David L. Severson

Insulin signaling coordinately regulates cardiac size, metabolism, and contractile protein isoform expression

To investigate the role of insulin signaling on postnatal cardiac development, physiology, and cardiac metabolism, we generated mice with a cardiomyocyte-selective insulin receptor knockout (CIRKO) using cre/loxP recombination. Hearts of CIRKO mice were reduced in size by 20-30% due to reduced cardiomyocyte size and had persistent expression of the fetal beta-myosin heavy chain isoform. In CIRKO hearts, glucose transporter 1 (GLUT1) expression was reduced by about 50%, but there was a twofold increase in GLUT4 expression as well as increased rates of cardiac glucose uptake in vivo and increased glycolysis in isolated working hearts. Fatty acid oxidation rates were diminished as a result of reduced expression of enzymes that catalyze mitochondrial beta-oxidation. Although basal rates of glucose oxidation were reduced, insulin unexpectedly stimulated glucose oxidation and glycogenolysis in CIRKO hearts. Cardiac performance in vivo and in isolated hearts was mildly impaired. Thus, insulin signaling plays an important developmental role in regulating postnatal cardiac size, myosin isoform expression, and the switching of cardiac substrate utilization from glucose to fatty acids. Insulin may also modulate cardiac myocyte metabolism through paracrine mechanisms by activating insulin receptors in other cell types within the heart.

Short-term diabetes alters K+ currents in rat ventricular myocytes.

The electrophysiological properties of single ventricular myocytes from control rats and from rats made diabetic by streptozotocin (STZ) injection (100 mg/kg body weight) have been investigated using whole-cell voltage-clamp measurements. Our major goal was to define the effects of diabetes on rate-dependent changes in action potential duration and the underlying outward K+ currents. As early as 4 to 6 days after STZ treatment, significant elevation of plasma glucose levels occurs, and the action potential duration increases. In both control and diabetic rats, when the stimulation rate is increased, the action potential is prolonged, but this lengthening is considerably more pronounced in myocytes from diabetic rats. In ventricular myocytes from diabetic rats, the Ca(2+)-independent transient outward K+ current (I(t)) is reduced in amplitude, and its reactivation kinetics are slowed. These changes result in a smaller I(t) at physiological heart rates. The steady-state outward K+ current (IK) also exhibits rate-dependent attenuation, and this phenomenon is more pronounced in cells from diabetic rats. These STZ-induced changes in I(t) and IK also develop when a lower dose (55 mg/kg) of STZ is used and measurements are made after 7 weeks of treatment. These electrophysiological effects are not related to the hypothyroid conditions that accompany the diabetic state, since they cannot be reversed by replacement of the hormone L-triiodothyronine to physiological levels. Direct effects of STZ could be ruled out, since preceding the STZ injection with a bolus injection of 3-O-methylglucose, which prevents development of hyperglycemia, prevents the electrophysiological changes.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucose and fatty acid metabolism in the isolated working mouse heart

Although isolated perfused mouse heart models have been developed to study mechanical function, energy substrate metabolism has not been examined despite the expectation that the metabolic rate for a heart from a small mammal should be increased. Consequently, glucose utilization (glycolysis, oxidation) and fatty acid oxidation were measured in isolated working mouse hearts perfused with radiolabeled substrates, 11 mM glucose, and either 0.4 or 1.2 mM palmitate. Heart rate, coronary flow, cardiac output, and cardiac power did not differ significantly between hearts perfused at 0.4 or 1.2 mM palmitate. Although the absolute values obtained for glycolysis and glucose oxidation and fatty acid oxidation are significantly higher than those reported for rat hearts, the pattern of substrate metabolism in mouse hearts is similar to that observed in hearts from larger mammals. The metabolism of mouse hearts can be altered by fatty acid concentration in a manner similar to that observed in larger animals; increasing palmitate concentration altered the balance of substrate metabolism to increase overall energy derived from fatty acids from 64 to 92%.

Thyroid status and diabetes modulate regional differences in potassium currents in rat ventricle.

