Mechanisms underlying rate-dependent remodeling of transient outward potassium current in canine ventricular myocytes.

Transient outward K+ current (I to) downregulation following sustained tachycardia in vivo is usually attributed to tachycardiomyopathy. This study assessed potential direct rate regulation of cardiac I(to) and underlying mechanisms. Cultured adult canine left ventricular cardiomyocytes (37 degrees C) were paced continuously at 1 or 3 Hz for 24 hours. I to was recorded with whole-cell patch clamp. The 3-Hz pacing reduced I to by 44% (P<0.01). Kv4.3 mRNA and protein expression were significantly reduced (by approximately 30% and approximately 40%, respectively) in 3-Hz paced cells relative to 1-Hz cells, but KChIP2 expression was unchanged. Prevention of Ca2+ loading with nimodipine or calmodulin inhibition with W-7, A-7, or W-13 eliminated 3-Hz pacing-induced I to downregulation, whereas downregulation was preserved in the presence of valsartan. Inhibition of Ca2+/calmodulin-dependent protein kinase (CaMK)II with KN93, or calcineurin with cyclosporin A, also prevented I to downregulation. CaMKII-mediated phospholamban phosphorylation at threonine 17 was increased in 3-Hz paced cells, compatible with enhanced CaMKII activity, with functional significance suggested by acceleration of the Ca2+i transient decay time constant (Indo 1-acetoxymethyl ester microfluorescence). Total phospholamban expression was unchanged, as was expression of Na+/Ca2+ exchange and sarcoplasmic reticulum Ca2+-ATPase proteins. Nuclear localization of the calcineurin-regulated nuclear factor of activated T cells (NFAT)c3 was increased in 3-Hz paced cells compared to 1-Hz (immunohistochemistry, immunoblot). INCA-6 inhibition of NFAT prevented I to reduction in 3-Hz paced cells. Calcineurin activity increased after 6 hours of 3-Hz pacing. CaMKII inhibition prevented calcineurin activation and NFATc3 nuclear translocation with 3-Hz pacing. We conclude that tachycardia downregulates I to expression, with the Ca2+/calmodulin-dependent CaMKII and calcineurin/NFAT systems playing key Ca2+-sensing and signal-transducing roles in rate-dependent I to control.

Cardiac-directed parvalbumin transgene expression in mice shows marked heart rate dependence of delayed Ca2+ buffering action.

Relaxation abnormalities are prevalent in heart failure and contribute to clinical outcomes. Disruption of Ca2+ homeostasis in heart failure delays relaxation by prolonging the intracellular Ca2+ transient. We sought to speed cardiac relaxation in vivo by cardiac-directed transgene expression of parvalbumin (Parv), a cytosolic Ca2+ buffer normally expressed in fast-twitch skeletal muscle. A key feature of Parv's function resides in its Ca2+/Mg2+ binding affinities that account for delayed Ca2+ buffering in response to the intracellular Ca2+ transient. Cardiac Parv expression decreased sarcoplasmic reticulum Ca2+ content without otherwise altering intracellular Ca2+ homeostasis. At high physiological mouse heart rates in vivo, Parv modestly accelerated relaxation without affecting cardiac morphology or systolic function. Ex vivo pacing of the isolated heart revealed a marked heart rate dependence of Parv's delayed Ca2+ buffering effects on myocardial performance. As the pacing frequency was lowered (7 to 2.5 Hz), the relaxation rates increased in Parv hearts. However, as pacing rates approached the dynamic range in humans, Parv hearts demonstrated decreased contractility, consistent with Parv buffering systolic Ca2+. Mathematical modeling and in vitro studies provide the underlying mechanism responsible for the frequency-dependent fractional Ca2+ buffering action of Parv. Future studies directed toward refining the dose and frequency-response relationships of Parv in the heart or engineering novel Parv-based Ca2+ buffers with modified Mg2+ and Ca2+ affinities to limit systolic Ca2+ buffering may hold promise for the development of new therapies to remediate relaxation abnormalities in heart failure.

Calcium-handling abnormalities underlying atrial arrhythmogenesis and contractile dysfunction in dogs with congestive heart failure.

