissue damage , we limiTo determine whether short-term pacing influenced synchrony of cardiac contraction, we examined mice prior to and during pacing using echocardiography. The septal-to-posterior wall motion delay (SPWMD), an echocardiographic index of dyssynchronous contraction ,23, incrAlthough global ventricular function was slightly diminished during pacing, fractional shortening was not significantly altered after cessation of pacing in the paced mice compared to matched sham-paced controls or compared to their baseline values. Similarly, left ventricular dimensions and wall thicknesses were not significantly changed in paced mice compared to matched sham-paced controls or baseline values Table . Thus, sTo characterize the effect of short-term pacing-induced dyssynchrony on gap junction distribution in the mouse heart, we paced mice for 6 hours  -26 and aPost-hoc analysis showed that the Cx43 signal area was decreased significantly only at the basal LV endocardial segments in paced hearts compared to sham-paced hearts . There were trends at the apical (6.76 ± 0.89% in paced vs. 7.97 ± 0.96% in sham) and mid-ventricular (7.15 ± 0.69% in paced vs. 9.10 ± 1.59% in sham) LV endocardial segments toward decreased Cx43 signal area in paced hearts, although these were not statistically significant.There was no difference in Cx43 signal area between sham (3.40 ± 0.25%) and paced (3.91 ± 0.45%) RV sections. Specifically at the RV apex where the stimulating electrode was placed, there was no statistically significant difference in Cx43 signal area between sham (3.51 ± 0.46%) and paced hearts  to 3 . We found that sham sections demonstrated extensive subjective lateralization of Cx43 signal (2.75 ± 0.25) and that the extent of lateralization did not appear significantly different in the paced hearts .Since Cx43 immunosignal area was reduced at the LV endocardium after 6 hours of pacing, we investigated the effects of short-term pacing on Cx43 mRNA levels in the LV free wall endocardial and epicardial regions of paced and sham-paced control hearts. As shown in Figure Since short-term pacing in the murine heart resulted in down-regulation of Cx43 mRNA levels in the LV and decreased immunosignal of Cx43 at the endocardium, we investigated whether endocardial Cx43 protein abundance was affected by pacing. Surprisingly, there was no significant change in Cx43 protein expression in the paced LV endocardial region compared to sham-paced controls (n = 11 sham and 11 paced; Figure Since total endocardial Cx43 protein levels in the paced hearts were unchanged despite reduced mRNA levels and immunosignal area, we considered that pacing might have resulted in redistribution of Cx43 from the sarcolemma into non-sarcolemmal pools. Fractionation of heart samples demonstrated significantly reduced Cx43 abundance in the sarcolemma-enriched fraction isolated from paced hearts compared with sham-paced controls (40.4 ± 8.7% decrease in the paced hearts; p < 0.05; n = 11 sham and 12 paced hearts; Figure With reduced Cx43 mRNA levels despite preserved total protein abundance and an apparent redistribution of Cx43 immunosignal from the sarcolemma, we predicted that the process of degradation of gap junction moieties might be disrupted, resulting in an accumulation of ubiquitinated Cx43 in the paced hearts. We tested this prediction by immunoprecipitating Cx43 in lysates from five sham-paced and five paced hearts, followed by gel electrophoresis and blotting for ubiquitin. We observed a substantially increased expression of ubiquitinated forms in the paced hearts Figure . These fWe next investigated whether pacing-induced changes in Cx43 expression were associated with persistent alterations in electrocardiographic and electrophysiologic indices. After cessation of pacing, we did not detect significant changes in electrocardiographic parameters in the paced mice when compared with baseline values or sham-paced controls (Table In this study, we investigated the effects of short-term, single-lead ventricular pacing on Cx43 expression and distribution. We found that pacing at the epicardial surface of the RV using the subdiaphragmatic approach was associated with dyssynchronous systolic contraction of the left ventricle. Pacing for only six hours at rates within 10% – 15% of sinus rhythm resulted in significantly reduced membrane-based Cx43 immunosignal in the endocardial regions of the LV free wall. While levels of Cx43 mRNA were significantly decreased, Cx43 protein abundance was unchanged by pacing in lysates isolated from the endocardial third of the LV free wall. Fractionation of ventricular lysates was consistent with a partial redistribution of Cx43 protein from the sarcolemma into non-sarcolemmal pools in the paced hearts compared to sham-paced controls. Immunoprecipitation of Cx43 and blotting for ubiquitin demonstrated a substantially increased accumulation of ubiquitinated Cx43 in the paced hearts. Thus, our studies suggest that short-term pacing results in significant reductions in Cx43 transcription and/or mRNA stability, altered intracellular distribution of gap junction protein, and a disruption in the process of degradation of Cx43.It is intriguing that in the absence of an underlying disease process, such as tachycardia-induced cardiomyopathy, hypertrophy or ischemia, short-term pacing of the heart at rates just above that of normal sinus rhythm should produce significantly decreased Cx43 mRNA levels and intracellular redistribution of Cx43 protein. Nevertheless, our observations are in keeping with findings from pacing studies in other animal models. Patel, et al, performed a study in which dogs were paced for 21 days at rates 10% – 15% above the normal sinus rate , in contIn another model of the effects of chronic dyssynchrony, dogs studied four weeks after radiofrequency ablation of the left bundle branch were found to have Cx43 lateralization that was associated with reductions in conduction velocity, action potential duration and refractory period . LateralWhile altered gap junction expression was observed after several weeks of pacing or experimentally induced left bundle branch block in the canine model, other data suggest that changes in gap junction expression may be induced over a much shorter time frame. Ischemia induced by cessation of perfusion for up to 40 min in the isolated rat heart resulted in dephosphorylation and redistribution of Cx43 from the intercalated disc, without a net loss of Cx43 protein abundance from the myocardium. Lateralized Cx43 immunosignal in the isolated ischemic rat heart appears to be mainly phosphorylated in those hearts that recovered contractile function, but nonphosphorylated in those hearts that did not recover function during reperfusion . Based oThe effect of short-term pacing on connexin expression has been previously described in the setting of strong electric currents, which were up to 100 times greater than those used in this study and represented a model of cardiac tissue damage . In thatOur results demonstrating a gradient of increasing epicardium to endocardium Cx43 immunostaining area in the sham-paced hearts are consistent with data presented by other groups. Yamada et al. observed a similar transmural gradient of Cx43 immunosignal distribution in mice of the same strain as that used for our study . A transPreviously, we and others have observed significant changes in ECG parameters and a substantially elevated risk of lethal ventricular tachyarrhythmias in conditional models of decreased cardiac Cx43 expression ,35-37. IThe Cx43 gap junction is known to be degraded along the ubiquitin proteasome pathway, with involvement of the endosome/lysosome either sequentially or in parallel ,39. UbiqWe intend to use the pacing model to broaden our understanding of the role of Cx43 and its regulation in cardiac disease. However, an important potential limitation of this model is that the mechanisms underlying pacing-induced changes in gap junction expression are not necessarily the same as those responsible for gap junction remodeling observed in ischemia, the peri-infarct zone or other pathological states. Nonetheless, defining the mechanisms responsible for pacing-induced alterations in the intracellular distribution of Cx43 will allow us to generate hypotheses that will be testable in models of cardiac disease.In summary, we report that short-term cardiac pacing at rates just fast enough to ensure capture, induces mechanical dyssynchrony of the left ventricle, significantly decreased Cx43 mRNA levels and partial redistribution of Cx43 protein in the murine heart. These data suggest that limited exposure to dyssynchronous activation results in remodeling of the cardiac gap junctions in the absence of sustained measurable effects on contractility or arrhythmic inducibility. However, in the setting of cardiac disease with decreased baseline Cx43 expression, dyssynchronous activation with its attendant effects on gap junction remodeling may further exacerbate arrhythmic complications and worsen cardiac performance.AK participated in the design of the study, carried out or assisted in all experiments and drafted the manuscript. RAK participated in the design of the study, carried out pacing, immunoblotting and immunofluorescence experiments and helped to draft the manuscript. EK carried out immunoblotting experiments and helped to draft the manuscript. JEF carried out the qRT-PCR experiments. PPG carried out the immunoprecipitation experiments. MP carried out the lysate fractionation experiments. FL carried out immunofluorescence experiments. MJR helped design, oversee and interpret the lysate fractionation and immunoprecipitation experiments. ALW helped conceive of the study, participated in its design and helped to draft the manuscript. EAF participated in the design of the study and coordinated the qRT-PCR experiments. NSP helped conceive of the study, participated in its design and helped to draft the manuscript. DEG conceived of the study, participated in its design, oversaw coordination and execution of the study and drafted the manuscript. All authors have read and approved the final manuscript.


