Thomas D. Scholz

CaMKII determines mitochondrial stress responses in heart

Myocardial cell death is initiated by excessive mitochondrial Ca2+ entry causing Ca2+ overload, mitochondrial permeability transition pore (mPTP) opening and dissipation of the mitochondrial inner membrane potential (ΔΨm). However, the signalling pathways that control mitochondrial Ca2+ entry through the inner membrane mitochondrial Ca2+ uniporter (MCU) are not known. The multifunctional Ca2+/calmodulin-dependent protein kinase II (CaMKII) is activated in ischaemia reperfusion, myocardial infarction and neurohumoral injury, common causes of myocardial death and heart failure; these findings suggest that CaMKII could couple disease stress to mitochondrial injury. Here we show that CaMKII promotes mPTP opening and myocardial death by increasing MCU current (IMCU). Mitochondrial-targeted CaMKII inhibitory protein or cyclosporin A, an mPTP antagonist with clinical efficacy in ischaemia reperfusion injury, equivalently prevent mPTP opening, ΔΨm deterioration and diminish mitochondrial disruption and programmed cell death in response to ischaemia reperfusion injury. Mice with myocardial and mitochondrial-targeted CaMKII inhibition have reduced IMCU and are resistant to ischaemia reperfusion injury, myocardial infarction and neurohumoral injury, suggesting that pathological actions of CaMKII are substantially mediated by increasing IMCU. Our findings identify CaMKII activity as a central mechanism for mitochondrial Ca2+ entry in myocardial cell death, and indicate that mitochondrial-targeted CaMKII inhibition could prevent or reduce myocardial death and heart failure in response to common experimental forms of pathophysiological stress.

4-D Cardiac MR Image Analysis: Left and Right Ventricular Morphology and Function

In this study, a combination of active shape model (ASM) and active appearance model (AAM) was used to segment the left and right ventricles of normal and Tetralogy of Fallot (TOF) hearts on 4-D (3-D+time) MR images. For each ventricle, a 4-D model was first used to achieve robust preliminary segmentation on all cardiac phases simultaneously and a 3-D model was then applied to each phase to improve local accuracy while maintaining the overall robustness of the 4-D segmentation. On 25 normal and 25 TOF hearts, in comparison to the expert traced independent standard, our comprehensive performance assessment showed subvoxel segmentation accuracy, high overlap ratios, good ventricular volume correlations, and small percent volume differences. Following 4-D segmentation, novel quantitative shape and motion features were extracted using shape information, volume-time and dV/dt curves, analyzed and used for disease status classification. Automated discrimination between normal/TOF subjects achieved 90%-100% sensitivity and specificity. The features obtained from TOF hearts show higher variability compared to normal subjects, suggesting their potential use as disease progression indicators. The abnormal shape and motion variations of the TOF hearts were accurately captured by both the segmentation and feature characterization.

Congenital aortic disease: 4D magnetic resonance segmentation and quantitative analysis

Automated and accurate segmentation of the aorta in 4D (3D+time) cardiovascular magnetic resonance (MR) image data is important for early detection of congenital aortic disease leading to aortic aneurysms and dissections. A computer-aided diagnosis (CAD) method is reported that allows one to objectively identify subjects with connective tissue disorders from 16-phase 4D aortic MR images. Starting with a step of multi-view image registration, our automated segmentation method combines level-set and optimal surface segmentation algorithms in a single optimization process so that the final aortic surfaces in all 16 cardiac phases are determined. The resulting aortic lumen surface is registered with an aortic model followed by calculation of modal indices of aortic shape and motion. The modal indices reflect the differences of any individual aortic shape and motion from an average aortic behavior. A Support Vector Machine (SVM) classifier is used for the discrimination between normal and connective tissue disorder subjects. 4D MR image data sets acquired from 104 normal volunteers and connective tissue disorder patients MR datasets were used for development and performance evaluation of our method. The automated 4D segmentation resulted in accurate aortic surfaces in all 16 cardiac phases, covering the aorta from the aortic annulus to the diaphragm, yielding subvoxel accuracy with signed surface positioning errors of -0.07+/-1.16 voxel (-0.10+/-2.05mm). The computer-aided diagnosis method distinguished between normal and connective tissue disorder subjects with a classification correctness of 90.4%.

