In numerical classification, four species of the Mycobacterium nonchromogenicum complex, Mycobacterium nonchromogenicum, M. terrae, M. novum, and M. triviale, formed one cluster. These four species appeared to be reduced to one species, Mycobacterium nonchromogenicum. Furthermore, relationships between the species were numerically analyzed by using the hypothetical median organism pattern. The results showed that the M. nonchromogenicum complex can be divided into two subgroups: M. nonchromogenicum and the other three. These two subgroups were differentiated from each other by scores based on two or more positive reactions in the following three characteristics: resistance to bleomycin (5 pgjml); heat-stable acid phosphatase activity; nicotinamidase or pyrazinamidase activity or both activities. M. nonchromogenicum gave two or three positive reactions among these three, and M. terrae, M. novum, and M. triviale gave two or three negative reactions.Three cases of lung infection due to M. nonchromogenicum, as well as three other cases of probable lung infection due to M. nonchromogenicum, were observed in this study. Only one organism isolated from one doubtful case was M. terrae. Up to now, M. nonchromogenicum was considered a nonpathogen. It was shown, however, that this organism causes lung infection in humans. Wayne 1966 (36), M. novum Tsukamura 1966, and M. triviale Kubica et al 1970 (8) are closely related, slowly growing, nonphotochromogenic mycobacteria (16, 24). They form one cluster in numerical classification (24), and, based on this finding, were named the Mycobacterium nonchromogenicum complex (28). These organisms have been considered nonpathogenic, but recently there appeared several papers reporting infections due to these organisms (2,3,5,6,26). As will be stated below, the causative organisms were not adequately identified in these studies. The reason is the uncertain knowledge of the taxonomy of these organisms. There exists, at present, no reliable method of identification of these organsims. The 219 Mycobacterium nonchromogenicum Tsukamura 1965 (17), M. terrae

Activation of CaMKII induces a myriad of biological processes and plays dominant roles in cardiac hypertrophy. Caveolar microdomain contains many calcium/calmodulin-dependent kinase II (CaMKII) targets, including L-type Ca channel (LTCC) complex, and serves as a signaling platform. The location of CaMKII activation is thought to be critical; however, the roles of CaMKII in caveolae are still elusive due to lack of methodology for the assessment of caveolae-specific activation. Our aim was to develop a novel tool for the specific analysis of CaMKII activation in caveolae and to determine the functional role of caveolar CaMKII in cardiac hypertrophy. To assess the caveolae-specific activation of CaMKII, we generated a fusion protein composed of phospholamban and caveolin-3 (cPLN-Cav3) and GFP fusion protein with caveolin-binding domain fused to CaMKII inhibitory peptide (CBD-GFP-AIP), which inhibits CaMKII activation specifically in caveolae. Caveolae-specific activation of CaMKII was detected using phosphospecific antibody for PLN (Thr). Furthermore, adenoviral overexpression of LTCC β-subunit (β) in NRCMs showed its constitutive phosphorylation by CaMKII, which induces hypertrophy, and that both phosphorylation and hypertrophy are abolished by CBD-GFP-AIP expression, indicating that β phosphorylation occurs specifically in caveolae. Finally, β phosphorylation was observed after phenylephrine stimulation in β-overexpressing mice, and attenuation of cardiac hypertrophy after chronic phenylephrine stimulation was observed in nonphosphorylated mutant of β-overexpressing mice. We developed novel tools for the evaluation and inhibition of caveolae-specific activation of CaMKII. We demonstrated that phosphorylated β dominantly localizes to caveolae and induces cardiac hypertrophy after α-adrenergic stimulation in mice. While signaling in caveolae is thought to be important in cardiac hypertrophy, direct evidence is missing due to lack of tools to assess caveolae-specific signaling. This is the first study to demonstrate caveolae-specific activation of CaMKII signaling in cardiac hypertrophy induced by α-adrenergic stimulation using an originally developed tool.

