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Introduction Calcium handling is generally impaired in heart failure (HF). Mechanisms that can restore cardiac relaxation improving cellular Ca2 + cycling, represent a promising therapeutic approach for HF. Istaroxime is a Na-K ATPase (NKA) inhibitor with the property of accelerating Ca2 + re-uptake into sarcoplasmic reticulum (SR) through the SR Ca2 + pump (SERCA) stimulation by displacing the interaction between SERCA and its inhibitor, phospholamban (PLB). Objective To characterize Istaroxime effects in a model of mild diabetes (type 1) with diastolic dysfunction and preserved global systolic function. Istaroxime was tested at a concentration mostly unaffecting NKA to isolate its effects dependent on SERCA only. Method Streptozotocin (STZ)-treated rats were evaluated at 9 weeks after STZ injection in comparison to control (CTR) ones. STZ-induced changes were evaluated in vivo (echocardiography), in isolated left ventricular myocytes and in SERCA2a-enriched microsomes. SERCA and PLB protein levels were measured by western blot and SERCA activity as 32P hydrolysis. Action potential rate-dependency and intracellular Ca2 + handling were evaluated in patch clamped or field-stimulated (2 Hz) cells. Results STZ-induced cardiomyopathy was characterized by impaired diastolic relaxation which was associated to reduced SERCA protein level and activity at the cellular level. Moreover, the monomeric PLB/SERCA ratio was increased. In STZ group, action potentials (AP) were significantly prolonged at each cycle length; Ca2 + transients were characterized by slower decay, delayed onset and increased diastolic Ca2 + . Istaroxime at a concentration of 100 nM significantly stimulated SERCA activity and SR Ca re-uptake after caffeine depletion in STZ group only. Moreover, Istaroxime reduced STZ-induced diastolic Ca2 + enhancement but not affected AP prolongation. Conclusion SERCA stimulation can be considered a promising therapeutic approach for diastolic dysfunction treatment.

Background: De novo mutations in calmodulin genes have been recently associated with markedly prolonged QT interval and life-threatening ventricular arrhythmias in infants. Biochemical evidences suggested impaired calcium ion binding to mutant calmodulins. Calcium dependent inactivation (CDI) of L-type calcium channel (ICaL) involves the interaction between Ca2+-calmodulin and the channel protein. Aims: To explore the effects of two CALM1 mutations (F142L and D130G) on electrical activity and ICaL properties in human induced-pluripotent stem cells (iPSC)-derived cardiomyocytes (CM). Methods: Skin fibroblasts of two patients carrying the mutation CALM1-F142L or CALM1-D130G were reprogrammed to generate iPSC and these cells differentiated into CM; a healthy donor was selected as control. Two cell clones for each mutation were analyzed to rule out clone specificity. Patch clamp and micro electrode arrays (MEA) analyses were performed on isolated iPSC-derived CM and beating clusters at about 40 days of differentiation respectively. ICaL was isolated in the presence of calcium or barium ions as charge carriers. Results: CALM1-F142L iPSC-derived CM showed prolonged field potential duration (FPD) with high beta-adrenergic sensitivity; peak ICaL density was unaltered, but CDI was markedly reduced and inactivation incomplete. CALM1-D130G iPSC-derived CM showed higher ICaL density and unaltered CDI. Conclusions: F142L and D130G calmodulin mutations differently affect ICaL properties in human iPSC-derived CM. F142L mutation is consistent with reduced calcium affinity of calmodulin; D130G abnormality suggests ICaL facilitation instead, possibly resulting from upregulation of alternative calmodulin isoforms. Overall, both calmodulin mutations might potentially cause arrhythmogenic calcium overload.

BACKGROUND: enhancement of the sustained component of the sodium current (INaL) is a major factor contributing to myocardial damage. A beneficial effect of the INaL blocker Ranolazine (RAN) on glycemic control was recently described in the MERLIN-TIMI 36 trial in diabetic patients. However, the mechanism underlying this finding hasn't been elucidated yet.AIM: To characterize INaL in insulin-secreting cells (INS-1E) and evaluate its role in glucose-stimulated insulin secretion (GSIS).METHODS: INaL, identified as the steady-state current blocked by 10 μM RAN (IRAN) or 0.5 μM TTX (ITTX), and its impact on membrane potential (Vm) were assessed by patch clamp. Veratridine (VERA) was used as INaL enhancer. INaL-dependent intracellular Ca2+ changes were detected by Fluo-4AM. To simulate hyperglycemic stress (CHG), INS-1E cells were exposed to 33 mM glucose for 24 hrs. GSIS was measured by HTRF assay.RESULTS: Glucose- and tolbutamide-triggered action potentials were suppressed by TTX, but not by RAN. VERA caused depolarization, countered by INaL blockade. The reversal potentials of ITTX and IRAN were negative to Na+ equilibrium potential, but they approached it when K+-channels were blocked. This revealed INaL coupling to Na+-activated K+ current (IKNa); expression of IKNa channels (Slo2.1/2.2) was confirmed by transcript analysis. INaL blockade blunted cytosolic Ca2+ response to depolarization. CHG enhanced INaL. Whereas acute INaL enhancement (VERA) increased GSIS, chronic one (CHG or VERA) depressed GSIS, which was partially restored by RAN.CONCLUSIONS: INaL is expressed in insulin-secreting cells, is coupled to IKNa , affects Ca2+ signalling and, when enhanced acutely, increases GSIS. However, constitutive INaL enhancement, induced by chronic hyperglycemic stress, and leads to GSIS depression instead. Overall, chronic INaL enhancement may represent a mechanism of damage progression common to myocardial and insulin-secreting cells.

