Recurrent arrhythmic storms and unsuccessful catheter ablation in chronic ischemic heart disease.

The prototypical substrate for reentrant ventricular tachycardia (VT) is post-myocardial infarction (MI) scar. Catheter ablation is an important therapeutic option for recurrent VT but sometimes it is not effective despite the technical advances. Here we describe the case of a 60-year-old man who suffered a MI in 1998 and presented with recurrent arrhythmic storms during his long-term follow-up. Twenty years later, he underwent two catheter ablations with bipolar electroanatomic voltage mapping (EVM) demonstrating only an area of low voltages in the lateral left ventricular free wall. Both procedures were unsuccessful and the patient eventually underwent cardiac transplantation in 2019. Pathology examination revealed circumferential subendocardial scar with hypertrabeculation, so that the reentry substrate was unreachable by ablation with the use of standard techniques. The comparison of EVM findings with the morphologic ones in patients with chronic ischemic heart disease can help to better understand the feasibility and effectiveness of VT substrate ablation.

Heart failure impairs the mechanotransduction properties of human cardiac pericytes.

The prominent impact that coronary microcirculation disease (CMD) exerts on heart failure symptoms and prognosis, even in the presence of macrovascular atherosclerosis, has been recently acknowledged. Experimental delivery of pericytes in non-revascularized myocardial infarction improves cardiac function by stimulating angiogenesis and myocardial perfusion. Aim of this work is to verify if pericytes (Pc) residing in ischemic failing human hearts display altered mechano-transduction properties and to assess which alterations of the mechano-sensing machinery are associated with the observed impaired response to mechanical cues. RESULTS: Microvascular rarefaction and defects of YAP/TAZ activation characterize failing human hearts. Although both donor (D-) and explanted (E-) heart derived cardiac Pc support angiogenesis, D-Pc exert this effect significantly better than E-Pc. The latter are characterized by reduced focal adhesion density, decreased activation of the focal adhesion kinase (FAK)/ Crk-associated substrate (CAS) pathway, low expression of caveolin-1, and defective transduction of extracellular stiffness into cytoskeletal stiffening, together with an impaired response to both fibronectin and lysophosphatidic acid. Importantly, Mitogen-activated protein kinase kinase inhibition restores YAP/TAZ nuclear translocation. CONCLUSION: Heart failure impairs Pc mechano-transduction properties, but this defect could be reversed pharmacologically.

Preservation by cold storage vs ex vivo normothermic perfusion of marginal donor hearts: clinical, histopathologic, and ultrastructural features.

BACKGROUND: The aim of this study was to match clinical outcomes of heart transplantation (HTx) against histopathologic and ultrastructural characteristics of marginal grafts preserved by cold storage (CS) or ex vivo normothermic perfusion. METHODS: Since 2011, 100 patients had undergone HTx at our institution by using marginal donors (aged ≥55 years, expected ischemic time of >4 hours, left ventricular ejection fraction of ≤50%, interventricular septum thickness of ≥14 mm, drug abuse history, episodes of cardiac arrest, and presence of mild coronary artery disease). CS was utilized in 79 cases (Group 1, 79%), and ex vivo perfusion was utilized in 21 (Group 2, 21%). Pre-operative data, survival, and complications in the first 5 years after HTx were analyzed. Myocardial biopsies were collected at graft harvesting, just before implantation, and immediately after aortic declamping. RESULTS: Pre-operative demographics were similar in the 2 groups. Graft utilization rate with ex vivo perfusion was 81%. Ischemic, cardiopulmonary bypass, and surgical times were shorter in Group 2 patients, who showed a lower incidence of overall complications (33% vs 13%, p = 0.04) and better 5-year survival (log-rank, p = 0.04). Moreover, restoration of hypertrophy-related sarcomere changes and mitigation of reperfusion-dependent myocardium injuries were more frequently observed in Group 2 hearts. CONCLUSIONS: Ex vivo perfusion allows for continuous evaluation of marginal donor hearts, favoring exclusion of unsuitable grafts, reduction of complications, and optimal survival of up to 5 years. Such results, supported by consistent histopathologic and ultrastructural findings, suggest better myocardial preservation.

