Claudia Altomare

Heteromeric HCN1–HCN4 channels: a comparison with native pacemaker channels from the rabbit sinoatrial node

‘Funny‐’ (f‐) channels of cardiac sino‐atrial node (SAN) cells are key players in the process of pacemaker generation and mediate the modulatory action of autonomic transmitters on heart rate. The molecular components of f‐channels are the hyperpolarization‐activated, cyclic nucleotide‐gated (HCN) channels. Of the four HCN isoforms known, two (HCN4 and HCN1) are expressed in the rabbit SAN at significant levels. However, the properties of f‐channels of SAN cells do not conform to specific features of the two isoforms expressed locally. For example, activation kinetics and cAMP sensitivity of native pacemaker channels are intermediate between those reported for HCN1 and HCN4. Here we have explored the possibility that both HCN4 and HCN1 isoforms contribute to the native If in SAN cells by co‐assembling into heteromeric channels. To this end, we used heterologous expression in human embryonic kidney (HEK) 293 cells to investigate the kinetics and cAMP response of the current generated by co‐transfected (HCN4 + HCN1) and concatenated (HCN4‐HCN1 (4–1) tandem or HCN1‐HCN4 (1–4) tandem) rabbit constructs and compared them with those of the native f‐current from rabbit SAN. 4–1 tandem, but not co‐transfected, currents had activation kinetics approaching those of If; however, the activation range of 4–1 tandem channels was more negative than that of the f‐channel and their cAMP sensitivity were poorer (although that of 1–4 tandem channels was normal). Co‐transfection of 4–1 tandem channels with minK‐related protein 1(MiRP1) did not alter their properties. HCN1 and HCN4 may contribute to native f‐channels, but a ‘context’‐dependent mechanism is also likely to modulate the channel properties in native tissues.

miR669a and miR669q prevent skeletal muscle differentiation in postnatal cardiac progenitors

miR669a and miR669q inhibit postnatal cardiac progenitor differentiation by directly targeting the 3′UTR of MyoD.

Functional characterisation and subcellular localisation of HCN1 channels in rabbit retinal rod photoreceptors

Gating of voltage‐dependent conductances in retinal photoreceptors is the first step of a process leading to the enhancement of the temporal performance of the visual system. The molecular components underlying voltage‐dependent gating in rods are presently poorly defined. In the present work we have investigated the isoform composition and the functional characteristics of hyperpolarisation‐activated cyclic nucleotide‐gated channels (HCN) in rabbit rods. Using immunocytochemistry we show the expression in the inner segment and cell body of the isoform 1 (HCN1). Electrophysiological investigations show that hyperpolarisation‐activated currents (Ih) can be measured only from the cell regions where HCN1 is expressed. Half‐activation voltage (–75.0 ± 0.3 mV) and kinetics (t1/2 of 101 ± 8 ms at –110 mV and 20 °C) of the Ih in rods are similar to those of the macroscopic current carried by homomeric rabbit HCN1 channels expressed in HEK 293 cells. The homomeric nature of HCN1 channels in rods is compatible with the observation that cAMP induces a small shift (2.3 ± 0.8 mV) in the half‐activation voltage of Ih. In addition, the observation that within the physiological range of membrane potentials, cAMP does not significantly affect the gain of the current‐to‐voltage conversion, may reflect the need to protect the first step in the processing of visual signals from changes in cAMP turnover.

Effects of dronedarone on Acetylcholine-activated current in rabbit SAN cells

The effect of the antiarrhythmic drug dronedarone on the Acetylcholine‐activated K+ current (IK(ACh)) was investigated in single cells isolated from sinoatrial node (SAN) tissue of rabbit hearts. Externally perfused dronedarone (0.001–1 μM) caused a potent, voltage independent block of IK(ACh). Fitting of the dose response curve of IK(ACh) block yielded an IC50 value of 63 nM, a value over one order of magnitude lower than those reported for dronedarone block of other cardiac currents. IK(ACh) block was not due to an inhibitory action of dronedarone on the muscarinic M2 receptor activation, since the drug was effective on IK(ACh) constitutively activated by intracellular perfusion with GTP‐γS. External cell perfusion with dronedarone inhibited the activity of IK(ACh) channels recorded from cell‐attached patches by reducing the channel open probability (from 0.56 to 0.11) without modification of the single‐channel conductance. These data suggest that dronedarone blocks IK(ACh) channels either by disrupting the G‐protein‐mediated activation or by a direct inhibitory interaction with the channel protein.

