Gender-specific effects of thyroid hormones on cardiopulmonary function in SHHF rats.

Spontaneously hypertensive heart failure (SHHF) rats develop hypertension and heart failure. We hypothesized that induction of hyperthyroidism should accelerate development of heart failure in male SHHF rats. Male and female SHHF rats received diets containing desiccated thyroid glands (DTG) or a control diet for 8 wk. Male and female Wistar-Kyoto rats were used as normotensive controls. DTG treatment reduced body weight in male, but not female, SHHF rats but increased body temperature and heart weight-to-body weight ratio in both genders. In DTG-treated male SHHF rats, serum triiodothyronine levels doubled relative to SHHF controls, whereas O2 consumption increased in DTG-treated SHHF rats. Frequency of breathing in air increased in DTG-treated female rats, and ventilation increased in DTG-treated male rats. Ventilatory equivalents exhibited gender differences in SHHF rats, were decreased in both genders by DTG treatment, and reached levels similar to those of Wistar-Kyoto rats. DTG increased heart rate, right ventricular pressure, and contractility in both genders and increased left ventricular pressure in SHHF male rats. These results refute our hypothesis and suggest that cardiopulmonary function of SHHF male rats may be improved by DTG treatment.     

Aging-associated changes in whole cell K(+) and L-type Ca(2+) currents in rat ventricular myocytes

The effect of aging on cardiac membrane currents remains unclear. This study examined the inward rectifier K(+) current (I(K1)), the transient outward K(+) current (I(to)), and the L-type Ca(2+) channel current (I(Ca,L)) in ventricular myocytes isolated from young adult (6 mo) and aged (>27 mo) Fischer 344 rats using whole cell patch-clamp techniques. Along with an increase in the cell size and membrane capacitance, aged myocytes had the same magnitude of peak I(K1) with a greater slope conductance but displayed smaller steady-state I(K1). Aged myocytes also had a greater I(to) with an increased rate of activation, but the I(to) inactivation kinetics, steady-state inactivation, and responsiveness to L-phenylephrine, an alpha(1)-adrenergic agonist, were unaltered. The magnitude of peak I(Ca,L) in aged myocytes was decreased and accompanied by a slower inactivation, but the I(Ca,L) steady-state inactivation was unaltered. Action potential duration in aged myocytes was prolonged only at 90% of full repolarization (APD(90)) when compared with the action potential duration of young adult myocytes. Aged myocytes from Long-Evans rats showed similar changes in I(to) and I(Ca,L) but an increased I(K1). These results demonstrate aging-associated changes in action potential, in morphology, and in I(K1), I(to), and I(Ca,L) of rat ventricular myocytes that possibly contribute to the decreased cardiac function of aged hearts.     

Selective contractile dysfunction of left,not right,ventricular myocardium in the SHHF rat

The progression of hypertension to cardiac failure involves systemic changes that may ultimately affect contractility throughout the heart. Spontaneous hypertensive heart failure (SHHF) rats have depressed left ventricular (LV) function, but right ventricular (RV) dysfunction is less well characterized. Ultrathin (87 +/- 5 mircom) trabeculae were isolated from end-stage failing SHHF rats and from age-matched controls. Under near-physiological conditions (1 mM Ca(2+), 37 degrees C, 4 Hz), developed force (in mN/mm(2)) was not significantly different in SHHF LV and RV trabeculae and those of controls. SHHF LV preparations displayed a negative force-frequency behavior (40 +/- 7 vs. 23 +/- 4 mN/mm(2), 2 vs. 7 Hz); this relationship was positive in SHHF RV preparations (27 +/- 5 vs. 40 +/- 6 mN/mm(2)) and controls (32 +/- 6 vs. 44 +/- 9 mN/mm(2)). The response to isoproterenol (10(-6) M, 4 Hz) was depressed in SHHF LV preparations. The inotropic response to hypothermia was lost in SHHF LV trabeculae but preserved in SHHF RV trabeculae. Intracellular calcium measurements revealed impaired calcium handling at higher frequencies in LV preparations. We conclude that in end-stage failing SHHF rats, RV function is only marginally affected, whereas a severe contractile dysfunction of LV myocardium is present.     

Gender modulates activation of renin-angiotensin and endothelin systems in hypertension and heart failure.

