Heart failure-associated alterations in troponin I phosphorylation impair ventricular relaxation-afterload and force-frequency responses and systolic function.

Recent studies have found that selective stimulation of troponin (Tn)I protein kinase A (PKA) phosphorylation enhances heart rate-dependent inotropy and blunts relaxation delay coupled to increased afterload. However, in failing hearts, TnI phosphorylation by PKA declines while protein kinase C (PKC) activity is enhanced, potentially augmenting TnI PKC phosphorylation. Accordingly, we hypothesized that these site-specific changes deleteriously affect both rate-responsive cardiac function and afterload dependence of relaxation, both prominent phenotypic features of the failing heart. A transgenic (TG) mouse model was generated in which PKA-TnI sites were mutated to mimic partial dephosphorylation (Ser22 to Ala; Ser23 to Asp) and dominant PKC sites were mutated to mimic constitutive phosphorylation (Ser42 and Ser44 to Asp). The two highest-expressing lines were further characterized. TG mice had reduced fractional shortening of 34.7 +/- 1.4% vs. 41.3 +/- 2.0% (P = 0.018) and slight chamber dilation on echocardiography. In vivo cardiac pressure-volume studies revealed near doubling of isovolumic relaxation prolongation with increasing afterload in TG animals (P < 0.001), and this remained elevated despite isoproterenol infusion (PKA stimulation). Increasing heart rate from 400 to 700 beats/min elevated contractility 13% in TG hearts, nearly half the response observed in nontransgenic animals (P = 0.005). This blunted frequency response was normalized by isoproterenol infusion. Abnormal TnI phosphorylation observed in cardiac failure may explain exacerbated relaxation delay in response to increased afterload and contribute to blunted chronotropic reserve.

Angiotensinergic stimulation of vascular endothelium in mice causes hypotension, bradycardia, and attenuated angiotensin response.

It is not clear whether endothelial cell (EC) activation by the hormone angiotensin II (Ang II) modulates contraction of vascular smooth muscle cells (VSMCs) in the vasculature and whether impairment of this regulation in vivo contributes to hypertension. Delineation of the actions of Ang II through the type 1 receptor (AT1R) on ECs in the blood vessels has been a challenging problem because of the predominance of the AT1R functions in VSMCs that lie underneath the endothelium. We have obviated this limitation by generating transgenic (TG) mice engineered to target expression of the constitutively active N111G mutant AT1R only in ECs. In these TG mice, the enhanced angiotensinergic signal in ECs without infusion of Ang II resulted in hypotension and bradycardia. The pressor response to acute infusion of Ang II was significantly reduced. Increased expression of endothelial nitric oxide synthase and production of hypotensive mediators, nitric oxide and cyclic guanosine monophosphate, cause these phenotypes. Hypotension and bradycardia observed in the TG mice could be rescued by treatment with an AT1R-selective antagonist. Our results imply that the Ang II action by means of EC-AT1R is antagonistic to vasoconstriction in general, and it may moderate the magnitude of functional response to Ang II in VSMCs. This control mechanism in vivo most likely is a determinant of altered hemodynamic regulation involved in endothelial dysfunction in hypertensive cardiovascular disease.

Murine strain differences in contractile function are temperature- and frequency-dependent.

Despite the widespread use of mice in the investigation of cardiac function, little is known as to what extent cardiac contractile function varies between different murine strains. We have investigated basic contractile function in isolated multicellular right ventricular trabeculae from three commonly used mouse strains (C57BL/6, SV129, and FVBN). Suitable trabeculae (<100 microm thick, >1 mm long) occurred rather frequently in FVBN and SV129 mice (on average about 2 per heart), but only sporadically in C57BL/6 mice (on average only 1 per 3-4 mice). However, using similar sized preparations under experimental conditions closely mimicking those in vivo (37 degrees C and frequencies between 8 and 12 Hz), contractile function was virtually identical. In addition, the magnitude of response to beta-adrenergic stimulation was also indistinguishable between the strains. However, at subphysiological frequency, FVBN mice consistently displayed more developed force compared to C57/BL6 and SV129 mice. Furthermore, contractile performance at a subphysiological temperature and frequency, where studies on isolated myocardium often are performed, was also strain-dependent. We conclude that basic contractile function under near physiological conditions is preserved throughout various strains, but subphysiological conditions can have a profound effect on contractile performance. Hence, choice of strain can have important implications for cardiac contractile function under nonphysiological conditions.

Xanthine oxidase inhibitors improve energetics and function after infarction in failing mouse hearts.

