Insulin-like effects of a physiologic concentration of carnitine on cardiac metabolism.

Pharmacologic (millimolar) levels of carnitine have been reported to increase myocardial glucose oxidation, but whether physiologically relevant concentrations of carnitine affect cardiac metabolism is not known. We employed the isolated, perfused rat heart to compare the effects of physiologic levels of carnitine (50 microM) and insulin (75 mU/l [0.5 nM]) on the following metabolic processes: (1) glycolysis (release of 3H2O from 5-3H-glucose); (2) oxidation of glucose and pyruvate (production of 14CO2 from U-14C-glucose, 1-14C-glucose, 3,4-14C-glucose, 1-14C-pyruvate, and 2-14C-pyruvate); and (3) oxidation of palmitate (release of 3H2O from 9,10-3H-palmitate). We found that addition of carnitine (50 microM) to a perfusate containing both glucose (10 mM) and palmitate (0.5 mM) stimulated glycolytic flux by 20%, nearly doubled the rate of glucose oxidation, and inhibited palmitate oxidation by 20%. These actions of carnitine were uniformly similar to those of insulin. When carnitine and insulin were administered together, their effects on the oxidation of glucose and palmitate, but not on glycolysis, were additive. When pyruvate (1 mM) was substituted for glucose, neither carnitine nor insulin influenced the rate of oxidation of pyruvate or palmitate. In combination, however, carnitine and insulin sharply suppressed pyruvate oxidation (75%) and doubled the rate of palmitate oxidation. None of the responses to carnitine or insulin was affected by varying the isotopic labeling of glucose or pyruvate. The results show that carnitine, at normal blood levels, exerts insulin-like effects on myocardial fuel utilization. They also suggest that plasma carnitine in vivo may interact with insulin both additively and permissively on the metabolism of carbohydrates and fatty acids.

The pathogenesis of familial hypertrophic cardiomyopathy: early and evolving effects from an alpha-cardiac myosin heavy chain missense mutation.

Familial hypertrophic cardiomyopathy (FHC) is a genetic disorder resulting from mutations in genes encoding sarcomeric proteins. This typically induces hyperdynamic ejection, impaired relaxation, delayed early filling, myocyte disarray and fibrosis, and increased chamber end-systolic stiffness. To better understand the disease pathogenesis, early (primary) abnormalities must be distinguished from evolving responses to the genetic defect. We did in vivo analysis using a mouse model of FHC with an Arg403Gln alpha-cardiac myosin heavy chain missense mutation, and used newly developed methods for assessing in situ pressure-volume relations. Hearts of young mutant mice (6 weeks old), which show no chamber morphologic or gross histologic abnormalities, had altered contraction kinetics, with considerably delayed pressure relaxation and chamber filling, yet accelerated systolic pressure rise. Older mutant mice (20 weeks old), which develop fiber disarray and fibrosis, had diastolic and systolic kinetic changes similar to if not slightly less than those of younger mice. However, the hearts of older mutant mice also showed hyperdynamic contraction, with increased end-systolic chamber stiffness, outflow tract pressure gradients and a lower cardiac index due to reduced chamber filling; all 'hallmarks' of human disease. These data provide new insights into the temporal evolution of FHC. Such data may help direct new therapeutic strategies to diminish disease progression.

Neonatal cardiomyopathy in mice homozygous for the Arg403Gln mutation in the alpha cardiac myosin heavy chain gene.

