Microtubule Actin Cross-linking Factor 1 regulates cardiomyocyte microtubule distribution and adaptation to hemodynamic overload

Abstract

Aberrant cardiomyocyte microtubule growth is a feature of pressure overload induced cardiac hypertrophy believed to contribute to left ventricular (LV) dysfunction. Microtubule Actin Cross-linking Factor 1 (MACF1/Acf7) is a 600 kd spectraplakin that stabilizes and guides microtubule growth along actin filaments. MACF1 is expressed in the heart, but its impact on cardiac microtubules, and how this influences cardiac structure, function, and adaptation to hemodynamic overload is unknown. Here we used inducible cardiac-specific MACF1 knockout mice (MACF1 KO) to determine the impact of MACF1 on cardiac microtubules and adaptation to pressure overload (transverse aortic constriction (TAC).In adult mouse hearts, MACF1 expression was low under basal conditions, but increased significantly in response to TAC. While MACF1 KO had no observable effect on heart size or function under basal conditions, MACF1 KO exacerbated TAC induced LV hypertrophy, LV dilation and contractile dysfunction. Interestingly, subcellular fractionation of ventricular lysates revealed that MACF1 KO altered microtubule distribution in response to TAC, so that more tubulin was associated with the cell membrane fraction. Moreover, TAC induced microtubule redistribution into this cell membrane fraction in both WT and MACF1 KO mice correlated strikingly with the level of contractile dysfunction (r(2) = 0.786, p<.001). MACF1 disruption also resulted in reduction of membrane caveolin 3 levels, and increased levels of membrane PKCα and β1 integrin after TAC, suggesting MACF1 function is important for spatial regulation of several physiologically relevant signaling proteins during hypertrophy. Together, these data identify for the first time, a role for MACF1 in cardiomyocyte microtubule distribution and in adaptation to hemodynamic overload.

Figure 1. MACF1 expression is increased in response to pressure overload.

RT-PCR was performed to amplify between exons 10 and 14 of MACF1. A 269 b.p. band corresponding to splicing between exon 10 and 14 (indicating loss of exons 11–13) is observed only in MACF1 KO mice, whereas WT mice express only the predicted size of 645 b.p. (Figure 1A) Western blot analysis of triton soluble (Figure 1B and 1C) and insoluble fractions (Figure 1B and 1D) of ventricular lysates of WT and MACF1 KO mice under control or after 3 weeks of TAC conditions (total of 7 weeks after tamoxifen treatment) demonstrates MACF1 expression increases in response to TAC, and is effectively silenced in MACF1 KO hearts (graphs represent averages of 3 WT or KO control mice, and 9 WT and KO TAC mice). Troponin I and vinculin were used as loading controls. (* indicates p<.05 as compared to control conditions, # indicates p<.05 as compared to WT under same conditions).

Figure 2. MACF1 deletion exacerbates pressure overload induced hypertrophy.

3 weeks after control or TAC conditions, WT or MACF1 KO hearts were collected and used to measure left ventricle(LV) weight to body weight ratio (Figure 2A), Left atria (LA) to body weight ratio (Figure 2B), Right ventricle (RV) weight to body weight ratio (Figure 2C) and right atria weight to body weight ratio (Figure 2D). Some hearts(n = 5 per condition) were used for measuring cardiomyocyte cross-sectional area (Figure 2E), or for Sirius red staining for fibrosis (Figure 2F) Graphs represent average tissue weights from 8 WT and 8 MACF1 KO mice under control conditions, and 19 WT and 12 MACF1 KO mice under TAC conditions. Cardiomyocyte cross sectional area was averaged from 5 mice (∼300 cells per heart) from each condition (* indicates p<.05 as compared to control conditions, # indicates p<.05 as compared to WT under same conditions).

Figure 3. MACF1 KO exacerbates pathological features of hypertrophy in response to TAC.

