Introduction

Heart failure (HF) is a leading cause of death in the US and worldwide, 1 and is generally characterized as an inability of the left ventricle to pump enough blood to meet the oxygen demands of body tissues. However, impaired function of the right ventricle (RV) contributes to the mortality and morbidity of multiple cardiovascular diseases, including pulmonary hypertension (PH), left v entricular failur e with pr eserv ed ejection fraction, and congenital heart disease. Unfortunately, there is still a lack of targeted treatment options for patients with RV dysfunction due in part to the poor understanding of its pathophysiology. The passi v e mechanical pr operties of RV tissue ar e known to contribute significantly to its physiology. For example, Jang et al. found a significant correlation between RV biomechanical stiffness and hemodynamic measurement of diastolic function. 2 Additionally, our own ovine study demonstrated marked correlations between RV tissue elasticity and geometrical/hemodynamical indices. 3 While these findings support the impact of RV elastic behavior on performance, the RV is a viscoelastic organ. This implies that both elastic and viscous r esistances ar e pr esent during deformation, but their impact on RV function and hemodynamics is incompletely understood.

Ther e is accum ulating evidence of viscoelasticity in both the left and right ventricles of neonatal porcine 4 and adult human, [5][6][7] ovine, 8,9 and rodent [10][11][12][13] m y ocardium. The RV free wall (RVFW) is a complex, mesh-like tissue comprised of m y ofibers, colla gen, v asculatur e , and extr acellular matrix (ECM) components, all of which contribute to the viscoelastic and anisotropic behavior of the tissue. Our group previously reported significant reductions of viscosity and elasticity in healthy and pr essur e-ov erloaded RV tissues after acute de pol ymerization of the microtubule (MT) network, a cytoskeletal structure in cardiom y ocytes. 10,11 In these ex vivo studies, the decrease in viscosity w as gr eater than that of elasticity and less directiondependent, indicating distinct molecular mechanisms of these mechanical properties. Moreover, the reduction in viscoelasticity w as str onger in the pr essur e-ov erloaded than healthy tissue, pr oba b l y due to the denser MT network associated with RV remodeling. 14 While these findings suggest a significant contribution of tissue viscoelasticity to RV mechanical behavior, onl y passi v e tissue mec hanics w er e inv estigated and the r ole of m y ocardial viscoelasticity in RV function in vivo remains unclear.

Recently, Caporizzo et al. found that MT depolymerization via colchicine (COL) treatment reduces cardiom y ocyte viscoelasticity and enhances the shortening velocity of the cells in healthy and failing LV, 5,6 demonstrating an impact of the cardiom y ocyte mec hanics on contr actile function. Additionally, in the LV, Hancock et al. and Pietsch et al. have investigated the contribution of MT polymerization and tyrosination on cardiac function. It was found that acute COL tr eatment incr eased the m y ocardial w ork of the healthy LV via enhanced force development and filling performance, 15 Mor eov er, in a mouse model of hypertrophic cardiom y opathy, Pietsc h et al. found that c hronic detyrosination of the MT network increased stroke volume and impr ov ed LV function during disease pr ogr ession, 16 indicating a therapeutic effect of COL on diseased L V. Similarly , in a rodent model of pulmonary arterial hypertension, Prins et al. administer ed chr onic tr eatment of COL (3-wk) and found significant impr ov ements in pulmonary vascular disease and metrics of RV function, such as tricuspid annular planar systolic excursion and cardiac output, 14 It was also found that MT structure became less organized in the pr essur e-ov erloaded RV, and COL treatment induced an increase in junctophilin-2 expression and reduction in m y ocyte diameter, thus indicating the direct impact of COL on MT organization and m y ocardial h ypertroph y, leading to further impr ov ement of RV function. While these preclinical studies point to the role of the MT network in ventricular performance, the impr ov ement is a combined outcome of chronic biological remodeling and mechanical changes in pulmonary and cardiac tissues, and thus the mechanical benefit of COL via reduction of m y ocardial viscoelasticity remain unclear.

Ther efor e, the aim of this study is to investigate the response of RV performance to acute COL treatment in vivo in healthy and diseased states. We measured the RV pressure-volume relationship in vivo in PH and healthy rats before and after intramyocardial injections of COL. Acute COL treatment has been demonstrated to reduce tissue viscoelasticity in our prior ex vivo studies, 10 , 11 and a similar dosage was applied to the RV in vivo in this study. We ther efor e expect minimal off-target effects of MT de pol ymerization in the RV due to the brief period. Our results demonstrate that acute COL treatment, which reduces m y ocardial viscoelasticity, impr ov es contractile and diastolic function of the diseased RV but has minimal effect on the healthy RV. These findings deepen our understanding of the role that m y ocardial tissue mec hanics pla y in the pathoph ysiology of RV dysfunction, and the impr ov ement in tissue viscoelasticity may be a potential therapeutic target in RV failure patients.

