Microtubules orchestrate local translation to enable cardiac growth - PubMed (original) (raw)
Microtubules orchestrate local translation to enable cardiac growth
Emily A Scarborough et al. Nat Commun. 2021.
Abstract
Hypertension, exercise, and pregnancy are common triggers of cardiac remodeling, which occurs primarily through the hypertrophy of individual cardiomyocytes. During hypertrophy, stress-induced signal transduction increases cardiomyocyte transcription and translation, which promotes the addition of new contractile units through poorly understood mechanisms. The cardiomyocyte microtubule network is also implicated in hypertrophy, but via an unknown role. Here, we show that microtubules are indispensable for cardiac growth via spatiotemporal control of the translational machinery. We find that the microtubule motor Kinesin-1 distributes mRNAs and ribosomes along microtubule tracks to discrete domains within the cardiomyocyte. Upon hypertrophic stimulation, microtubules redistribute mRNAs and new protein synthesis to sites of growth at the cell periphery. If the microtubule network is disrupted, mRNAs and ribosomes collapse around the nucleus, which results in mislocalized protein synthesis, the rapid degradation of new proteins, and a failure of growth, despite normally increased translation rates. Together, these data indicate that mRNAs and ribosomes are actively transported to specific sites to facilitate local translation and assembly of contractile units, and suggest that properly localized translation - and not simply translation rate - is a critical determinant of cardiac hypertrophy. In this work, we find that microtubule based-transport is essential to couple augmented transcription and translation to productive cardiomyocyte growth during cardiac stress.
Conflict of interest statement
The authors declare no competing interests.
Figures
Fig. 1. Microtubule depolymerization uncouples increased protein translation from cardiac growth.
a Experimental design for PE-induced hypertrophy mouse model. b Representative western blot images of free α-tubulin and loading control GAPDH (top left) and quantification of normalized free tubulin from each group (bottom left). Representative western blot images of polymerized α-tubulin and loading control Histone H3 (top right) and quantification of normalized polymerized tubulin from each group (bottom right). c Quantification of cardiac hypertrophy. d Quantification of Nppa expression. e Representative western blots for puromycinylated proteins (left) and total protein (center). Quantification of translation rate (right). For all panels, statistical significance determined via one-way ANOVA with post hoc Bonferroni comparisons. For all panels, PBS (N = 6), Colch (N = 8), PE + PBS (N = 8), and PE + Colch (N = 10). For all graphs shown in figure, the mean line is shown, with whiskers denoting standard error (SE) from the mean. Source data are provided as a Source data file.
Fig. 2. Microtubule depolymerization leads to mislocalized translation.
a Experimental design of NRVM isolation, patterning, and experiments. b Representative live-cell (left) or immunofluorescence (right) images of NRVMs treated with DMSO, PE, Colch, or PE + Colch. c Quantification of cell areas in NRVMs treated with (left) DMSO (n = 147), PE (n = 148), Colch (n = 138), or PE + Colch (n = 143) and (right) in NRVMs treated with DMSO (n = 145), PE (n = 148), Nocod (n = 150), or PE + Nocod (n = 151). Statistical significance determined via one-way ANOVA with post hoc Bonferroni comparison, each treatment repeated in at least 3 independent NRVM litters. d Measurement of translational activity in NRVMs. Representative western blots labeled for puromycinylated proteins (left) and total protein (center). (right) Quantification of translation rate, normalized to DMSO. One-sample, two-tailed _t_-test vs. mean = 1, unadjusted _p_-value reported. e Representative fluorescence images of NRVMs treated with DMSO, PE, Colch, or PE + Colch. Yellow arrows indicate nuclei. f Quantification of HPG intensity. DMSO (n = 137 NRVMs), PE (n = 126), Colch (n = 132), or PE + Colch (n = 140), each data point represents pooled single-cell values from a field of view, statistical significance determined via one-way ANOVA with post hoc Bonferroni comparison. g Averaged line scans of HPG intensity. DMSO (n = 30 NRVMs), PE (n = 30), Colch (n = 30), or PE + Colch (n = 30). For all graphs shown in figure, the mean line is shown, with whiskers denoting standard error (SE) from the mean. Source data are provided as a Source data file.
Fig. 3. Microtubule-dependent peripheralization of mRNA transcripts is concomitant with cardiac hypertrophy.
a Representative images of NRVMs treated with DMSO, PE, Colch, and PE + Colch and hybridized for Actc1 mRNA. b Quantitative line scan analysis of images from a. After normalization to the initial value, mean fluorescence intensities were binned every 5 µm from the center of the nucleus outward for each cell. DMSO (n = 96), PE (n = 85), Colch (n = 82), and PE + Colch (n = 103). Significance of PE group from either DMSO (*), PE + Colch (#) or initial PE measurement at distance = 0 (+) indicated by number of symbols, i.e., #p < 0.05, ##p < 0.01, and ###p < 0.001. Corresponding data points were omitted in overlay due to high n value and high variability, causing difficulty in visualization. c Representative images of NRVMs treated with DMSO, PE, Colch, and PE + Colch and hybridized for Dsp mRNA. Numbered insets show zoomed-in regions. d Quantification of c. DMSO (n = 82), PE (n = 88), Colch (n = 76), and PE + Colch (n = 87). Significance of PE group from either DMSO (*), PE + Colch (#) indicated by number of symbols, i.e., ##p < 0.01 and *** or ###p < 0.001. e (left) Experimental design for measurement of protein degradation rate using pulse HPG labeling. (middle) Representative western blots of HPG-labeled protein over time. (right) Quantification of relative remaining HPG over time. For all data in figure, statistical significance determined via two-way ANOVA with post hoc Bonferroni comparison, and data are from four independent NRVM litters. For all graphs shown in figure, whiskers denote standard error (SE) from the mean. Source data are provided as a Source data file.