Transfer of a human gene variant associated with exceptional longevity improves cardiac function in obese type 2 diabetic mice through induction of the SDF-1/CXCR4 signalling pathway.

AIMS: Homozygosity for a four-missense single-nucleotide polymorphism haplotype of the human BPIFB4 gene is enriched in long-living individuals. Delivery of this longevity-associated variant (LAV) improved revascularisation and reduced endothelial dysfunction and atherosclerosis in mice through a mechanism involving the stromal cell-derived factor-1 (SDF-1). Here, we investigated if delivery of the LAV-BPIFB4 gene may attenuate the progression of diabetic cardiomyopathy. METHODS AND RESULTS: Compared with age-matched lean controls, diabetic db/db mice showed altered echocardiographic indices of diastolic and systolic function and histological evidence of microvascular rarefaction, lipid accumulation, and fibrosis in the myocardium. All these alterations, as well as endothelial dysfunction, were prevented by systemic LAV-BPIFB4 gene therapy using an adeno-associated viral vector serotype 9 (AAV9). In contrast, AAV9 wild-type-BPIFB4 exerted no benefit. Interestingly, LAV-BPIFB4-treated mice showed increased SDF-1 levels in peripheral blood and myocardium and up-regulation of the cardiac myosin heavy chain isoform alpha, a contractile protein that was reduced in diabetic hearts. SDF-1 up-regulation was instrumental to LAV-BPIFB4-induced benefit as both haemodynamic and structural improvements were inhibited by an orally active antagonist of the SDF-1 CXCR4 receptor. CONCLUSIONS: In mice with type-2 diabetes, LAV-BPIFB4 gene therapy promotes an advantageous remodelling of the heart, allowing it to better withstand diabetes-induced stress. These results support the viability of transferring healthy characteristics of longevity to attenuate diabetic cardiac disease.

Fabry cardiomyopathy: Gb3-induced auto-reactive panmyocarditis requiring heart transplantation.

Resistance to enzyme replacement therapy (ERT) is a major therapeutic challenge in Fabry disease (FD). Recent reports attribute to immune-mediated inflammation a main role in promoting disease progression and resistance to ERT. Aim of the study is to report a Gb3-induced auto-reactive panmyocarditis causing inefficacy of ERT and severe electrical instability, which required cardiac transplantation. Examining the explanted heart from a 57-year-old man with FD cardiomyopathy (CM) on 3-year ERT presenting incoming ventricular fibrillation, we documented a severe virus-negative myocarditis extended to cardiomyocytes, intramural coronary vessels, conduction tissue, and subepicardial ganglia. Serology was positive for anti-Gb3, anti-heart, and anti-myosin antibodies. In vitro Gb3 stimulation of patient's peripheral blood mononuclear cells (PBMC) induced high amount production of inflammatory cytokine IL1-ß, IL-6, IL-8, and TNF-α. PBMC were stained using the monoclonal antibodies CD3-V500, CD4-V450, CD8-APCcy7, CD45RO-PerCPcy5.5 and CD27-FITC from BD Biosciences and CD56-PC7 from Bekman Coulter. The phenotypic analysis of PBMC showed a lower frequency of CD8 (9.2%) vs. 19.3% and NKT cells (1.6% vs. 2.4%) in Fabry patient respect to healthy donor, suggesting a possible homing to peripheral tissues. A Gb3-induced auto-reactive myocarditis is suggested as a possible cause of FDCM progression and ERT resistance. Immune-mediated inflammation of systemic Fabry cells may coexist and be controlled by implemental immunosuppressive therapy.

Heart transplantation in Danon disease: Long term single centre experience and review of the literature.

Danon disease is characterized by hypertrophic cardiomyopathy, skeletal myopathy, and intellectual disability due to deficiency of the lysosome-associated membrane protein-2 (LAMP-2). Although heart transplantation is considered an option for end stage Danon cardiomyopathy, scarce information is available about long term follow up. We report on long term follow up (14.7 years, IQ range 9-21 years) of 4 patients, transplanted for Danon disease cardiomyopathy, showing two LAMP-2 gene variants, the novel c.815T > C and the previously reported c.294G > A. We have also analysed previous published paper on this topic comparing available data from different follow up. Being a skeletal and cardiac muscle disease, with systemic effects, long term results about HTx are indispensable to justify any treatments in this subset of patients.