Pericardial adhesions are a pathophysiological marker of constrictive pericarditis (CP), which impairs cardiac filling by limiting the total cardiac volume compliance and diastolic filling function. We studied diastolic transmitral flow efficiency as a new parameter of filling function in a pericardial adhesion animal model. We hypothesized that vortex formation time (VFT), an index of optimal efficient diastolic transmitral flow, is altered by patchy pericardial-epicardial adhesions.In 8 open-chest pigs, the heart was exposed while preserving the pericardium. We experimentally simulated early pericardial constriction and patchy adhesions by instilling instant glue into the pericardial space and using pericardial-epicardial stitches. We studied left ventricular (LV) function and characterized intraventricular blood flow with conventional and Doppler echocardiography at baseline and following the experimental intervention.Significant decreases in end-diastolic volume, ejection fraction, stroke volume, and late diastolic filling velocity reflected the effects of the pericardial adhesions. The mean VFT value decreased from 3.61 ± 0.47 to 2.26 ± 0.45 (P = 0.0002). Hemodynamic variables indicated the inhibiting effect of pericardial adhesion on both contraction (decrease in systolic blood pressure and +dP/dt decreased) and relaxation (decrease in the magnitude of -dP/dt and prolongation of Tau) function.Patchy pericardial adhesions not only negatively impact LV mechanical functioning but the decrease of VFT from normal to suboptimal value suggests impairment of transmitral flow efficiency. Left ventricular (LV) diastolic filling and its alterations are important indicators of overall cardiac health status -4 and coDevelopment of pericardial adhesions is a pathophysiological marker of constrictive pericarditis (CP), which impairs cardiac filling by limiting the total cardiac volume compliance ,16. The The study was approved by the Mayo Clinic Institutional Animal Care and Use Committee. Eleven pigs weighing 40 to 45 kg were anesthetized with inhalation of 1.5% isoflurane and mechanically ventilated with a mechanical ventilator , and blood gases were periodically checked. Pressures in the LV and ascending aorta were measured with high-fidelity catheters  placed via internal carotid artery cannulation. Internal jugular vein cannulation allowed administration of fluids and medications. After median sternotomy, the heart was exposed, sparing the pericardium.We have described the pericardial adhesion model in detail previously  Figure . BrieflyLV end-diastolic pressure (LVEDP) was timed to the R-wave peak on the synchronous electrocardiographic (ECG) tracing. Besides peak positive dP/dt (+dP/dt) and peak negative dP/dt (-dP/dt), the time constant of LV pressure decay during the isovolumic relaxation period (Tau) was ascertained by using a validated zero-asymptote model .An Acuson Sequoia C512 ultrasound system  equipped with an 8V3C transducer set to 3.5 MHz was used to acquire echocardiographic images. The transducer was placed on the pericardium and acoustically coupled with a small amount of gel. Ultrasound scans for each pig included: 1) apical 2-chamber and 4-chamber views for measurements of LV cardiac output, end-diastolic volume (EDV), end-systolic volume, stroke volume, and ejection fraction; 2) Doppler spectral flow for peak early (E-wave) and atrial (A-wave) filling velocities; and 3) tissue Doppler for peak velocities of the mitral annulus in early (E') phases of LV filling , and the dimensionless ratio E/E' was computed ,20. For Transmitral flow can produce an intraventricular rotating fluid mass referred to as a vortex ring which supports a more efficient fluid transport compared to a straight jet alone ,12. The where β is the fraction of the stroke volume provided by the atrial component of LV filling and was calculated as the A-wave area divided by the sum of the A-wave and E-wave areas . In our However, vortex ring formation during diastole can also be defined as :E (cm). This leads to a simple definition of vortex formation time , which has been used in our study as follows:where al views . BecauseWe used this formula because of its practicality and we believe that this formula is a more convenient and more practical implication.Data were expressed as means ± standard deviation. Hemodynamic, 2 D, and Doppler echocardiographic data, as well as strain and strain rate (SR) measurements were obtained at baseline and during intervention. These data were compared using a 2-tailed paired t test. The relationship between different continuous variables was analyzed using single and multiple regression analyses. All differences were considered significant for P < 0.05.Data were obtained from 8 animals and were used to induce the pericardial adhesion model  and geneHemodynamic results are summarized in Table Conventional echocardiographic measurements of LV filling and mechanical function are summarized in Table E decreased and resulted in a significant decrease in VFT after the intervention  formation of a noncompliant pericardium with adhesions negatively affects transmitral flow efficiency, as expressed by a shift in VFT values out of their optimal range.Our animal model produced areas of pliable and rigid pericardium  and strong pericardial-epicardial adhesions. In a fully developed model of CP, the encasement of the heart by a rigid shell of noncompliant pericardium results in characteristic pathophysiologic effects, including impaired diastolic filling of the ventricles .Hemodynamic parameters Table  and funcIn our model, LV function was impaired in its mechanical performance by patchy pericardial-epicardial adhesions and spotty stiffening or hardening of the pericardium led to impairment of both systolic and diastolic myocardial function. The observed decreases in ejection fraction, stroke volume, cardiac output, and +dP/dt are consistent with impairment in systolic function, but the mechanism of reduced ejection performance may be secondary to impaired ventricular filling, especially in mid- and late diastole (reduced preload) .The decrease in the absolute value of -dP/dt and an increase in Tau are consistent with impairment in diastolic function. Impairments in LV filling  ,25 are tAlthough the possibility of injury and its effect on LV function cannot be excluded, Kaplan et al  showed tLV diastolic filling plays an important role in cardiac function and its optimal means of cardiac efficiency . LV diasThe diastolic vortex ring acts as a kinetic energy reservoir that facilitates propulsion of blood in systole , contribThe rapid development of the adhesions in our study impairs both systolic and diastolic myocardial function. Consequently, as our study shows, this alteration also negatively impacts conditions for vortex ring formation,  and implies the loss of optimal hemodynamic efficiency during LV filling .This is a starting point towards further investigations of VFT in chronic pericarditis both in animal experiments and clinical practice. Diagnosis of early stages of CP can be challenging because of variable hemodynamic presentations and the difficulty of distinguishing CP from other diseases with similar symptoms and signs . VFT maySpreading the tissue glue within the pericardial space was a manual process, which introduced variations in the distribution of adhesions and pericardial stiffening. However, despite the variability in this process, we observed a consistent and statistically significant decrease in VFT. We cannot conclude that this animal model of pericardial adhesion is the same as physiology in chronic pericarditis. We plan on conducting further studies, both acute and chronic, to continue the development of the animal model and to characterize its functional and histopathologic effects on myocardium. There is a slight inconsistency between the optimal VFT value range considered for experimental in vitro setting and clinical settings; the latter has a somewhat broader range . Thus, VIn our animal model, patchy pericardial stiffening and pericardial-epicardial adhesions resulted in systolic and diastolic dysfunction. However besides the mechanical effect, we found also detrimental effect of pericardial adhesion on diastolic vortex function, a decrease in the VFT value, suggesting impairment in the efficiency of diastolic filling.The authors declare that they have no competing interests.JP conceived the study and participated in the design and creation, performed detailed statistical analyses, and drafted manuscript . MA critically reviewed study and manuscript. AC critically reviewed study and manuscript. EM critically reviewed study and manuscript. MK critically reviewed the manuscript. JW critically reviewed VFT analyses. MM critically reviewed VFT analyses. JH critically reviewed VFT analyses. FM critical review of manuscript. MB participated in design and creation of study and critically reviewed statistical analyses and manuscript. All authors read and approved the final manuscript.


