Human Left Ventricular Myocytes Research Articles

A low voltage activated Ca2+ current found in a subset of human ventricular myocytes

ABSTRACT Low voltage activated (ICa-LVA) calcium currents including Cav1.3 and T-type calcium current (ICa-T) have not been reported in adult human left ventricular myocytes (HLVMs). We tried to examine their existence and possible correlation with etiology and patient characteristics in a big number of human LVMs isolated from explanted terminally failing (F) hearts, failing hearts with left ventricular assist device (F-LVAD) and nonfailing (NF) human hearts. LVA (ICa-LVA) was determined by subtracting L-type Ca2+ current (ICa-L) recorded with the holding potential of −50 mV from total Ca2+ current recorded with the holding potential of −90 mV or −70 mV. ICa- LVA was further tested with its sensitivity to 100 µM CdCl2 and tetrodotoxin. Three HLVMs (3 of 137 FHLVMs) from 2 (N = 30 hearts) failing human hearts, of which one was idiopathic and the other was due to primary pulmonary hypertension, were found with ICa-LVA. ICa-LVA in one FHLVM was not sensitive to 100 µM CdCl2 while ICa-LVA in another two FHLVMs was not sensitive to tetrodotoxin. It peaked at the voltage of −40~-20 mV and had a time-dependent decay faster than ICa-L but slower than sodium current (INa). ICa-LVA was not found in any HLVMs from NF (75 HLVMs from 17 hearts) or F-LVAD hearts (82 HLVMs from 18 hearts) but a statistically significant correlation could not be established. In conclusion, ICa-LVA was detected in some HLVMs of a small portion of human hearts that happened to be nonischemic failing hearts.

Calcium Activated Chloride Current in Mammalian Ventricular Myocytes

Background: Calcium activated Cl− current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood. Our goal was to study the current profile under normal calcium cycling and AP voltage-clamp condition as well as in case of altered intracellular calcium concentration ([Ca2+]i). The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied.Methods: Whole-cell configuration of the patch-clamp technique and action potential voltage-clamp were used to monitor ICl(Ca), detected as 9-anthracene carboxylic acid (9-AC)-sensitive current. FURA-2-AM dye was used to measure [Ca2+]i. Expression and cellular localization of Cav1.2, Bestrophin-3 and TMEM16A was analyzed with immunocytochemistry and confocal microscopy.Results: Under AP voltage-clamp conditions the profile of ICl(Ca) contained an early fast outward (1.62±0.06 A/F) and a late inward component (−0.16±0.02 A/F), overlapping early and terminal repolarizations, respectively. Both components were reduced by ryanodine (1.05±0.03 A/F; −0.07±0.03 A/F), while fully abolished by BAPTA (0.13±0.10 A/F; −0.08±0.02 A/F), but not EGTA (1.17±0.09 A/F; −0.13±0.02 A/F). Setting [Ca2+]i to the systolic level (1.1 µM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Both L-type Ca2+ current and ICl(Ca) were eliminated by nisoldipine. TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels both canine myocytes and human ventricular myocardium.Conclusions: Activation of ICl(Ca) in canine ventricular cells requires calcium entry through neighboring L-type Ca2+ channels and is only augmented by SR Ca2+-release. Substantial activation of ICl(Ca) requires high Ca2+ in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

Sarcolemmal Ca2+-entry through L-type Ca2+ channels controls the profile of Ca2+-activated Cl− current in canine ventricular myocytes

