Absence of cardiomyocyte differentiation following transplantation of adult cardiac-resident Sca-1+ cells into infarcted mouse hearts

Although several lines of evidence suggest that the glycosyl phosphatidylinositol-anchored cell surface protein Sca-1 marks cardiac-resident stem cells, a critical analysis of the literature raises some concerns regarding their cardiomyogenic potential.1 Here, isolated adult cardiac-resident Sca-1+ cells were engrafted into infarcted hearts and monitored for cardiomyogenic differentiation. Donor cells were prepared from ACT-EGFP; MHC-nLAC double-transgenic mice ([C57/Bl6J x DBA/2J]F1 genetic background; all procedures followed were in accordance with Institutional Guidelines). The ACT-EGFP transgene targets ubiquitous expression of an enhanced green fluorescent protein reporter, and the MHC-nLAC transgene targets cardiomyocyte-restricted expression of a nuclear-localized β-galactosidase reporter. Donor cell survival was monitored via EGFP fluorescence, while cardiomyogenic differentiation was monitored by reacting with the chromogenic β-galactosidase substrate 5-bromo-4-chloro-3-indolyl-β-D-galactoside (X-GAL), which gives rise to a blue product.2 Double-transgenic hearts were dispersed with Blendzyme and the resulting cells reacted with an APC-conjugated anti-Sca-1 antibody and a PE-conjugated cocktail of antibodies recognizing hematopoietic lineage markers.3 Sca-1+, EGFP+, lineage- cells were then isolated via fluorescence-activated cell sorting (FACS; characterization of the donor cells is provided in Figure 1A), and 100,000 cells were injected into the infarct border zone of non-transgenic [C57/Bl6J x DBA/2J]F1 mice immediately following permanent coronary artery occlusion

Synergy between CD26/DPP-IV Inhibition and G-CSF Improves Cardiac Function after Acute Myocardial Infarction

SummaryIschemic cardiomyopathy is one of the main causes of death, which may be prevented by stem cell-based therapies. SDF-1α is the major chemokine attracting stem cells to the heart. Since SDF-1α is cleaved and inactivated by CD26/dipeptidylpeptidase IV (DPP-IV), we established a therapeutic concept—applicable to ischemic disorders in general—by combining genetic and pharmacologic inhibition of DPP-IV with G-CSF-mediated stem cell mobilization after myocardial infarction in mice. This approach leads to (1) decreased myocardial DPP-IV activity, (2) increased myocardial homing of circulating CXCR-4+ stem cells, (3) reduced cardiac remodeling, and (4) improved heart function and survival. Indeed, CD26 depletion promoted posttranslational stabilization of active SDF-1α in heart lysates and preserved the cardiac SDF-1-CXCR4 homing axis. Therefore, we propose pharmacological DPP-IV inhibition and G-CSF-based stem cell mobilization as a therapeutic concept for future stem cell trials after myocardial infarction

High Body Mass Index is Associated with Elevated Blood Levels of Progerin mRNA

Obesity is a well-described risk factor resulting in premature aging of the cardiovascular system ultimately limiting longevity. Premature cardiac death and aging is the hallmark of Hutchinson–Gilford syndrome (HGPS), a disease caused by defined mutations in the lamin A gene leading to a shortened prelamin A protein known as progerin. Since small amounts of progerin are expressed in healthy individuals we aimed to investigate the association of Body-Mass-Index (BMI) with respect to expression of progerin mRNA in blood samples of patient with known cardiovascular disease. In this cross-sectional retrospective analysis, 111 patients were consecutively included of which 46 were normal (BMI < 25 kg/m2) and 65 overweight (BMI ≥ 25.0 kg/m2). Blood samples were analyzed for quantitative expression of progerin mRNA. Progerin as well as high-sensitive C-Reactive Protein (hs-CRP) levels were significantly upregulated in the overweight group. Linear regression analyses showed a significant positive correlation of BMI and progerin mRNA (n = 111; r = 0.265, p = 0.005), as well as for hs-CRP (n = 110; r = 0.300, p = 0.001) and for Hb1Ac (n = 110; r = 0.336, p = 0.0003). Our data suggest that BMI strongly correlates with progerin mRNA expression and inflammation. Progerin might contribute to well described accelerated biologic aging in obese individuals

Upregulation of the aging related LMNA splice variant progerin in dilated cardiomyopathy.

Mutations in the LMNA gene are a common cause (6-8%) of dilated cardiomyopathy (DCM) leading to heart failure, a growing health care problem worldwide. The premature aging disease Hutchinson-Gilford syndrome (HGPS) is also caused by defined mutations in the LMNA gene resulting in activation of a cryptic splice donor site leading to a defective truncated prelamin A protein called progerin. Low levels of progerin are expressed in healthy individuals associated with ageing. Here, we aimed to address the role of progerin in dilated cardiomyopathy.mRNA expression of progerin was analyzed in heart tissue of DCM (n = 15) and non-failing hearts (n = 10) as control and in blood samples from patients with DCM (n = 56) and healthy controls (n = 10). Sequencing confirmed the expression of progerin mRNA in the human heart. Progerin mRNA levels derived from DCM hearts were significantly upregulated compared to controls (1.27 ± 0.42 vs. 0.81 ± 0.24; p = 0.005). In contrast, progerin mRNA levels in whole blood cells were not significantly different in DCM patients compared to controls. Linear regression analyses revealed that progerin mRNA in the heart is significantly negatively correlated to ejection fraction (r = -0.567, p = 0.003) and positively correlated to left ventricular enddiastolic diameter (r = 0.551, p = 0.004) but not with age of the heart per se. Progerin mRNA levels were not influenced by inflammation in DCM hearts. Immunohistochemistry and Immunofluorescence analysis confirmed increased expression of progerin protein in cell nuclei of DCM hearts associated with increased TUNEL+ apoptotic cells.Our data suggest that progerin is upregulated in human DCM hearts and strongly correlates with left ventricular remodeling. Progerin might be involved in progression of heart failure and myocardial aging

Alignment of progerin PCR product cDNA confirms alternative splicing in the heart.

<p>The sequence of purified progerin PCR cDNA (red letters) was aligned to the genomic sequence of LMNA exon 11 (green) to exon 12 (yellow) with the 3´UTR sequence (blue) showing the expected gap of 150bp between exon 11 to exon 12 confirming alternative splicing of progerin.</p

Characteristics of hearts.

<p>Characteristics of hearts.</p

PCR strategy for the detection of progerin expression in heart and blood.

<p><b>A</b> Schematic overview of PCR based strategy to analyze progerin mRNA expression in human heart and the blood samples. Shown are the consensus donor splice sequence at the end of exon 11, the sequence of the normal LMNA cryptic splice site, and two common known mutated cryptic splice sites in HGPS patients. <b>B</b> First row: PCR gel showing total lamin A (first arrow) and small amounts of progerin expression (arrow showing Δ150bp band). Second row: Specific progerin expression utilizing primers spanning exon 11 to 12. Third row: RPL32 expression was used for relative quantification of progerin expression. G: Genomic DNA (G) did not reveal any significant LMNA bands in the gel verifying specific mRNA expression.</p

Progerin and inflammation in the heart.

<p>Progerin and inflammation in the heart.</p