Gender-specific potential inhibitory role of Ca2+/calmodulin dependent protein kinase phosphatase (CaMKP) in pressure-overloaded mouse heart.

BACKGROUND: Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) has been proposed as a potent regulator of multifunctional Ca2+/calmodulin-dependent protein kinases (i.e., CaMKII). The CaMKII-dependent activation of myocyte enhancer factor 2 (MEF2) disrupts interactions between MEF2-histone deacetylases (HDACs), thereby de-repressing downstream gene transcription. Whether CaMKP modulates the CaMKII- MEF2 pathway in the heart is unknown. Here, we investigated the molecular and functional consequences of left ventricular (LV) pressure overload in the mouse of both genders, and in particular we evaluated the expression levels and localization of CaMKP and its association with CaMKII-MEF2 signaling. METHODOLOGY AND PRINCIPAL FINDINGS: Five week-old B6D1/F1 mice of both genders underwent a sham-operation or thoracic aortic constriction (TAC). Thirty days later, TAC was associated with pathological LV hypertrophy characterized by systolic and diastolic dysfunction. Gene expression was assessed by real-time PCR. Fetal gene program re-expression comprised increased RNA levels of brain natriuretic peptide and alpha-skeletal actin. Mouse hearts of both genders expressed both CaMKP transcript and protein. Activation of signalling pathways was studied by Western blot in LV lysates or subcellular fractions (nuclear and cytoplasmic). TAC was associated with increased CaMKP expression in male LVs whereas it tended to be decreased in females. The DNA binding activity of MEF2 was determined by spectrophotometry. CaMKP compartmentalization differed according to gender. In male TAC mice, nuclear CaMKP was associated with inactive CaMKII resulting in less MEF2 activation. In female TAC mice, active CaMKII (phospho-CaMKII) detected in the nuclear fraction, was associated with a strong MEF2 transcription factor-binding activity. CONCLUSIONS/SIGNIFICANCE: Gender-specific CaMKP compartmentalization is associated with CaMKII-mediated MEF2 activation in pressure-overloaded hearts. Therefore, CaMKP could be considered as an important novel cellular target for the development of new therapeutic strategies for heart diseases

Gender-associated changes in CaMKP and in CaMKII activation in cardiac subcelullar fractions 30 days after TAC.

<p>(<b>A</b>) Representative immunoblots of Oct1, GAPDH, CaMKP, CaMKII (phospho-Trh286 CamKII and total CaMKII) and HDAC4 (phospho-HDAC4 (Ser246)/HDAC5 (Ser259)/HDAC7 (Ser155) and total HDAC) in cardiac subcellular fractions (nuclear (n) and cytoplasmic (c)) of mouse hearts of both genders 30 days after sham-operation (Sham) or thoracic aortic constriction (TAC). In histograms, blue bars indicate male mice (light blue bar: M Sham; dark blue bar: M TAC), and red bars indicate female mice (light red bar: F Sham; dark red bar: F TAC). (<b>B – C</b>) Quantification of CaMKP protein level in LV nuclear (<b>B</b>) or cytoplasmic (<b>C</b>) extracts reveals a marked gender-dependent up-regulation of CaMKP expression in LV samples after TAC. CaMKP protein levels are increased in both nuclear and cytoplasmic extracts in male mice only. (<b>D – E</b>) Phosphorylation status of CaMKII in LV nuclear (<b>D</b>) and cytoplasmic (<b>E</b>) extracts: phospho-Thr286 CaMKII (yellow bar); total CaMKII (green bar); pCaMKII/tCaMKII ratio (red bar). CaMKP and CaMKII protein levels are expressed as fold increases in TAC mice relative to their respective sham groups. N = 4–8 mice per group. *p<0.05, TAC <i>vs.</i> sham; ^# p<0.05, female <i>vs.</i> male.</p

Gender-associated changes in CaMKP and in CaMKII activation in cardiac subcelullar fractions 30 days after TAC.