Although previous studies have helped define the natural history of Duchenne Muscular Dystrophy (DMD)-associated cardiomyopathy, the myocardial pathobiology associated with functional impairment in DMD is not yet known.cc) was calculated using tagged CMR imaging. T2 maps of the LV were generated for all subjects using a black blood dual spin echo method at two echo times. The Full Width at Half Maximum (FWHM) was calculated from a histogram of LV T2 distribution constructed for each subject.The objective of this study was to assess the distribution of transverse relaxation time (T2) in the left ventricle (LV) of DMD patients, and to determine the association of myocardial T2 heterogeneity to the severity of cardiac dysfunction. DMD patients (n = 26) and normal control subjects (n = 13) were studied by Cardiovascular Magnetic Resonance (CMR). DMD subject data was stratified based on subject age and LV Ejection Fraction (EF) into the following groups: A ; B  and C . Controls were also stratified by age into Groups N1  and N2 . LV mid-slice circumferential myocardial strain . Further, FWHM was significantly higher in those with reduced circumferential strain (|εcc| ≤ 12%)  than those with |εcc| > 12% (Group A). Group A FWHM was not different from the two normal groups .In DMD subject groups, cc correlates well with increased T2 heterogeneity quantified by FWHM, indicating that subclinical functional impairments could be associated with pre-existing abnormalities in tissue structure in young DMD patients.Reduced EF and ε A profibrosis -6. Studifibrosis ,8. Previncing age. While tin-vivo pathobiology has, however, been available for quite some time. The measure of transverse relaxation time (T2) of tissue has been well-characterized; T2 weighted MR signal has been used to detect various pathologies of soft tissues [A CMR methodology for evaluating  tissues ,11. Fact tissues , increas tissues  are know tissues . In rece tissues -17, myoc tissues ,19, and  tissues .DMD is an example of a progressive disease process where fibrosis and edema related to inflammation coexist. In a previous study conducted at a magnetic field strength of 0.5 Tesla, Mavrogeni et al  showed tWe thus hypothesized that heterogeneity of T2 across an entire slice of the heart exist in the early stages of DMD-associated cardiomyopathy and are associated with the subclinical functional abnormalities quantified by myocardial circumferential strain. The purpose of this study was thus to evaluate the heterogeneity of T2 distribution in the LV in boys with various stages of DMD.cc| measurements were not available in the N2 control group. The Institutional Review Board at the Cincinnati Children's Hospital Medical Center approved the study.Data were analyzed from records of DMD subjects followed at Cincinnati Children's Hospital Medical Center. The diagnosis of DMD was confirmed by skeletal muscle biopsy showing absent dystrophin and/or DNA analysis demonstrating a characteristic dystrophin mutation in all patients. DMD patients who underwent clinical CMR between September 2005 and September 2007 were included in this analysis. Additionally, two control groups of young  and older  normal control males underwent an identical protocol. EF and |εAll subjects were > 5 years of age, thereby eliminating the need for sedation. CMR was performed at our center on 97 DMD patients between September 2005 and September 2007. Data from 71 was excluded due to absence of T2 images (n= 44), absence of tagged images (n = 18) or poor tag quality secondary to breathing artifact or patient movement (n = 9).The subject data were stratified into 5 groups (two groups of control subjects: N1(n = 6) and N2(n = 5)) and three groups of DMD patients (A (n = 12), B (n = 8), and C (n = 6)). Stratification in the control subjects was based on age. In the DMD subjects stratification was primarily by age and secondarily by LV Ejection Fraction (EF)  system.CMR was performed on a Siemens 3-T Cardiac functional imaging was performed as previously described . CMR was2, slice thickness 6 mm, flip angle 20°, TE/TR 3 ms/4.2 ms, views/segment 7-9.Tagged cine CMR were acquired in the short axis of the mid left ventricle at the level of the papillary muscles with an ECG-triggered segmented k-space fast gradient echo sequence with spatial modulation of magnetization in orthogonal planes. Grid tag spacing was 7 to 8 mm. The scan parameters used were: field of view (30 - 32) × (25 - 26) cm1 = 6 ms, TE2 = 34 ms. Data acquisition was done during late diastole by inserting a subject dependent delay.For T2 measurements, spin echo images of the left ventricle in the short axis plane were acquired using a black blood dual spin echo method. Imaging parameters were: Slice thickness = 5 mm, in plane resolution = 1.4 mm × 1.4 mm, echo train length = 5, Echo times: TEVentricular volumes, mass, and global function were assessed via standard planimetry techniques with semi-automated computer software  by expert readers .cc, to allow for comparison purposes. All HARP strain analyses were performed by an experienced reader (K.N.H.). To assess inter-observer variability of HARP analysis, a second experienced reader (W.M.G) performed the same analysis on a subset of DMD subjects (n = 6) and controls (n = 3).Tagged images were analyzed with the HARmonic Phase  technique, as previously described . Based oT2 values were calculated on a pixel-by-pixel basis according to the standard description of the signal behavior in spin echo magnetic resonance imaging as:1 and S2 are the signal intensity at echo times TE1 and TE2 respectively. Regions of interests were manually drawn (J.P.W.) to extract the T2 values. The LV was defined on the T2 weighted magnitude image (i.e. at TE = TE2) by manually drawing (J.P.W) the endo- and epicardial boarders with care taken to avoid signal from blood. Similarly, lateral wall and septum was segmented manually as defined by the American Heart Association 17-segmental model i.e. septum = regions 7 and 8; lateral wall = regions 11 and 12. Using these T2 values, a histogram of LV T2 distribution with bin size equal to 1 ms was constructed for each subject.where SFWHM) was calculated after applying box car averaging. The FWHM was defined as the width of the histogram at half the