At 2 and 3 weeks after sham or TAC surgery, echocardiography was used to measure end systolic diameter (Figure 3A), end diastolic diameter (Figure 3B) percent ejection fraction (Figure 3C) and posterior wall thickness at end systole (Figure 3D) and end diastole (Figure 3E). After 3 weeks TAC, tissue was collected and lung weight to body weight ratio was used to indicate pulmonary congestion (Figure 3F) Expression of stress responsive proteins atrial natrurietic protein (ANP) (Figure 3G), Serca2A (Figure 3H) β-Myosin heavy chain (Figure 3I) were measured by western blot. α-MHC was used as a loading control (Figure 3J) (* indicates p<.05 as compared to control conditions, # indicates p<.05 as compared to WT under same conditions. For LV function measurements, WT control (n = 8), MACF1 KO control (n = 8), WT TAC (n = 16), KO TAC (n = 11)).

Figure 4. Maladaptive response to pressure overload is associated with redistribution of microtubules into distinct subcellular fractions.

LV lysates from WT and MACF1 KO hearts exposed to control or TAC conditions were separated into cytosolic fractions (Free), Microtubule fractions (MT), trition soluble membrane fractions (Membrane), and triton insoluble cytoskeletal fraction(CSK) Equal amounts of fractionated proteins from each sample in a treatment group were pooled and the four different fractions were examined side by side by western blot to measure relative changes in levels and subcellular distribution of MACF1, Tubulin, Desmin and β-Actin (Figure 4A–4G) (* indicates p<.05 as compared to control conditions, # indicates p<.05 as compared to WT under same conditions).

Figure 5. Maladaptive response to pressure overload is associated with redistribution of microtubules into distinct subcellular fractions.

The relationship between LV ejection fraction and the levels of Tubulin, as well as β-actin, and Desmin, in different subcellular fractions (Figure 5A) was examined by linear regression analysis (Figure 5B–5G). The data was collected from 18 WT mice and 6 MACF1 KO mice exposed to TAC. Relative values of the protein levels were determined as fold increase over non-banded controls from the same experiment. Samples with a red x are MACF1 KO.

Figure 6. MACF1 KO alters distribution of β1 integrin, caveolin 3, and PKCα content in the cell membrane fraction.

Western analysis of focal adhesion proteins β1 integrin, FAK and SRC, cell/cell adhesion proteins N-cadherin, and α-catenin, as well as caveolin-3 and PKCα content from pooled subcellular fractions of WT and MACF1 KO hearts (Figure 6A). Levels of each protein were also examined in individual samples within a fraction to determine if observed differences were statistically significant. β1 integrin (Figure 6B) and caveolin 3 (Figure 6C) were mostly localized to the cell membrane. PKCα protein was most abundant in the cytosol, but was significantly elevated in the membrane fraction of MACf1 KO mice exposed to TAC. Immunofluorescence analysis of PKCα (green) and caveolin 3 (red) in heart cross-sections (Figure 6D) revealed that caveolin 3 membrane distribution is disrupted in MACF1 KO hearts after TAC. Hoescht stain (blue) identifies nucleii (* indicates p<.05 as compared to control conditions, # indicates p<.05 as compared to WT under same conditions, n = 4 to 6 per condition; n = 4–6 per group.)

Figure 7. MACF1 KO increases membrane abundance of PKCα.

Western blot analysis of PKCα in subcellular fractions of ventricular lysates from WT and MACF1 KO hearts (Figure 7A–E). (* indicates p<.05 as compared to control conditions, # indicates p<.05 as compared to WT under same conditions, n = 4 to 6 per condition; n = 4–6 per group.)

Figure 8. MACF1 depletion alters microtubule organization in neonatal rat cardiomyocytes.

Neonatal rat ventricular cardiomyocytes(NRVMs) transfected with MACF1 RNAi or non-targeting control RNAi were immunostained for tubulin (red). Hoescht stain (blue) was used to identify nuclei.