Sexual dimorphism may occur during the development of hypertension and congestive heart failure (CHF). Male and female spontaneous hypertension heart failure (SHHF) rats with established hypertension, but before CHF (age 5-8 mo) and during cardiac decompensation leading to CHF (age 18-20 mo in male rats and 22-24 mo in female rats), were studied. At 5-8 mo, male SHHF rats showed early activation of the renin-angiotensin system (RAS), as indicated by increased plasma renin activity (PRA) and higher serum angiotensin-converting enzyme activity compared with female rats. The increase in PRA in female rats was delayed compared with males rats, but it reached comparable levels just before CHF. Urinary endothelin excretion was significantly greater in 5- to 8-mo-old female rats compared with age-matched male rats. Urinary endothelin excretion increased in both male and female rats as CHF developed. Plasma atrial natriuretic peptide (ANP) was comparable at both time points, and both genders showed similar, marked increases as CHF developed. In conclusion, male rats show early activation of the RAS, whereas female rats show early activation of the endothelin vasopressor system. During cardiac decompensation, generalized activation of the RAS, endothelin, and ANP systems occurs and is similar in male and female SHHF rats.     

Thyroid hormones induce unique and potentially beneficial changes in cardiac myocyte shape in hypertensive rats near heart failure

We examined the effects of thyroid hormones (THs) on left ventricular (LV) function and myocyte remodeling in rats with spontaneously hypertensive heart failure (SHHF). SHHF rats were treated with three different TH doses from 20-21 mo of age. In terminal experiments, LV function (as determined by echocardiography and catheterization) and isolated myocyte shape were examined in SHHF rat groups and age-matched Wistar-Furth control animals. Compared with Wistar-Furth rats, the ratio of alpha- to beta-myosin was reduced in untreated SHHF rats. The alpha-to-beta-myosin ratio increased in all TH groups, which suggests a reversal of the fetal gene program. Low-dose TH produced no changes in LV myocyte size or function, but high-dose TH produced signs of hyperthyroidism (e.g., increased heart weight, tachycardia). The chamber diameter-to-wall thickness ratio declined with increasing dose due to reduced chamber diameter and increased wall thickness. This resulted in a 38% reduction in LV systolic wall stress in the middle- and high-dose groups despite sustained hypertension. Isolated myocyte data indicated that chamber remodeling and reduced wall stress were due to a unique alteration in myocyte transverse shape (e.g., reduced major diameter and increased minor diameter). Based on our present understanding of ventricular remodeling and wall stress, we believe these changes are likely beneficial. Results suggest that TH may be an important regulator of myocyte transverse shape in heart disease.     

Temperature-dependent expression of sarcolemmal K(+) currents in rainbow trout atrial and ventricular myocytes

Temperature has a strong influence on the excitability and the contractility of the ectothermic heart that can be alleviated in some species by temperature acclimation. The molecular mechanisms involved in the temperature-induced improvement of cardiac contractility and excitability are, however, still poorly known. The present study examines the role of sarcolemmal K(+) currents from rainbow trout (Oncorhynchus mykiss) cardiac myocytes after thermal acclimation. The two major K(+) conductances of the rainbow trout cardiac myocytes were identified as the Ba(2+)-sensitive background inward rectifier current (I(K1)) and the E-4031-sensitive delayed rectifier current (I(Kr)). In atrial cells, the density of I(K1) is very low and the density of I(Kr) is remarkably high. The opposite is true for ventricular cells. Acclimation to cold (4 degrees C) modified the two K(+) currents in opposite ways. Acclimation to cold increases the density of I(Kr) and depresses the density of I(K1). These changes in repolarizing K(+) currents alter the shape of the action potential, which is much shorter in cold-acclimated than warm-acclimated (17 degrees C) trout. These results provide the first concrete evidence that K(+) channels of trout cardiac myocytes are adaptable units that provide means to regulate cardiac excitability and contractility as a function of temperature.     