1. The rate dependence and recovery kinetics of the Ca(2+)‐independent transient (I(t)) and steady‐state or ‘pedestal’ (Iss) outward potassium (K+) currents were studied in single myocytes isolated from epicardial and endocardial regions of rat left ventricles. The whole‐cell, suction microelectrode method was used to measure baseline (fully reactivated) I(t), as well as its rate‐dependent attenuation. Results from a group of control animals were compared with data from three other groups having an experimentally altered hormonal status. 2. I(t) was significantly smaller in endocardial cells than in epicardial cells, in part due to a very large difference in the recovery kinetics of this current in endocardial cells. This was reflected in a pronounced rate‐dependent prolongation of endocardial action potentials. In contrast, the non‐inactivating ‘pedestal’ current, Iss, was very similar in magnitude and showed comparable rate dependence in cells from both epicardium and endocardium. 3. Changing the thyroid status had selective, differential actions on the amplitude and rate dependence of It in epicardial and endocardial cells. Under hypothyroid conditions there was a more pronounced reduction of baseline I(t) in epicardial than in endocardial cells. Moreover, a slowing of the recovery kinetics in epicardial cells resulted in an enhanced attenuation of this current at high rates. Changing thyroid status had no effect on the magnitude or rate dependence of Iss in cells from either region of the left ventricle. 4. Following establishment of hyperthyroid conditions, there was no significant change in I(t) magnitude at baseline. However, when compared with control data, the recovery of I(t) was considerably faster in endocardial cells, and marginally faster in epicardial cells. 5. Streptozotocin‐induced diabetic conditions resulted in a much greater attenuation of I(t) in epicardial cells than in endocardial cells. Epicardial action potentials in these conditions showed prominent rate‐dependent prolongation. Iss was reduced to a similar extent in cells from these two regions. 6. Our findings demonstrate that altered hormonal status can selectively change the amplitude and kinetics of It in the epi‐ and endocardium of rat left ventricle. These changes can reduce the epicardial‐endocardial gradients in the magnitude and recovery kinetics of It and hence diminish the intrinsic differences in both action potential duration and refractoriness.

Type I and II models of diabetes produce different modifications of K+ currents in rat heart: role of insulin

1 Several K+ currents were measured and compared in enzymatically dispersed ventricular myocytes from control and diabetic rats. 2 Diabetic conditions were established either with a single intravenous injection of streptozotocin (STZ, 100 mg kg−1; 6‐14 days duration) or by feeding with a fructose‐enriched diet for 4‐10 weeks. Both groups became hyperglycaemic, with the former having decreased and the latter having elevated levels of plasma insulin. These conditions therefore mimic type I (insulin‐dependent) and type II (non‐insulin‐dependent) diabetes mellitus, respectively. 3 As reported previously, a Ca2+‐independent transient outward K+ current, It, was attenuated in the type I model. This was not observed in the type II model. The two models differed greatly in the changes observed in a quasi‐steady‐state K+ current denoted Iss. In the STZ model Iss was substantially attenuated, whereas in the fructose‐fed model it was augmented. In both models, the background inwardly rectifying current, IK1, was unchanged. Concomitantly, there was a substantial prolongation of the action potential in the STZ model but not in the fructose‐fed model. 4 Incubation of control myocytes with insulin (100 nM) for 5‐9 h caused a significant augmentation of Iss, with no effect on It or on IK1. Incubation of myocytes from STZ‐diabetic rats with insulin reversed the attenuation of It, but not of Iss. 5 The effect of insulin was not blocked by wortmannin, an inhibitor of phosphatidylinositol 3‐kinase. However, inhibition of the mitogen‐activated protein kinase pathway with PD98059 prevented restoration of It. Insulin action on It may therefore involve changes in transcription or expression of channel proteins, rather than changes in cellular metabolism.

Mechanisms responsible for enhanced fatty acid utilization by perfused hearts from type 2 diabetic db/db mice

Abstract The aim of this study was to determine the biochemical mechanism(s) responsible for enhanced FA utilization (oxidation and esterification) by perfused hearts from type 2 diabetic db/db mice. The plasma membrane content of fatty acid transporters FAT/CD36 and FABPpm was elevated in db/db hearts. Mitochondrial mechanisms that could contribute to elevated rates of FA oxidation were also examined. Carnitine palmitoyl transferase-1 activity was unchanged in mitochondria from db/db hearts, and sensitivity to inhibition by malonyl-CoA was unchanged. Malonyl-CoA content was elevated and AMP kinase activity was decreased in db/db hearts, opposite to what would be expected in hearts exhibiting elevated rates of FA oxidation. Uncoupling protein-3 expression was unchanged in mitochondria from db/db hearts. Therefore, enhanced FA utilization in db/db hearts is most likely due to increased FA uptake caused by increased plasma membrane content of FA transporters; the mitochondrial mechanisms examined do not contribute to elevated FA oxidation observed in db/db hearts.

