Nicholas M.P. King

Altered Titin Expression, Myocardial Stiffness, and Left Ventricular Function in Patients With Dilated Cardiomyopathy

Background—The role of the giant protein titin in patients with heart failure is not well established. We investigated titin expression in patients with end-stage heart failure resulting from nonischemic dilated cardiomyopathy, in particular as it relates to left ventricular (LV) myocardial stiffness and LV function. Methods and Results—SDS-agarose gels revealed small N2B (stiff) and large N2BA (compliant) cardiac titin isoforms with a mean N2BA:N2B expression ratio that was significantly (P <0.003) increased in 20 heart failure patients versus 6 controls. However, total titin was unchanged. The coexpression ratio was highest in a subsample of patients with an impaired LV relaxation pattern (n=7), intermediate in those with pseudonormal filling (n=6), and lowest in the group with restrictive filling (n=7). Mechanical measurements on LV muscle strips dissected from these hearts (n=8) revealed that passive muscle stiffness was significantly reduced in patients with a high N2BA:N2B expression ratio. Clinical correlations support the relevance of these changes for LV function (assessed by invasive hemodynamics and Doppler echocardiography). A positive correlation between the N2BA:N2B titin isoform ratio and deceleration time of mitral E velocity, A wave transit time, and end diastolic volume/pressure ratio was found. These changes affect exercise tolerance, as indicated by the positive correlation between the N2BA:N2B isoform ratio and peak O2 consumption (n=10). Upregulated N2BA expression was accompanied by increased expression levels of titin-binding proteins (cardiac ankyrin repeat protein, ankrd2, and diabetes ankyrin repeat protein) that bind to the N2A element of N2BA titin (studied in 13 patients). Conclusions—Total titin content was unchanged in end-stage failing hearts and the more compliant N2BA isoform comprised a greater percentage of titin in these hearts. Changes in titin isoform expression in heart failure patients with dilated cardiomyopathy significantly impact diastolic filling by lowering myocardial stiffness. Upregulation of titin-binding proteins indicates that the importance of altered titin expression might extend to cell signaling and regulation of gene expression.

Truncation of Titin’s Elastic PEVK Region Leads to Cardiomyopathy With Diastolic Dysfunction

Rationale: The giant protein titin plays key roles in myofilament assembly and determines the passive mechanical properties of the sarcomere. The cardiac titin molecule has 2 mayor elastic elements, the N2B and the PEVK region. Both have been suggested to determine the elastic properties of the heart with loss of function data only available for the N2B region. Objective: The purpose of this study was to investigate the contribution of titin’s proline–glutamate–valine–lysine (PEVK) region to biomechanics and growth of the heart. Methods and Results: We removed a portion of the PEVK segment (exons 219 to 225; 282 aa) that corresponds to the PEVK element of N2B titin, the main cardiac titin isoform. Adult homozygous PEVK knockout (KO) mice developed diastolic dysfunction, as determined by pressure-volume loops, echocardiography, isolated heart experiments, and muscle mechanics. Immunoelectron microscopy revealed increased strain of the N2B element, a spring region retained in the PEVK-KO. Interestingly, the PEVK-KO mice had hypertrophied hearts with an induction of the hypertrophy and fetal gene response that includes upregulation of FHL proteins. This contrasts the cardiac atrophy phenotype with decreased FHL2 levels that result from the deletion of the N2B element. Conclusions: Titin’s PEVK region contributes to the elastic properties of the cardiac ventricle. Our findings are consistent with a model in which strain of the N2B spring element and expression of FHL proteins trigger cardiac hypertrophy. These novel findings provide a molecular basis for the future differential therapy of isolated diastolic dysfunction versus more complex cardiomyopathies.

Mouse intact cardiac myocyte mechanics: cross-bridge and titin-based stress in unactivated cells.