AUTOMATED IDENTIFICATION OF LEFT VENTRICULAR BORDERS FROM SPIN-ECHO MAGNETIC RESONANCE IMAGES : EXPERIMENTAL AND CLINICAL FEASIBILITY STUDIES

Gated cardiac magnetic resonance imaging (MRI) permits detailed evaluation of cardiac anatomy, including the calculation of left ventricular volume and mass. Current methods of deriving this information, however, require manual tracing of boundaries in several images; such manual methods are tedious, time consuming, and subjective. The purpose of this study is to apply a new computerized method to automatically identify endocardial and epicardial borders in MRIs. The authors obtained serial, short-axis, spin-echo MRIs of 13 excised animal hearts. Also obtained were selected short-axis, spin-echo ventricular images of 11 normal human volunteers. A method of automated edge detection based on graph-searching principles was applied to the ex vivo and in vivo images. Endocardial and epicardial areas were used to compute left ventricular mass and were compared with the anatomic left ventricular mass for the images of excised hearts. The endocardial and epicardial areas calculated from computer-derived borders were compared with areas from observer tracing. There was very close correspondence between computer-derived and observer tracings for excised hearts (r = 0.97 for endocardium, r = 0.99 for epicardium) and in vivo scans (r = 0.92 for endocardium, r = 0.90 for epicardium). There also was a close correspondence between computer-generated and actual left ventricular mass in the excised hearts (r = 0.99). These data suggest the feasibility of automated edge detection in MRIs. Although further validation is needed, this method may prove useful in clinical MRI.

Metabolic Adaptation of the Fetal and Postnatal Ovine Heart: Regulatory Role of Hypoxia-Inducible Factors and Nuclear Respiratory Factor-1

Numerous metabolic adaptations occur in the heart after birth. Important transcription factors that regulate expression of the glycolytic and mitochondrial oxidative genes are hypoxia-inducible factors (HIF-1α and -2α) and nuclear respiratory factor-1 (NRF-1). The goal of this study was to examine expression of HIF-1α, HIF-2α, and NRF-1 and the genes they regulate in pre- and postnatal myocardium. Ovine right and left ventricular myocardium was obtained at four time points: 95 and 140 d gestation (term = 145 d) and 7 d and 8 wk postnatally. Steady-state mRNA and protein levels of HIF-1α and NRF-1 and protein levels of HIF-2α were measured along with mRNA of HIF-1α-regulated genes (aldolase A, α- and β-enolase, lactate dehydrogenase A, liver and muscle phosphofructokinase) and NRF-1-regulated genes (cytochrome c, Va subunit of cytochrome oxidase, and carnitine palmitoyltransferase I ). HIF-1α protein was present in fetal myocardium but dropped below detectable levels at 7 d postnatally. HIF-2α protein levels were similar at the four time points. Steady-state mRNA levels of α-enolase, lactate dehydrogenase A, and liver phosphofructokinase declined significantly postnatally. Aldolase A, β-enolase, and muscle phosphofructokinase mRNA levels increased postnatally. Steady-state mRNA and protein levels of NRF-1 decreased postnatally in contrast to the postnatal increases in cytochrome c, subunit Va of cytochrome oxidase, and carnitine palmitoyltransferase I mRNA levels. The in vivo postnatal regulation of enzymes encoding glycolytic and mitochondrial enzymes is complex. As transactivation response elements for the genes encoding metabolic enzymes continue to be characterized, studies using the fetal-to-postnatal metabolic transition of the heart will continue to help define the in vivo role of these transcription factors.

Ontogeny of malate-aspartate shuttle capacity and gene expression in cardiac mitochondria

