Mechanisms of Cardiovascular Calcification and Experimental Models: Impact of Vitamin K Antagonists.

Cardiovascular calcification is a multifactorial and complex process involving an array of molecular mechanisms eventually leading to calcium deposition within the arterial walls. This process increases arterial stiffness, decreases elasticity, influences shear stress events and is related to an increased risk of morbidity and mortality associated with cardiovascular disease. In numerous in vivo and in vitro models, warfarin therapy has been shown to cause vascular calcification in the arterial wall. However, the exact mechanisms of calcification formation with warfarin remain largely unknown, although several molecular pathways have been identified. Circulating miRNA have been evaluated as biomarkers for a wide range of cardiovascular diseases, but their exact role in cardiovascular calcification is limited. This review aims to describe the current state-of-the-art research on the impact of warfarin treatment on the development of vascular calcification and to highlight potential molecular targets, including microRNA, within the implicated pathways.

Adult Multipotent Cardiac Progenitor-Derived Spheroids: A Reproducible Model of In Vitro Cardiomyocyte Commitment and Specification.

BACKGROUND: Three-dimensional cell culture systems hold great promise for bridging the gap between in vitro cell-based model systems and small animal models to study tissue biology and disease. Among 3D cell culture systems, stem-cell-derived spheroids have attracted significant interest as a strategy to better mimic in vivo conditions. Cardiac stem cell/progenitor (CSC)-derived spheroids (CSs) provide a relevant platform for cardiac regeneration. METHODS: We compared three different cell culture scaffold-free systems, (i) ultra-low attachment plates, (ii) hanging drops (both requiring a 2D/3D switch), and (iii) agarose micro-molds (entirely 3D), for CSC-derived CS formation and their cardiomyocyte commitment in vitro. RESULTS: The switch from a 2D to a 3D culture microenvironment per se guides cell plasticity and myogenic differentiation within CS and is necessary for robust cardiomyocyte differentiation. On the contrary, 2D monolayer CSC cultures show a significant reduced cardiomyocyte differentiation potential compared to 3D CS culture. Forced aggregation into spheroids using hanging drop improves CS myogenic differentiation when compared to ultra-low attachment plates. Performing CS formation and myogenic differentiation exclusively in 3D culture using agarose micro-molds maximizes the cardiomyocyte yield. CONCLUSIONS: A 3D culture system instructs CS myogenic differentiation, thus representing a valid model that can be used to study adult cardiac regenerative biology.

Polarizing Macrophage Functional Phenotype to Foster Cardiac Regeneration.

There is an increasing interest in understanding the connection between the immune and cardiovascular systems, which are highly integrated and communicate through finely regulated cross-talking mechanisms. Recent evidence has demonstrated that the immune system does indeed have a key role in the response to cardiac injury and in cardiac regeneration. Among the immune cells, macrophages appear to have a prominent role in this context, with different subtypes described so far that each have a specific influence on cardiac remodeling and repair. Similarly, there are significant differences in how the innate and adaptive immune systems affect the response to cardiac damage. Understanding all these mechanisms may have relevant clinical implications. Several studies have already demonstrated that stem cell-based therapies support myocardial repair. However, the exact role that cardiac macrophages and their modulation may have in this setting is still unclear. The current need to decipher the dual role of immunity in boosting both heart injury and repair is due, at least for a significant part, to unresolved questions related to the complexity of cardiac macrophage phenotypes. The aim of this review is to provide an overview on the role of the immune system, and of macrophages in particular, in the response to cardiac injury and to outline, through the modulation of the immune response, potential novel therapeutic strategies for cardiac regeneration.

A Mouse Model of Dilated Cardiomyopathy Produced by Isoproterenol Acute Exposure Followed by 5-Fluorouracil Administration.

