Mariappan Muthuchamy

Mouse Model of a Familial Hypertrophic Cardiomyopathy Mutation in α-Tropomyosin Manifests Cardiac Dysfunction

To investigate the functional consequences of a tropomyosin (TM) mutation associated with familial hypertrophic cardiomyopathy (FHC), we generated transgenic mice that express mutant alpha-TM in the adult heart. The missense mutation, which results in the substitution of asparagine for aspartic acid at amino acid position 175, occurs in a troponin T binding region of TM. S1 nuclease mapping and Western blot analyses demonstrate that increased expression of the alpha-TM 175 transgene in different lines causes a concomitant decrease in levels of endogenous alpha-TM mRNA and protein expression. In vivo physiological analyses show a severe impairment of both contractility and relaxation in hearts of the FHC mice, with a significant change in left ventricular fractional shortening. Myofilaments that contain alpha-TM 175 demonstrate an increased activation of the thin filament through enhanced Ca2+ sensitivity of steady-state force. Histological analyses show patchy areas of mild ventricular myocyte disorganization and hypertrophy, with occasional thrombi formation in the left atria. Thus, the FHC alpha-TM transgenic mouse can serve as a model system for the examination of pathological and physiological alterations imparted through aberrant TM isoforms.

Molecular and functional analyses of the contractile apparatus in lymphatic muscle

Lymphatics are necessary for the generation and regulation of lymph flow. Lymphatics use phasic contractions and extrinsic compressions to generate flow; tonic contractions alter resistance. Lymphatic muscle exhibits important differences from typical vascular smooth muscle. In this study, the thoracic duct exhibited significant functional differences from mesenteric lymphatics. To understand the molecular basis for these differences, we examined the profiles of contractile proteins and their messages in mesenteric lymphatics, thoracic duct, and arterioles. Results demonstrated that mesenteric lymphatics express only SMB smooth muscle myosin heavy chain (SM‐MHC), whereas thoracic duct and arterioles expressed both SMA and SMB isoforms. Both SM1 and SM2 isoforms of SM‐MHC were detected in arterioles and mesenteric and thoracic lymphatics. In addition, the fetal cardiac/skeletal slow‐twitch muscle‐specific β‐MHC message was detected only in mesenteric lymphatics. All four actin messages, cardiac α‐actin, vascular α‐actin, enteric γ‐actin, and skeletal α‐actin, were present in both mesenteric lymphatics and arterioles. However, in thoracic duct, predominantly cardiac α‐actin and vascular α‐actin were found. Western blot and immunohistochemical analyses corroborated the mRNA studies. However, in arterioles only vascular α‐actin protein was detected. These data indicate that lymphatics display genotypic and phenotypic characteristics of vascular, cardiac, and visceral myocytes, which are needed to fulfill the unique roles of the lymphatic system.

Molecular and Physiological Effects of α-Tropomyosin Ablation in the Mouse

Abstract —Tropomyosin (TM) is an integral component of the thin filament in muscle fibers and is involved in regulating actin-myosin interactions. TM is encoded by a family of four alternatively spliced genes that display highly conserved nucleotide and amino acid sequences. To assess the functional and developmental significance of α-TM, the murine α-TM gene was disrupted by homologous recombination. Homozygous α-TM null mice are embryonic lethal, dying between 8 and 11.5 days post coitum. Mice that are heterozygous for α-TM are viable and reproduce normally. Heterozygous knockout mouse hearts show a 50% reduction in cardiac muscle α-TM mRNA, with no compensatory increase in transcript levels by striated muscle β-TM or TM-30 isoforms. Surprisingly, this reduction in α-TM mRNA levels in heterozygous mice is not reflected at the protein level, where normal amounts of striated muscle α-TM protein are produced and integrated in the myofibril. Quantification of α-TM mRNA bound in polysomal fractions reveals that both wild-type and heterozygous knockout animals have similar levels. These data suggest that a change in steady-state level of α-TM mRNA does not affect the relative amount of mRNA translated and amount of protein synthesized. Physiological analyses of myocardial and myofilament function show no differences between heterozygous α-TM mice and control mice. The present study suggests that translational regulation plays a major role in the control of TM expression.

