Slower Calcium Handling Balances Faster Cross-Bridge Cycling in Human MYBPC3 HCM.

BACKGROUND: The pathogenesis of MYBPC3-associated hypertrophic cardiomyopathy (HCM) is still unresolved. In our HCM patient cohort, a large and well-characterized population carrying the MYBPC3:c772G>A variant (p.Glu258Lys, E258K) provides the unique opportunity to study the basic mechanisms of MYBPC3-HCM with a comprehensive translational approach. METHODS: We collected clinical and genetic data from 93 HCM patients carrying the MYBPC3:c772G>A variant. Functional perturbations were investigated using different biophysical techniques in left ventricular samples from 4 patients who underwent myectomy for refractory outflow obstruction, compared with samples from non-failing non-hypertrophic surgical patients and healthy donors. Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes and engineered heart tissues (EHTs) were also investigated. RESULTS: Haplotype analysis revealed MYBPC3:c772G>A as a founder mutation in Tuscany. In ventricular myocardium, the mutation leads to reduced cMyBP-C (cardiac myosin binding protein-C) expression, supporting haploinsufficiency as the main primary disease mechanism. Mechanical studies in single myofibrils and permeabilized muscle strips highlighted faster cross-bridge cycling, and higher energy cost of tension generation. A novel approach based on tissue clearing and advanced optical microscopy supported the idea that the sarcomere energetics dysfunction is intrinsically related with the reduction in cMyBP-C. Studies in single cardiomyocytes (native and hiPSC-derived), intact trabeculae and hiPSC-EHTs revealed prolonged action potentials, slower Ca2+ transients and preserved twitch duration, suggesting that the slower excitation-contraction coupling counterbalanced the faster sarcomere kinetics. This conclusion was strengthened by in silico simulations. CONCLUSIONS: HCM-related MYBPC3:c772G>A mutation invariably impairs sarcomere energetics and cross-bridge cycling. Compensatory electrophysiological changes (eg, reduced potassium channel expression) appear to preserve twitch contraction parameters, but may expose patients to greater arrhythmic propensity and disease progression. Therapeutic approaches correcting the primary sarcomeric defects may prevent secondary cardiomyocyte remodeling.

Pharmacological basis of the antifibrotic effects of pirfenidone: Mechanistic insights from cardiac in-vitro and in-vivo models.

Pirfenidone is a small drug with marked antifibrotic activity approved for the treatment of Idiopathic pulmonary fibrosis. Recently, its peculiar pharmacological profile has attracted attention for its potential therapeutic benefit for extra-pulmonary disorders characterized by pathological fibrosis, such as kidney, liver, and cardiac failure. A major pitfall of pirfenidone is the lack of consistent understanding of its mechanism of action, regardless of the target. In addition to the increasing attention to the role of inflammation and its mediators in several processes, a better knowledge of the variety of fibroblasts' population, of signals controlling their activation and trans-differentiation, and of crosstalk with other cell resident and non-resident cell types is needed for prevention, treatment and possibly reverse of fibrosis. This review will focus on pirfenidone's pharmacological profile and its effects on cardiac fibroblasts.

Genetic characterization of juvenile sudden cardiac arrest and death in Tuscany: The ToRSADE registry.

Background: Sudden cardiac arrest (SCA) in young people represents a dramatic event, often leading to severe neurologic outcomes or sudden cardiac death (SCD), and is frequently caused by genetic heart diseases. In this study, we report the results of the Tuscany registry of sudden cardiac death (ToRSADE) registry, aimed at monitoring the incidence and investigating the genetic basis of SCA and SCD occurring in subjects < 50 years of age in Tuscany, Italy. Methods and results: Creation of the ToRSADE registry allowed implementation of a repository for clinical, molecular and genetic data. For 22 patients, in whom a genetic substrate was documented or suspected, blood samples could be analyzed; 14 were collected at autopsy and 8 from resuscitated patients after SCA. Next generation sequencing (NGS) analysis revealed likely pathogenetic (LP) variants associated with cardiomyopathy (CM) or channelopathy in four patients (19%), while 17 (81%) carried variants of uncertain significance in relevant genes (VUS). In only one patient NGS confirmed the diagnosis obtained during autopsy: the p.(Asn480Lysfs*20) PKP2 mutation in a patient with arrhythmogenic cardiomyopathy (AC). Conclusion: Systematic genetic screening allowed identification of LP variants in 19% of consecutive patients with SCA/SCD, including subjects carrying variants associated with hypertrophic cardiomyopathy (HCM) or AC who had SCA/SCD in the absence of structural cardiomyopathy phenotype. Genetic analysis combined with clinical information in survived patients and post-mortem evaluation represent an essential multi-disciplinary approach to manage juvenile SCD and SCA, key to providing appropriate medical and genetic assistance to families, and advancing knowledge on the basis of arrhythmogenic mechanisms in inherited cardiomyopathies and channelopathies.

