Search

Cardiac stem cell-like populations exist in adult hearts, and their roles in cardiac repair remain to be defined. Sca-1 is an important surface marker for cardiac and other somatic stem cells. We hypothesized that heart-derived Sca-1(+)/CD31(-) cells may play a role in myocardial infarction-induced cardiac repair/remodeling. Mouse heart-derived Sca-1(+)/CD31(-) cells cultured in vitro could be induced to express both endothelial cell and cardiomyocyte markers. Immunofluorescence staining and fluorescence-activated cell sorting analysis indicated that endogenous Sca-1(+)/CD31(-) cells were significantly increased in the mouse heart 7 days after myocardial infarction (MI). Western blotting confirmed elevated Sca-1 protein expression in myocardium 7 days after MI. Transplantation of Sca-1(+)/CD31(-) cells into the acutely infarcted mouse heart attenuated the functional decline and adverse structural remodeling initiated by MI as evidenced by an increased left ventricular (LV) ejection fraction, a decreased LV end-diastolic dimension, a decreased LV end-systolic dimension, a significant increase of myocardial neovascularization, and modest cardiomyocyte regeneration. Attenuation of LV remodeling was accompanied by remarkably improved myocardial bioenergetic characteristics. The beneficial effects of cell transplantation appear to primarily depend on paracrine effects of the transplanted cells on new vessel formation and native cardiomyocyte function. Sca-1(+)/CD31(-) cells may hold therapeutic possibilities with regard to the treatment of ischemic heart disease.

In an established swine model of severe left ventricular (LV) hypertrophy (LVH), the bioenergetic and functional consequences of transplanting autologous mesenchymal stem cells (MSCs) overexpressing vascular endothelial growth factor (VEGF-MSCs) into the LV were evaluated; transplantation was accomplished by infusion of VEGF-MSCs into the interventricular cardiac vein. Specifically, the hypertrophic response to aortic banding was compared in seven pigs treated with 30 million VEGF-MSCs, eight pigs treated with 30 million MSCs without VEGF modification, and 19 untreated LVH pigs. Eight pigs without banding or cell transplantation (normal) were also studied. Four weeks postbanding, LV wall thickening (MRI), myocardial blood flow (MBF), high-energy phosphate levels (31P magnetic resonance spectroscopy), and hemodynamic measurements were obtained under basal conditions and during a catecholamine-induced high cardiac workstate (HCW). Although 9 of 19 untreated banded pigs developed clinical evidence of biventricular failure, no MSCs-treated animal developed heart failure. MSCs engraftment was present in both cell transplant groups, and both baseline and HCW MBF values were significantly increased in hearts receiving VEGF-MSCs compared with other groups ( P < 0.05). During HCW, cardiac inotropic reserve (defined as the percent increase of rate pressure product at HCW relative to baseline) was normal in the VEGF-MSCs group and significantly decreased in all other banded groups. Additionally, during HCW, the myocardial energetic state [reflected by the phosphocreatine-to-ATP ratio (PCr/ATP)] of VEGF-MSCs-treated hearts remained stable, whereas in all other groups, PCr/ATP decreased significantly from baseline values ( P < 0.05, each group). Myocardial von Willebrand factor and VEGF mRNA expressions and myocardial capillary density were significantly increased in VEGF-MSCs-treated hearts ( P < 0.05). Hence, in the pressure-overloaded LV, transplantation of VEGF-MSCs prevents LV decompensation, induces neovascularization, attenuates hypertrophy, and improves MBF, myocardial bioenergetic characteristics, and contractile performance.

NO and O2compete at cytochrome- c oxidase, thus potentially allowing NO to modulate mitochondrial respiration. We previously observed a decrease of myocardial phosphocreatine (PCr)/ATP during very high cardiac work states, corresponding to an increase in cytosolic free ADP. This study tested the hypothesis that NO inhibition of respiration contributes to this increase of ADP. Infusion of dobutamine + dopamine (DbDp, each 20 μg·kg−1·min−1iv) to more than double myocardial oxygen consumption (MV̇o2) in open-chest dogs caused a decrease of myocardial PCr/ATP measured with31P NMR from 2.04 ± 0.09 to 1.85 ± 0.08 ( P < 0.05). Inhibition of NO synthesis with Nω-nitro-l-arginine (l-NNA), while catecholamine infusion continued, caused PCr/ATP to increase to the control value. In a second group of animals, l-NNA administered before catecholamine stimulation (reverse intervention of the first group) increased PCr/ATP during basal conditions. In these animals l-NNA did not prevent a decrease of PCr/ATP at the high cardiac work state but, relative to MV̇o2, PCr/ATP was significantly higher after l-NNA. In a third group of animals, pharmacological coronary vasodilation with carbochromen was used to prevent changes in coronary flow that might alter endothelial NO production. In these animals l-NNA again restored depressed myocardial PCr/ATP during catecholamine infusion. The finding that inhibition of NO production increased PCr/ATP suggests that during very high work states NO inhibition of mitochondrial respiration requires ADP to increase to drive oxidative phosphorylation.

Inhibition of ATP-sensitive K+ (KATP) channel activity has previously been demonstrated to result in coronary vasoconstriction with decreased myocardial blood flow and loss of phosphocreatine (PCr). This study was performed to determine whether the high-energy phosphate abnormality during KATP channel blockade can be ascribed to oxygen insufficiency. Myocardial blood flow and oxygen extraction were measured in open-chest dogs during KATP channel blockade with intracoronary glibenclamide, whereas high-energy phosphates were examined with 31P magnetic resonance spectroscopy (MRS), and myocardial deoxymyoglobin (Mb-δ) was determined with 1H MRS. Glibenclamide resulted in a 20 ± 8% decrease of myocardial blood flow that was associated with a loss of phosphocreatine (PCr) and accumulation of inorganic phosphate. Mb-δ was undetectable during basal conditions but increased to 58 ± 5% of total myoglobin during glibenclamide administration. This degree of myoglobin desaturation during glibenclamide was far greater than we previously observed during a similar reduction of blood flow produced by a coronary stenosis (22% of myoglobin deoxygenated during stenosis). The findings suggest that reduction of coronary blood flow with an arterial stenosis was associated with a decrease of myocardial energy demands and that this response to hypoperfusion was inhibited by KATP channel blockade.

