Cytoskeletal protein 4.1R affects repolarization and regulates calcium handling in the heart.

The 4.1 proteins are a family of multifunctional adaptor proteins. They promote the mechanical stability of plasma membranes by interaction with the cytoskeletal proteins spectrin and actin and are required for the cell surface expression of a number of transmembrane proteins. Protein 4.1R is expressed in heart and upregulated in deteriorating human heart failure, but its functional role in myocardium is unknown. To investigate the role of protein 4.1R on myocardial contractility and electrophysiology, we studied 4.1R-deficient (knockout) mice (4.1R KO). ECG analysis revealed reduced heart rate with prolonged Q-T interval in 4.1R KO. No changes in ejection fraction and fractional shortening, assessed by echocardiography, were found. The action potential duration in isolated ventricular myocytes was prolonged in 4.1R KO. Ca(2+) transients were larger and slower to decay in 4.1R KO. The sarcoplasmic reticulum Ca(2+) content and Ca(2+) sparks frequency were increased. The Na(+)/Ca(2+) exchanger current density was reduced in 4.1R KO. The transient inward current inactivation was faster and the persistent Na(+) current density was increased in the 4.1R KO group, with possible effects on action potential duration. Although no major morphological changes were noted, 4.1R KO hearts showed reduced expression of NaV1.5alpha and increased expression of protein 4.1G. Our data indicate an unexpected and novel role for the cytoskeletal protein 4.1R in modulating the functional properties of several cardiac ion transporters with consequences on cardiac electrophysiology and with possible significant roles during normal cardiac function and disease.

Role and possible mechanisms of clenbuterol in enhancing reverse remodelling during mechanical unloading in murine heart failure.

AIMS: Combined left ventricular assist device (LVAD) and pharmacological therapy has been proposed to favour myocardial recovery in patients with end-stage heart failure (HF). Clenbuterol (Clen), a beta(2)-adrenoceptor (beta(2)-AR) agonist, has been used as a part of this strategy. In this study, we investigated the direct effects of clenbuterol on unloaded myocardium in HF. METHODS AND RESULTS: Left coronary artery ligation or sham operation was performed in male Lewis rats. After 4-6 weeks, heterotopic abdominal transplantation of the failing hearts into normal recipients was performed to induce LV unloading (UN). Recipient rats were treated with saline (Sal) or clenbuterol (2 mg/kg/day) via osmotic minipumps (HF + UN + Sal or HF + UN + Clen) for 7 days. Non-transplanted HF animals were treated with Sal (Sham + Sal, HF + Sal) or clenbuterol (HF + Clen). LV myocytes were isolated and studied using optical, fluorescence, and electrophysiological techniques. Clenbuterol treatment improved in vivo LV function measured with echocardiography (LVEF (%): HF 35.9 +/- 2 [16], HF + Clen 52.1 +/- 1.4 [16]; P < 0.001; mean +/- SEM [n]). In combination with unloading, clenbuterol increased sarcomere shortening (amplitude (microm): HF + UN + Clen 0.1 +/- 0.01 [50], HF + UN + Sal 0.07 +/- 0.01 [38]; P < 0.001) by normalizing the depressed myofilament sensitivity to Ca(2+) (slope of the linear relationship between Ca(2+) transient and sarcomere shortening hysteresis loop during relaxation (microm/ratio unit): HF + UN + Clen 2.13 +/- 0.2 [52], HF + UN + Sal 1.42 +/- 0.13 [38]; P < 0.05). CONCLUSION: Clenbuterol treatment of failing rat hearts, alone or in combination with mechanical unloading, improves LV function at the whole-heart and cellular levels by affecting cell morphology, excitation-contraction coupling, and myofilament sensitivity to calcium. This study supports the use of this drug in the strategy to enhance recovery in HF patients treated with LVADs and also begins to elucidate some of the possible cellular mechanisms responsible for the improvement in LV function.

The role of the cardiac Na+/Ca2+ exchanger in reverse remodeling: relevance for LVAD-recovery.