Correlation Between Myofilament Response to Ca2+ and Altered Dynamics of Contraction and Relaxation in Transgenic Cardiac Cells That Express β-Tropomyosin

We compared the dynamics of the contraction and relaxation of single myocytes isolated from nontransgenic (NTG) mouse hearts and from transgenic (TG-beta-Tm) mouse hearts that overexpress the skeletal isoform of tropomyosin (Tm). Compared with NTG controls, TG-beta-Tm myocytes showed significantly reduced maximal rates of contraction and relaxation with no change in the extent of shortening. This result indicated that the depression in contraction dynamics determined in TG-beta-Tm isolated hearts is intrinsic to the cells. To further investigate the effect of Tm isoform switching on myofilament activity and regulation, we measured myofilament force and ATPase rate as functions of pCa (-log of [Ca2+]). Compared with controls, force generated by myofilaments from TG-beta-Tm hearts and myofibrillar ATPase activity were both more sensitive to Ca2+. However, the shift in pCa50 (half-maximally activating pCa) caused by changing sarcomere length from 1.8 to 2.4 microm was not significantly different between NTG and TG-beta-Tm fiber preparations. To test directly whether isoform switching affected the economy of contraction, force versus ATPase rate relationships were measured in detergent-extracted fiber bundles. In both NTG and TG-beta-Tm preparations, force and ATPase rate were linear and identically correlated, which indicated that crossbridge turnover was unaffected by Tm isoform switching. However, detergent extracted fibers from TG-beta-Tm demonstrated significantly less maximum tension and ATPase activity than NTG controls. Our results provide the first evidence that the Tm isoform population modulates the dynamics of contraction and relaxation of single myocytes by a mechanism that does not alter the rate-limiting step of crossbridge detachment. Our results also indicate that differences in sarcomere-length dependence of activation between cardiac and skeletal muscle are not likely due to differences in the isoform population of Tm.

Roles of phosphorylation of myosin binding protein-C and troponin I in mouse cardiac muscle twitch dynamics.

A normal heart increases its contractile force with increasing heart rate. Although calcium handling and myofibrillar proteins have been implicated in maintaining this positive force–frequency relationship (FFR), the exact mechanisms by which it occurs have not been addressed. In this study, we have developed an analytical method to define the calcium–force loop data, which characterizes the function of the contractile proteins in response to calcium that is independent of the calcium handling proteins. Results demonstrate that increasing the stimulation frequency causes increased force production per unit calcium concentration and decreased frequency‐dependent calcium sensitivity during the relaxation phase. We hypothesize that phosphorylation of myosin binding protein‐C (MyBP‐C) and troponin I (TnI) acts coordinately to change the rates of force generation and relaxation, respectively. To test this hypothesis, we performed simultaneous calcium and force measurements on stimulated intact mouse papillary bundles before and after inhibition of MyBP‐C and TnI phosphorylation using the calcium/calmodulin kinase II (CaMK2) inhibitor autocamtide‐2 related inhibitory peptide, or the protein kinase A (PKA) inhibitor 14–22 amide. CaMK2 inhibition reduced both MyBP‐C and TnI phosphorylation and decreased active force without changing the magnitude of the [Ca2+]i transient. This reduced the normalized change in force per change in calcium by 19–39%. Data analyses demonstrated that CaMK2 inhibition changed the myofilament characteristics via a crossbridge feedback mechanism. These results strongly suggest that the phosphorylation of  MyBP‐C and TnI contributes significantly to the rates of force development and relaxation.

Molecular Regulation of Lymphatic Contractility

The lymphatic system plays critical roles in body fluid and macromolecular homeostasis, lipid absorption, immune function, and metastasis. To accomplish these tasks, the lymphatics must move lymph and its contents from the interstitial space through the lymph vessels and nodes and into the great veins. Contrary to popular belief, lymph does not passively “drain” down this pathway, because the net pressure gradients oppose flow. Instead, the lymphatics must act as both the conduits that direct and regulate lymph flow and the pumps that generate the lymph flow. Thus, to regulate lymph transport and function, both lymphatic pumping and flow resistance must be controlled. Both of these processes occur via regulation of lymphatic muscle contractions, which are classically thought to occur via the interaction of cell calcium with regulatory and contractile proteins. However, our knowledge of this regulation of lymphatic contractile function is far from complete. In this chapter we review our understanding of the important molecular mechanisms, the calcium regulation, and the contractile/regulatory proteins that control lymphatic contractions. A better understanding of these mechanisms could provide the basis for the development of better diagnostic and treatment modalities for lymphatic dysfunction. While progress has been made in our understanding of the molecular biology of lymphangiogenesis as a result of the development of potential lymphangiogenic therapeutic targets, there are currently no therapeutic agents that specifically modulate lymphatic pump function and lymph flow via lymphatic muscle. However, their development will not be possible until the molecular basis of lymphatic contractility is more fully understood.