Appropriate dilated cardiomyopathy (DCM) animal models are highly desirable considering the pathophysiological and clinical heterogeneity of DCM. Genetically modified mice are the most widely and intensively utilized research animals for DCM. However, to translate discoveries from basic science into new and personalized medical applications, research in non-genetically based DCM models remains a key issue. Here, we characterized a mouse model of non-ischemic DCM induced by a stepwise pharmacologic regime of Isoproterenol (ISO) high dose bolus followed by a low dose systemic injection of the chemotherapy agent, 5-Fluorouracil (5-FU). C57BL/6J mice were injected with ISO and, 3 days after, were randomly assigned to saline or 5-FU. Echocardiography and a strain analysis show that ISO + 5FU in mice induces progressive left ventricular (LV) dilation and reduced systolic function, along with diastolic dysfunction and a persistent global cardiac contractility depression through 56 days. While mice treated with ISO alone recover anatomically and functionally, ISO + 5-FU causes persistent cardiomyocyte death, ensuing in cardiomyocyte hypertrophy through 56 days. ISO + 5-FU-dependent damage was accompanied by significant myocardial disarray and fibrosis along with exaggerated oxidative stress, tissue inflammation and premature cell senescence accumulation. In conclusions, a combination of ISO + 5FU produces anatomical, histological and functional cardiac alterations typical of DCM, representing a widely available, affordable, and reproducible mouse model of this cardiomyopathy.

Clinical Application of Circular RNAs as Biomarkers in Acute Ischemic Stroke.

Despite the substantial improvement in diagnosis and treatment within the last decades, ischemic stroke still represents a challenge, responsible still for a high burden of morbidity and mortality. Among the unmet clinical needs are the difficulties in identifying those subjects with the greatest risk of developing a stroke, the challenges in obtaining a timely diagnosis, the prompt recognition of the different clinical forms of stroke, the assessment of the response to treatments and the prognostic assessment. All these issues might be improved with appropriate smart biomarkers that could better inform clinical management. The present article offers an overview of the potential role of circular RNAs as disease biomarkers in stroke. A systematic approach was adopted to gather all potentially relevant information in order to provide a panoramic view on this class of promising molecules.

COVID-19 and atrial fibrillation: Intercepting lines.

Almost 20% of COVID-19 patients have a history of atrial fibrillation (AF), but also a new-onset AF represents a frequent complication in COVID-19. Clinical evidence demonstrates that COVID-19, by promoting the evolution of a prothrombotic state, increases the susceptibility to arrhythmic events during the infective stages and presumably during post-recovery. AF itself is the most frequent form of arrhythmia and is associated with substantial morbidity and mortality. One of the molecular factors involved in COVID-19-related AF episodes is the angiotensin-converting enzyme (ACE) 2 availability. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uses ACE2 to enter and infect multiple cells. Atrial ACE2 internalization after binding to SARS-CoV-2 results in a raise of angiotensin (Ang) II, and in a suppression of cardioprotective Ang(1-7) formation, and thereby promoting cardiac hypertrophy, fibrosis and oxidative stress. Furthermore, several pharmacological agents used in COVID-19 patients may have a higher risk of inducing electrophysiological changes and cardiac dysfunction. Azithromycin, lopinavir/ritonavir, ibrutinib, and remdesivir, used in the treatment of COVID-19, may predispose to an increased risk of cardiac arrhythmia. In this review, putative mechanisms involved in COVID-19-related AF episodes and the cardiovascular safety profile of drugs used for the treatment of COVID-19 are summarized.

Endothelial Glycocalyx and Cardiomyocyte Damage Is Prevented by Recombinant Syndecan-1 in Acute Myocardial Infarction.

The outer layer of endothelial cells (ECs), consisting of the endothelial glycocalyx (eGC) and the cortex (CTX), provides a protective barrier against vascular diseases. Structural and functional impairments of their mechanical properties are recognized as hallmarks of endothelial dysfunction and can lead to cardiovascular events, such as acute myocardial infarction (AMI). This study investigated the effects of AMI on endothelial nanomechanics and function and the use of exogenous recombinant syndecan-1 (rSyn-1), a major component of the eGC, as recovering agent. ECs were exposed in vitro to serum samples collected from patients with AMI. In addition, in situ ECs of ex vivo aorta preparations derived from a mouse model for AMI were employed. Effects were quantified by using atomic force microscopy-based nanoindentation measurements, fluorescence staining, and histologic examination of the mouse hearts. AMI serum samples damaged eGC/CTX and augmented monocyte adhesion to the endothelial surface. In particular, the anaphylatoxins C3a and C5a played an important role in these processes. The impairment of endothelial function could be prevented by rSyn-1 treatment. In the mouse model of myocardial infarction, pretreatment with rSyn-1 alleviated eGC/CTX deterioration and reduced cardiomyocyte damage in histologic analyses. However, echocardiographic measurements did not indicate a functional benefit. These results provide new insights into the underlying mechanisms of AMI-induced endothelial dysfunction and perspectives for future studies on the benefit of rSyn-1 in post-AMI treatment.

