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

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.

Evans, J. M., Cox, M. L., Huska, J., Li, F., Gaitero, L., Guo, L. T., Casal, M. L., Granzier, H. L., Shelton, G. D., & Clark, L. A. (2016). Exome sequencing reveals a nebulin nonsense mutation in a dog model of nemaline myopathy. Mammalian genome : official journal of the International Mammalian Genome Society, 27(9-10), 495-502.

Nemaline myopathy (NM) is a congenital muscle disorder associated with muscle weakness, hypotonia, and rod bodies in the skeletal muscle fibers. Mutations in 10 genes have been implicated in human NM, but spontaneous cases in dogs have not been genetically characterized. We identified a novel recessive myopathy in a family of line-bred American bulldogs (ABDs); rod bodies in muscle biopsies established this as NM. Using SNP profiles from the nuclear family, we evaluated inheritance patterns at candidate loci and prioritized TNNT1 and NEB for further investigation. Whole exome sequencing of the dam, two affected littermates, and an unaffected littermate revealed a nonsense mutation in NEB (g.52734272 C>A, S8042X). Whole tissue gel electrophoresis and western blots confirmed a lack of full-length NEB in affected tissues, suggesting nonsense-mediated decay. The pathogenic variant was absent from 120 dogs of 24 other breeds and 100 unrelated ABDs, suggesting that it occurred recently and may be private to the family. This study presents the first molecularly characterized large animal model of NM, which could provide new opportunities for therapeutic approaches.

Granzier, H., Chung, C. S., Hutchinson, K. R., Methawasin, M., Saripalli, C., Smith, J. E., Hidalgo, C. G., Luo, X., Labeit, S., Guo, C., & Granzier, H. L. (2013). Shortening of the elastic tandem immunoglobulin segment of titin leads to diastolic dysfunction. Circulation, 128(1).

Diastolic dysfunction is a poorly understood but clinically pervasive syndrome that is characterized by increased diastolic stiffness. Titin is the main determinant of cellular passive stiffness. However, the physiological role that the tandem immunoglobulin (Ig) segment of titin plays in stiffness generation and whether shortening this segment is sufficient to cause diastolic dysfunction need to be established.

Granzier, H., Hidalgo, C. G., Chung, C. S., Saripalli, C., Methawasin, M., Hutchinson, K. R., Tsaprailis, G., Labeit, S., Mattiazzi, A., & Granzier, H. L. (2013). The multifunctional Ca(2+)/calmodulin-dependent protein kinase II delta (CaMKIIδ) phosphorylates cardiac titin's spring elements. Journal of molecular and cellular cardiology, 54.

Titin-based passive stiffness is post-translationally regulated by several kinases that phosphorylate specific spring elements located within titin's elastic I-band region. Whether titin is phosphorylated by calcium/calmodulin dependent protein kinase II (CaMKII), an important regulator of cardiac function and disease, has not been addressed. The aim of this work was to determine whether CaMKIIδ, the predominant CaMKII isoform in the heart, phosphorylates titin, and to use phosphorylation assays and mass spectrometry to study which of titin's spring elements might be targeted by CaMKIIδ. It was found that CaMKIIδ phosphorylates titin in mouse LV skinned fibers, that the CaMKIIδ sites can be dephosphorylated by protein phosphatase 1 (PP1), and that under baseline conditions, in both intact isolated hearts and skinned myocardium, about half of the CaMKIIδ sites are phosphorylated. Mass spectrometry revealed that both the N2B and PEVK segments are targeted by CaMKIIδ at several conserved serine residues. Whether phosphorylation of titin by CaMKIIδ occurs in vivo, was tested in several conditions using back phosphorylation assays and phospho-specific antibodies to CaMKIIδ sites. Reperfusion following global ischemia increased the phosphorylation level of CaMKIIδ sites on titin and this effect was abolished by the CaMKII inhibitor KN-93. No changes in the phosphorylation level of the PEVK element were found suggesting that the increased phosphorylation level of titin in IR (ischemia reperfusion) might be due to phosphorylation of the N2B element. The findings of these studies show for the first time that titin can be phosphoryalated by CaMKIIδ, both in vitro and in vivo, and that titin's molecular spring region that determines diastolic stiffness is a target of CaMKIIδ.

Pappas, C. T., Mayfield, R. M., Henderson, C., Jamilpour, N., Cover, C., Hernandez, Z., Hutchinson, K. R., Chu, M., Nam, K., Valdez, J. M., Wong, P. K., Granzier, H. L., & Gregorio, C. C. (2015). Knockout of Lmod2 results in shorter thin filaments followed by dilated cardiomyopathy and juvenile lethality. Proceedings of the National Academy of Sciences of the United States of America, 112(44), 13573-8.

Leiomodin 2 (Lmod2) is an actin-binding protein that has been implicated in the regulation of striated muscle thin filament assembly; its physiological function has yet to be studied. We found that knockout of Lmod2 in mice results in abnormally short thin filaments in the heart. We also discovered that Lmod2 functions to elongate thin filaments by promoting actin assembly and dynamics at thin filament pointed ends. Lmod2-KO mice die as juveniles with hearts displaying contractile dysfunction and ventricular chamber enlargement consistent with dilated cardiomyopathy. Lmod2-null cardiomyocytes produce less contractile force than wild type when plated on micropillar arrays. Introduction of GFP-Lmod2 via adeno-associated viral transduction elongates thin filaments and rescues structural and functional defects observed in Lmod2-KO mice, extending their lifespan to adulthood. Thus, to our knowledge, Lmod2 is the first identified mammalian protein that functions to elongate actin filaments in the heart; it is essential for cardiac thin filaments to reach a mature length and is required for efficient contractile force and proper heart function during development.