Heterogeneity of IK1 in the mouse heart

Previous studies have shown that cardiac inward rectifier potassium current (I(K1)) channels are heteromers of distinct Kir2 subunits and suggested that species- and tissue-dependent expression of these subunits may underlie variability of I(K1). In this study, we investigated the contribution of the slowly activating Kir2.3 subunit and free intracellular polyamines (PAs) to variability of I(K1) in the mouse heart. The kinetics of activation was measured in Kir2 concatemeric tetramers with known subunit stoichiometry. Inclusion of only one Kir2.3 subunit to a Kir2.1 channel led to an approximate threefold slowing of activation kinetics, with greater slowing on subsequent additions of Kir2.3 subunits. Activation kinetics of I(K1) in both ventricles and both atria was found to correspond to fast-activating Kir2.1/Kir2.2 channels, suggesting no major contribution of Kir2.3 subunits. In contrast, I(K1) displayed significant variation in both the current density and inward rectification, suggesting involvement of intracellular PAs. The total levels of PAs were similar across the mouse heart. Measurements of the free intracellular PAs in isolated myocytes, using transgenically expressed Kir2.1 channels as PA sensors, revealed "microheterogeneity" of I(K1) rectification as well as lower levels of free PAs in atrial myocytes compared with ventricular cells. These findings provide a quantitative explanation for the regional heterogeneity of I(K1).     

Effects of anandamide on potassium channels in rat ventricular myocytes: a suppression of I(to) and augmentation of K(ATP) channels

Anandamide is an endocannabinoid that has antiarrhythmic effects through inhibition of L-type Ca(2+) channels in cardiomyocytes. In this study, we investigated the electrophysiological effects of anandamide on K(+) channels in rat ventricular myocytes. Whole cell patch-clamp technique was used to record K(+) currents, including transient outward potassium current (I(to)), steady-state outward potassium current (I(ss)), inward rectifier potassium current (I(K1)), and ATP-sensitive potassium current (I(KATP)) in isolated rat cardiac ventricular myocytes. Anandamide decreased I(to) while increasing I(KATP) in a concentration-dependent manner but had no effect on I(ss) and I(K1) in isolated ventricular myocytes. Furthermore, anandamide shifted steady-state inactivation curve of I(to) to the left and shifted the recovery curve of I(to) to the right. However, neither cannabinoid 1 (CB(1)) receptor antagonist AM251 nor CB(2) receptor antagonist AM630 eliminated the inhibitory effect of anandamide on I(to). In addition, blockade of CB(2) receptors, but not CB(1) receptors, eliminated the augmentation effect of anandamide on I(KATP). These data suggest that anandamide suppresses I(to) through a non-CB(1) and non-CB(2) receptor-mediated pathway while augmenting I(KATP) through CB(2) receptors in ventricular myocytes.     

Protein kinase C isozymes in hypertension and hypertrophy: Insight from SHHF rat hearts

Chronic hypertension results in cardiac hypertrophy and may lead to congestive heart failure. The protein kinase C (PKC) family has been identified as a signaling component promoting cardiac hypertrophy. We hypothesized that PKC activation may play a role mediating hypertrophy in the spontaneously hypertensive heart failure (SHHF) rat heart. Six-month-old SHHF and normotensive control Wistar Furth (WF) rats were used. Hypertension and cardiac hypertrophy were confirmed in SHHF rats. PKC expression and activation were analyzed by Western blots using isozyme-specific antibodies. Compared to WF, untreated SHHF rats had increased phospho-active (10-fold), (4-fold), and (3-fold) isozyme expression. Furthermore, we analyzed the effect of an angiotensin II type 1 receptor blocker (ARB) and hydralazine (Hy) on PKC regulation in SHHF rat left ventricle (LV). Both the ARB and Hy normalized LV blood pressure, but only the ARB reduced heart mass. Neither treatment affected PKC expression or activity. Our data show differential activation of PKC in the hypertensive, hypertrophic SHHF rat heart. Regression of hypertrophy elicited by an ARB in this model occurred independently of changes in the expression and activity of the PKC isoforms examined. (Mol Cell Biochem 270: 63–69, 2005)     

