Hendrikus L Granzier
Professor, BIO5 Institute
Professor, Biomedical Engineering
Professor, Cellular and Molecular Medicine
Professor, Genetics - GIDP
Professor, Molecular and Cellular Biology
Professor, Physiological Sciences - GIDP
Professor, Physiology
Primary Department
Department Affiliations
(520) 626-3641
Work Summary
Our research is focused on elucidating the structure and function of titin and nebulin, two large filamentous proteins found in muscle. We use a range of model systems with a major focus on KO and TG mouse models. The techniques that we use range from single molecule mechanics, (immuno) electron microscopy, exon microarray analysis, in vitro motility assays, low angle X-ray diffraction, cell physiology (including calcium imaging), muscle mechanics, and isolated heart physiology.
Research Interest
Hendrikus Granzier, PhD, studies the mechanisms whereby the giant filamentous protein titin (the largest protein known) influence muscle structure and function. His lab has shown that titin functions as a molecular spring that mediates acute responses to changing pathophysiological states of the heart. They also study the role of titin in cardiac disease, using mouse models with specific modifications in the titin gene, including deciphering the mechanisms that are responsible for gender differences in diastolic dysfunction. An additional focus of Dr. Granzier’s lab is on nebulin, a major muscle protein that causes a severe skeletal muscle disease in humans. Based on previous work, they hypothesize that nebulin is a determinant of calcium sensitivity of contractile force. To test this and other concepts, he uses a nebulin knockout approach in the mouse. Research is multi-faceted and uses cutting-edge techniques at levels ranging across the single molecule, single cell, muscle, and the intact heart. His research group is diverse and has brought together individuals from several continents with expertise ranging from physics and chemistry to cell biology and physiology.

Publications

Granzier, H., Chung, C. S., & Granzier, H. L. (2011). Contribution of titin and extracellular matrix to passive pressure and measurement of sarcomere length in the mouse left ventricle. Journal of molecular and cellular cardiology, 50(4).

It remains to be established to what degree titin and the extracellular matrix (ECM) contribute to passive pressure in the left ventricle (LV). Thus, we aimed to elucidate the contribution of major molecular determinants of passive pressure in the normal mouse LV. Furthermore, we determined the working sarcomere length (SL) range of the LV to bridge our findings to earlier work in skinned muscle fibers. We utilized Frank-Starling type protocols to obtain diastolic pressure-volume relationships (PVR) in Langendorff perfused isolated LVs. To quantify the molecular contribution of titin and ECM, we innovated on methods of fiber mechanics to chemically permeabilize intact LVs and measure a fully passive PVR. To differentially dissect the contributions of the ECM and titin, we utilized myofilament extraction techniques in permeabilized LVs, measuring passive PVRs at each stage in the protocol. Myofilament extraction suggests that titin contributes ~80% of passive pressures in the heart. Langendorff perfusion was also used to chemically fix passive and BaCl(2) activated hearts at specific volumes to determine that the maximal working SL range of the midwall LV fibers is approximately 1.8-2.2 μm. A model of the passive SL-volume relationship was then used to estimate the pressure-SL relationships, indicating that the ECM contribution does not exceed titin's contribution until large volumes with SLs >~2.2 μm. In conclusion, within physiological volumes, titin is the dominant contributor to LV passive pressure, and ECM-based pressures dominate at larger volumes.

Begay, R. L., Graw, S., Sinagra, G., Merlo, M., Slavov, D., Gowan, K., Jones, K. L., Barbati, G., Spezzacatene, A., Brun, F., Di Lenarda, A., Smith, J. E., Granzier, H. L., Mestroni, L., Taylor, M., & , F. C. (2015). Role of Titin Missense Variants in Dilated Cardiomyopathy. Journal of the American Heart Association, 4(11).

The titin gene (TTN) encodes the largest human protein, which plays a central role in sarcomere organization and passive myocyte stiffness. TTN truncating mutations cause dilated cardiomyopathy (DCM); however, the role of TTN missense variants in DCM has been difficult to elucidate because of the presence of background TTN variation.