BACKGROUND: Congestive heart failure (CHF) is a common cause of atrial fibrillation. Focal sources of unknown mechanism have been described in CHF-related atrial fibrillation. The authors hypothesized that abnormal calcium (Ca(2+)) handling contributes to the CHF-related atrial arrhythmogenic substrate. METHODS AND RESULTS: CHF was induced in dogs by ventricular tachypacing (240 bpm x2 weeks). Cellular Ca(2+)-handling properties and expression/phosphorylation status of key Ca(2+) handling and myofilament proteins were assessed in control and CHF atria. CHF decreased cell shortening but increased left atrial diastolic intracellular Ca(2+) concentration ([Ca(2+)](i)), [Ca(2+)](i) transient amplitude, and sarcoplasmic reticulum (SR) Ca(2+) load (caffeine-induced [Ca(2+)](i) release). SR Ca(2+) overload was associated with spontaneous Ca(2+) transient events and triggered ectopic activity, which was suppressed by the inhibition of SR Ca(2+) release (ryanodine) or Na(+)/Ca(2+) exchange. Mechanisms underlying abnormal SR Ca(2+) handling were then studied. CHF increased atrial action potential duration and action potential voltage clamp showed that CHF-like action potentials enhance Ca(2+)(i) loading. CHF increased calmodulin-dependent protein kinase II phosphorylation of phospholamban by 120%, potentially enhancing SR Ca(2+) uptake by reducing phospholamban inhibition of SR Ca(2+) ATPase, but it did not affect phosphorylation of SR Ca(2+)-release channels (RyR2). Total RyR2 and calsequestrin (main SR Ca(2+)-binding protein) expression were significantly reduced, by 65% and 15%, potentially contributing to SR dysfunction. CHF decreased expression of total and protein kinase A-phosphorylated myosin-binding protein C (a key contractile filament regulator) by 27% and 74%, potentially accounting for decreased contractility despite increased Ca(2+) transients. Complex phosphorylation changes were explained by enhanced calmodulin-dependent protein kinase IIdelta expression and function and type-1 protein-phosphatase activity but downregulated regulatory protein kinase A subunits. CONCLUSIONS: CHF causes profound changes in Ca(2+)-handling and -regulatory proteins that produce atrial fibrillation-promoting atrial cardiomyocyte Ca(2+)-handling abnormalities, arrhythmogenic triggered activity, and contractile dysfunction.

Cardiac transgenic and gene transfer strategies converge to support an important role for troponin I in regulating relaxation in cardiac myocytes.

Elucidating the relative roles of cardiac troponin I (cTnI) and phospholamban (PLN) in beta-adrenergic-mediated hastening of cardiac relaxation has been challenging and controversial. To test the hypothesis that beta-adrenergic phosphorylation of cTnI has a prominent role in accelerating cardiac myocyte relaxation performance we used transgenic (Tg) mice bearing near complete replacement of native cTnI with a beta-adrenergic phospho-mimetic of cTnI whereby tandem serine codons 23/24 were converted to aspartic acids (cTnI S23/24D). Adult cardiac myocytes were isolated and contractility determined at physiological temperature under unloaded and loaded conditions using micro-carbon fibers. At baseline, cTnI S23/24D myocytes had significantly faster relaxation times relative to controls, and isoproterenol stimulation (Iso) had only a small effect to further speed relaxation in cTnI S23/24D myocytes (delta Iso: 7.2 ms) relative to the maximum Iso effect (31.2 ms) in control. The Ca(2+) transient decay rate was similarly accelerated by Iso in Tg and nontransgenic (Ntg) myocytes. Gene transfer of cTnI S23/24D to myocytes in primary culture showed comparable findings. Gene transfer of cTnI with both serines 23/24 converted to alanines (cTnI S23/24A), or gene transfer of slow skeletal TnI, both of which lack PKA phosphorylation sites, significantly blunted Iso-mediated enhanced relaxation compared with controls. Gene transfer of wild-type cTnI had no effect on relaxation. These findings support a key role of cTnI in myocyte relaxation and highlight a direct contribution of the myofilaments in modulating the dynamics of myocardial performance.