Developmental downregulation of the malate-aspartate shuttle has been observed in cardiac mitochondria. The goals of this study were to determine the time course of the postnatal decline and to identify potential regulatory sites by measuring steady-state myocardial mRNA and protein levels of the mitochondrial proteins involved in the shuttle. By use of isolated porcine cardiac mitochondria incubated with saturating concentrations of the cytosolic components of the malate-aspartate shuttle, shuttle capacity was found to decline by ∼50% during the first 5 wk of life (from 921 ± 48 to 531 ± 53 nmol ⋅ min-1 ⋅ mg protein-1). Mitochondrial aspartate aminotransferase mRNA levels were greater in adult than in newborn myocardium. mRNA levels of mitochondrial malate dehydrogenase in adult cardiac tissue were 224% of levels in newborn tissue, whereas protein levels were 54% greater in adult myocardium. Aspartate/glutamate carrier protein levels were also greater in adult than in newborn tissue. mRNA and protein levels of the oxoglutarate/malate carrier were increased in newborn myocardium. It was concluded that 1) myocardial malate-aspartate shuttle capacity declines rapidly after birth, 2) divergence of mitochondrial malate dehydrogenase mRNA and protein levels during development suggests posttranscriptional regulation of this protein, and 3) the developmental decline in malate-aspartate shuttle capacity is regulated by decreased oxoglutarate/malate carrier gene expression.Developmental downregulation of the malate-aspartate shuttle has been observed in cardiac mitochondria. The goals of this study were to determine the time course of the postnatal decline and to identify potential regulatory sites by measuring steady-state myocardial mRNA and protein levels of the mitochondrial proteins involved in the shuttle. By use of isolated porcine cardiac mitochondria incubated with saturating concentrations of the cytosolic components of the malate-aspartate shuttle, shuttle capacity was found to decline by approximately 50% during the first 5 wk of life (from 921 +/- 48 to 531 +/- 53 nmol.min-1.mg protein-1). Mitochondrial aspartate aminotransferase mRNA levels were greater in adult than in newborn myocardium. mRNA levels of mitochondrial malate dehydrogenase in adult cardiac tissue were 224% of levels in newborn tissue, whereas protein levels were 54% greater in adult myocardium. Aspartate/glutamate carrier protein levels were also greater in adult than in newborn tissue. mRNA and protein levels of the oxoglutarate/malate carrier were increased in newborn myocardium. It was concluded that 1) myocardial malate-aspartate shuttle capacity declines rapidly after birth, 2) divergence of mitochondrial malate dehydrogenase mRNA and protein levels during development suggests posttranscriptional regulation of this protein, and 3) the developmental decline in malate-aspartate shuttle capacity is regulated by decreased oxoglutarate/malate carrier gene expression.

Vascular nitric oxide and superoxide anion contribute to sex-specific programmed cardiovascular physiology in mice

Intrauterine environmental pertubations have been linked to the development of adult hypertension. We sought to evaluate the interrelated roles of sex, nitric oxide, and reactive oxygen species (ROS) in programmed cardiovascular disease. Programming was induced in mice by maternal dietary intervention (DI; partial substitution of protein with carbohydrates and fat) or carbenoxolone administration (CX, to increase fetal glucocorticoid exposure). Adult blood pressure and locomotor activity were recorded by radiotelemetry at baseline, after a week of high salt, and after a week of high salt plus nitric oxide synthase inhibition (by l-NAME). In male offspring, DI or CX programmed an elevation in blood pressure that was exacerbated by N(omega)-nitro-l-arginine methyl ester administration, but not high salt alone. Mesenteric resistance vessels from DI male offspring displayed impaired vasorelaxation to ACh and nitroprusside, which was blocked by catalase and superoxide dismutase. CX-exposed females were normotensive, while DI females had nitric oxide synthase-dependent hypotension and enhanced mesenteric dilation. Despite the disparate cardiovascular phenotypes, both male and female DI offspring displayed increases in locomotor activity and aortic superoxide production. Despite dissimilar blood pressures, DI and CX-exposed females had reductions in cardiac baroreflex sensitivity. In conclusion, both maternal malnutrition and fetal glucocorticoid exposure program increases in arterial pressure in male but not female offspring. While maternal DI increased both superoxide-mediated vasoconstriction and nitric oxide mediated vasodilation, the balance of these factors favored the development of hypertension in males and hypotension in females.