Ca2+-activated Cl− current (ICl(Ca)) mediated by TMEM16A and/or Bestrophin-3 may contribute to cardiac arrhythmias. The true profile of ICl(Ca) during an actual ventricular action potential (AP), however, is poorly understood.We aimed to study the profile of ICl(Ca) systematically under physiological conditions (normal Ca2+ cycling and AP voltage-clamp) as well as in conditions designed to change [Ca2+]i. The expression of TMEM16A and/or Bestrophin-3 in canine and human left ventricular myocytes was examined. The possible spatial distribution of these proteins and their co-localization with Cav1.2 was also studied.The profile of ICl(Ca), identified as a 9-anthracene carboxylic acid-sensitive current under AP voltage-clamp conditions, contained an early fast outward and a late inward component, overlapping early and terminal repolarizations, respectively. Both components were moderately reduced by ryanodine, while fully abolished by BAPTA, but not EGTA. [Ca2+]i was monitored using Fura-2-AM. Setting [Ca2+]i to the systolic level measured in the bulk cytoplasm (1.1μM) decreased ICl(Ca), while application of Bay K8644, isoproterenol, and faster stimulation rates increased the amplitude of ICl(Ca). Ca2+-entry through L-type Ca2+ channels was essential for activation of ICl(Ca). TMEM16A and Bestrophin-3 showed strong co-localization with one another and also with Cav1.2 channels, when assessed using immunolabeling and confocal microscopy in both canine myocytes and human ventricular myocardium.Activation of ICl(Ca) in canine ventricular cells requires Ca2+-entry through neighboring L-type Ca2+ channels and is only augmented by SR Ca2+-release. Substantial activation of ICl(Ca) requires high Ca2+ concentration in the dyadic clefts which can be effectively buffered by BAPTA, but not EGTA.

Select Articles Published on the Topic of Arrhythmia and Electrophysiogy in 2013

Select Articles Published on the Topic of Arrhythmia and Electrophysiogy in 2013

Myocardial Infarction-Induced N-Terminal Fragment of Cmybp-C Impairs Myofilament Function in Human Left Ventricular Myofibrils

Rationale: Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin binding protein-C (cMyBP-C), a cardiac-specific myofilament protein, is proteolyzed post-MI in humans and results in an N-terminal fragment, C0C1f. The presence of C0C1f in cultured adult cardiomyocytes results in decreased Ca2+ transients and cell shortening, in addition to the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear.Objective: To determine how C0C1f causes altered contractility in human cardiac myofilaments in vitro.Methods and Results: We generated recombinant human C0C1f (hC0C1f) and incorporated it into skinned human left ventricular myocytes. Mechanical properties were then studied at sarcomere lengths of 2.0 and 2.3 µm. Our data demonstrate that the presence of hC0C1f in the sarcomere decreased maximal force myofilament Ca2+ sensitivity, increased cooperative activation at short lengths and enhanced length-dependent activation. Furthermore, hC0C1f led to increased cross-bridge cycling kinetics and tension cost at both short and long sarcomere lengths. We further established that the detrimental effects of hC0C1f occur through direct interaction with the thin filament proteins actin and α-tropomyosin (α-TM).Conclusions: Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca2+ sensitivity in otherwise healthy human donor myofilament preparations. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-TM, suggesting that cleaved C0C1f could act as a poison peptide and disrupt the interaction of native cMyBP-C with the thin filament.Keywords: Cross-bridge cycling kinetics; length-dependent activation; cMyBP-C; C0C1f protein.