Autophagy and Inflammasome Activation in Dilated Cardiomyopathy.

BACKGROUND: The clinical outcome of patients affected by dilated cardiomyopathy (DCM) is heterogeneous, since its pathophysiology is only partially understood. Interleukin 1ß levels could predict the mortality and necessity of cardiac transplantation of DCM patients. OBJECTIVE: To investigate mechanisms triggering sterile inflammation in dilated cardiomyopathy (DCM). METHODS: Hearts explanted from 62 DCM patients were compared with 30 controls, employing immunohistochemistry, cellular and molecular biology, as well as metabolomics studies. RESULTS: Although misfolded protein accumulation and aggresome formation characterize DCM hearts, aggresomes failed to trigger the autophagy lysosomal pathway (ALP), with consequent accumulation of both p62SQSTM1 and dysfunctional mitochondria. In line, DCM hearts are characterized by accumulation of lipoperoxidation products and activation of both redox responsive pathways and inflammasome. Consistently with the fact that mTOR signaling may impair ALP, we observed, an increase in DCM activation, together with a reduction in the nuclear localization of Transcription Factor EB -TFEB- (a master regulator of lysosomal biogenesis). These alterations were coupled with metabolomic alterations, including accumulation of branched chain amino acids (BCAAs), known mTOR activators. Consistently, reduced levels of PP2Cm, a phosphatase that regulates the key catabolic step of BCAAs, coupled with increased levels of miR-22, a regulator of PP2Cm levels that triggers senescence, characterize DCM hearts. The same molecular defects were present in clinically relevant cells isolated from DCM hearts, but they could be reverted by downregulating miR-22. CONCLUSION: We identified, in human DCM, a complex series of events whose key players are miR-22, PP2Cm, BCAA, mTOR, and ALP, linking loss of proteostasis with inflammasome activation. These potential therapeutic targets deserve to be further investigated.

Coronary Dissection Discovered During Ex Vivo Organ Preservation: Avoiding a Fatal Complication.

This report describes the case of undiagnosed posttraumatic coronary artery dissection in a young multiorgan donor. Ex vivo preservation with the Organ Care System (TransMedics, Inc, Andover, MA) revealed the presence of coronary disease and avoided transplantation of an organ at high risk for failure.

Acromegalic Cardiomyopathy With Malignant Arrhythmogenic Pattern Successfully Treated With Mechanical Circulatory Support and Heart Transplantation.

Cardiovascular involvement is common in acromegaly and can lead to development of acromegalic cardiomyopathy, characterized by concentric biventricular hypertrophy with a progressive impairment of diastolic and systolic function. The onset of heart failure and arrhythmias are related to poor prognosis. We report on a case of a 48-year-old man with acromegalic cardiomyopathy caused by pituitary adenoma. Despite the successful trans-sphenoidal resection of the tumour, the patient was rehospitalized for ventricular arrhythmic storms that led to cardiogenic shock, which required mechanical hemodynamic support with intra-aortic balloon pump, venoarterial extracorporeal membrane oxygenation, and urgent heart transplantation.

Non-random spatial relationships between mast cells and microvessels in human endometrial carcinoma.

Mast cells (MCs) accumulate in the stroma surrounding tumors, where they secrete angiogenic cytokines and proteases, and an increased number of MCs have been demonstrated in angiogenesis associated with solid and hematological tumors. The aim of this study is to contribute to the knowledge of distribution of MCs in tumors, investigating the pattern of distribution of tryptase-positive MCs around the blood vessels in human endometrial carcinoma samples by introducing a quantitative approach to characterize their spatial distribution. The results have shown that in human endometrial cancer bioptic specimens the spatial distribution of MCs shows significant deviation from randomness as compared with control group in which, instead, the spatial distribution of MCs is consistent with a random distribution. These findings confirm that MCs enhance tumor angiogenesis and their preferential localization along blood vessels and sites of new vessel formation sustaining the suggestion for an association between MCs and angiogenesis. However, this spatial association between vessels and MCs might simply reflect migrating MCs from the blood stream at vessel growing sites.