maximum height. Additionally, the mean and the standard deviation of T2 of the entire LV as well as in the septum and in the lateral wall were also calculated. All image processing related to T2 measurements were done using custom developed software written in the Interactive Data Language .For each histogram, the Full Width at Half Maximum . The General Linear Model procedure was used to access group differences among the means for FWHM adjusting for age. Since age was not shown to be significant, it was removed from the model. The distribution of T2 was slightly skewed; therefore the Wilcoxon Rank sum test was used. All tests were two-sided and p-values ≤0.05 or confidence intervals that did not include zero were considered statistically significant.Analyses were performed using SASA summery of results is presented in Table cc| < 16%, consistent with our prior work [cc| was significantly different among the DMD groups and progressively decreased with age and EF. Control subjects in group N1 had normal EF and normal |εcc| (>16%).All DMD subjects had abnormal |εior work , despiteFWHM of the T2 histogram was significantly (p < 0.05) greater in Groups B (30.9 ± 5.3 ms) and C (33.0 ± 6.4 ms) compared to that of Group A (25.3 ± 3.8 ms) and the normal Groups N1 (25.3 ± 3.5 ms) and N2 (24.0 ± 7.3 ms)  had a significantly greater T2 heterogeneity than that of DMD patients with normal EF and |εcc| >12% (Group A).Figure 33.0 ± 6. ms compaincreasing FWHM with decreasing mean T2 of the LV (T2mean) was noticed. We characterized this trend by calculating the ratio FWHM/T2mean for all subjects of groups A and B. FWHM/T2mean was significantly (p < .0001) higher in group B (0.54 ± 0.08) compared to Group A (0.42 ± 0.06). Regression analysis showed moderate association between FWHM/mean T2 and |εcc| in group A and B , a trend towards roup B 0.4 ± 0.08 FWHM and εcc are shown in Table FWHM were higher in the septum than in the lateral wall. No difference in the septum and lateral wall was found in εcc. Although not statistically significant the lateral wall T2 FWHM followed the same trend as T2 FWHM of the entire LV: Group A (19 ± 6 ms) < Group B (21 ± 8 ms) < Group C (26 ± 6 ms).Results of regional analysis of mean T2, T2 FWHM of 6 subjects using the custom made software. For each subject, the mean and the standard deviation of the independent measurements were calculated. The inter-observer variability of T2 and T2 FWHM was assessed as the standard deviation/mean %, averaged among all the subjects. The mean percentage of standard deviation among the investigators was 3.2% for meant T2 and 3.1% for T2 FWHM.Three investigators blind to the identity of the subjects independently measured the mean T2 and T2 (FWHM) of T2 distribution in LV to quantify the myocardial structural heterogeneity in DMD patients. In DMD subject groups, FWHM of the T2 histogram rose progressively with age and decreasing EF. Further, FWHM was significantly higher in those with reduced circumferential strain (|εcc| ≤ 12%)  than those with |εcc| > 12% (Group A). The myocardial structural abnormality suggested by the observed trend is likely due to concomitant presence of micro-fibrosis  technique) and myocardial edema. Our results support the notion that the regional dysfunction depicted by depressed |εcc| is associated with the ultrastructural myocardial cell abnormality present in DMD patients.In this study, we used the Full Width of Half Maximum FWHM rather than the mean T2 is likely to better characterize this disease. Furthermore, LGE studies in muscular dystrophy show that distribution pattern of late enhancement is patchy and diffused unlike the well defined sub-endocardial LGE related to coronary territories found in myocardial infarction [FWHM proposed in this study quantify both spatial and pathologic heterogeneity of the DMD tissue.T2 is known to increase with intramuscular edema and inflammation that increases free water content in tissue  whereas farction ,24,27-29FWHM, the standard deviation of T2, calculated assuming that T2 is normally (Gaussian) distributed within the LV, was not significantly different between groups. This is not surprising since the histograms of T2, as seen in Figure Unlike the The mean myocardial T2 of the normal subjects (both N1 and N2 combined) in our study was 52 ± 11 ms which is same as that found by Giri et al (52 ± 3 ms) . RegionaWe used a turbo spin echo method with two acquisitions (echoes) to calculate T2. The dual echo method has been successfully used by others in cardiac and brain imaging applications in the past ,30,34. Wcc| and FWHM were demonstrated between young DMD patients with normal EF and older patients with reduced EF, this is a cross-sectional and not a serial study. Accordingly, repeat serial examinations would provide a more robust analysis of longitudinal |εcc| and FWHM in this patient population. In the current study, only the midventricular slice was analyzed secondary to our experience of limited reproducibility of the basal and apical slices.Although significant differences in |εFWHM in T2 points to an underlying pathobiology that is both microscopic and widely distributed within the myocardium. Unlike gross myocardial changes that arise in the later stages of the disease, these early abnormalities are not likely to be detected by T2 weighted imaging or LGE. Thus, T2 heterogeneity quantified by FWHM could provide an early surrogate marker for pathobiology in DMD patients to monitor activity of disease as well as to assess efficacy of pharmacological interventions.The increased cc: Myocardial Circumferential Strain; EF: Ejection Fraction; S1, S2: Signal Intensity of Spin Echo Images; TE1, TE1: Echo Times; FWHM: Full Width at Half Maximum; T2mean: mean T2 time of the Left ventricle; B0: Bore magnetic field strength; B1: Radio frequency transmit magnetic field strength.DMD: Duchenne Muscular Dystrophy; T2: Transverse Relaxation Time; εThe authors declare that they have no competing interests.JW, KH, and WG contributed to all aspects of the manuscript's conception, design, data analysis, collection, critical revision and final approval. SH contributed to inter-observer variability analysis. WB, WM and RF contributed in data analysis, critical revision and final approval of the manuscript.