A rare case of pediatric cardiomyopathy: Alström syndrome identified by gene panel analysis.

Genetic investigation of early-onset Dilatative cardiomyopathy phenotype, including molecular autopsy, is the key to appropriate recognition and management of rare etiologies and atypical presentations and to offer genetic counseling to the family.

Angiotensin-II Drives Human Satellite Cells Toward Hypertrophy and Myofibroblast Trans-Differentiation by Two Independent Pathways.

Skeletal muscle regeneration is ensured by satellite cells (SC), which upon activation undergo self-renewal and myogenesis. The correct sequence of healing events may be offset by inflammatory and/or fibrotic factors able to promote fibrosis and consequent muscle wasting. Angiotensin-II (Ang) is an effector peptide of the renin angiotensin system (RAS), of which the direct role in human SCs (hSCs) is still controversial. Based on the hypertrophic and fibrogenic effects of Ang via transient receptor potential canonical (TRPC) channels in cardiac and renal tissues, we hypothesized a similar axis in hSCs. Toward this aim, we demonstrated that hSCs respond to acute Ang stimulation, dose-dependently enhancing p-mTOR, p-AKT, p-ERK1/2 and p-P38. Additionally, sub-acute Ang conditioning increased cell size and promoted trans-differentiation into myofibroblasts. To provide a mechanistic hypothesis on TRPC channel involvement in the processes, we proved that TRPC channels mediate a basal calcium entry into hSCs that is stimulated by acute Ang and strongly amplified by sub-chronic Ang conditioning. Altogether, these findings demonstrate that Ang induces a fate shift of hSCs into myofibroblasts and provide a basis to support a benefit of RAS and TRPC channel blockade to oppose muscle fibrosis.

ß3-Adrenoreceptors Control Mitochondrial Dormancy in Melanoma and Embryonic Stem Cells.

The early phases of embryonic development and cancer share similar strategies to improve their survival in an inhospitable environment: both proliferate in a hypoxic and catecholamine-rich context, increasing aerobic glycolysis. Recent studies show that ß3-adrenergic receptor (ß3-AR) is involved in tumor progression, playing an important role in metastasis. Among ß-adrenergic receptors, ß3-AR is the last identified member of this family, and it is involved in cancer cell survival and induction of stromal reactivity in the tumor microenvironment. ß3-AR is well known as a strong activator of uncoupling protein 1 (UCP1) in brown fat tissue. Interestingly, ß3-AR is strongly expressed in early embryo development and in many cancer tissues. Induction of uncoupling protein 2 (UCP2) has been related to cancer metabolic switch, leading to accelerated glycolysis and reduced mitochondrial activity. In this study, for the first time, we demonstrate that ß3-AR is able to promote this metabolic shift in both cancer and embryonic stem cells, inducing specific glycolytic cytoplasmic enzymes and a sort of mitochondrial dormancy through the induction of UCP2. The ß3-AR/UCP2 axis induces a strong reduction of mitochondrial activity by reducing ATP synthesis and mitochondrial reactive oxygen species (mtROS) content. These effects are reverted by SR59230A, the specific ß3-AR antagonist, causing an increase in mtROS. The increased level of mtROS is neutralized by a strong antioxidant activity in embryonic stem cells, but not in cancer stem cells, where it causes a dramatic reduction in tumor cell viability. These results lead to the possibility of a selective antitumor therapeutic use of SR59230A. Notably, we demonstrate the presence of ß3-AR within the mitochondrial membrane in both cell lines, leading to the control of mitochondrial dormancy.

