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New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α1-Adrenoceptor Mediated Inotropic Response.

Taniguchi M, Okamoto R, Ito M, Goto I, Fujita S, Konishi K, Mizutani H, Dohi K, Hartshorne DJ, Itoh T - PLoS ONE (2015)

Bottom Line: The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J.Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N.There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15-30%.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology and Nephrology, Mie University Graduate School of Medicine, Edobashi, Tsu 514-8507, Mie, Japan.

ABSTRACT

Background & aims: Cardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated.

Methods and results: By Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15-30%.

Conclusions: We identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain.

No MeSH data available.


Related in: MedlinePlus

Effects of nifedipine, BAY K 8644 and ryanodine on phenylephrine-induced mechanical responses in papillary muscles.(A) The representative response to phenylephrine (10 μM) in C57BL/6J. Actions of nifedipine (0.3 μM, B), BAY K 8644 (1 μM, C) and ryanodine (0.3 μM, D) on twitch tension in the absence and presence of phenylephrine in C57BL/6J. Phenylephrine exerted positive inotropic response in the presence of ryanodine (i.e., in situations where SR-function is greatly reduced). Summary of the effects of BAY K 8644 (1 μM, E) and ryanodine (0.3 μM, F) on phenylephrine-induced response (n = 4–6, *P<0.05). (G) The levels of papillary muscle MLC2v phosphorylation in the presence and absence of BAY K 8644 or ryanodine in C57BL/6J and C57BL/6N. The % ratio of phosphorylated MLC2v vs. total MLC2v was calculated. BAY K 8644 and ryanodine did not modify MLC2v phosphorylation levels. The significance in the differences in the MLC2v phosphorylation levels was preserved in the presence of BAY K 8644 and ryanodine between C57BL/6J and C57BL/6N (n = 4–6, *P<0.05). Atenolol was present throughout the experiments for tension measurements.
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pone.0141130.g006: Effects of nifedipine, BAY K 8644 and ryanodine on phenylephrine-induced mechanical responses in papillary muscles.(A) The representative response to phenylephrine (10 μM) in C57BL/6J. Actions of nifedipine (0.3 μM, B), BAY K 8644 (1 μM, C) and ryanodine (0.3 μM, D) on twitch tension in the absence and presence of phenylephrine in C57BL/6J. Phenylephrine exerted positive inotropic response in the presence of ryanodine (i.e., in situations where SR-function is greatly reduced). Summary of the effects of BAY K 8644 (1 μM, E) and ryanodine (0.3 μM, F) on phenylephrine-induced response (n = 4–6, *P<0.05). (G) The levels of papillary muscle MLC2v phosphorylation in the presence and absence of BAY K 8644 or ryanodine in C57BL/6J and C57BL/6N. The % ratio of phosphorylated MLC2v vs. total MLC2v was calculated. BAY K 8644 and ryanodine did not modify MLC2v phosphorylation levels. The significance in the differences in the MLC2v phosphorylation levels was preserved in the presence of BAY K 8644 and ryanodine between C57BL/6J and C57BL/6N (n = 4–6, *P<0.05). Atenolol was present throughout the experiments for tension measurements.

Mentions: Pretreatment with nifedipine (L-type Ca2+ channel blocker, 0.3 μM) reduced not only the twitch tension but also the phenylephrine-induced enhancement of the twitch tension in C57BL/6J (Fig 6A vs. 6B). BAY K 8644 (L-type Ca2+ channel activator, 1 μM) enhanced the twitch tension in both C57BL/6J and C57BL/6N, but blocked phenylephrine-induced inotropic response in both C57BL/6J and C57BL/6N (Fig 6C and 6E). Ryanodine [a blocker of Ca2+-induced Ca2+ release in sarcoplasmic reticulum (SR)] greatly reduced the basal twitch tension. In the presence of ryanodine, phenylephrine induced an inotropic action in C57BL/6J and C57BL/6N (Fig 6D and 6F).


New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α1-Adrenoceptor Mediated Inotropic Response.

Taniguchi M, Okamoto R, Ito M, Goto I, Fujita S, Konishi K, Mizutani H, Dohi K, Hartshorne DJ, Itoh T - PLoS ONE (2015)

