Ken-Ichi Furukawa

Mutations in the gene encoding the type II collagen gene (COL2A1) have been found to affect the entire skeletal system. Recently, inheritable skeletal dysplasia caused by novel COL2A1 mutations has been linked to an inherited disease of the hip joint that neither involves the entire skeletal system nor is characterized by the presence of concomitant disorders, such as spinal or ocular abnormalities.

Aortic valve calcification can aggravate aortic stenoses, and it is a significant cause of sudden cardiac death. The increasing number of patients with age-related calcification is a problem in developed nations. However, the only treatment option currently available is highly invasive cardiac valve replacement. Therefore, clarification of the etiology of calcification is urgently needed to develop drug therapies and prevention methods. Recent studies have revealed that calcification is not a simple sedimentation of a mineral through a physicochemical phenomenon; various factors dynamically contribute to the mechanism. Further, we are finally beginning to understand the cellular origins of calcification, which had been unclear for a long time. Based on these findings that help to clarify potential drug targets, we expect to establish drug therapies that reduce the stress on patients. In this paper, I introduce the latest findings on cells that are most likely to contribute to calcification and on calcification-related factors that may lead to the development of drug therapies.

Previously, we showed that in adult rat cardiomyocytes, nitric oxide (NO) donors stimulate mitochondrial cGMP production, followed by cytochrome c release, independently of the mitochondrial permeable transition pore. We investigated whether mitochondrial cGMP-induced cytochrome c release from cardiac mitochondria is Ca²⁺-sensitive. Mitochondria and primary cultured cardiomyocytes were prepared from left ventricles of male Wistar rats. The cytosolic Ca²⁺ concentration was adjusted with Ca²⁺-EGTA buffers. Cytochrome c released from mitochondria was measured by Western blotting. Cardiomyocyte apoptosis was assessed by Annexin V staining. Cytochrome c release from cardiac mitochondria was evoked by buffered Ca²⁺ (1 μM); this was inhibited by NO-cGMP pathway inhibitors such as N^G-monomethyl-l-arginine monoacetate (inhibitor of NO synthase), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (NO scavenger), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, NO-sensitive guanylyl cyclase inhibitor) and voltage-dependent anion channel (VDAC) inhibitor, 4,4′-diisothiocyano-2,2′-disulfonic acid stilbene, but not by cyclosporin A (mitochondrial permeable transition pore inhibitor). Furthermore, this release was significantly and dose dependently inhibited by 0.3–3 μM KT5823 (protein kinase G inhibitor). At the cellular level, intracellular perfusion of cardiomyocytes with buffered Ca²⁺ (1 μM) also induced apoptosis, which was inhibited in the presence of ODQ. A membrane-permeable cGMP analog, 8-Br-cGMP, but not cGMP itself, mimicked buffered Ca²⁺ actions in both cardiac mitochondria and cardiomyocytes. We further confirmed an increase in protein kinase G activity by adding cGMP in mitochondrial protein fraction. Our results suggest that mitochondrial NO-cGMP pathway-induced cytochrome c release from cardiac mitochondria, triggered by increased cytosolic Ca²⁺, occurs through VDAC via the stimulation of an undiscovered mitochondrial protein kinase G.

When cardiac valve stenosis is accompanied by calcification, symptoms and prognosis become much worse and may cause sudden cardiac death. The prevalence of this disease has increased with the rapidly aging in Japanese society. It has recently been revealed that several genes which relate to physiological ossification and calcification play important roles in this process. To find a suitable target for medical treatment, the molecular mechanism for calcification of cardiac valves should be elucidated in detail. In this review, we summarize the current knowledge on the pathology and molecular mechanism for ectopic calcification of the cardiac valve.