Inositol 1, 4, 5-Trisphosphate Receptors and Human Left Ventricular Myocytes

Little is known about the function of inositol 1,4,5-trisphosphate receptors (IP3Rs) in the adult heart experimentally. Moreover, whether these Ca(2+) release channels are present and play a critical role in human cardiomyocytes remains to be defined. IP3Rs may be activated after Gαq-protein-coupled receptor stimulation, affecting Ca(2+) cycling, enhancing myocyte performance, and potentially favoring an increase in the incidence of arrhythmias. IP3R function was determined in human left ventricular myocytes, and this analysis was integrated with assays in mouse myocytes to identify the mechanisms by which IP3Rs influence the electric and mechanical properties of the myocardium. We report that IP3Rs are expressed and operative in human left ventricular myocytes. After Gαq-protein-coupled receptor activation, Ca(2+) mobilized from the sarcoplasmic reticulum via IP3Rs contributes to the decrease in resting membrane potential, prolongation of the action potential, and occurrence of early afterdepolarizations. Ca(2+) transient amplitude and cell shortening are enhanced, and extrasystolic and dysregulated Ca(2+) elevations and contractions become apparent. These alterations in the electromechanical behavior of human cardiomyocytes are coupled with increased isometric twitch of the myocardium and arrhythmic events, suggesting that Gαq-protein-coupled receptor activation provides inotropic reserve, which is hampered by electric instability and contractile abnormalities. Additionally, our findings support the notion that increases in Ca(2+) load by IP3Rs promote Ca(2+) extrusion by forward-mode Na(+)/Ca(2+) exchange, an important mechanism of arrhythmic events. The Gαq-protein/coupled receptor/IP3R axis modulates the electromechanical properties of the human myocardium and its propensity to develop arrhythmias.

Role of the R1135H KCNH2 mutation in Brugada syndrome

Recently, a novel mutation, R1135H, in KCNH2, the gene encoding the α-subunit of the rapid delayed rectifier K+ channel (IKr), has been identified in a patient with short QT interval as well as Brugada-type ECG. Voltage clamp experiments revealed larger tail currents and slowed deactivation of mutant IKr channels. We have carried out computer simulations to test the effects of the R1135H mutation on the action potential of human left and right ventricular myocytes. Our results demonstrate that this ‘gain-of-function’ mutation in KCNH2 shortens the action potential and enhances the susceptibility of right ventricular myocytes to all-or-none repolarization (epicardial loss of the action potential dome). Thus, the mutation induced changes in IKr cannot only explain the short QT interval but also the Brugada-type ECG of the patient.

Catenins and connexin43 gap junction remodelling in human congestive heart failure

We have recently shown that connexin43 (Cx43) gap junction remodelling in human congestive heart failure is associated with increased interaction of Cx43 with ZO-1. The action of ZO-1 in regulating this remodelling is proposed to involve other connexin-interacting partners, in particular adhesion junction proteins. Double-label immunoconfocal microscopy in human left ventricular myocytes shows that a proportion of the signal for the adhesion junction proteins β-catenin and γ-catenin co-localise with Cx43 at the intercalated disk.

Modeling of IK1 mutations in human left ventricular myocytes and tissue

Elucidation of the cellular basis of arrhythmias in ion channelopathy disorders is complicated by the inherent difficulties in studying human cardiac tissue. Thus we used a computer modeling approach to study the mechanisms of cellular dysfunction induced by mutations in inward rectifier potassium channel (K(ir))2.1 that cause Andersen-Tawil syndrome (ATS). ATS is an autosomal dominant disorder associated with ventricular arrhythmias that uncommonly degenerate into the lethal arrhythmia torsade de pointes. We simulated the cellular and tissue effects of a potent disease-causing mutation D71V K(ir)2.1 with mathematical models of human ventricular myocytes and a bidomain model of transmural conduction. The D71V K(ir)2.1 mutation caused significant action potential duration prolongation in subendocardial, midmyocardial, and subepicardial myocytes but did not significantly increase transmural dispersion of repolarization. Simulations of the D71V mutation at shorter cycle lengths induced stable action potential alternans in midmyocardial, but not subendocardial or subepicardial cells. The action potential alternans was manifested as an abbreviated QRS complex in the transmural ECG, the result of action potential propagation failure in the midmyocardial tissue. In addition, our simulations of D71V mutation recapitulate several key ECG features of ATS, including QT prolongation, T-wave flattening, and QRS widening. Thus our modeling approach faithfully recapitulates several features of ATS and provides a mechanistic explanation for the low frequency of torsade de pointes arrhythmia in ATS.

