The Influence of the Xin Repeat-Containing Proteins on the Development of Pressure Induced Cardiac Hypertrophy in Mice

The proteins of intercalated discs (ICDs) are components of the cardiac cytoskeleton which forms the scaffold of cardiomyocytes and thus helps to maintain cell shape, provide tissue integrity, and to stabilize the sarcomeric proteins. Part of the cytoskeleton are the Xin repeat containing proteins 1 and 2 (XIRP1 and XIRP2), which are located in ICDs of the mammalian heart. XIRP1 and XIRP2 have been detected in the adherens junctions of the ICDs and play critical roles in the cardiac development and the structural integrity of ICDs. XIRP1-null mice created by our group exhibited a mild cardiac phenotype with increasing non-terminal ICDs and an increased intraventricular conduction velocity. Also the application of elevated afterload to XIRP1 deficient mice to induce hypertrophic cardiac remodeling did not lead to a more severe phenotype. Possibly lack of XIRP1 protein was compensated by XIRP2 up-regulation. Therefore XIRP1 deficient mice were crossed with XIRP2 knockdown mice (XIRP1 XIRP2 dko). We hypothesized that an additional knock-down of XIRP2 may help to unravel the functions of both XIRPs in the murine heart in case of normal and elevated afterload. XIRP wild-type (XIRP WT) and XIRP1XIRP2 dko female mice of about 12 weeks or one year age were assigned to transverse aortic constriction , TAC, or sham surgery. Surgeries were performed on anesthetized (2 vol % isoflurane) mice provided i.p. with analgesia (buprenorphine 0.065 mg/kg body weight, BW). A 27 gauge needle was used to standardize the degree of aortic constriction. After 14 days of TAC or sham, hemodynamic parameters were recorded by a pressure catheter in mild anesthesia (heart rate, HR, ~500 min-1). Furthermore, surface electrocardiography was performed under the same conditions. Different morphometric parameters were measured: body weight, BW; heart weight, HW; left ventricular weight, LVW; lung weight, LW; tibia length, TL. From explanted hearts single cardiomyocytes were prepared by retrograde Langendorff-perfusion, proteins of interest were localized in the isolated cardiomyocytes by antibodies and visualized by immunofluorescence microscopy. Representative hearts from the different groups were embedded in paraffin and sections from 4 different areas were cut. In light microscopy the diameters of the septum and the ventricular wall were determined as well as the amount of cardiac fibrosis, visualized by Masson’s trichrome stain. 14 days after TAC surgery the XIRP WT and XIRP1XIRP2 dko mice of three month age had developed significant cardiac hypertrophy compared to the sham animals. However, both genotypes did not differ with respect to the measured morphological parameters (HW, HW/BW, HW/TL; LVW, LVW/BW, LVW/TL; LW, LW/BW; LW/TL). Also the diameters of septum and ventricular wall were significantly increased by the TAC but did not exhibit genotype differences. But TAC induced cardiac fibrosis was significantly higher in XIRP WT than in XIRP1XIRP2 dko mice. Protein localization performed by fluorescence microscopy revealed that XIRP1XIRP2 dko mice exhibited independently of TAC significantly less non-terminal ICDs than XIRP WT mice, whereas the number of terminally situated ICDs was not altered. Localization of proteins in the M-band, the Z-discs and the triads was neither influenced by genotype nor by TAC. The TAC elevated the systolic arterial pressure, SAP, and the left ventricular systolic pressure, LVSP, significantly by ~ 150% in both genotypes. Also the left ventricular end-diastolic pressure, LVEDP, HR, and the measures for contractility, maximal velocity of pressure increase, dP/dtmax, as well as maximal velocity of pressure decrease, dP/dtmin, were all up-regulated in response to TAC, however to the same degree in both genotypes. Abnormal ECG parameters were never recorded from XIRP WT mice neither in the sham nor in the TAC group. In contrast, ECG recordings from XIRP1XIRP2 dko sham mice exhibited biphasic P waves in 38.5% of the recordings. XIRP1XIRP2 dko TAC mice showed biphasic P waves in 18% of the evaluations, irregular HR, missing P waves and even ST elevations were detected. TAC induced a prolongation of P wave duration, PQ interval and QT interval in both genotypes. As the amount of prolongation was higher in the XIRP1XIRP2 dko TAC mice only these reached the level of significance. To find out whether there are genotype dependent phenomena which appear only in older age also 1 year-old mice of both genotypes underwent sham and TAC surgery. They were evaluated for the macroscopic morphological parameters and for the hemodynamics. The mice did not spontaneously develop any genotype dependent differences during aging. The cardiac growth gained within 14 days after TAC surgery was clearly smaller in the older mice than in the younger ones. Within this time span hypertrophic cardiac growth reached the level of significance only in the XIRP1XIRP2 dko mice, although both groups of mice responded to the excessive afterload by comparably increased levels of SAP, LVSP, and LVEDP. Taken together, only a few genotype-dependent differences could be detected. Regarding spontaneously appearing differences, a reduced amount of non-terminal ICDs were seen in fluorescence microscopy and a high rate of biphasic P waves were recorded in the XIRP1XIRP2 dko sham mice. As the reduced non-terminal ICDs were detected in ventricular cells and the biphasic P waves originate in the atrium these two observations could not be directly correlated. TAC dependent fibrosis was less distinct in XIRP1XIRP2 dko mice. This may possibly originate in a changed angiotensin II, AngII, signaling. As XIRP2 expression has been shown to be influenced by AngII, its absence in turn may disturb AngII signaling, which is known to enhance fibrosis. Prolongation of P wave duration, PQ interval, QRS complex, and QT interval may be taken signs for reduced conduction velocity in the XIRP1XIRP2 dko TAC mice. These mice also exhibit less non-terminal ICDs which could contribute to reduced conduction velocity. However, this hypothesis needs further substantiation by more refined ECG recordings

