Kurt W. Saupe

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.

ATP Synthesis During Low-Flow Ischemia Influence of Increased Glycolytic Substrate

BACKGROUND Our goals were to (1) simulate the degree of low-flow ischemia and mixed anaerobic and aerobic metabolism of an acutely infarcting region; (2) define changes in anaerobic glycolysis, oxidative phosphorylation, and the creatine kinase (CK) reaction velocity; and (3) determine whether and how increased glycolytic substrate alters the energetic profile, function, and recovery of the ischemic myocardium in the isolated blood-perfused rat heart. METHODS AND RESULTS Hearts had 60 minutes of low-flow ischemia (10% of baseline coronary flow) and 30 minutes of reperfusion with either control or high glucose and insulin (G+I) as substrate. In controls, during ischemia, rate-pressure product and oxygen consumption decreased by 84%. CK velocity decreased by 64%; ATP and phosphocreatine (PCr) concentrations decreased by 51% and 63%, respectively; inorganic phosphate (P(i)) concentration increased by 300%; and free [ADP] did not increase. During ischemia, relative to controls, the G+I group had similar CK velocity, oxygen consumption, and tissue acidosis but increased glycolysis, higher [ATP] and [PCr], and lower [P(i)] and therefore had a greater free energy yield from ATP hydrolysis. Ischemic systolic and diastolic function and postischemic recovery were better. CONCLUSIONS During low-flow ischemia simulating an acute myocardial infarction region, oxidative phosphorylation accounted for 90% of ATP synthesis. The CK velocity fell by 66%, and CK did not completely use available PCr to slow ATP depletion. G+I, by increasing glycolysis, slowed ATP depletion, maintained lower [P(i)], and maintained a higher free energy from ATP hydrolysis. This improved energetic profile resulted in better systolic and diastolic function during ischemia and reperfusion. These results support the clinical use of G+I in acute MI.

Compensatory Mechanisms Associated With the Hyperdynamic Function of Phospholamban-Deficient Mouse Hearts

Phospholamban ablation is associated with significant increases in the sarcoplasmic reticulum Ca(2+)-ATPase activity and the basal cardiac contractile parameters. To determine whether the observed phenotype is due to loss of phospholamban alone or to accompanying compensatory mechanisms, hearts from phospholamban-deficient and age-matched wild-type mice were characterized in parallel. There were no morphological alterations detected at the light microscope level. Assessment of the protein levels of the cardiac sarcoplasmic reticulum Ca(2+)-ATPase, calsequestrin, myosin, actin, troponin I, and troponin T revealed no significant differences between phospholamban-deficient and wild-type hearts. However, the ryanodine receptor protein levels were significantly decreased (25%) upon ablation of phospholamban, probably in an attempt to regulate the release of Ca2+ from the sarcoplasmic reticulum, which had a significantly higher diastolic Ca2+ content in phospholamban-deficient compared with wild-type hearts (16.0 +/- 2.2 versus 8.6 +/- 1.0 mmol Ca2+/kg dry wt, respectively). The increases in Ca2+ content were specific to junctional sarcoplasmic reticulum stores, as there were no alterations in the Ca2+ content of the mitochondria or A band. Assessment of ATP levels revealed no alterations, although oxygen consumption increased (1.6-fold) to meet the increased ATP utilization in the hyperdynamic phospholamban-deficient hearts. The increases in oxygen consumption were associated with increases (2.2-fold) in the active fraction of the mitochondrial pyruvate dehydrogenase, suggesting increased tricarboxylic acid cycle turnover and ATP synthesis. 31P nuclear magnetic resonance studies demonstrated decreases in phosphocreatine levels and increases in ADP and AMP levels in phospholamban-deficient compared with wild-type hearts. However, the creatine kinase activity and the creatine kinase reaction velocity were not different between phospholamban-deficient and wild-type hearts. These findings indicate that ablation of phospholamban is associated with downregulation of the ryanodine receptor to compensate for the increased Ca2+ content in the sarcoplasmic reticulum store and metabolic adaptations to establish a new energetic steady state to meet the increased ATP demand in the hyperdynamic phospholamban-deficient hearts.

Exercise training attenuates age-associated diastolic dysfunction in rats.