Targeted ablation of ILK from the murine heart results in dilated cardiomyopathy and spontaneous heart failure.

A requirement for integrin-mediated adhesion in cardiac physiology is revealed through targeted deletion of integrin-associated genes in the murine heart. Here we show that targeted ablation of the integrin-linked kinase (ILK) expression results in spontaneous cardiomyopathy and heart failure by 6 wk of age. Deletion of ILK results in disaggregation of cardiomyocytes, associated with disruption of adhesion signaling through the beta1-integrin/FAK (focal adhesion kinase) complex. Importantly, the loss of ILK is accompanied by a reduction in cardiac Akt phosphorylation, which normally provides a protective response against stress. Together, these results suggest that ILK plays a central role in protecting the mammalian heart against cardiomyopathy and failure.

Properties of a time-dependent potassium current in pig atrium: evidence for a role of kv1.5 in repolarization.

Cardiac electrical activity is modulated by potassium currents. Pigs have been used for antiarrhythmic drug testing, but only sparse data exist regarding porcine atrial ionic electrophysiology. Here, we used electrophysiological, molecular, and pharmacological tools to characterize a prominent porcine outward K(+) current (I(K,PO)) in atrial cardiomyocytes isolated from adult pigs. I(K,PO) activated rapidly (time to peak at +60 mV; 2.1 +/- 0.2 ms), inactivated slowly (tau(f) = 45 +/- 10; tau(s) = 215 +/- 28 ms), and showed very slow recovery (tau(f) = 1.54 +/- 0.73 s; tau(s) = 7.91 +/- 1.78 s; n = 9; 36 degrees C). Activation and inactivation were voltage-dependent, and current properties were consistent with predominant K(+) conductance. Neurotoxins (heteropodatoxin, hongatoxin, and blood depressing substance) that block K(v)4.x, K(v)1.1, -1.2, -1.3, and -3.4 in a highly selective manner as well as H(2)O(2) and tetraethylammonium, did not affect the current. Drugs with K(v)1.5-blocking properties (flecainide, perhexiline, and the novel atrial-selective antiarrhythmic 2'-{2-(4-methoxyphenyl)-acetylamino-methyl}-biphenyl-2-carboxylic acid (2-pyridin-3-yl-ethyl)-amide; AVE0118) inhibited I(K,PO) (IC(50) of 132 +/- 47, 17 +/- 10, and 1.25 +/- 0.62 microM, respectively). 4-Aminopyridine suppressed the current and accelerated its decay, reducing charge carriage with an IC(50) of 39 +/- 15 microM. Porcine-specific K(v) channel subunit sequences were cloned to permit real-time quantitative reverse transcription-polymerase chain reaction on RNA extracted from isolated cardiomyocytes, which showed much greater abundance of K(v)1.5 mRNA compared with K(v)1.4, K(v)4.2, and K(v)4.3. Action potential recordings showed that I(K,PO) inhibition with 0.1 mM 4-AP delayed repolarization (e.g., action potential duration at -50 mV increased from 45 +/- 9 to 69 +/- 5 ms at 3 Hz; P < 0.05). In conclusion, porcine atrium displays a current that is involved in repolarization, inactivates more slowly than classic transient outward current, is associated with strong K(v)1.5 expression, and shows a pharmacological profile typical of K(v)1.5-dependent currents.

Comparison of Ca2+-handling properties of canine pulmonary vein and left atrial cardiomyocytes.

