ArticlePDF AvailableLiterature Review

Keeping the beat against time: Mitochondrial fitness in the aging heart

Authors:

Abstract and Figures

The process of aging strongly correlates with maladaptive architectural, mechanical, and biochemical alterations that contribute to the decline in cardiac function. Consequently, aging is a major risk factor for the development of heart disease, the leading cause of death in the developed world. In this review, we will summarize the classic and recently uncovered pathological changes within the aged heart with an emphasis on the mitochondria. Specifically, we describe the metabolic changes that occur in the aging heart as well as the loss of mitochondrial fitness and function and how these factors contribute to the decline in cardiomyocyte number. In addition, we highlight recent pharmacological, genetic, or behavioral therapeutic intervention advancements that may alleviate age-related cardiac decline.
Content may be subject to copyright.
Keeping the beat against time:
Mitochondrial tness in the aging
heart
Arielys Mendoza
1
,
2
and Jason Karch
1
,
2
*
1
Department of Integrative Physiology, Baylor College of Medicine, Houston, TX, United States,
2
Cardiovascular Research Institute, Baylor College of Medicine, Houston, TX, United States
The process of aging strongly correlates with maladaptive architectural,
mechanical, and biochemical alterations that contribute to the decline in
cardiac function. Consequently, aging is a major risk factor for the
development of heart disease, the leading cause of death in the developed
world. In this review, we will summarize the classic and recently uncovered
pathological changes within the aged heart with an emphasis on the
mitochondria. Specically, we describe the metabolic changes that occur in
the aging heart as well as the loss of mitochondrial tness and function and how
these factors contribute to the decline in cardiomyocyte number. In addition,
we highlight recent pharmacological, genetic, or behavioral therapeutic
intervention advancements that may alleviate age-related cardiac decline.
KEYWORDS
mitochondria, cell death, mitophagy, autophagy, ROS, metabolism, mitochondrial
biogenesis, mitochondrial tness
1 Introduction
Extending life expectancy while attenuating the negative effects of aging is, arguably,
the overall goal of health sciences (Sierra et al., 2009). Reducing the maladaptive effects of
aging not only lengthens survival, but also preserves bodily functions and enhances overall
tness into the later stages of life. (Vaupel, 2010;Olshansky, 2018). Since the 19th century
we have successfully prolonged the average human lifespan to a natural maximum of
115 years, with the likelihood of individuals surviving an age greater than 125 years being
less than 1 in 10,000 (Dong et al., 2016). This increase in lifespan is due to the major
contributions of modern medical advancements which include preventative measures
such as immunizations and the administration of symptom-targeting therapies like
insulin (Ben-Haim et al., 2017). Inevitably, as the populations life expectancy
increased nearly two-fold within the last century, systematic conditions such as
cancer and heart disease now dominates the majority of late-age mortalities
(Crimmins, 2015). Undoubtedly, understanding age-related changes that occur in the
heart over time in order to therapeutically counter their effects is crucial for preventing
heart disease.
Cardiovascular disease (CVD) includes a collection of pathological heart disorders
such as heart failure (HF), ischemia-reperfusion (I/R) injury, atherosclerosis, and
arrhythmias, which together culminate into the majority of deaths in developed
OPEN ACCESS
EDITED BY
Gopi K Kolluru,
Ochsner LSU Health, United States
REVIEWED BY
Shaul Alam,
Louisiana State University Health
Shreveport, United States
Sergei Vatolin,
Case Western Reserve University,
United States
Chowdhury Sayef Abdullah,
Louisiana State University Health
Shreveport, United States
*CORRESPONDENCE
Jason Karch,
karch@bcm.edu
SPECIALTY SECTION
This article was submitted to Aging,
Metabolism and Redox Biology,
a section of the journal
Frontiers in Aging
RECEIVED 24 May 2022
ACCEPTED 30 June 2022
PUBLISHED 26 July 2022
CITATION
Mendoza A and Karch J (2022), Keeping
the beat against time: Mitochondrial
tness in the aging heart.
Front. Aging 3:951417.
doi: 10.3389/fragi.2022.951417
COPYRIGHT
© 2022 Mendoza and Karch. This is an
open-access article distributed under
the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the
original author(s) and the copyright
owner(s) are credited and that the
original publication in this journal is
cited, in accordance with accepted
academic practice. No use, distribution
or reproduction is permitted which does
not comply with these terms.
Frontiers in Aging frontiersin.org01
TYPE Review
PUBLISHED 26 July 2022
DOI 10.3389/fragi.2022.951417
countries (Tang X. et al., 2020). Cardiomyocyte viability is a
major contributing or initiating factor for many forms of CVD,
and mitochondrial health and function are critical to myocyte
contractility and survival. In this review, we will examine the
major ndings regarding the time-dependent decline of the heart
with respect to: 1) cardiomyocyte drop-out, 2) maladaptive shifts
in major signaling pathways, 3) the contributions of reactive
oxygen species (ROS) and 4) mitochondrial dysfunction. Finally,
we will highlight the current efforts to preserve heart health and
the novel developments in medical interventions, exercise, and
diet to delay the negative consequences of cardiac aging.
2 Cardiomyocyte drop-out over time
During embryonic development, cardiac progenitor cells
from the mesoderm arise and differentiate into their
cardiomyocyte fates, which then form the cardiac tube and
continue to populate what will become the chambers of the
primitive atria and ventricles (Asp et al., 2019). Shortly after
birth, the mammalian heart loses its regenerative capacity
(Figure 1)(Foley and Mercola, 2004). Unlike the embryonic
heart, the adolescent heart loses its ability to proliferate and
instead undergoes growth due to an increase in cell size (Figure 1)
(Günthel et al., 2018). This arrest of proliferative capacity
experienced by cardiomyocytes leads to a total withdrawal
from the cell cycle, induced by the downregulation of pro-
mitotic factors such as cyclins and cyclin-dependent kinases
(Hesse et al., 2018). In contrast, cyclin inhibitors such as p21,
p27, and p57 which antagonize cardiomyocyte mitosis also
become upregulated upon birth (Tane et al., 2014;Hesse
et al., 2018). Therefore, adult cardiomyocytes are post-mitotic
and terminally differentiated (Bergmann, 2019). Additionally,
the adult heart has an extremely low potential for regeneration
(Rizzo et al., 2018). There is evidence for low level regeneration
that occurs in the adult human heart which ranges between a rate
of 0.52% per year, however, this endogenous regenerative
capacity is not sufcient to overcome a catastrophic injury
such as a myocardial infarction (Senyo et al., 2013;Bergmann
et al., 2015). Earlier reports have claimed the existence of cardiac
progenitor pools in extra cardiac (i.e. bone marrow) or resident
tissues that can give rise to new cardiomyocytes (Beltrami et al.,
2003;Martin et al., 2004;Matsuura et al., 2004;Oyama et al.,
2007). Resident cardiac stem cells were identied by their
expression of hematopoietic stem cell markers such as c-kit
and stem cell antigen 1 (Beltrami et al., 2003). However, the
majority of these claims have been discredited and retracted,
causing much controversy within the eld (Li H. et al., 2020). The
classication of cardiac stem cells based on stem cell factor
expression alone was challenged (Sultana et al., 2015;
Vicinanza et al., 2017), and lineage-tracing techniques
demonstrated that previously described cardiac resident stem
FIGURE 1
Cardiac Aging. At the time of birth, the postnatal human heart exhibits a brief period of cell growth through proliferation. However, this
proliferative capacity is lost shortly after birth and heart growth proceeds by an increase in cardiomyocyte (CM) size throughout adolescence (ages
1224). During adulthood (ages 2564), a decline in heart function occurs due to the emergence of pathological age-related stresses, such as
imbalances in force distribution per myocyte as a result of natural CM dropout, altered metabolism, decreased ATP generation, and
accumulation of reactive oxygen species. These stresses consequently lead to mitochondrial dysfunction, increased CM death, inammation,
brosis, and the overall functional decline of the heart late in life.