Background: The cardiac action potential (AP) shape is a species-dependent feature related to differences in ionic currents underlying repolarization. In guinea pigs (GP), dogs and humans, the AP is prolonged by a pronounced plateau phase. In canine and human, but not GP, myocytes, a spike-and-dome profile characterizes repolarization of specific regions within the heart and to a different extent according to heart rate. It is unclear whether the response to β-adrenergic stimulation is dictated by peculiarities of ion channels properties, or may results from differences in AP contour.Aim: The aim of this project is to test whether the presence of the spike-and-dome in the AP contour is, by itself, able to modify the response of membrane current to β-adrenergic stimulation in a rate-dependent fashion.Methods: We performed AP-clamp on GP myocytes with dog epicardial and endocardial AP waveforms to assess the contribution of the spike-and-dome in isoprenaline (ISO) sensitive current (IISO) at diastolic intervals (DI) of 1750ms and 300ms. We also performed dynamic clamp experiments on GP myocytes with a computational simulated canine transient outward current (Ito) to evaluate the ISO response on the AP duration (APD) in presence of an artificial spike-and-dome.Results: We found that: 1) At DI1750, IISO is more inward with dog endocardial rather than epicardial waveform; this difference was not evidenced at DI300. 2) The injection of a simulated canine Ito is not sufficient by itself to affect the direction of APD changes during β-adrenergic stimulation.Conclusions: The differences between dog and GP in setting β-adrenergic stimulation response are a species-dependent feature not only related to Ito and might be explained as a more complex mechanism involving AP shape and a diverse contribution of Ca2+ and K+ channels during the AP.

Background: Calmodulin (CaM), a small essential Ca2+ binding protein, is encoded by 3 genes, namely CALM1, CALM2, CALM3, and exerts its modulatory role depending on intracellular Ca2+ concentration changes. In the heart, CaM mutations in these genes led to recurrent cardiac arrest in infants associated with severe QT prolongation (LQTS-CaM).Aims: To explore how the heterozygous CALM1-F142L mutation can support the reported extremely severe cardiac phenotype. To maintain the correct allele balance we studied the functional consequences of this mutation by using patient-specific human induced pluripotent stem cell (iPSC)-derived cardiomyocytes (CMs).Methods: Skin fibroblast of a patient carrying CALM1-F142L mutation were reprogrammed to iPSC and differentiated into CMs; control individuals were 2 unrelated healthy donors without history of cardiac disease. We focus our attention on ICaL biophysics, electrical activity and intracellular Ca2+ dynamics. To make the iPSC-CMs a more reliable model to investigate mechanisms underlying cardiac arrhythmia, in-silico IK1 was injected with a Dynamic-Clamp approach.Results: CALM1-F142L mutation induced impaired Ca2+-dependent inactivation (CDI) and incomplete inactivation of ICaL, thus causing increased Ca2+ influx and consequent action potential prolongation. Therapeutic concentration of verapamil markedly shortened QT in mutated cells. F142L iPSC-CMs failed to adapt to high pacing rates, pointing this as a likely arrhythmogenic mechanism for the mutation. Intracellular Ca2+ handling was instead marginally affected by the mutation. Indeed, sarcoplasmic reticulum (SR) Ca2+content and Ca2+ induced Ca2+ release (CICR)-gain were not affected by the mutation.Conclusions: LQTS-F142L CaM mutation is highly arrhythmogenic mainly because of impaired ICaL CDI; accordingly, its pathogenicity might be limited by the therapeutic use of Ca2+ channel blockers like verapamil.

