Impact of Chronic Fetal Hypoxia and Inflammation on Cardiac Pacemaker Cell Development

Chronic fetal hypoxia and infection are examples of adverse conditions during complicated pregnancy, which impact cardiac myogenesis and increase the lifetime risk of heart disease. However, the effects that chronic hypoxic or inflammatory environments exert on cardiac pacemaker cells are poorly understood. Here, we review the current evidence and novel avenues of bench-to-bed research in this field of perinatal cardiogenesis as well as its translational significance for early detection of future risk for cardiovascular disease.

Cardiac Niche Influences the Direct Reprogramming of Canine Fibroblasts into Cardiomyocyte-Like Cells

The Duchenne and Becker muscular dystrophies are caused by mutation of dystrophin gene and primarily affect skeletal and cardiac muscles. Cardiac involvement in dystrophic GRMD dogs has been demonstrated by electrocardiographic studies with the onset of a progressive cardiomyopathy similar to the cardiac disease in DMD patients. In this respect, GRMD is a useful model to explore cardiac and skeletal muscle pathogenesis and for developing new therapeutic protocols. Here we describe a protocol to convert GRMD canine fibroblasts isolated from heart and skin into induced cardiac-like myocytes (ciCLMs). We used a mix of transcription factors (GATA4, HAND2, TBX5, and MEF2C), known to be able to differentiate mouse and human somatic cells into ciCLMs. Exogenous gene expression was obtained using four lentiviral vectors carrying transcription factor genes and different resistance genes. Our data demonstrate a direct switch from fibroblast into ciCLMs with no activation of early cardiac genes. ciCLMs were unable to contract spontaneously, suggesting, differently from mouse and human cells, an incomplete differentiation process. However, when transplanted in neonatal hearts of SCID/Beige mice, ciCLMs participate in cardiac myogenesis.

Overexpression of TNNI3K, a cardiac-specific MAP kinase, promotes P19CL6-derived cardiac myogenesis and prevents myocardial infarction-induced injury

TNNI3K is a new cardiac-specific MAP kinase whose gene is localized to 1p31.1 and that belongs to a tyrosine kinase-like branch in the kinase tree of the human genome. In the present study we investigated the role of TNNI3K in the cardiac myogenesis process and in the repair of ischemic injury. Pluripotent P19CL6 cells with or without transfection by pcDNA6-TNNI3K plasmid were used to induce differentiation into beating cardiomyocytes. TNNI3K promoted the differentiation process, judging from the increasing beating mass and increased number of α-actinin-positive cells. TNNI3K improved cardiac function by enhancing beating frequency and increasing the contractile force and epinephrine response of spontaneous action potentials without an increase of the single-cell size. TNNI3K suppressed phosphorylation of cardiac troponin I, annexin-V+ cells, Bax protein, and p38/JNK-mediated apoptosis. Intramyocardial administration of TNNI3K-overexpressing P19CL6 cells in mice with myocardial infarction improved cardiac performance and attenuated ventricular remodeling compared with injection of wild-type P19CL6 cells. In conclusion, our study clearly indicates that TNNI3K promotes cardiomyogenesis, enhances cardiac performance, and protects the myocardium from ischemic injury by suppressing p38/JNK-mediated apoptosis. Therefore, modulation of TNNI3K activity would be a useful therapeutic approach for ischemic cardiac disease.

Abstract 578: Hex is Essential for Cardiac Myogenesis in Differentiating Embryonic Stem Cells

Background: We previously demonstrated that Sox17, an Sry-box-containing transcription factor that interacts with the Wnt/ β-catenin pathway, is essential for cardiac myogenesis in differentiating embryonic stem (ES) cells. Sox17 shRNA blocks cardiac myogenesis and selectively impairs the induction of Hex, a member of the homeobox family of transcription factors, many of which are involved in developmental processes. However, whether Hex is a direct target of Sox17 or not and the function of Hex in mouse ES cell differentiation to cardiomyocytes have not been defined. Hypothesis: Hex is a direct target of Sox17, and Hex is essential for cardiac myogenesis in ES cells. Methods: Cells were subjected to lentiviral transduction for a chimeric protein, contains Sox17 fused to protein A, using a protocol that reconstructs the native Sox17 expression pattern. Protein A-TEV-tagged Chromatin Immuno-precipitation (PAT-ChIP) technology was used to purify the DNA fragments bound to Sox17. Chromatin-immunoprecipitated DNA was subjected to PCR for promoter regions of Hex that contain putative Sox-binding motifs. Lentiviral vectors encoding shRNAs that knock down Hex were transduced into AB2.2 cells. Transduced cells, distinguished by expression of EGFP, were flow-sorted and subjected to embryonic body (EB) culture. Total RNA was extracted from EBs at 10 time points. Real-time QRT-PCR was carried out for representative genes related to mesoderm formation, mesoderm patterning, and cardiac myogenesis. Spontaneously beating EBs were scored. Results: Several Hex promoter regions containing predicted Sox17 binding sites were confirmed as potential direct targets of Sox17. The prevalence of beating EBs and the expression of cardiogenic transcription factors (Nkx2.5, Tbx5, Mef2c, Gata4 and myocardin) and cardiac structural genes (Ryr2 and α-MHC) both were suppressed by Hex shRNA. Hex shRNA did not impair the progressive down-regulation of Sox2 and Oct4 (master regulators of pluripotency) or the induction of Brachyury/T and Mesp1/2 (markers of primitive and precardiac mesoderm, respectively). Conclusion: Hex is potentially a direct target of Sox17, and is essential for cardiac myogenesis in differentiating ES cells at the stage of cardiac specification.