support-information-section).

In vitro analysis of primary isolated adult cardiomyocyte physiological processes often involves optical imaging of dye-loaded cells on a glass substrate. However, when exposed to rapid solution changes, primary cardiomyocytes often move to compromise quantitative measures. Improved immobilization of cells to glass would permit higher throughput assays. Here, we engineer the peripheral membrane of cardiomyocytes with biotin to anchor cardiomyocytes to borosilicate glass coverslips functionalized with streptavidin. We use a rat cardiac myoblast cell line to determine general relationships between processing conditions, ligand density on the cell and the glass substrate, cellular function, and cell retention under shear flow. Use of the streptavidin−biotin system allows for more than 80% retention of cardiac myoblasts under conventional rinsing procedures, while unmodified cells are largely rinsed away. The adhesion system enables the in-field retention of cardiac cells during rapid fluid changes using traditional pipetting or a modern microfluidic system at a flow rate of 160 mL/min. Under fluid flow, the surfaceengineered primary adult cardiomyocytes are retained in the field of view of the microscope, while unmodified cells are rinsed away. Importantly, the engineered cardiomyocytes are functional following adhesion to the glass substrate, where contractions are readily observed. When applying this adhesion system to cardiomyocyte functional analysis, we measure calcium release transients by caffeine induction at an 80% success rate compared to 20% without surface engineering.

This study examined the effects of curcumin and fenugreek soluble fiber supplementation on the ventilatory threshold (VT) and peak oxygen consumption ( V ˙ O2 peak). Methods: Forty-five untrained men and women were randomly assigned to one of three supplementation groups: placebo (PLA, n = 13), 500 mg·day−1 CurQfen® (CUR, n = 14), or 300 mg·day−1 fenugreek soluble fiber (FEN, n = 18). Participants completed a maximal graded exercise test on a cycle ergometer to determine the VT and V ˙ O2 peak before (PRE) and after (POST) 28 days of daily supplementation. Separate, one-way analyses of covariance (ANCOVAs) were used to examine the between-group differences for adjusted POST VT and V ˙ O2 peak values, covaried for the respective PRE-test values. Results: The adjusted POST VT V ˙ O2 values for the CUR (mean ± SD = 1.593 ± 0.157 L·min−1) and FEN (1.597 ± 0.157 L·min−1) groups were greater than (p = 0.039 and p = 0.025, respectively) the PLA (1.465 ± 0.155 L·min−1) group, but the FEN and CUR groups were not different (p = 0.943). There were no differences in the adjusted V ˙ O2 peak values (F = 0.613, p = 0.547) among groups. Conclusion: These findings indicated that fenugreek soluble fiber was responsible for the improvements in the submaximal performance index for both CUR and FEN groups.

Curcumin, a polyphenol, has been suggested to improve metabolic byproduct clearance and increase nitric oxide production in working muscle. These purported effects may delay neuromuscular fatigue. Therefore, the purpose of this study was to examine the effects of curcumin in combination with fenugreek (CUR) or fenugreek soluble fiber alone (FEN) on the neuromuscular fatigue threshold (PWCFT), time to exhaustion (Tlim) on a graded exercise test (GXT), and ̇O 2peak in untrained subjects. Forty-seven, college-aged, aerobically untrained individuals were randomly assigned to one of three supplementation groups; placebo (PLA, n=15), curcumin + fenugreek, CurQfen® (CUR, n=18), or fenugreek soluble fiber (FEN, n=14). All subjects performed a maximal GXT on a cycle ergometer to determine the PWCFT, Tlim, and V̇O2 peak before (PRE-test) and after (POST-test) 28 days of daily supplementation. Statistical analyses included 3 separate, one-way ANCOVAs to determine if there were any differences among the groups (PLA, CUR, FEN) for adjusted post-test scores for the PWCFT, V̇O2peak, and Tlim. The respective pre-test score was used as the covariate. In addition, reliability analyses (PRE-to POST-test) for the PLA group were used to calculate the minimal difference needed to be real (MD). The adjusted POST PWCFT values showed no statistical differences between groups (F= 3.141p= 0.053); however pairwise LSD comparisons indicated a significant difference between the CUR and PLA groups (p= 0.016), but not between the CUR and FEN groups. Therefore, separate one-way ANCOVAs were used to examine the adjusted PWCFT means for the PLA vs. CUR (F = 4.906, p =0.035) and the PLA vs. FEN (F = 2.969, p = 0.097). The one-way ANCOVA for ̇O 2 peak (F= 0.612 p= 0.547) and Tlim (F = 0.688, p = 0.508) values showed no statistical difference among the groups. Individual responses in each group showed ~ 20% of subjects in the CUR group, ~ 7% in the FEN group, and ~6% in the PLA group had values greater than the MD for the PWCFT, but none of the subjects in the PLA, FEN, or CUR groups exceeded the MD for ̇O 2 peak or Tlim. These findings indicated CurQfen® supplementation increased the PWCFT compared to a placebo, but not compared to fenugreek soluble fiber alone. However, there were no effects of CurQfen® on V̇O2 peak or Tlim. The mechanisms responsible for delaying time to neuromuscular fatigue may include increased NO production and increased blood flow to remove metabolic byproducts; however, the cellular changes which could lead to increases in Tlim and ̇O 2 peak may not have been sensitive to the GXT protocol or the given dosage of curcumin supplementation. Considering individual responses, CurQfen® supplementation resulted in a real change in the PWCFT for a small portion of the subjects (~20%). These findings suggested that CurQfen® supplementation without exercise training may help to improve time to neuromuscular fatigue in certain individuals, but the group mean analyses were not necessarily reflective of the responses for a majority of the...

The L-type Ca2+ channel (LTCC) provides trigger calcium to initiate cardiac contraction in a graded fashion that is regulated by L-type calcium current (ICa,L) amplitude and kinetics. Inactivation of LTCC is controlled to fine-tune calcium flux and is governed by voltage-dependent inactivation (VDI) and calcium-dependent inactivation (CDI). Rad is a monomeric G protein that regulates ICa,L and has recently been shown to be critical to β-adrenergic receptor (β-AR) modulation of ICa,L. Our previous work showed that cardiomyocyte-specific Rad knockout (cRadKO) resulted in elevated systolic function, underpinned by an increase in peak ICa,L, but without pathological remodeling. Here, we sought to test whether Rad-depleted LTCC contributes to the fight-or-flight response independently of β-AR function, resulting in ICa,L kinetic modifications to homeostatically balance cardiomyocyte function. We recorded whole-cell ICa,L from ventricular cardiomyocytes from inducible cRadKO and control (CTRL) mice. The kinetics of ICa,L stimulated with isoproterenol in CTRL cardiomyocytes were indistinguishable from those of unstimulated cRadKO cardiomyocytes. CDI and VDI are both enhanced in cRadKO cardiomyocytes without differences in action potential duration or QT interval. To confirm that Rad loss modulates LTCC independently of β-AR stimulation, we crossed a β1,β2-AR double-knockout mouse with cRadKO, resulting in a Rad-inducible triple-knockout mouse. Deletion of Rad in cardiomyocytes that do not express β1,β2-AR still yielded modulated ICa,L and elevated basal heart function. Thus, in the absence of Rad, increased Ca2+ influx is homeostatically balanced by accelerated CDI and VDI. Our results indicate that the absence of Rad can modulate the LTCC without contribution of β1,β2-AR signaling and that Rad deletion supersedes β-AR signaling to the LTCC to enhance in vivo heart function.