Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit^pos) cells. The adult heart indeed contains a heterogeneous mixture of c-kit^pos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit^pos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit^pos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit^pos sorting. The blood/endothelial lineage-committed (Lineage^pos) CD45^posc-kit^pos cardiac cells were compared to CD45^neg(Lineage^neg/Lin^neg) c-kit^pos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit^pos cardiac cells are blood/endothelial lineage-committed CD45^posCD31^posc-kit^pos cells. In contrast, the Lin^negCD45^negc-kit^pos cardiac cell cohort, which represents ⩽10% of the total c-kit^pos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit^neg and the blood/endothelial lineage-committed c-kit^pos cardiac cells. Single Lin^negc-kit^pos cell-derived clones, which represent only 1–2% of total c-kit^pos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Lin^negc-kit^pos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC’s myogenic regenerative capacity is dependent on commitment to the CM lineage through activation of the SMAD2 pathway. Such regeneration was not apparent when blood/endothelial lineage-committed c-kit^pos cardiac cells were injected. Thus, among the cardiac c-kit^pos cell cohort only a very small fraction has the phenotype and the differentiation/regenerative potential characteristics of true multipotent CSCs.

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.

Background: Diabetes mellitus ( DM ) has multifactorial detrimental effects on myocardial tissue. Recently, carbonic anhydrases ( CAs ) have been shown to play a major role in diabetic microangiopathy but their role in the diabetic cardiomyopathy is still unknown. Methods and Results: We obtained left ventricular samples from patients with DM type 2 (DM‐T2) and nondiabetic ( NDM ) patients with postinfarct heart failure who were undergoing surgical coronary revascularization. Myocardial levels of CA ‐I and CA‐ II were 6‐ and 11‐fold higher, respectively, in DM ‐T2 versus NDM patients. Elevated CA ‐I expression was mainly localized in the cardiac interstitium and endothelial cells. CA ‐I induced by high glucose levels hampers endothelial cell permeability and determines endothelial cell apoptosis in vitro. Accordingly, capillary density was significantly lower in the DM ‐T2 myocardial samples (mean± SE =2152±146 versus 4545±211/mm ² ). On the other hand, CA‐II was mainly upregulated in cardiomyocytes. The latter was associated with sodium‐hydrogen exchanger‐1 hyperphosphorylation, exaggerated myocyte hypertrophy (cross‐sectional area 565±34 versus 412±27 μm ² ), and apoptotic death (830±54 versus 470±34 per 10 ⁶ myocytes) in DM ‐T2 versus NDM patients. CA‐II is activated by high glucose levels and directly induces cardiomyocyte hypertrophy and death in vitro, which are prevented by sodium‐hydrogen exchanger‐1 inhibition. CA‐II was shown to be a direct target for repression by micro RNA ‐23b, which was downregulated in myocardial samples from DM ‐T2 patients. MicroRNA‐23b is regulated by p38 mitogen‐activated protein kinase, and it modulates high‐glucose CA‐II–dependent effects on cardiomyocyte survival in vitro. Conclusions: Myocardial CA activation is significantly elevated in human diabetic ischemic cardiomyopathy. These data may open new avenues for targeted treatment of diabetic heart failure.

Harnessing the mechanisms underlying the exacerbated vascular remodelling in Diabetes Mellitus (DM) is pivotal to prevent the high toll of vascular diseases in DM patients. microRNAs (miRNA) regulate vascular smooth muscle cell (VSMC) phenotypic switch. However, miRNA modulation of the detrimental diabetic VSMC phenotype is underexplored. Streptozotocin-induced Type 1 Diabetes (T1DM) Wistar rats and T2DM Zucker rats underwent right carotid artery experimental angioplasty and global miRNA/mRNA expression profiling was obtained by RNA sequencing (RNA-Seq). 2 days after injury a set of 6 miRs were found to be uniquely down-regulated or up-regulated both in VSMCs in T1DM and T2DM. Among these, miR-29c and miR-204 were the most significantly mis-regulated in atherosclerotic plaques from DM patients. miR-29c overexpression and miR-204 inhibition per se attenuated VSMC phenotypic switch in DM. Concomitant miR-29c overexpression and miR-204 inhibition fostered an additive reduction in VSMC proliferation. Epithelial membrane protein 2 (Emp2) and Caveolin-1 (Cav1) mRNAs were identified as direct targets of miR-29c and miR-204, respectively. Importantly, contemporary miR-29c overexpression and miR-204 inhibition in the injured artery robustly reduced arterial stenosis in DM rats. Thus, contemporaneous miR-29c activation and miR-204 inhibition in DM arterial tissues is necessary and sufficient to prevent the exaggerated VSMC growth upon injury.

Organoids are tiny, self-organized, three-dimensional tissue cultures that are derived from the differentiation of stem cells. The growing interest in the use of organoids arises from their ability to mimic the biology and physiology of specific tissue structures in vitro. Organoids indeed represent promising systems for the in vitro modeling of tissue morphogenesis and organogenesis, regenerative medicine and tissue engineering, drug therapy testing, toxicology screening, and disease modeling. Although 2D cell cultures have been used for more than 50 years, even for their simplicity and low-cost maintenance, recent years have witnessed a steep rise in the availability of organoid model systems. Exploiting the ability of cells to re-aggregate and reconstruct the original architecture of an organ makes it possible to overcome many limitations of 2D cell culture systems. In vitro replication of the cellular micro-environment of a specific tissue leads to reproducing the molecular, biochemical, and biomechanical mechanisms that directly influence cell behavior and fate within that specific tissue. Lineage-specific self-organizing organoids have now been generated for many organs. Currently, growing cardiac organoid (cardioids) from pluripotent stem cells and cardiac stem/progenitor cells remains an open challenge due to the complexity of the spreading, differentiation, and migration of cardiac muscle and vascular layers. Here, we summarize the evolution of biological model systems from the generation of 2D spheroids to 3D organoids by focusing on the generation of cardioids based on the currently available laboratory technologies and outline their high potential for cardiovascular research.