Fig. 4. Microtubules control the localization of mRNA, ribosomes, and new protein synthesis.
a Representative images of (left) newly synthesized protein, (middle) ribosomes and (right) mRNA in (top) DMSO or (bottom) Colch-treated ARVMs. White arrow indicates ICD. b Nuclear:cytosolic and ICD:cytosolic ratios of mean fluorescence intensities of experiments shown in a. Newly synthesized protein: DMSO (N = 3, n = 52), Colch (N = 3, n = 55); ribosomes: DMSO (N = 2, n = 54), Colch (N = 2, n = 57; mRNA: DMSO (N = 3, n = 82), Colch (N = 3, n = 90). Statistical significance determined via two sample, two-tailed _t_-test. c Cartoon (left, top) depicting representative placement of perinuclear, cytosolic, and ICD ROIs for analysis in b and c. Mean polyA fluorescence intensity in the cytosol (left, middle), ICD (left, bottom), and nucleus (right) as a function of time. DMSO, n = 47 (1 h), n = 51 (3 h), n = 54 (6 h), n = 40 (24 h). Colch, n = 49 (1 h), n = 47 (3 h), n = 50 (6 h), n = 47 (24 h). Statistical significance determined via one-way ANOVA with post hoc Bonferroni comparison. d Representative images of (left) newly synthesized protein and (right) mRNA in (top) DMSO or (bottom) Latrunculin A-treated ARVMs. White arrow indicates ICD. e Nuclear:cytosolic and ICD:cytosolic ratios of mean fluorescence intensities of experiments shown in d. Newly synthesized protein: DMSO (N = 2, n = 46), Blebb (N = 2, n = 49), Y27632 (N = 2, n = 48), DMSO (N = 3, n = 82), LatA (N = 3, n = 80); mRNA: DMSO (N = 3, n = 70), Blebb (N = 3, n = 63), Y27632 (N = 3, n = 74), and DMSO (N = 3, n = 77), LatA (N = 3, n = 59). Statistical significance determined via one-way ANOVA with post hoc Bonferroni comparision (DMSO, Blebb, Y27632) and two sample, two-tailed _t_-test (DMSO, LatA). For all graphs shown in figure, the mean line is shown, with whiskers denoting standard error (SE) from the mean. Source data are provided as a Source data file.
Fig. 5. Microtubules control the localization of ribosomes in vivo.
a Representative image of PBS-injected rat ventricular myocardium, with zoomed-in image below and white arrow highlighting ICD region. b Representative image of Colch-injected rat ventricular myocardium with similar zoom and highlight as in a. c (left) Zoomed-in image of PBS-injected rat ventricular myocardium. (right) Line scan analysis of the composite image shown at bottom left. d Nuclear:cytosolic and ICD:cytosolic ratios of 18S rRNA mean fluorescence intensities of experiment depicted in a and b. PBS (N = 2), (n = 71, N/C), (n = 137, ICD/C), Colch (N = 2), (n = 92, N/C), (n = 108, ICD/C). Statistical significance determined via two sample, two-tailed _t_-test. For all graphs shown in figure, the mean line is shown, with whiskers denoting standard error (SE) from the mean. Source data are provided as a Source data file.
Fig. 6. mRNA localization and cardiac hypertrophy depends on the microtubule motor Kinesin-1.
a (top) Representative western blot images of kinesin-1 and loading control GADPH in ARVMs. (bottom) Quantification of relative kinesin-1 expression, normalized to GAPDH. N = 3. b Representative immunofluorescence images of the microtubule network (left) and polyA mRNA (right) in Scramble and Kif5b KD cells. White arrow indicates ICD. c Nuclear:cytosolic and ICD:cytosolic ratios of polyA mean fluorescence (left) and 18s rRNA mean fluorescence (right) in Scramble and Kif5b KD cells. Scramble (N = 2, n = 62, polyA) (N = 2, n = 88, 18 s), Kif5b KD (N = 2, n = 61, polyA) (n = 2 , N = 76, 18 s). Statistical significance determined via two sample, two-tailed _t_-test. d (top) Representative western blot images of kinesin-1 and loading control GADPH in NRVMs. (bottom) Quantification of relative kinesin-1 expression, normalized to GAPDH. N = 3. e Representative live-cell images of (top row) Scramble or (bottom row) Kif5b KD NRVMS, treated with (left column) vehicle or (right column) PE. f Quantification of cell areas in NRVMs from experiment shown in e. Scramble+vehicle (N = 3, n = 160), Scramble+PE (N = 3, n = 160), Kif5b KD + vehicle (N = 3, n = 160), Kif5b KD + PE (N = 3, n = 160). For all graphs shown in figure, the mean line is shown, with whiskers denoting standard error (SE) from the mean. Source data are provided as a Source data file.
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