You are on page 1of 11

CARDIAC MUSCLE

Cardiac or heart muscle resembles skeletal muscle in some ways: it is striated and
each cell contains sarcomeres with sliding filaments of actin and myosin.

However, cardiac muscle has a number of unique features that reflect its function of
pumping blood.

• The myofibrils of each cell (and cardiac muscle is made of single cells — each
with a single nucleus) are branched.
• The branches interlock with those of adjacent fibers by adherens junctions. These
strong
• Junctions enable the heart to contract forcefully without ripping the fibers apart.

This electron micrograph (reproduced with permission from Keith R. Porter and Mary A.
Bonneville, An Introduction to the Fine Structure of Cells and Tissues, 4th ed., Lea &
Febiger, Philadelphia, 1973) shows an adherens junction and several of the other features
listed here.

• The action potential that triggers the heartbeat is generated within the heart itself.
Motor nerves (of the autonomic nervous system) do run to the heart, but their
effect is simply to modulate — increase or decrease — the intrinsic rate and the
strength of the heartbeat. Even if the nerves are destroyed (as they are in a
transplanted heart), the heart continues to beat.
• The action potential that drives contraction of the heart passes from fiber to fiber
through gap junctions.
o Significance: All the fibers contract in a synchronous wave that sweeps from
the atria down through the ventricles and pumps blood out of the heart.
Anything that interferes with this synchronous wave (such as damage to part
of the heart muscle from a heart attack) may cause the fibers of the heart to
beat at random — called fibrillation. Fibrillation is the ultimate cause of
most deaths and its reversal is the function of defibrillators that are part of
the equipment in ambulances, hospital emergency rooms, and — recently —
even on U.S. air lines.
• The refractory period in heart muscle is longer than the period it takes for the
muscle to contract (systole) and relax (diastole). Thus tetanus is not possible (a
good thing, too!).
• Cardiac muscle has a much richer supply of mitochondria than skeletal muscle.
This reflects its greater dependence on cellular respiration for ATP.
• Cardiac muscle has little glycogen and gets little benefit from glycolysis when the
supply of oxygen is limited.
o Thus anything that interrupts the flow of oxygenated blood to the heart leads
quickly to damage — even death — of the affected part. This is what
happens in heart attacks.

1. ARRANGEMENT OF CARDIAC MUSCLE

Characteristic of cardiac muscle are intercalated discs and branched striated cells.
Cardiac muscle cells do not form syncytia as doe’s skeletal muscle. Their large nucleus is
centrally located. By careful use of the fine focus you will see striations in regions of this
section where the fibers have been cut longitudinally. Note the branched appearance of
the cardiac myocytes. Intercalated discs appear as single thin dark lines.

In cross section, cardiac cell have a punctate or dotted appearance (due to the
arrangement of their myofibrils). The centrally located nucleus of the cardiac muscle cell
may not be seen if the section is made at either pole of the cell, missing the nucleus. In
sections where the nucleus is absent, you may see a clear central zone (lacking
myofibrils) surrounded by the punctate appearance of the crosscut myofibrils

On this composite slide of the three types of human and mammalian muscle find
the section of cardiac muscle and compare it with the section of skeletal muscle. Look for
intercalated discs and striated branching cells in the cardiac muscle.

What can be determined about the size of the cardiac myocytes in comparison with
the skeletal muscle fibers? Examine the arrangement of connective tissue surrounding the
cardiac myocytes and bundles of myocytes.
2. WALL OF THE HEART

The ventricular muscle and compare the thickness of the walls of the ventricle with that
of the atrium. Can you see any obvious differences in the muscle fibers of these two
regions? Observe capillaries and connective tissue between the muscle cells. Compare the
appearance of longitudinally sectioned cardiac muscle with that transversely sectioned.

3. PURKINJE FIBERS

Purkinje fibers which are modified myofibers that carry the impulse for contraction
into the myocardium. They are much larger in diameter than the myocytes and often willl
have two nuclei and a relatively pale sarcoplasm as myofibrils are displaced by the
nuclear and perinuclear organelles.

