Gene expression and genome sequence analysis of Mylk3 in C57BL/6N and identification of a new isoform of cMLCK.

<p>(A) Expressions of cMLCK mRNA in C57BL/6J and C57BL/6N hearts examined by Northern blot analysis. (B) Schematic representation of Mylk3 encoding for cMLCK and identification of point mutation in C57BL/6N. The highlight indicates T146A mutation in exon1 of Mylk3. (C) Identification of cMLCK isoform. Peptides that matched sequences of cMLCK isoform 1 are shown in underlined bold. Predicted sequences of cMLCK isoform X2 is also shown. Slash bar indicates the identical sequence to cMLCK (isoform 1). (D) RT-PCR of cMLCK isoforms using cardiomyocytes. The fragment (arrow) was sequenced to confirm the existence of cMLCK isoform X2.</p

Effects of calyculin A (10 nM) on MLC2v phosphorylation and on twitch tension in the presence and absence of phenylephrine in papillary muscles from C57BL/6J and C57BL/6N.

<p>(A) MLC2v phosphorylation. Percentage ratio of phosphorylated-MLC2v <i>vs</i>. total MLC2v was calculated. *<i>P</i><0.05, <i>n</i> = 4–7. (B) Twitch tension induced by phenylephrine in the presence and absence of calyculin A. *p<0.05, n = 5–6. (C) A relationship between phenylephrine-induced inotropic responses and MLC2v phosphorylation levels in the presence and absence of calyculin A. The increase in twitch tension by phenylephrine was plotted against the levels of MLC2v phosphorylation. Spearman rank correlation coefficient was calculated (by Statview software version 4.0).</p

Effects of nifedipine, BAY K 8644 and ryanodine on phenylephrine-induced mechanical responses in papillary muscles.

<p>(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, *<i>P</i><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 <i>vs</i>. 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 (<i>n</i> = 4–6, *P<0.05). Atenolol was present throughout the experiments for tension measurements.</p

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

<div><p>Background & Aims</p><p>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.</p><p>Methods and Results</p><p>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%.</p><p>Conclusions</p><p>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.</p></div

Effects of phenylephrine (PE) on twitch tension and MLC2v phosphorylation in papillary muscles from C57BL/6J and C57BL/6N.

<p>(A) Representative tension recordings. (B) The phosphorylation levels of MLC2v. The % ratio of phosphorylated MLC2v <i>vs</i>. total MLC2v was calculated (<i>n</i> = 5). (C) Concentration-dependent effects of phenylephrine in twitch tension. After basal twitch tension was recorded, phenylephrine was cumulatively applied. Atenolol was present throughout the experiments for tension measurements. *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001, <i>n</i> = 5–6.</p

New Isoform of Cardiac Myosin Light Chain Kinase and the Role of Cardiac Myosin Phosphorylation in α_{1 - Fig 3}-Adrenoceptor Mediated Inotropic Response

<p>(A) Phosphorylation levels of MLC2v determined by urea-glycerol-PAGE in C57BL/6J and C57BL/6N hearts. The ratio (%) of phosphorylated MLC2v <i>vs</i>. total MLC2v was calculated. The expression of smMLCK (B), ZIPK (C) and MYPT2 (myosin phosphatase target subunit 2) (D) in C57BL/6J and C57BL/6N hearts. Upper panels show representative Western blots and the lower panels summarize the densitometrical data. The expression level in C57BL/6J was expressed as 100%. *<i>P</i><0.05. <i>n</i> = 4.</p

Renal papillary tip extract stimulates BNP production and excretion from cardiomyocytes

<div><p>Background</p><p>Brain natriuretic peptide (BNP) is an important biomarker for patients with cardiovascular diseases, including heart failure, hypertension and cardiac hypertrophy. It is also known that BNP levels are relatively higher in patients with chronic kidney disease and no heart disease; however, the mechanism remains unclear.</p><p>Methods and results</p><p>We developed a BNP reporter mouse and occasionally found that this promoter was activated specifically in the papillary tip of the kidneys, and its activation was not accompanied by <i>BNP</i> mRNA expression. No evidence was found to support the existence of BNP isoforms or other nucleotide expression apart from BNP and tdTomato. The pBNP-tdTomato-positive cells were interstitial cells and were not proliferative. Unexpectedly, both the expression and secretion of BNP increased in primary cultured neonatal cardiomyocytes after their treatment with an extract of the renal papillary tip. Intraperitoneal injection of the extract of the papillary tips reduced blood pressure from 210 mmHg to 165 mmHg, the decrease being accompanied by an increase in serum BNP and urinary cGMP production in stroke-prone spontaneously hypertensive (SHR-SP) rats. Furthermore the induction of BNP by the papillary extract from rats with heart failure due to myocardial infarction was increased in cardiomyocytes.</p><p>Conclusions</p><p>These results suggested that the papillary tip express a substance that can stimulate BNP production and secretion from cardiomyocytes.</p></div

Mild activation of the BNP promoter in the heart, with no activation in the other organs, of pBNP-tdTomato Tg mice.

<p>The pictures are representative of three mice.</p

Characterization of papillary extracts by proteolysis and molecular weight fractionation.

<p>A, Papillary extracts were digested with 0.3 μg/ml Proteinase K for each indicated time and loaded into 10% and 15% gel. The pictures are representative of three experiments. B, BNP mRNA expression was examined by Northern blot analysis in cardiomyocytes treated with control buffer (MOCK) or each digested extract of the papillary tip. *P < 0.05 vs. cardiomyocytes treated with the buffer alone (MOCK). n = 4. C, Papillary extracts were fractionated according to their molecular weight range. The pictures are representative of three experiments. D, BNP mRNA expression was examined by Northern blot analysis in cardiomyocytes treated with control buffer or each molecular-weight-fractionated extract of the papillary tip. *P < 0.05 vs. cardiomyocytes treated with the buffer alone (MOCK). n = 3.</p

Effects of extracts of the papillary tip (●) and inner medulla (○) of the kidneys on stroke-prone spontaneously hypertensive rats (SHR-SP).

<p>A, Each extract was intraperitoneally administered to SHR-SP rats and their systolic blood pressure was followed for 24 h using the tail-cuff method. *P < 0.05 vs. basal blood pressure (at 0 h). n = 4 to 6. B, Measurement of serum BNP (n = 4 to 8) and C, cGMP in the urine 4h and 24 h after intraperitoneal injection of buffer alone or the extract of the papillary tip or the inner medulla. *P < 0.05 or n.s. = no significant difference vs. serum from SHR-SP rats treated with buffer alone (MOCK). n = 4 to 8. D, The ratio of urine volume to MOCK 4h and 24h after the injection. n.s. = no significant difference vs. the urine from SHR-SP rats treated with buffer alone (MOCK). n = 4 to 8. E, urine sodium excretion for 4h (0-4h) and 20h (4–24 h) after the injection of control buffer or extract of the papillary tip or the inner medulla into SHR-SP rats. n.s. = no significant difference vs. the urine from SHR-SP rats treated with buffer alone (MOCK). n = 4 to 8. For all figures, data indicate the mean +/- standard error of the mean.</p