Talin Is Required Continuously for Cardiomyocyte Remodeling during Heart Growth in <i>Drosophila</i>

<div><p>Mechanotransduction of tension can govern the remodeling of cardiomyocytes during growth or cardiomyopathy. Tension is signaled through the integrin adhesion complexes found at muscle insertions and costameres but the relative importance of signalling during cardiomyocyte growth versus remodelling has not been assessed. Employing the <i>Drosophila</i> cardiomyocyte as a genetically amenable model, we depleted the levels of Talin, a central component of the integrin adhesion complex, at different stages of heart growth and remodeling. We demonstrate a continuous requirement for Talin during heart growth to maintain the one-to-one apposition of myofibril ends between cardiomyocytes. Retracted myofibrils cannot regenerate appositions to adjacent cells after restoration of normal Talin expression, and the resulting deficit reduces heart contraction and lifespan. Reduction of Talin during heart remodeling after hatching or during metamorphosis results in pervasive degeneration of cell contacts, myofibril length and number, for which restored Talin expression is insufficient for regeneration. Resultant dilated cardiomyopathy results in a fibrillating heart with poor rhythmicity. Cardiomyocytes have poor capacity to regenerate deficits in myofibril orientation and insertion, despite an ongoing capacity to remodel integrin based adhesions.</p></div

Ultrastructure of Talin depleted larval hearts reveal dilation of the lumen.

<p>The wildtype LIII dorsal aorta (segment A4) was completely encompassed with myofibrils (A, B). Arrows mark Z lines of myofibrils. Extensions of a valve cell line the left and ventral lumen (asterisk, A). After continuous Talin depletion in UAS-dsTalinRNA; Hand-GAL4, the aorta lumen was dilated (C). The aorta wall had zones with myofibril (white arrowhead, C and E) and without (black arrowhead, C and D). Cell processes covered the perimeter of the aorta, and Z-lines are evident (arrowhead, D). Heart cardiomyocytes (cm) retract completely in a severely affected LIII (Segment A6) heart (asterisk, F), exposing naked fibrils of the heart ECM (arrowheads, F, G) The heart in (F) is flanked by fat cells (f). Scale 10 μm (A,C,F) and 1μm (B,D,E,G).</p

Cardiomyocytes do not re-connect after transient reduction of Talin function.

<p>In a wildtype adult, 6 days after eclosion, phalloidin labeled contractile fibres encompassed the entire heart, and integrin concentrated at cell insertions and the midline (arrowhead, A). This morphology was not altered after a further 6 weeks of aging (arrowhead, F). Talin was transiently depleted during larval, pupal or adult stages as shown by the coloured schema at the bottom of each panel, and stages labeled in (A). <i>UAS-talin RNAi; Hand-Gal4</i>, <i>tub- Gal80</i>^<i>ts</i> flies were at 18°C during grey timeline, and Talin levels were normal., Hearts express the RNAi transgene, and deplete Talin levels at 29°C during the green timeline. Depletion during L1 created large gaps in myofibril coverage seen in the adult (B). Depletion during L2 created small gaps, filled with a broadened zone of integrin adhesion to the heart ECM (C). Depletion during pupal stages only generated an intermediate phenotype (D), comparable to depletion during L2, L3 and pupal stages combined (E). Depletion of Talin for 6 weeks of adulthood triggered small changes in the width of integrin labeled insertions (G). In contrast, gaps triggered in L2 persisted in 6 week old adults (H). Confocal projections of immunolabled adult heart dissections.</p

Talin depletion in cardiomyocytes resulted in retraction of the muscle actin network.

<p>The third instar larval hearts lack longitudinal fibres, revealing the transverse fibres (phalloidin, red) and integrin rich cell insertions at the midline. Confocal projections of abdominal segments 5 and 6 of dissected, immunolabled control (Hand-GAL4/+) hearts are shown (arrowhead, β-integrin, green, in A). The contractile heart is flanked by integrin rich pericardial cells (pc). When imaging single myofibrils, Integrin antibody also labeled myocyte Z-lines (arrow in inset, A). When dsTalinRNA expression was generated in UAS-<i>dsTalinRNAi (Hm0516); Hand-Gal4</i> and examined in third instar larvae, partial myofibril retraction was observed (B). Integrin was concentrated at the cell periphery, beyond the limit of myofibril projection (arrow, B). Talin distribution in wildtype is similar to β-integrin (C), but after chronic depletion in the heart, levels are low in pericardial cells (pc) and cardiomyocytes (cm) but still abundant in hemocytes (h) (D). Zasp, a component of the costamere, aligns to heart and body wall muscle Z-lines, and in hemocytes (E, and inset). This pattern is maintained in myofibrils subsequent to Talin depletion (F). The larval heart ECM contains a latticework of Pericardin rich fibrils (G). After Talin depletion, this latticework becomes more dense in the pericardium (H). The myocytes of the adult control (Hand-Gal4/+) heart are comprised of longitudinal fibres (arrow) overlying the transverse fibres of the cardiomyocytes (I, phalloidin labeling). Relative to the embryonic heart at hatching (inset I, labeled with Hand-GFP), the adult heart has grown 4.7 times in length. dsTalinRNA expression was generated in UAS-<i>dsTalinRNA; HandGAL4 (VDRC 40399)</i> larvae, generating a more severe phenotype, resulting in adult escapers with a collapsed myofibril network, where actin bundles surround each myocyte nucleus (arrowhead, J). Posterior is at right. Scale: 50 μm in A-H; 120 μm in I,and J.</p

Talin reduction impairs cardiac rhythmicity but not heart rate.

<p>Cross-sectional area of the larval heart chamber (abdominal segment 6) was significantly dilated at diastole if Talin expression was suppressed during the first instar, but not other stages of larval growth (A, p<10⁻⁵, Mann-Whitney U test). The degree of contraction (ratio of diastolic to systolic cross-sectional area) was significantly reduced by depletion of Talin at any larval stage (B, L1- p<10⁻⁵; L2- p = 0.01; L2,3- p<10⁻⁵). The Rhythmicity Index (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131238#sec002" target="_blank">Methods</a>) was significantly reduced by depletion during L1 (p<0.005) and L2,3 (p<0.02). There were no significant differences in heart rate relative to control larvae for any treatment. OCT records of heart contraction were analysed for 6–20 <i>UAS-TalinRNAi/+; Hand-Gal4</i>, <i>tubGal80</i>^<i>ts</i><i>/+</i> larvae for each temperature treatment and compared to 11 <i>UAS-talinRNAi/+</i> raised at 29°C. Period of RNAi expression on abscissa: first instar (L1), second instar (L2) and both second and third instar (L2,3). SEM indicated.</p

Lifespan is reduced by transient Talin reduction.

<p>Longevity of adults, subsequent to transient depletion of Talin during larval growth was tracked for 150 <i>UAS-Talin/+</i> or <i>UAS-talin RNAi; Hand-Gal4</i>, <i>tub- Gal80</i>^<i>ts</i> adults for each treatment. Lifespan was dramatically shorter after Talin depletion in either L2 or in L2,3, and raised at 18°C (A), as well as raised with Talin depletion from L3 onwards or from pupation onwards at 29°C (B).</p