The micro- or nano-structured mold used for polymer embossing typically must be coated with an anti-adhesion material to reduce its interaction with the embossing. The mold is typically made by nickel sulphamate electroforming. For the anti-adhesion coating to adhere to the mold, the nickel mold surface must be clean and preferably unoxidized or possess reactive groups suitable for covalent bonding with the anti-adhesion coating. The effectiveness of plasma cleaning using mixtures of oxygen (O2) and tetrafluoromethane (CF4) with varying ratios versus liquid-only cleaning was investigated. To simulate the nickel mold, Ni200 alloy was used. Plasma treatment using mixtures of O2 and CF4 was found to be more effective in cleaning the Ni200 surface than liquid-only cleaning or pure O2 or pure CF4 plasma treatment. Using a 1 : 1 O2 /CF4 mixture plasma, the contact angles of water, glycerol and diiodomethane on Ni200 were the lowest and the calculated surface energy was the highest among the investigated treatme...

OBJECTIVESThis study was conducted to test hypotheses stating that: 1) altered signaling for mitochondrial membrane proteins occurs during postinfarction remodeling, and 2) successful myocardial adaptation relates to promotion of specific mitochondrial membrane components.BACKGROUNDAbnormalities in high-energy phosphate content and limitations in adenosine 5′-triphosphate (ATP) synthesis rate occur during the transition to contractile failure from compensatory remodeling after left ventricular infarction. The adenine nucleotide translocator (ANT) and F1-ATPase respectively regulate mitochondrial adenosine 5′-diphosphate (ADP)/ATP exchange and ADP-phosphorylation, which are key components of high-energy phosphate metabolism.METHODSSteady-state mRNA and protein expression for ANT isoform1 and the beta subunit of the F1-ATPase (betaF1) were analyzed in myocardium remote from the infarction zone eight weeks after left circumflex coronary artery ligation in pigs, demonstrating either successful left ventricular remodeling (LVR, n = 8) or congestive heart failure (CHF, n = 4) as determined by clinical and contractile performance parameters.RESULTSSubstantial reductions in steady-state mRNA expression for ANT1 and betaF1 relative to normal (n = 8) occur in CHF, p < 0.01, but not in LVR. Relative expression for both proteins coordinated with their respective steady-state mRNA levels; CHF at 40% normal, p < 0.05 for ANT and 70% normal for betaF1, p < 0.05.CONCLUSIONSMaintained signaling for major mitochondrial membrane proteins occurs in association with successful remodeling and adaptation after infarction. Reduced expression of these proteins relates to limited ATP synthesis capacity and high energy phosphate kinetic abnormalities previously demonstrated in CHF. These findings imply that mitochondrial processes participate in myocardial remodeling after infarction.

"P- and I H-nuclear magnetic resonance spectroscopy (MRS) are powerjiul tools for studying myocardial energy metabolism. The purpose of this review is to illustrate how these MRS techniques can be used to study complex bioenergetic issues in normal and abnormal in vivo myocardium. The results provide insight into the energetic alterations present in remodeled and hypertrophied myocardium. A detailed understanding of energy metabolism in normal and abnormal myocardium may point the way to improved preventive, diagnostic, and therapeutic modalities for left ventricular dysfunction.

This study tested the hypothesis that the loss of myocardial high-energy phosphates (HEP), which occurs during high cardiac work states [J. Zhang, D. J. Duncker, Y. Xu, Y. Zhang, G. Path, H. Merkle, K. Hendrich, A. H. L. From, R. Bache, and K. Uğurbil. Am. J. Physiol. 268: ( Heart Circ. Physiol. 37): H1891–H1905, 1995], is not the result of insufficient intracellular O2 availability. To evaluate the state of myocardial oxygenation, the proximal histidine signal of deoxymyoglobin (Mb-δ) was determined with1H nuclear magnetic resonance spectroscopy (MRS), whereas HEP were examined with31P MRS. Normal dogs ( n = 11) were studied under basal conditions and during combined infusion of dobutamine and dopamine (20 μg ⋅ kg−1 ⋅ min−1iv each), which increased rate-pressure products to >50,000 mmHg ⋅ beats ⋅ min−1. Creatine phosphate (CP) was expressed as CP/ATP, and myocardial myoglobin desaturation was normalized to the Mb-δ resonance present during total coronary artery occlusion. This Mb-δ resonance appeared at 71 parts per million downfield from the water resonance. CP/ATP decreased from 2.22 ± 0.12 during the basal state to 1.83 ± 0.09 during the high work state ( P < 0.01), whereas ΔPi/CP increased from 0 to 0.21 ± 0.04 ( P < 0.01). Despite these HEP changes, Mb-δ remained undetectable. In contrast, when a coronary stenosis was applied to produce a similar decrease in CP/ATP, Mb-δ reached 0.38 ± 0.10 of the value present during total coronary occlusion. These data demonstrate that Mb-δ is readily detected in vivo during limitation of coronary blood flow sufficient to cause a decrease of myocardial CP/ATP. However, similar HEP changes that occur at high work states in the absence of coronary occlusion are not associated with a detectable Mb-δ resonance. The findings support the hypothesis that the myocardial HEP changes observed at high work states are not due to inadequate O2 availability to the mitochondria and emphasize the limitations of interpreting HEP alterations in the absence of knowing the level of myocyte oxygenation.