Background—In contrast to systolic function, which is relatively well preserved with advancing age, diastolic function declines steadily after age 30. Our goal was to determine whether changes in diastolic function that occur with aging could be reversed with exercise training. Methods and Results—Adult (6-month-old) and old (24-month-old) Fischer 344/BNF1 rats were studied after either 12 weeks of treadmill training or normal sedentary cage life. Three aspects of diastolic function were studied: (1) left ventricular (LV) filling in vivo via Doppler echocardiograph, (2) LV passive compliance, and (3) the degree of ischemia-induced LV stiffening. Maximal exercise capacity was lower in the old rats (18±1 minutes to exhaustion on a standard treadmill) than in the adult rats (25±1 minutes). Training increased exercise capacity by 43% in the old rats and 46% in the adults (to 26±1 and 37±1 minutes, respectively). Echocardiographic indices of LV relaxation were significantly lower in the old rats, but with training, they increased back to the levels seen in the adults. LV stiffness measured in the isolated, perfused hearts was not affected by age or training. Also in the isolated hearts, the LV stiffened more rapidly during low-flow ischemia in the old hearts than in the adults, but training eliminated this age-associated difference in the response to ischemia. Conclusions—Our findings indicate that in rats, some age-associated changes in diastolic function are reversible and thus may not be intrinsic to aging but instead secondary to other processes, such as deconditioning.

Exercise Intolerance in Rats With Hypertensive Heart Disease Is Associated With Impaired Diastolic Relaxation

A decrease in functional capacity is one of the most important clinical manifestations of hypertensive heart disease, but its cause is poorly understood. Our purpose was to evaluate potential causes of hypertension-induced exercise intolerance, focusing on identifying the type(s) of cardiac dysfunction associated with the first signs of exercise intolerance during the course of hypertensive heart disease. Exercise capacity was measured weekly in Dahl salt-sensitive rats as they developed hypertension as well as in Dahl salt-resistant control rats. Exercise capacity was unchanged from baseline during the first 8 weeks of hypertension, suggesting that hypertension itself did not cause exercise intolerance. After 9 to 12 weeks of hypertension, exercise capacity decreased in salt-sensitive rats but not in control rats. After 10 weeks of hypertension, indices of diastolic function (early truncation of the E wave), as assessed by echocardiography at rest, were decreased in the salt-sensitive rats. When exercise capacity had decreased by ≈25% in a rat, the heart was isolated, and left ventricular (LV) compliance and systolic function were measured. At that time point, LV hypertrophy was modest (an ≈20% increase in LV mass), and systolic function was normal or supernormal, indicating that exercise intolerance began during “compensated” LV hypertrophy. Passive LV compliance remained normal in salt-sensitive rats. Thus, in this model of hypertensive heart disease, exercise intolerance develops during the compensated stage of LV hypertrophy and appears to be due to changes in diastolic rather than systolic function. However, studies in which LV function is assessed during exercise are needed to conclusively define the roles of systolic and diastolic dysfunction in causing exercise intolerance.

Hypoperfusion-induced contractile failure does not require changes in cardiac energetics.