This is a slide of the special fibers located in the heart called Purkinje fibers (white
arrows). The yellow arrows are pointing to the normal cardiac cells found in most of the
heart. Can you notice the different staining pattern of the purkinje fibers than the regular
cardiac cells?

White arrow - Purkinje Fibers Yellow arrow - Cardiac Muscle Cell
SODIUM ION-CALCIUM ION EXCHANGE

Researchers have been aware of the existence of a membrane transporter in cardiac
myocytes that exchanged calcium for sodium for more than 30 years. In that period of
time, even as the molecular details of the sodium-calcium exchanger (NaCaX) were being
worked out, its physiological role had still not been clearly defined. Early reports focused
on the role of the exchanger as an important source of Ca2+ influx and as a regulator of
contractile force, an important issue given that the most commonly used inotropic agent at
the time was digitalis. By the mid-1980s, when it had become clear that Ca2+ entry via L-
type Ca2+ channels was the major trigger for contractile Ca2+ release from the
sarcoplasmic reticulum (SR), it became widely accepted that the chief role of the
exchanger was to remove Ca2+ from the cytosol during diastole. Interest in the exchanger
as an important Ca2+ entry mechanism resurfaced early in this decade when LeBlanc and
Hume demonstrated sodium-current–dependent Ca2+ influx via the exchanger, capable of
triggering contraction; however, this idea has remained controversial. Now, as we
approach the new millennium and study excitation-contraction (E-C) coupling at the
subcellular level, the importance of the exchanger as a modulator of Ca2+ release and E-C
coupling gain is becoming clearer.

The pathophysiological roles of the exchanger have been much less controversial.
In the ischemic/reperfused heart, a rise in intracellular sodium frustrates the ability of the
exchanger to remove Ca2+.Stimulation of the exchanger by oxygen free radicals may
further accelerate the rise in cell Ca2+ under sodium-loaded conditions. The resulting Ca2+
overload is not only detrimental to contractile function, metabolism, and cellular
integrity, but it is also thought to be an important cause of spontaneous Ca2+ release from
the SR. This in turn triggers the transient inward current (ITI), of which NaCaX is a major
component.This inward current underlies delayed afterdepolarizations (DADs), the source
of deadly triggered ventricular arrhythmias. Ca2+ overload via NaCaX and subsequent
activation of ITI during spontaneous Ca2+ release from the SR is also a well-known
complication of overzealous digitalis treatment. This relatively rare complication is
characterized by a chaotic focal arrhythmia.

With regard to heart failure, a fairly common finding among several different
animal models, as well as in human tissue, is an increase in NaCaX mRNA and protein.
In heart failure, an upregulated exchanger has generally been regarded in a positive light,
for it has been assumed that the exchanger acts as a compensatory Ca2+ entry mechanism,
taking up the slack in an otherwise dysfunctional E-C coupling system. The exchanger is
also credited with helping to preserve diastolic function in failing hearts.
In this issue of Circulation Research, Pogwizd et al focus our attention on a
negative consequence of upregulated NaCaX in heart failure: arrhythmogenesis. Given
the prevalence of sudden death due to arrhythmias in the 4.6 million patients living with
heart failure in the United States, any information that helps unravel the pathogenesis of
this lethal aspect of congestive heart failure is of supreme importance. The authors have
used a multipronged approach in their study. Their elegant model of arrhythmogenic heart
failure in the rabbit is created by a combination of volume and pressure overload due to
mechanical injury of the aortic valve, followed 2 to 4 weeks later by aortic constriction.
The model is relevant to human valvular cardiomyopathy, and its attractiveness as a
general model of heart failure is further enhanced by its independence from the genetic
abnormalities so common to many other models, such as the spontaneously hypertensive
rat. The authors used echocardiography to validate the presence of aortic regurgitation and
subsequently to document the extent of left ventricular enlargement and dysfunction.
Continuous electrocardiographic monitoring was carried out on all animals to document
an impressive burden of ventricular ectopy, which is one of the unique features of this
model. Ten percent of the animals died suddenly over the 2-year course of the study,
similar to the annual incidence of sudden death in human dilated cardiomyopathy.