Myocardial apoptosis prevention by radical scavenging in patients undergoing cardiac surgery

BackgroundReactive oxygen-derived species, including those generated during myocardial ischemia and reperfusion induced by cardioplegia, have been suggested to be involved in myocardial apoptosis induction. The purpose of our study was to investigate (1) whether cardioplegic arrest initiates apoptosis in the hearts of cardiac surgery patients and (2) whether reactive oxygen-derived species scavenging with N-acetylcysteine attenuates myocardial apoptosis initiation. MethodsIn transmural left ventricular biopsy samples collected before and at the end of cardiopulmonary bypass, we densitometrically determined cardiac myocyte staining intensity for active caspases-3 and -7, the apoptosis signal pathway central effector enzymes. The left ventricular biopsy samples had been obtained from 36 coronary artery bypass graft patients randomized in a double-blind fashion to receive either N-acetylcysteine (100 mg/kg into cardiopulmonary bypass prime followed by infusion at 20 mg · kg−1 · h−1; n = 18) or placebo (n = 18). ResultsThe change in left ventricular cardiac myocyte staining (end of cardiopulmonary bypass minus before cardiopulmonary bypass) differed significantly between groups for both measures: caspase-3, −3.1 ± 4.5 gray units (mean ± SD; N-acetylcysteine group) versus 7.1 ± 8.1 gray units (placebo); 95% confidence interval, 6.4 to 14.4; P < .0001; caspase-7, −5.1 ± 6.1 gray units (N-acetylcysteine) versus 5.1 ± 5.7 gray units (placebo); 95% confidence interval, 6.3 to 15.0; P < .0001. Clinical outcome did not differ between N-acetylcysteine and placebo. ConclusionsOur data show that cardioplegic arrest initiates the apoptosis signal cascade in human left ventricular cardiac myocytes. This apoptosis induction can effectively be prevented by N-acetylcysteine.

Effects of adrenomedullin on human myocyte contractile function and beta-adrenergic response.

Adrenomedullin has been demonstrated to cause systemic vasodilation, and increased plasma adrenomedullin levels have been observed in cardiovascular disease states such as heart failure. While adrenomedullin receptors have been localized to the myocardium, the effects of adrenomedullin on human myocyte contractility remained unknown. Left ventricular myocytes were isolated from myocardial biopsies of patients (n = 16) undergoing elective coronary artery bypass surgery with normal left ventricular ejection fractions (51 +/- 1%). A total of 233 left ventricular myocytes were studied by videomicroscopy. Myocyte shortening velocity (microm/s) was measured at baseline and following the addition of either 3 nM, 30 nM, or 60 nM of adrenomedullin. The change in myocyte shortening velocity with increasing concentrations of adrenomedullin was computed. At all concentrations, adrenomedullin reduced myocyte shortening velocity from baseline values (P < 0.05). Next, the potential interaction of adrenomedullin with the beta-adrenergic receptor system was examined using 25 nM isoproterenol. The beta-adrenergic receptor-mediated increase in the myocyte shortening velocity was blunted with adrenomedullin (29 +/- 7 vs 63 +/- 13 microm/s, P < 0.05). These unique findings demonstrate that adrenomedullin reduced contractility in isolated human left ventricular myocytes and exhibited a negative interaction with the beta-adrenergic receptor system. Past studies have shown that adrenomedullin induces nitric oxide synthesis and that nitric oxide can uncouple myocyte metabolism. Thus, while adrenomedullin causes systemic vasodilation, this peptide can also exert a negative contractile effect in human left ventricular myocytes.

L-type Ca2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices.