Selective Blockade of HCN1/HCN2 Channels as a Potential Pharmacological Strategy Against Pain.

A prominent role of hyperpolarization-activated, cyclic nucleotide-gated (HCN) channels has been suggested based on their expression and (dys)function in dorsal root ganglion (DRG) neurons, being likely involved in peripheral nociception. Using HCN blockers as antinociceptive drugs is prevented by the widespread distribution of these channels. However, tissue-specific expression of HCN isoforms varies significantly, HCN1 and HCN2 being considered as major players in DRG excitability. We characterized the pharmacological effect of a novel compound, MEL55A, able to block selectively HCN1/HCN2 isoforms, on DRG neuron excitability in-vitro and for its antiallodynic properties in-vivo. HEK293 cells expressing HCN1, HCN2, or HCN4 isoforms were used to verify drug selectivity. The pharmacological profile of MEL55A was tested on mouse DRG neurons by patch-clamp recordings, and in-vivo in oxaliplatin-induced neuropathy by means of thermal hypersensitivity. Results were compared to the non-isoform-selective drug, ivabradine. MEL55A showed a marked preference toward HCN1 and HCN2 isoforms expressed in HEK293, with respect to HCN4. In cultured DRG, MEL55A reduced I h amplitude, both in basic conditions and after stimulation by forskolin, and cell excitability, its effect being quantitatively similar to that observed with ivabradine. MEL55A was able to relieve chemotherapy-induced neuropathic pain. In conclusion, selective blockade of HCN1/HCN2 channels, over HCN4 isoform, was able to modulate electrophysiological properties of DRG neurons similarly to that reported for classical I h blockers, ivabradine, resulting in a pain-relieving activity. The availability of small molecules with selectivity toward HCN channel isoforms involved in nociception might represent a safe and effective strategy against chronic pain.

Hyperpolarization-activated cyclic-nucleotide-gated channels: pathophysiological, developmental, and pharmacological insights into their function in cellular excitability.

The hyperpolarization-activated cyclic-nucleotide-gated (HCN) proteins are voltage-dependent ion channels, conducting both Na+ and K+, blocked by millimolar concentrations of extracellular Cs+ and modulated by cyclic nucleotides (mainly cAMP) that contribute crucially to the pacemaker activity in cardiac nodal cells and subsidiary pacemakers. Over the last decades, much attention has focused on HCN current, If, in non-pacemaker cardiac cells and its potential role in triggering arrhythmias. In fact, in addition to pacemakers, HCN current is constitutively present in the human atria and has long been proposed to sustain atrial arrhythmias associated to different cardiac pathologies or triggered by various modulatory signals (catecholamines, serotonin, natriuretic peptides). An atypical If occurs in diseased ventricular cardiomyocytes, its amplitude being linearly related to the severity of cardiac hypertrophy. The properties of atrial and ventricular If and its modulation by pharmacological interventions has been object of intense study, including the synthesis and characterization of new compounds able to block preferentially HCN1, HCN2, or HCN4 isoforms. Altogether, clues emerge for opportunities of future pharmacological strategies exploiting the unique properties of this channel family: the prevalence of different HCN subtypes in organs and tissues, the possibility to target HCN gain- or loss-of-function associated with disease, the feasibility of novel isoform-selective drugs, as well as the discovery of HCN-mediated effects for old medicines.

Pathogenesis of Hypertrophic Cardiomyopathy is Mutation Rather Than Disease Specific: A Comparison of the Cardiac Troponin T E163R and R92Q Mouse Models.