Cardiac NaCaX mRNA and protein levels were on average elevated in the heart
failure group, and the highest levels of NaCaX mRNA were found in the hearts with the
worst left ventricular function. However, half of the hearts had exchanger mRNA levels
that were no different from control. Functionally, patch-clamp experiments demonstrated
2-fold increase in NaCaX current, whereas Ca2+ current (ICa) was unchanged. Increased
NaCaX was also confirmed by a functional assay: an increase in the rate of relaxation and
rate of decline of the [Ca2+]i transient during continuous application of caffeine to disable
SR Ca2+ uptake. A surprising result was that there was no difference in the half time of
relaxation after an electrically stimulated twitch, despite the finding that NaCaX was
increased 2-fold. This is typical of other heart failure models, where SERCA2 levels are
generally decreased, accounting for a reduction in SR Ca2+ uptake. However, SERCA2
levels were not significantly lower in the rabbit heart failure model in the Pogwizd et al
study, although two of the animals with the worst left ventricular function apparently did
show a marked decrease in SERCA2. Considering that SERCA2 levels were on average
unchanged, one can only speculate that the function of the SR Ca2+ pump was reduced by
other mechanisms: either by altered expression of a regulatory protein such as
phospholamban (or its phosphorylation state) or perhaps a change in the metabolic state of
the cell. SR Ca2+ content appeared to be decreased, but this is presumably a consequence
of successful competition for Ca2+ by NaCaX over the SR Ca2+ pump.

An important feature of the Pogwizd et al study is that single cells isolated from the
failing hearts exhibited spontaneous contractions, suggestive of DADs, when treated with
isoproterenol. Unfortunately, confirmation of a "DADogenic" mechanism by membrane-
voltage recordings was not included. Nevertheless, the dependence of spontaneous
activity due to DADs on isoproterenol makes sense for two reasons. First, at the cellular
level, isoproterenol enhances both Ca2+ entry and SR Ca2+ uptake to promote Ca2+
overload. Second, at the clinical level, heart failure is associated with an increase in
circulating catecholamines, and chronic treatment with ß agonists is associated with an
increase in sudden death. The lack of spontaneous activity in the absence of isoproterenol
also serves to underscore the potential pitfalls of experiments in isolated cells, which at
best can only simulate the complex in vivo environment within which these clinical
syndromes occur.

Myriad models of heart failure exist, and the relevance of each to human heart
failure is of paramount importance when considering the significance of experimental
results. Pogwizd et al have already demonstrated in clinical studies that focal arrhythmias
predominate in idiopathic dilated cardiomyopathy, compared with ischemic
cardiomyopathy where reentrant arrhythmias are more common. Furthermore, they have
demonstrated in 3D mapping studies that the rabbit volume/pressure overload model of
heart failure appears to faithfully recapitulate the focal ventricular arrhythmias observed
in humans. Why other animal models of heart failure have not exhibited such arrhythmias
is uncertain, although perhaps we have simply not looked hard enough.

It is tempting to compare the rabbit heart failure model with a transgenic model of
NaCaX overexpression in mice. In the transgenics, postpartum females have a higher
incidence of heart failure and death compared with their wild-type postpartum littermates
(Ken Philipson, personal communication, October 1999). However, unlike the rabbit
heart failure model, the transgenic mice exhibit stable or even increased SR Ca2+ content
without any evidence for functional impairment of the SR Ca2+ pump. This implies that in
the background of an isolated genetic program to increase NaCaX, the SR Ca2+ pump can
compensate and effectively compete for Ca2+. Nevertheless, these mice do show a
predilection for premature death, and the increase in NaCaX is the obvious commonality
to the rabbit heart failure model. Although differences in species may confuse the issue,
these observations both implicate the exchanger and reaffirm the complexity of the failing
heart compared with the transgenic model. And just as one Ca2+ handling mechanism may
compensate for another in a model of heart failure, similar compensatory mechanisms
must also operate in transgenic animals. The challenge now is to take advantage of the
availability of newer methodologies, such as adenovirus vectors, which enable
investigators to acutely overexpress proteins such as NaCaX. This may evade the problem
of compensation, which should help to sort out the issue of whether exchanger
upregulation is a primary or secondary event in heart failure. Furthermore, this method
will enable investigators to test important hypotheses regarding the role of the exchanger
in arrhythmogenesis as well as modulation of E-C coupling.

