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 b 2 -adrenoceptor (b 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 þ UN þ Sal or HF þ UN þ Clen) for 7 days. Non-transplanted HF animals were treated with Sal (Sham þ Sal, HF þ Sal) or clenbuterol (HF þ Clen). LV myocytes were isolated and studied using optical, fluorescence, and electrophysiological techniques. 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

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 107 green fluorescence protein (GFP)-positive bone marrow mononuclear cells or 5 × 106 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, Ca2+ 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 Ca2+ transient observed in failing cardiomyocytes. The smaller and slower L-type Ca2+ current observed in heart failure normalized after skeletal myoblast, but not bone marrow cell, transplantation. Measurement of Ca2+ sparks suggested a normalization of sarcoplasmic reticulum Ca2+ leak after skeletal myoblast transplantation. The increased Ca2+ 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

Cytoskeletal Protein 4.1R Affects Sodium Current in Cardiomyocytes from Transgenic Mice with Prolonged QT Interval

The 4.1 proteins are part of the spectrin-associated cytoskeleton, promote mechanical stability of plasma membranes and are required for cell surface expression of several ion transporters. Protein 4.1R is expressed in the heart and upregulated in human heart failure but its functional role in the myocardium is unknown. 4.1R deficient mice (4.1RKO) have prolonged QT interval and, at cardiomyocyte level, prolonged action potential duration and Ca2+ regulation abnormalities. The causes for these changes remain elusive. We have measured, using whole-cell patch clamping of isolated cardiomyocytes from 4.1RKO, the persistent Na+ current (IpNa), implicated in long QT interval in patients. From a holding potential of -100 mV increasing voltage (20 mV) steps from –80mV to +20 mV for 1 s were applied. The protocol was repeated in the presence of 30 µM tetradotoxin (TTX), and the TTX-sensitive current was analysed. IpNa density was increased in 4.1RKO myocytes compared with wild type (WT) (average IpNa density between 50 and 100 ms after depolarisation for steps to –20 mV (pA/pF): WT = –0.53 ± 0.03 (16); 4.1R KO = –0.36 ± 0.05 (25) (Mean ± SEM (n)); p<0.01; IpNa integral between 50 and 300 ms after depolarisation (pC/pF): WT = –57 ± 9 (16); 4.1R KO = –98 ± 9 (25); p<0.01). To evaluate possible direct binding between SCN5a and 4.1R, a peptide biotin-EPITTTLRRKHEEVSA was synthesized (SCN5a-pep: corresponding to residues 1893–1909 of SCN5a and similar to a 4.1R binding sequence of anion exchanger AE1). A scrambled version of the wild type peptide was used as control. The peptides were mixed with extracts from COS7 cells that had been transfected with a mouse 120kDa cardiac 4.1R tagged with the HA epitope, or extract from cells transfected with vector only. After incubation, bound complexes were recovered on streptavidin beads, and analysed by Western blotting using anti-HA antibody. The 120kDa band corresponding to 4.1R was found only in the SCN5a-pep and not in the control peptide pull-down suggesting that SCN5a can bind 4.1R. Our data indicate a novel function for protein 4.1R in modulating the properties of the Na+ channel, possibly by direct interaction. This may underlie a significant role of proteins 4.1 on the electrophysiology of the heart in normal conditions and in disease

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. Ca2+ transients were larger and slower to decay in 4.1R KO. The sarcoplasmic reticulum Ca2+ content and Ca2+ sparks frequency were increased. The Na+/Ca2+ 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 NaV1.5{alpha} 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

Prolonged mechanical unloading affects cardiomyocyte excitation-contraction coupling, transverse-tubule structure, and the cell surface

Prolonged mechanical unloading (UN) of the heart is associated with detrimental changes to the structure and function of cardiomyocytes. The mechanisms underlying these changes are unknown. In this study, we report the influence of UN on excitation-contraction coupling, Ca2+-induced Ca2+ release (CICR) in particular, and transverse (t)-tubule structure. UN was induced in male Lewis rat hearts by heterotopic abdominal heart transplantation. Left ventricular cardiomyocytes were isolated from the transplanted hearts after 4 wk and studied using whole-cell patch clamping, confocal microscopy, and scanning ion conductance microscopy (SICM). Recipient hearts were used as control (C). UN reduced the volume of cardiomyocytes by 56.5% compared with C (UN, n=90; C, n=59; P<0.001). The variance of time-to-peak of the Ca2+ transients was significantly increased in unloaded cardiomyocytes (UN 227.4±24.9 ms2, n=42 vs. C 157.8±18.0 ms2, n=40; P<0.05). UN did not alter the action potential morphology or whole-cell L-type Ca2+ current compared with C, but caused a significantly higher Ca2+ spark frequency (UN 3.718±0.85 events/100 μm/s, n=47 vs. C 0.908±0.186 events/100 μm/s, n=45; P<0.05). Confocal studies showed irregular distribution of the t tubules (power of the normal t-tubule frequency: UN 8.13±1.12×105, n=57 vs. C 20.60± 3.174×105, n=56; P<0.001) and SICM studies revealed a profound disruption to the openings of the t tubules and the cell surface in unloaded cardiomyocytes. We show that UN leads to a functional uncoupling of the CICR process and identify disruption of the t-tubule-sarcoplasmic reticulum interaction as a possible mechanism.—Ibrahim, M., Al Masri, A., Navaratnarajah, M., Siedlecka, U., Soppa, G. K., Moshkov, A., Abou Al-Saud, S., Gorelik, J., Yacoub, M. H., Terracciano, C. M. N. Prolonged mechanical unloading affects cardiomyocyte excitation-contraction coupling, transverse-tubule structure, and the cell surface