Methods

Animal Model

All animal pr ocedur es wer e appr ov ed by the Colorado State University Institutional Animal Care and Use Committees (IACUC). Six-w eek-old male Spr ague-Da wle y (Charles Ri v er) rats ( n = 7) wer e administer ed a single subcutaneous injection (60 mg/kg) of monocrotaline (MCT) and housed in normal conditions for 3 wk to induce PH and RV failure. We followed the identical disease model protocol as described previously, in which we observed overt PH. 12 Healthy, age-matched rats ( n = 10) served as control (CTL).

In Vivo Assessment of Right Ventricular Hemodynamics

Animals were anesthetized via an intraperitoneal injection of urethane (1.2-1.5 g/mL) and then were intubated. The chest was opened to expose the heart, and the RV was catheterized through the m y ocardium using a 1.9F admittance catheter (Transonic/Scisense). After obtaining the sta b le pr essur e-v olume (PV) r elationships (base), thr ee intr am y ocardial injections of COL (0.3 m m , in a total bolus of 0.25 mL 10 , 11 ) wer e administer ed along the long axis of the right v entricle. PV r elationships wer e r ecorded and assessed within two minutes of COL treatment or a similar administration of saline as a vehicle control. The changes in PV relationships w ere tr ansient due to the fast washout of the COL bolus.

Standar d hemod ynamic v aria b les including RV end-diastolic and end-systolic pr essur es (EDP and ESP) and volumes (EDV and ESV) and deri v ed metrics of inotropic and lusitropic function (dP/dt max and min, r especti v el y) wer e measur ed using La bChart (AD Instruments, v ersion 8). These measur ements ena b led calculations of str oke v olume (SV; EDV-ESV), ejection fraction (EF; [EDV/SV] × 100), cardiac output (CO; SV × heart r ate), stroke w ork (SW, area within the PV loop), RV chamber compliance (defined as V/ P), total pulmonary vascular resistance (tPVR, estimated as mPAP/CO 17 , 18 ), and effecti v e arterial elastance (E a ; ESP/SV). Additionally, the RV end-diastolic and end-systolic pr essur e v olume r elationships (EDPVR and ESPVR) wer e deri v ed fr om PV loop data using the single beat method 19 , 20 ). Finally, the RV-pulmonary vascular interaction was e valuated b y ventricular-vascular coupling, determined by the ratio of end-systolic elastance (E es ; slope of the ESPVR) to E a . 20

Sta tistical Anal ysis

We performed unpaired student t -tests to compare the healthy and diseased intact RV performance, and paired student t -tests to compare the performance of the RV before and after acute COL treatment in each group. Statistical analysis was performed using GraphPad Prism, version 10. Results are presented as mean ± SEM.

Results

Impaired RV Function With PH Progression

We first examined the PV relationship of the healthy and pr essur e-ov erloaded RV. As expected, PH development led to pathologic concentric RV h ypertroph y, indicated b y marked r eductions in EDV ( Figur e 1 A) and ESV ( Figur e 1 B). This corr esponded to a significant reduction in RV stroke volume ( Figure 1 C) but not ejection fraction ( Figure 1 D), which is similar to diastolic RV heart failure with preserved ejection fraction (HFpEF). MCT treatment did not alter EDP ( Figure 1 E) but increased EDPVR ( F igure 1 F), whic h implied markedl y r educed RV compliance ( Figure 1 G). RV lusitropic function (dP/dt min) was -2105 mmHg/s in MCT compared to -1040mmg/s in CTL animals ( Figure 1 H), implicating a role of structural remodeling in the RV in diastolic dysfunction. 21 RV ESP ( Figure 1 22 , 23 Taken together, these results demonstrate marked RV dysfunction r esulting fr om pathologic RV hypertr ophy and w all stiffening in response to chronic pressure overload, consistent with our prior studies. 12 , 11 F igure 2. Health y (A) and pr essur e-ov erloaded (B) RV pr essur e-v olume r elation befor e (base) and after MT de pol ymerization (COL).

