Introduction

It is estimated that by 2035, nearly one in four individuals in the United States will be 65 years of age or older. Hypertension, atherosclerosis, and resultant chronic HF reach epidemic proportions among older persons, and the clinical manifestations and the prognoses of these worsen with increasing age. The reason is that, in older individuals, specific pathophysiological mechanisms that underlie these diseases become superimposed on heart and vascular substrates that are modified by the process of aging. In other words, cardiovascular aging is “risky.” An understanding of how age per se modifies cardiovascular structure and function is critical to the prevention or treatment of cardiovascular diseases in the older person.

Cardiac aging

Cellular and molecular mechanisms implicated in age-associated changes in myocardial structure and function in humans have also been studied largely in rodents (Table 1). The altered cardiac structural phenotype that evolves with aging in rodents includes an increase in LV mass due to an enlargement of myocyte size [17] and focal proliferation of the matrix in which the myocytes reside, which may be linked to an altered cardiac fibroblast number or function. The number of cardiac myocytes becomes reduced because of necrosis and apoptosis, with the former predominating [2]. Putative stimuli for cardiac cell enlargement with aging in rodents include an age-associated increase in vascular load due to arterial stiffening and stretching of cells caused by drop out of neighboring myocytes [39]. Stretch of cardiac myocytes and fibroblasts initiates growth factor signaling (e.g. angiotensin II/TGF-ß) that, in addition to modulating cell growth and matrix production, leads to apoptosis [13]. The expression of atrial natriuretic [78] and opioid [11] peptides, molecules that are usually produced in response to chronic stress, is increased in the senescent rodent heart.

Table 1 Myocardial changes with adult aging in rodents
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Reduced acute response to stress

Acute excess myocardial Ca2+ loading leads to dysregulation of Ca2+ homeostasis, impaired diastolic and systolic function, arrhythmias, and cell death [37]. The cell Ca2+ load is determined by membrane structure and permeability characteristics, the intensity of stimuli that modulate Ca2+ influx or efflux via their impact on regulatory function of proteins within membranes, and ROS, which affect both membrane structure and function. Excessive cytosolic Ca2+ loading occurs during physiological and pharmacological scenarios that increase Ca2+ influx (e.g. neurotransmitters, postischemic reperfusion, or oxidative stress) [23, 40]. In hearts or myocytes from the older heart, enhanced Ca2+ influx, impaired relaxation, and increased diastolic tone occur during pacing at an increased frequency [10, 45, 67, 79]. This is a “downside” of the aforementioned age-associated adaptation that occurs within the cells of senescent heart (and also of young animals chronically exposed to arterial pressure overload). Causes of reduced Ca2+ tolerance of the older heart include changes in the amounts of proteins that regulate Ca2+ handling, caused in part by altered gene expression (Tables 1 and 2), and an age-associated alteration in the composition of membranes in which Ca2+ regulatory proteins reside, which includes an increase in membrane ω6:ω3 polyunsaturated fatty acids (PUFAs) [55]. ω3 PUFAs are protective of cardiac Ca2+ regulation. An additional potential cause of the reduced threshold of senescent myocytes for Ca2+ overload is an enhanced likelihood for intracellular generation of ROS [40, 48] in cells from the senescent versus the younger adult heart during stress. In this regard, the older cardiac myocyte and endothelial cells [69] share common “risks” with aging.

Table 2 Age-associated changes in the expression of Ca2+-transporting proteins
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Cellular ß-adrenergic signaling

Age-associated deficits in the myocardial ßAR signaling cascade also occur with aging (see [38] for review). A reduced myocardial contractile response to either ß1AR or ß2AR stimulation is observed with aging [57, 73, 74]. This is due to failure of ß-adrenergic stimulation to augment Ca 2+i to the same extent in cells of senescent hearts that it does in those from younger adult hearts [73] an effect attributable to a deficient increase of L-type sarcolemmal Ca2+ channel availability, which leads to a lesser increase in Ca2+ influx [73]. The richly documented age-associated reduction in the postsynaptic response of myocardial cells to ß-adrenergic stimulation seems to be due to multiple changes in the molecular and biochemical steps that couple the receptor to postreceptor effectors. However, the major limiting modification of this signaling pathway that occurs with advancing age in rodents seems to be the coupling of the ß-AR to adenylyl cyclase via the Gs protein and changes in adenylyl cyclase protein, which lead to a reduction in the ability to sufficiently augment cell cAMP and to activate protein kinase A (PKA) to drive the phosphorylation of key proteins that are required to augment cardiac contractility [31, 74]. In contrast, the apparent desensitization of ß-adrenergic signaling that occurs with aging does not seem to be mediated via increased ß-AR kinase or increased Gi activity [74]. A blunted response to ß-adrenergic stimulation of the cells within older myocardium can, in one sense, be viewed as adaptive with respect to its effect to limit the risk of Ca2+ overload and cell death in these cells.