Dapagliflozin protects the kidney in a non-diabetic model of cardiorenal syndrome.

Cardiorenal syndrome encompasses a spectrum of disorders involving heart and kidney dysfunction, and sharing common risk factors, such as hypertension and diabetes. Clinical studies have shown that patients with and without diabetes may benefit from using sodium-glucose cotransporter 2 inhibitors to reduce the risk of heart failure and ameliorate renal endpoints. Because the underlying mechanisms remain elusive, we investigated the effects of dapagliflozin on the progression of renal damage, using a model of non-diabetic cardiorenal disease. Dahl salt-sensitive rats were fed a high-salt diet for five weeks and then randomized to dapagliflozin or vehicle for the following six weeks. After treatment with dapagliflozin, renal function resulted ameliorated as shown by decrease of albuminuria and urine albumin-to-creatinine ratio. Functional benefit was accompanied by a decreased accumulation of extracellular matrix and a reduced number of sclerotic glomeruli. Dapagliflozin significantly reduced expression of inflammatory and endothelial activation markers such as NF-κB and e-selectin. Upregulation of pro-oxidant-releasing NADPH oxidases 2 and 4 as well as downregulation of antioxidant enzymes were also counteracted by drug treatment. Our findings also evidenced the modulation of both classic and non-classic renin-angiotensin-aldosterone system (RAAS), and effects of dapagliflozin on gene expression of ion channels/transporters involved in renal homeostasis. Thus, in a non-diabetic model of cardiorenal syndrome, dapagliflozin provides renal protection by modulating inflammatory response, endothelial activation, fibrosis, oxidative stress, local RAAS and ion channels.

Streptozotocin-Induced Type 1 and 2 Diabetes Mellitus Mouse Models Show Different Functional, Cellular and Molecular Patterns of Diabetic Cardiomyopathy.

The main cause of morbidity and mortality in diabetes mellitus (DM) is cardiovascular complications. Diabetic cardiomyopathy (DCM) remains incompletely understood. Animal models have been crucial in exploring DCM pathophysiology while identifying potential therapeutic targets. Streptozotocin (STZ) has been widely used to produce experimental models of both type 1 and type 2 DM (T1DM and T2DM). Here, we compared these two models for their effects on cardiac structure, function and transcriptome. Different doses of STZ and diet chows were used to generate T1DM and T2DM in C57BL/6J mice. Normal euglycemic and nonobese sex- and age-matched mice served as controls (CTRL). Immunohistochemistry, RT-PCR and RNA-seq were employed to compare hearts from the three animal groups. STZ-induced T1DM and T2DM affected left ventricular function and myocardial performance differently. T1DM displayed exaggerated apoptotic cardiomyocyte (CM) death and reactive hypertrophy and fibrosis, along with increased cardiac oxidative stress, CM DNA damage and senescence, when compared to T2DM in mice. T1DM and T2DM affected the whole cardiac transcriptome differently. In conclusion, the STZ-induced T1DM and T2DM mouse models showed significant differences in cardiac remodeling, function and the whole transcriptome. These differences could be of key relevance when choosing an animal model to study specific features of DCM.

Initial Phase of Anthracycline Cardiotoxicity Involves Cardiac Fibroblasts Activation and Metabolic Switch.