Ca2+ influx through the L-type calcium channel (LTCC) induces Ca2+ release from the sarcoplasmic reticulum (SR) and maintains SR Ca2+ loading. Alterations in LTCC properties, their contribution to the blunted adrenergic responsiveness in failing hearts and their recovery after support with LV assist devices (LVAD) were studied. L-type Ca2+ current (I(Ca,L)) was measured under basal conditions and in the presence of isoproterenol (ISO), dibutyryl-cAMP (db-cAMP), Bay K 8644 (BayK), Okadaic acid (OA, a phosphatase inhibitor), and phosphatase 2A (PP2A) in nonfailing (NF), failing (F), and LVAD-supported human left ventricular myocytes (HVMs). Basal I(Ca,L) density was not different in the 3 groups but I(Ca,L) was activated at more negative voltages in F- and LVAD- versus NF-HVMs (V(0.5): -7.18+/-1.4 and -7.0+/-0.9 versus 0.46+/-1.1 mV). Both ISO and db-cAMP increased I(Ca,L) in NF- and LVAD- significantly more than in F-HVMs (NF >LVAD> F: ISO: 90+/-15% versus 77+/-19% versus 24+/-12%; db-cAMP: 235%>172%>90%). ISO caused a significant leftward shift of the I(Ca,L) activation curve in NF- and LVAD- but not in F-HVMs. After ISO and db-cAMP, the I(Ca,L) activation was not significantly different between groups. BayK also increased I(Ca,L) more in NF- (81+/-30%) and LVAD- (70+/-15%) than in F- (51+/-8%) HVMs. OA increased I(Ca, L) by 85.6% in NF-HVMs but had no effect in F-HVMs, while PP2A decreased I(Ca, L) in F-HVMs by 35% but had no effect in NF-HVMs. These results suggest that the density of LTCC is reduced in F-HVMs but basal I(Ca,L) density is maintained by increasing in LTCC phosphorylation.

Distinct patterns of dystrophin organization in myocyte sarcolemma and transverse tubules of normal and diseased human myocardium.

Genetic mutations of dystrophin and associated glycoproteins underlie cell degeneration in several inherited cardiomyopathies, although the precise physiological role of these proteins remains under discussion. We studied the distribution of dystrophin in relation to the force-transducing vinculin-rich costameres in left ventricular cardiomyocytes from normal and failing human hearts to further elucidate the function of this protein complex. Single- and double-label immunoconfocal microscopy and parallel high-resolution immunogold fracture-label electron microscopy were used to localize dystrophin and vinculin in human left ventricular myocytes from normal (n=6) and failing hearts (idiopathic dilated cardiomyopathy, n=7, or ischemic heart disease, n=5). In control cardiomyocytes, dystrophin had a continuous distribution at the peripheral sarcolemma, with concentrated bands corresponding to the vinculin-rich costameres. Intracellular labeling extended along transverse (T) tubule membranes. Fracture-label confirmed this distribution, showing significantly greater label on plasma membrane fractures overlying I-bands (I-band 4.1+/-0.3 gold particles/micrometer A-band 3.3+/-0.2 gold particles/micrometer mean+/-SE, P=0.02). Hypertrophied myocytes from failing hearts showed maintenance of this surface distribution except in degenerating cells; there was a clear increase in intracellular dystrophin label reflecting T-tubule hypertrophy. Dystrophin partially colocalizes with costameric vinculin in normal and hypertrophied myocytes, a distribution lost in degenerating cells. This suggests a primarily mechanical role for dystrophin in maintenance of cell membrane integrity in normal and hypertrophied myocytes. The presence of dystrophin in the cardiac T-tubule membrane, in contrast to its known absence in skeletal muscle T-tubules, implies additional roles for dystrophin in membrane domain organization.

Endothelin receptor pathway in human left ventricular myocytes: relation to contractility