Action of internal pronase on the f‐channel kinetics in the rabbit SA node

1 The hyperpolarization‐activated If current was recorded in inside‐out macropatches from sino‐atrial (SA) node myocytes during exposure of their intracellular side to pronase, in an attempt to verify if cytoplasmic f‐channel domains are involved in both voltage‐ and cAMP‐dependent gating. 2 Superfusion with pronase caused a quick, dramatic acceleration of channel opening upon hyperpolarization and slowing, rapidly progressing into full blockade, of channel closing upon depolarization; these changes persisted after wash off of pronase and were irreversible, indicating proteolytic cleavage of channel regions which contribute to gating. 3 I f recorded from patches normally responding to cAMP became totally insensitive to cAMP following pronase treatment, indicating partial or total removal of channel regions involved in the cAMP‐dependent activation. 4 The fully activated I‐V relationship was not modified by pronase, indicating that internal proteolysis did not affect the f‐channel conductance. 5 The changes in If kinetics induced by pronase were due to a large depolarizing shift of the f‐channel open probability curve (56.5 ± 1.1 mV, n= 7). 6 These results are consistent with the hypothesis that cytoplasmic f‐channel regions are implicated in dual voltage‐ and cAMP‐dependent gating; also, since pronase does not abolish hyperpolarization‐activated opening, an intrinsic voltage‐dependent gating mechanism must exist which is inaccessible to proteolytic cleavage. A model scheme able to account for these data thus includes an intrinsic gating mechanism operating at depolarized voltages, and a blocking mechanism coupled to cAMP binding to the channel.

Rate dependency of β‐adrenergic modulation of repolarizing currents in the guinea‐pig ventricle

β‐Adrenergic stimulation modulates ventricular currents and sinus cycle length (CL). We investigated how changes in CL affect the current induced by isoprenaline (Iso) during the action potential (AP) of guinea‐pig ventricular myocytes. Action‐potential clamp was applied at CLs of 250 and 1000 ms to measure: (1) the net current induced by 0.1 μm Iso (IIso); (2) the L‐type Ca2+ current ICaL and slow delayed rectifier current IKs components of IIso (IIsoCa and IIsoK), identified as the Iso‐induced current sensitive to nifedipine and HMR1556, respectively; and (3) IIso persisting after inhibition of both ICa and IKs (IisoR). The pause dependency of IKs and its modulation were evaluated in voltage‐clamp experiments. The rate dependency of the duration of the action potential at 90% repolarization (APD90) and its modulation by isoprenaline were tested in current‐clamp experiments. At a CL of 250 ms IIso was inward during initial repolarization and reversed at 59% of APD90. At a CL of 1000 ms IIso became mostly inward in all cells. Switching to shorter CL did not change IIsoCa and IIsoK amplitudes, but moved their peak amplitudes to earlier repolarization; IIsoR was independent of CL. Acceleration of IIsoK at shorter CL was based on faster pause dependency of IKs activation rate. The ‘restitution’ of activation rates was modulated by isoprenaline. The APD90–CL relation was rotated anticlockwise by isoprenaline and crossed the control curve at a CL of 150 ms (400 beats min−1). We conclude that: (1) isoprenaline induced markedly different current profiles according to pacing rate, involving CL‐dependent ICa and IKs modulation; (2) the effect of isoprenaline on APD90 was CL dependent, and negligible during tachycardia; and (3) during sympathetic activation, repolarization stability may involve matched modulation of sinus rate and repolarizing currents.

Elucidating arrhythmogenic mechanisms of long-QT syndrome CALM1-F142L mutation in patient-specific induced pluripotent stem cell-derived cardiomyocytes

Aims Calmodulin (CaM) is a small protein, encoded by three genes (CALM1-3), exerting multiple Ca2+-dependent modulatory roles. A mutation (F142L) affecting only one of the six CALM alleles is associated with long QT syndrome (LQTS) characterized by recurrent cardiac arrests. This phenotypic severity is unexpected from the predicted allelic balance. In this work, the effects of heterozygous CALM1-F142L have been investigated in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) obtained from a LQTS patient carrying the F142L mutation, i.e. in the context of native allelic ratio and potential gene modifiers. Methods and Results Skin fibroblasts of the mutation carrier and two unrelated healthy subjects (controls) were reprogrammed to hiPSC and differentiated into hiPSC-CMs. Scanty IK1 expression, an hiPSC-CMs feature potentially biasing repolarization, was corrected by addition of simulated IK1 (Dynamic-Clamp). Abnormalities in repolarization rate-dependency (in single cells and cell aggregates), membrane currents and intracellular Ca2+ dynamics were evaluated as putative arrhythmogenic factors. CALM1-F142L prolonged repolarization, altered its rate-dependency and its response to isoproterenol. This was associated with severe impairment of Ca2+-dependent inactivation (CDI) of ICaL, resulting in augmented inward current during the plateau phase. As a result, the repolarization of mutant cells failed to adapt to high pacing rates, a finding well reproduced by using a recent hiPSC-CM action potential model. The mutation failed to affect IKs and INaL and changed If only marginally. Intracellular Ca2+ dynamics and Ca2+ store stability were not significantly modified. Mutation-induced repolarization abnormalities were reversed by verapamil. Conclusion The main functional derangement in CALM1-F142L was prolonged repolarization with altered rate-dependency and sensitivity to &bgr;-adrenergic stimulation. Impaired CDI of ICaL underlined the electrical abnormality, which was sensitive to ICaL blockade. High mutation penetrance was confirmed in the presence of the native genotype, implying strong dominance of effects.

