Vital staining of cardiac myocytes during embryonic stem cell cardiogenesis in vitro.

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types, including spontaneously contracting cardiac myocytes. The primary aim of this work was to use vital stain techniques for real-time detection of developing cardiac myocytes in ES cell differentiation cultures. The -440 to +6 human cardiac alpha-actin promoter was used to direct expression of the Escherichia coli reporter gene lacZ (pHCActlacZ) into ES cell-derived cardiac myocytes during cardiogenesis in vitro. Undifferentiated ES cells were electroporated with HCActlacZ together with a plasmid containing the neomycin gene under the direction of the phosphoglycerate kinase promoter, and stable transformants were selected in G418. Individual clones were screened for activation of lacZ gene expression in cardiac myocytes developing in vitro. Results showed that expression of the HCActlacZ reporter construct was activated very early during the ES cell differentiation program, at a time point before the appearance of spontaneous contractile activity. The earliest detection was at day 6 of differentiation, when approximately 25% of the differentiation cultures expressed the reporter construct, with expression increasing to approximately 70% at day 9 and continuing throughout the duration of spontaneous contractile activity exhibited by the ES cell-derived cardiac myocytes. Indirect immunofluorescence assays provide evidence that expression was restricted to the cardiac myocytes in culture. In the present study, we show vital staining of transgene expression in living cardiac myocytes using lipophilic fluorogenic beta-galactopyranoside substrates for real-time detection of the reporter gene during continuous contraction of the ES cell myocytes in vitro. The vital stain approach used in the present study will permit the identification of differentiating ES cells that are committed to the cardiac lineage for analysis of gene expression at early time points of ES cell cardiogenesis and, in addition, will aid in selecting genetically modified ES cell cardiac myocytes for use in functional studies.

Myosin heavy chain expression in contracting myocytes isolated during embryonic stem cell cardiogenesis.

Mouse embryonic stem (ES) cells are totipotent cells derived from the inner cell mass of the preimplantation blastocyst and are capable of differentiating in vitro into cardiac myocytes. Attached cultures of differentiating ES cells were established to document the timing of contractile development by microscopic observation and to permit the microdissection of cardiac myocytes from culture. The onset of spontaneous contraction varied markedly in differentiation culture, with contraction being maintained on average for 9 days (range, 1 to 75 days). Indirect immunofluorescence in microscopy showed that myosin expression was localized to the contracting cardiac myocytes in culture. Myosin heavy chain (MHC) isoform expression in microdissected ES cell-derived cardiac myocytes was determined by means of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The distribution of MHC isoform expression in isolated ES cell cardiac myocytes was as follows: 27% expressed the beta-MHC isoform, 33% expressed both the alpha- and beta-MHC isoforms, and 40% expressed the alpha-MHC isoform. MHC phenotype was correlated to the duration of continuous contractile activity of the myocytes. Myocytes that had just initiated spontaneous contractile activity predominantly expressed the beta-MHC (average days of contraction before isolation, 2.5 +/- 0.7). The alpha-MHC isoform was detected after mouse prolonged contractile activity in vitro (1 to 5 weeks). A strong correlation was obtained between MHC phenotype and days of contraction of the cardiac myocyte preparations isolated from ES cell cultures (r = .93). The apparent transition in MHC isoform expression during ES cell differentiation parallels the beta- to alpha-MHC isoform transition characteristic of murine cardiac development in vivo. These findings are evidence that ES cell cardiac myocyte differentiation follows the normal developmental program of murine cardiogenesis.

Transition in cardiac contractile sensitivity to calcium during the in vitro differentiation of mouse embryonic stem cells.

Mouse embryonic stem (ES) cells differentiate in vitro into a variety of cell types including spontaneously contracting cardiac myocytes. We have utilized the ES cell differentiation culture system to study the development of the cardiac contractile apparatus in vitro. Difficulties associated with the cellular and developmental heterogeneity of this system have been overcome by establishing attached cultures of differentiating ES cells, and by the micro-dissection of the contracting cardiac myocytes from culture. The time of onset and duration of continuous contractile activity of the individual contracting myocytes was determined by daily visual inspection of the cultures. A functional assay was used to directly measure force production in ES cell-derived cardiac myocyte preparations. The forces produced during spontaneous contractions in the membrane intact preparation, and during activation by Ca2+ subsequent to chemical permeabilization of the surface membranes were determined in the same preparation. Results showed a transition in contractile sensitivity to Ca2+ in ES cell-derived cardiac myocytes during development in vitro. Cardiac preparations isolated from culture following the initiation of spontaneous contractile activity showed marked sensitivity of the contractile apparatus to activation by Ca2+. However, the Ca2+ sensitivity of tension development was significantly decreased in preparations isolated from culture following prolonged continuous contractile activity in vitro. The alteration in Ca2+ sensitivity obtained in vitro paralleled that observed during murine cardiac myocyte development in vivo. This provides functional evidence that ES cell-derived cardiac myocytes recapitulate cardiogenesis in vitro. Alterations in Ca2+ sensitivity could be important in optimizing the cardiac contractile response to variations in the myoplasmic Ca2+ transient during embryogenesis. The potential to stably transfect ES cells with cardiac regulatory genes, together with the availability of a functional assay using control and genetically modified ES cell-derived cardiac myocytes, will permit determination of the functional significance of altered cardiac gene expression during cardiogenesis in vitro.

Skeletal troponin C reduces contractile sensitivity to acidosis in cardiac myocytes from transgenic mice.

Depressed contractile function plays a primary role in the pathophysiology of acute myocardial ischemia. Intracellular acidification is an important factor underlying the inhibition of force production in the ischemic myocardium. The effect of acidosis to depress contractility is markedly greater in cardiac as compared to skeletal muscle; however, the molecular basis of this difference in sensitivity to acidosis is not clearly understood. In this report, we describe transgenic mice that express the fast skeletal isoform of troponin C (sTnC) in cardiac muscle. In permeabilized single cardiac myocytes the shift in the midpoint of the tension-pCa relationship (i.e., pCa50, where pCa is -log[Ca2+]) due to lowering pH from 7.00 to 6.20 was 1.27 +/- 0.03 (n = 7) pCa units in control cardiac TnC (cTnC) expressing myocytes and 0.96 +/- 0.04 (n = 11) pCa unit in transgenic cardiac myocytes (P < 0.001). The effect of pH to alter maximum Ca(2+)-activated tension was unchanged by TnC isoforms in these cardiac myocytes. In a reciprocal experiment, contractile sensitivity to acidosis was increased in fast skeletal muscle fibers following extraction of endogenous sTnC and reconstitution with purified cTnC in vitro. Our findings demonstrate that TnC plays an important role in determining the profound sensitivity of cardiac muscle to acidosis and identify cTnC as a target for therapeutic interventions designed to modify ischemia-induced myocardial contractile dysfunction.

Congenital multicystic dysplastic kidney in the adult.

Unilateral multicystic kidneys are not infrequent during infancy. However, few cases in adults are reported. A case is presented in which a unilateral multicystic kidney with atresia of the ureter was found as an incidental finding in an asymptomatic elderly patient. The literature is reviewed, and a nonoperative diagnostic approach is favored.