Decreasing coronary perfusion causes an immediate decrease in contractile function via unknown mechanisms. It has long been suspected that this contractile dysfunction is caused by ischemia-induced changes in cardiac energetics. Our goal was to determine whether changes in cardiac energetics necessarily precede the contractile dysfunction as one would expect if a causal relationship exists. In 14 isolated rat hearts, we gradually decreased coronary perfusion using a coronary perfusate with a normal hematocrit and normal concentrations of the major metabolic substrates. Using 31P NMR spectroscopy to measure ATP, phosphocreatine (PCr), Pi, and ADP concentrations ([ATP], [PCr], [Pi], [ADP]), pH, and amount of free energy released from ATP hydrolysis (|DeltaGATP|), we found that none of these variables changed significantly until several minutes after systolic pressure had significantly decreased. Even when developed pressure had decreased by over one-third, only very slight changes in [Pi], pH, and |DeltaGATP| had occurred, with no significant changes in [ATP], [PCr], or [ADP]. Additionally, the rate of high-energy phosphate transfer between ATP and PCr did not decrease enough during hypoperfusion to explain the contractile dysfunction. We conclude that nonenergetic factors are the dominant cause of the initial decrease in systolic function when myocardial perfusion is decreased.Decreasing coronary perfusion causes an immediate decrease in contractile function via unknown mechanisms. It has long been suspected that this contractile dysfunction is caused by ischemia-induced changes in cardiac energetics. Our goal was to determine whether changes in cardiac energetics necessarily precede the contractile dysfunction as one would expect if a causal relationship exists. In 14 isolated rat hearts, we gradually decreased coronary perfusion using a coronary perfusate with a normal hematocrit and normal concentrations of the major metabolic substrates. Using31P NMR spectroscopy to measure ATP, phosphocreatine (PCr), Pi, and ADP concentrations ([ATP], [PCr], [Pi], [ADP]), pH, and amount of free energy released from ATP hydrolysis (‖Δ G ATP‖), we found that none of these variables changed significantly until several minutes after systolic pressure had significantly decreased. Even when developed pressure had decreased by over one-third, only very slight changes in [Pi], pH, and ‖Δ G ATP‖ had occurred, with no significant changes in [ATP], [PCr], or [ADP]. Additionally, the rate of high-energy phosphate transfer between ATP and PCr did not decrease enough during hypoperfusion to explain the contractile dysfunction. We conclude that nonenergetic factors are the dominant cause of the initial decrease in systolic function when myocardial perfusion is decreased.

Effects of AT1 receptor block begun late in life on normal cardiac aging in rats.

The goal of this study was to determine how short-term (12 weeks) angiotensin type I (AT1) block begun late in life affects aspects of myocardial biology and physiologic function altered by normal aging. Exercise capacity, myocardial morphology, histopathology, and coronary vascular function (degree of coronary vasodilation in response to adenosine) were evaluated in 53 Fischer 344 rats. Adult (6 months of age) and old (21 months of age) rats were studied after 12 weeks of either control drinking water, a low dose of candesartan that did not significantly lower blood pressure (1 mg/kg/d), or a high dose of candesartan (10 mg/kg/d). Significant age-associated changes in exercise capacity (38% decrease), coronary dilation in response to adenosine (41% decrease), and histopathology occurred but were not affected by candesartan treatment. Age-associated myocardial hypertrophy occurred as indicated by an increase in heart weight–to–tibia length ratio from 0.27 g/cm ± 0.01 in the adult controls to 0.34 g/cm ± 0.02 in the old controls (P < 0.05). This hypertrophy in the aged hearts was significantly attenuated by both low-dose (0.30 g/cm ± 0.01) and high-dose (0.29 g/cm ± 0.01) candesartan (P < 0.05). Echocardiographic measurements indicate that the candesartan-induced decrease in hypertrophy occurred concomitantly with slight decreases in septal wall thickness and left ventricular (LV) chamber diameter. It is concluded that short-term AT1 block, even when initiated late in life, can decrease age-associated LV hypertrophy independent of blood pressure–lowering effects.

MR spectroscopy of transgenic mice

The rapidly growing information on the mouse genome and transgenic techniques that allow sophisticated genetic manipulations, including over-expression, ablation and mutation of specific gene products has placed the mouse at the forefront of cardiovascular research [1]. This has resulted in an increasing interest in applying invasive and non-invasive biophysical tools to characterize the cardiovascular phenotype of these transgenic animals [2]. The mouse in general and the mouse heart in particular, however, offers substantial challenges for MR spectroscopy, including its small size and rapid heart rate. Our example will focus on 31p-NMR spectroscopy but the spectra based on the other MR-observable nuclei such as 1H, 3tC and 23Na can also be obtained. The challenge of performing NMR experiments on a mouse heart may be best explained by comparing it to the isolated perfused rat heart preparation. The adult mouse heart weighs 90-140 mg, or about one-tenth of the size of a rat heart. Because the NMR signal is proportional to the number of nuclei in the sample, if all factors are equal, it would take 10-times longer to achieve the same signal for the mouse heart placed in the same (usually 20 ram) NMR probe. It goes without saying that this does not allow for many repetitive measures. The challenge is to increase the ratio of cytosolic volume to total NMR-sensitive volume. The obvious answer to this problem is to use a small probe. But the physical limitations imposed by the plumbing of the isolated heart are formidable. By perfusing the