Background: Ischemia ischaracterized by an overproduction of toxic metabolites well-known enhancers of the late sodium current (INaL). Ranolazine (RAN), a blocker of INaL, improves recovery during reperfusion. However, during ischemia diastolic potential depolarization might limit INa recruiting; thus, whether INaL is actually enhanced during acute ischemia is still unknown.Aim: To test the functional contribution of INaL in a cellular model of acute ischemia.Methods: Rat ventricular myocytes were exposed for 7 min to a (normoxic) ischemia-mimic solution (ISC); to evaluate the role of INaL, RAN (10μM) or TTX (1μM) were added throughout the protocol. INaL was isolated as TTX (1μM)-sensitive current during action potentials (APs) elicited at 1Hz. Cell shortening, cytosolic Na+ (Nai) and Ca2+ (Cai) were monitored in field-stimulated myocytes (1Hz). The sarcolemma (s) and mitochondrial (m) Na+/Ca2+exchanger (NCX) were blocked by SEA-0400 (1μM) and CGP37157 (1μM) respectively.Results: During ISC, loss of contraction was followed by partial recovery. While APs persisted throughout ISC, diastolic potential markedly depolarized and AP upstroke velocity decreased. In spite of this, ISC induced INaL enhancement, which was completely prevented by RAN. ISC increased Nai and Cai, which were partially prevented by RAN; TTX affected ISC-induced Nai enhancement only. SEA-0400 boosted cytosolic Cai and sarcoplasmic reticulum (SR) Ca2+content during ISC. SEA-0400 effects were sharply curtailed by RAN and partially prevented by TTX. CGP37157 prevented RAN effects during sNCX blockade.Conclusion: During ISC, INaL increased in spite of AP changes and contributed to Nai increment. Unexpectedly, sNCX blockade unrevealed RAN and TTX effects on Cai. These results suggest Na+-dependent, but sNCX-independent Ca2+ accumulation mechanisms during acute ischemia. Indeed, mNCX appears to be involved in the INaL induced cytosolic Ca2+ accumulation.

Background: Knock out of β-sarcoglycan (βSG-/-), a dystrophin-complex protein, leads to cardiomyopathy. Reportedly, cultured WT cardiac mesoangioblasts (cMABs) spontaneously undergo cardiogenic differentiation. We recently found that cMABs structural differentiation is switched to skeletal muscle (SKm) by βSG-/-, because of deletion of miRNAs encoded by the βSG gene (miR669a-p). Aims: To compare the functional properties of βSG-/-cMABs with those of SKm (C2C12) and neonatal cardiac cells (CMs). Methods: single-cell motion, Ca2+ transients (FLUO4 loaded cells), membrane potential (Vm) and currents were analyzed along with channel isoform expression; Vm was depolarized by 40 mM K+ (hK+). Results: in βSG-/-cMABs acetylcholine (ACh 30 μM) or nicotine (Nic 100 μM) elicited inward current, membrane depolarization, Ca2+ transients and contraction, all prevented by Nic-receptor (d-TbC 100 μM) and Ca2+ channel (nifedipine 30 μM) antagonists. This was similar to C2C12 myotubes, but different from CMs. Ca2+ current (ICaL) inactivation was similar between βSG-/-cMABs cells and C2C12 myotubes (tau=661±53 vs 497±55 ms, NS), but faster in CMs (tau =9.7±1.2 ms; p 90% for all stimuli); response to Nic was abolished by nifedipine. Notably, induction of Ca2+ transients by Nic did not affect the amplitude of subsequent Caffeine-induced transients. βSG-/-cMABs and C2C12 expressed mRNA for SKm channels Cav1.1, Cav1.3 and ryanodine receptor RyR1, CMs expressed Cav 1.1, Cav1.2 and RyR2; RyR3 was found only in C2C12 myotubes. Conclusions: 1) Differentiation of BSG-/--cMABs, but not of WT-cMABS, leads to a SK-muscle phenotype; 2) the BSG encoding sequence may effectively direct molecular and functional differentiation of cardiac mesoangioblasts.

Background: Pulmonary hypertension (PH) complicates many clinical conditions and may result in right ventricular (RV) hypertrophy and failure. Monocrotaline (MCT) induces PH by direct action on pulmonary microvasculature. Enhancement of the late sodium current (INaL) results from myocardial remodelling and may contribute to its evolution. Aims: To evaluate the effects of chronic INaL blockade in the prevention of PH-induced myocardial remodeling. Methods: PH was induced in adult rats by a single injection of MCT (60 mg/Kg i.p.); animals were studied 3 weeks later and untreated littermates served as controls. The INaL blocker ranolazine (RAN, 30 mg/Kg bid i.p.) was administered to a subset of MCT-treated rats over the 3 weeks period and completely washed-out before study. Results: MCT effects (vs control): MCT increased RV pressure. In the RV, weight, wall thickness, collagen content, myocyte electrical capacitance and cross sectional area were all increased, to indicate hypertrophy. The LV was slightly hypotrophyc instead. Nonetheless, both RV and LV myocytes showed INaL enhancement and signs of remodelling, such as α-myosin heavy chain (α-MHC) and Ito downregulation, prolonged action potential duration (APD) and delayed afterdepolarizations (DADs). IK1 was reduced in RV only. Chronic RAN effects (vs MCT alone): RAN reduced RV pressure and significantly blunted all signs of RV hypertrophy. In both RV and LV, INaL enhancement, DADs induction, α-MHC and Ito downregulation were reversed by RAN. However, APD and IK1 abnormalities persisted. Conclusions: MCT caused pressure-induced hypertrophy in the RV, but also remodelled the LV (by indirect strain?). Chronic INaL blockade prevented constitutive INaL enhancement and blunted many aspects of myocardial remodeling in both ventricles. In the RV, but not in the LV, this might partly reflect the unexpected inhibition by RAN of MCT effects on pulmonary vascular resistance.