Finally, how can we place these results in a clinical context? At the present time,
there are no satisfying therapies for preventing the onset of lethal ventricular arrhythmias
in patients with heart failure. The most effective therapies, eg, implantable cardioverter
defibrillators, are clumsy and expensive. Although it is unclear whether the dominant
mechanism of sudden death in humans is a "triggered" arrhythmia or "reentry," it is likely
that a premature ventricular contraction, initiated by a DAD, serves as the trigger for a
reentrant ventricular tachycardia that degenerates to ventricular fibrillation. Whether
NaCaX is an appropriate therapeutic target for preventing triggered arrhythmias is
uncertain, especially given the Cardiac Arrhythmia Suppression Trial (CAST), which
targeted arrhythmia initiation with disastrous results. However, a specific blocker of
NaCaX was not clinically available or tested in that trial. Effective blockers of NaCaX
(other than XIP) remain elusive, although isothiourea derivatives show some promise.
Whether these can be of any clinical benefit remains to be seen.

CALCIUM ION-ATPase EXCHANGE

Calcium, an ion acting as a cellular signal in virtually all cells, plays a special role
in muscles. It is the signal that stimulates muscles to contract. In the resting state, the
levels of Ca2+ near the muscle fibers are very low (approximately 0.1 M ), and nearly all
of the calcium ion in muscles is sequestered inside a complex network of vesicles called
the sarcoplasmic reticulum, or SR. Nerve impulses induce the sarcoplasmic reticulum
membrane to quickly release large amounts of Ca2+, with cytosolic levels rising to
approximately 10 M . At these levels, Ca2+ stimulates contraction. Relaxation of the
muscle requires that cytosolic Ca2+ levels be reduced to their resting levels. This is
accomplished by an ATP-driven Ca2+ transport protein known as the Ca2+-ATPase. This
enzyme is the most abundant protein in the SR membrane, accounting for 70 to 80% of
the SR protein. Ca2+-ATPase bears many similarities to the Na+,K+-ATPase. It has a -
subunit of the same approximate size, it forms a covalent E-P intermediate during ATP
hydrolysis, and its mechanism of ATP hydrolysis and ion transport is similar in many
ways to that of the sodium pump.
Fig: Some of the sequence homologies in the nucleotide binding and phosphorylation
domains of Na+,K+-ATPase , Ca2+-ATPase, and gastric H+, K+-ATPase.

The amino acid sequence of the -subunit is homologous with the sodium pump -
subunit, particularly around the phosphorylation site and the ATP-binding site (Fig). Ten
transmembrane helical segments are predicted from hydropathy analysis, as well as a
“stalk” consisting of five helical segments (Fig). This stalk lies between the membrane
surface and the globular cytoplasmic domain containing the nucleotide-binding domain
and the site of phosphorylation. The E-P formed by SR Ca2+-ATPase is an aspartyl
phosphate like
+ +
that of Na ,K -ATPase ,
in this case Asp residue
351.
Fig: The arrangement of Ca2+-ATPase in the sarcoplasmic reticulum membrane. Ten
transmembrane segments are postulated on the basis of hydropathy analysis.

Two Ca2+ ions are transported into the SR per ATP hydrolyzed by this enzyme,
and the mechanism (Figure 10.15) appears to involve two major conformations, E1 and
E2, just as the Na+,K+-ATPase mechanism does. Calcium ions are strongly occluded in the
E1-Ca2-P state, and these occluded ions do not dissociate from the enzyme until the
enzyme converts to the E2-Ca2-P state, which has a very low affinity for Ca2+. In the E1-
Ca2-P state, the transported Ca2+ ions are bound in the transport channel.

CHANNELOPATHY

MIXED SIGNALS IN HEART FAILURE: CANCER RULES

In the world according to Webster, heart failure is a form of cardiac cancer.
Although primary cardiac malignancies are among the rarest of human diseases, this
singular viewpoint is substantiated by the massive, abnormal, "tumor-like" growth in
cardiac muscle that accompanies heart failure. The biological principles of cell growth,
death, and survival are as important in the onset of heart failure as in tumor progression,
and the molecular signals for cell proliferation and cardiac myocyte hypertrophy are
highly conserved. Both diseases are inexorable and progressive, characterized by clinical
stages that predict survival and outcome, ultimately resulting in a terminal phase. There
are "multi-hit" pathways for both cancer and heart failure progression, largely based upon
the interplay between genetic susceptibility and environmental stimuli. Like cancer, heart
failure represents one of the most important unmet clinical needs in medicine today.