Postnatal development has a marked effect on ventricular repolarization in mice

To better understand the mechanisms that underlie cardiac repolarization abnormalities in the immature heart, this study characterized and compared K(+) currents in mouse ventricular myocytes from day 1, day 7, day 20, and adult CD1 mice to determine the effects of postnatal development on ventricular repolarization. Current- and patch-clamp techniques were used to examine action potentials and the K(+) currents underlying repolarization in isolated myocytes. RT-PCR was used to quantify mRNA expression for the K(+) channels of interest. This study found that action potential duration (APD) decreased as age increased, with the shortest APDs observed in adult myocytes. This study also showed that K(+) currents and the mRNA relative abundance for the various K(+) channels were significantly greater in adult myocytes compared with day 1 myocytes. Examination of the individual components of total K(+) current revealed that the inward rectifier K(+) current (I(K1)) developed by day 7, both the Ca(2+)-independent transient outward current (I(to)) and the steady-state outward K(+) current (I(ss)) developed by day 20, and the ultrarapid delayed rectifier K(+) current (I(Kur)) did not fully develop until the mouse reached maturity. Interestingly, the increase in I(Kur) was not associated with a decrease in APD. Comparison of atrial and ventricular K(+) currents showed that I(to) and I(Kur) density were significantly greater in day 7, day 20, and adult myocytes compared with age-matched atrial cells. Overall, it appears that, in mouse ventricle, developmental changes in APD are likely attributable to increases in I(to), I(ss), and I(K1), whereas the role of I(Kur) during postnatal development appears to be less critical to APD.     

Expression and redistribution of β-catenin in the cardiac myocytes of left ventricle of spontaneously hypertensive rat

Beta-catenin is not only an adhering junction protein, but also the central player of the canonical Wnt signalling pathway. In order to investigate the roles of β-catenin in the mechanism of myocardial hypertrophy, we determined the expression and distribution of β-catenin in the cardiomyocytes of spontaneously hypertensive heart failure (SHHF) rats and age-matched Wistar-Kyoto (WKY) rats. We identified the reducing of β-catenin expression in the membrane protein fraction but increasing in the nuclear protein in the 6 and 12 month-old SHHF rats as compared with the age-matched WKY rats by Western blotting. Immunolabeling of β-catenin colocalized with cadherin at the intercalated disc sites and showed nuclear accumulation in myocytes of SHHF rats. We also revealed that the association between glycogen synthase kinase-3β and β-catenin had weakened in the 6 month-old SHHF rats as compared with the age-matched WKY rats by immunoprecipitation. These findings suggested that nuclear translocation of β-catenin might play important roles in regulating signal transduction in the decompensated hypertrophy stage.     

Characterization and functional consequences of delayed rectifier current transient in ventricular repolarization

Although inactivation of the rapidly activating delayed rectifier current (I(Kr)) limits outward current on depolarization, the role of I(Kr) (and recovery from inactivation) during repolarization is uncertain. To characterize I(Kr) during ventricular repolarization (and compare with the inward rectifier current, I(K1)), voltage-clamp waveforms simulating the action potential were applied to canine ventricular, atrial, and Purkinje myocytes. In ventricular myocytes, I(Kr) was minimal at plateau potentials but transiently increased during repolarizing ramps. The I(Kr) transient was unaffected by repolarization rate and maximal after 150-ms depolarizations (+25 mV). Action potential clamps revealed the I(Kr) transient terminating the plateau. Although peak I(Kr) transient density was relatively uniform among myocytes, potentials characterizing the peak transients were widely dispersed. In contrast, peak inward rectifier current (I(K1)) density during repolarization was dispersed, whereas potentials characterizing I(K1) defined a narrower (more negative) voltage range. In summary, rapidly activating I(Kr) provides a delayed voltage-dependent (and functionally time-independent) outward transient during ventricular repolarization, consistent with rapid recovery from inactivation. The heterogeneous voltage dependence of I(Kr) provides a novel means for modulating the contribution of this current during repolarization.     

Developmental changes in functional expression and beta-adrenergic regulation of I(f) in the heart of mouse embryo

The hyperpolarization-activated current (I(f)) plays an important role in determining the spontaneous rate of cardiac pacemaker cells. The automatic rhythmicity also exists in working cells of embryonic heart, therefore we studied developmental changes in functional expression and beta-adrenergic regulation of I(f) in embryonic mouse heart. The expression of I(f) is high in early developmental stage (EDS) (10.5 d after coitus) ventricular myocytes, low in intermediate developmental stage (IDS) (13.5 d) atrial or ventricular myocytes and even lower in late developmental stage (LDS) (16.5 d) atrial or ventricular myocytes, indicating that these cells of the EDS embryonic heart have some properties of pacemaker cells. Beta-adrenergic agonist isoproterenol (ISO) stimulates I(f) in LDS but not in EDS cardiomyocytes, indicating that the beta-adrenergic regulation of I(f) is not mature in EDS embryonic heart. But forskolin (a direct activator of adenylate cyclase) and 8-Br-cAMP (a membrane-permeable analogue of cAMP) increase the amplitude of I(f) in EDS cells, indicating that adenylate cyclase and cAMP function fairly well at early stage of development. Furthermore, the results demonstrate that I(f) is modulated by phosphorylation via cAMP dependent PKA both in EDS and LDS cells.     