High cardiac workloads produced by catecholamine infusion result in loss of myocardial phosphocreatine (PCr) and accumulation of inorganic phosphate (Pi) which are more prominent in heart with left ventricular hypertrophy (LVH) than in normal hearts. Since ischemia can cause changes in phosphorylated compounds similar to those during catecholamine stimulation, this study tested the hypothesis that the exaggerated depletion of PCr and accumulation of Pi during high workloads in LVH is the result of impaired myocyte oxygenation.31P- and 1H-NMR spectroscopy were used to determine myocardial high energy phosphate levels and myoglobin desaturation, respectively, in eight normal dogs and nine dogs with LVH produced by ascending aortic banding. The mean LV weight/body weight ratio was approximately twice normal in the LVH group. Infusion of dobutamine (15 and 30 micrograms/kg/min), and dobutamine + dopamine (each 20 micrograms/kg/min) caused progressive similar increases in the heart rate x systolic LV pressure product to a maximum of 57.4 +/- 3.3 x 10(3) in normal and 63.9 +/- 2.7 x 10(3) in LVH animals, while myocardial oxygen consumption increased from 0.09 +/- 0.01 to 0.24 +/- 0.04 in normals and from 0.10 +/- 0.02 to 0.25 +/- 0.03 ml/min/g in LVH. PCr/ATP ratios during basal conditions were lower in LVH hearts (1.73 +/- 0.10, 1.61 +/- 0.09 and 1.51 +/- 0.09 in subepicardium, midwall and subendocardium, respectively) as compared with normals (2.24 +/- 0.09, 2.01 +/- 0.08 and 1.89 +/- 0.07; each p0.01 normal vs. LVH). Catecholamine infusions caused dose-related decreases in PCr/ATP and appearance of Pi which was more marked in LVH than in normal hearts. 1H-NMR spectroscopy did not detect deoxymyoglobin in either normal or LVH hearts even during the highest workloads. In contrast, occlusion of the anterior descending coronary artery resulted in a large deoxymyoglobin signal.Increases of cardiac work produced by catecholamine stimulation resulted in greater decreases of PCr and greater increases of Pi in hypertrophied than in normal hearts. These abnormalities were not the result of inadequate intracellular oxygen availability and consequently cannot be ascribed to demand ischemia.

Stroke volumes measured by impedance were compared with values obtained by dye dilution and an electromagnetic flowmeter (EMF) on 14 dogs during drug-induced changes in cardiac contraction strength and peripheral resistance changes. Grouping all data for a total of 305 points showed correlations between dye and EMF, dye and impedance, and EMF and impedance of 0.89, 0.68, and 0.72, respectively. Correlations for individual dogs between dye and EMF, dye and impedance, and EMF and impedance ranged from 0.60 to 0.99, −0.39 to 0.96, and −0.26 to 0.89, respectively. These data suggest that the use of impedance cardiac output measurements to make treatment decisions about individual patients could result in serious error.

During moderate reductions of blood flow, the myocardium downregulates contractile function and ATP utilization to result in reduced but stable ATP levels, recovery or stability of (reduced) creatine phosphate (CP), and preservation of myocyte viability. The intent of this study was to determine the influence of the level of ischemic blood flow and the major determinants of myocardial O2 consumption (MVO2) (heart rate and systolic blood pressure) on recovery of CP during prolonged moderate myocardial hypoperfusion. 31P-nuclear magnetic resonance spectroscopy was used to measure CP, ATP, and Pi in the subepicardium (Epi) and subendocardium (Endo) of 13 open-chest dogs. Wall thickening was measured with sonomicrometry. A coronary stenosis reduced mean myocardial blood flow (microspheres) from 1.10 +/- 0.07 to 0.71 +/- 0.06 ml.g-1.min-1 (P < 0.01) and the Endo-to-Epi blood flow ratio from 1.12 +/- 0.07 to 0.59 +/- 0.06 (P < 0.01), and dyskinesis developed. Coronary blood flow and systolic wall thickening did not change significantly during 4 h of hypoperfusion. Epi CP and ATP fell to 80 +/- 4% (P < 0.05) and 93 +/- 3% of control, respectively, at 30 min. Epi CP then recovered to 87 +/- 5% while ATP decreased further to 83 +/- 5% of baseline by the end of the 240-min ischemic period. Endo CP and ATP fell to 53 +/- 4 and 77 +/- 5% of control, respectively, at 30 min; then Endo CP recovered to 85 +/- 6% while ATP decreased further to 68 +/- 6% of baseline at 240 min of hypoperfusion. ADP levels were significantly increased at 30 min but recovered to baseline by 240 min of hypoperfusion. delta Pi/CP increased significantly (Endo > Epi) at the onset of ischemia and then progressively decreased. At 30 min, mild myocardial acidosis was observed in some hearts with variable pH recovery during continuing hypoperfusion. The data demonstrate that variations in blood flow cannot account for the magnitude of the initial fall in CP or for the final extent of recovery. However, the rate at which CP recovered was significantly correlated with the level of blood flow. Variations in the determinants of MVO2 did not account for differences in CP recovery.

In animal models of myocardial infarction (MI), transplantation of various types of progenitor cells has been reported to (i) improve left ventricular (LV) function, (ii) decrease LV remodeling, (iii) limit fibrosis of noninfarcted LV regions, and (iv) in some cases, reduce infarct scar size. Moreover, in some reports these beneficial effects were present despite very low rates of long-term engraftment and transdifferentiation of transplanted cells into cardiomyocytes. In contrast, in other reports, significant numbers of transplanted cells do appear to have transdifferentiated into cardiomyocytes and vascular cells. Paracrine signals emanating from transplanted cells also appear to be very important because they protect injured cardiomyocytes and may activate endogenous cardiac progenitor cells (CPCs) to generate cardiomyocytes and vascular cells. Herein, we review evidence that transplanted bone-marrow- or cardiac-derived CPCs and/or in situ CPCs can be stimulated to propagate, differentiate, and partially replace cardiomyocytes damaged during AMI. The possibility that preexisting cardiomyocytes can be induced to reenter the cell cycle and regenerate replacement cardiomyocytes is also discussed.