BACKGROUND: In cardiomyocytes from patients with hypertrophic cardiomyopathy, mechanical dysfunction and arrhythmogenicity are caused by mutation-driven changes in myofilament function combined with excitation-contraction (E-C) coupling abnormalities related to adverse remodeling. Whether myofilament or E-C coupling alterations are more relevant in disease development is unknown. Here, we aim to investigate whether the relative roles of myofilament dysfunction and E-C coupling remodeling in determining the hypertrophic cardiomyopathy phenotype are mutation specific. METHODS AND RESULTS: Two hypertrophic cardiomyopathy mouse models carrying the R92Q and the E163R TNNT2 mutations were investigated. Echocardiography showed left ventricular hypertrophy, enhanced contractility, and diastolic dysfunction in both models; however, these phenotypes were more pronounced in the R92Q mice. Both E163R and R92Q trabeculae showed prolonged twitch relaxation and increased occurrence of premature beats. In E163R ventricular myofibrils or skinned trabeculae, relaxation following Ca2+ removal was prolonged; resting tension and resting ATPase were higher; and isometric ATPase at maximal Ca2+ activation, the energy cost of tension generation, and myofilament Ca2+ sensitivity were increased compared with that in wild-type mice. No sarcomeric changes were observed in R92Q versus wild-type mice, except for a large increase in myofilament Ca2+ sensitivity. In R92Q myocardium, we found a blunted response to inotropic interventions, slower decay of Ca2+ transients, reduced SERCA function, and increased Ca2+/calmodulin kinase II activity. Contrarily, secondary alterations of E-C coupling and signaling were minimal in E163R myocardium. CONCLUSIONS: In E163R models, mutation-driven myofilament abnormalities directly cause myocardial dysfunction. In R92Q, diastolic dysfunction and arrhythmogenicity are mediated by profound cardiomyocyte signaling and E-C coupling changes. Similar hypertrophic cardiomyopathy phenotypes can be generated through different pathways, implying different strategies for a precision medicine approach to treatment.

Role of Nitric Oxide, Nitric Oxide Synthase, Soluble Guanylyl Cyclase, and cGMP-Dependent Protein Kinase I in Mouse Stem Cell Cardiac Development.

Introduction and Aim. Nitric oxide (NO) can trigger cardiac differentiation of embryonic stem cells (ESCs), indicating a cardiogenic function of the NO synthetizing enzyme(s) (NOS). However, the involvement of the NO/NOS downstream effectors soluble guanylyl cyclase (sGC) and cGMP activated protein kinase I (PKG-I) is less defined. Therefore, we assess the involvement of the entire NO/NOS/sGC/PKG-I pathway during cardiac differentiation process. Methods. Mouse ESCs were differentiated toward cardiac lineages by hanging drop methodology for 21 days. NOS/sGC/PKG-I pathway was studied quantifying genes, proteins, enzymatic activities, and effects of inhibition during differentiation. Percentages of beating embryoid bodies (mEBs) were evaluated as an index of cardiogenesis. Results and Discussion. Genes and protein expression of enzymes were increased during differentiation with distinctive kinetics and proteins possessed their enzymatic functions. Exogenous administered NO accelerated whereas the blockade of PKG-I strongly slowed cardiogenesis. sGC inhibition was effective only at early stages and NOS blockade ineffective. Of NOS/sGC/PKG-I pathway, PKG-I seems to play the prominent role in cardiac maturation. Conclusion. We concluded that exogenous administered NO and other pharmacological strategies able to increase the activity of PKG-I provide new tools to investigate and promote differentiation of cardiogenic precursors.

Biochemical and Electrophysiological Modification of Amyloid Transthyretin on Cardiomyocytes.

Transthyretin (TTR) amyloidoses are familial or sporadic degenerative conditions that often feature heavy cardiac involvement. Presently, no effective pharmacological therapy for TTR amyloidoses is available, mostly due to a substantial lack of knowledge about both the molecular mechanisms of TTR aggregation in tissue and the ensuing functional and viability modifications that occur in aggregate-exposed cells. TTR amyloidoses are of particular interest regarding the relation between functional and viability impairment in aggregate-exposed excitable cells such as peripheral neurons and cardiomyocytes. In particular, the latter cells provide an opportunity to investigate in parallel the electrophysiological and biochemical modifications that take place when the cells are exposed for various lengths of time to variously aggregated wild-type TTR, a condition that characterizes senile systemic amyloidosis. In this study, we investigated biochemical and electrophysiological modifications in cardiomyocytes exposed to amyloid oligomers or fibrils of wild-type TTR or to its T4-stabilized form, which resists tetramer disassembly, misfolding, and aggregation. Amyloid TTR cytotoxicity results in mitochondrial potential modification, oxidative stress, deregulation of cytoplasmic Ca2+ levels, and Ca2+ cycling. The altered intracellular Ca2+ cycling causes a prolongation of the action potential, as determined by whole-cell recordings of action potentials on isolated mouse ventricular myocytes, which may contribute to the development of cellular arrhythmias and conduction alterations often seen in patients with TTR amyloidosis. Our data add information about the biochemical, functional, and viability alterations that occur in cardiomyocytes exposed to aggregated TTR, and provide clues as to the molecular and physiological basis of heart dysfunction in sporadic senile systemic amyloidosis and familial amyloid cardiomyopathy forms of TTR amyloidoses.