Reduced chronic adaptive capacity of the older heart

Many of the multiple changes in cardiac structure, excitation, myofilament activation, contraction mechanisms, Ca2+ dysregulation, deficient ß-AR signaling, and altered gene expression of proteins involved in EC coupling that occur with aging (Tables 1 and 2) also occur in the hypertrophied myocardium of younger animals with experimentally induced chronic hypertension [38, 77] and in failing animal or human hearts, in which they have been construed as an adaptive response to a chronic increase in LV loading. When chronic mechanical stresses that evoke substantial myocardial hypertrophy (e.g. pressure or volume overload) are imposed on the older heart, the response in many instances is reduced. There is evidence that transcriptional events associated with hypertrophic stressors become altered with advancing age, e.g. the nuclear binding activity of the transcription factor nuclear factor-κB is increased and that of another transcription factor, Sp1, is diminished [24]. Because these transcription factors each influence expression of a number of genes, they may contribute to the pattern of gene expression observed in the hearts of senescent rodents and may also dictate the limits of adaptive responses to the imposition of additional chronic stress. The acute induction of both immediate early genes and later responding genes that are expressed during the hypertrophic response is blunted in hearts of aged rats after aortic constriction [60, 66]. Similarly, the acute induction of heat shock 70 protein genes in response to either ischemia or heat shock is reduced in hearts of senescent rats [9, 52]. A similar loss of adaptive capacity is observed in younger rats that have used a part of their reserve capacity before a growth factor challenge [61].

Molecular and functional changes of Ca2+-handling proteins in the aging heart

The SR Ca2+ release and uptake play key role in the regulation of cardiac contraction and relaxation. The SR Ca2+-transporting proteins include the sarcoplasmic reticular Ca2+-ATPase (SERCA2), its inhibitory protein phospholamban (PLB), the Ca2+-storage protein calsequestrin (CSQ), and the SR-Ca2+ release channel (ryanodine receptor; RYR). The SR Ca2+ cycling is further modulated by Ca2+ influx through LCC and by Ca2+ transport via NCX.

SR Ca2+ pump

Sequestration of Ca2+ by the SERCA2 pump serves a dual function: (1) to cause muscle relaxation by lowering the cytosolic Ca2+ and (2) to restore SR Ca2+ content necessary for muscle contraction. The age-associated reduction in SERCA2 mRNA levels is well documented (Table 1). As summarized in Table 2, the majority of studies in aging vs. younger rats have shown a significant reduction in protein levels of SERCA2 [5, 58, 65, 80]. On the other hand, most studies in aging mice have shown unchanged levels of SERCA2 [27, 46, 63]. Apart from the phosphorylation status of SERCA2-PLB complex, discussed later, SR Ca2+ uptake is dependent on the relative levels of both proteins, i.e. reduced at lower SERCA2/PLB ratio [50]. The majority of studies reporting expression levels of both SERCA2 and PLB showed reduced SERCA2/PLB ratios in aging rodent hearts (Table 2). Increasing SERCA/PLB ratio through in vivo gene transfer of SERCA2a markedly improved rate-dependent contractility and diastolic function in 26-months-old rat hearts [58]. Functional improvement consequent to increasing SERCA/PLB ratio by PLB suppression was also reported in human failing myocytes [14]. On the other hand, PLB ablation in transgenic mouse models of HF was beneficial only in some models (reviewed in [4] and [50]).

Age-associated decline in the Ca2+-sequestering activity of SERCA2 in rodent myocardium has been well documented both by biochemical studies in isolated SR vesicles and by biophysical studies in cardiac preparations [18, 35, 58, 65, 76]. Apart from reduced content of SERCA2 or SERCA2/PLB ratio, discussed earlier, lower pumping activity of SERCA2 in aging myocardium may result from reduced phosphorylation of the SERCA2-PLB complex. Specifically, in its unphosphorylated state, PLB interacts with SERCA2 exerting an inhibitory effect manifested largely through a decrease in the enzyme’s affinity for Ca2+. Phosphorylation of PLB by PKA and/or Ca2+/calmodulin-dependent protein kinase (CaMK) is thought to disrupt this interaction resulting in enhanced affinity of the ATPase for Ca2+ and stimulation of Ca2+ pump activity [50]. Besides PLB, CaMK has been suggested to modulate the SR Ca2+ uptake and release through direct phosphorylation of SERCA2 [75]. The well established deficits in ß-AR signaling that occur in aging humans and animals [38] include significantly lower PKA-dependent phosphorylation of PLB in aged vs. adult rat ventricular myocardium [31]. Recent studies have also shown that significant age-associated decrements occur in (1) the amount of CaMK (δ-isoform) in the rat heart, (2) the endogenous CaMK-mediated phosphorylation of SERCA and PLB, and (3) the phosphorylation-dependent stimulation of SR Ca2+ sequestration [76]. Increased activity of the SR-associated phosphatase PP1, which dephosphorylates PLB, had already been reported, and overexpression of PP1 in transgenic mice resulted in HF. PP1 activity was further shown to be regulated by the inhibitor I-1, and I-1 was found to be reduced in human HF (reviewed in [62]). However, potential age-related changes in the activity of cardiac phosphatases have yet to be examined.

