Cardiac fibroblasts convert to myofibroblasts with injury to mediate healing after acute myocardial infarction (MI) and to mediate long-standing fibrosis with chronic disease. Myofibroblasts remain a poorly defined cell type in terms of their origins and functional effects in vivo. Here we generate Postn (periostin) gene-targeted mice containing a tamoxifen-inducible Cre for cellular lineage-tracing analysis. This Postn allele identifies essentially all myofibroblasts within the heart and multiple other tissues. Lineage tracing with four additional Cre-expressing mouse lines shows that periostin-expressing myofibroblasts in the heart derive from tissue-resident fibroblasts of the Tcf21 lineage, but not endothelial, immune/myeloid or smooth muscle cells. Deletion of periostin+ myofibroblasts reduces collagen production and scar formation after MI. Periostin-traced myofibroblasts also revert back to a less-activated state upon injury resolution. Our results define the myofibroblast as a periostin-expressing cell type necessary for adaptive healing and fibrosis in the heart, which arises from Tcf21+ tissue-resident fibroblasts.

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine if endogenous c-kit+ cells contribute differentiated cardiomyocytes to the heart during development, with aging or after injury in adulthood. A cDNA encoding either Cre recombinase or a tamoxifen inducible MerCreMer chimeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit+ cells did produce new cardiomyocytes within the heart, although at a percentage of ≈0.03% or less, and if a preponderance towards cellular fusion is considered, the percentage falls below ≈0.008%. In contrast, c-kit+ cells amply generated cardiac endothelial cells. Thus, endogenous c-kit+ cells can generate cardiomyocytes within the heart, although likely at a functionally insignificant level.

reverse transcribed using random oligo-dT primers and a Verso cDNA Synthesis Kit (Thermo Fisher, AB1453) according to the manufacturer's instructions. Real-time PCR was performed using SsoAdvanced SYBR Green (Bio-Rad, 6090), and Rpl7 expression was used for normalization. The following primer sets were used to identify transcripts: collagen 1a1, 5′-AATGGCACGGCTGTGTGCGA and 5′-AACGGGTCCCCTTG-GGCCTT; collagen 3a1, 5′-TCCCCTGGAATCTGTGAATC and 5′-TGAGTCGAATTGGGGAGAAT; periostin, 5′-ACGGAGCTCAGG-GCTGAAGATG and 5′-GTTTGGGCCCTGATCCCGAC.Cell death analysis. At 90% confluence, primary skin fibroblasts were treated with 200 nM staurosporine for 36 hours or vehicle (DMSO). Cell death was determined by the Muse Count & Viability Assay (Millipore, MCH100102) as previously described (62). Briefly, the medium was collected with the trypsin-liberated cells, which were centrifuged and washed twice with PBS and then incubated with the Muse Count & Viability reagent. The cells were then quantified on a Muse cell analyzer (Millipore) at 5,000 counts per sample.Statistics. One-way ANOVA with post hoc Tukey's honest significant difference (HSD) or Student's t test was used to determine statistical significance, depending on the type of data analyzed and number of comparisons. P values of less than 0.05 were considered statistically significant. Averaged data are presented with SEM to indicate variability.Study approval. Mice were observed daily and cages changed weekly by certified veterinary technicians at Cincinnati Children's Hospital Medical Center. Mice were also closely assessed for their well-being, monitored by adequate physical activity and food intake on a daily basis. Housing conditions and husbandry conformed to AAALAC standards as well as the standard guidelines from the NIH Office of Laboratory Animal Welfare (http://grants.nih.gov/grants/olaw/animal_use. htm). The institution also retains ongoing certification by AAALAC.

Cyclophilin D (which is encoded by the Ppif gene) is a mitochondrial matrix peptidyl-prolyl isomerase known to modulate opening of the mitochondrial permeability transition pore (MPTP). Apart from regulating necrotic cell death, the physiologic function of the MPTP is largely unknown. Here we have shown that Ppif -/-mice exhibit substantially greater cardiac hypertrophy, fibrosis, and reduction in myocardial function in response to pressure overload stimulation than control mice. In addition, Ppif -/-mice showed greater hypertrophy and lung edema as well as reduced survival in response to sustained exercise stimulation. Cardiomyocyte-specific transgene expression of cyclophilin D in Ppif -/-mice rescued the enhanced hypertrophy, reduction in cardiac function, and rapid onset of heart failure following pressure overload stimulation. Mechanistically, the maladaptive phenotype in the hearts of Ppif -/-mice was associated with an alteration in MPTP-mediated Ca 2+ efflux resulting in elevated levels of mitochondrial matrix Ca 2+ and enhanced activation of Ca 2+ -dependent dehydrogenases. Elevated matrix Ca 2+ led to increased glucose oxidation relative to fatty acids, thereby limiting the metabolic flexibility of the heart that is critically involved in compensation during stress. These findings suggest that the MPTP maintains homeostatic mitochondrial Ca 2+ levels to match metabolism with alterations in myocardial workload, thereby suggesting a physiologic function for the MPTP.

