Kenneth T. MacLeod

Loss of T-tubules and other changes to surface topography in ventricular myocytes from failing human and rat heart

T-tubular invaginations of the sarcolemma of ventricular cardiomyocytes contain junctional structures functionally coupling L-type calcium channels to the sarcoplasmic reticulum calcium-release channels (the ryanodine receptors), and therefore their configuration controls the gain of calcium-induced calcium release (CICR). Studies primarily in rodent myocardium have shown the importance of T-tubular structures for calcium transient kinetics and have linked T-tubule disruption to delayed CICR. However, there is disagreement as to the nature of T-tubule changes in human heart failure. We studied isolated ventricular myocytes from patients with ischemic heart disease, idiopathic dilated cardiomyopathy, and hypertrophic obstructive cardiomyopathy and determined T-tubule structure with either the fluorescent membrane dye di-8-ANNEPs or the scanning ion conductance microscope (SICM). The SICM uses a scanning pipette to produce a topographic representation of the surface of the live cell by a non-optical method. We have also compared ventricular myocytes from a rat model of chronic heart failure after myocardial infarction. T-tubule loss, shown by both ANNEPs staining and SICM imaging, was pronounced in human myocytes from all etiologies of disease. SICM imaging showed additional changes in surface structure, with flattening and loss of Z-groove definition common to all etiologies. Rat myocytes from the chronic heart failure model also showed both T-tubule and Z-groove loss, as well as increased spark frequency and greater spark amplitude. This study confirms the loss of T-tubules as part of the phenotypic change in the failing human myocyte, but it also shows that this is part of a wider spectrum of alterations in surface morphology.

Effect of 17β‐oestradiol on contraction, Ca2+ current and intracellular free Ca2+ in guinea‐pig isolated cardiac myocytes

1 The effect of 17β‐oestradiol on cardiac cell contraction, inward Ca2+ current and intracellular free Ca2+ ([free Ca2+]i) was investigated in guinea‐pig single, isolated ventricular myocytes. The changes of cell length were measured by use of a photodiode array, the voltage‐clamp experiments were performed with a switch clamp system and [free Ca2+]i was measured with the Ca2+ indicator, Fura‐2. 2 17β‐Oestradiol (10, 30 μm) caused a decrease in cell shortening at both 22 and 35°C. This negative inotropic effect was accompanied by a decrease in action potential duration mainly brought about by a shortening of the plateau region of the action potential. 17β‐Oestradiol (10, 30 μm) induced a similar decrease in cell shortening in voltage‐clamped and current‐clamped cells. 3 In Fura‐2 loaded cells, 17β‐oestradiol (10 and 30 μm) decreased systolic Fura‐2 fluorescence to 72 ± 7% and 47 ± 4% (n = 6, P < 0.001) of control respectively. 17β‐Oestradiol (10 μm) had no significant effect on diastolic Fura‐2 fluorescence, but at higher concentration (30 μm) induced a slight decrease in resting Fura‐2 fluorescence. The effect of 17β‐oestradiol was reversible after 1–2 min of washout of the steroid. 4 17β‐Oestradiol (10 and 30 μm) decreased the peak inward Ca2+ current (ICa), which was sensitive to [Ca2+]o, dihydropyridines and isoprenaline, to 59 ± 3% and 39 ± 5% (n = 7∼9, P < 0.01) respectively, without producing any significant change in the shape of the current‐voltage relationship. 5 The recovery time of ICa from inactivation was delayed by 17β‐oestradiol (10 μm). The inhibitory effect of 17β‐oestradiol on ICa was less at a holding potential of −80 mV than at −40mV. 6 We conclude that 17β‐oestradiol has a negative inotropic effect on guinea‐pig single ventricular myocytes by inhibiting ICa and so reducing systolic [Ca2+]i. 17β‐Oestradiol may therefore have a Ca2+ channel blocking property in guinea‐pig isolated ventricular myocytes.

Na+ −Ca2+ exchange and sarcoplasmic reticular Ca2+ regulation in ventricular myocytes from transgenic mice overexpressing the Na+ −Ca2+ exchanger