After myocardial infarction, ventricular geometry and function, as well as energy metabolism, change markedly. In nonischemic heart failure, inhibition of xanthine oxidase (XO) improves mechanoenergetic coupling by improving contractile performance relative to a reduced energetic demand. However, the metabolic and contractile effects of XO inhibitors (XOIs) have not been characterized in failing hearts after infarction. After undergoing permanent coronary ligation, mice received a XOI (allopurinol or oxypurinol) or matching placebo in the daily drinking water. Four weeks later, 1H MRI and 31P magnetic resonance spectroscopy (MRS) were used to quantify in vivo functional and metabolic changes in postinfarction remodeled mouse myocardium and the effects of XOIs on that process. End-systolic (ESV) and end-diastolic volumes (EDV) were increased by more than sixfold after infarction, left ventricle (LV) mass doubled (P < 0.005), and the LV ejection fraction (EF) decreased (14 +/- 9%) compared with control hearts (59 +/- 8%, P < 0.005) at 1 mo. The myocardial phosphocreatine (PCr)-to-ATP ratio (PCr/ATP) was also significantly decreased in infarct remodeled hearts (1.4 +/- 0.6) compared with control animals (2.1 +/- 0.5, P < 0.02), in agreement with prior studies in larger animals. The XOIs allopurinol and oxypurinol did not change LV mass but limited the increase in ESV and EDV of infarct hearts by 50%, increased EF (23 +/- 9%, P = 0.01), and normalized cardiac PCr/ATP (2.0 +/- 0.5, P < 0.04). We conclude that XOIs improve ventricular function after infarction and normalize high-energy phosphate ratio in heart failure. Thus XOI therapy offers a new and potentially complementary approach to limit the adverse contractile and metabolic consequences after infarction.

Chronic xanthine oxidase inhibition prevents myofibrillar protein oxidation and preserves cardiac function in a transgenic mouse model of cardiomyopathy.

Heart failure is a clinical syndrome associated with elevated levels of oxygen-derived free radicals. Xanthine oxidase activity is believed to be one source of reactive oxygen species in the failing heart. Interventions designed to reduce oxidative stress are believed to have significant therapeutic potential in heart failure. This study tested the hypothesis that xanthine oxidase activity would be elevated in a mouse model of dilated cardiomyopathy and evaluated the effect of chronic oral allopurinol, an inhibitor of xanthine oxidase, on contractility and progressive ventricular dilation in these mice. Nontransgenic and transgenic mice containing a troponin I truncation were treated with oral allopurinol from 2-4 mo of age. Myocardial xanthine oxidase activity was threefold higher in untreated transgenic mice compared with nontransgenic mice. Analyses of myofilament proteins for modification of carbonyl groups demonstrated myofibrillar protein damage in untreated transgenic mice. Treatment with allopurinol for 2 mo suppressed xanthine oxidase activity and myofibrillar protein oxidation. Allopurinol treatment also alleviated ventricular dilation and preserved shortening fraction in the transgenic animals. In addition, cardiac muscle twitch tension was preserved to 70% of nontransgenic levels in allopurinol-treated transgenic mice, a significant improvement over untreated transgenic mice. These findings indicate that chronic inhibition of xanthine oxidase can alter the progression of heart failure in dilated cardiomyopathy.

Chronic treatment with allopurinol boosts survival and cardiac contractility in murine postischemic cardiomyopathy.

Oxidative stress is a hallmark of systemic illnesses, including heart failure. Nevertheless, the overall importance of radical production in the heart remains conjectural; is it merely a marker of illness, or can intervention alter the progression of disease? This question was addressed by blocking xanthine oxidase (XO), a superoxide-generating enzyme that is upregulated in animal models of heart failure. In a randomized prospective trial design, we administered the XO inhibitor allopurinol orally to mice that had undergone massive myocardial infarction (MI). Cardiac XO activity was elevated in untreated mice after MI; allopurinol suppressed the XO activity to levels comparable to those in sham-operated mice. Eighty-one percent of untreated mice died of advanced heart failure over 2 to 4 weeks of follow-up. Survival doubled in the allopurinol-treated mice, whereas cardiac contractile function (both in vivo and in isolated muscle) was markedly improved. Response to isoproterenol was restored to near-normal levels in the allopurinol group but was attenuated in untreated mice. Oxidative modifications to proteins were prevented in the allopurinol-treated mice. Our findings indicate that targeted blockade of just one source of oxidants, XO, impacts dramatically on the progression of postischemic cardiomyopathy in mice and prevents oxidative protein modifications.

Frequency- and afterload-dependent cardiac modulation in vivo by troponin I with constitutively active protein kinase A phosphorylation sites.