Heterogeneous ventricular chamber response to hypokalemia and inward rectifier potassium channel blockade underlies bifurcated T wave in guinea pig

It was previously demonstrated that transmural electrophysiological heterogeneities can inscribe the ECG T wave. However, the bifurcated T wave caused by loss of inward rectifier potassium current (I(K1)) function is not fully explained by transmural heterogeneities. Since right ventricular (RV) guinea pig myocytes have significantly lower I(K1) than left ventricular (LV) myocytes, we hypothesized that the complex ECG can be inscribed by heterogeneous chamber-specific responses to hypokalemia and partial I(K1) blockade. Ratiometric optical action potentials were recorded from the epicardial surface of the RV and LV. BaCl(2) (10 micromol/l) was perfused to partially block I(K1) in isolated guinea pig whole heart preparations. BaCl(2) or hypokalemia alone significantly increased RV basal (RV(B)) action potential duration (APD) by approximately 30% above control compared with LV apical (LV(A)) APD (14%, P<0.05). In the presence of BaCl(2), 2 mmol/l extracellular potassium (hypokalemia) further increased RV(B) APD to a greater extent (31%) than LV(A) APD (19%, P<0.05) compared with BaCl(2) perfusion alone. Maximal dispersion between RV(B) and LV(A) APD increased by 105% (P<0.05), and the QT interval prolonged by 55% (P<0.05) during hypokalemia and BaCl(2). Hypokalemia and BaCl(2) produced an ECG with a double repolarization wave. The first wave (QT1) corresponded to selective depression of apical LV plateau potentials, while the second wave (QT2) corresponded to the latest repolarizing RV(B) myocytes. These data suggest that final repolarization is more sensitive to extracellular potassium changes in regions with reduced I(K1), particularly when I(K1) availability is reduced. Furthermore, underlying I(K1) heterogeneities can potentially contribute to the complex ECG during I(K1) loss of function and hypokalemia.     

Mechanisms of impaired calcium handling underlying subclinical diastolic dysfunction in diabetes

Isolated diastolic dysfunction is found in almost half of asymptomatic patients with well-controlled diabetes and may precede diastolic heart failure. However, mechanisms that underlie diastolic dysfunction during diabetes are not well understood. We tested the hypothesis that isolated diastolic dysfunction is associated with impaired myocardial Ca(2+) handling during type 1 diabetes. Streptozotocin-induced diabetic rats were compared with age-matched placebo-treated rats. Global left ventricular myocardial performance and systolic function were preserved in diabetic animals. Diabetes-induced diastolic dysfunction was evident on Doppler flow imaging, based on the altered patterns of mitral inflow and pulmonary venous flows. In isolated ventricular myocytes, diabetes resulted in significant prolongation of action potential duration compared with controls, with afterdepolarizations occurring in diabetic myocytes (P < 0.05). Sustained outward K(+) current and peak outward component of the inward rectifier were reduced in diabetic myocytes, while transient outward current was increased. There was no significant change in L-type Ca(2+) current; however, Ca(2+) transient amplitude was reduced and transient decay was prolonged by 38% in diabetic compared with control myocytes (P < 0.05). Sarcoplasmic reticulum Ca(2+) load (estimated by measuring the integral of caffeine-evoked Na(+)-Ca(2+) exchanger current and Ca(2+) transient amplitudes) was reduced by approximately 50% in diabetic myocytes (P < 0.05). In permeabilized myocytes, Ca(2+) spark amplitude and frequency were reduced by 34 and 20%, respectively, in diabetic compared with control myocytes (P < 0.05). Sarco(endo)plasmic reticulum Ca(2+)-ATPase-2a protein levels were decreased during diabetes. These data suggest that in vitro impairment of Ca(2+) reuptake during myocyte relaxation contributes to in vivo diastolic dysfunction, with preserved global systolic function, during diabetes.     

