Hanne C. Gadeberg

Heterogeneity of T-Tubules in Pig Hearts

Background T-tubules are invaginations of the sarcolemma that play a key role in excitation-contraction coupling in mammalian cardiac myocytes. Although t-tubules were generally considered to be effectively absent in atrial myocytes, recent studies on atrial cells from larger mammals suggest that t-tubules may be more numerous than previously supposed. However, the degree of heterogeneity between cardiomyocytes in the extent of the t-tubule network remains unclear. The aim of the present study was to investigate the t-tubule network of pig atrial myocytes in comparison with ventricular tissue. Methods Cardiac tissue was obtained from young female Landrace White pigs (45–75 kg, 5–6 months old). Cardiomyocytes were isolated by arterial perfusion with a collagenase-containing solution. Ca2+ transients were examined in field-stimulated isolated cells loaded with fluo-4-AM. Membranes of isolated cells were visualized using di-8-ANEPPS. T-tubules were visualized in fixed-frozen tissue sections stained with Alexa-Fluor 488-conjugated WGA. Binary images were obtained by application of a threshold and t-tubule density (TTD) calculated. A distance mapping approach was used to calculate half-distance to nearest t-tubule (HDTT). Results & Conclusion The spatio-temporal properties of the Ca2+ transient appeared to be consistent with the absence of functional t-tubules in isolated atrial myocytes. However, t-tubules could be identified in a sub-population of atrial cells in frozen sections. While all ventricular myocytes had TTD >3% (mean TTD = 6.94±0.395%, n = 24), this was true of just 5/22 atrial cells. Mean atrial TTD (2.35±0.457%, n = 22) was lower than ventricular TTD (P<0.0001). TTD correlated with cell-width (r = 0.7756, n = 46, P<0.0001). HDTT was significantly greater in the atrial cells with TTD ≤3% (2.29±0.16 μm, n = 17) than in either ventricular cells (1.33±0.05 μm, n = 24, P<0.0001) or in atrial cells with TTD >3% (1.65±0.06 μm, n = 5, P<0.05). These data demonstrate considerable heterogeneity between pig cardiomyocytes in the extent of t-tubule network, which correlated with cell size.

Altered Na/Ca exchange distribution in ventricular myocytes from failing hearts

Na/Ca exchange (NCX) is normally located predominantly in the T tubules of cardiac ventricular myocytes. However, redistribution of NCX occurs in myocytes from failing hearts, resulting in more uniform distribution between T tubule and surface sarcolemma; this alters access of NCX to Ca released from sarcoplasmic reticulum and thus cellular Ca handling.

Cardiac ion channel current modulation by the CFTR inhibitor GlyH-101

The role in the heart of the cardiac isoform of the cystic fibrosis transmembrane conductance regulator (CFTR), which underlies a protein kinase A-dependent Cl(-) current (I(Cl.PKA)) in cardiomyocytes, remains unclear. The identification of a CFTR-selective inhibitor would provide an important tool for the investigation of the contribution of CFTR to cardiac electrophysiology. GlyH-101 is a glycine hydrazide that has recently been shown to block CFTR channels but its effects on cardiomyocytes are unknown. Here the action of GlyH-101 on cardiac I(Cl.PKA) and on other ion currents has been established. Whole-cell patch-clamp recordings were made from rabbit isolated ventricular myocytes. GlyH-101 blocked I(Cl.PKA) in a concentration- and voltage-dependent fashion (IC(50) at +100 mV=0.3 ± 1.5 μM and at -100 mV=5.1 ± 1.3 μM). Woodhull analysis suggested that GlyH-101 blocks the open pore of cardiac CFTR channels at an electrical distance of 0.15 ± 0.03 from the external membrane surface. A concentration of GlyH-101 maximally effective against I(Cl.PKA) (30 μM) was tested on other cardiac ion currents. Inward current at -120 mV, comprised predominantly of the inward-rectifier background K(+) current, I(K1), was reduced by ∼43% (n=5). Under selective recording conditions, the Na(+) current (I(Na)) was markedly inhibited by GlyH-101 over the entire voltage range (with a fractional block at -40 mV of ∼82%; n=8). GlyH-101 also produced a voltage-dependent inhibition of L-type Ca(2+) channel current (I(Ca,L)); fractional block at +10 mV of ∼49% and of ∼28% at -10 mV; n=11, with a ∼-3 mV shift in the voltage-dependence of I(Ca,L) activation. Thus, this study demonstrates for the first time that GlyH-101 blocks cardiac I(Cl.PKA) channels in a similar fashion to that reported for recombinant CFTR. However, inhibition of other cardiac conductances may limit its use as a CFTR-selective blocker in the heart.