Different strategies can, at least in certain conditions, prevent or reverse myocardial remodeling due to heart failure and induce myocardial functional improvement. Na+/Ca2+ exchanger (NCX) is considered a major player in the pathophysiology of heart failure but its role in reverse remodeling is unknown. A combination of mechanical unloading by left ventricular assist devices (LVADs) and pharmacological therapy has been shown to induce clinical recovery in a limited number of patients with end-stage heart failure. In myocytes isolated from these patients we found that, after LVAD treatment, NCX1/SERCA2a mRNA was 38% higher than at device implant. We studied the ability of NCX to extrude Ca2+ during caffeine-induced SR Ca2+ release in isolated ventricular myocytes from these patients. The time constant of decline was slower in heart failure. In myocytes from patients with clinical recovery following mechanical and pharmacological treatment, NCX1-mediated Ca2+ extrusion was faster compared with myocytes from patient who, despite identical treatment, did not recover. We propose that increased NCX function may be associated with reverse remodeling in patients and that factors that regulate NCX function (i.e., phosphorylation or intracellular [Na+]) other than NCX expression levels alone, may have detrimental consequences on cardiac function.

Evaluation of frequency, type, and function of gap junctions between skeletal myoblasts overexpressing connexin43 and cardiomyocytes: relevance to cell transplantation.

Cell transplantation of skeletal myoblasts (SMs) is one possible treatment for repairing cardiac tissue after myocardial injury. However, inappropriate electrical coupling between grafted SMs and host cardiomyocytes may be responsible for the arrhythmias observed in clinical trials of SM transplantation. Whether functional gap junctions occur between the two cell types remains controversial. We have studied the ability of SMs to electrically couple with isolated adult rat cardiomyocytes (CMs) and assessed whether connexin43 (Cx43) overexpression enhanced gap junctional conductance (Gj). C2C12 myoblast lines overexpressing Cx43 were generated by gene transfection and clonal selection. CMs were cocultured with either SMs overexpressing Cx43 (CM-SM(Cx43)) or control SMs (CM-SM(WT)) in vitro. Gj between pairs of SMs and CMs was quantified with dual whole cell patch clamping. Formation of Gj occurred between 22% of CM-SM(WT) pairs (n=73) and 48% of CM-SM(Cx43) pairs (n=71, P<0.001). The Gj of CM-SM(Cx43) pairs (29.7+/-4.3 nS, n=21) was greater than that of CM-SM(WT) pairs (14.8+/-2.0 nS, n=12, P<0.05). The overexpression of Cx43 in SMs increased the formation of electrical communication and the steady-state conductance between SMs and CMs. Enhanced gap junctional conductance may be useful to promote the integration of transplanted SMs into the myocardium.

The red wine polyphenol, resveratrol, exerts acute direct actions on guinea-pig ventricular myocytes.

Epidemiological evidence suggests that moderate consumption of red wine may be cardioprotective, although the precise mechanism(s) responsible remains poorly understood. We hypothesized that the red wine polyphenol, resveratrol, may exert direct actions on the heart and thus potentially contribute to cardioprotection. We show that resveratrol acutely decreases Ca2+ transient amplitude in isolated cardiac myocytes. Intriguingly, resveratrol simultaneously increases cell shortening in half the cells tested, while decreasing shortening in the other half. The former could be attributed to heightened myofilament Ca2+ sensitivity. This was no longer observed in myocytes that had been incubated with the oestrogen receptor antagonist, ICI 182,780, suggesting an oestrogen-receptor dependent mechanism of action. In addition, resveratrol significantly decreased action potential duration and the peak L-type Ca2+ current. Our findings provide evidence that resveratrol exerts multiple direct actions on cardiac myocytes, the net result of which is no overall change in cell contraction. The clinical significance of these results remains to be determined.

Gender determines the acute actions of genistein on intracellular calcium regulation in the guinea-pig heart.