Heterozygous mice bearing an Arg403Gln missense mutation in the alpha cardiac myosin heavy chain gene (alpha-MHC403/+) exhibit the histopathologic features of human familial hypertrophic cardiomyopathy. Surprisingly, homozygous alpha-MHC403/403 mice die by postnatal day 8. Here we report that neonatal lethality is caused by a fulminant dilated cardiomyopathy characterized by myocyte dysfunction and loss. Heart tissues from neonatal wild-type and alpha-MHC403/403 mice demonstrate equivalent switching of MHC isoforms; alpha isoforms in each increase from 30% at birth to 70% by day 6. Cardiac dimensions and function, studied for the first time in neonatal mice by high frequency (45 MHz) echocardiography, were normal at birth. Between days 4 and 6, alpha-MHC403/403 mice developed a rapidly progressive cardiomyopathy with left ventricular dilation, wall thinning, and reduced systolic contraction. Histopathology revealed myocardial necrosis with dystrophic calcification. Electron microscopy showed normal architecture intermixed with focal myofibrillar disarray. We conclude that 45-MHz echocardiography is an excellent tool for assessing cardiac physiology in neonatal mice and that the concentration of Gln403 alpha cardiac MHC in myocytes influences both cell function and cell viability. We speculate that variable incorporation of mutant and normal MHC into sarcomeres of heterozygotes may account for focal myocyte death in familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy mice display gender differences in electrophysiological abnormalities.

Genetically-manipulated mice harboring an alpha-myosin heavy chain Arg403Gln missense mutation (alpha-MHC403/+) display a phenotype characteristic of familial hypertrophic cardiomyopathy (FHC). Male and female (30 +/- 8 week old) heterozygous alpha-MHC403/+ mice and litter-mate controls were evaluated using a surface electrocardiogram (ECG) and an in vivo cardiac electrophysiology study (EPS). Wild type animals had normal intracardiac electrophysiology, with no significant differences between male and female control mice during EPS. The female wild-type mice did have slower heart rates and longer ECG intervals than their male wild-type counterparts. The female alpha-MHC403/+ mice had similar ECG's, cardiac conduction times, and refractory periods compared with female wild-type mice. In contrast, male FHC mice had distinctive ECG and electrophysiologic abnormalities including right axis deviation, prolonged ventricular repolarization and prolonged sinus node recovery times. During programmed ventricular stimulation, 62% of male alpha-MHC403/+ mice and 28% of female alpha-MHC403/+ mice had inducible ventricular tachycardia. These studies identify gender-specific electrophysiologic abnormalities in alpha-MHC403/+ FHC mice, concordant with the histological and hemodynamic derangements previously reported.

Diastolic dysfunction and altered energetics in the alphaMHC403/+ mouse model of familial hypertrophic cardiomyopathy.

An arginine to glutamine missense mutation at position 403 of the beta-cardiac myosin heavy chain causes familial hypertrophic cardiomyopathy. Here we study mice which have this same missense mutation (alphaMHC403/+) using an isolated, isovolumic heart preparation where cardiac performance is measured simultaneously with cardiac energetics using 31P nuclear magnetic resonance spectroscopy. We observed three major alterations in the physiology and bioenergetics of the alphaMHC403/+ mouse hearts. First, while there was no evidence of systolic dysfunction, diastolic function was impaired during inotropic stimulation. Diastolic dysfunction was manifest as both a decreased rate of left ventricular relaxation and an increase in end-diastolic pressure. Second, under baseline conditions alphaMHC403/+ hearts had lower phosphocreatine and increased inorganic phosphate contents resulting in a decrease in the calculated value for the free energy released from ATP hydrolysis. Third, hearts from alphaMHC403/+ hearts that were studied unpaced responded to increased perfusate calcium by decreasing heart rate approximately twice as much as wild types. We conclude that hearts from alphaMHC403/+ mice demonstrate work load-dependent diastolic dysfunction resembling the human form of familial hypertrophic cardiomyopathy. Changes in high-energy phosphate content suggest that an energy-requiring process may contribute to the observed diastolic dysfunction.

Electrophysiological abnormalities and arrhythmias in alpha MHC mutant familial hypertrophic cardiomyopathy mice.