In animal models of myocardial infarction (MI), transplantation of various types of progenitor cells has been reported to (i) improve left ventricular (LV) function, (ii) decrease LV remodeling, (iii) limit fibrosis of noninfarcted LV regions, and (iv) in some cases, reduce infarct scar size. Moreover, in some reports these beneficial effects were present despite very low rates of long-term engraftment and transdifferentiation of transplanted cells into cardiomyocytes. In contrast, in other reports, significant numbers of transplanted cells do appear to have transdifferentiated into cardiomyocytes and vascular cells. Paracrine signals emanating from transplanted cells also appear to be very important because they protect injured cardiomyocytes and may activate endogenous cardiac progenitor cells (CPCs) to generate cardiomyocytes and vascular cells. Herein, we review evidence that transplanted bone-marrow- or cardiac-derived CPCs and/or in situ CPCs can be stimulated to propagate, differentiate, and partially replace cardiomyocytes damaged during AMI. The possibility that preexisting cardiomyocytes can be induced to reenter the cell cycle and regenerate replacement cardiomyocytes is also discussed.

Magnetic resonance spectroscopy-based magnetization transfer techniques (MT) are commonly used to assess the rate of oxidative (i.e., mitochondrial) ATP synthesis in intact tissues. Physiologically appropriate interpretation of MT rate data depends on accurate appraisal of the biochemical events that contribute to a specific MT rate measurement. The relative contributions of the specific enzymatic reactions that can contribute to a MT Pi→ATP rate measurement are tissue dependent; nonrecognition of this fact can bias the interpretation of MT Pi→ATP rate data. The complexities of MT-based measurements of mitochondrial ATP synthesis rates made in striated muscle and other tissues are reviewed, following which, the adverse impacts of erroneous Pi→ATP rate data analyses on the physiological inferences presented in selected published studies of cardiac and skeletal muscle are considered.

This review explores the complex relationship between excess lipid exposure, mitochondrial dysfunction, and insulin resistance at the level of human skeletal muscle. Lipotoxicity — i.e., the elevation of lipids and/or associated lipid metabolites within blood and tissues with subsequent metabolic derangement — has been proposed as a possible mechanism of skeletal muscle insulin resistance. Intravenous lipid infusion is a well-documented method of inducing insulin resistance. Although IMCL content has been correlated with insulin resistance, there is increasing evidence that lipid metabolites such as 4-HNE, DAG, ceramide, and LC-CoA may play a more significant role than triglycerides in producing skeletal muscle insulin resistance. The association between mitochondrial dysfunction and insulin resistance is unclear, particularly due to the varied options for measuring mitochondrial function. The effect of acute lipid exposure producing skeletal muscle insulin resistance in humans is well documented. The effects of chronic lipid exposure from dietary ingestion on skeletal muscle insulin resistance and skeletal muscle mitochondrial function remain disputed. The effects of skeletal muscle mitochondrial dysfunction on accumulation of lipotoxic species and skeletal muscle insulin resistance also remain uncertain. Certainly, pursuit of the role of lipid metabolites and their roles in the generation of skeletal muscle insulin resistance remain an exciting area for future research. Moreover, alteration in skeletal muscle insulin resistance does not occur in isolation as functional perturbations of any component of the glucose homeostatic system may initiate development of insulin resistance in skeletal muscle through “cross talk” between tissues. Nevertheless, understanding pathophysiology of skeletal muscle insulin resistance remains critically important due to its role in preceding and facilitating the development of Type 2 diabetes.

This chapter reviews the metabolic pathways involved in transferring the chemical energy stored in dietary carbon substrates (primarily glucose and fatty acids) into adenosine triphosphate (ATP) and the regulatory systems that integrate the functions of these pathways and make them responsive to changes in energy demand. Normal cardiac function depends upon both the adequate delivery of carbon substrates and oxygen to the heart by the coronary circulation and the ability of the heart to extract and metabolize these substrates at rates sufficient to support a wide range of ATP demands. The effects of several physiological states and disease processes on cardiac metabolism are also discussed and the concept that the diseased heart may be energy limited is presented. Lastly, an example of a new therapeutic approach, based on a detailed understanding of an inherited metabolic pathway abnormality, emphasizes the importance of detailed knowledge of energetic pathways.

The relationships among myocardial ATP, intracellular pH, and ischemic contracture in Langendorff-perfused rat hearts were investigated by 31P nuclear magnetic resonance spectroscopy during total global normothermic ischemia while the left ventricular pressure was recorded continuously via an intraventricular balloon. Glucose-perfused hearts (n = 63) were divided into five groups based on the time of onset of contracture (TOC), and three other groups of hearts were treated to vary the ischemic glycogen availability. ATP levels, which showed no evidence of accelerated ATP depletion during contracture, were significant and variable at TOC. Intracellular pH initially declined and then leveled off at TOC, with lower final pH in hearts with later TOC. We conclude that contracture began when anaerobic glycolysis (and thus glycolytic ATP synthesis) stopped. These results, though consistent with the concept that ischemic contracture in normal hearts results from rigor bond formation due to low ATP levels at the myofibrils, suggest that TOC is more closely related to glycolytic ATP production than to total cellular ATP content, thus providing evidence of some degree of subcellular compartmentation or metabolite channeling. In glycolytically inhibited hearts, the quite early contracture may have a Ca2+ component.

Spatially localized phosphorus-31 nuclear magnetic resonance (31P NMR) spectroscopy has been applied to the study of the normal canine myocardium to measure the relative content of high energy phosphates across the left ventricular wall. Transmural NMR data were acquired in five voxels spanning the wall of the left ventricle using the FLAX-ISIS technique. The validity of the FLAX-ISIS approach in acquiring localized spectra for transmural studies and in providing quantitative information from the localized spectra was examined rigorously by studies involving phantoms, intact rats, and the canine myocardium in vivo. The results indicated that (1) this technique yields spatially resolved spectra with partial overlap between adjacent voxels and virtually no overlap between every other voxel; (2) in the canine heart, signals from subepicardium, midwall, and subendocardium can be detected separately without cross contamination; and (3) relative metabolite contents within a voxel and among voxels can be quantitated. Transmural 31P NMR spectra were acquired with cardiac gating on 29 separate animals either at early systole or late diastole, and at three different workloads with the heart rate peak systolic pressure product (RPP) increasing from 6000 mmHg/min to 35,000 mmHg/min. The data revealed that in the normal canine myocardium, the creatine phosphate (CP) content and the CP/ATP ratio was significantly lower in the subendocardium than in the subepicardium. ATP levels were transmurally constant. Both the CP content and the CP/ATP ratio measured for each voxel remained unaltered in relation to either the phase of the cardiac cycle or approximately fourfold increase in workload. Free ADP levels calculated for each voxel showed that ADP was relatively higher in the subendocardium than the subepicardium, and in all transmural layers was higher than its apparent Km for oxidative phosphorylation. In this domain changes in ADP content with workload and MVO2 are not expected and were not observed.