Cardiac tissue in the pulmonary vein sleeves plays an important role in clinical atrial fibrillation. Mechanisms leading to pulmonary vein activity in atrial fibrillation remain unclear. Indirect experimental evidence points to pulmonary vein Ca(2+) handling as a potential culprit, but there are no direct studies of pulmonary vein cardiomyocyte Ca(2+) handling in the literature. We used the Ca(2+)-sensitive dye indo-1 AM to study Ca(2+) handling in isolated canine pulmonary vein and left atrial myocytes. Results were obtained at 35 degrees C and room temperature in cells from control dogs and in cardiomyocytes from dogs subjected to 7-day rapid atrial pacing. We found that basic Ca(2+)-transient properties (amplitude: 186 +/- 28 vs. 216 +/- 25 nM; stimulus to half-decay time: 192 +/- 9 vs. 192 +/- 9 ms; atria vs. pulmonary vein, respectively, at 1 Hz), beat-to-beat regularity, propensity to alternans, beta-adrenergic response (amplitude increase at 0.4 Hz: 96 +/- 52 vs. 129 +/- 61%), number of spontaneous Ca(2+)-transient events after Ca(2+) loading (in normal Tyrode: 0.9 +/- 0.2 vs. 1.3 +/- 0.2; with 1 microM isoproterenol: 7.6 +/- 0.3 vs. 5.1 +/- 1.8 events/min), and caffeine-induced Ca(2+)-transient amplitudes were not significantly different between atrial and pulmonary vein cardiomyocytes. In an arrhythmia-promoting model (dogs subjected to 7-day atrial tachypacing), Ca(2+)-transient amplitude and kinetics were the same in cells from both pulmonary veins and atrium. In conclusion, the similar Ca(2+)-handling properties of canine pulmonary vein and left atrial cardiomyocytes that we observed do not support the hypothesis that intrinsic Ca(2+)-handling differences account for the role of pulmonary veins in atrial fibrillation.

Atrial fibrillation-associated minK38G/S polymorphism modulates delayed rectifier current and membrane localization.

BACKGROUND: Atrial fibrillation (AF) is a common acquired arrhythmia with multi-factorial pathogenesis. Recently, a single nucleotide polymorphism (SNP, A/G) at position 112 in the KCNE1 gene, resulting in a glycine/serine amino acid substitution at position 38 of the minK peptide, was associated with AF occurrence (AF more frequent with minK38G); however, the functional effect of this SNP is unknown. METHODS AND RESULTS: We used patch clamp recording, confocal microscopy and protein biochemistry to study the effect of this SNP on delayed-rectifier current expression and mathematical simulation to identify potential functional consequences. The density of slow delayed rectifier current (I(Ks)) resulting from co-expression with KvLQT1 was smaller with minK38G (e.g. at +10 mV: 50+/-7 pA/pF in Chinese hamster ovary (CHO) cells, 45+/-14 pA/pF for COS-7 cells) compared to minK38S (93+/-17 pA/pF, 104+/-23 pA/pF, respectively, P<0.05 for each). I(Ks) kinetics and voltage-dependence were unaffected. Currents resulting from co-expression of human ether-a-go-go-related gene (HERG) were similar for minK38G and minK38S, e.g. upon repolarization from +10 to -50 mV: tail currents 23+/-4 pA/pF versus 22+/-5 pA/pF (P=ns). KvLQT1 membrane immunofluorescence was less in CHO cells co-expressing minK38G versus minK38S, and surface expression of KvLQT1, as determined by labelling with streptavidin/biotin, was increased with minK38S co-expression. Computer simulations with a human atrial action potential model predicted that the minK38G SNP would slightly prolong the atrial action potential and reduce the frequency for alternans behaviour. In the presence of reduced repolarization reserve, these effects were enhanced and under specific conditions early afterdepolarizations occurred. CONCLUSIONS: The minK38G isoform is associated with reduced I(Ks), likely due to decreased KvLQT1 membrane expression. This study reveals a novel amino acid determinant of the minK-KvLQT1 interaction, and if the role of minK38G in AF is confirmed, would suggest mechanistic heterogeneity in genetic determinants of AF.

Genetic manipulation of calcium-handling proteins in cardiac myocytes. II. Mathematical modeling studies.