A new mouse cardiac electrophysiology method was used to study mice harboring an alpha-myosin heavy chain Arg403Gln missense mutation (alpha-MHC403/+), which results in histological and hemodynamic abnormalities characteristic of familial hypertrophic cardiomyopathy (FHC) and sudden death of uncertain etiology during exercise. Wild-type animals had completely normal cardiac electrophysiology. In contrast, FHC mice demonstrated (a) electrocardiographic abnormalities including prolonged repolarization intervals and rightward axis; (b) electrophysiological abnormalities including heterogeneous ventricular conduction properties and prolonged sinus node recovery time; and (c) inducible ventricular ectopy. These data identify distinct electrophysiologic abnormalities in FHC mice with a specific alpha-myosin mutation, and also validate a novel method to explore in vivo the relationship between specific genotypes and their electrophysiologic phenotypes.

Role of MgADP in the development of diastolic dysfunction in the intact beating rat heart.

Sarcomere relaxation depends on dissociation of actin and myosin, which is regulated by a number of factors, including intracellular [MgATP] as well as MgATP hydrolysis products [MgADP] and inorganic phosphate [Pi], pHi, and cytosolic calcium concentration ([Ca2+]c). To distinguish the contribution of MgADP from the other regulators in the development of diastolic dysfunction, we used a strategy to increase free [MgADP] without changing [MgATP], [Pi], or pHi. This was achieved by applying a low dose of iodoacetamide to selectively inhibit the creatine kinase activity in isolated perfused rat hearts. [MgATP], [MgADP], [Pi], and [H+] were determined using 31P NMR spectroscopy. The [Ca2+]c and the glycolytic rate were also measured. We observed an approximately threefold increase in left ventricular end diastolic pressure (LVEDP) and 38% increase in the time constant of pressure decay (P < 0.05) in these hearts, indicating a significant impairment of diastolic function. The increase in LVEDP was closely related to the increase in free [MgADP]. Rate of glycolysis was not changed, and [Ca2+]c increased by 16%, which cannot explain the severity of diastolic dysfunction. Thus, our data indicate that MgADP contributes significantly to diastolic dysfunction, possibly by slowing the rate of cross-bridge cycling.

Cardiac glucose and fatty acid oxidation in the streptozotocin-induced diabetic spontaneously hypertensive rat.

Hypertension intensifies the cardiac dysfunction of diabetes. We investigated the possible role of altered exogenous fuel oxidation in this phenomenon. Diabetes was induced by streptozotocin in spontaneously hypertensive rats and normotensive Sprague-Dawley rats. Two weeks later, mechanical performance and the oxidation of glucose and palmitate were quantified in working hearts ex vivo at intermediate and high workloads. The results showed that the nondiabetic spontaneously hypertensive rat hearts, compared with those of the normotensive controls, oxidized glucose at a higher rate but oxidized palmitate at a much lower rate, as reported previously. The effects of diabetes in the hypertensive rats, compared with its effects in the normotensive strain, were characterized by (1) a more pronounced decrease in heart performance, (2) either a similar or a less marked reduction in the rate of glucose oxidation, depending on the workload, and (3) a relatively greater increase in palmitate oxidation, particularly at the higher workload. These findings suggest that the exaggerated stimulation of fatty acid oxidation by diabetes in the hypertrophic left ventricle may be a more important contributor to the premature mechanical dysfunction than the inhibition of glucose oxidation. Possible mechanisms include antagonism of energetically favorable shifts in fuel oxidation or inhibition of accelerated membrane lipid biosynthesis in left ventricular hypertrophy.

Cilazapril treatment depresses ventricular function in spontaneously hypertensive rats.

