This study focused on the physiological impact of histidine-modified cTnI A164H in aged mice. These data provide new evidence that molecular manipulation of myofilament calcium sensitivity can preserve heart function during ageing. Specifically, this study showed that age-related decrements in baseline cardiac systolic function were significantly attenuated in Tg mice compared with Ntg littermates. Echocardiography studies also demonstrated that age-dependent diastolic dysfunction (elevated E/Ela) was attenuated in Tg animals. Importantly, when exposed to an acute hypoxic challenge in vivo, aged Tg mice maintained cardiac performance that significantly extended their survival compared with Ntg mice. These data support the hypothesis that myofilament-based enhanced function by single histidine-modified cTnI provides a mechanism for attenuating age-related decrements in cardiac function.
Increasing calcium sensitivity of the myofilament consequent to alterations in the biochemistry of cTnI has been well studied.10,12,13,15,16,28
A recent report of the crystal structure of cTnI places Ala164 at the critical switch domain that is key in regulating myofilament calcium activation.11
Codon 164 is situated at the interface between the amphiphilic switch region, H3, and the C-terminal actin-binding domain, H4. These regions are defined as the regulatory segment. In the calcium-saturated state, the entire regulatory segment of cTnI (residues 137–210) undergoes a conformational change concomitant with binding of the H3 domain to the conserved N-terminal hydrophobic patch of TnC. In the slow skeletal isoform of TnI (ssTnI), which is the foetal/neonatal isoform in mammals, a unique biochemical characteristic of the switch region was found to confer pH-dependent increases in calcium sensitivity to the myofilament.29,30
In initial studies, mutagenesis experiments identified a critical histidine at position 132 as responsible for this pH-dependent functional outcome.12,14,15
The physiological effects of this mutation in the heart directly reflect the unique biochemical properties of histidine’s imidazole moiety which, unique among all the amino acids, ionizes within the physiological range (pKR
= 6.0). Numerous studies have shown that under conditions of acidosis, ssTnI protects contractility of myocytes in vitro
and the whole heart in vivo
In light of these findings, we sought to introduce this pH sensitivity into the switch domain of cTnI. An engineered histidine modification in codon 164 of cTnI (A164H) takes advantage of this critical functional property of ssTnI transposed into the context of cTnI. This modification takes place without altering important physiological functions of cTnI in regulating global cardiac inotropic and lusitropic responses to β-adrenergic signalling.15
Cardiac TnI A164H provides a titratable molecular switch mechanism regulating myofilament tension development in response to biochemical changes in the adult myocyte. One of the central hypotheses of this study was that increasing myofilament calcium sensitivity by means of cTnI A164H is a powerful molecular therapy for attenuating morbidity and mortality associated with ischaemia/hypoxia-induced contractile failure in aged mice.
Declining cardiac function contributes to waning health in geriatric populations.5
In many cases these age-related changes are secondary to coronary artery disease.6
Of particular salience to this study is the propensity for chronic ischaemia associated with vascular stenosis to contribute to poor cardiovascular health of the elderly.4
A portion of this study was based on an experimental protocol to impose a controlled state of hypoxia in aged mice through low inhaled oxygen. In the clinical setting, hypoxia is usually secondary to cardio-pulmonary pathologies frequently seen in geriatric populations. Physiologically these conditions are usually due to hypoventilation, vascular shunting, ventilation/perfusion mismatch, and interstitial diffusion defects. Clinical diseases of the lungs such as COPD, pneumonia, interstitial lung disease, or pulmonary embolic disease are common conditions associated with hypoxia which may lead to secondary ischaemic cardiac injury. The aged heart is particularly at risk in these settings because of intrinsic muscle disease and underlying coronary artery disease with areas of marginally perfused myocardium. These clinically relevant aetiologies, known risk factors for CVD, provide contextual relevance for the key findings of this study. These important results include the finding that aged cTnI A164H Tg mice have enhanced cardiac function and extended survival capacity during an acute hypoxic challenge compared with Ntg mice.
Irrespective of the aetiology, systolic and diastolic dysfunction are strong predictors of heart failure in geriatric populations.33
In this study we show that cTnI A164H Tg mice have improved contractility during the ageing process compared with Ntg mice. The equalization of the HW/BW ratio at 2 years of age neutralizes any heart size-dependent variables that influence contractility measurements between groups at this time point. This lends credence to the conclusion that Tg mice, which retained the same relative increase in contractility over Ntg mice during the ageing process, actually underwent an age-dependent and heart size-independent increase in absolute contractility. This could be explained in part by the increase in stoichiometric incorporation of cTnI A164H in old vs. young Tg mice and could provide mechanistic basis for the observed increase in contractility at 2 years of age. Additionally, compared with Tg mice, increased LV cavitary dilation in aged Ntg mice may also, at least in part, contribute to the divergence of contractile efficiency at the 2-year time point. A shift towards higher sarcomere lengths, which occurs with dilated cardiomyopathies and may be a component of age-related dysfunction, could reduce the efficiency of contractility based on the Frank–Starling principle.