SR Ca2+ release channel

Besides SERCA2, the RYR is a major determinant of the SR Ca2+ release, regulated by its protein expression and gating properties during Ca2+-release triggered by Ca2+ influx through LCC, as well as during cardiac relaxation and diastole. Accordingly, alterations in the expression or function of RYR have been implicated in both systolic and diastolic dysfunction of the aging heart. Reduced protein expression of cardiac RYR has been reported in aging Wistar rats [5], but not Fisher 344 rats [58, 76, 80] (Table 2). The RYR is phosphorylated by PKA and CaMK, and a significant reduction in the CaMK-mediated phosphorylation of the RYR has been shown to occur in the aged compared with adult Fisher 344 rats [76].

Single channel properties of RYR and unitary SR Ca2+ release events (Ca2+ sparks) in ventricular cardiomyocytes were recently examined in hearts from 6- to 24-month-old Fisher 344 rat [80]. Senescent myocytes displayed a decreased Ca 2+i transient amplitude and an increased time constant of the Ca 2+i transient decay, both of which correlated with a reduced Ca2+ content of the SR. Senescent cardiomyocytes also had an increased frequency of spontaneous Ca2+ sparks and a slight but statistically significant decrease in their average amplitude, full-width-at-half-maximum and full-duration-at-half-maximum. Single channel recordings of RYR demonstrated that in aging hearts, the open probability of RYR was increased but the mean open time was shorter, providing a molecular correlate for the increased frequency of Ca2+ sparks and decreased size of sparks, respectively [80]. These results suggest modifications of normal RYR gating properties associated with increased sensitivity of RYR to resting and activating Ca2+ that may play a role in the altered Ca2+ homeostasis observed in senescent myocytes. Another recent study [26] examined the effects of aging on whole cell electrically stimulated Ca2+ transients and Ca2+ sparks at 37°C in ventricular myocytes isolated from young adult (~5 mo) and aged (~24 mo) B6SJLF1/J mice of both sexes. The results showed reduced amplitude and abbreviated rise time of the Ca 2+i transient in aged cells stimulated at 8 Hz and markedly higher incidence and frequency of spontaneous Ca2+ sparks in aged vs. young adult cells. Spark amplitudes and spatial widths were similar in both age groups. However, spark half-rise times and half-decay times were abbreviated in aged cells compared with younger cells. Neither resting Ca 2+i levels nor SR Ca2+ content differed between young adult and aged cells, indicating that increased spark frequency in aging cells was not attributable to increased SR Ca2+ stores and that decrease in the Ca 2+i transient amplitude was not due to a decrease in SR Ca2+ load. These results suggest that alterations in SR Ca2+ release units occur in aging ventricular myocytes and raise the possibility that alterations in Ca2+ release may reflect age-related changes in fundamental release events rather than changes in SR Ca2+ stores and/or diastolic Ca 2+i levels.

Differences in the characteristics of Ca2+ sparks (and the SR Ca2+ content) reported in these experiments [26, 80] might be partly related to differences in species and experimental conditions (e.g. temperature) employed. However, consistent with previous findings [23], both these studies [26, 80] have shown increased frequency of spontaneous Ca2+ sparks in aging ventricular myocytes. The resulting increased Ca2+ leak from the SR may reduce the net rate of SR Ca2+ sequestration. Functional consequences of the latter include (1) slower decline of the Ca 2+i transient and increased diastolic Ca 2+i (diastolic dysfunction), (2) reduced SR Ca2+ load available for release (systolic dysfunction), and (3) reduced threshold for myocardial cell Ca2+ intolerance [23, 37].

The PKA-mediated hyperphosphorylation of RYR, resulting in abnormal SR Ca2+ leak through the RYR, has been implicated in both diastolic and systolic dysfunction of the failing heart [51]. However, more recent evidence points to CaMKII site phosphorylation of RYR in normal cardiac tissue [22, 43], and a potential role and mechanism for PKA modulation of this process in the pathophysiology of HF associated with aging remains lacking.