A critical event in ischemia-based cell death is the opening of the mitochondrial permeability transition pore (MPTP). However, the molecular identity of the components of the MPTP remains unknown. Here, we determined that the Bcl-2 family members Bax and Bak, which are central regulators of apoptotic cell death, are also required for mitochondrial pore-dependent necrotic cell death by facilitating outer membrane permeability of the MPTP. Loss of Bax/Bak reduced outer mitochondrial membrane permeability and conductance without altering inner membrane MPTP function, resulting in resistance to mitochondrial calcium overload and necrotic cell death. Reconstitution with mutants of Bax that cannot oligomerize and form apoptotic pores, but still enhance outer membrane permeability, permitted MPTP-dependent mitochondrial swelling and restored necrotic cell death. Our data predict that the MPTP is an inner membrane regulated process, although in the absence of Bax/Bak the outer membrane resists swelling and prevents organelle rupture to prevent cell death.DOI: http://dx.doi.org/10.7554/eLife.00772.001

The mitochondrial phosphate carrier (PiC) is critical for ATP synthesis by serving as the primary means for mitochondrial phosphate import across the inner membrane. In addition to its role in energy production, PiC is hypothesized to have a role in cell death as either a component or a regulator of the mitochondrial permeability transition pore (MPTP) complex. Here, we have generated a mouse model with inducible and cardiac-specific deletion of the Slc25a3 gene (PiC protein). Loss of PiC protein did not prevent MPTP opening, suggesting it is not a direct pore-forming component of this complex. However, Slc25a3 deletion in the heart blunted MPTP opening in response to Ca 2 þ challenge and led to a greater Ca 2 þ uptake capacity. This desensitization of MPTP opening due to loss or reduction in PiC protein attenuated cardiac ischemic-reperfusion injury, as well as partially protected cells in culture from Ca 2 þ overload induced death. Intriguingly, deletion of the Slc25a3 gene from the heart long-term resulted in profound hypertrophy with ventricular dilation and depressed cardiac function, all features that reflect the cardiomyopathy observed in humans with mutations in SLC25A3. Together, these results demonstrate that although the PiC is not a direct component of the MPTP, it can regulate its activity, suggesting a novel therapeutic target for reducing necrotic cell death. In addition, mice lacking Slc25a3 in the heart serve as a novel model of metabolic, mitochondrial-driven cardiomyopathy. The mitochondrial oxidative phosphorylation (OXPHOS) system is the primary source of cellular energy production. Defects in OXPHOS occur with a frequency of 1 in 5000 live births 1 and underlie a wide range of mitochondrial disorders that often affect multiple organ systems and tissues with high oxidative energy demands, such as brain, skeletal muscle, and heart.2 Cardiac phenotypes associated with mitochondrial disease are diverse, and can range from cardiomyopathies to cardiac conduction defects. [3][4][5]

Purpose: Alternative CCND1 splicing results in cyclin D1b, which has specialized, protumorigenic functions in prostate not shared by the cyclin D1a (full length) isoform. Here, the frequency, tumor relevance, and mechanisms controlling cyclin D1b were challenged. Experimental Design: First, relative expression of both cyclin D1 isoforms was determined in prostate adenocarcinomas. Second, relevance of the androgen axis was determined. Third, minigenes were created to interrogate the role of the G/A870 polymorphism (within the splice site), and findings were validated in primary tissue. Fourth, the effect of G/A870 on cancer risk was assessed in two large case-control studies.Results: Cyclin D1b is induced in tumors, and a significant subset expressed this isoform in the absence of detectable cyclin D1a. Accordingly, the isoforms showed noncorrelated expression patterns, and hormone status did not alter splicing. Whereas G/ A870 was not independently predictive of cancer risk, A870 predisposed for transcript-b production in cells and in normal prostate. The influence of A870 on overall transcript-b levels was relieved in tumors, indicating that aberrations in tumorigenesis likely alter the influence of the polymorphism. Conclusions: These studies reveal that cyclin D1b is specifically elevated in prostate tumorigenesis. Cyclin D1b expression patterns are distinct from that observed with cyclin D1a. The A870 allele predisposes for transcript-b production in a context-specific manner. Although A870 does not independently predict cancer risk, tumor cells can bypass the influence of the polymorphism. These findings have major implications for the analyses of D-cyclin function in the prostate and provide the foundation for future studies directed at identifying potential modifiers of the G/A870 polymorphism. (Clin Cancer Res 2009;15(17):5338-49)

The mitochondrial permeability transition pore (MPTP) has resisted molecular identification. The original model of the MPTP that proposed the adenine nucleotide translocator (ANT) as the inner membrane pore-forming component was challenged when mitochondria from Ant1/2 double null mouse liver still had MPTP activity. Because mice express three Ant genes, we reinvestigated whether the ANTs comprise the MPTP. Liver mitochondria from Ant1, Ant2, and Ant4 deficient mice were highly refractory to Ca2+-induced MPTP formation, and when also given cyclosporine A (CsA), the MPTP was completely inhibited. Moreover, liver mitochondria from mice with quadruple deletion of Ant1, Ant2, Ant4, and Ppif (cyclophilin D, target of CsA) lacked Ca2+-induced MPTP formation. Inner-membrane patch clamping in mitochondria from Ant1, Ant2, and Ant4 triple null mouse embryonic fibroblasts showed a loss of MPTP activity. Our findings suggest a model for the MPTP consisting of two distinct molecular components: The ANTs and an unknown species requiring CypD.