A 49-year old patient presented late with an anterolateral ST-elevation myocardial infarction and was treated with rescue angioplasty to an occluded left anterior descending artery. Her recovery was complicated by low-grade pyrexia and raised inflammatory markers. Cardiovascular magnetic resonance 5 weeks after the acute presentation showed transmural infarction and global late gadolinium enhancement of the pericardium in keeping with Dressler's syndrome. Secondary pericarditis can occur following myocardial infarction; this is relatively common acutely between 2 and 4 days after myocardial infarction. In contrast Dressler's syndrome presents with a low-grade fever and chest pain 2 to 10 weeks after myocardial infarction, and is less common, affecting between 1 and 5% of patients , latent A 49 year old woman presented with jaw and arm pain to her local Emergency Department. She had the pain since the previous evening, approximately 14 hours, and had initially presented to her dentist before being redirected. Cardiac risk factors included hypertension, smoking and a positive family history of ischaemic heart disease in her father. The ECG showed anterolateral ST elevation and because of this and ongoing pain, she was thrombolysed with retaplase despite the late presentation. At 90 minutes the ECG changes and pain had not resolved and therefore she was transferred to our tertiary centre for rescue angioplasty. The left anterior descending artery (LAD) was occluded with a large thrombus load. The LAD was successfully opened and a Vision 3.5 × 15 mm bare metal stent was deployed, the procedure was covered with heparin and abciximab and an intra-aortic balloon pump. There was moderate circumflex and right coronary artery disease which was not intervened upon. Cardiac enzymes were elevated with peak CK of >4000 iu/L and troponin I of 98 μg/L. A transthoracic echocardiogram the following day showed a mildly dilated left ventricle (LV) with mild hypertrophy. The anterolateral wall was akinetic with moderate to severely impaired LV function. There was no significant valvular disease and a 1 cm pericardial effusion around the posterior, inferior and lateral walls and right heart with no haemodynamic compromise. Her recovery was complicated by low-grade pyrexia and raised inflammatory markers, peak CRP 321 mg/L. Blood, urine and sputum cultures were negative however she was treated empirically for a chest infection with doxycycline. An episode of hypoxia and chest pain without gross changes on chest radiograph resulted in a CT pulmonary angiogram which did not show any evidence of pulmonary embolism but some basal atelectasis. Two weeks after admission she was asymptomatic. Prior to discharge an exercise tolerance test was arranged to assess for ischaemia in view of the moderate untreated coronary disease. She completed 6 minutes of the Bruce protocol achieving 85% of her maximal predicted heart rate, there was no pain but mild ST depression inferiorly. The test was submaximal but did not show gross ischaemia. In view of this she was discharged home with a plan for outpatient myocardial perfusion imaging.She attended for a CMR stress perfusion study 5 weeks after her initial infarct. At this point she did not report any symptoms. The LV was moderately dilated with an akinetic anteroseptal wall (Additional files This patent presented late after ST elevation myocardial infarction with a resulting large rise in cardiac enzymes and significant transmural myocardial infarction. Her inpatient stay was complicated by low-grade pyrexia, raised inflammatory markers and a small pericardial effusion. In view of the negative cultures and subsequent CMR findings this was likely to have been due to pericardial inflammation. CMR is one of the most versatile modalities to assess pericardial disease. PericarWritten informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.The authors declare that they have no competing interests.CS drafted the manuscript and manipulated the CMR images for inclusion. JKhoo helped draft the manuscript. JKovac is the cardiologist responsible for the patient's care. GM acquired and interpreted the CMR images and helped draft the manuscript. All authors read and approved the final manuscript.4-chamber view. SSFP cine demonstrating moderate LV dilatation, apical dyskinesis and functional mitral regurgitation.Click here for fileShort axis view. SSFP cine at mid-ventricular level demonstrating thinned and akinetic anterior and anteroseptal segments.Click here for file


Cardiac resynchronization therapy (CRT) is now a well established treatment modality for adult patients with drug refractory symptomatic congestive heart failure. Multiple large-scale studies have clearly demonstrated reduction in heart failure-related morbidity with CRT -4. More More recently, small case series have shown the potential for benefit for CRT in patients with congenital heart disease including those with a systemic right ventricle and single ventricle physiology. Whether long-term benefit for extending CRT therapy to children with congenital heart disease exists is unclear.There are several challenges confronting the implanter when considering CRT therapy in children, and these are summarized in Despite these limitations in our present knowledge as to how indications, implant, and optimization of CRT devices should be amended in children, evidence is accumulating that suggests at least a similar benefit in children exists, as has been been demonstrated in adults.In children with left ventricular failure and left bundle-branch block, acute left ventricular hemodynamics has been shown to improve with biventricular stimulation . In longIn this article, we discuss commonly encountered causes for difficulty in implanting CRT devices in children with and without congenital heart disease. We suggest potential solutions and an approach in solving such difficulties. We further present guidelines for understanding differences in interpreting the standard indications for CRT implantation and optimization of CRT devices in adults to children .Implanting left ventricular leads in children is generally more challenging than in adults. The reasons for this, as expected, are largely a result of the smaller size of the vessels and cardiac chambers as well as the increased risk of cardiac perforation as with most pediatric cardiac procedures. Beyond this, however, there are specific challenges that are addressed in this section involving challenges often unique to the pediatric population.Entering the coronary sinus in children requires an accurate understanding of the standard fluoroscopic views and important anatomic landmarks in these views. Vagaries of the coronary ostium itself in the young and the immediately adjacent atrial myocardium is important to appreciate, particularly when selecting sheaths and manipulating the pacing lead. A prominent thebesian valve at the ostium of the coronary sinus likely occurs more frequently in this age group as well.Because the ventricular apex is to the left of the body, the fluoroscopic views must take this into consideration to obtain anatomic accuracy. The use of the left anterior oblique (LAO) and right anterior oblique (RAO) projections is highly recommended and can be used to avoid common complications . In the Performing this maneuver in every case in the standard fluoroscopic projections will significantly enhance the ease in which this important first step for CRT delivery is performed. As seen in Rarely, a completely occlusive imperforate thebesian valve may preclude standard cannulation of the coronary sinus. Even with near occlusive valves, typically the inferior-ventricular quadrant of the coronary sinus ostium will not be covered. This is one of the reasons why the maneuver described above starting from the ventricle as inferiorly as possible and pulling back to the coronary sinus is more likely to be successful in the difficult case. In the rare patient with a near occlusive valve, gentle force with a blunt catheter being certain that one is in the plane of the coronary sinus and using intracardiac or transesophageal ultrasound guidance is successful. In very rare circumstances, radiofrequency energy posteriorly applied  can be used to create a controlled perforation of the thebesian valve and allow entrance of the coronary sinus.An important anatomical fact should be appreciated when implanting left ventricular leads in children. The atrial myocardium between the eustachian ridge and the tricuspid valve immediately adjacent to the coronary sinus ostium is often aneurysmal or pouch-like in young children. This subeustachian pouch  can makeOnce the coronary sinus has been entered and the guiding sheath stabilized in its proximal portion, the next task that may be difficult in children is advancing the pacing lead within the coronary sinus to a ventricular vein that drains the lateral wall of the left ventricle.Particularly in the very young, the coronary vein can be quite friable, and even a relatively moderate manipulation can result