The purpose of this study was to characterize the effect of chronic treatment with an angiotensin-converting enzyme (ACE) inhibitor on left ventricular function in spontaneously hypertensive rats (SHR). Cilazapril (5 mg/kg) was administered in the drinking water continuously for 11 wk, beginning at 4 wk of age. Systolic arterial pressure (SAP) was monitored weekly. At the end of the 11-wk period, left ventricular function was quantified using the perfused working heart preparation. Cilazapril exerted a rapid, complete, and persistent antihypertensive effect in the SHR in vivo but had no effect on SAP in the normotensive Sprague-Dawley (S-D) group. Nevertheless, the drug reduced left ventricular weight to the same extent in both strains. Function of untreated SHR hearts was not different from that of the untreated S-D hearts. Cilazapril treatment depressed heart performance (28-35%) in SHR but had no effect in the S-D group. The decline in pump performance in SHR hearts was associated with diminished tension development and velocity of shortening of papillary muscles. These results demonstrate that an ACE inhibitor, administered to young SHR, produces a reduction in left ventricular contractile function, which may be due to a decline in muscle contractility and which cannot be explained exclusively by the reduction in left ventricular mass.

Altered glucose and fatty acid oxidation in hearts of the spontaneously hypertensive rat.

Metabolic fuel oxidation may be altered in left ventricular hypertrophy (LVH), but detailed characterizations are lacking. Although the spontaneously hypertensive rat (SHR) is a widely used experimental model of LVH, its myocardial fuel oxidation rates are unknown. The purpose of this study was to directly measure glucose and fatty acid (FA) oxidation in the SHR heart ex vivo under controlled loading conditions. Hearts from 15-week-old SHR and Sprague Dawley (SD) rats were perfused in a recirculating system and indices of cardiac performance were continuously monitored. The oxidation of glucose and palmitate were determined simultaneously at low and high workloads by the addition of U-14C-glucose and 9,10-3H-palmitate to the recirculating perfusate. The results demonstrate that FA oxidation of SHR hearts is profoundly suppressed (60-80%) relative to that of the normotensive SD strain, particularly at high workloads. Glucose oxidation is also moderately elevated, yielding a marked (four-to-five-fold) increase in the ratio of glucose/FA oxidation rates in the SHR hearts. Since more ATP is generated per mole of oxygen consumed when glucose is the fuel scource, these results are consistent with the hypothesis that a shift away from FA use toward glucose contributes to the preservation of energetic economy in stable, concentric LVH.

Cardiac ornithine decarboxylase of diabetic spontaneously hypertensive rat: effects of insulin and thyroid hormone treatment.

Diabetes mellitus causes a more profound reduction of left ventricular weight (LVW) in the spontaneously hypertensive rat (SHR) than it does in nonhypertensive strains. Diabetes also depresses the activity of cardiac ornithine decarboxylase (ODC), an index of cell growth. We measured ODC activity, of ventricular homogenates obtained from diabetic SHR and nonhypertensive WKY rats, with and without chronic treatment with insulin or triiodothyronine (T3). Left ventricular ODC activities of nondiabetic SHR and WKY rats were not different from each other. Streptozotocin-induced diabetes (8 weeks) reduced left ventricular ODC activity of SHR and WKY rats to the same extent; the effect was characterized by a reduction in apparent Vmax with no change in apparent Km. Both T3 and insulin therapy prevented the decline in ventricular ODC activity in both strains, although only the effect of insulin was correlated with LVW. The results suggest that the effects of diabetes on LVW and ODC activity are independent of attendant reductions in serum thyroid hormone levels. However, the results did not reveal any strain-selective effects of diabetes on ODC activity which might have contributed to the pronounced loss of LVW in the SHR strain.

Inhibition of rabbit liver arylsulfatase B by phosphate esters.

Arylsulfatase B (aryl-sulfate sulfohydrolase, EC 3.1.6.1) purified from rabbit liver is competitively inhibited at modest concentrations by a variety of phosphate esters derived from amino acids, amines and simple sugars. Phospho-L-serine coupled to Sepharose 4B could be used as an affinity column to enhance the purity of a crude preparation of the enzyme. It is suggested that phosphate esters containing functional groups can be used to obtain affinity reagents to purify arylsulfatases and also to probe their active sites.