A carbon fiber–based cell attachment and force measurement system was used to measure the diastolic stress–sarcomere length (SL) relation of mouse intact cardiomyocytes, before and after the addition of actomyosin inhibitors (2,3-butanedione monoxime [BDM] or blebbistatin). Stress was measured during the diastolic interval of twitching myocytes that were stretched at 100% base length/second. Diastolic stress increased close to linear from 0 at SL 1.85 µm to 4.2 mN/mm2 at SL 2.1 µm. The actomyosin inhibitors BDM and blebbistatin significantly lowered diastolic stress by ∼1.5 mN/mm2 (at SL 2.1 µm, ∼30% of total), suggesting that during diastole actomyosin interaction is not fully switched off. To test this further, calcium sensitivity of skinned myocytes was studied under conditions that simulate diastole: 37°C, presence of Dextran T500 to compress the myofilament lattice to the physiological level, and [Ca2+] from below to above 100 nM. Mean active stress was significantly increased at [Ca2+] > 55 nM (pCa 7.25) and was ∼0.7 mN/mm2 at 100 nM [Ca2+] (pCa 7.0) and ∼1.3 mN/mm2 at 175 nM Ca2+ (pCa 6.75). Inhibiting active stress in intact cells attached to carbon fibers at their resting SL and stretching the cells while first measuring restoring stress (pushing outward) and then passive stress (pulling inward) made it possible to determine the passive cell’s mechanical slack SL as ∼1.95 µm and the restoring stiffness and passive stiffness of the cells around the slack SL each as ∼17 mN/mm2/µm/SL. Comparison between the results of intact and skinned cells shows that titin is the main contributor to restoring stress and passive stress of intact cells, but that under physiological conditions, calcium sensitivity is sufficiently high for actomyosin interaction to contribute to diastolic stress. These findings are relevant for understanding diastolic function and for future studies of diastolic heart failure.

Muscle Interstitial Calcium During Head-Up Tilt in Humans

Background—During head-up tilt (HUT), peripheral vasoconstriction occurs. This response requires appropriate communication between the sympathetic nerve terminal and vascular smooth muscle cell in the neurovascular space. Both of these cell types require extracellular calcium ([Ca2+]o) for proper activation and function. We hypothesize that [Ca2+]o rises with tilt and in the process contributes to vasoconstriction. Methods and Results—We used microdialysis techniques in the lower-limb skeletal muscle to measure [Ca2+]o changes in this space with HUT. [Ca2+]o was measured in 10 healthy subjects during HUT. We found a 62% increase in the dialysate [Ca2+] (0.223±0.018 to 0.353±0.028 mmol/L) with HUT. Conclusions—This result implies a significant increase in [Ca2+]o in the neurovascular space during HUT. This represents the first report of such in situ [Ca2+]o measurements in humans. This rise in [Ca2+]o may provide a mechanism for proper cell-cell interaction, helping to promote peripheral vasoconstriction during HUT. How this [Ca2+]o transient affects the nerve terminal, vascular smooth muscle cells, or both remains to be determined.

Mouse Intact and Skinned Cardiac Myocyte Mechanics: Crossbridge and Titin-Based Stress in Unactivated Cells

We investigated the contribution of actomyosin interactions in unactivated intact and skinned cardiomyocytes in physiologic conditions.A carbon fiber based cell-attachment system was used to measure the diastolic stress-sarcomere length (SL) relation of murine intact cardiomyocytes, before and after the addition of actomyosin inhibitors (BDM or blebbistatin). Stress was measured during the diastolic interval of twitching myocytes that were stretched at 100% length/s. Diastolic stress increased nearly linearly from 0 at SL 1.85 µm to 4.2 mN/mm2 at SL 2.1 µm. Actomyosin inhibitors lowered diastolic stress by ∼1.5 mN/mm2 at SL 2.1 µm (∼30% of total), suggesting that during diastole actomyosin interaction is not fully switched off. Stretch-hold-release studies on skinned cardiomyocytes showed that as temperature changed from 24°C to 37°C, there was shortening of slack SL (from 1.90±0.01 μm to 1.89±0.01 μm) and increasing of both peak stress (∼35%) and steady state stress (∼26%). Shortening of slack SL and increasing stress could be inhibited by blebbistatin. This suggests that at physiologic temperature, crossbridge cycling takes place which contributes to diastolic stress. To extend this further, calcium sensitivity of skinned cardiomyocytes was studied under conditions that simulate physiologic diastole: 37°C, presence of Dextran T500 to compress the myofilament lattice to the physiological level, and [Ca2+] from below to above 100 nM. Mean active stress was increased at [Ca2+] >55 nM (pCa 7.25) and was ∼0.7 mN/mm2 at 100 nM [Ca2+] (pCa 7.0) and ∼1.3 mN/mm2 at 175 nM [Ca2+] (pCa 6.75). The presence of active stress at pCa 7, which is a physiologic Ca2+ concentration of cytoplasm during diastole, confirms the contribution of crossbridge cycling to diastolic stress. These findings are relevant for understanding diastolic function and for future studies of diastolic heart failure.