in coronary venous dissection. Although this is well tolerated by patients, once dissection occurs, further manipulation in the coronary sinus or placing the lead may be precluded. Perhaps the most important method to avoid coronary dissection in addition to applying very gentle force is to never advance an oversized sheath (inner diameter of the sheath larger than the outer diameter of the wire or guiding catheter within the sheath) in tandem into the coronary sinus. Once a guiding catheter has been placed relatively deep into the coronary vein, it should be pulled back as the sheath is being advanced. Similarly, when advancing the pacing lead once the guide wire has advanced to a vein of interest, when trying to advance the lead, the wire should be gently pulled back as the lead is advanced. After advancing about a centimeter, the wire can then be re-advanced into the ventricular vein of interest and the maneuver repeated until the lead is in the required position. This maneuver (pulling back on wire while pushing the lead) is particularly important to execute when negotiating tortuous vessels or at the point of branching in the ventricular veins and tributaries.A common reason for difficulty in advancing the pacing lead or causing venous damage is inadvertent sub-selection of an atrial or ventricular branch of the coronary sinus. If the operator is unfamiliar with the expected course of the coronary sinus in the RAO and LAO projections, an atrial branch may have been entered after engaging the coronary sinus. With continued forward force application or further counterclockwise torque, the lead/catheter will not only fail to advance but dissection may occur. Similarly, soon after engaging the coronory sinus, a posterior ventricular vein may have been entered. Again, without familiarity with this possibility, the operator may think that the coronary sinus has not been entered and the lead/catheter is in the right ventricle and pull out of the vein when simply advancing the lead or sheath over the catheter to allow lead placement may have given a satisfactory result.Just as the thebesian valve "guards" the ostium of the coronary sinus, various valves may be present, particularly in children, throughout the course of the coronary sinus including the junction with the great cardiac vein and at the ostia of the various ventricular venous branches. The most consistent of these valves is the so-called Vieussens valve located at the ostium of the posterolateral vein, typically at the same location of the vein of Marshall  approximately 2-3 cm from the coronary sinus ostium in children. This valve, if small, does not cause much difficulty once a wire has crossed the valve and entered either the posterolateral ventricular vein or great cardiac vein. When nearly circumferential, however, this may preclude entering the posterolateral vein, a typical target for left ventricular lead implantation . When thThe ideal target to place the left ventricular pacing lead in most patients is the midportion of the left ventricular free wall. There is typically a lateral cardiac vein draining this location into either the great cardiac vein or coronary sinus . SometimAnecdotal and in relatively small case series ,20, CRT Several congenital anomalies may have an associated persistent left superior vena cava. However, this anomaly often exists as a separate entity. The right superior vena cava may exist in addition to the persistent left superior vena cava, and one or more anastomotic veins between the vena cavae may be present. During initial experience with CRT, operators assumed that using the left superior vena cava would be easier for accessing the coronary sinus and a ventricular vein since this structure drains into the coronary sinus . HoweverWhen the left superior vena cava is not entirely patent, it may remain as a smaller oblique vein of Marshall . In certIn the setting of more complex congenital heart disease, the left superior vena cava may offer the best option to access both ventricles . Angiography from the vein and from the left superior vena cava is sometimes useful to understand the best target, guiding sheath, and catheter torque to obtain optimal results .A critical question that must be asked by the implanter and thoroughly researched with review of prior admitting data is where the coronary sinus drains in the patient being considered for CRT and with congenital heart disease . For exaExact surgical details and recent imaging data are essential before proceeding. In patients, for example, with AV canal defects, the closure of the septum primum defect and/or placement of a prosthetic tricuspid valve may be done in such a way that the coronary sinus actually drains into the right ventricle, distal to the tricuspid valve placement. For such patients, the catheter that is being used in an attempt to engage the coronary sinus must first access the ventricle and then appropriately maneuvered to cannulate the coronary sinus. Right ventricular as well as right atrial angiography may be useful, for even when the coronary sinus has been excluded from the right-sided structures accessible through the vena cavae, large thebesian veins may be draining into the basal right ventricle that can be cannulated and a left ventricular pacing lead placed either directly or via anastomoses to the coronary veins.In addition to clearly understanding the detailed anatomy of a patient's congenital defect, the operator should be aware of where the apex is located and the relative positions of the right and left ventricles.Knowledge of where the apex is located will allow the operator to adjust the fluoroscopic views accordingly. For example, in a patient with dextrocardia, the RAO angle is lined up along the axis of the apex and will in effect be used like the LAO projection to know when the coronary sinus is cannulated and whether the ventricular free wall is being accessed for pacing. The orthogonal view, in this case the LAO projection, will be used similarly to the RAO projection in the normal heart to follow the typical course of the coronary sinus and ventricular veins. If a patient has mesocardia, then the AP becomes the effective LAO view .Once the preimplant knowledge of the apex and coronary sinus drainage is known, it can be very useful to know the relative positions of the two ventricles. If the right ventricle is anterior, generally, coronary sinus lead placement is not distinctly more difficult than with normal anatomy. However, when the right ventricle is the posterior ventricle, careful venography, possible coronary arteriography to view the venous phase, and placement of a lead in the right ventricle prior to attempting coronary sinus cannulation can be useful.In patients with L-TGA and morphological right ventricular failure, CRT may be beneficial ,25. The In patients with a prosthetic tricuspid valve, it may not be possible  or inadvisable (bioprosthesis) to cross the valve to place an ICD lead in the right ventricle. In these patients, using similar techniques  describePatients with intracardiac shunts have a contraindication to placement of an endocardial system. The majority of experience with epicardial pacing leads for left ventricular stimulation comes from pediatric experience. Epicardial leads are frequently used in this situation because of the smaller ventricular veins in infants and small children. The use of epicardial leads is documented to have shorter longevity and higher incidence of associated exit block in comparison to endocardial steroid-eluting leads. Recently, however, the creation of steroid-eluting epicardial leads and longer duration follow-up have shown that acceptable results can be obtained with these pacing systems -28. The In recent years, CRT along with assist devices has been used as a bridge to heart transplant. Patients on heart transplant waiting lists have established criteria for CRT . Some paEven in the adult population, intensive study is presently being done to understand which exceptions to the standard criteria for recommending CRT therapy should be made. In the pediatric population, however, these issues are paramount to consider. Very few pediatric patients, both in the literature and in practice, meet the adult criteria for CRT, yet have benefited from this form of therapy .Present guidelines include having patients in New York Heart Association (NYHA) class III-IV, normal sinus rhythm, electrical evidence of ventricular dyssynchrony with QRS duration greater than 120 ms, and left ventricular ejection fraction less than 35% ,32. FurtElectrical dyssynchrony, as evidenced by bundle-branch block or wide QRS duration, is likely associated with mechanical dyssynchrony. This is not, however, a consistent relationship. This discordance (between QRS duration and likelihood of CRT benefit) is more pronounced in children since their normal QRS duration and intraventricular conduction time tend to be smaller ,31,34,35Investigations are ongoing as to whether earlier intervention with CRT in adults (NYHA class II) is beneficial. In