Abstract-Elucidating the relative roles of cardiac troponin I (cTnI) and phospholamban (PLN) in ␤-adrenergic-mediated hastening of cardiac relaxation has been challenging and controversial. To test the hypothesis that ␤-adrenergic phosphorylation of cTnI has a prominent role in accelerating cardiac myocyte relaxation performance we used transgenic (Tg) mice bearing near complete replacement of native cTnI with a ␤-adrenergic phospho-mimetic of cTnI whereby tandem serine codons 23/24 were converted to aspartic acids (cTnI S23/24D). Adult cardiac myocytes were isolated and contractility determined at physiological temperature under unloaded and loaded conditions using micro-carbon fibers. At baseline, cTnI S23/24D myocytes had significantly faster relaxation times relative to controls, and isoproterenol stimulation (Iso) had only a small effect to further speed relaxation in cTnI S23/24D myocytes (delta Iso: 7.2 ms) relative to the maximum Iso effect (31.2 ms) in control. The Ca 2ϩ transient decay rate was similarly accelerated by Iso in Tg and nontransgenic (Ntg) myocytes. Gene transfer of cTnI S23/24D to myocytes in primary culture showed comparable findings. Gene transfer of cTnI with both serines 23/24 converted to alanines (cTnI S23/24A), or gene transfer of slow skeletal TnI, both of which lack PKA phosphorylation sites, significantly blunted Iso-mediated enhanced relaxation compared with controls. Gene transfer of wild-type cTnI had no effect on relaxation. These findings support a key role of cTnI in myocyte relaxation and highlight a direct contribution of the myofilaments in modulating the dynamics of myocardial performance. Key Words: contraction Ⅲ calcium Ⅲ heart ␤ -adrenergic stimulation of the heart causes a dramatic increase in heart performance. 1 This represents a vital mechanism of the mammalian "fight or flight" response wherein modulation of heart performance responds on a beat-to-beat basis to variations in physiological demands. During ␤-adrenergic stimulation heart rate (chronotropy), pressure development (inotropy) and muscle relaxation rate (lusitropy) are all increased. To accomplish a marked increase in cardiac output in the face of reduced time for ventricular filling, the speed of myocardial relaxation must increase.The molecular determinants of enhanced cardiac muscle relaxation during ␤-adrenergic stimulation are important to define and are still being debated. Downstream of ␤-adrenergic receptor stimulation and subsequent activation of protein kinase A (PKA) are several cardiac phosphoproteins that have been implicated in hastening relaxation. 2,3 Phospholamban (PLN) is known to regulate the activity of the SERCA2a calcium ATPase pump on the sarcoplasmic reticulum (SR), such that pump activity is increased on phosphorylation of PLN leading to faster sequestration of calcium into the SR. 2,4 It is also well known that PKA phosphorylates cTnI in the sarcomere. 2,5 Troponin I is a sarcomeric protein that is essential for regulating myofilament function. 6,7 PKAmediated cTnI phosphoryla...

Recent reports have described a stem cell population termed stromal vascular cells (SVCs) derived from the stromal vascular fraction of adipose tissue, which are capable of intrinsic differentiation into spontaneously beating cardiomyocytes in vitro. The objective of this study was to further define the cardiac lineage differentiation potential of SVCs in vitro and to derive methods for enriching SVC-derived beating cardiac myocytes. SVCs were isolated from the stromal vascular fraction of murine adipose tissue. Cells were cultured in methylcellulose-based murine stem cell media. Analysis of SVC-derived beating myocytes included Western blot, and calcium imaging. Enrichment of acutely isolated SVCs was carried out using antibody tagged magnetic nanoparticles, and pharmacologic manipulation of Wnt and cytokine signaling. Under initial media conditions, spontaneously beating SVCs expressed both cardiac developmental and adult protein isoforms. Functionally, this specialized population can spontaneously contract and pace under field stimulation, and shows the presence of coordinated calcium transients. Importantly, this study provides evidence for two independent mechanisms of enriching the cardiac differentiation of SVCs. First, this study shows that differentiation of SVCs into cardiac myocytes is augmented by non-canonical Wnt agonists, canonical Wnt antagonists, and cytokines. Second, SVCs capable of cardiac lineage differentiation can be enriched by selection for stem cell-specific membrane markers Sca1 and c-kit. Adipose-derived SVCs are a unique population of stem cells that show evidence of cardiac lineage development making them a potential source for stem cell-based cardiac regeneration studies.

The MLC isoform can be a determinant of force-generating ability of cardiac myosin by modulating crossbridge kinetics without affecting the catalytic activity.