GIRK3 deletion facilitates kappa opioid signaling in chondrocytes, delays vascularization and promotes bone lengthening in mice

“Activation of G protein-coupled receptor (GPCR) signaling pathways is crucial for skeletal development and long bone growth.  protein-gated inwardly-rectifying K⁺ (GIRK) channel genes are key functional components and effectors of GPCR signaling pathways in excitable cells of the heart and brain, but their roles in non-excitable cells that directly contribute to endochondral bone formation have not been studied. In this study, we analyzed skeletal phenotypes of Girk2^−/−, Girk3^−/− and Girk2/3^−/− mice. Bones from 12-week-old Girk2^−/− mice were normal in length, but femurs and tibiae from Girk3^−/− and Girk2/3^−/− mice were longer than age-matched controls at 12-weeks-old. Epiphyseal chondrocytes from 5-day-old Girk3^−/− mice expressed higher levels of genes involved in collagen chain trimerization and collagen fibril assembly, lower levels of genes encoding VEGF receptors, and produced larger micromasses than wildtype chondrocytes in vitro. Girk3^−/− chondrocytes were also more responsive to the kappa opioid receptor (KOR) ligand dynorphin, as evidenced by greater pCREB expression, greater cAMP and GAG production, and upregulation of Col2a1 and Sox9 transcripts. Imaging studies showed that Kdr (Vegfr2) and endomucin expression was dramatically reduced in bones from young Girk3^−/− mice, supporting a role for delayed vasculogenesis and extended postnatal endochondral bone growth. Together these data indicate that GIRK3 controls several processes involved in bone lengthening.”

“GIRK channels are homo- and hetero-tetramers formed by four mammalian GIRK subunits (GIRK1/Kcnj3, GIRK2/Kcnj6, GIRK3/Kcnj9, and GIRK4/Kcnj5). GIRK channels are activated when GPCR ligands stimulate pertussis toxin-sensitive G_i/o-G proteins; the liberated Gβγ subunit then binds to GIRK channels and increases their gating. The resultant efflux of K⁺ reduces the excitability of neuronsand cardiomyocytes. Knockout studies in mice have shown that GIRK2-containing GIRK channels mediate pain relief evoked by opioids and other analgesics. In chondrocytes, K⁺ efflux reduces swelling during unloading and can affect proteoglycan secretion”<-so maybe GIRK2 and GIRK3 knockouts have more chondrocyte hypertrophy and are more responsive to mechanical loading.

“GIRK2 mutations in patients with Keppen Lubinsky syndrome, which is characterized by growth above the 50th to 75th percentile at birth with subsequent developmental delays and other phenotypes “

“Girk3 deletion increases femur and tibia lengths and augments kappa opioid signaling in chondrocytes. Thus, these data identify GIRK3 as a suppressor of bone lengthening and kappa opioid activity in developing skeletons.”

“Numerous GPCR-signaling pathways, including kappa opioids, influence growth.”

Need to get the full paper and research more on GIRK3.

I’m still working on height increase.  Just doing more independent research and learning more about anatomy and physiology and actions of cells as the research I’m looking at is a lot of the same old, same old.  Maybe the key is to look at older studies before things were set in their ways.  It seems in the old days they were willing to try new risks to get people to grow taller.

Essentially the study found that heat did not increase bone length however there was some promise in that decalcification could be caused by the heat and that could enable longitudinal bone growth.  And the study shows islands of cartilage which could be the creation of new growth plates which is quite promising.  But the heat itself actually didn’t stimulate the growth plate itself.  It was only the heat degeneration the calcified bone matrix and stimulating the creation of growth plate islands that incurred new growth.

The effect of heat upon the growth of bone

“GROWTH in length of long bones consists of two mutually independent processes, the division and palisade arrangement of cartilage cells, and the subsequent calcification of the matrix between these cells and its replacement by bone. It is generally agreed that growth is dominated by the activities of the cells of the reserve zone and those in the adjacent apex of the cartilage columns. The subsequent enmeshment, of hypertrophic cartilage cells within a calcified matrix must prevent elongation at other sites. Recent attempts to stimulate the growth of bone have been based upon the production of an irritative lesion within the metaphysis, stimulating ossification rather than cartilage-cell division. ”

“Chapchal and Zeltienrust (1947-48) reported an inconstant increase in the rate OF growth in the rabbit after the insertion of metal or ivory pins within the metaphysis. Wilson (1952) using copper and constantan wire in the dog produced similar results. The application of these methods to limb inequality in children has been reported by Pease (1962). Metal or ivory screws WPI’P inserted transversely into the metaphysis of the femur and tibia. Each of the seven children subjected to this procedure showed an increase in the rate of growth of the shorter limb, but equalization of length was not attained. ”

“The overgrowth of bone which is constantly seen after fractures, in the presence of arteriovenous aneurysms, and in association with bone and joint infections”

arteriovenous aneurysm an abnormal communication between an artery and a vein in which the blood flows directly into a neighboring vein or is carried into the vein by a connecting sac.<-Lateral loading can increase blood flow.  Though this probably won’t work without existing growth plates.