The Effects of Aging on the Regulation of T-Tubular ICa by Caveolin in Mouse Ventricular Myocytes

Abstract Aging is associated with diminished cardiac function in males. Cardiac excitation-contraction coupling in ventricular myocytes involves Ca influx via the Ca current (ICa) and Ca release from the sarcoplasmic reticulum, which occur predominantly at t-tubules. Caveolin-3 regulates t-tubular ICa, partly through protein kinase A (PKA), and both ICa and caveolin-3 decrease with age. We therefore investigated ICa and t-tubule structure and function in cardiomyocytes from male wild-type (WT) and caveolin-3-overexpressing (Cav-3OE) mice at 3 and 24 months of age. In WT cardiomyocytes, t-tubular ICa-density was reduced by ~50% with age while surface ICa density was unchanged. Although regulation by PKA was unaffected by age, inhibition of caveolin-3-binding reduced t-tubular ICa at 3 months, but not at 24 months. While Cav-3OE increased cardiac caveolin-3 protein expression ~2.5-fold at both ages, the age-dependent reduction in caveolin-3 (WT ~35%) was preserved in transgenic mice. Overexpression of caveolin-3 reduced t-tubular ICa density at 3 months but prevented further ICa loss with age. Measurement of Ca release at the t-tubules revealed that the triggering of local Ca release by t-tubular ICa was unaffected by age. In conclusion, the data suggest that the reduction in ICa density with age is associated with the loss of a caveolin-3-dependent mechanism that augments t-tubular ICa density.

Caveolin 3‐dependent loss of t‐tubular ICa during hypertrophy and heart failure in mice

What is the central question of this study? Heart failure is associated with redistribution of L‐type Ca2+ current (ICa) away from the t‐tubule membrane to the surface membrane of cardiac ventricular myocytes. However, the underlying mechanism and its dependence on severity of pathology (hypertrophy versus failure) are unclear. What is the main finding and its importance? Increasing severity of response to transverse aortic constriction, from hypertrophy to failure, was accompanied by graded loss of t‐tubular ICa and loss of regulation of ICa by caveolin 3. Thus, the pathological loss of t‐tubular ICa, which contributes to impaired excitation–contraction coupling and thereby cardiac function in vivo, appears to be attributable to loss of caveolin 3‐dependent stimulation of t‐tubular ICa.

Cholesterol depletion does not alter the capacitance or Ca handling of the surface or t-tubule membranes in mouse ventricular myocytes

Cholesterol is a key component of the cell plasma membrane. It has been suggested that the t‐tubule membrane of cardiac ventricular myocytes is enriched in cholesterol and that this plays a role in determining t‐tubule structure and function. We have used methyl‐β‐cyclodextrin (MβCD) to deplete cholesterol in intact and detubulated mouse ventricular myocytes to investigate the contribution of cholesterol to t‐tubule structure, membrane capacitance, and the distribution of Ca flux pathways. Depletion of membrane cholesterol was confirmed using filipin; however, di‐8‐ANEPPS staining showed no differences in t‐tubule structure following MβCD treatment. MβCD treatment had no significant effect on the capacitance:volume relationship of intact myocytes or on the decrease in capacitance:volume caused by detubulation. Similarly, Ca influx and efflux were not altered by MβCD treatment and were reduced by a similar amount following detubulation in untreated and MβCD‐treated cells. These data show that cholesterol depletion has similar effects on the surface and t‐tubule membranes and suggest that cholesterol plays no acute role in determining t‐tubule structure and function.