Background. Increased synthesis and release of the potent bioactive peptide endothelin-1 (ET-1) occurs during and after cardiac surgery. However, the cellular and molecular basis for the effects of ET-1 on human left ventricular (LV) myocyte contractility remains unknown.Methods. LV myocyte contractility was examined from myocardial biopsies taken from patients (n = 30) undergoing elective coronary artery bypass. LV myocytes (n = 997, > 30/patient) were isolated using microtrituration and contractility examined by videomicroscopy at baseline and after ET-1 exposure (200 pmol/L). In additional studies, myocytes were pretreated to inhibit either protein kinase C (PKC) (chelerythrine, 1 μmol/L), the sodium/hydrogen (Na/H) exchanger (EIPA, 1 μmol/L), both PKC and the Na/H exchanger, or the ETA receptor (BQ-123, 1 μmol/L), followed with ET-1 exposure.Results. Basal myocyte shortening increased 37.8 ± 6.3% with ET-1 (p < 0.05). Na/H exchanger, PKC, and dual inhibition all eliminated the effects of ET-1. Furthermore, ETA inhibition demonstrated that ET-1 effects on myocyte contractility were mediated through the ETA receptor subtype.Conclusions. ET-1 directly influences human LV myocyte contractility, which is mediated through the ETA receptor and requires intracellular activation of PKC and stimulation of the Na/H exchanger.

Identification of novel matrix metalloproteinase inducers and activators in normal adult human left ventricular myocytes

Identification of novel matrix metalloproteinase inducers and activators in normal adult human left ventricular myocytes

The structure and function of the cardiac myocyte: A review of fundamental concepts

The structure and function of the cardiac myocyte: A review of fundamental concepts

Na+-channel modulating effect of the inotropic compound S(-)BDF 9196 in human myocardium.

S(-)BDF 9196, the active enantiomer of racemic (+/-)BDF 9148, has been shown to increase force of contraction in myocardium from different species including humans. The present study aimed to investigate the mechanism of the positive inotropic action of the active enantiomer S(-)BDF 9196 in human myocardium. In electrically driven human left ventricular papillary muscle strips (dilated cardiomyopathy, NYHA IV, cardiac transplantation, n=9), S(-)BDF 9196 increased force of contraction concentration-dependently. The maximal positive inotropic effect remained unchanged after the addition of carbachol (1 mmol/l, indicating a cAMP-independent mode of action of S(-)BDF 9196. While [3H]ouabain binding in human myocardial membranes was not influenced by S(-)BDF 9196 up to 10 micromol/l, the inward Na(+)-current in isolated human left ventricular myocytes was increased significantly by S(-)BDF 9196 (1 micromol/l, n=5). These results provide evidence that S(-)BDF 9196 increases force of contraction in human myocardium primarily by enhancing Na(+)-influx, while cAMP-dependent or Na(+),K(+)-ATPase blocking effects do not seem to play a role.

Isolated left ventricular myocyte contractility in patients undergoing cardiac operations

Background: Because of methods required for obtaining isolated left ventricular myocytes, evaluation of the contractile function of isolated left ventricular myocytes in normal human patients has been limited. Accordingly, the goal of the present study was to develop a means to isolate human left ventricular myocytes from small myocardial biopsy specimens collected from patients undergoing elective coronary artery bypass operations and to characterize indices of myocyte contractile performance. Methods: Myocardial biopsy specimens were obtained from the anterior left ventricular free wall of 22 patients undergoing coronary artery bypass operations. Myocytes were isolated from these myocardial samples by means of a stepwise enzymatic digestion method and micro-trituration techniques. Isolated left ventricular myocyte contractile function was assessed by computer-assisted high-speed videomicroscopy under basal conditions and in response to β-adrenergic receptor stimulation with isoproterenol. Results: A total of 804 viable left ventricular myocytes were successfully examined from all of the myocardial biopsy specimens with an average of 37 ± 4 myocytes per patient. All myocytes contracted homogeneously at a field stimulation of 1 Hz with an average percent shortening of 3.7% ± 0.1% and shortening velocity of 51.3 ± 1.3 μm/s. After β-adrenergic receptor stimulation with isoproterenol, percent shortening and shortening velocity increased 149% and 118% above baseline, respectively ( P < .05). Conclusion: The unique results of the present study demonstrated that a high yield of myocytes could be obtained from human left ventricular biopsy specimens taken during cardiac operations. These myocytes exhibited stable contractile performance and maintained the capacity to respond to an inotropic stimulus. The methods described herein provide a basis by which future studies could investigate intrinsic and extrinsic influences on left ventricular myocyte contractility in human beings. (J Thorac Cardiovasc Surg 1998;116:495-502)