“increase in the length of a limb following the production of an arteriovenous fistula in the dog”

“One of the clinical features of most cases of limb elongation with an increase in the temperature of the skin, and in the arteriovenous aneurysm this is associated with an increase in the temperature of bone”

“The rate of growth of the ulna was in some animals slightly depressed, in rabbit 480 markedly SO. In this animal there was considerable formation of new bone around tJie resistor, although the epiphyseal cartilage appeared normal. The overgrowth of the ulna noted in rabbit 465 was not associated with any abnormal histological appearance of the epiphyseal cartilage and the radiographs suggest that the discrepancy may have been due to a disturbance of growth in the control limb. The radiographs also show a relative decalcification of the the whole of the treated forelimb and this may have been associated with a generalized hyperemia{excess of blood in the vessels} of this limb.”

“Where the heating level was high, the epiphyseal cartilage is affected. The earliest changes in this region are an irregularity of the cartilage columns and a granularity of the matrix. When cellular destruction occurs it is at first confined to the region of the apex of the cartilage columns and is associated with fibrillation of the cartilage matrix. ”

“In only one animal (465) did an increase in bone length occur. This animal was heated for two days and, the wires having broken, the resistor was left unheated for a further 36 days. Although the increase in length of the limb was considerable, aid was associated with a generalized decalcification of the forelimb, suggesting an increase in the vascularity of this member, the difference in length of the limbs may have been due to some abnormality of growth on the control side. “<-So decalcification is key to growing taller.  High levels of heat can was found to cause necrosis which reduced growth but if decalcification can enhance growth.  Then maybe heat could enhance growth by decalcifying bones.

“heat has been shown to stimulate the production of cartilage around the resistor and to produce islands of endochondral ossification within the bony epiphysis and along the shaft of the radius.”<-islands of endochondral ossification is promising because it shows new induction of bone growth.

“Islands of cartilage were produced within the bony epiphysis close to the resistor, and along the ulnar border of the radial shaft. Some of them islands showed endochondral ossification, but there was no increase of cellular activity in the epiphyseal cartilage. A large cartilaginous mass developed in the region where the resistor was buried, and, in many animals, the transverse diameter of the metaphysis was greatly increased. ”

Figure 8 is an image near the resistor so there should be islands of cartilage visible.  I can’t tell If there are cartilage islands or not and if there are cartilage islands we can’t say for sure whether they are not just broken off cartilage from the growth plate.  Cartilage islands within the diaphysis would be key to seeing if heat can induce new growth plates.

Here’s mentioned overgrowth.

I’m still working on my own device.   I’m not sure if the device listed here is stimulatory enough to induce longitudinal bone growth.  The device may be useful if you have existing growth plates.  But to grow taller you’d need bone breakdown to occur faster than bone buildup{so that cartilage has room to grow} and the device is optimized for bone buildup.

Development of an Artificial Finger-Like Knee Loading Device to Promote Bone Health.

“This study presents the development of an innovative artificial finger-like device that provides position specific mechanical loads at the end of the long bone and induces mechanotransduction in bone{So you could theoretically use your own fingers to see what such an a device would do; use your own fingers to press on the epiphysis of bone}. Bone cells such as osteoblasts are the mechanosensitive cells that regulate bone remodeling{in order to induce height growth you’d need a lot more than bone remodeling, you need to degrade cortical bone and induce MSC differentiation into chondrogenic cells}. When they receive gentle, periodic mechanical loads, new bone formation is promoted{how this bone formation is promoted is of importance of whether such a method can induce height growth}. The proposed device is an under-actuated multi-fingered artificial hand with 4 fingers, each having two phalanges. These fingers are connected by mechanical linkages and operated by a worm gearing mechanism. With the help of 3D printing technology, a prototype device was built mostly using plastic materials. The experimental validation results show that the device is capable of generating necessary forces at the desired frequencies, which are suitable for the stimulation of bone cells and the promotion of bone formation. It is recommended that the device be tested in a clinical study for confirming its safety and efficacy with patients.”