The isolated working mouse heart: methodological considerations

Abstract Our aim was to develop a working isolated murine heart model, as the extensive use of genetically engineered mice in cardiovascular research requires development of new miniaturized technology. Left ventricular (LV) function was assessed in the isolated working mouse heart perfused with recirculated oxygenated Krebs-Henseleit bicarbonate buffer (37 °C pH 7.4) containing 11.1 mM glucose and 0.4 mM palmitate bound to 3% albumin. The hearts worked against an afterload reservoir at a height equivalent to 50 mmHg, and heart rate was controlled by electrical pacing of the right atrium. LV pressure was measured with a micromanometer connected to a small steel cannula inserted through the apex of the heart. The experimental protocol consisted of two interventions. First, following instrumentation and stabilization, the preload reservoir was raised from a pressure equivalent of 7 to 22.5 mmHg, while pacing at 390 beats·min–1. Thereafter the height of the preload reservoir was set to 10 mmHg, and the pacing rate was varied from 260 to 600 beats·min–1. Aortic and coronary flows were measured by timed collections of effluent from the afterload line and that dripping from the heart, respectively [aortic+coronary flow=cardiac output (CO)]. Elevation of LV end-diastolic pressure (LVEDP) from approximately 5 to 10 mmHg resulted in a twofold increase in average cardiac power [product of LV developed pressure (LVDevP) and CO], whereas myocardial contractility (first derivative of LV pressure, dP/dt) and LVDevP (LV systolic pressure–LVEDP) increased only minimally (5–10%). Measured LVEDP was lower than the equivalent height of the preload reservoir by an amount that was related to the heart rate. Cardiac power, LVDevP and dP/dt were stable at heart rates up to 400 beats·min–1, but declined markedly with higher rates, consistent with the decrease in LVEDP. Thus, cardiac power was reduced to 50% of its maximum value when stimulated at approximately 500 beats·min–1, and at even higher rates there was little ejection. By systematic manipulation of the height of the preload reservoir and heart rate, we conclude that LV afterload and preload can be assessed only by high-fidelity measurement of intraventricular pressures. The heights of the afterload column and the preload reservoir are unreliable and potentially misleading indicators of LV afterload and preload.

Insulin stimulation of rat ventricular K+ currents depends on the integrity of the cytoskeleton

1 The effect of insulin on K+ currents was studied with enzymatically dispersed ventricular myocytes from insulin‐deficient (type I) diabetic rats. Diabetic conditions were induced by a single intravenous injection of streptozotocin (100 mg kg−1) given 8‐13 days before the experiments. Measurements of plasma glucose and insulin levels confirmed the diabetic status of the animals. 2 A Ca2+‐independent transient outward K+ current, It, and a slowly inactivating, quasi‐steady‐state current, Iss, which are depressed in diabetic myocytes, could be restored by exposure to 1, 10 or 100 nM insulin. This was only observed after a delay of 5‐6 h, although an insulin exposure of only 1 h was sufficient to initiate its stimulatory action on It and Iss. The stimulatory effect of insulin on these K+ currents was prevented by 2 μM cycloheximide, which in itself had no direct effect on these currents. 3 Disruption of the actin microfilament network with 1 μM cytochalasin D (CD) also prevented the stimulatory effect of 100 nM insulin on both It and Iss. Since CD was added 1 h after insulin, inhibitory effects on insulin signalling were ruled out. Adding CD (1 μM) 5‐9 h after insulin, when currents were already augmented, had no effect (up to 50 min exposure). Incubating control cells for 6‐10 h with 1 μM CD had no effect on any of the currents measured. 4 Stabilization of the actin network by pre‐exposure to 2·5 μM phalloidin restored the stimulatory effect of insulin, in the continued presence of CD, ruling out any effects of CD on components other than the cytoskeleton. 5 The stimulatory effect of insulin was also prevented by incubating cells with insulin in the presence of the microtubule‐disrupting agent colchicine (5 μM). 6 These results suggest that the insulin‐mediated augmentation of K+ currents in diabetic myocytes requires protein synthesis, possibly of K+ channels, as well as an intact cytoskeleton. The possibility that newly formed channels translocate to the plasma membrane in a process dependent on different elements of the cytoskeleton is discussed.