Calsequestrin

Reports have consistently shown that aging does not alter CSQ expression at either the transcriptional (Table 1) or protein level (Table 2) [58, 65, 76, 80].

L-type Ca2+ channel

Ca2+ influx of via LCC has a dual role in cardiac EC coupling: peak L-type Ca2+ current (ICaL) provides the primary “trigger” for SR Ca2+ release, while the integrated Ca2+ entry replenishes the SR Ca2+ content available for release. The effects of aging on protein expression of LCC have not been systematically studied. Electrophysiological examinations have shown that both the apparent number and the activity of individual cardiac LCCs increase with advanced age in the rat [32]. The age-associated changes in ICaL are summarized in Table 3.

Table 3 Age-associated changes in the L-type Ca2+ current
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Maintained peak density of ICaL has been shown in ventricular myocytes from senescent (21–25 months) vs. young (2–3 months) male Wistar rats [71, 73], and in 20–22 vs. 10- to 12-mo-old male FVB mice [68]. ICaL inactivates more slowly in myocytes from older vs. younger Wistar rats [32, 71], and this, as well as reductions in peak transient outward K+ current (ITO) [71], might partially account for prolongation of the AP reported in senescent Wistar and Fisher 344 rat hearts [71, 72]. Experiments in ventricular myocytes isolated from young adult (6 mo) and aged (>27 mo) Fischer 344 and Long-Evans rats showed an age-associated decrease in peak density of ICaL accompanied by a slower inactivation, and a greater amplitude of ITO [47]. Compared to young myocytes, AP duration in aged myocytes was longer at 90% of repolarization but shorter 20 and 75% of repolarization [47]. In ventricular myocytes isolated from young (2 mo) and senescent (20–27 months) C57/BL6 mice (sex unspecified), peak ICaL density was similar at stimulation rates of 2–8 Hz, but higher in aged group at 0.4 and 1 Hz [27]. These experiments also showed that the ICaL time integral (a function of peak amplitude and inactivation rate) normalized to cell capacitance was similar in both age groups under 2–6 Hz stimulation. Compared to young cells, ICaL time integral in aged myocytes was significantly smaller at 8 Hz and larger at 0.4 Hz [27]. Experiments in ventricular myocytes isolated from young adult (~7 mo) and aged (~24 mo) male and female B6SJLF1/J mice [21] stimulated at 2 Hz, showed a significant reduction in peak ICaL density, accompanied by a significantly slower inactivation, in aged vs. young adult myocytes from males. No age-related changes in ICaL characteristics were found in the female group. In myocytes isolated from the hearts of young (18 months) and aged (>8 years) female sheep, the AP duration and both the peak ICaL and integrated Ca2+ entry were significantly greater in aged cells [15].

Ca2+ influx via LCC is a complex function of several interdependent mechanisms, including (1) voltage-dependent modulation, (2) Ca2+-dependent modulation via direct binding to LCC of Ca2+-calmodulin and via CaMKII, and (3) β-adrenergic modulation via PKA. In aging myocardium, voltage-dependent changes may be consequent to prolongation of the AP duration and manifested by reduced peak amplitude accompanied by slower inactivation/larger time integral of ICaL (e.g. [29]). Ca2+-mediated effects may contribute to frequency-dependent reduction in the amplitude and time integral of ICaL (e.g. [27]) due to rate-dependent diastolic Ca2+ accumulation, which has been shown to slow the rate of LCC recovery from inactivation in both normal and failing cardiac myocytes (reviewed in [7]). In addition, Ca2+-mediated crosstalk between LCC and RYR, well established in normal myocardium [7], may facilitate Ca2+ influx via LCC in the presence of slower and/or smaller SR Ca2+ release in aging myocardium. For instance, buffering of Ca 2+i with EGTA eliminated age-related differences in the AP configuration and the time course of ICaL inactivation in myocytes from senescent and young rats [71]. As discussed earlier, aging is associated with deficits in the myocardial ß-AR signaling cascade [38] that includes reduced stimulation of the ICaL [73] through PKA-mediated changes in the availability and gating properties of LCC.

Na+–Ca2+ exchanger

The NCX serves as the main transsarcolemmal Ca2+ extrusion mechanism and is centrally involved in the beat-to-beat regulation of cellular Ca2+ content and cardiac contractile force, including regulation of the AP configuration in the late repolarization phase and the later Ca2+ clearance phase of the Ca 2+i transient. Thus, alterations in NCX activity may contribute to the prolongation of both the AP duration and relaxation in aging myocardium (Lakatta, 1993). Age-related increase in the NCX expression has been demonstrated at the transcriptional level (Table 1). However, protein levels of NCX reported in aging rodent hearts were unchanged [44, 49, 58] or reduced [5, 28, 46] (Table 2).