OBJECTIVES: The soy-isoflavone, genistein, appears to be cardioprotective partly through direct actions on the heart, although the relative benefits between men and women are not fully known. The purpose of the present study was to determine whether gender influences the acute electrophysiological actions of genistein at the level of isolated cardiac myocytes and to elucidate the mechanisms involved. METHODS: Left ventricular myocytes, isolated from weight-matched male and female guinea-pigs and rats, were field stimulated at a rate of 1 Hz in a superfusion chamber (37 degrees C). The effects of acute application of genistein on cell shortening and the Ca(2+) transients were measured. Electrophysiological recordings were performed using single electrode voltage-clamp. RESULTS: Genistein increased cell contraction and the Ca(2+) transients in a concentration-dependent manner in myocytes from male guinea-pigs [by 54+/-11% and 22+/-4%, respectively (mean+/-S.E.M., p<0.001, n=18) at 40 microM], while having no significant corresponding effect in those from females. In contrast, genistein increased both parameters in myocytes obtained from male and female rats. The changes in guinea-pigs occurred despite inhibition of the L-type Ca(2+) current in both sexes (n>23, p<0.001). In order to explain these observations, we measured sarcoplasmic reticulum (SR) Ca(2+) contents by integrating the Na(+)/Ca(2+) exchanger currents (I(NCX)) following rapid caffeine application. Genistein increased I(NCX) integrals by 27% in males (n=12, p<0.01) and 20% in females (n=14, p<0.01). The increased SR Ca(2+) load in males, but not females, could be related to an impaired ability of the Na(+)/Ca(2+) exchanger to extrude Ca(2+). CONCLUSIONS: We have demonstrated novel, gender-related differences in the acute cardiac actions of genistein, which can be attributed to actions at distinct points of the intracellular Ca(2+) cycle. Our results suggest that genistein may afford greater cardioprotection in females than in males.

The effects of overexpression of the Na+/Ca2+ exchanger on calcium regulation in hypertrophied mouse cardiac myocytes.

In cardiac hypertrophy and failure it has been shown that the amount of Na/Ca exchanger protein can increase. Several studies have investigated this modification in overt heart failure. However, the role of Na/Ca exchanger overexpression during the development of hypertrophy is unknown. To address this question we investigated Ca2+ regulation in an early stage of cardiac hypertrophy before signs of heart failure occurred and evaluated the role of Na/Ca exchanger overexpression. Cardiac hypertrophy was induced by a constant infusion of angiotensin II (Ang, 1 microg/min/kg) via an osmotic pump for 14 days. Thereafter, ventricular myocytes from either wild type (NON) or transgenic mice overexpressing the Na/Ca exchanger (TR) were isolated. Myocytes were loaded with indo-1 AM or fluo-4 AM to monitor cytoplasmic [Ca2+] with all experiments performed at 37 degrees C. In myocytes exposed to Ang there was an increase in cell capacitance of more than 20% indicating cellular hypertrophy. Ca2+ transients were prolonged in hypertrophied NON myocytes but not in TR myocytes. Action potentials had a less negative plateau in TR myocytes. Sarcoplasmic reticulum (SR) Ca2+ content, measured using rapid caffeine application, was greater in TR myocytes but unaffected by hypertrophy. Ca2+ spark frequency was significantly greater in TR. Na/Ca exchanger overexpression prevented the prolongation of the Ca2+ transient observed in hypertrophy and maintained a similar SR Ca2+ leak suggesting a compensatory role in Ca2+ regulation in hypertrophied cardiac myocytes from transgenic mice. We suggest this compensatory effect is mediated by increased SR Ca2+ content and faster Ca2+ removal via the Na/Ca exchanger.

Raloxifene acutely suppresses ventricular myocyte contractility through inhibition of the L-type calcium current.