1 The contribution of the sarcoplasmic reticulum (SR) and Na+−Ca2+ exchanger to intracellular Ca2+ regulation in mouse cardiac myocytes was investigated by measuring contraction after variable rest intervals, rapid cooling contractures (RCCs) and fast application of caffeine. The results obtained showed differences from other species in the roles played by the SR and the Na+−Ca2+ exchanger. They suggest that in mouse ventricular myocytes there is significant Ca2+ entry via the exchanger during rest and during the latter part of the Ca2+ transient. 2 In cardiac myocytes isolated from transgenic mice overexpressing the cardiac Na+−Ca2+ exchanger the time to peak and relaxation of twitches and RCCs were faster than in control littermates. The decline of Ca2+, assessed by indo‐1 fluorescence, was faster in transgenic myocytes even in the absence of Na+ and Ca2+ in the superfusing solution. This suggests that SR Ca2+ uptake is faster in these myocytes. However, no difference in the expression of SERCA2a, phospholamban or calsequestrin measured with Western blotting could be found in the two groups. 3 We measured SR Ca2+ content by integrating the caffeine‐induced transient inward current. The amount of Ca2+ stored in the SR of transgenic mouse myocytes was 69 % greater than in non‐transgenic littermates. The increased SR Ca2+ content may be responsible for the faster rate of SR Ca2+ release and uptake in cells from transgenic mice. 4 We performed experiments to assess whether the reversal potential of the Na+−Ca2+ exchanger (ENa‐Ca) was different in transgenic cardiac cells. We measured a Ni2+‐sensitive current elicited by voltage ramps in non‐dialysed myocytes. The current‐voltage relationship showed no difference in the reversal potential of the Na+−Ca2+ exchanger in transgenic and control myocytes. This suggests that the effects on the SR Ca2+ content in transgenic cardiac myocytes can be ascribed to the overexpression of the exchanger and are not secondary to changes in intracellular diastolic Ca2+ and Na+.

Sheep cardiac sarcoplasmic reticulum calcium-release channels: modification of conductance and gating by temperature.

1. The gating and conduction properties of single calcium‐release channels of sheep isolated cardiac junctional sarcoplasmic reticulum membranes incorporated into planar phospholipid bilayers were investigated under voltage clamp conditions at temperatures between 4 and 32 degrees C. 2. Single channel conductance was reduced linearly when temperature was decreased from 32 to 5 degrees C with a Q10 value of 1.5 between 10 and 20 degrees C. The apparent activation enthalpy for conductance between 32 and 5 degrees C was 6.16 +/‐ 1.2 kcal/mol. 3. Cooling the channel increased open probability (Po) when activating cytosolic calcium concentrations were varied within the range 0.1‐100 microM. At an activating free calcium concentration of 10 microM, channel Po increased from 0.13 +/‐ 0.05 at 23 degrees C to 0.69 +/‐ 0.07 at 5‐10 degrees C. 4. At sub‐activating calcium concentrations (100 pM) or high concentrations of calcium (greater than or equal to 1000 microM), the calcium‐release channel remained closed at 23 degrees C. Cooling the channel under these conditions did not increase Po. 5. Lifetime analysis indicates that with calcium as the sole activating ligand, the cooling‐induced increase in Po results from an increase in channel open lifetimes with no significant alteration in the frequency of channel opening. At 23 degrees C, the open and closed lifetime distributions of the calcium‐activated channel are best described by two and three exponentials respectively. At reduced temperatures (5‐10 degrees C), both open and closed lifetime distributions were best described by three exponentials. 6. At sub‐activating calcium concentrations, calcium‐independent channel openings could be induced by sulmazole (AR‐L 115 BS, 0.5‐10 mM). At 23 degrees C, with sulmazole as the sole activating ligand, the best fits to both open and closed lifetime distributions were obtained with three exponentials. At reduced temperatures (5‐10 degrees C), Po was increased. Gating was characterized by long open events, however the open and closed lifetime distributions were still best described by three exponentials. 7. The net effect of temperature reduction is an increase in calcium current through the channel. This finding is consistent with the suggestion that calcium release from the SR is a major factor in the initiation of rapid cooling contractures of mammalian cardiac muscle preparations.

Plant-derived estrogens relax coronary arteries in vitro by a calcium antagonistic mechanism

OBJECTIVES To investigate the potential for plant derived estrogens (phytoestrogens) genistein, phloretin, biochanin A and zearalanone to relax rabbit coronary arteries in vitro and to determine the mechanism(s) of such relaxation. BACKGROUND Epidemiological data suggests a reduction in the incidence of coronary heart disease in humans who have a high intake of phytoestrogens. METHODS Isolated rabbit coronary artery rings were suspended in individual organ baths, precontracted with potassium chloride (30 mM), and the relaxing effects and mechanisms of relaxation to genistein, phloretin, biochanin A and zearalanone were determined by measurement of isometric tension. RESULTS Genistein, phloretin and biochanin A induced significant gender-independent relaxation in rings with and without endothelium. Inhibition of nitric oxide and prostaglandin synthesis with L-NAME and indomethacin had no effect on genistein-induced relaxation. Relaxation was unaffected by the specific estrogen receptor antagonist ICI 182,780, the ATP-sensitive potassium channel inhibitor glibenclamide and the potassium channel inhibitor, barium chloride. Calcium concentration-dependent contraction curves in high potassium depolarization medium were significantly shifted to the right and downward after incubation with genistein and zearalanone. An inhibitory effect of genistein (2 microM) on L-type calcium current in guinea-pig ventricular myocytes confirmed a calcium antagonist relaxing mechanism of action. In healthy volunteers, plasma genistein levels of approximately 2 microM are achieved after ingestion of a commercially available soy protein drink (Supro) containing 37 mg genistein. CONCLUSIONS This study demonstrates that phytoestrogens induce endothelium-independent relaxation of coronary arteries; the mechanism involves calcium antagonism. These mechanisms may contribute to the potential long-term cardiovascular protective effect of these substances.