The results of experiments using enriched sarcolemmal vesicles or muscle strips isolated from rats were also inconsistent, i.e. the NCX activity in aged myocardium was decreased [25, 28], increased [19], or unchanged [1]. More recent functional assessments of NCX activity in cardiac myocytes isolated from young (14–15 mo) and aged (27–31 mo) male Fischer Brown Norway rats [49] showed that under conditions where membrane potential and intracellular [Na+] and [Ca2+] could be controlled, “forward” NCX activity was increased in aged vs. young cells. The increased “forward” NCX activity was interpreted as a factor contributing to the late AP prolongation in aging myocardium [49]. Such increased Ca2+ efflux via NCX would compensate for increased Ca2+ influx via LCC [15, 32, 71]. Prolongation of the AP consequent to reduced ITO [71] may temporarily limit “forward” NCX during relaxation, allowing better SR Ca2+ reuptake by SERCA2 [29] and Fig. 2.

Excitation–contraction coupling in the aging heart

Coordinated changes in the expression and function of proteins that regulate several key steps of the cardiac EC coupling process occur in the rodent heart with aging and typically result in a prolonged AP, a prolonged Ca 2+i transient, a prolonged contraction, and blunted inotropic and lusitropic responses to increased pacing rate (Figs. 1, 3 and 4).

Fig. 1
figure 1

Differences in various aspects of excitation–contraction coupling mechanisms measured between adult (6–9 months) and senescent (24–26 months) rat hearts. a Transmembrane action potential and b isometric contraction (from [72]). c Cytosolic calcium transient, measured by a change in the luminescence of aequorin injected into several cells comprising the muscle preparation (from [54]). d Sarcoplasmic reticulum Ca2+ uptake rate. (From [18].)

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Age-related prolongation of the AP [15, 49, 71, 72] is thought to stem, in part at least, from increased number and the activity of individual cardiac L-type Ca2+ channels [32], slower inactivation of ICaL [15, 32, 71], and reductions in outward K+ currents [71]. Additionally, the late repolarization phase of the AP may be prolonged by increased Ca2+ efflux via NCX [49]. Of particular interest is the role of ITO as an indirect modulator of EC coupling in cardiac cells (reviewed by Bassani [6]). Specifically, recent studies have provided evidence that the early repolarization phase may considerably influence the entire AP waveform and that ITO is the main current responsible for this phase. Decreased ITO density is observed in immature and aging myocardium, as well as during several types of cardiomyopathy and HF, i.e. under conditions in which SR function is depressed. The resulting AP prolongation favors Ca2+ influx during the depolarization and limits voltage-dependent Ca2+ efflux via NCX and thus may be adaptive since it provides partial compensation for SR deficiency, although possibly at the cost of asynchronous SR Ca2+ release and greater propensity to triggered arrhythmias [6]. The imposition of a shorter AP to myocytes from the old rat heart reduces the amplitude and the rate of decline of the Ca 2+i transient [29]. This is attributable to a reduction in the SR Ca2+ uptake and loading which, in the presence of a reduced rate of Ca2+ sequestration by SERCA2, is presumably due to a reduced ICaL time integral and likely also to an increased net Ca2+ extrusion via NCX (Fig. 2) [29].

Fig. 2
figure 2

Schematic representation of the role of age-dependent action potential (AP) prolongation on L-type Ca2+ current (ICa) and intracellular Ca2+ (Ca 2+i ) regulation in rat ventricular myocytes. Compared to young rats (6 mo; left) old rats (24 mo; middle) display a similar amplitude of the Ca 2+i transient triggered by a markedly prolonged AP. Relative to stimulation with their long, native AP, stimulation of old rat ventricular myocytes with a short, “young type” AP (right) increases the amplitude of L-type Ca2+ current (ICa) but reduces its time integral and diminishes the amplitude and rate of relaxation of the Ca 2+i transient. Thus, it appears that ventricular myocytes of old rats utilize AP prolongation to sustain youthful Ca 2+i regulation. (From [29])