BACKGROUND AND PURPOSE Stem cell transplantation therapy is a promising option for treatment of severe ischaemic heart disease. Dimethyl sulphoxide (DMSO) differentiates P19CL6 embryonic carcinoma cells into cardiomyocyte-like cells, but with low differentiation capacity. To improve the degree of this differentiation, we have assessed several derivatives of the differentiation-inducing factor-1 (DIF-1), originally found in the cellular slime mould Dictyostelium discoideum, on P19CL6 cells. EXPERIMENTAL APPROACH P19CL6 cells were cultured with each derivative and 1% DMSO for up to 16 days. Differentiation was assessed by measuring the number of beating and non-beating aggregates, and the expression of genes relevant to cardiac tissue. The mechanism of action was investigated using a T-type Ca²⁺ channel blocker. KEY RESULTS Of all the DIF-1 derivatives tested only Br-DIF-1 showed any effects on cardiomyocyte differentiation. In the presence of 1% DMSO, Br-DIF-1 (0.3–3 µM) significantly and dose-dependently increased the number of spontaneously beating aggregates compared with 1% DMSO alone, by day 16. Expression of mRNA for T-type calcium channels was significantly increased by Br-DIF-1 + 1% DMSO compared with 1% DMSO alone. Mibefradil (a T-type Ca²⁺ channel blocker; 100 nM) and a small interfering RNA for the T-type Ca²⁺ channel both significantly decreased the beating rate of aggregates induced by Br-DIF-1 + 1% DMSO. CONCLUSIONS AND IMPLICATIONS Br-DIF-1 accelerated the differentiation, induced by 1% DMSO, of P19CL6 cells into spontaneously beating cardiomyocyte-like cells, partly by enhancing the expression of the T-type Ca²⁺ channel gene.

Ossification of the posterior longitudinal ligament of the spine (OPLL) is characterized by ectopic bone formation in the spinal ligaments. We previously reported that P2 purinoceptor Y1 (P2Y1) expression is elevated in the spinal ligament cells of OPLL patients, but the role of P2Y1 in the spinal ligament calcification process is unknown. To verify the hypothesis that P2Y1 expression causes ossification of the spinal ligaments, we forced expression of P2Y1 in spinal ligament cells obtained from OPLL and non-OPLL patients using a cytomegaloviral vector. The expression of mRNA and protein was investigated by quantitative real-time polymerase chain reaction and immunofluorescence staining, respectively. After transfection, bone morphogenetic protein-2 (BMP-2) and Sox9 mRNA expression was significantly increased in spinal ligament cells derived from OPLL patients (4.36- and 6.44-fold, respectively) compared with cells from non-OPLL patients (0.57- and 3.64-fold, respectively) 2 days after P2Y1 transient transfection. Furthermore, a statistically significant correlation was observed between BMP-2 and P2Y1 mRNA expression levels in cells obtained from OPLL patients but not from non-OPLL patients. Immunofluorescence analysis showed that BMP-2 and P2Y1 expression was increased in OPLL patients only, while Sox9 expression was increased in OPLL and non-OPLL patients. MRS2279, a selective P2Y1 antagonist, blocked the upregulation of Sox9 and BMP-2 after forced expression of P2Y1. Furthermore, 4 days after transient transfection of P2Y1, mineralization was observed only in spinal ligament cells from OPLL patients. These results suggest that P2Y1 expression plays an important role in ectopic bone formation in the spinal ligaments of OPLL patients.

It has been reported that resveratrol (trans-3,5,4′-trihydroxystilbene) from Vitis plants has various cardioprotective effects. Vitis plants also include various resveratrol tetramers. The aim of our study is to clarify the pharmacological properties of resveratrol tetramers. We isolated two resveratrol tetramers as major products of Vitis plants. One is vitisin A, a complex of two resveratrol dimers, (+)-ϵ-viniferin and ampelopsin B, and the other is hopeaphenol, composed of 2 mol ampelopsin B. Vitisin A (30–300 nM) unexpectedly dose-dependently facilitated swelling and depolarization of mitochondria and cytochrome c release from mitochondria, which are indices of cardiomyocyte apoptosis. Furthermore, vitisin A induced apoptosis in the primary culture of adult rat ventricular myocytes. On the other hand, hopeaphenol (1–10 μM) dose-dependently inhibited Ca²⁺ (30 μM)-induced mitochondrial depolarization and cytochrome c release from mitochondria but had not affected mitochondrial swelling. Moreover, hopeaphenol inhibited vitisin A-induced apoptosis. In structural and functional studies, we further confirmed that vitisin B, one of the resveratrol tetramers having (+)-ϵ-viniferin unit, induces mitochondrial swelling and cytochrome c release from mitochondria like vitisin A and that vitisifuran A, one of the resveratrol tetramers having the ampelopsin B unit, inhibits Ca²⁺-induced cytochrome c release from mitochondria like hopeaphenol. These results show that resveratrol tetramers have at least two opposite effects on cardiomyocytes; the one having the (+)-ϵ-viniferin unit induces cardiomyocyte apoptosis, and the other having ampelopsin B but not (+)-ϵ-viniferin unit inhibits it.