Cortical bone is highly inhibitory towards longitudinal bone growth.  You need to generate sufficient fluid flow to induce degradation of the cortical bone or you likely will not be able to grow taller.  I think it would take a lot of fluid flow for that to happen.  Maybe with very high frequency and duration with this device it would be possible.

” if a small magnitude of mechanical stimuli is applied at a high frequency, an osteogenic response can be stimulated via mechanotransduction in bone cells.”<-we don’t care about stimulating bone cells EXCEPT for osteoclasts.  We need to simulate stem cells to differentiate into chondrocytes.

“Osteocytes are the most abundant type of cells in bone tissue, and they constitute more than 90% of the cells in bone matrix. They are rooted in the calcified bone medium, and communicate with each other and with bone-forming osteoblasts through slender processes and gap junctions. Osteocytes are highly mechanosensitive. Haversian system or osteon, one of the key components of a porous bone matrix, encloses a blood vessel in its center and sets up the canals known as Haversian canals or Volkmann’s canals. Osteogenesis is induced by the process of osteoinduction in which premature cells are recruited, stimulated and developed into pre-osteoblasts{we just need to induce a microenvironment where premature cells are induced into pre-chondrocytes instead and one way to do that is via a high hydrostatic pressure environment which could be induced by manipulating fluid flow}. Osteogenesis can also result from osteoconduction which is the passive process of bone growth on surfaces such as bone-implant surfaces”

“When rapid mechanical loading is applied at the end of long bone (e.g., knee), it is proposed that the interstitial fluid present around the osteocytes in the lacuna-canalicular network induces a pressure gradient and elevates nutrient transport throughout the porous network. “<-interstitial fluid flow could do more than this.  If you have a lot of fluid flow it could induce shear that degrades the cortical bone that prevents longitudinal bone growth.

Update:  I’m still hard at work for a methodology to grow taller.  It’s just mostly a lot of self experimentation.  Most scientific studies seem to be a lot of more of the same stuff like IGF2 is key.

Cartilage degeneration and excessive subchondral bone formation in spontaneous osteoarthritis involves altered TGF-β signaling.

The question is:  Can excessive subchondral bone formation make you taller?

Judging from this picture here yes it should.

“Transforming growth factor-β (TGF-β) has been demonstrated as a potential therapeutic target in osteoarthritis. However, beneficial effects of TGF-β supplement and inhibition have both been reported, suggesting characterization of the spatiotemporal distribution of TGF-β during the whole time course of osteoarthritis is important. To investigate the activity of TGF-β in osteoarthritis progression, we collected knee joints from Dunkin-Hartley (DH) guinea pigs at 3, 6, 9, and 12-month old (n = 8), which develop spontaneous osteoarthritis in a manner extraordinarily similar to humans. Via histology and micro-computed tomography (CT) analysis, we found that the joints exhibited gradual cartilage degeneration, subchondral plate sclerosis[a thickening of the subchondral bone where it begins to develop cysts, hardens, and thickens], and elevated bone remodeling during aging. The degenerating cartilage showed a progressive switch of the expression of phosphorylated Smad2/3 to Smad1/5/8, suggesting dual roles of TGF-β/Smad signaling during chondrocyte terminal differentiation in osteoarthritis progression. In subchondral bone, we found that the locations and age-related changes of osterix(+) osteoprogenitors were in parallel with active TGF-β, which implied the excessive osteogenesis may link to the activity of TGF-β. Our study, therefore, suggests an association of cartilage degeneration and excessive bone remodeling with altered TGF-β signaling in osteoarthritis progression of DH guinea pigs.”

“knee osteoarthritis is a disease of the entire joint, including synovitis, meniscal degeneration and malposition, periarticular bone overgrowth{periarticular bone is bone that surrounds the joint overgrowth of this bone should be good for height growth}, and articular cartilage destruction”

” In response to the altered mechanical environment, the bone–cartilage functional unit adjusts the architecture via cells’ adaptations. However, a discrepancy of repair capacity between the chondrocytes and other skeletal cells is thought to further accelerate the progression of osteoarthritis. Furthermore, it is widely appreciated that the subchondral plate and trabecular bone show different responses, where thickening of the plate can be identified along with osteopenic trabeculae at the advanced stage of osteoarthritis”