Effects of nifedipine, BAY K 8644 and ryanodine on phenylephrine-induced mechanical responses in papillary muscles.(A) The representative response to phenylephrine (10 μM) in C57BL/6J. Actions of nifedipine (0.3 μM, B), BAY K 8644 (1 μM, C) and ryanodine (0.3 μM, D) on twitch tension in the absence and presence of phenylephrine in C57BL/6J. Phenylephrine exerted positive inotropic response in the presence of ryanodine (i.e., in situations where SR-function is greatly reduced). Summary of the effects of BAY K 8644 (1 μM, E) and ryanodine (0.3 μM, F) on phenylephrine-induced response (n = 4–6, *P<0.05). (G) The levels of papillary muscle MLC2v phosphorylation in the presence and absence of BAY K 8644 or ryanodine in C57BL/6J and C57BL/6N. The % ratio of phosphorylated MLC2v vs. total MLC2v was calculated. BAY K 8644 and ryanodine did not modify MLC2v phosphorylation levels. The significance in the differences in the MLC2v phosphorylation levels was preserved in the presence of BAY K 8644 and ryanodine between C57BL/6J and C57BL/6N (n = 4–6, *P<0.05). Atenolol was present throughout the experiments for tension measurements.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4626101&req=5

pone.0141130.g006: Effects of nifedipine, BAY K 8644 and ryanodine on phenylephrine-induced mechanical responses in papillary muscles.(A) The representative response to phenylephrine (10 μM) in C57BL/6J. Actions of nifedipine (0.3 μM, B), BAY K 8644 (1 μM, C) and ryanodine (0.3 μM, D) on twitch tension in the absence and presence of phenylephrine in C57BL/6J. Phenylephrine exerted positive inotropic response in the presence of ryanodine (i.e., in situations where SR-function is greatly reduced). Summary of the effects of BAY K 8644 (1 μM, E) and ryanodine (0.3 μM, F) on phenylephrine-induced response (n = 4–6, *P<0.05). (G) The levels of papillary muscle MLC2v phosphorylation in the presence and absence of BAY K 8644 or ryanodine in C57BL/6J and C57BL/6N. The % ratio of phosphorylated MLC2v vs. total MLC2v was calculated. BAY K 8644 and ryanodine did not modify MLC2v phosphorylation levels. The significance in the differences in the MLC2v phosphorylation levels was preserved in the presence of BAY K 8644 and ryanodine between C57BL/6J and C57BL/6N (n = 4–6, *P<0.05). Atenolol was present throughout the experiments for tension measurements.
Mentions: Pretreatment with nifedipine (L-type Ca2+ channel blocker, 0.3 μM) reduced not only the twitch tension but also the phenylephrine-induced enhancement of the twitch tension in C57BL/6J (Fig 6A vs. 6B). BAY K 8644 (L-type Ca2+ channel activator, 1 μM) enhanced the twitch tension in both C57BL/6J and C57BL/6N, but blocked phenylephrine-induced inotropic response in both C57BL/6J and C57BL/6N (Fig 6C and 6E). Ryanodine [a blocker of Ca2+-induced Ca2+ release in sarcoplasmic reticulum (SR)] greatly reduced the basal twitch tension. In the presence of ryanodine, phenylephrine induced an inotropic action in C57BL/6J and C57BL/6N (Fig 6D and 6F).

Bottom Line: The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J.Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N.There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15-30%.

View Article: PubMed Central - PubMed

Affiliation: Department of Cardiology and Nephrology, Mie University Graduate School of Medicine, Edobashi, Tsu 514-8507, Mie, Japan.

ABSTRACT

Background & aims: Cardiac myosin light chain kinase (cMLCK) plays an obligatory role in maintaining the phosphorylation levels of regulatory myosin light chain (MLC2), which is thought to be crucial for regulation of cardiac function. To test this hypothesis, the role played by ventricular MLC2 (MLC2v) phosphorylation was investigated in the phenylephrine-induced increase in twitch tension using the naturally-occurring mouse strain, C57BL/6N, in which cMLCK is down regulated.

Methods and results: By Western blot and nanoLC-MS/MS analysis, cMLCKs with molecular mass of 61-kDa (cMLCK-2) and/or 86-kDa were identified in mice heart. Among various mouse strains, C57BL/6N expressed cMLCK-2 alone and the closest relative strain C57BL/6J expressed both cMLCKs. The levels of MLC2v phosphorylation was significantly lower in C57BL/6N than in C57BL/6J. The papillary muscle twitch tension induced by electrical field stimulation was smaller in C57BL/6N than C57BL/6J. Phenylephrine had no effect on MLC2v phosphorylation in either strains but increased the twitch tension more potently in C57BL/6J than in C57BL/6N. Calyculin A increased papillary muscle MLC2v phosphorylation to a similar extent in both strains but increased the phenylephrine-induced inotropic response only in C57BL/6N. There was a significant positive correlation between the phenylephrine-induced inotropic response and the levels of MLC2v phosphorylation within ranges of 15-30%.

Conclusions: We identified a new isoform of cMLCK with a molecular mass of 61kDa(cMLCK-2) in mouse heart. In the C57BL/6N strain, only cMLCK-2 was expressed and the basal MLC2v phosphorylation levels and the phenylephrine-induced inotropic response were both smaller. We suggest that a lower phenylephrine-induced inotropic response may be caused by the lower basal MLC2v phosphorylation levels in this strain.

No MeSH data available.


Related in: MedlinePlus