The contribution of the sarcoplasmic reticulum (SR) and Na+−Ca2+ exchanger to intracellular Ca2+ regulation in mouse cardiac myocytes was investigated by measuring contraction after variable rest intervals, rapid cooling contractures (RCCs) and fast application of caffeine. The results obtained showed differences from other species in the roles played by the SR and the Na+−Ca2+ exchanger. They suggest that in mouse ventricular myocytes there is significant Ca2+ entry via the exchanger during rest and during the latter part of the Ca2+ transient. In cardiac myocytes isolated from transgenic mice overexpressing the cardiac Na+−Ca2+ exchanger the time to peak and relaxation of twitches and RCCs were faster than in control littermates. The decline of Ca2+, assessed by indo‐1 fluorescence, was faster in transgenic myocytes even in the absence of Na+ and Ca2+ in the superfusing solution. This suggests that SR Ca2+ uptake is faster in these myocytes. However, no difference in the expression of SERCA2a, phospholamban or calsequestrin measured with Western blotting could be found in the two groups. We measured SR Ca2+ content by integrating the caffeine‐induced transient inward current. The amount of Ca2+ stored in the SR of transgenic mouse myocytes was 69 % greater than in non‐transgenic littermates. The increased SR Ca2+ content may be responsible for the faster rate of SR Ca2+ release and uptake in cells from transgenic mice. We performed experiments to assess whether the reversal potential of the Na+−Ca2+ exchanger (ENa‐Ca) was different in transgenic cardiac cells. We measured a Ni2+‐sensitive current elicited by voltage ramps in non‐dialysed myocytes. The current‐voltage relationship showed no difference in the reversal potential of the Na+−Ca2+ exchanger in transgenic and control myocytes. This suggests that the effects on the SR Ca2+ content in transgenic cardiac myocytes can be ascribed to the overexpression of the exchanger and are not secondary to changes in intracellular diastolic Ca2+ and Na+.

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Abstract-We have recently demonstrated that stem cell antigen 1-positive (Sca-1 ϩ ) progenitors exist in the vascular adventitia of apolipoprotein E-deficient (apoE Ϫ/Ϫ ) mice and contribute to smooth muscle cell (SMC) accumulation in vein graft atherosclerosis. Using a combined proteomic and metabolomic approach, we now characterize these local progenitors, which participate in the formation of native atherosclerotic lesions in chow-fed apoE Ϫ/Ϫ mice. Unlike Sca-1 ϩ progenitors from embryonic stem cells, the resident Sca-1 ϩ stem cell population from the vasculature acquired a mature aortic SMC phenotype after platelet-derived growth factor-BB stimulation. It shared proteomic and metabolomic characteristics of apoE Ϫ/Ϫ SMCs, which were clearly distinct from wild-type SMCs under normoxic and hypoxic conditions. Among the differentially expressed proteins were key enzymes in glucose metabolism, resulting in faster glucose consumption and a compensatory reduction in baseline interleukin-6 secretion. The latter was associated with a marked upregulation of insulin-like growth factor binding proteins (IGFBPs) 3 and 6. Notably, reconstitution of interleukin-6 to levels measured in the conditioned medium of wild-type SMCs attenuated the elevated IGFBP expression in apoE Ϫ/Ϫ SMCs and their vascular progenitors. This coregulation of apoE, interleukin-6, and IGFBPs was replicated in wild-type SMCs from hypercholesterolemic mice and confirmed by silencing apoE expression in SMCs from normocholesterolemic mice. In summary, we provide evidence that Sca-1 ϩ progenitors contribute to native atherosclerosis in apoE Ϫ/Ϫ mice, that apoE deficiency and hypercholesterolemia alter progenitor cell behavior, and that inflammatory cytokines such as interleukin-6 act as metabolic regulators in SMCs of hyperlipidemic mice. Key Words: atherosclerosis Ⅲ insulin-like growth factor-1 Ⅲ progenitor cells Ⅲ proteomics Ⅲ vascular smooth muscle W ith the introduction of apolipoprotein (apo)E-deficient strains, the mouse became the preferred animal model in cardiovascular research. 1 ApoE is a glycoprotein that is synthesized in the liver and the brain, but it is also produced locally in the vessel wall, mainly in infiltrating monocytes and macrophages, 2 and gets recruited from the circulation after vascular injury. 3 Besides apoE-mediated cholesterol transport, lipid-independent effects of apoE also have relevance in vitro and in vivo. For instance, apoE is synthesized in quiescent but not actively proliferating smooth muscle cells (SMCs) in culture 4 and suppresses growth factor and oxidized LDL-induced SMC migration and proliferation. 5 A possible role of apoE in modulation of SMC growth in vivo is supported by observations that the numbers of intimal SMCs are increased in fibroproliferative atherosclerotic plaques of chow-fed apoE Ϫ/Ϫ mice but reduced after vascular injury in transgenic mice overexpressing apoE. 1,6 Similarly, we found that vein grafts of apoE Ϫ/Ϫ mice showed increased neointima formation even if grafted to normolipidemic ...