We developed a mathematical model specific to rat ventricular myocytes that includes electrophysiological representation, ionic homeostasis, force production, and sarcomere movement. We used this model to interpret, analyze, and compare two genetic manipulations that have been shown to increase myocyte relaxation rates, parvalbumin (Parv) de novo expression, and sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a) overexpression. The model was used to seek mechanistic insights into 1) the relative contribution of two mechanisms by which SERCA2a overexpression modifies Ca2+ sequestration, i.e., more pumps and an increase in the SERCA2a-to-phospholamban ratio, 2) the mechanisms behind postrest potentiation and how Parv and SERCA2a influence this response, and 3) why Parv myocytes retain their fast kinetics when endogenous SERCA2a is partially impaired by thapsigargin (a condition used to mimic diastolic dysfunction). The model was also utilized to predict whether Parv metal-binding characteristics might be modified to improve diastolic and systolic functions and whether Parv or SERCA2a might affect diastolic Ca2+ levels and myocyte energetics. One outcome of the model was to demonstrate a higher peak and total ATP consumption in SERCA2a myocytes and more even distribution of ATP throughout the cardiac cycle in Parv myocytes. This may have implications for failing hearts that are energetically compromised.

Genetic manipulation of calcium-handling proteins in cardiac myocytes. I. Experimental studies.

Two genetic experimental approaches, de novo expression of parvalbumin (Parv) and overexpression of sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA2a), have been shown to increase relaxation rates in myocardial tissue. However, the relative effect of Parv and SERCA2a on systolic function and on beta-adrenergic responsiveness at varied pacing rates is unknown. We used gene transfer in isolated rat adult cardiac myocytes to gain a fuller understanding of Parv/SERCA2a function. As demonstrated previously, when Parv is expressed in elevated concentration (>0.1 mM), the transduced myocytes showed a reduction in sarcomere-shortening amplitude: 129 +/- 17, 81 +/- 8, and 149 +/- 14 nm for control, Parv, and SERCA2a, respectively. At physiological temperature, shortening amplitude responses of Parv and SERCA2a myocytes to the beta-adrenergic agonist isoproterenol (Iso) were not statistically different from that of control myocytes. However, in SERCA2a myocytes, in which baseline was slightly elevated and the Iso-stimulated value was slightly lower, the increase in shortening was slightly less than in Parv or control myocytes: 108 +/- 14, 169 +/- 39, and 34 +/- 12% for control, Parv, and SERCA2a, respectively. In another test set, Parv myocytes had the strongest early postrest potentiation among all groups studied (rest time = 2-10 s), and SERCA2a myocytes were the least sensitive to variations in stimulation rhythm. To replicate the deficient Ca2+ removal observed in heart failure, we used 150 nM thapsigargin. Under these conditions, control myocytes exhibited slowed relaxation, whereas Parv myocytes retained their rapid kinetics, showing that Parv is still able to control relaxation, even when SERCA2a function is impaired.

Parvalbumin corrects slowed relaxation in adult cardiac myocytes expressing hypertrophic cardiomyopathy-linked alpha-tropomyosin mutations.

Hypertrophic cardiomyopathy mutations A63V and E180G in alpha-tropomyosin (alpha-Tm) have been shown to cause slow cardiac muscle relaxation. In this study, we used two complementary genetic strategies, gene transfer in isolated rat myocytes and transgenesis in mice, to ascertain whether parvalbumin (Parv), a myoplasmic calcium buffer, could correct the diastolic dysfunction caused by these mutations. Sarcomere shortening measurements in rat cardiac myocytes expressing the alpha-Tm A63V mutant revealed a slower time to 50% relengthening (T50R: 44.2+/-1.4 ms in A63V, 36.8+/-1.0 ms in controls; n=96 to 108; P<0.001) when compared with controls. Dual gene transfer of alpha-Tm A63V and Parv caused a marked decrease in T50R (29.8+/-1.0 ms). However, this increase in relaxation rate was accompanied with a decrease in shortening amplitude (114.6+/-4.4 nm in A63+Parv, 137.8+/-5.3 nm in controls). Using an asynchronous gene transfer strategy, Parv expression was reduced (from approximately 0.12 to approximately 0.016 mmol/L), slow relaxation redressed, and shortening amplitude maintained (T50R=33.9+/-1.6 ms, sarcomere shortening amplitude=132.2+/-7.0 nm in A63V+PVdelayed; n=56). Transgenic mice expressing the E180G alpha-Tm mutation and mice expressing Parv in the heart were crossed. In isolated adult myocytes, the alpha-Tm mutation alone (E180G+/PV-) had slower sarcomere relengthening kinetics than the controls (T90R: 199+/-7 ms in E180G+/PV-, 130+/-4 ms in E180G-/PV-; n=71 to 72), but when coexpressed with Parv, cellular relaxation was faster (T90R: 36+/-4 ms in E180G+/PV+). Collectively, these findings show that slow relaxation caused by alpha-Tm mutants can be corrected by modifying calcium handling with Parv.