Repolarization abnormalities and afterdepolarizations in a canine model of sudden cardiac death

Ventricular tachyarrhythmias are the most common cause of sudden cardiac death (SCD); a healed myocardial infarction increases the risk of SCD. We determined the contribution of specific repolarization abnormalities to ventricular tachyarrhythmias in a postinfarction model of SCD. For our methods, we used a postinfarction canine model of SCD, where an exercise and ischemia test was used to stratify animals as either susceptible (VF(+)) or resistant (VF(-)) to sustained ventricular tachyarrhythmias. Our results show no changes in global left ventricular contractility or volumes occurred after infarction. At 8-10 wk postmyocardial infarction, myocytes were isolated from the left ventricular midmyocardial wall and studied. In the VF(+) animals, myocyte action potential (AP) prolongation occurred at 50 and 90% repolarization (P < 0.05) and was associated with increased variability of AP duration and afterdepolarizations. Multiple repolarizing K(+) currents (I(Kr), I(to)) and inward I(K1) were also reduced (P < 0.05) in myocytes from VF(+) animals compared with control, noninfarcted dogs. In contrast, only I(to) was reduced in VF(-) myocytes compared with controls (P < 0.05). While afterdepolarizations were not elicited at baseline in myocytes from VF(-) animals, afterdepolarizations were consistently elicited after the addition of an I(Kr) blocker. In conclusion, the loss of repolarization reserve via reductions in multiple repolarizing currents in the VF(+) myocytes leads to AP prolongation, repolarization instability, and afterdepolarizations in myocytes from animals susceptible to SCD. These abnormalities may provide a substrate for initiation of postmyocardial infarction ventricular tachyarrhythmias.     

DITPA restores the repolarizing potassium currents Itof and Iss in cardiac ventricular myocytes of diabetic rats

Diabetes Mellitus (DM) can produce an increase in the cardiac action potential duration and QT interval that can be associated with sudden death. These cardiac effects are due to a region-specific decrease in repolarizing outward K(+) currents. Some authors have suggested that the proarrhythmic effects of diabetes can be due to diabetes-induced hypothyroidism. Thus, we have examined the effect of the thyroid hormone analog diiodothyropropionic acid (DITPA) on calcium-independent outward potassium currents in ventricular myocytes from diabetic rats. Sustained (I(ss)) and fast transient outward (I(tof)) K(+) currents were recorded using the whole-cell configuration of the patch-clamp technique. Myocytes were enzymatically isolated from the free wall of the right ventricle, and the epicardial and endocardial layers of the left ventricle of healthy, diabetic and DITPA-treated diabetic rats. Circulating thyroid hormones were measured by electrochemiluminescence. DITPA-treatment of diabetic rats restored I(tof) and I(ss) current densities in cardiac myocytes from the three regions studied, but did not alter current densities in myocytes of control rats. T(3) and T(4) levels were reduced by diabetes, and DITPA-treatment increased circulating T(3) levels. T(3)-treatment of diabetic rats also restored current densities to control values. However, direct incubation of diabetic myocytes with DITPA did not restore current densities. In summary, DITPA-treatment of diabetic rats restored the potassium current (I(tof) and I(ss)) densities in myocytes from all ventricular regions.     

Biological pacemakers

Genetically engineered pacemakers could be a possible alternative to implantable electronic devices for the treatment of bradyarrhythmias. The strategies include upregulation of beta adrenergic receptors, conversion of myocytes into pacemaker cells and stem cell therapy. Pacemaker activity in adult ventricular myocytes is normally repressed by the inward rectifier potassium current (I(K1)). The I(K1) current is encoded by the Kir2 gene family. Use of a negative construct that suppresses current when expressed with wild-type Kir2.1 is an experimental approach for genesis of genetic pacemaker. Hyperpolarisation activated cyclic nucleotide gated (HCN) channels which generate If current, the pacemaker current of heart can be delivered to heart by using stem cell therapy approach and viral vectors. The unresolved issues include longevity and stability of pacemaker genes, limitations involved in adenoviral and stem cell therapy and creation of genetic pacemakers which can compete with the electronic units.