Electrophysiological and mechanical properties of failed human left ventricular myocytes

Studies on human ventricular myocytes have been limited due to the difficulty of isolating viable myocytes from diseased human explants. Calcium-tolerant left ventricular myocytes were isolated from midmyocardium of 5 ischemic (ICM), 8 non-ischemic/idiopathic (NICM) and I normal (N) donor heart tmsuitable for transplant. The diseased hearts had ejection fractions <20% and the patients were NYHA Class IV. All patients were receiving inotropic agents at the time of transplantation. A blood vessel perfusing a piece of left ventricular free wall (approx. 8 x 10 cm) was cannulated and perfused with calcium-free Krebs solution for 30 minutes followed by collagenasecontaining Krebs solution (180 units/ml) for 40-60 minutes. Isolated myocytes were filtered and resuspended in Krebs solution containing 200raM Ca2+ without high K+ or EGTA until use. Myocytes were superfused with Tyrodes solution containing lmM Ca z+. Using patch clamp techniques, action potentials were recorded following stimulation at .2-2 Hz. Action potential characteristics were not significantly different between groups (@ 1 Hz, resting membrane potential = -75.1+.1 mV (N), n=3, -74.4+.8 mV (ICM), n=21, -75.2+.7 mV (NICM), n=16, APD90 = 667+60 msec (N), 507+35 msec (ICM), 526+50 msec (NICM) n=27). Rate-dependent (.2-2 Hz) shortening of the action potential was evident in all cells. Contractions of single myocytes in response to action potentials were recorded using a video edge detector. Contractions were coordinated and well synchronized. The magnitude of contraction was significantly less in ICM and NICM compared to N. The time-to-peak, and time to 50% relaxation were significantly shorter in N compared to ICM and NICM. We conclude that myocytes from failed human hearts of various etiologies share similar electrophysiological properties while the mechanical properties of heart failure result in smaller and slower contractions in ICM and NICM compared to normal. The Second Annual Scientific Meeting • HFSA 13

Contribution of reverse-mode sodium-calcium exchange to contractions in failing human left ventricular myocytes.

To examine the contribution of reverse mode sodium-calcium (Na-Ca) exchange to contractions in isolated left-ventricular myocytes from failing human heart. Low resistance patch pipettes were used to dialyze cells with Na-free or high-Na pipette solution ([Na]pipette = 0 and 20 mmol/L, respectively) to reduce or enhance Na-Ca exchange. Whole-cell membrane-potential, membrane-current and cell-shortening data were simultaneously acquired during whole-cell voltage clamp protocols. Thapsigargin (100 nmol/L) and nifedipine (1 mumol/L) were also used to inhibit sarcoplasmic reticulum (SR) Ca-ATPase and L-type Ca channels, respectively. Two types of contractions were observed. Rapid phasic contractions were seen in both Na-free and high-Na cells. Slow tonic contractions were seen only in high-Na cells. Phasic contractions demonstrated bell-shaped voltage dependence over the voltage range that corresponds to the activity of the L-type Ca channel. Although the voltage dependence of phasic contractions were similar Na-free and high-Na cells, phasic contractions in high-Na cells were larger than phasic contractions in Na-free cells. Phasic contractions were sensitive to inhibition of SR Ca-ATPase and L-type Ca channels. Tonic contractions were not inhibited by either thapsigargin or nifedipine. In thapsigargin-treated high-Na cells, tonic contraction magnitude increased exponentially with test-potential. The increases in phasic contraction magnitude observed in high-Na cells compared to Na-free cells were most likely due to increased SR Ca loading resulting from increased reverse-mode Na-Ca exchange. Our results also suggest that tonic contractions in high-Na cells were mediated by Ca entry via reverse-mode Na-Ca exchange and were not the result of either SR Ca release or L-type Ca channel activity.