“ALK5/Smad2/3 route restrains terminal differentiation of chondrocytes, whereas the ALK1/Smad1/5/8 route induces the differentiation. The increase in ALK1/ALK5 ratio in chondrocytes may contribute to the cartilage degeneration.On the other hand, TGF‐β acts as a coupling factor to induce the migration of mesenchymal stem cells (MSCs) to bone resorption sites, implying its potential function in rebalancing bone resorption and formation.  Inhibition of TGF‐β signaling in subchondral bone resulted in higher bone quality and less cartilage degeneration in an induced‐osteoarthritis model”

If you look at figure 3 you can see increased subchondral bone height although it should be noted that these pigs were still growing.

“The tidemark advancement is a result of the pathological endochondral ossification at the calcified zone of cartilage”

“Smad2/3 signaling is essential to repress the hypertrophy of chondrocyte, whereas Smad1/5/8 route, namely bone morphogenetic protein (BMP) pathway, is required for chondrocyte terminal differentiation. Inhibition of the Smad1/5/8 signaling pathway led to reduced or delayed chondrocyte hypertrophy. Increase in ALK1/ALK5 ratio was associated with age and osteoarthritis and dominant ALK1/Smad1/5/8 pathway was found in advanced stage of induced osteoarthritis”

Here’s an interesting Jeffrey Baron paper who’s done a lot of growth research.  Essentially, smaller bones undergo senescence earlier so the chondrocytes undergo less proliferation cycles.

Differential aging of growth plate cartilage underlies differences in bone length and thus helps determine skeletal proportions.

“Bones at different anatomical locations vary dramatically in size. For example, human femurs are 20-fold longer than the phalanges in the fingers and toes. The mechanisms responsible for these size differences are poorly understood. Bone elongation occurs at the growth plates and advances rapidly in early life but then progressively slows due to a developmental program termed “growth plate senescence.”{estrogen may be responsible for this senescence} This developmental program includes declines in cell proliferation and hypertrophy, depletion of cells in all growth plate zones, and extensive underlying changes in the expression of growth-regulating genes. Here, we show evidence that these functional, structural, and molecular senescent changes occur earlier in the growth plates of smaller bones (metacarpals, phalanges) than in the growth plates of larger bones (femurs, tibias) and that this differential aging contributes to the disparities in bone length. We also show evidence that the molecular mechanisms that underlie the differential aging between different bones involve modulation of critical paracrine regulatory pathways, including insulin-like growth factor (Igf), bone morphogenetic protein (Bmp), and Wingless and Int-1 (Wnt) signaling. Taken together, the findings reveal that the striking disparities in the lengths of different bones, which characterize normal mammalian skeletal proportions, is achieved in part by modulating the progression of growth plate senescence.”

<-So if the change in body proportions is a result of senescence shouldn’t the body proportions be different in someone with different senescence such as someone with no estrogen receptors.  We know that body proportions are different in dwarfism.

“. The rate of long bone elongation (length/time) is primarily determined by the rate of chondrocyte proliferation (cells/time) per column multiplied by the cell height (length/cell) achieved after chondrocyte hypertrophy”

“During mammalian embryonic development, all long bones form from mesenchymal condensations of similar size. However, different long bones diverge in growth rate, ultimately leading to dramatic differences in bone length. ”

“the rates of bone elongation at the proximal tibia and the distal femur, measured by calcein labeling, were greater than those of the metacarpal bones and proximal phalanges. Some previous studies have attributed these differences in growth rate between bones to differences in the size attained by the hypertrophic chondrocytes of the growth plate. However, the rate of bone elongation is also dependent on chondrocyte proliferation and is approximated by the height of the terminal hypertrophic chondrocyte in the column multiplied by the chondrocyte proliferation rate per cell column ”

” a chondrocyte near the top of the growth plate in the larger bones would go through more rounds of cell division before slowing and ceasing proliferation compared with the smaller bones.”<-So how do we get chondrocytes to undergo more rounds of cell division?  There are number of factors.    A number of senescence related genes are mentioned in the study itself.  IGF2 is a key one.

“Growth plate senescence is characterized not only by a decline in proliferation rates but also by a gradual structural involution of the growth plate, including declines in the overall height of each growth plate zone and the number of chondrocytes in each zone.”

Note:  I’m still hard at work at finding a methodology of growing taller and am investigating non-LSJL method.

Finite-element analysis of the mouse proximal ulna in response to elbow loading.