Atrial-ventricular differences in rabbit cardiac voltage-gated Na+ currents: Basis for atrial-selective block by ranolazine

Background Class 1 antiarrhythmic drugs are highly effective in restoring and maintaining sinus rhythm in atrial fibrillation patients but carry a risk of ventricular tachyarrhythmia. The antianginal agent ranolazine is a prototypic atrial-selective voltage-gated Na+ channel blocker but the mechanisms underlying its atrial-selective action remain unclear. Objective The present study examined the mechanisms underlying the atrial-selective action of ranolazine. Methods Whole-cell voltage-gated Na+ currents (INa) were recorded at room temperature (∼22°C) from rabbit isolated left atrial and right ventricular myocytes. Results INa conductance density was ∼1.8-fold greater in atrial than in ventricular cells. Atrial INa was activated at command potentials ∼7 mV more negative and inactivated at conditioning potentials ∼11 mV more negative than ventricular INa. The onset of inactivation of INa was faster in atrial cells than in ventricular myocytes. Ranolazine (30 μM) inhibited INa in atrial and ventricular myocytes in a use-dependent manner consistent with preferential activated/inactivated state block. Ranolazine caused a significantly greater negative shift in voltage of half-maximal inactivation in atrial cells than in ventricular cells, the recovery from inactivation of INa was slowed by ranolazine to a greater extent in atrial myocytes than in ventricular cells, and ranolazine produced an instantaneous block that showed marked voltage dependence in atrial cells. Conclusion Differences exist between rabbit atrial and ventricular myocytes in the biophysical properties of INa. The more negative voltage dependence of INa activation and inactivation, together with trapping of the drug in the inactivated channel, underlies an atrial-selective action of ranolazine.

Caveolin-3 KO Disrupts T-Tububle Structure and Decreases T-Tubular ICa Density in Mouse Ventricular Myocytes

Caveolin-3 (Cav-3) is a protein that has been implicated in t-tubule formation and function in cardiac ventricular myocytes. In cardiac hypertrophy and failure, Cav-3 expression decreases, t-tubule structure is disrupted, and excitation-contraction coupling is impaired. However, the extent to which the decrease in Cav-3 expression underlies these changes is unclear. We therefore investigated the structure and function of myocytes isolated from the hearts of Cav-3 knockout (KO) mice. These mice showed cardiac dilatation and decreased ejection fraction in vivo compared with wild-type control mice. Isolated KO myocytes showed cellular hypertrophy, altered t-tubule structure, and decreased L-type Ca2+ channel current (ICa) density. This decrease in density occurred predominantly in the t-tubules, with no change in total ICa, and was therefore a consequence of the increase in membrane area. Cav-3 KO had no effect on L-type Ca2+ channel expression, and C3SD peptide, which mimics the scaffolding domain of Cav-3, had no effect on ICa in KO myocytes. However, inhibition of PKA using H-89 decreased ICa at the surface and t-tubule membranes in both KO and wild-type myocytes. Cav-3 KO had no significant effect on Na+/Ca2+ exchanger current or Ca2+ release. These data suggest that Cav-3 KO causes cellular hypertrophy, thereby decreasing t-tubular ICa density. NEW & NOTEWORTHY Caveolin-3 (Cav-3) is a protein that inhibits hypertrophic pathways, has been implicated in the formation and function of cardiac t-tubules, and shows decreased expression in heart failure. This study demonstrates that Cav-3 knockout mice show cardiac dysfunction in vivo, while isolated ventricular myocytes show cellular hypertrophy, changes in t-tubule structure, and decreased t-tubular L-type Ca2+ current density, suggesting that decreased Cav-3 expression contributes to these changes in cardiac hypertrophy and failure.