Control of myocardial energetics by Ca2؉ signal propagation to the mitochondrial matrix includes local Ca 2؉ delivery from sarcoplasmic reticulum (SR) ryanodine receptors (RyR2) to the inner mitochondrial membrane (IMM) Ca 2؉ uniporter (mtCU). mtCU activity in cardiac mitochondria is relatively low, whereas the IMM surface is large, due to extensive cristae folding. Hence, stochastically distributed mtCU may not suffice to support local Ca 2؉ transfer. We hypothesized that mtCU concentrated at mitochondria-SR associations would promote the effective Ca 2؉ transfer. mtCU distribution was determined by tracking MCU and EMRE, the proteins essential for channel formation. Both proteins were enriched in the IMM-outer mitochondrial membrane (OMM) contact point submitochondrial fraction and, as super-resolution microscopy revealed, located more to the mitochondrial periphery (inner boundary membrane) than inside the cristae, indicating high accessibility to cytosol-derived Ca 2؉ inputs. Furthermore, MCU immunofluorescence distribution was biased toward the mitochondria-SR interface (RyR2), and this bias was promoted by Ca 2؉ signaling activity in intact cardiomyocytes. The SR fraction of heart homogenate contains mitochondria with extensive SR associations, and these mitochondria are highly enriched in EMRE. Size exclusion chromatography suggested for EMRE-and MCU-containing complexes a wide size range and also revealed MCU-containing complexes devoid of EMRE (thus disabled) in the mitochondrial but not the SR fraction. Functional measurements suggested more effective mtCU-mediated Ca 2؉ uptake activity by the mitochondria of the SR than of the mitochondrial fraction. Thus, mtCU "hot spots" can be formed at the cardiac muscle mitochondria-SR associations via localization and assembly bias, serving local Ca 2؉ signaling and the excitation-energetics coupling.

Rationale: Adoptive transfer of multiple stem cell types has only had modest effects on the structure and function of failing human hearts. Despite increasing the use of stem cell therapies, consensus on the optimal stem cell type is not adequately defined. The modest cardiac repair and functional improvement in patients with cardiac disease warrants identification of a novel stem cell population that possesses properties that induce a more substantial improvement in patients with heart failure. Objective: To characterize and compare surface marker expression, proliferation, survival, migration, and differentiation capacity of cortical bone stem cells (CBSCs) relative to mesenchymal stem cells (MSCs) and cardiac-derived stem cells (CDCs), which have already been tested in early stage clinical trials. Methods and Results: CBSCs, MSCs, and CDCs were isolated from Gottingen miniswine or transgenic C57/BL6 mice expressing enhanced green fluorescent protein and were expanded in vitro. CBSCs possess a unique surface marker profile, including high expression of CD61 and integrin β4 versus CDCs and MSCs. In addition, CBSCs were morphologically distinct and showed enhanced proliferation capacity versus CDCs and MSCs. CBSCs had significantly better survival after exposure to an apoptotic stimuli when compared with MSCs. ATP and histamine induced a transient increase of intracellular Ca 2+ concentration in CBSCs versus CDCs and MSCs, which either respond to ATP or histamine only further documenting the differences between the 3 cell types. Conclusions: CBSCs are unique from CDCs and MSCs and possess enhanced proliferative, survival, and lineage commitment capacity that could account for the enhanced protective effects after cardiac injury.

Functional mitral regurgitation in the setting of an enlarged heart is challenging to repair surgically with an annular approach, and the need to develop subannular and ventricular approaches is recognized yet unrealized because of the lack of models for investigations. In this study, we report a novel model of functional mitral regurgitation induced by left ventricular thinning and distension in pig hearts. Seven pig hearts were explanted at a local slaughterhouse, and left ventricular distension induced by thinning the ventricular myocardium by 60–65% of its original thickness. Distension of the thinned hearts with a 120 mmHg column confirmed significant left ventricular dilatation and mitral valve tethering. These hearts were then mounted into a pulsatile flow model and animated at 120 mmHg left ventricular pressure, 5 L/min cardiac output at 70 beats/min. Echocardiography was used to assess valvular kinematics and hemodynamics. Left ventricular wall thickness reduced by 60.5% ± 10.1% at the basal plane, 64.8% ± 11.3% at the equatorial plane, and 64.0% ± 11.4% at the apical plane after thinning. Upon distension, ventricular volumes increased by 852.4% ± 639.8% after left ventricular thinning, with an 89.5% ± 33.9% increase in sphericity index. Mitral valve systolic tenting height increased from 7.92 ± 2.06 to 15.02 ± 3.89 mm, systolic tethering area increased from 130.7 ± 38.2 to 409.9 ± 124.6 mm2 and an average mitral regurgitation fraction of 24.4% ± 16.6% was measured. In a case study, use of multimodality imaging to test the efficacy of transcatheter mitral devices was confirmed. Ventricular wall thinning leading to passive left ventricular distension and dilatation is a reproducible ex vivo model of mitral valve tethering and functional mitral regurgitation, which in combination with multimodality imaging provides a good simulation model.

Rationale: Adoptive transfer of bone marrow and cardiac derived stem cells (CDCs) into failing human hearts has been shown to be safe, yet these cells have induced modest improvements in myocardial function. Recently we have shown in a mouse model that cortical bone derived stem cells (CBSCs) induced a greater enhancement of cardiac function after myocardial infarction than CDCs, possibly through enhanced paracrine signaling and transdifferentiation of the transplanted cells. However, the relative reparative potential of CBSCs relative to other known stem cell types including bone marrow derived mesenchymal stem cells (MSCs) and cardiac derived stem cells (CDCs) is not known. Objective: To characterize surface marker expression, proliferation, survival and differentiation capacity of swine CBSCs relative to MSCs and CDCs. Methods and Results: CBSCs, MSCs and CDCs were isolated from cortical bone, bone marrow and heart (left ventricle) of Gottingen miniswine. CBSCs were morphologically distinct from MSCs and CDCs, with differences in length to width ratio and overall cell surface area. Cell surface marker profiling using RT-PCR analysis revealed that CBSCs express some of the classical MSC markers such as CD106, CD271, CD105, CD90, CD44 and CD29 and are negative for CD45 and CD11-b. CBSCs had an enhanced proliferation capacity versus CPCs and MSCs, measured by CyQuant assay. Concurrently CBSCs had significantly a decreased population doubling time (3.57 and 1.26 fold decrease) as compared to MSC and CDCs respectively. CBSCs were also more hydrogen peroxide stress tolerant than CSCs and MSCs as measured by Annexin-V and propidium iodide (PI) labeling using flow cytometery. A significantly greater % of CBSCs expressed markers of cardiac lineage commitment when exposed to dexamethasone than did CSCs or MSCs. Markers of cardiac lineages including GATA-4, α SMA, Troponin T, Nkx2.5, sm22 were measured with RT-PCR and immunocytochemistry. Conclusion: CBSCs have enhanced proliferative capacity, survival in oxidative stress conditions and cardiac lineage commitment versus CSCs and MSCs. These features make them a promising cell source for cardiac regeneration after myocardial infarction.