Frontiers in Aging frontiersin.org02
Mendoza and Karch 10.3389/fragi.2022.951417
cells do not produce cardiomyocytes (Van Berlo et al., 2014;He
et al., 2017). A recent consensus statement was published to
redene the narrative on cardiomyocyte renewal and to assert
agreement of the low regenerative potential of adult myocardium
at both homeostatic and post-injury conditions (Figure 1)
(Eschenhagen et al., 2017). Therefore, cardiomyocyte viability
must stand the test of time, as the cardiomyocytes present at
young adulthood are the same cardiomyocytes present later
in life.
Since cardiomyocytes are naturally long-lived cells it is logical
to assume that they are naturally resistant to cell death. Indeed,
many cell death-regulating factors are suppressed at the
transcript level compared to other tissues in the body (Patel
and Karch, 2020). However, cardiomyocytes are not invincible
and throughout life, there is a slow but gradual loss of
cardiomyocytes, referred to as cardiomyocyte drop-out. It has
been estimated that roughly 33% of cardiomyocytes of the left
ventricle dropout naturally during a persons lifetime, according
to a study which reported that between the ages of 1790 years,
the human heart experiences a drop-out rate of 38 million
cardiomyocytes/yr within the left ventricle and a cell volume
enlargement of the surviving myocytes of 110 μm
3
/yr (Figure 1)
(Olivetti et al., 1991). As a highly contractile organ, the heart
must sustain appropriate levels of rigidity and elasticity to
maintain shape, distribute force, and efciently eject blood in
a controlled fashion (Vikhorev and Vikhoreva, 2018). The slow
decline in myocytes, which are the contractile unit of the heart, is
responsible for increasing mechanical stress per myocyte
(Bernhard and Laufer, 2008). Once a sufcient amount of
dropout has occurred, the heart may compensate by becoming
hypertrophic in order to maintain the workload (Lakatta, 2000;
Fajemiroye et al., 2018). In addition to myocyte hypertrophy,
activated broblasts (myobroblasts) will proliferate and
inltrate the heart to ll the residual space left by dead CMs
in a process known as brosis (Gourdie et al., 2016). A recent
study evaluated the physical tness of 104 healthy volunteers
ranging from 20 to 76 years of age and demonstrated that brosis
negatively impacts the exercise tness of older individuals, as
their hearts cannot eject an adequate volume of blood to the body
(Pandey et al., 2020).
Furthermore, the most common cause of heart failure is due
to a sudden loss of a large portion of cardiomyocytes due to
myocardial infarction (MI) (Hofstra et al., 2000). This occurs
when arteries that provide oxygenated blood to the heart become
blocked, which commonly requires a lifetime of plaque build-up
to occur. Preserving cardiomyocyte viability in the face of these
extreme events will limit the onset of age-related
cardiomyopathies, and a better understanding of what
pathways are engaged during cardiomyocyte drop-out may
provide insight on how to fortify myocytes against a life time
of stressors. The majority of cardiovascular diseases involve or
are initiated by the irreplaceable death of cardiomyocytes
(Benjamin et al., 2019). In terms of cardiac injury and disease
states, at least six types of cell death have been previously
described (Patel and Karch, 2020). Of these cell death
mechanisms, mainly two forms of cell death, apoptosis and
necrosis, occur in the aging heart (Figure 2)(Kajstura et al.,
1996). Apoptosis is regulated mainly by the B-cell lymphoma 2
(BCL-2) family at the level of the mitochondria, where pro-
apoptotic family member effectors, BAK and BAX, can form
homo/hetero-oligomeric pores on the outer mitochondrial
membrane (OMM) to induce mitochondrial outer membrane
permeability (MOMP). MOMP is prevented by anti-apoptotic
BCL-2 family members such as BCL-2. BCL-xL, MCL-1, which
directly interact with pro-apoptotic BCL-2 family members to
inhibit BAX/BAK oligomerization (Otera et al., 2013). Once
MOMP occurs, cytochrome c is released into the cytosol
which leads to the formation of the apoptosome and the
initiation of the caspase cascade (Garrido et al., 2006).
Activation of execution caspases 3 and 7 (CASP3, CASP7)
result in proteolysis, morphological membrane blebbing, and
cell shrinkage, which are all considered hallmarks of apoptosis
(Tait and Green, 2010).
A recent study determined that intrinsic apoptosis signaling
becomes altered in the cardiac muscles of old Fischer 344 rats
(20 months), such that pro-apoptotic Bax protein is increased by
at least 69% and anti-apoptotic protein levels of Bcl-2 were
reduced by 70% comparative to very young rat hearts
(1 month) (No et al., 2020a). This group also observed a
higher number of cleaved CASP3 and TUNEL-positive
myocytes within elderly rat hearts as well, which may indicate
that the change in expression of apoptotic regulators may
exacerbate aging-induced cardiac dysfunction. One team of
investigators concluded that subjecting senile rats to 12 weeks
of exercise helped attenuate age-induced apoptosis, cardiac
remodeling, and BAX/BCL-2 ratio in the heart (Kwak et al.,
2006). Currently, there lacks consensus on the contribution of
apoptosis to age-related cardiomyocyte drop-out (Li H. et al.,
2020). Early studies on elderly human patients determined that
levels of apoptotic death increase with age in the myocardium
(James, 1994), however, more recent investigations also using
human subjects failed to detect a correlation between this form of
age-related cardiomyocyte drop-out and apoptosis (Mallat et al.,
2001).
Necrotic cell death has also been implicated in cardiac aging.
In contrast to apoptosis, the necrotic death of the cardiomyocyte
results in the release of intracellular components due to plasma
membrane permeabilization, which causes an inammatory
response upon the neighboring myocardial tissue (Bernhard
and Laufer, 2008). One investigation determined that the
inhibition of regulated necrosis by administration of
necrostatin-1, which specically inhibits of necroptosis (a sub-
type of necrosis), can signicantly reduce infarct size by 37% of
I/R injury-induced aged rat hearts compared to controls (Garvin
et al., 2018). Necroptosis is governed by the receptor-interacting
protein kinases 1 and 3 (RIPK1, RIPK3) which together become
Frontiers in Aging frontiersin.org03
Mendoza and Karch 10.3389/fragi.2022.951417
the necrosome which phosphorylates mixed lineage kinase
domain-like protein (MLKL) which, upon activation, induces
detrimental membrane permeabilization and subsequent death
via necroptosis (Sun et al., 2012). In a recent investigation, an
anti-aging drug known as Metformin was administered to aged
WT mice for 4 weeks prior to being subjected to I/R injury which
resulted in a decrease in infarct size compared to aged hearts that
received the vehicle control (Li C. et al., 2020). This study
determined that Metformin could be targeting the necroptosis
pathway in a RIP1-RIP3-dependent mechanism, thus providing
anti-aging properties to injured and aged myocardium (Li C.
et al., 2020). In addition, the same group observed that RIPK3-
decient (Ripk3
/
) murine hearts became more resistant to
I/R-induced myocardial necrosis comparative to WT
counterparts. While the association of necroptosis to age-
related cardiomyocyte mortality is still relatively new in the
literature, these reports certainly support the hypothesis of
this form of regulated necrosis occurring in the old heart.
In addition to maturing cardiac tissue becoming increasingly
impaired due to cardiomyocyte drop-out, altered calcium (Ca
2+
)
handling has also been associated with the decline of
cardiomyocyte function. Physiologically, Ca
2+
is central to
various signaling pathways and is tightly regulated by inux/
efux channels and pumps (Modesti et al., 2021). It is well
understood, that the dysregulation of Ca
2+
within the connes
of the cell can lead to the overloading of the ion to toxic degrees.