1. The selective oestrogen (ER) receptor modulator, raloxifene, is widely used in the treatment of postmenopausal osteoporosis, but may also possess cardioprotective properties. We investigated whether it directly suppresses myocyte contractility through Ca(2+) channel antagonism in a similar way to 17beta-oestradiol. 2. Cell shortening and Ca(2+) transients were measured in single guinea-pig ventricular myocytes field-stimulated (1 Hz, 37 degrees C) in a superfusion chamber. Electrophysiological recordings were performed using single electrode voltage-clamp. 3. Raloxifene decreased cell shortening (EC(50) 2.4 microm) and the Ca(2+) transient amplitude (EC(50) 6.4 microm) in a concentration-dependent manner. At a concentration of 1 microm, raloxifene produced a 33+/-2% (mean+/-s.e.m) and 24+/-2% reduction, respectively (P<0.001, n=14 for both parameters). 4. These inhibitory actions were not observed in myocytes that had been incubated with the specific antagonist, ICI 182,780 (10 microm) (n=11). 5. Raloxifene (1 microm) shortened action potential durations at 50 and 90% repolarisation (P<0.05 and <0.001, respectively; n=27) and decreased peak L-type Ca(2+) current by 45%, from -5.1+/-0.5 pA/pF to -2.8+/-0.3 pA/pF (P<0.001, n=18). 6. Raloxifene did not significantly alter sarcoplasmic reticulum Ca(2+) content, as assessed by integrating the Na(+)/Ca(2+) exchanger currents following rapid caffeine application. 7. The present study provides evidence for direct inhibitory actions of raloxifene on ventricular myocyte contractility, mediated through Ca(2+) channel antagonism.

Acute actions of 3 alpha-hydroxy-tibolone on factors influencing contraction in guinea-pig ventricular myocytes.

We investigated whether one of the main estrogenic metabolites of the postmenopausal agent, tibolone (Org OD14), exerts direct cardiac actions in a similar way to 17beta-estradiol. 3alpha-OH-tibolone (40 microM) decreased both cell shortening and Ca2+ transient amplitude of field-stimulated (1 Hz, 37 degrees C) guinea-pig ventricular myocytes. These effects were still observed in cells that had been incubated with the specific estrogen receptor antagonist, ICI 182,780 (C(32)H(47)F(5)O(3)S), suggesting an estrogen receptor-independent mechanism of action. In addition, 3alpha-OH-tibolone inhibited the L-type Ca2+ current and shortened action potential durations (APD). This mechanism may contribute to a potential cardiovascular action of tibolone.

Adult progenitor cell transplantation influences contractile performance and calcium handling of recipient cardiomyocytes.

Adult progenitor cell transplantation has been proposed for the treatment of heart failure, but the mechanisms effecting functional improvements remain unknown. The aim of this study was to test the hypothesis that, in failing hearts treated with cell transplantation, the mechanical properties and excitation-contraction coupling of recipient cardiomyocytes are altered. Adult rats underwent coronary artery ligation, leading to myocardial infarction and chronic heart failure. After 3 wk, they received intramyocardial injections of either 10(7) green fluorescence protein (GFP)-positive bone marrow mononuclear cells or 5 x 10(6) GFP-positive skeletal myoblasts. Four weeks after injection, both cell types increased ejection fraction and reduced cardiomyocyte size. The contractility of isolated GFP-negative cardiomyocytes was monitored by sarcomere shortening assessment, Ca(2+) handling by indo-1 and fluo-4 fluorescence, and electrophysiology by patch-clamping techniques. Injection of either bone marrow cells or skeletal myoblasts normalized the impaired contractile performance and the prolonged time to peak of the Ca(2+) transient observed in failing cardiomyocytes. The smaller and slower L-type Ca(2+) current observed in heart failure normalized after skeletal myoblast, but not bone marrow cell, transplantation. Measurement of Ca(2+) sparks suggested a normalization of sarcoplasmic reticulum Ca(2+) leak after skeletal myoblast transplantation. The increased Ca(2+) wave frequency observed in failing myocytes was reduced by either bone marrow cells or skeletal myoblasts. In conclusion, the morphology, contractile performance, and excitation-contraction coupling of individual recipient cardiomyocytes are altered in failing hearts treated with adult progenitor cell transplantation.

Identification of cell-specific soluble mediators and cellular targets during cell therapy for the treatment of heart failure.