Buck, D., Smith, J. E., Chung, C. S., Ono, Y., Sorimachi, H., Labeit, S., & Granzier, H. L. (2014). Removal of immunoglobulin-like domains from titin's spring segment alters titin splicing in mouse skeletal muscle and causes myopathy. The Journal of general physiology, 143(2), 215-30.

Titin is a molecular spring that determines the passive stiffness of muscle cells. Changes in titin's stiffness occur in various myopathies, but whether these are a cause or an effect of the disease is unknown. We studied a novel mouse model in which titin's stiffness was slightly increased by deleting nine immunoglobulin (Ig)-like domains from titin's constitutively expressed proximal tandem Ig segment (IG KO). KO mice displayed mild kyphosis, a phenotype commonly associated with skeletal muscle myopathy. Slow muscles were atrophic with alterations in myosin isoform expression; functional studies in soleus muscle revealed a reduced specific twitch force. Exon expression analysis showed that KO mice underwent additional changes in titin splicing to yield smaller than expected titin isoforms that were much stiffer than expected. Additionally, splicing occurred in the PEVK region of titin, a finding confirmed at the protein level. The titin-binding protein Ankrd1 was highly increased in the IG KO, but this did not play a role in generating small titin isoforms because titin expression was unaltered in IG KO mice crossed with Ankrd1-deficient mice. In contrast, the splicing factor RBM20 (RNA-binding motif 20) was also significantly increased in IG KO mice, and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Thus, increasing titin's stiffness triggers pathological changes in skeletal muscle, with an important role played by RBM20.

Granzier, H., Lee, E., Peng, J., Radke, M., Gotthardt, M., & Granzier, H. L. (2010). Calcium sensitivity and the Frank-Starling mechanism of the heart are increased in titin N2B region-deficient mice. Journal of molecular and cellular cardiology, 49(3).

Previous work suggests that titin-based passive tension is a factor in the Frank-Starling mechanism of the heart, by increasing length-dependent activation (LDA) through an increase in calcium sensitivity at long sarcomere length. We tested this hypothesis in a mouse model (N2B KO model) in which titin-based passive tension is elevated as a result of the excision of the N2B element, one of cardiac titin's spring elements. LDA was assessed by measuring the active tension-pCa (-log[Ca(2+)]) relationship at sarcomere length (SLs) of 1.95, 2.10, and 2.30 microm in WT and N2B KO skinned myocardium. LDA was positively correlated with titin-based passive tension due to an increase in calcium sensitivity at the longer SLs in the KO. For example, at pCa 6.0, the KO:WT tension ratio was 1.28+/-0.07 and 1.42+/-0.04 at SLs of 2.1 and 2.3 microm, respectively. There was no difference in protein expression or total phosphorylation of sarcomeric proteins. We also measured the calcium sensitivity after PKA treating the skinned muscle and found that titin-based passive tension was also now correlated with LDA, with a slope that was significantly increased compared to no PKA treatment. Finally, we performed isolated heart experiments and measured the Frank-Starling relation (slope of developed wall stress-LV volume relation) as well as diastolic stiffness (slope of diastolic wall stress-volume relation). The FSM was more pronounced in the N2B KO hearts and the slope of the FSM correlated with diastolic stiffness. These findings support that titin-based passive tension triggers an increase in calcium sensitivity at long sarcomere length, thereby playing an important role in the Frank-Starling mechanism of the heart.

Methawasin, M., Hutchinson, K. R., Lee, E., Smith, J. E., Saripalli, C., Hidalgo, C. G., Ottenheijm, C. A., & Granzier, H. (2014). Experimentally increasing titin compliance in a novel mouse model attenuates the Frank-Starling mechanism but has a beneficial effect on diastole. Circulation, 129(19), 1924-36.

Experimentally upregulating compliant titins has been suggested as a therapeutic for lowering pathological diastolic stiffness levels. However, how increasing titin compliance impacts global cardiac function requires in-depth study. We investigate the effect of upregulating compliant titins in a novel mouse model with a genetically altered titin splicing factor; integrative approaches were used from intact cardiomyocyte mechanics to pressure-volume analysis and Doppler echocardiography.