Intrinsic increase in lymphangion muscle contractility in response to elevated afterload

Collecting lymphatic vessels share functional and biochemical characteristics with cardiac muscle; thus, we hypothesized that the lymphatic vessel pump would exhibit behavior analogous to homeometric regulation of the cardiac pump in its adaptation to elevated afterload, i.e., an increase in contractility. Single lymphangions containing two valves were isolated from the rat mesenteric microcirculation, cannulated, and pressurized for in vitro study. Pressures at either end of the lymphangion [input pressure (P(in)), preload; output pressure (P(out)), afterload] were set by a servo controller. Intralymphangion pressure (P(L)) was measured using a servo-null micropipette while internal diameter and valve positions were monitored using video methods. The responses to step- and ramp-wise increases in P(out) (at low, constant P(in)) were determined. P(L )and diameter data recorded during single contraction cycles were used to generate pressure-volume (P-V) relationships for the subsequent analysis of lymphangion pump behavior. Ramp-wise P(out) elevation led to progressive vessel constriction, a rise in end-systolic diameter, and an increase in contraction frequency. Step-wise P(out) elevation produced initial vessel distention followed by time-dependent declines in end-systolic and end-diastolic diameters. Significantly, a 30% leftward shift in the end-systolic P-V relationship accompanied an 84% increase in dP/dt after a step increase in P(out), consistent with an increase in contractility. Calculations of stroke work from the P-V loop area revealed that robust pumps produced net positive work to expel fluid throughout the entire afterload range, whereas weaker pumps exhibited progressively more negative work as gradual afterload elevation led to pump failure. We conclude that lymphatic muscle adapts to output pressure elevation with an intrinsic increase in contractility and that this compensatory mechanism facilitates the maintenance of lymph pump output in the face of edemagenic and/or gravitational loads.

Ectopic expression of tropomyosin promotes myofibrillogenesis in mutant axolotl hearts

Expression of tropomyosin protein, an essential component of the thin filament, has been found to be drastically reduced in cardiac mutant hearts of the Mexican axolotl (Ambystoma mexicanum) with no formation of sarcomeric myofibrils. Therefore, this naturally occurring cardiac mutation is an appropriate model to examine the effects of delivering tropomyosin protein or tropomyosin cDNA into the deficient tissue. In this study, we describe the replacement of tropomyosin by using a cationic liposome transfection technique applied to whole hearts in vitro. When mouse α‐tropomyosin cDNA under the control of a cardiac‐specific α‐myosin heavy chain promoter was transfected into the mutant hearts, tropomyosin expression was enhanced resulting in the formation of well‐organized sarcomeric myofibrils. Transfection of a β‐tropomyosin construct under control of the same promoter did not result in enhanced organization of the myofibrils. Transfection of a β‐galactosidase reporter gene did not result in the formation of organized myofibrils or increased tropomyosin expression. These results demonstrate the importance of α‐tropomyosin to the phenotype of this mutation and to normal myofibril formation. Moreover, we have shown that a crucial contractile protein can be ectopically expressed in cardiac muscle that is deficient in this protein, with the resulting formation of organized sarcomeres. Dev. Dyn. 1998;213:412–420.

Age-Related Alterations of Active Pumping Mechanisms in Rat Thoracic Duct

Objective: To evaluate the age‐related changes in active pumping in thoracic duct (TD) from 24‐month‐old Fisher‐344 rats comparing with TD pumping in 9‐month rats.

Independent and interactive effects of preload and afterload on the pump function of the isolated lymphangion

We tested the responses of single, isolated lymphangions to selective changes in preload and the effects of changing preload on the response to an imposed afterload. The methods used were similar to those described in our companion paper. Step-wise increases in input pressure (P(in); preload) over a pressure range between 0.5 and 3 cmH(2)O, at constant output pressure (P(out)), led to increases in end-diastolic diameter, decreases in end-systolic diameter, and increases in stroke volume. From a baseline of 1 cmH(2)O, P(in) elevation by 2-7 cmH(2)O consistently produced an immediate fall in stroke volume that subsequently recovered over a time course of 2-3 min. Surprisingly, this adaptation was associated with an increase in the slope of the end-systolic pressure-volume relationship, indicative of an increase in contractility. Lymphangions subjected to P(out) levels exceeding their initial ejection limit would often accommodate by increasing diastolic filling to strengthen contraction sufficiently to match P(out). The lymphangion adaptation to various pressure combinations (P(in) ramps with low or high levels of P(out), P(out) ramps at low or intermediate levels of P(in), and combined P(in) + P(out) ramps) were analyzed using pressure-volume data to calculate stroke work. Under relatively low imposed loads, stroke work was maximal at low preloads (P(in) ∼2 cmH(2)O), whereas at more elevated afterloads, the optimal preload for maximal work displayed a broad plateau over a P(in) range of 5-11 cmH(2)O. These results provide new insights into the normal operation of the lymphatic pump, its comparison with the cardiac pump, and its potential capacity to adapt to increased loads during edemagenic and/or gravitational stress.