Spatially localized nuclear magnetic resonance spectroscopy was used to investigate with transmural differentiation the response of myocardial high energy phosphate compounds and inorganic orthophosphate (Pi) to graded reductions in coronary blood flow caused by sustained coronary stenosis. In an open-chest model, localized 31P nuclear magnetic resonance spectra from five layers across the left ventricular wall were obtained simultaneously with transmural blood flow measurements during control conditions and during sustained graded reductions in intracoronary pressure. Both the blood flow, and high energy phosphate and Pi contents displayed transmural heterogeneity in response to decreases in intracoronary pressure. The subendocardial creatine phosphate (CP) level remained unchanged as blood flow was reduced to approximately 0.7 ml/min/g wet wt and decreased precipitously beyond this critical flow level. The relation between CP and flow in the midmyocardium and especially in the subepicardium was more complex. Subepicardial CP content did not correlate well with blood flow; however, in cases in which a coronary stenosis resulted in subendocardial hypoperfusion but subepicardial flow was near or above normal, a close correlation was present between subepicardial and subendocardial CP levels. ATP levels in all layers remained unaltered until blood flow was severely reduced. These results demonstrate that 1) the myocardial high energy phosphate and Pi levels at any transmural layer are not generally determined by O2 and blood flow limitation under basal conditions; 2) during subtotal coronary occlusion, increased oxygen extraction is able to meet myocardial needs until a critical level of stenosis is reached; 3) below a critical flow level, subendocardial CP and Pi contents are closely correlated with absolute subendocardial blood flow; and 4) in the presence of a coronary stenosis, subepicardial CP and Pi contents may change even in the absence of perfusion deficit secondary to loss of subendocardial function.

The ability to measure ATP synthesis rates using 31P-NMR spectroscopy is demonstrated in the normal, ischemic, and postischemic myocardium in vivo. Cardiopulmonary bypass (CBP) was employed to induce 20 min of global myocardial ischemia, and to conduct magnetization transfer measurements during the ischemic episode and following reperfusion and return to normal circulation. For the first few minutes of ischemia, transfer of magnetization from ATP gamma to Pi was extensive and the resultant fractional reduction (delta M/M0) in the Pi resonance intensity reached approximately 100%. Subsequent to reperfusion and stabilization off CPB and on normal circulation, both the fractional reduction and the spin-lattice relaxation time, T1*, of the Pi resonance were determined when ATP gamma spins were saturated. Under these conditions, the unidirectional ATP synthesis rate was 0.41 +/- 0.09 (SEM, N = 4) mumol/s/g wet wt. The data suggest that in the canine myocardium in vivo, glycolytic enzymes mediate a very rapid exchange between Pi and ATP gamma-phosphates during early phases of ischemia; in the postischemic reperfused myocardium, however, the glycolytic contribution to the unidirectional Pi----ATP rate measured by NMR in vivo is relatively small compared to that observed in glucose-perfused, postischemic rat hearts.

Mitochondrial uncoupling is often invoked as a mechanism underlying cellular dysfunction; however, it has not been possible to study this phenomenon directly in intact cells and tissues. In this paper, we report direct evaluation of mitochondrial uncoupling in the intact myocardium using 31P NMR magnetization transfer techniques. Langendorff perfused rat hearts were exposed to either a known uncoupler, 2,4-dinitrophenol (DNP), or a potential uncoupler, octanoate. Both DNP and octanoate decreased mechanical function as measured by the rate pressure product and caused an increase in the oxygen consumption rate (MVO2); with DNP this increase in MVO2 was dose-dependent. The ATP synthesis rate measured by 31P NMR, however, was not elevated commensurately with MVO2; instead, the P/O ratio declined. In contrast, the linear relationship between the ATP synthesis rate and rate pressure product was not altered by the uncoupling agents. These data demonstrate that 1) 31P NMR magnetization transfer can be utilized to measure uncoupling of oxidative phosphorylation in intact organs, 2) octanoate does not induce excess ATP utilization in the intact heart, and 3) high levels of octanoate induce mitochondrial uncoupling in the intact myocardium; and this may, in part, be the cause of the toxic effects associated with fatty acid exposure.

Background— The present study examined whether transplantation of adherent bone marrow–derived stem cells, termed pMultistem, induces neovascularization and cardiomyocyte regeneration that stabilizes bioenergetic and contractile function in the infarct zone and border zone (BZ) after coronary artery occlusion. Methods and Results— Permanent left anterior descending artery occlusion in swine caused left ventricular remodeling with a decrease of ejection fraction from 55±5.6% to 30±5.4% (magnetic resonance imaging). Four weeks after left anterior descending artery occlusion, BZ myocardium demonstrated profound bioenergetic abnormalities, with a marked decrease in subendocardial phosphocreatine/ATP ( 31 P magnetic resonance spectroscopy; 1.06±0.30 in infarcted hearts [n=9] versus 1.90±0.15 in normal hearts [n=8; P P P P P Conclusions— Thus, allogeneic pMultistem improved BZ energetics, regional contractile performance, and global left ventricular ejection fraction. These improvements may have resulted from paracrine effects that include increased vascular density in the BZ and spared myocytes in the infarct zone.