“Bone is a mechano-sensitive tissue that alters its structure and properties in response to mechanical loading. We have previously shown that application of lateral dynamic loads to a synovial joint, such as the knee and elbow, suppresses degradation of cartilage{We also should investigate inducing endochondral ossification of the cartilage, perhaps Lateral Joint Loading could be involved in that} and prevents bone loss in arthritis and postmenopausal mouse models, respectively. While loading effects on pathophysiology have been reported, mechanical effects on the loaded joint are not fully understood. Because the direction of joint loading is non-axial, not commonly observed in daily activities{This is what’s so interesting about lateral joint loading, any height increase method has to be something that people do not do in ordinary activities as otherwise people would already have discovered that it makes you taller}, strain distributions in the laterally loaded joint are of great interest. Using elbow loading, we herein characterized mechanical responses in the loaded ulna focusing on the distribution of compressive strain. In response to 1-N peak-to-peak loads, which elevate bone mineral density and bone volume in the proximal ulna in vivo, we conducted finite-element analysis and evaluated strain magnitude in three loading conditions. The results revealed that strain of ~ 1000 μstrain (equivalent to 0.1% compression) or above was observed in the limited region near the loading site, indicating that the minimum effective strain for bone formation is smaller with elbow loading than axial loading{This is likely due to increases in fluid flow and/or hydrostatic pressure resulting in most of the adaptive response}. Calcein staining indicated that elbow loading increased bone formation in the regions predicted to undergo higher strain.”

“dynamic strain in bone matrix and strain-induced fluid flow in the lacuno-canalicular network are two of the major contributors to mechanotransduction in bone”

“Unlike axial loading, such as in ulna and tibia loading, joint loading employs lateral loads that sandwich a synovial joint, such as the knee, ankle, and elbow”<-I have not heard joint loading described this way before.  This description emphasizes the lateral compression force.

” we determined the strain distribution to the ulna in response to elbow loading using a mouse model and finite-element (FE) analysis. Daily loading with 1-N peak-topeak force at 1 Hz for 5 min was applied to ovariectomized (OVX){OVX mice simulates osteoperiosis} mice as well as sham-OVX mice (control mice){we are more interested in this group}, and bone mineral density (BMD) was measured at the site of loading after 4 weeks of loading. ”

” We employed three loading configurations: load and a single point of support, load and a three-point support, and load across soft tissues that may mimic surrounding skin and muscle”

” The lateral loads to the elbow were given 5 min per day for 5 weeks using 1-N force (peak-to-peak) at 5 Hz to the right arm, and left arm data were used as a contralateral control. ”

You can see the bone formation in the control group.  Definitely doesn’t indicate that it increases height but it indicates that it does something which is a start.  There seems to be an increase in bone width as well which is harder than just increasing the trabecular bone from within which is also very good although these mice are 12 week old female mice.  But the increase in bone width indicates the possibility of abnormal effects going on which would be needed to induce height growth.

” We employed three loading and boundary conditions in response to elbow loading with 1-N loads: lateral loads applied at two opposing locations; lateral loads applied at a single site on one side and three supporting sites on the other side; and lateral loads on a pair of soft disks that sandwiched the elbow.”<-it’ll be interesting to see which is best.  I think the soft disks would really diminish the load.

“Compression of a pair of soft-tissue disks induced multiple strain spots that were widely distributed from the proximal tip to the whole elbow joint ”

“Elbow loading generates artificial lateral forces that are rarely encountered during routine physical activity. Unlike axial or bending loads with ulna loading or tibia loading, the mechanical response of synovial joints, such as the elbow, to lateral loads has not been fully characterized”You can’t really see any abnormal bone formation here like you could in the earlier image and they look to be similar sites.  “Calcein-stained cross sections of the proximal ulna distal to the trochlear notch” whereas the earlier one is “c μCT images of the cross sections of the proximal ulna, ~ 1 mm distal to the edge of the trochlear notch”

” Compared to strain at the single loading site, three supporting sites significantly reduced
the maximum compressive strain by ~tenfold (from ~ 2% in the dark blue label to ~ 0.1% in the cyan level). “<-So maybe one loading site is best.  Multiple loading sites would counter the deformation and act as a sort of stabilizing force [cancel each other out].  “strains exceeding 0.1% (1000 μstrain) are confined to the vicinity of the loading site and supporting points”<-So maybe in someone that is growing we would we want to load directly near the growth plate or in an adult maybe near the articular cartilage.

Not very much height stuff but good to see it still being worked on.