The mitochondrial calcium uniporter (MCU) is a known
transporter of Ca
2+
into the mitochondrial matrix, which
when exceeds physiological limits, may induce maladaptive
responses such as the opening of the mitochondrial
permeability transition pore (MPTP). The opening of the
MPTP, a non-specic voltage-dependent pore, results in a loss
of mitochondrial membrane potential, a reduction in adenosine
triphosphate (ATP) generation, and overall mitochondrial
dysfunction if prolonged opening occurs (Halestrap and
Pasdois, 2009). Although the molecular identity of the multi-
protein pore has yet to be fully elucidated, its known regulators
are the adenine nucleotide translocases (ANTs) and peptidyl-
prolyl isomerase cyclophilin D (CypD) (Karch and Molkentin,
2014). One previous study reported that deacetylation at lysine
166 of CypD can suppress mitochondrial dysfunction and age-
associated cardiac hypertrophy (Hafner et al., 2010).
There is a consensus that an overload of Ca
2+
perturbs aged
cardiac tissue, evidenced by several reports (Hunter et al., 2012;
No et al., 2018) Furthermore, aged myobrils have been
previously shown to grow insensitively to intracellular Ca
2+
concentrations, which is thought to reect a lack of
contractility and prolonged relaxation in older individuals
(Bernhard and Laufer, 2008). Accurately measuring the extent
of aging-dependent cardiomyocyte dropout in vivo is extremely
FIGURE 2
Mitochondrial Dysfunction in Aging. There are various intracellular pathways that become altered over time, especially with regard to
mitochondrial function. Between the stages of young and late adulthood, mitochondria within the cardiomyocytes experience a decreased level of
biogenesis due to decreased expression of PGC-1 and downregulation of NAD
+
-dependent sirtuins. A metabolic shift occurs in old hearts, resulting
in greater glucose and less fatty acids utilization for energy production. Mitochondrial quality control by mitophagy induction is also
compromised. Cell death levels increase in the form of apoptosis and necrosis, which utilize mitochondrial outer membrane permeabilization
(MOMP) through the Bcl-2 family and mitochondrial permeability transition pore (MPTP), respectively. Excess ROS is generated through increase
electron transport chain (ETC) leak, resulting in increased levels of mitochondrial DNA (mtDNA) damage, imbalance of CM redox states, damage to
lipids/proteins within the mitochondria, and the inability of antioxidants to clear ROS from the microenvironment. Together , these stressors lead to a
decrease in mitochondrial function and lower levels of ATP production.
Frontiers in Aging frontiersin.org04
Mendoza and Karch 10.3389/fragi.2022.951417
challenging and will require novel methods in order to investigate
the potential cell death mechanisms that are utilized. Once these
methods are established, genetic or pharmacological inhibition of
individual cell death pathways will reveal which mechanisms
contribute to age-related cardiomyocyte drop out.
3 Decline in mitochondrial ATP
generation
One organelle at the center of cell death and aging is the
mitochondrion. The mitochondrial theory of aging postulates
that the accumulation of damaged, genetically mutated,
respiratory dysfunctional, and excessively ROS-producing
mitochondria over time correlates strongly with age-related
heart disease (Kowald, 2001;Loeb et al., 2005). Thus, there is
a potential relationship between mitochondrial tness and
cardiomyocyte aging. There are two main types of
mitochondria within cardiomyocytes, one of which are located
beneath the sarcolemma (subsarcolemmal mitochondria, SSM)
or between myofribrils (interbrillar mitochondria, IFM)
(Palmer et al., 1977). Interestingly, there is a decreased
number of IFM in elderly rat hearts (2428 months) but no
change in SSM content comparative to younger adult hearts
(6 months old), which indicates that there is a potential change in
ATP levels within micro domains of the aged myocyte (Fannin
et al., 1999). Structurally, there are no apparent differences
between mitochondria of adult and aged rat hearts using
transmission electron microscopy (Palmer et al., 1985;
Lesnefsky et al., 2016). However, other groups have reported a
noticeable difference in the appearance of mitochondrial cristae
between adult and elderly myocyte mitochondria, as electron
microscopy revealed that SSM isolated from 24-month old rats
have normal lamelliform cristae, whereas IFM cristae display a
more tubular phenotype (Riva et al., 2005). Another study
identied an age-related decrease in the surface area of the
inner mitochondrial membrane (IMM) between 3-months and
24-month-old (Eldarov et al., 2015).
As the heart continuously works to supply blood throughout
the body, it must rely on mitochondria to generate a sufcient
amount of ATP to satisfy the high energetic demand of the
cardiomyocyte, which makes mitochondria a major contributing
factor to cardiac aging (Tang X. et al., 2020). Due to this absolute
necessity for energetic substrate, mitochondria form dense linear
networks along sarcomeres, occupying at least 3040% of
cardiomyocyte cell volume (Cao and Zheng, 2019). Within the
developing fetal and immediate postnatal heart, glucose is the
main source for energy production whereas the utilization of
fatty acids for substrate at this stage of life is very minimal due to
low circulation of fats and high availability of lactate (Girard
et al., 1992;Lopaschuk and Jaswal, 2010). However upon days
after birth, the mammalian heart no longer relies on anaerobic
glycolysis for its main form of energy transduction (Piquereau
and Ventura-Clapier, 2018). Over time, the maturing heart
transitions to metabolizing lipids as a primary source for ATP
in a process known as β-oxidation (fatty acid oxidation, FAO).
Adult cardiomyocytes preferentially utilize fatty acids, unlike
most other cells in the body which typically prefer carbohydrate
metabolism (Lionetti et al., 2011). From adolescence to
adulthood under normal conditions, the heart utilizes
approximately 7090% fatty acid as its primary metabolic
substrate, whereas carbohydrates can provide anywhere
between 1030% of the total acetyl-coenzyme A (CoA)
generated (Tuunanen et al., 2008;Martín-Fernández and
Gredilla, 2016;Piquereau and Ventura-Clapier, 2018).
Conversely, as the heart approaches old age, instead of
utilizing the major metabolite that drives β-oxidation known
as fatty acyl-CoA, pyruvate-derived metabolite acetyl-CoA
becomes the primary metabolite for energy production (Tang
J. X. et al., 2020). Previous reports demonstrated that there is a
40% decline of fatty acid utilization for ATP generation in the
aged mammalian cardiomyocyte, however there is no such
decline in the proportion of carbohydrates metabolized
(Figure 2)(Hansford, 1978;Lakatta and Yin, 1982). One study
determined that this switch to glycolysis occurs around the age of
65 in human subjects, a change that may contribute to the lower
amounts of ATP associated with longevity (Kates et al., 2003;Dai
et al., 2014). The alteration in metabolic behavior of
cardiomyocytes from adulthood to old age is understood to be
a response to stressful stimuli caused by pathological conditions
associated with aging (Picca et al., 2018). Carbohydrate
metabolism utilizes a lower amount of oxygen per ATP
molecule generated, and therefore may be more advantageous
for myocytes during states of hypoxic stress (Ussher et al., 2012).
This emphasizes how adaptive mitochondria must be in times of
stress in order to maintain ATP production (Stanley et al., 2005).
Therefore, the occurrence of a metabolic shift from β-oxidation
to glycolysis pathways is a classic cardiac aging phenotype. This
causes an imbalance that results in lipid toxicity in the aged
cardiomyocyte (Koonen et al., 2007). Indeed, lipotoxicity is a
well-established characteristic of an aged myocardium (Slawik
and Vidal-Puig, 2006).
Despite the time-dependent decrease in fatty acid utilization,
intracellular FA uptake is known to increase upon the rise of
sarcolemmal transporter CD36 expression, which is reported to
be responsible for 50% of all lipid uptake in the mammalian heart
(Koonen et al., 2007;Nagendran et al., 2013). Fatty acid oxidation
metabolite acyl-CoA cannot be utilized for energy production
until it is internalized into mitochondria by carnitine-palmitoyl
transferase-1 (CPT1) (Kolwicz et al., 2013). CPT1 is a rate-
limiting enzyme known to be dramatically downregulated in
old rat hearts, which may further explain the decrease in
oxidative phosphorylation during aging (Long et al., 2012;
Zhang X. et al., 2019). Without adequate CPT1 expression to
regulate mitochondrial fatty acid uptake, the likelihood of
cytoplasmic lipotoxicity increases as these metabolites are
Frontiers in Aging frontiersin.org05
Mendoza and Karch 10.3389/fragi.2022.951417
unable to be processed, leading to potential contractile
dysfunction, cardiac hypertrophy, and eventual heart failure
(Sharma et al., 2004;He L. et al., 2012).