SERCA2a Gene Transfer Decreases Sarcoplasmic Reticulum Calcium Leak and Reduces Ventricular Arrhythmias in a Model of Chronic Heart Failure

Background—Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure and is under evaluation in a clinical trial. A critical question is whether SERCA2a gene therapy predisposes to increased sarcoplasmic reticulum calcium (SR Ca2+) leak, cellular triggered activity, and ventricular arrhythmias in the failing heart. Methods and Results—We studied the influence of SERCA2a gene therapy on ventricular arrhythmogenesis in a rat chronic heart failure model. ECG telemetry studies revealed a significant antiarrhythmic effect of SERCA2a gene therapy with reduction of both spontaneous and catecholamine-induced arrhythmias in vivo. SERCA2a gene therapy also reduced susceptibility to reentry arrhythmias in ex vivo programmed electrical stimulation studies. Subcellular Ca2+ homeostasis and spontaneous SR Ca2+ leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca2+ leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca2+ load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca2+ leak threshold, leading to reduced triggered activity in vitro. Both indirect effects of reverse remodeling and direct SERCA2a effects appear to underlie the antiarrhythmic action. Conclusions—SERCA2a gene therapy stabilizes SR Ca2+ load, reduces ryanodine receptor phosphorylation and decreases SR Ca2+ leak, and reduces cellular triggered activity in vitro and spontaneous and catecholamine-induced ventricular arrhythmias in vivo in failing hearts. SERCA2a gene therapy did not therefore predispose to arrhythmias and may represent a novel antiarrhythmic strategy in heart failure.

SERCA2a gene therapy restores microRNA-1 expression in heart failure via an Akt/FoxO3A-dependent pathway.

Aims Impaired myocardial sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) activity is a hallmark of failing hearts, and SERCA2a gene therapy improves cardiac function in animals and patients with heart failure (HF). Deregulation of microRNAs has been demonstrated in HF pathophysiology. We studied the effects of therapeutic AAV9.SERCA2a gene therapy on cardiac miRNome expression and focused on regulation, expression, and function of miR-1 in reverse remodelled failing hearts. Methods and results We studied a chronic post-myocardial infarction HF model treated with AAV9.SERCA2a gene therapy. Heart failure resulted in a strong deregulation of the cardiac miRNome. miR-1 expression was decreased in failing hearts, but normalized in reverse remodelled hearts after AAV9.SERCA2a gene delivery. Increased Akt activation in cultured cardiomyocytes led to phosphorylation of FoxO3A and subsequent exclusion from the nucleus, resulting in miR-1 gene silencing. In vitro SERCA2a expression also rescued miR-1 in failing cardiomyocytes, whereas SERCA2a inhibition reduced miR-1 levels. In vivo, Akt and FoxO3A were highly phosphorylated in failing hearts, but reversed to normal by AAV9.SERCA2a, leading to cardiac miR-1 restoration. Likewise, enhanced sodium–calcium exchanger 1 (NCX1) expression during HF was normalized by SERCA2a gene therapy. Validation experiments identified NCX1 as a novel functional miR-1 target. Conclusion SERCA2a gene therapy of failing hearts restores miR-1 expression by an Akt/FoxO3A-dependent pathway, which is associated with normalized NCX1 expression and improved cardiac function.