Acute beta-adrenergic stimulation enhances cardiac contractility, accelerates muscle relaxation, and amplifies the inotropic and lusitropic response to increased stimulation frequency. These effects are modulated by phosphorylation of calcium handling and myofilament proteins such as troponin I (TnI) by protein kinase A (PKA). To more directly delineate the role of TnI PKA phosphorylation, transgenic mice were generated that overexpress cardiac TnI in which the serine residues normally targeted by PKA are mutated to aspartic acid to mimic constitutive phosphorylation (TnIDD22,23). Native cardiac TnI was near completely replaced in one transgenic line as assessed by in vitro phosphorylation, and this led to reduced calcium sensitivity of myofibrillar MgATPase, as expected. TnIDD22,23 mice had mildly enhanced basal systolic and diastolic function, and displayed marked augmentation of frequency-dependent inotropy and relaxation, with a peak frequency response 2-fold greater in mutants than controls (P<0.005). Increasing afterload prolonged relaxation more in nontransgenic than TnIDD22,23 (P<0.02), whereas contractile responses to afterload were similar between these strains. Isoproterenol treatment eliminated the differential force-frequency and afterload response between TnIDD22,23 and controls. In contrast to in vivo studies, isolated isometric trabeculae from nontransgenic and TnIDD22,23 mice had similar basal, isoproterenol-, and frequency-stimulated function, suggesting that muscle shortening may be important to TnI PKA effects. These results support a novel role for cardiac TnI PKA phosphorylation in the rate-dependent enhancement of systolic and diastolic function in vivo and afterload sensitivity of relaxation. These results have implications for cardiac failure in which force-frequency modulation is blunted and afterload relaxation sensitivity increased in association with diminished PKA TnI phosphorylation.

Augmented systolic response to the calcium sensitizer EMD-57033 in a transgenic model with troponin I truncation.

Myocardial stunning is a form of acute reversible cardiac dysfunction that occurs after brief periods of ischemia and reperfusion. In several animal models, stunning is associated with proteolytic truncation of troponin I (TnI). Mice expressing the same proteolytic TnI fragment [TnI-(1-193)] demonstrate cardiac depression with a decreased maximal calcium-activated tension. We therefore hypothesized preferential improvement in mice expressing TnI-(1-193) treated with the calcium-sensitizing drug EMD-57033. TnI-(1-193) and nontransgenic myofibrils exhibited significant sensitization to calcium in Mg-ATPase assays after EMD-57033 exposure. However, only transgenic myofibrils exhibited an increase in maximal activity (P = 0.023). EMD-57033 also increased maximal calcium-activated force in TnI-(1-193) muscle, such that it was comparable to nontransgenic cardiac muscle. EMD-57033 enhanced in vivo systolic function modestly in controls but had a marked effect in transgenic mice, with an almost threefold greater leftward shift of the end-systolic pressure-volume relation (P = 0.0005). These data indicate a targeted efficacy of EMD-57033 in offsetting the contractile defect in TnI-(1-193) mice, and this may have therapeutic implications in models displaying this myofilament defect.

Selective contractile dysfunction of left, not right, ventricular myocardium in the SHHF rat.

The progression of hypertension to cardiac failure involves systemic changes that may ultimately affect contractility throughout the heart. Spontaneous hypertensive heart failure (SHHF) rats have depressed left ventricular (LV) function, but right ventricular (RV) dysfunction is less well characterized. Ultrathin (87 +/- 5 mircom) trabeculae were isolated from end-stage failing SHHF rats and from age-matched controls. Under near-physiological conditions (1 mM Ca(2+), 37 degrees C, 4 Hz), developed force (in mN/mm(2)) was not significantly different in SHHF LV and RV trabeculae and those of controls. SHHF LV preparations displayed a negative force-frequency behavior (40 +/- 7 vs. 23 +/- 4 mN/mm(2), 2 vs. 7 Hz); this relationship was positive in SHHF RV preparations (27 +/- 5 vs. 40 +/- 6 mN/mm(2)) and controls (32 +/- 6 vs. 44 +/- 9 mN/mm(2)). The response to isoproterenol (10(-6) M, 4 Hz) was depressed in SHHF LV preparations. The inotropic response to hypothermia was lost in SHHF LV trabeculae but preserved in SHHF RV trabeculae. Intracellular calcium measurements revealed impaired calcium handling at higher frequencies in LV preparations. We conclude that in end-stage failing SHHF rats, RV function is only marginally affected, whereas a severe contractile dysfunction of LV myocardium is present.

A novel class II-binding motif selects peptides that mediate organ-specific autoimmune disease in SWXJ, SJL/J, and SWR/J mice.

