Hendrikus L Granzier

Hendrikus L Granzier

Professor, Cellular and Molecular Medicine
Professor, Molecular and Cellular Biology
Professor, Biomedical Engineering
Professor, Genetics - GIDP
Professor, Physiological Sciences - GIDP
Professor, Physiology
Professor, BIO5 Institute
Primary Department
Department Affiliations
Contact
(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., Hudson, B., Hidalgo, C., Saripalli, C., & Granzier, H. L. (2011). Hyperphosphorylation of mouse cardiac titin contributes to transverse aortic constriction-induced diastolic dysfunction. Circulation research, 109(8).

Mechanisms underlying diastolic dysfunction need to be better understood.

Hutchinson, K. R., Saripalli, C., Chung, C. S., & Granzier, H. (2015). Increased myocardial stiffness due to cardiac titin isoform switching in a mouse model of volume overload limits eccentric remodeling. Journal of molecular and cellular cardiology, 79, 104-14.

We investigated the cellular and molecular mechanisms of diastolic dysfunction in pure volume overload induced by aortocaval fistula (ACF) surgery in the mouse. Four weeks of volume overload resulted in significant biventricular hypertrophy; protein expression analysis in left ventricular (LV) tissue showed a marked decrease in titin's N2BA/N2B ratio with no change in phosphorylation of titin's spring region. Titin-based passive tensions were significantly increased; a result of the decreased N2BA/N2B ratio. Conscious echocardiography in ACF mice revealed eccentric remodeling and pressure volume analysis revealed systolic dysfunction: reductions in ejection fraction (EF), +dP/dt, and the slope of the end-systolic pressure volume relationships (ESPVR). ACF mice also had diastolic dysfunction: increased LV end-diastolic pressure and reduced relaxation rates. Additionally, a decrease in the slope of the end diastolic pressure volume relationship (EDPVR) was found. However, correcting for altered geometry of the LV normalized the change in EDPVR and revealed, in line with our skinned muscle data, increased myocardial stiffness in vivo. ACF mice also had increased expression of the signaling proteins FHL-1, FHL-2, and CARP that bind to titin's spring region suggesting that titin stiffening is important to the volume overload phenotype. To test this we investigated the effect of volume overload in the RBM20 heterozygous (HET) mouse model, which exhibits reduced titin stiffness. It was found that LV hypertrophy was attenuated and that LV eccentricity was exacerbated. We propose that pure volume overload induces an increase in titin stiffness that is beneficial and limits eccentric remodeling.

Bogomolovas, J., Fleming, J. R., Anderson, B. R., Williams, R., Lange, S., Simon, B., Khan, M. M., Rudolf, R., Franke, B., Bullard, B., Rigden, D. J., Granzier, H., Labeit, S., & Mayans, O. (2016). Exploration of pathomechanisms triggered by a single-nucleotide polymorphism in titin's I-band: the cardiomyopathy-linked mutation T2580I. Open biology, 6(9).

Missense single-nucleotide polymorphisms (mSNPs) in titin are emerging as a main causative factor of heart failure. However, distinguishing between benign and disease-causing mSNPs is a substantial challenge. Here, we research the question of whether a single mSNP in a generic domain of titin can affect heart function as a whole and, if so, how. For this, we studied the mSNP T2850I, seemingly linked to arrhythmogenic right ventricular cardiomyopathy (ARVC). We used structural biology, computational simulations and transgenic muscle in vivo methods to track the effect of the mutation from the molecular to the organismal level. The data show that the T2850I exchange is compatible with the domain three-dimensional fold, but that it strongly destabilizes it. Further, it induces a change in the conformational dynamics of the titin chain that alters its reactivity, causing the formation of aberrant interactions in the sarcomere. Echocardiography of knock-in mice indicated a mild diastolic dysfunction arising from increased myocardial stiffness. In conclusion, our data provide evidence that single mSNPs in titin's I-band can alter overall muscle behaviour. Our suggested mechanisms of disease are the development of non-native sarcomeric interactions and titin instability leading to a reduced I-band compliance. However, understanding the T2850I-induced ARVC pathology mechanistically remains a complex problem and will require a deeper understanding of the sarcomeric context of the titin region affected.

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.