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A slower decay of the Ca 2+i transient is a hallmark of aged cardiac myocyte (Figs. 1, 2, 3, 4, and Table 1). Reduction in the amplitude of the Ca 2+i transient in myocytes from aged hearts, compared to younger counterparts, has been reported in some studies already at low (≤2 Hz) stimulation rates [21, 80]. Examinations using a range of stimulation rates [27, 45] typically showed blunted force- and relaxation-frequency responses in myocytes from old vs. young hearts. Thus, while the age-related differences in the amplitude and the rate of decay of the Ca 2+i transients (and diastolic Ca 2+i levels) were small or absent at low stimulation rates, they became apparent and progressively larger at pacing rates approximating those in vivo (Fig. 3) [27, 45]. Likewise, abrupt changes in the stimulation rate reveal an impaired SR Ca2+ release in ventricular myocytes isolated from senescent vs. young rats (Fig. 4). Specifically, in the presence of similar kinetics of ICaL recovery, reduction in the amplitude of the Ca 2+i transients and the gain of ICaL-dependent Ca2+ release during premature depolarizations are attributable to a slower rate of SR Ca2+ reuptake in older myocytes (Fig. 4). Taken together, these results are consistent with a reduced adaptive capacity of the older heart to physiological stress, i.e. pacing rate and β-adrenergic stimulation (see [38] for review), and underscore the value of appropriate experimental design in examining potential age-related differences in cardiac phenotype. Another characteristic of age-related changes in the configuration of the Ca 2+i transient, reported in several (Fig. 1) and [27] but not all [21, 45] studies, is a prolonged time to peak Ca 2+i .

Fig. 3
figure 3

Top panels: a Averaged tracings of percentage cell shortening and [Ca2+]i recorded at 37°C from a representative adult myocyte paced at 2, 4, 7, and 9 Hz. B Normalized tracings of cell shortening (top panel) and [Ca2+]i (bottom panel) from a representative adult (black line) and senescent (gray line) myocyte paced at 9 Hz; tracings were normalized to peak value to show changes in time course. Cell shortening and [Ca2+]i were acquired at 4.2 and 2 ms sampling rates, respectively. To reduce the signal-to-noise ratio, each tracing was depicted as an average of ten original tracings. Percentage cell shortening (C), time constant (τ) of cell relengthening (D), calcium transient amplitude (E), and time constant (τ) of the intracellular calcium transient decay (F) in adult (solid line; n = 4) and senescent (dashed line; n = 5) myocytes at increasing pacing rates. Myocyte data from four adult and five senescent hearts were averaged and expressed as mean ± SEM (~4–5 myocytes were obtained from each heart). * P < 0.05 between adult and senescent curves (two-factor ANOVA). (From [45] with permission.)

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Fig. 4
figure 4

Recovery of the L-type Ca2+ current (ICaL) and the intracellular Ca2+ (Ca 2+i ) transient following a prior depolarization. A, Recordings of Ca 2+i transients (top) and ICaL (middle), induced by voltage-clamp depolarizations (bottom) from −75 to 0 mV (with Na+ current, K+ currents and “reverse” Na+–Ca2+ exchange blocked) in a representative ventricular myocyte isolated from young adult (6 mo) Wistar rat. Test pulse intervals of 75–300 ms duration were applied following a train of 9 conditioning voltage pulses (50 ms, from −75 to 0 mV at 0.5 Hz). B, Averaged data from these experiments in myocytes from young (n = 5) and old (24 mo; n = 7) rats, show slower rate of Ca 2+i decline during the last conditioning pulse, and similar kinetics of ICaL recovery but prolonged recovery time of the Ca 2+i transient and “gain” of ICaL-dependent Ca2+ release (Ca 2+i /ICaL) during premature depolarizations in old vs. young myocytes. (From AM Janczewski and EG Lakatta, unpublished results.)

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In cardiac myocytes, the development of the Ca 2+i transient is dependent primarily on the amount and the rate of Ca2+ release from the SR, and the decline of the Ca 2+i transient and the amount of Ca2+ available for subsequent release are dependent primarily on Ca2+ sequestration by the SR. Age-related reduction in the rate of rise and the amplitude of the Ca 2+i transient (systolic dysfunction), as well as the rate of decline of the Ca 2+i transient (diastolic dysfunction), appear to result, in large part, from impaired Ca2+ pumping by SERCA2. These changes have been extensively documented in biochemical and functional studies [18, 35, 58, 65, 76]. At the molecular level, they are attributable to a reduced protein expression of SERCA2 or its ratio to PLB (Table 2) and/or reduced phosphorylation of the SERCA2-PLB complex by PKA and CaMK [31, 75, 76]. A shift of SERCA2b distribution to the subsarcolemmal space has also been suggested [27]. Age-related alterations in the gating properties of RYR [26, 80], resulting in an increased SR Ca2+ leak, may also contribute to both diastolic and systolic dysfunction of the aging myocardium by limiting the net rate of SR Ca2+ sequestration and SR Ca2+ loading, respectively. Finally, a slower rate of development/longer time to peak of the Ca 2+i transient in aging myocytes is likely to result from reduced SR Ca2+ loading but may be also consequent to a longer time to peak ICaL [71], which synchronizes SR Ca2+ release.