Pharmacological perspectives in sarcopenia: a potential role for renin-angiotensin system blockers?

Sarcopenia represents a major health problem highly prevalent in elderly and age-related chronic diseases. Current pharmacological strategies available to prevent and reverse sarcopenia are largely unsatisfactory thus raising the need to identify novel targets for pharmacological intervention and possibly more effective and safe drugs. This review highlights the current knowledge of the potential benefits of renin-angiotensin system blockade in sarcopenia and discuss the main mechanisms underlying the effects.

Small interventions in outpatient surgery: Project of Advanced Nursing Skills.

The research analyzes data on the impact of the introduction of the figure nursing with advanced skills, "Advanced Skills" , in diagnostic and therapeutic surgical clinics within the Hospital of Piacenza. This is the application of the testing on the Project of Nursing "Skills"on advanced minor surgery "Minor Surgery", an outpatient procedure. They were given the satisfaction questionnaires to users and to the medical and nursing staff of the USL in Piacenza. The aim of the study was to verify the contention of the literature about the usefulness of an increased use of nursing skills within an ambulatory surgery to improve the quality of care and consequently to a higher user satisfaction. In light of the findings of the research, the ' impact of the implementation of the Project Advanced Nursing Skills has been positive, both among users is that health care providers. For a functional and effective management of the surgery may be useful to train a new nursing characterized by the possession of "Advanced Skills" whose "mission" is to develop more knowledge, share knowledge, as well as to foster innovation organizational and management to meet the demands of public health.

Human amniotic fluid stem cell differentiation along smooth muscle lineage.

Functional smooth muscle engineering requires isolation and expansion of smooth muscle cells (SMCs), and this process is particularly challenging for visceral smooth muscle tissue where progenitor cells have not been clearly identified. Herein we showed for the first time that efficient SMCs can be obtained from human amniotic fluid stem cells (hAFSCs). Clonal lines were generated from c-kit(+) hAFSCs. Differentiation toward SM lineage (SMhAFSCs) was obtained using a medium conditioned by PDGF-BB and TGF-ß1. Molecular assays revealed higher level of α smooth muscle actin (α-SMA), desmin, calponin, and smoothelin in SMhAFSCs when compared to hAFSCs. Ultrastructural analysis demonstrated that SMhAFSCs also presented in the cytoplasm increased intermediate filaments, dense bodies, and glycogen deposits like SMCs. SMhAFSC metabolism evaluated via mass spectrometry showed higher glucose oxidation and an enhanced response to mitogenic stimuli in comparison to hAFSCs. Patch clamp of transduced hAFSCs with lentiviral vectors encoding ZsGreen under the control of the α-SMA promoter was performed demonstrating that SMhAFSCs retained a smooth muscle cell-like electrophysiological fingerprint. Eventually SMhAFSCs contractility was evident both at single cell level and on a collagen gel. In conclusion, we showed here that hAFSCs under selective culture conditions are able to give rise to functional SMCs.

Induced pluripotent stem (iPS) cells from human fetal stem cells (hFSCs).