Plasticity of Surface Structures and β2-Adrenergic Receptor Localization in Failing Ventricular Cardiomyocytes During Recovery from Heart Failure

Background— Cardiomyocyte surface morphology and T-tubular structure are significantly disrupted in chronic heart failure, with important functional sequelae, including redistribution of sarcolemmal &bgr;2-adrenergic receptors (&bgr;2AR) and localized secondary messenger signaling. Plasticity of these changes in the reverse remodeled failing ventricle is unknown. We used AAV9.SERCA2a gene therapy to rescue failing rat hearts and measured z-groove index, T-tubule density, and compartmentalized &bgr;2AR-mediated cAMP signals, using a combined nanoscale scanning ion conductance microscopy-Förster resonance energy transfer technique. Methods and Results— Cardiomyocyte surface morphology, quantified by z-groove index and T-tubule density, was normalized in reverse-remodeled hearts after SERCA2a gene therapy. Recovery of sarcolemmal microstructure correlated with functional &bgr;2AR redistribution back into the z-groove and T-tubular network, whereas minimal cAMP responses were initiated after local &bgr;2AR stimulation of crest membrane, as observed in failing cardiomyocytes. Improvement of &bgr;2AR localization was associated with recovery of &bgr;AR-stimulated contractile responses in rescued cardiomyocytes. Retubulation was associated with reduced spatial heterogeneity of electrically stimulated calcium transients and recovery of myocardial BIN-1 and TCAP protein expression but not junctophilin-2. Conclusions— In summary, abnormalities of sarcolemmal structure in heart failure show plasticity with reappearance of z-grooves and T-tubules in reverse-remodeled hearts. Recovery of surface topology is necessary for normalization of &bgr;2AR location and signaling responses.

Measurements of Ca2+ entry and sarcoplasmic reticulum Ca2+ content during the cardiac cycle in guinea pig and rat ventricular myocytes

This study investigates the contribution of Ca2+ entry via sarcolemmal (SL) Ca2+ channels to the Ca2+ transient and its relationship with sarcoplasmic reticulum (SR) Ca2+ content during steady-state contraction in guinea pig and rat ventricular myocytes. The action potential clamp technique was used to obtain physiologically relevant changes in membrane potential. A method is shown that allows calculation of Ca2+ entry through the SL Ca2+ channels by measuring Cd(2+)-sensitive current during the whole cardiac cycle. SR Ca2+ content was calculated from caffeine-induced transient inward current. In guinea pig cardiac myocytes stimulated at 0.5 Hz and 0.2 Hz, Ca2+ entry through SL Ca2+ channels during a cardiac cycle was approximately 30% and approximately 50%, respectively, of the SR Ca2+ content. In rat myocytes Ca2+ entry via SL Ca2+ channels at 0.5 Hz was approximately 3.5% of the SR Ca2+ content. In the presence of 500 nM thapsigargin Ca2+ entry via SL Ca2+ channels in guinea pig cardiac cells was 39% greater than in controls, suggesting a larger contribution of this mechanism to the Ca2+ transient when the SR is depleted of Ca2+. These results provide quantitative support to the understanding of the relationship between Ca2+ entry and the SR Ca2+ content and may help to explain differences in the Ca2+ handling observed in different species.

SERCA2a Gene Transfer Decreases SR Calcium Leak and Reduces Ventricular Arrhythmias in a Model of Chronic Heart Failure

Background—Sarcoplasmic reticulum calcium ATPase 2a (SERCA2a) gene therapy improves mechanical function in heart failure and is under evaluation in a clinical trial. A critical question is whether SERCA2a gene therapy predisposes to increased sarcoplasmic reticulum calcium (SR Ca2+) leak, cellular triggered activity, and ventricular arrhythmias in the failing heart. Methods and Results—We studied the influence of SERCA2a gene therapy on ventricular arrhythmogenesis in a rat chronic heart failure model. ECG telemetry studies revealed a significant antiarrhythmic effect of SERCA2a gene therapy with reduction of both spontaneous and catecholamine-induced arrhythmias in vivo. SERCA2a gene therapy also reduced susceptibility to reentry arrhythmias in ex vivo programmed electrical stimulation studies. Subcellular Ca2+ homeostasis and spontaneous SR Ca2+ leak characteristics were measured in failing cardiomyocytes transfected in vivo with a novel AAV9.SERCA2a vector. SR Ca2+ leak was reduced after SERCA2a gene therapy, with reversal of the greater spark mass observed in the failing myocytes, despite normalization of SR Ca2+ load. SERCA2a reduced ryanodine receptor phosphorylation, thereby resetting SR Ca2+ leak threshold, leading to reduced triggered activity in vitro. Both indirect effects of reverse remodeling and direct SERCA2a effects appear to underlie the antiarrhythmic action. Conclusions—SERCA2a gene therapy stabilizes SR Ca2+ load, reduces ryanodine receptor phosphorylation and decreases SR Ca2+ leak, and reduces cellular triggered activity in vitro and spontaneous and catecholamine-induced ventricular arrhythmias in vivo in failing hearts. SERCA2a gene therapy did not therefore predispose to arrhythmias and may represent a novel antiarrhythmic strategy in heart failure.