In addition, components of the β-oxidation pathway are
transcriptionally downregulated in the aged heart due to the
decreased expression of their positively regulating transcription
factors such as PPAR, retinoid X receptor-α(RXRα), and PPAR
gamma co-activator 1 (PGC-1) (Lopaschuk et al., 2010;Dillon
et al., 2012). PGC-1 isoforms such as PGC-1αand PGC-1βare
originally both highly expressed in the young heart and are
considered master regulators of mitochondrial biogenesis;
however, cardiac PGC-1α/βexpression is commonly reported
to decrease during aging (Figure 2)(Dillon et al., 2012). One
study observed that the downregulation of PGC-1αand its target
gene estrogen-related receptor α(ERRα) are a key features of the
failing human heart, an observation which may be involved in the
age-related reduction of mitochondrial metabolic capacity (Sihag
et al., 2009). Deletion of either isoform in mice contributes to
decreased mitochondrial biogenesis, lower mitochondrial
volume, and a reduction in both nuclear and mitochondrial
encoded genes within heart and skeletal muscle (Arany et al.,
2005;Lelliott et al., 2006;Adhihetty et al., 2009). Indeed, genetic
ablation of both isoforms (PGC-1αβ
/
mice) leads to extreme
phenotypes such as smaller heart size, sudden cardiac arrest,
atypical mitochondrial morphology, and a detrimentally low
cardiac output, all of which results in low survival beyond a
few days post-birth (Lai et al., 2008). These ndings conrm that
PGC-1 is critical for the energy metabolism and overall health of
the adult heart.
Another major attribute of myocardial aging is the harmful
decrease in ATP generation with time (Figure 2)(No et al.,
2020b). Studies have reported that elderly human and mice
mitochondria express reduced levels of electron transport
chain (ETC) components (complexes I-V), thus lowering total
ATP synthesis rate (Zahn et al., 2006). In states of energy
depletion, ketones such as acetoacetate, acetone, and β-
hydroxybutyrate can also be utilized as alternate substrates for
the generation of energy in cardiomyocytes through a process
known as ketone oxidative phosphorylation (ketosis) (Selvaraj
et al., 2020). Various reports have established that the metabolic
utilization of ketones can therapeutically lessen the maladaptive
effects of cardiac aging by prevention of age-associated
myocardial remodeling, inhibition of apoptosis in elderly
murine myocardium, and contribution of additional oxidative
ATP production (Balietti et al., 2009;Sedej, 2018;Yu et al., 2020).
Notably, a ketone rich diet was also shown to lengthen the
lifespan of adult mice (Roberts et al., 2017). An increase in
ketone bodies, specically β-hydroxybutyrate dehydrogenase 1
(βDH1, also known as βOHB), was observed in murine hearts
showcasing classic features of heart failure, suggestive of a shift to
metabolizing ketone bodies to generate ATP in these contexts
(Aubert et al., 2016). Compared to glucose, βDH1 requires less
oxygen for ATP synthesis, which results in less oxidative stress,
lower inammation, and more efcient energy production (Bedi
et al., 2016). This may demonstrate that the increase of ketone
bodies as a source for energy, much like the metabolic switch
from FAO to glycolysis, may be a protective mechanism to
reduce the intracellular complications that correlate with age.
Nonetheless, there is no current consensus on whether ketosis is
truly a cardioprotective mechanism to prolong cardiomyocyte
life (Nilsson et al., 2016;McSwiney et al., 2018).
As cardiac metabolism becomes altered over time, responsive
enzymes facilitate energy production in order to prevent
cardiovascular deterioration. The silent mating type
information regulation (SIRT) family of proteins, also known
as sirtuins, are key regulators of sustaining metabolic function
and protection against myocardial stressors that commonly
emerge in the aging heart (Cencioni et al., 2015). As
metabolic sensors, sirtuins can sense the energy state of the
cell in order to enhance metabolic efciency as well as
mitochondrial function when necessary. Sirtuins can also
promote ATP generation, for example SIRT3, which is highly
expressed in the heart, increases the enzymatic activity of ATP
synthase β, a catalytic subunit of mitochondrial complex V of the
ETC, through its deactylation activity (Lombard et al., 2007;
Rahman et al., 2014). Indeed, genetic ablation of SIRT3 in the
heart leads to decreases in overall ATP synthesis, OXPHOS
activity, oxygen consumption, and rates of fatty acid and
glucose metabolism (Ahn et al., 2008).
With regards to aging, sirtuins have been observed to
ameliorate classic features of myocardial stresses that arise
over time. SIRT3 overexpression is protective against age-
related cardiac phenotypes (Sundaresan et al., 2009). This
protective role of SIRT3 was further conrmed by another
study, which determined that SIRT3 activity made aged
murine hearts more resistant to I/R-injury (Porter et al.,
2014). Genetic deletion of SIRT2 resulted in an accelerated
aging phenotype, such as spontaneous cardiac hypertrophy,
brosis, and overall dysfunction, whereas overexpression of
SIRT2 improved viability in cultured myocytes (Sarikhani
et al., 2018). SIRT1 expression is considered to be important
for maintaining cardiomyocyte health during aging,
demonstrated by cardiac-specic SIRT1 overexpression which
preserved systolic function and reduced hypertrophy, brosis,
and senescence marker expression in 18 month old mice
(Alcendor et al., 2007).
The ability of sirtuins to act as metabolic sensors is the result
of their activation being dependent on nicotinamide adenine
dinucleotide (NAD
+
), a redox carrier typically converted to
NADH by accepting a hydride group from either glycolysis,
TCA cycle, or fatty acid oxidation, which is crucial for driving
oxidative phosphorylation (Xie et al., 2020). Unfortunately, there
is an age-related decline of NAD
+
that is well known to contribute
to a myriad of aging hallmarks, such as metabolic imbalance,
mitochondrial dysfunction, oxidative stress, and pro-
inammatory conditions (Haigis and Sinclair, 2010;Nakagawa
Frontiers in Aging frontiersin.org06
Mendoza and Karch 10.3389/fragi.2022.951417
and Guarente, 2011). Due to the utilization of NAD
+
as a
substrate for SIRT enzymatic activity, as well as its utilization
by other enzymes such as DNA repair enzyme poly (ADP-
ribose)-polymerase 1 (PARP-1), the pool of available NAD
+
decreases within myocardium during aging (Figure 2)(Pillai
et al., 2005). Complex I governs the oxidation of NADH to
generate NAD
+
, however one study determined that a mouse
model of dysfunctional complex I (Ndufs4
/
) resulted in a
decreased NAD
+
/NADH ratio and heart failure (Karamanlidis
et al., 2013), therefore it is hypothesized that complex I
dysfunction may occur in aged hearts which prevents the
replenishment of NAD
+
stores (Bugger et al., 2016). Since
NAD
+
is consumed and may not be replenished, it is clear
why the sustained imbalance of NAD
+
levels would exacerbate
age-associated depletion of ATP as the metabolic-sensing
abilities of sirtuins would be rendered inactive (Xie et al.,
2020). Ultimately, the decline in sirtuin activity results in
many consequences for the onset and perpetuation of
maladaptive cardiac aging.