Angiotensin AT1 receptor blockade fails to attenuate pressure-overload cardiac hypertrophy in fetal sheep

We examined the hypothesis that endogenous angiotensin II and angiotensin type 1 (AT1) receptors participate in the development of fetal right ventricular hypertrophy by studying the effects of AT1 receptor blockade on cardiac growth in fetal sheep subjected to constrictive banding of the pulmonary artery (PA). Seven pairs of twin fetuses were studied beginning at 126 ± 1 days gestation (term = 145 days). One twin was given losartan (10 mg ⋅ kg-1 ⋅ day-1iv) for 7 consecutive days after PA banding, and the other twin served as a saline-treated, PA-banded control. Four additional pairs of twins served as sham-operated controls. Fetal heart rate (HR) and mean arterial blood pressure (MABP) were similar in the two groups of PA-banded animals before treatment and remained unchanged in the PA-banded control group. Losartan resulted in a significant decrease ( P < 0.05) in MABP between days 0 and 7, whereas HR was not affected. Total body weight of the losartan-treated animals was significantly less ( P < 0.05) than twin PA-banded controls and nonbanded fetuses. Right ventricle weight-to-body weight ratios were similar in saline (2.29 ± 0.34 g/kg) and losartan-treated (2.11 ± 0.15 g/kg) PA-banded animals and significantly greater than that in nonbanded fetuses (1.52 ± 0.07 g/kg). Similar differences were seen in the right ventricle weight-to-left ventricle weight ratios. Right and left ventricle AT1 receptor mRNA and protein expression were also similar among the three groups, as were AT2 receptor mRNA levels. These data suggest that endogenous angiotensin II does not contribute to the development of pressure overload-induced right ventricular hypertrophy during fetal life and that expression of angiotensin receptors is not altered by increased afterload in the ovine fetus.

Tissue determinants of nuclear magnetic resonance relaxation times. Effect of water and collagen content in muscle and tendon.

Previous studies have suggested a possible relationship between tissue collagen content and nuclear magnetic resonance (NMR) relaxation times. To further investigate this relationship, we studied skeletal muscle, tendon, and the muscle/tendon transition area of normal gastrocnemius muscle from 10 dogs, and determined tissue water and collagen (hydroxyproline) content and NMR T1 and T2 relaxation times at 20 MHz. Water and hydroxyproline contents and T1 and T2 were significantly different among the three tissues. Both spin-lattice and spin-spin relaxation times were linearly related to tissue water content. A significant curvilinear inverse relationship between T1 and hydroxyproline (r2 = 0.93) and a significant inverse curvilinear relationship between T2 and hydroxyproline (r2 = 0.92) were found. Statistically controlling for hydroxy-proline concentration eliminated differences in T1 and T2 among the muscle, muscle/tendon transition, and tendon groups. Thus, NMR relaxation times of skeletal muscle and tendon appear to be influenced by both tissue water and collagen content.

Essential Roles of an Intercalated Disc Protein, mXinβ, in Postnatal Heart Growth and Survival

Rationale: The Xin repeat-containing proteins mXin&agr; and mXin&bgr; localize to the intercalated disc of mouse heart and are implicated in cardiac development and function. The mXin&agr; directly interacts with &bgr;-catenin, p120-catenin, and actin filaments. Ablation of mXin&agr; results in adult late-onset cardiomyopathy with conduction defects. An upregulation of the mXin&bgr; in mXin&agr;-deficient hearts suggests a partial compensation. Objective: The essential roles of mXin&bgr; in cardiac development and intercalated disc maturation were investigated. Methods and Results: Ablation of mXin&bgr; led to abnormal heart shape, ventricular septal defects, severe growth retardation, and postnatal lethality with no upregulation of the mXin&agr;. Postnatal upregulation of mXin&bgr; in wild-type hearts, as well as altered apoptosis and proliferation in mXin&bgr;-null hearts, suggests that mXin&bgr; is required for postnatal heart remodeling. The mXin&bgr;-null hearts exhibited a misorganized myocardium as detected by histological and electron microscopic studies and an impaired diastolic function, as suggested by echocardiography and a delay in switching off the slow skeletal troponin I. Loss of mXin&bgr; resulted in the failure of forming mature intercalated discs and the mislocalization of mXin&agr; and N-cadherin. The mXin&bgr;-null hearts showed upregulation of active Stat3 (signal transducer and activator of transcription 3) and downregulation of the activities of Rac1, insulin-like growth factor 1 receptor, protein kinase B, and extracellular signal-regulated kinases 1 and 2. Conclusions: These findings identify not only an essential role of mXin&bgr; in the intercalated disc maturation but also mechanisms of mXin&bgr; modulating N-cadherin-mediated adhesion signaling and its crosstalk signaling for postnatal heart growth and animal survival.