Long noncoding RNA dysregulation in ischemic heart failure.

BACKGROUND: Long noncoding RNAs (lncRNAs) are non-protein coding transcripts regulating a variety of physiological and pathological functions. However, their implication in heart failure is still largely unknown. The aim of this study is to identify and characterize lncRNAs deregulated in patients affected by ischemic heart failure. METHODS: LncRNAs were profiled and validated in left ventricle biopsies of 18 patients affected by non end-stage dilated ischemic cardiomyopathy and 17 matched controls. Further validations were performed in left ventricle samples derived from explanted hearts of end-stage heart failure patients and in a mouse model of cardiac hypertrophy, obtained by transverse aortic constriction. Peripheral blood mononuclear cells of heart failure patients were also analyzed. LncRNA distribution in the heart was assessed by in situ hybridization. Function of the deregulated lncRNA was explored analyzing the expression of the neighbor mRNAs and by gene ontology analysis of the correlating coding transcripts. RESULTS: Fourteen lncRNAs were significantly modulated in non end-stage heart failure patients, identifying a heart failure lncRNA signature. Nine of these lncRNAs (CDKN2B-AS1/ANRIL, EGOT, H19, HOTAIR, LOC285194/TUSC7, RMRP, RNY5, SOX2-OT and SRA1) were also confirmed in end-stage failing hearts. Intriguingly, among the conserved lncRNAs, h19, rmrp and hotair were also induced in a mouse model of heart hypertrophy. CDKN2B-AS1/ANRIL, HOTAIR and LOC285194/TUSC7 showed similar modulation in peripheral blood mononuclear cells and heart tissue, suggesting a potential role as disease biomarkers. Interestingly, RMRP displayed a ubiquitous nuclear distribution, while H19 RNA was more abundant in blood vessels and was both cytoplasmic and nuclear. Gene ontology analysis of the mRNAs displaying a significant correlation in expression with heart failure lncRNAs identified numerous pathways and functions involved in heart failure progression. CONCLUSIONS: These data strongly suggest lncRNA implication in the molecular mechanisms underpinning HF.

Critical role of lysosomes in the dysfunction of human Cardiac Stem Cells obtained from failing hearts.

UNLABELLED: The in vivo reparative potential of Cardiac Stem Cells (CSC), cultured from explanted failing hearts (E-), is impaired by cellular senescence. Moreover, E-CSC are characterized, with respect to CSC obtained from healthy donors (D-), by an arrest in the autophagic degradation. Although the lysosome plays a pivotal role in cellular homeostasis and defects of this organelle may be associated with aging and heart failure, the lysosomal function of CSC has never been investigated. The aim of this work was to focus on the Lysosomal Compartment (LC) of E-CSC, evaluating elements that could jeopardize lysosome functionality. METHODS AND RESULTS: Bioinformatics analysis conducted on genes differentially expressed between D- and E-CSC identified lysosomal-related gene sets as significantly enriched. Moreover, 29 differentially expressed genes were part of CLEAR (Coordinated Lysosomal Expression and Regulation) gene network, by which Transcription Factor EB (TFEB) regulates cellular clearance. Consistently, live cell imaging and flow cytometry analyses showed that the lysosomes of E-CSC are less acidic than the D-CSC ones. Furthermore, confocal microscopy showed in E-CSC: an accumulation of intralysosomal lipofuscins, a reduction of cathepsin B activity, evidence of lysosome membrane permeabilization, and the reduction of the nuclear active TFEB. The use of Rapamycin (TORC1 inhibitor) was able on one hand to increase TFEB activation and, on the other hand, to reduce lipofuscin mass, potentiating the lysosomal functionality. CONCLUSIONS: This study demonstrated for the first time that E-CSC are characterized by a blunted activation of TFEB and an altered proteostasis. TORC1 hyperactivation plays a central role in this phenomenon.

Ex vivo molecular rejuvenation improves the therapeutic activity of senescent human cardiac stem cells in a mouse model of myocardial infarction.