Therapeutic strategies for management of patients with congestive heart failure (CHF) are now based on reduction of afterload and preload and the attenuation of neurohormonal activation. Recently, it has been suggested that excess myocardial dependence on lipid oxidation may have adverse effects in failing myocardium. Panchal et al. (1) have addressed this issue and report that, in dogs with coronary microembolism-induced CHF, metoprolol treatment for 12 weeks reduces left ventricular carnitine palmitoyl transferase I (CPT-I) activity in comparison to levels present in normal or untreated infarct animals. Metoprolol treatment did not affect the activities of citrate synthase and medium-chain acyl CoA dehydrogenase, which are other members of the group of mitochondrial enzymes involved in the oxidative metabolism of fatty acids, but it attenuated the progressive adverse changes in left ventricular mechanical performance and diastolic chamber size observed in the untreated animals with an infarct. Based on these observations and the higher myocardial triglyceride levels present in the metoprolol-treated group, these investigators postulated that partial suppression of myocardial fatty acid oxidation may be one mechanism underlying the beneficial effects of beta-adrenergic blocking agents on failing or remodeled hearts. If the excess fatty acid utilization hypothesis is correct, then primary efforts to suppress myocardial fatty acid metabolism may be an effective adjunct to optimal therapeutic regimens, which currently include the use of angiotonsin converting enzyme inhibitors, diuretic

Regions of myocardial infarct (MI) are surrounded by a border zone (BZ) of normally perfused but dysfunctional myocardium. Although systolic dysfunction has been attributed to elevated wall stress in this region, there is evidence that intrinsic abnormalities of contractile performance exist in BZ myocardium. This study examined whether decreases of high-energy phosphates (HEP) and mitochondrial F1F0-ATPase (mtATPase) subunits typical of failing myocardium exist in BZ myocardium of compensated postinfarct remodeled hearts. Eight pigs were studied 6 wk after MI was produced by ligation of the left anterior descending coronary artery (LAD) distal to the second diagonal. Animals developed compensated LV remodeling with a decrease of ejection fraction from 54.6 ± 5.4% to 31 ± 2.1% (MRI) 5 wk after LAD occlusion. The remote zone (RZ) myocardium demonstrated modest decreases of ATP and mtATPase components. In contrast, BZ myocardium demonstrated profound abnormalities with ATP levels decreased to 42% of normal, and phosphocreatine-to-ATP ratio (31P-magnetic resonance spectroscopy) decreased from 2.06 ± 0.19 in normal hearts to 1.07 ± 0.10, with decreases in α-, β-, OSCP, and IF1subunits of mtATPase, especially in the subendocardium. The reduction of myocardial creatine kinase isoform protein expression was also more severe in the BZ relative to the RZ myocardium. These abnormalities were independent of a change in mitochondrial content because the mitochondrial citrate synthase protein level was not different between the BZ and RZ. This regional heterogeneity of ATP content and expression of key enzymes in ATP production suggests that energetic insufficiency in the peri-infarct region may contribute to the transition from compensated LV remodeling to congestive heart failure.

Although high-energy phosphate metabolism is abnormal in failing hearts [congestive heart failure (CHF)], it is unclear whether oxidative capacity is impaired. This study used the mitochondrial uncoupling agent 2,4-dinitrophenol (DNP) to determine whether reserve oxidative capacity exists during the high workload produced by catecholamine infusion in hypertrophied and failing hearts. Left ventricular hypertrophy (LVH) was produced by ascending aortic banding in 21 swine; 9 animals developed CHF. Basal myocardial phosphocreatine (PCr)/ATP measured with31P NMR spectroscopy was decreased in both LVH and CHF hearts (corresponding to an increase in free [ADP]), whereas ATP was decreased in hearts with CHF. Infusion of dobutamine and dopamine (each 20 μg · kg–1· min–1iv) caused an approximate doubling of myocardial oxygen consumption ([Formula: see text]) in all groups and decreased PCr/ATP in the normal and LVH groups. During continuing catecholamine infusion, DNP (2–8 mg/kg iv) caused further increases of [Formula: see text] in normal and LVH hearts with no change in PCr/ATP. In contrast, DNP caused no increase in [Formula: see text] in the failing hearts; the associated decrease of PCr/ATP suggests that DNP decreased the mitochondrial proton gradient, thereby causing ADP to increase to maintain adequate ATP synthesis.

This editorial refers to ‘A novel chimeric natriuretic peptide reduces cardiomyocyte hypertrophy through the NHE-1–calcineurin pathway’ by A. Kilic et al. , doi:10.1093/cvr/cvq254. As is well known, intravenously infused nesiritide [a recombinant form of brain-type natriuretic peptide (BNP)] antagonizes renin–angiotensin–aldosterone system activation and induces natriuresis, diuresis, and venous and arterial dilation. When administered to patients with acutely decompensated heart failure, a rapid reduction of pulmonary capillary pressure and consequent relief of dyspnoea often results. However, BNP-induced dilation of resistance (arterioles) and capacitance (veins) vessels taken together with the diuresis-associated reduction of blood (plasma) volume causes clinically significant systemic hypotension in some patients. Unfortunately, this hypotension can be associated with decreased renal perfusion and worsening of renal function, and the latter has been associated with increased morbidity and mortality in some clinical trials.1 The hypotension and consequent complications can be attenuated by reducing the dose of nesiritide being administered.1 Recently, Burnett and colleagues speculated that they might be able to improve the therapeutic properties of natriuretic peptides by modifying their structures. Their goal was to generate a natriuretic peptide that retained the biological activities of BNP sans the capacity to dilate resistance vessels. Their first engineered chimeric natriuretic peptide (CD-NP) comprised the 15-amino acid C-terminus of the Dendroaspis peptide (a snake-derived natriuretic peptide) and the peptide ring component of the cardiac-type natriuretic peptide (CNP). CNP lacks renal actions but retains veno-dilator capacity and has only modest dilatory effects … *Corresponding author. Tel: +1 612 626 2001; fax: +1 612 626 2004, Email: fromx001{at}umn.edu

