diacetylmonoxime and Ventricular-Dysfunction--Left

To understand the functional consequences of the Lys184 deletion in murine cardiac troponin I (mcTnI(DeltaK184)), we have studied the primary effects of this mutation linked to familial hypertrophic cardiomyopathy (FHC) at the sarcomeric level.. Ca(2+) sensitivity and kinetics of force development and relaxation were investigated in cardiac myofibrils from transgenic mice expressing mcTnI(DeltaK184), as a model which co-segregates with FHC. Ca(2+)-dependent conformational changes (switch-on/off) of the fluorescence-labelled human troponin complex, containing either wild-type hcTnI or mutant hcTnI(DeltaK183), were investigated in myofibrils prepared from the guinea pig left ventricle. Ca(2+) sensitivity and maximum Ca(2+)-activated and passive forces were significantly enhanced and cooperativity was reduced in mutant myofibrils. At partial Ca(2+) activation, mutant but not wild-type myofibrils displayed spontaneous oscillatory contraction of sarcomeres. Both conformational switch-off rates of the incorporated troponin complex and the myofibrillar relaxation kinetics were slowed down by the mutation. Impaired relaxation kinetics and increased force at low [Ca(2+)] were reversed by 2,3-butanedione monoxime (BDM), which traps cross-bridges in non-force-generating states.. We conclude that these changes are not due to alterations of the intrinsic cross-bridge kinetics. The molecular mechanism of sarcomeric diastolic dysfunction in this FHC model is based on the impaired regulatory switch-off kinetics of cTnI, which induces incomplete inhibition of force-generating cross-bridges at low [Ca(2+)] and thereby slows down relaxation of sarcomeres. Ca(2+) sensitization and impairment of the relaxation of sarcomeres induced by this mutation may underlie the enhanced systolic function and diastolic dysfunction at the sarcomeric level.

Topics: Animals; Calcium Signaling; Cardiomyopathy, Hypertrophic, Familial; Diacetyl; Disease Models, Animal; Guinea Pigs; Humans; Kinetics; Lysine; Mice; Mice, Inbred C57BL; Mice, Transgenic; Muscle Contraction; Muscle Strength; Myofibrils; Papillary Muscles; Protein Conformation; Sarcomeres; Sequence Deletion; Troponin I; Ventricular Dysfunction, Left

Transmembrane voltage-sensitive fluorescent dyes are used to study electrical activity in hearts. Green and red fluorescence emissions from di-4-ANEPPS excited with 488 nm light indicate both transmembrane voltage changes and heart movement. We have previously shown that the ratio, green fluorescence divided by red fluorescence, indicates the transmembrane voltage without effects of movement. Here we examine the feasibility of measuring the movement, which is useful for the study of cardiac function, by subtracting this ratiometric signal from the red or green fluorescence signal. The results of this subtraction show tissue movement and its relative changes during cardiac ischemia and perfusion with an electromechanical uncoupling agent. By incorporating the spatial variations in fluorescence intensity from the heart, tissue movement can be qualitatively mapped to examine relative changes, however, with limited ability to quantify absolute displacement. Since these maps are obtained simultaneously with corresponding transmembrane potentials, the method allows study of spatiotemporal cardiac movement patterns and their relationship to the action potential.

Topics: Algorithms; Animals; Diacetyl; Feasibility Studies; Fluorescent Dyes; Heart Conduction System; Image Interpretation, Computer-Assisted; Microscopy, Fluorescence; Movement; Myocardial Contraction; Myocardial Ischemia; Rabbits; Spectrometry, Fluorescence; Ventricular Dysfunction, Left

Familial hypertrophic cardiomyopathy (FHC) is a human genetic disorder caused by mutations in sarcomeric proteins. It is generally characterized by cardiac hypertrophy, fibrosis, and myocyte disarray. A transgenic mouse model of FHC with mutations in the actin-binding domain of the alpha-myosin heavy chain (MyHC) gene displays many phenotypes similar to human FHC. At 4 months, male transgenic (TG) mice present with concentric cardiac hypertrophy that progresses to dilation with age. Accompanying this latter morphological change is systolic and diastolic dysfunction. Left ventricular (LV) myocytes from male TG and wild-type (WT) littermates at 5 and 12 months of age were isolated and used for morphological and functional studies. Myocytes from 5- and 12-month-old TG animals had shorter sarcomere lengths compared with WT. This sarcomere length difference was abolished in the presence of 2,3-butanedione monoxime, suggesting that the basal level of contractile element activation was increased in TG myocytes. Myocytes from 12-month-old TG mice were significantly longer than those from age-matched WT controls, and TG myocytes exhibited Z-band disorganization. When cells were paced at 0.5 Hz, TG myocyte relengthening and the fall in intracellular [Ca2+] were slowed when compared with cells from age-matched WT controls. Moreover, an increased amount of beta-myosin heavy chain protein was found in hearts from TG compared with WT. Thus, myocytes from the alpha-MyHC TG mouse model display many morphological and functional abnormalities that may help explain the LV dysfunction seen in this TG mouse model of FHC.

Topics: Age Factors; Amino Acid Substitution; Animals; Calcium; Cardiomyopathy, Hypertrophic, Familial; Diacetyl; Diastole; Gene Expression; Heart Ventricles; Male; Mice; Mice, Transgenic; Microscopy, Electron; Models, Animal; Mutation, Missense; Myocardial Contraction; Myocytes, Cardiac; Myosin Heavy Chains; Phenotype; Protein Isoforms; Sarcomeres; Systole; Ventricular Dysfunction, Left; Ventricular Myosins

Increased diastolic chamber stiffness (upward arrow DCS) during ischemia may result from increased diastolic calcium, rigor, or reduced velocity of relaxation. We tested these potential mechanisms during severe ischemia in isolated red blood cell-perfused isovolumic rabbit hearts. Ischemia (coronary flow reduced 83%) reduced left ventricular (LV) contractility by 70%, which then remained stable. DCS progressively increased. When LV end-diastolic pressure had increased 5 mmHg, myofilament calcium responsiveness was altered with 50 mmol/l NH(4)Cl or 10 mmol/l butanedione monoxime. These affected contractility (i.e., a calcium-mediated force) but not upward arrow DCS. Second, quick length changes reversed upward arrow DCS, supporting a rigor mechanism. Third, ischemia increased the time constant of isovolumic pressure decline from 47 +/- 3 to 58 +/- 3 ms (P < 0.02) but concomitantly abbreviated the contraction-relaxation cycle, i.e., pressure dissipation occurred earlier without diastolic tetanization. Finally, to assess any link between rate of relaxation and upward arrow DCS, hearts were exposed to 10 mmol/l calcium. Calcium doubled contractility and accelerated relaxation velocity, but without affecting upward arrow DCS. Thus upward arrow DCS developed during ischemia despite severely reduced contractility via a rigor (and not calcium mediated) mechanism. Calcium resequestration capacity was preserved, and reduced relaxation velocity was not linked to upward arrow DCS.

Topics: Ammonium Chloride; Animals; Blood Pressure; Calcium; Diacetyl; Diastole; Enzyme Inhibitors; Erythrocytes; Heart; In Vitro Techniques; Myocardial Contraction; Myocardial Ischemia; Perfusion; Rabbits; Ventricular Dysfunction, Left