children, considerations for earlier intervention may be important prior to adverse remodeling given the duration in which therapy will be required for heart failure and the higher relative incidence of transplant need when severe heart failure has its onset early in life .In adult patients who have drug refractory heart failure and ejection fraction less than 35% but are right ventricular paced (and as a result have a wide QRS), generally receive CRT therapy despite this situation not being specifically supported in randomized trials.The issue is more complex when considering CRT in children. Should the implanter wait until severe cardiomyopathy develops? The answer to this question is presently undefined. Some studies suggest that children with complete AV block and constantly paced from the right ventricle developed cardiomyopathy, and this can be prevented or treated by upgrading their devices to include left ventricular pacing ,37. HoweThe major randomized trials performed for CRT recruited adults in sinus rhythm. Whether or not a benefit exists for patients in atrial fibrillation or other atrial arrhythmias with CRT is being investigated. In children, the issues are even more complex. Cardiomyopathy in the setting of congenital heart disease is often complicated by scar-related atrial flutter that may be persistent in later stages. Clinical judgment must be individualized; however, in general, if ventricular rates can be well controlled , then the atrial flutter alone should not preclude CRT recommendation.Even when in sinus rhythm, children often have faster sinus rates and shorter atrioventricular conduction times. When sinus tachycardia is present, biventricular pacing that tracks the atrium may be rapid, and an element of tachycardia-related dysfunction may offset the benefits from resynchronization.Approximately a third of patients who receive CRT devices with appropriate and established indications for their placement do not respond to therapy. In certain cases, either the myocardial disease process is so far advanced or the substrate is worsening (new infarctions) that CRT benefit is far outweighed by these factors. In others, however, the left ventricular lead's position, function, and device programming may be suboptimal and result in the failure of benefit. A variety of techniques to try and optimize resynchronization devices in adults have been attempted, but none have yielded consistent and reproducible results.Since the expected result of CRT is mechanical resynchronization, echocardiographic parameters have been developed and tried to assess the extent of continuing dyssynchrony and then by reprogramming the device (offset between right and left ventricular stimulation) or repositioning the lead to check if optimal synchrony is occurring. A major issue with using these techniques in children is that of inadequate frame rates with presently existing systems. Conduction velocity, even in the diseased child's heart, is relatively rapid, and thus when frame rates are low, predicting the most dyssynchronous or latest to activate site can have unreliable spatial resolution.The effects of exercise and posture may also be more relative in children than in adults. Despite relatively advanced ventricular dysfunction, children may be more active and prone to changing their position. In these circumstances, the intracardiac conduction velocities and pacing vectors may change appreciably , making any attempt at optimization at rest in the supine position unreliable. Children may also have substantial changes in their underlying substrate, particularly the population waiting for transplantation. New surgical scars, atrial arrhythmias, and the effects of conduction slowing membrane active medications once again will not be helped with attempts at optimization prior to these changes.Since electrical stimulation must first occur prior to contraction of the myocardium, electrical synchrony has been used as a surrogate for mechanical synchrony, and attempts at using the 12-lead electrocardiogram to optimize CRT devices have arisen . The QRSNormalization of the QRS duration with biventricular stimulation is an inexact but usable surrogate for synchronization. As noted above, in children, however, the QRS is often in the normal range (as compared to adults) even with advanced cardiomyopathy and anecdotally shown benefit with resynchronization.Each pacing site gives rise to a characteristic vector that can be deduced with analysis of the 12-lead electrocardiogram. For example, right ventricular pacing from the apex results in a left bundle-branch block pattern with negative QRS complexes in leads II, III, aVF, and a tall R wave in lead I. On the other hand, left ventricular pacing from the midlateral wall results in a right bundle-branch block pattern and a negative QRS in lead I. It follows, therefore, that biventricular stimulation when optimally resynchronizing the heart , the electrocardiogram should be fused or intermediate between RV and LV pacing vectors . If, howAnother cause for the electrocardiogram to appear like right ventricular pacing alone even with biventricular stimulation is the phenomenon of anodal stimulation . This prJust as with adults, however, when CRT devices are placed in children, every effort to ensure close to 100% biventricular stimulation should be made. Causes that may be decreasing the amount of ventricular pacing including frequent PVCs, PACs that fall in the PVARP, atrial arrhythmias with rapid ventricular conduction, etc., should be sought and remedied.Cardiac resynchronization therapy has made a major impact in the lives of many adults with drug refractory congestive heart failure. While applying this therapy to children has been shown to be beneficial, distinct differences in how the physician should approach care in children exist. The standard indications often have to be modified with clinical judgment and techniques for optimization should be exactly understood prior to applying in the pediatric population.Specific difficulties associated with implanting a left ventricular pacing lead in children, and suggestions on how to overcome these difficulties have been reviewed in this paper.


The activation of the members of the myocyte enhancer factor-2 family  of transcription factors promotes cardiac hypertrophy and failure. However, the role of its individual components in the pathogenesis of cardiac hypertrophy remains unclear.In this study, we investigated whether MEF2C plays a role in mediating the left ventricular hypertrophy by pressure overload in mice. The knockdown of myocardial MEF2C induced by specific small interfering RNA (siRNA) has been shown to attenuate hypertrophy, interstitial fibrosis and the rise of ANP levels in aortic banded mice. We detected that the depletion of MEF2C also results in lowered levels of both PGC-1α and mitochondrial DNA in the overloaded left ventricle, associated with enhanced AMP:ATP ratio. Additionally, MEF2C depletion was accompanied by defective activation of S6K in response to pressure overload. Treatment with the amino acid leucine stimulated S6K and suppressed the attenuation of left ventricular hypertrophy and fibrosis in the aforementioned aortic banded mice.These findings represent new evidences that MEF2C depletion attenuates the hypertrophic responses to mechanical stress and highlight the potential of MEF2C to be a target for new therapies to cardiac hypertrophy and failure. Hypertrophy is a common feature of many forms of heart disease. While initially an adaptive response to increased workload and injury, in the long term cardiac hypertrophy predisposes to heart failuremyocyte enhancer factors-2, members A to D) transcription factors play prominent roles in the regulation of cardiac hypertrophy and remodelingAmong the intracellular pathways that integrate mechanical and hormonal signals, MEF2 RNA. Left ventricular hypertrophy induced by aortic banding in mice was used as a model system in this study.To define the potential function of MEF2C in the cardiac responses to pressure overload we conceived a strategy to deplete this factor in mouse heart by As an initial approach, experiments were set to test the efficacy of siMEF2C to knockdown MEF2C in cultured NRVMs . Transfection efficiency of siRNA in this model system was previously assessed with fluorescent oligonucleotideAfter the initial characterization in NRVMs, we then searched for an amount of siMEF2C that, when injected systemically via the jugular vein, could deplete myocardial MEF2C in mice left ventricle. We started off with 450 µg/Kg of siMEF2C based on a previous reportComplementary data on the ability of siMEF2C to deplete MEF2C protein levels was obtained by analyzing isolated cardiomyocytes. The expression of MEF2C was reduced in cardiomyocytes harvested from the left ventricle 24 hours after the treatment with siMEF2C to similar levels of myocardial extracts (∼75%), as depicted in th day after the administration of siMEF2C. However, by the 7th day after the siMEF2C myocardial MEF2C reached