Rosiglitazone treatment improves cardiac efficiency in hearts from diabetic mice

Abstract Isolated perfused hearts from type 2 diabetic (db/db) mice show impaired ventricular function, as well as altered cardiac metabolism. Assessment of the relationship between myocardial oxygen consumption (MVO2) and ventricular pressure-volume area (PVA) has also demonstrated reduced cardiac efficiency in db/db hearts. We hypothesized that lowering the plasma fatty acid supply and subsequent normalization of altered cardiac metabolism by chronic treatment with a peroxisome proliferator-activated receptor-γ (PPARγ) agonist will improve cardiac efficiency in db/db hearts. Rosiglitazone (23 mg/kg body weight/day) was administered as a food admixture to db/db mice for five weeks. Ventricular function and PVA were assessed using a miniaturized (1.4 Fr) pressure-volume catheter; MVO2 was measured using a fibre-optic oxygen sensor. Chronic rosiglitazone treatment of db/db mice normalized plasma glucose and lipid concentrations, restored rates of cardiac glucose and fatty acid oxidation, and improved cardiac efficiency. The improved cardiac efficiency was due to a significant decrease in unloaded MVO2, while contractile efficiency was unchanged. Rosiglitazone treatment also improved functional recovery after low-flow ischemia. In conclusion, the present study demonstrates that in vivo PPARγ-treatment restores cardiac efficiency and improves ventricular function in perfused hearts from type 2 diabetic mice.

Characteristics of nitric oxide-mediated cholinergic modulation of calcium current in rabbit sino-atrial node

1 We have previously shown that nitric oxide (NO) production is essential for cholinergic inhibition of the β‐adrenergic stimulated L‐type calcium current (ICa‐L) in rabbit pacemaker (sino‐atrial node (SAN)) cells. The present experiments demonstrate the presence of constitutive nitric oxide synthase (cNOS) in SAN cells, and characterize the NO‐mediated cholinergic response. 2 Immunohistochemical staining, using an antibody prepared against endothelial cNOS, demonstrated that this enzyme was present in single myocytes obtained from the SAN. 3 The activation of cNOS is known to be Ca2+ and calmodulin dependent. Strongly buffering intracellular Ca2+ with the membrane‐permeable chelator BAPTA‐AM (10 μM) significantly reduced (and in some cases abolished) the attenuation of ICa‐L by the muscarinic agonist carbamylcholine (CCh). In contrast, the CCh‐induced activation of an outward K+ current, IK,ACh, was unaffected by buffering of [Ca2+]i. The calmodulin inhibitor 48/80 (20 μM) also abolished the attenuation of ICa‐L by CCh, with no change in the activation of IK,ACh. 4 Neither thapsigargin nor ryanodine (5‐10 μM), agents which deplete intracellular Ca2+ stores, significantly changed the attenuation of ICa‐L by CCh. 5 Pertussis toxin (PTX) completely abolished both the inhibitory action of CCh on ICa‐L and the activation of IK,ACh. This establishes that a PTX‐sensitive GTP‐binding protein links the muscarinic receptor to NO synthase activation in SAN cells. 6 Our hypothesis is that NO leads to activation of a cyclic GMP (cGMP)‐activated phosphodiesterase (PDE II) as a mechanism for enhanced cyclic AMP breakdown and ICa‐L attenuation. This was supported by showing that a specific inhibitor of PDE II, erythro‐9‐(2‐hydroxy‐3‐nonyl) adenine (EHNA), blocks the effect of CCh on ICa‐L, but not on IK,ACh. Using reverse transcriptase‐polymerase chain reaction techniques, we have established that PDE II is the dominant cyclic nucleotide phosphodiesterase isoform in SAN cells.