Targeting diastolic dysfunction by genetic engineering of calcium handling proteins.

Diastolic heart failure (HF) is associated with significant morbidity and mortality, and is a growing medical problem in this country. Diastolic dysfunction is defined as an abnormality in myocardial relaxation that impairs filling during diastole and contributes to the clinical syndrome of HF. Effective clinical strategies to treat diastolic dysfunction are limited. This article focuses on the potential application of parvalbumin--a fast skeletal muscle calcium buffer--for remediation of slow relaxation in the failing heart.

Divergent abnormal muscle relaxation by hypertrophic cardiomyopathy and nemaline myopathy mutant tropomyosins.

Mutations in tropomyosin (Tm) have been linked to distinct inherited diseases of cardiac and skeletal muscle, hypertrophic cardiomyopathy (HCM), and nemaline myopathy (NM). How HCM and NM mutations in nearly identical Tm proteins produce the vastly divergent clinical phenotypes of heightened, prolonged cardiac muscle contraction in HCM and skeletal muscle weakness in NM is currently unknown. We report here a direct comparison of the effects of HCM (A63V) and NM (M9R) mutant Tm on membrane-intact myocyte contractile function as assessed by adenoviral gene transfer to fully differentiated cardiac muscle cells. Wild-type, and mutant HCM, and mutant NM proteins were expressed at similar levels in myocytes and incorporated into sarcomeres. Interestingly, HCM mutant Tm produced significantly longer contractions by slowing relaxation, whereas NM mutant Tm produced the opposite effect of accelerated muscle relaxation. We propose slowed relaxation caused by HCM mutant Tm can directly contribute to diastolic dysfunction seen in HCM even without secondary cardiac remodeling. Conversely, hastening of relaxation by NM mutant Tm may shift the force-frequency relationship in skeletal muscle and contribute to muscle weakness seen in NM. Together, these results implicate divergent, abnormal "turning off" of muscle contraction as a cellular basis for the differential pathogenesis of mutant Tm-associated HCM and NM.

Optimal range for parvalbumin as relaxing agent in adult cardiac myocytes: gene transfer and mathematical modeling.

Parvalbumin (PV) has recently been shown to increase the relaxation rate when expressed in intact isolated cardiac myocytes via adenovirus gene transfer. We report here a combined experimental and mathematical modeling approach to determine the dose-response and the sarcomere length (SL) shortening-frequency relationship of PV in adult rat cardiac myocytes in primary culture. The dose-response was obtained experimentally by observing the PV-transduced myocytes at different time points after gene transfer. Calcium transients and unloaded mechanical contractions were measured. The results were as follows. At low estimated [PV] (approximately 0.01 mM), contractile parameters were unchanged; at intermediate [PV], relaxation rate of the mechanical contraction and the decay rate of the calcium transient increased with little effects on amplitude; and at high [PV] (approximately 0.1 mM), relaxation rate was further increased, but the amplitudes of the mechanical contraction and the calcium transient were diminished when compared with control myocytes. The SL shortening-frequency relationship exhibited a biphasic response to increasing stimulus frequency in controls (decrease in amplitude and re-lengthening time from 0.2 to 1.0 Hz followed by an increase in these parameters from 2.0 to 4.0 Hz). The effect of PV was to flatten this frequency response. This flattening effect was partly explained by a reduction in the variation in fractional binding of PV to calcium during beats at high frequency. In conclusion, experimental results and mathematical modeling indicate that there is an optimal PV range for which relaxation rate is increased with little effect on contractile amplitude and that PV effectiveness decreases as the stimulus frequency increases.