Idiopathic dilated cardiomyopathy (DCM) is responsible for approximately 25% of all cases of congestive heart failure. We have recently shown that immunization of autoimmune-susceptible SWXJ mice with whole cardiac myosin leads to T cell-mediated experimental autoimmune myocarditis (EAMC) and DCM. We have now identified two disease-inducing peptides from cardiac alpha-myosin heavy chain (CAMHC). Our approach involved the use of a novel MHC class II-binding motif contained in several peptides known to be immunogenic in SWXJ (H-2(q,s)) mice or in the parental SJL/J (H-2(s)) or SWR/J (H-2(q)) mouse strains. Two of four CAMHC peptides containing the -KXXS- peptide motif were found to be immunogenic. Immunization of SWXJ or parental SJL/J and SWR/J mice with CAMHC peptides palpha406-425 or palpha1631-1650 resulted in EAMC and DCM, characterized by inflammation, fibrosis, and decompensated right-sided ventricular dilatation. Despite mediating high incidences of severe disease, both peptides were found to be cryptic determinants, thereby providing further evidence for the importance and perhaps predominance of self crypticity in autoimmunity. Both peptides showed dual parental I-A(q) and I-A(s) restriction and mediated passive transfer of disease with activated CD4(+) T cells. An intact motif was necessary for antigenicity because loss of activity occurred in peptides containing nonconservative substitutions at the motif's terminal lysine and serine residues. Our studies provide a new model for EAMC and DCM in strains of mice widely used in autoimmune studies. Moreover, the -KXXS- motif may be particularly useful in implicating previously overlooked proteins as autoimmune targets and in facilitating the development of new organ-specific autoimmune mouse models for human diseases.

Physiological determinants of contractile force generation and calcium handling in mouse myocardium.

Despite the fact that the mouse has become a common tool to study cardiac dysfunction, little is known regarding the regulation of murine cardiac contractility. We have investigated the three main mechanisms that regulate cardiac output (frequency-dependent activation, length-dependent activation, and beta-adrenergic stimulation) in ultra-thin right ventricular (RV) trabeculae from the mouse heart at body temperature (37 degrees C). [Ca(2+)](i) was recorded in a subset of trabeculae iontophoretically loaded with fura-2, and rapid cooling contractures were performed to estimate the sarcoplasmic reticulum (SR) calcium load. The force-frequency relationship was positive (2-12Hz); force increased, albeit slightly, while relaxation timing decreased. As expected, in response to beta-adrenergic stimulation, force development increased while contractile duration decreased, and increased muscle length led to increased force generation. Changes in SR calcium load and the calcium transient amplitude paralleled effects on active force generation. Despite several qualitative similarities with other mammalian species, the reserve for augmentation of force via either increased frequency or beta-adrenergic stimulation was considerably smaller in mouse than in other animals. Therefore, changes in preload, as opposed to increased HR or adrenergic tone, appears to be a much more important determinant of cardiac performance in the mouse than in larger mammals.

Myofilament properties comprise the rate-limiting step for cardiac relaxation at body temperature in the rat.

The majority of studies aimed at characterizing basic contractile mechanisms have been conducted at room temperature. To elucidate the mechanism of cardiac relaxation under more physiological conditions, we investigated contractile function and calcium handling in ultrathin rat cardiac trabeculae. Active developed tension was unaltered between 22.5 and 30.0 degrees C (from 89 +/- 10 to 86 +/- 11 mN/mm(2), P = not significant) but steeply declined at 37.5 degrees C (30 +/- 5 mN/mm(2)). Meanwhile, the speed of relaxation (time from peak force to 50% relaxation) declined from 22.5 to 30.0 degrees C (from 360 +/- 40 to 157 +/- 17 ms) and further declined at 37.5 degrees C to 76 +/- 13 ms. Phase-plane analysis of calcium versus force revealed that, with increasing temperature, the relaxation phase is shifted rightward, indicating that the rate-limiting step of relaxation tends to depend more on calcium kinetics as temperature rises. The force-frequency relationship, which was slightly negative at 22.5 degrees C (0.1 vs. 1 Hz: 77 +/- 12 vs. 66 +/- 7 mN/mm(2)), became clearly positive at 37.5 degrees C (1 vs. 10 Hz: 30 +/- 5 vs. 69 +/- 9 mN/mm(2)). Phase-plane analyses indicated that, with increasing frequency, the relaxation phase is shifted leftward. We conclude that temperature independently affects contraction and relaxation, and cross-bridge cycling kinetics become rate limiting for cardiac relaxation under experimental conditions closest to those in vivo.