MT Depol ymeriza tion Re verses Concentric Remodeling and Dysfunction in the Pressure-Overloaded RV

Ne xt, we e xamined the acute effect of intr am y ocardial COL injections on the RV pr essur e-v olume r elationship in contr ol ( Figure 2 A) and MCT groups ( Figure 2 B). From the representati v e PV loops, there was an overall right-ward shift of the PV loops in both groups after COL treatment, and the loop width became larger in the MCT group. We further compared the metrics deri v ed fr om the PV loops befor e and after COL tr eatment. In the MCT group, COL treatment markedly increased EDV ( Figure 3 A) and ESV ( Figur e 3 B), with onl y minor insignificant effects on contr ols. Impr ov ements in EDV wer e m uch gr eater than ESV following COL treatment, thus leading to a significant increase in SV ( Figure 3 C) and EF ( Figure 3 D). These parameters were unaffected by COL in contr ol RVs. While EDP w as unaffected by COL treatment in both groups ( Figure 3 baseline vs. 260 ± 29 bpm after COL, P = 0.2), whereas in the MCT group, a small but significant decrease in HR was noted (358 ± 17 bpm at baseline vs. 347 ± 17 bpm after COL, P < 0.001). Taken together, these results indicate that reducing RV viscoelasticity by MT de pol ymerization r ev erses pathologic RV dysfunction, restoring RV hemodynamics to near control values. Importantly, no marked changes wer e observ ed in the CTL group, indicating that the viscoelastic reduction is compensated by some homeostasis mechanism of the healthy RV at the organ level. Lastly, we confirmed negligible effects on the PV relationships following the administration of saline (vehicle) to healthy RVs ( Figure 4 ), ruling out impacts of m y ocardial fluid injections on the observed outcomes.

We further examined other hemodynamic parameters before and after COL treatment in the healthy and pressure-overloaded RVs. In the MCT group, an estimate of PVR ( Figure 5 A) and pulmonary arterial elastance (E a ; F igure 5 B) w ere significantly r educed by COL tr eatment, indicating some effect on the pulmonar y v asculatur e. The concomitant r eduction in E es ( Figur e 3 J) and E a resulted in a trend of improvement in RV-arterial coupling indicated by a higher E es /E a ( Figure 5 C).

Discussion

To our knowledge, this is the first study to investigate the in vivo response of RV performance to acute COL treatment in healthy and diseased states. Previous work by our group has demonstrated significant impacts of acute COL treatment on ex vivo anisotropic viscoelasticity of healthy and pr essur e-ov erloaded RV fr ee w all, 10 , 11 which complements evidence for viscoelastic reduction via COL treatment at the same dosage. As PHassociated RV remodeling leads to increases in RV viscoelasticity, 12 it is r easona b le to postulate a role of RV viscoelasticity in the RV function. The present study extends to r ev eal implications of RV viscoelasticity by demonstrating that acute COL treatment in vivo restores the RV mechanics and performance, improving both diastolic and systolic function and ventricularvascular coupling. COL also reduced RV afterload and heart rate in PH animals only, which may contribute to impr ov ed R V performance in addition to the mec hanical alter ation of the RV w all. These r esults adv ance our understanding of how the passi v e mechanical pr operties of the RV contribute to the pathophysiology and dysfunction in PH development, thus identifying a novel therapeutic target of RV biomechanics in RV failure patients.

In Vivo COL Treatment Improves Pressure-Overloaded RV Function

We first observed the establishment of RV concentric hypertrophy and diastolic dysfunction secondary to PH induced by MCT tr eatment. Str oke v olume and str oke w ork w er e both r educed along with elevated end-systolic pressure ( Figure 1 ), which are the hallmarks of RV dysfunction in PH. 3 , 12 , 24 More specifically, these changes along with pr eserv ed ejection fraction may suggest phenotypic similarities with heart failure with preserved ejection fraction (HFpEF).

COL tr eatment significantl y impr ov ed RV SV , SW , and EF in the MCT group, suggesting restoring of systolic and overall performance of the organ. We speculate that these largely resulted from the dramatic increase in RV preload (filling pressure or volume) and decrease in RV stiffness (EDPVR, RV compliance), as well as reductions in RV afterload (PVR and E a ). Pietsch et al. r ecentl y r e ported similar effects on the LV following detyrosination of the MT network in a mouse model of dilated cardiomyopath y. 16 The y concluded that the acute r emov al of the MT netw ork (ie , ventricular mec hanical impr ov ement in our study) impr ov ed LV function by normalizing LV h ypertroph y and reducing ventricular stiffness (EDPVR). 16 Another chronic treatment of COL in RVs of PH rats showed therapeutic effects, including the impr ov ement in systolic function (TAPSE, cardiac output) and normalization of RV h ypertroph y. 14 While these studies demonstrated organ level improvements after COL treatment, the chr onic tr eatment inv olv es long-term tissue r emodeling and thus the effect of viscoelastic (mechanical) reduction via COL is unclear. On the other hand, the effect of acute COL treatment on cardiom y ocytes or m y ocardial tissue slices has been r e ported, 5,6,15 but the investigations were limited to the LV and whether the beneficial outcome can be seen in PH-induced RV failur e r emains unclear. Thus, our present work pro vides no vel data on the response of RV to acute viscoelastic reduction via COL treatment in healthy and failing RVs.