In summary, there are various forms of mitochondrial
bioenergetics that generate ATP in the heart such as the ETC,
β-oxidation, glycolysis, and ketosis (López-Otín et al., 2013). As
these aforementioned studies have demonstrated, the
maintenance of fatty acid oxidation as the predominant
source of energy is a characteristic of normal adult heart
function and alterations to this metabolic programming can
accelerate aging phenotypes. Loss of homeostasis between
mitochondrial energy production pathways is due to the
altered expression of key regulatory enzymes such as PGC-1
and the sirtuin family, which results in the metabolic remodeling
and subsequent energy decit that occurs in aged hearts. Further
investigation is needed to determine the cause of age-related
metabolic switching and eventual cardiac decline.
4 Increase in mitochondrial ROS
generation
Reduction and oxidation (redox), a biochemical reaction that
involves the loss or gain of electrons between molecules, is the
source of oxidative stress in the heart which leads to age-related
pathologies (Cortassa et al., 2021). This transfer of electrons can
be mediated by ROS (Fuloria et al., 2021). The mitochondria are
the greatest source of ROS within a myocyte, with the majority of
ROS resulting from the ETC. Within the ETC, NADH and
FADH
2
are responsible for donating electrons to complex I
(NADH dehydrogenase) and complex II (succinate
dehydrogenase), which are then transferred to complex III
(cytochrome b-c1) by way of ubiquinone [coenzyme Q
(CoQ)], then to complex IV (cytochrome c-oxidase), until
they reach the nal acceptor, molecular O
2
(Jang and Javadov,
2018). ROS can be produced in various ways, however it is mainly
derived from the reverse electron transport through complex I by
way of either low CoQ availability or high proton-motive force
(Figure 2)(Robb et al., 2018). ROS can also become generated by
single electrons escaping the ETC in a passive process known as
electron leakage (Zhao et al., 2019). Electrons can interact with
and reduce O
2
to form an extremely unstable superoxide anion
(O
2
or O
2
)(Kausar et al., 2018). To counteract this form of
ROS generation, superoxide dismutase (SOD) initiates the
conversion of O
2
into a more stable form known as
hydrogen peroxide (H
2
O
2
)(Li X. et al., 2019). However, H
2
O
2
is readily interacts with metal atoms to generate a highly reactive
hydroxyl radical (OH
)(Wang et al., 2020). The common
hydroxyl radical (OH
) indiscriminately scavenges electrons
off biomolecules such as proteins, lipids, and nucleic acids,
thus damaging the myocyte.
While it is well established that ROS can be harmful inducers
of oxidative stress which contribute to impaired cardiac health, it
is important to emphasize that these molecular species are
essential for regulating a wide range of physiological
phenomena, as long as intracellular ROS concentrations are
controlled (Murphy et al., 2011). Mitochondrial-derived
oxidants can serve as mediators for a variety of ROS-mediated
signaling pathways important for baseline cardiomyocyte
function (Shadel and Horvath, 2015). An example of this can
be observed from the ability of O
2
and H
2
O
2
to promote
cardiomyocyte growth and viability (Finkel, 2003;Tsutsui
et al., 2009). Furthermore, nitric oxide (NO
, NO) has been
linked to the regulation of mitochondrial biogenesis, improving
OXPHOS function, and ATP content by signaling through its
second messenger, 3,5-cyclic guanosine monophosphate
(cGMP) (Nisoli and Carruba, 2006). NO is synthesized by
endothelial nitric oxide synthase (eNOS) and is derived from
the endothelium of coronary vasculature as well as from
cardiomyocytes (Gödecke et al., 2001). Upon stimulation of
eNOS, increases in NO activity enhance ventricular lling
capacity by lengthening diastolic intervals and lowering
frequency of contractility (Gao, 2010). The contributing
factors that determine which oxidation reaction or signaling
pathway becomes engaged by mitochondrial ROS depends on
the species of ROS generated, radical concentration, duration of
oxidant, and the microdomains in which ROS is produced/
present (Finkel and Holbrook, 2000).
Oxidative stress can be dened as the functional
dysregulation of molecules due to the removal of electrons
and introductions of unusual charges into their structures
(Sies, 2020). According to the radical theory of aging, which
was rst proposed in the mid-20th century, a main contributing
factor of aging is related to deleterious attacks by free radical
species within a cell over time, resulting in additive oxidative
stress unto biological structures that drives the functional decline
in aged animals (Figure 2)(Harman, 1956). Indeed, previous
investigations support this theory as they have demonstrated that
there is an increased production of ROS in the aged heart, such as
the nding that heart mitochondria isolated from elderly rats
Frontiers in Aging frontiersin.org07
Mendoza and Karch 10.3389/fragi.2022.951417
(23 months) produced 25% more super-oxide radicals than
young rats (3 months) (Nohl and Hegner, 1978). Additional
evidence in support of the radical theory of aging is shown by
a knock-out mouse model of the p66
Shc
gene (p66
Shc/
), which
encodes for a mitochondrial redox enzyme that generates ROS
from ETC by-products (Giorgio et al., 2005). The p66
Shc/
mice
study determined that targeting this enzymatic source of ROS
successfully lengthened the lifespan of these animals by 30%
(Migliaccio et al., 1999). Age-related increases of ROS production
results in the accumulation of damaged proteins and
dysfunctional mitochondria, of which exacerbate stress
(Anderson et al., 2018). The increased amounts of oxidized
macromolecules and ROS production levels contribute to a
multitude of age-associated cardiovascular diseases (Toba and
Lindsey, 2019). When this harmful process overwhelms the
cardiomyocyte, there are several known protective processes
that serve to maintain protein and organelle quality, which
will be discussed further in the sections below.
Besides ROS production, oxidative stress also is the result of a
decrease in homeostatic antioxidant activity (Balaban et al., 2005;
Dai et al., 2012). Biological antioxidants such as glutathione
peroxidase or catalase exist in the myocardium to provide
endogenous defenses against ROS (Figure 2)(Snezhkina et al.,
2019). In one previous study, an overexpression mouse model of
human antioxidant catalase (mCAT) increased life span due to
lower levels of oxidative stress, and exhibited reduced cardiac
aging phenotypes such as enhanced contractile function and
mitigated hypertrophy (Schriner et al., 2005). Additionally,
mCAT mice exhibited attenuated mitochondrial H
2
O
2
toxicity, oxidative DNA damage, and mitochondrial DNA
(mtDNA) mutation accumulation. Therefore, the ndings of
this study support the hypothesis that abnormal antioxidant
activity is a limiting factor to maintaining health in the aged
heart, and highlight a potential in utilizing antioxidants as a
therapeutic intervention.
The redox-state of a cardiomyocyte can be determined by
taking a ratio of oxidant to antioxidant levels, a balance that relies
on the conversion between the oxidized and reduced states
(Schafer and Buettner, 2001). Examples of redox couples
commonly measured to assess the redox-state of
cardiomyocytes are NADH:NAD
+
, cysteine:cystine, and GSH:
GSSG (Figure 2). The glutathione redox couple comprises the
majority of redox couples in the cell, therefore, this balance of
reduced GSH to oxidized GSSG has been utilized as a marker for
oxidative stress (Jones, 2002;Wang and Wang, 2021). Once
oxidized, GSSG can scavenge electrons from cysteinyl thiols
within the structures of various proteins in a reaction known
as S-thiolation or glutathiolation (Kouakou et al., 2019). An
imbalanced redox state where oxidizing species dominate results
in an upsurge of S-thiolation, and consequently, the disturbance
of physiological structures and functions of many critical
enzymes. A recent study performed an analysis of the effects
of hypoxia on redox biomarkers within young and aged Wistar
rats and determined that aged hearts were more prone to thiol
(T-SH) group oxidation, and thus, more susceptible to oxidative
stress (Ağaşcıoğlu et al., 2019). Another study supports the
notion that ROS levels are certainly increased in the aged
myocardium, as they observed that age-related ROS levels
only worsen the damaging effects of I/R injury, resulting in
signicantly greater mitochondrial dysfunction and
cardiomyocyte death (Zhang et al., 2020). Furthermore,
sirtuins (SIRT1 and SIRT3) have been associated with
efciently reducing ROS levels in a protective mechanism
against I/R stress in aged hearts, since the genetic deletion of
SIRT1/3 results in excessive oxidative stress, mitochondrial
dysfunction, and cardiomyocyte death (Zhang et al., 2020).