Prolonged time to peak and slower relaxation of contraction, typical for aged myocardium (Figs. 1 and 3) [21, 45, 46, 64, 67, 80] are attributable to both changes in the αMHC and βMHC protein ratio (Table 1) [38] and in the configuration of the Ca2+ transient. Clearly, the latter underlies impaired frequency-dependent inotropic and lusitropic responses [21, 45, 46, 58, 59] that largely contribute to the systolic and diastolic dysfunction of the aging heart. Consistent with the major role of SERCA2 in these effects, studies in rat isolated cardiac muscle preparations have shown that exercise training reverses age-associated slowing of contraction and relaxation [64, 67]. This was associated with increased Ca2+ transport by SERCA2 but not myosin ATPase activity in cardiac homogenates [67]. Likewise, overexpression of SERCA2 markedly improved rate-dependent contractility and contractile function in senescent rat hearts [58].

Sex-related differences

Biological sex is a well-recognized factor in the physiology and pathophysiology of the cardiovascular system, including the aging heart (reviewed in [41] and [36]). However, despite the growing number of reports in the literature identifying sex-related differences in cardiac function in both humans and rodents, the underlying mechanisms remain incompletely understood.

New insights into the molecular bases of sexual dimorphism in the context of aging and cardiovascular disease have been recently afforded by large-scale analyses of gene expression at the transcriptional level using microarrays. Specifically, Boheler and colleagues [8] have identified several HF-dependent gene products that may act as potential regulators for transducing mechanical, stress and neurohormonal stimuli into changes in gene expression. Most HF-candidate genes demonstrated significant changes in gene expression; however, the majority of differences among samples depended on variables such as sex and age, and not on HF alone. Some HF-responsive gene products also demonstrated highly significant changes in expression as a function of age and/or sex, but independent of HF. These results emphasize the need to account for biological variables (HF, sex, and age interactions) to elucidate genomic correlates that trigger molecular pathways responsible for the progression of HF syndromes. Subsequent analysis of gene expression differences by sex and age in left ventricular samples from patients with dilated cardiomyopathy has identified more than 1,800 genes displaying sexual dimorphism in the heart. A significant number of these genes were highly represented in gene ontology pathways involved in ion transport and G protein-coupled receptor signaling [16].

Postmortem morphometric assessments in non-failing human hearts have shown extensive age-related myocyte loss and hypertrophy of the surviving myocytes in males, but preserved ventricular myocardial mass, average cell diameter, and volume in aging female hearts [53]. These sex differences may stem, in part, from differences in the replicative potential of cardiac myocytes. For instance, the activity of telomerase, an enzyme present only during cell replication, was decreased 31% in aging male rat myocytes, but increased 72% in female counterparts [42]. Premature development of HF or death in males compared to matching females has been documented in rat models of pressure overload and/or myocardial infarction (reviewed in [36]). Sexually dimorphic cardiac phenotypes have been also discovered in some studies in genetically engineered mice (Table 4) [36, 41]. In general, transgenic models of HF present a more rapid onset and/or a greater severity of cardiac dysfunction in male vs. female hearts. For instance, mice with cardiac-specific overexpression of tumor necrosis factor-α (TNF-α) exhibit HF and increased mortality that is markedly higher in young males than females (~50% and 4%, respectively, by 20 weeks of age) [33]. At 12 weeks of age, female mice displayed LV hypertrophy without dilatation and only a small reduction of basal LV fractional shortening and response to isoproterenol (Iso), while male mice showed a large LV dilatation, reduced fractional shortening relative to both wild-type littermates and transgenic females, and minimal response to Iso [30]. Cardiac myocyte hypertrophy was similar in male and female transgenic mice. Compared to wild-type mice, myocytes from female TNF-α transgenic mice displayed a slower decline of the Ca 2+i transient, but similar amplitudes of Ca 2+i transients and contractions and the inotropic response to Iso. In contrast, the amplitude and the rate of decline of Ca 2+i transients and contractions, and the response to Iso were significantly reduced in myocytes from male transgenic TNF-α mice [30].

