The molecular components of the large-conductance Ca(2+)-activated K(+) channels that are functionally expressed in mitochondria (mitoK(Ca)) in cardiac myocytes have not been identified. Our experimental results show that the transcript corresponding to the large-conductance Ca(2+)-activated K(+) channel beta1-subunit (BK-beta1) is substantially expressed in mammalian heart. A yeast two-hybrid assay showed the BK-beta1 protein can interact with a mitochondrial protein, cytochrome c oxidase subunit I (Cco1). Results from immunocytochemical experiments also demonstrated that BK-beta1 interacted with Cco1 and colocalized in rat cardiac mitochondria. Furthermore, 17beta-estradiol, which enhances the activity of the BK channel alpha-subunit only in the presence of the beta1-subunit, significantly increased flavoprotein oxidation in rat ventricle myocytes and decreased the rate of cell death under simulated ischemia. Single-channel recordings from mitochondrial inner membrane indicated that the activity of mitoK(Ca), which had a conductance of approximately 270 pS, was enhanced by 17beta-estradiol and blocked by paxilline. In combination, the present study revealed a new mechanism for the cardioprotective effects of 17beta-estradiol, which include the activation of mitoK(Ca) via the interaction with BK-beta1. BK-beta1 may be an important molecular component that functionally couples with both Cco1 and mitoK(Ca) pore-forming alpha-subunit.

SummaryCardiac regenerative therapies utilizing human induced pluripotent stem cells (hiPSCs) are hampered by ineffective large-scale culture. hiPSCs were cultured in multilayer culture plates (CPs) with active gas ventilation (AGV), resulting in stable proliferation and pluripotency. Seeding of 1 × 106 hiPSCs per layer yielded 7.2 × 108 hiPSCs in 4-layer CPs and 1.7 × 109 hiPSCs in 10-layer CPs with pluripotency. hiPSCs were sequentially differentiated into cardiomyocytes (CMs) in a two-dimensional (2D) differentiation protocol. The efficiency of cardiac differentiation using 10-layer CPs with AGV was 66%–87%. Approximately 6.2–7.0 × 108 cells (4-layer) and 1.5–2.8 × 109 cells (10-layer) were obtained with AGV. After metabolic purification with glucose- and glutamine-depleted and lactate-supplemented media, a massive amount of purified CMs was prepared. Here, we present a scalable 2D culture system using multilayer CPs with AGV for hiPSC-derived CMs, which will facilitate clinical applications for severe heart failure in the near future.

Three-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, known as statins, induce skeletal muscle injury including myalgia, myositis, and rhabdomyolysis. The mechanism of this myotoxicity remains unknown. This study examined the effect of statins on single skeletal myofibers enzymatically isolated from the rat flexor digitorum brevis muscles. Fluvastatin and pravastatin induced the formation of numerous vacuoles in the myofibers after 72 h of treatment. This effect progressed in a time- and concentration-dependent manner and, consequently, cell death occurred after 120 h. Electron micrographs revealed craters along the sarcolemma and swelling of the sarcoplasmic reticula and mitochondria, in addition to intracellular vacuoles. When caffeine was added after 72 h of fluvastatin treatment, contractile shortening of statin-treated myofibers was significantly attenuated and blebs formed on the surface of the myofibers. The coapplication of geranylgeranylpyrophosphate (GGPP) with fluvastatin prevented the morphological changes, while that of farnesylpyrophosphate (FPP) was ineffective. Furthermore, perillyl alcohol, an inhibitor of Rab geranylgeranyl transferase and geranylgeranyl transferase-I (GGTase-I), mimicked the effect of statins, while a specific GGTase-I inhibitor (GGTI-298) or a farnesyl transferase inhibitor (FTI-277) failed to do so. These results suggest that the inactivation of Rab GTPase, which involved in intracellular membrane transport, is a crucial factor in statin-induced-morphological abnormality in skeletal muscle fibers.

Effects of pimaric acid (PiMA) and eight closely related compounds on large-conductance K ϩ (BK) channels were examined using human embryonic kidney (HEK) 293 cells, in which either the ␣ subunit of BK channel (HEKBK␣) or both ␣ and ␤1 (HEKBK␣␤1) subunits were heterologously expressed. Effects of these compounds (10 M) on the membrane potential of HEKBK␣␤1 were monitored by use of DiBAC 4 (3), a voltagesensitive dye. PiMA, isopimaric acid, sandaracoisopimaric acid, dihydropimaric acid, dihydroisopimaric acid, and dihydroisopimarinol induced substantial membrane hyperpolarization. The direct measurement of BK␣␤1 opening under whole-cell voltage clamp showed that these six compounds activated BK␣␤1 in a very similar concentration range (1-10 M); in contrast, abietic acid, sclareol, and methyl pimarate had no effect. PiMA did not affect the charybdotoxin-induced block of macroscopic BK␣␤1 current. Single channel recordings of BK␣␤1 in insideout patches showed that 10 M PiMA did not change channel conductance but significantly increased its open probability as a result of increase in sensitivity to Ca 2ϩ and voltage. Because coexpression of the ␤1 subunit did not affect PiMA-induced potentiation, the site of action for PiMA is suggested to be BK␣ subunit. PiMA was selective to BK over cloned small and intermediate Ca 2ϩ activated K ϩ channels. In conclusion, PiMA (Ͼ1 M) increases Ca 2ϩ and voltage-sensitivity of BK␣ when applied from either side of the cell membrane. The marked difference in potency as BK channel openers between PiMA and abietic acid, despite only very small differences in their chemical structures, may provide insight into the fundamental structure-activity relationship governing BK␣ activation.