INTRODUCTION: (1) Human embryonic stem (ES) cells are pluripotent but are difficult to be used for therapy because of immunological, oncological and ethical barriers. (2) Pluripotent cells exist in vivo, i.e., germ cells and epiblast cells but cannot be isolated without sacrificing the developing embryo. (3) Reprogramming to pluripotency is possible from adult cells using ectopic expression of OKSM and other integrative and non-integrative techniques. (4) Hurdles to overcome include i.e stability of the phenotype in relation to epigenetic memory. SOURCES OF DATA: We reviewed the literature related to reprogramming, pluripotency and fetal stem cells. AREAS OF AGREEMENT: (1) Fetal stem cells present some advantageous characteristics compared with their neonatal and postnatal counterparts, with regards to cell size, growth kinetics, and differentiation potential, as well as in vivo tissue repair capacity. (2) Amniotic fluid stem cells are more easily reprogrammed to pluripotency than adult fibroblast. (3) The parental population is heterogeneous and present an intermediate phenotype between ES and adult somatic stem cells, expressing markers of both. AREAS OF CONTROVERSY: (1) It is unclear whether induced pluripotent stem (iPS) derived from amniotic fluid stem cells are fully or partially reprogrammed. (2) Optimal protocols to ensure highest efficiency and phenotype stability remains to be determined. (3) The "level" of reprogramming, fully vs partial, of iPS derived from amniotic fluid stem cells remain to be determined. GROWING POINTS: Banking of fully reprogrammed cells may be important both for (1) autologous and allogenic applications in medicine, and (2) disease modeling.

Chronic atrial fibrillation alters the functional properties of If in the human atrium.

INTRODUCTION: Despite the evidence that the hyperpolarization-activated current (If) is highly modulated in human cardiomyopathies, no definite data exist in chronic atrial fibrillation (cAF). We investigated the expression, function, and modulation of If in human cAF. METHODS AND RESULTS: Right atrial samples were obtained from sinus rhythm (SR, n = 49) or cAF (duration >1 year, n = 31) patients undergoing corrective cardiac surgery. Among f-channel isoforms expressed in the human atrium (HCN1, 2 and 4), HCN4 mRNA levels measured by RT-PCR were significantly reduced. However, protein expression was preserved in cAF compared to SR (+85% for HCN4); concurrently, miR-1 expression was significantly reduced. In patch-clamped atrial myocytes, current-specific conductance (gf) was significantly increased in cAF at voltages around the threshold for If activation (-60 to -80 mV); accordingly, a 10-mV rightward shift of the activation curve occurred (P < 0.01). ß-Adrenergic and 5-HT4 receptor stimulation exerted similar effects on If in cAF and SR cells, while the ANP-mediated effect was significantly reduced (P < 0.02), suggesting downregulation of natriuretic peptide signaling. CONCLUSIONS: In human cAF modifications in transcriptional and posttranscriptional mechanisms of HCN channels occur, associated with a slight yet significant gain-of-function of If , which may contribute to enhanced atrial ectopy.

Molecular and functional evidence of HCN4 and caveolin-3 interaction during cardiomyocyte differentiation from human embryonic stem cells.

Maturation of human embryonic stem cell-derived cardiomyocytes (hESC-CM) is accompanied by changes in ion channel expression, with relevant electrophysiological consequences. In rodent CM, the properties of hyperpolarization-activated cyclic nucleotide-gated channel (HCN)4, a major f-channel isoform, depends on the association with caveolin-3 (Cav3). To date, no information exists on changes in Cav3 expression and its associative relationship with HCN4 upon hESC-CM maturation. We hypothesize that Cav3 expression and its compartmentalization with HCN4 channels during hESC-CM maturation accounts for the progression of f-current properties toward adult phenotypes. To address this, hESC were differentiated into spontaneously beating CM and examined at ∼30, ∼60, and ∼110 days of differentiation. Human adult and fetal CM served as references. HCN4 and Cav3 expression and localization were analyzed by real time PCR and immunocyto/histochemistry. F-current was measured in patch-clamped single cells. HCN4 and Cav3 colocalize in adult human atrial and ventricular CM, but not in fetal CM. Proteins and mRNA for Cav3 were not detected in undifferentiated hESC, but expression increased during hESC-CM maturation. At 110 days, HCN4 appeared to be colocalized with Cav3. Voltage-dependent activation of the f-current was significantly more positive in fetal CM and 60-day hESC-CM (midpoint activation, V1/2, ∼ -82 mV) than in 110-day hESC-CM or adult CM (V1/2∼-100 mV). In the latter cells, caveolae disruption reversed voltage dependence toward a more positive or an immature phenotype, with V1/2 at -75 mV, while in fetal CM voltage dependence was not affected. Our data show, for the first time, a developmental change in HCN4-Cav3 association in hESC-CM. Cav3 expression and its association with ionic channels likely represent a crucial step of cardiac maturation.