The enhanced systolic function in the pr essur e-ov erloaded RV by COL treatment may be partly explained by the F r ank Starling mechanism. The length-dependent activation (LDA) phenomenon at the cell level forms the basis of the F r ank Starling mechanism, which is applied to the tissue level. However, LDA refers to the relationship between the force during cell contraction and isometric cell length, 21 whereas the F r ank Starling mechanism refers to the relationship between preload and str oke v olume. Since the r emov al of MT w eakened the mec hanical resistance of the ventricle wall, the filling of the ventricle during diastole was improved, as e vident b y increased EDV. Thus, SV w as incr eased with a larger filling v olume of the RV due to a retention of the intrinsic systolic function described by the F r ank Starling mechanism.

In addition to changes in ventricular performance, we also observed changes in vascular function of the diseased animals after MT de pol ymerization. Specificall y, both pulmonar y v ascular r esistance (PVR) and effecti v e arterial elastance (E a ) wer e significantl y r educed ( Figur e 5 ). We suspect that the flushout of the COL bolus may have relaxed smooth muscle cells in the downstream pulmonary vasculature. Ochoa et al. has pr eviousl y found that the de pol ymerization of microtubules in pulmonary endothelia greatly affected endothelial shapeelongated shape to a round shape-which will confer altered v ascular tone. 25 Similarl y, Prins et al. observ ed a r eduction in total pulmonary resistance after a 3-wk treatment of COL. 14 COL and further observed enhanced v entricular-v ascular coupling after the treatment. Of course, the weaker afterload supports our finding of reduced ESP and dP/dt max, as there is a lesser demand of the ventricle. This coupled behavior was exacerbated, too, after MT r emov al, as we observed a strong trend of increase in the ventricular-vascular coupling (VVC) parameter ( Figure 5 C).

Prins et al. attributed this response to the antimitotic effects of

The decreases in RV contractility indicated by lower ESP, ESPVR and dP/dt max in MCT RVs following COL treatment likely r eflect r eductions in RV afterload (eg, PVR). We pr eviousl y did not expect a large effect of COL on non-cardiac tissues as only a total of 0.25 mL was delivered to the animal. But, in a human study, it was observed that acute treatment of COL led to a significantl y gr eater v asodilator r esponse. 26 Thus, it is possible that the pulmonary arteries were exposed to COL after the washout of the drug from the ventricular wall and responded quickly. In addition to the impact from PVR, we speculate that the reduction in RV ESP could result in part from a reduced viscoelastic resistance of the RV tissue. It is accepted that a more compliant ventricular wall would require less energy of the ventricle to eject the same volume of blood, thus leading to a lowered ESP. 27 As both elastic and viscous r esistance ar e decr eased after COL treatment, 10 , 11 the RV does not need high systolic pressures to maintain the cardiac output and the ESP is reduced.

Other Effects of Acute COL Treatment

We observed a small but significant reduction in HR in the diseased RVs after COL treatment. In the healthy rats, the HR r eduction w as insignificant thought. Various outcomes of COL on heart rate have been r e ported pr eviousl y. Earl y pr eclinical studies in rabbit car diomyoc ytes hav e r e ported incr eased heart rate or ventricular fibrillation with COL treatment in a dosedependent manner. [28][29][30] But the acute treatment of COL has also led to brad yd ysrhythmias. 31 Our result seems to support the direct effect of COL on accelerating electric firing of the heart, although it remains unclear why diseased and healthy RVs responded differently.

In addition, there is growing evidence of MTs' contribution to the functions of the nervous system, 32 including providing structural support for axons and dendrites, serving as longdistance rail w ays for pr oteins and organelles to be transported within axons and dendrites, and playing a role in the central nervous system by regulating cell migration 33 . The COL treatment may lead to de pol ymerization of the MT network in the neurons, thus impacting nervous system function. Reported neurotoxic effects of COL occur at much higher (20 ×) doses than the prescribed dose in this study and, thus, we can rule out neurotoxicity of COL in this study. 34,35 While MTs have not been linked to the behavior of the autonomic nervous system (ANS), their impact on axonal and dendritic function sur el y could affect the ANS (both para-and sympathetic) and lead to changes in HR indir ectl y. This hypothesis, though, awaits further investigation.