Heart failure remains poorly understood and is largely treated symptomatically by a
complex regimen of drugs whose actions we are neither entirely sure of nor comfortable
with. The lack of new biologically targeted therapy reflects the combined result of the
prohibitive cost of large-scale survival trials of adjunctive therapy designed to
incrementally slow heart failure progression, and the lack of clear clinical and/or
molecular surrogates that have predictive value. In short, the pipeline of heart failure
drugs is running dry.

Perhaps the time has come to attempt to dissect the mixed signals for heart failure
from the viewpoint of recent advances in cancer biology. On a molecular level, the failing
heart represents the end result of multiple cues for the growth, death, and survival of
cardiac myocytes, many of which are shared with signaling pathways in cancer biology.
In the failing heart, the underlying disease is also driven by multiple positive and negative
signaling pathways, but these are connected to specific phenotypic endpoints that are
more physiologically complex, e.g., electrical conduction, contractility, relaxation,
mechanical activation, and chamber morphogenesis. The development of new,
biologically targeted therapies for cancer has been fostered by placing therapy into the
framework of fundamental pathways that control the cell cycle and subsequent tumor cell
proliferation. In a similar manner, understanding the molecular logic of heart failure will
ultimately require forming mechanistic connections between defined clinical disease
surrogates with specific positive and negative molecular checkpoints that arise at specific
stages during the natural temporal progression of this chronic disease. In the current post-
genome world, a new wave of work in heart failure has begun to attack the integrative
biology of heart failure at multiple levels: genomic, genetic, and physiological in
creatures great and small. This brief review will highlight recent progress in the field and
point out new directions for research into this disease process.

MYOPATHY

MYOCARDIAL CELL DEATH IN HUMAN DIABETES

The renin-angiotensin system is upregulated with diabetes, and this may contribute
to the development of a dilated myopathy. Angiotensin II (Ang II) locally may lead to
oxidative damage, activating cardiac cell death. Moreover, diabetes and hypertension
could synergistically impair myocardial structure and function. Therefore, apoptosis and
necrosis were measured in ventricular myocardial biopsies obtained from diabetic and
diabetic-hypertensive patients. Accumulation of a marker of oxidative stress,
nitrotyrosine, and Ang II labeling were evaluated quantitatively. The diabetic heart
showed cardiac hypertrophy, cavitary dilation, and depressed ventricular performance.
These alterations were more severe with diabetes and hypertension. Diabetes was
characterized by an 85-fold, 61-fold, and 26-fold increase in apoptosis of myocytes,
endothelial cells, and fibroblasts, respectively. Apoptosis in cardiac cells did not increase
additionally with diabetes and hypertension. Diabetes increased necrosis by 4-fold in
myocytes, 9-fold in endothelial cells, and 6-fold in fibroblasts. However, diabetes and
hypertension increased necrosis by 7-fold in myocytes and 18-fold in endothelial cells.
Similarly, Ang II labeling in myocytes and endothelial cells increased more with diabetes
and hypertension than with diabetes alone. Nitrotyrosine localization in cardiac cells
followed a comparable pattern. In spite of the difference in the number of nitrotyrosine-
positive cells with diabetes and with diabetes and hypertension, apoptosis and necrosis of
myocytes, endothelial cells, and fibroblasts were detected only in cells containing this
modified amino acid. In conclusion, local increases in Ang II with diabetes and with
diabetes and hypertension may enhance oxidative damage, activating cardiac cell
apoptosis and necrosis.

Fig: Control (A) and DH (B) hearts stained with α-sarcomeric actin (red fluorescence;
myocyte cytoplasm), propidium iodide (blue fluorescence; nuclei), and laminin (yellow
fluorescence; interstitium). Cross sections of myocytes are apparent.