In addition to systolic dysfunction, decrements in diastolic function are also characteristic of the ageing process. LV dilation together with stiffening of the aged myocardium prevents sufficient relaxation of the heart during the filling phase of the cardiac cycle. Thus, another key finding of this study is that cTnI A164H protects diastolic function during the ageing process. These data indicate that Ntg mice show evidence of diastolic dysfunction based on a decrease in the velocity of the lateral annulus during the early filling phase (Ela) of the LV as well as an increase in the mitral valve E wave flow velocity to lateral annular E wave ratio (E/Ela). This latter parameter (E/Ela) shows the ratio of the inflow velocity to the tissue velocity providing insight into the elastic properties of the ventricle. In essence, the E wave velocity controls for cardiac output, heart rate, and filling so that the ratio (E/Ela) correlates with left atrial pressure. Here, the E/Ela shows evidence of the transgene altering the typical progression of diastolic dysfunction based on a significant interaction effect (P < 0.05). These data indicate that cTnI A164H Tg mice are able to retain improved diastolic function during the ageing process compared with Ntg mice which experience predictable age-related diastolic dysfunction.
The relationship between cTnI A164H and diastolic performance, however, is complex as indicated by differences in echocardiographic and micromanometry data at baseline. Conflicting data based on the measures of isovolumic relaxation (Tau) provide inconclusive evidence regarding baseline diastolic function in Tg mice, which is consistent with previous findings.15
However, the mechanical component of relaxation during the late (filling) phase of diastole, as measured non-invasively by DTI indicates that Ntg mice have compromised viscoelastic properties of the ventricle resulting in a decline in diastolic function during the ageing process which is significantly attenuated in Tg mice.
Taking this whole organ functional analysis into consideration, it has been established that, at the sub-cellular level, changes in the expression of calcium-handling proteins contribute to the progression of pump dysfunction during ageing.34
Our study supports these findings specifically with regard to the observation that SERCA2a levels are diminished at 2 years of age in Ntg and Tg mice. Reduced expression of SERCA2a has been implicated directly in the progression of age-induced cardiomyopathy26
and provides a sub-cellular basis, at least in part, for the whole organ diastolic dysfunction observed in this study during ageing. The lack of any genotypic
differences in CSQ, NCX and the SERCA2a to PLN stoichiometry during ageing suggests an alternative mechanism for improvement of cardiac function in the Tg mice during the ageing process. We propose that this mechanism is predominantly the result of increasing inotropy by molecular manipulation of myofilament performance by means of cTnI A164H.
The results of this study show that Tg hearts have reduced SR calcium loading. Previous reports have found that aged hearts are more susceptible to calcium overload than young hearts.3,35,36
This suggests that a reduced SR Ca2+
load may benefit cTnI A164H hearts in ageing, similar to our recent report in the context of myocardial injury such as ischaemia/reperfusion.15
We hypothesize that the lower SR Ca2+
load and Ca2+
transient are made possible by myofilament activation enhancement by cTnIA164H. We propose that there is an interplay between the myofilament enhancement and SR functionality that could account for higher SERCA2a levels in Tg hearts.
The cardiac functional readout of these findings at the sub-cellular level is seen in the whole organ serial echocardiographic analysis of Ntg and Tg mice during the 2-year ageing process. The decline in cardiac contractility (e.g. EF) increase in LV chamber geometry and development of diastolic dysfunction (e.g. increased E/Ela) particularly evident at the 2-year time point are likely the consequence, at least in part, of the changes observed in calcium-handling proteins.
In conclusion, ischaemia-related cardiac dysfunction in aged populations remains a significant cause of morbidity and mortality. Therapies for ischaemic heart disease and heart failure could be directed specifically towards improving myofilament function. The consequent improvement in contractility that results from increased myofilament performance may appear to counter the logic of commonly prescribed therapeutics, which call for the use of beta blockers that decrease contractility acutely, allowing for reduced oxygen consumption and energy expenditure. Although there is proven value in the diverse effects of beta blockers, we have shown that targeted alteration of the myofilaments to increase contractile performance is an effective mechanism for treatment of ischaemic heart disease and heart failure in small mammals.15
This is in concurrence with Mann and Bristow’s view that augmentation of myofilament responsiveness to calcium would improve the force-generating capacity of the sarcomere and thus redress global cardiac dysfunction.37
The present study, together with previous work,15
strengthens the hypothesis that altering the functionality of troponin I is an effective means of specifically augmenting myofilament function and consequently enhancing cardiac contractility. Adding to the growing evidence for the therapeutic role of histidine-modified TnI in the heart, this study provides new evidence that specific replacement of native cTnI with cTnI A164H in the adult heart protects cardiac function during ageing. We propose that the progression of pathologic and age-related diminutions in cardiac function may be improved by myofilament- based molecular therapeutics for increasing cardiac performance.