This study was performed to determine the myocyte Po 2 required to sustain normal high-energy phosphate (HEP) levels in the in vivo heart. In 10 normal dogs, myocyte Po 2 values were calculated from the myocardial deoxymyoglobin resonance (Mb-δ) intensity determined with 1H-NMR spectroscopy during sequential flow reductions produced by a hydraulic occluder that decreased coronary perfusion pressure to ∼60, 50, and 40 mmHg and, finally, during total occlusion. Myocardial blood flow was measured with microspheres, and HEP levels were determined with 31P magnetic resonance spectroscopy. During control conditions, Mb-δ was undetectable. Myocardial blood flow was 1.11 ± 0.06 ml · min−1 · g−1 during basal conditions and decreased with sequential graded occlusions to 0.78 ± 0.05, 0.58 ± 0.03, and 0.38 ± 0.04 ml · min−1 · g−1, respectively; blood flow during total occlusion was 0.07 ± 0.02 ml · min−1 · g−1. Reductions of blood flow caused progressive increases of Mb-δ, which were associated with decreases of phosphocreatine (PCr), ATP, and the PCr-to-ATP ratio, as well as progressive increases of the Pi-to-PCr ratio. There was a strong linear correlation between normalized blood flow and Mb-δ ( R 2 = 0.89, P < 0.01). Reductions of HEP and Po 2 were also highly correlated (although nonlinearly); with the assumption that myoglobin was 90% saturated with O2 during basal conditions and 5% saturated during total coronary occlusion, the intracellular Po 2 values for 20% reductions of PCr and ATP were ∼4.4 and ∼0.9 mmHg, respectively. The data indicate that O2 availability plays an increasing role in regulation of oxidative phosphorylation when mean intracellular Po 2 values fall below 5 mmHg in the in vivo heart.

Background —Postinfarction left ventricular remodeling (LVR) is associated with reductions in myocardial high-energy phosphate (HEP) levels, which are more severe in animals that develop overt congestive heart failure (CHF). During high work states, further HEP loss occurs, which suggests demand-induced ischemia. This study tested the hypothesis that inadequate myocyte oxygen availability is the basis for these HEP abnormalities. Methods and Results —Myocardial infarction was produced by left circumflex coronary artery ligation in swine. Studies were performed in 20 normal animals, 14 animals with compensated LVR, and 9 animals with CHF. Phosphocreatine (PCr)/ATP was determined with 31 P NMR and deoxymyoglobin (Mb-δ) with 1 H NMR in myocardium remote from the infarct. Basal PCr/ATP tended to be decreased in postinfarct hearts, and this was significant in animals with CHF. Infusion of dobutamine (20 μg · kg −1 · min −1 IV) caused doubling of the rate-pressure product in both normal and LVR hearts and resulted in comparable significant decreases of PCr/ATP in both groups. This decrease in PCr/ATP was not associated with detectable Mb-δ. In CHF hearts, rate-pressure product increased only 40% in response to dobutamine; this attenuated response also was not associated with detectable Mb-δ. Conclusions —Thus, the decrease of PCr/ATP during dobutamine infusion is not the result of insufficient myocardial oxygen availability. Furthermore, in CHF hearts, the low basal PCr/ATP and the attenuated response to dobutamine occurred in the absence of myocardial hypoxia, indicating that the HEP and contractile abnormalities were not the result of insufficient oxygen availability.

Background The underlying mechanisms by which left ventricular remodeling (LVR) leads to congestive heart failure (CHF) are unclear. This study examined the functional and bioenergetic abnormalities associated with postinfarction ventricular remodeling in a new, large animal model. Methods and Results Remodeling was induced by circumflex coronary artery ligation in young pigs. LV mass, volume, ejection fraction (EF), the ratio of scar surface area to LV surface area, and LV wall stresses were calculated from magnetic resonance imaging anatomic data and simultaneously measured LV pressure. Hemodynamics, transmural blood flow, and high-energy phosphates (spatially localized 31 P–nuclear magnetic resonance) were measured under basal conditions, during hyperperfusion induced by pharmacological vasodilation with adenosine, and during pyruvate infusion (11 mg/kg per minute IV). Six of 18 animals with coronary ligation developed clinical CHF while the remaining 12 animals had LV dilation (LVR) without CHF. The results were compared with 16 normal animals. EF decreased from 55.9±5.6% in normals to 34.6±2.3% in the LVR group ( P P P P P Conclusions Bioenergetic abnormalities in remodeled myocardium are related to the severity of LV dysfunction, which, in turn, is dependent on the severity of the initiating myocardial infarction.

Background This study tested the hypothesis that 31 P nuclear magnetic resonance (NMR)–detectable 2-deoxyglucose (2DG) uptake is increased in chronically pressure-overloaded hypertrophied left ventricular myocardium. Methods and Results 31 P NMR spectroscopy was used to determine the transmural distribution of high-energy phosphate levels and 2-deoxyglucose-6-phosphate (2DGP) accumulation during intracoronary infusion of 2DG (15 μmol · kg body wt −1 · min −1 ) in eight normal dogs and in eight dogs with severe left ventricular hypertrophy (LVH) produced by ascending aortic banding. The ratio of LV weight to body weight was 8.25±0.65 g/kg in the LVH group compared with 4.35±0.11 g/kg in the normal group ( P P r =.90 for subendocardial 2DGP versus LV weight/body weight). A transmural gradient of 2DGP was present, with greatest accumulation in the subendocardium (3.3±1.6, 5.8±2.3, and 7.9±2.2 μmol/g in Epi, midwall, and Endo of the LVH hearts, respectively; P Conclusions The pressure-overloaded hypertrophied left ventricle demonstrated increased accumulation of 2DGP detected with 31 P NMR spectroscopy. Accumulation of 2DGP was positively correlated with the degree of hypertrophy and was most marked in the subendocardium.

The response of myocardial high-energy and inorganic phosphates (HEP and Pi, respectively) and associated changes in myocardial blood flow, lactate uptake, and O2 consumption (MVo2) rates were examined in an open-chest canine model during progressively increasing workloads achieved by catecholamine infusion. HEP and Pi levels (measured with transmurally localized 31P-nuclear magnetic resonance spectroscopy) were unaffected by moderate increases in the level of energy expenditure but were significantly altered by high workloads, especially in the subepicardium. The MVo2 and HEP data from three different protocols that utilized pharmacological augmentation of blood flow demonstrated that the maximal rate of myocardial energy production during inotropic stimulation was dictated by perfusion limitation. This limitation was more severe in the subepicardial layer at the high workloads despite equivalent or even higher increases in blood flow to this layer, reflecting a preferential enhancement of demand in the outer layer by catecholamines. In contrast, under basal conditions, existence of a marginal perfusion limitation was evident in the inner but not in the outer layer.