In Vivo COL Treatment Impacts the Diseased, Not Health y R V

Our results show that the COL treatment in vivo affects only the pr essur e-ov erloaded RV, and not the healthy RV. At the micr ostructural lev el, prior r esear c h has shown an increase in MT network density in PH RVs, 14 as well as upregulated expression of the two subunits of the MT, αand β-tubulin. [36][37][38] The str onger r esponse of the pr essur e-ov erloaded RV than the health y RV ma y be explained b y tw o reasons. F irst, it may be due to higher content of the MT in the diseased tissue. Previous ex vivo mechanical studies have shown larger degree of mec hanical c hanges in diseased RVs after MT r emov al compared to the healthy RV. 11 Additionally, diseased RV tissue and LV m y ocardium exhibit higher viscoelasticity compared to healthy tissue, 5,11,12,39 so we would expect a greater effect of COL in these tissues. But in isolated healthy cardiom y ocytes or m y ocardial slices, the COL treatment is shown to improve contractile function, 5 , 6 , 15 which is absent in our study. This leads us to propose another explanation: at the organ level, the ventricular function is affected by some viscoelastic homeostasis mechanism, and the COL treatment did not change the homeostasis in healthy RVs.

Considering only the mechanical behavior of the m y ocardium, prior ex vivo studies, including our own, have shown that the de pol ymerization of the MT network via COL significantl y r educes the viscosity and elasticity of healthy m y ocardium, in both the LV and RV. 5,6,10,15,39 These studies, though, primarily focused on the individual changes of viscosity and elasticity, but not the r elati v e change of the two parameters. We pr eviousl y found that the ratio of viscosity to elasticity (V/E ratio) was unchanged in the healthy RV after COL treatment, suggesting a similar degree of reduction in the two mechanical properties. 11 However, in the diseased RV, the V/E ratio w as significantl y r educed after COL tr eatment, suggesting that it may not be the individual elasticity or viscosity, but the V/E ratio, that is linked to the organ function. Thus, there may be varied contributions of the viscous and elastic properties of the tissue, and it is the combined effect of them that is dictating the ov erall r ole of mechanics in ventricular function. Mor eov er, as previous cell studies have shown, reduced viscosity and elasticity led to significant enhancements of cell contractility (extent of shortening and shortening velocity). 5,6,40,41 The individual impact of these tw o mec hanical properties on contractile function, though, is unclear. Thus, understanding how viscosity and elasticity each r egulate v entricular function awaits further investigation.

Limitations

Ther e ar e a few limitations to our study. First, we induced a reduction of RV m y ocardial viscoelasticity via pharmaceutical interventions based on previous ex vivo studies. 10,11 However, w e w er e una b le to measur e RV biaxial viscoelasticity or ima ge microtubule network in vivo (as done in ex vivo studies) and only relate the main mechanism of acute COL treatment to reduced viscoelasticity. Furthermore, the improvement in PVR and E a after COL would lead to further impr ov ed v entricular function independent of the impact of reduced tissue viscoelasticity. We are not able, though, to determine the relative contributions of each of these effects in this current work. Additionally, the COL tr eatment w as administer ed via intr am y ocardial injections. Thus, the drug effects w ere tr ansient due to quick wash-out of the bolus and it precluded a vena cava occlusion to derive the ESPVR from a series of loops. A cor onar y infusion of COL may induce prolonged effect locally but is technically difficult for small animal hearts. Nevertheless, we measured ventricularvascular coupling using the single beat method. F inally, w e used only male rats in this study for initial assessment of the effect viscoelastic reduction on RV function, and acknowledge there may be sex differences in this response that limit the translation of our findings to the female RV.

Conclusions

To our knowledge, this is the first study to investigate the in vivo response of RV performance to acute COL treatment in healthy and diseased states. We found that the acute COL treatment led to significant impr ov ements in RV performance in the pr essur e-ov erloaded RV. Specifically, increased SV, EF, and SW, as well as reduced EDPVR, ESP and dP/dt max were observed. COL treatment also reduced RV afterload and heart r ate , whic h may contribute to impr ov ed RV performance in addition to the mechanical impact of the RV wall. In contrast, these beneficial effects were negligible in the healthy RV. These novel findings support the significant role of m y ocardial viscoelasticity in pr essur e-ov erloaded RV performance and implicate the m y ocardial mechanical impr ov ement as a potential therapeutic target for RV failure patients.