Bone marrow-derived cells can acquire cardiac stem cells properties in damaged heart

Experimental data suggest that cell‐based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit+ CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild‐type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate ‘cardiospheres’, a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit+ CSCs.

Modulation of sarcoplasmic reticulum function by PST2744 [istaroxime; (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride)] in a pressure-overload heart failure model.

PST2744 [Istaroxime; (E,Z)-3-((2-aminoethoxy)imino) androstane-6,17-dione hydrochloride)] is a novel inotropic agent that enhances sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA) 2 activity. We investigated the istaroxime effect on Ca2+ handling abnormalities in myocardial hypertrophy/failure (HF). Guinea pig myocytes were studied 12 weeks after aortic banding (AoB) and compared with those of sham-operated animals (sham). The gain of calcium-induced Ca2+ release (CICR), sarcoplasmic reticulum (SR) Ca2+ content, Na+/Ca2+ exchanger (NCX) function, and the rate of SR reloading after caffeine-induced depletion (SR Ca2+ uptake, measured during NCX blockade) were evaluated by measurement of cytosolic Ca2+ and membrane currents. HF characterization: AoB caused hypertrophy and failure in 100 and 25% of animals, respectively. Although CICR gain during constant pacing was preserved, SR Ca2+ content and SR Ca2+ uptake were strongly depressed. Resting Ca2+ and the slope of the Na+/Ca2+ exchanger current (INCX)/Ca2+ relationship were unchanged by AoB. Istaroxime effects: CICR gain, SR Ca2+ content, and SR Ca2+ uptake rate were increased by istaroxime in sham myocytes and, to a significantly larger extent, in AoB myocytes; this led to almost complete recovery of SR Ca2+ uptake in AoB myocytes. Istaroxime increased resting Ca2+ and the slope of the INCX/Ca2+ relationship similarly in sham and AoB myocytes. Istaroxime failed to increase SERCA activity in skeletal muscle microsomes devoid of phospholamban. Thus, clear-cut abnormalities in Ca2+ handling occurred in this model of hypertrophy, with mild decompensation. Istaroxime enhanced SR function more in HF myocytes than in normal ones; almost complete drug-induced recovery suggests a purely functional nature of SR dysfunction in this HF model.

Ranolazine prevents INaL enhancement and blunts myocardial remodelling in a model of pulmonary hypertension

AIMS Pulmonary arterial hypertension (PAH) reflects abnormal pulmonary vascular resistance and causes right ventricular (RV) hypertrophy. Enhancement of the late sodium current (INaL) may result from hypertrophic remodelling. The study tests whether: (i) constitutive INaL enhancement may occur as part of PAH-induced myocardial remodelling; (ii) ranolazine (RAN), a clinically available INaL blocker, may prevent constitutive INaL enhancement and PAH-induced myocardial remodelling. METHODS AND RESULTS PAH was induced in rats by a single monocrotaline (MCT) injection [60 mg/kg intraperitoneally (i.p.)]; studies were performed 3 weeks later. RAN (30 mg/kg bid i.p.) was administered 48 h after MCT and washed-out 15 h before studies. MCT increased RV systolic pressure and caused RV hypertrophy and loss of left ventricular (LV) mass. In the RV, collagen was increased; myocytes were enlarged with T-tubule disarray and displayed myosin heavy chain isoform switch. INaL was markedly enhanced; diastolic Ca(2+) was increased and Ca(2+) release was facilitated. K(+) currents were down-regulated and APD was prolonged. In the LV, INaL was enhanced to a lesser extent and cell Ca(2+) content was strongly depressed. Electrical remodelling was less prominent than in the RV. RAN completely prevented INaL enhancement and limited most aspects of PAH-induced remodelling, but failed to affect in vivo contractile performance. RAN blunted the MCT-induced increase in RV pressure and medial thickening in pulmonary arterioles. CONCLUSION PAH induced remodelling with chamber-specific aspects. RAN prevented constitutive INaL enhancement and blunted myocardial remodelling. Partial mechanical unloading, resulting from an unexpected effect of RAN on pulmonary vasculature, might contribute to this effect.