Cardiac stem cells (CSC) from explanted decompensated hearts (E-CSC) are, with respect to those obtained from healthy donors (D-CSC), senescent and functionally impaired. We aimed to identify alterations in signaling pathways that are associated with CSC senescence. Additionally, we investigated if pharmacological modulation of altered pathways can reduce CSC senescence in vitro and enhance their reparative ability in vivo. Measurement of secreted factors showed that E-CSC release larger amounts of proinflammatory cytokine IL1ß compared with D-CSC. Using blocking antibodies, we verified that IL1ß hampers the paracrine protective action of E-CSC on cardiomyocyte viability. IL1ß acts intracranially inducing IKKß signaling, a mechanism that via nuclear factor-κB upregulates the expression of IL1ß itself. Moreover, E-CSC show reduced levels of AMP protein kinase (AMPK) activating phosphorylation. This latter event, together with enhanced IKKß signaling, increases TORC1 activity, thereby impairing the autophagic flux and inhibiting the phosphorylation of Akt and cAMP response element-binding protein. The combined use of rapamycin and resveratrol enhanced AMPK, thereby restoring downstream signaling and reducing IL1ß secretion. These molecular corrections reduced E-CSC senescence, re-establishing their protective activity on cardiomyocytes. Moreover ex vivo treatment with rapamycin and resveratrol improved E-CSC capacity to induce cardiac repair upon injection in the mouse infarcted heart, leading to reduced cardiomyocyte senescence and apoptosis and increased abundance of endogenous c-Kit(+) CSC in the peri-infarct area. Molecular rejuvenation of patient-derived CSC by short pharmacologic conditioning boosts their in vivo reparative abilities. This approach might prove useful for refinement of CSC-based therapies.

Panencephalopathic Creutzfeldt-Jakob disease with distinct pattern of prion protein deposition in a patient with D178N mutation and homozygosity for valine at codon 129 of the prion protein Gene.

Prion diseases include sporadic, acquired and genetic forms linked to mutations of the prion protein (PrP) gene (PRNP). In subjects carrying the D178N PRNP mutation, distinct phenotypes can be observed, depending on the methionine/valine codon 129 polymorphism. We present here a 53-year-old woman with D178N mutation in the PRNP gene and homozygosity for valine at codon 129. The disease started at age 47 with memory deficits, progressive cognitive impairment and ataxia. The clinical picture slowly worsened to a state of akinetic mutism in about 2 years and the disease course was 6 years. The neuropathologic examination demonstrated severe diffuse cerebral atrophy with neuronal loss, spongiosis and marked myelin loss and tissue rarefaction in the hemispheric white matter, configuring panencephalopathic Creutzfeldt-Jakob disease. PrP deposition was present in the cerebral cortex, basal ganglia and cerebellum with diffuse synaptic-type pattern of immunoreactivity and clusters of countless, small PrP deposits, particularly evident in the lower cortical layers, in the striatum and in the molecular layer of the cerebellum. Western blot analysis showed the presence of type 1 PrP(Sc) (Parchi classification). These findings underline the clear-cut distinction between the neuropathological features of Creutzfeldt-Jakob disease associated with D178N PRNP mutation and those of fatal familial insomnia.

Absolute quantification of choline-related biomarkers in breast cancer biopsies by liquid chromatography electrospray ionization mass spectrometry.

It has been repeatedly demonstrated that choline metabolism is altered in a wide variety of cancers. In breast tumours, the choline metabolite profile is characterized by an elevation of phosphocholine and total choline-compounds. This pattern is increasingly being exploited as biomarker in cancer diagnosis. The majority of in vitro metabolomics studies, for biomarkers quantification in cell cultures or tissues, entail proton NMR spectroscopy. Although many "targeted" approaches have been proposed to quantify metabolites from standard one-dimensional (1D) NMR experiments, the task is often made difficult by the high degree of overlap characterizing 1H NMR spectra of biological samples. Here we present an optimized protocol for tissue extraction and absolute quantification of choline, phosphocholine and glycerophosphocholine by means of liquid chromatography electrospray ionization mass spectrometry (LC-ESI-MS). The selected chromatographic separation system with a HILIC (hydrophilic interaction chromatography) amide column effectively separates free choline and its phopshorylated derivatives, contrary to failure observed using standard reversed-phase chromatography. The metabolite absolute quantification is based on external calibration with commercial standards, and is validated by a parallel 1D proton NMR analysis. The LC-MS/NMR analysis is applied to three breast carcinoma specimens obtained by surgical excision, each one accompanied by a control tissue sample taken outside the tumor margin. The metabolite concentrations measured are in good agreement with previous results on metabolic profile changes of breast cancer. Each of the three cancerous biopsies, when compared with the control tissue, exhibit a highly increased levels phosphocholine, total choline and phosphocholine/glycerophosphocholine ratio.