Soluble adenylyl cyclase: A novel player in cardiac hypertrophy induced by isoprenaline or pressure overload

<div><p>Aims</p><p>In contrast to the membrane bound adenylyl cyclases, the soluble adenylyl cyclase (sAC) is activated by bicarbonate and divalent ions including calcium. sAC is located in the cytosol, nuclei and mitochondria of several tissues including cardiac muscle. However, its role in cardiac pathology is poorly understood. Here we investigate whether sAC is involved in hypertrophic growth using two different model systems.</p><p>Methods and results</p><p>In isolated adult rat cardiomyocytes hypertrophy was induced by 24 h β₁-adrenoceptor stimulation using isoprenaline (ISO) and a β₂-adrenoceptor antagonist (ICI118,551). To monitor hypertrophy cell size along with RNA/DNA- and protein/DNA ratios as well as the expression level of α-skeletal actin were analyzed. sAC activity was suppressed either by treatment with its specific inhibitor KH7 or by knockdown. Both pharmacological inhibition and knockdown blunted hypertrophic growth and reduced expression levels of α-skeletal actin in ISO/ICI treated rat cardiomyocytes. To analyze the underlying cellular mechanism expression levels of phosphorylated CREB, B-Raf and Erk1/2 were examined by western blot. The results suggest the involvement of B-Raf, but not of Erk or CREB in the pro-hypertrophic action of sAC. In wild type and sAC knockout mice pressure overload was induced by transverse aortic constriction. Hemodynamics, heart weight and the expression level of the atrial natriuretic peptide were analyzed. In accordance, transverse aortic constriction failed to induce hypertrophy in sAC knockout mice. Mechanistic analysis revealed a potential role of Erk1/2 in TAC-induced hypertrophy.</p><p>Conclusion</p><p>Soluble adenylyl cyclase might be a new pivotal player in the cardiac hypertrophic response either to long-term β₁-adrenoceptor stimulation or to pressure overload.</p></div

Effect of pharmacological sAC inhibition on ISO/ICI-induced hypertrophy of isolated cardiomyocytes.

<p>(A–D) Cardiomyocytes were cultured for 24 h under basal conditions or with the addition of ISO/ICI and/or KH7 (12.5 μmol/L). In (A) the cross sectional areas of 118 cells are given (n = 5). In (B) the RNA (ng/μL)/DNA (ng/μL) ratio (n = 3) and in (C) the protein (μg/μL)/DNA (ng/μL) ratio are presented (n = 3). (D) Western blot analysis followed by optical band density analysis of α-skeletal actin (n = 5). Data were normalized to GAPDH band density and to the control (no treatment, 100%). (A-D) Data are expressed as means ± SEM. *(P < 0.05) or ***(P < 0.001) vs. ISO/ICI-treated cells. (E) Isolated rat cardiomyocytes were incubated with ISO/ICI and KH7 (0.0, 6.0, 9.0, 12.5, 15.0 μmol/L) for 24 h. The protein (μg/μL)/ DNA (ng/μL) ratio was determined (n = 4 cardiomyocyte preparations). Data are expressed as means ± SEM. 6.0 and 9.0 μmol/L KH7 vs 0.0 μmol/L KH7: P<0.05; 12.5 and 15 μmol/L KH7 vs 0.0mKH7: P<0.01. n refers to the number of cardiomyocyte preparations.</p

Effect of TAC on haemodynamic parameters in WT and sAC-KO mice.

<p>Analyses of (A) heart rate (HR), (B) systolic (SAP), (C) diastolic aortic pressure (DAP), (D) left ventricular systolic pressure (LVSP), (E) left ventricular end-diastolic pressure (LVEDP), rates of (F) contraction (+dp/dt) and (G) of relaxation (–dp/dt) were performed in anaesthetised WT or sAC KO mice 2 weeks after TAC or sham surgery. Data are expressed as means ± SEM, n = 9–12, ***(P< 0.001), *(P<0.05).</p

Hypertrophic parameters in sham and TAC operated WT and sAC KO mice.