The application of doxorubicin (DOX) is hampered by cardiotoxicity, with diastolic dysfunction as the earliest manifestation. Fibrosis leads to impaired relaxation, but the mechanisms that operate shortly after DOX exposure are not clear. We asked whether the activation of cardiac fibroblasts (CFs) anticipates myocardial dysfunction and evaluated the effects of DOX on CF metabolism. CFs were isolated from the hearts of rats after the first injection of DOX. In another experiment, CFs were exposed to DOX in vitro. Cell phenotype and metabolism were determined. Early effects of DOX consisted of diastolic dysfunction and unchanged ejection fraction. Markers of pro-fibrotic remodeling and evidence of CF transformation were present immediately after treatment completion. Oxygen consumption rate and extracellular acidification revealed an increased metabolic activity of CFs and a switch to glycolytic energy production. These effects were consistent in CFs isolated from the hearts of DOX-treated animals and in naïve CFs exposed to DOX in vitro. The metabolic switch was paralleled with the phenotype change of CFs that upregulated markers of myofibroblast differentiation and the activation of pro-fibrotic signaling. In conclusion, the metabolic switch and activation of CFs anticipate DOX-induced damage and represent a novel target in the early phase of anthracycline cardiomyopathy.

Pharmacological clearance of senescent cells improves cardiac remodeling and function after myocardial infarction in female aged mice.

Cardiovascular diseases (CVD) are predominantly an aging disease. Important sex-specific differences exist and the mechanism(s) by which this sex-by-age interaction influences CVD development and progression remains elusive. Accordingly, it is still unknown whether cell senescence, a main feature of cardiac male aging, is a significant feature also of the female aged mouse heart and whether senolytics, senescence-clearing compounds, promote myocardial repair and regeneration after myocardial infarction (MI) in aged female mice. To this aim, the combination of two senolytics, dasatinib and quercetin (D+Q) or just their vehicle was administered to 22-24 months old C57BL/6 female mice after MI. D+Q improved global left ventricle function and myocardial performance after MI whereby female cardiac aging is characterized by accumulation of cardiac senescent cells that are further increased by MI. Despite their terminal differentiation nature, also cardiomyocytes acquire a senescent phenotype with age in females. D+Q removed senescent cardiac non-myocyte and myocyte cells ameliorating cardiac remodeling and regeneration. Senolytics removed aged dysfunctional cardiac stem/progenitor cells (CSCs), relieving healthy CSCs with normal proliferative and cardiomyogenic differentiation potential. In conclusions, cardiac senescent cells accumulate in the aged female hearts. Removing senescent cells is a key therapeutic target for efficient repair of the aged female heart.

The negative regulation of gene expression by microRNAs as key driver of inducers and repressors of cardiomyocyte differentiation.

Cardiac muscle damage-induced loss of cardiomyocytes (CMs) and dysfunction of the remaining ones leads to heart failure, which nowadays is the number one killer worldwide. Therapies fostering effective cardiac regeneration are the holy grail of cardiovascular research to stop the heart failure epidemic. The main goal of most myocardial regeneration protocols is the generation of new functional CMs through the differentiation of endogenous or exogenous cardiomyogenic cells. Understanding the cellular and molecular basis of cardiomyocyte commitment, specification, differentiation and maturation is needed to devise innovative approaches to replace the CMs lost after injury in the adult heart. The transcriptional regulation of CM differentiation is a highly conserved process that require sequential activation and/or repression of different genetic programs. Therefore, CM differentiation and specification have been depicted as a step-wise specific chemical and mechanical stimuli inducing complete myogenic commitment and cell-cycle exit. Yet, the demonstration that some microRNAs are sufficient to direct ESC differentiation into CMs and that four specific miRNAs reprogram fibroblasts into CMs show that CM differentiation must also involve negative regulatory instructions. Here, we review the mechanisms of CM differentiation during development and from regenerative stem cells with a focus on the involvement of microRNAs in the process, putting in perspective their negative gene regulation as a main modifier of effective CM regeneration in the adult heart.

Myocardial regeneration protocols towards the routine clinical scenario: An unseemly path from bench to bedside.