Together these studies demonstrate the importance of
conserving cardiomyocyte redox state, as disturbed redox
homeostasis can lead to various consequences regarding
aberrant signaling, mitochondrial dysfunction, and cell death.
The mitochondrion is the only organelle other than the
nucleus with its own genetic material. Mitochondrial DNA is
circular and comprised of 37 genes, 13 of which encode for
essential ETC subunits, housed within the matrix of the
mitochondrion (Ryzhkova et al., 2018). Notably, the vast
majority of proteins expressed at the level of the mitochondria
are nuclearly encoded and are imported to the mitochondria. The
occurrence of mtDNA point mutations and deletions have been
observed to increase with age in human heart as well as other
tissues (Figure 2)(Cortopassi and Arnheim, 1990). An increase
in the production of mitochondrial radicals presents a potential
risk to mtDNA stability (Lu and Finkel, 2008). One previous
report has shown that there is an increase in H
2
O
2
in SSM and
oxidative stress levels in IFM isolated from the hearts of old male
rats (24 months) compared to younger rats (6 months) (Judge
et al., 2005). As reported in another investigation, the senescence-
accelerated mouse model (SAMP8 mice) displayed an age-
associated increase in lipid peroxidation products indicative of
increase oxidative damage with respect to time (Rodríguez et al.,
2007). The phenomenon of age-related increase of ROS-
dependent mtDNA damage was highlighted by a previous
study in which investigators utilized an oxidative stress
biomarker known as 8-oxo-2-deoxyguanosine (8-OH-dG), a
major product of mtDNA oxidation, and measured this
biomarker in hearts of eight various mammalian species with
a spread of maximal life spans ranging from 3.5 to 46 years (Barja
and Herrero, 2000). This study determined that greater amounts
of 8-OH-dG were detected within short-lived animals, and vice
versa, suggestive that excessive mtDNA damage correlates with
shortened lifespans (Barja and Herrero, 2000). Overall, it is
apparent that oxidative damages upon mtDNA have been
shown by various sources to correlate with increasing age.
Further research has shown that a mouse model of a proof-
reading decient version of mitochondrial polymerase PolgA
(mtDNA mutator mouse, PolgA
mut/mut
) caused various
premature aging phenotypes such as reduced lifespan, weight
Frontiers in Aging frontiersin.org08
Mendoza and Karch 10.3389/fragi.2022.951417
loss, alopecia, and heart enlargement (Trifunovic et al., 2004).
Despite this, recent reports have suggested that mtDNA
deletions, not mtDNA point mutations, serve as the driving
force behind the shortened lifespan observed in these mice
(Vermulst et al., 2008). In contrast to this argument, other
researchers hypothesize that mtDNA replication errors made
by mtDNA polymerases are the driving factor in aging instead of
ROS-dependent mtDNA mutations (Kauppila et al., 2017). It is
also important to note that the mutator mouse model has been
challenged since these animals experience greater levels of
mtDNA mutations than aged humans, and therefore do not
recapitulate the natural aging processes (Khrapko et al., 2006).
Additionally, previous research efforts have shown that there are
very low levels of specic mtDNA deletions attributed to the
phenomenon of aging (Cortopassi and Arnheim, 1990). It is
currently debated whether mtDNA mutations directly cause
aging or simply correlate with the process (Vermulst et al.,
2007). Therefore, the origin of mtDNA mutations and their
contributions to the cardiac aging process remains to be fully
elucidated.
At baseline, mtDNA has a higher mutation rate than nuclear
DNA due to the high presence of radical species in the local
microenvironment (Hu et al., 2020). One mathematical model
demonstrated that 90% of cardiomyocytes will undergo a
minimum of 100 mtDNA mutations by the age of 70, which
likely contributes to age-related mitochondrial dysfunction (Li H.
et al., 2019). Although the nuclear genome is not impacted as
severely, poor nuclear genome stability can occur in the instance
of mitochondrial dysfunction, since imbalanced ROS-to-
antioxidant mode of oxidative stress can eventually surpass
the local microenvironment and induce an increase in nuclear
mutations as well (Veatch et al., 2009). In addition to SIRTs
metabolism-related functions, as previously mentioned in this
review, this family of deacetylases can also increase
cardiomyocyte levels of mitochondrial antioxidants such as
superoxide dismutase 2 (SOD2) in the event of detrimental
oxidative stress (Figure 2)(Imai and Guarente, 2014). Sirtuins
can also engage in nuclear as well as mtDNA damage repair as
they can induce deacetylation reactions directly upon hyper-
acetylated chromatin or correct DNA repair proteins (Tennen
and Chua, 2011).
In summary, there is a critical need to address the excessive
generation of radical species during aging. Due to the
nonselective damage that occurs due to oxidative stress,
radical species and antioxidants both serve as a major
therapeutic targets to deaccelerate age-associated cardiac
phenotypes. Additional research must be implemented to
reduce the oxidative burden of the old heart, and therefore
preserve mtDNA stability, sustained mitochondrial membrane
integrity, and maintain a balance of the overall redox state of the
cardiomyocyte. However, therapeutic attempts to reduce the
burden of ROS to prevent age-related decline have had little
success as discussed in section 7.
5 Decline in mitophagy during aging
Since mitochondria are the central generators of ROS within
the cardiomyocyte, they are constantly faced with the threat of
oxidative damage that can lead to the functional failure of the
organelle (Onishi et al., 2021). In this event, there is a need for
these dysfunctional organelles to be cleared from the cell to
preserve viability (Scheibye-Knudsen et al., 2015). Dysfunctional
mitochondria can be selected for degradation in a sub-type of
autophagy that is known as mitophagy, termed for its
mitochondrion-specic engulfment (Kubli and Gustafsson,
2012). Currently, there are two main forms of this
mitochondrial quality control mechanism that exist in the
mammalian heart: 1) ubiquitin-dependent mitophagy, and 2)
receptor-mediated mitophagy (Ding and Yin, 2012;Jin and
Youle, 2012).
Ubiquitin-dependent mitophagy is regulated by phosphatase
and tensin homolog (PTEN)-induced putative kinase protein 1
(PINK1) and Parkinson Protein 2 (Parkin). PINK1 is targeted to
the mitochondria, while Parkin is a cytosolic protein. Under
normal conditions, PINK1 activity remains negatively
constrained by mitochondrial processing peptidase (MPP) and
presenilin-associated rhomboid-like protease (PARL) via
consistent degradation through cleavage (Jin et al., 2010).
Upon the loss of mitochondrial membrane potential, which is
a classic feature of mitochondrial dysfunction, PINK1 will
translocate on the OMM and mediate the subsequent
translocation of Parkin, an E3 ubiquitin ligase, to the
dysregulated mitochondrion (Matsuda et al., 2010). Parkin
will then ubiquitinate outer membrane substrates such as
voltage-dependent anion channel (VDAC), translocase of the
outer membrane (TOM), mitofusins, BAK, and more (Sarraf
et al., 2013). Autophagy adaptors such as optineurin (OPTN) or
p62/SQSTM1 utilize their ubiquitin-binding domains (UBD) to
identify these ubiquitinated substrates, and consequently recruit
microtubule-associated protein light chain 3 (LC3) to enable
autophagosome engulfment of the mitochondrion for fated
lysosomal fusion and degradation (Ajoolabady et al., 2020).