This study examines the hypothesis that high-energy phosphate (HEP) compound levels in unstimulated in vivo myocardium are defined by 1) the level of perfusion and 2) non-perfusion-dependent metabolic characteristics. This hypothesis was tested by determining 1) the effects of pharmacological hyperperfusion of functioning myocardium on transmural HEP compound distribution, contractile function, and myocardial oxygen consumption rate (MVO2) as well as 2) the effect of KCl cardioplegia on transmural myocardial HEP compound distribution. Creatine phosphate (CP) and ATP were measured across the anterior left ventricular wall using spatially localized 31P-nuclear magnetic resonance (NMR). At baseline, the CP-to-ATP (CP/ATP) ratio was significantly lower in the subendocardium than in the subepicardium. This transmural HEP gradient was abolished by hyperperfusion without significant effects on contractile function or MVO2. Similarly, KCl arrest significantly increased CP and CP/ATP in all myocardial layers, and the transmural gradient of CP/ATP was abolished again. These studies indicate that in present experimental model 1) myocardial performance is not constrained by inadequate perfusion in any myocardial layer although modest oxygen limitation affects the kinetics of oxidative phosphorylation in the inner myocardial layers and 2) in all myocardial layers, submaximal activation of intermediary metabolism and oxidative phosphorylation reactions results in lower steady-state CP and higher ADP levels relative to their respective values when energy expenditure is markedly reduced by KCl arrest.

Spatially localized nuclear magnetic resonance (NMR) spectroscopy was used to examine the effect of tachycardia and inotropic stimulation on myocardial ATP, creatine phosphate (CrP), and inorganic phosphate (Pi) in animals with left ventricular hypertrophy (LVH). Studies were performed in eight normal dogs and seven dogs with moderate LVH produced by banding the ascending aorta. 31P-NMR spectra were obtained from five layers across the LV wall, while blood flow (BF) was measured with microspheres during control conditions, pacing at 200 and 240 beats/min, and during dobutamine infusion (Dob). Myocardial ATP and CrP levels were normal in the LVH hearts during control conditions. Pacing did not alter the transmural distribution of perfusion or the levels of CrP, ATP, and Pi in normal hearts. In contrast, in four of seven LVH hearts, pacing decreased the subendocardial/subepicardial (ENDO/EPI) BF ratio and caused depletion of CrP and appearance of Pi characteristic of ischemia in the subendocardium. Dob produced greater increases in the heart rate x LV systolic pressure product (RPP) and greater increases of Pi and decreases of CrP in LVH than in normal hearts; however, at comparable elevations of RPP the alterations of Pi and CrP were similar in both groups. Although Dob decreased the ENDO/EPI in LVH hearts, Dob-induced alterations in CrP and Pi were uniform across the LV wall. Increasing myocardial BF with adenosine or carbochromen did not reverse the alterations in Pi or CrP produced by Dob. We conclude that 1) ENDO perfusion abnormalities during tachycardia in LVH do produce ENDO subendocardial ischemia; 2) when the degree of augmentation of mechanical performance is considered, the metabolic changes induced by Dob were similar in normal and LVH hearts; 3) Dob-induced alterations in Pi and CrP were not related to inadequate perfusion, since increasing coronary BF did not reverse these changes; and 4) alterations of Pi and CrP during Dob infusion were not more prominent in the ENDO, indicating that the decreased ENDO/EPI flow did not cause ENDO ischemia but may reflect relatively lower O2 demands in this region during inotropic stimulation.

Publisher Summary This chapter focuses on (1) the mechanisms of respiratory regulation and the kinetic characteristics of high-energy phosphate (HEP) generation in in vitro and in vivo models; (2) the effects of myocardial abnormalities on regulation and kinetics; (3) the transmural heterogeneity of perfusion, contractile, and metabolic responses of in vivo myocardium and the interactions between altered perfusion patterns and HEP metabolism; and (4) applications of NMR techniques to the study of the human heart. The perfused rat heart studies have yielded invaluable information with regard to the mechanisms of respiratory regulation and the kinetic characteristics of H+ ATPase in intact myocardium. At present, spectroscopic studies of human myocardium are relatively few in number, they generally involve small numbers of patients, and they are complicated by technical limitations. Thus the utility of NMR spectroscopy as a cardiac diagnostic tool is at present limited.

Transmurally localized 31P-nuclear magnetic resonance spectroscopy (NMR) was used to study the effect of severe pressure overload left ventricular hypertrophy (LVH) on myocardial high energy phosphate content. Studies were performed on 8 normal dogs and 12 dogs with severe left ventricular hypertrophy produced by banding the ascending aorta at 8 wk of age. Spatially localized 31P-NMR spectroscopy provided measurements of the transmural distribution of myocardial ATP, phosphocreatine (CP), and inorganic phosphate (Pi); spectra were calibrated from measurements of ATP content in myocardial biopsies using HPLC. Blood flow was measured with microspheres. In hypertrophied hearts during basal conditions, ATP was decreased by 42%, CP by 58%, and the CP/ATP ratio by 32% in comparison with normal. Increasing myocardial blood flow with adenosine did not correct these abnormalities, indicating that they were not the result of persistent hypoperfusion. Atrial pacing at 200 and 240 beats per min caused no change in high energy phosphate content in normal hearts but resulted in further CP depletion with Pi accumulation in the inner left ventricular layers of the hypertrophied hearts. These changes were correlated with redistribution of blood flow away from the subendocardium in LVH hearts. These findings demonstrate that high energy phosphate levels and the CP/ATP ratio are significantly decreased in severe LVH. These abnormalities are proportional to the degree of hypertrophy but are not the result of persistent abnormalities of myocardial perfusion. In contrast, depletion of CP and accumulation of Pi during tachycardia in LVH are closely related to the pacing-induced perfusion abnormalities and likely reflect subendocardial ischemia.