<p>(A-C) Body weight, lung weight, heart weight and Tibia length (BW, LW, HW, TL, respectively) and their ratios (HW/BW;LW/BW and HW/TL) were analyzed 2 weeks after TAC or sham surgery in WT or sAC KO mice (n = 9–12). *(P< 0.05); **(P < 0.01). (D) Western blot analysis of ANP followed by optical band density analysis (n = 3 for sAC-WT and n = 5 for sAC-KO), ANP is normalized to tubulin. Equal experimental conditions as in A-C. Data are expressed as means ± SEM. **(P < 0.01) vs. TAC in wt mice. (E) Western blot analysis of pERK1/2 followed by optical band density analysis performed with cardiac tissue lysates from WT sham (n = 4), sAC WT TAC (n = 2), sAC KO sham (n = 4) and sAC KO TAC (n = 5) mice. Differences in total Erk1/2 band intensities are due to different total protein concentrations. Data are expressed as means ±SEM; **(P < 0.01) vs. TAC in WT mice.</p

CREB, B-Raf, Erk1/2 phosphorylation in adult rat cardiomyocytes.

<p>(A) Western blot analysis of phosphorylated CREB (pCREB,) from the lysates of untreated (-KH7) or KH7-treated (+KH7, 12.5 μmol/L) cardiomyocytes followed by optical band density analysis at different incubation times with ISO/ICI (n = 4). Data were normalized to the total proteins by gel staining as indicated in Methods; incubation times: 0, 15 min, 30 min, 1 h, 3 h, 6 h. Solid line indicates control, broken line—treatment with KH7. Western blot analysis of (B) phosphorylated B-Raf (pB-Raf; Ser445) and B-Raf (n = 3), of (C) phosphorylated Erk1/2 (pErk1, pErk2; TEY motif)) and Erk1/2 (n = 4) followed by the optical band density analysis (lower panel) are presented. Data were normalized to total protein and are expressed as means ± SEM. *(P<0.05), vs. ISO/ICI treated control cells. n refers to the number of cardiomyocyte preparations analyzed.</p

sAC knockdown in ISO/ICI treated isolated adult cardiomyocytes.

<p>(A) Representative western blots followed by the optical band analysis of sAC in lysates of cardiomyocytes after the following treatments: ISO/ICI (24h), adenoviral transfections either with scrambled shRNA (scRNA) or with sAC-targeted shRNA (shRNA). Data were normalized to actin band intensity. n = 5,*** (P<0.0001). (B-D) Statistical analyses of the cross sectional cell area of 110 cells from three cardiomyocyte preparations, the RNA/DNA ratio (n = 3) and the protein/DNA ratio (n = 3) are shown. *(P<0.05) and **(P<0.01),vs. ISO/ICI-treated cells expressing scRNA. <i>All</i> data are expressed as means ± SEM. n refers to the number of cardiomyocyte preparations analyzed.</p

Soluble adenylyl cyclase

$\bf Aims$ In contrast to the membrane bound adenylyl cyclases, the soluble adenylyl cyclase (sAC) is activated by bicarbonate and divalent ions including calcium. sAC is located in the cytosol, nuclei and mitochondria of several tissues including cardiac muscle. However, its role in cardiac pathology is poorly understood. Here we investigate whether sAC is involved in hypertrophic growth using two different model systems. $\textbf {Methods and results}$ In isolated adult rat cardiomyocytes hypertrophy was induced by 24 h $\beta_{1}$-adrenoceptor stimulation using isoprenaline (ISO) and a $\beta_{2}$-adrenoceptor antagonist (ICI118,551). To monitor hypertrophy cell size along with RNA/DNA- and protein/DNA ratios as well as the expression level of α-skeletal actin were analyzed. sAC activity was suppressed either by treatment with its specific inhibitor KH7 or by knockdown. Both pharmacological inhibition and knockdown blunted hypertrophic growth and reduced expression levels of α-skeletal actin in ISO/ICI treated rat cardiomyocytes. To analyze the underlying cellular mechanism expression levels of phosphorylated CREB, B-Raf and Erk1/2 were examined by western blot. The results suggest the involvement of B-Raf, but not of Erk or CREB in the pro-hypertrophic action of sAC. In wild type and sAC knockout mice pressure overload was induced by transverse aortic constriction. Hemodynamics, heart weight and the expression level of the atrial natriuretic peptide were analyzed. In accordance, transverse aortic constriction failed to induce hypertrophy in sAC knockout mice. Mechanistic analysis revealed a potential role of Erk1/2 in TAC-induced hypertrophy. $\bf Conclusion$ Soluble adenylyl cyclase might be a new pivotal player in the cardiac hypertrophic response either to long-term $\beta_{1}$-adrenoceptor stimulation or to pressure overload