Receptor-mediated mitophagy is mediated through
mitophagic receptors on the OMM such as FUN14 domain
containing 1 (FUNDC1), Bcl-2-like protein 13 (Bcl2L13/Bcl-
Rambo), FK 506-binding protein (FKBP8), Bcl2/adenovirus
E1B 19-kDa interacting protein 3 (BNIP3), and NIX/BNIP3L
(Novak et al., 2010;Hanna et al., 2012;Liu et al., 2012;Murakawa
et al., 2015;Bhujabal et al., 2017;Wei et al., 2017). All these
receptors are anchored to the OMM and bind directly to LC3 to
guide its tethered organelle to the autophagosome, bypassing the
need for ubiquitination (Liang and Gustafsson, 2020). Another
receptor, Prohibitin-2 (PHB2), found within the IMM also binds
with LC3, but this event typically occurs when the outer
membrane is permeabilized or ruptured (Wei et al., 2017).
Each of these mitophagy receptors can bind to LC3 for the
sequestration of mitochondria by their LC3-Interacting-Regions
Frontiers in Aging frontiersin.org09
Mendoza and Karch 10.3389/fragi.2022.951417
(LIRs), which are conserved domains located at various sites
specic to each regulator. Although these receptors are
established, their mechanisms of activation are still not well
understood (Liang and Gustafsson, 2020). Thus far, it is
understood that some of these receptors are regulated by
phosphorylation status to become activated and bind LC3.
It is interesting to note that BNIP3 and Bcl-Rambo are both
members of the Bcl-2 family of apoptotic regulators, based on the
presence of Bcl-2 homology (BH) domains within their peptide
structures. However unlike BNIP3, the role of Bcl-Rambo in
apoptotic cell death remains to be controversial (Meng et al.,
2021). Containing all four BH domains (BH1-4) Bcl-Rambo has
been shown to either induce cell death upon overexpression into
HEK 293T cells, or promote cell viability as in glioblastoma
cancers, resulting in controversial conclusions within the eld
(Kataoka et al., 2001;Jensen et al., 2014). Despite this ambiguity
in apoptosis, newly emerging studies have demonstrated that Bcl-
Rambo promotes mitochondrial ssion as a precursor to
mitophagy (Murakawa et al., 2015). It has yet to be
determined whether Bcl-Rambo is protective or maladaptive
with respect to the aging heart. BNIP3 is a BH3-only protein
that is typically inactive during physiological conditions, yet
upon stressful stimuli such as hypoxia, it has been previously
associated with the induction of cell death, since its activation has
been shown to correlate with a loss of membrane potential, and
an increase in oxidative stress (Burton and Gibson, 2009). Once
activated BNIP3 becomes upregulated and translocates to the
OMM as a stabilized homodimer, anchored by its C-terminus
domain (Hanna et al., 2012). Interestingly, a recent study
demonstrated that NIX (BNIP3L) and FUNDC1 are critical
for stimulating mitophagy in adult cardiac progenitor cells
(Lampert et al., 2019). Overall, the mitophagic functions of
these Bcl-2 proteins demonstrates that there are various
processes involved with this class of proteins beyond their
inuences on cell death.
There is an accumulation of dysfunctional mitochondria
within the aged myocyte which may be due to the loss of
efcient mitophagy in the old heart (Figure 2)(Linton et al.,
2015). Not only are defective mitochondria of the aged myocyte
not sufciently degraded, a majority of mitochondria become
structurally enlarged and exhibit reduced dynamics such as
ssion, resulting in an increasing number of these organelles
that are poorly engulfed by the autophagosome (Terman et al.,
2010). One study demonstrated that there is a decrease in
PINK1 protein expression in the hearts of middle-aged to
elderly human patients with end-stage heart failure when
compared to PINK1 levels in healthy controls (Billia et al.,
2011). Mitophagy was also determined to be diminished in
old hearts according to another study, where investigators
subjected old vs. young WT mice to a mitochondrial
uncoupler to induce a mitophagic response (Hoshino et al.,
2013). As a result of this study, investigators observed that
mitochondria within the hearts of adult WT mice (10 months)
appeared to be encapsulated within autophagosome vacuoles
more so than the mitochondria of aged mice (20 months)
(Hoshino et al., 2013). Evolutionarily conserved cysteine
residues on mitophagy regulator Parkin are prone to
oxidation by ROS, resulting in the loss of Parkins ubiquitin
ligase activity, misfolding, and subsequent clearance from the cell
(Wong et al., 2007;Meng et al., 2011). Therefore, it has been
postulated that the excess of oxidative stress, a classic feature of
the aged myocardium, may decrease the efciency of Parkin-
mediated mitophagy within the heart (Liang and Gustafsson,
2020). Unfortunately, less is known about the status of the
mitophagy receptors during aging, although BNIP3 is known
to be upregulated in aged hearts (Lee et al., 2011). Interestingly,
BNIP3 and Nix are thought to become maladaptive cell death
inducers during age-related myocardial stresses such as heart
failure and I/R injury, however this harmful shift from promoting
mitochondrial quality control is yet to be fully understood
(Hamacher-Brady et al., 2007;Baines, 2010;Shires and
Gustafsson, 2015).
Pharmacological or genetic alterations of the mitophagy
pathway inuences aging. Chemically inducing mitophagy by
natural compound, urolithin A (UA), can lead to the clearance of
damaged mitochondria and extend the lifespan of C. elegans (Ryu
et al., 2016). Importantly, this result has been translated into the
mouse model system through orally consumed UA which
protects against age-related muscle decline (Ryu et al., 2016).
Additionally, Parkin-decient mice (Parkin
/
) exhibit features of
accelerated cardiac aging such as accumulations of dysfunctional
mitochondria within their hearts and these mice experience
exaggerated cardiac damage and increased mortality when
stressed (Kubli et al., 2013). Conversely, Parkin overexpression
in mice delays cardiac aging and improves mitochondrial health
(Rana et al., 2013). Moreover, cardiac-specic Parkin
overexpression leads to a decrease in abnormal mitochondria,
as well as a resistance to an age-dependent decrease in oxygen
consumption (Hoshino et al., 2013). However, Parkin
overexpression leads to an increase in cardiac brosis
(Woodall et al., 2019), perhaps due to the over activation of
the autophagic pathway. This suggests, while increasing the rate
of mitophagy is protective, there is a threshold where too much
may be maladaptive. Interestingly, transgenic mice that
accumulate mtDNA mutations caused by defective mtDNA
polymerase were not protected by cardiac-specic
overexpression of Parkin (Woodall et al., 2019). This may
demonstrate that there is a minor role of Parkin-dependent
mitophagy in cardiomyocytes, at least concerning
mitochondrial instability genomic driven dysfunction.
Unfortunately, less is known about the status of the
mitophagy receptors during aging, although BNIP3 is known
to be upregulated in aged hearts (Lee et al., 2011).
A powerful genetic tool used as a reporter of mitophagy is the
mtKeima transgenic mice model, which express a pH-sensitive
and lysosome resistant-protein known as Keima which possesses
Frontiers in Aging frontiersin.org10
Mendoza and Karch 10.3389/fragi.2022.951417
dual-excitation uorescence (Sun et al., 2017). Fluorescent Keima
protein originates from coral and is localized to mitochondria
using the mitochondrial targeting sequence of inner-membrane
subunit Cox VIII (Sun et al., 2015). When at neutral pH within
uncompromised mitochondrial, Keima becomes excited at
approximately 458 nm recognized as green uorescence, yet
upon co-localization with the lysosome which contains an
acidic pH of 4.5, the probe gradually shifts its excitation
wavelength to 561 nm which appears as red (Sun et al., 2017).
Using this novel genetic tool, researchers have shown that both
the heart and brain contain higher levels of baseline mitophagy
compared to the other tissues in the body (Sun et al., 2017). In the
future, these mice will be a valuable asset to investigate changes in
mitophagy that occur during aging. Another group engineered a
mouse model known as the αMHC-MitoTimer mice, which
allows investigators to identify mitochondrial turnover with
respect to the passage of time (Stotland and Gottlieb, 2016).