Table 4 Summary of male- and female-related differences in genetically manipulated mice (From [41] with permission.)
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These studies underscore the value of considering biological sex and age in the assessment of cardiac phenotypes. However, inspection of the literature shows that the majority of studies in animal models of human cardiac disease has been carried out only in males or do not indicate which sex was studied. The same applies to studies of myocardial changes with adult aging, including molecular changes (Table 2). Remarkably, the available examinations of cardiac EC coupling in rodents of both sexes [21] or females only [70] show a lack of age-related changes in the configuration of the Ca 2+i transient in myocytes from female hearts. Specifically, Grandy and Howlett [21] have reported a significant reduction in the ICaL density, amplitudes of the Ca 2+i transients, fractional cell shortening, and the SR Ca2+ content in ventricular myocytes isolated from the hearts of aged (~24 mo) vs. young adult (~7 mo) male mice. In contrast, ICaL density, the amplitude of Ca 2+i transients and fractional cell shortening were similar in young adult and aged myocytes from female hearts, while the SR Ca2+ content was increased in the aged female group. Accordingly, fractional SR Ca2+ release was similar in both age groups of male myocytes but reduced in aged vs. young adult female myocytes. The gain of EC coupling was similar in young adult and aged myocytes, regardless of the sex of the animal. The somewhat unexpected findings of this study in aged female myocytes include (a) an apparent increase in the SR Ca2+ content, (b) the lack of the effect of the latter on the configuration of the Ca 2+i transients and contraction, and (c) a reduction of fractional SR Ca2+ release, in the presence of unchanged ICaL. Nevertheless, these results provide an initial direct demonstration of sexually dimorphic changes in cardiac EC coupling associated with normal adult aging. Interestingly, the ICaL and the amplitude and the rate of decline of the Ca 2+i transients were significantly increased in cardiac myocytes isolated from aged (>8 years) sheep vs. young (18 months) female sheep [15]. However, a potential sex aspect of this finding has not been explored.

Myocyte progenitors in the aging heart

Studies by Anversa and colleagues (reviewed in [3, 34]) have demonstrated that the heart is a self-renewing organ containing a pool of progenitor cells (PCs) that dictate cell turnover, organ homeostasis and myocardial aging.

Observations in humans and animals suggest that myocyte maturation and aging are characterized by loss of replicative potential, telomeric shortening and the expression of the senescence-associated protein/cell cycle inhibitor p16INK4a [12, 20, 56]. Telomeric shortening in PCs leads to generation of progeny that rapidly acquires the senescent phenotype. As discussed earlier, the latter involves progressive increase in the size of the cell (up to a critical volume beyond which myocyte hypertrophy is no longer possible), deficits in the electrical, Ca2+ cycling and mechanical properties, and cell death. Cardiac myocytes with senescent and non-senescent phenotypes already coexist at young age [56]. However, aging limits the growth and differentiation potential of PCs, thus interfering not only with their ability to sustain physiological cell turnover but also with their capacity to adapt to increases in pressure and volume loads [3, 34].

The loss of PC function with aging is mediated partly by an imbalance between factors enhancing oxidative stress, telomere attrition and death, and factors promoting growth, migration, and survival. Recent findings suggest a preeminent position of insulin-like growth factor-1 (IGF-1) among factors that interfere with cardiac cellular senescence. Specifically, cardiac-restricted overexpression of IGF-1 in transgenic mice has been shown to delay the aging myopathy and the manifestations of HF [68] and to restore SERCA2a expression and rescue age-associated impairment of cardiac myocyte contractile function [44]. The latter effect was also partly mimicked by short-term in vitro treatment with recombinant IGF-1 [44]. Furthermore, intramyocardial delivery of IGF-1 improved senescent heart phenotype in male Fisher 344 rats [20], including increased proliferation of functionally competent PCs and diminished angiotensin II-induced apoptosis. Myocardial regeneration mediated by PC activation attenuated ventricular dilation and the decrease in ventricular mass-to-chamber volume ratio, resulting in improvement of in vivo cardiac function in animals at 28 to 29 months of age [20].

Thus, understanding the biology of cardiac PCs, including factors enhancing the activation of the PC pool, their mobilization, and translocation may facilitate the development of novel strategies to prevent or reverse the diminished adaptive capacity to increases in pressure and volume loads (and perhaps HF) in the old population.

Summary

The occurrence, clinical manifestations and prognoses of HF worsen with increasing age because the underlying pathophysiological mechanisms become superimposed on heart and vascular substrates that are modified by the process of aging. Changes in the cardiac cell phenotype associated with aging include (1) enlargement of the myocyte size, consequent to increase in vascular load and loss of neighboring myocytes, (2) reduced acute response to stress, consequent to deficits in ß-AR signaling, changes in the composition of the cell membranes and enhanced intracellular generation of ROS, and (3) altered EC coupling process. The latter involves prolongation of the AP, Ca 2+i transient and contraction, and blunted force- and relaxation-frequency responses, consequent to changes in the expression and function of Ca2+ transport proteins involved in SR Ca2+ cycling, and in sarcolemmal K+ currents. Studies of sexual dimorphism in the context of cardiac aging and HF and the expansion of the concept of the heart as a continuously self-renewing organ may assist in our understanding the pathophysiology of cardiovascular aging and in the development of effective therapeutic strategies.