Cell therapy, the transplantation of progenitor cells into the myocardium, has been proposed as a possible treatment strategy for heart failure. Despite the lack of repopulation of the heart with progenitor cells, cell therapy induces a modest but well-documented functional improvement in patients. It is thought that paracrine mechanisms may account for the observed changes in heart function. However, there is little evidence that directly supports this hypothesis. We discuss the current views in the literature and present some preliminary data proposing that adult progenitor cells influence contractility and Ca2+ handling in neighboring failing cardiomyocytes by soluble mediators. This can be tested using a co-culture system. Our results suggest that soluble mediators from adult progenitor cells can enhance failing cardiomyocyte function, supporting the paracrine hypothesis. This co-culture strategy can be employed to identify cell-specific soluble mediators and their cellular targets during cell therapy for the treatment of heart disease.

Prolonged mechanical unloading reduces myofilament sensitivity to calcium and sarcoplasmic reticulum calcium uptake leading to contractile dysfunction.

BACKGROUND: Prolonged unloading using left ventricular (LV) assist devices (LVADs) leads to unloading-induced atrophy with altered cardiomyocyte contractility. The causes for this time-dependent deterioration of myocardial function are unclear. Our aim was to determine the effects of prolonged mechanical unloading on cardiomyocyte function and, more specifically, on Ca(2+) cycling and myofilament sensitivity to Ca(2+). METHODS: LV unloading was induced by heterotopic abdominal transplantation (UN) in rats for 5 weeks. Recipient hearts were used as controls (REC). LV myocytes were isolated and cardiomyocyte area measured by planimetry, sarcomere length measured by Fourier analysis of digitized cardiomyocyte images, and cytoplasmic [Ca(2+)] monitored using Indo-1. Myofilament sensitivity to Ca(2+) was assessed as the slope of the linear relationship between Indo-1 ratio and sarcomere shortening during relaxation. RESULTS: UN cardiomyocyte area was smaller compared with REC (mean +/- SEM: UN 2,503 +/- 78 microm(2) [n = 132], REC 3,856 +/- 89 microm(2) [n = 116]; p < 0.001). UN cardiomyocytes had a smaller sarcomere shortening amplitude (UN 0.08 +/- 0.01 microm [n = 37], REC 0.11 +/- 0.01 microm [n = 38]; p < 0.01), despite normal Ca(2+) transient amplitude (UN 0.13 +/- 0.01 Indo-1 ratio units [n = 37], REC 0.11 +/- 0.01 Indo-1 ratio units [n = 38]; p = non-significant). Myofilament sensitivity to Ca(2+) was reduced in UN (UN 2.0 +/- 1.2 microm/ratio unit [n = 20], REC 3.7 +/- 0.4 microm/ratio unit [n = 22]; p < 0.01). Sarcoplasmic reticulum (SR) Ca(2+) uptake (assessed by 20 mmol/liter caffeine) was also reduced in UN (UN 84.3 +/- 0.79% relative contribution [n = 22], REC 89.8 +/- 0.67% relative contribution [n = 24]; p < 0.001). CONCLUSIONS: Prolonged myocardial unloading causes depressed contractility due to reduced SR Ca(2+) uptake and myofilament sensitivity to Ca(2+). These effects may be relevant with regard to myocardial performance after prolonged LVAD support.

Direct effects of apelin on cardiomyocyte contractility and electrophysiology.

Apelin, the ligand for the angiotensin receptor like-1, has been implicated in the pathogenesis of atrial fibrillation and heart failure. However, it is unknown if apelin has direct effects on cardiomyocyte contractility and electrophysiology. APJ-like immunoreactivity was localized to T-tubules and intercalated disc area in isolated adult rat ventricular myocytes. Apelin (1 nM) significantly increased sarcomere shortening in normal as well as failing cardiomyocytes. The transient increase in shortening was not accompanied by increased [Ca(2+)] transient amplitude. Apelin significantly activated the sarcolemmal Na(+)/H(+) exchanger (NHE) and increased intracellular pH. Moreover, apelin (10 nM) increased conduction velocity in monolayers of cultured neonatal rat cardiac myocytes. Our results demonstrate for the first time that apelin has direct effects on the propagation of action potential and contractility in cardiomyocytes. One of the mechanisms involved in the inotropic effect may be an increased myofilament sensitivity to Ca(2+) as apelin enhanced the activity of NHE with consequent intracellular alkalinization.