This was accomplished by using MitoTimer, a mitochondrial-
targeted protein which can stably induce cardiomyocyte
mitochondria to uoresce from green-to-red over a course of
several hours, all of which was driven by a cardiac driven alpha-
MHC promoter (Williams et al., 2017). After comparing the
hearts of young (3 weeks) and old (16 weeks) αMHC-MitoTimer
mice, investigators observed a higher population of green-
uorescent mitochondria in young hearts compared to older
littermates, which demonstrated that the rate of mitochondrial
degradation was decreased in the aged cohort (Stotland and
Gottlieb, 2016). In all, the decline in efcient mitochondrial
clearance from the myocyte is one of many central features of
cardiac aging and further studies must be conducted to re-
establish quality control of this vital organelle.
6 The decline of autophagy and the
ubiquitin-proteasome system
It is critical for the non-proliferative cardiomyocyte to
maintain adequate protein quality control, which entails the
regulation of proper protein translation, folding, trafcking,
localization, and degradation. The quality control pathways
which occur commonly within the myocardium are autophagy
and the ubiquitin-proteasome system (UPS), which are
lysosomal- and proteasomal-dependent, respectively (Sun-
Wang et al., 2020). Here in this section we will review
previously published research of these protein degradation
mechanisms with special emphasis on the implications of the
aged myocardium.
Autophagy is a mechanism important for maintaining the
quality of proteins and organelle systems by selective lysosome-
dependent degradation of defective intracellular components
(Saha et al., 2018). There are three types of autophagy known
as macro-autophagy, micro-autophagy, and chaperone-mediated
autophagy. However, here we will focus primarily on macro-
autophagy as it is the most widely characterized form that exists
within the heart (Yamaguchi, 2019). Autophagy can serve either
protective or maladaptive roles when upregulated in response to
a stressful event, dependent on the pathological context
(Sciarretta et al., 2012). The functional unit of autophagy is
the autophagosome, a double lipid membrane that encapsulates
cargo destined for degradation once it fuses to a lysosome. The
initiation of autophagy begins with the seeding of an isolated
membrane known as the phagophore. Phagophore formation is
controlled by two multi-protein complexes, the Atg13/ULK-1/
Atg101/FIP200 heterotetratmer which is regulated tightly by
metabolic sensors mammalian target of rapamycin complex 1
(mTORC1) and AMP-activated protein kinase (AMPK) (Alers
et al., 2012). Once the ULK1 complex forms at designated
autophagophore assembly sites, it initiates phagophore
formation by the recruitment and subsequent activation of the
class III phosphoinositide 3-kinase (PI3K) complex I which is
composed of Vps34, Vps15, Beclin-1, and Atg14 (Russell et al.,
2013). The PI3K complex is responsible for producing
phosphatidylinositol 3-phosphates (PI3Ps) which recruits yet
another complex known as the ATG2-WIPI complex (Al-Bari
and Xu, 2020). The phagophore can expand in size as further
regulatory proteins are recruited at the assembly site such as the
ubiquitin-like proteins that behave like E1 (ATG7), E2
(ATG10 and ATG3), and E3 (ATG12-ATG5-
ATG16 L complex) UPS enzymes (Geng and Klionsky, 2008).
Altogether, these complexes complete the maturation of the
phagophore into the nascent autophagosome by way of
ATG4-induced conjugation of LC3-I (ATG8) to
phosphatidylethanolamine (PE) to form LC3-II (ATG8-II)
(Geng and Klionsky, 2008). The lipidation of cleaved LC3-I to
form LC3-II is critical for autophagy, hence, LC3-II is a common
marker of autophagy (Ghosh and Pattison, 2018). Adaptor
proteins such as p62/SQSTM1 link the damaged cargo to
LC3-II using ubiquitin-associated domains and LIRs,
respectively (Gatica et al., 2018;Johansen and Lamark, 2020).
This process nalizes once the autophagic machinery fully
engulfs damaged cargo and undergoes fusion with the
lysosome, causing all sequestered contents to become
degraded by lysosomal enzymes (Ravikumar et al., 2010).
Amino acids can then be released into the cytosol for nutrient
recycling through the amino acid transporter SLC38A9 and Rag
GTPase-Ragulator complex which each exist on the lysosomal
membrane (Wyant et al., 2017). The release of essential amino
acids from the lysosome ensures the continuous growth and
survival of the cell.
The UPS mediates the non-lysosomal degradation of
damaged or mutated proteins (Zheng and Shabek, 2017). This
mechanism initiates once ubiquitin-activating enzyme (E1)
activates ubiquitin in an ATP-dependent manner, then
ubiquitin conjugation enzymes (E2) attach multiple ubiquitin
molecules to the lysine residues of abnormal proteins fated for
degradation (Akutsu et al., 2016). After further interaction by
Frontiers in Aging frontiersin.org11
Mendoza and Karch 10.3389/fragi.2022.951417
E3 ligase enzymes, which acts as a scaffold for the targeted
substrate and the E2-ubiquitin complex to achieve a
signicant ubiquitination status (Pohl and Dikic, 2019). The
ubiquitinated protein is then ready for digestion by the 26S
proteasome (Roos-Mattjus and Sistonen, 2004). The 26S
proteasome is a 2.5 MDa multi-protein complex that consists
of a 33 subunit assembly known as the 20S core particle which is
capped by the 19S regulatory particle (Rousseau and Bertolotti,
2018). During physiological conditions, UPS is an ATP-
dependent quality control mechanism, yet in times of
pathological cardiac stress, the 26S proteasome can perform
degradative actions in an ATP-independent manner (Kumar
Deshmukh et al., 2019). The UPS system responsible for the
majority of intracellular protein maintenance via degradation, as
approximately 8090% of all dislocated, misfolded, oxidized, and
dysfunctional proteins become eliminated through UPS-
mediated clearance (Bhattacharyya et al., 2014). Therefore,
UPS is a highly efcient mechanism that maintains
cardiomyocyte health. It is important to note that while the
UPS mechanism shares similarities with the autophagic pathway
such as a similar goal and the use of ubiquitin-type enzymes, the
proteasome targets more transiently-active proteins such as
tumor suppressor p53, whereas the lysosome degrades larger,
more long-lived proteins such as the myosin heavy chains and
entire organelles that cannot t within the connes of the
proteasome core (Zhang et al., 2016).
The UPS system has been established to participate in the
turnover of proteins which exist on the OMM (Xu et al., 2011).
Originally it was proposed that proteins contained within the
mitochondria were not degraded by the UPS, but instead by
mitochondrial proteases, due the inaccessibility of the UPS
system (Langer and Neupert, 1996). However, this hypothesis
has been recently challenged by emerging evidence suggesting
that intramitochondrial proteins undergo ubiquitination (Udeshi
et al., 2013). It is important to note that 62% of the mitochondrial
proteome is ubiquitinated in humans, with a majority of these
proteins localized within the matrix and IMM (Lehmann et al.,
2016a). One group of investigators determined that degradation
of internally housed mitochondrial proteins can indeed be UPS-
dependent, since they observed that pharmacological inhibition
of the cytosolic 26S protesome led to the increase in levels of a
IMM protein known as uncoupling protein 2 (UCP2) (Azzu and
Brand, 2010). Other known matrix and IMM proteins have also
been hypothesized to undergo UPS-associated proteolysis
(Lehmann et al., 2016b;Lavie et al., 2018). The mechanism by
which inner membrane and matrix proteins can be degraded
has yet to be fully elucidated, however one potential hypothesis
is the mitochondria-associated degradation (MAD) pathway,
which lead to the extraction of these proteins out of the
organelle by Cdc48 (p97/VCP in higher eukaryotes) for
degradation by the proteasome (Franz et al., 2014;Liao
et al., 2020). Other than the shuttling of intramitochondrial
proteins to the proteasome, the proteasome can also become
recruited to the mitochondrial outer membrane via anchoring
onto FK506-binding protein 38 (FKBP38) (Nakagawa et al.,
2007). Further studies must be conducted to fully comprehend
the extend of UPS-dependent degradation of mitochondrial
contents with respect to aging.
Accordin