Background: L-type calcium channel (LTCC) localizes at T-tubules and caveolae in cardiomyocytes, and plays major roles in excitation-contraction coupling and cardiac hypertrophy. The expression of β2a subunit of LTCC (β2a) is increased in human failing heart. Recently, it was reported that phosphorylation of β2a by CaMKII enhanced LTCC activity. However, the functional role of β2a phosphorylation in heart failure remains to be elucidated. Objective: To make clear the functional role of β2a phosphorylation in cardiomyocytes. Methods and Results: Adenoviral overexpression of β2a demonstrated its constitutive phosphorylation by CaMKII and induced neonatal cardiomyocyte hypertrophy. Interestingly, phosphorylated β2a was co-localized with caveolin3. To assess caveolae-specific activation of CaMKII, we generated a fusion protein composed of phospholamban and caveolin3 (PLN-Cav3). This protein localized at caveolae, and caveolae-specific activation of CaMKII was detected using phospho-specific antibody for PLN (Thr17). In addition, to inhibit caveolae-specific CaMKII activity, we developed a GFP fusion protein with caveolin binding domain fused to CaMKII inhibitory peptide (CBD-GFP-AIP). We identified that this protein co-localized with caveolin3, and inhibited activation of CaMKII specifically at caveolae using PLN-Cav3 method. Moreover, CBD-GFP-AIP abolished β2a phosphorylation and attenuated β2a-induced cardiac hypertrophy. We found that phenylephrine (PE) stimulation activated caveolae-CaMKII and β2a in vitro and in vivo. Finally, we generated transgenic mice overexpressing wild-type (w-TG) or non-phospho mutant β2a (m-TG) in cardiomyocyte and evaluated cardiac hypertrophy after two weeks of chronic PE stimulation. The expression of β2a in both TG increased approximately 2.5 fold compared to control mice. PE-induced cardiac hypertrophy was attenuated in m-DTG compared to w-DTG mice (heart weight-body weight ratio: control; 4.8±0.2, *w-TG; 5.5±0.3, m-TG; 4.8±0.4, n=5-6, *p<0.05 vs others). Conclusion: We developed novel methods to evaluate and inhibit caveolae-specific activation of CaMKII. Using these methods, we revealed that phosphorylated β2a localized at caveolae, and exaggerates cardiac hypertrophy.

Rationale: Ca2+ influx via L-type Ca2+ channels (LTCCs) plays a pivotal role in excitation-contraction coupling and cardiac hypertrophy. Phosphorylation of LTCC β2a subunit (β2a) by CaMKII enhances channel activity. LTCCs are localized in both T-tubules and caveolae, but the functional role of phosphorylation of β2a in caveolae remains unelucidated. Objective: To develop a novel tool to analyze caveolae specific activation of CaMKII and to determine the functional roles of caveolae-specific CaMKII signaling in LTCC-related cardiac hypertrophy. Methods and Results: To evaluate caveolae-specific activation of CaMKII, we generated a fusion protein composed of the cytosolic domain of phospholamban (PLN) as a phosphopeptide tag and caveolin3 (cPLN-Cav3). Activation of CaMKII was assessed by phospho-specific antibody for PLN (Thr17). To inhibit caveolae-specific activation, we generated a GFP fusion protein with caveolae-targeting sequences fused to CaMKII inhibitory peptide (CTS-GFP-AIP). In neonatal rat cardiomyocytes (NRCM), adenoviral expression revealed that CTS-GFP-AIP co-localizes with caveolin3 and mediates caveolae specific inhibition of CaMKII, thus validating this novel method. CTS-GFP-AIP inhibited CaMKII phosphorylation of β2a in NRCM, thus suggesting that phosphorylation of β2a occurs exclusively in caveolae. Phenylephrine stimulation mediates CaMKII activation in caveolae, which leads to CaMKII-specific phosphorylation of β2a in vitro and in vivo. Finally, we generated non-phospho mutant β2a -overexpressing mice and assessed hypertrophic responses in both wild-type and mutant β2a transgenic animals (TG). Protein expression by transgenes in mutant TG was similar to those previously reported in wild-type β2a overexpressing mice. Wild-type β2a TG showed exaggerated cardiac hypertrophic responses at two weeks after phenylephrine stimulation when compared to controls (4.8 ± 0.2 vs. 5.5 ± 0.3 for heart weight to body weight ratio; p<0.05), which was completely abolished in mutant TG. Conclusions: We developed a novel method to analyze caveolae-specific activation of CaMKII and confirmed that caveolae-specific phosphorylation of β2a exaggerates cardiac hypertrophy caused by phenylephrine stimulation.