<p>(<b>A</b>) Representative immunoblots of Oct1, GAPDH, CaMKP, CaMKII (phospho-Trh286 CamKII and total CaMKII) and HDAC4 (phospho-HDAC4 (Ser246)/HDAC5 (Ser259)/HDAC7 (Ser155) and total HDAC) in cardiac subcellular fractions (nuclear (n) and cytoplasmic (c)) of mouse hearts of both genders 30 days after sham-operation (Sham) or thoracic aortic constriction (TAC). In histograms, blue bars indicate male mice (light blue bar: M Sham; dark blue bar: M TAC), and red bars indicate female mice (light red bar: F Sham; dark red bar: F TAC). (<b>B – C</b>) Quantification of CaMKP protein level in LV nuclear (<b>B</b>) or cytoplasmic (<b>C</b>) extracts reveals a marked gender-dependent up-regulation of CaMKP expression in LV samples after TAC. CaMKP protein levels are increased in both nuclear and cytoplasmic extracts in male mice only. (<b>D – E</b>) Phosphorylation status of CaMKII in LV nuclear (<b>D</b>) and cytoplasmic (<b>E</b>) extracts: phospho-Thr286 CaMKII (yellow bar); total CaMKII (green bar); pCaMKII/tCaMKII ratio (red bar). CaMKP and CaMKII protein levels are expressed as fold increases in TAC mice relative to their respective sham groups. N = 4–8 mice per group. *p<0.05, TAC <i>vs.</i> sham; ^# p<0.05, female <i>vs.</i> male.</p

Scenario of TAC-induced gender-dependent CaMKP compartmentalization associated with CaMKII-mediated MEF2 activation in the mouse heart.

<p>Diagrams summarizing some CaMKP actions induced by TAC in mouse heart of both genders: (<b>A</b>) Following TAC, CaMKP is mainly expressed in the cytoplasm in female LVs and so; the nuclear phospho-CaMKII (active form), pCaMKII, is able to induce HDAC export from the nucleus, relieving, as a consequence, the repression of the hypertrophic myocyte enhancer factor (MEF2). In turn, MEF2 activation results in increased BNP and α-SK gene transcription. (<b>B</b>) In males, nuclear CaMKP can dephosphorylate CaMKII. The resulting CaMKII inhibition maintains the repression of MEF2 by HDAC. Decreased MEF2 activation may facilitate the action of other often-associated transcription factors involved in left ventricular remodeling such as NFAT.</p

Activation of MEF2 transcription factor in cardiac nuclear extracts.

<p>Using the TransAm MEF2 kit, MEF2–DNA binding was quantified by spectrophotometry. (<b>A</b>) MEF2-DNA binding activity assayed in the presence of mutated consensus oligonucleotide in 10 µg of nuclear extracts from mouse hearts of both genders 30 days after sham-operation (Sham) or thoracic aortic constriction (TAC), (blue bars for male mice; light blue bar: M Sham; dark blue bar: M TAC; red bars for female mice; light red bar: F Sham; dark red bar: F TAC). The MEF2-DNA binding activity was shifted in presence of the wild-type consensus oligonucleotide at 20 pmol (green bar). (<b>B</b>) RNA quantification by real-time PCR of a gen driven by MEF2: alpha myosin heavy chain (α-MHC) mRNA levels were decreased in TAC mice of both genders; but α-MHC mRNA level was markedly higher in females (F TAC) than in males (M TAC). The number of mice is indicated in each column. *p<0.05, TAC <i>vs.</i> sham; ^# p,0.05, female <i>vs.</i> male. (<b>C</b>) Using the TransAm NFAT kit, NFAT–DNA binding quantified by spectrophotometry in mouse heart nuclear extracts, and assayed in the presence of the mutated consensus oligonucleotide (color code as for 7A) is significantly increased in M TAC. The NFATC1-DNA binding activity was shifted in presence of the wild-type consensus oligonucleotide at 20 pmol (green bar). (<b>D</b>) RNA quantification by real-time PCR of MCIP1 reflected that calcineurin-NFAT pathway activation was higher in male than in female mice after TAC. N = 3–10 mice per group. *p<0.05, TAC <i>vs.</i> sham; ^# p<0.05, female <i>vs.</i> male.</p

Quality control testing of recombinant mouse CaMKP (mCaMKP).