levels comparable to those of mice treated with siGFP and remained unchanged thereafter. Further analysis confirmed similar reductions in MEF2C transcripts in the myocardium over time after the administration of siMEF2C . Pretreatment with siMEF2C retarded the load-induced left ventricular hypertrophy as assessed by echocardiographic determination of left ventricular wall thickness . DepletiRepresentative examples of myocardial samples from sham-operated and banded mice treated with siGFP or siMEF2C are shown in MEF2 factors are important regulators of gene transcription involved in cardiac mitochondriogenesis and energy metabolismThe augmentation of mtDNA abundance during the myocardial hypertrophic growth involves regulation of mtDNA replication by mtTFA  by nuclear-encoded transcription factors (NRFs) and PGC-1 (peroxisome proliferator-activated receptor-gamma coactivator-1) transcriptional co-activatorsGiven that mitochondria are the major source of energy in the myocardium, we next examined whether changes in the levels of mtDNA:nDNA were accompanied by changes in the myocardial AMP:ATP ratio, as a mean to assess the energy levels of the myocardium. Previous studies have indicated that perturbing mitochondrial function can influence the activity of mTOR/S6 kinase, a signaling pathway critically involved in the reactive cardiac hypertrophyIn this study, we applied RNA interference technology to define the function of MEF2C in the adult heart. Our results demonstrate that the depletion of MEF2C by siRNA attenuates the hypertrophic growth of mice left ventricle in response to pressure overload, reduces hypertrophy in cardiomyocytes and diminishes interstitial fibrosis and attenuates the upregulation of ANP. These effects indicate that the downregulation of pathways controlled by MEF2C mitigates the adverse effects of pathologic cardiac hypertrophy and remodeling. Additionally, the results of the current study reveal that the anti-hypertrophic effects of MEF2C depletion may be related to a defective mobilization of mTOR/S6K by pressure overload, which may be associated with an intricate mechanism that involves negative modulation of the mtDNA replication and changes in myocardial energy metabolism caused by defective activation of PGC-1α.We observed a reduction of about 80% in the levels of myocardial MEF2C through siMEF2C delivery via the jugular vein. The gene silencing induced by siMEF2C was restricted to the lung and heart, indicating the efficiency of this route to achieve delivery of siRNA to the left ventricle, as well as the lack of significant exposure of tissues and organs distal to the left ventricle to siRNA. Besides the left ventricle, depletion of MEF2C was confirmed to occur in cardiomyocytes harvested from mice treated with siMEF2C, thereby demonstrating the susceptibility of this cell type to the effects of systemically delivered siRNA. Moreover, the specificity of MEF2C silencing was substantiated by the lack of effects of siMEF2C on MEF2A, MEF2B or MEF2D, as well as on the unrelated proteins FAK, JNK, SHP2 and GAPDH. The MEF2C depletion induced by the RNAi strategy used in the present study lasted for approximately 4 days. Such a prolonged effect of systemically delivered siRNA was previously observed for distinct targets in various tissuesA key finding in the current study is the attenuation of the load-induced left ventricular hypertrophy induced by MEF2C silencing. Remarkably, our results also show that depletion of MEF2C attenuated the interstitial fibrosis and the rise of ANP expression, without affecting the left ventricular function, indicating that depletion of MEF2C may reduce the adverse effects of pressure overload. Consistent with this hypothesis, data from previous studies indicate that activation of MEF2C may mediate myocardial adverse events invoked by pathological stress2+/Calcineurin activation of PGC-1α promoter and deletion of MEF2A in mice was demonstrated to result in perturbation of mitochondrial structureMEF2 transcription factors have been implicated in the direct and indirect regulation of multiple genesThe data from the present study indicate that MEF2C depletion is accompanied by downregulation of the mTOR/S6K pathway In conclusion, we used RNAi strategy to deplete MEF2C in the left ventricle of adult mice and understand the role of this factor in the responses of the left ventricle to mechanical stress. The data indicate that depletion of MEF2C is sufficient to markedly attenuate the hypertrophic growth and myocardial fibrosis of overloaded left ventricle by a mechanism dependent on defective activation of mTOR/S6K pathway. Overall, these data highlight the potential of MEF2C in the pathogenesis of cardiac hypertrophy and remodeling, and provide insights into novel therapeutic targets to heart disease.Expanded Animals were handled in compliance with the principles of laboratory animal care formulated by the Animal Care and Use Committee of University of Campinas. Procedures such as jugular vein catheterization, aortic banding, echocardiographic examination and arterial vessels catheterization for blood pressure monitoring were performed under anesthesia with a mixture of ketamine (100 mg/Kg) and xylazine (5 mg/Kg).Polyclonal mouse antibody against MEF2C was from Abcam (ab43796-100). Polyclonal rabbit antibodies against FAK (sc558), SHP2 (sc7384), JNK (sc571), GAPDH (sc25778), S6K p70 (sc230), pS6K p70 Thr389 (sc11759R) and PGC1-α (sc13067), were purchased from Santa Cruz Biotechnology (USA). Antibody against MEF2A was from Cell Signaling (9736). Antibodies were used following the manufacturer's recommendation. Colagenase type IA and trypsin were from Sigma (USA). Trizol, Phenol and Super Script II were from Invitrogen. Super Signal west Pico Cheluminescent Substract and Ampliscribe T7 high yield transcription were from Epicentre. L-Leucine was from Ajinomoto Aminoscience LLC.5 cells/well, as previously reportedSwiss mice (6–8 week old) and neonatal Wistar rats were obtained from the animal facility center of State University of Campinas. Animals were handled in compliance with the principles of laboratory animal care formulated by the university's Animal Care and Use Committee. Procedures such as jugular vein catheterization, aortic banding, echocardiographic examination and arterial vessels catheterization for blood pressure monitoring were performed under anesthesia with a mixture of ketamine (100 mg/Kg) and xylazine (5 mg/Kg) as previously reportedMouse cardiac myocytes were isolated from mice ventricle one day after treatment with siRNA targeted to MEF2C (siMEF2C) or GFP (siGFP), as previously reported2D M-mode echocardiography was performed with a 12-MHz probe connected to a Toshiba Power Vision system in anesthetized mice by a blinded observer at 15 minutes after the induction of anesthesia, as previously reportedFor blood pressure monitoring the right carotid and the right femoral arteries were cannulated with flame stretched PE-50 polyethylene tube. Blood pressure in the carotid and femoral arteries were simultaneously recorded for a 10 minute period to determine the transconstriction systolic gradient. The following parameters were computed: systemic systolic, mean and diastolic blood pressure (mmHg) and heart rate (bpm).Hearts were rapidly excised from fully anesthetized mice and washed in PBS. The left ventricles were fixed in 10% paraformaldehyde, embedded in paraffin and cut into 5 µm sections. Tissue sections stained with haematoxylin and eosin (HE) and Masson's trichrome underwent morphometric studies using an image analysis system .5′-GGTAATACGACTCACTATAG-3′. (ii): MEF2C 1187 sense: 5′-CCCACCUGGCAGCAAGAACAC-3′ (iii): MEF2C 1187 antisense: 5′-GUUCUUGCUGCCAGGUGGGAU-3′ (iv): GFP sense: 5′-GTGTCTTGTAGTTCCCGTCTATAGTGAGTCGTATTACC-3′. (v): GFP antisense: 5′-ATGACGGGAACTACAAACACCTATAGTGAGTCGTATTACC-3′ were ordered from IDT (USA). The oligonucleotide-directed production of small RNA transcripts with T7 RNA polymerase were made with Ampliscribe™ T7 transcription kit  according to manufacturer's instructions.siRNA targeted to mouse MEF2C gene was designed and synthesized as previously publishedNRVMs were transfected with siRNA as previously reported Tissue or cell extracts containing equal amounts of total protein (50 µg) were resolved by 8% SDS-PAGE. The membranes were incubated with primary antibodies. Detection was accomplished by using an enhanced chemiluminescence detection system.Myocardial MEF2A, MEF2C, MEF2D and ANP transcripts were quantified after reverse transcription to cDNA followed by real-time PCR. Primers are shown in Samples of myocardium were analyzed for AMP and ATP quantification by a method based on high performance liquid chromatography.t-test and 1-way repeated-measures ANOVA were used to compare groups. Post hoc analysis was performed with Bonferroni multiple-range test. A value of P<0.05 indicated statistical significance.Data are presented as means±SEM. Student's Text S1(0.08 MB DOC)Click here for additional data file.Table S1(0.04 MB DOC)Click here for additional data file.