Intracellular function of interleukin-1 receptor antagonist in ischemic cardiomyocytes.

BACKGROUND: Loss of cardiac myocytes due to apoptosis is a relevant feature of ischemic heart disease. It has been described in infarct and peri-infarct regions of the myocardium in coronary syndromes and in ischemia-linked heart remodeling. Previous studies have provided protection against ischemia-induced cardiomyocyte apoptosis by the anti-inflammatory cytokine interleukin-1 receptor-antagonist (IL-1Ra). Mitochondria triggering of caspases plays a central role in ischemia-induced apoptosis. We examined the production of IL-1Ra in the ischemic heart and, based on dual intra/extracellular function of some other interleukins, we hypothesized that IL-1Ra may also directly inhibit mitochondria-activated caspases and cardiomyocyte apoptosis. METHODOLOGY/PRINCIPAL FINDINGS: Synthesis of IL-1Ra was evidenced in the hearts explanted from patients with ischemic heart disease. In the mouse ischemic heart and in a mouse cardiomyocyte cell line exposed to long-lasting hypoxia, IL-1Ra bound and inhibited mitochondria-activated caspases, whereas inhibition of caspase activation was not observed in the heart of mice lacking IL-1Ra (Il-1ra-/-) or in siRNA to IL-1Ra-interfered cells. An impressive 6-fold increase of hypoxia-induced apoptosis was observed in cells lacking IL-1Ra. IL-1Ra down-regulated cells were not protected against caspase activation and apoptosis by knocking down of the IL-1 receptor, confirming the intracellular, receptor-independent, anti-apoptotic function of IL-1Ra. Notably, the inhibitory effect of IL-1Ra was not influenced by enduring ischemic conditions in which previously described physiologic inhibitors of apoptosis are neutralized. CONCLUSIONS/SIGNIFICANCE: These observations point to intracellular IL-1Ra as a critical mechanism of the cell self-protection against ischemia-induced apoptosis and suggest that this cytokine plays an important role in the remodeling of heart by promoting survival of cardiomyocytes in the ischemic regions.

Sudden death associated with danon disease in women.

Danon disease is an X-linked systemic disorder characterized by left ventricular hypertrophy, mental retardation, and skeletal myopathy affecting young men. Electrocardiogram usually displays a Wolff-Parkinson-White preexcitation pattern. Less has been reported about the phenotype in women, although later-onset cardiac symptoms have been described. The aim of this study was to expand the knowledge of the phenotype of Danon disease in women. We clinically followed and evaluated with echocardiography, cardiac magnetic resonance imaging (cMRI), and genetic testing a family affected by Danon disease in which 2 men and 6 women showed a severe arrhythmogenic phenotype. Affected family members carried a nucleotide substitution at position 294 in exon 3 (c.294 G → A) that changed a tryptophan residue to a stop codon at position W98X in the lysosome-associated membrane protein 2 (LAMP2) gene. Four women died suddenly (1 aborted) at 37 to 54 years of age. Wolff-Parkinson-White pattern with atrioventricular block was detected in 2 of 6 women. Four had successful pregnancies without symptoms of heart failure. cMRI showed late gadolinium enhancement areas in a clinically healthy woman who was a mutation carrier. Two patients underwent heart transplantation; histology of explanted hearts demonstrated severe interstitial fibrosis, hypertrophic cardiomyocytes with cytoplasmic vacuoles, and myofibrillar disarray. In conclusion, LAMP2 mutation can cause a severe arrhythmogenic phenotype in women that includes a high risk of sudden death. cMRI may be useful in women harboring LAMP2 mutations to permit early detection of cardiac involvement and guide timely considerations of implantable cardioverter-defibrillator therapy. Heart transplantation should be considered at onset of heart failure symptoms owing to rapid progression of the disease.