<p>(<b>A</b>) Purification of recombinant mCaMKP. Left panel: SDS-PAGE of GST-affinity chromatography-purified mCaMKP stained with Coomassie brilliant blue appearing as a protein band with apparent molecular weight of 49 kDa; right panel: immunoreactivity of purified mCaMKP detected by Western blotting; (<b>B</b>) In-gel Phosphatase activity of varying amounts of purified mCaMKP (lanes1–3: 4.5 µg; lanes 4–6: 9 µg; lane 7: none; lane 8: immunoprecipitated mCaMKP (IP)) detected using 4-MUP as a substrate; (<b>C</b>) Sensitivity of custom-made antibodies to N- and C-terminals detected by dot blotting using varying amounts of mCaMKP (1.5, 3, 6, 12, 24, 48, 96, 192 ng). Both antibodies recognized mCaMKP terminals, with a slightly stronger signal for the anti-C-terminus antibodies.</p

Gravimetric and echocardiographic data.

<p>Sham, sham-operated; TAC, thoracic aortic constriction; BW, body weight; HW, heart weight; TL, tibia length; HR, heart rate; IVSTD, interventricular septum thickness; PWTD, posterior wall thickness; LVEDD, left ventricular end diastolic diameter; LV mass, left ventricle mass; EF, LV ejection fraction; Spw, maximum systolic velocity of posterior wall; E/Ea, peak velocity of early mitral inflow/early diastolic velocity of the mitral annulus; Velocity ratio, stenotic jet velocity/ LV outflow velocity. *P<0.05, TAC <i>vs</i> sham; # P<0.05, female <i>vs</i> male.</p

Functional analysis of mouse hearts of both genders.

<p>Echocardiographic measurements were performed on mice anaesthetized with isoflurane 30(<b>A</b>) Representative echocardiographic images of the left ventricular function. A. Motion-mode of the left ventricle in parasternal long-axis view. IVS, Interventricular septum; PW, posterior wall; LVEDD, Left Ventricle End Diastolic Diameter; LVESD, Left Ventricle End Systolic Diameter. (<b>B</b>) Tissue Doppler spectra of the mitral annulus velocity. Ea, early diastolic wave of the mitral annulus (<b>C</b>) Pulsed-wave Doppler spectra of the transmitral flow velocity E, peak velocity of the early diastolic mitral wave. All measurements were gated on the ECG trace. (<b>D</b>) Ejection fraction evaluation from M-mode measurements. (E) Filling pressure evaluation of the left ventricle (E/Ea ratio). The ratio E/Ea increased similarly in both genders after TAC, indicating diastolic dysfunction. Mice of both genders showed similar systolic and diastolic dysfunction 30 days after TAC. The number of mice is indicated in each column. <b>M Sham</b>: male sham-operated mice; <b>M TAC</b>: male TAC mice; <b>F Sham</b>: female sham-operated mice; <b>F TAC</b>: female TAC mice. *p<0.05, TAC <i>vs.</i> sham; ^# p<0.05, female <i>vs.</i> male.</p

Gender difference in TAC-induced LV CaMKP expression and phosphatase activity.

<p>(<b>A</b>) Immunoblots from mouse hearts of both genders 30 days after sham-operation. (<b>B</b>) Densitometric analysis of Western blot of CaMKP protein levels normalized to GAPDH. At basal level, CaMKP protein level was higher in females (F Sham) than in males (M sham). (<b>C</b>) Immunoblots from mouse hearts of both genders 30 days after sham-operation (Sham) or thoracic aortic constriction (TAC). (<b>5D</b>) Densitometric analysis of Western blots of CaMKP in TAC mice of both genders. CaMKP protein levels in TAC mice are expressed as fold changes relative to their respective shams. (<b>E</b>) In-agarose gel detection of phosphatase activity, using 4-MUP as substrate, of CaMKP immunoprecipitated with anti-CaMKP antibodies from mouse LVs of both genders (left panel: male mice; right panel female mice). RP: CaMKP recombinant protein. (<b>F</b>) Phosphatase activity of IP-CaMKP was assayed using para-nitrophenyl phosphate as substrate. *p<0.05, TAC <i>vs.</i> sham; ^# p<0.05, female <i>vs.</i> male.</p