Heart failure secondary to cardiomyocyte loss and/or dysfunction is the number one killer worldwide. The field of myocardial regeneration with its far-reaching primary goal of cardiac remuscularization and its hard-to-accomplish translation from bench to bedside, has been filled with ups and downs, steps forward and steps backward, controversies galore and, unfortunately, scientific scandals. Despite the present morass in which cardiac remuscularization is stuck in, the search for clinically effective regenerative approaches remains keenly active. Starting with a concise overview of the still highly debated regenerative capacity of the adult mammalian heart, we focus on the main interventions, that have reached or are close to clinical use, critically discussing key findings, successes, and failures. Finally, some promising and innovative approaches for myocardial repair/regeneration still at the pre-clinical stage are discussed to offer a holistic view on the future of myocardial repair/regeneration for the prevention/management of heart failure in the clinical scenario. Funding: This research was funded by Grants from the Ministry of University and Research PRIN2015 2015ZTT5KB_004; PRIN2017NKB2N4_005; PON-AIM - 1829805-2.

Scn1b expression in the adult mouse heart modulates Na+ influx in myocytes and reveals a mechanistic link between Na+ entry and diastolic function.

Voltage-gated sodium channels (VGSCs) are macromolecular assemblies composed of a number of proteins regulating channel conductance and properties. VGSCs generate Na+ current (INa) in myocytes and play fundamental roles in excitability and impulse conduction in the heart. Moreover, VGSCs condition mechanical properties of the myocardium, a process that appears to involve the late component of INa. Variants in the gene SCN1B, encoding the VGSC ß1- and ß1B-subunits, result in inherited neurological disorders and cardiac arrhythmias. But the precise contributions of ß1/ß1B-subunits and VGSC integrity to the overall function of the adult heart remain to be clarified. For this purpose, adult mice with cardiac-restricted, inducible deletion of Scn1b (conditional knockout, cKO) were studied. Myocytes from cKO mice had increased densities of fast (+20%)- and slow (+140%)-inactivating components of INa, with respect to control cells. By echocardiography and invasive hemodynamics, systolic function was preserved in cKO mice, but diastolic properties and ventricular compliance were compromised, with respect to control animals. Importantly, inhibition of late INa with GS967 normalized left ventricular filling pattern and isovolumic relaxation time in cKO mice. At the cellular level, cKO myocytes presented delayed kinetics of Ca2+ transients and cell mechanics, defects that were corrected by inhibition of INa. Collectively, these results document that VGSC ß1/ß1B-subunits modulate electrical and mechanical function of the heart by regulating, at least in part, Na+ influx in cardiomyocytes.NEW & NOTEWORTHY We have investigated the consequences of deletion of Scn1b, the gene encoding voltage-gated sodium channel ß1-subunits, on myocyte and cardiac function. Our findings support the notion that Scn1b expression controls properties of Na+ influx and Ca2+ cycling in cardiomyocytes affecting the modality of cell contraction and relaxation. These effects at the cellular level condition electrical recovery and diastolic function in vivo, substantiating the multifunctional role of ß1-subunits in the physiology of the heart.

Unraveling and Targeting Myocardial Regeneration Deficit in Diabetes.

Cardiomyopathy is a common complication in diabetic patients. Ventricular dysfunction without coronary atherosclerosis and hypertension is driven by hyperglycemia, hyperinsulinemia and impaired insulin signaling. Cardiomyocyte death, hypertrophy, fibrosis, and cell signaling defects underlie cardiomyopathy. Notably, detrimental effects of the diabetic milieu are not limited to cardiomyocytes and vascular cells. The diabetic heart acquires a senescent phenotype and also suffers from altered cellular homeostasis and the insufficient replacement of dying cells. Chronic inflammation, oxidative stress, and metabolic dysregulation damage the population of endogenous cardiac stem cells, which contribute to myocardial cell turnover and repair after injury. Therefore, deficient myocardial repair and the progressive senescence and dysfunction of stem cells in the diabetic heart can represent potential therapeutic targets. While our knowledge of the effects of diabetes on stem cells is growing, several strategies to preserve, activate or restore cardiac stem cell compartments await to be tested in diabetic cardiomyopathy.